Environmental exposure to organophosphorus nerve agents

Accepted Manuscript Title: Environmental exposure to organophosphorus nerve agents Authors: Slavica Vucinic, Biljana Antonijevic, Aristidis M. Tsatsakis, Loukia Vassilopoulou, Anca Oana Docea, Alexander E. Nosyrev, Boris N. Izotov, Horst Thiermann, Nikolaos Drakoulis, Dragica Brkic PII: DOI: Reference: S1382-6689(17)30262-4 http://dx.doi.org/10.1016/j.etap.2017.09.004 ENVTOX 2877 To appear in: Environmental Toxicology and Pharmacology Received date: Revised date: Accepted date: 8-6-2017 4-9-2017 6-9-2017 Please cite this article as: Vucinic, Slavica, Antonijevic, Biljana, Tsatsakis, Aristidis M., Vassilopoulou, Loukia, Docea, Anca Oana, Nosyrev, Alexander E., Izotov, Boris N., Thiermann, Horst, Drakoulis, Nikolaos, Brkic, Dragica, Environmental exposure to organophosphorus nerve agents.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2017.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ENVIRONMENTAL EXPOSURE TO ORGANOPHOSPHORUS NERVE AGENTS Slavica Vucinic 1,*, Biljana Antonijevic 2, Aristidis M Tsatsakis 3, Loukia Vassilopoulou3, Anca Oana Docea 4,*, Alexander E. Nosyrev 5, Boris N. Izotov6 , Horst Thiermann 7, Nikolaos Drakoulis 8, Dragica Brkic 9 1 National Poison Control Centre, Military Medical Academy, Medical Faculty, University of Defense, Belgrade, Serbia, [email protected] 2 Department of Toxicology “Akademik Danilo Soldatovic”, Faculty of Pharmacy, University of Belgrade, Serbia, [email protected] 3 Research Centre, Department of Forensic Sciences of the Medical School, University of Crete, Greece, [email protected], [email protected] 4 Department of Toxicology, University of Medicine and Pharmacy, Faculty of Pharmacy, 2 Petru Rares, 200349, Craiova, Romania, [email protected] 5 Central Chemical Laboratory of Toxicology, I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation, [email protected] 6 Department of Analytical Toxicology Pharmaceutical Chemistry and Pharmacognosy, I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation, [email protected] 7 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany, [email protected] 8 Research Group of Clinical Pharmacology and Pharmacogenomics, Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Greece, [email protected] 9 Faculty of Agriculture, University of Belgrade, Institute for Phytomedicine, Serbia, [email protected] *Corresponding Author Anca Oana Docea Department of Toxicology, University of Medicine and Pharmacy, Faculty of Pharmacy, 2 Petru Rares, 200349, Craiova, Romania Email: [email protected] 1 Slavica Vucinic National Poison Control Centre, Military Medical Academy, Medical Faculty, University of Defense, Belgrade, Serbia Email: [email protected] Graphical abstract Highlights    Exposure to organophosphorus nerve agents is possible in a variety of situations Physical, chemical and toxicological properties of CWA are essential in practice Biological markers of exposure are essential for poison diagnosis and treatment Abstract Exposure to organophosphorus nerve agents, the most deadly chemical warfare agents, is possible in a variety of situations, such as destruction of chemical warfare agents, terrorist 2 attacks, armed conflicts or accidents in research laboratories and storage facilities. Hundreds of thousands of tons of chemical munitions were disposed of at the sea in the post World War II period, with European, Russian, Japanese and US coasts being the most affected. Sulfur mustard, Lewisite and nerve agents appear to be the most frequently chemical warfare agents disposed of at the sea. Addressing the overall environmental risk, it has been one of the priorities of the world community since that time. Aside from confirming exposure to nerve agents in the alleged use for forensic purposes, the detection and identification of biological markers of exposure are also needed for the diagnosis and treatment of poisoning, in addition to occupational health monitoring for specific profiles of workers. When estimating detrimental effects of acute or potential chronic sub-lethal doses of organophosphorus nerve agents, released accidentally or intentionally into the environment, it is necessary to understand the wide spectra of physical, chemical and toxicological properties of these agents, and predict their ultimate fate in environmental systems. Keywords: chemical warfare agents, biological markers, risk assessment, environmental persistence, field management and triage Introduction Bearing in mind that over 100 dumping sites in the Baltic region (Gotland Deep, Bornholm Deep, Little Belt, Skagerrak Strait) were used for depositing chemical warfare agents (CWAs) between 1945 and 1970, as well as over 60 sites in the Gulf of Mexico, the coasts of Japan and the oceans on both coasts of the USA – which has been confirmed in the CWA exposure reports by hundreds of fishermen who have caught dangerous cargo via their nets – this method of exposure is not to be underestimated. Moreover, at several places all over the world, e.g. in the US, China, and Europe, old chemical munition has been lost or buried and could be accidently released as it is shown in figure 1 (CHEMSEA 2013, Vučinić et al 2014). Figure 1 While a wide variety of chemical warfare agents including also sulfur mustard, Lewisite, have 3 been dumped or buried, this manuscript deals with problems that arise from nerve agents exposure. The aim of this review is to raise the atention of environmental exposure to NAs that could persist in different media long after initial exposure, producing a wide range of toxic effects. Better understanding of the time frame for detection of biological markers of exposure to NAs is critical for diagnosis and treatment in uncertain cases of exposure. After the first confirmed use of nerve agents (NAs) in the Iran-Iraq War (1980-1988), when the Iraqi army used tabun and sarin against the Iranian forces (Majnoon Island) and the civilian population (Halabjah) (Balali-Mood 2008), these deadly agents were used for the terrorist attacks in Japan. The terrorist attacks in Matsumoto in 1994, which claimed the lives of 7 people (Nakajima et al 1999) and Tokyo in 1995, with over 5500 injured and 13 dead (Nagao et al 1997, Murata et al 1997), showed the necessity for development of specific diagnostic methodology for identifying nerve agents, which had not existed until then (Salem et al 2008). The importance of the engagement of the Organisation for the Prohibition of Chemical Weapons (OPCW) and the chain-of-custody procedures had became obvious after the UN mission in the Syrian Arab Republic, performed by the OPCW and WHO teams which had investigated the Ghouta and other sites of attacks. Difficulties in establishing a credible epidemiological pattern through interviews with first responders, victims and medical personnel had confirmed the importance of designated laboratories (Pita and Domingo 2014). The core objective of the OPCW is the promotion and adherence to the Chemical Weapons Convention (CWC). Among other activities, the OPCW performs inspections of facilities for destroying CWAs, industry inspections, challenge inspections and investigations of alleged use, in order to verify destruction and non-proliferation of chemical agents. The OPCW technical secretary provides off-site analysis through a network of designated laboratories proficient in NAs analysis and their degradation products at concentrations greater than 1 ppm in environmental and man-made samples (OPCW 1994, Black and Read 2013). Since 2016 the OPCW has had 17 designated laboratories for analysis of biological samples. Some of them were already involved in the verification of poisoning induced by CWAs during the recent events in Syria. Aside from confirming exposure to NAs in the alleged use for forensic purposes, the detection and identification of biological markers of exposure is also needed for the diagnosis and therapy 4 of intentional or accidental poisoning, in addition to occupational health monitoring for specific worker profiles (Black and Read 2013). Nerve agents and Toxicity Chemical weapons, including OP NAs, have been used for decades in armed conflicts and terrorist attacks, but accidental leakage from industrial and storage facilities, laboratories, dumping and chemical weapon incineration sites, has also led to health issues and even deaths in those exposed. Although the scientific literature on the NAs is extensive (PubMed search listed 54.400 articles), less than 10% of articles deal with the environmental exposure to OP Nas (Vučinić et al 2014, Davisson et al 2005, Kingery and Allen 1995, Lionetto 2013, USACHPPM 1999). Published articles refer mainly to the significant alterations in ecosystems with CWA dumpsites, where the solubility and hydrolysis rates of CWAs’ innate toxicity had been assessed for marine environment risk prediction. There is indeed evidence of chronic toxicity, though studies in marine organisms have not exhibited concerning amount in tissues of these agents and their byproducts. Acute exposure to an agent presents the major human health risk, either by accidental recovery of a CWA on a fishing vessel or by munitions washed ashore onto beaches (CHEMSEA 2013). In order to provide successful medical protection it is essential to understand the toxicity of NAs (VX vapor is the most toxic with LCt50 of 10 mg/min/m3, and tabun is the least toxic with LCt50 of 400 mg/min/m3), but also the relevant physicochemical properties of NAs such as: volatility (for sarin 22,000 mg/m3 and VX 10.5 mg/m3), vapor pressure showing how quickly an NA will evaporate (for sarin at 200C 2.1 mmHg, while VX evaporates 2000 times slower), vapor density (VX has a high vapor density so it accumulates in low grounds), stability and persistence on soil and in water. While nerve agents are toxic by all routes of exposure, due to mentioned properties, G agents act primarily via inhalation, whereas the skin penetration is most commonly the route of exposure for V agents. Ingestion of NAs (from contaminated water) is highly unlikely, considering the persistence of NAs, the effect of hydrolysis, dilution and water treatment processes (Salem et al 2008). Environmental persistence of agents such as tabun can be found in 5 water from one (at 20°C) to six days (at 5°C); sarin evaporates as fast as the water; soman and cyclosarin are less volatile and more persistent, lingering up to two days, and both pose a significant threat if inhaled or if they reach the skin. The colorless, relatively non-volatile liquid thiophosphonate NA "venomous agent X" (VX) can stay in water for weeks to months due to its slow evaporation (approximately 2000 times slower than sarin) (Augerson 2000, Kikilo 2001, Capacio et al 2008, Fatz 2004, National research council of the National Academies 2003, Agency for Toxic Substances & Disease Registry 2014, Carnes and Watson 1989, Petrakis et al 2017 ) (Table 1). TABLE 1 In summary, there is a threat to both military and civilian population from the environmental NAs persistency. While sarin and tabun are very evaporable, which makes them more dangerous when inhaled, soman and cyclosarin are less evaporable and more persistent, making them dangerous with both inhalation and dermal exposure. However, soman might be more persistent by a “thickening agent” such as polymethymethacrylate. Sarin hydrolysis to isopropyl methylphosphonic acid and hydrofluoric acid depends on pH; at pH levels between 6.5 and 14, hydrolysis is mediated by hydroxide-ion catalysis, while at pH levels between 4 and 6.5, reaction occurs between sarin and water molecules. Temperature also effects hydrolysis, so at 250C under alkaline conditions, the half-life of sarin is 5.4 h. A principal hydrolysis product of soman is hydrofluoric acid, however soman is more stable in water than sarin. Efficient hydrolysis of tabun in water after 10 minutes of heating at 950C, allows using this water for technical purposes. VX is not easily evaporable, it is persistent and dangerous when dermally absorbed or inhaled as aerosol. Other important factors that are correlating with temperature and ground persistency are vapor pressure and vapor density (Augerson 2000) (Table 2). TABLE 2 In the different environmental conditions VX can have significant behavior variation: at pH 5, the half-life of VX is ≈100 days, whereas at pH 8 the half-life is 9 days. The hydrolysis rates depend on temperature as well. In seawater, the half-life of VX was about 5-14 days ate 250C, but at 40C 6 it may be several years. At pH 5, ethyl methylphosphonic acid (EMPA) and methylphosphonic acid (MPA), that show low toxicity, are the most abundant degradation products; but at pH 8, the most abundant degradation product is S-(2-diisopropylaminoethyl)methylphosphonic acid (which is considerably less toxic than VX). The fate of VX at trace-levels and whether adsorption on soil surfaces or complexation with natural organic matter may affect its degradation rate is also not fully clarified. It is thought that VX is more likely to persist in an adsorbed or complexed form, meaning that there is still potential for secondary release and exposure (Augerson 2000, Fatz 2004, Agency for Toxic Substances & Disease Registry 2014, US EPA 1995, Breyer et al 2010). Confirmation of exposure There are different requirements for confirmation of NAs exposure in certain scenarios. Hence, a comprehensive assessment of clinical picture, biological and environmental samples as well as reliable witness reports is crucial. Environmental samples are used to prove not only the identity, but possibly even the origin of NA. There are different portable detectors for rapid on-site detection and identification of CWAs and technologies of identification of agent such as ion mobility spectrometry, flamme photometry, photoionisation, Raman spectroscopy etc. (Black and Read 2013, Augerson 2000 National Research Council of the National Academies 2003, Breyer et al 2010, van der Schans 2008). The final proof of poisoning, however, is only possible through confirmation with forensic investigation of samples (blood, tissue, urine) from the victim. Such forensic analysis needs advanced analytical methods and complex technical equipment (Hernández et al 2014, Kavvalakis and Tsatsakis 2012, Tsatsakis et al 2009). Clinical picture and field treatment Based on their lipophilic qualities, the NAs permeate the skin, lungs and gastrointestinal tract easily and reach circulation whereupon they distribute to organs and tissues (Balali-Mood 2008, van der Schans 2008, David 2005). On the whole, the acute effects of exposure to organophosphorus compounds is well described and can be predominantly characterized by the cholinergic crisis (muscarinic, nicotinic and CNS effects), with the occurrence of the signs and 7 symptoms, as well as the time course of poisoning and severity based on the dosage, route and length of exposure (Kikilo et al 2001, Senanaykae and Karalliedde 1987, Crook et al 1983, Romano et al 2001, Dabisch et al 2001, Moshiri et al 2012). Military services and several special units are trained in skills necessary to manage Chemical, Biological, Radiological and Nuclear (CBRN) events. In detail, they may even be equipped with drugs for self and buddy aid. Such medical equipment is generally not available for civilian population and drug administration is the task of the medical service. In most countries, especially the administration of drugs, e.g. antidotes is the responsibility of a physician. Hence, the following recommendations on drug administration and dosing are directed to physicians. Figure 2 shows health effects of sarin, cyclosarin and VX. Figure 2 Minimal Exposure Minimal vapor exposure to NAs includes miosis, blurred vision, eye pain and rhinorrhea. Vapors and fumes can cause toxic effects within minutes. In contrast, when exposure arises from vapor or liquids via skin without inhalation, it may take hours for signs and symptoms to manifest. For the treatment of ocular symptoms, topical atropine or homatropine can be administered. First systemic sign might be tachycardia due to initial stimulation of sympathetic nicotinic receptors. As long as heart rate is markedly increase, any systemic atropine administration needs specific care (Balali-Mood 2008, Agency for Toxic Substances & Disease Registry 2014, Kassa 2005). Light Exposure In case of miosis, rhinorrhea, light dyspnea, nausea and vomiting, systemic effects can be diagnosed. In this case, atropine and an oxime should be administered. A starting dose of 2 mg atropine is generally recommended. Such a dose should be sufficient after light exposure (otherwise, see below). However, atropine only antagonises symptomatically NA induced effects. In contrast, oximes are used to restore NA inhibited acetylcholinesterase (AChE) thereby resolving the effects of the NA and coincidentally metabolising it. Therefore, military services 8 and several special forces are equipped with autoinjectors. They are filled either with atropine alone (most commonly with 2 mg) or with a combination of atropine (2 mg) and an oxime, e.g. pralidoxime or obidoxime. Two prominent devices are the Mark I kit - atropine (2 mg, 0.7 ml) and 2-PAM (600 mg) or the ATOX II consisted of atropine (2 mg, 0.7 ml) and obidoxime (220 mg). Other types of autoinjectors are commercially available. At present, the most effective strategy is the use of a combination of atropine (2 mg) and obidoxime (220-250 mg). Using this regimen, effective concentrations for substantial reactivation of AChE inhbited by a relative broad spectrum of NAs may be adjusted. At any case, autoinjectors can be used as soon as systemic signs and symptoms arise. However, young children are quite sensitive to atropine and administration needs caution. Apart from undressing, followed by showering (at best by using soapy water), no specific decontamination is required if the poisoning was caused by vapours. However, after exposure to liquids, thorough spot decontamination with reactive skin decontamination lotion (RSDL), followed by showering, is strongly recommended. In the case of dermal exposure, localized sweating and fasciculations are expected and one autoinjector containing atropine and an oxime should be administered. Moderate Exposure Moderate exposure is followed by miosis, rhinorrhea, moderately severe to severe dyspnea, nausea and vomiting. If the patient is attended by medical service personnel, in the first 5 minutes, 2 autoinjectors and diazepam should be administered. Following immediate atropinization, further doses of atropine should be titrated according to signs and symptoms. If a liquid NA exposure is suspected, extended observation for at least 18 hours is necessary. During such a potential latency period, repetitive determination of red blood cell AChE (RBC-AChE) activity may be extremely helpful to estimate the course of poisoning. At the best, effective oximes can be administered while AChE activity is decreasing and the signs and symptoms are not yet detectable. With such a strategy, even symptomatic poisoning could be prevented. Severe Exposure Generally it has to be mentioned that self protection of medical personnel and facilities is mandatory. The life-threatening signs of severe exposure are loss of consciousness, respiratory 9 depression, apnea, cardiovasular failure, convulsions, and flaccid paralysis. Three autoinjectors [e.g. 3x MARK I (2 mg atropine and 600 mg pralidoxime, each) or one ATOX II (220 mg obidoxime and 2 mg atropine) followed by two AtroPen (2 mg atropine)] should be given immediately, if available. Otherwise, fast atropinisation is recommended according to the schedule mentioned above and oxime treatment should be started. Alternatively, treatment has to be initiated as an initial dose of atropine of 6 mg, followed by 2-4 mg i.v. and oxime (if no autoinjector is availabe, e.g. 250 mg obidoxime i.m or better i.v.). Additionally, diazepam (e.g. 10 mg) should be administered for amelioration of central effects. Ventilatory support is of vital importance for survival (Figure 3). Figure 3 The time for initial response should be spared before proceeding with atropine dosing, and it is deemed wise to wait, before further atropine administration, until the secretion decreases and the breathing improves. The critical parameter is actually the restoration of oxygenation. In the case of dermal exposure, if similar, though delayed, signs as severe vapor exposure occur and the same treatment should be applied (Balali-Mood 2008, Moshiri et al 2012, Kassa 2005). Suspected exposure to NAs can sometimes occur in an environment where NAs can be found as liquids, albeit this could be still described as unconfirmed exposure, where perhaps not even the afflicted are cognizant that they were exposed to a harmful chemical (Claborn et al 2004). Following skin contact, the onset of signs and symptoms of NA poisoning may also be delayed up to 18 hours. In such scenarios, repetitive determination of AChE activity might be helpful. The longer the interval between the exposure and the symptoms, the less severe will the latter be – which does not imply that medical attention is not necessary (Agency for Toxic Substances & Disease Registry 2014). In any case of severe poisoning, especially with persisting agents such as VX, prolonged treatment (several days) may be necessary. Dosing of atropine should be performed according to signs and symptoms at best using an i.v. line. However, over-atropinisation should be avoided. As long as reactivation of inhibited AChE can be expected, sufficient oxime doses (e.g. 750 mg obidoxime per 24 hours) should be administered via continuous infusion. Simultaneously, 10 necessary symptomatic intensive care measures have to be applied. Especially in scenarios with NA mass casualties, oximes are expected to be of enormous help: they may enable survival on the spot when the resources for artificial ventilation are not available or run short, and they may contribute to the reduction of the period of necessity for intensive care treatment when the units are overwhelmed with patients. While in the times of Cold War, when troops of nations were facing each other, a certain level of protection was assumed, though at present, such scenarios have shifted towards asymmetric conflicts as well as terrorist attacks. Civilian population, unlike the army, was entirely unprotected against the exposure to NAs, as they did not possess personal protective equipment, autoinjectors and there was at least 30 minutes delay before they got the proper medical treatment. While the acute effects of exposure are well documented, it has been only for the last twenty years that a upsurge of interest in the delayed effects of single-dose poisoning and chronic effects of exposure (continuous or intermittent) to NAs has been reported. Most published epidemiological studies are scarce, revealing exposure evaluation, using adequate methodology as main problem. Studies with military personnel suffer from inaccurate exposure assessment and the clinical data are often insufficient. Over the last 15 years, studies with sarin attack survivors in Japan have provided additional data regarding the neurobehavioral effects and chronic decline of psychomotor skills and memory, along with neurophysiological function defects, which persisted for months and years after the exposure (Nakajima et al 1999, Murata et al 1997, Nagao 1991, Yokoyama et al 1998). The kind of exposure represents a significant difference between the civilian and military population. In the case of civilians, the most possible ways of exposure are either single or repeated, whereas for the military population a dose of decreased level, results in immediate effects (mostly miosis), or reduced performance. The long-term effects of NAs were thought to only be expected after sever intoxications but no consistent data exist on this subject of the repeated exposure to NAs. Thus, organophosphateinduced delayed neuropathy (OPIDN) has not been reported in Iranian chemical attack survivors. Asymptomatic sequelae of sarin poisoning in central and autonomic nerve system have been reported in Matsumoto and Tokyo attack survivors. However, it was concluded that they had no 11 clinical relevance. It has been suspected for a while that there are chronic effects of exposure to low doses if OP compounds, but that has yet to be ascertained (Nakajima et al 1999, Murata et al 1997, Nagao et al 1991, Yokoyama et al 1998, Duffy et al 1979, Duffy and Burchfield 1980, Burchfield etal 1976). Biological markers of exposure to NAs The diagnosis of acute exposure to high doses of NAs is confirmed by the aforementioned clinical picture, measuring of cholinesterase (ChE) activity, determination of OP NAs, their metabolites and reaction products in biological samples (Balali-Mood 2008, Lionetto et al 2013, Moshiri et al 2012). Determination of ChE activity inhibition is generally sufficient for initial treatment in clinical settings and occupational health monitoring. Established ChE baseline levels in occupational settings, when compared to ChE levels after exposure, might be indicative of significant exposure. Sensitivity can be clearly improved, when baseline levels of AChE are known prior a potential exposure towards inhibitors of cholinesterases. However, the specificity of these parameteres for NAs is low as ChE inhibition might be a result of OP and carbamate pesticides exposure. For diagnostic purposes, in case of poisoning, no baseline levels exist, so inhibition levels less than 20% per se do not provide sufficient evidence of exposure (US EPA 1995). The parent NA has a relatively short self-life (G-type agents: up to several hours, V-type agents up to few days) due to its rapid hydrolysis and affinity for binding to the proteins in plasma and tissues, although its determination has the highest specificity. After the processes of absorption and distribution in tissues, the NAs undergo spontaneous, subsequent enzymatic hydrolysis by endogenous hydrolases – e.g. paraoxonases. Through irreversible binding to serine esterases other than AChE, such as Butyrylcholinesterase (BuChE) and carboxylesterases (CaE), they become inactive. Additionally, they may form covalent bonds with albumin and other proteins in the blood and tissues (VX has low affinity for CaE, whereas sarin and soman’s affinity for CaE is high). In order to detect the non-metabolised agent, samples of blood from the casualties within hours of exposure should be collected; it is possible to detect V-agents for longer periods, possibly even days after exposure due to slow rate of absorption after dermal exposure and longer 12 half-lifes (Balali-Mood 2008, van der Schans 2003). Despite the highest specificity and a relatively simple analytical technique, non-metabolised agent is rarely useful due to its short lifetime in circulation (Balali-Mood 2008). NA metabolites are not expected to be present for more than a few days after exposure, e.g. Isopropyl methyl phosphonic acid (IMPA) was detected in the urine of one of the sarin attack victims in Japan on the seventh day after the incident (Nakajima et al 1999). Free metabolites have been used as biomarkers for confirming the NA exposure in terrorist attacks or other alleged uses as following: IMPA (identified in Matsumoto and Tokyo victims) and methylphosphonic acid – MPA (a secondary hydrolysis product also found after exposure to other NA) in urine and blood for sarin; pinacolyl MPA in urine and blood for soman; cyclohexyl MPA in urine and blood for cyclosarin, ethyl dimethylphosphoramidic acid (EDMPA) and ethyl phosphorocyanidic acid in urine for tabun; ethyl MPA in urine and blood for VX. Although detection methods for urinary metabolites have moderate to high specificity due to their occurrence in other sources, metabolites of tabun are not an unequivocal indicator of poisoning. The time frame for detection varies based on the severity of poisoning and the limits of detection of the method, but generally its durance is no more than a several days. Other analytical methods are also available: fluoride induced reactivation of BuChE and AChE/BuChE serine adducts in blood identified as phosphylated peptide; analysis of ChE adducts (albumin tyrosine adducts in plasma, keratin tyrosine adducts in the skin that is still being researched). The time window for adducts is no longer than for the free metabolites (up to several weeks or months), with limitations due to aging (albumin tyrosine adducts in plasma do not have this issue) (Nakajima et al 1999, Okudera 2002, Matsuda et al 1998, van der Schans 2004, Verstappen et al 2012). Three years ago, the OPCW faced the situation of alleged use of CWAs in Syria. As a historical precedent, the inspection teams of the UN and OPCW were to collect the samples within days of the incident, which, depending on the situation in the field, was not always an easy task. Nearly 90% of urinary metabolites of NAs is excreted within 72 hours, so the protein adducts were more significant because of their longer half-lives of up to several weeks. The chemical attacks happened in August 2013 in Ghouta/Syria but reaching the scene in time was impossible. Aside from taking environmental samples, which entailed the likelihood of having NAs and/or their degradation products, and performing analyses of the aforementioned, only the analysis of 13 biological samples could prove NA poisoning (including body tissues for the deceased). In contrast to armed conflicts, in terrorist attacks it may be assumed that samples could already be gathered within a few hours, as it was performed in Tokyo. When speaking of metabolites, it is desirable for them to have a good stability and not be subjects of hydrolysis or oxidation (primarily the issue with tabun, which hydrolyzes rapidly to the ubiquitary product of excretion, ethyl phosphoric acid). These substances can be detected in urine, blood and tissue samples by using liquid or gas chromatography. As they present a very high toxicity, very low concentrations in biological samples have to be assessed. Therefore, sofisticated GC-MS/MS or LC-MS/MS methods using single ion monitoring (SIM) or multiple reaction monitoring (MRM) modes are used. There is a specific demand for targeted analysis, for example, compounds such as IMPA. During the past decade there has been a significant development in off-site analysis as highresolution mass spectrometry is now available in several designated laboratories for retrospective trace analysis (Pita and Domingo 2014). In spite of these diagnostic techniques available, there is still a possibility for unidentified exposure especially in the case of very low levels of NAs without ChE inhibition, acute exposure followed by ChE inhibition but without symptoms, reduced clinical response if the patient had already been exposed to ChE inhibitors, or simple misidentification of symptoms of poisoning as a different diagnosis. Risk Assessment The parameters for risk assessment are no-observed-effect level (NOEL), lowest-observed-effect level (LOEL) and effective dose (ED50). The level at which no noteworthy depression in red blood cells cholinesterase activity (RBC-AchE) occurs, is defined as the maximum acceptable level of exposure for the NAs. If RBC-AChE inhibition is the same or less than 20% and when it is not correlated to clinical signs and symptoms, only evidence of organophosphorus agent exposure exists. According to the 1995a EPA report a statistically significant restraint of AChE in many organs and tissues is hazardous, provided there are clinical results. If clinical effects are not exhibited, this inhibition might be not of biological importance (Augerson 2000, US EPA 1995, Dabish et al 2001, Young et al 1999). 14 Health-Based Environmental Screening Levels (HBESLs) were derived from using chronic toxicity criteria with risk assessment models. They are used for assessing potential long-term human exposure to soil, water and waste contaminated from a liquid agent as it is not likely that ambient vapour alone would result in deposition or soil contamination (Auerson 2000, Agency for Toxic Substances & Disease Registry 2014). Specific exposure scenarios are estimated and shown in Table 3. The field drinking water standards were designed for a military scenario in which a small water container might be deliberately contaminated with signifiant amount of NA. The values <12 µg/L of GA, GB, GD and VX up to 7 days exposure at 5 L/day water consumption (higher rate of drinking in hot environments) are safe. Daily exposure for a lifetime to soil contaminated from liquid GB (via ingestion, inhalation and dermal contact) is safe up to the limit of 1.3 mg/kg. In hazardous waste treatment facilities, possible occasional exposure of workers to waste water that may contain 8.3 mg/L of GB will not endanger worker's health. Also risk assessment approaches that are used to determine the safe limits for chronic exposure to NAs should include all the possible synergism or potentiation effects that could appear in real life exposure from different sources between them and other chemicals with the same mechanism of action as organophosphorous compounds used as pesticides (Tsatsakis et al 2016a, Tsatsakis et al 2016b). TABLE 3 The so called „Gulf War“ syndrome has inspired numerous studies and extensive research related to NAs, and a few relevant questions have been raised: a) Is it possible to have NA effects from low-level exposures without the typical clinical picture? b) Are long-term effects possible after mild exposure? c) If the exposure goes unrecognized are the delayed effects possible? One cannot exclude the possibility seeing as how there are cases of occupationally exposed workers that were found to have very low ChE levels but without clinical picture. Also, impaired thinking, memory, calculating ability were documented in volunteers exposed to VX and were asymptomatic. There are case reports of workers with mild exposures who had fatigue, poor memory and 15 concentration difficulties 4-10 months after the exposure. Duffy and Burchfield (1980) found nonspecific EEG changes in workers exposed to sarin, but explained that it had no clinical significance (Duffy et al 1979, Duffy and Burchfiel 1980, Burchfiel et al 1976). No human studies exist that accurately describe that effects of NAs on neurotoxic esterase (NTE) caused delayed neurotoxicity. Effects on NTE were confirmed in experimental model on animals. However, the relevance of these effects in humans exposed to a single dose of NAs is unclear. So far there is no evidence that NAs can cause delayed effects similar to those associated with NTE in a military, asymetric conflict or terrist scenario, but the possibility is not excluded for atypical forms (Augerson 2000). Conclusion Suspected exposure to an OP NA can occur in an environment where the agent can be found as a liquid, and when perhaps not even the afflicted know they are exposed to a harmful chemical. 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Health effects of sarin, cyclosarin and VX Figure 3. CBRN Emergency Medical Treatment 22 23 24 Table 1. Physical, chemical and toxicological properties of OP Nas Property Tabun (GA) Sarin (GB) Soman (GD) VX Mol. weight/(Da) 162.3 140.1 182.2 267.4 Boiling point/(°C) 230 158 198 298 Melting point/(°C) Vapor pressure/mmHg Vapor Density Liquid Density (g/ml) (25 °C) Volatility (mg/m3) (25 °C) Solubility in water -49 -56 -80 -20 0.036 (20 °C) 2.1 (20 °C) 0.27 (20 °C) 0.00044 (20 °C) 5.6 4.9 6.3 9.2 1.8 1.10 1.02 1.01 610 22000 3900 10.5 9.8 g per 100 g Miscible Miscible 1-1.5 day (half-life) 2-24 h (5-20 °C) 2.1 g per 100 g Relatively persistent 400 100 50 10 1000 1700 100 6-10 13 h 3h 40 sec to 10 min 36 h Peristency on soil LCt50 in humans /(mg min m3) LD50 in humans /(mg per 70 kg) percutaneous Ageing half-life 2-6 days Table 2. Persistence of NAs Agent Persistence Sarin Heavily splashed liquid – 1 to 2 days. In water 1 day (20 0C) and 6 days (50C) Evaporates as fast as water Soman Heavily splashed liquid – 1 to 2 days. Thickeners can extend. Cyclosarin Heavily splashed liquid – 1 to 2 days. VX Splashed liquid can persist for weeks or months (evaporates 2000 times slower than sarin) Tabun Table 3. Summary of Multi-Media Chemical Agent Toxicity and Exposure Values: Existing Information as of 8/03/04 POC.V.Hauschild, USACHPPM, 410-436-5213. https://www.osha.gov./SLTC/emergencypreparedness/guides/nerve_cwa_othermedia_table_08032004.pdf accessed 10.10.2016. Media Standard/Guideline Name Population Exposure Scenario WATER FDWS (Field Drinking Water Standards) µg/L designed for military * but can have civilian applications safe for up to 7 days H/HD/HT GA (Mustard) (Tabun) 200 20 GB (Sarin) GD/GF VX Lewisite 20 20 20 200 25 SOIL WASTE Chronic Toxicity Reference Criteria (for use in risk assessment calculations) normal/humid climate - 5 L/day ingestion rate (140) (12*) (12*) (12*) (12*) (80) dry/arid climate - 15 L/day ingestion rate (47) (4*) (4*) (4*) (4*) (27) HBESL Residential (Health-Based Environmental Screening Levels) mg/kg civilian general population: adults and children daily exposure via ingestion, inhalation, and dermal contact for a lifetime 0.01 2.8 1.3 0.22 0.042 0.3 HBESL - Industrial mg/kg civilian general adult population frequent exposures via ingestion, inhalation, and dermal contact: 250 days/ year for 30 years 0.3 68 32 5.2 1.1 3.7 HWCLsol (solid hazardous waste control limit) mg/kg civilian/DoD worker possible occasional exposure at HW treatment facility 6.7 680 320 52 10 37 HWCL (liquid hazardous waste control limit) mg/L worker civilian/ DoD possible occasional exposure at HW treatment facility 0.7 20 8.3 0.3 0.08 3.3 NHWCL (non-hazardous waste control limit (haz waste exemption level) mg/kg worker civilian/ DoD possible occasional exposures at a nonHW land disposal facility 0.3 68 32 5.2 1.1 3.7 RfDo (Oral Reference Dose) mg/kg-day General population: adults and children CSFo Oral Cancer Slope Factor (mg/kg/day)-1 General population: adults and children Inhalation Unit Risk (µg/m3)-1 General population: adults and children chronic (lifetime) ingested dose at or below which no adverse health effects are expected represents the potency of the agent by ingestion to cause increased cancer risk represents the potency of the agent by inhalation to cause increased cancer risk 0.000007 0.00004 0.00002 0.000004 0.0000006 0.0001 7.7 Not determined to be a carcinogen 0.0041 * Military application 26