the application of bacteriophage as tracers of chalk aquifer systems

Journal of Applied Bacteriology 1989, 66, 549-557 2860/07/88

The application of bacteriophage as tracers of chalk aquifer systems

HELEN SKILTON & D. WHEELER* Department of Microbiology, University of Surrey and *Robens Institute of Health & Safety, University of Surrey, Guildford GU2 5 X H , U K

Received 6 July 1988, revised 6 January 1989 and accepted 12 January 1989

SKiL.roN, H. & WHEELER, D. 1989. The application of bacteriophage as tracers of chalk aquifer systems. Journal of Applied Bacteriology 66, 549-557.

Three tracer experiments were performed at two chalk groundwater sites. In the first experiment three bacteriophage were injected at three different depths within a chalk aquifer, 16.5 m from the pumping well. Two of the bacteriophage were recovered from the abstraction point giving a fastest migration rate of between 17 and 34.5 cm/h. One of the bacteriophage tracers, however, was not detected. In the second experiment, three bacteriophage were injected at different depths within a second chalk aquifer site, 1 km distance from the pumping well. A small percentage of original inoculum (0.055 and 0.002%) of two of the bacteriophage traversed the 1 km distance and was detected within 6 months, demonstrating a fastest migration rate of 30 and 29.5 cm/h. At the same site, two bacteriophage were injected into a different borehole, 50 m away from the pumping well. Neither of the two bacteriophage were recovered from the abstraction point. These bacteriophage tracer experiments expose several interesting hydrogeological features about each chalk aquifer system and re-assert bacteriophage as excellent tracers at groundwater sites.

The disposal of wastewater and sewage sludge on land is widespread and has been practised for thousands of years. Sludge production in the UK is in the order of 1 million tonnes of dry solids per year (Bowden 1987) and 39% of it is disposed of on agricultural land either by the direct application of treated or untreated sludge to arable land and grassland or by sub-surface injection (Bruce & Davis 1988). Thirty-one per cent of sludge is disposed of by other land outlets such as landfill sites, reclaimed land and forestry. The third major option for sludge disposal is by the marine environment. Disposal of sludge to sanitary landfills is used for more than 40% of sludge in Europe and about 20% of sludge in the USA and Canada.

There have been a few investigations in the USA of the vertical migration of viruses through soil and the unsaturated zone beneath land application sites. These have been reviewed

Address for correspondence: Dr H.E. Skilton, Part- wick, West End Lane, Henfield, Sussex BN5 9RE, UK.

by Keswick & Gerba (1980). Penetration by enterovirus to depths as great as 67 m has been reported. In several cases human enteric viruses have migrated vertically through the unsaturated zone to reach the groundwater table. Upon reaching the saturated zone horizontal migration may be considerable. For example, Fletcher & Myers (1974) used T4 coliphage as a tracer of water movement in the carbonate rock terrain of southern Missouri and detected bacteriophage approximately 1 mile from the injection site. Enteroviruses such as poliovirus, echovirus and coxsackievirus have also been shown to migrate 250 m vertically through sand and coarse gravel in saturated conditions (Keswick & Gerba 1980). Thus it is conceivable that the application of sewage to land may result in the viral contamination of potable water sources.

Bitton et al (1985) collated 17 published records of viral isolations from groundwater. Those recovered were mostly poliovirus, cox- sackie B virus and echovirus. Slade (1977)

Helen Skilton and D. Wheeler isolated human enteric viruses from raw and partially purified river, stored and well waters. A notable feature of his results was the apparent lack of any correlation between the numbers of bacteria, including Escherichia coli 1, and viruses. However, no viruses were isolated in water from which E. coli was absent. Slade (1985) also isolated human enteric virus from a chalk well but this time in the absence of bacte- riological indicators (coliform bacteria, faecal streptococci and clostridial species). A propor- tion of these viruses survived disinfection treat- ment which consisted of a nominal 1 mg/l of free chlorine maintained for a minimum contact period of 15 min at a pH value of 7.2.

Viruses have also been found in groundwaters suspected of causing outbreaks of disease. A study of the incidence of waterborne disease in the USA (Lippy & Waltrip 1984) revealed that 35.3% of outbreaks from 1946 to 1980 were caused by contaminated or untreated groundwater. Of these 11.8% were of viral origin. In a further 52.1% of the outbreaks, no bacterial agent was demonstrated.

It is of some importance in the development of groundwater protection policy to establish the likelihood of pollution of drinking water supplies from known or potential contaminant sources. However, a significant difficulty associ- ated with the study of viral migration in groundwater is the unique chemical and physical nature of each groundwater site. The main geological components of the aquifer, the hydraulic conductivity of the aquifer material and the age and source of water will all affect the chemical and physical qualities of the water and thus the behaviour of viral contaminants within it. Therefore, at each site many factors may have to be considered in order for a proper assessment to be made of the potential risk of viral contamination of drinking water. Further- more, investigations of the properties of aquifers are difficult and expensive, usually requiring drilling and sophisticated monitoring equipment.

Tracers are used in groundwater investigations with increasing frequency, both for study- ing the direction and velocity of the groundwater and of any potential contaminant within it. Of these tracers bacteriophage appear to be the most promising microbiological agents because of their size, ease of assay and lack of pathogenicity (Keswick et al. 1982). Clearly,

they are particularily suitable for indexing the risk of viral contaminants. Bacterial viruses have previously been used successfully at several groundwater sites (Martin & Thomas 1974; Noonan & McNabb 1979; Skilton & Wheeler 1988 ; J. Watkins, personal communication). We describe here experiments conducted using four bacteriophage at two chalk groundwater sites.

Materials and Methods

Four bacteriophage tracers were used: Serratia marcescens bacteriophage; Enterobacter cloacae bacteriophage; and two Escherichia coli bacteriophage, K12 and MS2 coliphage. They were prepared and donated by the Yorkshire Water Authority and assayed by methods we have described previously (Wheeler et al. 1988).

S A M P L I N G

At both experimental sites, the water abstraction point had a sample tap which allowed a small volume of the water pumped from the aquifer to be collected for analysis. An automatic sampler (Warren Jones Surveyor Sampler) was connected to the sample tap over- flow and set to take samples at required inter- vals. At the first site (Site A) water samples were taken from the injection piezometers, and at the second site (Site B) water samples were taken from the injection boreholes, where bacteriophage were introduced. This was done using flow-through depth samplers.

All samples were stored in sterile 25 ml screw- capped bottles at 4°C until they were examined for plaque-forming units (pfu).

SITE A

The intergranular permeability of the chalk at site A is low (10-3-10-4 m/d) with a porosity of 3040%. Pumping tests and dye dispersion tests have demonstrated that groundwater flow is mainly horizontal and occurs in discrete horizons which are confined between layers of lower Permeability. Groundwater flow is greatest in the upper 20-40 m of the saturated zone. To minimize pollution by nitrates the pumping borehole was lined with 20 cm diameter steel casing to a depth of 75 m to exclude the upper horizons of groundwater high in nitrates.

0

20

40

60

00

100

I20

Bacteriophage tracers in chalk aquifers Abstract ion point

Woter level

Piezorneters

16.5 rn Fig. 1. Site A. Not to scale.

551

115rn

Two observation boreholes had been drilled 16.5 m from the water abstraction point to monitor changes in groundwater quality and flow with depth in the aquifer. In each borehole four 5 cm diameter piezometers were installed at several depths. The water level was 32-39 m. This unique arrangement (Fig. 1) provided an ideal opportunity to perform a bacteriophage tracer experiment on the site at the same time as investigating the efficacy of the steel lining on the pumping station. The pumping borehole is 30 cm in diameter and 119 m deep. The pump extracted water at a rate of 3 x lo5 ml/min for the duration of the experiment. During the experiment the water was of good microbiological and chemical quality providing potable water to a surrounding population.

E X P E R I M E N T 1

The three bacteriophage were injected into three piezometers at different depths. Approximately 1 x 1014 pfu of Ent. cloacae bacteriophage were injected into a piezometer 53 m in depth; 1 x 1014 pfu of K12 coliphage were injected into a piezometer 75 m in depth; and 9 x lot4 pfu of S. marcexens bacteriophage were injected into a 115 m depth piezometer. In each case, the method of injection was simple; tracer was introduced into the piezometers through a funnel which was subsequently flushed with

approximately 1 1 of groundwater. Samples were taken from the pumping station at regular inter- vals for a period of 6.5 weeks. Samples were also taken from within the piezometers for examination after injection of tracer bacteriophage.

S I T E B

This study area lies in a chalk river valley. Drift deposits consisting of sands, clays and gravels some 2-7 m thick overlie middle lower chalk. Gault clay forms an impermeable base to the chalk aquifer. The chalk aquifer is unconfined and the water-table fluctuates within the drift deposits. The drift probably acts as a minor aquifer although the quantities of water trans- mitted in comparison to those within the chalk would be very small. Chalk porosity is 3 M O % . Geophysical logging of two boreholes 1 and 2 (Fig. 2) indicated that major groundwater movement is restricted to the upper two-thirds of the aquifer. Groundwater flow is in a north-western direction and velocities are assumed to be in the range 1&20 m/d. The water at this site was not required for potable use but, nevertheless, water was being pumped at a rate of 2.3 x lo6 ml/min. Both boreholes were 10 cm in diameter. Borehole 1 was approximately 1 km distant from the abstraction point whereas borehole 2 was only 50 m distant.

552 Helen Skilton and D. Wheeler I km

Pumping Borehole station no. I

Borehole no. 2

Fig. 2. Site B. Not to scale.


The three bacteriophage were injected into borehole 1 by a 25 mm hose pipe followed by thorough flushing. Each bacteriophage was injected at a different depth. Approximately 1 x 1014 pfu of Ent. cloacae bacteriophage were injected at a 15 m depth within the borehole; 9 x 1014 pfu of S. marcescens bacteriophage were injected at a 38 m depth; and 1 x l O I 4 pfu of K12 coliphage were injected at a 44 m depth. Samples were taken from within this borehole after bacteriophage injection on two separate occasions. Samples were taken by automatic

sampler every hour and composited every 4 h over a period of 6 months.


Approximately 4 x 10” pfu of MS2 coliphage were injected into borehole 2 at a depth of 15 m. The sampling regime was the same as in Experi- ment 2.

Five 1014 pfu of S . marcescens bacteriophage were also injected into this borehole at a depth of 15 m. In this case, samples were taken from the pumping station every 5 min after injection for 2 h and then every 10 min for 1 h.

Results

By calculating the number of pfu/ml and the overall volume of water extracted by the pumping station, an estimate of the total numbers of bacteriophage migrating through the chalk aquifer as well as the rate of migration and pattern of recovery could be made. By calculating the volume of water within the observation boreholes and relating this to the number of pfu/ml at different depths an estima- tion of the total number of bacteriophage remaining in the boreholes could also be made.

Time ( d )

Fig. 3. Experiment 1 (Site A). Bacteriophage remaining within injection piezometers. A, Serratia marcescens bacteriophage; W, Enterobacter cloacae bacteriophage; 0, K12 coliphage.

S I T E A ( E X P E R I M E N T 1)

The results of analysis of piezometers used for the injection of bacteriophage show that a small amount of the tracers remained at the injection

Bacteriophage tracers in chalk aqugers 553 point for some time (Fig. 3). After 13 d, 0.13% of S. marcexens bacteriophage, 2% of K12 coliphage and 4% of Ent. cloacae bacteriophage tracer still remained. These numbers do decrease with time over the sampling period, in particular S. marcescens bacteriophage.

Serratia marcescens bacteriophage and K 12 coliphage succeeded in migrating to the abstraction point. The first arrival occurred between 47 and 101.5 h (a gap of 54.5 h in sampling occurred due to equipment failure), rep- resenting a fastest migration rate of between 17 and 34.5 cm/h (Table 1). A higher percentage of the S . marcescens bacteriophage injected at 115 m depth was recovered than for the K12 coli-

Table 1. Fastest migration rates observed at Site A and Site B groundwater sites

Experiment 1 Experiment 2 Bacteriophage (Site A) (Site B)

Serrat ia

Escherichia

host (cmih) (cmP)

marcescens 17-34.5 29.5

coli K12 17-34.5 30

phage injected at 75 m depth (0.16% and 0.01% respectively) (Table 2). The pattern of recovery for the two bacteriophage suggests that peak numbers were detected for the S . marcescens bacteriophage but may have been missed for the K12 coliphage due to omission of sampling (Figs 4 & 5). The Ent. cloacae bacteriophage injected at 53 m was not detected.

SITE B ( E X P E R I M E N T S 2 A N D 3)

Three bacteriophage were injected into the aquifer at a point 1 km away from the abstraction well (Experiment 2), and on a separate occasion two bacteriophage were injected by a borehole only 50 m away from the abstraction point (Experiment 3). Positive results were obtained at the abstraction point for two of the bacteriophage introduced into borehole 1, but none were detected for the bacteriophage injected via borehole 2 (Table 2). The two bacteriophage detected were the S. marcescens bacteriophage, which was injected at 38 m within borehole 1 and the K12 coliphage, injected at 44 m. The Ent. cloacae bacteriophage, injected at 15 m, was not detected

Table 2. Percentage of original inoculum of bacteriophage recovered from Site A and Site B abstraction points

Experiment 1 Experiment 2 Experiment 3 Bacteriophage (Site A) (Site 8) (Site B)

host (Yo) (%) (X)

Serratia marcexens >0.16 0.002 0 Escherichia coli K12 >0.0104 0.055 ND Enterobacter cloacae 0 0 ND E . coli MS2 ND ND 0

ND, not done.

Time ( d ) Fig. 4. Experiment 1 (Site A). Recovery pattern of Serratia marcescens phage, injected at a 115 m depth, 16.5 m from abstraction point.

554 Helen Skilton and D. Wheeler

0 10 20 30 40 50 Time (d)

Fig. 5. Experiment 1 (Site A). Recovery pattern of K12 coliphage, injected at a 75 m depth, 16.5 m from abstraction point.

(Table 2). The first bacteriophage to be recovered at the abstraction well was the K12 coliphage (Fig. 6) after 135 d. This tracer had a migration rate of 30 cm/h (Table 1) and 0.055%

of total K12 coliphage was recovered (Table 2). The S. marcescens bacteriophage was detected after 141 d (Fig. 7) Only 0.002% was recovered, and its fastest migration rate was 29.5 cm/h.

Time (d) Fig. 6. Experiment 2 (Site B). Recovery pattern of K12 coliphage, injected at a 44 m depth, 1 km from abstraction point.

Time (d)

Fig. 7. Experiment 2 (Site B). Recovery pattern of Serratia marcescens phage, injected at a 38 m depth, 1 km from abstraction point.

Bacteriophage tracers in chalk aquifers 555

u . 0

- -15 12 20 30 40 50

Depth (rn) Fig. 8. Experiment 2 (Site B). Bacteriophage remaining within injection borehole after 220 h. ., Serratia marcescens bacteriophage; A, Enterobacter cloacae bacteriophage; 0, K12 coliphage.

Samples were also taken from injection borehole 1, 212 and 220 h after introduction of the tracer. At both times a similar pattern of bacteriophage recovery occurred. Figure 8 shows bacteriophage remaining within the borehole after 220 h. Only very small percentages of original inoculum were observed; higher numbers of S. marcescens and Ent. cloacae bacteriophage were found at the lower depths and higher numbers of K12 coliphage were found at the higher depths.

In Experiment 3, two tracers were injected into borehole 2 on two separate occasions. The MS2 coliphage was injected and the pumping well water sampled thereafter every hour for 5.5 months. The S . marcescens was injected at a much later date and sampled at the abstraction well every 5-10 min for 3 h. They were both introduced into the aquifer at a 15 m depth but neither was detected at the abstraction point.

Discussion

Bacteriophage were observed to migrate through chalk at two groundwater sites.

In Experiment 1, three bacteriophage were injected, 16.5 m from a pumping well, at different depths within the aquifer. Two of the bacterial viruses migrated the distance; one bacteriophage, however, that of Ent. cloacae was not detected. This was expected as the bacteriophage was injected at a depth of 53 m within the aquifer and the abstracting borehole has a steel lining down to 75 m in order to

exclude groundwater from these upper horizons because of high nitrate concentrations. It would appear that in this case the steel casing was effective. Horizontal layers of lower permeability within the aquifer prevented vertical movement of the tracer at this close distance (16.5 m from abstraction well). Unfortunately no conclusions can be drawn about the hydrogeological conditions at depths of 75 and 11 5 m because of omission of samples.

A small percentage of the original inocula remained in the injection piezometers for several days. This indicates the absence of a strong hydrological gradient around the piezometer drawing the bacteriophage into the main stream of groundwater. However, the majority of bacteriophage did escape from the piezometer into the surrounding aquifer and so the portion remaining would not necessarily have a significant effect on the percentage of original inoculum recovered from the abstraction point.

In Experiment 2, three bacteriophage were injected at a distance of 1 km from the abstraction point. Two of the three bacteriophage were detected, the Ent. cloacae bacteriophage, injected at a 15 m depth, was not detected.

Injection at different depths within borehole 1 did not guarantee that the tracer would only enter the aquifer at this depth. Unlike Experi- ment 1, where the piezometers were of different lengths (thus preventing the bacteriophage from entering the rock bed from lower depths), tracer injected within this borehole may well move up or down before entering the aquifer system. It appears from the injection borehole results (Fig. 8), that the S. marcescens bacteriophage (38 m) and the Ent. cloacae phage (15 m) tracers followed a similar route within the borehole, i.e. downwards. Conversely, the K12 (44 m) coliphage left very low numbers at the greater depths and the majority of the remaining bacteriophage was found in the upper levels. Poss- ibly, the chalk was more permeable at 44 m. When K12 coliphage was injected at this point the majority was drawn, therefore, through to the aquifer immediately. In contrast, the other two tracers had to depend on movement within the borehole to allow them to come in close contact with a suitable access point to the aquifer. When used in the field, tracers are mixed with glycerol. Before this is diluted within the aquifer it adheres to the tracer and increases

Helen Skilton and D . Wheeler its density. It is, therefore, probable that the reason for the high numbers of S. maicescens bacteriophage and Ent. cloacae phage observed at the lower depths is this greater density. The Ent. cloacae bacteriophage was injected at 15 m, 29 m above the depth of K12 injection (44 m). This slower route of entry into the aquifer and thus greater dilution, may explain its non- recovery. Alternatively it could be due to certain characteristics of the Ent. cloacae bacteriophage which prevented it from migrating to the abstraction well in sufficient quantities to be detected.

In Experiment 3, neither of the two tracers, injected at a distance of 50 m from the abstraction point, were recovered. This suggests that, even though there must be considerable pressure on the groundwater within this area to move towards the pumping station, there is no connection between borehole 2 and the abstraction well, 50 m away. It is possible, although extremely unlikely, that the peak of bacteriophage reached the abstraction site but was not sampled at the appropriate time, i.e. if the entire detectable peak of bacteriophage arrived after 3 h over a period of less than 1 h. It is expected, however, that after a delay of 3 h, dispersion, diffusion and adsorption affects within the aquifer would cause the tracer to arrive over a substantially longer period than 1 h (as in all previous experiments) and so would be detected.

In both Experiments 1 and 2, the tracer not detected was the same, i.e. the Ent. cloacae bacteriophage. It is possible that this is due to an inherent feature of this bacteriophage, even though in three previous experiments (Skilton & Wheeler 1988) the Ent. cloacae bacteriophage behaved similarly to the K12 coliphage and the S. rnarcescens bacteriophage, under identical conditions. Another explanation is the influence of depth of injection within the aquifer on transport and recovery of tracer. This was especially true for Experiment 1.

The bacteriophage tracers revealed important features about both chalk aquifer sites. Experi- ment 1 at Site A exposed the influence of a steel casement lining the pumping well to a depth of 75 m. In Experiments 2 and 3 at Site B, a network of connections extending over a distance of 1 km but not 50 m, was confirmed.

This investigation has highlighted the adapt- ability and suitability of bacteriophage as

tracers at chalk groundwater sites. The high titres of tracer introduced allowed detection of bacteriophage even after massive dilution and transport over long distances and long periods of time. The long-term stability of the bacteriophage (Skilton 1987) permitted detection even after 6 months in the environment, and the specificity of the bacteriophage to their bacterial host allowed all three to be included in his investigation without adversely affecting each other. Finally their non-pathogenicity allowed the bacteriophage to be used as tracers at groundwater sites while the water was actually supplying the local population.

This work was funded by a grant from the Natural Environmental Research Council. Special thanks are due to the British Geological Survey and the U K water industry for their valuable assistance. The authors are particularly grateful to the Yorkshire Water Authority for the supply of bacteriophage tracers.

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the application of bacteriophage as tracers of chalk aquifer systems

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