Transcript Water Analysis Capabilities for Homeland Security

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
Chemical and Biological Method
Development
• Chemical
• Biological
• Laboratory Response
Network Ultrafiltration
(LRN UF) QC Criteria
• EPA Field Portable UF
Device
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*) 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
4
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 multilaboratory 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
Analytical results show that LC-MS screening can
detect 13 priority contaminants, 6 of which are not
included in 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 (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)
7
Biological Single-Laboratory
Verification Studies
• E. coli O157:H7
• Non-typhoidal Salmonella
• Salmonella Typhi
• Vibrio cholerae O1 and O139
Salmonella spp. produce halos
indicating motility on MSRV
plates
8
Next Steps: Biological MultiLaboratory 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
9
LRN Ultrafiltration (UF) QC
Criteria Study – Background
• 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
10
Use of Surrogates for Development
of UF Criteria
• 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
11
UF Study Surrogate Selection
• 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
12
UF QC Criteria Study Objectives
• 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
13
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%
14
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
15
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
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
June 16-17, 2010
San Francisco, CA
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
Not meant to represent “The List”
18
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
From: www.selectagents.gov
• Tetrodotoxin
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
19
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
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
20
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
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)
22
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
Potential Tangential Filtration
Schematics
To
waste
To
waste
Filter
Filter
Concentrated
sample
Pump
Pump
Sample
Concentrated sample
Sample
24
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
25
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
26
Prototype Concentrator Device, cont
Interior of prototype
Control screen
for prototype
27
Comparison of Recovery Efficiency:
Automated versus Manual Systems
Automated
Prototype
Trial
Manual
Version
% B. globigii recovery
Automated
Prototype
Manual
Version
% 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
28
Recovery of Organisms from Finished
Waters using a Laboratory Based System
Average Percent Recovery1, 2
Water Source
n = 3 to 5
Bacillus
anthracis
Sterne
[106]
Yersinia
pestis
CO92
[107]
Francisella
tularensis
LVS
[107]
MS2
PhiX174
[106]
[105]
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
2
Cryptosporidium
parvum
[103]
Spiked amount per approximately 100 liters in [brackets]
Standard Deviation in (parenthesis)
Source: Holowecky, P., et al. Evaluation of Ultrafiltration Cartridges for a Water Sampling Device. Journal of Microbiological Methods (2009)
29
Comparison of EPA and CDC Ultrafiltration
Techniques for Recovering Biothreat Agents in Water
Microbe
B. anthracis
Sterne spores
Y. pestis
F. tularensis
E. faecalis
C. perfringens
spores
UF Method
N
% Recovery
Std Dev.
Cv
EPA
10
100
13
12
CDC/LRN
10
85
17
20
EPA
9
70
18
26
CDC/LRN
9
70
16
23
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
EPA
10
100
10
10
CDC/LRN
10
97
12
13
EPA
9
110
27
24
CDC/LRN
9
100
22
22
Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention
30
EPA and CDC Ultrafiltration:
Viral and Parasitic Microbes
Microbe
Echovirus 1
MS2
Phi X174
C. parvum (High Dose)
C. parvum (ColorSeed)
G. intestinalis (High
Dose)
G. intestinalis
(ColorSeed)
UF Method
N
% Recovery
Std. Dev.
Cv
EPA
11
47
15
33
CDC/LRN
11
68
26
38
EPA
11
120
33
28
CDC/LRN
11
110
38
34
EPA
11
95
11
12
CDC/LRN
11
100
13
13
EPA
11
73
28
39
CDC/LRN
11
82
29
36
EPA
10
30
22
72
CDC/LRN
10
38
12
33
EPA
11
85
14
17
CDC/LRN
11
99
18
18
EPA
10
44
24
53
CDC/LRN
10
42
11
25
Source: Vincent Hill, Suresh Pai, Tina Lusk. Centers for Disease Control and Prevention
31
Status
• This technology is
patent pending
• Has been licensed to
Teledyne-ISCO
• Prototypes are being
tested for compatibility
with current field and
laboratory processes
32
Questions?
Contact Information:
Alan Lindquist
[email protected]
Acknowledgments:
• EPA:
– Latisha Mapp
– Malik Raynor
– Vincente 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
33