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 ResponseJune 16-17, 2010 San Francisco, CA

Water Laboratory Alliance Security Summit:Chemical and Biological Analysis

for Drinking Water ResponseJune 16-17, 2010 San Francisco, CA

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Chemical and Biological Method Development

• Chemical

• Biological

• Laboratory Response Network Ultrafiltration (LRN UF) QC Criteria

• EPA Field Portable UF Device

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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

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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

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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

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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

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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)

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Biological Single-Laboratory Verification Studies

• E. coli O157:H7

• Non-typhoidal Salmonella

• Salmonella Typhi

• Vibrio cholerae O1 and O139

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Salmonella spp. produce halos indicating motility on MSRV plates

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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

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• 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

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• 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

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• 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

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• 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 analysesE. faecalis colonies with distinct blue halos on mEI agar

UF QC Criteria Study Objectives

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E. faecalisDraft 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%

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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

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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

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Water Analysis Capabilities for Homeland Security – Biological Agents

Water Laboratory Alliance Security SummitWater Analysis Capabilities for Homeland

SecurityJune 16-17, 2010San Francisco, CA

Water Laboratory Alliance Security SummitWater Analysis Capabilities for Homeland

SecurityJune 16-17, 2010San Francisco, CA

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

Protection Agency

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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

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Not meant to represent “The List”

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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 (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

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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)• -Amanitin• Anatoxin-a• Brevetoxins (B form)• Cylindrospermopsin• Microcystins (Principal isoforms: LA, LR, YR,

RR, LW)• Picrotoxin

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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

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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 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)

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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 – 30 psi

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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

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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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Comparison of EPA and CDC Ultrafiltration Techniques for Recovering Biothreat Agents in

Water

Microbe UF Method N % Recovery Std Dev. Cv

B. anthracis Sterne spores

EPA 10 100 13 12

CDC/LRN 10 85 17 20

Y. pestis EPA 9 70 18 26

CDC/LRN 9 70 16 23

F. tularensis EPA 8 29 15 52

CDC/LRN 8 17 6.9 41

EPA w/NH4Cl 8 39 15 38

CDC/LRN w/ NH4Cl

8 23 8.8 38

E. faecalis EPA 10 100 10 10

CDC/LRN 10 97 12 13

C. perfringens spores

EPA 9 110 27 24

CDC/LRN 9 100 22 22

30Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention

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EPA and CDC Ultrafiltration: Viral and Parasitic Microbes

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Microbe UF Method N % Recovery Std. Dev. Cv

Echovirus 1 EPA 11 47 15 33

CDC/LRN 11 68 26 38

MS2 EPA 11 120 33 28

CDC/LRN 11 110 38 34

Phi X174 EPA 11 95 11 12

CDC/LRN 11 100 13 13

C. parvum (High Dose) EPA 11 73 28 39

CDC/LRN 11 82 29 36

C. parvum (ColorSeed) EPA 10 30 22 72

CDC/LRN 10 38 12 33

G. intestinalis (High Dose)

EPA 11 85 14 17

CDC/LRN 11 99 18 18

G. intestinalis (ColorSeed)

EPA 10 44 24 53

CDC/LRN 10 42 11 25

Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention

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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

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Questions?

Contact Information:Alan Lindquist lindquist.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

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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