The reactions of chemical warfare agent simulants, 2-chloroethyl ethyl sulfide (2-CEES) and di-
Use of chemical and biological warfare agents (CWAs and BWAs) in conventional or terrorism related incidents is not new [
The handling and transport of contaminated human remains is an important consideration of both combative and humanitarian military actions. Current protocol has remained largely unchanged for decades, although significant advances in decontamination and incident response have been made. Handlers of contaminated and potentially contaminated human remains face significant biological and chemical threats [
In recognition of this need, NanoScale Corporation (NanoScale), in collaboration with Kappler, Inc., has recently produced a human remains pouch which was specifically designed to allow for safe handling and transport of contaminated human remains (
The objective of this study was thus twofold: (1) to develop highly efficient, dually active (against CWAs and BWAs) solid decontaminants based on nanocrystalline metal oxides, and (2) to develop reliable analytical methods and generate data to quantify the residual CWA simulants and degradation products that may be present in contaminated human remains. Specifically, we have investigated the degradation of CWA simulants in Gamble’s fluid, a synthetic equivalent of physiological fluid, with and without the inclusion of novel dually active solid decontaminants. Over the past several years, NanoScale has developed a series of highly reactive nanocrystalline metal oxides and their derivatives, which have been shown to be effective in destructive adsorption of a number of toxic compounds, including the CWAs, soman (GD), VX, and mustard (HD) [
Decomposition of nerve/blister agents on metal oxide surfaces.
Herein, efforts to evaluate the fate of CWA simulants di-
Deuterium oxide (99.9% D) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Deuterated chloroform (99.8% D) was purchased from ACROS Organics (Morris Plains, NJ, USA). 2-CEES (98% pure), ethyl vinyl sulfide (EVS, 96% pure), and DFP were purchased from Aldrich (St. Louis, MO, USA). 2-(ethylthio) ethanol (96% pure), also known as hydroxylethyl ethyl sulfide (HEES), was purchased from Alfa Aesar (Ward Hill, MA, USA). Isopropanol (>99% pure) was purchased from Barton Solvent, Inc. (Des Moines, IA, USA). Sodium chloride (ACS grade), ammonium chloride (ACS grade), sodium citrate tribasic (ACS grade), sodium bicarbonate (ACS grade), glycine (ACS grade), calcium chloride (ACS grade), and sodium phosphate dibasic (ACS grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Titanium (IV) oxide (certified grade, FS-TiO2) and diethyl ether (lab grade) were purchased from Fisher (Pittsburg, PA, USA). NanoActive TiO2 (NA-TiO2) and NanoActive® CaO (NA-CaO) were manufactured by NanoScale Corporation (Manhattan, KS, USA). All chemicals were used as received with no further purification.
Three silver based metal oxide materials were prepared and assessed for their CWA simulant decontamination ability. These materials are referred to as Exceptional Hazard Attenuation Materials (EHAMs), designated EHAM 1, EHAM 2, and EHAM 4. EHAM 1 (SSA = 264 m2/g) was based on Ag-impregnated NanoActive TiO2 ([Ag]NA-TiO2). EHAM 2 (SSA = 213 m2/g) contains [Ag]NA-TiO2 and [Ag]NA-CaO. EHAM 4 (SSA = 88 m2/g) contains [Ag]FS-TiO2, NA-CaO and NA-TiO2.
Saline solutions were prepared at a concentration of 0.9% (w/w). Gamble’s fluid was prepared according to the published literature [
The fates of the individual simulants, 2-CEES and DFP, in fluids were investigated by measuring pH of the solutions as a function of time, as likely reaction products were expected to increase the acidity of a given simulant solution. Thus, pH measurements were made on aqueous, 0.9% saline, and Gamble’s fluid solutions of 2-CEES and DFP with a Fisher Scientific, accumet AB15 pH meter. In a representative procedure, 20
GC analysis was conducted to determine the fate of the simulant in fluids with and without any EHAM present. A Hewlett Packard 5890 Series II GC equipped with a Hewlett Packard 5972 Mass Selective Detector and a HP-5MS capillary column was used for sample analysis. Since 2-CEES and HEES coeluted on this column, quantitation of 2-CEES and HEES was performed using a Hewlett Packard 5890 Series II GC equipped with a Flame Photometric Detector utilizing a 5% SE-30 ON CW-HP 80/100 packed column. In a typical experiment, 5
FTIR spectra of the powder samples were recorded in the absorbance mode using a Thermo Scientific Nicolet 6700 spectrophotometer. The FTIR unit was equipped with a HeNe laser source, and an MCT detector cooled with liquid nitrogen. A Praying Mantis Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFTS) unit with purging nitrogen was used for the sample analysis at ambient temperature. All spectra were collected by coadding 1032 scans at a resolution of 4 cm−1. The spectra were recorded from 4000 to 500 cm−1.
Due to the instrumentation detection limit, a higher agent concentration was needed to perform the DRIFTS experiments. As a result, all of the FTIR related samples were prepared in duplicate with a 2-CEES to sorbent (EHAM 1, EHAM 2, or EHAM 4) ratio of 1 : 10 (wt : wt). In a typical reaction, 9.3
Solution NMR experiments were performed at Kansas State University, Manhattan, KS, USA, using a11.75 T (500 MHz for 1H) Varian UNITY
1H NMR spectra were acquired at room temperature, using a spectral width of 6010 Hz over 8 K data points. A relaxation delay of 1 s was employed for a 45 degree flip angle. Four scans were coadded for each sample and Fourier-transformed with a line-broadening factor of 0.5 Hz to improve the S/N ratio. Chemical shifts were referenced against the solvent peak (7.24 ppm for CDCl3 and 4.8 ppm for HOD). Relative amounts of various products formed were estimated from corresponding methyl peak intensities expressed as percentages of the total intensity of all methyl peaks in a spectrum. This permitted comparison of NMR data as a function of time not only for a given sample, but also between samples to obtain semiquantitative estimates of reaction rates.
Solid state proton-decoupled 31P NMR spectra were obtained at Iowa State University, Ames, IA, USA, with a Bruker AVANCE II 14.1 T (242.9 MHz for 31P) equipped with a Bruker PH MASDVT600WB H/X/Y probe under the condition of magic angle spinning (MAS) at a rate of 5–10 KHz, using a spectral width of 73529 Hz over 4 k time domain points; a relaxation delay of 10 s was used for a 90 degree pulse; 32 scans were collected for each sample, a line-broadening factor of 20 Hz was employed to improve the S/N ratio. Chemical shifts are reported relative to the external standard, phosphoric acid, which is assigned a value of 0 ppm. Typically, 20
pH values of individual solutions of 2-CEES and DFP in water, 0.9% saline solution, and Gamble’s fluid were measured after 10 min, 1 h, 96 h, and 8 days of preparation. The results (Figure
pH of simulant-containing fluids at various time periods.
Agent-contacted fluid and sorbent were extracted with ethyl ether after 1, 24, and 96 h of preparation and analyzed by gas chromatography. Results were analyzed in terms of all the possible 2-CEES and DFP degradation products, as summarized in Schemes
Relative (to external 2-CEES control) amounts of extractable products from various fluids.
Fluid | Contact time (hours) | 2-CEES | HEES | DEDS | NEES | EVS | BEE | Trimer |
---|---|---|---|---|---|---|---|---|
1 | 2.1 | 13 | ≤0.24 |
|
||||
Water | 24 | 0.91 | 42 | BDL |
|
|
||
96 | 1.1 | 84 | BDL | BDL | ||||
| ||||||||
1 | 5.6 | 26 | ≤0.14 | 5.6 | ||||
Saline | 24 | 1.9 | 37 | BDL | BDL |
|
|
|
96 | 3.5 | 73 | BDL | |||||
| ||||||||
1 | 2.7 | 14 | ≤0.32 | |||||
Gamble’s | 24 | 1.9 | 28 | BDL | BDL |
|
|
|
96 | 7.1 | 78 | BDL |
Relative (to external 2-CEES control) amounts of extractable products from Gamble’s fluid in the presence of EHAM.
EHAM | Contact time (hours) | 2-CEES | HEES | DEDS | NEES | EVS | BEE | Trimer |
---|---|---|---|---|---|---|---|---|
1 | 28 | 1.1 | ||||||
1 | 24 |
|
1.4 |
|
|
BDL | ≤0.0007 | ≤0.79 |
96 | 1.5 | |||||||
| ||||||||
1 | 28 | |||||||
2 | 24 |
|
18 | BDL | BDL | ≤1.3 | ≤1.9 | ≤0.09 |
96 | 60 | |||||||
| ||||||||
1 | 34 | |||||||
4 | 24 |
|
62 | BDL | BDL | ≤0.47 | ≤0.40 | ≤0.09 |
96 | 64 |
Degradation products of 2-CEES.
Degradation products of DFP.
Similarly, the fluids and sorbent reacted with DFP within the first hour of contact and the amount of DFP present continued to decrease during the 96 h period (Figures
(a) Fate of DFP in fluids, (b) fate of DFP in Gamble’s fluid with EHAMs.
The FTIR spectrum of neat 2-CEES is shown in Figure
FTIR spectrum of 2-CEES.
Subtracted FTIR spectra of EHAM 1 and 2-CEES.
In contrast to EHAM 1, EHAM 2-treated samples (Figure
Subtracted FTIR spectra of EHAM 2 and 2-CEES.
Subtracted FTIR spectra of EHAM 4 and 2-CEES.
An assigned 1H NMR spectrum of 2-CEES in CDCl3 is shown in Figure
Comparison of methyl-peak intensities of 2-CEES reaction products.
Relative intensity percentage | |||||||||
---|---|---|---|---|---|---|---|---|---|
Peak | D2O | Saline | Gamble’s | ||||||
1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | |
b | 13 | 11 | 28 | 16 | 10 | 34 | 25 | 34 | 61 |
a | 40 | 45 | 34 | 35 | 43 | 35 | 33 | 33 | 18 |
c | 34 | 40 | 38 | 37 | 42 | 41 | 28 | 33 | 22 |
d | 13 | 4 | 0 | 13 | 5 | 0 | 15 | 0 | 0 |
| |||||||||
Assignment | HEES (b) | 2-CEES (a) | Dimer and/or trimer, (c and d) |
1H NMR spectrum of 2-CEES in CDCl3.
1H NMR spectra of 2-CEES in Gamble’s fluid.
1H NMR spectra of 2-CEES in fluids in the presence of EHAMs are shown in Figures
Comparison of methyl-peak intensities of 2-CEES reaction products.
Relative intensity percentage | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Peak | Gamble’s | EHAM 1 | EHAM 2 | EHAM 4 | ||||||||
1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | |
b | 25 | 34 | 61 | 52 | 50 | 50 | 55 | 66 | 56 | |||
a | 33 | 33 | 18 | 9 | 10 | 13 | 10 | 14 | 11 | |||
e | 0 | 0 | 0 | 100 | 100 | 100 | 0 | 0 | 0 | 0 | 0 | 0 |
c | 28 | 33 | 22 | 18 | 20 | 13 | 17 | 6 | 11 | |||
d | 15 | 0 | 0 | 21 | 20 | 24 | 19 | 14 | 22 | |||
| ||||||||||||
Assignment | HEES (b) | 2-CEES (a) | NEES (e) | Dimer and/or trimer (c and d) |
1H NMR spectra of 2-CEES in Gamble’s fluid with (a) EHAM 1, (b) EHAM 2, and (c) EHAM 4.
1H NMR spectra of DFP in CDCl3, D2O, saline, and Gamble’s fluid are shown in Figures
Comparison of methyl-peak intensities of DFP reaction products.
Relative intensity percentage | |||||||||
---|---|---|---|---|---|---|---|---|---|
Peak | D2O | Saline | Gamble’s | ||||||
1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | |
g | 0.9 | 4 | 5 | 1 | 4 | 4 | 0.7 | 4.4 | 11 |
h | 2 | 18 | 5 | 2 | 25 | 0 | 2.2 | 15 | 20 |
i | 1 | 3 | 89 | 1 | 4 | 94 | 0.7 | 4.2 | 11 |
j | 2 | 2 | 1.5 | 2 | 2 | 1 | 1.8 | 1.8 | 1.8 |
f | 94 | 73 | 0 | 94 | 64 | 0.5 | 94 | 74 | 56 |
| |||||||||
Assignment | IPA (g) | TIPPP (h) | DIPP (i) | IPP (j) | DFP (f) |
1H NMR spectra of DFP in (a) CDCl3, (b) D2O, (c) saline, and (d) Gamble’s fluid.
Time-dependent 1H NMR spectra of DFP in Gamble’s fluid containing EHAMs 1, 2, and 4 are displayed in Figures
Comparison of methyl-peak intensities of DFP reaction products.
Relative intensity percentage | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
peak | Gamble’s | EHAM 1 | EHAM 2 | EHAM 4 | ||||||||
1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | 1 h | 24 h | 96 h | |
g | 0.7 | 4.4 | 11 | 2 | 15 | 24 | 0.8 | 1.6 | 2 | 6 | 7 | 1 |
h | 2.2 | 15 | 20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
i | 0.7 | 4.2 | 11 | 3 | 51 | 65 | 97 | 96 | 96 | 93 | 91 | 96 |
j | 1.8 | 1.8 | 1.8 | 3 | 11 | 12 | 2 | 2 | 2 | 1 | 1 | 2 |
f | 94 | 74 | 56 | 92 | 22 | 0 | 0.8 | 0.8 | 0.9 | 0.5 | 0.7 | 1 |
| ||||||||||||
Assignment | IPA (g) | TIPPP (h) | DIPP (i) | IPP (j) | DFP (f) |
1H NMR spectra of DFP in Gamble’s fluid with (a) EHAM 1, (b) EHAM 2, and (c) EHAM 4.
A reference proton-decoupled solid state 31P NMR of DFP was collected by adding DFP to low surface area conventional TiO2 (FS-TiO2, SSA = 11 m2/g, shown as DFP control in Figure
31P MAS NMR spectra of DFP with (a) EHAM 1, (b) EHAM 2, and (c) EHAM 4.
Changes in pH of the simulant-contacted fluids (water, saline, and Gamble’s fluid) indicate the occurrence of a reaction or reactions that produce acidic compounds. These changes were observed for both 2-CEES and DFP. Gamble’s fluid slows down the rate of drop in pH, likely due to the buffering effect of salts and amino acids present in the solution.
As seen from Table
When experiments were performed using EHAMs in Gamble’s fluid (Table
As attested by GC analysis (Figure
The negative absorption feature at ~3700 cm−1 from reaction of 2-CEES and EHAM 1 (Figure
2-CEES undergoes hydrolysis to yield at least three products after 96 h. One of the products is identified as HEES, while the other two products are likely to be the two sulfonium condensation products (dimer and/or trimer in Scheme
Very similar changes are observed for 2-CEES in all the three fluids (Table
Differences in the amounts of reactions products formed, as monitored by the two methods, may arise out of degradation of the condensed products, back into 2-CEES and HEES on the GC column [
Interestingly, EHAM 1-contacted Gamble’s fluid contained only one product, namely, NEES. Additional products might have formed and been trapped on the sorbent. EHAM 2 and EHAM 4 showed similar degradation results with the amount of HEES formed being around 50% after 96 h. However, in both cases, higher levels of the condensed products were observed after 96 h, as compared to 2-CEES in Gamble’s fluid or EHAM 1. The NMR results are consistent with the GC-MS data, especially those related to extractable amounts of HEES from all the EHAMs (Tables
In the case of DFP, analysis of its degradation products in D2O, saline, and Gamble’s fluid, as deduced from relative intensities of various peaks (Table
The relative intensities of various methyl peaks in the spectra of DFP and EHAMs are shown in Table
Solid state 31P NMR spectra of DFP treated with EHAM 1 are consistent with the formation of hydrolysis products as observed in solution 1H NMR spectra. The initial spectrum of the DFP control shows a very sharp doublet and does not appear to be bound to the low surface area TiO2 support. Broadening of the two peaks after 1 h suggests binding of the compound to the EHAM surface. After 16 h, the peaks broaden further and begin to merge into a broad band at −10.8 ppm, thus indicating the cleavage of fluorine atom to yield DIPP [
Reactions of DFP with EHAMs 2 and 4 yielded similar results to EHAM 1, except that the reactions occurred much faster, as noted from solution 1 H NMR studies. Slight variations in chemical shifts are expected for the reaction products based on different metal oxide surfaces [
pH measurement of fluids is a good indicator of CWA simulant degradation, since acid by-products result from hydrolysis of these compounds. Similarly, solvent extraction of CWA simulant-contacted fluids and subsequent GC analysis provide an overview of various degradation products. For the safe handling of contaminated human remains, it is necessary to use pouches enhanced with EHAMs because contaminated body fluids are expected to contain appreciable amounts of intact agent (as seen by 2-CEES and DFP analysis) or their derivatives (2-CEES analysis). NMR analysis of simulant-contacted fluids revealed that the rate of degradation is highly fluid dependent for DFP (Gamble’s < D2O ~ saline) and 2-CEES (Gamble’s > D2O ~ saline). Inclusion of EHAMs facilitates degradation of both DFP and 2-CEES in these fluids, especially during the early hours of contamination. The toxicity of the contaminated Gamble’s fluid can be minimized by including an appropriately formulated EHAM sorbent. The ongoing threat of homicidal use of CWAs during military conflicts and by terrorists underlines the necessity for development of clear guidelines that will guarantee foolproof protection of personnel dealing with victims of these serious incidents. Early recognition and protective measures are essential when dealing with CWA-related incidents, otherwise, responding human remains handlers will only add to the list of victims.
1, 2-bis(ethylthio) ethane
Biological Warfare Agents
Deuterated choroform
Contaminated human remain pouch
Continental United States
Chemical Warfare Agents
Diethyl disulfide
Defense Threat Reduction Agency
Diisopropyl fluoro phosphonate
Diisopropyl phosphoric acid
Diffuse Reflectance Infrared Fourier Transform Spectroscopy
Exceptional Hazard Attenuation Materials
Ethyl vinyl sulfide
Fourier Transform Infrared
Fisher Scientific titanium (IV) oxide
Gas Chromatography
Soman
Mustard
Hydroxyl ethyl ethyl sulfide
Improvised Explosive Device
Isopropyl alcohol
Isopropyl phosphoric acid
Mortuary Affairs Decontamination Collection Point
Magic Angle Spinning
NanoActive CaO
NanoActive Ti
2-Nitroethyl ethyl sulfide
Phosphoric acid
Specific surface area
Tetra isopropoxy pyrophosphate
Nuclear Magnetic Resonance
Toxic Industrial Chemicals
US Army Research Office
2-Chloro ethyl ethyl sulfide.
The authors report no conflict of interests.
This paper was funded by the Defense Threat Reduction Agency (DTRA) and US Army Research Office (USARO) W911NF-09-C-0116. The authors thank Mr. Alvaro Herrera (Biomolecular NMR Facility, Kansas State University) and Dr. Shu Xu (Magnetic Resonance Facility, Iowa State University) for assistance in acquiring NMR data.