Decontamination &

Chlorine Dioxide Gas

Dr. Henry S. Luftman Consultant – Odor Science

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

Microbial Agents microscopic, potential pathogens

•  Prions •  Bacterial endospores •  Protozoan cysts •  Mycobacteria •  Non-enveloped (naked) viruses •  Fungi and fungal spores •  Vegetative bacteria •  Enveloped viruses

Increasing Resistance

•  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

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)

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

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.

Ascospores  

Sexual  Reproduction  Hyphal  Growth  

Germination  

Conidiospores  

Spores  

Asexual  Reproduction  

Mold Life

Cycle

Vegetative Bacteria Active bacteria are called vegetative.

Examples: Staphylococcus aureus Streptococcus pyogenes Geobacillus

stearothermophilus Escherichia coli Neisseria meningitidis Salmonella spp.

Types of Disinfection

•  Chemical – our focus •  Radiation – ultraviolet light •  Thermal – an autoclave •  Filtration – a liquid filter

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

•  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.

Disinfectant Active Sites

Cytoplasmic Membrane

Peroxide/Peracetic acid Chlorine dioxide

Phenolics Quats

Proteins Peroxide/Peracetic acid

Chlorine dioxide Alcohols

Aldehydes Halogens Phenolics

DNA Aldehydes

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.

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.

•  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.

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

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

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.

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.

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

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

Items Within Space

•  Furniture •  Technical equipment •  Refrigerators, freezers •  Computers and other electronic devices •  Sinks, showers

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

Requirements for a Successful Decontamination (Cont.)

•  Disposal of disinfectant or residue? – Vent, isloate, clean

•  Validation of disinfection – Biological indicators

•  Material compatibility •  Safety

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

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

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

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, …

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

Formaldehyde Gas CH2OH– Issues

•  “Fall-out” residue – Added clean-up time

•  Carcinogen •  Potential residual odor •  Polymerization on cold surfaces •  Deactivated by some fungi

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

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

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

Chlorine Dioxide (CD) Properties:

Ø Yellow-Green Gas

Ø Water Soluble

Ø Boiling Point 11oC

Ø  Tri-atomic Molecule

Ø Molecular Weight 67.5

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

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)

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

•  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

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)

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

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.

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)

Issue Formaldehyde Gas

Hydrogen Peroxide Vapor

Chlorine Dioxide Gas

Sporocidal effectiveness + + +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 ScrubbingLimited development effort + - +Limited cost + - - / +Cycle Time (hr) 9 to 15 4 to 7 3 to 4

Non-corrosive +

Comparison

Widener Large Animal ICU/NICU

The 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.

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

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

Interior Views

Biological Indicators

20 Bacillus atrophaeus 40 Geobacillus stearothermophilus 40 Salmonella Newport (109 bacteria)

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)

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

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

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

Test BI Locations

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

Preparation BI    PLACEMENT  

SEALED  CABINET  

LEAK  TESTING  

Preparation

BI    PLACEMENT  

BI    RETRIEVAL  

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

Results

•  Method successfully validated on A2 console, A2 bench top, B1 and B2 cabinets

•  Chlorine dioxide added to National Sanitation Foundation protocol (2009)

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

Published works about Chlorine Dioxide as a

Decontaminant

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

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

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.

Aqueous Chlorine Dioxide

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

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.

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

Other CD Applications

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