Cutting the mustard: novel detection of Chemical Warfare Agents

Successful detection of chemical warfare agents is a difficult but vital task. Scientists at Dstl Porton Down have developed a new and unlikely tool in the fight against these abhorrent weapons Chemical warfare is the use of toxic chemicals in conflict. Chemical warfare agents (CWAs) have been condemned as abhorrent since their first modern use during World War 1. Their utilisation was prolific on both sides with the French first deploying the tear gases ethyl bromoacetate and chloroacetone and the Germans the irritants methylbenzyl bromide and dianisidine chlorosulfonate. A harrowing example of full-scale deployment of a chemical weapon occurred during the Second Battle of Ypres when German troops released chlorine gas on French, Canadian and Algerian troops, resulting in mass casualties. Most recently chemical weapons have been used, with detrimental effects, by Iraq during the Iran-Iraq War, by the Aum Shinrikyo cult in Matsumoto in 1994 and on the Tokyo Underground in 1995, and in the Syrian Arab Republic in Ghouta in 2013. Nerve agents are a particular class of CWAs that are extremely toxic and act by inhibiting the conduction of signals across the nerve synapse causing death. VX, an especially toxic liquid organophosphorus nerve agent, has been shown to be fatal when an amount equivalent in weight to about 10 sugar crystals (~5 mg) enters the skin. Due to these devastating characteristics intensive international diplomacy has sought to eliminate this class of weapons of mass destruction. The Organisation for the Prohibition of Chemical Weapons (OPCW), an international body based in The Hague, monitors the implementation of the Chemical Weapons Convention (CWC) by Member States (190 at present). The CWC prohibits the development, production, acquisition, stockpiling, retention and transfer or use of chemical weapons by its Member States. Proof of compliance and investigations of use, an important component of the CWC, with Member States subject to a challenge inspection at any time if called for by another member state, requires the accurate detection of CWAs and their degradation products. The OPCW designates a series of national laboratories globally that have the ability to detect CWAs, their precursors and degradation products, at concentrations down to parts per billion. The Defence Science and Technology Laboratory (Dstl) houses the UK Designated Laboratory. It has maintained this designation since the CWC entered into force in 1997 – one of only five laboratories in the world to do so – and has received the maximum ‘A grade’ in seven consecutive proficiency tests organised by the OPCW. The importance and far reaching social impact of the work of the OPCW was recognised by the award of the Nobel Peace Prize in 2013 to the organisation. Gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS), are the main techniques for the identification of nerve agents and related compounds, due to the requisite combination of high selectivity and sensitivity. Current applications of these technologies for detection of CWAs in the environment rely upon the analysis of soil samples and debris suspected of contamination. Successful detection depends upon the sampling strategy (where in a large field of soil is it best to sample?) and efficient extraction and sample preparation (soil organic matter provides a complex matrix from which it is difficult to extract nerve agents). Due to these reasons, in collaboration with my colleagues, Matthew Gravett and Christopher Timperley from Dstl Porton Down, in Wiltshire UK, we investigated the ability of plant material to absorb and store nerve agents, which would hopefully be amenable to a simple extraction procedure. Over the course of two studies^1,2, the first of their kind, we contaminated white mustard seed, Sinapis alba, and cultivated the resulting plants in different soil types and over different time periods to develop a new detection capability. Our procedure used 1 ml of a 250 ?g ml^-1 aqueous solution of VX applied to the seed. The seeds were grown and watered with 10 ml of local borehole water daily out to 45 days. White mustard was chosen as the study plant as it grows cosmopolitically and tolerates a wide variety of natural growth conditions. At set times, plants where harvested, pulverised by pestle and mortar with aliquots of ethanol, and analysed by GC-MS and LC-MS. This simple extraction procedure is an important step which allows the deployment of our plant detection methodology in the field. As VX degrades in the environment it follows a hydrolysis pathway, first hydrolysing to ethyl methylphosphonic acid (EMPA) and then to methylphosphonic acid (MPA). Detection of intact CWA and degradation products is considered evidence of CWA use and as such our detection targets were VX, EMPA and MPA. Our results show that vegetation absorbs intact nerve agents and their degradation products from complex environmental matrices and acts as a time capsule whose interrogation can reveal nerve agent use. We were able to detect intact VX and its degradation products after 45 days from initial contamination, using plant pulverisation and ethanol extraction. Importantly this was possible by working up plants grown in three different types of soil: sand, loam, and clay. This confirmed that soil type does not impact upon the detection of VX using plant material. Interestingly, when analysing soil samples, it was not possible to detect the degradation products EMPA and MPA. VX was detectable but consistent with other studies its soil concentration decreased dramatically after day nine, as in the environment VX hydrolysis, once started, occurs autocatalytically. Thus, the study provided a technique that produced evidence of CWA use that was not possible via standard procedures. A potential major benefit of this research is the ability of plant material to act as a phytoremediation technique for contaminated land, enabling the return of this land for arable usage. Our method for detecting nerve agent residues in the environment will help the OPCW and CWC member states confirm their presence in future. Localisation of such residues in plants and a simple extraction procedure will increase the probability of discovering nerve agent use. The ability of plants such as S. alba to absorb nerve agents and marker compounds protects against the deliberate removal of evidence of use, as CWAs can leach from soil over time. Matthew Gravett, a senior scientist at Dstl Porton Down, states: “The simple extraction procedure combined with the storage of the nerve agent evidence by the plants, which shields it from environmental weathering, vastly improves the ability to discover nerve agent use, and also crucially deters the use of CWAs in future.” References

1. Gravett MR, Hopkins FB, Main MJ, Self AJ, Timperley CM, Webb AJ, Baker MJ, Detection of the organophosphorus nerve agent VX and its hydrolysis products in white mustard plants grown in contaminated soil, Anal. Methods, 2013, 5(1), 50.
2. Gravett MR, Hopkins FB, Self AJ, Webb AJ, Timperley CM, Baker MJ, Evidence of VX nerve agent use from contaminated white mustard plants, Proc. Roy. Soc. A, 2014, 470, 20140076, published open access on 21 May 2014.

Author Matthew J. Baker, a former Research Fellow at Dstl Porton Down, conducted studies on the detection of chemical and biological warfare agents, before moving to the University of Central Lancashire (UCLan), to lead its Bioanalytical Sciences Research Group. He has recently been appointed as Senior Lecturer in Chemistry in the Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow, Scotland.