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a U.S. Dep. of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Waste Pathogens Laboratory, Bldg. 173, BARC-East, 10300 Baltimore Ave, Beltsville, MD 20705-2350
b U.S. Dep. of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Environmental Quality Laboratory
c U.S. Dep. of Agriculture, Agricultural Research Service, Pasture Systems and Watershed Management Research Laboratory
* Corresponding author (ypachepsky{at}anri.barc.usda.gov)
Received 6 November 2002.
ABSTRACT
Manures are sources of several human pathogens that can potentially contribute to surface and groundwater contamination. Microorganisms must first be released from the manure matrix before they can infiltrate into and leach through the vadose zone. The objective of this study was to estimate rates of rainfall-induced release of fecal coliform (FC) from surface-applied bovine manure. Simulated rainfall of 7.1 cm h-1 was applied to the surface of 90-cm-long lysimeters filled with the undisturbed stony soil. When the steady state was reached, clumps of manure were placed on the surface. Rainfall was continued for about 5 h after application of manure, and 10-min leachate portions were analyzed for turbidity and FC. The convective–dispersive equation with linear adsorption–exclusion and the first-order removal–regrowth terms was used as a model of the coliform transport in soil. Asymptotic properties of the solution of this equation with the exponentially decreasing boundary concentration were used to infer the release rate constant from the FC breakthrough curves. A value of 0.0054 ± 0.0015 min-1 was found for the FC release rate constant. The regression line of reduced coliform concentrations on reduced turbidity values was not significantly different from the one-to-one line; R2 was 0.807. Assuming that turbidity can be used as a measure of concentration of manure particulates in leachates, we found that average values for the release rate constants were not significantly different for FC and manure particulates. The average velocity of bacteria and manure particulates transport was about seven times larger than the average pore velocity. The proposed technique of estimating FC and manure release rates shows promise for use in further studies needed to elucidate and assess factors affecting release rate.
Abbreviations: CFU, colony forming units • FC, fecal coliform • NTU, nephelometric turbity units
THERE IS GROWING CONCERN regarding the potential for contamination of surface and ground water by pathogens from bovine manures. Even though they are considered to be a beneficial fertilizer and soil amendment, bovine manures are a substantial agricultural source of several human parasites/pathogens. Escherichia coli O157 and other EHEC strains are commonly found in beef and dairy cattle (Bos taurus L.) (Elder et al., 2000; Hancock et al., 1998; Porter et al., 1997). On-farm monitoring of E. coli O157:H7 suggests that shedding occurs episodically (up to 105 organisms g-1 feces) and can persist for variable periods of time ranging from about 1 to 5 mo (Shere et al., 1998; Zhao et al., 1995).
Pathogens applied onto soil surfaces may enter the soil and travel through the vadose zone until they reach groundwater (McMurry et al., 1998). A substantial body of literature exists describing leaching of microorganisms through soil. Early studies documenting subsurface bacterial transport from septic effluents were reviewed by Hagedorn et al. (1981) and Bitton and Harvey (1992). These studies describe bacterial transport from a few meters to 830 m, depending on soil or sediment texture and permeability, water saturation degree, and length of time. More recent studies have quantified the leaching potential of fecal bacteria and genetically modified bacteria. Although leaching rates were influenced to some extent by all soil and microbial properties, the predominant factors in disturbed soil cores appear to be soil structure and texture and porosity and bulk density, in conjunction with bacterial size (Gannon et al., 1991a,b; Huysman and Veerstraete, 1993; Tan et al., 1991). In intact soil cores, distribution and continuity of macropores (preferential flow pathways) in conjunction with initial water content appeared to be the predominant factors (Abu-Ashour et al., 1998; McMurry et al., 1998; Paterson et al., 1993). In general, higher leaching rates were observed in undisturbed soils as compared with disturbed soils (Smith et al., 1985; van Elsas et al., 1991), presumably due to the destruction of macropores during the repacking of soil.
The majority of studies of pathogen bacteria transport in soils used bacterial cells that were suspended in solutions and then applied to soil surface or mixed with the influent solution (Bitton et al., 1974; Abu-Ashour et al., 1998; Devare and Alexander, 1995; Goldschmid et al., 1973; Gannon et al., 1991a,b; Germann et al., 1987; Hendry et al., 1999; Hekman et al., 1995; Huysman and Verstraete, 1993; Hornberger et al., 1992; Lahlou et al., 2000; Paterson et al., 1993; Smith et al., 1985; Tan et al., 1991; van Elsas et al., 1991). Results of those studies are only partly applicable to microbial leaching from either land-applied or naturally deposited manures, since microorganisms must first be released from the manure matrix before they can infiltrate into and leach through the soil profile. We are not aware of any data on bacteria release rates from manure subject to rainfall, and could find only one recent paper (Bradford and Schijven, 2002) that contains data on manure dissolution in laboratory conditions. It is not known whether rates of manure dissolution and bacteria release can be equated since the bacteria distribution within manure material has not been studied. At the same time, knowledge of the release rates is a precondition of bacteria and manure transport simulations.
The objective of this study was to estimate rates of fecal coliform and manure release due to rainfall action. We obtained breakthrough curves of bacteria and manure particulates from soil columns and used the analytical solution (van Genuchten, 1985) of the convective–dispersive equation with exponentially decreasing inlet concentration to derive the release rates from tail sections of the breakthrough curves.
MATERIALS AND METHODS
Experimental
The lysimeters were located at the USDA-ARS experimental research site near the town of Leck Kill, PA. The soil in lysimeters is a loamy-skeletal, mixed, active, mesic Typic Hapludult. This soil is channery; that is, it has rock fragments of shale or thin flat sandstone or siltstone up to 20 cm long. Illite and kaolinite are the most common clay minerals. This is a deep, well-drained soil with rapid permeability. Percolation is dominated by the macropore flow. Soil properties are given in Table 1 (Stout et al., 1998). Lysimeters were 60 cm in diameter and 90 cm long. They were built according to the design of Moyer et al. (1996) and had a steel plate welded to the bottom with about twenty 6-mm holes in the middle 75 mm of the bottom. A 1-mm wire mesh was placed over the holes and about 4 kg of coarse gravel was placed over the mesh. The middle of the bottom of the soil core was hollowed out to accommodate the gravel. Detailed descriptions of the site and monolith lysimeters can be found elsewhere (Gburek and Folmar, 1999; Stout et al., 1998).
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20 mL) for 5 h. Manure was collected from the lot of a local dairy farm on the previous day. Percentage dry weight was 28%. Manure was applied to the lysimeter surfaces in 10 to 12 lumps approximately in nodes of a grid.
Leachate samples were refrigerated continuously (4°C) until analysis, except during transport from Leck Kill, PA to Beltsville, MD when samples were packed in ice. Microbial analyses were initiated the next day after transporting samples to the laboratory and required 3 d (two lysimeters per day). Samples were plated on MacConkeys Agar using an Autoplate 4000 spiral plater (50 µL) manufactured by Spiral Biotech (Bethesda, MD). Total FC colony forming units (CFUs) were counted using a Protocol plate reader (Synoptics, Cambridge, UK). Turbidity (nephelometric turbity units, NTU) was determined using a Model 965-10A Turbidimeter manufactured by Orbeco Analytical Systems, Inc. (Farmingdale, NY).
Data Analysis
The FC transport was simulated assuming steady state water flow and neglecting variations in water content and pore structure along the soil profile. The model was chosen in the form
 | [1] |
Here c is the volume-averaged bacteria concentration in the soil solution (cells cm-3), 
a is the porosity available for bacteria transport in soil (m3 m-3), 
is soil bulk density (g cm-3), s is the adsorbed amount of bacteria (cells g-1), D is the dispersion coefficient (cm2 min-1), Jw is the volumetric water flux density (cm min-1), µl and µs (min-1) are first-order decay coefficients to simulate removal of bacteria from solution and solid phase as a result of deposition and entrainment (Hornberger et al., 1992). If a regrowth occurs, values of µl and µs also include growth rate constants with negative signs.
Bacteria adsorption by the solid phase is described with a linear isotherm as
 | [2] |
 | [3] |
(cm min-1) is the average pore-water velocity, (cm3 cm-3) is the volumetric soil water content, Ra is the retardation factor given by
 | [4] |
 | [5] |
The third-type boundary condition was used for the bacteria flux on the surface:
 | [6] |
The concentration of bacteria released from manure cm (cells cm-3) was assumed to decrease exponentially with time:
 | [7] |
(min-1) is the bacteria release rate constant.
Using dimensionless variables C = c/c0, Z = x/L, T = vt/L, P = vL/(Daa), µE = µL/v, and E = L/v, Toride et al. (1999) presented the solution of the boundary problem (Eq. [3]–[7]) for the infinite domain with zero initial concentration for arbitrary X and T values:
 | [8] |
 | [9] |
. This expression is valid when 
> -P/4 (i.e., µE > RE - P/4 and 
0). There exists also a solution for relatively small deposition and/or entrainment rates when µE < RE - P/4 (van Genuchten, 1985). This solution is expressed in the form of integrals that need to be evaluated using quadratures.
Using values of the erfc function at infinity, one can show that when T becomes large, the function GE1(1, T, ) given by Eq. [9] approaches the limit 2exp[P(1 - u)/2]/(1 + u). Therefore the asymptotic behavior of concentrations for large T is
 | [10] |
 | [11] |
Therefore breakthrough curves obtained from the solution (Eq. [9]) have their tails well approximated with straight lines in the log10(concentration)–time coordinate plane. Slopes of those lines are values of the release rate constant divided by ln10 
2.303.
We could not find an analytical expression for the asymptotic behavior of concentrations for the case of small µE values, when µE < RE - P/4 and values of u become complex numbers. However, multiple experiments with computing GE1 by numerical evaluation of integrals using the subroutine F from the computer code given by van Genuchten (1985) showed that the loglinear asymptotic behavior of the breakthrough curves exists also for µE < RE - P/4.
Figure 1 shows examples of the breakthrough curves computed from Eq. [9] with sets of parameters that are realistic for the experiment in this work. The length L = 90 cm and the pore velocity v = 0.5 cm min-1 were used in simulations. Figure 1a shows that large (5–50 cm2 min-1) dispersion coefficient values will delay the beginning of the linear decrease of the log10(c) with time. Figure 1b shows that the linear decrease begins soon after the minimum is reached for values of the retardation coefficient R less than one (i.e., when only part of the water-filled pore space is available for the solute transport). The effect of the release rate constant value on the concentration–time dependencies is shown in Fig. 1c. The FC concentration on the surface decreases very slowly when the release rate is small (0.001 min-1), and this is reflected in the shape of the breakthrough curve showing almost constant concentration as the time progresses. The effect of the deposition–entrainment rate constant µ on effluent concentration is shown in Fig. 1d. An increase in value of µ causes vertical shift of the linear part of the breakthrough curve and does not affect slope of this linear part.
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RESULTS
Data on FC in and turbidity of the effluent are shown in Fig. 2. Maximum FC concentrations were observed between 60 and 80 min. All FC breakthrough curves had a period of a steep increase followed by the gradual decrease phase.
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= 0.0054 ± 0.0015 min-1. The average half-time for the release of FC from manure was ln(2)/ 128 min.
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mp was 0.0053 ± 0.0006 min-1. The average value was very close to and not statistically significantly different from the average FC release rate constant 0.0054 min-1. Although the accurate estimation of parameters in the model (Eq. [3]–[7]) is possible only if the initial FC concentration is known in the very first influent portion, a crude estimation of values of the transport parameters D and R can be made from comparison of graphs shown in Fig. 1 and 4a. Figure 1a shows that the initial steep concentration increase, as observed in Fig. 4a, corresponds to values of D about 0.5 to 1 cm2min-1. For such values of D, the graph in Fig. 1d shows that the entrapment–entrainment rate µ does not have much of an effect on the time at which the breakthrough curve begins the steep rise, and the graph in Fig. 1b shows that this time is approximately equal to RL/v, where L is the length of the monolith and v is the average pore-water velocity. The steep rise in Fig. 4a begins around 20 to 40 min; the average v value is about 0.42 cm min-1. Therefore value of the retardation factor R appears to be in the range from 0.09 to 0.19.
DISCUSSION
Bacteria and manure particulates moved relatively fast through the soil profile in this work. The pore water velocity was seven times less than the average velocity of bacteria transport. This ratio of velocities is given by the value 1/R, where R is the retardation coefficient that was about 0.15 in this work. Bitton and Harvey (1992) reviewed the values of retardation coefficient found in studies of E. coli in soil and groundwater and reported the range from 0.1 to 1.0. These authors attributed the apparent accelerated bacteria transport to the preferential transport of microorganisms along preferred flow paths (large pores, fractures and channels) because bacteria may be excluded from the smaller pores on the basis of their size. Values of the retardation coefficient R as given by Eq. [4] reflect both the unavailability of the part of pore space for the bacteria transport (first term) and the delay in bacteria transport caused by the adsorption on soil particles (second term). It is possible that bacteria adsorption on soil particles did not affect the bacteria transport in this work because this adsorption was slow. Bengtsson (1995) reported time scales to reach adsorption equilibrium for 13 bacteria species from 10 to 15 h to several days. Assuming the small effect of the adsorption on soil particles, we could estimate the porosity available for bacteria transport as 15% of the total water content.
Manure particulates composed of microbial biomass and partially digested food were transported along with FC. Using reduced values enhanced the similarity between dependencies of the effluent turbidity and the FC concentrations on time (Fig. 3), probably because reduced concentrations eliminated the effect of the initial concentration of FC and particulates on the breakthrough curves. The release and transport models for particulates apparently could be the same as for FC, and that assumption led to statistically indistinguishable release rate constants for manure particulates and for FC. Observed FC concentrations in leachate samples would not give a measurable turbidity. Consequently, turbidity was due to non-FC microbial biomass and other particulates. A relatively strong correlation between reduced FC concentrations in effluent and the effluent turbidity makes turbidity a plausible candidate for an easily measurable proxy variable to estimate the level of bacteria concentration in leachates. This correlation also raises the question of whether the transport bacteria released from manure to soil can be a facilitated transport. Manure amendment has been shown to affect transport of bacteria through soil (Gagliardi and Karns, 2000). Jin et al. (2000) presented the first experimental example of colloid-facilitated virus transport in sandy columns. It might be important to find out what percentage of bacteria travels in soil solution being attached to manure particulates.
An interesting feature of the FC transport in this experiment was a very low longitudinal dispersion. Fecal coliform transport developed almost as if it followed piston flow or a kinematic wave (Germann et al., 1987). Pore water velocities obviously did not have substantial variability in the pore space available for the bacteria transport, and that might be a consequence of the presence of manure material in solution affecting the soil water viscosity and density. Low dispersion allowed the asymptotic regime (Eq. [11]) to appear relatively early in the experiment, and we had enough data to estimate the release rate constants as shown in Fig. 4b and 4d.
Using properties of the analytical solution (van Genuchten, 1985) allowed us to obtain the release rates while avoiding the need of knowing the initial concentration c0 in Eq. [7] for both FC and manure particulates. Measurements of the initial concentration in the experimental setup of this work would be very uncertain. We note that intercepts of the regression equations (Eq. [11]) are not suitable to compute the c0 value because those intercepts include the value of u = 
, which depends on the removal–entrainment rate constant µE. Data in Fig. 1d illustrate this point. Extending the linear sections of the breakthrough curves to the concentration axis leads to values of c/c0 less than one.
We note that FCs represent only a small fraction of total microbial biomass in manure. It remains to be seen whether release and transport of bacterial pathogens (Salmonella, E. coli O157) as well as release of protozoan parasites, such as Cryptosporidium and Giardia can be mimicked by FC.
The exponential release model (Eq. [7]) performed reasonably well both for fecal coliforms and for manure particulates (Fig. 4). The release rate constants should depend on manure source, composition and management, as well as on rain parameters. The values of 0.005 min-1 found in this work should be treated as estimates applicable to this experiment but by no means the absolute values. We computed the manure release rates from the data of Bradford and Schijven (2002) and found values ranging from 0.002 to 0.003 min-1. The difference between release rates in this and Bradford and Shijven's works may be related both to the differences in water application to manure and to the quality of manure used. Because knowledge of microorganism release rates is essential for simulations of pathogen dissemination from manures, further studies are needed to elucidate manure and microorganism release factors. Such studies may take advantage of the promise shown by the breakthrough-based technique developed in this work.
ACKNOWLEDGMENTS
We thank Valerie McPhatter and Kerry Sefton for technical assistance.
REFERENCES
This article has been cited by other articles:
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