EPA/600/R-16/132 I July 2016 www.epa.gov/homeland-security-research United States Environmental Protection Agency &EPA Expedient Approaches for the Management of Wastes Generated from Biological Decontamination Operations in an Indoor Environment Office of Research and Development National Homeland Security Research Center ------- This page is intentionally left blank ------- EPA 600/R-16/132 July 2016 Expedient Approaches for the Management of Wastes Generated from Biological Decontamination Operations in an Indoor Environment Evaluation of Waste Sampling and Decontamination Procedures - Part II Assessment and Evaluation Report National Homeland Security Research Center Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, NC 27711 ------- This page is intentionally left blank ------- Disclaimer The United States Environmental Protection Agency (EPA), through its Office of Research and Development's National Homeland Security Research Center, funded and directed this investigation through contract EP-C-09-027 with ARCADIS U.S., Inc, and contract EP-C-15-008 with Jacobs Technology Inc. This report has been peer and administratively reviewed and has been approved for publication as an EPA document. It does not necessarily reflect the views of EPA. 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. EPA does not endorse the purchase or sale of any commercial products or services. Questions concerning this document or its application should be addressed to: M. Worth Calfee, Ph.D. Decontamination and Consequence Management Division National Homeland Security Research Center U.S. Environmental Protection Agency (MD-E343-06) Office of Research and Development 109 T.W. Alexander Drive Research Triangle Park, NC 27711 Phone:919-541-7600 Fax: 919-541-0496 E-mail: Calfee.Worth@epa.gov ------- Acknowledgments This effort was directed by the principal investigator from the Office of Research and Development's (ORD) National Homeland Research Center (NHSRC), Decontamination and Consequence Management Division (DCMD) utilizing support from the U.S. Environmental Protection Agency's (EPA) Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence Management Advisory Division (CMAD) within the Office of Emergency Management (OEM). The contributions of the entire team are acknowledged. Project Team: Worth Calfee, Ph.D. (Principal Investigator) U.S. EPA, Office of Research and Development, NHSRC, DCMD Research Triangle Park, NC 27711 Paul Lemieux, Ph.D. U.S. EPA, Office of Research and Development, NHSRC, DCMD Research Triangle Park, NC 27711 Mario lerardi U.S. EPA, Land and Emergency Management, OLEM, Office of Resource Conservation and Recovery, Materials Recovery and Waste Management Division, Water Compliance Branch Arlington, VA 22202 Paul Kudarauskas U.S. EPA, Land and Emergency Management, OLEM, OEM, CBRN CMAD Washington, DC 20004 R. Leroy Mickelsen, M.S., P.E. U.S. EPA, Office of Land and Emergency Management, OLEM, OEM, CBRN CMAD Research Triangle Park, NC 27711 Randy Schademann U.S. EPA, Federal On-Scene Coordinator, Region 7 Lenexa, KS 66219 This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc, and Jacobs under contract EP-C-15-008. The support and efforts provided by ARCADIS-US, Inc., and Jacobs are acknowledged. ii ------- Table of Contents Disclaimer i Acknowledgments ii List of Figures v List of Tables vi List of Acronyms and Abbreviations vii Executive Summary viii 1 Introduction 1 1.1 Process 2 1.2 Project Objectives 3 1.3 Experimental Approach 3 1.3.1 Testing Sequence 4 1.3.2 Decontamination Strategy 4 1.3.3 Method Development for Neutralization 4 2 Materials and Methods 5 2.1 Facility Design 5 2.2 Agitation 5 2.3 Test Coupon Preparation 6 2.3.1 Carpet and Upholstery 6 2.3.2 Paper 7 2.3.3 Nitrile Gloves 8 2.4 Spore Preparation and Coupon Inoculation 8 2.5 Decontamination Solutions 8 2.6 Decontamination Procedure 9 2.7 Method Development for Neutralization 10 2.8 Test Matrix 10 pAB, pH-adjusted bleach; PPE, personal protective equipment 11 3 Sampling Methods 12 3.1 Sampling Approach 12 3.1.1 Sponge-Stick™ Sampling 12 3.1.2 Extractive Sampling 13 3.2 Sample Type 13 3.2.1 Carpet and Upholstery 13 3.2.2 Paper Samples 13 3.2.3 PPE Samples 14 3.3 Sample Preservation 14 iii ------- 3.4 Sampling Points 14 PPE, personal protective equipment 15 3.5 Sampling Frequency and Sample Quantities 15 dEach finger of a glove is considered one sample; note that, for the gloves, runoff samples were also collected 16 3.6 Measurement Methods 16 3.6.1 Microbiological Samples 16 3.6.1.1 Sample Extraction 16 3.6.1.2 Sample Analysis 16 3.7 Data Analysis 17 3.7.1 Sampling Efficiency 17 3.7.2 Surface Decontamination Efficacy 17 3.7.3 Statistical Analysis 18 4 Results and Discussion 19 4.1 Sampling Methods Evaluation 19 4.1.1 Carpet Material 19 4.1.1.1 Positive Controls 19 4.1.1.2 Post-Decontamination Sample Recoveries 20 4.1.2 Upholstery Material 22 4.1.3 Paper Material 23 4.1.4 PPE Material 24 4.2 Neutralization Method Evaluation 24 4.3 Dunking/Immersion Decontamination Test Results 25 4.3.1 Carpet Decontamination Results 25 4.3.2 Upholstery Decontamination Results 27 4.3.3 Paper Decontamination Results 28 4.3.4 PPE Decontamination Results 29 5 Quality Assurance 31 5.1 Sampling, Monitoring, and Analysis Equipment Calibration 31 5.2 Data Quality 31 5.3 Acceptance Criteria for Critical Measurements 32 5.4 QA/QC Checks 34 5.4.1 Quality Control Management 34 5.4.2 Quality Control Evaluation 35 6 Summary 37 7 References 39 iv ------- List of Figures Figure 2.1: Poly Hog Trough® 5 Figure 2.2: Top view of the immersion vessel retrofitted for agitation 6 Figure 2.3: Carpet tile 6 Figure 2.4: Front of assembled upholstered coupon 7 Figure 2.5: Paper material 7 Figure 2.6: White disposable nitrile glove 8 Figure 3.1: Material section shown with template during sampling with Sponge-Stick™ and 18-mm coupon removal 13 Figure 3.2: PPE sample (finger) excision 14 Figure 3.3: Sampling timeline 15 Figure 4.1: Spore recoveries (± SD) from carpet using the extractive and Sponge-Stick™ methods 20 Figure 4.2: Post-decontamination spore recoveries (± SD) for carpet as a function of decontaminant procedure and sampling method 21 Figure 4.3: Effect of waste storage time on positive control recoveries (± SD) from upholstered material for the extractive and Sponge-Stick™ methods 22 Figure 4.4: Effect of waste storage time on positive control recoveries (± SD) from paper (PM, middle page; PF, front page) for the extractive method 23 Figure 4.5: Effect of waste storage time on positive control recoveries (± SD) from personal protective equipment for the extractive method 24 Figure 4.6: Decontamination efficacy (± SE) versus decontamination treatment for carpet 27 Figure 4.7: Decontamination Efficacy (± SE) for Upholstered Coupon Using Spor-Klenz® 28 Figure 4.8: Decontamination efficacy (± SE) for Spor-Klenz® and paper coupons for front page (PF) and middle page (PM) 29 Figure 4.9: Decontamination efficacy (± SE) versus decontamination treatment for personal protective equipment (PPE) material 30 v ------- List of Tables Table 2.1: Decontaminants and Accessibility 11 Table 2.2: Decontamination Procedures and Intensity 11 Table 2.3: Decontamination Test Sequence Event 11 Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures 15 Table 3.2: Number of Sample Types per Material Section per Sampling Sequence 16 Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the Extractive and Sponge-Stick™ Sampling Methods 20 Table 4-2: Post-Decontamination Spore Recoveries (CFU) for Carpet as a Function of Decontaminant Procedure and Sampling Method 21 Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstery for the Extractive and Sponge-Stick™ Sampling Methods 22 Table 4.4: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the Extractive and Sponge-Stick™ Sampling Methods 23 Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal Protective Equipment 24 Table 4.6: Preliminary Neutralization Optimization 25 Table 4.7: Decontamination Efficacy versus Type of Decontamination Treatment for Carpet Material 26 Table 4.8: Decontamination Efficacy (Log Reduction) versus Type of Decontamination Treatment for Paper Material 28 Table 4.9: Decontamination Efficacy (CFU LR) versus Type of Decontamination Treatment for PPE Material 30 Table 5.1: Instrument Calibration Requirements 31 Table 5.2: Critical Measurement Acceptance Criteria 32 Table 5.3: Data Quality Assessment 33 Table 5.4: Quality Control Checks 35 Table 5.5: Quality Control Evaluation 36 Table 6.1: Summary of Waste Decontamination Results 38 vi ------- List of Acronyms and Abbreviations ATCC American Type Culture Collection BOTE Bio-response Operational Testing and Evaluation CBRN chemical, biological, radiological, and nuclear CFU colony-forming unit CMAD Consequence Management Advisory Division (EPA/OLEM/OEM) DCMD Decontamination and Consequence Management Division (EPA/ORD/NHSRC)) EPA U.S. Environmental Protection Agency FAC free available chlorine FIFRA Federal Insecticide, Fungicide, and Rodenticide Act HSPD Homeland Security Presidential Directive HSRP Homeland Security Research Program ISO International Organization for Standardization LR log reduction MOP miscellaneous operating procedure NHSRC National Homeland Security Research Center (EPA/ORD) NIST National Institute of Standards and Technology OEM Office of Emergency Management (EPA/OLEM) OLEM Office Land and Emergency Management (EPA) OPP Office of Pesticides Programs (EPA) ORD Office of Research and Development (EPA) pAB pH-adjusted bleach PAA peroxyacetic acid PBST phosphate-buffered saline with Tween®20 PF paper front PM paper middle PPE personal protective equipment QA quality assurance QAPP quality assurance project plan QC quality control RH relative humidity RTP Research Triangle Park SD standard deviation SE sampling efficiency STS sodium thiosulfate TSA Tryptic Soy Agar VHP® vaporized hydrogen peroxide vii ------- Executive Summary 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 remediation of areas contaminated with biological agents. Transporting waste contaminated with biological agents to remote treatment/disposal facilities incurs costs and risks. If waste from a biological contamination incident could be shown to have no residual biological agent present, then it might be able to be dealt with as conventional solid waste, which would incur less transportation risk and less cost. The primary objective of this investigation was to determine the effectiveness of an expedient on-site approach to waste decontamination. In this report, data are frequently presented as the average log reduction (LR) for a particular test. In laboratory tests, if a particular set of decontamination conditions achieves > 6 LR (against a 6-7 log challenge), the decontamination is generally considered "effective." This benchmark is consistent with sporicidal efficacy tests used to register sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Achieving complete kill (no viable agent recovered following the decontamination treatment) is considered "highly effective." A number of waste decontamination and management approaches have been used in previous bioterror remediation operations, although their effectiveness has yet to be determined experimentally. Previously, a study was conducted to determine the effectiveness of an immersion-based approach to decontaminating waste materials contaminated with Bacillus atrophaeus spores (surrogate for Bacillus anthracis). The study evaluated effectiveness of the immersion approach on high-traffic commercial carpet tile, nitrile gloves (personal protective equipment [PPE]), books, and upholstered seat pans, which are typical of porous materials found in an indoor office or items expected to be contaminated during sampling and remediation (i.e., PPE). Most waste materials, with the exception of carpet, were effectively decontaminated (greater than 6 log reduction [LR]) by a 15-minute immersion in pH-adjusted bleach (pAB). Longer immersion times increased decontamination efficacy on carpet, but a 60-minute immersion failed to provide more than a 4 log reduction in viable spores. In the previous study, diluted bleach and pAB were unable to achieve complete kill for any of the material types tested. The current study continued the investigation and sought to determine waste decontamination conditions that would achieve effective or highly effective decontamination of all material types. For these tests, more stringent decontamination procedures were evaluated specifically using pAB amended with a surfactant and/or waste submersion procedures involving agitation. Further, an off-the-shelf decontaminant solution (Spor-Klenz®) was evaluated for a comparative analysis with pAB submersion procedures. Test materials were inoculated with Bacillus spores at known locations and concentrations and subjected to the prescribed decontamination procedure (i.e., immersion in decontaminant). After the decontamination procedure, a subset of the test materials was sampled immediately, and then the items were bagged and stored (to simulate waste handling/staging during a response). The simulated waste items were resampled in a waste staging area after a drying time of 7 days. A subset of bagged, inoculated waste samples was left untreated to serve as positive controls. ------- The efficacies of three decontamination solutions (pAB, pAB with a surfactant, and Spor-Klenz®) were determined using a single immersion time of 1 hour, with and without agitation of the solution. Two sampling methods were used for carpet and upholstery: extractive and surface sampling with 3M Sponge- Stick™. Only extraction-based methods were used for PPE and books. The decontamination efficacy results were as follows: • The efficacy of the decontamination treatments using pAB on carpet materials, with or without surfactant or agitation, and increasing the storage time up to 7 days was found in the range 2.0 to 3.4 LR, which is less than a 6.0 LR that is generally accepted to be effective during laboratory evaluations. • The efficacy of Spor-Klenz® was found to increase over the 7-day period to reach a greater than 7 LR in colony-forming units (CFUs). Spor-Klenz® was found to be a more effective decontaminant than pAB for bundled/bagged waste using an immersion/dunking approach. Spor-Klenz® achieved complete kill of spores for almost all upholstery and paper material samples. • The results of the PPE (gloves) decontamination tests showed greater variability than the results of the tests on office environment materials. In some of the gloves, full decontamination was achieved in some of the glove fingers, but not in the others. The glove fingers where limited or no decontamination is achieved might be the result of spores confined in areas (e.g., inside finger tips) with no contact with the decontamination solution. In addition to decontamination efficacy, the collection efficiency of the two sampling methods (extractive and Sponge-Stick™) used in this study were compared as a function of material and elapsed time from inoculation and from when the sample was collected. Analysis showed no significant effect of sample storage time of up to 30 days on spore recovery when using either sampling method. The extractive sampling approach was found to be superior, particularly for wet, porous materials. The results of this study can inform decision makers regarding the utility of expedient waste decontamination techniques. However, other inherent risks associated with the shipment of the contaminated waste, post-decontamination material/decontaminant disposal, and landfill acceptance criteria should be considered prior to implementation of such decontamination approaches in actual remediation operations involving infectious or hazardous waste items. ix ------- 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 (HSPDs) 5, 7, 8, and 10, 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. NHSRC's mission includes providing expertise and guidance on the selection and implementation of decontamination methods and providing the scientific basis for a significant reduction in time, cost, and complexity of decontamination and waste handling activities. NHSRC's research supports EPA's Office of Land and Emergency Management (OLEM), Office of Chemical Safety and Pesticide Programs, and the regional offices. Close collaboration among the different program offices with homeland security responsibilities is sought in order to rapidly increase EPA's capabilities to help the nation recover from a terrorist event involving the intentional release of chemical, biological, radiological, and nuclear (CBRN) materials. In 2001, the introduction of a few letters containing Bacillus anthracis (anthrax) spores into the U.S. Postal Service system resulted in the contamination of several facilities and the deaths of two postal employees. Although most of the facilities in which these letters were processed or received were heavily contaminated, they were successfully decontaminated with approaches such as fumigation with chlorine dioxide or vaporized hydrogen peroxide (VHP®). Fumigation was used primarily in heavily contaminated facilities. Other cleaning methods were used in less heavily contaminated facilities such as those that were secondarily contaminated or those primarily contaminated facilities that showed a minimal presence of anthrax spores. These other "expedient" or "low-tech" methods included removing contaminated items from a contaminated area prior to ultimate treatment and disposal of the items at a specialized facility (i.e., incineration then landfilling of the ash), and/or decontaminating them on-site (within or in proximity to the contaminated structure) by inactivating the spores. In these incidents, decision makers' selection of on-site or off-site destruction of spores was facility-dependent and took into account many issues (e.g., physical state of the facility). One important factor complicating the decision-making process was that such decontamination was unprecedented for the United States government and no sporicidal technologies had been proven or registered for use against B. anthracis spores at the time. The cost of waste management for the 2001 incident proved to be very significant and was complicated by the nature of the waste. Additional quick, effective and economical decontamination methods with the capacity to be employed over wide areas (outdoor and indoor) are required to increase preparedness for an incident involving the release of a biological agent. If the waste from a biological contamination incident could be shown to have no residual biological agent present, then it might not require any special packaging for transport to remote treatment/disposal facilities, and may be able to be dealt with as conventional solid waste. So, since 2001, the emphasis for facility decontamination research has been to identify and characterize efficacious on-site decontamination methods and to optimize the 1 ------- decontamination/waste management paradigm; this optimization could reduce the overall time and cost required for the entire remediation. If proven effective, a lower tech approach to decontaminating waste on-site could reduce overall decontamination costs by reducing the amount of waste treatment required by off-site, specialized facilities (e.g., medical waste incinerators). On-site waste treatment could also reduce the risk and complications associated with transporting contaminated materials to such facilities. Developing and demonstrating on-site waste management solutions could increase EPA's readiness to respond to a wide-area release that would generate contaminated waste volumes much larger than those previously managed. Response and remediation activities associated with this type of incident will require additional waste handling, segregation, and staging. These additional requirements illustrate the need for efficacy data and improved process knowledge to support assessments regarding decontamination operations and waste management. Waste items (ceiling tile and carpet) generated during a recent facility-scale test (Bio-response Operational Testing and Evaluation (BOTE study) [1] that were decontaminated during the study by expedient methods (liquid bleach spray orspritz), bagged/managed in a manner typical of a real remediation effort, stored (~ 6 months), and subsequently sampled, showed that significant quantities of the test organism (B. atrophaeus) had survived the treatment and subsequent 6-month storage duration. This finding indicates that current waste management techniques used during expedient decontamination efforts can generate waste items that have residual contamination. Since the willingness of waste treatment/disposal facilities to accept waste items might depend partly on the contamination level of the items, identification and demonstration of methodologies that effectively decontaminate waste on-site during low-tech decontamination activities are needed. This study evaluated several waste decontamination strategies that could be conducted on-site. For waste generated during the decontamination of typical indoor office or indoor residential settings, no sampling methods have been standardized. Many waste items generated from the decontamination of building interiors are expected to consist largely of porous materials [1], which pose challenges to currently available sampling methods. To provide the data necessary to standardize a waste sampling method, extraction-based and surface-wipe-based sampling methods were evaluated. Extraction-based sampling consisted of excision and subsequent extraction of a portion of the material. Surface-wipe- based sampling consisted of collection of the contaminant from the material surface with a Sponge- Stick™ (3M, St. Paul, MN). 1.1 Process The purpose of this study was to identify effective and efficient means to decontaminate waste on-site and to compare the efficiency of the two sampling methods used. The decontamination and sampling strategies utilized were selected by an EPA project team, which consisted of staff from the NHSRC within EPA's Office of Research and Development, OLEM, and Region 7 Office. Effectiveness of each decontamination approach was evaluated in terms of Logio Reduction (LR). Approaches that achieved > 6 LR (against a 6-7 log challenge), were considered "effective." This benchmark is consistent with sporicidal efficacy tests used to register sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Achieving complete kill (no viable agent recovered following the decontamination treatment) was considered "highly effective." This study investigated decontamination of selected materials by an immersion (dunking) approach of waste materials contaminated with B. atrophaeus spore inoculum (i.e., surrogate for B. anthracis). The 2 ------- effectiveness of this decontamination approach was evaluated for high-traffic commercial carpet tile, books, and upholstered seat pans that are typical of materials found in indoor offices, or for items such as nitrile gloves (personal protective equipment [PPE]) that would be expected to be contaminated during sampling and decontamination operations. Replicate sections of test materials were inoculated at known locations with a targeted number of Bacillus spores. After decontamination, sections of the test material were sampled immediately and then bagged and stored (to simulate field waste handling procedures) in the waste staging area. The decontaminated test materials were resampled after a drying time of 7 days to determine if storage duration could increase the effectiveness of the decontamination treatment (where decontamination treatment is defined, for the purpose of this study, as a prescribed decontamination procedure). A subset of inoculated bagged waste samples was left untreated to serve as positive controls. The sampling strategies discussed herein have been selected and optimized to determine the survival of B. atrophaeus spores following decontamination treatment. 1.2 Project Objectives The primary objective of this work was to continue efforts from a previous study that estimated the efficacy of liquid-based decontamination approaches for on-site treatment of bundled or bagged waste items typically generated during a Bacillus anthracis cleanup response for an indoor office setting. The waste items, such as contaminated indoor office items, would generally be placed in bags or bundled for transportation for off-site treatment and disposal during the removal process, however, this previous study evaluated on-site alternatives. Within that study it was demonstrated that pH-adjusted bleach (pAB) solution was efficacious (greater than 6 LR) for most of the materials tested (nitrile gloves, books, and upholstered seat pans), it was less effective for carpet (4 LR in viable spores with 1-hour immersion time), failing the generally accepted criterion of 6 LR to consider an approach effective during laboratory evaluations of sporicides. Recovery of no viable spores following treatment was considered highly effective. None of the waste decontamination procedures evaluated previously achieved complete kill for any of the tested material types. Waste acceptance criteria are likely to be unknown prior to an incident, therefore determining conditions that achieve complete kill are of interest. In an effort to achieve complete kill for all material types, more rigorous tests incorporating a surfactant and/or agitation have been designed and performed under this study. Additionally, submersion testing using Spor-Klenz® (STERIS, Mentor, OH), with and without agitation, was performed on all materials for a comparative analysis with the pAB submersion procedures. The ultimate objective was to provide data to support development of a stepwise procedure(s) for on- scene responders and decontamination teams to use for on-site waste treatment during responses involving expedient approaches for the indoor environment. Demonstrated waste decontamination procedures could reduce the cost and time of a response by reducing the number of waste characterization samples required and increasing the acceptance of these types of wastes from more waste management facilities, thereby providing a great capability for wide-area incidents. 1.3 Experimental Approach The experimental approaches described throughout this document are consistent with a previous study[2], whereby waste decontamination and sampling methods were evaluated. 3 ------- 1.3.1 Testing Sequence The following is the testing sequence used to meet the objectives of this project: 1. Prepare material sections for each test material as described in Section 2.3. 2. Pre-punch material sections (carpet, upholstery, and book materials), and retain the excised sections for use as 18-mm (0.7-in) diameter coupons for extraction-based sampling procedure. The nitrile gloves did not require the use of 18-mm punch coupons, but rather the 25.4-mm (1 -in.) tip of each finger was used for extraction-based analysis. 3. Assemble the material sections by reinserting the 18-mm excised coupons into their respective void areas of the material section. 4. Sterilize the materials prior to inoculation using ethylene oxide or VHP® sterilization. Wait a minimum of 7 days after sterilization before inoculating materials (See Section 2.4.1). 5. Inoculate 18-mm positive control and test material coupons (without removing from larger material section), each fingertip of each nitrile glove PPE, and the target page samples. 6. Allow inoculum carrier to dry for at least 18 hours. 7. Apply the decontamination procedure to the procedural blank material section batch then to the test material section batch. 8. Collect the samples from material sections immediately upon completion of the decontamination procedure. 9. After sample collection, bag the material batch such that the total weight does not exceed 35 lb per bag. 10. Sample remaining bagged material sections in the waste staging area after a drying time of 1 day (at least 18 hours), 7 days, and 30 days. 1.3.2 Decontamination Strategy The decontamination procedures used for carpet and PPE materials included submersion in pAB with surfactant and/or agitation. For all materials, decontamination efficacy was evaluated for Spor-Klenz® (1.0% by wt. hydrogen peroxide, 0.08% peracetic acid, and <10% acetic acid), for a duration of 60 minutes submersion, with no agitation. 1.3.3 Method Development for Neutralization The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the coupon) could negatively bias colony-forming unit (CFU) quantification results. Prior to the decontamination testing sequence, neutralization tests were performed to determine the amount of neutralizer liquid needed to quench each decontaminant/application combination. Decontaminant neutralizers were added to liquid samples immediately after collection to quench their activity, resulting in precise chemical exposure durations and lower recovery bias. 4 ------- 2 Materials and Methods 2.1 Facility Design All decontamination activities were conducted inside the spray booth area located in the EPA's Research Triangle Park (RTP), NC facility in room H122A of the High Bay building, a room with a single access point and ventilation independent of the High Bay building. The spray booth also served as the waste staging area. The immersion tank was a 0.28 m3(10 ft3) Poly Hog Trough® (EZ Grout Corp., Waterford, OH) made of virgin polyethylene with steel legs (http://www.ezgrout.com/index.php/products/supply/hog-trough) (Figure 2.1). The overall dimensions of the tank were 66.04 cm x 138.43 cm x 60.96 cm (26 in. x 54% in. x 24 in.). 2.2 Agitation Tests that prescribed agitation during the immersion step utilized a low-pressure air pneumatic apparatus (Figure 2.2) to simulate physical agitation of waste items during decontamination procedures. It was hypothesized that agitation may increase decontaminant contact with waste items and thereby increase treatment efficacy. House air was forced through four 1,9-crn (%-in.) tubes, positioned equidistant along the length of the submersion vessel, at a rate of 20 L/min. Air was supplied to the decontamination solution during the entire submersion procedure. Figure 2.1: Poly Hog Trough®. 5 ------- !¦¦¦ Figure 2.2: Top view of the immersion vessel retrofitted for agitation. 2.3 Test Coupon Preparation All coupons were sterilized prior to use to prevent any background organisms from confounding the tests. The books and the glove materials were fumigated with ethylene oxide using an Andersen (Haw River, NC) EO-Gas® 333 sterilization system. The carpet and the upholstered coupons were fumigated via a 250 ppm, 4 hours vaporous hydrogen peroxide (VHP®) sterilization cycle, followed by an overnight aeration process. To prevent cross contamination, on the day of testing, procedural blank coupons were moved into the spray chamber, and procedural blank decontamination occurred before decontamination of any inoculated coupons. 2.3,1 Carpet and Upholstery The carpet coupons were ready-made 0.61 -m x 0.61-m (24-in. x 24-in.), 100% nylon tiles (P/N Multiplicity Tile 54594, Shaw Industries, Dalton, GA, USA), exuberant 00310 color type (Figure 2.3). Upholstered 0.51-m x 0.51-m (20-in. x 20-in.) coupons were prepared in-house with layers of foam and fabric adhered together (Figure 2.4). Each set of five material coupons were bundled and tested together. Figure 2.3: Carpet tile. 6 ------- Figure 2.4: Front of assembled upholstered coupon. 2.3.2 Paper Paper samples consisted of the entire front cover (inside cover inoculated) along with the first page of the Merck Manual of Medical Information (Second Home Edition, 2004) (Figure 2.5) and pages 955 (inoculated) plus one page before and two pages after (953-960). Each page measured 22.8 cm x 16.5 cm (9 in. x 6.5 in.). - > THE MERCK MANUAL OF MEDICAL INFORMATION SECOND HOME EDITION The world's most widely used medical reference for the twenty-first century The complete and unabridged national bestseller Figure 2.5: Paper material. 7 ------- 2.3.3 Nitrile Gloves The material chosen to represent PPE waste consisted of powder-free white disposable nitrile gloves, 5.5 mm (0.21 in.) thick and 22.8 cm (9 in.) in length (McMaster part #52555T 15, www.mcmaster.com'). illustrated in Figure 2.6. The powder-free gloves are considered superior for applications where particulate contamination is a concern. Whole gloves were utilized; however, the tips of each finger were inoculated and served as replicates. Gloves were inverted following inoculation, yet prior to testing, to simulate field conditions where gloves are turned inside out during doffing procedures. Figure 2.6: White disposable nitrile glove. 2.4 Spore Preparation and Coupon Inoculation The test organism for this investigation was a liquid spore suspension of B. atrophaeus (strain: ATCC® 9372) in 29% ethanol solution. This bacterial species was formerly known as B. subtilis var niger and subsequently B. globigii. The spores were purchased from Yakibou, Inc. (Holly Springs, NC) at a population density of 1 x 109 CFU per mL. The titer of the stock was confirmed at the start of each testing event. Inoculation of the 18-mm coupons (carpet and upholstery) and the front cover and middle pages of the books was performed by aseptically applying 100 jj.L of a diluted spore solution to reach a target recovery of 5 x 107 CFU from each positive control sample. Fingertips of the nitrile gloves were inoculated to reach the same target concentration. Gloves were inoculated on the exterior surface of each fingertip, allowed to dry for 18-24 hours, and then turned inside out prior to use in testing. 2.5 Decontamination Solutions The pH-adjusted bleach was prepared as follows: one part Clorox® concentrated germicidal bleach (Clorox Corp., Oakland, CA) was diluted with eight parts of deionized water and one part 5% (v/v) acetic acid (Fisher Scientific, Pittsburgh, PA; Part# 13025, or equivalent). The pH was adjusted to 6.5-7.0 with 5% acetic acid, and the free available chlorine (FAC) content was adjusted to 6000-6700 ppm with deionized water after preparation. The pH-adjusted bleach was used within 3 hours of preparation. The diluted bleach was prepared fresh prior to testing by mixing one part Clorox® concentrated regular bleach with approximately 14 parts of deionized water to reach a target FAC of approximately 6000 ppm. Safety 8 ------- precautions were taken to protect personnel from liberated chlorine gas produced as a result of pH reduction of the bleach solution. The pH-adjusted bleach with surfactant was prepared by adding 1.8 oz. (50 mL) of the surfactant (original Tide® [Procter and Gamble, Cincinnati, OH]) to every 20 gallons of pH- adjusted bleach solution. The FAC concentration of bleach formulations was measured based on ASTM Method D2022-89 [3] . A 5-mL aliquot was mixed with a buffered potassium iodide solution and iodometrically titrated with sodium thiosulfate (STS) to a colorless end point. The aliquot was taken and analyzed immediately after formulation and mixing. The validity of the FAC measurement equipment (Hach® [Hach Company, Loveland, CO] high-range bleach test kit, Method 10100 [model CN-HRDT]) was confirmed through the titration of a chlorite ion standard. The pH of each solution was measured with an Oakton Acorn® series pH 5 meter (Oakton Instruments, Vernon Hills, IL). This meter was calibrated daily. Spor-Klenz® solutions were prepared from Spor-Klenz® concentrate as directed by the manufacturer for sterilization by diluting 1 part Spor-Klenz® with 99 parts deionized water. Final hydrogen peroxide and acetic acid concentrations were measured as follows: A 5-gram sample was mixed with 50 mL of 1 N ice- cooled sulfuric acid. Ferroin indicator was added, and then the sample was titrated with 0.1 N eerie sulfate to an orange-pink end point. Next, 4 grams of potassium iodide (Kl) was added and a red color was allowed to develop. Starch indicator was then added, and the sample was titrated with 0.0375 N STS to a pale orange end point. 2.6 Decontamination Procedure The general decontamination procedure consisted of "dunking" a batch of coupons in the immersion tank containing the decontamination solution for a prescribed immersion time. This decontamination procedure is described below: 1. Prepared decontaminant bath in chemical resistant container. Performed all required quality control (QC) checks listed in Table 5.2. 2. Collected the material batch for immersion, which consisted of three pre-punched sterile material sections (containing the test samples) and enough non-punched sterile material sections (did not contain test samples) to fill the waste storage bag (not to exceed 16 kg [35 lb] when wet; amounts varied per material). Of the material sections in each batch, only three sections of the decontaminated material were inoculated with Bacillus spores. For example, of a 16 kg batch of carpet tiles, only three tiles contained inoculated 18-mm coupons. 3. The material batch (not to exceed 35 lb when wet) was submerged in the decontaminant bath and subjected to the prescribed decontamination procedure. 4. Removed material sections and allowed them to drain briefly (15 minutes) over the decontaminant bath, and then immediately collected the post-decontamination (To) samples per material type. 5. Aseptically transferred decontaminated materials to a labeled 55-60 gallon contractor's storage bag (Uline model S-19876 [Uline, Pleasant Prairie, Wl]), which remained closed in the waste staging area until the next sampling event. Material types were bagged separately and arranged such that one inoculated material section was located at the bottom of the batch, one was located in the middle, and one was located on the top. 9 ------- This procedure was repeated for each material using a single immersion container. Therefore, the immersion container was sanitized between tests by removing all debris, wiping interior surfaces with Dispatch® hospital cleaner disinfectant wipes with bleach (The Clorox Company, Oakland, CA), rinsing interior surfaces with deionized water, and then drying them with 70% ethanol prior to the start of each test. Containers were sampled by swab prior to test initiation to confirm sterility. Testing was performed using a "clean team/dirty team" technique. The "dirty team" was responsible for moving the material sections into and out of the immersion tank and performing the decontamination procedure. The "clean team" was used for procedural blank, control, and test sampling. Only dirty team members handled contaminated items, and only clean team members handled procedural blank coupons and samples. New disposable lab coats were worn for each new material or contamination level. Fresh gloves were donned prior to performing the decontamination procedure and then changed before handling the material section after completion of the decontamination procedure. Dirty team members could become clean team members by donning a new set of protective garb (inner and outer gloves, lab coat, P95 mask, and hair net). 2.7 Method Development for Neutralization The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the sample) could negatively impact bacterial culture-based assays and therefore bias spore recovery estimates. Prior to the decontamination testing sequence, neutralization tests were performed to determine the amount of neutralizer liquid required to quench residual decontaminant produced from each decontaminant/application combination. HACH® Method 10100 (http://www.hachco.ca; Hach Company, Loveland, CO) for high-range bleach was used to experimentally determine the amount of STS required to neutralize in excess the active ingredient (i.e., free available chlorine [FAC]) in pAB. Hydrogen peroxide (H202)-peracetic acid (PAA) titration was used to determine the residual hydrogen peroxide and peracetic acid concentrations in exposed samples to determine the volume of STS required for neutralization. Due to the variation of the amount of decontaminant solution on the coupons, excess of stoichiometric neutralization was evaluated to ensure that it did not affect spore recovery estimates. Analyses of the spores in the optimized excess neutralizer solution was also evaluated at 1-hour and 24-hour hold times to see if the lag time for processing the samples had an effect on the viable spore recoveries. Finally, the effect of spore inoculation concentration and the decontamination time on the neutralization test recoveries were also evaluated. 2.8 Test Matrix The test matrix was initially devised to optimize the on-site decontamination procedures by maximizing efficacy and minimizing the manual effort and hazard level of the procedure. The decontaminant optimization process was designed to test decontaminants in order of accessibility (i.e., order of chemical and equipment availability) and simplicity of execution. This order is shown in Table 2.1. The planned decontamination procedures, in order of increasing intensity, were immersion and then immersion with agitation, as shown in Table 2.2. Table 2.3 shows the test matrix that was performed and described in this report. When a decontaminant procedure was found to be effective for a material type, subsequent tests with more stringency were forgone for that material type. 10 ------- Table 2.1: Decontaminants and Accessibility Decontaminant Decreasing Accessibility pH-adjusted bleach i } pH-adjusted bleach with surfactant Spor-Klenz® Table 2.2: Decontamination Procedures and Intensity Decontamination Procedure Increasing Intensity Immersion ) Immersion with agitation Table 2.3: Decontamination Test Sequence Event Decontamination Procedure Decontaminant Solution Material Type Exposure Time Test Date (DayO) End Date (Day 7) Immersion with agitation pAB PPE 60 minutes Nov 10, 2014 Nov 17, 2014 Immersion pAB with surfactant PPE 60 minutes Nov 18, 2014 Nov 25, 2014 Immersion pAB with surfactant Carpet 60 minutes Jan 6, 2015 Jan 13, 2015 Immersion with agitation pAB Carpet 60 minutes Jan 8, 2015 Jan 15, 2015 Immersion with agitation pAB with surfactant Carpet 60 minutes Jan 20, 2015 Jan 27, 2015 Immersion Spor-Klenz® Carpet 60 minutes Jan 27, 2015 Feb 3, 2015 Immersion with agitation pAB with surfactant PPE 60 minutes Jan 29, 2015 Feb 5, 2015 Immersion Spor-Klenz® PPE 60 minutes Feb 5, 2015 Feb 12, 2015 Immersion Spor-Klenz® Upholstery 60 minutes May 13, 2015 May 20, 2015 Immersion Spor-Klenz® Paper 60 minutes May 21, 2015 May 29, 2015 pAB, pH-adjusted bleach; PPE, personal protective equipment 11 ------- 3 Sampling Methods 3.1 Sampling Approach Prior to each sampling event, all materials needed for sampling were prepared using aseptic techniques and placed in a bin containing enough sampling kits, gloves, and bleach wipes to accommodate all required samples for the specific test. The materials specific to each protocol are included in the relevant sections below. In an effort to minimize the potential for cross contamination during sampling, and in accordance with aseptic technique, a sampling team was utilized. The team was made up of a "sampler" (handling only the sampling media), a "coupon handler" (the only person to handle material coupons during the sampling event), and a "support person" (who in addition to being responsible for handing sterile templates to the sampler was also responsible for handling sealed samples and disinfecting outer bags and containers for transport). Within a single test, sampling of the material sections was completed for all procedural blank coupons first before sampling of any test material or control sections. Sampling was done by collecting the coupons for extraction first, then sponge sampling the remaining coupons according to the protocols documented below. The surface area for sponge samples was approximately 103 cm2 (16 in.2) for carpet material and 79 cm2 (12.25 in.2) for the upholstery material. The extraction coupons were 18 mm (0.71 in.) in diameter. Once sampling was complete, material sections were returned to their original waste storage bags. Sponge-stick and stub sample integrity was maintained by storage of samples in triple containers (1 - sample collection container, 2 - sterile bag, 3 - disinfected container holding all samples from a test). All individual sample containers remained sealed while in the decontamination lab or in transport after the introduction of the sample. Since the current sampling techniques are intrusive, each coupon was sampled only once. However, each test was replicated 3-5 times, as described in Section 3.5. Test coupons and positive controls were sampled in parallel for each sampling time sequence. Temperature, pH, and active ingredient measurements of each decontaminant solution were performed prior to each decontamination procedure. The temperature and relative humidity (RH) of the waste staging area were recorded by three strategically placed, calibrated HOBO® data loggers (Onset Computer Corporation, Bourne, MA). Additional measurements included QC checks on the reagents and equipment used in the decontamination procedure. 3.1.1 Sponge-Stick™ Sampling 3M™ Sponge-Sticks with Neutralizing Buffer (part number SSL10 NB; 3M, St. Paul, MN) were used to aseptically sample the surface of carpet and upholstery materials. Sampling templates with 25 individual sampling areas were used as a guide. The sampling process was performed according to the standardized Centers for Disease Control and Prevention method "Surface sampling procedures for Bacillus anthracis spores from smooth, non-porous surfaces" [4]. 12 ------- 3.1.2 Extractive Sampling The 18-mm coupons for extractive sampling were removed from the sampling area and transferred to the into 50-mL sterile vials containing 10 mL phosphate-buffered saline with Tween®20 (PBST) (Sigma- Aldrich, St. Louis, MO) and the predetermined amount of neutralization liquid (STS). For PPE samples, excess pAB was captured in a separate vial for subsequent analysis. 3.2 Sample Type 3.2.1 Carpet and Upholstery Samples were collected from a 5 x 5 pre-drawn sampling grid consisting of 10.2-cm x 10.2-cm (4-in. x 4-in.) grid size sections (Figure 3-1). An 18-mm diameter coupon was excised from the center of each grid section for sample inoculation. For each sampling event, the 18-mm coupons were either removed for extraction or left in place to be part of the grid section that was sampled by the Sponge-Stick™ approach. When possible, Sponge-Stick™ and extraction samples were taken from areas representing different parts of the coupon (center, sides, and corners). Figure 3.1: Material section shown with template during sampling with Sponge-Stick™ and 18-mm coupon removal. 3.2.2 Paper Samples Paper samples, designated paper front (PF) and paper middle (PM), included the front cover and first and middle pages, as described previously. The additional pages adjacent to those inoculated were collected to account for any spores being relocated (via capillary action, direct transfer, etc.) to the adjacent pages during inoculation and/or decontamination. Sterile razor cutters were used to excise the paper samples after decontamination testing. Once excised, the paper samples were put inside a sterilized pre-labeled Stomacher® bag (Seward, Sussex, UK) along with 80 mL of PBST and a pre-determined volume of STS Sampling template 18 mm coupon (installed) Sponge Stick ~ 18 mm coupon (removed) Material section 13 ------- neutralizer arid mixed together. Eight books were used for each sampling sequence (three books for test samples: front and middle pages and inside cover page; two books for positive controls: front and middle pages; two books for field blank samples: front and middle pages; and one book for laboratory blank: front and middle pages. A total of 32 books were used for the four test sequences for each decontamination method. 3.2.3 PPE Samples Gloves were inoculated on the outside and then aseptically turned inside out to mimic removal and placement into a decontamination line waste stream. Three gloves were inoculated for each test (three samples from three inoculated fingers—thumb, middle, and pinky—for each glove, resulting in nine samples), one glove for positive controls (five samples from all five inoculated fingers), one glove for field blank sample (three non-inoculated fingers), and one glove for a laboratory blank sample (one sample from one non-inoculated finger) for a total of six gloves per test sampling sequence, or 24 gloves for the four test sequences for each decontamination method. Following decontamination, the terminal 2.5 cm (1 in.) of each finger was excised (Figure 3.2) and collected as an individual sample. Figure 3.2: PPE sample (finger) excision. 3.3 Sample Preservation After sample collection for a single test was complete, all biological samples were transported to the NHSRC RTF Microbiology Laboratory immediately, with appropriate chain of custody form(s), and stored at 4 ± 2 °C until processed. All samples were allowed to equilibrate at room temperature for 1 hour prior to extraction and plating. Liquid samples were stored no longer than 24 hours prior to analysis. Samples of other matrices were stored no longer than 5 days before the primary analysis. A typical holding time was 2 days prior to analyses for most biological samples. 3.4 Sampling Points All samples were collected from wet materials immediately after application of the decontamination procedure or after the required hold time (as bagged waste in the waste staging area) and neutralized 14 ------- immediately after sample collection. Table 3.1 lists the coupon types and the respective sampling procedures. Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures Material Porous or Non-porous Material Description Coupon/Sample Size Sampling Procedure(s) Carpet Porous Building material, high-traffic commercial carpet tile, 61 cm x 61 cm (24 in. x 24 in.) 18-mm punch /101 mm x 101-mm square Extraction / Sponge-Stick™ Upholstered furniture Porous Upholstered seat pan, 51 cm x 51 cm (20 in. x 20 in.) 18-mm punch / 89-mm x 89-mm square Extraction / Sponge-Stick™ Paper Porous Book pages Whole front and middle pages/22.8 cmx 16.5 cm (9 in. x6.5 in.) Extraction PPE materials Non-porous Nitrile, powder-free, disposable exam gloves 2.5 cm (1 in.) tip of finger Extraction PPE, personal protective equipment 3.5 Sampling Frequency and Sample Quantities After the waste decontamination procedure was executed, material sections were either sampled immediately or bagged and stored in the waste staging area for subsequent sampling after a drying period of 7 days. Figure 3.3 outlines the sampling timeline for both sampling approaches (extraction and Sponge-Stick™). The indicated number of test samples was collected from each of the three inoculated material sections as well as from procedural blanks (non-inoculated coupons that were exposed to test procedures) and positive control sections (inoculated coupons that were not exposed to test procedures). This timeline was developed to model the hold times decontaminated materials might be subject to on- site prior to being transported off-site for final disposal. Three positive control and three procedural blank samples were collected during each sampling event. Positive control coupons were inoculated concurrently with test coupons so they would have the same bacterial aging times as the samples. The synchronization of inoculation and sampling of positive control and test coupons was critical for accurate log reduction analysis. Bagged untreated positive control materials were sampled at the same time (within 8 hours) as their decontaminated counterparts. Immediately After Decon (T0) • 3 test coupons removed for extraction • 3 test coupons Sponge- Stick™ sampled • 3 positive controls, 3 procedural blanks 7 Days After Decon (T7 • 3 test coupons removed for extraction • 3 test coupons Sponge- Stick™ sampled • 3 positive controls, 3 procedural blanks Figure 3.3: Sampling timeline. 15 ------- The number of samples collected for both the neutralization and on-site decontamination tests are outlined in Table 3.2. This table includes not only the biological samples, but also samples collected to describe the decontamination process for each test in the test matrix. Some tests required 18-mm samples, sponge samples, or both. For tests that indicate both 18-mm and sponge samples were collected, the sample quantities for sterility blanks, positive control samples, procedural blanks, and test samples for both sample types were identical. Table 3.2: Number of Sample Types per Material Section per Sampling Sequence Test Type of Sample Material Laboratory Blank Positive Control Samples Field Blanks Test Samples Extractive Samples (Y/N) Sponge Samples (Y/N) Ea S» E S E S E S STSC 0 0 5 0 0 0 5 0 Y N On-Site Carpet coupons 1 1 3 3 3 3 3 3 Y Y Decontamination Upholstered coupons 1 1 3 3 3 3 3 3 Y Y Day (To Days, T7Days) PPEd 1 0 5 0 3 0 15 0 Y N Paper 2 0 6 0 2 0 6 0 Y N aNumber of extractive samples bNumber of Sponge-Stick™ samples cTests performed for the neutralization tests that span all four materials. dEach finger of a glove is considered one sample; note that, for the gloves, runoff samples were also collected. 3.6 Measurement Methods In addition to the collection of material samples, temperature, pH, and active ingredient measurements of each decontaminant solution were performed prior to each decontamination procedure. The temperature and RH of the waste staging area were recorded by three strategically placed, calibrated HOBO® data loggers. Additional measurements included quality control checks on the reagents and equipment used in the decontamination procedure. 3.6.1 Microbiological Samples General aseptic laboratory technique was followed to prevent cross contamination. Additionally, the order of analysis (consistent with the above) was as follows: (1) all blank coupons, (2) all decontaminated coupons, and then (3) all positive control coupons. Both coupon and Sponge-Stick™ extract samples were diluted, plated, and manually enumerated. Details of the extraction and analytical procedures are provided below. 3.6.1.1 Sample Extraction Extraction sample vials containing 18-mm coupons, PBST (Sigma-Aldrich, St. Louis, MO, P/N P3563- 10PAK), and neutralizer were vortexed for 2 minutes to dislodge viable spores from the coupon. Each vial was briefly re-vortexed immediately before any solution was withdrawn for dilution or filter plating. 3.6.1.2 Sample Analysis Experimental samples were subjected to up to five-stage serial dilutions (10-1 to 10-5), plated in triplicate onto Tryptic Soy Agar (TSA) and incubated overnight at 35 ± 2 °C. Following incubation, CFUs were manually enumerated. Samples that had fewer than the reportable limit of 30 CFU/plate of the undiluted sample underwent further analysis filter-plating, and subsequent attempted cultivation of surviving 16 ------- bacterial spores in liquid growth media. This method allowed a lower limit of detection for bacterial recovery/survivorship assays. 3.7 Data Analysis The total spore recovery for each method, material, and time point was calculated by multiplying the mean CFU 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 (X mL for Sponge-Stick™ samples and Y mL for extracted stubs). 3.7.1 Sampling Efficiency To determine which of the two sampling methods employed in the study was more efficient at recovering viable spores on the waste materials tested, the sampling efficiency (SE) for each method, all time points, and material types was calculated. SE is defined as the ratio of the measured mean sampled CFU (CFUm) to that of the inoculums (CFUo): CFU SE-as? <3-1) 3.7.2 Surface Decontamination Efficacy The efficacy of each decontaminant was assessed by determining the number of viable organisms remaining on each inoculated test coupon after decontamination and comparing this result to the number of viable organisms extracted from the positive control coupons, which were inoculated but not decontaminated. Excess decontamination solutions (in the form of rinsate) were also analyzed from PPE samples to determine if the representative decontamination application washed the spores from the surface of the PPE coupons or if the decontaminant inactivated the spores. The surface decontamination efficacy is defined as the extent (as logio reduction) by which viable spores extracted from test coupons after decontamination were less numerous than the viable spores extracted from positive control coupons. First, the logarithm of the CFU abundance from each coupon extract was determined, and then the mean of those logarithm values was determined for each set of control and associated test coupons, respectively. This value is reported as a log reduction on the specific material surface as defined in Equation 3-2. Łlog (CFUck) J>g (CFUsk) rj=——- ——- (3-2) >' Nc Ns where: Surface decontamination effectiveness; the average log T], = reduction of spores on a specific material surface (surface material designated by /). 17 ------- f>g(c™cl) k=1 The average of the logarithm (or geometric mean) of the number of viable spores (determined by CFU) recovered on the control coupons (C indicates control and Nc is the number of control coupons). f>g(c™„) k=1 N, The average of the logarithm (or geometric mean) of the number of viable spores (determined by CFU) remaining on the surface of a decontaminated coupon (S indicates a decontaminated coupon and Ns is the number of coupons tested). When no viable spores were detected, a value of 0.5 CFU was assigned for CFUs,k, and the efficacy was reported as greater than or equal to the value calculated by Equation 3-2. The cumulative standard deviation for the LR is calculated as follows: Let SDUn and SDTr denote the standard deviations of the log reduction values for the untreated carriers (positive controls) and the treated carriers (post-decontamination samples), respectively. Then, the cumulative standard deviation is calculated as follows: Where: nun and ntr designate the number of control and post-decontamination samples, respectively. 3.7.3 Statistical Analysis To determine if either the extraction or sponge sampling method was better for collecting spores, a paired independent Student's t-test using the two-tailed distribution was performed for each decontamination/ material combination. The null hypothesis of this test is that no statistical significance is observed between the paired populations (extraction versus sponge stick methods). In other words, if the p-value is less than 0.05 (95% confidence interval), the null hypothesis is rejected and there is evidence that the data from the sampling methods are different. On the other hand, if the p-value is greater than 0.05, then the two sampling methods are comparable. (3-3) 18 ------- 4 Results and Discussion This section presents the results of the overall effectiveness of the dunking or immersion of the waste to reduce and/or inactivate spores of B. atrophaeus from contaminated materials typical of indoor environments and of PPE waste items generated during sampling and decontamination operations. Effectiveness was determined by sampling the waste contents following decontamination and comparing to sampling controls that did not undergo the decontamination treatment. A 7 log spore challenge (inoculation of simulated waste items with ~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current study utilized the generally accepted criterion of 6 LR to consider an approach effective. Recovery of no viable spores following treatment was considered highly effective. Evaluation of the two sampling methods (extraction versus Sponge-Stick™) is discussed in Section 4.1. The results for the neutralization tests performed prior to each decontamination sequence are presented in Section 4.2. The results of the decontamination approach that utilized dunking or immersion of the waste are reported in Section 4.3. 4.1 Sampling Methods Evaluation The performance of the two sampling methods (extractive and Sponge-Stick™) was evaluated by comparing positive controls and post-decontamination recoveries at Day 0 and Day 7 for carpet, upholstery, paper materials, and PPE. 4.1.1 Carpet Material 4.1.1.1 Positive Controls Spore recoveries of positive control samples from carpet as a function of sampling method and over two simulated waste storage times (sampling time delay) are shown in Figure 4.1 and summarized in Table 4.1. The average recoveries, independent of storage time, were 1.21 x 107 CFU (SD = 5.69 x 106, n = 24 samples) using the extractive method (removal of a coupon from a larger sample) and 1.79 x 105 CFU (SD = 1.55 x 105, n = 24 samples) using the Sponge-Stick™ sampling approach. These results confirm the results from the previous study [2], which showed that the extractive sampling approach is more efficient than the Sponge-Stick™ sampling method. A two-sample independent t-test for these data show a p-value less than 0.001, confirming that the populations means between the two sampling methods are significantly different. No significant effects of storage time on recoveries were detected for either sampling method (p-value = 0.08 and 0.33 for the extractive and Sponge-Stick™ sampling techniques, respectively). 19 ------- I I Extractive Method Sponge-Stick™ Method Day 0 Day 7 Waste Storage (Days) Figure 4.1: Spore recoveries (± SD) from carpet using the extractive and Sponge-Stick™ methods. Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the Extractive and Sponge-Stick™ Sampling Methods Waste Storage Time Spore Recovery (CFU) Extraction Sponge-Stick™ Average SD Average SD Day 0 1.36E+07 6.11E+06 1.93E+05 1.61E+05 Day 7 1.05E+07 5.04E+06 1.85E+05 1.47E+05 4.1.1.2 Post-Decontamination Sample Recoveries The results presented in this section report the overall effectiveness of each sampling method/decontaminant procedure combination applied to carpet coupons. Sections of the test materials were sampled immediately after the decontamination treatment and were bagged and resampled and reanalyzed after a drying time of 7 days. The post-decontamination results are presented in Figure 4.2 and summarized in Table 4.2. 20 ------- 10- o 0 § 01 Ł o CL C/D c 8 0 Q 1)3 R. 10"; 1Q2 ¦¦ 101": 10° A ffl of® of® *2* ] Extractive Sampling (Day 0) ] Extractive Sampling (Day 7) ] Sponge-Stick™ (DayO) I Sponge-Stick™ (Day 7) r#' Decontamination Treatment 9?° Figure 4.2: Post-decontamination spore recoveries (± SD) for carpet as a function of decontaminant procedure and sampling method. Table 4-2: Post-Decontamination Spore Recoveries (CFU) for Carpet as a Function of Decontaminant Procedure and Sampling Method Decontamination Solution Extraction Sponge-Stick™ DayO Day 7 DayO Day 7 Average SD Average SD Average SD Average SD pAB with surfactant 5.65E+03 3.86E+03 5.07E+03 3.29E+03 7.24E-01 3.17E-02 1.54E+00 2.03E+00 pAB with su rfactant-agitati on 6.84E+04 4.42E+04 4.87E+04 1.99E+04 6.45E-01 1.47E-02 3.41 E+03 4.84E+03 pAB with agitation 2.12E+04 1.50E+04 2.75E+04 1.09E+04 7.17E-01 8.01 E-02 2.05E+00 2.83E+00 Spor-Klenz® 3.41 E+04 1.80E+04 S.7.35E-01* 2.72E-01 S.7.09E-01* 4.68E-02 S.8.52E-01* 8.52E-01 *Less than or equal to the detection limit. pAB, pH-adjusted bleach The post-decontamination results confirm that extractive sampling achieves higher recoveries than Sponge-Stick™ sampling for carpet. The wetness of the samples (Day 0) seems to reduce sampling efficiency of the Sponge-Stick™ when compared to higher recoveries achieved after the 7-day drying period. No significant effects of storage time on recoveries for the extraction method were observed for the decontamination solutions other than Spor-Klenz®. The Spor-Klenz® low post-decontamination recoveries after a 7-day period indicate a residual decontamination of the coupon during the drying period, suggesting that the residual decontamination was still ongoing during the 7-day waste storage. 21 ------- 4.1.2 Upholstery Material Spore recoveries from upholstery material as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.3 and summarized in Table 4.3. The average recoveries were 9.45 x 107 CFU (SD = 9.00 x 107, n = 6 samples) using the extractive method (removal of a coupon from a larger sample) and 9.24 x 106 CFU (SD = 1.93 x 106, n = 6 samples) using the Sponge-Stick™ sampling approach. A two-sample independent t-test showed that, at the 95% confidence interval, the difference of the population means is not significant (p = 0.07). Extractive Method Sponge-Stick™ Method Day 0 Day 7 Waste Storage (Days) Figure 4.3: Effect of waste storage time on positive control recoveries (± SD) from upholstered material for the extractive and Sponge-Stick™ methods. Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstery for the Extractive and Sponge-Stick™ Sampling Methods Waste Storage Time Spore Recovery (CFU) Extraction Sponge-Stick™ Average SD Average SD Day 0 1.75E+08 2.49E+07 1.01E+07 2.61 E+06 Day 7 1.44E+07 1.92E+07 8.42E+06 6.95E+05 22 ------- 4.1.3 Paper Material Spore recoveries from the front page paper (PF) and the middle page paper (PM) as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.4 and summarized in Table 4.4. Due to the highly porous and absorptive nature of paper, spores were expected to migrate from the original location to subsequent pages in the book. As such, extraction-based methods were used on the inoculated page and the adjacent pages to optimize spore recovery. The average CFU recoveries for the PF and PM samples are 9.45 x 107 (SD = 9.00 x 107, n = 6 samples) and 9.24 x 106 (SD = 1.93 x 106, n = 6 samples), respectively. A two-sample independent t-test showed, that at the 95% confidence interval, the difference of the spore recovery population means between the PF and PM samples is not significant (p = 0.07). PM Sample PF Sample O 10 Day 0 Day 7 Waste Storage (Days) Figure 4.4: Effect of waste storage time on positive control recoveries (± SD) from paper (PM, middle page; PF, front page) for the extractive method. Table 4.4: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the Extractive and Sponge-Stick™ Sampling Methods Waste Storage Time Spore Recovery (CFU) Front Page Middle Page Average SD Average SD Day 0 1.60E+07 4.65E+06 4.62E+06 2.33E+06 Day 7 5.46E+07 8.16E+06 2.08E+07 1.56E+07 CFU, colony-forming unit 23 ------- 4.1.4 PPE Material Spore recoveries from PPE material as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.5 and summarized in Table 4.5. Due to the irregular shape and small size of the PPE samples, only extractive sampling methods were used. The average CFU recoveries for two tests, which included four sampling events with five samples (each finger of nitrile glove is considered a single sample), were 1.27 x 107 (SD = 5.47 x 106, n = 40 samples). A paired independent Student's t-test using the two- tailed distribution for these data show no significant effects of storage time on recoveries (p-value = 0.59). & ^ 2 103 o O | 102 '(/) O o- 101 m Day 0 Day 7 Waste Storage (Days) Figure 4.5: Effect of waste storage time on positive control recoveries (± SD) from personal protective equipment for the extractive method. Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal Protective Equipment Waste Storage Time Spore Recovery (CFU) Extraction Average SD Day 0 1.35E+07 1.96E+07 Day 7 1.19E+07 1.27E+07 4.2 Neutralization Method Evaluation The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the sample) could negatively bias spore recoveries via residual decontamination. Prior to each decontamination testing sequence, neutralization tests were performed to determine the optimal neutralization concentration (neutralizerto decontaminant). 24 ------- To determine the optimal amount of neutralizer (STS) for each material/decontaminant, preliminary neutralization tests were conducted. The samples were neutralized at different stoichiometric ratios of STS to decontaminant (X); then the solutions (with the samples) were spiked with either approximately 2 x 102 spores (low concentration) or 5 x 107 spores (high concentration) before analysis. The results are presented in Table 4.6. The data collected showed that complete spore recovery can be obtained when the required amount of STS, based on stoichiometric ratio, is applied for each material/decontaminant combination. The neutralization tests determined that optimal recoveries of the spores were obtained with 2.5 X (stoichiometric ratio) for both Spor-Klenz® and pAB decontamination solutions. The time lag between the time the sample was neutralized and the time it was processed over a 24-hour period, the immersion time, and the spore inoculum concentration did not appear to bias the spore recoveries if adequately neutralized. Table 4.6: Preliminary Neutralization Optimization Decontaminant Stoichiometric Ratio Material Type Spore Recovery (CFU) Recovery (%) Positive Controls Neutralized Samples Mean SD Mean SD Mean SD pAB 1.3-1.5X Carpet 3.43E+07 1.12E+07 2.57 x 107 8.84 x10s 74.9 35.6 Upholstery 5.23E+07 6.75E+06 4.46 x 107 2.94 x10s 85.2 12.3 2.5X Carpet 3.76E+07 2.03E+06 3.89 x 107 7.79x10® 104 21.5 1.3-1.5X Carpet 2.74E+02 3.38E+01 2.78 x 102 4.91 x 101 101 21.8 Upholstery 4.00E+02 6.13E+01 3.24 x 102 4.42 x101 81.1 16.6 2.5X Carpet 2.74E+02 3.38E+01 2.40 x 102 1.75 x 101 87.5 12.5 Spor-Klenz® 1.5X Carpet 3.70E+07 4.71 E+05 2.83 x 107 1.50x10® 76.6 4.2 2.5 X Paper 6.43E+07 1.75E+06 4.04 x 107 4.34x10® 93.0 10.7 PPE 4.17E+07 2.13E+06 4.03 x 107 9.67x10® 96.7 23.7 2.5X Carpet 4.49E+01 9.22E+00 5.35 x 101 3.50x10° 119 25.7 Upholstery 9.04E+01 1.06E+01 5.34 x 101 8.68x10° 59.6 11.8 Paper 6.19E+03 3.78E+02 8.51 x 103 8.40 x102 138 16.0 PPE 4.23E+03 5.22E+01 4.14 x 103 2.42.x 102 97.8 5.8 pAB, pH-adjusted bleach, PPE, personal protective equipment 4.3 Dunking/Immersion Decontamination Test Results This section reports the results for overall effectiveness of the decontamination treatment for each material/decontaminant/procedure combination. Sections of the test materials were sampled immediately after the decontamination treatment and were bagged and resampled and reanalyzed after a drying time of 7 days. A subset of bagged waste samples was left untreated to serve as positive controls. 4.3.1 Carpet Decontamination Results Evaluation of the two sampling techniques (section 4.1) showed that the extraction method was much more effective than the Sponge-Stick™ method for carpet material, which can lead to an over/under estimate of decontamination efficacy for the tested decontaminant. In order to avoid any bias in the 25 ------- calculation of the decontamination efficacy of the decontaminant/procedure combination, only the results of the extraction method are presented in this section. The results of the carpet decontamination tests are presented in Table 4.7 and Figure 4.6. The decontamination effectiveness is presented as the mean Logio reduction in CFUs recovered from all samples as a function of the decontamination treatment. A paired independent Student's t-test using the two-tailed distribution for these data show no significant effects of storage time on the decontamination efficacy for all pAB-related decontamination treatments (p > 0.33). The efficacy of the Spor-Klenz® method was found to increase after the 7-day storage period to reach greater than 7 LR. The efficacy of the pAB-related decontamination treatments was in the range 2.0 to 3.4 LR. The addition of surfactant, and/or agitation seemed to have little or no effect, or even a negative effect, on the overall decontamination efficacy. No significant effects of storage time on recoveries were observed for pAB- based decontamination treatments for both carpet and upholstery materials. Table 4.7: Decontamination Efficacy versus Type of Decontamination Treatment for Carpet Material Decontamination Solution Decontamination Efficacy (LR)a Day 0 Day 7 Average SD Average SE pAB with surfactant 3.04 0.18 3.14 0.26 pAB with surfactant-agitation 2.24 0.26 2.37 0.16 pAB with agitation 3.05 0.31 2.82 0.13 Spor-Klenz® 2.74 0.17 7.03 0.11 a Based on extraction sampling pAB, pH-adjusted bleach; LR, log reduction; SE, sampling efficiency 26 ------- c o t5 "O CD a: Ł 2- 03 c "E 03 -t—< C 8 1- CD Q I Extraction_Day 0 I Extraction_Day 7 O 5- O d) o 03 O S c o •\a^ Decxintamination Treatments svdf Figure 4.6: Decontamination efficacy (± SE) versus decontamination treatment for carpet. 4.3.2 Upholstery Decontamination Results The results of the upholstery tests using Spor-Klenz® as the decontaminant are presented in Figure 4.7. A paired independent Student's t-test using the two-tailed distribution for this limited data show no significant effects of storage time on the decontamination efficacy (p > 0.5). The decontamination efficacy is presented as the mean Logio reduction in CFUs recovered from all samples for the two sampling periods (Day 0 and Day 7). This aggregate approach was utilized since the t-test indicated no significant interaction between sample storage time and recovery. The average combined log-io CFU decontamination efficacy for the 60-minute immersion time is 7.35 + 0.20 (n = 6 samples), nearly achieving complete kill. 27 ------- Day 0 Day 7 Waste Storage (Days) Figure 4.7: Decontamination Efficacy (± SE) for Upholstered Coupon Using Spor-Klenz®. 4.3.3 Paper Decontamination Results The results of the paper decontamination tests, using Spor-Klenz® as decontaminant, are presented in Table 4.8 and in Figure 4.8. The mean combined decontamination efficacies (Logio CFU reductions) for the front and middle pages after a 60-minute immersion time were greater than 5.3 + 0.3 (n = 6 samples) and 5.6 + 0.25 (n = 6), respectively. The seemingly low recoveries are due to the extraction method; the paper samples were put inside a sterilized pre-labeled Stomacher bag along with 80 mL of PBST and a pre-determined volume ofSTS neutralizer, and only a 1-mL aliquot was analyzed, resulting in a high detection limit of 40 CFU per sample. Only two of the 12 samples resulted in detection of viable spores following treatment. Table 4.8: Decontamination Efficacy (Log Reduction) versus Type of Decontamination Treatment for Paper Material Samples Decontamination Efficacy (LR)C Day 0 Day 7 Average SD Average SE Front page 5.6a 0.0 5.93a 0.0 Middle page 5.6a 0.0 5.72" 0.25 a All non-detects; b4 out of 6 non-detects;c Efficacy determined by extraction-based sampling. LR, log reduction; SE, sampling efficiency 28 ------- IPF Samples IPM Samples Day 0 Day 7 Waste Storage (Days) Figure 4.8: Decontamination efficacy (± SE) for Spor-Klenz®and paper coupons for front page (PF) and middle page (PM). 4.3.4 PPE Decontamination Results The PPE sampling technique consisted of collecting three inoculated fingers (1st, 3rd, and 5th fingers starting with the thumb) from each glove separately. Spores were recovered by extraction of individual glove fingers in 10 mL of PBST. Five gloves were used to produce 15 test samples (five samples from three inoculated fingers for each glove) for each decontamination treatment (pAB with surfactant, pAB with surfactant-agitation, pAB with agitation, and Spor-Klenz®). The results of the PPE decontamination tests are presented in Table 4.9 and Figure 4.9. The decontamination efficacy is presented as the mean logio reduction in recoveries (CFU) from all samples within a particular material and treatment. Only extraction-based sampling methods were utilized for PPE. These data suggest that achieving complete coverage of all PPE surfaces with the decontaminant is challenging; full decontamination (complete kill) of one finger of a glove sample might be obtained, while no decontamination is seen on the other fingers from the same glove. As stated in the previous report [2] , this is likely to occur as the inside of contaminated glove fingers may have no contact with the decontamination solution. All decontamination treatments achieved full kill in at least one finger, which presumably had contact with the decontamination solution. As a result, the high variability observed in the results did not allow a strong comparative evaluation across the decontamination treatments used in this study. 29 ------- Table 4.9: Decontamination Efficacy (CFU LR) versus Type of Decontamination Treatment for PPE Material Decontamination Solution Decontamination Efficacy (LR)a Day 0 Day 7 Average SE Average SE pAB with surfactant 6.41 0.38 2.30 3.23 pAB with surfactant-agitation 2.62 3.32 2.48 3.65 pAB with agitation 6.48 2.08 6.29 2.08 Spor-Klenz® 2.25 3.00 2.46 2.77 aBased on extraction sampling CFU LR, colony-forming unit log reduction; pAB, pH-adjusted bleach; PPE, personal protection equipment; SE, sampling efficiency Extraction_Day 0 Extraction_Day 7 ^Decontamination Treatments Figure 4.9: Decontamination efficacy (± SE) versus decontamination treatment for personal protective equipment (PPE) material. 30 ------- 5 Quality Assurance This project was performed according to an approved Category III quality assurance project plan (QAPP). Sufficient detail of the methods outlined in the QAPP are provided within this report. 5.1 Sampling, Monitoring, and Analysis Equipment Calibration Operating procedures for the maintenance and calibration of all laboratory and NHSRC RTP Microbiology Laboratory equipment were followed. All equipment was verified as being certified calibrated or having the calibration verified by EPA's Air Pollution Prevention and Control Division on-site (Research Triangle Park, NC) Metrology Laboratory at the time of use. Standard laboratory equipment such as balances, pH meters, biological safety cabinets, and incubators were routinely monitored for proper performance. Data gathered with the HOBO thermistors were processed using the factory calibration. Calibration of instruments was done at the frequency shown in Table 5.1. Any deficiencies were noted. If tolerances were not met after recalibration, additional corrective action was taken, possibly including, recalibration and/or replacement of the equipment. Table 5.1: Instrument Calibration Requirements Equipment Calibration/Certification Expected Tolerance Thermometer Compare to independent National Institute of Standards and Technology (NIST) thermometer (this is a thermometer that is recertified annually by either NIST or an International Organization for Standardization (ISO) 17025 facility) value once per quarter. ±1°C pH Meter Perform a two-point calibration with standard buffers that bracket the target pH before each use. ±0.1 pH units HOBO® RH sensor Compare to calibrated RH sensor prior to use. ± 5% Stopwatch Compare against NIST official U.S. time at httD://nist.time.aov/timezone.cai?Eastern/d/-5/iava once every 30 days. ±1 min/30 days Micropipettes All micropipettes certified as calibrated at least once per year. Rainin™ pipette liquid handling devices are recalibrated by gravimetric evaluation of pipette performance to manufacturer's specifications every 6 months by the supplier (Rainin Instruments, Mettler Toledo, Greifensee, Switzerland). ± 5% Scale Check calibration with Class 2 weights ±0.1% weight 5.2 Data Quality The primary objective of this project was to estimate the efficacy of liquid-based decontamination approaches for on-site treatment of bundled or bagged waste items typical of an indoor office setting that had been contaminated with B. anthracis spores. The QAPP in place for this project was followed with deviations that have been documented in the laboratory notebook. These deviations did not affect data quality. 31 ------- 5.3 Acceptance Criteria for Critical Measurements The data quality objectives define 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: • Chlorine concentration, determined by measuring FAC in decontaminant solutions • pH • Temperature • Relative Humidity (RH) • Decontamination time • Plated volume Data quality indicators for the critical measurements were used to determine if the collected data met the data quality objectives. The critical measurement acceptance criteria are shown in Table 5.2. The target values and actual test parameters for each run are shown in Table 5.3. The tests were conducted so that all the critical parameters were within the acceptance criteria described in the next section. When a test parameter did not meet the test target value, the test method was repeated or modified to reach the test target value and therefore achieve 100% completeness for the task. For example, if the target FAC concentration in the bleach (decontaminant) solution was not met, the solution was either re-prepared or adjusted. Test RH values were adjusted with data from calibrated RH sensors. Similarly, if the CFU count for bacterial growth did not fall within the target range, the sample was either filtered or replated. Table 5.2: Critical Measurement Acceptance Criteria Critical Measurement Measurement Device Accuracy Detection Limit Completeness Plated volume Pipette ±2% Not applicable 100% CFU/plate Hand counting ±10 % (between 2 counters) 1 CFU 100% FAC HACH® Method 10100 - digital titrator ± 1 % 1 digit (0.5 g/L chlorine) 100% Exposure time Timer ±1 second 1 second 100% pH Oakton® pH meter ± 0.01 pH Not applicable 90% RH/temp of chamber HOBO® U12 sensor ± 2.5 % from 10-90 % Not applicable 40% CFU, colony-forming unit; FAC, free available chlorine; RH, relative humidity 32 ------- Table 5.3: Data Quality Assessment Decontaminant Material Type Chlorine Concentration (FAC) - per 5 mL Bleach titrated pH Chamber Parameters (HOBO®) Target Value (PPm) Test Value (PPm) Frequency Target Value Test Value Frequency Avg. RH (%) Avg. Temp (°C) Frequency pAB + surfactant Carpet 6000-6700 6129 Once before testing 6.5-7.0 6.87 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test PPE 6000-6700 6129 Once before testing 6.5-7.0 6.70 Once before testing 26.1 19 Data recorded at 5 min intervals for the duration of the test pAB + agitation Carpet 6000 -6700 6209 Once before testing 6.5-7.0 6.89 Once before testing 19.5 17 Data recorded at 5 min intervals for the duration of the test PPE 6000-6700 6430 Once before testing 6.5-7.0 6.90 Once before testing 28.2 19 Data recorded at 5 min intervals for the duration of the test pAB + surfactant and agitation Carpet 6000-6700 6530 Once before testing 6.5-7.0 7.02 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test PPE 6000-6700 6089 Once before testing 6.5-7.0 6.67 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test Decontaminant Material Type PAA/H2O2 concentration pH Chamber Parameters (HOBO®) Test Value (%/%) Frequency Test Value Frequency RH (%) Temp °C Frequency Spor-Klenz® Carpet 0.20/0.44 Once before testing 2.99 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test PPE 0.26/0.10 Once before testing 3.95 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test Upholstery 0.26/0.09 Once before testing 2.75 Once before testing 42.6 23 Data recorded at 5 min intervals for the duration of the test Paper 0.30/0.19 Once before testing 2.70 Once before testing NA NA Data recorded at 5 min intervals for the duration of the test FAC, free available chlorine; NA = Not available; pAB, pH-adjusted bleach; PPE, personal protective equipment; RH, relative humidity 33 ------- 5.4 QA/QC Checks 5.4.1 Quality Control Management Several management controls were put in place in order to prevent cross contamination. This project was labor intensive and required that many activities be performed on material sections or coupons that were intentionally contaminated (test samples and positive controls) or intentionally not-contaminated (procedural blanks). The treatment of these three groups of test areas (positive control, test, and procedural blank) varied for each group. Hence, specific procedures were put in place to prevent cross- contamination among the groups. Adequate cleaning of all common materials and equipment was critical in preventing cross contamination; therefore, all common materials were fumigated using a VHP® or ethylene oxide sterilant and the swab sampled for sterility prior to each use. 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, CFUs were enumerated manually and recorded. QC checks for critical measurements/parameters are shown in Table 5.4. These checks also served as data quality indicator goals. The acceptance criteria were set at the most stringent level that can be routinely achieved. Tests with conditions falling outside of these criteria were rejected or repeated. 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. The confirmation procedure, controls, blanks, and method validation efforts were the basis of support for biological investigation results. Background contamination was controlled by sterilization of test 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 CFUs, 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). 34 ------- Table 5.4: Quality Control Checks QC Sample Information Provided Frequency Acceptance Criteria Corrective Action Procedural blank (coupon without biological agent) Controls for sterility of materials and methods used in the procedure. 1 per test No observed CFUs. Reject results of test coupons on the same order of magnitude. Identify and remove source of contamination. Positive control (sample from material coupon contaminated with biological agent but not subjected to the test conditions) Initial contamination level on the coupons; allows for determination of log reduction; controls for confounds arising from history impacting bioactivity; controls for special causes. Shows plate's ability to support growth. 3 or more replicates per test For high inoculation, target loading of 1 x 107 CFU per sample with a standard deviation of < 0.5 log. (5 x 106 - 5 x 107 CFU/sample). For low inoculation, target loading of 1 x 102 CFU per sample with a standard deviation of < 0.25 log (56 -177 CFU/sample); Grubbs' outlier test (or equivalent). Outside target range: correct loading procedure for next test and repeat depending on decided impact. Outlier: evaluate/exclude value. Blank plating of microbiological supplies Controls for sterility of supplies used in dilution plating. 3 of each supply per plating event No observed growth following incubation. Sterilize or dispose of source of contamination. Replate samples. Blank tryptic soy agar sterility control (plate incubated, but not inoculated) Controls for sterility of plates. Each plate No observed growth following incubation. All plates are incubated prior to use, so any contaminated ones will be discarded. Chlorine concentration Concentration of FAC in the fresh pAB or diluted bleach solution. 1 per use 6000-6700 ppm for fresh pH adjusted bleach or diluted bleach. Reject solution; replace reagents and prepare a new solution. pH Effective concentration of hydrogen ions in solution. 1 per use > 6.5 and < 7.0 for fresh pH adjusted bleach. Reject solution; replace reagents and prepare a new solution. Field blank samples The level of contamination present during sampling. 1 per sampling event Non-detect. Clean up environment. Sterilize sampling materials before use. 5.4.2 Quality Control Evaluation Table 5.5 illustrates how well QC objectives for maintaining sterility, minimizing cross contamination, and spore recovery were met. Only carpet and upholstery materials were sampled using the sponge stick method; therefore, sponge stick sample data are not available for other materials. Positive control data for each test show that the coupons were dosed with the targeted 1 x107 CFU. The extractive sampling method proved effective with a median recovery of 8.2 x 106 CFU from all material surfaces. The sponge stick sampling method performed poorly on carpet surfaces, with a median recovery of only 2.2 x 105 CFU; however, recovery improved when used on the relatively smoother surface of the upholstery material. The available negative control data suggest the sterilization procedures used for each material were effective. Sampling data collected from the procedural blanks prove cross contamination prevention efforts were successful. Temperature and RH data for the decontaminants was not collected on several occasions (Table 5.3), however these were not critical measures and quality of the efficacy or recovery data were not affected. 35 ------- Table 5.5: Quality Control Evaluation Decontaminant Material Type Negative Controls (Avg CFU) Procedural Blanks (Avg CFU) Positive Controls (Avg CFU) Extractive Samples Sponge Stick Samples Extractive Samples SD Sponge Stick Samples SD Extractive Samples SD Sponge Stick Samples SD pAB + surfactant Carpet 6.67E-01 7.38E-01 6.08E-01 1.46E-02 7.20E-01 2.39E-02 5.82E+06 1.92E+06 2.20E+05 8.54E+04 PPE 7.04E-01 NA 6.75E-01 4.55E-03 NA NA 3.18E+06 1.39E+06 NA NA pAB + agitation Carpet 6.41 E-01 6.63E-01 6.00E-01 8.38E-03 5.98E-01 1.75E-01 2.01 E+07 1.13E+06 3.05E+05 1.34E+05 PPE 6.25E-01 NA 3.70E+00 5.36E+00 NA NA 4.29E+07 1.93E+07 NA NA pAB + surfactant and agitation Carpet 8.62E-01 6.70E-01 6.30E-01 0.00E+00 6.57E-01 1.53E-02 1.06E+07 7.85E+05 2.24E+05 2.31 E+05 PPE NA NA 6.03E-01 0.00E+00 NA NA 3.41 E+06 1.88E+06 NA NA Spor-Klenz® Carpet 7.72E-01 1.30E+00 5.83E-01 1.71E-02 6.54E-01 5.15E-02 1.77E+07 2.64E+06 2.48E+04 2.15E+04 PPE NA NA 2.18E+00 2.55E+00 NA NA 4.60E+06 3.28E+06 NA NA Upholstery 6.67E+00 6.67E-01 6.67E+00 1.09E-15 1.84E+01 3.07E+01 1.75E+08 2.49E+07 1.01 E+07 2.61 E+06 Paper NA NA 4.00E+01 NA NA NA 4.62E+06 2.33E+06 NA NA CFU, colony-forming unit; NA = Not available; pAB, pH-adjusted bleach; PPE, personal protective equipment 36 ------- 6 Summary The evaluation of the two sampling techniques (Section 4.1) showed that the extraction method was more effective than the Sponge-Stick™ method for carpet material. Use of Sponge-Sticks™ to sample carpet could result in an over estimation of efficacy. In order to avoid any bias in the estimation of the decontamination efficacy of the decontaminant/procedure combination, only the results of the extraction method are presented in this section. The following summarizes these results: • The efficacy of the pAB-related decontamination treatments on carpet materials was in the range 2.0 to 3.4 LR. The addition of surfactant, and/or agitation or increasing the storage time from Day 0 (within 1 hour from the immersion) to seven days (Day 7) showed no significant effect on the overall decontamination efficacy of pAB-related treatment. The efficacy of Spor-Klenz® was found to increase over the 7-day period to reach greater than 7 LR. These results show that the use of Spor-Klenz® as a decontamination treatment over time might result in an effective approach for decontaminating carpet materials (see Table 6.1). However, even with Spor-Klenz®, four of the six carpet samples yielded viable spores following the decontamination treatment. • The results of the upholstery and paper tests using Spor-Klenz® as the decontaminant showed high efficacy, nearly achieving complete kill of the spores (only 1 out of 6 samples and 2 out of 12 samples showed any spore detection for upholstery and paper, respectively). • PPE decontamination, independent of the material/decontaminant/ procedure combination, was found to be challenging. All the decontamination treatments achieved full kill in at least one glove finger, while poor decontamination efficacy was observed on the others of the same glove. The high variability observed in the results hindered our ability to compare across treatments used in this study. • The two sampling methods (extractive and Sponge-Stick™) were evaluated for both carpet and upholstered materials. For these materials, the extraction-based method consistently achieved higher recoveries. As a consequence of its lower recoveries, the Sponge-Stick™ method resulted in overestimation of the decontamination efficacies. If possible, utilization of extraction-based methods for waste sampling provides improved sensitivity of detection. However, these methods might not be easily deployed in the field, and could generate samples that are not as easily analyzed in the laboratory. 37 ------- Table 6.1: Summary of Waste Decontamination Results Material Type Decontamination Treatment Post-Decon Samples with >6 LR / Total Number of Samples Collected Post-Decon Samples with No Viable Spores Detected / Total Number of Samples Collected Carpet pAB + surfactant 0/6 0/6 pAB + surfactant + agitation 0/6 0/6 pAB + agitation 0/6 0/6 Spor-Klenz® 3/6 1/6 Upholstery Spor-Klenz® 6/6 5/6 Paper Spor-Klenz® 0/12a 10/12 PPEb pAB + surfactant 24/30 23/30 pAB + surfactant + agitation 10/30 10/30 pAB + agitation 10/30 25/30 Spor-Klenz® 11/30 14/30 alow postitive control recoveries resulted in low LR values, >5 LR achieved for all 12 test samples beach glove finger considered a separate replicate for this table pAB, pH-adjusted bleach; PPE, personal protective equipment 38 ------- References US Environmental Protection Agency. Technical Brief - Bio-response Operational Testing and Evaluation (BOTE) Project Washington, DC. EPA/600/S-12/001 US Environmental Protection Agency. Expedient Approaches for the Management of Wastes Generated from Biological Decontamination Operations in an Indoor Environment- Evaluation of Waste Sampling and Decontamination Procedures. Washington, DC. EPA 600/R-14/262 D2022-89, A., Standard Test Methods of Sampling and Chemical Analysis of Chlorine-Containing Bleaches. Book of Standards 2008.15.04. US Centers for Disease Control and Prevenetion. Surface sampling procedures for Bacillus anthracis spores from smooth, non-porous surfaces. NIOSH - Workplace Safety & Health Topics 2012; Available from: http://www.cdc.aov/niosh/topics/emres/surface-samplina-bacillus- anthracis.html. 39 ------- vvEPA 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 ------- |