Half-life of chlorpyrifos oxon and other organophosphorus esters in aqueous solution

Article abstract of DOI:10.1016/j.cbi.2019.108788

Aqueous solutions of chlorpyrifos oxon are used to study the ability of chlorpyrifos oxon to catalyze protein crosslinking. Assays for protein crosslinking can avoid artifacts by using information on the stability of chlorpyrifos oxon in solution. We unde

Full text of DOI:10.1016/j.cbi.2019.108788

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Half-life of chlorpyrifos oxon and other organophosphorus esters in aqueous solution
Oksana Lockridge, Laurent Verdier, Lawrence M. Schopfer
PII:
S0009-2797(19)31003-8
https://doi.org/10.1016/j.cbi.2019.108788
CBI 108788
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 7 June 2019
Revised Date: 14 July 2019
Accepted Date: 7 August 2019
Please cite this article as: O. Lockridge, L. Verdier, L.M. Schopfer, Half-life of chlorpyrifos oxon and
other organophosphorus esters in aqueous solution, Chemico-Biological Interactions (2019), doi: https://
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Graphical abstract

Half-life of chlorpyrifos oxon and other organophosphorus esters in
aqueous solution
Oksana Lockridge1, Laurent Verdier2, Lawrence M. Schopfer1
1 Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5900 USA
2 DGA CBRN Defense, Le Bouchet 91710 Vert le Petit, France
olockrid@unmc.edu, lmschopf@unmc.edu,
Corresponding author
Oksana Lockridge
Eppley Institute
University of Nebraska Medical Center
Omaha, NE 68198-5900
olockrid@unmc.edu
phone 1 402 559 6032
Abstract
Aqueous solutions of chlorpyrifos oxon are used to study the ability of chlorpyrifos oxon to
catalyze protein crosslinking. Assays for protein crosslinking can avoid artifacts by using
information on the stability of chlorpyrifos oxon in solution. We undertook to determine
the half-life of chlorpyrifos oxon in aqueous solution because literature values do not exist.
The rate of conversion of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol was measured at
23˚C in 20 mM TrisCl pH 8 and pH 9 by recording loss of absorbance at 290 nm for
chlorpyrifos oxon and increase in absorbance at 320 nm for 3,5,6-trichloro-2-pyridinol.
The half-life of chlorpyrifos oxon was 20.9 days at pH 8 and 6.7 days at pH 9. Literature
reports for the stability of other organophosphorus toxicants were summarized because
our current studies suggest that other organophosphorus toxicants are also crosslinking
agents.
1

Keywords: chlorpyrifos oxon, stability, half-life, extinction coefficient, absorbance spectra
Abbreviations
CPO, chlorpyrifos oxon
OP, organophosphorus toxicants
TCP, 3,5,6-trichloro-2-pyridinol
1. Introduction
Neurotoxic symptoms are associated with chronic, low-dose exposure to
organophosphorus pesticides (Jamal et al., 2002; Kamel et al., 2007; Wang et al., 2014;
Jokanovic, 2018; Naughton and Terry, 2018). Low dose exposure to the organophosphorus
nerve agent, sarin, is hypothesized to explain Gulf War Illness (Golomb, 2008). We have
developed a mechanism to explain neurotoxicity resulting from low dose exposure to
organophosphorus toxicants (OP). We find that organophosphorus esters are crosslinking
agents. Mass spectrometry analysis shows that proteins treated with chlorpyrifos oxon
form stable covalent crosslinks between lysine and glutamic acid or lysine and aspartic acid
(Schopfer and Lockridge, 2018; Schopfer and Lockridge, 2019). We hypothesize that
crosslinked proteins form insoluble aggregates that disrupt neuronal function.
Our experimental protocol incubates proteins with OP in aqueous buffer. The
question we addressed in this report is the stability of OP in aqueous buffer. We needed to
know whether or not OP esters spontaneously degrade in a few hours. The published
literature has information on the stability of many OP, but no information on the stability of
chlorpyrifos oxon. We report that the half-life of chlorpyrifos oxon in pH 8 and pH 9
buffers at 23˚C is 20.9 and 6.7 days, respectively.
2

2. Materials and Methods
2.1. Materials:
Chlorpyrifos oxon Chem Service MET 11459B
3,5,6-trichloro-2-pyridinol Chem Service MET-674A
Paraoxon ethyl Chem Service N-12816
Paraoxon methyl Chem Service N-11775
Diazoxon Chem Service MET-11621A
Human butyrylcholinesterase purified in house (Schopfer et al., 2019)
YM-10 centrifugal filters, 10,000 MW cut off (Merck Millipore MRCPRT010)
Slide-A-Lyzer cassettes, 7000 MW cut off, 0.5-3 mL capacity (Pierce 66370)
Cellulose dialysis tubing 12,000-14,000 MW cut off (Spectrapor 132700)
2.2. Absorbance spectra of chlorpyrifos oxon and 3,5,6-trichloro-2-pyridinol.
Stock solutions of 2 mM 3,5,6-trichloro-2-pyridinol (TCP) and 2 mM chlorpyrifos oxon
(CPO) in acetonitrile were diluted into 20 mM TrisCl pH 8.0 to make 20 µM. Absorbance
was read against water in matched 4 ml quartz cuvettes over the range 265 nm to 350 nm
on a Gilford single-beam spectrophotometer at 23˚C. Extinction coefficients were
calculated from absorbance at 290 nm for CPO and 320 nm for 3,5,6-trichloro-2-pyridinol
(TCP). Data in Figure 1.
2.3. Hydrolysis of CPO by sodium hydroxide.
TCP was confirmed as the major hydrolysis product of CPO by acquiring a series of
absorbance spectra over time for 28.8 µM CPO in 10 mM sodium hydroxide pH 11.8 at 23˚C
in a Cary 3 Bio UV-Visible double-beam spectrophotometer. The reference cuvette
contained 0.1 M potassium phosphate pH 7.0. Data in Figure 2.
3

2.4. Stability of CPO in aqueous buffer
In our hands 300 mM CPO solutions in ethanol or acetonitrile are stable for years at -80˚C.
However, we suspected that CPO is unstable in aqueous buffers. We measured the stability
of 20 µM CPO by recording the increase in absorbance at 320 nm, the wavelength for
maximum absorbance of the TCP hydrolysis product. Duplicate 100 mL solutions of 20 µM
CPO in 20 mM TrisCl, 0.01% azide pH 8.0 and pH 9.0 were prepared in tightly closed glass
bottles stored in the dark at 23˚C. Absorbance at 320 nm was read against water over a
period of one month for aliquots transferred from the bottles to matched quartz cuvettes.
2.5. Calculation of decay rate constant and half-life
Absorbance at 320 nm of 20 µM TCP was 0.137 at pH 8 and at pH 9. The natural logarithm
for the difference between 0.137 and the observed absorbance at 320 nm of the
chlorpyrifos oxon solution was plotted as a function of days of incubation at 23˚C. The
slope of the resulting lines yielded the first order rate constant k for decay of chlorpyrifos
oxon. The half-life was calculated using the equation t½ = 0.693/k. Duplicate absorbance
readings were essentially identical for each point. Data in Figure 3.
The first order rate constant for decay of chlorpyrifos oxon at pH 11.8 and the half-
life at pH 11.8 in 10 mM sodium hydroxide were calculated from the absorbance spectra in
Figure 2.
The rate constants and half-lives for decay of paraoxon ethyl, paraoxon methyl, and
diazoxon were measured in 100 mM sodium hydroxide pH 13.
2.6. Methods for removing excess CPO
Our publications on the crosslinking action of CPO have used dialysis and diafiltration to
remove excess CPO. Small protein volumes, less than 3 mL, were dialyzed in Slide-A-Lyzer
4

cassettes against 3 x 4 L of 10 mM ammonium bicarbonate pH 8 at 4˚C. Alternatively 0.5 ml
protein volumes were centrifuged through YM-10 centrifugal filters that allowed CPO to
pass through, but retained protein. Repeated dilution and centrifugation cleared the
protein sample of CPO. Large protein volumes, 3 to 10 mL, were placed into cellulose
dialysis bags and dialyzed at 4˚C against 3 x 4 L of 10 mM ammonium bicarbonate pH 8.
2.7. Test for successful removal of excess CPO
The activity of human and equine butyrylcholinesterase is inhibited by CPO concentrations
as low as 0.5 x 10-9 M (Amitai et al., 1998; Heilmair et al., 2008). Spent dialysis buffer and
the final dialyzed protein were tested for the presence of CPO by adding an aliquot to
purified human butyrylcholinesterase and comparing butyrylcholinesterase activity before
and after addition of the test sample. For example, 100 µl of purified human
butyrylcholinesterase with an activity of 1.1 u/ml (18 x 10-9 M) was incubated with 100 µl
of spent dialysis buffer for 30 min to 2 h at room temperature. A 50 µl aliquot of the
incubation mixture was added to a 4 ml quartz cuvette containing 2 ml of 1 mM
butyrylthiocholine and 0.5 mM 5,5’-dithiobis(2-nitrobenzoic acid) in 0.1 M potassium
phosphate pH 7.0. Increase in absorbance at 412 nm was recorded for 1 min in a Gilford
spectrophotometer at 25˚C. The absorbance change per min was converted to µmoles
butyrylthiocholine hydrolyzed per min using the Extinction coefficient of 13,600 M-1 cm-1
for the reaction product (Ellman et al., 1961). The ∆Absorbance at 412 nm per min was
0.177 for control and for samples that were free of OP, but was zero for samples that
contained more than 18 nM OP.
3. Results
5

3.1. Background information for measuring stability of chlorpyrifos oxon in aqueous
solution
Absorbance spectra of freshly prepared 20 µM solutions of CPO and 3,5,6-trichloro-2-
pyridinol (TCP) in 20 mM TrisCl pH 8.0 distinguished between the two compounds. Figure
1 shows a peak absorbance for CPO at 290 nm with an extinction coefficient of 6800 M-1
cm-1. Peak absorbance for TCP was at 320 nm with an extinction coefficient of 6700 M-1
cm-1.
Figure 1. Absorbance spectra of 20 µM chlorpyrifos oxon (CPO) and 20 µM 3,5,6-trichloro-
2-pyridinol (TCP) in 20 mM TrisCl pH 8.0 at 23˚C, measured in a single-beam Gilford
spectrophotometer in 4 ml quartz cuvettes. The symbols indicate the wavelengths at which
absorbance was measured manually. Curves were drawn through the points with Excel software.
Diamond symbols are absorbance values for 20 µM chlorpyrifos oxon. Squares are
absorbance values for 20 µM 3,5,6-trichloro-2-pyridinol.
6

The time course for hydrolysis of CPO at alkaline pH in Figure 2 shows that CPO is
converted to a product with the characteristic absorbance spectrum of TCP, in an isosbestic
manner indicating that there are no intermediate species. The half-life of CPO at pH 11.8
was 14 min. The extinction coefficient of TCP was pH sensitive, being slightly higher at pH
11.8 (7100 M-1 cm-1) than at pH 8.0 (6700 M-1 cm-1).
Figure 2. Hydrolysis of 28.8 µM chlorpyrifos oxon (CPO) to 3,5,6-trichloro-2-pyridinol
(TCP) by 10 mM sodium hydroxide at 23˚C. Arrows indicate disappearance of CPO at 290
nm and appearance of TCP at 320 nm. Conversion to TCP was complete in 103 min. The
half-life of CPO at alkaline pH was 14 min. The extinction coefficient for TCP at 320 nm pH
11.8 was 7100 M-1 cm-1.
7

3.2. Stability of chlorpyrifos oxon in aqueous buffers
The information in Figures 1 and 2 led to the conclusion that the stability of CPO can be
monitored by measuring increase in absorbance at 320 nm. We wanted to know the
stability of CPO in more-neutral, aqueous buffers to allow us to estimate the concentration
of intact CPO in protein solutions treated with CPO for extended periods of time. The
results in Figure 3 show that CPO has a half-life of 20.9 days at pH 8.0 and 6.7 days at pH
9.0. We treat proteins with CPO in pH 8 to pH 9 buffers. The long half-life of CPO in pH 8
and pH 9 buffers makes it important to remove excess CPO from CPO-treated protein
solutions before processing samples for SDS gel electrophoresis and mass spectrometry.
Our protocol for studying the crosslinking action of CPO and other organophosphorus
toxicants always includes removal of excess toxicant before analyzing proteins for
crosslinked peptides.
Figure 3. Rate of decay of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol (TCP) at 23˚C.
Chlorpyrifos oxon (20 µM) in 100 mL of 20 mM TrisCl, 0.01% sodium azide pH 8.0 and pH
9.0 was stored in the dark. Spontaneous decay of chlorpyrifos oxon to TCP was monitored
by the increase in absorbance at 320 nm. The half-life for chlorpyrifos oxon at pH 8.0 was
8

20.9 days, and at pH 9.0 was 6.7 days. Duplicate absorbance readings were essentially
identical for each point.
3.3. Stability of OP at alkaline pH
The first order rate constant of 0.0499 per min and half-life of 13.9 min for chlorpyrifos
oxon in 10 mM sodium hydroxide pH 11.8 were calculated by plotting the natural log of
absorbance change versus time in Figure 4. We used a similar protocol to determine the
first order rate constant of decay and the half-life of diazoxon (t½ = 2.5 min), paraoxon-
ethyl (t½ = 11.5 min), and paraoxon methyl (t½ = 2.3 min) in 100 mM sodium hydroxide
pH 13, see Table 1. Our values for paraoxon ethyl are similar to the results of Ginjaar and
Vel (Ginjaar and Vel, 1958). Table 1 includes literature values for decay of chlorpyrifos and
dichlorvos at alkaline pH. It was concluded that organophosphorus esters are rapidly
inactivated by 10 to 100 mM sodium hydroxide solutions in water.
Figure 4. Rate of decay of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol at pH 11.8 and
23˚C. Absorbance change at 320 nm as a function of time in 10 mM sodium hydroxide was
from Figure 2. The half-life of chlorpyrifos oxon was 13.9 min at pH 11.8.
9

Table 1. OP half-life in sodium hydroxide
OP
pH
t½ , min k, min-1 ˚C Solvent
Reference
Chlorpyrifos
12.9 18.2
0.038
0.05
0.015
0.28
0.06
0.3
25 NaOH in water (Macalady and Wolfe, 1983)
Chlorpyrifos oxon 11.8 13.9
23 10 mM NaOH
Present report
Dichlorvos
Diazoxon
11.5 46.2
25 NaOH in water (Grechko et al., 1983)
23 100 mM NaOH Present report
23 100 mM NaOH Present report;
23 100 mM NaOH Present report;
13
13
2.5
Paraoxon-ethyl
11.5
2.3
Paraoxon-methyl 13
4. Discussion
4.1. Chlorpyrifos oxon
Our study shows that chlorpyrifos oxon, the toxic metabolite of the pesticide chlorpyrifos,
persists for days in aqueous solution. This finding necessitates removal of excess CPO
before CPO-treated protein samples are processed for SDS gel electrophoresis to look for
aggregation, and before CPO-treated proteins are digested with trypsin for mass
spectrometry analysis of protein crosslinks. Excess OP could accelerate crosslinking during
the heat-denaturation step of proteins prior to SDS gel electrophoresis. Excess OP could
induce random crosslinks between tryptic peptides that would not represent crosslinks
between peptides in native proteins.
4.2. Inactivation at high pH
OP pesticides and nerve agents are rapidly destroyed at pH 10 to 13 as shown in Figures 5
and 6. Fifty percent of an aqueous solution of paraoxon methyl degrades to nontoxic
products in 2.3 min and 99% in about 30 min in the presence of 100 mM sodium
10

hydroxide. The most stable OP in Table 1 is dichlorvos which is 50% degraded in 46.2 min
and is 99% inactivated in about 4 hours at pH 11.5.
4.3. OP stability information in the literature
To date only chlorpyrifos oxon and the nerve agent VX have been described as crosslinking
agents (Schmidt et al., 2014; Schopfer and Lockridge, 2018; Schopfer and Lockridge, 2019).
The toxicants listed in Table 2 are under investigation as crosslinking agents and therefore
information on their stability in aqueous solution is needed.
Table 2 gives half-lives as a function of OP structure, pH, temperature, and buffer
composition. As can be seen from the table, the reaction condition information is
incomplete in several publications. This is likely the reason for differences in the half-life
values reported by different authors. For example, chlorpyrifos at pH 7 and 25˚C was
reported to have a half-life of 29 days by Chapman and Cole and 72 days by Solomon et al.
(Chapman and Cole, 1982; Solomon et al., 2014), but the reaction buffer was not stipulated
by Solomon et al. Reaction conditions clearly affect the measured hydrolysis rates. For
example, dichlorvos at pH 7 and 25˚C has a half-life of 5 days in buffered water, but 0.07
days in cacodylate buffer (Lim, 1996; Benoit-Marquié et al., 2004). Spontaneous hydrolysis
of O-hexyl 2,5-dichlorophenyl phosphoramidate was significant in 100 mM phosphate
buffer pH 7.4, but negligible in 100 mM Tris/citrate pH 7.4 (Sogorb et al., 1993). The
presence of copper (II) ions increases the rate of hydrolysis for OP pesticides and nerve
agents (Epstein, 1974; Meikle and Youngson, 1978; Smolen and Stone, 1997; Liu et al.,
2001).
Figure 5A shows that chlorpyrifos is relatively stable below pH 6, with a half-life at
pH 5 of 77 days. Figure 5B shows that parathion ethyl and paraoxon ethyl are stable in
11

aqueous solution at pH below 7. The half-life at pH 5 for parathion ethyl is 150 days, and
for paraoxon ethyl is 170 days. However at pH 10 the half-lives of all three OP are a few
minutes.
Figure 5. Chlorpyrifos (Panel A), parathion ethyl, and paraoxon ethyl (Panel B) are stable
at acid to neutral pH (4 to 7) for days, but decay rapidly at alkaline pH (Gomaa and Faust,
1972; Freed et al., 1979; Chapman and Cole, 1982; Macalady and Wolfe, 1983; Solomon et
al., 2014).
The oxons (chlorpyrifos oxon, paraoxon, and diazoxon) are orders of magnitude
more reactive with acetylcholinesterase than the parent pesticides chlorpyrifos, parathion,
and diazinon. This brings up the question of whether the oxons are also more reactive with
water. Figure 5B supports the observation of Faust and Gomaa that parathion hydrolysis is
slightly faster than paraoxon hydrolysis under acidic or neutral conditions, while paraoxon
hydrolysis is 7 times faster than parathion at pH 9.0 and five times faster at pH 10.4 (Faust
and Gomaa, 1972). Figure 6A shows that an aqueous solution of diazinon is more stable at
12

all pH values than an aqueous solution of diazoxon. Thus, though there is a tendency for
oxons to be more susceptible to aqueous hydrolysis than thions, the differences in
reactivity are only a few fold not orders of magnitude as is seen with reactivity with
acethycholinesterase.
Figure 6. Diazinon and diazoxon (Panel A), sarin and soman (Panel B) are unstable at pH 3
and pH 10. Sarin and soman are most stable at pH 4-6 where sarin has a half-life of 461
hours, and soman 144 hours. Note the half-life data for sarin and soman are in hours in the
figure, but in days in Table 1. Diazinon and diazoxon are most stable at pH 7-8. Figure 6A
was constructed from information in (Gomaa et al., 1969). Figure 6B was constructed from
information in (Epstein, 1974) and (Clark, 1989).
Figures 6A and 6B show the stability patterns for diazinon, diazoxon, sarin, and
soman in aqueous solution. These patterns are different from those in Figures 5A and 5B
for chlorpyrifos, parathion, and paraoxon. Figures 6A and 6B display bell shaped stability
13

curves. Diazinon and diazoxon are most stable at pH 7 to 8. The nerve agents sarin and
soman are most stable at pH 4-6. All of these organophosphylates show decreasing
stability at both acidic and basic pH extremes. Even under the most stable conditions, sarin
is less stable than diazinon, i.e. at pH 5, an aqueous solution of sarin is about 10-fold less
stable than an aqueous solution of diazinon at pH 7.4.
The foregoing observations illustrate that stability of OP in aqueous solution is
highly dependent on the pH of the solution, is markedly dependent upon the structure of
the OP, and is susceptible to buffer catalysis.
Table 2. Half-life of OP in aqueous solution
OP
pH
t ½ ,
days
89.1
˚C Solvent
Reference
Chlorpyrifos
1
25 Sterile water or carbonate (Macalady and
1.2
1.2
5.9
6.1
9.8
9.8
9.7
9.7
123.4 25 buffer
Wolfe, 1983)
CAS
2921-88-2
54.7
53.5
25
25
141.5 25
10.1
10.2
10.5
10
25
25
25
25
10.3 1.6
10.3 1.4
10.3 1.6
10.7 0.8
25
25
25
25
11
0.5
25
11
0.5
25
11.2 0.4
11.2 0.4
11.9 0.2
12.4 0.04
12.4 0.04
12.9 0.01
12.9 0.01
25
25
25
25
25
25
25
Chlorpyrifos
Chlorpyrifos
5
7
9
4.5
5
6
7
8
73
72
16
77
77
49
29.4
18.9
25 Not specified
25
25
(Solomon et al.,
2014)
25 Sterile 0.2 M phosphate
(Chapman and
Cole, 1982)
25
25
25
25
14

Chlorpyrifos
4.7
6.9
8.1
6.9
7.3
7.1
63
35
23
51.8
76.6
110
25 0.02 M phosphate
25
25
22 Pond water
22
22 water
(Meikle and
Youngson, 1978)
Chlorpyrifos
Chlorpyrifos
Chlorpyrifos
Chlorpyrifos
Chlorpyrifos
(Lu et al., 2006)
(Mansour et al.,
1999)
(Sharom et al.,
1980)
(Freed et al.,
1979)
(Liu et al., 2001)
7.1
70
21 Distilled water
6.1
7.4
5.7
7.66 56
7.93 126
7.99 26
8.15 24
120
53
45.9
20 Phosphate buffer
20
Deionized water
River water
Chlorpyrifos
Chlorpyrifos
6
16.8
2.5
9.7
1
0.1
0.2
62
28 Tap water
28 River water
(Thomas and
Mansingh, 2002)
8.5
8.7
6
8.5
8.7
8
28 Brackish water
28 Aerated tap
28 Aerated river
28 Aerated brackish
29 Sterile 0.02 M phosphate
(Noblet et al.,
1996)
Chlorpyrifos
oxon
8
9
20.9
6.7
23 20 mM TrisCl, 0.01% NaN3 Present report
23
CAS
5598-15-2
Chlorpyrifos
oxon
11.8 0.01
23 10 mM NaOH for pH 11.8
Present work
Diazinon
3.1
5.0
7.4
9.0
10.4
0.5
31
185
136
6
20 0.02 M phosphate
(Gomaa et al.,
1969)
20
20
20
20
CAS
333-41-5
Diazinon
5
12
Not specified
(EPA, 2006)
7
9
138
77
Diazinon
Diazinon
Diazinon
Diazinon
Diazinon
7.3
171
21 Not specified
21 Distilled water
(Mansour et al.,
1999)
(Sharom et al.,
1980)
(Lartiges and
Garrigues, 1995)
(Lu et al., 2006)
7.1
70
6.1
7.3
6.9
7.3
4.5
5
69
52
69.5
22 MilliQ water
22 filtered river water
22 Pond water
134.4 22 Pond water
3.1
14
25 Sterile 0.2 M phosphate
25
(Chapman and
Cole, 1982)
6
7
54.6
70
25
25
8
2.2
53.9
0.25
25
Diazinon
28 Mineral growth medium
(Drufovka et al.,
15

3.2
4.2
5.2
6.2
7.5
8
0.5
8
16.6
16.6
16.6
130
28
28
28
28
2008)
28
Diazinon
Diazoxon
29 Sterile 0.02 M phosphate
(Noblet et al.,
1996)
(Gomaa et al.,
1969)
3.1
5.0
7.4
9.0
0.017 20 0.02 M phosphate
1.27
29
20
20
20
20
CAS
962-58-3
18
10.4 0.42
Diazoxon
Dichlorvos
13
5
7
0.002 23 100 mM NaOH
Present report
(Lim, 1996)
12
5
25 Buffered water
25
CAS
9
0.9
25
62-73-7
Dichlorvos
2.6
5.6
7.0
9.0
6.1
>1.7
0.31
0.07
0.40
<81
25 Water
(Benoit-Marquié
et al., 2004)
25 Cacodylate
25 Cacodylate
25 Tetraborate
22 Not specified
Dichlorvos
Dichlorvos
(Lartiges and
Garrigues, 1995)
6
7
8.5
4.0
6.9
9.3
12.7
14.8
13.6
30
4.5
0.9
25 Well water near Ljubljana (Druzina and
25
25
25 Buffered water
25
25
Stegu, 2007)
Dichlorvos
(Grechko et al.,
1983)
11.5 0.03
25
Methamido
phos
CAS
7
9
5
2.9
22 Not specified
22
(Tomlin, 1997)
(Tomlin, 1997)
10265-92-6
Monocrotop
hos
CAS
5
7
9
96
66
17
20 Not specified
20
20
6923-22-4
Parathion
ethyl
3.1
5
7.4
9
174
153
108
22
20 0.2 M phosphate
20
20
20
(Gomaa and
Faust, 1972)
CAS
56-38-2
Parathion
ethyl
Parathion
ethyl
10.4 1.4
20
6.1
7.4
7.2
170
130
60
20 Phosphate buffer
20
20 Not specified
(Freed et al.,
1979)
(Mansour et al.,
1999)
Parathion
ethyl
4
7
9
6.1
7.3
272
260
130
84
22 Not specified
22
22
22 Milli-Q water
22 Filtered river water
(Tomlin, 1997)
Parathion
ethyl
(Lartiges and
Garrigues, 1995)
33
16

Parathion
ethyl
4.5
5
6
7
8
273
301
231
168
105
120
25 Sterile 0.2 M phosphate
25
25
25
(Chapman and
Cole, 1982)
25
Parathion
ethyl
7
25 Not specified
(Coates, 1949)
Parathion
ethyl
6
7
8.5
6
7
8.5
23.9
33.5
31.4
45.5
47.2
47.6
25 Well water near Ljubljana (Druzina and
25
25
Stegu, 2007)
Parathion-
methyl
CAS
25 Well water near Ljubljana (Druzina and
25
25
Stegu, 2007)
298-00-0
Paraoxon
ethyl
3.1
5
7.4
9
197
173
144
2.9
20 0.2 M phosphate
20
20
20
(Gomaa and
Faust, 1972)
CAS
311-45-5
Paraoxon
ethyl
Paraoxon
ethyl
Paraoxon
methyl
CAS
10.4 0.25
20
7
87
25 Not specified
(Coates, 1949)
Present report
Present report
13
13
0.008 23 100 mM NaOH
0.002 23 100 mM NaOH
950-35-6
Sarin
2.0
4.0
6.5
7.0
7.5
8.0
9.0
2
0.1
19.2
19.2
6.1
1.9
0.6
0.06
0.1
1
20 Buffered water
20
20
20
20
20
20
(Epstein, 1974)
(Clark, 1989)
CAS
107-44-8
Soman
20 Buffered water
20
3
CAS
4
6
20
96-64-0
5
6
6
6
20
20
7
7.6
9
3.3
0.28
0.02
0.02
18.5
15.9
2.6
6-14
0.3
0.1
0.1
0.35
20
20
20
20
10
4.5
4.5
4.5
6.4
7.4
8.0
8.6
7
Soman
Soman
25 Dilute acid
25 Water
25 Acetate
27 0.15 M NaCl
27 0.02 M phosphate
27 0.02 M phosphate
27 0.02 M phosphate
20 water
(Ellin et al., 1981)
(Broomfield et al.,
1986)
Tabun
(Clark, 1989)
CAS
77-81-6
17

VX
7
16-
42
25 Not specified
(National.Researc
h.Council.US,
1999)
CAS
50782-69-9
5. Conclusion
Organophosphorus toxicants, including chlorpyrifos oxon, persist in aqueous buffers at
neutral pH and 20-25˚C for days to months. They are completely degraded in minutes by
10 to 100 mM sodium hydroxide.
Conflict of interest statement
The authors have no competing interests
Funding
Supported by Fred & Pamela Buffett Cancer Center Support Grant P30CA036727 from NIH,
and Direction Générale de l’Armement (DGA) under grant PEA ENVAU2.
Acknowledgement
The findings and conclusions in this article are those of the authors and do not necessarily
represent the views of the French Ministry of Armed Forces or the U.S. Department of
Health and Human Services. Use of trade names is for identification only and does not
imply endorsement by the French Ministry of Armed Forces or the U.S. Department of
Health and Human Services.
References
Amitai G, Moorad D, Adani R and Doctor BP (1998) Inhibition of acetylcholinesterase and
butyrylcholinesterase by chlorpyrifos-oxon. Biochem Pharmacol 56:293-299.
Benoit-Marquié F, de Montety C, Gilard V, Martino R, Maurette MT and Malet-Martino M
(2004) Dichlorvos degradation studied by 31P-NMR. Environ Chem Lett 2:93-97.
Broomfield CA, Lenz DE and MacIver B (1986) The stability of soman and its stereoisomers
in aqueous solution: toxicological considerations. Arch Toxicol 59:261-265.
18

Chapman RA and Cole CM (1982) Observations on the influence of water and soil pH on the
persistence of insecticides. Journal of environmental science and health Part B,
Pesticides, food contaminants, and agricultural wastes 17:487-504.
Clark DN (1989) Review of reactions of chemical agents in water. Defense Technical
Information Center, Alexandria, VA AD-A213 287
.
Coates H (1949) The chemistry of phosphorus insecticides. The Annals of applied biology
36:156-159.
Drufovka K, Danevcic T, Trebse P and Stopar D (2008) Microorganisms trigger chemical
degradation of diazinon. International Biodeterioration & Biopdegradation 62:293-
296.
Druzina B and Stegu M (2007) Degradation study of selected organophosphorus
insecticides in natural waters. Intern J Environ Anal Chem 87:1079-1093.
Ellin RI, Groff WA and Kaminskis A (1981) The stability of sarin and soman in dilute
aqueous solutions and the catalytic effect of acetate ion. Journal of environmental
science and health Part B, Pesticides, food contaminants, and agricultural wastes
16:713-717.
Ellman GL, Courtney KD, Andres V, Jr. and Feather-Stone RM (1961) A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol
7
:88-95.
EPA (2006) Reregistration eligibility decision for diazinon. US Environmental Protection
Agency Office of Pesticide Programs EPA 738-R-04-006:23.
Epstein J (1974) Properties of GB in water. J Am Water Works Assoc 66:31-37.
Faust SD and Gomaa HM (1972) Chemical hydrolysis of some organic phosphorus and
carbamate pesticides in aquatic environments. Environmental letters 3:171-201.
Freed VH, Chiou CT and Schmedding DW (1979) Degradation of selected organophosphate
pesticides in water and soil. J Agric Food Chem 27:706-708.
Ginjaar L and Vel S (1958) Reactivity of organophosphorous compounds. I. Alkaline
hydrolysis of dialkyl p-nitrophenyl phosphates. Recueil des Travaux Chimiques des
Pays Bas 77:929-956.
Golomb BA (2008) Acetylcholinesterase inhibitors and Gulf War illnesses. Proc Natl Acad
Sci U S A 105:4295-4300.
Gomaa HM and Faust SD (1972) Chemical hydrolysis and oxidationof parathion and
paraoxon in aquatic environments. Fate of Organic Pesticides in the Aquatic
Environment, Advances in Chemistry Series 111:189-209.
Gomaa HM, Suffet IH and Faust SD (1969) Kinetics of hydrolysis of diazinon and diazoxon.
Residue reviews 29:171-190.
Grechko AV, Nabolotnaya OM and Marchenko PV (1983) The hydrolysis of dichlorvos.
Soviet journal of water chemistry and technology (Khimiya i Tekhnologiya Vody)
5
:164-165; translated 187-189.
Heilmair R, Eyer F and Eyer P (2008) Enzyme-based assay for quantification of chlorpyrifos
oxon in human plasma. Toxicol Lett 181:19-24.
Jamal GA, Hansen S and Julu PO (2002) Low level exposures to organophosphorus esters
may cause neurotoxicity. Toxicology 181-182:23-33.
Jokanovic M (2018) Neurotoxic effects of organophosphorus pesticides and possible
association with neurodegenerative diseases in man: A review. Toxicology 410:125-
131.
19

Kamel F, Engel LS, Gladen BC, Hoppin JA, Alavanja MC and Sandler DP (2007) Neurologic
symptoms in licensed pesticide applicators in the Agricultural Health Study. Human
& experimental toxicology 26:243-250.
Lartiges SB and Garrigues PP (1995) Degradation Kinetics of Organophosphorus and
Organonitrogen Pesticides in Different Waters under Various Environmental
Conditions. Environ Sci Technol 29:1246-1254.
Lim LO (1996) Dichlorvos Risk Characterization Document. California Environmental
Protection Agency:8.
Liu B, McConnell LL and Torrents A (2001) Hydrolysis of chlorpyrifos in natural waters of
the Chesapeake Bay. Chemosphere 44:1315-1323.
Lu J, Wu L, Newman J, Faber B and Gan J (2006) Degradation of pesticides in nursery
recycling pond waters. J Agric Food Chem 54:2658-2663.
Macalady DL and Wolfe NL (1983) New perspectives on the hydrolytic degradation of the
organophosphorothioate insecticide chlorpyrifos. J Agric Food Chem 31:1139-1147.
Mansour M, Feicht EA, Behechti A, Schramm KW and Kettrup A (1999) Determination
photostability of selected agrochemicals in water and soil. Chemosphere 39:575-585.
Meikle RW and Youngson CR (1978) The hydrolysis rate of chlorpyrifos, O-O-diethyl O-
(3,5,6-trichloro-2-pyridyl) phosphorothioate, and its dimethyl analog, chlorpyrifos-
methyl, in dilute aqueous solution. Arch Environ Contam Toxicol 7:13-22.
National.Research.Council.US (1999) Review of the US Army's Health Risk Assessments for
oral exposure to six chemical-warfare agents. National Academies Press (US)
Washington, DC
.
Naughton SX and Terry AV, Jr. (2018) Neurotoxicity in acute and repeated
organophosphate exposure. Toxicology 408:101-112.
Noblet JA, Smith LA and Suffet IH (1996) Influence of natural dissolved organic matter,
temperature, and mixing on the abiotic hydrolysis of triazine and organophosphate
pesticides. J Agric Food Chem 44:3685-3693.
Schmidt C, Breyer F, Blum MM, Thiermann H, Worek F and John H (2014) V-type nerve
agents phosphonylate ubiquitin at biologically relevant lysine residues and induce
intramolecular cyclization by an isopeptide bond. Anal Bioanal Chem 406:5171-
5185.
Schopfer LM and Lockridge O (2018) Chlorpyrifos oxon promotes tubulin aggregation via
isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications
for neurotoxicity. The Journal of biological chemistry 293:13566-13577.
Schopfer LM and Lockridge O (2019) Mass Spectrometry Identifies Isopeptide Cross-Links
Promoted by Diethylphosphorylated Lysine in Proteins Treated with Chlorpyrifos
Oxon. Chem Res Toxicol 32:762-772.
Schopfer LM, Lockridge O, David E and Hinrichs SH (2019) Purification of human
butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin
affinity chromatography. PLoS One 14:e0209795.
Sharom MS, Miles JRW, Harris CR and McEwen FL (1980) Persistence of 12 insecticides in
water. Water Research 14:1089-1093.
Smolen JM and Stone AT (1997) Divalent metal ion-catalyzed hydrolysis of
phosphorothionate ester pesticides and their corresponding oxonates. Environ Sci
Technol 31:1664-1673.
20

Sogorb MA, Vilanova E and Diaz-Alejo N (1993) The kinetics of O-hexyl O-2,5-
dichlorophenyl phosphoramidate hydrolysing activity in hen plasma. Chem Biol
Interact 87:117-125.
Solomon KR, Williams WM, Mackay D, Purdy J, Giddings JM and Giesy JP (2014) Properties
and uses of chlorpyrifos in the United States. Reviews of environmental
contamination and toxicology 231:13-34.
Thomas C and Mansingh A (2002) Dissipation of chlorpyrifos from tap, river and brackish
waters in glass aquaria. Environmental technology 23:1219-1227.
Tomlin CDS (1997) (ed) The Pesticide Manual. The British Crop Protection Council, 11th ed
Surrey, UK
.
Wang A, Cockburn M, Ly TT, Bronstein JM and Ritz B (2014) The association between
ambient exposure to organophosphates and Parkinson's disease risk. Occup Environ
Med 71:275-281.
21

Highlights
•
•
•
Chlorpyrifos oxon has a half-life of 21 days in pH 8 buffer at 23˚C
Chlorpyrifos oxon has a half-life of 7 days in pH 9 buffer at 23˚C
Nerve agents and pesticides are degraded within minutes in sodium hydroxide

Declaration of interests
X The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: