Environ. Sci. Technol. 2005, 39, 2636-2644
Settling Behavior of Unpurified Cryptosporidium Oocysts in Laboratory Settling Columns PAMELA L. YOUNG* AND SIMEON J. KOMISAR Department of Civil and Environmental Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180
The settling behavior of fresh and aged unpurified oocysts was examined in settling column suspensions with varied ionic strengths and concentrations of calcium and magnesium. Independent measurements of the size and density of unpurified oocysts were performed to determine a theoretical settling velocity for the test populations. Viability of the oocysts was assessed using a dye permeability assay. Latex microspheres were included to provide a standard by which to assess the settling conditions in the columns. Mean settling velocities for viable oocysts measured in this work were faster than predicted and faster than measured for purified oocysts in other work: 1.31 ((0.21) µm/s for viable oocysts from populations having a low percentage of viable oocysts and 1.05 ((0.20) µm/s for viable oocysts from populations with a high percentage of viable oocysts. Results were attributed to the higher than previously reported densities measured for oocysts in this study and the presence of fecal material, which allowed opportunity for particle agglomeration. Settling velocity of oocysts was significantly related to the viability of the population, particle concentration, ionic strength, and presence of calcium and magnesium in the suspending medium. Behavior of the latex microspheres was not entirely predictive of the behavior of the oocysts under the test conditions. Viable oocysts may have a greater probability of settling than previously assumed; however, nonviable, and especially nonintact, oocysts have the potential to be significantly transported in water. This work underscores the importance of assessing the viability of oocysts to predict their response to environmental and experimental conditions.
controlling the transport and fate of Cryptosporidium in the environment and in water treatment plants. The settling velocity of Cryptosporidium oocysts has been estimated on the basis of Stoke’s Law and has been measured in laboratory settling columns. Ives (1) predicted that oocysts would settle at a rate of 0.5 µm/s, and Chapra (2) predicted a rate of 0.92 µm/s, given temperature and viscosity of the suspending medium and published measurements of the diameter and density of purified oocysts. Medema et al. (3) indirectly measured the settling velocity of purified oocysts in settling tubes, assigning an initial settling velocity of 0.35 µm/s to their test population. Several studies have noted that Cryptosporidium oocysts may not occur as discrete particles in the environment but may have a tendency to stick to other particles or occur in clumps (4-7). The tendency for oocysts to adhere to other particles might be conferred by the surface chemistry of the oocyst wall. Brush et al. (8) presented evidence that purification methods employing ethyl acetate, formalin, and Percoll may alter the surface characteristics of the oocyst, changing their net surface charge from neutral to negative. Brush et al. (8) also found that the hydrophobicity of oocysts purified in deionized water and sucrose (DIS) varies as a function of oocyst age and ionic strength of the suspending medium. The work of Brush et al. (8) suggests that the process of purifying Cryptosporidium oocysts and aging may alter their surface characteristics. In addition, Medema et al. (3) have demonstrated that the settling behavior of oocysts is impacted by contact with other particles in suspension. Since Cryptosporidium does not occur in nature as artificially purified oocysts or in absence of other particles, the research presented here was designed to examine the settling behavior of unprocessed oocysts in the presence of the particles with which they are originally deposited (i.e., oocysts unpurified in their native fecal material). Settling velocities were derived for fresh and aged unpurified Cryptosporidium oocysts under quiescent settling conditions in aqueous suspensions of oocyst-containing bovine fecal material. Suspensions in laboratory settling columns were varied to represent ionic strengths and concentrations of calcium and magnesium likely to be encountered in the natural environment. Independent measurements of the size and density of unpurified oocysts were also performed to determine a theoretical settling velocity for the test populations. Latex microspheres were added to the test columns to provide a standard by which to assess the settling conditions in the columns. Settling velocities observed for the test populations of oocysts were compared to one another, to their theoretical velocities, and to the velocities observed for the microspheres to assess the impacts of the various experimental treatments.
Introduction
Materials and Methods
The resistance of the waterborne pathogen Cryptosporidium parvum to conventional disinfection practices has shifted focus to source water protection and removal techniques for minimizing the occurrence of these microorganisms in drinking water. The extent to which land-deposited Cryptosporidium oocysts might be transported to a raw water intake is determined in part by their settling characteristics. As well, the effectiveness of removal processes in water treatment is in part dependent on the extent to which Cryptosporidium oocysts will settle. For these reasons, understanding the potential for oocysts to settle is integral to predicting and
Unpurified Cryptosporidium Oocysts. Fecal material was obtained from five less than 10-day-old calves that were infected with Cryptosporidium parvum (Excelsior Sentinel, Inc., Ithaca, NY). Oocysts described as “fresh” in this study were from feces that had been stored 1 month or less in tightly capped plastic tubes at 4 °C; “aged” oocysts were stored for 2 months or more. Detection of Oocysts in Samples. The concentrations of oocysts in each batch of feces and in samples from settling experiments were determined using an immunofluorescence assay (IFA). For the fecal material, the concentration of oocysts in each batch was determined by preparing suspensions of known dilution of the fecal material in distilled (DI) water. For both the fecal dilutions and the samples from the
* Corresponding author phone: (518)584-1780; e-mail: ply01@ health.state.ny.us 2636
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10.1021/es040470j CCC: $30.25
2005 American Chemical Society Published on Web 03/15/2005
settling columns, two to four (typically three) 100-µL subsamples were filtered onto 13-mm Whatman Nucleopore membrane filters, pore size 0.8 µm. A filtered sample was incubated for 40 min in the dark at room temperature with 100 µL of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody for Cryptosporidium parvum (AquaGlo, Waterborne Inc., LA) diluted 1:20 with AusFlow mAbBuffer (0.89 g tetra-sodium pyrophosphate, 500 µL 0.05% w/v Tween 80 solution, 500 µL 0.05% w/v sodium azide, and 5 g BSA albumin, in 1 L DI water, adjusted to pH 8 with HCl, and filtered through 0.2-µm filter). At the end of the incubation period, the filter was rinsed with a few drops of AusFlow FlowBuffer (same as AusFlow mAbBuffer without the BSA albumin added) and then placed on 5 µL of AusFlow mounting medium (2 mL glycerol, 2.4 mL DI water with 100 mg/mL DABCO, 4.8 mL 1 M TRIS buffer, 0.5 mL formalin, and 0.5 mL 5 M NaCl, adjusted to pH 8.6 using 1 M HCl) on a glass microscope slide. Entire filters were examined for oocysts at 400× magnification using a Nikon epifluorescent microscope (excitation at 480/30 nm and emission at 535/40 nm). Oocysts were identified as bright apple-green fluorescing ovoid to spherical objects with brightly highlighted perimeters and 3-6 µm in diameter, as measured by a calibrated ocular grid. Assessment of Viability of Cryptosporidium Oocysts. Oocysts in the fecal material and in the settling column samples drawn initially and at the end of the tests from the upper and lower column were exposed to a dye permeability assay, as described by Anguish and Ghiorse (6). Briefly, 100µL samples were incubated with 10 µL each of solutions of 4′,6-diamidino-2-phenylindole (DAPI) (2 mg/mL in HPLCgrade methanol) and propidium iodide (PI) (1 mg/mL in 0.1 M phosphate buffered saline (PBS) (8.1 mM Na2HPO4‚12 H2O, 1.47 mM KH2PO4, 0.138 M NaCl, 2.7 mM KCl at pH 7.4)) at 37 °C for 1.5 h and then with 100 µL of diluted Aqua-Glo for an additional 0.5 h. Samples were washed twice in 0.1 M PBS and resuspended in 100 µL of 0.3 M 1,4-diazabicyclo[2.2.2]octane in 0.1 M PBS. Ten-microliter samples were pipetted onto 1% agar-coated microscope slides for examination under the epifluorescent microscope. Oocysts identified by FITC staining were examined with appropriate microscope filter sets for inclusion of DAPI (excitation 330380 nm and emission 420 nm) and PI (excitation 510-560 nm and emission 590 nm). FITC-positive oocysts that did not exhibit inclusion of DAPI or PI were further examined under differential interference contrast (DIC) for the presence of internal structures. Oocysts with internal structures were classified as intact (I), while those without internal structures were identified as nonintact (NI). While various relationships have been demonstrated between the permeability of oocysts to vital dyes and their infectivity (e.g., 9-13), for the purposes of this work, we adopted a classification system based on dye permeability to describe the “viability” of our oocysts (i.e., the nomenclature used by Anguish and Ghiorse (6)): DAPI-PI-, I and DAPI+PI-, I oocysts are referred to as viable; DAPI+PI+, I and DAPI-PI-, NI oocysts are considered nonviable. Size Measurement of Unpurified Cryptosporidium Oocysts. Latex microspheres were measured using both the epifluorescent microscope and a Beckman Coulter Multisizer. When viewed under the microscope, the diameters of these fluorescent objects appeared larger than their specified sizes, while the Multisizer results accurately reflected the specified sizes. The diameters of oocysts from fecal material were therefore assessed using the Multisizer (n ) 1000). To make these measurements, oocysts were first separated from 3-month-old fecal material using a Becton Dickinson FACSCalibur Flow Cytometer (14). Oocysts were identified and sorted from the samples on the basis of their characteristic fluorescence and light-scattering responses, which had been
established by examining FITC-stained DIS-purified oocysts using the flow cytometer. The numbers of oocysts sorted from the samples were within the range expected on the basis of direct counts of oocysts in the fecal material, indicating that the sorting criteria were not selectively eliminating some segment of the oocyst population. Determination of Specific Gravity of Cryptosporidium Oocysts. The specific gravity of unpurified Cryptosporidium oocysts was determined using an isopycnic gradient centrifugation technique (15). Specific gravity measurements were performed on unpurified 11-week-old oocysts, unpurified 12-week-old oocysts, and unpurified 10-day-old oocysts. Settling Column Tests. Sedimentation rates for Cryptosporidium oocysts in fecal material were measured in side-byside duplicate settling columns. Each column was constructed of glass tubing with sample ports mounted vertically in a poly(vinyl chloride) (PVC) and metal gate valve sealed with a PVC base (Figure 1). The settling columns were clamped to metal stands, which were mounted on a pneumatically damped vibration-free table (Melles Griot StableTop 150 optical table). Each column was wrapped with vinyl tubing through which 26 °C water was pumped by a circulating water bath (Neslab model RTE221). A Styrofoam cap and fiberglass insulation encasing each apparatus further aided thermal stability to within (0.2 °C. Oocyst-containing fecal material was gently stirred into 200 mL of DI water along with fluorescent latex microspheres. Diameters of the latex microspheres were 2.977 ((0.101) µm, 3.92 or 4.0 (standard deviation N/A) µm, and 5.75 ((0.262) µm or 5.895 µm (2.977, 5.75, and 5.895 µm spheres manufactured by Polysciences, Inc., Warrington, PA; 3.92 and 4.0 µm spheres by Spherotech, Inc., Libertyville, IL). Density of all latex microspheres was reported to be 1.05 g/cm3. Microspheres could be detected in the samples using the appropriate filter sets on the epifluorescent microscope. The suspension of fecal material and latex microspheres was stirred into approximately 10.5 L of DI water, which had been warmed to 26 °C and mixed with NaHCO3, KCl, MgSO4, and CaCl2 to produce the test conditions of ionic strength and divalent cation concentration. Ionic strength was formulated to simulate the ionic strength of freshwater (low, conductivity approximating 100 µS/cm) or that of domestic wastewater (high, conductivity approximating 1600 µS/cm) (16). When present, divalent cations Ca2+ and Mg2+ were in concentrations typical of a low alkalinity surface water. In Test #10, 0.1 × Hank’s Balanced Salt Solution (HBSS) was used as the suspending medium to more closely simulate conditions used by Medema et al. (3). The test conditions used in each experiment are given in Table 1. The suspension was poured into the settling column, 0.5 mL of column contents was flushed through the sample needles, and 1.5 mL of sample was immediately drawn from a sample port at each depth in the column using the syringes. Sample ports were flushed and samples were collected at 24, 48, 72, and 96 h after the start of a test. Forty-five minutes after the start of a test, the gate valve in the base of the column was closed to segregate the fastest settling particles in the suspension. All samples were immediately chilled to 4 °C until analysis. As initial results (data not shown) indicated that there was no significant difference between concentrations of oocysts or latex beads at the various depths for a given sample time, the eight samples drawn from a column at a given sample time were combined into a single glass vial. After the 96-h sample had been collected, the remaining column contents from the upper (above the drain tube), middle, and lower (below the gate valve) column were collected separately. Concentrations of oocysts and latex microspheres in suspension in each of these sections were VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Settling column apparatus for determination of settling characteristics of unpurified Cryptosporidium oocysts. Detail of syringe samplers shown on one port only. (Not to scale.)
TABLE 1. Conditions of Fecal Material and Suspending Media for Settling Column Tests test #
oocyst age(wk)
mass of feces (g)
particle concentrated (#/mL × 106)
% viable at to
ionic strength
1 2 3 4 5 6 7 8 9 10
0.3 3.5 5 0.3 13 16 4.7 10 21 14
3 3 3 29 3 3 8 2 2 2
1.26 1.10 1.01 2.39 0.92 1.05 1.09 0.62 0.60 0.73
75 66 66 91 20 20 74 75 28 NA
low high high low low high high high high high
determined to calculate a mass balance for these particles in the settling column and thereby assess if particles had been lost to the test apparatus. Determination of Settling Velocities in a Completely Mixed Settling Column. Settling theory would predict that in a quiescent column initially containing a uniform suspension of particles with various settling velocities, one would observe the concentration of particles in the upper column to decrease with time, while the concentration in the lower column would increase. Although this study was designed to produce quiescent settling conditions, in initial settling tests (data not shown), the concentrations of particles at different depths were not found to be significantly different from one another at a given sample time, yet the overall concentration in the suspension decreased with time (i.e., the settling column contents were mixing but were also settling). Given these results, the model of Einstein (17) (eq 1) for settling of particles in a completely mixed column of fluid was employed to assign a Stoke’s settling velocity to the particles examined in this work, where nt is the number concentration of particles at time t, no is the initial number 2638
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divalent cations
X X X X X
NaHCO3 (mg/L)
KCl (mg/L)
57 57 57 57 57 57 57 57 57 35
13 76 76 13 13 76 76 76 76 40
CaCl2 (mg/L)
MgSO4 (mg/L)
4.1
1.6
5.1 5.1 5.1 18.5
2.0 2.0 2.0 20
concentration of particles, vs is the settling velocity, and H is the height of the fluid column.
vs t n t ) n o eH
(1)
The number concentrations of suspended oocysts or latex beads at each sample time divided by their initial concentrations were plotted against the sample time divided by the column height (96 cm). Using the nonlinear regression function in SigmaStat (Jandel Scientific, San Rafael, CA) and the model for settling in a completely mixed column (eq 1), vs was selected to produce a best fit of the model to the experimental data from each settling column. Determination of Settling Velocity of Viable Oocysts Only. Initial analysis of settling results suggested that oocyst settling velocity decreased as the age of the fecal material increased. At the same time, the proportion of viable oocysts in the fecal material used in these experiments declined as the fecal material aged. Our concurrent measurement of oocyst specific gravity (15) revealed that viable oocysts had
TABLE 2. Modal Density Ranges and Mean Densities for Populations of Unpurified Cryptosporidium Oocysts Determined Using Isopycnic Gradient Centrifugation age of feces (wks)
modal density range (kg/m3)
mean density of population ((SD) (kg/m3)
1.4 11 11 12
1070-1073 1010-1016 (lower density peak) 1079-1108 (higher density peak) 1077-1080 (entire gradient)
1074 ((12) 1077 ((37)
a higher specific gravity than both intact nonviable oocysts and nonintact oocysts. In addition, the specific gravity of viable oocysts varied as a function of the percentage of viable oocysts in the source fecal material (i.e., viable oocysts from populations that had a low percentage of viability were denser than those from populations with a higher percentage of viability). Since we were primarily interested in the settling characteristics of viable oocysts, the settling test results were reanalyzed to isolate the settling behavior of only the viable oocysts. Loss of viability of oocysts has been modeled in other work as a first-order reaction (3). Assuming the decrease in viability of oocysts with time followed a first-order decay function in our experiments, the initial concentration of viable oocysts and the final concentration of viable oocysts measured in the bottom of the column (contents of the bottom of the column were isolated by the gate valve at the beginning of each test and then were mixed before being sampled) could be used to calculate a first-order decay constant (k) for loss of viability over the course of the experiment:
nbt ) noe-kt
(2)
where no is the initial number concentration of viable oocysts in the column suspension, nbt is the concentration of viable oocysts in the bottom of the column at the end of the experiment, and t is the duration of the experiment. Since the change in number of viable oocysts (nvt) in suspension was due to both decay and settling, eq 1 and eq 2 were combined:
nvt ) noe-
( ) k+
vs H
t
(3)
For each sample time, the difference between the number of oocysts in the suspension at the previous sample time and the sum of the number detected plus the number predicted to have decayed in the time interval (eq 2) would represent the number of viable oocysts that had settled during the time interval. Nonlinear regression was then performed to select a vs that produced a best fit of eq 1 to the change in the number of viable oocysts because of settling. The settling velocities derived for the unpurified Cryptosporidium oocysts and latex microspheres were statistically compared using one-way and two-way Analysis of Variance (ANOVA) (SigmaStat, Jandel Scientific, San Rafael, CA) to determine if settling velocities varied in relation to the test conditions. One-sample t-tests, using a significance level of P ) 0.05, were used to determine if settling velocities in the columns were different from velocities predicted by Stoke’s Law on the basis of measured diameters and densities. Particle Size Analysis. Particle size analysis was performed on three 0.01-0.03 mL unvortexed aliquots from each column at each sample time using a Beckman Coulter Multisizer, which assigns an equivalent spherical diameter to a particle on the basis of a measurement of volume displacement. To compare the rate of disappearance of particles in the different settling experiments, the change in volume of particles in three size ranges, 1.5-4.4 µm, 4.4-7.4 µm, and all sizes, was measured with time. A first-order decay function (eq 4) was
1067 ((22)
fit to the particle size data from each column:
Vt ) Voe-kpt
(4)
where Vo is the initial volume of particles of a given size range, Vt is the volume of particles of that size with time (t), and kp is the first-order decay constant for disappearance of particles in the size range from suspension. The resulting kp values from each test were then compared using one-way or two-way ANOVA to determine if the rate of disappearance of the particle size groups was significantly related to any of the test variables.
Results Size and Specific Gravity of Unpurified Oocysts. The modal diameter of oocysts measured using the Multisizer was 4.3 µm. The modal density ranges (i.e., the range of densities represented in the fraction of the density gradient that contained the greatest number of oocysts per unit density) for unpurified and purified oocysts are given in Table 2 (specific gravities have been converted to densities using 996.8 kg/m3 as the density of water at 26 °C). The 11-weekold unpurified oocysts exhibited a bimodal distribution of densities. More than 75% of the oocysts whose densities were in the 1010-1016 kg/m3 range were determined to be nonintact when examined by DIC microscopy. For the 12week-old fecal material, greater than 75% of oocysts in the 1005-1024 kg/m3 fraction of the gradient were also nonintact, while 68% of the oocysts in the fraction representing the densities 1024-1041 kg/m3 were nonviable but had internal structures (DAPI+PI+). For all the fecal batches, the oocyst populations in the denser fractions had a greater percentage of viable oocysts than the original population that had been centrifuged. Mass Balance and Rapid Settling. The percentage of recovery of oocysts from the columns at the end of the tests ranged from 75 to 117% with a mean of 89 ((19)%. Mean recoveries for the microspheres ranged from 72 to 82%. Concentrations of oocysts and microspheres detected in suspension samples were adjusted accordingly to account for losses to the experimental apparatus in each test. The concentration of oocysts in the bottom section of the settling columns, which was isolated from the upper column by a gate valve 45 min after the columns were filled, was not significantly different from the initial concentration, indicating that the number of oocysts that settled faster than approximately 400 µm/s under the test conditions was minimal. Settling Velocities of Unpurified Oocysts and Latex Microspheres. A representative plot of the change in concentration of oocysts and latex microspheres remaining in suspension with time divided by column height is shown in Figure 2. Einstein’s (17) model for settling in a completely mixed column (eq 1) has been fit to these data. To avoid sampling errors due to varying initial concentrations of oocysts in the test columns, the amount of fecal material used in each settling test was selected to provide a consistent number concentration of oocysts in each test. However, batch-to-batch variability in the concentration of oocysts in fecal material (concentrations ranged from 1.6 × VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Fractions of initial concentrations of Cryptosporidium oocysts and latex microspheres remaining in suspension in settling columns as a function of time divided by settling column height. This test used fresh fecal material with a high percentage of viable oocysts, high ionic strength, and no Ca2+ or Mg2+. Model for settling in a completely mixed column (17) has been fit to the data.
TABLE 3. Summary of Relationships between Test Variables and Settling Velocities of Oocysts and Latex Microspheres in Laboratory Settling Columnse % viability high low
test particle all oocysts (viable and nonviable) viable oocystsb 2.97 µm microspheres 3.92 µm microspheres 5.8 µm microspheres
higher particle concentration
ionic strength high low
Xa
X
X
Ca2+ and Mg2+ added X
Xc X
X
Xd X
When combined with and or fresh fecal material. Smaller data set limited statistical analysis. c P < 0.10. d Low percent viability covaried with absence of Ca2+ and Mg2+. e X ) settling velocity of test particle faster in the presence of test variable (P < 0.05) a
Ca2+
Mg2+
b
105 to 1.8 × 107 oocysts/g of wet fecal material) resulted in differences in initial particle number concentrations among the tests. In particular, Tests #8 and #9 had much lower particle concentrations than Tests #1-7 (Table 1). Although the number concentration of particles was not originally a test variable, ANOVA tests indicated that settling velocities of the oocysts and the 5.8-µm microspheres were significantly related to the number of particles in suspension. Inclusion of this inadvertent variable subsequently reduced the statistical power of the data set. However, significant relationships between settling velocities of the oocysts and latex spheres and the test variables were detected (Table 3). Results for Latex Microspheres. When the results from all the tests were compared, two-way ANOVA revealed the 5.8µm beads settled significantly faster (P < 0.05) in suspensions containing high particle numbers. Within the group of tests having high particle concentration, no significant differences in the settling velocities of the 5.8-µm spheres that corresponded to oocyst viability, ionic strength, or divalent cation level were detected. Within this group, the 3.92-µm microspheres settled significantly faster in suspensions containing fecal material with a high percentage of viable oocysts when the effect of ionic strength was accounted for (P ) 0.033). However, in this group of tests, a low percentage of viability covaried with absence of Ca2+ and Mg2+, which may have confounded the results. In the tests that had higher particle counts, the 2.977-µm microspheres settled fastest in the presence of high ionic strength and divalent cations. In general, the settling velocities of the microspheres in the settling columns were greater than predicted by Stoke’s 2640
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Law. The mean settling velocities for all tests were 0.50 ((0.21) µm/s for the 2.977-µm spheres, 0.64 ((0.19) µm/s for the 3.92-µm spheres, and 1.17 ((0.31) µm/s for the 5.8-µm microspheres, compared to the predicted settling velocities for these sized spheres: 0.29, 0.51, and 1.09 (5.75 µm) or 1.15 µm/s (5.89 µm), respectively. One-sample t-tests showed that the mean derived velocities for the 2.977- and the 3.92-µm spheres were significantly faster than their predicted velocities, while the mean settling velocity derived for the 5.8-µm spheres was not significantly different from their theoretical velocity. If one assumes that the settling conditions in the test columns influenced the settling behavior of the Cryptosporidium oocysts in the same way that the latex microspheres were impacted, then the relationship between the settling velocities derived for the spheres and their theoretical velocities could be used as a model to predict the observed velocities of the oocysts on the basis of their theoretical velocities. The mean observed settling velocities for the microspheres were plotted against their theoretical velocities (Figure 3). The relationship described by the linear regression line fit to the data was significant at the P < 0.001 level (eq 5).
mean observed vs in settling column ) 0.839 × (theoretical vs) + 0.2377 (5) Results for Whole Populations of Cryptosporidium Oocysts (Viable and Nonviable). The mean settling velocities derived
FIGURE 3. Mean observed settling velocity (vs) ((1 SD) for latex microspheres and Cryptosporidium oocysts in settling column tests versus theoretical Stoke’s velocity. Linear regression line has been fit to data for latex microspheres only.
TABLE 4. Settling Velocities (vs) Observed for Unpurified Cryptosporidium Oocysts in Settling Columns and Results of One-Sample t-Test Results Comparing Observed and Predicted Velocities
oocyst population type Whole Populations all tests low % viability populations high % viability populations Viable Populations all tests from populations with low % viability from populations with high % viability
mean observed vsa (µm/s) ((SD, range)
theoretical vsb (µm/s)
theoretical vs significantly different from observed? (P < 0.05)
0.67 ((0.29, 0.22-1.06) 0.36 ((0.12, 0.22-0.47) 0.83 ((0.21, 0.59-1.06)
0.65 0.51 0.73
no yes no
0.78 0.67 0.85
no yes no
1.11 ((0.21, 0.87-1.44) 1.31 ((0.21, 1.18-1.44)
0.96 1.09
yes yes
1.04 1.15
no no
1.05 ((0.20, 0.87-1.19)
0.89
yes
0.98
no
a Test #10 results excluded. v for Test #10 was 1.78 µm/s. s theoretical Stoke’s Law velocity.
b
theoretical vs adjustedc (µm/s)
adjusted vs significantly different from observed? (P < 0.05)
Velocity predicted by Stoke’s Law. c Velocity predicted by eq 5 on the basis of
from the best fit of eq 1 to the data are presented in Table 4. The fastest settling velocities observed for the latex microspheres and oocysts occurred in Test #10, in which the suspending medium was 0.1 × HBSS. The pH of the suspension in this test decreased from 6.2 to 3.5 over 96 h. Since pH was not a controlled variable in Test #10 (suspensions were buffered against significant changes in pH in the other settling tests), the results of this test were not included in the statistical analyses of settling test results. When the other variables were accounted for, ANOVA tests indicated that settling velocities were significantly faster for whole populations of oocysts (viable and nonviable together) that had a high initial percentage of viability, had been stored for less than 1 month, and were suspended in a medium of low ionic strength or in a medium containing Ca2+ and Mg2+. Settling velocities of oocysts were also significantly faster in suspensions with higher particle number concentrations that also contained Ca2+ and Mg2+ or fresh fecal material. When only the tests that used less than 1-month-old (fresh) populations of oocysts (viable and nonviable) were compared (all these tests also had higher particle numbers), these populations settled fastest under conditions of low ionic strength combined with Ca2+ and Mg2+(P ) 0.044). When settling velocities of just the populations of oocysts in aged
fecal material were compared, these oocysts settled faster in the presence of Ca2+ and Mg2+ combined with high ionic strength and lower particle numbers as compared to aged oocysts in suspensions with no divalent cations added, low ionic strength, and high particle counts and in suspensions with no divalent cations added, high ionic strength, and high particle counts (P ) 0.057). However, since the presence of the divalent cations, the level of particle numbers, and the ionic strength covaried within this group, the effects of these variables could not be analyzed separately from one another. Using the densities derived in our laboratory and the mean diameter measured by the Multisizer, the theoretical settling velocities for viable oocysts from a population with a high percentage of viability, viable oocysts from a population with a low percentage of viability, nonviable intact oocysts, and nonintact oocysts were calculated from Stoke’s Law. On the basis of these predicted velocities and the mean proportions of the oocyst types in each test population, the composite velocities for the whole populations of oocysts were predicted (Table 4). Equation 5 was then used to convert these theoretical velocities to velocities predicted to be observed in the settling columns (Table 4). One-sample t-tests indicated that whole populations of oocysts from low viability fecal material settled significantly slower than predicted by Stoke’s Law, while settling velocities for populations with a high VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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percentage of viability and for all tests combined were not significantly different from theoretical velocities or from the velocities obtained when theoretical velocities were adjusted according to eq 5 (P < 0.05) (Table 4). Results for Viable Cryptosporidium Oocysts. The mean derived settling velocities for viable oocysts are shown in Table 4. When the effect of either particle concentration, ionic strength, or Ca2+ and Mg2+ was accounted for, two-way ANOVA indicated that the results for viable oocysts were reversed from those obtained for the whole populations of oocysts; the settling velocity of viable oocysts from a population with a low percentage of viability was greater than that of viable oocysts from a population with a high percentage of viability at a significance level of P < 0.10. No significant differences in settling velocities were detected in relation to varying particle number concentration, ionic strength, or Ca2+ and Mg2+ levels (P > 0.10 for all). However, the results of Tests #3 and #6 did not allow for isolation of the viable oocysts only. Consequently, the data set was limited for statistical analysis of the settling velocities of the viable oocysts and may not have allowed detection of differences in velocities that resulted from the various treatments. Using measured densities of 1074 and 1092 kg/m3 and a mean diameter of 4.3 µm, theoretical Stoke’s settling velocities of 0.89 and 1.09 µm/s were assigned to viable oocysts from a population with a high percentage of viable oocysts and to viable oocysts from a population with a low percentage of viable oocysts, respectively. Adjusting these velocities using eq 5, velocities of 0.98 µm/s and 1.15 µm/s are predicted for the high percent viability and the low percent viability populations, respectively. One-sample t-tests indicated that viable oocysts from fecal material that had either a low or high percentage of viable oocysts settled significantly faster than predicted by Stoke’s Law but did not settle significantly differently from the velocities predicted by eq 5 (Table 4). Results of Particle Size Analysis. Measurements of fecal particle sizes using the Multisizer indicated that 95% of the particles were less than or equal to 4.4 µm, and 99% were less than or equal to 7.4 µm. One- and two-way ANOVA tests revealed that the initial number of particles in the column suspension significantly affected the rate of disappearance of the 4.4-7.4 µm particles and all sizes combined (P < 0.05), when at least one of the other test variables was accounted for. When only the tests that had higher numbers of particles were compared (Tests #1-7), the rate of disappearance for each of the particle size groups was faster in the presence of Ca2+ and Mg2+ when the effect of ionic strength was accounted for (P < 0.001, P ) 0.002, and P < 0.001 for the 1.5-4.4 µm, 4.4-7.4 µm, and all sizes, respectively). The 1.54.4 µm particles also disappeared faster in high ionic strength suspensions (P ) 0.014).
Discussion The settling column results suggested that agglomeration of particles was occurring in the test suspensions. This conclusion was supported by the observations of greater than predicted settling velocities for the latex microspheres in the settling tests and faster settling velocities for the microspheres and fecal particles in the presence of higher particle numbers, higher ionic strength, and divalent cations, conditions that generally promote particle agglomeration. Derived settling rates for whole populations of unpurified Cryptosporidium oocysts ranged from 0.22 to 1.06 µm/s in nearly neutral pH suspensions. Except for the settling velocity of the whole population of oocysts in aged fecal material, the settling velocities derived for the unpurified Cryptosporidium oocysts in our tests were significantly faster than those predicted by Stoke’s Law, using the values for density and size measured in our laboratories. When predicted settling velocities were adjusted to reflect the faster-than-predicted 2642
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velocities of the latex spheres in the test columns, the observed settling velocities for the oocysts were not significantly different from what was predicted. These results suggest that the oocysts in the settling column suspensions were also attached to other particles. This finding supports the observations of Medema et al. (3), who reported that purified oocysts mixed with a settled sewage effluent settled with velocities characteristic of the larger particles in the effluent, implying that the oocysts were attaching to the sewage particles. However, settling velocities of the oocysts in our work were not increased to the extent observed by Medema et al. (3). The extent to which naturally occurring oocysts will attach to other particles under environmental conditions should be examined further. When the settling results for all tests were averaged, except for the results of whole populations of oocysts with a low percentage of viability, the settling velocity of the oocysts deviated from their predicted velocities in a manner that was similar to the latex microspheres. However, the behavior of the oocysts was markedly different from that of the microspheres in some important respects. First, the settling velocity of the whole populations with a low percentage of viability was significantly slower than their theoretical velocity, even when adjusted for the behavior of the microspheres. Second, while the latex microspheres exhibited faster settling velocities in the presence of high ionic strength, the whole populations of oocysts in less than 1-month-old fecal material were observed to have significantly faster velocities in a low ionic strength suspension. Finally, unlike for the microspheres, the combination of the oocysts having a range of sizes and densities, and having differential tendencies to attach to other particles, produces a range of settling velocities for the oocysts within a given population. While the model used in this study assigned a characteristic composite velocity to a test population, for a heterogeneous population, the model would tend to underestimate the velocity that characterizes the population at initial times and overestimate the velocity at later times (i.e., as the population of particles left in suspension becomes increasingly composed of slower settling particles). As can be seen in Figure 2, this was especially true for the oocysts. This effect was less apparent for the more homogeneous microsphere populations. These differences in behavior suggest that latex microspheres should not be used as a surrogate for Cryptosporidium oocysts in studies that seek to examine the settling and attachment behavior of oocysts, as well as transport mechanisms and treatment techniques that would be impacted by this behavior. In addition, these findings indicate that the heterogeneity of oocyst populations in regard to settling and attachment behavior should be controlled or accounted for when working with these microorganisms. While the observation of faster velocities for fresh oocysts in low ionic strength was not consistent with what one would predict on the basis of agglomeration theory, it is consistent with the observations of Brush et al. (8). In their study, which measured the hydrophobicity of DIS-purified oocysts as a function of oocyst age and ionic strength of the suspending medium, greater than 80% of fresh (2-week-old) Cryptosporidium oocysts attached to a hydrophobic surface in low ionic strength (0-20 mM or with conductivity less than ∼740 µS/cm), while only 10-20% adhered under conditions of higher ionic strength (20-95 mM or conductivity ∼7401850 µS/cm). The physical explanation for these observations is not clear at this time and bears further investigation. Although our limited data set did not allow discrimination between the effects of the divalent cations and ionic strength on the settling velocity of aged oocysts in our work, we did observe that fastest settling velocities for populations in aged fecal material occurred under conditions of high ionic strength combined with the presence of Ca2+ and Mg2+ and
that aged viable oocysts settled significantly faster than fresh viable oocysts. While this latter result would be expected on the basis of our measurement of greater densities associated with aged viable oocysts, it may also be a consequence of aged oocysts having a greater affinity than fresh oocysts for attachment to other particles, at least under some conditions. Brush et al. (8) also found that a higher percentage of aged oocysts than fresh oocysts attached to a hydrophobic substrate under conditions of high ionic strength. In their work, approximately 60-80% of aged oocysts (>2 months old) adhered to a hydrophobic substrate in all the ionic strengths tested. While most of the test particles in the columns settled more quickly than predicted by settling theory for discrete particles, the whole populations of oocysts in aged fecal material settled significantly more slowly than predicted, even when the lower density measured for nonviable oocysts was accounted for. Two possible explanations may account for this result: (1) although our results provided evidence that aged viable oocysts may have a greater affinity for attachment to other particles, aged nonviable oocysts may have a lesser tendency to attach to other particles and (2) the shape of nonintact oocysts (that of a split-open spheroid) may confer a greater coefficient of drag, which would decrease their settling velocity. The first explanation is supported by the results of Walker and Montemagno (18). While both desiccated and frozen oocysts adhered to Al2O3 in their work, 61% of the frozen oocysts, of which 4% were nonintact, adhered, while a lower percentage, 43%, of the desiccated oocysts, of which 29% were nonintact, adhered, suggesting that nonintact oocysts did not adhere to the Al2O3. An additional observation from our study suggests that nonintact oocysts do not readily settle: in most cases, the concentration of nonintact oocysts remaining in suspension in the settling columns at the end of a settling test was either greater than or similar to the concentration detected in the valved off bottom of the settling column, where decay of oocysts, but not settling, occurred (i.e., the contents of the bottom section were resuspended before sampling). The mean velocity derived for oocysts from whole populations that had a low initial percentage of viability was 0.36 ((0.12) µm/s. This velocity is similar to that determined by Medema et al. (3), who assigned an initial composite settling velocity of 0.35 µm/s to purified oocysts suspended in particle-free 1 × HBSS. Although the population of oocysts used in Medema et al.’s (3) work would be considered aged by our definition (2-8 months old), and would therefore be expected to contain a high percentage of nonviable and nonintact oocysts, 99% of the population they used showed internal structures under DIC microscopy and 78% of their Giardia cysts showed viable type morphology (the percentage of viable type morphology seen in their Cryptosporidium was not given). Given this high percentage of intact (and possibly viable) oocysts, one would have expected their population of oocysts to have exhibited a faster settling rate, comparable to that predicted for our population that had a high percentage of viability. A high percentage of nonviable oocysts in their initial population or a rapid decay of their aged oocysts after they were introduced to their settling column conditions (23 °C for 72 h) may have contributed to the slower velocities observed by Medema et al. (3). Unpurified oocysts in our work in a suspension of 0.1 × HBSS, with pH that decreased to 3.5, exhibited the fastest settling velocity observed in any treatment (Test #10, 1.78 µm/s). Again, this result demonstrates the impact that the presence of fecal particles can have on the settling behavior of at least aged oocysts, particularly in the presence of conditions that are conducive to agglomeration of negatively charged particles (i.e., higher ionic strength, higher levels of divalent cations, and lower pH). However, even under the
relatively quiescent settling conditions provided in our study, after 96 h only 52% of the total population of oocysts was removed from the water column by sedimentation in this test. Since little settling of nonintact oocysts was observed in our work, the percent removal of viable oocysts may have been greater than that seen for the total population in this test (this could not be evaluated since dye permeability tests were not performed in Test #10). Derived settling velocities for unpurified viable oocysts ranged from 0.87 to 1.44 µm/s. While the settling velocities derived for unpurified Cryptosporidium oocysts in this study were faster than have been previously measured and were somewhat faster than the values that have been used previously for oocyst settling in models of Cryptosporidium transport (e.g., 19, 20), sedimentation velocities of this magnitude are not likely to lead to significant removal of oocysts from water columns. The results of this study suggest that a high percentage of Cryptosporidium oocysts introduced to waterways in fecal material can remain suspended in the water column for a long time and therefore have the potential to be significantly transported by water in the environment. However, the potential for transport is greater for slower settling nonintact, and presumably noninfective oocysts, emphasizing the importance of assessing the viability of Cryptosporidium oocysts detected at the drinking water intake. Since the viability of an oocyst and the overall viability of the population with which it is associated (as assessed by dye permeability and DIC microscopy) significantly influenced the density and perhaps attachment behavior of an oocyst, researchers investigating oocyst behavior that is related to settling and attachment should use these tools to assess the viability of their test populations both before and after experimental treatment.
Acknowledgments We would like to thank the National Science Foundation and the New York State Water Resources Institute whose support made this research possible.
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(16) American Public Health Association. In Standard Methods for the Examination of Water and Wastewater, 18th ed.; American Public Health Association: Washington, DC, 1992. (17) Einstein, H. A. Deposition of suspended particles in a gravel bed. J. Hydraul. Div., Am. Soc. Civ. Eng. 1968, 94 (HY5), 11971205. (18) Walker, M. J.; Montemagno, C. Sorption of Cryptosporidium parvum oocysts in aqueous solution to metal oxide and hydrophobic substrates. Environ. Sci. Technol. 1999, 33 (18), 3134-3139. (19) Auer, M. T.; Bagley, S. T.; Stern, D. A.; Babiera, M. J. A framework for modeling the fate and transport of Giardia and Cryptosporidium in surface waters. Lake Reservoir Manage. 1998, 14(23), 393-400. (20) Anderson, M. A.; Stewart, M. H.; Yates, M. V.; Gerba, C. P. Modeling the impact of body-contact recreation on pathogen concentrations in a source drinking water reservoir. Wat. Res. 1998, 32 (11), 3293-3306.
Received for review July 12, 2004. Revised manuscript received January 25, 2005. Accepted January 27, 2005. ES040470J
