Looking for evidence of chemical attack

The use of chemical weapons in a conflict is often difficult to prove. TNO is working on analytical methods that can determine exactly what substances were used - and even by whom - up to months later.In defiance of all kinds of international treaties, the use of toxic substances as weapons by parties in military conflicts or terrorist violence is by no means out of the question. In Ukraine alone, more than nine thousand chemical weapons have been used in recent years, the intelligence services AIVD and MIVD reported last year. Chemical weapons were also frequently used in the Syrian civil war, says Mirjam de Bruin-Hoegée, a chemist and forensic researcher at TNO. 'When the use of chemical weapons is suspected, forensic research is needed to determine whether that is true and what chemicals would be involved.' That could be anything from nerve gases, blister agents and synthetic opioids to compounds containing chlorine.Within TNO's Chemical, Biological, Radiological and Nuclear (CBRN) Protection Department, where research includes protection against chemical and biological threats, De Bruin-Hoegée is developing new analytical techniques for the detection and identification of chemical weapons and their possible origin. This mainly involves retrospective validation in the laboratory. Her colleague Jelle de Koning focuses on detection with small, mobile devices(see Rapid test for detection of chemical weapons). He is developing a sampling and measurement system to measure substances directly in the field. Suspicious samples can then go on to the lab for further verification.BiomarkersTNO is the only lab in the Netherlands allowed to produce chemical warfare agents in small quantities for research. The research institute works on behalf of the Organisation for the Prohibition of Chemical Weapons (OPCW), which also promotes the development of new analytical methods. Together with some 30 more labs worldwide, TNO may be asked to conduct research after a possible attack. Who the samples are placed with and what the results are is not known: only the OPCW communicates about it. 'So we actually never talk about what we do or don't do. With one exception in 2013. Then we were allowed to publish about research in Syria where the nerve agent sarin had been used. 'Methods to demonstrate these kinds of substances, we are constantly trying to improve,' says De Bruin-Hoegée.In an attack, the toxic substance is spread over a large area. When forensic investigators are on the scene - looking for traces too soon after an attack is not safe - the reactive substance has often already broken down in the environment or been converted in the body. With analysis techniques that the TNO researchers have now developed, very low concentrations of specific biomarkers can still be found in blood. These might be substances in the body formed by a reaction of the pesticide with specific proteins or DNA. People exposed to these substances are therefore an important source of evidence. But biomarkers can also be found in plants in the war zone and even in concrete.Finger prickAdvanced laboratory equipment can be used to determine in blood the exact substance to which a person has been exposed. Nerve gases, for example, react in blood with a specific piece of the enzyme acetylcholinesterase, crucial for the breakdown of neurotransmitters in the nervous system. When this enzyme stops working, it results in overstimulated muscles, muscle twitching, fluid accumulation and eventually paralysis of the respiratory muscles. In war zones, it is difficult to transfer a tube of blood cooled and stable to the lab. But De Bruin-Hoegée saw an alternative. 'In young babies, blood is taken via a heel prick. Droplets of blood appear on a filter paper and then dry out. We wanted to test that idea for our application, but via a finger prick.'Filter papers with dried blood droplets. Photo: TNO She left the filter papers containing dried blood drops, about 50 microlitres, for weeks, meanwhile measuring the biomarkers at different times. 'Even a month later, we still found the biomarker. The stability of such a sample has many advantages. Apart from the fact that the blood samples no longer need to be refrigerated, no doctor is needed to take a tube.'Earlier, TNO developed a mobile device that can hold a drop of blood to instantly determine whether someone has been exposed to a nerve agent. 'Then someone can then immediately administer an antibody, via one of those pens you put in a leg. That works well and quickly. In the lab, we can then use the dried blood spots to determine which nerve agent is involved: sarin, novichok or something else.'Like De Koning, De Bruin-Hoegée is trying to bring the techniques she is developing closer to the field, to be able to measure quickly close to the front line. Then the right protective measures can be taken right away. 'For example, with a simple test where you can already see what is going on with a bit of blood or saliva. That is not there yet for nerve gas and chlorine compounds.'Setup in a fume hood in the lab to expose a plant to chemical warfare agents, in this case sarin. The plant is placed in a glass container with inlet and outlet tubes for administering chemical warfare agents, such as chlorine gas, nerve gas, and mustard gas, in various concentrations. At low concentrations, nothing is visible and the plant continues to thrive. “At higher concentrations, it did turn a bit black; with chlorine, it developed yellow spots,” says Mirjam de Bruin-Hoegée. Photos: TNOBasil and bay leafChemical combatants also react with proteins in plants. 'We investigated this for the first time a few years ago,' says De Bruin-Hoegée. This soon proved promising. The reaction of a toxic substance with plant protein yields a stable biomarker that, three months later, is still found in the living plant, as well as in dried leaves. This is rubisco, which is essential for photosynthesis.For her research, she chose plants found everywhere, such as nettles, but also basil, bay leaf and grass. 'Each time, we managed to find stable biomarkers. That was reassuring.'In the lab, exposure to chemical warfare agents is easy to mimic. 'In humans, of course, this is not possible, there we worked with blood from, for example, blood bank Sanquin.'The plant's leaves are pulverized and the substances extracted. After processing—which includes breaking down large proteins into smaller pieces—the material is ready for the analytical equipment. This is halfway through the process: the fresher the plant, the greener the color. Photo: TNO Chlorine gasTo extract the proteins from the plant, De Bruin-Hoegée grinds the plant finely. The protein molecules are then still intact and quite large. 'Small protein pieces we can measure better with our liquid chromatograph and mass spectrometer than large intact proteins. Therefore, we first cut the proteins into pieces by adding enzymes, just as happens in your stomach after you eat something.'The biomarkers found are the result of the reaction between the chemical agent and the plant itself and therefore very specific. This makes it possible to distinguish whether it is nerve gas, mustard gas or chlorine gas. 'With chlorine gas, we found indicators that also occur when you throw household bleach over a plant. So in a follow-up study, we looked at whether specific markers could be found for chlorine gas, which you don't find with bleach. We bought all kinds of bleach, from those jerry cans you throw in the swimming pool to household bleach. That yielded interesting results. Bleach is alkaline and chlorine gas is more like acid, and they react differently.'Four types of concrete from Europe and the Middle East are exposed to chlorine gas. Photo: TNO ConcreteMeanwhile, the protocol for detecting the use of chemical pesticides via plants has been adopted by laboratories affiliated to the OPCW. A new standard method is thus a reality. 'Great that it can now be used more widely for forensic investigations. That is the beauty of this work. When it comes down to it, we can make a difference in detection and legal processing.'In the middle of the city, there are fewer plants and weeds, but there is concrete everywhere. The researchers are now working with laboratories in Finland and Sweden, among others, to see if markers can be detected in it. Recent initial tests showed that substances in concrete, such as acetone or phenols, react with chlorine gas. 'We also saw a reaction with tin oxide, a mineral found in rocks that can serve as a raw material for concrete. Colleagues in Sweden discovered that lignin, woody plant residues found as impurities in concrete, react specifically with chlorine gas.'Chemical fingerprintMore forensic information can be extracted from the measurement data. For example, in which factory and how the chemical was made, and from which raw materials. For example, such a substance consists of 90 per cent poison and 10 per cent other substances, such as contamination from the production process or residues from one of the raw materials. These give a lot of information about how and possibly where the poison was made. Thus, every chemical weapon has its own fingerprint.'The easiest way is if you have samples from a crime scene and from a suspected production facility, so you can match them. But that is usually not the case. Still, chemical composition in itself can also give information,' says De Bruin-Hoegée. This has been reported, for example, by the OPCW, which identified the terror group IS as a possible perpetrator of an attack, because the composition appeared 'amateurish' and very different from that used in previous attacks by the Syrian government.King adds: 'It is also possible to tell from the purity of the by-products whether it was professionally done or not. That already provides a lot of forensic information.'Offender profilesA chemical weapon includes not only the toxic substance but also the means by which it is distributed. 'These too are regulated and therefore trackable,' says De Bruin-Hoegée. Think plastic from a drone, for example, or an improvised explosive device made of PVC pipes with some tape and electrical wires. 'From a small piece of plastic it is already possible to extract forensic information, such as where the parts were bought.'Researchers went to buy hundreds of materials from DIY stores, from jerry cans and PVC pipes to electrical wires and tape to determine their element composition - i.e. how much zinc, copper and iron is in them and in what concentration. For example, has a catalyst containing zinc been used? What inorganic substance was used for the colour white? This is not the composition of the plastic, but any residue from the production process that can still be found. This varies from brand to brand. This data is used to build a database at the Netherlands Forensic Institute (NFI) and the National Institute of Standards and Technology (NIST) in the US, partners in the project. 'At the moment, the database is widely used by the NFI for the provenance of glass. For example, when a window has been smashed by a burglar and they later find some glass on the suspect's clothes, if those profiles match, it can be used as evidence. The idea now is to set this up further for plastics.'Opening photo: TNO researcher Mirjam de Bruin-Hoegée at a liquid chromatograph, which serves to separate substances in processed plant extracts, for example, before going into the mass spectrometer. Image: TNO

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