Microbial Risk Analysis xxx (xxxx) xxxx

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Microbial Risk Analysis

journal homepage: www.elsevier.com/locate/mran

Refined ambient water quality thresholds for human-associated fecal

indicator HF183 for recreational waters with and without co-occurring gull
fecal contamination
A.B. Boehma, J.A. Sollerb,
⁎

a
Department of Civil & Environmental Engineering, Stanford University, Stanford, CA, United States 94305-4020
b
Soller Environmental, Berkeley, California, United States

ARTICLE INFO ABSTRACT

Keywords: A number of studies have used quantitative microbial risk assessment (QMRA) to derive risk-based water quality
Microbial risk assessment thresholds (RBTs) for a diverse set of novel water quality indicators in recreational waters. Over time, the QMRA
Recreational water approach has been refined with respect to model inputs and hazard characterization. Recent work considered
HF183 differential decay of pathogens and indicators, and mixtures of contamination of diverse ages. In the present
Alternative indicator
study, we present a refined ambient water quality RBT for the human-associated marker HF183. The new es­
timate updates previous work as it specifically considers contamination aging through the inclusion of tem­
perature-specific organism decay, and the presence of mixtures of human sewage contamination of diverse ages.
Based on these analyses we derived a RBT of 525 HF183 copies/100 mL as representative of conditions con­
sistent with those described in the 2012 Recreational Water Quality Criteria (32 illnesses /1000). In recreational
waters, microbial contamination due to gulls is also common. To account for this, we consider the case where
human contamination from sewage co-occurs with contamination from gull feces. The resultant proposed RBTs
for HF183 range from 1 to 525 copies/100 ml and are a function of the amount of gull fecal contamination that is
present in the water. The proposed RBTs can be considered for use in the evaluation of recreational water
quality.

1. Introduction health from recreational exposures as compared to fecal contamination

The protection of public health is the cornerstone of recreational
water management worldwide (WHO, 2003; USEPA, 2012a). Histori­
cally, epidemiological studies have been used to establish water quality
criteria values or recommendations to ensure public health protection
(Pruss, 1998; Zmirou et al., 2003; Wade et al., 2003). Most recreational
water epidemiological studies and subsequent water quality criteria or
regulations for recreational waters have relied on fecal indicator bac­
teria (FIB) such as fecal coliform or enterococci as indices of water
quality, since those FIB have generally demonstrated good correspon­
dence with adverse health effects, and are inexpensive and convenient
to enumerate. Those water quality criteria and related regulations are
considered to be protective of public health under a wide variety of
conditions (USEPA, 2012a; USEPA, 1986). However, the scientific un­
derstanding of risk to human health from recreational water exposure
has evolved significantly over the last decade – it is now generally ac­
cepted that human fecal contamination poses the greatest risk to human
from other sources, such as gulls, whereas FIB sources can be diverse
and widespread (Soller et al., 2010a; Soller et al., 2010b; Korajkic et al.,
2018). It is also accepted that there can be site specific conditions
where alternative indicators may yield better estimates of adverse
health outcomes, depending on the sources of contamination to a par­
ticular waterbody (USEPA, 2012b; USEPA, 2014a; USEPA, 2014b;
USEPA, 2015).
To address the disparity between potential FIB contributions from
wide-ranging sources and the most important contributors of risk to
human health, scientists have advanced the use of alternative in­
dicators, such as molecular markers that are specific to the source of
contamination (Boehm et al., 2013; Shanks and Korajkic, 2020; Bae and
Wuertz, 2012; Staley et al., 2012; Symonds et al., 2016). These mole­
cular markers were originally developed for microbial source tracking
purposes, and have gained broad acceptance among water quality sci­
entists and water quality managers. Success with molecular markers
within the context of microbial source tracking has led to interest in

⁎
Corresponding author.
E-mail address: jsoller@sollerenvironmental.com (J.A. Soller).

https://doi.org/10.1016/j.mran.2020.100139
Received 24 April 2020; Received in revised form 31 August 2020; Accepted 5 September 2020
2352-3522/ © 2020 Elsevier B.V. All rights reserved.

Please cite this article as: A.B. Boehm and J.A. Soller, Microbial Risk Analysis, https://doi.org/10.1016/j.mran.2020.100139
A.B. Boehm and J.A. Soller
their wider use as potential water quality management tools. Their use
could allow remediation efforts to focus on sources of fecal con­
tamination that are the greatest source of risk to human health. The
Recreational Water Quality Criteria in the United States (USEPA,
2012a), are now based on specific levels of human health protection (32
or 36 illnesses per 1000) rather than specific levels of FIB, as was the
case previously (USEPA, 1986). Therefore, there is now the possibility
for a site-specific, cost-effective, and health protective approach to
water quality management (Given et al., 2006) that focuses on specific
sources of contamination of critical interest rather than on generic FIB
(Environmental Incentives and EcoNorthwest 2017). In addition to the
potential use of a viral indicator (USEPA, 2015), molecular markers are
another promising tool that can be used to help facilitate and imple­
ment this new approach.
Boehm et al. (2015) developed quantitative estimates of the risk
associated with specific levels of molecular markers in recreational
waters. They used quantitative microbial risk assessment (QMRA) to
simulate the risk of gastrointestinal (GI) illness associated with re­
creating in waters containing different concentrations of human-asso­
ciated fecal markers (HF183 and HumM2) from raw sewage. HF183
was of particular interest in that study and is currently considered to be
the state-of-the-art human molecular marker because it has been eval­
uated in a number of studies and been shown to be highly specific to
human feces with little to no cross reactivity with other host feces
(Boehm et al., 2013; Layton et al., 2013; Shanks et al., 2009;
Shanks et al., 2010; Griffith et al., 2003). Further justification for the
use of HF183 in this regard is provided by a recent review article which
summarized the sensitivity and specificity metrics for the HF183 assay -
a collective sensitivity of 83.1% calculated from 1242 human waste
samples and specificity of 94.6% based on a collection of 2966 non­
human fecal samples was reported (Ahmed et al., 2016). Additionally, a
national study from the US reported 100% sensitivity of HF183 qPCR
DNA target determined from 54 sewage samples collected from 39
different states (Shanks et al., 2010). Albeit rare, the HF183 DNA target
has been reported in a small proportion of chicken, dog, gull, and deer
reference fecal samples almost always at concentrations considerably
lower than levels observed in human waste (Shanks and Korajkic, 2020;
Nguyen et al., 2018; Ahmed et al., 2012).
Since the Boehm et al. (2015) study, a number of other studies have
used the QMRA approach to identify concentrations for human-asso­
ciated genetic markers that correspond to defined public health risk
benchmarks for sewage-impacted recreational water (Zhang et al.,
2019; Boehm et al., 2018; Ahmed et al., 2018). These risk-based
threshold estimates (RBT) are valuable research tools to compare the
utility of various indicators to each other and across different experi­
mental scenarios based on a set of defined model assumptions. A recent
review review indicates that a diverse set of novel water quality in­
dicators have been used for this purpose under ever broadening en­
vironmental conditions (Zhang et al., 2019). Recent work has con­
sidered differential decay of pathogens and indicators and mixtures of
contamination of diverse ages. For example, Boehm et al. (2018) ex­
tended their earlier work to consider aging of contamination;
Brown et al. (2017a) developed RBTs for gull-associated fecal marker
concentrations for the case that gull feces were not aged in the en­
vironment; Brown et al. (2017b) considered mixtures of un-aged human
and gull-associated contamination and marker levels; and Crank et al.
(2019) developed RBTs with exposure to the crAssphage human marker
and pepper mild mottle virus from fresh, unaged sewage. Wu et al.
(2020) considered the risk associated with exposure to feces with a
range of ages.
In the present study, we present refined ambient water quality RBTs
for human-associated HF183 and the gull and pigeon-associated marker
Catellicoccus marimammalium (Sinigalliano et al., 2013; Lee et al.,
2013). The new estimates update previous work by adding adenovirus
to the previous reference pathogens for human-associated fecal con­
tamination, specifically considering contamination aging through the
2
Microbial Risk Analysis xxx (xxxx) xxxx
inclusion of temperature-specific organism decay, and considering a
scenario where human contamination from sewage co-occurs with
contamination from gull feces. These new scenarios were considered in
the present study to address a growing desire from the regulatory
community for guidance on the use of alternative fecal indicators for
monitoring bathing water quality for the protection of swimmer health.
2. Methods
Exposure to untreated, raw sewage of known age: HF183. A
static QMRA was used to estimate gastrointestinal illness risk from
swimming in surface waters with varying concentrations of HF183 from
untreated sewage of different ages using R (Version 1.1.463). The in­
fluence of immunity and secondary transmission was not considered in
the models (Soller and Eisenberg, 2008). In the QMRA, HF183 serves as
an index for the amount of sewage present in surface water. The QMRA
considers the cumulative risk from exposure to reference pathogens
adenovirus (not considered by Boehm et al. (2015)), norovirus, Giardia,
Cryptosporidium, E. coli O157:H7, Campylobacter, and Salmonella as re­
commended by USEPA and used extensively in bathing water QMRAs
(Soller et al., 2010a; Soller et al., 2017a; Soller et al., 2015d;
Schoen and Ashbolt, 2010).
The volume fraction of raw sewage in surface water (Vs) serves as an
input to the model and varies between 10−8 and 1 in increments of 0.5
log10 units. The age of the contamination (the time it has spent in
surface water after being released from an untreated, raw sewage
source) is τ. τ serves as a model input and varies between 0 (unaged)
and 15 days (d) in 0.5 d increments. A total of 527 Vs-τ combinations
(31 distinct ages and 17 distinct Vs) were modeled. The increment size
for Vs and τ were chosen to be fine enough to elucidate trends in the
results without requiring excessive computational time; choosing dif­
ferent increment sizes did not affect the results (data not shown). The
choice of 15 days for the largest age was arbitrary and chosen to be
large enough to illustrate the effect of aging.
After Vs and τ are specified as model inputs, the concentration of ith
reference pathogen in surface waters (Ci_surface) is modeled as follows:
Ci _ surface = Vs Ci _ sewage e ki
(1)
where Ci_sewage and ki are, respectively, the ith reference pathogen
concentration in sewage, and ki is the ith reference pathogen's first order
decay rate constant. In Eq. (1), Ci_sewage are described by distributions
(Table 1) that were obtained from the literature and have been used in
previous QMRA studies (Brown et al., 2017b; Soller et al., 2017b;
Boehm, 2019).
Two systematic reviews and meta-analysis of decay rate constants in
surface waters indicated that decay rate constants of allochthonous
microorganisms in surface waters are a function of water temperature
(Boehm et al., 2018; Boehm et al., 2019). Those meta-analyses in­
dicated little evidence that salinity or nutrient content of the water
significantly affected decay rate constants, so those factors are not
considered herein. Here we considered ki values at 20°C. This tem­
perature represents an annual average temperature of waters off San
Diego, CA based on data obtained from Scripps pier (data not shown).
An annual average temperature was chosen for this work to identify
risk-based thresholds that would be appropriate any time of year.
Previous work compiled raw surface water decay rate constants for
HF183 and the reference pathogens used in this study for surface waters
and noted the experimental conditions, including temperatures, at
which the rate constants were derived (Boehm et al., 2018;
Boehm et al., 2019). Those systematic reviews only included results
from experiments conducted with raw water and excluded experiments
that were conducted with sterilized water or artificial waters; therefore,
the decay rate constants represent estimates of decay in real environ­
mental waters. We derived distributions for the rate constants needed in
Eq. (1) (ki) for temperatures of 20 °C using the methods outlined in
A.B. Boehm and J.A. Soller Microbial Risk Analysis xxx (xxxx) xxxx

probability of infection, Pill|inf is probability of becoming ill after infection. Note that units of pathogens are per liter and for indicators is per ml to reflect the units used in the literature for these parameters. CFU is colony
forming unit, MPN is most probable number, copy refers to gene copy number, IU is infectious unit, and PFU is plaque forming unit. 1F1 is the hypergeometric function. When specified, Pill|inf are represented by a range of
parameters, as indicated, drawn from a uniform distribution. Pill|inf for Campylobacter is dose-dependent with r = 2.44 × 108 and n = 3.63 × 10−9. References (Refs) for Pinf and Pill|inf are provided in the last column.
Untreated sewage concentrations for reference enteric pathogens and indicators, and dose-response relations, and Pill|inf for reference enteric pathogens. Unit (refs) is the concentration in sewage, µ is the dose, Pinf is
Table 2
Normal distributions of log10-transformed k for the organisms and indicators

Teunis et al., 2008; Ludwig et al., 2002; Werber et al., 2008;

used in the QMRA. Mean and standard deviation (SD) are provided. Units of k
before they were log10-transformed are d− 1. CAT k is described by a uniform
distribution bounded by 0.11 and 3.5 per day, as described in the text.

Haas et al., 1999; Fazil, 1996; Teunis et al., 1999

Organism/ mean SD

Rose and Gerba, 1991; Eisenberg et al., 1996

Indicator log10-k log10-k

Salmonella spp. −0.13 0.058

Campylobacter 0.45 0.16
E. coli O157:H7 −0.36 0.044
Cryptosporidium −1.01 0.16
Giardia −1.59 0.21
Bielaszewska et al., 1997

norovirus −1.01 0.14

adenovirus −0.34 0.15
Teunis et al., 2005

Teunis et al., 2008

Teunis et al., 2016
HF183 0.063 0.34
enterococci 0.068 0.49
US EPA 2006

Boehm (2019). Briefly, a statistical model relating ki to temperature was

Refs

used to generate predictions for ki at the chosen temperature. The

prediction includes a mean and variance describing a predicted dis­
Pill|inf (distribution)

0.17–0.4 (uniform)

tribution. ki were subsequently, randomly drawn from their respective

0.2–0.6 (uniform)

0.2–0.7 (uniform)
0.3–0.8 (uniform)
0.3–0.7(uniform)

distributions (Table 2). Note that these ki values are estimates for the
0.5 (uniform)
1-(1+nµ)−r

pathogens as enumerated using culture-based methods (Boehm, 2019).

In deriving Eq. (1), it is assumed that the advection and dispersion of
indicator and reference pathogens are identical, and any non-con­
NA
NA

servative behavior of water quality targets is adequately captured by

1- 1F1 (0.04, 0.04+ 0.055, -µ)

first order kinetics.

1–1F1(0.024,0.024+0.011,-µ)

1- 1F1 (5.11, 5.11+ 2.8, -µ)

It is assumed that the volume (V) of water ingested by a swimmer

per swimming event follows the log10-normal distribution with a mean
−0.3126

1-(1+ µ/48.8)−0.248

1 - exp(−0.0199 µ)

of 1.20 and standard deviation of 0.68; units of V are ml (DeFlorio-

1 - exp(−0.09 µ)
1-(1+ µ/2884)

Barker et al., 2018). The dose of pathogen i, µi, is given by Ci_surfaceV.

The dose was used as input to the reference pathogen dose-response
functions (Table 1) to determine the probability of infection (Pinf_i). The
The two values separated by a comma are the mean and standard deviation of a log10-normal distribution.
The two values separated by a comma are the minimum and maximum of the log10-uniform distribution.
Pinf

probability of illness (Pill_i) was calculated by multiplying the prob­

NA
NA

ability of infection by the probability of illness given infection Pill|inf_i

(Table 1). Pill|inf_i was randomly drawn from a uniform distribution for
each model iteration except for the case of Campylobacter which used a
dose-dependent formula (Table 1). The cumulative risk of illness from
oocysts/L (Jian et al., 2015; Crockett, 2007; Harwood et al., 2005;

Lower range is not detected and −1 is used as a lower bound. NA means not applicable.
IU/L Soller et al., 2017b; Hurst et al., 1988; Hewitt et al., 2011
CFU/L (Koivunen et al., 2003; Lemarchand and Lebaron, 2003)

exposure to all reference pathogens (Pill) is given by

Pill = 1 (1 Pilli)
(2)
cysts/L (Harwood et al., 2005; Kitajima et al., 2014)

It was assumed that infection and illness for each pathogen is in­
References for sewage concentration range are provided adjacent to the unit.

dependent. 50,000 iterations were obtained for each Vs-τ combination

(Staley et al., 2012; Sinigalliano et al., 2013). The median, interquartile
range, and 10th and 90th percentiles of Pill for each Vs -τ combination
Nasser, 2016; Schoen et al., 2017)
CFU/L (García-Aljaro et al., 2004)

were calculated from the respective 50,000 iterations. Pill was com­
copies/ml (Shanks et al., 2010)

pared to the value 32/1000 which is equal to one of the key risk
CFU/ml (Soller et al., 2010a)
MPN/L (Stampi et al., 1993)

copy/L (Eftim et al., 2017)

thresholds published by USEPA for bathing waters (USEPA, 2012). For

each τ, the Vs for which the median simulated risk was 32/1000 was
determined – this value of Vs is hereafter referred to as “risk-based
threshold Vs”. Note that Vs is independent of the indicator used to
measure fecal contamination.
Unit (refs)

The concentration of HF183 in the surface water when the volume

fraction of sewage is equal to risk based threshold Vs and aged a time τ
was determined as follows:
[5.212, 0.566]^
#

[1.75,3.84] #
[−0.52, 3.7]
[−1,3.3] #,*

(3)
CHF183 = Vs CHF183_ sewage e k HF183
#
[2.9,4.6] #

[4.0,1.1] ^
[0.51,4.2]
Ci_sewage

[2.8, 5]#
#

where CHF183_sewage and kHF183 are, respectively, the concentration of

[0.5,5]

HF183 in sewage and the decay rate constant of HF183 in surface

waters, and Vs is the risk based threshold Vs. In Eq. (3), CHF183_sewage is
Organism/Target

described by a distribution (Table 1), and kHF183 is also described by a

E. coli O157:H7

Cryptosporidium
Salmonella spp.
Campylobacter

distribution obtained using the same methods as described for ki

enterococci
adenovirus
norovirus

(Table 2). To find CHF183, values were drawn from the distributions
Giardia
Table 1

HF183

50,000 times and the median CHF183 as well as other percentiles de­
#

scribing the resultant distribution were recorded. The median of this

^

3
A.B. Boehm and J.A. Soller
distribution is referred to as the “risk-based threshold for HF183”.
Exposure to untreated sewage of unknown age: HF183. The age
of sewage in surface water is usually unknown and furthermore, surface
water contamination may represent a mixture of contamination of di­
verse ages. We repeated the QMRA for a scenario where the indicator
HF183 is measured in surface waters, and the contamination age is
unknown. We used the same methods as described by Boehm et al.
(2018; 2019). The concentration of HF183 as measured in surface water
(CHF183) was specified as an input to the model and varied between 100
and 105 copies/100 ml in increments of 0.2 log10 units. The con­
centrations of pathogens in surface waters (Ci_surface) was modeled as
follows:
CHF183 Ci_ sewage k
Ci _ surface = e
CHF183_ sewage (4)
where Δk = kHF183 - ki, and Ci_sewage and CHF183_sewage are, respectively,
the ith reference pathogen and HF183 concentrations in sewage, and ki
and kHF183 are their first order decay rate constants. These variables
were all drawn from their respective distributions (Tables 1 and 2). τ
was drawn from a uniform distribution ranging from 0 to a maximum
realistic value (τmax) given the specified Cmeas:
max = 1/m [log10Cmeas b] (5)
where b is the log10-transformed median concentration of HF183 in raw
sewage (CHF183), and m is -kmed*log10e where kmed is the median HF183
decay rate constant (kHF183) in surface waters (Table 4). Further con­
ceptual discussion of τmax can be found in Boehm (2019). Ci_surface was
used to determine dose, the dose was input into the dose-response
equations, and then Pill was determined, as described in the previous
section.
Model output, including calculation of the risk-based threshold, was
handled as described in the previous section. Note that this model re­
quires Cmeas to be input rather than Vs since τmax is dependent speci­
fically on Cmeas (Eq. (5)).
Exposure to untreated sewage of unknown age: ENT. We re­
peated the analysis described in the previous section, but instead of
using HF183 as an index for the amount of sewage present in the sur­
face water, we used culturable enterococci (ENT). The modeling
method is the same except CENT was used in place of CHF183, kENT was
used in place of kHF183, and CENT_sewage was used in place of
CHF183_sewage in Eq. (4); and the m and b in Eq. (5) are those for ENT
(Table 4). CENT_sewage and kENT are described by distributions (Tables 1
and 2). kENT was extracted from the literature review completed by
Brooks and Field (2016). We downloaded their supplementary data of
compiled kENT. After removing suspected duplicates, and converting
reported T90 and T99 values to k values, we log10-transformed the kENT
and took their mean and standard deviation to define a log-normal
distribution for modeling (Table 2). We were unable to account for the
effect of temperature using their data as the appropriate meta-data were
not provided .
Exposure to gull feces of known age: CAT. A molecular marker
for a bacterium Catellicoccus marimammalium (CAT) (Sinigalliano et al.,
2013; Lee et al., 2013; Lu et al., 2008) has proven to be a sensitive and
specific marker of bird feces (gull and pigeon) in surface waters
(Boehm et al., 2013). We investigated the risk-based threshold for CAT
in recreational waters assuming they are contaminated with gull feces
of a known age. This method mirrors that described above for in­
vestigation exposure to raw sewage using HF183 as an indicator, except
reference pathogens are Salmonella and Campylobacter (Soller et al.,
2010a; Brown et al., 2017a; Schoen and Ashbolt, 2010). This approach
mirrors that used by Brown et al. (2017a) with slight modifications.
The mass concentration of bird feces in surface water (Mb) serves as
an input to the model and varies between 10−8 and 100 g/100 ml in
increments of 0.5 log10 units. The upper limit of 100 g/ 100 ml implies
the water is ~ 100% gull feces by mass and represents a maximum
4
Microbial Risk Analysis xxx (xxxx) xxxx
Table 3
Concentrations of Salmonella, Campylobacter, and CAT in gull feces.
Organism/indicator C_i_feces units ref
#
Salmonella [2.3,9] CFU/g (Lévesque et al., 2000)
Campylobacter [3.3,6]# CFU/g (Lévesque et al., 2000)
CAT [8.26,8.73]* copies/g (Brown et al., 2017a)
#
The two values separated by a comma are the minimum and maximum of
the log10-uniform distribution.
⁎
The two values separated by a comma are the shape and scale of a Weibell
distribution where the concentrations were log10-transformed prior to being fit
to the distribution. CAT was measured using the LeeSeaGull assay (Lee et al.,
2013).
possible amount of feces. The age of the contamination τ serves as a
model input and varies between 0 (unaged) and 7 d in 0.5 d increments.
A total of 315 Mb -τ combinations (15 distinct ages and 21 distinct Mb)
were modeled.
After Mb and τ are specified as model inputs, the concentration of ith
reference pathogen in surface waters (Ci_surface) is modeled as follows:
(6)
Ci _ surface = Mb Ci _ feces e ki
th
where Ci_feces and ki are, respectively, the i reference pathogen con­
centration in gull feces, and ki is the ith pathogen's first order decay rate
constant, both described by distributions (Tables 2 and 3). The dose of
pathogen i (µi) was subsequently determined by the following equation:
µi = Ci _ surface Vpi (7)
where V was drawn from the same distribution described in the pre­
vious sections, and pi represents the fraction of that pathogen from gull
feces that is human infectious. pi can vary between 0.01 and 0.4 and is
drawn from a uniform distribution; as described in our previous work
(Brown et al., 2017a). The dose was subsequently used in the dose-
response equations and a cumulative illness risk (Pill) was determined.
Model output, including calculation of the risk-based threshold for
Mb, was handled as described in the previous sections.
The concentration of CAT in the surface water (CCAT) when the mass
concentration of feces is equal to the risk-based threshold Mb and aged a
time τ was determined as follows:
CCAT = Mb CCAT_ feces e k CAT
(8)
where CCAT_feces and kCAT are respectively, the concentration of CAT in
gull feces and the decay rate constant of CAT in surface waters, and Mb
is the risk-based threshold Mb. CCAT_feces and kCAT are described by
distributions (Tables 2 and 3). There are limited data on kCAT in the
literature. Brown and Boehm (2015) report kCAT for seawater under
dark, and intensely sunlit conditions. For the present study, we deemed
the kCAT reported by those authors for sunlit conditions would be un­
realistic for field conditions – they would need to be corrected for flu­
ence and screening of photons by the water column to be applicable to a
field setting (Nelson et al., 2018). A well-mixed water column of a
natural water is likey to have low incident UVA and UVB, the most
germicidal wavelengths of sunlight (Nelson et al., 2018), and thus may
be better approximated as dark rather than intensely sunlit. Therefore,
we opted to use kCAT they report for dark conditions. We used the lower
estimate and upper estimate for dark kCAT as bounds on a uniform
distribution for kCAT for the QMRA model, 0.11 and 3.5 per day, re­
spectively.
To find CCAT, values were drawn from the distributions 50,000 times
and the median CCAT as well as other distribution percentiles were re­
corded. The median of this distribution is referred to as the “risk-based
threshold for CAT”.
2.1. Exposure to gull feces unknown age: CAT
We repeated the QMRA for a scenario where the CAT is measured in
A.B. Boehm and J.A. Soller
surface waters, and the gull fecal contamination age is unknown. The
methods are similar as described above for unknown age exposure
scenarios. The concentration of CAT as measured in surface water was
specified as an input to the model and varied between 100 and 108
copies/100 ml in increments of 0.5 log10 units. The concentrations of
reference pathogens in surface waters (Ci_surface) was modeled as fol­
lows:
CCAT Ci_ feces k 1
Ci _ surface = e
CCAT _ feces
where Δk = kCAT - ki, and Ci_feces and CCAT_feces are, respectively, the ith
reference pathogen and CAT concentrations in feces, and ki and kCAT
are their first order decay rate constants. These variables were all drawn
from their respective distributions (Tables 2 and 3). τ was drawn from a
uniform distribution ranging from 0 to a maximum realistic value (τmax)
given the specified Cmeas as described in Eq. (5). Here b is the log10-
transformed median concentration of CAT in feces (Cfeces) multiplied by
a maximum realistic mass concentration of feces in water Mbmax, and m
is -kmed*log10e where kmed is the median CAT decay rate constant (kCAT)
in surface waters (Table 4). We assumed Mbmax was 100 g/ 100 ml.
Ci_surface was used to determine dose, accounting for pi (as in Eq. (7)),
the dose was input into the dose-response equations, and then Pill was
determined.
Model output, including calculation of the risk-based threshold, was
handled as described in the previous sections. Note that this model
requires Cmeas to be input rather than Mb since τmax is dependent spe­
cifically on Cmeas (Eq. (5)).
2.2. Exposure to a mixture of gull feces and sewage of unknown ages:
CATand HF183
This QMRA model estimates risk from swimming in surface waters
containing a mixture of gull feces and sewage, measured using CAT and
HF183, respectively, where the ages of gull and raw sewage con­
tamination are both unknown and independent. The concentrations of
CAT and HF183 measured in surface waters are used as inputs to the
model. CCAT is varied from 100 to 108 copies/100 ml, and CHF183 is
varied from 100 to 105 copies/100 ml, both in 0.5 log10 units resulting
in 187 different simulations that combined the different CAT and
HF183 concentrations. As described above, each simulation included
50,000 Monte Carlo iterations.
The concentrations of pathogens in surface waters from sewage
Ci_surface_sewage are computed using Eq. (4) where τ is drawn from a
uniform distribution between 0 and τmax_HF183. The concentration of
pathogens in surface waters from gull feces Ci_surface_feces is calculated
using Eq. (9) where τ is drawn from a uniform distribution between 0
and τmax_CAT. τmax_HF183 and τmax_CAT are computed using Eq. (5) using
the indicator specified m and b (Table 5) described in the previous
sections for the non-mixture exposure scenario for HF183 and CAT,
respectively. The doses of Cryptosporidium, Giardia, E. coli O157:H7,
norovirus and adenovirus are computed as µi= Ci_surface_sewageV as there
is no contribution from gull feces. The doses of Salmonella and Cam­
pylobacter are calculated as µi= (Ci_surface_sewage + Ci_surface_fecespi) V as
there are contributions from both pathogen sources. The doses are used
to calculate cumulative risk (Pill) as described above.
The median Pillof each of the simulations (each representing a
Table 4
m and b for calculating tmax via Eq. (5). These values presume that the units
ofτmax are days, and the unit of CHF183, CENT, and CCAT in Eq. (5) are per
100 ml.
Indicator b m
HF183 7.2 −0.50
ENT 5.9 −0.51
CAT 10.35 −0.78
Microbial Risk Analysis xxx (xxxx) xxxx
Table 5
The risk-based threshold for HF183 given CCAT. The values in all rows come
from the line of constant risk in Fig. 6 of 32/1000. The value for the first row
(when CCAT = 0) is from the simulation for HF183 when raw sewage con­
tributes exclusively to risk and the age is unknown (Fig. 2).
CCAT [copies / 100 ml] Risk-based threshold for HF183 [copies/100 ml]
0 525
370
(9) 10 275
100 175
300 120
1000 70
3000 30
10,000 7
22,500 1
different CCAT and CHF183) was extracted and used to create a contour
plot illustrating median simulated risk as a function CCAT and CHF183.
The data were interpolated to identify the risk contour representing 32/
1000 to elucidate the risk-based threshold for HF183 as a function of
CAT concentration.
3. Results
Exposure to untreated, raw sewage of known and unknown
age. The risk-based volume fraction of sewage (Vs) increases with the
age of contamination (Fig. 1) because pathogen concentrations de­
crease as the sewage ages. In contrast, the risk-based threshold for
HF183 declines with the age of contamination, as reported previously
(Boehm et al., 2018). The decline occurs because the decay rate con­
stant of norovirus, on average, is smaller than that of HF183 and even
with the addition of adenovirus in these simulations, norovirus con­
tinued to dominate risk (Figure S1). When sewage is fresh (unaged,
τ = 0), the risk-based threshold is approximately 9000 copies/100 ml.
After approximately 9 days, the risk-based threshold declines to ~ 1
copy/100 ml, which is at a possible, but very sensitive, limit of quan­
tification for the assay. This implies that after about sewage has aged
approximately 9 days, the risk-based threshold for HF183 is lower that
what can be readily measured in surface waters. As indicated above,
norovirus is the pathogen that contributes the most to risk at all ages, as
reported in previous studies (Boehm et al., 2015; Boehm et al., 2018).
When the age of contamination is unknown per the scenario de­
scribed in the methods, the risk-based threshold for HF183 is 525 co­
pies/100 ml (Fig. 2). If the risk-based threshold is defined as where the
55th or 45th percentile of risk is equal to 32/1000, then the threshold
would be 240 or 1000 copies/100 ml, respectively. These alternative
percentiles are chosen arbitrarily; any percentile could be chosen by a
policy maker depending on their needs. This scenario assumes that the
age of contamination could be any number between 0 and τmax where
τmax is the oldest age contamination could be in surface waters. As for
the known age scenario, exposure to norovirus accounts for the ma­
jority of the risk (Boehm et al., 2018).
We repeated the QMRA to calculate risk of illness from exposure to
ENT from raw sewage of unknown age to evaluate the approach and
assumptions used for the HF183 analysis. The risk-based threshold for
ENT is calculated to be 47 CFU/100 ml (Fig. 3). This number is close to
the USEPA surface water criterion for ENT (at the same level of health
protection) of 32 CFU/100 ml (USEPA, 2012a).
3.1. Exposure to gull feces known and unknown age: CAT
The risk-based mass concentration of gull feces (Mb) increases with
the age of contamination (Fig. 4) because associated pathogen con­
centrations decrease as the fecal contamination ages. The increase is
biphasic because initially, exposure to Campylobacter contributes to the
total risk (~60% of the risk, averaged all concentrations for t = 0 days),
5
A.B. Boehm and J.A. Soller
Fig. 1. Panel A. The risk-based threshold for Vs (volume fraction of sewage) as a function of the age of sewage contamination. Panel B. The risk-based threshold of
HF183 as a function of age of sewage contamination. The 45th and 55th percentiles are shown as well as the median to give a sense of variability in the model output.
The horizontal dotted line shows a sensitive lower limit of quantification of the HF183 assay for comparison purposes.
Fig. 2. The median, 45th, and 55th percentiles of calculated risk for a measured
concentration of HF183 in surface waters when the age of contamination is
unknown, and thus assumed to be any possible age in the Monte Carlo simu­
lations. The median risk in the figure is the median of 50,000 Monte Carlo
simulations for each CHF183. Reference line corresponds to 32/1000 illness
level.
but after ~2.5 days, due to its relatively high decay rate constant
(Table 2, median 2.8 day−1), Campylobacter contributes little to the risk
(average 25% or less of the total risk, averaged across concentrations),
and most of the risk is then attributable to Salmonella.
The risk-based threshold for CAT is relatively constant, compared to
that of HF183, with the age of contamination (Fig. 4). There is an initial
increase in the RBT and then a decrease after 2.5 days. The trend can be
explained by differences in the relative decay of the pathogens and
CAT. The initial increase occurs because the pathogen contributing
most of the risk, Campylobacter, has a higher decay rate constant, on
average, than CAT (medians of 2.8 per day for Campylobacter compared
to 1.8 day−1 for CAT). After about 2.5 days, when Salmonella overtakes
Campylobacter as contributing most of the risk, the RBT begins to de­
crease because the decay rate constant for Salmonella (median of 0.74
day−1) is smaller, on average than the decay rate constant for CAT.
When gull fecal contaminations is fresh (unaged, τ = 0), the risk-based
threshold is approximately 45,000 copies/100 ml. After 7 days, the risk-
based threshold declines to approximately 8500 copies/100 ml. The
geometric mean RBT for CAT over all ages considered in the simulation
is 94,000 copies/100 ml.
We conducted a QMRA assuming that the age was unknown as
6
Microbial Risk Analysis xxx (xxxx) xxxx
Fig. 3. The median, 45th, and 55th percentiles of calculated risk for a measured
concentration of ENT in surface waters when the age of contamination is un­
known, and thus assumed to be any possible age in the Monte Carlo simula­
tions. The median risk in the figure is the median of 50,000 Monte Carlo si­
mulations for each CENT. Reference line corresponds to 32/1000 illness level.
described in the methods (Fig. 5). Based on this analysis, the risk based
threshold for CAT is 200,000 copies/100 ml. If the risk-based threshold
were redefined to be the CAT concentration at which the 55th or 45th
percentile of risk were equal to 32/1000, then the threshold would be
100,000 or 400,000 copies/100 ml. Since the CAT RBT for gull fecal
contamination of known age is a relative weak function of age (Fig. 4),
the CAT RBT for the unknown age case is not very sensitive to the
functional relationship between τmax and CCAT. Changing Mbmax from 1
to 10 to 100 g/ 100 ml does not substantially change the CAT RBT for
unknown age (results not shown).
3.2. Exposure to a mixture of gull feces and sewage of unknown ages:
CATand HF183
We considered a hazard scenario where surface water contains both
gull feces and raw sewage, detected using the CAT marker and HF183,
respectively. The age of contamination is unknown in each case. The
resultant median calculated risk given different co-occurring con­
centrations of CAT and HF183 is shown in the form of a contour plot
(Fig. 6). The constant risk line of 32/1000 is shown in black (it is la­
beled with the log10-transform 32/1000). This line shows different risk-
based thresholds for HF183 given CCAT. A summary of the values
A.B. Boehm and J.A. Soller
Fig. 4. Panel A. The risk-based threshold for Mb as a function of the age of fecal contamination. Panel B. The risk-based threshold of CAT as a function of age of
contamination. The 45th and 55th percentiles are shown as well as the median – which is the risk-based threshold for CAT.
Fig. 5. The median, 45th, and 55th percentiles of calculated risk for a measured
concentration of CAT in surface waters when the age of contamination is un­
known, and thus assumed to be any possible age in the Monte Carlo simula­
tions. The median risk in the figure is the median of 50,000 Monte Carlo si­
mulations for each CCAT.
corresponding to a risk of 32/1000 is provided in Table 5. As CCAT
increases from 0 to 10,000 copies/100 ml, the risk-based threshold for
HF183 decreases from 525 to 7 copies/100 ml. In other words, as the
amount of gull contamination (of unknown age) increases in the water,
the amount of raw sewage (of unknown age) that can be present de­
creases in order to ensure the median calculated risk does not exceed
32/1000.
4. Discussion
The risk-based threshold (RBT) for a water quality indicator is de­
fined as the concentration of the indicator for which the median si­
mulated illness risk is 32/1000. The RBT of HF183 is a strong function
of the age of contamination, as described previously (Boehm et al.,
2018). This is due to the fact that norovirus, the pathogen to which
exposure drives the risk estimates, has a smaller decay rate constant on
average, than HF183 (Boehm et al., 2018). Sewage contamination in a
water body is rarely of one single known age, unless there was a spe­
cific, short duration release of sewage into an otherwise clean water­
body. If there is a mixture of sewage contamination of diverse ages
7
Microbial Risk Analysis xxx (xxxx) xxxx
Fig. 6. Median calculated risk as a function of CHF183 and CCAT. The lines re­
present lines of constant median risk and are labeled with the log10-transform of
the risk value. The line of constant risk = 32/1000 is shown in black and is
labeled with the log10(32/1000)= −1.49485. The symbols represent the va­
lues used to create Table 5. Similar contours for the 45th and 55th percentiles of
risk can be found in the supporting material (Figures S3 and S4).
present in surface waters, and the proportion of each component of the
mixture and its age is known, it is possible to estimate the RBT for
HF183. However, it is rarely the case that anything specific is known
about the age of contamination, and the proportion of each aged
component that constitute the mixture. Given these uncertainties, we
considered the case where the age of contamination is unknown, and
bound by 0 and a maximum possible age (τmax). The maximum possible
age is a function of the concentration of HF183; it is the age for which
that HF183 concentration would exist in the water if the water were
100% sewage decaying with a rate constant equivalent to the median k
for HF183. In this scenario, it is assumed the sewage contamination is
present in equal proportions of each possible age. Under this scenario,
which we term hereafter “the unknown age” scenario, we derived a RBT
of 525 copies/100 ml for HF183. This represents an update to the RBT
reported by Boehm et al. (2018) of 4100 copies/ 100 ml for a similar
hazard scenario. The present RBT derivation includes refined tem­
perature-adjusted decay rate constants, and additional risk from ex­
posure to adenovirus, updated pathogen concentrations in sewage for
Cryptosporidium, and, in our opinion, a more defensible formulation for
A.B. Boehm and J.A. Soller
the maximum age of contamination. The difference between the ap­
proach used herein for estimating the maximum age of contamination
and that used by Boehm et al. (2018) is discussed in detail elsewhere
(Boehm, 2019).
The RBT for HF183 for the unknown scenario is sensitive to the
relationship between τmax and CHF183. This sensitivity was described by
Boehm (2019) in her analysis of RBTs for coliphage. The RBT for HF183
decreases when τmax is larger for a specific CHF183. For the present
analysis, τmax represents the age for which that HF183 concentration
would exist in the water if the water were 100% sewage decaying with
a rate constant equivalent to the median for HF183. Given that we
understand that recreational water would never be 100% raw sewage,
and a lower percent of raw sewage at time 0 would decrease τmax for a
given CHF183 by reducing “b” in Eq. (5), this formulation for τmax pro­
vides a margin of safety around the estimate of the risk-based threshold.
An additional margin of safety allowed for in our analysis is our use of
the non-aggregated hypergeometric norovirus dose-response curve
published by Teunis et al. (2008) which predicts a larger probability of
infection given low doses of norovirus than the fractional beta-poisson
model described by Messner et al. (2014) by additional margin of
safety, we mean that we provide an estimate for the RBT that assumes a
relatively high level of risk from exposure to norovirus. Using the
fractional beta-poisson model to describe the norovirus dose-response
provides a lower risk estimate for exposure to norovirus, and adeno­
virus contributes the most to the cumulative risk (data not shown).
Using a dose-response curve that weights the contributions of both the
norovirus dose response curves gives a similar estimate of risk as using
the non-aggregated hypergeometric norovirus dose-response
(Boehm, 2019).
To evaluate the credibility of the approach for calculating the
HF183 RBT for the unknown age scenario, we repeated the analysis
using culturable ENT instead of HF183. We found that the RBT for ENT
for the unknown age scenario was 47 CFU/100 ml, a value close to, but
higher than, the USEPA ambient water quality criterion of 30 CFU/
100 ml, derived via a completely independent method. The fact that the
unknown age scenario-derived RBT for ENT and the national ambient
water quality criteria are the same order of magnitude, and quite close
together, adds credence to this numerical simulation-based approach.
Depending on the concentration of HF183, the maximum age of
contamination can sometimes be quite large (for example 10 days when
HF183 is 160 copies/100 ml). We considered the case where the
maximum age of contamination was capped at 3 days, and reran a
scenario similar to the unknown age scenario (hereafter, “capped age”
scenario) (Figure S2). This duration may represent, for example, when
there is a local source of contamination (and no non-local source that
would introduce aged contamination) in a surface water that has a
maximum residence time of 3 d The capped age scenario resulted in a
HF183 RBT of 2400 copies /100 ml. However, it is unclear the extent to
which this or similar scenarios can be applied, as they require that no
non-local source has the potential to introduce aged contamination into
the waterbody.
Many coastal and inland waterbodies have bird fecal contamination
present. For example, along the California coast, several recent studies
have measured bird-specific fecal source tracking markers at California
beaches. For example, Russell et al. (2013) measured the LeeSeaGull
bird fecal marker (CAT) at Cowell Beach in Santa Cruz and measured
between 102 and 105 copies/100 ml in the seawater. Ervin et al. (2014)
measured a bird fecal marker (Gull2) at a beach in Santa Barbara and
found ~103 copies/100 ml. Steele et al. (2018) report the LeeSeaGull
marker (CAT) in stormwater in San Diego between below their detec­
tion limit and 103 copies/100 ml.
We were interested in determining how much untreated human
sewage could be present in a waterbody if that waterbody also con­
tained bird feces while maintaining a calculated risk less than 32/1000.
We first updated previous work by Brown et al. (2017a; 2017b) to
consider the effect of bird feces aging on the RBT for the LeeSeaGull
8
Microbial Risk Analysis xxx (xxxx) xxxx
bird fecal marker (hereafter, CAT). We used risks from gull con­
tamination as our basis since gulls are commonly present near coastal
waters and pathogen data in gull feces were previously reported
(Soller et al., 2010a). We found that the CAT RBT varied with fecal age
in a bimodal fashion; initially increasing with age and then decreasing
owing to different pathogen exposures dominating the risk as the
contamination ages (first Campylobacter and then Salmonella). Overall,
however, the RBT is a much weaker function of contamination age than
HF183 in sewage contaminated waters because of the more similar
decay rate constants of the indicator CAT and the pathogens Salmonella
and Campylobacter compared to the decay rates of HF183 and nor­
ovirus. As with the human sewage hazard, it is often unknown how
aged bird fecal contamination is in a surface water, or if the con­
tamination is composed of a diverse mixture of ages. We therefore
considered the unknown age scenario for gull fecal contamination and
found that CAT RBT to be 200,000 copies/100 ml.
We then considered a hazard of a mixture of gull feces and human
sewage in surface waters, considering the unknown age scenario for
both. The results of that analysis indicate that RBT for HF183 declines
as more CAT is present in the water body. This is because as CAT in­
creases in the water, exposure to bird feces adds risk meaning that less
human sewage can be in the water if the allowable risk is capped at 32/
1000 illnesses. For example, if CAT is present at 1000 copies/100 ml,
and the manager wishes to ensure that the total risk from exposure to
gull feces and human sewage is less than 32/1000, then HF183 RBT
would be 70 copies/100 ml.
All the hazards we considered in this work included contributions of
untreated sewage. Another potential source of pathogens to the en­
vironmental is treated wastewater effluent. While treated effluent cer­
tainly contains far fewer pathogens than untreated sewage, it may
contain viruses or other hardy pathogens that persist through the
treatment train (Brown et al., 2017b), and it also can contain HF183
(~104 copies/ml) (Brown et al., 2017b). We previously conducted a
QMRA to calculate the RBT for HF183 when the source of contamina­
tion is unaged, treated effluent; we found that the RBT was approxi­
mately 10 times higher than it was if the source of contamination is
unaged, untreated sewage (Brown et al., 2017b). We thus expect that
RBT established for untreated sewage to be protective for exposure to
treated effluent.
This work, along with prior publications evaluating the potential for
molecular marker-based RBTs (Boehm et al., 2015; Boehm et al., 2018;
Brown et al., 2017a; Brown et al., 2017b; Crank et al., 2019), indicates
that molecular markers have potential to be valuable tools in recrea­
tional water management and potentially for water quality regulations.
These molecular markers exhibit desirable characteristics which could
allow water quality managers to efficiently identify salient sources of
contamination in a waterbody and focus their remediation efforts on
contamination sources that can yield the greatest impact on improved
human health risk – the basis for current recreational water quality
criteria, recommendations, and/or regulations worldwide. This overall
approach – focusing on risk-relevant contamination is consistent with
recent revisions to water quality recommendations in the USA (USEPA,
2012b). In fact, to facilitate this overall approach, the USEPA has
published guidelines describing the development of site-specific alter­
native recreational water quality criteria which include alternative in­
dicators and methods (USEPA, 2014a; USEPA, 2014b). This work fun­
damentally fits within the overarching framework described there and
provides water quality managers with an additional tool that could be
useful in specific circumstances. We acknowledge that making HF183
measurements requires qualified laboratory staff and specialized
equipment. While some agencies that conduct water quality measure­
ments may have reliable access to the appropriate staff and equipment,
others may not.
We understand that this field is quickly evolving and that refine­
ments to this work will undoubtedly be published in both near and
longer terms. This raises an important and philosophically interesting
A.B. Boehm and J.A. Soller
question about when a new approach or indicator should be adopted
and/or how much information is required. Although there is no uni­
versal answer to this question, we believe that, in this case, the science
has sufficiently advanced and the potential benefits of focusing on risk-
relevant contamination outweigh the drawbacks associated with un­
certainties and future iterative refinements, and thus encourage the
consideration of the proposed RBTs for use in the evaluation of re­
creational water quality for those agencies capable of conducting mo­
lecular measurements. Future efforts might also consider monitoring for
viral pathogens which appear to be the main drivers of risk in recrea­
tional waters.
Sensitivity analyses of the models described in this paper have lar­
gely been completed previously (Boehm et al., 2015; Boehm et al.,
2018; Brown et al., 2017a; Brown et al., 2017b) and the sensitivity of
the HF183 RBT to certain assumptions and parameter choices have
already been discussed herein. An important parameter that strongly
affects the HF183 RBT is the decay rate constant for norovirus. Since
norovirus is difficult to culture using a cell line, estimates for this
parameter come from studies that quantify the virus using RT-QPCR or
by using a viral surrogate like murine norovirus (Boehm et al., 2019).
Future refinement of our understanding of norovirus persistence in
environmental waters can improve the QMRA described herein. The
decay rate constants used in the QMRA models were derived from
published systematic reviews of decay rate constants of pathogens in
raw waters and this represent the best available estimates to date;
however, further refinements on those estimates are encouraged. Ad­
ditionally, there are few data on CAT decay in water and additional
data are needed. It is also important to note that the HF183 RBT is for
HF183 measured in the manner used to obtain the HF183 distribution
in raw sewage (Shanks et al., 2010). HF183 measured using another
method (including both the concentration and quantification steps)
would need to be shown to be equivalent to the method of Shanks et al.
(2010) for the HF183 RBTs reported herein to be applicable.
Acknowledgements
This work was funded via an MOU between the City and County of
San Diego, and County of Orange. Funding was provided through the
City of San Diego's contract with Tetra Tech, Inc. We thank Ruth Kolb,
Victoria Kalkirtz, and Drew Kleis (City of San Diego), Todd Snyder and
Jo Ann Weber (County of San Diego), and Grant Sharp and Cindy Rivers
(County of Orange) for their support of this work. Helen Yu from the
San Diego Water Quality Control Board provided technical direction
and oversight of this work. Clint Boschen (TetraTech, Inc.) and Ken
Schiff (Southern California Coastal Water Research Project) were on the
technical team and provided review and thoughtful feedback
throughout the course of this work. Clint Boschen was the Project
Manager for the TetraTech contract and served as liaison with the
funders.
The authors declare no conflict of interest in the conduct of this
work.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.mran.2020.100139.
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