EPA/600/R-20/439 | May 2021 www.epa.gov/emergency-response-research United States Environmental Protection Agency oEPA Collection of Microbiological Agent Samples from Potentially Contaminated Porous Surfaces Using Microvacuum Techniques Office of Research and Development Homeland Security Research Program ------- EPA/600/R-20/439 May 2021 * \ Collection of Microbiological Agent Samples from Potentially Contaminated Porous Surfaces Using Micro vacuum Techniques U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- Disclaimer The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development, funded and managed the information described here, supported by CSRA under Contract No. EP-C-15- 012. This document is underwent review prior to approval for publication. Note that approval does not necessarily signify that the contents reflect the views of the Agency. Mention of trade names, products or services does not convey official EPA approval, endorsement or recommendation. The method described for collection of microbiological agents in this document have not been validated or verified at the time of publication. The document will be updated or replaced with validated steps for collection upon availability. Questions concerning this document or its application should be addressed to: Erin Silvestri U.S. Environmental Protection Agency Center for Environmental Solutions and Emergency Response 26 W. Martin Luther King Drive, MS NG16 Cincinnati, OH 45268 513-569-7619 Silvestri.Erin@,EPA.gov ------- Foreword The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the nation's land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to formulate and implement actions leading to a compatible balance between human activities and the ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is providing data and technical support for solving environmental problems today and building a science knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect our health, and prevent or reduce environmental risks in the future. The EPA's Center for Environmental Solutions and Emergency Response (CESER) within the Office of Research and Development (ORD) conducts applied stakeholder-driven research and provides responsive technical support to help solve the nation's environmental challenges. The Center's research focuses on innovative approaches to address environmental challenges associated with the built environment. We develop technologies and decision-support tools to help safeguard public water systems and groundwater, guide sustainable materials management, remediate sites from traditional contamination sources and emerging environmental stressors, and address potential threats from terrorism and natural disasters. CESER collaborates with both public and private sector partners to foster technologies that improve the effectiveness and reduce the cost of compliance, while anticipating emerging problems. We provide technical support to EPA regions and programs, states, tribal nations, and federal partners, and serve as the interagency liaison for EPA in homeland security research and technology. The Center is a leader in providing scientific solutions to protect human health and the environment. When an environmental contamination involving a microbiological agent occurs, whether resulting from intentional or an unintentional incident, collection and analysis of numerous numbers of environmental samples will be needed to determine the extent of contamination and to make informed decisions regarding remediation. Sample collection procedures can be used during site characterization and remediation activities in support of EPA's post-incident responsibilities in order to provide instructions regarding the collection of samples from indoor/outdoor environmental, building, and infrastructure materials that will be analyzed for contaminants. This document provides step-by-step instructions for the use of 37-mm vacuum filter cassettes, to be used in conjunction with a microvacuum, to collect samples from surfaces potentially contaminated with select microbiological agents. This document provides information on the materials and equipment needed for sample collection; the assembly of sampling kits; step-by-step instructions for taking field and quality control (QC) samples; and information on sample packaging, storage, and transport. Use of the procedure by EPA, or EPA contracted sample collectors, will help ensure that samples are collected in a consistent manner prior to laboratory analysis. Gregory Sayles, Director Center for Environmental Solutions and Emergency Response iii ------- Acknowledgments This document was developed by the U.S. Environmental Protection Agency's (EPA) Homeland Security Research Program within EPA's Office of Research and Development under a task order with General Dynamics Information Technology. Contributions of the following individuals and organizations to this report are gratefully acknowledged: US Environmental Protection Agency (EPA) Project Team Erin Silvestri (Project Lead) John Archer Worth Calfee General Dynamics Information Technology (GDIT) Project Team John Chandler Yildiz Chambers-Velarde Joan Cuddeback Emily King US EPA Technical Reviewers of Report Shannon Serre Helen Buse US EPA Quality Assurance Ramona Sherman External Peer Reviewers Lisa Delaney, Centers for Disease Control and Prevention (CDC) Chad Dowell, CDC Laura Rose, CDC ------- Executive Summary This document provides step-by-step instructions for the use using a 37-mm filter cassette, air sampling pump, and microvacuuming techniques to collect samples from porous surfaces potentially contaminated with Bacillus anthracis spores. Although recovery efficiency and performance are not known for its use with other microbiological agents, this document could potentially be applied for use with those agents. It is intended to be used in conjunction with the analytical methods listed in U.S. Environmental Protection Agency's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (EPA/600/R-17/356) document and in the Environmental Sampling and Analysis Method Program online query tools for SAM, following contamination incidents. The document includes instructions for use of either a mixed cellulose ester (MCE) filter or a polytetrafluoroethylene (PTFE) filter. Although testing has not been completed and collection efficiencies are unknown, these instructions might also be applicable to other pathogens. Product Development Quality Assurance Literature used for this procedure came from recognized, reputable and credible secondary sources including: peer-reviewed journals, scientific manuals and other scientific publications; federal agency websites, publications and regulations; industry providers of equipment and materials (i.e., vendors); and nationally recognized scientific, technical or response organizations. Citations are provided throughout the document. Full citations and/or access to each source used are provided in the references section. No deficiencies were noted with this review. The document completed several review cycles prior to publication including EPA project lead review, internal EPA technical review, Homeland Security and Materials Management Division (HSMMD) quality assurance and technical edit reviews, external technical review, and HSMMD management reviews. All comments from reviewers have been tracked and are maintained by EPA and General Dynamics Information Technology (GDIT), along with the revisions and adjustments made to address the comments. vi ------- Acronyms (.un micrometer CDC U.S. Centers for Disease Control and Prevention CESER Center for Environmental Solutions and Emergency Response CFR Code of Federal Regulations COC chain of custody cm centimeter DGR Dangerous Goods Regulation DOL U.S. Department of Labor DOT U.S. Department of Transportation EPA U.S. Environmental Protection Agency ESAM Environmental Sampling and Analytical Methods (Program) GPS Global Positioning System HASP health and safety plan HAZMAT hazardous material HAZWOPER Hazardous Waste Operations and Emergency Response IATA International Air Transport Association I-WASTE DST Incident Waste Decision Support Tool ID identification L liter MCE mixed cellulose ester mL milliliter mm millimeter NIOSH National Institute for Occupational Safety and Health ORD Office of Research and Development OSHA Occupational Safety and Health Administration PPE personal protection equipment PPM Policy and Procedure Manual psi pounds per square inch PTFE polytetrafluoroethylene PVC polyvinylchloride QAPP quality assurance project plan QC quality control QR quick response SAM Selected Analytical Methods (for Environmental Remediation and Recovery) SAP sampling and analysis plan SCID Sample Collection Information Document WMP waste management plan ------- Table of Contents Disclaimer ii Foreword iii Acknowledgments v Executive Summary vi Acronyms vii I.0 Introduction 1 2.0 Limitations and Interferences 3 3.0 Health and Safety Considerations 4 4.0 General Considerations for Porous Surface Sampling 6 5.0 Waste Management 7 6.0 Sample Documentation 8 6.1 Sample Identification 8 6.2 Sample Labels 8 6.3 Sample Documentation Information 9 6.4 Sample Control and Chain of Custody 9 6.5 Custody Seals 10 7.0 Sampling Equipment and Supplies 11 7.1 Sampling Equipment 11 7.2 Sampling Kit Supplies 11 7.3 General Supplies 12 7.4 Sample Transport Containers and Packing Materials 12 8.0 Preparation for Sample Collection 13 8.1 Sampling Teams 13 8.2 Techniques to Minimize Potential Cross Contamination 14 8.3 Sampling Kits 14 8.4 Decontamination 16 9.0 Quality Control Samples 18 9.1 Field Blanks 18 9.2 Trip Blanks 18 9.3 Field Replicates 18 10.0 Sample Collection 19 10.1 Materials, Supplies and Equipment 19 10.2 Collection 20 10.3 QC Sampling 22 II.0 Sample Packaging and Transport 23 11.1 Sample Holding Time and Temperature 23 11.2 Sample Container Transport and Labeling 24 11.3 Sample Packaging 24 11.4 Transfer of Custody 26 12.0 References 27 Appendix A: Applicable Microbiological Agents for this Document 30 Appendix B: Supplemental Plans 32 Appendix C: Example Chain of Custody Form 36 viii ------- List of Figures Figure 1. 37-milliliter filter cassette (left), sample pump (center) for microvacuuming, and performing sampling (right) 2 List of Tables Table 2-1. Available Recovery Efficiency Data for Mixed Cellulose Ester and Polytetrafluoroethylene Filters 3 Table 4-1. Microvacuum Sampling Uses and Considerations 6 Table 7-1. Filter Cassette Options 11 Table 8-1. Sampling Kit Check List 16 Table 11-1. Transport Conditions and Holding Times 23 ix ------- 1.0 Introduction When an environmental catastrophe resulting in contamination occurs, emergency responders and decision-makers need timely and accurate data as well as robust and well-defined methods for data collection. Catastrophic contamination can occur from an intentional incident such as a terrorist attack or an unintentional incident such as an industrial spill. Incidents can require collection and analysis of numerous amounts of samples, which will provide scientific data needed to make evidence-based decisions on the extent of contamination and subsequent remediation. The U.S. Environmental Protection Agency (EPA)'s Environmental Sampling & Analytical Methods (ESAM) Program (U.S. EPA 2017b) is intended to facilitate a coordinated response to a chemical, radiochemical, biotoxin, or microbiological (including pathogenic organisms) contamination incident by providing a comprehensive resource for sampling and analysis needs before, during, and after a contamination incident. The ESAM Program provides field- and laboratory-ready documents and web-based tools that focus on sample collection, processing, and analysis to facilitate site characterization, as well as remediation, waste disposal, and clearance decisions. As part of the ESAM Program, Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (SAM) (U.S. EPA 2017a) provides a compendium of analytical methods that have been selected specifically for use during environmental response activities, by work groups consisting of methods experts from within EPA, as well as other federal, state and local agencies, and public utilities.1 SAM identifies a single selected method or suite of methods for each analyte/sample type. A SAM companion document (Sample Collection Information Document [SCID1 for Pathogens: Companion to Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (SCID) (Chattopadhyay 2017) provides complementary information on sample containers, preservation, size, and packaging, as well as additional resources that support collection of samples to be analyzed specifically for the selected pathogens, using the methods listed in SAM. This document provides step-by-step instructions for the use of 37-mm filter cassettes (hereafter called filter cassettes), to be used in conjunction with an air sampling pump (Figure 1) and microvacuuming techniques, to collect samples from porous surfaces (e.g., wood, carpet, cloth) potentially contaminated with select pathogens. It is intended to be used in conjunction with the analytical methods listed in SAM, as well as the corresponding SCID for pathogens. This document provides information on the materials and equipment needed for sample collection and the assembly of sampling kits. The information includes step-by-step instructions for taking field and quality control (QC) samples and information on sample packaging, storage, and transport. This document is applicable to collection of particulate samples from porous surfaces for detection of Bacillus anthracis spores using a 37-millimeter (mm) filter cassette, an air sampling pump, and microvacuuming techniques, with either a mixed cellulose ester (MCE) filter or a polytetrafluoroethylene (PTFE) filter. Although testing has not been completed and efficiencies are unknown, these procedures may also be applicable to other microbiological agents (See Appendix A for a list of SAM microbiological agents for which this document is applicable). The filter medium is sealed within a plastic case, thus reducing the potential for exposure of sampling personnel and laboratory personnel to the sampling media. Sterilized 37-mm filter cassettes are commercially available from multiple sources. 1 For more information on SAM workgroups, see: for more information on the workgroups: https://www.epa.gov/esam/basic-information-epas-selected-analvtical-methods-environmental-remediation-and- recoverv-sam (last accessed 9/29/20201 1 ------- Figure 1. 37-milliliter filter cassette (left), sample pump (center) for micro vacuuming, and performing sampling (right). In summary, this document: Is applicable for collection of samples potentially contaminated with the microbiological agents listed in EPAs SAM (see Appendix A). Addresses sample collection only and is intended for use by sampling personnel who have been sufficiently trained in microbiological sampling techniques and corresponding safety protocols. Is intended to support collection of environmental samples at the point where remediation activities are turned over to the U .S. EPA. It is applicable to the following sampling phases of a remediation event: site characterization and post-decontamination sampling. While this document was not specifically developed to support sample collection during the public health response phase, it could be used for that purpose. Assumes that collected samples will be analyzed using analytical methods and protocols consistent with those listed in EPA"s SAM. The laboratory(ies) analyzing samples should be consulted prior to preparation of the sampling and analysis plan (SAP) to ensure they will accept samples, the number of samples they will accept, and whether they can process and analyze samples in accordance with SAM. SAPs (and other site- and incident-specific plans and procedures) should be consulted to determine additional procedures and data needs beyond what is discussed in this document, including whether additional sampling is needed for QC should low concentrations of microbiological agents be suspected. In addition, the SAP and analyzing laboratory should be consulted prior to sample collection to determine if additional modifications to the steps described in this document will be needed to accommodate laboratory capacity, analysis of the target microbiological agent, the data quality objectives, and the number of samples needed. This document does not provide information that is typically included in the following documents, which are described briefly in Appendix B: Sampling and analysis plan (SAP) Quality assurance project plan (QAPP) Health and safety plan (HASP) Analytical methods Waste management plan (WMP) 2 ------- 2.0 Limitations and Interferences This document includes information based on sampling techniques that were available at the time of publication; not all procedures or protocols have been fully evaluated or validated. In addition, more research is needed to determine appropriate preservation and holding times for many of the microbiological agents, as well as collection efficiencies for agents other than Bacillus anthracis (the causative agent for the disease anthrax). Information on recovery efficiency of this method that was available at the time of this publication are included in Table 2-1. The document is expected to be updated on a periodic basis to include advances in technologies and results of validation studies. Table 2-1. Available Recovery Efficiency Data for Mixed Cellulose Ester and Polytetrafluoroethylene Filters Reference" Organism 37 mm Filter Type Pump Used (flow rate) Inoculation Concentration (CFU/sample) Matrix size (cm2) Type of Matrix Analysis Method Extraction Liquid Extraction Method Recovery Efficiency % Calfee et al. 2013 Bacillus atrophaeus (Bg) Mixed cellulose ester (MCE) Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Carpet Culturable Plate Count Phosphate buffered saline with 0.05% Tween 20 (PBST) Sonication/ Vortex/ Rinse/ Sonication 47.4 Calfee et al. 2013 Bg MCE Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Concrete Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 124.2 Calfee et al. 2013 Bg MCE Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Upholstery Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 35 Calfee et al. 2014 Bg MCE Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') ~1E8 929 HVAC Filter (Electrostatic and Mechanical) Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 2-19 Calfee et al. 2013 Bg Polytetraflu- oroethylene (PTFE) Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Carpet Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 20.2 Calfee et al. 2013 Bg PTFE Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Concrete Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 23.8 Calfee et al. 2013 Bg PTFE Filter Cassette Vac-U-Go pump SKC, Int.; part# 228-9605, (20 L mill ') 9.29E+07 929 Upholstery Culturable Plate Count PBST Sonication/ Vortex/ Rinse/ Sonication 12.9 *Full references are found at the end of report. Various substances, such as non-target background microorganisms, organic matter (e.g., dust, humic acid), decontamination agents, and certain trace elements, can interfere with sample analysis. In addition, particulates can adhere to the inner walls of the nozzle or clog the filter, decreasing the flowrate. Any observed potential interferences should be noted in the sample documentation at the time of collection. 3 ------- 3.0 Health and Safety This document does not address all health and safety issues associated with sample collection. The importance of training, medical monitoring, required vaccinations (if applicable) and information included in the site- or incident- specific HASP should be emphasized. Sampling personnel must refer to their site-specific HASP for health, safety, and personal protective equipment (PPE) considerations specific to the sample collection event. A job hazard analysis should be conducted for the site-specific sampling procedures. In addition to potential harm to staff, unsafe conditions in the field can indirectly impact the ability to collect representative samples, which may affect the resulting analytical data. A summary of health and safety considerations are included below: Training - The importance of training cannot be overstated; training is critical, and in some cases mandatory, in order to ensure appropriate safety and health conditions for sampling personnel. Training requirements for Hazardous Waste Operations and Emergency Response (HAZWOPER) are outlined in Occupational Safety and Health Administration's (OSHA's) HAZWOPER standard (29 Code of Federal Regulations [CFR] 1910.120). Training elements are specified in section 1910.120(e)(2) and should include specific training on biohazards and microbiological agents. The safety officer and/or sampling lead should be consulted for required training that will be needed to operate equipment and perform techniques prior to their use in the field, to use methods to minimize cross-contamination, and to appropriately don and doff PPE. Training requirements for respiratory protection can be found in OSHA's Respiratory Protection standard (1910.134; OSHA 2011). Training requirements for packaging, documenting, and shipping infections substances can be found in the U.S. Department of Transportations (DOT's) Transporting Infectious Substances Safely (U.S. DOT 2020). Safety Officer - The safety officer should be appropriately trained and is responsible for the development and implementation of safety requirements and the HASP; assessing all site activities for potential safety concerns; ensuring that personnel are informed as to the potential hazards in a sampling area and dictating the requirements for safely working in the area; and stopping any sampling activity if necessary to protect personnel from a dangerous situation. Medical Examination - Medical examinations to assess fitness to conduct sampling should include clearance for wearing respiratory protection (see OSHA's Respiratory Protection standard (1910.134; OSHA 2011). In addition, the examination should assess fitness for working with specific microbiological agents, vaccinations (if applicable), and prophylactics, if available, for the microbiological agent. Sampling personnel should be monitored for fatigue, stress, behavior changes, and general health during sampling events. First Aid - First aid kits must be available at all times during a sampling event. At least one kit should be available to the sampling team at the sampling site. The HASP(s) must state the requirements for reporting all injuries, and for examination by medical personnel when injuries occur. PPE - PPE should be used during all sample collection and equipment decontamination activities, as required in the HASP. The type and level of PPE should be selected based on the potential hazard to provide the optimal personal protection and mobility for the task being performed. The HASP should also be consulted to determine if personal monitoring is required for the sampling event. Sampling personnel should familiarize themselves with the HASP and SAP for required PPE. Sampling personnel can also review specific guidance for levels of protection and protective gear developed by OSHA, provided in Appendix B of 29 CFR 1910.120 (U.S. Department of Labor (DOL) and OSHA, 2013). The National Institute for Occupational Safety and Health (NIOSH) has also developed recommendations (Interim Recommendations for the Selection and Use of Respirators and Protective Clothing for Protection Against Biological Agents: NIOSH 4 ------- 2009) for the selection and use of respirators and PPE for protection against microbiological agents (NIOSH 2009). Incident-specific PPE requirements must be included in the HASP and SAP. General overarching considerations include the following: o In all cases, new, unused powder-free disposable nitrile gloves must be worn to protect hands and to protect samples from contact with potentially contaminated surfaces and samples. Wearing two pairs of gloves is recommended but should not compromise dexterity. Gloves should be changed between samples, when they become contaminated, and when the integrity of the gloves is compromised (e.g., torn). o Care should be taken to ensure that PPE is not compromised. If PPE is suspect or compromised, sample collection must be stopped. Compromised PPE can result in contamination of personnel or contamination of collected samples. o After use, PPE (e.g., gloves, protective clothing) should be appropriately disposed of or decontaminated. 5 ------- 4.0 General Considerations for Porous Surface Sampling Selection of the technique required for collection of particulates is based on site-specific sampling objectives and strategies and on the physical nature of a sampling site (such as the size, complexity, and texture of a potentially contaminated porous surface). For example, the primary method for collecting samples of B. anthracis spores from porous surfaces such as carpet, ceiling tiles, ventilation system filters, keyboards, and upholstered furniture is microvacuum sampling using 37-mm filter cassettes and an air sampling pump. Microvacuum sampling is also recommended for sampling sensitive items, such as electronics or personal items, since the technique is less likely to cause damage compared to pre- moistened swabs and wipes. The standard sampling area for 37-mm filter cassettes is typically 1 ft2, (~ 0.0929 square meters) and a template can be used, if applicable. In addition to the information included in this document, sampling personnel should consult the site- and incident-specific SAP for sampling and laboratory requirements, which might include the: Number, type, location, and area of samples that will be needed to support QC requirements Appropriate sample containers, preservation, and holding times Sample packaging requirements (e.g., primary and secondary containment) Sample receipt requirements During sampling, particulate material is trapped on a filter using an air sampling pump to draw air through a filter cassette. This type of sampling also works well for capturing powder or dust in hard-to- reach places. Some general considerations regarding microvacuum sample collection are included below and in Table 4-1. Table 4-1. Microvacuum Sampling Uses and Considerations loch it i(|ii e Description Polcnlhil Problems Considci'iilions Microvacuum with MCE or PTFE filter 37-mm filter in 3-stage plastic cassette and air sampling pump for sampling Used to collect microbiological material and dust deposited onto surfaces such as carpet, fabrics, draperies, and other porous surfaces Ambient particulates can interfere with sample analysis Cross-contamination can occur from mishandling while packaging for shipment Particulates on the surface, other than the microbiological agent of interest or high concentrations of the microbiological agent of interest, can block filters and interfere with airflow and reduce sampling volume Foot traffic should be controlled and minimized as much as possible prior to sample collection in order to minimize re-aerosolization of spores and to avoid stepping in areas that have not yet been sampled Unknown recovery efficiencies for microbiological agents other than Bacillus spores (See Table 2-1 fox Bacillus spore recovery efficiency information) MCE, mixed cellulose ester; PTFE, polytetrafluoroethylene 6 ------- 5.0 Waste Management Waste generation and management begin as soon as the response to a contamination incident is initiated. Used PPE, materials from sampling activities, and liquids from decontamination associated with sample collection activities can be generated by sampling personnel, and generation of these waste streams will continue throughout the response and recovery phases. Planning for waste management is critical. It is the responsibility of all sampling personnel to comply with the site- or incident-specific WMP (see Appendix B) and with federal, state, and local regulations governing waste management, including biohazard and hazardous waste identification, tracking and reporting, accumulation documentation, and land disposal restrictions. It is also the responsibility of sampling personnel to minimize and control all releases. Sampling personnel should refer to the site- or incident-specific WMP for instructions on anticipated waste generation due to sampling, as well as waste management requirements and procedures. Sampling personnel and planners also can refer to EPA's Waste Management Options for Homeland Security Incidents website and EPA's Incident Waste Decision Support Tool (I-WASTE DST), which provides information regarding regulations and guidance to support decision-making regarding waste treatment and disposal (websites last accessed 9/29/2020).2 In general: Excess sample materials, reference materials, accumulated waste, and equipment that will not be reused must be placed in appropriate waste containers separating solid waste from liquid waste, and stored separately from collected samples and sampling equipment prior to removal from a contaminated site. The site-specific WMP should be consulted regarding whether decontamination of these materials will be conducted prior to removal from a contaminated site or at a facility designated for decontamination or disposal. Unused and uncontaminated sample collection materials such as cassettes/filter assemblies should be shipped to the laboratory with each batch of samples. (These filter materials serve as field blanks (Section 9.0) providing information to determine if analytical results might be impacted by interferences contained in the sampling media used [Section 9.0].) Media blanks are used to assess the sampler for contamination. 2 I-WASTE is a decision support tool that organizes information related to waste management. The tool also provides access to technical information, regulations, and guidance to work through waste management issues to facilitate safe and efficient removal, transport, and management of waste materials. Pre-registration is needed to access EPA's I-WASTE Tool and Disposal Decision Tool at http://www2.ergweb.com/bdrtool/login.asp (last accessed 09/29/2020) 7 ------- 6.0 Sample Documentation Documentation collected associated with sample collection is necessary to determine how the contaminant was disseminated, determine the extent of contamination, inform the investigation and drive the need for sampling, and to help understand and evaluate the analytical results associated with each sample. It is also necessary for validating those results; documenting the protocols used, sampling conditions, sample location, and individual sampling personnel; and tracking the sample during transfer to ensure sample integrity. This section summarizes some of the key components of documentation that should be implemented during sample collection. Additional guidance is provided in EPA's Sampling. Laboratory and Data Considerations for Microbial Data Collected in the Field (Silvestri et al. 2018) and the EPA's Office of Research and Development (ORD) Policy and Procedure Manual (PPM) Chapter 13 (Quality Assurance Policies and Procedures) (U.S. EPA 2017d). Electronic data recording devices are also available for use, and it is EPA policy (Stanislaus 2016) to use Scribe (U.S. EPA 2018) wherever practical to collect, store, and report sampling and analytical data. Scribe is a database management tool developed by EPA's Environmental Response Team for managing environmental data. Scribe was designed to capture sampling data, observational information, monitoring field data, and analytical data. Documentation produced during collection and processing of samples is considered a legal record by the sampling team and by EPA. Training is required for samplers in order to accurately generate/maintain legal records. Legibility and permanence of the records must be maintained. If an error is made, the error should either be struck out using a single line, initialed, and dated, or rewritten and checked for accuracy, initialed, dated, and attached to the original for record keeping. 6.1 Sample Identification Each sample collected must include an identification (ID) label, including QC samples (9.0). Each field and QC sample, sometimes referred to as blanks, must have a unique ID, and the ID must be recorded on all field documentation, sample container labels, chain of custody (COC) forms, and any other documents pertaining to the sample. This recording ties all sample collection, handling, and transport information directly to the sample and is critical for sample tracking and data analysis. The ID is used to track information linked to the sample, including sample location and type, date and time of collection, name of person collecting samples, and associated QC samples. Sample IDs are site- or incident-specific; sampling personnel should consult the SAP to determine sample ID assignments. 6.2 Sample Labels A unique sample label must be applied to each individual sample container, with information that identifies and describes the sample. Sample information is added in waterproof ink, and the label is covered with clear tape. Alternatively, pre-prepared labels that uniquely identify the sample, such as a bar or quick response (QR) code that tracks the sample information that uniquely identify the sample, can be affixed to each container. Sample container labels will be incident- and site-specific and must, at a minimum, include the sample ID. Additional information that can be included on these labels includes: Time and date of sample collection Sample matrix (e.g., particulate) Sample volume (flow rate and collection time) Preservation, if applicable Sample collection location (Global Positioning System [GPS] coordinates, local positioning system [LPS] coordinates or brief description) Signature or initials of the sample collector 8 ------- Sample labels are placed on the outside of each primary and secondary sample container (see Section 8.3). 6.3 Sample Documentation Information During sample collection, information associated with each sampling event is recorded and maintained in logbooks, on sample tracking forms, or in other sample documentation designated in the incident SAP and data management plan. These field records are completed at the time each sample is collected, and copies must accompany samples during shipment. The information recorded on these forms is essential to data validation, is extremely useful to laboratories and data users, and includes, at a minimum: Unique sample ID Date and time of sample collection Sampling location (including GPS coordinates, if appropriate) Sample type and collection method Sample collection start and stop times Collection equipment settings (e.g., flow rate) Names of sampling team members Additional information that might be requested and recorded could include site conditions, field analyses, and other pertinent observations. Electronic devices may also be used as a means of recording information in the field. If electronic recording devices are to be used, they should be selected based on durability, accuracy, backup capability, and ease of decontamination. If photographs are included as part of the sampling documentation, the name of the photographer, corresponding sample ID, date, time, site location, and site description are recorded sequentially in the logbook as each photograph is taken. Once photographs are transferred to hard copy, the associated information included in the sample documentation is electronically associated with the photograph or written on the back of the photograph. 6.4 Sample Control and Chain of Custody Once samples are collected, they must be maintained under controlled and secured conditions until transport to the laboratory. This control is required to ensure that samples are not compromised, and that analytical data are representative of site conditions. The COC forms create a written record that can be used to trace the creation, possession, and handling of the sample from the moment of its collection through analysis. A COC form accompanies each sample or group of samples as custody is transferred from one custodian to another. Sample progress is tracked and recorded at each step of sample handling, from collection through processing, packaging, and shipment. Sampling teams are responsible for initiation, maintenance, and completion of COC forms. The individual(s) performing each step of sample transfer is required to record their initials or signature on the sample label, field records, COC form, and any other document associated with the sample to qualify the condition of the sample at that point of sample progression. For example, the sample collector will sign off (e.g., electronically or on documentation) to relinquish the samples after collection for packaging and shipment. One copy of the form is retained by the original sample collector. If multiple laboratories are receiving samples, individual COCs are provided to each individual laboratory, each COC representing the contents of the sample shipment. Although COC forms vary in style, format and detail, the forms shall contain the same minimal information required to identify the sample and document custody. In cases where multiple samples are transferred as a group, the COC 9 ------- form should account for each individual sample. EPA policy (Stanislaus 2016) is to use Scribe wherever practical to generate COC forms.3 At a minimum, sampling teams are responsible for providing the following information: General incident information (sample owners, contact information, site name) Detailed site map for locating sampling points Sample information (e.g., sample IDs, sample types, number and type of sample containers, and date/time samples were collected) Date and time the samples were relinquished Signature of persons transferring the samples 6.5 Custody Seals Custody seals are part of the COC process and are attached over the sealed opening of sample containers to ensure that the samples have not been opened or tampered with after collection and packaging. A custody seal also can be placed over the shipping or transport container, making it impossible to open the container without ripping the seal. Typically, there is one seal per sample container and two seals are placed on opposite sides of the transport container. Custody seals contain the signature of the person responsible for packing the container and the date sealed. The seal must be sufficiently sturdy to resist incidental contact but be able to break when the cap or lid is moved. Sample collectors should sign and date the sample custody seal (usually a 1 x 3-inch white paper label with adhesive backing) using waterproof ink. 3 Scribe is a software tool developed by EPA's Environmental Response Team to assist in managing environmental data. For additional information regarding this tool see https://www.epa.gov/ert/environmental-response-team- information-management (last accessed 09/29/2020). 10 ------- 7.0 Sampling Equipment and Supplies Samples collected in response to a microbiological contamination incident should be collected using dedicated and clean, or new, sampling equipment to minimize interferences and cross-contamination. In some cases, pre-packaged equipment and materials are available and can be used. 7.1 Sampling Equipment Sampling equipment should be operated in accordance with manufacturers' instructions (unless otherwise specified in the SAP). Sampling equipment must undergo routine maintenance, calibration, and reliability checks according to manufacturers' instructions and be verified to be in working order before deployment and use in the field. Equipment calibration and reliability checks must be documented to reduce the likelihood that malfunctioning equipment will be used for sampling and to allow any problems to be more quickly diagnosed and corrected. Equipment used for microvacuum sampling includes the following: Calibrated, portable air sampling pump with batteries (e.g., Gilian 12 [Sensidyne] personal sampling pump, or equivalent) capable of sampling at a flow rates > 5 L/minute; please note recovery efficiency data in Table 2-1 is based on a flow rate of 20 L/minute Calibrated field rotameter (SKC, Catalog No. 320-100, or equivalent) or electronic dry cell calibrator (SKC, Catalog No. 717-5 3 OH, or equivalent) capable of measuring a flow rate of up to 30 L/minute Timer 7.2 Sampling Kit Supplies Sampling kits are prepared prior to field sampling (see Section 8.3.1) and consist of the following components: Filter cassettes - Filter cassettes will vary in design according to the manufacturer. Table 7-1 lists several filter cassette options that have been evaluated for microvacuum collection of particulate samples from porous surfaces to be analyzed for B. anthracis spores and B. anthracis surrogates (Calfee et al. 2013; Calfee et al. 2014; U.S. EPA 2013a; U.S. EPA 2013b; U.S. EPA 2017c). Table 7-1. Filter Cassette Options Filter Cassette Options 0.8-micrometer (|im) pore size MCE or 0.3-|im pore size PTFE* 0.45 |im pore size MCE, with attached nozzle New 37-mm filter cassette with 0,8-|im pore size MCE membrane (SKC, Catalog No. 225- 3-01, or equivalent) or 0.3-|im pore size PTFE membrane preloaded in 3-piece cassettes (SKC, Catalog No. 2251723, or equivalent)* New 37-mm filter cassette with 0.45-(im pore size MCE membrane (Zefon, Catalog No. 7345CC, or equivalent) * Note: The polytetrafluoroethylene (PTFE) filters can induce a higher pressure drop than the mixed cellulose ester (MCE) filters due to the smaller pore size, which could cause pumps to fail sooner than larger pore-size filters. 11 ------- Luer taper cassette adaptors (SKC, Catalog No. 225-132A; Zefon, Catalog No. ZA0005, or equivalent) Tygonฎ tubing, 1/4-inch inside diameter, 3/8-inch outside diameter (SKC, Catalog No. 225-1345, or equivalent) 12" x 12" disposable paper sampling template (SKC, Catalog No. 225-2416, or equivalent) Sterile or new/clean, leak-proof, sealable 15- mL polypropylene conical tube (Fisher Scientific, Part No. 15-959-70C, or equivalent) if needed for containment of filter nozzles 4" x 6" clean sealable polyethylene bags 1-quart clean sealable polyethylene bags 1-gallon clean sealable polyethylene bags 7.3 General Supplies New, unused powder-free nitrile gloves for the sample collector, supplier, and support person PPE (consult the SAP and HASP for the required PPE for the sampling event) Pre-printed labels for sample containers (see Section 6.2) Permanent marker(s) and indelible ink pens Disinfectant wipes (e.g., Dispatchฎ Wipes) Sample documentation materials (e.g., digital camera, electronic tablet, forms, and/or logbook [Section 6.0]) Custody seals (see Section 6.5) 1-gallon sealable plastic bags (for contaminated equipment) Sealable waste containers (e.g., 5-gallon or 20-gallon buckets with lids) 7.4 Sample Transport Containers and Packing Materials (see Section 11.0 for more information) Transport container - Rigid insulated cooler able to withstand an internal pressure of 14 pounds per square inch (psi) (-96526.6 pascals), with a secure, sealable lid. The container must be capable of 1) surviving impacts without being compromised or damaged, and 2) containing and maintaining ice packs (See Section 11.2.2) Durable absorbent packing material (see Section 11.2) Self-contained ice or cold packs Sealing tape Custody seals (see Section 6.5) Shipping documentation (see Section 11.0) COC forms (see Section 6.4) 12 ------- 8.0 Preparation for Sample Collection Adequate and appropriate preparation for sample collection is critical for ensuring that representative samples are collected properly and as needed to meet analytical requirements. In addition, preparation is critical for ensuring the safety of sampling personnel, transporters, and laboratory technicians. This section summarizes several of the activities that should be done prior to initiating the sampling steps described in Section 10.0. Sampling personnel also should work closely with the incident commander or site/project managers to ensure that sampling activities are conducted in accordance with the SAP. 8.1 Sampling Teams Any sampling effort requiring the collection of multiple samples, particularly those involving hazardous conditions and/or collection of samples containing microbiological agents, should involve a sampling team consisting of at least two personnel. Additional personnel may be required for large-scale sampling efforts or when site-specific hazards may be encountered. Individual team members must be trained to assume specific activities or duties related to the sampling effort. Depending on the size of the sampling event and the number of samples required, a three-person sampling team, consisting of a collector, a supplier, and a support person, is recommended for microvacuum sampling. While not as optimal as a three-person team, a two-person sampling team could be acceptable in some situations with the supplier conducting the duties of the support person. Collector - responsible for operating the sampling equipment and collecting the sample and post- sampling disassembly of the filter apparatus Supplier - responsible for providing the collector with the pre-labeled sample kit and materials needed to collect the sample, helps with the assembly of the vacuum filter apparatus, and decontaminates sample containers. The supplier does not come into direct contact with any of the materials or equipment that will come into direct contact with the sample. Support Person - responsible for sample documentation, documenting flow rate and sampling duration, and radio communication This team approach can reduce the time required for sample collection and adds an additional layer of quality assurance to the overall process. Importantly, sampling teams also provide an additional level of safety. Each team member must be trained in the collection of samples using techniques designed to minimize potential cross contamination (see Section 8.2). Prior to initiating sample collection activities, sampling personnel will: Review and understand all specifications and requirements that are included in the incident SAP and/or QAPP, HASP and WMP (see Appendix B) Communicate with all sampling team members to ensure roles and responsibilities have been established and are understood Ensure that the sampling teams have access to a detailed site map to assist in locating sampling points. Evaluate and prepare sampling equipment and PPE prior to site entry. Equipment that can be prepared outside the sampling area should be pre-assembled, cleaned, calibrated (e.g., checking flow rates of sample pumps), and protected from contamination prior to site entry. Pre-prepared equipment (e.g., sampling kits) should be contained (e.g., in an overpack bag) prior to site entry and until use. Assemble and don the appropriate PPE prior to site entry, as directed in the HASP. Summary information regarding PPE for use during sample collection following a microbiological contamination incident is provided in Section 3.0. 13 ------- Understand site egress procedures, which address decontamination and transfer of sample containers and contained waste in accordance with the SAP and HASP. Ensure procedures for equipment decontamination have been established, and appropriate materials are available. Summary information regarding decontamination of equipment is provided in Section 8.4. Sampling personnel are required to decontaminate prior to exiting the contaminated area, as instructed in the HASP, to ensure contamination is not spread outside the area. Required decontamination is outside the scope of this document, however, information regarding the and decontamination line procedures and required decontamination for all areas on the site will be included in the site-specific HASP. For additional personnel decontamination information, see EPA's Decontamination Line Protocol Evaluation for Biological Contamination Incidents Assessment and Evaluation Report. (U.S. EPA 2015). 8.2 Techniques to Minimize Potential Cross Contamination The clean and dedicated sampling equipment, and appropriate PPE should prevent contamination during sample collection. As sources of contamination may or may not be obvious, inclusion of the appropriate field QC samples, as described in Section 9.0, can help identify the presence and sources of contamination. Cross contamination between samples can usually be avoided by adherence to techniques to minimize potential cross contamination, by changing gloves between samples, and by avoiding contact between sampling equipment and contaminated surfaces. Sampling personnel should receive training in these techniques prior to collecting samples. To limit possible contamination, templates should only be taped to the surface if truly necessary and should remain in place after sampling (Calfee et al. 2016). Techniques should be used to collect samples to reduce the risk of exposure to sampling personnel, the risk of contaminating the samples, and the risk of spreading contaminants in the environment. The use of these techniques is the first and most important step in ensuring consistent and accurate sampling results. The following practices can be used as a guideline: Minimize the amount of time sample containers are open. Hold open containers away from sources of contamination (e.g., blowing air, other possibly contaminated objects). Do not touch the inside of sample containers or caps. Once a container is filled, do not touch the contents. Work as quickly as possible, without compromising techniques, to minimize potential cross- contamination. Change gloves as prescribed by the SAP and/or HASP, using appropriate doffing/donning procedures. Avoid touching areas of the collection device that come into contact with surfaces or that concentrate contaminants. Avoid contact between the sampling equipment and contaminated surfaces. Keep the work area (if applicable) uncluttered. 8.3 Sampling Kits It is essential that sampling personnel use pre-packaged sampling kits for sample collection, and that these kits be properly equipped, maintained, and organized before deployment of sampling teams. Information regarding the equipment and materials included in these kits is provided in Section 7.2. However, samplers should consult with the incident commander or site/project managers and with the SAP to determine what equipment and materials will be required. 14 ------- 8.3.1 Sampling Kit Assembly Sampling kit assembly, which includes assembly of the filter cassette, nozzle, and tubing, must be conducted in a controlled, clean area, preferably in a laboratory or similar controlled location, using clean disposable powder-free nitrile gloves. Once prepared, the kits are stored in a clean and dry location prior to use. A unique sampling kit is required for each microvacuum sample and each QC sample (field blanks and trip blanks). Note that disposable powder-free nitrile gloves should also be included in the sampling box for the supplier, collector, and support personnel, and is included in the general supply list. Note: Some filter cassettes are supplied by the manufacturer with sampling nozzles attached, and the nozzles can be difficult to remove under field conditions. When these filter cassettes are used, nozzles do not need to be prepared or attached to the filter cassette and are not removed following sample collection. Sampling kit assembly steps are: Don clean gloves prior to kit assembly. If the filter cassette is not provided with a manufacturer-attached nozzle, prepare and attach a nozzle as follows: o Aseptically remove the filter cassette plugs and place a polyvinylchloride (PVC) luer adaptor onto each end of the cassette. Label a 1-quart plastic bag and place the cassette plugs in the bag. o Prepare a sampling nozzle from the tubing by cutting a 1-inch (2.5-cm) section of tubing, then cutting one end of the tubing at approximately a 45-degree angle. [Note: Extra nozzles should be prepared in the event they might be required during sample collection. Extra nozzles stored in 15 mL conical tubes can be packaged in a separate sampling kits for later use.] o Attach the sampling nozzle (1-inch section of tubing) to the PVC luer adapter on the upstream (inlet) end of the cassette with the angled side farthest from the cassette. Attach a 20-inch piece of tubing onto the luer adapter on the downstream (outlet) end of the cassette. [Note: If the filter cassette has a manufacturer-attached nozzle, ensure the plugs are removed from both the nozzle and outlet before attaching the tubing, and place the plugs in a pre- labeled 1-quart plastic bag.] Label a sealable 4" x 6" plastic bag with the same unique sample ID as the 1-quart plastic bag. Place the following into a sealable, pre-labeled 1-quart bag: filter cassette assembly, a 4" x 6" pre-labeled sealable plastic bag, original filter cassette plugs, and one pre-labeled 15-mL polypropylene conical tube (if nozzles have been prepared and attached as described above) (Figure 2-1). Make sure the end of the tubing attached to the filter cassette outlet is closest to the bag opening and seal the 1-quart filter cassette assembly bag. Place the filter cassette assembly bag and a 12" * 12" paper sampling template in a pre-labeled 1- gallon plastic overpack bag and seal the overpack bag (Figure 2-1). Prior to sample collection, samplers must check sampling kits to confirm they are complete. A sampling kit check list is provided in Table 8-1. 15 ------- Figure 2- Example Sample Collection Kit Table 8-1. Sampling Kit Check List Filter cassettes with manufacturer-attached nozzles Filter cassettes with nozzles prepared and attached as described in Section 8.3.1 Labeled filter cassette with attached tubing and nozzle in pre-labeled 1-quart plastic bag Filter cassette plugs Labeled sealable 4" x 6" plastic bag 12" x 12" sampling template Pre-labeled 1-gallon plastic overpack bag Labeled filter cassette with attached tubing and nozzle in pre-labeled 1 -quart plastic bag Filter cassette plugs 15-mL conical tube Labeled sealable 4" x 6" plastic bag 12" x 12" sampling template Pre-labeled 1-gallon plastic overpack bag 8.3.2 Supplemental Supplies and Materials In addition to the sampling kits, samplers should ensure they have all the additional materials, supplies and equipment needed for sample collection, decontamination, documentation, tracking, and packaging activities. At a minimum, the following should be made available to sampling teams: Pre-assembled sampling kit (one per sample, see Section 8.3.1) PPE (e.g., protective clothing, powder-free disposable nitrile gloves for sampler, supplier, and support personnel) as required by the SAP and HASP Site maps Air sampling pump (with pre-assembled filter cassette and tubing for calibration and batteries) Sample preservation supplies (e.g., self-contained ice packs) Sample documentation (see Section 6.0) Sample packaging supplies Decontamination materials (see Section 8.4) 8.4 Decontamination Decontamination of the primary sample receptacle using wipes should be completed prior to removal from the exclusion zone to minimize contaminant transfer (Calfee 2016; CDC 2012). While it is outside the scope of this document to provide full instructions on decontamination of sampling equipment, the list below provides general considerations: 16 ------- The HASP and/or SAP should be referred to for the personnel assigned for equipment decontamination and for specific procedures to be followed for equipment decontamination or for transport of contaminated equipment offsite for decontamination. Additional information regarding decontamination is provided in EPA's Decontamination Line Protocol Evaluation for Biological Contamination Incidents. Assessment and Evaluation Report (U.S. EPA 2015). Equipment that is contaminated in the field can be cleaned with disinfectant wipes and either reused or bagged for transport to a facility that can clean the equipment for re-use. Refer to the HASP and or/SAP for packaging and labeling instructions if the equipment needs to be transported prior to decontamination. Note: Do not transport contaminated sampling equipment or supplies in the same transport container as the samples. The sample outer packaging should be decontaminated upon arrival at the receiving laboratory. Unless determined to be free of contamination, materials used for decontamination must be collected as waste and removed from the sampling site for proper disposal. Five- or 20-gallon buckets with lids can be used for containment. Drums or large garbage cans can be used to contain contaminated PPE, accumulated wastes, sampling materials or equipment. 17 ------- 9.0 Quality Control Samples Additional samples should be collected to assess the validity of the analytical results, as well as possible analytical interferences, contamination, and sample integrity (QC). For samples collected using filter cassettes, QC samples typically consist of field blanks, trip blanks, and field replicates. The sampling task leader or on scene coordinator should consult with the laboratory or refer to the SAP to determine the type and number (or frequency) of QC samples that should be collected. 9.1 Field Blanks Field blanks are used to monitor contamination that may be introduced into samples during sample collection. Field blanks are typically prepared prior to sample collection at the sample collection site, then transported to the laboratory along with the field samples for analysis. Field blanks for microvacuum samples are prepared in the field by opening a new or unused filter assembly (37-mm filter cassette and tubing prepared as described in Section 8.3.1), connecting the sampling pump, plugging the ends of the cassette, and placing the unused filter assembly directly into a clean labeled sample container (e.g., plastic bag), without pulling any air through the filter. 9.2 Trip Blanks Trip blanks are used to monitor contamination that might be introduced into samples during handling and transport. Trip blanks are prepared prior to going into the field, taken to the sampling site, and shipped back to the laboratory, unopened, with the samples. Unlike field blanks, the filter assembly in the trip blank is not exposed to field conditions. At no time after their preparation are the sample containers of trip blanks (e.g., plastic bags) opened before they reach the laboratory. Note: In some cases, a trip blank might also be used as a media blank by the laboratory. 9.3 Field Replicates Field replicates are collected in conjunction with the initial field sample. Replicates are samples of the same material that are collected in the same manner and as close in proximity as possible to the initial sample. These samples can be used for replicate analyses, confirmation of results, or archived for repeat analyses. Sampling personnel must ensure that replicates are as equivalent as possible. QC samples are often submitted blind to the laboratory to increase objectivity (i.e., sample documentation received by the laboratory does not identify which QC samples correspond to which field samples). Sampling personnel should refer to the SAP to determine how all samples (field and QC) are to be identified and labeled for transport to the laboratory. 18 ------- 10.0 Sample Collection Tli is section outlines activities specific to the collection of representative porous surface samples using 37-mm filter cassettes and air sampling pump to be analyzed for microbiological contamination. Prior to field sampling, all sampling personnel should be familiar with the incident-specific SAP, HASP and WMP, in addition to the following incident-specific information: Health and safety (Section 3.0) Waste management (Section 5.0) Sample documentation (Section 6.0) Equipment and supplies (Section 7.0) Sample collection preparation (Section 8.0) QC requirements (Section 9.0) Packaging and transport (Section 11.0) Sampling personnel should understand their role(s) and responsibilities regarding sample collection and should practice sampling activities as a team to ensure consistent and efficient collection of representative samples while taking into account the pattern and duration of sampling. In addition, sampling personnel must be aware of any specific laboratory sample acceptance requirements (e.g., appropriate primary and secondary containers, preservation, holding time, integrity) and ensure that samples are collected and handled in accordance with these requirements. These requirements must be detailed in the incident- specific SAP. However, if there is any uncertainty or lack of clarity regarding sampling activities, sampling personnel should contact the incident commander or site/project managers for clarification prior to initiating field sampling. 10.1 Materials, Supplies and Equipment In addition to being familiar with the documentation and information noted above, sampling teams must have access to all necessary equipment prior to entering the area of contamination and initiating sample collection activities. Required equipment and supplies include: Appropriate PPE (Section 3.0) Powder free nitrile gloves for sample supplier, collector, and support person Sample documentation (Section 6.3) Air sampling pump and power source (Section 7.1) Calibrated rotameter or electronic dry cell calibrator (Section 7.1) Timer (Section 7.1) Sample transport containers and packing materials (Section 7.4) Sampling kit (Section 8.3) Decontamination supplies (Sections 8.4) Sampling personnel must ensure that the sampling kits and documentation are specific for the sample(s) that are to be collected. 19 ------- 10.2 Collection As noted in Section 8.1, in most cases, a three-person sampling team, consisting of a collector, a supplier and a support person, is recommended for microvacuum sampling. The number of individuals needed, however, will depend on the size of the sampling event and number of samples required. The sample collection process described below is based on a three-person sampling team, a collector, a supplier, and a support person, with roles as previously described in Section 8.1. Once the sampling team has arrived at the sampling site and determined the required sampling location(s), each team member should don clean powder-free disposable nitrile gloves and calibrate the air sampling pump outside the area of contamination. The pump should be calibrated following instructions in the SAP or from the manufacturer, using a clean filter cassette and tubing assembly that will not be used for sample collection. Once the pump has been calibrated, the sampling team proceeds to the sampling location and follows the procedures described below, using a new sampling kit and a new pair of gloves for each field and QC sample collected. Pre- and post-sampling flow rates should be recorded. I. Equipment: Preparation and Setup Supplier and Collector: Don new, clean sampling gloves (Note: the support person should also don new, clean gloves if they accidently touch the sample or contaminated area). Supplier: Remove a sampling kit from the sampling supply bin. Support Person: Scan the bar code, record the sample ID, or radio the ID information to a data recorder in the support zone. Supplier: Open the sample kit outer bag and remove the 12" * 12" sampling template. Hand the template to the collector. Collector: Place the 12" x 12" sampling template over the area to be sampled Support Person: If required, photograph the sampled area, and/or draw a map of the location in the logbook. II. Collection Supplier: Supplier: Collector: Collector: Collector: Collector: Remove the filter cassette assembly bag from the sampling kit outer bag. Open the filter cassette assembly bag and hold the bag so that the collector can remove the filter cassette assembly. Remove the filter cassette assembly using the 20" tubing closest to the bag opening and ensure that the tubing and sampling nozzle are correctly placed on the filter cassette. Make any necessary adjustments. Connect the tubing to the pump inlet. Hold the sampling nozzle at an angle so that the tapered opening of the nozzle is flush with the sample area surface. Note: The inlet of the nozzle (cut at a 45ฐ angle) should be approximately parallel to the surface being sampled. Keep the nozzle lightly in touch with the surface; do not press the nozzle hard against the surface being sampled. Activate the pump air flow. Support Person: Initiate timer and record flow rate and start time. Collector: Collect the sample using the horizontal and vertical patterns described below. The duration of sampling should approximate the following: 20 ------- - Total sampling duration - 300 seconds (5 minutes) - Single pass duration - 3 to 5 seconds - Number of passes per direction - approximately 50 Note: If filter overloading becomes evident (e.g., reduced flow rate or pump failure), stop sample collection. Partial collection, including the sampling start and stop times, should then be recorded on the sample documentation form by the support person. Vacuum "horizontally," using S-strokes to cover the entire area delineated by the template. Pace the speed of the nozzle such that the target sampling time is achieved. Vacuum the same area "vertically" using the same technique, while keeping the sampling nozzle angled so that the tapered opening of the nozzle is flush with the sample area surface. n Microvacuum Sampling Pattern Example Support Person: Once sampling is completed, record final pump flow rate and total sampling time. Collector: Turn off the pump. III. After Collection of Each Sample Note: Some filter cassettes are supplied by the manufacturer with sampling nozzles attached (see Section 8.0), and the nozzles can be difficult to remove under field conditions. When these filter cassettes are used, it is recommended that the nozzle and outlet plugs supplied with the filter cassette be reinserted after sample collection and the entire sealed filter be placed directly into the 4" x 6" pre-labeled resealable plastic bag for primary containment. If the nozzle has been attached during filter assembly (see Section 8.0), a conical tube will be included in the sampling kit. In this case, follow the disassembly instructions in the box below. If the nozzle is attached to cassette with PVC luer adapter (e.g., SKC filter cassette) Supplier: Remove the 15-mL tube from the sampling kit and open the conical tube. Collector: Remove the nozzle and luer adapter from the filter cassette, and place it into the tube, with the PVC luer adapter end down. Supplier: Seal the tube and place it in the 4" * 6" pre-labeled resealable plastic bag. The supplier should not touch the device after it has been used to collect a sample. 21 ------- Collector: Supplier: Collector: Supplier: Collector: Supplier: Supplier: Supplier: Supplier: Supplier: Supplier: Support Person Collector: All Personnel: Remove the tubing and luer adapter from the outlet side of the filter cassette and place in a disposal container. Hold the 4" x 6" plastic bag containing the filter cassette plugs (and nozzle plug if required) for collector to remove plugs. Remove plugs from the bag, and seal both ends of the cassette (and nozzle if attached) with the two plugs supplied with the filter cassette. Hold the 4" * 6" pre-labeled resealable plastic bag (primary sample container) open. Place the sealed filter cassette into the bag. Seal the 4" x 6" bag and place it into the pre-labeled 1-quart bag (secondary container). The supplier should not touch the device after it has been used to collect a sample. Seal the 1-quart bag and decontaminate the outer surface of the bag (Section 8.4). Open the pre-labeled 1-gallon bag and place the 1-quart bag into it. Seal the 1-gallon bag and decontaminate the outer surface with a disinfectant wipe. Apply a custody seal over the sealed 1-gallon bag opening. Place the double-bagged sample into the sample collection bin for transfer to sample packaging and transport (see Section 11.0). Complete sample documentation (see Section 6.3). Gather the sampling template, tubing, and any additional contaminated equipment or supplies for proper disposal. Doff sampling gloves prior to moving to the next location. 10.3 QC Sampling QC samples that might be requested for microvacuum sampling include negative controls (field blanks, trip blanks) and field replicates (Section 9.0). The number, type and locations of QC samples required will be specified in the SAP. Field blanks (Section 9.1) are prepared and handled as described above for field samples with the exception that the pump is not turned on and the sampling nozzle is not allowed to touch a potentially contaminated surface. Trip blanks (Section 9.2) are supplied as unopened/unused sampling kit assemblies and are transferred and transported along with the field samples. Field replicates (Section 9.3) are prepared and handled as described above for field samples. 22 ------- 11.0 Sample Packaging and Transport This section provides general information on packaging and preparing samples for transport and to help ensure that sample integrity is maintained during these processes. Information regarding packaging for select agents is not covered in this document and can be found in CDC's Federal Select Agent Program Guidance on the Shipment and Receipt of Packages with Select Agents and Toxins (CDC 2014). Since microbiological samples often degrade quickly over time, it is imperative to have procedures for sample storage (short and long term), packaging, and transport that are efficient and preserve sample integrity. Laboratories should receive samples that are properly preserved, packaged, and received within the holding time requirements needed to support analysis, and receiving laboratories can and will reject samples if sample packaging and transport requirements are not met. Note: While section 11.1 and Table 11-1 give general information on storage holding time and temperature, the laboratory that will be conducting the laboratory analysis should be consulted to determine the exact temperature requirements that samples should be stored and shipped at prior to analysis. Samples also must be accompanied by the appropriate documentation. 11.1 Sample Holding Time and Temperature Many variables go into making decisions regarding sample storage temperatures and holding time. Caution should be taken when applying these requirements that were developed for other sampling media or organisms. The best storage temperature for a given microbiological sample often varies depending on the microbiological organism, the sample matrix, the sample's intended use, and storage time. When storing samples, it is also important to consider the molecular structure of the microbiological agent (Holland et al. 2003; Budowle et al. 2006; NRC 2014; Shabihkhani et al. 2014) and the degree of microbiological integrity required for analysis. Some general considerations for holding times and temperatures are included in Table 11-1 and Sections 11.1.1 (Sample Holding Times) and 11.1.2 (Temperature) below: Table 11-1. Transport Conditions and Holding Times Afii'iil l>|K' S(ปr;iปe 1 OllipOI'illlllV KccomiiK'iHk'ri Molding Time Oilier (onsidoi'iilions Bacteria 2-8 ฐC; do not freeze Vegetative bacterial samples should be analyzed as soon as possible. (Maximum holding time of 24^18 hours.) Samples containing bacterial spores should be analyzed within 48 hours (CDC, 2012). Samples should not come in direct contact with ice or ice packs Ice packs should be placed outside the secondary receptable Consult the sampling and analysis plan (SAP) for temperature monitoring or other transport requirements designated by the receiving laboratory Viruses 2-8 ฐC; do not freeze Samples should be analyzed as soon as possible. (Maximum holding time of 24-72 hours.) Samples should not come in direct contact with ice or ice packs Ice packs should be placed outside the secondary receptable Consult the SAP for temperature monitoring or other transport requirements designated by the receiving laboratory Protozoa/ Helminths 2-8 ฐC; do not freeze Samples should be analyzed as soon as possible. (Maximum holding time of 96 hours.) Samples should not come in direct contact with ice or ice packs Ice packs should be placed outside the secondary receptable Consult the SAP for temperature monitoring or other transport requirements designated by the receiving laboratory Vibrio cholera* Room temperature Samples should be analyzed as soon as possible. Consult the SAP for temperature monitoring or other transport requirements designated by the receiving laboratory Full reference at the end of the report. *Note: Samples to be analyzed for Vibrio cholerae should be kept at room temperature and must not be cooled. 23 ------- 11.1.1 Sample Holding Time Maximum sample holding time, a critical aspect to consider when making decisions regarding sample packaging and transport, is the sum of the time between sample collection and receipt at the laboratory and the time between sample receipt at the laboratory and sample analysis. In all cases, samples should be transported to the laboratory and analyzed as quickly as possible following collection, in a manner that stabilizes the sample and minimizes the loss of viability. 11.1.2 Sample Temperature In all cases, sample collectors should consult the SAP for information regarding requirements for sample preservation (temperature) and transport conditions. For example, a receiving laboratory may require temperature blanks or temperature monitoring devices to be placed in transport coolers to evaluate whether an appropriate temperature has been maintained throughout transport. The procedure to be followed for sample preservation and transport conditions should be resolved with the laboratory prior to initiation of sample collection and included as part of the SAP. If the SAP does not provide the requirements for sample temperature and transport conditions, the sampling team leader should consult with the project team leader or designee who in turn should consult with the laboratory to resolve any questions. In general: Samples should be stored at 2ฐC-8ฐC without freezing prior to processing (CDC, 2012), and analyzed as soon as possible (see Table 11-1). An exception is made for samples that will be analyzed for Vibrio cholerae; these samples should be maintained at room temperature and should not be cooled. Samples often degrade over time when stored at room temperature, but some samples can lose integrity even at low temperatures if subjected to multiple freeze-thaw cycles. Samples that require low temperature preservation shall be considered acceptable if the arrival temperature of a representative sample container meets the temperature requirement. 11.2 Sample Container Transport and Labeling Biological agents (including certain microbiological agents) are classified as hazardous materials (HAZMATs) and require transportation in accordance with International Air Transport Association (IATA) Dangerous Goods Regulations (DGRs) for shipment by air or DOT requirements at 49 CFR Parts 171 through 180 (IATA 2015; U.S. DOT, 2012), Hazardous Materials Regulations for movement in public rights-of-way within the U.S. (U.S. DOT 2012; U.S. DOT 2020). IATA and DOT publications are revised frequently, and individuals should consult the most current publications, the Federal Register, and publications of other governing agencies for complete instructions. It is the sample originator's responsibility to ensure adherence to all regulations. Only trained and certified HAZMAT shippers may ship the biological agents. Sampling personnel can refer to the following websites for information regarding the shipping of infectious substances and biological agents: International Air Transport Association (last accessed 09/28/2020) U.S. Department of Transportation (last accessed 09/04/2020) American Society for Microbiology (ASM 2017) American Biological Safety Association (last accessed 09/28/2020) WHO "Guidance on Regulations for the Transport of Infections Substances 201.7201.8" (WHO 2017) 11.3 Sample Packaging This section provides general guidance on sample packaging; however, level of packaging is dependent 24 ------- on whether samples are considered a dangerous goods shipment or not and the most recent IATA and DOT publications should be consulted. Samples should be shipped with appropriate chain of custody forms and labels. Packaging kits should include inner and outer packaging, coolers, labels, and absorbent material. Battery packs for sampling pumps should be removed from the pump prior to shipment. 11.3.1 Primary and Secondary Sample Containment Following sample collection and prior to leaving the contaminated area, all field and QC samples are packaged in primary and secondary sample containers, as described in Section 10.2. For samples collected into filter cassettes using the procedures described: Primary sample containers are clean, leak-proof, and sealable 15- mL polypropylene conical tubes for containment of the filter nozzle (if needed, see Section 8.3.1), and/or 4" x 6" sealable plastic bags for containment of the filter cassette Secondary sample containers are clean sealable plastic bags Each primary and secondary sample container is sealed and labeled with the Sample ID (see Sections 6.3 and 10.2). Prior to leaving the area of contamination, ensure that the surface of the outermost container is decontaminated and placed into a clean bag or box for transfer to the container packaging area. 11.3.2 Packing Sample Transport Containers Transport containers must be sufficiently durable and constructed of material that will ensure sample integrity, including maintaining appropriate temperatures. If the proper containers, packing materials, and labels are used incorrectly, damage to the samples can occur and sample integrity could be compromised. If the samples will be shipped by air, the container must be able to withstand an internal pressure of 14 pounds per square inch (psi). Rigid, insulated coolers with secure, sealable lids, that are capable of 1) surviving impacts without being compromised or damaged, and 2) containing and maintaining self- contained ice or cold packs are recommended. Inner containers should be cushioned with enough absorbent material to absorb any fluids (e.g., melted ice). Remove the contained sample(s) from the sample transfer bag or box and decontaminate the outside of the secondary sample container(s) using decontamination wipes (Section 8.4). Pack secondary sample containers into a transport container with sufficient absorbent packing material to absorb any fluids (e.g., condensation from ice packs) and to ensure they are protected and will not shift during transport. Self-contained ice or cold packs are recommended for cooling samples during transport. When multiple sample containers are packaged within a single transport container, absorbent packing material absorbent material (such as paper towels or absorbent gel sheets) should be used to separate containers and to ensure there is no contact between the containers. Complete a COC form, seal the form in a plastic bag along with other pertinent sample documentation, and adhere the bag to the inside of the transport container lid. Retain a copy of all documentation, including the COC. Seal the transport container, and place at least two custody seals on the transport container lid, in a manner such that the container cannot be opened without breaking the seals. CAUTION: Samples must not be frozen (see Table 11-1). To avoid freezing all or portions of the sample(s), samples should not be packed in direct contact with ice or ice packs. 25 ------- 11.3.3 Transport Documents All sample transport containers should be accompanied by sample documentation, including COC forms and field records (see Section 6.0). To maintain COC, the COC form must be readily accessible when transferring samples from one individual to another. Therefore, COC forms should be placed inside a waterproof self-sealing bag, which is adhered to the inside of the transport container lid. One copy of these forms should be retained by the sampler. If the transport container is being shipped, the shipping receipt should be retained by the sampling personnel for the permanent record. Common carrier documents should be included with each shipment and completed as required by the individual carrier. All packages must securely display the following: Sampling contact information, mailing address, and phone number Laboratory name(s), mailing address, and phone number Quantity and description of contents Date of shipment 11.4 Transfer of Custody An unbroken COC must be maintained for all samples from collection through analysis and archiving. Information that is included in a COC form is discussed in Section 6.4, and an example COC form is provided in Appendix C. Laboratories should be notified in advance of any shipments, and an accurate description of the contents of the shipment and the expected date of arrival should be included. Once received at the laboratory, each individual releasing and receiving the samples must sign the COC form to provide evidence of the custody transfer. Upon receipt of samples, the laboratory will document the condition of each sample container received and should report this information to the analytical services requestor to confirm that the package was not damaged or tampered with during transport. 26 ------- 12.0 References American Society for Microbiology (ASM). (2017) Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases. https://www.asm.org/Articles/Policv/Laboratorv-Response-Network-LRN-Sentinel-Level-C (last accessed 09/28/2020). Budowle, B., S.E. Schutzer, J.P. Burans, D.J. Beecher, T.A. Cebula, R. Chakraborty, W.T. Cobb, J. Fletcher, M.L. Hale, R.B. Harris, M.A. Heitkamp, F.P. Keller, C. Kuske, J.E. LeClerc, B.L. Marrone, T.S. McKennaq, S.A. Morse, L.L. Rodriguez, N.B. Valentine and J. Yadev. (2006) Quality sample collection, handling, and preservation for an effective microbial forensics program. Appl Environ Microbiol. 72(10): 6431-6438. doi: 10.1128/AEM.01165-06 Calfee, M.W., L.J. Rose, S. Morse, D. Mattorano, M. Clayton, A. Touati, N. Griffin-Gatchalian, C. Slone and N. McSweeney. (2013) Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores. J Microbiol Methods. 95(3): 389-396. doi:10.1016/j.mimet.2013.10.015 Calfee, M.W., L.J. Rose, S. Morse, D. Mattorano, M. Clayton, A. Touati, N. Griffin-Gatchalian, C. Slone and N. McSweeney. (2014) Evaluation of sampling methods for Bacillus spore- contaminated HVAC filters. J Microbiol Methods. 96:1-5. doi:10.1016/j.mimet.2013.10.012 Calfee, M. W., J. Tufts, K. Meyer, K. McConkey , L. Mickelsen, L. Rose, C. Dowell, L. Delaney , A. Weber, S. Morse, J. Chaitram, and M. Gray. (2016) Evaluation of standardized sample collection, packaging, and decontamination procedures to assess cross-contamination potential during Bacillus anthracis incident response operations . J Occup Environ Hyg. December; 13(12): 980- 992. doi: 10.1080/15459624.2016.1200725 Centers for Disease Control and Prevention (CDC). (2012) Emergency Response Resources: Surface sampling procedures for Bacillus anthracis spores from smooth, non-porous surfaces. Centers for Disease Control and Prevention: Atlanta, GA. https://www.cdc.gov/niosh/topics/emres/surface- sampling-bacillus-anthracis.html#e (last accessed 10/02/2020). CDC. (2014) Federal Select Agent Program Guidance on the Shipment and Receipt of Packages with Select Agents and Toxins. Centers for Disease Control and Prevention and U.S. Department of Agriculture. Chattopadhyay, S. (2017) Sample Collection Information Document for Pathogens: Companion to Selected Analytical Methods for Environmental Remediation and Recovery (SAM). EPA/600/R- 17/374. U.S. Environmental Protection Agency: Washington, DC. https://www.epa.gov/esam/sample-collection-information-documents-scids (last accessed 09/29/2020). Holland, N.T., M.T. Smith, BB. Eskenazi and M. Bastaki. (2003) Biological sample collection and processing for molecular epidemiological studies. MutatRes. 543(3):217-34. doi:10.1016/sl383- 5742(02)00090-x International Air Transport Association (IATA). (2015) IATA Dangerous Goods Regulations Manual. International Air Transport Association: Washington, DC. https://www.iata.org/en/publications/dgr/ (last accessed 09/04/2020). National Institute for Occupational Safety and Health (NIOSH). (2009) Recommendations for the Selection and Use of Respirators and Protective Clothing for Protection Against Biological Agents. National Institute for Occupational Safety and Health: Atlanta, GA. DHHS (NIOSH) Publication number 2009-132. https://emergencv.cdc.gov/documentsapp/Anthrax/Protective/10242Q01Protect.asp (last accessed 02/01/2021). 27 ------- National Research Council (NRC). (2014) Science Needs for Microbial Forensics: Developing Initial International Research Priorities. Board on Life Sciences, Division on Earth and Life Studies, National Research Council, The National Academies Press: Washington, DC. Shabihkhani, M., G.M. Lucey, B. Wei, S. Marenisov, J.J. Lou, H.V. Vinters, E.J. Singer, T.F. McCloughesy and W.H. Yong. (2014) The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings. Clin Biochem. 47(4-5):258-266. doi: 10.1016/j.clinbiochem.2014.01.002 Silvestri, E., K. Hall, Y. Chambers-Velarde, J. Chandler, J. Cuddeback, and K. Jones. (2018) Sampling, Laboratory, and Data Considerations for Microbial Data Collected in the Field. U.S. Environmental Protection Agency, Cincinnati, OH. EPA/600/R-18-164. https://cfpub.epa.gov/si/si public record report.cfm?Lab=NHSRC&dirEntrvId=341832 (last accessed 09/29/21). Stanislaus, M. (2016) Memorandum from Mathy Stanislaus, Office of Land and Emergency Management, U.S. Environmental Protection Agency to Office of Land and Emergency Management Employees regarding subject "Scribe - Exclusive Agency Database during a National Response," March 8, 2016. U.S. Environmental Protection Agency: Washington DC. U.S. Department of Labor (DOL), Occupational Safety and Health Administration. 1910.134 - Respiratory Protection. Occupational Safety and Health Administration. 1910.134 - Respiratory Protection. https://www.osha.gOv/laws-regs/regulations/standardnumber/l910/1910.134 (last accessed 02/01/2020). U.S. DOL, OSHA (2013) Hazardous Waste Operations and Emergency Response. 29 CFR 1910.120. Occupational Safety and Health Administration: Washington, DC. https://www.osha.gov/laws- regs/regulations/standardnumber/1910/1910.120 (last accessed 09/29/2020). U.S. Department of Transportation (U.S. DOT). (2012) Hazardous Materials Regulations 49 CFR Parts 171-180. https://gov.ecfr.io/cgi-bin/text- idx?SID=51627edf054ac9506f03a5729aa8cbb9&mc=true&tpl=/ecfrbrowse/Title49/49tab 02.tpl (last accessed 09/04/2020). U.S. DOT. (2020) Transporting Infectious Substances Safely. https://www.phmsa.dot.gov/sites/phmsa.dot.gov/files/2020-04/Transporting-Infectious- Substances-Safely.pdf (last accessed 02/03/2021). U.S. Environmental Protection Agency (U.S. EPA). (2013a) Bio-Response Operational Testing and Evaluation (BOTE) Project - Phase 1: Decontamination Assessment. U.S. Environmental Protection Agency: Washington, DC. EPA/600/R-13/168 U.S. EPA. (2013b) Evaluation of Vacuum-based Sampling Devices for Collection of Bacillus Spores from Environmental Surfaces. U.S. Environmental Protection Agency: Washington, DC. EPA 600/R-13/137 U.S. EPA. (2015) Decontamination Line Protocol Evaluation for Biological Contamination Incidents Assessment and Evaluation Report. U.S. Environmental Protection Agency: Washington, DC. EPA/600/R-14/476. https://cfpub.epa.gov/si/si public file download.cfm?p download id=522240&Lab=NHSRC (last accessed 03/16/21). U.S. EPA. (2017a) Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017. U.S. Environmental Protection Agency: Washington, DC. EPA/600/R-17/356. https://www.epa.gov/esam/selected-analvtical-methods-environmental-remediation-and-recoverv- sam (last accessed 9/29/2020). 28 ------- U.S. EPA. (2017b) Environmental Sampling and Analytical Methods (ESAM) Program. https://www.epa.gov/esam (last accessed 02/03/2021). U.S. EPA. (2017c) Underground Transport Restoration (UTR) Operational Technology Demonstration (OTD). U.S. Environmental Protection Agency: Cincinnati, OH. EPA/600/R-17/272 U.S. EPA, Office of Research and Development. (2017d) PPM 13.14 - ORD Implementation of the EPA Quality Assurance Field Activities Procedure. EPA ORD Policy and Procedure Manual (PPM) Chapter 13 - Quality Assurance Policies and Procedures. EPA Office of Research and Development. U.S. EPA. (2018) Scribe Product Overview. U.S. Environmental Protection Agency, ERT [Environmental Support Team] Software Support. https://www.ertsupport.org/Scribe (last accessed 09/09/2020). World Health Organization (WHO). (2017) Guidance on Regulations for the Transport of Infectious Substances 2017-2018, World Health Organization: Geneva, Switzerland. (WHO/WHE/CPI/2017.8). https://apps.who.int/iris/bitstream/handle/10665/254788/WHO-WHE- CPI-2017.8-eng.pdf (last accessed 09/28/2020). 29 ------- Appendix A: Applicable Microbiological Agents for this Document This document is applicable for sampling porous surfaces potentially contaminated with Bacillus anthracis spores using a 37-mm filter cassette, air sampling pump, and microvacuuming techniques. Although testing has not been completed and efficiencies are unknown, these procedures may also be applicable to other microbiological agents listed in the Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (SAM) (U.S. EPA 2017) document (see Tables A-l and A-2 for list of applicable microbiological agents) that provides a compendium of analytical methods that have been selected specifically for use during environmental response activities, by work groups consisting of methods experts from within EPA, as well as other federal, state and local agencies, and public utilities.4 SAM identifies a single selected method or suite of methods for each analyte/sample type. This document is to be used in conjunction with laboratory analysis methods listed in SAM. Table A-l. Example Subset of Bacterial Microbiological Agents1 Bacteria Disease Typical Exposure Reservoir in United States 2 Bacillus anthracis Anthrax Mammals, humans and soil Brucella spp. Brucellosis, Undulant Fever Animals and by-products (e.g., contaminated milk) Burkholderia mallei Glanders Not known to occur in U.S. Disease primarily affects animals, although exposure in humans can occur. Burkholderia pseudomallei Melioidosis Water and soil Campylobacter jejuni Campylobacteriosis Food and water Chlamydophila psittaci (formerly known as Chlamydia psittaci) Parrot Fever Pet birds and by-products (e.g., cage debris) Coxiella burnetii Q-Fever Animals and by-products (e.g., contaminated milk) Escherichia coli 0157:H7 Enterohemorrhagic E. coli or EHEC Animals, humans, soil, water and food Francisella tularensis Tularemia, Rabbit Fever Animals, insects, soil, water and vegetation Legionella pneumophila Legionellosis Water Leptospira interrogans Leptospirosis Animals, soil and water Listeria monocytogenes Listeriosis Food Non-typhoidal Salmonella (Not applicable to S. Typhi) Salmonellosis Animals, humans and food Salmonella enterica serovar Typhi (S. Typhi) Typhoid Fever Humans, food and water Shigella spp. Shigellosis Humans, water and food Staphylococcus aureus Staphylococcal Food Poisoning Animals, humans, soil, water and food Vibrio cholerae Cholera Shellfish, humans, water and food Yersinia pestis Plague Animals and insects 1 List from EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (U.S. EPA 2017) fhttps://www.epa. gov/homeland-securitv-research/sam (last accessed 09/29/2020) 2 The occurrence and reservoirs listed for the bacteria are those described by the CDC ditto: //www. cdc. gov/ and http://www.cdc.gov/biosafetv/publications/bmbl5/. last accessed 10/02/2020) 4 For more information on SAM workgroups, see: for more information on the workgroups: https://www.epa.gov/esam/basic-information-epas-selected-analvtical-methods-environmental-remediation-and- recoverv-sam (last accessed 9/29/2020) 30 ------- Table A-2. Example Subset of Viruses, Protozoa and Helminths1 Viruses Adenoviruses: Enteric and Non-enteric (A-F) Astroviruses Caliciviruses: Noroviruses Caliciviruses: Sapovirus Coronaviruses: Severe Acute Respiratory Syndrome (SARS)-associated Human Coronavirus (HCoV) Hepatitis E Virus (HEV) Influenza H5N1 virus Picornaviruses: Enteroviruses Picornaviruses: Hepatitis A Virus (HAV) Reoviruses: Rotavirus (Group A) Protozoa Disease Cryptosporidium spp. Cryptosporidiosis Entamoeba histolytica Amebiasis Giardia spp. Giardiasis Naeqleria fowleri Naeqleriasis Toxoplasma gondii Toxoplasmosis Helminths Disease Baylisascaris procyonis Raccoon roundworm infection 1 EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (U. S. EPA 2017 dittosV/www.epa.gov/homeland-securitv-research/sam. last accessed 10/01/2020) and Canadian governmental agencies ditto://www.ehs.Columbia.edu/Policv2.2.html, last accessed 10/01/2020) References for Appendix A U.S. EPA. (2017) Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017. U.S. Environmental Protection Agency: Washington, DC. EPA/600/R-17/356. https://www.epa.gov/esam/selected-analvtical-methods-environmental-remediation-and-recoverv- sam (last accessed 09/29/2020). 31 ------- Appendix B: Supplemental Plans The sampling strategy developed in support of sample collection activities following a contamination incident needs several site- and incident-specific supplemental documented plans. These documents should be consulted to determine which sample collection techniques to use. These supplemental items are necessary to support the sampling strategy and are addressed below. B.1 Quality Assurance Project Plan The quality assurance project plan (QAPP) is a comprehensive document describing in detail the activities that must be implemented to ensure that the results of the data and information collected satisfy the project performance criteria (U.S. EPA 2000). For a specific incident, it is possible that a QAPP will be developed for the overall incident, and then a more detailed sampling and analysis plan (SAP) could be developed for each specific sampling activity to be conducted. The elements of a QAPP address aspects of project management, quality assurance and quality control (QC), and data collection, production, and use (U.S. EPA 2001). Guidance on the technical requirements of a QAPP is provided in Requirements for Quality Assurance Project Plans EPA QA/R-5 (U.S. EPA 2001) and Guidance on Quality Assurance Project Plans EPA QA/G-5 (U.S. EPA 2002a), which present advice intended to help prepare a QAPP. At a minimum, QAPPs should address the following elements: Project Management - key personnel and their roles; organization chart; project description and background; data quality objectives and criteria for measurement data; documentation and records Data Generation and Acquisition - sampling design, methods, and handling; analytical methods; quality control; instrument and equipment inspection, maintenance, and calibration; data management Assessment and Oversight - assessments and response actions; reports to management Data Validation and Usability - data review, verification, and validation; verification and validation methods B.2 Sampling and Analysis Plan For collection of samples, a well-defined and thorough SAP needs to be developed and implemented. The SAP is specific for the site being evaluated and outlines the sampling and analysis strategies that should be in place prior to initiating the sample collection. The information included in the SAP provides detailed site-specific instructions and requirements that should be used in conjunction with sample collection and analysis. The SAP is important because analytical results can be used by the Incident Command, local health departments, decontamination teams, decision makers, and attorneys. For this reason, laboratories performing the analyses should be consulted regarding sample sizes, containers, and shipment when developing the SAP and prior to sampling. The SAP should also include consideration of data quality objectives (DQO), which are used to ensure that collected data are of known and documented quality for their intended use. Information of specific importance to sampling teams includes: Types of samples to be collected or measurements to be performed (check with analyzing laboratory to see what can be accepted) Target microbiological agent and sample types Potential interferences, including environmental conditions and weather impacts Number of field samples to be collected Amount of material to be collected for each sample Required sample container size and type 32 ------- Sample locations and frequencies Sample collection techniques and procedures Data quality objectives of the sampling and analysis activities Type and frequency of field QC samples to be collected QC requirements and measurement quality objectives Sample preservation and holding time requirements Sample packaging and shipping requirements Documentation requirements Guidance for information to be included when developing a SAP is provided in EPA's Sampling, Laboratory, and Data Considerations for Microbial Data Collected in the Field, EPA/600/R-18/164 (U.S. EPA 2018). This document summarizes elements that should be considered when planning, developing, and implementing the SAP for microbiological contamination incidents in which the EPA would be responsible for supporting sampling and analysis. The SAP template provided in EPA's Interim Draft Outline: Sampling and Analysis Plan for Environmental Sample Potentially Containing Pathogens, EPA/600/R-17/129 (U.S. EPA 2017a) can be used as a "ready to go" outline for creating a SAP and associated data quality objectives. Additional information on sample collection strategies and designs can be found in: Guidance on Choosing a Sampling Design for Environmental Data Collection for use in Developing a Quality Assurance Project Plan, EPA QA/G-5S (EPA 2002b). Additional information on sample preservation, holding times, and packaging and shipping requirements, are included in EPA's Sample Collection Information Document for Pathogens (last accessed 10/01/2020). B.3 Health and Safety Plan Safety is a primary consideration for any sampling event, and the health and safety plan (HASP) should be specific to a site and incident. Each microbiological agent and contamination incident pose specific health hazards, and an incident-specific HASP should be available to sampling personnel. HASPs will vary depending on the site, the sampling phase (site assessment, remediation, or post-decontamination) and the responsible organization. The purpose of these plans is to ensure maximum protection to workers, the environment, and surrounding communities, in a way that is consistent with requirements needed to perform operational activities. HASPs should follow guidelines provided by U.S. Department of Labor Occupational Safety and Health Administration (OSHA) (U.S. DOL 2008). At a minimum, HASPs should include instructions and guidelines regarding: Names, positions, and contact information of key personnel and health and safety personnel and emergency services Site- or incident-specific risk assessment and hazard analysis addressing sample collection activities Training requirements PPE on site and usage requirements Medical screening requirements (maintain confidential documents properly and securely) Site or incident control Emergency response plan, containing off-site emergency contact information such as local hazardous materials response teams or additional trained rescue personnel (U.S. DOL 2002) Entry and egress procedures Spill containment Personnel decontamination procedures 33 ------- Personnel safety requirements and considerations for a particular site might extend beyond concerns related to exposure to microbiological agents and can include exposure to physical hazards and chemicals that are toxic, corrosive, emit harmful or explosive vapors, or are incompatible when mixed. General health and safety considerations that should be considered when implementing sample collection described in this document are provided in Section 4.0. B. 4 Analytical Protocols and Laboratories Analytical protocols describe the methods that will be used in the laboratory to analyze the collected samples. These protocols often include information that should be considered by individuals collecting samples (e.g., the types of QC samples required, sample holding times and conditions, use of dechlorinating or neutralizing agent, and sample sizes). Analytical protocols also often include procedures that might be required to prepare various sample types prior to implementing procedures for microbiological agent detection and measurement. Sample collection procedures described in this document are intended to be used in conjunction with the analytical methods that are included in EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (U.S. EPA 2017b; last accessed 10/01/2020). Sampling personnel should consult the SAP for specifications on the following sampling requirements that affect subsequent sample analysis: Allowable sample holding times Required sample volumes and containers Preferred sampling device and collection reagents (e.g., wetting agents, selective media) Sample packaging and shipping/delivery requirements QC samples Sample decontamination procedures B.5 Waste Management Plan A Waste Management Plan (WMP) that outlines waste management requirements, procedures, strategies, and processes from the point of generation to final deposition should be in place prior to an incident. Ideally, a general WMP will be in place that can be used to prepare an incident-specific WMP. This incident-specific plan should address federal, state and local waste management requirements for the different waste streams, waste characterization and waste acceptance sampling and analysis, identification of waste management facilities, on-site waste management and minimization strategies and tactics, off- site waste management, waste transportation, health and safety, as well as tracking and reporting of waste sampling results. State and local waste management officials should be contacted as early in the development process as possible. For more information on WMPs, see EPA's Waste Management Benefits, Planning and Mitigation Activities for Homeland Security Incidents website (available at: https://www.epa.gov/homeland-securitv-waste/waste-management-benefits-planning-and-mitigation- activities-homeland. last accessed 10/01/2020). References for Appendix B U.S. DOL. (2002) Emergency Action Plans. 29 CFR 1910.38. U.S. Department of Labor, Occupational Safety and Health Administration: Washington, DC. U.S. DOL. (2008) 29 CFR 1926.65. Hazardous Waste Operations and Emergency Response. U.S. Department of Labor, Occupational Safety and Health Administration: Washington, DC. U.S. DOL (2008) Hazardous Waste Operations and Emergency Response. U.S. Department of Labor, Occupational Safety and Health Administration: Washington, DC. OSHA 3114-07R. 34 ------- U.S. Environmental Protection Agency (U.S. EPA). (2000) EPA Quality Manual for Environmental Programs, CIO 2105-P-01-0. U.S. Environmental Protection Agency: Washington, DC. U.S. EPA. (2001) EPA Requirements for Quality Assurance Project Plans, EPA QA/R-5. U.S. Environmental Protection Agency: Washington, DC. EPA/240/B-01/003 U.S. EPA. (2002a) Guidance for Quality Assurance Project Plans, EPA QA/G-5. U.S. Environmental Protection Agency: Washington, DC. EPA/240/R-02/009 U.S. EPA. (2002b) Guidance on Choosing a Sampling Design for Environmental Data Collection for Use in Developing a Quality Assurance Project Plan, EPA QA/G-5S. U.S. Environmental Protection Agency: Washington, DC. EPA/240/R-02/005 U.S. EPA. (2017a) Interim Draft Outline: Sampling and Analysis Plan for Environmental Samples Potentially Containing Pathogens. U.S. Environmental Protection Agency: Cincinnati, OH. EPA/600/R-17/129 U.S. EPA. (2017b) Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017. U.S. Environmental Protection Agency: Washington, DC. EPA/600/R-17/356 U.S. EPA. (2018) Sampling, Laboratory, and Data Considerations for Microbial Data Collected in the Field. U.S. Environmental Protection Agency: Cincinnati, OH. EPA/600/R-18-16 35 ------- Appendix C: Example Chain of Custody Form 0% \jV JJ EPA USEPA Sample Traffic Report & Chain of Custody Record Case No.: DAS No.: SDG No.: Date Shipped Chain of Custody Record: Sample Collector Signature: For Lab Use Only Carrier Name Relinquished By: (Date/Time) Received By: (Date / Time) Lab Contract No.: Airbill: 1) Unit Price: Shipped To: 2) Transfer To: 3) Lab Contract No.: 4) Unit Price: Sample Identification Code Sample Collector Matrix / Type Volume / Mass Analysis Required Sampling Location / Sample Depth Date / Time Laboratory Sample No. FOR LAB USE ONLY Sample Condition on Receipt 1 2 3 4 Additional Sample Collector Signature(s): Sample(s) to be used for laboratory QC? Temperature Upon Receipt: Chain of Custody Seal Number: Shipment Iced? (Yes/No) Custody Seal Intact? (Yes/No) Matrix codes: SO - Soil; DW - Drinking Water; AF - Air Filter; AI - Air Impinger; P - Particulate; WI - Wipe; SW - Swab; DCW - Decontamination Wastewater; 37F -37 mm Filter DAS: Delivery as Analytical Services SDG: Sample Delivery Group 36 ------- 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 ------- |