This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.Standardized methods used for surface and drinking waters are available to evaluate the occurrence of protozoan pathogens in water supplies, wastewater effluents, and reclaimed effluents. Reclaimed effluents can be defined as those waters that have received a combination of physical, chemical, and biological processes and operations to remove settleable, suspended, and dissolved solids, organic matter, metals, nutrients, and pathogens from wastewater. The major uses of reclaimed effluents include agricultural and landscape irrigation, groundwater recharge, industrial use, and surface water replenishment (2, 16).Cryptosporidium parvum and Giardia lamblia, according to some public health officials, are no more prevalent in reclaimed effluents than in other irrigation waters. Occasional findings of (oo)cysts in reclaimed water may, however, present a health risk due to the potential high viability and various routes of exposure. Public concern about the health risk has prompted many states, including Florida, to add periodic sampling for Giardia and Cryptosporidium to present reuse rules (10).Monitoring for protozoan pathogens is necessary for estimating the risk of infection resulting from exposure to reclaimed water. Presently, there are no approved methods for sampling and detecting protozoan pathogens in reclaimed effluents. With the development of new methods for detecting waterborne Cryptosporidium and Giardia, there is a great intere...

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.Reclaimed water (treated wastewater) is being utilized in the United States and throughout the world as an alternative nonpotable water source. In the United States, 18 states currently have standards and another 18 have guidelines for reclaimed water (8). These standards, for the most part, are based on total suspended solids and fecal coliforms. A variety of microbial pathogens are present in wastewater and can be detected in reclaimed water. Therefore, advanced treatment, including filtration and disinfection, is required to produce reclaimed water that does not have a negative impact on public health. In regulatory language, this means that pathogens are to be less than the limit of detection of the assay (8).Cryptosporidium parvum, a coccidian protozoan parasite, is a potential contaminant of reclaimed water. C. parvum oocysts have been found to be persistent in the environment and resistant to chlorination. Because of this, physical removal by chemical pretreatment and filtration is the primary means of reducing the level of oocysts in environmental water (6). A possible risk to human health exists if filtration fails to function efficiently. This risk is greater still with reclaimed water, as to date no monitoring for C. parvum oocysts has been required and little information is available on the filtration efficiency in these facilities. Recently, in the state of Florida, monitoring for protozoan parasites, including Cryptosporidium, once every 2 years for larger facilities and once every 5 years for smaller facilities has been mandated. Sampling is recommended at a single point following disinfection (2).In one study, C. parvum oocysts were detected in untreated wastewater (67% of the samples were positive) and in reclaimed water (25% of final effluent samples were positive) (5). However, only the presence of the oocysts was evaluated using fluorescence microscopy. Robertson et al. evaluated wastewater samples for viable C. parvum by using vital stains; 35% of the influent samples and 46% of the effluent samples contained viable oocysts (4). In the last few years, the focus detection method-most-probable-number (FDM-MPN) cell culture technique has been developed to test the oocyst infectivity because the previously employed methods did not accurately reflect the infectious nature of the oocysts (7). The objective of the present study was to demonstrate the presence of infectious C. parvum oocysts in final reclaimed effluent from six reclamation facilities in the United States by using the FDM-MPN cell culture technique.Samples were coll...

Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen.

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The need for alternative sources of water, especially for non-potable purposes, has been met in many states in the US and throughout the world through the use of reclaimed wastewater. Wastewater contains a wide variety of microbial pathogens that may pose a risk to human health if not properly controlled; however, there are currently no national standards for microorganisms or consistent treatment requirements in the US with regards to reclaimed water. Besides the routine monitoring for TC and FC bacteria that has been used for assessing water quality, several types of alternative microorganisms have been suggested as indicators of water quality, fecal pollution, and public health risks. These include enterococci, Clostridium perfringens, and coliphage among others. This project evaluated the removal of both indicators (TC and FC, enterococci, C. perfringens and coliphage) and pathogens (enteroviruses, Giardia spp. and Cryptosporidium spp.) at 3 water reclamation facilities with varying treatment designs and operations. The facilities evaluated range in size from 20 to 44 million Liters per day capacity, with both shallow and deep bed sand/anthracite filters. Differences also existed in the disinfection processes for the 3 reclamation plants. The TC and FC showed a 5 to 7 log reduction throughout the treatment processes with no detectable levels in the final effluent. However, even though the alternative indicators (enterococci, C. perfringens and coliphage) showed reductions that varied from 2 to 6 logs, some levels of these indicators were consistently detected in 2 of the 3 facilities in the final reclaimed effluent. Both Cryptosporidium spp. and Giardia spp. were detected in the final effluent in all 3 facilities, even when no indicators were present. Virus detection was seasonal, and associated with cooler temperatures and less disinfection. The results of this study indicate that differences in filter design, operations, and disinfection approaches were responsible for differences in inter and intra facility water quality variability.

The presence of infectious protozoan pathogens in reclaimed water may present an unacceptable health risk. This study was designed similar to a study reported by Garcia et al. (2002), which detected no infectious Giardia cysts in the final effluent of a tertiary treatment facility as determined by animal infectivity (dose 1000 cysts/gerbil). This study also included evaluation of Cryptosporidium oocyst infectivity. Infectious Giardia cysts were detected in the final effluent with 1 gerbil out of 3 inoculated with 250 cysts from reclaimed water showing signs of infection 15 days postinoculation. None of the Cryptosporidium oocysts concentrated from the reclaimed water samples appeared to be infectious. Water Environ. Res., 78, 2297Res., 78, (2006.

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