EPA-R2-72-010
. Environmental Protection Technology Series
Rapid Detection System
for Organophosphates and
Carbamate Insecticides in Water
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were'established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are;
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
H. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
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.
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EPA-R2-72-010
August 1972
RAPID DETECTION SYSTEM FOR ORGANOPHOSPHATES
AND CARBAMATE INSECTICIDES IN WATER
By
Midwest Research Institute
^25 Volker Boulevard
Kansas City, Missouri 6U110
Contract No. 68-01-0038
Project 15090 GLU
Project Officer
Dr. Thomas B. Hoover
Southeast Water Laboratory - EPA
College Station Road
Athens, Georgia 30601
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
ii
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ABSTRACT
An apparatus for the detection and monitoring of water supplies for hazardous
spills of organophosphate and carbamate insecticides has now been designed
and fabricated. The new unit is called the Cholinesterase Antagonist Monitor,
CAM-1, because it produces an alarm in 3 min. when toxic or subtoxic levels
of cholinesterase antagonists are present in water. Response of this appara-
tus to subtoxic levels of azodrin, sevin, dimetilan, malathion, parathion and
DDVP has already been demonstrated. CAM-1 uses immobilized cholinesterase
for the collection of cholinesterase inhibitors from the water supplies. The
activity of the immobilized cholinesterase is determined automatically in an
electrochemical cell by passing a substrate solution over the enzyme at regu-
lar 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 situa-
tion which occurs in the presence of organophosphate and carbamate insecticides
in the water sampled.
This report was submitted in fulfillment of Project #15090-GLU, Contract
#68-01-0038, under sponsorship of the Water Quality Office, Environmental
Protection Agency.
iii
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV THE DETECTION PRINCIPLE 9
V IMMOBILIZED ENZYME STUDIES 13
Assay Procedure 13
Preparations 15
Covalently Bound Immobilized Enzyme Products 15
Starch Gel Entrapped Immobilized Enzyme Products .... 17
An Electrochemical Cell Using Immobilized Enzymes .... 19
VI OPERATING PARAMETERS FOR THE ELECTROCHEMICAL ENZYME
SENSOR 25
VII DETECTION CYCLES FOR WATER MONITORING 27
VIII DESIGN AND FABRICATION OF THE CHOLINESTERASE ANTAGONIST
MONITOR (CAM-1) 35
IX OPERATION OF THE MONITOR, CAM-1 55
X RESPONSE OF CAM-1 TO SEVERAL INSECTICIDES 59
XI ACKNOWLEDGEMENTS 63
XII REFERENCES 65
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FIGURES
Page
1 Cross Section of an Electrochemical Cell Developed for Water
Monitoring 10
2 Reaction Sequences Used for the Preparation of the Immobilized
Enzyme Products 16
3. Experimental Electrochemical Cell for Investigating
Immobilized Enzyme Products for Use in Water Monitoring ... 21
4 Proposed 2-Pad Water Monitoring System 28
5 Breadboard Detector for Toxic Substances in Water 29
6 CAM-1 3-Min Operating Cycle 31
7 Response of the Electrochemical Cell Operating on the 3-Min
Cycles to Water Containing 0.2 ppm DDVP . 32
8 The Cholinesterase Antagonist Monitor (CAM-1) With Side
Panels and Recorder Cover Removed 36
9 Automatic Enzyme Pad Changer—Electrochemical Cell Assembly
From CAM-1 37
10 Components of CAM-1: (A) the Case; (B) Rear View of CAM-1
Showing the Relay Board and the Integrated Circuit Boards on
the Upper Level, the Air Pump and the 5V DC Power Supply on
the Second Level, and the Peristaltic Pump on the Lowest
Level; (C) Pulse Generator for Operating, the Stepping Motors;
(D) Relay Board 39
11 Components of CAM-1: (A) Automatic Enzyme Pad Changer-
Electrochemical Cell Inside of Case; Note Manual Controls
to the Left: (B) and (D) Two Views of the Computer and Logic
Circuitry Boards; (C) View of the Substrate and Water Pumps
Inside of Case 40
vi
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FIGURES (Concluded)
Page
12 Simplified Block Diagram of CAM-1 45
13 Wiring Diagram of CAM-1 (Part I) 46
14 Wiring Diagram of CAM-1 (Part II) 48
15 Wiring Diagram of CAM-1 (Part III) 50
via.
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TABLES
Page
1 Half-Life of Some Organophosphate Insecticides in Water
at pH 1-5 5
2 Activity of Horse Serum Cholinesterase Used in Immobilization
Studies 14
3 Covalently Bound Immobilized Enzyme Products 18
4 Immobilized Enzyme Products in Which Aluminum Hydroxide and
Starch Gels are Used to Hold Cholinesterase on Polyurethane
Foam 20
5 Comparison of Four Types of Immobilized Enzyme Products
Prepared for Use in the Electrochemical Cell 23
6 Suppliers for CAM-1 Components 52
7 Detection of Insecticides in Water 60
viii
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SECTION I
CONCLUSIONS
On the basis of the present investigation of the use of immobilized enzymes
for the detection of toxic organophosphate and carbamate insecticides in
water the following conclusions have been made:
1. An automatic system for monitoring water supplies for the presence of
organophosphate and carbamate insecticides has been designed and fabricated.
The new system has been designated as the Cholinesterase Antagonist Monitor,
CAM-1, since it responds rapidly to low levels of cholinesterase antagonists
in water supplies.
2. The complete CAM-1 detection system and several of its component parts,
including specifically the enzyme cell for water monitoring, the automatic
enzyme pad changer, and certain circuit design features, are novel and
patentable.
3. CAM-1 responds to toxic and subtoxic levels of organophosphate and
carbamate insecticides (i.e., based on rat and animal toxicity data). In one
response test with city tap water to which 0.2 ppm of DDVP (dimethyl-2,2-
dichlorovinyl phosphate) had been added, CAM-1 provided both visible and
audible alarms repeatedly. On this basis it is concluded that CAM-1 has
adequate sensitivity to prevent accidental poisoning of human and other ani-
mal species by cholinesterase inhibitors in water supplies.
4. CAM-1 responds to toxic and subtoxic levels of other organophosphate
and carbamate insecticides including the following which have been tested:
azodrin, sevin, paraoxon, dimetilan, malathion and parathion.
5. The response data presented in this report suggest that there is a
rough correlation between the rat toxicities of the various insecticides
and the levels of these insecticides which can be detected by CAM-1. This
observation is not surprising since the inhibition of cholinesterase is the
cause of the rat toxicity and also the basis of the detection of the in-
hibitors. Differences in the affinity of specific insecticides for cholin-
esterase explains why their toxicities are different and why the levels de-
tectable by CAM-1 were observed to be different.
6. The sensitivity of CAM-1 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 concentrator of the materials
to be detected.
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7. CAM-1 is the first instrument which has successfully used an immobilized
enzyme product for the automatic monitoring of water supplies for the presence
of enzyme inhibitors.
8. CAM-1 operates satisfactorily with enzyme pads made of open-pore urethane
foam, starch gel, and cholinesterase complexed with aluminum hydroxide gel.
A single pad of this type performed satisfactorily for 56 hr with water flow
rates of 1,200 ml/min during the water sampling portion of the detection cycle.
9. An inanoblized enzyme product made by covalent bonding of cholinesterase
to cheesecloth has been used successfully in CAM-1 but packing of the wet
cheesecloth tended to restrict the flow of water through the enzyme product.
10. The present CAM-1 operates on a 3-min detection cycle in which water is
sampled for 2 min and the pad activity is determined during the third minute.
The sensitivity of the system can be increased by manually increasing the
water sampling period using the controls provided.
11. CAM-1 will continue to function even after a single enzyme pad becomes
unsatisfactory due to loss in activity since electrical sensing and automa-
tic pad change mechanisms have been provided to replace a nearly exhausted
enzyme pad with a new one.
12. CAM-1 will operate for 48 hr without servicing. Continuous unattended
operation for even longer periods could be achieved through the addition of
a mechanism to convert the dry substrate ingredients into fresh substrate
solution.
13. Continuous sampling of a water supply can be achieved by operating two
CAM-1 units out-of-phase so that one unit samples water while the second unit
tests the enzyme activity of its pad. A sync cable to keep the two detectors
operating out-of-phase has been provided. The use of two CAM-1 units simul-
taneously provides redundancy and, if an alarm is signalled, it can be con-
firmed in 90 sec.
14. It was necessary to incorporate a minicomputer in CAM-1 to program the
detection cycle, to compare the differences in cell voltage for successive
cycles, to compare the voltage differences between cycles with the preset
alarm threshold, to signal an alarm when the rate of enzyme inhibition ex-
ceeds the sensitivity setting, to change enzyme pads before they fail and
to permit unattended operation.
15. The CAM-1 unit which has been developed on this program can be useful
to operators of water treatment facilities to warn them of hazardous spills
of insecticides so that they can take appropriate action.
16. CAM-1 *s ability to detect insecticides in water is based upon a bio-
chemical reaction known to be involved in animal toxicity.
2
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SECTION II
RECOMMENDATIONS
1. The response profile of CAM-1 should be further evaluated by an exposure
of the detector to a wide variety of potential water pollutants. This study
should be concerned not only with the detection of compounds but also with
establishing the thresholds at which CAM-1 will detect them.
2. Studies on the effects of various water pollutants and environmental
parameters on the system and its components should be extended. For example,
the effects of salt water, and of various organic and inorganic water pollu-
tants, should be studied in relation to the performance of the system.
3. Studies should be conducted in which the objectives are (1) to determine
the reliability of the various components, and (2) to improve reliability
for components where failures or malfunctions occur. They would evaluate
enzyme pads under long periods of use under a variety of conditions. Testing
of this type is needed to provide the assurance of trouble-free operation
under anticipated use conditions.
4. A rugged version of CAM-1 should be fabricated for stream monitoring.
It should withstand rain, dust, and salt spray, and operate reliably with
a minimum of servicing under extremes of environmental conditions.
5. A research program should be initiated to determine if the capabilities
of CAM-1 could be expanded by using different enzymes and different sub-
strates in it.
6. The present study adequately demonstrates the possibility of fabricating
a toxic hazards detector'which will respond to most (if not all) of the toxic
substances which are likely to occur at toxic levels in the environment. As
the first step in the fabrication of such a toxic hazards detector system,
a search for enzymes which are inhibited by specific types of toxic materials
should be initiated. The enzymes selected from such a study could be im-
mobilized and incorporated into a multi-enzyme detection system. Although
the present CAM-1 uses an electrochemical system for monitoring the activity
of the immobilized enzyme after exposure to water suspected of containing
the inhibitors, there would be no need to restrict an enzyme-substrate search
to systems which could be used in the present electrochemical cell.
7. The unit which has been developed should be evaluated to determine its
potential for routine use in the Water Monitoring Network.
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SECTION III
INTRODUCTION
Spills of hazardous materials into rivers, streams or lakes create the danger
that toxic levels of chemicals will be pumped into the distribution network
of municipal water supplies. As one example, a train wreck might well lead
to the spill of a tank car of a toxic chemical such as an insecticide into
a stream above the water intake of a city water plant. Such hazardous spills
must be detected in time to close off the intake valves of these water sup-
plies until the toxic material has flowed past the inlet.
The possibility of hazardous material spills is increasing because of the
switch from the chlorinated hydrocarbon insecticides to the organophosphate
insecticides which possess greater acute toxicities. However, most environ-
mentalists believe that organophosphates are safer than the chlorinated
hydrocarbons because they decompose in soil and water, and because they do
not concentrate in food chains or accumulate in the body fat of man or ani-
mals. Under certain environmental conditions, particularly low temperature,
neutral or slightly acid waters or the absence of water, some of these per-
sist for prolonged periods of time. For example, Thoman and Nicholsooi/(1963)
reported that organophosphate wastes containing parathion discharged into a
river in the southeastern part of the United States caused an extensive fish
kill for 25 miles and affected fish in a reservoir 100 miles downstream.
An important review of the recovery, separation and identification of organic
pesticides from natural and potable waters has been published by Faust and
Suffet.2/ The following information on the persistance of organophosphates
in water has been adapted from the work of Miihlmann and Schrader3-/ (Table 1).
TABLE 1
HALF-LIFE OF SOME ORGANOPHOSPHATE INSECTICIDES IN WATER AT pH 1-5
Temperature Methyl
(°C) Paraoxon Parathion Dipterex DDVP Parathion
10 1,200 3,000 2,400 240 760
20 320 690 526 61.5 175
30 93 180 140 17.3 45
50 9.6 15 10.7 1.66 4.0
70 1.2 1.65 1.13 0.164 0.47
Hours
70
pH 3 23.0 21.0 33.0 3.4 11.2
pH 5 24.4 19.5 15.3 2.8 10.7
pH 7 11.5 7.8 0.7 0,45 6.9
pH 9 2.1 2.7 0.1 ~ 1.5
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Both water temperature and pH are important factors in the stability of the
organic phosphate insecticides in water. Some of these compounds persist
at hazardous levels in water supplies for months, or even years, if the water
is cool and the pH is on the neutral or acid side. In waters of high alka-
linity, the persistence of the organophosphates is much reduced; but they
may, at low temperatures, have half-lives measured in days and weeks instead
of months and years. Since some organophosphates persist for long periods
of time under conditions common in streams, lakes and ponds, it is impera-
tive that we have the capability of detecting hazardous spills of these
materials into water supplies prior to their ingestion by fish, wildlife
and humans.
A number of analytical methods for the detection and identification of these
compounds have been reported. Because these compounds are toxic at such low
levels, it is usually necessary to pass large volumes of water through a column
of activated charcoal and to elute with an organic solvent to get a large
enough sample to permit gas chromatography, mass spectrometry, enzyme analysis,
etc.
4 /
For example, Davis and Malaney—' collected the acetylcholinesterase inhibitors
from water supplies using a charcoal column; chloroform was then used to ex-
tract the organophosphates from the column before measuring the enzyme in-
hibition capacity of the chloroform extracts.
Guilbault, et al.,—' reported an electrochemical assay procedure for organo-
phosphate insecticides based on their inhibition of cholinesterase and its
conversion of a substrate to a hydrolysis product which depolarized platinum
electrodes to which a constant current was applied. Guilbault, et al.,— also
showed that cholinesterases from different insect species were inhibited to
different degrees by different organophosphates, and that both sensitivity
and selectivity could be used to achieve trace analyses of pesticides by
enzymatic methods.
In his discussion of equipment for monitoring pollutants in water,
PorterfieldZ/ enumerated some of the common water pollutants for which there
was no monitoring equipment available. He 'implied that the lack of standards
for organophosphates in water was tied to a lack of suitable monitoring equip-
ment. Some investigators have felt that perhaps the important thing for them
to know was whether there were any toxic substances in the water supplies
rather than just the concentration of one or more specific insecticides. To
o /
accomplish this goal, Cairns, et al. ,—' set up equipment for continuous moni-
toring of the activity of aquatic life; in one system he monitored the heart
rate and breathing rate of bluegill sunfish for the detection of sublethal
concentrations of zinc ions and other toxicants. In spite of their success,
a nonliving toxic hazard monitoring system could have many advantages.
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In trying to develop a system which could be used to monitor water supplies
for the presence of toxic substances, a statement in the report of the
National Technical Advisory Committee—' should be kept in mind: "The most
important mechanism of toxic action is thought to be the poisoning of enzyme
systems." Although this statement may be an oversimplification of toxic
phenomena, enzymes offer a great potential for the detection of toxic sub-
stances. It was for this reason we proposed, to the Federal Water Quality
Office, that (1) they sponsor a search for a group of enzymes which could
be used in the detection of toxic substances in water supplies, and (2) a
water monitoring apparatus based upon the use of immobilized cholinesterase
as the sensor should be constructed for the purpose of detecting those toxic
substances which inhibit cholinesterase, e.g., organophosphate insecticides,
some carbamate insecticides, and some heavy metals.
Previous investigation in these laboratories by Bauman, et al.,i2' had shown
that cholinesterase immobilized by starch gel on the surface of open-pore
urethane foam could be used in an electrochemical cell for the continuous
monitoring of air for the presence of cholinesterase inhibitors. In this
system the air being sampled and a solution of substrate for the enzyme
were pumped simultaneously and continuously through the immobilized enzyme
product; activity of the enzyme was then monitored continuously by passing
an electric current through platinum electrodes in contact with the immobil-
ized enzyme product and observing changes in the electrode potentials.
Initial experiments, on the current project, concentrated efforts in determining
whether the air monitoring system could be adapted for the detection of toxic
substances in water, and were conducted with small bench type electrochemical
cells. We were unable to monitor the residual enzyme activity in the presence
of large volumes of water, and for this reason developed a detection cycle
in which the enzyme inhibitors were collected on the immobilized enzymes
in the first part of the cycle, and then the activity of the enzyme was de-
termined during the second part of the cycle. After demonstrating the fea-
sibility of the two-cycle system, we collected information about the electro-
chemical cell design, the proper buffer concentration, pH, and flow rate of
the substrate solution, and other information necessary for the fabrication
of an integrated detection and monitoring unit which would operate with a
minimum of attention.
This report describes the various individual studies which resulted in the
development of the Cholinesterase Antagonist Monitor, CAM-1: specifically,
the immobilization of the cholinesterase on open-pore polyurethane foam, the
design of two electrochemical cells, the selection of the buffer, substrate
concentration, flow rate, applied electric current, and a detection cycle;
the electrical and mechanical work included design and fabrication of logic
and memory circuits, a digital clock, automatic and manual controls, selection
of alarm logic, size, shape, and materials of construction of the case, place-
ment of components, etc.
7
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After completion of the assembly, it was necessary to conduct studies on the
performance of the detector in both the absence and also in the presence of
enzyme inhibitors. After shakedown runs and completion of minor modifications,
the CAM-1 was operated continuously for 56 hr to show that its components
were functioning in a reliable manner. Following this, the unit was taken
to the Edison Water Quality Laboratory, where its response to 1 ppm of DDVP
was demonstrated to the sponsor.
In the following sections of this report the proposed detection principle
and the experiments which led to its incorporation into a functioning water
monitoring apparatus identified as CAM-1 are described. A variety of im-
mobilized enzyme products were fabricated and several electrochemical cell
configurations were investigated in our attempts to perfect an electrochemi-
cal enzyme cell suitable for use in water monitoring. A breadboard apparatus
was constructed which possessed the components which were necessary for water
monitoring. This apparatus permitted consideration of both continuous water
sampling and intermittent water sampling and experimentation with various en-
zyme cell configurations. Also it permitted a selection of operating param-
eters for the electrochemical cell and a determination of the effectiveness
of different detector cycles for the intermittent-type water sampling system.
The need for unattended operation resulted in the design and fabrication of
an automatic enzyme pad changer-electrochemical cell assembly and a programmer
which would control the various detector functions. Data gathered with this
breadboard apparatus facilitated the fabrication of the computerized water
monitoring apparatus known as CAM-1. Information is presented showing how
CAM-1 is operated and how it responds to a group of organophosphate and car-
bamate insecticides. These experiments and related information are provided
in this report.
8
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SECTION IV
THE DETECTION PRINCIPLE
The detection principle used in the monitoring of water for the presence of
toxic substance in water can be divided into the following parts: (1) the
collection of the enzyme inhibitors on immobilized cholinesterase, (2) the
chemical reaction of immobilized cholinesterase with its substrate, and (3)
the electrochemical monitoring of substrate hydrolysis products. The mecha-
nisms by which enzyme inhibitors block the activities of enzymes have been
extensively investigated. In most, if not all, of the known examples of en-
zyme inhibition the inhibitor becomes attached (either reversibly or non-
reversibly) to the active sites of the enzyme and blocks the substrate from
these sites. For this reason immobilized cholinesterase is able to capture
pure or mixed organophosphates from very dilute aqueous solutions; measure-
ment of residual enzyme activity gives an indication of the presence of
enzyme inhibitors even when very low levels of inhibitors are sampled. We
think that noncompetitive inhibitors may also be collected by immobilized
enzymes but perhaps to a lesser degree particularly from dilute solutions.
The chemical reaction which we have chosen for determining the activity of
the immobilized enzyme is shown by the following equation:
CH3
C3H7COSCH2CH2-N-CH3
CH3
Butyrylthiocholine Iodide (BuSChI)
CH3
C3H7COOH + HS-CH2CH2~tN-CH3 I"
CH3
Thiocholine Iodide
In this reaction a thioester is cleaved by the enzyme, cholinesterase (ChE),
to give a product with a free thiol group, thiocholine iodide. As will be
explained more fully in the description of the electrochemical cell, the
formation of thiol from the passage of the substrate over the immobilized
enzyme product is evidence for the absence of enzyme inhibitors.
The design of an electrochemical cell adapted for water monitoring of enzyme
inhibitors is shown in Figure 1. An immobilized enzyme pad is located be-
tween two perforated platinum electrodes. A constant current is supplied by
a battery (e.g., 9V) or other source in series with a resistance (e.g.,
4.7 meg) so that a current of about 2 uA flows through the circuit. A high
impedance voltmeter or electrometer is used to monitor the voltage. During
the water pumping part of the cycle only water is passed through the enzyme
pad, but during the substrate pumping cycle both air and substrate solution
are pumped through the cell. The air flow is needed to push most of the
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SVJ*Sl
l&l
WATER/AIR INLET
-ENZYME PAD HOLDER
ENZYME PAD
PLATINUM ELECTRODE
WASTE
Figure 1 - 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.
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water from the cell and reduce the dilution of the substrate solution; also
the air helps to control the amount of liquid in the enzyme pad during the
voltage measurements.
During the water pumping part of the cycle, water is pumped through the
immobilized enzyme pad, at which time enzyme inhibitors, if present, are col-
lected on the active sites of the enzyme. During the enzyme-activity-testing
part of the cycle, a solution of BuSChI is pumped over the immobilized en-
zyme product; if enzyme activity remains, then a small part of the substrate
is cleaved to give a thiol which then comes in contact with the positive
platinum electrode (anode). The lower electrochemical voltage produced
between the electrodes when thiol is present (around 200 mV) is due partly
to a depolarization of the anode and partly to the electrochemical oxidation
of the thiol to the corresponding disulfide. On the other hand, if the im-
mobilized enzyme pad was inactivated by the inhibitors it collected, then
there would be no thiol formed, and a higher voltage (around 400 mV) would be
observed between the platinum electrodes. Thus by passing a substrate and a
current through an electrochemical enzyme cell it is possible to monitor
the activity of the enzyme—and also the presence or absence of enzyme in-
hibitors in the water pumped through the enzyme pad.
We have found no other electrodes which work as well as platinum, and there
is a conditioning period for newly prepared electrodes, during which time
a coating is formed on the anode; as a result of this invisible coating
on the anode, it is different from the cathode, and the two electrodes can
function as a battery. The chemical nature of the coating on the anode
of a working cell is not known; however, it is formed when current is applied
to the cell when thiol groups are present.
Although we have usually used an applied current of 2 pA, either higher or
lower currents can also be used. In such cases different voltages will be
noted at the electrodes when either substrate or hydrolyzed substrate is
present; also, the speed of response to chemical enzyme inhibitors will be
different. For example, if lower currents are used, then there is too little
electrooxidation of free thiol at the anode, and response to inhibitors is
much slower. At higher currents the spread of voltage between hydrolyzed
and unhydrolyzed substrate is less. Only a part of the substrate solution
passing over the immobilized enzyme is cleaved, and the moles of electrons
applied to the cell do not balance the moles of substrate present. The
system is quite satisfactory for monitoring of the enzyme activity, although
some theoretical aspects of the principles involved are not absolutely clear.
11
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SECTION V
IMMOBILIZED ENZYME STUDIES
The heart of the detection system is the immobilized enzyme product, which
removes and concentrates the enzyme inhibitors from the water sampled, and
which forms an integral part of the electrochemical cell detection system.
For our present investigation we used horse serum cholinesterase (acylcholine
acyl-hydrolase, E.G. No. 3.1.1.8 obtained from the Sigma Chemical Company)
since we already knew that it was readily inhibited by the organophosphate
insecticides. Analyses of the commercial cholinesterase used for the im-
mobilization experiments are shown in Table 2; the procedure for the assay
of the soluble enzyme was essentially the same as that reported by Ellman,
et al.,—' except that BuSChI was substituted for acetylthiocholine iodide.
It is described below:
Assay Procedure
Apparatus: A Beckman DB-G recording double-beam grating spectrophotom-
eter was used for the assay. We operated the strip chart recorder at
1 in/min and measured the change in absorbancy at 410 mil as a function
of time.
Buffer solution; 0.08 M tris buffer was prepared by dissolving 96.8 g
of tris-(hydroxymethyl)-aminomethane in 10 liters of distilled water and
adjusting the pH to 7.4 with approximately 33 ml of concentrated HCl.
Indicator solution: 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB),
396 mg, was dissolved in 10 ml of tris buffer by adding sufficient sodium
bicarbonate to give a clear solution. This solution was diluted to a
volume of 100 ml with buffer and stored in the refrigerator until used.
Enzyme solution: A weighed sample of horse serum cholinesterase was
placed in a volumetric flask and diluted with tris buffer to give a
solution containing approximately 1 mg/ml. The activity of this solu-
tion increases slightly during storage at 4°C for periods up to 48 hr.
Vigorous shaking of this solution was avoided.
Procedure: Measured quantities of the following solutions were placed
in the cuvettes in the order shown:
0.02 ml of BuSChI solution,
0.1 ml of DTNB solution,
2.0 to 2.98 ml of tris buffer (enzyme + buffer = 3.00 ml),
0.020 to 1.0 ml of enzyme solution depending upon the activity
of the enzyme present.
13
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TABLE 2
ACTIVITY OF HORSE SERUM CHOLINESTERASE(a)
Amount
20
40
60
Mean (X)
Relative Standard
Deviation
USED IN IMMOBILIZATION STUDIES
Activity (°) (iiM/min/mg)
3-18-71
2.96
2.77
2.91
2.83
2.86
2.84
2.85
2.87
3.09
2.887
tion(SD) ±0.093
ard 0.032
10-6-71
3.53
3.26
3.10
3.09
3.15
3.09
3.11
3.18
3.12
3.18
±0.140
0.045
(a) Sigma Chemical Company, Type IVA, Lot 120C-2240.
(b) Quantity of enzyme solution (1 mg/ml) added to the cuvette; see text
for analytical procedure.
(c) The assays were conducted by Ellman's procedure using butyrylthiocholine
iodide (BuSChI) as substrate.
14
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The blank contained the same volume of solutions but the enzyme solution
was replaced with buffer.
Immediately (i.e., within 15 sec) after the addition of the enzyme, the
sample was mixed rapidly in the cuvette with a square plastic plunger.
Changes in absorbance units per minute (AA) were calculated from the per
cent transmission recorded on the strip chart.
Calculations: The activity of the acylcholine acyl-hydrolase was ex
pressed as micromoles of BuSChI hydrolyzed per minute by 1 mg of the
original enzyme preparation:
Activity = Qg ? v uM/min/mg
* J • v J*, vv
where
AA = change in absorbance/min
V = volume of liquid in cuvette (ml)
w = weight of enzyme product (mg) placed in cuvette.
Preparations
A number of different immobilized enzyme products were prepared for trial
in the electrochemical cell during our enzyme studies. These products can
be divided into two general classes: (1) those in which the enzyme was
covalently bound to the support, and (2) those in which the enzyme was com-
plexed with aluminum hydroxide gel, and then entrapped on a polyurethane
foam support with starch gel. Both types of products are described in the
following paragraphs.
Covalently Bound Immobilized Enzyme Products
A number of methods for the attachment of enzymes to insoluble supports
through the use of covalent bonds have been reported in a review by Goldman,
et al.—' Of principal interest to the present investigation, however, are
the reports on the attachment of enzymes to insoluble materials with the
help of the 2-chloro-s-triazines. .13-"AV The chemical reactions involved
in the covalent binding of the enzyme to ion exchange cotton is shown in
Figure 2. In the first step, diethylaminoethyl cellulose (DEAE-cellulose)
is combined with a dichloro-s-triazine dyestuff (Procion Brilliant Blue M-3GS
Trademark of ICI and distributed in the USA by Colab) to form a deep blue
addition compound. The last chlorine group is reacted with N-methyl morpholine
in an effort to facilitate the reaction with the amino groups on the enzyme
molecule. The covalently bound immobilized enzyme products made by this
15
-------
Et
j
—OCH2CH2-hjl
DEAE-Cellulose
+ PBB-
Immobilized Enzyme
Figure 2 - Reaction Sequences Used for the Preparation of the Immobilized
Enzyme Products. PBB is Procion Brilliant Blue and NH2~
enzyme is horse serum cholinesterase.
16
-------
general procedure are shown in Table 3. Examination of the column identified
as "Supporting Matrix" will reveal that the first product was made from ab-
sorbent cotton to which the DEAE groups had been attached. The second and
.third products were made in a similar fashion by attaching DEAE groups to
woven cheesecloth. The remaining five products were made by attaching com-
mercially available fibrous DEAE-collulose (Whatman's DE-23) to the surface
of open-pore urethane foam with an adhesive and then coupling the enzyme to
the outer surface with PBB using the same basic chemical reaction shown in
Figure 2.
The covalently bound enzyme products made with the ion exchange cellulose
and PBB were evaluated in the electrochemical cells. Attaching the enzyme
to absorbent cotton or to cheesecloth gave enzyme pads which presented a
good deal of resistance to the flow of water, and tended to pull away from
the upper electrode so that changes in electrical contact of the enzyme
materials with the electrode tended to produce noise in our system. In an
effort to obtain better water flow through the cell, improved contact of
the pads with the electrodes and improved uniformity of enzyme activity per
pad, we prepared a number of the products in which 45 pores/in.open-pore
polyurethane foam (Scott Paper Company) served as the support for the ion
exchange cellulose and also the enzyme. Although these products appeared
promising, we did not have time to optimize their development nor to conduct
exhaustive stability and performance tests with them. Instead, we chose
the starch gel immobilized enzyme products described below for use in CAM-1.
Starch Gel Entrapped Immobilized Enzyme Products
In connection with other studies conducted at MRI we developed an enzyme
product in which aluminum hydroxide was first used to complex the enzyme,
after which the complex was entrapped in starch gel on the surface of open-
pore polyurethane foam.1^7 The procedure for preparation of enzyme pads
for use in the operation of CAM-1 is given in the following paragraphs.
Aluminum chlorohydroxide solution was made by carefully dissolving aluminum
chloride (8.7g) in 100 ml of distilled water and gradually adding aqueous
ammonium hydroxide until the pH was 7.0. The resulting gel was spun down
in a centrifuge and washed with six portions of water totaling 600 ml. The
residue was resuspended in 100 ml of water and the pH was adjusted to 7.01.
After standing 1 month the precipitate had dissolved completely, leaving an
opalescent solution, pH 5.0. The dissolved material was primarily aluminum
chlorohydroxide. The enzyme pads were made in this manner: horse serum
cholinesterase (40 mg) was dissolved in 6 ml of tris buffer (0.08 M, pH 7.4)
and to this was added with stirring 4.0 ml of the above solution of aluminum
chlorohydroxide (note: at this point the aluminum hydroxide gel precipi-
tates and adsorbs the enzyme from the solution) ' In a separate container
2 g of partially hydrolyzed potato starch (recommended for use in starch
gel electrophoresis) was suspended in 10 ml of cold tris buffer pH 7.4
and added to 30 ml of boiling buffer. Heating was continued until the
17
-------
00
TABLE 3
COVALENTLY BOUND IMMOBILIZED ENZYME PRODUCTS
Matrix Preparation
Enzyme Supporting
Preparation^*) Matrix
3-15-71
7-12-71
9-7-71
9-28-71
10-11-71
10-21-71
11-22-71A
11-22-71B
DEAE-Cotton
DEAE-Cheesecloth
DEAE-Cheesecloth
DE-23-Varnish-
Foam
DE-23-Vamish-
Foam
1.77
0.310
0.179
0.847
0.402
0.142
0.205
0.091
(a)
(b)
(c)
(e)
(f)
(g)
The reaction sequences for the preparation of these products are given in Figure 2.
Enzyme activity was determined In a flowing system by pumping BuSChI solution at constant rate over a known weight of product and then measuring
the color produced with DTNB by the Ellman method.
NMM is N-methyl morpholine; a 1% aqueous solution was used In these tests.
DEAE-cellulose (Whatman DE-23) with an ion exchange capacity of 1.0 meq/g has been attached to urethane foam circles (6 in. dia x 1/4 in. thick)
using binders to form a laminated support matrix for subsequent attachment of PBB and cholinesterase.
These three products used Sear's Interior Polyurethane Varnish (linseed oil modified urethane varnish) as binder for the DE-23. The quantity of
the varnish, the method of application and different drying procedures were the variables.
The binder was Penny's Polyurethane Varnish (safflower alkyd toluene diisocyanate).
The binder was Sylgard 184 slllcone potting and encapsulating resin catalyzed with 107. of Sylgard 184 curing agent (Dow Corning Corporation).
-------
solution cleared; then the mixture was stirred with a magnetic stirrer to
prevent the formation of a scum or lumps in the starch while the mixture
was cooling to 45°C. At this temperature the suspension of horse serum
cholinesterase complexed with aluminum hydroxide gel was added all at once
and carefully mixed with the starch solution. A 10-ml aliquot was with-
drawn with a pipette and deposited on a 4 in. x 6 in. x 1/4 in. sheet of
45 pore/in, open-cell urethane foam lying on a piece of warm plate glass.
The starch-enzyme mixture was distributed through the urethane foam as
uniformly as possible with the aid of a plastic rolling pin filled with
47°C water. In this same manner three other 10-ml aliquots of the warm
starch-enzyme slurry were added to other urethane foam sheets as before.
The coated sheets were placed on edge in a wooden rack and allowed to gel
for 1 hr at room temperature before drying overnight at 115°F. The dried
sheets were then cut into 3/8 in. dia enzyme pads with a motor-driven
stainless-steel cutter. The pads were stored in glass bottles with calcium
sulfate desiccant in a refrigerator until assayed or used. Additional
information about four gel entrapped enzyme products made by this proce-
dure is given in Table 4.
A modification of the procedure of Ellman, et al.,—' was used for deter-
mination of the cholinesterase activity of these enzyme pads. In this case,
we solubilized the enzyme from the urethane foam pads by mascerating them
in 3 ml of tris buffer, pH 7.4 with 10 units of a-amylase (hog pancreas)
and then incubating the mixture for 1 hr at 34°C. The resulting solution
of cholinesterase was then assayed by the procedure described for the com-
mercial cholinesterase products (see page 13). g-Amylase does not hydrolyze
BuSChl.
The enzyme products made by this procedure were stored over a desiccant in
the refrigerator until used. Similar enzyme pads made on another project
and stored in this way for 5 years were shown to perform well in CAM-1. In
an accelerated aging test, water at 47°C was pumped through the enzyme pad
for 2 hr; we found that 48.6% of the initial enzyme activity remained after
this extraction. At lower temperatures the enzyme product is more resistant
to extraction and/or inactivation.
An Electrochemical Cell Using Immobilized Enzymes
For the design of an integrated electrochemical cell-automatic enzyme pad
changer assembly (for use in monitoring of water for the presence of enzyme
inhibitors), the new cell must be compatible with the new enzyme products.
The first step was to fabricate an electrochemical cell with which experi-
mental immobilized enzyme products could be studied. For this cell, Figure
3, perforated platinum sheet electrodes, through which water and substrate
solution could be pumped sequentially or simultaneously, was chosen. We then
19
-------
TABLE 4
IMMOBILIZED ENZYME PRODUCTS IN WHICH ALUMINUM HYDROXIDE AND
STARCH GELS ARE USED TO HOLD CHOLINESTERASE
ON POLYURETHANE FOAM^
Pad
Preparation
11-10-71
11-12-71
1-19-72
1-31-72
Cholinesterase(k) Starch^0)
Source
120C-2240
120C-2240
120C-2240
120C-2240
(mg)
80.0
80.8
79.1
79.9
(g)
4.0
4.0
4.0
4.0
Aluminum
Chlorohydroxide
Dry Wt (mg)
300 (d)
300 (d)
300 (e)
300 (e)
Enzyme
Activity
(uM/min/g)
0.959
0.764
1.201
1.065
(a) Quantities shown are for a batch consisting of four sheets, 4 x 6 x
1/4 in. and yielding 250-300 enzyme pads.
(b) Sigma Chemical Company, Type IV, cholinesterase from horse serum.
(c) Connaught starch-hydrolyzed for electrophoresis, Connaught Medical
Research Laboratories, University of Toronto, Canada.
(d) Initial pH of the solution was 3.5.
(e) Initial pH of the solution was 5.2.
20
-------
SUBSTRATE SOLUTION
WATER INLET
OUTLET
CATHODE CONNECTOR
—MOVEABLE PORTION OF CELL BODY
STAND
Figure 3 - Experimental Electrochemical Cell for Investigating Immobilized
Enzyme Products for Use in Water Monitoring.
21
-------
tested four types of enzyme products which we described on the basis of the
physical support for the enzyme: (1) balls of cotton, (2) woven cotton,
(3) fibers laminated on urethane foam, and (4) starch gel on urethane foam.
Table 5 gives a comparison of the performance of these same enzyme products
but lists them on the basis of their actual preparation. The enzyme was
covalently bonded to ion exchange cellulose in the first three of the types
and held by physical entrapment in the fourth type shown in the table. As
noted earlier, the selection of the urethane foam coated with starch gel
and aluminum hydroxide gel entrapped cholinesterase, for routine use in the
CAM-1, was based mainly on the advantages shown in Table 5. Information
about individual enzyme preparations is presented in Tables 3 and 4.
For the experimental electrochemical cell (Figure 1) we chose dimensions
for the water passageways, the perforated platinum electrodes and the en-
zyme pad so that water flow rates of up to 1,200 ml/min could be achieved
with pressures of less than 1 lb/in2. In this system the enzyme pad was
arbitrarily set at 3/8 in. in dia and 1/4 in. thick; probably other sizes
would have worked equally as well, but they were not tried.
The electrodes in this cell were made of perforated platinum sheet which
had been platinized by electroplating with a 3% solution of chloroplatinic
acid which contained 0.1% of lead acetate.
For this platinizing step approximately 2.5 volts was applied across the two
electrodes located 1 cm apart and the polarity was reversed every 30 sec
until an even black coating of platinum black was obtained (approximately
5 min). The black electrodes were washed in nitric acid and distilled water
and then heated to redness in a Bunsen burner to convert the coating to
platinum gray, which retains much of its surface area but which is much more
resistant to abrasion.
22
-------
ISJ
TABLE 5
COMPARISON OF FOUR TYPES OF IMMOBILIZED ENZYME PRODUCTS PREPARED
FOR USE IN THE ELECTROCHEMICAL CELL
No.
1
2
3
Type of Enzyme Support
Ion exchange cotton
(long fibers)
Ion exchange cheesecloth
Short ion exchange
Retention of
Enzyme
Activity
During Use
Excellent
Excellent
Good
Ease of Preparing
Enzyme Pads With
Equal Activity
Difficult
Less difficult
Less difficult
Contact With
Both Electrodes Resistance to
of the Cell Water Flow
Poor High
Fair Medium
Excellent Low
Relative
Cost of
Preparation
High
High
High
cotton fibers lami-
nated on open-pore
urethane foam
Urethane foam coated
with starch gel +
A1(OH)3
Good
Easy
Excellent
Low
Low
-------
SECTION VI
OPERATING PARAMETERS FOR THE ELECTROCHEMICAL ENZYME SENSOR
An explanation has been given of the basic principles involved in the opera-
tion of an electrochemical cell for the detection of low levels of organophos-
phates in water supplies (see Section IV). In order to make this electro-
chemical enzyme cell both sensitive and reliable for the detection of enzyme
inhibitors, it was necessary to control those variables which affected the
response of the cell: (1) the buffer solution, (2) the substrate, (3) the
applied current, (4) the rate of water sampling, (5) time of water sampling
and the like. Some of the variables turned out to be interdependent; others
were not.
Tris buffer 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 concentration 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
was adequate for our system. The pH of 7.4 for the buffer was a compromise
between a mildly acidic pH where the substrate is very stable and pH 8.6
where the cholinesterase is especially active in hydrolyzing the substrate,
BuSChl. 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.
The selection of substrate concentration and substrate flow rate should be
considered together, since the quantity of substrate reaching the electrodes
per unit time is the important quantity affecting the base line voltage and
sensitivity of the detection system. Greatest sensitivity to low levels of
enzyme inhibitors is obtained when an excess of the substrate is avoided;
obviously enough substrate solution is needed to produce a lower electrode
voltage when the enzyme is active. At the same time, more inhibitor will
be required to cause an increase in cell voltage if there is a large excess
of substrate. Although several substrate concentrations were investigated
2.5 x 10"4 M BuSChl in 0.08 M tris buffer, pH 7.4 is about the optimum for
a pumping rate of about 1 ml/min. The substrate used in these experiments,
BuSChl, had a mp of 172°-174°C and was supplied by Pierce Chemical Company
or Eastman Organic Chemicals.
As mentioned earlier, an applied current of about 2 uA has been used. This
current appears to give a good spread of cell voltages between those en-
countered 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 of the substrate solution were increased.
25
-------
In selecting a combination of applied current of 2.0 uA, a buffer concentra-
tion of 0.08 M, a pH of 7.4, a substrate concentration of 2.5 x 10~^ M,
a substrate flow rate of 1.0 ml/min and an enzyme pad with approximately
0.02 to 0.04 uM/min/g activity, we were attempting to optimize the various
cell operating parameters so as to provide good sensitivity, reliability,
and fast voltage responses when the enzyme was inhibited.
26
-------
SECTION VII
DETECTION CYCLES FOR WATER MONITORING
Originally it seemed possible to mix substrate solution continuously with
the water being sampled and obtain a continuous record of the activity of
the immobilized enzyme product, but experiments soon showed that this approach
was not feasible because of the large quantities of buffer and substrates
which would be required.
A second approach to continuous aqueous monitoring is shown in Figure 4.
Here two enzyme pads are used alternately for collection of enzyme inhibi-
tors and for measurement of residual enzyme activity. Although the automa-
tic system shown was not built, some studies, which we believe approximated
the results to be expected with such a system, were conducted. In these
experiments two enzyme pads, A and B, were used; water was passed through
pad A for 2 min, while the enzyme activity of pad B was being monitored in
an electrochemical cell. Pads A and B were then'manually switched for many
2-min cycles, while simulating the two-pad detection system. The voltages
observed with pad A were about 50 mV different from the voltages obtained
with pad B; as a result of this, we concluded that it would be advantageous
to compare each pad only with itself. This conclusion was based upon an
inability to make or keep pairs of enzyme pads which had exactly the same
amount of enzyme activity or even provided the same electrical pathways be-
tween the electrodes. Although a logic circuit could be devised for use
with the two-pad system which would permit a comparison of the previous and
present voltages of pad A and similarly for pad B, we elected, in the interest
of simplicity, to adopt an intermittent monitoring system with only one enzyme
pad in use at a time.
Following the decision to build an intermittent sampling type of aqueous
monitoring apparatus, the breadboard apparatus shown in Figure 5 was con-
structed. In this particular system the Haydon electrical timer was adjust-
able so that we could vary the duration of any particular part of the cycle.
The substrate pump was a Bolter peristaltic pump with adjustable flow rate
control; the electrometer used for most of these experiments was the Keithley
Model 602B which had an input impedance of up to 10 ^ ohms. Less expensive
high impedance voltage measuring instruments could have been used, but an
input impedance of 10 megohms or greater is necessary to prevent masking of
the signal. The air pump used in some of the later experiments was a simple
vibrator diaphragm pump intended for aeration of aquaria. Detailed con-
struction of the electrochemical cell is shown in Figure 3.
In most of the early investigations with the breadboard detection system
a 6-min cycle, in which water was sampled from 3-5 min and the activity of the
27
-------
WATER WITH
INHIBITOR
SUBSTRATE
SOLUTION
n
ENZYME PAD
NO. 1
N)
00
AUTOMATIC
PAD CHANGER
I 1 I \
ENZYME PAD
NO. 2
rr
WATER
1
ftn
I
ELECTROCHEMICAL
DETECTION SYSTEM
HYDROLYZER OR
UNHYDROLYZED
SUBSTRATE
Figure 4 - Proposed 2-Pad Water Monitoring System Showing How Alternate Use of Two Enzyme Pads
Might Permit Simultaneous Agent Collection and Readout of Enzyme Inhibitor
-------
110V A.C.
AIR PUMP-
N5
SEQUENCE
TIMER
Figure 5 - Breadboard Detector for Toxic Substances in Water
-------
enzyme was monitored during the balance of the cycle, was used. In these
studies substrate concentrations of 1.25, 2.50 and 5.0 x 10~^ M BuSChI in
0.08 M tris buffer, pH 7.4, and flow rates of 0.5, 0.8, 1.0, 1.5 and 2.0
ml/min, were used. Water flow rates varied from about 450 to 1,200 ml/min;
the lower flow rates were mostly encountered while the PBB-cotton and PBB-
cheesecloth enzyme products were used, a condition which resulted in partial
plugging of the cell and the low flow rates. For pumping the water through
the cell an electric fuel pump (diaphragm type), an oscillating pump with
Hypalon impeller which was self-priming at a 50-in. depth, a Gelber Model PQ
gear pump with a capacity of about 1,200 ml/min, and a Flotec pump made to
our requirements with a capacity of approximately 1,200 ml/min (self-priming
when wet) were evaluated for use in CAM-1.
One of the most important problems investigated with this breadboard system
was a way to obtain a rapid determination of the immobilized enzyme activity
after exposure to the water which had been sampled. In the initial experi-
ments, 2-3 min were required for estimation of the residual enzyme activity
due to the time required for the electrodes to reach (or approach) an
equilibrium voltage after exposure to the water. However, by turning off
the current during all of the water sampling portion of the cycle and also
during the first part of the substrate pumping cycle, it was possible to
shorten the electrode equilibration time. Further shortening of this time
was accomplished by blowing the excess water out of the cell with air im-
mediately after turning the water off; increasing of the substrate pumping
rate to 1.0 ml/min also speeded the equilibration time. These changes made
it possible to obtain uniform voltages from cycle to cycle with 1 min for
the electrode equilibration. The detection cycle developed with this bread-
board apparatus and used in the CAM-1 is shown in Figure 6. The sponsor
selected the 2-min water sampling part of the cycle so that the entire cycle
would be complete within 3 min. As shown in subsequent experiments this
2-min water sampling period was adequate for obtaining response of the CAM-1
to both subtoxic and toxic levels of enzyme inhibitors in water supplies.
Figure 7 shows the voltage tracing obtained with the electrochemical enzyme
cell operating on the 3-min cycle shown in Figure 6. During the water-
pumping part of the cycle, the applied current is turned off and the voltage
falls rapidly at first and then levels off somewhat. When the water is turned
off and the air is blown out of the cell,there is often a spike in the curve
which is of no consequence so far as the operation of the monitoring appa-
ratus is concerned. After the substrate pump has been pumping the substrate
solution at the rate of 1.0 ml/min for 20 sec, the current is applied to the
cell, and there is a sharp rise in voltage which levels off (and in some cases
falls a little from the maximum) at a voltage which is indicative of the
activity of the enzyme product in the electrochemical cell. Even though the
voltage may not reach a true equilibrium in 1 min, experience has shown
30
-------
WATER SAMPLING
AIR AND SUBSTRATE
PUMPS ON
APPLIED CURRENT
START
DIGITAL
CLOCK
60
120
SYNC
PULSE
180 SEC
SAMPLE
VOLTAGE
HOLD READING AND
COMPARE WITH
EARLIER CYCLE
Figure 6 - CAM-1 3-Min Operating Cycle
-------
UJ
N)
300
> 200
i= 100
LJJ
to
-100
15
TIME (Min)
30
45
Figure 7 - Response of the Electrochemical Cell Operating on the 3-Min
Cycle to Water Containing 0.2 ppm DDVP
-------
that the voltage obtained 55 sec after starting the flow of the substrate
and 35 sec after applying the cell current is reproducible, and is satisfac-
tory for monitoring of the enzyme activity. In the figure the difference in
peak heights is exaggerated in order to show the kind of noise which can
be expected in a system of this type. The straight line over the top of the
voltage peaks obtained during the first seven cycles shows a gradual slope
upward with time. This slope is due partly to the slow loss of enzyme ac-
tivity which occurs with the use of the enzyme pads and which does not inter-
fere with the response of the detector so long as enough enzyme activity
remains to cause a voltage rise when more of the enzyme is inhibited. As
will be noted later, we have provided for automatic enzyme pad rejection when
the quantity of residual enzyme activity is marginal to that required for
reliable detector performance.
Figure 7 also shows the kind of change in the voltage tracings which can be
expected when an enzyme inhibitor is present in the water sampled. The
arrow located at the end of the first seven cycles represents the point
at which the water supply was changed to a solution containing 0.2 ppm of
DDVP. As may be seen from the right-hand portion of the curve, the cumula-
tive alarm signal (i.e., the increase in cell voltage over and above the ex-
pected voltage shown by the extrapolated straight line) was 182 mV or
18.2 mV/cycle. If an alarm threshold of 12 mV increase were used as the
measure of the presence or absence of significant quantities of inhibitors
then it is clear that (1) there would have been an alarm within 3 min after
introduction of the DDVP, (2) a single enzyme pad would have given 10 alarms,
and (3) there would have been no false alarms during the six preceding cycles
of operation. Although not shown on the curve, challenge of the electro-
chemical enzyme system with higher concentrations of DDVP results in larger
(perhaps 100 mV) changes in baseline voltages between cycles.
33
-------
SECTION VIII
DESIGN AND FABRICATION OF THE CHOLINESTERASE ANTAGONIST
MONITOR (CAM-1)
Before designing or constructing the water monitoring system based upon
the use of immobilized cholinesterase as the sensor, it was necessary to
consider the intended use of the detector, the manipulations and skills to
be required of the operators, the period of unattended operation desired,
the sensitivity, the selectivity, the permissible size, weight and cost,
the need for maintenance, and other parameters affecting the design. From
the beginning it was planned that 110 V AC power would be available; that
the initial model would be operated indoors so that there would be no need
to provide insulation and case heaters; that unattended operation might be
as long as a week, although 48 hr was chosen as the immediate goal; and
that the weight could be kept below 50 Ib and the volume below 2 cu ft
because a smaller instrument would be much easier for the operator to use.
^
If the instrument were to function unattended for long periods of time,
every operation should be made as nearly automatic as possible. Of particu-
lar importance was a provision for an automatic enzyme pad changer which
would change enzyme pads before all of the activity was used up, so that
an automatic detection cycle which should signal an alarm when toxic hazards
were present in the water sampled could go into operation. The enzyme it-
self would probably give the unit selectivity, since materials which inhibit
this enzyme are likely to be toxic. Speed of response to inhibitors and
sensitivity to low levels of inhibitors are dependent variables; it was ob-
vious that longer sampling times would give the greater sensitivity, but
as was shown after the unit was fabricated, a 2-min water sampling period
is adequate for the detection of subtoxic levels of organophosphates.
The Cholinesterase Antagonist Monitor (CAM-1) which we have designed and
fabricated for the rapid detection of organophosphates in water supplies is
shown in Figure 8. Basically this apparatus is made up of the same kinds
of components as the breadboard detector shown in Figure 5, except that an
alarm has been added and operation of the various components has been auto-
mated through the addition of logic circuits, interlocks, controls and read-
out devices. Additional information about the design and construction of
CAM-1 and its components is given in the following paragraphs.
Insofar as possible commercially available parts for the fabrication of
CAM-1 have been used. However it was necessary to design and fabricate the
automatic enzyme pad changer-electrochemical cell assembly shown in Figure 9.
The cross-section of the electrochemical cell, Figure 1, shows the shape and
configuration of the perforated platinum electrodes which were made from
0.008-in. platinum sheet so that they would have some mechanical strength
35
-------
to
ALARM TOTALIZER
ALARM THRESHOLD
SWITCH
CURRENT LIGHT
SUBSTRATE LIGHT
WATER LIGHT
HORN
ALARM LIGHT
POWER ON/OFF
CLOCK HOLD
RESET
PAD CHANGE THRESHOLD
SWITCH
MANUAL CONTROL
PANEL
..
CELL VOLTAGE
INDICATOR
ALARM LIGHT
CELL VOLTAGE
RECORDER
WATER PUMP MOTOR
ELECTROCHEMICAL
CELL-PAD CHANGER
ASSEMBLY
Figure 8 - The Cholinesterase Antagonist Monitor (CAM-1) With Side Panels and Recorder Cover Removed
-------
u>
-1
PAD EJECTOR
SUBSTRATE INLET
UPPER CELL BODY
ENZYME PAD
BETWEEN ELECTRODES
ENZYME PAD HOLDER
LOWER CELL BODY
PAD POSITIONING
STEPPING MOTOR
MOTOR FOR OPENING
AND CLOSING CELL
Figure 9 - Automatic Enzyme Pad Changer—Electrochemical Cell Assembly From CAM-1
-------
and retain their shape. The wires attached to these electrodes are platinum
and they are welded on. Approximately 34 holes (1 mm in dia) were drilled
in the flat surface of the electrode prior to coating it with platinum black
and heating to make the platinum gray surface. As may be seen in Figure 9,
twin screws driven by a stepping motor through sprockets and a chain are
used to open and close the electrochemical cell. When the cell is open, the
enzyme pad holder may be removed and loaded with enzyme pads; although there
are 12 holes in this plastic holder no more than 11 pads should be inserted
at one time. On closing of the electrochemical cell, the pad positioning
motor advances the pad holder one space and then ejects the pad which had
been placed directly over the electrodes. An operator will, as a consequence,
not think that he has good pads in his pad holder when in reality some or
all of them may have been exhausted during previous use. Thus every time
that a new pad is inserted automatically into the cell, the old pad is pushed
out of the enzyme holder and onto the floor of the instrument.
The upper and lower cell bodies are made of delrin, the 0-rings for sealing
the cell are made of silastic and the substrate inlet is made of stainless
steel. Wherever possible corrosion resistant materials have been used in
the construction of the pad changer mechanism and also for the case.
Most of the case is made of aluminum; its method of construction is shown
in Figure 10(A). The detector is purposely arranged vertically so that the
wet components can be localized on the bottom away from the electronic
parts—or at least most of them. Figure 10(B) shows the back of CAM-1 with
both of the doors open and the accessibility of the various components for
servicing and repair, if necessary. The 24 V DC and the 5 V DC power sup-
plies are located on the second level along with the air pump and the re-
corder (not visible from the back). These two power supplies are fused
individually. Figure 10(C) shows the pulse generator used for operating the
two stepping motors on the pad changer. Figure 10(D) shows a transformer
and three relays; the transformer is used as the source of 6 V AC for opera-
ting the digital clock; Relay 1 turns the water on and the air off; Relay 2
controls the substrate flow and Relay 3 is part of the interlock system which
keeps the water turned off when the cell is open. Figure 11(A) shows the pad
changer assembly installed inside of the CAM-1 case. At the extreme left
of this picture are four toggle switches which are used for the manual con-
trol of the various components; counting down from the top, Switch 1 opens
the cell, Switch 2 controls the water pump, Switch 3 controls the substrate
pump and Switch 4 turns the current on and off. All of these switches must
be in the down (off) position for CAM-1 to operate in the automatic mode.
When the cell is opened electrically, there is a delay before it opens; this
delay was built-in to allow time for air to blow the water from the lines
before the cell opened. The small electrical connector attached to the
pad changer assembly provides an easy way to disconnect the anode and cathode
38
-------
D
Figure 10 - Components of CAM-1: (A) the case; (B) rear view of CAM-1 showing
the relay board and the integrated circuit boards on the upper
level, the air pump and the 5V DC power supply on the second
level, and the peristaltic pump on the lowest level; (C) pulse
generator for operating the stepping motors; (D) relay board.
39
-------
D
Figure 11 - Components of CAM-1: (A) automatic enzyme pad changer-electro-
chemical cell inside of case; note manual controls to the left;
(B) and (D) two views of the computer and logic circuitry
boards; (C) view of the substrate and water pumps inside of
case.
40
-------
when the whole assembly is removed for cleaning or other servicing. The
cable attached to the pad changer assembly leads to the recorder housing
where the operation amplifier and the constant current power source are
located.
Figures 11(B) and 11(D) show two sides of the same circuit boards. The
sockets into which the integrated circuits, gates, memory devices, relays,
counters, dividers and other solid state logic circuit devices are plugged,
are connected by a solderless wirewrap technique developed by Bell Labs.
The digital clock for providing all time functions is mounted on one of these
boards. Figure 11(C) shows the rear of the lowest level in CAM-1. The
peristaltic pump is shown at the lower right, the Flotec pump is shown in
the center, and the connector for obtaining a remote readout of cell voltage
or an alarm signal is shown in the upper left.
Some of the remaining design features may be seen best by reference to
Figure 8. A Digilin digital voltmeter has been mounted on the front of the
CAM-1 case for two reasons. The more important is that it is necessary to
convert the electrochemical cell voltages into a binary code decimal (BCD)
voltage so that it can in turn -be processed by the memory and logic cir-
cuitry; the digital voltmeter makes this conversion; second, it is useful
to be able to read the cell voltage to the nearest millivolt without reading
a needle on a meter or looking at a tracing on a recorder. At one point in
the detection cycle a voltage-hold function is activated which enables the
same cell voltage fed into the memory bank to be held on the meter until
either (1) the current is turned on in the next cycle, or (2) the reset
button is depressed. In the later case it is possible to obtain exact cell
voltage readings during the water pumping part of the detection cycle without
reference to the recorder.
A strip chart recorder (Simpson) is provided to make a permanent time-base
recording of the cell voltage. Recordings of cell voltage can be particularly
valuable to the operator if he should be away from the instrument for a pro=
longed period of time—particularly if the alarm totalizer should indicate
that there had been one or more alarm signals since the totalizer reset
button (not shown) had been pushed. The recording would show not only when
the alarm occurred,but it would also give an indication as to whether the
alarm was the result of a massive spill or merely a false alarm due perhaps
to setting the alarm threshold too low.
The alarm threshold switch is an adjustable digital switch which is used
to control the sensitivity of the CAM-1 unit. The operator may select
alarm thresholds of 0 to 99 mV as the voltage change between successive
cycles to which he would like to have the alarm lights flash and the
Sonalert horn give its 4-sec whistle. For best results, the alarm thresh-
old should be set high enough so that it does not give false alarms and low
41
-------
enough so that hazardous material spills are not missed. Our experience
indicates that 8-20 mV is usually a good place to set this threshold switch.
Next to the alarm threshold switch is the pad-change threshold switch which
is used to activate the automatic-enzyme pad-changer. In order to avoid the
possibility that all of the enzyme activity in an enzyme pad has been destroyed
and that the pad can no longer respond to enzyme inhibitors, a mechanism by
which pads can be replaced automatically before they have lost all of their
enzyme activity, has been provided. One way to select a setting for this
switch is to place a plain starch-coated urethane foam pad (no enzyme) in
the electrochemical cell and operate the CAM-1 for several cycles; following
this activity the pad change threshold switch is set 50 mV or more below the
voltage obtained with the starch pad in the electrochemical cell. By following
this procedure, one can be sure that good pads (i.e., pads which have enough
enzyme activity to respond to inhibitors) are always kept in the electro-
chemical cell.
The detection cycle was shown graphically in Figure 6. The following de-
scription of the automatic operation of the detection cycle in CAM-1 shows
the timing sequence activated by the minicomputer:
0 sec Everything off (except for air which comes on
when the water is turned off).
1 sec Turn water pump on and air pump off.
90 sec Generate sync pulse which can be used to keep
the slave detector system out-of-phase and
provide total sampling.
120 sec Turn water off; start air and substrate flow.
140 sec Apply constant current to electrochemical cell.
175 sec Enter cell voltage into memory circuit, process
this voltage (i.e., compare voltage with voltage
from previous cycle); signal an alarm if the
voltage increase is in excess of the sensitivity
setting; compare cell voltage with amplitude
set point and activate automatic pad changer
if pad needs changing.
180 sec Turn off substrate solution and applied current
and reset clock to 0.
42
-------
The three blue lights located above the horn are connected to the water
pump, the substrate pump, and the applied current relay. These lights come
on in sequence,and indicate which part of the detection cycle is then in
progress. If these lights fail to cycle, it may be an indication that one
of the manual control switches has been left in the up position, or that there
is some malfunction.
The three lights below the horn are also push button switches. The first of
these switches on the left turns the power on and off. If the detector is
stopped in the middle of a cycle and then turned on again, the memory will
be erased and the unit will start the first of a new cycle. Depressing the
second switch stops the clock at the part of the cycle where the detector
happens to be at that instant; pressing this button a second time releases
the clock-hold mechanism, and the clock continues with the cycle where it
left off. This button has value if the operator should like to increase
the duration of any cycle; for example, if there were a desire to increase
the water sampling period from 2 min to 6 min, the operator could stop the
clock for 4 min during the water pumping cycle and the rest of the cycle
would be unaffected. The reset button sets the clock back to zero; it
releases the voltage hold on the digital voltmeter but it does not erase
the memory of the last voltage entered into the memory bank.
When detector operation is first started with an enzyme pad, there should
be no alarm at the end of the first cycle because no voltage is stored in
the memory. Even if the voltage exceeds the previous cell voltage reading
by an amount equal to or in excess to the alarm threshold, there will be no
alarm on the second cycle since CAM-1 was designed to ignore an alarm on the
second cycle. On the third and subsequent cycles there will be an alarm
every time the alarm threshold is exceeded, except that if the enzyme pad
should be replaced automatically, there would be no alarm for the first two
cycles whether or not the alarm threshold is exceeded. This provision for
no alarm at the end of the second cycle can be changed, but possible false
alarms due to the finite time required for new pads to equilibrate can be
avoided, and the provision seems wise.
After an alarm signal, the flashing red lights will continue to flash until
one of the lights is pushed. The alarms are counted on the alarm totalizer
lights which are light-emitting diodes (LED). Since individual segments
of this counter could burn out, these lights flash "88" each time the sync
signal is generated, i.e., 90 sec after the start of the new cycle to show
that all segments are lighting properly.
43
-------
A block diagram of CAM-1 is presented in Figure 12. This diagram shows in
a general way how the various components are interrelated but obviously it
cannot show all of the detailed relationships needed to make the unit func-
tion. The complete wiring diagrams in a greatly reduced format are included
in this report. Figure 13 shows a number of components including: (1) pulse
generator for operation of the two stepping motors on the automatic enzyme
pad changer, (2) the constant current power supply and high impedance ampli-
fier for the electrochemical enzyme cell (located inside the recorder case),
(3) the function control switches, (4) the alarm counter, (5) the relay cir-
cuits for operating the various pumps, etc., (located on the relay board
shown in Figure 10(D)), etc. Figure 14 shows additional circuit features
including: (1) digital clock, (2) counting circuits, (3) analog to digital
to binary code decimal converters, programmer, etc. Figure 15 shows still
more of the CAM-1 circuitry including memory circuits, flip-flops, inverters,
decade counters, binary counters, latches, adders, dividers, comparators and
other computer logic components. Together these three figures show the ad-
vanced solid state circuitry which enables CAM-1 to operate automatically
unattended and to make decisions as to when to change enzyme pads or when
to signal an alarm situation. The circuits given on opposite pages of this
report may be cut out and pasted together to simplify the tracing of signals.
Table 6 gives a list of the suppliers of components in CAM-1 and these are
arranged by position of these components on the three levels in CAM-1.
44
-------
r
Sequential Tinner
Water
Pump
Substrate
Pump
Air
Pump
Cell
Current
Digital
Control
Constant
Current
Source
Recorder
Enzyme
Cell
High Impedance,
Zero Gain,
Amplifier
Ln
Digital
Meter
Memory
Subtracter
Adjustable
Pad Change
Threshold Switch
Comparator
Pad Change
Mechanism
Comparator
Alarm
Adjustable
Alarm Threshold
Switch
Figure 12 - Simplified Block Diagram of CAM-1 Showing the Principal
Relationships of the Logic and Decision Circuits to
the Other Components
-------
PAGE NOT
AVAILABLE
DIGITALLY
-------
TABLE 6
SUPPLIERS FOR CAM-1 COMPONENTS
(Lower Level)
Capacitor
Capacitor
Capacitor
Capacitor
Chain
Chain Connector
Connector
Connector
Connector
Connector
Connector
Connector
Connector
Connector
Fuse Holder
Gear
Gear
Knurled Nut
Magnet
Magnetic Reed Switch
Motor
Motor
Resistors
Solenoid
Solenoid Valve
Sprocket
Sprocket
Substrate Pump
Switch
Switch
Transistor
Transistor
Water Pump
Kemet 1 uf 35 V DC
Kemet 100 uf 6 V DC
Mallory 50 uf 25 V DC
Mallory 100 jif 35 V PC
Pic Design Corporation —
Pic Design Corporation
Amphenol Mil 3100A - 145
Mil 3106 - 145
Mil 3102 - 145
Mil 3106A - 145
Mil 3102A - 145
Mil 3106A - 145
50 - 20 - A -
TR2A
Amphenol
Amphenol
Amphenol
Amphenol
Amphenol
Cinch Jones
Switch Craft
2P
2S
5P
5S
6P
6S
30
Littel Fuse
Pic Design Corporation G3-84
Pic Design Corporation G3-21
Pic Design Corporation
Hamlin H-31-604
Kamiin MINI-2-115
A. W. Haydon K82201
A. W. Haydon K82101
IRC - Ohmite
Guardian 3.5 x 9
ASCO 8360-62£/
Pic Design Corporation EM-5-36
Pic Design Corporation EM-1-9
Extra Corporeal Corporation
Alco 305D
Alco 205N
Fairchild 2N3569
Fairchild 2N4237
Flotec, Inc. FV 158 - 1085£/
53
-------
TABLE 6 (Concluded)
(Second Level)
(Top Level)
Air Pump
Diode
Diode
Indicator Cover (Amber)
Indicator Cover (Blue)
Indicator Cover (Green)
Indicator Cover (Red)
Indicator
Indicator Switch
Indicator Switch
Indicator Switch
Integrated Circuits
Lamp (28 V)
Lamp (6 V)
Lamp (14 V)
Power Supply (5 V)
Power Supply (24 V)
Recorder
Relays (24 V)
Sonalert
Terminal Strip
Trimpot
BCD Switch
Digital Panel Meter
Integrated Circuits
Integrated Circuits
Integrated Circuits
Metaframe Hush !
-------
SECTION IX
OPERATION OF THE MONITOR, CAM-1
Water from the Kansas City Municipal Water Supply, raw Missouri River water,
and city water in Edison, New Jersey, have all been used with the CAM-1 with
no difficulty. It is advisable, however, that the CAM-1 not be connected
directly to any pressurized water source since hoses may come off and the
electrochemical cell may leak. The water should be run into a beaker or
other container in a sink near the CAM-1, and the pump in CAM-1 used to draw
the water into the system. The Flotec pump in CAM-1 is self-priming if there
is a little water in the pump; if the pump is dry, it is best to prime the
pump before operation of the detector. Raw water supplies should be filtered
through sand or a similar coarse filter to remove things which might cause
plugging of the platinum electrodes or of the enzyme pad.
If two CAM-1 units are used together for the purpose of obtaining continuous
monitoring of a water supply, it is necessary to use a sync cable between the
two CAM-1 units. When the two units are connected together, one unit becomes
the master and the other unit becomes the slave. After the two units have
been operated together for 90 sec a sync pulse from the master unit resets
the clock in the slave unit to time zero; additional sync pulses are sent
from master to slave each 180 sec thereafter. Either CAM-1 unit may be
master or slave, depending on which end of the sync cable is plugged into
the detector. If the master CAM-1 should change its pad, the second unit
would continue to operate without the sync pulse until the master unit was
again operating and another sync pulse was sent to the slave. Thus the
units would stay out of sync and provide continuous monitoring as planned.
The simplified operating instructions given below should be of value to an
operator who is unfamiliar with the instrument.
CAM-1 Operating Instructions
To Start:
1. Prepare fresh substrate solution using 40 mg of butyrylthiocholine iodide
and 500 ml of 0.08 M tris buffer, pH 7.4 and insert end of small plastic tube,
2. Turn on power (green light switch), and if cell is not open, open it with
manual switch.
3. Load pad holder with 1 to 11 enzyme pads.
4. Insert pad holder into the cell with an empty hole between the cell
electrodes. Be sure that there is a pad to the left of the empty hole as
the pad holder advances clockwise one space before the cell closes.
55
-------
5. Close the electrochemical cell by moving the top switch to the down
position.
6. Prime the substrate lines by moving the substrate switch (third from
top) to the upper position for 5 min and then lowering it.
7. The sound of water being pumped indicates that the unit is now opera-
ting automatically. If it is not, be sure that the clock-hold light is off,
the cell is all the way closed, and all manual controls are in the down
position.
8. Adjust alarm threshold to about 12 mV. Greater sensitivity is obtained
at 8 mV, whereas lesser sensitivity is obtained at 20 mV.
9. The pad change threshold switch should be set 50 mV below the maximum
voltage that is obtained by pumping substrate solution through a plain
starch (no enzyme) pad. This setting is usually about 150 mV above the
lowest voltage obtainable with a good enzyme pad.
To Stop:
1. Move manual control for the electrochemical cell to the open position
(up). (Note: there is a 13.6-sec delay so that water may be blown from the
waterlines before the cell will open.)
2. When the cell is all the way open, turn off the power by pressing the
green light switch.
3. Remove the plastic disc containing the unused enzyme pads, if any, so
that the cell may dry out while not in use.
In an effort to determine if CAM-1 would operate automatically for 48 hr
unattended, we conducted a study in which enough substrate solution to last
for 2 days without replenishment was made up and enzyme pads were put into
the pad holder. CAM-1 was started and observed continuously for 56 hr; a
record of the digital voltmeter readings was made for each cycle for the en-
tire period. During this period no one touched CAM-1. The individuals who
watched CAM-1 for this period reported that the water and substrate pumps
started and ran as expected for every cycle, and that there were no malfunc-
tions of the electronic circuitry. Three false alarms were scattered over
the 56-hr period which, judging from the digital voltmeter readings, re-
sulted from abnormally low cell voltage readings and an alarm when the vol-
tage returned to normal voltage on the next cycle. An examination of the
voltage tracing showed that no sudden inhibition of the enzyme occurred
during this sampling period. If these false alarms should be considered
a serious problem, a change in alarm logic could be made to eliminate them;
56
-------
one such change might be the requirement that the voltage rise for two
successive cycles before signalling an alarm. Other alternatives could be
worked out if this continues to be a problem.
This 56-hr test did not provide testing of the enzyme pad changer since one
enzyme pad gave satisfactory base line voltages for the entire period; how-
ever, examination of this enzyme pad after this test revealed that it was
near exhaustion, and should have been replaced at about the 56th hr.
The second CAM-1 was also operated during this 56-hr test but some difficulty
was encountered due to the numerous alarms produced with it; the difficulty
was caused by the use of the sync cable. After disconnection of the sync
cable, both of the CAM-1 units performed well. At the conclusion of the
test, an error in the wiring of the sync cable connector was corrected. Our
experience in operating the two units together with the sync cable is limited,
but so far both units have worked well either alone or as slave and master.
The first CAM-1 unit was operated about 300 hr before its delivery to the
Edison Water Quality Laboratories.
57
-------
SECTION X
RESPONSE OF CAM-1 TO SEVERAL INSECTICIDES
In the initial studies conducted on the response of the electrochemical
enzyme system to an organophosphate, the experimental cell shown in Figure 3,
and a solution of tetraethylpyrophosphate (TEPP), were used. Problems with
this system developed because of the rapid hydrolysis of the TEPP. In some
very crude experiments, three dilutions of Tetron-100 (American Potash and
Chemical Company, 40% TEPP and 60% other ethyl phosphates) were prepared
in tap water, and stored at room temperature for periods of up to 6 days;
these solutions were assayed after various periods of time to determine their
ability to inhibit a known quantity of cholinesterase. Although the 1:1,000
solution had some activity after 6 days, the 1:1,000,000 solution had lost
more than 25% of its activity after only 24 hr. We decided to pick an
organophosphate which was more stable in solution, and which could be used
for determining sensitivity of our system. In a study of 2,2-dichlorovinyl
dimethyl phosphate (DDVP), we found that a 1:10,000,000 solution of DDVP
in distilled water and stored at room temperature retained 63% of its ac-
tivity after 7 days. This observation led to the conclusion that DDVP
would be a better compound to use in testing the CAM-1 than TEPP because of
the greater stability of the DDVP in water. Some of the data obtained on
the response of an electrochemical cell to DDVP are shown in Figure 7.
Some of the other data collected in a study of response to low concentra-
tions of insecticides in water are presented in Table 7. In Part A of the
table some of the results obtained with the experimental electrochemical
enzyme cell used with standard laboratory instruments, and operated on a
cycle like the one selected for use in CAM-1, are given. Both malathion
and parathion can be detected, although the sensitivity to parathion is
several times greater. Although time did not permit tests of CAM-1 with
these insecticides, we are confident that it will detect them at approxi-
mately the same levels as we have reported here.
In Part B of the table the response of CAM-1 to a number of commercially
important insecticides, including both organophosphates and also carbamates,
is reported. CAM-1 responded to all of them except Ronnel; this failure to
respond was probably due to the insolubility of the Ronnel in tap water.
Since Ronnel is both insoluble in water and reasonably nontoxic, the failure
of CAM-1 to alarm is probably of no consequence. With the compound Sevin,
when CAM-1 was set at a threshold sensitivity of 12 mV per cycle, there were
five alarm signals prior to the inactivation of the enzyme pad.
The question was raisedj "What would happen if the toxic substance were re-
moved after the first or second alarm?" In order to find out, a solution
59
-------
TABLE 7
DETECTION OF INSECTICIDES IN
Part A - Using an Experimental Enzyme Cell
Insecticide
(source)
Malathion (Prentiss Drug) ^ '
Parathion (Chevron Chemical) (d)
Chemical Type
Phosphate
Phosphate
LD50 (rats)
(mg/kg)
1,640
56
Concentration of
Inhibitor (ppm)
16.5
3-5
Number of
Alarms
(c)
(c)
Ronnel (Dow)(e)
Sevin (Union Carbide)
Dimetilan (Geigy)
Azodrin (Shell)<*>
Paraoxon (Cyanamid)
DDVP (Shell)
(g)
Part B - Using CAM-1
Phosphate
Carbamate
Carbamate
Phosphate
Phosphate
Phosphate
1,720
560
64
21
3.5
56.0
80 (f)
50
10
20
1
1
None
5
3
5
(a) All measurements were made with city tap water at 5° ± 3°C.
(b) Dimethyl S-(l,2~dicarbethoxyethyl)phosphorodithioate.
(c) There was no alarm mechanism in this system; responses of 12 mV or more per cycle were obtained for
several cycles.
(d) Diethyl 4-nitrophenyl phosphorothioate.
(e) 0,0-Dimethyl 0-2,4j5-trichlorophenyl phosphate.
(f) The Ronnel was dissolved in warm methanol and added to water at room temperature. Most of the Ronnel
came out of solution and plugged up the electrodes and the pad. The concentration in solution was
only a small fraction of this value.
(g) N-Methyl-1-naphthyl carbamate.
(h) 2-Dimethylcarbamoy1-3-methylpyrazoyl-5-dimethyl carbamate.
(i) 3-(Dimethoxyphosphinoxy) -N-Ttiethyl-cis-crotonamide.
(j) Diethyl p-nitrophenyl phosphate.
(k) The voltage increase for one cycle was 147 mV indicating that much lower levels could be detected.
(1) 2,2-Dichlorovinyl dimethyl phosphate.
-------
of 0.2 ppm of DDVP was prepared, and exposed to CAM-1. After two alarms
on successive cycles, the inlet water was switched to tap water. On the
third cycle there was an alarm, but on cycles 4, 5, and 6 there was no
alarm. The inlet hose was switched back to the jug containing the DDVP
and alarms on cycles 7, 8, and 9 were obtained; for all of these 9 cycles,
the same enzyme pad was used. The alarm on the third cycle was probably
due to the inhibitor's remaining in the waterlines from the previous cycle.
This experiment indicates that the alarm can be stopped when the enzyme
pad is partially inhibited, and that it can still provide alarms when more
inhibitor is encountered. With DDVP and the other enzyme inhibitors in-
vestigated so far, the enzyme activity does not return after these inhibited
enzyme pads are washed.
The greatest response we have encountered (i.e., the greatest voltage in-
crease between successive detection cycles) occurred when we challenged
CAM-1 to 1 ppm of paraoxon and obtained a 147-mV rise in one cycle. It is
concluded that CAM-1 will respond to much lower levels of this insecticide,
although these have not yet been tried.
There is a rough correlation between the toxicity of the insecticides and
the levels which can be detected with the immobilized enzyme system (see
Table 7). We believe that the more toxic compounds are collected from the
water with greater efficiency due to their greater affinity for the enzyme.
At the same time compounds possessing the greatest affinity for enzymes
in vivo are probably the ones which are going to be more toxic. Thus the
similarity of the mechanism of detection in CAM-1 with the mechanism of
toxicity of the inhibitors in vivo explains why we see the correlation. If
the mechanism of action were different in the two systems, and we expect
it to be so for some other insecticides, the correlation would probably not
exist.
The experiments reported in Table 7 were conducted with tap water at 5° ±
3°C. The response data were collected under temperature conditions much less
favorable for the collection of the enzyme inhibitors than 25°C. In some
preliminary studies with DDVP, the system was four to five times more sensi-
tive at 25°C than at 5°C. However, the system still possesses enough sensi-
tivity at 5°C to provide adequate protection against accidental poisoning.
The following rough calculation shows that this sensitivity is more than
adequate for the protection of individuals using the water supplies: If
we assume the LD50 of DDVP in man is 6 mg/kg (Heath—' reports the LD50 for
rats is 6 mg/kg and the Canada Department of Agriculture!^/ reports the LD5Q
for rats is 56-170 mg/kg) and also that a man weighs 70 kg, then we see that
a man would need to drink at least 4,200 liters of water containing 0.2 ppm
of DDVP in order to receive the U>5Q dose—an obvious impossibility. CAM-1
need not be operated at this high sensitivity; to lower the sensitivity all
that is necessary is to increase the allowable voltage change between suc-
cessive voltage measurements by adjusting the digital switch on the front
of the instrument.
61
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SECTION XI
ACKNOWLEDGEMENTS
This work was sponsored by the Environmental Protection Agency Water Quality
Office under Contract No. 68-01-0038.
The authors wish to thank Mr. Robert W. Schafer, Mr. Walter D. Hodge,
Mr. Edward T. Fago, Mr. Jack Breen, Mr. Frank J. Barker, Miss Margo L.
Wilkinson and Mr. Lorren D. Kurtz for their help in the design, fabrication
and evaluation of the CAM-1.
Dr. Thomas B. Hoover, Research Chemist, Southeast Water Laboratory, Athens,
Georgia, was the Project Officer for this contract.
63
-------
SECTION XII
REFERENCES
1. Thoman, J. R., and H. P. Nicholson, Pesticides and Water Quality.
Proc. 2nd Sanit. Eng. Conf., Vanderbilt University, May 1963.
2. Faust, S. D., and I. H. Suffet, "Recovery, Separation and Identification
of Organic Pesticides from Natural and Potable Waters," Residue Reviews,
F. A. Gunther, ed. Springer-Verlag, New York, pp. 44-116 (1966).
3. Muhlmann, R., and G. Schrader, Hydrolyse der insektiziden
Phosphorsaureester, Z^ JNa_turforsch. T_12_B, 196 (1957).
4. Davis, T. J., and G. W. Malaney, "Acetylcholinesterase Inhibition--
A New Parameter of Water Pollution," Water and Sewage Works, 114, (7)
pp. 272-274 (1967).
5. Guilbault, G. G., D. N. Kramer and P. L. Cannon, Jr., "Electrochemical
Determination of Organophosphorous Compounds," Anal. Chem. 34, (11)
pp. 1437-1439 (1962).
6. Guilbault, G. G., M. H. Sadar, S. S. Kuan and D. Casey, "Enzymatic
Methods of Analysis. Trace Analysis of Various Pesticides with Insect
Cholinesterase," Anal. Chem. Acta, 52, pp. 75-82 (1970).
7. Porterfield, H. W., Water Pollution Analyzers: "A Changing Market,"
Oceanology Intl., pp. 22-24, October 1970.
8. Cairns, J., Jr., R. E. Sparks and W. T. Waller, "Biological Systems as
Pollution Monitors," Research/Development, pp. 22-24, September 1970.
9. National Technical Advisory Committee to the Secretary of the Interior,
Report, Water Quality Criteria, Federal Water Pollution Control
Administration, p. 85, April 1, 1968.
10. Bauman, E, K., L. H. Goodson, G. G. Guilbault and D. N. Kramer,
"Preparation of Immobilized Cholinesterase for Use in Analytical Chemistry,"
Anal. Chem., 37, (11) pp. 1378-1381 (1965).
11. Ellman, G. L., K. D. Courtney, V. Andres, Jr., and R. M. Featherstone,
"A New and Rapid Colorimetric Determination of Acetylcholinesterase
Activity," Biochem. Pharmacol. 7, pp. 88-95 (1961).
65
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12. Goldman, R., L. Goldstein, and E. Katchalski, in Biochemical Aspects
of Reactions on Solid Supports, G. R. Stark, ed., Academic Press,
New York (1971).
13. Kay, G., and E. M. Crook, "Coupling of Enzymes to Cellulose Using
Chloro-s-triazines," NATURE. 216, 514 (1967).
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of Lactic Dehydrogenase Attached to Water-Insoluble Particles and
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66
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Accession Number
Subject Field & Group
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Midwest Research Institute, Kansas City, Missouri,
Life Sciences Division
Title
RAPID DETECTION SYSTP-M FOR ORGANOPHOSPRATES AND CARBAMATE INSECTICIDES IN WATER
1 Q Authors)
Goodson Louis H.
Jacobs, William B.
16
21
Project Designation
EPA WQO Contract No. 68-01-0038
15090 GUI
Note
22
Citation
Environmental Protection Agency report
number EPA-R2-72-010, August 1972.
23
Descriptors (Starred First)
Pesticide Detector,* Organophosphates, Carbamates, Anticholinesterases,
Immobilized Enzyme Detector
25
Identifiers (Starred First)
Water Monitoring Device, Cholinesterase Antagonist Monitor, CAM-1
JO
Abstract
An apparatus for the detection and monitoring of water supplies for hazardous spills
of organophosphate and carbamate insecticides has now been designed and fabricated.
The new unit is called the Cholinesterase Antagonist Monitor, CAM-1, because it
produces an alarm in 3 min when toxic or subtoxic levels of Cholinesterase antago-
nists are present in water. Response of this apparatus to subtoxic levels of
azodrin, sevin, dimetilan, malathion, parathion and DDVP has already been demon-
strated. CAM-1 uses immobilized Cholinesterase for the collection of Cholinesterase
inhibitors from the water supplies. The activity of the immobilized Cholinesterase
is determined automatically in an electrochemical cell by passing a substrate 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 which occurs in the presence of organophosphate and carba-
mate insecticides in the water sampled.
Abstractor
Institution
WR:102 (REV. JULY 1969)
WRSIC
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 20240
if U.S. GOVERNMENT PRINTING OFFICE: 1972-724-046/53 3-1
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