Pathways for the hydrolysis of phorate: Product studies by 31P NMR and GC-MS

Article abstract of DOI:10.1021/jf990558u

A new intramolecular mechanism is proposed for the hydrolysis of phorate. ³¹P NMR was used to study the formation of P-containing products of phorate hydrolysis in situ. When hydrolysis was followed by ³¹P NMR, a dominant P-containing product was found and was identified to be diethyl dithiophosphate using methylation and GC-MS. Combining the data from phorate hydrolysis at three different temperatures, thermodynamic parameters were calculated. The contributions of various possible pathways to phorate hydrolysis are discussed.

Full text of DOI:10.1021/jf990558u

J. Agric. Food Chem. 2000, 48, 3013−3017
3013
P a th w a ys for th e Hyd r olysis of P h or a te: P r od u ct Stu d ies by 31P
NMR a n d GC-MS
Feng Hong,† Simo O. Pehkonen,*,† and Elwood Brooks‡
Departments of Civil and Environmental Engineering and Chemistry, University of Cincinnati,
Cincinnati, Ohio 45221-0071
A new intramolecular mechanism is proposed for the hydrolysis of phorate. 31P NMR was used to
study the formation of P-containing products of phorate hydrolysis in situ. When hydrolysis was
followed by 31P NMR, a dominant P-containing product was found and was identified to be diethyl
dithiophosphate using methylation and GC-MS. Combining the data from phorate hydrolysis at
three different temperatures, thermodynamic parameters were calculated. The contributions of
various possible pathways to phorate hydrolysis are discussed.
Keyw or d s: Phorate; diethyl dithiophosphate; 31P NMR; intramolecular nucleophilic attack;
activation entropy; activation enthalpy; GC-MS
INTRODUCTION
Organophosphorus compounds are widely used as
broad-spectrum pesticides, taking advantage of their
inhibitory action on cholinesterase, a key enzyme in-
volved in the metabolism of acetylcholine, one of the key
neurotransmitters (Coats, 1993). In recent years, under
F igu r e 1. Structure of phorate and disulfoton.
mounting pressure from public and environmental
health professionals, the U.S. EPA has decided to
reassess the impact of organophosphorus pesticides on
public health (Cooney, 1999). The data will be used to
further regulate the application of organophosphorus
pesticide, and may assist in determining if their use
must ultimately be discontinued.
Phorate (1) (Figure 1) belongs to the dithioate sub-
group organophosphorus pesticides, as does its struc-
tural cousin, disulfoton (2). It is a systemic pesticide that
targets chewing and sucking pests on a variety of plants
(Szeto and Brown, 1982). It can be oxidized to phorate
oxon, phorate sulfoxide, phorate sulfone, etc. via either
abiotic or biotic pathways (Eto, 1974; Chapman et al.,
1982; Szeto et al., 1990). It can also undergo rapid
hydrolysis at the pH of natural water systems (i.e., 5-9)
(American Cyanamid Corp. data, 1990; Hong and Pe-
hkonen, 1998). Two possible hydrolysis mechanisms
were proposed earlier following the identification of
several unusual, previously unreported hydrolysis prod-
ucts such as HCHO and diethyl disulfide (Hong and
Pehkonen, 1998). Thermodynamic and kinetic studies
are the two most important tools for the elucidation of
reaction mechanisms in addition to product identifica-
tion. Thermodynamic studies are very useful in estimat-
ing the relative contribution of each pathway and
disproving certain pathways especially when multiple
reaction pathways are present.
F igu r e 2. SNi mechanism for demeton-S hydrolysis.
Nucleophilic attacks on the central phosphorus atom
or on a side chain carbon atom are the main modes for
hydrolysis of organophosphorus compounds including
phorate. While an SN2 mechanism usually dominates
with a water molecule and/or hydroxide as the nucleo-
phile (Schwarzenbach et al., 1993), the SNi mechanism
has also been proposed for several pesticides where
internal nucleophiles are present, such as demeton-S
(Schwarzenbach et al., 1993). In demeton-S, a sulfur
atom can serve as an internal nucleophile and the
hydrolysis pathway is relatively independent of pH
(Figure 2). It is important to note that very little
experimental evidence (e.g., product studies) exists to
support an SNi mechanism for the hydrolysis of orga-
nophosphorus pesticides (Muhlmann and Schrader,
1957; Schwarzenbach et al., 1993).
The progress of chemical transformations of organo-
phosphorus pesticides and their derivatives is monitored
mainly by liquid or gas chromatography in combination
with UV-vis or mass spectrometric detection. However,
some of the derivatives are thermally labile or too polar
for GC analysis. Phosphorus 31 NMR (31P NMR) can
be a very useful technique for the analysis of these types
of compounds (Gurley and Ritchey, 1976; Lang and
* To whom correspondence should be addressed at the
Department of Chemical and Environmental Engineering,
National University of Singapore, 10 Kent Ridge Crescent,
Singapore 119260. Fax: (65) 779-1936. E-mail: chesop@
nus.edu.sg.
Martin, 1986; Lai et al., 1995). The disadvantage of 31
NMR is its lower sensitivity compared to GC or LC.
However, if concentration levels are high enough, the
P
† Department of Civil and Environmental Engineering.
‡ Department of Chemistry.
10.1021/jf990558u CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/28/2000

3014 J. Agric. Food Chem., Vol. 48, No. 7, 2000
Hong et al.
Ta ble 1. NMR Da ta of P h or a te Hyd r olysis
time, ha
31/3
151/3 271/3 391/3 511/3 631/3 751/3 871/3 991/3 1111/3 1231/3 1351/3 1471/3 1591/3
NMR signalb (phorate) 17.0
NMR signal (productc) N/Ad
17.2
1.6
14.5
2.6
11.2
3.2
11.5
4.0
8.7
5.4
6.9
5.8
6.5
6.6
5.0
6.6
4.8
7.5
4.4
9.0
3.9
8.5
3.4
10.0
3.2
12.3
a
b
Time between the onset of the experiment and the middle of the acquisition. NMR spectra were printed; NMR peaks were cut and
weighed to represent NMR signals (in mg). c Product refers to O,O-diethyl dithiophosphate. Not distinguishable from the background
d
noise.
Meth yla tion a n d GC-MS. Diazomethane was synthesized
by using a MNNG diazomethane-generation apparatus (Al-
drich Chemical Co.). The manufacturer’s protocol was followed.
Samples for the methylation originated from phorate hydroly-
sis experiments (pH 8.5) at room temperature and the 7-day-
long 31P NMR experiment. Technical-grade diethyl dithiophos-
phate (3) (Aldrich Chemical Co.) was also methylated to serve
as a GC-MS standard. The diethyl ether layers from the
methylation experiments were used as the GC-MS samples.
A HP 5890 Series II GC equipped with a HP 5970 MS detector
(Hewlett-Packard Co.) was used for GC-MS analysis. GC-MS
conditions were the following: 30-m × 0.25-mm i.d. fused silica
capillary column with 0.25-µm film thickness (DB-5ms, J &W
Scientific) and a carrier gas of helium (5.5 psi) were used;
initial temperature was 40 °C; temperature increased at 8.5
°C/min up to 240 °C; injector port temperature was 225 °C;
detector temperature was 250 °C.
technique is robust for kinetics studies because a
reaction system can be continuously monitored in situ
without being disturbed by sampling and various P-
containing products can be identified without the need
for isolation or derivatization.
In this work, 31P NMR and GC-MS were used to
further investigate phorate hydrolysis. The results from
a series of kinetic, thermodynamic, and product studies
together provide a plausible scenario for phorate hy-
drolysis under simulated natural water conditions.
EXPERIMENTAL METHODS
31P NMR An a lysis. The 31P NMR analysis was performed
at 161.98 MHz on a Bruker AMX 400 wide bore spectrometer
equipped with a B-VT 1000 variable-temperature unit. A 30
mm nonspin phosphorus probe was used for the analysis. An
external reference capillary of 1% (w/w) Na2HPO4 was used.
This reference standard was calibrated against 85% phosphoric
acid (Fisher Scientific, Pittsburgh, PA) and found to be 3.45
ppm downfield from phosphoric acid. A 45.0-µs, 90° pulse was
used with a 0.5-s recycle delay time. Immediately after the
onset of the experiment, 32000 16K spectra were acquired with
a spectral window of 33 333.33 Hz (205.78 ppm) using the
Bruker Kinet Experiment pulse sequence. The acquisition took
about 6.67 h and was repeated every 12 h 13 more times. The
temperature was maintained at 310.1 ( 0.1 K and the data
were processed with a line broadening of 20 Hz.
Dimethyl sulfoxide, boric acid, NaOH, and Na2HPO4 were
obtained from Fisher Scientific. Neat phorate (98%) was
obtained from Chem Service (West Chester, PA). Deuterium
oxide (Cambridge Isotope Laboratories, Inc., Andover, MA) was
used for all aqueous solutions. A DMSO:D2O (1:3 v/v) mixed
solvent (containing 0.01 M boric acid, pH adjusted to ∼9.0 with
0.1 M NaOH) was used to dissolve 385 µM (100 ppm) of
phorate in a 30-PP-7 (30-mm outside diameter and 7-in.
length) NMR sample tube, which was purchased from Wilmad
Glass (Buena, NJ ). A coaxial insert tube (520-3 from Wilmad
Glass) was filled with 1% (w/w) aqueous solution of Na2HPO4
to serve as an external reference. One hundred milligrams per
liter of diethyl dithiophosphate (Aldrich Chemical Co., Mil-
waukee, WI) was dissolved in DMSO:D2O (1:3 v/v) mixed
solvent and analyzed with the same 31P NMR protocol.
Hyd r olysis Exp er im en ts. The setup for hydrolysis experi-
ments of phorate was the same as previously described (Hong
and Pehkonen, 1998). Three temperature ranges were cho-
sen: 24.5-27.2 °C (room temperature), 11.4-14.0 °C (low
temperature), 35.5-36.5 °C (high temperature). The temper-
ature range reflects all the replicates, while the temperature
for any given experiment varies no more than 2 °C. All
experiments were homogeneous and carried out through at
least three hydrolysis half-lives of phorate. Six to seven 10-
mL samples were taken for each hydrolysis experiment. Each
sample was immediately extracted with 2 mL of benzene
containing 50 mg/L 4-chloro-3-methylphenol (internal standard
for GC, obtained from Aldrich Chemical Co.) and stored at 4
°C prior to GC analysis. A HP 5890 Series II GC with an FID
detector (Hewlett-Packard Co., Palo Alto, CA) was used for
GC analysis. GC conditions were the following: a 30 m × 0.53-
mm i.d. fused silica capillary column with 1.5-µm film thick-
ness (DB-5, J &W Scientific, Folsom, CA) and a carrier gas of
nitrogen (10 psi) were used; initial temperature was 105 °C
for 2 min; temperature increased at 13 °C/min up to 215 °C
for 3 min; injector port temperature was 250 °C; detector
temperature was 300 °C.
RESULTS AND DISCUSSION
31P NMR An a lysis. The kinetics profile of phorate
hydrolysis is shown in Figure 3 and the peak areas are
summarized in Table 1. The reduction of the phorate
peak (∼90.35 ppm) with time fits well with pseudo-first-
order kinetics. However, the increase of the peak area
of the only significant product peak (∼108.60 ppm) does
not exactly satisfy the mass balance (Table 1). There
are two possibilities: multiple pathways exist for phor-
ate hydrolysis under the experimental condition, or the
initial P-containing hydrolysis product can undergo
further degradation. Based on an earlier study (Hong
and Pehkonen, 1998), two possible P-containing prod-
ucts were expected: diethyl dithiophosphate and diethyl
phosphorothioate. The P-containing product shown here
in the NMR spectrum was later identified via methyl-
ation and GC-MS to be diethyl dithiophosphate. When
diethyl dithiophosphate was dissolved in the same
DMSO/D2O mixed solvent and analyzed using the same
31P NMR parameters, the same chemical shift (∼108.6
ppm) was observed as compared to that of the dominant
product peak in the hydrolysis kinetics study of phorate
(see Figure 3). Hence the P-S bond was left intact
during the hydrolysis under our experimental condi-
tions. It is interesting to note that 31P NMR results by
Lai et al. (1995) show that catalytic hydrolysis by
organophosphorus hydrolase does result in the cleavage
of the P-S bond for five organophosphorus pesticides
although the kcat values for malathion and azinophos-
ethyl, the two phosphorodithioates, are smaller than the
kcat of the other three organophosphorus pesticides. The
kcat value for malathion is especially small (more than
103 times smaller than those of acephate, demeton-S,
and phosalone). This aspect will be discussed further
in the Methylation and GC-MS section.
Hyd r olysis Exp er im en ts. Hydrolysis rate constants
(kobs) are listed in Table 2. Apparently, kobs of phorate
hydrolysis increases with temperature at both pH 5.7
and 8.5. The activation enthalpy, ∆Hq, was determined
by plotting ln(kobs/T) vs 1/T (Figure 4) and measuring
the slope (eq 1), while the activation entropy, ∆Sq, was
calculated from eq 2 (Carey and Sundberg, 1984):

Phorate Hydrolysis Mechanisms
J. Agric. Food Chem., Vol. 48, No. 7, 2000 3015
F igu r e 3. 31P NMR spectrum of phorate hydrolytic kinetics.
F igu r e 4. Determination of activation enthalpy for phorate hydrolysis.
Ta ble 2. kobs (in s-1) for Hom ogen eou s Hyd r olysis of P h or a te
11.4-14.0 °C
24.5-27.2 °C
35.5-36.5 °C
pH 5.7
pH 8.5
4.17 × 10-7 ( 3.93 × 10-8
3.71 × 10-6 ( 3.69 × 10-7
1.50 × 10-5 ( 5.70 × 10-7
3.75 × 10-7 ( 1.96 × 10-8
3.15 × 10-6 ( 5.03 × 10-7
1.47 × 10-5 ( 2.67 × 10-6
ln(kobs/T) ) -∆Hq/RT + constant
∆Sq ) (∆Hq/T) + R ln[hkobs/(kκT)]
(1)
(2)
entropy reflects the loss or gain of degrees of freedom
between the starting compounds and the transition
state (i.e., the activated complex). However, because
multiple pathways may simultaneously contribute to
the hydrolysis of phorate, the magnitude and the sign
of ∆Sq can be used to evaluate the relative significance
of the individual pathways. The negative ∆Sq for phor-
ate hydrolysis at pH 8.5 suggests that pathways other
than the common SN2 (Schwarzenbach et al., 1993) play
an important role in phorate hydrolysis at that pH.
where h is the Planck’s constant, k is the Boltzmann’s
constant, κ is the transmission coefficient (∼1), and kobs
is the observed hydrolysis rate constant in s-1. The
Arrhenius activation energy, Ea, can be obtained from
Ea ) ∆Hq + RT
(3)
Meth yla tion a n d GC-MS. The one dominant prod-
uct peak (∼108.60 ppm) from 31P NMR data suggests a
dominant P-containing hydrolysis product. When the
The obtained activation parameters for homogeneous
hydrolysis of phorate are listed in Table 3. Activation

3016 J. Agric. Food Chem., Vol. 48, No. 7, 2000
Hong et al.
Ta ble 3. Activa tion P a r a m eter s for Hom ogen eou s
Hyd r olysis of P h or a te a n d Disu lfoton
culprit for the absence of the thermodynamically favored
P-S to P-O transition. The identification of diethyl
dithiophosphate provides further support for the hy-
drolysis mechanism proposed earlier by Hong and
Pehkonen (1998).
∆Hq,
∆Gq,
∆Sq,
Ea,
kJ /mol
pesticide
phorate
pH
kJ /mol
kJ /mol
J /mol K
5.7
8.5
5.7
8.5
120
104
65
130
103
78
39
-2.8
-150
-54
120
110
68
There is another possible P-containing product, O,O,S-
triethyl dithiophosphate (5) (Figure 5). This compound
was tentatively identified from several hydrolysis mix-
tures of phorate at pH 8.5 (at room temperature),
accounting for approximately 10% of the initial phorate
concentration based on relative GC peak areas. Using
the Wiley 275L searchable library of mass spectra, a
90% match between the experimentally obtained mass
spectrum and the library mass spectrum of O,O,S-
triethyl dithiophosphate (5) resulted from the search.
Unfortunately, the pure compound is not commercially
available, which makes a more conclusive identification
difficult. The mechanism leading to this compound is
not so straightforward by just examining the structure
of phorate (1). After extensive consultation and insight-
ful input from several colleagues, two intriguing mech-
anisms (Figure 6) can be proposed to account for this
never-reported product. All mechanisms proposed for
phorate hydrolysis so far are also summarized in Figure
6. The SN2 pathway in Figure 6 is supported by the
identification of diethyl dithiophosphate, HCHO, and
diethyl disulfide (Hong and Pehkonen, 1998). A ther-
modynamically stable six-membered ring transition
state (Figure 6) builds a strong case for the intramo-
lecular nucleophilic attack pathway (SNi). In the mean-
time, the activation entropy data from phorate hydrol-
ysis at pH 5.7 and 8.5 seem to support the SN1 pathway
(Figure 6) for the production of O,O,S-triethyl dithio-
phosphate (5). Both SN2 and SNi pathways would lower
disulfotona
95
80
98
a
Data from Dannenberg and Pehkonen, 1998.
F igu r e 5. Structure of selected phorate hydrolysis products.
hydrolysis mixture was methylated following the 7-day
31P NMR experiment, GC-MS identified the product as
diethyl methyl dithiophosphate, which is the logical
methylation product of O,O-diethyl dithiophosphate (3)
(Figure 5). It is well-known that phosphorus has a
strong tendency to bond with oxygen and that the P-O
bond is about 40% stronger than the P-S bond (Lide
and Frederikse, 1997), yet diethyl dithiophosphate
seems to be rather stable in the mixed solvent applied
here. One possibility is that the hydrolysis of diethyl
dithiophosphate (3) to diethyl phosphorothionate (4)
may not be kinetically favored. The other possibility is
that DMSO interferes with the hydrolysis of diethyl
dithiophosphate. While DMSO was found to slow the
hydrolysis of phorate at pH 9.0 by 60% at room tem-
perature (that is the reason for the elevated tempera-
ture in the 31P NMR experiment), its polar and inert
nature and its low nucleophilicity makes it an unlikely
F igu r e 6. Summary of phorate hydrolysis pathways.

Phorate Hydrolysis Mechanisms
J. Agric. Food Chem., Vol. 48, No. 7, 2000 3017
the entropy of the system in going from the reactant
ground state to the transition state; in fact, the loss of
translational freedom in the SN2 pathway may lead to
an entropy loss of as much as 100 J /K-mol (Isaacs, 1995),
while the entropy loss in closing rings of six members
or less (SNi) would be smaller than that of the SN2
pathway (March, 1992). For phorate hydrolysis, the
proposed SN1 pathway might reduce or increase the
entropy of the system, depending on the rate-determin-
ing step: at acidic pH, the protonation step should be
much faster than the formation of the carbocation and
the ∆Sq contribution should be positive (Figure 6); at
basic pH, the ∆Sq contribution would be negative if the
second step is the rate-determining step (Figure 6). The
positive ∆Sq at pH 5.7 and the negative ∆Sq at pH 8.5
for phorate hydrolysis suggest a large SN1 pathway at
pH 5.7. By way of comparison, the ∆Sq for disulfoton
hydrolysis (Table 3) is negative at both pH 5.7 and 8.5.
Apparently, no stable carbocation intermediate forms
in disulfoton hydrolysis, precluding an SN1 pathway. In
fact, the SN1 pathway is rarely suggested for the
hydrolysis of (thio)phosphate esters (Schwarzenbach et
al., 1993).
Department of Chemistry, University of Cincinnati:
Prof. Koka J ayasimhulu for his input on SNi mechanism,
Prof. George Kreishman for his generous help on 31P-
NMR analysis, and Prof. Allan Pinhas for stimulating
discussions.
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latter (Figure 1). However, a similar intramolecular
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CONCLUSIONS
31P NMR analysis of phorate hydrolysis at pH 8.5
suggested one dominant reaction pathway. The disap-
pearance of the pesticide peak fits extremely well with
the appearance of the dominant product peak. The
dominant product was later identified via methylation
and GC-MS as diethyl dithiophosphate.
An interesting intramolecular nucleophilic pathway
was proposed for phorate hydrolysis based upon the
product studies using GC-MS. The six-membered ring
within the transition state makes the hypothetical
pathway plausible. Additionally, this pathway is inde-
pendent of pH and, if dominant, should result in the
degradation of phorate even in the absence of external
nucleophiles such as OH- and H2O.
Activation parameters were determined for homoge-
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vation free energy, and activation energy of phorate
hydrolysis are all similar to those of other structurally
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ACKNOWLEDGMENT
Received for review May 24, 1999. Revised manuscript received
March 13, 2000. Accepted March 28, 2000.
We owe credits to Mr. Andy Zureick for his help in
hydrolysis experiments, Dr. Barry Austern of US EPA
for his help in GC-MS analysis, and three colleagues in
J F990558U