EPA/600/R-21/007 | May 2021 www.epa.gov/emergency-response-research United States Environmental Protection Agency oEPA Collection of Air Samples Potentially Contaminated with Microbiological Agents Using Impingers, Impactors, and Low-Volume Filters Office of Research and Development Homeland Security Research Program ------- EPA/600/R-21/007 May 2021 ฃ i \ \X! O Collection of Air Samples Potentially Contaminated with Microbiological Agents Using Impingers, Impactors, and Low-Volume Filters 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 has undergone 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 methods 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 collection of collection of air samples potentially contaminated with microbiological agents using low- volume filter, impactor (collected on agar plates), and impinger sampling devices. 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 Sales, Director Center for Environmental Solutions and Emergency Response Hi ------- Acknowledgments This document was developed by the U.S. Environmental Protection Agency's (EPA) Homeland Security Research Program (HSRP) within EPA's Office of Research and Development under 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 Katherine Ratliff Shannon Serre US EPA Quality Assurance Ramona Sherman External Peer Reviewers Nancy Burton, Centers for Disease Control and Prevention (CDC), National Institute for Occupational Safety and Health (NIOSH) Ginger Chew, CDC Brett Green, CDC NIOSH Technical Editing Marti Sinclair, General Dynamics Information Technology, Inc. iv ------- Executive Summary This document provides step-by-step instructions for the collection of air samples potentially contaminated with microbiological agents (including the bacteria, viruses, protozoa, and helminths listed in EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017) using low-volume filter, impactor (collected on agar plates), and impinger sampling devices. Impingers draw airborne particles through a tube into liquid collection medium for culturing or molecular analysis while impactors draw airborne particles through a slit or sieve to be collected on a solid or adhesive media. This document is intended to be used in conjunction with the analytical methods listed in EPA's SAM document and in the Environmental Sampling and Analysis Method Program online query tools for SAM. Information is provided on: the materials and equipment needed for sample collection; the assembly of sampling kits; step-by-step instructions for taking field and quality control samples; and sample packaging, storage, and transport. 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. ------- Acronyms CDC U.S. Centers for Disease Control and Prevention CFR Code of Federal Regulations COC chain of custody DGR Dangerous Goods Regulations DOL U.S. Department of Labor DOT U.S. Department of Transportation EHEC Enterohemorrhagic Escherichia coli EPA U.S. Environmental Protection Agency ESAM Environmental Sampling and Analysis Methods Program GPS Global Positioning System HASP health and safety plan HAZMAT hazardous material HAZWOPER Hazardous Waste Operations and Emergency Response HEPA high efficiency particulate air IATA International Air Transport Association i.d. inner diameter ID [sample] identification LRN Laboratory Response Network MCE mixed cellulose ester NIOSH National Institute for Occupational Safety and Health o.d. outer diameter OSHA Occupational Safety and Health Administration PC polycarbonate PCR polymerase chain reaction PPE personal protective equipment PTFE polytetrafluoroethylene PVC polyvinylchloride QA quality assurance QAPP quality assurance project plan QC quality control SAM Selected Analytical Methods for Environmental Remediation and Recovery 2017 SAP sampling and analysis plan SCID Sample Collection Information Document UN United Nations WMP waste management plan ------- Table of Contents Disclaimer ii Foreword iii Acknowledgments iv Executive Summary v Acronyms vi I.0 Scope and Application 1 2.0 Limitation and Interferences 2 3.0 Health and Safety Considerations 3 4.0 General Considerations for Collection of Air Samples 4 4.1 General Considerations 4 4.2 Sampling Techniques 5 5.0 Waste Management 8 6.0 Sample Documentation 9 6.1 Sample Identification 9 6.2 Sample Labels 9 6.3 Sample Documentation Information 10 6.4 Sample Control and Chain of Custody (COC) 10 6.5 Custody Seals 11 7.0 Sampling Equipment and Supplies 12 7.1 Sampling Equipment 12 7.2 Filters/Button Samplers 13 7.3 Agar Plate Impactor Supplies and Reagents 13 7.4 Liquid Impinger Supplies and Reagents 13 7.5 General Supplies (required for each sampling device) 14 7.6 Sample Transport Containers and Packing Materials 14 8.0 Preparation for Sample Collection 15 8.1 Sampling Teams 15 8.2 Techniques to Minimize Potential Cross Contamination 16 8.3 Sampling Kits 16 8.4 Calibration Kits 20 8.5 Decontamination 22 8.6 Equipment Blanks 22 9.0 Quality Control Samples 23 9.1 Field Blanks 23 9.2 Transport Blanks 23 9.3 Equipment Blanks 23 9.4 Media Blanks 24 10.0 Sample Collection 24 10.1 Air Filters/Samplers 25 10.2 Agar Plate Impactors 27 10.3 Liquid Impingers 30 10.4 Sampler Disassembly 32 II.0 Sample Packaging and Transport 34 11.1 Sample Holding Time and Temperature 34 11.2 Sample Container Transport and Labeling 36 11.3 Sample Packaging 36 11.4 Transfer of Custody 37 vii ------- 12.0 References 39 Appendix A: Applicable Microbiological Agents 43 Appendix B. Supplemental Plans 45 Appendix C. Example Chain of Custody Form 49 Tables Table 4.1 Air Sampling Techniques 5 Table 9.1 Field Blanks 23 Table 10-1 Summary of Air Sampling Approaches 25 Table 11-1. Transport Conditions and Holding Times 34 Figures Figure 8.1. Example impactor calibration set up 21 Figure 8.2. Example impinger calibration set up 22 viii ------- 1.0 Scope andApplication 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 the collection and analysis of large amounts of samples. The resulting data from the samples provide important scientific data needed to support evidence-based decisions to determine 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 one-stop 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 2017 (SAM) (SAM) (U.S. EPA 2017a) provides a compendium of analytical methods that have been selected specifically for use during environmental response activities, by workgroups 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 ISCIDI for Pathogens; Chattopadhvav 2017) provides complementary information on sample containers, preservation, size and packaging, and additional resources that support collection of samples to be analyzed specifically for the selected microbiological agents, using the methods listed in SAM. This document provides step-by-step instructions for the use of impingers, impactors and low-volume filter sampling devices to collect samples from air potentially contaminated with select microbiological agents. Impingers draw airborne particles through a tube into liquid collection medium for culturing or molecular analysis while impactors draw airborne particles through a slit or sieve to be collected on a solid or adhesive media. For both impingers and impactors, air sample volumes can be calculated, and quantitative sampling results produced. These instructions are intended to be used in conjunction with the analytical methods listed in SAM (Appendix A), 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 including step-by-step instructions for taking field and quality control (QC) samples and information on sample packaging, storage, and transport. In summary, this document: Is applicable for collection of samples of indoor air potentially contaminated microbiological agents listed in EPA's SAM (see Appendix A). Collection efficiencies of these methods have not been fully determined for all agents listed in Appendix A. Provides instructions for devices that can be used to collect viable and/or non-viable pathogens, depending on the media used. 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. 1 For more information on SAM microbiological agents, methods and workgroups, see: https://www.epa.gov/esam/basic-information-epas-selected-analvtical-methods-environmental-remediation-and- recoverv-sam (last accessed 9/29/20201. 1 ------- Is intended to support collection of environmental samples at the point where remediation activities are turned over to the EPA and 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, 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, number of samples they will accept, and whether the samples can be processed and analyzed according to the SAM. Although additional media are available and high-volume sampling devices are available, this document focuses on the use of agar plate media for sample collection using impactors and on low-volume sampling using button samplers. SAPs (and other site- and incident-specific plans and procedures) should be consulted to determine if additional procedures and databeyond what is discussed in this documentare needed. Additional needs could include the need for additional sampling for QC purposes or to ensure an accurate count when low concentrations of microbiological agents are suspected. In addition, SAPs (and all) should be consulted to determine additional modifications to plans and procedures are needed to accommodate laboratory capacity, target agent, incident background information, data quality objectives, and sample locations and amounts. 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) 2.0 Limitation and Interferences This document includes information based on sampling techniques that were available at the time of publication and not all procedures or protocols which have been included in this document 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 listed in Appendix A, as well as collection efficiencies for agents other than Bacillus anthracis. The document is expected to be updated to include advances in technologies and results of validation studies on a periodic basis. Factors that can influence collection of pathogens in indoor environments include relative humidity, temperature, oxygen levels, indoor pollutants, sampling flow rate/face velocity, concentration (breakthrough capacity/breakthrough volume), and indoor atmospheric stability (degree to which the atmosphere can dampen vertical and horizontal motion). Stable ambient conditions generally result in low dispersion, and unstable ambient conditions (for example, hot conditions) generally result in higher dispersion. Appendix D lists the quality assurance for development of this protocol. 2 ------- 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. In addition to potential harm posed to the individuals involved, unsafe conditions in the field can indirectly impact 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 to be covered are specified in 1910.120(e)(2) and should include specific training on biohazards and microbiological agent awareness. 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; U.S. DOL 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 is responsible for 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; U.S. DOL OSHA 2011), 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 sampling team at the primary sampling site. The HASP(s) must state the requirements for reporting injuries and for examination by medical personnel when injuries occur. PPE - PPE must 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 maximum personal protection and mobility for the task being performed. Sampling personnel must 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 U.S. DOL 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 2009) for selection and use of respirators and PPE for protection against biological agents 3 ------- (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 from contact with potentially contaminated samples. Wearing two pairs of gloves is recommended but should not compromise needed dexterity. Gloves should be changed between samples or whenever they become visibly contaminated or the integrity of the gloves is compromised. o Care should be taken to ensure that PPE is not compromised. If PPE is suspect or is compromised, sample collection must be stopped. Compromised PPE can result in contamination of personnel. o After use, PPE (e.g., gloves, protective clothing) must be placed in sealed bags for appropriate disposal or decontamination. 4.0 General Considerations for Collection ofAir Samples Selection of the technique required for air sampling is based primarily on site-specific sampling objectives and strategies, the analyses to be performed, conditions of the indoor environment, fate and transport of the pathogen, and the physiological characteristics of the pathogen including pathogen size. Other aspects that might be considered include the: concentrations of pathogens and other particulate matter (high levels may overload some samplers, which may lead to shortened sampling time); comprehensive quantitative and qualitative analysis (which might require the use of multiple sampling and analytical methods); and practical constraints (such as spatial restrictions, proximity to the source, proximity to the ventilation systems, and other logistical considerations). 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: number, type, and location 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) specific areas to be sampled sample receipt requirements 4.1 General Considerations Air samplers are designed for sampling under various lengths of sampling cycles and temperatures. Most air sampling equipment consists of a minimum of a sampling pump with an air flow inlet and includes the use of a collection medium (e.g., filters, agar plate or collection fluid). Use of isokinetic sampling techniques (velocity of air flow into the sampler nozzle is equal to the velocity of the air that has not been disturbed) help prevent overrepresentation of larger particles (Wilcox 1965). Sampling parameters include: Constant flow rates Stable ambient conditions (humidity and temperature) Appropriate flow rates (sufficient to collect and retain target organisms) 4 ------- 4.2 Sampling Techniques Table 4.1 provides general information regarding the sampling techniques/devices discussed in this document, along with their potential uses and potential problems or considerations. In general, liquid impingers can be used for the collection of most pathogens, including viruses, while filters and impactors are typically used to collect and culture bacterial pathogens. Additional information on these types of sample collection devices can be found in Haig et al. 2016. Table 4.1 Air Sampling Techniques S;i in pi ill vi l)c\ ice Description ;ind Poicnlhil I scs Pulcnliiil Problems or (unsi(lcr;ilions Filters (designed to collect bacteria and viruses) Airborne particulates are captured on filters by impaction or diffusional forces Typically used for <10 |im and <2.5 |im fractions Common and robust form of high-volume sampling at low cost Filter types: Gelatin filters (3|im pore size) are used to collect microbiological agents for microscopic, culture and other assays. Gelatin filters dissolve easily onto agar plates or into liquid for analysis. Collection times range from 20 to 45 minutes at 1 to 2 L/minute* Mixed cellulose ester (MCE; 0.45- to 5.0-|im). polytetrafluoroethylene (PTFE; 0.5- to 5.0-|im) or polycarbonate (PC; 0.4- to 0,8-|im) filters are designed to collect microbiological agents for microscopic and molecular assays. Collection times range from approximately 15 minutes to 8 hours at 1-4 L/minute* Negatively impacted by high temperature Airborne particulate matter other than the target analyte can block filters, interfering with airflow and reducing overall sample volume Gelatin filters are negatively impacted by high humidity and should not be used past the recommended sampling times Exposure of gelatin filters to air can reduce bacterial survivability Gelatin filters are dry and brittle when placed into and removed from samplers and can easily be broken Use of dry filters can impact viability of some microbiological agents PC filters tend to be electrostatic and can be difficult to install in the sampling device Contaminant concentration and particle size can affect collection efficiency Sampling time can affect physical extraction efficiency during analysis Impactors Airborne particulate matter is drawn into an impactor and collected on various media (e.g., agar plates, filters, greased/mineral oil plates, adhesive backed tape/microscope slides, scanning electron microscopy stubs); samples collected using agar plates can be used for collecting and enumerating viable bacteria Many varieties are available (e.g., BioStageฎ impactor, multi-stage Anderson cascade impactor, or equivalent) Multi-stage cascade impactors provide stage- specific particle size separation Sampling rate is approximately 28 L/minute (typical)* Widely used to define particle size distribution Require proper sterilization before use Require trained personnel to operate Low humidity and/or high temperatures will reduce the length of time that agar plates can be used due to potential for desiccation Agent-specific media may have a short shelf life High cost, especially for high volume sampling Not ideal for low concentration sampling as total sampled mass is divided among multiple stages May not support assessment of virus infectivity For non-spore forming pathogens, collection media other than agar plates may not support viability assessments Back pressure could potentially cause the pump to suck up impinger liquid into the tubing and pump. However, pumps should be equipped with filters and back pressure gauges to prevent this from happening. Impingers Airborne particulate matter is collected in liquid media that can be used for culturing or molecular analysis 10-14 L/minute* Require proper sterilization before use Require trained personnel to operate Negatively impacted by high temperatures and humidity 5 ------- S;i in pi ill vi l)c\ ice Description ;iihI Poien 1 i;i 1 I ses Polcnlhil Problems or ( onsidernlions Efficiency drops in low-volume glass impingers for collection of particles < 1 |im Low cost for low flow glass impingers Specific media can have a short shelf life. Inertial forces and impact can injure fragile vegetative bacteria if using collection flow rates greater than that recommended by the manufacturer Extended collection times may negatively impact viability and reduce collection efficiency due to fluid evaporation Glass impingers are fragile and must be handled with care If a cascade impactor is used, the final stage includes a filter which should serve to collect bioaerosols and prevent contamination of the pump and tubing. Some pumps also include filters as well. If a single stage impactor such as the SKC BioStage is used, the collection media is an agar plate which is seated into the BioStage which should prevent contamination of tubing and pump. MCE, mixed cellulose ester; PC, polycarbonate; PTFE, polytetrafluoroethylene * Flow rates are based on manufacturer's recommendations and literature references (Cao et al. 2011; Burton et al. 2005; Grinshpun et al. 1997; Grinshpun et al. 2017; Haig et al. 2016; Wang et al. 2015) and are provided for information only; actual sampling flow rates depend on the equipment used (e.g., filter size, pore size). In some instances, higher flow rates and longer sampling durations can be used to increase sample volume and should be based on instructions provided in the SAP or from the manufacturer. 4.2.1 Air Filters/Cassettes Low-volume air filtration devices (e.g., personal aerosol samplers) are generally used for collection of bacterial pathogens. Air filtration devices (filter holders, cassettes) use filters as collection media, which are available in multiple "dry" filter types: gelatin, mixed cellulose ester (MCE), polytetrafluoroethylene (PTFE), and polycarbonate (PC). Use of "dry filters" could impact the viability of some pathogens (e.g., non-spore forming bacteria). As a result, these filters should be analyzed using analytical techniques other than culture, such as polymerase chain reaction (PCR) and immunological assays to detection microbiological organisms. In addition, exposure of gelatin filters to air can reduce bacterial survivability (Koller and Rotter 1974). Burton et al. 2005 noted collection efficiencies for Bacillus subtilis var. niger endospores in air filter samples to be 94% or greater using MCE, 1 |_im PTFE, and gelatin filters. PC filters and 3 (.un PTFE filters had collection efficiencies of 61.4% and 63.6%, respectively (Burton et al. 2005). Another study noted >99.95% collection efficiency when gelatin filters were used to collect particles 0.5-3.0 (im in size (Koller and Rotter 1974). Samples can be collected using MCE, PTFE and PC filters for longer periods of time (up to 8 hours) for collection of low concentrations of bacteria. However, relative culturability and physical extraction efficiency can be affected by sample collection time used (see Koller and Rotter 1974 and Burton et al. 2005 for more information). Although this document focuses on the use of low-volume filtration devices, high-volume dry filter units (e.g., DFU-1000 dry filter unit) and portable sampling units (e.g., PSU-2) also are available.2 2 The use of portable sampling units is discussed in Garza, A.G. et al., "Detection of the Urban Release of a Bacillus anthracis Simulant by Air Sampling" (November 2014) and Van Cuyk, S. et al. "Transport of Bacillus thuringiensis var. Kurstaki Via Fomites" (November 2011) 6 ------- 4.2.2 Impactors Impactors with agar plate collection media can be used for the collection of viable bacterial pathogens, which can be directly enumerated after incubation of the agar plate. Collection media other than agar plates (e.g., greased plates, filters, tape) can be analyzed using molecular or immunological assays for non-spore forming bacteria and might support viability assessments of spores. Impactors are made of metal, used for short lengths of time (10-30 minutes) at a moderate airflow rate (approximately 28 L/minute), and use multiple collection media (e.g., agar plates, filters). Agar plates or other collection media (e.g., filters, greased plates, tape) are used in single- stage impactors, whereas multiple agar plates or collection media are stacked in multi-stage (e.g., six-stage) cascade impactors. The jet holes on each stage of the multi-stage impactors are successively smaller, allowing for sizing of airborne particulates, based on their aerodynamic diameter. For example, multi-stage or cascade impactors, such as the Andersen six-stage cascade impactor, provides stage-specific size separation and collection of particulates (e.g., 7 micrometers [Stage 1], 3.3 micrometers [Stage 3], 2.1 micrometers [Stage 4], 1.1 micrometers [Stage 5], and 0.65 micrometers [Stage 6]). Positive hole corrections should be used to adjust calculations for multiple impactions through a single hole (see Macher 1989 for more information). Ideally, the agar plates used will be specific (i.e., selective media) for the target bacteria; however, non-selective media can be used if selective media are not available for the target bacteria. The SAP should specify the appropriate sample collection media to be used. 4.2.3 Liquid Impingers Liquid impingers can be used for the collection of most pathogens, including viruses. Traditional liquid impingers are made of glass and are used for short lengths of time (< 20 minutes); used with low airflow rates; and use water, mineral oil, or a buffer as the sample collection fluid. The use of liquid impingers minimizes the potential desiccation of microbiological agents, making it possible to assess pathogen viability using culture techniques. Evaporation of impinger liquid over increased sampling times can lead to particle bounce and reduced collection efficiency. Sampling personnel should consult the SAP regarding the appropriate collection fluid, which is based on the target pathogen and the analytical technique to be used. 4.2.4 Sampling Pumps A calibrated sampling pump with an air flow inlet and power source is needed in conjunction with the air filtration device, impinger or impactor. Sampling pumps used to collect samples must be compatible with the sampling needs and the media used and must be capable of maintaining the desired flow rate throughout sampling period. Calibration of pumps should be performed before and after use (McCammon 1998). 7 ------- 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 are 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. 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).3 In general: Excess sample materials, reference materials, accumulated waste, and equipment that will not be reused must be placed in appropriate waste container(s) 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 unused impinger fluid or agar plates, can be retained for additional sampling or shipped to the laboratory with each batch of samples. Unused and uncontaminated sample collection materials can serve as QC samples (field blanks [see Section 9.1] and transportation blanks [see Section 9.2]) and provide information to determine if analytical results might be impacted by interferences resulting from the equipment used [Section 9.0].) Media blanks are used to assess the sampler for contamination. 3 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) 8 ------- 6.0 Sample Documentation Documentation associated with sample collection is necessary for understanding and evaluating the analytical results associated with each sample. It is also necessary for validating those results, documenting the protocols used, documenting sampling conditions and names of individual sampling personnel, and tracking the sample during transport to ensure sample integrity. This section summarizes some of the key components of documentation that should be implemented and maintained by individuals involved in 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 2017c). 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, and was designed to capture sampling data, observational information, monitoring field data and analytical data. Documentation produced during collection and processing of samples are considered a legal record by the sampling team and EPA. Training is required for samplers in order to accurately generate/maintain legal records. Legibility and permanence must be maintained. If an error is made, the error should either be struck out using a single line, initialed, and dated, or re-written and checked for accuracy, initialed, dated, and attached to the original documentation for record keeping. 6.1 Sample Identification Each sample collected must include an identification (ID) label, including QC samples. Each field and QC sample 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 record 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, sample collector and associated QC samples. Determination of 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 and should include 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 (a type of bar code) that tracks the sample information, can be affixed to each container. Cryostorage labels (e.g., from Thomas Scientific, Swedesboro, NJ) can be used for prolonged storage. 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 sample collected Sample matrix (e.g., air) Sample volume (flow rate and collection time) Preservation, if applicable Sample collection location (Global Positioning System [GPS] coordinates or brief description) 9 ------- Signature or initials of the sample collector 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. 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 used 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 but is not limited to 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 written on the back of the photograph. 6.4 Sample Control and Chain of Custody (COC) Once samples are collected, they must be maintained under controlled and secured conditions until transported to the laboratory. This control is required to ensure that samples are not compromised, and that analytical data are representative of site conditions. 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. 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 should account for each 10 ------- individual sample. EPA policy is to use Scribe wherever practical to generate COC forms.4 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 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. 4 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). 11 ------- 7.0 Sampling Equipment and Supplies Samples collected in response to a microbiological contamination incident are collected using dedicated and sterile (e.g., filter devices, impactors and impingers) or decontaminated (e.g., pumps and tubing) sampling equipment to minimize interferences and cross-contamination. In some cases, pre-packaged equipment and materials are available and can be used. This section provides general information regarding requirements and considerations associated with the equipment and materials needed for sample collection. 7.1 Sampling Equipment Note: Equipment cited as "sterile" throughout this section, should be certified as sterile by the manufacturer or be sterilized following manufacturer's instructions for sterilization prior to use. Sampling equipment should be operated in accordance with manufacturers' operating 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 air sampling includes the following: Filters/Personal Aerosol Samplers o Button Personal Inhalable Aerosol Sampler (SKC Catalog No. 225-360, or equivalent). Note: While the Institute of Occupational Medicine Inhalable sampler (SKC Catalog No. 225-76A, or equivalent) or other personal inhalable aerosol samplers could also be used for sampling, this document only includes instructions for button samplers. Selection of the type of sampler to use is dependent on the microbiological agent being sampled and corresponding pore size needed for sampling. In addition, instructions for sampling kit assembly (section 8.3.1), sample collection (10.1), and flow rate would need to be adjusted according to the sampler chosen if a sampler other than the button sampler were to be utilized with this document, o Calibrated sampling pump capable of flows up to >5 L/minute (e.g., AirChekฎ XR5000 air sampling pump [SKC Catalog No. 210-5000, or equivalent], Gilian 12 air sampling pump [Sensidyne], or equivalent) and power source o Calibration adapter (SKC Catalog No. 225-361, or equivalent) Agar Plate Impactors o Calibrated sampling pump capable of flows up to 30 L/minute (e.g., QuickTake 30ฎ high flow pump [SKC Catalog No. 228-9530A], or equivalent) and power source o Impactor - Sterile, calibrated single-stage (e.g., SKC BioStageฎ impactor, Catalog No. 225-9611, or equivalent) or multi-stage (e.g., Six-Stage Viable Andersen cascade impactor, Thermo Scientific Catalog No. 10830-EPD, or equivalent). Note: A sterile, pre-packaged single-use impactor also is commercially available for clean rooms and controlled environments but has not been evaluated for the intended use described in this document (e.g., BioCaptฎ impactor, particle measuring systems, or equivalent), o Calibration adapter (e.g., SKC P33100, or equivalent) to be used with rotameter Liquid Impingers o Calibrated sampling pump capable of flows up to 30 L/minute (e.g., QuickTake 30 high flow pump [SKC Catalog No. 228-9530A], or equivalent) and power source o Sterile calibrated liquid impinger (SKC BioSamplerฎ impinger, Catalog No. 225-9595, AGI-30, or equivalent) and pump attachment 12 ------- General Equipment o Calibrated field rotameter (SKC Catalog No. 320-100, or equivalent) or electronic dry cell calibrator (SKC Catalog No. 717-530H, or equivalent) capable of reading of at least 30 L/minute o High efficiency particulate air (HEPA) filter (Pall Life Sciences Catalog No. 12144, or equivalent) for pump exhaust outlet o Timer Sampling kits are prepared prior to field sampling (see Section 8.3.1) and consist of the following components as described in Sections 7.2-7.4. 7.2 Filters/Button Samplers Sterile filters - Note: Filter pore sizes are provided as examples (Cao et al. 2011; Grinshpun et al. 2017; Wang et al. 2015) pore sizes should be selected based on the target pathogen(s). o Gelatin filters (pre-sterilized gelatin [water soluble] filter, 25 mm [SKC Catalog No. 225- 9551, or equivalent] o MCE 0.45- to 5.0-(im 25-mm filters (e.g., \.2-\im, 25-mm filters [SKC Catalog No. 225- 1912, or equivalent]) o PTFE 0.5- to 5.0-(im 25-mm filters (e.g., 3.0-(im, 25-mm filters [SKC Catalog No. 225- 1711, or equivalent]) o PC 0.4- to 0.8-(im, 25-mm filters (e.g., 0.8-(im, 25-mm filters [SKC Catalog No. 225- 1601, or equivalent]) Tygonฎ tubing, 1/4-inch inside diameter (i.d.), 3/8-inch outside diameter (o.d.) (SKC Catalog No. 225-1345, or equivalent) Sterile forceps Sterile, sealable, leak-proof containers (e.g., 50-mL, Fisher Scientific Catalog No. 06-443-20, or equivalent) - for filters Sealing tape (e.g., Parafilmฎ, or equivalent) 4" x 6" clean, sealable plastic bags 1-quart clean, sealable plastic bags 1-gallon clean, sealable plastic bags 7.3 Agar Plate Impactor Supplies an d Reagents Sterile agar plates containing selective (laboratory-prepared, agent-specific) or non-selective (e.g., Trypticase soy agar [BBL Catalog No. 221185, or equivalent] or blood agar [BBL 221239, or equivalent]) media for each stage of the impactor Clean, flexible tubing (e.g., Nalgeneฎ, Tygon, or equivalent) Tygonฎ tubing, 1/4-inch i.d. (SKC Catalog No. 225-1345, or equivalent) Tygonฎ tubing, 3/8-inch i.d. (SKC Catalog No. 225-1351, or equivalent) Luer adapters for 1/4 and 3/8-inch tubing 1-quart clean, sealable plastic bags 7.4 Liquid Impinger Supplies and Reagents Sterile impinger collection fluid (e.g., mineral oil [Sigma-Aldrich Catalog No. M5310, or equivalent], distilled reagent grade water, or 10 mM phosphate buffer, pH 7.4 [Sigma-Aldrich P3619-1GA, or equivalent], diluted 1:100 into reagent grade water and filter sterilized) Sterile silicon stopcock grease 13 ------- Sealing tape Tygonฎ tubing, 1/4-inch i.d., 3/8-inch o.d. (SKC Catalog No. 225-1345, or equivalent) for attaching rotameter for calibration Silicon tubing, 3/8-inch i.d., 5/8-inch o.d. (Cole Parmer Catalog No. 95802-20, or equivalent) 3/8 to 1/4-inch reducer/adapter Liquid trap (e.g., Impinger trap [SKC Catalog No. 225-22, or equivalent] or plastic Erlenmeyer flask [Thermo Scientific Catalog No. 41030250, or equivalent] with No. 6 two-hole stopper [Thomas Scientific Catalog No. 1145C75, or equivalent) Stainless steel single or double impinger holders (e.g., SKC Catalog No. 225-20-01 or 225- 20-02, or equivalent, for AGI-30; SKC Catalog No. 225-9606, or equivalent, for BioSamplerฎ impinger; or ring stand with clamps sufficient to hold impinger during sampling) Sterile, sealable, leak-proof sample container (e.g., 50-mL centrifuge tubes, Fisher Scientific Catalog No. 06-443-20, or equivalent) Clean plastic sheeting (large enough to set equipment on without contaminating it) Impinger transport container (e.g., Pelican case with foam interiors) 7.5 General Supplies (required for each sampling device) New, unused powder-free nitrile gloves for the sample collector, supplier, and support person PPE (refer to the SAP and HASP for required PPE for the sampling event) Pre-printed labels for sample container (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) 7.6 Sample Transport Containers and Packing Materials Transport container - Rigid, insulated cooler able to withstand an internal pressure of 14 pounds per square inch (psi), with a secure, sealable lid. 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) 14 ------- 8.0 Preparation for Sample Collection Adequate and appropriate preparation for sample collection is critical to ensuring that representative samples are collected properly and as needed to meet analytical requirements, as well as 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, including designation of sampling teams (Section 8.1), use of aseptic techniques (Section 8.2), preparation of sampling kits (Section 8.3), preparation of calibration kits (Section 8.4) (Note that calibration and reliability checks according to manufacturers' instructions should be completed before deployment and use in the field), equipment decontamination (Section 8.5) and preparation of equipment blanks (Section 8.6). Sampling personnel also should work closely with the environmental unit leader or site/project managers to ensure that sampling activities are conducted in accordance with the SAP. 8.1 Sampling Teams Prior to initiating sample collection activities all 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. Sampling teams should have access to a detailed site map to assist in locating sample collection points. Evaluate and prepare sampling equipment and PPE prior to site entry. Equipment that can be prepared outside the sampling area should be preassembled, cleaned, and calibrated (e.g., check flow rates of sample pumps) 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. Understand site egress procedures, which address decontamination and the 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 Sections 8.5. Any sampling effort requiring the collection of multiple samples, particularly those involving hazardous conditions and/or collection of samples containing pathogens, should involve a sampling team consisting of at least two personnel. Additional personnel might be required for large-scale sampling efforts or when site-specific hazards might be encountered. Individual team members must be trained to assume specific activities or duties related to the sampling effort. A three-person sampling team consisting of a collector, a supplier, and a support person, is recommended for air sampling, although the ideal size of the sampling team will depend on the size of the sampling event and the number of samples required. 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 collection apparatus. 15 ------- Supplier - provides the collector with the pre-labeled sample kit and materials needed to collect the sample, helps with the assembly of the collection 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 tracking/safeguarding sample documentation, documenting flow rate and sampling duration, and setting up/maintaining 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). Sampling personnel are required to follow decontamination procedures prior to exiting the contaminated area (hot zone), as instructed in the HASP, to ensure contamination is not spread outside the area. A prescribed level of personal monitoring might also be required. 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 use of clean and dedicated sampling equipment, and appropriate PPE, should prevent cross- contamination of samples during sample collection. As sources of contamination might or might not be obvious, inclusion of the appropriate field quality control (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 appropriate sampling techniques 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. Techniques should be used to collect samples to reduce exposure risk to sampling personnel, to reduce risk of contaminating the samples, or to reduce 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. 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 16 ------- regarding the equipment and materials included in these kits is provided in Sections 1.2-1 A. However, sampling personnel should consult with the incident commander or site/project managers and the SAP to determine what equipment and materials will be required. 8.3.1 Filter Sampling Kit Assembly Sampling kit assembly must be conducted in a controlled clean area, preferably in a laboratory biological safety cabinet, or similar controlled location, using clean disposable laboratory gloves and appropriate PPE. Once prepared, the kits are stored in a clean and dry location prior to use. A unique sampling kit is required for each air sample and QC sample (field blanks and trip blanks). Note: Disposable powder-free nitrile gloves are also included in the sampling box for the supplier, for the sampler, and for support personnel. Steps for sampling kit assembly include: Don new, unused gloves prior to kit assembly. Remove cap from a sterile button sampler. Unscrew the inlet of the sampler counterclockwise until it comes off and remove the PTFE O- ring. Place the 25-mm filter (i.e., MCE, PTFE, PC) on top of the support screen inside the sampler using sterile forceps. Note: Gelatin filters are placed into the button samplers immediately prior to entering the contaminated area. Place the PTFE O-ring on top of the filter, replace inlet, and very gently turn the inlet clockwise until moderately tight. Note: Excessive tightening of the inlet can twist and tear the filter. Replace cap. Attach flexible (e.g., Tygonฎ) tubing, 1/4-inch i.d., onto the outlet of the button sampler. Label a sterile, sealable, leak-proof 50-mL centrifuge tube with a unique sample ID. Label sealable 4"x 6", 1-quart, and 1-gallon plastic bags with the same unique sample ID. Place the following into a sealable, pre-labeled 1-quart bag: button sampler assembly, a 50- mL centrifuge tube, and 4"/ 6" and 1-quart pre-labeled sealable plastic bags. Place the button sampler assembly bag, clean plastic sheeting, and Parafilmฎ in a pre-labeled 1-gallon plastic overpack bag and seal the overpack bag. Prior to sample collection, sampling personnel should check sampling kits to confirm they are complete. 17 ------- Air Filter Sampling Kit Check List Button sampler, sterile, with 25-mm filter (PC, PTFE, MCE) Note: Gelatin filters are prepared and provided separately from the sampling kit and placed in the sampler in a clean area immediately prior to sample collection. Flexible tubing Labeled sterile 50-mL centrifuge tube Labeled sealable 4"* 6" plastic bag Labeled sealable 1-quart sealable plastic bag Labeled sealable 1-gallon plastic overpack bag Clean plastic sheeting Parafilmฎ Sterile forceps 8.3.2 Agar Impactor Sampling Kit Assembly Agar impactor sampling kit assembly must be conducted in a controlled clean area, preferably in a laboratory biological safety cabinet, or similar controlled location, using new and unused powder-free gloves. Once prepared, the kits are stored in a clean and dry location prior to use. A unique sampling kit is required for each field and QC sample. Due to storage requirements (e.g., refrigeration) and relatively short shelf life, agar plates are not included in the sampling kit. Note: Agar plates should not be frozen and cannot be used to culture bacteria if frozen. Agar plates are prepared and provided separately and placed in the impactors during prestaging (see Section 10.2). Steps for sampling kit assembly include: Don new, unused gloves prior to kit assembly. Prepare Tygonฎ tubing 3/8-inch i.d. Prepare Tygonฎ tubing 1/4-inch i.d. Label sealable 1-quart and 1-gallon plastic bags with the same unique sample ID. Place the following into a sealable, pre-labeled 1-gallon bag: a sterile impactor, 1/4 and 3/8- inch tubing, 1/4 and 3/8-inch Luer adapter, a 1-quart pre-labeled sealable plastic bag, Parafilmฎ, clean plastic sheeting, and 1-gallon bags. Agar plates are prepared, stored, and provided separately for transport to the sampling site. Plastic bin for impactor transport. Prior to sample collection, sampling personnel should check sampling kits to confirm they are complete and confirm the number and type of agar plates are appropriate. 18 ------- Agar Impactor Sampling Kit Check List* Sterile impactor Flexible tubing 1/4 and 3/8-inch (i.d.) Luer adapters for 1/4 and 3/8-inch tubing Labeled sealable 1-quart sealable plastic bag Labeled sealable 1-gallon plastic overpack bag Clean plastic sheeting Parafilmฎ 1-gallon plastic bags Agar plates are prepared, stored, and provided separately for transport to the sampling site. 8.3.3 Impinger Sampling Kit Assembly Sampling kit assembly must be conducted in a controlled, clean area, preferably in a laboratory biosafety cabinet or similar controlled location, using new, unused powder-free gloves. Once prepared, the kits are stored in a clean and dry location prior to use. A unique sampling kit is required for each field and QC sample. Steps for sampling kit assembly include: Don new, unused gloves prior to kit assembly. Dispense 20 mL of impinger collection fluid into a sterile 50-mL sterile centrifuge tube and seal with Parafilmฎ. Prepare pieces of silicon tubing to connect impinger to liquid trap and to connect the liquid trap to the sampling pump. Label a sterile, sealable, leak-proof 50 mL centrifuge tube with the unique sample ID. Label a 1-quart and 1-gallon sealable plastic bag with the same unique sample ID. Place the following into a sealable, pre-labeled 1-quart bag: 50-mL centrifuge tube and a 1- quart pre-labeled sealable plastic bag. Place the following into a sealable, pre-labeled 1-gallon overpack bag: sterile impinger, impinger fluid, liquid trap, silicon tubing, clean plastic sheeting, sterile silicon stopcock grease and Parafilmฎ, and seal the overpack bag. Prior to sample collection, sampling personnel must check sampling kits to confirm they are complete. 19 ------- Impinger Sampling Kit Check List Sterile impinger Impinger holder Impinger collection fluid (sealed in sterile centrifuge tube) Silicon tubing Silicon stopcock grease Liquid trap Labeled sterile 50-mL centrifuge tube Labeled sealable 1-quart sealable plastic bag Labeled sealable 1-gallon plastic overpack bag Clean plastic sheeting Parafilmฎ Pelican case for transporting assembled impinger to sampling location 8.3.4 Supplemental Supplies and Materials In addition to the sampling kits, sampling personnel should ensure they have all the additional materials, supplies and equipment needed for sample collection, decontamination, documentation, and packaging activities. Materials needed for documentation of activities, sample tracking and sample packaging should accompany the sampling kits. At a minimum, the following should be made available to sampling teams: Powder-free disposable nitrile gloves for sampler, supplier, and support personnel Pre-assembled sampling kit (one per sample, see Sections 8.3.1- 8.3.3) Pre-assembled calibration kit (see Section 8.4) PPE (e.g., protective clothing, gloves) as required by the SAP and HASP Site maps GPS for tracking sample locations if required by SAP Sampling media: Agar plates, gelatin filters Sampling pump Sample preservation (e.g., self-contained ice or cold packs) Sample documentation (see Section 6.0) Sample packaging supplies (e.g., containers, bubble wrap, clear sealing tape, custody seals) Decontamination materials (see Section 8.5) Extension cord Roll of aluminum foil (to create a "clean" workspace if one is not available in disaster settings) 8.4 Calibration Kits In addition to the sampling kits, sampling personnel must ensure they have all the additional materials, supplies and equipment needed to calibrate each type of sampling device by consulting the SAP and sampling lead. Sections 8.4.1-8.4.3 provide the components required in each calibration kit for the sampling devices included in this document. Note: Equipment and materials in the calibration kits are used only to calibrate the sampling pumps; they are not used for sample collection. Calibration should be completed prior to deployment to the field before use and after use. Example impactor calibration set-up and example impinger calibration set-up are shown in Figures 8.1 and 8.2, respectively. 20 ------- 8.4.1 Filter Calibration Kit Button sampler and appropriate filter medium (e.g.s MCE, PTFE, PC, gelatin filter stored in separate containers) Calibration adapter Rotameter, or wet or dry cell calibration unit Tygonฎ tubing, 1/4-inch i.d. 8.4.2 Agar Impactor Calibration Kit Impactor Agar plate Calibration adapter Rotameter, or wet or dry cell calibration unit Tygonฎ tubing, 1/4-inch i.d. Tygonฎ tubing, 3/8-inch i.d. Two, 3/8- to 1/4-inch (i.d.) Luer adapters Calibration adapter 3/8-inch Tubing Impactor outlet Luer adapter 1/4-inch Tubing Tubing Rotameter outlet 3/8-inch Adapter tubing Figure 8.1. Example impactor calibration set up. 8.4.3 Impinger Calibration Kit Impinger Liquid trap Collection fluid Rotameter, or wet or dry cell calibration unit Tygonฎ tubing, 1/4-inch i.d., 3/8-inch o.d. (attached to the rotameter) Silicon tubing, 3/8-inch i.d. 3/8 to 1/4-inch reducer/adapter 21 ------- DnHaf Reducer/adapter Inlet Vacuum .gauge Tygon tubing f 4- ฉ) Inlet Silicon tubing Flow adjuster Rotameter Inlet Figure 8.2. Example impinger calibration set up. 8.5 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). If tubing or pumps are suspected to be contaminated, it should be disposed of appropriately. Specific procedures for equipment decontamination should be included in the HASP. While it is outside the scope of this document to provide full instructions on decontamination of sampling equipment (i.e., the sampling pumps), the list below provides general considerations. The HASP and/or SAP should designate the personnel responsible for equipment decontamination and should be referred to for specific procedures to be followed for equipment decontamination or transport of contaminated equipment offsite for decontamination. 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 or field sampling task leader 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 with polyethylene liner can be used to contain contaminated PPE, accumulated wastes, containers, or equipment. 8.6 Equipment Blanks In addition to field blanks and transport blanks (which accompany sampling teams in the field, and are used to evaluate potential contamination introduced during sample collection), equipment blanks are collected in a clean and controlled environment and used to confirm sterilization or decontamination procedures for the equipment were effective (see Section 9.3). These blanks are collected prior to using equipment for sample collection and are not taken into the field. Equipment blanks are prepared in a contaminant-free area, by individuals involved in assembling the equipment that will be provided to the sample collection teams. The blanks consist of either sterile rinsate water that has been run over the surface of the equipment or media that has been attached to the equipment and then processed to determine if the sterilization or decontamination procedure was appropriate. 22 ------- 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 air sampling devices and techniques, QC samples typically consist of field blanks, trip blanks, and media blanks. 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. Results of QC samples can be used to provide information regarding the accuracy of both the sampling and analytical procedures. For this reason, 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 sample). Samplers should refer to the SAP to determine how all samples (field and QC) are to be identified and labeled for transport to the laboratory. 9.1 Field Blanks Field blanks are used to monitor contamination that may be introduced into samples during sample collection. If required, 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 air sampling will be dependent on the device/technique as described below in Table 9.1. Table 9.1 Field Blanks Device Field Blank Filters A sterile filter or filter assembly (filter cassette and tubing) is opened and placed in a sterile container, and the container is securely sealed and labeled. For gelatin filters, add 10 mL of sterile distilled water to the container is added to the container. Impactors The medium (e.g., agar plate) is opened and then closed and sealed with sealing tape. The plate is then labeled and placed in a clean sealable bag. Impingers Liquid medium (20 mL) is placed in a sterile sample container and the container is securely tightened, sealed and labeled. 9.2 Transport Blanks Transport blanks are used to monitor contamination that might be introduced into samples during handling and transport. Transport 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 transport blanks (i.e., filters, agar plates, and collection fluid) are not exposed to field conditions. At no time after their preparation are the sample containers (e.g., plastic bags, sterile containers) opened before they reach the laboratory. Note: In some cases, a transport blank might also be used as a media blank by the laboratory. 9.3 Equipment Blanks Equipment blanks are used to assess the adequacy of equipment decontamination and/or ensure the equipment is sufficiently clean for use in sample collection. These blanks are prepared in a contaminant- free area, by individuals involved in assembling the equipment that will be provided to the sample collection teams (see Section 8.6). The blanks consist of either sterile rinsate water that has been run over the surface of the equipment or of media that has been attached to the equipment. The collected rinsate or 23 ------- media is then processed to determine if the sterilization or decontamination procedure was appropriate. 9.4 Media Blanks Media blanks are used to assess the sampler for contamination. If required, these blanks are provided to the laboratory, unopened as provided by the manufacturer. The blanks are shipped directly to the laboratory and not taken into the field. 10.0 Sample Collection This section outlines step by step instructions for the collection of representative environmental air samples to be analyzed for pathogens during site characterization and post-decontamination sampling following a microbiological contamination incident. The sampling techniques provided should not be considered all-inclusive of the techniques that exist but might be more commonly used for remediation during a contamination incident in which EPA is responsible for sample collection. Prior to field sampling, all sampling personnel should be familiar with the incident-specific SAP, HASP and WMP, in addition to the following information in this guide: 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 also must understand their role(s) and responsibilities regarding sample collection and should practice the sampling activities as a team to ensure that consistent and efficient collection of representative samples while taking into account the pattern and duration of sampling. As noted in Section 8.1, in most cases, a three-person sampling team is recommended, consisting of a collector, a supplier and a support person. The number of individuals needed, however, will depend on the size of the sampling event and number of samples being collected. 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. All requirements must be detailed in the incident-specific SAP. However, if there is any uncertainty regarding sampling activities, sampling personnel should contact the environmental unit leader or site/project managers for clarification. Note: The flow rates noted in this section are based on manufacturer recommendations and provided for information only; actual sampling flow rates will depend on the specific equipment used. In some instances, higher flow rates and/or longer sampling durations can be used to increase sample volume and should be based on instructions provided in the SAP or from the manufacturer. A summary of the sample collection approaches described in this section is provided in Table 10-1. 24 ------- Table 10-1 Summary of Air Sampling Approaches Sampling Approach Pathogen Type Description Section Reference Filters Bacteria Particulate matter is are collected on a filter. 10.1 Impactors Bacteria Particulate matter is deposited on the surface of an agar plate. 10.2 Liquid impingers All Particulate matter is collected into a liquid medium. 10.3 10.1 Air Filters/Samplers Instructions for collection of air samples using gelatin, PC, MCE and PTFE filters are provided below. Filters/Button samplers Used for collection of pathogens other than viruses 10.1.1 Materials, Supplies, and Equipment In addition to being familiar with the documentation and information noted above, sampling teams must have access to all necessary sampling equipment prior to entering the area of contamination and initiating sample collection activities. Required equipment and supplies include, but are not limited to: Appropriate PPE (Section 3.0) Powder free nitrile gloves for sample supplier, collector, and support person Sampling kit (Section 8.3) Calibration kit (Section 8.4) Calibrated pump and power source (Section 7.1) Calibrated rotameter or electronic dry/wet cell calibrator (Section 7.1) Timer (Section 7.1) Sample documentation (Section 6.3) Decontamination supplies (Sections 7.5 and 7.6) Sample transport containers and packing materials (Section 7.4) Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample(s) that are to be collected. 10.1.2 Pre-staging Once the sampling team has arrived at the sampling site and determined the required sampling location(s), each team member should don new, unused gloves and ensure sampling equipment is assembled properly and pump flow rates are adjusted as needed for sample collection. 10.1.2.1 Sampler Assembly Filters should be inserted into the sampler in a clean area prior to entry to the site for sample collection using techniques that minimize contamination and the materials included in the sampling kit. Samplers containing these filters can be prepared ahead of time as described in 25 ------- Section 8.3.1. Cap the sampler and place it back it the bag for transport to the sample collection site. 10.1.2.2 Calibration Check The pump flow rate should be checked outside of the contaminated area following instructions in the SAP or from the calibration equipment manufacturer by the personnel in charge of sampling kit assembly or other designated personnel as noted in the SAP. The flow rate of the pump should be checked and adjusted, if needed, to the flow rate that will be used for sample collection, using a complete sampling train (i.e., calibration kit, Section 8.4). Post-sampling flow rates must be checked once sample collection has been completed, and both pre- and post-sampling flow rates should be recorded. General guidelines regarding the flow rates to use during sample collection and calibration are as follows: Gelatin filters and holders: 1-2 L/minute MCE filters and holders: 1-4 L/minute PTFE filters and holders: 1-4 L/minute PC filters and holders: 1-4 L/minute 10.1.3 Collection Once the pump(s) has been calibrated and checked outside of the sampling area, the sampling team proceeds to the sampling location, and follows the procedures described below. Note: New, unused gloves and a sampling train (calibrated pump and sampling kit [sterile filter assembly and clean tubing]) are required for each field and QC sample collected. Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample(s) that are to be collected. I. Equipment: Preparation and Setup Supplier and Collector: Supplier: Support Person: Supplier: Supplier: Collector: Collector: Don or put on new, clean sampling gloves (Note: the support person should also don or put on new, clean gloves if they accidently touch the sample or contaminated area). Obtain a calibrated pump and clean sampling kit from supply bin. Record the sample ID. Place the calibrated pump onto a clean piece of plastic to protect it from contamination. Open the plastic bag containing the sampling kit for the collector to remove the filter assembly and tubing. Remove inlet cap and outlet plug from sampler. Attach clean tubing between the pump inlet and the filter holder/cassette outlet. Note: The orientation of the filter cassettes should be 90 degrees (or slightly less) to the ground to prevent oversampling of larger particles. Depending on the goal of the sample collection event, the sampler can be placed near where the breathing zone of potentially exposed persons might be, or if more general sampling is desired, the samplers can be placed at several different locations at differing heights to provide expanded sampling coverage (Haig et al. 2016). 26 ------- II. Collection Collector: Activate the pump and collect sample for the specified time. Supplier: Start timer. Support Person: Record flow rate and start time. Note: If samples are to be collected for more than 4 hours, re-check the pump at midpoint for appropriate airflow and the filter for blockage. Collector: Once sample collection is completed, turn off the pump. Supplier: Stop timer. Support Person: Record flow rate and stop time. I r Collection of Each Sample Collector: Supplier: Collector: Supplier: Support Person Collector: Collector: All Personnel: Disconnect the tubing from the sampler and replace the inlet cap and outlet plug to seal the sampler contents. Open and hold a pre-labeled clean plastic sealable bag for the collector to place the filter assembly into the bag. Place the filter assembly into the pre-labeled bag and seal the bag. Decontaminate the outer surface of the bag and hand-off to support person (e.g., using Dispatchฎ Wipes). Complete sample documentation and provide the documentation and filter assembly to personnel outside the contaminated area for sampler disassembly (Section 10.4), filter retrieval and packaging. Once all samples have been collected, place used equipment to be decontaminated into a bag and seal the bag. Gather the tubing and any additional contaminated equipment or supplies for proper disposal. Doff sampling gloves prior to moving to the next location. 10.1.4 QC Samples QC samples that might be requested for air sampling include field blanks and transport blanks. 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. Transport blanks (Section 9.2) are supplied as unopened/unused sampling kit assemblies and are transferred and transported along with the field samples. 10.2 Agar Plate Impactors Instructions for collection of aerosol samples using agar plate impactors are provided below. Collection is similar for single-stage and multi-stage impactors. Agar Plate Impactor Used for collection of pathogens other than viruses 27 ------- 10.2.1 Materials, Supplies and Equipment In addition to being familiar with the documentation and information noted above, sampling teams must have access to all necessary sampling equipment prior to entering the area of contamination and initiating sample collection activities. Required equipment and supplies include, but are not limited to: Appropriate PPE (Section 3.0) Powder free nitrile gloves for sample supplier, collector, and support person Sampling kit (Section 8.3) Agar plates (Section 7.3) Calibration kit (Section 8.4) Calibrated pump and power source (Section 7.1) Calibrated rotameter or electronic dry/wet cell calibrator (Section 7.1) Timer (Section 7.1) Sample documentation (Section 6.3) Decontamination supplies (Sections 7.5 and 7.6) Sample transport containers and packing materials (Section 7.4) Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample(s) that are to be collected. Note: Agar plates should not be used if they have been frozen. 10.2.2 Pre-staging Once the sampling team has arrived at the sampling site and determined the required sampling location(s), each team member should don new, unused gloves and ensure sampling equipment is assembled appropriately and pump flow rates are adjusted as needed for sample collection. 10.2.2.1 Sampler Assembly Sterile agar plate(s) are provided separately from the sampling kits and placed into a clean impactor(s), using aseptic techniques, immediately prior to entering the contaminated area and collecting samples. Reseal the sampler and place it into 1-gallon bag, then place the bag into a plastic bin for transport to the sample collection site. Note: The impactor assembly should remain upright during transport and use. 10.2.2.2 Calibration Check The pump flow rate should be checked outside of the contaminated area following instructions in the SAP or from the pump manufacturer. The flow rate of the pump should be checked and adjusted, if needed, to the flow rate that will be used for sample collection, using a complete sampling train (i.e., calibration kit, Section 8.4). Post-sampling flow rates should be checked once sample collection has been completed, and both pre- and post-sampling flow rates should be recorded. General guidelines regarding the flow rates to use during sample collection and calibration approximate 28 L/minute. 10.2.3 Collection Once the pump(s) has been calibrated and checked outside of the sampling area, the sampling team proceeds to the sampling location and follows the procedures described below. 28 ------- Note: New, unused gloves and sampling trains (calibrated pump and sampling kit [sterile impactor assembly and clean tubing]) are required for each sample collected. Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample (s) that are to be collected. I. Equipment: Preparation and Setup Supplier and Collector: Supplier: Support Person: Supplier: Supplier: Collector: Collector: Don or put on new, clean sampling gloves (Note: the support person should also don or put on new, clean gloves if they accidently touch the sample or contaminated area). Obtain a calibrated pump and clean sampling kit from supply bin. Record the sample ID. Place the calibrated pump onto a clean piece of plastic to protect it from contamination. Open the plastic bag containing the sampling kit for the collector to remove the impactor assembly and tubing. Remove the impactor assembly and tubing. Attach clean tubing between the pump inlet and the impactor outlet. II. Collection Collector: Activate the pump and collect sample for the specified time. Supplier: Start timer. Support Person: Record flow rate and start time. Collector: Once sample collection is completed, turn off the pump. [Note: Recommended sampling times range from 10 to 15 minutes.] Supplier: Stop timer. Support Person: Record stop time and final flow rate. I r Collection of Each Sample Collector: Disconnect the tubing from the impactor assembly outlet and seal the assembly inlet and outlet with sealing tape (Parafilmฎ). Supplier: Open and hold a pre-labeled clean plastic sealable bag for the collector to place the impactor assembly into the pre-labeled bag. Collector: Place the impactor assembly into the bag and seal the bag. Supplier: Decontaminate the outer surface of the bag and hand-off to support person. Support Person: Complete sample documentation and provide the documentation and filter assembly to personnel outside the contaminated area for sampler disassembly (Section 10.4), filter retrieval and packaging. Collector: Once all samples have been collected, place used equipment to be decontaminated into a bag and seal and decontaminate the bag. Collector: Gather the tubing and any additional contaminated equipment or supplies for proper disposal. All Personnel: Doff sampling gloves prior to moving to the next location. 10.2.4 QC Samples 29 ------- QC samples that might be requested for air sampling include field blanks and transport blanks. 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. Transport blanks (Section 9.2) are supplied as unopened/unused sampling kit assemblies and are transferred and transported along with the field samples. 10.3 Liquid Impingers Instructions for collection of air samples using a typical glass liquid impinger are provided below. Liquid Impinger Used for viruses and other pathogens Supports viability assessments 10.3.1 Materials, Supplies and Equipment In addition to being familiar with the documentation and information noted above, sampling teams must have access to all necessary sampling equipment prior to entering the area of contamination and initiating sample collection activities. Required equipment and supplies include, but are not limited to: Appropriate PPE (Section 3.0) Powder free nitrile gloves for sample supplier, collector, and support person Sampling kit (Section 8.3) Calibration kit (Section 8.4) Calibrated pump and power source (Section 7.1) Calibrated rotameter or electronic dry/wet cell calibrator (Section 7.1) Timer (Section 7.1) Sample documentation (Section 6.3) Decontamination supplies (Sections 7.5 and 7.6) Sample transport containers and packing materials (Section 7.4) Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample(s) that are to be collected. 10.3.2 Pre-staging Once the sampling team has arrived at the sampling site and determined the required sampling location(s), each team member should don new, unused gloves and should ensure sampling equipment is assembled appropriately and pumps flow rates are adjusted as needed for sample collection. 10.3.2.1 Sampler Assembly Sterile impinger fluid is added to a clean impinger immediately prior to site entry and prior to sample collection using materials and supplies provided in the sampling kit. Remove the 50-mL centrifuge tube with 20 mL of impinger fluid from the sampling kit and, using aseptic technique, pour the premeasured volume of collection fluid that will be used for sampling into the impinger. Apply sterile stopcock grease to the ground glass connector at the top of the impinger and seal the 30 ------- inlet and outlet of the impinger with Parafilmฎ. Secure the filled impinger back in the transport container for transport to the sample collection site. 10.3.2.2 Calibration Check The pump flow rate should be checked outside of the contaminated area following instructions in the SAP or from the pump manufacturer. The flow rate of the pump must be checked and adjusted, if needed, to the flow rate that will be used for sample collection, using a complete sampling train (i.e., calibration kit, Section 8.4). Post-sampling flow rates must be checked once sample collection has been completed, and both pre- and post-sampling flow rates should be recorded. General guidelines regarding the flow rates to use during sample collection and calibration range from 10 to 14 L/minute. 10.3.3 Collection Once the pump(s) has been calibrated and checked outside of the sampling area, the sampling team proceeds to the sampling location, and follows the procedures described below. Note: New, unused gloves and a sampling train (calibrated pump, sterile impinger assembly with collection fluid, and clean tubing) are required for each sample collected. Sampling personnel must ensure that the sampling kits and documentation are complete and specific for the sample (s) that are to be collected. I. Equipment: Preparation and Setup Supplier and Collector: Don or put on new, clean sampling gloves (Note: the support person should also don or put on new, clean gloves if they accidently touch the sample or contaminated area). Supplier: Obtain a calibrated pump and sampling kit from supply bin. Support Person: Record the sample ID. Supplier: Place the calibrated pump onto a clean piece of plastic sheeting to protect it from contamination. Supplier: Open the plastic bag containing the sampling kit for the collector to remove the tubing. Collector: Attach clean tubing between the pump inlet and the liquid trap outlet. Supplier: Open the impinger transport container for the collector to remove the impinger. Collector: Remove the impinger and stabilize it in the impinger holder for sampling. Collector: Attach clean tubing between the liquid trap inlet and the impinger outlet. II. Collection Collector: Place the calibrated sampling train in the desired location and turn on the pump. [Note: Impingers must be kept upright to prevent loss of fluid due to leaking or spillage.] Supplier: Start timer. Support Person: Record the flow rate and start time. Collector: After sample collection is completed, turn off the pump. [Note: 31 ------- Recommended sampling time is approximately 20 minutes (Hermann et al. 2006).] Supplier: Stop timer. Support Person: Record flow rate and stop time. I r Collect ion of Each Sample Collector: Collector: Supplier: Collector: Supplier: Supplier: Support Person: Collector: Collector: All Personnel: Don a new and unused pair of gloves and remove the impinger from the impinger holder and sampling train. Seal the impinger inlet and outlet with Parafilmฎ or sealing tape. Open and hold a pre-labeled clean plastic sealable bag for the collector to place the impinger into the bag. Place the impinger into the pre-labeled bag and seal the bag. Decontaminate the outer surface of the bag and secure the bag containing the impinger in an upright position in the transport container. Hand-off to support person for final sample documentation and off-site filter collection. Provide sample documentation and filter assembly to personnel outside the contaminated area for filter disassembly, filter retrieval and packaging. Once all samples have been collected, place used equipment to be decontaminated into a bag and seal the bag. Ensure that all solid and liquid waste is contained (e.g., solid wastes in a plastic bag and liquid wastes in a durable sealable container) for proper disposal or decontamination. Doff sampling gloves prior to moving to the next location. 10.3.4 QC Sampling QC samples that might be requested for air sampling include field blanks and transport blanks. 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. Transport blanks (Section 9.2) are supplied as unopened/unused sampling kit assemblies and are transferred and transported along with the field samples. 10.4 Sampler Disassembly Filters, impactors and impingers that have been used to collect air samples are transferred to a clean area outside the contaminated area for disassembly and packaging of samples (i.e., filters, impact media or impinger fluid) into appropriate primary and secondary sample containers. In all cases, disassembly is performed using new, unused gloves and aseptic techniques to ensure the collected samples are not compromised. Disassembly should not be done in an area where new sampling media is stored. 32 ------- 10.4.1 Filters Using new, unused gloves and aseptic techniques, remove the filtration apparatus from the pre- labeled bag, and disassemble the apparatus following manufacturer instructions and/or by referring to the assembly instructions provided in Section 8.3.1. Once disassembled: Remove the filter with sterile forceps and place the filter into a sterile primary sample container (e.g., 50-mL tube or plastic bag). Note: For gelatin filters, depending on the microbiological agent being sampled, add approximately 10 mL of sterile distilled water or Tween to the container. Securely tighten, seal and label the primary sample container (e.g., unique sample identifier, sample location, initials of sample collector(s) and date and time sample was collected). Note: The information included on the primary sample container label should be identical to the information included on the pre-labeled bag that contained the filtration apparatus. Place the labeled container into a secondary sample container (i.e., clean, sealable plastic bag). Seal and label the secondary container with a label identical to the label placed on the primary container. Place the contained and labeled sample into the sample collection bin for transfer to sample packaging and transport (see Section 11.0). 10.4.2 Impactors Using new, unused gloves and aseptic techniques, remove the impactor assembly from the pre- labeled bag, and disassemble the impactor sampler following manufacturer instructions and/or by referring to the assembly instructions provided in Section 8.3.2. Once disassembled: Remove the agar plate(s), cover each plate with a plate lid, and seal each plate with sealing tape (e.g., Parafilmฎ). Label each plate (e.g., unique sample identifier, sample location, initials of sample collector(s) and date and time sample was collected) including the appropriate impactor stage number if applicable. Place plates so agar is oriented down (to prevent condensation from forming on the top of the lid) into a clean sealable plastic bag). Securely seal and label the bag. Note: The information included on this label should be identical to the information included on the pre-labeled bag that contained the filtration apparatus. Place the contained and labeled sample into the sample collection bin for transfer to sample packaging and transport (see Section 11.0). 10.4.3 Impingers Note: Impinger fluid transfer must be conducted using appropriate containment practices, such as a biosafety cabinet or fume hood. Please consult SAP or site lead if a biosafety cabinet or fume hood are not available. Using new, unused gloves and aseptic techniques, remove the impinger assembly from the pre-labeled bag. Transfer the impinger fluid to a sterile primary sample container (e.g., 50-mL tube). Securely tighten, seal and label the sample container (e.g., unique sample identifier, sample location, initials of sample collector(s) and date and time sample was collected). Note: The 33 ------- information included on the primary sample container label should be identical to the information included on the pre-labeled bag that contained the filtration apparatus. Place the labeled container into a secondary sample container (i.e., clean, sealable plastic bag). Seal and label the secondary container with a label identical to the label placed on the primary container. Place the contained and labeled sample into the sample collection bin for transfer to sample packaging and transport (see Section 11.0). 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 the transport process. 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 ofPackas.es with Select Agents and Toxins (CDC 2014). Since microbiological samples often degrade quickly overtime, 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 including COC. 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 requirements that were developed for other sampling media or organisms. The best storage temperature for a given microbiological sample often varies depending on the type of microbiological organism, the sample matrix, the sample's intended use, and how long the sample will be stored. When storing samples, it is also important to consider the molecular structure of the microbiological organism (Holland et al. 2003, Budowle et al. 2006, NRC 2014, Shabihkhani et al. 2014) and the degree of biological 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 Piilho^on Siปr;iปe 1 OllipCI'illlllV Ki'CommciHk'ri Molding Time Oilier ConsiriiTiilions 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 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. 34 ------- Piilho^on Siปr;iปe 1 OllipOI'illlllV Ki'CommciHk'ri Molding Time Oilier Coiisidoi'iilioiis 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. *Note: Samples to be analyzed for Vibrio cholerae should be kept at room temperature and must not be cooled. 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. See also U.S. EPA 2005. 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: Microbiological 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. Microbiological 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. 35 ------- 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 (IATA 2015) or DOT requirements at 49 CFR Parts 171 through 180 (U.S. DOT, 2012), Hazardous Materials Regulations for movement in public right-of-ways 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; last accessed 09/28/2020) American Biological Safety Association (last accessed 09/28/2020) WHO"( inick nee on Regulations for the Transport of Infections Substanc 18" (WHO 2017; last accessed 09/28/2020) 11.3 Sample Packaging This section provides general guidance on sample packaging; however, level of packaging is dependent 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. 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. 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 using the procedures described in this document: Primary sample containers for filters and impactor plates are 4"* 6" sealable plastic bags Primary sample containers for impinger fluid are sterile centrifuge tubes Secondary sample containers are clean sealable plastic bags Each primary and secondary sample container is sealed and labeled with the Sample ID (see Section 6.2). Prior to leaving the area of contamination, the outer surface of the outermost container is decontaminated and placed into a clean bag or box for transfer to the transport 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, it can cause damage to the samples and 36 ------- compromise sample integrity. 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 ice packs are recommended. Well-constructed packing materials should be used to cushion the inner containers and 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 disinfectant wipes. Pack secondary sample containers into a transport container with sufficient padding to ensure they are protected and will not shift during transport. Sample containers holding impinger fluid are packed separately from other sample types, along with sufficient absorbent material (such as paper towels or absorbent gel sheets) to absorb the entire contents of any spillage. Self-contained ice packs or frozen packs are recommended for cooling samples during transport. When multiple sample containers are packaged within a single transport container, padding 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 their seals. 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). In order 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 Appropriate U.S. Department of Transportation (DOT) and/or International Air Transport Association (IATA) labeling 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 notification 37 ------- should include an accurate description of the contents of the shipment and the expected date of arrival. 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. If custody seal is broken during transport, resulting analytical data might not be accurate or results might not be legally defensible. 38 ------- 12.0 References American Society for Microbiology (ASM). 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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 40 ------- Employees regarding subject "Scribe - Exclusive Agency Database during a National Response," March 8, 2016. 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DOI: 10.1080/00966665.1956.10467715 42 ------- Appendix A: Applicable Microbiological Agents This document is to be used in conjunction with laboratory analysis methods listed in the Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2017 (SAM) (U.S. EPA 2017; last accessed 9/29/2020) which 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. SAM identifies a single selected method or suite of methods for each analyte/sample type. Table A-l lists the SAM bacterial microbiological agents and Table A-2 lists SAM viruses, protozoa, and helminths. Table A-l. Example Subset of Bacterial Microbiological Agents Bacteria Disease Typical Exposure Reservoir in United States 1 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 (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 The occurrence and reservoirs listed for the bacteria are those described by the CDC fhttpV/www.cdc.gov/ and http://www.cdc.gov/biosafetv/publications/bmbl5/. last accessed 10/02/2020) 43 ------- Table A-2. Example Subset of Viruses, Protozoa and Helminths 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 Naeqieria fowieri Naegleriasis Toxoplasma gondii Toxoplasmosis Helminths Disease Baylisascaris procyonis Raccoon roundworm infection 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 44 ------- 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 (QAPP) 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 (QA) 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 - sample 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 (SAP) For collection of samples, a well-defined and thorough sampling and analysis plan (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 agents 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 Working Draft - Do not cite, quote, or distribute 45 ------- required sample container size and type sample locations and frequencies sample collection techniques and procedures data quality objectives (DQO's) 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 (Silvestri et al. 2018). This document summarizes elements that should be considered when planning, developing and implementing a 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 DQOs. 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 (HASP) Safety is a primary consideration for any sampling event. The Health and Safety Plan (HASP) is developed to 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 Site- or incident-specific risk assessment addressing sample collection activities Training requirements Personal protective equipment (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 Working Draft - Do not cite, quote, or distribute 46 ------- Personnel safety requirements and considerations for a particular site might extend beyond concerns related to exposure to microbiological agents and can include exposures 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 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). Sampling personnel should consult the SAP to for specifications on the following sampling requirements which might 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 Sample throughput (number of samples a lab can process per unit time) B.5 Waste Management Plan (WMP) A Waste Management Plan (WMP) that outlines waste management requirements, procedures, strategies, and processes from the point of generation to final deposition. Ideally, the WMP 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 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-18/16 U.S. DOL. (2002) Emergency Action Plans. 29 CFR 1910.38. U.S. Department of Labor, Occupational Safety and Health Administration: Washington, DC. Working Draft - Do not cite, quote, or distribute 47 ------- 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. 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 Working Draft - Do not cite, quote, or distribute 48 ------- Appendix C. Example Chain of Custody Form o EPA USEPA Sample Traffic Report & Chain of Custody Record Case No.: DAS No.: SDG No.: Pase of 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 Waste Water; VF - Vacuum Filter DAS: Delivery as Analytical Services SDG: Sample Delivery Group Working Draft - Do not cite, quote, or distribute 49 ------- 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 ------- |