Kinetics and Mechanism of Amitraz Hydrolysis

Article abstract of DOI:10.1021/jf970049x

As a precursor to the development of effective vat management and waste disposal strategies, the kinetics and basic mechanisms of amitraz, N′-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]- methyl]-N-methylmethanimidamide, hydrolysis were examined as was the effect of cosolvents and metal ions. Amitraz was readily hydrolyzed at low pH values, forming acid-stable 2,4-dimethylphenylformamide, which can be further hydrolyzed to 2,4-dimethylaniline. The hydrolysis of 2,4- dimethylphenylformamide was faster under basic conditions. Thus, the addition of lime, a management technique used to stabilize the amitraz, will enhance the hydrolysis of its degradation products to aniline.

Full text of DOI:10.1021/jf970049x

J. Agric. Food Chem. 1997, 45, 1937
−1939
1937
Kin etics a n d Mech a n ism of Am itr a z Hyd r olysis
Anthony C. Pierpoint,† Cathleen J . Hapeman,‡ and Alba Torrents*,†
Environmental Engineering Program, Department of Civil Engineering, University of Maryland,
College Park, Maryland 20742, and Environmental Chemistry Laboratory, Natural Resources Institute,
Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705
As a precursor to the development of effective vat management and waste disposal strategies, the
kinetics and basic mechanisms of amitraz, N′-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]-
methyl]-N-methylmethanimidamide, hydrolysis were examined as was the effect of cosolvents and
metal ions. Amitraz was readily hydrolyzed at low pH values, forming acid-stable 2,4-dimeth-
ylphenylformamide, which can be further hydrolyzed to 2,4-dimethylaniline. The hydrolysis of 2,4-
dimethylphenylformamide was faster under basic conditions. Thus, the addition of lime, a
management technique used to stabilize the amitraz, will enhance the hydrolysis of its degradation
products to aniline.
Keyw or d s: Amitraz; hydrolysis; pesticide waste; dip-vats
INTRODUCTION
MATERIALS AND METHODS
Sta n d a r d s a n d Rea gen ts. Amitraz, 2,4-dimethylphenyl-
formamide, and N′-(2,4-dimethylphenyl)-N-methylformami-
dine were obtained gratis from AgrEvo, Analytical Services
(Wilmington, DE). 2,4-Dimethylaniline and salts (all of ana-
lytical grade) were purchased from Aldrich (Milwaukee, WI)
and used without further purification. Tactic EC was pur-
chased from Animal Medic, Inc. (Manchester, PA). Buffered
solutions were prepared by combining appropriate volumes of
0.067 M potassium dihydrogen phosphate and 0.067 M diso-
dium phosphate for pH values between 5 and 8 and 0.0125 M
sodium tetraborate for pH values of 8-10 with high-purity
water. Solutions were adjusted with hydrochloric acid (0.1 M)
or sodium hydroxide (0.1 M) to the required pH.
Hyd r olysis of Am itr a z. Experiments were conducted
three to five times. To determine the effects of cosolvents,
solutions of 8 ppm of amitraz in 25, 30, 40, and 50% methanol
or acetonitrile in buffered water were prepared and the loss
of amitraz was monitored. A slight increase in the initial
solution pH was observed when acetonitrile was used as a
cosolvent. This was likely due to a cosolvent-induced increase
in electrode liquid-junction potential, and, as such, the hy-
drolysis rates reported reflect the pH measured prior to
acetonitrile addition.
Hydrolysis experiments were conducted using 8-16 ppm of
amitraz in 25% acetonitrile/buffered or nonbuffered water and
monitored by HPLC. The effect of metal ions was examined
by adding one of the following metal sulfates or nitrates to
pH 5 buffered acetonitrile/water: ZnSO4‚7H2O, MgSO4, Cu-
(NO3)2‚3H2O, Fe(NH4)(SO4)2‚12H2O, Ni(NO3)2‚6H2O, and
MnSO4‚H2O, for a final metal concentration of 10-4 M.
Some experiments were conducted with commercial prod-
uct: 1 mL of Tactic EC was diluted in 500 mL of tap water
(no acetonitrile) to achieve a concentration equivalent to the
recommended amitraz treatment dosage of 250 ppm. The pH
was determined and the reaction monitored. During the first
10 days of the experiment, a precipitate appeared, which was
collected by filtering a 10 mL aliquot through a 5 µm filter.
The filtered solid was dissolved in acetonitrile and analyzed
by HPLC.
HP LC An a lysis. Samples were analyzed directly by HPLC
employing (1) two Gilson (Middleton, WI) Model 303 HPLC
pumps equipped with a Model 715 controller, a Model 210
autosampler, and a Model 116 UV detector, monitoring at 288
and 240 nm, or (2) a Waters (Milford, MA) Model 616 LC and
Millennium system equipped with two 510 pumps, a Model
717 autosampler, and a Model 996 photodiode array detector.
Separations were achieved using a sequence of linear gradi-
ents, 40% (6 min), 40-85% (1 min), 85% (9 min) acetonitrile
Amitraz, N′-(2,4-dimethylphenyl)-N-[[(2,4-dimeth-
ylphenyl)imino]methyl]-N- methylmethanimidamide, a
formamidine pesticide initially developed for use on
deciduous fruit and citrus mites, is also effective against
mange mites on livestock and ticks on cattle (Bonsall
and Turnbull, 1983; Ware, 1989). Amitraz has moder-
ate mammalian toxicity, is acutely toxic to fish, and may
affect avian reproduction (Aziz and Knowles, 1973;
Benezet and Knowles, 1976; Benezet et al., 1978; Rieger
et al., 1980; Bonsall and Turnbull, 1983; J ones, 1990).
Tactic EC, a formulated product of amitraz, is widely
used in Puerto Rico to control ticks, specifically Boo-
philus microplus and Amblyomma variegatum. Mobile
and stationary spray vats of up to 200 gal are used to
apply the pesticide to cattle and livestock; however,
large quantities of semiconcentrated (ca. 250 ppm)
pesticide waste are generated. Amitraz is being evalu-
ated as an alternative for tick eradication where cou-
maphos is currently used. Approximately 100 000 gal
of pesticide waste is generated annually from 42 dip-
vats on the Texas-Mexico border (Agricultural Re-
search Service, 1996).
Amitraz can also be hydrolyzed, giving rise to 2,4-
dimethylphenylformamide and N-2,4-dimethylphenyl-
N-methylformamidine, both of which can be further
hydrolyzed to 2,4-dimethylaniline (Figure 1) (U.S. En-
vironmental Protection Agency, 1996). Thus, stoichio-
metrically, 1 mol of amitraz will give rise to 2 mol of
2,4-dimethylaniline. More importantly, 2,4-dimethyl-
aniline is also toxic, with an acute oral LD50 of 467 mg/
kg for rats, almost half that of the parent pesticide
(Vernot et al., 1977). To develop effective vat manage-
ment and waste disposal strategies, the fate of amitraz
in treatment vats must be studied. The purpose of this
study was to examine and quantitate the kinetics and
mechanisms of amitraz hydrolysis and the effect of
cosolvents and metal ions on the hydrolysis of amitraz.
* Corresponding author [fax (301) 405-1979; e-mail
alba@eng.umd.edu].
† University of Maryland.
‡ U.S. Department of Agriculture.
S0021-8561(97)00049-6 CCC: $14.00
© 1997 American Chemical Society

1938 J. Agric. Food Chem., Vol. 45, No. 5, 1997
Pierpoint et al.
F igu r e 1. Hydrolysis pathway of amitraz in aqueous solutions.
Ta ble 1. P seu d o-F ir st-Or d er Ra te Con sta n ts for Am itr a z
pH
% cosolvent
k (h-1)/r2
1.57/0.997
t1/2 (h)
3.24
4.15
5.09
5.09
5.09
5.09
5.63
7.02
7.57
9.12
25
25
25
30
40
50
25
25
25
25
0.4
3.1
8.6
8.7
9.3
11
13
59
74
112
2.22 × 10-1/0.982
8.09 × 10-2/0.997
7.93 × 10-2/0.993
7.47 × 10-2/0.998
6.51 × 10-2/0.997
5.42 × 10-2/0.990
1.18 × 10-2/0.987
9.39 × 10-3/0.999
6.18 × 10-3/0.992
in water, at a flow rate of 1.5 mL/min on a Beckman (Fullerton,
CA) C18 (ODS 5 µm), end-capped 4.6 mm × 25 cm steel-
jacketed column. Peak identification was established by
comparison of the retention times and UV spectra with
standards.
RESULTS AND DISCUSSION
Am itr a z Hyd r olysis a n d P r od u ct F a te. Solubility
limitations of analytical grade amitraz required the use
of a cosolvent to achieve suitable concentrations for
analysis of the parent material and its products. Ami-
traz was unstable in pure methanol, rapidly hydrolyzing
to form 2,4-dimethylphenylformamide, N′-(2,4-dimeth-
ylphenyl)-N-methylformamidine, and an unknown prod-
uct (data not shown). However, amitraz was stable in
acetonitrile. Negligible effects on the rate and product
profile were observed at cosolvent concentrations of 25,
30, and 40%. A marked decrease was seen in the
hydrolysis rate at 50%, probably due to protective
dehydration of amitraz (Table 1). Reductions in cosol-
vent concentration below 25% resulted in parent com-
pound precipitation.
The hydrolysis of amitraz was more rapid under acidic
conditions and afforded 2,4-dimethylphenylformamide
via cleavage of the carbon-nitrogen bond of the metha-
nimidamide (Figure 2). After an initial delay of 22 h to
62 days, 2,4-dimethylaniline was also observed. The
simultaneous formation of N′-(2,4-dimethylphenyl)-N-
methylformamidine was contraindicated as the com-
pound was not detected. It is likely that the instability
of the formamidine, which can degrade to the forma-
mide, precluded any detectable accumulation of forma-
midine.
F igu r e 2. Product profile of amitraz hydrolysis: (a) pH 5.09,
(b) pH 7.02, and (c) pH 9.12; (0) amitraz, (O) 2,4-dimethylphen-
ylformamide, (4) 2,4-dimethylaniline.
illustrates, no changes in the concentrations of 2,4-
dimethylphenylformamide and 2,4-dimethylaniline were
observed after 175 days at pH 5.09 and 7.02. Form-
amide hydrolysis is base-catalyzed and is much slower
than amitraz hydrolysis: t1/2 at pH 9.12 ca. 300 days.
Throughout all of the hydrolysis experiments, the total
amount of 2,4-dimethylaniline and the formamide was
approximately equal to twice the concentration of the
amitraz lost.
The rate of amitraz hydrolysis is pseudo-first-order
as shown in the linear plots of ln([amitraz]/[amitraz]0)
versus time, eq 1 (Figure 3). The pseudo-first-order rate
ln[amitraz]/[amitraz]0 ) -kobs
t
(1)
constants (kobs in h-1) and r2 values are shown in Table
1. The observed rate constant, kobs, is described in eq
2. Since no base-catalyzed hydrolysis was observed for
-
-
2,4-Dimethylphenylformamide can further hydrolyze
to yield 2,4-dimethylaniline. However, as Figure 2
amitraz, the term kOH [OH ] can be neglected. Per-
+
forming a least-squares fit of the data, kH (acid-

Amitraz Hydrolysis
J. Agric. Food Chem., Vol. 45, No. 5, 1997 1939
formulation components, such as organic cosolvents and
surfactants, may have been responsible for the de-
creased hydrolysis rate.
Sign ifica n ce. Amitraz is readily hydrolyzed at low
pH values, forming acid-stable 2,4-dimethylphenylfor-
mamide. A common management method for dip-vats
is the addition of lime, which neutralizes uric acid,
increases pH, and stabilizes the parent compound.
Unfortunately, hydrolysis of the formamide is base-
catalyzed, forming 2,4-dimethylaniline, which is stable
in aqueous systems. Thus, despite the decreased hy-
drolysis rate of amitraz associated with the formulation
components and possible lime addition, an environmen-
tal impact or risk may remain due to the presence 2,4-
dimethylphenylformamide and 2,4-dimethylaniline.
Additional studies are necessary to identify manage-
ment strategies and develop disposal options for spray-
dip and dip-vat waste.
F igu r e 3. First-order fit for amitraz hydrolysis at pH 5.09,
5.63, 7.02, and 9.12.
LITERATURE CITED
Agricultural Research Service, U.S. Department of Agricul-
ture. Fighting Back at Biting Flies. Agric. Res. 1996, 44,
10-15.
Aziz, S. A.; Knowles, C. O. Inhibition of Monoamine Oxidase
by Pesticide Chlordimeform and Related Compounds. Na-
ture 1973, 242, 417-418.
Benezet, H. J .; Knowles, C. O. Inhibition of Rat Brain
Monoamine Oxidase by Formamidines and Related Com-
pounds. Int. J . Neuropharmacol. 1976, 15, 369-373.
Benezet, H. J .; Chang, K. M.; Knowles, C. O. Formamidine
Pesticides: Metabolic Aspects of Neurotoxicity. In Pesticide
and Venom Neurotoxicity; Shankland, D. C., Hollingworth,
R. M., Smyth, T., Eds.; Plenum: New York, 1978; pp 189-
206.
Bonsall, J . L.; Turnbull, G. J . Extrapolation From Safety Data
to Management of Poisoning with Reference to Amitraz and
Xylene. Hum. Toxicol. 1983, 2, 587-592.
J ones, R. D. Xylene/Amitraz: A Pharmacologic Review and
Profile. Vet. Hum. Toxicol. 1990, 32, 446-448.
F igu r e 4. Least-squares fit of kobs values at pH 3.24, 4.15,
5.09, 5.63, 7.02, 7.57, and 9.12.
Reiger, J . A.; Robinson, C. P.; Gherezghiher, T.; Leung, T.
Inhibition of Mammalian Monoamine Oxidase by Two
Formamidine Pesticides. Pharmacologist 1980, 32, 172.
U.S. Environmental Protection Agency. Reregistration Eligi-
bility Decision: Amitraz; Publication EPA-738-R-96-031;
Office of Prevention, Pesticides and Toxic Substances:
Washington, DC, Nov 1996.
catalyzed hydrolysis) equals 2862 ( 553 M-1 h-1 and
kn (unassisted or neutral hydrolysis) equals (1.13 ×
10-2) ( 0.006 h-1. Figure 4 shows the least-squares fit
curve with the observed values.
Vernot, E. H.; MacEwen, J . D.; Haun, C. C.; Kenkead, E. R.
Acute Toxicity and Skin Corrosion Data for Some Organic
and Inorganic Compounds and Aqueous Solutions. Toxicol.
Appl. Pharmacol. 1977, 42, 417-23.
kobs ) kH+[H+] + k + k -[OH-]
(2)
n
OH
The addition of 10-4 M Zn2+, Mg2+, Cu2+, Al3+, Ni2+
,
Ware, G. W. Formamidines. In The Pesticide Book; Thompson
Publishers: Fresno, CA, 1989; p 49.
and Mn2+ did not have a significant effect on the
hydrolysis of amitraz, at 10-5 M, or on the degradation
of its products (data not shown). Further study is
necessary to determine the fate of amitraz following
treatment of cattle and livestock, since spray-dip and
dip-vat conditions, i.e. suspended solids, organic matter,
soil, etc., would also affect hydrolysis.
Received for review J anuary 21, 1997. Revised manuscript
received February 10, 1997. Accepted February 10, 1997.X
Mention of specific products or supplier is for identification
and does not imply endorsement by U.S. Department of
Agriculture to the exclusion of other suitable products or
suppliers.
Finally, the hydrolysis rate of amitraz in the diluted
formulated product, Tactic EC, was substantially slower
than predicted: t1/2 ) 80 days at pH 8.3. A precipitate
formed during the reaction, which was found to be
amitraz. Precipitation of amitraz during the reaction
and possible dehydration of the active ingredient by
J F970049X
X Abstract published in Advance ACS Abstracts, April
1, 1997.