VALIDATION OF METHODS TO DETECT COLIPHAGES IN GROUNDWATER Prepared for Health and Ecological Criteria Division Office of Science & Technology Nena Nwachuku, Ph. D., Work Assignment Manager Prepared by Mark Sobsey1 Sagar Goyal2, Aaron Margolin3, and Suresh Pillai4 With assistance from:

Nicola Ballester3, Baldev R. Gulati2, Sigrun Haugerud2, Gregory Lovelace1 Sunil Maherchandani2, Yashpal Malik2, Dorothy Thompson1, and Douglas Wait1 1University of North Carolina, CB# 7431, McGavran Greenberg Hall, Chapel Hill, NC 27599 7431 2University of Minnesota, Department of Veterinary Diagnostic Medicine, College of Veterinary Medicine, 1333 Gortner Avenue, St. Paul, MN 55108 3University of New Hampshire, Department of Microbiology, Rudman Hall. 46 College Rd. Durham, NH 03824 4Texas A& M University, Poultry Science Department 418 D Kleberg Center, MS 2472, College Station, Texas 77843 2472 2 Table of Contents Cover Page 1 Table of Contents 2 INTRODUCTION AND BACKGROUND 5 Background 5 Experimental Approach 6 Coliphages and their Detection Methods 6 EPA Methods for Coliphage Detection in Ground water 8 Method 1601 9 Method 1602 10 Confirmation of Positive Results by Methods 1601 and 1602 12 Simultaneous Detection of both Somatic and Male specific Coliphages on a Single Host 12 Survival of Coliphages in Groundwater 13 Field Application of Methods 1601 and 1602 to Detection of Coliphages as Indicators of Fecal Contamination in Vulnerable Groundwater 14 PHASE I STUDIES 15 PURPOSES, GOALS, AND TASKS OF PHASE I STUDIES 15 PHASE I METHODS AND MATERIALS 18 Method 1601 18 3 Method 1602 19 PHASE I RESULTS 20 Coliphage Recovery by Method 1602 20 Confirmation of Plaques in Method 1602 25 Summary of Method 1602 Results 29 Coliphage Recovery by Method 1601 30 Summary of Method 1601 Results 36 PHASE I CONCLUSIONS 38 PHASE II 40 Statement of Work: Coliphage Method 1601 and 1602 Validation and Field Testing 40 Background 40 Purpose and Objectives of the Study 41 Specific Objectives 41 Schedule of Deliverables 45 PHASE II METHODS AND MATERIALS 48 Groundwater Samples and Wells 48 Coliphage Analysis of Groundwater 52 Coliphage Isolate Characterization 53 Bacteriological Analysis of Groundwater 55 Analysis of Groundwater for Human Enteric Viruses 57 Primary virus concentration from groundwater 57 4 Virus isolation in cell cultures 58 Virus detection by nucleic acid amplification 61 Nucleic acid amplification by ( RT) PCR 62 PHASE II RESULTS AND DISCUSSION 75 Introduction 75 Results of Field Sample Analysis of Coliphage and Bacterial Indicators in Groundwater 75 Comparative Detection of Two Indicators in Groundwater Samples 80 Statistical Comparisons of Fecal Indicators in Groundwater Samples 89 Analysis for Enteric Viruses in Groundwater 91 Survival of Coliphage in Seeded Groundwater 92 Comparison of Coliphage, Bacterial Indicator and Enteric Virus Detection in This Study and in Previous Studies in the USA 95 Responses to Questions and Comments of the April 2004 Coliphage Workshop 98 SUMMARY AND CONCLUSIONS 102 REFERENCES 105 APPENDIX I: Summary Report of the Northeast Region 108 APPENDIX II: Summary Report of the Southwest Region 122 APPENDIX III: Summary Report of the Upper Midwest 135 5 INTRODUCTION AND BACKGROUND Background This report summarizes the results of Phase I and phase studies on a project to evaluate and, if necessary further improve, EPA Methods 1601 and 1602 to detect coliphages in groundwater. In the first phase of the study samples of groundwater were seeded with known quantities of naturally occurring coliphages from sewage and the recovery efficiency of the methods in detecting these added coliphages was determined in a series of controlled experiments performed concurrently by four participating laboratories located in different regions of the country. The data from the seeded sample recovery experiments were used to further establish and quantify the performance characteristics of the methods.

In the second phase of the study EPA Methods 1601 and 1602 were applied to geographically representative samples of groundwater potentially vulnerable to fecal contamination in order to compare the performance of the different coliphage methods and to compare their ability to detect fecally contaminated groundwater relative to the detection of fecal indicator bacteria and the detection of culturable enteric viruses. Each of the four geographically representative laboratories ( southeast, northeast, upper Midwest and southwest) was to analyze at least 16 groundwater samples for coliphages, indicator bacteria and enteric viruses, for a total target number of 64 samples to analyzed for second and final phase of the project. Several other tasks were linked to this effort to further validate and improve the coliphage methods and their ability to detect and characterize coliphages in groundwater 6 Experimental Approach Coliphages and their detection methods. Coliphages are viruses infecting Escherichia coli bacteria. Coliphages are present at high concentrations in sewage and other fecal wastes and they are indicators of fecal contamination of groundwater, other waters and other environmental media. There are two main groups of coliphages: somatic and male specific. The relationships between these coliphages and their host bacteria, showing specific bacterial strains as examples,

are summarized in Figure 1. The conventional method to detect coliphages is by their ability to infect host cells in which they replicate ( proliferate), producing large numbers of progeny viruses and lysing ( killing) the host cells in the process. It is this killing and lysis of host cells that forms the basis of most coliphage infectivity assay methods, including those employed for coliphage analysis by the EPA methods.

Figure 1. Somatic and Male specific ( F+) Coliphages and their Relationship to Host Bacteria 7 Somatic coliphages infect host bacteria by attaching directly to the outer cell wall ( outer cell membrane). The male specific coliphages infect only male F+ strains of bacteria by attaching to the hair like appendages projecting from the cell surface, called F pili or fimbrae, that are the characteristic male trait. Somatic coliphage hosts lack the F pili and cannot be infected by F+

coliphages. F+ coliphage hosts differ in their ability to be infected by somatic coliphages. Some F+ coliphage hosts are very resistant to somatic coliphage infection because they have an outer cell membrane that differs from those of E. coli ( such as the Salmonella typhimurium strain WG49) and E. coli Famp ( which was experimentally selected as a somatic coliphage resistant mutant). Other F+ coliphage hosts such as E. coli C3000 have not been subjected to selection for resistance to somatic coliphages and are susceptible to F+ coliphage infection as well as somatic coliphage infection. Therefore, some host bacteria are infected only by somatic coliphages ( E.

coli C and CN13), others only by male specific coliphages ( E. coli Famp and Salmonella typhimurium WG49) and yet others by both groups of coliphages ( E. coli C3000).

There are still questions about which groups of coliphages, somatic, male specific or both groups together, are the appropriate and preferred indicators of fecal contamination. There is evidence in support of both somatic and male specific coliphages as being effective and useful virus indicators of fecal contamination. Some have suggested that both somatic and male specific coliphages should be detected as fecal indicator viruses of contamination of groundwater and other waters.

It is the understanding of the authors that EPA has so far not made any final decisions about which of the coliphage groups to target for detection in future guidelines or regulations. It also been suggested that both groups of coliphages, somatic and male specific, could be 8 simultaneously detected on a single coliphage host, thereby giving the greatest probability and highest sensitivity in detecting any coliphage indicative of fecal contamination.

EPA Methods for Coliphage Detection in Ground Water. EPA Methods 1601 and 1602 were developed to detect somatic and male specific coliphages in large volumes of groundwater, with target sample volumes of up to 1000 mL in Method 1601 ( an enrichment method) and 100 mL in method 1602 ( a Single Agar Layer plaque assay method), respectively. The methods are based upon the ability of the coliphages to infect host bacteria, which results in the lysis of the host bacteria. This a widely used approach to detect coliphages. In plaque assays or other assays on solid media, such as those containing agar, the lysis of the host bacteria is visualized as zones of lysis or clearing of the bacteria as discrete, circular areas ( called a lysis zones or plaques) in a confluent layer ( or " lawn") of host bacteria in a solid nutrient medium. In liquid enrichment cultures in broth media, the lysis of host bacteria can in principle be observed as the clearing of turbidity from the culture as the bacteria are lysed and their resulting cell debris settles out of suspension. Because such clearing of broth cultures as evidence of host cell lysis can be hard to observe due to interference from other bacteria that may grow in the broth culture, other ways to confirm the presence of phages are often used. One of the most common ways is to take some of the enrichment culture containing phages, apply it to a lawn of bacteria in an agar medium, and allow the phages to infect and lyse the host cells in the lawn to produce a clear zone of lysis that can be readily observed. 9 Method 1601. Method 1601 is a so called two step " enrichment" method and the steps of the method are outlines in Figure 2. In the first step of this method, liquid bacterial media,

magnesium chloride ( to promote coliphage attachment to the host bacteria), and the E. coli host are added to the water sample, making a liquid ( broth) culture for coliphage infection of the E.

coli host bacteria. After allowing for coliphage infection and lysis of the host bacteria during overnight incubation, a small volume ( several microliters) of the enrichment culture is placed on the surface of a Petri dish of agar medium containing E. coli host bacteria ( a spot). This is the second step of the method. If the applied sample contains coliphages able to infect the host bacteria, a circular zone of host cell lysis ( clearing) develops after several hours of incubation in the spot where the sample was applied. Such a lysis zone in the spot indicates coliphage presence in the enrichment broth and is a positive result. If no such lysis zone develops in the sample spot on the plate, the enrichment culture of the sample is considered negative for coliphages. 10 Figure 2.

Method 1601   Two Step Enrichment Spot Plate Method for Coliphage Presence Absence When Method 1601 is applied to a single sample volume, the analysis provides a determination of the presence or absence of coliphages in the sample volume analyzed. If the method is applied to multiple sample volumes, each in separate enrichment cultures, the method is capable of giving an estimation of the concentration of coliphages in the water sample, based on which sample enrichment volumes become positive and negative for coliphages.

Method 1602. EPA Method 1602 is a so called single agar layer method for the enumeration of coliphage plaques ( discrete clear zones of lysis of host bacteria) developing in a culture of host bacteria in an agar medium in a Petri dish. As shown in Figure 3, a 100 mL sample of 11 groundwater is supplemented with magnesium chloride, host bacteria and then combined with molten agar medium. The mixture is then distributed into Petri plates, the agar medium is allowed to solidify and the plates are incubated overnight for the development of coliphage plaques, which are clear, circular zones of lysis, each produced by a separate or individual coliphage. The plaques are then counted to determine the total number of number of coliphages in the sample,

assuming each plaque arose from an individual infectious coliphage.

Figure 3.

Method 1602   Single Agar Layer ( SAL) Plaque Assay Method for Coliphage Enumeration 12 Confirmation of Positive Results by Methods 1601 and 1602. For both Methods 1601 and 1602, EPA has proposed a method to confirm positive results. For confirmation of positive results, material is removed ( picked or aspirated with a capillary pipette or a micropipettor) from the lysis zones of enrichment spots on agar medium host cell plates ( Method 1601) or from the plaques that develop in agar medium host cell plates of Method 1602. The recovered material is transferred to a small volume of buffered water, mixed briefly, and then a small volume ( several microliters) of the material is placed (" spotted") on the surface of a Petri dish of agar medium containing E. coli host bacteria. If the applied material contains coliphages capable of infecting the host bacteria, a circular zone of host cell lysis develops after incubation ( for several hours or overnight) in the spot where the sample was applied. Such a lysis zone in the spot is indicative of coliphage presence in the material recovered from either a lysis zone on the spot plate of an enrichment broth ( Method 1601) or from the plaque of a Single Agar Layer plate ( Method 1602).

If no such lysis zone develops in the sample spot on the confirmation plate, the sample ( presumptive lysis zone from an enrichment culture or presumptive plaque from an SAL plate) is considered negative for coliphages.

Simultaneous Detection of both Somatic and Male specific Coliphages on a Single Host.

EPA Methods 1601 and 1602 were originally developed to separately detect somatic and malespecific coliphages using separate E. coli hosts able to support the growth of only one or the other coliphage group ( somatic or male specific, respectively). E. coli CN13 is used to detect somatic coliphages and E. coli Famp is used to detect male specific coliphages ( Figure 1). It was later suggested that perhaps a single E. coli host could be used to simultaneously detect both 13 somatic and male specific coliphages present in a groundwater samples rather than having to use two separate E. coli hosts to separately detect each coliphage group ( Figure 1). If the presence of either or both groups of coliphages indicates fecal contamination, simultaneous detection of both on one host would reduce time, effort, materials and cost and provide appropriate data about coliphage presence in a sample. As previously noted, E. coli C3000 is such a host. However use of a single E. coli host bacterium capable of detecting both somatic and male specific coliphages had not been adequately tested for its performance characteristics in previous studies on the development and evaluation of Methods 1601 and 1602 and their application to either seeded samples or field samples of groundwater.

Survival of Coliphages in Groundwater. In the development and evaluation of methods for coliphage detection in groundwater, the question has been raised as to how long samples can be held before being subjected to analysis. It has been suggested that samples may have to be collected and sent to a distant lab capable of coliphage analyses, but that the time between sample collection and analysis may be more than 1 or 2 days. If the sample holding time is 2 or more days will the coliphages still be present and be detectable? To address this question additional experiments were done as an added task in Phase II of this study at the request of the EPA project manager. Groundwater was seeded with known, low level amounts of mixed populations of sewage derived coliphages and aliquots of these samples were subjected to coliphage analysis by Methods 1601 and 1602 on days 0, 2, 3 and 6. These assay days were chosen to model those that might be used if samples were shipped to a lab for coliphage analysis and even held overnight before analysis once received by the lab. The resulting data on coliphage concentrations were 14 analyzed to determine if the coliphages were stable and still detectable for periods ranging from 1 to 6 days.

Field Application of Methods 1601 and 1602 to Detection of Coliphages as Indicators of Fecal Contamination in Vulnerable Groundwater. An important test of the newly developed EPA methods to detect coliphages in groundwater, Methods 1601 and 1602, would be to validate their performance for coliphage detection in vulnerable groundwater, in comparison with the detection of fecal indicator bacteria and human enteric viruses in the same samples. Preferably such studies would apply the methods to different, geographically representative groundwater in order to make sure that the methods were not adversely affected by interfering constituents in the groundwater, or so called " matrix effects". Furthermore, the concurrent detection of coliphages by Methods 1601 and 1602 in the same groundwater samples would provide an opportunity to compare their relative detection sensitivities and lower limits of coliphage detection. In addition,

the concurrent detection of coliphages as well as fecal indicator bacteria and enteric viruses in the same groundwater samples would make it possible to determine if coliphages were as good or better than fecal indicator bacteria or enteric viruses in identify fecally contaminated ground water. Such analysis would make it possible to determine if one of these microbe groups was a superior indicator of fecal contamination because it was detected more frequently and/ or at higher concentrations. Such analyses were done in Phase II of this study. 15 PHASE I STUDIES PURPOSES, GOALS AND TASKS OF PHASE I STUDIES The overall purposes and goals of Phase I studies were to determine the performance characteristics of Methods 1601 and 1602 in detecting and quantifying somatic and male specific coliphages in ground water samples seeded with known quantities of natural, mixed populations of coliphages obtained from municipal sewage. These studies were done using certain modifications and additions to Methods 1601 and 1602 in order to address recommendations suggested for the methods after their original development, evaluation and multi laboratory testing. Specifically, host E. coli C3000 was tested for simultaneous detection and quantification of both somatic and male specific coliphages in addition to testing the methods with the individual hosts previously specified for separate detection of somatic ( E. coli C3000) and male specific ( E.

coli Famp) coliphages. In addition, Methods 1601 and 1602 were tested using the confirmation procedure, as proposed by EPA for all methods to detect microbes in ground water.

The key tasks and activities of the Phase I studies are listed below.

1. Recruit a total of 4 experienced laboratories, each from a different region of the country, to test Methods 1601 and 1602 using the standard protocols with the modifications indicated: a) include host E. coli 3000 for simultaneous detection of both somatic and male specific coliphages, and b)

include confirmation of presumptive positive results obtained from samples. The 4 laboratories 16 are:

University of North Carolina ( UNC), under the direction of Mark D. Sobsey ( southeast)

University of New Hampshire ( UNH), under the direction of Aaron Margolin ( northeast)

Texas A& M University ( TAMU), under the direction of Suresh Pillai ( southwest)

Wisconsin State Hygiene Lab ( WSHL), under the direction of David Battigelli ( upper Midwest)

2. Develop bench sheets ( bench laboratory aids or protocols in easy to follow format) to be used by analysts performing the methods in these repeated, weekly experimental trials.

3. Perform weekly experimental tests ( trials) of the methods using the developed bench sheets.

4. Test each method ( 1601 and 1602) simultaneously by the 4 laboratories on a weekly basis,

using locally collected ground waters seeded with the same stock of sewage derived coliphages prepared and distributed weekly by the lead or reference laboratory ( UNC) and all three E. coli hosts ( CN13 for somatic, Famp for male specifics and C3000 for both).

5. Perform repeated trials of each method and submit the results to the lead ( UNC) laboratory for compilation and data analysis in order to develop and evaluate a sufficient database to characterize the performance of the methods. 17 6. Identify any deficiencies or limitations encountered with the methods. If possible within a short time period ( no more than a few weeks), devise and implement modifications or corrective measures to improve the performance characteristics of the methods.

7. Based on the compiled data from the 4 laboratories, determine if the performance characteristics of the methods are of sufficient quality to recommend the use of the methods to detect coliphages in ground water samples.

8. Save ( archive) representative coliphages detected by each method on each E. coli host for further characterization by the UNC laboratory to determine if the coliphage isolates have properties consistent with a fecal origin. These properties include bacterial host range, growth temperatures and taxonomic group ( sub set of representative isolates only). 18 PHASE I METHODS AND MATERIALS The methods and materials used in this project are those specified in the documents for US EPA Methods 1601 and 1602. Stepwise procedural steps in the application of these methods for the specific purpose of this study are also given in the laboratory bench sheets ( laboratory bench protocols) presented in the Appendix to this report. The only departures or modifications to Methods 1601 and 1602 employed in this study are: ( a) the addition of E. coli C3000 as a host bacterium for the simultaneous detection of both somatic and male specific coliphages, and ( b) the addition of the newly proposed confirmation procedure for plaques from plates of Method 1602 and from lysis zones of plates from Method 1601.

Method 1601 For Method 1601, the two step enrichment method, the goal was for each of the 4 participating laboratories to seed 30+ liters of ground water with a quantity of coliphage stock ( filtered sewage) to achieve between 1 and 2 infectious units of coliphages per liter of water. The seeded water was then aliquotted into 30 1 liter volumes. Groups of 10 1 liter volumes were subjected to the enrichment assay method using one of the three host bacteria, thereby testing each host bacterium for coliphage detection using 10 replicate 1 liter volumes per host bacterium per weekly experiment. As negative control samples, three additional 1 liter volumes of unseeded ground water were also subjected to coliphage analysis by the two step enrichment method using each of the three different E. coli host bacteria. As negative controls, these samples were intended to demonstrate no background level of coliphages were present in the ground water 19 prior to seeding with sewage derived coliphages. A total of 8 replicate experiments were conducted, one experiment per week, between May and July, 2001.

Method 1602 For Method 1602, the single agar layer ( SAL) method, the goal was for each of the 4 participating laboratories to seed replicate 300 mL volumes of water with a quantity of coliphage stock ( filtered sewage) to give about 100 infectious units of coliphages per 100 mL of ground water. The seeded water was aliquotted as 3 100 mL volumes and each of these volumes was assayed by the single agar layer method using one of the three different E. coli host bacteria. As negative controls, 3 100 mL volumes of unseeded ground water were subjected to coliphage analysis by the SAL method using each of the three different host bacteria. As negative controls,

these samples were intended to demonstrate no background level of coliphages were present in the ground water prior to seeding with sewage derived coliphages. A total of 10 replicate experiments were performed, once experiment per week, during February and April, 2001. 20 PHASE I RESULTS Coliphage Recovery by Method 1602 Table 1 shows the recovery of seeded coliphages by method 1602 ( single agar layer assay) for the total of 10 successive trials performed weekly. In some initial weekly trials no data were available from the WSLH laboratory. This was due to other obligations that precluded their participation.

In the interest of time, the initial three experiments were performed among the other three laboratories in order to initiate the project and to begin addressing potential logistical issues of coordination among laboratories. No serious logistical problems arose among the three labs participating initially. This indicated a reliable system for concurrent method performance among the labs using the same coliphage stocks prepared by UNC lab to seed test groundwater.

The WSLH also was unable to participate another week due to a state mandated holiday. 21 Table 1: Recovery of Seeded Coliphages in 100 mL Groundwater Samples by Method 1602 ( Single Agar Layer Assay)

Date Host UNC TAMU WSLH UNH 21 Feb 01 C3000 2% 14% no data 22% CN13 80% 90% no data 65% Famp 246% 160% no data 24% 27 Feb 01 C3000 9% 79% no data 131% CN13 43% 43% no data 127% Famp 22% 15% no data 53% 6 Mar 01 C3000 9% 6% no data 45% CN13 37% 19% no data 50% Famp 101% 18% no data 35% 13 Mar 01 C3000 9% 0% 20% 26% CN13 19% 0% 35% 23% Famp 72% 0% 28% 33% 20 Mar 01 C3000 12% 8% 56% 37% CN13 20% 9% 94% 39% Famp 91% 68% 92% 45% 27 Mar 01 C3000 4% 8% 39% 81% CN13 73% 30% 120% 118% Famp 48% 20% 40% 70% 3 Apr 01 C3000 10% 16% 28% 88% CN13 30% 36% 70% 77% Famp 49% 47% 67% 72% 10 Apr 01 C3000 5% 17% 34% 93% CN13 21% 63% 64% 96% Famp 26% 10% 32% 77% 17 Apr 01 C3000 44% no data 49% 86% CN13 139% no data 77% 84% Famp 72% no data 94% 98% 24 Apr 01 C3000 33% 55% 32% 77% CN13 117% 88% 76% 89% Famp 36% 33% 26% 84%

The percent coliphage recovery data were subjected to Analysis of Variance ( ANOVA) to discover if there were significant recovery differences among the hosts and/ or the laboratories ( Table 2) . As shown in Table 2, there were significant differences in recovery among the 3 hosts ( p= 0.00008), and significant differences in recovery among the four labs ( p= 0.000002). The highest recovery ( 73%) was obtained using E coli CN13, the lowest ( 39%) was obtained using E coli C3000 and an intermediate recovery of 46% was obtained with E coli Famp. The differences among host bacteria were consistent ( not significantly different) within each laboratory ( p= 0.42). 22 This latter result suggests that recoveries among the three host bacteria are generally similar within a lab and therefore, the hosts are equivalent on a within lab basis. In other words, the three different E. coli hosts will give similar recovery efficiencies when used by an individual lab to analyze mixed populations of coliphages of sewage ( fecal) origin in a groundwater matrix.

Table 2: Descriptive Statistics for Seeded Coliphage Recovery by Method 1602 OVERALL C3000 UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 49% 35% 56% 68% 14% 23% 37% 69% Median 35% 19% 49% 75% 9% 14% 34% 79% Mode NONE 0% NONE NONE NONE NONE NONE NONE Std. Dev. 52% 37% 28% 31% 14% 26% 12% 35% Var. 27% 14% 8% 10% 2% 7% 2% 12% Minimum 2% 0% 20% 21% 2% 0% 20% 22% Maximum 246% 160% 120% 131% 44% 79% 56% 131% Count 30 27 21 30 10 9 7 10 95% CI 19% 15% 13% 12% 10% 20% 11% 25% CN13 Famp UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 58% 42% 77% 77% 76% 41% 54% 59% Median 40% 36% 76% 81% 61% 20% 40% 62% Mode NONE NONE NONE NONE NONE NONE NONE NONE Std. Dev. 43% 33% 26% 33% 65% 49% 30% 25% Var. 18% 11% 7% 11% 43% 24% 9% 6% Minimum 19% 0% 35% 23% 22% 0% 26% 24% Maximum 139% 91% 120% 127% 246% 160% 94% 98% Count 10 9 7 10 10 9 7 10 95% CI 31% 25% 24% 24% 47% 38% 28% 18%

The differences in coliphage recovery efficiency among the laboratory groups led us to question whether there were differences in the groundwater of each region ( i. e., a " matrix" effect) which might account for those observed differences in recovery. Further experiments were conducted using regional groundwater and additionally reagent water ( as a control measure) in an attempt to answer this question. Four replicate experiments were conducted and these data are summarized in Table 3, with descriptive statistics in Tables 4a, 4b and 4c for hosts E. coli C3000, CN13 and 23 Famp, respectively..

Table 3: Coliphages Recovery Efficiency of Method 1602 Concurrently Applied to Seeded Groundwater and Reagent Water Date Matrix Host UNC TAMU WSLH UNH 3 Apr 01 ground C3000 10% 16% 28% 88% reagent C3000 51% 22% 26% 87% ground CN13 30% 36% 70% 77% reagent CN13 61% 49% 57% 74% ground Famp 49% 47% 67% 72% reagent Famp 64% 88% 48% 72% 10 Apr 01 ground C3000 5% 17% 34% 93% reagent C3000 52% 13% 37% 92% ground CN13 21% 63% 64% 96% reagent CN13 76% 66% 63% 78% ground Famp 26% 10% 32% 77% reagent Famp 66% 18% 47% 75% 17 Apr 01 ground C3000 44% no data 49% 86% reagent C3000 73% no data 67% 76% ground CN13 139% no data 76% 84% reagent CN13 135% no data 68% 82% ground Famp 72% no data 94% 98% reagent Famp 87% no data 138% 97% 24 Apr 01 ground C3000 33% 55% 32% 77% reagent C3000 31% 13% 51% 79% ground CN13 117% 88% 76% 89% reagent CN13 103% 48% 65% 83% ground Famp 36% 33% 26% 84% reagent Famp 59% 49% 78% 79% 24 Table 4a: Descriptive Statistics: Coliphage Recovery by Method 1602 for Seeded Groundwater and Reagent Water on Host E. coli C3000 GROUNDWATER REAGENT WATER UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 23% 29% 36% 86% 52% 16% 45% 84% Median 21% 17% 33% 87% 52% 13% 44% 83% Std. Dev. 18% 22% 9% 7% 17% 5% 18% 7% Var. 3% 5% 1% 0% 3% 0% 3% 1% Minimum 5% 16% 28% 77% 31% 13% 26% 76% Maximum 44% 55% 49% 93% 73% 22% 67% 92% Count 4 3 4 4 4 3 4 4 95% CI 29% 55% 14% 11% 27% 13% 28% 12%

Table 4b: Descriptive Statistics: Coliphage Recovery by Method 1602 for Seeded Groundwater and Reagent Water on Host E. coli CN 13 GROUNDWATER REAGENT WATER UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 77% 63% 72% 86% 94% 54% 63% 79% Median 73% 63% 73% 86% 90% 49% 64% 80% Std. Dev. 60% 26% 6% 8% 32% 10% 5% 4% Var. 36% 7% 0% 1% 11% 1% 0% 0% Minimum 21% 36% 64% 77% 61% 48% 57% 74% Maximum 139% 88% 77% 96% 135% 66% 68% 83% Count 4 3 4 4 4 3 4 4 95% CI 95% 65% 9% 12% 52% 25% 8% 6%

Table 4c: Descriptive Statistics: Coliphage Recovery by Method 1602 for Seeded Groundwater and Reagent Water on Host E. coli Famp GROUND REAGENT UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 46% 30% 55% 83% 69% 52% 78% 81% Median 43% 33% 49% 81% 65% 49% 63% 77% Std. Dev. 20% 19% 32% 11% 12% 35% 42% 11% Var. 4% 4% 10% 1% 2% 12% 18% 1% Minimum 26% 10% 26% 72% 59% 18% 47% 72% Maximum 72% 47% 94% 98% 87% 88% 138% 97% Count 4 3 4 4 4 3 4 4 95% CI 32% 47% 51% 18% 20% 87% 67% 18% 25 These coliphage recovery data were subjected to ANOVA. No interaction effects were indicated ( p> 0.09 in all cases), implying that any observed factor differences were consistent throughout the experiment. As expected, there were significant differences in recovery among the host bacteria ( p= 0.00007), with recovery for E. coli C3000 ( 45%) being lower than recoveries for E. coli CN13 ( 73%) and E. coli Famp ( 63%). There were also significant differences in recovery among the laboratory groups ( p= 0.0000006), with UNH having the highest overall recovery ( 83%),

followed by UNC ( 60%), WSLH ( 58%), and TAMU showing the lowest overall recovery ( 40%).

But there was no significant difference due to a possible matrix effect ( p= 0.17), leaving unexplained the previously observed differences among the laboratory groups.

Confirmation of Plaques in Method 1602 The laboratories also applied several modified versions of a confirmation procedure for plaques isolated from the SAL plates of Method 1602 when applied to the detection of coliphages in seeded water samples. Plaques were picked from the SAL plates using a variety of methods ( i. e.,

with Pasteur pipettes, with Eppendorf pipettes, etc.), resuspended in Tryptic Soy Broth, and spotted onto pre poured gridded plates of Tryptic Soy Agar containing host bacteria ( as used in the Two Step Enrichment procedure). The confirmation percentages are presented in Table 5a,

which summarizes all data by experiment date, lab and host and in Table 5b, and which summarizes the descriptive statistics for the plaque confirmations. The results of these attempts at coliphage plaque confirmation ranged from excellent ( average 99 100% at UNH) to moderate ( 38 68% at UNC). Overall, there are high likelihoods that the plaques detected on assay plates for Method 1602 are indeed produced by coliphages, with a 78% average or a nearly 4 out of 5 26 plaque confirmation rate. It is likely that confirmation rates can be further improved to give a greater confirmation efficiency by minor modifications in the plaque recovery and re spotting procedure. 27 Table 5a: Percent Confirmation of Picked Plaques Isolated using Method 1602 Date Matrix Host UNC TAMU WSLH UNH 21 Feb 01 ground C3000 33% not done 95% 100% ground CN13 85% no data 100% 100% ground Famp 15% not done 90% 100% 27 Feb 01 ground C3000 13% not done 100% 100% ground CN13 75% no data 80% 100% ground Famp 15% not done 45% 100% 6 Mar 01 ground C3000 60% 80% 100% 100% ground CN13 45% 100% 60% 100% ground Famp 30% 88% 67% 100% 13 Mar 01 ground C3000 21% not done 100% 100% ground CN13 35% no data 75% 95% ground Famp 15% not done 80% 95% 20 Mar 01 ground C3000 78% no data 95% 90% ground CN13 50% 25% 90% 100% ground Famp 50% 0% 80% 95% 27 Mar 01 ground C3000 40% no data 100% 95% ground CN13 80% 0% 80% 100% ground Famp 35% 53% 95% 95% 3 Apr 01 ground C3000 50% 70% 100% 100% reagent C3000 65% 90% No data 100% ground CN13 45% 80% 95% 100% reagent CN13 75% 90% No data 100% ground Famp 0% 20% 100% 100% reagent Famp 0% 10% No data 100% 10 Apr 01 ground C3000 93% 80% 100% 100% reagent C3000 100% 80% 100% 100% ground CN13 15% 90% 85% 100% reagent CN13 90% 80% 95% 100% ground Famp 10% 50% 90% 100% reagent Famp 60% 30% 100% 100% 17 Apr 01 ground C3000 80% not done 100% 100% reagent C3000 95% not done 100% 100% ground CN13 95% no data 85% 100% reagent CN13 70% not done 100% 100% ground Famp 70% not done 100% 100% reagent Famp 70% not done 95% 100% 24 Apr 01 ground C3000 90% 100% 100% 100% reagent C3000 90% 100% 100% 100% ground CN13 100% 100% 100% 100% reagent CN13 95% 100% 100% 100% ground Famp 85% 80% 95% 100% reagent Famp 80% 90% 95% 100% 28 Table 5b: Descriptive Statistics for Plaque Confirmation of Coliphages Isolated by Method 1602 ( Percent Confirmation Overall and by E. coli Host for each Laboratory)

Overall C3000 UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 57% 67% 91% 99% 65% 86% 99% 99% Median 63% 80% 95% 100% 71% 80% 100% 100% Mode 15% 80% 100% 100% 90% 80% 100% 100% Std. Dev. 31% 34% 13% 2% 29% 11% 2% 3% Variance 10% 11% 2% 0% 9% 1% 0% 0% Minimum 0% 0% 45% 90% 13% 70% 95% 90% Maximum 100% 100% 100% 100% 100% 100% 100% 100% Count 42 25 39 42 14 7 13 14 95% CI 10% 14% 4% 1% 17% 10% 1% 2% CN13 Famp UNC TAMU WSLH UNH UNC TAMU WSLH UNH Mean 68% 74% 88% 100% 38% 47% 87% 99% Median 75% 90% 90% 100% 33% 50% 95% 100% Mode 75% 100% 100% 100% 15% NONE 95% 100% Std. Dev. 26% 36% 12% 1% 30% 34% 16% 2% Variance 7% 13% 1% 0% 9% 12% 3% 0% Minimum 15% 0% 60% 95% 0% 0% 45% 95% Maximum 100% 100% 100% 100% 85% 90% 100% 100% Count 14 9 13 14 14 9 13 14 95% CI 15% 28% 7% 1% 18% 26% 10% 1%

Subsequent efforts to improve confirmation rates for plaques picked from SAL plates or lysis zones picked from spot plates of the enrichment method compared the original EPA conformation method described above to a modified method. In the modified method the picked material from plaques or lysis zones was re enriched by culturing in host bacteria again. The picked plaque or lysis zone material was transferred to 5 mL of Tryptic Soy Broth to which had been added host bacteria and the mixture was incubated overnight at 37 /

C. Volumes of 10 microliters were withdrawn from the resulting overnight enrichments and spotted onto prepoured lawns of host cells in nutrient agar media. The spot plates were incubate at 37 /

C for a minimum for 4 hours and then observed for lysis zones indicative of coliphage positivity. The results of a side by side comparison of the original confirmation method with the modified method are summarized in 29 Table 5c.

Summary of Method 1602 Results Results from a series of 10 replicate experiments by all four participating laboratories on the performance of the SAL method have been presented. The method was applied to replicate 100

mL volumes of groundwater seeded with sewage coliphages and detected with each of three E.

coli host bacteria. The summarized results of these experiments ( Table 6) show efficient coliphage detection ( average 53%) and confirmation ( average 78%) in 100 mL volumes of ground water. There were differences in recoveries based on the host used, and there were unexplained differences in recovery among the laboratories. Confirmation of plaque isolates gave success rates ranging from moderate to excellent among labs. Individual adaptations or modifications of confirmation methods somewhat improved low confirmation rates. Method 1602 gave generally acceptable detection of coliphages in seeded ground water and the majority of plaques detected by the method could be easily confirmed by a simple procedure. Overall, the results of these studies indicate that there is high likelihood of detecting even low levels of coliphages in 100 mL volumes of ground water using Method 1602.

Table 6. Coliphage Detection in 100 mL Volumes of Seeded Ground Water by Method 1602 Coliphage Group Estimated Phages/ 100 mL Coliphage Recovery (%) Plaque Confirmation (%)

Somatic ( E. coli CN13) 100 64 82 Male specific ( E. coli Famp) 100 58 68 Both ( E. coli C3000) 100 38 87 30 Coliphage Recovery by Method 1601 The Two Step Enrichment ( SAL) validation study consisted of 8 replicate experiments performed by the four participating laboratories. In each experiment a small volume of the assayed sewage was added to a 30 liter volume of groundwater and mixed well to disperse the inoculum evenly.

This inoculated groundwater was then dispensed into 30 1 liter bottles to which were added the enrichment media and the proper host bacteria ( 10 bottles per host). The bottles were incubated overnight, and small portions were spotted onto gridded TSA plates as described above. After incubation, these plates were examined for zones of lysis. Positive zones of lysis were considered positive for coliphage, and these were counted and recorded for each host. Based on the volume and the titer of the inoculated sewage, an expected coliphage titer per bottle was calculated for each host. Based on the number of positive bottles, the MPN ( Most Probable Number) of coliphages per bottle was calculated using Thomas's MPN equation and taken as the number of coliphages recovered ( observed number of coliphages). Using this observed MPN and the expected coliphage titer per bottle, percent recoveries were calculated.

The coliphage recovery rates for experiments in which replicate ten 1 liter volumes of groundwater were seeded with about 1.5 to 3 infectious units of coliphages are presented in Table 7a as percentage recoveries based on the observed ( calculated) MPN coliphage concentrations per liter and in Table 7b as a comparison of expected and observed number of coliphage positive 1

liter samples out of 10. Descriptive statistics presented in Tables 8a and 8b. 31 Table 7a: Recovery of Seeded Coliphages in 1 Liter Groundwater Samples by Method 1601 ( Two step enrichment)

Date Host UNC TAMU WSLH UNH 9 May 01 C3000 4% no data 13% > 63% CN13 134% no data > 122% 131% Famp 26% no data 19% > 70% 14 May 01 C3000 30% 281% 6% 62% CN13 193% 92% > 189% 9% Famp > 39% 348% 7% > 21% 21 May 01 C3000 10% no data 134% no data CN13 101% no data 148% no data Famp 26% no data 35% no data 4 Jun 01 C3000 41% no data 23% 122% CN13 372% no data > 359% 22% Famp 143% no data 62% 99% 11 Jun 01 C3000 20% 79% 24% 82% CN13 215% <= 5% > 204% 304% Famp 44% <= 14% 15% 123% 18 Jun 01 C3000 15% 39% 28% 130% CN13 > 137% 163% > 124% 77% Famp 13% 112% 10% 21% 25 Jun 01 C3000 no data 125% 6% 131% CN13 18% 122% 39% 220% Famp no data 45% 56% 224% 9 Jul 01 C3000 261% 220% 24% 108% CN13 61% 191% 187% 230% Famp <= 518% 57% 33% 2317% 32 Table 7b: Descriptive Statistics for Recovery of Seeded Coliphages by Method 1601 C3000 UNC TAMU WSLH UNH Mean 54% 149% 33% 109% Median 20% 125% 24% 122% Std. Dev. 92% 100% 42% 27% Variance 84% 100% 18% 7% Minimum 4% 39% 6% 62% Maximum 261% 281% 134% 131% Count 7 5 8 7 95% CI 85% 124% 35% 25%

CN13 UNC TAMU WSLH UNH Mean 171% 114% 296% 142% Median 163% 122% 247% 131% Std. Dev. 117% 74% 208% 113% Variance 136% 55% 431% 127% Minimum 18% 0% 39% 9% Maximum 372% 191% 718% 304% Count 8 5 8 7 95% CI 97% 92% 174% 104%

Famp UNC TAMU WSLH UNH Mean 47% 112% 30% 424% Median 26% 57% 26% 123% Std. Dev. 49% 138% 21% 838% Variance 24% 190% 4% 7018% Minimum 0% 0% 7% 21% Maximum 143% 348% 62% 2317% Count 7 5 8 7 95% CI 45% 171% 17% 775%

The results in Tables 7a and 7b indicate that when 1 liter volumes of ground water seeded with 1

2 PFU of coliphages are analyzed by the enrichment method, there is a very high likelihood that coliphages will be detected with relatively high efficiency. Average coliphage recoveries from 8 replicate trials per coliphage host per lab ranged were 86% for combined coliphages on host E.

coli C3000, 181% for somatic coliphages on host E. coli CN 13, and 153% for male specific coliphages on host E. coli Famp. The results in Tables 7a and 7b indicate variability in coliphage recoveries from trial to trial. However, this extent of variability is to be expected because 33 coliphage recoveries are based on MPN estimates of the number of positive 1 liter enrichment culture bottles out of ten. Based on calculated 95% confidence intervals ( CIs), the observed degree of variability was within the range expected for a 10 culture, single dilution Most Probable Number method. Examination of the 10 replicate single dilution MPN table in Standard Methods for the Examination of Water and Wastewater indicates that MPN estimates can have 95% CIs that vary by nearly 6 fold ( 600%) at low rates of positivity and almost always 3 fold ( 300%) or more at intermediate and high levels of positivity. Probably more important is that of the total 82 trials in which 10 1 liter bottles of seeded ground water were used per trial, coliphages were not detected in only 3 trials. Therefore, there is a very high probability of detecting low levels of ( 1 to 3) coliphages in 1 liter volumes of groundwater when using this method.

As in the statistical analyses for Method 1602 described earlier, the data for Method 1601 were subjected to ANOVA. This analysis detected no significant differences in recovery among the laboratories ( p= 0.38); nor did it detect any significant differences in recovery among the hosts ( p= 0.41).

The results from this series of 8 replicate experiments per lab are also summarized in Table 8 and Figure 1 based on the observed and expected number of positive 1 liter enrichment culture bottles out of 10. These results indicate sensitive coliphage detection that is close to the theoretical level of detection. 34 Table 8. Comparison of Observed and Expected Coliphage Detection in 1 Liter Volumes of Seeded Ground Water by Method 1601 Coliphage Group Estimated Coliphages/ L # Positive Bottles out of 10 Expected* Observed ( Average) Somatic ( E. coli CN13) 1.7 8.2 (~ 8) 4.8 (~ 5) Male specific ( E. coli Famp) 3.1 9.5 ( 9 10) 7.1 (~ 7) Both ( E. coli C3000) 1.7 8.5 ( 8 9) 5.7 (~ 6)

Figure 4. Coliphage Detection in 1 Liter Volumes of Seeded Ground Water by Method 1601 35 Based on direct analysis ( plaque assay) of the sewage derived coliphage stocks added to the ground water samples, the average concentrations of coliphages per 1 liter enrichment bottle were: 1.7 for somatic coliphages ( detected on host E. coli CN13), 3.1 for male specific coliphages ( detected on E. coli Famp) and 1.9 for both groups of coliphages ( detected on host E.

coli C3000). According to these estimated coliphage concentrations per liter of seeded ground water, the expected numbers of positive enrichment bottles out of a total 10 enrichment bottles per coliphage host are computed. These estimates of expected numbers of positive enrichment bottles out of 10 were based on the principles of Poisson statistics ( the Poisson Distribution and Poisson probabilities). The estimates were computed directly from the Poisson probability equation using the estimated coliphage concentrations per liter ( based on direct assay of the sewage coliphages seeded into groundwater as an estimate of the mean number of coliphages per bottle. The expected numbers of positive bottles out of 10 were: about 8 for somatic coliphages,

9 to 10 for male specific coliphages and 8 to 9 for both groups of coliphages. The experimentally observed numbers of positive enrichment bottles out of 10 for each group of coliphages are shown in Table 8 and Figure 1. Rounded to the nearest whole bottle, the numbers of positive enrichment bottles out of 10 were: 5 for somatic coliphages, 7 for male specific coliphages and 6 for both groups of coliphages. These actual results indicate that the likelihood of detecting coliphages when analyzing 1 liter volumes of ground water containing only about 1.5 3 coliphages per liter by the enrichment method are very high and very close to the theoretically expected results. It is noteworthy that out of a total of 87 trials with 10 1 liter enrichment bottles per trial there were only 4 occasions when all 10 bottles were negative. This is well within the expected number of times when all 10 bottles would yield negative results, based on statistical 36 considerations.

The expected probability of getting 10 negative bottles in a 10 bottle enrichment test when there is an average of 1.5 to 3 infectious coliphages per bottle was actually higher than the observed numbers of 10 negative enrichment bottles in a test. It should be noted that the detection of both somatic and/ or male specific coliphages together on host E. coli C3000 was similar to the detection of either somatic or male specific coliphages on their respective E. coli hosts. Although the numbers of positive enrichment bottles out of 10 were no higher on host E. coli C3000 than on the other two hosts, the ability to simultaneously detect both groups of coliphages using this host was not appreciably different than the detection of each coliphage group alone. Based on the number of times that all 10 bottles in an enrichment test were negative, E. coli C3000 was the same as or better than the other two hosts. The negativity rates per 30 trials of the method were 1, 1 and 2 per 30 trials for E. coli C3000 ( both), CN 13( somatic) and Famp ( male specific)

respectively. These results indicate that E. coli C3000 can be successfully and reliably used to simultaneously detect low levels of both somatic and male specific coliphages in 1 liter volumes of ground water using the two step enrichment method ( Method 1601).

Summary of Method 1601 Results In summary, recoveries of somatic, male specific and total ( somatic plus male specific) coliphages from 1 liter volumes of ground water were efficient using Method 1601. Coliphage recoveries at input levels of about 1.5 to 3 infectious units per liter of ground water were somewhat variable but close to those expected based on the infectivity titer of the sewage seed and to the expected 37 number of positive 1 liter enrichment bottles out of a total of 10. The observed variability of coliphage detection was no more variable than would be predicted for a 10 sample MPN test.

Coliphage recoveries were not significantly different among E. coli hosts and participating labs using ANOVA. Therefore, there is a high likelihood of detecting as few as 1 3 coliphages in 1

liter volumes of water using the two step enrichment methods of Method 1601. 38 PHASE I CONCLUSIONS In comparing the two coliphage recovery and detection methods, the participating laboratory groups tended to favor the Two Step Enrichment over the Single Agar Layer method. This is because the former test was considered easier to perform, sensitive in detecting low numbers of coliphages and more consistent in its results. The Single Agar Layer ( SAL) assay proved to be cumbersome when assaying multiple samples, and the time constraints imposed by the method were difficult to adhere to. The Two Step Enrichment method is simpler to set up and much easier to carry out. The statistical analyses showed it to be more consistent among different laboratory groups. In addition, the results for the enrichment method showed that somatic and male specific coliphages can be detected simultaneously on a single host, E. coli C3000, at a sensitivity comparable to detecting either somatic or male specific coliphages individually. The simultaneous detection of both somatic and male specific coliphages simplifies the method as well as lowers costs.

Further studies were done to characterize the performance of Methods 1601 and 1602 when applied to the detection of coliphages in unseeded samples of fecally contaminated ground water in Phase II of this study. The presence and concentrations of coliphages in field samples of groundwater were compared and also were to also be compared to the presence and concentrations of human enteric viruses in these fecally contaminated ground water samples. This information was to be used to determine if somatic, male specific and total coliphages are 39 sensitive and reliable indicators of fecal contamination and the presence of human enteric viruses in groundwater. 40 PHASE II STUDIES Statement of Work: Coliphage Method 1601 and 1602 Validation and Field Testing Background. The US EPA's proposed Ground Water Rule may propose the examination of ground waters for coliphages. Coliphages have been found to be reliable indicators of fecal contamination of ground and surface water and of the fate of human enteric viruses in the subsurface environment. Recently, two different EPA methods were developed to detect somatic and male specific coliphages in ground water. Method 1601 detects and quantifies coliphages by liquid enrichment culture method and Method 1602 detects and enumerates coliphages by a single agar layer ( SAL) plaque assay. The original methods round robin studies analyzed for somatic and male specific coliphages separately, and did not evaluate E. coli C3000 as host bacterium for detection and quantification of both somatic and male specific coliphage. The SAL method for coliphage detection did not require a confirmation step for the plaques that were observed in the agar host cell medium. There was some concern about the detection of " false positives" based on simply scoring plaques. Therefore, the methods needed to be further substantiated and validated and scientifically supportable to: ( 1) show that coliphages detected by these methods are of likely fecal origin and have characteristics and properties consistent with fecal origin, ( 2) reduce the cost and burden of using two different hosts to separately analyze somatic and male specific coliphage by measuring both simultaneously in a single E. coli host bacterium, ( 3) include a simple confirmation step. 41 Purpose and Objectives of the Study. The purpose of this study was to further validate EPA method 1601 and 1602 and to test these methods in groundwater samples in four geographically representative regional laboratories in the USA ( North Carolina, Minnesota, New Hampshire and Texas). The study was to determine if coliphages detected by Methods 1601 and 1602 can be confirmed and show properties and characteristics consistent with a fecal origin. The labs that conducted the studies were equipped and experienced to conduct the tasks required in the SOW.

The study was headed by a coliphage expert who has at least 20 years experience in coliphage virology, who has experience in round robin testing, in developing research procedures and in method 1601 and 1602. The expert participated in the original EPA round robin testing for method 1601 and 1602, is knowledgeable about EPA programs, and the proposed Ground Water Rule.

Specific Objectives. The specific objectives of the study were to: ( 1) conduct a field validation of Methods 1601 and 1602 for coliphage detection, ( 2) determine the ability of coliphage indicators in predicting the presence of human enteric viruses in the same ground water samples,

( 3) confirm the presence of coliphages detected in ground water samples, ( 4) characterize the properties of these coliphages to confirm that they are of likely fecal origin, ( 5) determine the correlation and the reliability of such correlation in detecting fecal contamination and the presence of human enteric viruses in ground water, and ( 6) address some key questions about these coliphage methods and their use for coliphage detection in groundwater that arose in the April 2004 EPA International Workshop on Coliphages as Indicators of Fecal Contamination in Water and Other Environmental Media. An additional task was added to the study at the request of the 42 EPA project manager. This task was: ( 7) to determine the stability or survival of coliphages in groundwater samples that held for up to several days before analysis by Methods 1601 and 1602.

Task 1. Characterization and determination of properties of confirmed coliphage isolates.

A total of 800 coliphages ( 200 from each of the four participating laboratories) were to be characterized of which 400 ( 100 from each laboratory) were to be from field samples and 400 ( 100 from each laboratory) were to be from sewage seeded ground water samples used in methods validation studies in Phase I. The contractor was to subject confirmed coliphage isolates to the analyses described below.

( a) bacterial host range analyses. Determine the ability of test coliphage isolates to be grown in both E. coli and non E. coli coliform hosts and other bacteria by spotting onto pre poured agar medium host cell lawns of the following 23 different host bacteria, if available: E. coli strains C,

CN13, C3000, K12F, K12F and Famp, S. typhimurium WG45 and WG49, Klebsiella pneumoniae ATCC strains 23356 and 23357, Enterobacter cloacae ATCC strain 223355,

Citrobacter braakii ( formerly Citrobacter freundil) ATTC strain6570 ATCC strain 12012,

Serratia marcescens ATCC strain 14764, Shigella sp. ATCC 23354, Shigella flexneri ATCC 12661, Yersinia pseudotuberculosis ATCC strain 23207, Proteus mirabilis ATCC strain 9921,

Yersinia enterocolitica ATCC strains 9610, 29913, Pseudomonas aeruginosa ATCC s train 12175, Aeromonas hydrophila ATCC strain 23211.

( b) growth temperature range. Test the coliphage isolates for their ability to grow at 43 temperatures of 25, 36, 42 and 44.5 /

C on E. coli hosts.

( c ) nucleic acid analyses. Examine coliphage isolates for taxonomy. Male specific coliphages were be tested to determine the type of nucleic acid as either DNA or RNA.

The contractor shall analyze 32 geographically representative ground water samples ( 8 per laboratory in four geographically representative laboratories) for coliphages and human enteric viruses by combined cell culture and nucleic acid amplification methods.

Task 2. Cell culture RT PCR or cell culture PCR. The contractor shall analyze 32 geographically representative ground water samples ( 8 per laboratory in four geographically representative laboratories) for coliphages and human enteric viruses by combined cell culture and nucleic acid amplification methods.

Task 3. Coliphages and enteric viruses from groundwater. Each of the four participating laboratories shall analyze an additional 8 samples of ground water for culturable human enteric viruses and for coliphages. Four different ground waters shall be analyzed on two different occasions. A total of 36 ground water samples shall be analyzed for coliphages and human enteric viruses. The extent to which somatic and male specific coliphages detected by Methods 1601 ( in 1 liter sample volumes) and 1602 ( in 0.1 liter sample volumes) are associated with the occurrence of human enteric viruses in 100 1000 liter sample volumes of fecally contaminated ground water will be determined. The contractor shall statistically analyze data on the occurrence of coliphages 44 and human enteric viruses in field ground water samples to determine if there is a co occurrence and the extent to which they co occur.

Task 4. Coliphage and bacterial analyses. Each lab will sample, process, and analyze ground water by Methods 1601 and 1602 using hosts E. coli Famp, E. coli CN 13 and E. coli C3000 according to the established methods. In parallel to the coliphage analysis, each lab will sample and process the same fecally contaminated ground waters by the EPA ICR methods for human enteric viruses. Also, in parallel each lab will sample and process the same fecally contaminated ground waters for E. coli and enterococci.

Task 5. Enteric virus analyses. Processed ground water samples will be analyzed for culturable human enteric viruses by observation for cytopathogenic effects ( CPE) in BGMK cells according to EPA ICR method. In addition, the inoculated cell cultures also will be examined for noncytopathogenic enteroviruses, caliciviruses, adenoviruses, hepatitis A, rotaviruses, reoviruses by combined cell culture and nucleic acid amplification methods, as previously described. The labs also will analyze the samples for culturable human enteric viruses in CaCo2 cells. The data on the occurrence and concentrations of coliphages and concentrations of human enteric viruses as detected by CPE and by PCR will be statistically analyzed to determine co occurrence with coliphages.

Task 6. Report. The contractor shall prepare a consolidated draft report of all the data generated in all the tasks in all 4 laboratories and statistically analyzed. The report shall include an 45 interpretation of the results and recommendations to EPA. The report shall be submitted in 3 double spaced hard copies and a 3 ½ diskette in WordPerfect, version 9/ 8.0 for Windows. A summary fact sheet of the study and results shall accompany the draft report.

Task 7. Peer review. The contractor shall incorporate internal and external peer review comments and a workshop input comments in a final report.

The EPA Work Assignment Manager will give technical direction in this study. The contractor shall not cite, quote or distribute the results of this EPA study until EPA publishes it. Publications from any aspect of this EPA research study will be subjected to EPA review and will be published jointly with EPA. A monthly conference call shall be scheduled by the contractor until completion of the study. An on site visit will be conducted by the EPAWAM on a mutually acceptable date with the technical lead.

Schedule of Deliverables The deliverables will include a consolidated 1st draft report with data from all the completed 7 tasks listed above and a final peer review report.

NOTE: These project objectives were not fully met due to extenuating circumstances.

Specifically, Task 1 could not be completed due to circumstances beyond the control of the project investigators. Although more than 800 coliphage isolates were obtained during this study,

the vast majority of the inventory of these coliphages were lost to due failures of ultracold 46 freezers in which the isolates were stored at the UNC laboratories for subsequent characterization. These freezer failures were due to both freezer malfunctions and to power outages at the laboratories that were due to natural disasters ( ice storms and hurricanes) beyond the control of the project investigators. Furthermore, these freezer failures caused the loss of the majority of bacterial hosts on which the coliphages isolates were to be characterized as to host range in order to fulfill Task 1a. Most of these bacterial strains had been previously purchased from the American Type Culture Collection and the costs of replacing them were prohibitive and had not been included in the project budget.

To compensate for the loss of these coliphage isolate samples and their further characterization,

the project labs undertook additional work in support of meeting other project objectives and tasks. Specifically, the participating labs analyzed more samples of groundwater in the Phase II studies than were originally specified. Contract specifications called for the analysis of a total of 64 samples ( 16 per laboratory) for coliphages, bacterial indicators and human enteric viruses. The eventual number of samples analyzed was actually 106 samples ( 27 by three laboratories and 25 by the fourth laboratory). It was believed that the analysis of extra field samples would provide more representative data for determining if coliphages were effective indicators of fecal contamination of groundwater and of human enteric viruses. Additionally, the analysis of human enteric viruses in groundwater samples was expanded to include astroviruses, which were not originally included in the specifications for human enteric viruses to be analyzed. Hence, this additional task was taken on to further improve the opportunities to detect human enteric viruses in groundwater as part of the effort to obtain more definitive data. 47 At the request of the UNC project manager, the UNC lab also took an additional task that was not in the original scope of work or its budget. This additional task was to determine the survival of coliphages in groundwater samples to be analyzed for viruses by Methods 1601 ( two step enrichment spot plate) and Method 1602 ( SAL plaque assay). Groundwater samples seeded with mixed populations of sewage derived viruses were analyzed for coliphages initially ( on day zero)

and also after 2, 3 and 6 days of storage at 4oC. These survival experiments were done to determine if collected samples held for several days prior to analysis due shipping and storage still had most of their initial coliphages that still could be detected by Methods 1601 and 1602. 48 PHASE II METHODS AND MATERIALS Groundwater Samples and Wells The original goal of this study was for each of the four, regionally representative laboratories ( southeast, northeast, upper midwest and southwest) to collect and analyze 27 ground water samples from public water supply wells. Efforts were made to identify candidate public water supplies that previously had coliform bacteria violations or other evidence of vulnerability to fecal contamination. In some cases candidate wells were prescreened by bacteriological and coliphage analyses for evidence of fecal contamination. Because not all participating labs could identify and get access to 27 public water supply wells, some labs also included non public and private wells in their sampling. Three labs obtained 27 ground water samples and one lab obtained a total of 25 samples for a total of 106 samples overall. The characteristics of the wells that were sampled are presented below, by region.

Southeast. Of the 27 wells in the Southeast, 13 were in North Carolina and 4 were in Florida.

The Florida wells were all public water supply wells. Florida Well UNC # 1 is in Orange County,

FL. There is no history of that well ever being disinfected. Florida Well UNC # 2 is in Orange County, Fl. The pump was taken out of service for repairs ( rebuilt pump), and it was disinfected in January  February of 2002. Prior to placing the well back into production it was disinfected with chlorine. Approximately 30 gallons of 12% liquid bleach was placed into the well for 24 hours ( 100ppm). Then water was discharged for a minimum of 4 hours and bacteriological samples were taken to confirm their absence. Theses Florida wells were samples in June and 49 September, 2002, which was 4 and 7 months after Well # 2 had been chlorinated.

Two other Florida wells, designated UNC # 3 and UNC # 4 and located in Ocala County also were sampled. Both wells had periodic coliform positivity during the and prior to the study period ( 2002). They serve a population of about 57,000 in the Ocala area. UNC # 3 and # 4 were not disinfected prior to or during sampling for this study. However, the utility currently ( year 2004) adds calcium hypochlorite ( granular chlorine) to control the total coliforms they are getting ( and have been getting a lot more since the hurricanes this year  2004). The chlorine is now added weekly to both wells to achieve the CT for 4 log virus removal through their treatment process, including chlorination.

There were 13 wells in North Carolina, and the characteristics of these wells are summarized below.

Type County Well Identification Private industrial Cartaret BF Community water supply Cartaret BMHP Community water supply Carteret SB MHP Non community water supply ( private campgrounds) Pamlico Camp DL Non community water supply ( private campgrounds) Pamlico Camp SF # 1 Non community water supply ( private campground) Pamlico Camp SF # 2 50 Private Carteret TGUMC Private Carteret GL Private Pender SC Private Pender VE Private Duplin RH Private Duplin KC Private Duplin TH Southwest. Only PWS wells were investigated in this study, and a total of eleven different PWS wells were included. The sampling sites were located in the San Antonio region of Texas ( wells RS, KK, and HCR) and along the US Mexico border in southern New Mexico ( wells MHPa,

MHPb, MHPc, FVE, AVC, SME, and LME). The wells in the San Antonio region were part of a karst aquifer and were previously implicated in a documented groundwater contamination event.

Also, during the initial pre screening of the wells some of the samples were positive for somatic and male specific coliphages. The wells in southern New Mexico were identified as being vulnerable to groundwater contamination based on parameters such as closeness to septic tanks,

proximity to the Rio Grande river and the aquifer in question. These wells were part of a previous EPA funded project on the microbiological quality of wells in the shallow aquifer along the US Mexico border during which some of the wells in the sampling area were positive for enterococci, E . coli, male specific coliphages and somatic coliphages. The wells were in the 100

150 feet depth range. The static water levels were around 10 20 feet and in terms of their hydrogeologic setting, they were located in the Rio Grande alluvium/ Hueco Tularosa aquifers. 51 Groundwater samples were collected between June 2002 and January 2003. Multiple samples were collected from each of the wells to be representative of the aquifer and the sampling location. During each sampling adequate volumes were collected for the coliphage analysis as well as for the enteric virus analysis. Grab samples were collected for the coliphage and bacterial analysis while the 1MDS filters were used for collecting the large volume enteric virus samples.

Upper Midwest. A total of 27 groundwater samples were collected from 25 wells. Two wells were tested twice. Details of these wells are provided in the report of the Upper Midwest lab,

which is in the Appendix). All wells except 6 private ones in Minnesota were considered noncommunity public water supplies by the State of Minnesota and none were disinfected.

Noncommunity transient public water supplies ( i. e., groundwater) are monitored for nitrate and total coliform bacteria as required by the SDWA. Private water systems including those places of business not meeting the federal definition of PWS have no long term monitoring requirements.

Northeast. All sample sites were located in New England. Eight well sites were public water sources and 17 were private wells. A total of 25 wells samples were collected instead of 27 due to a very severe and harsh winter. NH had its first snowfall at the end of October and a second snowfall at the beginning of November, 2002. Plans to sample two additional wells as soon as the weather permitted could not be carried out because New England experienced one of the snowiest winters ever. Therefore, only 25 well samples were collected and analyzed. Of the 25 wells, there were 12 sample sites in New Hampshire, two of which were from public wells that were approximately 500 and 700 ft deep, respectively. None of these wells had any form of 52 disinfection. The other wells from NH were all private wells. These wells also were not disinfected. One well from NH was a private, very shallow well, less then 35 feet deep and lined with stone. This was not considered a potable well but was used for farm irrigation. Four sites in Maine were all privately owned wells and not disinfected. Three sites were in Vermont, and they were all privately owned wells and not disinfected. All of the privately owned wells were drilled wells, excepted for the one in NH as indicated above, and they were of varying depths that were unknown to the homeowner at the time samples were collected. There were 6 samples from public water supply wells in Massachusetts. The public water supplies in Massachusetts were chosen due to positive results previously found for total and fecal coliforms, enterococci, and male specific coliphages. Additionally 3 of the 6 locations had positives previously reported for rotavirus and enterovirus, by molecular methods.

Coliphage Analysis of Groundwater Groundwater samples were analyzed by Method 1601, the two step spot plate enrichment method and by Method 1602, the Single Agar Layer ( SAL) plaque assay using sample volumes of 1 liter and 100 mL, respectively, for each target group of coliphages ( male specific, somatic and " total" coliphages). Host bacteria for the target groups of coliphages were E. coli CN 13 for somatic coliphages, E. coli Famp for F+ coliphages and E. coli C3000 for " total" ( somatic plus F+) coliphages. Coliphage analyses were performed according to the EPA approved methods,

except lysis zones from enrichment spot plates and plaques from SAL plates were confirmed using the proposed EPA confirmation method. In this method, material from individual SAL plaques or lysis zones on spot plates was removed ( aspirated) with a Pasteur pipette, micropipette tip, or 53 other device and the recovered material was resuspended in 0.5 mL of tryptic soy broth. These suspensions were held briefly for coliphages to diffuse out of the agar and then the samples were vortex mixed vigorously to disperse the coliphages. Then, 10: l aliquots were removed from the suspension and spotted onto pre poured spot plates of the appropriate E. coli host bacterium as in the enrichment procedure. The spot plates were incubated overnight and checked for zones of lysis. Any spots showing lysis were scored as confirmed coliphages.

Coliphage Isolate Characterization A total of 800 coliphages ( 200 from each of the four participating laboratories) were to be characterized for their properties to determine if they were of likely fecal origin. For each of the four participating laboratories 100 coliphage isolates from the phase I studies with groundwater samples seeded with sewage derived coliphages and another 100 isolates from the unseeded field groundwater samples of each laboratory were to be subjected to characterization by bacterial host range analysis, growth temperature range analysis and determination of type of nucleic acid ( for F+ coliphages).

For bacterial host range analyses coliphage isolates were to be tested for their ability to grow in both E. coli and non E. coli coliform hosts and other bacteria by spotting onto pre poured agar medium host cell lawns of the following 23 different host bacteria if available: E. coli strains C,

CN13, C3000, K12F, K12F and Famp, S. typhimurium WG45 and WG49, Klebsiella pneumoniae ATCC strains 23356 and 23357, Enterobacter cloacae ATCC strain 223355,

Citrobacter braakii ( formerly Citrobacter freundil) ATTC strain6570 ATCC strain 12012, 54 Serratia marcescens ATCC strain 14764, Shigella sp. ATCC 23354, Shigella flexneri ATCC 12661, Yersinia pseudotuberculosis ATCC strain 23207, Proteus mirabilis ATCC strain 9921,

Yersinia enterocolitica ATCC strains 9610, 29913, Pseudomonas aeruginosa ATCC s train 12175, Aeromonas hydrophila ATCC strain 23211. Spotted plates are incubated at 370C overnight and observed for evidence of lysis of the host bacteria in each spot as evidence of growth on each host bacterium.

For growth temperature range characterization, coliphage isolates were to be tested for their ability to grow at temperatures of 25, 36, 42 and 44.5 /

C on E. coli hosts. Coliphage isolates were to be serially diluted 10 fold and several dilution were to be spotted in 10 uL amounts onto replicate pre poured lawns of E. coli host bacteria in agar media Petri dishes. Each replicate plate was to be incubated at the aforementioned temperatures overnight and then the spots n these plates were to be observed and quantified for coliphage growth at each of the 4 test temperatures.

Coliphage growth at temperatures of not only 36oC but also growth at the temperatures of 42 and or 44.5oC was considered evidence of thermotolerance and of a likely fecal origin.

For nucleic acid analyses of F+ coliphages, isolates were to be examined for taxonomy as F+

DNA or F+ RNA coliphages using previously described methods ( Hsu et al., 1995). Briefly, 10 : l volumes of F+ coliphage suspensions were to be spotted onto duplicate pre poured lawns of E.

coli host bacteria in agar medium. One plate contained Rnase at 100 : g/ mL and the other plate did not. Plates were to be incubated overnight at 37oC and then they were to be observed for lysis or the appearance of plaques in the spots of applied coliphage suspensions. Presence of a lysis 55 zone or plaques in the spot on the plate without Rnase and the absence of such lysis or plaques in the spot on the plate with Rnase were considered evidence of an RNA coliphage. The presence of lysis or plaques in the spots of plates with and without Rnase was considered evidence of an F+

DNA coliphage.

As indicated above, these coliphage characterization activities were not completed due to extenuating circumstances beyond the control of the project investigators. Ultracold freezer failures caused the loss of archived coliphage isolates to be characterized and also the loss of most of the bacterial hosts that were to be used for host range characterization studies of these coliphage isolates.

Bacteriological Analysis of Groundwater Field groundwater samples were analyzed for E. coli and enterococci and in some cases for fecal coliforms using EPA approved methods. For E. coli, some labs used mFC agar for fecal coliforms, with incubation at 44.5oC for 20 22 hours, followed by transfer or membranes to nutrient agar MUG medium, re incubation for several hours, and observation for colonies fluorescing blue under long wavelength UV light as evidence of E. coli colonies. Another lab used mEC medium for E. coli with incubation at 44.5 oC ( APHA, 1998). Another lab used mColiBlue agar for simultaneous detection of total coliforms and E. coli, according to USEPAapproved methods. For enterococcus analysis, labs used standard membrane filter methods and with either modified ME agar or MEI agar and incubation conditions as specified in the EPA method ( APHA, 1995; Levin et al., 1975; USEPA, 2002). Samples for E. coli analysis were 100 56 mL, although one lab also analyzed volumes of 1000 mL. Data for the results of this larger 1000 mL volume were not included in the compilation and analysis of data for all labs, as no other lab analyzed this volume and it is not a standard volume used for bacteriological analysis of water.

The results for this larger volume are in the report from this participating laboratory, which is in the Appendix to this report. 57 Analysis of Groundwater for Human Enteric Viruses Primary virus concentration from groundwater. Ground water from candidate wells was sampled using the EPA ICR method, with minor modifications ( USEPA, 1996). Groundwater sample volumes of 1,500 liters ( 397 gallons) were to be filtered through a 1 MDS pleated cartridge filter ( CUNO) at pH 6 8. The filter was eluted with 1.5% beef extract ( Becton Dickinson # 212303) buffered with 0.05 M glycine at pH 9.5. Viruses in the resulting beef extract eluate were further concentrated by organic flocculation ( acid precipitation) as specified by the ICR methods. The only significant change to the ICR procedure was that the acid precipitate was resuspended in 20 about mL of sodium phosphate rather than 30 mL in order to reduce the concentrate volume and the number of cell cultures required to assay the concentrate. The entire concentrate was filter sterilized using a 0.2 micrometer pore size Gelman Serum Acrodisc filter (# 4525) which has been pretreated with a small volume of beef extract eluent to minimize viral adsorption.

The filter sterilized concentrate was subdivided into the following aliquots prior to being frozen at  80 ° :

  Equivalent of 500 L of water sample for assay of viruses in Caco 2 cell cultures   Equivalent of 500 L of water sample for assay of viruses in BGMK cell cultures, further subdivided into a 1.5 mL subsample, and the remainder. The 1.5 mL subsample was used in a pre test for cytotoxicity in BGMK cultures.

  Equivalent of 100 L of water sample for assay of HAV in FRhK 4 cell cultures. 58   Equivalent of 100 L of water sample for assay by direct RT PCR for caliciviruses ( noroviruses).

The remainder of the sample concentrate ( 20%, equivalent to 300 L) was archived at  80 ° C.

Virus isolation in cell cultures. Three cell lines were used to detect a range of infectious enteric viruses. The BGMK cell line was used to propagate adenoviruses, enteroviruses, and reoviruses according to the procedure of ( Chapron et al. ( 2000). Caco 2 cells were used for the detection of astroviruses and rotaviruses according to Chapron et al. ( 2000). The FRhK 4 cell line was used to detect hepatitis A virus ( HAV).

Cell culture infectivity assays were performed in a minimal number of 75 cm2 flasks ( generally 4 or 5). A pretest to screen each sample concentrate for cytotoxicity was performed in 25 cm2 BGMK culture flasks, with one flask being inoculated with 1.0 mL of sample concentrate and a second flask being inoculated with 0.5 mL of sample concentrate. The pretest cultures were observed microscopically for evidence of cytotoxicity ( or CPE) for one week, before the remainder of the sample was inoculated into cell cultures.

Sample concentrates inoculated into BGMK and Caco 2 cultures were pre activated by treatment with the proteolytic enzyme trypsin prior to inoculation. This was done because previous studies had indicated enhanced enteric viruses detection using this trypsin pre treatment. Each 59 concentrate was mixed with a solution of type IX trypsin ( Sigma T 0303) yielding a final 10 : g/ mL concentration, then incubated 30 minutes at 37 ° C. Pre activation was not necessary and therefore not employed for HAV propagation in FRhK 4 cultures. Because divalent cations enhance attachment of HAV and many other enteric viruses to cells, sample concentrates were diluted with an equal volume of Dulbecco =

s PBS before being inoculated into FRhK 4 cultures.

Cell cultures in 75 cm2 ( confluent monolayers for BGMK cells and 90 95% confluency for Caco 2 and FRhK 4 cell cultures) were drained and rinsed three times with Dulbecco =

s phosphate buffered saline ( Sigma # D 8662, Gibco # 14040 or equivalent) to remove residual serum. FRhK 4 cell cultures were rinsed once. Replicate cultures were inoculated with sample concentrates, and incubated at 37 ° C for 90 minutes, while being rocked every 15 20 minutes to re distribute the inoculum, to allow for virus adsorption to cells. One negative control flask was inoculated with PBS before any flasks were inoculated with sample concentrates, and a second negative control culture was similarly be inoculated at the end of the sample inoculation step. No enteric virus positive control flasks were to be prepared at this time in order to avoid possible laboratory virus contamination. Maintenance medium was then added to each flask, and the cultures were incubated at 37 ° C. The maintenance medium for BGMK and Caco 2 cultures consisted of serum free Eagle's minimum essential medium with Earle's salts ( MEM) supplemented with 5 : g/ mL trypsin. The maintenance medium used in FRhK 4 cultures consisted of Eagle's minimum essential medium with Earle's salts supplemented with 2% serum and 30 mM MgCl 2 . 60 Cultures were observed microscopically on days 1 and 2, then at least every other day following inoculation. The occurrence of cytopathology or cytopathic effects ( CPE) on the first two days was tentatively assumed to be evidence of sample cytotoxicity or the release of cells from the bottom surface of the tissue culture flask by the action of the trypsin. If cytotoxicity thought to be associated with sample inocula was not too far advanced, the affected cultures were given a change of maintenance medium to saved them from possible destruction by sample cytotoxicity.

Alternative approaches for cytotoxicity reduction included removing the inoculum following the 90 minute incubation period and then rinsing the cell layer with PBS, diluting the sample concentrate in Dulbecco =

s PBS, or inoculating less concentrate into each cell culture. Every reasonable effort was made to reduce sample cytotoxicity and maximize enteric virus detection.

BGMK and Caco 2 cultures were incubated for 7 at 370C days following inoculation. All cultures were freeze thawed, and 10% of the lysate from each flask was inoculated into fresh cultures for a second 7 day passage. If a flask exhibited possible viral cytopathology ( CPE), the lysate was passed through a 0.22: m pore size, sterilizing filter into a fresh culture to confirm the presence of viruses and the absence of bacterial or fungal contamination. FRhK 4 cultures were incubated for two 14 day passages, with the maintenance medium being changed after seven days, to maximize the detection of typically slow growing HAV.

At the end of the final cell culture passage, flasks were frozen and thawed twice. A 1 mL aliquot from each of the first and second passage BGMK and Caco 2 flasks inoculated with a given sample was pooled in a centrifuge tube. A half volume of chloroform was added, and the tube was vortex mixed at high speed for two minutes. The tube was centrifuged at 1,200 1,800 x g for 61 20 minutes, then the supernatant extract was removed and split into aliquots for viral analysis or archiving.

Virus detection by nucleic acid amplification. Chloroform extracted cell culture lysate pools were examined for adenoviruses, astroviruses, enteroviruses, HAV, reoviruses and rotaviruses by RT PCR or PCR. Viral nucleic acids were extracted from lysates using the QIAamp Viral RNA Mini Kit ( Qiagen # 52904). By modifying two steps of the QIAamp protocol, adenovirus DNA could be efficiently recovered without compromising extraction of viral RNA:

In step # 8 of the Qiagen protocol, The sample column was incubated for one minute after adding buffer AW1, before centrifuging the column.

In step # 9, the sample column was incubated for one minute after adding buffer AW2,

before centrifuging the column.

Prior to RNA extraction, enteric viruses in 2 milliliter aliquots of pooled, chloroform extracted cell culture lysates were concentrated using polyethylene glycol ( PEG) precipitation.

Polyethylene glycol ( Sigma # P2139, molecular weight = 8,000) was added to a final 8%

concentration. Sodium chloride was added to a 0.3 M concentration, and the sample was mixed until the additives dissolved. The solution was incubated for two hours at room temperature and then centrifuged at 6,700 x g for 20 minutes at 4 ° C. The pellet was resuspended in 300 : L of Dulbecco =

s PBS, then extracted with 300 : L of chloroform. 62 An aliquot of each water sample concentrate was assayed directly for human caliciviruses ( noroviruses) by nucleic acid amplification using RT PCR because these viruses cannot be propagated in cell cultures. Each sample was reconcentrated using polyethylene glycol precipitation. PEG was added to a final 10% w/ v concentration. Sodium chloride was added to a final 0.3 M concentration. Since the sample has previously been adjusted to pH 7.0 7.5, no further pH adjustment was necessary. The mixture was shaken until the additives had dissolved.

The solution was incubated at room temperature for two hours or at 4 ° C overnight. The solution was centrifuged at 6,000 to 10,000 x g for 15 minutes, and the supernatant was removed by aspiration and discarded. The pellet, which may not be visible, was resuspended in a maximum of 140 : L of Dulbecco =

s PBS containing magnesium and calcium ions.

Prior to RNA extraction, the resuspended pellet was extracted with chloroform. A 100: L volume of chloroform was added, and the sample vortex mixed for one minute. The sample was centrifuged at about 3,000 x g for 5 10 minutes. The supernatant was removed by aspiration and recovered. Viral RNA was extracted from the recovered, chloroform extracted supernatant using the standard QIAamp Viral RNA Mini Kit protocol.

Nucleic acid amplification by ( RT) PCR Introduction. Viruses in chloroform extracted cell culture lysates that had been inoculated with water sample concentrates and human caliciviruses ( noroviruses) in aliquots of water sample concentrates were analyzed by either PCR for adenoviruses or RT PCR for astroviruses,

caliciviruses, enteroviruses, hepatitis A virus ( HAV), reoviruses, and rotaviruses. The procedures 63 for combined cell culture and ( RT) PCR were based on those previously used by Chapron et al.

( 2000) with minor modifications. The nucleic acid extraction procedures and the ( RT) PCR primers and amplification procedures applied to each virus or virus group are described in more detail in the sections that follow and in the individual report of the other three participating laboratories ( see Appendix). RT PCR for HAV and caliciviruses was done by the University of North Carolina lab ( Southeast), RT PCR for enteroviruses was done by the University of Minnesota lab ( Upper Midwest), RT PCR for reovirus and rotavirus was done by the TAMU lab ( Southwest), and ( RT) PCR for adenoviruses and astroviruses was done by the University of New Hampshire lab ( Northeast). The details of the virus ( RT) PCR procedures of the participating laboratories are given below and also in more detail in the individual project reports of the other three participating labs, which appear in the Appendix of this report.

Enterovirus RT PCR The primers for RT PCR amplification of enteroviruses were:

3' pan enterovirus primer: 5' ACC GGA TGG CCA ATC CAA 5' pan enterovirus primer: 5' CCT CCG GCC CCT GAA TG Random hexamers may also be used as the primer for enteroviruses for reverse transcription.

The reaction mixtures for a 3.5 : L sample were as follows. ( For larger sample volumes the amounts were increased proportionally). ( Note: These mixtures utilized reagents from the GeneAmp RNA PCR Core Kit, Applied Biosystems # N808 0143.) 64 RT master Mix Stock conc. reaction/ reaction Final conc.

MgCl2 25 mM 4 5 mM 10x PCR Buffer II, pH 8.3 each dNTP 10x 10 mM 2 2 each 1x 1 mM 3' primer or random hexamers 50 : M 0.5 1.26 : M MuLV reverse transcriptase 50 U/: L 0.9 45 units Rnase inhibitor 20 U/ µ L 0.9 18 units The RT conditions were: 95 ° for 5 minutes, 42 ° for 60 minutes, and 95 ° for 5 minutes.

PCR master mix Stock conc. reaction/ reaction Final conc.

MgCl2 25 mM 4 2 mM 10x PCR Buffer II, pH 8.3 10x 8 1x Water ( Sigma # W 4502) 66 Ampli Taq DNA polymerase 5 U/: L 0.5 2.5 units 5' primer ( and 3' primer if used random hexamers) 50 : M 0.5 0.25 : M The PCR conditions were per cycle: 95 ° C for 1.5 minutes, 55 ° C for 1.5 minutes, and 72 ° C for 1.5 minutes, for a total of 40 cycles. The expected product ( amplicon) size was 197 bp. The internal oligonucleotide probe for hybridization was: 5' TAC TTT GGG TGT CCG TGT TTC.

Hybridization was at 55 ° C. 65 Hepatitis A virus RT PCR The primers for RT PCR amplification of HAV were:

3' HAV primer: 5' CTC CAG AAT CAT CTC CAA C 5' HAV primer: 5' CAG CAC ATC AGA AAG GTG AG ( VP1 VP3 capsid protein interface region)

The RT PVR reaction mixtures and reaction conditions were the same as for enteroviruses, and the expected product ( amplicon) size was192 bp. The internal oligonucleotide probe for HAV was: 5' TGC TCC TCT TTA TCA TGC TAT G. and the hybridization temperature was 55 ° C Rotavirus RT PCR The primers for RT PCR amplification of rotaviruses were those for Group A, gene 9:

3' rotavirus primer: 5' GGT CAC ATC ATA CAA TTC T 5' rotavirus primer: 5' GAT ATA ACA GCT GAT CCA ACA AC The reaction mixtures and reaction conditions were the same as for enteroviruses, and the expected product ( amplicon) size was 208 bp. The internal probe that could be used for product confirmation by hybridization was as follows: 5' AAT TGG AAA AAA TGG TGG CAA GT.

The hybridization temperature was 55 ° C. 66 Adenovirus and Astrovirus ( RT) PCR Nested PCR was performed on UNH, UNC, UMN and TAMU samples for both astrovirus and adenovirus type 40 and 41. The equivalent volume of original water sample examined for each virus was 500 liters. Positive controls were at the level of ( RT) PCR. Virus was added to cell culture lysate to act as a positive control for ( RT PCR) PCR Astrovirus. All molecular techniques were done as specified in the methods and materials developed by the project team in communication with the EPA project manager. Astrovirus RTPCR was done according to Chapron et al. ( 2000). The primers used were specific for human astrovirus:

RT primer 5' GTAAGATTCCCAGATTGGT 3', and PCR primer 5' CCTGCCCCGAGAACAACCAAG 3'.

An 11: L sample of the combined ( pooled) cell lysate was denatured with 0.5 : L each of 0.05 M EDTA and downstream primer at 99 /

C for 8 min. Eighteen : L of the RT mixture was then added and run for 42 min. at 42 /

C to reverse transcribe, followed by 5 min. at 99 ° C. The RT mixture per sample consisted of 2.5 : L 10X buffer II, 8.5 : L of 25mM MgCl 2, 1.25 : L of each 10mM dNTP, 0.5 : L of 100mM DTT ( Promega), 10 units of Rnasin, and 50 units of RT.

After the RT step, 28.5 : L of a PCR master mix was added. The PCR mixture per sample consisted of: 3 : L of 10X buffer II, 1 : L of the PCR primer, 0.5 : L of the RT primer, 24 : L of 67 molecular grade water, and 2.5 units of Ampli Taq DNA polymerase. The PCR amplification parameters were 95 ° C for 5 minute hot start, followed by 35 cycles of: 95 ° C for 30 seconds, 56 /

C for 30 seconds, 72 ° C for 30 seconds, with a final extension at 72 ° C for 5 minutes. These primers yielded a 193 and/ or 243 bp amplicon.

For nested PCR, 1 µ L from each RT PCR reaction was added to a new tube containing 90 µ L of a nested PCR reaction mixture, which contained 8 mM MgCl 2, 10 : L 10x buffer, 1mM of each dNTP, 2.5 units of Ampli Taq DNA polymerase and 1 : M of each primer. The primers used were: 5' CCTTGCCCCGAGCCAGAA 3' and 5' TTGTTGCCATAAGTTTGTGAATA 3'.

These primers yield a 143 and/ or 183 bp amplicon. Twelve µ l of each RT PCR product as well as 12 : L of the nested PCR product was resolved and sized by electrophoresis on an 1.8%

agarose gel, stained with ethidium bromide. Molecular weights were determined by comparison with a 1 Kb DNA ladder ( Life Technologies). Astrovirus serotype 2 was used as a positive control.

Adenovirus. All molecular techniques were done as specified in the methods and materials developed by the project team and communicated to the EPA project manager. Adenovirus Hexon PCR was done generally according to the procedures of Xu et al. ( 2000).

The PCR primers used were:

Ad1 5' CCCTGGTA( G/ T) CC( A/ G) AT( A/ G) TTGTA 3' and Ad2 5' TTCCCCATGGC( Inosine) CA( C/ T) AACAC 3'. 68 A 5: L sample of the combined cell lysates was added to 47.5 : L final volume PCR master mix.

Final concentrations in the PCR master mix per sample were 1.5mM MgCl 2 , 1x ( 10x Buffer II),

0.2mM dNTP mix, 0.6: M of each primer, and 2.5 units of Ampli Taq DNA polymerase. The PCR parameters were 95 ° C for 5 minutes, followed by 40 cycles of: 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes, with a final extension at 74 ° C for 5 minutes. These primers yielded a 482 bp amplicon.

For nested PCR, 1 µ l from each PCR reaction was added to a new tube containing 90 µ l of a nested PCR reaction mixture, which contained 8 mM MgCl 2, 10 : L 10x buffer, 1mM of each dNTP, and 1 : M of each primer. The primers used were:

5' GCCACCGAGACGTACTTCAGCCTG 3' and 5' TTGTACGAGTACGCGGTATCCTCGCGGTC 3.

These nested primers were specific for Adenovirus type 40 and 41. Samples were run for 35 cycles of: 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, yielding a 142 bp amplicon. Twelve : L of each nested PCR product was resolved and sized by electrophoresis on 1.8% agarose gels and stained with ethidium bromide. Molecular weights were determined by comparison with a 1 Kb DNA ladder ( Life Technologies). Adenovirus 40 and 41 were used as positive controls. 69 Reovirus RT PCR The primers for RT PCR amplification of reoviruses and kindly provided by Shay Fout of the US EPA, Cincinnati, were:

3' pan reovirus primer: 5' GTG CTG AGA TTG TTT TGT CCC AT 5' pan reovirus primer: 5' ACG TTG TCG CAA TGG AGG TGT Reaction mixtures for 5 : L samples were:

RT master mix Stock conc. reaction/ reaction Final conc.

MgCl 2 25 mM 1.8 1.5 mM 10X PCR Buffer II 10X 3 1X ( Applied Biosystems)

dNTP mix 10 mM each 2 0.7 mM 3' primer 10 : M 5 1.7 : M Water 11.45 The initial RT PCR reaction conditions were: 99 ° C for 5 minutes; then tubes were placed in ice.

Enzyme mix Stock conc. reaction/ reaction Final conc.

RNasin 30 units/: L 0.75 22 units ( Promega N2511)

MuLV RT 50 units/: L 1 50 units ( Applied Biosystems)

The RT reaction conditions were: 43 ° C for 60 minutes, 95 ° C for 5 minutes, and then tubes were placed in ice. 70 PCR master mix Stock conc. reaction/ reaction Final conc.

MgCl 2 25 mM 4.2 1.5 mM 10X PCR Buffer II 10X 7 1X 5' primer 10 : M 5 0.5 : M AmpliTaq Gold 1 ( pH 8.3 buffer only)

The PCR reaction conditions were per cycle: 95 ° C for 1 minute, 55 ° C for 1.5 minutes, and 72 °

C for 1.5 minutes, for a total of 40 cycles. The expected product ( amplicon) size was125 bp.

Internal oligonucleotide probes for the individual reovirus types 1, 2 and 3 were kindly provided by Shay Fout, and the hybridization temperature was 51 ° C.

Calicivirus direct RT PCR Calicivirus ( Norovirus) RT PCR analysis of concentrated virus samples from groundwater was done with the modified generic primers designated JV12/ JV13 ( Vinje et al., 2001; Hamidjaja et al.

2004).

3' RegA primer: 5' CTC ( A/ G) TC ATC ( Inosine) CC ATA ( A/ G) AA ( Inosine) GA 5' MJV12 primer: 5' TA( C/ T) CA( C/ T) TAT GAT GC( A/ C/ T) GA( C/ T) TA The RT PCR reaction mixture for 5: l samples had the following composition:

Antisense mix Stock conc. reaction/ reaction Final conc.

RegA primer 50 : M 1.2 60 pM Water 2.8 71 The initial RT reaction conditions were as follows: 94 ° C for 2 minute and then chilling in ice.

RT master mix Stock conc. reaction/ reaction Final conc.

MgCl2 25 mM 1.8 3 mM 10X PCR Buffer II 10X 1.5 1X ( pH 8.3)

dNTP mix 10 mM each 1.5 1 mM Water 0.2 AMV RT 10 units/: L 0.5 5 units Rnase inhibitor 40 units/: L 0.5 20 units The subsequent RT reaction conditions were: 42 ° C for 60 minutes and then 94 ° C for 5 minutes,

followed by chilling in ice.

PCR master mix Stock conc. reaction/ reaction Final conc.

MgCl2 25 mM 2.4 1.5 mM 10X PCR Buffer ( pH 9.0) 10X 4.5 dNTPs 10 mM each 0.5 0.2 mM MJV12 primer 50 : M 1 50 pM RegA primer 50 : M 0.6 50 pM Taq polymerase 5 units/: L 0.5 2.5 units Water 35.5 72 The PCR reaction conditions were an initial 94 ° C for 3 minutes, followed by 40 cycles with each cycle consisting of: 94 ° C for 1 minute, 50 ° C for 1.5 minutes, and 74 ° C for 1 minute. This was followed by 74 ° C for 7 minutes. The expected product ( amplicon) size was 327 bp.

The internal oligonucleotide probe for human caliciviruses was a mixture of one Group 1 probe ( GGI) and three Group II probes as follows:

GGI probe: 5' ATG GA( CT) GTT GG( CT) GA( C/ T) TAT GT ( 20 pM)

GGIId probe: 5' TGG AAC TCC ATC GCC CAC TGG ( 40 pM)

GGIIe probe: 5' TGG AAC TCC ATC ACA CAT TGG ( 80 pM)

GGLeeds probe: 5' TCA CCA GAT GTT GTC CAA GC The hybridization temperature was 42 ° C.

MS 2 RT PCR Coliphage MS2 was used as the positive control for RT PCR analyses by some laboratories to avoid introduction of potential human enteric virus contamination. About 100 pfu of MS2 per reaction tube was to be used and RT PCR was done according to Meschke and Sobsey ( 1998).

The RT PCR primers for MS2 were as follows:

3' ( downstream) MS2 primer: 5' CCC TAC AAC GAG CCT AAA TTC 5' ( upstream) MS2 primer: 5' GCA ACC TCC TCT CTG GCT AC 73 Random hexamers could be used as the primer MS2 reverse transcription. Reaction mixtures and reaction conditions for MS2 were the same as for enteroviruses. The expected PCR product size was 220 bp.

RT PCR and PCR controls To reduce the possibility of cross contamination of field samples during nucleic acid amplification,

RNA coliphage MS 2 was selected to act as a positive control for RT PCR procedures. This positive control with its own pair of primers was included in each set of reactions done by some participating labs. Other labs already had their own RT PCR and PCR controls and they used those existing QA/ QC control procedures and reagents that were already in place. The details of those measures can be found in the reports of the other 3 participating laboratories, which are in the Appendix to this report.

The minimal negative control samples that were to be run as part of each set of RT PCR and PCR samples included the following: ( 1) combined master mixes, enzymes and water done twice, one tube placed at the beginning of the set, and one tube placed at the end of the set, and ( 2) a cell culture negative control. Because the same pooled cell cultures were tested for multiple groups of human enteric viruses, cell culture negative control RNA extracts needed to be assayed using all of the appropriate virus primer pairs used by a given participating laboratory. RT PCR for HAV and Caliciviruses was done by the UNC lab ( Southeast), RT PCR for enteroviruses was done by the University of Minnesota lab ( Upper Midwest), RT PCR for reovirus and rotavirus was done by the TAMU lab ( Southwest), and ( RT) PCR for adenoviruses and astroviruses was done by the University of New Hampshire lab ( Northeast). Further details of the virus ( RT) PCR 74 methods of the participating laboratories are given in their individual project reports, which appear in the Appendix of this report.

Confirmation of presumptive ( RT) PCR positive samples Amplified PCR products from non nested protocols were to be examined by agarose gel electrophoresis. If cDNA bands of the appropriate size were detected, the presence of enteric virus sequences was to be confirmed using a labeled oligonucleotide probe internal to the original amplicon, as specified above. If enteric virus cDNA was detected, it was to be preserved for possible nucleotide sequencing. 75 PHASE II RESULTS AND DISCUSSION Introduction The Phase II studies of the project consisted of both additional lab studies as well as field studies.

Lab studies were performed to further characterize and improve the method to confirm coliphage isolates from plaques on SAL plates and lysis zones on spot plates from the enrichment method.

Lab studies also were conducted to determine the survival of coliphages in groundwater held at 4oC for up to 6 days prior to coliphage assay by Methods 1601 and 1602. Field studies consisted of the analysis of groundwater samples from wells for F+, somatic and " total" coliphages ( by Methods 1601 and 1602), fecal indicator bacteria ( E. coli and enterococci), and human enteric viruses by each of the four regional labs. Each lab collected and analyzed groundwater samples in its region. The samples concentrated for recovery of human enteric viruses by each of the 4 labs were divided into aliquots so that individual aliquots could be sent to other participating labs for centralized analysis of one or two the different target groups of human enteric viruses. The data for coliphages and fecal indicator bacteria in groundwater were analyzed to determine if the analysis of both coliphages and fecal indicator together in the same sample of groundwater gave greater detection of fecally contaminated groundwater than the analysis of only one indicator,

either a bacterium or a coliphage.

Results of Field Sample Analysis of Coliphage and Bacterial Indicators in Groundwater Table 9 contains all of the data for the presence and concentrations coliphages and fecal indicator bacteria in samples of groundwater from all four laboratories. 76 Table 9. Coliphages Detected by Methods 1601 and 1602 and Indicator Bacteria in Groundwater Lab* and Samples SAL (#/ 100 mL) Enrichment ( 1 L) Bacteria/ 100 mL) Famp CN 13 C3000 Famp CN 13 C3000 Fec. Colif. E. coli Ent. TAMU RS ( 1) 0 0 0 0 1 0 No Data 0 0 TAMU HCR ( 1) 0 0 0 0 0 0 No Data 0 0 TAMU RS ( 2) 0 0 0 0 0 0 No Data 0 0 TAMU BM ( 1) 0 0 0 0 0 0 No Data 0 0 TAMU KK ( 1) 0 0 0 0 0 0 No Data 0 0 TAMU RS ( 3) 0 0 0 0 0 0 No Data 0 0 TAMU KK ( 2) 0 0 0 0 1 0 No Data 0 0 TAMU HCR ( 2) 0 1 0 0 1 1 No Data 0 1 TAMU RS ( 4) 0 0 0 0 0 0 No Data 0 0 TAMU RS( 5) 0 0 0 0 0 0 No Data 1 0 TAMU MHP1a 0 0 0 0 0 0 No Data 1 5 TAMU MHP1b 0 0 0 0 0 0 No Data 0 0 TAMU AVC1 0 0 0 0 1 0 No Data 0 0 TAMU FVE1 0 0 0 0 0 0 No Data 0 0 TAMU AVC2 0 0 0 0 0 0 No Data 0 0 TAMU FVE2 0 0 0 0 0 0 No Data 0 0 TAMU FVE3 0 0 0 0 0 0 No Data 0 0 TAMU AVC3 0 0 0 1 1 1 No Data 0 0 TAMU MHP1c 0 0 0 0 0 0 No Data 0 0 TAMU MHP2a 0 0 0 0 0 0 No Data 0 0 TAMU MHP2c 0 0 0 0 0 0 No Data 0 0 TAMU MHP3a 0 0 0 0 0 0 No Data 0 0 TAMU MHP2b 0 0 0 0 0 0 No Data 0 0 TAMU SME1 0 0 0 0 0 0 No Data 0 0 TAMU SME2 0 0 0 0 0 0 No Data 0 0 TAMU LME1 0 0 0 0 0 0 No Data 0 0 TAMU MHP3b 0 0 0 0 0 0 No Data 0 0 UNH 1 0 0 0 0 0 0 1 0 0 UNH 2 0 0 0 0 0 0 0 0 0 UNH 3 4 0 0 1 0 1 0 0 0 UNH 4 0 0 0 0 0 0 0 0 0 UNH 5 0 0 0 0 0 0 0 0 0 UNH 6 0 0 0 0 0 0 0 0 0 UNH 7 0 0 0 0 0 0 0 0 0 UNH 8 0 0 0 0 0 0 0 0 0 UNH 9 0 0 0 0 0 0 0 0 0 UNH 10 0 0 0 0 0 0 0 0 0 UNH 11 0 0 0 0 0 0 0 0 0 UNH 12 0 0 0 0 0 0 0 0 0 UNH 13 0 0 0 0 0 0 0 0 0 UNH 14 0 0 0 0 0 0 0 0 0 UNH 15 0 0 0 0 0 0 35 0 0 UNH 16 0 0 0 0 0 0 0 0 0 UNH 17 0 0 0 0 0 0 0 0 0 UNH 18 0 0 0 0 0 0 0 0 1 UNH 19 0 0 0 0 0 0 2 0 13 77 UNH 20 0 0 0 0 0 0 0 0 0 UNH 21 0 0 0 0 0 0 0 0 2 UNH 22 0 0 0 0 0 0 0 0 0 UNH 23 0 0 0 0 0 0 5 0 2 UNH 24 0 0 0 0 0 0 0 0 0 UNH 25 0 0 0 0 0 0 0 0 0 MN 01 Amu 0 TNTC 0 0 0 No data 0 0 0 MN 02 Ger 0 4 5 0 0 0 0 0 0 MN 03 Rou 4 2 4 0 0 0 0 0 0 MN 04 Tur 2 2 3 0 1 1 0 0 0 MN 05 Bro 0 0 0 0 0 0 0 0 0 MN 06 OG 0 0 0 0 0 0 0 0 0 MN 07 KM 1 58 4 0 0 0 0 0 0 MN 08 KM 40 12 7 1 0 1 0 0 1 MN 09 Ham 0 0 0 0 0 0 0 0 0 MN 10 Nor 9 28 0 0 0 0 0 0 0 MN 11 Pre 0 0 0 0 0 0 0 0 0 MN 12 Imm 0 0 0 0 0 0 30 0 1 MN 13 Cen 0 0 0 0 0 0 0 0 0 MN 14 His 0 0 0 0 0 0 1 0 0 MN 15 Nor 0 0 0 0 0 0 0 0 0 MN 16 Lak 1 234 574 0 1 0 0 1 0 0 MN 17 Lak M 0 0 0 0 0 0 0 0 0 MN 18 Lak M 0 0 0 0 0 0 0 0 0 MN 19 Al 0 0 0 0 0 0 0 0 0 MN 20 Day 0 0 0 0 0 0 0 0 0 MN 21 Lak M 0 9 1 0 0 0 0 0 0 MN 22 GF 0 2 2 0 0 0 0 0 1 MN 23 TA 3 2 7 0 1 1 1 0 0 MN 24 Mil 11 6 1 0 0 0 17 15 12 MN 25 Jay 0 6 3 0 0 0 3 1 1 MN 12 ChR 3 3 4 0 0 0 0 0 0 MN 16 Cem R 6 5 6 0 0 0 0 0 0 MN 24 Mil R 2 5 0 0 0 0 248 3 20 UNC 1 BMH 0 0 0 0 0 0 0 0 0 UNC 2 GL 0 0 0 0 0 0 0 0 0 UNC 3 VE 0 0 0.4 0 0 0 0 0 0 UNC 4 KC 0 0 0.4 0 1 0 0 0 0.5 UNC 5 OC FL# 1 0 0 0 0 0 0 0 0 0 UNC 6 OC FL# 2 0 0 0 0 0 0 0 0 1.5 UNC 7 KC 0 0.4 0 0 0 0 0 0 0 UNC 8 VE 0 0.4 0 0 0 0 0 0 0 UNC 9 BF 0 0 0 0 0 0 0 0 0 UNC 10_ SB MHP 0 0 0 0 0 0 0 0 0 UNC 11 BMH1 0 0 0 0 0 0 0 0 0 UNC 12_ GL 0 0 0 0 0 0 0 0 0 UNC 13_ OC FL# 1 0 0 0 0 0 0 0 0 0 UNC 14_ OC FL # 2 0 0 0 0 0 0 0 0 0 UNC 15_ GL 0 0 0 0 0 0 0 0 0 UNC 16 CDL 0 0 0 0 0 0 0 0 0 78 UNC 17 BF 0 0 0 0 0 0 0 0 0 UNC 18 SB MHP 0 0 0 0 0 0 0 0 0 UNC 19 CDL 0 0 0 0 0 0 0 0 0 UNC 20 CSF # 1 0 0 0 0 0 0 0 0 0 UNC 21 CSF # 2 0 0 0 0 0 0 0 0 0 UNC 22 Oca FL# 1 0 0 0 0 0 0 0 0 0 UNC 23 Oca FL# 2 0.4 0 0 0 0 0 0 0 0 UNC 24 CSF # 1 0 0 0 0 0 0 0 0 0 UNC 25 CSF # 2 0 0 0 0 0 0 0 0 0 UNC 26 Oca FL # 1 0 0 0 0 0 0 0 0 0 UNC 27 Oca FL # 2 0 0 0 0 0 0 0 0 0 * TAMU = Texas Agricultural and Mechanical University, UNH = University of New Hampshire,

MN = University of Minnesota and UNC = University of North Carolina Table 10 summarizes these data on the presence of fecal indicator microbes, including somatic coliphages, male specific coliphages, " total" coliphages ( detected on host E. coli C3000), fecal coliform bacteria, E. coli and enterococci in groundwater samples in this study on the basis of positive samples, regardless of microbe concentration. A total of 107 samples were analyzed and these samples correspond to the samples that were also analyzed for human enteric viruses.

Additional groundwater samples were analyzed by some laboratories in the initial screening of groundwater wells for possible inclusion in the study. However, these samples are not included in the table because not all microbial indicators were measured by all methods during this prescreening analysis effort and there was no concurrent analysis of human enteric viruses for possible comparison. 79 Table 10. Frequency of Occurrence of Fecal Indicator Microbes in Field Ground Water Samples #. Pos./# Tested at: Total # Pos./ Total # Tested, All Labs % Positive Indicator TAMU UMN UNC UNH Somatic Coliphage  SAL 1/ 27 16/ 28 2/ 27 0/ 25 19/ 116 16.4% F+ Coliphage by SAL 0/ 27 11/ 28 1/ 27 1/ 25 13/ 116 11.2% " Total" Coliphage"  SAL 0/ 27 12/ 28 2/ 27 0/ 25 14/ 116 12% Somatic Coliphage Enrichment 5/ 27 2/ 28 1/ 27 0 / 25 8/ 116 6.9%

F+ Coliphage Enrichment 1/ 27 2/ 28 0/ 27 1/ 25 4/ 116 3.4% " Total" Coliphage Enrichment 2/ 27 3/ 28 0/ 27 1/ 25 6/ 116 5.2%

Fecal Coliform Not done 7/ 28 0/ 27 4/ 25 11/ 80 13.8% E. coli 2/ 7 3/ 28 0/ 27 0/ 25 5/ 116 4.3% Fecal Coliform and/ or E. coli 2/ 27 7/ 28 0/ 27 4/ 25 13/ 116 11.2% Enterococci 2/ 27 6/ 28 2/ 27 4/ 25 14/ 116 12.1%

As shown in Table 10, The frequency of detection of any single fecal indicator microbe was highest for somatic coliphages as measured by the SAL method at 16.4% and second highest for fecal coliform at 13.8%. However, the frequency of detection of somatic coliphage by the SAL method and of fecal coliform was not significantly different ( P = 0.768 by Mann Whitney U test).

Interpretation of these statistical results for comparative detection of somatic coliphages by SAL and fecal coliforms is limited. This is because not all samples were analyzed for both of these fecal indicators and therefore a paired statistical analysis of the results was not possible.

Enterococci and " total coliphage" by the SAL method were tied for third in detection frequency at 12.1%. Overall, these results indicate the rate of detection of any single fecal indicator was higher for coliphages, specifically somatic coliphage detected by SAL, than any other single indicator tested. It is also noteworthy that the simultaneous detection of both somatic and male specific coliphages as " total coliphages" by the SAL method on a single host bacterium, E. coli C3000, 80 gave a high frequency of detecting fecal contamination at 12.1%, making it one of the best indicators tested.

Examination of the results of coliphage analyses in Table 10 indicate that each coliphage group was detected more frequently by the single agar layer ( SAL) method than by the two step enrichment spot plate method. This finding is striking given that the sample volume for the SAL method was only 100 mL and for the enrichment method it was 1 liter. The comparative detection of coliphages by SAL and enrichment methods was 16.4% versus 6.9% for somatic coliphage, 11.2% versus 3.4% for F+ coliphages and 12.1% versus 5.2% for " total" coliphages.

The results for the frequency of detection of the different coliphage groups by the SAL or enrichment method were statistically compared by a non parametric, paired t test ( Wilcoxon matched pairs signed ranks test). The detections frequencies between SAL and enrichment methods were significantly different for somatic coliphages ( P = 0.137) and for F+ coliphages ( P = 0.0351) and they were nearly significant for " total" coliphages ( P = 0.580). Overall, these results indicate that the SAL method gave significantly better detection coliphages than did the enrichment method. The reasons for this are not known and probably deserve further investigation. It should be remembered that both methods were highly efficient in detecting coliphages when tested in phase I studies on seeded samples of groundwater.

Comparative Detection of Two Indicators in Groundwater Samples It was of interest to consider the simultaneous detection of two indicators in groundwater samples. This is because the proposed groundwater rule has considered the possibility of measuring only one indicator in a sample ( either bacterium or a coliphage indicator) versus 81 measuring both a bacterial and a coliphage indicator. Therefore, a fundamental consideration is whether or not the dual measurement of two indicators improves the detection of fecal contamination in groundwater samples by increasing the frequency of fecal indicator ( a positive sample). The results for selected pairs of fecal indicators that gave the highest detection frequencies are summarize in Table 11.

Table 11. Frequency of Occurrence of Dual Indicators in Field Ground Water Samples Indicator Pair #. Pos./# Tested at: Total # Pos./ Total # Tested, All Labs % Positive Somatic and/ or F+ Coliphage  SAL 1/ 27 16/ 28 3/ 27 1/ 25 21/ 116 18.1% Somatic and/ or F+ Coliphage  Enrichment 5/ 27 4/ 28 1/ 27 1/ 25 11/ 116 9.5% Enterococci and Fecal Coliform and/ or E. coli 3/ 27 9/ 28 2/ 27 6/ 25 20/ 116 17.2% Somatic Coliphage  SAL and/ or Enterococci 2/ 27 16/ 28 2/ 27 4/ 25 24/ 116 20.7% Somatic Coliphage  SAL and/ or Fecal Coliform or E. coli 3/ 27 17/ 28 2/ 27 4/ 25 26/ 116 22.4%

Fecal Coliform and/ or E. coli 2/ 27 7/ 28 0/ 27 4/ 25 13/ 116 11.2% Enterococci 2/ 27 6/ 28 2/ 27 4/ 25 14/ 116 12.1% Enterococci and Fecal Coliform and/ or E. coli 3/ 27 9/ 28 2/ 27 6/ 25 20/ 116 17.2% Somatic Coliphage  SAL and/ or Enterococci 2/ 27 16/ 28 2/ 27 4/ 25 24/ 116 20.7% Somatic Coliphage  SAL and/ or Fecal Coliform or E. coli 3/ 27 17/ 28 2/ 27 4/ 25 26/ 116 22.4%

Dual versus individual detection of somatic and F+ coliphages by SAL. As a first case of comparing the detection of positive samples with pairs of indicators versus single indicators is the SAL detection of somatic and/ or male specific coliphages in groundwater samples. This coliphage indicator pair was considered because the measurement of both of these two groups of coliphages in an option in the proposed groundwater rule. Currently, there is no clear basis for choosing one coliphage group over the other and therefore, the measurement of both coliphage groups in a sample on their respective E. coli hosts is an option. The frequency of detecting a 82 positive sample ( positive for one or the other or both) was 18.1% ( Table 11). For each group alone, the SAL detection frequency was 16.4% for somatic coliphages and 11.2% for F+

coliphages ( Table 10). The SAL detection of either or both of these coliphage indicators in a sample by dual analysis ( 18.1%) was compared to the frequency of detection of each of them alone ( F+ SAL = 11.2% and somatic SAL = 16.4%) using the Friedman test, a non parametric Analysis of Variance ( ANOVA). The P value was very significant ( 0.0055), indicating the detection frequencies were significantly different and were highest for the detection of either or both coliphage groups when both are measured in a sample.

When the individual SAL detection frequencies of F+ coliphages ( 11.2%) and somatic coliphages ( 16.4%) were compared by a non parametric t test ( Wilcoxon matched pairs signed ranks test),

there was no significant difference ( P= 0.105), indicating equivalent detection of either of these two coliphage groups alone. Furthermore, when the SAL detection frequencies of F+ coliphages alone ( 11%) were compared to the dual detection of either F+ coliphages and/ or somatic coliphages ( 18.1%) by the Wilcoxon matched pairs signed ranks test, the difference was very significant ( P = 0.0078). This indicates that SAL detection of both coliphage groups is better than detecting F+ coliphages alone. A similar comparison for SAL detection of somatic coliphages alone ( 16%) versus the SAL detection of either or both coliphages when both are measured in a sample ( 18.1%) indicated no significant difference because the sample size was too small.

Overall, SAL detection of both groups of coliphages ( F+ and somatic) is better than SAL detection of either group alone. 83 Dual versus individual detection of bacterial indicator pairs. When the detection of two fecal indicator bacteria such as fecal coliforms and/ or E. coli versus enterococci is considered because both groups are measured simultaneously, the frequency of detecting a positive sample ( positive for one or the other or both) was also high at 17.2% ( Table 11). For each group alone, the detection frequency was 12.1% for enterococci and 11.2% for fecal coliforms and/ or E. coli ( Table 10). The dual detection of either or both of these bacterial indicator groups in a sample ( 18.1%) was compared to the frequency of detection of each of them alone ( enterococci = 12.1%

and fecal coliforms and/ or E. coli = 11.2%) using the Friedman test. The P value was significant ( 0.0366), indicating the detection frequencies were significantly different and were highest for the detection of either or both indicator bacteria groups when both are measured in a sample. When the individual detection frequencies of enterococci ( 12.1%) and fecal coliforms and/ or E. coli ( 11.2%) were compared by the non parametric Wilcoxon matched pairs signed ranks test, there was no significant difference ( P= 0.839), indicating equivalent detection of either of these bacterial indicator groups alone. Furthermore, when the detection frequencies of enterococcus alone ( 12.1%) was compared to the dual detection of either or both enterococcus and/ or fecal coliforms and/ or E. coli ( 17.2%) by the Wilcoxon matched pairs signed ranks test, the difference was significant ( P = 0.031). This indicates that dual detection of both bacterial indicator groups is better than detecting enterococci alone. A similar comparison for detection of fecal coliforms and/ or E. coli alone ( 11.2%) versus the dual detection of either or both enterococcus and/ or fecal coliforms and/ or E. coli ( 17.2%) by the Wilcoxon matched pairs signed ranks test also was significant ( P = 0.016). This indicates that detection of both bacterial indicator groups is better than detecting fecal coliforms and/ or E. coli alone. Overall, detection of both groups of bacteria is better than detection of either group of bacteria alone. 84 Dual versus individual detection of bacterial and coliphage indicator pairs. The extent to which dual detection of a coliphage indicator and bacterial indicator versus individual detection of either one alone was considered. This is because the detection of both a coliphage indicator and a bacterial indicator in a groundwater sample has been an option for the proposed groundwater rule. The first pair of coliphage and bacteria indicators to compare was somatic coliphages detected by the SAL method ( 16.4%) and enterococci ( 12.1%) ( Table 10). This pair was chosen because these were the individual coliphage and bacterial indicators measured in all samples and detected most frequently. For SAL somatic coliphages and/ or enterococcus being measured together in samples, the frequency of detecting a positive sample ( positive for one or the other or both) was 20.7% ( Table 11), which was higher than either indicator alone or any coliphage pair or bacterial pair. Furthermore, a statistical comparison of measuring either indicator alone or both indicators together showed significant improvement in detecting fecal contamination of groundwater. The detection of either or both of these indicator groups in a sample ( 20.7%) was statistically compared to the frequency of detection of each of them alone ( enterococci = 12.1%

and SAL somatic coliphages = 16.4%) using the Friedman test. The P value was significant ( 0.028), indicating detection frequencies were significantly different and were highest for the detection of either or both indicator groups ( enterococci and/ or SAL somatic coliphages) when both are measured in a sample. When the individual detection frequencies of enterococci ( 12.1%)

and SAL somatic coliphages ( 16.4%) were compared by the non parametric Wilcoxon matchedpairs signed ranks test, there was no significant difference ( P= 0.355), indicating equivalent detection of either of these indicator groups alone. Furthermore, when the detection frequencies of enterococcus alone ( 12.1%) were compared to the detection of either or both enterococcus and/ or SAL somatic coliphages ( 20.7%) by the Wilcoxon matched pairs signed ranks test, the 85 difference was very significant ( P = 0.002). This indicates that detection of both indicator groups is better than detecting enterococci alone. A similar comparison for detection of SAL somatic coliphages alone ( 16.4%) versus the detection of either or both enterococcus and/ or SAL somatic coliphages ( 20.7%) by the Wilcoxon matched pairs signed ranks test was not significantly different ( P = 0.206). Overall, the detection of both groups of indicators ( SAL somatic coliphages and enterococci) was generally better than detection of either indicator group alone.

When the coliphage with the highest detection frequency, which was SAL somatic coliphages ( 16.4%), and the bacterial indicator with the highest detection frequency ( and measured in any project samples), which was fecal coliforms and/ or E. coli ( 11.2%), were being measured together in samples, the frequency of detecting a positive sample ( positive for one or the other or both)

was 22.4%% ( Table 11). This indicator detection frequency was even higher than either indicator alone or any coliphage pair or any bacterial pair. Furthermore, a statistical comparison of measuring either indicator alone or both indicators together showed significant improvement in detecting fecal contamination of groundwater. The detection of either or both of these indicator groups in a sample ( SAL somatic coliphages and/ or fecal coliforms and/ or E. coli = 22.4%) was statistically compared to the frequency of detection of each of them alone ( SAL somatic coliphages = 16.4% and fecal coliforms and/ or E. coli = 11.2%) using the Friedman test). The P value was very significant ( 0.003), indicating the detection frequencies were significantly different.

When the individual detection frequencies of fecal coliforms and/ or E. coli ( 11.2%) and SAL somatic coliphages ( 16.4%) were compared by the non parametric Wilcoxon matched pairs signed ranks test, there was no significant difference ( P= 0.276), indicating equivalent detection of either of these indicator groups alone. Furthermore, when the detection frequency of SAL 86 somatic coliphages alone ( 16.4%) was compared to the combined detection of either or both fecal coliforms and/ or E. coli and/ or SAL somatic coliphages ( 22.4%) by the Wilcoxon matched pairs signed ranks test, the difference was significant ( P = 0.039). This indicates that detection of both indicator groups is better than detecting SAL somatic coliphages alone. A similar comparison for detection of fecal coliforms and/ or E. coli alone ( 11.2%) versus the combined detection of either or both fecal coliforms and/ or E. coli and/ or SAL somatic coliphages ( 22.4%) by the Wilcoxon matched pairs signed ranks test was extremely significantly different ( P = 0.0002). Therefore,

the dual detection of both a coliphage indicator and a bacteria indicator in groundwater samples is better than detecting either indicator group alone.

Because there is interest in using a single coliphage host to simultaneously detect both somatic and male specific ( or " total") coliphages, it also was of interest to examine the dual detection of total coliphages and a bacterial indicator. When " total" coliphages detected by the SAL method ( detection frequency = 12.1%) and the bacterial indicator of enterococci ( detection frequency =

12.1%) are considered together, the frequency of detecting a positive sample ( for either or both indicators) was 19.8% ( Table 11). A statistical comparison of measuring either indicator alone or both indicators together showed significant improvement in detecting fecal contamination of groundwater. The detection of either or both of these indicator groups in a sample ( 19.8%) was statistically compared to the frequency of detection of each of them alone ( SAL total coliphages =

12.1% and enterococci = 12.1%) using the Friedman test. The P value was significant ( 0.011),

indicating the detection frequencies were significantly different. When the individual detection frequencies of enterococci ( 12.1%) and SAL total coliphages ( 12.1%) were compared by a nonparametric t test ( Wilcoxon matched pairs signed ranks test), there was no significant difference 87 ( P> 0.999), indicating equivalent detection of either of these indicator groups alone. Furthermore,

when the detection frequencies of SAL total coliphages alone ( 12.1%) was compared to the dual detection of either or both SAL total coliphages and enterococci ( 19.8%) by the Wilcoxon matched pairs signed ranks test, the difference was very significant ( P = 0.0039). This indicates that detection of both indicator groups is better than detecting SAL total coliphages alone. A similar comparison for detection of enterococci alone ( 12.1%) versus the dual detection of either or both enterococcus and/ or SAL total coliphages ( 19.8%) by the Wilcoxon matched pairs signed ranks test the difference also was very significant ( P = 0.0039). Therefore, dual detection of both a coliphage indicator (" total" coliphages by SAL) and a bacteria indicator ( enterococci) in groundwater samples is better than detecting either indicator group alone. This is the case even for the single coliphage indicator capable of detecting both male specific and somatic coliphages (" total" coliphages).

Detection of " total" coliphages by SAL ( detection frequency = 12.1%) and the bacterial indicator of fecal coliforms and/ or E. coli ( detection frequency = 11.2%) also were considered together,

and the frequency of detecting a positive sample ( for either or both indicators) was 20.7% ( Table 11). A statistical comparison of measuring either indicator alone or both indicators together showed significant improvement in detecting fecal contamination of groundwater. The detection of either or both of these indicator groups in a sample ( 20.7%) was statistically compared to the frequency of detection of each of them alone ( SAL total coliphages = 12.1% and fecal coliforms/ E. coli = 11.2%) using the Friedman test. The P value was very significant ( 0.005),

indicating the detection frequencies were significantly different and were highest for the detection of either or both indicator groups ( fecal coliforms/ E. coli and SAL total coliphages) when are 88 both measured in a sample. When the individual detection frequencies of the bacterial indicator ( fecal coliforms/ E. coli = 12.1%) and SAL total coliphages ( 11.2%) were compared by the nonparametric Wilcoxon matched pairs signed ranks test, there was no significant difference ( P =

0.865), indicating equivalent detection of either of these indicator groups alone. Furthermore,

when the detection frequencies of SAL total coliphages alone ( 12.1%) was compared to the detection of either or both SAL total coliphages and/ or fecal coliforms/ E. coli ( 20.7%) by the Wilcoxon matched pairs signed ranks test, the difference was very significant ( P = 0.002). This indicates that detection of both indicator groups is better than detecting SAL total coliphages alone. A similar comparison for detection of fecal coliforms/ E. coli alone ( 11.2%) versus the dual detection of either or both fecal coliforms/ E. coli and/ or SAL total coliphages ( 20.7%) by the Wilcoxon matched pairs signed ranks test also was extremely significant ( P = 0.001). Therefore,

these results again show that detection of both a coliphage indicator (" total" coliphages) and a bacterial indicator ( fecal coliforms and/ or E. coli) in groundwater samples is better than detecting either indicator group alone.

It is noteworthy that the frequency of detecting fecal contamination in a groundwater sample was nearly as high with " total" coliphages detected by SAL and a bacteria indicator ( with either enterococci or fecal coliforms/ E. coli at 19.8 and 20.7%, respectively) as with somatic coliphages detected by SAL with a bacteria indicator ( with either enterococci or fecal coliforms/ E. coli;