*>EPA United States Environmental Protection Agency EPA 600/S-24/011 I March 2024 I Summary of Decontamination Techniques for Materials Contaminated with Ricin Office of Research and Development Center For Environmental Solutions and Emergency Response ------- EPA 600/S-24/011 I March 2024 I www.epa.gov/research This page is intentionally left blank ------- EPA 600/S-24/011 I March 2024 I www.epa.gov/research Summary of Decontamination Techniques for Materials Contaminated with Ricin Joseph Wood Center for Environmental Solutions and Emergency Response Research Triangle Park, NC 27711 ------- EPA 600/S-24/011 I March 2024 I www.epa.gov/research Foreword The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to formulate and implement actions leading to a compatible balance between human activities and the ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is providing data and technical support for solving environmental problems today and building a science knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect our health, and prevent or reduce environmental risks in the future. The 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 summarizes previous research conducted by EPA/ORD to evaluate decontamination options for materials contaminated with ricin. This report summarizes the efficacy of several decontamination techniques to neutralize both crude and pure ricin on several different surfaces and materials. Gregory Sayles, Ph.D., Director Center for Environmental Solutions and Emergency Response Notice and Disclaimer This technical summary document has been subjected to the Agency's peer and administrative review and has been approved for publication as an EPA document. Any mention of trade names, products, or services does not imply an endorsement or recommendation for use. The views expressed here are the authors' own and do not necessarily reflect the views or policies of USEPA. ------- EPA 600/S-24/011 I March 2024 I www.epa.gov/research Table of Contents Foreword ii Notice and Disclaimer ii List of abbreviations and acronyms iv Acknowledgments v 1 Introduction 1 2 Natural Attenuation/Climate Control 3 3 Spray-applied liquid-based decontaminants 6 4 Hydrogen Peroxide Vapor 8 5 Chlorine dioxide gas 10 6 Conclusions/Summary 11 7 References 12 List of Figures Figure 1. Typical liquid inoculation of ricin suspension onto coupons using a micropipette 1 Figure 2. Coupon types 4 Figure 3. Spray apparatus used to apply liquid decontaminants to coupons 6 Figure 4. Average percent reduction of crude ricin ± 95% confidence interval 7 List of Tables Table 1. Time required to attenuate > 99% of pure ricin 3 Table 2. Test and environmental conditions in which > 99% attenuation of ricin achieved 4 Table 3. Average percent attenuation obtained for each environmental condition at 14 and 28 days 5 Table 4. Parameters required to achieve >99% reduction on all materials using HPV 8 Table 5. Average percent reduction of pure ricin with CIO2 at 200 ppm and 30 minutes 10 iii ------- EPA 600/S-24/011 I March 2024 I www.epa.gov/research List of abbreviations and acronyms ABS acrylonitrile butadiene styrene C Celsius CESER Center for Environmental Solutions and Emergency Response CIO2 chlorine dioxide COTS commercial-off-the-shelf EPA United States Environmental Protection Agency HP hydrogen peroxide HPV hydrogen peroxide vapor Hr or h hour LCHPV low concentration hydrogen peroxide vapor min minute MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ORD Office of Research and Development PAA peracetic acid PPM parts per million QAC quaternary ammonium compound RH relative humidity SH sodium hypochlorite (chlorine bleach) SP sodium percarbonate |jg microgram iv ------- Acknowledgments This brief technical report is a summary of US EPA studies that have evaluated the efficacy of decontamination techniques to neutralize ricin toxin on a variety of different materials. The following individuals are acknowledged for their contributions to these studies: Shannon Serre, US EPA Office of Emergency Management Shawn Ryan, US EPA Office of Research and Development William Richter, Battelle Memorial Institute James Rogers, Battelle Memorial Institute M. Autumn Smiley, Battelle Memorial Institute Bailey Weston, Battelle Memorial Institute Michelle Sunderman, Battelle Memorial Institute Zach Wllenberg, Battelle Memorial Institute v ------- 1 Introduction This technical summary report provides a review of the U.S. EPA Office of Research arid Development's (ORD) laboratory studies that evaluated the efficacy of decontamination techniques to neutralize ricin on a variety of different surfaces. Since material can greatly impact the efficacy of a decontamination technique, decontamination tests are typically performed using several types of realistic indoor and outdoor, porous and nonporous materials. Additionally, tests were conducted with either a vendor-provided pure (refined, with extraneous materials removed) ricin, a crude form of ricin produced in the laboratory from castor beans (which is more representative of what would be used in an actual incident), or both. Efficacy results for a decontaminant are typically reported as the percent reduction of ricin toxicity and are calculated based on the ricin recovered from test material coupons (after decontamination) relative to the ricin recovered from positive controls (coupon materials inoculated with ricin but not exposed to the decontaminant). Although the focus of this summary report is on the efficacy of a decontaminant as a function of material and other operating parameters, other criteria such as material compatibility (e.g., see US EPA, 2022) may also be considered in the selection of a decontaminant. For the lab tests described in this report, a ricin suspension was inoculated onto the test and positive control coupon materials typically using one or two streaks (See Figure 1 below). Following decontamination, ricin from the positive controls and any remaining ricin toxin from the test coupons were extracted from the coupon materials using a growth medium and then quantified. The cytotoxicity assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to quantify ricin in the extraction liquid and utilizes Vero cells and ricin reference standards. Further details on the methods used can be found in the reports referenced in this summary. Figure 1. Typical liquid inoculation of ricin suspension onto coupons using a micropipette The decontamination techniques and their efficacy for neutralizing ricin summarized in this report include the following: • Natural attenuation or climate control • Liquid decontaminants and cleaners, applied as a spray: o Chlorine bleach at three concentrations o Peracetic acid (PAA) 1 ------- o Aqueous hydrogen peroxide o A commercial, off-the-shelf (COTS) cleaner using a quaternary ammonium compound (QAC) as its active ingredient o A COTS cleaner with sodium percarbonate as its active ingredient • Hydrogen peroxide vapor (HPV) • Chlorine dioxide (CIO2) gas The liquid spray decontaminants may be useful when the specific location(s) of the ricin contamination is known. When the ricin is suspected of being dispersed in a large area and/or the specific indoor location of the contamination is not known, a volumetric decontamination approach (such as CIO2 gas, HPV, or natural attenuation/climate control) may be a more appropriate option. 2 ------- 2 Natural Attenuation/Climate Control In the EPA's first decontamination study for ricin (US EPA 2006), natural attenuation/climate control was evaluated, in which 25 |jg of pure ricin were inoculated onto coupons of galvanized steel and painted concrete. Ambient temperature and relative humidity (RH) were the experimental variables and evaluated over several time points to assess their impact on ricin attenuation (loss). Table 1 summarizes the results and shows the time required to achieve at least 99% reduction for the three environmental conditions and time points evaluated for the two materials. The 99% reduction benchmark was not achieved in any of the tests for the painted concrete. When comparing the results for the tests conducted at 30 °C, the tests at the elevated RH levels resulted in a higher reduction of ricin. Table 1. Time required to attenuate > 99% of pure ricin Material 20 °C, 40-70% RH 30 °C, > 70% RH 30 °C, < 40% RH Galvanized metal 9 days 14 days NA (86%) Painted concrete NA (32%) NA (72%) NA (38%) NA= did not achieve 99% reduction at the longest time point (14 days), with highest % reduction achieved noted in parentheses A more comprehensive ricin attenuation study was conducted several years later (Wood et al., 2018) and included both crude (target inoculation of 320 |jg per coupon) and pure (target inoculation of 250 |jg per coupon) forms of ricin inoculated onto six different materials: mild steel, neoprene rubber, paper, optical grade plastic, bare pine wood, and industrial carpet. Several ambient temperatures (20, 25, 30, 40 and 50 °C), RH levels (20, 45, or 75%), and attenuation times (7-28 days at the 20-30 °C temperatures, and 0.25-14 days at the higher temperatures) were evaluated. For the attenuation temperatures evaluated at 40 and 50 °C, these would most likely require additional heating equipment within a building but would not be expected to be overly detrimental to interior building materials. See Figure 2 for a photograph of the coupon materials used in the study. Table 2 shows the environmental conditions and materials in which 99% reduction of ricin was achieved. Over the entire study, there were only seven cases (out of over 200 test combinations of ricin type, temperature, RH, material, and contact time) in which 99% reduction was achieved. Specifically, there were no cases in which any form of ricin was attenuated > 99% at 20 °C (up to 28 days) or at 25 °C and 45% RH (up to 14 days). There was only one case (25 °C, 75% RH, 7 days, on paper) in which the crude ricin preparation was attenuated > 99%. With respect to material, >99 % reduction occurred most often on the mild steel and paper coupons. 3 ------- Figure 2. Coupon types from Left to Right: Mild Steel, Neoprene Rubber, Optical Grade Plastic, Pine Wood, Industrial Carpet, Paper Table 2. Test and environmental conditions in which > 99% attenuation of ricin was achieved Contact Ricin Form Temp °C %RH Time (Days) Material Pure 25 75 7 Mild Steel Crude 25 75 7 Paper Pure 25 75 14 Mild Steel Pure 30 45 7 Carpet Pure 30 45 7 Paper Pure 50 20 6 Mild Steel Pure 50 20 7 Mild Steel Table 3 shows the average percent reduction of ricin across ail materials, for the tests conducted at 20-30 °C, and provides an indication of the effect of temperature and RH on ricin attenuation. 4 ------- Table 3. Average percent attenuation obtained for each environmental condition at 14 and 28 days Temperature °C %RH Test duration (days) Average % Attenuation for Pure Ricin Average % Attenuation for Crude Ricin 20 45 14 61 ± 36 % 7 ± 16% 20 45 28 72 ± 37% 75 ±11% 20 75 14 57 ± 32% 49 ± 37% 25 45 14 87 ± 14% 66 ±21% 25 75 14 87 ± 15% 49 ± 52% 30 45 14 83 ± 13% 68 ± 24% 30 75 14 62 ± 32% 35 ± 42% Overall, the results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent with more variable results. There was minimal attenuation of the crude ricin after two weeks at typical indoor environmental conditions (20 °C, 45% RH), except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40 °C for 5 days or 50 °C for 2-3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40 °C after two weeks. 5 ------- 3 Spray-applied liquid-based decontaminants A comprehensive study was conducted that evaluated ricin (only crude ricin was used) neutralization efficacy for seven decontaminants that were spray-applied to four types of coupon materials (Richter et al., 2024). The seven decontaminants evaluated included three different dilutions of chlorine bleach (sodium hypochlorite), two COTS cleaning solutions (a QAC-based cleaner and a cleaning solution with sodium percarbonate as the active ingredient), aqueous hydrogen peroxide, and PAA. (Note: PAA may also be referred to as peroxyacetic acid.) The four materials evaluated were bare pine wood, laminated countertop, joint tape, and industrial carpet. The decontaminants were applied to the coupons using a spray apparatus that allowed for precise quantities to be applied (see Figure 3). Contact times ranged from 15-120 minutes. When a contact time of 120 minutes was used, the decontaminant was spray-applied to the coupons a second time at the 1-hr mark. Figure 3. Spray apparatus used to apply liquid decontaminants to coupons The results of the study, in terms of percent reductions of crude ricin, are summarized in Figure 4 by material, decontaminant, and contact time. Results showed that decontamination efficacy varied by decontaminant and material, and that efficacy generally improved as the number of spray applications and/or contact time increased. The solutions of 0.45% PAA and the 20,000- part per million (ppm) sodium hypochlorite bleach (this would be a 1 in 4 dilution of bleach containing 8% hypochlorite, a concentration typically used in germicidal bleach) provided the overall best decontamination efficacy. For the three bleach solutions, efficacy generally 6 ------- improved with increasing concentration of the hypochlorite. The 0.45% PAA solution achieved 97.8 to 99.8% reduction with only a 30-min contact time. With respect to material, the laminated countertop was generally the easiest to successfully decontaminate, presumably due to its nonporous nature. 15 30 60 Time (min) Percent Reduction 0 25 50 75 100 Figure 4. Average percent reduction of crude ricin ± 95% confidence interval SP= sodium percarbonate cleaner; QAC= quaternary ammonium compound cleaner; HP= 3% aqueous hydrogen peroxide solution; SH= sodium hypochlorite (chlorine bleach), at three concentrations noted in parts per million (ppm); PAA= 0.45% peracetic acid. 7 ------- 4 Hydrogen Peroxide Vapor In the first study (US EPA, 2015) conducted to evaluate the efficacy of hydrogen peroxide vapor (HPV) to neutralize ricin, seven types of materials were used to represent those found in a mail sorting facility. These included aluminum, industrial carpet, ceramic tile, neoprene rubber, optical plastic, paper, and stainless steel. (Although for some of the tests, not all seven materials were included.) Decontamination efficacy tests were conducted using two different types of HPV generators - a STERIS 1000ED and a Bioquell Clarus C - against two forms of ricin toxin: a commercially-available purified version and a crude version prepared from castor beans. (The Steris generator operates at a somewhat lower RH, i.e., less than 40%, while the Bioquell generator is designed to operate at a higher RH [~ 70-80%], such that micro-condensation of the HPV occurs on surfaces.) The effect of the mass of ricin inoculated onto the test coupons (250 and 500 |jg) was also evaluated as part of the test matrix. The HPV was effective in reducing ricin toxin under several test conditions. Table 4 summarizes the test parameters required to achieve > 99% reduction on the materials tested. Eight hours was the minimum contact time required to achieve > 99% reduction. Table 4. Parameters required to achieve >99% reduction on all materials using HPV Hydrogen peroxide generation technology Type of ricin/ quantity (pg) Average hydrogen peroxide vapor concentration (ppm) Contact time (hr:min) Bioquell Pure/250 279 8:00 Bioquell Crude/250 301 16:00 Bioquell* Pure/500 240 16:00 Steris* Pure/500 398 13:40 Steris* Crude/500 398 13:40 Steris§ Crude/500 392 13:40 *Limited materials tested were industrial carpet, optical plastic, paper, and steel §Limited materials tested were neoprene rubber, aluminum, ceramic tile, and unpainted concrete 8 ------- A follow-on study was conducted to evaluate relatively lower concentrations of HPV (US EPA, 2023), coupled with longer contact times, to neutralize crude ricin on materials. Achieving and maintaining relatively high concentrations of HPV within a building may be challenging and require expensive generation equipment, relatively high (and hazardous) concentrations of aqueous hydrogen peroxide solution, and additional technical personnel. In contrast, relatively low concentrations of HPV (LCHPV) can be achieved using COTS 3% aqueous solutions of hydrogen peroxide disseminated as a vapor using COTS humidifiers. In this study, only crude ricin was used, and inoculated onto four materials (pine wood, ceramic tile, industrial carpet, and ABS plastic). The LCHPV efficacy was evaluated at two concentrations (25 and 50 ppm) and three contact times (from 1-4 days). The results of the study are summarized in Figure 5. For most of the test conditions, the use of LCHPV was an effective method to neutralize crude ricin toxin on the evaluated materials. Greater than 90% reduction in the toxin was achieved using 25 and 50 ppm HPV for all materials tested at exposure times of 96 and 48 hours, respectively. 25 ppm Pine Wood 92.0 ±6.8 94.1 ±3.7 97.0 ±3.3 Ceramic Tile 77.2 ±3.7 88.2 ± 1.3 95.6 ±0.3 Carpet 91.3 ± 1.8 92.2 ±0.6 94.9 ±0.9 ABS Plastic 74.6 ± 4.4 67.5 ±6.4 90.9 ±0.7 Pine Wood Ceramic Tile Carpet ABS Plastic 50 ppm 68.7 ±24.4 93.0 ±2.5 89.6 ±3.6 82.6 ±2.8 97.5 ±0.5 98.0 ±0.2 72.3 ±9.7 93.0 ±3.4 93.9 ±2.3 80.0 ±5.4 97.5 ± 0.3 97.8 ±0.2 24 48 72 96 Time (h) Percent Reduction 0 25 50 75 100 Figure 5. Summary of average percent crude ricin toxin reduction ± 95% confidence interval for LCHPV 9 ------- 5 Chlorine dioxide gas The neutralization of pure ricin using chlorine dioxide (CIO2) gas was evaluated as part of a larger systematic decontamination study (US EPA, 2011). 25 |jg of pure ricin were inoculated onto seven types of materials. The materials included glass, painted concrete, galvanized metal, decorative laminate, cellulose insulation, particle board, and industrial carpet. Tests were conducted at 1,500 ppm CIO2 with a contact time of 20 minutes and at 200 ppm with a contact time of 30 minutes. Ambient temperatures during testing ranged between 23-25 °C and the RH was elevated to a range of 80-84%. For the test with CIO2 fumigation conducted at 1,500 ppm for 20 minutes, ricin was reduced by >99% from all materials except cellulose insulation, which exhibited a 93% reduction. For the fumigation test conducted at 200 ppm CIO2 for 30 minutes, the percent reduction in ricin was > 93% for all materials. The results for this test are summarized in Table 5 for each material. Table 5. Average percent reduction of pure ricin with CIO2 at 200 ppm and 30 minutes Material Average Percent Reduction Glass 99.8 Painted concrete 99.9 Galvanized metal 98.5 Decorative laminate 99.7 Cellulose insulation 93.4 Particle board 95.8 Industrial carpet 98.3 10 ------- 6 Conclusions/Summary There is no standard method for determining the efficacy of a decontamination method for neutralizing ricin. Therefore, the EPA studies described in this report used a variety of different materials and variable amounts of ricin (both crude and pure forms) inoculated onto the test materials. Nonetheless, the data and information summarized in this technical brief may be useful to officials needing to make decisions about how to best remediate indoor areas contaminated with ricin. For the tests in which both the crude and pure forms of ricin were used, the crude form was generally neutralized at a lower efficacy than the pure form. Since crude forms of ricin are expected to be encountered in an actual incident, the decontamination efficacy results for crude ricin presented in this summary may be more relevant or representative. Nonetheless, the efficacy test results for the pure form of ricin are useful and will also inform decision-making. For incidents when the ricin is suspected of being dispersed throughout an area, and/or if the location of the ricin contamination is unknown, volumetric decontamination approaches may be better suited. In these cases, and if time is not a factor, decontamination via attenuation at elevated temperatures or LCHPV may be easy-to-implement options. If a more rapid, volumetric decontamination approach is needed, CIO2 gas at 200 ppm was shown to be effective on many materials using only a 30-minute contact time. While this contact time may be short, the time required for upfront planning to implement the CIO2 fumigation may be lengthy, since its use requires certain technology and professional expertise not widely available. A similar barrier exists with using HPV at the higher concentrations discussed in this technical brief (e.g., > 250 ppm), i.e., the shorter contact times for the actual fumigation (on the order of a few hours) may be offset by longer upfront planning times required to obtain the expensive HPV generating technology and associated required expertise. For simplicity and short contact times, and if the contamination locations on surfaces are known, the spray application of decontaminants is a good option. With respect to which liquid decontaminants to use, the 0.45% PAA and 20,000 ppm SH (1 in 4 dilution of germicidal chlorine bleach) were generally the most effective in neutralizing ricin in the laboratory tests conducted. Finally, while most of the results have been described here as "effective" if the percent reduction was > 90 or 99% in lab tests, the remediation goals in an actual ricin contamination incident will be site-specific to sufficiently mitigate exposure and health risks. 11 ------- 7 References Richter RR, Weston BL, Sunderman MM, Willenberg Z, Ratliff K, Wood JP. 2024. Neutralization of Ricin Toxin on Building Interior Surfaces Using Liquid Decontaminants. Submitted to, and currently under review at, PLoS One. U.S. Environmental Protection Agency. 2006. Impact of Temperature and Humidity on the Persistence of Vaccinia Virus and Ricin Toxin on Indoor Surfaces. EPA/600/R-08/002. U.S. Environmental Protection Agency. 2011. Systematic Investigation of Liquid and Fumigant Decontamination Efficacy Against Biological Agents Deposited on Test Coupons of Common Indoor Materials. EPA/600/R-11/076. U.S. Environmental Protection Agency. 2015. Evaluation of the Inactivation of Ricin Toxin on Surfaces Using Vapor Phase Hydrogen Peroxide. EPA/600/R-15/131. U.S. Environmental Protection Agency. 2022. Update on Material Compatibility Testing for Decontamination Methods Used for Bacillus anthracis (Anthrax). EPA/600/S-22/008. U.S. Environmental Protection Agency. 2023. Neutralization of Ricin Toxin on Surfaces using Low Concentration Hydrogen Peroxide Vapor. EPA/600/R-23/096. Wood JP, Richter W, Smiley MA, Rogers JV. 2018. Influence of environmental conditions on the attenuation of ricin toxin on surfaces. PLoS ONE, 13(8): e0201857. https://doi.org/10.1371/journal.pone.0201857. 12 ------- v> EPA United States Environmental Protection Agency EPA 600/S-24/011 I March 2024 I Office of Research and Development (8101R)Washington, DC 20460 PRESORTED STANDARD POSTAGE & FEES PAID EPA PERMIT NO. 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