Carbophos - As an Imitator in the Indication of Organofosforous Chemical Warfare Agents

By Prof. Col. (Ret.) Mihail Haralampiev, Professor Engineer, Vasil Levski National Military University of Bulgaria

Although chemical weapons have been almost destroyed (5), their relevance remains. This is because the ability to synthesize old and new toxic substances is determined by the powerful chemical industry of many countries. Therefore, CBRN specialists must have a solid engineering chemical and physical education and appropriate practical training for the detection of combat poisons, as well as their timely disposal in case of possible use. The training of the cadet chemists at the Vasil Levski National High School, Veliko Tarnovo, Bulgaria takes place in a five-year engineering course. After studying the main chemical disciplines: inorganic chemistry, organic chemistry, physicochemistry, analytical chemistry, the cadets study the properties of Chemical Warfare Agents (CWA), their rapid indication, and protection from them.

Much attention is paid to the indication of CWA through traditional methods of analytical chemistry. This laboratory practice then allows them to easily study instrumental methods for identification and analysis (1;2). Therefore, the aim of our study was to look for a suitable and inexpensive simulator of CWA, through which to perform basic reactions for qualitative and quantitative detection of neuroparalytic CWA. The organophosphate insecticide carbophos (malathion) turned out to be such a possible imitator. It is one of the most common organophosphorus insecticides all over the world (3,6,7).

Diethyl 2-[ethoxy (hydroxy) phosphinothioyl] sulfanylbutanedioate

The gross formula of carbophos (malathion) is C10H19O6PS2. It has the following physical properties: boiling point - 120C °, m.p. 2.8-3.7° C, water solubility 145 mg / l; the molar mass is approximately 330.4 g / mol. It is an acetylcholinesterase inhibitor, Organothiophosphate.

The experiment: The reaction for indication with this substance was performed with the assumption that C10H19O6PS2 binds (inactivates) the enzyme cholinesterase and its property to accelerate (catalyze) the hydrolysis of acetylcholine to choline and acetic acid according to the scheme. Interaction of organophosphorus warfare agents with the enzyme cholinesterase (E - H), for example with sarin, proceeds as follows:

Hydrolysis of acetylcholine under the action of other non-inactivated by the toxic substance cholinesterase proceeds as follows:

The reagents we used to perform the biochemical reaction using the Diethyl 2- [ethoxy (hydroxy) phosphinothioyl] sulfanylbutanedioate simulator are as follows: reagent № 1-aqueous solution of dry horse blood plasma; reagent № 2- bromothymol blue indicator; reagent № 3- indicator acetylcholine. As a standard for comparison, we used a tube with a similar mixture in which acetylcholine was replaced with a corresponding amount of acetic acid. We placed the tubes in a thermostat-comparator to maintain a constant temperature in the range of 38°C. The contents of the reaction mixtures are shown in the following table:

It is known that the rate of hydrolysis of acetylcholine is directly proportional to the amount of free cholinesterase available. The greater this amount, the greater the rate of hydrolysis, i.e., more acetylcholine is hydrolyzed at the same time, and more acetic acid is released. The change in the pH of the medium, which is determined by an indicator, serves as an indicator of the rate of the reaction. The bromothymolblau indicator is blue in alkaline environments and yellow in acidic environments. This is evident from the following reaction (1:2 ):

Bromothymolblau- quinoid form (blue color): Bromothymolblau- lactone form (yellow color) In this experiment, we practically compared the activity of equal amounts of cholinesterase, after one of them came into contact with the sample infected with carbophos, and the other did not. If this object is contaminated with organophosphorus poisons (in this case carbophos), depending on their amount, more or less of the cholinesterase in contact with it is inactivated. As a result, the amount of free cholinesterase decreases compared to the amount of non-contacted cholinesterase, which has a negative effect on the rate of hydrolysis of acetylcholine and, accordingly, on the change in color of the indicator. As a result of the acetic acid released, the color of the bromothymolblau indicator changes from blue to yellow. First, we prepared the standard or the final coloring. Initially, it was slightly yellow and, for this, we had to add 5 drops of 0.01 N sodium hydroxide solution to achieve a yellowish-green color (4). Therefore, in all other tubes, we added 5 drops of 0.01 N sodium hydroxide. Then in the other two tubes, we introduced 1 ml of cholinesterase - reagent №1, 0.5 ml of uncontaminated water (control sample) and contaminated water (experimental sample with carbophos) and 0.2 ml of indicator (reagent №2).

We observed the time in minutes for which the color of the control and test sample matched the color of the standard.

We shook the tubes to homogenize by inverting several times and dipped all the tubes and kept for 10 minutes in the comparator thermostat, half-filled with heated water in the range of 38-40°C. Then, for 10-15 s, 0.2 ml of 0.5% acetylcholine solution (reagent №3) was added to the control and test tubes. We shook the tubes closed with polished plugs by inverting them and placing them in the comparator thermostat. We observed the time in minutes for which the color of the control and test sample matched the color of the standard. Usually at a temperature of 38°C the color of the control samples is equal to the color of the standard for no more than 9 minutes, and the experimental ones, depending on the contamination of the tested solution - for a longer time. It is considered that the water is not contaminated if the colors of the control and test samples are aligned with the standard at the same time or the difference between them does not exceed 0.5 minutes according to the equation: Тexper. – Тcontr. . = 0,5 min If this difference is greater than 0.5 minutes, the water is considered contaminated The data for the quantitative determination of carbophos according to the conducted experiment were calculated by the formula:

Degree of cholinesterase inactivation – У =

We conducted 5 experiments and the experimental times were as follows: 12 min ; 12:15 min ;12 min; 11:45 min; 12 min. The average control time was 12 min. Therefore У = inactivation of cholinesterase

This percentage of cholinesterase inactivation indicates that in this case the concentration of carbophos corresponds to the following concentrations:4.7.10-7 mg / Vx; 1.7.10-5 mg / l sarin; 5.1.10-6 mg / l zoman; 5.95.10-6 mg / tabun. This experiment convincingly shows that carbophos can be used as an imitator of a nerve agent.

Conclusion: The conducted experiment showed that carbophos (malathion) can be used as an imitator for qualitative and quantitative determination of real war poisons of neuroparalytic nature. This creates laboratory confidence and training, as well as creating conditions for savings, as no real war poisons are purchased that are under the control of the Organization for the Prohibition and Destruction of Chemical Weapons (5).


1.Haralampiev, M., The Chemical Weapon, Veliko Turnovo, Bulgaria, Faber, 2017 2. Haralampiev, M.,, V. Manolov, Chemistry Warfare Agents, Sofia, Military Printing House, 1991. 3.Ivanov, V., М. Haralampiev, Z. Raykov., Bulgarian Patent № 64 922 от 06.10.2006 г. Sorbent for capturing toxic gases. 4. Manual of the Field Laboratory PHL -54. 5.Convention on the Prohibition of the Development, Production, Stockpiling and use of Chemical Weapons and their Destruction, 29 April 1997, OPCW, The Hague, The Netherlands. 6.Compton, J. Military Chemical and Biological Agents (Chemical and Toxicological Properties),The Telford Press, USA, 1987. 7.Ghosh, R., J. F. Newman., A new group of organophosphorus pesticides. Chemistry and industry, 1955, p. 118.


Professor Colonel (retd.) Mihail Stefanov Haralampiev started his career in September, 1962 as an Officer cadet with the CBRN Defense Department at the ‘Vasil Levski’ National Military University Veliko Turnovo, graduating in 1967 with the rank of Lieutenant and civil engineer of ‘Organic Synthesis & Technology of Macromolecular Compounds’. In the period September 1977- July 1980 he graduated from the Russian Federation CBRN Academy with a Master Degree in the specialisation ‘Operational and Tactical Actions of the CBRN Corps. In 1967 he became a platoon commander in the divisional CBRN Defence company. He spent 30 years with the CBRN Corps, covering different assignments (Company Commander, Chief of the CBRN department in the Motorised Rifle Regiment, Deputy Chief of the CBRN department in the Motorised Rifle Division; also appointed to the mobilisation positions of Chief of the CBRN department and Chief of Staff Management of CBRN troops of the Bulgarian Army, CBRN Defence Department at ‘Vasil Levski’ National Military University. In the period 1971 to1976 he developed a doctoral dissertation related to the behaviour of polymeric materials under explosive loads with a PhD rewarded at the University of Chemical Technology and Metallurgy, Sofia Bulgaria. At the same time he worked as a lecturer at the Vasil Levski National Military University. In 1993, he completed a pilot course as an inspector at the future Organisation for the Prohibition of Chemical Weapons (OPCW), Den Hague, The Netherlands and at the United Kingdom Military Research College at Shrivenham (Cranfield University). In 1995, he completed pilot courses at the U.S. CBRN Defense School and Academy in Aniston, Alabama and a specialized technology course at Pine Bluff Arsenal, Arkansas, USA. In the period January to May 1997 he completed specialised courses in the CBRN Defence Centres of The Netherlands, the Russian Federation, France, China, the United Kingdom, Germany and Italy. From mid 1997 to March 2007 he worked as a Chemical Weapons Specialist at the OPCW in another of different roles including Inspector P-4, Chief of Inspection Teams, Leader of the Group responsible for writing the work instructions and the textbook for carrying out inspections and for two years he was an assistant to the Director of the Inspectorate responsible for the quality control of inspection activities. Since 2008 he has been working in the Department of ‘Protection of the Population and Infrastructure’ at the Vasil Levski National Military University Veliko Turnovo. He teaches the subjects: General Chemistry, Organic Chemistry, Physical Chemistry, Ecology, Chemical Weapons and Defence, Biological Weapons and Defence and Nuclear Weapons and Defence. He has published as author and co-author fifteen authoritative textbooks and manuals on these themes and more than two hundred and fifteen papers and articles related to Polymers, Organic Chemistry, Ecology, WMD and CBRN. His monograph ‘The Chemical Weapon’ is considered a standard work on the subject. Professor Haralampiev is a member of the Union of Scientists in Bulgaria. In 2013, he received a Certificate of Contribution to the Nobel Peace Prize awarded to the OPCW. In 2013 he was awarded the title: Doctor Honorius Cause of Vasil Levski National Military University, Veliko Turnovo, Bulgaria.