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Movement of viruses after artificial recharge
War. Res. Vol. 23, No. 3, pp. 293-299, 1989 Printed in Great Britain. All rights reserved
0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Pr~s pie
MOVEMENT OF VIRUSES AFTER ARTIFICIAL RECHARGE JANIS JANSONSt'*, LINDSAY W. EDMONDS2, BRENT SPEIGHT2 and MARION R. BUCENS3 'Arthur Webster Pry Ltd, P.O. Box 234, Bauikharn 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 December 1987; accepted in revised form October 1988) Abstract--Results of human enteric virus movement through soil and groundwater aquifers after artificial recharge using wastewater are presented. The penetration through the recharge soil of indigenous viruses from treatment plant effluent was found to be much greater than that of a seeded vaccine poliovirus. Echovirus type 11 from wastewater was detected at a depth of 9.0 m in groundwater from a bore located 14 m from the recharge basin whereas seed poliovirus was not isolated beyond a depth of 1.5 m below the recharge basin. It was concluded that data on enteric virus survival in groundwater were required if safe abstraction distances were to be determined.
Key words--artificial recharge, enteric viruses, groundwater, wastewater
INTRODUCTION The removal o f viruses from wastewater after land application has been widely investigated, with conflicting results. Some investigators have failed to detect virus penetration into the aquifer (Gilbert et ai., 1976) whereas others have demonstrated considerable vertical and lateral migration of viruses (Wellings et al., 1975; Vaughn et al., 1983). In this study, virus penetration through the soil and aquifer after land application o f wastewater was investigated. Vertical m o v e m e n t o f viruses through soil was determined by sampling from pans under the recharge basin and lateral m o v e m e n t through the aquifer was determined by the sampling of groundwater for viruses in sampling bores at various distances from the recharge basins. The monitoring of indigenous virus removal by percolation through the soil was carried out during 1981-1982. This was followed by an experiment on the m o v e m e n t of a seeded vaccine strain of poliovirus through the recharge basin soil after seeding.
sendean Sand overlaying an impervious layer known as the Osborne formation some 41 m below the recharge basins. Bassendean Sand has a high silica content and a very low pH (McArthur and Bettenay, 1974). The depth to groundwater below the recharge basins was approx. 9 m. Sampling
23.00
,.o
MATERIALS AND METHODS
Description of recharge site The groundwater recharge site was situated at Canning Vale, Western Australia. Secondary effluent was provided by the Canning Vale wastewater treatment plant which treated wastewater from domestic sources. After initial settling carried out to reduce the level of suspended solids, the effluent was pumped to a high level tank where it gravitated to six rectangular soakage basins (Fig. 1). Each basin had a total area of 800 m 2. The recharge site was situated on an unconfined aquifer which consisted of Bas*Author to whom all correspondence should be addressed.
24.00
Fig. 1. Site plan showing monitoring bores 1-15 and groundwater contour lines. 293
JANIS JANSONSet al.
294
inplant biological nutrient removal. Flows to the treatment plant were reduced from 3 to 1 M1/day resulting in a significant increase in detention time and these changes led to a reduction in total nitrogen of > 50% but little change in phosphorus levels. Effluent quality for both periods is shown in Table 1.
Basin
Flool
.~.;-:": Slml:
Cl~
Sampling of effluent and pans for bacteria and viruses Two hundred ml samples were collected from effluent and pans for bacterial studies. These samples were processed within 4 h of collection. Five hundred ml samples were collected from effluent and pans for viral studies. These samples were stored at - 28°C and assayed for virus content within 2 weeks of collection. Pans were completely emptied 30 min prior to collection of samples.
Isolation and enumeration of indigenous viruses from effluent and pans
directly under the basins was achieved by a system of sampling pans located at various depths (Fig. 2). These were used to monitor changes in the effluent in the upper soil layers. The aquifer was sampled by 15 monitoring bores located around the recharge basin (Fig. 1). The bores were screened at varying depths to enable sampling of the upper and lower levels of the aquifer. The depth of the aquifer was approx. 32 m. During the first part of the experiment, 3 MI of wastewater were disposed of per day on a 5 day flooding, 9 day drying cycle which was aimed at encouraging denitrification of the effluent in the soil. After 1982, the effluent volume was reduced to 1 M1/day. The direct soakage rate for both periods ranged from initial rates of 9 down to 0.3m/day and averaged 4m/day.
Effluent and pan samples were placed in pre-soaked 50 cm lengths of 76 mm dialysis membrane (Union Carbide Corp., II1., U.S.A.). The dialysis membranes containing the samples were surrounded with flakes of polyethylene glycol 6000 (ICI, Sydney, Australia) and left at 4°C overnight. This process concentrated the sample volume approx. 10-fold. The concentrated samples were inoculated onto 6 different cell cultures; primary monkey kidney cells, a locally derived strain of human diploid embryo lung fibroblasts, LLC-MK2, FL-amion, Vero and RD (human rhabdomyosarcoma cells). The cell cultures were grown in Eagles MEM (Commonwealth Serum Labs, Parkville, Australia) containing 10% v/v foetal calf serum (Flow Labs, Australia), HEPES buffer (Calbiochem, La Jolla, Calif., U.S.A.) to a final concentration of 28 mM and sodium bicarbonate to a final concentration of 0.14%. Antibiotics were added as follows: penicillin (Commonwealth Serum Labs, Victoria, Australia) 0.06 mg/ml; neomycin (Upjohn, Ontario, Canada) 0.05mg/ml; streptomycin (Glaxo, Australia) 0.1 mg/ml. The ceils were maintained in Eagles MEM containing 2% foetal calf serum (FCS). All other ingredients were the same as the growth medium. Quantitation of indigenous viruses in effluent and pans was carried out by the most probable number method as described by Chang (1958), and the concentration was expressed as the most probable number of eytopathic units per litre (MPNCU/1).
Effluent quality
Identification of indigenous viruses
During 1981 and 1982 there were periods when primary effluent was blended with the secondary effluent in a ratio of 70:30 to improve nutrient removal at the recharge site. However, adequate nutrient removal could not be achieved at the recharge site and from 1983 process changes were made at the wastewater treatment plant to encourage
Virus isolates were initially grouped on the basis of characteristic cytopathic effect (CPE) and then further identified by neutralization tests. Enteroviruses were typed using the World Health Organization antisera pools described by Melnick et al. (1973). Adenoviruses were typed in a similar manner to enteroviruses; specific antisera
Fig. 2. Sampling pan system.
Table 1. Et~uent quality 1981-1982 Mean
Conductivity* pH BOD5 Chloride TDSt Organic nitrogen (as N) Ammonia (as N) Nitrite (as N) Nitrate (as N) Total nitrogen (as N) Total phosphorus Colour:~ Turbidity§
86 30 145 450 2.1 15 1.0 3.5 21.6 10.6 88 7.6
Range 67-128 6.2-7.2 10-55 125-255 440-460 I-5 1.9-30 0.05-6.8 0.1-14 15-35 7.7-15 70-110 1-20
1983-1986 Mean
76 5 115 418 1.6 1.6 0.4 6.8 12 9.5 NT¶I NT
Range 75-80 6.9-7.6 5-20 110-125 413-440 1.3-2.2 1.3-2.2 0-1.10 0.5~-16 23-30 9-1 I NT NT
Units in mg/I unless staled. *mS m-~ at 25°C. tConductivity x 5.5. SHazen units. §NTU, ¶lNot tested.
Movement of viruses after artificial recharge (Microbiological Associates, Md, U.S.A.) for types 1-7 were used in the initial attempt to identify an isolate. If these failed, the isolate was tested against other adenovirus antisera (Types 8-30).
Recovery of indigenous viruses from groundwater During the period of 1981-1982, groundwater samples were processed using the filter adsorption-elution technique which was originally described by Wallis et al. (1972). Many difficulties were experienced using this method, (Jansons and Bucens, 1986) and therefore ultraflltration was adopted as an alternate technique. In the latter part of 1982, 201. volumes of groundwater were processed by ultraflltration using an Amicon DC2 (Amicon Corp., Lexington, Mass. U.S.A.).
Procedurefor poliovirus seeding of effluent In 1983, process changes in wastewater treatment greatly reduced the number of human enteric viruses subsequently isolated from the plant effluent. This reduction in virus concentration made it difficult to evaluate further the capacity of the soil to remove indigenous viruses during groundwater recharge. Artificial seeding of the recharge basins with a vaccine strain of poliovirus was then carried out to quantify the virus adsorbing properties of the soil. To avoid any risks to public health associated with the release of large amounts of viable virus into the environment, the commercially available Sabin oral trivalent vaccine (Smith Kline-RIT, Rixensart, Belgium) was selected. The vaccine contained different concentrations of the three serotypes of poliovirus. Each dose (approx. 0.05m l) of undiluted vaccine contained approx. 106 TCIDso poliovirus type 1, l0 s TCIDso poliovirus type 2 and I0 s's TCIDs0 poliovirus type 3. The final concentration of seed virus was estimated to be 4000 MPNCU/l. The contents of the header tank were allowed to flow into the basin and normal dosing with effluent was resumed. The seeding was carried out in the late afternoon to reduce the risk of solar inactivation of the virus and to avoid exposure to the virus to high ambient temperatures. Due to the relatively small volume of seeded effluent the contents of the header tank were allowed to flow into the basins via a system of levee banks which directed the flow of the seeded effluent to an area over the five sampling pans to a depth of approx. 150 ram. Normal dosing with effluent followed the seeded effluent dosing. The basin floor was free from grass and weeds. Prior studies using fluorescein dye revealed an even hydraulic flow into the basin with a uniform distribution of dye in all the sampling pans.
Recovery of seeded poliovirus from groundwater Groundwater samples were processed by an ultrafiltration method using the Amicon DC30 and DC2 as previously described (Jansons and Bucens, 1986).
Assay of seed poliovirus
295
Estimation of bacterial numbers in effluent and pans Estimation of bacterial numbers was carried out by the method recommended by HMSO (1982) except that membrane enriched Teepol broth was replaced with membrane enriched sodium lauryl sulphate as recommended by the JCPHLSSCA (1980). Cultures for faecal coliforms were incubated for 4h at 30°C followed by 14 at 44°C. Bacterial counts were performed using membrane filter techniques on selective media. RESULTS
Movement of indigenous viruses and bacteria beneath the recharge basin The m o v e m e n t of indigenous viruses beneath the recharge basins at the various soil levels was investigated by means of pan samples. During 1981-1982, effluent and pans were sampled twice weekly. However, on a number of occasions, blockages in the pan sampling system prevented sampling. A comparison of the total virus numbers in effluent and pans during this period showed a 2-fold reduction after travel through the first 0.5 m of sand to pan 1 (Fig. 3). Total virus numbers were reduced by a further 3-fold at pan 2 which was located I m below the basin floor after which the virus concentration remained constant for the remaining sampling levels. This indicated that Bassendean Sand showed a limited capacity for the removal of indigenous viruses. The concentration of virus in the effluent peaked at 351 M P N C U / i in December 1981. Penetration of virus through soil generally coincided with virus concentration peaks in the effluent. During this sampling period the movement of faecal coliforms was also investigated. The reduction in bacterial concentration after travel through the first 0.5 m of sand was greater than that found for indigenous viruses with at least a 10-fold decrease in total bacterial numbers in pan l compared with effluent (Table 2), Thereafter, bacterial numbers were constant at the remaining sampling depths.
Distribution of indigenous virus serotypes at different soil levels Differences were observed in the distribution o f enteric viruses at the various sampling levels (Table 3). Echovirus types 11, 14, 24, 29 and 30, coxsackievirus type B4, untyped adenovirus and adenovirus type 3 and untyped enterovirus all showed an ability to penetrate to 3 m into the soil. This contrasted with the lesser penetration of poliovirus, echovirus types 6 and 17, coxsackievirus types B2 and BS, adenovirus type 7 and reovirus. However, these differences may have been due to chance distribution of virus during sampling.
Effluent and pan samples collected during the poliovirus seeding experiment were thawed and extracted with Freon R113 (Daikin, Osaka, Japan) to reduce the level of bacterial contamination. The virus was quantitated using a method described by Joret et al. (1980) in which three replicate sample volumes of 10, 1.0 and 0.1 ml were inoculated into 75, 25 and 25 cm2 flasks, respectively, containing confluent monolayers of Vero ceils. Virus isolation studies prior to the poliovirus seeding experiment indicated that indigenous virus concentrations in the effluent had fallen below detectable limits. Isolation of indigenous viruses from groundwater The concentrated groundwater samples were processed in The penetration of indigenous viruses into the the same manner as effluent and pan samples except that the total sample volume was assayed for virus. The most aquifer was evaluated by sampling of groundwater probable number of cytopathic units (MPNCU)/I. was for the presence of viruses. During 1981-1982 a calculated using the method described by Chang (1958). total of 45 groundwater samples was taken from
JANIS JANSONSet al.
296 EFFLUENT
300,
the shallow bores closest to the recharge basin (1, 2, 3 and 5) and processed using the filter adsorption-elution method. The deep and shallow bores were sampled in the initial experiments but chemical indicators suggested that the effluent plume was localized in the upper aquifer during initial stages of the recharge study (Table 4) and therefore deep bore sampling was discontinued in subsequent experiments. Considerable difficulties were experienced with the filter adsorption-elution method due to interference by humic acids present in the groundwater (Jansons and Bucens, 1986) in these early experiments and therefore failure to demonstrate virus penetration into the aquifer may have been due to the unsuitability of this technique for Canning Vale groundwater. During the same period, thirty 20 1. groundwater samples were processed by ultrafiltration. Echovirus type I 1 was isolated on one occasion from a shallow bore (bore 3) which was located 14 m from recharge basin 4.
351
2ooi
182
100. ;14 40:
42
30, 20, 10
300 ,PAN 1 200,
215
100,
4o!
°ll
I
20,
10, 0
I
I
. . . . . . . . . . .
I Lj 40 PAN 2
20
1
. . . . . . . .
'° 1 0
j . . . . . . .
. . . . . . . .
oLi
30 PAN 4
10
0 . . . . . . . .
Movement of seed poliovirus beneath the recharge basins The total numbers of seed poliovirus were reduced 40-fold after the first 0.5 m of travel through the soil (Table 5). There was evidence of a further reduction in virus concentration below this sampling level with no virus detected in pans 2, 4 or 5, although virus was observed on one occasion in pan 3 at a concentration similar to that found in pan 1. Sampling of pan 5 continued for 156 h after release of virus. Earlier experiments had shown no loss of infectivity of vaccine poliovirus over a 14 h period, and a 10-fold reduction over a 30 h period when suspended in secondary effluent. This would suggest that reduction of poliovirus concentration in pan samples after travel through soil in this experiment was largely due to adsorption. Pans were again sampled at the onset of the first winter rains some 16 weeks after seeding to determine whether polioviruses could be recovered from pan samples. All these samples were found to be negative for viruses indicating either loss of viability or that adsorption of seed poliovirus to the soil could not be reversed by rainfall.
Isolation of seed poliovirus from groundwater ].
.
., I I
40 PAN 5
:1 II ,I 10
o l I
A'S'O'N'D'J'F'M'A'M'J'J'A 1981 1982
J
The penetration of the seeded poliovirus into the aquifer was determined by sampling of groundwater in the bores adjacent to the seeded basin. A total of thirty 400 1. groundwater samples from bores 1-6 was processed by ultrafiltration using the Amicon DC 30 and DC2 (Jansons and Bucens, 1986). All were negative for virus when tested for the presence of the seed poliovirus. Sampling was continued for 12 weeks after poliovirus seeding.
S'ON'D"
Fig. 3. Concentration of indigenous viruses of various soil levels (virus concentration exprcsscd as MPNCU/I).
DISCUSSION
Several factors have now been described which affect the adsorption of virus to soil including soil
M o v e m e n t of viruses after artificial recharge
297
Table 2. Bacterial concentration beneath the recharge basins during 1981-1982 recharge period (faecal coliforms/100 ml) Final effluent
Pan 1
Pan 2
Pan 3
Pan 4
104 > l0 s > l0 s
2.3 x 103 2.3 x 103 1.1 X 103
4
> l0 s
> 105
5 6 7
>105 >105 > 105 > 105
NT >105 > 105 5.4 X 10 3
1.5 x 103 1.3 x 103 8.0 X 102 1 . 7 X 104 6.7 x 103 1.5x 104 3.6 × 103 ST
2.5 x 103 6.3 x 103 9.0 x 103 4.5 X 103 NT 6.0 x 104 5.0 X 103 NT
1.9 x 103 8.4 × 103 2.7 x 103 1.3 X 104 8.0 x 102 3.8 x 103 5.0 × 103 5.2 X 10 3
Sample 1
2 3
8
Pan 5 102 < 102 I(F NT*
6.0 x 102 2.6 x 102 NT 3.2 X 10 3
*Not tested.
Table 3. Number of isolations of indigenous virus serotypes in effluent and at various soil levels Virus serotypes Effluent Pan 1 Pan 2 Pan 3 Pan 4 Pan 5 Eehoviruses Type 6 Type 11 Type 14 Type 17 Type 24 Type 29 Type 30
3 13 2 I 12 . 4
Polioviruses Type 1 Type 2 Type 3 Coxsaekieviruses Type B2 Type 134 Type B5
.
Reovirus Untyped
.
.
.
Upper aquifer
.
--
. 1
.
.
.
. --
.
.
. --
8
6
1
4
--
4
t y p e ( D r e w r y a n d E l i a s s e n , 1968; B u r g e a n d E n k i r i , 1978; G o y a l a n d G-erba, 1979; S o b s e y et al., 1980), s p e c i e s o f v i r u s ( G o y a l a n d G e r b a , 1979), i n f i l t r a t i o n
--
--
1
. .
.
i
3
0.05 0.40 <0.05 0.15 0.65 < 0.05 95
--
.
.
Lower aquifer
.
.
. --
3.45 7.05 0.30 3.85 14.65 < 0.05 I I0
1 1 1
1
--
2
.
--
1 .
.
-1 1
.
.
2 1 .
-2 1
. --
Table 4. Chemical indicators for effluent plume localization during recharge experiments (units in rag/l) Organic nitrogen (as N) A m m o n i a (as N) Nitrite (as N) Nitrate (as N) Total nitrogen (as N) Phosphorus Chloride
.
.
.
1 1
-3 -. --
I
. 1 --
I 6 3 .
1 1 2
--
3 1
Adenoviruses Type 3 Type 7 Untyped Enteroviruses Untyped
-7 2 . 5
. 2
I
2
1
2
--
--
--
r a t e ( V a u g h n et al., 1981), p r e s e n c e o f o r g a n i c m a t t e r ( S c h e u e r m a n et al., 1979) a n d e l e c t r o l y t e c o n c e n t r a t i o n ( L a n c e a n d G e r b a , 1984). The Canning Vale study results show a conside r a b l e v a r i a t i o n in t h e r e m o v a l o f d i f f e r e n t e n t e r i c v i r u s e s b y t h e soil. S e e d e d v a c c i n e s t r a i n p o l i o v i r u s w a s s h o w n to b e s t r o n g l y a d s o r b e d b y t h e soil, w i t h a 4 0 - f o l d r e d u c t i o n in t o t a l v i r u s n u m b e r s o c c u r r i n g in t h e first 0.5 m o f t r a v e l t h r o u g h t h e soil, a n d n o p e n e t r a t i o n b e y o n d 1.5 m . I n c o n t r a s t , i n d i g e n o u s v i r u s e s s u c h a s e c h o v i r u s t y p e s 11, 14, 24, 29 a n d 30, a s well a s c o x s a c k i e v i r u s t y p e B4, u n t y p e d a d e n o v i r u s a n d a d e n o v i r u s t y p e 3, p e n e t r a t e d 3.0 m i n t o t h e soil,
Table 5. Movement of seeded poliovirus through soil. (Virus concentration expressed as MPNCU/I) Time (h) after seeding Effluent Pan 1 Pan 2
0 Neg --
0.1 759 --
0.5 1898 35 --
1.0 2712 35 Neg
--
--
Pan
3
--
--
--
35
Pan Pan
4
--
--
--
5
--
--
--
Neg Neg
1.5 Neg 35 Neg
2.0 Neg 35 Ne8
3.0 Neg Neg Neg
12 Neg Neg Neg
Neg
Ne8
Neg
Neg
Neg Neg
Ne8 Neg
Neg Neg
Neg Neg
36 60 Neg Neg . . . . . . .
.
.
. . . Neg Neg
108 Ne 8 . .
156 Neg
.
. Neg
Neg
298
JANIS JANSONSet al.
with a concentration of virus as high as 37.2 MPNCU/I on one occasion. A single isolation of echovirus type 11 demonstrated penetration of this virus into the groundwater aquifer some 9.0 m below the basin floor. The virus distribution profile for indigenous viruses at the various sampling levels was different from that found for the seeded poliovirus. The reduction of indigenous viruses was less marked, with a 2-fold reduction in concentration occurring after 0.5m. The average virus concentration at the sampling points below 1.0 m remained almost constant. The concentration of seed poliovirus in the effluent was 100-fold greater than the average concentration of indigenous virus found in the effluent, so it can be inferred that the failure of poliovirus to penetrate beyond 1.5 m during the seeding experiment was not related to insufficient concentration of seed virus. The wide variation in the adsorption of indigenous viruses compared with seeded poliovirus may be due to a number of factors. The differences may be based solely on serotypic associated variations of surface charges on enteroviruses. Polioviruses have been previously reported to have a strong affinity for a majority of soils (Goyal and Gerba, 1979). The variation may reflect differences between field isolates and laboratory grown strains. Goyal and Gerba (1979) first noted a variation in soil adsorption characteristics between reference strains of virus compared with recent isolates. More evidence for this hypothesis has been provided by Hodes et al. (1960) who reported variable avidity of virulent (Mahoney) compared with attenuated (LSc, 2ab) poliovirus type 1 onto DEAE cellulose. Similar findings have been reported by Thomssen and Majer (1964). Lance and Gerba (1980) suggested that virus distribution profiles and therefore their soil penetration depths were related to variations in virus charge strength in virus populations. They argued that viruses with a net negative charge below a certain level are immediately adsorbed while viruses with a stronger negative charge move further down the column. Therefore the more uniform the virus population the more compact the virus distribution profile. The results of the Canning Vale study may be consistent with this hypothesis as populations of indigenous viruses excreted by the community would be more likely to be variable than viruses adapted in the laboratory to grow in uniform cell lines. Comparison of the virus distribution profiles for indigenous viruses and seeded poliovirus reveals a far more compact distribution for the laboratory grown virus. Finally, indigenous viruses in treatment plant effluent may be associated with organic materials which may alter their ability to adsorb to certain surfaces. The soil characteristics which increase virus adsorption have been well described. However, several workers have reported difficulties in describing characteristics of a single soil that would
adsorb all viruses efficiently (Drewry and Eliassen, 1968; Goyai and Gerba, 1979). Burge and Enkiri (1978) reported that cation exchange capacity and specific surface area correlated with virus adsorption rates. However, one soil tested which ranked highest in these two characteristics was found to have a poor virus adsorption rate. Similar observations have been made by Drewry and Eliassen (1968). They reported that virus adsorption by soil increases with increasing ion-exchange capacity, clay content, organic carbon and glycerol-retention capacity. However, they described a soil which ranked lowest in these various properties, but proved to have greater adsorption power for viruses than most of the other soils tested. Wastewater contains many virus types which are likely to have heterogeneous properties and may also to be associated with organic material. Therefore, it would be difficult to find a soil which could efficiently adsorb all enteric viruses during groundwater recharge procedures. Virus adsorption by the soil does not necessarily result in virus inactivation, and adsorption has been shown to be reversible after a change in conditions such as the ionic environment (Duboise et aL, 1976; Landry et al., 1979). Hurst et al. (1980) suggested that soils which had a high virus adsorption capacity might in fact favour virus survival. It was concluded that the only method by which a reliable determination of virus removal in the aquifer could be made would be to study the survival of enteric viruses in groundwater. Once the virus survival time and local groundwater flow characteristics are known, safe abstraction distances from the recharge basin may then be determined. A study on enteric virus survival in groundwater has been recently completed by this laboratory. Acknowledgements--The authors wish to thank the Water Authority of Western Australia for financial assistance provided during this study. The authors also acknowledge the assistance of Mr K. Xanthis for the chemical characterization of effluent obtained from Canning Vale and Mr R. Curtis for estimating bacteria numbers in effluent and pans.
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0043-1354/89 $3.00+0.00 Copyright © 1989 Pergamon Pr~s pie
MOVEMENT OF VIRUSES AFTER ARTIFICIAL RECHARGE JANIS JANSONSt'*, LINDSAY W. EDMONDS2, BRENT SPEIGHT2 and MARION R. BUCENS3 'Arthur Webster Pry Ltd, P.O. Box 234, Bauikharn 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 December 1987; accepted in revised form October 1988) Abstract--Results of human enteric virus movement through soil and groundwater aquifers after artificial recharge using wastewater are presented. The penetration through the recharge soil of indigenous viruses from treatment plant effluent was found to be much greater than that of a seeded vaccine poliovirus. Echovirus type 11 from wastewater was detected at a depth of 9.0 m in groundwater from a bore located 14 m from the recharge basin whereas seed poliovirus was not isolated beyond a depth of 1.5 m below the recharge basin. It was concluded that data on enteric virus survival in groundwater were required if safe abstraction distances were to be determined.
Key words--artificial recharge, enteric viruses, groundwater, wastewater
INTRODUCTION The removal o f viruses from wastewater after land application has been widely investigated, with conflicting results. Some investigators have failed to detect virus penetration into the aquifer (Gilbert et ai., 1976) whereas others have demonstrated considerable vertical and lateral migration of viruses (Wellings et al., 1975; Vaughn et al., 1983). In this study, virus penetration through the soil and aquifer after land application o f wastewater was investigated. Vertical m o v e m e n t o f viruses through soil was determined by sampling from pans under the recharge basin and lateral m o v e m e n t through the aquifer was determined by the sampling of groundwater for viruses in sampling bores at various distances from the recharge basins. The monitoring of indigenous virus removal by percolation through the soil was carried out during 1981-1982. This was followed by an experiment on the m o v e m e n t of a seeded vaccine strain of poliovirus through the recharge basin soil after seeding.
sendean Sand overlaying an impervious layer known as the Osborne formation some 41 m below the recharge basins. Bassendean Sand has a high silica content and a very low pH (McArthur and Bettenay, 1974). The depth to groundwater below the recharge basins was approx. 9 m. Sampling
23.00
,.o
MATERIALS AND METHODS
Description of recharge site The groundwater recharge site was situated at Canning Vale, Western Australia. Secondary effluent was provided by the Canning Vale wastewater treatment plant which treated wastewater from domestic sources. After initial settling carried out to reduce the level of suspended solids, the effluent was pumped to a high level tank where it gravitated to six rectangular soakage basins (Fig. 1). Each basin had a total area of 800 m 2. The recharge site was situated on an unconfined aquifer which consisted of Bas*Author to whom all correspondence should be addressed.
24.00
Fig. 1. Site plan showing monitoring bores 1-15 and groundwater contour lines. 293
JANIS JANSONSet al.
294
inplant biological nutrient removal. Flows to the treatment plant were reduced from 3 to 1 M1/day resulting in a significant increase in detention time and these changes led to a reduction in total nitrogen of > 50% but little change in phosphorus levels. Effluent quality for both periods is shown in Table 1.
Basin
Flool
.~.;-:": Slml:
Cl~
Sampling of effluent and pans for bacteria and viruses Two hundred ml samples were collected from effluent and pans for bacterial studies. These samples were processed within 4 h of collection. Five hundred ml samples were collected from effluent and pans for viral studies. These samples were stored at - 28°C and assayed for virus content within 2 weeks of collection. Pans were completely emptied 30 min prior to collection of samples.
Isolation and enumeration of indigenous viruses from effluent and pans
directly under the basins was achieved by a system of sampling pans located at various depths (Fig. 2). These were used to monitor changes in the effluent in the upper soil layers. The aquifer was sampled by 15 monitoring bores located around the recharge basin (Fig. 1). The bores were screened at varying depths to enable sampling of the upper and lower levels of the aquifer. The depth of the aquifer was approx. 32 m. During the first part of the experiment, 3 MI of wastewater were disposed of per day on a 5 day flooding, 9 day drying cycle which was aimed at encouraging denitrification of the effluent in the soil. After 1982, the effluent volume was reduced to 1 M1/day. The direct soakage rate for both periods ranged from initial rates of 9 down to 0.3m/day and averaged 4m/day.
Effluent and pan samples were placed in pre-soaked 50 cm lengths of 76 mm dialysis membrane (Union Carbide Corp., II1., U.S.A.). The dialysis membranes containing the samples were surrounded with flakes of polyethylene glycol 6000 (ICI, Sydney, Australia) and left at 4°C overnight. This process concentrated the sample volume approx. 10-fold. The concentrated samples were inoculated onto 6 different cell cultures; primary monkey kidney cells, a locally derived strain of human diploid embryo lung fibroblasts, LLC-MK2, FL-amion, Vero and RD (human rhabdomyosarcoma cells). The cell cultures were grown in Eagles MEM (Commonwealth Serum Labs, Parkville, Australia) containing 10% v/v foetal calf serum (Flow Labs, Australia), HEPES buffer (Calbiochem, La Jolla, Calif., U.S.A.) to a final concentration of 28 mM and sodium bicarbonate to a final concentration of 0.14%. Antibiotics were added as follows: penicillin (Commonwealth Serum Labs, Victoria, Australia) 0.06 mg/ml; neomycin (Upjohn, Ontario, Canada) 0.05mg/ml; streptomycin (Glaxo, Australia) 0.1 mg/ml. The ceils were maintained in Eagles MEM containing 2% foetal calf serum (FCS). All other ingredients were the same as the growth medium. Quantitation of indigenous viruses in effluent and pans was carried out by the most probable number method as described by Chang (1958), and the concentration was expressed as the most probable number of eytopathic units per litre (MPNCU/1).
Effluent quality
Identification of indigenous viruses
During 1981 and 1982 there were periods when primary effluent was blended with the secondary effluent in a ratio of 70:30 to improve nutrient removal at the recharge site. However, adequate nutrient removal could not be achieved at the recharge site and from 1983 process changes were made at the wastewater treatment plant to encourage
Virus isolates were initially grouped on the basis of characteristic cytopathic effect (CPE) and then further identified by neutralization tests. Enteroviruses were typed using the World Health Organization antisera pools described by Melnick et al. (1973). Adenoviruses were typed in a similar manner to enteroviruses; specific antisera
Fig. 2. Sampling pan system.
Table 1. Et~uent quality 1981-1982 Mean
Conductivity* pH BOD5 Chloride TDSt Organic nitrogen (as N) Ammonia (as N) Nitrite (as N) Nitrate (as N) Total nitrogen (as N) Total phosphorus Colour:~ Turbidity§
86 30 145 450 2.1 15 1.0 3.5 21.6 10.6 88 7.6
Range 67-128 6.2-7.2 10-55 125-255 440-460 I-5 1.9-30 0.05-6.8 0.1-14 15-35 7.7-15 70-110 1-20
1983-1986 Mean
76 5 115 418 1.6 1.6 0.4 6.8 12 9.5 NT¶I NT
Range 75-80 6.9-7.6 5-20 110-125 413-440 1.3-2.2 1.3-2.2 0-1.10 0.5~-16 23-30 9-1 I NT NT
Units in mg/I unless staled. *mS m-~ at 25°C. tConductivity x 5.5. SHazen units. §NTU, ¶lNot tested.
Movement of viruses after artificial recharge (Microbiological Associates, Md, U.S.A.) for types 1-7 were used in the initial attempt to identify an isolate. If these failed, the isolate was tested against other adenovirus antisera (Types 8-30).
Recovery of indigenous viruses from groundwater During the period of 1981-1982, groundwater samples were processed using the filter adsorption-elution technique which was originally described by Wallis et al. (1972). Many difficulties were experienced using this method, (Jansons and Bucens, 1986) and therefore ultraflltration was adopted as an alternate technique. In the latter part of 1982, 201. volumes of groundwater were processed by ultraflltration using an Amicon DC2 (Amicon Corp., Lexington, Mass. U.S.A.).
Procedurefor poliovirus seeding of effluent In 1983, process changes in wastewater treatment greatly reduced the number of human enteric viruses subsequently isolated from the plant effluent. This reduction in virus concentration made it difficult to evaluate further the capacity of the soil to remove indigenous viruses during groundwater recharge. Artificial seeding of the recharge basins with a vaccine strain of poliovirus was then carried out to quantify the virus adsorbing properties of the soil. To avoid any risks to public health associated with the release of large amounts of viable virus into the environment, the commercially available Sabin oral trivalent vaccine (Smith Kline-RIT, Rixensart, Belgium) was selected. The vaccine contained different concentrations of the three serotypes of poliovirus. Each dose (approx. 0.05m l) of undiluted vaccine contained approx. 106 TCIDso poliovirus type 1, l0 s TCIDso poliovirus type 2 and I0 s's TCIDs0 poliovirus type 3. The final concentration of seed virus was estimated to be 4000 MPNCU/l. The contents of the header tank were allowed to flow into the basin and normal dosing with effluent was resumed. The seeding was carried out in the late afternoon to reduce the risk of solar inactivation of the virus and to avoid exposure to the virus to high ambient temperatures. Due to the relatively small volume of seeded effluent the contents of the header tank were allowed to flow into the basins via a system of levee banks which directed the flow of the seeded effluent to an area over the five sampling pans to a depth of approx. 150 ram. Normal dosing with effluent followed the seeded effluent dosing. The basin floor was free from grass and weeds. Prior studies using fluorescein dye revealed an even hydraulic flow into the basin with a uniform distribution of dye in all the sampling pans.
Recovery of seeded poliovirus from groundwater Groundwater samples were processed by an ultrafiltration method using the Amicon DC30 and DC2 as previously described (Jansons and Bucens, 1986).
Assay of seed poliovirus
295
Estimation of bacterial numbers in effluent and pans Estimation of bacterial numbers was carried out by the method recommended by HMSO (1982) except that membrane enriched Teepol broth was replaced with membrane enriched sodium lauryl sulphate as recommended by the JCPHLSSCA (1980). Cultures for faecal coliforms were incubated for 4h at 30°C followed by 14 at 44°C. Bacterial counts were performed using membrane filter techniques on selective media. RESULTS
Movement of indigenous viruses and bacteria beneath the recharge basin The m o v e m e n t of indigenous viruses beneath the recharge basins at the various soil levels was investigated by means of pan samples. During 1981-1982, effluent and pans were sampled twice weekly. However, on a number of occasions, blockages in the pan sampling system prevented sampling. A comparison of the total virus numbers in effluent and pans during this period showed a 2-fold reduction after travel through the first 0.5 m of sand to pan 1 (Fig. 3). Total virus numbers were reduced by a further 3-fold at pan 2 which was located I m below the basin floor after which the virus concentration remained constant for the remaining sampling levels. This indicated that Bassendean Sand showed a limited capacity for the removal of indigenous viruses. The concentration of virus in the effluent peaked at 351 M P N C U / i in December 1981. Penetration of virus through soil generally coincided with virus concentration peaks in the effluent. During this sampling period the movement of faecal coliforms was also investigated. The reduction in bacterial concentration after travel through the first 0.5 m of sand was greater than that found for indigenous viruses with at least a 10-fold decrease in total bacterial numbers in pan l compared with effluent (Table 2), Thereafter, bacterial numbers were constant at the remaining sampling depths.
Distribution of indigenous virus serotypes at different soil levels Differences were observed in the distribution o f enteric viruses at the various sampling levels (Table 3). Echovirus types 11, 14, 24, 29 and 30, coxsackievirus type B4, untyped adenovirus and adenovirus type 3 and untyped enterovirus all showed an ability to penetrate to 3 m into the soil. This contrasted with the lesser penetration of poliovirus, echovirus types 6 and 17, coxsackievirus types B2 and BS, adenovirus type 7 and reovirus. However, these differences may have been due to chance distribution of virus during sampling.
Effluent and pan samples collected during the poliovirus seeding experiment were thawed and extracted with Freon R113 (Daikin, Osaka, Japan) to reduce the level of bacterial contamination. The virus was quantitated using a method described by Joret et al. (1980) in which three replicate sample volumes of 10, 1.0 and 0.1 ml were inoculated into 75, 25 and 25 cm2 flasks, respectively, containing confluent monolayers of Vero ceils. Virus isolation studies prior to the poliovirus seeding experiment indicated that indigenous virus concentrations in the effluent had fallen below detectable limits. Isolation of indigenous viruses from groundwater The concentrated groundwater samples were processed in The penetration of indigenous viruses into the the same manner as effluent and pan samples except that the total sample volume was assayed for virus. The most aquifer was evaluated by sampling of groundwater probable number of cytopathic units (MPNCU)/I. was for the presence of viruses. During 1981-1982 a calculated using the method described by Chang (1958). total of 45 groundwater samples was taken from
JANIS JANSONSet al.
296 EFFLUENT
300,
the shallow bores closest to the recharge basin (1, 2, 3 and 5) and processed using the filter adsorption-elution method. The deep and shallow bores were sampled in the initial experiments but chemical indicators suggested that the effluent plume was localized in the upper aquifer during initial stages of the recharge study (Table 4) and therefore deep bore sampling was discontinued in subsequent experiments. Considerable difficulties were experienced with the filter adsorption-elution method due to interference by humic acids present in the groundwater (Jansons and Bucens, 1986) in these early experiments and therefore failure to demonstrate virus penetration into the aquifer may have been due to the unsuitability of this technique for Canning Vale groundwater. During the same period, thirty 20 1. groundwater samples were processed by ultrafiltration. Echovirus type I 1 was isolated on one occasion from a shallow bore (bore 3) which was located 14 m from recharge basin 4.
351
2ooi
182
100. ;14 40:
42
30, 20, 10
300 ,PAN 1 200,
215
100,
4o!
°ll
I
20,
10, 0
I
I
. . . . . . . . . . .
I Lj 40 PAN 2
20
1
. . . . . . . .
'° 1 0
j . . . . . . .
. . . . . . . .
oLi
30 PAN 4
10
0 . . . . . . . .
Movement of seed poliovirus beneath the recharge basins The total numbers of seed poliovirus were reduced 40-fold after the first 0.5 m of travel through the soil (Table 5). There was evidence of a further reduction in virus concentration below this sampling level with no virus detected in pans 2, 4 or 5, although virus was observed on one occasion in pan 3 at a concentration similar to that found in pan 1. Sampling of pan 5 continued for 156 h after release of virus. Earlier experiments had shown no loss of infectivity of vaccine poliovirus over a 14 h period, and a 10-fold reduction over a 30 h period when suspended in secondary effluent. This would suggest that reduction of poliovirus concentration in pan samples after travel through soil in this experiment was largely due to adsorption. Pans were again sampled at the onset of the first winter rains some 16 weeks after seeding to determine whether polioviruses could be recovered from pan samples. All these samples were found to be negative for viruses indicating either loss of viability or that adsorption of seed poliovirus to the soil could not be reversed by rainfall.
Isolation of seed poliovirus from groundwater ].
.
., I I
40 PAN 5
:1 II ,I 10
o l I
A'S'O'N'D'J'F'M'A'M'J'J'A 1981 1982
J
The penetration of the seeded poliovirus into the aquifer was determined by sampling of groundwater in the bores adjacent to the seeded basin. A total of thirty 400 1. groundwater samples from bores 1-6 was processed by ultrafiltration using the Amicon DC 30 and DC2 (Jansons and Bucens, 1986). All were negative for virus when tested for the presence of the seed poliovirus. Sampling was continued for 12 weeks after poliovirus seeding.
S'ON'D"
Fig. 3. Concentration of indigenous viruses of various soil levels (virus concentration exprcsscd as MPNCU/I).
DISCUSSION
Several factors have now been described which affect the adsorption of virus to soil including soil
M o v e m e n t of viruses after artificial recharge
297
Table 2. Bacterial concentration beneath the recharge basins during 1981-1982 recharge period (faecal coliforms/100 ml) Final effluent
Pan 1
Pan 2
Pan 3
Pan 4
104 > l0 s > l0 s
2.3 x 103 2.3 x 103 1.1 X 103
4
> l0 s
> 105
5 6 7
>105 >105 > 105 > 105
NT >105 > 105 5.4 X 10 3
1.5 x 103 1.3 x 103 8.0 X 102 1 . 7 X 104 6.7 x 103 1.5x 104 3.6 × 103 ST
2.5 x 103 6.3 x 103 9.0 x 103 4.5 X 103 NT 6.0 x 104 5.0 X 103 NT
1.9 x 103 8.4 × 103 2.7 x 103 1.3 X 104 8.0 x 102 3.8 x 103 5.0 × 103 5.2 X 10 3
Sample 1
2 3
8
Pan 5 102 < 102 I(F NT*
6.0 x 102 2.6 x 102 NT 3.2 X 10 3
*Not tested.
Table 3. Number of isolations of indigenous virus serotypes in effluent and at various soil levels Virus serotypes Effluent Pan 1 Pan 2 Pan 3 Pan 4 Pan 5 Eehoviruses Type 6 Type 11 Type 14 Type 17 Type 24 Type 29 Type 30
3 13 2 I 12 . 4
Polioviruses Type 1 Type 2 Type 3 Coxsaekieviruses Type B2 Type 134 Type B5
.
Reovirus Untyped
.
.
.
Upper aquifer
.
--
. 1
.
.
.
. --
.
.
. --
8
6
1
4
--
4
t y p e ( D r e w r y a n d E l i a s s e n , 1968; B u r g e a n d E n k i r i , 1978; G o y a l a n d G-erba, 1979; S o b s e y et al., 1980), s p e c i e s o f v i r u s ( G o y a l a n d G e r b a , 1979), i n f i l t r a t i o n
--
--
1
. .
.
i
3
0.05 0.40 <0.05 0.15 0.65 < 0.05 95
--
.
.
Lower aquifer
.
.
. --
3.45 7.05 0.30 3.85 14.65 < 0.05 I I0
1 1 1
1
--
2
.
--
1 .
.
-1 1
.
.
2 1 .
-2 1
. --
Table 4. Chemical indicators for effluent plume localization during recharge experiments (units in rag/l) Organic nitrogen (as N) A m m o n i a (as N) Nitrite (as N) Nitrate (as N) Total nitrogen (as N) Phosphorus Chloride
.
.
.
1 1
-3 -. --
I
. 1 --
I 6 3 .
1 1 2
--
3 1
Adenoviruses Type 3 Type 7 Untyped Enteroviruses Untyped
-7 2 . 5
. 2
I
2
1
2
--
--
--
r a t e ( V a u g h n et al., 1981), p r e s e n c e o f o r g a n i c m a t t e r ( S c h e u e r m a n et al., 1979) a n d e l e c t r o l y t e c o n c e n t r a t i o n ( L a n c e a n d G e r b a , 1984). The Canning Vale study results show a conside r a b l e v a r i a t i o n in t h e r e m o v a l o f d i f f e r e n t e n t e r i c v i r u s e s b y t h e soil. S e e d e d v a c c i n e s t r a i n p o l i o v i r u s w a s s h o w n to b e s t r o n g l y a d s o r b e d b y t h e soil, w i t h a 4 0 - f o l d r e d u c t i o n in t o t a l v i r u s n u m b e r s o c c u r r i n g in t h e first 0.5 m o f t r a v e l t h r o u g h t h e soil, a n d n o p e n e t r a t i o n b e y o n d 1.5 m . I n c o n t r a s t , i n d i g e n o u s v i r u s e s s u c h a s e c h o v i r u s t y p e s 11, 14, 24, 29 a n d 30, a s well a s c o x s a c k i e v i r u s t y p e B4, u n t y p e d a d e n o v i r u s a n d a d e n o v i r u s t y p e 3, p e n e t r a t e d 3.0 m i n t o t h e soil,
Table 5. Movement of seeded poliovirus through soil. (Virus concentration expressed as MPNCU/I) Time (h) after seeding Effluent Pan 1 Pan 2
0 Neg --
0.1 759 --
0.5 1898 35 --
1.0 2712 35 Neg
--
--
Pan
3
--
--
--
35
Pan Pan
4
--
--
--
5
--
--
--
Neg Neg
1.5 Neg 35 Neg
2.0 Neg 35 Ne8
3.0 Neg Neg Neg
12 Neg Neg Neg
Neg
Ne8
Neg
Neg
Neg Neg
Ne8 Neg
Neg Neg
Neg Neg
36 60 Neg Neg . . . . . . .
.
.
. . . Neg Neg
108 Ne 8 . .
156 Neg
.
. Neg
Neg
298
JANIS JANSONSet al.
with a concentration of virus as high as 37.2 MPNCU/I on one occasion. A single isolation of echovirus type 11 demonstrated penetration of this virus into the groundwater aquifer some 9.0 m below the basin floor. The virus distribution profile for indigenous viruses at the various sampling levels was different from that found for the seeded poliovirus. The reduction of indigenous viruses was less marked, with a 2-fold reduction in concentration occurring after 0.5m. The average virus concentration at the sampling points below 1.0 m remained almost constant. The concentration of seed poliovirus in the effluent was 100-fold greater than the average concentration of indigenous virus found in the effluent, so it can be inferred that the failure of poliovirus to penetrate beyond 1.5 m during the seeding experiment was not related to insufficient concentration of seed virus. The wide variation in the adsorption of indigenous viruses compared with seeded poliovirus may be due to a number of factors. The differences may be based solely on serotypic associated variations of surface charges on enteroviruses. Polioviruses have been previously reported to have a strong affinity for a majority of soils (Goyal and Gerba, 1979). The variation may reflect differences between field isolates and laboratory grown strains. Goyal and Gerba (1979) first noted a variation in soil adsorption characteristics between reference strains of virus compared with recent isolates. More evidence for this hypothesis has been provided by Hodes et al. (1960) who reported variable avidity of virulent (Mahoney) compared with attenuated (LSc, 2ab) poliovirus type 1 onto DEAE cellulose. Similar findings have been reported by Thomssen and Majer (1964). Lance and Gerba (1980) suggested that virus distribution profiles and therefore their soil penetration depths were related to variations in virus charge strength in virus populations. They argued that viruses with a net negative charge below a certain level are immediately adsorbed while viruses with a stronger negative charge move further down the column. Therefore the more uniform the virus population the more compact the virus distribution profile. The results of the Canning Vale study may be consistent with this hypothesis as populations of indigenous viruses excreted by the community would be more likely to be variable than viruses adapted in the laboratory to grow in uniform cell lines. Comparison of the virus distribution profiles for indigenous viruses and seeded poliovirus reveals a far more compact distribution for the laboratory grown virus. Finally, indigenous viruses in treatment plant effluent may be associated with organic materials which may alter their ability to adsorb to certain surfaces. The soil characteristics which increase virus adsorption have been well described. However, several workers have reported difficulties in describing characteristics of a single soil that would
adsorb all viruses efficiently (Drewry and Eliassen, 1968; Goyai and Gerba, 1979). Burge and Enkiri (1978) reported that cation exchange capacity and specific surface area correlated with virus adsorption rates. However, one soil tested which ranked highest in these two characteristics was found to have a poor virus adsorption rate. Similar observations have been made by Drewry and Eliassen (1968). They reported that virus adsorption by soil increases with increasing ion-exchange capacity, clay content, organic carbon and glycerol-retention capacity. However, they described a soil which ranked lowest in these various properties, but proved to have greater adsorption power for viruses than most of the other soils tested. Wastewater contains many virus types which are likely to have heterogeneous properties and may also to be associated with organic material. Therefore, it would be difficult to find a soil which could efficiently adsorb all enteric viruses during groundwater recharge procedures. Virus adsorption by the soil does not necessarily result in virus inactivation, and adsorption has been shown to be reversible after a change in conditions such as the ionic environment (Duboise et aL, 1976; Landry et al., 1979). Hurst et al. (1980) suggested that soils which had a high virus adsorption capacity might in fact favour virus survival. It was concluded that the only method by which a reliable determination of virus removal in the aquifer could be made would be to study the survival of enteric viruses in groundwater. Once the virus survival time and local groundwater flow characteristics are known, safe abstraction distances from the recharge basin may then be determined. A study on enteric virus survival in groundwater has been recently completed by this laboratory. Acknowledgements--The authors wish to thank the Water Authority of Western Australia for financial assistance provided during this study. The authors also acknowledge the assistance of Mr K. Xanthis for the chemical characterization of effluent obtained from Canning Vale and Mr R. Curtis for estimating bacteria numbers in effluent and pans.
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