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Systematic Review and Meta-Analysis of the Persistence and

Disinfection of Human Coronaviruses and Their Viral Surrogates in
Water and Wastewater
Andrea I. Silverman* and Alexandria B. Boehm
Cite This: Environ. Sci. Technol. Lett. 2020, 7, 544−553 Read Online

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ABSTRACT: A systematic review and meta-analysis was con-

ducted to identify decay rate constants (k) of human coronaviruses
and their viral surrogates (i.e., animal coronaviruses and the
Downloaded via 201.244.96.9 on November 3, 2020 at 20:44:36 (UTC).

enveloped bacteriophage Phi6) in water and wastewater and

disinfection rates with exposure to free chlorine and germicidal
ultraviolet light (UV254). Here, 73 k were identified, with only 12
for human coronaviruses, as opposed to animal coronaviruses or
Phi6. In the absence of disinfectants, k increased with temperature.
Between 22 and 25 °C, mean k for coronaviruses ranged from 0.19
± 0.06 d−1 in laboratory buffer (n = 4) to 2.9 ± 0.03 d−1 in
sterilized wastewater (n = 3), which are within the ranges observed
for Phi6 and nonenveloped viruses. No free chlorine or UV254
disinfection studies for coronaviruses were identified that met the
systematic review inclusion criteria, although evidence from the literature suggests that coronaviruses would be inactivated if
disinfectant doses recommended for nonenveloped viruses were applied. Three disinfection experiments were identified for Phi6.
However, given different genome compositions and virion structures between coronaviruses and Phi6, it is not clear whether Phi6
should be used as a surrogate for evaluating free chlorine or UV254 k. Therefore, there is a critical need for additional studies that
specifically evaluate disinfection kinetics of coronaviruses in the aqueous environment.

■ INTRODUCTION
Over the last two decades, novel coronaviruses (CoV) have
captured the world’s attention by causing local outbreaks and
global pandemics.1,2 The severe acute respiratory syndrome
coronavirus (SARS-CoV-1) outbreak of 2003 and Middle
Eastern respiratory syndrome coronavirus (MERS-CoV)
outbreak of 2012, for example, caused thousands of infections
with mortality rates of 10% and 40%, respectively. The
emergence of SARS-CoV-2, the causative agent of COVID-19,
in December 2019 has caused a global pandemic with
unprecedented and ongoing global health and economic
impacts. At the time of submission of this article (April 17,
2020), there have been an estimated 2.2 million clinically
diagnosed illnesses and 149,000 deaths across the globe,3 more
than one-third of the world’s population is under stay-at-home
orders to reduce transmission rates,4 and the incidence of
illness is continuing to grow.
While primary transmission of coronaviruses is through
respiratory droplets, viral RNA can be excreted fecally.5−12 The
RNA of SARS-CoV-1,5,6 SARS-CoV-2,7−10 MERS-CoV,11 and
human coronavirus HUK1,12 for example, have been detected
in the stool of some infected patients. Furthermore, researchers
in Australia13 and The Netherlands14 observed SARS-CoV-2
RNA in raw sewage samples collected during the first few
weeks of their respective outbreaks. It has been suggested that
coronaviruses can replicate in the gastrointestinal tract;15
however, there is limited evidence as to whether infectious
virions can be excreted in feces. One study to date has reported
observing infectious SARS-CoV-2 virions in feces,16 whereas
two others did not find evidence of this, despite detection of
high virus RNA concentrations in the stool samples,10,17 and
data from Zang et al.17 suggest that SARS-CoV-2 is inactivated
in fluids of the gastrointestinal tract.
While it is not believed that water and wastewater will play
an important role in coronavirus transmission,18 there is still a
need to understand the fate of these viruses outside the human
host, including their persistence in water and wastewater, and
inactivation with exposure to commonly used disinfectants. A
greater knowledge of coronavirus decay rates in wastewater can
also inform wastewater epidemiology, which has been
Received: April 17, 2020
Revised: May 28, 2020
Accepted: May 28, 2020
Published: May 28, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.estlett.0c00313

544 Environ. Sci. Technol. Lett. 2020, 7, 544−553
Environmental Science & Technology Letters pubs.acs.org/journal/estlcu Review

suggested as a way to estimate disease burden within the

Number of k extracted is divided into k from persistence studies (i.e., in the absence of a disinfectant) and k with exposure to disinfectants. Sc = Scopus, WOS = Web of Science core collection, and PM
Persistence: 46, UV254: 0,

Persistence: 24, UV254: 1,

population.13,14

Number k extracted
Coronaviruses are 120−160 nm diameter, enveloped viruses

Free chlorine: 0,

Free chlorine: 2,
with a single-stranded, nonsegmented RNA genome between

Chloramines: 0

Chloramines: 0
26.4 and 31.7 kilobases long.19 The presence of a lipid-
containing envelope and relatively large ssRNA genome are
attributes that set coronaviruses apart from the nonenveloped
waterborne viruses that are more commonly evaluated for
persistence and disinfection in water and wastewater (e.g.,

Number of
adenoviruses, enteroviruses, coliphage). Properties of the viral

included
papers
genome and envelope can have an outsized influence on virus

5
decay rates in aqueous environments;20,21 therefore, corona-
viruses are hypothesized to have persistence and disinfection
behaviors that differ from smaller, nonenveloped viruses.

Number of papers
subject to full text
The disinfection of nonenveloped waterborne viruses and

review
fecal indicator bacteria has traditionally been used to

15

11
determine required disinfection doses for water and wastewater
unit processes, to monitor disinfection unit process perform-
ance, and to evaluate pathogen persistence in natural
waters.22−27 Therefore, it is of interest to understand the

papers identified during

Number of additional
disinfection and persistence of coronaviruses relative to these
organisms. Thus, the goal of this work was to conduct a

review
systematic review and meta-analysis of the peer-reviewed

0
literature to collate rates of decay of coronaviruses in aqueous
environmentsincluding water and wastewatersand with
exposure to free chlorine and germicidal ultraviolet light
(UV254).
Given safety challenges in conducting laboratory work with
unique papers
Number of
identified
highly infectious human coronaviruses, many researchers have 69

23
instead evaluated closely related viral surrogates, including
animal coronaviruses [e.g., murine hepatitis virus (MHV),
transmissible gastroenteritis virus (TGEV), feline infectious
peritonitis virus (FIPV)]28−31 and the enveloped bacterio-
Number of papers

coronavir* OR SARS OR MERS OR “mouse hepatitis virus” OR Sc: 33, WOS: 30,

Sc: 9, WOS: 17,

phage Phi6.20,31−34 These surrogates were included in the

identified in
searches

systematic review. MHV, TGEV, and FIPV are viruses within

PM: 37

PM: 8

the Coronaviridae family that infect mice, pigs, and cats,

respectively, and have been used as surrogates for human
coronaviruses given similar size, composition, and morphol-
ogy.28−31 Phi6 (family Cystoviridae) is an 85 nm diameter,
“murine hepatitis virus” OR “transmissible gastroenteritis virus”

enveloped bacteriophage that infects Pseudomonas spp.

bacteria. Phi6 has a double capsid structure and a segmented,
Table 1. Search Terms and Statistics for Systematic Reviewa

dsRNA genome, with a total length of approximately 13.4

kilobases.35 There are therefore key differences in the capsid
and genome structure between coronaviruses and Phi6 (see
cystovir* OR phi6 OR “phi 6” OR “phi-6”

the abstract graphic) that may affect their relative persistence

Search terms (“X”)

and disinfection in water.

■ METHODS
The systematic review and meta-analysis followed PRISMA
guidelines.36 The goal of the review was to compile
quantitative information from the peer-reviewed literature on
the decay rates of human coronaviruses and their viral
surrogates in water and wastewater, with and without exposure
OR TGEV

to common water and wastewater disinfectants [i.e., chlorine,

chloramines, and germicidal ultraviolet light (UV254)]. Viruses
included in the review were coronaviruses (family Coronavir-
idae) and the enveloped bacteriophage Phi6 (family Cystovir-
Bacteriophage Phi6

idae), which has been suggested as a surrogate for mammalian

enveloped viruses.20,31−34
Organism
Coronaviruses

Web of Science core collection (search field = topic), Scopus

= PubMed.

(search field = article title, abstract, keyword), and PubMed

(search field = all fields) were searched on March 18, 2020
(Table 1). Two searches were conducted: one targeting virus
a

545 https://dx.doi.org/10.1021/acs.estlett.0c00313
Environ. Sci. Technol. Lett. 2020, 7, 544−553
Environmental Science & Technology Letters
Table 2. Number of Decay Rate Constants Identified for Persistence of Each Virus in Each Experimental Matrix (i.e., in the
absence of disinfectant exposure) for All Temperatures between 4 and 56 °Ca
Laboratory Cell Culture
Water/Buffer Supernatant
Mouse hepatitis virus (MHV) 2 −
Feline infectious peritonitis virus (FIPV) − −
Transmissible gastroenteritis virus 14 − 2
(TGEV)
Human coronavirus (HCoV) 229E 1 1
HCoV OC43 1 1
SARS-CoV-1 − 2
Coronaviruses (total) 18 4 4
Bacteriophage Phi6 10 4 2
a
“−” denotes that no k values were identified.
persistence in water and wastewater and the other targeting
virus inactivation with exposure to disinfectants. For the
persistence search, the search terms were “(X) AND (water
OR seawater OR stormwater OR groundwater OR waste-
water) AND (die-off OR persistence OR survival OR
inactivat* OR decay)” where X is the target-specific text
(“search term” in Table 1). For the disinfection search, the
search terms were “(X) AND (water OR seawater OR
stormwater OR groundwater OR wastewater) AND (chlorine
OR chloramine OR UV OR UV254 OR ultraviolet OR
UVC)”.
Identified articles were assembled, and duplicates were
removed. Details of the review process, which involved two
independent full-text reviews of papers, are provided in Boehm
et al.24 The inclusion criteria were that the paper: (1)
contained quantitative data on the decay of a target virus in
raw (unaltered), filtered, or pasteurized water or wastewater, or
in laboratory buffer or cell culture supernatant (i.e., not on
surfaces or in aerosols), (2) was in English, (3) was not a
review paper, presented primary data, and was peer-reviewed,
(4) contained data on decay either without exposure to
disinfectants, or with exposure to free chlorine, chloramines, or
UV254, (5) described methods to enumerate the target that are
logical and justifiable, and (6) if the paper presented
disinfection data, it was included only if adequate information
was provided to allow calculation of the virus inactivation rate
as a function of the disinfection dose applied (e.g., free chlorine
concentration in solution or UV254 irradiance or fluence,
corrected for light attenuation in the experimental solution).
Decay rate constants were extracted from papers by a single
reviewer. First-order decay rate constants (k), in units of per
day (d−1), calculated from natural log (ln)-transformed
concentration data as used in Chick’s law, were sought. If a
study presented k values, then they were extracted from the
paper along with any reported errors and model fit values [R2
and/or root-mean-square error (RMSE)], and unit conversions
were applied where appropriate. If a study only reported t90 or
t99, (i.e., time to 90% or 99% reduction in concentration,
respectively), or other times for a specific amount of
inactivation, then those times were converted to first-order
decay rate constants assuming Chick’s law applied. If no first-
order decay rate constant was reported by the study authors,
but data were available in graphs, then Plot Digitizer (http://
plotdigitizer.sourceforge.net) was used to digitize the concen-
tration time series appearing in graphs within the publication,
and k was calculated as the regression slope of ln(C/C0) versus
time (in days) using linear least-squares regression in R. In this
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pubs.acs.org/journal/estlcu Review
Fresh Tap Raw Sterilized
Water Water Wastewater Wastewater
2 − 4 2
− 3 2 1
− 2 −
− 3 2 1
− − − −
− − − −
6 10 4
1 2 5
formulation, C is the virus concentration at time t, and C0 is the
concentration at the start of the experiment (i.e., t = 0). k and
its associated error, as well as model fit parameters, were
recorded.
In addition to extracting information on the decay of the
viral target, a record of each experiment was kept, including
(1) properties of the experimental solution (i.e., the type of
water or wastewater used, whether it was filtered or disinfected,
salinity, pH, concentrations of organic and inorganic
constituents), (2) experimental conditions (i.e., temperature,
experimental reactor volume and sample size, disinfectant
concentration, starting concentration of the target virus,
whether experiments were conducted with light exposure or
in the dark), (3) virus enumeration method, and (4) the model
used to calculate virus decay rates.
Fifteen percent of the papers from which data were extracted
by a single reviewer were randomly chosen for a second round
of data extraction by a different reviewer. Data extracted by the
two reviewers were compared to ensure consistency. A single
reviewer conducted detailed review of all data sets to identify
missing data, data outliers, and data entry mistakes.
When necessary, k values were log10-transformed (log10 k) to
facilitate comparison and visualization of decay rate constants
over multiple orders of magnitude. Data compiled from this
review are available in the Supporting Information.
■ RESULTS AND DISCUSSION
Systematic Review. The systematic review identified 46
decay rate constants (k) for coronaviruses (from seven
publications)28−31,37−39 and 27 decay rate constants (from
five publications)20,31−34 for bacteriophage Phi6, which has
been suggested as a surrogate for coronaviruses (Table 1).
Each k is from a different “experiment”, where experiment is
defined as one viral target exposed to a unique set of
conditions (e.g., type of experimental matrix, temperature,
disinfectant exposure). All inactivation data from the identified
experiments were determined using culture-based assays [i.e.,
cell culture (TCID 50 , MPN, and plaque assays) for
coronaviruses and double agar layer plaque assays for Phi6],
thereby indicating loss of virus viability.
Seventy of the identified experiments were conducted
without exposing the viruses to disinfectants; these experi-
ments are designated as persistence experiments. One study was
identified that evaluated chlorine disinfection of SARS-CoV-1
in wastewater,40 and one evaluated UV254 disinfection of
SARS-CoV-1 in cell culture supernatant.39 However, neither
study provided enough information on the disinfectant dose
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Environmental Science & Technology Letters
applied to meet inclusion criteria and allow for extraction of k
values; therefore, these papers were not included in the total
experiment count. Only three experiments were identified that
evaluated Phi6 disinfection: two with exposure to chlorine20,32
and one with exposure to UV254.20
Of the 46 persistence experiments identified for coronavi-
ruses, 10 were conducted with murine hepatitis virus (MHV)
as a target, six with feline infectious peritonitis virus (FIPV), 18
with transmissible gastroenteritis virus (TGEV), eight with
human coronavirus (HCoV) 229E, two with HCoV OC43,
and two with SARS-CoV-1 (Table 2). No experiments were
identified that examined the persistence of SARS-CoV-2 or
MERS in aqueous solution.
Of the 70 coronavirus and Phi6 persistence experiments, 28
were conducted in distilled water or laboratory buffer (i.e.,
phosphate or HEPES buffer), eight in cell culture supernatants,
six in environmental fresh waters (i.e., river or lake water),
seven in tap water, 12 in raw wastewater, and nine in sterilized
(i.e., filtered or pasteurized) wastewater (Table 2). Experi-
ments were conducted at temperatures between 4 and 56 °C.
No experiment was indicated as being conducted with
exposure to light; we therefore assumed that all experiments
were conducted in the absence of sunlight, which could have
impacted decay rates (see discussion below).
For all experiments except for one, the studies either
provided figures illustrating that there was a log−linear (i.e.,
first-order) decay curve or noted within the text that virus
inactivation followed log−linear decay. The one experiment
that deviated from log−linear decay was for Phi6 inactivation
in pasteurized wastewater at 22 °C, which had a biphasic decay
curve.34 Data for this experiment were extracted and fit with a
log−linear decay curve, which resulted in k = 1.5 d−1 (log10 k =
0.18) and R2 = 0.49; for consistency, we used this calculated
first-order rate constant in the meta-analysis. Two of the
identified experiments (i.e., MHV decay at 4 °C in laboratory
water and in lake water) had negligible decay rates over the
course of the experimental time frame (49 and 14 d,
respectively). These experiments were assigned k = 0.01 d−1,
given that a value of zero cannot be log10-transformed, and the
lowest identified k at this temperature was 0.012 d−1. The two
experiments with these assigned k values are designated in
Figures 1 and 2.
Persistence in Water and Wastewater. In the absence
of exposure to disinfectants, the decay rate constants (k) of
coronaviruses and bacteriophage Phi6 were observed to
increase with temperature regardless of the experimental
matrix (Figure 1). This trend of increased k with increased
temperature was also previously observed for nonenveloped
viruses.24 The slope of the best-fit linear regression line for the
coronavirus log10 k data versus temperature was 0.065 ± 0.006
(y-intercept = −1.66; R2 = 0.75), meaning that log10 k (where
original units of k are d−1) increased by 0.065 for every 1 °C
increase in temperature. This slope is within the range
previously reported for nonenveloped viruses (i.e., 0.03−
0.07).24 The best-fit linear regression line for Phi6 log10 k data
versus temperature was not found to be significantly different
from that of the coronaviruses (p = 0.6).
Within the range of environmentally relevant temperatures
(i.e., 4−30 °C), the identified coronavirus experiments were
conducted at 4, 10 °C, and room temperature (defined as 22−
25 °C); therefore, mean log10 k values were calculated for each
of these ranges and for each experimental water type (Table 3).
Within each temperature range, mean and median log10 k were
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pubs.acs.org/journal/estlcu Review
Figure 1. Log10-transformed first-order decay rate constants (log10 k)
of coronaviruses (orange circles) and bacteriophage Phi6 (blue
circles) as a function of temperature. Each marker represents one
log10 k value extracted from the literature. These data include all
experiments identified in the review that were not conducted in the
presence of a disinfectant (n = 70). Solid line is the best-fit linear
regression line for the coronavirus data (slope = 0.065 ± 0.006; y-
intercept = −1.66; R2 = 0.75). Dashed line is the best-fit linear
regression line for the Phi6 data (slope = 0.070 ± 0.011; y-intercept =
−1.72; R2 = 0.65). The coronavirus markers with a cross (n = 2)
designate experiments with minimal virus decay that were assigned k
= 0.01 d−1. t99 is the time (d) required for 99% inactivation.
greatest in wastewater and lowest in laboratory water and
buffer (Figure 2). It has been suggested that increased k of the
enveloped viruses in wastewater could be attributed to a variety
of factors, including enzymatic activity, predation, and the
presence of solvents, detergents, and organic matter in
wastewater,28,29,31 although there has yet to be specific
evidence to definitively attribute these factors to virus decay.
To put coronavirus and Phi6 persistence data into context,
these data were compared to log10 k of nonenveloped
waterborne viruses (i.e., enteroviruses, rotaviruses, noroviruses,
hepatitis A virus, adenoviruses, astrovirus, and F+ and somatic
coliphages; Figure 2), which were compiled in a previously
conducted systematic review by Boehm et al.24 The log10 k
values for the nonenveloped viruses presented herein were
determined in unaltered environmental surface waters (i.e.,
fresh, estuarine, and marine waters) in the dark (i.e., without
sunlight exposure) and quantified using infectivity assays
(Table 4). There was more variability in the temperatures used
for the nonenveloped virus decay experiments; therefore, log10
k values from experiments conducted at a range of temper-
atures ±2 °C around each target temperature were included
for the nonenveloped viruses.
A similar range of log10 k values were identified for
coronaviruses, Phi6, and the nonenveloped viruses. It was
hypothesized that the lipid-containing envelope that surrounds
the coronavirus nucleocapsid and Phi6 capsids would make
these viruses more susceptible to degradation than the
nonenveloped viruses. However, the virus decay data identified
in this review suggests that enveloped and nonenveloped
viruses have similar rates of persistence in the aqueous
environment in the dark, despite different structures.
All identified coronavirus and Phi6 persistence experiments
were conducted under dark conditions. Sunlight exposure was
previously found to result in greater average k for the
nonenveloped viruses,24 as compared to in the dark, likely
owing to the ability of absorbed photons in the UVB region of
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Figure 2. Log10-transformed first-order decay rate constants (log10 k) of viruses for persistence experiments (without exposure to disinfectants) at
(a) 22−25 °C, (b) 9−12 °C, and (c) 4−6 °C. Each coronavirus (orange circles) and Phi6 (blue circles) marker represents one log10 k value
extracted from the literature; the horizontal bar represents the median value. Coronavirus markers with a cross (n = 2) designate experiments with
minimal virus decay that were assigned k = 0.01 d−1. The log10 k values for the nonenveloped viruses (i.e., enteroviruses, rotaviruses, noroviruses,
hepatitis A virus, adenoviruses, astrovirus, and F+ and somatic coliphages; green circles) were collected from the systematic review by Boehm et
al.24 and are decay rate constants determined in unaltered environmental surface waters (i.e., fresh, estuarine, and marine waters) in the dark and
quantified using culture-based methods (n for each is presented in Table 4). t99 is the time (d) required for 99% inactivation.

Table 3. Mean Log10-Transformed First-Order Decay Rate Constants (log10 k) for Coronaviruses and Bacteriophage Phi6 in
Each Experimental Matrix and Each Temperaturea
Coronaviruses [mean log10k ± stdev (n)] Bacteriophage Phi6 [mean log10k ± stdev (n)]
4 °C 10 °C 22−25 °C 4 °C 10 °C 22−25 °C
Laboratory Water/Buffer −1.84 ± 0.23 (2) − −0.74 ± 0.14 (4) −1.46 (1) − 0.16 (1)
Cell Culture Supernatant − − −0.24 ± 0.19 (3) −1.60 ± 0.15 (2) − −0.46 ± 0.14 (2)
Fresh Water −1.39 ± 0.86 (2) − −0.70 ± 0.08 (2) − − −0.31 ± 0.25 (2)
Tap Water −1.60 ± 0.46 (2) − −0.21 ± 0.05 (4) − − −0.13 (1)
Raw Wastewater −1.10 ± 0.12 (2) 0.15 (1) 0.25 ± 0.30 (7) − 0.34 (1) 0.88 (1)
Sterilized Wastewater − −0.30 (1) 0.46 ± 0.01 (3) − −0.39 (1) 0.06 ± 0.11 (3)
a
Units on k before transformation are day−1. “−” denotes that no k values were identified. n is the number of experiments identified. k values
determined outside of the designated temperature ranges were not included in this analysis.

the solar spectrum to damage genomic DNA and RNA.41−43 It
is therefore expected that coronavirus and Phi6 k would be
greater than those presented herein if measured in sunlit
waters. While not proven, there is a possibility that sunlight
exposure would have a larger impact on increasing coronavirus
k than it would on k of the waterborne viruses, given that
coronaviruses have a ssRNA genome that is much larger than
the genomes of the nonenveloped ssRNA viruses (e.g.,
enteroviruses ∼7−8.5 kb).21 The large coronavirus genome
548
presents more targets for direct sunlight-induced RNA damage,
which could result in faster rates of inactivation. This would
not necessarily be the case for Phi6, given that dsRNA
genomes have been found to be more resistant to UV
damage.44 A further discussion of this is provided in the UV254
disinfection section below.
Disinfection with UV254. Only one experiment was
identified for Phi6 disinfection with UV254 (conducted in
laboratory buffer at 23 °C; k = 0.067 cm2/mJ; log10 k = −1.17;
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Environmental Science & Technology Letters
Table 4. Mean Log10-Transformed First-Order Decay Rate Constants (log10 k) for Nonenveloped Waterborne Virusesa
4−6 °C
Enteroviruses −0.51 ± 0.41 (35)
Rotaviruses −1.05 ± 0.45 (5)
Noroviruses −1.70 (1)
Hepatitis A virus −1.26 ± 0.14 (2)
Adenoviruses −1.29 ± 0.13 (3)
Astrovirus −1.19 (1)
F+ coliphages −1.15 ± 1.01 (21)
Somatic coliphages −1.87 ± 1.47 (7)
a
Units on k before transformation are day−1. Data were originally collected in the systematic review by Boehm et al.24 and are decay rate constants
determined in unaltered environmental surface waters (i.e., fresh, estuarine, and marine waters) in the dark and quantified using culture-based
methods. “−” denotes that no k values were identified. n is the number of experiments identified.
Figure 3. Log10-transformed first-order decay rate constants (log10 k)
with exposure to germicidal ultraviolet light (UV254). The Phi6 log10 k
value (blue circle) is from Ye et al.20 The remaining log10 k values
were extracted from the UV254 disinfection review paper by Hijnen et
al.48 (bacteria represented by gray circles and nonenveloped viruses
represented by green circles). Only k determined with low pressure
UV254 light (as opposed to medium pressure UV) were included. Gray
blocks for the human viruses and Phi6 are the range of log10 k values
predicted by Lytle and Sagripanti (see main text for details).21 The
secondary y-axis represents the UV254 dose (i.e., UV254 irradiance ×
time) required for 99.99% microorganism inactivation. The gray
horizontal line represents the USEPA recommended UV254 dose for
99.99% virus inactivation.22
Figure 3),20 and no experiments were identified for UV254
disinfection of coronaviruses that met the inclusion criteria. To
supplement the limited data available on UV254 disinfection
kinetics, we reviewed UV254 disinfection rates predicted for
human viruses by Lytle and Sagripanti (hereafter, LS).21 The
LS model uses the nucleic acid type of the viral genome (e.g.,
ssRNA) and the genome length to predict UV254 inactivation
rates.21 Briefly, eq 1 was used to calculate k, where D37 is the
UV254 dose required for a 37% reduction in virus infectivity (J/
m2), SnS is the genome size-normalized sensitivity of the virus
to UV254 (J/m2·kilobase; SnS values were calculated and
reported by LS and are correlated with nucleic acid type), and
GS is the genome length (kilobase).21 LS reported virus-
specific values for D37, which we converted to k.
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Mean log10k ± stdev (n)
9−12 °C 22−25 °C
−0.23 ± 0.37 (7) −0.05 ± 0.33 (95)
− −0.32 ± 0.10 (2)
− −0.75 ± 0.44 (2)
− −0.63 ± 0.41 (5)
−0.77 (1) −0.69 ± 0.81 (2)
− −0.74 ± 0.30 (2)
−0.26 ± 0.16 (5) 0.19 ± 0.32 (31)
−0.43 ± 0.20 (4) −0.25 ± 0.47 (13)
1 SnS
= D37 =
k GS (1)
LS predicted coronavirus UV254 k to be between 2.6 and 4.0
cm2/mJ (log10 k = 0.41−0.60; based on a genome length of
between 20 and 31 kilobases; Figure 3) and validated these
values through comparison with a previously measured
inactivation rate constant of a torovirus (i.e., Berne virus).21
Toroviruses are a genus of enveloped ssRNA viruses with a
virion size, genome length, and morphology similar to
coronaviruses.45 In fact, toroviruses were classified as a genus
within the Coronaviridae family until 2018, when the genus was
reclassified under the Tobaniviridae family.46 The Berne virus
used for validation of modeled UV254 k had a measured k of 1.9
cm2/mJ (log10 k = 0.29; data extracted from Figure 2 in Weiss
and Horzinek47), which was close to, but slightly lower than,
the range of k predicted by LS.
Lytle and Sagripanti21 focused their effort on viruses
infecting vertebrates. As such, the authors did not include
predicted UV254 disinfection rates for bacteriophage, such as
Phi6. Therefore, we calculated a range of estimated k for Phi6
using eq 1, with GS equal to 13.4 kilobases and two values of
SnS representing median SnS values for the two dsRNA viruses
included in the analysis by LS (i.e., Birnaviridae and Reoviridae;
median SnS = 1400 and 3800 J/m2·kilobase, respectively). The
resulting predicted Phi6 k ranged between 0.035 and 0.096
cm2/mJ (log10 k = −1.45 to −1.02), which overlaps with
measured Phi6 UV254 k.
To compare Phi6 and predicted coronavirus UV254 k with
those of fecal indicator bacteria and nonenveloped viruses, k
for the latter were extracted from the UV254 disinfection review
paper by Hijnen et al.48 In that review, the researchers
compiled UV254 disinfection data from studies that (i) used
culture-based assays to quantify microorganism viability, (ii)
provided data on the UV254 fluence applied, and (iii) corrected
the fluence for light attenuation in the experimental solution.
Hijnen et al.48 used the compiled data to calculate k values as a
function of fluence; these values were calculated using log10
inactivation data. We calculated true first-order k by converting
these values to a ln scale [i.e., the values reported by Hijnen et
al.48 were multiplied by ln(10)]; only k values measured with
monochromatic UV254 light were included.
The k values from Hijnen et al.48 were as follows (error is
presented as the 95% confidence interval; n = number of
studies included by Hijnen et al.48 in calculating k for each
microorganism; Figure 3): E. coli = 1.2 ± 0.11 cm2/mJ (not
including O157:H7, n = 6), Enterococcus faecalis = 0.72 ± 0.074
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Environmental Science & Technology Letters pubs.acs.org/journal/estlcu Review

Figure 4. Log10-transformed first-order decay rate constants (log10 k) with exposure to free chlorine. No coronavirus free chlorine disinfection
experiments with quantifiable k were identified. The Phi6 log10 k values (blue markers) are from Ye et al.20 and Adcock et al.32 Laboratory-strain
bacteria log10 k are represented by gray markers;51−54 nonenveloped viruses are represented by green markers.55−59 All experiments were conducted
between pH 7 and 8. Circles represent experiments conducted at a temperature of 5 °C. Squares represent experiments between 20 and 23 °C.
Diamonds represent experiments where the temperature was not indicated. The secondary y-axis represents the free chlorine dose (i.e., free
chlorine concentration × time) required for 99.99% microorganism inactivation. The gray horizontal band represents the USEPA recommended
chlorine dose for 99.99% virus removal at pH between 6 and 9 and temperatures between 20 and 25 °C.23

cm2/mJ (identified in the review by its former name,
Streptococcus faecalis; n = 2), hepatitis A virus = 0.42 ±
0.065 cm2/mJ (n = 3), poliovirus type 1 = 0.31 ± 0.016 cm2/
mJ (n = 6), coxsackievirus B5 = 0.27 ± 0.014 cm2/mJ (n = 2),
calicivirus = 0.24 ± cm2/mJ (feline, canine; n = 3), rotavirus
SA-11 = 0.24 ± cm2/mJ (n = 5), bacteriophage MS2 = 0.13 ±
0.005 cm2/mJ (n = 5), and adenovirus = 0.055 ± 0.002 cm2/
mJ (types 2, 15, 40 and 41; n = 5).48 For comparison, k
predicted by LS21 for the human viruses are also presented in
Figure 3.
The predicted range of coronavirus UV254 k is greater than
the k of Phi6, the nonenveloped viruses, and the fecal indicator
bacteria E. coli and Enterococcus faecalis. The main target of
UV254 damage to viruses is their nucleic acids, and the large
ssRNA genome of coronaviruses likely makes them particularly
susceptible. The UV 254 degradation rates of free and
encapsidated ssRNA have been found to be greater than
those of dsDNA and dsRNA, when normalized by the number
of bases.44 Additionally, larger genomes have more targets of
damage, leading to greater decay rate constants,21,44 and
coronaviruses have larger genomes than most other ssRNA and
ssDNA viruses. The single measured UV254 k of Phi6 was
smaller than those of the other included bacteria and viruses,
with the exception of adenoviruses, suggesting that it is
particularly resistant to UV254. This is likely due to the dsRNA
nature of the Phi6 genome; dsRNA has been found to be more
resistant to UV254 than the other nucleic acid types.44
One paper 39 was identified that described a UV 254
inactivation experiment conducted with SARS-CoV-1 in cell
culture supernatant. This experiment was not included in the
analysis because there was no description of how the reported
UV254 irradiance used in experiments was measured or if it was
corrected for light attenuation in the experimental solution.
Nonetheless, the study39 suggested a lower coronavirus UV254
k than the range predicted by LS.21 This discrepancy,
combined with limited UV254 disinfection data available for
coronaviruses, highlight a critical need for more research to
generate additional data on UV254 inactivation of actual
coronaviruses. Future experiments should be conducted with
550
careful measurement of the UV254 fluence applied.49 Addition-
ally, given different nucleic acid types and genome length, it is
unclear whether Phi6 is a good surrogate for coronaviruses
when evaluating or monitoring UV254 disinfection.
Disinfection with Free Chlorine. No experiments of
coronavirus k with exposure to free chlorine were identified
that met the systematic review inclusion criteria. Two
experiments were identified that evaluated Phi6 disinfection
with exposure to chlorine.20,32 Both experiments were
conducted in laboratory buffers: one was conducted at pH 7
and 5 °C and the other at pH 7.4 and 23 °C. A greater free
chlorine k value was observed for Phi6 at 23 °C than at 5 °C,
which is expected, given that increased temperatures typically
result in greater observed chlorine disinfection rates.50
Free chlorine decay rate constants for indicator bacteria51−54
and nonenveloped viruses55−59 (all quantified using culture-
based viability assays) were identified from the literature to
place Phi6 free chlorine k in perspective. While the review of
the literature describing free chlorine disinfection of these
additional microorganisms was not systematic, they at least
give an indication of relative free chlorine susceptibility. The
Phi6 free chlorine k at 5 °C was within the range of k measured
for the nonenveloped viruses at the same temperature: Phi6 k
was lower than those of the adenoviruses but greater than
those of MS2 and the picornaviruses (i.e., echoviruses,
coxsackieviruses). Interestingly, Phi6 free chlorine k at 23 °C
was greater than those of E. coli and Enterococcus faecalis
measured around the same temperature, suggesting greater free
chlorine susceptibility of the enveloped bacteriophage
compared to the indicator bacteria.
One paper evaluated free chlorine disinfection of SARS-
CoV-1 in wastewater.40 While the data provided in the paper
were not presented in a manner that allowed for extraction of a
disinfection rate constant that could be compared to other
values, the study did find that when exposed to free chlorine,
SARS-CoV-1 was inactivated at a rate faster than those of E.
coli and bacteriophage f2 (a nonenveloped, ssRNA phage that
is similar to MS2).
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Environmental Science & Technology Letters
Ye et al.20 evaluated the targets of oxidative damage for
bacteriophage Phi6 exposed to free chlorine. While some
oxidation of the genome and membrane-bound lipids and
proteins was observed, the main driver of free chlorine
inactivation of Phi6 was found to be oxidation of proteins in
the capsid (which is located within the lipid envelope). It is
unclear whether this disinfection mechanism, and subsequent
decay rate constants, can be extrapolated to coronaviruses.
Implications. The United States Environmental Protection
Agency (USEPA) provides tables outlining the disinfectant
doses [i.e., UV254 irradiance × time (mJ/cm2) or chlorine
concentration × time (mg-min/L)] required for 4-log (i.e.,
99.99%) inactivation of viruses with exposure to UV25422 and
free chlorine.23 We converted k reported above to disinfectant
doses for 4-log inactivation using the following equation
ln(0.0001)
disinfectant dose99.99% = −
k (2)
The coronavirus UV254 dose required for 4-log virus
disinfection (based on log10 k predicted by LS21) was lower
than the USEPA recommended value (disinfectant dose99.99% =
186 mJ/cm2; Figure 3), suggesting that coronaviruses would be
efficiently inactivated if following the USEPA recommended
UV254 dose for virus disinfection. The free chlorine dose
required for 4-log disinfection of Phi6 was lower than the
USEPA recommended dose (e.g., disinfectant dose99.99% = 3
mg-min/L at 20 °C and pH between 6 and 9; Figure 4);
however, there is no direct evidence of what this would mean
for coronavirus disinfection by free chlorine.
Overall, there is very little data available in the literature on
coronavirus inactivation in aqueous solution with exposure to
disinfectants. There is therefore a critical need for additional
studies that specifically evaluate the disinfection kinetics of
human coronaviruses and their surrogates that infect animals
(e.g., MHV, TGEV, and FIPV). Given different genome
compositions and virion structures between coronaviruses and
bacteriophage Phi6, it is not clear whether Phi6 should be used
as a surrogate for evaluating coronavirus free chlorine or UV254
k.
Alternatively, the available data on coronavirus survival in
the absence of disinfectants suggest that these viruses are as
persistent as nonenveloped viruses and bacteriophage Phi6 in
the aqueous environment, including in natural waters and
wastewaters. Additional research on the persistence of
coronaviruses in water, as well as in the presence of sunlight,
will further our understanding of their fate outside of the
human body.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.estlett.0c00313.
Systematic review metadata for coronaviruses and Phi6
(XLSX)
■ AUTHOR INFORMATION
Corresponding Author
Andrea I. Silverman − Department of Civil and Urban
Engineering, New York University Tandon School of
Engineering, Brooklyn, New York 11201, United States;
Department of Global Health, New York University School of
Global Public Health, New York, New York 10012, United
551
pubs.acs.org/journal/estlcu Review
States; orcid.org/0000-0001-8199-5860; Phone: 646-997-
3018; Email: andrea.silverman@nyu.edu
Author
Alexandria B. Boehm − Department of Civil and
Environmental Engineering, Stanford University, Stanford,
California 94305, United States; orcid.org/0000-0002-
8162-5090
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.estlett.0c00313
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was partially supported by the National Science
Foundation (CBET-2022877 awarded to A.B.B.).
■
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