Role of Divalent Cations on Deposition of Cryptosporidium parvum

May 13, 2010 - ... barriers to oocyst deposition on a SRNOM-coated surface disappeared at 30 ... In contrast, in sandy aquifer with low hardness groun...
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Environ. Sci. Technol. 2010, 44, 4519–4524

Role of Divalent Cations on Deposition of Cryptosporidium parvum Oocysts on Natural Organic Matter Surfaces DAO JANJAROEN,† YUANYUAN LIU,† MARK S. KUHLENSCHMIDT,‡ THERESA B. KUHLENSCHMIDT,‡ AND T H A N H H . N G U Y E N * ,† Department of Civil and Environmental Engineering, The Center of Advanced Materials for the Purification of Water with Systems, and Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana Illinois 61801

Received December 20, 2009. Revised manuscript received April 13, 2010. Accepted April 16, 2010.

A Radial Stagnation Point Flow (RSPF) system coupled with a microscope was used to study deposition of Cryptosporidium parvum oocysts on quartz and Suwannee River Natural Organic Matter (SRNOM)-coated surfaces in solutions with different Ca2+ or Mg2+ concentrations. Both untreated and proteinase K-treated oocysts were used. Deposition of oocysts on a SRNOM surface in Ca2+ solution was higher than in Mg2+ solution, even though the energy barriers calculated from Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for Ca2+ solution were higher than for Mg2+ solution. On the other hand, the attachment of oocysts on a quartz surface was the same in both Ca2+ and Mg2+ solution and in qualitative agreement with the DLVO energy profiles. Inductive coupled plasma (ICP) was employed to measure the free divalent cation concentration in solutions containing oocysts. ICP data showed more Ca2+ bound to oocyst surface than Mg2+. Moreover, proteinase K treatment of oocysts led to a significant decrease in deposition rate due to less binding of Ca2+ to the surface of the treated oocysts as shown by the ICP data. The deposition and ICP results suggested that inner-sphere complexation of Ca2+ with carboxylate groups on both SRNOM and oocyst surfaces enhanced deposition of oocysts on a SRNOM surface.

Introduction Cryptosporidium parvum is a protozoan parasite causing a disease called cryptosporidiosis, which has primary symptoms of acute, watery, and nonbloody diarrhea. Cryptosporidiosis is one of the most common waterborne diseases found worldwide and is of great concern for immunocompromised patients (1, 2). Cryptosporidium is ubiquitous in natural waters partially because C. parvum from infected cattle and humans has a spore phase (oocyst) which protects and prolongs the survival of the parasite in the environment. The hardy oocysts are * Corresponding author phone: (217)244-5965; fax: (217)333-6968; e-mail: [email protected]. † The Center of Advanced Materials for the Purification of Water with Systems. ‡ University of Illinois at Urbana-Champaign. 10.1021/es9038566

 2010 American Chemical Society

Published on Web 05/13/2010

also resistant to conventional water treatment processes such as chlorine-based disinfection. Therefore, physical removal processes such as granular filtration and UV disinfection are the primary barrier to oocyst passage in water treatment plants (3, 4). In addition, natural removal of oocysts in the subsurface environment such as bank filtration is being considered as a promising process (5, 6). Previous studies have shown the importance of surface properties of oocysts and bacteria on the deposition kinetics (7-10). Oocysts treated by heat or formalin or proteinase K have reduced steric interaction and had a higher deposition rate on quartz surface compared to untreated oocysts (7, 8). The role of steric interaction due to the hairy layer on the oocyst surface has been shown by atomic force microscope (AFM) technique (11-13). Oocyst wall was determined to be approximately 40 nm thick (14). The wall outer layer is composed of acidic glycoprotein, whereas the apparently rigid central wall is a complex lipid (14). The thick inner layer is a filamentous glycoprotein (14). Most previous deposition studies involving oocysts were conducted only on a quartz surface in the presence of either monovalent or divalent solutions (7, 8, 15). In the subsurface environment, iron oxide, which has positively charged components at near-neutral pH, can be covered with negatively charged macromolecules such as natural organic matter (NOM), proteins, or polysaccharides, which come from microbial activity and natural decay (16, 17). Surfaces covered with these macromolecules or biofilm are abundant and have a significant effect on oocyst transport and adhesion in the subsurface environment (18, 19). A recent study has shown that NOM has a significant effect on adsorption of plasmid DNA to a NOM-coated silica surface in the presence of monovalent electrolyte solution (20). Another study has found that deposition of bacteriophage MS2 on a NOMcoated surface is much lower than on a clean quartz surface (21). Thus, to better and thoroughly understand the fate and transport of oocysts in the environment is essential to expand our understanding of the deposition of oocysts onto surfaces coated with macromolecules in solution with hardness (i.e., Ca2+/Mg2+). Our objective was to study the role of hardness on the deposition of oocysts onto quartz and NOM-coated surfaces. We hypothesized that the specific interaction formed by Ca2+ between oocyst and NOM controlled the deposition of oocysts on a NOM-coated surface. A radial stagnation point flow (RSPF) system with well-defined hydrodynamic conditions was used to observe the real-time deposition of oocysts on ultrapure quartz or NOM surfaces (7, 13, 22, 23). To study the effect of specific interaction between oocyst surface macromolecules and NOM surfaces on deposition, we used oocysts treated with proteinase K enzyme.

Materials and Methods Preparation of C. parvum Oocysts. C. parvum oocysts were isolated from feces of male Holstein calf at University of Illinois Veterinary Medicine School, as described in our previous work (24). The oocysts were washed through centrifugation in tris-ethylenediamine-tetraacetic acid (EDTA) solution and stored at 4 °C in a solution of 50% Hanks’ balanced salt solution (HBSS) and 50% antibiotic-antimycotic solution (0.6% penicillin, 1% streptomycin, 0.0025% amphotericin, and 0.85% NaCl in sterile water). No further treatments to inactivate oocysts were used. All experiments were conducted with the same batch of oocysts. Before being used in each experiment, oocysts were washed by centrifugation twice at 17 000g for 3 min. The VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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desired C. parvum oocyst concentration was prepared by suspending the clean particles in electrolyte solution of either Ca2+ or Mg2+ (1-30 mM) at ambient pH (pH 5.6-5.8). For each experiment, the number of oocysts was counted with a hemocytometer (Hausser Scientific, cat. no. 3100). C. parvum Oocyst Treatment with Proteinase K. The proteinase K treatment protocol was modified from the method described in ref 14. Proteinase K was prepared at a concentration of 1 mg/mL in 10 mM Tris and 1 mM CaCl2 at pH 8. In every step involving oocysts, AXYGEN MCT-060L-C tube was used to minimize the adsorption of oocysts to a tube. Before the treatment, the oocyst solution at concentration of 2 × 108 oocysts/mL was thoroughly washed by centrifugation at 17 000g for 3 min. Proteinase K treatment was conducted at 27 °C for 3 h. After the treatment, oocysts were washed twice with DI water by centrifugation and subsequently resuspended in 1 mM NaCl and stored at 4 °C. After every treatment, we used discontinuous CsCl density centrifugation modified from established protocols to separate intact oocysts from broken oocysts (25). The CsCl gradient solutions were prepared with measured reflective indexes of 1.3500, 1.3465, and 1.3440. We used 13 × 51 mm ultraclear centrifuge tubes (Beckman cat. no. 344057) precoated with bovine serum albumin (BSA). Oocysts were centrifuged at 20 537g for 65 min at 4 °C using a precooled (4 °C) ultracentrifuge. After centrifugation, the oocyst band was collected separately in BSA-coated 15 mL conical tubes. These tubes were then diluted with cold stock Tris/EDTA buffer and centrifuged at 1250g for 30 min. The supernatant was carefully suctioned off and the pellet was resuspended in 5 mL HBSS. One drop from each tube was stained by propidium iodide and examined under a microscope to determine the presence/absence of broken oocysts. This verification experiment showed that treated oocysts were intact after CsCl step gradient centrifugation. All intact treated oocysts were pooled into 15 mL BSA-coated conical tubes and stored at 4 °C in a 50% mixture of HBSS and 50% of antibioticantimycotic solution. Before being used in each experiment, proteinase K-treated oocysts were washed and suspended in desired electrolyte solution at ambient pH (pH 5.6-5.8). Electrophoretic Mobility Measurement. A Zetasizer Nano ZS90 instrument (Malvern Instruments, Southborough, MA) was used to measure electrophoretic mobilities of oocysts, SRNOM-coated surfaces, and quartz surfaces in divalent cation solutions at 25 °C. Ooocyst concentration of 2.5 × 106 oocysts/mL in each of the studied electrolyte solutions with unadjusted pH of 5.6-5.8 was used in electrophoretic mobility measurements. SRNOM-coated silica beads were used as a surrogate for a SRNOM-coated surface for electrophoretic mobility measurements. Poly-L-lysine (PLL) with average molecular weight of 150 000 and Suwannee River Natural Organic Matter (SRNOM) were obtained from Sigma and International Humic Substances Society (IHSS), respectively. Silica particles (1.6 µm in diameter, wt. 10%, Bangs Laboratories Inc.) were used in preparation for SRNOM coating, as described previously (22). Briefly, the silica particles were sequentially coated with PLL and SRNOM. SRNOM-coated silica particles were diluted in 3 mL electrolyte solution at concentration of interest and were used for electrophoretic mobility measurements. Clean pulverized quartz surfaces were dispersed in the electrolyte solution of interest. The solution was sonicated for 10 min, and then the supernatant was taken for electrophoretic mobility measurements. At least three replicates were conducted for each condition. Preparation of Substrates. Three types of collector surfaces were prepared for deposition experiments. A PLLcoated surface represented nonrepulsive conditions, whereas SRNOM-coated and quartz surfaces were used to study 4520

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repulsive conditions. Glass bottom Petri dishes were used to hold a flow cell for the deposition experiments (22). PLL and SRNOM coating of the collector surface was described previously (22). For deposition experiments on a quartz surface, the glass slide at the bottom of the Petri dish was replaced with a clean quartz slide (19 × 19 mm × 0.5 mm thick, TED PELLA, INC.). The quartz surface was cleaned sequentially by Hellmanex and Chronomix solutions (22). PLL-coated surfaces were used for favorable conditions and for SRNOM coating. For deposition experiments on SRNOM surfaces, we further coated PLL-coated glass-bottom Petri dishes with SRNOM solution. Coated surfaces were rinsed with DI water, dried and stored in a desiccator at room temperature. Deposition of C. parvum Oocysts in a RSPF System. Deposition of C. parvum oocysts on quartz, PLL-coated, and SRNOM-coated surfaces were studied in a RSPF system with an injection capillary radius of 1 mm and a distance between the capillary outlet to the collector surface of 0.7 mm. Deposition experiments were performed in different cation concentrations (0.1-30 mM CaCl2/MgCl2) at ambient pH (pH 5.6-5.8) and at 2.2 × 106 oocysts/mL and a constant flow of 1 mL/min. This concentration of oocysts was selected so that no aggregation of oocysts was observed during the deposition experiments. Deposited oocysts were observed and counted in a rectangular viewing area of 296 × 222 µm every 15 s for 30 min using an electronic inverted microscope (Leica DM15000 M) equipped with a phase filter at bright field. The microscope images were recorded using the QIMAGING RETIGA 2000R Fast 1394 and analyzed with ImagePro 6.2 software. At the end of each deposition experiment, electrolyte solution with 0.1 mM CaCl2 or MgCl2 was introduced into the RSPF at the same flow rate for observation of possible detachment. The deposition rate coefficient, kd, was calculated from oocyst deposition flux (number of deposited particles per viewing area per time) divided by the initial particle concentration. Deposition were represented by attachment efficiency, R, which was obtained by normalizing oocyst deposition rate coefficients under repulsive conditions (such as onto quartz or SRNOM-coated surfaces) with particle deposition rate coefficient under favorable conditions (onto PLL-coated surfaces) at a given cation concentration. Unadjusted pH of 5.6-5.8 was used so that high Ca2+ concentration conditions can be studied. At least three replicates were conducted for each condition. DLVO Energy Profiles. The total interaction energy between charged particle and plate surface was calculated as a sum (ΦT) of electrostatic (ΦE) and van der Waals (ΦVDW) interactions using the Hogg et al. (26) expression. The retarded van der Waals attractive interaction energy was calculated using Gregory (27) approximate expression. A Hamaker constant for C. parvum oocyst deposition is 6.5 × 10-21 J, as suggested in ref 7. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) Analysis for Free Calcium and Magnesium Cation Concentration in Deposition Solutions. Oocysts suspended at concentration of 2 × 106 oocysts/mL were added to 1.5 mL of either Ca2+ or Mg2+ electrolyte solution at different concentrations. The oocysts were equilibrated with Ca2+/Mg2+ solution for 5 min. After equilibration oocysts were removed from the solution by centrifugation at 17 000g for 2 min. The aliquot was analyzed for either Ca2+ or Mg2+ concentration left in the solution by ICP-OES (PerkinElmer Optima 2000DV ICP-OES) at the School of Chemical Sciences, University of Illinois. The sample was diluted 10 times with 1% trace mineral grade HNO3, which was also used to prepare blanks and standards. Ca2+ was measured at two wavelengths, 317.933 and 315.887 nm, whereas Mg2+ was measured at only 285.213 nm. Three

FIGURE 1. Electrophoretic mobilities and zeta potentials of C. parvum oocysts (circle), pulverized quartz coverslip (square), SRNOM-coated silica particles (triangle) and digested C. parvum oocysts (diamond/star) in the presence of (a) CaCl2 and (b) MgCl2. Experiments were carried out at ambient pH (pH 5.6-5.8) and a temperature of 25 °C. Shown data are averages and standard deviations of at least three replicates. iterations were tried per wavelength. In case of multiple wavelengths, the average of both results was considered. At the end of an analysis, the instrument reanalyzed standard solutions for a quality control test. All studied samples passed the quality control test. The Ca2+ or Mg2+ percentage reduction was calculated by subtraction between initial Ca2+/ Mg2+ concentrations and final Ca2+/Mg2+ concentration measured from ICP-OES.

Results Surface Potentials of Collector Surfaces and C. parvum Oocysts. The electrophoretic mobilities (EPMs) and the corresponding zeta potentials of C. parvum oocysts, quartz, and SRNOM-coated silica beads in the presence of CaCl2 and MgCl2 are presented in Figure 1. For all studied surfaces at the experimental pH conditions of 5.6-5.8, the electrophoretic mobilities were negative and became less negative with increasing concentrations of divalent cations. Electrophoretic mobilities of PLL-coated silica beads were positive (i.e., 2.1 µmVS-1cm-1 at 30 mM Ca2+ and 2.3 µmVS-1cm-1 at 30 mM Mg2+). The EMPs of PLL-coated silica beads were in agreement with our previous study (28). As shown in Figure 1, the electrophoretic mobilities (EMPs) of quartz and SRNOM-coated silica beads did not show significant deviation from one another at most cation concentrations of either Ca2+ or Mg2+. Similar values and trends for EPMs have been reported for silica beads in ref (28, 29). The electrophoretic mobilities of original and proteinase K-treated oocysts in CaCl2 and MgCl2 are shown in Figure 1a and b, respectively. Interestingly, Ca2+ and Mg2+ had a comparable impact on EMPs of oocysts at almost all concentrations. Specifically, EMPs at 1 mM Ca2+ and Mg2+

were -0.36 ( 0.03 and -0.39 ( 0.03 µmVS-1cm-1, respectively. In 10 mM Ca2+ or Mg2+ solutions, EMPs of proteinase K-treated oocysts were statistically the same (-0.18 ( 0.05 in Ca2+ vs -0.20 ( 0.03 µmVS-1cm-1 in Mg2+). The electrophoretic mobilities shown in Figure 1 were converted to zeta potentials by Smoluchowski equation and would later be used to calculate interaction energies between oocyst surfaces and the collector surfaces using DerjaguinLandau-Verwey-Overbeek (DLVO) theory. Divalent Cation Binding to Oocyst Surfaces. ICP measurement was conducted to quantify the amount of divalent cation binding to oocyst surfaces. Higher percentage of divalent cation reduction suggests stronger binding of the divalent cation to oocysts. The divalent cation percentage reduction was calculated by subtraction between initial divalent cation concentrations and final divalent cation concentration measured by ICP-OES technique. The results of ICP-OES measurement showed that the percentage of Ca2+ reduction increased with the Ca2+ concentration in solution (i.e., from 2.5 ( 0.7% at 1 mM Ca2+ to 9.0 ( 1% at 30 mM Ca2+). In contrast, the percentage of Mg2+ reduction in the solution with the same oocyst concentration was not detectable until at 10 mM Mg2+. The percentage of Ca2+ reduction due to binding to the original oocysts at 10 mM Ca2+ was 2 times higher than that of treated oocysts (7.8 ( 0.3% vs 3.6 ( 1.2%). However, the percentage of Mg2+ reduction due to binding to the treated oocysts was not detectable in 10 mM Mg2+ solution. Thus, compared to Mg2+, Ca2+ had higher binding affinity to oocyst surfaces. Treatment of oocysts with proteinase K resulted in lower binding of both Ca2+ and Mg2+ to oocyst surfaces. Deposition of C. parvum Oocysts under Nonrepulsive Conditions. Deposition rate coefficients (kRSPF) for oocysts under nonrepulsive conditions (i.e., on PLL-coated surfaces) are shown in Figure 2. The deposition rate coefficients of oocysts decreased from 3.5 ( 0.1 × 10-7 m/s at 0.3 mM Ca2+ to 2.6 × 10-7 m/s at 30 mM Ca2+. In Mg2+ solution, kRSPF decreased from 2.2 × 10-7 m/s at 10 mM Mg2+ to 1.1 × 10-7 m/s at 30 mM Mg2+. As previously suggested (22, 29, 30), the decrease in kRSPF with ionic strength under nonrepulsive condition is a result of short-range attractive electric-double layer force at high ionic strength due to compression of electrostatic double-layer and electrostatic shielding by cations. Deposition of C. parvum Oocysts under Repulsive Conditions: Quartz and SRNOM-Coated Surfaces. The deposition rate coefficient and the corresponding attachment efficiency (R) on quartz and SRNOM surfaces are presented in Figures 2 and 3, respectively. For all studied Ca2+ concentration, the deposition rate coefficients (kRSPF) and the attachment efficiencies of oocysts on a SRNOM surface were higher than those on quartz. Specifically, the highest alpha of 0.82 ( 0.02 was observed on a SRNOM surface at 10 mM Ca2+ and of 0.25 ( 0.1 on a quartz surface. However, in Mg2+ solution, the attachment efficiencies on a SRNOM surface and on quartz were comparable up to 10 mM Mg2+, and at 30 mM Mg2+ attachment efficiencies on a SRNOM surface were twice as high as that on a quartz surface. The attachment efficiencies on a quartz surface were comparable within the entire range of studied concentrations of Ca2+ and Mg2+ (Figure 3). On a SRNOM surface, however, the attachment efficiencies in Ca2+ solution were 0.2-5 times higher than those obtained for Mg2+ solution. For example, alpha in 10 mM Ca2+ was 0.82 ( 0.02, while alpha in the same Mg2+concentration was only 0.25 ( 0.1. Similar results showing higher deposition on a SRNOM-coated surface compared to that on a silica surface in Ca2+ solution and comparable deposition on both surfaces in Mg2+ solution were reported for bacteriophage MS2 (28). In addition, the detachment experiments using solutions with VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Deposition rate coefficient (kRSPF) of C. parvum oocysts on to PLL-coated SRNOM-coated, and quartz surfaces in the presence of (a) CaCl2 and (b) MgCl2. Experimental conditions were as follows: capillary flow rate ) 1 mL/min (average velocity ) 0.0053 m/s with Re ) 5.29), pH 5.6-5.8, and temperature ) 25 °C. Shown data are averages and standard deviations of at least three replicates. 0.1 mM CaCl2 or MgCl2 revealed that the oocysts irreversibly attached to the collector surfaces. Comparison of Deposition Rate Coefficient of Treated C. parvum Oocysts on Quartz and SRNOM-Coated Surfaces. The role of oocyst surface macromolecules on oocyst deposition on SRNOM and quartz surfaces was investigated using proteinase K-treated oocysts. This treatment to remove oocyst surface functional groups has been reported in ref 8. The deposition experiments for proteinase K-treated oocysts were carried out at 10 mM Ca2+ and Mg2+. The deposition rate coefficients (kRSPF) for original and treated oocysts in either 10 mM Ca2+ or Mg2+ solution are shown in Figure 4. kRSPF for treated oocysts on a quartz surface in Ca2+ and Mg2+ was 5 times lower than those for untreated oocysts (e.g., 6.9 ( 0.3 × 10-8 vs 1.3 ( 0.5 × 10-8 m/s in Ca2+ solution). kRSPF for treated oocysts on a SRNOM surface was 10 times lower than those for untreated oocysts in Ca2+ solution. In Mg2+ solution significant difference of kRSPF for treated and untreated oocysts on a SRNOM surface was not observed.

Discussion Role of Divalent Cation on Oocyst Deposition. In contrast to the expectation based on the energy barrier calculated from DLVO theory (Table 1), oocyst attachment efficiencies on a SRNOM surface in Ca2+ solution were 0.2-5 times higher than those in Mg2+ solution (Figure 3a). Specifically, almost at every studied cation concentration except 1 mM, the energy barrier interactions calculated from DLVO theory between 4522

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FIGURE 3. Experimental attachment efficiencies for C. parvum oocysts on to SRNOM-coated, and quartz surfaces. Experimental conditions were as follows: capillary flow rate ) 1 mL/ min (average velocity ) 0.0053 m/s with Re ) 5.29), pH 5.6-5.8, and temperature ) 25 °C. Shown data are averages and standard deviations of at least three replicates. oocysts and a SRNOM surface in Ca2+ solution were higher than those in Mg2+ solution. Higher calculated energy barriers suggest that the attachment efficiencies of oocysts on a SRNOM surface in Ca2+ solution should be lower than those in Mg2+ solution. In addition, the attachment efficiencies of oocysts on quartz were less than those on SRNOM in Ca2+ solution, despite similar energy barriers for both surfaces. We hypothesize that the discrepancy between DLVO theory and experimental data for oocyst deposition in Ca2+ and Mg2+ solution can be explained by inner-sphere complexation by Ca2+ and outer-sphere complexation by Mg2+ with carboxylate groups on the oocyst and NOM-coated surfaces. Similar to the results on oocyst deposition presented here, previous study has shown significantly higher attachment efficiencies of bacteriophage MS2 on a SRNOM surface in solutions of 1 mM Ca2+ compared to those in solutions of 1 mM Mg2+ (28). Enhanced bacteria deposition or nanoparticle aggregation by divalent cations and alginate solution was also reported by other researchers (28, 29, 31-35). These previous studies consistently show the importance of divalent cation specific interactions with carboxylate groups on both colloid and collector surfaces. The role of divalent cation specific interaction with oocyst surface was further illustrated by the deposition result with proteinase K-treated oocysts. Although proteinase K treatment for oocysts did not lead to different zeta potential between original and treated oocysts, the deposition kinetics on SRNOM surfaces in Ca2+ after proteinase K treatment were one magnitude lower than those of untreated ones

FIGURE 4. Deposition rate coefficient (kRSPF) of treated C. parvum oocysts on SRNOM-coated, and quartz surfaces in (a) 10 mM CaCl2, (b) 10 mM MgCl2. Experimental conditions were as follows: capillary flow rate ) 1 mL/min (average velocity ) 0.0053 m/s with Re ) 5.29), pH 5.6-5.8, and temperature ) 25 °C. Shown data are averages and standard deviations of at least three replicates.

TABLE 1. Values of Energy Barriers and Attachment Efficiency for C. parvum Oocysts on the SRNOM-Coated and Quartz Surfacesa concentration (mM) 0.1 0.3 1 10 30 0.3 1 3 10 a

energy barrier (kT) CaCl2

MgCl2

NOM 743 241 200 124 58 81 5.4 0 0 0 quartz 201 127 64 82 21 34 10 4.3

attachment efficiency CaCl2

MgCl2 NOM

0.014 0.11 0.65 0.82 0.53

NA 0.049 0.20 0.25 0.35 quartz

0.014 0.059 0.16 0.25

0.031 0.13 NA 0.33

NA: no data available.

(Figure 4). Based on the pKa fitting value of 2.5, Karaman et al. (36) suggested that oocyst surfaces comprise of carboxyl groups. The percentage of cation reduction results further confirms that Ca2+ ions bind to the original oocyst surface more than treated-oocysts at 10 mM Ca2+ (7.8 ( 0.3% vs 3.6 ( 1.2%). As a broad spectrum proteinase, proteinase K is able to cleave the peptide bonds adjacent to the carboxyl group of amino acids (37). Thus, for oocysts, proteinase K treatment removed carboxyl groups that can form complexes with divalent cations.

Different affinities of Ca2+ and Mg2+ to carboxylate groups were shown in a molecular simulation study by Kalinichev and Kirkpartrick (38). Specifically, the tendency of forming metal-NOM complexation is strongly dependent on charge/ radius (z/R) and the size of the cation. Ca2+ is found to form inner-sphere complexes with NOM, whereas Mg2+ form outer-sphere complexes with NOM. Because Ca2+ is bigger than Mg2+ (RCa ) 1.61 Å vs RMg ) 0.92 Å), the hydration sphere of Ca2+ is more loosely held and can be released to form inner-sphere complexes (33, 38). Newman and McCormick also found that poly(sodium acrylate) bind more strongly with Ca2+ than Mg2+ or Na+ because of inner-sphere complexes with Ca2+ rather than outer-sphere complexes with Mg2+ and charge shielding by Na+ (39). The presence of carboxylate groups in both NOM and oocyst wall (16, 36) allows Ca2+ and Mg2+ to form inner-sphere and outer-sphere complexation, respectively. The ICP data and theoretical simulation studies by others (38, 39) suggest that the higher deposition of oocysts on a SRNOM surface in Ca2+ solution compared to that in Mg2+ solution can be explained by stronger affinity of Ca2+ to oocyst and SRNOM surfaces. The ability of Ca2+ to bind to both carboxylate groups of SRNOM and oocyst surfaces allows the formation of cation bridging between these surfaces and leads to deposition enhancement. On the other hand, Mg2+ weakly associates with carboxyl groups and tend to yield only charge neutralization. Steric Repulsion Interaction. Another discrepancy between observed deposition and expectation based on the energy barrier calculated from DLVO theory should be mentioned. As shown in Table 1 the energy barriers to oocyst deposition on a SRNOM-coated surface disappeared at 30 mM for both Ca2+ and Mg2+ solution. The attachment efficiency is expected to reach one at this divalent cation concentration. The measured values of alpha in both solutions containing 30 mM of either Ca2+ or Mg2+ were 0.53 and 0.35, respectively. Lower deposition than predicted by DLVO has been reported in previous studies (7, 22). As suggested before, the oocyst surface causes steric interaction between oocyst surfaces and collector surfaces (12, 22). Steric repulsion results from the compression of surface polymer upon contact with another surface (7). The oocyst surface is composed of many functional groups and proteins such as anchored glycoprotein (13, 14). These macromolecules can stretch into the solution yielding steric repulsion between oocysts and collector surfaces (12). Liu et al. (22) has found the attachment efficiencies of oocysts on the quartz collector in the presence of monovalent cation did not reach unity even though the calculated energy barrier was absent at NaCl concentration of 30 mM. The steric interaction caused by the soft layer on oocysts prevented deposition of oocysts on the collector surface. Environmental Implication. In subsurface environments, where NOM-coated surfaces and Ca2+ hardness are present, the deposition of oocysts tends to be enhanced due to the cation bridging mechanism between oocyst surfaces and collector surfaces. In contrast, in sandy aquifer with low hardness groundwater, oocysts will be more mobile. Moreover, pH of the solution also plays an important role in oocyst fate and transport in the subsurface environment (40). Besides the solution ionic composition and the physicochemical properties of collector surfaces, macromolecules on an oocyst outer layer also control the deposition kinetics of oocysts on either quartz or SRNOM surfaces. Aging in the natural environment may lead to removal of oocyst surface functional groups and as a result lead to a change in oocyst mobility in porous media environments. In addition, recent work has shown that the presence of dissolved NOM would also increase mobility of oocysts in subsurface environment (41). VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The results of this study added to the growing number of studies on deposition of oocysts to eventually allow accurate prediction of oocyst fate and transport in environmental systems.

Acknowledgments This work was partially supported by the NSF WaterCAMPWS (CTS-0120978), a Royal Thai Government Fellowship to D.J., USDA Grant No. 2008-35102-19143, a Illinois Water Resources Center grant USGS 06HQGR0083, and a NSF CAREER award to T.H.N. (0954501). Deposition and characterization experiments for oocysts were conducted by D.J. Digestion method was developed by Y.L. Oocysts were purified by T.B.K. and M.S.K. T.H.N. assisted with experimental planning, data interpretation and manuscript preparation.

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