Published online May 3, 2018

Journal of Environmental Quality

Special Section
Microbial Water Quality—Monitoring and Modeling

Minimizing Virus Transport in Porous Media by Optimizing
Solid Phase Inactivation
Salini Sasidharan,* Scott A. Bradford, Jiří Šimůnek, and Saeed Torkzaban
Abstract
The influence of virus type (PRD1 and FX174), temperature (flowat 4 and 20°C), a no-flow storage duration (0, 36, 46, and 70 d),
and temperature cycling (flow at 20°C and storage at 4°C) on virustransport and fate were investigated in saturated sand-packed
columns. The vast majority (84–99.5%) of viruses were irreversibly
retained on the sand, even in the presence of deionized waterand beef extract at pH = 11. The reversibly retained virus fraction(fr) was small (1.6 × 10-5 to 0.047) but poses a risk of long-term
virus contamination. The value of fr and associated transportrisk was lower at a higher temperature and for increases in the
no-flow storage period due to the temperature dependencyof the solid phase inactivation. A model that considered
advective–dispersive transport, attachment (katt), detachment(kdet), solid phase inactivation (ms), and liquid phase inactivation(ml) coefficients, and a Langmuirian blocking function provided agood description of the early portion of the breakthrough curve.
The removal parameters were found to be in the order of katt > ms>> ml. Furthermore, ms was an order of magnitude higher than ml for
PRD1, whereas ms was two and three orders of magnitude higher
than ml for FX174 at 4 and 20°C, respectively. Transport modelingwith two retention, release, and inactivation sites demonstrated
that a small fraction of viruses exhibited a much slower releaseand solid phase inactivation rate, presumably because variationsin the sand and virus surface roughness caused differences in thestrength of adhesion. These findings demonstrate the importanceof solid phase inactivation, temperature, and storage periods ineliminating virus transport in porous media. This research has
potential implications for managed aquifer recharge applications
and guidelines to enhance the virus removal by controlling thetemperature and aquifer residence time.

Core Ideas• Solid phase inactivation is 2–3 orders of magnitude higher thanliquid phase inactivation.
• Solid phase inactivation increases with temperature and column storage duration.
• The solid phase inactivation was higher for FX174 than PRD1.
• Solid phase inactivation reduced the reversible virus fraction by
1 to 2 orders of magnitude.

Copyright © American Society of Agronomy, Crop Science Society of America, andSoil Science Society of America. 5585 Guilford Rd., Madison, WI 53711 USA.
All rights reserved.
J. Environ. Qual.
doi:10.2134/jeq2018.01.0027
Supplemental material is available online for this article.
Received 19 Jan. 2018.
Accepted 2 Apr. 2018.
*Corresponding author (salinis@ucr.edu).

Water reclamation, recycling, and reuse are

low-cost, low-emission, and low-energy technologies that have received a great deal of interest inindustrialized nations to achieve sustainable water supplies for
growing populations (Asano and Levine, 1996). A number ofmanaged aquifer recharge (MAR) operations have been designed
to purposely inject or infiltrate rainwater, stormwater, reclaimed
water, or water from other aquifers into selected aquifers forstorage and later recovery (Dillon et al., 2010; Gordon and Toze,
2003). One of the major risks associated with MAR operations
is the presence of microbial pathogens in recovered drinking
water and their potential risk to human health (Dillon et al.,2010; NHMRC, 2011; NRMMC–EPHC–NHMRC, 2009).
If the groundwater already meets drinking water standards or therecovered water is to be used as drinking water, then the source orrecovered water requires a higher level of treatment, which adds
considerable expense to MAR operations (Dillon et al., 2010).
A growing body of research is continuously evaluating andvalidating processes involved in pathogen attenuation in aquifers. Most studies have used laboratory-scale columns packed
with clean sand (Sasidharan et al., 2017b; Schijven et al., 2002)or sediment collected from potential MAR sites (Hijnen et al.,2005; Sasidharan et al., 2017a), whereas only a few field-scalestudies have been conducted (Page et al., 2015; Pieper et al.,
1997; Sidhu et al., 2010). Viruses are generally considered tobe the microbial pathogen of greatest risk because of their low
infectious dose and potential to travel long distances in the subsurface (Reynolds et al., 2008). Mechanisms that will lead tovirus removal during MAR include inactivation in the liquid
phase, attachment on the solid surface, and subsequent inactivation on the solid surface (Harvey and Ryan, 2004; Sasidharan
et al., 2017a; Schijven and Hassanizadeh, 2000). However, current MAR guidelines only consider liquid phase inactivation
as a reliable mechanism to remove viruses (Hannappel et al.,2014; Johnson, 1989; NRMMC–EPHC–NHMRC, 2009).
Factors influencing liquid phase inactivation (Keswick et al.,
1982; Schijven and Hassanizadeh, 2000) of pathogens in aquifers include temperature (Anders and Chrysikopoulos, 2006;

S. Sasidharan and J. Šimůnek, Dep. of Environmental Sciences, Univ. of California,
Riverside, Riverside, CA 92521, USA; S. Sasidharan, CSIRO Land and Water, Glen
Osmond, SA 5064, Australia; S. Sasidharan and S. Torkzaban, Flinders Univ.,
Adelaide, SA 5001, Australia; S.A. Bradford, USDA–ARS, US Salinity Lab., Riverside,
CA 92507, USA. Assigned to Associate Editor Hyunjung Kim.
Abbreviations: ASR, aquifer storage and recovery; ASTR, aquifer storage transfer
and recovery; BTC, breakthrough curve; DLA, double layer agar; MAR, managed
aquifer recharge; PFU, plaque forming unit.

Chrysikopoulos and Aravantinou, 2012; Sasidharan et al.,2017b; Schijven et al., 2016), dissolved oxygen ( Jansons et al.,1989), water chemistry (Katzenelson, 1978), and the presenceof indigenous microorganisms (van Leeuwen, 1996). It may takemonths to years to achieve one-log virus removal by liquid phaseinactivation depending on the system conditions and virus type
(Gordon and Toze, 2003; Page et al., 2012; Toze et al., 2010).
Guidelines for MAR have focused on virus removal by liquid
phase inactivation because reversible virus attachment andrelease can create potential risks for long-term virus transport.
The objective of this research is to better understand and quantify factors that minimize the risks from reversible virus attachment and release. In particular, we focus on the role of solid
phase inactivation to eliminate the release and transport of infective viruses. Many researchers have reported that virus inactivation is greater in the solid phase than the liquid phase (Bradford
et al., 2006; Chu et al., 2001; Pieper et al., 1997; Ryan et al.,
2002; Schijven et al., 1999). This has been attributed to strong
adhesive forces between negatively charged viruses and positively
charged metal oxide surfaces (Harvey and Ryan, 2004). In contrast, several other investigators reported that attached viruses
can be protected from solid phase inactivation by the presence oforganic matter and clay-sized particles in soils (Liew and Gerba,
1980; Schmoll, 2006; Stagg et al., 1977; Yates et al., 1988).
Consequently, the relative importance of solid phase inactivation remains a controversial (or perhaps case-specific) issue. In
addition, the amount of virus attachment and inactivation will
increase with the residence time because they are kinetic processes (Schijven and Hassanizadeh, 2000). The strength of theadhesive force may increase with residence time (Mondon et al.,
2003; Stuart and Hlady, 1995; Vadillo-Rodríguez et al., 2004;
Xu and Logan, 2006), and this may further enhance irreversible
attachment and solid phase inactivation. The temperature of a
MAR operation can also influence the rate of attachment andinactivation. Higher temperatures have been reported to increaseattachment by decreasing the energy barrier (Sasidharan et al.,2017b) and increase inactivation by enhancing degradation of
the viral genome and conformational changes in proteins associated with host recognition (Harvey and Ryan, 2004).
The microbial water quality of recovered water from MAR
operations could be improved by optimizing temperature andstorage conditions that promote irreversible virus attachment
and solid phase inactivation and thereby decrease costs associated with expensive pre- or post-treatment. However, no studies
have systematically investigated the influence of temperature andstorage time on irreversible attachment and solid phase inactivation under conditions that are relevant for MAR. In addition,
the temperature of groundwater can fluctuate with seasonal variations in groundwater recharge and flow (Gunawardhana et al.,2009). This has an important implication on virus survival during
the aquifer storage period and on potential health risks associated with active viruses being released into the recovered water.
To date, no research has investigated the effect of temperature
cycling on reversible–irreversible virus attachment, solid phaseinactivation, release during transient chemistry, and the longterm (2–3 mo) virus infectivity during transport experiments.
The objective of this research was to experimentally and theoretically investigate the influence of virus type, temperature, temperature cycling, and storage duration on the extent and kinetics

of reversible–irreversible virus attachment and solid phase inactivation. For this purpose, PRD1 and FX174 viruses were usedin saturated transport (deposition, storage, and release) experiments under different temperatures (4 and/or 20°C), no-flow
storage durations (0, 36, 46, and 70 d), and temperature cycling(flow at 20°C and storage at 4°C) conditions. A mass balance
was conducted to quantify reversible and irreversible virus fractions. Numerical modeling of the observed breakthrough curves(BTCs) was used to inversely determine the rates of attachment,
detachment, and solid phase inactivation. Results from this work
provide valuable insight into the underlying mechanisms that
control the transport and fate of viruses, as well as the design of
MAR operations to eliminate viruses by solid phase inactivation.

Materials and Methods

Porous Medium and Electrolyte Solution
Ultrapure quartz sand (Charles B. Chrystal Co., Inc.) withsize ranging from 125 to 300 mm was used in transport experiments. The sand was cleaned using an acid wash and boilingprocedure described by Sasidharan et al. (2014). An electrolyte
solution of 10 mM was prepared for transport studies using analytical grade NaCl and Milli-Q water at unadjusted pH of 5.5
to 5.8 as in previous studies (Walker et al., 2004; Zhang et al.,2010).

Viruses
Bacteriophages PRD1 and FX174 were used as the model
viruses in this study. The bacteriophages were analyzed using
their respective Escherichia coli host. The characteristics of theseviruses, their production, and quantification using the double
layer agar (DLA) method are described in Sasidharan et al.
(2016). The detection limit of the DLA technique is around 30
plaque forming unit (PFU) mL-1 (ISO, 2000). Stock solutionsof viruses were diluted in the electrolyte solution and equilibrated at the experimental temperature (4 or 20°C). An average
input concentration (C0) of 3.98 × 106 PFU mL-1 for PRD1 and7.17 × 105 PFU mL-1 for FX174 were used. The liquid phaseinactivation rate of PRD1 and FX174 was determined over a
period of 70 d in 10 mM NaCl at 4 and 20°C.

Transport Experiments
The column experiments were conducted in temperaturecontrolled laboratories (4 ± 1 and 20 ± 1°C). Sterilized polycarbonate columns (1.9 cm i.d. and 5 cm height) were wet-packed
using clean quartz sand while the column was being vibrated. The
packed column has a porosity of 0.4. After packing, the column
was preconditioned with 10 pore volumes of a 10 mM NaCl solution using a syringe pump (Model 22, Harvard Apparatus) at apore-water velocity of 5 m d-1. The columns were equilibrated tothe selected temperature (4 and 20°C) for 12 h before starting theexperiment. The virus transport experiments consisted of a deposition phase (Phase I), a storage phase (Phase II), and four release
phases (Phases III–VI). A virus (PRD1 or FX174) suspension in
10 mM NaCl solution at a temperature of 4 or 20°C was introduced into the column at an average pore-water velocity of 0.1
m d-1 during Phase I. Various storage periods and temperatures
were implemented during Phase II that are discussed in the following paragraph. Phase III consisted of eluting the column with
Journal of Environmental Qualityvirus-free 10 mM NaCl solution at a pore-water velocity of 0.1
m d-1. Columns were eluted with Milli-Q water and beef extract
with pH 11 (Sasidharan et al., 2017a) during Phases IV and V,
respectively, at a pore-water velocity of 5 m d-1. The column effluent samples were collected using a Spectra/Chrom CF-1 Fraction
Collector (Spectrum), and the concentration of viruses was quantified using the DLA method (Sasidharan et al., 2016).
Three sets of virus transport experiments were conductedto mimic different MAR strategies and to better differentiate
between liquid phase inactivation, reversible and irreversible
attachment on the solid phase, and solid phase inactivation.
The first set of experiments did not implement a storage period
during Phase II. These experiments focused on the influence ofconstant temperature conditions (4 or 20°C) on virus transportand release. The second set of experiments included a storage
period of 0, 46, or 70 d during Phase II to study the effects ofsolid phase inactivation under constant temperature conditions(4 or 20°C). A 70-d storage period was used for comparison to
field-scale pathogen decay studies (Sidhu et al., 2015) and toinvestigate the worst-case (minimum) residence time scenario(when winter, spring, and summer storms are harvested) during
MAR. Columns were closed, wrapped within thin aluminum
foil sheets, and stored under constant temperature conditions
during this storage period. The third set of experiments implemented temperature cycling and a storage period by conducting
Phases I and III to VI at 20°C and Phase II at 4°C with a storage
period of 0, 36, 46, or 70 d. These experiments were designed to
study the influence of seasonal variations in temperature on solid
phase inactivation.
Following completion of Phases I to V, the quartz sand fromthe column was decanted into a test tube and washed with 10
mL pH 11 solution twice, and the eluted virus concentration
was determined using the DLA method (Phase VI). In addition,
spot tests were conducted after completion of Phases I to VI for
the storage at constant temperature experiments to determine
whether remaining viruses on the solid phase were still capableof infecting and reproducing on their host bacterium. First, 5 gof washed (pH = 11) quartz sand was incubated with 25 mL of
Tryptone Soy Broth (Oxoid, Thermo Scientific, CM0129) andthe E. coli hosts (separate flasks for each host) at 37°C for 12
h. The broth was collected, 2.5 mL of chloroform was added tothe broth, and the broth was stored in the refrigerator overnight.
The aqueous phase was centrifuged at 3000 × g for 20 min andthe supernatant was collected. The spot test was conducted asfollows. One milliliter of E. coli host grown to the log phase
was mixed with 40 mL of Tryptone Soy agar (Oxoid, Thermo
Scientific, CM0131) that had 10 mM CaCl2, poured into a Petridish, and allowed to dry completely. Next, 10 mL of virus supernatant was placed onto the Petri dish as a spot (seven spots intotal), allowed to dry completely, and incubated at 37°C overnight. The plates were observed for the presence of spots (i.e.,
clear/lysis zone or plaques).
The observed BTCs were plotted as a dimensionless concentration (C/C0) of viruses as a function of pore volumes. The total
mass of retained viruses during Phase I (MS) in the column experiment was determined by calculating the difference between themass of injected viruses into the column in Phase I (MIn) and themass of viruses that was recovered in the effluent BTC (MBTC)
during Phase I. The percentage of injected viruses that was
Journal of Environmental Quality

recovered during Phases III, IV, V, and VI were denoted as MIII,
MIV, MV, and MVI, respectively. The percentage of the total massof the viruses recovered (MIII + MIV + MV + MVI) was denoted
as MR . The percentage of injected viruses that were irreversibly
retained on the solid phase (MIrr) was determined as 100 − MBTC
− MR . The total fraction of the viruses that were released from
MS (fr = MR/MS) and the total fraction of viruses that were notreleased from MS (fnr = MIrr/MS) were also calculated.

Mathematical ModelingExperimental BTCs for viruses were simulated by numerically solving the following aqueous phase and solid phase massbalance equations using the HYDRUS-1D model (Šimůnek etal., 2016):
¶C
¶ 2C
¶C
= lv 2 - v-( katt1 y1 + katt2 )C
¶t
¶z
¶z
r
+ b ( kdet1S1 + kdet2 S2 ) -m lC
q[1]

r b ¶S1r= katt1 y1C - b ( kdet1 +m s1 )S1
q ¶t
q

[2]

r b ¶S2r= katt2C - b ( kdet2 +m s2 )S2
q ¶t
q

[3]

where t (T; T denotes unit of time) is time, z (L; L denotes
units of length) is the direction of mean water flow, C (N L-3; Ndenotes the virus number) is the aqueous phase virus concentration, l (L) is the dispersivity, v (L T-1) is the average pore-watervelocity, rb (M L-3; M denotes the unit of mass) is the bulk density, q is the water content, S (N M-1) is the solid phase virus
concentration, katt (T-1) is the virus attachment rate coefficient,kdet (T-1) is the virus detachment rate coefficient, ms (T-1) is thesolid phase virus inactivation rate, ml (T-1) is the liquid phasevirus inactivation rate, and subscripts 1 and 2 on parametersindicates the solid phase sites. The parameter yl is a dimensionless Langmuirian blocking function that is given as (Adamczyk
et al., 1994)æS ö
y1 = ççç1- 1 ÷÷÷
çè Smax1 ø÷[4]where Smax1 (N M-1) is the maximum solid phase concentrationof retained virus on Site 1.
Note that the above model considers two kinetic retention
and inactivation sites on the solid phase, and Langmuirian
blocking only on Site 1. The justification for the selection of thismodel formation will be given in the “Mathematical Modeling”
section under “Results and Discussion.” The value of m1 was calculated from the 70-d liquid phase inactivation study. Other
model parameters were determined from available experimental information (e.g., v, q, rb, l) or by optimization to thevirus BTCs using the nonlinear least squares fitting routine in
HYDRUS-1D. The possibility of non-unique parameter fits
increases with the complexity of the model and the numberof fitted model parameters. Our modeling analysis of the virustransport data was therefore conducted in a stepwise manner toconstraint model parameters and to minimize the potential for
non-unique parameter fits. Initially, only a one-site retention andinactivation model was used to describe the early-time portion of
the BTC by setting parameters for Site 2 to zero and using thefollowing sequence. In the first step, katt1 and Smax1 were fitted toonly the deposition phase (Phase I), and the tailing portion of
the BTC was neglected. These values of katt1 and Smax1 were kept
as constant during step 2, and kdet1 and ms1 were fitted to the deposition (Phase I) and the initial tailing portion the BTC. The full
two-site model was needed to accurately describe the additional
complexity of the long-term portion of the BTC. In step 3, thevalues of Smax1, kdet1, and ms1 from step 2 were constants while fitting values of katt1, katt2, kdet2, and ms2.
The fraction of the solid surface area that is available for retention (Sf ) was calculated from Smax1 as follows (Kim et al., 2009;
Sasidharan et al., 2014):
Sf =Ac r b Smax1(1-g) As

PRD1. Furthermore, the value of ml was 121 and 388% higher
at 20°C than at 4°C for viruses PRD1 and FX174, respectively.
Field-scale pathogen decay studies at MAR site (average temp
= 23–25°C) using human pathogenic viruses showed ~109 to
185 d for 1 log removal of viruses (Sidhu et al., 2015). Hence,
human pathogenic viruses and the bacteriophages used in thisstudy exhibited similar decay rates. Viruses are submicron tonanosized particles that contain genetic materials (nucleic acid)such as RNA or DNA surrounded by a protein coat capsid. The
major mechanisms in viral inactivation are nucleic acid degradation, loss of nucleic acid, fracture of the capsid protein, andloss of host reorganization sites, which will lead to their complete degradation and/or loss of infectivity (Harvey and Ryan,
2004). Previous researchers demonstrated that temperature plays
a dominant role in liquid phase virus inactivation (Blanc andNasser, 1996; Harvey and Ryan, 2004; Schijven et al., 2016).
The relatively low values of R2 for ml in Table 1 reflect a deviation from the first-order model and scatter in the data. To overcome this limitation, previous studies used different models tosimulate liquid phase inactivation. Sim and Chrysikopoulos(1996), for example, used a time-dependent inactivation modelto account for virus subpopulations with different inactivation
rates. We did not investigate this issue further here because
results shown below demonstrated that liquid phase inactivation
had an insignificant influence in our column-scale studies.

[5]

where Ac (L2 N-1) is the cross-sectional area of a virus, As (L-1)
is the solid surface geometric area per unit volume, and g is theporosity of a monolayer packing of viruses on the solid surface that was taken from the literature to be 0.5 ( Johnson andElimelech, 1995).
The value of the sticking efficiency (a) was determined fromthe fitted katt = katt1 + katt2 value and filtration theory as follows(Schijven and Hassanizadeh, 2000; Yao et al., 1971):a=

2dc katt3(1- n)vh

Virus Transport Experiments[6]

Figure 1 presents breakthrough and release curves for PRD1
and FX174 under constant temperature conditions of 4 and
20°C. Table 2 shows mass balance data for the virus breakthroughand release curves. Similar to Sasidharan et al. (2016), the retention of viruses was higher at 20 than at 4°C, and for FX174 than
PRD1. For example, the mass retained on the solid phase (MS)for PRD1 and FX174 was 90.6 and 99.2% at 20°C comparedwith 88.1 and 98.9%, respectively, at 4°C. The release of PRD1
and FX174 was initiated during Phases III (~51 d of virus-free
10 mM NaCl solution at 0.1 m d-1), IV (Milli-Q water at 5 md-1), and V (beef extract with pH 11 at 5 m d-1) at 4 and 20°C.
Figure 1 shows that only a small number of the injected viruses
were released (<4.17%) during Phases III, IV, and V at both 4

where n is the porosity (0.4) and dc (L) is the collector (mediangrain) diameter. The value of the single collector-efficiency,h, was calculated using the correlation equation presented by
Messina et al. (2015).

Results and Discussion
Liquid Phase Virus Inactivation

Table 1 shows the calculated first-order liquid phase inactivation coefficient (ml) for PRD1 and FX174 in 10 mM NaCl
at 4 and 20°C over a 70-d period. The result shows that theliquid phase inactivation increased for FX174 in comparison to

Table 1. A summary of the experimental condition (temperature) and model parameters that were fitted to the viruses PRD1 and FX174 breakthrough curves using one-site and two-site kinetic models (the attachment coefficient, katt; the detachment coefficient, kdet; the maximum solid
phase virus concentration, Smax/C0; and solid phase inactivation, ms). The subscripts 1 and 2 represent the first and second sites, respectively. The
parameters a, Sf, and h were calculated from katt, Smax/C0, and the correlation equation of Messina et al. (2015), respectively. The table also includes
the coefficient of determination (R2) for the goodness of model fit and liquid phase inactivation coefficient, ml. All experiments were conducted attemperature = 4 or 20°C, ionic strength = 10 mM NaCl, and flow velocity = 0.1 m d-1. Observed and simulated breakthrough curves are shown in Fig.
1B.†
Virus

Temp.

FX174

°C
4
20

PRD1

4
20

Model

katt1

one-sitetwo-site
one-sitetwo-site
one-sitetwo-site
one-sitetwo-site

min-1
1.5 × 10-2
1.1 × 10-2
1.2 × 10-2
1.1 × 10-2
1.4 × 10-2
1.5 × 10-2
2.5 × 10-2
2.8 × 10-2

kdet1

Smax1/C0

ms1

min-1
no. g-1
min-1
4.9 × 10-5 0.763 4.7 × 10-4
4.9 × 10-4 0.953
5.0 × 10-4
4.8 × 10-8

0.46
2.43

katt2

kdet2

min-1
NA‡

min-1
NA

1.5 × 10-3

4.3 × 10-4 1.2 × 10-6NANA

5.9 × 10-4

7.6 × 10-5 4.8 × 10-7NANA

4.2 × 10-5

4.0 × 10-5 9.2 × 10-7NANA
1.0 × 10-7 1.0 × 10-12

R2 (Model)
0.65
0.62
0.50
0.68
0.70
0.71
0.76
0.77

Sf

h

ml

R2 (ml)7.8 × 10-9 0.031

0.65

min-1
2.2 × 10-6

0.37

9.8 × 10-9 0.028

0.71

8.6 × 10-6

0.87

1.6 × 10-7 0.049

0.54

1.3 × 10-6

0.37

8.5 × 10-7 0.081

0.60

1.6 × 10-6

0.28

a

† Note that the fitted values of ms2 were always less than 1.0 × 10-12.
‡ NA, not applicable. The parameter is not fitted during the simulation.
Journal of Environmental Quality

Fig. 1. (A) The observed breakthrough curve for viruses PRD1 and FX174 for the experiments conducted at temperature = 4 and 20°C, ionicstrength = 10 mM NaCl, and flow velocity = 0.1 m d−1. (B) The observed (markers) and fitted (solid line) breakthrough curve for viruses PRD1 andFX174 for Phase I and Phase III. C/C0, output concentration/input concentration; PV, pore volume. Table 1, Supplemental Table S1, and Table 2
present fitted model parameters, standard error coefficient, and mass balance information, respectively.

and 20°C, even in the presence of Milli-Q water (Phase IV) or
especially beef extract at pH = 11 (Phase V), which produced
higher release peak concentrations. Similarly, mass balance information (Table 2) indicates that 95 to 99% of the injected viruses
remained irreversibly attached or inactivated on the solid surface. However, virus release was greater for PRD1 than FX174
and at 4 than 20°C; this suggests a potential transport risk forsome viruses at lower temperatures.
One way to potentially minimize the risk from virus transport during MAR operations such as aquifer storage and recovery (ASR) and aquifer storage transfer and recovery (ASTR) isto provide an adequate residence time for liquid and especiallysolid phase inactivation. Additional transport experiments with
PRD1 and FX174 at constant temperatures of 4 and 20°C were
therefore conducted to investigate the influence of different storage periods before initiating release during Phases III, IV, and
V. Figures 2A and 2B present breakthrough and release curves
for PRD1 and FX174 at 4°C with a storage time of 0 or 70 d.
Figures 2C and 2D present similar results for PRD1 and FX174
at 20°C when a 0- and 70-d storage period was implemented.
Table 2 summarizes experimental conditions and mass balancedata during these experiments. An increase in the storage period
lowered the effluent virus concentrations during the release
Phases III, IV, and V. The difference in effluent concentration
with storage time was greater for FX174 than PRD1 and for 20
than 4°C, due to differences in solid phase inactivation. Similarly,
the reversible virus fraction was reduced by one to two orders ofmagnitude when the storage period was increased, a drop thatwas enhanced at higher temperatures. These results demonstratethat immediate release does not give adequate time for solid
phase inactivation. In contrast, longer storage periods can significantly contribute to virus removal by solid phase inactivation,
Journal of Environmental Quality

especially at higher temperatures. Indeed, the value of C/C0 was
below the detection limit for FX174 at 20°C during all release
phases following a 70-d storage period. Consequently, a lower
storage time is needed at a higher temperature because of anincrease in the solid phase inactivation. However, solid phaseinactivation over a 70-d storage period can still contribute to a
one order of magnitude reduction in a reversible virus fraction
even under a worst-case scenario of PRD1 at 4°C.
Following completion of the virus transport and storage
experiments shown in Fig. 2, sand samples from the columns
were collected, washed twice with pH 11 solution, and thenanalyzed for infective virus concentrations using the spot test
procedure described in section “Transport Experiments” under“Materials and Methods” above. The sand from transport andstorage experiments at 4 and 20°C after 0 d was positive (presence of a few PFUs) for the PRD1 virus. This indicates thatsome of the irreversibly retained viruses were still infectious on
the solid surface under these conditions. In contrast, sand fromexperiments at 20°C was negative (no PFU) for PRD1 after 70 d,and negative for FX174 after 0 and 70 d. However, both FX174
and PRD1 were positive (a few PFUs) at 4°C after 0 and 70 dof storage period. This provides convincing evidence that given
sufficient storage time, irreversibly attached viruses can be inactivated on the solid phase at a higher temperature. However, thelower temperature conditions need more time for complete solid
phase inactivation and may pose a health risk, and therefore, thetemperature effect should be taken into account in MAR designs.
Seasonal temperature variations initiate and drive thermal
groundwater convection, especially during the transition fromsummer to winter months (Engström and Nordell, 2016). In natural systems, thermally driven convection is initiated by various processes such as varying groundwater flow and infiltration of rain andsnowmelt water, which may lead to a decline in groundwater temperature. Such changes in groundwater temperature will influence
the amounts of virus retention and inactivation (Fig. 1). The next
set of FX174 and PRD1 transport experiments, therefore, investigated the combined influence of temperature cycling and storage
period. In particular, Phases I, III, IV, and V were all conducted
at 20°C. Virus release was initiated immediately following Phase I
in some experiments (storage period of 0 d), whereas others were
subjected to a 36-, 46-, or 70-d storage period at 4°C before initiating release at 20°C. Figure 3 presents the breakthrough and release
curves for these temperatures cycling and storage experiments.
Table 2 presents a summary of the experimental conditions andmass balance data. Similar to constant temperature storage experiments (Fig. 2, Table 2), Fig. 3 and Table 2 revealed small amountsof virus release and that the reversible virus fraction decreased for
longer storage periods and for FX174 in comparison to PRD1.
However, temperature cycling and storage experiments showintermediate release behavior to the storage experiments that wereconducted at a constant temperature of 4 or 20°C (Fig. 2, Table2). The temperature cycling and storage study, therefore, confirmsthat the solid phase inactivation is a function of temperature andthat the required storage period to remove viruses by solid phaseinactivation will need to account for temperature fluctuations.

Mathematical Modeling
Mathematical modeling of FX174 and PRD1 transport
during Phases I and III was undertaken to better understandand quantify the relative importance of different processes at 4
and 20°C. Initially, the virus breakthrough curves shown in Fig.
1 were modeled using only a one-site kinetic retention modelthat included Langmuirian blocking and liquid and solid phaseinactivation. Blocking was included in the model because somebreakthrough curves exhibited a delay in breakthrough and thenan increase in the effluent virus concentration with continued
injection due to the filling of available retention sites. Fitted(katt1, kdet1, Smax1, and ms1), measured (ml), and calculated (a, h, andSf ) model parameters, as well as statistical measures of the goodness of fit, are provided in Table 1. The standard error coefficientsfor fitted parameters are given in Supplemental Table S1.
The one-site model captured the main processes controllingthe early portion of the breakthrough curve. Fitted (katt1 andSmax1) and calculated (h and Sf ) retention parameters were higher
with increasing temperature. Sasidharan et al. (2017b) previously attributed this temperature dependency to slight increases
in the adhesive interaction on heterogeneous surfaces and slightdifferences in mass transfer (h in Table 1). Similar to Sasidharan

Table 2. The mass balance information for viruses column experiments shown in Fig. 1, 2, and 3.
FigureFig. 1A

Retention†

Release‡

Virus

Temp.

Storageperiod

PRD1

°C
20

d
0

————— % —————
9.4
90.61
90.4

0.998

0.001

——————————— % ———————————
0.18
1.1 × 10-4 2.9 × 10-3 1.9 × 10-4 0.18

0.953
0.999
0.997
0.980
0.995
0.982
0.992
0.995
1
0.992
1
0.977
0.993
0.995
0.999
0.977
0.991
0.992
0.995
0.999
0.999
0.995
0.999
0.992
0.999

0.047

3.9

0
70
0
70
0
70
0
70
0
46
0
46
0
36
46
0
36
46
0
70
0
70

11.8
0.8
1
8.6
10.1
1.79
1.99
0.8
4.6
0.03
0.55
5.37
7.0
0.11
0.73
5.37
6.57
7.08
0.11
0.54
0.37
0.8
4.5
0.03
0.55

FX174
Fig. 2A

PRD1Fig. 2B

FX174

Fig. 2C

PRD1Fig. 2D

FX174

NA§

PRD1

4
20
4
4

20

20

FX174
Fig. 3A

PRD1 20–4Storage–20

Fig. 3B

FX174

Fig. 3C

PRD1

Fig. 3D

FX174

MBTC

MS

88.14
99.18
98.99
91.38
89.87
98.21
98.01
99.20
95.44
99.97
99.44
94.63
93.00
99.89
99.27
94.63
93.43
92.92
99.89
99.46
99.63
99.20
95.49
99.97
99.45

MIrr

84
99.2
98.6
89.37
89.53
96.40
97.19
98.73
95.44
99.14
99.44
92.45
92.36
99.41
99.25
92.45
92.57
92.11
99.41
99.33
99.57
98.73
95.43
99.14
99.38

fnr

fr

MIII

1.1 × 10-4 0.01
0.004
0.14
0.02
1.42
0.003
0.03
0.018
0.15
0.008
0.29
0.004
0.09
2.1 × 10-5 0.002
0.008
0.001

MIV

MV

MVI

0.02
0.25
6.4 × 10-4
4.0 × 10-4 5.1 × 10-4 0
0.05
0.24
0.01
0.44
0.13
0.01
0.19
0.11
1.5 × 10-3
0.2
0.88
0.58
0.37
0.16
7.4 × 10-4
0.10
0.26
5.7 × 10-3
3.9 × 10-4 4.5 × 10-4 1.6 × 10-4
0.03
0.78
0.02

1.6 × 10-5 2.4 × 10-4 4.6 × 10-4 5.5 × 10-4 5.6 × 10-4
0.02
1.07
0.19
0.89
0.005
0.007
0.63
1.6 × 10-4 2.9 × 10-4 6.5 × 10-4
0.004
0.03
0.07
0.35
0.06
2.0 × 10-4 0.02
9.7 × 10-5 1.1 × 10-4 0
0.02
1.07
0.19
0.89
0.005
0.009
0.85
2.7 × 10-4 3.8 × 10-4 5.4 × 10-4
0.009
0.79
2.5 × 10-3 8.1 × 10-3 8.3 × 10-4
0.004
0.03
0.07
0.35
0.06
0.001
0.12
2.4 × 10-3 4.7 × 10-3 0
6.0 × 10-4 0.05
1.2 × 10-4 7.3 × 10-5 0
0.004
0.09
0.10
0.26
5.7 × 10-3
0.04
0.2
6.1 × 10-4 0.003
5.2 × 10-4
0.008
0.001
0.03
0.78
0.02
0.02
0.04
0.003
7.1 × 10-4 0.001

MR

4.17
0.01
0.44
2.0
0.3
1.81
0.82
0.47
0.003
0.83
0.001
2.17
0.64
0.48
0.02
2.17
0.86
0.81
0.48
0.13
0.05
0.47
0.06
0.83
0.07

† MBTC, mass of viruses recovered in the effluent breakthrough curve (BTC); MS, total mass of retained viruses during Phase I; MIrr, percentage of injectedviruses irreversibly retained on the solid phase.
‡ MIII, MIV, MV, and MV, percentage of injected viruses recovered during Phases III, IV, V, and VI, respectively; MR, percentage of the total mass of the viruses
recovered (MIII + MIV + MV + MVI).
§ NA, not applicable. These data are not presented as a figure in the text.
Journal of Environmental Quality

Fig. 2. The observed breakthrough curves for viruses PRD1 and FX174 at temperature = 4 and 20°C, ionic strength = 10 mM NaCl, and flow velocity= 0.1 m d−1. The release phases (Phases III, IV, and V) were conducted either immediately after the retention phase or after storing the columns for
70 d under the respective experimental temperature. (A) PRD1 and (B) FX174 at 4°C, and (C) PRD1 and (D) FX174 at 20°C. C/C0, output concentration/input concentration; PV, pore volume. Table 2 shows the corresponding mass balance data.

Fig. 3. The observed breakthrough curve for viruses PRD1 and FX174 at ionic strength = 10 mM NaCl, flow velocity = 0.1 m d−1, and temperature =20°C. The first set of columns were released immediately after the retention phase (Phase I). Remaining columns were stored at 4°C (Phase II) andthe release phases (Phases III, IV, and V) were conducted after various storage durations. (A) PRD1 and (B) FX174 after 0, 36, or 46 d of storage. (C)PRD1 and (D) FX174 after 0 and 70 d of storage. Phases III, IV, and V were conducted at temperature = 20°C and flow velocity = 5 m d−1 using thefollowing solution chemistry: Phase III = 10 mM NaCl, Phase IV = Milli-Q water, and Phase V = beef extract with pH 11. C/C0, output concentration/
input concentration; PV, pore volume. The corresponding mass balance data is given in Table 2.
Journal of Environmental Quality

et al. (2017a), it was found that katt1 > ms1 >> ml. Significantly, ms1
was two and one orders of magnitude higher than ml for PRD1,
whereas ms1 was two and three orders of magnitude higher thanml for FX174 at 4 and 20°C, respectively. Generally, the inactivation parameters ms1 and ml were higher with increasing temperature and for FX174 than PRD1 (Tables 1 and 2). The standard
error coefficients on fitted one-site parameters were generally
low but were somewhat higher for some parameter (katt1 andSmax1) associated with FX174 at 20°C because of uncertainty inblocking.
Calculations shown in Table 2 indicate that a small fraction of the injected virus was reversibly retained and not inactivated (<4.17%), and the remaining fractions were irreversibly
retained or inactivated on the solid phase. The one-site retention
model did not account for the reversible virus fraction that was
not inactivated, and therefore this model could not provide an
accurate description of the observed low concentration tailingthat poses an important long-term transport risk of viruses in
MAR systems. To account for this behavior, a model with two
kinetic retention sites and separate solid phase inactivation rates
was needed. The simulated breakthrough curves for this twosite model are shown in Fig. 1B. Fitted model parameters anda statistical measure of the goodness of fit are shown in Table
1. Standard error coefficients for fitted parameters are given in
Supplemental Table S1. Note that katt1 >> katt2, ms1 >> ms2, andkdet1 > kdet2. This suggests that attached viruses on Site 2 detachat a smaller rate than Site 1 and a very small fraction of theseviruses was inactivated at a much slower rate on Site 2 than on
Site 1. The standard error coefficient for some fitted two-site
model parameters was high (Supplemental Table S1) due to theincreased complexity and parameter uncertainty of this model.
Nevertheless, R2 values between measured and simulated breakthrough curves were always >0.62; this agreement suggests that
long-term virus transport and fate processes were correctly captured by the two-site model.
A small fraction of viruses was released at a very small rateduring Phases III to V (Tables 1 and 2). This could be explainedby the difference in adhesive strength of the viruses at a specificattachment location. Previous studies demonstrated that thepresence of nanoscale roughness and chemical heterogeneity onsand (Han et al., 2016; Choo et al., 2015) and virus (Kazumori,
1981; McKenna et al., 1992; Merckel et al., 2005; Peralta et al.,2013) are expected to reduce the magnitude of the energy barrier to attachment/detachment and the depth of the primary
minimum (Bradford et al., 2017; Bradford and Torkzaban,
2013, 2015; Torkzaban and Bradford, 2016). The observationof low rates of virus detachment (Table 1) is consistent withslow, diffusion-controlled, virus release from a primary minimum with a low probability of release (e.g., the energy barrier
to detachment of 5 to 10 kT) (Bradford et al., 2017). Previous
studies demonstrated that a strong adhesive interaction of viruses
to a solid surface can lead to structural damage and the leakageof virus genetic material (adsorption of influenza virus on thehydrophobic polycation immobilized surface) (Hsu et al., 2011),
and spontaneous virion disassembly and physical disruption of
the virion (Murray and Laband, 1979) that will influence virus
inactivation. Consequently, differences in the depth of the primary minimum for viruses on heterogeneous surfaces may also

explain the need to use separate solid phase inactivation rates inthe two-site model.
Reversible and irreversible virus attachment is therefore
highly sensitive to the chemical heterogeneity and roughness
conditions on both the solid and virus surface. For example,
irreversible virus attachment may occur at smooth surfaces withchemical heterogeneity due to the presence of deep primary
minimum, whereas reversible virus interaction was expected onrough surfaces due to the presence of a shallow primary minimum (Bradford et al., 2017; Bradford and Torkzaban, 2012).
This implies that only a very small fraction of the solid surface
may contribute to irreversible virus attachment in a deep primary
minimum since nanoscale roughness is ubiquitous on solid andvirus surfaces (Bradford et al., 2017; Bradford and Torkzaban,2015). These findings are consistent with the small values of a(0.028–0.081) and Sf (8.5 × 10-7–7.8 × 10-9) shown in Table 1,
and the observed blocking behavior for both viruses in the BTCs
(Fig. 1) and our previous study (Sasidharan et al., 2017a). An
increase in temperature decreases the energy barrier height andincreases the depth of the primary minimum (Sasidharan et al.,2017b). A slight increase in the depth of the primary minimum
may increase the fraction of the surface that is favorable to irreversible attachment and may explain the increased retention andsolid phase inactivation of viruses at a higher temperature.

Conclusions and Implications
Experimental results demonstrated that virus attachment
increased with temperature, and 95% of the injected viruses
were irreversibly retained on the solid surface. An advective–dispersive transport model with one-site attachment, detachment,
blocking, and solid and liquid phase inactivation provided anadequate description of the initial BTC. Liquid phase inactivation had a negligible effect on observed virus BTCs comparedwith irreversible attachment and solid phase inactivation, i.e.,katt1 > ms1 >> ml. However, a two-site model was needed to accurately describe low levels of long-term virus release; i.e., katt1 >>katt2, ms1 >> ms2, and kdet1 > kdet2. The calculated values of Sf and awere always very small, which implies that only a small fractionof the solid surface was favorable for attachment. This observation could be explained by the presence of nanoscale roughness
on surfaces of viruses and sand, which decreases the energy barrier and depth of the primary minimum.
Although the vast majority of viruses were irreversibly
retained on the solid phase, a small percentage (<4.17%) werereleased into flowing water, especially during transient solution
chemistry conditions, which can pose a potential risk of longterm transport and to human health. However, the reversible
virus fraction decreased with increasing temperature (4 to 20°C),
increasing storage duration (0 to 70 d), and for FX174 in comparison to PRD1 due to increases in solid phase inactivation. In
addition, temperature cycling (flow at 20°C and storage at 4°C)
demonstrated that solid phase inactivation was a function oftemperature.
Acknowledging irreversible attachment, solid phase inactivation, and liquid phase inactivation in MAR guidelines cansignificantly reduce the number of expensive treatments suchas ultraviolet, reverse osmosis, and micro-membrane filtration.
In particular, the risk of virus release during MAR can be minimized or eliminated by selecting an adequate residence time for
Journal of Environmental Quality

liquid and especially solid phase inactivation under expected
groundwater flow and temperature conditions. For example, ourlong-term experimental results suggest that a 2 to 3 mo residence
time will be sufficient to remove one to three logs of viruses via
irreversible attachment and solid phase inactivation in aquifers
with a temperature above 20°C. Note that the residence time forcurrent MAR applications such as ASR and ASTR in Australia
and United States is 6 to 9 mo when the winter storm is collected
and recovered during summer (Donald et al., 2011; Kremer etal., 2008; NRMMC–EPHC–NHMRC, 2009; Rahman et al.,2012; Toze et al., 2010). However, the residence time may be
shorter in the event of harvesting spring and summer storms. In
some instances, cold stormwater may be injected into a warm
groundwater. In this case, the time required for the mixing andequilibrium of both waters should be considered in the residence
time calculation. This would be especially beneficial for the
ASTR technique (e.g., injected water is mixed with the groundwater and transferred to the recovery well) because it would
provide a greater opportunity for virus removal via solid phaseattachment and inactivation.
Conducting laboratory-scale column experiments can help togain a mechanistic understanding of the process and factors that
control virus removal in saturated porous media. However, it isimportant to translate this knowledge to field-scale applications.
Several column studies have demonstrated the importance ofsite-specific differences such as virus type, sediment mineralogy,
injection water and groundwater composition, flow regimes,
temperature, residence time, and native microbial community
on virus removal in porous media. For example, metal oxides
and calcite-rich sediments have been demonstrated to produce
a three to four log removal of viruses via solid phase attachment
and inactivation (Sasidharan et al., 2017a). The selection ofpotential MAR sites with favorable sediment mineralogy will
therefore also play a key role in determining the efficacy of theASTR applications. Conversely, one should be cautious about
the potential health risk associated viruses with low inactivation
rates, groundwater with low temperature, and aquifers with highpermeability zones with low residence times.
Future MAR guidelines could be based on a database of column-scale and field-scale virus transport and fate experiments.
Column-scale studies for site-specific conditions are relatively
cheap and easy to conduct. Results from such column-scalestudies could be used to aid the design of a limited number offield-scale experiments to validate whether an acceptable level ofthe virus removal can be achieved via ASR or ASTR techniques.
Conducting several site-specific column and field-scale studies
could lead to the development of a database of results for various
geologic conditions to inform MAR guidelines. This information could be further extended through numerical modeling andcalibration of pathogen retention and inactivation parameters.

Supplemental Material
The standard error coefficients for fitted parameters are given
in Supplemental Table S1, available online.

Acknowledgments

The project was completed in collaboration with CSIRO Land andWater program and the Flinders University of South Australia. The
experimental work was conducted in the CSIRO Laboratories at the
Waite Campus, Adelaide, South Australia.
Journal of Environmental Quality

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