United States Health Effects Research
Environmental Protection Laboratory
Agency Research Triangle Park NC 27711
EPA-600/1-80-019
May 1980
Research and Development
Rapid Field
Measurements of
Organophosphorus
Pesticide Residues
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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 nine series These nine broad cate-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-80-019
May 1980
RAPID FIELD MEASUREMENTS
OF ORGANOPHOSPHORUS PESTICIDE RESIDUES
by
Francis A. Gunther, Ben Berck, and Yutaka Iwata
Department of Entomology
University of California
Riverside, California 92521
R805 642-01
Project Officer
Ronald L. Baron
Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
*
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environ-
mental Protection Agency, now does mention of trade names or
commercial products constitute endorsement or recommendation for
use.
,.,
11
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FOREWARD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the rela-
tive risk of existing and new man-made environmental hazards is
necessary for the establishment of sound regulatory policy. These
regulations serve to enhance the quality of our environment in order
to promote the public health and welfare and the productive capacity
of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in
toxicology, epidemiology, and clinical studies using human volunteer
subjects. These studies address problems in air pollution, non-
ionizing radiation, environmental carcinogenesis and the toxicology
of pesticides as well as other chemical pollutants. The Laboratory
participates in the development and revision of air quality criteria
documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration
of new pesticides or proposed suspension of those already in use,
conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency
is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure
the adequacy of health care and surveillance of persons having suffered
imminent and substantial endangerment of their health.
111
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This report describes a simple, low-cost Rapid Field Method
(RFM) for the on-site determination of organophosphorus insecticide
residues on foliage and in surface soil. The RFM will help ensure
the safety of workers reentering fields following application of
organophosphorus insecticides.
F. G. Hueter, Ph.D.
Acting Director
Health Effects Research Laboratory
IV
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PREFACE
Rapid analytical methods are needed to monitor and reduce the
occupational hazards of exposure to insecticide residues, such as
are involved in the Worker Reentry Problem (Gunther et al., 1977),
and to ascertain that the residue levels on the foliage of fruit
and vegetable crops and on the surface soil in the worker pathways
are sufficiently low to permit safe reentry of field workers engaged
to cultivate, prune, thin, and harvest argicultural crops.
Organophosphorus (OP) insecticides and acaricides are used on
a worldwide scale to control pests of fruit, vegetable, cereal and
oil crops, cotton, nuts, etc. Globally, the amount of land area for
crop production treated with OP and other agricultural chemicals is
large, and the considerable number of field laborers (over 300,COO
in California alone) underlines the need to obtain worker-transfer-
able residue data quickly and accurately to ensure the occupational
health of field workers through prevantive measures.
Current state and federally prescribed "reentry intervals" used
to protect workers have been criticized as being arbitrary and over-
simplistic for not taking into account modifying factors such as
regional differences in temperature, rainfall, soil moisture,
relative humidity, wind speed and direction, nature of formulation,
dosage rate, and method of application. Thus, the wide use, for
economic reasons, of concentrated OP formulations for low-volume and
ultra low-volume spray application raises OP residue levels and
extends the residue disappearance curves. Also, in contrast to
v
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conventional direct application as dusts and sprays, controlled-
release formulations of OP compounds encapsulated within a polymeric
matrix prolong the ultimate disappearance of the OP molecules.
The rate of disappearance of OP residues is variable. Depend-
ing on the type of soil, the persistence patterns of OP compounds
may be prolonged due to physical binding (sorption) by soil particles
(particulates) on soil and foliage that stabilize the residues and
protract their ultimate dissipation. These particulates become
airborne as a result of air currents, the normal movement of workers,
and farm machinery in action. They are thus transferred from orchard
soil and leaf surfaces to the clothing, lungs, hair, and skin surfaces
of field workers.
Monitoring chronic effects of sustained or periodic intake or
metabolism of OP residues by measuring their inactivation effects on
enzyme functions, e.g., inhibition of cholinesterase or carboxyl-
esterase activity, is slow, costly on a mass scale, variable in
response depending on the subject's age, physical condition and
past pathological history among other factors, and requires medical
surveillance because of the requirement for drawing blood samples.
One could add more facets to the complexities of preventing
poisoning of field workers through exposure to dislodgable OP residues
on foliage or on airborne particulates, but in each case one is con-
fronted with optional methods of OP residue analysis, each with
advantages and disadvantages for getting answers rapidly and accurate-
ly to the question "Is it safe to reenter the workplace?" Towards
this question we offer the following condensed comparison of method-
ology and response between the colorumetic Rapid Field Method (RFM)
VI
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described herein and a gas chromatography (GC) method also described
herein.
The RFM depends on the alkylation reaction of OP compounds with
NBP [4-(p-nitrobenzyl)pyridine], accelerated to 3 min at 150°C, to
form a magenta color in an alkaline medium. With the RFM and a
measurement of the developed color intensities with a portable spec-
trophotometer, one can determine the dislodgable residues on foliage
or soil of a broad range of OP compounds directly in the field.
Using a portable setup, one can process within 30 min/6 samples,
50 min/12 samples or 90 min/24 samples after the collected samples
are ready for processing. This method is non-specific in that it
does not distinguish between the thion (P=S) and oxon (P=O) forms
of OP insecticides. Although not all OP compounds react equally in
the RFM procedure, the response for each compound is linear in the
range 1-30 yg for the more reactive compounds. Because of its non-
specificity, the method will register presence of OP residues
remaining from previous treatments or from airborne drift or from
runoff in soil. It has a lower limit of detection of 0.005 ug OP
2
residue/cm leaf surface and 0.1 ppm OP residue on surface soil,
both of which are considered to be well below the lower limit of
significance for the Worker Reentry Problem.
When coupled to GC with appropriate instrumental parameters and
columns, the flame photometric detector is a widely used detector
system for measuring OP residues with specificity and sensitivity.
In our experience, the linear range of detector response traverses
only the range 0.5-16 ng OP insecticide. To work within these
range limits, considerable dilution of the sample extract may be
VI1
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necessary, in which case the resulting high dilution factor may
cancel the intrinsic advantage of reproducible measurement of
nanogram or subnanogram levels of OP residues. In real terms,
the total volume of sample extract encompasses all the OP residue
being sought. Thus, ability to register 1 ng OP residue from a
10-yL injection of a 10-mL total volume of extract translates to
1 yg of the particular OP compound present in the sample, an
amount that can readily be measured by the RFM. The sample
preparation and extraction procedures needed for GC are considerably
longer than for the RFM. P=S and P=0 analogues of a given OP
species may each be determined in a single injection of an aliquot,
provided their respective concentration levels are within the working
range. Since the P=O analogue, if present, is generally only 0.2-40%
of that of the thion, the extract volume may require additional
concentration to bring the P=0 fraction into range. Taking dilution
2
factors into account, amounts as low as 0.002 yg OP residue/cm leaf
surface could be measured, depending on the nature of the compound.
With the possible exception of a GC setup in a mobile laboratory,
the GC system is unsuitable for use directly in the field and is more
time consuming than the RFM. In addition, the RFM is reproducible,
adequately sensitive and modest in cost. With practice and minimal
instruction, on-the-spot field tests could readily be conducted by
farm or orchard managers, or by personnel of regulatory agencies.
The blanket response of the RFM to mixtures of OP compounds and lack
of specificity for individual OP species present in a mixture is of
no great consequence because all OP residues are toxic, some much more
so than others. In contrast, GC would not identify components of a
Vlll
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mixture were known in advance, and appropriate methods and columns
were available for their measurement.
The RFM has a large potential of other applications. One
important use would be for checking for OP residues before releasing
biological control predators to ensure their survival in an inte-
grated pest management program.
IX
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ABSTRACT
A rapid field method (RFM) for on-the-spot determination of
organophosphorus (OP) insecticide residues on crop foliage and
surface soil dust was developed., The RFM is applicable to the
data needs of the Worker Reentry Problem (Gunther et al., 1977)
for which rapid assessment of dislodgable OP residues on foliage
and in surface soil is needed prior to clearance for reentry of
• »
workers in sprayed fields or groves. The method is based on the
alkylation reaction of OP compounds with NBP [4-(p_-nitrobenzyl)-
pyridine] to form a magenta color in an alkaline medium. The
method for foliage consists of adding Nad solution to dislodge
the OP residues of the leaf sample by shaking, transfer of the
aqueous wash to a 50-mL tube, adding hexane for partitioning
purposes, removal of a hexane aliquot to a prepared reaction tube,
evaporation of the hexane, reacting the OP residue at 150°C for
3 min with the NBP added in advance to each tube, and adding
alkaline reagents for color development. The color intensity
(absorbance) is measured with a portable mini-spectrophotometer.
It is also possible to conduct the OP-NBP reaction at 100°C for
30 min but the colors formed for the 1-30 i_ig level are not as
intense, although the standard curve is equally linear. Although
the method reacts with nearly all OP compounds that have been
tested to date, the ratio of absorbance unit per ng of OP com-
L
pound (the slope of the standard curve) varies with the particular
molecular species. By determining the ratio of the slopes of the
curves obtained at 100° for 30 min"vs. 150° for 3 min, one obtains
values that are characteristic or relatively constant for a given
-------
OP species, and thus contributes to identification of the species
provided that only one species is present in the sample. The
lower limits and linear range of measurement of 32 OP compounds
including 8 oxons were investigated. Amounts as small as 0.005 yg
o
OP residue/cm leaf surface and 0.1 ppm OP residue on surface soil
could be determined. After the samples are obtained, 12 samples
can be processed and the OP residue levels determined in the field
within 50 min. The recovery of OP compounds added to fortified
leaf extracts ranged from 76 to 102%, depending on the species,
except for low recoveries of 40-45% registered by water-soluble OP
species such as dimethoate and some oxons. Recoveries from forti-
fied soil in the range 10-450 ppm ranged from 94-103% for 3 thions
and 3 oxons. Recoveries were reproducibly obtained for each
compound and results are accurate when the treatment history of the
sample is known. In addition to the Worker Reentry Problem, involv-
ing over 300,000 workers in California alone, the RFM is useful for
the testing of foliage in a given area for OP residues prior to
release of parasites and predators for biological control in an
integrated pest management program.
This report was submitted in fulfillment of Contract No.
R805 64201 to the University of California, Riverside (F. A. Gunther,
Principal Investigator) under the sponsorship of the U.S. Environ-
mental Protection Agency. This report covers the period October 1,
1978 through September 30, 1979.
XI
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CONTENTS
Foreward
Preface ..... -V
Abstract x
Contents xii
Figures and Illustrations xv
Tables xvi
Acknowledgements xviii
Section 1. Introduction 1
Section 2. Conclusions-. .. 8
Section 3. Recommendations " 16
Section 4. Materials and Equipment
1. Equipment and supplies needed to determine dislodgable
OP residues of foliage and surface soil, collated in
Table 2, with supplementary comments 18
2. OP compounds used to explore the analytical scope of
the RFM (Table 3) 30
3. GC equipment and supplies used 32
Section 5. Methods
1. The 4-stage sequence of the RFM and time required to
process foliar and soil residues, collated in Table 4,
with supplementary comments 33
2. Procedure for processing leaf-punch samples ... 36
3. Procedure for processing samples of surface soil . 39
4. Standard solutions for calibration of methods. . . 41
5. Determination of % recovery from fortified leaf
extracts 42
6. Determination of % recovery from fortified soil. . 43
7. GC methods 46
8. Determination of storage stability of prepared
tubes 47
9. Determination of mean absorbance unit/ng ratios of
39 OP compounds 48
10. Fading of color intensity 49
11. Determination of foliar and soil residues of
phenthoate 50
xii
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12. Dissipation curves for dislodgable foliar residues
of malathion, parathion and methidathion after
application to orange trees 51
13. Distribution patterns of methidathion on leaves
after application to lemon trees 53
Section 6^. Results and Discussion
1. Optimum time-temperature combination for rapid OP-NBP
reaction 55
2. Two different temperature-time combinations to assist
identification of OP compounds 59
3. Effect of amounts of 'oxalic acid and NBP on the color
formation 59
4. Effect of duration and temperature of storage of
prepared tubes on the reaction with parathion 63
5. Effect of elapsed time between hexane evaporation and
high temperature NBP reaction . 64
6. -Effect of temperature and heating time on the reaction
of parathion with NBP 65
7. Effect of elapsed time between NBP reaction and color
development 68
8. Absorbance values after reaction of 24 OP compounds with
NBP at 150°C for 3 min 68
9. Absorbance values after reaction of 24 OP compounds with
NBP at 100°C for 30 min 72
10. Linear regression analysis values for the data of
Tables 10 and 11 . . 72
11. Mean absorbance unit/ng ratios of 39 OP compounds after
NBP reaction at 150° for 3 min 76
12. Absorption maxima of the products resulting from NBP
reaction with OP compounds 78
13. Decrease in absorbance with time 79
14. Recovery of OP compounds after fortification of 20%
sodium chloride solution 82
15. Recovery of OP compounds after fortification of aqueous
leaf washes 83
Xlll
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16. Comparative recovery of parathion from fortified dry
vs. fresh, moist soil 85
17. Modifications in processing soil samples to improve %
recovery of OP compounds 90
18. Foliar residues of phenthoate by the RFM and by GC . 93
19. Determination of phenthoate residues of surface soil 96
20. Foliar residues of malathion, parathion and methidathion
by GC and the RFM 98
21. Foliar residues of methidathion at various locations of
sprayed lemon trees after application as dilute and
low-volume sprays . .' Ill
xiv
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FIGURES
Number page
1 Comparative standard curves for methidathion ( • ), 57
parathion ( O ), and demeton ( A ) ; reacted at 150°C
for 3 min; mean absorbance of duplicate determinations
2 Comparative standard curves for methidathion ( • ) , 58
parathion ( O ), and demeton ( A ); reacted at 100°C
for 30 min; mean of duplicate determinations
3 Dissipation curves for malathion WP and parathion WP, 103
each applied as dilute and low-volume sprays,
respectively, to orange trees, and determined over a
62-day period by the'RFM as dislodgable foliar OP
residues
4 Dissipation curves for methidathion WP and methida- 104
thion EC, each applied as dilute application and
low-volume sprays, respectively, to orange trees and
determined over a 62-day period by the RFM as
dislodgable foliar OP residues
5 'Correlation between total (thion and oxon) dislodgable 106
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of a wettable powder
formulation of malathion. The line is described by
In y = 0.87 In x - 0.380 (one point omitted) and the
correlation coefficient is 0.99
6 Correlation between total (thion and oxon) dislodgable 107
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying
trees with low-volume and dilute sprays of a wettable
powder formulation of parathion. The line is described
by In y = 0.67 In x - 1.09 and the correlation
coefficient is 0.96
7 Correlation between total (thion and oxon) dislodgable 108
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of a wettable powder
formulation of methidathion. The line is described by
In y = In x - 0.133 and the correlation coefficient is
0.99
8 Correlation between total (thion and oxon) dislodgable 109
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of an emulsifiable
concentrate formulation of methidathion. The line is
described by In y = In x - 0.313 and the correlation
coefficient is 0.93
xv
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TABLES
Number Paqe
1 Summary of Reported Organophosphorus Insecticide Usage 7
in California in 1977
2 Equipment and Supplies for Determining Dislodgable OP 18
Residues of Foliage and Surface Soil
3 Organophosphorus Compounds Used in Exploring the 31
Analytical Scope of the RFM
4 The 4-Stage Sequence and Time Needed to Process Six 33
Determinations of OP Residues of Foliage or of Surface
Soil
5 Effect of Varying Amounts of Oxalic Acid and 61
4-(p_=Nitrobenzyl)pyridine on the Color Formation Step
6 Effect of Storage of Test Tubes Containing Oxalic Acid 62
and 4- (p_-Nitrobenzyl) pyridine on the Reaction With
Parathion
7 Effect of Elapsed Time Between Hexane Evaporation and 66
4-(£-Nitrobenzyl)pyridine Reaction on Absorbance of
the Solution at 560 nm
8 Effect of Temperature and Keating Time on the Reaction 67
of Parathion with 4-(p_-Nitrobenzyl) pyridine
9 Effect of Elapsed Time Between 4-(p_-Nitrobenzyl) - 69
pyridine Reaction and Color Development With Base on
Absorbance of the Solution at 560 nm
10 Absorbance at 560 nm of Solutions After Reaction of 71
Compounds With 4- (p_-Nitrobenzyl) pyridine at 150°C
for 3 Min
11 Absorbance of 560 nm of Solutions After Reaction of 73
Compounds with 4-(p_-Nitrobenzyl) pyridine at 100 °C
for 30 Min
12 Linear Regression Analysis Values for the Data Given 74
in Tables 10 and 11
13 Mean Absorbance Unit/ng Ratios of 39 OP Compounds 77
After NBP Reaction at 150° for 3 Min
14 Absorption Spectra of the Products From the Reaction 80
of 4-(p_-Nitrobenzyl) pyridine With Organophosphorus
Compounds
xvi
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Number TABLES (Cont'd) Page
15 Decrease in Absorbance (560 ran) With Time 81
16 Recovery of Compounds After Fortification of 84
20% Nad Solutions
17 Recovery of Insecticides After Fortification of 86
Aqueous Leaf Washes
18 Comparison % Recovery of Parathion from Fortified 89
Dry vs Fresh, Moist Soil (<100 Mesh)
19 Effect of Changes in the Water :0rganic Solvent Ratio 92
on % Recovery of 3 Thions and 3 Oxons From Fortified
Soil
20 Foliar Residues After Application of Phenthoate to 94
Orange Trees, Determined by the RFM and by GC
21 Phenthoate Residues (ppm) of Surface Soil Dust and 97
tag/ft2 Soil Surface, at the Dripline Area of Sprayed
Orange Trees Determined by the RFM
22 Dislodgable Foliar Residues (|ag/cm2) of Parathion, 100
Malathion and Methidathion After Application of
Dilute and LV Sprays to Orange Trees
23 Dislodgable Foliar Residues (ng/cm) of Methidation 112
at Various Locations of Sprayed Lemon Trees After
Application as Dilute and Low -Volume Sprays
xvn
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ACKNOWLEDGEMENTS
We gratefully acknowledge the technical assistance of
J. Virzi, J. Barkley, G. E. Carman, J. Pappas, T. Dinoff,
• *
D. Aitken and M. Wells. We are especially indebted to
E. Papadopoulou who assisted in the development of all the
basic features of the method which is described herein.
XVIXI
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SECTION 1
INTRODUCTION
This report deals with the development and some field
applications of a simple, portable, low-cost Rapid Field Method
(RFM) for on-site determination of organophosphorus (OP) insecti-
cide residues on foliage and in surface soil.
The RFM apparatus and supplies as herein used to conduct 48
tests in the field, not including the weight of sample jars,
•
weigh approximately 22 lb. The RFM procedure involves four easy
steps (see Table 4), and can readily be learned and used by persons
other than trained residue chemists. After leaf samples have been
taken or soil samples have been obtained and sieved, 6 tests can
be executed within 30 min, 12 tests within 50 min, and 24 tests
within 90 min by one person.
Measurement of OP residues by the RFM is based on the
alkylation of the pyridine nitrogen of NBP [4-(;p_-nitrobenzyl)-
pyridine] (6,7). For rapid, reproducible OP-NBP reactions we
found the optimum temperature-time combination to be 150°C for
3 min. The reaction is conducted in test tubes in a heated
6-hole aluminum block containing ethylene glycol for uniform
heat transfer. The alkylated NBP in an acid medium is colorless,
but in an alkaline environment a blue or magenta color is formed,
depending on the base used. We used triethylamine and sodium
carbonate solutions in sequence to develop a magenta color, the
intensity of which is directly proportional to the OP concentration.
The absorbance of the color at a wavelength of 560 nm is measured
with a portable mini-spectrophotometer. The standard curve is
-1-
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consistently linear over the range 1-30 ng, except for highly
reactive OP compounds that yield standard curves with large slope
values and for which the practical working range is 1-20 ng. It
is also possible to conduct the OP-NBP reaction at 100°C for 30
min but the colors formed are not as intense (have lower absorbance
values), although the standard curve is equally linear. By
determining the ratio of the slopes obtained at 100°C for 30 min
and at 150°C for 3 min respectively, values are obtained that are
•
characteristic or relatively constant for a given OP species. The
ratio may thus be used to assist identification of an unknown OP
species, provided only one species is present in the sample.
The main objective of this research was to meet the needs of
the Worker Reentry Problem (11,15) for which a fast, simple method
of measuring OP residues that could be used by an orchard or farm
foreman or regulatory officer is needed to ascertain whether it is
safe for orchard or field crop workers to enter previously sprayed
work areas in order to cultivate and harvest fruit and vegetable
crops.
Some comments on aspects of determining safety of worker re-
entry may help to provide perspective. All OP compounds are toxic,
the toxicity varying in degree for a unit amount and the route of
entry. Different OP pesticides are used in agricultural practice
for chemical control of many insect pests. Under field conditions,
OP compounds such as parathion, methidathion, azinphosmethyl and
others can be transformed into more toxic oxygen analogues (oxons)
believed to result from their interaction with atmospheric ozone
(11,29) or sunlight. The rate of disappearance of OP residues in
-2-
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the field is variable (1,2,11). Depending on the type of soil,
the persistence of OP compounds may be prolonged due to physical
binding (sorption) by soil particles that stabilize the residues
and protract their ultimate dissipation (1,2,9-11). Under field
conditions, conversion on foliage of parathion to the more toxic
paraoxon is influenced more by the amount of foliar dust particles
than by the oxidant (ozone) levels _n the atmosphere (28). The
particles (particulates) become airborne by air currents (wind),
by normal movement of field workers and by farm equipment in action.
They are thus translocated from orchard soil and leaf surfaces to
the clothing, lungs, hair and skin surfaces of workers. Case
histories of OP poisoning of field workers have been documented
(3,4,17,19,21,22) .
The type of application influences the OP residue levels.
Thus, low-volume and ultra low-volume spray applications raise OP
residue levels and extend the residue persistence curves signifi-
cantly (5). In contrast to application as dusts and sprays,
controlled-release formulations of OP compounds (23) encapsulated
within a small polymeric matrix would, by slow, sustained release,
prolong the ultimate disappearance of the OP molecules. In
either case, on-site data on OP levels are needed to ensure that
prescribed levels and tentative safety limits for foliar and soil
residues are not exceeded.
It should be noted that numerically defined Threshold Limit
Values developed by occupational health professionals are not yet
available for OP residues that remain in the work environment.
The current state and federally prescribed "reentry interval"
-3-
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concept (24,25) has been criticized (14) as being arbitrary and
over-simplistic for not taking into account modifying factors
such as regional differences in temperature, rainfall, soil
moisture, relative humidity, wind speed and direction, nature of
formulation, dosage rate and method of application.
Foliar dust is regarded as the main source of transfer of OP
residues to field workers (11,13). Estimates of aerosol vs.
dermal exposure showed that 98-99°' of the workers' dose was
• *
dermal, predominantly to the hands and upper extremities (20).
Low-volume spraying results in higher but more variable OP
residues on fruit and leaf surfaces than dilute (oscillating
boom) applications, and yields higher OP levels on the sides of
the trees that face the spray application unit (5) .
Assessment of the impairment of the health of field workers
repeatedly exposed to traces of OP compounds by determining the
degree of inactivation of cholinesterase in the serum or red
blood cell fraction of the subject's blood is not proportional to
OP concentration because of variable response (19,20). Response
by enzyme inactivation is affected by the time elapsed since spray
application, the nature of the OP compound, and physical condition
of the subject and his response to work and heat stress, among
other factors (19,20). Testing of blood samples from an individual
taken at 3 different times improves the validity of this test
i
procedure. Depending on the nature of the OP compound, serum
carboxylesterase is more susceptible to inhibition, and is
therefore a more sensitive indicator of OP poisoning than is
serum cholinesterase (16).
—4-
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We have referred to the thion-oxon conversion and the greater
mammalian toxicity of oxons. Depending on the soil type, paraoxon
levels as high as 35% of the initia: parathion residues have been
formed (1,2). The rate of disappearance of oxons is slowed
considerably by clay soils (2). Different metallic oxides in
soils may have a catalysing influence in accelerating the thion-
oxon conversion by UV radiation (11).
The foregoing aspects ajce indicative of some of the
difficulties of linking toxic effects to a single OP species
since other species, some generated after spray application due
to various environmental combinations, might also be contributors
to the toxicity syndrome.
Assessment of worker reentry hazards by the determination of
OP residues present in foliage and surface soil becomes increasingly
difficult when one considers the combinations and permutations that
are available today for chemical control of insect pests. Thus,
of the multiplicity of pesticidal formulations available
(thousands registered in the U.S.A. comprising over 550 different
a.i. chemicals, not including adjuvants, synergists, emulsifiers,
solvents, stabilizers, etc.), an appreciable number consist of two
or more OP or other insecticidal chemicals combined for specific
use requirements. Knowing what OP insecticides were applied in
a mixture is a prerequisite for correct interpretation of RFM
results.
The apparently simple RFM has a useful fact-finding potential,
particularly when knowledge of the formulation used and chrono-
logical history of a given application are known. The RFM responds
-5-
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to nearly all OP compounds. The basic premise is that all OP
residues are toxic, but vary in toxicity. How much "total" OP
residue is present in leaf or soil samples at different times
accordingly is useful information.
We have referred briefly to prescribed reentry intervals
and their intrinsic oversimplification of an interlocking mixture
of shifting variables. Table 1 gives a summary of reported OP
insecticide usage in Ca.lifor.nia for crops for which reentry
intervals are currently assigned. These are the compounds that
an analyst might encounter under field conditions. Although the
extent of the reentry problem is difficult to assess nationwide,
incidents of poisonings appear to be mainly located in California.
The prescribed reentry intervals vary with the OP insecticide and
the specific crop to take into account the mammalian toxicity of
the insecticide and particular features of the crop. For example,
citrus leaves may accumulate more foliar dust than peach leaves
because citrus trees are non-deciduous. No reentry intervals have
as yet been assigned for monocrotophos and trichlorfon.
-6-
-------
TABLE 1. SUMMARY OF REPORTED ORGANOPHOSPHORUS INSECTICIDE USAGE (POUNDS OF ACTIVE
INGREDIENT) IN CALIFORNIA IN 1977-S/
Insecticide
azinphosmethyl
carbophenothion
demeton
dialifor
diazinon
dimethoate
dioxathion
ethion
malathion
methidathion
methyl parathion
mevinphos
monocrotophos
naled
oxy d eme t on-me thy 1
parathion
phosalone
phosmet
phosphamidon
trichlorfon
grapefruit
226
-
-
-
-
16,671
319
-
2,247
6,589
-
-
-
40
-
3,409
-
-
576
24
Citrus
lemon
718
-
-
-
-
52,753
1,585
1,279
6,486
22,320
-
94
975
36
-
14,555
-
-
803
-
orange
8,950
12
-
-
117
210,815
7,649
5,925
23,888
95,522
-
158
223
4,282
252
90,240
42
-
3,077
1,234
grape
1,332
1,673
7,280
945
830
271,042
4,281
20,503
1,133
-
4,987
5,839
-
122,283
3,668
21,190
14,483
-
-
-
nectarine peach
188 78,166
129
2
-
-
- .
-
3,171
9
8,001
50
127
3,064 11,342
16 2,411
-
27,831 64,844
104
1,694 15,988
-
-
apple
8,639
3,827
-
20
1,828
-
-
9,377
25
-
-
-
-
-
-
215
579
6,170
1,850
-
a/ ...
agencies and from growers applying restricted material*. This summary reflects only a portion of the
pesticide use in California (California Department of Food and Agriculture, 1977).
-------
SECTION 2
CONCLUSIONS
i
1. Portability, Speed and Economy in Determination of Dislodgable
OP Residues. A portable, low-cost rapid field method (RFM)
suitable for on-site determination of dislodgable organopho-
sphorus (OP) residues on foliage and in surface soil was
developed. The method meets the needs of the Worker Reentry
Problem for rapid on-site data acquisition on dislodgable OP
residues to ascertain the occupational safety status of
sprayed orchards and fields prior to permitting entry of field
workers to execute their work assignments. After samples of
foliage (leaf disks) and of sieved soil (< 100 mesh) have
been taken, samples of either foliage or soil can be processed
and analyzed at a rate of 6 in 30 min, or 24 in 90 min.
2. Non-Specific, but Nevertheless Useful For Worker Protection.
The RFM responds linearly to a wide range of OP compounds in
the range 1-30 ng for most and in the range 1-20 ug for the
more reactive compounds, and is accordingly non-specific.
The non-selectivity, however, does not cancel the usefulness of
the RFM since all OP species are toxic, some more than others.
For worker protection, the speed and reproducibility of data
acquisition, albeit on a total OP basis, outweighs the non-
specificity factor. The impact of non-specificity is reduced
when the history of the spray treatment is known beforehand,
and a specific rather than an average factor could then be
used in the calculation of OP levels. It is also possible to
-8-
-------
reduce the slate of candidate OP insecticides in a sample of
unknown history by determining the ratio of the slopes of the
curves derived from OP-NBP reactions at 100°C for 30 min and
at 150°C for 3 min, respectively, to provide values that are
characteristic or relatively constant for a given OP species.
The latter procedure would be useful only if one OP species
was present or predominant in the sample.
3. RFM Responds to a Broad .Spectrum of Potential Contributors to
Overall Toxic Effects. The RFM will respond also to (a) OP
residues that remain from previous spray treatments, or
(b) that may have been deposited through spray drift from
applications to adjacent areas, or (c) from dust storms that
provide free aerial transport and translocation for long
distances to OP residues physically bound to dust particles.
Other alternate sources of positive response by the RFM
include OP conversion products, e.g., highly toxic oxons
resulting from oxidation of thions by ozone and other oxidants.
4. Lightweight Kit, Relatively Inexpensive, Simple to Use. The
RFM kit, including 6 sample jars, a 2-screen set for sieving
soil, and 24 discardable prepared tubes, weighs about 21 Ib.
The kit is simple to use after 1 or 2 practice trials. With
about 2 h of supervised training and typed instructions and
charts for calculating OP levels, orchard and farm managers .
with no experience in residue analysis could be taught the
mechanics of operation of the kit in order to provide data
on OP levels comparable to those of regulatory personnel using
an identical kit.
-9-
-------
5. Higher Values Obtained by GC vs RFM on Foliage 3-16 Days
After Spray Application Ncvt Resolved. After treatment of
orange trees using LV and dilute sprays at equivalent
commercial dosage rates of parathion, malathion, and
methidathion (see Table 22 and Figures 5-8), results obtained
over a 62-day period by GC were higher than by RFM between
Days 3 and 16, and particularly for parathion LV between
Days 3-9. The disparity was less for the malathion and
*
methidathion formulations. The more exhaustive multiple
extractions and dislodgement methods used for the GC pro-
cedure (12) would contribute to better recovery and larger
values particularly at the early part of the disappearance
curves. It should be noted that concurrent repetitive tests
of standard curve points at each sampling were reproducible,
linear, accurate and equivalent for both the GC and the RFM
methods. If repeat field sampling of foliage should show a
consistent difference pattern between GC and the RFM over a
given dissipation time period, correction factors (about 1.2)
would be applied to the RFM results to obtain equivalence to
those of GC used as reference standards.
6. Residue Levels on Citrus Foliage Influenced by Sample Location
and Method of Application. In a field experiment in which LV
and dilute sprays of methidathion were applied to ascertain
the possible correlation of differences in OP residue levels
on citrus foliage as a function of different sampling locations
and type of spray application (Table 23) , it was found that:
-10-
-------
(a) among the 4 tree perimeter locations at 6,4 and 1 1/2
ft above ground, the highest residues were found at the lowest
height (1 1/2 ft) 7 to 11 days after application. However,
in the period 14-17 days after application, the highest levels
were at the 4-ft height, but in lesser amounts.
(b) the LV spray treatment yielded higher residues than the
dilute treatment, as had been expected, and
(c) the residues in the case of the dilute spray application
*
were invariably higher on the tree sides parallel to (facing)
the between-row pathways used during the mechanical application
of the spray. In the case of the LV treatment, only the 6-ft
locations showed the highest residues to be at the tree sides
facing the pathways used in applying the spray, but the
ranking order shifted at the 4-ft and 1 1/2-ft locations.
It was concluded from the results (Table 23) that sampling
of foliage at 4-5 ft above ground (chest height) was the
"best" general and convenient location for obtaining a
maximum sample, and that each tree should be traversed in a
circular pattern to obtain 8 leaf punches, one disk from each
of the cardinal direction points, thus to provide a sample
consisting of 40 leaf disks from 5 trees.
7. Improved Recovery of OP Residues From Soil by Maintaining a
• Low Water Content. In the exploratory stages of methods
development for determination of dislodgable OP residues from
sieved surface soil, procedures analogous to those for foliage
were used. Recoveries at the time from fortified soil varied
from low (35-55% range) to medium (56-80%), and were lowest
-11-
-------
and more variable when oxons were involved. In subsequent
testing programs that included systematic reduction of the
volume of the water phase (20% NaCl solution) combined with
use of 20 mL/sample of acetone-hexane 15:85 v/v, it was found
that considerably improved recoveries (89-103%) were obtained
with 3 thions and 3 oxons in the range 10-400 ppm when only
0.25 mL of salt solution was added per soil dust sample.
The latter addition was adequate to prevent the formation of
*
a finely dispersed suspension of soil particles when solvent
alone was used, and concomitantly to provide a higher
efficiency of recovery.
8. Convenience of Prepared Tubes and Their Storage Stability
at 73 and 1_10°F. The convenience of using for RFM purposes
reaction tubes prepared in advance in 10-dozen batches was
readily demonstrated. Preparation consisted of adding to each
18x150 mm culture tube 0.1 mL of 10% NBP in acetone, 0.1 mL
of 0.04% oxalic acid in acetone, and approximately 25 mg of
salt (NaCl) crystals, and allowing the acetone to evaporate
at room temperature. When required, aliquots of solvent
extracts were deposited in the tubes for evaporation and
subsequent OP-NBP reaction. The prepared tubes in groups of
24-30 were readily transported. Because of their relatively
low cost, they were considered discardable after use in order
to save costs of washing and drying time, particularly when
suitable facilities were absent and the quality of washing
could not be guaranteed.
-12-
-------
Tests of the storage stability of prepared tubes at 23
and 43°C (73 and 110°F) respectively showed no change in net
absorbance value after reaction with known OP standards when
the tubes were stored at about 23°C for up to 6 months (end
of test period). After 6-8 weeks of storage at 43°C, reduced
and erratic absorbance values were obtained.
9. Standing Time After Hexane Evaporation and Before OP-NBP
Reaction. After the hexane has been evaporated from reaction
tubes, the subsequent steps of OP-NBP reaction and of color
development may for convenience be postponed for at least 6
days without effective change in the original OP levels.
Thus, where multiple locations in an integrated series of
orchards or fields are to be sampled in a 2-day period,
samples of each group could be processed to the hexane
evaporation stage until, say, 48-72 evaporated tubes have
accumulated. Processing to the final stage could then ensue
on the following day, thereby achieving a significant overall
reduction in downtime. Tubes are also safer to transport
between locations when hexane is absent.
10. Standing Time After OP-NBP Reaction. After the OP-NBP re-
action has been completed, two days may elapse without
affecting the intensity of color development and the final
• measurement. Thus, if a batch of 72 or more samples are to
be taken in one day, the samples can be processed to the
completion of OP-NBP reaction (Stage 3) on the first day,
leaving color development and calculation of results for
the second day.
-13-
-------
11. Stability of the Developed Color. The magenta color is not
too stable and should be read immediately upon co lor develop-
ment. Fading is accelerated rapidly upon exposure of the
color to direct sunlight, but proceeds at a slow rate when
readings are conducted in the shade, or indoors under
fluorescent lighting.
12. Contributing Factors That Accelerate the RFM and Increase
the Output of Needed Data. The relative speed and simplicity
of the RFM stem from a combination of factors, such as:
a. The use of prepared tubes, each containing salt crystals
for smooth boiling of hexane.
b. Simplified process of dislodgment of OP residues from
foliage and surface soil.
c. Rapid evaporation of solvent extracts (1.5 min for 12
tests) .
d. Rapid OP-NBP reaction (3 min at 150°C).
2
e. Use of 1/8 ft template with small dustpan and non-
magnetic brush to obtain surface soil sample, and
precalibrated volume (e.g., a level 0.5 teaspoon) to
"weigh" sieved soil.
f. Use of propane-burning (preferred) or gasoline-operated
campstove as a rapid, portable, easily regulated heat
source to heat 6-hole aluminum blocks (heat sink)
containing ethylene glycol as a heat-transfer medium.
g. Use of precalibrated, readily cleaned, glass syringes,
where possible, to complement or replace disposable
pipets to dispense specified volumes of solvents and
reagents.
-14-
-------
h. Use of predetermined absorbance unit/ng ratios and
pocket calculator for calculation of dislodgable OP
residues as ng/cm leaf surface or ppm of surface soil.
i. Miscellaneous improvisations to simplify field operations:
egg-timer as field stopwatch; salt bed for tube rack;
padded tin cans for holding empty or hot tubes; poly-
propylene squeeze bottle with precalibrated reservoir
for dispensing 15 mL salt solution; improvised field
workbench (tailgate of station wagon?).
13. General Conclusion. The RFM is ready for use to ascertain
the safety status for worker reentry into previously sprayed
orchards, fields, groves and vineyards.
-15-
-------
SECTION 3
RECOMMENDATIONS
Based on the overall experience gained from research invested
to date in the analytical and application phases of the RFM, we
recommend the following:
1. Test Combination of Surfactants with Salt Solution to Improve
Recovery of Residues of Foliage. The possible increase of
recovery of OP residues of foliage by adding 2 drops of diluted
surfactant to the 20-mL amounts of salt solution/sample should
be determined. For cross reference, 4 drops of surfactant
(Sur-Ten, 1:50 dilution) are added to 3 successive 100-mL
amounts of water used in washing leaf disks (12) for subsequent
analysis by GC for OP residues.
2. Extend Research on Improved Recovery of OP Residues from Soil
to Additional OP Compounds. Confirmation of the increased
efficiency of recovery from fortified soil, as was obtained
in testing 3 thions and 3 oxons, should be extended to
additional OP compounds. We found that OP compounds vary in
individual response in recovery trials, and their upper and
lower limits in relation to sharply reduced water content
(0.25 mL/sample) should accordingly be ascertained.
3. Extend Storage Stability Tests on Prepared Tubes to 120 and
130°F (49 and 54.5°C). Storage stability tests undertaken
to date on prepared tubes stored at 73 and 110°F respectively
should be extended to 120 and 130°F (49 and 54.5°C). The
latter temperatures can be attained and exceeded in the trunk
-16-
-------
of a car on a warm (100°F) sunny day. To assist dependable,
reproducible analytical performance, it is important to know
the temperature-time limits of storage of prepared tubes, so
that they could be used well within prescribed storage limits,
-17-
-------
SECTION 4
MATERIALS AND EQUIPMENT
1. Equipment and Supplies Needed for Determining Dislodqable
OP Residues and Surface Soil. The equipment and supplies
needed for determination of OP residues on foliage and in
'•>
surface soil in the field by the RFM are collated in Table 2,
which is followed by a section entitled "Supplementary
• •
Comments" pertaining to the items of Table 2.
TABLE 2. EQUIPMENT AND SUPPLIES FOR DETERMINING DISLODGABLE OP
RESIDUES OF FOLIAGE AND SURFACE SOIL, WITH SUPPLEMENTARY
COMMENTS*
Item Nature of item or requirement Comments*
1 Portable campstove options: (a) Propane fuel type, Note 1
e.g., Kangaroo Trail Boss, Model No. 200024 or
Kangaroo Slimline One, or (b) Gasoline fuel type,
e.g., Coleman 2-burner, Model 425E499. Note:
Propane type preferred.
2 Spare fuel for Item 1, either (a) disposable propane
16.4-oz cylinder, or (b) 1 qt "white gas" or
equivalent campstove fuel
3 Matches or cigarette lighter
4 Two 6-hole aluminum blocks to specifications Note 2
5 Two thermometers, either (a) metal type, with 5-in. Note 3
metal probe and 2-in. diameter dial, in the range
0-210°C, or (b) yellow glass back mercury type
6 Egg timer, 3 min, flowing sand or salt type Note 4
7 Leaf punch apparatus, with attached screw-cap to Note 5
accommodate 8-oz jars
' 8 Sample jars, 8-oz size, screw-cap, with thin
Teflon liner in each cap
-18-
-------
TABLE 2 (cont'd). EQUIPMENT AND SUPPLIES FOR DETERMINING
DISLODGABLE OP RESIDUES OF FOLIAGE AND SURFACE SOIL, WITH
SUPPLEMENTARY COMMENTS*
Item Nature of item or requirement Comments*
9 Polypropylene (PP) centrifuge tubes, screw-cap, Note 6
50-mL size, 5-mL gradations
10 Prepared glass reaction tubes, 18x150 mm, Note 7
containing NBP, oxalic acid and salt
11 Improvised tube rack for field, consisting of salt Note 8
bed in a small plastic dishpan to hold PP tubes in
either a vertical or 30-45° slant position. About
3 Ib salt required
12 For processing surface soil, one 100-mesh and one
10-mesh Tyler U.S. standard brass screen with fitted
catchpan and lid
13 For simplified measurement of sieved soil samples Note 9
by volume, one set of 4 U.S. standard measuring
spoons, comprising 1/4-, 1/2-, 1- and 3-teaspoon
(1 tablespoon) sizes
14 Reagents and solvents: (a) ethylene glycol, Note 10
commercial grade, for aluminum heating blocks,
(b) 20% salt solution for dislodging OP residues
from foliage, (c) distilled or deionized water,
(d) hexane, reagent grade, (e) acetone, reagent
grade, (f) acetone-hexane, 15:85 v/v, and (g) poly-
propylene squeeze bottles, 60-500 mL capacity range,
with dispenser tubes to expedite dispensing required
amounts of solvent or reagent
15 Color-developing reagents: (a) Triethylamine, 20% Note 11
w/v in acetone, and (b) sodium carbonate solution,
12% in 15% Nad aqueous solution
16 Portable mini-spectrophotometer, Bausch & Lomb Mini
Spectronic 20 with rectangular 2-mL cuvettes, 10-mm
path length, and rechargable battery
17 Glass syringes, 2-, 5-, 10- and 20-mL sizes, pre- Note 12
calibratedjWith 2-inch 18-gauge B-D "Luer-lok"
needles
18 Op insecticide standards in hexane in 4 con- Note 13
centration ranges: (a) 100 ng/mL, (b) 10 ng/mL,
(c) 2.5ng/mL and (d) 1 ug/mL
-18-
-------
TABLE 2 (cont'd). EQUIPMENT AMD SUPPLIES FOR DETERMINING
DISLODGABLE OP RESIDUES OF FOLIAGE AND SURFACE SOIL, WITH
SUPPLEMENTARY COMMENTS*
Item Nature of item or requirement Comments*
2
19 Assembly with metal template for sampling 1/8 ft Note 14
of surface soil, with anti-magnetic nylon brush
and dustpan
20 Miscellaneous items. Polyethylene bags, 1-lb size,
each to hold a composite of soil samples taken at
8 cardinal points (N,NE,E, etc); bag ties or elastic
bands; grease pencil or marking pen; labels (optional);
tissue; paper towelling; two 100-mL beakers; open-top
cans (1-lb coffee cans or equivalent) to hold prepared
tubes before and after the NBP reaction step; notebook
to record sample data and analytical results; kitbag,
knapsack or back-pack unit to accommodate Items 1-2O,
of combined weight, including 12 sample jars, of
approximately 22. Ib
21 Improvised movable workbench for field operations, Note 15
consisting for us of one plywood board, 18 in. x
6 ft x 1/2 in., supported on 2 stacked empty 5-gal
cans at each end to provide a bench height of
approximately 36 in. above ground
*Supplementary Comments on Specific Items in Table 2
Note 1. The Kangaroo Trail Boss propane carapstove (easily dis-
assembled, and of a gross weight including the 16.4-oz size propane
cylinder of 3-1/8 Ib) proved to be better for i.ield and laboratory
use than the Coleman 2-burner gasoline-operated campstove which
had been used in our developmental program. In the latter regard,
after heating the aluminum blocks to 160° in preparation for
Stage 2 (Table 3), the Coleman burner valve is closed. The con-
comitant afterburn in the burner ring lingers for about 1.5 min.
•To avoid possible flash ignition of emerging hexane vapors by the
residual open flame, the aluminum blocks were removed to the work-
bench 20 ft away for safety in evaporating the hexane aliquots
-19-
-------
contained in the prepared tubes. The blocks were then returned
to the Coleman for reheating to 150°C for Stage 3 (Table 3).
This procedure added about 3 to 4 min of downtime to the RFM.
In contrast, afterburn is completely absent when the burner
valve of the Kangaroo propane campstove is closed, permitting
immediate placement of 6 or 12 tubes for evaporation of the hexane
in the hot aluminum blocks, thus reducing downtime. After Stage 2
is completed (1.5 min), the blocks are reheated to 150°C (1.5 min)
• •
and Stage 3 is completed (3 min) with the burner control in a
simmer or "hold" position. The heat distribution via the 1/4-in.
aluminum plate above the burner is uniform, flame management over
a range of settings is simple, no priming is required, and the
fuel consumption per test is modest.
Note 2. Aluminum heating blocks made from 2-in. thick bar stock
can readily be made to the following specifications in any machine
shop. Either of 2 types of heating blocks are suitable for the
RFM: (a) Two aluminum blocks, 3x4x2 in. (7.6x10.2x5.1 cm) each
having six 23.4 mm diameter holes (59/64 in. drill used) and
three 8 mm diameter holes (5/16 in. drill used) placed between
the larger holes; all holes were drilled to a aepth of 1-5/8 in.
(4.1 cm). The large holes accommodate the prepared tubes both
for the hexane evaporation step and the subsequent OF-NBP reaction
stage; the smaller holes hold a thermometer and are used in moving
or positioning the heated blocks with a long-nosed plier. Ethylene
glycol (Item 14, Table 2) is placed in the holes and serves as a
•heat transfer medium for uniform heating of the tubes. Sufficient
amounts are added such that when a.tube or thermometer is in place
the ethylene glycol is not forced out of the hole.
-20-
-------
Note 3. Metal type thermometers have the advantage of being
resistant to breakage. Alcohol type thermometers respond slowly
to temperature changes, and in our experience were less accurate
than mercury bulb type thermometers. The latter respond rapidly
and provide accurate readings at any point in time. The yellow
back type makes reading easier in bright sunlight. With red or
black grease pencils or glass marking pens, reference marks in
contrasting colors can be made at the 150 and 160°C graduations
and with the thermometer placed in a heater block, are readjusted
if necessary against parallel readings of a precalibrated mercury
thermometer used as a reference standard.
Note 4. A conventional 3-min egg timer serves as a simple stop-
watch for timing the 3 min required to conduct the OP-NBP reaction
at 150°C. In selecting one egg timer from 8 that were available
for purchase, we found significant differences, several requiring
up to 3 min 38 sec to discharge completely from the upper chamber.
Egg timers for the RFM should be checked for accuracy to within
10 sec of 3 min at periodic intervals.
Note 5. A suitable leaf punch sampler described by Iwata et al.
(12) is available from the Birkestrand Co., 2705 Lee Ave., South
El Monte, CA 91733, U.S.A. A sample comprises 40 leaf disks per
jar, with a total surface area (both sides) of 400 cm2. To prevent
cross-contamination, the cutting surface and plunger of the leaf
punch apparatus are washed between samples with a stream of water
from a squeeze bottle and then dried with tissue.
Note 6. Polypropylene (PP)centrifuge tubes, 50 mL capacity, with
screw caps, and embossed and numbered in ascending 5-mL increments,
-21-
-------
are available from laboratory supply firms. The PP tubes are
resistant to hexane and acetone.
Even when the caps are screwed on tightly, some PP tubes
may leak somewhat during the shaking step. By holding the tubes
in an inverted position while shaking them vigorously, chance
leakage is eliminated.
Note 7. For convenience in the field, the borosilicate glass
reaction tubes, 18x150 mm size, are prepared in advance. Culture
*
(rimless) tubes are preferred to conventional test tubes because
they are less subject to chipping of the rim during handling and
transport. To each tube are added (a) 100 nL of 10% w/v NBP in
acetone solution, (b) 100 nL of 0.04% w/v oxalic acid in acetone
solution, and (c) 25-50 grains of plain table salt. The salt is
readily dispensed by using a narrow-mouth scoop or spatula, or a
V-shaped narrow-mouth piece of heavy gauge aluminum foil made into
a small scoop. The salt crystals ensure smooth evaporation of
hexane and prevent superheating with resultant sudden, potentially
dangerous, expulsion of hot hexane into the working environment.
Reaction tubes may be used either immediately after pre-
paration or after the acetone evaporates. The latter are
preferred for safety and convenience in handling during transport.
Note 8. An aluminum meatloaf pan or bread tin or equivalent can
be used to contain common salt to serve as an improvised tube rack
in the field for both 18x150 mm reaction tubes and for the PP
tubes used in processing leaf and soil samples. The tubes are
readily supported by pushing them into the salt bed. The rack is
especially useful for the PP tubes, which can be positioned at a
-22-
-------
30° angle, thus facilitating water-hexane phase separation and
the taking of hexane aliquots therefrom.
Alternatively, open-top coffee cans or equivalent are useful
in holding reaction tubes, both cold or hot, and for transport.
Similarly, a rectangular plastic or metal container, supported at
the rear bottom edge on a block of wood to make a 30° angle on PP
tubes placed therein, can also be used.
Note 9. Standard measuring .spoons of the type used in domestic
cooking procedures are well-suited for transferring known volumes
of sieved surface soil (< 100 mesh size) into PP tubes. The
weight in grams of sieved dust per levelled volume (the edge of
a prewashed knife, spatula or plastic ruler can be used in
leveling) per spoon size is determined by weighing consecutively
8 volumed replicates beforehand. Thus, we found that with clay
soil dust in the Riverside area, the mean weights delivered by
1/4, 1/2 and 1 tsp sizes were 1.45 ± 0.05, 2.9 ± 0.1 and 6.0 ±
0.1 g. A spoon after each use is wiped with tissue to remove
adhering dust.
Note 10. We have these comments regarding two of the items
listed under Item 14 in Table 2:
A. Ethylene glycol, commercial quality was stored in a 125-mL
polyethylene squeeze bottle for convenience in dispensing amounts
as needed to the various hole cavities of the aluminum blocks.
Ethylene glycol is readily available, non-toxic, .not readily
flammable and easily washed away with water for cleaning of the
block or glass tubes. Allowance is made for expansion of the
liquid upon heating of the blocks. After the reaction step is
-23-
-------
completed, removal of the tubes from the hot glycol bath is
accompanied by momentary appearance of "steam clouds" on the
periphery of the tube bottoms, particularly on a humid day, but
which subsides as the tube cools. Diluted glycol "steams"
excessively and should be replaced. The glycol levels in the
holes need periodic supplements to replace losses incurred in
removing the tubes and by slow volatilization of glycol at
elevated temperatures. •
B. Salt solution (20% w/v) was stored in a 500-mL polyethylene-
polymethylpentene variable-volume dispenser (Nalge Co., Chicago,
IL) which upon squeezing enabled filling the attached 40-mL
reservoir graduated in 5-mL divisions to a desired level. This
item expedited rapid loading of the sample jars with 20-mL each
of salt solution during Stage 1. It is recommended that the
graduations be checked beforehand for accuracy of delivery of
the stated amounts.
Note 11. To develop the magenta color after the OP-NBP reaction
(Stage 3) has been completed, two base solutions are added sepa-
rately by precalibrated syringes to each tube :!n this order:
(a) 2.5 mL of 20% w/v triethylamine in acetone, and (b) 1.0 mL
of 12% w/v sodium carbonate in 15% w/w aqueous sodium chloride.
It is best to treat only 4 tubes at a time and then determine
their absorbance values within 4 min in a shaded area in order to
minimize color fading that occurs after prolonged exposure to
light.
After addition of the base solutions, each tube is oscillated
for about 2 sec by short rapid wris.t motion to mix the contents,
-24-
-------
resulting in a clear phase separation. If the upper phase is
not crystal clear and has traces of residual cloudiness, additional
oscillation will clear it. The absorbance value of a reagent
blank, which is deducted from the values obtained for the pro-
cessed samples, depends on the storage age of the triethylamine
reagent. The latter, when not in use, should be stored in an
amber glass bottle. Slow oxidation of the reagent occurs upon
storage at room temperature* After storage for a 4-5 wk period,
the absorbance value of the reagent blank increases from a normal
0.03-0.05 absorbance unit of freshly prepared triethylamine
solution to above 0.10 absorbance unit. When the absorbance of
the reagent blank reaches 0.1, it is recommended that a new batch
be made. In usage during a workday, triethylamine from the stock
solution is transferred to a 125-mL 4-oz'clear glass bottle with
screw-cap containing an aluminum foil or polyethylene insert. The
smaller bottle facilitates loading of a 5-mL syringe with 18
gauge needle to discharge 2 aliquots per loading.
Note 12. Glass syringes in 2-, 5-, 10-, and 20-m.Tj sizes, each
with 2-in., 18-gauge needles, and precalibrateq. for accuracy of
delivery were used to dispense reagents and standards, and to
transfer aliquots. A 100-nL syringe was used to dispense 0.1 mL
amounts of NBP and oxalic acid solutions in preparing reaction
tubes.
Syringes are more rapid and convenient to use, clean and
dry than pipets. After use, the syringes were rinsed with
acetone from a wash-bottle, rinsed well with warm water, followed
by a few mL of acetone, and were then pumped to near-dryness and
were disassembled to drain and air dry for about 30 min.
-25-
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Note 13. OP insecticide standards are essential to assess the
measurement limits and to periodically check the RFM for accuracy
and reproducibility of spectrophotometric response when known
amounts of an OP compound are present in a sample. The instrument
response is in terms of absorbance units measured at a wavelength
setting of 560 nm. The practical upper limit for measuring
absorbance with accuracy is 1.0, although our instrument meter
showed 2.0 as the upper .limit.
A satisfactory standard curve shows a linear relationship
between the absorbance values and the amounts (ng) of OP compound
present in the reaction tubes. A standard curve is made by
depositing in reaction tubes a series of OP amounts in duplicate,
e.g., 0,1,2,4,10,15,20 and 25 ng, where "zero" represents the
reagent blank. The standard additions are then evaporated,
reacted at 150°C for 3 min, the colors are developed, and the mean
absorbance (uncorrected) is determined for each level added. The
absorbance value of the controls are deducted to provide corrected
absorbance values, which are then plotted as a straight line on
graph paper against the corresponding OP levels reacted. The
standard curve enables determination of OP residues of foliage and
soil by superimposing the absorbance values obtained in the
analyses and locating the corresponding concentration values on
the graph. Alternatively, the spectrophotometer scale could be
2
manufactured to read ppm or ng/cm directly.
As is shown in a subsequent section, the slope of the standard
curve was found to vary with the nature of the OP compound (see
Table 12). To calculate OP residue levels correctly, we need to
-26-
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know which OP compound was used in the spray application. This
prerequisite is complicated when two or more OP compounds co-
exist in the sample. Thus, the RFM does not differentiate between
the parent thion (P=S) and the oxon (P=0) analogue that may be
formed through oxidation. These aspects have been previously
discussed.
In closing Note 13, a simpler method of calculating OP residues
is by means of the mean absoorbance unit/^g ratio (Table 12) .
Thus, the absorbance unit/Vg ratio is calculated for each of the
seven concentration points in the example given above. The mean
value is the slope of the curve expressed as a number. Graphing
as such is unnecessary. A table of mean ratios to encompass the
OP insecticides of interest is prepared in advance. In application,
the absorbance value obtained for unknown amounts of OP residues
present in a sample is divided by the mean absorbance unit/ug
ratio of the OP compound.known or assumed to be present, and the
resulting number is the micrograms present in the sample. More
exact information is obtained by deducting the mean value in (jg
of pretreatment samples, if available, taken from the same area
and locations of the post-treatment areas from which the samples
of foliage or soil originated.
Note 14. This note deals with simplified soil sampling, processing
of soil samples, and a simple method of fortifying sieved soil dust
to determine efficiency of recovery of OP residues from surface
soil.
Since a direct linkage exists between soil particles dis-
lodged from leaves of fruits and vegetables and the adjacent soil,
-27-
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the need to examine surface soil for OP residues needs no
elaboration. For citrus trees, the dripline area has the greatest
v
OP residue levels and is also the area of greatest worker activity
(30). OP deposits on orchard soil may reach residue levels as
high as 800 ng/g (800 ppm). Longevity of deposit depends in part
on soil type (11).
Surface soil dust can be sampled by vacuuming with a portable
vacuum cleaner through .a 100-mesh screen placed over the soil
surface (30), but a gasoline-powered field generator is needed
to power the vacuum cleaner.
A simple, reasonably rapid alternative that enables sampling
by unit area consists of a metal or cardboard template with an
2 2
opening 4x4 1/2 in. (- 18 in. , or 1/8 ft ) placed on the area
to be sampled. The metal template can be made out of an aluminum
cookie pan or a piece of sheet metal. In use, the template opening
is placed against the rim of a rigid polyethylene dustpan obtain-
able at hardware stores complete with a nylon fiber anti-magnetic
2
brush 4 1/2-in. wide. Surface soil within the 1/8 ft area is
swept into the dustpan, and the sweepings are emptied into labeled
bags. The process is repeated at the 8 cardinal points (N, NE,
2
E, etc.) of a tree to traverse a combined area of 1 ft within
one composite sample.
Residual dust is not retained by the nylon brush. The
composite soil sample contains extraneous material (dried leaves,
small pebbles, twig fragments, etc.) which is sieved out by the
10-mesh screen nested on top of the 100-mesh screen and catchpan.
After vigorous shaking for about 45 sec, the soil dust in the
-28-
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catchpan (^ 100 mesh) is transferred to a small labeled bag, the
rest of the sample is discarded, and the catchpan is wiped with
tissue in readiness for the next sample. A range-finder test is
then made with a level 1/2-tsp of sieved soil to find a suitable
combination of spoon size and acetone: hexane aliquot fraction
to use for the determinations. After obtaining a suitable com-
bination (with 25 ng OP residue in the aliquot as the upper limit),
the results can be reported both'as ppm (ng OP/g soil) in the
2
samples and also as ng OP/ft of'surface soil, since the sieved
sample is a composite of the contributions of 8 subsamples each
2
representing 1/8 ft . In our experience, the mean weight of soil
2
dust/ft was approximately 21 g (3 1/2 level tsp), so that, for
example' a value of 50 ppm OP would thus be equivalent to 1050 jag
2
OP residue/ft of surface soil taken from the dripline or other
tree area for which information on OP residues may be sought.
The efficiency of recovery of OP compounds from fortified
soil samples was determined as follows: OP standards in hexane
are added to a duplicate series of PP tubes containing 1- and 2-g
samples of sieved soil. The standards are added to obtain con-
centrations corresponding to 0 (none added), 10,20,50,75,100,200,
300,450 and 600 ppm. The tubes are gently swirled for about 8 sec
to obtain a uniform slurry or suspension, after which they are
placed overnight in the fumehood. The hexane evaporates completely
and the dry fortified soil is then processed as indicated above.
The % recovery is calculated as the ratio of the ng of OP residue
recovered compared to the ug OP incorporated into the soil,
multiplied by lOOx.
-29-
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Note 15. A portable workbench or equivalent is needed to accommo-
date the items required to conduct the RFM in the field (Table 2).
A 1/2-in. thick plywood board, 18 in.x6 ft, supported at the ends
on empty 5-gal cans or on crates, is adequate for field work.
The tailgate of a pick-up truck or station wagon can also be used
as a workbench. Light-weight portable tables with a telescoping
surface and fold-up legs are commercially available, but not in
table heights of 36-42 in. that are suited for stand-up work.
The campstove (either propane or gasoline type) should be
located on a pair of stacked empty 5-gal cans or equivalent about
20 ft away from the workbench to avoid the risk of chance ignition
of flammable vapors stemming from organic solvents on the work-
bench.
During Stage 4, the workbench should be located in a well-
shaded area to minimize the fading effect of bright sunlight on
the magenta colors developed upon completion of the OP-NBP reaction.
2. OP Compounds Used to Explore the Analytical Scope of the RFM.
Table 3 lists 43 OP compounds that were tested in the range
1-30 (jg in most cases, and in the range 1-20 |j.g for the more
reactive species. All the compounds were tested for response
by reaction at (A)150°C for 3 min. However, 24 of the 43
compounds were reacted also at (B)100°C for 30 min to obtain
a comparison set of mean absorbance unit/Vg ratios to comple-
ment those resulting from reaction at 150° for 3 min. This
experimental procedure was considered useful since the
relationship of Ratio B/Ratio A appeared to be relatively
constant for a given compound and might therefore be of value
-30-
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TABLE 3. ORGANOPHOSPHORUS COMPOUNDS USED IN EXPLORING THE
ANALYTICAL SCOPE OF THE RFM
Cpd
no.
Common name
Cpd
no.
Common name
1 acephate
2 azinphosmetnyl
3 azinphosmethyl oxon
4 carbophenothion
5 chlorpyrifos
6 chlorpyrifos oxo'n
7 DDVP
8 DEF
9 demeton
10 diazinon
11 0,O-diethyl S-methyl
phosphorothioate
12 0,0-dimethyl
phosphorodithioic acid
13 dimethoate
14 dimethoate oxon
15 dioxathion
16 EPN
17 ethion
18 0-ethyl 0-nitrophenyl
cyclohexyl phosphonate
19 O-ethyl O-nitrophenyl
phenyl phosphonate
20 fenthion
2l glyphosate
22 isomalathion
23 isopropyl parathion
24 isopropyl parathion
oxon
25 malathion
26 malathion oxon
27 methidathion
28 methidathion oxon
29 methyl parathion
30 mevinphos
31 monocrotophos
32 naled
33 parathion
34 parathion oxon
35 phenthoate
36 phenthoate oxon
37 phosphamidon
38 O,O,O,O-tetramethyl
pyrophosphorodithioate
39 trichlorfon
40 0, S,S-trimethyl
phosphorodithioate
41 O,O,S-trimethyl
phosphorothioate
42 0,O,S-trimethyl
phosphorodi thioate
43 o,O,O-trimethyl
phosphorothioate
-31-
-------
in assisting in the identification of the OP compound in a
sample, providing only one OP species was involved.
Of the 43 compounds shown, 4 reacted weakly or not at
all at 150°C for 3 min. Additional comments on the variable
reactivity are made in Section 6, in the Results and Discussion
section.
3. GC Equipment and Supplies Used. Gas chromatography (GC) was
used in particular aspects of the field evaluation program to
supplement the RFM, but mainly to obtain comparative data on
the longevity and levels of residues resulting from spray
applications of parathion, malathion, methidathion and phen-
thoate to citrus trees in field experiments. GC was also used
to measure as coexisting species the oxon levels environmentally
generated during the disappearance of their parent thions from
previously sprayed foliage.
A Tracer MT-222 Gas Chromatograph equipped with a flame
photometric detecto'r was used. The instrument was fitted
with a carrier gas by-pass valve to enable venting of the
carrier gas (nitrogen) at desired points during the progress
of the chromatogram.
Two different glass columns were used for OP separation
from the hexane extracts of samples that were injected, namely:
Column A, 150 cm x 4 mm i.d., packed with 4% OV-101 on 80/100
mesh Gas Chrom Q and used for samples containing residues of
parathion, malathion and phenthoate and their oxons; Column B,
40 cm x 4 mm i.d., packed with 5% Apiezon N on 80/100 mesh Gas
Chrom Q and used for residues of methidathion and methidathion
oxon.
-32-
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SECTION 5
METHODS
1. The 4-Stage Sequence of the -RFM. The 4-stage sequence and
time required to process 6 foliage or soil samples by the RFM
are indicated in Table 4, with Supplementary Note numbers 16
and 17 appended to this Table.
TABLE 4. THE 4-STAGE SEQUENCE AND TIME NEEDED TO PROCESS SIX
DETERMINATIONS OF OP RESIDUES OF FOLIAGE OR OF
SURFACE SOIL
Stage
no.
Nature
General operational procedure
Mean time
(min) for
6 tests
Sample
preparation
Hexane
evaporation
OP-NBP
reaction
Measure
color
intensity
& calculate
OP levels
Add 20 mL 20% NaCl solution to sample 12
jars containing leaf punch disks, cap
and shake jars vigorously for 30 sec
to dislodge OP residues. Decant the
extract into 50-mL graduated poly-
propylene (PP) tubes to the 15-mL mark.
Add 15 mL hexane, cap the PP tubes,
shake 20 sec to partition OP residues
into hexane layer. Transfer 10-mL
aliquots of hexane layer to prepared
test tubes.
Evaporate hexane solutions to near- 1 1/2
dryness in aluminum heating block
containing ethylene glycol and pre-
heated to about 160°C. Note 16
Heat evaporated extracts at 150°C for 3
3 min to react OP molecules in the
extract with NBP in the prepared tubes.
Add triethylamine and sodium carbonate 8
solutions to reacted tubes to develop
a magenta color. Shake to clear. Trans-
fer aliquot to spectrophotometer cuvette
and determine color intensity 'absorbance)
at 560 nm. Deduct absorbance value of
reagent blank and calculate OP levels by
table or graph. Note 17
Add downtime used between Stages 1-4 5 1/2
Total time needed for 6 tests of
foliar OP residues30 min
-33-
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Preparation
of soil
samples*
2-4 Same as 2-4
above
Based on preliminary tests for approximate
OP levels in soil, measure 1/4-3 level tsp
sieved soil ( < 100 mesh) and deposit in PP
tubes. Add 1/4 mL 20% salt solution and
20 mL acetone-hexane 15:85 v/v. Shake
capped tubes vigorously for 20 sec. Transfer
by syringe aliquots ranging from 1 to 10 mL
of extract into prepared tubes.
Stages 2-4 for surface soil are the same
as for foliage above. Calculate OP residues
as ppm (|ag OP residue/g soil) and/or as
Hg/ft2, taking into account the predeter-
mined weight of soil per unit volume of
level spoon size used in Stage 1, and the
aliquot size (fraction of 20 mL extract
volume) used for Stage 2.
*Total time
needed for
6 soil samples
= 26 min.
Note 16 (re hexane evaporation). With the burner flame completely
extinguished and within 7-8 sec after placement in the heated
aluminum blocks, the hexane in the reaction tubes commences to
boil rapidly and within 1-1 1/4 min. is reduced to near dryness,
leaving about 100|jL of residual hexane resulting from condensation
of hexane vapor at the upper part of the tube when the tube is
removed from the hot block.
To promote uniform heating times during Stage 3 that follows
hexane evaporation, it is recommended that the tubes undergoing
evaporation in Stage 2 be removed about 3 sec after hexane evapor-
ation visibly ceases. This minimizes the hexane condensate that
forms (and which poses no hazard in Stage 3), and also ensures
that the tubes get a uniform heating time for the OP-NBP reaction
step that follows. If more than 0.1-0.2 mL remains in the tube,
-34-
-------
replace in the heater block for about 6-7 sec. Evaporation to a
dry state is acceptable if the period of complete dryness is short,
not exceeding 8 sec.
Note 17 (re color measurement and calculations). Charging the
spectrophotometer battery overnight one day prior to use ensures
quicker attainment of equilibrium in the absorbance readings.
As was indicated, advance knowledge of the nature of the OP
compound that is the active ingredient (a.i.) in the spray treat-
ment applied makes it possible to use the appropriate absorbance
unit/ng ratio, such as one from Table 12 herein, or one obtained
independently by on-the-spot tests with standards, in calculating
OP residues. A graph is not necessary. After deducting the
absorbance value of the reagent blank from the uncorrected
absorbance values, the resulting corrected values are divided by
the mean absorbance unit/Vg ratio of the insecticide in question
to obtain the |ag present in the sample, and then by 200 to obtain
2
ug/cm leaf surface, if 10-mL aliquots and 40-leaf disk samples
were used.
For soil, the (jig of a.i. present are divided by the weight
of soil used to obtain a result expressed as ppm (ug/g), which
o
in turn is multiplied by 21 to obtain ng/ft of surface soil.
Because soil types and moisture contents differ according to
source, the 21x factor should be checked and adjusted to fit the
individual soil sampling program.
To monitor for research purposes the dissipation or dis-
appearance rate of the active ingredient, the mean absorbance
value of pretreatment samples is also deducted from the uncorrected
-35-
-------
values. However, to assess foliage or soil for worker safety
status, it is not deducted, since the residual OP levels present
in the pretreatment samples augment the residual levels stemming
from the most recent spray application.
The non-specificity of the RFM has previously been.stated,
but for the express purpose of monitoring risks from OP residues
to worker safety this is not regarded as an insurmountable obstacle,
since it would be useful to know that measurable amounts of OP
compounds were present, since all are toxic, some more than others.
2. Procedure for Processing Leaf-Punch Samples. After the leaf-
punch samples have been taken (Table 2, Item 7, Note 5), remove
the screw caps containing the Teflon disk inserts, and add
20 mL 20% salt solution to each jar, using the 500-mL squeeze
bottle with precalibrated dispensing reservoir (Table 2,
Item 14g). Cap the jars tightly.
Shake the jars vigorously for 30 sec, allow to settle for
about 15 sec, and decant 15 mL of the leafwash suspension into
50-mL graduated polypropylene (PP) screw-cap centrifuge tubes.
Add 15 mL hexane by syringe, shake the capp ,d PP tubes vigo-
rously for 20 sec, held with the caps facing the ground during
the shaking to minimize chance of leakage from a cap with
defective screw threading. If a stable emulsion is formed
due to the vigorous shake procedure, tap the sides of the PP
tube hard with the forefinger and then whip the tube sharply
V
downwards twice. Repeat 3 times, if necessary.
Place the PP tubes at approximately a 30° angle into the
salt bed tube rack. With a 10-mL syringe, fitted with a
-36-
-------
2-inch 18-gauge needle, remove a 10-mL aliquot of the clear
hexane layer from each tube,keeping the needle point away
from the salt layer or any floating leaf fragments. Transfer
each aliquot to pencil-numbered prepared reaction tubes.
While the foregoing preparatory tasks are in progress,
heat the 6-hole aluminum blocks containing ethylene glycol
to 155-160°C on the campstove. The campstove should be
located upwind about 20 ft from the hexane-handling-and-use
area of the field workbench (Table 2, Note 15). Smoking or
any open flame within the immedic,te vicinity (8-ft radius) of
hexane or acetone open to the air is a fire hazard.
Shut off the burner valve as soon as the block temperature
reaches 155-160°C. If a propane burner was used, place* the
reaction tubes into the block for rapid (1 1/2 min) evaporation
of the hexane. If a gasoline-operated burner was used, (a) wait
1 1/2 min until the lingering after-burn flame is self-
extinguished before placing the reaction tubes into the heated
blocks; alternatively, (b) convey the hot blocks, using needle-
nosed pliers for gripping and moving the blocks to the workbench,
and then insert the reaction tubes.
Remove the tubes from the hot blocks about 3 sec after
evaporation visibly stops. This leaves about 6-8 drops of
liquid hexane in the tube bottom resulting from the conden-
sation of residual hexane vapor in the upper portion of the
tube when the tubes are removed. Place the tubes in padded
open-top coffee cans or equivalent to serve as tube racks.
Add some ethylene glycol from the squeeze bottle to the block
-37-
-------
holes to replenish glycol removed by adhesion to the bottoms
of the reaction tubes. Add a fe^ drops of glycol to the
thermometer wells to replenish glycol lost by slow evaporation.
Reheat the aluminum blocks to 150 ± 1°C, at which point
put the burner valve in a "simmer" or "hold" position. (With
nominal practice, 150° can be maintained by burner management
for as long as is required.) Place the reaction tubes, two at
a time, into the heated blocks, and react them for 3 min, using
the egg-timer as a 3-min timing device. Remove the tubes from
the block in the same order as they were placed therein. Cool
the tubes in the can racks for about 2 min. (End of Stage 3.)
To each tube add by syringe 2.5 mL of triethylamine 20%
in acetone solution, and 1 mL sodium carbonate (12% w/v in
15% salt solution), including the reagent blank tubes, in
that order. A magenta color varying in intensity with the
amount of OP compound in the residue develops when the tri-
ethylamine is added. Shake the mixture by short, rapid wrist
oscillation for a few seconds until a crystal clear super-
natant solution is obtained, free from cloudiness or haze
bands.
Pour an aliquot (about 1 1/2 mL) of the clear supernatant
solution into the spectrophotometer cuvette (square 10-mm cell
preferred) after zeroing the instrument, and determine the
absorbance at a wavelength setting of 560 nm on the portable
spectrophotometer. Deduct the absorbance value of the reagent
blank from those of the samples to obtain corrected readings.
Calculate the ng levels from the corrected values using the
-38-
-------
absorbance unit/ug ratio for the OP compound in question, or
a table or graph pertaining thereto. Divide the net ug level
2
by 200 to obtain ng/cm leaf surface.
3. Procedure for Processing Samples of Surface Soil Dust. With
a polyethylene dustpan, a fitted non-magnetic 3 1/2-in. nylon
fibre brush and a 1/8 ft template (Table 2, Note 14), sweep
into the dustpan the surface soil at the dripline area of
each designated tree at the 8 cardinal points (N,NE,E,etc.).
Transfer the succession of sweepings to a numbered polyethylene
2
bag, thus obtaining a composite sample representing one ft of
surface soil.
Using two Tyler U.S. standard screens with a 10-mesh
screen size mounted above a 100-mesh size fitted to a catch-
pan, sift the composite sample vigorously for about 1 min
and discard the coarse soil particles, twigs and pebbles
caught by the no. 10 screen and the soil particles that remain
on the 100-mesh screen, but returning and transferring the
fine dust (< 100 mesh) in the catchpan to polyethylene bags
for subsequent weighing and storage at or near 2°C for future
reference. The sifted soil dust (-^100 mesh) is of importance
because of its potential of becoming airborne as a result of
worker activity and agricultural machinery and orchard equip-
ment in operation.
Using a precalibrated set of standard spoons (Table 2,
Note 9), transfer a level 1/2-teaspoon of sifted soil to a PP
tube. Add 0.25 mL 20% salt solution and 20 mL of mixed solvent
(acetone: hexane 15:85 v/v) by syringe. Shake the capped PP
-39-
-------
tube vigorously for 20 sec. Place each PP tube at approxi-
mately a 30° angle in the salt bed tube rack. Remove from
each rack by syringe three test aliquots of l-,4- and 10-mL
size respectively, and transfer to 3 prepared reaction tubes.
(Aliquots of the foregoing sizes correspond to factors of
20x, 5x and 2x respectively, to be applied to the subsequent
net absorbance values after color development of the subsamples.)
Evaporate the solvent (1 1/2 min approximately), react
(150° for 3 min) and develop the magenta color (Stages 2-4)
as outlined in the previous section on processing dislodgable
OP residues of foliage. Observe the previously stated safety
precautions against possible flash ignition of rapid, high-
volume generation of solvent vapors from a lingering burner
flame in the immediate proximity (8-ft radius) of the
evaporation (Stage 2) procedure.
Calculations. To calculate the ppm level (|jg OP residue/g
surface soil dust), first multiply the uncorrected absorbance
values by the appropriate factor for the aliquot size used,
then deduct from this augmented absorbance the absorbance
value of the reagent blank to ge : a corrected value, and then
convert the corrected absorbance reading into total (jg levels
by using a standard curve or simply by. dividing by a pre-
determined mean absorbance unit/ug ratio corresponding to the
OP compound being sought (see Table 13).
The weight/volume relationship of the particular soil
dust is invoked next, e.g., if one level 1/2-tsp of sieved
soil has a mean weight of 2.9 g, divide the total ^g by 2.9
-40-
-------
to obtain ppm values. To convert: ppm to the more useful
ug/ft2 of soil surface, first determine the mean weight of
sieved (<100 mesh) soil by weighing 6 composite samples of
2
sifted soil from a given location, each representing 1 ft
of surface, and record for future calculation purposes the
o
average wt/ft . Then multiply the ppm value by the mean
9 2
wt/ft . For example, if the mean weight was 29 g/ft , and
results of 30 ppm (30 ng/g) had been obtained, then an
2
average of 870 ng OP pesticide/ft of surface soil dust was
present at the time of sampling the particular location.
It should be noted that absorbance values that register
higher than 1.0 on the spectrophotometer are beyond the
linearity of instrumental response, and are therefore inaccurate
and invalid. When excessively high readings are obtained,
repeat the test by using a smaller aliquot size, or lesser
sample weight (smaller spoon size). In general, a suitable
aliquot size selected by the test method above is applicable
also to the various samples in a given sampling program,
assuming that the sample history (type of treatment, dosage,
application date, etc.) is the same for all.
4. Standard Solutions for Calibration of Methods. To ascertain
the efficiency of recovery of OP residues by a particular
method of processing foliage or soil samples, or to determine
on a daily or weekly basis the precision and reproducibility
of the instrumental response, or to determine the range of
linear response under particular conditions, standard solutions
of OP compounds of known identity and concentration are used.
-41-
-------
5. Determination of % Recovery From Fortified Leaf Extracts.
Fortification of leaf extracts requires at least 12 x 40-leaf
disk samples taken from foliage that shows low net absorbance
in the sample blank (e.g., 0.009 to 0.06) after the reagent
blank is deducted. Before considering the mathematical
interrelation in preparing leafwashes fortified with known
amounts of OP compounds for subsequent tests of the effici-
ency of this recovery by the RFM, preliminary comments and
recapitulation are perhaps in order. Thus, the method
previously outlined for determination of dislodgable OP
residues on foliage has these basic parts: (a) Add 20 mL
salt solution to each sample jar containing 40 leaf disks,
2
which have a combined area (both sides) of 400 cm . After
shaking, (b) decant 15 mL of leafwash (15/20 or 3/4 of the
20 mL salt solution used) into PP tubes and add 15 mL hexane
(extract). After shaking, (c) transfer to 10 mL of hexane
supernatant (2/3 of the 15 mL hexane previously added) to
prepared reaction tubes. Then evaporate, react, and color-
develop as indicated. The corrected absorbance values are
then converted to ug of OP compound and are divided by 200
2
to obtain ug/cm leaf surface. The denominator 200 stems
2 2
from: (a) 3/4 of the 400 cm leaf disk sample (=300 cm
leaf area) which is decanted into the PP tubes, from which
2
(b) 2/3 .or 200 cm leaf area are actually analysed.
Fortification. In adding variable amounts of standards
to the leaf wash in PP tubes and adjusting the hexane volume
to 15 mL, 50% more of the standard than the amount to be
-42-
-------
analysed is added. This allows for the fact that 2/3 of the
volume (10 raL) will be used.
Calculation of % Recovery. The corrected absorbance
values of the amounts of OP recovered from the fortified
samples are divided by the predetermined absorbance unit/[ag
ratio to obtain "micrograms recovered." The latter values are
then divided by the tag added, and multiplied by 100 to obtain
% recovery.
6. Determination of % Recovery From Fortified Soil. The forti-
fication procedure for soil dust (<100 mesh) is more direct
than that for foliage. It involves different soil sample
weights and OP standards to span an adequately wide ppm range,
and the following steps:
a. Obtain about 150-200 g of soil dust (•<• 100 mesh) from an
area untreated with OP pesticide for over one year, that in
any event is low in net absorbance (0.10 or less) after the
reagent blank absorbance has been deducted, and of a type
and moisture content analogous to that of the designated
treated areas.
b. To traverse the range 0-400 ppm (6 concentrations, each
in duplicate, see Column 2 in Table following), deposit 3-g
and 1-g amounts of the soil dust into PP tubes (Table,
Column 1). Add standards in hexane in amounts shown in
Column 3. Swirl the PP tubes gently by hand to get a uniform
suspension of soil-hexane, and allow to settle. The hexane
evaporates to dryness in storing the tubes overnight at room
temperature.
-43-
-------
c. Add to each tube 0.25 mL 20% salt solution and 20 mL of
mixed solvent (acetone; hexane 15:85) (Column 6), and shake
the PP tubes vigorously for 20 sec. Transfer aliquots of the
size indicated in Column 7 to reaction tubes. (Note: Two
separate soil blanks are shown in the Table for 3-g and 1-g
amounts of soil, respectively, (Column 1) . From the 3-g soil
blanks, transfer 1, 2 and 4 mL aliquots to reaction tubes,
and from the 1-g soil blanks transfer 1- and 2-mL aliquots.
Their respective absorbance values are deducted from those
of the fortified soils that require similar aliquot sizes.
(See Calculations.) After raising the hexane volumes in the
reaction tubes to approximately 10 mL, evaporate, react and
color-develop as was previously indicated.
-44-
-------
Table for Preparing Fortified Soil Samples for Determination of Efficiency
of Recovery
in
(1) (2) (3) (4)
Grams of ppm of \ig of std. mL of mixed
soil dust OP incorpora- solvent to
in PP tube Cpd. ted in soil add after
evaporation
3 Soil
1 Blanks
3
3
3
1
1
*Aliquot
0
0
25
50
100
200
400
sizes
0
0
75
150
300
200
400
(Column 5)
20
20
20
•20
20
20
20
transferred from the
(5) (6) (7)
mL to trans- |ag of std. Multiplication
fer to present in factor
reaction aliquot
tubes
variable *
variable *
4
2
1
2
1
soil blanks
0
0
15 5
15 10
15 20
20 10
20 20
vary, and are taken to
correspond in size to those transferred to reaction tubes from the fortified soils.
From the 3-g blank, a total of 7 mL would be transferred among 3 tubes, and from
the 1-g blank, a total of 3 mL among 2 tubes would be needed.
Calculations; The uncorrected absorbance values of the fortified samples are first
multiplied by the appropriate factor 'Column 7), and the appropriate absorbance
value of the soil blank is then deducted from the augmented absorbance value. The
corrected absorbance numbers are then divided by the mean absorbance unit/Vg ratio
for the particular OP pesticide, which yields "micrograms recovered." The latter
values are then divided by the "ng added" (Column 3), and the resulting ratio or
quotient is multiplied by 100 to obtain % recovery.
-------
7. GC Methods. GC equipment and supplies used in conjunction
with the GC method were specified in Section 4, subsection 3.
Analyses by GC were confined to multiple samples of citrus
foliage and surface soil from the dripline area.
No changes were made in the processing of soil samples
for analysis by GC, except for occasional dilution of the
hexane extracts that were necessary to bring the OP levels
down to the instrumental working range. Processing of foliage
samples, however, was different and more exhaustive in the
extraction than that used for the RFM, and followed the method
of iwata et al. (12) .
The processing of foliage samples for analysis of dis-
ledgable OP residues were conducted as follows:
a. With the leaf-punch apparatus (Table 2, Notes) take a
series of leaf disk samples in duplicate, obtaining 40 disks/
sample comprising 8 disks per tree punched at 45° intervals
around the tree perimeter at about 4 ft above ground level,
and sampling only mature, second-cycle leaves, cutting a disk
from the center portion of each leaf sampled. To offset
possible chemical changes that conceivably could change the
thion-oxon ratio, temporarily store.the sample jars in ice
immediately after the samples have been taken, then process
as soon as possible.
b. To each jar add 100 mL water and 4 drops of a 1:49 dilution
of Sur-Ten (70% dioctylsulfosuccinate, sodium salt) or equiva-
lent wetting agent. Cap each jar tightly and shake on a
reciprocating shaker at 200 shakes/min for 20 min.
-46-
-------
c. Decant the leafwash including dislodged leaf dust present
into a 500-mL separatory funnel or an interim holding bottle
(12-16 oz size), but retaining the leaf disks in the jar.
d. Repeat the foregoing dislodgement process two more times,
adding the leafwashes to the separatory funnel or bottle.
e. Add 50 mL dichloromethane to the combined leafwashes in
the separatory funnel and shake for 1 min.
f. Drain the lower layer through a glass funnel containing
approximately 10 g of sodium sulfate into a 300-mL Erlenmeyer
flask or sample storage bottle.
g. Repeat the partitioning above with a second 50-mL of
CH-Cl- and a 1-min shake. The water in the separatory funnel,
upon standing, builds up residual CH2C12 that settles out from
the partly emulsified water and accumulates above the stopcock.
The accumulated solvent should be added to the portion pre-
viously drained through the sodium sulfate.
h. Wash the receiving funnel and sodium sulfate with about
10 mL CH2C12.
i. Remove the CH2C12 by evaporation under partial vacuum using
a Buchler Roto-vap or equivalent apparatus.
j. Dissolve the residue in the flask in acetone or hexane and
transfer the solution quantitatively to a labeled 15-mL
graduated glass-stoppered centrifuge tube, from which sub-
samples are taken for analysis.
8. Determination of Storage Stability of Prepared Tubes. While the
convenience and contribution to RFM speed of an ample supply of
prepared reaction tubes was readily demonstrated, a question was
-47-
-------
posed regarding their suitability for reproducible OP-NBP
reaction after prolonged storage at ambient or elevated
(e.g., 110°F) temperatures.
An experiment was accordingly set up with parathion.
Salt crystals were added during the preparation of the reaction
tubes and triplicate tubes were used. Thus:
2 storage temperatures: 22 and 43°C
7 storage .periods: 0, 2, 4, 6, 8, 10 and 12 weeks
1 reaction temperature: 150° for 3 rain
3 parathion amounts: 0, 5 and 10 pig (added after
each storage period)
3 replicates for each condition: each prepared
tube in triplicate
After each storage period, tubes were removed from storage,
and the designated amounts of parathion standards were
introduced. Tubes were then reacted at 150°.
The findings of this experiment are reported in Section 6.
9. Determination of Mean Absorbance unit/^g Ratios of 39 OP
Compounds. Reference has been made to the analytical useful-
ness and simplicity of using the mean absorbance unit/ng ratio
to calculate the residue levels of a specific OP compound on
foliage or surface soil after the corrected absorbance of the
- sample is determined. Because this ratio expresses
mathematically the slope of the standard curve, wherein
experimentally obtained absorbance values at 560 nm are
plotted against a range of concentrations (ug), it can also
be used to compare the relative reactivity of OP compounds
with NBP, either at 150° for 3 min or at any other temperature-
time combination. Thus, a high -"reactivity index" would be
-48-
-------
shown by a relatively steep slope and an accordingly high
absorbance unit/pig ratio.
With the objective of ranking OP compounds along lines
of relative reactivity towards N3P, 43 compounds (Table 3)
were each reacted at 150° for 3 min over a range of at least
5 and generally 7 amounts and the mean absorbance unit/|ag
ratios were determined. Four compounds reacted weakly or not
at all and were accordingly discontinued, although reaction at
165° for 4 min or 150° for 8 min raised their reactivity
appreciably.
Of the remaining 39 compounds, 24 were reacted also at
100° for 30 min to test the possibility of using ratios to
help characterize and identify a given OP compound.
Interim results of these endeavors are tabulated in
Tables 10, 11 and 13, and are discussed in Section 6. As
will be seen, there is considerable variation in the com-
templated "reactivity index."
10. Fading of Color Intensity. In preliminary field sampling of
foliage after application of phenthoate to orange trees,
the RFM results were lower than their GC counterparts. This
was traced to induced fading of the developed color by direct
sunlight, which fact was not evident until a well-shaded area
was used during the color-development step and subsequent
absorbance measurement.
This observation posed the question of the effect of
quality and intensity of light on color fading. To examine
this point, comparative tests using parathion standards over
-49-
-------
the 2-20 ug range were conducted under the following conditions:
(a) fluorescent light (laboratory), (b) indirect light (well-
shaded area), (c) near absence of light (a dark room with a
limited amount of light), and (d) direct sunlight.
The tubes after OP-NBP reaction were color-developed under
the 4 conditions of light. After absorbance readings had been
made of the 8 tubes in a given group, the absorbances were
redetermined in the same order, repeating this process at 4-min
intervals until 24 min had elapsed.
Results and significance of the tests are discussed in
Section 6.
11. Determination of Foliar and Soil Residues of Phenthoate. Tests
were conducted by the RFM and by GC to obtain dissipation curves
for phenthoate applied to orange trees. Phenthoate was applied
by two methods: (a) low-volume (LV) spray, and (b) oscillating
boom (conventional method) spray, using 1 pt of 4EC formulation/
100 gal of spray at 6 Ib a.i./acre. The amount of a.i. was
identical for both methods of application, but previous ex-
perience had shown that LV sprays yielded comparably higher
residues. It was also an opportunity to use the RFM on the
determination of phenthoate residues on surface soil.
For the foliage samples, the design was:
1. Two methods of application
2. Two methods of residue determination (RFM and GC)
3. Twelve sampling times (1 pre-application; 6 post-
application, twice weekly for 3 weeks; 5 post-
application, once each week for 5 weeks)
-50-
-------
4. Duplicate samples for each analytical method, each a
composite (40 disks) of 4 trees.
The methods used for determination of phenthoate by the
RFM and by GC have been indicated herein for the RFM. The
GC method for phenthoate and phenthoate oxon employed a
1.5 m x 4 mm i.d. glass column packed with 4% OV-101 on 80/100
mesh Gas Chrom Q. Column, inlet and detector temperatures were
210, 215 and 195°C, respectively; nitrogen carrier gas flow was
80 cc/min.
Results are discussed in Section 6.
12. Dissipation Curves for Dislodgable Foliar Residues of Malathion,
Parathion, and Methidathion After Application to Orange Trees.
The main objectives were: (a) to test the speed, accuracy,
capabilities, and limitations of the RFM developed to this
point for application in the field, and (b) to map the compara-
tive dissipation rates of malathion WP, parathion WP, and
methidathion, all applied on the same day and in the same
general location to orange trees. With methidathion, two
formulations, WP and EC, were applied.
The analytical methods employed (a) the RFM conducted at
the field-sampling site, and (b) GC conducted at the laboratory
for the determination of the parent compounds and their oxons.
The advantage of application of the three insecticides (four
formulations) by two methods of application (dilute and LV) on
the same day was that diurnal weather variations throughout the
field exposure period would be essentially the same, and would
thus enhance the reliability of' the intercomparisons.
-51-
-------
The design was as follows:
1. Four OP treatments: malathion WP, parathion WP,
Supracide WP, Supracide EC
2. Two methods of application: (a) diluted as for
conventional oscillating boom spray but applied
manually, and (b) LV spray. Six to 12 trees were
used for each method of application for the 4 OP
treatments.
3. Two samples per method of application per sampling
4. Twelve sampling dates, spanning a time interval from
the pretreatment sampling to 62 days postapplication.
5. Two methods of determining OP residues: (a) RFM, for
determining the total OP residue present, and (b) GC
for determining the parent insecticide and its oxygen
analogue.
Orange trees were sprayed at the highest rate likely to be
made under agricultural practice in California. Formulations
used were Phoskil 25WP (parathion), Malathion 25WP, and
Supracide 40WP and 2EC (methidathion). LV applications were
made with a Kinkelder sprayer equipped with an air tower.
Rates were 7.2 Ib a.i. parathion, 12 lb a.i. malathion or
4.8 lb a.i. methidathion/100 gal of spray/A. Dilute full-
coverage applications were made manually using spray mixtures
of 0.38 lb a.i. parathion, 0.63 lb a.i. malathion, or 0.25 lb
a.i. methidathion/100 gal of water.
At each sampling date, 4 sets of leaf-punch samples (40
disks per sample) were taken for each of the 8 treatments.
-52-
-------
Two sets of samples were processed in the field by RFM,
yielding results of 20 tests within 1-3/4 h of termination of
sampling. The other two sets were analyzed by the GC method.
Samples were immediately stored in a portable ice chest.
Samples were then mechanically shaken with 100 mL of water
containing a small amount of surfactant, the dislodgement-
washing was repeated 3 times. Residues in the combined aqueous
extracts were partitioned into dichloromethane, the partitioning
step was repeated twice, and the combined dichloromethane
extract was then dried by passage through sodium sulfate.
After evaporation of the solvent, the residue was dissolved
in acetone and transferred to graduated tubes. The 4% OV-101
column at 210°C column temperature was used for analysis of
parathion and malathion and their oxons, and the 5% Apiezon N
column at a column temperature of 215° was used for mcthidathion
and its oxon.
Results are discussed in Section 6.
13. Distribution Patterns of Methidathion on Leaves After Application
to Lemon Trees. mhe objective was to ascertain the areas on
citrus trees of the highest dislodgable foliar residues after
application of sprays. The RFM was selected to determine the
levels of OP residues on leaves along both a vertical and
horizontal axis at two postapplication periods. Supracide WP
was applied as dilute and LV sprays of the same total a.i.
levels/acre to lemon trees.
The design was as follows:
1. Three vertical-axis locations on each of 16 trees
coded as H=high, 6 ft above ground level, M=medium,
-53-
-------
4 ft above ground level, and 1^=1.5 ft above ground
level; all three locations at the outer periphery
of each tree.
2. Four horizontal-axis directional points: N,E,S, and
W sides of each tree.
3. Two Supracide WP treatments: (a) dilute, using
conventional oscillating boom, and (b) LV applications
using a Kinkelder sprayer with an air tower.
4. Two post-application sampling periods.
5. Three replicates of each sample location, directional
point and treatment of each period. Samples consisted
of 40 leaf disks each.
Analytical results were corrected for the control values
of pretreatment samples.
Results are summarized in Table 24 and discussed in
Section 6.
-54-
-------
SECTION 6
RESULTS AND DISCUSSION
1. Optimum Time-Temperature Combinations for Rapid OP-NBP
Reaction. Fast, reproducible OP-NBP alkylation reactions
were obtained at 150°C for 3 min of heating, which temperature-
time combination was selected through the systematic exploration
of the temperature range 95-1.65 °C and heating intervals of
from 1-40 min.
For examination of reaction temperatures above 100°, the
tubes were heated in 6-hole aluminum blocks. With a gasoline-
operated campstove, temperature control to ± 1° was readily
obtained by burner management. In the event of overheating,
the hot blocks are transferred with long-nosed pliers to an
adjacent aluminum plate for about 6 sec to lower the block
temperature, and are then returned to the burner plate to
complete the heating time to the 3-min termination.
Among various options that were tested for suitability
as a heat transfer medium, ethylene glycol was safe, miscible
with water in all proportions (an advantage in cleaning), and
has a sufficiently high boiling range (193-205°C) to be suitable.
As a second option for field use, one may conduct the
reaction in a boiling water bath for 30 min. This option
requires more time but is simpler as no manual temperature
control is required. The developed magenta colors are less
intense than by the preferred rapid 3-min method. Thus, the
slope of the absorbance vs |ag curves resulting from reaction
-55-
-------
at 100° for 30 min is shallower and therefore less sensitive
for measuring OP residue than the preferred reaction at 150°
t
for 3 min; both sets of curves, however, are equally linear
and reproducible.
Differences between slopes or absorbance unit/ug ratios
are illustrated in Figures 1 and 2, in which standard curves
for methidathion, parathion and demeton reacted at 150° for
3 min (Figure 1) can be compared with the same OP compounds
reacted at 100° for 30 min (Figure 2). However, before con-
sidering the possible economic advantage of using a 30-min
reaction period at a constant boiling point, one should include
as a prior step the prolonged time needed to evaporate tubes of
hexane to near-dryness in a pot of hot water. Thus, when the
water temperature reaches 100° and the burner is extinguished
to prevent fires, the tubes containing hexane soon reduce the
temperature to 90° or less, which prolongs the evaporation
process considerably, depending on the number of tubes introduced.
Nevertheless, the absorbance unit/ng value of the 100° reaction
can be used as an adjunct to the recommended 150° reaction
temperature to assist in identifying specific OP compounds,
which aspect is further discussed in Subsection 2 following.
Figures 1 and 2 also show that OP compounds vary in the
degree of response to reaction with NBP. This aspect is
illustrated in Tables 10 and 13, and further discussed in
Subsections 8 and 11.
-56-
-------
2.0 r
5 10 15 20
MICROGRAMS OF INSECTICIDE
25
Figure 1. Comparative standard curves for methidathion ( • ),
parathion ( O ), and demeton ( A ); reacted at 150°C for
3 min; mean absorbance of duplicate determinations.
-57-
-------
2.0 r-
5 10 15 20
MICROGRAMS OF INSECTICIDE
25
Figure 2. Comparative standard curves for methidathion ( • ),
parathion ( O ), and demeton (A ); reacted at 100°C for
30 min; mean of duplicate determinations.
-58-
-------
2. Two Different Temperature-Time Combinations to Assist
Identification of OP Compounds. To ascertain the slope
(mean absorbance unit/jag value) of 24 OP compounds, a series
of 5 to 7 amounts (ng) in duplicate in hexane to traverse the
range 1-20 or 1-30 ug were reacted after evaporation of the
hexane at (A) 150° for 3 min and (B) 100° for 30 min,
respectively. The respective slope values were then compared
for each compound as the ratio of their B/A values.
Differences in the B/A ratios were found (Table 12) with
a sufficiently wide range of ratios to indicate a potential
for identifying and confirming the presence of a specific OP
compound in residues. Such a method would presuppose that
only one OP species was present, or was predominant, in the
residues of unknown compositon. Such an assumption would be
unwarranted without prior information that might reduce the
number of prospective OP candidates.
3. Effect of the Amounts of Oxalic Acid and MBP on Color Formation.
For convenience in use in the field or laboratory, reaction tubes
are best prepared in advance in batches of 24-240 at a time,
depending on the number of samples to be processed in a given
period. The recommended proportions of reagents per tube
(0.1 mL NBP, 10% w/v in acetone, 0.1 mL oxalic acid, 0.04%
w/v in acetone, and about 25 mg of salt added to promote smooth
evaporation of hexane) were obtained empirically. As the
acetone volatilizes (6 h to dryness), crystals form on the
walls on the lower half of each tube. The prepared tubes can
be stored at room temperature covered with aluminum foil.
-59-
-------
Since preparation of reaction tubes is intended to be
done by personnel unfamiliar with the procedure, data on the
effects of inadvertent departures from the recommended amounts
of NBP and oxalic acid on absorbance after reaction with
parathion standards were sought. Results of various departures
are shown in Table 5.
The procedure directs that 0.1 mL of each reagent be added
to each reaction tube. If NBP is unintentionally omitted, no
color will form. If the oxalic acid is unintentionally omitted,
a color will develop but results will be unreliable. Addition
of an extra dose of either or both reagents above the recommended
amounts will, in general, yield values that are at least 80%
of the absorbance values obtained by using the recommended
amounts.
Salt crystals (25-50 mg, about 50-100 crystals) are added
to each tube with a small V-shaped spatula. Salt induces a
smooth boil start-up within 8 sec of placing hexane-containing
tubes in the hot aluminum blocks. In the absence of salt,
induction of boiling is retarded, resulting in superheating
and a violent boil-up of the hexane after about 30 sec.
Since no measurable color may be interpreted as no
determinable OP residues present, and low, erratic absorbance
values may be attributed to low field residues or to normal
variability of field samples, it is essential that each batch
of prepared tubes be checked for quality of preparation by
reproducibility of response to 2, 5 and 10 yg amounts of OP
standard.
-60-
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TABLE 5. EFFECT OF VARYING AMOUNTS OF OXALIC ACID AND
4- (jl-NITROBENZYL) PYRIDINE ON THE COLOR FORMATION STEP
a/
Amount—
NBP
1
2
1
2
1
2
Oxalic
acid
0
0
1
1
2
2
Absorbance at 560 nm—
Parathion (jig)
0
0.03
0.03
0.04
0.05
0.05
0.05
4
0.12
0.10
0.12
0.10
0.11
0.09
6
0.09
0.14
0.18
0.14
0.16
0.16
8
0.19
0.17
0.23
0.17
0.20
0.21
a/
— 1 = 100 pL of solution added, 2 = 200 yL of solution added.
— All values are means of duplicate tests. Absorbance values
for 4, 6,and 8 yg have been corrected for the reagent blank
(0 yg) reading. Heating was conducted at 150°C for 3 min.
-61-
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TABLE 6. EFFECT OF STORAGE OF TEST TUBES CONTAINING OXALIC ACID
AND 4- (ja-NITROBENZYL) PYRIDINE ON THE REACTION WITH
PARATHION
Absorbance—
Storage Storage
temp. (°C) time (wk)
22 0
2
5
7
8
10
12
43 0
2
5
7
8
10
12
Parathion (yg)
None
0.05
0.02
0.06
0.06
0.09
0.10
0.07
0.04
0.02
0.07
0.06
0.09
0.09
0.08
«£/
0.12
0.15
0.11
0.12
0.15
0.15
0.14
0.12
0.14
0.11
0.10
0.15
0.10
0.11
10*/
0.25
0.28
0.22
0.24
0.28
0.26
0.28
0.23
0.28
0.21
0.22
0.27
0.24
0.24
— Reaction conducted at 150° for 3 min. Values are means of 3 tubes
— Values have been corrected using the control tube values.
-62-
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4. Effect of Duration and Temperature of Storage of Prepared
Tubes on Reaction with Parathion. The convenience of pre-
paring large batches (200-600) of reaction tubes in advance
of their use in the field is readily apparent. As was indicated
in Section 5, subsection 8, the question of the effects of
duration and temperature of storage of prepared tubes on
reproducibility of response (absorbance) was investigated at
22 and 43°C (82 and 110°F) over a 12-week period. Table 6
shows results obtained when the tubes were removed from
storage and were reacted in triplicate tests with 0, 5 and
10 ng of parathion standards.
Reaction using tubes stored for longer than 2 weeks
yielded higher absorbance values. When parathion standards
were added and then reacted at 150° for 3 min, no adverse
effects due to storage were evident when corrections for
higher absorbance values of the reagent blanks were made.
The reagents in the tubes stored at 43°C were visibly yellow
in color after 7 weeks, the color intensity of which increased
with increase in storage duration. After reaction with para-
thion, the 10- and the 12-week tubes stored at 43°C were
visually off-color due to the strong yellow background present.
However, this did not interfere with the spectrophotometric
measurements of 560 run. Use of tubes stored at 43 °C for
periods up to 12 weeks is satisfactory since the higher
absorbance values would err on the side of workplace safety.
Additional comments regarding the effects of duration and
temperature of storage: (a) Performance tests with 2, 5 and
-63-
-------
10 (jig parathion standard made on prepared tubes stored for 10
months at 72°F (22°C) showed no significant change in absorbance
values or in absorbance unit/jag ratio, indicating that sustained
storage of prepared tubes at 72°F, at least for a 10-month
period, does not diminish or change their performance quality;
(b) It is unlikely that tubes would be stored continuously for
12 weeks at 100°F (43°C).
5. Effect of Elapsed Time Between Hexane Evaporation and High
Temperature NBP Reaction. For sample programming in the field,
it was considered useful to know whether prolonging the interval
between completion of hexane evaporation (Stage 2) and commence-
ment of the OP-NBP reaction (Stage 3) would affect the ultimate
results. For this purpose, 72 tubes (6 series in the range
0-10 pg parathion, each series in duplicate) were evaporated
after standards had been added, and were then reacted with NBP
at 150° for 3 min daily in groups of 12 up to 6 days after
preparation.
Table 7 shows that after the hexane has been evaporated,
the subsequent NBP reaction can be postponed up to 6 days, if
necessary, without any effect on the final determination. Thus,
the person responsible for taking and processing samples in the
field could, during a 4-day period, accumulate and process
samples up to Stage 2 completion, and then conduct the OP-NBP
reaction and color development more conveniently in a single
session at the ranch house or orchard office, provided that
obtaining results immediately after sampling was not a primary
objective at the time.
-64-
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When tubes are to be stored after the hexane is evaporated,
the numbered tubes should be covered with aluminum foil.
6. Effect of Temperature and Heating Time on the Reaction of
Parathion with NBP. Table 8 provides data on the reaction of
parathion at temperatures ranging from 100 to 160°C and heating
times from 2 to 40 min. The various slope, intercept and
correlation coefficient values are also shown. The reaction at
100°C, (actually 99.5° at Riverside, CA elevation) was con-
ducted in a boiling water bath; reactions above 100° were
conducted in the aluminum blocks as previously described. All
values shown are corrected for the reagent blanks used at each
individual temperature-time combination.
In the temperature test range 140-160°C, the temperature-
time combination of 150° for 3 min was selected as being the
most rapid, reproducible and generally suitable to meet the
objective of obtaining the most sensitive response (highest
absorbance) in a short time period suitable for field use.
This temperature-time combination was used as a standard
procedure in examining the relative OP-NBP reactivity of 43
compounds.
Table 8 shows also that the OP-NBP reaction can be con-
ducted at 100°, but absorbance values significantly lower than
those in the 140-160° range were obtained. Based on the
reproducible results, a temperature-time combination of 100°
for 30 min was selected as being suitable as a second option.
Heating time beyond 30 min is excessive for a rapid field
method. In considering the second option for field application,
-65-
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TABLE 7. EFFECT OF ELAPSED TIME BETWEEN HEXANE EVAPORATION AND
4-(£-NITROBENZYL)PYRIDINE REACTION ON ABSORBANCE OF
THE SOLUTION AT 560 nm
Elapsed
time
Ch)
1
24
48
120
144
Absorbance at 560 nm
Parathion (yg)
0
0.06
0.06
0.06
0.06
0.05
2
0.05
0.04
0.05
0.05
0.04
4
0.10
0.10
0.10
0.09
0.09
6
0.14
0.15 .
0.15
0.14
0.13
8
0.20
0.20
0.20
0.20
0.19
Slope
0.025
0.027
0.025
0.025
0.025
Intercept
0
-0.01
0
-0.01
-0.01
Corr.
coef f .
0.997
0.999
1.000
0.996
0.997
-66-
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TABLE 8 EFFECT OF TEMPERATURE AND HEATING TIME ON THE REACTION
OF PARATHION WITH 4-(£-NITROBENZYL)PYRIDINE
Absorbance (560
Temp . Time
(•C) (min)
140 2
3
4
5
145 2
3
4
5
150 2
3
4
5
155 2
3
4
5
160 2
3
4
5
100 5
10
15
20
25
30
35
40
ran)
Parathion (pg)
2.5
0.08
0.09
0.09
0.07
0.08
0.10
0.09
0.08
0.09
0.09
0.08
0.08
0.07
0.08
0.07
0.09
0.08
0.08
0.09
0.07
0.03
0.05
0.05
0.06
0.05
0.06
0.06
0.05
5
0.17
0.18
0.18
0.18
0.16
0.18
0.17
0.14
0.16
0.17
0.16
0.16
0.16
0.16
0.11
0.15
0.16
0.16
0.12
0.14
0.07
Q.09
0.10
0.12
0.12
0.12
0.11
0.10
7.5
0.23
0.25
0.26
0.25
0.25
' 0.28
0.26
0.25
0.22
0.27
0.23
0.22
0.21
0.25
0.19
0.17
0.22
0.22
0.21
0.21
0.11
0.13
0.17
0.16
0.18
0.20
0.18
0.16
10
0.31
0.34
0.37
0.32
0.34
0.35
0.36
0.29
0.35
0.35
0.31
0.26
0.28
0.31
0.32
0.22
0.31
0.28
0.28
0.23
0.12
0.16
0.20
0.21
0.24
0.25 .
0.24
0.23
Slope
0.030
0.033
0.037
0.033
0.035
0.034
0.036
0.030
0.034
0.035
0.030
0.024
0.027
0.031
0.033
0.016
0.030
0.026
0.026
0.022
0.012
0.015
0.021
0.020
0.022
0.026
. 0.024
0.024
Intercept
0.01
0.01
-0.01
0
-0.01
0.02
-0.01
0.01
-0.01
0
0.01
0.03
0.01
0.01
-0.04
0.06
0.01
0.02
0.01
0.03
0.01
0.02
0
0.02
0
-0.01
-0.01
-0.02
•Corr.
Coeff .
0.997
0.999
0.998
0.993
1.000
0.998
0.999
0.985
0.983
0.999
1.000
0.989
0.994
0.997
0.972
0.984
0.997
0.997
0.984
0.976
0.973
0.999
0.990
0.997
0.988
0.996
0.998
0.997
-67-
-------
one should also consider the longer time required to evaporate
the hexane from the reaction tubes (Stage 2) , a point pre-
viously discussed (Section 6, subsection 1).
7. Effect of Elapsed Time Between NBP Reaction and Color
Development. After the OP-NBP reaction is completed, it is
possible that some time may elapse before the base solutions
(triethylamine and sodium carbonate) are added to produce the
magenta color. To examine the effect of this possibility, 4
sets of tubes were each reacted with parathion in amounts of
0, 2, 4, 6 and 8 |ag in duplicate at 150° for 3 min. The
colors were subsequently developed after periods of 1, 24,
48 and 72 h after the reaction had been completed.
Table 9 provides data on comparative absorbance values,
and slope, intercept and correlation coefficient values. The
data in Table 9 show that 1 to 3 days may elapse prior to
color development without affecting the absorbance significantly.
8. Absorbance Values After Reaction of 24 OP Compounds With NBP
at 150° for 3 Minutes. All OP pesticides can, at least in
theory, alkylate the pyridine nitrogen of NBP, which upon
adding an alkaline solution assumes a blue, reddish purple, or
i
magenta color depending on the base, and can thus be measured
spectrophotometrically. It was considered useful to determine
under our test conditions the linearity of response and
comparative reactivities judged by the slope of the standard
curves of various OP pesticides including the oxons.
For the foregoing purposes, two sets of standard curves
were made for each of 24 OP compounds tested, comprising
-68-
-------
TABLE 9. EFFECT OF ELAPSED TIME BETWEEN 4- (g-NITROBENZYL)PYRIDINE
REACTION AND COLOR DEVELOPMENT WITH BASE ON ABSORBANCE
OF THE SOLUTION AT 560 nm
Elapsed
time
(h) 0
1 0.06
24 0.06
48 0.06
72 0.05
Absorbance at 560 ran—
Parathion (yg)
2468 Slope
0.06 0.12 0.18 0.24 0.030
0.06 0.10 0.16 0.21 0.026
0.06 0.11 0.15 . 0.23 0.028
0.05 0.11 0.14 0.21 0.026
Intercept
0
0.01
0
0
Corr .
coeff .
1.000
0.997
0.989
0.990
a/
are corrected for the "blank" reading.
-69-
-------
duplicate determinations at 7 OP amounts in the range 1-25 ng,
conducted at two reaction temperatures, namely, 150° for 3 min
and 100° for 30 min,respectively.
Tests were also made to check for possible interference
from presence in OP residue extracts of two widely used non-OP
insecticides, namely, (a) carbaryl, a major carbamate insecti-
cide, and (b) dicofol, a DDT analogue. Carbaryl and dicofol
were tested at levels ranging from 10-250 ng, and no response
above the reagent blank values were obtained. A concentration
of 250 (ag would be extremely high and not likely to be
encountered in a normal residue.
Table 10 shows absorbance values obtained after reaction
with NBP at 150° for 3 min of 24 OP compounds which may be
present in environmental samples, each compound tested in the
range 1-25 ng. The 24 thions and oxons listed in Table 10
include 0_-ethyl and p_-methyl phosphate esters, and thio- and
dithiophosphates. EPN and trichlorfon are phosphonates.
Demeton and carbophenothion have sulfide sidechains. In the
latter regard, Getz and Watts (7) reported similar reactivities
for carbophenothion, its sulfoxide, and its sulfone under their
reaction conditions.
Diazinon was the least responsive (lowest mean absorbance
unit/(jg ratio) to the RFM among the 24 compounds shown. This
was due to the formation of a reddish color instead of the
normal magenta shade. A red color formed by diazinon was also
found by Watts (34) under their conditions.
-70-
-------
TABLE 10. ABSORBANCE AT 560 run OF SOLUTIONS AFTER REACTION OF
COMPOUNDS WITH 4-(g-NITROBENZYL) PYRIDINE AT 150°C
FOR 3 MINUTES^/-
Micrograms of compound
Compound
azinphosmethyl
azinphosmethyl oxon
carbophenothion
chlorpyrifos
chlorpyrifos oxon
demeton
diazinon
dimethoate
dimethoate oxon
dioxathion
EPN
ethion
malathion
malathion oxon
methidathion
methidathion oxon
mevinphos
naled
parathion
parathion oxon
phenthoate
phenthoate oxon
phosphamidon
trichlorfon
0^
0.05
0.04
0.03
0.03
0.05
0.04
0.05
0.04
0.04
0.04
0.04 '
0.04
0.03
0.05
0.03
0.06
0.04
0.03
0.04
0.04
0.06
0.04
0.04
0.03
1
0.03
0.04
0.02
0.04
0.02
0.02
0.01
0.04
0.03
0.02
0.02
0.01
0.04
0.04
0.07
0.05
0.08
0.03
0.03
0.03
0.03
0.04
0.04
0.03
2
0.06
0,08
0.05
0,08
0.04
0.03
0.03
0.10
0.08
0.06
0.06
0.04
0.07
0.07
0.13
0.11
0.16
0.05
0.06
0.05
0.06
0.06
0.07
0.07
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
5
13
23
09
19
10
05
06
22
19
15
07
09
18
20
31
26
40
11
16
09
16
16
15
14
10
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
30
41
17
40
21
07
11
40
38
32
14
20
37
41
66
51
80
22
34
14
29
30
23
32
15
0.45
0.65
0.26
0.60
0.29
0.12
0.13
0.62
0.53
0.41
0.19
0.28
0.46
0.50
0.93
0.73
1.1
0.29
0.40
0.21
0.45
0.36
0.34
0.45
20
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
0.
1.
0.
0.
0.
0.
0.
0.
0.
65
84
32
83
39
17
17
84
76
51
25
37
76
64
5
99
7
41
57
27
58
46
45
57
25
0.78
1.3
0.38
1.2
0.50
0.21
0.23
1.2
0.89
0.71
0.30
0.52
0.93
0.75
1.9
1.6
1.8
0.51
0.66
0.36
0.77
0.61
0.64
0.78
a/
— Absorbance values are means of duplicate sample determinations and
were corrected for background values given in the column
labeled "0".
— This column gives the background values used to correct values obtained
for samples.
-71-
-------
As may be deduced from Table 10, the reactivities of the
24 compounds towards NBP at 150° for 3 min varied considerably.
Except for azinphosmethyl oxon, the oxygen analogs produced
absorbance values of 60 to 80% of that obtained from an
equivalent weight of the parent thion insecticide. This is
contrary to the results of Getz and Watts (7) but their
reaction conditions were slightly different.
With regard to reactivity differences, Table 13 lists
39 OP compounds reacted at 150° for 3 min that include the
24 compounds shown in T?ble 10, from which data a tentative
categorization into groups of high, medium and low reactivity
was made.
9. Absorbance Values After Reaction of 24 OP Compounds With NBP
at 100° for 30 Minutes. Table 11, showing a range of absorb-
ance values of 24 OP compounds after reaction with NBP at
100° for 30 min, is a counterpart to Table 10, since the
second option for reaction was used on the same compounds.
Table 11 also shows variable response (absorbance values)
but the reactivity levels compared to those of Table 10 are
less, as may be expected due to the lower reaction temperature,
albeit longer reaction time, used. The standard curves
obtained from the data in both Tables 10 and 11 show good
linearity of response, but the slopes are significantly higher
for the higher reaction temperature. The data of Table 11 are
nevertheless useful, as is discussed next.
10. Linear Regression Analysis Values for the Data of Tables 10 and 11
Table 12 gives the slope, intercept and correlation coefficient
-72-
-------
TABLE 11. ABSORBANCE OF 560 nm OF SOLUTIONS AFTER REACTION OF
COMPOUNDS WITH 4-(£-NlTROBENZYL^PYRIDINE AT 100°C
FOR 30 MIN§7
Micrograms of
Compound
azinphosmethyl
azinphosniethyl oxon
carbophenothion
chlorpyrifos
chlorpyrifos oxon
demeton
diazinon
dimethoate
dimethoate oxon
dioxathion
EPN
ethion
malathion
malathion oxon
methidathion
methidathion oxon
mevinphos
naled
parathion
parathion oxon
phenthoate
phenthoate oxon
phosphamidon
trichlorfon
oS/
0.05
0.04
0.04
0.05
0.06
0.04
0.06
0.06
0.05
0.05
0.03
0.04
0.04
0.06
0.04
0.05
0.05
0.04
0.05
0.05
0.05
0.05
0.05
0.03
1
0.03
0.06
0.03
0.04
0.03
0.04
0.02
0.05
0.05
0.04
0.05
0.03
0.06
0.05
0.09
0.07
0.09
0.03
0.04
0.03
0.05
0.03
0.04
0.04
2
0.08
0.12
0.05
0.08
0.05
0.05
0.03
0.10
0.10
0.07
0.06
0.06
0.09
0.11
0.18
0.13
0.20
0.06
0.08
0.06
0.09
0.08
0.09
0.08
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
o.
0.
0.
0.
0.
5
18
29
14
19
13
08
06
26
24
16
13
14
21
25
38
35
47
16
20
13
19.
20
23
20
compound
10
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
36
58
26
38
25
20
13
55
45
33
24
31
39
47
80
65
91
32
35
28
35
37
40
39
15
0.54
0.77
0.32
0.57
0.34
0.25
0.15
0.80
0.66
0.46
0.33
0.39
0.58
0.54
-
1.0
1.6
0.37
0.56
0.39
0.58
0.49
0.71
0.54
20
0.83
1.3
0.42
0.73
0.43
0.34
0.20
0.96
0.83
0.54
0.44
0.53
0.78
0.66
-
1.7
1.8
0.59
0.70
0.51
0.80
0.62
0.90
0.84
25
0
1
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
.99
.6
.54
.95
.58
.35
.27
-
.2
.62
.51
.71
.1
.84
-
.8
-
.67
.91
.63
.91
0.77
0
-
.92
a/
— Absorbance values are means of duplicate sample determinations and
were corrected for background values given in the column
labeled "0".
— This column gives the background values used to correct values
obtained for samples.
-73-
-------
TABLE 12. LINEAR REGRESSION ANALYSIS VALUES FOR THE DATA GIVEN
IN TABLES 10 AND 11
150° for 3 min
azinphosmethyl
azinphosmethyl oxon
carbophenothion
chlorpyrifos
chlorpyrifos oxon
demeton
diazinon
dimethoate
dimethoate oxon
dioxathion
EPN
ethion
malathion
malathion oxon
methidathion
methidathion oxon
mevinphos
naled
paiathion
parathion oxon
phenthoate
phenthoate oxon
phosphamidon
trichlorfon
Slope A
0.040
0.064
0.021
0.037
0.022
0.014
0.010
0.050
0.045
0.025
0.020
0.027
0.042
0.031
0.078
0.076
0.095
0.027
0.036
0.025
0.037
0.030
0.046
0.038
Intercept
-0.02
-0.04
0.02
0
0.01
0.03
0.01
0.02
0
0.04
0.03
0.01
0
0.07
•0.01
-0.04
0.01
0.01
0.01
0.01
0.01
0.03
-0.01
0.01
r
0.997
0.993
0.996
1.000
0.998
0.985
0.993
0.996
0.995
0.990
0.998
0.997
0.995
0.988
0.999
0.989
0.992
0.992
0.999
0.999
0.997
0.996
0.997
0.995
100°
Slope B
0.032
0.049
0.015
0.046
0.020
0.0079
0.0085
0.046
0.036
0.027
0.011
0.020
0.037
0.030
0.076
0.059
0.076
0.020
0.026
0.013
0.030
0.023
0.023
0.030
for 30 min
Intercept
-0.01
-0.04
0.02
-0.04
0
0.01
0.01
-0.02
0.01
0.01
0.02
-0.01
-0.01
0.04
-0.06
-0.04
0.02
0.01
0.02
0.02
0
0.03
0.01
0
r
0.999
0.988
0.997
0.993
0.999
0.993
0.992
0.994
0.999
0.995
0.996
0.996
0.994
0.991
0.994
0.982
0.993
0.999
0.993
0.997
0.999
0.994
0.993
0.998
Slope 1
Slope I
0.80
0.76
0.71
1.2'
0.91
0.56
0.85
0.92
0.80
1.1
0.55
0.74
0.88
0.93
0.97
0.78
0.80
0.74
0.72
0.52
0.81
0.77
0.50
0.79
-74-
-------
(r) values obtained after linear regression analysis of the
data of (A) Table 10, based on reaction at 150° for 30 min
and (B_) Table 11, based on reaction at 100° for 30 min,
respectively. The high r values, ranging from 0.988 to 1.000
in Group "A", and from 0.982 to 0.999 in Group "B", indicate
a good correlation of absorbance with OP amounts.
Slope "A" values, equivalent to mean absorbance unit/pig
ratios after reaction at 150° for 3 min, range from a high of
0.095 (mevinphos) to a low of. 0.010 (diazinon) , which calculates
to a factor in relative reactivity of 9.5 between the most
reactive and least reactive compound in the 24-compound series.
By the same token, slope "B" values, equivalent to mean
absorbance unit/|jg ratios after reaction at 100° for 30 min,
range from a high of -0.076 (mevinphos) to a low of 0.0079
(demeton) , indicating a reactivity factor of these extremes
of 9.6.
A new set of 24 values is also shown in Table 12, namely,
the (Slope "B")/( Slope "A") ratio, which can be designated for
purposes of discussion as the B/A ratio, equivalent to the new
range of values, ranging from a high of 1.2 (chlorpyrifos) to
a low of 0.50 (phosphamidon) .
The importance of the B/A ratio is not that additional
differences between reactivities of 24 OP compounds are shown,
but that the B/A ratios are sufficiently different between
compounds to be used as a new parameter or constant to assist
identification of an unknown OP compound in a residue of
-75-
-------
unknown history, or at least to narrow the field of investi-
gation of possible OP candidates. Practical use of the B/A
ratio assumes that only one OP compound is present regarding
which there could normally be no guarantee. Other problems
are that two or more OP compounds could have the same, or
nearly the same, B/A ratio, and there are many more commercially
available OP compounds than the 24 listed in Tables 10-12.
11. Mean Absorbance Unit/pg Ratios of 39 OP Compounds After NBP
Reaction at 150°C for 3 Minutes. Table 13 shows the mean .
absorbance unit/ug ratios of 39 OP compounds after reaction
at 150° for 3 min. Of 43 compounds that were tested (Table 3),
4 compounds reacted slightly or not at all. Only 24 of the 39
compounds- shown in Table 13 were reacted at both 150 and 100°C
(Tables 10-12) .
The main purpose of Table 13 is to catalogue a limited
extension of OP reactivities on the basis of mean absorbance
unit/ug ratios after reaction at 150° for 3 min, and from the
ratios to group the 39 compounds into 3 tentative categories,
namely: High (above 0.045), Medium (0.020-0.045), and Low
(less than 0.020). Within these tentative groups, the compound
numbers assigned in Table 13 are used to conserve space:
A.- High reactivity B. Medium reactivity C. Low Reactivity
1,3,11,18,23-27,33,34, 2,4-7,12-15,21,22, 8-10,16,17,19,20
36,38 28-32,35,37,39
13 compounds 19 compounds 7 compounds
The category boundaries in this preliminary group are
quite arbitrary and amenable to change. The intent is to
-76-
-------
TABLE 13. MEAN ABSORBANCE UNIT/ng RATIOS OF 39 OP COMPOUNDS
AFTER NBP REACTION AT 150° FOR 3 MINUTES^/
Cpd.
no.
1
2
3
4
.5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Common name or Mean A/pig
chemical name ratio
acephate
azinphosmethyl
azinphosmethyl oxon
carbophenothion
chlorpyrifos
chlorpyrifos oxon
dichlorvos
demeton (demeton-O +
demeton-S)
diazinon
0_, 0_-dimethyl
phosphorodithioic acid
dimethoate
dimethoate oxon
dioxathion
EPN
ethion
0-ethyl jO-nitrophenyl
cyclohexyl phosphonate
0-ethyl jD-nitrophenyl
phenyl phosphonate
fenthion
isopropyl parathion
isopropyl paraoxon
0.047
0.040
0.064
0.021
0.037
0.022
0.037
0.014
0.010
0.018
Cpd
no
21
22
23
24
25
26
27
28
29
30
31
0.050 32
0.045
0.025
0.020
0.027
0.006
0.011
0.066
0.016
0.009
33
34
35
36
37
38
39
Common name or Mean A/jag
chemical name ratio
malathion
malathion oxon
methidathion
methidathion oxon
methyl parathion
mevinphos
monocrotophos
naled
parathion
parathion oxon
phenthoate
phenthoate oxon
phosphamidon
O,O,g,O-tetramethyl
pyrophosphorodithioate
trichlorfon
0, S,J3-tr imethy 1
phosphorodithioate
0 » 0 ' JL-tr imethy 1
phosphorothioate
0 ,0 , £-tr imethy 1
phosphorodithioate
0,0,0-tr imethy 1
phosphorothioate
0.042
0.031
0.078
0.076
0.051
0.095
0.064
0.027
0.036
0.025
0.037
0.030
0.046
0.057
0.038
0.046
0.043
0.055
0.023
a/ Of 43 OP compounds tested (see Table 3), 4 compounds responded
slightly or not at all after reaction at 150°C for 3 min. The
compounds were:
a.
b.
c.
d.
DBF - no reaction
0_,O-diethyl S_-methyl phosphorothioate - reaction not linear;
variable A/ng response
Glyphosate - no reaction
Isomalathion - weak reactivity (0.0.02) at 150°C for 3 min,
but rose to 0.016 after reacting at 150°C for 8 min
-77-
-------
provide a clearer view of the impact of results obtained by the
RFM when neither the nature of the spray application treatment
or the postapplication history ij known.
The categories reflect the finding that OP compounds react
with different intensities under RFM test conditions. For
example, a residue of 10 ng OP compound/40-disk sample of
2
foliage (=0.05 ug/cm leaf surface) could, if placed in the
High Reactivity group, yield absorbance values that are from
3 to 6 times those if the 10 |ug residue was in the Low
Reactivity group. By the same token, a small absorbance
unit/tag value, which is normal for a compound in the Low
Reactivity group, might nevertheless stem from a compound of
high toxicity. This underlines the need to know the nature
of the compounds being sought and the postapplication history
of the samples. These points have been made in previous
sections.
12. Absorption Maxima of the Products Resulting from NBP Reaction
with OP Compounds. The optimum wavelength, extinction co-
efficient (absorptivity) and the half-life of the colored species
formed when a base solution is added to the OP-NBP alkylation
products depend considerably on the nature of the alkaline
solution that is added. The RFM enables measurement of amounts
as small as 1 ng of OP compound present in residues, (0.5 ng if
the compound has high reactivity with NBP, such as methidathion,
mevinphos, or monocrotophos, and 1.5 ug if the compound is in
the low reactivity group (Table 13, discussion). After tri-
ethylamine and sodium carbonate solution are added, absorbance
-78-
-------
readings should be made immediately due to gradual fading of
the developed color.
The absorption maximum (560 nm) setting at which the
reacted residues were measured after color development in an
alkaline medium was checked with 12 different OP compounds,
with results shown in Table 14.
Table 14 gives absorbance data between 535 and 580 nm
for solutions prepared from OP compounds that included 0_-
methyl and 0-ethyl esters, phosphorothioates, phosphorodi-
thioates, phosphates and a phosphonate.
Table 14 shows that the maximum absorbance values
occurred in the region 555-560 nm, with the exception of
azinphosmethyl oxon and dimethoate which showed maxima at
555 nm. The 560 nm wavelength was selected for general use
for all OP compounds.
In developing the color, the organic base solution
(triethylamine in acetone) is added first, followed by the
aqueous inorganic base solution (sodium carbonate in salt
solution). When the order of addition was reversed, the
absorbance unit/|ag ratios obtained with parathion used as a
reference standard were 11 to 25% lower than the values
obtained when the standard order of addition was used.
13. Decrease in Absorbance with Time. The magenta color developed
upon adding the two base solutions is not stable, and accordingly
the absorbance values after OP-NBP reaction and color development
should be measured immediately after addition of the base
solutions. Table 15 gives data obtained with parathion that
illustrate the decrease in absorbance with time.
-79-
-------
TABLE 14. ABSORPTION SPECTRA OF THE PRODUCTS FROM THE REACTION OF
4-(£-NITROBENZYL)PYRIDINE WITH ORGANOPHOSPHORUS COMPOUNDS-/
Wavelength (run)
Compound—
azinphosmethyl
azinphosmethyl oxon
carbophenothion
dimethoate
ethion
malathion
malathion oxon
methidathion
' methidathion oxon
parathion
parathion oxon
trichlorfon
535
0
0
0
0
0
0
0
0
0
0
0
0
.60
.84
.45
.80
.56
.59
.49
.69
.70
.53
.40
.67
540
0.62
0.87
0.47
0.82
0.58
0.61
0.50
0.70
0.72
0.55-
0.41
0.69
545
0.63
0.89
0.48
0.84
0.59
0.62
0.51
0.72
0.73
0.56
0.42
0.71
550
0.64
0.90
0.49
0.85
0.60
0.64
0.52
0.73
0.74
0.57
0.43
0.73
555
0.65
0.91
0.49
0.86
0.60
0.64
0.53
0.74
0.75
0.58
0.43
0.73
560
0.65
0.90
0.49
0.85
0.60
0.64
0.53
0.74
0.76
0.58
0.43
0.73
565
0.64
0.89
0.49
0.84
0.59
0.63
0.52
0.74
0.75
0.57
0.42
0.72
570
0.62
0.87
0.48
0.81
0.58
0.61
0.51
0.71
0.73
0.55
0.41
0.70
575
0.59
0.83
0.46
0.77
0.56
0.58
0.49
0.69
0.70
0.53
0.40
0.67
580
0.56
0.78
0.43
0.73
0.53
0.56
0.46
0.65
0.66
0.50
0.38
0.64
a/
— Amount of compound used was 20 yg except for methidathion and its oxon where 10 pg
each was used. Corrections were made for minor absorbance of the reagent blank
solution.
-80-
-------
TABLE 15. DECREASE IN ABSORBANCE (560 nm) WITH TIME-/
Parathion
(yg)
2
4
6
8
10
2
4
6
8
10
0.5
0.05
0.11
0.17
0.20
0.26
0.06
0.12
0.18
0.24
0.29
Time
1.0
0.05
0.09
0.14
0.19
0.25
0.05
0.11
0.17
0.22
0.28
elapsed (h)
1.5
0.05
0.10
0.16
0.21
0.25
0.05
0.09
0.13
0.20
0.25
2.0
0.03
0.07
0.11
0.14
0.17
0.03
0.06
0.10
0.16
0.21
2.5
0.01
0.06
0.10
0.12
0.14
0.02
0.04
0.07
0.12
0.18
3.0
0.02
0.05
6.10
0.11
0.15
0.02
0.05
0.08
0.11
0.17
Slope
-
0.32
0.23
0.27
0.28
-
0.45
0.40
0.34
0.24
Intercept
0.
0.
0.
0.
—
0.
0.
0.
0.
-
13
19
25
32
16
23
30
34
Corr.
coef f .
- •
0.954
0.907
0.912
0.918
_
0.933
0.953
0.980
0.984
\
-
2.2
3.0
2.6
2.5
_
1.5
1.7
2.0
2.9
a/
— Each value is a mean of duplicate samples. Values corrected for background.
-81-
-------
After addition of base to the parathion-NBP reaction
product, absorbance readings were taken after 0.5, 1.0, 1.5,
2.0, 2.5 and 3.0 h. Each value is a mean of duplicate samples
and was corrected for the absorbance value of the reagent
blanks. Each measurement represents a different reaction tube.
Table 15 also shows values of the slopes [logarithm (In) of
concentration ((jig) vs time (h)], intercepts, correlation co-
efficients, and half-lives (t, ,^) .
The decrease in absorbance appears to be a first-order
process with a half-life of 1.5 to 3 h 'mean 2.2 h). Turner
(33) stated that the stability of the color varies with the
pesticide used. Half-life values calculated from his data
obtained after addition of tetraethylenepentamine in acetone
to NBP adducts with m'alathion, dichlorvos, tetrachlorvinphos
and fenchlorphos were 1.7, 1.2, 2.0 and 1.5 h, respectively.
He recommended that exposure of the developed colors to bright
sunlight be avoided, as was confirmed independently in the
present investigation.
14. Recovery of OP Compounds After Fortification of 20% Sodium
Chloride Solutions. In processing samples of foliage, each
consisting of 40 leaf disks (2.54 cm diameter), each sample
is shaken with 20 mL aqueous NaCl solution to remove the
surface residues. A 15-mL aliquot of the leaf wash is then
decanted into a PP tube and the OP residues present in the
leaf wash are partitioned into 15 mL hexane. After shaking
20 sec and partitioning, a 10-mL aliquot of the hexane extract
is removed and analysed for OP residues by the RFM.
-82-
-------
To determine the efficiency of recovery from 20% sodium
chloride solution as a prior step before repeating the pro-
cedure using actual leaf-wash solutions, 14 OP compounds were
added in the amounts shown in Table 16. The mean recoveries
and standard deviations are shown. Standard curves were made
and the linear absorbance unit/|jg ratios were calculated and
designated as "recoveries in the absence of leaves."
The recoveries were satisfactory except for azinphosmethyl
oxon, dimethoate and phosphamidon, which showed low recoveries
because they are strongly water-soluble. On this point, as in
other instances of suboptimal recoveries, low but consistent
recoveries can be "normalized" by applying an appropriate
multiplication factor to the analytical results.
15. Recovery of OP Compounds After Fortification of Aqueous Leaf
Washes. Fourteen groups designated for 14 OP compounds, each
group consisting of 14 jars, each jar containing 40 leaf disks
of citrus foliage were given the standard dislodgment treatment
with 20% Nad as described. After 15 mL of each leaf-wash
suspension were decanted into PP tubes, OP standards in hexane
were added in duplicate to the PP tubes to provide levels of 0
(reagent blank), 1.5, 3, 7.5, 15, 22.5 and 30 pig. For some
compounds the latter two levels were substituted by 24 and 36 jjg '
of the OP standard. Additional hexane was added, where necessary,
to yield a total volume of 15 mL-hexane solution in each tube.
The capped tubes were then shaken vigorously for 20 sec, and
10-mL aliquots of each tube were transferred to reaction tubes
for evaporation and reaction. After reaction at 150°C for 3 min,
-83-
-------
TABLE 16. RECOVERY OF COMPOUNDS AFTER FORTIFICATION OF 207, NaCl SOLUTIONS
Recovery (%)
Fortification (yg)
Compound
azinphosmethyl
azinphosmethyl oxon
carbophenothion
chlorpyrifos
dimethoate
dioxathion
EPN
ethion
malathion
malathion oxon
methidathion
parathion
parathion oxon
phosphamidon
1.5
130
-
100
100
25
100
67
67
100
130
100
67
-
50
3
150
36
.100
130
29
100
60
83
71
120
92
67
130
57
7.5
110
33
110
95
21
110
69
94
82
100
93
73
91
56
15
110
26
95
100
11
85
64
94
91
90
93
77
86
47
22.5
100
25
100
110
11
-
-
-
86
-
97
84
91
~"
24 30 36
110
24
110
100
11
90 - 95
70 - 75
84 - 95
85
91 - 90
110
77
93
45 - 44
Mean
120
29
100
110
18
97
68.
86
86
100
98
74
98
50
Std.
dev.
18
5
6
13
8
9
5
11
10
17
7
7
18
6
-84-
-------
the color intensities were measured for each group of 14 tubes
and the % recoveries were determined from the corrected
absorbance values in relation to standard curve points.
Table 17 shows the % recoveries of each concentration
added, the mean % recovery for each OP compound used in the
fortification, and the standard deviations. Also shown are
the slope values of standard curves made at the same time
from additions to reaction tubes in duplicate of 0, 1, 2, 5,
10, 15 and 20 ng of OP standard, and the correlation coefficient
(r) values of the standard additions.
The mean % recoveries of the 14 OP compounds in the
presence of leaf-wash extractions (Table 17) compare reasonably
well with the mean % recoveries of the same compounds in the
absence of leaf wash extractions (Table 16), except for azin-
phosmethyl oxon, dimethoate, and parathion oxon (recoveries of
20, 32 and 80% in the presence vs 29, 18 and 98 in the absence
of leaf extracts). Phosphamidon showed similar recovery
efficiencies (50 vs 49%) in both tubes. It was apparent that
the water solubility factor reduced the efficiency of recovery
of the latter 4 compounds, exerting a greater diminution on
some than on others.
16. Comparative Recovery of Parathion From Fortified Dry vs Fresh,
Moist Soil. Before embarking on discussion of results obtained
on comparative recoveries of parathion residues from fortified
dry vs fresh, moist soil **• 100 mesh, a brief review of methods
of sample preparation and some comments on the orientation and
development of improvements that were made later to achieve higher
recoveries of oxons would be useful at this point.
-85-
-------
TABLE 17. RECOVERY OF INSECTICIDES AFTER FORTIFICATION OF AQUEOUS LEAF WASHES
Recovery ('•£)
Fortification fp£)
Insecticide
azinphosmethyl
azinphosmethyl oxon
carbophenothion
chlorpyrifos
dimethoate
dioxathion
EPN
ethion
malathion
malathion oxon
methidathion
parathion
parathion oxon
phosphamidon
1.5
130
-
100
100
75
67
-
99
67
100
83
67
-
100
3
120
18
100
100
57
120
60
100
86
120
92
83
75
71
7.5
110
19
90
110
21
120
54
88
82
100
97
87
64
33
15
110
19
110
100
14
85
68
100
110
97
85
83
82
28
22.5 24
98
21
110 -
110
13. -
- 93
- 73
- 92
86
- 89
93
82
94
- 33
30 36
100
22
110
100
11
97
77
77
98
- 86
110
77
86
30
Mean
110
20
100
100
32
97
66
93
88
99
93
80
80
49
Std.
dev.
12
2
8
5
27
21
9
9
15
12
10
7
11
30
Standard curve
Slope
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
031
053
020
038
037
026
025
031
033
029
060
030
022
036
Corr.
coeff .
0.996
0.999
0.997
0.996
0.994
0.991
0.993
0.998
0.998
0.997
0.998
0.999
0.997
0.999
-86-
-------
ft. I'rr-.l iniin^ry Corfmontr;^ Exploratory tests on sieved soil
du/it fortjfiod with parathion in the range 1-400 ppm showed
that higher recoveries were obtained when a 12:20 v/v ratio
of water (20% Nad) : hexane was used in the PP tubes instead
of the 15:15 water: hexane ratio used for foliage residues.
In a systematic series of trials and evolution of methods
aimed at improving the low recovery of oxons and of dimethoate
from soils, mixtures of acetone: hexane of 1:19, 2:18 and
3:17 v/v were found to increase recoveries of oxons signifi-
cantly, particularly when the water: solvent (mixed solvent)
ratio was concomitantly altered to 4:20, 2:20, 1:20 and
ultimately to 0.25:20 v/v. The latter ratio is our current
recommendation for soil residue analysis by the RFM, and was
tested on 3 thions and 3 oxons. The tests with dry vs moist
soils fortified with parathion were conducted at an earlier
stage of development of the soil section of this project,
when the ratio in the PP tubes was 12:20 v/v (12 mL 20% salt
solution + 20 mL hexane).
b. Summary of Method. This aspect of methods development was
conducted in the laboratory. A parallel series of dry sieved
surface soil and later fresh, moist sieved surface soil were
weighed out into PP tubes in duplicate in amounts of 1, 2 and
4 g. Parathion standards in hexane were added to provide a
series of concentrations for the range 1-400 ppm. The tubes
were swirled gently to obtain uniform suspensions and the
series were allowed to evaporate to dryness overnight at room
temperature. Twelve mL 20% NaCl and 20 mL hexane were added
-87-
-------
to the air-dried soils, which were then shaken vigorously in
the capped tubes for 20 sec. The tubes were centrifuged for
30 sec at about 1200 rpm in a clinical centrifuge to break the
emulsions. Appropriate aliquots of the supernatant hexane
were transferred to reaction tubes, followed by evaporation
and reaction in heated aluminum blocks, and color development,
as described. The absorbance values at 560 run of the various
aliquot sizes (mL subsample) were multiplied by the corres-
ponding factors, the absorbance value of the soil blanks (no
parathion added) were subtracted therefrom, and the absorbance
unit/(ag ratios and % recovery of parathion were determined.
c. Objective. The main objective was to ascertain whether
recovery tests on fortified dry, 1-year old surface soil
obtained from an unsprayed area gave results different from
those of a parallel series of tests executed on fresh, moist
surface soil sieved to < 100 mesh particle size. Confirmation
was sought for a preliminary test showing that the moisture
level of orchard soil did not affect the % recovery of parathion.
Table 18 shows the % recoveries obtained from dry vs moist
surface soil freshly sampled, both taken from previously un-
sprayed areas, and fortified with parathion standards at 10
concentrations in the range 1-400 ppm. The mean recoveries
obtained from dry vs moist soil were 94 and 93%, respectively.
As may be seen in Table 18, dry soil showed significant
deviations from the mean recovery of the 1, 20 and 400 ng
levels, and moist soil at the 50 |ag level. It was concluded
that the recoveries were satisfactory, and that either dry
-88-
-------
TABLE 18. COMPARATION % RECOVERY OF PARATHION FROM FORTIFIED
DRY VS FRESH, MOIST SOIL (-=100 MESH)
Soil
wt. , g
4
4
4
2
2
4
2
2
1
1
Parathion added
^g
4
8
20
20
40
100
100
200
200
400
Mean %
ppm
1
2
5
10
20
25
50
100
200
400
Recovery
Dry surface soil
% recovery
100
90
88
91
110
83
84
93
94
104
94
Fresh, moist surface soil
% recovery
92
86
87
94
90
93
102
92
92
96
S>3_
-89-
-------
soil or freshly obtained moist surface soil would give
essentially the same % recovery of OP residues, assuming that
the same amounts of residue were present.
To broaden the scope somewhat, the same general procedure
above-mentioned applied to dry soil fortified in the range
10-320 ppm with malathion, paraoxon and azinphosmethyl oxon
yielded mean % recoveries of 90, 54 and 28%, respectively.
Other tests in the range 10-400 ppm with dimethoate applied
to dry soil with the above-mentioned procedure yielded
recoveries from 10-16%, whereas with phenthoate in the same
range the mean recovery was 90%.
The variable results on soil correlated well with our
more extensive experience pertaining to recovery tests on
foliage, indicating in both instances that the nature of the
OP compound could influence the efficiency of its recovery.
It was concluded that the sample preparation stage for
surface soil should be modified to raise the recovery levels
of oxons, dimethoate and other relatively water-soluble OP
compounds. To implement the latter objective tests involving
systematic alteration of the water: solvent ratio were made,
as are described next.
17. Modifications in Processing Soil Samples to Improve % Recovery
of OP Compounds. In the course of exploratory tests with soil
fortified with parathion, it was found that the recovery would
be increased when the ratio 15/15 of 20% Nad solution hexane,
used in processing dislodged OP residues of foliage was altered
to 15/20 and later to 12/20. The latter ratio was subsequently
-90-
-------
changed to 9/20 and 6/20 to achieve increased % recovery, at
which time soil fortified with paraoxon was used for contrast.
The change to an oxon corroborated the general finding that
the % increase in recovery depended on the nature of the OP
.•i
compound used.
Other experimental trials with fortified soil showed .
that acetone mixed with hexane in 5:95, 10:90 and particularly
15:85 admixture yielded improved recoveries, but a 20:80
mixture yielded excessively high soil blank values, as did
100% acetone. The 15:85 v/v acetone: hexane mixture was
adopted for a more extensive series of tests on comparative
recovery from soil fortified with 6 OP compounds indicated in
Table 19, comprising 3 thions (parathion, azinphosmethyl and
malathion) and 3 oxons (paraoxon, azinphosmethyl oxon and
chlorpyrifos oxon). Table 19 shows the effects on mean %
recovery from soil fortified with the 6 OP compounds in the
range 10-350 ppm of systematic change in the water (20% Nad)/
solvent ratio from 12/20 to 0/20. It should be noted that
100% hexane was used as extracting solvent in the 12/20, 9/20
and 6/20 ratios, but 15:85 acetone: hexane was used thereafter.
The comparative series indicated in Table 19 showed that
the highest overall recoveries (92-109%) were obtained at a
0/20 water/mixed solvent ratio. However, use of the 0/20
proportions yielded a yellow-colored extract and a fine
suspension of soil dust that required about 1 h standing to
clear, or alternatively use of a centrifuge. When 0.25 mL
of salt solution was added, both the soil suspension and the
-91-
-------
TABLE 19. EFFECT OF CHANGES IN THE WATERrORGANIC SOLVENT RATIO ON % RECOVERY
OF 3 THIONS AND 3 OXONS FROM FORTIFIED SOIL
Mean % recovery
I
VD
to
1
Water/
Extracting solvent
solvent ratio
100% 12/20
Hexane 9/20
6/20
4/20
3/20
Acetone/ 2/20
hexane 1/20
15:85 0.5/20
v/v 0.25/20
0/20
Parathion
91
90
93
92
94
96
99
98
99
99
Azinphos-
methyl Malathion
65
69
72
81
83
88
93
96
97
99
80
84
88
92
91
95
96
96
98
102
Azinphos-
Parathion methyl
oxon oxon
58
61
66
76
80
85
93
97
101
109
18
23
25
37
29
70
78
86
93
96
Chlorpyrifos
oxon
24
28
30
40
37
45
51
73
87
92
-------
yellow color disappeared. We accordingly adopted a 0.25/20
ratio of 20% salt solution/mixed solvent at the expense of a
slightly lower mean recovery (range 87-101%) but with a gain
of less downtime and lower soil blank values than if a 0/20
ratio had been used. Emulsion formation was completely absent
at ratios smaller than 4/20.
18. Foliar Residues of Phenthoate by the RFM and by GC. For a
preliminary' exercise in comparing the results obtained by the
RFM and the GC methods, respectively, phenthoate was applied
to orange trees as dilute (oscillating boom) and LV sprays at
commercially used dosages, both spray treatments having the
identical a.i. content. The objective was to ascertain the
dislodgable foliar residues over a 31-day postapplication
period by the RFM and GC procedures previously outlined in
Section 5 herein.
Duplicate sets of leaf samples were taken for each
analytical method at 9 time intervals, including the pre-
treatment samples for foliage blanks that were taken one day
prior to spraying. All the RFM samples were processed in the
field. Seven samplings of soil were also taken (one for
pretreatment levels), with results described in the next
subsection.
Table 20 shows the comparative results, expressed as
2
micrograms of phenthoate/cm leaf surface, obtained by use
of the RFM and GC, respectively, in processing the duplicate
sets of samples taken.
-93-
-------
TABLE 20. FOLIAR RESIDUES AFTER APPLICATION OF P1IENTHOATE TO ORANGE TREES.
DETERMINED BY TILE RFM AND BY GC
Spray
treatment
none
none
dil.
LV
dil.
LV
dil.
LV
dil.
LV
dil .
LV
dil.
LV
dil.
LV
dil.
LV
Days — — —
0
Phenthoate residues, jag/era
elapsed- RFM
-1 0.02
0.01
3 0.19
0.27
6 0.12
0.18
10 0.06
0.12
13 0.08
0.09
17 0.06
0.08
20 0.04
0.09
24 0.03
0.04
31 0.01
0.02
0.02
0.01
0.14
0.29
0.09
0.22
0.05
0.16
0.04
0.15
0:08
0.10
0.07
0.06
0.03
0.05
0.02
0.01
Mean
0.02
0.01
0.17
0.28
0.11
0.20
0.06
0.14
0.06
0.12
0.07
0.09
On £.
. WU
0.08
0.03
0.05
0.02
0.02
GC
0.01
0.01
0.20
0.37
0.16
0.54
0.11
0.11
0.09
0.06
0.04
0.06
n no
U . \J £.
0.03
0.01
0.02
0.006
0.005
0.01
0.01
0.23
0.31
0.10
0.31
0.05
0.09
0.04
0.01
0.08
0.05
0.06
0.04
0.02
0.01
0.008
0.008
Mean
0.01
0.01
0.22
0.34
0.13
0.43
0.08
0. 10
0.07
0.04
0.06
0.06
r, r\ t
\J . U--f
0.04
0.02
0.02
0.007
0.007
leaf blank values (|jg/cm leaf surface) ,obtained from the pretreatment
samples of foliage taken one day before spray application, were deducted from
the residue levels determined by the RFM and by GC, respectively. The
considerable differences between the RFM blanks and the GC blanks are
discussed in text.
-94-
-------
The pretreatrnent samples processed by the RFM showed mean
values of 0.015 and 0.014 ug OP residue expressed as phenthoate,
t
which were deducted from those obtained for the postapplication
2
samples. Less than 0.01 ng/cm phenthoate were obtained by
the GC method on the pretreatment samples.
Table 20 shows a descending concentration gradient, as is
expected, in phenthoate residues over a 31-day postapplication
period by both analytical methods for the dilute and LV spray
applications, with the exception of the LV treatment after 6
days had elapsed, which instead showed a substantial increase
due to the comparative nonuniforrnity of spray distribution
when LV sprays are applied.
The phenthoate residues stemming from the LV spray treat-
ment were, with several minor exceptions shown by GC after 17
days postapplication, considerably higher than those obtained
from the dilute treatment. Differences in residue levels
between the two treatments were generally small after 17-20
days, with greater differences being shown by the RFM. Results
for the LV residues at 3 and 6 days after application show
the greatest difference between the two methods. It was sub-
s equently found that other OP pesticides showed this pattern
also, suggesting that the more exhaustive extraction procedure
used in the GC method would recover substantially larger pro-
portions of phenthoate from the leaf-wash suspensions, and
would register higher residues particularly within the first
week of postapplication sampling.
-95-
-------
Since the phenthoate applications and sampling were
intended as preliminary, the foliage sampling program was
terminated at 31 days.
19. Determination of Phenthoate Residues of Surface Soil. The
dilute and LV sprays applied to orange trees for purposes of
intercomparison and testing of analytical methods (see Sub-
section 18) also provided an opportunity to sample surface
soil at the dripline area of the sprayed trees to ascertain
the phenthoate residues over a 24-day postapplication period.
Each designated tree sample represented "sweepable"
surface soil and associated debris on the orchard floor from
2
1 ft of surface, and was a composite of 8 subsamples, each of
2
1/8 ft of soil surface, and taken in the manner previously
described. Each composite sample was sieved to <100 mesh
size. The sieved soil was deposited in 1/2-lb size poly-
ethylene bags, weighed, recorded, and stored at room
temperature for approximately 4 months. The samples were
analysed by the RFM, wherein a 2/20 ratio of 20% salt solution/
mixed solvent (acetone:hexane, 15:85) was used, after a mean
recovery of 89% from soil fortified with phenthoate standards
("normalizing" factor=l.l) was obtained. (In subsequent
tests as part of an intermittent search for methods of
improving recovery of OP compounds from soil, tests with a
0.25/20 ratio yielded a mean recovery of 96% from fortified
soil.)
Table 21 shows the ppm phenthoate in sieved soil dust from
samples at the dripline area of 4 trees representing the dilute
-96-
-------
.TABLE 21. PHENTHOATE RESIDUES (ppm) OF SURFACE SOIL DUST AND pg/ftr SOIL SURFACE, AT THE
DRIPLINE AREA OF SPRAYED ORANGE TREES DETERMINED BY THE RFM5/
Days
after
application
3
6
10
Spray
application
Dll.
LV
Dll.
LV
Dll.
LV
Mean
residue,
ppm
48±9
24±2
46±10
22±4
30±2
17±3
Mean
residue,
g/ft2
(calc.)
610
300
550
270
320
220
Days
after Spray
application application
13 Oil.
LV
17 Oil.
LV
24 Dll.
LV
Mean
residue,
ppm
17±7
7 ±4
16±3
5±1
18±5
7±1
Mean
residue,
g/ft2
(calc.)
330
100
220
75
210
90
a/Each residue value was corrected for the soil blanks (pretreatment samples) taken at the
.respective tree locations before the phenthoate sprays were applied. Each residue (ppm) is
the mean of duplicate 1-gram samples of soil sieved to pass a Tyler 100-mesh U.S. standard
screen. Each tree was sampled at 8 different points (N,NE,E,etc.) at the dripline area,
2
using a template with 1/8 ft of surface exposed for sweeping with a non-magnetic nylon fiber
brush. The grams of each composite sample of sieved dust was determined, and the U g
2 2
phenthoate/ft of surface was calculated as the mean ppm x mean weight/ft sample for each
of the 12 groups above.
-------
treatment and of 4 trees representing the LV spray treatment.
Each value shown is corrected for the soil blank (pretreatment
sample) of the designated tree. Also shown are values for the
2
mean residue expressed in ug/ft soil surface, calculated as
the product of ppm x g of sieved dust obtained in the original
2
1 ft sample from each tree.
In both cases, the phenthoate residues of the soil surface
at the dripline area of trees treated with dilute spray were
substantially higher than those treated with LV sprays. This
reversal in order of magnitude from the findings previously
obtained with the corresponding foliage samples (see Table 20)
is related to the lOx greater volume of diluted spray used with
the greated wetting power and subsequent runoff compared to the
reduced wetting obtained by the more concentrated LV treatment.
Table 21 also shows that the dissipation rate of phenthoate
residues is slower and more protracted on surface soil dust
2
than on citrus foliage, and that the residue levels/ft of soil
2
dust can be considerable, exceeding 600 pig/ft in some instances.
20. Foliar Residues of Malathion, Parathion and Methidathion by GC
and RFM. An outline of the objectives and design was provided
in Section 5, subsection 12. This experiment involved the use
of commercial dosages of malathion, parathion and methidathion
each applied as dilute and LV sprays, respectively, with
resulting foliar residues determined over a 62-day period in
duplicate sets of leaf-punch samples for analysis by the RFM
method in the field and by GC in the laboratory. The oxon and
thion levels were determined separately for each OP compound by
GC.
-98-
-------
Table 22 shows the dislodgable foliar residue values,
2
expressed in terms of yg/cm of leaf surface, over a 62-day
period after application of malathion WP, parathion WP, and
methidathion WP and EC, applied as LV sprays and as dilute or
full-coverage sprays. The column on the GC side wherein the
mean thion and oxon values determined by GC are combined to
give a "total" OP value for a particular OP application can
be used for comparison with the mean total OP residue levels
determined in the field by the RFM, as is expressed in the
last column of Table 22.
The first surprising finding in the case of the RFM was
the relatively high absorbance values of the pretreatment
samples which registered a mean absorbance of 0.17 compared
to previous "normal" levels of 0.08-0.08 in that area. In
contrast, all the pretreatment samples processed by GC
2
registered less than 0.01 yg/cm of malathion, parathion, or
methidathion.
Figures 3 and 4 show the dissipation curves of dislodgable
OP foliar residues determined by the RFM of the 8 treatments
above-mentioned spanning the 62-day postapplication period.
All 8 curves show a dashed "bridge" line between 20 and 28
days. In each instance, the lower half of the curve (28-62
days) show a lateral shift that is more or less parallel to
the upper portion (3-20 days), and is reflected in more time
2
needed to reach 0.002 yg/cm levels.
Figure 4 shows that the lateral shifts for the LV and
dilute applications of the methidathion WP and EC formulations,
-99-
-------
TABLE 22. DISLODGABLE FOLIAR RESIDUES ( Mg/cn/) OF PARATHION, MALATHION AND METHIDATHION AFTER APPLICATION OF DILUTE
AND LV SPRAYS TO ORANGE TREES
Determined by the GC procedure
Application
Parathion
WP, LV
7.2 Ib a.i.
per 100 gal/A
Malathion
I WP, LV
O
o 12 Ib a.i.
per 100 gal/A
Methidathion
WP, LV
4.8 Ib a.i.
per 100 gal/A
Days
elapsed
3
6
9
13
16
20
28
34
42
50
62
3
6
9
13
16
20
28
34
42
50
62
3
6
9
13
16
20
28
34
42
50
62
1
3.91
2.03
0.71
0.17
0.05
0.020
0.007
0.004
<. 0 . 004
0.005
<0.004
7.32
3.40
4.10
0.60
.0.47
0.51
0.65
0.080
0.004
0.014
< 0.004
1.46
1.07
0.86
0.18
0.08
0.116
0.014
0.011
0.004
0.004
<0.004
2
5.44
2.82
1.39
0.19
0.1.6
0.049
0.004
0.005
<0.004
< 0.004
< 0.004
5.00
4.;38
2.56
0.08
0.72
0.18
0.06
0.056
0.052
0.002
<0.004
2.55
2.13
1.37
0.63
0.16
0.121
0.083
0.011
0.008
0.004
< 0 . 004
Thion
mean
4.68
2.42
0.94
0.18
0.11
0.035
0.006
0.005
0.004
0.004
0.004
6.16
4.14
3.33
0.34
0.60
0.35
0.35
0.068
0.028
0.008
0.004
2.00
1.60
1.12
0.41
0.12
0.118
0.049
0.011
0.008
0.004
<0.004
1
0.102
0.109
0.145
0.123
0.097
0.131
0.064
0.029
0.015
0.032
0.004
0.153
0.114
0.091
0.149
0.187
0.314
0.250
0.113
0.044
0.099
0.017
0.004
0.092
0.035
0.056
0.042
0.040
0,016
0.007
0.008
0.006
<0.004
2
0.102
0.166
0.160
0.153
0.176
0.129
0.041
0.035
0.035
0.027
0.064
0.117
0.107
0.074
0.135
0.231
0.220
0.149
0.108
0.12.7
0.047
0.036
0.205
< 0.004
< 0.004
<0.004
0.089
0.056
0.047
0.011
0.013
0.011
<0.004
Oxon
mean
0.102
0.138
0.153
0.138
0.137
0.130
0.053
0.032
0.025
0.030
0.032
0.135
0.111
0.083
0.142
0.209
0.267
0.200
0.111
0.086
0.073
0.027
0.103
0.046
0.018
0.028
0.066
0.049
0.032
0.009
0.011
0.009
<0.004
Thicn-t-
oxon
(mean)
4.78 •
2.56
1.09
0.32
0.25
0.17
0.059
0.037
0.025
0.030
0.032
6.30
4.25
3.41
0.48
0.81
0.62
0.45
0.18
0.11
0.081
0.027
2.10
1.65
1.14
0.44
0.19
0.17
0.081
0.020
0.017
0.013
<0.004
By the
RFM procedure
Total OP in residues (corr.
1
0.73
0.53
0.27
0.19
0.13
0.060
0.091
0.036 ,
0.017
<0.004
<0.004
4.26
3.35
2.27
1.10
0.44
0.44
0.13
0.066
0.081
0.053
0.024
1.63
1.12
0.80
0.44
0.19
0.091
0.058
0.016
0.006
< 0.004
*0.004
2
0.99
0.57
0.37
0.20
0.16
0.081
0.103
0.038
0.021
< 0.004
<0.004
4.71
3.75
2.02
1.14
0.45
0.39
0.32
0.175
0.114
0.040
0.030
• 2.28
1.51
1.02
0.50
0.16
0.114
0.06
0.037
0.020
< 0.004
<0.004
Mean
0.86
0.55
0.32
0.20
0.15
0.071
0.097
0.037
0.019
< 0.004
<0.004
4.49
3.55
2.15
1.12
0.45
0.42
0.225
0.121
0.098 '
0.047
0.027
1.96
1.32
0.91
0.47
0.18
0.103
0.064
0.027
0.013
<0.004
<0.004
Ratio of
) GC : RFM
means
5.6
4.7
3.4
0.6
1.7
• 2,-f
0.6 '
1.0
1.3
-
•
1.4
1.2
1.6
. 0.4
1.8
1.5
2.0
l.Z
1.1
1.7
1.0
1.1
1.3 .
1.3
0.9
1.1
1.7
1.3
0.7.
1.3
-
1.0 .
-------
TABLE 22 (Cont'd). DISLODGABLE FOLIAR RESIDUES ( Mg/cm2) OF PARATHION, MALATHION AND METHIDATHION AFTER APPLICATION 'OF
DILUTE AND LV SPRAYS TO ORANGE TREES
I
M
O
M.
I
Application
Methidathion
EC, LV
4.8 Ib a.i./
100 gal/A
Parathion WP,
dilute
0.38 Ib a.i./
100 gal (full
coverage)
Malathion WP,
dilute
0.63 Ib a.i./
100 gal (full
coverage)
Days
elapsed
3
6
9
13
16
20
28
34
42
.50
62
3
6
9
13
16
20
28
34
42
50
' 62
3
6
9
13
16
20
28
34
42
50
62
1
1.60
0.42
0.49
0.08
0.061
0.039
0.011
0.005
0.010
0.010
0.008
0.81
0.43
0.27
0.028
0.013
o.ao?
0.002
<0.004
<0.004
<0.004
<0.004
1.38
0.57
0.24
0.10
0.029
0.035
0.005
< 0.004
< 0.004-
< 0.004
<0.004
Determined by
Thion
2 mean
1.84 1.72
0.66 0.54
0.33 0.43
0.21 0.15
0.078 0.070
0.047 0.043
0.026 0.019
0.015 0.010
O.OL1 0.011
0.008 0.009
0.006 0.007
0.84 0.83
0.36 0.40
0.10 0.18
0.024 0.026
0.014 0.014
0.006 0.007
0.004 0.003
<0.004 <0.004
<0.004 <0.004
<0.004 <;0.004
<0.004 <0.004
1.14 1.26
0.60 0.58
0.53 0.41
0.15 0.13
0.055 0.047
0.025 0.030
0.005 0.005
<0.004 <0.004
<0.004 <0.004
<0.004 <0.004
<0.004 ^0.004
the GC
1
0.182
0.047
0.100
0.037
0.024
0.026
0.014
0.014
0.008
0.004
0.004
0.023
0.020
0.024
0.018
0.016
0.015
0.004
<0.004
<0.004
<0.004
<0.004
0.115
0.023
0.018
0.039
0.021
0.052
0.015
0.018
0.017
0.015
<0.004
procedure
2
0.171
0.033
0.053
0.051
0.038
0.036
0.019
0.031
0.012
0.007
<0.004
0.030
0.027
0.020
0.018
• 0.016
0.012
0.006
<0.004
<0.004
<0.004
<0.004
0.029
0.030
0.036
0.058
0.034
0.052
0.017
0.011
0.012
0.009
<0.004
Oxon
mean
0.177
0.040
0.077
0.044
0.031
0.031
0.017
0.033
0.010
0.006
<0.004
0.027
0.024
0.022
0.018
0.016
0.014
0.005
<0.004
<0.004
<0.004
<0.004
0.072
0.027
0.027
0.049
0.033
0.052
0.016
0.015
0.015
0.012
<0.004
Thion+
oxon
(mean)
1.90
0.58
0.51
0.19
0.10
0.074
0.036
0.033
0.021
0.015
0.007
0.86
0.42
0.20
0.044
0.030
0.021
0.008
0.004
0.004
0.004
0.004
1.33
0.61
0.44
0.18
0.080
0.082
0.021
0.015
0.015
0.012
0.004
By the
RFM procedure
Total OP in residues (corr.]
1
0.84
0.61
0.33
0.26
0.12
0.073
0.084
0.011
0.003
<0.004
<0.004
0.06
0.22
0.07
0.048
0.029
0.010
0.016
< 0 . 004
<0.004
<0.004
< 0.004
0.67
0.49
0.31
0.17
0.104
0.080
0.041
0.019 ,
0.024
<0.004
<0.004
2
0.79
0.62
0.33
0.31
0.10
'0.093
0.049
0.016
0.023
< 0.004
< 0.004
0.40
0.19
0.055
0.045
0.031
0.004
0.009
0.009
<0.004
<0.004
<0.004
0.74
0.42
0.24
0.21
0.087
0.072
0.032
0.012
0.022
< 0.004
< 0.004
Mean
0'.82
0.62
0.33
0.28
0.11
0.083
0.067
0.014
0.013
<0.004
<0.004
0.50
0.21
0.063
0.047
0.030
0.007
0.012
0.005
<0.004 '
< 0.004
<0.004
0.70
0.46
0.28
0.19
0.096
0.076
0.037
0.016
0.023
< 0.004
<0.004
Ratio of
) GC:RFM
means
2.3
0.9
1.5
0.7
0.9
0.8
0.5
2.3 -
1.6
..
• -
1.7
2.0
3.0
0.9
1.0
3.0
0.7
0.8
1.0
1.0
1.0
1.8
1.3 ',
1.6
0.9
0.8
I.I
0.6
0.9
0.7
3.0
1.0
-------
-TABLE 22 (Cont'd). DISLODGABLE FOLIAR RESIDUES ( Mg/cm2) OF PARATHION, MALATHION AND METHIDATHION AFTER APPLICATION
OF DILUTE AND LV SPRAYS TO ORANGE TREES
Deter nined by the GC procedure
1
t-1
o
M.
1
Application
Methid-
athion WP,
dilute
0.25 Ib a.i./
100 gal (full
coverage)
Methida-
thion EC,
dilute
0.25 Ib a.i./
100 gal (full
coverage)
Days
elapsed
3
6
9
13
16
20
28
34
42
' 50
62
3
6
9
13
16
20
28
34
42
50
62
1
0.62
0.38
0.21
0.093
0.039
0.024
0.020
0.004
0.004
0.005
0.004.
0.23
0.12
0.079
0.034
0.028
0.017
0.029
0.005
<0.004
<0.004
<0.004
2
0.58
0.33
0.20
0.084
0.045
0.034
0.016
< 0.004
<0.004
0.005
<0.004
0.71
0.07
0.113
0.030
0.028
0.019
0.036
0.006
<0.004
<0.004
<0.004
Thion
mean 1
0.60 <0.004
0.355 <0.004
0.205 <0.004
0.089 0.028
0.042 0.023
0.029 0.017'
0.018 0.012
<0.004 0.005
<0.004 0.005
0.005 0.004
<0.004 <0.004
0.47 0.182
0.095 0.037
0.096 0.032
0.032 0.031
0.028 0.018
0.018 0.018
0.033 0.016
0.006 0.009
<0.004 0.005
<0.004 0.006
<0.004 <0.004
2
<0.004
< 0.004
0.022
0.022
0.024
0.020
0.009
0.005
0.006
0.005
<0.004
0.165
0.054
0.047
. 0.026
0.016
0.016
0.014
0.012
0.004
0.002
<0.004
Oxon
mean
< 0.004
<0.004
0.011
0.025
0.024
0.019
0.011
0.005
0.006
0.005
<0.004
0.173
0.046
0.040
0.029
0.017
0.017
0.015
0.011
0.005
0.004
<0.004
Thion-t-
oxon
(mean)
0.60
0.36
0.22
0.11
0.066
0.048
0.029
0.005
0.006
0.005
<0.004
0.64
0.15
0.14
0.061
0.045
0.035
0.048
0.017
0.005
0.004
<0.004
By the
RFM procedure
Total OP in residues (corr.]
1
0.55
0.31
0.18
0.078
0.047
0.036
0.025
0.006
0.004
< 0.004
<0.004
0.43
0.16
0.079
0.063
0.027
0.012
0.019
0.011
< 0.004
<0.004
<0.004
2
6.65
0.32
0.25
0.069
0.042
0.026
0.019
0.011
0.007
<0.004
<.0.004
0.35
0.16
0.055
0.047
0.027
0.017
0.008
0.004
< 0.004
< 0.004
<0.004
Mean
0.60
0.32
0.215
0.073
0.045
0.031
0.022
0.009
0.006
< 0.004
<0.004
0.39
0.16
0.067
0.055
0.027
0.015
0.014
0.008 '
<0.004
<0.004
<0.004
Ratio of
1 GC : RFM
means ,
1.0
1.1
1.0
1.5
' 1.5
1.6
1.3
0.6
1.0
•
1.0
1.6
0.9
2.0
1.1
1.7
2.3
3.4
2.1
1.3
1.0
1.0
-------
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O MALATHION WP, LV
• MALATHION WP, DILUTE
D PARATHION WP, LV
• PARATHION WP, DILUTE
0
_L
10 20 30 40 50
DAYS AFTER APPLICATION
60
Figure 3. Dissipation curves for malathion WP and parathion WP, each
applied as dilute and low-volume sprays, respectively, to
orange trees, and determined over a 62-day period by the
RFM as dislodgable foliar OP residues.
-103-
-------
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respectively, are remarkably similar. The downward slide of
methidathion EC residues resulting from the dilute and LV
2
applications is faster (reaches 0.002 ^g/cm sooner) than their
WP counterparts. Figure 3 shows similar trends between mala-
thion and parathion perhaps due to different structural charac-
teristics of the malathion and parathion molecules or to
differences in their formulation makeup. Malathion WP applied
in LV form lasts longest. In all cases, the LV applications
show greater residual longevity than those of the dilute
treatments, as expected.
Figures 5-8 show the correlations between the RFM (y
axis) and GC values (x axis), with calculations of the various
slopes and regression values. Not shown are the closeness of
2
the values below 0.01 |ag/cm between the two analytical methods,
On figures 5-8 the GC values are the sum of the thion and oxon
components. The regression values are considered to be quite
acceptable.
The best correlation (i.e., the closest to the mathe-
matically ideal slope of 45°) is shown by methidathion WP in
the LV and dilute applications (Fig. 7). Reasons for the
greater disparity in results of dislodgable residue levels
shown in Figs. 4, 5 and 8 by the two methods are not entirely
clear. Although amounts of OP residues on leaf surfaces as
2
low as 0.002 ng/cm are measured by both the RFM and the GC
2
methods, residue levels below 0.004 pig/cm are considered to
be insignificant from a Worker Reentry perspective, and are
reported in Table 22 as < 0. 004 ng/cm2.
-105-
-------
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13
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MALATHION
o LOW-VOLUME
• DILUTE
I I
_L_
i 1.1 I 1 1
_1 L.
0.01 0.1 I
GAS CHROMATOGRAPHIC VALUES
Figure 5. Correlation between total (thion and oxon) dislodgable
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of a wettable powder
formulation of malathion. The line is described by
In y = 0.87 In x - 0.380 (one point omitted) and the
correlation coefficient is 0.99.
-106-
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i.
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PARATHI ON
o LOW-VOLUME
• DILUTE
J 1 i i i i i i
0.01 O.I |
GAS CHROMATOGRAPHIC VALUES
Figure 6. Correlation between total (thion and oxon) dislodgable
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of a wettable powder
formulation of parathion. The line is described by
In y = 0.67 In x - 1.09 and the correlation coefficient
is 0.96.
-107-
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CM
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METHIDATHION
O LOW-VOLUME
• DILUTE
t i i i i i
i i i i i i
0.01
O.I
GAS CHROMATOGRAPHIC VALUES (/zq/cm2)
Figure 7. Correlation between total (thion and oxon) dislodgable
foliar OP residues obtained by the colorimetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of a wettable powder
formulation of methidathion. The line is described by
In y = In x - 0.133 and the correlation coefficient is
0.99.
-108-
-------
__ I
CJ
if)
LU
Q
O
I
I-
UJ
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METHIDATHION
o LOW-VOLUME
• DILUTE
9 •
J I I I
I I L
0.01
O.I
GAS CHROMATOGRAPHIC VALUES (/zg/cm2)
Figure 8. Correlation between total (thion and oxon) dislodgable
foliar OP residues obtained by the coloriraetric field
method and the GC laboratory method after spraying trees
with low-volume and dilute sprays of an emulsifiable
concentrate formulation of methidathion. The line is
described by In y = In x - 0.313 and the correlation
coefficient is 0.93.
-109-
-------
In Table 22, the major differences between the total OP
residue obtained by the GC and the RFM, respectively, may be
seen for parathion WP, LV spray at 3, 6, 9, 13 and 16 days
after application where the differences were 1.6-5.6 times
greater for the values obtained by GC compared to those by
the RFM. In the case of malathion LV, the differences in
magnitude of the total OP were reduced to 1.2-1.6x for the
first 3 sampling dates in favor of GC, and for methidathion
LV, the differences in magnitude were further reduced to
ratios of 1.07-1.25x. It was believed that the nature of the
OP compound, the type of spray application and the higher
extraction efficiency of the 3 OP compounds by the GC method
were factors in the disparity noted, and that differences in
this regard would be more evident during the upper ranges of
the dissipation curves (0-16 days postapplication), particularly
by the LV treatments with their higher residue levels. This
was indeed the case, since Table 22 shows smaller differences-
in-magnitude ratios in favor of the GC method at 3-16 days
when the dilute sprays were used.
Table 22 shows that the percentage of oxon in relation to
the thion + oxon totals rises from about 2-4% at 3 days to 100%
of the total residue after 42-62 days postapplication, but the
nature of the OP compound has a bearing on the rate and amount
of change from thion to oxon. The presence of a high percentage
of oxons has greater toxicological impact than the same amount
of their thion analogues. If the rate of thion-to-oxon
conversion can be shown to be reproducible and predictable, it
-110-
-------
is conceivable that it could be extrapolated to results
obtained by the RFM and assi.st in interpretation of OP residue
levels and their relative importance in assessing the health
hazard potential of the analytical findings.
21. Foliar Residues of Methidathion at Various Locations of Sprayed
Lemon Trees After Application as Dilute and LV Sprays. The
objectives, outline and design of this experiment were given
in Section 5, subsection 13. . The overall objective was to
ascertain whether the residue levels are affected by the
height from the ground of the foliage sampling point, arid by
the directional point (W,N,E,S). The dilute and LV sprays
were applied to lemon trees. Samples were taken in triplicate
sets, and analytical results were corrected for the blank
(control) values of pretreatment samples taken at 36 points.
In Table 23, the replicate and mean values are reported
2
'as micrograms of me th i da th ion/cm leaf surface. The locations
of each sampling date are ranked for each group from the
highest mean to the lowest mean value. Each value shown is
the mean of two subsamples. It should be noted that a light
rain had fallen 10 days after application.
In the two sampling periods, 7 and 14 days after appli-
cation, respectively, of the dilute spray application, the
foliage of the E and W directions at the 3 locations [H,M and
L (see Table)], were invaribly higher in methidathion residues
than the N and S locations. This reflects the fact that the
roadways (inter-row throughways) at the application site face
East and West, and accordingly the trees received a larger
amount of spray.
-Ill-
-------
TABLE 23. DISLODGABLE FOLIAR RESIDUES ( ug/cmz) OF METHIDATION AT VARIOUS LOCATIONS OF SPRAYED
LEMON TREES AFTER APPLICATION AS DILUTE AND LOW-VOLUME SPRAYS
N)
I
Dilute spray, 7 days post-application
Sample
information
6 ft (H)
above
ground
4 ft (M)
above
ground •
1.5 ft (L)
above
ground
Replicate
E
W
N
S
E
W
S
N
E
W
S
N
' 1
0.10
0.10
0.071
0.081
0.15
0.12
0.10
0.11
0.20
0.14
0.12
0.11
2
0.12
0.13
0.063
0.061
0.18
0.12
0.11
0.10
0.18
0.12
0.13
0:11
LV spray, 11
Sample
information
6 ft (H)
above
ground
4 ft (M)
above
ground
1.5 ft (L)
above
ground
3
0.12
0.10
0.076
0.054
0.18
0.09
0.12
0.09
0.18
0.15
0.15
0.13
j Overall
Mean mean
0.11 0.09
0.11
0.070
0.065
0.17 0.12
0.11
0.11
0.10
0.19 0.15
0.14
0.13
0.12
Dilate
spray,
, 14 days
post-application
Replicate
E
W
S
N
E
W
S
N
E
W
N
S
days post-application
Replicate
E
W
S
N
S
W
N
E
S
N
E
W
1
0.12
0.13
0.12
0.11
0.28
-
0.11
0.12
0.25
0.22
0.18
0.067
2
•0.18
0.13
0.12
0.08
0.20
0.17
0.13
0.12
0.25
0.26
0.11
0.082
3
0.24
0.09
0.10
0.13
0.22
0.13
0.13
0.11
0.29
0.25
0.12
0.084
Overall
Mean mean
0.18 0.13
0.12
0.11
0.11
0.23 0.16
0.15
0.12
0.12
0.26 0.18
0.24
0.14
0.078
1
0.037
0.031
0.028
0.024
0.092
0.039
0.059
0.049
0.074
0.068
0.042
0.038
2
0.042
0.041
0.033
0.029
0.067
0.039
0 . 034
0.033
0 . 044
0.033
0.033
0.047
LV spray, 17
3
0.049
0.032
0.030
0.019
0.067
0.061
0.043
0.040
0.057
0.039
0.035
0.035
Mean
0.043
0.035
0.030
0.024
0.075
0.046
0.045
0.042
0.058
0.048
0.038
0.040
Overall
mean
0.033
0.052
0.046
days post-application
Repl icate
E
W
N
S
S
N
E
W
N
E
W
S
1
0.042
0.060
0.052
0.060
0.116
0.046
0.026
0.036
0.123
0.040
0.032
0.065
2
0.045
0.045
0.049
0.029
0.097
0.037
0.077
0 . 034
0.067
0.067
0.031
0.011
3
0.065
0.048
0.045
0.044
0.066
0.061
0.043
0.040
0.116
0.027
0.037
0.011
Moan
0.051
0.051
0.044
0.044
0.093
0.065
0.049
0.035
0.102
0.037
0.033
0.029
Overall
mean
0.049
0.061
0.050
.
-------
The overall means show that the foliage sampled 4 ft
above ground is in between the residue values of the 6-ft (H)
and 1 1/2-ft (L) samplings at 7 days•postapplication, but at
14 days after application the L samples assume the in-between
values. The same trend is shown by the LV samples in this
regard at the two sampling periods (11 and 17 days after
application) indicated in Table 23.
The high-to-low residue ranking shown by the LV application
changed with time and location. Clear-cut reasons for this
are not apparent. In retrospect, it wovld have been preferable
to have only two sampling dates instead of 4 different ones for
the intercomparison purposes. However, circumstances were not
favorable for handling such a large volume of samples at one
time.
The results indicate that, the directional location of the
sampling point, the height from the ground, the type of spray
application and the days of postapplication are factors that
influence the values obtained. In practice, sampling of a
4-ft height from the ground in a circular pattern would appear
to provide nearly maximum residue levels from which interpre-
tations of health hazard potential could be made.
-113-
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REFERENCES
1. Adams, J. D., Y. Iwata, and F. A. Gunther. Worker environment research.
IV. The effect of dust derived from several soil types on the dissipation
of paraoxon dislodp,able residues on citrus foliage. Bull. Environ.
Contain. Toxicol. _15, 547-554, 1976.
2. Adams, J. D., Y. Iwata, and F. A. Gunther. Worker environment research.
V. Effect of soil dusts on dissipation of paraoxon dislodgable residues
on citrus foliage. Bull. Environ. Contam. Toxicol. 18, 445-451, 1977.
3. Bailey, B. The effects of pesticide residues on farm laborers. Agric. Age
2,8, 6, 1972.
4. Baker, E. L., Jr., M. Zack, J. W. Miles, L. Alderman, W. McWilson, R. D.
Dobbin, S. Miller, and W. R. Teeters. Epidemic malathicn poisoning in
Pakistan malaria workers. Lancet. _!, 31-34, 1978.
5. Carman, G. E., W. E. Westlake, and F. A. Gunther. Potential residue problems
associated with low volume sprays on citrus in California. Bull.
Environ. Contam. Toxicol. 8^ 38-45, 1972.
6. Epstein, J., R. W. Rosenthal, and R. J. Ess. Use of 4-(4-nitrobenzyl)pyridine
as an analytical reagent for ethyleneimines and alkylating agents.
Anal. Chem. 2_7^, 1435-1439, 1955.
7. Getz, M. E., and R. R. Watts. Application of 4-(p-nitrobenzyl)pyridine as
a rapid quantitative reagent for organophosphate pesticides. J.
Assoc. Official Anal. Chem. 4_7_, 1094-1096, 1964.
8. Grvmwell, J. R., and R. H. Erickson. Photolysis of parathion (0,0-diethyl
O-(p-nitrophenyl)phosphorothioate). New products. J. Agr. Food Chem.
21., 929-931, 1973.
9. Gunther, F. A., W. E. Westlake, J. H. Barkley, W. Winterlin, and L. Langbehn.
Establishing dislodgable pesticide residues on leaf surfaces. Bull.
Environ. Contam. Toxicol. _9_, 243-249, 1973.
10. Gunther, F. A., J. H. Barkley, and W. E. Westlake. Worker environment
research. II. Sampling and processing techniques for determining
dislodgable pesticide residues on leaf surfaces. Bull. Environ.
Contam. Toxicol. 12, 641-644, 1974.
11. Gunther, F. A., Y. Iwata, G. E. Carman, and C. A. Smith. The citrus reentry
problem: Research on its causes and effects, and approaches to its
minimization. Residue Reviews 6JZ, 1-139, 1977.
12. Iwata, Y., J. B. Knaak, R. C. Spear, and R. J. Foster. Procedure for the
determination of dislodgable pesticide residues on foliage. Bull.
Environ. Contam. Toxicol. 18, 649-655, 1977.
13. Iwata, Y., G. E. Carman, and F. A. Gunther. Worker environment research:
Methidathion applied to orange trees. J. Agr. Food Chem. 27, 119-129,
1979.
-114-
-------
14. Kahn, E. Worker reentry safety. V. Reentry intervals as health standards.
Residue Reviews 62, 35-40, 1976.
15. Maddy, K. T. Worker reentry safety. IV. The position of the California
Dept. of Food and Agriculture on pesticide reentry safety intervals.
Residue Rev. 62, 21-34, 1976.
]6. Mallipudi, N. M., N. Umetsu, R. F. Tuia, R. E. Talcott, and T. R. Fukuto.
Toxicity of 0,0,S-trimethyl and triethyl phosphorothioate to the rat.
J. Agr. Food Chem. _27_, 463-466, 1979.
17. Milby, T. H., F. Ottoboni, and H. W. Mitchell. Parathion residue poisoning
among orchard workers. J. Amer. Med. Assoc. 389, 351-356, 1964.
18. Pellegrini, G., and Santi, .R. Potentiation of toxicity of organophosphorus
compounds containing carboxylic ester functions toward warm-blooded
animals by some organophosphorus impurities. J. Agr. Food Chem. 20,
944-950, 1972.
19. Popendorf, W. J., and R. C. Spear. Preliminary survey of factors affecting
the exposure of harvesters to pesticide residues. Amer. Ind. Hyg.
Assoc. J. _35_, 374-380, 1974.
20. Popendorf, W. J., R. C. Spear, J. T. Leffingwell, J. Yager, and E. Kahn.
Harvester exposure to Zolone (phosalone) residues in peach orchards.
J. Occup. Med. 21, 189-194, 1979.
21. Quinby, G. E., and A. B. Lemmon. Parathion residues as a cause of poisoning
in crop workers. J. Amer. Med. Assoc. 166, 740-746, 1958.
22. Savage, E. P. A study of hospitalized acute pesticide poisoning in the
United States, 1971-1973. EPA Contract 68-02-1271 and 68-01-3138,
1975.
23. Scher, H. H., ed. Controlled Release Pesticides. ACS Symposium Series 53.
Symposium at 173rd ACS Meeting, New Orleans, LA., Mar. 21-27, 1977.
American Chemical Society, Washington, D.C., 1977.
24. Serat, W. F. Calculation of a safe reentry time into an orchard treated
with a pesticide chemical which produces a measurable physiological
response. Arch. Environ. Contam. Toxicol. 1, 170-181, 1973.
25. Serat, W. F., H. P. Anderson, E. Kahn, and J. B. Bailey. On the estimation
of worker reentry intervals into pesticide treated fields with and
wothout the exposure of human subjects. Bull. Environ. Contam. Toxicol.
JJ, 506-512, 1975.
26. Smith, C. A., and F. A. Gunther. Worker environment research. VI. Rapid
estimation of organophosphorus pesticide residues in citrus grove
v ' . soil. Bull. Environ. Contam. Toxicol. 19, 571-577, 1978.
Smith, C. A., F. A. Gunther, and J. D. Adams. Worker environment research.
III. A rapid method for the semi-quantitative determination of some
dislodgable pesticide residues on citrus foliage. Bull. Environ.
Contam. Toxicol. 15. 305-319, 1976.
-115-
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28. Spo.ar, R. C., Y.Lee, J. T. Leffingvell, and D. Jenkins. Conversion of
parathion to paraoxon in foliar residues: Effects of dust level and
ozone concentration. J. Agr. Food Chem. 26, 434-436, 1978.
29. Spencer, W. F., M. M. Cliath, R. D. Davis, R. C. Spear, and W. J. Popendorf.
Persistence of parathion and its oxidation to paraoxon on the soil
surface as related to worker reentry into treated crops. Bull. Environ.
Contain'. Toxicol. 14_, 265-272, 1975.
30. Spencer, W. F., Y. Iwata, W. W. Kilgore, and J. B. Knaak. Worker Reentry
into PesticideTreated Crops. II. Procedures for the determination of
pesticide residues on the soil surface. Bull. Environ. Contam. and
Toxicol. 1_8, 656-662, 1977.
31. Talcott, R. E., N. M. Mallipudi, N. Umetsu, and T. R. Fukuto. Tnactivation
of esterases by impurities isolated from technical malathion. Toxicol.
and Appl. Pharmacol. _49_, 107-112, 1979.
32. Talcott, R. E., H. Denk, and N. M. Mallipudi. Malathion carboxylesterase
activity in human liver and its inactiviation by isomalathion. Toxicol,
and Appl. Pharmacol. 49, 373-376, 1979.
33. Turner,. C. A. Spectrophotometric determination of organophosphates with 4-
C4-nitrobenzyl)pyridine. Analyst _99, 431-434, 1974.
34. Watts, R. R. 4- (p_~Nitrobenzyl) pyridine, a new chromogenic reagent for the
organophosphorus pesticides. .7. Assor. Off, Anal= Chem. £8, 1161-1163,
1965.
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TECHNICAL R£> OPT DATA
/Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-80-019
13. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Rapid Field Measurements of Organophosphorus
Pesticide Residues
5. REPORT DATE
May 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
Francis A. Gunther, Ben Berck, and Yutaka Iwata
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADDRESS
Department of Entomology
University of California
Riverside, California 92521
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R805 642-01
12. SPONSORING AGENCY NAME ANO ADDRESS
Office of Research and Development
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT ANO PERIOD COVERED
14. SPONSORING AGENCY CODE
600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A rapid field method (RFM) for on-the-spot determination of organphosphorus (OP)
insecticide residues on crop foliage and surface soil dust was developed. The RFM
is applicable to the data needs of the Worker Reentry Problem for which rapid
assessment of dislodgable OP residues on foliage and in surface soil is needed prior
to clearance for reentry of workers in sprayed fields or groves. The method is
based on the alkylation reaction of OP compounds with NBP [_p_-nitrobenzyl)~
pyridine] to form a magenta color in an alkaline medium. The color intensity
(absorbance) is measured with a portable mini-spectrophotometer. The ratio of
absorbance unit per yg of OP compound (the slope of the standard curve) varies with
the particular molecular species. By determining the ratio of the slopes of the
curves obtained at 100° for 30 min vs. 150° for 3 min, one obtains values that are
characteristic or relatively constant for a given OP species, and thus has value for
identification of the species provided that only one species is present in the sample.
In addition to the Worker Reentry Problem, involving over 300,000 workers in California
alone, the RFM is useful for the testing of foliage in a given area for OP residues
prior to release of parasites and predators for biological control in an integrated
pest management program.
17. KEY WORDS AND DC
a. DESCRIPTORS
Rapid field method (RFM)
Organophosphorus (OP) residue
Worker Reentry Problem
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
<;UMENT ANALYSIS
b.lOENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS ,Tha Rsporri
TTNfTr.ASSTFTKn
20. SECURITY CLASS , This pa?ei
UNCLASSIFIED
c. COSATi Field/Group
06F,T
21 NO. OF PAGE3
134
22. PRICE
EPA Form 2220-1 j3«v. 4-77) PREVIOUS EDITION is OBSOLETE
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