&EPA
United States
Environmental Protection
Agency
EPA/600/R-16/368 I December 2016
www.epa.gov/homeland-security-research
Evaluation of Persistence of Viruses in
Landfill Leachate
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-16/368
December 2016
Evaluation of Persistence of Viruses in
Landfill Leachate
National Homeland Security Research
Center Office of Research and Development
U.S. Environmental Protection Agency
26 West Martin Luther King Drive Cincinnati,
OH 45268
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this
technology evaluation through Task Order 0002 of Contract No. EP-C-15-002 with Battelle. This
report has been peer and administratively reviewed and has been approved for publication as an
EPA document. Mention of trade names or commercial products does not constitute endorsement
or recommendation for use of a specific product.
Questions concerning this document or its application should be addressed to:
Dr. Paul M. Lemieux
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
109 T.W. Alexander Drive
Mail Code: E343-06
Research Triangle Park, NC 27709
919-541-0962
lemieux.paul@epa.gov
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Acknowledgments
Contributions of the following individuals and organizations to the development of this
document are gratefully acknowledged.
United States Environmental Protection Agency (EPA)
Paul Lemieux, Task Order Contracting Officer's Representative
Eletha Brady-Roberts, National Homeland Security Research Center
Battelle Memorial Institute
Meg Howard
Nola Bliss
Ryan James
Zachary Willenberg
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Table of Contents
Page
Disclaimer i
Acknowledgments ii
Abbreviations/Acronyms v
Executive Summary 1
1.0 Introduction 4
2.0 Approach 6
2.1 Task 1 - Landfill Leachate Acquisition and Characterization 6
2.2 Method Development 7
2.3 Virus Persistence Testing 8
2.4 Microbial Activity 9
3.0 Procedures 10
3.1 Landfill Leachate Acquisition and Characterization 10
3.1.1 Landfill Selection 10
3.1.2 Logistics 10
3.1.3 Leachate Collection 11
3.1.4 Microbial Activity 15
3.2 Virus Propagation 15
3.2.1 TGEV Propagation 15
3.2.2 Bacteriophage Propagation 17
3.3 Persistence Testing 18
3.3.1 Sample Preparation 18
3.3.2 Incubation 20
3.3.3 Sample Analysis 21
3.3.4 Data Analysis and Interpretation 27
4.0 Quality Assurance/Quality Control 29
4.1 Performance Evaluation Audit 29
4.2 Technical System Audit 29
4.3 Data Quality Audit 29
4.4 QA/QC Reporting 30
5.0 Results 31
5.1 Landfill Leachate Characterization 31
5.2 TGEV Persistence 33
5.3 MS2 Persistence 35
5.4 Phi6 Persistence 38
5.5 Microbial Activity 41
5.6 Evaporation 45
6.0 Discussion 46
7.0 Conclusions 48
8.0 References 50
Appendix A: Method Development Summary Report 51
Appendix B: Miscellaneous Operating Procedure 59
in
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List of Figures
Page
Figure 1. Landfill A Leachate Accumulation Area 12
Figure 2. Landfill B leachate sump area 12
Figure 3. Landfill C leachate accumulation area 13
Figure 4. Landfill Leachates A, B, and C in 3.78 L containers 14
Figure 5. Persistence samples in 5-mL cryovials placed within cryobox 20
Figure 6. Sample incubators 21
Figure 7. TGEV CPE on ST Cells 23
Figure 8. TCID50 Titer Calculation adapted from [3] 24
Figure 9. MS2 plaques on E. coli 26
Figure 10. Phi6 plaques oni5. syringae 26
Figure 11. TGEV Persistence at 12°C 34
Figure 12. TGEV Decay Curves at 12°C 35
Figure 13. MS2 Persistence at 12°C 36
Figure 14. MS2 Persistence at 37°C 37
Figure 15. MS2 Decay Rate at 12°C 37
Figure 16. MS2 Decay Rate at 37°C 38
Figure 17. Phi6 Persistence at 12°C 39
Figure 18. Phi6 Persistence at 37°C 40
Figure 19. Phi Decay Rates at 12°C 40
Figure 20. Phi6 Decay Rates at 37°C 41
Figure 21. Bacterial and Fungal Growth on TSA and PDA growth media 43
Figure 22. Bacterial and Fungal Growth on TSA and PDA growth media 44
List of Tables
Executive Summary Table 1. Persistence of Various Viruses in Three Landfill Leachates 2
Executive Summary Table 2. Decay Rates of Viral Agents in Three Landfill Leachates 3
Table 1. Test Matrix for Virus Persistence Evaluation in Landfill Leachates 9
Table 2. Landfill Characteristics 10
Table 3. Sample Analysis Time Points 22
Table 4. Landfill Leachate Characterization Data 32
Table 5. TGEV Measured D-values at 12°C 34
Table 6. MS2 D-values and Persistence 36
Table 7. Phi6 D-Values and Persistence 39
Table 8. Microbial Activity in Landfill Leachates 42
iv
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Abbreviations/Acronyms
AHA activity hazard analysis
APHIS U.S. Department of Agriculture Animal and Plant Health Inspection
Service
ATCC American Type Culture Collection
BOD biological oxygen demand
BSL Biosafety Level
BW biological warfare
°C degree(s) Celsius
CAA Clean Air Act
CFU colony forming unit(s)
CFU/mL colony forming unit(s) per milliliter
cm centimeter(s)
CPE cytopathic effect
CoA Certificate of Analysis
COC chain of custody
COD chemical oxygen demand
DAL double agar layer
DHL DHL Analytical, Inc.
EDTA Ethylenediaminetetraacetic acid
EMEM Eagle's Minimum Essential Medium
E. coli Escherichia coli
EPA U.S. Environmental Protection Agency
°F degree(s) Fahrenheit
FBS Fetal Bovine Serum
FSMA Food Safety Modernization Act
g gram(s)
HDPE high density polyethylene
L liter(s)
LB A Luria Bertani Agar
LBB Luria Bertani Broth
LBTA Luria Bertani Top Agar
In natural logarithm
LOD limit of detection
MERS
Middle East Respiratory Syndrome
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MHV
mouse hepatitis virus
|im
micrometer(s)
mg
milligram(s)
|iL
microliter(s)
mL
milliliter(s)
min
minute(s)
MOI
multiplicity of infection
MOP
miscellaneous operating procedure
MS2
MS2 bacteriophage
MSW
Municipal Solid Waste
NHSRC
EPA National Homeland Security Research Center
ORP
oxidation reduction potential
Phi 6
Phi6 bacteriophage
PBS
Phosphate Buffered Saline
PD
proportional distance
PDA
potato dextrose agar
PE
performance evaluation
PFU
plaque forming unit(s)
PPE
personal protective equipment
ppm
parts per million
P. syringiae
Pseudomonas syringiae
QA
quality assurance
QAPP
quality assurance project plan
QC
quality control
QMP
Quality Management Plan
rcf
relative centrifugal force
RCRA
Resource Conservation and Recovery Act
RNA
ribonucleic acid
SARS
Severe Acute Respiratory Syndrome
ST
swine testicular
T&EII
Testing and Evaluation II Contract
TCIDso
50 % Tissue Culture Infectious Dose
TDS
total dissolved solids
TGEV
Transmissible Gastroenteritis Virus
TNTC
too numerous to count
TO
task order
TOC
total organic carbon
TOCOR
Task Order Contracting Officer's Representative
TOL
Task Order Leader
TSA
Tryptic Soy Agar
vi
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TSB
Tryptic Soy Broth
TSS
total suspended solids
TSTA
Tryptic Soy Top Agar
USD A
U.S. Department of Agriculture
VEEV
Venezuelan Equine Encephalitis Virus
vii
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Executive Summary
The purpose of this effort was to assess the persistence of viruses in landfill leachate. Due to the
limited capacity of incinerators and hazardous waste sites, municipal solid waste (MSW) landfills,
or landfills of a similar design, may receive decontaminated building materials following a terrorist
attack with biological warfare (BW) agents, a natural outbreak of a highly infectious viral pathogen
(i.e., Ebola virus) and/or an unintentional release. The ultimate fate of those materials is of great
concern, particularly if the materials were incompletely decontaminated and contain residual
amounts of BW agents. To determine whether active viruses could pose a threat to human and
environmental health once introduced into a landfill, laboratory testing was performed to measure
the decay rate of viral agents in landfill leachate. This effort was performed using surrogate test
agents similar to BW agents following the well-established hypothesis that, though the diversity
of viral contaminants may be quite large, a limited list of viral surrogates can be chosen that
qualitatively represent the likely BW threat agents of interest.
This effort evaluated the persistence of Transmissible Gastroenteritis Virus (TGEV), MS2
bacteriophage (MS2), and Phi-6 bacteriophage (Phi6) in landfill leachates collected from three
landfills. Each of the three landfill leachates was individually spiked with a known quantity of
virus, dispensed into replicate screw-top vials, and statically incubated. One set of all three virus-
spiked samples was incubated at 12°C, and one set of MS2 and Phi6-spiked samples was incubated
at 37°C; TGEV was not analyzed at 37°C. Throughout the course of the study, triplicate samples
were assayed for infectious viruses (via infectious viral titer) at up to seven time points post To.
TGEV-spiked samples were assayed using a 50% tissue culture infectious dose assay (TCID50), in
which samples were serially diluted, and a range of dilutions was assessed for infectivity on Swine
Testicular (ST) cells. For MS2 and Phi6-spiked samples, triplicate samples were assayed using the
standard Double Agar Layer (DAL) method, in which serial dilutions of samples were analyzed
on Escherichia coli (MS2-specific) and Pseudomonas syringae (Phi6-specific) host strains. Data
were graphed as concentration versus time, and best fit regression curves were used to calculate
persistence (the time where the measured linear rate of decay intersects with the assay limit of
detection) and decay rate (D-value, time required for the viral titer to reduce by 90%, or to 10% of
the starting titer).
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Data generated from this study included viral persistence and decay rates in landfill leachates for
three surrogate viral test agents. Viral persistence and decay rates in three unique landfill leachates
were determined under two temperature conditions (illustrated in Executive Summary Tables 1
and 2, respectively). Data indicated viral surrogate agents (TGEV, MS2 and Phi6) can persist for
weeks to months in landfill leachates, and persistence varies by environmental condition; viruses
persisted longer at mild temperatures (12 degrees Celsius [°C]) and decayed far more rapidly at
warmer temperatures (37°C). The study results suggest that viruses may persist in landfill leachates
for a lengthy period of time (weeks to months) under the mild conditions present in the majority
of the US. Should waste from an attack with viral agents still containing residual agent be disposed
of in a landfill, knowledge of the persistence of the virus in the leachate will allow landfill
operations to be adapted to minimize potential exposures to waste management workers and the
public.
Executive Summary Table 1. Persistence of Various Viruses in Three Landfill Leachates
Virus
Temperature
lost Condition
Calculated Days I mil No Longer Detected'1
Leacliale A
Leacliale li
Leacliale C
Control
Matrix1'
Transmissible Gastroenteritis
Virus (enveloped RNA virus)
12°C
5
17
7
43
MS2 Bacteriophage
(non-enveloped phage)
12°C
113
75
87
NRC
37°C
3
2
2
NRC
Phi6 Bacteriophage
(enveloped phage)
12°C
55
66
81
122
37°C
0.3
0.2
0.2
2
'Calculated time (days) when measured linear decay rate intersects with assay limit of detection.
bTGEV in sterile incomplete Eagle's Minimum Essential Medium (EMEM) medium; bacteriophage in sterile phosphate
buffered saline.
°No decay, or minimal, observed within incubation period tested.
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Executive Summary Table 2. Decay Rates of Viral Agents in Three Landfill Leachates
Virus
Temperature
Measured l)-\:tluc ill Dsiys
lost Condition
l,eachate A
l.eachate li
l.eachate ('
Control
Matrix-'
Transmissible Gastroenteritis
Virus (enveloped RNA virus)
12°C
1
4
2
7
MS2 Bacteriophage
12°C
10
7
8
189
(non-enveloped phage)
37°C
0.3
0.2
0.2
NRb
Phi6 Bacteriophage
12°C
6
10
12
17
(enveloped phage)
37°C
<0.1
<0.1
<0.1
0.15
aTGEV in sterile incomplete EMEM; bacteriophage in sterile phosphate buffered saline (PBS).
bNo decay observed within incubation period tested.
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1.0 Introduction
U.S. Environmental Protection Agency (EPA) is designated as a coordinating Agency under the
National Response Framework to prepare for, respond to, and recover from threats to public health,
welfare, or the environment caused by actual or potential oil and hazardous materials incidents.
Hazardous materials include accidentally or intentionally released chemical, biological, and
radiological substances. The EPA is also designated as a support Agency, to support the U.S.
Department of Agriculture's (USDA's) Animal and Plant Health Inspection Service (APHIS)
activities in agricultural emergency response. In addition, the EPA is a lead agency under Section
208 of the Food Safety Modernization Act (FSMA), tasked with developing model plans for
protecting the nation's food and agricultural infrastructure to safeguard human health and the
environment. Management of waste resulting from cleanup after incidents involving
contamination with biological agents typically involves some sort of treatment process (e.g.,
decontamination, incineration, autoclaving) followed by disposal of the treatment residues in a
secure landfill. Secure landfills include Resource Conservation and Recovery Act (RCRA) Subtitle
C (hazardous waste) or RCRA Subtitle D (municipal waste) landfills, depending on the decisions
that are made, typically at the state level.
This study assessed the persistence and decay rate of viral surrogates in landfill leachate. Waste
generated during natural outbreaks (i.e., Ebola virus waste), clean-up of unintentional releases, or
following a terrorist attack involving biological agents may be placed in MSW landfills. The
ultimate fate of the BW agent(s), in this case viral agents, is of concern. Although these materials
will be decontaminated, large quantities, heterogeneous materials, and laboratory limitations may
lead to residual biological contamination. To evaluate whether infectious viruses in landfill
leachate could survive to be a risk to human and environmental health, laboratory testing was
performed to measure the decay rate of viral surrogates in landfill leachates.
A scientific basis to assess this concern was developed to assess the potential for residual agent to:
1) persist in the landfill environment; and 2) be transported within the landfill environment to
different media (e.g., waste, leachate, gas). This study aimed to evaluate survivability and/or
persistence of viral agents in landfill leachate. Data from this study provide a good framework for
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estimating and determining the fate of residual viral agents that may be placed into a landfill. This
study provides confidence in the ability to effectively predict the fate of infectious viral BW agents
in these types of waste and reduce the need for characterization of highly infectious BW viral
agents represented by these surrogates.
This study used three viral agents as surrogates for highly pathogenic BW viral agents. Three
ribonucleic acid (RNA) viruses were selected: one enveloped mammalian virus (TGEV), one
enveloped bacteriophage (Phi6) and one non-enveloped bacteriophage (MS2). These surrogates
were selected because they represent three common classes of viruses, all which can easily be
manipulated in biosafety level (BSL)-2 facilities, and do not include human infectious agents.
TGEV is an Alphacoronavirus that causes severe disease in young swine (mortality close to 100%
in piglets) and is related to several human coronaviruses. TGEV was used here as a model for Risk
Group 3 Coronaviruses, including Severe Acute Respiratory Syndrome (SARS) and Middle East
Respiratory Syndrome (MERS) coronaviruses, as well as other emerging human enveloped RNA
viruses (e.g., influenza). Risk Group 3 agents are agents associated with serious or lethal human
disease, for which preventative or therapeutic interventions may not be available. These agents
represent high individual risk, and low community risk if released from a laboratory, and often (as
is the case of MERS and SARS) are manipulated in BSL-3 facilities. Phi6 is an enveloped RNA
bacteriophage that infects Pseudomonas syringae. This phage contains a tripartite double-stranded
RNA genome and was used in this study as an intermediate stability enveloped RNA virus. MS2
is a non-enveloped single-stranded RNA virus that infects Escherichia coli. This phage was used
as a surrogate for non-enveloped human infectious viral agents, including poliovirus, norovirus,
parvovirus, rotavirus, hepatitis A and E viruses, and Coxsackievirus.
Study deliverables included time-course survivability data and specific persistence and D-values
(i.e., decay rates) for the three surrogate viruses in three unique landfill leachates under two
temperature conditions. These deliverables are included as appendices to this report.
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2.0 Approach
The study was divided into the following three tasks consecutively performed.
Task 1: Landfill Leachate Acquisition and Characterization.
Task 2: Method Development
Task 3: Virus Persistence Testing
2.1 Task 1 - Landfill Leachate Acquisition and Characterization
Three landfill facilities were used to support this evaluation. These facilities were selected based
on meeting the following acceptance criteria:
• "Large" in size (capacity of > 2.5 million tons MSW) and subject to Clean Air Act
(CAA) requirements;
• RCRA Subtitle D-type waste (with RCRA Subtitle C-type construction if possible);
• Operational for at least five years;
• All three with similar design and operating characteristics such as waste composition
and gas extraction and capture, including no active leachate recirculation;
• Steady leachate composition and quantity (demonstrated by available historical
monitoring data);
• Not under any enforcement action (for any local, state, or federal regulations); and
• Willing to allow access to research staff to collect leachate from an accessible leachate
collection point representative of leachate across the landfill.
Approximately 10 liters (L) of landfill leachate was collected and returned the laboratory where a
portion of each was sent to an analytical laboratory for characterization analysis. The remaining
portion was stored under refrigeration and used as needed for virus persistence testing. The details
of the procedures used for landfill leachate facility selection, landfill leachate acquisition
processes, and characterization testing are described in Section 3.0, and the results are presented
in Section 5.1 . The three landfills selected have been kept anonymous in this report and are
referred to herein as Landfills A, B, and C. Each of these landfills met the primary selection
criteria, including Landfill B which accepts animal carcasses.
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2.2 Method Development
This task involved acquiring and preparing three viral agents (Table 1), establishing virus
quantitation assays (mammalian-based TCID50 and double agar layer [DAL] plaque assays),
assessing each landfill leachate for the presence of indigenous viral agents that could cause false
positive results, identifying whether each landfill leachate causes assay inhibition, and developing
sample analysis procedures (extraction and quantitation) so that assay inhibition is minimized or
eliminated while viral concentration is accurately measured. This task was not intended to be an
exhaustive method optimization activity but was intended to develop virus recovery methods for
generating accurate, defensible virus persistence data from landfill leachates using existing
standard methods.
Methods to recover live viral agent from each of the three leachates, while minimizing mammalian
cell cytotoxicity were evaluated. Landfill leachate is a very complex matrix, consisting of many
chemical and biological constituents. Therefore, tests were performed to determine if leachates
induced cytotoxicity on mammalian cell monolayers used for the TGEV quantification assay, or
adversely effected bacterial cells used in the DAL assay for the bacteriophage quantification
assays. The most robust method for minimizing assay inhibition/interference and cytotoxic effects
was dilution. Further testing of sample processing procedures were proposed (including filtration
and precipitation) if dilution was not successful, but fortunately dilution adequately eliminated the
majority of the effects of the leachate on the TGEV TCID50 assay. The leachate also interfered
with the Phi6 DAL assay, and a short slow-speed centrifugation step applied to the leachate sample
prior to initiating the DAL assay was effective in eliminating assay inhibition. No leachate-induced
adverse effects were observed on the MS2 DAL assay. In addition to inducing cytotoxicity or other
adverse effects, leachate may have harbored indigenous viruses that could interfere with detecting
surrogate agents, thus generating false positive results. Each leachate was screened for indigenous
viruses using the appropriate assay system; no indigenous viruses were identified using either
assay system.
Upon completion of preliminary evaluation, analytical methods for each viral agent were identified
and shown to be effective in efficiently and accurately determining target viral agent
concentrations from each landfill leachate. A detailed summary of the method development
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activities performed and results are provided in Appendix A. The final methods that were utilized
for the persistence study were written into a Miscellaneous Operating Procedure (MOP). The MOP
(provided in Appendix B) included sample preparation, incubation, and processing procedures and
is discussed in detail in Section 3.0.
2.3 Virus Persistence Testing
Persistence of three viral surrogates (Table 1) was evaluated in the three landfill leachates over
time (12°C or 37°C) using methods described in Section 2.2 and procedures described in Section
3.0 (also refer to MOP in Appendix B). To accurately measure the persistence and decay rate of
the surrogate agents in the landfill leachate, a decay (kill) curve was generated. To generate this
curve, samples of each agent in each leachate were prepared (4 milliliter [mL] aliquots in 5 mL
screw-top tubes spiked with virus) and incubated at the test temperature. Triplicate test samples,
triplicate positive controls (TGEV spiked into Eagle's Minimum Essential Medium [EMEM]
without fetal bovine serum [FBS] or MS2/Phi6 spiked into phosphate buffered saline [PBS]) and
negative controls per leachate (leachate without virus) were removed and analyzed for viral titer
at each time point. Samples were analyzed over eight weeks or until the samples were below the
limit of detection for two sequential time points. The analysis included up to seven time points
after To.
Persistence testing was designed to capture decreasing agent viability at each incubation
temperature, and time points were selected to capture decay across at least three sequential time
points. Initially, time points were chosen to capture viable agent over one to three days, and time
between sample time points was adjusted based on immediately preceding results. MS2 and Phi6
assay results were obtained 24 hours after samples were analyzed, and the data were used to make
informed decisions regarding subsequent time points. The TGEV assay has a longer read-out time,
and assay results were thus acquired three to four days after the samples were processed,
preventing informed day-to-day decision making early in the persistence study. Time points for
TGEV analysis were selected using an assumption of rapid viral decay for the first three time
points, and then refined using these data thereafter. Details of sample preparation, incubation,
quantitation assay and data analysis are described in Section 3.0.
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Table 1. Test Matrix for Virus Persistence Evaluation in Landfill Leachates.
I'jinimoler
Description
Virus surrogates (3)
TGEV coronavirus (enveloped)
Phi 6 (enveloped bacteriophage)
MS2 (non-enveloped bacteriophage)
Landfill Leachate
Three; each from different landfill facilities
Incubation Temperature
12°C for TGEVa; 12°C and 37°C for MS2 and Phi6
Time Points'3
0 (baseline), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56 days
a TGEV persistence was not measured at 37°C, as its survival at 12°C suggested that it would not survive for more than several
hours at 37°C.
bSubject to change throughout test and amended as deemed necessary based on results of previous time points. Actual time points
tested are shown in Table 3.
2.4 Microbial Activity
Microbial activity intrinsic to each leachate was analyzed using standard plate count methods.
Heterotrophic bacterial and fungal concentrations in each leachate were characterized
approximately months apart, once at the onset of each test. Microbial diversity was
qualitatively assessed by analyzing bacterial and fungal colonies on non-selective media for
heterotrophic bacteria (Tryptic Soy Agar, a non-selective medium) and fungi (Potato Dextrose
Agar, a reduced pH medium to support fungal growth and minimize bacterial growth). Colonies
were enumerated on spread plates to calculate colony forming units per milliliter (CFU/mL), and
colony morphology was observed at the same time. Colony morphology was recorded during both
heterotrophic plate counts; however, recovered colonies were not identified or characterized.
Results are described in section 5.5 and Table 8.
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3.0 Procedures
3.1 Landfill Leachate Acquisition and Characterization
3,1.1 Landfill Selection
Potentially suitable landfills were identified using the EPA's Incident Waste Decision Support
Tool (I-WASTE) tool [1] (V6.4; http://www2.ergweb.com/bdrtool/login.asp), selecting "MSW
Landfills" and "Large Landfills" as search terms, and collating results for facilities meeting the
capacity criterion for the States of Indiana, Michigan, Pennsylvania, and Ohio. These states were
selected to achieve operational efficiency for project personnel located in Newtown, Pennsylvania,
and Columbus, Ohio. After multiple potentially suitable landfills were identified, the short-listed
facilities were contacted to obtain commitment from the owners/operators to participate in the
study. Three landfills were selected that met the primary selection criteria, including Landfill B
which accepts animal carcasses. Basic characteristics about each landfill are provided in Table 2.
Table 2. Landfill Characteristics
Chsirsiclcrislic
l.iiiuirill A
l.iiiuirill B
i.iHHiriii (
Waste Acceptance Rate
In 2014, accepted
approximately 3,200
tons per day
3,500 to 5,000 tons per
day, Approximately
1,000,000 tons of waste
received in 2014
Average 1,400 tons/day
Footprint
100 acres permitted to
accept waste
283 acres permitted to
accept waste
168 acres permitted to
accept waste
Year Opened
1997
1995
1995
Expected Closure Date
2023 or 2024 (pursuing
an expansion which
could extend life by 25
years)
2030 to 2045
Information not
provided
Gas collection system
Yes
Yes
(approximately 190 gas
collection wells or
points)
No
3.1.2 Logistics
For the leachate sampling, a field sampling technician traveled to the sampling sites with coolers,
sampling equipment, and supplies. The coolers contained unpreserved 1.89 L (1/2 U.S. -gallon)
and 3.78 L (one U.S. gallon) high density polyethylene (HDPE) containers, laboratory sample
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containers provided by the selected study laboratory, DHL Analytical, Inc. (DHL) of Round Rock,
Texas, and frozen blue-ice packs to keep the samples cold after collection. Equipment included a
multi-parameter water quality instrument with pH, oxidation reduction potential (ORP), and
temperature probes, disposable Teflon bailers, a peristaltic pump, and a sample collection rod.
Supplies included chain of custody (COC) forms, sample bottle labels, deionized water, nylon
rope, tubing, disposable beakers, labels, a spray bottle with bleach solution, a 9.47 L (2.5 U.S.
gallon) bucket, Ziploc® bags, nitrile gloves, Tyvek coats, hard hat, face shield, paper towels, and
trash bags.
Project health and safety was governed by a project-specific activity hazard analysis (AHA). The
AHA was strictly adhered to during the course of performing landfill leachate sampling activities,
including the use of appropriate personal protective equipment (PPE).
3,1,3 Leachate Collection
All three leachates were collected within a two-day window, October 7 - 8, 2015. Leachate was
collected directly from an accessible leachate collection point into two 3.78-L and two 1.89-L
HDPE containers. The containers were completely filled and sealed with minimal headspace to
avoid oxygenation of the leachate.
At Landfill A, leachate was collected from a leachate accumulation area, consisting of three above-
ground 8,976 L (34,000 U.S. gallon) storage tanks situated in a cement containment area (Figure
1). A three-inch discharge line connected to the tanks is routinely used by Landfill A personnel to
collect leachate samples for analysis. The leachate flow from the tanks is controlled by a ball valve.
Sample collection began at approximately 9:00 AM. The discharge line was purged at a high flow
rate for approximately five minutes. After purging, the flow rate was reduced using the ball valve
to a controlled laminar flow for sample collection. The leachate appeared yellow and without
significant particulate matter. The collection time was written on the labels, and the labels were
placed on the appropriate sample containers.
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Figure 1. Landfill A Leachate Accumulation Area
At Landfill B, leachate was collected from a leachate sump area (Figure 2). Sample collection
began at approximately 12:00 PM. The leachate level was approximately three meters below the
surface of the sump. Nylon rope was tied to the 9.47 L bucket and dropped into the sump. The
leachate appeared brown with significant particulate matter. The collection time was written on
the labels, and the labels were placed on the appropriate sample containers.
Figure 2. Landfill B leachate sump area.
At Landfill C, leachate was collected from a leachate accumulation area in which the leachate is
stored in a 22,176 L (84,000 U.S. gallon) tank surrounded by a containment area (Figure 3). A
three-inch discharge line was connected to the tank routinely used by Landfill C personnel to
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collect leachate samples for analysis. The leachate flow from the tank was controlled by a ball
valve. Sample collection began at approximately 11:00 AM. The leachate line was purged at a
high flow rate for five minutes before sample collection. After purging the line, the flow rate was
reduced using the ball valve to a controlled laminar flow for sample collection. The leachate
appeared dark brown and contained significant particulate matter. The collection time was written
on the labels, and labels were placed on the appropriate sample containers.
Figure 3. Landfill C leachate accumulation area.
At each site, a disposable beaker was filled with leachate, and the pH, oxi dation reduction potenti al
(ORP), and temperature were measured using the multi-parameter water quality instrument
immediately after leachate collection. Following the measurements, the leachate was immediately
placed in a cooler packed with ice packs and delivered to the contractor laboratory. Upon receipt,
leachate samples were inspected, photographed, assigned a unique lot number, inventoried, and
stored at 4°C. Figure 4 illustrates the visual appearance of the leachates in 3.78 L bottles.
Page 13 of 67
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Landfill Leachate
Site C
Landfill Leachate
Site A
Landfill Leachate
Site B
Figure 4. Landfill Leachates A, B, and C in 3.78 L containers.
For each landfill leachate, one 1.89 L bottle of leachate was shipped to a commercial analytical
laboratory (DHL) in coolers loaded with wet ice on the same day as sample collection (expedited
overnight shipping) for characterization testing. The remaining 1.89 L and two 3.78 L HDPE
containers were relinquished to the custody of virology laboratory staff for method development
activities and the persistence study.
Leachate from each of the three landfill facilities selected for the study was submitted to DHL for
analysis of the following parameters (unless otherwise noted):
• Alkalinity (total)
• Ammonia
• Anions
o Chloride
o Nitrate
o Sulfate
• Biological oxygen demand (BOD)
• Chemical oxygen demand (COD)
• Metals
o Calcium
o Iron
o Magnesium
o Manganese
o Potassium
o Sodium
o Zinc
• pH (performed in the field by Battelle)
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• ORP; performed in the field by Battelle Technical Staff
• Total dissolved solids (TDS)
• Total organic carbon (TOC)
• Total suspended solids (TSS)
The analytical data received from DHL are summarized in Table 4 in Section 5.1, with data derived
in the field at the time of sample collection from a multi-parameter water quality instrument (ORP,
pH, and temperature) and visual characteristics.
3,1,4 Microbial Activity
Heterotrophic microbial activity in each leachate sample was measured at two points during this
study: (1) start of the initial persistence testing and (2) start of second persistence testing.
Microbial activity was determined using a standard plate count assay. Leachate samples were
serially diluted in PBS out to 10"4 dilution and 100 microliter (|iL) samples of each dilution were
plated in triplicate on Tryptic Soy Agar (TSA) and Potato Dextrose Agar (PDA). Bacterial activity
was quantitated using TSA and fungal activity using PDA. Samples were incubated at 25°C for
60-72 hours, and colonies were enumerated. Colony morphology on each media was noted,
however colonies were not identified or characterized. Microbial activity was determined as
colony-forming units per mL (CFU/mL) using the equation:
CFU Average number of colonies per dilution
mL (dilution) x (volume added to plate)
3.2 Virus Propagation
3,2,1 TGEV Propagation
TGEV Purdue strain (ATCC, VR-763 lot 4) was acquired from the American Type Culture
Collection (ATCC). The ATCC stock is the only known commercially available source of TGEV..
As the ATCC TGEV stock is contaminated with bacteria [2], filtration was used to generate a
bacteria-free sample prior to seed stock generation. TGEV was propagated on swine testicular (ST)
cells (ATCC CRL1746). The virus was thawed on ice, diluted 1:2 in incomplete growth medium
(Modified Eagle's Medium supplemented with Earle's buffered salt solution, L-glutamine and 1
% antibiotic/antimycotic) and sterile-filtered using a 0.22 micrometer (|im) low-protein binding
Page 15 of 67
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filter. An additional 1 mL of sterile incomplete growth medium was passed through the filter to
ensure the virus was completely removed from the filter. Filtered virus was centrifuged (12,000
relative centrifugal force [rcf], four minutes [min]) to remove any residual cells and debris, and
inoculated onto a healthy confluent monolayer of ST cells in a 25 cm2 tissue culture flask (T25)
and incubated for one hour (37°C, 5% CO2); the flask was rocked every 15 minutes. Additional
incomplete growth medium (5 mL) was added, and the flask was returned to the incubator (37°C,
5% CO2). Inoculated cells were observed for cytopathic effect (CPE), and virus was harvested
when the cells exhibited 70-90% CPE (approximately 24 hours post-inoculation). Virus was
harvested in a two-step process: the flask was frozen at -15 to -30°C for at least one hour, then
thawed at room temperature, and virus seed stock was prepared by centrifugation (4°C, 400 rcf for
20 min), and the supernatant was aliquoted and frozen at -75 to -80°C.
TGEV seed stock was used to prepare a second pass stock (TGEV p2). Undiluted seed stock was
directly inoculated onto a healthy confluent monolayer of ST cells in a 75 cm2 tissue culture flask
(T75) and incubated for one hour (37°C, 5% CO2); the flask was rocked every 15 minutes.
Additional incomplete growth medium (8 mL) was added, and the flask was returned to the
incubator (37°C, 5% CO2). Virus was identically harvested to the seed stock at 21 hours post-
inoculation. Virus supernatant was aliquoted and stored at -75 to -80°C. TGEV p2 virus titer was
4.6><106 TCIDso/mL via TCID50 assay on ST cells (see section 3.3.3).
A working stock (third pass) of TGEV was prepared from TGEV p2 (TGEV p3). TGEV p2 was
diluted in incomplete medium to a multiplicity of infection (MOI) of 0.1 and inoculated onto a
healthy 70 % confluent monolayer of ST cells in 150 cm2 tissue culture flasks (T150). Flasks were
incubated for one hour (37°C, 5% CO2) and rocked every 15 minutes. Additional incomplete
growth medium (25 mL) was added, and the flask was returned to the incubator (37°C, 5% CO2).
Virus was identically harvested to the seed stock at 21 hours post-inoculation. Virus supernatant
was frozen in 1 mL aliquots at -65 to -80°C. TGEV p3 virus titer was 4.1><106 TCIDso/mL via
TCID50 assay on ST cells (see Section 3.3.3). TGEV p3 was used for all persistence studies.
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3.2,2 Bacteriophage Propagation
Master stocks of MS2 (ATCC 155597-B1) and Phi6 (EPA-provided) bacteriophages were
propagated from infected agar cultures of Escherichia coli (E. coli) (ATCC 700891) and
Pseudomonas syringae (P. syringae) LM2489 (EPA-provided), respectively.
To propagate MS2, a log phase broth E. coli culture was grown in Luria Bertani Broth (LBB) for
approximately 3.5 hours. This culture was added to molten Luria Burtani Top Agar (LBTA) and
overlaid onto Luria Bertani agar (LBA). A lyophilized pellet of MS2 (ATCC) was rehydrated in
0.5 mL LBB and overlaid onto E. co/z'-inoculated LBTA and incubated for approximately 24 hours
at 35-37°C. The soft top agar, consisting of MS2-infected coli cells, was scraped off, transferred
to a tube containing 15 mL SM buffer, and centrifuged at 7000 rcf for 15 minutes. Virus
supernatant was filtered through a 0.2 |im syringe filter to remove residual bacterial cells. Filtered
supernatant was designated the MS2 master stock (assigned lot number MS2091515) and was
stored as replicate 0.5 mL aliquots at < -70°C. MS2 master stock titer was measured using a
standard DAL method (see Section 3.3.3) and determined to be 4.0><109 plaque forming units per
milliliter (PFU/mL).
To propagate Phi6, a 100 |iL aliquot of the EPA Phi6 stock was suspended in 30 mL Tryptic Soy
Broth (TSB) supplemented with magnesium (TSB-Mg) and combined with 6 mL of a P. syringae
culture grown for approximately 24 hours in TSB-Mg stock and 90 mL of molten Tryptic Soy Top
Agar (TSTA) supplemented with magnesium (TSTA-Mg). Phi6 suspension was gently mixed,
overlaid as 4 mL aliquots onto each of approximately 30 TSTA-Mg plates, and incubated for
approximately 20 hours at 25-27°C. Phi6 was harvested by adding 5 mL TSB-Mg to each plate,
incubating at 25-27°C for two hours, and resuspended by gently swirling the plates. The Phi6 stock
was filtered through a 0.2 |im syringe filter to remove residual bacterial cells. Filtered supernatant
was designated the Phi6 master stock (assigned lot number PHI6092516) and was stored as
replicate 1 mL and 5 mL aliquots at < -70°C. Phi6 master stock titer was measured using a standard
DAL method (see Section 3.3.3) and determined to be 1.8><1010 PFU/mL.
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3.3 Persistence Testing
The persistence of TGEV, MS2, and Phi6 viruses in landfill leachates were evaluated as follows:
leachates were individually spiked with a known quantity of virus, dispensed into replicate screw-
top vials, and statically incubated for up to 56 days or until the virus was no longer detected. One
set of TGEV spiked samples was incubated at 12°C, and two sets of each bacteriophage samples
(MS2 and Phi6) were incubated (one at 12°C and the second set at 37°C). TGEV persistence was
not measured at 37°C, as its survival at 12°C suggested it would not survive for more than several
hours at 37°C. Throughout the incubation period, triplicate samples were assayed at the initiation
of testing (To) and up to seven following time points.
TGEV spiked samples were assayed using an end-point dilution TCID50 assay on ST cells. Briefly,
each replicate sample was serially diluted and plated on ST cells. TCID50 assays measure infectious
virus and identified the dilution of the virus at which 50 % of cell cultures were infected. MS2-
and Phi6-spiked samples were assayed using a standard DAL method. Triplicate samples were
serially diluted in a buffer, and dilutions were used to infect either E. coll (MS2) or P. syringae
(Phi6), followed by the DAL assay.
Results from each time point were assessed as concentration versus time, and these data were
subsequently used to determine the time at which the viral agent was no longer detectable. Decay
rates were expressed as D-values: the time required for the reduction of the infectious virus titer
by 90 %. The key activities associated with persistence testing included sample preparation,
incubation, analysis, and data analysis; each is discussed in detail in the following sections.
3,3-1 Sample Preparation
Aliquots of Leachates A, B, and C were dispensed into replicate tubes approximately 24 hours
prior to the initiation of persistence testing. Each leachate was mixed well by manually and
vigorously swirling a 3.78 L container of leachate and immediately dispensing a 500 mL aliquot
into a sterile 1 L flask. Leachate was then continuously mixed on medium-high speed for five
minutes, and 4 mL aliquots were dispensed into replicate 5 mL cryotubes. All sample tubes were
labeled and stored overnight at 2-8°C.
Page 18 of 67
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3.3.1.1 Test Samples
TGEV, MS2 and Phi6 samples were prepared in pre-dispensed leachate samples (Section 3.3.1).
Persistence test samples were separately prepared for each virus in triplicate for each leachate and
each incubation test condition.
TGEV Samples: TGEV virus was rapidly thawed and spiked into each 4-mL leachate sample at a
final concentration of 5 * 104 TCIDso/mL (80 |iL of pooled TGEV p3). Each sample was mixed by
swirling and inverting three times. Samples were incubated upright at 12 ± 1°C with no mixing.
Samples were incubated without mixing to ensure that all effects on viral infectivity were due to
the matrix, and not mechanical stress from periodic mixing.
Bacteriophage Samples: Bacteriophage (MS2 and Phi6) master stocks were thawed and diluted in
PBS to generate 1x10s PFU/mL working stocks. Each bacteriophage was separately spiked into
each 4-mL leachate sample at a final concentration of 1 x 106 PFU/mL (40 |j,L of the working stock).
Samples were incubated upright as described in Section 3.3.2. Three test samples per virus were
assessed per persistence time point and incubation temperature.
3.3.1.2 Negative Controls
Negative controls were generated from pre-dispensed leachate samples (Section 3.3.1). Leachate
samples were removed from refrigerated storage and incubated with the respective test and positive
control samples. One negative sample was assessed per persistence time point.
3.3.1.3 Positive Controls
Positive samples were generated in sterile media. TGEV positive samples were generated in sterile
incomplete medium (EMEM supplemented with 1 % antibiotic/antimycotic). MS2 and Phi6
samples were prepared in the PBS in the same manner as the test samples (Section 3.3.1.1). TGEV
positive samples were generated at the same time and with the same pooled virus as the test
samples (Section 3.3.1.1). Each sample was mixed by swirling and inverting three times. These
controls were incubated with their respective test and negative control samples.
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3.3.1.4 Evaporation Controls
To assess the role of evaporation, pre-dispensed leachate samples (Section 3.3.1) were spiked with
40 |iL of incomplete medium (EM EM supplemented with 1 % antibiotic/antimycotic) or 40 |iL of
PBS and incubated with each set of test samples. Each sample was weighed on an analytical
balance to the nearest 0.0001 g at each time point and returned to the incubators.
3.3.2 Incubation
Test samples were statically incubated upright with negative (one per time point) and positive
(three per time point) samples and evaporation controls in cryoboxes (Figure 5). Samples were
incubated within incubators (Figure 6) set to operate at 10 - 14°C or 35 - 39°C with desired set
points of 12°C and 37°C, respectively. Incubator temperatures were monitored throughout the
incubation period using calibrated thermometers or via a calibrated electronic temperature
monitoring system.
Figure 5. Persistence samples in 5-niL cryovials placed within cryobox.
Page 20 of 67
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innovate
Figure 6. Sample incubators.
3.3.3 Sample Analysis
Persistence testing on all three landfill leachates was simultaneously conducted. Initial persistence
testing evaluated TGEV persistence at 12°C, and MS2 and Phi6 at 12°C and 37°C. Initial tests
successfully evaluated TGEV, MS2 and Phi6 at 12°C; however, the decay rate at 37°C was too
rapid to be captured within the initial tested timeframe (3 days). In fact, no MS2 or Phi6 virus was
detected after To. A second MS2 and Phi6 persistence test was performed at 37°C using a shorter
series of time points to capture the linear decay rate (Table 3); however, TGEV persistence was
not measured at 37°C, as its survival at 12°C suggested it would not survive for more than several
hours at 37°C.
Page 21 of 67
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Table 3. Sample Analysis Time Points
Viral A»cnl
Temperature
Sample Analysis l ime Points
TGEV
12°C
0,3,7, 10, 14,21,28, 42 days
MS2determined fo
12°C
0,3,7, 14, 21, 35, 56 days
37°C
0, 3, and 7 days
37°C (2nd test)
0, 6, 12, 24, 34 hours
Phi6
12°C
0,3,7, 14,21,35,42, 56 days
37°C
0, 3, and 7 days
37°C (2nd test)
0, 6, 12, 24, 30 hours
The baseline (starting concentration) of each analysis set was determined by immediately
analyzing one set of samples (the To set). Per leachate type (A, B, and C), three test samples, three
positive samples and one negative sample were analyzed within two hours of dosing using the
MOPs generated in method development. All remaining test, positive control, and negative control
samples were incubated and analyzed over time (Table 3). At each test point, evaporation samples
were weighed to the nearest 0.0001 g to assess evaporation during testing.
3.3.3.1 TGEVSample Analysis
Per leachate, triplicate test samples and single negative control samples, along with triplicate
positive control samples, were analyzed using a TCID50 assay. The TGEV TCID50 assay was
adapted from a standard mouse hepatitis virus (MHV) TCID50 assay. All samples were serially
diluted using a twofold and/or tenfold dilution in complete medium (EMEM, supplemented with
10% FBS and 1% antibiotic/antimycotic). Each dilution was added to a healthy confluent
monolayer of ST-cells across one row (12 wells) of a 96-well plate at 0.1 mL diluted sample/well.
The lowest dilution able to be used without showing leachate-induced cytotoxicity was 10"1. The
calculated assay limit of detection was determined for both natural logarithm (In) and base-10
logarithm (log) to be: 230 TCIDso/mL (2.3 log (TCID50) or 5.4 In (TCID50)). The 96-well plates
were incubated for two days (37°C, 5% CO2) and manually scored by experienced virologists for
CPE using a phase-contract microscope. All bacterial or fungal contaminations were noted, and
those wells were not included in titer calculations. TGEV titer (concentration) calculated via
TCID50 reflects the concentration of virus in a sample in which 50 % of the sample wells were
infected. Positive wells were scored and documented for all dilutions plated, and viral titer was
Page 22 of 67
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determined as TCID50 using the Reed-Muench method [3] in Excel 2013. Examples of viral CPE
are shown in Figure 7, and TCID50 calculations are shown in Figure 8.
Mi
•*, •
ST-cell morphology andTGEV infection CPE. (A) Control ST-cells at 100 %
confluence, (B) ST-cells infected with low levels of TGEV and(C) ST-cells
demonstrating CPE due to TGEV infection.
Figure 7. TGEV CPE on ST Cells
Page 23 of 67
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1) Calculate the percentage of wells infected for each dilution.
For example, if 4 of 12 wells are scored infected
* iQQ o/o = 33.3 o/o weiis infected
12 total wells
If there is contamination, calculate the total of infected wells without those wells. For
example, if there are four infected wells and one with fungal contamination.
4 infected wells
- 100 % = 36.4 % wells infected
11 total wells counted
2) Calculate the proportional distance (PD).
i. Choose two dilutions
The dilution that has just above 50 % of wells infected: (a)
The next highest dilution (more dilute): (b)
ii. Calculate proportional distance.
(% of wells infected at (a I} — 50 %
PD
j % of wells infected at (a)) - < % of wells infected at Co))
iii. Calculate TCID50.
Log(TCIDSO) = log(//) + log (dilution series) * PD
-(b) refers to (b) identified in step 2i.
- PD is the proportional distance calculated in step 2ii.
- Dilution series. This will determine the correction factor to
accurately calculate titer. Use 10 for a tenfold series and 2 for a two-
fold series. The correction factor for a ten-fold dilution series is 1.0,
for a two-fold dilution series is 0.3.
Figure 8. TCID50 Titer Calculation adapted from |3|
3.3.3.2 MS2 andPhi6 Sample Analysis
Per leach ate, triplicate test samples and single negative control samples, along with triplicate
positive control samples, were analyzed using the DAL assay. Samples were each vortexed at
moderate speed for 30 seconds and serially diluted using a tenfold dilution series in PBS no further
than the 10~5 dilution. MS2 samples were diluted neat, and Phi6 samples were vortexed briefly
and centrifuged at 12,000 rcf for two min prior to dilution. Appropriate serial dilutions were
selected based on initial viral titer and previousiy-analyzed time points to select dilutions that
would most likely provide plaque counts within the desired countable range of 25-250 plaques per
Page 24 of 67
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plate. In general, positive control samples were plated with 10"3' ~4' "5 dilutions, and negative
controls were plated without dilution.
Per dilution, triplicate suspensions of the following mixture were prepared in individual 50 mL
conical tubes, as follows:
1) MS2 analysis: 0.1 mL of a log-phase E. coli culture (approximately three-six hours old)
grown in LBB, 0.1 mL of sample (undiluted or diluted), and 5 mL of molten (50°C) LBTA
supplemented with 15 mg/mL streptomycin and 15 mg/mL ampicillin (LBTA+S+A).
2) Phi6 analysis: triplicate suspensions of the following mixture were prepared in individual
50 mL conical tubes: 0.1 mL of an overnight P. syringae culture grown in TSB-Mg, 0.1
mL of sample (undiluted or diluted), and 5 mL of molten TSTA supplemented with 20
mg/mL ampicillin (TSTA+A20).
Conical tubes containing virus, agar and bacteria mixtures were promptly swirled and overlaid
onto 10-cm Petri dishes. MS2/A". coli was overlaid onto LBA-S+A, and Phi6//J. syringae was
overlaid onto Tryptic Soy Agar (TSA) supplemented with magnesium and 20 mg/mL ampicillin
(TSA-Mg+A20). Plates were incubated overnight (MS2 at 37±2°C; Phi6 at 25±2°C). Viral titers
were determined using the standard PFU calculation (see below). Plates having 0-250 plaques
were counted and recorded. Plaque counts were used to calculate decay rates and persistence
values as described later in this section. The appearance of typical MS2 plaques are shown in
Figure 9, and typical Phi6 plaques are shown in Figure 10.
Calculation of viral titer:
Plates having 25-250 plaques were used to calculate the viral titer in PFU/mL. The PFU/mL was
calculated by multiplying the mean PFU/plate by the dilution factor. Total PFU recovered per
sample were calculated by multiplying the PFU/mL recovered by the total sample volume (4 mL).
These values were converted to log PFU and natural log (In) PFU and plotted versus time. Log
graphs are shown in Section 5.0 and represent virus persistence in each leachate and positive
control matrix. Graphs of In PFU versus time were used to calculate decay rates. Viral persistence
was calculated to be the time at which the linear decay rate intersects the assay theoretical limit of
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detection (LOD). The calculated LOD for the DAL assay is 40 PFU, equivalent to 1.6 log PFU or
3.7 In PFU. This value was calculated using a detection limit of 1 PFU per 0.1 mL plated,
equivalent to 10 PFU/mL and 40 PFU/sample in a 4-mL leachate test sample. Decay rate and
persistence calculations are detailed in Section 3.3.4.
sr
if
i
h
IM
P-
¦r
m
1502-3
MS2 Leaciiate A 37C
66087 TN
H «
i 5 *i
•*
!?"p
Figure 9. MS2 plaques on E. coli.
Figure 10. Phi6 plaques on P. syringae.
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3,3,4 Data Analysis and Interpretation
Viral decay rate was determined by measuring the decrease of infectivity and was measured using
a first order decay equation and calculating decimal reduction times (D-values, the time required
for the viable concentration to be reduced to 10 % of the starting concentration). These decay
kinetics are commonly used to measure biological agent decay, making this a practical and
appropriate approach to measure viral persistence. D-values were calculated using persistence data
plotted as In PFU recovered (for MS2 and Phi6) or In TCIDso/mL (for TGEV) versus time. A linear
decay curve was fitted to data points that included data points in which viral recovery was detected
in at least two or three replicates, including the initial To time point.
A linear regression of the data was generated using the following formula:
y = mx + b
where:
y = concentration (In PFU recovered for MS2 and Phi6, or In TCIDso/mL for TGEV)
m = slope
x = time (days)
b = y-intercept
The slope (m) from the linear regression was used to calculate D-values using the following
formula:
1
D — value = —X — 1
m
where:
m= slope
Viral persistence time (x) was calculated as the time (in days) required for the rate of linear
decay to intercept the assay LOD using the following formula:
Cy~b)
x =
m
where
y = In (assay LOD)
b = y-intercept of linear decay rate
m = slope of linear decay rate
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in which Y equals:
5.4 In per mL for TGEV (eq. to 230 TCIDso/mL)
3.7 In (eq. to 40 PFU) for MS2 and Phi6
Persistence (log (viral titer) versus time) and decay curves (linear regression plots used to
calculate D-value and persistence) are shown and discussed in Section 5.0.
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4.0 Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the previously approved quality
documents for this evaluation.
4.1 Performance Evaluation Audit
A Performance Evaluation (PE) audit was inadvertently not performed for temperature, volume,
and PFU calculation measurements in project documentation. However, assurance that these
measurements were accurate throughout this study can be demonstrated based on the following:
• All analysts were technically competent, several with many years of experience with these
assays. All equipment used was calibrated.
• Calibrated micropipettes were used to perform all sample enumerations as documented in
the daily worksheets used to perform the study. All micropipettes were used within their
calibration due date.
• There is sufficient evidence in the raw data that volume, temperature and PFU calculations
were accurately recorded. All PFU calculations were verified on the completed worksheets
as indicated by the reviewer signatures on these worksheets. The Quality Assurance (QA)
representative verified that the raw data and calculations transcribed into the Excel
spreadsheets were accurate.
A deviation report was written and included in the study file. The impact was deemed as "minimal"
as there was sufficient documented evidence that these measurements were accurate.
4.2 Technical System Audit
The QA Manager performed a technical systems audit on February 23 through 25, 2016, to confirm
compliance with both TO and program level quality documents. The audit focused on both virus
and bacteriophage sample preparation, plating, and reading of results. Procedures followed
requirements in the MOP developed under this task order (see Appendix B).
4.3 Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. The QA Officer traced the
data from the initial acquisition, through reduction and statistical analysis, to final reporting, to
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ensure the integrity of the reported results. All calculations performed on the audited data were
checked for accuracy. The audit revealed three activity measurement transcription errors that were
corrected in the report and data spreadsheets.
4.4 QA/QC Reporting
Each assessment and audit was documented in accordance with the quality system developed for
the testing and evaluation program.
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5.0 Results
5.1 Landfill Leachate Characterization
The landfill leachate characterization data are summarized in Table 4. Although Leachate A was
chemically markedly different from Leachates B and C (Figure 4), analytical data illustrated that
all three leachates showed numerous similarities and distinct differences in composition and
characteristics. Similarities between all three leachates included pH (varying only between 7.1 to
7.9) and temperature (ranging between 20 to 25°C). Chemical characteristics that did not vary by
>1 order of magnitude included iron, magnesium, potassium, sodium, chloride, ammonia
concentrations, alkalinity, COD, TDS, TOC, and TSS. Distinct differences (varying by >1 order
of magnitude) in one leachate versus the other two included calcium, zinc, nitrate, sulfate
concentrations, BOD, and ORP.
Viral persistence data and calculated decay rates in leachate (discussed in Sections 5.2 to 5.4),
identified that MS2 and Phi6 decay rates were only slightly different at each temperature.
Interestingly, leachate effects on viruses were not consistent between viruses. Decay rates for MS2
(at 12°C and 37°C) and Phi6 (at 37°C) were slowest for Leachate A, followed by Leachate C, and
most rapid in Leachate B. However, decay rates of Phi6 at 12°C showed the opposite trend: slowest
in Leachate C, followed by Leachate B, and most rapid in Leachate A. The decay rates for TGEV
(at 12°C) showed a trend different from both MS2 and Phi6, decaying slowest in Leachate B (four
days), followed by Leachate C (two days) and decaying fastest in Leachate A. Results from all
tests are summarized in Executive Summary Tables 1 and 2. Unsurprisingly, all viruses persisted
significantly longer in control matrices (incomplete EMEM for TGEV, PBS for MS2 and Phi6)
than leachate matrices.
Leachate constituents clearly contributed to viral inactivation; however, this study did not assess
the contribution of individual constituents of the leachate to viral inactivation. Further persistence
testing with viral agents in a much larger sample of different leachates from different landfills, or
artificial leachate substitutes varying in constituents, would be required to gain further insight into
the root cause(s) of viral inactivation and chemical markers that could indicate the rate of viral
inactivation in landfill leachate.
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Table 4. Landfill Leachate Characterization Data
Analvtc
Leachate A
l.eachale li
Leachate ('
Metals (mg/L)
Calcium
11.6
200
312
Iron
6.36
17.4
31.5
Magnesium
130
84.3
297
Manganese
0.0468
0.152
2.26
Potassium
468
260
937
Sodium
1,880
1,500
2,360
Zinc
0.140
0.0199
0.0711
Biological Oxygen Demand (mg/L)
BOD
187
2,020
2,350
Anions (mg/L)
Chloride
2,070
1,980
2,810
Nitrate-N
4.00
3.08
<1.00
Sulfate
3.19
10.1
33.0
Total Alkalinity as CaCOs (mg/L)
Total Alkalinity
6,100
2,600
8,040
Ammonia as Nitrogen (mg/L)
Ammonia
1,050
386
1,370
Chemical Oxygen Demand (mg/L)
COD
1,500
2,470
9,060
pH (Standard Units)
pH
7.76
7.06
7.55
pH (field)
7.88
7.14
7.36
Oxidation Reduction Potential (millivolts)
ORP (field)
47.4
-60.7
-96.8
Temperature (°C)
Temperature (field)
21.8
25.0
20.0
Total Dissolved Solids (mg/L)
TDS
6,680
5,980
13,500
Total Organic Carbon (mg/L)
TOC
448
796
2,960
Total Suspended Solids (mg/L)
TSS
12.3
82.0
72.0
Visual Observations
Color
yellow
brown
dark brown
Particulates
not significant
significant
significant
Page 32 of 67
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5.2 TGEV Persistence
TGEV persistence was analyzed in all three different leachates (A, B and C) at intervals over 42
days at 12 ± 1°C. Samples were removed from incubation and quantified via TCID50 assay at 0, 3,
7, 10, 14, 21, 28 and 42 days post inoculation. Viral titer in TCID50 was used to generate decay
curves. Viral titer, expressed as mean log (TCIDso/mL) or In (TCIDso/mL) versus time (Figure 11
and Figure 12 respectively), and a linear regression of the data were used to calculate D-values
and persistence time (Table 5).
Results indicated that TGEV persisted for 4.6 to 16.6 days at 12°C in leachate (Figure 11). While
viral concentrations close to the limit of detection can result in varying results (e.g., concentration
reporting at 0 or close to the limit of detection) at low dilutions (e.g., 10"1), viral CPE was
distinguishable from leachate-induced toxicity. TGEV was observed to be inactivated fastest in
Leachate A (4.6 days), followed by Leachate C (6.7 days), and slowest in Leachate B (16.6 days).
Leachates A and C initially showed similar decay rates; however, virus incubated in Leachate C
did result in a measurable titer at 14 days after dropping below the limit of detection on day 10.
This rebound in infectious virus may have been due to difficulties in separating leachate-induced
cytotoxicity from viral CPE. Data from Day 14 for Leachate C were obtained from the 10"1 dilution
and were only observed in the 2"5 and 2"6 dilution (1:32 and 1:64 dilutions from neat sample), with
results very close to the limit of detection. It is likely the Day 10 data in Leachate C were occluded
by leachate cytotoxicity and were at a similar titer.
TGEV also degraded in the control matrix (EMEM supplemented with 1 % antibiotic/
antimycotic), reducing the viral titer to the neat assay detection limit on Day 42 (the final time
point). Viral inactivation in the control matrix was expected; however, viral survival greater than
one-two days in the leachate was unexpected.
Page 33 of 67
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Table 5. TGEV Measured D-values at 12°C
Matrix
Slope
Measured D-Value
(days)
Persistence"
(days)
Leachate A
-1.2003
0.8
4.6
Leachate B
-0.2654
3.8
16.6
Leachate C
-0.4580
2.2
6.7
Incomplete EMEM Medium
(Positive Control)
-0.1428
7.0
43.1
"¦Calculated time in days at which measured linear decay rate intersects with assay limit of detection.
LOG TCID50/mL reduction TGEV
6.00
5.00
4.00
o
I/)
9 3.00
a
00
O
Z.00
1.00
0.00
L.%
Linear Trendiines calculated with data for:
EMEM, Leachate B: Day 0-42
Leachate A: Day 0-10
%\\
V:
\
Leachate C
Day 0-21
k
xV-
\
s
V
^
V
%
\
-v
V
1
1 ^
\
\
v
Limit of Accurate Quantitation for a
single
*
\
w
Y\
L
\ /
K
\
\
V
1
•v
TCID50
a$$»y (2_3 T.og TCIT>50)
1
\
\
\ / "• •••••
, A /
\
\
\
—^ L*
«v
5 10 15
f- ¦ Leachate A — •- Leachate B
20 25
Days Post-Incubation
—• -Leachate C
30
35
40
y = -0.5213X + 4,7461 y = -0.1153*+ 4.2576
RJ = 0.9504 Ra = 0.9235
y = -0.1989X + 3.6866
R2 = 0.6148
-¦•--Positive Control (EMEM)
y = -0.062X+ 5.0172
R1 = 0.898
Figure 11. TGEV Persistence at 12°C
Page 34 of 67
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Ln TCID50/mL TGEV in Leachate
• Positive
D12C = 7 days
y=-0.1428x+11.552
R2 = 0.8979
10 15 20 25 30
Days post-inoculation
• Leachate A • Leachate B
Duc= 0.8313 days Due = 1.08 days
y = -1.2003X + 10.928 y = -0.2654x + 9.8033
R2 = 0.9504 R2 = 0.9235
• Leachate C
Due = 2.18 days
y= -0.458X+8.4888
R2 = 0.6148
Figure 12. TGEV Decay Curves at 12°C
5.3 MS2 Persistence
MS2 persistence in leachate was expressed as mean log (PFU recovered) versus time at 12°C and
37°C (Figure 13 and Figure 14, respectively). Mean ln (PFU recovered) was calculated and
expressed versus time to determine linear decay rates (Figure 15 and Figure 16, respectively).
Linear regression of the data and the slope of the decay curves was used to calculate D-values and
persistence time (Table 6).
MS2 was deactivated very rapidly in leachate at 37°C, therefore data from the initial MS2 37°C
persistence test are not presented, as no virus was recovered after the initial time point (To; the first
subsequent time point was on Day 3). Data presented for MS2 persistence at 37°C are from the
second test performed several months later, using the same landfill leachates (stored refrigerated).
Page 35 of 67
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As shown in Figure 13 and Figure 14, respectively, results indicated that MS2 persisted much
longer at 12°C (approximately 2.5 to four months) than at 37°C (two to three days). While
temperature did affect the decay rate, decay was slowest in Leachate A, followed by Leachate C,
and fastest in Leachate B at both 12°C and 37°C. In the control matrix (PBS), MS2 was very stable
at both temperatures during test duration (35-52 days) with no discernible decay observed.
Table 6. MS2 D-values and Persistence
Matrix
12°C
37°C
Slope
D-Value
(days)
Persistence"
(days)
Slope
D-Value
(days)
Persistence3
(days)
Leachate A
-0.1002
10.0
113
-0.1643
0.3
3
Leachate B
-0.1377
7.3
75
-0.2384
0.2
2
Leachate C
-0.1216
8.2
87
-0.1989
0.2
2
PBS
-0.0053
188.7
NRb
0.0147
NRb
NRb
"¦Calculated time in days at which measured linear decay rate intersects with assay limit of detection.
bNo decay observed.
18.0
16.0
T3
0> 14.0
1-
QJ
o 12.0
u
V
~£ 10.0
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Q. S.O
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6.0
4.0
2.0
0.0
MS2 Decay Rates in Landfill Leachatesat 12C
N
s
1
-—*—'
4- - -
A"
"A
"-f
¦»
LIMIT OF DETECTION
12
Leachate A
y- -0.1002x+ 14.987
R2 = 0.9819
16 20 24 28 32 36
Time (days)
40
44
48
52
56
- ~ - Leachate B
y = -0.1377X+ 14.066
R2 = 0.9224
—- Leachate C
y = -0.1216X+ 14.29
R2 = 0.9775
-*— Positive Controls
y=-0.0053x+15.181
R2 = 0.0644
60
Figure 13. MS2 Persistence at 12°C
Page 36 of 67
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MS2 Decay Rates in Landfill Leachatesat 37C
18.0
17,0
16.0
15.0
14.0
"3 13.0
a! 12.0
8 no
10.0
3
LL.
a.
z
__i
c
m
4)
5
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
1... *
> - -f. ~
""' - ¦
~
* ^
U ~ *
LIMIT OF DETECTION
Leachate A
12 18 24 30
Time (hours)
Leachate B -~-Leachate C —Positive Controls
36
Figure 14. MS2 Persistence at 37°C
18.0
16.0
T3
0) 14.0
L_
-------
MS2 Decay Rates in Landfill Leachates at 37C
18.0
17.0
16.0
15.0 *
14.0
XI 13.0
QJ
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o 11.0
| 10.0
D
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9.0
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7.0
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5.0
4.0
3.0
2.0
1.0
0.0
¦ LeachateA
Linear (Leachate A)
y=-0.1643x + 15.322
R* = 0.9682
~
~
LIMIT OF DETECTION
12
* Leachate B
Linear (Leachate B)
y=-0.2384X + 15.316
R» = 0.987
24
18
Time (hours)
~ Leachate C
Linear (Leachate C)
y=-0.1989x+14.517
R2 = 0.9744
30
36
a Positive Controls
— Linear (Positive Controls)
y=0.0147x+ 15.005
R* = 0.1339
Figure 16. MS2 Decay Rate at 37°C
5.4 Phi6 Persistence
Phi6 persistence in leachate was expressed as mean log (PFU recovered) versus time at 12°C and
37°C (Figure 17 and Figure 18, respectively). Mean In (PFU recovered) was calculated and
expressed versus time in each leachate at 12°C and 37°C to determine linear decay rate (Figure 19
and Figure 20, respectively). Linear regression of the data and the measured slope of the decay
curves were used to calculate D-values and persistence (results summarized in Table 7).
Like with the MS2 vims, Phi6 virus was deactivated very rapidly at 37°C, therefore data from the
initial Phi6 37°C persistence test are not presented, as no virus was recovered after the initial time
point (To; the first subsequent time point was on day 3). Data presented for Phi6 persistence at
37°C are from the second test performed several months later using the same landfill leachates
(stored refrigerated).
Results demonstrated that Phi6 persisted much longer at 12°C (approximately two to three months)
than at 37°C with rapid decay to the assay detection limit within six hours. While temperature also
Page 38 of 67
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affected the decay rate, decay was noticeably fastest in Leachate A, with decay in Leachates B and
C occurring slower. Unlike MS2, Phi6 did decay in the control matrix (PBS) at both temperatures,
significantly more so at 37°C.
Table 7. Phi6 D-Values and Persistence
Matrix
12°C
37°C
Slope
Measured
D-Value
(days)
Persistence"
(days)
Slope
Measured
D-Value
(days)
Persistence3
(days)
Leachate A
-0.1584
6.3
55
-1.5867
0.03
0.3
Leachate B
-0.0981
10.2
66
-1.7500
0.02
0.2
Leachate C
-0.0819
12.2
81
-2.0968
0.02
0.2
PBS
-0.0604
16.6
122
-0.2701
0.15
1.8
"¦Calculated time in days at which measured linear decay rate intersects with assay limit of detection
18.0
1G.0
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0)
g 12.0
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5 4.0
2.0
0.0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
Time (days)
Leachate A Leachate B —~ ¦ Leachate C Positive Controls
Figure 17. Phi6 Persistence at 12°C
Phi6 Decay Rates in Landfill Leachates at 12C
V"
\\
«.
V
"V
V.
•s
% '
v
s
" \
s
*
*'*¦
LIMIT OF DETECTION
Page 39 of 67
-------
8.0
Phi6 Persistence in Landfill Leachateat37C
6.0
aj 5.0
[I 4.0
TO 2.0
Leachate A
12 18 24 30
Time (hours)
>—Leachate B -• • Leachate C > Positive Controls ¦ Negative Controls
Figure 18. Phi6 Persistence at 37°C
Phi6 Decay Rates in Landfill Leachates at 12C
18.0
LIMITOF DETECTION
2.0
16.0
Tf
O
u
a>
cc
14.0
12.0
10.0
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c
—I
c
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cu
8.0
0.0
0 4 8
¦ Leachate A
Linear (Leachate A)
y= -0.1584x + 12.478
R2 = 0.9267
12
16
20
24
28
32
36
40
* Leachate 8
Linear (Leachate B)
y =-0.0981k+10.166
R2 = 0.621
Time (days)
~ Leachate C
Linear (Leachate C)
y =-0.0819*+10.356
R2 = 0.6889
44 48
52
56
60
A Positive Controls
Linear (Positive Controls)
y=-0.0604x+11.047
R2 = 0.4891
Figure 19. Phi Decay Rates at 12°C
Page 40 of 67
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18.0
16.0
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14.0
CD
CD
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12.0
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Q£
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CD
5
4.0
2.0
0.0
•Leachate A
Linear (Leachate A)
y = -1.5867x+ 15.001
R2 = 1
Phi6 Decay Rates in Landfill Leachatesat 37C
~ \
V
LIMIT OF DETECTION
12
18
24 30 36
Time (hours)
~ Leachate B ~ LeachateC
— Linear (Leachate B) Linear (Leachate C)
42
48
54
60
y = -1.75x + 13.191
R*= 1
y= -2.0968X + 14.989
R2 = 1
A Positive Controls
— Linear (Positive Controls)
y=-0.2701*+15.297
R" = 0.9982
Figure 20. Phi6 Decay Rates at 37°C
5.5 Microbial Activity
Heterotrophic bacterial and fungal concentrations in each leachate were characterized
approximately 2V2 months apart, once at the onset of each test (Table 8). Results indicated that
landfill leachates were biologically active, with Leachate A showing less fungal activity than
Leachates B and C. Based on these limited data, leachates generally remain biologically active
between the first and second persistence tests, a period of approximately 2V2 months. Heterotrophic
bacterial concentration remained approximately the same (105 to 107 CFU/mL) throughout the
incubation. Heterotrophic fungal concentration did appear to increase slightly from 102-104 to 105
CFU/mL over the 2Vi month incubation.
Fungal and bacterial colony types observed on plates differed between leachates, and over time.
Although recovered colonies from heterotrophic plate counts (representative images in Figure 21
and Figure 22) were notes, they were not identified or characterized, so this analysis was purely
qualitative and was not intended to measure population or diversity shifts.
Page 41 of 67
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Another indication that microbial activity altered between the first and second persistence tests
was identified during method development. During initial testing, Leachate B interfered with the
Phi6 DAL assay, preventing plaques from forming. However, Phi6 could be recovered from
autoclaved Leachate B, suggesting that the assay inhibition was caused by a biological constituent.
This issue was resolved by adding a centrifugation step post-spiking, prior to the DAL assay, that
allowed a high percentage of Phi6 to be recovered from the supernatant using TSA supplemented
with magnesium and 20 ppm ampicillin (Appendix A, Table 3). Interestingly, interference of
Leachate B with Phi6 was not evident in a follow-on experiment, suggesting that microbial
activity, and/or the population of specific microbes, changed during storage from the time the
leachate was collected.
Table 8. Microbial Activity in Landfill Leachates
Test
Start
Leachate
pH
CFU/mL
Date
Bacteria
Fungi
Initial Test with all
2/15/16
A
8.02
3xl06
3xl02
three viruses
(3-5 colony types)
(3 colony types)
B
7.37
9xl05
(3-4 colony types)
8xl04
(10-20 colony types)
C
7.73
8xl05
(8-10 colony types)
9xl03
(10-20 colony types)
MS2 2nd Iteration at
4/27/16
A
ND
>3xl07a
6xl05
37°C
(~5 colony types)
(3 colony types)
B
ND
lxlO6
(4 colony types)
6xl05
(5-10 colony types)
C
ND
5xl06
(~5 colony types)
3xl03
(-10 colony types)
ND= not determined (note: pH of the leachates was measured in July, 2016, and did not shown discernible changes, having pH
readings of 8.09, 7.36, and 7.55 for Leachates A, B, and C, respectively.)
a: Colonies on all plates of all dilutions returned were too numerous to count. As the maximum number of countable colonies per
plate is 300, the CFU/mL of the sample is greater than this quantity.
Page 42 of 67
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Colonies on Potato Dextrose Agar
(PDA)
Leachate
Colonies on Tryptic Soy Agar
(TSA)
Figure 21. Bacterial and Fungal Growth on TSA and PDA growth media
(initial analysis on 2/15/2016).
Page 43 of 67
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Colonies on Tryptic Soy Agar
(TSA)
Colonies on Potato Dextrose Agar
(PDA)
Leachate
Figure 22. Bacterial and Fungal Growth on TSA and PDA growth media
(second analysis on 4/27/2016).
Page 44 of 67
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5.6 Evaporation
The weights of "evaporation control" samples were measured at each time point and showed no
change, demonstrating that evaporation of the leachate within the vials did not occur. Therefore,
observed changes in virus concentration over time could not be attributed to evaporation as the
total volume in the sample tubes had not changed.
Page 45 of 67
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6.0 Discussion
Viral persistence varied by agent and temperature. At 12°C, MS2 persisted the longest in the
leachate (three-four months), Phi6 persisted for a slightly shorter period (two-three months) and
TGEV persisted the shortest period (5-16 days). At 37°C, MS2 survived only a few days, and
Phi6 decayed very rapidly, within the first six hours. TGEV persistence was not measured at 37°C,
as its survival at 12°C suggested it would not survive for more than several hours at 37°C.
Variation in agent survival time was expected as each virus has a distinctive structure. MS2 and
Phi6 are bacteriophages (viruses that infect bacteria). MS2 is a non-enveloped single-stranded
RNA virus with a very small 3.5 kilobase genome, while Phi6 is an enveloped double-stranded
RNA virus with a 13.5 kilobase genome. TGEV is an enveloped single-stranded positive sense
RNA mammalian virus (a Coronavirus) with a 28.6 kilobase RNA genome. Non-enveloped
viruses are generally more environmentally stable than enveloped viruses. Enveloped viruses are
thought to be less stable due in part to the outer lipid layer, which is susceptible to dehydration and
disruption by a variety of environmental and chemical factors (e.g., pH, humidity or water activity,
heavy metals). Data from this study were in accordance with this general trend, showing the most
stable virus (MS2) to be the non-enveloped virus, while the enveloped viruses (TGEV and Phi6)
were less stable. In addition, Phi6 was substantially more stable than TGEV, even though both
are enveloped viruses, suggesting that viral characteristics beyond basic structure can influence
viral stability.
Interestingly, leachate effects on viruses were not consistent between agents: for example, TGEV
persisted the longest in Leachate B, MS2 persisted the longest in Leachate A, and Phi6 persisted
the longest in Leachate C (see Executive Summary Tables 1 and 2), although both enveloped
viruses were most affected by Leachate A. This observation suggests that each class of virus is
affected by leachate constituents in unique ways, and that non-enveloped and enveloped viruses
may maintain stability in different types of leachate. While pH can inactivate viruses, primarily by
altering surface protein structure and/or the lipid envelope, all three leachates displayed pH
readings similar to environments that do not affect viral infectivity (within physiological levels
Page 46 of 67
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[pH 7.1-7.9]). The data suggests that viral agents were affected primarily by the varying chemical
constituents in the leachate.
Statistical analysis was attempted to try to distinguish any trends with respect to the chemical
analysis of the leachate samples and the respective persistence of viruses in those leachate, but no
statistically significant trends were identified.
Differences in decay rates between leachates was not surprising, as each landfill used in this study
varied in intake and management, so each leachate showed its own biological and chemical
fingerprint, sometimes substantially varying in constituent concentration. In fact, Leachate B
interfered with the Phi6 plaque assay but not the MS2 plaque assay, and Leachate C interfered
with the TGEV TCID50 assay but not the MS2 or Phi6 plaque assays.
Page 47 of 67
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7.0 Conclusions
This controlled laboratory study evaluated the persistence of three uniquely different viral agents:
one enveloped single-stranded RNA virus (Coronavirus, TGEV), one enveloped bacteriophage
(Phi6), and one non-enveloped bacteriophage (MS2) in three unique landfill leachates. Viral
inactivation in each landfill leachate was measured at 12°C, and bacteriophage inactivation was
also evaluated at 37°C. These data were used to establish decay rates and estimate viral persistence
in leachate (persistence: time at which a viral agent reached the assay limit of detection). This
study showed that, in general, viral agents can persist for weeks or months in landfill leachate if
the leachate remains at a mild temperature, and that viral decay rates increase rapidly as leachate
warms. These results also suggest that the chemical characteristics of a specific landfill can affect
viral decay rates although we were not able to statistically identify any relevant trends with respect
to chemical composition of leachate and the persistence of viruses.
This study showed that live infectious viral agents can persist for days, weeks, even months in the
landfill leachate under certain environmental conditions.
Key findings and observations from this study are the following:
• Some viral biological agents likely can persist in landfill leachate for several months;
especially at lower temperatures.
• Moderately elevated temperatures, such as 37°C (99°F), can drastically reduce viral
persistence and infectivity and can decrease D-values to < 1 day.
• Leachate composition likely dramatically effect viral decay rates and persistence. This
study did not identify how leachate composition affects viral inactivation, however
differences in viral persistence between leachates is likely due to chemical constituents and
concentrations. As this study investigated leachates from only three landfills, it is unknown
how these data correspond to leachate from other landfills across the U.S. Further
persistence analysis of these viral agents in a much larger number of landfill leachates
would be needed to gain insight into the key characteristics that effect viral decay rates,
and generate actionable data for use in waste management.
Page 48 of 67
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Non-enveloped viruses were found to persist longer than enveloped viruses. This result
was expected based on the persistence of both of these viral types in other environments.
Page 49 of 67
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8.0 References
1. Thornelow, S. A., et al., EPA's Suite of Homeland Security Decision Support Tools For
Managing Disaster-Generated Waste and Debris, in Proceedings Global Waste
Management Symposium, Promoting Technology and Scientific Innovation200& : Copper
Mountain Conference Center, Colorado USA.
2. (ATCC), A.T.C.C. Transmissible gastroenteritis virus (ATCC VR-763). 2016 [cited 2016
10/05/2016]; ATCC Catalogue],
3. Reed, L.J. and H. Muench, A Simple Method of Estimating Fifty Per Cent Endpoints.
American Journal of Epidemiology, 1938. 27(3): p. 493-497.
Page 50 of 67
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Appendix A:
Method Development Summary Report
Page 51 of 67
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1/28/2016
Landfill Leachate Virus Persistence Study under Contract No. EP-C-15-002
Task 3 (Method Development) Summary
Described below is summary of the key results of laboratory activities performed for developing
recovery assays of TGEV, MS2, and Phi6 bacteriophage from three landfill leachates designated
A, B, and C. Based on these results, the recommended persistence study design and methods is
summarized in Table 6.
TGEV
Assay Inhibition/Interference
Prior to testing the recovery of TGEV from leachate, a cytotoxicity assay was performed to assess
if there was a toxic effect on the ST-cells due to the unique matrix of Leachate A, B and C.
Additionally, a comparison of autoclaved versus untreated leachates allowed for a screening for
virus within the leachate matrix which could potentially interfere with the assay. Three treatments
of leachate (untreated, autoclaved and EDTA at 0.1M) were inoculated onto a monolayer ST-cells
at six dilutions (neat, 1:5, 1:10, 1:20, 1:50 and 1:100). Cells were observed at 18, 24, 48, 72 and
96 hours post inoculation for cytopathic effect (CPE).
No evidence of mammalian virus (viral-induced CPE) was observed on ST-1 cells in untreated
leachate matrix A, B or C. No evidence of cytotoxcity was observed on ST-1 cells when cellular
monolayers were exposed to a 1:0 dilution of untreated leachate, however concentrated leachate
did cause cytotoxic effects. The 1:10 dilution of leachate was determined sufficient to prevent cell
death due to leachate toxicity. Previous studies used 0.1M EDTA treated Leachate to reduce
mammalian cytotoxcity, however this treatment resulted in increased cytotoxcity over untreated
leachate matrix, and thus was not explored further.
Recovery Efficiency
To assess the recovery of TGEV from Leachates A, B and C, three TCID50 assays were performed
per leachate. Leachate aliquots were spiked at a 1:100 ratio with stock TGEV (lot#: TGEV-P3-
121515) for an estimated titer of 4e+4 TCID50/mL (i.e., 4.6 log TCID50/mL). Leachate aliquots
were then immediately serially diluted in complete growth media (5% FBS in Eagles Minimum
Essential Media (EMEM)) and plated on ST-cells for TCID50 assays. Assays were scored for CPE
at 48 hours post-infection. Positive, leachate and negative controls were ran with the experiment
for comparison. As shown in table 1, TGEV was recovered at >90% from all three leachates. The
recovery % is calculated using empirical data from the positive control results (an average of 4.68
log TCID50 inoculated per sample).
Page 52 of 67
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Table 1. Recovery of TGEV
Leachate
Spiked (log
TCID50)
Recovered
Coefficient of
Variance
Recovery
with 95%
CI
A
4.68
95.19%
6.03%
95% +/- 44%
B
4.68
96%
2.05%
96% +/- 15%
C
4.68
98.62%
4.17%
99%+/-31%
Limit of Detection (LOD) Assay and Inter-Analyst Observations
The LOD of the TGEV TCID50 assay, TGEV (lot# TGEV-P3-121515) was determined in two
independent experiments, each of which was read by three Battelle analysts. Results from replicate
1 and 2 were used to evaluate the limit of detection and results from each analyst were used to
determine inter-analyst variation. The LOD was determined in each assay by diluting stock TGEV
to extinction in Complete Growth Medium (5% FBS in EMEM), followed by triplicate TCID50
assays on low concentrations of TGEV (see Table 2). All TCID50 assays were read separately by
three analysts at 48 hours post-infection. Results are shown in Table 2.
The limit of detection was estimated by comparing the concentration of virus in each high dilution
to the calculated titer present in each sample. The coefficient of variance (variability) was used to
assess the reliability of each result. Calculated titers (shown in Table 2) were determined using
positive control samples run with each replicate for accuracy. Conservatively, the LOD was
estimated to be approximately 200 TCID50/mL.
As the TCID50 assay is dependent on analyst ability to differentiate cytopathic effect (CPE) of
TGEV from any other cellular damage/death on ST-cell monolayers (including leachate-induced
cytotoxcity), and results are often dependent on sequential reading of many assay plates (up to 27
plates per assay), analyst calls for positive CPE wells and overall results from analyst results were
compared to determine if there was significant inter-analyst variation in the results.
Analyst observations were compared using a Paired Two Sample for Means t-Test (Microsoft
Excel 2007). No significant differences between the mean numbers of TICD50/ml observations
was perceived between three analysts (significant differences defined as p<0.01).
Table 2: Limit of Detection results for replicate TCID50 assays using both 2-fold (Table 2A) and
10-fold (Table 2B) dilution schemes. The 2-fold dilution scheme was able to identify lower
concentrations of virus, however the 10-fold dilution scheme resulted in less variable results.
Legend: * Dilution was not repeated during the second run of the assay; - N/A; 1 High CV reflects
substantial increase in variation in TCID50 assays when using low concentration of virus. TCID50
calculations are dependent on at least 50% + CPE in at least one dilution of starting material (e.g.
one row on one plate). At a concentration of 40 TCID50/mL, it is likely that only a small number
of wells were inoculated with enough virus to cause + CPE.
Page 53 of 67
-------
Table 2A: LOD results using 2-fold dilution series
Limit of Detection Results for TGEV TCID50 assay using 2-fold dilution series
Replicate Experiment 1 Replicate Experiment 2
Dilution
Series used
for TCID50
assay
Dilution
from Stock
for
TCID50
Calculated
titer
TCID50/mL
Empirically
determined
TCID50 of
stock
solution
Standard
Deviation
Coefficient
of
Variation
Empirical
Titer /
Stock
Titer
Calculated
titer
TCID50/mL
Empirically
determined
TCID50 of
stock
solution
Standard
Deviation
Coefficient
of
Variation
Empirical
Titer /
Stock Titer
2-fold
1.00E-07
0.28
0.00
0
-
-
0.21
*
*
*
-
2-fold
1.00E-06
2.82
0.00
0
-
-
2.07
0.00
0
-
-
2-fold
1.00E-05
28.20
37.36
9.46
25.32%
132.48%
20.70
5.85
10.13
173.2P/01
28.25%
2-fold
1.00E-04
282.00
339.66
56.58
16.66%
120.45%
207.00
91.60
21.71
23.71%
44.25%
10-fold
Stock
2.82E+06
2.82E+06
2.94E+05
10.46%
100.00%
2.07E+06
2.07E+06
1.77E+06
85.77%
100.00%
Table 2B: LOD results using 10-fold dilution series
Limit of Detection Results for TGEV TCID50 assay using 10-fold dilution series
Replicate Experiment 1 Replicate Experiment 2
Dilution
Series used
for TCID50
assay
Dilution
from Stock
for
TCID50
Calculated
titer
TCID50/mL
Empirically
determined
TCID50 of
stock
solution
Standard
Deviation
Coefficient
of
Variation
Empirical
Titer /
Stock
Titer
Calculated
titer
TCID50/mL
Empirically
determined
TCID50 of
stock
solution
Standard
Deviation
Coefficient
of
Variation
Empirical
Titer /
Stock
Titer
10-Fold
1.00E-07
0.28
0.00
0
-
-
0.21
*
*
*
10-Fold
1.00E-06
2.82
0.00
0
-
-
2.07
*
*
*
-
10-Fold
1.00E-05
28.20
32.35
0.67
7.77%
114.72%
20.70
*
*
*
-
10-Fold
1.00E-04
282.00
321.24
24.95
6.27%
113.91%
207.00
147.83
39.18
26.50%
71.42%
10-fold
Stock
2.82E+06
2.82E+06
2.95E+05
10.46%
100.00%
2.07E+06
2.07E+06
1.77E+06
85.77%
100.00%
Page 54 of 67
-------
Proposed Recovery Method
For the persistence study, the following test methods are proposed:
• Spike 3 mL leachate samples at a ratio of 1:50 with TGEV stock to achieve a starting
concentration of 5 loglO TCID50/mL. Spike EMEM without FBS, with antibiotics
(Incomplete growth medium) as Positive Controls. Include non-spiked leachate as
Negative Controls.
• Incubate samples at 12C in screw-top, polypropylene tubes.
• Assay triplicate test samples per time point along with triplicate Positive Control sample
(stock virus of the same lot as spiked sample), and a single Negative Control per leachate.
BACTERIOPHAGE
The Double Agar Overlay (DAL) method was used to measure the concentration of MS2 (E. coli
ATCC 700891 as host) and Phi6 (Pseudomonas syringae ATCC LM2489 as host). Luria Bertani
Agar (LBA) was used for MS2 testing and Tryptic Soy Agar supplemented with MgC12
(TSA+Mg) was used for Phi6 testing. Working stocks of MS2 and Phi6 were prepared at 3.6e+9
and 1.0E+10 pfu/mL respectively. These stocks were stored frozen as 1 ml aliquots and used as
needed.
Assay Inhibition/Interference
Leachate samples that were non-diluted and diluted 1:5 and 1:10 were plated using the DAL
method and showed that that none of the leachates caused assay inhibition for MS2 nor did
leachates A and C for Phi6. Leachate B did however did contain constituents that prevented Phi6
plaques from forming. This same inhibition effect of Leachate B with Phi6 was observed when
all three leachates were spike with MS2 and Phi6 at 104 pfu/mL as there was virtually no reduction
with MS2 and Phi6 with leachates A and C, and no Phi6 was recovered from Leachate B. A
subsequent test with autoclaved Leachate B showed that assay inhibition was caused by a
biological constituent as Phi6 was recovered from the autoclaved material.
The use of antibiotics in the bottom and top agar was also evaluated. For MS2, Luria Bertani agar
supplemented at 15 ppm streptomycin and 15 ppm ampicillin (LA+S+A) was evaluated. For Phi,
TSA+Mg supplemented with ampicillin at 20, 50, 100, 150, and 200 ppm was evaluated. In short,
the addition of antibiotics in the media for MS2 neither aided in recovery nor adversely affected
recovery. The addition of ampicillin didn't eliminate Leachate B inhibition. The Phi6 plaques are
also less distinct and more difficult to count using media containing > 50 ppm ampicillin.
A follow-on experiment showed that if Leachate B was centrifuged post-spiking to pellet the
microbes and particulates from the bacteriophage, a high percentage of Phi6 (Table 3) was
recovered in the supernatant using TSA+Mg+A20 medium. Interestingly, Leachate B no longer
showed assay inhibition. One explanation may be that the concentration of microbe(s) that caused
the inhibition may have significantly declined during storage from the time the leachate was
collected.
Page 55 of 67
-------
Table 3. Recovery of Phi6 from Leachate B post-centrifugation
Matrix
PFU/mL Recovered
Pre-C entrifugati on
Post-Centrifugation
PBS
2.7e+3
n/d
Leachate B
1.9E+3
1.6E+3
Recovery
Leachates A, B, and C were spiked at le+104 pfu/mL (i.e., 4 logio/mL), serial diluted with PBS,
and the assayed using the DAL method. The results (Table 4) illustrate that the phage can be
recovered very well in leachates A, B, and C as the log reduction observed was insignificant with
the exception of Phi6 from Leachate B.
Table 4. Phage Recovery
Reduction of Recovered Phage Observed (logio
Leachate
PFU/mL)
MS2
Phi 6
A
0
No data (assay inhibition)
B
0.2
-0.3
C
0.2
-0.2
LOD and Intra-assay Precision
To LOD of the DAL method was determined assaying five replicate 0.1 aliquots of MS2 and Phi6
prepared in PBS at 10, 50, 100, 500, and 1000 PFU/mL. LA+S+A medium was used for MS2
quantitation and LA+Mg+A20 was used for Phi 6 quantitation. Two analyst assayed each
suspension.
In short, the results suggest the LOD for the bacteriophage is approximately 500 pfu/mL (i.e., 2.7
log pfu/mL) based on the phage was detected in all replicate sample from both analysts at this
concentration. The results (Table 5) also illustrate the assay has a high precision as the results
between the two analyst were very comparable. The recovery efficiency was lower than desired
50% but since the initial spike concentration for the persistence study will be 6 logio pfu/mL, a
range of 4 logs of decay will still be able to be measured.
Proposed Bacteriophage Recovery Methods
Based on these results, the proposed recovery assay for MS2 is to serial dilute the test samples
with phosphate buffered saline, and assay triplicate aliquots of various dilutions with the DAL
method using bottom and top Luria Bertani Agar supplemented with 15 ppm streptomycin and 15
ppm ampicillin. These antibiotic concentrations are often used with the E. coli host strain and did
not adversely affect recovery of MS2.
The proposed method for recovering Phi6 is centrifuge two 1 mL aliquots at 12,000 xg for 2
minutes, combine the supernatants, serial dilute with PBS, then using DAL method, assay triplicate
0.1 ml aliquots per dilution using TSA+Mg+A20 medium.
Page 56 of 67
-------
Table 5. LO]
) of DAT; Method
Phage
Analyst
Theoretical
Concentration
(PFU/mL)
Measured
Cone.
% Recovery
(PFU/mL)
MS2
1
10
2
20%
50
4
8%
100
10
10%
500
64
13%
1000
158
16%
2
10
0
0%
50
6
12%
100
8
8%
500
70
14%
1000
138
14%
Phage
Analyst
Theoretical
Concentration
(PFU/mL)
Measured
Cone.
% Recovery
(PFU/mL)
Phi 6
1
10
2
20%
50
4
8%
100
14
14%
500
100
20%
1000
252
25%
2
10
6
60%
50
10
20%
100
20
20%
500
84
17%
1000
176
18%
Page 57 of 67
-------
PROPOSED DESIGN OF PERSISTENCE STUDY
Table 6. Recommended Test Design for Persistence Study
Test Parameter
TGEV
MS2 and Phi6
Spike Concentration and Sample
Volume
Spike at a ratio of 1:50
• 60 |tL virus stock into 2.96 mL leachate to achieve starting
conc. of 4.9 logio TCID50/mL
• Sample volume= 3 mL
Sample Tube: 5 mL, screw-top polypropylene
Spike at ratio of 1:100
• Spike with lE+8pfu/mL suspension prepare in PBS to achieve
starting conc. of 1E+6 pfu/mL (or 6 logio pfu/mL)
• Spike 0.3 ml phage into 2.7 mL leachate
• Sample volume= 3 mL
Spiking
Option A: Add replicate 3 ml aliquots of leachate per 5 mL screw-top polypropylene tubes, then spike each tube with virus or phage.
Prepare three replicates per each time point listed below. Incubate tubes in test tube racks with caps tightly fastened in incubators set at
temperatures indicated below.
Option B: Spike leachate with virus or phage, mix well, and dispense into individual tubes. Then incubate as described in Option A.
Controls
Per Time Point:
1 Neg. Control Per Leachate (non-spiked leachate)
3 Positive Controls* (spiked EMEM without FBS, with
Antibiotics Complete Growth)
Per Time Point:
1 Neg. Control Per Leachate (non-spiked leachate)
3 Positive Controls (spiked PBS)
Incubation Temperature
12°C
12°C and 37°C
Time Points
(subject to change throughout testing
based on real-time results)
0, 3, 7, 14, 21, 28, 35, and 42 days
0, 3, 7, 14,21, 28, 35, and 42 days
Recovery Assay
Assay triplicate samples per time point
Mix sample well via repeated pipeting then dilute sample 1:10
in Complete Growth Medium. Continue with serial dilutions
(1:2 and/or 1:10 as deemed appropriate) using 96-well plate.
Assay triplicate wells per dilution using 2-day TCID50 assay.
Persistence assays will be performed in triplicate. A second
analyst will score results for 50% of the experiments. This will
give second observations for 50% of the persistence
experiments. Data will be reported as an average of both
analysts for those experiments. Recommend spiking virus into
leachate at a minimum of 1:50.
Mix sample well via moderate vortexing.
• MS2: serial dilute with PBS to 10-4, then using DAL method,
assay triplicate 0.1 ml aliquots per dilution using LA+15S/A
medium.
• Phi6: centrifuge two 1 mL aliquots at 12,000 xg for 2 minutes,
combine supernatants, serial dilute to 10-4, then using DAL
method, assay triplicate 0.1 ml aliquots per dilution using
TSA+Mg+A20 medium.
Data will reported as mean pfu/sample
*3 reps at T0 to determine baseline spiking concentration but may be reduced to one replicate in subsequent time points.
Page 58 of 67
-------
Appendix B:
Miscellaneous Operating Procedure
Page 59 of 78
-------
MOl'IdWi "Ui-fXJ
I t'sUivc Date 02-n-'?Olo
Page 1 of 1ft
Miscellaneous Operating Procedure (MOP) io Fvaluate the Persistence of
Transmissible Gastroenteritis Virus (ICF V), >1S2 bacteriophage, and Phi6
bacteriophage in ! arulfUl Leachate
LB Purpose and Scope
To C"'.iloau* the ptrMSfctv e nil iat*s"«v>thli't iaMuvntentis v" i JOL V p VIS"1
Kvfenfolsa^e and fhi.t tMtxitophaae "i 1 1 caJ^ie vd'n red norr fmx landhiK
F.ici) ot t';e time la idftli i> achate < vul' n«> spiked itnlivitltnll'v with kioviti ^uartitv ol «ru»
oi bivtawpkigo diswnsul laid upluMU' ,*ciew-rop wis. and incubated statically o\c tlie
aavse o'" jppto\una*cK f> v»cUk-> All it>FV-«ptkca ijmp'c> w'l lx- ifn,ufxits.d «f ! ' d. giv ,,
i elsius ('O, ind one s«>* of the bactvnophagc soiked m uplcs m iP be mcub. led a i " I it J
anoihtt sii at 1 / '£ "\r point, implicate samples «ili be s*^ ivw p^noUicali) me> uiTit
ioi up lo fi;;ht time point tndl.tiitu' T, TGf V -<.pik 'J ^etmpir, will K a^av'v-d rut lt'ffctm*
( n Libit * \\tls in <.enajh diluting iiic sampk in tmie uiltuit -nidium aad tlitn jssayng a
ot rtih i Atsiruii a Et LDvj cT-vy oi Swmp Te-mcolat (VI ! ceils Fm tnderujphage-
-.pikul sampK's-. tnpKai * NdPPflt\ » ill be v>utll diluted ui a buffer mi rssasid ioi udeotn e
Kactertopfiape tug d otdfilaid Double IV t> Vj;a Chtrlai (DAI t »ra\N>d jMnt I rend \i
. ¦»/' and fir v.K«if cvr;r^m a.-, the Host snaiiiL 'or MS2 and PbiO kmvU1 ael\ Santrie;.
w >11 be as^ayid r>vvt t me ami cilhti flit, \ I n? bactenopha^t is no lortgt'i detected or until
>¦>" <-.ur>p'is tat all .1,4111 Utiik potnU Ivvt Ken iv»a\ed
2.0 Materials and Reagents
*1 'lcvnlu- S\>\ Bottom Ajjar supplemented \uth M-wncSiUirt '"'hHride and AnpitiJl'n n ?0
opm SA LVS^>+AJ0)
U hvptit Sm T'»p Vgai ^applcmcnted vvdb Ma^ti^-mnu Chloride and AmptcilKn ai 20 pom
^TST\.Mpi A2(«i'
t"s I ana Hcr\.ru ltottnm A£ai siippleirenfal Streptom)cm ard Amntcdlin at 1 .*• ppm
(iBA-H-t A)
D, l.iru tJcrtani Uv Asr;;rsiipplancntcd JticpKanxcni and 4mp«cUlin at 1^ ppm
(LBTA+S+A)
E I rypfk Suj Broth > f'SB)
T Tnpfu Soy Ap^T ( FSAl
G i una Ben mi Bwili ( lRBI
Fi Potato Dexrose >gai (PDA'S
1. I'bospbaii; Bi tTer^l Salwe pH to 7 2. «tenlc li^hS)
J 7(t#o lH>pit>py5 \Ico1 i>! Spia; {'PA!
K. IP A Wipes, Sterile
1 Bleach. (o. (1 sodmm hypr>vhlutdo julutitMi)
M Bleach, hxu^'holt la bS'-i sodrntn li>podilontc)
>i At/it c't/i Ainencan 1 ypt Cnltutc Callechon ( ATCC) 700891
O buctcrtaphagv A~CC S5<55y"-BJ
? (,<'IN1
SUid\ Number W>0S7
Page 60 of 67
-------
11 fecti ve l>att 02/12/201 o
Pagt 2 »! Ut
Q Phu> "hacterit iplugt i tP \ ptovidod i
R 1 c Khati A. Iff! r.wnbcr AI < tft" 1 i, collected 111 7'.*01 *>
t>. Lv-aclwtc B, latiwiiobrr fliUO/11, ctlkvHtJ lO 1
f I eavbdtw C, lot rmmh-r tlObll collected !0/>V-01 S
l< ( omjilcir Gtovth Medium IX tagSe <; M'Pinuicn tsrctUMl Mod mm iFMFM* containing
5' .'¦> v \ te\*>i bevnte -icsoni (FBsS) and 1% v \ unttbi- tfir-a.)iimjcotie sulutian (Anti-Anti!
\ ii»coi!i|sbu- itrt'wr't Medium L X L \IF\! W'th i"o v,v aniibU'tw-
.mtinv.c.itic tn
\iV 7 rummif.NibV OaMiocimutis V aus (TGL\ K All C VR-7&3
7v Svufit TeNfiuilax (NTt teiis (ATCC f'RI !"4()) iruini{urK*t in Complete Ormvia Medium
3.0 Equipment and Supplies
A 'ih uhiiLif 36 °C
B Refnfciatoi Inouatot U-i'T
C Waki k:lh, 45-50 °C
ft. t '!as& 1) Biological Sitetv C abjnct (BM't
fc. Vortex
K Lock n I ock Bo\e• ot equn atom plasiiewarv i-ortasnzr
U L"5w(«;Utllent rur nc-nh i b «tv tv* s\v tru. te„4^ (ST)-cells
Q I issue cuttuic reservoirs
R ^6 deep well pktt-
S. McfU>, P-20 P HlO P-1 Odd
T. 2' 10 miciohtc Tvhiltfdvnnei pipette, I, 200
f i Assorted acnle Flfced mpttie tip*
V Aifiiatot
%. .\W-tiUnixi pipette lips
X Pi pet did
V A^otled column sieine disposable pipettes, yidautid
/ Analytical Balance
.\\. (Vibiatcd weight set
4.0 Acronyms
A. ATCC - AnKMCdti T)pe Cultrrn Coll'vtion
B BSC - Biological Safety t .
-------
MOP 20 i <>-001-00
T ffci'hvi- 02/ S J/2016
Page J of 1(1
C Hi.) - HtolOjTica' K^feH i fid
I). CFlT CoitMn Iarming Umt
• 1 COM t'oirpltle' Jrrwth Medium
t. ("Ft t >%>pii{htc Ptfe<..
(j o Al - 'A'bbk' Top Ayjt Ovui\
H fMfjVt Pig'e ° tsviwal Medium
1 I fcsS ! etal Bo\ ;nt Strum
1 IP \ 'ott3 lvipts>{«y! Uc>M\o)
k. I H\- Luna Bert,mi H,»iloTn Agar
L LBB Luia Douani 8r> MS ttom Landfill i t. achate
I I Worktihcjt i»f R xovcnng Fhtd trow t qatifill t eattosr __
W.UKsbcf! to l'tepaie ti Sev Rufturo \£Ji \upp'enitnic*4
, with M igneMum Chionde and Ampiolllin at 20 pfvy I! S \-
' Mu ¦ A70) _ _
1 Worksheet Piepaic t'rvptic Agar supplunenutt with
Magnesium Chloride and Anptalhr at ?0 ppm (TS < \-Mjj-' V Ptcpait" I una Beitatii Boitooi Afur supplemented i
I Stieptorrvun ar>d_ \ ,it 15_ppm i LRA A.-* J
Studv Number 6o087
Page 62 of 67
-------
6
Worksheet to Prepare Luria Bertani Top Agar suppl
u and 'VmpKil'in ai i5_pptn it Itil*
W.jrtilicri co Pujnjy * ryptic »>o\ Bioth 11 SPi
A)
Workstvu to Vicfwv Lito Welfare Broth HroltiU BBi
I* WorUhe«t« Preane !>ot.,fo l>r\tu>K Agar iPDAJ _
( WoiK^ht-ei to Fiepiirt- Fiynu, S»n 4gor(lNAl
TGF.V Woikshcetj'M P>«.pj>nn«;v4uK.e
Woiksntcts V»oikjIh a for Ptcixngali^n "»¦' i Gi'V
! orkshce, foi Recovery ut Ti I X l"'o»n IvWtok vnth ^ 1
j Woikshec* to; Seeding Jb Weil FUut. *Jh R r-t elk
: "^eon'alion T1 < cynnlelr «_m>wth Mcnlijn (CUM) tot SI 1
i Pit i\iiatior> or Im oniRlfl' 1 >to**h Mui'iitu tat ST C elu
B Sample Preparation -MS2 and Phi6
.Vo/c / Perform all manipulations with virus and bacteriophage in a certified, class II
/W>/» J .* < nor/" e>" » url to1!' fi/t" j> wj/i~ L u r f H jwu'e'mc:
\ot i ( iKiftni: »/-t< f -Wi/»-wrhcec ?/rc ^ *lA >f w/i •«;
persistence study using the DAL method.
Vn'i / f, i -/( h. •/'
v- iHtL J \ r;tT'lf ID put It< u/>.' i /* >?i;r >};> i\hJa\n' *l v oi 'awV.i <• P>
n t jk njM t'ic,¥(i'/w' i /V V"* ir kC» tm i if "jWi h>,n<1vr
1 Kit Samples (yi bacteriophage)
D Per if itlut- (tin > sn d >. ?*» K. pa f ^nut to -> stcriL II I'llcnrnejei
flask containing a stir bar.
in \ti\ lc.tch:uc on a s-tn plak M at nsriiiifti-hiidi fin a* k.xst nimuies
iv ft h'le leacha^e conii^ue t" tn* v a si!? olrfie v'v a 5 uil jdtluat'Yl pipr* *o
dispells 4 m' oi kschaic imr prc !aHel«S Uibts
\ Stor sawoK-!. 'ein^etd.cj n"(he^ J'e U< be ipiicei4 the following, dr\ U ^j day pirccet' to ntxt step
b) R^mo\e bjctvT'«plijgt"»tocl- Som us id
c> Diiuft bavtetiophsigs tu I > C tunning unlL (PP'J i, ii»f x»,ing PBS,
rim. strum;: tt«c !-pil..nc vuipcnvifHis Ni*x s-uspcm.ons ^cll bv \ tiri^xnii; ai im-
moderate speed.
tl) »u •* uuihrateit P J00, jtnle txh 4 nil leartwte s^mole ^ Pi 40 pi oi tie
pppcoiinifr- *pt'/vtig ,-iHAHiior >o achioe.ilitgci stdt>inii\,« ucvpIiaIiouut H i-T
pT I ,ntl , Afn nsionJfcqi't nlv <:• waincm u
if i nrfenfu' .«ht ,»•!»" L
Sluih Nti)ftbc»
Page 63 of 67
-------
MOF1OttH,01-uO
;/2016
tV;'v 5 OS fit
iStened, then mix each sample by
w irhi'g and jrt\eito>g fN- tannic ^ nine an to
') H itc uilv> in a idck,.nd il« ei mmsfJk teH pi tcc in an incubator set to operate at
eithci 3 ' C 01 „*" c
p Ft>»mpil> sauii e>i»V'lie It"'1 !Tl nf spikaig ^!«fl and triplicutr sptLc IcacMatc
samples as described in step E.
2 Controls (pet l.actvnoph-tgO
+ die cppnrpnate
"¦p'kuiji ¦sitjH.iMon ». achieve a tdtgei staling Anivauiatiort of h Hf PT i1 «nl.
Intj-tficH A/?rfcne w/vp.vfiw 'i^menr.) ft> c fn'>n<e tij,hth fattened, then i n\ cr,R.h samplo b\
swtHing and inciting tilt- ^awple 1 timo
rt one sc> pei bacteriophage aiul pc mcubrthon
lemtjeramre.
h) 1 tisure caps are fVtencd s,. carol >-
<.) Wt-ipl. eawi .sample ou an ana -ytiud fwl.'inco Record weight out to 0,f>Wl g,jins
d) Mace lobes w a raok, ^nd then iiroricdiatel} plac.- m an i'.cobator s,ct to outtate ai
oithfr 1 ? T cr I"1 T
Note 1 'iosi .samples mil be weiglved at s:adi tine point thior.ghout the
pcrsisrcncc s,t«d} wtighcd and tbet< returned k> the ippropri&io incuhatot
Studv Nuinbtr 7
Page 64 of 67
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vSO
L«..tPC lliiK 0,> I 720! 6
r^e 6 ..f U)
C P.anifli* Preparation - TGEV
1. Test humpies
a I i'n teachate ohs? sttp can ht, pmormut up to 1 diy prior to spiking wun \na-»).
ktrnove cr>c o»~;he lti'cs uClrtchiik- 'ttm ictiigera&rt '-.It-rate and hut
very we1,1 bv s~vnbrtg \ ".oron.iiv
it IimntAjt.")} dispuisc appir.xmiatoh '>00 mL itiM a -tcnle ' L r s'n pLuc, \m a uaduiied 5 ml pipet to
cbspar-w 4 trl ot kit-hate into tne-tolvk'ti rubes
i' Siott u nplcs tefhgewteJ if thtv are to he spiked llic fulluu itig dc y It soiled
flit <. tuie tb) p'oceed to rfep
bs Thaw rCiE-V aliquot^ ?.nd b jo" nii.joiie i5 ml tube. iT-saw enough FCE\
to spike all lutchaie j>i-i posiitv; tvnftol to the
nieaia lor zws.
ct] Spike each 4 mi tw^ohate sample. tliqu-'Uditi *-(«"{> C,U>, Hit!) 80 (J TOFV .m
achicse a taiget {stirtin^ ..o'KcnUation or at k\i«;t i>\ I ft' T< IDv/ml,
e) Fusuie set e»*<-caps c f cadi sample art- u^htly "astened, thei> mix inch sample by
^wiri .Hid iruviiitiv; ftic sample- 1 tiri.un.
Ptact nibs': in i "mil- and .ncukilc at L1 \
2. PoMfr.e CmrtF rai pspci, accurate! v dispense I inL of »te«lie ukunnpktt
ing the same swtinp •.uspitision raep.itet! m 4t-p C.t i\ and usm« a c.ihbrjied
miciopipe'tc, spike eacti 4 »-,il lncontpk u I WVf maniple v nil ^0 fit of
iht spiling sus-pcnsioi, (o acliie^e a Liigcl .starting lOiKeirhat'on > Ijigt'i sumo,!
concentration >>i ,it IraM. 5*. 10* Tf fl"k, in( .
ci I iiMiK- screw-uips or e ich -sample a t- usitilh tlien miv c^ch scrapie bv
Pwitling .«id mvtrttng tht* samples 5 urncs without mtroducinj.* bubnip^miv thr
samplc.
d) PI.ki tubes m u> openik at
12
a) k\w|tii} a-.s iv ihrco ^ismve contif spiking «tock' liwn? llic I (ID^O methvxi
vief<^be.l m >ectiw H
3. "Ne^ativi.: Controls
a) I-sing (hetertch.mi thai ,kc pn pwed in skp r I a <->3 that tire nnung a i acl and then imiSKJidtciy place ,n ati incubatoi hI to operate at
cilhej 1} "C
Study Number
Page 65 of 67
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F.f'cctivc •i") i2'J0l«.
7 oi lt>
4 1 \ ,*f>irt4iiuii S.tmj-tlcs
hi Pi^pcnso ur •? mi oi I oai'utc \,\l mj ' , md 4 ml of IriKYtnplett. Urovth
\1«i ned ^e<*iui*iy
v) Wtv*fi c.xh x«mpk oil an an,il\iica! balance Record "uatdrt out to 0 00>M gj arns
d> Haco tubes, m ,< rati .tuf then inupfiidtelv pi.itT in ar ina'bat*- j -e< to i^rite a
cither 12 °C.
Note I hov s ciinlc* wl11x jx caib tim^ poini throughout tb~
p^tstfcjc,"- snub tvcijj. jit. and tren ictun.rJ U< the (twbati r
1). Incubation
1 Pl.ue tcvt ".wiiplts prisiii .e .ontr.Ms and ne^at.vt eoit*rois» in an rppn piiHtc
inc"hj4or kc\.riV stdil «ipn% dau\ ;vrd u^npetalutc.
2 Kiontm temmatur. o* in* ubalot .u U,jv H pei ^cei ut lnonuoi c^nlmuousiv if a
temperctrte Aiti logjjtt i< ataihlie
1 Remove tnpluvtr km -arrplts, triplicate positive eorttoSs. jnc* oik fxg«*inr eenliot
pet th>, 1oHov»uit'Uine OjMt. \ I, 14, 11, 1>J ij. 41 aays Sctf Tone pumt< ut\
*ui/Cl• n<. hiX.sM or ieii' 'jw ipsu;l\ 1h< pM •> tj <> v<\ c can/ r , jt at.QC of Mk3\
4. RccutJ date, turn* of da\, ,ma tciawamre -vher; «amp)ts arc removed per t,me pomt,
5 Ai e.ich liint piiMir, lunuve f (up«jiaij»i 'luobdiot, rruawrt n uple
\\ ri'iht-, wd t cu'to tlvrp to the ipeak ten.
1' S^mok Aral/sis MS2 and Phiu
\c,t Fft ,-iiiv •'/ fwjiV'x- jt ( on* th< « «.>ot.>c rjnhoh fir\' ,t,Uowsd t»j ,ht > si
\u fipL > aid dun ^insk m i'ic vo>it
1. cuitoit PrepuotiCM!
tor .VS2, on the dav of sample aiMiysK, motuljic 10 mE I F-B with 1-2
cvtk'ws uf i *. ' a ay uld ^ cot< ciliuic gmsn on lR*\ (neub.He broih mlture sn
m mcobtito: \ old f wnnfiaT cvltute gimvn on 1S V lrn,vbate broth
euhurt .>\cnjf;h4 >t> a« -nciibauw ^e' at 23 25 ditd 200 it«n. Use ^rt>tb euhurt
\dcjll> wiiea i> has been iricubatmn, foi 1 t to "*+ houis.
2 Removed bouoro agai p!:rt» troin reP igcwtwd sioraf c and allr»,\ to tome to roopi
temperature
3. Prt-paie rnollcu u
-------
MOf-JUiMKli-Of!
C.ifcctuc Date: '•?•( .'/AH6
Pifr- *> ^ l'i
4. Prepare sample:
< ) I at MS.' S aitplo (tt^t iiKl un ruh)
t Vntux sample ai kuod,mte ^pcec1 f« r HI second •>
n ^"cnaliv Jti'Kt to 10 ' u m* PBS Iprr dilution ?od i> 5 ml plmy-laden
sanpk w 4 5 >nl I'Hbj
1.) »" »i Phi(> fU-mpks fit -.i ,tn.' eontioKr
\ rni'x vimpk* .u moderate "itsecd tut SO vixoiius
*i D'spejsse two 1 niL ilsqtiit; inu> mncuxcotntju tabes
tit. t "wr.fujv ,u K',o0i^pfoi I rn'mlis
0 T\>ol (t S >n' u»"sin>emjtarti twin both ruKt> u> a %•« iur*
Srulh dilute to '0 1 u^ti^ TBS (r>u dilution add 0 5 raL phri'c-laJcp
> ample to 4 5 .pi PBS")
\r»v i i x h". i uiphu-r -u throi'iji ul ihc t ta^iJiox ns , i.Y S>».-qutu'd 17 «< Prf.ip if 1 :¦ > \tt\ulor *. J1
ih u rmiht ¦< hit h Jdut. ?}>', ate t< ... '/•' t :v .-w ik ih
\,,t, \, .r,ly ^r( < ,Ci ruT {/..>»( J I' til "l^Ww
Vv < I •>' t\ \i .»•» t s« ti 'th «/iV:r l > It' 1 and ^r'y tJ 4 as mc b-Mum agar and
I tft i- i L !S (' Rip ugrt/.
\oi? K<> us\ P untied - >i5 i;ie host < hd fS-s M%: • as the bottom agar
iihJ IS/'A Mg • t20,', 'hz t>)/<
Di^ienst 0J ml K-otli culture prepared! in step F 1 'nto mplicsie 50 ml conical
tube«
bl liispentc 0 ml ofhiivtti'oplkigi samnk iunJilak-a oi dilated) into each ttilx.
with br>»th culture.
c' Withia 10 ['nm.tCi, ,x!d c tuL >if nwlien top to {.nth tube, pn-mptlj n'\ uibc
bv sw irhng (av.^c1 atw«g bubble <-t anil putn eirtnc co«i«n» unM >b<; b.)ttpni
aeav ntaic.
i!) -\Ilow mp n,K,i to foli'ln^' Sjiwei.t" y |uw a t,nv rntimit> nf-cJud), and then
rocubifc MS2 plat's a' V" '€ and fbif- pi aw- at 2^-21 C uptil plaque1! rt n^b
1 c.miU-ble 'idt fgmerall v 1 i to 30 h ">5'i PFU'plai^ l-'ecoid rcsuU^ .'ml tdleuiuted
PUJ'wl vt^rp the mean nt plw-, rdtunp^frcm PH 'plate Record plates
It ivm$r .'5U PFU pel p'at". Fiks Numeunt\ k> <\'UTit ilNJi ),
s.) i\; tnpbcaics (or 5sivh iime tx;iiL vaWulat.* >ticott PFU rctovrtv't! and the, log
lini tvccn^rtil Plot t.i.'in Ic;, P" t1 reonertd vcrsu:, tunc.
Stud} KuEsbcr 6t-i087
Page 67 of 67
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\lOP~"(tl 6~ fi< 10
F ScUMple - 1CF\
1 t c'l Culture Prcprtiviwi
as ST-Cella hat s e-.t<;vl ono da; prior to urr j point testing,
bi Cchs counted and SvCtkd ai appropriate cotwnL'Jtwrs m % weU tissue tuiSnre
pi ale tc Itln"., CfntliisrH r>c«\fJaye> ST-ceih ihe !olio\' ni;- a:(> NUTf
Seed hetv-tx n 1 *0 0t >0 i Jl< pet well eta ')t>-w ;11 p)««; o\crnigiit
Ptmr to inoculation fot 1 t rcme\w ncriis and w \sh cells vMh 200 pf of
t omplefe Growh medium
2. Sample Preparation
3* R«no»t* s impk positive and rtegaiive centmi \ > a I *iom appropriate jntuitilw.
b) Work Sivps II',.1 c w.th nt£, ds\e ^onunl<. fitji. then tess sample: and then
tvMtisc ci>n»r>xts hi 'jintf nos-s conttmirsition K'twern ^irrplcs.
cj ScnaUv j.'utt t.ampWposmve iocu.kInejMiue contu'f to exnntfKm i>siii<: j 10-
fold \r»d4>r d dilution scheme I ^ Union end pomt will he ha^d on ih; hfork
conwntratu>r ot TCt V a>Kt tbi, prior ts.cos.ety dutmg ihc pit nous Mm. DOiut
l>t. I \ Lorr.pk'i..' C-iowh Vkdium as tlit dilatn* apJ pmurat dilutee is in ,i
deep wdl pHk' on tcc
•\) Remove i\a^( ti'>in ST-cc ll> plated m ^ wcl) pi »ks.
h> Irtcrjlatf v«its dilutions prctMivd m step r ' b
c; Record inocuUa>>n tunc tot each rtate and itKiib-ste a 37 °C and 5% COi for 48
hours.
dl Observe T)]ate,^ for cvtonfithic Hfcci lOPPs 48 hour posr-'&ociitoien
e) Calculate tnwn IC IDm ittevwed .'ad jty ft iDm> rt\ut,i Tceo'-'crca mitsu^ itmc
6.0 References
A BiO >V -ti'i* Sup lor tk. Opa?tmii a.irt Mamienance of i B^tloycal ("abnie*
K. Kio tmui hOP i<« Recap. Mtttnt'e. f w\p(wt, ^hipii'tiu t1" Pink>t«iv J1 ?¦1
C. RIO >VM >P f n the I'Se 'jid M^mlcr-aiue ot Imc nfc^tois
7.0 Revision History
None. TTiis i;- unjmn! \'ersion.
Study Number 66087
Page 68 of 67
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/2016
Miscellaneous Operating Procedure (MOP* to F\ aluale the PLr\iste»u'o
of'lC£\ . MS2 bacteriophage, and Phi6 bacteriophage in Landfill 1 om bate
tJdie-
Nola Ba<-s
CHRM »«fA*1 v -\{*|'« CtS UH.OKH S Jl.d !$K «()KV
-¦¦¦¦ \ ¦ ;
Rr,\ «t A*®5 b>" J- *t_;. _ _ ®^-"r " hj-L
Vejjjn Ho^4ro
Pi man,>1 lii/e^tig.v.vx
R.en^\\ cd hv ^
!);in LorcK
TjsV Urdu Lsjwior
. t
Hcv icwcJ bv
vehjt\ Wiii^
Ou*'U\ •ViMUTjUln.
/ '« 5'^ ^JUlA'i^
Effective Date: <7y ' **f 9pf u
T\ ji> K*t
Page 69 of 67
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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