Overview and Status of EPA collaborations for Detection of Selected Biotoxins in Drinking Water, Soils, and Wipes
Matthew L. Magnuson, Ph.D. US Environmental Protection Agency National Homeland Security Research Center
Office of Research and Development NHSRC Mission
To conduct research and develop scientific products that improve the capability of the Agency to carry out its homeland security responsibilities
ADVANCING OUR NATION’S SECURITY THROUGH SCIENCE
Office of Research and Development NHSRC Research Projects
Homeland Security
Multi Use Cross agency
“Normal” Environmental Operations
Many homeland security practices may also benefit day to day operation. For example, emerging analytical techniques to monitor water quality might be used during other water emergencies and/or clean-up after contamination.
Office of Research and Development NHSRC Products
• 125 reports and journal articles since 2003 (including classified) • Results presented many other ways—stakeholder meetings, symposia, workshops, etc. • Products and research plans receive rigorous quality reviews
Most scientists regarded the new streamlined peer-review process as ‘quite an improvement.’
Office of Research and Development Overview of Detection of Biotoxins
• Laboratory methods – SAM method compendium – Collaborative projects – Future directions
Office of Research and Development Laboratory methods
• Method development and study – Documentation: Methods and study reports
• Methods aim to have DQOs fit for their intended use by – EPA/Water Security Division through the Water Laboratory Alliance – EPA/Office of Emergency Management through the Environmental Response Laboratory Network (ERLN)
• Availability of Methods: Most on-going studies or in clearance – Availability announced on website and we also maintain a list of stakeholders who are specifically informed – Register to get updates at http://www.epa.gov/nhsrc
Office of Research and Development Standardized Analytical Methods (SAM) Document
• Cornerstone document of Environmental Response Laboratory Network (ERLN)
• Identifies chemical, biological and radiological (CBR) agents of concern and preferred analytical methods to be used for ENVIRONMENTAL RESTORATION NOT EMERGENCY RESPONSE
• Originally published in 2005, updated annually
• SAM 5.0 published in October 2009
• Searchable SAM website: www.epa.gov/sam
Office of Research and Development Generic Response Analytical Performance Considerations for Environmental Restoration
• Sample Magnitude: 10,000’s of samples for weeks to months.
• Multiple Analysis Types: Field and laboratory
• Multiple Sample Types: Clinical, Environmental, Forensic
• Multiple Sample Matrices: Clinical, food, solids, liquids, etc.
Office of Research and Development Analysis needs in Generic Response Timeline
Office of Research and Development SAM Revision 5.0 Analytical Methods
•Chemical Methods •Radiochemistry Methods •135 analytes •23 analytes •6 sample matrices •5 sample matrices •Sample preparation and •Qualitative determination and determinative methods confirmatory methods
•Biotoxin Methods •Pathogen Methods •18 analytes •32 analytes •5 sample matrices •5 sample matrices •Presumptive, confirmatory, •General remediation efficacy added and biological activity methods using spore strips
Office of Research and Development SAM Analytical Methods
• Sample matrix types – Solid Samples (e.g. soil) – Aqueous liquid samples (e.g. surface water, waste water, etc) – Drinking water samples – Air samples – Wipes (from surfaces)
• These are NOT compliance monitoring methods. But the goal is to meet similar DQOs as regulated analytes.
Office of Research and Development Method Development Process
Goal: All steps between sample collection and data reporting
Optimize Instrumentation Determine Best Calibration (chromatography, mass calibrate, (linear/quadratic, internal tune, evaluate instrument stability) standards)
Determine Interferences (Are DQOs Determine Preservatives met in various difficult matrices?) (e.g. select antimicrobial & dechlor)
YES Determine Does Method Meet Holding Time DQOs? Write Method
NO
Revise Technical Approach Use in ERLN, WLA
Office of Research and Development Adapted from J. Shoemaker and B. Boutin, USEPA/ORD at WQTC 2008 Analytical Target Levels for Methods
– Water and Air Risk-based Exposure Guidelines Are Used to Estimate Appropriate Target Levels for Analytical Methods
– Risk-Based Criteria to Support Validation of Detection Methods for Drinking Water and Air
http://www.epa.gov/nhsrc/pubs/600r08021.pdf
Office of Research and Development Detection of Biotoxins in environmental matrices— Biotoxins and SAM
• Expert input through SAM Biotoxin Working Group • Proteins – Abrine, Ricin, Bot (A, B, E, F), Shiga 1 & 2, SEA, SEB, SEC • Small molecule – Aflatoxin B1, α-Amanitin, anatoxin-a, brevetoxin B, α-Conotoxin, Cylindrospermopsin, Diacetoxyscirpenol, microcystins (LA, LR, LW, RR, YR), picrotoxin, saxitoxins, T2 mycotoxin, tetrodotoxin
Office of Research and Development Detection of Biotoxins in environmental matrices – Biotoxins and SAM
• Analytical techniques – Antibody based detection schemes • ELISA • Lateral flow devices • Various antibody-capture-release detectors (fluorescence, electrochemiluminescence, etc. – Instrumental analysis • Liquid chromatography-mass spectrometry • Gas chromatography-mass spectrometry • others
Office of Research and Development Meeting throughput requirements
Multi-tier analysis approach • Environmental restoration hopefully is effective, so while there are many samples, fewer will be positive after initial decontamination activities. • Samples initially subjected to higher throughput, screening methods • Followed by analysis of selected samples with lower throughput, but more definitive, techniques • Application approach of techniques listed will appear in future version of SAM (e.g. see techniques of ricin in table below)
Office of Research and Development Previous biotoxin projects
Dahlgren Naval Surface Warfare Center • Included both literature review and experimental components. • Develop an improved scientific understanding of the nature of biotoxins potentially used to intentionally contaminate drinking water systems. • Methods of reducing the risks from biotoxin contamination • Target applications: – Decontamination – Threat analysis
Office of Research and Development Experimental Approach
Designed to reflect possible operational paradigm during water system contamination/decontamination
• Different oxidants •chlorine, chloramine, chlorine dioxide, ozone • Fate process •Hydrolysis, Heat inactivation, Coagulation
Office of Research and Development Previous biotoxin projects
EPA Technology Testing and Evaluation Program (TTEP)
Test, evaluate, and report on the performance of homeland security-related technologies • building and outdoor area decontamination • air, water, and wastewater treatment • water security technologies • detectors/monitors for air and water
Desired Outcome:
Informed decision making by our customers to detect, contain, decontaminate, and manage hazardous chemical, biological, and radiological materials purposefully or accidentally introduced into structures, facilities, drinking water systems, or the environment.
Office of Research and Development Reports for various technologies at www.epa.gov/etv
• Immunoassay Test Kits for Biotoxins – BADD Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Strips – BioVerify Botulinum Toxin A and Ricin Test Kits and M-Series M1M Analyzer – EzyBot A and EzyBot B Test Kits – QTL Biosensor – RAMP Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Cartridges – BioThreat Alert Anthrax, Botulinum Toxin, and Ricin Immunoassay Test Strips (Tetracore) – Anthrax, Botulinum Toxin, and Ricin Enzyme-Linked Immunosorbent Assay (ELISA, Tetracore)
• Rapid Toxicity Testing Systems (included ricin and bot) – AbraTox Kit – Chem-IQ Tox Test Kit – IQ Toxicity Test Rapid Toxicity Testing System – ToxScreen II Rapid Toxicity Testing System (Round 1) – ToxScreen II Test Kit (Round 2) – Toxi-Chromotest – ToxTrak Rapid Toxicity Testing System – BioTox Rapid Toxicity Testing System – POLYTOX Rapid Toxicity Testing System – LuminoTox SAPS Test Kit – Eclox Rapid Toxicity Testing System
Office of Research and Development Current biotoxin detection projects
Centers for Disease Control and Prevention • The CDC’s Chemical Terrorism Analytical Response Laboratory is currently developing assays for the quantitation of biotoxins in clinical matrices such as urine. These clinical assays are expected to be compatible with drinking water matrices. • LC/MS/MS methods used in chemical laboratory response network (LRN) for clinical samples • Addition of drinking water matrices to experimental design • Stability, extraction, chromatography, MS tuning • High throughput, small samples, automation, IT
Office of Research and Development Biotoxins will be extracted from water samples, separated, and analyzed
HPLC Solid Phase Extraction
Data Analysis Mass Spectrometry Office of Research and Development ine ol pr xy ro yd -h e 4 cin OH leu so ne yi gi ox ra dr pa y as O dih O 5- HO 4, H2N N O OH O NH HN O Features of Adapted Methods cysteine O S n ha HN HN top HN yp ytr ox dr hy O 4-
e cin NH HO O gly • 100-1000 samples/day H O N NH e cin O gly e cin leu iso • Analytes Exact Mass: 918.35 –α, β -amanitin –Mycotoxins –Marine Toxins Native –TETS –Biomarkers for ricin and abrin • Stable Isotopic Internal Standards • Availability: Fall 2010 Labeled ISTD
Office of Research and Development Current Projects
Centers for Disease Control and Prevention (very recent) • Adaptation of the LRN Botulinum Toxin ELISA assay for water. • Adaptation of the Endopep MS assay for water. – Antibody based cleanup, SNAP-25 cleavage with MS detection • Investigate the ability of UF concentration device(s) to concentrate botulinum toxin. • Several analytical approach adaptations for ricin.
EPA CDC SAM environmental clinical matrices matrices
Office of Research and Development Current Projects
Food Safety Inspection Service (USDA) on-going project • Adaption of immunoassay with electrochemiluminescence (ECL) detection methods from Food Emergency Response Network (FERN) to environmental matrices • Plant and bacterial toxins (botulinum, ricin, others) • Civil Support Teams also have this equipment and reagents
Office of Research and Development Future directions for biotoxin detection
Future collaborative work • Analysis approaches for other matrices • Additional biotoxins • Refinement of existing methods • Decontamination-focused studies (and associated detection challenges) • Enhanced federal collaborations – recent agreement between EPA, DHS, and DOD.
Method verification • Methods may be evaluated in 2nd or more labs. • May be compatible with current public health lab operations
Office of Research and Development Acknowledgements
• Centers for Disease Control and Prevention • Naval Surface Warfare Center – Rudy Johnson, Ph.D. – Elaine Strauss, Ph.D. – Jennifer Links, Ph.D. – Wynn Vo – Stephen Morse, Ph.D. – Andrew Slaterbeck, Ph.D. – CT method development group – Bradford Gutting, Ph.D.
• Food Safety Inspection Service • Environmental Protection Agency – Mark Campbell, Ph.D. – Michelle Burgess, Ph.D., OEM – Marcus Head, Ph.D. – Sanjiv Shah, Ph.D., NHSRC – Jim Jones, Ph.D. – Erin Silvestri, MPH, NHSRC – Anne Hurley, DVM, MPH – Sarah Perkins, Ph.D., NHSRC
Office of Research and Development Questions?
Matthew Magnuson, Ph.D. Research Chemist National Homeland Security Research Center Water Infrastructure Protection Division Cincinnati, OH 45268
513 569 7321 [email protected] http://www.epa.gov/nhsrc
Disclaimer: The U.S. Environmental Protection Agency funded, partially funded, managed, and/or collaborated in the research described in this presentation. It has been subject to an administrative review but does not necessarily reflect the views of the Agency. No official endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial or non-commercial products or services.
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