SEPA EPA Report Number EPA 600/R-22/009 January 2022 Development of Field-Executable Sample Collection Protocols for USCG Assets following a Biological Incident ------- Development of Field-Executable Sample Collection Protocols for USCG Assets following a Biological Incident U.S. EPA Principal Investigator: Worth Calfee U.S. Environmental Protection Agency Research Triangle Park, NC 27711 Prepared by: Abderrahmane Touati, Barbara Wyrzykowska-Ceradini, Jonathan Sawyer, Timothy Chamberlain, and Eric Morris. Jacobs ^BBF^ ^BP* ^BP ^BB^ ^BBF* Contract Number: 68HERC20D0018 Task Order Number: 68HERC20F0440 1 ------- Disclaimer This study was funded through the Analysis for Coastal Operational Resiliency (AnCOR) Project by the U.S. Department of Homeland Security Science and Technology Directorate under interagency agreement IA 70RSAT18KPM000084. The U.S. Environmental Protection Agency (EPA), through its Center for Environmental Solutions and Emergency Response (CESER) within the Office of Research and Development (ORD), the U.S. Department of Homeland Security (DHS), and the U.S. Coast Guard (USCG), collaborated in the research described herein under EPA Contract No. EP- C-15-008 to Jacobs Technology, Inc. It has been subjected to the Agency's review and approved for publication. Note that approval does not signify that the contents necessarily reflect the views of the Agency. Any mention of trade names, products, or services does not imply an endorsement by the U.S. Government or EPA. The EPA does not endorse any commercial products, services, or enterprises. The contractor role did not include establishing Agency policy. Questions concerning this document or its application should be addressed to the following individual: Dr. M. Worth Calfee Homeland Security and Materials Management Division Center for Environmental Solutions and Emergency Response (CESER) U.S. Environmental Protection Agency (MD-E343-06) 109 T.W. Alexander Drive Research Triangle Park, NC 27711 E-mail Address: calfee.worth@epa.gov ii ------- Forward 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 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. This report outlines sample collection procedures for United States Coast Guard (USCG) assets and bases. These procedures are in a format consistent with the USCG Maintenance Procedure Cards (MPC) and can be used to better plan and execute the sampling operations for USCG small response boats, vessels, a USCG port/base, or any large outdoor urban area, following a biological contamination incident. This work was coordinated with and managed by the EPA's Homeland Security Research Program (HSRP) under the Department of Homeland Security Science and Technology Directorate funded Analysis for Coastal Operational Resiliency (AnCOR) project. Gregory Sales, Director Center for Environmental Solutions and Emergency Response 111 ------- Table of Contents Disclaimer ii Forward iii Table of Contents iv Figures iv Acronyms/Abbreviations v Acknowledgments viii Executive Summary ix 1.0 Introduction 1 2.0 Field Demonstration 1 3.0 Field Evaluation Summary 2 4.0 Quality Assurance / Quality Control 5 5.0 Summary of findings 5 References 7 Appendix A. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING CELLULOSE SPONGE-STICK 8 Appendix B. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING VACUUM FILTER CASSETTES 18 Appendix C. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING PRESSURIZED WATER WASH-DOWN 30 Figures Figure 1. Phase 2 field trial sampling locations on RBS-I boat 2 Figure 2. Examples of Phase 2 field trial sampling photo-documentation 4 iv ------- Acronyms/Abbreviations ACMS Asset Computerized Maintenance System AnCOR Analysis for Coastal Operational Resiliency Ba Bacillus anthracis CESER Center for Environmental Solutions and Emergency Response (EPA) CFR Code of Federal Regulations CLP Contract Laboratory Program CMAD Consequence Management Advisory Division COC chain of custody COTS commercial off-the-shelf DE decontamination efficiency DHSS&T Department of Homeland Security Science and Technology Directorate DQI data quality indicator DQO data quality objective EPA Environmental Protection Agency FMF Fluid Modeling Facility ft. Feet HASP health and safety plan HSMMD Homeland Security and Materials Management Division (EPA) HSRP Homeland Security Research Program (EPA) IA Interagency IC Incident Command ------- ID (sample) identification in. inch(es) MCE Mixed Cellulose Ester MPC USCG Maintenance Procedure Card MOP miscellaneous operating procedure NIST National Institute of Standards and Technology NRF National Response Framework OSHA Occupational Safety and Health Administration PPE Personal Protective Equipment OLEM Office of Land and Emergency Management OP operating procedure(s) ORD Office of Research and Development (ORD) PPM ORD QA Policies and Procedure Manual PTFE Polytetrafluoroethylene QA quality assurance QAO Quality Assurance Officer QAPP quality assurance project plan QC quality control RBS Response Boat-Small RBM Response Boat-Medium RLS Research Laboratory Support RSD relative standard deviation RTP Research Triangle Park VI ------- SAP sampling and analysis plan SHEM Safety, Health, and Environmental Management SST Sponge-stick protocol STICS Scientific and Technical Information Clearance System T temperature TAT turnaround time TBD to be determined TLP Technical Lead Person TO task order TOCOR Task Order Contracting Officer's Representative TOL Task Order Lead TOM Task Order Manager TOP Task Order Plan TSA technical systems audit UAC Unified Area Command UC Unified Command USCG United States Coast Guard VFC vacuum filter cassette protocol WI Work Instruction Vll ------- Acknowledgments The principal investigator from the U.S. Environmental Protection Agency (EPA) directed this effort with the support of a project team from across EPA. The contributions of the individuals listed below have been a valued asset throughout this effort. Project Team Worth Calfee, CESER/HSMMD (Center for Environmental Solutions and Emergency Response/ Homeland Security and Materials Management Division) Task Order Contracting Officer's Representative (TOCOR) Leroy Mickelsen, OLEM/CMAD (Office of Land and Emergency Management/Consequence Management Advisory Division) Alt TOCOR Shannon Serre, OLEM/CMAD Anne Mikelonis, CESER/HSMMD John Archer, CESER/HSMMD Lukas Oudejans, CESER/HSMMD Emile Benard, U.S. Coast Guard LCDR Clifton Graham, U.S. Coast Guard EPA Quality Assurance Ramona Sherman, CESER/HSMMD Jacobs Technology, Inc. Abderrahmane Touati, Task Order Manager Barbara Wyrzykowska-Ceradini, Task Order Lead Jonathan Sawyer, Technical Support Timothy Chamberlain, Technical Support Eric Morris, Science Systems and Applications, Inc., Technical Support (Sub-contractor) Vlll ------- Executive Summary The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's land, air, and water resources. EPA is designated as a coordinating agency under the National Response Framework to prepare for, respond to, and recover from a threat to public health, welfare, or the environment caused by actual or potential oil and hazardous materials incidents. Hazardous materials include chemical, biological, and radiological substances, whether accidentally or intentionally released. EPA is expected to respond to outdoor contamination incidents to characterize and remediate the impacted sites, and thus, determining the performance of available sampling and analysis methods for outdoor environments is a high priority. The United States Coast Guard (USCG) shares similar responsibilities under the National Response Framework for response to hazardous material incidents. USCG bases and ports are potential targets for biological terror, and by nature of their mission and location, may have unique surfaces and/or environments that could complicate response activities. The diversity and magnitude of surfaces at a USCG base that would be impacted during bioagent remediation necessitate proactive sample collection procedures for priority assets, to define the extent of contamination, evaluate the effectiveness of decontamination, and inform waste disposal. EPA and USCG have formed an Interagency Project Team to support research under the Analysis for Coastal Operational Resiliency (AnCOR) program, of which this study is a part. This study was conducted to help the USCG recover rapidly following a biological contamination incident and return assets to duty. Determination of asset contamination status is necessary to make decisions regarding asset safety and deployability. The outcome of the study described in this report provides data and information that can be used to inform sampling operations following an outdoor biological contamination incident impacting a USCG base. Ultimately, it is desired that these procedures will facilitate a rapid recovery following a large- scale biological incident. IX ------- 1.0 Introduction The U.S. Environmental Protection Agency (EPA) and U.S. Coast Guard (USCG) have a need to assess contamination extent in order to recover rapidly following a biological incident. This work entailed adapting three surface sample collection operating procedures (OPs) into a format common and familiar to USCG personnel. The three sample collection OPs were experimentally evaluated and developed under related research efforts of EPA and USCG under the Analysis for Coastal Operational Resiliency (AnCOR) Program (USEPA, 2011). These procedures are meant to increase preparedness for sample collection operations, following a biological contamination incident. This project focused on preparation of step-by-step field protocols/operating procedures for three [non-traditional and standard] surface sampling methods for selected USCG maritime assets. To maximize USCG responder familiarity with the sampling procedures, increase just-in-time training effectiveness, and minimize response delays, the OPs were written in a common USCG procedure format: USCG Maintenance Procedure Card (MPC). Part of the work, and the subject of this summary, includes an assessment of the OPs developed, in a realistic setting and with appropriate Personal Protective Equipment (PPE) being worn to simulate real-world conditions. The following draft OPs were used for sampling of selected smooth/non-porous and porous surfaces of the USCG Response Boat Small (RBS-1): 1. OP 1. Procedure for Bacillus Anthracis Surface Sampling using Cellulose Sponge (Rose et al.. 2011); the procedure was used to perform sampling of two types of smooth materials: (a) glass windows (rear cabin), and (b) rubber covering bumper material (front sideboard); sampling area was 10 x 10-in (100 in2 [645 cm2]). 2. OP 2. Procedure tor Bacillus Anthracis Surface Sampling using 37-mm Vacuum Cassettes (Calfee et al.. 2013); the procedure was used to perform simulated sampling of the anti-skid material of the RBS-I floor (rear floor); sampling area was 12 x 12-in (144 in2 [929 cm2]). 3. OP 3. Procedure for Bacillus Anthracis Surface Sampling Using Pressurized Water Washdown (Calfee. 2021); the procedure was used to perform simulated wash-down of the front half of the RBS-I; the run-off water was collected from the front forward drain. 2.0 Field Demonstration The draft OPs were field tested on a decommissioned RBS-1 boat located outside of the auxiliary EPA RTP, NC facility on Page Rd. (Fluid Modeling Facility [FMF]). The field-testing locations on the RBS-I boat are shown in Figure 1. Field demonstrations were performed in either a two- or three-person configuration with adherence to necessary health and safety protocols. Mock samples were collected for representation of actual sampling times, as described in OPs written in the USCG MPC format. All procedural steps of OPs were evaluated for field usability (e.g., ease of use in performing portions of the procedure, potential for cross-contamination of surfaces and/or samples) and adherence to standard methods, where applicable. Collection procedures were 1 ------- executed several types each, with multiple personnel. Procedures were provided to the sampling team well in advance of the Field demonstration, to maximize their familiarity with the procedures. No sample analysis plans (SAPs) or chain of custody (COC) documents were prepared for field demonstrations. Biological analyses of simulated field samples were not performed. Samples were disposed of per an approved Health and Safety Plan (HASP). Figure 1. Phase 2 field trial sampling locations on RBS-I boat. The sampling protocols were evaluated as follows on the RBS-I boat locations listed below: 1. OP 1. Sponge Stick Sampling Locations A. Cabin window (rear) B. Rubber bumper material (front sideboard) 2. OP 2. Vacuum Filter Cassette (VFC) Sampling C. Anti-skid flooring 3. OP 3. Wash-down Sampling D. The front half of the boat, with runoff collected through the front forward drain (E) 3.0 Field Evaluation Summary 2 ------- The findings of field evaluations are summarized in Section 2. Examples of photo-documentation for each OP are shown in Figure 2. Note: The comments from the EPA Team observers regarding OPs and possible sample procedure improvements were submitted to Jacobs by email within one business day from the completion of sampling and are included in Section 4. The photo-documentation of each sampling type material surface, shown in Figure 2, is summarized below: 1. OP 1. Sponge Stick Sampling A-F Rubber G-I Glass 2. OP 2. Vacuum Filter Cassette (VFC) Sampling J-L Anti-skid flooring 3. OP 3. Wash-down Sampling M-N Wash-down of the front half of the boat O Run-off collection through the front forward drain 3 ------- ------- 4.0 Quality Assurance / Quality Control Research activities for this effort were documented according to the requirements of ORD QA Policies and Procedure Manual (PPM) Sections 13.2 and 13.4, entitled "Scientific Recordkeeping: Paper"; and "Scientific Recordkeeping: Electronic and Quality Assurance / Quality Control Practices for ORD Laboratory and Field-Based Research", respectively, as well as requirements defined in the approved QA Project Plan entitled "Field-Executable Sample Collection Protocols for USCG Assets". Sampling protocols were prepared using USCG MPC format. A physical laboratory notebook was used to document daily test activities. Electronic project records are maintained on the ORD network drive: (L:\Lab\DTRL\TO 440). All records are maintained in a manner that maximizes the confidentiality, accessibility, and integrity of the data. Records that are generated under this research effort will be retained per EPA Records Schedule 1035, and as required by Section 5.1 of the ORD Quality Management Plan for QA Category B Projects. The data quality indicators (DQIs) for test measurements are provided in Table 4-1. No results/tests were outside the acceptance criteria as defined in the project-specific quality assurance project plan. There was no indication of systematic errors introduced into the final procedures developed under this study. Table 4-1. Critical Measurements Acceptance Criteria and QA check results. Critical Measurement Measurement Device Acceptance Criteria Accuracy/Precision Observed Time NIST-traceable stopwatch RSD <30 % between sampled areas of identical size RSD <30 % between sampled areas of identical size 5.0 Summary of findings The main findings from the Phase 2 demonstration included modifications to the draft OP procedural steps for improved ease of surface sampling and sample handling on the actual boat, and—hypothetically—in the port. The main technical changes suggested for each OP were as follows: 1. OP 1 (Sponge Stick Sampling) a. Addition of the sampling kit preparation step to the procedure. b. Use of sterile specimen containers for collecting the sponge samples; using a rigid (non-collapsible) container is a standard method requirement for transport of samples from the field to the analytical laboratory. c. Addition of another type of the sponge stick sampler (Solar-Cult® Sponge- Stick, Neutralizing Buffer Cellulose Sponge, Hardy Diagnostics, USA; P/N 5 ------- SH10NB1) to the test supplies list. Unlike the sponge stick currently listed in the OP (3M™ Sponge-Stick, 3M, USA; P/N SSL-10NB), the handle sampler does not require breaking off the sponge tip but allows for quick release into a sampling container (specimen cup). 2. OP 2 (37-mm Vacuum Sampling) a. Change the name of the procedure to VFC-based sampling rather than 37-mm cassette sampling. b. Addition of the preparation step for calibration check of the sampling pump at the beginning of the sampling. c. Addition of the warning statement about possible sampling interruptions due to filter clogging and/or portable pump malfunctions. d. Addition of a more detailed sampling technique description, indicating that the nozzle should gently touch the surface to be sampled, with an angle such that the nozzle flow and orifice aren't blocked by the surface. e. Addition of instructions for re-use of Tygon tubing downstream of the cassette for improved throughput of multi-location sampling. f. Addition of the filter cassette handling technique by the support person to allow easy retrieval by the sampler. g. Addition of the VFC plugs handling technique before and after sample collection by the sampler and support person. 3. OP 3 (Wash-down) a. Identification of the boat sampling locations, indicating the water drains accessible for water runoff collection during on-the-water (boat-in-the-dock) and on-the-land (boat-on-the-water) sampling operations; locations for different types of the USCG boats should be included. b. Addition of the maximum sampling bottle fill with run-off (90% fill, with a small amount of headspace to allow sample homogenization prior to analysis). Other proposed modifications included changes related to the PPE donning, with glove changes signaled by the use of pictograms in the OP text. In addition, for the 'Tool/Equipment' section, the replacement of 'timer' with 'electronic timekeeping device' was recommended, as the latter is more applicable to the field sampling. The OPs can easily be applied to surface sample collection on other USCG vessels, including the RBS II, RBM, and 87-ft cutter. 6 ------- References Rose, L. J., Hodges, L., O'Connell, H., and Noble-Wang, J. (2011). "National validation study of a cellulose sponge wipe-processing method for use after sampling Bacillus anthracis spores from surfaces." Applied and Environmental Microbiology. 77(23), 8355-8359. https://doi.org/10.1128/AEM.05377-11 USEPA (2021). "Development of Field-Executable Sample Collection Protocols for USCG Assets following a Biological Incident." Quality Assurance Project Plan QAPP-0J21-004.0. Calfee, M. W., Rose, L. J., Morse, S., Mattorano, D., Clayton, M., Touati, A., Griffin-Gatchalian, N., Slone, C., and McSweeney, N. (2013). "Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores." Journal of Microbiological Methods. 95(3), 389-396. https://doi.Org/10.1016/i.mimet.2013.10.015 Calfee, M. W. (2021). "Evaluation and Testing of Outdoor Surface Sample Collection and Analysis Methods." U.S. Environmental Protection Agency, Washington, DC, EPA/600R-21- 212. 7 ------- Appendix A. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING CELLULOSE SPONGE-STICK ------- ASSET NUMBER OPERATING ACTIVITY MAINTENANCE ACCOMPLISHED ASSET HOURS MAINTENANCE DUE ASSET HOURS PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING CELLULOSE SPONGE-STICK SCHEDULED UNSCHEDULED DISCREPANCIES FOUND: NO YES MAN HOURS: MK DC EM ET IT GM BM FN SN OTHER REMARKS: TECHNICIAN'S SIGNATURE and DATE TECHNICIAN'S ID ' ASTERISK INDICATES QA REQUIRED QA REFERENCES: CDC (2012). "Surface sampling procedures for Bacillus anthracis spores from smooth, non- porous surfaces." Emergency Response Resources. REVIEWED BY and DATE DATA ENTRY COMPLETED 9 ------- PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING CELLULOSE SPONGE-STICK TOOLS/EQUIPMENT: Quantity Item Name 1 Sponge, sterile, pre-moistened with 10 mL neutralizing buffer solution, 1.5-in by 3- in cellulose sponge folded over a handle (such as the 3M™ Sponge-Stick [3M, St. Paul, Minnesota; P/NSSL-10NB] or Solar-Cult® Sponge-Stick, Neutralizing Buffer Cellulose Sponge [Hardy Diagnostics, USA; P/N SH10NB1], or equivalent) 1 Medium-size sterile sampling bag with flat-wire closures (Thermo Fisher Scientific, Waltham, MA; P/N 14-955-187 or equivalent) 1 Screw-cap specimen container, sterile, 4 oz 1 Disposable sample template 10-in x 10-in (sample area size 100 in2 [645 cm2]); or disposable ruler and masking tape 1 Sample kit container (e.g., storage bin, backpack, or another container that will be used for transport and storage of sample kits prior to sampling) 1 Sampling log CONSUMABLES: Quantity Item Name 1 Box of nitrile gloves or equivalent 1 Disinfecting wipes (Dispatch® Disinfectant Towels with Bleach, Clorox Company, Oakland, CA; P/N 69150 or equivalent) 1 Sample labels or permanent marker 10 ------- A. PRELIMINARY STEPS WARNING THE RISK OF FATALITY ASSOCIATED WITH THE INHALATION OF BACILLUS ANTHRACIS SPORES CAN BE EXTREMELY HIGH. TO AVOID THE RISK OF EXPOSURE DURING SAMPLING OF POTENTIALLY CONTAMINATED AREAS, APPROPRIATE PROTECTIVE MEASURES MUST BE INITIATED AS PART OF COORDINATING RESPONSE ACTIONS. IT IS RECOMMENDED THAT A COMPREHENSIVE HEALTH AND SAFETY PLAN BE DEVELOPED TO ADDRESS SAMPLING PERSONNEL SAFETY ISSUES PRIOR TO INITIATION OF SAMPLING ACTIVITIES. TO REDUCE THE RISK OF ILLNESS AND DEATH, PROPER PERSONAL PROTECTIVE EQUIPMENT (PPE) MUST BE USED WHEN WORKING IN A POTENTIALLY CONTAMINATED ENVIRONMENT. PPE INCLUDES PROPER RESPIRATORY PROTECTION, PROTECTIVE GARMENTS, AND GLOVES. CONSULT WITH YOUR SAFETY OFFICER FOR PROPER PPE SELECTION. When mandated by the Incident Commander (IC) as part of a Unified Command (UC) or other incident management system, PPE, including respiratory protection, needs to be employed under OSHA standards and regulations found in 29 CFR Part 1910 Subpart I (1910.132—1910.138) or the equivalent construction standards (29 CFR Part 1926). These standards require training on the proper selection, use, removal, and disposal of PPE; a medical evaluation and fit testing must be conducted before the use of any respiratory protection and annually thereafter. Contact the Unified Command (UC), Operations Section Chief, and/or Safety Officer for PPE requirements, and for selecting applicable sampling procedures, stations, and routes. B. SAMPLING WARNING ALL TEAM MEMBERS MUST WEAR SUITABLE PPE. AFTER APPROPRIATE DECONTAMINATION PROCEDURES, PPE MUST BE CORRECTLY REMOVED AND DISPOSED OF. FAILURE TO COMPLY MAY RESULT IN PERSONAL INJURY OR DEATH. CAUTION TO PREVENT THE SPREAD OF CONTAMINATION, DO NOT PERFORM SAMPLING OF THE BOAT IN THE VICINITY OF WATER INTAKES, ENVIRONMENTALLY SENSITIVE AREAS, OR IN AREAS WHERE THE PUBLIC COULD BE AFFECTED. 11 ------- 1. Identify sampling locations on the exterior and interior of the boat. This procedure is intended for sampling of (10-in x 10-in) non-porous surfaces (e.g., aluminum, painted aluminum, glass). For non-smooth, porous surface sampling, refer to the vacuum filter cassette (VFC) sampling procedure (MPC # TBD). For large area sampling of porous and non-porous surfaces using non- specialized equipment and fresh or seawater sources, refer to the pressurized water spray procedure (MPC # TBD). At a minimum, a two-person team must be used. The team will consist of a sampler (person who will perform sampling of contaminated surfaces, i.e., "dirty person") and a support person (person who will be responsible for taking notes, labeling, and other sample handling procedures, i.e., "clean person"). CAUTION ALL MEMBERS OF THE SAMPLING TEAM WILL WEAR APPROPRIATE PPE THROUGHOUT THE DURATION OF SAMPLING TO AVOID POTENTIAL EXPOSURE. CHANGE GLOVES BETWEEN EACH SAMPLE COLLECTION. TO AVOID CROSS-CONTAMINATION OF SAMPLES, FOLLOW APPROPRIATE PPE DONNING AND DOFFING PROCEDURES. DISPOSE OF PPE PROPERLY. NOTE: Clean outer nitrile gloves should be worn for the collection of each new sample to prevent cross-contamination. Points at which gloves must be changed during the sampling procedure are indicated using the "Glove change" graphic below 12 ------- 2.1. Sampling Preparation 2.1.1. Sampling Kit Preparation Sampling kits can be prepared in advance and stored in a clean, dry environment. Examples of a fixed and quick release handle are shown below in Figure A-l. Figure A-l. Sponge-stick sampling kits (fixed handle [3M® sampler, 3M Corporation] kit (on the left) and quick release handle [Solar-Cult® sampler, Hardy Diagnostics] kit (on the right)). A sample kit for each sponge-stick sampler will be prepared in a laboratory setting using the following procedure: 1. Don nitrile gloves. 2. Assemble the sponge-stick kit. • Label the original sponge-stick overpack with a unique sample ID (consistent with an approved Sampling and Analysis Plan). • Label the specimen container with the same unique sample ID. • Place both sampling kit components into the large overpack bag. 3. Label the large bag with the sample ID and place it in a clean sample kit storage container. 4. Store the sample container with sample kits in a clean, dry environment. 2.1.2. Pre-sampling procedures 1. The sampler will don a pair of nitrile gloves over existing gloves. Glove change 13 ------- 2. The support person will don a pair of nitrile gloves over existing gloves, unwrap a template from the bag, and present it to the sampler, taking care not to touch the template. 3. The sampler will then place the template over the surface to be sampled. The template will be secured with tape if preferred. NOTE: If a template cannot be used, measure the target 10-in x 10-in sampling area with a disposable ruler and delineate the area to be sampled (100 in2 [645 cm2]) with masking tape. Change gloves after taping off the area. NOTE: Barcoding and automated linking of sampling kit ID and sample location may be utilized instead of manual recording. 5. The support person will retrieve the corresponding sampling kit for the current sampling location. 6. Without touching the outside of the bag, the sampler will remove the sterile sponge from its package and immediately proceed to sampling. NOTE: Always grasp the sponge stick near the tip/end of the handle. Do not grasp past the thumb stop, near the sponge itself. The left portion of Figure A-2 shows the proper technique for grasping the sponge stick handle during the sampling. ------- \ Figure A-2. Proper technique for grasping the sponge-stick handle during the sampling (left) versus the improper technique (right). 3. Sampling Protocol The sampler will wipe the surface to be sampled using the moistened sterile sponge using the following sampling sequence: 1. Lay the widest part of the sponge on the surface, leaving the leading edge slightly lifted. Apply gentle but firm pressure and use an overlapping 'S' pattern to cover the entire surface with horizontal strokes (Figure A-3). Use the other hand to hold the template during sampling, being careful not to touch the sampling surface with the gloved hand. If no template is used, sample only within the taped area. D 7X? Figure A-3. First pass with sponge - horizontal strokes using one side of the sponge (CDC, 2012). 2. Turn the sponge over and wipe the same area again using vertical 'S'-strokes (Figure A-4). 15 ------- Figure A-4. Second pass with sponge - vertical strokes using the other side of the sponge (CDC, 2012). 3. Turn the sponge by 90° and use the edge of the sponge (narrow side) to wipe the same area using diagonal 'S'-strokes (Figure A-5). Next, flip the sponge by 180° to use the opposite narrow side immediately after the longest stroke midway through the sampling surface (use opposite side at the red dot on Figure A-5). Figure A-5. Third pass with sponge - diagonal strokes using the edges of the sponge (CDC, 2012). 4. Lastly, use the tip of the sponge to wipe the perimeter of the sampling area (Figure A- 6). Figure A-6. Final pass with sponge - perimeter wipe using the tip of the sponge (CDC, 2012). 4. Post Sampling Procedures 16 ------- If a fixed handle sponge stick was used, the sampler will place the head of the sponge directly into a sterile specimen container and break off the head of the sponge by bending the handle and rotating and twisting the head. For the quick- release sponge sticks, the sampler will place the tip of the sponge stick into a sterile container and release the cellulose tip by simultaneously pulling back the quick release mechanism located on both sides of the handle. The end of the sponge handle, touched by the sampler, should not touch the inside of the specimen container. 2. The support person will securely seal and label the container using a unique sample identifier from the sample collection log, the sampler's initials, and the date and time the sample was collected. 3. The support person will place the sample container in the overpack bag and securely seal and label the bag with a unique sample identifier (e.g., sample location, date and time sample was collected, and name of individual collecting the sample). 4. The support person should remove excessive air from the re-sealable plastic bags to increase the number of samples that can be shipped in one container. 5. The support person will then seal the labeled overpack bag and wipe the outside with a disinfecting wipe(s). 6. The sampler will remove and dispose of the tape or the template if used. 7. All sampling team members will remove their outer gloves and discard them. Specimen containers and re-sealable bags may be pre-labeled to assist with sampling Collect a photo of the sampled area, with template in place, if possible to document the sampling location. Clean outer gloves should be worn for each new sample. NOTE: NOTE: efficiency. Glove change ------- Appendix B. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING VACUUM FILTER CASSETTES 18 ------- ASSET NUMBER OPERATING ACTIVITY MAINTENANCE ACCOMPLISHED ASSET HOURS MAINTENANCE DUE ASSET HOURS PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING VACUUM FILTER CASSETTES SCHEDULED UNSCHEDULED DISCREPANCIES FOUND: NO YES MAN HOURS: MK DC EM ET IT GM BM FN SN OTHER REMARKS: TECHNICIAN'S SIGNATURE and DATE TECHNICIAN'S ID ' ASTERISK INDICATES QA REQUIRED QA REFERENCES: Silvestri, E., J. Chandler, Y. Chambers-Velarde, J. Cuddeback, J. Archer, and W. Calfee (2021). "Collection of Microbiological Agent Samples from Potentially Contaminated Porous Surfaces Using Microvacuum Techniques." U.S. EPA Office of Research and Development, Washington, DC, EPA/600/R-20/439. EPA (2017). "Sample Collection Information Document for Pathogens—Companion to Selected Analytical Methods for Environmental Remediation and Recovery (SAM)." U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-17/374. Calfee, M.W. et al. (2013). "Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores "Journal of Microbiological Methods. 95(3): 389-396. REVIEWED BY and DATE DATA ENTRY COMPLETED 19 ------- PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING VACUUM FILTER CASSETTES TOOLS/EQUIPMENT: Quantity Item Name 1 Vacuum sampling kit consisting of: (a) one 37-mm vacuum filter cassette (VFC) loaded with filter. NOTE: Various cassette-filter combinations can be utilized: - 0.8 (mi pore size MCE membrane filter (SKC Inc., Eighty-Four, PA; P/N SKC 225-3-01, or equivalent) - 0.3 |im pore size PTFE membrane (SKC Inc., Eighty-Four, PA; P/N SKC 225-1723, or equivalent) - 0.45 |im pore size filter membrane (Zefon International, Ocala, FL, P/N 7345CC, or equivalent) (b)two PVC Luer adaptors (SKC Inc., Eighty-Four, PA; P/N 225- 132A, Zefon International, P/N ZA0005) NOTE: two adaptors required for SKC cassette, one required for Zefon cassette (c) one sterile 15-mL polypropylene conical tube (Thermo Fisher Scientific, Waltham, MA; P/N 225-352196 or equivalent) (d) one small-size sterile sampling bag (Thermo Fisher Scientific, Waltham, MA; P/N 14-955-176, or equivalent) (e) one medium-size sterile sampling bag with flat-wire closures (Thermo Fisher Scientific, Waltham, MA; P/N 14-955-191, or equivalent) (f) one large overpack sample bag with round wire enclosure (TWIRL' EM ECOLO, Sainte-Julie Quebec, CN; P/N EFR- 1015E, or equivalent) 1 2.5 cm length of Tygon® tubing (0.635 cm inside diameter, SKC Int.; P/N 225-1345, or equivalent); needed for SKC kit only 1 20 cm length of Tygon® tubing (0.635 cm inside diameter, SKC Int.; P/N 225-1345, or equivalent) 1 Gilian 12 Air Sampling Pumps, (Sensidyne, St. Petersburg, FL, P/N 610- 1601-01-R, or equivalent calibrated pump capable of pulling a minimum 5 lpm) 20 ------- 1 Disposable 12 in x 12 in sample template (sample area size 144 in2 [930 cm2]; SKC, Inc., Eighty-Four, PA; P/N 225-2416 or equivalent) or disposable ruler and masking tape 1 Sample kit container (e.g., storage bin, backpack, or other container that will be used for transport and storage of sample kits prior to sampling) 1 Electronic time keeping device 1 Sampling log CONSUMABLES: Quantity Item Name 1 Box of nitrile gloves or equivalent 1 Disinfecting wipes (Dispatch® Disinfectant Towels with Bleach, Clorox Company, Oakland, CA; P/N 69150 or equivalent) 1 Sample labels or permanent marker A. PRELIMINARY STEPS WARNING THE RISK OF FATALITY ASSOCIATED WITH THE INHALATION OF BACILLUS ANTHRACIS SPORES CAN BE EXTREMELY HIGH. TO AVOID THE RISK OF EXPOSURE DURING SAMPLING OF POTENTIALLY CONTAMINATED AREAS, APPROPRIATE PROTECTIVE MEASURES MUST BE INITIATED AS PART OF COORDINATING RESPONSE ACTIONS. IT IS RECOMMENDED THAT A COMPREHENSIVE HEALTH AND SAFETY PLAN BE DEVELOPED TO ADDRESS SAMPLING PERSONNEL SAFETY ISSUES PRIOR TO INITIATION OF SAMPLING ACTIVITIES. TO ELIMINATE THE RISK OF ILLNESS AND DEATH, PROPER PERSONAL PROTECTIVE EQUIPMENT (PPE) MUST BE USED WHEN WORKING IN A POTENTIALLY CONTAMINATED ENVIRONMENT. PPE INCLUDES PROPER RESPIRATORY PROTECTION, PROTECTIVE GARMENTS, AND GLOVES. CONSULT WITH YOUR SAFETY OFFICER FOR PROPER PPE SELECTION. When mandated by the Incident Commander (IC) as part of a Unified Command (UC) or other incident management system, PPE, including respiratory protection, needs to be employed under OSHA standards and regulations found in 29 CFR Part 1910 Subpart I (1910.132—1910.138) or the equivalent construction standards (29 CFR Part 1926). These standards require training on the proper selection, use, removal, and disposal of PPE; a medical evaluation and fit testing 21 ------- must be conducted before the use of any respiratory protection and annually thereafter. Contact the Unified Command (UC), Operations Section Chief, and/or Safety Officer for PPE requirements, and for selecting applicable sampling procedures, stations, and routes. B. SAMPLING WARNING ALL TEAM MEMBERS MUST WEAR SUITABLE PPE. AFTER APPROPRIATE DECONTAMINATION PROCEDURES, PPE MUST BE CORRECTLY REMOVED AND DISPOSED OF. FAILURE TO COMPLY MAY RESULT IN PERSONAL INJURY OR DEATH. CAUTION TO PREVENT THE SPREAD OF CONTAMINATION, DO NOT PERFORM SAMPLING OF THE BOAT IN THE VICINITY OF WATER INTAKES, ENVIRONMENTALLY SENSITIVE AREAS, OR IN AREAS WHERE THE PUBLIC COULD BE AFFECTED. Identify sampling locations on the exterior and interior of the boat. The procedure is intended for the sampling of 12-in x 12-in, non-smooth, porous surface areas (e.g., non-skid surfaces, concrete, etc.). For smooth, non-porous surface sampling, refer to the cellulose sponge-stick sampling procedure (MPC # TBD). For larger area sampling of porous and non-porous surfaces using non- specialized equipment and fresh or seawater sources, refer to the pressurized water spray procedure (MPC # TBD). At a minimum, a two-person team must be used. The team will consist of a sampler (person who will perform sampling of contaminated surfaces; i.e., "dirty person") and a support person (person who will be responsible for taking notes, labeling, and other sample handling procedures; i.e., "clean person"). 22 ------- CAUTION ALL MEMBERS OF THE SAMPLING TEAM WILL WEAR APPROPRIATE PPE THROUGHOUT THE DURATION OF SAMPLING TO AVOID POTENTIAL EXPOSURE. CHANGE GLOVES BETWEEN EACH SAMPLE COLLECTION. TO AVOID CROSS-CONTAMINATION OF SAMPLES, FOLLOW APPROPRIATE PPE DONNING AND DOFFING PROCEDURES. DISPOSE OF PPE PROPERLY. NOTE: Clean outer nitrile gloves should be worn for the collection of each new sample to prevent cross-contamination. Points at which gloves must be changed during the sampling procedure are indicated using the "Glove change" graphic below: 2. Sampling Preparation 2.1. Sampling Kit Preparation Sampling kits can be prepared in advance and stored in a clean, dry environment. See examples of both SKC and Zefon cassette kits below in Figure B-l. 23 ------- Figure B-l. Disassembled VFC sampling kits (SKC cassette kit (on the left) and Zefon cassette kit (on the right)). A sample kit for a single cassette vacuum sample will be prepared in a laboratory setting using the following procedure: 1. Don nitrile gloves. 2. Assemble nozzles for the SKC cassettes; Zefon cassettes come with a nozzle already attached. • For SKC Cassette only: Cut a sampling nozzle, using scissors, from a section of Tygon tubing 2.5 cm long. Then cut one end of the tubing at approximately a 45-degree angle. NOTE: Prepare 25% more nozzles than required by the testing protocol. Sterilize the nozzles with a 15-minute gravity autoclave cycle. 3. Assemble VFC kit. • Label the 37-mm cassette with a unique sample ID. • Label the 15-mL conical tube with the same unique sample ID. • For SKC Cassette only: Remove the cassette plugs and place a PVC adaptor onto each end of the cassette. Save the removed red plugs for inclusion in a later step. • For Zefon Cassettes only: No PVC nozzle is included in the sampling kit. Rather, a PVC adaptor is attached to the vacuum pump tubing and not required to be replaced between samples. • Cut a 20 cm long piece of Tygon tubing with scissors. • Connect the 20-cm tubing onto the adaptor on the downstream end of the cassette. • For SKC Cassette only: Place the sampling nozzle (2.5 cm section 24 ------- of tubing) onto the upstream end (labeled inlet on cassette) of the cassette with the angled side furthest from the cassette. • Place the cassette assembly into the labeled medium size overpack bag (8-in x 10-in bag in Figure 1). The cassette assembly consists of: (for SKC) the attached PVC adaptors, 20 cm tubing, and nozzle; (for Zefon) the cassette and nozzle are pre-assembled and contained within a small bag. For SKC, the end of the cassette that attaches to the vacuum tubing (downstream-most end) should be closest to the bag opening, for easy retrieval in the field. o SKC Only. Place the red plugs previously removed from the cassette into the medium size bag. o Place the 15-mL conical tube and the medium size bag with 37-mm cassette assembly into the large overpack bag (10-in x 12-in bag in Figure 1). o Place an additional, labeled small sterile bag (4-in x 6-in bag in Figure 1) into the large overpack bag. o Label the large bag with the sample ID and place in a clean sample kit storage container. 4. Store the sample container with sample kits in a clean, dry environment. 2.2. Pre-Sampling Procedures 1. The support person will verify the calibration of the sampling pump. The pre-sampling daily calibration check should be performed "according to the manufacturer's instructions," as specified in the equipment operation manual. 2. The support person will record the sample ID number and location in the sampling log. NOTE: Barcoding and automated linking of sampling kit ID and sample location may be utilized instead of manual recording. 3. The support person will turn on the sampling pump and adjust the flow rate to >5 liters per minute (1pm) and record the flow rate in the sampling log. 4. The support person and sampler will don a pair of nitrile gloves over existing gloves. 25 ------- 5. The support person will unwrap a template from the bag and present it to the sampler, taking care not to touch the template. 6. The sampler will then place the template over the surface to be sampled. The template will be secured with tape, if preferred. NOTE: If a template cannot be used, measure the target 12-in x 12-in sampling area with a disposable ruler and delineate the area to be sampled (144 in2 [929 cm2]) with masking tape. Change gloves after taping off the area. 7. The support person will retrieve the corresponding sampling kit for the current sampling location. 8. The support person will record the sample kit ID and sample location number on the sampling log sheet or scan the sample kit ID barcode if automated logging is available. 9. The support person, touching only outside of the largest sample bag, will open the large bag and work the sampling kit bag toward the large bag opening. The support person will then open the sample kit bag, and while keeping hands only on the outside of the largest bag, position the vacuum cassette near the opening of the sample kit bag. 10. The sampler will then remove the 37-mm cassette from the inner sampling kit bag. NOTE: Zefon Cassettes will require the removal of red caps (2) from the tube and the back of the cassette. After removal, the sampler will place the plugs into the open/flat hand of the support person. The support person will then place the plugs into small overpack bag for the duration of sampling. After sample collection, the sampler re-attaches the plugs to the cassette. 11. If the tubing has become unattached to the cassette during retrieval from the kit bag, the support person will hold the tubing for the sampler to place the 37-mm cassette assembly onto the tubing. 12. The sampler places the 37-mm cassette assembly onto the tubing. • For SKC Cassette only: the sampler will remove the 37-mm cassette assembly from the bag and attach it to the Tygon vacuum 26 ------- tube held by the support person. 3. Sampling Protocol Before sampling, the sampler will ensure that the VFC is correctly connected to the Tygon vacuum tube and adjust, if necessary. NOTE: The sampling pump stops occasionally and restarts after 15 seconds. It is a normal self-check function when flow control is being re-zeroed. If the flow rate of the pump cannot be maintained within the constant flow rate specification or the sample media has increased in flow resistance (i.e., back pressure)—due to the accumulated sample or a blockage in the sample train—the pump will go into a fault condition and stop operating. Refer to the pump manufacturer's manual for corrective action. 1. The target sample duration is 300 seconds (5 minutes) per each 12-in x 12- in sampling area, with 50 passes of the sampling nozzle performed horizontally and vertically. 2. The support person will record the duration of sampling. Prompts should be given to the sampler to avoid surpassing the target duration (e.g., half-way, or 2.5 minutes elapsed time). 3. The sampler will turn on the vacuum and sample the surface using the following sampling sequence: Step 1. Vacuum "horizontally" using Z-strokes (left-to-right, right-to-left, etc.) to cover the entire area of the material surface not covered by the template. Step 2. Vacuum the same area "vertically" using the same technique, bottom-to-top, top-to-bottom (N strokes). NOTE: Keep the sampling nozzle angled so that the tapered opening of the sampling nozzle is flush with the sampled surface (Figure B-2). The nozzle should gently touch the surface to be sampled, with an angle such that the nozzle flow and orifice are not blocked by the surface. 27 ------- 1 Figure B-2. Position of a sampling nozzle during VFC sampling. 4. After the conclusion of sampling, each cassette will be disassembled as follows: • SKC cassette procedure a) The sampler will hold the nozzle and remove it from the cassette. b) The support person will remove the 15-mL collection tube from the sample kit and open the tube. c) The sampler will place the nozzle into the 15-ml collection tube with the adapter end down while holding the cassette in the opposite hand. d) The support person will seal the 15-ml collection tube and place it in the small unlabeled bag. e) The sampler will use the "nozzle" hand to remove the tubing from the downstream end of the cassette. f) The sampler will then seal the cassette with the two red plugs found in the small unlabeled sample collection bag. g) The support person will open the small unlabeled sample collection bag. h) The sampler will place the secured 37 mm cassette inside the bag with the 15 mL conical tube. • Zefon cassette procedure a) The sample person will remove the downstream end of the tubing from the back of the cassette. 28 ------- b) The support person will open the bag containing the two red caps for the sampler to retrieve. c) The sampler will then seal the end of the cassette and nozzle with the two red caps found in the small sample collection bag. d) The sampler will place the secured 37-mm cassette inside the small unlabeled sample collection bag. 4. Post Sampling Procedures 1. The support person will seal the small, labeled sample collection bag and wipe the outside with a disinfecting wipe(s). 2. The support person will open the labeled overpack bag and place the smaller unlabeled collection bag containing the cassette inside. 3. The sampler will remove the used 20-cm length of tubing attached to the pump and discard it. 4. The support person should remove excessive air from the re-sealable plastic bags to increase the number of samples that can be shipped in one container. 5. The support person will then seal the labeled overpack bag and wipe the outside with a disinfecting wipe(s). 6. The sampler will remove and dispose of the tape, and the template if used. 7. All sampling team members will remove their outer gloves and discard them. NOTE: Tygon tubing downstream of the cassette can be re-used between samples; however, it should be bleach-wiped between sample locations (at the end of the collection procedure) and should be replaced if any part touches a surface in the exclusion zone. Collection bags may be pre-labeled or barcoded to assist with sampling efficiency. Collect a photo if possible, with template in place, to document the sampling location. Clean outer gloves should be worn for each new sample. Glove change 29 ------- Appendix C. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING PRESSURIZED WATER WASH-DOWN 30 ------- ASSET NUMBER OPERATING ACTIVITY MAINTENANCE ACCOMPLISHED MAINTENANCE DUE ASSET HOURS ASSET HOURS PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING PRESSURIZED WATER WASH-DOWN SCHEDULED UNSCHEDULED DISCREPANCIES FOUND: NO YES MAN HOURS: MK . DC EM . ET . IT . GM . BM FN SN OTHER REMARKS: TECHNICIAN'S SIGNATURE and DATE TECHNICIAN'S ID * ASTERISK INDICATES QA REQUIRED QA REFERENCES: None. REVIEWED BY and DATE DATA ENTRY COMPLETED 31 ------- PROCEDURE FOR BACILLUS ANTHRACIS SURFACE SAMPLING USING PRESSURIZED WATER WASH-DOWN TOOLS/EQUIPMENT: Quantity Item Name 1 5/8" Water hose (Home Depot, Atlanta, GA; P/N 506517 or equivalent) 1 Heavy-duty adjustable brass nozzle (Home Depot, Atlanta, GA; P/N 14033591 or equivalent) 1 Large (4,000 mL or larger) graduated cylinder 1 1-L Nalgene™ Wide-Mouth Lab Quality high-density polyethylene (HDPE) bottles with screw caps, leakproof (Thermo Fisher Scientific, Waltham, MA; P/N 2199- 0032 or equivalent) 1 Overpack sample bag with round wire enclosure ('TWIRL'EM ECOLO, Sainte-Julie Quebec, CN; P/N EFR-1015E or equivalent) 1 Electronic time keeping device 1 Sample kit container (e.g., storage bin, backpack or other container that will be used for transport and storage of sample bottles and overpack sampling bags) 1 Sampling log 1 Bottle holder (optional), e.g., Nasco 6" to 12" extendible swing sampler (Nasco Sampling LLC, Fort Atkinson, WI; P/N B01310WA M or equivalent) CONSUMABLES: Quantity Item Name 1 Box of nitrile gloves or equivalent 1 Disinfecting wipes (Dispatch® Disinfectant Towels with Bleach, Clorox Company, Oakland, CA; P/N 69150 or equivalent) ------- 1 Sample labels or permanent marker A. PRELIMINARY STEPS WARNING THE RISK OF FATALITY ASSOCIATED WITH THE INHALATION OF BACILLUS ANTHRACIS SPORES CAN BE EXTREMELY HIGH. TO AVOID THE RISK OF EXPOSURE DURING SAMPLING OF POTENTIALLY CONTAMINATED AREAS, APPROPRIATE PROTECTIVE MEASURES MUST BE INITIATED AS PART OF COORDINATING RESPONSE ACTIONS. IT IS RECOMMENDED THAT A COMPREHENSIVE HEALTH AND SAFETY PLAN BE DEVELOPED TO ADDRESS SAMPLING PERSONNEL SAFETY ISSUES PRIOR TO INITIATION OF SAMPLING ACTIVITIES. TO REDUCE THE RISK OF ILLNESS AND DEATH, PROPER PERSONAL PROTECTIVE EQUIPMENT (PPE) MUST BE USED WHEN WORKING IN A POTENTIALLY CONTAMINATED ENVIRONMENT. PPE INCLUDES PROPER RESPIRATORY PROTECTION, PROTECTIVE GARMENTS, AND GLOVES. CONSULT WITH YOUR SAFETY OFFICER FOR PROPER PPE SELECTION. When mandated by Incident Commander (IC) as part of a Unified Command (UC) or other incident management system, PPE, including respiratory protection, should be employed under OSHA standards and regulations found in 29 CFR Part 1910 Subpart I (1910.132—1910.138) or the equivalent construction standards (29 CFR Part 1926). These standards require training on the proper selection, use, removal, and disposal of PPE; a medical evaluation and fit testing must be conducted before the use of any respiratory protection and annually thereafter. Contact the Unified Command (UC), Operations Section Chief, and/or Safety Officer for PPE requirements, and for selecting applicable sampling procedures, stations, and routes. B. SAMPLING WARNING ALL TEAM MEMBERS MUST WEAR SUITABLE PPE. AFTER APPROPRIATE DECONTAMINATION PROCEDURES, PPE MUST BE CORRECTLY REMOVED AND DISPOSED OF. FAILURE TO COMPLY MAY RESULT IN PERSONAL INJURY OR DEATH. ------- CAUTION TO PREVENT THE SPREAD OF CONTAMINATION, DO NOT PERFORM SAMPLING OF THE BOAT IN THE VICINITY OF WATER INTAKES, ENVIRONMENTALLY SENSITIVE AREAS, OR IN AREAS WHERE THE PUBLIC COULD BE AFFECTED. 1. Identify sampling locations on the exterior and interior of the boat. This procedure is intended for large area sampling of porous and non-porous surfaces using non-specialized equipment and fresh or seawater sources. For smaller areas that involve discrete sampling using conventional sampling methods, refer to the cellulose sponge-stick sampling procedure (MPC # TBD) and vacuum filter cassette sampling (MPC # TBD). At a minimum, a three-person team must be used. The team will consist of a sampler (person who will perform wash-down of surfaces), a sample support person (person who will be responsible for taking notes, labeling, and other preparatory procedures), and a sample handler (who will collect water run-off or sampling liquid). The sample handler will also assist the sample support person for data recording and shipment of samples to the analytical laboratory. CAUTION ALL MEMBERS OF THE SAMPLING TEAM WILL WEAR APPROPRIATE PPE THROUGHOUT THE DURATION OF SAMPLING TO AVOID POTENTIAL EXPOSURE. CHANGE GLOVES BETWEEN EACH SAMPLE COLLECTION. TO AVOID CROSS-CONTAMINATION OF SAMPLES, FOLLOW APPROPRIATE PPE DONNING AND DOFFING PROCEDURES. DISPOSE OF PPE PROPERLY. NOTE: ------- Clean outer nitrile gloves should be worn for the collection of each new sample to prevent cross-contamination. Points at which gloves must be changed during the sampling procedure are indicated using the "Glove change" graphic below: 1. Sampling preparation 1. Each member of the sampling team will don a clean pair of gloves over existing gloves. 2. The sample support person will attach the hose adapter to the water source spigot and hand-tighten the connection. NOTE: The water hose length should allow for sufficient operational dexterity on the boat. Any fresh or seawater supply equipped with a spigot compatible with the water hose can be used for wash- down. 3. The sample support person will attach the spray nozzle to the hose and hand-tighten the connection. 4. After the spray system is assembled, the sampler will open the nozzle and adjust the spray flow to produce a water cone of approximately 12- to 14-in diameter, at approximately 2 ft from the surface to be sampled, to minimize the contaminated water overspray to the operator. These parameters may need to be modified depending on the water hose pressure and type of nozzle used. 5. After verification of the flow pattern, perform the flow rate measurement using the graduated cylinder and electronic time-keeping device (stopwatch). The sample support person will record the time and amount of liquid collected and calculate the spray rate. 6. The nozzle should be directly above the graduated cylinder. ------- Simultaneously, as the sampler opens the nozzle, the sample support person will start the stopwatch and give prompts to the sampler to spray for a predetermined amount of time (15 seconds). After the time lapses, the sampler will shut the nozzle off and read the volume of liquid dispensed into the graduated cylinder. The nozzle's flow rate (expressed in liters per minute, 1pm) is the amount of liquid dispensed (in liters, 1) divided by the spray time, in minutes (min). The sample support person will record the flow rate measurements in the sampling log. 7. The sample support person will record the sample ID number and location in the sampling log. NOTE: Barcoding and automated linking of sampling kit ID and sample location may be utilized instead of manual recording. 8. The sample support person will retrieve the corresponding sampling kit for the current sampling location and prepare for the wash-down of the first half of the boat. NOTE: Typically, a wash-down of the forward and aft half of the boat will be performed. For larger vessels, additional segments can be sampled (e.g., forward, mid, and aft sections), depending on the accessibility of the sample collection points using drains or scuppers. Some run-off collection locations may only be used when the vessel is trailered, but not while afloat. The operational accessibility of the run-off collection locations must be determined prior to wash-down for each area to be sampled. 9. The water runoff should be collected into a sterile 1-L Nalgene bottle directly from the deck drains or scuppers located closest to the sampled area. The run-off collection locations for the RB-S class vessel are shown in Figure C-l. ------- TiS-dAL FUEL TAMK ' HIKJ, 7 m W.I.1S1/1 • WT 16 - BtACHWC PLATE (HULL DDI JBLER) Figure C-l. The run-off collection locations for the RB-S class boat: the forward bow drain (A) and aft drain (or stern scupper) (B). 2. Sampling Protocol The target water spray duration is approximately 15 seconds per each 2-ft x 2-ft surface area, with several passes of the spray nozzle performed using horizontal overlapping strokes. This spray rate results in approximately 2 min-long wash- downs for each boat section to be sampled for an RB-S class boat. Spray durations for larger vessels can be modified as needed. 1. When ready to begin sampling, the sampler will position the nozzle approximately 2 ft above the sample surface. The nozzle should generate a cone that is approximately 12-14-in in diameter at this height. 2. The sample handler will don nitrile gloves and prepare to collect the run-off from the scupper drain closest to the sampled area. NOTE: The sample handler must be ready to collect the sample when spraying starts, as the first flush (first 1-L flow runoff) is the most desirable to collect. Bottle holders (e.g., grip sticks) may be utilized when needed. ------- 3. 4. 5. The sampler will start spraying and prompt the sample handler to start collecting the sample. The sampler will spray the surface using horizontal S-strokes (left-to- right, right-to-left, etc.), working from midship forward (forward half sample) or from midship toward aft (for aft section sampling), washing the vessel from the windshields, towards the deck walls, and driving run-off toward the deck surface and floor drains. The passes should be positioned so that the spray cone overlaps the edges of the outboard by 2-3 in. The sample handler will begin collecting surface run-off from the front or rear scupper drains directly into a 1-L sterilized Nalgene bottle until the sampling container is 90% full (or the run-off stops, whichever occurs sooner). A small amount of headspace should be left so that samples can be homogenized prior to analysis. The sample handler will then close the sample collection bottle carefully. Figure 2 shows an example of the run-off collection from the forward (bow deck) drain and aft drain (stern scupper) of the RB-S class boat. Figure C-2. Wash-down run-off collection from the stern scupper (left) and bow deck drain (right) of the trailed RB-S type vessel. 4. Post Sampling Procedures 1. The sample handler will wipe the outside of the closed collection bottle with disinfecting wipe(s) and mark the surface sampling liquid (water run-off) level in the bottle with the permanent marker. 2. The sample support person will open the labeled overpack bag and place the bottle inside. 3. The sample support person will then seal the labeled overpack bag, wipe it with a disinfecting wipe, and place it in the shipping container for transport to the analytical laboratory. 4. The sample support person should remove excessive air from the re- sealable plastic bags to increase the number of samples that can be ------- shipped in one container. 5. The sampler will wipe the nozzle with a disinfecting wipe. The sampler will then wipe the outside of the other spray kit components (hoses, connectors, and water pump). 6. All sampling team members will remove their outer gloves and discard them. Collection bags may be pre-labeled to assist with sampling efficiency. Collect a photo of the sampled area if possible, to document the sampling location. Clean outer gloves should be worn for each new sample. NOTE: Glove change ------- |