EPA 600/R-14/332 I October 2014 I www.epa.gov/research United States Environmental Protection Agency Parametric Testing of Decontamination Chemistries to Guide Decontaminant Selection I: Peracetic Acid • * Office of Research and Development National Homeland Security Research Center ------- EPA/600/R-14/332 October 2014 Parametric Testing of Decontamination Chemistries to Guide Decontaminant Selection I: Peracetic Acid National Homeland Security Research Center Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, NC 27711 ------- Disclaimer The United States Environmental Protection Agency, through its Office of Research and Development's National Homeland Security Research Center, funded and directed this investigation through EP-C-09- 027 WAs 4-76 and 5-76 with ARCADIS U.S., Inc. This report has been peer and administratively reviewed and has been approved for publication as an Environmental Protection Agency document. It does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be inferred. This report includes photographs of commercially available products. The photographs are included for purposes of illustration only and are not intended to imply that EPA approves or endorses the product or its manufacturer. Environmental Protection Agency does not endorse the purchase or sale of any commercial products or services. Questions concerning this document or its application should be addressed to: Sang Don Lee, Ph.D. Decontamination and Consequence Management Division National Homeland Security Research Center U.S. Environmental Protection Agency (E-311-K) Office of Research and Development 109. T.W. Alexander Drive Research Triangle Park, NC 27711 Phone:919-541-2973 Fax:919-541-4531 E-mail:lee.sangdon@epa.gov ------- Acknowledgments Contributions of the following individuals and organization to this report are gratefully acknowledged: EPA Project Team: Lukas Oudejans, EPA Alternate Work Assignment Manager, EPA National Homeland Security Research Center, Decontamination and Consequence Management Division, Research Triangle Park, NC. Worth Calfee, EPA Biocontaminant Laboratory Manager. National Homeland Security Research Center, Decontamination and Consequence Management Division, Research Triangle Park, NC. EPA Peer reviewers: Tim Dean, EPA National Risk Management Research Laboratory (NRMRL), Air Pollution Prevention and Control Division, Research Triangle Park, NC. Matthew Magnuson EPA National Homeland Security Research Center Water Infrastructure Protection Division, Cincinnati, OH Tiffany Yelverton, EPA National Risk Management Research Laboratory (NRMRL), Air Pollution Prevention and Control Division, Research Triangle Park, NC. ARCADIS US, Inc. Abderrahmane Touati, Project Manager. ARCADIS U.S., Inc. Gayatri Snigdha, Engineer. ARCADIS U.S., Inc. ------- Table of Contents Disclaimer 1 Acknowledgments 2 Table of Contents 3 List of Figures 5 List of Tables 6 List of Acronyms and Abbreviations 7 Executive Summary 9 1 Introduction 11 1.1 Process 12 1.2 Project Objectives 12 1.3 Experimental Approach 13 2 Materials and Methods 14 2.1 Task 1: PAA Formulation Characterization 14 2.1.1 In-house PAA Formulations with Sulfuric Acid 15 2.1.2 In-house PAA Formulations without Sulfuric Acid 17 2.1.3 In-house PAA Formulations with Sulfuric Acid Derived from Chemicals Available OTC 18 2.1.4 Modified AHP Decontamination Formulation Developed by SNL 20 2.1.5 Diluted Hydrogen Peroxide Solution 20 2.2 Task 2: Method Development for Neutralization 20 2.2.1 Standard Neutralization Test Method 21 2.2.1.1 Test A: Neutralizer Effectiveness 21 2.2.1.2 Test B: Neutralizer Toxicity 22 2.2.1.3 TestC: Organism Viability 22 2.2.1.4 Test D: Test Material Control 22 2.2.2 Sampling Frequency for Neutralization Testing 22 2.3 Task 3: Liquid-Liquid Decontamination Testing 23 2.3.1 Efficacy Testing 23 2.3.2 Sampling Frequency for Decontamination Testing 24 2.4 Task 4: Surface Decontamination Testing 26 2.4.1 Spray Down Technical Approach 26 2.4.2 Test Coupon Preparation and Inoculation - Spray Down Approach 27 2.4.3 Spray Down Neutralization Method Evaluation 27 ------- 2.4.4 Spray Down Decontamination Procedure 28 2.4.5 Spray Down Decontamination Test Matrix 29 2.5 Task 5: Material Compatibility Evaluation 31 3 Sampling and Analytical Procedures 32 3.1 Sampling Procedures 32 3.2 Sample Extraction/Analysis 32 3.2.1 Liquid-Liquid Decontamination 32 3.2.2 Surface Decontamination 32 3.3 Sample Analysis 32 3.4 Data Analysis 33 3.5 Efficacy Testing 33 3.6 Decontamination Efficacy 33 3.7 Sample Preservation 35 3.8 Holding Times 35 3.9 Sample Archival 35 3.10 Steady State Conditions 35 3.11 PAA and HP Characterization Measurements 35 4 Results and Discussion 36 4.1 Formulation Characterization Results 36 4.1.1 "In-House" PAA Formulations 36 4.1.2 Modified AHP Decontamination Formulation 38 4.1.3 Over-The-Counter (OTC) In-house PAA Formulation 38 4.2 Neutralization Testing Results 40 4.2.1 Neutralization Effectiveness 40 4.2.2 Neutralizer Toxicity 41 4.2.3 Test Material Control 42 4.3 Task 3: Liquid-Liquid Decontamination Testing 43 4.3.1 In-House PAA Formulation with Sulfuric Acid 43 4.3.2 In-House PAA Formulation without Sulfuric Acid 45 4.3.3 Modified AHP Formulation 47 4.3.4 Diluted Hydrogen Peroxide Formulation 48 4.3.5 OTC Formulation 49 ------- 4.3.5.1 Liquid-Liquid Decontamination Approach 49 4.3.5.2 Surface Decontamination Approach 50 4.4 Material Compatibility Tests 53 4.5 Results Summary 56 5 Quality Assurance 57 5.1 Sampling, Monitoring, and Analysis Equipment Calibration 57 5.2 Data Quality Objectives 58 5.3 Acceptance Criteria for Critical Measurements 58 5.4 Quality Assurance (QA)/Quality Control (QC) Checks 60 5.5 Data Quality Audits 61 5.6 QA/QC Reporting 61 6 References 62 List of Figures Figure 2-1. Temporal Product/Reactant Evolution (modeled) for PAA Formulation with Acetic Acid 16 Figure 2-2. Temporal Product/Reactant Evolution (modeled) for PAA Formulation without Acetic Acid 18 Figure 2-3. Temporal Product/Reactant Evolution (modeled) for the OTC PAA Formulation 19 Figure 2-4. Efficacy Testing Sequence Flow Chart 25 Figure 2-5. Front View of Spray Apparatus with Orifice Plate 26 Figure 4-1. Temporal Product/Reactant Evolution for the In-House PAA Formulation with SulfuricAcid 37 Figure 4-2. Temporal Product/Reactant Evolution - In-House PAA without SulfuricAcid 37 Figure 4-3. PAA and HP Concentration for the Modified AHP Formulation 38 Figure 4-4. Temporal Product/Reactant Evolution - OTC PAA 39 Figure 4-5. Temporal PAA Degradation over Time for the OTC and PAA with Sulfuric Acid Formulations 40 Figure 4-6. Temporal Efficacy of the In-House PAA Formulations with SulfuricAcid 45 Figure 4-7. Temporal Efficacy of the In-House PAA Formulations wthout SulfuricAcid 47 Figure 4-8. Corrosion on Carbon Steel with pH ~2 using Procedure 1 (Post-Rinse) 53 Figure 4-9. Front Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse) 53 ------- Figure 4-10. Back Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse) 54 Figure 4-11. Front Surface of Carbon Steel with pH 6 using Procedure 2 (No Post-Rinse) 54 Figure 4-12. Galvanized Metal with pH 2 using Procedure 1 (Post Rinse) 55 Figure 4-13. Salt Deposition on Carbon Steel and Stainless Steel with pH 6 using Procedure 1 55 Figure 4-14. Salt Deposition on Stainless Steel and Aluminum with pH 6 using Procedure 2 56 List of Tables Table 2-1. Description and Sources of Compounds used in the PAA Formulations 15 Table 2-2. Determination of Recipe Formulation - PAA with Sulfuric Acid Formulation (SA) 16 Table 2-3. Determination of Recipe Formulation - PAA without Sulfuric Acid Formulation (AD) 17 Table 2-4. Determination of Recipe Formulation - OTC PAA Formulation 19 Table 2-5. AHP Decontamination Solution Recipe 20 Table 2-6. Components of Tests Ato D 21 Table 2-7. Sampling Measurements for Neutralization Testing 22 Table 2-8. Sampling Measurements for Decontamination Testing 24 Table 2-9. Coupon Material Specifications 27 Table 2-10. OTC PAA Formulation -Spray Duration and Frequency (Series 1) 29 Table 2-11. OTC PAA Formulation -Spray Duration and Frequency (Series 2) 30 Table 2-12. Neutralizer Amounts Added To Samples -Spray Down Approach (Series 1) 31 Table 4-2. Neutralizer Toxicity (Test B) 42 Table 4-3. Test Material Control (Test D) 42 Table 4-4. Test Parameters Measurements Verification 43 Table 4-5. In-house PAA Formulation with Sulfuric Acid - Efficacy Testing 44 Table 4-6. In-house PAA Formulation without Sulfuric Acid - Efficacy Testing 46 Table 4-7. Modified AHP Formulation - Efficacy Testing 48 Table 4-8. Diluted HP Formulation - Efficacy Testing 49 Table 4-9. OTC PAA Formulation - Efficacy testing (Liquid-Liquid Decontamination) 49 Table 4-10. Test Parameters Measurements Verification (Series 1) 50 Table 4-11. Test Parameter Measurement Verification (Series 2) 50 Table 4-12. OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series 1) 51 Table 4-13. OTC PAA Formulation - Efficacy testing (Surface Decontamination -Series 2) 52 Table 5-1. Analysis Equipment Calibration Frequency 57 6 ------- Table 5-2. Critical Measurement Acceptance Criteria 59 Table 5-3: QA/QC Sample Acceptance Criteria 61 List of Acronyms and Abbreviations AA AD AHP APPCD ATCC B. Ba BSC CPU DCMD D-Value Dl DQI DQO DTRL EPA EtO HP HP HSRP K2C03 NaHC03 OH- OOH" LR MOP N NA NE NHSRC NIST NT Acetic Acid "In-house" PAA formulation without sulfuric acid Activated Hydrogen Peroxide Air Pollution Prevention and Control Division American Type Culture Collection Bacillus Bacillus atrophaeus Biosafety Cabinet Colony Forming Unit(s) Decontamination and Consequence Management Division Refers to decimal reduction time and is the time required at a certain temperature to kill 90% of the organisms being studied Deionized Data Quality Indicator Data Quality Objective Decontamination Technologies Research Laboratory U. S. Environmental Protection Agency Ethylene Oxide Hydrogen Peroxide Hydrogen Peroxide solution alone without PAA Homeland Security Research Program Potassium Carbonate Sodium bicarbonate (baking soda) hydroxyl free radicals Peroxy anion Log Reduction Miscellaneous Operating Procedure Normal (Equivalent per liter) Not Applicable Neutralizer Effectiveness National Homeland Security Research Center National Institute of Standards and Technology Neutralizer Toxicity ------- oc OTC PAA pAB PBST PPE QA QAC QAPP QC RH RSD RTP SA SD SNL STS ISA UV WA WACOR X "In-house" PAA formulation with sulfuric acid derived from chemicals available over-the-counter Over-the-Counter Peracetic Acid pH-Adjusted Bleach Phosphate Buffered Saline with 0.05% TWEEN®20 Personal Protective Equipment Quality Assurance Quaternary Ammonium Compound Quality Assurance Project Plan Quality Control Relative Humidity Relative Standard Deviation Research Triangle Park "In-house" PAA formulation with sulfuric acid Modified AHP decontamination formulation developed by SNL Sandia National Laboratories Sodium Thiosulfate Tryptic Soy Agar Ultraviolet Work Assignment Work Assignment Contracting Officer Representative Stoichiometric equivalent ------- Executive Summary The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program (HSRP) helps protect human health and the environment from adverse impacts of terrorist acts by carrying out performance tests on homeland security technologies. The primary objective of this effort is to develop a low-tech Bacillus anthracis decontamination method that would be beneficial for numerous building interiors for effective decontamination under various environmental conditions. This research effort focuses on low-tech peracetic acid (PAA) formulations that can be used as a green alternative to low-tech chlorine-based decontamination applications and the less potent antimicrobial activated hydrogen peroxide (AHP). The low-tech PAA must be effective against microorganisms including bacterial spores and can be prepared in situ from over-the-counter (OTC) ingredients prior to use for a decontamination event. This project evaluated key parameters to decontaminate a nonpathogenic bacterial species Bacillus atrophaeus (Ba), a simulant of virulent and toxic B. anthracis, using "low-tech" expedient decontaminants such as PAA and AHP. Four PAA formulations, as well as a hydrogen peroxide (HP) solution without PAA, were tested to determine their respective decontamination effectiveness when tested against direct inoculation of Bacillus spores. The PAA formulations included an in-house solution prepared with and without sulfuric acid, a modified AHP solution developed by Sandia National Laboratories (SNL), and a fourth solution derived from OTC. The target stock solution concentration for the in-house and the modified AHP formulations were set at 1.5 % PAA, which was then diluted successively to encompass a full range of PAA test concentrations from 0.001 % to 1 %. The in-house PAA formulations with sulfuric acid were found to reach maximum PAA concentration and equilibrium within six days, while the formulation without sulfuric acid reached the maximum target PAA concentration within 31 days. The OTC PAA formulation was prepared in a single 1- liter batch and reached a maximum PAA equilibrium concentration of 0.07 % PAA in just over one day. The modified AHP formulation reached its target PAA concentration of 1.5 % immediately after mixing. However, the solution was highly unstable and the concentration of both PAA and HP dropped within four hours. The diluted HP antimicrobial solution was tested at 4.5 % HP concentration. This value is the same estimated concentration of HP in the 1 % PAA solutions. Once equilibrium was reached, all diluted solutions were observed to be stable overtime with minor shifts in PAA and HP concentrations (less than 10 % change in concentration over a period of weeks); note that the AHP solution was not monitored beyond the four-hour suggested use time). The first phase of the project evaluated the liquid inoculation/liquid decontamination solution approach for decontamination efficiency of all five formulations at different PAA concentrations. The results from the first phase of the project demonstrated a near complete decontamination at a two-minute exposure time with the 1 % PAA in-house formulation. The colony forming unit (CPU) recovery between the two- and eight-minute exposure time lies within the uncertainty of the measurement technique, which suggests a calculated D-value(linear interpolation) for this formulation of less than 0.26 min. The 0.1 % PAA also showed promise with an average D-value calculated to be 1.8 min, which suggests a 6-Log CPU reduction (LR) within an exposure time of about 10 minutes. The 0.01 % and 0.001 % PAA formulation were found to be ineffective with D-values greater than 18 minutes. ------- The liquid-liquid decontamination approach using the modified AHP formulation resulted in greater than 6 LR of spores when the concentration of the formulation was at 1 % PAA. However, the 0.1 % dilution proved to be less effective, even with extended exposure times. The diluted HP (4.5 % HP, no PAA) formulation was found to be ineffective with an LR of less than 0.5, suggesting that the sporicidal ingredient of the PAA solutions is the PAA itself. The averaged D-value for the diluted HP formulation was 18 min, which suggests that this product may not be a practical sporicide for field application. The liquid- liquid decontamination approach using the OTC formulation (0.07 % PAA) provided greater than 6 LR for exposure times of 8 minutes or longer. As this solution is made from OTC common household products, this formulation could be a promising decontaminant for use by the general population. However, the PAA solution is highly acidic (pH 1.7), which could potentially corrode some surfaces and potentially be hazardous to the users. The OTC PAA (0.07 % PAA) surface decontamination efficacy was evaluated using a spray-down approach on coupons of carpet, wood, glass, vinyl flooring, and concrete. These tests demonstrated that increasing exposure time combined with frequent spraying resulted in greater decontamination efficacy as compared to multiple sprays at the same time. However, the overall surface decontamination efficacy for the OTC formulation was not sufficient to achieve 6 LR. These lower surface decontamination efficiencies were due to the high degradation of the OTC PAA concentration observed during the spraying. The surface decontamination approach can be improved significantly if the OTC formulation is further stabilized by using laboratory grade ingredients or stabilizing the formulation by using other OTC ingredients. Further studies will be necessary to determine the cause of the OTC degradation. The in-house PAA formulations (PAA with and without sulfuric acid and OTC components) are acidic (pH 1.7). To analyze the acidity effect of these formulations, a material compatibility test was designed to assess the corrosion or changes of low PAA formulations at different pH levels. This study was performed on coupons made of stainless steel, galvanized metal, aluminum, and carbon steel. These coupons, tested in duplicate, followed two test procedures: (1) Post-rinse procedure and (2) No post-rinse procedure. In the post-rinse procedure, two coupons of each material were immersed in the original OTC PAA (0.07 % PAA) formulation. They were then rinsed with deionized (Dl) water and left to dry after a contact time of 30 minutes with the decontaminant. In the no post-rinse procedure, two coupons of each material were immersed in OTC formulation and then left to dry without a Dl water rinse after a contact time of 30 minutes with the decontaminant. No post-rinse materials appeared to have salt deposits (arising from the food grade baking soda added as an acid neutralizerto the OTC PAA formulation to bring the solution to a neutral pH). Materials that did follow a post-rinse procedure did not show any significant except for the carbon steel which was observed to have reacted most vigorously to the decontamination solution (with both procedures), with visible amounts of rust and corrosion on its surface. Little change was observed on the surfaces of stainless steel and aluminum coupons that were post- rinsed after being treated with the decontamination solutions. Typical indoor room conditions (71 °F and 37 % RH) were observed throughout the duration of this material compatibility test. 10 ------- 1 Introduction This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of Research and Development's (ORD) Homeland Security Research Program (HSRP) by providing information relevant to the decontamination of areas contaminated as a result of an act of terrorism. Under Homeland Security Presidential Directives (HSPD)-5, 7, 8, and 10, the EPA, in a coordinated effort with other federal agencies, is responsible for "developing strategies, guidelines, and plans for decontamination of equipment, and facilities" to mitigate the risks of contamination following a biological agent contamination incident. EPA's National Homeland Security Research Center (NHSRC) aims to help EPA address the mission of the HSRP by providing expertise and products that can be widely used to prevent, prepare for, and recover from public health and environmental emergencies arising from terrorist threats and incidents. One of NHSRC's missions is to provide expertise and guidance on the selection and implementation of decontamination methods and provide the scientific basis for a significant reduction in the time, cost, and to address the complexity of decontamination activities. Quick, effective, and economical decontamination methods that have the capacity to be employed over wide areas (outdoor and indoor) are one specific focus of this research program. This project's aim is to assess the effectiveness of in situ decontamination methods using peracetic acid (PAA) solutions formulated from over-the-counter (OTC) products. The detrimental effect these decontamination solutions have on various surfaces (e.g., corrosion) is also being assessed. Decontamination can be defined as the process of inactivating or reducing contaminants in or on humans, animals, plants, food, water, soil, air, areas, or items through physical, chemical, or other methods to meet a cleanup goal. In terms of the surface of a material, decontamination can be accomplished by physical removal of the contaminant or via inactivation of the contaminant with antimicrobial chemicals, heat, ultraviolet (UV) light, etc. Physical removal could be accomplished via in situ removal of the contaminants from the material or by physical removal of the material itself (i.e., disposal) [1~51. Similarly, inactivation of the contaminant can be conducted in situ or after removal of the material for ultimate disposal[1 2 4 5I. Following the 2001 anthrax incidents, a combination of removal and in situ decontamination was used [1 2I. The balance between the two procedures was facility-dependent and factored in many issues (e.g., the physical state of the facility). Such remediation was unprecedented for the United States Government, and no wide-area decontamination techniques had been proven effective at the time against spores of Bacillus anthracis. The cost of disposal proved to be significant, and finding an ultimate disposal site was complicated by the nature of the waste. Since 2001, a primary focus for facility remediation has been improving the effectiveness and practical application of in situ decontamination methods and evaluating waste treatment options to be able to provide the information necessary to optimize the decontamination/disposal paradigm. This optimization has a significant impact on reducing the cost of and time for the remediation effort. Low-tech PAA formulations can be used as a method alternative to low-tech pH Adjusted Bleach (pAB) and other chlorine-based decontamination applications and to the less effective antimicrobial activated hydrogen peroxide (AHP). Peroxides (compounds which contain two oxygen atoms bonded together) are strong oxidants that offer an environmentally friendly alternative to the toxic and corrosive chlorine-based decontaminants [6 7I. The sporicidal effectiveness of hydrogen peroxide (HP) solutions is due to the peroxy anion (OOH~) and hydroxyl free radicals (OH-)[7 8I. Another peroxy compound that is commonly 11 ------- used is PAA (CH3CO(OOH))[61. PAA is often added as a supplemental oxidizing agent in mixtures with HP. PAA is effective against all microorganisms, including bacterial spores, because of its high oxidizing potential, and is often added as an oxidizing agent in mixtures with HP. The co-presence of HP and PAA have a synergistic sporicidal efficacy [6]. Due to stringent shipping and handling requirements, PAA is often generated in situ, prior to decontamination, using AHP solutions. This method has many variations, but the foundation of the chemistry is the same: generation of PAA through the hydrolysis of an acetyl donor, with a specifically formulated HP solution as an oxygen source [7 8I. A significant variable affecting practical application of PAA is that different formulations take different amounts of time for the PAA to be formed, because formation of PAA is an equilibrium process. 1.1 Process This project examined the ability of five PAA formulations to remove B. anthracis surrogate spores (8. atrophaeus (Ba)) from samples: three in-house PAA formulations (with and without sulfuric acid, and with sulfuric acid derived from chemicals available OTC); a modified AHP decontamination formulation developed by Sandia National Laboratories (SNL) under the Wide Area Recovery and Resiliency Program research efforts; and a hydrogen peroxide (HP) solution alone without PAA (with the same estimated HP concentration as the 1 % PAA solution). In the first phase of the project, the spores were directly inoculated into the decontaminant samples and exposed to the decontaminant for contact times ranging from two minutes to 20 minutes. Because these formulations were generated in s/fujust prior to decontamination testing, an understanding of the time to reach equilibrium for simple PAA and the relationship between PAA concentration, exposure time, and sporicidal effectiveness was developed. In addition to looking at the decontamination efficacy, this effort also qualitatively assessed the tendency of the decontaminant to corrode different building materials by visual inspection and gravimetric analysis. The subsequent phase of the project evaluated the decontamination potential and physical effects of the OTC decontamination formulation on designated test coupons prepared from materials typical of surfaces found in an indoor environment. 1.2 Project Objectives The primary objectives were: • To develop a simple PAA solution that could easily be generated in the field, within a realistic time (i.e., a few hours) to reach equilibrium; • To develop an understanding of the time to reach equilibrium for simple PAA and the relationship between PAA concentration, exposure time, and sporicidal effectiveness; • To determine the fate of the spores during the decontamination process -LR of B. anthracis surrogate spores on contaminated liquid media; and • To assess the corrosion produced by application of low PAA formulations derived from OTC products. The operational parameters of decontamination (e.g., pH and temperature of decontamination solution within the pot life of the decontamination solution, the concentration of the PAA formulations, and the exposure times) were considered important for understanding the sporicidal activity of the PAA-based decontamination process and were characterized in addition to sporicidal effectiveness. 12 ------- 1.3 Experimental Approach The general testing approach consisted of the following Tasks: • Task 1: PAA Formulation Characterization The concentration of active ingredients and other characteristics were measured overtime for four formulations of PAA. • Task 2: Method Development for Neutralization of Decontaminant Solutions Neutralization methods to inactivate the sporicides were tested for compatibility with spore enumeration methods. • Task 3: Liquid-Liquid Decontamination Testing The direct liquid inoculation/liquid decontamination solution approach was evaluated forsporicidal effectiveness of the target decontamination solutions at different concentrations. • Task 4: Surface Decontamination Testing Surface decontamination testing was conducted using an OTC formulation that was applied to coupons of carpet, wood, glass, vinyl flooring and concrete in the form of spray. • Task 5: Material Compatibility Evaluation Visual and gravimetric assessments of the PAA formulation material compatibility were undertaken to qualitatively investigate any changes/corrosion on the surfaces of different material types. 13 ------- 2 Materials and Methods This section describes the test facilities and equipment, general decontamination approach, and test conditions, test materials and methods that were used to evaluate the data related to the project objectives. Testing was conducted at the EPA RTP facility. The research presented in this report characterized the decontamination efficacy of diluted PAA formulations against Ba (American Type Culture Collection [ATCC] 9372) spores. The overall effectiveness of the methods used in this study was determined by the ability of the method to reduce the number of and/or to inactivate the spores of Ba in the PAA solution. 2.1 Task 1: PAA Formulation Characterization Each stock solution PAA formulation was prepared in a single one-liter batch, and the PAA and HP concentration measurements of the stock solutions were monitored overtime until each of the solutions reached a maximum PAA equilibrium concentration for a set of initial mixture conditions. The target stock solution concentration was set in excess of the highest target test PAA solution, which was then diluted successively to encompass a full range of PAA concentrations from 0.001 % to 1 %. The stock solutions and the corresponding diluted solutions were measured overtime to determine the kinetics and stability of the mixture. Stability, in this case, is defined as a pseudo-steady state of a target solution in which variation of the PAA concentration is less than 10 % over a period of one hour. The following five formulations were characterized in this study: 1) In-house PAA Formulation with Sulfuric Acid (SA) 2) In-house PAA Formulation without Sulfuric Acid (AD) 3) In-house PAA Formulation with Sulfuric Acid Derived from Chemicals Available OTC (OTC) 4) Modified AHP Decontamination Formulation Developed by SNL (SD) 5) HP solution alone without PAA (same estimated HP concentration as the 1 % PAA solution) Descriptions and sources of the ingredients used in the formulations are provided in Table 2-1. The formulations are described further in the following subsections. 14 ------- Table 2-1. Description and Sources of Compounds used in the PAA Formulations Formulat ion 1.5% Peracetic Acid (SA and AD) OTC Modified AHPby SNL(SD) Diluted HP (HP) Ingredients Hydrogen Peroxide Glacial Acetic Acid Sulfuric Acid Dl Water Hydrogen Peroxide Topical Solution White Distilled Vinegar Battery Acid (Sulfuric Acid) Hydrogen Peroxide Maquat(MC1412-80%E) Potassium Carbonate (K2C03) Ethanol Triacetin Hydrogen Peroxide CAS Number 7722-84-1 64-19-7 7664-93-9 7732-18-5 7722-84-1 64-19-7 7664-93-9 7722-84-1 648424-85-1 64-17-5 68439-90-3 7732-18-5 584-08-7 64-17-5 102-76-1 7722-84-1 Label Concentration 50% 99.7+ % 1 Normal (N) 100% 3% 5% 30^3 % (by wt) 50% 80-82 % (by wt) Quaternary Ammonium Compounds (QACs) >99 % 99.5 % 99% 50% Origin / Manufacturer Fisher Scientific Acros Organ ics Fisher Scientific Produced in-house using Dl Water System from Purologix Water Services Inc. Walgreens Heinz East Penn Manufacturing Company, Inc Fisher Scientific Mason Chemical Fisher Scientific Acros Organics Acros Organics Fisher Scientific City/State Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Holly Springs, NC Deerfield, IL Pittsburgh, PA Lyons, PA Pittsburgh, PA Arlington Heights, IL Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA Pittsburgh, PA 2.1.1 In-house PAA Formulations with Sulfuric Acid This in-house PAA formulation was prepared by reaction of acetic acid (AA) and HP [9 10 13]. The rate at which equilibrium is achieved is accelerated by adding a strong acid catalyst (sulfuric acid). The target decontamination formulation was prepared by adding the AA to a specially cleaned volumetric flask (triple rinsed with Dl water followed by a one-hour autoclave sterilization cycle at 121 °C) with concentrated sulfuric acid, before adding HP to the flask contents. An initial stock solution batch of one liter of the PAA formulation was prepared to achieve a high PAA concentration of 1.5 %. This solution was then diluted to achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001 % forsporicidal testing. A mathematical kinetic model based on rate constants determined by other researchers [13] was developed in this study to estimate the time when the equilibrium between the reactants and products could be established. The input parameters for this model were the HP concentration, the molar ratio of AAto HP, and the loading of the sulfuric acid. The recipe formulation for the target decontamination solution was also established through this model to determine the amount of reactants needed to achieve the target PAA at equilibrium, as illustrated in Table 2-2 for the PAA with sulfuric acid formulation. The time to reach the target equilibrium of 1.5 % (0.2 mol/L) PAA is shown in Figure 2-1. 15 ------- Table 2-2. Determination of Recipe Formulation - PAA with Sulfuric Acid Formulation (SA) Recipe Formulation Recipe HP Acetic acid Water Sulfuric Acid Input Parameters 15 100 1 %wt/wt % <-N v/v %-> Volume Calculated (mL) 403 128.34 404.2 62.5 Initial Set Conditions Recipe PAA HP Acetic acid Water Sulfuric Acid mol/L 0.00 1.82 2.18 45.24 0.13 wt(g) 0.00 61.74 130.79 814.26 12.26 wt% 0.00 6 13 80 1 Expected Equilibrium Concentration Recipe PAA HP Acetic acid Water Sulfuric Acid mol/L 0.20 1.61 1.98 45.44 0.13 wt(g) 15.24 54.92 118.75 817.87 12.26 wt% 1.50 5.39 11.65 80.26 1.20 1.75. 1.50- 1.25- ° LOO- IS 0.75- 0.50- 0.25- 0.00- 10 20 30 40 50 60 Reaction Time (Hours) 70 6.2 6.0 5.8:5 5.6 5.4 ' CL 5.2 5.0 80 Figure 2-1. Temporal Product/Reactant Evolution (modeled) for PAA Formulation with Acetic Acid 16 ------- 2.1.2 In-house PA A Formulations without Sulfuric Acid The in-house PAA formulation without sulfuric acid (SD) was prepared in the same way as the in-house PAA acid solution, with the exception that no sulfuric acid was added as a catalyst to speed up the reaction. As a result, the solution required approximately a month to reach the target maximum PAA concentration (1.5 %) and equilibrium. A 15 % HP concentrated solution was used to prepare this decontamination solution. The volumetric ratio of AA initial solution to HP initial solution was kept at 1.2:1 as well. This solution was then diluted to achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001 % for sporicidal testing. The recipe for the target SD formulation is shown in Table 2-3. The time to reach target equilibrium of 1.5 % (0.2 mol/L) PAA is shown in Figure 2-2. Table 2-3. Determination of Recipe Formulation - PAA without Sulfuric Acid Formulation (AD) Recipe Formulation Recipe HP Acetic acid Water Input Parameters 15 100 % wt/wt % Volume Calculated (ml_) 403 129.05 461.0 Initial Set Conditions Recipe PAA HP Acetic acid Water mol/L 0.00 1.82 2.18 45.61 wt(g) 0.00 61.74 130.79 820.93 wt% 0.00 6.10 12.90 81.00 Expected Equilibrium Concentration Recipe PAA HP Acetic acid Water mol/L 0.20 1.62 1.98 45.81 wt(g) 15.14 54.96 118.83 824.51 wt% 1.49 5.42 11.73 81.36 17 ------- 200 400 600 800 1000 1200 1400 Reaction Time (Hours) 6.2 6.0 5.8 5.6 5.4 ' Q_ 5.2 5.0 Figure 2-2. Temporal Product/Reactant Evolution (modeled) for PAA Formulation without Acetic Acid 2.1.3 In-house PAA Formulations with Sulfuric Acid Derived from Chemicals Available OTC The OTC PAA formulation was prepared in a single one-liter batch. The PAA and HP concentrations, along with the pH values, of the stock solution were monitored overtime until the solution reached a maximum PAA equilibrium concentration (0.07 %) for a set of initial conditions. This solution was derived from products that are available OTC or off-the-shelf so that they are easily accessible to the general population. Battery acid (sulfuric acid) was used as the catalyst in this recipe to enable equilibrium to be reached in hours. The volumetric ratio of the AA (from vinegar) initial solution to HP initial solution was kept at 1.2:1 as well. A material compatibility study using the OTC solution was performed on metal coupons and is discussed in Section 2.4. The recipe for the target OTC decontamination solution is shown in Table 2-4. The time to reach target equilibrium of 0.07 % PAA is shown in Figure 2-3. 18 ------- Table 2-4. Determination of Recipe Formulation - OTC PAA Formulation Recipe Formulation Recipe HP Acetic acid Water Sulfuric Acid Input Parameters 3 5 9.4 %wt/wt % <-N v/v %-> Volume Added (mL) 439 559 0 3.5 Initial Set Conditions Recipe PAA HP Acetic acid Water Battery acid mol/L 0.00 0.39 0.47 53.23 0.066 wt(g) 0.00 13.22 28.00 958.14 6.47 wt% 0.00 1.30 2.80 95.30 0.60 Expected Equilibrium Concentration Recipe mol/L PAA 0.009 HP 0.38 Acetic acid 0.46 Water 53.23 Sulfuric Acid 0.066 wt(g) 0.71 12.90 27.44 958.31 6.47 wt% 0.07 1.28 2.73 95.28 0.64 0.075- 0.060- •S 0.045- o 0.030- 0.015- 0.000 1.280 10 20 30 40 50 60 70 80 Reaction Time (Hours) Figure 2-3. Temporal Product/Reactant Evolution (modeled) for the OTC PAA Formulation 19 ------- 2.1.4 Modified AHP Decontamination Formulation Developed by SNL The AHP decontamination formulation was developed by SNL. The original recipe for preparation of AHP provided by SNL is given in Table 2-5. For preparation of large batches of AHP, the original recipe was modified as shown. Variquat was not available at the commencement of this study and was replaced by Maquat. Variquat and Maquat are both quaternary ammonium compounds (QACs) that have microbicidal properties. Table 2-5. AHP Decontamination Solution Recipe Part A PartB PartC Original Recipe Dl water [mL] K2C03 [g] Variquat [mL] Ethanol [mL] 50 % HP [mL] Triacetin [mL] 76.9 3 0.1 5 12 3 Modified Recipe Dl water [mL] K2C03 [g] Maquat [mL] Ethanol [mL] 15%HP[mL] Triacetin [mL] 489 30 1 50 400 30 The stock AHP solution was then diluted to achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001 % for sporicidal testing. The target maximum PAA concentration (1.5 %) was achieved almost immediately post-mixing. The solution has a shelf life of only four hours and thus had to be used in testing within that time frame. A new solution was prepared fresh for each test sequence. 2.1.5 Diluted Hydrogen Peroxide Solution The diluted HP antimicrobial solution was tested at 4.5 % HP concentration. This value is the same concentration of HP estimated to be in the 1 % PAA solutions. 2.2 Task 2: Method Development for Neutralization The presence of decontamination solution components in the test solution after the contact time could negatively bias colony forming unit (CPU) quantification. Sporicide neutralization tests of a Ba spore solution were carried out in advance of the decontamination solution evaluation to determine the most suitable neutralization solutions required for complete inactivation of the microbicidal properties for two PAA decontamination solutions (0.001 % and 1 %). Sodium thiosulfate (STS) was evaluated as a neutralizing agent for PAA and HP in an aqueous solution containing 0.1 g potassium carbonate (K2CO3), phosphate buffered saline with 0.05% TWEEN®80 (PBST), and a specific decontamination solution. Because the PAA formulations are acidic, K2CO3 was added to the neutralizer solution to raise the neutralizer-decontamination solution mixture to a pH between 5.5 - 8.5. If 0.1 g of K2CO3 was insufficient to raise the pH within the test parameter range, more K2CO3 was added as needed to satisfy the pH criteria. The STS solution was evaluated at 1X, 2X, and 5X molar excess for the 1 % PAA, and at 1000X stoichiometric excess for the 0.001 % PAA solution (where X represents stoichiometric neutralizing conditions of PAA and HP). In addition to STS, a 2-mL lecithin/Tween solution was added to neutralize the QACs present in the AHP formulation. The following recipe was used to prepare 30 mL of the lecithin/ TWEEN® 80 solution: 20 ------- 5 g Soybean Lecithin (Fisher Scientific, CAS No. 8002-43-5) • 5 ml TWEEN® 80 (Fisher Scientific, CAS No. 9005-65-6) • 25 ml Dl Water (In-house Dl Water System, by Purologix Water Services Inc) Contents were thoroughly mixed in a sterile 125 ml media bottle and autoclaved for 20 minutes at 121°C. The neutralization solution with STS was prepared once the lecithin/ TWEEN® 80 solution cooled to room temperature. 2. 2. 1 Standard Neutralization Test Method Due to the specific interest in the exposure period of Bacillus spores to the test formulation, the neutralization solution was evaluated according to the "Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents," ASTM E1 054-8mi with recovery on semi-solid media. Table 2-6 shows that the test components for Tests A through D were kept the same, with an emphasis on maintaining the same total solution volumes and ratios throughout all tests. Table 2-6. Components of Tests A to D Components Microbial Suspension Decontaminant Neutralizer PBST Test A X X X TestB X X X TestC X X TestD X X X The microbial suspension was comprised of Ba ATCC 9372 and was manipulated so that the microbial suspension used in Tests A through D resulted in the total components containing 25 to 250 CFU/ ml, with the target concentration being 100 CFU/mL. 2.2.1.1 Test A: Neutralizer Effectiveness This test determined the NE to quench the activity of the decontamination solution by simulating the conditions of the efficacy test (i.e., neutralizer volume, decontamination solution volume, challenge microbial solution volume, decontamination solution to neutralizer ratio, contact time). After the concentrations of PAA and HP for the target decontamination solution were measured, the stoichiometric equivalent (X) for STS to neutralize the decontaminant was calculated. Five ml of the target neutralizer (an aqueous solution containing PBST and 0.1 g baking soda with 1X, 2X, and 5X STS for the 1 % PAA, and 1000X for the 0.001 % PAA solution STS) were added to 5 ml of the decontamination solution (0.001 % PAA or 1 % PAA).. Once the pH of the neutralizer/decontamination solution had been aseptically checked, the solution was inoculated with 0.1 ml of 1xio4 CFU/mL challenge microbial suspension to result in a final suspension that contains approximately 100 CFU/mL (1000 total CFU) of the microorganism. The decontamination/neutralizer/microbial suspension was allowed to stand for a total holding time of 30 minutes. This test was conducted in triplicate A 1 mL aliquot was removed from each of the decontamination/neutralizer/microbial suspension samples and transferred, in triplicate, to a tube with 10 mL PBST. Once homogenized, each 11 mL sample was 21 ------- filtered using a Nalgene filter unit (cat no. 130-4020) and the filters were aseptically removed and placed onto a pre-labeled Tryptic Soy Agar (ISA) media plate and incubated at 35 ± 2 °C overnight. 2.2.1.2 Test B: Neutralizer Toxicity Test B was designed to determine whether the presence of neutralizer in excess was biostatic or biocidal. This test sequence was a repeat of Test A, with the exception that the decontamination solution was replaced with a sterile PBST solution that resulted in the same total sample volume as that used in Test A. 2.2.1.3 Test C: Organism Viability The organism viability test determined the recovery of the microorganisms exposed to the sterile buffer (PBST) only. This test sequence was a repeat of Test A, with the exception that the decontamination/ neutralizer solution mixture was replaced with a sterile PBST solution that resulted in the same dilution ratio as that used in Tests A and B. Samples from this test also serve as positive controls to calculate the recoveries from Test A, B, and D. 2.2.1.4 Test D: Test Material Control This test sequence was a repeat of Test A, with the exception that no STS was added to the neutralizer solution, resulting in the same dilution ratio as Tests A and B. 2.2.2 Sampling Frequency for Neutralization Testing The neutralization testing sequence for each formulation is listed in Table 2-7. Table 2-7. Sampling Measurements for Neutralization Testing Testing Sequence For Each Decontamination Formulation Measurement Titration (PAA concentration) Titration (HP concentration) pH Probe Decontamination Solution Negative Controls CFU Laboratory Blank Solution CFU Neutralization Test Solution CFU Measurement Range 0.001-1 % 0-5 % 0-9.5 0-1 00 CFU per filter 30-1 00 CFU per filter Frequency Once before and after completion of the neutralization testing One sample per decontamination solution (PAA 1 % and PAA 0.001 %) One sample per neutralization essay Three samples per decontamination solution per neutralizer concentration per neutralization assay type 22 ------- 2.3 Task 3: Liquid-Liquid Decontamination Testing The testing sequence included both the decontamination formulation evaluation prior to testing (checks for stability, concentration of active ingredients, and solution neutralization,) and the sporicidal efficacy based on direct inoculation as a function of reaction time. The microbial suspension was tested for sporicidal effectiveness, in quintuplicate, at four decontamination exposure times (2, 4, 6, and 8 minutes) for the 1 % PAA formulations and at six decontamination exposure times (2, 4, 6, 8, 16 and 20 minutes) for further dilutions of the PAA formulations. For each microbial suspension test, there was one negative test control using the same approach as the efficacy testing, three positive test control tests using a neutralizer/PBST solution, and one inoculum control test using a PBST solution alone. 2.3.1 Efficacy Testing This test was designed to determine the sporicidal effectiveness of each decontamination formulation at a prescribed reaction time. After the concentrations of PAA and HP for the target decontamination solution were measured, the target neutralizer containing appropriate amounts of STS (determined during the neutralizer effectiveness test), PBST with baking soda was prepared. Lecithin was also added to the neutralizer solution when testing the AHP formulation. Five ml samples of the decontamination solution were inoculated with 0.1 ml of 5.0 x 108 CFU/mL challenge microbial suspension to result in a final suspension (decontamination/neutralizer/microbial suspension) that contained approximately 5.0 xio7 CFU of the microorganism. After the required exposure time had passed, 5 ml of the neutralizer solution was added to each sample to stop the decontamination reaction. The decontamination/neutralizer/microbial suspension was held at room conditions for 30 minutes and the mixture was then immediately homogenized for 15 seconds using a vortex mixer prior to a tenfold serial dilution and plating onto TSA. In addition, a 1-mL aliquot was removed and aseptically transferred to a sterile, pre-labeled tube containing 10 ml of PBST. The tube containing the 1 ml aliquot and the 10 ml of PBST was homogenized by vortex mixer for 15 seconds and then aseptically transferred to an individual Nalgene filter unit, where the sample was filtered. Immediately following filtration, the filter unit was rinsed using another 10 ml of PBST, which was also filtered. The filters were aseptically removed and placed onto a pre-labeled TSA media plate that was incubated at 35 ± 2 °C overnight. Following incubation, CFU were enumerated. The pH of the decontamination/neutralizer solution mixture was required to be within a pH range of 5.5 - 8.5 to provide an optimum medium for the spores post-neutralization. A separate test vial containing 5 ml of decontamination solution and 5 ml of neutralization solution was used to test the pH of the solution mixture to maintain sterility of the solutions and to avoid contaminating the pH meter probe with the spores. The pH of all other samples used in efficacy testing was assumed to be the same as this test vial. 23 ------- 2.3.2 Sampling Frequency for Decontamination Testing The decontamination testing sequence for each formulation is listed in Table 2-8. Figure 2-4 presents a flowchart of the efficacy testing sequence. Table 2-8. Sampling Measurements for Decontamination Testing Testing Sequence For Each Decontamination Formulation Measurement Titration (PAA concentration) Titration (HP concentration) pH Probe Decontamination Solution Negative Controls CFU Laboratory Blank Solution CFU Positive Controls CFU Inoculum Controls CFU Test Samples CFU Concentration Range 0.001 - 1 % 0-5 % 0-9.5 0-1 00 CFU per filter ~5.0xlo7CFUper ml_, 30-300 per plate 30-300 per plate for dilution plating; 0-100 CFU per filter Frequency Once before and after completion of the efficacy testing One sample per decontamination solution One sample per neutralization assay Three samples per decontamination solution One sample in PBST per decontamination solution Five samples per decontamination solution per solution concentration per reaction time 24 ------- Make the Diluted Decon Solution Assess concentration of active ingredients using titrations (eerie sulfate, followed by iodiometric determination of PAA) After 1 hour, reassess concentration of active ingredients using titrations (eerie sulfate, followed by iodiometric determination of PAA) Is solution stable (± 10% drift in PAA)? Is solution within 50% of desired PAA target No Determine STS equivalents and use to prepare neutralization solution from Test A-D Remove 5 ml aliquot of decon solution. Spike with inoculum Allow required reaction time Add 5 ml neutralizer solution Add 10 ml extraction buffer solution, vortex, and plate within 30 minutes Figure 2-4. Efficacy Testing Sequence Flow Chart 25 ------- 2.4 Task 4: Surface Decontamination Testing The study conditions for decontaminant solutions generated in-house during the first phase of the project do not reflect the way in which decontaminants are applied. While laboratory studies are largely conducted in solution, technical decontamination requires more readily available procedures such as spraying for surface decontamination. The spraying approach using readily available equipment may increase readiness to respond to a wide-area contaminant release. 2.4.1 Spray Down Technical Approach The OTC PAA decontamination formulation was applied to coupons of carpet, wood, glass, vinyl flooring, and concrete in the form of spray. Two series of tests were designed under this approach. The first series of tests required a 10 minute coupon-decontaminant exposure time. The second series of tests required a 30 minute coupon-decontaminant exposure time and more frequent spraying to determine if these factors had an effect on the surface decontamination efficacy. The spray application was conducted using a custom-built testing apparatus shown in Figure 2-5. The spray testing apparatus was previously employed in EPA decontamination testing, and a test method was developed for the use of this apparatus. Decontamination solution was sprayed onto coupons using a 0.5-gallon chemical- and break- resistant adjustable commercial hand sprayer (RL FloMaster Model # 1985VI, Lowell, Ml) made of high- density polyethylene with a Viton® seal. The adjustable sprayer included a pump trigger that can provide a controlled delivery and was set on fine mist to minimize runoff and drips of the decontaminant. The sprayer is also fitted with a pressure gauge that reads up to 30 psi. Figure 2-5. Front View of Spray Apparatus with Orifice Plate 26 ------- 2.4.2 Test Coupon Preparation and Inoculation - Spray Down Approach Test coupons were made from five different materials that represent typical indoor materials consisting of porous materials (carpet, wood and concrete) and nonporous materials (glass and vinyl flooring). The size of the test coupons was approximately 1.7 cm x 5 cm to fit into the 50 ml conical tubes used during the spore extraction process. Concrete coupons were coated with a waterproof epoxy coating on five sides leaving only one 1.7 cm x 5 cm surface of the coupon absorbent to the decontaminant solution (MOP 3202). Similarly, wood coupons were coated with a polyurethane coating on five sides. Carpet, vinyl and wood coupons were sterilized prior to use with ethylene oxide (EtO) from an Andersen EOGas 333 sterilization system (Andersen Products, Haw River, N.C), while concrete and glass coupons were sterilized in an autoclave. Supplier and specification for all five coupon materials are shown in Table 2-9. Table 2-9. Coupon Material Specifications Material Glass Wood Concrete Carpet Vinyl Flooring Origin Prism Research Glass Maple Dowels, Cindoco Wood Products Made in-house Beaulieu Solutions Peel and Stick Vinyl Tile, Home Depot Location Raleigh, N.C Mt. Orab, OH RTP, N.C Dalton, GA Gary, N.C Specification Dimensions: 1 .7 cm x 5 cm x 1 cm Dimensions: 3/4" x 36" Dimensions: 1 .7 cm x 5 cm x 1 cm Model #657985861200AB; Dimensions: 18"x18" Model #26295061; Dimensions: 12"x12" Final Coupon Size 1 .7 cm x 5 cm The test coupons were inoculated with 100 uL of a Ba ATCC 9372 spore stock suspension. Coupons were placed flat in a sterile Petri dish inside the biosafety cabinet (BSC) and inoculated with approximately 1 x 104 viable spores per coupon for method development for neutralization, and 1 x 108 viable spores per coupon for the decontamination testing. Ten (10) droplets (each 10 uL in volume) of the spore stock suspension were dispensed uniformly across the surface of the test coupon using a micropipette. After inoculation, the coupons were allowed to dry overnight in the BSC. 2.4.3 Spray Down Neutralization Method Evaluation A series of method development tests was performed to determine the required volume and strength of the neutralizerto be preloaded in the 50 ml extraction conical tubes (BD Falcon cat. no. 352098, Franklin Lakes, NJ) following the decontamination spray. This testing was conducted as follows: a. The spray apparatus, with coupons and rinsate collection vials, was aseptically assembled. The vials were pre-weighed before spraying. b. The decontamination solution was sprayed onto the coupons (spray frequency for each coupon and series of tests is detailed in Tables 2-10 and 2-11). c. For Series 1 Tests, coupons were to remain wetted for the entire duration of the exposure time (10 min). If the coupons appeared to dry before the 10 minutes were up, they were sprayed again. For Series 2 Tests, a 30-minute exposure time was required. The coupons were not required to remain wet for the entire duration of the exposure time. 27 ------- d. After the specified exposure time (10 minutes or 30 minutes), the coupons were extracted aseptically using sterile thumb forceps and transferred to the coupon collection vial. e. Final weights of the collection vials were recorded. The net volume of runoff collected was calculated from the recorded weights. A stoichiometric equivalent (X) portion of neutralization solution was determined for the highest sprayed volume of runoff collected independent of material type. The neutralization solution volume (containing the required mass of baking soda to achieve a pH between 5 and 8.5) was adjusted to achieve a 10X STS strength. The funnels were rinsed with sterile Dl until a total of 20 ml was reached. Four additional preliminary tests (Tests A-D) were conducted to determine the proper neutralization procedures: • Test A: Neutralization Effectiveness • Test B: Neutralizer Toxicity • Test C: Organism Viability • Test D: Material Control Tests (A-D) were similar to the method development tests that were employed in the first phase of this project. 2.4.4 Spray Down Decontamination Procedure The general decontamination procedure consisted of spraying the coupons set up in the spray apparatus and was conducted as follows: a. The concentrations of PAA and HP for the decontamination solution were measured. b. The neutralizer solution (an aqueous solution containing baking soda and the appropriate amount of STS solution) was pre-loaded onto the coupon and rinsate collection vials. The neutralization solution volume (containing the required amount of baking soda to achieve a pH range of 5.5-8.5) was manipulated so that the STS was at 10X strength, and the coupons were completely immersed in the decontamination/neutralizer solution mix without risk of overflow in the conical tubes. c. Coupons were then sprayed with the decontamination solution. d. After the required contact time with the decontaminant solution, the coupons were aseptically transferred into each of their respective coupon collection vials (pre-loaded with PBST and the required amount of neutralizer totaling a volume of 26 ml). e. The funnels were rinsed with sterile Dl until a total of 20 ml was reached. After an overall holding time of 30 minutes post extraction, the samples were subjected to a ten-minute sonication step, followed immediately by two minutes of continuous vortexing on setting 10. 28 ------- f. All samples were then homogenized for one 15-second burst using a vortex mixer on setting 10 prior to a tenfold serial dilution with spread-plating. In addition, a 1ml_ aliquot was removed for filter-plating. g. The plates were placed into an incubator for 18 to 24 hours at 35 ± 2 °C, following which the CPU were enumerated. 2.4.5 Spray Down Decontamination Test Matrix Two series of spray down decontamination tests were performed as part of this study. In both series of tests, OTC PAA decontamination formulation was sprayed onto coupons of carpet, wood, glass, vinyl flooring and concrete. The duration and frequency of each decontaminant spray in both series of tests is listed in Table 2-10 and 2-11. The adjustable spray wash bottle used in these approaches was maintained at 20 psi to ensure a consistent spray each time. Because the coupons were to remain wetted with the decontaminant solution for the entire exposure time of 10 minutes (for Series 1 tests only), concrete (a porous, fast absorbing material) required more sprays as compared to the other materials. Tables 2-12 and 2-13 list the neutralizer amounts added to each of the five material sample types for Series Tests 1 and 2, respectively. Table 2-10. OTC PAA Formulation - Spray Duration and Frequency (Series 1) Material Glass Wood Concrete Carpet Vinyl Flooring Duration of Spray (sec) 2 2 2 2 2 Frequency 1 -Once every 10 minutes 1 -Once every 10 minutes 3 - Once every 3 minutes 1 -Once every 10 minutes 1 -Once every 10 minutes 29 ------- Table 2-11. OTC PAA Formulation - Spray Duration and Frequency (Series 2) Material type concrete wood carpet glass vinyl flooring Number of Sprays (#) 1 2 2 4 3 4 1 2 2 4 3 4 1 2 2 4 3 4 1 2 2 4 3 4 1 2 2 4 3 4 Spray Frequency (min) 1 at time 0 1 at time 0 and 1 at 15 min 2 at time 0 2 at time 0, and 2 at 15 min 3 at time 0 4 at time 0 1 at time 0 1 at time 0 and 1 at 15 min 2 at time 0 2 at time 0, and 2 at 15 min 3 at time 0 4 at time 0 1 at time 0 1 at time 0 and 1 at 15 min 2 at time 0 2 at time 0, and 2 at 15 min 3 at time 0 2 at time 0, and 2 at 15 min 1 at time 0 1 at time 0 and 1 at 15 min 2 at time 0 2 at time 0, and 2 at 15 min 3 at time 0 4 at time 0 1 at time 0 1 at time 0 and 1 at 15 min 2 at time 0 2 at time 0, and 2 at 15 min 3 at time 0 4 at time 0 30 ------- Table 2-12. Neutralizer Amounts Added To Samples -Spray Down Approach (Series 1) Material Glass Wood Concrete Carpet Vinyl Flooring STS Strength 10X 10X 10X 10X 10X Coupon (ml) STS (0.1 N) 0.3 0.8 1.75 2.0 0.3 PBST 25.7 25.2 23.25 24.0 25.7 Total Volume (ml) 26 26 26 26 26 Rinsate (ml) STS(1.5N) 0.8 1.2 1.9 0.8 0.9 Baking Soda (g) 0.1 0.2 0.35 0.1 0.1 Total Volume (ml) 20 20 20 20 20 2.5 Task 5: Material Compatibility Evaluation The in-house PAA formulations are very acidic (pH <2). To analyze the effects of the acidity of these formulations, a material compatibility test was designed to assess the corrosion or changes of the OTC PAA formulation at different pH levels. This study was performed on 10 cm x 10 cm coupons made of stainless steel, galvanized metal, aluminum and carbon steel. These coupons, tested in duplicate, followed two test procedures: (1) post-rinse procedure, and (2) no post-rinse procedure. In the post-rinse procedure, two coupons of each material were immersed in the original OTC PAA formulation. The coupons were rinsed with Dl water and left to dry after a contact time of 30 minutes with the decontaminant. In the no post-rinse procedure, two coupons of each material were immersed in OTC solution and left to dry without a Dl water rinse after a contact time of 30 minutes with the decontaminant. The original OTC solution has a pH of 1.7 -1.8 (~2). Further assessment on the coupons was conducted using the OTC solution at a higher pH of 4 and 6 to determine the extent of the acidity of the solution on material compatibility. These higher pH values were achieved by adding baking soda (sodium bicarbonate) to the OC solution. Visual and gravimetric analyses were performed on the coupons on a weekly basis for a total assessment period of eight weeks. Temperature and relative humidity (RH) inside the laboratory were monitored using a HOBO Data Logger U12 Series (Onset Computer Corporation, Bourne, MA) throughout the eight-week assessment period. 31 ------- 3 Sampling and Analytical Procedures 3.1 Sampling Procedures Within a single test, sampling was completed first for all procedural blank samples before sampling of any test material. Prior to the sampling event, all materials needed for sampling were prepared using aseptic techniques. The order of sampling was performed as follows: (1) all blank samples, (2) all decontaminated samples, and (3) all positive control samples. This order ensures that coupons are handled in an order from the least level of contamination to the most.. The general sampling supplies were purchased sterile or were sterilized/disinfected prior to each sampling event. After sample collection for a single test was complete, all biological samples were extracted typically within ten minutes. 3.2 Sample Extraction/Analysis 3.2.1 Liquid-Liquid Decontamination For the neutralizer evaluation testing, the microbial decontamination/sterile PBST/neutralizer microbial suspension solution was immediately homogenized for 15 seconds using a vortex mixer. A 1-mL undiluted aliquot was removed and aseptically transferred in triplicate to three tubes with 10 ml of PBST. These three 11-mL tubes were homogenized using a vortex mixer for 15 seconds and then each solution was aseptically transferred to an individual Nalgene filter unit, cat no. 130-4020 (Thermo Fisher Scientific, Waltham, MA) where the samples were filtered. Immediately following filtration, the filter unit was rinsed using another 10 ml of PBST, which was also filtered. Each filter was aseptically removed using sterile thumb forceps and placed onto a pre-labeled TSA media plate. For the decontamination effectiveness testing (starting with a high microbial concentration), the solution was immediately homogenized for 15 seconds using a vortex mixer. A 0.1-ml aliquot was removed and aseptically dilution-plated onto a pre-labeled TSA media plate. Further, three 1-mL undiluted aliquots were removed in parallel with the 0.1-ml aliquot and aseptically transferred in triplicate to three tubes with 10 ml of PBST. These three 11 -ml tubes were homogenized for 15-seconds using a vortex mixer and then aseptically transferred to an individual Nalgene filter unit for the filter plating sequence. Samples from the neutralization Tests A through D were filtered in triplicate and the plates were incubated overnight at 35 ±2 °C. 3.2.2 Surface Decontamination After the required contact time of 10 minutes with the decontaminant solution, the coupons were aseptically transferred into each of their respective coupon collection vials (pre-loaded with PBST and the required amount of neutralizer totaling a volume of 26 ml). After an overall holding time of 30 minutes post extraction, the samples were subjected to a 10-minute sonication step, followed immediately by two minutes of continuous vortexing at the maximum rotational speed. Prior to plating, all samples were then homogenized for one 15-second burst using a vortex mixer at the maximum rotational speed. 3.3 Sample Analysis Decontamination samples were subjected to up to five-stage tenfold serial dilutions (10~1 to 10~5). The resulting samples were plated in triplicate and incubated overnight at 35 ± 2 °C. Following incubation, CFU were manually enumerated. The samples resulting in fewer than the reportable limit of 30 CFU/plate on the undiluted sample underwent further analysis. When no detectable spores were detected from the 32 ------- filter-plating, a value of 0.5 CFU/sample was assigned as the detection limit for efficacy determinations for purposes of calculating LR. 3.4 Data Analysis The total spore recovery for each test method was calculated by multiplying the mean CPU counts from triplicate plates by the inverse of the volume plated (e.g., 1/0.1 ml or 10), by the dilution factor, and finally by the volume of the sample extract as illustrated in Equation 3-1. average CPU from CPU replicate dilution plates or on filter 1 , . . . r x ,„ ,. N r = —; ;—. ,., -H-:— x —:— x(extract vo lume, ml) (3-1) sample volume plated or filtered,mL (tube dilution factor) 3.5 Efficacy Testing D-value, defined here as the time required to reduce the population of the target organism by 1 log (90 %) and temporal CPU LR were used to determine the efficacy of each decontamination solution. 3.6 Decontamination Efficacy The sporicidal effectiveness (efficacy) of a decontamination technique is a measure of the ability of the method to inactivate the spores. The sporicidal effectiveness is evaluated by measuring the difference in the logarithm of the measured CPU before decontamination (determined from sampling positive control coupons) and after decontamination (determined from the sampling of the test samples) for the same type of material (represented by the neutralizer/decontamination suspension). The number of viable spores recovered was measured as CPU and reported as a LR for each specific decontamination solution as defined in the Equation 3-2. n =^ -- -^ - (3-2) '' Nc Ns where: j, _ Decontamination effectiveness; the average LR of spores for a '' specific solution (designated by ;) NC V loe,(CFU } ~*~he avera9e °f tne logarithm (or geometric mean) of the number of _i=i _ _ = viable spores (determined by CPU) recovered on the positive controls Nc (C indicates control and Nc is the number of positive controls) N, The average of the logarithm (or geometric mean) of the number of 2_\og(CFU sk} ^ viable spores (determined by CPU) remaining in the decontamination — - solution (S indicates a decontaminated solution and Ns is the number N s of test samples). 33 ------- The standard deviation of the average log reduction of spores corresponding to a specific decontamination solution is calculated by Equation 3-3. Ns-l (3-3) where: SZ> „ _ '' Standard deviation of r|j, the average LR of spores for a specific solution The average LR of spores for a specific solution type designated by ; The average of the LR for a specific test sample (Equation 3- 3) Number of test samples for a specific decontamination solution and, where: log(CFUc) = — £log(CTC7Cjt) N c CFUS k Ns Represents the "mean of the logs" (geometric mean), the average of the logarithm-transformed number of viable spores (determined by CPU) recovered on the positive controls (C = control samples, Nc = number of control samples, k = test sample number and Ns is the number of test samples) = Number of CPU in the kth decontaminated sample = Total number (1 ,k) of decontaminated samples. (3-4) 34 ------- In this report, decontamination efficacy is generally reported in terms of LR for a particular decontamination formulation. We also occasionally report results by noting whether the average LR for a particular test is > 6.0, since a decontaminant that achieves > 6 LR is considered effective ^ 3.7 Sample Preservation The presence of decontamination solution components in the test solution could negatively bias spore recovery tests. Therefore, the coupon solutions were neutralized immediately after the exposure time (ten minutes) and plated within 30 minutes, which is the longest holding time set for the neutralization tests. The prepared decontamination samples were stored in sealed 1-L jars and aseptically opened only for sample retrieval for chemical analysis or testing. All samples were stored in the refrigerator at the EPA RTP facility Microbiology Laboratory at 4 °C ± 2 °C until further analysis was required. 3.8 Holding Times The sample collection and analysis occurred in the NHSRC RTP Microbiology Laboratory. After sample collection for a single test was complete, all biological samples were transferred to the microbiology analyst for immediate extraction with typical holding times not exceeding ten minutes. 3.9 Sample Archival Samples were archived by maintaining the primary extract at 4 °C ± 2 °C in a sealed extraction tube until the data set had been quality controlled and the sample released for disposal. Any deviations from sampling protocols were documented in the laboratory notebook. Sampling duration, time of day, and observations were also recorded in the laboratory notebook. 3.10 Steady State Conditions Decontamination solutions are not expected to be stable overtime. Solutions were prepared and monitored overtime until they reached a pseudo-steady state with a drift in PAA concentration not exceeding 10 % of the maximum concentration within one hour of testing. The prepared decontamination samples were stored in sealed 1 -L jars and opened only for aseptic samples retrieval for chemical analysis or testing. 3.11 PAA and HP Characterization Measurements The concentration of PAA and HP in the PAA formulations was determined using an iodometric titration [12]. 35 ------- 4 Results and Discussion This section discusses results of the decontamination solution characterization tests (Section 4.1), neutralization characterization testing (Section 4.2), liquid-liquid decontamination testing (Section 4.3), and material compatibility testing (Section 4.4). 4.1 Formulation Characterization Results The pH and concentration of HP and PAA in the working decontamination solutions were considered critical to the understanding of the dynamic changes of key decontamination agents (HP, PAA) in the formulations. These parameters were monitored throughout the entire testing (immediately after mixing and before each application of decontamination solution). 4.1.1 "In-House" PAA Formulations The target maximum concentration for the in-house PAA formulations (with and without sulfuric acid) was set at 1.5 % PAA (0.2 mol/L). The diluted HP formulation reached its target concentration of 4.5 % HP immediately post-mixing. The pH of this decontamination formulation was 4.7. The in-house PAA with sulfuric acid formulation reached its target maximum concentration and equilibrium in approximately six days, while the in-house PAA without sulfuric acid formulation reached its target maximum concentration and equilibrium in 31 days. Once equilibrium was reached, the solutions were observed to be stable over the duration of the tests (over two months) with minor shifts in PAA and HP concentrations (less than 10 % deviation). The average pH of the PAA with sulfuric acid formulation was 1.05, while the average pH of the PAA without sulfuric acid formulation was 1.9 during testing. The temporal product/reactant evolution for the in-house PAA formulations with and without sulfuric acid is presented in Figures 4-1 and 4-2, respectively. 36 ------- • Model HP results • Model PAA results ^ Actual HP solution results o Actual PAA solution results 0 100 200 300 400 500 600 700 Time since start of the formulation preparation (hrs) Figure 4-1. Temporal Product/Reactant Evolution for the In-House PAA Formulation with Sulfuric Acid 7- 6- ^ s-\ KM* U — 1 4^ 8 : re a- i-\ 0- Model HP results Model PAA results Actual HP solution results Actual PAA solution results 0 100 200 300 400 500 600 700 Time since start of the formulation preparartion (hrs) Figure 4-2. Temporal Product/Reactant Evolution - In-House PAA without Sulfuric Acid 37 ------- 4.1.2 Modified AHP Decontamination Formulation The modified AHP decontamination formulation developed by SNL reached its target PAA concentration of 1.5 % immediately after mixing. However, the solution was highly unstable, and the concentration of both PAA and HP dropped continuously, as illustrated in Figure 4-3. This drop is operationally important, as low PAA concentrations affect the decontamination efficacy of the modified AHP solution against 8. atrophaeus (Ba) spores (Section 4.3.3). PAA concentrations below 0.1 % produced inadequate spore reductions (< 6LR). These results are discussed in further detail in Section 4.3. 6- 5- T3 I 3- 2- 1- 0- -•- HP for target PAA of 1.5% -•- HP for target PAA of 1.0% -o- PAA target of 1.5% -»- PAA target of 1.0% PH = 7.4| 5 10 15 20 Time since start of the formulation (hrs) 25 Figure 4-3. PAA and HP Concentration for the Modified AHP Formulation 4.1.3 Over-The-Counter (OTC) In-house PAA Formulation PAA, pH, and HP concentrations of the OTC formulation were measured overtime to determine the kinetics and stability of the mixture (potential loss of active ingredients by decomposition and evaporation). The developed kinetic model reasonably predicted the PAA formation rate from solution prepared from the OTC products (Figure 4-4). The target maximum concentration (0.1 %) and equilibrium were achieved in 25 hours. The OTC solution had a pH of 1.7 during decontamination testing. 38 ------- 0.12- 0.10- 0.08- o.oe- 0.04- 0.02-1 0.00- Actual PAA results Model PAA results 0 20 40 60 80 100 120 140 Time since start of the formulation preparation (hrs) Figure 4-4. Temporal Product/Reactant Evolution - OTC PAA The stability of the PAA formulations made, respectively, from laboratory grade ingredients (in-house PAA with sulfuric acid) and from OTC ingredients was evaluated during the spraying down procedure. Initial HP and PAA concentrations of both formulations were measured before proceeding to the spray application. Each formulation was then sprayed using an adjustable spray wash bottle fitted with a pressure gauge to help ensure uniform pressure during each spray. The sprayer pressure was kept constant at 20 psi. The sprayer-dispersed liquid was collected in conical tubes, which were also used for runoff/rinsate collection during decontamination testing. To determine the residual concentration of the active ingredients in the two formulations, the collected solutions were titrated immediately post-spraying (0 minutes) and subsequently at 15 minutes, 30 minutes and 60 minutes. The spraying is shown to have a remarkable effect on the degradation of the PAA concentration from the OTC formulation compared with negligible to no degradation of the PAA from the formulation made from laboratory grade ingredients (Figure 4-5). This OTC PAA degradation during the spraying will be shown later to result in dramatic reduction in the sporicidal efficacy of the OTC formulation during surface decontamination. 39 ------- 0.11 0.10- 0.09- 0.08- 0.07- O 0.06- 0.05- 0.04- 0.03. SA Formulation OTC Formulation Time delay after the spraying process Figure 4-5. Temporal PAA Degradation over Time for the OTC and PAA with Sulfuric Acid Formulations 4.2 Neutralization Testing Results Tables 4-1 through 4-3 in the following sections show the results from method development for neutralization using STS as the neutralizing agent for the decontamination formulations. Neutralization tests (Tests A-D, as described in Section 2.2) were conducted with three of the five decontamination formulations used in this project: in-house formulation with sulfuric acid, modified AHP formulation, and the diluted HP formulation. Neutralization testing was not performed on the in-house PAA formulation without sulfuric acid or on the OTC PAA formulation since it was assumed that these solutions would follow the same decontamination trend as the PAA without sulfuric acid formulation. The samples tested were allowed to stand for a holding time of 30 minutes before being filter plated. For neutralization tests, all PAA stock solutions tested were diluted to two concentrations, 1 % PAA and 0.001 % PAA. The values represent the highest and the lowest PAA concentrations used in efficacy testing for this work. Diluted HP stock solution was tested as is and with 4.5 % HP. 4.2.1 Neutralization Effectiveness Neutralization Test A determined the NE to stop the activity of the decontamination solution. The results for these tests are presented in Table 4-1 for the three formulations cited above. Initial Neutralizer Effectiveness (Test A) tests for the AHP formulation also showed that the 2X neutralizer with STS and PBST alone was insufficient. In addition to PAA and HP, the modified AHP formulation contains QACs that have microbicidal properties. To neutralize these QACs, a solution of lecithin in TWEEN®80 was used. Analysis of the data from neutralization tests determined that 2X neutralizer 40 ------- worked effectively, inactivating 1 % PAA concentrations in all formulations evaluated. For the 0.001 % dilution, STS neutralizer of strength 1000X was also found to be an effective neutralizing agent. Table 4-1. Neutralization Effectiveness (Test A, In-house PAA Formulation with Sulfuric Acid (SA), Modified AHP Decontamination Formulation Developed by SNL (SD), HP solution alone without PAA (HP)) Formulation Type SA SD+ HP Concentration 1.0% 0.001 % 1.0% 0.001 % 4.5 % Positive Controls (three replicates) Average 7.41 x102 8.01 x102 9.02 x102 Stdev 6.09 x101 5.92 x101 1.74x102 Stoichiometric Ratio 1X 2X* 5X 1000X 1X 2X 5X 1000X 1X 2X 5X Recovery (CFU) (five replicates) Average 2.76 x102 7.53 x102 3.13x102 5.39 x102 4.92 x102 9.60 x102 8.28 x102 7.71 x102 7.10x102 8.65 x102 5.96 x102 Stdev 1.76x102 1.38x102 3.03 x101 2.10x101 1.63 x102 1.31 x102 3.64 x101 4.77 x101 6.41 x102 1.08x102 3.64 x101 % 37 100 42 73 61 120 100 96 79 96 66 *Represent CFU counts from Test A repeat for 1 % +Test A for SD was repeated with lecithin added to PAA (SA) evaluated at 2X. neutralize QACs present in decontaminant 4.2.2 Neutralizer Toxicity The neutralizer toxicity (Test B) determined the growth inhibition caused by the neutralizer on the test organisms. The neutralizer toxicity (NT) was determined as the ratio between the recoveries of a population exposed to the neutralizer to a population that was not exposed to the neutralizer. The results of the neutralizer toxicity are presented in Table 4-2. The results confirm that the neutralizer used in these tests did not have a sporicidal or growth-inhibiting effect on the spores. 41 ------- Table 4-2. Neutralizer Toxicity (Test B) Formulation Type SA SD HP Positive Controls (three replicates) Avg CPU 6.50 x102 7.91 x102 9.02 x102 Stdev 5.74 x101 5.74 x101 1.74x102 Stoichiometric Ratio 1X 2X 5X 1X 2X 5X 1X 2X 5X Recovery (CPU) (five replicates) Avg CPU 5.66 x102 5.79 x102 6.03 x102 7.64 x102 7.44 x102 8.28 x102 9.29 x102 8.25 x102 7.98 x102 Stdev 8.80 x101 6.72 x101 1.20x102 2.54 x101 1.11x102 7.28 x101 5.25 x102 1.08x102 7.07 x101 % 87 89 93 97 94 110 100 92 89 4.2.3 Test Material Control Test D determined the efficacy of the decontamination solution and demonstrated that the neutralizer was effective in deactivating the decontamination solution. Analysis of the data from neutralization tests (Table 4-3) demonstrated that the 0.001 % PAA solution was an ineffective decontaminant, while the 1 % PAA solutions and the diluted HP solution proved to be effective when no neutralizer was added. The combined results from Test A and Test D demonstrate that STS effectively quenched the decontaminant solution. Table 4-3. Test Material Control (Test D) Formulation Type SA SD HP Concentration 1.0% 0.001 % 1.0% 0.001 % 4.5 % Positive Controls (three replicates) Avg CFY 6.50 x102 7.91 x102 9.02 x102 Stdev 5.74 x101 5.74 x101 1.74x102 Stoichiometric Ratio OX OX ox ox ox Recovery (CPU) (five replicates) Avg CPU ND 6.30 x102 ND 7.24 x102 ND Stdev - 1.26x102 - 7.65 x101 - % 0.00 97.0 0.00 92.0 0.00 ND = non-detect 42 ------- 4.3 Task 3: Liquid-Liquid Decontamination Testing Prior to each liquid-liquid decontamination efficacy test, the formulations were checked for stability and compared to the target set conditions. The results of these measurements are presented in Table 4-4. Table 4-4. Test Parameters Measurements Verification Decontamination PAAwith sulfuric acid PAAwith sulfuric acid PAAwith sulfuric acid PAAwith sulfuric acid HP Modified AHP Modified AHP PAA without sulfuric acid PAA without sulfuric acid PAA without sulfuric acid OTC Test ID 1 2 3 4 0 1 2 1 2 3 0 PAA Concentration Target Value (%) 1 0.1 0.01 0.001 4.5 1 0.1 1 0.1 0.01 0.1 Test Value (%) 1.04 0.11 0.012 0.0014 4.52 1.08 0.11 1.04 0.11 0.012 0.09 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing PH Test Value 1.42 2.38 3.09 3.62 4.68 8.7 8.9 2.15 2.6 3.4 1.7 Temp °C 25 25 25 25 25 21.3 25 25 25 25 25 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing 4.3.1 In-House PAA Formulation with Sulfuric Acid Decontamination efficacy is dependent upon the concentration of active ingredients in the sporicidal solution (PAA and HP, in this case), pH, and exposure times. The results of the liquid-liquid decontamination approach are presented in Table 4-5 and illustrated in Figure 4-6. An 8 LR was achieved with the high PAA concentration (1 %) even for an exposure time of two minutes. The pH of the 1 % PAA with sulfuric acid formulation was 1. For the 0.1% PAA with sulfuric acid dilution, longer exposure times helped achieve a greater than 6 LR. The pH of the 0.1 % PAA with sulfuric acid solution was 2.4 during efficacy testing. The 0.01 % dilutions (pH 3.1) and 0.001 % dilutions (pH 3.6) were less compelling as decontaminants, with LR values less than 1. 43 ------- Table 4-5. In-house PAA Formulation with Sulfuric Acid - Efficacy Testing In House SA Formulation Decontamination Results Concentration 0.001 % 0.01 % 0.1 % 1.0% Positive Controls(CFU) (three replicates) Average 4.00 x107 5.17 x107 3.87 x107 4.24 x107 Stdev 5.74x1 0s 1.40 x 10s 6.73x1 0s 2.63x1 0s Time (min) 2 4 6 8 2 4 6 8 2 4 6 8 16 20 2 4 6 8 Avg. Recovery (CFU) (five replicates) Average 1.74x107 1.67 x107 1.72x107 1.51 x107 4.30 x107 3.04 x107 2.76 x107 2.61 x107 4.88 x 10s 3.04 x107 1.09x102 1.83x102 <1. 01x10° <0.67x10° <1. 40x10° <0.60x10° <2.10x10° <3.70x10° Stdev 2.76x1 0s 1.10x106 2.97 x 10s 2.08 x 10s 2.85 x107 3.18x106 3.05 x 10s 1.49x106 4.29 x105 3.18x106 8.02 x101 3.25 x102 <0.73x10° <0.25x10° <2.01x10° <0.22x10° <1. 24x10° <6.88x10° LR Average 0.37 0.38 0.37 0.43 0.14 0.23 0.28 0.30 0.90 2.3 5.7 6.4 7.7 7.8 7.7 7.9 7.4 7.6 Stdev 0.07 0.03 0.08 0.07 0.22 0.04 0.05 0.02 0.04 0.15 0.54 1.4 0.27 0.13 0.45 0.13 0.33 0.65 D-Value (min) 5.5 11 16 19 15 17 22 27 2.2 1.7 1.1 1.2 2.1 2.5 0.26 0.51 0.81 1.1 44 ------- 8- •2 7 "£i " 3 "S (£ 6- |, § S4- 12^ 8 o- 10 15 Exposure Time (minutes) 20 Figure 4-6. Temporal Efficacy of the In-House PAA Formulations with Sulfuric Acid When 0.1 % and 0.01 % PAA solutions (in-house PAA with and without sulfuric acid) were mixed with their neutralizer solutions (10X and 100X, respectively), the pH of the combined decontaminant/ neutralizer mix was significantly higher (pH > 9) than the individual components, even with no baking soda added. The pH rise suggested that the PBST buffer in the neutralizer solution could have been overwhelmed when mixed with the decontamination solution, causing the pH to rise drastically. Adding an additional 10 ml of PBST to the neutralizer/decontamination solution mix solved this problem, and a stable neutral pH was achieved. Based on the decontamination efficacy trend, a near complete decontamination was observed at the two- minute exposure time with the 1 % PAA formulation. The CPU recovery between the two and eight minute exposure time lies within the uncertainty of the measurement technique, which suggests a calculated D- value for this formulation of less than 0.26 min. The 0.1 % PAA also showed promise with an average D- value calculated to be around 1.8 min, which suggests a 6-Log CPU reduction within an exposure time of approximately 10 minutes. The 0.01 % and 0.001 % PAA formulations were found to be too dilute to have an effective sporicidal activity, with D-values calculated at the maximum exposure time of eight minutes being greater than 18 minutes. 4.3.2 In-House PAA Formulation without Sulfuric Acid The decontamination efficacy of the AD solution (Table 4-6, and Figure 4-7) was found to be very similar to that observed for PAA with sulfuric acid formulation. Complete kill was achieved with the 1 % PAA dilution (pH 1.9), while longer exposure times resulted in greater than 6 LR for the 0.1 % PAA dilution (pH 2.6). Because the 0.01 % PAA dilution (pH 3.4) did not have very effective sporicidal activity, the lowest PAA dilution (0.001 %) was not tested for efficacy. 45 ------- Table 4-6. In-house PAA Formulation without Sulfuric Acid - Efficacy Testing "In House" AD Formulation Decontamination Results Concentration 0.01 % 0.1 % 1.0% Positive Controls(CFU) (three replicates) Average 4.48 x107 1.30x107 4.39 x107 Stdev 5.23x1 0s 1.31 x106 5.10x106 Time (min) 2 4 6 8 16 20 2 4 6 8 16 20 2 4 6 8 Avg. Recovery (CFU) (five replicates) Average 6.21 x106 6.81 x 10s 5.45x1 0s 4.82x1 0s 4.67x1 0s 4.61 x 10s 2.14 x105 6.81 x 10s 5.03x10° 1.21 x101 0.50x10° 1.20x10° 0.50x10° 0.50x10° 0.50x10° 0.50x10° Stdev 7.52 x105 6.55 x105 5.86 x105 5.23 x105 3.94 x105 8.67 x105 2.99 x104 6.55 x105 0.00x10° 1.57x101 0.00x10° 1.04x10° 0.00x10° 0.00x10° 0.00x10° 0.00x10° LR Average 0.86 0.82 0.92 0.97 0.98 0.99 1.7 6.7 6.4 6.2 7.4 7.1 7.9 7.9 7.9 7.9 Stdev 0.05 0.04 0.05 0.05 0.04 0.08 0.06 0.97 0.00 0.40 0.00 0.32 0.00 0.00 0.00 0.00 D-Value (min) 2.3 4.9 6.5 8.3 16 20 1.1 0.59 0.94 1.3 2.2 2.8 0.25 0.50 0.75 1.0 46 ------- 7- 3- 2- I 0.01 %PAA I I0.1%PAA 1.0% PAA 10 15 Exposure Time (minutes) Figure 4-7. Temporal Efficacy of the In-House PAA Formulations wthout Sulfuric Acid 4.3.3 Modified AHP Formulation Table 4-7 summarizes LR values for the SNL-modified AHP formulation. These data suggest that AHP- based liquid sporicide provides greater than 6 LR of spores when the concentration of the formulation is at 1 % PAA. However, the 0.1 % dilution proved to be ineffective, even with extended exposure times. Because the 0.1 % solution was ineffective at complete decontamination, lower dilutions of 0.01 % PAA and 0.001 % PAA were not tested for decontamination efficacy. The pH of the 1 % PAA dilution and 0.1 % PAA dilution during efficacy testing were 8.7 and 9.1, respectively. 47 ------- Table 4-7. Modified AHP Formulation - Efficacy Testing Modified AHP Formulation Decontamination Results Concentration 0.1 % 1 % Positive Controls(CFU) (three replicates) Average 1.64x107 5.70 x107 Stdev 1.17x105 3.05 x 10s Time (min) 2 4 6 8 16 20 2 4 6 8 Avg. Recovery (CFU) (five replicates) Average 2.27 x107 1.86x107 1.68 x107 1.52x107 1.61 x107 1.57x107 1.91x101 3.77x10° 1.91x10° 4.35 x101 Stdev 7.57x1 0s 2.88x1 0s 1.09x106 1.24x106 1.20x106 2.39x1 0s 3.34 x101 5.07x10° 1.33x10° 7.54 x101 LR Average -0.13 -0.05 -0.01 0.03 0.01 0.02 6.9 7.5 7.6 6.7 Stdev 0.13 0.06 0.03 0.03 0.03 0.06 0.66 0.58 0.38 0.81 D-Value (min) - - - 240 2000 870 0.29 0.53 0.79 1.2 4.3.4 Diluted Hydrogen Peroxide Formulation Table 4-8 summarizes LR values for the diluted HP formulation. The diluted HP formulation is not effective at decontaminating the spores. The pH of this formulation during decontamination testing was 4.7, suggesting that the sporicidal activity of the PAA solutions is due largely to the individual or synergistic effects of the PAA. The D-value averaged approximately 18 min/log CFU, so longer contact times will be required compared to other PAA formulations to achieve effective decontamination (>6 LR). 48 ------- Table 4-8. Diluted HP Formulation - Efficacy Testing Diluted HP Formulation Decontamination Results Concentration 4.510% Positive Controls(CFU) (three replicates) Average 4.79 x107 Stdev 4.16x106 Time (min) 2 4 6 8 Avg. Recovery (CFU) (five replicates) Average 4.23 x107 2.27 x107 1.88x107 1.78x107 Stdev 5.01 x 10s 2.75 x 10s 2.62x1 0s 1.01x106 LR Average 0.06 0.33 0.41 0.43 Stdev 0.05 0.05 0.06 0.02 D-Value (min) 25 12 15 19 4.3.5 OTC Formulation 4.3.5.1 Liquid-Liquid Decontamination Approach The OTC formulation provided nearly complete kill for exposure times of eight minutes or longer (Table 4- 9). Further extending the contact time of spores with the decontamination solution should result in complete kill based on the efficacy trend. As this solution is made from OTC and off-the-shelf common household products, this formulation could be a promising decontaminantthat can be readily and widely available sporicide. . However, the pH of this solution is low (1.7), indicating that the solution could potentially corrode some surfaces and be hazardous to the users. The 0.1 % OTC solution showed promise with greater than 6-Log CFU reduction found with eight minutes exposure time. Table 4-9. OTC PAA Formulation - Efficacy testing (Liquid-Liquid Decontamination) In-house OTC Liquid-Liquid Decontamination Results Concentration 0.100% Positive Controls(CFU) (three replicates) Average 5.17x107 Stdev 4.84x1 0s Time (min) 2 4 8 16 20 Avg. Recovery (CFU) (five replicates) Average 8.69 x105 1.76x104 0.57x10° 0.56x10° 0.56x10° Stdev 1.01 x105 9.15x103 0.70x10'2 0.00x10° 0.00x10° LR Average 0.18 3.5 8.0 8.0 8.0 Stdev 3.5 0.21 0.00 0.00 0.00 D-Value (min) 1.1 1.1 1.0 2.0 2.5 49 ------- 4.3.5.2 Surface Decontamination Approach Prior to each surface decontamination efficacy test, the formulations were checked for stability and compared to the target set conditions. The results of these measurements are presented in Tables 4-10 and 4-11. As the results in these tables establish, there was no significant difference in how the decontamination solution changed with respect to test parameters (PAA concentration and pH). Table 4-10. Test Parameters Measurements Verification (Series 1) Decon OTC OTC OTC OTC OTC Test Material WO VF GL CO CA PAA Concentration Target Value (%) 0.07 0.07 0.07 0.07 0.07 Test Value (%) 0.08 0.08 0.076 0.076 0.069 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing PH Test Value 1.82 1.82 1.9 1.9 1.79 Temp °C 21.9 21.9 21.9 21.9 21.7 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing Table 4-11. Test Parameter Measurement Verification (Series 2) Decontamination OTC OTC OTC OTC OTC OTC OTC OTC OTC OTC OTC OTC Test Material WO WO wo VF VF VF GL GL CO CO CA CA Test ID 11, 12 21,22,31 41 22,31 11, 12 21,41 11, 12,21 22,31,41 11, 12,21 22,31,41 11, 12,21 22,31,41 PAA Concentration Target Value (%) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 Test Value (%) 0.076 0.076 0.072 0.065 0.071 0.072 0.64 0.067 0.070 0.068 0.066 0.068 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing PH Test Value 1.89 1.82 1.91 1.84 1.90 1.91 1.84 1.87 1.82 1.79 1.85 1.81 Temp °C 21.9 22.1 22.2 20.9 21.7 22.2 22.0 21.9 21.4 21.9 22.3 22.1 Frequency Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing Once before testing The efficacy test results for Series 1 Tests, as outlined in Table 4-12, demonstrated that a ten-minute exposure time was not sufficient to achieve full decontamination using the spray-down approach. Thus a second series of tests (Series 2) was conducted to determine the effect of increased exposure time and frequency of spraying. The efficacy test results for Series 2 Tests are outlined in Table 4-13. These tests demonstrated that the increased exposure time (30 minutes) also contributed to the overall surface 50 ------- decontamination against the spores. Further, frequent spraying was observed to result in greater decontamination efficacy as compared to multiple sprays at the same time. Both series of tests using the spray-down approach didn't prove sufficiently efficacious in achieving the target 6 LR. These lower surface decontamination efficiencies were due to the high degradation of the OTC PAA concentration observed during the spraying. The surface decontamination approach can be improved significantly if the OTC formulation is further stabilized by using laboratory grade ingredients or stabilizing the formulation by using other OTC ingredients. Further studies would have to be conducted to determine the cause of the OTC degradation. Table 4-12. OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series 1) In-house OTC Surface Decontamination Results Material Type Carpet Concrete Wood Vinyl Glass Exposure Time (min) 10 Positive Controls (CFU) Average 3.94 x107 5.82 x107 2.39 x107 3.51 x107 2.91 x107 Stdev 6.34x1 0s 6.31 x 10s 1.77x106 1.63x107 1.57x107 Avg. Recovery (CFU) Average 1.56x107 2.57x1 0s 3.36 x105 3.60x1 0s 1.37x106 Stdev 6.56x1 0s 1.41 x 10s 1.66 x105 6.15x106 1.01x106 LR Average 0.33 1.4 1.9 1.5 1.4 Stdev 0.27 0.21 0.18 0.75 0.33 51 ------- Table 4-13. OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series 2) In House OTC Surface Decontamination Results -Amendment 3 Material Carpet Concrete Glass Vinyl Flooring Wood Exposure Time (min) 30 Number and Frequency of Sprays 1 at 0 min 1 at 0 min, 1 at 15 min 2 at 0 min 2 at 0 min, 2 at 15 min 3 at 0 min 4 at 0 min 2 at 0 min, 2 at 15 min 3 at 0 min 4 at 0 min 1 at 0 min 1 at 0 min, 1 at 15 min 2 at 0 min* 2 at 0 min, 2 at 15 min 3 at 0 min 4 at 0 min 1 at 0 min 1 atO min, 1 at 15 min** 2 at 0 min 4 at 0 min 3 at 0 min 2 at 0 min, 2 at 15 min 1 at 0 min 1 at 0 min, 1 at 15 min 2 at 0 min 2 at 0 min, 2 at 15 min 3 at 0 min 4 at 0 min Positive controls (CFU) avg 2.75 x107 3.21 x107 4.68 x107 2.09 x107 1.18x108 3.45 x107 1.81 x107 3.26 x107 5.48 x107 3.43 x107 stdev 4.97x1 0s 3.44x1 0s 1.11 x106 3.00x1 0s 1.21 x107 5.16 x 10s 4.84x1 0s 1.04x107 7.52x1 0s 4.39x1 0s Avg CFU Recovered avg 1.35x106 1.41 x105 1.57x106 2.68 x105 6.54 x105 8.11 x105 1.40 x107 2.29 x107 2.29 x107 4.39 x104 1.18x104 9.62 x104 1.62 x104 4.66 x104 1.02x106 3.30 x105 3.06 x103 3.41 x104 5.04 x104 1.44 x104 8.29 x104 1.21 x106 3.11 x104 2.35 x105 4.32 x104 4.04 x105 7.27 x104 stdev 1.40x106 4.78 x104 1.41x106 6.34 x104 3.71 x105 7.53 x105 1.12x107 6.34x1 0s 3.01 x 10s 7.98 x104 1.61x104 1.89x105 3.27 x104 2.35 x105 1.82x106 5.97 x105 2.68 x103 3.44 x104 4.01 x104 1.58x104 9.77 x104 1.27x106 3.80 x104 1.21x105 3.44 x104 4.32 x105 4.46 x104 LR avg 1.5 2.3 1.4 2.1 1.8 1.7 0.7 0.3 0.3 3.3 4.9 3.5 5.3 3.5 2.6 2.6 4.2 2.9 2.6 3.7 3.0 2.0 3.7 2.2 3.0 2.1 2.7 stdev 0.49 0.14 0.45 0.09 0.26 0.32 0.47 0.12 0.06 0.80 2.0 1.2 1.8 0.61 0.73 0.72 0.33 0.38 0.30 0.80 0.73 0.86 0.89 0.30 0.32 0.48 0.25 'Average calculated from only four samples instead of five (possible contamination of one of the samples). **28 and 29 CFU counted as valid results on one of the five samples. 52 ------- 4.4 Material Compatibility Tests Figures 4-8 through 4-14 show the results of the material compatibility tests. None of the test coupons (subjected to either Post-Rinse or No Post-Rinse procedures) showed significant changes in mass. Any changes in mass recorded were within 0.02 % of the initial coupon masses. Materials that followed No Post-Rinse procedure appeared to have salt deposits (possibly arising from baking soda) on them. Carbon steel was observed to have reacted most to the decontamination solution (with both procedures), with a visible amount of rust and corrosion on its surface (Figures 4-8 and 4-10). Little change was observed on the surfaces of stainless steel and aluminum coupons that were post rinsed after being treated with the decontamination solutions. Typical indoor room conditions (70.8 °F and 37.1 % RH) were observed throughout the duration of the Material Compatibility test. Figure 4-8. Corrosion on Carbon Steel with pH ~2 using Procedure 1 (Post-Rinse) t> Figure 4-9. Front Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse) 53 ------- Figure 4-10. Back Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse) Figure 4-11. Front Surface of Carbon Steel with pH 6 using Procedure 2 (No Post-Rinse) 54 ------- Figure 4-12. Galvanized Metal with pH 2 using Procedure 1 (Post Rinse) Figure 4-13. Salt Deposition on Carbon Steel and Stainless Steel with pH 6 using Procedure 1 55 ------- Figure 4-14. Salt Deposition on Stainless Steel and Aluminum with pH 6 using Procedure 2 4.5 Results Summary Based on the liquid-liquid decontamination testing approach evaluated under this study, PAA (at 0.1 % or higher concentrations) shows strong efficacy against Ba spores. However, the techniques and procedures used to decontaminate materials contaminated with Ba spores using this approach do not effectively reflect the way in which decontaminants are applied. The spraying approach using readily available equipment may increase readiness to respond to a wide-area containment release. However, a concern regarding PAA usage, in the form of a spray, is its stability. When prepared using chemicals available off-the-shelf (OTC PAA formulation), the PAA breaks down quickly (within an hour). This deterioration has a direct effect on the potency of the solution overtime. Future study and research is needed to improve upon the spraying technique and stability of the OTC PAA formulation. Although the PAA spray prepared using laboratory grade chemicals was observed to be stable overtime, the practical use of this formulation is limited. Access to these laboratory grade chemicals is restricted. Production and wide consumer access to supplies such as sulfuric acid would have to be enhanced. Furthermore, PAA is a powerful oxidizer, and users will have to be thoroughly briefed on the safety aspects of handling these chemicals. The materials compatibility test performed under this study also suggests that users need to sufficiently neutralize the decontaminant to prevent potential damage to the exposed surfaces. Environmental precautions such as diluting the PAA with large amounts of water before allowing it to enter drains and sewers will have to be addressed to reduce the risk of decomposition. 56 ------- 5 Quality Assurance This project was performed under an approved Category III Quality Assurance Project Plan (QAPP) entitled, "Parametric Testing of Decontamination Chemistries to Guide Decontamination Selection I: Peracetic Acid" (July 2013). All test activities were documented via narratives in laboratory notebooks and the use of digital photography. The documentation included, but was not limited to, a record for each decontamination procedure, any deviations from the Quality Assurance Project Plan (QAPP), and physical impacts on materials. 5.1 Sampling, Monitoring, and Analysis Equipment Calibration There were standard operating procedures for the maintenance and calibration of all laboratory and microbiology laboratory equipment. All equipment was verified as being certified calibrated or having the calibration validated by EPA's on-site (Research Triangle Park [RTP], NC) Metrology Laboratory at the time of use. Standard laboratory equipment such as balances, pH meters, BSCs, and incubators were routinely monitored for proper performance. Calibration of instruments was done at the frequency shown in Table 5-1. If deficiencies were noted, the instrument was adjusted to meet calibration tolerances and recalibrated within 24 hours. If tolerances were not met after recalibration, additional corrective action was taken, possibly including, recalibration or/and replacement of the equipment. Table 5-1. Analysis Equipment Calibration Frequency Equipment pH meter HOBO RH Sensor Stopwatch Micropipettes Clock Scale Buret Temperature of Incubation Chamber Calibration/Certification Perform a two-point calibration with standard buffers that bracket the target pH before each use Compare to calibrated RH sensor prior to use. Compare against NIST* Official U.S. time at http://nist.time.gov/timezone.cgi?Eastern/d/- 5/java once every 30 days All micropipettes will be certified as calibrated at least once per year. Pipettes are recalibrated by gravimetric evaluation of pipette performance to manufacturer's specifications every six months by supplier (Rainin Instruments) Compare to office U.S. Time @ time.gov every 30 days Check calibration with Class 2 weights Manufacturer certified Once a year against an NIST-traceable thermometer Expected Tolerance ± 0.1 pH units ±5% ±1 min/30 days ±5% ±1 min/30 days + 0.1 % weight 0.1 ml_ +2°C *NIST = National Institute of Standards and Technology 57 ------- 5.2 Data Quality Objectives The primary objective of this project was to develop an effective sporicidal low tech PAA formulation that could easily be generated in the field, using over-the-counter ingredients. This formulation needs to reach equilibrium within realistic (i.e., a few hours) time. The Data Quality Objectives (DQOs) assigned to this study are summarized below: 1. Characterizing the decontamination solutions (four PAA formulations and one Hydrogen Peroxide formulation) by developing an understanding of the time to reach equilibrium. 2. Assessing the relationship between PAA concentration, hydrogen peroxide concentration, exposure time, and sporicidal effectiveness. 3. Determining the fate of the spores during the decontamination processes (liquid-liquid and surface decontamination) and providing a 6-LR of 8. anthracis surrogate spores. 4. Assessing the corrosion of low PAA formulations derived from OTC products. The operational parameters of decontamination (e.g., pH and temperature of decontamination solution within the pot life of the decontamination solution, the concentration of the PAA formulations, the exposure times) were considered important in understanding the sporicidal activity of the PAA-based decontamination process and were characterized in addition to sporicidal effectiveness. 5.3 Acceptance Criteria for Critical Measurements The DQOs are used to determine the critical measurements needed to address the stated objectives and specify tolerable levels of potential errors associated with simulating the prescribed decontamination environments. The following measurements were deemed to be critical to accomplish part or all of the project objectives: • PAA concentration in the decontamination solutions • HP concentration in the decontamination solutions • pH • Volume of the decontamination solution • Volume of HP in the decontamination solution • Volume of AA in the decontamination solution • Volume of sulfuric acid in the decontamination solution • Volumetric ratio of AA initial solution to HP initial solution • Volume of the neutralizer solution • CPU abundance per plate • Decontamination time • Decontamination solution stability overtime (drift) 58 ------- • Weight during titration • Weight of the coupons from the material compatibility testing Data Quality Indicators (DQIs) for the critical measurements were used to determine if the collected data met the QAPP objectives. The critical measurement acceptance criteria is shown in Table 5-2. Table 5-2. Critical Measurement Acceptance Criteria Measurement Parameter Time Plated Volume Dilution Ratio Volumes Weight Counts of CFU/plate PH PAA HP Analysis Method NIST-calibrated stopwatch Pipette Volumetric Serological pipette tips Burettes Scale Manual counting NIST-traceable buffer solutions lodometric Titration per MOP 3200 lodometric Titration per MOP 3200 Accuracy ± 1 minute per hour 2% 0.2 % 0.1 ml_ 0.1 ml_ NA ±10% of all CPU per plate between first and second count ±0.1 pH units NA NA Precision/ Repeatability NA 1 % NA NA NA 0.0001 g 50 % RSD between triplicates for each plate NA 10% 10% Detection Limit NA NA NA NA NA NA 1 CPU NA 0.001 % NA Completeness (%) 100 100 100 100 100 100 100 100 100 100 NA = not applicable RSD = relative standard deviation Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside contractor (Calibrate, Inc, Carrboro, N.C). Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results (per test), a second count was performed on 25 percent of the plates within the quantification range (plates with 30-300 CFU). All second counts were found to be within 10 percent of the original count. Many QA/QC checks were used to validate spore recovery measurements. These QA/QC checks include samples that demonstrate the ability of the EPAS's onsite microbiology laboratory to culture and grow spores as well as to demonstrate that materials used in this effort do not themselves contain spores. The checks included: • Laboratory material coupons: includes all materials, individually, used by the on-site microbiology laboratory in sample analysis; materials were confirmed sterile. 59 ------- • Positive control coupons: coupons inoculated but not decontaminated. • Inoculation control coupons: PBST samples inoculated but not decontaminated to assess the variability of the inoculation operation. • Procedural blank coupons: Spores were detected on some blank coupons. The potential impact on the decontamination efficacy was considered negligible. 5.4 Quality Assurance (QA)/Quality Control (QC) Checks Uniformity of the material sections was a critical attribute to assure reliable test results. Uniformity was maintained by obtaining a large enough quantity of material that multiple material sections and coupons could be constructed with presumably uniform characteristics. Samples and test chemicals were maintained to ensure their integrity. Samples were stored away from standards or other samples that could cross-contaminate them. Supplies and consumables were acquired from reputable sources and were NIST-traceable when possible. Supplies and consumables were examined for evidence of tampering or damage upon receipt and prior to use, as appropriate. Supplies and consumables showing evidence of tampering or damage were not used. All examinations were documented, and supplies were appropriately labeled. Project personnel checked supplies and consumables prior to use to verify that they met specified task quality objectives and did not exceed expiration dates. Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this investigation did not involve the use of analytical measurement devices. Rather, the CPU were enumerated manually and recorded. QC checks for critical measurements/parameters are shown in Table 5-2. The acceptance criteria were set at the most stringent level that can routinely be achieved. Positive controls and procedural blanks were included along with the test samples in the experiments so that well- controlled quantitative values could be obtained. Background checks were also included as part of the standard protocol. Replicate coupons were included for each set of test conditions. Standard operating procedures using qualified, trained and experienced personnel were used to ensure data collection consistency. The confirmation procedure, controls, blanks, and method validation efforts were the basis of support for biological investigation results. If necessary, training sessions were conducted by knowledgeable parties, and in-house practice runs were used to gain expertise and proficiency prior to initiating the research. 60 ------- Table 5-3: QA/QC Sample Acceptance Criteria Coupon or Sample Type Decontamination Solution PAA Stability (drift) Decontamination Solution Negative Controls (solution without biological agent) Positive Control Coupons Laboratory Blank Blank ISA, Sterility Control (plate incubated, but not inoculated) Acceptance Criteria + 10 % Drift over an hour of testing No observed CPU Target recovery of 1 *1 o7 CPU per coupon (sample) with a standard deviation of < 0.5. (5x1 06 through 5x1 07 CPU/coupon); 90 % recovery of inoculum; Grubbs outlier test (or equivalent) No observed CPU No observed growth following incubation Corrective Action After 1 hour, reassess concentration of active ingredients using titrations (eerie sulfate, followed by iodiometric determination of PAA) If detected, discuss possible implications with the EPA WACOR*to improve the source of cross-contamination Outside target range: discuss potential impact on results with EPA WACOR; check inoculum and prepare new inoculum if necessary Trace and remove source of contamination. Reject results of the same order of magnitude as the laboratory blank recovery Discard plate. Review plate making procedures to identify source of contamination *WACOR = Work Assignment Contracting Officer Representative Potential confounding organisms were excluded or controlled by sterilization of the materials and use of aseptic technique, procedural blank controls, and a pure initial culture. Aseptic technique was used to ensure that the culture remains pure. Procedural blank controls were run in parallel with the contaminated materials. Assuming that the procedural blank controls showed no CPU, the observed colonies from inoculated coupons indicated surviving spores from the inoculated organisms provided they were consistent with the expected colony morphology (i.e., orange color, round form, flat elevation, rough texture, and undulate margin). 5.5 Data Quality Audits This project was assigned QA Category III and did not require technical systems, performance evaluation or data quality audits by EPA or contractor QA personnel. 5.6 QA/QC Reporting QA/QC procedures were performed in accordance with the QAPP for this investigation. 61 ------- References 1. Canter, D.A., Sgroi, T. J., O'Connor, L, and Kempter, C.J. Source reduction in an anthrax- contaminated mail facility. Biosecurity and Bioterrorism : Biodefense Strategy, Practice, and Science, 2009. 7(4): 405-12. 2. Canter, D.A., Gunning, D., Rodgers, P., O'Connor, L., Traunero, C. and Kempter, C.J. Remediation of Bacillus anthracis contamination in the U.S. Department of Justice mail facility. Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science, 2005. 3(2): 119-127. 3. Canter, D.A., Remediating anthrax-contaminated sites: Learning from the past to protect the future. Chemical Health and Safety, 2005.12(4): 13-19. 4. Calfee, M.W., Ryan, S.P., Wood, J.P., Mickelsen, L., Kempter, C, Miller, L., Colby, M., Touati, A., Clayton, M., Griffin-Gatchalian, N. McDonald, S., and Delafield, R. Laboratory evaluation of large-scale decontamination approaches. Journal of Applied Microbiology, 2012.112(5): 874-82. 5. U.S. Environmental Protection Agency, After Action Report - Danbury Anthrax Incident, National Decontamination Team, Editor 2008: Erlanger, KY. 6. Rutala, W., Disinfection, Sterilization and Antisepsis: Principles, Practices, Current Issues, and New Research, ed. W. Rutala. 2007, Washington, D.C.: Association for Professionals in Infection Control and Epidemiology. 281. 7. Heisig, C.C., Kaiser, H.J., Klein, D.A., Linder, J.S., Frey, K.J., Kaiser, N.E., and Newman, J.L. Low odor, hard surface sporicide. Inorganic active ingredient containing peroxide or compositions of or releasing gaseous oxygen or ozone hydrogen peroxide, U.S.Patent Office, Editor 2010: United States. 8. Gupta, A., Reedy, C.J., Moriarty, B.E., and Atkins, J.M. Strategy for on-site in situ generation of oxidizing compounds and application of the oxidizing compound for microbial control, U.S. Patent Office, Editor 2012: United States. 9. Block, S.S. Disinfection, sterilization, and preservation. 4th ed. Philadelphia: Lea & Febiger Pubs., 1991. 10. Rangarajan B., Havey, A., Grulke E. A., and Culnan, P.O. Kinetic parameters of a two-phase model for in situ epoxidation of soybean oil. Journal of the American Oil Chemists' Society, 1995.72(10): 1161-1169. 11. ASTM E1054-8, A.S., Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agentsl. ASTM International, 100 Barr Harbor Drive, P.O. Box C700, West Conshohocken, PA 19428-2959. United States, 2013. 12. Greenspan, F & Mackellar, Anal. Chem. 20, 1061 (1948) FMC. Technical Bulletin 4, peracetic acid, 35%, page 10. 13. Zhao, X-B, Zhang, T., Zhou, Y.-J., and Liu, D.-H., Preparation of Peracetic Acid from Acetic Acid and Hydrogen Peroxide: Experimentation and Modeling. The Chinese Journal of Process Engineering, 2008. 8(1): 35-41. 62 ------- 14. U.S.EPA, U. (2010). "Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination Studies for Bacillus anthracis using Multiple Test Methods." 63 ------- United States Environmental Protection Agency PRESORTED STANDARD POSTAGE & FEES PAID EPA PERMIT NO. G-35 Office of Research and Development (8101R) Washington, DC 20460 Official Business Penalty for Private Use $300 ------- |