Colloids and Surfaces B: Biointerfaces 122 (2014) 778–784

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Relative transport of human adenovirus and MS2 in porous media Kelvin Wong ∗ , Dermont Bouchard, Marirosa Molina ∗∗ USEPA Office of Research and Development, National Exposure Research Laboratory, 960 College Station Road, Athens, GA 30605, United States

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Article history: Received 21 April 2014 Received in revised form 17 July 2014 Accepted 13 August 2014 Available online 22 August 2014 Keywords: Adenovirus MS2 Fate and transport Filtration Ionic strength Deposition

a b s t r a c t Human adenovirus (HAdV) is the most prevalent enteric virus found in the water environment by numerous monitoring studies and MS2 is the most common surrogate used for previous virus transport studies. However, the current knowledge on the transport behavior of HAdV in porous media and the transport relationship between HAdV and MS2 is very limited. In this study, we investigated the influence of ionic strength (IS) on the transport behaviors of HAdV, MS2, and pilus-associated MS2 (p-MS2) in saturated quartz sand columns. Retention of HAdV was higher than MS2 in all three IS (1, 10 and 100 mM NaCl), especially in 10 and 100 mM where virus recoveries in the effluent samples were ≤1% for HAdV, but ≥55% for MS2. Derjaguin and Landau, Verwey and Overbeek (DLVO) theory alone cannot explain why the deposition of HAdV was so much higher. HAdV retention may be strongly enhanced by attaching its long fibers to the sand surface and this deposition mechanism is supported by DLVO energy profiles which show that HAdV can approach the sand surface within reach of its fibers at 10 and 100 mM NaCl. Results of transmission electron microscopy, dynamic light scattering and 0.05 ␮m membrane filtration suggest that the majority of MS2 cultured by Escherichia coli Famp were associated with a residue of pili. Although retention of pilus-associated MS2 (p-MS2) in the column was just slightly higher than individual MS2 particles, membrane filtration results indicated potentially important differences between removal of MS2 and p-MS2 by filtration with finer pore sizes. This is the first study reporting (1) increasing differences in the transport of HAdV and MS2 in porous media with an increase in ionic strength; (2) significant influence of pilus-association to MS2 removal by membrane and porous media filtration; and (3) a mechanistic explanation for the deposition differences of HAdV and MS2 using virus morphology information and DLVO theory. Published by Elsevier B.V.

1. Introduction Fecal pollution of environmental waters is a major concern for the general public since it can have severe impacts on health and can create economic and societal burdens. Enteric viruses have been found in both raw and treated wastewater and sewage sludge [1–3] and with typical dimensions of no more than 100 nm [4] these viruses have potential for significant transport in porous media. Because of their mobility, viruses have been selected as the biological agent to model transport of waterborne pathogens in subsurface environments [5]. Human adenoviruses (HAdVs) are a common cause of gastroenteritis, upper and lower respiratory system infections, and conjunctivitis [6]. They are widespread in the environment and are

∗ Corresponding author. Tel.: +1 706 355 8133; fax: +1 706 355 8104. ∗∗ Corresponding author. Tel.: +1 706 355 8113; fax: +1 706 355 8104. E-mail addresses: [email protected] (K. Wong), [email protected] (M. Molina). http://dx.doi.org/10.1016/j.colsurfb.2014.08.020 0927-7765/Published by Elsevier B.V.

found in marine, river, ground, drinking, recreational and wastewaters [7]. Due to its prevalence in environmental waters, HAdV is a promising fecal source tracker [7] and has been included in the U.S. Environmental Protection Agency’s contaminant candidate lists one, two and three in 1998, 2005 and 2007, respectively [8]. Most viral transport studies, however, focus on bacteriophages [9–13], with MS2 being the most common [5,9–11,13–15]. For the first time, a sorption isotherm approach to evaluate the influence of ion composition and organic carbon on the sorption behavior of HAdV to soil and sand particles [16,17] was utilized by our research group. Nevertheless, there is still very little literature reporting the mobility of HAdV under porous media filtration and the relative transport behavior of HAdV and bacteriophages such as MS2; this lack of information about their transport relationship leads to uncertainty about applying previous MS2 transport data to predict the mobility of HAdV and utilizing MS2 as a transport surrogate for HAdV in future studies. Pili on Escherichia coli cells are the receptors for MS2 during infection [4]. Previous studies reported formation of a virusreceptor complex when isolated cell-free pili were added to MS2

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(ATCC 700891) using the double-layer agar method [24]. After infecting E. coli, the top layer of agar was scraped off, vortexed, and suspended in tryptic soy broth for about 3 h. The cell debris and agar was pelleted by centrifuging at 3000 g for 15 min and the supernatant was filtered by 0.22 ␮m membrane. Since we found the majority of MS2 was associated with the pilus of E. coli even after 0.22 ␮m filtration (see Section 3), two methods, (1) density gradient by the cesium chloride (CsCl) [25] and (2) filtering by 0.05 ␮m membrane, were used to further purify the virus culture to determine if any of these procedures can produce a culture batch with only individual MS2 particles. Diafiltration with 1 mM NaCl was performed on both CsCl purified and 0.05 ␮m-filtered MS2 culture using 100 kDa Amicon (Millipore) to remove the nutrient and other impurities and to increase virus concentration by reducing the volume. The concentration of purified HAdV and MS2 as determined by plaque assay was about 107 and 1011 PFU/ml, respectively. 2.2. Transmission electron microscopy (TEM) Fig. 1. TEM image of pilus-associated MS2 (p-MS2). The long rod-shaped structure is the pilus. Length and thickness of pilus in this image were about 430 and 6 nm, respectively.

[18]. In the work reported herein, pilus-associated MS2 (p-MS2) was identified in the virus culture propagated by E. coli Famp (Fig. 1). Formation of virus aggregate from pilus-association likely can affect MS2 removal by porous media and membrane filtration; however, this effect has never been reported in the literature. Ionic strength (IS) is known to have significant influence on microbial and virus transport behaviors [19–22] and a wide range of IS is present in different environmental waters such as rivers, lakes, estuaries and oceans. Previous studies found the breakthroughs of MS2 were more retarded [20] and recoveries of MS2 were lower [21] under higher IS conditions. However, the attachment of different viruses may respond differently to IS, for example, the recovery of X174 remained the same in higher IS buffer [21]. Other studies have found that IS can enhance the transport of virus under favorable conditions; Zhuang and Jin [22] found retention of both MS2 and X174 by Al-oxide coated sand column reduced under higher IS conditions. Although these studies have shown that the influence of IS on virus transport is significant, no study, to the best of our knowledge, has investigated the effect of IS on HAdV mobility during porous sand filtration. Therefore, the main objectives of this study are to gain knowledge about the influence of IS on HAdV transport in porous media, the relative transport of HAdV and MS2, as well as the removal of p-MS2 by membrane filtration and influence of pili-association on MS2 transport. For the first time, the 96-deep well plate columns packed with Iota quartz sand [23] and quantitative polymerase chain reaction (qPCR), were applied together to evaluate and compare the mobility of viruses in the porous media. Finally, the interfacial potential energies of virus and sand were determined using classic Derjaguin and Landau, Verwey and Overbeek (DLVO) theory to provide mechanistic interpretations to the column observations. 2. Methods and materials 2.1. Virus propagation and purification HAdV2 (strain 6) was obtained from the Centers for Disease Control (Atlanta, GA) and propagated in A549 cells. Propagation and purification of HAdV followed the procedure described in our previous study [16] and are described in the Supplementary data. Bacteriophage MS2 was provided by Professor Joan Rose’s laboratory (Michigan State University) and propagated in E. coli Famp

TEM imaging was performed using the procedure described in our previous study [16] to determine the size of MS2 and p-MS2 used in this study. Approximately 10–20 drops of the viral suspension were dropped, one at a time, onto a carbon-coated formvar grid of 400-mesh and later stained with 3% aqueous phosphotungistic acid, pH 7.0. The grid was then dried in a desiccator and TEM images were acquired using JEM-1210 (JEOL, Tokyo, Japan), coupled with a XR41C Bottom-Mount CCD Camera (AMT, Danvers, MA). 2.3. Zeta potential and hydrodynamic diameter Effect of IS (1, 10, 100 mM NaCl at pH 6) on the zeta potential () and hydrodynamic diameter (Dh ) of HAdV, MS2 and p-MS2 was determined using a ZetaSizer Nano ZS (Malvern Instrument Inc., UK) at 25 ◦ C. NaOH and H2 SO4 were used to adjust the pH. The Dh measured at time zero was compared to the Dh after 15 min of dynamic light scattering (DLS) measurement, or when no further changes in size were observed. Each  and Dh value is the average of four and ten repeated measurements, respectively. Detailed procedures for  and Dh measurements are described in our previous study [16]. 2.4. Well plate column transport studies High purity Iota quartz sand (99.9% silica, Unimin Corporation, New Canaan, CT) was used as porous media in the transport studies and this media has been extensively characterized and its properties are summarized in a previous study [26]. Briefly, the sand was washed with deionized water, oven-dried at 42 ◦ C, and sieved to yield a particle size ranging from 125 to 300 ␮m with an organic carbon percentage of 0.015% by weight, and 4.9 and 0.43 mg/kg of Al and Fe hydroxide, respectively. The well plate column method used here was adopted from Bouchard et al. [23], and has been used successfully to study the transport of bacteria [27] and nanomaterials [23]. Bouchard et al. [23] found that the breakthrough curve (BTC) variability observed for single-walled nanotubes was comparable to that observed in transport studies using traditional packed column techniques [28]. The well plate columns were fabricated by drilling an approximately 2-mm diameter hole in the bottom of each well of a 96-position 2-mL deep well plate (Nalge Nunc, Rochester, NY), and then placing a 20-␮m stainless steel frit (ChromTech, Apple Valley, MN) in each well to support the porous media. Porous media (2 g) were wet-packed into each well which yielded a packed column length of 3.4 cm and left about 0.5 cm of open space at the top of the packed well. Column porosity () was determined gravimetrically assuming a particle density of 2.65 g/cm3 and subsequently a  value of 0.45 was obtained for

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both porous media. The tracer test results described in Bouchard et al. [23] illustrate that the well-plate columns have high columnto-column hydrodynamic reproducibility and are characterized by a convection dominant flow regime with Peclet number means and 95% confidence limits of 60 ± 6. The column experiments were run at three different IS: 1, 10, and 100 mM NaCl at pH 6, and three different virus suspensions (HAdV, MS2, and pilus associated-MS2 (p-MS2)) were run at each IS. It should be noted that “p-MS2” was composed of approximately a 9:1 ratio of p-MS2 to individual MS2 particles (see Section 3). To determine whether the difference between the deposition behaviors of the two viruses was due to charge density of carboxyl groups, column experiments with the aqueous conditions of 3 mM CaCl2 and 1 mM NaCl were run for HAdV and MS2. Virus influent concentrations for all column studies were around 107 PFU/ml. A multi-channel pipettor delivered all liquid volumes to the columns so they were simultaneously eluted by gravity at a mean pore water velocity of 2.1 cm/min. Replicate columns were run for each matrix condition. Columns were prepared by first eluting with a minimum of 5 ml (equivalent to approximately 8 pore volumes) of experimental matrix, followed immediately by seven 200-␮L pulses of virus suspension (approximately 2 pore volumes), and concluded with 17 200-␮L pulses of background solution. Using this elution technique, the degree of saturation was 92% for the Iota quartz. Each 200-␮L pulse resulted in a corresponding 200-␮L eluate fraction which was collected in a well plate. After quantifying the virus concentration, BTCs were plotted as the normalized effluent concentrations (effluent concentration [C] divided by the influent concentration [Co ]) versus pore volumes; the recovery of the virus in the effluent was calculated by the trapezoidal rule [29]. Due to the wide range of C values, the normalized effluent concentrations were plotted in log scale.

Table 1 MS2 concentration in the filtrate and recovery from filtrate after 0.05 ␮m filtration.

Replicate 1 (PFU/ml) Replicate 2 (PFU/ml) Recovery from filtrate (%)

1st filtration

2nd filtration

2.00 × 1010 1.20 × 1010 12.8a

2.30 × 1010 1.15 × 1010 107.8b

a Recovery is calculated as average virus concentration in the filtrate after 1st filtration divided by pre-filtered culture (concentration = 1.25 × 1011 PFU/ml). b Recovery is calculated as average virus concentration in the filtrate after 2nd filtration divided by average virus concentration in the filtrate after 1st filtration.

qPCR measurement, no DNA and RNA controls are included during the column experiment. 2.6. DLVO modeling Classic DLVO theory was used to determine the interfacial potential energy between virus and sand surface [16]. It is calculated by the sum of potential energy contributed by van der Waals (˚vdW ) and the electrostatic double layer (˚EdI ) interaction energies. The calculation of ˚vdW and ˚EdI are summarized in the Supplementary data. 2.7. Statistical analysis To determine whether different conditions can cause significant changes in the Dh of virus, analysis of variance (ANOVA) single tests were performed using Sigma Plot 11.0; a p-value of ≤0.05 indicates a significant difference. 3. Results and discussion 3.1. Identification and removal of pilus-associated MS2 (p-MS2)

2.5. Quantitative PCR (qPCR) analysis qPCR was used to quantify viral particles in the column experiment samples. For MS2, samples and standards were diluted 10× with water, then heated to 99 ◦ C for 5 min to lyse the capsid and release the RNA. For HAdV, 90 ␮L of QuickExtract DNA (Epicenter WI) was added to 10 ␮L samples and standards and then heated to 65 ◦ C for 15 min, followed by heating at 98 ◦ C for 10 min to release the DNA. A standard curve for each IS matrix was generated to quantify the virus number. The R2 values for most standard curves (7 out of 8) were above 0.99 and slopes ranged from 3.24 to 3.78 (Fig. S1). At least one concentration point in the standard curve was included in the sample run to monitor stability of the instrument. The qPCR assay for HAdV and MS2 was adopted from Heim et al. [30] and O’Connell et al. [31], respectively. Details of the reaction mix and amplification conditions are described in the Supplementary data. Capsid-free HAdV-DNA and MS2-RNA can be present in virus culture and can influence the accuracy of column results if there is a high proportion of nucleic acid present in the virus stock. To determine the ratio of virus to nucleic acid numbers, six serial dilutions of virus stocks were prepared. Prior to qPCR analysis, each dilution was separated into two batches: one batch had the capsid lysis step described in the previous paragraph and the other batch had none. For both HAdV and MS2 stocks, the average copy numbers with lysis were more than 3 logs (99.9%) higher than the ones without lysis (Table S1), which means the intact virus number was at least 3 logs higher than the number of capsid-free nucleic acids. The actual ratio of virus to nucleic acid numbers is most likely greater than those presented in Table S1 because copy numbers obtained from batches without lysis were not all from capsid-free nucleic acid due to the heating cycle during qPCR, which could also cause certain numbers of capsid lysis, especially for MS2. Because of the insignificant contribution by the capsid-free nucleic acid to

Many MS2 particles were attached to pilus residues after cell lysis and a TEM image of p-MS2 is illustrated in Fig. 1. The length of p-MS2 in Fig. 1 is around 430 nm; based on our observations, most pilus attached with MS2 were 300–700 nm, with some as long as 1000 nm. As mentioned earlier, previous studies have reported the formation of p-MS2 when isolated cell-free pili were added to MS2 [18], however, this phenomenon has not been taken into consideration in virus transport, to the best of our knowledge. Virus purification by gradient density using CsCl is widely believed to be the most effective method for virus purification, but many p-MS2 were still observed in the post-CsCl purified virus culture under TEM imaging (results not shown), indicating that this method was unable to separate pilus from MS2 or MS2 from p-MS2. We found that filtering with a 0.05 ␮m membrane could obtain a culture with only individual MS2 virus since no p-MS2 was found by TEM in the filtrate. Virus concentration in the filtrate was 87% lower (Table 1) than the pre-filtered culture, indicating that most MS2 before 0.05 ␮m filtration were pilus-associated since a 0.05 ␮m pore is not small enough to filter individual MS2 particles and MS2 did not form large aggregates (see Section 3.2). In addition, we determined whether the removal could be due to MS2 binding to membrane surface by re-filtering the filtrate with a 0.05 ␮m membrane; results showed the virus concentration in the filtrate after second filtration remained the same (Table 1), indicating that all individual virus particles passed through the filter without adhering to the membrane surface. MS2 can also be propagated using E. coli C3000 and this strain has been used in many previous virus transport studies [15,32–34]. However, DLS measurements of MS2 propagated by E. coli C3000 in those studies were about the actual size of MS2 even without 0.05 ␮m filtration. We did not investigate further to determine the influence of E. coli strain on the formation of p-MS2 since it is not

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(a)

500

MS2 p-MS2 HAdV

400

100

1mM, R=31 % 10mM, R=1.0% 100mM, R=0.44 %

10-1

300

C/Co

Hydrodynamic diameter (nm)

600

200 100

10-2 10-3 10-4

0 1mM NaCl

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10-6

-5 -10 -15 -20

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10-2

-45 Fig. 2. Hydrodynamic diameters (Dh ) and zeta potentials () of HAdV and MS2 in 1, 10, and 100 mM of NaCl at pH 6. Error bars represent the standard deviation of 10 and 4 repeated measurements for Dh and , respectively.

within the scope of this study. Because the infection mechanism of E. coli Famp is only via their pili and E. coli C3000 can be infected via both pili and the cell wall [35], it is possible that E. coli Famp has a higher number of pili than E. coli C3000. Further investigation is needed to confirm this hypothesis. In addition, this phage can also be propagated in broth culture, followed by a chloroform extraction [18] and further studies can investigate whether this method can be effective for removing p-MS2. 3.2. Virus aggregation Fig. 2 illustrates the  and Dh measurements of MS2, p-MS2 and HAdV at three different IS. As expected,  of viruses became less negative in higher IS due to electrostatic charge shielding [16]. When IS increased from 1 mM to 100 mM,  increased by 9 mV for p-MS2 and by 22 mV for MS2, indicating that IS has less influence on the  of pilus associated MS2 than free MS2. Similar to previous findings [16,32], MS2 did not aggregate (P > 0.05) when NaCl concentration increased, but HAdV did. The significant aggregation of HAdV in 100 mM can be the result of the  becoming much closer to neutral value (Fig. 2). In addition to the TEM imaging, DLS analysis also indicated that most MS2 without 0.05 ␮m filtration were pilus-associated because Dh values ranged from ∼300 to ∼500 nm, much larger than individual MS2 particles. While the  of p-MS2 became less negative at higher IS, Dh decreased significantly (P ≤ 0.05) (Fig. 2). TEM was not able to determine structural changes of p-MS2 in different IS since the technique used was negative gram stain which requires freeze-drying to remove liquid in the samples. 3.3. Transport of HAdV and MS2 The BTCs of HAdV and MS2 are illustrated in Fig. 3. The peak C/Co at 1, 10 and 100 mM is 0.3, 0.01 and 0.005 for HAdV and 0.8, 0.6 and 0.4 for MS2, respectively, and the recovery at 1, 10 and 100 mM

1

2

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4

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8

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8

Pore Volume

100

-25

-40

0

(b)

C/Co

Zeta potenal (mV)

781

10-3 10-4

1 mM, R=79 % 10mM, R=56 % 100mM, R=55 %

10-5 10-6

0

1

2

3

4

5

Pore Volume Fig. 3. Breakthrough curves of (a) HAdV and (b) MS2 in 1, 10, 100 mM of NaCl at pH 6. The percentage values represent virus recovery (R) from the effluent samples. C/Co was plotted in log scale. Error bars represent the standard deviation from replicate column experiments.

is 31%, 1.0% and 0.44% for HAdV and 79%, 56% and 55% for MS2, respectively. Recoveries of both HAdV and MS2 in the effluent were reduced when IS increased, indicating that higher IS decreased their transport in porous media. However, higher IS conditions reduced the transport of HAdV much more than MS2: recovery of HAdV was reduced by 2 and 3 orders of magnitude when IS increased to 10 and 100 mM, respectively, but MS2 recovery decreased no more than 30%. Both recovery and peak C/Co values at 10 and 100 mM for HAdV are about 2 and 3 logs lower than MS2, respectively. These results clearly show that deposition of HAdV to quartz sand was much more sensitive than MS2 to increased IS. This observed difference in HAdV and MS2 deposition behavior is consistent with aggregation data which indicated that HAdV has greater sensitivity to increases in IS (Fig. 2). Generally, lesser differences between peak and tail-end C/Co values indicate a stronger tailing effect which is a result of reversible deposition. For MS2, there is a direct relationship between the peak C/Co and tailing effect (Fig. 3b): while the peak C/Co values decreased as IS increased, the tailing effect became more pronounced. HAdV had a different trend: the tail-end C/Co at both 1 and 10 mM NaCl are about 2 logs lower than the peak C/Co , but there is almost a 3 log difference between the peak and tail-end C/Co for 100 mM NaCl; the difference in 100 mM is probably larger than what is represented in Fig. 2 since concentrations of HAdV after 4 pore volumes were calculated at the detection limit. The rapid decline of HAdV after the peak C/Co at 100 mM indicates an irreversible deposition of HAdV to sand particles.

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HAdV 10mM 0.1

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70 80 90 100 110 120 130 140

15 20 25 30 35 40 45 50 55 60 -0.005

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Distance (nm) -1 Fig. 4. Interface energy profile between virus and sand particles in 1, 10, 100 mM of NaCl at. pH 6: (a) overall energy profile and (b) energy profile at the secondary minimum.

DLVO interaction energies of viruses and the quartz sand surface were calculated to provide a mechanistic interpretation of the column results (Fig. 4). As expected, the energy barriers for both viruses are reduced as the IS increases, which is due to lowering the electrostatic repulsion by charge shielding with Na+ . The higher deposition of each virus in higher IS generally agrees with the energy barrier profile. The energy barriers of HAdV are higher than MS2 at 1 and 10 mM, but at 100 mM there is no energy barrier for HAdV while MS2 still has a slight energy barrier (∼1 KBT). No energy barrier between HAdV and sand particle at 100 mM agrees with the irreversible deposition shown in the column experiments since, theoretically, deposition in the primary minimum should be irreversible. The reversible deposition of MS2 at 100 mM is possibly due to significant retention in the secondary minimum (Fig. 3b). At

1 and 10 mM the primary energy barriers of HAdV are higher than MS2, but the depositions of HAdV are higher than MS2 – especially at 10 mM (Figs. 2 and 3). DLVO theory thus fails to explain why retention of HAdV was much higher than MS2 at 1 and 10 mM. The hydrophobic interaction is not accounted for here since previous studies have shown hydrophobic interaction between sand and HAdV/MS2 is not significant [14,16]; however, hydrophobic interaction should be considered if the future studies are conducted in clay colloids [36]. Shi et al. [37] suggested that strong deposition of HAdV was due to the high isoelectric point (pHiep ) of HAdV’s fibers. Our results showed that this is unlikely since all three IS had the same pH (∼6), but only 10 and 100 mM produced high deposition of HAdV. Since the function of HAdV fibers is to mediate HAdV attachment

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100

C/Co

10-1 10-2 10-3 10-4 MS2, R=58% HAdV, R=0.8%

10-5 10-6

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Pore Volume Fig. 5. Breakthrough curves of HAdV and MS2 in 3 mM CaCl2 ; 1 mM NaCl at pH 6. The. percentage values represent virus recovery (R) from the effluent samples. Error bars represent the standard deviation from replicate column experiments.

to the host cell [37,38], it is possible that the long (34 nm) and short (20 nm) HAdV fibers have the ability to mediate attachment to the sand surface for attachment. As mentioned earlier, the deposition of HAdV at 10 mM is very high; interestingly, the secondary minimum maximum depth in 10 mM NaCl occurs around 23 nm, which is within the reach of long fibers. At 1 mM, the deposition of HAdV is much lower and there are significant energy barriers at 20 and 30 nm (Fig. 4); the secondary minimum maximum depth occurs near 110 nm, indicating that most HAdV at 1 mM are likely separated from sand surface at a distance beyond the fiber’s length. Therefore, the DLVO profiles support the hypothesis of fibers enhancing HAdV deposition since they indicate HAdV can approach the sand surface close enough for fiber attachment at 10 and 100 mM NaCl (where the deposition is very high), but not at 1 mM NaCl where most HAdV and sand particles are likely separated beyond the fiber’s length. It should also be noted that, although we believe most HAdV and sand particles are far apart in 1 mM, the energy barrier at 30 nm is not very high (∼4 KBT) (Fig. 4); therefore, a portion of HAdVs might be able to approach sand surface close enough to enhance deposition by their fibers, resulting in higher deposition of HAdV than MS2 in 1 mM (Fig. 3). Finally, MS2 has short loops (1 nm) on its capsid [39]; although we do not know whether they can enhance the deposition of MS2 through the same mechanism as the fibers on HAdV, both energy barrier and secondary minimum distance show that MS2 was not likely to reach a distance within reach of its loops (Fig. 4). Previous studies have shown that charge density can influence deposition behaviors of carbon nanotubes and fecal indicator bacteria significantly [40,41]. The loops on MS2 capsid are much denser than the fibers on HAdV capsid [16,39], which can result in higher charge density on the MS2 capsid and increase its steric hindrance. To investigate if the difference between the depositions of HAdV and MS2 is mainly caused by steric hindrance, we utilized the approach described by Yi and Chen [41] which showed that Ca2+ is able to bridge the adjacent carboxyl groups on highly oxidized multiwalled carbon nanotubes better than isolated carboxyl groups on lowly oxidized multiwalled carbon nanotubes, reducing the effect of steric hindrance. Results indicate recovery of HAdV (0.8%) was still much lower than MS2 (58%) in 3 mM CaCl2 , 1 mM NaCl (Fig. 5); therefore, charge density on functional groups was not the major factor causing the different deposition behaviors of the two viruses. Shi et al. [37] evaluated the removal of MS2, X174, Aichi virus and HAdV from drinking water using sand and zero-valent iron (ZVI)-coated sand. After sand filtration, the mass recovery of HAdV in the effluent was about 7.5 percent, but there was almost no removal of MS2; their results, therefore, agree qualitatively

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with ours. Except for studies by Shi et al. [37], to the best of our knowledge there are no others comparing the mobility of HAdV and a bacteriophage in porous media. Prior studies on virus recovery using electropositive filters indicated that HAdV attached to filter materials more strongly than other viruses [7]. Our previous study also indicated that HAdV was highly sorbed on a polypropylene surface when IS increased [17]. Therefore, the consensus of this and previous studies indicates that HAdV is a very “attachable” and “undetachable” virus to different solid surfaces, especially in conditions of minimum electrostatic repulsion such as a high IS aqueous phase or a positively charged absorbent. Because of easy set-up and less analytical time, future studies can consider combining the wellplate column and qPCR to perform a rapid assessment of the deposition behaviors of virus and other forms of microbes such as bacteria, protozoa and antibiotic resistance genes before conducing the large-scale column and field experiments. However, fine control of the flow rate is difficult to achieve in the well plate column and therefore the flow rate of wellplate column is significantly higher than the common groundwater flow rate (0.01–1.05 cm/min) [42]. In addition, using qPCR as the analytical method may require higher concentration of viruses than culture assay since only a few microliter of sample can be put into qPCR reaction compared to culture assay, where the analytical volume can be up to 1–2 ml. 3.4. Transport of p-MS2 The BTCs of p-MS2 are illustrated in Fig. S3, with MS2 BTCs included for comparison. Like MS2, IS did not have a significant impact on p-MS2 transport behavior, and recovery differences between different IS were no more than 25 percent. Both recovery results and peak C/Co values indicate that MS2 was slightly more mobile than p-MS2 (Fig. S2). The mechanism causing more retention of p-MS2 in the column could be straining [10], although it is likely the straining effect is not very pronounced due to the large pore size in the sand column. Although differences between removals of MS2 and p-MS2 in sand column experiments are low, membrane filtration results (Table 1) indicate potentially important differences in other types of filtration studies with pore sizes between the dimensions of MS2 and p-MS2. Therefore, future studies should investigate whether there is a significant difference between the removals of MS2 and p-MS2 by other kinds of filtration processes with finer pores. Also, enteric viruses such as norovirus and enterovirus occur as individual virus particles and are not associated with any pilus-like structure. To better mimic enteric viruses, future transport, filtration or other studies using MS2 as the surrogate should use only the virus culture with individual MS2 particles which requires removing p-MS2 by 0.05 ␮m membrane filtration, particularly for MS2 propagated by E. coli Famp. 3.5. Environmental implications and future perspectives Overall, this study provided important insight into the transport behavior of HAdV and the transport relationship of HAdV and MS2. Results indicated that MS2 can be a conservative but unlikely an accurate transport surrogate for HAdV since IS can influence the fate of HAdV in sand media of the subsurface environment much more than MS2. Even though HAdV is the most prevalent enteric virus in biosolids, its high retention in sand is protective of aqueous resources since it is more likely to be removed during soil infiltration. Unlike MS2, HAdV is very resistant to UV inactivation in water and wastewater treatment [6,43], but this study shows sand filtration may be able to remove it effectively. On the other hand, HAdV might not be a good indicator for viral removal by sand filtration since its removal could overestimate removal of other viruses.

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Previous studies showed that the presence of organic matter (OM) also plays a critical role in hindering the attachment of HAdV and other viruses to solid surface [13,17], and future studies should investigate the influence of OM on the transport of HAdV in different IS conditions and on the relative transport of HAdV and other viruses. In addition, co-presence of MS2 and clay colloid can affect the transport of MS2 [44] and a similar study should be performed on HAdV. Finally, as more column studies of HAdV are conducted in the future, mathematical modeling of the breakthrough curve should be performed to determine critical parameters such as attachment and detachment coefficients for modeling the transport behavior of HAdV in the field. Acknowledgments The authors would like to thank Dr. Vincent Hill and Amy Kahler from the Centers for Disease Control and Prevention (Atlanta, GA) for providing the adenovirus stock, Dr. Maricarmen Garcia and Sylvia S. Riblet from the Poultry Diagnostic Research Center at the University of Georgia (Athens, GA) and Dr. Bruce Seal and Johnna Garish from the United State Department of Agriculture for their assistance in viral purification, and Dr. Indranil Chowdhury of National Research Council for valuable review comments. This report has been subjected to the U.S. EPA’s peer and administrative review and has been approved for publication. The mention of trade names or commercial products in this report does not constitute endorsement or recommendation for use. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.08.020. References [1] K. Wong, B.M. Onan, I. Xagoraraki, Quantification of enteric viruses, pathogen indicators, and salmonella bacteria in class B anaerobically digested biosolids by culture and molecular methods, Appl. Environ. Microbiol. 76 (2010) 6441–6448. [2] F.J. Simmons, D.H.W. Kuo, I. Xagoraraki, Removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing, Water Res. 45 (2011) 2739–2750. [3] K. Wong, I. Xagoraraki, A perspective on the prevalence of DNA enteric virus genomes in anaerobic-digested biological wastes, Environ. Monit. Assess. 184 (2012) 5009–5016. [4] R.M. Maier, I.L. Pepper, C.P. Gerba, Environmental Microbiology, Academic Press, 2008. [5] J.F. Schijven, S.M. Hassanizadeh, Removal of viruses by soil passage: overview of modeling, processes, and parameters, Crit. Rev. Environ. Sci. 30 (2000) 49–127. [6] S.C. Jiang, Human adenoviruses in water: occurrence and health implications: a critical review, Environ. Sci. Technol. 40 (2006) 7132–7140. [7] K. Wong, T.T. Fong, K. Bibby, M. Molina, Application of enteric viruses for fecal pollution source tracking in environmental waters, Environ. Int. 45 (2012) 151–164. [8] EPA, Water Contaminant Candidate List, 2012, http://water.epa.gov/scitech/ drinkingwater/dws/ccl/ [9] L. Cheng, A.S. Chetochine, I.L. Pepper, M.L. Brusseau, Influence of DOC on MS2 bacteriophage transport in a sandy soil, Water Air Soil Pollut. 178 (2007) 315–322. [10] Y. Jin, M. Flury, Fate and transport of viruses in porous media, Adv. Agron 77 (2002) 39–100. [11] D.K. Powelson, J.R. Simpson, C.P. Gerba, Effects of organic matter on virus transport in unsaturated flow, Appl. Environ. Microbiol. 57 (1991) 2192–2196. [12] M.P. Tong, Y. Shen, H.Y. Yang, H. Kim, Deposition kinetics of MS2 bacteriophages on clay mineral surfaces, Colloids Surf. B Biointerfaces 92 (2012) 340–347. [13] J. Zhuang, Y. Jin, Virus retention and transport as influenced by different forms of soil organic matter, J. Environ. Qual. 32 (2003) 816–823. [14] R. Attinti, J. Wei, K. Kniel, J.T. Sims, Y. Jin, Virus’ (MS2, phi X174, and Aichi) attachment on sand measured by atomic force microscopy and their transport through sand columns, Environ. Sci. Technol. 44 (2010) 2426–2432. [15] B. Michen, F. Meder, A. Rust, J. Fritsch, C. Aneziris, T. Graule, Virus removal in ceramic depth filters based on diatomaceous earth, Environ. Sci. Technol. 46 (2012) 1170–1177.

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