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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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.
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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.
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Appendix A. PROCEDURE FOR BACILLUS ANTHRACIS SURFACE
SAMPLING USING CELLULOSE SPONGE-STICK
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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
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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
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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.
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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
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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
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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.
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\
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).
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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.
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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
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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
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