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a Department of Biological and Environmental Engineering, Riley-Robb Hall, Cornell University, Ithaca, NY 14853
b INRA, Unité d'Agronomie de Laon-Peronne, 02007 Laon Cedex, France
c USDA-ARS, Campbell Natural Resource Conservation Center, Watkinsville, GA 30677
d Department of Microbiology, Cornell University, Ithaca, NY 14853
e Laboratory of Geoenvironmental Engineering and Science, Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853
* Corresponding author (tss1{at}cornell.edu).
Received 27 June 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: AWS, air–water–solid • PBS, phosphate-buffered saline
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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During the past two decades, the presence of C. parvum in surface- and groundwaters in the United States and Great Britain (Galbraith et al., 1987; Rose et al., 1991; Craun et al., 1998) has been associated with several major outbreaks of cryptosporidiosis (Hayes et al., 1989; MacKenzie et al., 1994). Among the different pathways for the transport of oocysts to drinking water sources, downward percolation is usually considered to be insignificant, because soils are generally assumed to be an effective filter for a wide range of pathogens. Studies of packed columns with saturated flow by Brush et al. (1999) and Harter et al. (2000) and undisturbed columns with unsaturated flow (Mawdsley et al., 1996), however, showed that C. parvum oocysts could be transported rapidly downward through the soil. Although transport of C. parvum oocysts in saturated flow has been studied experimentally and described mathematically (Brush et al., 1999; Harter et al., 2000), detailed observations of the transport and persistence of C. parvum oocysts in unsaturated soils with preferential flow are still lacking, particularly in the presence of preferential flow processes.
In this context, the overall objective of the present research was to investigate and model the transport of C. parvum oocysts through preferential flow paths in the vadose zone. Both fingered flow and macropore flow were investigated under simulated steady-state rainfall at rates much less than the saturated conductivity. The experiments represented a worst-case scenario where feces of calves containing C. parvum oocysts were applied during rainfall at the soil surface.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In Exp. I and II, the columns consisted of 50 segments of Plexiglas tubing (14-cm i.d.), 1 cm high, held together by two large metal frames and secured by four hose clamps that were tightened to keep the rings in place (Fig. 1) . At the bottom, the effluent flowed through a metal screen with a 0.5-mm mesh drop-through funnel into a sample container located immediately below. Sand was poured into the columns and packed to uniform density using a vibrator. In Exp. I, the columns contained industrial-quartz 12/20 silica sand (Union Corp.) with an average particle diameter of 1.1 mm. For Exp. II, the same 12/20 sand was used but made hydrophobic to different degrees by mixing in different portions of strongly water-repellent sand that was made by coating the regular 12/20 sand with octadecyltrichlorosilane according to the method described by Bauters et al. (1998). Two types of sand columns were used. The first type consisted of uniform hydrophobic sand made by mixing in three parts of the strongly hydrophobic sand with 97 parts of regular sand. For the second type the sand columns were filled with regular silica sand except for two 7-cm-thick hydrophobic 12/20 silica sand layers positioned between the 12- to 19- and 31- to 38-cm depths. The hydrophobic layers consisted of a mixture of one part of the strongly water-repellent sand and four parts of regular silica sand. In Exp. II, the columns were wrapped with parafilm to prevent leakage from the water ponded on top of the hydrophobic layer.
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Fecal samples were collected from Holstein calves (6–21 d old) exhibiting diagnostic signs of cryptosporidiosis and housed in hutches at the Cornell University Teaching and Research Center in Harford, NY. The samples were obtained by palpating the rectum to induce defecation into sample cups and were then sieved through a food strainer with a 2- to 3-mm mesh to remove large solids and mucus. All collected feces were mixed together, stored in a refrigerator at 4°C until needed, and then mixed again before each use. This procedure ensured uniformity of the input concentration. The oocyst concentration in the fecal samples was determined before the experiments in 100-µL samples (five replicates) via immunofluorescence staining and microscopic counting (method described below). The oocyst concentration in the mixed fecal sample was 3.5 x 108 oocysts L–1 and is representative of that naturally found in calf feces.
In all experiments, distilled water was used as artificial rainfall and applied with a laboratory rainfall simulator slightly modified from Andreini and Steenhuis (1990). The simulator had six needles installed on a frame with rotating patterns in two directions to randomize raindrop distributions. In each experiment, the rain simulator was calibrated using volumetric gauges. In Exp. I, the application rates were 0.3, 1, and 2 cm h–1. For Exp. II and III, the rainfall intensity was 1 cm h–1.
Experimental Procedures
In Exp. I and II, two replicate columns were placed simultaneously under the rainfall simulator. One hundred milliliters of feces with 2 g of NaCl salt was applied once on the top surface of all the columns after steady-state flow was reached. At the end of the experiment, using a thin aluminum plate, the soil contained in each ring was carefully separated from the rest of the column and analyzed for both water content and number of oocysts. After the finger flow pattern in each ring was observed and identified, the water content was determined by drying for 24 h at 105°C. Subsamples consisting of porous media and fluid materials were taken within the finger and placed in plastic tubes for microbiological analysis. After the quantity of oocysts was determined, an estimation of the total amount of oocysts in the whole cross section was obtained by multiplying the ratio of the mass of water in the cross section by the mass of water in the subsample by the amount of oocysts in the subsample.
To prevent contamination from previous runs, the rings and funnel were cleaned with chlorine and Ajax (Colgate-Palmolive, New York) commercial solutions using scratch sponges, followed by drying at 105°C for 30 min. Before application of the oocyst–feces mix, effluent samples were analyzed to assess whether oocysts were present.
In Exp. III, 100 mL of feces was mixed with a sufficient amount of KCl to give a concentration of 12.5 g Cl– L–1 suspension. The mixture was applied on top of soil columns after steady-state flow was reached. Each 50 mL of effluent was sampled, collected in a plastic tube, and analyzed for the number of oocysts and Cl concentration. Soil extractions were only done for the 12-cm-tall column.
Characterization of Preferential Flow Paths
The characterization of preferential flow paths was performed qualitatively in Exp. I and II by adding FD&C blue dye #1 to the infiltrating water, using the same procedure as in Baveye et al. (1998). After the experiments were finished, blue stains were observed in successive horizontal cross sections. Color photographs of the horizontal cross sections were taken. These pictures were scanned in and the image format was changed from red–green–blue (RGB) to cyan–yellow–magenta–black (CYMK). The cyan channel was retained and converted to black and white images using Adobe Photoshop (Adobe, San Jose, CA).
Chemical and Microbiological Analysis
The Cl– concentration in the effluent was determined with a digital chloridometer (Buchler Instruments) to obtain the Cl– breakthrough curves.
The microbiological analysis of C. parvum oocysts consisted of immunofluorescence staining to visualize and enumerate oocysts in the samples (Anguish and Ghiorse, 1997). Effluent samples were either directly analyzed or concentrated by centrifugation at 10000 g for 2 and 15 min for Exp. I and II, respectively. The resulting pellets were resuspended in a fraction of the original liquid and subjected to analysis and counting.
Enumeration of C. parvum oocysts in the columns at different depths required a preliminary extraction using the following procedure. About 15- to 20-cm3 samples of porous medium containing entrapped feces and C. parvum were placed in a 50-mL centrifuge tube, to which 15 mL of an extraction solution was added. This solution consisted of 0.1 M phosphate-buffered saline [PBS; 0.028 M Na2HPO4 H2O, 0.072 M NaH2PO4, 0.145 M NaCl, pH 7.2] and 0.1% by weight of a commercial surfactant, Tween 80 [Polyoxyethylene (20) Sorbitan Monooleate] from J.T. Baker Chemical Co. (Phillipsburg, NJ). The tubes were placed on a horizontal shaker set on low speed (180 rpm) for 20 min and then on the same shaker at high speed (300 rpm) for 10 min. Coarse particles were eliminated in each case by siphoning the slurry into a new centrifuge tube. Fifteen milliliters of a cold sucrose solution (5°C, specific gravity 1.18) was then injected into the centrifuge tubes with a syringe hypodermic needle (18 G 1 1/2) in such a way that the soil–water slurry was on top of, and did not mix with, the sucrose. After centrifugation at 2700 g for 20 min (without automatic braking at the end), the bilayer system in each tube evolved into a trilayer one, with most of the organic matter accumulating into a layer of distinct yellow color. Ten-milliliter aliquots of this intermediate layer were removed using the syringe hypodermic needle, diluted four times with 0.1 M PBS to obtain a total volume of 50 mL, and homogenized by hand. The aliquots were then centrifuged at 2700 g for 30 min (brake on). The bulk (roughly 49 mL) of the supernatant was discarded and the remaining liquid and pellet vortexed on a Fisher (Pittsburgh, PA) vortex genie 2, transferred into a 1.5-mL Eppendorf tube and vortexed again. In each Eppendorf tube, a 100-µL sample was taken for enumeration.
Samples were examined with a Zeiss (Stuttgart, Germany) LSM-210 microscope and observed with an 100X (NA,1.3) oil immersion Neofluar objective lens under both conventional DIC (Differential Interference Contrast) and epifluorescence mode with a triple excitation-emission filter (Chroma Technology Corp., Brattleboro, VT). For optimal imaging, the top element of the 1.4 NA condenser lens was also immersed in oil.
Cryptosporidium parvum oocysts were counted within a smear of a 10-µL sample spread over the slide. The concentration of C. parvum, C, in oocysts/L, was calculated as
 | [1] |
Mathematical Modeling
Preferential water and solute transport through the profiles has been modeled by assuming that the soil consists of a distribution zone overlaying a conveyance zone (Steenhuis et al., 1994). The distribution zone funnels water and solutes into distinct flow paths of the preferential flow zone. In the transport model for nonadsorbing solutes, the distribution zone acts as a linear reservoir, resulting in an exponential loss of solutes from this zone, and the transport of solutes through the preferential zone is governed by the convective–dispersive equation (Steenhuis et al., 1994; Kim et al., unpublished data, 2003).
The concentration of a nonadsorbing solute in and leaving the distribution zone can be described as (Steenhuis et al., 1994)
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In the conveyance zone, water and solutes flow with an average velocity, v, through the preferential flow paths. If the solute flux in the finger is described with the convective–dispersive equation, the steady-state solution of Eq. [2] at x = 0 as a boundary condition and no solutes at the column at t = 0 can be found for a conservative solute using Laplace transforms for 4D
/V2 (Toride et al., 1995; Kim et al., unpublished data, 2003) as
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= 0 and C 
C0. The last term can usually be neglected when x or t are sufficiently large; that is, (x + vt
)/(4Dt)1/2 > 3.
To model the transport of a nonconservative substance like C. parvum, a sink term is introduced that describes the removal of C. parvum due to adsorbance to the solid–water or AWS interfaces. Assuming that the removal is irreversible and proportional to the concentration in solution, the loss of C. parvum from the distribution zone becomes
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)/(4Dt)1/2 > 3 as
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Figure 4 displays the moisture content, as well as the concentration and mass of C. parvum oocysts retained in the sand columns. The moisture content (as percentage of saturation) was again similar for all of the sand columns. Moisture contents were elevated in the top 3 to 5 cm where the distribution zone was located and near the bottom of the column in the capillary fringe. In addition, the moisture contents were greater in the capillary fringes above both water-repellent layers in Exp. II. In the conveyance zone, the average water content was low. As expected, the average water content increased for the increasing flow rates within Exp. I because a larger portion of the columns was wetted, resulting in a higher average moisture content. Since the water contents were taken after the water application was stopped, some drainage took place before the samples were taken and, consequently, the moisture contents in the distribution and conveyance zones are underestimated.
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If we compare the breakthrough curves for Exp. III (Fig. 5) and Exp. I and II (Fig. 3), it took a longer time for the Cl– to initially appear but the overall shape of the breakthrough curves was approximately the same. The differences in C. parvum oocysts were more significant. In the Hudson column, application of the manure at the soil surface resulted in a rapid decrease of the water outflow rate (Fig. 6) . After about 3 cm of rain, this decrease stopped and the outflow rate began to increase. By the end of the experiment, it had returned to 75% of its original value (Fig. 6). The time where the outflow of the column started to increase coincided with the appearance of oocysts in the effluent (Fig. 5b), significantly later than the peak in Cl– concentration. In the Arkport soil column (Fig. 5c), oocysts appeared in the effluent during the tailing of the Cl– breakthrough curve. Thereafter, the oocyst breakthrough had an erratic pattern and continued after the Cl concentration had subsided.
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Mass Balance
Despite the fact that recovery of C. parvum oocysts in soils was uncertain (Mawdsley et al., 1996; McElroy et al., 2001), the numbers of oocysts in the columns were measured after the columns were taken apart. By adding the quantity in the column with what was lost in the drainage water and comparing with the amount originally added, the percentage recovered could be calculated (Table 1). The wide range of recoveries from 14 to 86% is the same as reported in the literature (Brush et al., 1999). This variability might have been caused by any one of the various steps involved in the cumbersome procedure used for the enumeration of the oocysts. In addition, variability may be caused by the necessity of taking subsamples. Particularly in the distribution zone where fingers develop, this sampling may have introduced artifacts that were, unfortunately, impossible to avoid at this stage, for lack of a better enumeration technique.
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Dispersivities for all sand columns were in the reasonable range of 1.0 to 6.7 cm. The model predicts Cl– breakthrough curves well, with high R2 values of >0.97 for all columns. Using these parameters, we estimated a ß (Eq. [4]) that would best fit the oocysts concentrations. The ß values found ranged from 0.6 to 1.8 h–1 and were higher than literature values of 0.004 and 0.09 h–1 for the medium and fine sand, respectively, by Wan and Tokunaga (1997), and 0.1 h–1 for experiments of saturated porous media with E. coli. by Stevik et al. (1999). The higher ß values in this study were a result of a higher retention of C. parvum in the unsaturated columns compared with the saturated columns.
Illustrations of the fit are shown in Fig. 3. The model did not fit the oocyst breakthrough curves as well as the Cl– curves, but was overall predictable with R2 values of >0.70, although some exceptions occurred. The largest discrepancy was observed for a 1 cm h–1 rainfall experiment, in which only one-half the observed oocysts were predicted. The better fitting results for the other two rainfall intensity columns in Exp. I using the same parameters obtained from the Cl– breakthrough curves suggest that the observed oocyst data were overestimated by the technical error. For all columns, peaks occurred at the same time for C. parvum and Cl–, but C. parvum showed a much faster decrease in concentration. One exception is replicate A of the experiment with two water-repellent interfaces in Exp. II, in which an oocyst increase was delayed a little compared with the Cl– breakthrough. This resulted in a low R2 value for modeling this column.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The results shown in Fig. 5 suggest that in clay soils, oocyst breakthrough may be limited compared with that in other soils, in spite of the presence of macropores. However, the drastic reduction of outflow rate immediately after addition of the feces indicated that some of the macropores became severely clogged with the feces material. Unclogging of the macropores and resumption of flow permitted the passage of oocysts in large numbers shortly thereafter.
Despite the uncertainty of the initial oocyst number applied, it is clear that the oocyst numbers predicted through 50-cm sand columns were reduced by two orders of magnitude from hundreds of millions to several millions. Therefore, if the rain water has to percolate through several meters of soil before reaching the groundwater, contamination risks should be minimal. However, in the cases where the groundwater is shallow and the source of the C. parvum oocysts and the drinking water well are close together, contamination of well water with C. parvum oocysts could occur on a sandy soil after a rainstorm. A realistic example could be a sick animal defecating near a well during a county fair with temporary shallow wells on a sandy flood plain soil.
It is of interest to compare our results of breakthrough experiments of C. parvum oocysts in unsaturated columns with those performed by Brush et al. (1999) and Harter et al. (2000), where the columns were saturated. Comparing the two flow regimes, the main difference is that under unsaturated conditions, the C. parvum oocysts concentration in the effluent decreases faster than the Cl–, while for the saturated columns there was a significant tailing in the latter part of the breakthrough curve and a measurable concentration was observed throughout the duration of the experiment (Harter et al., 2000). Therefore, to simulate the breakthrough of C. parvum oocysts, Harter et al. (2000) had to assume that the attachment of the oocysts to grains was reversible, while for unsaturated conditions we assumed that the attachment was irreversible. The latter is consistent with the results of Crist et al. (2003) who recorded the pore-scale distribution of colloids in still and video images. They found that the hydrophilic, negatively charged carboxylated latex microspheres, which have similar characteristics as C. parvum oocysts, were retained primarily in a thin film of water at the edge of the menisci at the AWS interface. Under steady-state flow conditions (as was the case in our experiments), the microspheres stayed in place and did not go back into the solution. Under saturated conditions, meniscus (and the AWS interfaces) do not exist, and other mechanisms of retention become important such as filtration in narrow pore spaces. Retention by filtration is likely less permanent and allows remobilization. Consequently, it is not unexpected that the modeling retention of C. parvum oocysts under saturated and unsaturated conditions is different.
Finally, it is of interest to note that the flow rate was independent of the time it took for the C. parvum oocysts to reach the bottom of the columns in Exp. I (i.e., velocity and flow rate are not related in Table 2). Since the velocity of the fingers only depends on the type of porous media, mass balance consideration dictates that more fingers form under increasing flow rates in initially dry sand (Steenhuis et al., 2001). Thus, the likelihood of pollution of aquifers depends partly on the duration of the rainstorm.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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, dispersivity (d/v); ß, rate of removal from the solution; R2, correlation coefficients.