How Clean is Clean?

By Lt. Col. Alan Gavel, Biological Analyst and Trainer,

Population Protection Institute of the Czech Republic

The decontamination process is a set of activities that should lead to the removal of unwanted contamination. Whereas importance and residual level requirements differ among agencies, there is one fact that should never be forgotten. Validation of the process for field conditions cannot be complete without verifying developed methods out of the laboratory. All involved factors including, e.g., material compatibility, dirt burden, operators, or commonly used technical means need to be tested. Decontamination is often only shown during trainings and exercises, but it is a crucial element of real CBRN response. This should be highlighted in case of B incidents where we cannot evaluate residual contamination immediately. We were challenged by two major biological risks during the recent decade – Ebola and SARS-CoV-2. Both caused the spread of contagious illnesses that also required countermeasures from multiple response agencies where proper decontamination of persons, responders, and buildings was interdisciplinary. This article describes experiences from verification and optimization of biological decontamination methods of responders and testing of technical means with different disinfection solutions for indoor spaces.

The Czech Republic relies on a system of so-called Integrated Rescue System (IRS) which means the cooperation of response agencies based on IRS Act No. 239/2000 coll. Basic response agencies are police, fire service, and emergency medical service, which are supported by other agencies. Experts from these organizations met and established an unofficial working group to discuss existing procedures and methods shortly before the West Africa Ebola outbreak occurred. There were no real Ebola cases in the Czech Republic. Experience from exercises and real suspected cases showed the lack of verification of effectivity of biological agents decontamination method in the field which was reflected by the national joint security research project DEKOVRAT (Czech Ministry of Interior) by the Population Protection Institute (Czech Fire Rescue service, FRS), Military Medical Institute and Public Health Institute.

The goal of the DEKOVRAT project was to verify the existing decontamination method of responders from the battlefield, optimize it and finally test it in real environments with real responders and their own technical means. We used harmless Bacillus Subtilis spores as surrogates to anthrax in the project. We considered their similarity in resistance to anthrax spores as the worst-case scenario that should cover the decontamination safety of all biological agents. Project staff worked with volunteers (responders) from fire services, army, and customs. We started at controlled conditions of BSL4 military hospital of the Czech Army to eliminate environmental variables. We used proper controls to guarantee quality in all results. Bovine Serum Albumin (BSA) simulated biological burden. A decontamination solution with active substance approx. 0.7% peroxoacetic acid (Persteril 36 or Persteril 15) was used as the basic disinfectant from the battle regulation list.1 We used a point source sprayer and the exposure time of 2 minutes with three areas contaminated on the splash suit type 3B with 100 independent repetitions. We chose an efficacy threshold of 100 CFU for decontamination of responders in splash suits when the original contamination solution CFU was 107.

We clearly identified that the official decontamination method is not efficient in this way as 43% (95% confidence interval 0.3-0.57) of swab samples remained positive at least on one controlled area. Optimization with time and concentration variables showed that even a concentration twice as high (4% Persteril 36) cannot be accepted, whereas exposure extension to 4 minutes with the original 2% solution was 95% effective (95% confidence interval 0.89-0.98). It was no shock to us as we experienced that during a 2-minutes timespan we could hardly reach any spot of splash suit twice. The liquid does not remain on the splash suit in higher quantity and, thus, real exposure time is far lower than total time. It already clearly illustrates the need for operational testing of laboratory-based methods before they are accepted for the battlefield. We developed a testing method for further use in the project, which the Czech Fire Rescue Service DG certified at the end of the project. It is based on a set of 90 marked squares (10x10 cm) painted all over the suit where 3 of them are randomly selected to be contaminated with the spores. We left contaminated surfaces to dry for at least 12 hours, which could seem very strict, but we were still able to reach 95 % efficacy by optimized methods without any mechanical cleaning. Operators should not estimate which spots were contaminated and they continuously sprayed the solution over the whole responder’s body in cycles for 4 minutes. Staff immediately neutralized and sampled contaminated squares after exposure.

We tested all disinfectant solutions from our battle regulations (chlorine based and binary, liquid, and foaming) and compared them with Persteril. None of them succeeded in a similar time. Contrary, we tested other commercial formulations based on the peroxoacetic acid (Vanodox) in equal concentration and time and succeeded. Choosing reasonable time for exposure of the responders is important because of the heat stress in suits and limited air supply if the SCBA is used. Exposure time longer than 5 minutes thus seems to be too long from an operational point of view.

It is hard to estimate effectiveness against less resistant biological agents, but first responders should have at least one solution and method that works even if they do not know the biological background of the incident and there are no experts at hand yet.

We continued the research with the evaluation of the human factor (training and active approach of responders), conditions, and technical means at regional fire stations with volunteers. We observed that the training of the operator on decontamination, active cooperation of the decontaminated responder, and air drafts are significant factors. Wind could take away the spray of the disinfection droplets in the case of point source decontamination as well as in the unclosed decontamination shower. It could be clear for experts, but our responders saw the quantification of their effort in numbers for the first time. Further details of the experiments were published by Rybka et al. 2021 - 10.1111/jam.15041.

Space decontamination difficulties

SARS-CoV-2 appeared just after the DEKOVRAT project finished. This virus is far less resistant than spores but the total number of spaces for decontamination suddenly created a heavy load on many actors. Czech FRS are not legally in charge of performing specialized disinfection in a normal situation according to the Public Health Act No. 258/2000 coll. The Czech Fire and Rescue Service has, however, responsibility for civil protection tasks and, thus, we were involved in many decontamination operations, especially when the government declared the state of emergency.

Many companies started to offer their services or products during the spring of 2020 and some of them asked the FRS DG for a public recommendation. Companies often did not have any history or qualification in the area of specialized disinfection, but they were often equipped with some certificates and devices for indoor spaces with special emphasis on aerosol or gas decontamination. We did not find binding norms nor recognized testing laboratories for the use of disinfectants (biocides) in this way in the spring of 2020, which was later addressed by EN 17272:2020. Czech FRS DG made a series of tests in cooperation with State Health Institute during which decontamination efficacy in public transport and other indoor spaces were verified with a series of tests with bioindicators (bacteria, bacteriophage, spores, molds) on different carriers (glass, metal, fabric, paper, or agar).

Ozone, which was available to many regional fire brigades, appeared as very ineffective in concentration and represented many health and material compatibility concerns. There were contradictory results with using thermal fog generators (Swingfog) because some disinfectants decompose in higher temperatures and using combustion engines indoors is problematic. Some people even reported irritation from Virkon S residuals in indoor spaces after application. The most promising results came with an ultrasound cold fog generator (ECA 400 QC) and fumigation with hydrogen peroxide (Cleamix). All of the methods were sensitive to biological burden (BSA), which creates a shield against disinfectants. The Population Protection Institute continues in the verification of hydrogen peroxide fumigation with very promising results. There are clear limitations identified so far: the minimal temperature of at least 18-20°C, overall exposure in hours, and material compatibility (fabrics and absorptive materials and peroxide decay catalysators in some wall paintings). This method seems to be very promising with sensitive devices because we can avoid harmful condensation by monitoring the process. We successfully tested controlled hydrogen peroxide fumigation in a hospital environment, and we are currently undergoing negotiations on the aircraft decontamination test.

We could conclude the results of field tests of aerosol decontamination or fumigation by several findings. Starting with wet contact disinfection (which removes any remaining matrix) is crucial for overall effectivity on affected surfaces. A critical amount of disinfectant together with proper exposure time is necessary to efficacious decon. Complications come through air physics, which may cause the effective decontamination solutions to be ineffective. Bigger aerosol particles tend to settle from the air only a few meters from the source whereas small ones fly further but they carry a low volume of disinfectant and remain easier in the air for a long time. It is necessary to reach homogenous filling of space by aerosol or gas and combine it with indicators. We recommend a combination of chemical indicators with biological ones at least during optimization of the method for specific decontamination spaces.


How clean is clean should be defined, first, by the response agency in charge of the response for the expected purpose of use. Laboratory-developed decontamination methods need verification by field tests involving all significant factors. We recommend reviewing existing methods if they do not have evidence that they were already evaluated by this testing. Classical spraying techniques could be very efficient if significant variables are properly set. Modern methods of aerosol or gas decontamination could seem promising; however, they should be tuned for exact application to prevent investments in ineffective solutions. We recommend using bioindicators and biological burdens for this setup. Our experiments showed the need for mechanical wet decontamination of critical areas of indoor spaces prior to these methods.


Alan Gavel was born 1977 in Levoča, former Czechoslovakia. Alan graduated in 2001 at Masaryk University in Brno, Czech Republic in ecotoxicology and continued research programme in environmental chemistry. After short period on department of experimental phycology and toxicology at Institute of Botany of Czech Academy of Sciences his carrier turned to the public safety, radiological and biological preparedness and countermeasures. He joined the Population Protection Institute (part of the Czech Fire Rescue Service DG - Ministry of interior of the Czech Republic) in 2004. His work is divided among education of first responders, applied research and specialised intervention in the field (hazmat team). Subject matter is the detection of radioactive and nuclear materials and biopreparedness for hazardous biological agents (biological terorism). He is deputy commader and instructor of Czech OPCW courses „Advanced training course in civil defence against chemical weapons“.