Human Virus and Bacteriophage Inactivation in Clear Water by

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Environ. Sci. Technol. 2010, 44, 6965–6970

Human Virus and Bacteriophage Inactivation in Clear Water by Simulated Sunlight Compared to Bacteriophage Inactivation at a Southern California Beach DAVID C. LOVE,* ANDREA SILVERMAN, AND KARA L. NELSON* Department of Civil and Environmental Engineering, University of California Berkeley, Berkeley, California 94720

Received January 18, 2010. Revised manuscript received August 8, 2010. Accepted August 12, 2010.

Few quantitative data exist on human virus inactivation by sunlight and the relationship between human and indicator viruses under sunlit conditions. We investigated the effects of sunlight on human viruses (adenovirus type 2, poliovirus type 3) and bacteriophages (MS2, Q-Beta SP, Fi, M13, PRD1, PhiX174, and coliphages isolated from Avalon Bay, California). Viruses were inoculated into phosphate buffered saline or seawater, exposed to a laboratory solar simulator for e12 h, and enumerated by double agar layer or cell culture to derive first-order inactivation rate constants (kobs, h-1). The viruses most resistant to sunlight were adenovirus type 2 (kobs) 0.59 ( 0.04 h-1) and bacteriophage MS2 (kobs) 0.43 ( 0.02 h-1), which suggests MS2 may be a conservative indicator for sunlight resistant human viruses in clear water when sunlight inactivation is the main removal mechanism. Reasonable agreement was observed between somatic coliphage inactivation rates measured in the solar simulator (kmean ) 1.81 h-1) and somatic coliphages measured in the surf zone during a field campaign at Avalon Bay during similar sunlight intensity (k ) 0.75 h-1 at log-RMSE minimum; krange ) 0.54 h-1 to >1.88 h-1; Boehm, A. B. et al. Environ. Sci. Technol. 2009, 43, (21), 8046-8052). Hence, measuring sunlight inactivation rates of viruses in the laboratory can be used to estimate inactivation in the environment under similar sunlight and water quality conditions.

Introduction Natural sunlight that reaches the surface of the Earth is composed of medium and long wavelength UV light [UVB (280-320 nm); UVA (320-400 nm)], visible light (400-700 nm), and longer wavelengths. Sunlight can damage microorganisms directly, for example, when DNA and RNA absorb photons resulting in the formation of pyrimidine dimers and other photoproducts (2). In addition, indirect damage can occur if endogenous or exogenous photosensitizers absorb photons leading to the formation of reactive intermediates that damage cell or viral components (3-5). In marine water, sunlight has a dramatic and measurable impact on populations of eukaryotic primary producers such * Address correspondence to either author. Phone: 443.287.4761 (D.C.L.); 510.643.5023 (K.L.N.). E-mail: [email protected] (D.C.L.); [email protected] (K.L.N.). 10.1021/es1001924

 2010 American Chemical Society

Published on Web 08/20/2010

as phytoplankton (6), fecal bacteria (7, 8), and bacteriophages (9-11). Sunlight can be used to inactivate human viruses and other microbial pathogens as a beneficial and low-cost treatment method for drinking water (e.g., SODIS) (12-15) or in waste stabilization ponds (3, 4, 16, 17) and for “disinfecting” recreational bathing water (18). Many waterborne diseases are of viral etiology (19), and yet, sunlight inactivation of human viruses is understudied (12, 20). In a recent review of virus inactivation by sunlight, findings were extrapolated instead from germicidal UVC studies because few published studies with sunlight wavelengths exist (21). The few sunlight studies that do exist use bacteriophages (viruses of bacteria), but sunlight inactivation of bacteriophages has not been compared to inactivation of human viruses (8, 22-24). The goal of this study was to quantify the effects of simulated sunlight on a large (n ) 26) and diverse panel of ssRNA, ssDNA, and dsDNA bacteriophage and human viruses in PBS and seawater. Inactivation rates measured in the laboratory of field isolated bacteriophages are compared to bacteriophage reference strains, human viruses, and an empirical deterministic model of bacteriophage inactivation developed from a field study in Avalon Bay, California (1). To generalize findings, the inactivation rates for bacteriophages and human viruses in sunlight are compared to inactivation rates previously reported for UVC.

Materials and Methods Human Viruses and Host Cells. Adenovirus type 2 was kindly provided by Mark Sobsey (University of North Carolina) and cultured in A549 cells (ATCC CCL-185). Attenuated poliovirus type 3 was kindly provided by Ali Boehm (Stanford University) and was cultured in HeLa cells (ATCC CCL-2). Virus plaque assays were performed in 6-well plates with duplicate 100 µL virus inocula; plaque forming units (PFUs) were enumerated after 6 day of incubation for adenovirus and 3 day of incubation for poliovirus. Virus propagation methods are further described in the Supporting Information; purification methods are described here. Crude virus stocks were prepared by 3× freeze/thaw of flasks to release intracellular viruses, followed by chloroform extraction (1:3, vol/vol) and centrifugation (4,000g for 10 min) in 50 mL conical bottom centrifuge tubes (Fisher Scientific) to remove cell debris, and filtered through a 0.22 µm filter. Virus stocks were stored at -80 °C. Bacteriophages and Host Cells. Bacteriophage strains MS2, QB, Sp, Fi, M13, PRD1, and Phi-X174 were kindly provided by Mark Sobsey. Field isolates were picked from somatic and F+ coliphage plaques recovered from Avalon, CA, marine water as described previously, and a representative subset of isolates was selected for inactivation experiments based on viral nucleic acid content, time of collection, plaque size, and titer (1). Bacteriophages were propagated in respective hosts (Table 1) by broth enrichment (25) and prepared as crude virus stocks similar to human viruses though without freeze/thaw steps (see Supporting Information for additional details). In some experiments (Figures 3 and S5, Supporting Information), bacteriophages were prepared as purified virus stocks, in which crude virus stocks were polyethylene glycol (PEG 6000) precipitated overnight at 4 °C, centrifuged at 23 000g for 30 min to produce virus pellets that were then resuspended in phosphate buffered saline (PBS: 5 mM phosphate, 10 mM NaCl, pH 7.5), chloroform extracted again as above, and filtered through a 0.22 µm filter. PRD1 was not chloroform extracted for purification because chloroform partially inactivates PRD1 VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Nomenclature, Characteristics, and Culture Methods for Reference Human Viruses and Bacteriophages Used in This Study “colloquial name” virus family: strain(s) used Tectiviridae: PRD1 “somatic coliphage” I. Microviridae: Phi-X174 II. Podoviridae III. Siphoviridae IV. Myoviridae “F+ coliphages” I. Leviviridae “F+ RNA coliphage”: MS2, QB, Sp, Fi II. Inoviridae “F+ DNA coliphage”: M13 Picornaviridae enteroviruses: poliovirus 3 Adenoviridae adenoviruses: adenovirus 2 a

genome length (kb)a

nucleic acid typea

147-157

dsDNA, linear

4.4-5.4 40-42 48.5 33.6-170

ssDNA, circular

icosahedral

dsDNA, linear

icosahedral with tails

3.5-4.3

ssRNA, linear

icosahedral

4.4-8.5 7-8.5 35.8-36.2

ssDNA, circular ssRNA, linear dsDNA, linear

filamentous icosahedral icosahedral with spikes

International Committee on the Taxonomy of Viruses (41).

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icosahedral with spikes

methods used (host/cell line; ATCC #) DALb (Salmonella LT2; 19585)

DAL (E. coli CN13; 700609)

DAL (E. coli Famp; 700891)

(data not shown). Bacteriophage stocks were stored at -80 °C. Bacteriophages were enumerated as PFUs using the double agar layer (DAL) method (26) with 100 µL of virus inocula. Solar Simulator Experiments. Experiments were conducted with a 1000 W Xe lamp producing a collimated beam in an Oriel solar simulator (model # 91194-1000, Newport Co., Irvine, CA) equipped with an Oriel AM 1.5:G:A “global” filter. Additional light filters were used (i) to mimic full spectrum natural sunlight (atmospheric attenuation filter, Oriel part # 81088, Newport Co.) and (ii) to block the UVB portion of the full spectrum (UVB blocking filter, Oriel part # 81017, Newport Co.; Figure S1, Supporting Information). The resulting light spectra were measured using spectroradiometers (RPS 200 and 380, International Light). Experiments were conducted in 150 mL glass beakers (reactors) painted black, with 100 mL solutions (PBS, Avalon, CA, seawater, or 0.45 µm filtered Avalon, CA, seawater) stirred by magnetic stir bars and cooled to 20 °C by partial submersion in a water bath with a recirculating chiller (Thermo Electron). Viruses were inoculated into reactors at titers of 104-106 PFU/mL. Duplicate 0.5 mL subsamples were removed at intervals, with no more than 10% of the total reactor volume removed during any experiment. Seawater samples were analyzed immediately, because of poor recovery in frozen samples (data not shown). All samples in PBS were frozen at -80 °C before analysis. Virus inactivation was not observed in dark controls, except for one trial (Table S1, Supporting Information). Bacteriophage Typing and Pulse Field Gel Electrophoresis. Coliphages isolated from Avalon Bay, CA, were characterized by several methods: RNase test (25), genotyping (26) for F+ RNA and F+ DNA coliphages, and pulse field gel electrophoresis (PFGE) to determine somatic coliphage genome length (27). PFGE was only used for somatic coliphage because F+ RNA and F+ DNA coliphage families have fairly well-defined genome size ranges, while somatic coliphages do not (Table 1). Coliphage typing and PFGE methods are further described in the Supporting Information. Data Analysis. Virus inactivation rate constants (kobs, h-1) were calculated as the slope of a linear regression trendline of ln(N/No) versus time. R2 for linear regression lines and standard deviation of kobs from replicate experiments are reported (Table S2, Supporting Information). Statistical tests were performed in R (http://www.r-project.org/) or Prism (v5.0b, GraphPad Software, San Diego, CA). 6966

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b

cell culture (HeLa; CCL-13) cell culture (A549; CCL-185)

DAL ) double agar layer method (26).

FIGURE 1. Inactivation rate of (kobs, h-1) somatic coliphage field isolates from Avalon Bay, CA, compared to somatic coliphage genome length (kilobases) measured by pulse field gel electrophoresis.

Results Solar Simulator and Natural Sunlight. The characteristics of light from the solar simulator and field sunlight data from Avalon Bay, CA, are presented in the Supporting Information (Figures S1,S2; Table S3) to illustrate that the solar simulator produces realistic sunlight and to introduce the basis of comparison between laboratory and field virus data. Solar Simulator Inactivation of Coliphages Recovered from Avalon Bay, CA. The inactivation of a representative number of coliphage isolates recovered from Avalon Bay, CA, water (Table S4, Supporting Information) was studied in a solar simulator to characterize intrapopulation variability at the field site and for comparison to other viruses. To better interpret variability, the somatic coliphage field isolates were then analyzed by PFGE to determine their genome sizes, which ranged from 33.5 to 121.7 kb (Figure 1). Somatic coliphage inactivation rate constants were roughly correlated with genome size, with higher inactivation rate constants for larger viral genomes (Figure 1). The linear regression slope of PFGE genome size vs kobs was significant (p ) 0.0053, F ) 15.89) with R2 ) 0.69. The time of sample collection was not correlated with somatic coliphage inactivation rate constants (p ) 0.1742, F ) 2.287, R2 ) 0.25). Inactivation rate constants were similar among four F+ DNA coliphages collected over a 5 h period (kobs ) 1.58-1.94 h-1; R2 ) 0.97-0.99; Figure S3A, Supporting Information) and among four F+ RNA coliphages collected over a 4 h period (kobs ) 0.57-1.02 h-1; R2 ) 0.97-0.99; Figure S3B, Supporting Information). There was a significant difference in inactivation rate constants between F+ DNA and F+ RNA coliphages (p ) 0.0007, 2-tailed t test; Figure S3, Supporting

FIGURE 2. Solar simulator inactivation of (A) adenovirus type 2 and (B) poliovirus type 3 inoculated into phosphate buffered saline (pH 7.5). The solar simulator was operated with an atmospheric filter (open circles; n ) 4) or a UVB blocking filter (open squares; n ) 3). Standard deviation error bars may be obscured by some data points. Information). In partial genome sequencing, F+ RNA coliphage isolates were at most two nucleotides different from each other in the 228 base-pair replicase region sequenced and were genogrouped as MS2-like Leviviruses (Figure S4, Supporting Information). All F+ DNA coliphage isolates were identical in the 194 base-pair gene IV region sequenced and were genogrouped as f1-like Inoviruses (Figure S5, Supporting Information). Solar Simulator Inactivation of Human Viruses in PBS. With full spectrum simulated sunlight, the adenovirus type 2 inactivation rate constant was kobs ) 0.59 ( 0.04 h-1 (R2 ) 0.96-1.00; n ) 3; Figure 2A), and the poliovirus type 3 inactivation rate constant was kobs ) 2.16 ( 0.08 h-1 (R2 ) 0.97-0.99; n ) 3; Figure 2B). When only UVA and visible light were present, inactivation of poliovirus and adenovirus was insignificant over the 8 h exposure period, although the slopes of the inactivation curves were nonzero (Figure 2A adenovirus p ) 0.0159, F ) 7.676; Figure 2B poliovirus p ) 0.034, F ) 5.582). We did not attempt to determine whether host-mediated repair of adenovirus occurred in the A549 cells (28). Solar Simulator Inactivation of Bacteriophage Reference Strains MS2, PRD1, and Phi-X174 in Seawater and PBS. The relative inactivation of three bacteriophage reference strains under all conditions studied was as follows: Phi-X174 > PRD1 > MS2. Significantly less inactivation was observed when UVB wavelengths were blocked (Figure 3D-F) than when under full spectrum sunlight (Figure 3A-C). Inactivation rate constants were similar in the three matrixes (PBS and filtered and unfiltered Avalon seawater) in the presence and absence of UVB for MS2 (Figure 3A,D,G) and PRD1 (Figure 3B,E,H). Experiments with UVA and visible light using crude virus

stocks (Figure 3D-F) were repeated using purified virus stocks (Figure 3G-I). Although no difference was seen for MS2 or PRD1, less inactivation of purified stocks of Phi-X174 was observed (Figure 3I) than of the crude stocks of PhiX174 for all three matrixes (Figure 3F). Further experiments determined that the small amount of culture media present in the unpurified stocks of Phi-X174 contributed to inactivation (Figure S6, Supporting Information). However, matrix effects were not evident with Phi-X174 purified stocks (Figure 3F). Virus Inactivation Constants for Human Viruses, Bacteriophage Reference Strains, and Field Isolates. Using full spectrum sunlight, the range of solar simulator inactivation rates varied more widely for ssDNA and dsDNA viruses than for ssRNA viruses (Figure 4). Significant differences were observed among all viruses (p < 0.0001, ANOVA). The relative sensitivity of virus types to simulated sunlight was as follows: Poliovirus type 3 ) [F+ DNA coliphage field isolates]avg ) [somatic coliphage field isolates]avg > PRD1 g [F+ RNA field isolates]avg g [F+ RNA lab strains (MS2, Q-Beta, Sp, Fi)]avg ) Adenovirus type 2 (p < 0.05 Tukey’s post-test) (Figures 4 and S2, Supporting Information). Statistical comparison of M13 and Phi-X174 was not possible because only one replicate was used. The mean inactivation rates for somatic coliphage field isolates in a laboratory solar simulator (kmean ) 1.81 h-1 with an outlier of 7.51 h-1) were the same order of magnitude as model predictions for somatic coliphage inactivation rates in Avalon Bay, CA (klog-RMSE min ) 0.75 h-1; krange ) 0.536 to >1.88 h-1), using the time of day (10:00 a.m., IUVB,SMARTS ) 0.65 W/m2, ksun ) 28 d-1 IUVB-1 (1)) when intensity in Avalon, CA, ost resembled laboratory solar simulator intensity (Figures 4 and S2, Supporting Information). Comparison of Virus Inactivation by Simulated Sunlight and Monochromatic UV 254 nm. Inactivation rate constants and 95% confidence intervals were plotted for six viruses exposed to simulated sunlight in this study and UV 254 nm as compiled by Hijnen and colleagues (29) (Figure 5). Similar rankings of UV sensitivities between viruses exposed to monochromatic UV 254 nm and simulated sunlight (atmospheric filter) were revealed, which may indicate some similarities in the mechanisms of inactivation by UVC and sunlight in clear water.

Discussion Laboratory and Field Comparison. This research builds on a field study of virus and bacteria concentrations over 72 h with a 1 h sampling interval at a swimming beach in Avalon Bay, CA (1). In that study, sunlight inactivation rates for somatic coliphage and fecal indicator bacteria were estimated from the field data by accounting for various sources and lossprocessesinthenearshorewithanempirical-deterministic model. It was not possible to estimate field inactivation rates of F+ coliphage and human virus concentrations in Avalon Bay, CA, because concentrations were too low in the 1 L samples. To overcome this limitation, in the present work, we propagated virus stocks in the laboratory and exposed seeded reactors to simulated sunlight. To allow us to compare these two different but complementary approaches with each other, we collected somatic coliphage isolates during the field study and measured their inactivation using the laboratory solar simulator. We found reasonable agreement between the measured inactivation rates in the field and the lab (Figure 4). The mean somatic coliphage inactivation rate measured in the solar simulator was about 2.5-fold higher than the best-fit value determined from the Avalon model, but there is considerable uncertainty associated with several model parameters, such that we cannot rule out that the actual mean value was several times higher (as indicated by the dashed arrow in Figure 4, described further in ref 1). In VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Solar simulator inactivation using an atmospheric filter (A, B, C) or a UVB blocking filter (D, E, F, G, H, I) with bacteriophages MS2, PRD1, and Phi-X174. Bacteriophages were inoculated into PBS (triangles; n ) 1), 0.45 µm filtered Avalon, CA, seawater (circles; n ) 1), or unfiltered Avalon, CA, seawater (squares; n ) 1). Solar simulator inactivation was performed with crude bacteriophage stocks (A, B, C, D, E, F) and purified bacteriophage stocks (G, H, I). addition, one somatic isolate was much more sensitive to sunlight than the other eight isolates (Figure 4). It is possible that more differences would be observed if a larger pool of isolates was studied; thus, the isolates studied may not accurately represent the diverse population present in Avalon Bay during the field study. Another important difference is that the coliphages in the laboratory were exposed to constant intensity sunlight, compared to variable intensity in the field; small differences in the UVB spectra may also be important. Nonetheless, we consider the level of agreement between the field and laboratory-measured rates to be good enough to give us confidence that the experiments conducted in the laboratory with other viruses can be useful for understanding their inactivation in the field under similar sunlight and water quality conditions. Human Viruses. Adenovirus type 2 (dsDNA) was more resistant to sunlight than poliovirus type 3 (ssRNA) and the different types of bacteriophages, except for the F+ RNA coliphage lab strains (ssRNA), which were inactivated at about the same rate (Figure 4). Poliovirus type 3 (ssRNA) was inactivated at about the same rate as the somatic field isolates in the laboratory. If we extrapolate these results to the field conditions at Avalon beach, where we observed about a 3 log decrease in somatic coliphage during sunlit hours (August, 2008), we might expect poliovirus to also be decreased by about 3 logs, which agrees well with the 3 log inactivation of poliovirus reported in seawater microcosms in Hawaii during the summer (20). We would expect 3 to 4 times lower inactivation of adenoviruses, or less than one log. Virus Factors. Differences in virus inactivation rates among strains we exposed to sunlight were not simply a 6968

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function of nucleic acid type; adenovirus (dsDNA) was more resistant than poliovirus (ssRNA), whereas F+ RNA coliphages (ssRNA) were more resistant than somatic coliphages (dsDNA; Figure 4). Rather, as indicated in the literature, many additional factors influence the sunlight inactivation rate, including genome length and morphology. In this study, the inactivation rate of the somatic coliphage isolates roughly increased as the genome length increased (Figure 1; Table S4); however, this result needs to be verified with a larger number of isolates with more variability in genome length. Others have shown that naked viral DNA and RNA with increasing strand length was associated with increased DNA or RNA lesions (30, 31) and that intact viruses (vesicular stomata virus and Q Beta) are more resistant to UV inactivation than the same viruses with defective, smaller genomes (21, 32). Thus, within a virus family, genome length may correlate with sensitivity to sunlight. For example, Lytle and Sagripanti (21) found that viruses with the same type of genome have similar sensitivities to UVC once normalized by viral genome size. In our limited study of field isolated viruses and laboratory reference strains exposed to sunlight, we found that F+ RNA and F+ DNA coliphage isolates from the field and laboratory had similar inactivation kinetics within these virus families (Figure S3, Supporting Information). More research is needed to characterize the variability among isolates of F+ and somatic coliphage from other field sites. On virus morphology, Rauth (33) found that an icosahedral-shaped virus (Phi-X174) was inactivated faster than a rod-shaped virus (fd), although both have circular ssDNA of similar genomic length. Thus, genomic packaging

FIGURE 4. Box and whisker plot of inactivation rate constants (kobs, h-1) for viruses in a laboratory solar simulator using the full sunlight spectrum and a range of rate constants from a model of field observations at Avalon Bay, CA (i.e., “somatic_ field_model”). The somatic_field_model was calculated using 10:00 a.m. sunlight intensity (see Figure S2A, Supporting Information) and was a range of modeled kobs, h-1, values minimizing log RMSE (open circle marks kobs, h-1, at log-RMSE minimum). More on the somatic_field_model is in Boehm et al. (1). N ) # strains and n ) # replicates per strain. Letters indicate significant different rate constants with A g B g C > D (p < 0.05 Tukey’s multiple comparison test).

reduces adenoviruses’ intracellular host repair. If similar protein damage occurs with exposure to sunlight, then intracellular host repair may also be reduced or prevented. Mechanism of Damage in Clear Water. The water from Avalon beach was exceptionally clear compared to most coastal ocean waters (35) with a 90% absorbance depth of 12.3 m for 305 nm light and 20.3 m for 340 nm light (Figure S7, Supporting Information; absorption in the 4.6 cm deep reactors was minimal). For MS2 and PRD1, we did not see a difference between inactivation in seawater and PBS in our laboratory microcosms, and for Phi-X174, the matrix affects were eliminated when purified virus was used. We conclude that indirect inactivation involving exogenous sensitizers did not contribute significantly to virus inactivation in the Avalon seawater or PBS. Inactivation was dominated by UVB wavelengths, suggesting that direct damage to nucleic acids was a dominant inactivation mechanism. However, damage to the protein capsid is also possible. For example, it was recently shown that low pressure UV (254 nm) damaged the protein capsid of MS2, possibly via both direct and indirect mechanisms involving endogenous sensitizers (36); also, as mentioned above, medium pressure UV has been suggested to damage the adenovirus capsid (34). Our results should be extendable to other clear waters and also complement studies on bacteriophage ecology in the ocean, in which others report direct nucleic acid damage by UVB wavelengths in sunlight to be an important inactivation mechanism (9, 37-39). However, inactivation mechanisms and relative inactivation rates among viruses are expected to be different in waters containing higher levels of sensitizers that produce reactive oxygen species (ROS). For example, in water containing high concentrations of algae or natural organic matter (NOM), MS2 is very sensitive to 1O2 (4, 40), and wavelengths longer than UVB contribute significantly to inactivation (4, 8, 24).

Acknowledgments This researched was supported by an NSF CAREER/PECASE Award to K.L.N. (BES-0239144). We thank Jee Yeon Kim, Kevan Yamahara, Ali Boehm, Kris McNeill, and Brit Peterson for their work during the Avalon, CA, field campaign. Khalid Khadir and Mike Fisher, University of California Berkeley (UCB), provided technical support on the laboratory solar simulator. Isaac Ward, Hillary Smith, and Seth Nickell were helpful laboratory and field volunteers. We thank Lee Riley, Sara Tartof, and Owen Solberg (UCB) for assistance with PFGE, Andy Jackson (UCB) for his ultracentrifuge, and Ann Fisher and her tissue culture facility at UCB.

Supporting Information Available -1

FIGURE 5. Comparison of inactivation rate constants (kobs, h ) for viruses by laboratory simulated sunlight (i.e., UVB, UVA, and visible light) and by monochromatic UV 254 nm (29). X-axis and Y-axis 95% confidence intervals are presented, except for Phi-X174 and Q-Beta that only have X-axis error bars. Viruses in order of most-to-least UV 254 nm resistance: adenovirus > MS2 > Q-Beta > PRD1 > poliovirus > Phi-X174. within viruses of different morphologies may impact susceptibility to sunlight. Adenovirus may be more resistant to sunlight than other waterborne viruses because its double stranded DNA genome has redundancies that single stranded genomes lack; damage to dsDNA can also be repaired by cell host machinery (28) unlike some DNA or RNA viruses. Interestingly, Eischeid and colleagues (34) showed that adenovirus is more susceptible to medium-pressure (MP) polychromatic UV than low-pressure (LP) monochromatic UV, despite causing similar genome damage, possibly because MP causes protein damage which

A description of human virus and bacteriophage propagation, and bacteriophage typing methods and findings. Figures on the solar simulator spectrum, ambient sunlight spectrum, and UV absorbance depth in water. A figure on the inactivation of F+ RNA and F+ DNA coliphage field isolates, as well as figure on Phi-X174 inactivation following different purification methods. A table lists inactivation in dark controls in all experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Boehm, A. B.; Yamahara, K. M.; Love, D. C.; Peterson, B. M.; McNeill, K.; Nelson, K. L. Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage-impacted marine beach. Environ. Sci. Technol. 2009, 43 (21), 8046–8052. (2) Jagger, J., Solar-UV actions on living cells. Praeger: New York, 1985; p 1-202. (3) Davies-Colley, R.; Donnison, A.; Speed, D.; Ross, C. Inactivation of faecal indicator micro-organisms in waste stabilisation ponds. Water Res. 1999, 35, 219–225. VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(4) Kohn, T.; Nelson, K. L. Sunlight-mediated inactivation of MS2 coliphage via exogenous singlet oxygen produced by sensitizers in natural waters. Environ. Sci. Technol. 2007, 41 (1), 192–197. (5) Sammartano, L. J.; Tuveson, R. W. The effects of exogenous catalase on broad-spectrum near-UV (300-400 nm) treated Escherichia coli cells. Photochem. Photobiol. 1984, 40 (5), 607–612. (6) Smith, R. C.; Pre´zelin, B. B.; Baker, K. S.; Bidigare, R. R.; Boucher, N. P.; Coley, T.; Karentz, D.; MacIntyre, S.; Matlick, H. A.; Menzies, D. Ozone depletion: ultraviolet radiation and phytoplankton biology in antarctic waters. Science 1992, 255 (5047), 952–959. (7) Davies-Colley, R.; Bell, R.; Donnison, A. Sunlight Inactivation of Enterococci and Fecal Coliforms in Sewage Effluent Diluted in Seawater. Appl. Environ. Microbiol. 1994, 60 (6), 2049–2058. (8) Sinton, L. W.; Finlay, R. K.; Lynch, P. A. Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater. Appl. Environ. Microbiol. 1999, 65 (8), 3605–3613. (9) Regan, J. D.; Carrier, W. L.; Gucinski, H.; Olla, B. L.; Yoshida, H.; Fujimura, R. K.; Wicklund, R. I. DNA as a solar dosimeter in the ocean. Photochem. Photobiol. 1992, 56 (1), 35–42. (10) Weinbauer, M.; Wilhelm, S.; Suttle, C.; Pledger, R.; Mitchell, D. Sunlight-induced DNA damage and resistance in natural viral communities. Aquat. Microb. Ecol. 1999, 17 (2), 111–120. (11) Wilhelm, S. W.; Jeffrey, W. H.; Suttle, C. A.; Mitchell, D. L. Estimation of biologically damaging UV levels in marine surface waters with DNA and viral dosimeters. Photochem. Photobiol. 2002, 76 (3), 268–273. (12) Heaselgrave, W.; Patel, N.; Kilvington, S.; Kehoe, S. C.; McGuigan, K. G. Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water-a laboratory study using simulated sunlight. Lett. Appl. Microbiol. 2006, 43 (2), 125–130. (13) Sommer, B.; Marino, A.; Solarte, Y.; Salas, M. SODIS-an emerging water treatment process. J. Water S.R.T., Aqua 1997, 46 (3), 127–137. (14) Reed, R. H. The inactivation of microbes by sunlight: solar disinfection as a water treatment process. Adv. Appl. Microbiol. 2004, 54, 333–365. (15) Fisher, M. B.; Keenan, C. R.; Nelson, K. L.; Voelker, B. M. Speeding up solar disinfection (SODIS): effects of hydrogen peroxide, temperature, pH, and copper plus ascorbate on the photoinactivation of E. coli. J. Water Health 2008, 6 (1), 35–51. (16) Curtis, T.; Mara, D.; Silva, S. Influence of pH, Oxygen, and Humic Substances on Ability of Sunlight To Damage Fecal Coliforms in Waste Stabilization Pond Water. Appl. Environ. Microbiol. 1992, 58 (4), 1335–1343. (17) Davies-Colley, R.; Donnison, A.; Speed, D. Sunlight wavelengths inactivating faecal indicator microorganisms in waste stabilisation ponds. Water Sci. Technol. 1997, 35 (11-12), 219– 225. (18) Boehm, A.; Grant, S.; Kim, J.; Mowbray, S. Decadal and shorter period variability of surf zone water quality at Huntington Beach, California. Environ. Sci. Technol. 2002, 36 (18), 3885– 3892. (19) WHO, Guidelines for drinking-water quality: microbiological agents in drinking water, 2 ed.; World Health Organization: Geneva, 2002. (20) Fujioka, R. S.; Yoneyama, B. S. Sunlight inactivation of human enteric viruses and fecal bacteria. Water. Sci. Technol. 2002, 46 (11-12), 291–295. (21) Lytle, C. D.; Sagripanti, J.-L. Predicted inactivation of viruses of relevance to biodefense by solar radiation. J. Virol. 2005, 79 (22), 14244–14252. (22) Campos, C.; Guerrero, A.; Cardenas, M. Removal of bacterial and viral faecal indicator organisms in a waste stabilization pond system in Choconta, Cundinamarca (Colombia). Water Sci. Technol. 2002, 45 (1), 61–66.

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(23) Davies, C. M.; Roser, D. J.; Feitz, A. J.; Ashbolt, N. J. Solar radiation disinfection of drinking water at temperate latitudes: Inactivation rates for an optimized reactor configuration. Water Res. 2009, 43 (3), 643–652. (24) Sinton, L.; Hall, C.; Lynch, P.; Davies-Colley, R. Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters. Appl. Environ. Microbiol. 2002, 68 (3), 1122–1131. (25) EPA, Method 1601: male-specific (F+) and somatic coliphage in water by two-step enrichment procedure; 821-R-01-030; EPA: Washington, DC, 2001; p 1-25. (26) Adams, M. H. Bacteriophages; Interscience Publishers: New York, 1959. (27) Steward, G.; Montiel, J.; Azam, F. Genome size distributions indicate variability and similarities among marine viral assemblages from diverse environments. Limnol. Oceanogr. 2000, 45 (8), 1697. (28) Day, R. S. Studies on repair of adenovirus 2 by human fibroblasts using normal, xeroderma pigmentosum, and xeroderma pigmentosum heterozygous strains. Cancer Res. 1974, 34 (8), 1965– 70. (29) Hijnen, W. A. M.; Beerendonk, E. F.; Medema, G. J. Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: a review. Water Res. 2006, 40 (1), 3–22. (30) Simonet, J.; Gantzer, C. Inactivation of Poliovirus 1 and F-Specific RNA Phages and Degradation of Their Genomes by UV Irradiation at 254 Nanometers. Appl. Environ. Microbiol. 2006, 72 (12), 7671–7677. (31) Yoshida, H.; Regan, J. D. Solar UVB dosimetry by amplification of short and long segments in phage lambda DNA. Photochem. Photobiol. 1997, 66 (5), 672–675. (32) Bay, P.; Reichmann, M. UV inactivation of the biological activity of defective interfering particles generated by vesicular stomatitis virus. J. Virol. 1979, 32 (3), 876–884. (33) Rauth, A. The physical state of viral nucleic acid and the sensitivity of viruses to ultraviolet light. Biophys. J. 1965, 5 (3), 257–273. (34) Eischeid, A. C.; Meyer, J. N.; Linden, K. G. UV disinfection of adenoviruses: molecular indications of DNA damage efficiency. Appl. Environ. Microbiol. 2009, 75 (1), 23–28. (35) Tedetti, M.; Sempe´re´, R. Penetration of ultraviolet radiation in the marine environment. A review. Photochem. Photobiol. 2006, 389–397. (36) Rule Wigginton, K.; Menin, L.; Montoya, J.; Kohn, T. Oxidation of virus proteins during UV(254) and singlet oxygen mediated inactivation. Environ. Sci. Technol. 2010, 44 (14), 5437–5443. (37) Regan, J. D.; Yoshida, H. DNA UVB dosimeters. J. Photochem. Photobiol., B: Biol. 1995, 31 (1-2), 57–61. (38) Wilhelm, S.; Weinbauer, M.; Suttle, C.; Jeffrey, W. The role of sunlight in the removal and repair of viruses in the sea. Limnol. Oceanogr. 1998, 586–592. (39) Smith, R.; Baker, K. Penetration of UV-B and biologically effective dose-rates in natural waters. Photochem. Photobiol. 1979, 29 (2), 311–323. (40) Kohn, T.; Grandbois, M.; McNeill, K.; Nelson, K. L. Association with natural organic matter enhances the sunlight-mediated inactivation of MS2 coliphage by singlet oxygen. Environ. Sci. Technol. 2007, 41 (13), 4626–4632. (41) Fauquet, C. Virus taxonomy: classification and nomenclature of viruses: eighth report of the international committee on taxonomy of viruses. Elsevier Academic Press: Hong Kong, 2005; 1-1259.

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