Enzymatic Determination of Organic Phosphorus Insecticides PAUL A. GIANG AND S . A. HALL Bureau of Entomology and Plant Quarantine, United States Department of Agriculture, Beltsville, M d . The wide use of toxic organic phosphorus insecticides has created a need for sensitive methods for their detection as spray residues on plant materials. As the organic phosphorus insecticides inhibit the enzyme cholinesterase in varying degree, this property was used as the basis for an analytical method. Ether extractives of plant materials that had been sprayed with insecticide were reacted for 30 minutes at 25” C. with standard cholinesterase and buffer solution to effect p-artial inhibition of the enzyme; standard acetylcholine chloride solution as the substrate was added and reacted for 60 minutes at 25’ C.; the pEI change was measured and converted
W
ITH the increasing use of organic phosphorus insecticides
there has arisen a need for sensitive methods of detecting and measuring minute quantities of these toxic compounds on plants and in animal tissues. The colorimetric method of Averell and Norris ( 2 ) has been widely used for the determination of parathion. By this method other organic phosphorus insecticides that possess a n aromatic nitro group-such as 0-ethyl 0-p-nitrophenyl benzenethiophosphonate (EPN), 0,O-dimethyl 0-p-nitrophenyl thiophosphate (the methyl homolog of parathion), and diethyl p-nitrophenyl phosphate (para-oxon or the oxygen analog of parathion)-can also be determined. The Averell and h’orris method is sensitive to the determination of about 20 micrograms of any of these compounds, but i t does not distinguish between them-for example, parathion cannot be distinguished from diethyl p-nitrophenyl phosphate, which is more toxic than parathion. Analytical methods are lacking for the determination of microquantities of tetraethyl pyrophosphate (TEPP), tetraethyl dithiopyrophosphate (eulfotepp), a trialkyl thiophosphate (E-lO59), and a number of other organic phosphorus Compounds that are in experimental use as insecticides. Tetraethyl pyrophosphate as a sprag residue on fruits and vegetables does not ordinarily present a problem, as in the presence of moisture it hydrolyzes rapidly t o nontoxic watersoluble products. The same can be said of hexaethyl tetraphosphate (HETP), which contains tetraethyl pyrophosphate as its principal toxic ingredient ( 5 ) . HoBever, there still exists a need t o determine minute amounts of tetraethyl pyrophosphate in animal and plant tissues and in the atmosphere under certain conditions of application or manufacture. Organic phosphorus insecticides in varying degree are inhibitors of the enzymes cholinesterase and pseudo-cholinesterase (3, 6). On the basis of this property the authors have devised a highly sensitive method for the determination of insecticides that inhibit cholinesterase. Although the method is in a general sense nonspecific, one can distinguish between certain of the organic phosphorus insecticides that have large differences in inhibitory pon-er. For example, diethyl p-nitrophenyl phosphate was found to be an extremely poJverful inhibitor, greatly exceeding parathion in this respect on cholinesterase. Furthermore, parathion could be readily converted in the minute amounts present as a spray residue to diethyl p-nitrophenyl phosphate by oxidation in the cold with ,a mixture of concentrated nitric and fuming nitric acids. Thus, it was possible t o distinguish b e h e e n
to per cent inhibition, which is related to micrograms of the insecticide in question. The method has been used to determine spray residues of tetraethyl dithiopyrophosphate, which is a fairly strong inhibitor of cholinesterase. Other insecticides that can be determined by this method are tetraethyl pyrophosphate, compound E-1059 (a trialkyl thiophosphate), 0-ethyl 0-p-nitrophenyl benzene thiophosphate, parathaon, and the oxygen analogof parathion (para-oxon). Parathion, which is not a strong inhibitor, was readily converted to para-oxon, a very strong inhibitor, and thus measured in hundredths of a microgram by the enzyme method.
parathion and diethyl p-nitrophenyl phosphate. By the enzymatic method the authors could distinguish between parathion and methyl parathion, as the latter is a much weaker inhibitor. On the other hand, they could not easily distinguish bet.rceen parathion and 0-ethyl 0-p-nitrophenylthiophosphate, as they did not find sufficient difference in the inhibitory potencies of these related compounds. The enzyme-inhibition method is based on Michel’s ( 7 ) simplified method of measuring the cholinesterase activity of human blood cells and blood plasma. He used a p H meter in place of the much more elaborate and costly Warburg apparatus (I), and his method is more convenient and faster than the titrimetric method described by Glick (3). In Michel’s method the acetic acid produced by the splitting action of cholinesterase on the substrate acetylcholine is measured in terms of the change in pH in the presence of a standard buffer over a definite time interval. In the adaptation of his method tetraethyl dithiopyrophosphate was determined in a number of plant materials, particularly lettuce and tomatoes, that had been treated with aerosols containing this insecticide. The method is described in detail for tetraethyl dithiopyrophosphate. Essentially the same procedure can be applied to the determination of other organic phosphorus insecticides that are generally stronger inhibitors than parathion. The structural formulas of the seven organic phosphorus insecticides that were tested are shown below.
S
Parathion 0,O-Diethyl 0-p-nitrophenyl thiophosphate
8
Methyl Parathion (Methyl homolog) 0,O-Dimethyl 0-p-nitrophenyl thiophosphate
1830
0
Para-Oson (Oxygen analog of parathion) Diethyl p-nitrophenyl phosphate
S
EPN 0-Ethyl 0-p-nitrophenyl benzene thiophosphonate
V O L U M E 23, NO; 1 2 , D E C E M B E R 1 9 5 1 Table I .
1831
ChoIinesteraJAInhibitiveEffects of Organic Phosphorus Insecticides
Quantity to Produce Inhibition of: 50% 80% Mol. Weight. Weight, Weight y Molar oonon. Molar concn. 1 . 4 8 X 10.4 2 . 4 3 X 10258 291.0 42.5 3 . 5 0 x 10-8 0.001 2~0.2 0.023 1 . 3 2 x io-$ 275.0 0.031 1.88 x 100.098 5 . 9 4 x IO7 . 7 5 X 1012.0 2 . 2 7 X 10258.2 3.52 0.05 x 1 0.. 11.7 322.3 3.78 1 . 9 5 x 10-8 323.1 15.4 7 . 9 4 X 10BOIO 3 . 0 9 X 10' 1.08 x IO-LC 263.0 1.90 1.20 X 1 U - S 170 mg. 286.3 30.0 1 . 7 5 x 10-2 zz?? 1 . 3 3 x IO-,e
-,
Insecticide Parathion TEPPb Pars-Oron E-1059 Sulfotepp EPN Methylparathion (methyl homolog) OMPA
~
Relative Potency 88 Inhibitor (Referred to Parathion) At 80% Level At 50% Level Weight basis Molar basis Weight basis Molsr basis 1 1 1 1 1850 4230 4230 1850 2490 1370 1290 2030 12.1 10.7 21.5 19.1 11.2 12.5 22.0 24.5 2.8 3.1 4.3 4.8 2 . 2 x 10-1 2.0 x IO-* 1.5 x I O - 1 ~ 1 . 4 x 10-8.
__
Slope' 2.01 1.41 1.67 1.78 1.63 1.97 3.17 2.04
1.4
x
10-8
1.4 x
10-8
1.1
x
in-ae
1.1 x 10-r
The enzymatic-inhihitian method includes the following operations: extracting the plant material with ether; washing the extract wkth 10% sodium bicarbonate solution and with saturated sodium chloride solution; chilling the ether extract and m k i n g t o volume with cold ether; evaporating a n aliquot of t h e extract to dryness; "incubating" the extractive aliquot by stirring i t with a standard cholinesterase solution and a standard buffer a t 25" C. for 30 minutes in order t o effect the inhibitory reaction; adding a standmd acetylcholine chloride solution and, after 60 minutes a t 25", measuring the pH change; and converting the pHchange toper cent ofinhibition, which isrelated hyastandard curve to micrograms of the inhibitor. APPARATUS
Soxhlet extractors. pH meter (Beekman Model G or equivalent). Apparatus (Figure 1). Three crystallization dishes (9 em. in diameter) serving as small constantAemperature baths (25' C.), each placed upon a magnetic stirrer. Magnetic stirring "fleas." (A flea is a small piece of iron wire sealed in glass tubing 10 mm. long and 2 mm. in outside diameta .\ "-1
Figure 1. Constant-Temperature A p p a r a t u s A.
B.
Rubber mbing connected to iource of nompresaed air
C. D. E.
Air-lift pump copper cooling coil Heating element Thermometer
F.
E*:*-*-
G.
Mcroury therrnostsf
""...".
l..+....~...
Rheostat control € I . Small insulated ~~~stsnf-femperafure bath, cwatsllisafion I. dish. 9 cm. in diameter J . COPP.. eoil
K. Magnetic atirrer Miorobenker, 10-ml. capaoit). .I M.
N.
0.
-
M a ~ n e t i cstirring Rea Lead coil Water inlet tubing
n
,.
Microbeakem, 10-ml. capacity each with an enclosing leadsolder wire coil to hold the b e a k d i n place in the water hath. ConstanLtemperature hath and air-lift pump to circulate water through the three small baths. Serologicltl syringes, 2- and 25-ml. capacity. Pipets (1-d. graduated and 3-ml. capacity), volumetric flasks (10- and 100-ml. capacity). Interval timer. Electric fan.
n
R
s
. .
TEPP Tetraethyl pyrophosphate
0
Sulfotepp Tetraethyl dithiopyrophosphate
0
OMPA Ootamethyl pyrophasphoramide The choliuesteraseinhihitive effects of these insectioides are shown in Table I. After parathion, which is assigned a relative potency of I , these materids are listed in decreasing order of potency.
1832
ANALYTICAL CHEMISTRY
will keep for 6 months without appreciable loss in activity. K i t h a sterilized and chilled 2-ml. syringe withdraw slightly more than 1 nil. of the ice-cold stock solution. Plug the point of the syringe needle by sticking it upright into a clean rubber stopper and then remove the piston of the syringe. With a clean precision pipet transfer 1.0 ml. from the syringe barrel to a 50-ml. volumetric flask, make to the mark with ice-cold saline solution, and mix. This constitutes the trial solution ivhich is used to determine the strength of the cholinesterase preparation. From this determination the required dilution for making up the standard working sulution can be calculated.
60
-
50
-
a40
-
I
i
I: C W v)
c
3
the washings are neutral to litmus. Shake twice with saturated sodium chloride solution, draw off the lower layer, and pass the ether solution of tetraethyl dithiopyrophosphate into a 125-ml. Erlenmeyer flask through a plug of dried cotton contained in a small cylindrical funnel. Drop in two glass beads and remove the ether on the steam bath in a current of dried air. Transfer the residual pale-yellow oil to a small glass-stoppered bottle. Store the bottle in a closed jar in the refrigerator. By means of a dropper with capillary tip weight accurately into a 0.2-ml. cup or microbeaker 50 mg. of the purified tetraethyl dithiopyrophosphate. Drop the cup into a 100-ml. volumetric flask, chill in the refrigerator, and make to the mark with cold ether. After mixing the cold solution, pipet 10 ml. of it into another 100-ml. volumetric flask, make to the mark while cold, and mix in the same way. Repeat this operation to give a solution containing 5 micrograms per milliliter (mark this solution 8). In the same way make a solution containing 0.5 microgram per milliliter (mark this solution W ) . Store the solutions in the refrigerator. Preparation of Standard Curve. Place 10 numbered microbeakers, 10-ml. capacity, in the hood. Place in each beaker a magnetic flea. Remove from the refrigerator the two standard solutions ii3 and W ) of tetraethyl dithiopyrophosphate and the bottle of ether and place them in an ice bath. Then pipet these materials into the beakers as follows:
W v)
2 30 W
Beaker so.
W v)
z
$20
-
1 2 3
0
4 10
5 6
-
7 8 9
10 7.5
7.0
6.5
6.0
5.5
1,6
2.0
2.5 5.0
10.0 15.0 20.0 None
MI. 'None 2 W 3w 4 w
5W 1s
Ether, 111. 5 3 2
1 None 4
3s
3 2 1
Sone
5
2 s 4 s
5.0
PH
Figure 2.
Solution Tetraethyl Dithiopyrophosphate, y Sone 1.0
Cholinesterase Hydrolysis of Acetylcholine Chloride in 1 Hour at 25' C.
3 ml. of buffer, 3 ml. of cholinesterase, 0.6 ml. of substrate
Place three niicrobeakers, 10-ml. capacity, on the laboratory bench and in each place a stirring flea. In the following manipulations all solutions must be cold. Into beaker 1 put 3.0 ml. each of saline solution and standard buffer. Place this beaker in the constant-temperature bath (25" C.) and start the magnetic stirrer and the interval timer (zero time). Into beaker 2 put 3.0 ml. each of the trial solution of cholinesterase and standard buffer. At 2 minutes (all time referred from zero time) place beaker 2 in the bath with magnetic stirring. At 4 minutes start beaker 3, which is a replicate of No. 2. At 30 minutes remove beaker 1 from the bath and immediately take the p H reading. This is taken as the initial pH; it should read 8.00 A 0.05. At 32 and 34 minutes put into beakers 2 and 3, respectively, 0.6 ml. of acetylcholine chloride substrate solution and a t 92 and 94 minutes read the respective pH's of these beakers. The last two readings should check within 0.02 pH, Refer now to Figure 2, a curve which relates the p H reading to arbitrary units of cholinesterase per milliliter. Read off the number of cholinesterase units per milliliter corresponding to the pH reading (average of beakers 2 and 3) and from this calculate the volume, V (in ml.), of cold saline solution to put into 1.0 ml. of the concentrated stock solution: V = 49/38 X number of cholinesterase units per milliliter in trial solution. Make up the standard working solution of cholinesterase from 1.0 ml. of the concentrated stock solution by adding the calculated volume of cold saline. The standard cholinesterase solution thus made up should produce a change of 2.0 p H units when tested with buffer and substrate under the conditions described above. Put 2 drops of toluene as a preservative into each of the following reagents: buffer, cholinesterase working solution, and substrate solution. Store them in the refrigerator when not in use. To keep the cholinesterase solutions cold a t all times have an ice bath on hand during the manipulations a t the bench. TETRAETHYL DITHIOPYROPHOSPHATE
Preparation of Standard Solution. Purify a technical grade of tetraethyl dithiopyrophosphate by dissolving about 1 gram in 35 ml. of ether and washing it in a separatory funnel with 10% sodium bicarbonate solution and then w-ith distilled water until
Without heating and by directing a gentle breeze from an electric fan into the hood, evaporate the ether from the beakers until they are just dry. Kormally this takes about 10 minutes. Into beaker 1 put 3 ml. of cold standard buffer and 3 ml. of cold standard cholinesterase solution. Place in the constant-temperature bath (25' C,), start the magnetic stirrer, and set the interval timer a t zero. Repeat this operation on beaker 2, placing it in the constant-temperature bath a t 2 minutes (from zero time) and so on, a t intervals of 2 minutes, through beaker 10. A4t30 minutes remove beaker 1 from the constant-temperature bath, insert the microelectrodes (rinsed with water and dried with a piece of cotton or lens paper immediately before being used) of the p H meter and read the pH. This reading (8.00 0.05) may be considered as the initial p H of all beakers in a given run. At 32 minutes put 0.6 ml. of acetylcholine chloride substrate solution into beaker 2, and likewise a t 2-minute intervals into beakers 3 through 10. Remove the beakers from the constant-temperature bath in regular order to take the pH reading, commencing with beaker 2 a t 92 minutes and proceeding a t 2minute intervals through beaker 10 a t 108 minutes from zero time. Experiment has shown that the pH reading of beaker 10 (final pH after 30 minutes' incubation without inhibitor but with 60 minutes' reaction of the cholinesterase upon the substrate) is close to the value 6 (6.00 =t0.05). Calculate the per cent inhibition as follows:
*
15
here pH, = initial H 8.00 d= 0.05 (beaker 1) pH, = final pff ==6.00 i 0.05 (beaker 10) ApH = pHi - pH
Table I1 shows a typical set of values for tetraethyl dithiopyrophosphate. A standard curve is obtained by plotting on semilogarithmic paper the per cent inhibition on the linear scale against micrograms of tetraethyl dithiopyrophosphate on the logarithmic scale and drawing a straight line best fitting the points (Figure 3). Extraction from Plant Material. Place a weighed representative sample (1 to 200 grams) of the plant material in a Soxhlet extractor, add diethyl ether, and extract on the steam bath for 3 hours or more. Concentrate the ether extract to a volume of about 50 nil., transfer to a separatory funnel, draw off the aqueous
V O L U M E 2 3 , NO. 1 2 , D E C E M B E R 1 9 5 1 Table 11. Beaker KO.
1833
Data for Standard Curve of Tetraethyl Dithiopyrophosphate Tetraethyl Dithiopyrophosphate, 7 0.0 1.0
1,s
2.0 2.5 5.0 10.0 15.0 20.0 0.0
pII
ApH
8 . 0 0 (initial! 6.30 6.52 6 66 6.76 7.10 1.54 I 74 7.88 6 00 {final,
0.00 1.70
7.
Inhibition
1.48 1.34 1.24
0.90 0.46 0.26 0.12 2.00
..
15 26
33 38 55 77 87 94 0
General Precautions. Cse a vacuum line or rubber bulb in pipetting solutions containing tetraethyl dithiopyrophosphate or other toxic inhibitors. Store all solutions and reagents in the refrigerator xhen not in use, and while working with them place in an ice bath. Clean all glassware with hot cleaning solution and wash thoroughly with water. Designate individual pipets for measuring out the cholinesterase, buffer, and acetylcholine chloride solutions. When delivering solutions from these pipets, take care not to make contact with the sides of the microbeakers. Work systematically to keep the proper time intervals, bearing in mind that the method is dependent on the rate of inhibition and the rate of enzymatic hydrolysis during definite periods of time. PARATHIOB AYD DIETHYL p-NITROPHEXYL PHOSPHATL
A standard cum? for parathion (Figure 4) is prepared by the same procedure as that for tetraethyldithiopyrophosphate,with cold ether solutions of pure parathion. Because parathion is a weaker inhibitor than tetraethyldithiopyrophosphate,it can be directly determined only at the high level of 15 to 500 micrograms as compared with 1.5 t o 20 micrograms for tetraethyldithiopyrophosphate. Diethyl p-nitrophenyl phosphate is an extremely powerful inhibitor (see Table I), in contrast t o parathiou. Parathion can be oxidized t o diethyl p-nitrophenyl phosphate most conveniently by treating parathion in the cold with a 1 to 1 (by volume) mixture of concentrated and fuming nitric acida. A standard curve for oxidized parathion ran be made as follo~vs: Prepaie a standard solution of parathion in cold ether containing 5.0 micrograms per milliliter. Transfer 10 ml. (50 micrograms) of the cold solution into a 30-ml. long-necked, roundhottonied flask: evaporate to drync.ss, nithout heating. nith a
Figure 3. Inhibitive Effect of Tetraethyl Dithiopyrophosphate on Cholinesterase Activity
layer, wash twice with 10-ml. portions of 10% sodium bicarbonate solution, and then wash with 10-ml. portions of saturated sodium chloride solution until the washings are neutral to litmus. Filter the neutral extract through a plug of dried cotton into a 100-ml. volumetric flask. Make up approximately to the mark with ether, which is also washed through the cotton. Chill the flask by placing it in an ice bath for 20 minutes, make to the mark with ice-cold ether, and mix. Estimation of Tetraethyl Dithiopyrophosphate in Plant Extract. For tetraethyl dithiopyrophosphate it is desirable to have the final extract made up to contain about 0.5 microgram per milliliter. If necessary, make a preliminary determination by transferring 2 ml. of the cold extract into a 10-ml. beaker with a magnetic flea and evaporating to dryness in a current of air. In the beaker put 3 ml. each of cold standard cholinesterase and buffer solutions, transfer to the 25" C. constant-temperature bath, and start the magnetic stirring for 30 minutes' incubation; put in 0.6 ml. of substrate solution and read the pH after exactly 10 minutes' reaction time-i.e., from the moment the substrate is added. Calculate the approximate ApH by subtracting the reading from 8.0 and multiplying by 6. From this figure and the standard graph estimate the potency of the plant extract and dilute with cold ether to contain about 0.5 microgram per milliliter. Then run replicate determinations following the procedure used in making up the standard curve. Four different extracts of plant material can be determined during a single run of 10 beakers, beakers 1 and 10 being used for the initial and final blanks (untreated plant material), and pairs of beakers, Kos. 2 and 3 , 4 and 5 , 6 and 7,8and 9, respectively, for replicate determinations of extracts. In measuring out the plant extract into the microbeaker, use a 5-ml. ali uot if possible; if you take a smaller aliquot, make to 5 ml. wi& the required cold ether, as in the procedure used to prepare the stantfwd curve.
Figure 4. Inhibitive Effect of Parathion on Cholinesterase Activity
gentle stream of air; chill the flask in an ice bath; and add slowly 5 ml. of nitric acid reagent (1 to 1 by volume mixture of concentrated and fuming nitric acids). Swirl to wet the walls of the flask, remove from the ice bath, and let stand for 5 minutes. Cautiously put about 25 ml. of cold distilled water into the flask and transfer the contents to a 125-ml. separatory funnel. Rinse the flask twice with a little cold distilled water and then with two
1834
ANALYTICAL CHEMISTRY
25-ml. portions of cold ether, transferring all rinsings to the separatory funnel. Shake well and then draw off the aqueous layer and discard. Wash the ether layer with 10-ml. portions of 10% sodium bicarbonate solution until the washings are alkaline to litmus. Finally wash twice with saturated sodium chloride solution, and filter the ethereal solution through a small plug of dried cotton into a 100-ml. volumetric flask. Rinse the separatory funnel twice with 20-ml. portions of ether, filtering through the cotton into the flask. Cool the flask in an ice bath for 20 minutes and then make to the mark with cold ether. Make two solutions in cold ether, containing 0.05 and 0.005 microgram per milliliter of oxidized parathion calculated as parathion. From this point follow the same procedure as in the preparation of the standard curve for tetraethyl dithiopyrophosphate.
0.06
0.04
Sample
Date NO. Collected Dec. 12 Dee. 12 Dee. 15 Dec. 19 Dec. 22 Dec. 29 Jan. 3 Untreated sample.
Weight, Grams 266 124 130 181 190 161 173
Tetraethyl Dithiop rophosphate 6ound Total, y P.p.m. 0 11,400 5,000 2,270 1,630 940 520
OQ
92.0 38.5 12.5 8.6 5.8 3.0
A greenhouse of 7000 cubic feet in which lettuce was being grown was treated by Floyd F. Smith of this bureau with an aerosol containing 3.5 grams of tetraethyl dithiopyrophosphate. Samples of lettuce were then harvested and analyzed by the enzyme-inhibition method for spray residues of tetraethyl dithiopyrophosphate. A4check sample of untreated lettuce mas run through the etherextraction procedure to provide checks for beakers 1 and 10, corresponding to the initial and final pH. In experiments where known amounts of tetraethyl dithiopyrophosphate were added to lettuce and extracted in a Soxhlet with ether for 3 hours, 95 to 98% of the added inhibitor was recovered.
- I n 0.I z 0.09 U 0.08 0.07 - u
0.05
Table 111. Spray Residues on Lettuce Treated with Tetraethyl Dithiopyrophosphate on December 12, 1950
-
Typical results of the spray-residue studies are shown in Table 0.03
111. A number of plant materials as well as a series of common
0.02
insecticides were tested for their possible interfering effects on the enzyme-inhibition method. S o interferences n ere encountered. The following materials did not affect cholinesterase:
0.01
IO
20
30 4 0 S O 60 70 PERCENT INHIBITION
80
90
Figure 5. Inhibitive Effect of Diethyl pNitrophenyl Phosphate and Oxidized Parathion on Cholinesterase Activity
Figure 5 shows a standard curve for pure diethyl p-nitrophenyl phosphate and a standard curve for oxidized parathion read directly in micrograms of parathion. DISCUSSION
The authors have changed somewhat Michel’s (’7) buffer and substrate solutions with respect t o their concentration and volume used. They arrived a t the proper values for their purpose by a process of trial and error until they obtained a satisfactory linear relationship between change of pH Lvhen plotted against time within the limits of p H 8 to pH 6 over a period of 60 minutes from the moment the substrate n-as added. They also found that it was unnecessary t o use hlichel’s two correction factors, b and j , which he calculated for the purpose of determining the activities of red blood cell and plasma cholinesterases. blost of the experimental work 4 ith the enzymatic method was done in connection n ith tetraethyl dithiopyrophosphate, a hich a t the present time finds its widest use in aerosol applications in greenhouses.
Plant Materials. Bean leaves (snap beans and limas), lettuce, cabbage, tomato, barley, alfalfa, strawberry leaves, onion (wild), pine and spruce needles, tobacco (cured), chrysanthemum, geranium, and petunia. Insecticides. DDT, BHC (technical benzene hexachloride, gamma isomer 207,), chlordan, toxaphene, dieldrin, aldrin, rotenone, and pyrethrum (20% extract). Of the materials tested, methyl parathion and octamethyl pyrophosphoramide (ORIPA) are too weakly inhibitive to make their determination practicable by the enzymatic method. The weak inhibitors generally exhibit steeper slopes than the strong inhibitors. LITERATURE CITED
(1) Xmmon, R., Pflugers Arch. Physiol., 233, 486 (1934). (2) Averell, P. R., and Norris, hl. V., ANAL.CHEM.,20, 753-6 (1948). (3) Glick, D., Biochem. J . , 31, 521 (1937); J . Gen. Physiol., 21, 289,
297 (1938). (4) Hall, S. A., Advances in Chem. Series, 1 , 150-9 (1950). (5) Hall, S.rl., and Jacobson, SI.,Ind. Eng. Chem., 40, 694-9 (1948). (6) Metcalf, R. L., and hlarch, R. B., J . Econ. Entomol., 42, 721-8 (1949). (7) Michel, H. O., J . Lab. Clin. Med., 34, 1564-8 (1949). RECEIVED June 29, 1951. Presented before the Divisions of Agricultural and Food Chemistry and Analytical Chemistry, Symposium on Methods of Analysis for Micro Quantities of Pesticides, a t the 119th Meeting of t h e AMERICASCHEMICAL SOCIETY, Boston, Mass. Taken i n part from a thesis presented by Paul A. Giang t o the Graduate School of Georgetown University in partial fulfillment of the requirements for the degree of doctor of philosophy.
