In this study, three full-scale, operational stormwater harvesting systems located in Melbourne, Australia were evaluated with respect to water yields; pathogen removal performance by analysis of native surrogate data (Escherichiacoli, somatic coliphages and Clostridium perfringens); and potential human health risk associated with exposures to faecal pathogens using Quantitative Microbial Risk Assessment (QMRA). The water yield assessment confirmed variation between design and measured yields. Faecal contamination of urban stormwater was site specific and variable. Different treatment removal performance was observed between each of the microbial surrogates and varied between event and baseline conditions, with negligible removal of viruses during event conditions. Open storages that provide a habitat for waterfowl may lead to elevated risk due to the potential for zoonotic transmission. Nevertheless, in the Australian urban setting studied, the potential for human faecal contamination of the separated stormwater system was a critical driver of risk. If the integrity of the sewerage system can be ensured, then predicted health risks are dramatically reduced.

Solar radiation-driven inactivation of bacteria, virus and protozoan pathogen models was quantified in simulated drinking water at a temperate latitude (34 degrees S). The water was seeded with Enterococcus faecalis, Clostridium sporogenes spores, and P22 bacteriophage, each at ca 1x10(5) mL(-1), and exposed to natural sunlight in 30-L reaction vessels. Water temperature ranged from 17 to 39 degrees C during the experiments lasting up to 6h. Dark controls showed little inactivation and so it was concluded that the inactivation observed was primarily driven by non-thermal processes. The optimised reactor design achieved S90 values (cumulative exposure required for 90% reduction) for the test microorganisms in the range 0.63-1.82 MJ m(-2) of Global Solar Exposure (GSX) without the need for TiO2 as a catalyst. High turbidity (840-920 NTU) only reduced the S(90) value by <40%. Further, when all S90 means were compared this decrease was not statistically significant (prob.>0.05). However, inactivation was significantly reduced for E. faecalis and P22 when the transmittance of UV wavelengths was attenuated by water with high colour (140 PtCo units) or a suboptimally transparent reactor lid (prob.<0.05). S90 values were consistent with those measured by other researchers (ca 1-10 MJ m(-2)) for a range of waters and microorganisms. Although temperatures required for SODIS type pasteurization were not produced, non-thermal inactivation alone appeared to offer a viable means for reliably disinfecting low colour source waters by greater than 4 orders of magnitude on sunny days at 34 degrees S latitude.

Studies undertaken to assess the performance of filter materials to remove phosphorus in decentralised sewage systems have not reported on the broader performance of these systems. This study aimed to identify virus fate and transport mechanisms at the laboratory scale for comparison with field experiments on a mound system amended with blast furnace slag. Inactivation was a significant removal mechanism for MS2 bacteriophage, but not for PRD1 bacteriophage. Column studies identified rapid transport of PRD1. Laboratory studies predicted lower removal of PRD1 in a full scale system than was experienced in the field study, highlighting the importance of considering pH and flow rate in pathogen removal estimates. The results highlight the necessity for studying a range of organisms when assessing the potential for pathogen transport.

Pathogenic microorganisms have been identified as the main human health risks associated with the reuse of treated urban stormwater (runoff from paved and unpaved urban areas). As part of the Smart Water initiative (Victorian Government, Australia), a collaborative evaluation of three existing integrated stormwater recycling systems, and the risks involved in non-potable reuse of treated urban stormwater is being undertaken. Three stormwater recycling systems were selected at urban locations to provide a range of barriers including biofiltration, storage tanks, UV disinfection, a constructed wetland, and retention ponds. Recycled water from each of the systems is used for open space irrigation. In order to adequately undertake exposure assessments, it was necessary to quantify the efficacy of key barriers in each exposure pathway. Given that none of the selected treatment systems had previously been evaluated for their treatment efficiency, experimental work was carried out comprising dry and wet weather monitoring of each system (for a period of 12 months), as well as challenging the barriers with model microbes (for viruses, bacteria and parasitic protozoa) to provide input data for use in Quantitative Microbial Risk Assessment.

The dispersion and transport of Cryptosporidium parvum oocysts, Escherichia coli and PRD1 bacteriophage seeded into artificial bovine faecal pats was studied during simulated rainfall events. Experimental soil plots were divided in two, one sub-plot with bare soil and the other with natural vegetation. Simulated rainfall events of 55 mm.h(-1) for 30 min were then applied to the soil plots. Each experimental treatment was performed in duplicate and consisted of three sequential artificial rainfall events ('Runs'): a control run (no faecal pats); a fresh faecal pat run (fresh faecal pats); and an aged faecal pat run (one week aged faecal pats). Transportation efficiency increased with decreasing size of the microorganism studied; Cryptosporidium oocysts were the least mobile followed by E. coli and then PRD1 phage. Rainfall events mobilised 0.5 to 0.9% of the Cryptosporidium oocysts, 1.3-1.4% of E. coli bacteria, and 0.03-0.6% of PRD1 bacteriophages from the fresh faecal pats and transported them a distance of 10 m across the bare soil sub-plots. Subsequent rainfall events applied to aged faecal pats only mobilised 0.01-0.06% of the original Cryptosporidium oocyst load, between 0.04 and 15% of the E. coli load and 0.0006-0.06% of PRD1 bacteriophages, respectively.

Commercial feedlots for beef cattle finishing are potential sources of a range of trace chemicals which have human health or environmental significance. To ensure adequate protection of human and environmental health from exposure to these chemicals, the application of effective manure and effluent management practices is warranted. The Australian meat and livestock industry has adopted a proactive approach to the identification of best management practices. Accordingly, this review was undertaken to identify key chemical species that may require consideration in the development of guidelines for feedlot manure and effluent management practices in Australia. Important classes of trace chemicals identified include steroidal hormones, antibiotics, ectoparasiticides, mycotoxins, heavy metals and dioxins. These are described in terms of their likely sources, expected concentrations and public health or environmental significance based on international data and research. Androgenic hormones such as testosterone and trenbolone are significantly active in feedlot wastes, but they are poorly understood in terms of fate and environmental implications. The careful management of residues of antibiotics including virginiamycin, tylosin and oxytetracycline appears prudent in terms of minimising the risk of potential public health impacts from resistant strains of bacteria. Good management of ectoparasiticides including synthetic pyrethroids, macrocyclic lactones, fluazuron, and amitraz is important for the prevention of potential ecological implications, particularly towards dung beetles. Very few of these individual chemical contaminants have been thoroughly investigated in terms of concentrations, effects and attenuation in Australian feedlot wastes.

New WHO and Australian guidelines promote a risk-management approach for minimising exposure to pathogens in recreational waters. Between 2003 and 2005, they were applied to Lake Parramatta (10 ha, 450 ML), a potential recreation site in Sydney, Australia. A three stage approach was developed involving (1) initial suitability assessment using historic data, (2) revised suitability assessment based on new data and (3) characterisation of hazardous (especially wet weather) events. Contrary to the stage 1 suitability classification, stage 2 baseline data indicated that during dry weather the lake had water quality sufficient for primary contact recreation (95th percentiles for enterococci = 19 MPN/100, n = 50) and the major pathogen source was wildfowl. Guideline principles provided a rationale for collecting microbiological and geographic data needed to understand local cycles of lake contamination/recovery. The concept of hazardous events was particularly useful. Studies of stormwater events led us to identify a transition point (> 10 mm rainfall in 24 h) where human-faecal pathogen risks increased and access needed to be controlled. Together baseline and event data yielded operational tools (i.e. event detection methods, action triggers, auditing criteria, remediation priorities) for minimising bather exposure.

Artificial cow pats were seeded with Cryptosporidium oocysts and subjected to a simulated rainfall event. The runoff from the faecal pat was collected and different particle size fractions were collected within settling columns by exploiting the size-dependent settling velocities. Particle size and Cryptosporidium concentration distribution at 10 cm below the surface was measured at regular intervals over 24 h. Initially a large proportion of the total volume of particles belonged to the larger size classes (> 17 microm). However, throughout the course of the experiment, there was a sequential loss of the larger size classes from the sampling depth and a predominance of smaller particles (< 17 microm). The Cryptosporidium concentration at 10 cm depth did not change throughout the experiment. In the second experiment samples were taken from different depths within the settling column. Initially 26% of particles were in the size range 124-492 microm. However, as these large particles settled there was an enrichment at 30 cm after one hour (36.5-49.3%). There was a concomitant enrichment of smaller particles near the surface after 1 h and 24 h. For Pat 1 there was no difference in Cryptosporidium concentration with depth after 1 h and 24 h. In Pat 2 there was a difference in concentration between the surface and 30 cm after 24 h. However, this could be explained by the settling velocity of a single oocyst. The results suggested that oocysts are not associated with large particles, but exist in faecal runoff as single oocysts and hence have a low (0.1 m(d-1)) settling velocity. The implications of this low settling velocity on Cryptosporidium risk reduction within water supply reservoirs was investigated through the application of a three-dimensional model of oocyst fate and transport to a moderately sized reservoir (26 GL). The model indicated that the role of settling on oocyst concentration reduction within the water column is between one and three orders of magnitude less than that caused by advection and dilution, depending on the strength of hydrodynamic forcing.

This paper introduces the culture preparation of ovine, bovine and human cancellous bone cores to be used in an explants model Zetos.The three dimensional (3D) bone cores were prepared and evaluated for all three animals.Bone cells in vivo constantly interact with each other, migratory cells, surrounding extracellular matrix (ECM) and interstitial fluid in a microenvironment, which continuously responds to various endogenous and exogenous stimuli.The Zetos system was designed to culture and mechanically load viable cancellous bone explants in their near natural microenvironment.This 3D ex vivo system bridges the current gap between in vitro and in vivo methods.One aim of this work was to compare the macro and micro-architecture of ovine, bovine and human cancellous bone tissue in preparation for culture within the Zetos system in order to determine the optimal source of experimental material.A second aim was to optimise the preparations of the bone cores as well as develop techniques involved during tissue maintenance.Bone core response was visualised using histological and immunohistochemical methods.The results demonstrate that cancellous bone explants vary greatly in trabecular density and bone volume depending on species, age and location.Sheep and human samples displayed the greatest variation between bones cores when compared to bovine.Even cores taken from the same animal possessed very different characteristics.The histology demonstrated normal bone and cell structure after the core preparation.Immunohistochemistry results demonstrated antigen retention after preparation methods.

Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 mum unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 mum thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure and enabled marrow viability to be assessed as a factor of volume occupied by viable marrow. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.

There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.

AIMS:To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage.METHODS AND RESULTS:Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage.CONCLUSIONS:Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport.SIGNIFICANCE AND IMPACT OF THE STUDY:A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.

Environmental Context. It is well known that bacteria can significantly accelerate oxidation rates of copper sulfide minerals under acidic conditions. However, this process is not thought to be important at higher pH. This study reports the bacterially assisted oxidation of copper sulfide minerals in slightly alkaline (pH 8.0–8.3) tropical river waters downstream of a large copper mine. This process leads to significant solubilization of copper from particulate phases. Abstract. Field and laboratory experiments are reported which demonstrate the bacterially facilitated oxidation of copper sulfide minerals in the water column of tropical rivers. When river water samples, collected downstream of a large copper mine, were incubated under controlled laboratory conditions (pH 8.0–8.3) significant dissolved copper release was observed. This was accompanied by an increase in cold acid soluble particulate copper, indicating oxidation of copper sulfides. Dissolved copper release and copper sulfide oxidation were markedly lower in sterile control samples demonstrating biological mediation. In samples collected close to the mine discharge, dissolved copper release ceased after the first 150 h of incubation, presumably following the consumption of easily oxidizable solid copper sulfide phases or armouring of particles with insoluble oxidation products. Attempts to isolate the bacteria responsible were unsuccessful. However, oxidation rates could be enhanced by culturing aliquots of unfiltered river water samples in simple mineral media (pH 7.0) amended only with sulfide. This provided strong evidence that the copper sulfide oxidizing bacteria were chemolithotrophs. Our results suggest that such bacteria are naturally present in mineralized areas and are actively involved in the cycling of particulate copper between sulfide and hydroxy-carbonate forms, thus influencing the solubility and bioavailability of copper.

Aims: To generate field-relevant inactivation rates for Cryptosporidium oocysts in soil that may serve as parameter values in models to predict the terrestrial fate and transport of oocysts in catchments.Methods and Results: The inactivation of Cryptosporidium oocysts in closed soil microcosms over time was monitored using fluorescence in situ hybridization (FISH) as an estimate of oocyst 'viability'. Inactivation rates for Cryptosporidium in two soils were determined under a range of temperature, moisture and biotic status regimes. Temperature and soil type emerged as significantly influential factors (P < 0.05) for Cryptosporidium inactivation. In particular, temperatures as high as 35degreesC may result in enhanced inactivation.Conclusions: When modelling the fate of Cryptosporidium oocysts in catchment soils, the use of inactivation rates that are appropriate for the specific catchment climate and soil types is essential. FISH was considered cost-effective and appropriate for determining oocyst inactivation rates in soil.Significance and Impact of the Study: Previous models for predicting the fate of pathogens in catchments have either made nonvalidated assumptions regarding inactivation of Cryptosporidium in the terrestrial environment or have not considered it at all. Field-relevant inactivation data are presented, with significant implications for the management of catchments in warm temperate and tropical environments.

A rapid, highly sensitive bacterial bioassay to determine copper toxicity in freshwaters was developed based on the inhibition of cellular assimilation of radiolabeled glucose. The test used a copper-sensitive bacterium isolated from a freshwater stream. Employing sensitive radiochemical techniques enabled environmentally relevant concentrations of the test bacterium (105 cells mL−1) and a short incubation period (4 hours) to be used, which minimized the potential for changes in copper speciation during the test. The 4-hour median effective concentration (EC50) for inorganic copper at pH 7.5 in synthetic freshwater was 0.6 μg L−1 (95% confidence limits 0.4 to 1.0 μg L−1). This compared well with chronic growth inhibition of this bacterium in minimal medium (48-hour EC50 of 0.9 μg L−1 [95% confidence limits 0.7 to 1.0 μg L−1 ]). MINEQL + software (Environmental Research Software) was used to calculate copper (II) ion concentrations in synthetic freshwater at pH 7.5, giving an EC50 value of pCu2+ 8.8. However, using nitrilotriacetic acid metal-ion buffers (Cu-NTA), 50% inhibition occurred at a pCu2+ of 9.7, suggesting this bacterium was markedly more inhibited by copper in these Cu2+-buffered solutions. This may indicate that the Cu-NTA species was contributing to toxicity. The radiochemical bioassay was evaluated further using freshwater samples from both copper-impacted and pristine environments. Measured EC50 values ranged from 3.4 to 34.0 μg L−1 inorganic copper and were strongly correlated with dissolved organic carbon (DOC) concentrations (r = 0.88, p < 0.05).

ABSTRACT The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h −1 for 30 min and 25 mm h −1 for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 10 7 oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5° and 10°) was evaluated twice. Generally, a fivefold increase ( P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks ( P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 10 0.2 oocysts from vegetated loam soil (25-mm h −1 , 180-min event on 10° slope) to up to 10 4.5 oocysts from unvegetated soil (55-mm h −1 , 30-min event on 10° slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds.

Constructed wetland and detention pond as a treatment system was applied for stormwater management in two adjacent areas in western Sydney. F-specific RNA and somatic coliphages were used as a model for assessing two systems for removal of viral pollution, fate, behavior and survival of viruses in the sediment. Water samples were collected weekly in sterile containers and sediment samples were collected three times using a box dredge sampler via a boat at the inlet, middle and outlet areas of the systems. F-specific RNA coliphages were enumerated using the double layer plaque assay (ISO 1995) with Salmonella typhimurium WG49 as a host. Survival test continued 28 d for each sub-sample. Viral removal in constructed wetland was more effective than the detention pond system. Survival of somatic coliphages in the inlet and middle of the systems was similar. Slope of declining for outlet of two systems was very slow and completely stable in whole of test duration. Constructed wetland may offer an attractive alternative to stormwater management for reducing the load of disease-causing viruses to the receiving waters.

ABSTRACT Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C. parvum loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 10 2 oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces −1 must be present to be detected.

Aims: To investigate the occurrence of coliphages in, and their removal from, urban stormwater.Methods and Results: Inflow and outflow concentrations of somatic and f-specific RNA coliphages to two stormwater treatment systems were determined on 21 occasions over a period of 5 months. Somatic coliphages were detected in 19 (90%) of the constructed wetland inlet samples, 13 (62%) of the pond inlet samples, and less frequently at the outlets of the two systems. F-specific RNA coliphages were detected at the inlets but only occasionally at the pond outlet. Somatic coliphages were found to attach preferentially to particles < 5 mu m in size and persisted in the sediments of the two systems.Conclusions: Treatment systems providing conditions that are conducive to the settlement of fine particles may effectively remove sediment-bound coliphages and, therefore, possibly enteric viruses from stormwater.Significance and Impact of the Study: The results will aid the design of systems for effective removal of viral contaminants from urban stormwater.

Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20 degrees C. In this study, Technovit 9100 New-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7 microm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry.