Large-scale comparison of E. coli levels determined by culture and a qPCR method (EPA Draft Method C) in Michigan towards the implementation of rapid, multi-site beach testing.

Fecal pollution remains a challenge for water quality managers at Great Lakes and inland recreational beaches. The fecal indicator of choice at these beaches is typically Escherichia coli (E. coli), determined by culture-based methods that require over 18 h to obtain results. Researchers at the United States Environmental Protection Agency (EPA) have developed a rapid E. coli qPCR methodology (EPA Draft Method C) that can provide same-day results for improving public health protection with demonstrated sensitivity, specificity, and data acceptance criteria. However, limited information is currently available to compare the occurrence of E. coli determined by cultivation and by EPA Draft Method C (Method C). This study provides a large-scale data collection effort to compare the occurrence of E. coli determined by these alternative methods at more than 100 Michigan recreational beach and other sites using the complete set of quantitative data pairings and selected subsets of the data and sites meeting various eligibility requirements. Simple linear regression analyses of composite (pooled) data indicated a correlation between results of the E. coli monitoring approaches for each of the multi-site datasets as evidenced by Pearson R-squared values ranging from 0.452 to 0.641. Theoretical Method C threshold values, expressed as mean log10 target gene copies per reaction, that corresponded to an established E. coli culture method water quality standard of 300 MPN or CFU /100 mL varied only from 1.817 to 1.908 for the different datasets using this model. Different modeling and derivation approaches that incorporated within and between-site variability in the estimates also gave Method C threshold values in this range but only when relatively well-correlated datasets were used to minimize the error. A hypothetical exercise to evaluate the frequency of water impairments based on theoretical qPCR thresholds corresponding to the E. coli water quality standard for culture methods suggested that the methods may provide the same beach notification outcomes over 90% of the time with Method C results differing from culture method results that indicated acceptable and unacceptable water quality at overall rates of 1.9% and 6.6%, respectively. Results from this study provide useful information about the relationships between E. coli determined by culture and qPCR methods across many diverse freshwater sites and should facilitate efforts to implement qPCR-based E. coli detection for rapid recreational water quality monitoring on a large scale in the State of Michigan.

Antibiotic Resistance in Recreational Waters: State of the Science.

Ambient recreational waters can act as both recipients and natural reservoirs for antimicrobial resistant (AMR) bacteria and antimicrobial resistant genes (ARGs), where they may persist and replicate. Contact with AMR bacteria and ARGs potentially puts recreators at risk, which can thus decrease their ability to fight infections. A variety of point and nonpoint sources, including contaminated wastewater effluents, runoff from animal feeding operations, and sewer overflow events, can contribute to environmental loading of AMR bacteria and ARGs. The overall goal of this article is to provide the state of the science related to recreational exposure and AMR, which has been an area of increasing interest. Specific objectives of the review include (1) a description of potential sources of antibiotics, AMR bacteria, and ARGs in recreational waters, as documented in the available literature; (2) a discussion of what is known about human recreational exposures to AMR bacteria and ARGs, using findings from health studies and exposure assessments; and (3) identification of knowledge gaps and future research needs. To better understand the dynamics related to AMR and associated recreational water risks, future research should focus on source contribution, fate and transport-across treatment and in the environment; human health risk assessment; and standardized methods.

Evaluating health risks associated with exposure to ambient surface waters during recreational activities: A systematic review and meta-analysis.

Recreational water quality guidelines protect the public from health risks associated with water recreation by helping to prevent unacceptable concentrations of pathogenic organisms in ambient water. However, illness risk is associated with both the concentration of pathogens in the water and the degree of contact with those pathogens. Different recreational activities can result in different levels of contact with ambient water containing water-borne pathogens. We conducted a systematic literature review and meta-analysis to evaluate risks of illness associated with different recreational activities and different levels of contact to ambient surface waters. We screened 8,618 potentially relevant studies for quantitative measures of risk using inclusion/exclusion criteria established in advance. We categorized recreational activities as swimming, sports-related contact, minimal contact, and sand contact. We combined relative risks using a random effects meta-analysis for adverse health outcome categories representing gastrointestinal illness, respiratory illness, skin, eye, ear, nose, throat, and cold/flu illness. We identified 92 studies meeting our inclusion criteria. Pooled risk estimates indicate significant elevation of gastrointestinal illness with the recreational activity categories swimming (2.19, 95% CI: 1.82, 2.63) and sports-related contact (2.69, 95% CI: 1.04, 6.92), and nonsignificant elevation of gastrointestinal illness with minimal contact (1.27, 95% CI: 0.74, 2.16). We also found a significant elevation of respiratory illness with swimming (1.78, 95% CI: 1.38, 2.29) and sports-related contact (1.49, 95% CI: 1.00, 2.24), and no elevation of respiratory illness with minimal contact (0.90, 95% CI: 0.71, 1.14). This study suggests that exposures associated with different types of recreational activities are important characteristics of the exposure pathway when assessing illness risk associated with recreation in ambient surface waters.

Comparison of Predicted Microbiological Human Health Risks Associated with de Facto, Indirect, and Direct Potable Water Reuse.

Increasing interest in recycling water for potable purposes makes understanding the risks associated with potential acute microbial hazards important. We compared risks from de facto reuse, indirect potable reuse (IPR), and direct potable reuse (DPR) scenarios using a previously published quantitative microbial risk assessment methodology and literature review results. The de facto reuse simulation results are compared to a Cryptosporidium spp. database collected for the Long Term 2 Enhanced Surface Water Treatment Rule's information collection rule (ICR) and to a literature review of norovirus (NoV) densities in ambient surface waters. The de facto simulation results with a treated wastewater effluent contribution of 1% in surface waters and a residence time of 30 days most closely match the ICR dataset. The de facto simulations also suggest that using NoV monitoring data from surface waters may overestimate microbial risks, compared to NoV data from raw sewage coupled with wastewater treatment reduction estimates. The predicted risks from IPR and DPR are consistently lower than those for the de facto reuse scenarios assuming the AWTFs are operating within design specifications. These analyses provide insight into the microbial risks associated with various potable reuse scenarios and highlight the need to carefully consider drinking water treatment choices when wastewater effluent is a component of any drinking water supply.

Advancements in mitigating interference in quantitative polymerase chain reaction (qPCR) for microbial water quality monitoring.

The United States Environmental Protection Agency's (EPA)1 2012 Recreational Water Quality Criteria included an Enterococcus spp. quantitative polymerase chain reaction (qPCR) method as a supplemental indicator-method. In 2012, performance of qPCR for beach monitoring remained limited, specifically with addressing interference. A systematic literature search of peer-reviewed publications was conducted to identify where Enterococcus spp. and E. coli qPCR methods have been applied in ambient waters. In the present study, we evaluated interference rates, contributing factors resulting in increased interference in these methods, and method improvements that reduced interference. Information on qPCR methods of interest and interference controls were reported in 16 papers for Enterococcus spp. and 13 papers for E. coli. Of the Enterococcus spp. qPCR methods assessed in this effort, the lowest frequencies of interference were reported in samples using Method 1609. Low frequencies of sample interference were also reported EPA's modified E. coli qPCR method, which incorporates the same reagents and interference controls as Method 1609. The literature indicates that more work is needed to demonstrate the utility of E. coli qPCR for widespread beach monitoring purposes, whereas more broad use of Method 1609 for Enterococcus spp. is appropriate when the required and suggested controls are employed.

Occurrence of coliphage in raw wastewater and in ambient water: A meta-analysis.

Coliphage have been proposed as indicators of fecal contamination in recreational waters because they better reflect the persistence of pathogenic viruses in the environment and through wastewater treatment than traditional fecal indicator bacteria. Herein, we conducted a systematic literature search of peer-reviewed publications to identify coliphage density data (somatic and male-specific, or MSC) in raw wastewater and ambient waters. The literature review inclusion criteria included scope, study quality, and data availability. A non-parametric two-stage bootstrap analysis was used to estimate the coliphage distributions in raw wastewater and account for geographic region and season. Additionally, two statistical methodologies were explored for developing coliphage density distributions in ambient waters, to account for the nondetects in the datasets. In raw wastewater, the analysis resulted in seasonal density distributions of somatic coliphage (SC) (mean 6.5 log10 plaque forming units (PFU)/L; 95% confidence interval (CI): 6.2-6.8) and MSC (mean 5.9 log10 PFU/L; 95% CI: 5.5-6.1). In ambient waters, 49% of MSC samples were nondetects, compared with less than 5% for SC. Overall distributional estimates of ambient densities of coliphage were statistically higher for SC than for MSC (mean 3.4 and 1.0 log10 PFU/L, respectively). Distributions of coliphage in raw wastewater and ambient water will be useful for future microbial risk assessments.

Comparison of somatic and F+ coliphage enumeration methods with large volume surface water samples.

Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects [(ND); 10.8%] and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 [standard deviation, SD] log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 [SD] log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.

Potable Water Reuse: What Are the Microbiological Risks?

PURPOSE OF REVIEW: With the increasing interest in recycling water for potable reuse purposes, it is important to understand the microbial risks associated with potable reuse. This review focuses on potable reuse systems that use high-level treatment and de facto reuse scenarios that include a quantifiable wastewater effluent component. RECENT FINDINGS: In this article, we summarize the published human health studies related to potable reuse, including both epidemiology studies and quantitative microbial risk assessments (QMRA). Overall, there have been relatively few health-based studies evaluating the microbial risks associated with potable reuse. Several microbial risk assessments focused on risks associated with unplanned (or de facto) reuse, while others evaluated planned potable reuse, such as indirect potable reuse (IPR) or direct potable reuse (DPR). The reported QMRA-based risks for planned potable reuse varied substantially, indicating there is a need for risk assessors to use consistent input parameters and transparent assumptions, so that risk results are easily translated across studies. However, the current results overall indicate that predicted risks associated with planned potable reuse scenarios may be lower than those for de facto reuse scenarios. Overall, there is a clear need to carefully consider water treatment train choices when wastewater is a component of the drinking water supply (whether de facto, IPR, or DPR). More data from full-scale water treatment facilities would be helpful to quantify levels of viruses in raw sewage and reductions across unit treatment processes for both culturable and molecular detection methods.

Dose-Response Models for Eastern US, Western US and Venezuelan Equine Encephalitis Viruses in Mice - Part II: Quantification of the Effects of Host Age on the Dose Response.

Many infectious disease hazards demonstrate higher susceptibility with regards to younger host ages. This trend of increased susceptibility with decreasing host age can also lead to an increased likelihood of mortality, and prolonged/chronic health effects. For quantitative microbial risk assessment (QMRA) modeling, the ability to quantify the effect of host age in the dose response model can allow modelers to account for these effects mechanistically. Additionally, QMRA modelers using age-dependent dose response models can model entire populations within the dose-response itself rather than modeling age ranges using susceptibility factors. This research developed host-age dependent exponential and beta Poisson dose response models for Eastern, Western and Venezuelan encephalitis viruses (EEV, WEV and VEV respectively) for two routes - intracranial and intraperitoneal. Improvement in fit was statistically tested as a means of assessing the benefit of including age dependency into the dose response models. EEV demonstrated improvement in fit using host-age dependency only for the exponential model except for intracranial exposure. EEV demonstrated an improvement in fit when using age dependency in the beta Poisson dose response model for both exposure routes. VEV demonstrated an improvement in fit using age dependency for both exposure routes. WEV demonstrated an improvement in fit for intracranial exposure, but neither of the age dependent dose-response models provided a good fit for WEV intraperitoneal exposure.

Direct potable reuse microbial risk assessment methodology: Sensitivity analysis and application to State log credit allocations.

Understanding pathogen risks is a critically important consideration in the design of water treatment, particularly for potable reuse projects. As an extension to our published microbial risk assessment methodology to estimate infection risks associated with Direct Potable Reuse (DPR) treatment train unit process combinations, herein, we (1) provide an updated compilation of pathogen density data in raw wastewater and dose-response models; (2) conduct a series of sensitivity analyses to consider potential risk implications using updated data; (3) evaluate the risks associated with log credit allocations in the United States; and (4) identify reference pathogen reductions needed to consistently meet currently applied benchmark risk levels. Sensitivity analyses illustrated changes in cumulative annual risks estimates, the significance of which depends on the pathogen group driving the risk for a given treatment train. For example, updates to norovirus (NoV) raw wastewater values and use of a NoV dose-response approach, capturing the full range of uncertainty, increased risks associated with one of the treatment trains evaluated, but not the other. Additionally, compared to traditional log-credit allocation approaches, our results indicate that the risk methodology provides more nuanced information about how consistently public health benchmarks are achieved. Our results indicate that viruses need to be reduced by 14 logs or more to consistently achieve currently applied benchmark levels of protection associated with DPR. The refined methodology, updated model inputs, and log credit allocation comparisons will be useful to regulators considering DPR projects and design engineers as they consider which unit treatment processes should be employed for particular projects.

Occurrence of norovirus in raw sewage - A systematic literature review and meta-analysis.

Human noroviruses (NoV) are a leading cause of recreational waterborne illnesses and responsible for the majority of viral-associated gastrointestinal illnesses nationwide. We conducted a systematic literature review of published peer-reviewed publications to identify NoV density data in wastewater influent, and provided an approach for developing pathogen density distributions, using the NoV data. Literature review inclusion criteria included scope, study quality, and data availability. A non-parametric bootstrap statistical model was used to estimate the NoV distribution in wastewater influent. The approach used accounts for heterogeneity in study-specific distribution curves, sampling locations, and sampling season and provides a comprehensive representation of the data. Study results illustrate that pooling all of the available NoV data together in a meta-analysis provides a more comprehensive understanding of the technical literature than what could be appreciated from individual studies. The studies included in this analysis indicate a high density of NoV in wastewater influent (overall mean = 4.6 log10 genome copies (GC)/liter (L)), with a higher density of NoV genogroup (G) II (overall mean = 4.9 log10 GC/L) than for GI (overall mean = 4.4 log10 GC/L for GI). The bootstrapping approach was also used to account for differences in seasonal and geographical occurrences of NoV GI and GII. The methods presented are reproducible and can be used to develop QMRA-ready density distributions for other viral pathogens in wastewater influent, effluent, and ambient waters. To our knowledge, our results are the first to quantitatively characterize seasonal and geographic differences, which could be particularly useful for future risk assessments.

Fractional poisson--a simple dose-response model for human norovirus.

This study utilizes old and new Norovirus (NoV) human challenge data to model the dose-response relationship for human NoV infection. The combined data set is used to update estimates from a previously published beta-Poisson dose-response model that includes parameters for virus aggregation and for a beta-distribution that describes variable susceptibility among hosts. The quality of the beta-Poisson model is examined and a simpler model is proposed. The new model (fractional Poisson) characterizes hosts as either perfectly susceptible or perfectly immune, requiring a single parameter (the fraction of perfectly susceptible hosts) in place of the two-parameter beta-distribution. A second parameter is included to account for virus aggregation in the same fashion as it is added to the beta-Poisson model. Infection probability is simply the product of the probability of nonzero exposure (at least one virus or aggregate is ingested) and the fraction of susceptible hosts. The model is computationally simple and appears to be well suited to the data from the NoV human challenge studies. The model's deviance is similar to that of the beta-Poisson, but with one parameter, rather than two. As a result, the Akaike information criterion favors the fractional Poisson over the beta-Poisson model. At low, environmentally relevant exposure levels (<100), estimation error is small for the fractional Poisson model; however, caution is advised because no subjects were challenged at such a low dose. New low-dose data would be of great value to further clarify the NoV dose-response relationship and to support improved risk assessment for environmentally relevant exposures.

Recreational use assessment of water-based activities, using time-lapse construction cameras.

Recreational exposure to surface waters during periods of increased pathogen concentration may lead to a significantly higher risk of illness. However, estimates of elementary exposure factors necessary to evaluate health risk (i.e., usage distributions and exposure durations) are not available for many non-swimming water-related activities. No prior studies have assessed non-swimming water exposure with respect to factors leading to impaired water quality from increased pathogen concentration, such as weather condition (rain events produce increased runoff and sewer overflows) and type of day (heavy recreational periods). We measured usage patterns and evaluated the effect of weather and type of day at eight water sites located within Philadelphia, by using a novel "time lapse photography" technology during three peak recreational seasons (May-September) 2008-2010. Camera observations validated with simultaneous in-person surveys exhibited a strong correlation (R(2)=0.81 to 0.96) between the two survey techniques, indicating that the application of remote photography in collecting human exposure data was appropriate. Recreational activities usage varied more on a temporal basis than due to inclement weather. Only 14% (6 out of 44) of the site-specific activity combinations showed dry weather preference, whereas 41.5% (17 out of 41) of the combinations indicated greater usage on weekends as compared with weekday. In general, the log normal distribution described the playing and wading duration distribution, while the gamma distribution was the best fit for fishing durations. Remote photography provided unbiased, real-time human exposure data and was less personnel intensive compared with traditional survey methods. However, there are potential limitations associated with remote surveillance data related to its limited view. This is the first study to report that time lapse cameras can be successfully applied to assess water-based human recreational patterns and can provide precise exposure statistics for non-swimming recreational exposures.

Norovirus highly prevalent cause of endemic acute diarrhea in children in the peruvian Amazon.

To determine the burden of norovirus infections in children stools from a longitudinal community cohort were evaluated using reverse transcription polymerase chain reaction. Norovirus was detected in 21.3% of diarrheal and 8.0% of nondiarrheal stools (P < 0.01). Norovirus diarrhea was highly associated with age and the odds ratio for norovirus diarrhea fell by 2.8% per month (OR = 0.97, 95% CI: 0.95-0.99). Norovirus seems to be an important etiology of community acquired diarrhea in this study population.

Bioaccumulation, retention, and depuration of enteric viruses by Crassostrea virginica and Crassostrea ariakensis oysters.

Crassostrea ariakensis oysters are under review for introduction into the Chesapeake Bay. However, the human health implications of the introduction have not been fully addressed. This study evaluated rates of bioaccumulation, retention, and depuration of viruses by Crassostrea virginica and C. ariakensis when the two oyster species were maintained in separate tanks containing synthetic seawater of various salinities (8, 12, or 20 ppt). Oyster bioaccumulation tanks were seeded with 10(3) PFU/ml of hepatitis A virus (HAV), poliovirus, male-specific bacteriophage (MS2), and murine norovirus 1 (MNV-1) and 10(3) PCR units/ml of human norovirus (NoV). After 24 h, depuration commenced as oysters (n = 255) were placed in pathogen-free seawater under continuous filtration. Oysters (n = 6) were sampled weekly for 1 month from each tank. Viral RNA was recovered using a modified proteinase K, guanidine, and glassmilk method and analyzed by quantitative reverse transcription-PCR. The odds of C. ariakensis oysters harboring NoV, MNV-1, or HAV were statistically greater than the odds of C. virginica oysters harboring the same viruses (MNV-1 odds ratio [OR], 4.5; P = 0.01; NoV OR, 8.4; P < 0.001; HAV OR, 11.4; P < 0.001). Unlike C. virginica, C. ariakensis bioaccumulated and retained NoV, MNV-1, and HAV for 1 month at all salinities. Additionally, the odds of an oyster testing positive for NoV was 25.5 times greater (P < 0.001) when the oyster also tested positive for MNV-1. This research helps assess the threat of C. ariakensis as a vehicle for viral pathogens due to the consumption of raw oysters and validates the role for MNV-1 as a surrogate for NoV.

Recovery, bioaccumulation, and inactivation of human waterborne pathogens by the Chesapeake Bay nonnative oyster, Crassostrea ariakensis.

The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 x 10(5) transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters.

Male-specific coliphages as indicators of thermal inactivation of pathogens in biosolids.

Male-specific (F+) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F+ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F+ DNA and F+ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F+ RNA phages. Separate experiments on individual isolates at 53 degrees C confirmed the apparent heat resistance of group I F+ RNA coliphages as well as the susceptibility of group III F+ RNA coliphages. Although few F+ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F+ RNA phages. Hence, F+ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F+ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.

Laboratory evaluation of thermophilic-anaerobic digestion to produce Class A biosolids. 2. Inactivation of pathogens and indicator organisms in a continuous-flow reactor followed by batch treatment.

Thermophilic-anaerobic digestion in a single-stage, mixed, continuous-flow reactor is not approved in the United States as a process capable of producing Class A biosolids for land application. This study was designed to evaluate the inactivation of pathogens and indicator organisms in such a reactor followed by batch treatment in a smaller reactor. The combined process was evaluated at 53 degrees C with sludges from three different sources and at 51 and 55 degrees C with sludge from one of the sources. Feed sludge to the continuous-flow reactor was spiked with the pathogen surrogates Ascaris suum and vaccine-strain poliovirus. Feed and effluent were analyzed for these organisms and for indigenous Salmonella spp., fecal coliforms, Clostridium perfringens spores, and somatic and male-specific coliphages. No viable Ascaris eggs were observed in the effluent from the continuous reactor at 53 or 55 degrees C, with greater than 2-log removals across the digester in all cases. Approximately 2-log removal was observed at 51 degrees C, but all samples of effluent biosolids contained at least one viable Ascaris egg at 51 degrees C. No viable poliovirus was found in the digester effluent at any of the operating conditions, and viable Salmonella spp. were measured in the digester effluent in only one sample throughout the study. The ability of the continuous reactor to remove fecal coliforms to below the Class A monitoring limit depended on the concentration in the feed sludge. There was no significant removal of Clostridium perfringens across the continuous reactor under any condition, and there also was limited removal of somatic coliphages. The removal of male-specific coliphages across the continuous reactor appeared to be related to temperature. Overall, at least one of the Class A pathogen criteria or the fecal coliform limit was exceeded in at least one sample in the continuous-reactor effluent at each temperature. Over the range of temperatures evaluated, the maximum time required to meet the Class A criteria by batch treatment of the continuous-reactor effluent was 1 hour for Ascaris suum and Salmonella spp. and 2 hours for fecal coliforms.