Environ. Sci. Technol. 2008, 42, 4589–4594
Removal of Bacteriophages MS2 and ΦX174 during Transport in a Sandy Anoxic Aquifer PAUL W. J. J. VAN DER WIELEN,* WIEL J. M. K. SENDEN, AND GERTJAN MEDEMA Kiwa Water Research, P.O. Box 1072, 3433BB Nieuwegein, The Netherlands
Received January 16, 2008. Revised manuscript received March 27, 2008. Accepted April 1, 2008.
The objectives of our study were to determine (i) removal of bacteriophage MS2 and ΦX174, as surrogates for human pathogenic viruses, in an anoxic aquifer and (ii) the safe length of the microbial protection zone in anoxic aquifers. 3.5 Log units of MS2 were removed by adsorption and inactivation during 63 days residence time, which was 1.4 log units lower than removal of ΦX174. These removal rates were considerably lower than previously reported for MS2 and ΦX174 in oxic aquifers and consequently longer protection zones around anoxic aquifers might be needed. Therefore, the observed log removal of MS2 was used in a risk assessment approach to determine the required safe length of the microbial protection zone. In case of a leaking sewer in the vicinity of a well in an anoxic aquifer, the risk assessment demonstrated that a microbial protection zone of 110 m may be needed to meet a risk of infection of 10-4 persons per year. This length can be two to three times larger than the length of the protection zone currently used in a number of countries.
Introduction Groundwater abstraction wells are protected against microbial contamination by microbial protection zones around the abstraction well. The length of the microbial protection zone differs among countries, but mostly a water residence time of at least 50-60 days between the edge of the zone and abstraction well is used (1, 2). The 50-60 days water residence time is based on research in the 1930s by Knorr, who showed that removal of Escherichia coli during 50- 60 days transport in an aquifer was sufficient to protect abstraction wells against breakthrough (3). However, even at that time Knorr remarked that it was uncertain whether 60 days residence time protected abstraction wells against virus contamination. Since the 1930s, knowledge about microbial transport through soil has increased considerably and many studies have shown that bacteria (including E. coli) were removed at higher rates than viruses during transport through saturated soil (reviewed in refs 4, 5). Consequently, field studies were conducted to study removal of bacteriophages, as surrogates for human pathogenic viruses, in sandy aquifers (6–12). These studies all demonstrated that high removal rates (up to 8 log removal) were achieved within 30 days residence time in the aquifer. As a result, a microbial protection zone based on 50-60 days residence time would * Corresponding author phone: +31 (0)306069642; fax: +31(030)6061165; e-mail:
[email protected]. 10.1021/es800156c CCC: $40.75
Published on Web 05/14/2008
2008 American Chemical Society
be sufficient to protect the abstraction well against virus breakthrough from a contamination source. Recently, the length of the protection zone around a number of groundwater abstraction wells in The Netherlands was calculated using a modeling approach (13, 14). The conclusion from these studies was that a microbial protection zone based on 60 days residence time, the guideline for the microbial protection zone around groundwater abstraction wells in The Netherlands, protected abstraction wells in oxic aquifers sufficiently. In contrast, the microbial protection zone around anoxic aquifers should be based on 2-2.5 years residence time to stay with 95% certainty under an infection risk of 1 out of 10,000 persons per year; the tolerable infection risk for drinking water according to the current Dutch legislation (15). A sensitivity analysis showed that the virus transport model was most sensitive for inactivation rate and collision efficiency of viruses (13, 14). The value of both parameters under anoxic conditions is unknown and was assumed, because no field studies have investigated virus transport entirely under anoxic conditions. Hence, the model results of both studies are more indicative rather than definite. The objective of the current study was to determine removal of bacteriophage MS2 and ΦX174 in an anoxic sandy aquifer. To achieve this objective, anoxic groundwater containing high titers of MS2 and ΦX174, as surrogates for human pathogenic viruses, was injected under anoxic conditions in an anoxic aquifer with a relatively high pH, and removal of both bacteriophages during transport was determined. The results obtained in our field study were also used to determine the required length of the microbial protection zone around groundwater abstraction wells under a worst case scenario using a risk assessment approach.
Materials and Methods Field Location. The removal of bacteriophages in an anoxic aquifer was studied at a field location in The Netherlands. The aquifer is positioned at 9 m below ground level and consists of moderately coarse sand. A 9 m confining layer of clay and loam overlies the aquifer. An abstraction well was placed in the aquifer at a depth of 10 to 15 m below ground level (Figure S1), and groundwater was abstracted with 11.32 m3 h-1. One injection well and two monitoring wells, with the screen depth at 11 to 13 m below surface level, were placed at a horizontal distance of 20.5, 29.9, and 37.7 m from the abstraction well, in line with the groundwater flow direction. The groundwater is anoxic (O2 < 0.5 mg L-1; NO3 < 0.5 mg L-1; Fe g 0.1 mg L-1, and Mn g 0.1 mg L-1; (16)), has a relatively high pH of 7.5, and a temperature of 12.9 °C. The majority (97%) of the aquifer consists of sand; the remaining 3% is silt (2.4%) and clay (0.6%). The porosity of the aquifer is 0.32 and the geometric mean of the grain size is 405 µm. The percentage organic matter in the aquifer is low (∼ 0.15%) and the cation exchange capacity varies between 5.6 and 19.3 meq kg-1. Seeding Experiments. Before sodium bromide was injected as a conservative tracer, the abstraction well was in operation for two months and had abstracted a volume of 15,000 m3 water. Bromide tracer injectate was prepared by dissolving 500 g NaBr in 1 L of demineralized water. The tracer was diluted to 809 mg L-1 bromide with anoxic groundwater in a seeding tank under a continuous flow of nitrogen gas to keep the solution anoxic. The bromide tracer was injected in the injection well at 12 m depth with a flow of 18.2 L h-1 for 96 h, resulting in a total injected volume of 1747 L of tracer solution. During injection of the bromide tracer, the seeding tank was kept under anoxic conditions as VOL. 42, NO. 12, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Breakthrough Characteristics of Bromide and Bacteriophage MS2 and ΦX174 at the Two Monitoring Wells and Abstraction Well well
C0 or Cmaxa (mg L-1; pfu mL-1)
ta (days)
xa (m)
dilution
vb (m day-1)
rLb (m)
0 3.2 6.2 5287
0.33 0.38 0.56
0.19 0.26 0.23
Log C/C0
bromide injection well monitoring well 1 monitoring well 2 abstraction well
809 252 130 0.153
0 25 47 72
0 7.8 17.2 37.7
MS2 injection well monitoring well 1 monitoring well 2 abstraction well
6.9 × 109 1.4 × 108 5.9 × 106 4.5 × 102
0 18 36 63
0 7.8 17.2 37.7
-1.7 -3.1 -7.2
ΦX174 injection well monitoring well 1 monitoring well 2 abstraction well
2.5 × 106 4.6 × 103 2.8 × 102 6.9 × 10-3
0 19 37 62
0 7.8 17.2 37.7
-2.7 -4.0 -8.6
a C0 is the concentration injected at the injection well; Cmax is the maximum breakthrough concentration at the monitoring and abstraction wells; t is the residence time from the injection well; x is the distance to the injection well. b The average interstitial velocity (v) and the longitudinal dispersivity (RL) were determined using the curve-fitting program CXTFIT (19) and were determined for the transport from injection well to each monitoring well or abstraction well.
well. The two monitoring wells and abstraction well were sampled for the next 93 days. High titer solutions of bacteriophage MS2 and ΦX174 were obtained from GAP Enviromicrobial Services (London, Canada). One liter of MS2 and 1 L of ΦX174 solutions were diluted together in a seeding tank with anoxic groundwater under anoxic conditions. After dilution, MS2 concentration in the seeding tank was 6.9 × 109 pfu mL-1, whereas ΦX174 concentration was 2.5 × 106 pfu mL-1. The bacteriophage solution was injected under anoxic conditions for 96 h at 12 m depth in the injection well with a flow of 19.8 L h-1, resulting in a total volume of 1901 L of bacteriophage solution. The injection well, two monitoring wells, and abstraction well were sampled for the next 163 days. Analytical Methods. Samples were taken from injection and monitoring wells using a mobile pump. To sample groundwater, two times the volume of stagnant water in the well was pumped out before a sample was taken for bromide or bacteriophage analyses. Samples from the abstraction well were taken at a tap located on the abstraction well. Because low concentrations of bacteriophages were expected at the abstraction well, volumes up to 10 L were analyzed for MS2 and ΦX174. Bromide was analyzed using ion chromatography. Bacteriophages were determined by enumerating plaque forming units (pfu) using the agar overlay technique. Bacteriophage MS2 was determined as described in ISO 10705-1 using Salmonella typhimurium WG49 as host strain, and bacteriophage ΦX174 was determined according to ISO 10705-2 using E. coli WG5 as host strain. Inactivation Rate of Free Bacteriophages in Groundwater. The inactivation rate of free bacteriophages in groundwater was determined by sampling (i) water from the seeding tank, (ii) groundwater from the first monitoring well during maximum breakthrough of the bacteriophages (18 days after bacteriophages were injected in the injection well), and (iii) groundwater from the second monitoring well 43 days after maximum breakthrough of both bacteriophages (80 days after bacteriophages were injected in the injection well). The three water samples were stored in bottles under strict anoxic conditions at 12 °C, conditions identical to the redox and temperature conditions of the groundwater. For 100 days, the bottles with anoxic groundwater were sampled weekly and numbers of bacteriophage MS2 and ΦX174 were 4590
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determined using the methods as described above. Inactivation followed first order kinetics and the inactivation rate was determined using log linear correlation. Calculation of Collision Efficiencies. The collision efficiency represents the fraction of collisions (contacts) between suspended bacteriophages and collector soil grains that result in attachment. This collision efficiency is described by the following equation (17): R)
2 dc katt 1 3 (1 - ε) v η
(1)
where R is the collision efficiency, dc is the average diameter of the collector (grains), is the porosity, katt is the attachment rate coefficient, v is the average interstitial velocity, and η is the single collector efficiency. The attachment rate coefficient was calculated using the equation described by Schijven et al. (10) (see Supporting Information) and the single collector efficiency η was calculated using the extended multiregression equation of Tufjenki and Elimelech (18) (see Supporting Information).
Results Tracer Bromide. To determine the water residence time between injection well, two monitoring wells, and abstraction well, bromide was injected as a conservative tracer. In addition to the water residence time, breakthrough characteristics of the conservative tracer bromide can be used to calculate the interstitial flow velocity, dispersivity of the porous medium, and dilution. The characteristics of bromide breakthrough are presented in Table 1; the breakthrough curves are shown in Figure S2. Maximum breakthrough at the first and second monitoring well and the abstraction well was noticed 25, 47, and 72 days after bromide was injected at the injection well, respectively (Table 1). Maximum breakthrough concentration was 252 mg L-1 bromide (31.3% of injected concentration) at the first monitoring well, 130 mg L-1 (16.1% of injected concentration) at the second monitoring well, and 0.153 mg L-1 (1.89 × 10-4% of injected concentration) at the abstraction well (Table 1). Because the abstraction well abstracted groundwater from radial surroundings, the injected bromide plume was diluted with surrounding groundwater at the abstraction well explaining the low bromide concentration at the abstraction well.
FIGURE 1. Removal of bacteriophage MS2 and ΦX174 versus residence time. Symbols: b, MS2 removal; 9, ΦX174 removal; ( MS2 removal without dilution; 2, ΦX174 removal without dilution; - - - is the linear regression line for the removal of bacteriophage MS2. The interstitial velocity and longitudinal dispersivity of the water were estimated from the breakthrough curves of bromide using the curve-fitting program CXTFIT (19). The interstitial velocity from injection well to the first or second monitoring well was similar, but was higher from injection well to abstraction well (Table 1). Apparently, the velocity in the second section (monitoring well 2 to abstraction well) was slightly higher than in the first section (injection well to monitoring well 2). The longitudinal dispersivity values were low and similar for monitoring and abstraction wells (Table 1), indicating that the sandy layer of the aquifer was relatively homogeneous. Bacteriophage MS2 and ΦX174. Although it was concluded from the bromide tracer experiment that the water residence time between injection and abstraction well was slightly higher than 50-60 days, the bacteriophage data are sufficient to determine the removal of viruses during 50-60 days residence time (which is used as length for the microbial protection zone in many countries) in an anoxic aquifer. Characteristics of the breakthrough of bacteriophage MS2 and ΦX174 are presented in Table 1 and Figure S3. The residence time of the bacteriophage MS2 and ΦX174 from injection well to monitoring and abstraction wells was similar, but lower than the residence time of the conservative tracer bromide (Table 1). This difference in residence time demonstrates that MS2 and ΦX174 traveled faster through the saturated soil than bromide, which has been observed as well in previous field studies (reviewed in ref (20)). This phenomenon was named pore size exclusion and refers to the assumption that MS2 and ΦX174 did not travel through the smallest pore sizes in an aquifer, whereas bromide did. Consequently, a part of the injected bromide in our study traveled through the smallest pore sizes with the lowest velocity, whereas bacteriophages traveled through bigger pore sizes where higher velocities occurred. The conservative tracer was only reduced by dispersion and dilution, whereas both bacteriophages were reduced by dispersion, dilution, inactivation, and adsorption. Therefore, C/C0 values of bacteriophages were lower than those for bromide. C/C0 values of ΦX174 were lower than C/C0 values of MS2, indicating higher removal of ΦX174 compared to MS2 (Table 1). The log removal of both bacteriophages was also plotted against residence time (Figure 1) and distance (Figure S4).The total reduction of bacteriophages was low from injection well to the second monitoring well, and high from the second monitoring well to abstraction well (Figure 1). The high reduction rate at the abstraction well (7.2 log units for MS2 and 8.6 log units for ΦX174) was observed with bromide as well (Table 1), and was mainly caused by dilution of the injected plume at the abstraction well with surrounding
abstracted groundwater. The removal of MS2 or ΦX174 by inactivation and adsorption was estimated by correcting the reduction values for dilution, using the dilution+dispersion factor of bromide. Removal of bacteriophage ΦX174 by adsorption and inactivation was 4.9 log units, whereas 3.5 log units of bacteriophage MS2 were removed (Figure 1). Furthermore, removal of ΦX174 was higher in the first part (injection well to monitoring well 1) and lower in the second part (monitoring well 1 to abstraction well), whereas log removal of MS2 was linear (R2 ) 0.99) over the whole transect (Figure 1). The amount of removed bacteriophages was split in removal by inactivation and removal by adsorption. Inactivation rates from the survival experiments were used to calculate removal by inactivation; subsequently total, dilution, and inactivation reduction values were used to calculate removal by adsorption. Results from these calculations showed that 2.3 log units of MS2 were removed by inactivation and 1.2 log units were removed by adsorption. For ΦX174, 0.8 log units were removed by inactivation and 4.1 log units were removed by adsorption. These calculations showed that adsorption behavior of MS2 was more conservative, but for ΦX174 inactivation was more conservative. Overall, removal of MS2 was lower and thus more conservative than removal of ΦX174 during transport in an anoxic aquifer with a relatively high pH. Inactivation of Bacteriophage MS2 and ΦX174. The inactivation of the bacteriophages followed first-order kinetics in all samples and inactivation rates were calculated by log linear regression (Figure S5). The inactivation rate of ΦX174 was lower than the inactivation rate of MS2 (Table 2), indicating that ΦX174 is a more conservative virus regarding inactivation than MS2. The inactivation of MS2 and ΦX174 did not differ much among the three sample locations (Table 2). We conclude that inactivation kinetics were similar along the transect where bacteriophages moved through the aquifer and that there were no subpopulations of phages in relation to inactivation. The inactivation of attached bacteriophages was determined as described by Schijven et al. (10), which is by calculating the tail slope of the breakthrough curves of MS2 and ΦX174 at logarithmic scale. Inactivation rates of attached MS2 or ΦX174 phages were comparable with inactivation rates of free MS2 or ΦX174 phages (Table 2), demonstrating that inactivation behavior was comparable for attached and free bacteriophages. Only the inactivation rate of attached MS2 determined from the tail of the breakthrough curve at the second monitoring well and of attached ΦX174 determined from the curve at the injection well showed higher values (Table 2). However, these high values were less reliable, since regression coefficients of the tail slope were lower (R2 is 0.92 or 0.84) compared to regression coefficients of the other tail slopes (R2 > 0.95). Collision Efficiencies. The collision efficiency holds information about the adsorption capacity of phages to soil particles. The collision efficiency of MS2 or ΦX174 was similar at each monitoring or abstraction well (Table 2). Thus, adsorption of MS2 and ΦX174 was constant over the whole transect from injection to abstraction well, which indicates that available attachment sites were homogenously distributed throughout the aquifer. The collision efficiency of bacteriophage MS2 at each well was 3.5-5 times lower than the collision efficiency of ΦX174 (Table 2). Consequently, we conclude that adsorption behavior of phage MS2 was more conservative than that of ΦX174. This can be explained by the lower iso-electric point of MS2 (21) resulting in a more negatively charged phage and a higher repulsion by the negatively charged soil particles than the less negatively charged ΦX174 phages (21). VOL. 42, NO. 12, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Inactivation Rates and Collision Efficiency of Bacteriophage MS 2 and ΦX174 bacteriophage MS2 inactivation (log a
bacteriophage ΦX174
day-1)
location
free
attached
seeding tank monitoring well 1 monitoring well 2 abstraction well average
0.031 0.040 0.037
0.038 0.036 0.055
0.036
0.043
inactivation (log day-1) collision efficiency 3.4 × 10-5 2.9 × 10-5 2.8 × 10-5 3.0 × 10-5
free
attached
0.008 0.013 0.018
0.020 0.017 0.019
0.013
0.019
collision efficiency 1.7 × 10-4 1.1 × 10-4 1.0 × 10-4 1.3 × 10-4
a
For the inactivation of free bacteriophages samples were taken from the seeding tank, monitoring well 1 during breakthrough, and monitoring well 2 43 days after maximum breakthrough.
FIGURE 2. Removal of MS2 (A) and ΦX174 (B) observed at different field locations. Symbols: 9, this study; [ ref (8); b, ref (10); 2, ref (11); 4, ref (7); 0, ref (6); O, ref (12).
Discussion Adsorption and Inactivation. Removal of bacteriophages in the saturated zone of sandy aquifers has been studied at different field locations (6–8, 10–12). At these other locations, high removal rates of both bacteriophage MS2 and ΦX174 were observed during relatively short residence times in the aquifer. The low removal rates of MS2 and ΦX174 observed in our study were clearly in contrast with results from these other studies (Figure 2). It is well-known that grain size and groundwater pH can impact removal of phages (reviewed in refs 21, 22). Grain sizes of sand and groundwater pH in the studied aquifers were in most cases similar to grain sizes and pH at the field location used in our study. Hence, grain size and pH were not responsible for the observed difference between our study and other field studies. Five of the six studies that investigated removal of bacteriophages MS2 and/ or ΦX174 were done in aquifers that contained oxic groundwater (6–8, 10, 12) and one study investigated removal of MS2 in an anoxic aquifer where oxic surface water was infiltrated, resulting in oxic conditions around the infiltration site (11). Consequently, it seems obvious that the anoxic redox 4592
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condition in the aquifer used in our study was the apt cause for the observed lower removal rates. The conclusion that anoxic conditions in the aquifer were responsible for the low removal rate of MS2 and ΦX174 was supported by laboratory studies. It has been observed that inactivation of viruses in groundwater was lower under anoxic conditions (23), and column studies demonstrated that the presence of oxidized metal ions like ferric oxihydroxides, which are present under oxic conditions but are likely to be absent under anoxic conditions, resulted in higher virus adsorption (24–26). In conclusion, the anoxic aquifer with a relatively high pH and low organic content we used in our transport study has unfavorable conditions for virus removal and can be seen as a worst case sandy aquifer. The seeding concentration of bacteriophage MS2 was 30,000 times higher than that of bacteriophage ΦX174. In a previous study, it was demonstrated that the removal rate of MS2 in slow sand filters was not affected when the seeding concentration of MS2 increased 1,000 times (27). Consequently, the difference in seeding concentration of MS2 and ΦX174 in our study is unlikely to be the cause for the observed difference in the removal rate of MS2 and ΦX174. As stated earlier, the lower iso-electric point of MS2 compared to ΦX174 (21) results in a more negatively charged MS2 than ΦX174 and consequently in a higher repulsion by the negatively charged soil particles. In conclusion, the difference in surface charge is the apt cause for the different removal rates of MS2 and ΦX174 observed in our study. Inactivation rates obtained for MS2 and ΦX174 at 5-13 °C in other studies were generally higher than inactivation rates obtained in our study (7, 10, 11, 28–31). The lower inactivation rates observed in our study confirm a previous laboratory study that demonstrated that anoxic conditions resulted in lower inactivation rates (23). Collision efficiencies of MS2 and ΦX174 have been calculated in other field studies as well, but these studies were mainly performed in oxic aquifers (6, 9–11). The collision efficiencies obtained in those studies were 10-100 times higher than the collision efficiencies observed in our study, indicating that adsorption of bacteriophages under oxic conditions was higher than that under anoxic conditions. A similar low collision efficiency was reported for MS2 during transport in the anoxic part of an aquifer that changed from anoxic to oxic during infiltration of oxic surface water (11, 13), supporting our observation that transport in an anoxic aquifer results in low collision efficiencies. In conclusion, the removal rate of bacteriophages MS2 and ΦX174 in an anoxic aquifer was low compared to removal rates of both phages in oxic aquifers. The low removal rate in anoxic aquifers was caused by a lower inactivation rate and a lower adsorption rate of both phages at anoxic conditions. Surrogates for Human Pathogenic Viruses. We have used bacteriophages MS2 and ΦX174 as surrogates for human pathogenic viruses. Compared to human pathogenic viruses
the adsorption kinetics of MS2 were more conservative and can be considered worst case when soil organic content was low (6, 12, 21, 32), as was the case at our field site (organic content ∼0.15%). The inactivation rate of bacteriophage ΦX174 was lower than that of MS2. Hence, the inactivation rate of MS2 is not worst case compared to other bacteriophages. Therefore, the inactivation rate of MS2 obtained in our study was compared with inactivation rates published for human pathogenic viruses (33). Inactivation rates of human pathogenic viruses varied between 0.0043 and 0.52 log day-1. The low inactivation rates reported for human pathogenic viruses are lower than the inactivation rate for MS2 observed in our study. However, low inactivation rates for human pathogenic viruses were obtained in studies where groundwater was not incubated under the environmental conditions of the aquifer (34), where inactivation did not follow first-order kinetics (35), or where incubation of viruses without host cells showed an increase in virus numbers (36, 37). The low inactivation rates for a number of pathogenic viruses were reported only once, mostly in a study that has not been published in a peer-reviewed journal (35). It can not be deduced from that study whether the groundwater incubations were under similar conditions as observed in the aquifer (35). Moreover, a higher inactivation was observed during the first 60 days, followed by a lower inactivation over the next 200 days (35). Consequently, the published low inactivation rates for human pathogenic viruses seems unreliable compared to the higher published inactivation rates, which were equal to or higher than the inactivation rate obtained for MS2 in our study. As a result, we conclude that the inactivation rate observed for MS2 in our study could be considered worst case when related to human pathogenic viruses, as was concluded before (21). Microbial Protection Zone. In previous field studies, 8 log removal of bacteriophages was achieved within 30 days residence time (6–8, 10–12). As a result, a microbial protection zone based on 50-60 days residence time seemed more than sufficient to protect the abstraction well against virus breakthrough from a contamination source. However, we observed only 3.5 log removal of MS2 during 63 days residence time and it was unclear if such low removal rates would protect the abstraction well against virus breakthrough. Therefore, the required length of the protection zone in case of a worst case scenario and Dutch legislation was calculated using data from our field study. The highest risk of contamination of groundwater with human pathogenic viruses is related to a leaking sewer in the vicinity of the abstraction well. In the Dutch water decree, it is stated that drinking water should not exceed an infection risk of 1 out of 10,000 persons per year (15). Because groundwater has a high hygienic quality, there are no treatment steps to remove pathogenic microorganisms from groundwater, nor is drinking water chemically disinfected in The Netherlands. Consequently, abstracted groundwater should have a virus concentration below 1.2 × 10-6 N L-1 to remain below the infection risk of 10-4 persons per year (38). This means that the virus concentration should be reduced below 1.2 × 10-6 N L-1 during transport in the aquifer from a leaking sewer to abstraction well. The highest Entero- and Reovirus concentration in raw sewage from a sewage plant in Apeldoorn, The Netherlands was 833 and 2143 N L-1, respectively (39). Thus, during transport from leaking sewer to abstraction well in an anoxic aquifer, 8.8 log removal of Enterovirus and 9.3 log removal of Reovirus should be achieved. Viruses that move from leaking sewer to abstraction well are diminished by inactivation, adsorption, and dilution. Schijven et al. (13) assumed in their modeling studies that a leaking rate of 1 m3 day-1 would be a realistic value for sewage leaks in The Netherlands. An abstraction rate of 1000
m3 day-1 is obtained by a small groundwater well that is used for drinking water production in The Netherlands. A leaking rate of 1 m3 day-1 and an abstraction rate of 1000 m3 day-1 result in 3 log reduction caused by dilution. Consequently, 5.8 log units of Enterovirus and 6.3 log units of Reovirus have to be removed by inactivation and adsorption. The removal rate of Reovirus and Enterovirus during transport in an anoxic aquifer is unknown. Since the removal of bacteriophage MS can be considered as a surrogate for conservative virus transport (reviewed in (21) and observations from this study), we used MS2 removal obtained in our study to calculate removal of Reovirus and Enterovirus during transport in an anoxic aquifer. We demonstrated in our study that removal (inactivation and adsorption) of MS2 in an anoxic aquifer with a relatively high pH was log linear with residence time and can be described by: log (C/C0) ) 0.0576 × t, where log (C/C0) is log removal and t is residence time in the aquifer. Using this equation, a 5.8 log removal of Enterovirus will be obtained with a residence time of 101 days, and a 6.3 log removal of Reovirus with a residence time of 109 days. Thus, the currently used guideline in The Netherlands for the length of the microbial protection zone (based on 60 days residence time) may not protect the abstraction well sufficiently under a worst case scenario of a sewer leaking directly in a shallow anoxic aquifer with a relatively high pH. However, the length of the protection zone should not have a residence time of 1-2 years as was proposed in studies that used a virus transport model to calculate the length of the microbial protection zones around Dutch anoxic aquifers (13, 14). The reason for the discrepancy between our field study and the modeling study is that values of the most sensitive model parameters, inactivation and collision efficiency, were unrealistically low in the modeling studies (13, 14). Overall, our study demonstrated that removal of viruses during transport in an anoxic aquifer was considerably lower than that in oxic aquifers. The low removal of viruses in an anoxic aquifer was caused by a lower inactivation and adsorption rate under anoxic conditions. Moreover, we conclude from our study that, under a worst case scenario of a leaking sewer, the currently used microbial protection zones in some countries, which are based on 50-60 days residence time (1, 2), may not be sufficient to produce drinking water that meets an infection risk of 10-4 persons per year. Consequently, the currently used length of microbial protection zones around shallow abstraction wells in anoxic sandy aquifers should be reconsidered and the total travel time between contamination sources and abstraction well should preferably be extended to 110 days.
Acknowledgments This study was financed by the Dutch water supply companies as part of the joint research program (BTO). We thank Kees Maas and Gijsbert Cirkel for help on the geohydrology and Anke Brouwer and Anita Lugtenberg for technical assistance.
Supporting Information Available Detailed description of the collision efficiency calculations, a schematic representation of the aquifer, breakthrough curves of bromide, MS2, and ΦX174 at the monitoring and abstraction wells, inactivation curves of free MS2 and ΦX174, and removal of MS2 and ΦX174 as function of distance. This material is available free of charge via the Internet at http:// pubs.acs.org.
Literature Cited (1) Chave, P.; Howard, G.; Schijven, J. F.; Appleyard, S.; Fladerer, F.; Schimon, W. Groundwater protection zones. In Protecting Groundwater for Health; Schmoll, O., Howard, G., Chilton, J., Chorus, J., Eds.; World Health Organization: Cornwall, UK, 2006; pp 465-492. VOL. 42, NO. 12, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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