1. U.S. EPA Office of Ground Water and Drinking Water U.S. EPA Office of Ground Water and Drinking Water Water Laboratory Alliance Security Summit: Chemical and Biological Analysis for Drinking Water Response June 16-17, 2010 San Francisco, CA Water Laboratory Alliance Security Summit: Chemical and Biological Analysis for Drinking Water Response June 16-17, 2010 San Francisco, CA

2. 2 Chemical and Biological Method Development Chemical Biological Laboratory Response Network Ultrafiltration (LRN UF) QC Criteria EPA Field Portable UF Device 2

3. 3 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

4. 4 Existing Drinking Water Methods 20 of the 33 priority chemical contaminants (or components*) 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 using existing methods routinely used for drinking water

5. 5 Validation for Chemical Contaminants in Drinking Water The first attempt to validate methods for 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

6. 6 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 Analytical results show that LC-MS screening can detect 13 priority contaminants, 6 of which are not included in any drinking water method LC-MS Screening Single Laboratory Validation Study

7. 7 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 (e.g., CWAs) 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)

8. 8 Biological Single-Laboratory Verification Studies E. coli O157:H7 Non-typhoidal Salmonella Salmonella Typhi Vibrio cholerae O1 and O139 8 Salmonella spp. produce halos indicating motility on MSRV plates

9. 9 Next Steps: Biological Multi- Laboratory Validation Studies Non-typhoidal Salmonella – 10 volunteer laboratories – Drinking water and surface water – Assess method performance and reproducibility – Develop quantitative quality control (QC) criteria E. coli O157:H7 – Preliminary analyses prior to multi-laboratory validation: Strain evaluation Evaluation of Rainbow ® agar

10. 10 WLA utilizes CDC’s Laboratory Response Network (LRN) protocol for concentrating large volumes (>100 L) of drinking water – LRN Filter Concentration for the Detection of Bioterrorism Threat Agents in Potable Water Samples – Potential contaminants concentrated include vegetative bacteria, bacterial spores, viruses, and some toxins (e.g., ricin) Requires comprehensive training and practice to achieve and maintain proficiency QC criteria did not exist for the UF protocol; therefore, it was difficult to determine if laboratory was proficient QC criteria can be used by laboratories to maintain proficiency between PT samples, identify issues (e.g., equipment or reagent problems), and problematic matrices LRN Ultrafiltration (UF) QC Criteria Study – Background 10

11. 11 Example agents of concern concentrated using the LRN ultrafiltration protocol: – Vegetative Bacteria (Francisella tularensis, Brucella spp., Salmonella Typhi) – Spore-forming Bacteria (Bacillus anthracis) – Viruses (Orthopoxviruses, Enteroviruses, Caliciviruses) Surrogates utilized to mitigate safety hazards during routine use and to reduce logistical challenges Use of Surrogates for Development of UF Criteria 11

12. 12 Vegetative bacteria: Enterococcus faecalis – Easy to work with, EPA Method 1600 available, commercially available BioBall spikes, previous data generated through WSi pilot at GCWW Bacterial spore: Bacillus atrophaeus – Commercially available BioBall spikes, Standard Methods 9218 available, produces orange colonies, making it distinguishable from background Bacillus in drinking water samples Virus: Male-specific (MS2) coliphage – Commercially available spikes, EPA Method 1602, used by UF researchers UF Study Surrogate Selection 12

13. 13 Develop quantitative QC criteria for UF procedure using surrogates Develop quantitative QC criteria for analytical surrogate methods Develop QA guidelines for implementation of the LRN UF procedure in support of the WLA – Positive and negative controls – Frequency of QC analyses E. faecalis colonies with distinct blue halos on mEI agar UF QC Criteria Study Objectives 13

14. 14 E. faecalis Draft QC Criteria for Ultrafiltration Initial precision and recovery (IPR) criteria based on 4, 40-L PBS samples – Recovery range: 52% − 100% – Precision, as maximum Relative Standard Deviation: 41% Ongoing precision and recovery (OPR) criteria based on 1, 40-L PBS sample – Recovery range: 36% − 112% Matrix spike (MS) criteria for E. faecalis based on 1, 100-L drinking water sample – Recovery range: 21% − 128%

15. 15 Concentration of Large-Volume Biological Samples Advantages of LRN Ultrafiltration Protocol Protocol has undergone multi-center validation by CDC QC criteria have been developed to help ensure laboratory proficiency Disadvantage Requires transfer of large volume (100-L) samples that are potentially contaminated from the field to the laboratory

16. 16 Next Steps: Increase WLA Select Agent Capability and Capacity Implement QC criteria for the LRN UF protocol Collaborate with CDC to optimize the LRN Filter Concentration for the Detection of Bioterrorism Threat Agents in Potable Water Samples protocol Expand the number of laboratories that are approved to evaluate water samples for select agents Continue EPA’s collaboration with CDC and others to implement a field-portable UF device 16

17. Water Analysis Capabilities for Homeland Security – Biological Agents Water Laboratory Alliance Security Summit Water Analysis Capabilities for Homeland Security June 16-17, 2010 San Francisco, CA Water Laboratory Alliance Security Summit Water Analysis Capabilities for Homeland Security June 16-17, 2010 San Francisco, CA H. D. Alan Lindquist, Water Infrastructure Protection Division, Office of Research and Development, U. S. Environmental Protection Agency

18. 18 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 and –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 18 Not meant to represent “The List”

19. 19 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 19 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 (Reconstructed1918 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

20. 20 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 20 Protozoa Cryptosporidium spp. Entamoeba histolytica Giardia spp. Toxoplasma gondii Helminths Baylisascaris procyonis Biotoxins Aflatoxin (Type B1)  -Amanitin Anatoxin-a Brevetoxins (B form) Cylindrospermopsin Microcystins (Principal isoforms: LA, LR, YR, RR, LW) Picrotoxin

21. 21 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 – The SAM document lists at least one method or assay per analyte – Not all assays are appropriate for all sample types – Intelligent decision making must be used in method selection – The next version of the SAM document will feature major changes 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 21

22. 22 Motivation for Developing Device 1.Standard microbiological sample concentration techniques may not allow detection of 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)

23. 23 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 23

24. 24 Potential Tangential Filtration Schematics 24 Filter Concentrated sample Pump Sample To waste Filter Concentrated sample Sample To waste Pump

25. 25 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 – 30 psi

26. 26 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

27. 27 Prototype Concentrator Device, cont 27 Interior of prototype Control screen for prototype

28. 28 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 28

29. 29 Recovery of Organisms from Finished Waters using a Laboratory Based System 29 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)

30. 30 Comparison of EPA and CDC Ultrafiltration Techniques for Recovering Biothreat Agents in Water MicrobeUF MethodN% RecoveryStd Dev.CvCv B. anthracis Sterne spores EPA101001312 CDC/LRN10851720 Y. pestisEPA9701826 CDC/LRN9701623 F. tularensisEPA8291552 CDC/LRN8176.941 EPA w/NH 4 Cl8391538 CDC/LRN w/ NH 4 Cl 8238.838 E. faecalisEPA1010010 CDC/LRN10971213 C. perfringens spores EPA91102724 CDC/LRN910022 30 Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention

31. 31 EPA and CDC Ultrafiltration: Viral and Parasitic Microbes 31 MicrobeUF MethodN% RecoveryStd. Dev.CvCv Echovirus 1EPA11471533 CDC/LRN11682638 MS2EPA111203328 CDC/LRN111103834 Phi X174EPA11951112 CDC/LRN1110013 C. parvum (High Dose)EPA11732839 CDC/LRN11822936 C. parvum (ColorSeed)EPA10302272 CDC/LRN10381233 G. intestinalis (High Dose) EPA11851417 CDC/LRN119918 G. intestinalis (ColorSeed) EPA10442453 CDC/LRN10421125 Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention

32. 32 Status This technology is patent pending Has been licensed to Teledyne-ISCO Prototypes are being tested for compatibility with current field and laboratory processes

33. 33 Questions? Contact Information: Alan Lindquist lindquist.alan@epa.govlindquist.alan@epa.gov Acknowledgments: EPA: – Latisha Mapp – Malik Raynor – Vincente Gallardo Idaho National Laboratory, managed by Battelle Energy Alliance: – Michael Carpenter – Lyle Roybal – Paul Tremblay 33 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