United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Cincinnati OH 45268
EPA-600/2-80-033
January 1980
Research and Development
CAM-4, A Portable
Warning Device for
Organophosphate
Hazardous Material
Spills
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution-sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-80-033
January 1980
CAM-4, A PORTABLE WARNING DEVICE FOR ORGANOPHOSPHATE
HAZARDOUS MATERIAL SPILLS
by
Louis H. Goodson
Brian R. Cage
Midwest Research Institute
Kansas City, Missouri 64110
Contract No. 68-03-0299
Project Officer
John E. Brugger
Oil & Hazardous Materials 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
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DISCLAIMER
This report has been reviewed by the Industrial Environmental
Research Laboratory - Cincinnati, U.S. Environmental Protection
Agency, and its publication approved. 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 of commercial products constitute endorsement or recommenda-
tion for use.
ii
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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 (lERL-Ci) assists in developing and demonstrating new and improved
methodologies that will meet these needs both efficiently and economically.
This report describes the design, fabrication, and preliminary evalua-
tion of CAM-4, a portable version of the earlier Cholinesterase Antagonist
Monitor, CAM-1. Both instruments use immobilized cholinesterase in an elec-
trochemical cell in order to sense the presence of organophosphates and
carbamates in water. CAM-4 operates equally well from a 110-v AC line at a
fixed facility or from a 12-v battery. Battery-powered units have been used
repeatedly on land and from boats. CAM-4 is useful for locating pesticide
discharges into streams and also for following the movement of pesticide
spills in lakes and streams. Information on this subject beyond that sup-
plied here may be obtained by contacting the Oil and Hazardous Materials
Spills Branch, lERL-Ci, U.S. EPA, Edison, NJ 08817.
David G. Stephan
Director
Industrial Environmental Research Laboratory
Cincinnati
iii
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ABSTRACT
Previously, an instrument designated as CAM-1 ("CAM" is an acronym for
"cholinesterase antagonist monitor") was constructed to continuously monitor
the levels of organophosphate and carbamate pesticides in water (ponds,
streams, plant outfalls, etc.). CAM-1 is a sophisticated research instrument
that cannot be conveniently used in the field or from a boat because of its
non-ruggedized construction. To meet the requirements for an equally sensi-
tive but portable system, CAM-4 was developed. CAM-4 is a field version of
CAM-1 that will operate continuously in the field from a 12-v DC power supply
for eight hours, or from 110-v AC. The present report describes the design,
fabrication, and evaluation of CAM-4.
Operation of the CAM instruments is based on inactivation of the enzyme
cholinesterase by organophosphate and carbamate pesticides. The extent of
inactivation, which is proportional to the amount of inhibitor present, is
determined by measuring the response of the system to a substrate readily
hydrolyzed by the enzyme.
The conventional method for detecting organophosphates and carbamates in
water requires the addition of cholinesterase, a buffer, and an enzyme-hydro-
lyzable substrate, e.g., butyrylthiocholine iodide, to a water sample, fol-
lowed by spectrophotometric determination of the residual substrate concen-
tration. The spectrophctometric method is disadvantageous because: (a) a
significant quantity of costly enzyme is needed for each test, and (b) the
system is not easily adapted to continuous use since the optical windows be-
come dirtied by the flow of contaminated water. It is necessary to correct
this problem by comparing the response of the contaminated sample, plus re-
agents, to that obtained with a contaminated sample alone. However, even
with this correction, frequent cleaning is necessary. The CAM systems elim-
inate these problems. Entrapping a given amount of enzyme in starch gel on
the surface of open-pore polyurethane foam makes repeated use possible over
extended periods of time. Residual activity of the enzyme is measured
electrochemically.
In the CAM system, residual activity is determined during a sampling
cycle by the level of substrate hydrolysis product present in the electro-
chemical cell. Presence of a cholinesterase inhibitor reduces the rate of
substrate hydrolysis (thiol formation) and produces an increase in cell volt-
age. The magnitude of the increase above the enzyme pad potential is a func-
tion of the residual enzyme activity; that is, the voltage rises as the
enzyme is inhibited. A voltage increase from one sampling cycle to the next,
above a designated alarm threshold, is used to trigger an "alarm" indicating
the presence of cholinesterase inhibitors.
iv
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Development of a portable system necessitated elimination of the follow-
ing convenience features from CAM-1: the computer logic circuits, automatic
pad changer, digital voltmeter, strip chart recorder, and audible alarm com-
ponents. CAM-4 contains an inverter to transform DC to AC current and a
digital printer to record cell voltage. An operator must read the digital
printout to determine when there is an "alarm" condition and when it is
necessary to replace the enzyme pad.
CAM-4 possesses detection and monitoring capabilities equal to those of
CAM-1. The sensitivity of CAM-4 to subtoxic levels of DDVP, SystoxR, Fura-
dan^, malathion, Sevin^, and other pesticides in water is comparable to the
sensitivity of CAM-1 for the same pesticide solutions.
This report was submitted in fulfillment of Contract No. 68-03-0299,
Task No. 2, by Midwest Research Institute under the sponsorship of the. U.S.
Environmental Protection Agency. This report covers the period from April 3,
1975 to May 3, 1976, and work was completed as of July 3, 1976.
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CONTENTS
Foreword ^..... iii
Abstract iv
Figures viii
Tables ix
Acknowledgments x
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Design and Fabrication of the Portable Cholinesterase
Antagonist Monitor (CAM-4) 6
5. CAM-4 Operating Principle 19
6. Operating Parameters for the Electrochemical Enzyme Sensor 22
7. Operating Procedures for CAM-4 25
8. Laboratory Studies with CAM-4 31
9. Field Testing of CAM-4 35
10. Appendices
A. Abstract of contract on CAM-1 development 37
B. Abstract of Task 1 report on this contract 38
C. CAM-4 field test report 39
D. CAM-4 parts list 43
E. Mechanism of electrochemical detection process in CAM-4 45
F. Enzyme pad preparation procedure 47
VII
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FIGURES
No. Page
1 CAM-4, showing fiberglass case 7
2 Internal view of portable Cbolinesterase Antagonist Monitor,
CAM-4 9
3 View of manual enzyme pad changer and motors in CAM-4 (behind
left panel) 10
4 CAM-4 3-min operating cycle for collecting enzyme inhibitors
on an immobilized enzyme pad and subsequently measuring a
voltage related to the enzyme activity of the pad 11
5a Analog-to-digital converter and cell signal amplifier and cur-
rent source 13
5b Power supply board providing regulated +5 and ±15-v DC for
logic boards and power for printer 13
6 Wiring diagram showing 12-v DC to 110-v AC inverter and DC/AC
switching 14
7 Timer and optically coupled triac switching circuits for pumps . 15
8 Wiring diagram showing interconnection of panel switches, digi-
tal printer, printed circuit boards, and other components .... 16
9 Cross section of an electrochemical cell developed for water
monitoring showing the platinum electrodes above and below
the enzyme pad to which a constant current is applied 17
10 Response of the electrochemical cell operating on the 3-min
cycle to water containing 0.2 ppm DDVP 21
11 Digital printout in volts from CAM-4 showing cycle-to-cycle
variation (noise) obtained when operating on a 3-min detec-
tion cycle in the absence of inhibitor 28
12 Typical calibration of CAM-4 showing cycle-to-cycle voltages
before (lower portion of tape) and after (upper portion)
exposure to 0.2 ppm DDVP for three cycles 30
viii
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TABLES
No. Page
1 Response of CAM-4 to Carbamate Pesticide Solutions 32
2 Response of CAM-4 to Organophosphate Pesticide Solutions 33
3 Comparative Response of CAM-4 and CAM-1 to Pesticide
Solutions 34
ix
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ACKNOWLEDGMENTS
The work upon which this publication is based was performed pursuant
to Contracts Nos. 68-01-0038 and 68-03-0299 with the Environmental Protec-
tion Agency. This report describes the work done on Task II of the
latter contract. Task I was concerned with the evaluation of the Cholin-
esterase Antagonist Monitor Model No. 1 (CAM-1) with a series of commer-
cially available pesticides including both organophosphates and carbam-
ates. Task II is concerned with the design, fabrication, and evaluation
of a portable version of CAM-1, which has been designated as CAM-4. Task
III is concerned with the investigation of alternate enzyme systems for
use in CAM-1 and CAM-4; this work has been partially funded and is now in
progress.
The authors wish to thank Mr. William B. Jacobs, Mrs. Julie Kelly,
Miss Vicki Appleman, and Mrs. Margo Rogers of Midwest Research Institute
and Mr. Edward T. Fago, Jr., Teletron Company, 5733 Kenwood Street,
Kansas City, MO 64110, for their technical assistance. We also wish to
thank Dr. Thomas Hoover of EPA's Southeast Water Quality Laboratory,
Athens, GA, and Dr. John E. Brugger of EPA's Industrial Environmental
Research Laboratory-Cincinnati, Edison, NJ, for their technical assistance
and encouragement.
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SECTION 1
INTRODUCTION
Spills or discharges of hazardous materials into rivers, streams, or
lakes create the danger that toxic levels of chemicals could result in a
massive fish kill or that the polluted water could enter a municipal water
supply. Hazardous material spills must be detected promptly and followed so
as to minimize health and environmental effects.
In an earlier report (EPA/R2-72-010), the Cholinesterase Antagonist
Monitor (CAM-1), which utilized cholinesterase immobilized on a polyurethane
pad in an electrochemical cell for detecting and/or monitoring cholinester-
ase antagonists in water, was described. This instrument provided a response
on a real-time basis to organophosphate and carbamate pesticide's in water.
An abstract of Report EPA/R2-72-010 is included as Appendix A.
Task I on the present contract reported the sensitivity of CAM-1 to a
series of pesticides and was approved for publication in August 1977 (EPA-
600/2-77-219). Such a study was necessary since the sensitivity of the
instrument was directly related to the affinity of cholinesterase for the
pesticide being detected. In most cases, the sensitivity of the instrument
was also related to the toxicity of the material being detected, since the
mechanism of toxicity and the mechanism of detection are both concerned with
the binding of the toxic material to cholinesterase. The abstract of EPA-
600/2-77-219 is included as Appendix B.
CAM-1 was designed for use at fixed locations where power was readily
available and where mobility was not a major consideration. One such appli-
cation that had been envisioned was its use at the intake of a municipal
water supply. Here, the instrument would detect pesticide spills occurring
upstream from the water inlet upon their arrival at the inlet pipe and pro-
vide a warning so that the inlet valve could be shut until the spill had
passed.
The objective of Task II on this contract has been the construction and
preliminary evaluation of a portable version of CAM-1 to which we have as-
signed a code designation CAM-4. It was planned that this instrument could
be operable from a boat using a 12-v DC power supply and that it could be
taken as needed to monitor the movement of spills and the effectiveness of
clean-up procedures. The present report describes the construction and
operation of CAM-4, the portable version of CAM-1.
In the CAM systems, the immobilized horse serum cholinesterase serves
as a dosimeter in that it collects pesticides (irreversible cholinesterase
inhibitors) on its active sites with a resulting decrease in enzyme activity.
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The enzyme itself is entrapped on open-pore polyurethane foam pads with.
aluminum hydroxide and starch gel; this permits its use for extended periods
of time when inhibitors are absent from the water sampled.
In addition to being a pesticide collector, the immobilized enzyme also
serves as a biological amplifier for the measurement of residual enzyme
activity following its exposure to an unknown water sample; thereby, it
enables the CAM instruments to determine the presence or absence of cholin-
esterase inhibitors in the. water sampled.
x\lthough either spectrophotometric or electrochemical methods could
have been used to monitor the rate of formation of substrate hydrolysis
products by the immobilized enzyme pad, we chose to use the electrochemical
method since it was cheaper to fabricate and less subject to problems caused
by dirt and air bubbJ.es in the water samples. In the CAM systems, the sub-
strate, i.e., butyrylthiocholine iodide, is hydrolyzed by the enzyme to give
the easily oxidizable substance, thiocholine iodide. This latter compound
is then detected by the application of a constant current (^ 2 yA) to plat-
inum electrodes in contact with the enzyme pad; thus, when enzyme inhibitors
are absent, the enzyme is active and there is an abundance of thiocholine
iodide present, which causes a low potential (^ 200 mv) between the two
electrodes. On the other hand, when enzyme inhibitors are present, the
activity of the enzyme is low, the quantity of thiocholine iodide formed is
small, and the potential between the electrodes is ^ 500 mv. Thus, it is
possible to tell whether the sampled water contained enzyme inhibitors by
observing the potential between the electrodes.
The reason for the voltage change in the CAM electrochemical cell is
not entirely understood. One hypothesis suggests that the low voltage be-
tween the electrodes when enzymes are present is due to the simple electro-
oxidation of the thiocholine iodide to the corresponding disulfide. The
other explanation says that the anode is coated with a layer of platinum
oxides or sulfides and that exposure of this coating to the thiol compounds
results in a partial depolarization of the anode. The arguments for and
against the two theories explaining the generation of characteristic volt-
ages are given in Appendix E (page 53) and in the Task I report on this
contract (EPA-600/2-77-219). In the latter report may be found detailed
discussions of the operating principle of the electrochemical enzyme cell,
the studies related to the selection of buffer, pH and concentration, the
effect of temperature on the operation of the system, the response of CAM-1
to many additional organophosphate and carbamate pesticides, the response to
reversible inhibitors, the response to possible interfering substances, a
procedure for fabricating the enzyme pads for the electrochemical cell, and
other subjects related to operation of CAM-type monitors.
The operating principles for CAM-1 and CAM-4 are identical even though
there are some differences in the degree of automation, the specific opera-
ting procedures, and the power requirements. The following sections of this
report describe the construction, operation, and preliminary evaluation of
the portable, battery-operated pesticide monitor known as CAM-4.
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SECTION 2
CONCLUSIONS
On the basis of the present investigation of immobilized enzymes for
the detection of toxic organophosphate and carbamate insecticides in water,
the following conclusions have been made:
1. A portable enzyme-based detection system to monitor water supplies for
the presence of organophosphate and carbamate insecticides has been
designed, fabricated, and tested. The new portable system has been
designated as the Cholinesterase Antagonist Monitor Model No. 4, CAM-4.
This unit is designed specifically for field use and, like CAM-1,
responds rapidly to low levels of cholinesterase antagonists in water
supplies, streams, and impoundments.
2. The complete CAM-4 detection system and several of its component parts,
including specifically the enzyme cell for water monitoring, have been
redesigned to reduce weight and power requirements and to permit easy
replacement of the electrochemical cell if this should become necessary.
3. CAM-4 responds to toxic and subtoxic levels of organophosphate and car-
bamate insecticides (the levels referred to are based on rat and animal
toxicity data). In a response test series with city tap water at 25 C
to which 0.2 ppm of DDVP (dimethyl 2,2-dichlorovinyl phosphate) had
been added, CAM-4 provided repeated voltage increases equal to or
exceeding the 10-mv alarm threshold. On this basis, it is concluded
that CAM-4 has adequate sensitivity to provide adequate warning of sit-
uations that may lead to accidental poisoning of human and other animal
species by cholinesterase inhibitors in water supplies.
4. Like CAM-1, CAM-4 responds to toxic and subtoxic levels of many organo-
phosphate and carbamate insecticides, including the following that have
been tested: Baytex^, Sevin^, Mesurol&, GuthionR, malathion, FuradanR,
Baygon^, Systox*^, and Nemacur^.
5. The sensitivity of CAM-4 to low levels of insecticides in water is the
result of the affinity of these insecticides for the reactive sites on
the surfaces of the immobilized enzyme used in the electrochemical
enzyme cell. Thus, the enzyme is functioning as a selective concentra-
tor (or dosimeter) for the materials to be detected.
6. CAM-4 is the first portable detection instrument that has successfully
used an immobilized enzyme product for the automatic monitoring of
water supplies for the presence of enzyme inhibitors.
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7. CAM-4 operates satisfactorily with enzyme pads fabricated from open-
pore urethane foam, starch gel, and cholinesterase complexed with
aluminum hydroxide gel.
8. The present CAM-4 operates on a 3-min detection cycle in which water is
sampled for 2 min and the pad activity (measured electrochemically) is
determined during the third minute. The sensitivity of the system can
be increased by manually increasing the water sampling period to 6 min
using the controls provided.
9. The CAM-1 developed earlier is recommended for fixed installations and
is suggested for use by operators of water treatment facilities to warn
of pesticides in water supplies; CAM-4, however, is portable and is
recommended for detecting or following the movement of spills in lakes
or streams so that appropriate action may be taken at the spill or
discharge site.
10. CAM-41s ability to detect insecticides in water is based upon a bio-
chemical reaction known to be involved in animal toxicity.
11. CAM-4 is not fully automated, but it is portable and can be operated by
one person in a small boat or at any location where either 12-v DC or
110-v AC power is available.
12. CAM-4 will operate continuously for at least 8 hrs from a 12-v auto-
mobile battery (55 amp-hr capacity).
13. Unlike CAM-1, CAM-4 requires an operator in attendance during its use
in water monitoring. The digital printer on CAM-4 provides the oper-
ator with information about the condition of the enzyme pad and the
presence or absence of inhibitors in the water sampled once each de-
tection cycle. Although the operator must decide when to change enzyme
pads and when toxic levels of pesticides are sampled, the omission of
some memory and logic circuits—incorporated in CAM-1—has greatly
simplified the construction of the instrument and lowered the cost
without reducing its sensitivity or response time. It also provides a
permanent time-based recording showing when spills are detected.
14. CAM-4 can probably be modified to reduce its power consumption when it
is operated on battery power if the power requirements prove to be
excessive. It is visualized that power can be saved if the various
pumps and circuits are designed to operate on direct current without an
inverter. Operation on alternating current could then be accomplished
with a 110-v AC to 12-v DC power supply; with this design, the major
power loss would occur when CAM-4 is connected to a 110-v AC line.
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SECTION 3
RECOMMENDATIONS
1. CAM-4, the portable version of the Cholinesterase Antagonist Monitor,
CAM-1, should be tested more extensively in the field to establish its
versatility and reliability for monitoring of pesticide spills in lakes
and rivers. Such further testing is needed primarily to encourage the
use of CAM-4 in the solution of practical problems encountered by gov-
ernmental agencies and private industries. For example, the sampling of
outfalls from manufacturing and formulating plants can be conveniently
accomplished by one -man in a boat equipped with CAM-4. Another applica-
tion is to use CAM-4 from a boat to map the extent of a pesticide spill
in a lake or to monitor the effectiveness of a cleanup procedure in
removing pesticide from a body of water.
2. It is recommended that CAM-4 be used to detect those pesticides detec-
table by CAM-1 where portability is an advantage.
3. Additional studies of the performance of either CAM-1 or CAM-4 should be
conducted to determine the possible adverse effects of various organic
and inorganic water pollutants that may occur in industrial or agricul-
tural wastewaters.
4. The response profile of CAM-1 and/or CAM-4 should be further evaluated
by an exposure of the detector to a wide variety of pesticides or other
potential water pollutants. This study should be concerned not only
with the detection of compounds, but also with establishing the thresh-
olds at which these chemicals are detected.
5. Studies should be conducted in which the objectives are: (a) to deter-
mine the reliability of the various CAM-4 components, and (b) to improve
reliability of the components and whole system when failures or malfunc-
tions occur. Testing of this type is needed to provide the assurance of
trouble-free operation under anticipated field conditions and to gener-
ate recommended servicing procedures and schedules.
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SECTION 4
DESIGN AND FABRICATION OF THE
PORTABLE CHOLINESTERASE ANTAGONIST MONITOR (CAM-4)
Before designing or constructing the portable water monitoring system
based upon the use of immobilized cholinesterase as the sensor, it was neces-
sary to consider the intended use of the detector and the manipulations and
skills required of the operators, as well as the sensitivity, selectivity,
permissible size, weight, cost, need for maintenance, and other parameters
affecting the design.
From the start, it was planned that the CAM-4 should be as much like
CAM-1 as possible, but it should be operable from 110-v AC and 12-v DC power
sources. Capability for operation of the instrument from a 12-v DC power
source was expected to expand its usefulness in the field since power would
be available from cars, trucks, boats, or inexpensive storage batteries. It
was required that the instrument in its case be watertight, occupy a space of
approximately 1 cu ft (0.03 m^) and weigh less than 30 Ib (14 kg) excluding
the battery.
A large share of the cost and complexity of the laboratory model (CAM-1)
is directly related to components that have been eliminated from the field
model (CAM-4). The items eliminated include: the automatic enzyme pad
changer, the strip chart recorder, the digital voltmeter, the horn, and the
computer logic circuits. Since there will always be an operator in atten-
dance with the CAM-4, he will decide when to change the enzyme pads or when
to signal an alarm. The digital printer is set to print the cell voltage at
the end of each 3-min detection cycle; also the printer provides a permanent
record of the exact voltages obtained during instrument operation, thus elim-
inating the need for either a strip chart recorder or a digital voltmeter.
The sensitivity of CAM-4 was expected to be essentially the same as the
laboratory model (CAM-1) since the enzyme pads, substrate, electrodes, and
tiding cycle would be the same in both instruments. As far as possible, the
individual operating procedures were to be as simple as possible so that a
minimum of effort or thought would be required from the operator in order to
obtain reliable data.
The portable Cholinesterase Antagonist Monitor (CAM-4), which has been
designed and fabricated for rapid detection of organophosphates and carbam-
ates in water supplies, is shown in its fiberglass carrying case in Figure 1.
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Figure 1. CAJ1-4, showing fiberglass case
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The instrument is designed so that most of the electrical components are
located in the right half of the case while the -mechanical components (pumps,
motors, cell holder, etc.) are located in the left half of the case. Figure
2 shows the location of these components.
In the upper right corner are the manual control switches for water,
substrate, and current. Each has a red indicator light above it that comes
on in sequence and tells which part of the detection cycle is in progress.
Any part of the cycle may be extended by manually flipping the proper toggle
switch up, but all of these switches must be in the "down" position for the
CAM-4 detection to operate automatically.
To the left of the manual controls on the front panel is the digital
printer; it has a manual print-paper advance switch, a low-paper indicator,
and a thumbscrew for removal of the assembly during paper reloading. In the
middle of the panel are located the fan for cooling the electronics, the AC-
DC on-off switch, and separate fuses for AC and DC operation. The 10 A fuse
is used when operating from an AC power source. At the bottom of the panel
are located the AC input plug, the DC input plugs, and the power inverter for
generating 110-v AC during battery operations.
The left panel of the instrument contains the mechanical components.
These include the cell open lever, the pad holder, and the cell voltage jack
from which an external recorder may be operated, if desired. The substrate
pump is immediately accessible from the front of the instrument, while the
water pump is reached by removal of the small pump access panel, located at
the lower left of the instrument. Also shown in the picture is the location
of the substrate supply bottle. Insofar as possible, commercially available
parts for the fabrication of CAM-4 have been used. However, it was necessary
to design and fabricate the manual enzyme pad changer-electrochemical cell
assembly, a view of which is shown in Figure 3.
The detection cycle is presented graphically in Figure 4. The following
description of the automatic operation of the detection cycle in CAM-4 shows
the time sequence activated by the electronic circuitry.
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Cell Voltage
Jack
Cell Open Lever
!
Pa.| Holder
Substrate
Pump
Digital Printer
1 "' i "v"" n
Cycle Lights
Pump Access Water
Panel Inlet
/ Substrate
Drain' Supply Bottle
Switches
Fan
ff Switch
Inverter
DC Input Plugs
AC Input Plug
Figure 2. Internal view of portable Cholinesterase Antagonist Monitor, CAM-4.
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Manual =|i »
Pad
Changer
Substrate
Pump
Motor
Water
Pump
Motor!
Figure 3. View of manual enzyme pad changer and motors in
CAM-4 (behind left panel).
I
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Water Sampling Pump On
Air and Substrate
Pumps On
Current Applied
0 60 120
t * - (sec)
Start
Timing
Cycle
180
t
Measure
Voltage
and
Print
Value
Figure 4. CAM-4 3-min operating cycle for collecting enzyme inhibitors
on an immobilized enzyme pad and subsequently measuring a
voltage related to the enzyme activity of the pad.
11
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0 sec Everything off.
1 sec Turn water pump on.
120 sec Turn water off; start air and substrate flow.
140 sec Apply constant current to electrochemical cell.
180 sec Print voltage on digital printer and advance paper one
line. Turn off substrate solution and applied current
and reset clock to "0".
As mentioned earlier, the decision to incorporate a 12-v DC to 110-v AC
inverter was based upon several considerations. Principally, the water,
air, and substrate pumps and the digital printer all require 110-v AC or DC
voltages differing from 12 v. Although DC motors might have been obtained
for these applications and the printer modified to operate on 12 v, it was
easier to obtain and service commercially available 110-v equipment than 12-
v equipment. A further consideration involved the need to operate the
substrate pump at constant speed to assure a flat baseline voltage. Con-
stant speed DC pump motors in the appropriate size are more expensive and
more difficult to control than are commercial AC motors.
Figures 5a, 5b, 6, 7, and 8 are the wiring diagrams for the CAM-4
system. Figures 5a and 5b show the "DVM" board, which provides the analog-
to-digital conversion for the signal obtained from the cell voltage ampli-
fier. Also shown in these drawings are the constant current source for the
electrochemical cell and the regulated power supplies for the remainder of
the circuits.
Figure 6 presents the wiring of the 12-v DC to 110-v AC inverter and
also the switching gear for operation of CAM-4 from either AC or DC power.
Figure 7 depicts the circuitry for the time circuit and the optically
coupled triac switching circuits for the pumps. Figure 8 shows the inter-
connections of the panel switches, the digital printer, the printed circuit
boards, and the other components.
The electrochemical cell design adapted for use in the CAM-4 is pre-
sented in Figure 9. The immobilized enzyme pad is placed between the two
perforated platinum electrodes, which are held in the injection molded
holders made of Cyclolac plastic. These electrode holders are held under
constant spring tension against the enzyme pad holder to form a leak proof
seal—the 0-ring between each surface is not shown in the drawing. The
constant current of ^ 2 yA applied to the electrodes once each cycle is
supplied by a regulated 8.5-v DC power supply with a 2.7-megohm resistor and
a 1-megohm potentiometer in series.
12
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Figure 5a,
Power
Ground
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12 ISVUnreg.
22 ) 5V Reg.
Power
13 )0n
Reset
POWER SUPPLY BOARD
Figure 5b.
Analog to digital converter and cell signal Power supply board providing regulated +5 and ±15-v
amplifier and current source. DC for logic boards and power for printer.
-------
H
n>
3 H-
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Binding Post
Red o
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Black 9
Power Switch
DC Off AC
Green
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=f=470^j/16V
Inverter
12V DC to 115V AC
1
5 VAC
y to Power
j Supply Board
40VAC C.T.
o > to Power
I Supply Board
BA Type
18A1487
n
INVERTER
-------
R17
R5
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1/2 MC
3J5EL.cn
IMil
7410,
IC6 \X IC7
83 |6 2
Current Power Resef
Control On
Reset
Pump
TIMER
Figure 7. Timer and optically coupled tiriaic switching circuits for pumps.
-------
Data Lines .
1248 II Printer
10 20 40 80 V ^ Connectors
100200400800 [C1&C2 CONNECTORS TO PRINTER
1000 J a a
DVM A B A B
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
PS
Gnrf
Gnd
A Gnd&
Trans C-T
t-15
-15
AC 15
AC 15
5 Unreg
Power
On Reset
AC 5
ACS
Power
Gnd
Power
Gnd
ACS
ACS
Power
Power
5
+5
J
J
J
J
1
J
]
"1
J
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LM309 -
TIMER
Reset
Out
Power
On Reset
S.S.
Sw
Cur
Sw
H2O
Sw
Cur
Control
S.S.
Control
Reset
In
H2O
Control
H2O
Indicator
S.S.
Indicator
*5V
AC
Common
H2O
Pump
S.S.
Pump
AC
Hot
1
1_
1
L
L-
L_
L-
DVM
+15
-15
Cell
A Gnd
Reset &
Print
Cur
Control
1
2
4
8
10
20
40
80
100
200
400
800
1000
Current
Indicator
+5
-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Gnd
Gnd
Black
Gnd
Gnd
Gnd
20
Yellow/Wht
2
Red/Wht
40
Greem/Wht
400
Green
Gnd
Gnd
Gnd
Gnd
80
Orange
8
Violet
Gnd
+5
Red
Print
White
4
Blue
Gnd
Gnd
>
1000
Purple
100
Green
Gnd
800
Green/Wht
Gnd
200
Orange
+5
Red
10
Blue
1
Black
+ 5 Gnd's Tied Internally
Power 5
O
Switches (Rear View)
O O O
O O—
0
?
Current Substrate H2O
OLED's
J(XX
1 JUC
YVX
'Wv
*~*-|A_A-
1/2 of CAM-4
Includes Cell
and Motors
Figure 8. Wiring diagram showing interconnection of panel switches,
digital printer, printed circuit boards, and other components.
16
-------
Injection Molded
Plastic Electrode
Holder
Enzyme Pod
Platinum Electrode
Waste
Figure 9. Cross section of an electrochemical cell developed for water
monitoring showing the platinum electrodes above and below the
enzyme pad to which a constant current is applied.
17
-------
A brief description of the operation of the electrochemical cell for
the generation of electrochemical voltages related to the activity of the
enzyme pad between the electrodes follows:
During the 2-min water sampling portion of the 3-min detection cycle,
the water is pumped through the immobilized enzyme pad at * 200 ml/min.*
Enzyme inhibitors, if present, are collected on the enzyme pad, where they
inhibit all or part of the active sites on the enzyme pad. During the
enzyme testing portion of the detection cycle, air at ^ 2 liters/min is
pumped through the cell continuously to remove the excess liquid from the
enzyme pad. At the same time, a solution of butyrylthiocholine iodide
(BuSChI) is pumped through the enzyme pad; during the last 40 sec of the
enzyme testing part of the cycle, a 2-uA constant current is applied and the
resulting cell voltage is recorded by the digital printer. (See Figure 4
for the complete detection cycle.)
When this system is functioning normally in the absence of inhibitors,
the substrate is cleaved by the enzyme to produce a thiol, which contacts
the lower platinum electrode (the anode). In this case (no inhibitors
present), a voltage of approximately 200 mv is produced, which is charac-
teristic of a new working enzyme pad. In the case where the enzyme is
completely inhibited (or when a blank pad is isolated in the electrochemical
cell), voltages of 400 mv or more are obtained. Intermediate voltages are
generated in proportion to the extent of inhibition of the enzyme. In
actual operation of the system, a voltage rise of 10 mv or more per cycle is
considered to be an "alarm" condition; a single enzyme pad may signal ten or
more alarms before it must be replaced with a fresh enzyme pad. However,
exposure of an enzyme pad to high levels of inhibitor could inhibit all of
the enzyme in one or two cycles.
* This pumping rate is less than half of the water flow rate in CAM-1. How-
ever, there is little loss in sensitivity with the lower pumping rate
since the diffusion of the inhibitor to the active sites on the enzyme is
the rate limiting reaction.
18
-------
SECTION 5
CAM-4 OPERATING PRINCIPLE
The operating principle for CAM-4 is as follows: an electrochemical
nrocess 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 9, the porous enzyme pad is
located between two porous platinum electrodes. First, the water sample is
pumped through the enzyme pad, at approximately 200 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 the 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 any residual water. Next, a solution of substrate,
consisting of butyrylthiocholine 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 yA is
applied 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
thiocholine iodide, which possesses a thiol group that produces character-
istic low voltages in the electrochemical cell.
+ - ChE +
C3H7COSCH2CH2N(CH ) I — ^ C^COOH + HSCH2CH2N(CH3)3I
electrochemical oxidation
+ I +
(CH3)3N-CH2CH2-S-S-CH2CH2N(CH3)32I
On the other hand, in the presence of enzyme inhibitors, the substrate is not
hydrolyzed; there is no thiol formation and the cell voltage rises *> 200 mv
(from ^ 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 present
in the sampled water.
The cell voltage changes observed in the operation of CAM-4 resemble
those observed for CAM-1. Consider CAM-1 response to water containing 0.2
ppm of DDVP (dimethyl dichlorovinyl phosphate). In this case, the voltage
tracing shown in Figure 10 was generated by applying a constant current to
the enzyme pad during each cycle. Upon encountering a cholinesterase inhib-
itor, a change in voltage is recorded. The voltage change from cycle to
cycle is used to trigger an alarm. As shown in Figure 10, during the first
24 min there is a very slow steady voltage rise from cycle to cycle, indica-
ting the gradual deterioration of the immobilized enzyme pad. The alarm
-------
level is set so that these changes are too small to trigger an alarm. How-
ever, when 0.2 ppm of DDVP is added to tap water, a sharp increase in the
height of the voltage peak occurs. Each cell voltage increase of 10 mv or
more between cycles is considered to be an alarm. (The 10-mv alarm thresh-
old can be changed, if desired.) In the present example, 10 individual
alarms from the same enzyme pad resulted from sampling the DDVP. If a
higher concentration of DDVP—perhaps 2 ppm—had been used, the cycle-to-
cycle voltage increases would have been much greater.
Enzyme pads cannot be used indefinitely. After a time, when there is
insufficient enzyme activity on the enzyme pad to allow a 50-mv voltage rise
when inhibitors are sampled, the used enzyme pad must be rejected as a
safety factor and a new pad inserted into the system by the operator.
Like CAM-1, the portable water monitor produces voltage increases when
cholinesterase inhibitors are present in the water. Unlike CAM-1, however,
the portable CAM-4 does not trigger an alarm, but relies on the operator to
determine when inhibitors are present and when to replace enzyme pads.
20
-------
300
> 200
LU
i= 100
1/1
0
-100
L_
0
15
30
45
TIME (Min)
Figure 10. Response of the electrochemical cell operating on the 3-min cycle to water
containing 0.2 ppm DDVP.
-------
SECTION 6
OPERATING PARAMETERS FOR THE ELECTROCHEMICAL ENZYME SENSOR
An explanation has been given of the basic principles involved in the
operation of an electrochemical cell for the detection of low levels of or-
ganophosphates in water supplies (Sections 1 and 4, and Appendix E). In
order to make this electrochemical enzyme cell both sensitive and reliable
for the detection of enzyme inhibitors, it was necessary to control those
variables that affected the response of the cell: (a) the buffer solution;
(b) the substrate; (c) the applied current; (d) the rate of water sampling;
(e) the time of water sampling, and the like. Some of these variables were
interdependent; others were not.
Tris buffer {tris(hydroxymethyl)aminomethane-hydrogen chloride} was
selected for use in our system because it was compatible with the platinum
electrodes, the enzyme, and the substrate. The selection of the buffer con-
centration at 0.08 M was arbitrary, and could have been changed to 0.10 or
0.15 M with little effect on the response of the unit to enzyme inhibitors.
However, the buffering capacity of the 0.08 M substrate is adequate for this
system. The pH of 7.4 for the'buffer is a compromise between a mildly
acidic pH where the substrate is very stable and pH 8.6 where the cholin-
esterase was especially active in hydrolyzing the substrate, butyrylthio-
choline iodide (BuSChI). At pH 7.4, the enzyme is quite active and the
spontaneous hydrolysis of the substrate is slow enough at room temperature
so that it usually is not a problem. That is, a substrate solution prepared
for use in the CAM-4 is still usable after 12 hr at 25°C. A procedure for
making the enzyme pads used in the electrochemical cell is given in Appendix
F.
Although much information regarding the theory of operation of CAM-type
pesticide monitors is given in the present report, the reader is urged to
consult the formal report on Task I of the present contract (EPA-600/2-77-
219, November 1977) for details relating to CAM-1 performance. In that
report may be found detailed discussions of the operating principle of the
electrochemical enzyme cell, the studies related to the selection of buffer,
pH and concentration, the effect of temperature on the operation of the sys-
tem, the response of CAM-1 to many additional organophosphate and carbamate
pesticides, the response to reversible inhibitors, the response to possible
interfering substances, a procedure for fabricating the enzyme pads for the
electrochemical cell, and other subjects related to operation of CAM-type
monitors.
22
-------
The following CAM-4 operating parameters have been selected to provide
sensitivity, reliability, and rapid voltage responses when the enzyme is
inhibited:
-4
Substrate: 2.5 x 10 M butyrylthiocholine iodide in 0.08 M tris(hy-
droxymethyl)aminomethane-HCl buffer, pH 7.4
Substrate . . .
Flow Rate: X ml substrate/minute
Enzyme Pad: 0.4-0.8 units of horse serum cholinesterase—entrapped in
aluminum hydroxide and starch gels—per enzyme pad
Applied
r, i_ 2 . 0 UA
Current: ^
Water Sam- . .
pling Rate: * 2°° ml/min
Optimization of substrate concentration and flow rate is crucial since
the quantity of substrate reaching the enzyme during each cycle affects the
alarm potential, the baseline voltage, and the sensitivity of the detection
system.
To maximize sensitivity, on one hand, addition of excess substrate is
undesirable, as illustrated by the following example. During each sampling
cycle under normal operation, water, contaminated by low levels of inhibitor,
flows through an electrochemical cell containing either a fresh or partially
inactivated enzyme pad. Then, substrate, in excess amounts, is passed
through the cell, as a constant current is applied. Because of the ready
availability of substrate, a small amount of enzyme inhibition will have
little effect on the rate and extent of hydrolysis (thiol formation). The
cell voltage remains- low, a characteristic of the absence of enzyme inhibi-
tion, in proportion to the amount of enzyme activity. Since an increase in
voltage from one cycle to the next is required to signal an "alarm," the
presence of the inhibitor is masked.
On the other hand, the use of very small amounts of substrate is also
undesirable, as illustrated by the following example. In the case in which
low substrate levels are used, very little thiol is formed per cycle and the
cell voltage rises, independent of the presence of inhibitors. This voltage
increase is seen as a "false alarm." To avoid a continuous "alarm" situa-
tion, the alarm threshold could be increased, but low levels of inhibitors
would still remain undetected since they cause minimal change in cell voltage.
A detailed step-by-step procedure for the preparation of enzyme pads for
use in both the CAM-1 and CAM-4 electrochemical cells is given in Appendix F.
Enzyme pads with activities in the range of 0.4 to 0.8 ymoles/min/pad are
recommended since pads with higher activities are likely to be less respon-
sive to,the inhibitors, and pads with lower activities are likely to give
intermediate baseline voltages (perhaps 300 mv) and are likely to fail sooner
than the pads with mare activity.
23
-------
An applied current of 2 yA is chosen to yield a good spread of cell
voltages between those encountered in the absence of enzyme inhibitors and
those encountered in the presence of inhibitors. The use of higher cell
currents could probably be tolerated if the concentration or flow rate of the
substrate solution were increased.
To insure that sufficient pesticide contacts the enzyme immobilized on
the pad and thus inactivates an appropriate fraction of the enzyme, the water
sample is pumped through the pad at a rate of ^ 200 ml/min. The pumping rate
is not extremely critical, since—at this pumping rate—the amount of inhibi-
tion occurring in a single cycle is limited by the rate of diffusion of the
inhibitor to the active sites of the gel-entrapped enzyme.
The following chemicals were purchased for use in the CAM-4 system:
- Butyryl cholinesterase Type IV-S from horse serum, Sigma Chemical
Company, Product :No. C-7512, approximately 15 units/mg (1 unit will
hydrolyze butyryl choline to choline and butyrate at a rate of 1
ymole/min at pH 8.0 and 25°C.
- Butyrylthiocholine iodide, A Grade, Calbiochem Product No. 2049,
melting point 172-173°C.
- Tris(hydroxymethyl)aminomethane, THAM, certified primary standard,
Fischer Scientific Company, Product No. T-295. (Note: Several dif-
ferent lot numbers were submitted and we chose the best one on the
basis of electrochemical cell performance, i.e., lowest in iron,
copper, and other heavy metals.) Analytical grade hydrochloric-acid
from Mallinckrodt was used to adjust the pH of the 0.08 M solution
in deionized water to prepare buffer. In previous studies, the sub-
strate was found to be unstable because of a reaction occurring in
the buffer. Although the chemistry of the reaction is unknown, un-
desirable changes can be avoided by treatment of the buffer with
petroleum-based pelletized charcoal, MCB Company.
24
-------
SECTION 7
OPERATING PROCEDURES FOR CAM-4
As explained in the previous sections, CAM-4 is not automatic but re-
quires an operator in attendance all of the time when rapid detection of
toxic materials is desired. Specifically, the operator must make up the sub-
strate solution (one day's supply at a time), install the enzyme pads in the
electrochemical cell when they are needed, determine when the enzyme pads are
nearly exhausted so that he can replace them, and signal the presence of
enzyme inhibitors when the baseline voltage increases 10 or more millivolts
in one cycle. Most of these tasks are handled automatically in CAM-1, which
is somewhat heavier, requires more power, and is more expensive than CAM-4.
When testing the CAM-4 instrument in the laboratory, it is necessary to
connect the inlet hose to the water source and the outlet water hose to the
drain; the unit is then connected to the appropriate power supply (either
12-v DC or 110-v AC). As a precaution, the water hose inlet for CAM-4 should
not be connected directly to any pressurized water source since hoses may
come off or the electrochemical cell may leak because of overpressurization.
Water should be sampled from an overflowing beaker or similar container in a
sink near the CAM-4, with the pump in CAM-4 used to suck the water into the
system. The drain hose must be arranged so that the liquid drains freely
and, accordingly, does not back up to fill the cell with liquid during the
enzyme activity measurement part of the detection cycle. The simplified op-
erating instructions given below will be of value to the reader or potential
operator who is unfamiliar with the instrument.
STARTING PROCEDURE
1. Insure that the power is off.
2. Prepare fresh substrate solution by dissolving 40 mg of butyrylthiocho-
line iodide in 500 ml of 0.08 M Tris buffer, pH 7.4 (9.7 g of tris(hy-
droxymethyl)aminomethane in 992 ml of distilled or deionized water with
enough concentrated HC1, -\» 8 ml, to bring the pH to 7.4). Place the
substrate solution in the plastic bottle in the lower left hand side of
the instrument with the inlet end of the small plastic tube reaching the
bottom of the bottle.
3. Open the electrochemical cell using the "cell open lever" shown in Fig-
ure 2 and remove the plastic enzyme pad holder. Place a fresh enzyme
pad in the pad holder and insert into the electrochemical cell. Close
the cell by pushing the "cell open lever" up.
25
-------
4. Move the "AC or DC switch lever" up for AC or down for DC depending upon
which power source is used.
5. Prime the substrate lines by moving the "substrate toggle switch" to the
"up" position (red light comes on) for ^ 3 to 5 min. When the line will
not prime, replace the pump tube with a new silicone rubber pump tube '
(Scientific Industries, Tube No. SR-094).
6. Prime the sample water inlet line as follows:
a. Place water inlet tube (Figure 2) into the water to be monitored.
(If monitoring water in a lake or stream, place a screen or filter
over the intake end of the tube. Open-pore urethane foam sponge
makes a satisfactory filter.)
b. Push "water toggle switch" to the "up" position (red indicator
light should come on and the pump motor should start).
c. Prime the pump by attaching a rubber vacuum/pressure bulb (similar
to Fischer No. 14-087) to the drain line so as to suck water into
the line and prime the pump.
7. Disconnect the rubber bulb and arrange the drain line so that water will
drain out of it freely. (Caution: Back pressure on this line will
cause water to remain in the cell during the voltage measuring part of
the cycle and will make the system inoperative.)
8. After the water and substrate pumps are primed, place all toggle switches
in the "down" or "automatic" position. The timing cycle will start and
the indicator lights will come on in sequence to indicate the water sam-
pling, the substrate pumping, and the applied current portions of the
detection cycle are operating properly.
DISCUSSION OF CAM-4 OPERATION
Although the preceding steps put the instrument into operation, addi-
tional steps are necessary for the operator to interpret the numbers being
generated by the digital printer. Specifically, the. operator needs to know
if his enzyme pad has any activity and how he can check the response of the
instrument to specific solutions of enzyme inhibitors in order to calibrate
its performance. Also, he needs to know when to replace the enzyme pad with
a fresh one. The following paragraphs will help the operator understand what
the instrument can do and what he must do.
Three definitions that will assist in the discussion to follow are
these: starch pad potential, enzyme pad potential, and alarm potential. The
starch pad potential is the voltage obtained when CAM-4 is operated with an
exhausted enzyme pad or with a pad made like an enzyme pad but with no enzyme
added to it. The voltage obtained with it is the highest voltage obtainable
on the CAM-4 and is called starch pad potential or Vo (i.e., voltage with
zero enzyme). Similarly, the enzyme pad voltage is the voltage obtained by
CAM-4 when an enzyme pad is used in the electrochemical cell; it is desig-
nated as Venz and varies with the amount of enzyme on the pad. The difference
26
-------
between Venz and V0 is called the alarm potential. By definition, the alarm
potential is the change in voltage that would occur if all of the enzyme in
an operating CAM-4 were inhibited. Normally, with good enzyme pads, the
alarm potential varies from 200 to 300 mv. It is obvious that a spent enzyme
pad cannot be used to detect enzyme inhibitors. To make certain that a work-
ing enzyme pad is always present in the electrochemical cell, the operator is
instructed to replace the enzyme pad when the cell voltage comes within 50 mv
of the V0. For example, if an operator puts a starch pad in the electro-
chemical cell and determines that V0 is 350 mv, then he should make a record
of this value and replace the working enzyme pad* before the cell voltage
reaches 300 mv.
Because CAM-4 is not temperature-compensated and because there is often
a difference in the dissolved solids in the water sampled, it is necessary to
determine V0 at least once or possibly twice each day that the instrument is
used.
Figure 11 shows typical printed cell voltages (in volts) obtained in the
absence of inhibitors with the electrochemical cell operating on the 3-min
cycle shown in Figure 4. During the water pumping part of the cycle (first
120 sec) the sampled water is pumped through the enzyme pad in the electro-
chemical cell at a rate of 200 ml/min. Some of the active sites on the
enzyme pad will become inhibited by the inhibitors in the water, if they are
present. For the last minute of the detection cycle, the water is turned off
and air is blown through the cell. At the same time, the substrate flow
starts. The rate of substrate hydrolysis is a function of the amount of
enzyme activity left on the enzyme pad. A build-up of substrate solution in
the cell is prevented by the stream of air. After the air and substrate
pumps have been on for 20 sec, current is continuously applied to the elec-
trodes in contact with the enzyme pad for 40 sec. Just before the current is
turned off and the water pump is turned on again, the voltage at the elec-
trodes is measured and printed on the paper tape. This voltage is low when
the enzyme pad is active and increases when inhibitors are present. For the
present study, we have assumed that a 10-mv increase or more in cell voltage
in one 3-min detection cycle constitutes evidence for the presence of inhib-
itors in the water sample. This sudden increase in voltage is considered to
be an "alarm" situation and is to be recognized by the operator since the
instrument (unlike CAM-1) has no automatic method of flashing a light or
sounding a horn.
Fresh enzyme pads will normally produce cell voltages, Venz, that are
at least 200 mv less than V0. A new enzyme pad should keep the CAM-4 system
in operation for a day or more, providing that inhibitors are absent from the
water sampled; this is usually the case in a clean environment. However, the
life of the pads is reduced in hot weather and they should also be protected
from exposure to direct sunlight or humid environments. Thus, enzyme pads
are normally stored in a dry, sealed container in the refrigerator.
* Caution:Always turn system off before opening cell to change pads.
27
-------
*
1)
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Figure 11. Digital printout in volts from CAM-4 showing cycle-to-cycle
variation (noise) obtained when operating on a 3-min detec-
tion cycle in the absence of inhibitor.
28
-------
MONITORING PROCEDURE
9. Follow the "Starting Procedure" (Steps 1-8) but insert a starch pad (no
enzyme) into the electrochemical cell and sample uncontaminated or char-
coal-filtered water.
10. Allow instrument to run for five 3-min cycles and record the last starch
pad voltage, Vo.
11. Shut off CAM-4, insert fresh enzyme pad in cell, close cell, and turn on
power.
12. Observe cell voltage, Venz, for at least three cycles. Replace enzyme
pad with fresh enzyme pad if Venz is less than 150 mv lower than V0.
13. Turn power off momentarily. Remove inlet hose from uncontaminated water
and place in water to be monitored for inhibitors. Turn power on.
14. Observe paper tape to determine sudden increases of cell voltages, 10 mv
or more per cycle, which indicate presence of inhibitors.
15. From time to time, check cell voltage, Venz, to determine whether enzyme
pad should be replaced, i.e., compare with V0 (Step 10 above) and re-
place if the difference is 50 mv or less.
16. Optional. To calibrate the system, prepare a gallon of water (3,785 ml)
containing 0.2 ppm of DDVP (3.8 mg in 1 gal) at the same temperature as
the water being sampled by the CAM-4. Switch from the uncontaminated
water to the DDVP solution for a minimum of three water sampling cycles.
The voltage response should average at least 10 mv/cycle. In the exam-
ple, Figure 12, the average increase per cycle is 13 mv.
SHUT-DOWN PROCEDURE
17. Remove substrate and water inlet lines from their respective solutions
and allow them to pump dry.
18. Remove the plastic substrate supply bottle from the case, discard the
unused substrate solution, and rinse and dry the bottle.
19. Turn AC-DC power switch to "off."
20. Open electrochemical cell and remove enzyme pad holder; discard pad and
wash pad holder.
21. For extended periods of disuse it is advisable to release the tension on
the pumping tube of the substrate pump. This is easily achieved by
lifting the tube off the rollers and pulling the tube toward the oper-
ator.
29
-------
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1 3 S'
1 3D
! 3 5
1 ff 9
Iff 9
/ 3 1
Iff 1
Iff 9
13 I
Iff S
Iff S
Iff 9
13?
1
"
0.178 After
0.138 Before
0.040 Change
av. change/cycle = 0.013 v
Figure 12. Typical calibration of CAM-4 showing cycle-to-cycle
voltages before (lower portion of tape) and after
(upper portion) exposure to 0.2 ppm DDVP for
three cycles.
(Note: Since starch pad voltage for this run was 327 mv, the CAM-4 was
able to continue monitoring with the same enzyme pad.)
30
-------
SECTION 8
LABORATORY STUDIES WITH CAM-4
Testing of the two CAM-4 instruments built on this contract, namely,
CAM-4-A and CAM-4-B, has centered around three specific goals: (a) response
of CAM-4 to eight selected organophosphate and carbamate pesticides and sub-
sequent determination of the sensitivity limit for each compound; (b) com-
parison of CAM-4 sensitivity to CAM-1 sensitivity for several representative
pesticides; and (c) operation of CAM-4 under widely diverse field conditions
for which CAM-4 was designed. All three of these goals were achieved during
the laboratory and field tests of CAM-4 and are described in this and subse-
quent sections of this report.
The response of CAM-4 to eight commercial pesticides, presented in
Tables 1 and 2, was determined in laboratory tests. Various quantities of
each pesticide were dissolved in city tap water (15 C) and pumped through the
instrument for five cycles. Voltage change was recorded for each of these
cycles, plus one additional cycle; the responses were recorded for three
trials. An average voltage increase of 10-mv or more per cycle (the total
response divided by five) was considered sufficient for detection. The lower
level of detection was considered to be that pesticide concentration that
produced two or more 10-mv increases during the five cycle exposure period.
Table 1 shows the response of CAM-4 (both systems A and B) to representative
carbamate pesticides, while Table 2 shows the response of CAM-4 systems to
representative organophosphate pesticides. In each of the tables, the lowest
level of pesticide tested was considered to be the minimum detectable under
the test conditions.
Sensitivities of the portable detection model, CAM-4, and of the labora-
tory model, CAM-1, were expected to be very nearly alike since the electro-
chemical and biochemical components of each are essentially the same, al-
though the flow rates differ somewhat. Still, it seemed desirable to conduct
a side-by-side comparison of response of the two systems to several pesti-
cides. Table 3 presents the comparative study of the two systems to several
carbamates and various organophosphates. As can be seen from the table, the
total response in millivolts for each of the systems was very nearly the same
during each of the three trials. One possible exception is the response of
CAM-1 to Mesurol, which was slightly greater than any response on either CAM-
4-A or CAM-4-B. No explanation for this difference is offered.
31
-------
TABLE 1. RESPONSE OF CAM-4 TO CARBAMATE PESTICIDE SOLUTIONS0
System
CAM-4A
CAM-4B
CAM-4A
CAM-4B
CAM-4A
CAM-4B
Pesticide
tested
a
Mesurol
Mesurol
Baygon
Baygon
Sevin1
Sevin
Level
ppm°
12
8
12
8
20
15
20
15
20
10
5
20
10
5
Voltage response,
for each cycle, mv
11
7
9
12
7
8
4
11
27
17
0
10
20
0
13
13
14
8
18
18
28
10
33
10
14
38
20
7
15
14
23
15
13
9
16
7
51
20
12
34
17
10
14
11
21
16
18
13
13
11
36
25
10
22
23
9
18
10
19
15
13
10
16
12
40
20
9
26
13
8
^
6
0
-2
-1
2
2
6
5
0
0
2
-3
-2
10
3
Total voltage change after
five cycle exposure, mv
Trial
71
53
85
68
71
64
82
51
187
94
42
128
103
37
Trial
57
56
83
73
101
54
54
51
168
102
45
136
86
28
Trial
66
41
87
72
78
53
63
61
175
98
43
115
90
31
Mean
standard
deviation
65 ± 7.1
50 ± 7.9
85 ± 2.0
71 ± 2.7
83 ± 16.0
57 ± 6.1
66 ± 14.0
54 ± 5.8
177 ± 9.6
98 ± 4.0
43 ± 1.5
126 ± 10.6
93 ± 8.9
32 ± 4.6
Alarm
potential
average
297
290
301
299
260
283
280
260
255
280
290
205
275
280
CO
to
Insoluble pesticides
The cor-
a Two CAM-4 units (A and B) were operated at 15 C using horse serum cholinesterase enzyme pads
(4-5-76, 0.500 units activity). Substrate was 2.5 x 10 M butyrylthiocholine iodide in 0.08 M
Fisher THAM, pH 7.4.
b A 1-liter stock solution of 1,000 ppm was made fresh for each pesticide.
were dissolved in a small amount of alcohol before mixing with 1 liter of pH 5.7 H_0.
rect amount of stock solution was poured slowly into a stirred carboy of tap H?0 for sampling.
c Level tested is based on active ingredient.
d Exposure to"stirred solution was for 5 cycles and voltage recorded for 6 cycles. Responses shown
are for Trial 1.
e Three trials were run at each level for each system.
f Alarm potential is an average of all three trials and is calculated from the initial baseline
using a fresh enzyme pad.
g MesurolR = 4-(methylthio)-3,5-xylyl methylcarbamate.
h Baygon = 0-isopropoxyphenyl methylcarbamate.
i Sevin = 1-naphthyl N-methylcarbamate.
-------
TABLE 2. RESPONSE OF CAM-4 TO ORGANOPHOSPHATE PESTICIDE SOLUTIONS*
System
CAM-4A
CAM-4B
CAM-4A
CAM-4B
CAM-4A
CAM-4B
Pesticide
Tested
Nemacur8
Nemacur
Baytex
Baytex
Systox-1
Systox
Level
ppm
0.4
0.2
0.15
0.1
0.4
0.2
0.15
0.1
20
10
6
5
20
10
6
5
2
0.5
0.1
0.05
0.025
2
0.5
0.1
0.05
0.025
Voltage Response,
For Each Cycle, mv
1 1 2
17
13
•j
6
21
17
10
10
19 ] 35
16 i 30
9
O
37
29
25
24
27
38
23
19 _,
185
68
27
10
10
109
41
26
6
10
11
13
66
22
15
C
36
31
15
15
56
98
29
9
11
48
44
28
14.
11
3
23
15
9
9
40
24
19
5
14
20
19
5
12
15
12
9
12
57
22
17
23
25
25
16
8
3
4
19
14
13
14
32
25
16
15
i
10
9
4
11
15
11
13
3
13
25
12
3
22
17
15
11
11
•j
19
15
10
10
5
2
19
30
_—
10
8
11
4
6
17
13
1
5
18
5
12
3
1
10
6
7
6
-3
-2
-1
-1
-26
-19
0
0
—
-16
-10
0
0
-30
-14
-22
-9
-17
-1
21
-19
-9
-8
-2
6
6
Total Voltage Change After
Five Cycle Exposure^ mve
Trial
1
96
72
48
48
105
78
74
65
__
75
66
49
90
75
64
47
249
224
120
74
40
198
120
93
51
48
Trial
2
90
62
61
39
119
80
66
67
___
85
60
39
99
72
70
54
257
229
128
72
70
212
132
100
63
57
Trial
3
88
68
49
44
152
95
71
59
__
70
63
42
98
57
62
43
229
215
105
78
51
209
141
98
67
46
Mean
Standard
Deviation
91 ± 4.2
67 ± 5.0
53 ± 7.2
44 + 4.5
125 ±24.1
84 ± 9.3
70 ± 4.0
I 64 ± 4.2
+
77 ± 7.6
63 ± 3.0
43 ± 5.1
96 ± 4.9
68 ± 9.6
65 ± 4.2
48 + 5.6
245 ±14.4
223 ± 7.10
118 ±11.7
75 ± 3.1
54 ±15.0
206 ± 7.37
131 ±10.5
97 ± 3.6
60 ± 8.3
50 ± 5.9
Alarm
3otential
Average
284
249
285
281
283
280
307
251
200
252
290
283
262
256
250
232
273
285
310
280
238
266
273
266
200
202
a Two CAM-4 units (A and B) were operated at 15 C using horse serum cholinesterase enzyme pads
(4_5_76) 0.500 units activity)(responses to Systox used HSChE enzyme pads, 1-21-75, 0.513 units
activity). Substrate was 2.5 x 10 M butyrylthiocholine iodide in 0.08 M Fisher THAM, pH 7.4.
b A 1-liter stock solution of 1,000 ppm was made fresh for each pesticide. Insoluble pesticides
were dissolved in a small amount of alcohol before mixing with 1 liter of pH 5.7 1^0. The cor-
rect amount of stock solution was poured slowly into a stirred carboy of tap H^O for sampling.
c Level tested is based on active ingredient.
d Exposure to stirred solution was for 5 cycles and voltage recorded for 6 cycles. The responses
shown are for Trial 1.
e Three trials were run at each level for each system.
f Alarm potential is an average of all three trials and is calculated from the initial baseline
using a fresh enzyme pad.
g Nemacur = ethyl 4-(methylthio)-M-tolyl isopropylphosphoramidate.
h Baytex = 0,0-dimethyl 0-{4-(methylthio)-M-tolyl}phosphorothioate.
i Electrical problems—system cycling erratically—did not finish response.
j SystoxR = mixture (2:1) of 0,0-diethyl 0-2-(ethylthio)ethyl phosphorothioate and 0,0-diethyl
S-2-(ethylthio)ethyl phosphorothioate.
33
-------
TABLE 3. COMPARATIVE RESPONSE OF CAM-4 AND CAM-1 TO PESTICIDE SOLUTIONS'
System
CAM-4A
CAM-4B
CAM-1
CAM-4A
CAM-4B
CAM-1
CAM-4A
CAM-4B
CAM-1
CAM-4A
CAM-4B
CAM-1
Pesticide
tested
DDVPS
DDVP
DDVP
Furadan h
Furadan
Furadan
"D
Systox i
Systox
Systox
Meaur.ol^-3
Mes.ur.ol
Mes.ur.el
Level
c
ppm
0.3
0.3
0.3
0.7
0.7
0.7
0.15
0.15
0.15
8
8
8
Voltage response
for each cycle, mv
1
10
9
8
5
10
5
19
21
20
8
11
12
2
10
11
12
8
7
18
12
9
18
14
11
19
3
11
7
8
7
13
10
4
13
22
12
15
16
4
7
12
8
12
8
9
13
26
5
12
8
0.6
5
9
14
15
9
8
3
15
12
17
12
17
19
6
-3
-3
2
2
-1
5
10
2
-10
8
-4
-6
Total voltage change after
five cycle exposure, mv
Trial
1
44
50
53
43
45
50
73
83
72
66
58
76
Trial
2
50
60
53
38
40
39
47
50
49
51
40
60
Trial
3
53
49
62
43
37
44
60
64
75
50
63
68
Mean
standard
deviation
49 ± 4.6
53 ± 6.1
56 ± 5.2
41 ± 2.9
41 ± 4.0
44 ± 5.5
60 ± 13
66 ± 17
65 ± 14
56 ± 9.0
54 ± 12
68 ± 8.0
Alarm
potential
average
240
235
286
257
223
260
267
250
250
245
255
275
u>
•e-
horse serum cholinesterase
M butyrylthiocholine
a Two CAM-4 units (A and B) and one CAM-1 unit were operated at 10 C usin;
enzyme pads (5-24-76, 0.326 units activity). Substrate was 2.5 x 10-
iodide in 0.08 M Fisher THAM, pH 7.4 (Tris buffer).
b A 1-liter stock solution of 1,000 ppm was made fresh for each pesticide. Insoluble pesticides were
dissolved in a small amount of alcohol before mixing with 1 liter of pH 5.7 1^0. The correct
amount of stock solution was poured slowly into a stirred carboy of tap F^O for sampling.
c Level tested is based on active ingredient.
d Exposure to stirred solution was for 5 cycles and voltage change recorded for 6 cycles. The
responses shown are for Trial 1.
e Three trials were run at each level for each system.
f Alarm potential is an average of all three trials and is calculated from the initial baseline using
a fresh enzyme pad.
g DDVP = 0,0-dimethyl 0-2,2-dichlorovinyl phosphate.
h Furadan^ = 2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate.
i SystoxR = mixture (2:1) of 0,0-diethyl 0-2-(ethylthio)-ethyl phosphorothioate and 0,0-diethyl
S-2-(ethylthio)ethyl phosphorothioate.
j MesurolR = 4-(methylthio)-3,5-xylyl methylcarbamate.
-------
SECTION 9
FIELD TESTING OF CAM-4
Both CAM-4 instruments were operated under a wide variety of field condi-
tions during their evaluation. The purpose of the field tests was two-fold:
(a) to gain experience in transportation of the system and the auxiliary
equipment required to operate and maintain the unit, including the 12-v power
source, filter sponge, tubing, solutions, pH paper, thermometer, tools, pads,
etc.; and (b) to monitor the performance of the CAM-4's under natural environ-
mental conditions. Several parameters were recorded at each location during
the field operations. These included water temperature and pH, air tempera-
ture, condition of the sample water (clean, turbid, debris, flowing or stag-
nant), and any other environmental conditions specific to each site.
Observations were made with respect to false alarms, baseline stability,
and alarm potentials at each location. A log was kept during the monitoring
of each location and observations were entered approximately every hour.
Problems encountered during each field test were recorded so that these could
be evaluated and corrected before initiation of subsequent tests. Part of
the test protocol at each sample site was an exposure to a standard solution
of DDVP (2,2-dichlorovinyl dimethyl phosphate) made up in a large container
with the water at the site. This system check was done to ascertain the
sensitivity of the system with the raw water actually being sampled. A brief
description of five field tests with CAM-4 is given in Appendix C of this
report.
Samples obtained from the Kansas City Municipal Water Treatment Plant,
the Missouri River (raw, untreated), several areas of a farm pond, a storm
drainage ditch, and the effluent from a local pesticide manufacturing plant
were tested with the CAM-4. No significant difficulties were encountered.
When CAM-4 is used under field conditions, such as those described below, it
is essential to attach a filter to the water inlet line of CAM-4 to remove
debris that might cause plugging of the platinum electrodes or of the one-way
valve in the sample line. With the sample line inserted into the center of a
small cube of urethane foam (5 in. on an edge), all field tests with the
instrument proved to be satisfactory. The urethane foam served both as a
filter and as a float to keep the inlet line just below the water surface,
thereby preventing air bubbles from entering the water pump. When CAM-4 is
operated from a small boat, it is essential that no part of the drain tube
from the cell be higher than the bottom of the cell, i.e., water must not be
allowed to collect in the drain tube since this produces back pressure on the
electrochemical cell and results in an erratic baseline. Good drainage is
easily achieved by setting the instrument on the boat seat rather than on the
floor of the boat.
35
-------
On the basis of the field tests, it is concluded that the CAM-4 instru-
ments performed well under a wide variety of environmental conditions and
were able to detect the presence of toxic levels of cholinesterase inhib-
itors in water downstream from the discharge of a pesticide manufacturing
plant. The systems would be easier to operate if self-priming water pumps
were used to replace the present gear pumps, but such pumps in the appro-
priate size and weight do not seem to be available commercially at this
time. (See Appendix C for Field Test with CAM-4.)
SUMMARY OF CAM-4 OPERATION IN THE FIELD
1. Both CAM-4's were operated in the field in an air temperature range of
6°C to 21°C.
2. Water temperature tested at the five locations ranged from 8 C to 23 C
while pH varied from 5 to 7.5.
3. CAM-4 was operated for a total of 31-1/2 hr in the field.
4. During the field tests, a floating urethane sponge filter was used on
the water inlet line to prevent plugging and proved satisfactory under
widely varying conditions.
5. No false alarms occurred during the 31-1/2 hr of testing although a
minor electrical problem was encountered with one printer.
6. No electrical or chemical interference problems were encountered during
the field tests.
7. At each location during field operation, CAM-4 was exposed to a stan-
dard sample of DDVP. A rapid increase in the voltage demonstrated that
the system was functioning properly.
8. CAM-4 proved to be portable, was easily operated from a river bank or
from a small boat, and performed well in clear, drizzly, or humid
weather.
9. A fully charged 12-v automobile battery provided sufficient DC power to
operate the CAM-4 continuously for 8 hr.
10. No problems were encountered in pad changing and the substrate solution
was easily prepared in the field.
36
-------
APPENDIX A
ABSTRACT OF CONTRACT ON CAM-1 DEVELOPMENT
Rapid Detection System for Organophosphates and
Carbamate Insecticides in Water
Environmental Protection Technology Series EPA-R2-72-010, August 1972
(Final Report, Contract No. 68-01-0038)
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 Antagon-
ist 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, mala-
thion, parathion, and DDVP has already been demonstrated. CAM-1 uses immo-
bilized cholinesterase for the collection of cholinesterase inhibitors from
the water supplies. The activity of the immobilized cholinesterase is deter-
mined automatically in an electrochemical cell by passing a substrate solu-
tion 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 that occurs in the presence of organo-
phosphate 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.
37
-------
APPENDIX B
ABSTRACT OF TASK I REPORT ON THIS CONTRACT
Evaluation of "CAM-1," A Warning Device for Organophosphate
Hazardous Material Spills
Environmental Protection Technology Series EPA-600/77-219, November 1977
(Final Report on Task I, Contract No. 68-03-0299)
The Cholinesterase Antagonist Monitor (CAM-1) has been operated with
water containing a variety of pollutants including organophosphates, carbarn- '
ates, chlorinated hydrocarbons, and various other economic poisons, and its
sensitivity to many of these materials has been measured. With few exceptions,
only the organophosphates and carbamates are detectable with CAM-1. One of
these exceptions is zinc, at 10 ppm, which inactivates cholinesterase and
behaves in CAM-1 like the organophosphates. Another compound detectable under
certain conditions is the reversible cholinesterase antagonist, tributyl amine
hydrochloride; it is detectable for only one or possibly two cycles when a
sudden increase in the concentration of the reversible inhibitor occurs. The
non-reversible enzyme inhibitors, on the other hand, produce repeated voltage
increases until the enzyme in CAM-1 is completely inactivated. CAM-1 is recom-
mended only for the detection of non-reversible 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 phosphorothioate
(-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.
Operation of CAM-1 in simulated sea water (3% NaCl) changes the voltages
registered on the digital voltmeter, but it does not change the sensitivity
of CAM-1 for compounds like DDVP; thus, CAM-1 is suitable for the detection of
cholinesterase inhibitors in either sea or brackish waters. CAM-1 has much
promise for monitoring of water supplies and plant effluents for cholinesterase
inhibitors, but it is so new that it should be investigated under the condi-
tions of intended usage prior to putting it into regular service.
38
-------
APPENDIX C
CAM-4 FIELD TEST REPORT
The first site for field testing was a 7-acre lake located on an experi-
mental farm in Grandview, Missouri. The lake is spring fed, contains fish
and weeds, and remains completely natural except for a large aerator located
at its center. In this test, CAM-4 was operated in a row boat from a 12-v
automobile battery at various points in the lake. The water temperature
ranged from 19 C at the center of the lake to 23°C in the shallow areas at
the edge. The pH of the water, determined with test paper, was 6.0.
For the initial testing, a thin open-pore polyurethane foam filter was
placed on the water inlet line to keep out lake scum and debris. The filter-
ing capacity of the foam was satisfactory, but the filter did not float and
had to be attached to the boat. Problems were encountered with this method
of filtering. The filter had a tendency to drag along the bottom and fill
with mud in the very shallow areas. The problem was corrected by raising
the line closer to the water surface; however, excess movement of the boat
allowed air to enter the line and stop the water flow. The operator was
required to re-prime the water pump. Floating inlet filters, as described
earlier in Section 8, were used for subsequent testing.
Baselines were smooth throughout the 6-hr test period, no inhibitor was
detected, and no false alarms occurred. An exposure to 0.3 ppm DDVP in lake
water gave a total change of 60 mv in five cycles showing that the system
was performing in the same manner that it had performed in the laboratory.
One minor problem was encountered in operating the portable detector
from a row boat; the drain tube retained water during the substrate pumping
part of the cycle and produced an erratic baseline. The problem was solved
by operating the detector from the boat seat rather than from the floor of
the boat, such that the drain tube was lower than the electrochemical cell.
The second test site was in Brush Creek which runs through the business
and residential area of Kansas City, Missouri. This creek collects much of
the storm drain runoff of the area and may even contain sewage at times.
CAM-4 was operated on a 12-v automobile battery from the creek bank and
remained at one location throughout the 7-1/2 hr test period.
At this site, the initial water temperature was 6 C with a pH of 5, as
measured by test paper, and air temperature was 9 C. During the test Q
period, the water temperature increased to 10 C and air temperature to 19 C.
This temperature change had no effect on the baseline stability and no false
alarms occurred. The response of CAM-4 to 0.3 ppm DDVP mixed with the creek
water was slightly lower than that obtained in the laboratory (52 mv), but
39
-------
the responses signalled in two cycles, 4 mv and 10 mv, were enough to con-
sider the instrument to be operational. No inhibitors were detected in the
creek at this location.
A new water inlet line filter was tried at the Brush Creek site. The
inlet filter was a 5-in. square cube of urethane foam sponge with a hole
drilled in the center into which the inlet tube was inserted. This cubic
sponge floated just below the surface and proved to be very satisfactory,
both as a filter and a float.
The water at this creek site was running rapidly and was very clear.
To test the filtering efficiency of the cube sponge, large clouds of dirt,
silt, leaves, and other debris were stirred up by agitating the creek bottom
20 yards (18 m) upstream from the CAM-4 and allowed to travel downstream
past the instrument. The filter became dark brown during the 7-1/2 hr run,
but no visible material entered the water line, and the baseline was un-
affected.
A minor electrical problem was encountered with the digital printer at
this location. Spurious extra digits, other than those showing the cell
voltage, were printed in columns. This did not seem to interfere with the
correct voltage reading and when CAM-4 was returned to the laboratory for
examination, the fault was traced to a bad connection.
The next field location to which the CAM-4 was taken was again the
experimental farm lake in Grandview, Missouri. The weather was much cooler
than during the previous tests here, allowing an assessment of the effect of
temperature on CAM-4. The new floating filter could be evaluated while
moving in a boat along the edges and shallow areas of the lake where there
was much debris. These areas had been inaccessible to monitoring using the
previous non-floating water inlet-line filter.
The CAM-4 was again operated from a 12-v automobile battery and was
moved to various points around the lake in a small row boat. The water
temperature here was 10 C, with a pH of 6 (test paper) at all locations.
Air temperature was 10 C throughout the 7-hr test period; the air was very
humid, and there was a slight drizzle for about 1 hr during the testing.
The CAM-4 monitor was next taken to the shallow end of the lake where
much floating debris (leaves, bark, duckweed) and large amounts of bottom
algae were present. The filter was tested in a stationary position among
this debris for 1 hr. No effect was seen on the baseline during this per-
iod, but the filter became covered with scum after 1 hr and a slight reduc-
tion in water flow rate was noticed (about 50 ml/min). Removal of the
filter and a quick rinsing in lake water alleviated this problem. The
filter was also tested at the spillway of the lake where there was a large
accumulation of a very fine green silt-like material, both floating and
suspended in the water. This material passed through the filter, as evi-
denced by the greenishness in the tubing, and resulted in a slightly more
erratic baseline though no false alarms occurred. Excessive foaming was
noticed at the drain tube as this material passed through the system. The
40
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filter floated satisfactorily during the test period independently of whether
the boat was moving or stationary.
In the field, the standard exposure test to 0.3 ppm DDVP was performed.
Total response was 40 mv, while one 10-mv "alarm" situation occurred. This
response was less than that obtained during laboratory testing and may pos-
sibly be attributed to the colder testing conditions. Although CAM-4 be-
comes less sensitive as the temperature decreases, it retains sufficient
sensitivity to detect subtoxic levels of all those compounds tested. With-
out changing the enzyme pad, its use was continued for several cycles and
then exposure to 5 ppm DDVP was tested. The result was a 136-mv increase in
three cycles and complete inhibition of the enzyme pad.
This 136-mv voltage increase demonstrated in the field that a partially
inhibited enzyme pad still has the ability to respond well when high levels
of inhibitor are encountered.
CAM-4 was taken to another small stream as its fourth field test site.
This stream (Rock Creek) runs through the back property of residences in
suburban Mission, Kansas, and is actually little more than a drainage ditch.
At this location, the water temperature was 8°C initially and rose to 10°C
during theQ5-l/2 hr test period (pH of 7.5). Air temperature was 6°C and
rose to 10 C during the testing. The stream contained dead twigs and tree
branches. There were numerous cardboard containers, beer cans, and bottles
along the bank and in the stream. The water was moving very slowly and was
almost completely covered with floating leaves (birch, cherry, black oak,
weeping willow, and apple). The stream passed through a landfill/garbage
dump 50 yds upstream from where the CAM-4 was located, and also caught
runoff water from residences in the immediate area.
The standard exposure test to 0.3 ppm DDVP was done at this location,
as on all the other field tests. Response was a total change of 68 mv with
three alarms of 10 mv or more—slightly larger than expected for such cold
weather conditions.
No significant problems were encountered during the entire 6 hr of
testing at the Rock Creek location. No inhibitor was detected, the baseline
was very stable, and no false alarms occurred. Plugging of the water line
filter, which was not unexpected since the majority of the debris being
filtered was very large, was not observed. There were no electrical or
mechanical failures. All in all, this was a very satisfactory field test.
CAM-4 was taken to one final field location for testing. The instru-
ment was used to monitor the effluent of a local pesticide manufacturing
plant where pr6cess wastewater poured into a major tributary of the Missouri
River (Little Blue River) in Kansas City, Missouri. The manufacturing plant
is located in an industrial district traversed by the stream. Upstream from
the pesticide manufacturer's facility are several other industrial establish-
ments including a grain elevator and feed packing plant, a chemical company,
a steel works, an electric generating station, and a sewage treatment plant.
41
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CAM-4 was operated from the river bank using a 12-v automobile battery
during the 6-hr test period. Two separate areas were monitored at this site:
the point at which the effluent entered the stream, and a point approximately
15 yards (14 m) upstream. Access to these locations was made by foraging
through thick brush and some dense undergrowth along the upper bank. The
effluent itself was pink/red and foaming, and had a strong unidentifiable
chemical smell. The dark brown muddy stream flowed slowly but was clear of
floating debris. Visible amounts of a black oily sludge-like material had
accumulated along the edge of the bank, on rocks, and on tree stumps up and
down the stream from the monitoring location. The water temperature was 12 C
with a pH of 6. The pesticide plant effluent pH was 4 to 5 asQdetermined by
test paper. Air temperature was initially 11 C and rose to 21 C during the
period of testing.
No mechanical or electrical problems were encountered during this test,
but high concentrations of enzyme inhibitor were present at the effluent
monitoring site. Three enzyme pads were used and completely inhibited during
the first 1-1/2 hr of operation. The alarm potential was slightly low at the
start of testing at the effluent site (160 mv versus 240 mv in the lab) but
this was attributed to the partial inhibition of the enzyme pad due to the
contaminated water used to determine the baseline voltage (Venz). Monitoring
of the effluent as it moved downstream would have been interesting, but no
boat was available. Relocation of the detector downstream from the effluent
gates was also impossible because the river bank was inaccessible for several
miles.
The portable detection apparatus was moved 15 yards (14 m) upstream from
the first location and monitoring was again initiated. The alarm potential
at this location was more acceptable (225 mv) and much less inhibitor was
detected as compared to direct sampling of the effluent. Since the enzyme
pad baseline continually rose at about 5 mv/cycle, it was evident that low
levels of inhibitor were present but no alarms occurred. Moving the detector
and storage battery from one stationary location to another along the over-
grown bank was difficult and time consuming for one operator. In the future,
when it is desired to sample several locations in a short period of time, it
would be more efficient to operate the detector from a small boat.
42
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APPENDIX D
CAM-4 PARTS LIST *
Parts
Description
DVM BOARD (FIGURE 5a)
1
Printed Circuit Board
ADC-1100
IC1
IC2
IC3
Tl
T2
T3
T4
Zl
01, D2
Cl, C2
C3
Rl, R5
R2
R3
R4
R6
PI
P2
Analog to Digital Converter
LM741CN Operational Amplifier
7400 Quad Name Gate
MC846P
2N3904 NPN
2N3905 PNP
2N3565 NPN
MPF102 FET
ZB82A Zener
1N914 Diodes
22 uf/25 v Tantalum caps.
0.02 uf Mylar cap.
3.3 Kfl 1/4 w Resistor
1.5 Kfl 1/4 w Resistor
1.0 Kfl 1/4 w Resistor
2.7 MB 1/4 w Resistor
470 S2 1/4 w Resistor
50 Kfl Trimpot 3006P-1-503
1 MJ2 Trimpot 3006P-1-105
Manufacturer or Supplier
Teletron
Analog Devices
National Semiconductor
National Semiconductor
Motorola
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Newark
Newark
Newark
Newark
Newark
Newark
Newark
•Bourne Newark
Bourne Newark
* Resistors are 1/4 w unless otherwise identified.
POWER SUPPLY BOARD (FIGURE 5b)
Printed Circuit Board
BR1
BR2t BR3
Regulator 1
Regulator 2
IC1
Cl, C2
C3
C4
Rl
R2
Bridge Rectifier WHO
Bridge Rectifier KBPC8005, KBPC1005
7805
4195
LM555CN
220 uf/35 v Electrolytic
5,000 uf/10 v Electrolytic
0.1 vf Electrolytic
3.3 n, 1/2 w
220 f! 1/4 w
TIMER AND TRIAC SWITCH BOARD (FIGURE 7)
Printed Circuit Board
IC1
IC2, 1C3, 1C4
IC5
IC6
IC7
IC8
IC9, IC10
IC11
. Tl
T2 T3 T4 T5
16
T7. T8
Dl, D2
D3
Timer LM555CN
TTL Decade Counter 7490
Decade Decoder 7442
Triple 3 input Nand 7410
Dual 4 input Nand 7420
8 input Nand 7430
Optical Isolators 7N28
nTL Gates 660P
2N3905
2N3565
2N3906
RCA Triac Type 40526
1 Trigger Diode MB54991 1
IR170 Rectifier
Teletron
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
National Service
Teletron
National Semiconductor
' National Semiconductor
National Semiconductor
National Semiconductor
National Semiconductor
National Semiconductor
Motorola
Motorola
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
43
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Parts
Description
Manufacturer or Supplier
TIMER AND TRIAC SWITCH BOARD (FIGURE 7) Contd.
Zl
PI
P2
Cl
C2, C3, C6
C4
C5
Rl, R2, R3, R4
R5
R6
R7, R8, R9, RIO
Rll, R12, R13, R14
R15, R16
R17, R18
R19
Zener (15 v) ZB15A
1 MI2 Trimpot Spectral Type 43P105
50 Kfl Trimpot Spectral Type 43P504
1 pf Mylar
0.005 yf
4 yf/250 v Electrolytic
0.022 uf Electrolytic
1.2 K Electrolytic
10 K Electrolytic
1,500 ft Electrolytic
3.3 K Electrolytic
180 n Electrolytic
42 K Electrolytic
8.2 K Electrolytic
3 Kfi 6 w Electrolytic
Semiconductor Specialists
Semiconductor Specialists
Semiconductor Specialists
Newark
Newark
Newark
Newark
Newark
Newark
Newark
Newark
Newark
Newark
Newark
Newark
INVERTER AND OTHER PASTS (FIGURES 2, 3, 6, 8)
Case
Hardware Mounting Panels
Printer
Inverter
Transformer
Transformer
Regulator (5 v)
PC Sockets
Card Guide (6 req.)
Spacers
Extractor (6 req.)
Extrusions for card cage
Binding Posts
SW1
SW2, SW3, SW4
Leds (3 req.)
AC Connector
AC Power Cord
Water Pump, Gear
Air Blower
'Substrate Pump
Substrate Pump
Substrate Pump
Air Pump
Electrochemical Cell Holder
Electrochemical Cell Injec-
tion Molded with Platinum
Anode and Cathode
Model 92500
DPP-7
Model 12-115
UP6377
40 v CT Type 18A1487
LM309
225-22221-401(117)
T-309-48
T-101-300
S-200
XTS-802-36
Type 29-1
Type 7693K2 4PDT
MTA106D
Delrin Plastic No. 7012
Sprite Tubeaxia/fan
Rotor
Tubing Support
Outboard Bearing Plate
Aquarium Type
Skydyne, Inc.
Teletron
Datel
Nucleonic Products, Inc.
Burstein Applebee
Amphenol
Cambion
Cambion
Cambion
Cambion
Grayhill
Cu.tler Hammer
Alco
Cole Farmer
Rotron
Scientific Industries
Scientific Industries
Scientific Industries
Hush
Teletron
MRI
44
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APPENDIX E
MECHANISM OF ELECTROCHEMICAL DETECTION PROCESS IN CAM-4
OXIDATION POTENTIAL THEORY
This theory proposes that cholinesterase is able to convert butyrylthio-
choline iodide (BuSChI) into butyric acid and thiocholine iodide (HSChI).
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 sub-
strate, BuSChI, is not readily oxidized. It has been proposed that the
voltage decrease is due to the oxidation of I~ to 12- This explanation has
appeal, but it does not explain (a) how this iodide-to-iodine oxidation
potential can vary from 100 mv to 600 mv as the electrodes are conditioned;
(b) why no trace of iodine color has ever been detected on the starch covered
enzyme pad; and (c) 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 concen-
trations. We have not found alternate electrode materials that give as
large voltage changes as platinum.
ANODE DEPOLARIZATION THEORY
This theory suggests that the anode is coated with a layer of platinum
oxides or sulfides, and perhaps other materials, tend to reduce its elec-
trical conductivity. Exposure of this coated anode to a solution containing
a trace of thiol results in a controlled depolarization, 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) produces two
stable, but dissimilar electrodes that generate voltage like a battery when
they are placed in an electrolyte. The electrodes 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, indica-
ting excellent conductivity. The electrodes recover after- the halogen is
gone.
In beaker experiments, a standard calomel electrode is used as a refer-
ence 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
45
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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 electrochemi-
cal 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 inhibitors.
The disulfide of thiocholine iodide found in the products coming from
the cell could arise either from air or electrochemical oxidation of the
HSChI. No evidence has yet been gained for either theory. With freshly-
plated platinum 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 when replacing an enzyme pad
with a starch pad (no enzyme) is 200 mv or more whether the electrodes are
new or conditioned. This suggests that the enzyme pad voltages obtained are
not characteristic of the oxidation potential or thiocholine iodide since
they range from 0 to 300 mv.
In summary, the exact mechanism of the electrochemical reaction is un-
known; both electrode polarization and thiocholine iodide oxidation may be
occurring simultaneously in the electrochemical cell. The mechanisms en-
abling electrochemical estimation of enzyme pad activity are worthy of
further investigation.
Further support to the anode depolarization theory is given by Kramer,
et al.,(l) who reported constant current experiments in which depolarization
of a platinum anode by thiocholine iodide resulted in increased conductivity
of the anode. This electrochemical reaction forms the basis of their pro-
cedure for analysis of cholinesterase and thiocholine esters.
(1) Kramer, D.N., P.L. Cannon, Jr., and G.G. Guilbaut. Electrochemical
Determination of Cholinesterase and Thiocholine Esters. Anal. Chem.
34(7):842-845, 1962.
46
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APPENDIX F
ENZYME PAD PREPARATION PROCEDURE
Care must be taken in the preparation of the enzyme pad for use in CAM-4
since the sensitivity of CAM-4 and the repeatability of the tests are a func-
tion 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 pro-
cedure for uniform distribution of the starch applied to the foam. After
gelling and drying, the starch-coated foam should then b^ cut into pads.
since starch pads are also needed for the determination of CAM-4*s alarm
potential at the beginning and ending of each day's tests.
(The alarm potential is the voltage increase which occurs when all of
the enzyme on the pad is inhibited or when 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 inch (ppi) x 1/4 in., Scott Industrial Foam, Scott
Paper Company, Chester, Pennsylvania (distributed by Crofton, Inc., 1801 West
Fourth Street, Marion, Indiana 46952); partially hydrolyzed potato starch
recommended for gel electrophoresis, Connaught Medical Research Laboratory,
Toronto, Canada; Chlorhydrol^ (aluminum chlorhydroxide complex, 50% w/w solu-
tion), Reheis Chemical Company, Chicago, Illinois; horse serum cholinester-
ase, Sigma Chemical Company, Type IV, approximately 15 M units/mg; and Tris
buffer, "THAM"R Fischer 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, adjust-
ment of the pH to 7.4 with concentrated HC1, and then adjustment of
the volume to 1 liter.
Step 2 One hundred twenty-five milligrams of horse serum cholinesterase
were dissolved in 6 ml of Tris buffer. To this solution were added,
with mechanical stirring, 4 ml of a dilute solution of aluminum
chlorhydroxide complex (0.03 g of ChlorhydrolR in 4 ml of water).
At this point the aluminum hydroxide gel precipitated and adsorbed
the enzyme from the solution (adjusted pH to 7.4-8.0 to ensure that
the precipitation was complete). This suspension was set aside at
ambient temperature until needed in Step 3.
47
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Step 3 Two grams of potato starch were suspended in 10 ml of cool Tris
buffer, added to 30 ml of boiling Tris buffer, and heated until the
suspension cleared. Care was taken to avoid the formation 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 de-
posited on a pre-cut sheet of open-pore urethane foam (4 x 6 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 distri-
buted throughout the sheet as uniformly as possible 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 dis-
tributed 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 fin-
ally 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. diameter pads with a stainless steel cutter moun-
ted in an electric drill press. The procedure yielded approxi-
mately 350 enzyme pads, which possessed an average activity of 0.5
units/pad (analysis based on the rate of hydrolysis of butyrylthio-
choline iodide and measured by a modification of the Ellman Pro-
cedure) . (-*-'
Step 7 The enzyme pads were then placed in a screw-capped bottle and
stored in a second container, with dessicant, in a refrigerator
until needed. Pads made and stored in this manner retain their
usefulness for more than a year.
(1) Ellman, G.L., K.D. Courtney, V. Andres and R.M. Featherstone. A New
and Rapid Colorimetric Determination of Acetylcholinesterase Activity.
Biochem. Pharmacol., 7, 88-95, 1961.
48
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
NO.
EPA-600/2-80-053
3. RECIPIENT'S ACCESSION-NO.
1. TITLE AND SUBTITLE
CAM-4, A PORTABLE WARNING DEVICE FOR ORGANOPHOSPHATE
HAZARDOUS MATERIAL SPILLS
5. REPORT DATE
January 1980 issuing date
6. PERFORMING ORGANIZATION CODE
Louis H. Goodson
Brian R. Cage
8. PERFORMING ORGANIZATION REPORT NO
MRI 3820-B Final Rpt Task II
>. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. PROGRAM ELEMENT NO.
1BB610 (Project No. 00202)
11. CONTRACT/GRANT NO.
68-03-0299
12. SPONSORING AGENCY NAME AND ADDRESS
Industrial Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Task II, Final Rpt 4/75-12/76
14. SPONSORING AGENCY CODE
EPA/600/12
15. SUPPLEMENTARY NOTES
This report describes work on Task II of Contract No. 68-03-0299.
16. ABSTRACT
CAM-4 is a completely portable, battery-operated, field version of the earlier
cholinesterase antagonist monitor, CAM-1, which senses organophosphates and carba-
mates in water supplies. The present report describes the design, fabrication, and
evaluation of the CAM-4 device. Like CAM-1, this device uses immobilized cholines-
terase in an electrochemical cell for the detection of cholinesterase inhibitors in
water supplies. CAM-4, however, is not fully automated and therefore requires an
operator to observe the cell voltages recorded by the digital printer and to decide
whether toxic levels of pesticides have been sampled. The elimination of automation
provided in CAM-1 was a trade-off to provide 8-hr operation from a standard size
12-v automobile battery and to keep the weight and cost of the instrument low. Sub-
toxic levels of MesurolR, NemacurR, BaytexR, DDVP, SystoxR, FuradanR, and SevinR
were all detected by CAM-4 at sensitivities comparable to those obtained with CAM-1
(see Task 1 report on this contract, EPA-600/2-77-219, November 1977).
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Warning system
Enzyme sensor
Electrochemistry
Pollution monitor
Insect control
Organophosphate and
carbamate detector
Immobilized enzyme as a
sensor
Insecticide spills
Water monitor
13B
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
59
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
49
U.S. GOVERNMENT HUNTING OFFICE I9BO-657-146/5569
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