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Survival of viruses in groundwater
War. Res. Vol. 23, No. 3, pp. 301-306, 1989 Printed in Great Britain. All rights reserved
0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Press pie
SURVIVAL OF VIRUSES IN GROUNDWATER JANIS JANSONS,1'* LINDSAY W. EDMONDS,2 BRENT SPEIGHT2 a n d MARION R. BUCENS3 ~Arthur Webster Pty Ltd, P.O. Box 234, Baulkham Hills, New South Wales 2153, 2Water Authority of Western Australia, 629 Newcastle Street, Leederville, Western Australia 6007 and 3Virus Laboratory, Combined Microbiology Service, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia
(First received January 1988; accepted in revised form October 1988) Abstract--Survival in groundwater of echovirus types 6, 11 and 24, coxsackievirus type B5 and poliovirus type 1 was determined. Enterovirus survival in groundwater was found to be variable and appeared to be influenced by a number of factors: temperature, dissolved oxygen concentration and possibly the presence of microorganisms. Dissolved oxygen concentration was the most significant factor in loss of virus infectivity in groundwater. Poliovirus type 1 incubated in groundwater with a mean dissolved oxygen concentration of 0.2 mg/1 decreased in infectivity by 100-fold in 50 days compared with 20 days for a decrease of the same magnitude when incubated in groundwater with a mean dissolved oxygen concentration of 5.4 mg/1. Echovirus type 6 was found to be least stable, and poliovirus type 1 was found to be most stable, although virus stability may have been due to conditions existing in individual groundwater bores.
Key words--groundwater, artificial recharge, temperature, wastewater, dissolved oxygen, enteric viruses
INTRODUCTION In a previous c o m m u n i c a t i o n (Jansons et al., 1989), it was concluded t h a t Bassendean Sand was u n a b l e to efficiently a d s o r b all enteric viruses d u r i n g artificial recharge a n d t h a t indigenous viruses were able to p e n e t r a t e into the aquifer. T o determine the possible extent o f viral c o n t a m i n a t i o n o f the aquifer d u r i n g artificial recharge with effluent, experiments o n the longevity o f virus survival in the aquifer were carried out. Viruses were placed in dialysis bags a n d lowered into the aquifer t h r o u g h boreholes. Virus samples were retrieved at various time intervals a n d the loss o f virus infectivity was determined over a 56 day period. MATERIALS AND METHODS
Description of study site The virus survival study was carried out at the Canning Vale Groundwater Recharge site, as previously described (Jansons et al., 1989). Preparation of virus suspensions for survival studies Virus suspensions for survival studies were prepared by mixing 35 ml of laboratory grown virus with 500 ml of groundwater from respective bores. This mixture was placed in 50 cm of dialysis tubing and sealed at both ends. The virus-containing dialysis tubing was then lowered into the aquifer through boreholes in the configuration described in Table 1. Six dialysis bags were used for each virus type. The bags were retrieved at varying time intervals up to 56 days and stored at - 7 0 ° C until assayed. Echovirus types 6, 11 and 24, and coxsackievirus type B5 were all previously isolated from effluent at the Canning Vale Groundwater Recharge Site (Jansons et al., 1989). Echoviruses, 6, 11 and 24 were grown and titrated in RD cells and coxsackievirus type B5 was grown and titrated in Vvro cells. Poliovirus *Author to w h o m all correspondence should be addressed. 301
1 w a s a Sabin oral vaccine strain (Smith Kline-RIT, Rixensart, Belgium) which had been passaged and titrated in Vero cell cultures. All viruses were stored at - 7 0 ° C until titrated. An experiment was also set up in the laboratory to determine the inactivation of poliovirus at 4 and 22°C in sterile phosphate buffered saline (PBS). The same batch of poliovirus and experimental methods were used in this study as for the groundwater study.
Groundwater quality Groundwater characteristics which were thought to be significant for virus survival, namely temperature and dissolved oxygen, were investigated for each bore (Table I). The dissolved oxygen concentrations of groundwater and effluent were determined using a dissolved oxygen and temperature meter (Delta Scientific, Envirotech Corp., New York, U.S.A.). The variations in groundwater temperature were measured using a maximum/minimum thermometer. The groundwater bores were slotted to sample the upper and lower levels of the aquifer (Fig. 1). A total of seven bores was used in this study. Estimation of bacterial numbers Total coliforms were incubated at 30°C for 4 h followed by 14h at 35°C. Faecal coliforms were estimated as described by Jansons et al. (1989). Faecal streptococci were incubated at 35°C for 4 h followed by 40 h at 45°C. The total bacterial count at 22°C was estimated by incubation of samples at 22°C for 18 h. The total bacterial count at 35°C was processed the same as total coliforms, and the total bacterial count at 44°C was estimated the same as faecal coliforms. Estimation of virus survival in groundwater The viruses were titrated by 10-fold dilutions in 96 well microplates (NUNC, Roskilde, Denmark) containing a confluent monolayer of either RD or Veto cells. After inoculation, the plates were incubated at 35°C in a humidified incubator containing 7.5% CO 2 in air. After 6 days the number of wells showing virus-induced cytopathic effect was recorded. The virus titre was calculated using the Karber method.
302
JANIS JANSONS et al. Table 1. Groundwater quality and location of viruses for survival studies Temperature °C
Dissolved oxygen (rag/I) pH
Bore
Mean
Range
Mean
Range
range
Virus
I 2 3 4 5 6 7 Effluent
19.4 21.9 16.2 16.4 15.7 15.9 21.7 16.2
19-21 21.5-22 15.5-17.5 16-17.5 14.5-17 15-18 21-22 16-16.5
1.2 0.2 2.3 1.6 5.4 0.2 0.06 6.5
0.1-2.6 0.1-0.4 0.2-5.9 0.1-4.7 2.7~.8 0.1-0.3 0.05-0.08 4.8-8.0
6.5~.7 6.4-6.8 6.6-6.8 6.6~.8 6.6-6.8 6.0~.8 5.9~6.2
Coxsackievirus B5 Eehovirus 6 Eehovirus 1 I
Statistical methods Average changes in virus inactivation in groundwater were determined by calculating the linear regression coefficient. The regression equation was determined using an Epistat statistical software package (Gustafson, 1984). The significance of the slope was tested using the t-test. A comparison of the regression lines was performed by the method recommended by Snedecor and Cochran (1980). Correlation coefficients between virus inactivation rates and groundwater quality were determined by the method of Clarke (1969).
out, the results of an integrity test of the dialysis tubing using polyvinyl-pyrrolidone K90 suggested an acceptable stability in groundwater after 33 days of exposure (data not shown). Virus loss by adsorption onto dialysis membrane An experiment performed prior to that on virus survival in groundwater showed that there was no detectable loss of poliovirus titre after either 1 day or after 7 days of incubation in dialysis tubing compared with a control. Ward and Winston (1985) showed that virus adsorption onto surfaces i s instantaneous. Therefore, virus loss by adsorption to dialysis membrane was not considered a significant factor in the virus survival experiments. O'Brien and Newman (1977) have reported virus adsorption to dialysis membranes to be <2%.
Use o f dialysis tubing in virus survival experiments The use of dialysis tubing and diffusion chambers for the study of virus survival has been reported by a number of workers (LaBelle and Gerba, 1980; Herrmann et al., 1974). This method is preferable to laboratory studies since it presents a more accurate picture of inactivating factors in environmental waters. The use of dialysis membranes has been criticised by some workers due to their degradation by microorganisms (Vargo et al., 1975). This resulted in the introduction of survival chambers for the study of bacterial (Vasconcelos and Swartz, 1976) and viral survival (LaBelle and Gerba, 1980). These chambers have the advantage of increased stability in environmental waters as they incorporate a polycarbonate membrane which is more resilient than a dialysis membrane. However, the use of similar survival chambers would have been impractical for the Canning Vale study due to the restriction of space in the bore hole casing. LaBelle and Gerba (1980) have shown the virus inactivation rates to be similar for dialysis tubing and viral survival chambers. During the virus survival study, it was noted that some of the virus-containing dialysis bags were becoming fragile after approx. 30 days of exposure to groundwater and would occasionally rupture while being lifted from the bore. Although virus loss from individual bags cannot be ruled Bore No.
1
2
3
4
5
6
7
Echovims 24 Poliovirus 1 Poliovirus 1 Poliovirus 1
RESULTS AND DISCUSSION
Groundwater quality
The temperature of natural groundwater in the aquifer at the study site ranged from 21 to 23°C and the dissolved oxygen level was < 1.0 mg/l. However, recharge of the aquifer with effluent altered these two parameters. Effluent temperature during the study averaged 16.2°C and the dissolved oxygen level ranged from 4.8 to 8.0 mg/l (Table 1) due to aeration during effluent treatment. Generally, the effluent plume was localised in the upper part of the aquifer and its lateral movement through the aquifer was estimated to be 0.3 m/day. Therefore, groundwater from the shallow slotted bores closest to the recharge basin was most influenced by the effluent. Bores 2 and 8
9
10
11
12
13
14
15
16 - 35m .30
w2=,., . . b , .
.2S -20 .15 .10 -5
Base of Aquifer
....
| ....
J-T-i--i
...........
" / / / / / / / / / / / / / / / / / / / / / / / / / , ' / / / / / / / / / / / / / / / / / / / / / / / / / Fig. I. Sampling depths of bore holes.
J-1 s~,~,d ' s,=~o. /
/ / / / /
AHD -S -10
Survival of viruses in groundwater
303
Table 2. Significance of regression slopes of virus survival Virus
Bore
Echovirus 6 Echovirus I I Poliovirus 1 Poliovirus l Coxsackievirus B5 Echovirus 24 Poliovirus I Poliovirus I Potiovirus I
Slope
2 3 5 7 1 4 6 22°C PBS 4°C PBS
-0.11 -0.10 -0.09 -0.07 -0.05 -0.05 -0.03 -0.01 +0.0005
t
Residual d.f.
9.6 4.6 2.3 25.0 68.8 3.4 8.7 4.1 0.18
1 3 2 2 2 4 3 2 2
P* 0.06 0.02 0.146 0.001 0.0002 0.02 0.003 0.05 0.87
*Level of significance.
7 were least affected by artificial recharge and bore 1 was slightly affected (Table 1). This was reflected by low dissolved oxygen concentrations and higher temperatures of groundwater in these bores compared with bores 3, 4 and 5 which were significantly influenced by the effluent plume. Bore 6 was slotted to sample the base of the aquifer (Fig. 1) and was therefore outside the influence of the effluent plume. This is reflected in the low dissolved oxygen recorded for this bore. However, t h e temperature of the groundwater for bore 6 was similar to that of groundwater in bores influenced by the effluent. This was thought to be due to the cooling effect produced by the effluent surrounding the unslotted section of the bore casing. During the course of the virus survival experiment, effluent movement towards bores 3 and 4 decreased, and this influenced the temperature and dissolved oxygen in the bores adjacent to basin 4.
0 .......
0
Echovirus Wire II
~&
Echovirus Wire 6
e-- -- -- -4
E(:hovirus WI~ 24
~*********A
Coxllcklevirul type B8
7
Virus survival in groundwater and P B S
Regression curves were calculated for the viability for each virus against time. Linear regressions gave adequate fit to the available data in each case with gradient significantly different from zero, with the exception of those for echovirus type 6 (P = 0.06), poliovirus type 1 in bore 5 (P = 0.146) and that for the poliovirus type l control in PBS incubated at 4°C (P = 0.87) (Table 2). The plot of echovirus type 6 was linear (Fig. 2) and the absence of significance for the regression curve may have been due to the small number of observations. The virus inactivation curve for poliovirus 1 in bore 5 was found to be very variable (Fig. 3). This was typical of the virus inactivation curves for the other virus types in bores which were significantly influenced by the effluent plume (echovirus type 11 and echovirus type 24) (Fig. 2). Linear virus inactivation curves as found in this study are consistent with the findings of other workers (O'Brien and Newman, 1977; McDaniels et al., 1983). The stability of poliovirus type 1 in Canning Vale groundwater was found to be greater than the stability previously reported. Keswick et al. (1982) described a 10-fold decrease
0
i'
"
0 0" . . . .
0 Poliovirus Wire I (Bore 6} • Poliovirus type I (Bore 5)
b ........
i& Poliovirus type I (Bore 7)
i'"
i
:
% %
•
%
1 I
10
I
I
2o
30
l
40 Time (dlys)
l
so
•
so
Fig. 2. Survival of coxsackieviruscs and echoviruses in groundwater.
*
10
e 20
. 30
,
40 Time (dlyl)
I
rio
I 60
Fig. 3. S u r v i v a l o f p o l i o v i r u s e s in g r o u n d w a t e r .
304
JANIS JJ~.'qsoyset al.
in poliovirus titre every 5 days at groundwater temperatures between 3-15°C, whereas the same decrease in virus titre required 26 days in the Canning Vale study. Virus survival in Canning Vale groundwater was much greater than the reported stability of this virus in surface waters. O'Brian and Newman (1977) reported a 10-fold decrease in poliovirus titre after 25 h compared with 13 days for the same decrease in poliovirus titre at similar temperatures in Canning Vale groundwater. These differences between surface and ground waters may be largely due to the virus inactivating effects of solar radiation on the surface waters (Bitton et al., 1979). The viruses in the Canning Vale study were completely protected from sunlight.
Poliovirus type I 14°C)
e- ....
•
0
0 Poliovirus type I (22oc)
0
-'_:s =
2
:>
I 10
I 20
I I 30 40 Time (days)
I 50
I 60
Effect o f temperature and dissolved oxygen concentration on virus survival
Fig. 4. Survival of polioviruses in sterile PBS.
The effect of temperature on virus survival in environmental waters has been widely reported (Won and Ross, 1973; O'Brien and Newman, 1977; Fujioka et al., 1980; McDaniels et al., 1983; Yates et al., 1985). In this study, the effect of temperature on virus survival in groundwater was examined by placing poliovirus into two bores which had different ambient temperatures. The rate of poliovirus inactivation in sterile PBS at different temperatures was also determined in a laboratory experiment. In both these experiments, increased temperature was related to increased virus inactivation in that poliovirus inactivation was more rapid in bore 7 (temperature range 21-22°C) than in bore 6 (temperature range 15-18°C). However, this effect may have also been due to an unknown factor in groundwater. The slopes of the two curves were shown to be - 0 . 0 7 and - 0 . 0 3 for bores 7 and 6, respectively (Table 2). However, these two curves could not be shown to be statistically different (P = 0.16) using the method of Snedecor and Cochran (1980) and this was probably due to insufficient observations. Temperature was also found to influence virus survival under laboratory conditions. When poliovirus type 1 was incubated in sterile PBS there was an increase in the rate of virus inactivation between 22 and 4°C (Fig. 4). After 56 days at 4°C there was no detectable virus inactivation. The slight increase in titre in the intermediate samples may have been due to virus deaggregation. However, the two virus survival curves could not be compared statistically since the curve of virus survival at 4°C in PBS could not be shown to be a linear regression (P = 0.87). Increased virus inactivation in groundwater at higher temperatures may have been due to increases in the activity of antagonistic microorganisms. However, the observation of more rapid virus inactivation at higher temperatures in sterile PBS would suggest that temperature-associated loss of virus titre may be independent from the activities of microorganisms. Katzenelson (1978) suggested that the inactivation o f
viruses in water is associated with the movement of water molecules. Some workers have shown that aerobic conditions can increase the rate of virus inactivation. Lund and Nissen (1983) reported a significant increase in the rate of virus inactivation in aerated animal waste compared with non-aerated conditions. Hurst et al. (1980) showed that aerobic soil microorganisms adversely affect virus survival while anaerobic microorganisms had no effect. In this study, poliovirus was placed in groundwater in two adjacent bores where dissolved oxygen concentration was different. The results of this experiment suggest that dissolved oxygen concentration in groundwater affected the rate of virus inactivation. The regression slopes for poliovirus survival in bores 5 (dissolved oxygen range 2.7-6.8 mg/l) and bore 6 (dissolved oxygen 0.06mg/l) were - 0 . 0 9 and -0.03, respectively. However, the two curves could not be compared statistically since poliovirus survival in bore 5 could not be shown to be a linear regression (P = 0.146). The differences in virus survival in these two bores may have been caused by an unknown factor present in bore 5. There did not appear to be the same association with decrease of virus titre and dissolved oxygen concentration in bores 2, 3 and 4, but unlike conditions in bore 5 where the dissolved oxygen was maintained at a high level, decreased effluent infiltration rates onto the recharge basin during the course of the virus survival experiments caused a rapid decrease in dissolved oxygen concentration in bores 3 and 4 (Table 1). The apparent absence of association between virus inactivation rates and dissolved oxygen concentration in bores 2, 3 and 4 may reflect varying individual stability amongst echoviruses types 6, 11 and 24. Echovirus type 24 was the most stable and eehovirus types 6 and 11 the least stable. Any association between dissolved oxygen concentration and virus inactivation may be a direct effect or may be due to an unknown factor present in the groundwater in bore 5.
Survival of viruses in groundwater Table
3. Bacterialconcentrationof groundwaterboresprior to virussurvivalstudy(concentrationexpressed as bacterial numbers/100ml) Total Faecal Faecal Total bacterialcount coliforms coliforms streptococci 22°C 35°C 44°C
Bore 1 Bore 2 Bore 3 Bore 4 Bore 5 Bore 6 Bore 7 Effluent
6 0 6 0 0 2 2 5 x 104
0 0 0 0 0 0 0 16 x 102
0 0 0 6 0 2 0 5 x 102
The effect of the dissolved oxygen may be a direct oxidation of components of the virus capsid or possibly as with temperature, the level of dissolved oxygen may influence the activity of antagonistic microorganisms. This would suggest that wastewater should have a low biochemical oxygen demand (BOD) and be aerated prior to land disposal to encourage virus inactivation. The significance of dissolved oxygen concentration on virus survival may be reflected in the findings of Hurst et al. (1980) who observed that echovirus type 7 survived better in polluted water compared with non-polluted water. Effect o f bacteria on virus survival A number of workers has reported an association of microbial populations with virus inactivation. Mitchell (1971) observed a direct relationship between rate of decline of bacteriophage and the size of the population of indigenous marine microorganisms. In another study, Mitchell and Jannasch (1969) demonstrated that the virus inactivating agent in seawater was filterable indicating a chemical substance. Cliver and Herrmann (1972) showed that some enteroviruses were susceptible to inactivation by proteolytic enzymes. The authors also demonstrated the virucidal activity of the bacteria Pseudomonas aeruginosa. In subsequent experiments, Herrmann et al. (1974) reported a more rapid inactivation of virus in lakewater compared with filtered, sterile lakewater, suggesting that viruses were degraded in the environment by microbial activity. Similar virusinactivating microorganisms have been reported by Fujioka et al. (1980) who found that they were present in seawater but not in fresh mountain stream waters. The concentration of bacteria was found to be variable for bores 1-7 (Table 3). However, in this study there was no association with rate of virus inactivation in groundwater and bacterial numbers. The correlation coefficient (r) was not significant (P > 0.05) when total bacterial numbers at 22°C (r = 0.072) and at 35°C ( r - - 0 . 2 3 ) were compared with the virus inactivation rate. However, increases in the virus inactivation rate due to the influence of microorganisms may be limited to specific species. The poliovirus samples from bore 5 were found to contain large numbers of Pseudomonas maltophilia which probably originated from the diluent groundwater. This organism was not found in the other WR 23/3---D
305
II 31 2 17 35 25 35 22
x x x x x x × x
103 102 l0 s 102 102 l04 102 104
30 60 460 190 70 710 5200 4500
0 0 0 0 0 l0 80 2600
bores and its finding in bore 5 may have been related to the higher average dissolved oxygen concentration in this bore. The presence of this organism may have contributed to the more rapid inactivation of poliovirus in bore 5 compared with bores 6 and 7, where there was no evidence of this organism. Therefore, virus inactivation could be due to specific organisms present in varying numbers in some of the groundwater bores. A comparison of poliovirus survival at 22°C in non-sterile groundwater in bore 7 (Fig. 3) with sterile PBS (Fig. 4) revealed a more rapid inactivation of virus in the non-sterile conditions with regression slopes for bore 7 and sterile PBS of - 0 . 0 7 and - 0 . 0 1 , respectively (Table 2). This may suggest that microorganisms play a role in virus inactivation. Calculation o f safe abstraction points As concluded in a previous communication (Jansons et al., 1989), the determination of safe abstraction points in the aquifer after recharge with wastewater based solely on soil/virus adsorption studies may prove unreliable in field situations due to virus breakthrough occurring unpredictably. As an added safeguard, safe abstraction distances should take into consideration virus inactivation rates and groundwater flow characteristics. A similar approach has been recently proposed by Yates et al. (1986) to determine safe septic tank set-back distances. Acknowledgements--Tl~s work was supported by a grant provided by the Water Authority of Western Australia. The authors acknowledge assistance provided by Mr K. Xanthis, Mr S. Medley and Mr R. Curtis. The authors also wish to thank Dr N. A. Goodchild and Dr I. Wright for statistical advice.
REFERENCES
Bitton G., Fraxedas R. and Gifford O. E. (1979) Effect of solar radiation on poliovirus: preliminary experiments. ;Vat. Res. 13, 225-228. Clarke O. M. (1969) Statistics and Experimental Design (Edited by Barrington E. and Willis A.). Arnold, L o n d o n . Cliver D. O. and Herrmann J. E. (1972) Proteolytic and microbial inactivation of enteroviruses. ;Vat. Res. 6, 797-805. Fujioka R. S., Loh P. C. and Lau L. S. (1980) Survival of human enteroviruses in the Hawaiian ocean environment:
306
JANIS JANSONSel al.
evidence for virus inactivating microorganisms. Appl. envir. Microbiol. 39, 1105-1110. Gustafson T. L. (1984) Epistat Statistical Package. Round Rock, Tex., U.S.A. Herrmann J. E., Kostenbader K. D. Jr and Cliver D. O. (1974) Persistence of enteroviruses in lake water. Appl. Microbiol. 28, 895-896. Hurst C. J., Gerba C. P. and Cech I. (1980) Effects of environmental variables and soil characteristics on virus survival in soil. Appl. envir. Microbiol. 40, 1067-1079. Jansons J., Edmonds L. W., Speight B. and Bucens M. R. (1989) Movement of viruses after artificial recharge. War. Res. 23, 293-297. Katzenelson E. (1978) Survival of viruses. In Indicators of Viruses in Water and Food (Edited by Berg G.). Ann Arbor Science, Ann Arbor, Mich., U.S.A. Keswick B. H., Gerba C. P.~ Secor S. L. and Cech I. (1982) Survival of enteric viruses and indicator bacteria in groundwater. J. envir. Sci. Hlth A17, 903-912. LaBelle R. L. and Gerba C. P. (1980) Influence of estuarine sediment on virus survival under field conditions. Appl. envir. Microbiol. 39, 749-755. Lund E. and Nissen B. (1983) The survival of enteroviruses in aerated and unaerated cattle and pig slurry. Agric. Wastes 7, 221-223. McDaniels A. E., Cochran K. W., Gannon J. J. and Williams G. W. (1983) Rotavirus and reovirus stability in microorganism-free distilled and wastewaters. Wat. Res. 17, 1349-1353. Mitchell R. (1971) Destruction of bacteria and viruses in
seawater. J. sanit. Engng. Div., Am. Soc. cir. Engrs 97, 425-432. Mitchell R. and Jannasch H. W. (1969) Processes controlling virus inactivation in seawater. Envir. Sci. Technol. 3, 941-943. O'Brien R. T. and Newman J. S. (1977) Inactivation of polioviruses and coxsackieviruses in surface water. Appl. envir. Microbiol. 33, 334-340. Snedecor G. W. and Cochran W. G. (1980) Statistical Methods. Iowa State University Press, Ames, Iowa, U.S.A. Vargo, G. A., Hargraves P. E. and Johnson P. (1975) Scanning electron microscopy of dialysis tubes incubated in flowing seawater. Mar. Biol. 31, 113-120. Vasconcelos G. J. and Swartz R. G. (1976) Survival of bacteria in seawater using a diffusion chamber apparatus in situ. AppL envir. Microbiol. 31, 913-920. Ward R. L. and Winston P. E. (1985) Development of methods to measure virus inactivation in fresh waters. Appl. envir. Microbiol. 50, 1144-1148. Won W. D. and Ross H. (1973) Persistence of bacteria and viruses in seawater. J. envir. Engng Div., Am. Soc. cir. Engrs 99, 205-211. Yates M. V., Gerba C. P. and Kelley L. M. (1985) Virus persistence in groundwater. Appl. envir. Microbiol. 49, 778-781. Yates M. V., Yates S. R., Warrick A. W. and Gerba C. P. (1986) Use of geostatistics to predict virus decay rates for determination of septic tank setback distances. Appl. envir. Microbiol. 52, 479483.
0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Press pie
SURVIVAL OF VIRUSES IN GROUNDWATER JANIS JANSONS,1'* LINDSAY W. EDMONDS,2 BRENT SPEIGHT2 a n d MARION R. BUCENS3 ~Arthur Webster Pty Ltd, P.O. Box 234, Baulkham Hills, New South Wales 2153, 2Water Authority of Western Australia, 629 Newcastle Street, Leederville, Western Australia 6007 and 3Virus Laboratory, Combined Microbiology Service, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia
(First received January 1988; accepted in revised form October 1988) Abstract--Survival in groundwater of echovirus types 6, 11 and 24, coxsackievirus type B5 and poliovirus type 1 was determined. Enterovirus survival in groundwater was found to be variable and appeared to be influenced by a number of factors: temperature, dissolved oxygen concentration and possibly the presence of microorganisms. Dissolved oxygen concentration was the most significant factor in loss of virus infectivity in groundwater. Poliovirus type 1 incubated in groundwater with a mean dissolved oxygen concentration of 0.2 mg/1 decreased in infectivity by 100-fold in 50 days compared with 20 days for a decrease of the same magnitude when incubated in groundwater with a mean dissolved oxygen concentration of 5.4 mg/1. Echovirus type 6 was found to be least stable, and poliovirus type 1 was found to be most stable, although virus stability may have been due to conditions existing in individual groundwater bores.
Key words--groundwater, artificial recharge, temperature, wastewater, dissolved oxygen, enteric viruses
INTRODUCTION In a previous c o m m u n i c a t i o n (Jansons et al., 1989), it was concluded t h a t Bassendean Sand was u n a b l e to efficiently a d s o r b all enteric viruses d u r i n g artificial recharge a n d t h a t indigenous viruses were able to p e n e t r a t e into the aquifer. T o determine the possible extent o f viral c o n t a m i n a t i o n o f the aquifer d u r i n g artificial recharge with effluent, experiments o n the longevity o f virus survival in the aquifer were carried out. Viruses were placed in dialysis bags a n d lowered into the aquifer t h r o u g h boreholes. Virus samples were retrieved at various time intervals a n d the loss o f virus infectivity was determined over a 56 day period. MATERIALS AND METHODS
Description of study site The virus survival study was carried out at the Canning Vale Groundwater Recharge site, as previously described (Jansons et al., 1989). Preparation of virus suspensions for survival studies Virus suspensions for survival studies were prepared by mixing 35 ml of laboratory grown virus with 500 ml of groundwater from respective bores. This mixture was placed in 50 cm of dialysis tubing and sealed at both ends. The virus-containing dialysis tubing was then lowered into the aquifer through boreholes in the configuration described in Table 1. Six dialysis bags were used for each virus type. The bags were retrieved at varying time intervals up to 56 days and stored at - 7 0 ° C until assayed. Echovirus types 6, 11 and 24, and coxsackievirus type B5 were all previously isolated from effluent at the Canning Vale Groundwater Recharge Site (Jansons et al., 1989). Echoviruses, 6, 11 and 24 were grown and titrated in RD cells and coxsackievirus type B5 was grown and titrated in Vvro cells. Poliovirus *Author to w h o m all correspondence should be addressed. 301
1 w a s a Sabin oral vaccine strain (Smith Kline-RIT, Rixensart, Belgium) which had been passaged and titrated in Vero cell cultures. All viruses were stored at - 7 0 ° C until titrated. An experiment was also set up in the laboratory to determine the inactivation of poliovirus at 4 and 22°C in sterile phosphate buffered saline (PBS). The same batch of poliovirus and experimental methods were used in this study as for the groundwater study.
Groundwater quality Groundwater characteristics which were thought to be significant for virus survival, namely temperature and dissolved oxygen, were investigated for each bore (Table I). The dissolved oxygen concentrations of groundwater and effluent were determined using a dissolved oxygen and temperature meter (Delta Scientific, Envirotech Corp., New York, U.S.A.). The variations in groundwater temperature were measured using a maximum/minimum thermometer. The groundwater bores were slotted to sample the upper and lower levels of the aquifer (Fig. 1). A total of seven bores was used in this study. Estimation of bacterial numbers Total coliforms were incubated at 30°C for 4 h followed by 14h at 35°C. Faecal coliforms were estimated as described by Jansons et al. (1989). Faecal streptococci were incubated at 35°C for 4 h followed by 40 h at 45°C. The total bacterial count at 22°C was estimated by incubation of samples at 22°C for 18 h. The total bacterial count at 35°C was processed the same as total coliforms, and the total bacterial count at 44°C was estimated the same as faecal coliforms. Estimation of virus survival in groundwater The viruses were titrated by 10-fold dilutions in 96 well microplates (NUNC, Roskilde, Denmark) containing a confluent monolayer of either RD or Veto cells. After inoculation, the plates were incubated at 35°C in a humidified incubator containing 7.5% CO 2 in air. After 6 days the number of wells showing virus-induced cytopathic effect was recorded. The virus titre was calculated using the Karber method.
302
JANIS JANSONS et al. Table 1. Groundwater quality and location of viruses for survival studies Temperature °C
Dissolved oxygen (rag/I) pH
Bore
Mean
Range
Mean
Range
range
Virus
I 2 3 4 5 6 7 Effluent
19.4 21.9 16.2 16.4 15.7 15.9 21.7 16.2
19-21 21.5-22 15.5-17.5 16-17.5 14.5-17 15-18 21-22 16-16.5
1.2 0.2 2.3 1.6 5.4 0.2 0.06 6.5
0.1-2.6 0.1-0.4 0.2-5.9 0.1-4.7 2.7~.8 0.1-0.3 0.05-0.08 4.8-8.0
6.5~.7 6.4-6.8 6.6-6.8 6.6~.8 6.6-6.8 6.0~.8 5.9~6.2
Coxsackievirus B5 Eehovirus 6 Eehovirus 1 I
Statistical methods Average changes in virus inactivation in groundwater were determined by calculating the linear regression coefficient. The regression equation was determined using an Epistat statistical software package (Gustafson, 1984). The significance of the slope was tested using the t-test. A comparison of the regression lines was performed by the method recommended by Snedecor and Cochran (1980). Correlation coefficients between virus inactivation rates and groundwater quality were determined by the method of Clarke (1969).
out, the results of an integrity test of the dialysis tubing using polyvinyl-pyrrolidone K90 suggested an acceptable stability in groundwater after 33 days of exposure (data not shown). Virus loss by adsorption onto dialysis membrane An experiment performed prior to that on virus survival in groundwater showed that there was no detectable loss of poliovirus titre after either 1 day or after 7 days of incubation in dialysis tubing compared with a control. Ward and Winston (1985) showed that virus adsorption onto surfaces i s instantaneous. Therefore, virus loss by adsorption to dialysis membrane was not considered a significant factor in the virus survival experiments. O'Brien and Newman (1977) have reported virus adsorption to dialysis membranes to be <2%.
Use o f dialysis tubing in virus survival experiments The use of dialysis tubing and diffusion chambers for the study of virus survival has been reported by a number of workers (LaBelle and Gerba, 1980; Herrmann et al., 1974). This method is preferable to laboratory studies since it presents a more accurate picture of inactivating factors in environmental waters. The use of dialysis membranes has been criticised by some workers due to their degradation by microorganisms (Vargo et al., 1975). This resulted in the introduction of survival chambers for the study of bacterial (Vasconcelos and Swartz, 1976) and viral survival (LaBelle and Gerba, 1980). These chambers have the advantage of increased stability in environmental waters as they incorporate a polycarbonate membrane which is more resilient than a dialysis membrane. However, the use of similar survival chambers would have been impractical for the Canning Vale study due to the restriction of space in the bore hole casing. LaBelle and Gerba (1980) have shown the virus inactivation rates to be similar for dialysis tubing and viral survival chambers. During the virus survival study, it was noted that some of the virus-containing dialysis bags were becoming fragile after approx. 30 days of exposure to groundwater and would occasionally rupture while being lifted from the bore. Although virus loss from individual bags cannot be ruled Bore No.
1
2
3
4
5
6
7
Echovims 24 Poliovirus 1 Poliovirus 1 Poliovirus 1
RESULTS AND DISCUSSION
Groundwater quality
The temperature of natural groundwater in the aquifer at the study site ranged from 21 to 23°C and the dissolved oxygen level was < 1.0 mg/l. However, recharge of the aquifer with effluent altered these two parameters. Effluent temperature during the study averaged 16.2°C and the dissolved oxygen level ranged from 4.8 to 8.0 mg/l (Table 1) due to aeration during effluent treatment. Generally, the effluent plume was localised in the upper part of the aquifer and its lateral movement through the aquifer was estimated to be 0.3 m/day. Therefore, groundwater from the shallow slotted bores closest to the recharge basin was most influenced by the effluent. Bores 2 and 8
9
10
11
12
13
14
15
16 - 35m .30
w2=,., . . b , .
.2S -20 .15 .10 -5
Base of Aquifer
....
| ....
J-T-i--i
...........
" / / / / / / / / / / / / / / / / / / / / / / / / / , ' / / / / / / / / / / / / / / / / / / / / / / / / / Fig. I. Sampling depths of bore holes.
J-1 s~,~,d ' s,=~o. /
/ / / / /
AHD -S -10
Survival of viruses in groundwater
303
Table 2. Significance of regression slopes of virus survival Virus
Bore
Echovirus 6 Echovirus I I Poliovirus 1 Poliovirus l Coxsackievirus B5 Echovirus 24 Poliovirus I Poliovirus I Potiovirus I
Slope
2 3 5 7 1 4 6 22°C PBS 4°C PBS
-0.11 -0.10 -0.09 -0.07 -0.05 -0.05 -0.03 -0.01 +0.0005
t
Residual d.f.
9.6 4.6 2.3 25.0 68.8 3.4 8.7 4.1 0.18
1 3 2 2 2 4 3 2 2
P* 0.06 0.02 0.146 0.001 0.0002 0.02 0.003 0.05 0.87
*Level of significance.
7 were least affected by artificial recharge and bore 1 was slightly affected (Table 1). This was reflected by low dissolved oxygen concentrations and higher temperatures of groundwater in these bores compared with bores 3, 4 and 5 which were significantly influenced by the effluent plume. Bore 6 was slotted to sample the base of the aquifer (Fig. 1) and was therefore outside the influence of the effluent plume. This is reflected in the low dissolved oxygen recorded for this bore. However, t h e temperature of the groundwater for bore 6 was similar to that of groundwater in bores influenced by the effluent. This was thought to be due to the cooling effect produced by the effluent surrounding the unslotted section of the bore casing. During the course of the virus survival experiment, effluent movement towards bores 3 and 4 decreased, and this influenced the temperature and dissolved oxygen in the bores adjacent to basin 4.
0 .......
0
Echovirus Wire II
~&
Echovirus Wire 6
e-- -- -- -4
E(:hovirus WI~ 24
~*********A
Coxllcklevirul type B8
7
Virus survival in groundwater and P B S
Regression curves were calculated for the viability for each virus against time. Linear regressions gave adequate fit to the available data in each case with gradient significantly different from zero, with the exception of those for echovirus type 6 (P = 0.06), poliovirus type 1 in bore 5 (P = 0.146) and that for the poliovirus type l control in PBS incubated at 4°C (P = 0.87) (Table 2). The plot of echovirus type 6 was linear (Fig. 2) and the absence of significance for the regression curve may have been due to the small number of observations. The virus inactivation curve for poliovirus 1 in bore 5 was found to be very variable (Fig. 3). This was typical of the virus inactivation curves for the other virus types in bores which were significantly influenced by the effluent plume (echovirus type 11 and echovirus type 24) (Fig. 2). Linear virus inactivation curves as found in this study are consistent with the findings of other workers (O'Brien and Newman, 1977; McDaniels et al., 1983). The stability of poliovirus type 1 in Canning Vale groundwater was found to be greater than the stability previously reported. Keswick et al. (1982) described a 10-fold decrease
0
i'
"
0 0" . . . .
0 Poliovirus Wire I (Bore 6} • Poliovirus type I (Bore 5)
b ........
i& Poliovirus type I (Bore 7)
i'"
i
:
% %
•
%
1 I
10
I
I
2o
30
l
40 Time (dlys)
l
so
•
so
Fig. 2. Survival of coxsackieviruscs and echoviruses in groundwater.
*
10
e 20
. 30
,
40 Time (dlyl)
I
rio
I 60
Fig. 3. S u r v i v a l o f p o l i o v i r u s e s in g r o u n d w a t e r .
304
JANIS JJ~.'qsoyset al.
in poliovirus titre every 5 days at groundwater temperatures between 3-15°C, whereas the same decrease in virus titre required 26 days in the Canning Vale study. Virus survival in Canning Vale groundwater was much greater than the reported stability of this virus in surface waters. O'Brian and Newman (1977) reported a 10-fold decrease in poliovirus titre after 25 h compared with 13 days for the same decrease in poliovirus titre at similar temperatures in Canning Vale groundwater. These differences between surface and ground waters may be largely due to the virus inactivating effects of solar radiation on the surface waters (Bitton et al., 1979). The viruses in the Canning Vale study were completely protected from sunlight.
Poliovirus type I 14°C)
e- ....
•
0
0 Poliovirus type I (22oc)
0
-'_:s =
2
:>
I 10
I 20
I I 30 40 Time (days)
I 50
I 60
Effect o f temperature and dissolved oxygen concentration on virus survival
Fig. 4. Survival of polioviruses in sterile PBS.
The effect of temperature on virus survival in environmental waters has been widely reported (Won and Ross, 1973; O'Brien and Newman, 1977; Fujioka et al., 1980; McDaniels et al., 1983; Yates et al., 1985). In this study, the effect of temperature on virus survival in groundwater was examined by placing poliovirus into two bores which had different ambient temperatures. The rate of poliovirus inactivation in sterile PBS at different temperatures was also determined in a laboratory experiment. In both these experiments, increased temperature was related to increased virus inactivation in that poliovirus inactivation was more rapid in bore 7 (temperature range 21-22°C) than in bore 6 (temperature range 15-18°C). However, this effect may have also been due to an unknown factor in groundwater. The slopes of the two curves were shown to be - 0 . 0 7 and - 0 . 0 3 for bores 7 and 6, respectively (Table 2). However, these two curves could not be shown to be statistically different (P = 0.16) using the method of Snedecor and Cochran (1980) and this was probably due to insufficient observations. Temperature was also found to influence virus survival under laboratory conditions. When poliovirus type 1 was incubated in sterile PBS there was an increase in the rate of virus inactivation between 22 and 4°C (Fig. 4). After 56 days at 4°C there was no detectable virus inactivation. The slight increase in titre in the intermediate samples may have been due to virus deaggregation. However, the two virus survival curves could not be compared statistically since the curve of virus survival at 4°C in PBS could not be shown to be a linear regression (P = 0.87). Increased virus inactivation in groundwater at higher temperatures may have been due to increases in the activity of antagonistic microorganisms. However, the observation of more rapid virus inactivation at higher temperatures in sterile PBS would suggest that temperature-associated loss of virus titre may be independent from the activities of microorganisms. Katzenelson (1978) suggested that the inactivation o f
viruses in water is associated with the movement of water molecules. Some workers have shown that aerobic conditions can increase the rate of virus inactivation. Lund and Nissen (1983) reported a significant increase in the rate of virus inactivation in aerated animal waste compared with non-aerated conditions. Hurst et al. (1980) showed that aerobic soil microorganisms adversely affect virus survival while anaerobic microorganisms had no effect. In this study, poliovirus was placed in groundwater in two adjacent bores where dissolved oxygen concentration was different. The results of this experiment suggest that dissolved oxygen concentration in groundwater affected the rate of virus inactivation. The regression slopes for poliovirus survival in bores 5 (dissolved oxygen range 2.7-6.8 mg/l) and bore 6 (dissolved oxygen 0.06mg/l) were - 0 . 0 9 and -0.03, respectively. However, the two curves could not be compared statistically since poliovirus survival in bore 5 could not be shown to be a linear regression (P = 0.146). The differences in virus survival in these two bores may have been caused by an unknown factor present in bore 5. There did not appear to be the same association with decrease of virus titre and dissolved oxygen concentration in bores 2, 3 and 4, but unlike conditions in bore 5 where the dissolved oxygen was maintained at a high level, decreased effluent infiltration rates onto the recharge basin during the course of the virus survival experiments caused a rapid decrease in dissolved oxygen concentration in bores 3 and 4 (Table 1). The apparent absence of association between virus inactivation rates and dissolved oxygen concentration in bores 2, 3 and 4 may reflect varying individual stability amongst echoviruses types 6, 11 and 24. Echovirus type 24 was the most stable and eehovirus types 6 and 11 the least stable. Any association between dissolved oxygen concentration and virus inactivation may be a direct effect or may be due to an unknown factor present in the groundwater in bore 5.
Survival of viruses in groundwater Table
3. Bacterialconcentrationof groundwaterboresprior to virussurvivalstudy(concentrationexpressed as bacterial numbers/100ml) Total Faecal Faecal Total bacterialcount coliforms coliforms streptococci 22°C 35°C 44°C
Bore 1 Bore 2 Bore 3 Bore 4 Bore 5 Bore 6 Bore 7 Effluent
6 0 6 0 0 2 2 5 x 104
0 0 0 0 0 0 0 16 x 102
0 0 0 6 0 2 0 5 x 102
The effect of the dissolved oxygen may be a direct oxidation of components of the virus capsid or possibly as with temperature, the level of dissolved oxygen may influence the activity of antagonistic microorganisms. This would suggest that wastewater should have a low biochemical oxygen demand (BOD) and be aerated prior to land disposal to encourage virus inactivation. The significance of dissolved oxygen concentration on virus survival may be reflected in the findings of Hurst et al. (1980) who observed that echovirus type 7 survived better in polluted water compared with non-polluted water. Effect o f bacteria on virus survival A number of workers has reported an association of microbial populations with virus inactivation. Mitchell (1971) observed a direct relationship between rate of decline of bacteriophage and the size of the population of indigenous marine microorganisms. In another study, Mitchell and Jannasch (1969) demonstrated that the virus inactivating agent in seawater was filterable indicating a chemical substance. Cliver and Herrmann (1972) showed that some enteroviruses were susceptible to inactivation by proteolytic enzymes. The authors also demonstrated the virucidal activity of the bacteria Pseudomonas aeruginosa. In subsequent experiments, Herrmann et al. (1974) reported a more rapid inactivation of virus in lakewater compared with filtered, sterile lakewater, suggesting that viruses were degraded in the environment by microbial activity. Similar virusinactivating microorganisms have been reported by Fujioka et al. (1980) who found that they were present in seawater but not in fresh mountain stream waters. The concentration of bacteria was found to be variable for bores 1-7 (Table 3). However, in this study there was no association with rate of virus inactivation in groundwater and bacterial numbers. The correlation coefficient (r) was not significant (P > 0.05) when total bacterial numbers at 22°C (r = 0.072) and at 35°C ( r - - 0 . 2 3 ) were compared with the virus inactivation rate. However, increases in the virus inactivation rate due to the influence of microorganisms may be limited to specific species. The poliovirus samples from bore 5 were found to contain large numbers of Pseudomonas maltophilia which probably originated from the diluent groundwater. This organism was not found in the other WR 23/3---D
305
II 31 2 17 35 25 35 22
x x x x x x × x
103 102 l0 s 102 102 l04 102 104
30 60 460 190 70 710 5200 4500
0 0 0 0 0 l0 80 2600
bores and its finding in bore 5 may have been related to the higher average dissolved oxygen concentration in this bore. The presence of this organism may have contributed to the more rapid inactivation of poliovirus in bore 5 compared with bores 6 and 7, where there was no evidence of this organism. Therefore, virus inactivation could be due to specific organisms present in varying numbers in some of the groundwater bores. A comparison of poliovirus survival at 22°C in non-sterile groundwater in bore 7 (Fig. 3) with sterile PBS (Fig. 4) revealed a more rapid inactivation of virus in the non-sterile conditions with regression slopes for bore 7 and sterile PBS of - 0 . 0 7 and - 0 . 0 1 , respectively (Table 2). This may suggest that microorganisms play a role in virus inactivation. Calculation o f safe abstraction points As concluded in a previous communication (Jansons et al., 1989), the determination of safe abstraction points in the aquifer after recharge with wastewater based solely on soil/virus adsorption studies may prove unreliable in field situations due to virus breakthrough occurring unpredictably. As an added safeguard, safe abstraction distances should take into consideration virus inactivation rates and groundwater flow characteristics. A similar approach has been recently proposed by Yates et al. (1986) to determine safe septic tank set-back distances. Acknowledgements--Tl~s work was supported by a grant provided by the Water Authority of Western Australia. The authors acknowledge assistance provided by Mr K. Xanthis, Mr S. Medley and Mr R. Curtis. The authors also wish to thank Dr N. A. Goodchild and Dr I. Wright for statistical advice.
REFERENCES
Bitton G., Fraxedas R. and Gifford O. E. (1979) Effect of solar radiation on poliovirus: preliminary experiments. ;Vat. Res. 13, 225-228. Clarke O. M. (1969) Statistics and Experimental Design (Edited by Barrington E. and Willis A.). Arnold, L o n d o n . Cliver D. O. and Herrmann J. E. (1972) Proteolytic and microbial inactivation of enteroviruses. ;Vat. Res. 6, 797-805. Fujioka R. S., Loh P. C. and Lau L. S. (1980) Survival of human enteroviruses in the Hawaiian ocean environment:
306
JANIS JANSONSel al.
evidence for virus inactivating microorganisms. Appl. envir. Microbiol. 39, 1105-1110. Gustafson T. L. (1984) Epistat Statistical Package. Round Rock, Tex., U.S.A. Herrmann J. E., Kostenbader K. D. Jr and Cliver D. O. (1974) Persistence of enteroviruses in lake water. Appl. Microbiol. 28, 895-896. Hurst C. J., Gerba C. P. and Cech I. (1980) Effects of environmental variables and soil characteristics on virus survival in soil. Appl. envir. Microbiol. 40, 1067-1079. Jansons J., Edmonds L. W., Speight B. and Bucens M. R. (1989) Movement of viruses after artificial recharge. War. Res. 23, 293-297. Katzenelson E. (1978) Survival of viruses. In Indicators of Viruses in Water and Food (Edited by Berg G.). Ann Arbor Science, Ann Arbor, Mich., U.S.A. Keswick B. H., Gerba C. P.~ Secor S. L. and Cech I. (1982) Survival of enteric viruses and indicator bacteria in groundwater. J. envir. Sci. Hlth A17, 903-912. LaBelle R. L. and Gerba C. P. (1980) Influence of estuarine sediment on virus survival under field conditions. Appl. envir. Microbiol. 39, 749-755. Lund E. and Nissen B. (1983) The survival of enteroviruses in aerated and unaerated cattle and pig slurry. Agric. Wastes 7, 221-223. McDaniels A. E., Cochran K. W., Gannon J. J. and Williams G. W. (1983) Rotavirus and reovirus stability in microorganism-free distilled and wastewaters. Wat. Res. 17, 1349-1353. Mitchell R. (1971) Destruction of bacteria and viruses in
seawater. J. sanit. Engng. Div., Am. Soc. cir. Engrs 97, 425-432. Mitchell R. and Jannasch H. W. (1969) Processes controlling virus inactivation in seawater. Envir. Sci. Technol. 3, 941-943. O'Brien R. T. and Newman J. S. (1977) Inactivation of polioviruses and coxsackieviruses in surface water. Appl. envir. Microbiol. 33, 334-340. Snedecor G. W. and Cochran W. G. (1980) Statistical Methods. Iowa State University Press, Ames, Iowa, U.S.A. Vargo, G. A., Hargraves P. E. and Johnson P. (1975) Scanning electron microscopy of dialysis tubes incubated in flowing seawater. Mar. Biol. 31, 113-120. Vasconcelos G. J. and Swartz R. G. (1976) Survival of bacteria in seawater using a diffusion chamber apparatus in situ. AppL envir. Microbiol. 31, 913-920. Ward R. L. and Winston P. E. (1985) Development of methods to measure virus inactivation in fresh waters. Appl. envir. Microbiol. 50, 1144-1148. Won W. D. and Ross H. (1973) Persistence of bacteria and viruses in seawater. J. envir. Engng Div., Am. Soc. cir. Engrs 99, 205-211. Yates M. V., Gerba C. P. and Kelley L. M. (1985) Virus persistence in groundwater. Appl. envir. Microbiol. 49, 778-781. Yates M. V., Yates S. R., Warrick A. W. and Gerba C. P. (1986) Use of geostatistics to predict virus decay rates for determination of septic tank setback distances. Appl. envir. Microbiol. 52, 479483.