Journal of Environmental Management 131 (2013) 121e128
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Fate of Cryptosporidium parvum oocysts within soil, water, and Plant environment Stephen J. McLaughlin a, Prasanta K. Kalita b, *, Mark S. Kuhlenschmidt c a
Hanson Professional Services Inc., Oak Brook, IL 60523, USA Department of Agricultural and Biological Engineering, University of Illinois, Agricultural Engineering Sciences Building, 332P AESB, 1304 W. Pennsylvania Avenue, Urbana, IL 61801, USA c Department of Pathobiology, University of Illinois, Urbana, IL 61802, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2013 Received in revised form 11 September 2013 Accepted 13 September 2013 Available online 22 October 2013
Vegetative Filter Strips (VFS) have long been used to control the movement of agricultural nutrients and prevent them from reaching receiving waters. Earlier studies have shown that VFS also dramatically reduce both the kinetics and extent of Cryptosporidium parvum (C. parvum) oocysts overland transport. In this study, we investigated possible mechanisms responsible for the ability of VFS to reduce oocyst overland transport. Measurement of the kinetics of C. parvum adhesion to individual sand, silt, and clay soil particles revealed that oocysts associate over time, albeit relatively slow, with clay but not silt or sand particles. Measurement of oocyst overland transport kinetics, soil infiltration depth, distance of travel, and adhesion to vegetation on bare and vegetated soil surfaces indicate that oocysts move more slowly, and penetrate the soil profile to a greater extent on a vegetated surface than on a bare soil surface. Furthermore, we demonstrate a small fraction of the oocysts become attached to vegetation at the soilvegetation interface on VFS. These results suggest VFS function to reduce oocyst overland transport by primarily decreasing oocyst surface flow enough to allow penetration within the soil profile followed by subsequent adhesion to or entrapment within clay particle aggregates, and to a lesser extent, adhesion to the surface vegetation. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Water quality Pathogen Transport Vegetative filter strips
1. Introduction It is difficult to imagine any resource of more value to society than clean water. The United States has over 330 million acres of agricultural land that produce an abundant supply of low-cost, nutritious food and other products. American agriculture is noted worldwide for its high productivity, quality, and efficiency in delivering goods to the consumer. However, when improperly managed, agricultural activities can affect water quality (Barwick et al., 2003; Brady and Weil, 1999). Animal agriculture farms are potential sources of microbial contamination of the water supply (Miller et al., 2008). Microbial pathogens present in agricultural runoff impose a significant hazard on human health when acquired directly via the fecal-oral route or indirectly as a waterborne contaminant (Gagliardi and Karns, 2000; Hill et al., 2005; Miller et al., 2007; Thurston-Enriquez et al., 2005). In certain individuals, such as the immunocompromised or those with AIDS or AIDS-related syndromes, waterborne infectious diseases, such as
* Corresponding author. Tel.: þ1 217 333 0945; fax: þ1 217 244 0323. E-mail address: [email protected] (P.K. Kalita). 0301-4797/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.09.017
cryptosporidiosis, may be lethal. Infected calves can excrete in excess of 3 1010 Cryptosporidium oocysts over approximately a two week period (Nydam et al., 2001). Considering the mean human infective dose is approximately 130 oocysts (Chappell et al., 1999), only a few infected animals are needed to produce enough oocysts to contaminate a large watershed (Olsen et al., 1999). The risk of such contamination is not only a human health hazard but also a potential threat to the continued existence of small to medium-sized dairy farms and feedlots. To design and develop control strategies, microbial transport processes in surface and near-surface runoff need to be properly understood and quantified along with various factors that affect pathogen transmission in the environment. Recent research results measuring Cryptosporidium parvum oocyst transport in soil, using small and large scale, field-applicable experimental systems, indicate vegetative filter strips (VFS) are effective at reducing oocyst overland transport (Atwill et al., 2002; Davies et al., 2004; Tate et al., 2004; Trask et al., 2004). Although VFS appear to be a fieldapplicable and cost-effective best management practice, the mechanism by which they reduce oocyst overland transport is not understood. Furthermore, the ultimate design of optimally effective VFS for a particular watershed and set of contaminating microbial
122
S.J. McLaughlin et al. / Journal of Environmental Management 131 (2013) 121e128
pathogens, e.g., viruses as well as bacteria and protozoa, requires knowledge of the mechanism of interaction of the particular pathogen with VFS. Our previous results demonstrated VFS dramatically reduce both the kinetics and number of oocysts transported in surface and near-surface runoff (Trask et al., 2004). The objectives of this study were to investigate the oocysts adhesion to individual soil particles and compare of their movement on and within the soil profile of both bare and vegetated soil beds. 2. Materials and Methods 2.1. Preparation of Cryptosporidium parvum oocysts Oocysts were propagated, collected and purified from experimentally infected neonatal calves as previously described in Johnson et al. (2004) and Trask et al. (2004). Feces containing oocysts were collected in plastic drop pans which were maintained over melting ice. A100 mL pen/strep/amphotericin (containing 6 g penicillin, 10 g streptomycin, and 25 mg amphotericin per liter of 0.85% sterile saline) and 100 mL nystatin (containing 1.98 g per liter sterile saline) per 1 to 1.5 L of feces were added to the collection pans. The oocysts containing antibiotics were stored at 4 C until use. All procedures involving animals were part of protocols approved by the University of Illinois Institutional Animal Care and Use Committee (IACUC). 2.2. Soil particle separation A moderately drained silt-loam (Catlin series, mesic Oxyaquic Argiudolls) was collected from Champaign, IL. The soil was sifted using 3 mm screen to break up the larger pieces into more uniform sizes and remove large organic matter and rocks. The sifted soil sample was air-dried for 48 h. The air-dried soil was then ground down using a mortar and pestle. The ground soil was passed through a series of sieves with sieve openings that corresponded to the USDA classification of soil fractions according to the particle diameter. None of the samples contained any particles that were classified as gravel. Soil samples were separated into six categories using the sieves; very coarse sand, coarse sand, medium sand, fine sand, very fine sand and a combination of silt and clay. The sieves are #18, which corresponds to a 1 mm opening to hold the very coarse sand, #35 with a 500 mm opening that holds the coarse sand, #60 with a 250 mm opening that holds the medium sand, #140 with an opening of 106 mm which will hold the fine sand, and #270 with an opening of 53 mm which will hold the very fine sand. Anything that passed through the #270 sieve was classified as silt and clay. The soil was placed in the top sieve and left on a vibrating auto sifter for 30 min. The bottom collection pan contained the silt and clay. The silt and clay were then separated using the principles of Stokes Law based on the settling time of the clay particles (diameter of 2 mm or less). The soil was placed in a beaker with distilled water and stirred for 30 s, then left to settle for over 7 h. The soil that was settled at the bottom of the suspended clay was classified as silt, which was then oven dried. The suspended clay was transferred to a large beaker, oven dried, and the clay collected. This process was repeated numerous times to ensure adequate amount of clay particles were collected for microbial adhesion experiments. 2.3. Oocysts-soil adhesion Oocyst-soil adhesion experiments were conducted using two methods. The first method uses a Coulter electronic particle counter (Model XM) to measure the rate of disappearance of single oocysts as the form aggregates with individual soil particles. The Coulter
electronic particle counter discriminates between particles of different volumes, and can determine the size distribution and the number of particles in the suspension. The method had been previously adapted for use in cell adhesion experiments (Obrink et al., 1977; Orr and Roseman, 1969; Schmell et al., 1982). In the Coulter Counter oocyst-soil particle adhesion experiments, the adhesion of oocysts to a particular soil particle is measured by quantifying the loss of oocysts from an oocyst “window” calibrated to count only single, non-aggregated oocysts. As the oocysts adhere to a soil particle they increase in size (volume) and thus disappear from the oocysts window. The Coulter counter settings defining the oocysts window were: upper threshold (Ut) ¼ 3.4 mm, lower threshold (Lt) ¼ 4.9 mm; attenuation ¼ 2, aperture current ¼ 400 mA; using a 100 m aperture probe and 500 ml manometer volume. In the second method, used to qualitatively estimate oocyst adhesion to the larger sand particles (sand particles are too large to accurately measure using the Coulter Counter), as well as confirm results obtained with the Coulter Counter method, oocysts were mixed and incubated with the soil particles as described in method 1, but visualized using differential contrast and fluorescent microscopy following staining of the oocysts with a FITC-labeled, oocystspecific monoclonal antibody (Crypt-a-Glo; Waterborne, Inc). Fluorescent staining of oocyst-soil particle incubations was performed by placing an aliquot (40 ml) of the incubation onto a precleaned (70% ethanol) microscope slide, air drying the slide on a slide warmer, and incubating with the FITC-Mab for 30 min at 37 C followed by washing according to manufacturer’s suggestions. The initial number of soil particles and oocysts added to adhesion incubations was determined either by Coulter counting or by enumeration in a hemocytometer. Approximately 2.5 104 oocysts were mixed with 5 104 soil particles in a final volume of 300 ml in a 1.5 ml microfuge tube and incubated at room temperature for various times by end over end rotation at 5.5 rpm. 2.4. Radiolabeling of oocysts Gradient purified oocysts [1.5 107] were washed in PBS three times and brought to a final volume of 100 ml. One mCi of Na125I (IMS 30, Amersham Biosciences, NJ) was placed in an IODO-GEN (Pierce Biotechnology, IL) pre-coated iodination tube with 20 mL of 0.5M Sodium Phosphate (NaHPO4) buffer with a pH of 7.1. The oocysts were then added to the IODO-GEN tube to start the labeling reaction. The IODO-GEN tube was agitated every few minutes to help deter settling of oocysts during incubation. The reaction was stopped after 15 min by removing the mixture from IODO-GEN tube and adding 2 mL of 1.0 g/ml potassium iodide. The oocysts are washed free of unbound 125I by centrifuging (15s, 11,000 g) three times with 1 mL PBS, and then stored in 500 ml final volume, 4 C until use. Before application to bed, the radioactive oocysts suspension was diluted to 10 ml with PBS. An aliquot of the final solution of oocysts was counted in a gamma counter to determine the specific radioactivity (cpm/oocyst) of the final oocyst suspension. 2.5. Oocysts overland transport on small-scale soil beds For measurement of oocyst overland transport and soil distribution following simulated rainfall, small scale tilting soil beds were constructed using 1.91 cm plywood boxes measuring 0.91 m long by 0.30 m wide and 4.45 cm deep. The boxes were sealed with silicone and then lined with isotope paper to prevent leaking or contamination to other surfaces. A 32 mm piece of clear plexiglass was used to split the bed into two chambers (one for vegetation and the other for bare soil). Boxes were then filled with soil (Catlin series, mesic Oxyaquic Argiudolls). The grass chamber was seeded
S.J. McLaughlin et al. / Journal of Environmental Management 131 (2013) 121e128
with tall fescue at a high seeding rate to ensure that adequate coverage was achieved. A collection system was installed to collect all surface and shallow subsurface runoff from the bed. The runoff from the surface and subsurface drains was collected in separate containers so the number of oocysts lost in runoff during the experiments could be quantified. The water (simulated rain) was applied with a watering can by pouring at a slow rate over the entire bed placed at a 3% slope. Radioactive oocysts were placed on the slightly damp surface 12.7 cm from the top of the bed (i.e., the top of the slope) in a small band across both the vegetated and bare soil sides of the bed. Immediately following the application, 7.1 L of rainfall was applied to the bed to ensure adequate runoff. When runoff ceased, representative runoff and soil samples were taken and counted for radioactivity as described below. The number of oocysts (1.5 107) and amount of rainfall applied was proportional to the amount used in our previous studies (Trask et al., 2001, 2004) employing a large-scale soil bed and rainfall simulator and based on a comparison of soil surface areas of both types of soil beds. 2.6. Measurement of oocyst distribution following overland transport To determine the oocyst distribution in the soil beds and adhesion to surface vegetation following overland transport, soil samples were taken with a cork bore every 4.5 cm on the left, right, and center along the length of the vegetated and bare soil beds after the rainfall ceased, placed in a test tube, and counted for radioactivity. Following quantification of radioactivity, core samples of highest oocyst concentrations were cross-sectioned from the beds for further analysis. A 2.54 cm by 5.08 cm core sample was taken from these areas so that the depth of infiltration of oocysts could be compared between vegetated and bare soil. Soil sections were divided (cut into cross-sections) every 0.5 cm from the soil surface to the bottom of the bed and counted for radioactivity. Vegetation was divided into three sections: 0e3 cm, 3e6 cm and greater than 6 cm from the soil surface, and each section was counted for
123
radioactivity. The grass root structure was included in the 0.5 cm sectioning of the soil profile. 2.7. Oocyst transport in vertical soil columns Vertical soil columns of 0.9 m height and 0.4 m diameter were constructed with rigid PVC pipe (Fig. 1). Two soils (Catlin and Newberry series) and three vegetations (Brome, Reed Canary, and Kentucky Blue grass) were tested using low (1 108) and high (1 109) doses of oocysts per column. Oocyst recoveries were determined as previously described in Trask et al. (2004) following a water load of 3 applications (10 L per application) per column. 3. Results 3.1. Adhesion of oocysts to silt, sand and clay soil particles Individual silt and clay particles were harvested, incubated with oocysts at 22 C for varying times, and adhesion to each other or oocysts was measured using a Coulter counter. The size profile of C. parvum oocysts as well as clay and silt particles that was used to define the lower and upper thresholds of a single cell (oocysts) window is shown in Fig. 2A. The diameter of both purified clay and silt particles (
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Fate of Cryptosporidium parvum oocysts within soil, water, and Plant environment Stephen J. McLaughlin a, Prasanta K. Kalita b, *, Mark S. Kuhlenschmidt c a
Hanson Professional Services Inc., Oak Brook, IL 60523, USA Department of Agricultural and Biological Engineering, University of Illinois, Agricultural Engineering Sciences Building, 332P AESB, 1304 W. Pennsylvania Avenue, Urbana, IL 61801, USA c Department of Pathobiology, University of Illinois, Urbana, IL 61802, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2013 Received in revised form 11 September 2013 Accepted 13 September 2013 Available online 22 October 2013
Vegetative Filter Strips (VFS) have long been used to control the movement of agricultural nutrients and prevent them from reaching receiving waters. Earlier studies have shown that VFS also dramatically reduce both the kinetics and extent of Cryptosporidium parvum (C. parvum) oocysts overland transport. In this study, we investigated possible mechanisms responsible for the ability of VFS to reduce oocyst overland transport. Measurement of the kinetics of C. parvum adhesion to individual sand, silt, and clay soil particles revealed that oocysts associate over time, albeit relatively slow, with clay but not silt or sand particles. Measurement of oocyst overland transport kinetics, soil infiltration depth, distance of travel, and adhesion to vegetation on bare and vegetated soil surfaces indicate that oocysts move more slowly, and penetrate the soil profile to a greater extent on a vegetated surface than on a bare soil surface. Furthermore, we demonstrate a small fraction of the oocysts become attached to vegetation at the soilvegetation interface on VFS. These results suggest VFS function to reduce oocyst overland transport by primarily decreasing oocyst surface flow enough to allow penetration within the soil profile followed by subsequent adhesion to or entrapment within clay particle aggregates, and to a lesser extent, adhesion to the surface vegetation. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Water quality Pathogen Transport Vegetative filter strips
1. Introduction It is difficult to imagine any resource of more value to society than clean water. The United States has over 330 million acres of agricultural land that produce an abundant supply of low-cost, nutritious food and other products. American agriculture is noted worldwide for its high productivity, quality, and efficiency in delivering goods to the consumer. However, when improperly managed, agricultural activities can affect water quality (Barwick et al., 2003; Brady and Weil, 1999). Animal agriculture farms are potential sources of microbial contamination of the water supply (Miller et al., 2008). Microbial pathogens present in agricultural runoff impose a significant hazard on human health when acquired directly via the fecal-oral route or indirectly as a waterborne contaminant (Gagliardi and Karns, 2000; Hill et al., 2005; Miller et al., 2007; Thurston-Enriquez et al., 2005). In certain individuals, such as the immunocompromised or those with AIDS or AIDS-related syndromes, waterborne infectious diseases, such as
* Corresponding author. Tel.: þ1 217 333 0945; fax: þ1 217 244 0323. E-mail address: [email protected] (P.K. Kalita). 0301-4797/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jenvman.2013.09.017
cryptosporidiosis, may be lethal. Infected calves can excrete in excess of 3 1010 Cryptosporidium oocysts over approximately a two week period (Nydam et al., 2001). Considering the mean human infective dose is approximately 130 oocysts (Chappell et al., 1999), only a few infected animals are needed to produce enough oocysts to contaminate a large watershed (Olsen et al., 1999). The risk of such contamination is not only a human health hazard but also a potential threat to the continued existence of small to medium-sized dairy farms and feedlots. To design and develop control strategies, microbial transport processes in surface and near-surface runoff need to be properly understood and quantified along with various factors that affect pathogen transmission in the environment. Recent research results measuring Cryptosporidium parvum oocyst transport in soil, using small and large scale, field-applicable experimental systems, indicate vegetative filter strips (VFS) are effective at reducing oocyst overland transport (Atwill et al., 2002; Davies et al., 2004; Tate et al., 2004; Trask et al., 2004). Although VFS appear to be a fieldapplicable and cost-effective best management practice, the mechanism by which they reduce oocyst overland transport is not understood. Furthermore, the ultimate design of optimally effective VFS for a particular watershed and set of contaminating microbial
122
S.J. McLaughlin et al. / Journal of Environmental Management 131 (2013) 121e128
pathogens, e.g., viruses as well as bacteria and protozoa, requires knowledge of the mechanism of interaction of the particular pathogen with VFS. Our previous results demonstrated VFS dramatically reduce both the kinetics and number of oocysts transported in surface and near-surface runoff (Trask et al., 2004). The objectives of this study were to investigate the oocysts adhesion to individual soil particles and compare of their movement on and within the soil profile of both bare and vegetated soil beds. 2. Materials and Methods 2.1. Preparation of Cryptosporidium parvum oocysts Oocysts were propagated, collected and purified from experimentally infected neonatal calves as previously described in Johnson et al. (2004) and Trask et al. (2004). Feces containing oocysts were collected in plastic drop pans which were maintained over melting ice. A100 mL pen/strep/amphotericin (containing 6 g penicillin, 10 g streptomycin, and 25 mg amphotericin per liter of 0.85% sterile saline) and 100 mL nystatin (containing 1.98 g per liter sterile saline) per 1 to 1.5 L of feces were added to the collection pans. The oocysts containing antibiotics were stored at 4 C until use. All procedures involving animals were part of protocols approved by the University of Illinois Institutional Animal Care and Use Committee (IACUC). 2.2. Soil particle separation A moderately drained silt-loam (Catlin series, mesic Oxyaquic Argiudolls) was collected from Champaign, IL. The soil was sifted using 3 mm screen to break up the larger pieces into more uniform sizes and remove large organic matter and rocks. The sifted soil sample was air-dried for 48 h. The air-dried soil was then ground down using a mortar and pestle. The ground soil was passed through a series of sieves with sieve openings that corresponded to the USDA classification of soil fractions according to the particle diameter. None of the samples contained any particles that were classified as gravel. Soil samples were separated into six categories using the sieves; very coarse sand, coarse sand, medium sand, fine sand, very fine sand and a combination of silt and clay. The sieves are #18, which corresponds to a 1 mm opening to hold the very coarse sand, #35 with a 500 mm opening that holds the coarse sand, #60 with a 250 mm opening that holds the medium sand, #140 with an opening of 106 mm which will hold the fine sand, and #270 with an opening of 53 mm which will hold the very fine sand. Anything that passed through the #270 sieve was classified as silt and clay. The soil was placed in the top sieve and left on a vibrating auto sifter for 30 min. The bottom collection pan contained the silt and clay. The silt and clay were then separated using the principles of Stokes Law based on the settling time of the clay particles (diameter of 2 mm or less). The soil was placed in a beaker with distilled water and stirred for 30 s, then left to settle for over 7 h. The soil that was settled at the bottom of the suspended clay was classified as silt, which was then oven dried. The suspended clay was transferred to a large beaker, oven dried, and the clay collected. This process was repeated numerous times to ensure adequate amount of clay particles were collected for microbial adhesion experiments. 2.3. Oocysts-soil adhesion Oocyst-soil adhesion experiments were conducted using two methods. The first method uses a Coulter electronic particle counter (Model XM) to measure the rate of disappearance of single oocysts as the form aggregates with individual soil particles. The Coulter
electronic particle counter discriminates between particles of different volumes, and can determine the size distribution and the number of particles in the suspension. The method had been previously adapted for use in cell adhesion experiments (Obrink et al., 1977; Orr and Roseman, 1969; Schmell et al., 1982). In the Coulter Counter oocyst-soil particle adhesion experiments, the adhesion of oocysts to a particular soil particle is measured by quantifying the loss of oocysts from an oocyst “window” calibrated to count only single, non-aggregated oocysts. As the oocysts adhere to a soil particle they increase in size (volume) and thus disappear from the oocysts window. The Coulter counter settings defining the oocysts window were: upper threshold (Ut) ¼ 3.4 mm, lower threshold (Lt) ¼ 4.9 mm; attenuation ¼ 2, aperture current ¼ 400 mA; using a 100 m aperture probe and 500 ml manometer volume. In the second method, used to qualitatively estimate oocyst adhesion to the larger sand particles (sand particles are too large to accurately measure using the Coulter Counter), as well as confirm results obtained with the Coulter Counter method, oocysts were mixed and incubated with the soil particles as described in method 1, but visualized using differential contrast and fluorescent microscopy following staining of the oocysts with a FITC-labeled, oocystspecific monoclonal antibody (Crypt-a-Glo; Waterborne, Inc). Fluorescent staining of oocyst-soil particle incubations was performed by placing an aliquot (40 ml) of the incubation onto a precleaned (70% ethanol) microscope slide, air drying the slide on a slide warmer, and incubating with the FITC-Mab for 30 min at 37 C followed by washing according to manufacturer’s suggestions. The initial number of soil particles and oocysts added to adhesion incubations was determined either by Coulter counting or by enumeration in a hemocytometer. Approximately 2.5 104 oocysts were mixed with 5 104 soil particles in a final volume of 300 ml in a 1.5 ml microfuge tube and incubated at room temperature for various times by end over end rotation at 5.5 rpm. 2.4. Radiolabeling of oocysts Gradient purified oocysts [1.5 107] were washed in PBS three times and brought to a final volume of 100 ml. One mCi of Na125I (IMS 30, Amersham Biosciences, NJ) was placed in an IODO-GEN (Pierce Biotechnology, IL) pre-coated iodination tube with 20 mL of 0.5M Sodium Phosphate (NaHPO4) buffer with a pH of 7.1. The oocysts were then added to the IODO-GEN tube to start the labeling reaction. The IODO-GEN tube was agitated every few minutes to help deter settling of oocysts during incubation. The reaction was stopped after 15 min by removing the mixture from IODO-GEN tube and adding 2 mL of 1.0 g/ml potassium iodide. The oocysts are washed free of unbound 125I by centrifuging (15s, 11,000 g) three times with 1 mL PBS, and then stored in 500 ml final volume, 4 C until use. Before application to bed, the radioactive oocysts suspension was diluted to 10 ml with PBS. An aliquot of the final solution of oocysts was counted in a gamma counter to determine the specific radioactivity (cpm/oocyst) of the final oocyst suspension. 2.5. Oocysts overland transport on small-scale soil beds For measurement of oocyst overland transport and soil distribution following simulated rainfall, small scale tilting soil beds were constructed using 1.91 cm plywood boxes measuring 0.91 m long by 0.30 m wide and 4.45 cm deep. The boxes were sealed with silicone and then lined with isotope paper to prevent leaking or contamination to other surfaces. A 32 mm piece of clear plexiglass was used to split the bed into two chambers (one for vegetation and the other for bare soil). Boxes were then filled with soil (Catlin series, mesic Oxyaquic Argiudolls). The grass chamber was seeded
S.J. McLaughlin et al. / Journal of Environmental Management 131 (2013) 121e128
with tall fescue at a high seeding rate to ensure that adequate coverage was achieved. A collection system was installed to collect all surface and shallow subsurface runoff from the bed. The runoff from the surface and subsurface drains was collected in separate containers so the number of oocysts lost in runoff during the experiments could be quantified. The water (simulated rain) was applied with a watering can by pouring at a slow rate over the entire bed placed at a 3% slope. Radioactive oocysts were placed on the slightly damp surface 12.7 cm from the top of the bed (i.e., the top of the slope) in a small band across both the vegetated and bare soil sides of the bed. Immediately following the application, 7.1 L of rainfall was applied to the bed to ensure adequate runoff. When runoff ceased, representative runoff and soil samples were taken and counted for radioactivity as described below. The number of oocysts (1.5 107) and amount of rainfall applied was proportional to the amount used in our previous studies (Trask et al., 2001, 2004) employing a large-scale soil bed and rainfall simulator and based on a comparison of soil surface areas of both types of soil beds. 2.6. Measurement of oocyst distribution following overland transport To determine the oocyst distribution in the soil beds and adhesion to surface vegetation following overland transport, soil samples were taken with a cork bore every 4.5 cm on the left, right, and center along the length of the vegetated and bare soil beds after the rainfall ceased, placed in a test tube, and counted for radioactivity. Following quantification of radioactivity, core samples of highest oocyst concentrations were cross-sectioned from the beds for further analysis. A 2.54 cm by 5.08 cm core sample was taken from these areas so that the depth of infiltration of oocysts could be compared between vegetated and bare soil. Soil sections were divided (cut into cross-sections) every 0.5 cm from the soil surface to the bottom of the bed and counted for radioactivity. Vegetation was divided into three sections: 0e3 cm, 3e6 cm and greater than 6 cm from the soil surface, and each section was counted for
123
radioactivity. The grass root structure was included in the 0.5 cm sectioning of the soil profile. 2.7. Oocyst transport in vertical soil columns Vertical soil columns of 0.9 m height and 0.4 m diameter were constructed with rigid PVC pipe (Fig. 1). Two soils (Catlin and Newberry series) and three vegetations (Brome, Reed Canary, and Kentucky Blue grass) were tested using low (1 108) and high (1 109) doses of oocysts per column. Oocyst recoveries were determined as previously described in Trask et al. (2004) following a water load of 3 applications (10 L per application) per column. 3. Results 3.1. Adhesion of oocysts to silt, sand and clay soil particles Individual silt and clay particles were harvested, incubated with oocysts at 22 C for varying times, and adhesion to each other or oocysts was measured using a Coulter counter. The size profile of C. parvum oocysts as well as clay and silt particles that was used to define the lower and upper thresholds of a single cell (oocysts) window is shown in Fig. 2A. The diameter of both purified clay and silt particles (
