The document discusses the properties and applications of chlorine dioxide gas for decontamination, highlighting its effectiveness against various pathogens, including spores, bacteria, and viruses. It compares chlorine dioxide with other disinfectants in terms of efficiency, safety, and application methods, emphasizing its role in bio-terrorism cleanup and environmental safety in controlled environments. Additionally, it outlines the requirements and challenges of effectively utilizing chlorine dioxide in various decontamination scenarios.

1. Decontamination
&
Chlorine Dioxide Gas
Dr. Henry S. Luftman
Consultant – Odor Science

2. Presentation
• Microbes – particularly fungi
• Decontamination
– Terms
– Types
– Decontaminants
• Chlorine Dioxide
– Properties
– Compared to Hydrogen Peroxide and others
• Chlorine Dioxide applications
– Non-fungal
– Fungal
• Review and Compare

3. Microbial Agents
microscopic, potential pathogens
• Prions
• Bacterial endospores
• Protozoan cysts
• Mycobacteria
• Non-enveloped (naked) viruses
• Fungi and fungal spores
• Vegetative bacteria
• Enveloped viruses
IncreasingResistance

4. • Thick-walled dormant form of some bacteria.
They are among the most resistant of all
microbes to:
– Chemicals
– UV light
– Drying
– Heat
• Think anthrax spores from 9-11
• Often used as a standard for validating sterilants and
high-level disinfectants
Bacterial Endospores

5. Fungi
• Fungi are plant-like organisms that lack
chlorophyll and do not photosynthesize. They
usually live on dead tissue, but can be
infectious. Examples include:
Mushrooms
Yeast
Filamentous (mold)

6. Fungi Terms
• Eukaryotic cells (nuclei, DNA)
• Cell wall with glycoproteins and polysacharides
(80%)
– Adds resistance to chemicals
• Requirements
– Organic nutrients: decaying matter, paper or
cardboard
– Moisture
• Mycoses – fungal infectious diseases

7. Fungi terms (cont.)
• Filamentous – molds (0.5 x 5 µm)
– Hyphae à mycelia (accumulation of cell walls)
– Trichophyton – dermatology issues
– Asperillus – infections. Produces aflatoxinà food
(contaminant, carcinogen) – most resistant to
chemicals
– Penicillium – rubratoxins à liver and kidney disease
– Stachybotys – mycotoxins à headaches, allergies,
sick building
• Unicellular – yeasts (8-10 µm)
• Spores
– Resistance > veg. mold > veg. yeast > veg. bacteria
– Conidia: asexual spore
– Ascospore: sexual (meiotic) spore, more resistant.

8. Ascospores
Sexual
Reproduction
Hyphal
Growth
Germination
Conidiospores
Spores
Asexual
Reproduction
Mold
Life
Cycle

9. Vegetative Bacteria
Active bacteria are called vegetative.
Examples:
Staphylococcus aureus
Streptococcus pyogenes
Geobacillus
stearothermophilus
Escherichia coli
Neisseria meningitidis
Salmonella spp.

10. Types of Disinfection
• Chemical – our focus
• Radiation – ultraviolet light
• Thermal – an autoclave
• Filtration – a liquid filter

11. Types of Disinfectants
• Sterilants can kill all microbes, spores and
viruses, given enough time.
• High-Level disinfectants kill all viruses,
most fungi and vegetative cells, but they may
not kill endospores reliably.
• Intermediate-Level disinfectants destroy all
vegetative cells including mycobacteria, fungi,
and most, but not all viruses. They cannot kill
endospores.
• Low-Level (General Purpose) disinfectants
destroy vegetative bacteria, except
mycobacteria, fungi and enveloped viruses

12. • Antiseptic – A substance that prevents or inhibits
the growth of microorganisms on living tissues.
• Sanitizer – A chemical agent that kills 99.999% of
a specific test bacterium in 30 seconds under
specified test conditions.

13. Disinfectant Active Sites
Cytoplasmic
Membrane
Peroxide/Peracetic acid
Chlorine dioxide
Phenolics
Quats
Proteins
Peroxide/Peracetic acid
Chlorine dioxide
Alcohols
Aldehydes
Halogens
Phenolics
DNA
Aldehydes

14. Disinfectant Vocabulary
• Viable – capable of living
• Pathogen – microorganism capable of
causing disease
– Bacteria - vegetative and sporal
– Virus
– Fungus – vegetative and sporal
– Prion
• Disinfection
– Chemical or physical inactivation of pathogenic
micro-organisms on inanimate surfaces.

15. Vocabulary (Continued)
• Decontamination
The use of physical or chemical means to remove,
inactivate, or destroy bloodborne pathogens on a
surface or item to the point where they are no
longer capable of transmitting infectious particles
and the surface or item is render safe for handling,
use, or disposal.
• Sterilization
Destruction of ALL microorganisms by procedure or
exposure to chemical or physical agents, or to
ionizing radiation.

16. • Biocide - (germicide) Kills all living organisms,
pathogenic and harmless.
• Sporicide - Destroys bacterial spores.
• Tuberculocide - Kills Mycobacterium tuberculosis
• Fungicide – Kills fungal spores.
• Bactericide – Kill pathogenic and harmless
bacteria, but not necessarily spores.
-Disinfection, Sterilization and Preservation, 5th ed., Block, S.S.

17. Vocabulary (Continued)
• Colony Forming Unit (CFU) – single
macroscopic colony formed after introducing
microorganisms into growth medium
• Biological Indicator (BI) – a monitor
impregnated with microbes to test the efficacy
of a decontamination event
• Log Reduction – Reduction of a microbial
population by 90%
– Example:Starting point 1,000,000
– 1 log 100,000
– 2 log 10,000
– 4 log 100
– 6 log 1

18. Properties of Ideal Disinfectants
• Broad spectrum
• High efficiency
• Unaffected by contaminants
• Nontoxic, non-corrosive, nonflammable
• Odorless
• Cheap
• Stable
• Environmentally friendly

19. What affects a disinfectant s efficiency?
• Surface condition – Rough and/or porous surfaces will be
more difficult to disinfect.
• Surface temperature – Higher temperatures will speed up
disinfection rates, but it will also increase evaporation.
• Organic load – Blood, sputum, bodily fluids, media, urine,
feces, etc. can protect and stabilize microorganisms.
Organics may also react with the disinfectant, consuming it.

20. What affects a disinfectant s efficiency?
• Contact time – Disinfectants that evaporate quickly may
require multiple applications to complete the contact time.
• Dried spills – Dried media, blood, etc. will decrease a
disinfectant's efficiency.
• Dirt, grease, or oils – All can protect microbes, and
grease and oil can repel water-based disinfectants.

21. Surface Decontamination
• Cleaning
– Physically removing dirt to towels or rinse
– Detergents best
• Sanitization
– Killing viables
• Bacteria – vegetative
• Viruses
• Mold / Fungus
• Bacteria - spores
– Alcohol, bleach, quats, phenols, peroxide, ClO2

22. Space Decontamination
• Exposing a confined space to a gas or vapor
with sporicidal capability
• Space can be a
– Room(s) or laboratory(ies)
– Building
– HVAC component or system
– Combination of any of the above
• Non technical space
– Home
– Business

23. Items Within Space
• Furniture
• Technical equipment
• Refrigerators, freezers
• Computers and other electronic devices
• Sinks, showers

24. Requirements for a
Successful Decontamination
• Disinfection – To what degree?
• Choice of disinfectant
• Penetration to all surfaces (optional ?)
• Penetration through HVAC system
(optional)
• Temperature and humidity control
• Containment of fumigant

25. Requirements for a Successful
Decontamination (Cont.)
• Disposal of disinfectant or residue?
– Vent, isloate, clean
• Validation of disinfection
– Biological indicators
• Material compatibility
• Safety

26. Safety
• If a decontaminant at a specified concentration
can kill bacterial spores, it cannot be good for
people
• Relevant Material Safety Data Sheets (MSDS s)
• Appropriate personal protective equipment for
the application
• Appropriate isolation from bystanders
• Monitors for residual gases

27. Disinfectant Phase
• Liquid (Beach, peroxide, aqueous CD,
quats, phenols, oils, alcohol, …)
– Ideal for accessible surfaces
– Can spray, line-of-sight decontamination
• Gas (CD, formaldehyde,
hydrogen peroxide (?))
– Ideal for spaces, 3 dimensional objects
– Containment?
• Aerosol – a middle ground

28. When are Gas
Decontaminations Needed?
• Cannot reach some surfaces with liquid or
fog
– Contaminated plenums
– Internal surfaces within equipment sets
– Porous surfaces
– Unassembled ductwork and HVAC systems
– Interstitial spaces
– Other hidden spaces

29. When are Gas Decontaminations
Needed? (cont.)
• Bacterial endospore contamination
– Difficult to maintain adequate contact time
with sporicidal liquid disinfectants
– Easier to validate decon via biological
indicators
• Material compatibility issues
– Liquids are wet and often corrosive
– Electronics, certain metals, …

30. Choice of Gas Decontaminants
• Formaldehyde Gas
• Hydrogen Peroxide Vapor
• Chlorine Dioxide Gas
• Others
– Methyl Bromide – Green House, bad with Al
– Ethylene Oxide – carcinogen, explosive
– Ozone – very corrosive, unstable
– Peracetic Acid Vapor – corrosive, unstable, high BP

31. Formaldehyde Gas CH2OH–
Issues
• Fall-out residue
– Added clean-up time
• Carcinogen
• Potential residual odor
• Polymerization on cold surfaces
• Deactivated by some fungi

32. Hydrogen Peroxide Vapor
(H2O2)
• Typically delivered by flash vaporization of
aqueous peroxide mixture
– The mixture is generally close to or above
saturation in air
• Mechanism: oxidation
• Required contact time less than
formaldehyde

33. HP Vapor Issues
• Instability of HP toward decomposition
– Flow pattern is critical to beat
decomposition rate
• Decomposition may block access of
decontaminant
• Condensation may cause control issues
– Heat-tracing or pre-thermal treatment

34. HP Vapor Issues (cont.)
• Cellulose materials absorb or decompose
– May effect decontamination or aeration
• Some material issues – nylon, cellulose,
copper, lead, iron oxide, epoxy
– Condensation may effect painted surfaces
• Capital equipment cost
• If delivered as aerosol, line-of-sight decon

35. Chlorine Dioxide (CD)
Properties:
Ø Yellow-Green Gas
Ø Water Soluble
Ø Boiling Point 11oC
Ø Tri-atomic Molecule
Ø Molecular Weight
67.5

36. Chlorine Dioxide Gas (ClO2)
• Mechanism: Selective oxidation (no
chloridation)
– alcohols, aldehydes, ketones, tertiary amines
and sulfur-containing amino acids
• Generated on site via reaction:
– Cl2(g) + 2NaClO2 à 2ClO2(g) + 2NaCl
– 5NaClO2 + 4H+(aq)à
4ClO2(g) + 2H2O + NaCl + 4Na+(aq)
• Humidification required for sporicidal work,
65-90% RH

37. Chlorine Dioxide Applications
Ø National Security Issues
Ø Post office anthrax cleanup
Ø Bio-terrorism
Ø Contamination elimination
Ø Samonella newport contamination (16 fatalities) at University of
Pennsylvania
Ø BioSafety Level applications (BSL1-2-3-4)
Ø Products and equipment entering/leaving controlled
environments
Ø Experiment change
Ø Food Safety and Food Spoilage Reduction
Ø Fungicide after Katrina
Ø Water Treatment
Ø Pulp and Paper (water treatment and paper bleaching)

38. Chlorine Dioxide Gas –
Advantages
• Safe by-products (oxygen and salt)
• No residue
• Not flammable / explosive
• True gas – no condensation issues
• Reputation for use in Anthrax
decontamination

39. • Is toxic – and it is an irritant to the eyes,
skin and respiratory system.
• Is noncorrosive to stainless steel, and is
compatible with most rubber and
plastics, and is nonflammable.
• It is not stable after preparation.
• Cost is more than bleach.
• It can be disposed of by
dilution in the sewer.
Chlorine dioxide

40. Chlorine Dioxide - Issues
• Less well-known or characterized
• Mild corrosion/discoloring to cold steel,
copper, brass at high concentrations
– Particularly in the presence of water
• Low PEL limit (0.1 ppm)

41. Sample CD Antimicrobial
Spectrum of Activity
Vegetative Bacteria:
Ø Staphylococcus aureus
Ø Pseudomonas aeruginosa
Ø Salmonella cholerasuis
Ø Mycobacterium smegmatis
Fungi:
Ø Aspergillus niger
Ø Candida albicans
Ø Trychophyton mentagrophytes
Bacterial Spores:
Ø Bacillus atropheus
Ø Bacillus stearothermophilus
Ø Bacillus pumilus
Ø Clostridium sporogenes
Viruses:
Ø Herpes simplex Type I (lipid)
Ø Polio Type II (non-lipid)
Ø Parvo Virus

42. Environmental Technology
Verification (ETV) Program
In 2002, EPA established the Building
Decontamination Technology Center at
Battelle. Battelle plans, coordinates, and
conducts verification tests of decontamination
technologies and reports the results to the
community at large. Information concerning
this specific environmental technology area
can be found on the Internet at http://
www.epa.gov/etv/centers/center9.html.

43. Environmental Technology
Verification (ETV) Program
Building Materials
• Industrial-grade carpet (IC)
• Bare wood (pine lumber) (BWD)
• Glass (GS)
• Decorative laminate (DL)
• Galvanized metal ductwork (GM)
• Painted (latex, flat) wallboard
paper (PW)
• Painted (latex, semi-gloss)
concrete cinder block (PC)

44. Issue Formaldehyde
Gas
Hydrogen Peroxide
Vapor
Chlorine Dioxide
Gas
Sporocidaleffectiveness + + +
Effective through HEPA
filters
+ + / ? +
Non Carcinogenic - + +
Toxicity (TWA PEL) 0.75 ppm 1.0 ppm 0.1 ppm
Humidity requirement (RH)
60-90%
30% (VHP) or
ambient (Clarus)
65-90%
No residue - + +
+ (VHP) / + /
? (Clarus) - (with chlorine)
Method of removal Neutralizer Catalytic breakdown Scrubbing
Limited development effort + - +
Limited cost + - - / +
Cycle Time (hr) 9 to 15 4 to 7 3 to 4
Non-corrosive +
Comparison

45. Widener Large Animal ICU/
NICUThe image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

46. George D. Widener
Large Animal Hospital,
New Bolton Center
• Part of University of Pennsylvania
• Largest equine hospital in U.S.
~ 6000 patients per year
• Possible Salmonella outbreak detected in
March 2004
– Increased environmental surveillance
– Fecal samples from all animals
– Improved collection and bacteria isolation methods

47. Widener Large Animal ICU/
NICU
• Center quarantined in May 2004, following
16 equine fatalities
• Multi-drug resistant Salmonella Newport
identified
– Gram-negative, non-spore forming bacteria
– No human infections noted
• Multiple attempts at surface
decontamination of ICU/NICU

48. Interior Views

49. Biological Indicators
20 Bacillus atrophaeus
40 Geobacillus stearothermophilus
40 Salmonella Newport (109 bacteria)

50. Biological Indicator Results
• G. stearothermophilus
(log reduction)
– 32/40 strips – no growth (>log 5.4
kill)
– 7/40 – 1 CFU (log 5.4 kill)
– 1/40 – 2 CFU (log 5.1 kill)

51. Validation Study for Biological
Safety Cabinets (BSCs)
• BSCs used to simultaneously
– protect biological material inside from external
contamination
– protect user and lab from biological material
being manipulated
• HEPA filtration, blower(s), plenums
• Found in medical, veterinary,
pharmaceutical, and biotech labs

52. BSC Decontamination
• Formaldehyde gas had been the standard
method for >30 yrs
• National Sanitation Foundation holds the
standard for BSC maintenance
• Until 2009, only formaldehyde considered
pre-validated
• Study to confirm similar status to CD

53. NSF Biological Indicator
Criteria (for each trial)
• 6 pairs of BIs
– 3 pairs in downstream side of exhaust
HEPA filter (center and opposite corners)
– 1 pair within positive pressure plenum
– 1 pair beneath cabinet workspace
– 1 pair in center of upstream (dirty) side of
down flow HEPA

54. Test BI Locations

55. Validation Experiments
• Experimenters – Micro-Clean
• BSC Manufacturer – NuAire
• Location – NuAire (Plymouth, MN)
• Conditions:
– 60 – 75% RH
– CD amount - 0.1 g/ft3
– Exposure time – 80 min

56. Preparation
BI
PLACEMENT
SEALED
CABINET
LEAK
TESTING

57. Preparation
BI
PLACEMENT
BI
RETRIEVAL

58. Results – Method 1
Repeat Study
Trial # 15 17 19 14 16 18 20
Dose 3.0 3.7 3.7 2.6 3.7 3.3 3.3
Position
1 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0
4 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0
6 0 0 0 0 0 0 0
P P P P P P P 0, 1, 2 = # positive
BIs out of 2
A2 -6' Console B1 – 6' Console
Dose – mg-hr/L
PASS PASS

59. Results
• Method successfully validated on A2
console, A2 bench top, B1 and B2
cabinets
• Chlorine dioxide added to National
Sanitation Foundation protocol (2009)

60. Other Large Scale CD Events
• 9-11 anthrax spore decontamnations
– Hart Senate Building, Miami Media Center, NJ
Post Office
• Hospital decontamination (~2008)
• Multiple anti-fungal fumigations in New
Orleans following Katrina
Note: It s all the same Chlorine Dioxide

61. Published works about
Chlorine Dioxide as a
Decontaminant

62. Comparison of Multiple Systems for Laboratory
Whole Room Fumigation
• Applied Biosafety 16 (2011) 139-157
• Alan Beswick et al., The Health and Safety
Laboratory, UK
• Tested HP(3), formaldehyde, CD and ozone vs 3
bacterial spore and virus.
• Tested on bench, in centrifuge, on floor, within
spill
• CD at top for almost all, beating HP by more
than x100 on Mycobacterium fortuitum,
particularly in spill

63. Effect of CD Gas on Fungi and Mycotoxins
Associated with Sick Building Syndrome
• Appl. Environ Microbiol 71 (2005) 5399
• S.C.Wilson et al, Texas Tech Univ.
• Stachybotrys chartarum, Cladosporium
cladosporiodes, Penicillium chrysogenum,
Chaetomium globosum
• 500 to 1000 ppm CD via dry chemicals in water
• 100% inactivation of conidia (asexual spores),
90% of ascospores (sexual, meiotic).
• While Stachy inactivated, remains toxic

64. Inactivation of Stachybotrys chartarum
grown on gypsum board using aerosolized
chemical agents.
• J. Environ. Eng. Sci. 5 (2006) 75
• Andrew Wagner et al., Univ. of Cincinnati
• 10% bleach (0.6% NaOCl), thiabendazole,
0.05% CD (aq), copper(II) sulfate – 4 and 8 hr.
• Only bleach and CD worked (~100%)
• Aerosols delivered directly to dry wall surface –
not real world for regular building.

65. Aqueous Chlorine Dioxide

66. Microorganisms Treatment
conditions
Log
Reduction
Surfaces
E. coli
L. monocytogenes
0.6 mg/l - 30 min 7.3
6.3
Green peppers (Han et al.
2000, 2001)
E. coli
L. monocytogenes
4.0 mg/l – 10 min
4.8 mg/l – 10 min
5.5
4.8
Apples (Du et al.
2002a and b)
E. coli
L. monocytogenes
0.6 mg/l –15 min 5.6 Strawberries (Han and
Linton 2002)
Salmonella spp.
E. coli
0.5-1 mg/l –10 min 3-5 Cantaloupes (Han et al.)
L. monocytogenes 0.2 mg/l - 30 min 2 Lettuce (Dlima and Linton
2002)
Efficacy of ClO2 gas treatment in reducing
microorganisms

67. Treated with 10 mg/l Chlorine
dioxide gas for 10 min and
stored for 6 weeks at 4oC
Untreated and stored for 6
weeks at 4oC
Han Y., Linton, R.H., and Nelson, P.E., Inactivation of Escherichia coli O157: H7 and Listeria
monocytogenes on strawberry by chlorine dioxide gas, annual meeting of Institute of Food
Technologists, Anaheim, CA, 2002.

68. Non-Experimental CD/MODEC Comparison
• Both have been shown as fungicidal under
laboratory conditions
• The real world is not under laboratory
conditions
– Surfaces are porous: Liquids and most
aerosols will not penetrate
– Surfaces are not all glass: Some chemicals,
such as hydrogen peroxide, decompose on
paper
– Water based decontaminants corrode

69. Other CD Applications
• Janitorial sanitation • Antiseptic
• Odor elimination • Produce protection
• Restoration • Water purification
• Insecticide
• Instrument sterilization
• Hospitals, Locker rooms, Restaurants
• Anti-Fungal station