Anand MudambiU.S. EPA

Office of Ground Water and Drinking Water

Anand MudambiU.S. EPA

Office of Ground Water and Drinking Water

Water Laboratory Alliance Security SummitChemical and Biological Analysis

for Drinking Water ResponseOctober 22-23, 2009, Philadelphia, PA

Water Laboratory Alliance Security SummitChemical and Biological Analysis

for Drinking Water ResponseOctober 22-23, 2009, Philadelphia, PA

Priority Drinking Water Chemical & Radioactive

ContaminantsWSD 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

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

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

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

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)

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

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

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

Water Analysis Capabilities for Homeland Security – Biological Agents

Water Laboratory Alliance Security SummitWater Analysis Capabilities for Homeland

SecurityOctober 22-23, 2009, Philadelphia, PA

Water Laboratory Alliance Security SummitWater Analysis Capabilities for Homeland

SecurityOctober 22-23, 2009, Philadelphia, PA

H. D. Alan Lindquist, Water Infrastructure Protection Division, Office of Research and Development, U. S. Environmental

Protection Agency

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

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Not meant to represent “The List”

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

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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

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

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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

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 concentrator14

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

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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 watera) 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. Goalsa) Safeb) Efficient, operator friendlyc) Fastd) Portable (take to sample location, versus moving sample)

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

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Potential Tangential Filtration Schematics

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Filter

Concentrated sample

Pump

Sample

To waste

Filter

Concentrated sample

Sample

To waste

Pump

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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

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

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Prototype Concentrator Device, cont

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Interior of prototype

Control screen for prototype

Comparison of recovery efficiency

automated versus manual systems

Automated Prototype

ManualVersion

Automated Prototype

Manual Version

Trial % B. globigii recovery % E. coli Recovery

1 42 38 41 48

2 50 48 58 73

3 27 34 56 64

4 37 37 46 44

5 49 44 47 46

6 33 50 56 49

7 55 60 69 54

Average 41.8 44.3 53.3 54.1

St. Dev. 10.1 9.1 9.5 10.8

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Recovery of organisms from finished waters using a laboratory

based system

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Average Percent Recovery1, 2

Water Sourcen = 3 to 5

Bacillus anthracis

Sterne[106]

Yersinia pestis CO92[107]

Francisella tularensis

LVS[107]

MS2

[106]

Phi-X174

[105]

Cryptosporidium parvum

[103]

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)

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Status

• This technology is patent pending

• Has been licensed to Teledyne-ISCO

• Prototypes are being tested for compatibility with current field and laboratory processes

Questions?

Contact Information:Alan Lindquist lindquist.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

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• 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