4
Methods for Assessing Health Risks of Reclaimed Water

Any plan to augment potable water supplies with reclaimed water must include an evaluation of the potential health risks. Yet as described in earlier chapters, such assessment is complicated by several factors, including uncertainties about the potential contaminants and contaminant combinations that may be found in reclaimed water and about the human health effects those contaminants may cause.

This chapter discusses methods and strategies for assessing the health risks of drinking reclaimed water. Previous National Research Council (NRC) reports have provided similar guidance on assessing health risks of reclaimed water (see, most recently, Ground Water Recharge Using Waters of Impaired Quality, published in 1994). This chapter updates and expands on information in those earlier reports. The chapter also discusses the complications of and alternative strategies for using epidemiological studies to evaluate health risks of potable water reuse.

Evaluating Microbial Contaminants

Efforts to monitor water quality for microbiological safety have historically relied on measurements of one or more groups of coliform bacteria as indicators of fecal contamination, treatment efficiency, and the integrity of the water distribution systems. Fecal coliform bacteria are indicative of fecal contamination and associated health risks; however, the measurement and control of total coliforms (rather than only fecal coliforms) during disinfection is considered to be a more stringent treat-



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--> 4 Methods for Assessing Health Risks of Reclaimed Water Any plan to augment potable water supplies with reclaimed water must include an evaluation of the potential health risks. Yet as described in earlier chapters, such assessment is complicated by several factors, including uncertainties about the potential contaminants and contaminant combinations that may be found in reclaimed water and about the human health effects those contaminants may cause. This chapter discusses methods and strategies for assessing the health risks of drinking reclaimed water. Previous National Research Council (NRC) reports have provided similar guidance on assessing health risks of reclaimed water (see, most recently, Ground Water Recharge Using Waters of Impaired Quality, published in 1994). This chapter updates and expands on information in those earlier reports. The chapter also discusses the complications of and alternative strategies for using epidemiological studies to evaluate health risks of potable water reuse. Evaluating Microbial Contaminants Efforts to monitor water quality for microbiological safety have historically relied on measurements of one or more groups of coliform bacteria as indicators of fecal contamination, treatment efficiency, and the integrity of the water distribution systems. Fecal coliform bacteria are indicative of fecal contamination and associated health risks; however, the measurement and control of total coliforms (rather than only fecal coliforms) during disinfection is considered to be a more stringent treat-

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--> ment goal. Water quality standards have used either or both measures depending on the type of water use. While coliform bacteria serve well as indicators of bacterial pathogens, they do not predict the inactivation or removal of enteric protozoa and viruses (Gerba and Rose, 1990; LeChevallier and Norton, 1993; LeChevallier et al., 1991a, 1991b; Rose et al., 1991). For instance, LeChevallier and Norton (1993) found that multiple linear regression models using coliforms and temperature could predict only 57 percent of the variation in Giardia cyst concentration, whereas no model using indicator bacteria could adequately predict Cryptosporidium oocyst levels. Methods to detect viruses in water were first developed by Paul and Trask (1947) for measuring enteroviruses in untreated wastewater. Coin (1966) was one of the first to detect viruses in finished drinking water meeting the existing coliform standard. In the 1970s, improvements in collection filters and the use of antibodies led to the first isolation of rotavirus and hepatitis A virus from water. Today, standard methods for the detection of enteric viruses are based on the ability of viable enteric viruses to destroy monkey kidney cells grown in vitro; this cell-destroying ability is known as cytopathic effect or CPE (Benenson, 1995). Methods for detecting microbial protozoa were first developed for Entamoeba in the 1940s. Starting in 1965, research focused on the detection of Giardia. In the 1980s, a standardized approach for Giardia detection was developed that used filtration for collection and antibodies labeled with a fluorescent isothiocyanate (FITC) for enhanced microscopic detection (Rose et al., 1988a, 1988b). This approach was applied to the detection of Cryptosporidium after its first recorded waterborne outbreak in the United States in 1985. As more protozoan and viral waterborne outbreaks occurred in waters meeting existing water quality standards, the limitations of using indicator bacteria became apparent. In response to these health concerns, the Environmental Protection Agency (EPA) promulgated the Surface Water Treatment Rule for drinking water in 1989 (U.S. EPA, 1989a, 1989b). The rule established treatment-based performance goals of 99.99 percent reductions of viruses and 99.9 percent reduction of Giardia. The rule also emphasized the use of sand or multimedia filtration for the removal of Giardia and the use of improved disinfection methods for the control of both viruses and Giardia. The target reduction level was based on anticipated levels of pathogens in ambient surface waters, and the performance goals were derived from a desired annual risk of microbial disease of not greater than 1 in 10,000. An Enhanced Surface Water Treatment Rule (U.S. EPA, 1996) is under development; the enhanced rule will include an assessment of Cryptosporidium in source waters and its removal by treatment processes.

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--> Arizona is the only state that has standards for the concentration of enteric viruses and Giardia in reclaimed water; it is also the only state in which water reuse is regulated by a laboratory certification program, although specialized studies have been undertaken in Florida and California for viruses and protozoa. To address the lack of information nationwide on levels of viral and protozoan pathogens and the efficacy of water treatment, EPA promulgated the Information Collection Rule (ICR) in 1996. Detection processes for the enteroviruses by cell culture and for protozoa by microscopy have been standardized for this rule, and laboratories are undergoing an approval process. The data will be used in future risk analyses to establish the necessary drinking water treatment performance criteria for the protection of public health. The results will be applicable to potable reclamation projects as well. However, when considering wastewater as a source of drinking water, particular attention should be paid to current limitations and other issues involved with the methodology used to detect microbial pathogens. Microbial Detection Methods Microbial detection methods can be described and compared in terms of recovery (the efficiency of the method for collecting microorganisms from water samples), sensitivity (a measure of the minimum number of microorganisms that can be detected per unit volume), and specificity (the proper taxonomic identification of the microbial agent). No method is 100 percent efficient; estimates of recovery tend to range from 5 to 60 percent. A method's sensitivity is often expressed as a detection limit, such as 1/100 ml, meaning that it is able to detect one microorganism in a 100 ml sample. In untreated wastewater, concentrations of microorganisms can be high enough to be readily detected in small test volumes. However, such methods are not sufficiently sensitive for testing the highly treated reclaimed water typically produced by potable reuse projects. With highly treated reclaimed water, larger volumes of water are needed for analysis, and microorganisms may occur at concentrations too low to be detected. Table 4-1 presents the major microbial detection techniques as they are applied to the detection and quantification of bacteria, viruses, and protozoa. Culture techniques have long been used for the detection and enumeration of viable bacteria and viruses, while microscopy techniques have a long history in the identification of bacteria and protozoa. Table 4-2 summarizes the advantages and disadvantages of some methods for evaluating the microbiological quality of reclaimed water. The polymerase chain reaction (PCR) has only recently been applied to

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--> TABLE 4-1 Methods and Issues for Detection of Microorganisms in Water Microbe Collection Identification Enumeration Major Concerns Bacteria Low volumes: 100 to 4000 ml grab samples Differential media and growth; biochemical testing Colony counts; MPN Viable but nonculturable and stressed bacteria are not accounted for. Staining and microscopy methods now available, but rarely used. Viruses Large volume collection: 10 to 1000 liters by filtration/adsorption Generally, antiserum is used to identify the viruses. Most often, viruses are broadly described as either cultivable enteric viruses or enteroviruses. PFU in cells or by an MPN method utilizing cell destruction (CPE) Standards methods are available for enteroviruses, rotaviruses, adenoviruses, and hepatitis A virus. Only a small percentage of the viruses will cause CPE. Most studies underestimate the level of viruses. Protozoa Large volume: 10 to 1000 liters by filtration/entrapment Based on antibody staining and microscopic visualization (size, shape, morphology) Counts under the microscope No available test for speciation or viability   NOTE: CPE = cytopathic effects; MPN = most probable number; PFU = plaque-forming units.

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--> TABLE 4-2 Advantages and Disadvantages of Microbiological Methods for Detecting Microorganisms in Reclaimed Water Tool Advantages Disadvantages Application Culture of bacteria and viruses Widespread use In some cases, lacks specificity (i.e., generic for enteroviruses; HPC) To evaluate water quality and treatment, retrospectively   Standardized   For comparison to new methods and old databases   Detects viable microorganisms Results take days or weeks When viability is of major concern     Difficult to quantitate in some cases       Measures only a fraction of the types of microorganisms present; no ability to detect those that are viable but nonculturable  

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--> Microscopy for bacteria and protozoa Can be used with specific stains (monoclonal antibodies, probes, fluorogenic dyes, and viability stains) Rapid, quantitative Sample preparation can interfere with detection Less sensitive than culture as smaller volumes are examined Can be tedious Only method readily available for protozoa Assessment of physical removal For use when stressed bacteria are an issue Polymerase chain reaction Highly specific, rapid Measures nonviable microorganisms Can be inhibited by sample constituents Nonquantitative Less sensitive due to low volumes processed Excellent for presence/absence assessment Only method available for key viruses of concern Can be applied with cell culture techniques for rapid and specific identification NOTE: HPC = heterotrophic plate count.

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--> wastewater to identify a variety of microorganisms based on their species-specific nucleic acid sequence. Use of Polymerase Chain Reaction Techniques Polymerase chain reaction, or PCR, is a molecular technique used to detect a variety of microorganisms in environmental samples (Atlas et al., 1992; Bej et al., 1990; Johnson et al., 1995; Kopecka et al., 1993; Mahbubani et al., 1991; Tsai and Olson, 1991). PCR can rapidly identify a specific organism. However, before PCR can be used routinely for environmental monitoring, several issues must be addressed, including the test's sensitivity, the viability of detected pathogens, and assay interference by inhibitors in the spectrum. The sensitivity (or the limit of detection) of PCR is constrained by the technology. In most cases, only very small volumes (100 µl or less) can be processed through the thermal cyclers (machines that control the sample temperatures during processing) used in PCR assays. Therefore, concentration of samples is necessary. (Alternatively, larger-capacity machines could be developed in order to increase the sample volume.) Further, because PCR does not distinguish live from dead microorganisms, a cell cultivation procedure must be performed before the results have relevance to health risks. This is especially true for samples taken from water that has undergone disinfection. PCR may therefore be most useful in untreated waters (source waters, recreational waters, shellfish harvesting waters, ground waters) where viability can be assumed, or in the evaluation of processes designed to physically remove microbiological particles (such as membrane processes). Water quality is also an issue, since physical and chemical constituents in water can mask the target nucleic acid or inhibit the enzyme reaction that the PCR process uses to amplify the target DNA, creating a false negative result. Recently developed antibody capture procedures appear to have great promise in addressing the problem of interference for both protozoa (Johnson et al., 1995) and viruses (Deng et al., 1994). Finally, PCR remains only qualitative in that the results are presented as positive or negative. The development of quantitative techniques using PCR would be very useful for assessments of the microbiological quality of drinking water. Detection of Bacteria Several well-established methods for detecting and enumerating coliform and fecal coliform bacteria indicators exist and are useful for evaluating the effectiveness of disinfection in water and wastewater treat-

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--> ment. Generally, any treatment process that inactivates the indicator bacteria also inactivates pathogenic enteric bacteria to similar degrees. Nevertheless, the use of indicator bacteria has limitations. For instance, recent findings suggest that very low levels (10 to 100 colony-forming units (CFU) per liter) of Salmonella may be related to incidences of reactive arthritis (Smith et al., 1993), suggesting that Salmonella should be measured directly. In addition, Singh and McFeters (1990) reported a noncultivable but viable state for Yersinia bacteria after disinfection. The public health significance of these noncultivable bacteria has not been fully assessed. Future regulations may dictate that greater reductions of pathogenic bacteria be achieved and documented. Directly detecting pathogenic bacteria has traditionally been a tedious process, requiring many biochemical tests to identify the genus and/or species. Most testing has been done with presence/absence tests or the most probable number (MPN) approach. A standard MPN procedure has been developed for detecting Salmonella in sludge (U.S. EPA, 1992); however, the sensitivity of the test has been questioned (National Research Council, 1996). No standard procedure exists for Salmonella testing in reclaimed water. As described above, PCR is a rapid detection method that can be combined with more traditional cell culture techniques to assess viability of bacterial pathogens. This approach has been successfully applied in the food industry (Fung, 1994) and may hold promise for use in reclaimed water as well. Detection of Protozoa Detecting low concentrations of protozoan cysts or oocysts in highly treated reclaimed water requires passing large volumes of water through a filter with an appropriate pore size (typically 1.0 µm). Unfortunately, this method also concentrates unwanted constituents in the sampled water (e.g., particulates, precipitated minerals), and these constituents must then be separated in the subsequent analysis using a concentration and clarification procedure (LeChevallier and Trok, 1990; Rose et al., 1989). The semipurified sample, consisting of the larger organisms with some of the unwanted constituents removed, is then mixed with fluorescent-tagged monoclonal antibodies specific to the cyst and oocyst wall using an indirect fluorescent antibody (IFA) procedure. The sample can then be examined and the protozoa identified by one or more techniques, such as epifluorescence or differential interference contrast (DIC) microscopy (LeChevallier et al., 1991a). The efficiency of cyst/oocyst recovery for Cryptosporidium and Giardia is quite variable, and losses occur at each of the various steps of the detection process; thus many of the organisms contained in the original

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--> water sample will be lost. Reported efficiencies are: 88-99 percent for sample collection; 16-78 percent for filter elution; 66-77 percent for concentration and clarification; and 9-59 percent for microscopic detection using the IFA method (LeChevallier and Trok, 1990; LeChevallier et al., 1991a; Ongerth and Stibbs, 1987; Rose et al., 1988b, 1989, 1991b). Given these variations in recovery, current methods for detecting protozoa tend to underestimate the true concentrations in environmental samples. Indirect fluorescent antibody techniques using antibodies labeled with fluorescent isothiocyanate have greatly enhanced the ability to detect Cryptosporidium and Giardia in environmental samples. However, most antibody techniques provide no species identification (e.g., bird versus mammalian isolates), nor do they determine whether cysts and oocysts are viable. LeChevallier et al. (1991a, 1991b) reported that 10 to 30 percent of the organisms detected by IFA were not empty and therefore viable. Other inaccuracies may occur due to background fluorescence from naturally fluorescing organisms and from nonspecific binding of the antibody. Such problems may produce either false positives or false negatives. Although false positives can create inconvenience or necessitate further testing, Clancy et al. (1994) noted that false negatives (failing to detect protozoa that are present) pose more serious problems. A number of IFA systems have been developed for Cryptosporidium and Giardia (Garcia et al., 1987; Rose et al., 1989; Stibbs et al., 1988). New methods under development should allow a better assessment of enteric protozoa. These methods include the use of cell cultures to detect viable and infectious oocysts, the use of immunomagnetic separation (IMS) techniques to enhance the recovery of cysts/oocysts (Jakubowski et al., 1996; Linquist, 1997; Slifko et al., 1997), and the use of internal stains for improved identification and detection. Detection of Viruses Viruses may be concentrated from water using either electropositive or electronegative filters. Either can concentrate viruses from large volumes of water, but clogging, particularly in positively charged filters, may occur if the water is high in suspended solids or turbidity (Rose et al., 1989). The adsorbed viruses are eluted and concentrated to a smaller volume using an organic flocculation procedure. This final concentrate may be inoculated into cell cultures to detect cytopathic effects, or it may be tested by PCR or other types of tests. Detection methods have primarily been developed for the enterovirus group, which consists of poliovirus, echoviruses, and coxsackieviruses. Microbiological studies in the San Diego potable reuse project demonstrated the feasibility of recovering 1 virus in 1000 liters of water.

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--> When large volumes of water are sampled, one of the unresolved technical issues is whether a given filter will adsorb an equivalent amount of viruses per unit volume of water as more water is passed through the filter or whether the filter's performance will decrease as the quantity of water sampled increases. Further, some research has suggested that not all enteroviruses are adsorbed by filters at the same rate (Powelson and Gerba, 1995). Cell culture techniques using CPE (cytopathic effect, or cell destruction) on monkey kidney cell lines provide the principal method for virus detection. In contrast to processing procedures for detecting protozoa, the debris collected along with viruses is more easily separated before cell culture. The types of viruses are further identified by the use of specific antibodies to neutralize the virus's cytopathic effect. However, these cell culture methods may detect only a small percentage of the viruses found in polluted waters because not all viruses will cause CPE. When Payment and Trudel (1987) used labeled antibodies to determine the numbers of infectious foci in the cell culture, they found levels of viruses up to 100 times greater than what they detected by CPE. While enteroviruses are the most heavily studied waterborne viruses, the limited data on virus identification indicate that retroviruses typically occur at higher levels in wastewater (Gerba and Rose, 1990). Hurst et al. (1988) found that adenoviruses could be detected at levels 94 times higher than enteroviruses when using molecular techniques (gene probes) to verify the results of cell cultures. In sum, although methods exist for detecting some viruses, we lack sufficient data to determine the concentrations and diversity of the many viruses of concern in wastewater, reclaimed water, and ambient waters (Hurst et al., 1989). These include emerging viruses of concern such as Norwalk viruses. Indicator Techniques Several types of microorganisms have been suggested as alternatives to coliform bacteria as indicators of water quality, fecal pollution, and public health risks. These include the bacteria Enterococcus and Clostridium perfringens and the F-specific coliphage bacterial virus. The Enterococcus bacteria comprise a subgroup of the fecal Streptococci bacteria and include S. faecium, S. faecalis, S. durans, and related biotypes (Clausen et al., 1977). Enterococci are generally more resistant to water treatment than bacterial pathogens or fecal coliforms, and membrane filtration procedures are available for sampling them (Cohen and Shuval, 1973; Davies-Colley et al., 1994; Sinton et al., 1994). Some epidemiological investigations found that levels of Enterococcus correlated with

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--> an increased incidence of gastrointestinal symptoms associated with exposure to polluted recreational waters (Cabelli et al., 1979; Dufour, 1984). The EPA has suggested that Enterococcus may be the best microbial indicator for ambient waters (U.S. EPA, 1986). A few states have adopted the use of Enterococcus, but most continue to use total and/or fecal coliforms for evaluating recreational water quality and the wastewater effluents that may impact these waters. Clostridium perfringens is a pathogenic bacterium found in human and animal feces. Because it forms resistant spores, Clostridium has been recommended as a conservative indicator of water quality and as a valuable supplement to other water quality tests, particularly in situations where the detection of viruses or fecal contamination is desirable (Fujioka and Shizumura, 1985; Payment and Franco, 1993). This microorganism is consistently present in municipal wastewater at concentrations of 103 to 104 CFU/100 ml. Fairly rapid and simple membrane filtration methods are available for the enumeration of C. perfringens. Researchers have found a significant correlation between levels of C. perfringens and enteric viruses and protozoa in evaluations of the treatment efficiency of filtration and disinfection (Fujioka and Shizumura, 1985; Payment and Franco, 1993). These authors suggested that the removal of this indicator by a factor 7 to 8 log10 essentially ensures the removal of enteric protozoa and viruses at current limits of detection. However, no jurisdictions in the United States are known to have adopted Clostridium as a regulatory standard (Cabelli, 1997). The coliphages are viruses that infect Escherichia coli, and therefore the presence of coliphages in water indicates the presence of their host E. coli, which is excreted by animals and humans. Coliphages may serve as better indicators for human enteric viruses than bacterial indicators do because coliphages more closely resemble human enteric viruses in size, shape, and resistance to treatment processes. In a comparison of untreated and treated wastewater, river water, treated river water, and treated lake water, Havelaar et al. (1993) found significant correlations between levels of coliphage and levels of enteric viruses. However, this correlation was not evident for the untreated and treated wastewater samples, which suggests that other unknown factors may complicate the use of this indicator when evaluating recent wastewater inputs into a water body. Payment and Franco (1993) examined the removal of coliphages and enteroviruses in drinking water treatment. The total removal and/or inactivation of enteroviruses by the complete drinking water process was estimated at 7 log10, based on coliphage removal. Coliphages have also been used as biological tracers in the environment to evaluate the movement of septic tank effluent (Paul et al., 1995). An advantage of using

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--> controls are selected is critical to the correct design of the study. Cases and controls must be selected without knowledge of their exposure. The strengths and limitations of various recruitment strategies need to be carefully considered. For example, choosing prevalent cases of cancer rather than incident cases will favor survivors; choosing controls from a hospital may select people with disease conditions that could also be influenced by environmental factors; and choosing community controls via telephone recruitment will favor those who have telephones and are available to answer the telephone (i.e., those who spend more time in the home). In cohort studies, the study population must be selected without knowledge of disease. Loss of certain members of the cohort during the follow-up period may result in bias if those who drop out of the cohort are unique in a certain way and become underrepresented in the final cohort. An example of such a unique group is families with young children; these families may find that they do not have time to participate in studies that require maintaining health diaries or responding to extensive interviews. Information or misclassification bias can occur in the determination of exposure or of health outcome. This bias results from systematic errors in measuring either the exposure or the disease. In water studies, misclassification of exposure is a major concern. Information on an individual's exposure to a specific water supply may be based on information from death certificates or water utility bills. However, this measure only indicates exposure at a specific point in time that may not be the exposure time frame relevant to the disease of interest. For example, risks of bladder cancer may be strongly associated with the type of water supply an individual was exposed to 20 years previously and may not be related to that person's current water supply. When evaluating this type of bias, it is important to determine whether it is "differential" or "nondifferential." Differential misclassification occurs when either (1) exposure was measured differently for cases than for noncases or (2) health outcomes were measured differently for exposed than for unexposed people. Differential misclassification will result in either an overestimate or an underestimate of the measure of association between exposure and disease. Nondifferential misclassification occurs when either (1) exposure is measured equally incorrectly for both cases and controls or (2) health outcomes are determined equally incorrectly for the exposed and unexposed people. In these situations, the measure of association between exposure and disease is underestimated. Recall bias can be a concern when data are gathered retrospectively and is most likely to occur in exposure assessment. For example, lifetime water histories may be recalled differently by cases than by controls, be-

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--> cause ill people may be more likely to remember (and blame) specific exposures for their illness. Confounding bias is a problem when the exposed and unexposed groups differ in the occurrence of some factor or factors that affect the development of the health outcome of interest. Common confounders are demographic characteristics such as age, gender, race, and occupation or behavioral characteristics such as smoking or alcohol abuse. For example, in an ecologic study, the study community may house a chemical manufacturing plant, and therefore it may have more individuals with occupational exposure to hazardous chemicals than the control community. This difference, rather than water supply, may account for higher cancer rates in the study community. The effect of confounding bias on the measure of association can be controlled in the study design either by restricting the study population to persons with a narrow range of a confounder (such as white, male, nonsmokers between the ages of 30 and 50) or by matching study groups based on confounders (such as matching cases and controls for age, gender, race); or confounding bias can be controlled in the analysis phase of the study by using techniques that account for the effect of the bias. All of these control methods rely on being able to identify and measure the relevant confounders for the health outcome of interest. Conclusions and Recommendations A number of methodological issues make it difficult to definitively determine the public health risks of drinking reclaimed water. Despite these uncertainties, any utility considering the implementation of a potable reuse project should estimate the increased risk from microbial and chemical contaminants in reclaimed water relative to those from other available sources of water. Microbiological Methods and Risk Assessment There is a lack of information nationwide on the levels of viral and protozoan pathogens in all waters and the efficacy of both conventional water treatment and wastewater treatment for water reclamation in reducing these levels. The Information Collection Rule, promulgated in 1996 by the EPA, is a first step toward providing some of the exposure data needed for more effective risk assessments, but additional steps are needed to improve methods for assessing risks posed by microbial pathogens in water reuse projects. Potable reuse projects should consider using some of the newer analytical methods, such as biomolecular methods, as well as new in-

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--> dicator microorganisms, such as Clostridium perfringens and the F-specific coliphage virus, to screen drinking water sources derived from treated wastewaters. The microbial methods currently used for detecting bacterial, viral, and protozoan pathogens in water all have limitations when used to detect pathogens in reclaimed water. Bacterial techniques do not account for viable, noncultivable bacteria, and the new techniques for assessing the viability of protozoan cysts or oocysts will require evaluation. Standard cell culture methods employing tests of cytopathic effects have been limited to the detection of well-known enteroviruses and do not account for many other enteric viruses that may be found in wastewater. New analytical methods for rapid measurement of health-related microbial contaminants, such as the polymerase chain reaction, are being developed. However, treatment performance and water quality goals have not been developed for these methods. In addition, several new indicator microorganisms are available, including Clostridium perfringens and the F-specific coliphage virus, and should be considered as alternatives to using the coliform bacteria as indicators for water quality. In particular, F-specific coliphage can be used in seeded studies to provide useful data on unit process removals. The EPA should include data on the concentrations of waterborne pathogens in source water in the new Drinking Water National Contaminant Occurrence Data Base and should develop better data on reductions of waterborne pathogens by various levels of treatment. The lack of monitoring data for evaluating exposure remains the greatest single barrier to the development of risk assessment for microbial pathogens. Microbial risk assessment requires better estimates of exposure, which should be based on monitoring data, to identify the concentration of microbial pathogens in raw wastewater, wastewater treated with various processes, ambient water, and drinking water treated with various processes. State officials, water utilities, and water research scientists should document survival rates of relevant protozoa in natural environments. Indirect potable reuse projects may rely on dilution in the environment and reduction by natural processes (i.e., die-off in ambient waters and removal by soil infiltration systems) to remove pathogens of all kinds; however, while reductions of bacteria and viruses have been well documented, the information on protozoa survival in ambient waters remains inadequate. More research is needed to fill that gap. Risk estimates should consider the effects on sensitive populations and the potential for secondary spread of infectious disease within a community. This precaution is necessary to prevent pathogens from infecting sensitive populations (the elderly or very young, or those with

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--> suppressed immune systems) in whom mortality may be high and from whom diseases might spread to others. The research community should conduct further studies to document the removal of pathogens of all types by membrane processes. Membrane systems, such as microfiltration, ultrafiltration, nanofiltration, and reverse osmosis, show the potential for nearly complete rejection of pathogens above certain size classes (in the case of the latter three processes, all size classes). More work is needed to demonstrate the suitability of these processes for potable reuse applications and to develop monitoring methods capable of continuously assessing process performance. Chemical Risk Assessment Analysis of reclaimed water is complicated by the fact that toxicological data on the wide variety of organic compounds in wastewater are much less complete than those for inorganic compounds. Thus, even if the organic material in the water could be analyzed completely, there would be no basis for assigning risks to most of the identifiable compounds present. This situation complicates the management of risks from chemical contaminants. A conventional toxicological safety testing strategy developed in the food and drug industries uses both live animal (in vivo) and cell culture (in vitro) testing. While this approach has been used to develop risk assessments and regulations for recognized chemical contaminants in drinking water, there has been little experience in applying the strategy to determine health risks posed by the poorly characterized mixtures of organic chemicals in reclaimed water. The current state of knowledge in toxicology is too limited to make meaningful risk assessments based on in vitro data alone. So far, most toxicological studies of potable reuse have focused on bacterial and/or in vitro mammalian tests of genotoxicity of product waters rather than comprehensive testing on live animals. These bacterial or in vitro tests do not accurately evaluate the risks posed by the complex mixtures of contaminants in reclaimed wastewater. Because of the uncertainty in the organic composition of reclaimed water, toxicological testing should be the primary component of chemical risk assessments of potable reuse systems. Attempting to ensure the safety of reclaimed water by analyzing only for known chemical contaminants, such as those regulated under the Safe Drinking Water Act, will not provide adequate protection of public health. In waters where toxicological testing appears to be important for determining health risks, emphasis should be placed on live animal test systems capable of expressing a wide variety of toxicological ef-

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--> fects. Chapter 5 presents a proposed system using fish in ambient waters. Further, toxicological testing standards for reclaimed water should be supplemented by strict regulation of the processes for ''manufacturing" the water. Regulators should review the processes for manufacturing the reclaimed water (that is, the treatment systems and environmental storage employed) on a plant-by-plant basis. Epidemiological Methods Several methodological challenges complicate epidemiologic investigations of the health effects of potable water reuse. These challenges include (1) obtaining accurate measures of individual or group exposure to the waterborne agent of interest, (2) selecting the appropriate health outcomes to monitor, (3) accurately measuring health outcomes through either a surveillance system or individual health records, (4) estimating the fraction of disease cases due to waterborne exposure, (5) selecting an appropriate comparison group, and (6) recruiting a study population large enough to detect a true effect. Given these challenges, the results of epidemiologic studies should be interpreted with caution and a recognition of the potential for systematic and random error and potential biases. The strongest observational study design for establishing a cause-and-effect relationship between exposure to waterborne disease agents and disease occurrences is a cohort study, which compares the disease rates over time among individuals who are exposed to reclaimed water to disease rates among individuals who use a different water source. References Asano, T., L. Y. C. Leong, M. G. Rigby, and R. H. Skaaji. 1992. Evaluation of the California wastewater reclamation criteria using enteric virus monitoring data. Water Science and Technology 26:1513-1524. Ashby, J., and R. W. Tennant. 1988. Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by U.S. NCI/NTP. Mutation Research 204:17-115. Atlas, R. M., G. Sayler, R. S. Burlage, and A. K. Bej. 1992. Molecular approaches for environmental monitoring of microorganisms. BioTechniques 12:706-717. Bailey, N. T. J. 1975. The Mathematical Theory of Infectious Diseases and Its Applications. New York: Oxford University Press. Bailey, N. T. J. 1986. Macro-modeling and prediction of epidemic spread at community level. Mathematical Modeling 7: 698-717.

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