1. Anand Mudambi U.S. EPA Office of Ground Water and Drinking Water Anand Mudambi U.S. EPA Office of Ground Water and Drinking Water Water Laboratory Alliance Security Summit Chemical and Biological Analysis for Drinking Water Response October 22-23, 2009, Philadelphia, PA Water Laboratory Alliance Security Summit Chemical and Biological Analysis for Drinking Water Response October 22-23, 2009, Philadelphia, PA

2. Priority Drinking Water Chemical & Radioactive Contaminants WSD identified Priority Contaminants in 2005 33 Chemical Contaminants – Pesticides, rodenticides, herbicides, cyanide compounds, organometallic compounds, CWAs, metal salts, pharmaceuticals, PCBs, fuels, fluorinated compounds 7 Radioactive Isotopes – Alpha, beta, and gamma emitters Selected based upon – Potency – Stability in drinking water – Solubility – Availability

3. Existing Drinking Water Methods 20 of the 33 priority chemical contaminants (or components of them*) were already on the list of analytes for existing drinking water methods *e.g., sodium arsenite can be detected by ICP/MS as arsenic. All 7 radioactive isotopes could be either detected or screened for using existing methods routinely used for drinking water

4. Drinking Water Validation of Chemical Contaminants The first attempt to validate the remaining 13 chemical contaminants was to analyze using existing methods Some of the methods were adequate for screening One method was successfully single and multi- laboratory validated for the two fluorinated organic compounds

5. Initiated to address gaps in capability not resolved by previous method development work Direct injection LC-MS in full scan mode allows for rapid screening of many contaminants with little preparation time Initial results show that LC-MS screening can detect 12 priority contaminants, 6 of which are not part of any drinking water method LC-MS Screening Single Laboratory Validation Study

6. NHSRC Method Development Studies EPA National Homeland Security Research Center (NHSRC) is currently testing several methods which can be used with drinking water, many of which include WSD Priority Contaminants Both single and multi-laboratory testing has been completed, additional methods are currently being tested A variety of separation and analysis techniques are utilized in these methods (LC-MS-MS, GC-MS, IC-MS, ICP-MS)

7. EPA WSD Ultrafiltration Study (UF) EPA’s WLA currently relies on CDC’s Laboratory Response Network (LRN) for select agent analyses using a LRN ultrafiltration (UF) lab-based protocol for concentration of large water volumes (10 – 100 L) This study will result in the development of QC criteria for the LRN UF protocol using non-select surrogates for: – Vegetative bacteria (Enterococcus faecalis) – Spore-forming bacteria (Bacillus atrophaeus) – Virus surrogate (MS2) EPA appreciates the 13 LRN labs that are participating in this study as volunteers

8. Ultrafiltration Study (UF), cont QC criteria will allow LRN labs to: Confirm acceptable performance Maintain proficiency between rounds of CDC’s Proficiency Testing (PT) program Identify method and lab issues Identify potentially problematic matrices Preliminary quality control (QC) criteria have already been developed and used in the EPA Region 1 and Region 2 Full Scale Exercise (conducted in September 2009) Laboratory analyses anticipated to be complete by October 2009

9. UF Participant LRN Labs Hawaii Department of Health, State Laboratories Division Bioterrorism Response Laboratory Indiana State Department of Health Laboratories Michigan Department of Community Health ATDC, Upper Peninsula Regional Laboratory Minnesota Department of Health Nebraska Public Health Environmental Laboratory Pennsylvania Department of Health Bureau of Laboratories Sacramento County Public Health Laboratory State of Idaho Bureau of Laboratories University of Iowa Hygienic Laboratory US Food and Drug Administration Northeast Regional Laboratory US Food and Drug Administration Southeast Regional Laboratory Wadsworth Center – NYSDOH Biodefense Laboratory Wisconsin State Laboratory of Hygiene

10. Water Analysis Capabilities for Homeland Security – Biological Agents Water Laboratory Alliance Security Summit Water Analysis Capabilities for Homeland Security October 22-23, 2009, Philadelphia, PA Water Laboratory Alliance Security Summit Water Analysis Capabilities for Homeland Security October 22-23, 2009, Philadelphia, PA H. D. Alan Lindquist, Water Infrastructure Protection Division, Office of Research and Development, U. S. Environmental Protection Agency

11. Biological Contaminants of Concern Select Agents Lists from HHS, DoA and “Overlap Agents” includes a list of plant pathogens HHS agents are human diseases DoA agents are animal or plant diseases −Some animal or plant diseases may become human diseases under particular conditions (e.g. BSE, HPAI) Overlap agents are of both veterinary (or plant) concern and concern for human health Includes bacteria, fungi, chromista, viruses, a prion, and toxins of biological origin Other contaminants of concern During the development of the Select Agent list, the CDC cited “water safety threats” in the “Category B” list Examples: –Vibrio cholerae –Cryptosporidium parvum SAM list (Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events Revision 5.0) Includes the CDC examples for water threats Excluding select agents for brevity 11 Not meant to represent “The List”

12. Select Agents (human and overlap) Bacteria Bacillus anthracis Brucella abortus Brucella melitensis Brucella suis Burkholderia mallei (formerly Pseudomonas mallei) Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) Botulinum neurotoxin producing species of Clostridium Coxiella burnetii Francisella tularensis Rickettsia prowazekii Rickettsia rickettsii Yersinia pestis Fungi Coccidioides posadasii/Coccidioides immitis Biotoxins Abrin Botulinum neurotoxins Clostridium perfringens epsilon toxin Conotoxins Diacetoxyscirpenol Ricin Saxitoxin Shiga-like ribosome inactivating proteins Shigatoxin Staphylococcal enterotoxins T-2 toxin Tetrodotoxin 12 Viruses Cercopithecine herpesvirus 1 (Herpes B virus) Crimean-Congo haemorrhagic fever virus Eastern Equine Encephalitis virus Ebola virus Hendra virus Reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (Reconstructed 1918 Influenza virus) Lassa fever virus Marburg virus Monkeypox virus Nipah virus Rift Valley fever virus South American Haemorrhagic Fever viruses –Flexal –Guanarito –Junin –Machupo –Sabia Tick-borne encephalitis complex (flavi) viruses –Central European Tick-borne encephalitis –Kyasanur Forest disease –Omsk Hemorrhagic Fever –Russian Spring and Summer encephalitis Variola major virus (Smallpox virus) Variola minor virus (Alastrim) Venezuelan Equine Encephalitis virus Animal and plant diseases Not listed here From: www.selectagents.gov

13. SAM Pathogens and Biotoxins (Select Agents Omitted) Bacteria Campylobacter jejuni Chlamydophila psittaci Escherichia coli O157:H7 Leptospira spp. Listeria monocytogenes Non-typhoidal Salmonella spp. Salmonella Typhi spp. Shigella spp. Staphylococcus aureus Vibrio cholerae O1 and O139 Viruses Adenoviruses A-F Astroviruses Caliciviruses: Noroviruses Caliciviruses: Sapoviruses Coronaviruses: SARS Hepatitis E Virus Picornaviruses: Enteroviruses Picornaviruses: Hepatitis A Virus Reoviruses: Rotaviruses 13 Protozoa Cryptosporidium spp. Entamoeba histolytica Giardia spp. Toxoplasma gondii Helminths Baylisascaris procyonis Biotoxins Aflatoxin (Type B1)  -Aminitin Anatoxin-a Brevetoxins (B form) Cylindrospermopsin Microcystins (Principal isoforms: LA, LR, YR, RR, LW) Picrotoxin

14. Methods Development Updates – Current Capabilities Analytical Assays Select Agents – Confirmatory assays available through LRN – Once confirmed, must be handled as a Select Agent – LRN laboratory may establish acceptance criteria for samples Non-select agents on SAM list – SAM lists at least one method or assay – Not all assays are appropriate for all sample types – Intelligent decision making must be used in method selection Sampling Techniques LRN (ship sample to appropriate confirmatory tier laboratory). Response Protocol Toolbox – More complete description published (Lindquist et al. 2007. J. Microbiol. Methods. 70(3):484-492) Portable semi-automated water sample concentrator 14

15. Non-Select Single-Laboratory Verification Studies E. coli O157:H7 – Project Completed Method optimized and verified for phosphate buffered saline (PBS, reference matrix), surface water, and drinking water matrices Mean recoveries for surface water and drinking water were 103% and 214%, respectively Vibrio cholerae O1 and O139 Optimization and verification in PBS and drinking water completed Additional optimization required for surface water Salmonella Typhi Optimization and verification in PBS and drinking water completed Additional optimization required for surface water 15

16. 16 Motivation for Developing Device 1.Standard microbiological sample concentration techniques may not allow detection some pathogens at levels of concern for public health impacts in water a)Increasing the concentration of microorganisms in a sample improves detection 2.Nearly all techniques for the detection of microorganisms in water require some type of concentration step, most often filtration 3.Develop one device that can concentrate bacteria, viruses, and protozoa, including microorganisms for which there are no existing methods 4.Goals a)Safe b)Efficient, operator friendly c)Fast d)Portable (take to sample location, versus moving sample)

17. Target Sample Volume and Typical Volume Reduction 100 liters down to 400 ml 250 fold increase in concentration of microorganisms Final volume may be tailored for specific needs 17

18. Potential Tangential Filtration Schematics 18 Filter Concentrated sample Pump Sample To waste Filter Concentrated sample Sample To waste Pump

19. 19 Typical Process Parameters Processing Flow rate: 1,750 – 2,500 mL/min Volume processed: 100 L of drinking water Processing time, including pretreatment: 1 hour Filter inlet pressure: 15 – 25 psi

20. Prototype Concentrator Device 31" long, 20" deep, 16" high 85 pounds Tubing assembly Dialysis filter Tubing Check valve Fittings Bottle and cap HEPA filter Cable ties Quick disconnect fittings Pressure transducer and cable All items considered disposable 20

21. Prototype Concentrator Device, cont 21 Interior of prototype Control screen for prototype

22. Comparison of recovery efficiency automated versus manual systems Automated Prototype Manual Version Automated Prototype Manual Version Trial% B. globigii recovery% E. coli Recovery 142384148 250485873 327345664 437 4644 549444746 633505649 755606954 Average41.844.353.354.1 St. Dev.10.19.19.510.8 22

23. Recovery of organisms from finished waters using a laboratory based system 23 Average Percent Recovery 1, 2 Water Source n = 3 to 5 Bacillus anthracis Sterne [10 6 ] Yersinia pestis CO92 [10 7 ] Francisella tularensis LVS [10 7 ] MS2 [10 6 ] Phi- X174 [10 5 ] Cryptosporidium parvum [10 3 ] Columbus OH, (Surface water source) 60% (44) 61% (5) 17% (10) 89% (32) 83% (34) 36% (27) Columbus OH (Groundwater source) 57% (11) 81% (13) 6% (5) 40% (47) 104% (6) 81% (34) New York City (Unfiltered surface water) 77% (28) 40% (39) 56% (84) 28% (2) 73% (101) Not Determined 1 Spiked amount per approximately 100 liters in [brackets] 2 Standard Deviation in (parenthesis) Source: Holowecky, P., et al. Evaluation of Ultrafiltration Cartridges for a Water Sampling Device. Journal of Microbiological Methods (2009)

24. 24 Status This technology is patent pending Has been licensed to Teledyne-ISCO Prototypes are being tested for compatibility with current field and laboratory processes

25. Questions? Contact Information: Alan Lindquist lindquist.alan@epa.govlindquist.alan@epa.gov Acknowledgments: EPA: – Latisha Mapp – Malik Raynor – Vincent Gallardo Idaho National Laboratory, managed by Battelle Energy Alliance: – Michael Carpenter – Lyle Roybal – Paul Tremblay 25 Pegasus Technical Services, Contractor to US EPA: – Ben Humrighouse – Adin Pemberton – William Kovacik – Margaret Hartzel – Sasha Lucas – Diana Riner Battelle Memorial Institute: – Patricia Holowecky – James Ryan – Scott Straka – Daniel Lorch