Chlorine (Cl₂) is an extremely toxic gas that not only is difficult to detect, it is dangerous to human life in both instances of accidental industrial releases and its intentional use as a chemical weapon. In such cases of alleged intentional use of chlorine gas as a chemical warfare agent (CWA), it is difficult to detect in the environment because it rapidly decomposes and dissipates. The lack of forensic approaches aimed at confirming chlorine gas releases in the environment leaves a major gap in operations seeking to prohibit its use as a CWA. Identification of chlorinated biomarkers generated by exposure to chlorine gas in vegetative tissues commonly found in the environment addresses this gap directly.

In this study, extractions of both protein and low molecular weight molecules from Cl₂ exposed vegetative tissue were used identify and characterize chlorine gas-associated biomarkers by mass spectrometric analysis. A. tauschii plants were grown in the lab and exposed to chlorine gas for variable periods of time and percentages of chlorine gas. Exposed tissues were photographed, then processed by a protein and small molecule coextraction procedure that was optimized and developed to provide quality fractions for analytical mass spectrometric analysis. Using a combination of gas chromatography- and high-resolution liquid chromatography-based mass spectrometry, chlorinated molecules generated during chlorine gas exposure were identified. Relative abundances were compared across the different samples as a function of exposure time and percentage of chlorine gas.

Chlorinated compounds were identified from vegetative tissue exposed to Cl₂ and confirmed using commercial reference materials. Additionally, a chlorinated peptide from the ubiquitous protein ribulose-1,5-bisphosphate carboxylase/oxygenase (common to all green plant tissue) was also identified and confirmed with a synthetic peptide standard. Results from this study establish that vegetative tissues can serve as a source of small molecule and protein biomarkers in investigations aimed at confirming chlorine gas release in the environment. Further analysis of these biomarkers including their persistence over time after exposure event, limit of detection, and applicability to other plant species would further establish the methodology and its reliability to confirm exposures to chlorine gas in the environment.

Mark Dreyer

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