Environ. Sci. Technol.

2005, 39, 8614-8621

Relative Value of Surrogate Indicators for Detecting Pathogens in Lakes and Reservoirs
J U S T I N D . B R O O K E S , * ,,,|,# MATTHEW R. HIPSEY, M I C H A E L D . B U R C H , ,| R U D I H . R E G E L , L E O N G . L I N D E N , ,|,# C H R I S T O B E L M . F E R G U S O N , , A N D JASON P. ANTENUCCI CRC for Water Quality and Treatment, PMB 3 Salisbury 5108, South Australia, SA Water Centre for Science and Systems, The University of South Australia, Mawson Lakes, South Australia, Centre for Water Research, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia, 6009, The Australian Water Quality Centre, PMB 3, Salisbury 5108, South Australia, Ecowise Environmental, P O Box 1834, Fyshwick, Australian Capital Territory 2609, and School of Earth and Environmental Sciences, The University of Adelaide, South Australia 5005

the dam wall, whereas enterococci were reduced by a factor of 10.

Introduction
The detection and enumeration of protozoan pathogens in environmental samples is complex, costly, and incurs lengthy delays due to the nature of the analytical tests. This limits the capacity of water utilities to confidently determine the fate and distribution of pathogens in large water bodies. In turn, this leads to an inability to properly characterize and anticipate the risk associated with the movement of pathogens through reservoirs to water supply offtakes following storm events, which are typically known to carry significant microbial contaminant loads (1-3). The alternative has been to consider the use of surrogates such as microbial indicator organisms, turbidity, or particle counting within lakes and reservoirs. There is evidence that bacterial indicators of fecal contamination (fecal coliforms and enterococci), bacteriophages, Clostridium spp. spores, and turbidity are useful surrogates under defined circumstances (4). However, there is ongoing uncertainty as to the most appropriate surrogate, or combination of surrogates, for determining pathogen risk in water supply reservoirs. Identification of the mechanisms that control pathogen fate and transport in lakes and reservoirs may indicate the most suitable surrogates for prediction of risk of pathogen presence. The principal controlling mechanisms include sedimentation, inactivation, which is influenced by temperature, visible and ultraviolet (UV) light, and predation (4). A small number of studies have examined the release and transport of pathogens from fecal material (5) or their subsequent transport across land surfaces (6-9). These studies highlight the importance of rainfall intensity, duration, slope, and vegetation cover in determining the efficiency of pathogen transport to surface waters. Consequently, although the microbiological quality of surface water inflows is highly variable, inflows following storm events represent the major mechanism for pathogen seeding in reservoirs. The behavior of inflows entering a water body is controlled by the density of the inflow water relative to that of the lake, the volume of the inflow, the slope at entry, and the bottom friction (10). Warm inflows will be buoyant surface intrusions and cold, dense inflows will intrude as interflows or, in the case of very dense inflows, will sink beneath the lake water where they will flow along the lake bed toward the deepest point. In either case the inflow will entrain water from the lake, increasing its volume, changing its density, and diluting the concentration of pathogens and other constituents. The speed at which an inflow travels through a lake, entrainment with ambient lake water, dilution, and insertion depth are all important in determining the distribution of pathogens in lakes and reservoirs. The distribution of microorganisms is also a function of their settling rate and consequently may vary for different organisms. This is further influenced by the potential for pathogens and other microorganisms to differentially aggregate with organic or inorganic particles and hence settle at higher rates than those of unattached microorganisms. Furthermore, the inactivation of pathogens and microbial indicators is influenced by environmental factors, such as temperature and visible and UV light, and will therefore vary between species. The aim of this study was to investigate the relative behavior of pathogens, fecal indicator organisms, and particles of varying size on entering a reservoir, and to
10.1021/es050821+ CCC: $30.25 2005 American Chemical Society Published on Web 10/18/2005

This study investigated the relative behavior of pathogens, fecal indicator organisms, and particles of varying size during transport through a reservoir following a storm event inflow in Myponga Reservoir, South Australia. During the inflow, samples were collected from the river and at various locations within the reservoir to determine the fate and transport of microroganisms as they progressed through the water body. Microbiological analysis included the indicator organisms Escherichia coli, enterococci, Clostridium perfringens, aerobic spores, and somatic coliphages, the protozoan pathogens Cryptosporidium spp. and Giardia spp., and the potential physical surrogates of pathogen contamination including particle size and turbidity. Of the microbial indicator groups, C. perfringens spores were the most highly correlated with Cryptosporidium spp. concentrations (Spearman Rho ) 0.58), closely followed by enterococci (Spearman Rho ) 0.57). Cryptosporidium spp. oocysts were predominantly associated with small sized particles (range of 14.3-27.7 m). All of the microbial indicator groups tested were associated with larger sized particle ranges (>63.3 m) except C. perfringens spores which were associated with particles in the size range of 45.5-63.3 m. Although indicators may rank correlate with Cryptosporidium spp., the variation in settling rates of different microorganisms has significant implications for the use of surrogates to estimate pathogen attenuation within reservoirs. For example, concentrations of Cryptosporidium spp. oocysts were reduced by a factor of 3 on reaching

* Corresponding author phone: 61-8-8259 0222; fax: 61-8-8259 0228; e-mail: justin.brookes@sawater.com.au. CRC for Water Quality and Treatment. University of South Australia. University of Western Australia. | The Australian Water Quality Centre. Ecowise Environmental. # The University of Adelaide.
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FIGURE 1. Myponga Reservoir bathymetry (m) showing the inflow locations, 1 and 2, the dam wall where the offtake is situated and the locations of the two meteorological stations including thermistor chains (Met1 and Met2). determine their appropriateness as indicators for assessing Cryptosporidium spp. concentrations. A comprehensive field experiment was conducted to track the passage of a storm event inflow through Myponga Reservoir in South Australia. During the event, samples were collected from the river and reservoir to determine the concentration of the microbial indicator organisms Escherichia coli, enterococci, Clostridium perfringens, aerobic spores, somatic coliphages, and the protozoan pathogens Cryptosporidium spp. and Giardia spp., as well as potential physical surrogates of contamination such as particle size and turbidity.

TABLE 1. Sampling Times (Day of Year in 2003) for Microbiological Samples

transect T1 T2 T3 T4 T5 inflow 178.042 178.625 178.702 179.800 180.571 Met2 178.042 178.601 178.688 179.743 180.500 Met1 178.104 178.622 178.733 179.792 180.542 dam wall 178.146 178.649 178.875 180.042 180.604

Materials and Methods

Myponga Reservoir. The experiment was undertaken at Myponga Reservoir, an impounded, flooded river valley located 70 km south of Adelaide, South Australia (S 35 24 13, E 138 25 29). The reservoir has a capacity of 26.8 106 m3 and a maximum depth of 42 m at full supply level. The mean retention time based on abstraction rates is approximately 3 years and the surface area is 2.8 km2. The catchment is approximately 124 km2 of mixed land use, including pasture for dairy, beef, and hay production, with patchy remnant vegetation. Recent estimates of dominant land uses are 62% grazing and 24% dairying (11). Sampling Design. A sampling program was structured to track the passage of a riverine intrusion, including its physical properties and the passage of pathogens and indicators, through the reservoir during a large runoff event following intense rainfall. The experiment was conducted from June 26-29, 2003 (days of year: 178-180), during which time the total rainfall was 58.4 mm. Tracking the Riverine Intrusion. The reservoir has two meteorological stations that detect wind and solar radiation and collect data at 10-min intervals. The stations also have thermistor chains that are used to capture the hydrodynamic conditions within the water body. One station (Met1) is located in the main basin and the other (Met2) is located in a long narrow sidearm of the reservoir (Figure 1). Flow and temperature were also measured in Myponga Creek at 10min intervals. Flow from a second minor tributary (Subcatchment 2) that enters near Myponga Creek was gauged using a logging doppler velocity meter (Starflow, Unidata Pty. Ltd.) operating in one of two stormwater pipes of 1.5-m internal diameter which channel the entire creek flow for a short distance just prior to entry into the reservoir. The characteristics of the riverine intrusion and the ambient reservoir conditions were measured by regular profiling at several sites along the length of the reservoir with a multi-probe water quality analyzer (Hydrolab with temperature, conductivity, dissolved oxygen, turbidity and

depth sensors) and a LISST (Laser In Situ Scattering and Transmissometry) particle profiler (12, 13), which measures the relative volume of particles in each of 32 size-classes between 2.5 and 500 m. Microbiological Analysis. Microbiological sampling was undertaken at the surface and at depth (2.5 m above the bottom) at three sites in the reservoir (Met 1, Met 2, and adjacent to the dam wall), and in the inflows (Figure 1). Sampling was timed to capture the storm event inflow and to precede and capture it within the reservoir (Table 1). Multiple samples were collected (3 for protozoans and 5 for all other microbes) at each location during five time periods (denoted T1-T5), and analyzed for the following: E. coli, enterococci, somatic bacteriophages, C. perfringens, and aerobic spores on all five occasions, and at three times (T1, T3, and T4) for Cryptosporidium spp. and Giardia spp. (Table 1). The Australian Water Quality Centre, a NATA and ISO 9000 accredited laboratory, performed the following microbiological analyses. E. coli. E. coli was determined using Colilert 18 (IDEXX). Coliform positives were recorded as yellow wells and these were tested for E. coli by checking for blue fluorescence under UV at 365 nm. Enterococci. Samples were filtered through a 0.45-m membrane before placing the membrane on m-Enterrococcus agar and incubating at 35 C for 48 h. Confirmation of Enterococcus species was achieved by plating of colonies from m-Enterococcus agar onto Glucosidase agar, further incubation, and counting under UV. Somatic Bacteriophages. Host E. coli broth was inoculated onto predried phage bottom agar with autoclaved phage top agar containing antifoam B solution (Sigma A-5757). Sample was added to the bacterial host plate and incubated for 18 h at 36 C. Somatic coliphages were counted as clear zones in the lawn of E. coli. A negative control of sterile diluent water and a positive control of diluted somatic coliphages were analyzed with each batch of samples. C. perfringens and Sulfite-Reducing Clostridia. An aliquot of sample was heat-treated for 20 min. at 70 C to eliminate nonspore-forming microorganisms prior to filtration through
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FIGURE 2. Flow rate and temperature of Myponga Creek and the Subcatchment 2 tributary monitored during the inflow event in June 2003, days 178-182. The average reservoir temperature was calculated from thermistors suspended vertically at 20 depths; on day 178 the reservoir was fully mixed. a 0.45-m membrane and placement of the membrane on a plate of Tryptose Sulfite Cycloserine agar. Incubation for 24 h was at 36 C in an atmosphere of H2 and N2. Samples were Gram-stained, and positive C. perfringens verified with MUP-ONPG (4-methyl-umbelliferyl phosphate + o-nitrophenyl). Aerobic Spores. Samples were heat-treated at 80 C for 30 min and then filtered through a 0.45-m membrane and placed onto Trytone Soya Agar. After 48 h incubation at 30 C, colonies were selected for Gram staining. Aerobic spores were identified as Gram positive bacilli. Cryptosporidium spp. and Giardia spp. Primary Concentration: Samples (10-L) were concentrated by CaCO3 flocculation (settled overnight and the supernatant aspirated, with recovery of the settled particulates by dissolving the CaCO3 using sulfamic acid) or by filtration using Envirochek filter capsules. ColorSeed (BTF Decisive Microbiology) was added to each sample as an internal control for each protozoan assay to allow determination of oocyst/cyst recovery efficiency. Secondary Concentration: The material collected in the primary concentration was further concentrated by centrifugation to reduce the pellet to approximately 10 mL. All samples were processed with the Dynal IMS kit following standard instructions. Staining and Microscopic Detection: The final concentrates were stained on Dynal spot-on slides using the DNAstaining fluorochrome DAPI and FITC-conjugated monoclonal antibodies against both Cryptosporidium and Giardia. The samples were scanned on glass microscope slides. Fluorescence microscopy, using blue-light excitation, was used to scan the slides for characteristic FITC-fluorescence of cells with the size and shape characteristics of Cryptosporidium or Giardia. Identification of organisms was confirmed using DAPI fluorescence (UV excitation) to judge structural integrity and count nuclei, and DIC (differential interference contrast) to determine internal structures such as sporozoites in Cryptosporidium and median bodies in Giardia, particularly in DAPI-negative cells. Data Analysis. Statistical analysis was performed using the JMP statistical package from the SAS Institute. The degree of association among Cryptosporidium spp. concentration, the concentration of surrogate microorganisms, and individual particle size classes was determined by Spearman rank correlation using corresponding concentrations of each parameter for all sampling times, sites, and depths. The Spearman correlation compares relative similarity by ranking the value for each sampling and determining the correlation between the ranked observations. Higher Spearman Rho values indicate a stronger correlation. The critical Rho for n ) 63, significance of R ) (0.05) is 0.248.
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Results
Inflow Characteristics. The high rainfall early on day 178 (42 mm) generated considerable runoff and elevated flow in Myponga Creek. The measured maximum total flow was 9.8 m3s-1 recorded after midday of day 178 (Figure 2). The riverine water entered the reservoir as an underflow since it was substantially cooler, contained more solutes, and therefore had a greater density than the ambient reservoir water (Figure 3). There was a general warming of the river water during the period, which acted to decrease the density of the incoming riverine water. Nevertheless, the inflow signal moved through the reservoir as a distinct pulse over 36 h. The underflow was first detected at Met2 in the sidearm as a general cooling of the bottom waters at 12:10 on day 178, approximately 9 h after the Myponga Creek hydrograph began to rise. Cooling at depth occurred subsequently at Met1 in the main basin, approximately 4 h after the intrusion passed Met2. The inflow pulse was first seen at the dam wall during T4, 36 h after the hydrograph peak in the inflow stream. Occurrence and Magnitude of Pathogens and Surrogates in the Intrusion. Both the Myponga Creek and Subcatchment 2 inflows delivered substantial concentrations of Cryptosporidium spp. to the reservoir. Concentrations in Myponga Creek ranged between 16 and 100 oocysts (10 L-1) and concentrations from Sub-catchment 2 were 19-95 oocysts (10 L-1). As the riverine intrusion moved through the reservoir, Cryptosporidium spp., the main pathogen of interest, was sequentially detected at lower concentrations in the bottom depth samples at locations further from the inflow (Table 2a). Very low concentrations of Giardia spp. were found, although they tended to also occur in the bottom waters that were influenced by the riverine intrusion (Table 2b). The other microbiological indicator organisms, (E. coli, Enterococci, somatic bacteriophages, C. perfringens, and aerobic spores) all displayed elevated concentrations within the riverine intrusion (Table 2c-h). As with Cryptosporidium spp., there was a relative decrease in surrogate concentration as the intrusion travelled through the reservoir; the rate of dilution, however, differed among the various organisms (Table 2). The general trend evident for all pathogens and surrogates was that they were in high concentrations in the incoming river water and appeared in the reservoir at the sites influenced by the riverine intrusion. The data were analyzed by nonparametric Spearman rank correlation to determine which of the microbial indicators had fate and transport characteristics most similar to those of Cryptosporidium spp. (Table 3). Giardia spp. was found in the most similar rank order to Cryptosporidium spp. but the very low Giardia spp. concentrations reduce the confidence in these correlations. Of the fecal indicator groups, C. perfringens spores and

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FIGURE 3. Temperatures (C) measured during the five transects as indicated by the day of year, preceded by year (yyyyddd). Transects were constructed by contouring around the profile locations (indicated by the dot-dash lines) using a rectangular grid with a sigma coordinate transformation. Enterococci showed the highest correlation with Cryptosporidium spp. oocyst concentrations (Spearman Rho ) 0.58 and 0.57, respectively). Concentrations of the traditional indicator organism E. coli showed a weaker correlation with Cryptosporidium spp. (Spearman Rho ) 0.48), although this was higher than the correlation with either somatic bacteriophages or aerobic spores (Spearman Rho ) 0.34 and 0.21, respectively). The correlation analysis also indicates the strength of relationship among all indicators, to determine whether one single surrogate was representative of the other surrogates, within the context of transport, settling, and decay losses within a riverine intrusion. E. coli and Enterococci were the most highly correlated (Spearman Rho ) 0.92). This means that throughout the hydrograph, and transport in the riverine intrusion, these two organisms displayed the most similar relative changes in concentration due to settling and dilution. The organism that showed the most consistently high correlation with indicators other than Cryptosporidium spp. was E. coli. C. perfringens spores showed the weakest correlation with E. coli (Spearman Rho ) 0.60). This is interesting given that both C. perfringens and Cryptosporidium spp. showed the highest rank correlations. The data for the microbial indicators were also correlated against particle size to try to deduce their behavior relative to particles. This analysis correlates each particle size class within the selected bins of the LISST profiler with each organism for all sampling times and depths. All organisms correlated moderately well with particle volume concentrations ranging between 4.48 and 259 m (Figure 4) because they shared the intrusion as the dominant controlling mechanism. The strongest correlations of organisms with particle size classes were aerobic spores (74.7-128 m), somatic bacteriophages (45.5-74.7 m), C. perfringens spores (45.5-63.3 m); E. coli and Enterococci (74.7-157 m), and sulfite-reducing clostridia (63.3-128 m). This contrasts with Cryptosporidium spp., which was most highly correlated with smaller particles in the range 16.8-27.7 m (Spearman Rho > 0.70). Cryptosporidium spp. showed only a weak correlation with turbidity (Spearman Rho ) 0.52) and total particle volume concentration (Spearman Rho ) 0.55). All surrogates
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TABLE 2. Mean Microorganism Measurements for the Five Transects (T1-T5)

T1 inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow 49 19 0 0 2 0 0 0 3 0 0 0 0 0 0 0 4067 2400 23 129 26 270 33 27 T2 100 T3 33 71 41 2 2 0 2 2 1 0 3 0 0 0 0 0 9000 23000 2767 77 35 37 95 21 T4 16 95 81 0 25 2 33 1 2 2 3 0 2 0 1 0 5233 2600 5300 63 1900 34 1267 34 4433 3700 1633 567 547 55 1030 78 T5

showed very poor correlation with particles greater than 332 m.

Discussion
Pathogen Similarity. This study measured a variety of microbiological and physical parameter concentrations and examined their correlations with each other within a riverine intrusion. The Spearman rank nonparametric correlation procedure was used to determine how similarly pathogens, surrogates, and particles behaved throughout the course of the experiment. However, a high correlation does not unequivocally prove that a particular surrogate of a pathogen is universally applicable. In this case the pathogens Cryptosporidium spp. and Giardia spp. were the most highly correlated biological parameters (Spearman Rho ) 0.65). Although the data and relationships related to Giardia spp. in this experiment must be treated with caution due to the very low concentrations, the results were similar to those of Ferguson et al. (14) who reported a partial correlation coefficient of 0.73 (p < 0.05) between Giardia spp. and Cryptosporidium spp. in waters of the Georges River, Sydney. Of the other microbial indicator groups Cryptosporidium spp. was most closely correlated with C. perfringens spores, albeit weakly (Spearman Rho ) 0.58). This was also comparable to the results of Ferguson et al. (14), who reported that C. perfringens spores were the microbial indicator group most highly correlated with Cryptosporidium spp. and Giardia spp. (Correlation coefficient R values of 0.27 and 0.41, respectively). In their study other fecal indicator groups showed weak correlations with Cryptosporidium spp. (fecal coliforms R ) 0.03, fecal streptococci R ) 0.08, F-RNA bacteriophages R ) -0.39). All microorganisms experience the same dilution due to entrainment within the inflow, implying the differences between microrganisms as they progress through the reservoir are due to differential inactivation or sedimentation rates. The time for the intrusion to move from the inflow to the offtake was less than 36 h, and considering the cool temperatures (<10 C) and high light attenuation (due to high dissolved organic carbon concentrations, >10 mg DOC L-1), there was likely to be little inactivation during that time. Consequently, the major difference between organisms is deemed to be sedimentation, and this data set therefore emphasizes that the existence of similar settling characteristics is important when seeking appropriate surrogates for pathogens. The different settling rates exhibited by the various microorganisms reflect their varied surface charge and affinity for attaching to particles, as well as the size and density of the particle aggregate. For example, the present study indicates that oocysts tend not to associate with larger/heavier particles and therefore settling provides little additional attenuation than dilution alone, whereas E. coli was attenuated to a much greater extent. Hipsey et al. (15) also found a similar high attachment and settling rate for E. coli in Sugarloaf Reservoir, near Melbourne, Australia. Turbidity as a Surrogate for Pathogen Transport. It is feasible that if the size of the particle and microorganism aggregate is known, then turbidity may well be a useful surrogate. As indicated above, the aggregation of particles with organic matter or clay minerals plays a major role in pathogen/surrogate transport, settling (15), and survival (16). The surface charge of the particles plays a key role in particleparticle interactions in water (17). It appears that Cryptosporidium spp. oocysts may be adequately aggregated and flocculated during conventional water treatment but may not adsorb well to natural negatively charged clays within the natural environment (18, 19). Considine et al. (20, 21) supported this hypothesis but concluded that protein linked tethering between silica and oocysts can occur and may facilitate adhesion. Although this suggests that oocysts would

(a) Cryptosporidium spp. (oocysts/10 L)

(b) Giardia (cysts/10 L)

3

(c) E. coli (/100 mL)

22667 20000 1933 25 75 42 116 15

(d) Aerobic Spores (/100 mL)

Myp Crk 21667 176667 320000 26333 24667 Subcatch 2 53000 200000 220000 14000 50000 Met2 bottom 2900 7600 136667 30000 7033 surface 5567 1500 4933 4033 1900 Met1 bottom 6067 8733 22000 29333 1900 surface 3867 1433 10667 2150 2433 dam wall bottom 5400 3367 19333 21000 4933 surface 4933 2633 92333 2667 2533

(e) Somatic Bacteriophages (/100 mL)

inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface inflow Myp Crk Subcatch 2 Met2 bottom surface Met1 bottom surface dam wall bottom surface
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167 310 7 14 11 27 10 13 109 320 1 7 0 0 1 1 4800 2800 9 319 10 497 47 27 173 320 17 23 20 16 13 17

630 820 133 23 12 11 16 9 107 10 40 0 0 3 2 0 12400 10000 5567 114 125 44 70 11 267 10 143 24 26 26 19 19

247 620 65 11 7 10 12 7 57 580 2 0 2 0 0 2 7233 9500 3833 9699 51 34 62 12 257 340 44 15 21 13 22 15

777 590 410 25 107 7 76 12 87 80 155 2 43 2 82 0 1950 560 3467 40 940 27 1230 27 141 80 155 33 89 6 96 13

363 530 110 28 10 6 14 4 40 100 50 1 48 0 2 0 737 780 930 327 405 62 570 53 62 440 76 27 99 19 13 17

(f) C. perfringens spores (/100 mL)

(g) Enterococci (/100 mL)

(h) Sulfite-Reducing Clostridia (/100 mL)

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TABLE 3. Spearman Rank Correlation Among Microbiological Surrogates

Cryptosporidium Cryptosporidium aerobic spores somatic bacteriophages C. perfringens spores E. coli Enterococci sulfite-reducing Clostridia
1.00 0.21 0.34 0.58 0.48 0.57 0.39 aerobic spores 0.21 1.00 0.53 0.41 0.82 0.84 0.86 somatic bacteriophages 0.34 0.53 1.00 0.72 0.78 0.66 0.72

C. perfringens spores
0.58 0.41 0.72 1.0 0.60 0.60 0.74

E. coli
0.48 0.82 0.78 0.60 1.00 0.92 0.84

Enterococci 0.57 0.84 0.66 0.60 0.92 1.00 0.91

sulfite-reducing Clostridia 0.39 0.86 0.72 0.74 0.84 0.91 1.00

FIGURE 4. Spearman rank correlation of microorganisms with particle size as measured by the LISST profiler. not adsorb readily to inorganic particles, there is some evidence of high settling velocities for Cryptosporidium spp. in field monitoring situations (22, 23). This suggests that, at least in certain situations, oocysts must be associated with larger particles, and it is possible that oocysts show an affinity for organic (e.g., sewage effluent) rather than inorganic particles. An alternative is that the oocysts may be physically enmeshed within an organic matrix of fecal material and/or soil particles during entrainment in surface water runoff, although Brookes et al. (24) found that this effect was not significant. By contrast, viruses appear to readily attach to particles (25-27), and this is reflected by their greater reduction relative to dilution over the course of the experiment. The correlation of Cryptosporidium spp. and the microbial indicators in this study with different particle size classes indicated that Cryptosporidium spp. tends to be transported similarly to small particles, whereas the bacterial and viral indicators tend to be transported like the relatively large particles. The association of microorganisms with particles is governed by their electrophoretic mobility which varies with strain (28) and species, pH, organic matter, and ionic strength (29). In field situations these parameters will vary with soil type, as will particle size and soil composition. Consequently, some variations in the relationship between microorganisms and particle size would be expected in different catchments and reservoirs. This study suggests that for a surrogate to be a useful indicator of pathogen risk in reservoirs it must share similar aggregation and settling characteristics to the pathogen of concern. The use of nonspecific measures of particle concentration, such as turbidity or total particle volume concentration, in monitoring may provide potentially erroneous predictions of pathogen risk depending on the particle size distribution. Nonetheless, a turbidity pulse will serve as an early warning of a potential pathogen threat if the origin of the turbidity and pathogenic contamination is similar. Implications for Monitoring and Pathogen Risk Assessment. The storm event that was monitored as part of this experiment delivered a considerable pathogen load to Myponga Reservoir in a distinct intrusion that travelled rapidly through the reservoir. This intrusion arrived at the offtake location at the dam wall within 36 h of the commencement of rainfall in the catchment. The response time for a normal routine microbial monitoring program to collect samples, return them to a laboratory for analysis, process the samples, and generate results for interpretation typically approaches 48 h. The detection of the inflow containing pathogens at the offtake point in less than 48 h following a modest rain event in this catchment highlights the need to reassess the ability of monitoring programs to adequately gauge pathogen risks. The use of surrogates has frequently been discussed as a more rapid alternative to measurement of pathogen concentrations to estimate pathogen transport, fate, and ultimately risk. However, for surrogates to be of value they must be rapidly measurable, representative of the pathogen of concern, and display similar fate and transport characteristics. For the case presented here, the commonly used coliform indicators would have significantly underesVOL. 39, NO. 22, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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timated the actual risk of Cryptosporidium spp. concentrations. The best and safest strategy for risk assessment of any given pathogen in a water supply reservoirs is to sample and analyze for the target organism in a systematic way, while taking into account the biological, physical, and chemical factors that control transport, distribution, and inactivation in natural source waters. Modeling tools have been developed to assess the inflow characteristics of a riverine intrusion to a reservoir prior to sampling for pathogens or surrogates. The minimum sampling strategy can be designed with assistance from the web-based model INFLOW (30, available at http://www.cwr.uwa.edu.au/ttfadmin/model/inflow/). This model uses flow and temperature of the river inflow, reservoir bathymetry, and density profiles to calculate the transport time, dilution, and depth of the inflow. A priori information of where the inflow can be located within the reservoir will enable sampling to target the water with highest pathogen concentrations for quantitative risk assessment. For example, in Myponga Reservoir the routine operational sampling program has involved the sampling of surface waters between locations Met2 and Met1 (Figure 1). It is clear from this study that the inflow had a negligible impact on the surface waters near this location (see Figure 3), and these data would be falsely negative and provide incorrect inputs to any quantitative microbial risk assessment. This study investigated the potential for microbial indicator organisms and particle concentrations (i.e., surrogates) to be utilized for pathogen risk assessments in reservoirs. The results indicate that microorganisms do not behave as similarly as would be expected based on hydrodynamic transport alone. The different settling rates of the microorganisms have significant implications for the attenuation of pathogens within the reservoir. For example, Cryptosporidium spp. oocysts were reduced by a factor of 3 on reaching the dam wall, whereas the Enterococcus spp. concentration was reduced by a factor of 10. It is unlikely that any single water quality indicator will reliably assess the risk of bacterial, protozoan, and viral contamination in aquatic environments, but it is feasible that a suite of surrogates can be used to estimate levels of microbial contamination within defined circumstances, such as within a storage reservoir with well-characterized inputs. However, this study highlights that surrogates should be carefully selected to ensure that they represent an accurate assessment of pathogen risks. While surrogates may provide a useful indicator of fecal contamination, their use for quantitative microbial risk assessment is complicated by their different kinetic, and settling characteristics relative to pathogens. Although turbidity and total particle volume displayed a relatively weak correlation with Cryptosporidium spp. concentrations, they are still useful parameters that can be measured on-line to indicate the presence and location of a riverine intrusion, enabling water quality managers to react to catchment rainfall events in near real-time.

Acknowledgments
The research has been made possible through funding from the American Water Works Association Research Foundation (AwwaRF Project 2752), the Cooperative Research Centre for Water Quality and Treatment (Project 2.2.0.1), and Melbourne Water Corporation. The project team thanks Warwick Grooby, Kerry Carabretta, and the Microbiology and Protozoology laboratories at the Australian Water Quality Centre, for microbiological analysis. Rod Boothy, Alan Brown, and Viv Allan provided excellent logistical support at Myponga Reservoir.

Literature Cited
(1) Atherholt, T. B.; LeChevalier, M. W.; Norton, W. D.; Rosen, J. S. Effect of rainfall on Giardia and Crypto. J. Am. Water Works Assoc. 1998, 90, 66-80.
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Received for review April 29, 2005. Revised manuscript received September 1, 2005. Accepted September 9, 2005. ES050821+

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