United States Environmental Protection Agency ------- ------- EPA/600/R-10/026 April 2010 www.epa.gov/ord INVESTIGATION REPORT Persistence Testing of Brucella suis on Outdoor Materials UNITED STATES ENVIRONMENTAL PROTECTION AGENCY RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711 Office of Research and Development National Homeland Security Research Center, Decontamination and Consequence Management Division ------- Disclaimer The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development, National Homeland Security Research Center, funded and directed this investigation through a Blanket Purchase Agreement under General Services Administration contract number GS23F0011L-3 with Battelle. This document has been subjected to the Agency's review and has been approved for publication. Note that approval does not signify that the contents necessarily reflect the views of the Agency. Mention of trade names or commercial products in this document or in the methods referenced in this document does not constitute endorsement or recommendation for use. Questions concerning this document or its application should be addressed to: Joseph P. Wood U.S. Environmental Protection Agency Decontamination and Consequence Management Division Mail Code E343-06 Research Triangle Park, NC 27711 (919) 541-5029 wood.ioseph(@,epa.gov ------- Acknowledgments Contributions of the following individuals and organizations to the development of this document are acknowledged. United States Environmental Protection Agency (EPA) Office of Research and Development, National Homeland Security Research Center Alan Lindquist Sanjiv Shah Sarah Taft Deborah McKean Frank Schaefer Office of Solid Waste and Emergency Response Curtis Snook (Office of Emergency Management, National Decontamination Team) David Wright (Office of Superfund Remediation and Technology Innovation, Environmental Response Team) EPA Regions Jon Rauscher (Region 6) Richard Rupert (Region 3) Randy Schademann (Region 7) United States Department of Energy Paula Krauter (Sandia National Laboratories) Battelle Karen Riggs James Rogers Harry Stone ------- Contents Disclaimer iv Acknowledgments v Abbreviations/Acronyms viii Executive Summary ix 1.0 Introduction 1 2.0 Investigation Approach 3 2.1 Biological Agent 3 2.2 lest Materials 3 2.3 Spiking Coupons 4 2.4 Environmental Conditions 4 2.5 lest Matrix 6 2.6 B. suis Recovery 7 3.0 Quality Assurance/Quality Control 9 3.1 Instrument/Equipment Testing, Inspection, and Maintenance 9 3.2 Inspection/Acceptance of Supplies and Consumables 9 3.3 Data Management 9 3.4 Assessment and Response Actions 9 3.4.1 Technical Systems Audit 9 3.4.2 Performance Evaluation Audit 9 3.5 Data Quality Audit 9 3.6 Reports to Management 9 3.7 Data Review 10 3.8 Performance Criteria 10 3.9 Deviation 10 4.0 Test Results 13 4.1 Method Demonstration - B. suis Recovery 13 4.2 Persistence Results 13 4.2.1 Aluminum 13 4.2.2 Concrete 15 4.2.3 Glass 15 4.2.4 Soil 18 4.2.5 Wood 20 5.0 Summary 21 6.0 References 23 ------- Figures Figure 2-1. Spiking coupon using a multichannel pipette 4 Figure 2-2. Refrigerator configured with ultraviolet-A/B lamps 5 Figure 2-3. Schematic (top view) of ultraviolet radiation sampling locations 6 Figure 4-1. B. suis persistence on aluminum 15 Figure 4-2. B. suis persistence on glass 18 Figure 4-3. B. suis persistence on soil 20 Tables Table ES-l. Summary of persistence test conditions andB. suis recoveries x Table 2-1. Test materials 3 Table 2-2. Ultraviolet radiation levels 6 Table 2-3. B. suis persistence test matrix 7 Table 3-1. Performance evaluation audits 10 Table 3-2. Spike and positive control recovery data from persistence testing 11 Table 4-1. Recovery data from method demonstration 13 Table 4-2. B. suis persistence on aluminum 14 Table 4-3. B. suis persistence on concrete 16 Table 4-4. B. suis persistence on glass 17 Table 4-5. B. suis persistence on soil 19 Table 4-6. B. suis persistence on wood 20 Table 5-1. Summary of B. suis persistence 22 ------- Abbreviations/Acronyms BHI brain heart infusion °C degrees Celsius CPU colony-forming unit(s) cm centimeter EPA U.S. Environmental Protection Agency g gram mL milliliter NHSRC National Homeland Security Research Center nm nanometer OD optical density PBS phosphate-buffered saline QA quality assurance RH relative humidity TSA technical systems audit uL microliter UV ultraviolet UV-A ultraviolet radiation within the wavelength range of 320 to 400 nanometers UV-A/B ultraviolet radiation within the wavelength range of 290 to 400 nanometers (i.e., UV-A and UV-B) UV-B ultraviolet radiation within the wavelength range of 290 to 320 nanometers UV-C ultraviolet radiation within the wavelength range of 180 to 290 nanometers uW microwatt ------- Executive Summary The persistence of biological agents is influenced by environmental conditions and the materials with which these biological agents are in contact. The generation of scientifically defensible persistence data is useful for the proper planning of decontamination efficacy tests and helps formulate response or remediation plans in preparation for possible natural occurrences or intentional releases of biological agents. This report presents the results of an investigation to evaluate Brucella suis persistence on five materials (typically found in the outdoor environment) under various environmental conditions and exposure durations. Persistence (recovery of viable organisms) was assessed for B. suis spiked onto five materials (aluminum, concrete, glass, soil, and wood). The spiked materials were then exposed to controlled environmental conditions. The environmental conditions comprised moderate temperature (about 22 °C) or low temperature (about 5 °C), and the absence or presence of ultraviolet (UV) radiation within the wavelength range of 290 to 400 nm (UV-A/B, which was intended to simulate natural sunlight). Relative humidity (RH) was ambient and not controlled. The persistence investigation lasted up to 28 days. Persistence was determined by the recovery of B. suis as colony-forming units (CPU) from the materials at the completion of the exposure duration. The persistence investigation results are summarized in Table ES-1. In the absence of UV-A/B, B. suis persisted on aluminum, glass, and soil for at least 28 days (the longest duration tested), at both low and moderate temperatures. On concrete, B. suis was only recovered from the seven day test at the "low temperature, no UV" environmental condition. Because B. suis showed low persistence on concrete, the concrete coupons were replaced with wood coupons for the longer persistence tests. Persistence testing with B. suis on wood resulted in recoveries after 21 and 28 days of exposures to the "low temperature, no UV environmental condition, but B. suis was not recovered from wood after 21 and 28 days of exposures to the "moderate temperature, no UV environmental condition. In general B. suis had higher CPU recoveries (persisted longer) at low temperature than at moderate temperature. In the presence of UV-A/B, the length of time that B. suis persisted on aluminum and glass was reduced, so that the longest exposure durations from which B. suis were recovered ranged from 2 to 7 days. Such reductions in the duration of B. suis persistence were not as pronounced when B. suis was spiked onto soil and exposed to UV-A/B. In the presence of UV-A/B, B. suis was recovered from soil at the longest exposure durations tested (for at least 14 days at "moderate temperature, UV-A/B" and for at least 14 days at "low temperature, UV-A/B"). When exposed to UV-A/B, B. suis was not recovered from concrete. Testing was not conducted with B. suis on wood in the presence of UV-A/B. ------- Table ES-1. Summary of persistence test conditions and B, suis recoveries Duration (Days) 7 14 21 28 Temperature (°C)t 23 23 23 23 BiffE 39 29 39 39 'MM ^Ei^^n^^^^l Moderate Temperature 7.71 x 104 2.29 x 104 1.38x 104 5.19x 102 Moderate Temperature, 1 2 4 7 10 14 22 23 22 22 22 23 50 50 50 50 50 48 NT NT l.SOx 102 2.27 x 102 ND ND , NoUV 0 0 NT NT UV-A/B§ ND ND ND ND NT NT • rr*i-i it . suis (LrU/cc Glass 3.32 x 104 7.36 x 103 6.26x 102 7.87x 102 2.53x 102 ND ND ND NT NT >upon) by Material* Soil 2.66x 104 9.29x 103 6.88x 102 1.13x 102 NT NT 5.03x 104 3.49 x 103 1.67x 103 4.33 x 102 Wood NT NT ND ND NT NT NT NT NT NT Low Temperature, No UV 7 14 21 28 5.3 5.8 4.1 4.4 52 50 11 12 8.75x 106 1.81 x 106 9.82 x 105 3.48 x 105 5.32 x 101 ND NT NT 2.23x 106 2.07x 105 7.75x 105 3.05x 105 9.67x 104 5.41 x 104 9.35 x 104 4.14x 104 NT NT 1.73x 105 1.57x 105 Low Temperature, UV-A/B5 1 2 2# 5 5# 14 6.5 7.6 4.9 4.9 4.5 4.2 53 46 58 59 58 57 4.10 x 103 2.20 x 102 8.68 x 101 ND 1.32 x 101 NT ND ND ND ND NT NT 4.23 x 103 4.21 x 102 ND ND NT NT 4.24 x 106 4.05 x 106 4.57 x 106 1.01 x 106 7.69x 105 7.11 x 103 NT NT NT NT NT NT # Spike controls ranged from 5.67 x 107 to 1.22 x 108 CPU/coupon. Mean temperature and RH values based on continuous monitoring at 1 to 2 minute intervals. "ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is approximately 33 CPU/coupon (see Section 2.6). UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. Tests were repeated to improve the quantification of the UV measurements. "NT" is a condition/material that was not tested. ------- 1.0 Introduction The U.S. Environmental Protection Agency (EPA) is investigating the persistence of biological and chemical agents in the absence of decontamination. For biological agents, persistence reflects the extent to which viability is retained over a defined period of time. Some biological agents are unstable and lose viability within minutes of their release, thereby diminishing the risk to human health and the environment; other agents can remain viable for weeks, months, or years. The persistence of biological agents is influenced by environmental conditions and the materials with which they are in contact. The generation of scientifically defensible persistence data is useful for the proper planning of decontamination efficacy tests and helps formulate response plans in preparation for possible natural occurrences or intentional releases of biological agents. This investigation focused on the persistence ofBmcella suis, a bacterium that can cause a debilitating influenza-like illness in humans. The intent was to determine the length of time that B. suis remained viable on various materials found in the outdoors (aluminum, concrete, glass, soil, and wood) under various environmental conditions, primarily while exposed to moderate or low temperatures and in the presence or absence of ultraviolet (UV) radiation levels typical of those associated with sunlight. ------- ------- 2.0 Investigation Approach This report describes the investigation of the persistence of B. suis on various materials exposed to various temperature and UV radiation conditions. Briefly, B. suis was spiked onto materials (aluminum, concrete, glass, soil, and wood) that could become contaminated in the outdoor environment and then be exposed to environmental conditions for controlled exposure durations. Persistence was then assessed by quantifying the recovery of B. suis as colony-forming units (CPU) from each tested combination of material, environmental condition, and exposure duration. All testing was performed in accordance with the peer-reviewed and EPA-approved Quality Assurance/Test Plan for Persistence Testing ofBrucella suis on Outdoor Materials.^ 2.1 Biological Agent The biological agent B. suis is a gram-negative, aerobic, non-spore-forming coccobacillus.(2) For this investigation, B. suis biotype I (Battelle BRU163) was used. The B. suis biotype I was originally obtained from American Type Culture Collection (Manassas, VA) and maintained in pure culture by Battelle. Fresh B. suis culture was prepared in advance of each day that coupons were spiked by transferring colonies from a streak plate (freshly growing or stored less than two weeks at 2 °C to 8 °C) into 10 mL of brain heart infusion (BHI) broth (BD Diagnostic Systems, Sparks, MD) and incubated overnight at 37 °C ± 2 °C on an orbital shaker set to 200 revolutions per minute, until an increase in turbidity was observed. The bacterial culture (late log phase of growth) was diluted with BHI broth to an optical density (OD) reading at 600 nm (OD600 nm) of approximately 0.1 to 0.2 OD units. The 0.1 to 0.2 OD600 nm has previously Table 2-1. Test materials been shown to yield average CFU/mL that would meet the stock suspension spore concentration requirements for this investigation (1 x 107 - 1 x 109 CFU/mL). The viable B. suis bacteria in the stock suspension were enumerated to determine CFU/mL ("spike control") by analyzing serial dilutions (serial 1:10 dilutions) of the stock suspension prepared using phosphate-buffered saline (PBS) and plated onto BHI agar for CPU determination. 2.2 Test Materials Materials typically found in the outdoors that were used for B. suis persistence testing are described in Table 2-1. Test coupons of the outdoor materials were generally cut to the sizes indicated in Table 2-1 from larger pieces of stock material. Concrete coupons were poured into molds rather than being cut to size. Soil "coupons" consisted of 3.5 cm diameter Petri dishes with a height of 1 cm lined with Parafilm® and filled with equal masses (7 g ± 1 g) of uncompacted soil. Coupons were sterilized by autoclaving or gamma irradiation (no pre-cleaning of the coupons occurred prior to sterilization). The selected approach, as shown in Table 2-1, was based on cost-effectiveness and minimization of physical alterations of the material. Autoclaving followed Battelle's standard operating procedure.(3) Gamma-irradiation at 40 kilogray was conducted by STERIS Isomedix Services (Libertyville, IL). Prior to gamma irradiation, coupons were sealed in 6 mL Urine® poly tubing (Urine, Chicago, IL) to preserve sterility until the coupons were ready for use. Test coupons were each visually inspected prior to being used in any experiment or test. Coupons with anomalies on the application surface were discarded and not used. Material Aluminum (finished) Concrete (unpainted) Glass Soil (topsoil) Wood (untreated pine) Lot, Batch, or Observation Aluminum alloy 2024 5 parts sand: 2 parts cement American Society for Testing and Materials International C1036 Batch No. PY1A0597 Generic molding Soil "coupons" consisted of a 3.5 cm diameter Petri • Manufacturer or Supplier Name Adept Products, Inc., West Jefferson, OH Wysong Concrete, Fairfield, OH Brooks Brothers Glass and Mirror Service, Columbus, OH GardenScape, Inc., Eau Claire, PA West Jefferson Hardware, West Jefferson, OH dish with a height of 1 cm (or equivalent) Coupon Size, Width x Length 1.9 cm x 7.5 cm 1.9 cm x 7.6 cm 1.9 cm x 7.5 cm * 1.9 cm x 7.5 cm lined with Parafilm8 and Coupon Thickness 0.2 cm 1.3cm 0.3 cm * 1 cm filled with 7 g ± 1 g c Material Preparation Autoclave Autoclave Autoclave Gamma irradiation Gamma irradiation if uncompacted soil. ------- 2.3 Spiking Coupons The stock suspension (approximately 1 x 108 CFU/mL, prepared as described in Section 2.1) was used to spike the coupons. Test and positive control coupons were placed lying flat in the cabinet and spiked with a 100 uL aliquot of stock suspension of approximately 1 x 108 CFU/mL of B. suis generally using a multichannel micropipette as two rows of five droplets (10 uL per droplet) across the surface of the coupons (Figure 2-1); soil coupons were spiked using a single channel pipette to apply ten 10 uL droplets across the material surface. The 100 uL aliquot of stock suspension yielded approximately 1 x 107 CPU/coupon. Spiked coupons were immediately exposed to the test conditions without a drying period. Spiking of test coupons with B. suis was performed in a Class III biological safety cabinet. To ensure further cleanliness and prevent contamination of test surfaces, sterile techniques following Battelle policies and guidelines(4"6)were exercised during all phases of handling the test coupons. Prior to spiking, each coupon was assigned a unique identifier code by the test personnel for traceability. The identifier code was placed on the coupons and test tubes in indelible ink. 2.4 Environmental Conditions The four environmental conditions tested were based on temperature (moderate and low) and UV radiation (absent or present). UV radiation, within the wavelength range of 290 nm to 400 nm (UV-A/B) was intended to simulate natural sunlight. Relative humidity (RH) was ambient; efforts were not taken to achieve an especially low, high, or stable RH level during any of the tests. The four environmental conditions included: • Moderate temperature, ambient (about 22 °C), no UV • Moderate temperature, ambient (about 22 °C), UV-A/B (12-hour on/off cycle) • Low temperature, about 4 °C, no UV • Low temperature, about 7 °C, UV-A/B (12-hour on/off cycle). The target temperature for the "low temperature, UV-A/B" environmental condition was 7 °C, rather than 4 °C which was the target temperature for the "low temperature, no UV environmental condition. The 7 °C target temperature was selected to avoid potential freezing/thawing cycles that might have been generated with the use of heat-producing lamps inside a refrigerator. Persistence testing was conducted inside a compact glove box (Plas Labs, Inc., Lansing, MI) for moderate temperature tests and inside a refrigerator modified with glove ports for the low temperature tests. In the low temperature testing, temperature and RH were monitored (every one to two minutes) using a Yokogawa DX2010 data logger (Yokogawa Electric Corporation, Tokyo) connected to an Omega HX93 AC temperature/RH probe (Omega Engineering Inc., Stamford, CT); for moderate-temperature testing a HOBO U10 data logger (Onset Computer Corporation, Bourne, MA) was used to measure temperature and RH. Although RH was monitored, efforts to manipulate the RH level were not undertaken. The actual temperatures and RH levels observed during testing are documented in the test matrix (Table 2-3) presented in Section 2.5. Fluorescent UV-A/B lamps inside the glove box or refrigerator were used to generate the UV-A/B (see Figure 2-2 for a photograph of the refrigerator configured with the UV-A/B lamps). UV radiation conditions were designed to approximate the intensity and wavelengths of UV radiation (especially UV-B, with UV radiation within the wavelength range of 290 to 320 nm) encountered in natural sunlight at the earth's surface. UV-B is reported to be the primary component of sunlight which inhibits biological activities.(7) The spectrum and intensity of terrestrial UV radiation is variable and is affected by time of day, day of year, geographical location, atmospheric pollution, and clouds. Naturally- occurring UV-B levels, observed around noon, range from 19.5 to 150 uW/cm2.(8-n) The target UV-B level of 70 uW/cm2 was generated using ReptiSunTM 10.0 Linear Fluorescent UV-B lamps (Zoo Med Laboratories, Inc., San Luis Obispo, CA). The amount of UV-A (UV radiation within the wavelength range of 320 to 400 nm) generated Figure 2-1. Spiking coupon using a multichannel pipette ------- during testing was approximately 110 uW/cm2, which was within the range of UV-A observed in natural sunlight (0 to 4,500 uW/cm2).(12) The level of UV-C (UV radiation within the wavelength range of 180 to 290 nm) generated during testing was 0 uW/cm2; solar UV-C does not generally reach the earth's surface/12'13) UV radiation was conducted for 12 hours (UV-A/B lamps on), and then the UV-A/B lamps were turned off for 12 hours to simulate diurnal conditions. UV radiation was measured with Solarmeter® Model 5.7 for total UV radiation, Model 6.2 for UV-B, and Model 8.0 for UV-C, from Solartech, Inc. (Harrison Township, MI). UV radiation measurements were made from five positions beneath the UV-A/B lamps (Figure 2-3). UV radiation measurements were taken beneath the UV-A/B lamps at the same distance the test coupons were from the UV-A/B lamps. UV-B, UV-C, and total UV radiation were generally monitored twice a day (at least four hours apart) during persistence testing when the UV-A/B lamps were operating (UV-A was calculated as total UV radiation minus the UV-B and UV-C levels). UV radiation measurements were generally monitored twice daily (at least four hours apart) during the persistence testing when the UV-A/B lamps were operating. UV radiation was not measured on Saturdays, Sundays, or at night. In only one instance, testing was initiated in the afternoon and therefore only one UV radiation measurement event occurred on the first test day. During the low temperature tests a downward drift in the UV-B measurements was discovered. This drift was evident as a decreasing trend in measured UV-B levels during the five-day test at low temperature. After the test, once the UV radiation meters were allowed to warm to room temperature, the measured irradiation inside the refrigerator showed typical UV-B levels (approximately 70 uW/cm2). Therefore, the downward trend in measurements was attributed to the prolonged exposure of the UV radiation meters to the low temperature conditions. Subsequent low temperature tests were conducted with UV radiation measurements taken only pre- and post-test, which enabled the UV radiation meters to be stored at room temperature when not in use. Although all of the UV radiation levels measured during each test are provided in Table 2-2, as footnoted, some of the low temperature measurements are believed to be inaccurate. The mean and range calculated included all irradiation measurements taken during a given persistence test. For example, the seven-day test shown in Table 2-2 resulted in 10 measurement events with five individual measurements taken at each event; the mean and range in Table 2-2 were for all fifty measurements (10 measurement events x five individual measurements per event = 50 measurements). Figure 2-2. Refrigerator configured with ultraviolet-A/B lamps ------- Work surface (top view) Five points measured for UV-B, UV-C and total UV radiation during persistence testing All coupons located within dashed line (irradiated area) Figure 2-3. Schematic (top view) of ultraviolet radiation sampling locations (not to scale) Table 2-2. Ultraviolet radiation levels* Duration (Days) UV Radiation Measurement Events UV-A (uW/cm2)' Moderate Temperature, UV-A/B* 1 2 4 7 10 14 1 2 2 5 5 14 2 (twice daily) 4 (twice daily) 4 (twice daily) 10 (twice daily) 16 (twice daily) 20 (twice daily) Low Temperature, 2 (pre- and post-testing) 4 (twice daily) 2 (pre- and post-testing) 9 (twice daily) 2 (pre- and post-testing) 2 (pre- and post-testing) 94 ±7.4 94 ±5. 8 96 ±5. 5 97 ±5. 7 96 ± 5.3 96 ± 5.3 UV-A/B* 110±6.0 113 ±5.6 109 ±6.9 101 ± 12 111 ±7.6 110±6.9 UV-B (uW/cm2)* 1 68 ± 4.2 68 ± 4.4 68 ± 4.0 69 ±4.1 68 ±4.1 69 ± 4.0 67 ±5.9 63 ± 8.3§ 67 ±7.1 60 ± 8.8§ 67 ±5.0 66 ±5.6 * The target UV-C level was 0 pW/cm2 and all measurements were 0 pW/cm2. ' Data are expressed as mean ± standard deviation. * The target UV-A level was 110 pW/cm2 and target UV-B level was 70 pW/cm2; UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. 8 These measurements are believed to be inaccurate, due to the effect of low temperature on the meter. The actual UV-B levels are believed to be in the range of the tests in which pre- and post-testing measurements were conducted. 2.5 Test Matrix The test matrix for persistence testing with B. suis is provided in Table 2-3; the order of the testing was not always done in the sequence shown in the table. After spiking the coupons, at least four non-zero exposure durations were monitored for each environmental condition. The range of exposure durations was determined in consultation with the EPA Task Order Project Officer and was based on the outcome of initial persistence test results. Positive controls (coupons spiked with B. suis and extracted at time-zero, i.e., immediately after spiking) and blanks (coupons not spiked with B. suis) were also run for each material, environmental condition, and exposure duration combination. A brief method demonstration ensured that the methods previously used(14) were adequate to obtain sufficient recovery of B. suis from the materials, with the exception of aluminum for which adequate recoveries were already established. Testing with wood was only occasionally conducted (i.e., in some cases B. suis would not persist on concrete, so persistence was tested on wood as an alternative material). ------- 2.6 B. suis Recovery For sample extraction, test coupons, positive controls, and blanks were transferred aseptically to sterile individual 50 mL conical vials containing 10 mL of sterile extraction buffer (i.e., PBS). For the soil "coupon", soil and the Parafilm liner (which prevented soil from adhering to the Petri dish) were removed from the Petri dish and placed into the vial containing the extraction buffer. The vials were agitated on an orbital shaker for 15 minutes at approximately 200 revolutions per minute at room temperature. Following extraction from the coupons, the extracts were removed and a series of dilutions (serial 1:10 dilutions) were prepared using PBS. During this investigation, B. suis was not recovered from any of the associated blanks. An aliquot (0.1 mL) of the selected dilutions and, when necessary, the undiluted extract were plated onto BHI agar (BD Diagnostic Systems, Sparks, MD) in triplicate. The cultures were incubated for up to 72 hours at 37 °C ± 2 °C. The colonies were counted manually and the CPU/ mL determined. Traditionally plates having colony counts between 25 and 250 are typically used for calculating the CFU/mL. However, under certain circumstances (i.e., poor recovery, reduced persistence over time, efficient decontamination, etc.) there were less than 25 colonies per plate from the undiluted extract. In these cases the number of colonies was counted and recorded even if there were 25 colonies or less per plate. The CPU/coupon were calculated Table 2-3. B, suis persistence test matrix by multiplying the CFU/mL by the volume of the extraction buffer used for each coupon (10 mL per coupon). The total CPU extracted from a coupon was calculated as: Total CPU/coupon = [(mean CPUplate count x I/dilution factor)'/plated volume] x (extraction buffer volume) (1) Where: Mean CPU plate count Plated volume Dilution factor Extraction buffer volume = average number of colonies counted in three replicate plates = volume that was applied to each plate, in this case 0.1 mL = portion of the total extraction buffer that was used to prepare the dilutions = volume of extraction buffer used to extract coupon, in this case 10 mL. A single viable bacterium present in a plated aliquot of sample would be expected to be observed as a CPU. Considering one CPU observed on one of the three plates of undiluted extract, the individual coupon detection limit is approximately 33 CPU/coupon based on Equation 1. Since only a portion (i.e., 0.1 mL aliquot per plate) of undiluted extract is cultured, viable bacteria could be present in the Target Environmental Condition Moderate temperature (22 °C), ambient RH, no UV Moderate temperature (22 °C), ambient RH, UV-A/B* Low temperature (4 °C), ambient RH, noUV Low temperature (7 °C), ambient RH, UV-A/B* Material Aluminum, concrete*, glass, soil, and wood§ Aluminum*, concrete11, glass11, and soil* Aluminum, concrete*, glass, soil, and wood§ Aluminum, concrete, glass, and soil1 Duration (Days) 7 14 21 28 1 2 4 7 10 14 7 14 21 28 1 2 2 5 5 14 Temperature (°O* 22.8 ±0.35 22.8 ±0.33 23.3 ± 0.83 23.3 ± 0.83 22.1 ±0.92 22.6 ± 1.25 22.3 ±0.95 22.3 ±0.96 22.4± 1.15 22. 7 ± 1.21 5.29 ±0.95 5.79± 1.00 4.08 ±3. 97 4.43 ±3. 25 6.47 ±2. 22 7. 56 ±4.30 4.87 ±2.38 4.92 ± 2.47 4.48 ±2. 61 4.22 ± 2.94 RH (%)* 38. 6 ±22. 5 29. 2 ±21.3 38. 8 ±2.88 38. 8 ±2.83 49. 9 ±2.48 50.4 ±4.82 50.1 ±4.36 50. 2 ±3.94 49. 6 ±3.66 48.4 ±4.15 52. 0± 15.0 50. 0± 14.0 10. 7 ±5.87 11.5±5.59 53.0 ±24.0 45. 5 ±23.0 58.4 ±25. 7 58. 7 ±20.9 58. 2 ±25. 7 56. 6 ±25.9 * Data are presented as mean ± standard deviation. ' The target UV-A level was 110 pW/cm2 and target UV-B level was 70 pW/cm2; UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. * Concrete was tested only on days 7 and 14. 8 Wood was tested only on days 21 and 28. * Aluminum and soil were tested on days 4, 7, 10, and 14. 1 Concrete and glass were tested on days 1, 2, 4, and 7. Only soil was tested on day 14. ------- extract that were not collected for plating. However, given combination by dividing the total number of viable organisms the number of replicate coupons (five) and replicate plates extracted from all five test coupons by the number of (three) per undiluted coupon extract it is unlikely that the replicate coupons (i.e., five). Persistence (recovery) curves, presence of viable bacteria would go undetected. out to 28 days, were also developed for B. suis for several The recovery of B. suis bacteria (quantified as mean CPU/ material wd environmental condition combinations, by coupon ± standard deviation) was calculated for each ^P1^ B' suis recovered (quantified as CPU/coupon) material/environmental condition/exposure duration agams ime. ------- 3.0 Quality Assurance/Quality Control Quality assurance (QA)/quality control procedures were performed in accordance with the program quality management plan(15) and the test/QA plan(1) for this investigation. QA/quality control procedures are summarized below. 3.1 Instrument/Equipment Testing, Inspection, and Maintenance All equipment (e.g., pipettes, incubators, biological safety cabinets) used was verified as being certified, calibrated, or validated at the time of the investigation and was maintained and operated according to the quality requirements of the Battelle Biomedical Research Center. 3.2 Inspection/Acceptance of Supplies and Consumables Supplies and consumables were acquired from reputable sources and were National Institute of Standards and Technology-traceable when possible. The source and purity were documented in the investigation records. Supplies and consumables were examined for evidence of tampering or damage; coupons were examined for anomalies on the test surface. Any suspect material was not used. In addition, expiration dates were noted and recorded. Solutions were prepared following Battelle Biomedical Research Center protocols and documented in reagent preparation forms. 3.3 Data Management Data acquisition during the investigation included proper recording of the procedures used in the testing to assure consistency in the investigation and adherence to the test/QA plan(1); documentation of sampling/testing conditions; and recording of analytical results and investigation conditions. Data acquisition was carried out electronically by the data logger (e.g., temperature, RH, and time) or manually by Battelle test personnel. Manually-acquired data were recorded immediately in a consistent format throughout the investigation. All written records were in ink and any corrections to recorded data were made with a single line through the original entry and the correction entered, initialed, and dated by the person making the correction. Non-obvious corrections included a reason for the correction. Whether collected manually or electronically, relevant data were entered into an electronic spreadsheet set up to organize the data in a clear and consistent manner. The accuracy of entering manually recorded data into the spreadsheets was checked at the time the data were entered, and a portion of the data was checked by the Battelle QA Manager as part of the data quality audit. 3.4 Assessment and Response Actions 3.4.1 Technical Systems Audit Battelle QA staff conducted a technical systems audit (TSA) during April, May, and August 2009 to ensure that the investigation was being conducted in accordance with the test/QAplan(1) and associated amendments and the quality management plan.(15) As part of the TSA, test procedures were compared to those specified in the test/QA plan and data acquisition and handling procedures were reviewed. Observations and findings from the TSA were documented and submitted to the Battelle Task Order Leader for response. None of the findings of the TSA required corrective action. TSA records were permanently stored with the QA Manager. 3.4.2 Performance Evaluation Audit Performance evaluation audits were performed for those measurements that factored into the data used in quantitative analysis. Table 3-1 summarizes the performance evaluation audits that were performed. The spectrophotometric absorbance was audited using a SpectraTest™ Absorbance Validation Package (MDS, Inc., Toronto, Canada) which provides a National Institute of Standards and Technology- traceable solution for validating optical performance. The test measurements and calculations are performed by SoftMax® Pro software (MDS, Inc., Toronto, Canada). No performance evaluation audit was performed for B. suis because quantitative standards for this biological material do not exist; however, the associated spike and positive controls and blanks form the basis of support for conclusions drawn from the investigation. 3.5 Data Quality Audit At least 10% of the data acquired during the investigation were audited. A Battelle QA auditor traced the data from the initial acquisition, through reduction and statistical analysis, to final reporting to ensure the integrity of the reported results. All calculations performed on the data undergoing the audit were checked. 3.6 Reports to Management Each audit was documented in accordance with the quality management plan.(15) The results of the TSA and data quality audit were submitted to EPA. ------- Table 3-1. Performance evaluation audits Volume Spectrophotometric absorbance Temperature RH Total UV radiation UV-B UV-C Time f^n iV^Q Micropipettes checked by gravimetric evaluation SpectraTest™ Absorbance Validation Package with calculations by SoftMax® Pro software Compared to independent calibrated thermometer value Compared to independent calibrated hygrometer value Compared to independent calibrated total UV radiation value Compared to independent calibrated UV-B value Compared to independent calibrated UV-C value Compared to independent clock or watch value RIIF ^Erat ± 10% ± 10% OD ±2°C ± 10% (full scale) ± 10% ± 10% ± 10% 2 seconds/hour Actual Tolerance Done 3 times, all <0.3% Done 7 times, all <10% OD Done 4 times, all <0.4 °C Done 4 times, all <1. 7% (full scale) Done 4 times, all <0.6% Done 4 times, all <1.4% Done 4 times, all = 0.0% Done 4 times, all = 0 seconds/hr 3.7 Data Review Records generated in the investigation received a quality control/technical review and a QA review before they were used to calculate, evaluate, or report investigation results. All data were recorded by Battelle staff. The person performing the review was involved in the experiments and added his/ her initials and the date to a hard copy of the record being reviewed. This hard copy was returned to the Battelle staff member who stored the record. 3.8 Performance Criteria As shown in Table 3-2, except for one stock suspension (shown in bold), all spiked amounts were within the target range of 1 x 107 - 1 x 109 CFTJ/mL (1 x 106 - 1 x 108 CPU/ coupon). One stock suspension slightly exceeded the target at 1.22 x 109 CFU/mL. The concentrations of stock suspensions ranged from 4.90 x 107 - 1.22 x 109 CFU/mL. As shown in Table 3-2 for the persistence tests, percent recoveries of viable and culturable bacteria from positive controls were within the target range for all materials except for some concrete samples (shown in bold). Although the recovery of B. suis from concrete during the method demonstration tests was adequate (see Table 4-1), the recovery from concrete positive controls was less than 5% for most of the subsequent persistence tests; the reason for this difference is not known. Nevertheless an appreciable amount of B. suis was recovered (generally >1 x 105 CPU/coupon) from concrete to assess persistence over time. No CPU were recovered from any blank coupons. No results were excluded as outliers. 3.9 Deviation The test/Q A plan specified that the RH was to be ambient conditions, and monitored, but not controlled. However, the data quality objectives specified in the test/QA plan set an allowable test measurement tolerance for RH in the chamber of ±20% (full scale). Because the RH range exceeded this specification, the deviation is noted. The level of RH may impact persistence, but RH was not a controlled or independent variable for this investigation. Although the RH range was higher than anticipated, mean RH levels were generally similar and ranged from 38.6% to 58.7% for all tests except three: the 14-day test at "moderate temperature, no UV" (29.2% RH) and the 21- and 28-day tests at "low temperature, no UV" (10.7% RH and 11.5% RH, respectively). ------- Table 3-2. Spike and positive control recovery data from persistence testing* Material Spike Control (CPU/Coupon) Aluminum 6.33 x 107 1.22 x 108 6.27 x 107 8.30 x 107 5.73x 107 l.OOx 108 7.07 x 107 4.90 x 107 7.60 x 107 8.07 x 107 5.67 x 107 Concrete 6.33 x 107 8.30 x 107 6.27 x 107 5.73x 107 7.07 x 107 4.90 x 107 7.60 x 107 8.07 x 107 Glass 6.33 x 107 1.22 x 108 8.30 x 107 6.27 x 107 5.73x 107 l.OOx 108 7.07 x 107 4.90 x 107 7.60 x 107 8.07 x 107 Soil 6.33 xlO7 1.22 x 108 6.27 x 107 8.30 x 107 5.73x 107 l.OOx 108 7.07 x 107 7.60 x 107 8.07 x 107 5.67 x 107 6.13 x 107 Wood 1.22 x 108 l.OOx 108 BIRliIiHSHlHHIliHWHHnBM from Positive Controls 7.32 ±0. 67 x 107 9.99 ±0. 78 x 107 5. 76 ±0.41 x 107 9.07 ± 1.69x 107 3.53± 1.79x 107 9.42 ± 1.99x 107 5.74+ i.44x 107 4.78 ±0. 53 x 107 5.71 ±0.64x 107 7.27 ± 1.22x 107 5.33 ± 1.75x 107 9.27 ±20. 5 x 106 3.32 ±7. 04 x 106 1.91 ±2.64x 105 3. 54 ±7. 64 x 106 1.81 ±3.49x 103 2.07 ±2. 94 x 105 1.64 ±2.31 x 105 2. 73 ±6. 08 x 106 6.49 ±0. 98 x 107 8.65 ± 0.49 x 107 1.03 ±0. 20 x 108 6.30 ± 0.49 x 107 5.18± 1.60x 107 9.86 ± l.SOx 107 5.57± l.OOx 107 6.13±0.79x 107 6.37 ±0. 70 x 107 8.33 ±0. 75 x 107 6.09 ±0. 65 x 107 7.74±0.53x 107 5.52±0.99x 107 8.24 ±0. 80 x 107 3.02 ±2. 03 x 107 6.93 ± 1.63x 107 4.44 ± 1.53x 107 5.98 ±0. 59 x 107 7.67 ±0. 69 x 107 4.01 ±0.48x 107 4.31 ±0.42x 107 3.60 ±2. 70 x 107 3.34 ± 2.47 x 107 Recovery as Percentage of 116% 82% 92% 109% 62% 94% 81% 98% 75% 90% 94% 15% 4.0% 0.3% 6.2% 0.003% 0.4% 0.2% 3.4% 103% 71% 124% 100% 90% 99% 79% 125% 84% 103% 96% 63% 88% 99% 53% 69% 63% 79% 95% 71% 70% 30% 33% *Spike and positive control recovery data associated with the method demonstration are presented in Table 4-1. Bolded values are those outside of the performance criteria ranges of 1 x 106 - 1 x 108 CPU/coupon based on spike control values or mean CPU >5% of spike levels recovered from the positive control coupons. ------- ------- 4.0 Test Results For this investigation, persistence data were generated for B. suis in contact with up to five different materials exposed to four environmental conditions for controlled exposure durations. Persistence curves were also generated, where applicable, by graphing the B. suis CFU/mL derived from bacteria recovered from each material against time for each set of environmental conditions. The following sections summarize the results of the method demonstration (tests conducted initially to ensure sufficient B. suis recovery from each spiked material) and the persistence testing investigation. 4.1 Method Demonstration - B. suis Recovery As noted in Section 2.5, a brief method demonstration was performed to ensure that previously used methods for extracting biological agents from materials were applicable for the combinations of B. suis and material. Results of the method demonstration are presented in Table 4-1. The B. suis recoveries, enumerated as CPU/coupon, attained the recovery performance criterion (mean CPU/coupon >5% of the spiked level) specified in the test/QAplan.(1) Recoveries of >5% of the bacteria spiked onto the coupon (i.e., >5.0 x 104 CPU) allow for a sufficient amount of initial bacteria to assess persistence. 4.2 Persistence Results Persistence results for each material/environmental condition combination are summarized in Tables 4-2 through 4-6 and Figures 4-1 through 4-3. Where spike controls are identical, the coupons were spiked with the same stock suspension on the same day. An "ND" for mean recovered CPU/coupon in the result tables indicates that no CPU were detected in the undiluted extract sample plated for any of five replicate coupons. A "0" was used in the calculations of mean recovered CPU/coupon for any replicate coupon having no CPU detected. A single viable bacterium present in the plated extract would be expected to be observed as a CPU. Also note, testing at the "low temperature, UV-A/B" environmental condition was repeated for the durations of 2 and 5 days because the low temperatures appeared to adversely affect the performance of the UV meters. 4.2.1 Aluminum The results obtained for B. suis persistence on aluminum are summarized in Table 4-2 and Figure 4-1. In the absence of UV-A/B, B. suis persisted for at least 28 days (the longest duration tested) at the "moderate temperature, no UV" and "low temperature, no UV environmental conditions; the associated bacteria recoveries were higher at low temperature (e.g., 3.48 x 105 CPU/coupon at 28 days) than at moderate temperature (e.g., 5.19 x 102 CPU/coupon at 28 days). The duration of B. suis persistence on aluminum decreased in the presence of UV-A/B. At the "moderate temperature, UV-A/B" environmental condition B. suis persisted less than 10 days, and B. suis did not persist or persisted at a relatively low level, i.e., 1.32 x 101 CPU/coupon, following the five- day duration at the "low temperature, UV-A/B" condition. Table 4-1. Recovery data from method demonstration Material Concrete Glass Soil Wood B. suis Spike Control (CPU/coupon) 9.33 x 107 (all spiked the same day with the same stock suspension) (CPU/coupon)* 7.98 ± 10.4 x 107 4.50 ± 0.55 x 107 4.29 ± 2.48 x 107 2.29 ± 1.77 x 107 Recovered B. suis 85.6 ± 112 48.2 ±5.87 46.0 ±26.6 24.5 ± 18.9 ' Data are expressed as mean ± standard deviation of five replicate coupons. ------- Table 4-2. B, suis persistence on aluminum Duration (Days)* Spike Contr (CFU/coupo 1 IVIcdM rxcCOVci 6Q D, SUt n) Positive Control* fsiuru/couponr Test Coupon§ Moderate Temperature, No UV 7 14 21 28 6.33 x 107 6.33 x 107 1.22 x 108 1.22 x 108 7.32 ±0. 67 x 107 7.32 ±0. 67 x 107 9.99 ±0. 78 x 107 9.99 ±0. 78 x 107 7.71 ±3.03x 104 2.29± 1.15x 104 1.38± 1.95x 104 5. 19 ± 4.09 x 102 Moderate Temperature, UV-A/B* 4 7 10 14 7 14 21 28 1 2 2 5 5 6.27 x 107 6.27 x 107 8.30 x 107 8.30 x 107 5.73x 107 5.73x 107 l.OOx 108 l.OOx 108 7.07 x 107 4.90 x 107 7.60 x 107 8.07 x 107 5.67 x 107 5. 76 ±0.41 x 107 5. 76 ±0.41 x 107 9.07 ± 1.69x 107 9.07 ± 1.69x 107 Low Temperature, No UV 3.53± 1.79x 107 3.53± 1.79x 107 9.42 ± 1.99x 107 9.42 ± 1.99x 107 Low Temperature, UV-A/B* 5.74+ i.44x 107 4.78 ±0. 53 x 107 5.71 ±0.64x 107 7.27 ± 1.22x 107 5.33 ± 1.75x 107 1.80 ± 2.06 x 102 2.27 ± 4.88 x 102 ND ND 8. 75 ± 2.89 x 106 1.81 ±0.49x 106 9.82 ± 1.18x 106 3.48 ±0. 57 x 105 4.10 ± 5.60 x 103 2.20 ±3.31 x 102 8.68 ± 12.4 x 101 ND 1.32 ± 1.81 x 101 * Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of testing was not always conducted sequentially. ' Data are expressed as mean ± standard deviation of five replicates. * Positive control coupons are spiked and extracted at time zero (i.e., immediately after spiking); one set of positive controls was used for test durations initiated at the same time. 8 Test coupons are spiked and exposed to the environmental condition for the exposure duration. * UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. "ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is approximately 33 CPU/coupon (see Section 2.6). ------- 0 2 4 6 S 10 i: 14 16 IS 20 22 24 26 2S 30 Duration (Days) ^^—Moderate Temperature.Xo UV ^^^Moderate Temperature. UY-A B A Lew Temperature. No UV ') ( Lcnv Temperature. UY-A B Notes: Only the highest persistence results are graphed for the replicate testing on days two and five for "Low Temperature, UV-A/B" * No CPU were detected on days 10 and 14 for "Moderate Temperature, UV-A/B". Figure 4-1. B. suis persistence on aluminum 4.2.2 Concrete The results obtained for persistence of B. suis on concrete are summarized in Table 4-3. On concrete, B. suis persisted only at the "low temperature, no UV" environmental condition, and, at that condition, B. suis was recovered (mean of 5.32 x 101 CPU/coupon) only from the seven-day exposure duration. 4.2.3 Glass The results obtained for persistence of B. suis on glass are summarized in Table 4-4 and Figure 4-2. In the absence of UV-A/B, B. suis persisted for at least 28 days (the longest duration tested) at the "moderate temperature, no UV" and "low temperature, no UV" environmental conditions; the associated bacteria recoveries were higher at low temperature (e.g., 3.05 x 105 CPU/coupon at 28 days) than at moderate temperature (e.g., 7.87 x 102 CPU/coupon at 28 days). The duration of B. suis persistence on glass decreased in the presence of UV-A/B. At the "moderate temperature, UV- A/B" environmental condition B. suis persisted on glass less than 2 days, and B. suis persisted less than 5 days at the "low temperature, UV-A/B" environmental condition. ------- Table 4-3. B, suis persistence on concrete . Duration (Days) 7 14 1 2 4 7 7 14 1 2 2 5 Spike Control mean Kecoverea °- (CPU/coupon) Positive Control Moderate Temperature, No UV 6.33 xlO7 9. 27 ±20. 5 x 106 6.33 xlO7 9. 27 ±20. 5 x 106 Moderate Temperature, UV-A/B* 8.30 xlO7 3.32 ± 7.04 x 106 8.30 xlO7 3.32 ± 7.04 x 106 6. 27 xlO7 1.91 ±2.64 x 105 6. 27 xlO7 1.91 ±2.64 x 105 Low Temperature, No UV 5. 73 xlO7 3. 54 ± 7.64 x 106 5. 73 xlO7 3. 54 ± 7.64 x 106 Low Temperature, UV-A/B* 7. 07 xlO7 1.81 ±3.49 x 103 4.90 xlO7 2. 07 ± 2.94 x 105 7. 60 xlO7 1.64 ±2.31 x 105 8. 07 xlO7 2. 73 ± 6.08 x 106 suis luru/coupon;1 Test Coupon5 ND ND ND ND ND ND 5.32 ± 10. 2 x 101 ND ND ND ND ND Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of testing was not always conducted sequentially. Data are expressed as mean ± standard deviation of five replicates. Positive control coupons were spiked and extracted at time zero (i.e., immediately after spiking); one set of positive controls was used for test durations # initiated at the same time. Test coupons were spiked and exposed to the environmental condition for the exposure duration. UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. "ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is approximately 33 CPU/coupon (see Section 2.6). ------- Table 4-4. B, suis persistence on glass Duration (Days)* Kffil ffffla 'coupon) Positive Control"'" suis (CPU/coupon)1 Moderate Temperature, No UV 7 14 21 28 1 2 4 7 6.33 x 107 6.33 x 107 1.22x 108 1.22x 108 6.49 ± 0.98 x 107 6.49 ± 0.98 x 107 8.65 ± 0.49 x 107 8.65 ± 0.49 x 107 3.32 ± 2.37 x 104 7.36 ± 14.0 x 103 6.26 ± 2.99 x 102 7.87± 11.2 x 102 Moderate Temperature, UV-A/B* 8.30 x 107 8.30 x 107 6.27 x 107 6.27 x 107 1.03±0.20x 108 1.03±0.20x 108 6.30 ± 0.49 x 107 6.30 ± 0.49 x 107 2. 53 ± 3.04 x 102 ND ND ND Low Temperature, No UV 7 14 21 28 5.73x 107 5.73x 107 l.OOx 108 l.OOx 108 5.18± 1.60x 107 5.18± 1.60x 107 9.86 ± 1.80 x 107 9.86 ± 1.80 x 107 2.23± 1.22 x 106 2.07±0.59x 105 7. 75 ±3. 57 x 105 3.05± 1.27 x 105 Low Temperature, UV-A/B* 1 2 2 5 7.07 x 107 4.90 x 107 7.60 x 107 8.07 x 107 5.57± l.OOx 107 6.13±0.79x 107 6.37 ±0. 70 x 107 8.33 ±0. 75 x 107 4.23 ±9. 16 x 103 4.21 ±9.22x 102 ND ND * Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of testing was not always conducted sequentially. ' Data are expressed as mean ± standard deviation of five replicates. * Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations initiated at the same time. 8 Test coupons were spiked and exposed to the environmental condition for the exposure duration. * UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. "ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is approximately 33 CPU/coupon (see Section 2.6). ------- 10 12 14 16 IS 20 22 24 26 IS 30 Duration (Days) •Moderate Temperature. Xo UV v Temperature.Xo UV HModerate Temperature. UV-A B ^Low Temperature. UV-A B Notes: Only the highest persistence results are graphed for the replicate testing on day two for "Low Temperature, UV-A/B". * No CPU were detected on days two, four, and seven for "Moderate Temperature, UV-A/B" or on day five for "Low Temperature, UV-A/B". Figure 4-2. B. suis persistence on glass 4.2.4 Soil The results obtained for persistence of B. suis on soil are summarized in Table 4-5 and Figure 4-3. On soil, B. suis persisted under all environmental conditions for the longest durations tested: 28 days at the "moderate temperature, no UV" and "low temperature, no UV" environmental conditions, 7 days at the "moderate temperature, UV- A/B" environmental condition, and 14 days at the "low temperature, UV-A/B" environmental condition. ------- Table 4-5. B, suis persistence on soil Duration (Days)* Spike Control (CPU s luru/cuupun; icai Coupon Moderate Temperature, No UV 7 14 21 28 6.33 x 107 6.33 x 107 1.22 x 108 1.22 x 108 6.09 ± 0.65 x 107 6.09 ± 0.65 x 107 7.74±0.53x 107 7.74±0.53x 107 2.66± 1.46x 104 9.29 ± 14.2 x 103 6.88 ±3. 78 x 102 1.13±0.90x 102 Moderate Temperature, UV-A/B* 4 7 10 14 6.27x 107 6.27x 107 8.30 x 107 8.30 x 107 5. 52 ± 0.99 x 107 5. 52 ± 0.99 x 107 8.24 ± 0.80 x 107 8.24 ± 0.80 x 107 5.03 ± 4.36 x 104 3.49 ± 1.14x 103 1.67 ± 1.08 x 103 4.33 ± 1.05x 102 Low Temperature, No UV 7 14 21 28 5.73x 107 5.73x 107 l.OOx 108 l.OOx 108 3.02 ± 2.03 x 107 3.02 ± 2.03 x 107 6.93 ± 1.63x 107 6.93 ± 1.63x 107 9.67 ± 1.28 x 104 5.41 ± 1.72 x 104 9.35 ± 2.09 x 104 4.14 ± 1.29 x 104 Low Temperature, UV-A/B* 1 2 2 5 5 14 7.07x 107 4.90 x 107 7.60x 107 8.07x 107 5.67x 107 6.13 x 107 4.44 ± 1.53x 107 4.57 ± 0.35 x 107 5.98 ±0. 59 x 107 7.67 ± 0.69 x 107 4.01 ±0.48x 107 4.31 ±0.42x 107 4.24 ± 1.28 x 106 4.05 ±2. 12 x 106 4.57 ± 1.28 x 106 1.01 ±0.46x 106 7.69± 1.01 x 105 7.11 ± 1.05x 103 * Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of testing was not always conducted sequentially. ' Data are expressed as mean ± standard deviation of five replicates. * Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations initiated at the same time. 8 Test coupons were spiked and exposed to the environmental condition for the exposure duration. * UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions. ------- 0 2 4 6 S 10 i: 14 16 IS 20 22 24 26 2S 30 Duration (Days} ^^—Moderate Temperature.Xo UV ^^^Moderate Temperature. UV-A B A Lo^v Temperature. No UV ) ( Low Temperature. UV-A B Note: Only the highest persistence results are graphed for the replicate testing on days two and five for "Low Temperature, UV-A/B" Figure 4-3. B, suis persistence on soil 4.2.5 Wood The results obtained for persistence of B. suis on wood are summarized in Table 4-6. Persistence testing of B. suis on wood was conducted only occasionally. Wood coupons were used in place of the concrete coupons in the longer persistence tests because of the lack of persistence of B. suis on concrete at the shorter time points. At the "moderate temperature, no UV" environmental condition, B. suis was not recovered from wood after a 21-day exposure duration (the shortest duration tested), but B. suis was recovered after 28 days of exposure to the "low temperature, no UV" environmental condition. Table 4-6. B. suis persistence on wood Duration (Days)' 21 28 Spike Control (CPU/coupon) Moderate Temperature, 1.22 x 108 1.22 x 108 Mean Recovered B. NoUV 3.60 ± 2.70 x 107 3.60 ±2. 70 x 107 suis (CPU/coupon^ ND ND Low Temperature, No UV 21 28 l.OOx 108 l.OOx 108 3.34 ± 2.47 x 107 3.34 ± 2.47 x 107 1.73± 1.43 x 105 1.57 ± 1.22 x 105 * Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of testing was not always conducted sequentially. ' Data are expressed as mean ± standard deviation of five replicates. * Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations initiated at the same time. 8 Test coupons were spiked and exposed to the environmental condition for the exposure duration. "ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is approximately 33 CPU/coupon (see Section 2.6). ------- 5.0 Summary The B. suis persistence results are summarized in Table 5-1. These data denote the longest exposure duration (days) that B. suis was recovered (persisted) and the shortest exposure duration that B. suis was not recovered to bracket the length of time that B. suis remained viable for each material and environmental condition. The shortest duration without B. suis recovery provides, where possible, an upper bound on the persistence of B. suis per materials and environmental conditions tested. In general, greater B. suis persistence occurred at lower temperatures (especially without exposure to UV), and B. suis persisted longer without exposure to simulated sunlight (although UV had less of an effect on the persistence of B. suis on soil). On aluminum, glass, and soil, B. suis persisted for at least 28 days (the longest duration tested) under the "moderate temperature, no UV" and the "low temperature, no UV environmental conditions. On concrete, B. suis did not persist following a 7-day exposure duration (the shortest duration tested) at the "moderate temperature, no UV environmental condition, but B. suis on concrete did persist 7 days when exposed to the "low temperature, no U V environmental condition. On wood, B. suis did not persist following a 21- day exposure duration (the shortest duration tested) at the "moderate temperature, no UV environmental condition, but B. suis did persist on wood for 28 days when exposed to the "low temperature, no UV environmental condition. The incorporation of UV-A/B into the environmental conditions shortened the duration that B. suis persisted on aluminum and glass, but had less of an effect on soil. At the "moderate temperature, UV-A/B" environmental condition, the longest exposure durations associated with recovered B. suis were 7 days for aluminum and one day for glass. At the "low temperature, UV-A/B" environmental condition, the longest durations from which B. suis was recovered were 2 days on glass and 5 days on aluminum. When exposed to UV-A/B, B. suis persisted on soil for 14 days (the longest duration tested) at the "moderate temperature, UV-A/B" environmental condition, and B. suis persisted on soil for 14 days (the longest duration tested) at the "low temperature, UV-A/B" environmental condition. On concrete, B. suis was not recovered at any of the environmental conditions that incorporated UV-A/B. Persistence testing with B. suis on wood was not conducted in the presence of UV-A/B. ------- Table 5-1. Summary of B, suis persistence iviaienai ana Lnvironmeniai Condition Aluminum Moderate temperature, No UV Moderate temperature, UV-A/B Low temperature, No UV Low temperature, UV-A/B Concrete Moderate temperature, No UV Moderate temperature, UV-A/B Low temperature, No UV Low temperature, UV-A/B Glass Moderate temperature, No UV Moderate temperature, UV-A/B Low temperature, No UV Low temperature, UV-A/B Soil Moderate temperature, No UV Moderate temperature, UV-A/B Low temperature, No UV Low temperature, UV-A/B Wood Moderate temperature, No UV Moderate temperature, UV-A/B Low temperature, No UV Low temperature, UV-A/B Longesi uurauon luays; with B. suis Recovery 28 7 28 5* ND ND 7 ND 28 1 28 2t 28 14 28 14 ND Not tested 28 Not tested 5SBB without B. suis Recovery AD 10 AD 5* 7 1 14 1 AD 2 AD 2t AD AD AD AD 21 Not tested AD Not tested AD = B. su/swas detected at all durations tested. ND = B. su/swas not detected at the shortest duration tested. * B. su/swas recovered during the 5-day test conducted 9/24/09 - 9/29/09 but not during the 5-day test conducted 8/23/09 - 8/28/09. • B. su/swas recovered during the 2-day test conducted 9/2/09 - 9/4/09 but not during the 2-day test conducted 9/18/09 - 9/20/09. ------- 6.0 References 1. Technology Testing and Evaluation Program Quality Assurance/Test Plan for Persistence Testing of Brucella suis on Outdoor Materials, Version 1, Battelle, Columbus, Ohio, October 2008. 2. Sarinas, P.S.A. and R.K. Chitkara, Brucellosis. Semin. Respir. Infect, 2003(18): p. 168-182. 3. Battelle, MREF Standard Operating Procedure for the Operation and Maintenance of Primus General Purpose Steam Sterilizer Model: PSS5-A-MSSD, 2006. 4. Battelle, MREF Facility Safety Plan Annex 12 to Appendix B, Guidelines for the Use of Class II and Class III Biological Safety Cabinets in the MREF Biofacility, July 2006. 5. Battelle, FSP Annex 5 to Appendix B, Guidelines for Safe Handling and Storage of Etiologic Agents at the MREF, July 2006. 6. Battelle, FSP Annex 7 to Appendix B, Guidelines for Disinfection/Decontamination of Etiological Agents at the MREF Biofacilities, July 2006. 7. Weinbauer, M.G., et al., Photoreactivation Compensates for UV Damage and Restores Infectivity to Natural Marine Virus Communities. Appl. Environ. Microbiol., 1997(63): p. 2200-2205. 8. Balasaraswathy, P., et al., UVA and UVB in Sunlight, Optimal Utilization of UV Rays in Sunlight for Phototherapy. Indian J. Dermatol. Venereol. Leprol., 2002(68): p. 198-201. 9. Jeanmougin, M. and J. Civatte, Dosimetry of Solar Ultraviolet Radiation. Daily and Monthly Changes in Paris. [Article inFrench] Ann. Dermatol. Venereol., 1987(114): p. 671-676. 10. Kolari, P.J., et al., Midsummer Solar UV-Radiation in Finland Compared with the UV-Radiation from Phototherapeutic Devices Measured by Different Technique. Photodermatol., 1986(3): p. 340-345. 11. McNamara, A.E. and W.R. Hill, UV-B Irradiance Gradient Affects Photosynthesis and Pigments but Not Food Quality of Periphyton. Freshwater Biology, 2000(43): p. 649-662. 12. Diffey, B.L., Sources and Measurement of Ultraviolet Radiation. Methods, 2002(28): p. 4-13 13. Qui, X., et al., Survival ofShewanella oneidensis MR-1 after UV Radiation Exposure. Appl. Environ. Microbiol., 2004(70): p. 6435-6443. 14. Technology Testing and Evaluation Program Test/QA Plan for Systematic Investigation ofFumigant Technologies for Decontamination of Biological Agents from Contaminated Building Materials, Version 1, Battelle, Columbus, Ohio, May 2007. 15. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP), Version 3, Battelle, Columbus, Ohio, January 2008. ------- &EPA United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center Cincinnati, OH 45268 Official Business Penalty for Private Use $300 Recycled/Recyclable Printed with vegetable-based ink on paper that contains a minimum of 50% post-consumer fiber content processed chlorine free PRESORTED STANDARD POSTAGES FEES PAID EPA PERMIT NO. G-35 ------- |