Anand Mudambi

1
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
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
LC-MS Screening Single Laboratory Validation
Study

• 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

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)

• EPAs WLA currently relies on CDCs 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 CDCs
  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
H. D. Alan Lindquist, Water Infrastructure
Protection Division, Office of Research and
Development, U. S. Environmental Protection Agency
Water Laboratory Alliance Security Summit Water
Analysis Capabilities for Homeland
Security October 22-23, 2009, Philadelphia, PA
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

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

• 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

From www.selectagents.gov
13
SAM Pathogens and Biotoxins (Select Agents
Omitted)

• Bacteria
• Campylobacter jejuni
• Chlamydophila psittaci
• Escherichia coli O157H7
• 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

• 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

15
Non-Select Single-Laboratory Verification Studies

• E. coli O157H7 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

16
Motivation for Developing Device

• Standard microbiological sample concentration
  techniques may not allow detection some pathogens
  at levels of concern for public health impacts in
  water
• Increasing the concentration of microorganisms in
  a sample improves detection
• Nearly all techniques for the detection of
  microorganisms in water require some type of
  concentration step, most often filtration
• Develop one device that can concentrate bacteria,
  viruses, and protozoa, including microorganisms
  for which there are no existing methods
• Goals
• Safe
• Efficient, operator friendly
• Fast
• 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

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

21
Prototype Concentrator Device, cont

• Interior of prototype

Control screen for prototype
22
Comparison of recovery efficiencyautomated
versus manual systems
Automated Prototype Manual Version Automated Prototype Manual Version
Trial B. globigii recovery B. globigii recovery E. coli 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
23
Recovery of organisms from finished waters using
a laboratory based system
Average Percent Recovery1, 2 Average Percent Recovery1, 2 Average Percent Recovery1, 2 Average Percent Recovery1, 2 Average Percent Recovery1, 2 Average Percent Recovery1, 2
Water Source n 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) 1 Spiked amount per approximately 100 liters in brackets 2 Standard Deviation in (parenthesis) 1 Spiked amount per approximately 100 liters in brackets 2 Standard Deviation in (parenthesis) 1 Spiked amount per approximately 100 liters in brackets 2 Standard Deviation in (parenthesis) 1 Spiked amount per approximately 100 liters in brackets 2 Standard Deviation in (parenthesis) 1 Spiked amount per approximately 100 liters in brackets 2 Standard Deviation in (parenthesis) 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
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_at_epa.gov
• Acknowledgments
• EPA
• Latisha Mapp
• Malik Raynor
• Vincent Gallardo
• Idaho National Laboratory, managed by Battelle
  Energy Alliance
• Michael Carpenter
• Lyle Roybal
• Paul Tremblay

• 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