CALCULATION OF A SAFE REENTRY TIME

INTO AN ORCHARD TREATED WITH A

PESTICIDE CHEMICAL WHICH PRODUCES A
MEASURABLE PHYSIOLOGICAL RESPONSE
WILLIAM F. SERAT
Community Studies on Pesticides
State Department of Public Health
Berkeley, Calif. 94704

A mathematical equation is presented for the calculation of a safe reentry time into
an orange orchard which has been treated with a pesticide chemical, the residues of which
produce a dose--dependent response in humans. The equation relates the kinetics of the
rate of loss of pesticide residues, which remain on the foliage, of pesticide--treated trees,
and the kinetics of the rate of change in a measurable physiological response (blood
cholinesterase activity), brought about by exposure of humans to the decreasing residue
levels, to the time of reentry of workers into the orchard.

Introduction
The use of organic phosphorus pesticides has been associated with many documented
incidences of poisoning of agricultural workers. In many cases, the workers have been
sent into fields or orchards at a time when sufficient pesticide chemical residue still
remained on the foliage to endanger persons who must contact the plants or inhale
dislodged particular matter. Attempts have been made to establish safe time intervals
between the time of application of known amounts of a pesticide and the time of entry
into the fields (the "reentry" time) but these intervals have often been determined sub-
jectively because well-designed, quantitative methods for their establishment generally
have not been used (California Department of Agriculture 1971). It is the purpose of
this paper to present a method for calculating safe reentry times for workers into
orchards based on the rate of loss of pesticide chemical residues on tree foliage and
on the rate of change of a measurable physiological effect or response brought about
by exposure to the decreasing residue levels on the foliage.

Many reports exist which show the rate of decay of certain pesticide chemicals
applied to crops. Some indicate an initial rapid loss of residues attributed to mechanical
erosion or flaking from leaf surfaces followed by a first order decay of the remaining
surface material (Gunther 1969); some residue decay is completely first order (or pseudo
first order) (Gunther 1969, MiSllhoff 1968). Residue decay has also been depicted as

170
Archives o f Environmental Contamination and Toxicology,
Vol. 1, No. 2, 1973, ~)1973 by
Springer-Verlag New York Inc.
 Calculation of Reentry Time Into Pesticide-Treated Orchard t71

a more complex event (Linskens et al. 1965), controlled probably by rate--limiting

physical processes which bring the pesticide chemical into contact with environmental
factors (oxygen, light, humidity, etc,) that modify or affect decay or which account
for its removal by translocation from leaf surlhces into the tissues. Each mathematical
expression for decay, in which the rate can be defined in terms of measurable quantities,
can be incorporated into the calculation of safe reentry time. Any determinable
physiological effect which can be quantitated in terms of 1) the level of pesticide chemi-
cal residue to which individuals are exposed and 2) the length of time defining that
exposure can be employed in estimating safe reentry times, At present the inhibition
of blood cholinesterase activity appears to be the only practical effect which is respon-
sive to the presence of organic phosphate pesticide chemicals, in general, and whose
sensitivity of measurement is such that it can be estimated within reason.

The kinetics of cholinesterase inhibition in vitro have been investigated thoroughly

by a number of workers (O'Brien 1960, Nachmansohn et al. 1948, Main 1964).
Generally, it is known that, at excess inhibitor (pesticide chemical) concentrations and
assuming a low concentration of any reversible enzyme--inhibitor complex, the rate
of loss of cholinesterase activity with time follows first order kinetics. However, there
are few reports of definitive kinetic in vivo studies with man because of the high level
of toxicity of many pesticide chemicals. In contrast to the known concentrations of
inhibitors used with in vitro studies, in vivo rates of percutaneous penetration (dermal
exposure}, hydrolysis, nonspecific binding, enzyme reactivation in the absence of
irreversible inhibition, enzyme synthesis, intestinal absorption (oral exposure), losses
by partitioning, etc. all tend to alter the in vivo levels of pesticide chemicals relative
to applied levels, and different pesticide chemicals have very different capacities to
be affected by these variables. Moreover, a pesticide chemical continues to enter the
exposed individual in the field, throughout the work day, by a probable combination
of three entry routes (oral, dermal, and respiratory) whose relative magnitudes differ
with different pesticide chemicals but, for any such chemical, remains virtually
unknown. The contribution each route exerts is influenced by the method of pesticide
application, the formulation used, and conditions which govern the pesticide removal
from leaves in the form of various sized particles.

Field data, from which the rate of pesticide decay and the simultaneous effect on
field workers can be established, are very sparse. One group of investigators, who
have collected some field data, has kindly made them available for use in illustrating
reentry--time calculation methods given here. While this use of the field data is per-
mitted, the author has agreed not to identify the investigators or the organophosphate
pesticide chemical used, pending additional field work by the group.

Methods
Pesticide Application. A commercial wettable powder formulation of an
172 William F, Serat

organophosphate pesticide chemical was applied at 7 pounds active material per acre
to a 5--acre plot of producing orange trees in California.

Treatment of Field Workers. Individuals who were to harvest the crop were inter-
viewed to determine their interest and willingness to participate in the study. W o r k - -
and medical histories were obtained from those participating in the study and blood
samples were drawn 2 and 3 days prior to entry into the fields for plasma and RBC
cholinesterase assays. Blood samples were again drawn from the participants after 1 -
and 5 days activity in harvesting fruit. (More frequent blood sampling was not possible
because of the workers' reluctance to having blood drawn. Moreover, not all workers
donated blood samples each of the four times mentioned above.) Workers entered the
fields 3 complete days after the application of pesticide and remained in the field for
approximately eight hours. All operations and procedures were within the limits
described by the registered label at the time of the study and medical supervision was
available throughout the study,

Cholinesterase assays. Blood cholinesterase activities (plasma and red blood cell)
were all determined in one laboratory by the pH-stat technique reported by Nabb and
Whitefield (1967) except that acetycholine iodide rather than acetylcholine perchlorate
was used as the substrate.

L e a f sampling and residue determinations. Leaf punches were removed from the
foliage on the day of pesticide application and at 2, 4, 8, and 26 days thereafter; the
punches were taken from the center of randomly selected leaves and were 14 cm 2 in
area. Each sample, which comprised 20 punches, was placed in a 4--ounce jar, and
the residue was extracted by adding 20 ml of benzene and shaking for 15 minutes,
The solvent was decanted and the sample was washed several times with small portions
of benzene, The extract and washings were combined, filtered through anhydrous
sodium sulphate, and stored under refrigeration in screw cap bottles until analyzed;
an aliquot was used for analysis.

Pesticide chemical residue analyses were performed with a Tracor Model MT--220Q
gas chromatograph using a pyrex column (2 ft x 1/4 in.) packed with 10 percent by
weight of D C - - 2 0 0 on washed 80/100 mesh Chromosorb W.H.P. A Melpar flame
photometric detector, using a 526--micron filter, was used to quantitate the residue
concentrations in the combined extract and washings,

Results
Decay of pesticide chemical residues. The pesticide chemical residues extracted
from the samples of leaf punches (largely, though probably not entirely, from
the leaf---punch surfaces) are given, as a function of time after pesticide application,
in Table I. A plot of the data in Table I (Fig. 1) depicts a first order, or pseudo first
order, decay from the second day following application. That there should be an
 Calculation of Reentry Time Into Pesticide-Treated Orchard 173

Table I, Pesticide Chemical Residues Remaining on Orange Tree Foliage

as a Function o f Post-application Time

Time after pesticide Residue level

application, days p~g/em2 ppm

0 (by analysis) 1.87 131

0 (by extrapolation ~) 1.31 91.7
2 1.12 78.4
4 1.01 70.7
8 0.67 46.9
26 0.21 14.7

~See Fig. I.

initial rapid loss of some residue from orange--tree foliage is not surprising in the
light of comparable findings noted in other reports (Gunther 1969). An initial deposit
level for calculation purposes was determined by extrapolation to the time of appli-
cation. This extrapolated value is more reasonable than the observed value, in terms
of the total--residue decay data, and it adds a safety factor by presenting the overall
decay as being less rapid than it would appear otherwise. The first--order rate constant,
k, for the decay of the pesticide chemical was determined to be - 7 . 1 6 x 10 -2 days -1

The decrease in the level of pesticide chemical residue is not significant over the
working day. Indeed, over a whole day, the residue loss amounts to only 4 ppm.
Residue level found on the fruit average slightly more than 1 ppm on the second and
fourth days after application. In view of the fact that the weight of an orange far exceeds
that of a leaf and tbr a given weight of residue on each, the actual exposure to workers
from handling the fruit, in addition to contacting foliage, might be notably higher than
the leaf--residue level would suggest. In the absence of information as to the number
of oranges contacted by workers, it is assumed that this means of exposure is in propor-
tion to that obtained from contacting foliage. The results obtained for the decrease in
cholinesterase activity, as a function of total potential exposure from leaves, makes
this assumption reasonable but they suggest that the decrease in enzyme activity is
dependent on a higher level of exposure than that determined. In any event, such an
assumption does not invalidate the results in establishing safe reentry times from the
lowering of cholinesterase activity as a function of leaf--residue levels.

Changes in Blood Cholinesterase Activities. Although 10 workers started in the

study, adequate data regarding cholinesterase activity were obtained from only four of
 130~-

--\
80 \

70

60

~ 50
g
O

40

30

2O

10
0 5 10 15 20 25 30

Time after pesticide application (days)

Fig. 1. Decrease in pesticide chemical residue level with time after application of pesticide to
orange--tree foliage
174
 Calculation of Reentry Time Into Pesticide-Treated Orchard 175

them. The data, shown in Table II, indicate that, as a group, only the plasma enzyme
activities underwent a significant change as the workers were exposed. Worker number
2 (Table II) showed some lowering of his red blood cell cholinesterase activity by the
fifth day of exposure. None of the workers exhibited signs clinically associated with
poisoning.

Table II. Changes in Blood Cholinesterase Activities o f Workers upon Exposure

to Pesticide Chemical Residues on Orange- Tree Foliage

Enzyme Activity, /~m/min/ml

Before exposure After exposure
Work -3 Days -2 Days 1 Day 5 Days
Number RBC ~' Plasma RBC Plasma RBC Plasma RBC Plasma

2 9.26 2.87 8.74 3.10 10.28 1.03 6.58 0.42

3 11,96 2.20 11.99 2.29 8.40 t .99 11.09 0.83
7 8.47 2.73 12.64 1.57 8.52 0.60
12 10.11 2.13 9.87 2, 11 13.27 1.69 9.91 0.46

dRed blood cell fraction.

big. 2 depicts the plasma cholinesterase activities as a function of the total

leaf~residue levels to which the workers were exposed (values obtained from Fig.
1). Since the participants could not be prevented from working fields immediately prior
to being utilized in this study, their "'beR~re exposure" cholinesterase activity levels
are not assumed to represent a true baseline. However, sequential plasma values on
the days before exposure indicate no adverse effects either in magnitude or direction
of any changes. Worker number 2 possibly shows the results of prior exposure by the
change in his red blood cell enzyme activity but this change is not significant in
the light of the additional change in the first-day exposure in this study. All
"before exposure" values fall within the so--called normal range for cholinesterase
activity.

Where two " b e f o r e e x p o s u r e " cholinesterase activity values are available for a
worker, the average value is plotted (Fig. 2). Although only two sets of cholinesterase
activity values could be obtained during the time of pesticide exposure (i,e., on the
first and fifth day after entering the field, corresponding to the third and seventh days
after spraying), their graphical representation (Fig. 2) suggests a first order loss of activ-
ity with potential exposure from foliage or - d E = k'E, where E, is the enzyme activity
dP
level associated with a residue level of P. Such a relationship implies that the same
 10.0 m

8.0
m

6.0 -

4.0--

E
E 2.0'

"6
~_~ 1.0 O

.8

if)

e-- .6

t-
o

0
E
•4 - -

,2

I 1 I I t I
50 100 150 200 250 300
Total pesticide chemical residue to which the workers were exposed (ppm)
Fig. 2. Decrease in plasma cholinesterase activity of workers as a function of total pesticide
chemical residue on orange--tree foliage to which workers were exposed. The working time
was approximately constant for each of 5 days for each of 4 workers. (The total residue is calcu-
lated from Fig. 1 .)
t76
 Calculation of Reentry Time Into Pesticide-Treated Orchard 177

fraction of the available pesticide chemical residue was absorbed on each of the days
of exposure. The value of the specific rate constant, k', is determined (from Fig. 2)
as -4.75 x 10.3 ppm -~.

Data depicting a first order loss of enzyme activity with total pesticide chemical
residue on leaves suggest either that there is no recovery of the enzyme activity between
exposures or that the logarithm of the fractional increase by recovery is constant for
each of the days of exposure. Some recovery is expected because Grob and Harvey
(1949, 1958) and Grob et al. (1947), working with TEPP, sarin, and DFP (respectively),
found similar first order losses in blood cholinesterase activities with dosage even
though they measured some degree of recovery within a day following the cessation
of administered doses, Their finding suggests that some recovery occurred between
doses.

C a l c u l a t i o n o f r e e n t r y time
Within the limits that decreases in cholinesterase activity can be related quantatively
to pesticide chemical residue levels, it is a simple matter to establish reentry times
from data such as those shown in Fig. 1 and Fig, 2. Calculations are facilitated by
the first order relationships found for both sets of data but are not exlusive to such
relationships. For example, if the plasma cholinesterase activity can be allowed to
decrease 30 percent [from its initial value (average) of 2.45 /,m rain -1 ml -t to 1.7t
/zm rain "1 m1-1] then, from Fig. 2, it is noted that the exposure accommodating this
decrease is 74 ppm of the residues on foliage. With reference to Fig. I, the 74--ppm
residue level would exist on the third day after spraying and would be the safe reentry
time if workers were to be in the fields for one day only. The workers in this study
harvested crops over a five--day period. In order for them to sustain no more than
a 30 percent loss in enzyme activity during this period, the total consecutive five--day
exposure to available residues must not exceed 74 ppm and the safe reentry time must
be determined as the length of time after spraying until the first day of the five--day
period.

From the integrated expression for a first order loss of pesticide residues:

P = Po lO-kt/2.303 (I)

[where P, Po, and k are the pesticide chemical level at time t, the initial (deposit)
level of pesticide chemical (in this study, as determined by extrapolation), and the
specific rate constant, respectively], it can be shown that the value of each day's residue
level is 10-k/2.303 times that of the previous day. Thus, for a consecutive five--day
exposure to a total of 74 ppm of residues, and with PE equal to the residue level on
the day of reentry:

PE + PE (10-k/2.303) + PE (t0-k/2"303) 2 + PE (10-k/2"303) 3 +

178 William F. Serat

PE (10-k/2"303) 4 = P = 74
(II)
or
PE (i + 10-k/2.3o3 + 10-2k/2.303 + 10-3k/2.30a + 10-4k/2.303) = p = 74

With k - 7 . 1 6 x 102 days -1, PE is calculated as 17 ppm for the day of reentry.
From Fig. l or by calculation from the expression for first order loss, the time of
safe reentry becomes 23Vz days after pesticide application.

In the above example, the use of Fig. 1 and Fig. 2 is necessary in order to establish
rate constants and is convenient in estimating total residue levels corresponding to given
losses in enzyme activity. However, the expressions from which Fig. 1 and Fig. 2
are derived can be used to calculate the reentry time. Once the specific rate constants
for loss of pesticide residue and physiological function are established, then it is only
necessary to know the initial (deposit) level of the pesticide chemical, Po, in order
to calculate the reentry time. The calculations, as well as the data plotted in Fig. 2,
assume the same daily exposure time for each of the days in the field.

From the integrated expression for the first order loss of enzyme activity:

2.303 E2 _ p
- k ' log Eo

[where Ec~ is the enzyme activity before exposure (P = 0) and Ez is the activity at
some value for P 4= 0].

By combination with equation II:

2,303
- k ' log E2 = (1 + 10-k/2-303 + 10-2k/2.303 + 10-3k/2.303 + 10-4/2.303) PE

Since, by equation I, PE can be related to a time period required for its appearance:

E2 (III)
2.303 log ~ o

- k ' (1 + 1 0 - k / 2 . 3 0 3 + 10--2k/2.303 + 10-3k/2-303 + 10-4k/2-303, = Po 10-kt/2"303

Thus the reentry time, t, can be calculated for the first of the five--day sequence
in which workers can enter the fields to sustain some permissible cholinesterase loss.
Applying equation IH to the data above, the safety reentry time becomes 23.4 days.
Discussion
In establishing safe reentry times into pesticide--treated fields, the goal is to limit
 Calculation of Reentry Time Into Pesticide-Treated Orchard 179

exposure to pesticide chemical residues beneath that found to cause a detrimental

effect. In attempting to do this, two points are implicit: 1) the detrimental effect must
be known to be a function of exposure and 2) if any quantitative means are to be
established in determining such reentry, some pertinent physiological function (other
than the detrimental effect which is known to respond in a predictable way to total
pesticide chemical exposure) must be available and it must be sensitive enough for
measurement. The detrimental effect upon overexposure to many pesticides (such as
organophosphates) is well characterized and it is attributable to the inhibition o f
acetylcholinesterase in the central nervous system.

As mentioned before, the only measurable physiological function now known to

vary with exposure to certain pesticide chemicals, in a quantitative way, is the activity
of blood cholinesterases. Inherent difficulties in the methodology of obtaining blood
samples (from workers in the field) and determining the activity of the blood enzymes,
under well controlled conditions, are apparent to anyone who has attempted, with
adequate forethought, a field study on the effects of pesticides on humans. The need
is acute for at least one other measurable physiological function whose relative change
is dependent on pesticide chemical exposure. Such a function, in its own right, need
not depict actual harm. The quantitative excretion of a pesticide chemical or its metab-
olites represent one example.

When change of blood cholinestrase activity is used as an index of exposure,

some safe limit on the relative change allowed must be established. In this paper, that
limit is 30 percent, chosen as being reasonable by a concensus of opinions. Care must
be exercised in differentiating between allowable decreases in cholinesterase activity
in individuals and for groups because limits for the latter, based on averages, could
mask values which imperil individuals. For the purpose of illustrating quantitative
methods, the philosophy of the choice of limits is not relevant.

In field studies conducted to gain sufficient information for setting safe reentry, times
into pesticide--treated orchards, one must remember that the rate constants for pesticide
residue dissipation and, thus, the decrease in cholinesterase activity pertain to the condi-
tions o f the study. Different crops, climatological conditions, protection afforded, etc.
are tully expected to affect the results.

Equation 111 was derived on the basis of first order decrease in both pesticide residue
level and enzyme activity. Had other kinetics been found, from which the rate constants
could be derived in terms of measurable quantities, probably other expressions could
have been derived for calculating the safe reentry time.

The pesticide deposit (residue) level for the day of application, in this study, was
high (92 ppm). The particular pesticide applied is usually used in o n e - - t h i r d to
one--fourth the amount used here, Equation II1 readily allows the determination of
t80 William F. Serat

safe reentry times for other levels of initial deposit. For example, had that level been
20 ppm, the safe reentry time would have been 2 days as the first of a five--con-
secutive-day working time. Similarly, the reentry time can be calculated tbr differing
work regimens, allowable cholinesterase activity decreases, and rates of pesticide chemi-
cal residue or enzyme--activity decreases. These variables could be plotted in a nomo-
graph for the rapid setting of safe reentry times.

In developing the expression for safe reentry time based on sequential exposures,
the linear first order relationships lead to the assumption that the decrease in cholinester-
ase activity is dependent on the total dose of pesticide chemical received by a worker,
regardless of dose rate. This assumption is questionable in the light of rapid changes
in pesticide chemical residue levels in vivo, and in an expected partial recovery in
enzyme activity between exposures, It is expected that a certain total dose received
once would be more detrimental than if it were received in increments over a few
days. Grob and Harvey (1949) show, however, that, fbr administered doses of TEPP,
the loss in red blood cell chotinesterase activity is dependent on the total dose whether
given at one time or sequentially. Since there is a lack of clarification on these issues,
it seems more hazardous to select, by the method of calculation reported above, a safe
reentry time for one or two working days than lbr longer times.

The data obtained in this study are all based on an eight--hour work day each of
the days of harvest. Since the rate constant for enzyme activity decrease is a function
of total exposure, the results are expected to vary with exposure time during the day.
It is necessary to measure the enzyme activity decrease under other daily exposure con-
ditions (maintaining constant exposure time per day) but, considering the difficulties
in performing field tests, it is more reasonable to conduct them on the basis of an
eight--hour work day. A safe reentry time calculated for this period obviously offers
a safety factor should shorter daily working times be used.

Acknowledgement

The study was supported by the Environmental Protection Agency Contract No.
68--03--0096, for the analytical work described.

References

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 Calculation of Reentry Time Into Pesticide-Treated Orchard 181

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in man, and its use in the treatment of myasthenia gravis. Johns Hopkins Hosp.
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Grob, D., and A. M. Harvey: Effects in man of the anticholinesterase compound sarin
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Linskens, H. F., W. Heinen, and A. L. Stoffers: Cuticula of leaves and the residue
problem. Residue Reviews 8, 136 (1965).
Main, A. R.: Affinity and phosphorylation constants for the inhibition of esterases by
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Manuscript received August 16, 1972; accepted January 17, 1973