EPA/600/R-18/077 | August 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
&EPA
Viral Persistence in
Landfill Leachate
Office of Research and Development
Homeland Security Research Program
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EP-C-15-002
TO-0002
August 2018
Testing and Evaluation Report
Evaluation of Viral Persistence 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
Worth Calfee, National Homeland Security Research Center
Mario Ierardi, Office of Land and Emergency Management
Susan Thorneloe, National Risk Management Research Laboratory
Doug Hamilton, ORISE Research Participant, National Homeland Security Research
Center
Battelle Memorial Institute
Megan Howard
Nola Bliss
Teara Stickey
Ryan James
Zachary Willenberg
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Table of Contents
Page
Disclaimer ii
Acknowledgments iii
Abbreviations/Acronyms vi
Executive Summary 1
1.0 Introduction 2
2.0 Approach 4
2.1 Task 1: Landfill Leachate Acquisition and Characterization 4
2.2 Task 2: MS2 and Phi6 Persistence Testing 5
2.3 Task 3: ZIKV Method Testing 6
2.4 ZIKV Persistence Testing 8
2.5 Heterotrophic Microbial Counts in Leachate 9
3.0 Procedures 9
3.1 Landfill Leachate Acquisition and Characterization 9
3.1.1 Landfill Selection 9
3.1.2 Logistics 11
3.1.3 Leachate Collection 12
3.1.4 Heterotrophic Microbial Counts 14
3.2 Virus Propagation 14
3.2.1 Zika virus Propagation 14
3.2.2 Bacteriophage Propagation 15
3.3 Persistence Testing 16
3.3.1 Sample Preparation 17
3.3.2 Incubation and Analysis 18
3.3.3 Data Analysis and Interpretation 23
4.0 Quality Assurance/Quality Control 25
4.1 Performance Evaluation Audit 25
4.2 Technical System Audit 25
4.3 Data Quality Audit 25
4.4 QA/QC Reporting 26
5.0 Results 29
5.1 Landfill Leachate Characterization 29
5.2 ZIKV Persistence 32
5.3 MS2 Persistence 35
5.4 Phi6 Persistence 40
5.5 Evaporation 43
6.0 Discussion 44
7.0 References 48
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List of Figures
Page
Figure 1. Leachate Accumulation Area at Landfills A, B and C 13
Figure 2. TCID50 Titer Calculation 21
Figure 3. ZIKV Persistence (A) and Decay (B) at 12 °C 34
Figure 4. MS2 Persistence at 12 °C 38
Figure 5. MS2 Persistence at 37 °C 39
Figure 6. Phi6 Persistence at 37 °C 42
List of Tables
Executive Summary Table 1. Persistence of Various Viruses in Three Landfill Leachates .Error!
Bookmark not defined.
Executive Summary Table 2. Decay Rates of Viral Agents in Three Landfill Leachates Error!
Bookmark not defined.
Table 1. Test Matrix for MS2 and Phi6 Persistence Evaluation in Landfill Leachates 6
Table 2. Test Matrix for ZIKV Persistence Evaluation in Landfill Leachates 8
Table 3. Landfill Characteristics 10
Table 4. Leachate Collection and Analysis Date Summary 11
Table 5. Sample Analysis Time Points 19
Table 6. Data Quality Objectives 26
Table 7. Landfill Leachate Analysis Data 31
Table 8. ZIKV 12 C Data Table 33
Table 9. ZIKV D-Values and Persistence 33
Table 10. MS2 37 C Data Table 36
Table 11. MS2 12 C Data Table 36
Table 12. MS2 D-values and Persistence 37
Table 13. Phi6 37 T Data Table 40
Table 14. Phi6 D-Values and Persistence 41
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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
BEI Resources Biodefense and Emerging Infections Research Resources Repository
BOD biological oxygen demand
BSL Biosafety Level
BWA biological warfare agent
°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
COC chain of custody
COD chemical oxygen demand
DHL DHL Analytical, Inc.
DMEM Dulbecco's Modified Eagle's Medium.
DNA Deoxyribonucleic acid
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)
I-WASTE Incident Waste Decision Support Tool
L liter(s)
LB Luria Bertani
LB A Luria Bertani Agar
LBB Luria Bertani Broth
LBTA Luria Bertani Top Agar
LDPE low density polyethylene
In natural logarithm
LOD limit of detection
log base-10 logarithm
|im micrometer(s)
mg milligram(s)
mM millimolar
min minute(s)
|iL microliter(s)
mL milliliter(s)
MERS Middle East Respiratory Syndrome
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MOP
miscellaneous operating procedure
MS2
Enterobacteria phage MS2
MSW
Municipal Solid Waste
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
P. syringae
Pseudomonas syringae
QA
quality assurance
QAPP
quality assurance project plan
QC
quality control
RCF
relative centrifugal force
RCRA
Resource Conservation and Recovery Act
RG3
Risk Group 3
RNA
ribonucleic acid
SARS
Severe Acute Respiratory Syndrome
TCIDso
50 % Tissue Culture Infectious Dose
TDS
total dissolved solids
TGEV
Transmissible Gastroenteritis Virus
TO
task order
TOC
total organic carbon
TOCOR
Task Order Contracting Officer's Representative
TOL
Task Order Leader
TSA
Tryptic Soy Agar
TSB
Tryptic Soy Broth
TSB-Mg
Tryptic Soy Broth supplemented with magnesium
TSS
total suspended solids
TSTA
Tryptic Soy Top Agar
TSTA-Mg
Tryptic Soy Top Agar supplemented with magnesium
USD A
U.S. Department of Agriculture
VEEV
Venezuelan Equine Encephalitis Virus
VERO
Cercopithecus aethiops (African Green Monkey) Vero E6 cell line
ZIKV
Zika virus
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Executive Summary
Waste generated during natural disease outbreaks (i.e., the 2014-2015 Ebola virus outbreak),
unintentional releases, or following an intentional release (i.e., a terrorist attack) will likely be
initially decontaminated/treated and then disposed as waste in a landfill. It is possible that residual
biological agent (e.g., a virus) could migrate from the waste in the landfill into the landfill leachate,
presenting a potential exposure risk to landfill workers or workers handling the leachate after it is
removed from the landfill. To fill knowledge gaps surrounding virus persistence in landfill
leachate, this study assessed the potential impacts from the waste management option of landfill
disposal.
To determine how long viral pathogens introduced into landfills could potentially survive, this
study performed tests under laboratory conditions to evaluate the persistence and decay of viral
infectivity in samples of landfill leachate from three currently-operating municipal solid waste
landfills. This study was performed using viral surrogates of biological warfare (BW) agents and
Zika virus (ZIKV), a human pathogen recently responsible for a global pandemic. Surrogate agents
used in this study include MS2 bacteriophage (MS2), and Phi 6 bacteriophage (Phi6), surrogates
commonly used in decontamination testing, that were used during the 2015-2016 landfill
persistence study [1],
Data indicated viral surrogate agents (ZIKV, MS2 and Phi6) can persist for days to months in
landfill leachates, and viral persistence varies according to environmental conditions. MS2
persisted longer at mild temperatures (12 °C) and decayed far more rapidly at warmer temperatures
(37 °C). Study results suggest that viruses may persist in landfill leachates for days to months
under the mild conditions present in most of the U. S., and that viral structure or type plays a critical
role in survival time. Should waste containing residual agent be disposed 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.
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1.0 Introduction
The 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. The EPA is also a lead agency for overseeing the Emergency Support Function 10 (Oil
and Hazardous Materials Response Annex) that coordinates the federal response to oil and
hazardous material incidents, which would likely generate wastes similar to the wastes investigated
in this project. Hazardous materials include accidentally or intentionally released chemical,
biological, or radiological substances. The EPA is also designated as a support Agency to 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.
This study evaluated the persistence and decay rate of viruses and viral surrogates in landfill
leachate. Waste generated during natural outbreaks (i.e., Ebola virus waste), cleanup of
unintentional releases, or following a terrorist attack involving BW agents may be placed in MSW
landfills. The ultimate fate of the BW agent(s), in this case ribonucleic acid (RNA) viral BW
agents, is of concern. Although these materials will be decontaminated, large quantities,
heterogeneous materials, and laboratory limitations may lead to incomplete decontamination and
residual biological contamination. It is possible that residual biological agent (e.g., a virus) could
migrate from the waste in the landfill into the landfill leachate, presenting a potential exposure risk
to landfill workers or workers handling the leachate after it is removed from the landfill. To
evaluate whether infectious viruses could survive once disposed in landfills, laboratory persistence
tests were performed to measure the decay rate of viral infectivity in landfill leachates.
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This study aimed to evaluate survivability and/or persistence of viral agents in landfill leachate.
Data from this study provide a good framework for estimating and determining the fate of residual
viruses that may be placed into a landfill.
This study used three viral agents as surrogates for highly pathogenic BW viral agents in three
different leachate samples. Three RNA viruses were selected by Battelle and the EPA: one
enveloped mammalian virus (ZIKV), one enveloped bacteriophage (Phi6) and one non-enveloped
bacteriophage (MS2). These viruses were selected because they represent three common classes
of viruses, all of which can easily be manipulated in biosafety level (BSL)-2 facilities and include
an existing pandemic virus (ZIKV) and two well-established surrogates commonly used in
decontamination testing (MS2 and Phi6). This study provides confidence in the ability to estimate
a temporal range representing the fate of infectious viral BW agents in different landfill leachates
at different operating temperatures, potentially reducing the need for sampling and analysis of
these difficult matrices.
ZIKV is a flavivirus, in the family Flaviviridae, that emerged in North and South America from
Africa in 2015-2016. ZIKV is an enveloped single stranded, positive sense RNA virus transmitted
primarily by th eAedes species of mosquitoes. While the majority of human ZIKV infections cause
mild symptoms (influenza-like illness) or are subclinical, severe effects of infection can manifest
(e.g., Guillain-Barre syndrome, microencephaly). ZIKV is closely related to several threat agents,
including West Nile virus, Japanese encephalitis virus, Dengue virus and the Yellow Fever virus
[2], ZIKV was used here to represent human RNA viruses, vector-borne viruses, and emerging
human enveloped RNA viruses similar in structure to ZIKV (e.g., Influenza, Chikungunya virus,
Severe Acute Respiratory Syndrome (SARS) coronavirus). Many emerging viruses are associated
with serious or lethal human disease, for which preventive or therapeutic interventions may not be
available [3], Many of these agents are classified as Risk Group 3 (RG3) agents, as they represent
high individual risk and therefore must be manipulated in BSL3 facilities. ZIKV is a Risk Group
2 agent, and an appropriate test agent to represent emerging RNA viruses without incurring the
increased risks and costs associated with RG3 agents.
Phi6 is an enveloped RNA bacteriophage that infects Pseudomonas syringae. Phi6 contains a
tripartite double-stranded RNA genome and was used in this study as an intermediate
stability
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enveloped RNA virus [4, 5], MS2 is a non-enveloped single-stranded RNA virus that infects
Escherichia coli and was used as a surrogate for non-enveloped human RNA viruses, including
poliovirus, norovirus, parvovirus, rotavirus, hepatitis A and E viruses, and coxsackievirus [4],
In 2015-2016, the survival and persistence of three viral surrogates (Transmissible Gastroenteritis
virus [TGEV], Phi6 and MS2) were evaluated in landfill leachate. The 2015-2016 study showed
that incubation temperature, viral structure and leachate source all contributed to viral inactivation
in landfill leachate [1], In 2017, this study was repeated using three viral surrogates: repeat testing
with Phi6 and MS2, and initial testing with Zika virus. This report addresses results from the 2017
study only. Previous data and results can be found in the earlier 2015-2016 landfill persistence
study [1],
Study deliverables include 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.
2.0 Approach
The study was divided into the following five tasks consecutively performed.
Task 1: Landfill Leachate Acquisition and Characterization
Task 2: MS2 and Phi6 Persistence Testing
Task 3: ZIKV Method Testing
Task 4: ZIKV Persistence Testing
Task 5: Microbial Activity in Leachate
2.1 Task 1: Landfill Leachate Acquisition and Characterization
Three landfill facilities were used to support this evaluation. These facilities were selected by
Battelle under the guidance and approval of the EPA Task Order Contracting Officer's
Representative (TOCOR) and met the following acceptance criteria:
• "Large" in size (capacity of > 2.5 million tons MSW) and subject to Clean Air Act
(CAA) requirements;
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• 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 Battelle staff to collect leachate from an accessible leachate
collection point representative of leachate across the landfill.
Approximately 20 liters (L) of landfill leachate was collected by Battelle or landfill staff and
returned to Battelle's laboratory. A portion of each leachate was sent to an analytical laboratory
for characterization and analysis. The remaining portion was stored under refrigeration at Battelle
and used for method development and 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. Per EPA direction, 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, and all were
approved by EPA. The landfills used in this study were identical to those used in a previous study
of viral persistence in the 2015-2016 landfill persistence study [1],
2.2 Task 2: MS2 and Phi6 Persistence Testing
Persistence of MS2 and Phi6 was evaluated in the three landfill leachates over time (12 °C and/or
37 °C) using an approach and methods described in Miscellaneous Operating Procedure (MOP) #
2016-001-00 (MOP in Appendix A, approach and procedures discussed in Section 2.2 and Section
3.0). To calculate persistence and decay of MS2 and Phi6 viruses in landfill leachate, decay (kill)
curves were generated from persistence data. Data were acquired from time course persistence
tests where samples of each agent were prepared in each leachate (4 milliliter [mL] aliquots of
leachate were spiked with virus) and incubated at test temperatures. Triplicate test samples,
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triplicate positive controls (MS2 or 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 throughout a time-course (several days to eight weeks);
analysis was halted after seven time points after To, or once samples were below the limit of
detection for two consecutive time points (Table 1).
Testing was designed to capture decreasing viral infectivity during each time course; time points
were selected to capture viral decay across at least three sequential time points. Initially, time
points were selected to best capture viral decay on the day-to-week timescale for 12 °C persistence
testing, and hour-to-day timescale for 37 °C testing. Assay results for MS2 and Phi6 were obtained
24 hours after samples were analyzed, allowing informed day-to-day decision making by the Task
Order Leader (TOL) regarding subsequent time points, and allowing modification of sample time
points as needed. However, MS2 and Phi6 were expected to inactivate rapidly at 37 °C, thus
preventing informed day-to-day decision making. During the 37 °C persistence testing, time points
were chosen to accommodate rapid viral decay over the first one to three days (first five time
points), and the final time points were refined using data gathered over those initial time points.
Details of sample preparation, incubation, quantitation assay and data analysis are described in
Section 3.0 and results discussed in Sections 5.3 and 5.4.
Table 1. Test Matrix for MS2 and Phi6 Persistence Evaluation in Landfill Leachates.
I'siriimclcr
Description
Virus Surrogates
Phi 6 (enveloped bacteriophage)
MS2 (non-enveloped bacteriophage)
Landfill Leachate
Leachate from 3 different landfill facilities
Incubation Temperature
37 °C for Phi6a; 12 °C and 37 °C for MS2
Time Pointsb
0 (baseline), 3, 7, 14, 21, 28, and 42 days @ 12 °C (MS2)
0, 2, 4, 8, 24, 30, 56, and 96 hours @ 37 °C (Phi6)
0, 4, 8, 24, 30, 56, 96 and 168 hours @ 37 °C (MS2)
a Phi6 persistence was not measured at 12 °C, only a 37 °C persistence test was conducted.
b Estimated times were subject to change throughout test; amended as deemed necessary based on results of previous time points.
2.3 Task 3: ZIKV Method Testing
This task involved preparing and establishing a virus quantitation assay (mammalian cell-based
TCID50 assay) for ZIKV, assessing each landfill leachate for the presence of indigenous viral
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agents that could cause false positive results in the assay, identifying whether landfill leachate can
inhibit the TCID50 assay, and developing sample analysis procedures to minimize or eliminate
assay inhibition. This task was not intended to be an exhaustive method optimization activity but
to develop a general recovery method for generating accurate, repeatable, defensible virus
persistence data from landfill leachates using standard methods.
Several methods were evaluated to recover live ZIKV from each leachate while minimizing
cytotoxicity. Landfill leachate is a complex matrix, consisting of numerous chemical and
biological constituents that have the potential to inhibit biological processes. Therefore, an initial
evaluation of each leachate was performed to measure inhibitory or cytotoxic effects on
mammalian cells used for ZIKV quantification (TCID50 assay). The initial evaluation tested end-
point dilution, and the secondary evaluation tested short low-speed centrifugation of leachate to
identify (and reduce) any potential assay inhibition/interference and/or cytotoxic effects on cell
monolayers. Additional methods to reduce leachate-induced assay interference were proposed
(including filtration and precipitation during sample processing), but short low-speed
centrifugation adequately reduced the majority of the cytotoxic effects of leachate on the ZIKV
TCID50 assay. Despite incorporating a centrifugation step to minimize leachate-induced
cytotoxicity, application of leachate B to cells caused cytotoxic effects and interfered with ZIKV
recovery. No ZIKV was recoverable from leachate B during this study.
In addition to inducing cytotoxicity, leachate may harbor indigenous viruses that could interfere
with detecting surrogate agents, potentially generating false positive results in the TCID50 assay.
Each leachate was screened for indigenous viruses in the TCID50 assay, and no cytopathic effect
(CPE, sign of viral infection) was identified. No other overt signs of indigenous viral infection
were observed from leachates A and C. However, this test was restricted to observing cells
microscopically and was not intended to be an exhaustive test. Landfill leachate may yet harbor
viruses that were not detected in this screen.
Once leachate-induced cytotoxicity was evaluated, the ZIKV TCID50 method was refined and
demonstrated to be an effective method to efficiently and accurately determine ZIKV
concentrations in leachates A and C. Discussions between the TOCOR and TOL resulted in the
de-prioritization of method refinement to recover ZIKV from leachate B. Final methods used in
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the ZIKV persistence study were written into MOP 2017-001-00 (Appendix B) and included
sample preparation, incubation, and processing procedures and were submitted to the EPA
TOCOR. Methods are discussed in detail in Section 3.0.
2.4 ZIKV Persistence Testing
ZIKV persistence was evaluated in two landfill leachates over a one-week incubation period at 12
°C, using the approach described in Section 2.3 and procedures described in Section 3.0 (refer also
to MOP 2017-001-00 in Appendix B). Persistence and the rate of ZIKV inactivation (decay rate)
in landfill leachate were calculated from decay (kill) curves generated from persistence data.
Results were gathered during ZIKV persistence testing, where ZIKV-spiked leachates were
prepared (4 mL leachate spiked with virus) and incubated at 12 °C. Triplicate test samples,
triplicate positive controls (ZIKV spiked into DMEM without fetal bovine serum [FBS]) and
negative controls per leachate (unspiked leachate) were removed and analyzed for viral titer at
each time point. Samples were analyzed over one week. The analysis included up to four time
points after To.
Table 2. Test Matrix for ZIKV Persistence Evaluation in Landfill Leachates.
I'iinimeter
Description
Virus Surrogates
ZIKV flavivirus (enveloped)
Landfill Leachate
Leachate from 2 different landfill facilities
Incubation Temperature
12°Ca
Time Points'3
0 (baseline), 6, 12, 24, 48 and 96 hours @ 12 °C
a ZIKV persistence was not measured
at 37 °C, as its survival at 12 °C suggested that it would not survive lor more than several
hours at 37 °C.
b Represents actual time points tested.
The persistence time course was designed to capture decreasing ZIKV viability, so time points
were selected to capture viral inactivation across at least three closely clustered sequential
sampling times. Rapid decay of ZIKV in leachate was expected, so time points were chosen to
capture infectious ZIKV over five days. As the ZIKV TCID50 has a seven-day read-out time
(results were acquired seven days after each time point), real-time day-to-day decision making was
not possible. Time points for ZIKV analysis were selected using an assumption of rapid decay
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over the first two to three days. Details of sample preparation, incubation, quantitation assay and
data analysis are described in Section 3.0.
2.5 Heterotrophic Microbial Counts in Leachate
Heterotrophic microbial counts intrinsic to each leachate were analyzed using standard plate count
methods. Heterotrophic bacterial and fungal concentrations in each leachate were determined prior
to persistence testing at 9-16 days after leachate acquisition (Table 4). Microbial counts were
qualitatively assessed by enumerating bacterial and fungal colonies on nonselective media for
heterotrophic bacteria (Tryptic Soy Agar (TSA), a nonselective medium) and heterotrophic fungi
(Potato Dextrose Agar (PDA), 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). Colony morphology was recorded during both heterotrophic plate counts.
However, recovered colonies were not identified or characterized. Results are described in Section
5.
3.0 Procedures
3.1 Landfill Leachate Acquisition and Characterization
3,1,1 Landfill Selection
Between July and September 2015, Battelle and the EPA jointly developed criteria for evaluating
and selecting landfill facilities to acquire leachate samples. The criteria for selecting appropriate
landfills appropriate for leachate collection was determined through discussions with EPA while
identifying suitable locations. The EPA-approved criteria required that landfills selected for this
study be:
• RCRA Subtitle D facilities (i.e., non-hazardous MSW landfill);
• "Large" in size (i.e., capacity of at least 2.5 million tons of waste);
• Operational for at least 5 years;
• Subj ect to CAA requirements;
• Known to have a steady leachate composition/quantity (demonstrated by available
historical monitoring data);
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• In regulatory compliance and therefore not under any enforcement action (relative to any
local, state, or federal regulations); and
• Willing/able to provide EPA/Battelle access to their facility for collection of a
representative leachate sample (without use of invasive sampling such as installation of a
monitoring well).
In 2015, a list of suitable landfills was identified using the EPA's Incident Waste Decision Support
Tool (I-WASTE) [6] (V6.4; http://www2.ergweb.com/bdrtool/login.asp accessed March 15.
2018), 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 Battelle project personnel
located in Columbus, Ohio. After multiple potentially suitable landfills were identified, Battelle
contacted the short-listed facilities to obtain commitment from the owners/operators to participate
in the study. EPA requested that a landfill facility actively accepting animal carcasses be included
in the study. Three landfills were selected that met the primary selection criteria, including landfill
B which accepts animal carcasses; all selections were approved by EPA. Basic characteristics of
each landfill are provided in Table 3.
Table 3. Landfill Characteristics
C h:ir:ictoristic
l.iiiuirill A
i.iiiuiriii is
Liiiuiriii c
Waste Acceptance
Rate3
~3,200b
3,500 to 5,000c
1,400
Footprint
100 acres
283 acres
168 acres
Year Opened
1997
1995
1995
Expected Closure Date
2023 or 2024®
2030 to 2045
Information not
provided
Gas collection system
Yes
Yesf
No
a Measurement in average tons of waste/day.
b Landfill A waste acceptance in 2014.
c Overall, -1,000,000 tons of waste received in 2014.
d Area in acres permitted to accept waste.
e Landfill A is pursuing an expansion which could extend life by 25 years.
f Landfill B contains -190 gas collection wells/points.
Per the direction of EPA, 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
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criteria, including landfill B which accepts animal carcasses, and all were approved by EPA.
Samples collected in 2017 were obtained from the identical landfills used in the 2015-2016 landfill
persistence study [1],
3.1.2 Logistics
Leachate sampling was conducted by either Battelle or landfill staff. In 2017, Battelle staff
collected leachate samples at landfill A, and landfill staff collected leachate samples at landfills B
and C.
Collection kits were transported to the sites, samples were collected, kept cold and returned to
Battelle within 24 hours of collection. Collection kits included coolers, sampling equipment, and
supplies (low density polyethylene (LDPE) containers, ice-packs, and safety supplies). Additional
supplies included chain of custody (COC) forms, labels, Ziploc® bags, nitrile gloves, safety glasses
or face shields, paper towels, and trash bags. Samples were transported on ice back to Battelle in
Columbus Ohio, where they were processed as directed for analysis by the selected study
laboratory, DHL Analytical, Inc. (DHL) of Round Rock, Texas. Samples were enumerated for
microbial activity at Battelle. Landfill leachate was collected and analyzed as summarized in Table
4.
Project health and safety was governed by a project-specific activity hazard analysis (AHA)
approved by the Battelle health and safety manager. The AHA was strictly adhered to while
performing landfill leachate sampling activities, including the use of appropriate personal
protective equipment (PPE).
Table 4. Leachate Collection and Analysis Date Summary
i.iiiuiriii
l-:icililv
Dsite
Collected
Dsite
Relumed to
liiillelle
l):ite
SenI to
DHL
Time Stored ;il
4 "C Prior to
Shipment
l$iolo<>ic;il
Kniinienilion
Time Stored ;il
4 "C Prior to
Kniimeriition
Landfill A
May 17,
2017
May 17,
2017
May
25,
2017
8 days
June 2, 2017
16 days
Landfill B
May 22,
2017
May 23,
2017
3 days
10 days
Landfill C
May 24,
2017
May 24,
2017
2 days
9 days
Page 11
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3.1.3 Leachate Collection
All three leachates were collected within a seven-day window (May 17-24, 2017). Leachate was
collected directly from an accessible leachate collection point into a 20-L container. The container
was filled and sealed with minimal headspace to avoid oxygenation of the leachate. All samples
were labeled with the collection time, date and location at the point of collection. Samples were
collected as described.
Landfill A . On May 17, 2017, Battelle staff collected samples from a leachate accumulation area,
consisting of three above-ground 8,976 L (34,000 U.S. gallons) storage tanks situated in a cement
containment area (Figure 1). A 3-inch discharge line was connected to the tanks and purged at a
slow-to-medium flow rate for approximately 1-2 minutes prior to collecting the sample. The
leachate appeared gray and without significant particulate matter, with an odor like hot wet asphalt.
After the sample was collected, the container was wrapped in a carrier bag with adsorbent paper,
the bag was closed and placed in a cooler with blue ice.
Landfill B: On May 22, 2017, landfill staff collected a sample of leachate at the leachate sump
area (Figure 1) at the main lift station. Sampled leachate appeared light gray with significant
particulate matter. After filling the 20 L-container, the sample was wrapped in a carrier bag with
adsorbent paper, the bag was closed and placed in a cooler with blue ice.
Landfill C. On May 24, 2017 landfill staff collected a sample of leachate at the leachate collection
area. Leachate was collected from an 84,000-gallon tank storage location surrounded by a
containment area (Figure 1). The leachate was collected through a 3-inch discharge line (controlled
by a ball valve) connected to the tank. The line was purged briefly (15-30 seconds) prior to
collection. The leachate appeared golden brown with no evident particulate matter. After filling
the 5-gallon (20 L) sample container, the container was wrapped in a carrier bag with adsorbent
paper, the bag was closed and placed in a cooler with blue ice.
Sample Custody and Shipping. Leachate samples were transported on ice to Battelle's
Columbus, Ohio headquarters within 24 hours of collection. Upon sample receipt, samples were
stored at 4°C and strict COC procedures were followed while samples were being prepared and
shipped to DHL. Leachate samples were shipped to DHL on May 25, 2017 via expedited overnight
Page 12
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shipping in coolers with blue ice, arriving at DHL on May 26, 2017. All samples were shipped
within 10 days of sample collection (Table 4). The remaining samples in LDPE containers were
relinquished to the custody of Battelle's virology laboratory staff in Columbus, Ohio for use in
Tasks 6 and 7 of the study. These samples were maintained at 4 °C.
Sample Analysis. In accordance with the project quality assurance project plan (QAPP), leachate
from each of the three landfill facilities selected for the study was submitted to DHL for analysis
of the following parameters: total alkalinity, ammonia, anion concentration (chloride, nitrate,
sulfate), biological oxygen demand (BOD), chemical oxygen demand (COD), metal concentration
(calcium, iron, magnesium, manganese, potassium, sodium, zinc), pH, total dissolved solids
(TDS), total organic carbon (TOC), and total suspended solids (TSS).
Landfill B
Landfill
Landfill C
Figure 1. Leachate Accumulation Area at
Landfills A, B and C
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The analytical data received from DHL are summarized in Section 5.0, along with characteristics
observed at the time of sample collection and/or receipt at Battelle's Columbus Laboratories. Data
from the 2015 collection are included for completeness. The analytical data report provided by
DHL can be provided upon request.
3,1,4 Heterotrophic Microbial Counts
Samples were analyzed for pH and microbial load on June 2, 2017. Standard plate count assays
were used to enumerate microbial numbers. Heterotrophic bacterial enumeration was performed
on TSA and heterotrophic fungal enumeration on PDA. Samples from well-mixed leachate
collection jugs were serially diluted with PBS, and 100 |iL of each dilution was plated in triplicate.
Samples were incubated at room temperature (27 °C ± 2 °C). Plates were monitored daily, and
counted at 1, 2, 4 and 7 days post-inoculation. Microbial colonies were counted from plates over
time. Colony morphology on each medium was noted. However, colonies were not identified or
characterized. Microbial enumeration was calculated as CFU/mL from plate counts recorded
between one and four days post-inoculation using Equation 1 [Eq (1)]:
Microbial Enumeration is represented as CFU/mL;
Dilution is the dilution from the neat sample added to each plate for the calculation.
3.2 Virus Propagation
3,2,1 Zika virus Propagation
Zika virus (ZIKV) used in this study was originally acquired from the Biodefense and Emerging
Infections Research Resources Repository (BEI Resources). Zika virus strain MR-766 (BEI
Resources, NR-50065) was propagated from a Battelle stock prepared in low passage
Cercopithecus aethiops normal kidney cells (Vero E6, American Type Culture Collection (ATCC)
CCL-81).
Eq (1): Microbial Enumeration
{Average number of colonies at dilution Y)
0.1 mL x DilutionY
where:
Page 14
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Virus stocks were prepared in DMEM with 4.5 grams (g)/L glucose supplemented with 3.7 g/L
sodium pyruvate, 4 milliMolar (mM) L-glutamine, 10% FBS, 100 units/mL penicillin and 0.1
milligrams (mg)/mL streptomycin. Virus was adsorbed onto cells for one to two hours at 37 °C,
5% CO2 and infected flasks were rocked every 15 minutes. Additional growth medium was added
to each flask and infected cells were incubated at 37 °C/5% CO2 until cells exhibited 70-90% CPE
(approximately five days post-inoculation). Crude virus stocks were harvested from the
supernatant via a short clarifying centrifugation step (4 °C, 400 relative centrifugal force (RCF)
for 20 min), the supernatant was aliquoted and frozen at -75 to -80 °C. The Zika MR-766 stock
titer used in this study was 4.6x 106 TCIDso/mL via a TCID50 assay on Vero E6 cells (see Sections
3.2.1 and 3.3).
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.
MS2 was propagated in log phase E. coli broth culture in Luria Bertani (LB) Broth (LBB), added
to molten LB Top Agar (LBTA) and overlaid onto LB 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 (containing MS2-infected E. coli cells)
was scraped off, resuspended in 15 mL SM buffer, and clarified via centrifugation (7000 RCF, 15
minutes). Supernatant was filtered (0.2 jam) to remove residual bacterial cells, and filtered
supernatant was designated the MS2 master stock (lot number MS2091515) and stored in 0.5 mL
aliquots at <-70°C. MS2 master stock titer was 4.0><109 plaque forming units (PFU)/mL via
standard plaque assay.
Phi6 was propagated in P. syringae. Initially, a 100 |iL aliquot of the EPA Phi6 stock was
suspended in 30 mL TSB supplemented with magnesium (TSB-Mg). Suspended Phi6 in TSB-Mg
was added to 6 mL of a 24-hour P. syringae culture (in TSB-Mg) and 90 mL of molten Tryptic
Soy Top Agar (TSTA) supplemented with magnesium (TSTA-Mg). The suspension was gently
mixed and overlaid on each of approximately 30 TSTA-M g plates (4 mL per plate) and incubated
Page 15
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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 liquid/overlay removed by gently swirling each plate and
pooling the suspension. The Phi6 stock was filtered (0.2 micrometer [|im]) to remove residual
bacterial cells. Filtered supernatant was designated the Phi6 master stock (lot number
PHI6092516) and stored in 1 mL and 5 mL aliquots at <-70 °C. Phi6 master stock titer was
1.8xl010 PFU/mL via standard plaque assay.
3.3 Persistence Testing
Viral persistence in landfill leachate was evaluated in individual spiked samples of leachate (spiked
with a known quantity of virus) dispensed into replicate screw-top vials and statically incubated
across the time course for each virus (time points summarized in Table 1 and detailed in Table 5).
ZIKV-spiked samples were incubated at 12 °C, Phi6-spiked samples were incubated at 37 °C, and
MS2-spiked samples were incubated at 12 °C and 37 °C. ZIKV virus persistence was measured
only at 12 °C. Data from the previous 2015-2016 landfill persistence study with TGEV suggested
that the survival of mammalian enveloped RNA viruses at 37 °C would be less than five days [1],
Throughout the incubation period, triplicate samples were assayed at the initiation of testing (To)
and at each additional time point. ZIKV decay was analyzed at To and up to four additional time
points; MS2 and Phi6 decay was analyzed at To and up to seven additional time points.
ZIKV spiked samples were assayed using an end-point dilution TCID50 assay. Briefly, each
replicate sample was serially diluted and plated on 80-100% confluent Vero E6 cells. The TCID50
assay is read via CPE, measures infectious virus, and is used to calculate the dilution of the virus
at which 50% of cell cultures are infected. MS2- and Phi6-spiked samples were assayed using a
standard plaque assay. Triplicate samples were serially diluted in PBS, and dilutions were used to
infect either E. coll (MS2) or P. syringae (Phi6), followed by the plaque 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 infectious virus was unable to be detected.
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.
Page 16
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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 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 or used the same day.
3.3.1.1 Test Samples
ZIKV, MS2 and Phi6 samples were prepared in pre-dispensed leachate samples (Section 3.3.1).
Persistence test samples were separately prepared in triplicate for each virus for each leachate and
each incubation test condition.
ZIKV Samples: ZIKV virus was rapidly thawed and spiked into each 4mL leachate sample at a
final concentration of 5 /104 TCIDso/mL (80 microliters (|iL) ZIKV stock per 4-mL leachate
sample). Each sample was mixed by swirling and inverting three times. Samples were incubated
upright at 12 ± 1°C with no 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, then spiked into each 4-mL leachate sample at a final concentration of 1.25><106 PFU/mL
(40|jL of the working stock per 4mL leachate sample). 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 control sample was assessed per persistence time point.
Page 17
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3.3.1.3 Positive Controls
Positive control samples were generated in sterile media. ZIKV positive control samples were
generated in sterile incomplete medium (DMEM without FBS, with 4.5 g/L glucose supplemented
with 3.7 g/L sodium pyruvate, 4 mM L-glutamine, 100 units/mL penicillin and 0.1 mg/mL
streptomycin). MS2 and Phi6 samples were prepared in the PBS in the same manner and at the
same time, as the test samples (Section 3.3.1.1). ZIKV 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.
3.3.1.4 Evaporation Controls
To assess the role of evaporation, pre-dispensed leachate samples (Section 3.3.1) were spiked with
40-80 |iL of incomplete medium (DMEM without FBS, with 4.5 g/L glucose supplemented with
3.7 g/L sodium pyruvate, 4 mM L-glutamine, 100 units/mL penicillin and 0.1 mg/mL
streptomycin) or 40 |iL of PBS and incubated with each set of test samples. Each sample was
weighed on an analytical balance to the nearest 0.001 g at each time point, or once per analysis
day, and returned to the incubators. Deviations to evaporation measurements are discussed in
Section 4.0.
3,3,2 Incubation and Analysis
Test samples were statically incubated upright with negative (one per time point) and positive
(three per time point) samples and evaporation controls. Samples were incubated within incubators
set to operate at 12 °C or 37 °C ± 2°C. Incubator temperatures were monitored throughout the
incubation period using calibrated thermometers or via a calibrated electronic temperature
monitoring system.
Sample Analysis
Persistence testing on all three landfill leachates was conducted simultaneously. Initial persistence
testing evaluated MS2 and Phi6 at 37 °C, then ZIKV and MS2 persistence at 12 °C. Based on data
from the 2015-2016 landfill persistence study [1], rapid sample decay was expected at 37 °C for
Page 18
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MS2 and Phi6. Time points were pre-selected to capture the decay rate of MS2 and Phi6 at 37 °C,
and ZIKV at 12 °C. Sample analysis time points are detailed in Table 5.
The baseline (starting concentration) of each analysis set was determined by immediately
analyzing one set of samples (designated the To set). Per leachate type (A, B, and/or C), three test
samples, three positive control samples and one negative control sample were analyzed using the
appropriate MOP. All remaining test, positive control, and negative control samples were
incubated and analyzed over time (Table 5). Evaporation samples were weighed once each testing
day to the nearest 0.001 g to assess water loss throughout each time course.
Table 5. Sample Analysis Time Points
Viral A»enl
TcmpcraluiT
Sample Analysis Time Points
ZIKV
12 °C
0, 6, 24, 48 and 96 hours
MS2
12 °C
0,3,7, 14,21,28, 42 days
37 °C
0, 4, 8, 24, 30, 56, 96 and 168 hours
Phi6
37 °C
0, 2, 4, 8, 24, 30, 56, and 96 hours
3.3.2.1 ZIKV Sample Analysis
Per leachate, triplicate test samples and single negative control samples, along with triplicate
positive control samples, were analyzed using a TCID50 assay. The ZIKV TCID50 assay used was
adapted from a standard TCID50 assay developed at Battelle. All samples were clarified of
bacterial contamination by centrifugation at 8,000 RCF for 5 min at 4 °C, then serially diluted
using a two-fold and/or ten-fold dilution in complete medium (DMEM with 4.5 g/L glucose
supplemented with 3.7 g/L sodium pyruvate, 4 mM L-glutamine, 10% FBS, 100 units/mL
penicillin and 0.1 mg/mL streptomycin). Each dilution was added to a healthy 80-100% confluent
monolayer of Vero E6 cells across one row (12 wells) of a 96-well plate at 0.1 mL diluted
sample/well.
Calculation of Viral Titer: The lowest dilution able to be used without showing leachate-induced
cytotoxicity was 10"2. The calculated assay limit of detection (LOD) for the unadjusted TCID50
using base-10 logarithm (log) and natural logarithm (In) is 304 TCIDso/mL (2.5 log (TCIDso/mL)
or 5.7 In (TCIDso/mL) respectively). Due to cytotoxic effects on Vero cells caused by landfill
leachate, the LOD for the ZIKV TCID50 is 304 TCIDso/mL or 5.7 In TCIDso/mL. TCID50 assays
Page 19
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were incubated for seven days (37 °C, 5% CO2) and manually scored for CPE using a phase
contrast microscope. No bacterial or fungal contamination was identified. ZIKV 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 determined as TCID50 using the Reed-Muench method [7], TCID50
calculations are shown in Figure 2.
Viral persistence was calculated to be the time at which the linear decay rate intersects the assay
LOD. The LOD for the ZIKV assay is 304 TCIDso/mL, equivalent to 5.7 In (TCIDso/mL). Data
from all time points, viral persistence and D-values are shown in Section 5.0 and include virus
persistence in each leachate and the positive control matrix. Graphs of In PFU/mL versus time
were used to calculate decay rates. Decay rate and persistence calculations are detailed in Section
3.3.3
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1) Calculate the percentage of wells infected for each dilution. For example, if 4 of 12 wells
are scored infected:
4 infected wells # 1QQ% = ^ 3% 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 just above 50% of wells infected: (a)
The next highest dilution (more dilute): (b)
ii. Calculate proportional distance.
(% of wells infected at (a)) — 50%
(% of wells infected at (a)) — (% of wells infected at (b))
iii. Calculate TCID50.
Log(TCIDS0) = log(b) + log(dilution series) * PD
- (b) refers to (b) identified in step 2i.
- PD is the proportional distance calculated in step 2ii.
- Dilution series. This value 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 2. TCID50 Titer Calculation
3.3.2.2 MS2 and Phi6 Sample Analysis
Per leachate, triplicate test samples and single negative control samples, along with triplicate
positive control samples, were analyzed via plaque assay. Samples were inverted or vortexed at
moderate speed for 30 seconds and serially diluted using a tenfold dilution series in PBS. MS2
samples were diluted from neat sample, while Phi6 samples were vortexed briefly and centrifuged
at 12,000 RCF for two min prior to dilution. Sample serial dilutions were selected based on the
initial viral titer, testing results from the 2015-2016 landfill persistence study [1] and previously-
analyzed timepoints to select dilutions most likely to provide plaque counts within the
desired
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countable range of 25-250 plaques per 100 mm diameter plate. In general, positive control samples
were plated with 10"1 to 10"4 dilutions, and negative controls were plated without dilution.
Per dilution, triplicate suspensions of the following mixtures were prepared as follows:
1) MS2 analysis: 0.1 mL of a log-phase E. coli culture grown in LBB (approximately three-
six hours old), 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: 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 mixtures were overlaid onto LBA-S+A, and Phi6//J. syringae
mixtures were overlaid onto TSA supplemented with magnesium and 20 mg/mL ampicillin (TSA-
Mg+A20). Solidified plates were incubated overnight (MS2 at 37±2°C; Phi6 at 25±2°C), and 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 viral titer, which was
used to calculate decay rates and persistence values (described later in this section).
Calculation of viral titer: Viral titer was calculated from plates containing between 0 and 250
plaques. Viral titer was calculated by determining the PFU/mL in each sample using
PFU Mean PFU/plate
mL dilution x volume plated
PFU/mL values were converted to log PFU/mL and In PFU/mL and plotted versus time. Data from
each time point at all temperature tested, viral persistence and D-values are shown in Section 5.0
and include virus persistence in each leachate and positive control matrix. Graphs of In PFU/mL
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 LOD. The calculated LOD for the MS2
and Phi6 plaque assay is 10 PFU, equivalent to 1 log PFU/mL or 2.3 In PFU/mL. This value was
calculated using a detection limit of 1 PFU per 0.1 mL plated, equivalent to 10 PFU/mL and 40
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PFU/sample in a 4-mL leachate test sample. Decay rate and persistence calculations are detailed
in Section 3.3.3.
3.3.3 Data Analysis and Interpretation
Viral decay rate was determined by measuring the reduction in viral infectivity, and calculating
decimal reduction times (D-values, the time required for the viable concentration to be reduced to
10% of the starting concentration). D-value and 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 the natural log (In) of persistence titers over time.
Persistence curves were plotted as In PFU/mL recovered (for MS2 and Phi6) or In TCIDso/mL (for
ZIKV) versus time. A linear decay curve was fitted to all positive data points where infectious
virus was detected in at least two or three replicates, including the initial To time point.
Calculate D-value and persistence time: Linear regression of the data was generated using the
following formula:
y = mx + b
where:
y = natural log (viral concentration) [In (PFU/mL) or In (TCIDso/mL)]
m = slope
x = time (days or hours)
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 (days or hours) required for the rate of
linear decay to intercept the assay LOD using the following formula:
Page 23
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Cy~b)
X =
m
where
y = In (assay LOD)
b = y-intercept of linear decay rate
m = slope of linear decay rate
in which y equals:
5.7 In TCID5o/mL for ZIKV (equivalent to 304 TCIDso/mL or 1216 TCIDso/sample)
2.3 In PFU/mL for MS2 and Phi6 (equivalent to 10 PFU/mL or 40 PFU/sample)
Persistence (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
Quality assurance/quality control (QA/QC) procedures were performed in accordance with the
Testing and Evaluation II Program and task order (TO) quality documents for this evaluation.
4.1 Performance Evaluation Audit
The Battelle TOL performed Performance Evaluation (PE) audits to confirm compliance with
QAPP Table 6. PE audits for temperature were performed on June 10 and June 27, 2017 to confirm
compliance for temperature. The audit focused on incubation temperature for persistence samples,
mammalian cell culture and incubation of plaque and TCID50 assays.
In August and September 2017, micropipette calibration records were checked, and all but one
pipette were within their calibration due date. The out-of-date pipet was within five days of
calibration, used to plate mammalian cells during the ZIKV persistence assay and was recalibrated
without modifications. No impact was found for this finding.
Viral titer calculations (PFU and TCID50) were performed by the TOL, and calculations were
checked by the project task lead.
4.2 Technical System Audit
The Battelle QA Manager performed a technical systems audit on July 21, 2017, 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 revised MOP developed under this study (see Appendices A and B).
4.3 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. The Battelle QA Manager
traced the data from the initial acquisition, through reduction and statistical analysis, to final
reporting, to ensure the integrity of the reported results. All calculations performed on the audited
data were checked for accuracy. Minor transcription errors were noted, but no issues were noted
with the calculations or reported results.
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4.4 QA/QC Reporting
Each assessment and audit were documented in accordance with the quality system developed for
the testing and evaluation program. Once the assessment report was prepared by the Battelle QA
Manager, the report was routed to the Task Order Leader and Battelle T&E II Program Manager
for review and approval. Copies of all assessment reports were provided to the EPA.
Project data quality objectives (DQI's) were met. Table 6 details project DQI's and tolerance
recorded.
Table 6. Data Quality Objectives
Mcsisiircmcnl
Audil Procedure
Allowsihle Tolcrsincc
Aclll.ll TollTillUT
Quantification
of Virus
Stock
Quantify using
standard plaque or
quantal assay
Titer > 108 PFU/mL;
determined at time
preparation and confirmed
prior to persistence testing.
Virus stock used was
highest reasonable titer
obtained. Zika virus
used was at least 1 x 106
TCIDso/mL
concentration. Phage
(MS2 and Phi6) were
used at a minimum
concentration of 108
PFU/mL
Background
Contaminants
Analyze for
background virus
using assays
developed in method
development
No background virus
detected.
No background virus
was detected
Positive
Controls
Measure using assay
developed in method
development
Challenge virus detected
in positive controls at each
time point (concentration
to be determined in
method development)
Discuss with TOCOR to
determine corrective
action.
Negative
Controls
Measure using assay
developed in method
development
No challenge virus
detected in negative
controls at each time point
No challenge virus was
detected.
Temperature
Use calibrated
thermometers or
calibrated
Temperature of incubators
used for sample incubation
should be within ±2 °C of
desired set point.
All thermometers and
incubators were within
tolerance.
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temperature
monitoring system
Storage and
use of
reagents and
media
Manufacturer
provided storage
conditions and
expiration date
Store all reagents and
media appropriately and
use by expiration date
All reagents and media
were used within the
appropriate period
A deviation report was written and included in the study file. Deviations during this evaluation are
listed below.
Deviations from the MOP 2016-001-00 in the 2017 evaluation are as follows:
1) Deviation from MOP section 5.E.5.a and b. Incorrect sample volume used during the MS2 12
°C persistence testing. This deviation slightly increased the assay LOD due to lower sample
volume. All but one sample returned countable plaques; other replicates for this sample
returned values <10 plaques/plate. Adjustments were made to compensate for the pipetting
error by the TOL when calculating PFU/mL for impacted plates. The impact was minimal.
2) Deviation from MOP section 5.E. 1 .a. During MS2 12 °C persistence testing, theE. coli stock
used to prepare the broth culture for the plaque assay was prepared on LBB+S+A (for T=3
days) or on expired LBA (for T=0). In both cases, there was no impact. First, the E. coli
stock used in this study grows well on LBA+S+A, and LBA+S+A medium was used at all
time points to determine viral titer from samples. Second, the E. coli stock grew clear on the
expired media with no contamination.
3) Deviation from MOP Section 5.B.4.C and 5.D.5. The evaporation sample mass was recorded
to three decimal places instead of four, which resulted in a decrease in sensitivity by 0.00049
grams (or 0.06% of total sample mass). No decrease in mass over the five-day time course
was measured. The impact was minimal.
4) Deviation from MOP section 5.D.4.a. During the 37 °C persistence study, one set of
evaporation samples was used rather than two. The MS2 and Phi6 37°C persistence tests were
performed concurrently, using identical equipment and analysts. No impact was found.
5) Deviation from MOP Section 5.B.4.d. During the 37°C persistence study, evaporation
samples were measured once per day of testing, and not during each time point. No impact
was found as no decrease in mass over the five-day time course was measured.
Page 27
-------
6) Deviation from MOP Section 5.E.l.a and 5.E.l.b. During 37°C persistence testing, E. coli
and I\ syringae were propagated in 15-50 mL cultures in 50-250 mL flasks rather than 10 mL
cultures. This deviation was performed to ensure that sufficient culture volume was available
for all samples processed each day. There was no impact.
Deviations from the MOP 2017-001-00 in 2017 are as follows:
7) Deviation from MOP Section 5.D.l.b. The analyst plated mammalian cell cultures using an
expired (by five to six days) multichannel pipette. The pipette was recalibrated without
modifications. There was no impact.
8) Deviation from MOP Section 5.C.l.b. Evaporation samples were measured once per day of
testing and not for each time point. There was no impact as no decrease in mass over the five-
day time course was measured.
9) Deviation from MOP Section 5.D. 1 .b. During the Zika 12 °C persistence test, the cell seeding
range varied (15.5-20.5 x 103/96-well vs 15 x 103/96-well as specified in the MOP). The
adjustments were made under TOL direction to ensure adequate cell density for ZIKV
infection. Plating density in the MOP was too low to ensure adequate monolayer density by
16-20 hours. There was no impact.
Page 28
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5.0 Results
5.1 Landfill Leachate Characterization
Landfill leachates were collected in May 2017 and were analyzed within three weeks for chemical
characteristics and microbiological productivity (see Table 4 for exact dates). Leachate
characterization results are summarized in Table 7.
All three leachate samples were markedly different from each other analytically, with varied
composition and characteristics (Table 7). All leachates were similar in pH (6.57-7.95). However,
samples varied substantially in most chemical components. Leachates from landfills A and C
showed similar levels of chloride, magnesium, sodium, COD, TDS, TOC and BOD, while leachate
from landfill B varied substantially from A and C in these characteristics.
For the 2015-2016 landfill persistence study, leachate samples were acquired from the same
landfills in 2015. Although leachate samples were collected from identical locations in 2015 and
2017, differences in leachate composition were observed in all three leachates. In 2017, leachate
A showed increased levels of magnesium and calcium, TDS, and BOD concentrations and
decreased zinc concentration. Leachate B collected in 2017 showed increases in nitrate, calcium,
iron, manganese, sulfate, COD, TSS and TOC concentrations. Leachate C collected in 2017
showed decreased metal concentrations (zinc excepted) and substantially decreased COD, TOC
and TDS. Results from 2015 were obtained from the 2015-2016 landfill persistence study [1],
Summary results from 2015 and 2017 are in Table 7.
Heterotrophic bacterial and fungal concentrations in each leachate were characterized within 16
days of receipt (Table 7). All leachates contained higher levels of both heterotrophic bacteria and
fungi in Spring of 2017 than in Fall of 2015 (Table 7). While heterotrophic bacterial concentrations
were equivalent in all leachates in 2017 (2.69 x 106 - 6.07 x 106), the fungal concentration in
leachate B was 102 CFU/mL higher than the fungal concentration observed in leachates A and C
(7.6 x 106 vs 1.64-5.73 x 104). Fungal and bacterial colony morphologies differed between
leachates.
Page 29
-------
Leachate samples collected in 2017 showed higher microbial concentrations than leachate samples
collected from the same locations in 2015 and reported in the 2015-2016 landfill persistence study
[1] (Table 7). An increase in heterotrophic microbial concentration was observed in all three
leachates: bacterial activity in leachates A and C increased by a factor of 10, and in leachate B by
a factor of 100, from 2015 to 2017, while fungal productivity in all leachates was increased by 10
to 100-fold from 2015 to 2017. The increased temperature and rainfall in the spring months likely
resulted in increased environmental microbial concentrations when compared to the fall and winter
timeframe. Results are summarized in Tables 7 and 11. Results from the 2015-2016 study can be
located in the report [1],
While the causes of chemical changes in the leachate are not known, and this study only represents
two data points, and these data suggest that landfills are dynamic environments with the potential
for substantial environmental changes over short periods.
Page 30
-------
Table 7. Landfill Leachate Analysis Data
Aiiiilvto
Liiiuirill A
l.nnillill li l.iiiullill (
20I5'1
201 "7
2015'1
20 r
2015'1
20 r
Metals (mg/L)
Calcium
11.6
45
200
536
312
83.1
Iron
6.36
5.60
17.4
120
31.5
7.87
Magnesium
130
245
84.3
79.5
297
175
Manganese
0.0468
0.0951
0.152
3.78
2.26
0.246
Potassium
468
367
260
151
937
747
Sodium
1,880
1,870
1,500
549
2,360
2,090
Zinc
0.140
0.0699
0.0199
0.259
0.0711
0.255
Anions (mg/L)
Chloride
2,070
2,280
1,980
533
2,810
2,530
Nitrate-N
4.00
6.40
3.08
1346
<1.00
<0.500
Sulfate
3.19
5.40
10.1
168
33.0
58.6
Total Alkalinity as CaCCb (m
g/L)
Total Alkalinity
6,100
5,120
2,600
2,020
8,040
8,450
Ammonia as Nitrogen (mg/L)
Ammonia
1,050
828
386
298
1,370
1,550
Oxygen Demand (mg/L)
COD
1,500
1,880
2,470
3,270
9,060
1,290
BOD
187
421
2,020
2,070
2,350
198
pH (Standard Units), Oxidation Reduction Potential (millivolts) and Temperature ("Celsius)
pH (field)3
7.88
ND
7.14
ND
7.36
ND
pH (lab)b
7.76
7.95
7.06
6.57
7.55
7.95
ORP (field)3
47.4
ND
-60.7
ND
-96.8
ND
Temperature (field)3
21.8
ND
25.0
ND
20.0
ND
Total Dissolved Solids, Total Organic Carbon, Total Suspended Solids (all in mg/L)
TDS
6,680
8,440
5,980
5,000
13,500
9,420
TOC
448
632
796
1,330
2,960
500
TSS
12.3
69.0
82.0
122
72.0
26.4
Visual Observations
Color
yellow
dark
gray/black
brown
light gray
dark brown
golden
brown
Particulates
not
significant
not
significant
significant
not
significant
significant
not
significant
Microbial Enumeration via Standard Plate Count (CFU/mL)
Bacterial0
3 x 106
6.07 x 107
9x 105
1.35 x 107
8x 105
2.69 x 106
Fungal0
3 x 102
1.64 x 104
8x 104
7.60 x 106
9x 103
5.73 x 104
ND: Not Determined
aField measurements not taken during 2017 sampling as samples sent to Battelle directly from landfill personnel. Oxidation-
Reduction potential (ORP).
bpH measurements taken on 6/2/2017.
cMicrobial loads enumerated via standard plate count on TSA for bacterial load, and PDA for fungal load. Samples incubated at 27
°C±2 °C. Microbial load determined on 6/2/2017 or 2/15/2016.
d2015 results from leachate collected October 7-8 2015, and analyzed within 2 days of sample collection [1],
Page 31
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5.2 ZIKV Persistence
ZIKV persistence was analyzed in two of the three landfill leachates (A and C) over five days at
12 ± 1°C at varying intervals. Samples were removed from incubation and quantified via TCID50
assay at 0, 6, 24, 48 and 96 hours post inoculation. Viral titer recovered from all time points was
used to generate decay curves. Data from each time point are listed in Table 8. Viral titer was
expressed as mean TCIDso/mL or In (TCIDso/mL) and plotted versus time (Figure 11 and Figure
12 respectively); a linear regression of the In (TCIDso/mL) data was used to calculate D-values
and persistence time (Table 9).
The calculated TCID50 assay LOD is between 90-160 TCIDso/mL. However, the cytotoxic effects
of the leachate samples decreased the LOD during this study. Leachate-induced toxicity was
evident in cell culture, resulting in an empirical LOD for the ZIKV assay of 304 ± 59 TCIDso/mL
when performed with ZIKV and leachates collected in 2017. Leachate collected in 2017 showed
increased cytotoxicity over leachate collected in 2015, likely due to changes in chemical
constituents and/or microbial population or activity (see Sections 5.1 and 5.5, respectively).
Results indicated that ZIKV persisted between 4.18 and 5.04 days at 12 °C in leachate (Table 9).
These data are similar to TGEV persistence in landfill leachate (4.6-16.6 days at 12 °C) reported
in the 2015-2016 landfill persistence study [1], During the ZIKV time-course, all time points
returned results greater than the LOD, and ZIKV CPE was distinguishable from leachate
cytotoxicity in all cases. Negative controls (leachate alone) were analyzed at each time point to
ensure that viral CPE was distinguishable from leachate toxicity. TCID50 values for leachate A and
C were negative at T=0 and T=6 hours (data not shown). The empirical ZIKV TCID50 LOD was
calculated from negative controls at each time point (Figure 3).
ZIKV was inactivated at similar rates in leachates A (4.18 days) and C (5.04 days) and did not
inactivate in the control matrix (DMEM supplemented with penicillin/streptomycin) over the
testing period.
Page 32
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Table 8. ZIKV 12 °C Data Table
Time
point
(Hours)
/ik:i \ iins
l.e;ich;ile A
l.e;ieh;ile (
l.iniiid Conirol (PliS)
Tiler1
SI)1'
(\ i':;.)'
Tiler1
SI)1'
(\ r ;.)'
Tiler1
SI)1'
( \
0
8.93E+04
4.65E+04
52.14%
2.77E+05
9.37E+04
33.80%
3.10E+05
9.15E+04
29.52%
6
1.61E+05
1.06E+05
65.80%
1.70E+05
1.33E+05
77.92%
2.82E+05
3.14E+04
11.15%
24
7.72E+04
3.95E+04
51.13%
8.12E+04
5.48E+04
67.41%
1.88E+06
2.39E+06
127.48%
48
4.10E+03
2.25E+03
54.92%
1.22E+04
1.00E+04
38.46%
1.60E+06
2.06E+06
129.20%
96
4.86E+02
1.87E+02
82.04%
1.97E+03
2.20E+03
111.47%
3.27E+05
1.01E+05
31.02%
aViral titer reported as average TCID50/mL over triplicate replicates.
bStandard deviation of data used to determine average titer at each time point.
Coefficient of Variation reported as the ratio of the standard deviation to the mean for each time point.
Note: Leachate B had a toxic effect on Zika virus, thus persistence testing was not performed.
Table 9. ZIKV D-Values and Persistence
Msilrix
Slope
Measured D-
Vsilue (hours)
Measured
l)-\ nine
(diiys)
Persistence-1
(hours)
Persistence"'
(diiys)
Leachate A
-0.0611
16.4
0.68
100.3
4.18
Leachate B
NDb
ND
ND
ND
ND
Leachate C
-0.0551
18.2
0.76
121.1
5.04
DMEM Medium
(Positive Control)0
0.0009
N/A
N/A
N/A
N/A
Calculated time at which measured linear decay rate intersects with assay limit of detection.
bND = Not Done. Leachate B had a toxic effect on Zika virus, thus persistence testing was not performed.
°Zika virus in positive control did not appreciably degrade over testing period. Persistence and D-values were not
calculated due to the positive slope.
Page 33
-------
LIMIT OF DETECTION
(304 TCID50/mL)
LIMIT OF DETECTION
(5,72 LN TCID50/mL)
A
1.00E+07
1.00E+06
1.00E+05
Sl.OOE+04
q
£
1.00E+03
1.00E+02
1.00E+01
B
16.00
14.00
12.00
10.00
E
o"
S 8.00
¥
c
6.00
4.00
2.00
v
10 20 30 40 50 60 70 80 90
Incubation time (hours)
-•— Positive -•— Leachate A —Leachate C -•- Leachate A Negative Leachate C Negative
• 0.0009x +13.038 y =-0.061 lx +11.847 V = -0.0551x +12.391
R1 - 0.0032 = 0,9385 Rl = 0.9947
Zika Virus Persistence in Landfill Leachate at 12°C
10 20 30 40 50 60 70 80 90
Incubation time (hours)
-•-Positive -»-LeachateA Leachate C -•-Leachate A Negative -•— Leachate C Negative
* - Standard deviation for 24 hour timepoint is 2.39 x 106TCID5o/mL
** - Standard deviation for 30 hour timepointis 2.06x 106TCID5o/mL.
Both 24 and 48 hours timepoint deviation is due to one of three replicates
Natural Log of Zika Virus Decay in Landfill Leachate at 12°C
Figure 3. ZIKV Persistence (A) and Decay (B) at 12 °C
Page 34
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5.3 MS2 Persistence
MS2 persistence in landfill leachate was evaluated at 12 ± 1°C and 37 ± l°Cat varying intervals.
Data from all time points and temperatures are reported in Tables 10 and 11. During incubation,
samples were removed from incubation and quantified via plaque assay. Linear regression of viral
titer over time was used to calculate D-values and persistence time (Table 12).
MS2 persisted longer during static incubation at 12 °C (1 to 1 '/2 months) than at 37 °C (1 to 2.2
days) [Table 12, Figure 4, Figure 5], Not surprisingly, MS2 decay was more rapid at higher
temperatures, with all leachate samples returning no viable virus by 4.3 days (52 hours) post-
incubation at 37 °C, while MS2 virus was recoverable at 28-42 days post-incubation at 12 °C.
When MS2 was incubated at 37 °C, no leachate-specific effects were observed; all leachates
showed similar reduction in MS2 persistence and nearly equal MS2 decay rates in all three
leachates (Figure 5). Interestingly, differences in MS2 inactivation were observed during
incubation at 12 °C (Figure 4). At 12 °C, MS2 was inactivated most rapidly in leachate B (27.4
days), followed by leachates C (35.7 days) and A (48.3 days). Surprisingly, MS2 was inactivated
when incubated in the positive control liquid (PBS) at 12 °C (persistence 34.4 days, Table 12),
suggesting that MS2 degradation in leachate may be due to long-term storage at 12 °C, rather than
the effects of the leachate constituents. No decay was observed during incubation in PBS at 37 °C
(persistence to 31.31 days, Table 12).
Page 35
-------
Table 10. MS2 37 °C Data Table
Time
Point'1
(Hours)
MS2 ill 37 °C
Lciichiile A
l.eiichiile li
l.eiichiile ('
Positi\c Control (IMiS)
Tiler1'
SI)'
CY'1
Tiler1'
SI)'
C Y'1
Titer1'
SI)'
(V
liter1'
SI)'
C Y'1
0.00
5.3
0.4
7.6%
5.3
0.1
1.9%
5.4
0.2
3.7%
5.4
0.1
1.9%
4.37
5.4
0.1
1.9%
5.5
0.0
e
5.8
0.1
1.7%
5.8
0.1
1.7%
7.73
5.2
0.1
1.9%
5.2
0.0
e
5.3
0.0
e
5.4
0.1
1.9%
23.73
3.2
0.3
9.4%
3.7
0.0
e
3.6
0.1
e
5.3
0.0
e
29.70
2.2
0.5
22.7%
2.8
0.2
7.1%
2.7
0.5
18.5%
5.4
0.0
e
50.40
0.0
0.0
e
0.0
0.0
e
0.0
0.0
e
5.2
0.0
e
77.03
0.0
0.0
e
0.0
0.0
e
0.0
0.0
e
5.2
0.2
3.9%
"Time post-inoculation (in hours) sample was analyzed.
bViral titer reported as average log (PFU/mL) from triplicate replicates.
"Standard deviation of data used to determine average titer at each time point.
Coefficient of variation reported as the ratio of the standard deviation to the mean for each time point.
"Coefficient of variation not calculated as no titer recovered from sample.
Table 11. MS2 12 °C Data Table
lime
Point'1
(Dnvs)
MS2 :il 12 °C
l.eiichiile A
l.eiichiile IJ
l.eiichiile A
Posilne Control (l>US)
liter1'
sir
C Y'1
liter1'
SI)'
CY'1
liter1'
sir
C Y'1
liter1'
sir
CY'1
5.7
0 0
n5",
5 <•>
".I
1.4%
5 X
0 0
0 5%
5 X
0 0
<)5%
3.00
4.6
0.1
1.9%
3.3
0.3
7.6%
4.3
0.2
4.8%
4.1
0.3
7.1%
7.00
4.4
0.2
3.5%
2.2
0.1
3.2%
4.7
0.1
1.2%
4.0
0.1
2.5%
14.00
4.6
0.0
0.9%
2.0
0.1
3.3%
4.2
0.1
2.0%
3.4
0.2
6.5%
21.00
3.6
0.1
1.5%
1.6
0.3
17.4%
2.5
0.1
5.9%
2.3
0.2
6.9%
28.00
2.6
0.1
3.3%
1.6
0.2
11.4%
1.2
1.0
87.9%
2.1
0.1
5.6%
42.00
0.0
0.0
e
0.0
0.0
e
0.0
0.0
e
0.00
0.0
e
aTime post-inoculation (in days) sample was analyzed.
bViral titer reported as average log (PFU/mL) from triplicate replicates.
"Standard deviation of data used to determine average titer at each time point.
Coefficient of variation reported as the ratio of the standard deviation to the mean for each time point.
"Coefficient of variation not calculated as no titer recovered from sample.
Page 36
-------
Table 12. MS2 D-values and Persistence
Matrix
12 °(
O
q
Slope
l)-\ illllO
(days)
Persistence1'
(days)
Slope
I)-Value
(days)
Persistence1'
(days)
Leachate A
-0.206
4.85
48.3
-0.253
0.16
1.78
Leachate B
-0.261
3.84
27.4
-0.203
0.21
2.21
Leachate C
-0.285
3.51
35.7
-0.231
0.18
2.03
PBS
-0.268
3.73
34.4
-0.014
3.0
31.31
aD-values and persistence calculated from all positive values: leachate A, B, and positive samples analyzed from
time points T=0, 6 hours, and 3, 7, 14, 21 and 28 days post incubation; leachate C samples analyzed from time
points T=0, 6 hours, 3, 7, 14 and 21 days post-incubation.
Calculated time in days at which measured linear decay rate intersects with assay limit of detection.
°D-values and persistence calculated from all positive values: all samples analyzed from time points T=0, 4, 8, 24
and 30 hours post incubation.
Page 37
-------
LIMIT OF DETECTION
(2.3 LN PFU/mL if count all plaques)
2017 MS2 Persistence in Landfill Leachate at 12°C
Leachate A
y = -0.2063x + 12.27
RJ = 0.8579
y = -0.2607* ~ 9.4547
ft1 = 06239
14.00
Time (days post-incubation)
Leachate C
y =-0.2851* + 12.485
R2 = 0.8126
Positive Controls
y = -0.268* + 11.518
Rl = 0.8753
- Negative Controls
0.0
0.00
Leachate A
14.00
—Leachate B
21.00 28.00
Time (days post-incubation)
—Leachate C ~ Positive Controls
35.00 42.00
- Negative Controls
2017 MS2 Decay Rate in Landfill Leachate at 12°C
LIMIT OF DETECTION
(2.3 IN PFU/mL if count all plaques)
Figure 4. MS2 Persistence at 12 °C
Page 38
-------
2 10.0
2017 MS2 Persistence in Landfill Leachate at 37°C
LIMIT OF DETECTION
(2.3 LN PFU/mL if count all plaques)
-•—Leachate A —Leachate B Leachate C -A Positive Controls -M-Negative Controls
10.00
20.00
30.00 40.00 50.00
Time (hours post-incubation)
60.00
70.00
80.00
Figure 5. MS2 Persistence at 37 °C
0.0
-•-Leachate A
y = -0.2534x + 13.109
R2 = 0.949
-~-Leachate B
y = -0.2025x+13.055
R2 = 0.9388
12.00 16,00 20.00
Time (hours post-incubation)
Leachate C Positive Controls
24.00 28.00 32.00
-Negative Controls
y = -0.2306X + 13.532
RJ = 0.9299
y = -0.0139x+ 12.745
Ra =0.1567
LIMIT OF DETECTION
(2.3 LN PFU/mL if count all plaques}
2017 MS2 Decay in Landfill Leachate at 37°C
Page 39
-------
5.4 Phi6 Persistence
Phi6 persistence in landfill leachates A and C was evaluated at 37 ± 1°C over 56 hours. Data for
all time points are in Table 13. Phi6 persistence and decay were not evaluated in leachate B as
Phi6 was unrecoverable from leachate B at all time points tested. During incubation, samples were
removed from incubation and quantified for surviving virus via plaque assay. Linear regression of
viral titer over time was used to calculate D-values and persistence time (Table 14).
Phi6 was inactivated rapidly at 37 °C and was unrecoverable after 8-24 hours in all leachate
samples (Figure 6). While Phi6 incubated in leachate A did return viable virus (just above assay
LOD at 24 (0.2 log PFU/mL) and 30 hours post-incubation (0.7 log PFU/mL), all plates were
below the countable number of plaques (25 plaques/10 cm plate). Phi6 persistence in leachates A
and C was less than 12 hours (0.46 and 0.43 days for landfill A and C, respectively, Table 14).
Unlike MS2, Phi6 maintained higher titer in the control matrix (PBS) than in the landfill leachate,
showing a steady decline between 8 and 56 hours, but maintaining titers higher than all test samples
throughout the time-course (persistence 7.54 days in PBS vs < 0.5 days in leachate). (Figure 6)
Table 13. Phi6 37 °C Data Table
Time
point
(lloiirs)
I»hi6 ;il 37 °C
l.i'iichiilc A
l.oiichiiU-1}
l.ciichiilc (
Posili\e Control
Tiler1
SI)1'
c v.
Tiler1
SI)1'
c v
Tiler'
SI)1'
C V
Tiler'
SI)1'
C \ 1
h h
".I
(I S"„
0
I)
—
fv5
0 0
(13%
fv5
(i (i
—
2.27
5.6
0.2
3.8%
0
0
—
5.5
0.2
4.0%
6.3
0.1
1.8%
4.30
3.8
0.9
23.6%
0
0
—
4.5
0.4
9.1%
6.5
0.1
--
8.30
2.6
0.4
13.4%
0.2
0.3
173.2%
2.0
0.3
13.0%
6.1
0.0
0.4%
23.87
0.2
0.3
173.2%
0
0
—
0
0
—
5.1
0.1
--
29.90
0.7
1.3
173.2%
0
0
—
0
0
—
4.3
0.1
2.3%
55.45
0.0
0.0
--
0
0
—
0
0
—
2.0
0.1
--
78.17
0.0
0.0
--
0
0
--
0
0
--
0.0
0.0
--
aViral titer reported as average log (PFU/mL) from triplicate replicates.
bStandard deviation of data used to determine average titer at each time point.
Coefficient of variation reported as the ratio of the standard deviation to the mean for each time point.
Note: Phi6 not recoverable from leachate B at the majority of time points.
Page 40
-------
Table 14. Phi6 D-Values and Persistence
M:ilri\
Slope
D-VilllllV1
(il:ivs / hours)
Persistence11,,h
(dsivs / hours)
Leachate A
-1.1356
0.04/0.96
0.46/11.2
Leachate B
NRC
NRC
NRC
Leachate C
-1.2573
0.03/0.72
0.43/10.4
PBS
-0.0694
0.60/14.41
7.54/ 180.9
aD-values and Persistence calculated from all positive values: all samples analyzed from time points T=0, 2.3, 4.3,
and 8.3 hours post incubation.
Calculated time in days at which measured linear decay rate intersects with assay limit of detection.
CNR = Phi6 not recoverable from leachate B, no persistence test performed.
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A 2017 Phi6 Persistence in Landfill Leachate at 37°C
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5.5 Evaporation
The weights of "evaporation control" samples were measured during each time-course, to evaluate
whether virus-spiked leachate samples were reduced in volume over time. All samples maintained
mass through the time-courses, 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.
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6.0 Discussion
This controlled laboratory study evaluated the persistence of three viral agents: one enveloped
single-stranded RNA virus (Flavivirus, ZIKV), one enveloped bacteriophage (Phi6), and one non-
enveloped bacteriophage (MS2) in three landfill leachates. This study obtained leachate from the
three landfills used in the 2016 landfill persistence study. This report discusses results from the
2017 study; data from the previous study can be found in the 2015-2016 landfill persistence report
[1].
Viral persistence and decay in each leachate were measured at 12 °C (MS2 and ZIKV), and/or 37
°C (MS2 and Phi6). Data showed that viruses can persist for days to months in landfill leachate at
mild temperatures (12 °C), with bacteriophages (MS2) persisting longer than mammalian viruses
(ZIKV). Viral persistence in landfill leachate varied by agent and temperature. At 12 °C, MS2
persisted longer than ZIKV in landfill leachate (one to two months vs
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stranded RNA vims. In terms of viral structure, MS2 is a surrogate for nonenveloped human
pathogens (e.g., polio virus, norovirus) while Phi6 is a surrogate for enveloped viruses (e.g., Ebola,
Influenza, SARS). ZIKV is an enveloped single-stranded positive sense RNA mammalian virus
(Flavivirus) and similar in structure to other pandemic human pathogens (e.g., Ebola, Influenza,
SARS).
The variation in agent survival time may be related to virus structure (enveloped vs nonenveloped),
genomic material (deoxyribonucleic acid [DNA] vs RNA) or other factors yet to be determined.
The environmental survival of viruses is influenced by many factors: environmental parameters
(pH, radiation, humidity, temperature), medium (e.g., leachate, respiratory secretions, soil, nasal
mucus, etc.), presence of other organisms, the physical state of the virus (aggregated, adsorbed to
surfaces, etc.). Each factor affects each agent to varying degrees and in different ways, rendering
generalizing and surrogate choice difficult. That being said, non-enveloped viruses (e.g., MS2,
norovirus) are thought to have increased environmental stability over enveloped viruses, due in
part to the outer protein layer. Enveloped viruses (e.g., Phi6, ZIKV, Ebola) have an outer lipid
layer that can be more susceptible to dehydration and disruption.
Data from this study were in accordance with this trend, with the non-enveloped virus showing
increased persistence and stability (MS2) and enveloped viruses (ZIKV, Phi6) showing decreased
stability when compared to MS2 at both tested temperatures. This study measured enveloped virus
persistence at different temperatures (ZIKV at 12 °C, Phi6 at 37 °C), but did not measure both
enveloped viruses at the same temperature, so a direct comparison between mammalian and
bacterial viruses is not possible with these data (Executive Summary Tables 1 and 2).
Differences in viral persistence in leachates sourced from different landfills suggests that viruses
may be affected by landfill leachate constituents in unique ways, and that nonenveloped and
enveloped viruses may maintain stability in different types of leachate. During this TO, viral
recovery from leachate varied by virus: MS2 was recoverable from all three leachates with no
issue, while Phi6 and ZIKV were unrecoverable from leachate B and required centrifugation for
effective recovery from leachates A and C.
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Variations in viral survival and inactivation were expected since variations in viral environmental
survival are already known. For instance Venezuelan Equine Encephalitis virus (VEEV) is more
stable than Lassa or Ebola viruses (also enveloped RNA viruses) under similar conditions, despite
all three agents being enveloped RNA viruses [8], Thus, differences in viral persistence and decay
identified in this study were not surprising, as MS2, Phi6 and ZIKV are diverse structurally.
An additional factor in viral survival is the viral environment. This study explored viral persistence
in multiple landfill leachates, each from a landfill varying in waste intake (volume and makeup)
and management (see Table 3). Thus, each landfill leachate has a specific leachate chemical and
biological fingerprints for each landfill (see section 5.1, Table 7). While all three leachates
displayed pH within physiological levels (leachate pH between 6.6-8.0), leachate chemistry,
microbial activity and microbial diversity differed, resulting in virus exposure to three diverse
environments. Thus, variations in virus survival in different leachates, as observed with MS2
during the 12 °C time-course (see Executive Table 1 and Section 5.3) was unsurprising, given the
different environments in each leachate.
Results from this study suggest that in the event of a significant adversarial attack on the U.S.
population using viral BW agents, an unintentional release, or a natural outbreak (i.e., Zika virus,
Ebola virus) where biological waste is disposed of without adequate decontamination, there can
be a potential threat to human health and the environment if the virus reaches the landfill leachate.
This study showed that 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 viruses can persist in landfill leachate for several months; especially at lower
temperatures;
• Moderately elevated temperatures, such as 37 °C (99 degrees Fahrenheit [°F]), can
drastically reduce viral persistence and infectivity and can decrease D-values to < 1 day in
some cases;
• Leachate composition likely effects viral decay rates and persistence dramatically. While
this study did not explore how leachate composition affects viral inactivation, differences
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in persistence and decay between leachates and viruses suggest that this is at least in part
due to chemical constituents and concentrations;
This study investigated leachates from only three landfills. Thus, how viral persistence in
these samples relates to viral persistence in other landfills across the U.S. is unknown.
Further persistence analysis of viral agents in a larger number of landfill leachates would
be needed to gain insight into the key characteristics (chemical or otherwise) that affect
viral decay rates and generate actionable data and predictive measurements for use in waste
management;
Non-enveloped viruses were found to persist longer than enveloped viruses;
Choice of surrogate agents is vital for studies that explore the survival of BW agents, as
data suggest that viral structure and virus-specific characteristics are key for viral
persistence in leachate samples. Choosing a surrogate with characteristics as similar as
possible to the agent of interest is recommended (e.g., mammalian RNA virus as a
surrogate for a similar virus), as poor surrogate choice may lead to results that misrepresent
agent survival.
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7.0 References
1. Evaluation of Persistence of Biological Agents in Landfill Leachate, U. S E. NHSRC,
Editor. February, 2017.
2. Plourde, A.R. and E.M. Bloch, A Literature Review ofZika Virus. Emerging Infectious
Diseases, 2016. 22(7): p. 1185-1192.
3. McCloskey, B., et al., Emerging infectious diseases and pandemic potential: status quo
and reducing risk of global spread. The Lancet Infectious Diseases, 2014. 14(10): p.
1001-1010.
4. Gallandat, K. and D. Lantagne, Selection of a Biosafety Level 1 (BSL-1) surrogate to
evaluate surface disinfection efficacy in Ebola outbreaks: Comparison of four
bacteriophages. PLOS ONE, 2017. 12(5): p. e0177943.
5. Aquino de Carvalho, N., et al., Evaluation ofPhi6 Persistence and Suitability as an
Enveloped Virus Surrogate. Environmental Science & Technology, 2017. 51(15): p.
8692-8700.
6. 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 Innovation. 2008: Copper
Mountain Conference Center, Colorado USA.
7. 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.
8. Sagripanti, J.-L., A.M. Rom, and L.E. Holland, Persistence in darkness of virulent
alphaviruses, Ebola virus, and Lassa virus deposited on solid surfaces. Archives of
Virology, 2010. 155(12): p. 2035-2039.
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