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a Landcare Research, Private Bag 3127, Hamilton, New Zealand
b Montana Microbiological Services, P.O. Box 4570, Bozeman, MT 59772
* Corresponding author (mcleodm{at}landcare.cri.nz)
Received for publication October 3, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: PV, pore volume • TSB, Tryptic Soy Broth • PFU, plaque forming units • BTC, breakthrough curve
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Geologically New Zealand is very young and mobile with numerous volcanoes resulting in a diverse landscape with a wide range of contrasting soil types occurring over short distances. There is a paucity of data on the fate of the microbial loads applied to New Zealand soils. Wells (1973) rated the properties of New Zealand soils in relation to effluent disposal and concluded that fine-grained soils from young (2000–7000 yr old) tephra had the ideal combination of soil properties for effluent disposal. However, there were no supporting experimental data. A study of coliforms in leachates from soil cores irrigated with secondary treated municipal effluent by Childs et al. (1977) is difficult to interpret as issues of edge-flow, inadequate core diameter (150 mm), and the use of some repacked cores were not addressed.
In a large field study, Sinton et al. (1996) studied movement of fecal coliforms and F-RNA coliphages through an alluvial aquifer. They reported percolation rates through the 13-m vadose zone 1.4 to 3.4 times greater for the bacteriophage, when compared with bacteria. At a nearby site, Pang et al. (1998) studied rhodamine WT and Bacillus subtilius endospore transport along preferential flowpaths within a gravel aquifer. The B. subtilis endospores exhibited greater transport velocities compared with the dye. This was attributed to the endospores being excluded from small pores where flow velocity was lower.
Given this history of little information on the transport of microbes through different soils, we examined the simultaneous fates of bacteriophage and Br- tracer applied to large (500 mm diam. by 700 mm high), undisturbed lysimeter soil cores of typical North Island New Zealand soil types commonly used for effluent disposal. Tracer solution was applied as a pulse followed immediately by rainfall applied via an overhead rainfall simulator.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Netherton soil (Grid ref. NZMS 260 T13 453 247) (Typic Endoaquept [Soil Survey Staff, 1996]). The clayey Netherton soil (McLeod, 1992a) developed in estuarine alluvium has coarse prismatic subsoil structures with low porosity within the prisms. Organic matter often lines structural cracks. Topsoils have medium or coarse polyhedral structure. The soil has mixed clay mineralogy.
Atiamuri soil (Grid ref. NZMS 260 U18 809 780) (Typic Udivitrand [Soil Survey Staff, 1996]). The Atiamuri soil is developed in sandy rhyolitic tephra grading into massive but porous, welded pumice flow tephra at about 40 cm depth (Rijkse 1992), which is inimical to root development.
Waihou soil (Grid ref. NZMS 260 T15 476 635) (Typic Hapludand [Soil Survey Staff, 1996]). The Waihou soil developed in rhyolitic tephra has allophanic clay mineralogy (McLeod, 1992b) with variable charge, and has some positive surface charge at typical field pH.
Waitarere soil (Grid ref. NZMS 260 S25 965 694) (Typic Udipsamment [Soil Survey Staff, 1996]). The young Waiterere soil developed in dune sand (Cowie and Rijkse, 1977) has minor pedological development with single grain structure throughout, except for some weak aggregation in the topsoil.
Lysimeters
Three replicate, undisturbed lysimeter cores 500 mm diameter by 700 mm high from each soil were used in this study. Soil lysimeters from farm paddocks were hand carved in situ and the lysimeter casing progressively pressed down over the soil monolith. The lysimeters had a 10-mm internal annulus filled with petroleum jelly to prevent water preferentially flowing at the soil–casing interface (Cameron et al., 1992). A sampling port was installed in the center of the base of the lysimeter to allow collection of leachate. The pore volume (PV) for each set of lysimeter cores was established from the same sites by extracting replicate undisturbed soil cores (55 mm diam. by 30 mm high) from each soil horizon and determining total porosity (Gradwell, 1972).
Virus Propagation and Assay
The bacteriophage Salmonella typhimurium 28B (Lilleengen, 1948) (size ca. 50 nm) was used as a tracer in this study. The phage, grown overnight on its host strain S. typhimurium Type 5 in Tryptic Soy Broth (TSB) (Difco) at 37°C, was isolated by chloroform lysis of the bacterial host then passed through a 0.45-µm mixed cellulose ester-based membrane filter to remove cell debris. To obtain a clean virus preparation free of organic material, the filtrate was centrifuged at 25000 x g (Sorval T21) for 2 h at 4°C, the supernatant was poured off, and the phage resuspended in 1 to 2 mL of phage storage buffer (Sambrook et al., 1989) and stored at 4°C until required. Phage stocks were enumerated using the soft agar overlay method, which resulted in plaque forming units per milliliter (PFU/mL). Leachate samples (1 mL), diluted in phosphate buffer (pH 7.0) as required, were mixed with 0.5 mL of a 4-h host-strain culture in 8 mL molten soft-agar and poured onto nutrient agar plates (Difco). After 18 to 24 h incubation at 37°C, well-formed, clear plaques were counted and reported as PFU/mL. Each reported phage concentration is the average of three replicate soft agar enumerations.
Samples of dairy-shed effluent and lysimeter leachate before tracer application showed no indigenous phages in 1-mL samples capable of forming plaques on S. typhimurium Type 5 host strain.
Bromide Analysis
Bromide concentration in the leachate samples was measured using an ion selective electrode (Metrohm 6.0502.100 Switzerland).
Leaching Experiments
Lysimeters were irrigated with tap water for 2 d to bring the cores to field saturation with leachate emanating from the sampling port, then allowed to drain for 7 d before application of the bacteriophage/Br- tracer solution. Seven days is similar to the return period commonly used at effluent irrigation sites. Each lysimeter was irrigated (30-mm depth of application) with tracer solution containing bacteriophage (109 PFU /mL) and Br- (2000 ppm) at 5 mm h-1. The lysimeters were then irrigated continuously with water at a rate of 5 mm h-1 using a drip-type rainfall simulator with drippers spaced on a 20-mm triangular grid approximately 170 mm above the soil surface. A diagram of the lysimeter and irrigation setup is shown in Fig. 1
. Background levels of host-specific Salmonella bacteriophage and bromide (Br-) in leachate were determined from samples taken at the end of the wetting-up period. Determination of any ponding during application and subsequent irrigation was by visual observation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Recoveries of applied Br- were 68 and 72% at 1.1 PV for the two lysimeters that did not have surface ponding, and 46% for the ponded lysimeter. Recoveries of bacteriophage were 12 and 38% for nonponded lysimeters, and 6% for the ponded lysimeter.
Atiamuri Lysimeters
Bromide tracer replicates peaked in a range between about 0.6 to 0.7 pore volume, but had a relatively symmetric BTC, suggesting matrix flow is the dominant flow regime (Magesan et al., 1999a) (Fig. 2B). The bacteriophage tracer was present at very low levels in all of the replicates' leachates. The pattern of bacteriophage tracer occurrence in leachates from the lysimeters was irregular, with some samples containing only a few virions, followed by other samples where the tracer was not detected. The peak bacteriophage C/Co ratio detected in the leachate was 9.4 x 10-12 at 0.9 PV.
Recoveries of applied Br- ranged between 87 and 91% at 1 pore volume, with <0.15% of the bacteriophage tracer recovered from each lysimeter.
Waihou Lysimeters
The bacteriophage tracer was not detected in leachate samples collected to about 1.8 PV for Waihou lysimeters (Fig. 2C). All Br- BTCs were relatively symmetrical, indicating a predominance of matrix flow over bypass flow. The BTCs all peaked at about 1.3 pore volumes, suggesting some retardation of the Br- tracer.
Recoveries of applied Br- ranged between 78 and 95% at 1.8 pore volumes.
Waitarere Lysimeters
In the sandy Waitarere soil, the Br- tracer was present in the leachate at maximum levels of about 30 to 40% of application concentration early on in the BTC, at about 0.2 pore volumes (Fig. 2D). As in the Netherton soil, this early high peak and long tail is typical of conditions where bypass flow is significant.
The bacteriophage tracer showed similar flow characteristics to the Br- tracer. Bacteriophage peaked early at about 0.3 to 0.4 pore volume with peak concentrations of approximately 4% of the application concentration. Phage tracer values then progressively decreased to about 0.001% of the application concentration when sampling was discontinued (Fig. 2D).
Recoveries of applied Br- ranged between 89 and 107% at 1.4 pore volumes, while recovery of the bacteriophage tracer ranged between 2 and 9%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The early peak and long tail to the BTCs of microbes and Br- on the Netherton and Waitarere lysimeters are indicative of bypass flow, although actual flow mechanisms are likely to be different. The clayey Netherton soil has large structural cracks lined with organic matter and clay cutans, as well as large bio-pores (worm holes and root channels). Natusch et al. (1996) demonstrated that microbes can rapidly be transmitted to depth via macropores such as these. The large difference between Ksat and K-40, and the slow K-40 value in the Netherton subsoil (Table 1) indicates preferential flow is likely (Magesan et al., 1999a). Gerba and Bitton (1984) indicate it is generally agreed that fine-grained soils retain viruses more efficiently than sandy soils since the clay mineral fraction has a greater surface area and ion-exchange capacity. However, this must be tempered by the physical soil structure and the ability of percolating waters to hydrodynamically interact with the soil particles. In the case of the Netherton soil, large soil cracks rapidly transmit percolating waters. Consequently there is little interaction with much of the fine-grained soil matrix.
In contrast to the clayey Netherton soil, it is common for young dune sand soils such as the Waitarere soil to be water repellent (Wallis et al., 1991), and consequently develop finger-flow pathways through the soil (Bauters et al., 1998) with Br- transport velocities up to three times faster than estimated piston flow velocities (Ritsema and Dekker, 1996). This different flow mechanism is also suggested in this study by the concentrations of the bacteriophage tracer in the leachates. The Netherton soil structure suggested predominantly inter-pedal flow, whereas in the Waitarere soil flow is likely to be through areas of wet sand. However, even with strong preferential flow characteristics, after 1 PV both Netherton and Waitarere soils continued to shed large numbers of the bacteriophage tracer, about 106 PFU/mL in the Netherton and about 105 PFU/mL in the Waitarere soil.
If all binding sites within a soil become saturated with virus, the result can be the early arrival of a virus tracer peak. This mechanism is referred to as blocking, and initially we hypothesised that this mechanism might have caused the early arrival of the bacteriophage peak in the Waitarere soil. However, the Waitarere soil contains sand, silt, and minor clay, and is expected to have considerably more binding sites available than Ottawa sand, a uniform 0.6 to 0.8 mm quartz sand that Moore et al. (1981) concluded has an enormous capacity for virus sorption (2.2 x1012 PFU/kg) and is unlikely to become saturated. Consequently, for the Waitarere soil, it is unlikely that the early peak in the bacteriophage tracer (Fig. 2D) was a result of blocking. Collected leachate was light yellow, presumably from dissolved humic substances that may play a role in mobilizing viruses (Sobsey et al., 1980). In the Waitarere soil, we suggest elution of the virus by dissolved organic matter combined with finger-flow characteristics allowed leaching of large numbers of the bacteriophage tracer. Although organic C plays an important role in predicting virus removal (Schijven and Hassanizadeh, 2000), we believe that because the C values in the topsoils of the soils we studied are similar (Table 1) and our soils had very different physical and mineralogical properties, C was not the primary factor influencing the difference in leaching of viruses from these soils.
Generally the Br- tracer peak arrived before the bacteriophage tracer peak. This was not the case for one Netherton lysimeter, where the bacteriophage peak arrived before the Br- peak (Fig. 2A). Elsewhere, earlier arrival of microorganisms over chemical tracers has also been observed and attributed to pore-size exclusion (Sinton et al., 1996; Harvey, 1997; Pang et al., 1998). Pore-size exclusion is where bacteriophage tracer is restricted to larger pores because of its physical size, and in these larger pores flow velocity is faster. Because the Br- ion is smaller, it is able to flow through a wider range of pore sizes, with consequent lower overall transport velocity.
Results from the allophanic Waihou soil, where no bacteriophage was detected, were not unexpected (Fig. 2C). The soil has a fine polyhedral structure throughout, and McLeod et al. (1998) demonstrated only minor bypass flow of pyranine dye tracer on similar allophanic soils under slow overhead irrigation rates. The difference between Ksat and K-40 values indicates this soil has a uniform pore-size distribution and bypass flow is less likely (Magesan et al., 1999a). Amorphous allophanic clays have a high isoelectric point of pH 6.0 (Cooper and Morgan, 1979), and Waihou soils have a topsoil field pH typically less than pH 6, where allophane possesses a net positive surface charge. Because the allophane and bacteriophage possess opposite net surface charge, adsorption should be facilitated. Gerba (1984) considered soils with a high isoelectric point better virus absorbents than those with low isoelectric points. Furthermore, allophane has a very large surface area (700000–900000 m2/kg). Moore et al. (1982) have shown virus sorption to be greater in soils with higher specific surface areas.
Retardation of the Br- peak for the allophanic Waihou soil is attributed to adsorption of Br- onto positively charged sites (Fig. 2C). Retardation has been observed by Church (2000) in similar allophanic soils, and by Brooks et al. (1998) onto ferrihydrite, which can also exhibit positive surface charge.
The early arrival of the relatively symmetrical Br- peak in the pumiceous Atiamuri soil was unexpected (Fig. 2B). The irrigation rate of 5 mm h-1 is relatively slow compared with the saturated hydraulic conductivity of the slowest conducting soil layer in the profile of about 30 mm h-1. This, combined with the Ksat/K-40 ratio and fine polyhedral soil structure, suggests bypass flow would not be dominant. Given the lack of weathering and particularly the welded nature of the flow tephra in the subsoil, we hypothesise that not all the pore volume was contacted by the leachate, with some of the pumice fragments acting as impermeable stones. We suggest this physical exclusion is more likely than anion exclusion. An improved method of sampling for total porosity using larger volume cores to accommodate larger pumice fragments may be appropriate for these soils. Although the bacteriophage replicates show minor differences in flow characteristics, numbers of bacteriophage in the leachate were extremely low compared with the application concentration. The uppermost Atiamuri subsoil layer showed a weak Fieldes and Perrott (1966) reactive-aluminium test reaction. This indicated the presence of minor amounts of reactive hydroxy–aluminium groups, which occur, for example, in allophane, and affects virus sorption. This may provide an explanation for the high virus sorption (low leaching) apparent in the Atiamuri soils.
The BTCs in this study were generated by continuous simulated rainfall that does not replicate the sorption–desorption that may take place under irregular natural rainfall. Even so, differences in the bacteriophage BTCs between soil classes gives us useful information regarding the relative abilities of various soils typically used to renovate surface-applied effluent before it reaches shallow ground water. From the bacteriophage tracer recovery data, we judge it is likely that at least three of these soil classes (Netherton, Atiamuri, Waitarere) will continue to shed the bacteriophage tracer for some time. With increasing pressure for land application for the disposal of effluent, we may see increased application onto less suitable land in the future, with consequent declines in leachate quality. This poses risks for chemical and biological contamination of associated ground water resources.
We concur with Wells (1973) that young, fine-grained tephric soils have the best characteristics for effluent disposal. We would also include Pumice soils that have similar properties to those we examined. Wells (1973) determined that Gley Soils were unsuitable for effluent disposal because of anaerobic conditions. Although we concur with this general conclusion, we attribute the poor performance in our study to significant bypass flow.
While one may reach general conclusions regarding virus movement through soils using indicator bacteriophage tracers (ST5, coliphages f2, T4, 
X 174, etc.), it should be emphasized that many factors influence viral adsorption and transport through soils (Gerba, 1984; Gerba and Bitton, 1984). These include soil pH, virus type and isoelectric point(s), water chemistry (conductivity and organic matter), saturated vs. unsaturated water flow, downward flow rate, and virus survival. However, as results presented here illustrate, the use of bacteriophage tracers is preferable to chemical tracer dyes, which may be mutagenic, and may not be indicative of virus movement (Nestmann et al., 1980). Indeed, in this study, Br- and bacteriophage movement was similar in only two of four soils (Netherton and Waitarere). Thus, with further work on its survival and general characteristics, the Salmonella phage used in this study may prove useful as a general nonpathogenic, simple to assay, tracer of the retention in soils of similar types of viruses. Furthermore, the general shape of the breakthrough curves for this bacteriophage were similar to those of fecal coliforms and fecal enterococci generated after application of dairy farm effluent on the same soils (Aislabie et al., 2001).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Large numbers of a bacteriophage tracer rapidly reached the subsoil of a well-structured clayey Gley Soil, and a sandy Recent Soil (Fig. 2A and 2D). In contrast, under identical application and irrigation conditions, the bacteriophage tracer was not detected in leachate from an Allophanic Soil, and only minor amounts detected in leachates from a Pumice Soil (Fig. 2B and 2C).
Results of this study have useful implications when sites for land disposal or treatment of effluent, including municipal and dairy shed wastes, are being considered. The BTC studies on large, intact cores are useful as one of the procedures for defining suitability criteria of a particular soil for land disposal of effluent. In the future, regionalization of this type of data may be accomplished using a pedotransfer function.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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