-.- .. --LORIDA AGRICULTURAL FXPEPIMENT STATIONS
HUMvi UiL; y Gainesville, Florida
DEC 12 1966 Dairy Science Himeo Report DY67-1
December 1, 1l66
I.F.A.S. Univ. of Florida
FFECT O OA GANIC PHOSPHORUS INSECTICIDE APPLICATION UPON BLOOD
CHOLINESTERASE LEVELS OF DAIRY CATTLE
C. J. Wilcox and 1. 17. Fead1l
Insecticide use in agricultural production has been widespread in recent years.
A major problem, however, is how to control pests with chemicals and yet spare
man, domestic animals and plants (9). The organic phosphorus insecticides have
been designed and synthesized to a large measure with specificity of action a
major consideration (6). Because of this, and their effectiveness in attaining
the desired objectives, their use has been widespread.
The organic phosphorus insecticides have the following general molecular
RI 0 or S
R2 acidic group
with R1 and R2 being alkoxy or substituted amino groups (4). These insecticides
have the common property of inhibiting the activity of esterases. Of the
esterases, cholinesterase is the most widely known and among the more important.
The enzymes which catalyze the hydrolysis of acetylcholine are known as cholin-
esterases and are found in red blood cells (RRC) and plasma or serum, as well as
in most of the body tissues. They are particularly active in nervous tissue.
The normal function of the true cholinesterases, such as acetylcholinesterase,
is to hydrolyze acetylcholine to acetic acid and choline (a), (2,5).
acetylcholine > CH3- -OK + CH2-CH2-N(CH3)3
The true cholinesterases (Group I) are found principally in RBC and in the central
nervous system. These enzymes hydrolyze acetylcholine more rapidly than they do
other choline esters such as butyrylcholine and benzoylcholine. The normal
function of these enzymes, catalyzing the hydrolysis of acetylcholine, is extremely
important physiologically since acetylcholine is the transmitting agent of the
nerve impulse to effector cells in the neuromuscular junction. The pseudocholin-
esterase enzymes (Group II) are found principally in the blood plasma or serum.
Their physiological importance is not clear but they are classified as such
because they hydrolyze choline esters other than acetylcholine more rapidly than
they do acetylcholine (3,5).
Associate Geneticist and Assistant Physiologist, respectively.
It is generally agreed that organic phosphorus insecticide toxicity to mammals
is associated with the inhibition of the cholinesterase enzymes (6,7). The
cholinesterase activity in whole blood may be considered as an index of the
overall activity present in the central nervous system. Hence, reduction in
cholinesterase levels associated with the presence of the organic phosphorus
insecticides is a serious problem and has been used as a measure of insecticide
contamination in humans and livestock. These insecticides are serious and
important inhibitors of enzyme activity since they have an affinity for the
active site and react at the esteratic site of the enzyme forming a phosphorylated
complex and yielding an inactive enzyme (6,7). Natural reactivation is negligible
or very slow. However, reactivation can be enhanced by hydroxylamine or with
good efficiency by 2-pyridine aldoxime methiodide (2-PAM). The latter substance,
used in combination with atropine, serves as a potent antidote for organic
phosphorus insecticide poisoning (5).
Two organic phosphorus insecticides which have been widely used in control of
flies around livestock are dimethyl phosphate of alpha-methyl benzyl 3-hydroxy-
cis crotonate (Ciodrin)1l and 2,2,dichlorovinyl dimethyl phosphate (Vapona)1l
Separately, each has been found to be safe and effective. A 2 ounce per day
oil spray of the mixture (1% Ciodrin, 0.257 Vapona) on cows after the morning
milking was found to be effective against the bloodsucking horn fly and the face
fly. Vapona (1%), though effective in killing face flies, did not give day-long
protection. Cheng et al. (1) sprayed cattle with both oil and water based
Ciodrin (2%) and found that it provided good control against stable flies and
face flies. Significant reductions in fly populations for 52-76 hours after
spray application were noted.
The objectives of the present investigation were to compare the cholinesterase
levels in man and animals before and after exposure to a combination of two
organic phosphorus insecticides.2.
Method of Procedure
Five mature Jersey females (age 3-5 years) and three Jersey calves (age 60 days)
were confined in a dairy barn, in stanchions or pens, and in which there was
minimum air movement. The insecticide was dispensed twice daily at the rate of
1 pint per 8000 cubic feet by means of an electric fogger over a time period of
7-10 minutes. The cows were then removed from the barn within 10 minutes after
fogging was discontinued. The calves were retained in their closed barn
continuously. The operator was equipped with a respirator.
Exposure of man and animals to the insecticide lasted 10 days and followed a
6-day pre-exposure period. Blood samples were taken from all subjects at -6,
-4, -2, +1, +3, +5, +8, and +10 days from the initial foging. Subsequently,
an operator not equipped with a respirator repeated the experiment, with blood
samples taken only from him.
. Shell Chemical Company.
Dimethyl Phosphate of Alpha-Methylbenzyl
2,2 Dichlorovinyl Dimethyl Phosphate 0.23
Related Compounds 0.02
Petroleum Hydrocarbons 98.57
Inert Ingredients 0.18
The method of Michel (5) was used to assay RBC and plasma for cholinesterase
activity. For this method, the enzyme in an aliquot of diluted red cell
hemolysate or plasma is allowed to act on a Riven concentration of acetylcholine
in a standard buffer solution for a -easured tire (usually 1 to 1.5 hours). The
pH of the mixture is measured at the beginning and end of the time. The action
of the enzyme produces acetic acid which lowers the pH of the mixture. The rate
of pH change is a measure of the enzyme activity.
Standard analysis of variance techniques were used in the statistical analysis
of data. Since time trends and treatment effects were perfectly confounded,
comparisons have been referred to as period effects, representing cholinesterase
levels before and after initial exposure to the insecticide.
Results and Discussion
Mean cholinesterase levels are shown in Table 1. The RBC cholinesterase levels
observed in cattle and calves were within the range reported by Radeleff and
Table 1. Cholinesterase levels in blood of experimental subjects
Subject Pre-exposure Post-exposure
Plasma RBC Plasma RBC
Mature 0.125 0.37P 0.121 0.406
Immature 0.130 0.407 0.121 0.455
Combined 0.127 0.389 0.121 0.424
A 1.247 0.763 0.970 0.742
B 1.027 0.710 0.942 0.728
a. A, equipped with respirator: B, not equipped.
These workers reported no significant cholinesterase activity present in the
plasma of cattle or sheep. Similarly, very low levels of cholinesterase activity
were recorded during the course of the present studies for cattle and calves.
The wide range of values observed indicate that care needs to be exercised in
interpretation of cholinesterase levels. Doubtless any number of environmental
and nutritional problems can result in altered RPC and plasma levels aside
from those changes associated with insecticide contamination or poisoning.
The data show clearly that dairy cattle have considerably lower cholinesterase
levels than do men. The difference between plasma levels of the two is especially
dramatic. However, the biological meaning of this is not known. Similarly, RBC
cholinesterase levels of cattle were only about 50% of those observed in man.
Period effects were not statistically significant (Table 2). Nor were differences
in levels between young and mature animals significant. Within the limits of
the experimental design and the possibility of committing a Type II error (real
effect present but undetected), exposure of the animals to insectide did not
result in decreased cholinesterase activity.
Table 2. Analysis of variance of cholinesterase levels.
Source d.f. SS MS SS MS
Period pa. 1 .00053 .00053 .01890 .01890
Age Ab. 1 .00006 .00006 .02625 .02625
P x A 1 .00010 .00010 .00156 .00156
Animals (in A) 6 .03029 .00505 .06759 .01127
P x Animals (in A) 6 .04896 .00816** .17696 .02949**
Error 48 .04483 .00060 .00868 .00124
Total 63 .07994 .29994
a. Comparison: pre-exposure vs post-exposure.
b. Comparison: mature vs young
A stringent test of cholinesterase activity in man was not possible. Means in
Table 1 show a decline in plasma for both men but not in RBC. No symptoms of
intoxication were noted in men or animals. More sensitive experiments are
required to define clearly the effects of the compounds used.
After administration of a combination of two organic phosphorus insecticides
(Ciodrin and Vapona), it was not possible to detect a significant change in
RBC or plasma cholinesterase levels. Additional research is required, however,
before it can be stated with certainty that such changes would not occur or that
they would be without biological significance.
The authors wish to express appreciation to the Shell Chemical Company for
materials and financial support, and to C. H. Van Middelem, B. B. Byham,
H. L. Somers, M. Kersey, and C. Sheffield for technical assistance. Chemical
analyses were performed by Patterson and Coleman Laboratories, Tampa, Florida.
(1) Cheng, Tien-Hsi, A. A. Hower, and P. K. Sprenkel. 1965. Oil Based and
Water Based Ciodrin Sprays for Fly Control on Dairy Cattle. J. Econ.
(2) Fukuto, T. R. 1957. The Chemistry and Action of Organophosphorus Insecticides.
Adv. in Pest Control Res., 1:147. Interscience Publ., Inc., New York.
(3) Gage, J. L. 1961. Residue Determination of Cholinesterase Inhibition
Analysis. Adv. in Pest Control Res., 4:183. Interscience Publ., Inc.,
(4) Hassal, K. A. 1965. Pesticides: Their Properties, Uses and Disadvantages.
Part I. General Introduction, Insecticides and Related Compounds. Brit.
Vet. J., 121:105.
(5) Hawk's Physiological Chemistry, 14th Ed., pp 1129-1131, 1965. McGraw-
Hill, New York.
(6) O'Brien, R. D. 1961. Selective Toxicity of Insecticides. Adv. in Pest
Control Res., 4:75. Interscience Publ., Inc., New York.
(7) O'Brien, R. D. 1966. Mode of Action of Insecticides. Ann. Rev. Entomol.,
(8) Radeleff, R. D. and G. T. Woodard. 1956. Cholinesterase Activity of
Normal Blood of Cattle and Sheep. Vet. Med., 51:512.
(9) Rogoff, W. M. 1961. Chemical Control of Insect Pests of Domestic Animals.
Adv. in Pest Control Res., 4:153. Interscience Publ., Inc., New York.