Toxic Chemical and Explosives Facilities - ACS Publications

18 D e v e l o p m e n t of

Highly

Sensitive M o n i t o r s f o r t h e

D e t e c t i o n of A n t i c h o l i n e s t e r a s e C o m p o u n d s

DONALD C. BEHRINGER, FREDERICK C. BALADUF, and VINCENT N. CAMMARATA

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: April 6, 1979 | doi: 10.1021/bk-1979-0096.ch018

Chemical Systems Laboratory, Aberdeen Proving Ground, MD 21010

Two highly sensitive monitors for the detection of anticholinesterase compounds were developed. One monitor was specifically developed to meet stringent time concentration requirements established by the Surgeon General of the Public Health Service and provides rapid and continuous detection of trace quantities of compounds of the type noted in Figure 1. As will be seen, however, the monitors could be useful devices for other enzyme-inhibiting compounds. The monitors will be evaluated by the Army's Project Manager for Chemical Demilitarization and Installation Restoration at the new Chemical Agent Munitions Disposal System (CAMDS), Tooele Army Depot, Utah, during execution of the program to destroy obsolete stocks of toxic compounds. The rapid monitor was developed to automatically monitor exhaust stacks and work areas while the other monitor is a personal dosimeter to be worn by plant workers. The well-known enzyme colorimetric method of analysis is employed as the basis for the determination of the anticholinesterase compound. The enzyme selected for determination of the presence of the compound is an acetylcholinesterase. As shown in Figure 2, without an inhibitor present, the series of reactions are: The acetylthiocholine (ATCI) is hydrolyzed by the enzyme acetylcholinesterase to produce acetic acid and thiocholine. The thiocholine then reacts with 5,5'-dithio-bis-2,2'-nitrobenzoic acid (DTNB) to form the colored anion, 5-thio-2-nitrobenzoate, production of which is monitored colorimetrically. With an inhibitor present, the enzyme hydrolysis of the ATCI does not occur resulting in a reduction or loss of color which is the basis of the sensitivity of the monitor. As such, this device is a measure of the toxicity of the enzyme inhibitors. A small amount of a highly toxic compound or a larger amount of a less toxic compound will produce similar results. As shown in Figure 3, the analytical reactions are performed in two glass coils immersed in a water bath at 32°C. In the first coil, the sample, enzyme, and segmentation air are introduced. If agent is present in the sample, inhibition will occur. The contact time is about 3 minutes. The stream then flows to the second coil where the substrate, ATCI, and the color developer, DTNB, are introduced. The hydrolysis of the substrate proceeds to yield acetic acid and thiocholine iodide (TCI). As the TCI is produced, it reacts with the DTNB to form the colored anion, 5-thio-2-nitrobenzoate. The reaction stream is then pumped to the colorimeter. The time for this stage is 2 minutes 40 seconds. The total time from This chapter not subject to U.S. copyright. Published 1979 American Chemical Society Scott; Toxic Chemical and Explosives Facilities ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

302

TOXIC C H E M I C A L A N D EXPLOSIVES FACILITIES

0

R

0

\

// P


Figure 1.

Anticholinesterase nant

contami-

/

\

R

F

"INHIBITOR" /

/ / / (CH )-N-CH -CH -S-C-CH3 3

2

U

h '

2

ACETYLTHIOCHOLINE (ATCI)

I

ENZYME

COO

"

[(CH^g-NL

/

-CH -CH -S 2

2

1

W-DITHIO BIS-2,2' NITRO BENZOIC ACID -

COO N0

3

-

3

COO

3

COO

+ CH -C-OH

THIOCHOLINE

(CH )

-

2

0 N 2

N-CH -CH -S-S 2

2

5-THIO-2-NITRO BENZOATE COLORED SPECIES 420 mji Figure 2.

Monitor detection concept

Scott; Toxic Chemical and Explosives Facilities ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


1

REFRIGERATOR

TO STACK.32

AIR

HO

Figure 3.

C5

Monitor system

GRAVITY DEBUBBLER

FLOWCELL

TO WASTE

AUX. DEBUBBLER .23

1.20

:OLORIM\ETER

REACTION COIL

Dl

REACTION COIL

TEMPERATURE BATH (32°C)

CELL PULL THRU

PROPORTIONING PUMP

DTNB .32

.32

ATCI

ENZ

.6

HCI

.6

SAMPLE

SCRUBBER-CONC.


RECORDER

ALARM HORN

L2

ALARM LIGHT


304

TOXIC CHEMICAL AND EXPLOSIVES FACILITIES

probe inlet to colorimeter is about 7 minutes. The agent response time varies from 8-1/2 to 11-1/2 minutes depending upon at what point in the 3-minute cycle the agent is first absorbed. The analysis is completed by a colorimeter that electronically compares the color intensity of the analytical stream against an air reference. The ratio signal is sent to a strip chart recorder and to a counter and logic card. Activation of visual and audible alarms will occur if predetermined agent levels exist. The analytical precision and accuracy are controlled by a peristaltic pump that meters out exact amounts of the various reagent solutions. The flow rates for the absorbent liquid, probe sample, enzyme, acid, ATCI, and DTNB, as well as the waste products, are all precisely controlled so the analytical process can be continually repeated. The color thus formed in the absence of inhibitor and the color loss when inhibitor is present are always predictable (±1%). A timer-activated solenoid valve replaces the enzyme with dilute acid (HCI) for 27 seconds during each 180-second cycle. The use of the acid wash removes all residual color that was produced during the previous 3-minute cycle. We found that this step was necessary to avoid the troublesome color "buildup" that is encountered in most automated continuous systems where color is formed. A block diagram of the system is shown in Figure 4 to further indicate the air, solution, and readout processes. A typical recorder printout is shown in Figure 5. Color is produced by uninhibited enzyme and extinguished by acid during each cycle. The colorimeter converts the color into an electric signal which is received by the record where the signals are manifested as a series of dark and light peaks. The counter and logic electronics evaluate the signals and will activate the alarm systems if the amount of color does not meet a preset level during any cycle. The color of the solution resulting from the reaction is inversely proportional to the concentration of inhibitor in the air sample. Three typical responses resulting from three exposures of the system to an inhibitor are shown in Figure 5. It should be noted that very good baseline stability has been demonstrated during evaluation of the system in the polluted atmospheres found in plant exhaust stacks. We have demonstrated the sensitivity of the system by introducing the low levels of the inhibitor while the system has sampled plant or stack atmospheres. Calibration of the monitor is performed using standard solutions of the compounds of interest. Reagents utilized in the system are shown in Figure 6. Servicing on 7-day intervals is possible using the amounts as indicated. The solutions are kept in a refrigerator which is part of the system. The solutions are stable and usable for periods of at least 2 weeks after preparation if kept cold. With this capability, the monitor can be operated intermittently without the need to continually prepare fresh solutions. A feature of the device is that the refrigerator continues to operate and maintain temperature even when the monitoring functions are not operating. The system as developed is shown in Figure 7. The device operates from standard 110 v ac; it weighs 450 pounds and measures 6 by 2.5 by 2.5 ft. Shown is the strip chart recorder, refrigerator, and alarm-light indicator. A unique air-sampling probe shown in Figure 8 was developed to eliminate sampling problems. The absorption and concentration of low volatile compounds had been a particular problem due to the propensity of the materials to adhere to


BEHRINGER ET AL.

Anticholinesterase

Compounds

ABSORBER PROBE

AIR EXHAUST-

-ABSORBENT

(H 0) 2

AIR/LIQUID SEPARATOR

INCUBATION OF SAMPLE A N D ENZYME (5MIN.)


HYDROLYSIS OF SUBSTRATE (ATCI) AND COLOR DEVELOPMENT (2 MIN.)

COLOf UMETER RECORDER

DIRECT & REMOTE ALARM SYSTEMS

Figure 4.

Monitor block system

100 MAX. ENZ. R A N G E -

80 A. 2ppt ENZ. INHIBITION60 O > ^

B. 4 p p t 40 C. 6 ppt

20

ACID BASELINE0

30

60

90

120

OPERATING TIME (MINUTES)

Figure 5.

Inhibitor response



DISTILLED

WATER

DTNB

WATER

( p H 8.4)

FEED

BUFFER

FOR PROBE

IN .05 M T R I S

50/i/«

0.2N

7.4)

ENZYME

HCI

(pH

. 0 0 0 4 M IN . 0 5 M T R I S

BUFFER

2.00

Monitor reagents

2.880

0.734

0.60

0.461

0.461

0.130

Figure 6.

DAILY REQUIREMENT

0.60

0.32

0.32

.0008MIN

ATCI

RATE

(mls/min)

REAGENT

FLOW


(liters)

D A Y WEEK

20.160

5.141

0.907

3.226

3.226

REQUIREMENT

7

(liters)

Anticholinesterase

Compounds


BEHRINGER E T A L .

Figure 7.

Work area monitor


TOXIC CHEMICAL AND EXPLOSIVES FACILITIES


308

Figure 8.

Monitor and sample probe



18.

BEHRINGER E T A L .

Anticholinesterase

Compounds

309

sampling lines or any internal equipment involved in the transfer of the vapor to the monitor. The air-sampling device used with the subject monitor minimizes the above problems. The device is simply two flexible tubes: one tube draws the air sample and the other tube supplies an absorbent liquid at the air inlet of the first tube. The air and liquid make contact and are drawn concurrently along the inside wall where absorption occurs. The air/liquid stream then empties into a calming chamber which reduces the velocity, allowing the liquid to gravitate into a reservoir where it is available for continuous analysis. In practice, the air/liquid contact is achieved as the liquid "wets" the tube wall surface with a thin-moving liquid layer. The large surface area of the liquid layer presents adequate opportunity for intimate contact and efficient absorption. The development of the personal dosimeter involved the design, construction, testing, and performance evaluation of a system which fulfills these specific requirements: (1) designed for lightweight and unencumbered attachment to the worker; (2) presents no danger to the health, safety, or welfare of the worker; (3) samples ambient air in the worker's breathing zone in the temperature range of 32° to 105°F for 8 hours; and (4) generates quantitative determinations of individual exposures. The requirement for sensitivity of the dosimeter system is to detect a concentration of 18 parts per trillion in 8 hours. At this safe limit concentration, the bubbler will collect 1.5 X 10~ g of contaminant when sampling air at 30 cc/min for an 8-hour period. A sufficient amount of contaminant in solution must be retained for analysis. The dosimeter design concept follows the principle of commonly used sampling devices which extract trace amounts of toxic substances from air samples drawn through a liquid absorber by solution. The sample collected is sent to the laboratory and the liquid is analyzed by a suitable quantitative procedure such as the colorimetric method just described. The essential components of the dosimeter are an aeration bubbler, a sampling pump, and an interconnecting plastic sampling line. Miniature connectors at each end provide a positive air seal and a "quick-disconnect" feature. A carrier is provided for wearing the dosimeter. The pump is located on the waist belt and the shoulder strap contains a pouch for placing the bubbler in the breathing zone. The dosimeter configuration is shown in Figure 9. Figure 10 shows the dosimeter on the wearer. The bubbler is a miniaturized two-piece glass unit. The lower piece is the aeration chamber containing the absorbent solution which is pH 3.7 distilled water and sulfuric acid, unbuffered. The top piece is an air chamber designed to prevent migration of the solution from the aeration chamber. A "trap" feature built into the air chamber provides confinement of the solution within the bubbler in any position or attitude. The two-piece construction facilitates the loading and unloading of the absorbent and simplifies the handling and analytical procedures in the laboratory. Extensive field testing determined that the most suitable and accurate pump for the dosimeter application was one designated "Accuhaler 808," which is manufactured by MDA Corporation. The principle of operation is based upon an aspiration cycle which draws a constant volume of sample air through a limiting type of orifice for each stroke of the pump. A counter reads out the pump strokes and the total sample volume is computed for a given sampling period. The dosimeter 9


TOXIC C H E M I C A L A N D EXPLOSIVES FACILITIES


310

Figure 9.

Personal dosimeter system


Anticholinesterase

Compounds


BEHRINGER E T A L .

Figure 10.

Dosimeter in wearing mode


TOXIC C H E M I C A L A N D EXPLOSIVES

FACILITIES


312

Figure 11.

Bubbler influence of airflow rate on collecting efficiency as a function of temperature for low concentrations .07 to .10 ng/L



BEHRINGER E T A L .

Figure 12.

Anticholinesterase

Compounds

313

Bubbler average collection efficiency as a function of temperature for 30 cm /min sampling rate and low generated concentrations 3


TOXIC C H E M I C A L A N D EXPLOSIVES


314


FACILITIES


18.

BEHRINGER E T A L .

Anticholinesterase

Compounds

315

samples at a very low flow of 30cc/min or 14.4 liters of air for an 8-hour period, which is 500 times less than the average breathing rate of a man at 15 £/min. The collection efficiency for the bubbler design maximizes at approximately 30 cc/min as shown in Figure 11. A data base for predicting the chemical behavior of the bubbler was established. The generated data base is used to characterize the sensitivity and stability of the bubbler in the presence of known quantities of toxic vapors under ambient stress conditions. The data base is used to correlate the exact relationship between field sampling results and a worker's daily exposure. The chemical characterization work included the following essential determinations: 1. The experimental data permitted the prediction that the average collection efficiency for the aerated bubbler system will be above 90% for the temperature range sampled. This is shown in Figure 12. 2. The desorption of the bubbler was quantified as a function of sample volume and temperature as shown in Figure 13. Retention of the contaminant in the bubbler after 8 hours of aeration is sufficient for analytical evaluation. 3. The optimum pH of the absorbent was found to be in the range of 3.7 to 4.5 where the absorbed contaminant is most stable over the sampling temperatures of 32° to 105°F. 4. The colorimetric method of enzymatic analysis was found to be specific and sensitive to the levels of absorbed contaminant collected when sampling around the limit concentration of 10" g/m . 5. A statistical analysis indicated a high confidence level in the relevancy of the data regarding reliability and reproducibility. The system inaccuracy results from agent desorption at elevated temperatures as shown in Figure 13. These devices are now being tested in demilitarization plants as monitors of stack exhaust atmospheres and work areas. We have not had the opportunity to determine the sensitivity of the devices to the numerous enzyme-inhibiting compounds which would be of interest to others. Generally, however, it is reasonable to expect, for instance, less sensitivity to P=S and P—S linkages and conversely better sensitivity to compounds where the sulfur is replaced by oxygen due to greater toxicity of these compounds. Such compounds as the carbamates, due to their inhibiting qualities, would also be expected to be detected with these systems. It is expected that these systems could find application as highly sensitive monitors during manufacturing, filling, and storage of specific compounds. 8

RECEIVED November 30,

3

1978.


Toxic Chemical and Explosives Facilities - ACS Publications

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