EPA-600/2-77-219
November 1977
EVALUATION OF "CAM-1," A WARNING DEVICE FOR
ORGANOPHOSPHATE HAZARDOUS MATERIAL SPILLS
by
Louis H. Goodson
William B. Jacobs
Midwest Research Institute
Kansas City, Missouri 64110
Contract No. 68-03-0299
Project Officer
John E. Brugger
Oil & Hazardous Material Spills Branch
Industrial Environmental Research Laboratory - Cincinnati
Edison, New Jersey 08817
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Industrial Environmental Research
Laboratory-Cincinnati, 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.. Environmental Protection Agency,
nor does mention of trade names or commercial products constitute endorsement
or recommendation for use.
ii
-------
FOREWORD
When energy and material resources are extracted, processed, converted, and
used, the related pollutional impacts on our environment and even on our health
often require that new and increasingly more efficient pollution control methods
be used. The Industrial Environmental Research Laboratory - Cincinnati (1ERL-
Ci) assists in developing and demonstrating new and improved methodologies that
will meet these needs both efficiently and economically.
This report describes the performance of the Cholinesterase Antagonist Monitor
(CAM-1) for the detection of toxic and subtoxic levels of organophosphate and
carbamate pesticides in water supplies on a real time basis. CAM-1 is intended
to be used in the laboratory environment for monitoring raw water supplies en-
tering a water treatment facility and checking effluents from pesticide manu-
facturing plants or related applications. It has the capability to warn of
pesticide spills in time to permit corrective action to be taken. Informa-
tion on this subject beyond that supplied here may be obtained from the Oil
and Hazardous Materials Spills Branch (IERL), Edison, New Jersey 08817.
David G. Stephan
Director
Industrial Environmental Research Laboratory
Cincinnati
iii
-------
ABSTRACT
The Cholinesterase Antagonist Monitor (CAM-1) uses a pad of immobilized cholin-
esterase for the collection and detection of pesticides in water. A 3-min de-
tection cycle provides an electrical readout proportional to the activity of
the enzyme before and after its exposure to the pesticide sample. CAM-1 has
been operated with water containing a variety of pollutants including organo-
phosphates, carbamates, chlorinated hydrocarbons, and various other economic
poisons; its sensitivity to these materials has been measured. With few ex-
ceptions, only the organophosphates and carbamates, which are nonreversible
inactivators of cholinesterase, are detectable with CAM-1. One of these ex-
ceptions is zinc at 10 ppm, which inactivates cholinesterase and behaves in
CAM-1 like the organophosphates. However, the zinc interference can be re-
moved or taken into account. Another compound detectable under certain con-
ditions is the reversible cholinesterase antagonist, tributylamine hydrochlo-
ride; it is detectable for only one or possibly two detection cycles when a
sudden increase in the concentration of the reversible inhibitor occurs.
The nonreversible enzyme inhibitors, on the other hand, produce repeated vol-
tage increases until the enzyme in CAM-1 is completely inactivated. CAM-1
is especially suitable for the detection of nonreversible inhibitors.
Correlation of the sensitivity of CAM-1 with the chemical structures of a
group of organophosphate (pesticides has shown that CAM-1 is generally more
sensitive for the phosphate (-0-P=0) compounds than for the phosphorothioates
(-0-P=S) or the phosphorodithioate (-S-P=S) compounds even though the animal
toxicities of these different types of compounds may be very close. A
technique for the conversion of the -0-P=S and -S-P=S moieties into the corres-
i i i
ponding -0-P=0 form is proposed as a method to increase the sensitivity of
CAM-1 to these particular types of compounds but this has not yet been studied.
A portable device using the CAM-1 principle could be fabricated and used to
locate spills, follow the spill plume, and to test treated water for its
freedom of insecticides.
Operation of CAM-1 in salt water (3% NaCl) changes the voltage baseline (regis-
tered on the digital voltmeter and also recorded) but it does not change the
sensitivity of CAM-1 for compounds like DDVP (dimethyl-dichlorovinyl-phosphate);
thus CAM-1 is suitable for responding to cholinesterase inhibitors in either
sea or brackish waters. CAM-1 has much promise for assessing the quality of
water supplies and of plant effluents that may have been contaminated by spills
or even chronic discharges of cholinesterase inhibitors, but obviously, prior
to putting CAM-1 into regular service, more extensive testing is advisable
under the conditions of intended usage.
iv
-------
CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Abbreviations viii
Acknowledgments ix
I. Introduction 1
II. Conclusions 7
III. Recommendations 9
IV. Enzyme Pad Preparation Procedure 11
V. Buffer and Substrate Studies 14
VI. CAM-1 Operation and Performance Data 19
VII. Effect of Temperature on Sensitivity of CAM-1 22
VIII. Response of CAM-1 to Organophosphates and Carbamates 25
IX. Correlation of Pesticide Detectability With Pesticide
Toxicity 31
X. Response of CAM-1 to Reversible Cholinesterase Inhibitors 34
XI. Studies of CAM-1 With Other Economic Poisons and
Dissolved Salts 36
References 39
Appendix A 40
Appendix B 41
v
-------
LIST OF FIGURES
Number
1 The Cholinesterase Antagonist Monitor, CAM-1, With
Cover Removed
2 Cross Section of Electrochemical Enzyme Cell Showing
Connections for the Constant Current Supply and the
Electrometer
3 Response of the Elctrochemical Cell Operating on the
3-Min Cycle to Water Containing 0.2 ppm DDVP 5
4 Enzyme Pad Cutter 13
5 Comparison of Toxicity and Detectability of Parathion
and Paraoxon 32
vi
-------
LIST OF TABLES
Number Page
1 Record of CAM-1 Operation Showing Substrate Failure
on 8-7-73 and Effect of Buffer Purification 17
2 Response of CAM-1 on Repeated^Challenge with Water
Containing 2.5 ppm Diazinon.' 21
3 Effect of Water Temperature on the Sensitivity of
CAM-1 to DDVP 23
4 Correlation of Organophosphate Insecticide Structures
and Detectability With CAM-1 26
5 Representative Carbamates Detected by CAM-1 30
6 Response of CAM-1 to Reversible Cholinesterase
Inhibitors 35
7 Effect of Inorganic Salts on Performance of CAM-1 37
vii
-------
LIST OF ABBREVIATIONS
BuSChI -- Butyrylthiocholine iodide
CAM-1 -- Acronym for Cholinesterase Antagonist Monitor
ChE -- Horse serum cholinesterase
DDVP -- 0,0-Dimethyl 2,2-dichlorovinyl phosphate
HSChI -- Thiocholine iodide
HEPES -- N-Hydroxyethylpiperazine-N'-2-ethane sulfonic acid
THAM -- Acronym for tris(hydroxymethyl)amino methane
Tris -- THAM (see above)
viii
-------
ACKNOWLEDGMENTS
The work upon which this publication is based was performed pursuant to Con-
tracts Nos. 68-01-0038 and 68-03-0299 with the Environmental Protection Agency
and describes the work on Task I for the latter contract. Task II on this con-
tract, "Construction of a Portable Enzyme Detection Apparatus," is in progress;
Task III, "Alternative Enzyme Systems for Use in CAM-1" is authorized and is
expected to be funded.
The authors wish to thank Mr. Lorren Kurtz, Mrs. Margo Rogers, and Miss Sandra
Puent of Midwest Research Institute for their technical assistance. Also we
wish to thank Dr. Thomas Hoover of EPA's Southeast Water Quality Laboratory,
Athens, Georgia, and Dr. John E. Brugger of EPA's Industrial Environmental Re-
search Laboratory - Cincinnati, Edison, New Jersey, for their technical assis-
tance and encouragement. The generosity of the pesticide manufacturers in
supplying samples for this study is appreciated.
IX
-------
SECTION I
INTRODUCTION
At the 1972 National Conference on Control of Hazardous Material Spills, MRI
reported the development of a rapid detection system for organophosphates in
water. A subsequent report, essentially on the material contained in this
n
document, was presented at the 1974 Conference. The apparatus using this
system (Figure 1) was identified as the Cholinesterase Antagonist Monitor
(CAM-1) since it uses cholinesterase for collection, concentration, and de-
tection of cholinesterase inhibitors present in water. At that time, this
use of immobilized cholinesterase for automatic detection of organophosphates
and carbamates in water was new. The final report of MRI's initial contract
with EPA contained the data in Reference 1 plus some additional details of
construction of CAM-1 and its performance; the abstract from this document is
reproduced as Appendix A of this report.
The objective of Task I was to determine the sensitivity of the CAM-1 instru-
ment for specific pesticides including not only the organophosphates and car-
bamates but also the chlorinated hydrocarbons and various potential interfering
substances. The studies were also intended to answer questions about the rela-
tive response of CAM-1 to reversible inhibitors vs nonreversible inhibitors.
In addition, this contract provided the experience with an immobilized enzyme
detection system necessary to prove its reliability and suitability for use in
both laboratory and field applications. As shown in this report, CAM-1 does
have application for the rapid detection of toxic or subtoxic levels of certain
pesticides in water.
Work on Task II is in progress; no work has yet been done on Task III on this
contract since it has not been funded. Task II is concerned with the fabrica-
tion of a portable model of CAM-1 and Task III is concerned with the use of a
CAM-1 type apparatus with another enzyme system.
The gel-entrapped cholinesterase product used in the CAM-1 instrument for the
detection of cholinesterase antagonists has already been described in the
literature. This product was prepared by coprecipitation of cholinesterase
with aluminum hydroxide gel followed by entrapment in starch gel on the surface
of open-pore polyurethane foam. The resulting sheets of treated foam were cut
into pads which were 1/4 in. thick and 3/8 in. diameter so that they would fit
into the CAM-1 electrochemical cell. These pads retain a major portion of their
-------
0)
I
0)
s:
O
O
4-1
•H
a
o
4-1
c
o
00
CO
4J
c
-------
activity after having been washed with water at 1,000 ml/min for 24 hr provided
that the water temperature is 25°C or below and provided enzyme inhibitors are
absent. Pads may also be used at higher water temperatures but their useful
life is thereby reduced. A detailed procedure for the preparation of these
enzyme pads is given elsewhere in this report.
Very briefly, the operating principle for CAM-1 is as follows: an electro-
chemical process is used for the automatic determination of the activity of
the enzyme pad once during each 3-min cycle. As may be seen from the cross
section of the electrochemical enzyme cell shown in Figure 2, the porous
enzyme pad is located betwen two porous platinum electrodes. First the water
sample is pumped through the enzyme pad at about 1,000 ml/min for 2 min during
which time a portion of the enzyme inhibitor in the water combines with the
active sites on the enzyme to reduce its enzyme activity. At the end of the
2-min water sampling period, the water is turned off and air is blown through
the enzyme pad to displace the residual water. Next, a solution of substrate
consisting of butyryl thiocholine iodide in Tris buffer is pumped through the
enzyme pad at the rate of 1 ml/min for a period of 1 min; during the last
two-thirds of this substrate pumping cycle a constant current of 2 uA is ap-
plied to the platinum electrodes so that the lower electrode is positive
(anode) and the upper electrode is negative (cathode). In the absence of
enzyme inhibitors, the cholinesterase (ChE) hydrolyzes the substrate to give
thiocholine iodide, which possesses a thiol group that gives characteristic
low voltages in the electrochemical cell.
electrochemical »oxidation
(CH3) 1j-CH2CH2-S-S-CH2CH2N(CH3) 21"
On the other hand, in the case of total enzyme inhibition, substrate is not
hydrolyzed; there is no thiol formation and the cell voltage rises about 200 mV
(from about 200 mV to 400 mV). Thus, a low cell voltage is indicative of the
absence of enzyme inhibitors, while an increase in cell voltage means that all
or part of the enzyme activity has been removed by an inhibitor from the water
sampled. Per cycle, increasing cell voltage means increasing inhibition.
The operation of CAM-1 is further illustrated by considering an example in
which water containing 0.2 ppm of DDVP (dimethyl-dichlorovinyl-phosphate) is
detected. The voltage tracing shown in Figure 3 was generated by applying a
constant current to the enzyme pad once each cycle. The difference in voltage
peak heights from cycle to cycle is used to trigger an alarm upon reaction of
-------
cu
.c
f-l
o
CO
d
o
O
^ 4J
N O
C
-------
•o
hr
o
CO
-10
*
u
c
•1-1
e
CO
Cl
o
00
c;
•H
U
0)
Cu
O
0)
o
ctf
o
•H
e
•u O
o o
" g
01 D-
o.
01
J2 cvj
4-)
O
M-l
O 00
Ci
O) ft
03 d
c -^
o rt
CU 4J
to a
a) o
P4 U
CO
01
M
3
00
•r-l
-------
a cholinesterase inhibitor such as DDVP. (Note: Only the peaks are of in-
terest. The decrease to the "resting" value of ca. -50 mV is not of impor-
tance in this discussion.) As shown in Figure 3, during the first 24 min,
there is a very slow steady voltage rise from cycle to cycle, indicating the
gradual deterioration of the immobilized enzyme pad. The alarm level is set
so that these changes are too small to trigger an alarm. However, when 0.2
ppm of DDVP is added to tap water, a sharp increase in the height of the vol-
tage peaks occurs. When the alarm threshold (or sensitivity adjustment) is
set at 10 mV, then an alarm will be sounded each time the voltage increases
by 10 mV or more between cycles. In the present example, 10 individual alarms
from the same enzyme pad resulted from sampling the DDVP. If a higher concen-
tration of DDVP--perhaps 2 ppm--had been used, the cycle-to-cycle voltage in-
creases would have been much greater.
Enzyme pads cannot be used indefinitely. After a time, when there is insuf-
ficient enzyme activity on the enzyme pad to allow a 50 mV voltage rise when
inhibitors are sampled, the used enzyme pad is rejected as a safety factor
and a new pad is automatically inserted into the system by means of an enzyme-
pad-changing mechanism built into the electrochemical cell. The system holds
a total of 11 pads.
-------
SECTION I
CONCLUSIONS
1. CAM-1 is essentially specific for organophosphate and carbamate insecti-
cides and is able to detect spills of these hazardous materials at subtoxic
or toxic levels within 3 min. CAM-1 can also be used to determine insecti-
cide levels in continuous discharges such as manufacturing plant effluents.
2. Compounds with high affinity for cholinesterase are detectable at low con-
centrations, whereas compounds with lesser affinities are detectable at
higher concentrations.
3. The sensitivity of CAM-1 to organic phosphates (-O-P=O) is often great-
er than to phosphorothioates (-O-P=S) and phosphorodithioates (-S-P=S),
4. CAM-1 at ambient temperature (20-25°C) responds to organic phosphate and
carbamate pesticides in water at temperatures ranging from 5° to 35°C. At
elevated temperatures, the CAM-1 cell voltages--a measure of enzyme activ-
ity—observed on the digital voltmeter are lower due to increased conduc-
tivity of warm water. DDVP (0,0-dimethyl 2,2-dichlorovinyl phosphate, acute
oral LDrQ to rats 56-80 mg/kg) can be detected at 1.0 ppm at 5°C and at 0.2
ppm at 25°C. Sensitivities for other pesticides were shown to exhibit simi-
lar changes with temperature.
5. Mixtures of two different pesticides each at one-half of its detectable
concentration became detectable indicating the additivity of the response.
6. Operation of CAM-1 on raw Missouri River water, as it was entering the
Kansas City, Missouri Municipal Water Plant, showed that there were no
detectable quantities of organophosphates or carbamates in the water supply
during a day of monitoring. Also, no interferences from species in the
raw water to the electrochemical cell were noted.
7. The aluminum hydroxide/starch gel-entrapped cholinsterase on open-pore
polyurethane foam products (referred to in this report as "enzyme pads")
were made by different laboratory technicians working only with written
instructions. The resulting pads functioned satisfactorily in CAM-1. In
the present experiments, these pads kept CAM-1 operational for periods
ranging from a few min to 24 hr (tests were limited to 24 hr), depending
-------
upon the water temperature anH the level of cholinesterase inhibitors
present. In earlier tests, single enzyme pads lasted as long as 56 hr
when used with tap water.
8. The butyrylthiocholine iodide used as the substrate was dissolved in Tris
buffer, pH 704. When compounded with commercial Tris buffer from different
suppliers, the substrate solution had only a useful life ranging from 6 to
12 hr. This useful life could be extended to 96 hr by the simple expedient
of treating the Tris buffer with activated charcoal within a day or two
prior to the preparation of the substrate solution.
9. A variety of possible interfering substances was tested to see whether
they behaved like the organophosphate and carbamate cholinesterase inhibi-
tors. The substances tested included other insecticides, herbicides, mo-
luscicides, nematocides, and fungicides. None of these affected the CAM-1
baseline voltage sufficiently to produce an alarm.
10. Studies of the effect of inorganic salts were also conducted. In the case
of zinc salts, there was nonreversible inhibition of cholinesterase and an
alarm signaled at 10 ppm and greater concentrations. Although the presence
of 3% salt (NaCl) lowered the cell voltages (because conductivity of the
cell increased while the cell current was maintained at a constant value,
namely, 2 uA) it did not change the sensitivity of the instrument to DDVP.
This indicates that CAM-1 could be used to assess the level of organophos-
phate insecticides in brackish waters.
11. The present experiments with CAM-1 have demonstrated the feasibility of
using immobilized cholinesterase to detect toxic levels of pesticides in
water and suggest the need for a more portable, battery operated system
for use at spills.
12. During this study CAM-1 instruments were operated for ~ 2,500 hr. There
were no breakdowns or significant maintenance problems. These units even
performed satisfactorily after shipment by air freight for four 1,000-mile
trips.
Note: In the text—to conserve space and avoid repetition--organo-
phosphate and carbamate pesticides (insecticides) are frequently
referred to collectively as "organophosphates". Further, unless the
text or context indicates otherwise, the specific term "organo-
phosphates" includes organic phosphates (-O-^=O), organic phosphoro-
thioates (-O-P=S), and organic phosphorodithioates (-S-P=S).
-------
SECTION III
RECOMMENDATIONS
On the basis of data reported in this document, CAM-1 has been found suitable
for assessing the quality of water to determine whether the water has been
accidentally contaminated by organophosphate or carbamate insecticides. Use
of CAM-1 is therefore recommended for checking water suspected of having been
contaminated by spills of insecticides, as well as by the effluent from insecti-
cide manufacturing operations, and by agricultural run-off, chronic discharges,
etc. It has been shown that the sensitivity of CAM-1 varies with the individual
organophosphate or carbamate; however, in each case the pesticides have been
detected in water at both subtoxic and toxic levels so that water users may be
protected from receiving an acute lethal dose of these pesticides.
Because field use of CAM-1 has been limited, it is recommended that CAM-1 be
subjected to field testing under the conditions of anticipated use prior to
putting it into regular service at a particular location.
In its present configuration, CAM-1 is not readily usable where electrical ser-
vice is absent. MRI has recommended that a battery-operated model of CAM-1
be fabricated and field tested. This lightweight, portable device would be
specifically designed to give rapid, in-field indications of cholinesterase
inhibitors in water, to trace the contaminated plume of spilled organophosphate
as it moved downstream, and to ascertain the level of organophosphates in the
effluent from processing equipment that has been employed to strip the spilled
pollutant from water.
Since horse serum cholinesterase has a lower affinity for those organophosphate
pesticides which possess the phosphorothioate (-S-^S) structures than for
-0-P=0 structures, it is recommended that further studies be conducted to opti-
mize the sensitivity of 'CAM-1 to these -S-P=S materials. Several approaches
to this problem which should be investigated include the following: (1) use
of an oxidant in the pesticide solution, (2) pass the pesticide solution over
an immobilized oxidase enzyme, (3) pass the pesticide solution over a solid
oxidizer, or (4) find an enzyme which is more readily inhibited by these phos-
phorous-sulfur compounds and yet retain sensitivity to the -0-P=0 compounds.
Eel cholinesterase (E.G. No. 3.1.1.7) is known to be more readily inhibited
by some organophosphates than horse serum cholinesterase; for this reason,
-------
eel cholinesterase is judged to be one of those enzymes that should be inves-
tigated as a method for obtaining an increase in sensitivity of CAM-1.
Finally, since the basic CAM-1 concept may be useful in detecting other pollu-
tants by using a different enzyme/substrate system, it is recommended that
other specific enzyme systems can be studied. For example, it may be possible to
utilize the pyruvate dehydrogenase/pyruvate/lipoic acid system in CAM-1 for
the detection of pyruvate dehydrogenase inhibitors; in this system the -S-S-
grouping of lipoic acid is reduced by pyruvate to -SH,-SH and this should be
detectable electrochemically.
10
-------
SECTION IV
ENZYME PAD PREPARATION PROCEDURE
Care must be taken in the preparation of the enzyme pad for use in CAM-1 since
the sensitivity of CAM-1 and the repeatability of the tests are a function of
the uniformity of the enzyme pads. Timing and manual dexterity are important
procedural factors in the preparation of the pads. It is suggested that the
individual selected to prepare the pads should practice the handling of the
starch and its application to urethane foam as mentioned below, but with a
water-soluble dye substituted for the enzyme. In this way, it will be possible
to anticipate changes in starch viscosity and to check out the procedure for
uniform distribution of the starch applied to the foam. After gelling and
drying, the starch-coated foam should then be cut into pads, since starch pads
are also needed for the determination of CAM-1's alarm potential at the be-
ginning and ending of each days tests.
(The alarm potential is the voltage increase which occurs when all of the
enzyme on the pad is inhibited or if the enzyme pad is replaced with a "starch
pad" to which no enzyme has been applied. Daily checks of the alarm potential
are suggested as a means to prove that the instrument is working.)
The following materials were used: open-pore polyurethane foam sheets, 44 to
45 pores per linear in. (ppi) x 1/4 in., Scott Industrial Foam, Scott Paper
Company, Chester, Pennsylvania; partially hydrolyzed potato starch recommended
for gel electrophoresis, Connaught Medical Research Laboratory, Toronto, Canada;
Chlorhydrol (aluminum chlorhydroxide complex, 50% w/w solution), Reheis Chem-
ical Company, Chicago, Illinois; horse serum cholinesterase, Sigma Chemical
Company, Type IV, approximately 15 uM units/mg; and Tris buffer, "THAM~ ,"
Fisher Scientific.
Step 1. A solution of Tris buffer, 0.08 M, was prepared by dissolution
of 9.7 g of tris(hydroxymethyl)amino methane in 900 ml of water,
adjustment of the pH to 7.4 with concentrated HCl and then ad-
justment of the volume to 1 liter.
Step 2. Forty milligrams of horse serum cholinesterase were dissolved
in 6 ml of Tris buffer. To this solution was added with mechan-
ical stirring 4 ml of a dilute solution of aluminum chlorhydroxide
/3\
complex (0.5 g of Chlorhydrol in 4 ml of water). At this point
11
-------
the aluminum hydroxide gel precipitates and adsorbs the enzyme
from the solution (check pH and adjust to 7.4 to ensure that
the precipitation is complete). This suspension is set aside
at ambient temperature until needed in Step 3.
Step 3. Two grams of potato starch were suspended in 10 ml of cool Tris
buffer and added to 30 ml of boiling Tris buffer and heated
until the suspension cleared. Care was taken to avoid the for-
mation of scum or lumps (start Step 3 over if lumps are obtained)
and the starch slurry was stirred with a magnetic stirrer while
it cooled slowly to 45°C. At this point the aluminum hydroxide
gel-entrapped cholinesterase (Step 2) was added, all at once and
quickly mixed. (Note: Step 4 and its three replications must
be done quickly before the starch gels.)
Step 4. A 10-ml portion of the warm starch gel slurry from Step 3 was
deposited on a pre-cut sheet of open-pore urethane foam (4x6
x 1/4 in. sheet) lying on a warmed glass or plastic surface
(usually over a pan of warm water). The starch-enzyme material
was now distributed throughout the sheet as uniformly as possi-
ble with a plastic rolling pin filled with warm water; the sheet
was rolled in all directions and turned over several times. In
this same manner, three other 10-ml portions of the starch-enzyme
product were distributed over three additional urethane foam
sheets.
Step 5. The coated sheets were placed on edge in a wooden rack (made
with dowel rods), dried at least an hour at room temperature and
finally dried overnight in an oven at 37°C.
Step 6. The resulting sheets were examined carefully to ensure that all
areas of all pads were evenly coated. Poorly-coated areas were
trimmed away. The sheets were handled carefully to avoid breakage
of the starch film on the dried sheets. The enzyme pads were
next cut into 3/8-in. pads with a stainless steel cutter (Figure
4) mounted in an electric drill press operated at about 600 rpm.
The procedure yielded approximately 270 enzyme pads, which pos-
sessed an average activity of 0.045 umoles/min/pad (analysis
based on the rate of hydrolysis of butyrylthiocholine iodide
and was measured by a modification of the Ellman Procedure).
12
-------
Figure 4. Enzyme Pad Cutter
13
-------
SECTION V
BUFFER AND SUBSTRATE STUDIES
During the initial laboratory studies with CAM-1, it was observed that there
was a deterioration of performance of the instrument when it was operated for
6 to 10 hr at a time. However, the performance appeared perfectly normal on
subsequent days when the instrument was recharged with fresh substrate solu-
tion and a new enzyme pad. The observed deterioration of performance took the
form of decreased starch pad and alarm potentials at the end of 8 hr of oper-
ation. At the start of one run, the initial voltage with a starch pad was
400 mV, the enzyme pad potential was 120 mV, and the alarm potential was
280 mV. After 8 hr of detector operation, the enzyme pad potential was 220
mV, the starch pad potential was 220 mV, and the alarm potential was 0 mV--
obviously a dangerous situation since the sampling of an enzyme inhibitor at
this time would give no voltage increase and no alarm signal. The problem
was shown not to be related to the enzyme pad since insertion of a new pad
did not restore the alarm potential. However, replacement of the substrate
solution with freshly-prepared substrate solution restored the alarm potential
to useful levels. For this reason, laboratory investigations of the substrate
were undertaken.
One proposed explanation for the loss of alarm potential was that the sub-
strate solution had undergone spontaneous hydrolysis and that the thiocholine
iodide liberated by this process was keeping the starch pad voltage low. Two
experiments were conducted to see whether the useful life of the substrate
solution could be prolonged.
In one approach, a gentle stream of air was bubbled through the substrate
solution for 8 hr during its use with CAM-1; this aeration of the solution
was expected to oxidize the thiol as fast as it was formed by spontaneous
hydrolysis and to keep both the starch pad voltage and the alarm potentials
high. Aeration had no effect.
In the second approach, the substrate solution was cooled in ice with the
expectation that the spontaneous hydrolysis of the thioester would be retarded
at the lower temperature. Improvement in alarm potential during longer runs
was noted; but, still, both the starch pad voltage and the alarm potentials
decreased during the longer runs. (Note that some decrease in alarm potential
due to the loss of enzyme activity of the enzyme pad is normal but does not
lower the starch pad voltage or interfere with the operation of CAM-1 as now
programmed.)
14
-------
The third approach was based on the premise that the poor stability of the
substrate solution arose from the presence of an impurity in the substrate,
butyrylthiocholine iodide (BuSChI). A new sample of BuSChI was obtained from
a second supplier and tested in CAM-1; this substrate solution also deterior-
ated in 6 to 8 hr. One commercial lot of BuSChI was recrystallized from iso-
propanol, washed with isopropanol, and dried in a vacuum; the white, odorless,
crystalline product melted at 174°-176°C and was believed to be. very pure based
upon previous studies at MRI with this product, which included examination by
thin-layer chromatography. The recrystallized BuSChI performed as well as,
but no better than, the unrecrystallized BuSChI from which it had been pre-
pared.
The Tris buffer was next evaluated as a possible source of the problem; three
alternative buffers were investigated as replacements for the Tris buffer.
The buffers investigated were 0.1 M phosphate, Veronal (sodium barbital),
and N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, Calbiochem).
These buffers were adjusted to pH 7.4 and then used for dissolving the BuSChI
at a concentration of 2.5 x 10 M. Substitution of each of these substrate
solutions for the corresponding substrate solution made with 0.08 M Tris buffer,
pH 7.4, produced an operating detector system but resulted in the introduction
of other problems, particularly an increased variability of the cycle-to-cycle
pad voltages. For example, with the phosphate buffer and an enzyme pad, the
cell voltage started at 104 mV and increased 72 mV during the first hour.
With the HEPES buffer the enzyme pad voltage increased 72 mV in 45 min (with
Tris buffer the voltage usually rises < 20 mV/hr). With the Veronal buffer,
the buffer crystallized from the solution and plugged the substrate lines.
These buffers offered no improvement over the Tris buffer.
It was learned that substrate (i.e., BuSChI) dissolved in Tris buffer whidh
has been freshly-treated with activated petroleum-base charcoal gave satis-
factory CAM-1 performance for at least 79 hr.
Several possibilities for the troublesome instability of the substrate solu-
tion were considered: (1) there was microbial growth in the buffer, (2) the
dry buffer contained an impurity which caused the trouble, and (3) there was
a slow chemical reaction occurring in the buffer solution when it was stored
for a long period in air and exposed to light. The first explanation appeared
to be incorrect since signs of microbial growth were not observed. The second
explanation appeared to be wrong, since the initial treatment of the buffer
with charcoal should have removed the impurity. The possibility of a slow
(1 month or more) chemical reaction of the Tris buffer with air and light seems
to fit the present observations. Additional studies are needed to obtain a
more definite chemical explanation for the substrate instability observed in
the initial experiments.
15
-------
Data showing the reproducibility of CAM-1 performance from day to day and also
the solution to the substrate instability problem are given in Table 1. The
starch pad voltage, the enzyme voltage, and the alarm potentials both at the
start and at the conclusion of the test runs are given for each day. The
footnotes to the table give explanations for the meaning of the potentials
measured. At the start of each run the alarm potentials were all over 250 mV,
which is considered quite satisfactory. Comparison of the final starch pad
voltages reveals that there were two runs in which there was a significant
drop in potential after 8 to 8-1/2 hr of CAM-1 operation. In the first case,
there was a drop in starch pad voltage from 410 to 218 mV; in this test, the
charcoal treatment of the old Tris buffer solution within a few days of the
run had been omitted. In the second case, the starch pad voltage fell from
435 to 344 mV; in this case freshly-made Tris buffer was used but the charcoal
treatment was omitted. These experiments demonstrate that with some lots of
THAM buffer charcoal treatment prior to use in making the substrate solution
is essential to good CAM-1 operation. Even though the chemistry of the changes
occurring in the substrate-buffer solution is unknown, the changes may be de-
layed for a day or two by a simple charcoal treatment of the buffer prior to
the addition of the substrate.
Petroleum-based pelletized charcoal (MCB Co.) was used for the purification
of the buffer. In this process the weight of charcoal was 1 to 2 times the
weight of the Tris dissolved in the water and the mixture was stirred from
2 to 8 hr before filtering.
16
-------
§
M
H
U
M
fa
M
«
e
o
H
U
a
O
M
H
O
06
tJ
B B
N «
" |
/B
u
n «
4J ctf
cn C4
•U (U
en -u
QJ cfl
H Q
fMenenmencncocn
i i i i i i i i
cTtcocooocoooaooo
i i i i i i i i
r-4
U O O
o o o
O O 1 O O O H
I A «
to «ro
1 II
oicaocatacotoco
tu0c;a>aj ^> P^ ^> ^i ^» ^>
*O T3 T3 T3 *O 'O "O "O
oooooooo
-------
2
S
H
5
S
H
a
w
CXl
O
Q
OS
co
H
r-l
0)
y-i
u-t
5
c
0
•r4
4J
CO
H
S
r-l
CO
B
•r4
fu
4J
J-l
CO
4-J
C/J
01
a -o
>> CO
N 04
c
w
— 4 i — 1
CO CO
> 0
H 0
CJ U
4J CO
C J3
M U
4_J
r— 1 C
CO
oj B
S
>i •
N T3
C CO
Ed 04
SJ
t>
.C E
u
r-l
CO 13
4J CO
C/} fM
TJ
1
E i-f
h CS
CO -H
r-4 4-J
< C
OJ
o
04
0
OJ >
E B
ix
N «
c -a
W CO
04
.0
* 1
CJ
n
ctj 'O
4J CO
c-o &j
4-J CU
CO 4-1
CU CO
H Q
m m m en ft en
r-.. i i i iii
1 00 CO CO CO 00 00
r-l 1 | | III
r^.
CJ
0
m
CM 1
O O O O O 1
- 1
CO
>•>
cO
'O
in
co en co to co co o
cu cu cu a; cu cu c
r*1 F>> >1 ,>! >•* r*1
T3 T3 13 T3 *O ? £
rH rH rH rH <-l 0) 0)
o o o o o c c
m
r^ co oo oo oo co co
'•c en oo oo o CT\ cyi
CTI in ^o r-i m CM CM
CM CM r-l CM r-l CM r-|
r-. r^- VD r-^ o ^ in
CM r^ in co f*"* ^ *~*
r-| r-( CNI rH ^ CM CM
WD
en o CO
en CM rn en en CM CM
in r-* r*. r~^ o r*. m
o -j- rH t^ r-H en in
, i , | , | , — i , — i , — i
rH oo en CM o o~i m
en rH CM o> rH CM en
,
N
C
OJ
0
c
•^^
rC
CJ
r-l
CO
4-J
CO
CO
&
4-J
•r-l
S
13
(U
C
•r-|
CO
4-J
-JO
O
CU
60
CO
4J
rH
O
rH
CU
u
T3
to
CL
CU
e
>.
N
C
01
a
to
^
4-J
•H
&
13
CU
C
•rH
CO
4-1
rO
0
CJ
00
CO
4-J
rH
O
r-H
CU
U
in a pad is completely inhibited.
§
>,
N
c
cu
CU
_c
•u
c
cu
rC
s
13
CU
4J
U
CU
ex
X
cu
cu
CO
CO
cu
)-l
o
c
cu
60
CO
4J
0
>
cu
J^
J_l
to
•r-4
i — 1
CO
•H
4J
C
CU
4J
O
CL
e
rH
to
1
rH
rH
CO
E
rH
O
c
J3
CO
O)
VH
CO
CO
cu
00
CO
4-)
i— 1
o
>
"O
CO
CX
X
U
rH
CO
4-J
CO
01
CO
cu
fS
18
-------
SECTION VI
CAM-1 OPERATION AND PERFORMANCE DATA
The length of time that CAM-1 may be operated without servicing depends upon
several variables. The automatic pad changer has slots for only 11 enzyme
pads, but this situation is not expected to restrict the period of operation
between servicing periods, except in unusual circumstances where multiple
spills occur in a short period of time or when a high level of cholinesterase
inhibitor persists for a long period of time. The pads themselves possess
sufficient enzyme so that each will operate the detector for 24 to 48 hr so
long as nonreversible enzyme inhibitors are absent. (Enzyme is slowly washed
out, limiting the useful performance of a pad.) Under ideal conditions
(i.e., when no inhibitors are present), 11 enzyme pads should last 11 to 22
days. In the presence of highly-polluted water, the pads will not last so
long. The substrate used in this instrument is butyrylthiocholine iodide
(BuSChI) at a concentration of 2.5 x 10"4 M in 0.08 M Tris buffer, pH 7.4.
Unfortunately the substrate undergoes changes in solution and these spontan-
eous changes probably will limit the period of service-free operation. When
the substrate is prepared carefully in Tris buffer which has been freshly
treated with activated charcoal, and when the temperature is kept at 25°C or
below, the substrate will provide good alarm potentials for 48 to 96 hr. For
longer operation it might be better to refrigerate the substrate solution or
to devise an automatic solution maker that will add solid substrate to fresh
buffer at intervals of a day or less. In this way, operation for a week be-
tween service periods could be assured. In the laboratory, it was convenient
to prepare new substrate solution and add new enzyme pads daily.
The cost of the materials used for operation of the instrument is of interest.
The horse serum cholinesterase used for our laboratory studies cost $67.50
for 1,000 uM units; however, since each enzyme pad contains only 0.01 to 0.05
units/pad, it is clear that the enzyme for a single pad costs less than $0.01.
When labor for pad fabrication and analysis is included with the cost of all
materials, the enzyme pads might cost from $0.03 to $0.10 each depending on
the quantities prepared in a single batch. The ingredients for the substrate
solution are expected to cost no more than $0.20/day and most of this cost is
for the buffer.
19
-------
Preliminary field testing has been conducted with CAM-1 primarily to determine
the background response that might be encountered with raw water supplies.
In one case, water from a freshwater lake was monitored from a floating dock
extending 30 ft from the shore; there was no sign of enzyme inhibition and one
enzyme pad provided satisfactory baseline voltages for more than 8 hr. In
another test, CAM-1 was taken to the Kansas City Municipal Water Works where
it was used to monitor the incoming raw water for an 8-hr day. In this case,
there was no evidence of enzyme inhibitors entering the water supply and no
false alarms when the threshold sensitivity was set at 10 mV. The next step
in field testing will be to evaluate the performance of the system with ef-
fluents from pesticide manufacturing operations.
Studies on the repeatability of CAM-1's response to constant levels of pest-
icides in water have been made. These studies were primarily aimed at finding
out whether the sensitivity of the instrument changed with time.
In some of the initial studies with Baygon it was observed that 16 ppm was
detectable with fresh solutions whereas solutions five times as concentrated
were barely detectable after 24 hr. The loss in sensitivity was attributed
to the partial hydrolysis of the Baygon®.
In a 79-hr test of substrate stability, CAM-1 was exposed repeatedly to a
solution of 2.5 ppm of Diazinon®. Table 2 shows the changes in the electro-
chemical cell potentials at the end of each 3-min detection cycle.
From the data in Table 2, it is clear that there was either a slight decrease
in sensitivity as the substrate solution became old or that there was some
hydrolysis of the stock solution used in making the 19-liter batch of Diazinon®
test solution. The unusually large voltage changes occurring after the 5th
cycle in Test 3 and after the second cycle in Test 7 suggest that mixing of
the solution was inadequate; in subsequent experiments, the pesticide solutions
were mechanically stirred and there was greater uniformity of cycle-to-cycle
voltage changes. However, examination of the voltage changes for each test
makes it clear that enzyme inhibition occurred in all of the tests.
During the course of the study the CAM-1 instruments were operated for approxi-
mately 2,500 hr. Very few mechanical or electrical problems were encountered.
Occasionally it was necessary to "free" (manually turn) the water pump before
startup when the unit has been turned off for several days. The rubber shaft
seals on the water pumps had to be replaced once. In one of the CAM-1 units,
there was a failure of a digital in-line packaged integrated circuit (DIP 1C)
compatible relay and a new relay had only to be plugged in to restore the
normal detection cycle. On one occasion a fuse in the water pump circuit
blew; replacement with a "slow-blow" fuse put the system back in operation
and no additional troubles were observed. Changes in the wiring diagrams or
components were not required.
20
-------
w
o
2
fd
IH
Q N
W <£
H M
M
W O
4-1
O 0)
0) CO
CO O
PI P<
0 X
a. w
CO
jltage Re
\)1 lowing
Temperature
Detectable at
>
4J
CO
0)
H
0
Q
Q
m
CM
VO
m
sr
CO
CM
CO
1 — 1
o
CO CO CO rH rH
CM CM CM CM CM
01
fi
•rl
CO CO CO CO rH
0) 0) 0) 0) M
JH EH >-i JH 0)
O
pq
rH VO 1 CO 1
CM rH 1 m CM m
CO -Ct m CTi rH •<(•
* rH
CM 1
CM CT>
rH rH
CO Ol
1 1
vo r^
rH rH
rH
CM
Borderline
rH
rH
1
rH
O
rH
00
CM
00
rH
O
CO
rH
01
,c
4-1
o
4J
01
3
01
rH
CO
•H
rH
cu
o
c
CO
co
0>
0
o
d)
$
•H
3
T3
01
00
C
CO
O
00
CO
4J
>
CU
CO
o E
CO
CO CO
)-i
0> 0)
C J3
•H 4-1
CO
4J >-,
§rH,
4-1
O O
CO
0) K
& 01
4J
4J
4-1 CO
CO
,C CO
4-> >i
CO
4-1 !3
O rH
MH
4J
01 O
4-1
01
T3 r4
a oi
CO &
S O
U -H
N
C CO
•r-l -r-l
Q
a
•H ^3
4-1 4J
CO
CO 4-1
C 3
0) O
QJ n
0 -H
0 IS
CU "O
!-i C
3 co
temperal
er with
4J
m co
0 13
perature
b
0)
4-1
0)
rH
O
U
I
4J
01
CO
S
T3
rH
O
CO
01
S
CO
rH
CO
01
4J
CO
3
ca
co
21
-------
SECTION VII
EFFECT OF TEMPERATURE ON SENSITIVITY OF CAM-1
Since CAM-1 is expected to operate with water ranging in temperatures from
5° to 35°C, it is important to know how temperature affects the electrode
potentials and also the sensitivity of CAM-1 to various pesticides. It is
well known that water with dissolved salts is a better electrical conductor
when warm than when cold due to the greater mobility of ions in the warm
solution. In the same manner, the movement of ions through the enzyme pad
to the electrodes in contact with it is faster with warm water and slower with
cold water. As a result of this conductivity effect and of the constant-
current system design, higher cell voltages are obtained when the cell is cold
and lower cell voltages when the cell is warm. Within the normal operating
temperatures, these relationships are of no concern unless there is a sudden
temperature change of the water sampled; a decrease of 3°C in the water temp-
erature within one detection cycle (3 min) could result in a 10-mV increase
in baseline voltage and a false alarm, provided the alarm threshold was set
at 10 mV. Because of the high heat capacity of water, it is not expected that
rapid changes of water temperature will be a problem. Temperature compensa-
tion of the baseline voltage is possible but it is not included in the present
CAM-1.
When the CAM-1 instrument is operated at 25°C with water at 5°C, it is likely
that the temperature of the electrochemical cell during the last minute of
the detection cycle is significantly higher than 5° since the substrate solution
is also at 25°C.
Quite a different problem is the effect of temperature on the reaction of the
enzyme with the substrate and with the inhibitors. A thorough study of this
problem has not been made since classical biochemical investigations have
already established the mathematical relationships of temperature to enzyme
reactivity. However, limited studies have been made on the effect of tempera-
ture on the total CAM-1 system—that is, the sum total of all temperature
effects occurring in the total detection system. The kinds of temperature
effects to be seen are best illustrated by considering one specific example,
i.e., the effect of temperature on the sensitivity of CAM-1 to DDVP as shown
in Table 3,
22
-------
D
P
O
H
o
e
r-l
H
H
r-l
C/l
W
w
§
w
PS
w
H
Pi
U
H
H
U
CO
CU
CO
H
o
I-l
01
cu
3
CO <->
r4 X-N
CU O
Cu o
H
4-1
ttf r-l
CU
CU >
r-< CU
CO
4J 4-1
U CO
0) CU
4-1 H
P
>>
6
*
CU
I™-l
O
o
i>Ci
o
ca
r4
o
CO
cu
o
0
M
cu
w
cci
4J
r-l
0
>
m
^J-
CO
CN
1-1
CO
S F;
0 P
Q a
4-1
CO •
CU O
H •&
vo vo •<)• » r-» CM
CO CO vO O"* CO CO vO OO OO
CO CO f**» ON rH O"\ 00 !**• CN
i— ICMOOOcMvO
(U
C
•H
j
!-i
CU
•a
t-i
o
oo
m
vD
m
o\
r4
•
o
t— i
i— i
in
CO
CO
cu
^-1
O
r-l
m
r-l
r-4
m
oo
00
r-4
CS|
r-4
m
CO
CO
CU
^-{
00
oo
r-i
r-i
vO
r-(
r^
r-(
CM
O
CO
i— 1
in
CO
cu
ti
, i
(U
*T3
J-4
o
o
r-4
C7N
oo
t-t
r-l
O
r-l
r-l
•
O
P
P
g^°
m
&
*"O
cu
0
CO
4-1
0
O
O
4J
0
^
•r4
CU
a,
X
cu
CO
•H
4J
0
'
•a
CU
CO
3
®co co'
0 CU
O 4J
a, co
CO 42
> CL
CO
cu o
f! tf~{
H tt.
CO
rQ
e
CO
cu
r-l
CO
£
CU
ca
J3
0
CO
•i-l
0
0
•H
4J
•r4
.0
•H
rj-]
0
•r4
CU
e
^
N
0
cu
cu
^
»H
4-)
cn
t-i
3
g
3
U
4-1
CO
ca
0
co
cu
•1-1
r-i
}_i
cu
13
S-l
O
CQ
,0
>
e
m
0
CO
r!
4J
CU
}-j
CO
CU
60
CO
V-I
CU
^
CO
ca
cu
r-i
O
^*»
o
0
cu
CU
£
4-1
CU
JZ
CU
CO
CO
CU
^1
o
0
•rH
cu
60
CO
4-1
i — t
O
CU
,£
4-1
4J
CO
t~{
4J
•
U
o
m
CN
4J
CO
^i
(-1
O
4J
CO
}-t
o
^1
CO
r-l
0
•r4
CU
0
O
*o
cu
vi
cu
&
co
cu
•H
T3
3
4J
CO
CU
^)
3
4J
CO
CU
Q-
H
cu
4-1
^1
cu
4J
to
12
CJ
23
-------
Comparison of the cycle-to-cycle voltage increases produced by DDVP at 1 ppm
and at temperatures of 6°, 14°, 25°, and 35°C shows sharply increased voltage
changes at the higher temperature; this is attibuted to an increase in the
rate of enzyme inhibition at the higher temperatures. In Test No. 12, it
will be seen that large voltage changes were observed for the first three
detection cycles but that the magnitude of the changes decreased for subse-
quent cycles, even though DDVP at 1 ppm was still present for all cycles.
The explanation for this anomaly is that nearly all of the enzyme was inhibited
during the first three cycles and the cell was approaching the maximum voltage
which could be obtained when no enzyme is present. Under normal CAM-1 opera-
tion, the pad change threshold would have been set so that a new pad would
have been inserted automatically after the third detection cycle.
Based on studies with DDVP and also other suitable insecticides, it is con-
cluded that CAM-1 is about two times more sensitive at 25°C than it is at
5°C. However, even at the lower temperature, CAM-1 retains sufficient activity
to detect both toxic and subtoxic levels of most insecticides with anticholin-
esterase activity.
24
-------
SECTION VIII
RESPONSE OF CAM-1 TO ORGANOPHOSPHATES AND CARBAMATES
As mentioned earlier, the principal objective of the present study was to de-
termine the potential usefulness of CAM-1 for which the sensitive element is
immobilized cholinesterase. A previously uninvestigated area was whether the
sensitivity of CAM-1 for a specific insecticide is a function of the affinity
of cholinesterase for that insecticide. In other words, is it true that com-
pounds with a high affinity for cholinesterase are detectable at low concen-
trations, whereas, compounds with lesser affinities for cholinesterase are
detectable only at higher concentrations. CAM-1 has now been operated with
solutions or suspensions of about 40 economic poisons to determine CAM-1"s
ability to detect the poisons.
Table 4 presents the CAM-1 response data collected with a group of 22 organo-
phosphates. The data are arranged so that it is convenient to make correla-
tions between the structural characteristics of the pesticides and the minimum
detectable levels. From the table it is apparent that the greatest sensitivity
was obtained with paraoxon (detectable at 0.1 ppm) and the least sensitivity
(R)
to Di-Syston (detectable at 65 ppm). In general, CAM-1 exhibited greater
sensitivity for the phosphates and phosphonates (-0-PO-compounds) than for
the phosphorothioates and dithioates (-OPS- and -S-PS-compounds). The rela-
tive insolubility of these compounds makes it difficult to know in every case
that the enzyme inhibition was due to dissolved, rather than dispersed, pesti-
cide; for this reason, caution is urged in making comparisons of relative con-
centrations producing enzyme inhibition.
For these response studies, commercial grades of the named compounds were dis-
solved or suspended in city tap water at several concentrations and pumped
through CAM-1 at known temperatures. For some of the experimental evaluations,
the very insoluble materials were dissolved in alcohol and added slowly to a
stirred container of water. The resulting clear solutions, opalescent solu-
tions, or milky suspensions were then pumped through CAM-1 at about 1,000 ml/
min. The lowest concentration of organophosphate producing a cell voltage in-
crease of at least 10 mV/cycle was considered to be detectable at this level
and at the temperature of the test. After each test, the pesticide solutions
were hydrolyzed with caustic prior to their disposal. Tests with carbamates,
chlorinated hydrocarbons, and other pollutants were conducted in much the same
manner, although caustic could not be used to destroy the dilute chlorinated
hydrocarbon wastes.
25
-------
<
o
H
cj
u
H
U
O
Q
H
CO
u
Q
H
CJ
CO
•z,
w
H
1
O
H
a
o
o
,0
CO
H
\\/
EM
/\
f— t X
CO CO
O to 00
CJ -^
•U O 60
3 10 B
0 Q ^
< iJ
• O
H o
Q) 2
i-f CX
43 CX
CO
•U x
O r-4
01 111
0) HI
Q iJ
to
Pi
CM
,— t
1><
CM
K
r-t
OS
01
«j
•jr
C
g
Q
U
1— 1
CM
i— 1
CM
r-H
r-4
O
)
8
I O
Ljf
^SjX^
,_t
CJ
1
O
i
CO
o
CM
CJ
O
CM
O
1
O
4J
to
§0)
iw
«
en
II
Cj
;^1
to
a
i
o
1
C/3
i
O
10
CM
CJ
o
m
CM
CJ
4J
•H
C
to
CO
CO
Q
0
CO
vO
>n
in
CM
CM
O
1
Q
CJ
1— 1 I— t
O
i
O
i
O
CO
g
g
Q_,
Q
O
CM
CM
1
O
in
i— i
en
CM
CM
f— 1
\S^s*
l' 1!
8
/ V
co X co
a a
CJ CJ
o
1
CO
o
in
CM
CJ
0
in
a
CM
CJ
C
o
c
•H
N
CO
Q
O
in
CM
g
CM
ffl
CJ
1
CO
1
in
CM
u
CO
\
[
CO
o
in
a
CM
0
m
a
CM
CJ
C
o
w
CO
•rH
Q
C
o
u
26
-------
s
<
o
-O
ffl
H
ora
ity
rats
me/kg
6
CU
a
O i-l
HI HI
a ri
X
Crf
£9
o
IT
*CV
CJ
ba
°m
§
CO
o
33 33
a-o
I.
O-CJ-O
I
I—I
o
fa
o
33
CJ
g
g
0
•a
H
a
o
§
0)
o
en
D
o
§
o
g
•o
01
3
B
•H
.u
C
O
CJ
o
m
33
U
g
>,
J3
g
Parathion
27
-------
<
CJ
H
M
rJ
M
m
cj
H
O
SB
CJ
g
H
Ed
O
H
CJ
O
z
<
8
o
fr.
o
§
M
H
a
cA
o
CJ
4J X t,
3 O I
a a
—i a.
xi a.
O rH
CO D
4J >
-------
Table 5 shows the sensitivity of CAM-1 to seven carbamate pesticides.
Furadan was detectable at 0.75 pptn and Temik was detected at 0.5 ppm, which
indicates very strong anti-cholinesterase activity for these carbamates--approx-
imating the toxicity of the organophosphates with the P=0 linkage. The other
five carbamates tested in this study were detected at concentrations low
enough to protect personnel using the water supplies from acute toxic doses
of these carbamates.
The previous tests were conducted using water solutions of pure pesti-
cides. The addition effects of two or more pesticides were demonstrated
by challenging CAM-1 with a solution containing one-half the detectable
concentration of DDVP and one-half the detectable concentration of
paraoxon. The response of CAM-1 to the combination was equivalent to
challenges by detectable concentrations of either. Experiments using
other pairs of pesticides confirmed the additivity rule, as did one
experiment in which a combination of three pesticides was used:
parathion, paraoxon, and DDVP.
29
-------
U
Q
W
H
CJ
W
H
W
Q
CO
w
H
CJ
p-l
m
01
r-l
43
CO
H
i-H «
CO CO
,j 4J x-s
O CO M
^ .y
0) \
4-1 O bl
3 m E
0 Q <-<
•<3 r-l
Ol
4-1
O
01
4J
Ol
Q
d
o E
•H Q
4J Q
CO
4-1
e
Ol
o
C
0
CJ
01
CO
1 — 1
CO
CJ
E
01
4:
§
CO
IS
Ol
T3
CO
H
m
«<)• o^
0
i— 1 01
1— 1
Ol eg
r— 1 &
CO 0)
ej it i
O
CN
01
4->
CO
E
CO
43
CO
0
r-l
^
42
4J
0)
Q
I
£5
i— 1
a
01
CL
^
X
0
M
ex
0
CO
M
i
O
'5)
**
O
bO
«
M
sj-
VO
1
r^
^-
O
r-l
1
O
N
CO
H
^>
cx
rH
j^
4-1
0)
E
1 0)
m 4J
r*» CO
CO CO
43 O
CO >>
O 42
rH 4J
>> 01
4J -i-l
01 T)
•H rH
Q >-.
1 1
CN m
C
CO
rH
•H
4-1
01
E
•H
Q
LO
m
•
o
1
(2
CO
^4
3
14-1
O
N
(2
Ol
43
rH
K*-,
42 OJ
4J 4J
0) eg
E c
•H CO
T3 43
CN CO
« O
CN i— 1
1 ^
0 42
^1 4J
T3 Q)
r*t C
42 I
n
i i-i
ro >^
» i
CN r~-
©
^p
CO
•o
CO
}-(
H
£
o
o
rH
CN
i— 1
1
i— 1
^)
42
4J
OJ
E
Z
i— i
r*^
rH
^
X
1
m
CO*
i
0
•H
42
4J
rH qj
t^, 4J
42 CO
01 CO
O "Q
" CO
•* 0
"S)
r— 1
o
3
CO
Ol
S
o
o
o
CN
0)
4-1
2
CO
43
CO
O
r- 1
4J
Ol
E
^
r-l
^
42
4J
1
CO
1
rH
ffi,
•i-l
^
4)
rH
m
o
01
">>
42
01
T3
rH
CO
(2
O
•H
CX
O
S-l
CX
1 01
/^S E
O -H
*r^ ^
42 0
4J /-s
rH rH
42 Q
4J E
01 CO
E 43
I co
CN CJ
1 rH
i — 1 >•>
r*"! r^
42 4-1
4-1 01
01 E
S s^
1 1
CN O
d^
^5
•H
E
Ol
H
O
CO
1^
Ol
4-1
2
CO
43
M
CO
O
rH
S^
42
4-1
1
J3
rH
^
rH
0
4-1
-------
SECTION IX
CORRELATION OF PESTICIDE DETECTABILITY WITH PESTICIDE TOXICITY
For most organophosphate and carbamate insecticides, both their toxicities
in warm blooded animals and their detectability on CAM-1 are directly related
to the affinity of the insecticide for the enzyme. This relationship exists
because the mechanism of toxicity and the mechanism of the detection process
are both dependent upon the blockage of active sites on the cholinesterase
molecule. For example, a compound such as paraoxon is quite toxic in animals
and is detectable in low concentrations in water with the CAM-1 system.
The mechanism by which these insecticides are able to inhibit cholinesterase
is well known and is reported in literature reviews of cholinesterase prepared
O fi
by Froede and Wilson and by Metcalf. In man and also in CAM-1, the organo-
phosphate insecticides phosphorylate one of these active sites (i.e. the es-
teratic site) on cholinesterase to inhibit its enzymatic activity. In man,
the cholinesterase is essential for the hydrolysis of the acetyl choline which
hydrolysis results in the relaxation of muscles after contraction. Inhibition
of the action of this enzyme in man, therefore, produces toxic symptoms which
include contraction of the pupils, nausea, muscular rigidity, convulsions, etc.
For some organophosphate and carbamate pesticides, however, the correlation
between animal toxicities and detectabilities by CAM-1 is poor. This is
especially true for those compounds that are classed as phosphoro-thioates,
phosphoro-dithioates and thiocarbamates. These compounds are less strongly
attracted to cholinesterase and are therefore less readily detectable by the
CAM-1 system. On the other hand, compounds in this group retain their toxicity
to warm blooded animals since their metabolism in the liver (oxidation) produces
the more toxic oxygen analogues. As shown in Figure 5, organo.phosphates such
as parathion and malathion are very toxic because they are oxidized to the oxygen
analogues in vivo. As shown in Table 4, many of the commercial organophosphates
do have the phosphoro-thioate and dithioate structures (i.e., -0-P=S and
-S-P=S). Chemically their reduced affinity for cholinesterase is due to the
fact that compounds with the P=S linkage are less electrophilic than compounds
with the P=0 linkage.6
31
-------
is
§
00 00
M 60
S B
in m
B
Cu
CL
U
C
•H
C
o
x
o
cd
M
a)
ex
•o
C
C!
O
•i-l
43
4J
cd
M
cd
a.
M-I
o
o
P5
M
13
C 0)
co B
Vi
a
•r-l
32
-------
It should be noted that a common impurity in the commerical phosphoro-thioate
and dithioate insecticide products is the oxygen analogue of the insecticide;
because of the greater affinity of the impurity for the enzyme, significant
differences in the detectability of the commercial pesticides can exist.
Parathion (P=S type) from two suppliers has been detected at 5 to 10 ppm,
whereas the corresponding oxidized material (i.e., paraoxon, P=0 type) is
readily detected at 0.1 ppm. On the other hand, the toxicities of these two
compounds in rats are essentially the same. The sensitivity of CAM-1 to the
P=S type of organophosphates is less than that to the P=0 type of compounds.
Nevertheless, CAM-1 can detect the P=S type of insecticides at concentrations
that will prevent water supply users from receiving acute oral doses of these
pesticides. Promising ideas for increasing the sensitivity of CAM-1 to the
P=S compounds by an order of magnitude have been generated but have not yet
been investigated sufficiently to report on at this time. Basically, the
idea is to pre-oxidize the P=S type compounds to the P=0 type.
33
-------
SECTION X
RESPONSE OF CAM-1 TO REVERSIBLE CHOLINESTERASE INHIBITORS
Several reversible inhibitors of cholinesterase have been encountered in this
study of the response of CAM-1 to a variety of potential water pollutants.
Reversible inhibitors have the ability to inhibit cholinesterase when these
inhibitors are present in moderate concentrations; however, they neither bind
tightly nor do they phosphorylate the enzyme; therefore, they are not concen-
trated in the enzyme pad to the same extent as the nonreversible inhibitors.
Table 6 provides some response data on three such reversible cholinesterase
inhibitors. Thiban" 75 (tetramethylthiuram disulfide) is an animal .repellent
and fungicide, and, judging from its chemical structure, it would not be ex-
pected to form covalent bonds with the active sites of the enzyme. However,
at 30 ppm this material produced a voltage increase of 45 mV during the first
cycle, with very small increases during subsequent cycles. Changing of the
CAM-1 inlet hose to freshwater resulted in the washing out of the inhibitor,
the reactivation of the enzyme, and the return of the electrochemical voltage
to its pre-exposure value. A similar result was obtained with tributyl amine
(TEA) hydrochloride, which should add reversibly to the "anionic site" (i.e.,
cation attracting site) of cholinesterase (i.e., the quaternary nitrogen of
acetylcholine chloride). As shown, 100 ppm of tributyl amine caused a 38-mV
increase on the first cycle and only small increases on subsequent cycles.
On introducing freshwater into CAM-1, the TEA was washed out of the enzyme
pad slowly and five wash cycles were required to obtain the original pre-
exposure voltage. A third example of a reversible inhibitor is Sutan , which
is S-ethyl diisobutylthiocarbamate, The voltage increase in the first detec-
tion cycle is a function of the concentration; little or no voltage increase
is noted in subsequent detection cycles. From these data (Table 6), it is
apparent that detection of reversible inhibitors is likely to occur only when
there is a sudden change from no inhibitors to fairly high levels of the
reversible inhibitors. In such cases, only one cycle will show a large vol-
tage increase and subsequent cycles will show either small responses or none.
A voltage drop with no alarm signal will be noted when the reversible inhibitor
is removed by washing the freshwater. In passing, it should be emphasized
that low levels of reversible inhibitors do not prevent the detection of the
nonreversible cholinesterase inhibitors.
34
-------
"3
Pi
o
H
M
PQ
h-l
PC
w
CO
H
CO
W
O
W
u
H
hJ
PQ
M
CO
Pi
W
>•
Ed
o
H
W
o
PM
co
w
Pi
vD
01
as
H
h
a)
PL,
CO
co
00 >
s B
J3 1
U ,
CO CJ
4J
r— 1
O
E>
tH
1
S
<
0
s
0
o
.
*
0
c
o
o
v£)
r»
CT\
(J>
1 1^.
co
1191
JS-J
cn-^ "=
«3=O CO
43
1 -H
A e
en \ en
sc w
0 0
rH
g
•
ja
CO
/••%
Oi
a
«*
o
S_s
CM
A
CM
A
ON
ft
i-l
1
•\
r^
n
00
co
0
o
1-1
t-t
to
•H
1 1 *j-\
ffi cu
J3
CU U
c
1 I
•H t~~i
E co
CO -i-l
•~4 t-i
>-. w
U CO
3 3
,0 T3
•H C
t-l •<->
H N-'
CO
t
CM
1
rt
O
<)•
1
o
CM
CM
o\
**
r*1*
i
0
1— (
0
co
o
o
1 — 1
cu
T3
•H
O
.,_)
r^
S-l
CU
rC
^J
M
to
U
CO
35
-------
SECTION XI
STUDIES OF CAM-1 WITH OTHER ECONOMIC POISONS AND DISSOLVED SALTS
The various non-phosphate, non-carbamate pesticides investigated included
herbicides, defoliants, insecticides, nematocides, fungicides, rodenticides,
repellents, and others. Some specific compounds tested with CAM-1 included
aldrin, chlordane, dieldrin, lindane, chlorophenoxyacetate salts, piperonyl
butoxide with pyrethrins, Daconil (tetrachloroisophthalonitrile), Thiophanate
[diethyl 4,4'-0-phenylenebis (3-thioallophonate)], Fumasol-Cr [sodium salt
of 3(a-acetonylfurfuryl)-4 hydroxycoumarin], Phaltan (N-trichloro-methyl-
thiophthalamide), etc. None of these economic poisons had any effect on the
cell voltage in CAM-1 and thus were not detectable.
Only very preliminary studies of the effect of dissolved inorganic materials
in water on the performance of CAM-1 have been made and these are shown in
Table 7. Surprisingly, zinc sulfate at 10 ppm caused repeated alarms until
the enzyme pad was exhausted. Reports of zinc as an inhibitor of cholines-
terase were not found; however, there are some enzymes, such as carbonic an-
hydrase which require zinc and other enzyme systems which require either cal-
cium or magnesium ions in which inhibition by zinc occurs because of a dis-
placement of the calcium or magnesium ions. Since cholinesterase does re-
quire calcium ions, its replacement by zinc may be the mechanism of cholin-
esterase inhibition but this has not been investigated. Detection of potas-
sium dichromate by CAM-1, if it had occurred, would not have been surprising
since dichromate is known to denature proteins by a tanning reaction and since
it could also produce a rise in the electrochemical cell voltage simply by
oxidation of the thiol liberated from the substrate of the enzyme. No alarms
were produced by this compound; neither was the enzyme denatured nor was the
substrate oxidized (most of the dichromate was apparently removed from the
electrochemical cell prior to the addition of the substrate). Experiments
with sodium arsenate showed that it could not be detected at 10 ppm.
Mercuric chloride at 10 ppm produced alarms for the first two out of six de-
tection cycles. The effect on the system was similar to that observed for
the reversible enzyme inhibitors. Low levels of mercuric ions in water would
not produce an alarm in CAM-1 although a situation in which there were rapidly
fluctuating levels of mercury might cause an occasional alarm. Field tests
on polluted streams will be required to determine whether reversible inhibitors
in the water cause significant problems, and these are recommended.
36
-------
H
U
Cu
W
CO
H
^
4J 0|
CU O
•-H CU
fl rJ "
c0 Q-
4J 4J B
U CO CU
CU CU H
4J H ^
CU
U
0 1
rj
0 -
CO CU
W V-i
3
H CO
o o
M-I a
cu w
CO
C OC
o c
CO &
cu o
1-1
cu o
60 fn
CO
4-1
1— (
o
vO
m
^j-
CO
CM
[
cu
> B
T) »rl O.
CU 4-1 0.
4-1 U
CO CO «
CU 4-1
H C C
o cu
cu -a -a
> a) cu
CU CO J-l
i-J CO M
"-' -H
•o
4J
CO
CU
H
CO
-o
c
3
o
a
B
o
o
G"
0
O
CXI
x*/
O
z
,
vO
1
O
CN
1
V4-,
1
oo
i
o
t-H
-------
Sodium chloride has but little effect on cholinesterase activity of the en-
zyme pads. However, switching of the CAM-1 rapidly from freshwater to 3%
salt water causes a 40-mV drop in cell voltage. After this voltage drop, the
voltage remained essentially unchanged for the next five cycles. The observed
voltage change is attributed to the presence of more ions in the enzyme pad
after the thorough washing in 3% salt water. Because of the porous nature
of the starch gel, this extra ionic material is not all removed in 1 mln
during the enzyme pad testing portion of the cycle. So long as the salinity
of the water being tested changes slowly, no problems in monitoring fresh,
brackish, or sea water for the presence of enzyme inhibitors are anticipated.
Tests with 0.1 ppm DDVP in 3% salt water at 9°C showed that it was detectable
by CAM-1; this is the same sensitivity we observed in freshwater at 6°C.
38
-------
REFERENCES
1. Goodson, L. H. and W. B. Jacobs. A Rapid Detection System for
Organophosphates in Water. In: Proceedings of the 1972 National
Conference on Control of Hazardous Material Spills. U.S. Envir-
onmental Protection Agency and the University of Houston, Houston,
Texas, March 21-23, 1972.
2. Goodson, L. H. and W. B. Jacobs. Use of Immobilized Enzyme Product
in Water Monitoring. In: Proceedings of the 1974 National Con-
ference on Control of Hazardous Material Spills. American Insti-
tute of Chemical Engineers and the U.S. Environmental Protection
Agency (Industrial Waste Treatment Research Laboratory, Edison,
New Jersey), San Francisco, California, August 25-28, 1974.
3. Goodson, L. H. and W. B. Jacobs. Rapid Detection System for Organo-
phosphates and Carbamate Insecticides in Water. Environmental
Protection Technology Series, EPA-R2-72-010, Final Report on
Contract 68-01-0038, Office of Research and Monitoring, U.S. En-
vironmental Protection Agency, Washington, D.C. 20460, August
1972.
4. Goodson, L. H., W. B. Jacobs, and A. W. Davis. An Immobilized
Cholinesterase Product for Use in the Rapid Detection of Enzyme
Inhibitors in Air or Water. Anal Biochem. !51_ (2): 362-367, 1973.
5. Frear, D. E. H. Pesticide Index. Fourth Edition, College
Science Publishers, State College, Pennsylvania, 1969. 399 pp.
6. Metcalf. R. L. The Chemistry and Biology of Pesticides. In:
Pesticides in the Environment. R. White-Stevens, Editor, Mar-
cel Dekker, Inc., New York. I:102-110, 1971.
7. Kramer D. N., P. L. Cannon, Jr., and G. G. Guilbault. Electro-
chemical Determination of Cholinesterase and Thiocholine Esters.
Anal. Chem., 34_ (7) : 842-845, 1962.
8. Froede, H. C. and I. B. Wilson. Acetylcholinesterase. In: The Enzymes,
Vol 5, 3rd Ed. P. D. Boyer, Editor, Academic Press, New York, 1971, esp.
pp 93-109.
39
-------
APPENDIX A
A RAPID DETECTION SYSTEM FOR ORGANOPHOSPHATES
AND CARBAMATE INSECTICIDES^/ IN WATER
ABSTRACT
An apparatus for the detection and monitoring of water supplies for hazardous
spills of organophosphate and carbamate insecticides has now been designed
and fabricated. The new unit is called the Cholinesterase Antagonist Monitor,
CAM-1, because it produces an alarm in 3 min when toxic or subtoxic levels of
cholinesterase antagonists are present in water. Response of this apparatus
to subtoxic levels of Azodrin , Sevin , dimetilan, malathion, parathion, and
DDVP has already been demonstrated. CAM-1 uses immobilized cholinesterase for
the collection of cholinesterase inhibitors from the water supplies. The
activity of the immobilized cholinesterase is determined automatically in an
electrochemical cell by passing a substrate solution over the enzyme at regular
time periods. A minicomputer is used to automate the detection process and
to signal an alarm when there is a rapid loss of enzyme activity—a situation
which occurs in the presence of organophosphate and carbamate insecticides
in the water sampled.
This report was submitted in fulfillment of Project No. 15090-GLU, Contract
No. 68-01-0038, under sponsorship of the Water Quality Office, Environmental
Protection Agency.
40
-------
APPENDIX B
MECHANISM OF ELECTROCHEMICAL DETECTION PROCESS IN CAM-1
1. Oxidation Potential Theory. This theory proposes that cholinesterase is
able to convert butyrylthiocholine iodide (BuSChI) into butyric acid and
thiocholine iodide (HSChI) and since the thiol is easily oxidized a low
voltage is measured by the constant current electrochemical system. In
the absence of enzyme there is no thiol present and the voltage rises--
usually about 250 mV since the original substrate BuSChI is not readily
oxidized. It has been proposed that the voltage setting reaction is the
oxidation of I" to ~L<^\ this explanation has appeal but it does not explain
(1) how this iodide-to-iodine oxidation potential can vary from 100 mV to
600 tnV as the electrodes are conditioned; (2) no trace of iodine color
has ever been detected on the starch covered enzyme pad and (3) why the
voltage does not immediately fall to zero since traces of iodine completely
depolarize the electrodes.
Supporting this theory is the knowledge that HSChI is readily oxidizable
to the disulfide and it can be found among the products coming through the
electrochemical cell. If this theory is correct, the voltage change should
be observable with a number of electrode pairs at equivalent solution con-
centrations. We have not found alternate electrode materials which work
as well as platinum.
2. Anode Depolarization Theory. This theory suggests that the anode is coated
with a layer of platinum oxides or sulfides and perhaps other materials
which tend to reduce its electrical conductivity. Exposure of this coated
anode to a solution containing a trace of thiol results in a depolariza-
tion or increase in conductivity of the anode coating. In favor of this
theory is the finding that application of a direct current to two identical
electrodes for a few minutes in the presence of hydrolyzed substrate (i.e.,
HSChI) results in the making of stable dissimilar electrodes which generate
voltage like a battery when they are placed in an electrolyte. The elec-
trodes are readily made alike or depolarized by treatment with a trace of
free iodine or chlorine; such treatment in our electrochemical system drives
the voltage to zero indicating excellent conductivity. The electrodes
recover after the halogen is gone.
41
-------
In beaker experiments a standard calomel electrode is used as a reference
electrode while current is applied to two identical platinum electrodes in
a solution; when the solution is changed, from HSChI to BuSChI, it is noted
that nearly all of the voltage change occurs at the anode. This suggests
that the conductivity of the anode surface is changing with the change of
material in the beaker. Measurement of applied current in the electro-
chemical cell with an enzyme pad showed that it was sufficient to oxidize
only about 5% of the HSChI produced by the enzyme pad. Presumably a
close balance between coulombs of applied current and moles of HSChI
would be required to obtain rapid response of the system to enzyme inhi-
bitors.
The disulfide of thiocholine iodide found in the products coming from the
cell could arise either from air or electrochemical oxidation of the HSChI.
Hence no evidence is gained for either theory. With freshly-plated plat-
inum electrodes, the voltages obtained with enzyme pads are often as low
as 0 mV at first, and after the electrodes have been used for a while
(e.g., a day or two), the enzyme pad voltage may be as high as 250 to
300 mV; at the same time the voltage change obtained on replacing an en-
zyme pad with a pad without enzyme is 200 mV or more whether the elec-
trodes are new or conditioned. This suggests that the enzyme pad voltages
obtained are not characteristic of the oxidation potential of thiocholine
iodide since they range from 0 to 300 mV. In summary, the exact mechanism
of the electrochemical reaction is unknown and both electrode polarization
and thiocholine iodide oxidation may be occurring simultaneously in the
electrochemical cell. The mechanisms enabling electrochemical estimation
of enzyme pad activity are worthy of further investigation.
Further support to the anode depolarization theory is given by Kramer,
et al.,—' who reported constant current experiments in which depolariza-
tion of a platinum anode by thiocholine iodide resulted in increased con-
ductivity of the anode. This electrochemical reaction forms the basis of
their procedure for anlaysis of cholinesterase and thiocholine esters.
Measurement of Cell Voltages. During the last minute of the 3-min detec-
tion cycle, the relative activity of the enzyme pad is determined electro-
chemically. While the substrate solution is pumped continuously over the
enzyme pad at ~ 1 ml/min, air at ~ 1 liter/min is also passed through the
enzyme pad. During the final 40 sec of this substrate pumping cycle, a
constant current of ~ 2 uA is applied to the platinum electrodes with the
lower electrode being made positive. The cell voltage (as measured with
a high-impedance voltmeter or electrometer) rises rapidly at first but
then either approaches or in some cases passes a maximum voltage prior
to the end of the cycle. Whether the voltage is sampled and shifted into
the memory immediately before or after the peak voltage occurs makes
42
-------
little difference in the performance of the CAM-1 provided the substrate
flow is constant and provided that the constant current is applied for a
constant period of time. Repeatability rather than attainment of a true
equilibrium voltage was the basis for applying the constant current for
40 sec during each detection cycle.
43
-------
TECHNICAL REPORT DATA
(Please read iHiimctions on the reverse bcjore completing)
REPORT NO.
EPA-600/2-77-219
3. RECIPIENT'S ACCESSI ON-NO.
. TITLE AND SUBTITLE
IVALUATION OF "CAM-1," A WARNING DEVICE FOR ORGANO-
PHOSPHATE HAZARDOUS MATERIAL SPILLS
5. REPORT DATE
November 1977 issuing date
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
uis H. Goodson
William B. Jacobs
PE-RFOt^MINiJ ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
------- |