EPA #823

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## Applicability of Great Lakes NEEAR Dataset to Inland Recreational Water Criteria: Summary of Key Studies

U.S. Environmental Protection Agency

November 2010Inland Waters Summary Report November 2010



### Contents

Contents ....................................................................................................................... ..................iii
Tables......................................................................................................................... .................... iv Figures........................................................................................................................ .................... iv Abbreviations and Acronyms ......................................................................................................... v Executive Summary........................................................................................................................ 1
1. Introduction................................................................................................................... ...... 3
1.1. Objective............................................................................................................................. 3 1.2. Methodology....................................................................................................................... 4 1.2.1. Documents reviewed.................................................................................................. 4 1.2.2. Topics featured in document review.......................................................................... 5 1.3. General descriptions of inland flowin g, inland impounded, and Great Lakes coastal settings ................................................................................................................................ 7 1.3.1. Qualitative comparison of indicator pro cesses in inland and coastal waters............. 7 1.3.2. Brief review of epidemiology studi es conducted for inland waters .......................... 9 2. Documents review ............................................................................................................ 12
2.1. Summary of findings......................................................................................................... 12 2.2. Detailed reviews............................................................................................................... . 19 2.2.1. Final Report on the Experts Scientific Workshop on Criti cal Research and Science Needs for the Development of Recreati onal Water Quality Criteria for Inland Waters (WERF 2009) .............................................................................................. 19 2.2.2. Meeting Report: Knowledge and Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dor evitch et al. 2010)............................................................ 27 2.2.3. Literature Review of Assessment of th e Applicability of Existing Epidemiology Data to Inland Waters (USEPA 2010a) ................................................................... 31 2.2.4. Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b) ................................................................. 34 2.2.5. Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters (WERF 2010a) ....................................................................... 36 2.3. Methodology Performance and Relevance to Applicability of Criteri a to Inland Waters (compilation of findings fr om multiple reports) ............................................................... 38 References..................................................................................................................................... 43

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### Tables

Table 1. Key areas that the reviewed reports a nd publications used to compare and contrast Great Lakes and inland waters.........................................................................................6 Table 2. Study purpose, methodology, findings and limitations for the Final Report on the Experts Scientific Work shop on Critical Research and Science Needs for the Development of Recreational Water Quality Criteria for Inland Waters (WERF 2009) ..............................................................................................................................12 Table 3. Study purpose, methodol ogy, findings and limitations for Meeting Report: Knowledge Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dorevitch et al. 2010).......................................................................................14 Table 4. Study purpose, methodol ogy, findings and limitations for Literature Review of Assessment of the Applicabili ty of Existing Epidemiology Data to Inland Waters (USEPA 2010a) .............................................................................................................15 Table 5. Study purpose, methodology, ma jor findings and limitations for Sampling and Consideration of Variability (Tem poral and Spatial) for Monitoring of Recreational Waters (USEPA 2010b) ...........................................................................16 Table 6. Purpose, methodology, major findings, and limitations of draft final report. Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters (WERF 2010a).............................................................................17 Table 7. Purpose, methodology, major fi ndings, and limitations of Methodology Performance and Relevance to Applicab ility of Criteria to Inland Waters (compilation of findings from multiple reports) ............................................................18 Table 8. Priority research activities from the 2009 Inland Waters Workshop (WERF 2009).......24 Table 9. Comprehensive list of proposed rese arch activities from the 2009 Inland Waters Workshop (WERF 2009) ...............................................................................................24



### Figures
Figure 1. Illustration of fecal indicator organism sources for coastal waters ................................. 8 Figure 2. Illustration of f ecal indicator organism sources for inland waters .................................. 8 Figure 3. Paired qPCR and Culture Enterococcus Data: Huntington Beach: 8 AM Samples Only ...............................................................................................................................40 Figure 4. Paired qPCR and Culture Enterococcus Data: Huntington Beach: 3 PM Samples Only ...............................................................................................................................40

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### Abbreviations and Acronyms

AWQC ambient water quality criteria BMP best management practice BEACH Act Beaches Environmental Assessment and Coastal Health Act CFU colony forming unit CPSP Critical Path Science Plan CSO combined sewer overflow CWA Clean Water Act DNA deoxyribonucleic acid EPA U.S. Environmental Protection Agency EC European Commission FIB fecal indicator bacteria GI gastrointestinal MF membrane filtration MPN most probable number MST microbial source tracking NEEAR National Epidemiological and Envi ronmental Assessment of Recreational Water Study NPDES National Pollutant Discharge Elimination System PC prospective cohort PCR polymerase chain reaction POTW publicly-owned treatment works QMRA quantitative microbial risk assessment qPCR quantitative polymerase chain reaction RCT randomized control trial TMDL total maximum daily load(s) UV ultraviolet (light) VBNC viable but not culturable WERF Water Environment Research Foundation WHO World Health Organization WWTP wastewater treatment plant

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### Executive Summary
This report evaluates the applicability of NEEAR Great Lakes data to inland waters and assesses the similarities and differences between coastal fre shwaters and inland freshwaters to establish if there are significant differences to justify additional stud ies to support applicab ility of criteria to inland waters. Thirteen reports and peer-reviewed key publications pertaining to the establishment of new or revised recreational water quality criteria appropriate to U.S. inland waters were reviewed in this Inland Waters Summary Report . Focused observations and conclusions were extracted and organized into se veral finding categories in the table below. The reports and articles provide (1) overall assessmen ts on whether criteria developed on the basis of studies of coastal waters (marine/estuarine a nd Great Lakes waters) ar e applicable to inland waters; (2) findings concerning differences in the microbial ecology, fate, and transport of indicators in inland and coastal waters; and (3) discussions of likely diffe rences in implementing new or revised criteria to inland and coastal wa ters, including indicator detection and monitoring schemes. Current ongoing and future research EPA is pursuing and planning is also addressed in individual report sections, along with the sugge stions provided by each report’s authors. Major findings as reported in the reports for each of th e categories are summarized below (not in order of importance). Note that the ta ble presents the major findings as proposed by the study authors. The findings differ in the degree to which they ar e supported either through data or citations of other studies. In some cases, the findings might reflect a conclusion drawn by the study authors based on best scientific judgment when data was insufficient to offer a complete scientific assessment.
Finding category Conclusions and observations Fecal pollution source is the main driver of health risk at inland and coastal sites, before specific setting (e.g. phy sical and biological processes). This observation is consistent with findi ngs from quantitative microbial risk assessment (QMRA) studies and the lim ited number of epidemiology studies conducted in both inland and coastal settings and for sites with different fecal pollution sources. Application of coastal water-based criteria to inland recreational waters is expected to result in sporadic, mild illness at rates no higher and probably lower than those experienced in Great Lakes/coastal waters.

Inland and Great Lakes/coastal waters might pose very different risks of severe diseases. Data and health effects relationships developed for Great Lakes waters that are primarily affected by publicly-owned treatment works (POTW) effluent are generally believed to be applicable to inland waters primarily affected by POTW effluent. Overall assessments pertaining to the extension of coastal water criteria to inland waters

Different studies interpret the state of t he science differently from each other. All the studies directly assessing the extension of criteria on the basis of epidemiology studies of POTW-impacted Great Lakes waters to inland waters note that there are no definitive epidemiology studies to support or preclude making the extension.

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Finding category Conclusions and observations Experts participating in the 2009 Wate r Environment Research Foundation (WERF) inland waters workshop speculat e that livestock and wildlife fecal pollution sources affect a greater proportion of inland waters than coastal waters. However, inland waters comprise a diverse set and individual sites might be affected by POTW, diffuse human pollution, livestock fecal pollution, or fecal pollution from wildlife. Fecal pollution sources discharge directly to inland waters, and fecal pollution undergoes less dilution in inland waters than in Great Lakes/coastal waters. Understanding that runoff and streamflow ar e highly variable, the result of less dilution capacity in inland waters results in higher densities of pathogens in inland waters under some flow conditions and higher risk of disease from exposure to inland waters. Reported ranges of indicator densities in inland and Great Lakes/coastal waters are comparable. While relatively few data are available for systematic comparison of response of coastal and inland sites to rain events, it is likely that indicator variability within storm events is greater for inland waters than for coastal waters. Indicator decay rates in Great Lakes and inland waters fall within comparable ranges.
Escherichia coli growth has been observed in water columns and sediments of both inland and coastal waters. Enterococcus growth has also been observed in soils and sediments, though fewer studies have assessed Enterococcus potential, and differences in growth in inland and coastal settings cannot be assessed. Growth might be more likely in sediments and soils of inland waters than in those of coastal waters. Resuspension mechanics of sediment and soil-associated indicators and pathogens differ for coastal and inland waters. Resuspension might be more important in inland waters because turbulent shear at the sediment-water interface results in large loads of suspended organisms and particles and because dilution is lower than that of resuspended indicator organisms at coastal sites. Differences in occurrences, fate, and transport of indicators in inland and coastal waters

Quantitative polymerase chai n reaction (qPCR) and cultural enumerations of indicator organisms tend to be better correlated for fresh fecal material and very poorly correlated for aged fecal pollution, indicators that have been subjected to sunlight, and chlorinated waters. Inland waters are more widely dispersed geographically and cover more territory overall (as stream miles) than do coastal waters (as coastal miles). Inland waters typically have use patterns different from developed coastal beaches. Monitoring for such inland waters likely entails assessing water quality on the basis of fewer samples. For inland waters, the ratio of qPCR counts of indicators to culture counts of indicators likely differs from that typica l of coastal sites because of differences in age of fecal pollution, presence and c oncentration of chlorinated secondary effluent, and exposure of fecal pollution to solar radiation. Findings related to implementation of criteria

Standardized sanitary survey tools should be developed for both inland and coastal waters. Completion of sanitary surveys should be an integral component in development of monitoring schemes.

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### 1. Introduction

1.1. Objective
New or revised ambient water quality criteria (AWQC) for the protection of primary contact recreation are likely to be based on health eff ects observed in epidemiology studies of coastal waters. A decision to extend or not extend those criteria to inland wate rs must be supported by analysis of the scien ce of inland and coastal waters as well as analysis of practical concerns, such as likely differences in use and sampling of inla nd and coastal waters. Recognizing those needs, the U.S. Environmental Protection Agency’s (EPA’s) Critical Path Science Plan for the Development of New or Revised Criteria for Recreational Waters (the CPSP; USEPA 2007) proposed studies to develop the information n ecessary to inform ex trapolation of AWQC. This report summarizes the knowledge that EPA collected and produced in response to the CPSP based on a review of thirteen reports and key research articles. Each report and article is summarized, and the key concepts, results, or conc lusions are highlighted. Three reports and five publications were pooled to condense all aspects of monitoring methodology relevant to this report’s subject matter. Findings from these report s will be used to develop analyses that will support a decision of whether and how AWQC can be extended to inland waters on the basis of epidemiology studies conducted as part of EPA’ s National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study, which include the Great Lakes epidemiology studies. Stakeholder and scientific community concerns In stakeholder meetings and in the scientific li terature, concerns have been expressed regarding both the scientific and practical considerations of extending AWQC developed for coastal waters to inland waters. Concerns about the science of extending AWQC ge nerally relate to perceptions or findings that indicator organi sms perform differently in inland waters than in coastal waters. In this report, performance is taken as (1) the relationship between indicator density and the observed rates of illness in a particular type of wa ter and (2) the fate and transport of indicators relative to pathogens and loads from specific feca l pollution sources. Practical concerns relate to the development of monitoring schemes and interp retation of sample results for inland waters. Paramount among those concerns is the interpretation for all waters of indicat or densities from samples collected during and immediately afte r precipitation (especia lly rain) events (NRC 2004). To develop context for this report, PowerPoint presentations from stakeholder meetings and correspondence between EPA and st akeholders were reviewed. Questions drawn from those communications are presented below. The que stions provide a focus for reviewing the documents presented in this study. Some of these questions do not relate to differences between inland and coastal waters per se. Questions related to the science of indicators

 How does the persistence of fecal indicator organisms in typical inland soils and sediments differ from that in coastal soils and sediments?

 How does the fecal pollution source affect th e performance of indicators in inland and coastal waters? What are altern ative criteria appropriate for fecal pollution sources other than publicly owned treatment works (POTW) effluent?

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 Does indicator performance differ in fl owing waters, impounded inland and coastal waters?

 Does clim ate (i.e., tropical, temperate, and subtropical) affect indicator performance?

 If E. coli is a better indicator of feca l pollution in freshwaters, could Enterococcus criteria be equally protective of human heal th for inland and coastal waters? Questions related to impl ementing indicator-based criteria to inland waters

 How do indicator-based AWQC for beaches, where monitoring occurs frequently, apply to inland waters, where recreational use is likely intermittent and where water quality assessments are based on much less frequent sampling?

 What criterion relates to long- term water quality and eliminates undue influence of spikes in fecal pollution and indicator density?

 Can/should criteria be relaxe d during extreme-flow events?

 How will culture methods be integrated with new or revised criteria? Is a rapid method needed for inland waters, partic ularly those with low use?

1.2. Methodology

1.2.1. Documents reviewed Eight studies were originally propos ed for review for this report. Of those, seven were available for review during preparation of the report, and one further study was added. The added study (number 2 below) is a condensed version of the Water Environmental Research Foundation (WERF) experts workshop report (number 1 belo w). The condensed version of the workshop proceedings was reviewed separately because it ha s been distributed widely in the scientific community (it is a peer-reviewed publication) and because it makes more definitive statements than the full workshop report. Key documents reviewed for this report were as follows:
1. Final Report on the Experts Scientific Work shop on Critical Research and Science Needs for the Development of Recreational Wate r Quality Criteria for Inland Waters (WERF 2009) 2. Meeting Report: Knowledge Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dorevitch et al. 2010) 3. Final report Literature Review of Assessment of the Applicability of Existing Epidemiology Data to Inland Waters (USEPA 2010a) 4. Final report Sampling and Consideration of Vari ability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b)
5. Interim draft report Comparison of Different Methodologies for the Enumeration of Fecal Indicator Organisms (USEPA 2010c) 6. Draft final report Quantification of Pathogens and Sour ces of Microbia l Indicators for QMRA in Recreational Waters (WERF 2010a)
7. Final report Comparative Evaluation of Molecular and Culture Methods for Fecal Indicator Bacteria for use in Inland Recreational Waters (WERF 2010b)

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8. Results of the Single-Laboratory Valida tion of EPA Method A for Enterococci and Method B for Bacteroidales in Water s by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay (USEPA 2010d) In addition, the following five ke y articles were selected and revi ewed for their direct relevance to the subject matter:
9. “Covariation and Photoinactivation of Traditional and Novel Indicator Organisms and Human Viruses at a Sewage-Impacted Marine Beach” (Boehm et al. 2009)
10. “Persistence of Nucleic Acid Markers of Health-Relevant Organisms in Seawater Microcosms” (Walters et al. 2009)
11. “Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide” (Bae and Wuertz 2009)
12. “Linking Non-Culturable (qPCR) and Cu lturable Enterococci Densities with Hydrometeorological Conditions” ( Byappanahalli et al. 2010)
13. “A Cross Comparison of QPCR to Agar-Bas ed or Defined Substrate Methods for Determination of Escherichia coli and Enterococci in Municipal Water Quality Monitoring Programs” (Lavender and Kinzelman 2009) Note that five out of eight reports (#1 to 4 and 6) are discussed individually in this report, while the other reports (#5, 7, and 8), as well as the fi ve published research arti cles (#9 to 13), are discussed together in Section 2.3 to condens e all aspects of methodology performance with respect to applicability of criteria to inland waters.

1.2.2. Topics featured in document review The stakeholder questions presented in Section 1.1.2 and the physical differences between inland and coastal waters presented in Section 1.3 provide topic areas that were emphasized for review and synthesis from the key reports and articles. Those areas can be divi ded into topics that inform the performance of indicators and topics re lated to differences in implementing criteria in inland and coastal waters. Topics related to the performance of indicators in inland and coastal wa ters include findings on the intrinsic physical, biological, and hydrologic differences betwee n inland and coastal waters; findings related to the incidence of pathogens or the association of illness in inland and coastal waters; and findings on differences in method performance for inland and coastal waters. Topics related to implementing criteria relate to differences in the content of sanitary surveys for inland and coastal waters, practical constraints causing inland water monitoring plans to differ from those for coastal waters, and differences in how water quality sample results are interpreted.
Section 1.3 describes the features of inland and co astal waters with the potential to result in differences in the association of indicator levels with health effects. A thorough exploration of the differences in criteria for inland and co astal waters entails re view of epidemiology, hydrodynamics, fecal pollution sources, use patter ns, and performance of indicator measurement techniques. The reports and research articles reviewed herein were selected according to their coverage of all those subject areas. The specifi c factors pertinent to extending coastal water criteria to inland waters review ed in each study are presented in Table 1

.

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Table 1. Key areas that the reviewed reports and publications used to compare and contrast Great Lakes and inland waters
Report/publication Inland vs. coastal water factors addressed Final Report on the Experts Scientific Workshop on Critical Research and Science Needs for the Development of Recreational Water Quality Criteria for Inland Waters (WERF 2009) Epidemiology studies of inland and coastal sites; differences related to hydrology and resuspension; fecal source differences; differences related to performance of detection techniques; monitoring strategies for inland and coastal sites; data gaps and research prioritization Meeting Report: Knowledge Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dorevitch et al. 2010) Epidemiology studies of inland and coastal sites; differences related to hydrology and resuspension; fecal source differences; data gaps and research prioritization
Literature Review of Assessment of the Applicability of Existing Epidemiology Data to Inland Waters (USEPA 2010a) Epidemiology and watershed-scale studies of inland and coastal sites; differences related to hydrology and resuspension; fate and transport characteristics of indicators in coastal and inland settings Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b) The hydrology of inland and coastal sites; variability in indicator density at inland and coastal sites; sampling schemes and their association with inland and coastal sites Comparison of Different Methodologies for the enumeration of Fecal Indicator Organisms (USEPA 2010c) Differences in performance of enumeration techniques; identification of site features impacting comparison of qPCR and culture counts of fecal indicator organisms Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters (WERF 2010a) Performance of enumeration techniques for different water matrices, indicators and settings; use of water quality data in a quantitative microbial risk assessment (QMRA) framework for assessing health risks (in the absence of epidemiology studies) Comparative Evaluation of Molecular and Culture Methods for Fecal Indicator Bacteria for use in Inland Recreational Waters (WERF 2010b) Performance of qPCR enumeration of fecal indicators for waters from different settings and for different laboratories; evaluation of the uncertainty of qPCR in enumeration of fecal indicator bacteria Single Lab Validation Study of Enterococcus qPCR and Bacteroidales qPCR (USEPA 2010d) Method performance of qPCR in a variety of fresh and marine water settings

“Covariation and Photoinactivation of Traditional and Novel Indicator Organisms and Human Viruses at a Sewage-Impacted Marine Beach” (Boehm et al. 2009) Impact of photoinactivation on method performance for fecal indicator bacteria (FIB) monitoring

“Persistence of Nucleic Acid Markers of Health-Relevant Organisms in Seawater Microcosms” (Walters et al. 2009) Impact of seawater qualit y and environmental factors on FIB DNA persistence in marine settings

“Discrimination of Viable and Dead Fecal Bacteroidales Bacter ia by Quantitative PCR with Propidium Monoazide” (Bae and Wuertz 2009) Comparison of method performance and significance of FIB viability for qPCR and culture- based assays in marine settings

“Linking Non-Culturable (qPCR) and Culturable Enterococci Densities with Hydrometeorological Conditions” ( Byappanahalli et al. 2010) Impact of hydrometeorological factors on method performance for FIB monitoring

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Report/publication Inland vs. coastal water factors addressed“A Cross Comparison of QPCR to AgarBased or Defined Substrate Methods for Determination of Escherichia coli and Enterococci in Municipal Water Quality Monitoring Programs” (Lavender and Kinzelman 2009) Comparison of method performance for qPCR and culturebased assays in surface waters, municipal stormwater and wastewater

1.3. General descriptions of inland flowi ng, inland impounded, and Great Lakes coastal settings
Inland waters are waterbodies that are not coastal recreational waters as defined by th e Clean Water Act (CWA). They are typically freshwater but can include some saltwater (estuarine) waterbodies (e.g., streams with tidal influences). They include flowing (rivers and streams) and impounded (lakes and reservoirs) wa terbodies, but not the Great Lakes, which are defined as coastal waters under the Beaches Environmenta l Assessment and Coasta l Health (BEACH) Act of 2000. This section provides qualitative comparisons of coastal and inland waters. It is intended as background material and is included to help r eaders understand and inte rpret findings of the studies reviewed. Two types of comparisons are provided. In Section 1.3.1, the physical, hydrological, and biological differences in co astal and inland sites are described. Such differences influence indicator fate and transport and the association of in dicators with specific fecal pollution sources. In Section 1.3.2, the findings of epidemiology studies of inland waters are reviewed. Epidemiology studies provide the best indication of the risks associated with recreation in inland and coastal waters and association of in land sites with specific fecal pollution sources and pathogens.

1.3.1. Qualitative comparison of indicator processes in inland and coastal waters With the understanding that inla nd and coastal waters are divers e, generalizations about these waters are provided in this secti on as an introduction to the findi ngs of the studies reviewed for this report. Intrinsic physical differences between inland and coastal settings include the following:

 the ways in which the sites ar e loaded with fecal pollution;

 the mechanisms by which indicators ar e advected into and out of sites;

 hydrograph and indicator organism de nsity responses to rain events;

 the locations and mechanisms important in resuspension of se diment indicators;

 the dilution of fecal pollution loads; and

 average insolation (average incident so lar radiation per wa ter surface area).
Those differences are illustrated in Figure 1 and Figure 2 and are described below. Other important differences between recreational si tes include the predominant f ecal pollution source(s) and the frequency of use for recreation. Those differences are not intrinsic to inland and coastal waters, and no data sources providi ng means for quantitative assessment of their importance were reviewed for this report.

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Figure 1. Illustration of fecal indicator organism sources for coastal waters

Figure 2. Illustration of fecal indicator organism sources for inland waters

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Inland and coastal waters receive f ecal indicator bacteria (FIB) from point sources, diffuse sources including nonpoint sources, direct depositi on (e.g., gulls or cattle in a stream), and resuspension of FIB in sediments or overbanks. It is likely that the ov erall predominant sources in inland waters differ from those in coastal wate rs. However, for a specific stream compared to a specific coastal water, the sources might be th e same or very similar. Human fecal pollution and nonhuman fecal pollution are associated with different types of pathogens. Those pathogens, in turn, pose different hazards, and the exposur e necessary to observe a given response (e.g., adverse health effect) in the exposed population also differs. FIB loading and hydrodynamics in POTW-impacte d inland and coastal waters are generally similar. POTW discharges to both inland and co astal sites are relatively steady and have FIB densities that are variable but not dependent on whether a site is coastal or inland. Loading from non-POTW sources might differ significantly fo r inland and coastal si tes. Non-POTW fecal pollution is loaded to receiving waters primar ily during and immediately following rain events. Because non-POTW sources are usually in closer proximity to inland waters than to coastal waters and because the volumes of inland waters r eceiving fecal pollution are lower than those of coastal waters, fecal pollu tion in inland waters is expected to be generally less dilute than that in coastal waters. Because dose-re sponse functions for individu al pathogens are not linear, differences in dilution in coastal and inland wate rs might result in differences in incidence of illness typical of the two sites. Differences in in cidence of illness are particularly important for pathogens such as E. coli O157:H7, which can result in very serious illness. Inland and coastal waters differ in typical soils a nd sediments, the ratio of the sediment area to the water volume, and the mechanisms most respons ible for resuspension of sediment-associated indicator organisms. Together , those factors can cause diffe rences in the abundance of resuspended indicator organisms in the two set tings. Because resuspended indicator organisms are not associated with a specific fecal pollution source, they are not good indicators of fecal pollution sources and might confound interpretation of microbial water qu ality from indicator density measurements. None of the studies review ed for this report sought to associate inland or coastal waters with specific soil and sediment t ypes or to compare and contrast the extent to which growth is likely in either setting, although one report (WERF 2009) asse rts that growth is more likely in the soils and sediments of inland wa ters and at the water-sediment interface. In inland waters, resuspension is by means of turbulen t shear at the stream bottom, while in coastal waters, resuspension is primarily due to wave action. The net resuspende d indicator load at a given site is a function of both the abundance of sediment-associa ted indicators and the processes by which free or particle-associated organisms are drawn from the sediments. Although a quantitative comparison of resuspen sion in inland and coastal waters is not possible, it is likely that resuspension is different in inland and coas tal waters because of the different sediments typical of the sites and the very different mechan isms that cause resuspen sion in the two settings. No information in the documents reviewed indi cates whether the differe nces would affect the relationship between indicator density and a dverse health outcomes in exposed persons.

1.3.2. Brief review of epidemiology studies conducted for inland waters At present, epidemiology studies provide the be st means for comparing recreation-associated risks in inland and coastal sites. However, due to the complex and variable processes associated with the transport of fecal pollution and indicators to recreational sites, only a few studies have demonstrated a correlation between indicators an d health risks via modeling. Recently published

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QMRA studies indicate the potential for connect ing indicator densities and health effects via modeling (Schoen and Ashbolt 2010 ; Soller et el. 2010a, 2010b).
As context for the studies reviewed in this doc um ent, a brief review of epidemiology studies conducted at inland sites is pres ented below. These studies were conducted for different purposes and with different methodologies, and these diffe rences hamper direct comparison of their results. In general, the studies have resulted in two types of data. First, they produce a measure of the increase in odds (or likelihood) of some health endpoint (usually gastrointestinal [GI] illness) for swimmers as compared to non-swimmers. Such an odds ratio does not relate to the water quality or indicator level. Second, some of the studies produce an associati on of the incidence of health endpoints with the level (density) of indi cators to which swimmers were exposed. In some cases, no statistically significant a ssociation is observed. That lack of association could relate to a lack of association between the indicator and a specific fecal pollution sour ce or could relate to the epidemiology study design. In a limited numbe r of cases, studies have produced health effects curves predicting the incidence of illness as a function of indicator density. To date, eight sets of U.S. and international epidemiological studies ha ve been conducted to evaluate the association of swimming in inland surface waters with the incidence of GI illness (Stevenson 1953; Dufour 1984; Seyfried et al. 1985a, 1985b; Ferley et al. 1989; Calderon 1991; Wiedenmann et al. 2006; European Commissi on [EC] 2009a, 2009b [referred to as Epibathe studies]; Marion et al. 2010). They are grouped below as follows:

 investigation of (presumably) POTW-imp acted inland sites (Dufour 1984; Weidenmann et al. 2006; EC 2009a, 2009b; Marion et al. 2010);

 investigation of untreated domestic sewage a ffected inland flowing wa ters (Ferley et al. 1989)

 investigation of unspecified inland waters 1 with unspecified fecal sources (Seyfried et al., 1985a, 1985b)

 investigation of flowing a nd impounded inland waters (Duf our 1984; Stevenson et al. 1953; Weidenmann et al. 2006; EC 2009a, 2009b; Marion et al. 2010);

 investigation of avian and wildlife affected sites (Calderon 1991);

 use of randomized control trial study design (Weidenmann et al. 2006; EC 2009a, 2009b);

 use of prospective cohort study design (Cal deron 1991; Dufour 1984; Marion et al. 2010; Stevenson et al. 1953; Seyf ried et al. 1985a, 1985b); and

 use of retrospective cohort study design (Ferley et al. 1989). In all of those epidemiology studi es, statistically significant differe nces in the incidence of GI illness (and in some cases other health endpoints such as respiratory illness) were observed between swimmers and non-swimmers. In studies of POTW-impacted waters, three studies identified an association of the increased incide nce of GI illness with indicator density (Dufour

1 Seyfried et al. 1985b describes the locations of the study as being conducted “at various Ontario lakes.” It unclear whether or not this may include Lake Ontario, which would be considered a Great Lake and not an inland water for the purposes of applying recreational criteria.

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1984; Seyfried et al. 1985b; Mari on et al. 2010), and one study re lated indicator density to GI illness risk via estimation of a no adverse eff ect level of indicator organism density (Wiedenmann et al. 2006). No such relationships have been proposed for livestock and wildlifeaffected inland waters, although two studies (S eyfried et al. 1985a, 1985b; Wiedenmann et al. 2006) pooled illness rates and indicator densitie s without regard to fecal pollution source. Calderon (1991) found no association between indi cator level and incidence of GI illness in recreational waters with exclusivel y nonhuman impacts for the indicators E. coli
, Enterococcus
, fecal coliforms, and Staphylococcus —despite higher incidence of illness among swimmers than non-swimmers. The Epibathe (EC 2009a, 2009b) study of four beach es on inland waters in Hungary is difficult to compare with the other epidemiology studies because the fecal pollution sources for the study sites are not characterized and they employed a randomized cont rol trial (RCT) study design. At present, results from PC and RCT study desi gns cannot be used interchangeably, and no techniques currently exist for c onverting their statistical outputs to allow meaningful quantitative comparisons across study designs. Based on these studies, E. coli (as measured by the culture-bas ed method) seems a better index of GI illness in swimmers using fresh recreational waters than Enterococcus (as measured by the culture-based method), which appears to be the best predictor of such symptoms in marine waters (see also reviews by Pruss 1998; Wade et al. 2003; Zmirou et al. 2003).

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### 2. Documents review
This section is comprised of a summary of findi ngs (Tables 2 through 7) an d a detailed review of the documents (Section 2.2). Five ou t of eight reports (#1 to 4 a nd 6) are discussed individually hereafter (Sections 2.2.1 to 2.2.5). The remaining reports (#5, 7, and 8), as well as the five published research articles (#9 to 13), are discussed together in S ection 2.3 to compile aspects of methodology performance with resp ect to applicability of crit eria to inland waters.

2.1. Summary of findings

Table 2. Study purpose, methodology, findings and limitations for the Final Report on the Experts Scientific Workshop on Critical Research and Science Needs for the Development of Recreational Water Quality Criteria for Inland Waters (WERF 2009)
Study purpose(s) 1. Determine if or how marine co astal and Great Lakes recreational water research can be extrapolated to apply to inland waters 2. Identify additional research that could aid in the development of water quality criteria applicable to inland waters in both the near and the longer term Methodology Findings are drawn from results of an experts workshop. Five major topic areas— indicators and pathogens, health effects, water matrix, sources, and implementation realities—were evaluated by separate teams of experts. Teams were asked to assess the state of the sc ience in their topic area and use that information to assess the applicability of water quality criteria developed using Great Lakes epidemiology studies for inland waters. Experts also identified and prioritized data gaps, short-term, and long-term research needs. Major findings Indicators and pathogens group findings

 The group found that insufficient evidence exists for direct extrapolation of criteria based on Great Lakes studies for use for inland waters. The group speculated that swimmers in POTW-impacted Great Lakes and inland waters likely face similar risks but that the pr esence of non-fecal indicator sources at sites could result in differences in the meaning of indicator levels at inland and coastal sites.

 Inland waters are generally more diverse, shallower (greater bacterial redistribution), and better suited to tree growth (creating additional shading and protection from sunlight inac tivation) than coastal waters.

 Soils and physical conditions in inland waters appear more conducive to extraenteric indicator growth than those of coastal waters. Health effects group findings

 The group recognized the im perative that new or revised criteria be developed and the low likelihood that additional epidemiology studies will be conducted in time for use in developing new or revised criteria. Under those circumstances, the group generally supported the positi on that AWQC derived from Great Lakes studies would likely be protective of public health at inland waters.

 Epidemiology studies indicate that fecal source is more important than water type (marine or fresh) or setting (inland vs. coastal) in determining health effects related to swimming.

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Water matrix group findings

 Inland water conditions that appear to favor indicator occurrence, growth and resuspension from sediments were thought to be the following: (1) higher ratio of sediment-water interface area to wate r volume for inland waters, (2) finer sediment sizes typical in inland waters, (3) greater presence of wetting and drying areas (per volume of water) at inland sites, and (4) relatively high velocities and higher potential for resuspension at inland water sediment-water interfaces.

 Models appear to be the best avenue for exploring the impact of setting on indicator and pathogen occurrence. Models that should be developed for this purpose are regression models, mechanistic (watershed) models, and QMRA. Sources group findings

 Even within a particular fecal pollution source (human treated, human nonpoint, livestock, companion animal, livestock) t he relationship between indicator level and health effects for different fecal pollutions sources differs with the level of treatment of the waste, the proximity of the waste to the receiving water, the prevalence and abundance of pathogens in the fecal pollution, and the persistence of pathogens in the fecal polluti on source relative to the persistence of indicators.

 Inland waters are believed to be in closer proximity to sources and more influenced by on-site wastewater treat ment facilities than coastal waters. Implementation realities group findings

 Approaches that have been used or co uld be used to introduce flexibility into implementation of new or revised water quality criteria are the following:

o different criteria for beaches with different use patterns;

o discounting water quality measurements taken after rain events in concert with implementing risk management strategies for protecting human health;

o using multiple metrics (sample anal ysis approaches and statistical measures) for assessing water quality; and

o using temporary or perma nent site-specific criteria. Priority short- and longer-term research recommendations

 Short-term

o Identify and quantify human pathogens in animal feces

o Examine relationships between qPCR and culture-based FIB

o Optimize and anchor QMRA models to observed health effects data obtained from epidemiologic studies

o Develop QMRA tools for implementation of new AWQC

 Longer-term

o Characterize fate and transport of animal pathogens in relation to indicators Conduct epidemiology studies in inland waters

Limitations In many instances, findings were ba sed on the best estimates or hypotheses of the experts and could not be assessed against actual data because they were asked to use their judgment in the absence of s pecific data. The major limitation/data gap identified in the WERF report was the scar city of epidemiology studies of inland waters.

13Inland Waters Summary Report November 2010

Table 3. Study purpose, methodology, findings and limitations for Meeting Report: Knowledge Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dorevitch et al. 2010)
Study purpose(s) Summarize the WERF Inland Waters Workshop Results (WERF 2009) in a peerreviewed, widely distributed publication Methodology Principal investigators distilled the experts workshop report into a peer-reviewed publication and added contextual materials Major findings  The physical and biological processes that are most likely to cause FIB to relate to different risks in inland and coastal waters are the following:

o differences in source types, suites of potential pathogens in the source materials, and severity of the di seases associated with the pathogens typical of inland and coastal waters;

o closer proximity of inland waters to fecal pollution sources and lower dilution of fecal pollution in inland waters than in coastal waters;

o differences in the potential for FIB growth in sediments typical of inland and coastal waters; and

o differences in resuspension rates of sediment-associated indicators and pathogens.

 Application of coastal water-based criteria to inland waters is expected to result in sporadic, mild illnesses at rates no higher and possibly lower than those experienced in coastal waters. However, coastal and inland waters might pose very different risks of severe illness.

 Near-term research activities that will improve the understanding of indicator performance in inland waters are the following:

o characterizing the spatiotemporal variab ility of indicator in inland waters by assessing the literature, collecting field data, and/or developing mechanistic models of indicator and pathogen fate and transport;

o developing and standardizing a sanita ry survey tool to use for inland waters;

o anchoring QMRA using data and relationships from epidemiological studies;

o developing a database describing the setting-specific relationships between molecular- and culture-based determinations of indicator densities; and

o evaluating the viability of regression and mechanistic models for predicting indicator density and water quality for inland waters.

Limitations This study is subject to the same limitations as WERF (2009)

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Table 4. Study purpose, methodology, findings and limitations for Literature Review of Assessment of the Applicability of Existi ng Epidemiology Data to Inland Waters (USEPA 2010a)
Study purpose(s) To assess and compare the performance of FIB in inland and coastal settings Methodology A comprehensive literature survey was conducted to assemble studies reporting the following:

 epidemiology studies of inland waters;

 occurrence of indicators in inland and coastal waters;

 persistence of indicators in inland and coastal waters; and

 co-occurrence of indicators and pa thogens in inland and coastal waters. Reports were reviewed, synthesized, and used in a qualitative comparison of inland and coastal sites Major findings  The data and relationships developed for Great Lakes waters studies, which are affected primarily by POTW effluents, c an be applied to inland waters that are also affected primarily by POTW effluents.

 For inland waters that are affected pr edominantly by sources other than POTW effluent, the available science is not suffi cient to support the extension of the relationships developed in the Great Lak es because of potentially important differences in fecal sources and hydrodynamics.

 The reported occurrence ranges of FIB for coastal and inland waters are not substantially different; indicator densities are widely variable in both settings.

 Reported decay rates for inland and coastal freshwaters are not substantially different. In all cases, predation, inso lation, and the presence of sediments are the most important determinants of indicator organism persistence.

 Growth of E. coli has been reported in sediments of both inland and coastal waters; insufficient data were obtained to assess the growth potential for Enterococcus in inland and coastal waters.

 As anticipated, no consistent correlations between indicator and pathogen densities were observed, irrespective of setting.

Limitations The study was based on a literat ure survey and significant data gaps exist, including the following:

 epidemiology studies of agriculture-affected coastal and inland waters;

 quantitative assessments of the relative importance of resuspension in inland and coastal settings;

 quantitative assessment of the prevalence of different fecal pollution source types in inland and coastal waters; and

 large-scale studies of indicator densities as determined by molecular methods and other alternatives to cult ure-based membrane filtration.

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Table 5. Study purpose, methodology, major findings and limitations for Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b)
Study purpose(s)  Describe and quantify temporal and spatial variability of FIB density at inland and coastal sites and the implications of variability for the design of sampling plans Methodology  A comprehensive literature survey was conducted to assemble physical and biological processes at all relevant temporal and spatial scales, and to assess FIB variability for coastal and inland waters. Major findings  Temporal variability: discrete events (e.g ., precipitation events, CSOs) produce the greatest impact

o Other factors affecting temporal variability include the following: diurnal, tidal, seasonal, and short-time-scale variability

 Spatial variability: sample depth and along-stream sampling have the greatest impact for coastal and inland sites, respec tively. Other factors affecting spatial variability are the following:

o For coastal sites: site features, along shore variations, depth below surface at which samples are collected

o For inland sites: depth below surface at which samples are collected, and cross-stream variations

 Sanitary surveys and pilot monitori ng are important components in the development of beach monitoring plans. Thes e activities establish the likely fecal pollution sources and allow estimation of spatial variability in indicator density.

 Monitoring considerations

o Pilot monitoring studies and sanitary surveys are the best tools available for collecting data required to devel op effective site-specific monitoring plans.

o Where: area allowing most efficient characterization

o When: morning samples most conservative; sampling frequency is sitespecific, and providing best correl ation between qPCR and culture-based results

o How: approaches for choice of location and number of samples based on site-specific constraints and historical data

Limitations  The study was based on a literature su rvey and limited to the studies reviewed.

 Most of the studies reviewed reported re sults from studies of limited duration or

spatial extent. Extrapolating data from those studies to the diverse set of recreational waters might not be warranted.

 Few data are available to allow char acterization of variability in qPCR FIB

estimates.

 No studies assembled as part of the literature survey proposed protocols for

discounting or otherwise accounting for elevated indicator organism density counts during rain events; flexibility in accounting for rain events was a stated concern among stakeholders

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Table 6. Purpose, methodology, major findings, and limitations of draft final report. Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters (WERF 2010a)
Purpose(s)  Identify and address data gaps pertaining to loadings and concentrations of waterborne pathogens and indicators in di scharges to recreational waters that are affected by fecal pollution

 Compile, analyze and synthesize the data in QMRA models and waterborne risk management frameworks Methodology  Compilation of existing pathogen data and collection of a new, comprehensive suite of bacteria, protozoa, and virus me asurements for a variety of dischargesof-concern to recreational waters acro ss the U.S. These data can serve as inputs for QMRA models.

 Collection of additional pathogen and indicator data through field studies and surveys of water and wastewater professionals.

 Evaluation of microbial source tracking tools, such as the source identifier bacteria Bacteroidales , for quantitative source apportionment and as a component of QMRA

 Critical review and analysis of QMRA as a risk analysis tool, determination of the potential risks associated with measured discharges-of-concern, and consideration of the role of QMRA for implementation of new recreational water quality criteria. Major findings  Data gaps pertaining to waterborne pathogens and indicators in fecally-impacted discharges to recreational waters were identified and filled by targeted monitoring campaigns in three geographic regions.

 QMRA analyses revealed norovirus as the most dominant health risk followed by rotavirus, regardless of setting (inland v. coastal).

 Norovirus and Enterococcus both had significant correlations with a number of pathogens in discharges.

 Using qPCR data on the fecal source identifier Bacteroidales a new model can predict the true amount of human fecal contamination in a water sample by relating a human-associated genetic marker to a universal assay for fecal sources. The model output can then be used to implement and evaluate management options intended to restore microbial water quality.

Limitations  Inhibition and, for select pathogens, poor or widely varying recoveries must be identified and overcome

 Weather conditions prevented some monitoring events

 Resource limitations did not allow for flow-weighted composite sampling (instead, grab sampling was used), but for each sample the timing of the sampling with respect to the hydrograph was estimated.

 The inability of molecular-based methods to distinguish between dead and viable cells is highlighted as a major limitation for risk assessment.

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Table 7. Purpose, methodology, major findings, and limitations of Methodology Performance and Relevance to Applicability of Criteria to Inland Waters (compilation of findings from multiple reports)
Purpose(s)  Describe important factors that in fluence enumeration method performance

 Describe relevant differences in method performance between culture-based and qPCR assays with respect to POTWs, solar inactivation, persistence and other environmental factors

 Review findings of studies comparing the relationship between qPCR and culture indicator densities for inland and coastal sites. Methodology  Review of relevant findings reports #5, 7, and 8, and peer-reviewed publications #9 through 13 (Section 1.2.1) Major findings  Accounting for bacterial viability and DNA persistence is an important consideration when comparing culture-ba sed methods with qPCR assays. This leads to the following:

o qPCR cell equivalent counts are cons istently greater than culture-based CFU inventories due to the comprehensive detection of all DNA by qPCR (commonly, the culturable fraction of cells is relatively marginal with respect to the total count), prompti ng the design of live-only qPCR assays, such as PMA-qPCR;

o accounting for different persistenc e in qPCR and culture targets when evaluating site water quality; and

o coastal site morning samples analyzed by qPCR and culture-based assays exhibit better correlations than samples collected in the afternoon (less photoinactivation).

 qPCR and culture-based results are well correlated at high densities but the correlation is lost at low density due to high uncertainty of the qPCR assay in this range and high variability in relationship between culture and qPCR density at low cell densities. One study review ed indicated better correlation between qPCR and culture counts in Lake Michigan waters than in other waters including inland lakes and rivers.

 Quantitative polymerase chain reaction (qPCR) and cultural enumerations of indicator organisms tend to be better correlated for fresh fecal material and very poorly correlated for aged fecal pollution, indicators that have been subjected to sunlight, and chlorinated waters.

 qPCR Enterococcus and qPCR Bacteroidales methods are suitable for multi-lab validation.

 Culture-based bacterial densities are st rongly reduced (2-5 orders of magnitude) through wastewater treatment processes, especially by secondary treatment and disinfection; qPCR inventories experien ce smaller reductions or remain unchanged. This is probably due to the strong impact of disinfection on culturable cells.

 qPCR results are not greatly affected by insolation while culture-based methods (e.g., membrane filtration [MF]) experience strong reduction by photoinactivation during sunlight exposure. If coastal recr eational waters receive greater fluxes per volume of solar radiation (due to higher surface area-to-volume ratio compared with streams), afternoon diffe rences in qPCR and culture counts for coastal waters are expected to be greater than for inland waters.

 Inhibition under specific conditions (e.g., high salinity) can potentially affect the qPCR assay and lead to lower counts. This inhibition differs among inland and coastal sites and is not an intrinsic feature of either site type.

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Limitations  Data and experience with qPCR are limited; no single epidemiological studyreviewed for this report has produced dose-response curves for both molecularand culture-based methods.

 Inhibition of the qPCR reaction likely differs among environmental waters and, at present, is not thoroughly characterized.

 qPCR assays vary from one study to another with respect to bacterial targets and primer sets used, highlighting the nee d for harmonization/standardization for comparison of molecular-based results.

 Finite number and variety of sites sampled.

2.2. Detailed reviews

2.2.1. Final Report on the Experts Scientific Workshop on Critical Research and Science Needs for the Development of Recreational Water Quality Criteria for Inland Waters (WERF 2009) In February 2009, EPA and WERF conducted the Inland Waters Expert Workshop with the following objectives:
1. Determine if or how marine coastal and Great Lakes recrea tional water research can be extrapolated to appl y to inland waters.
2. Identify additional near- and long-term resear ch that could aid in the development of water quality criteria applicable to inland waters. The workshop was organized around th e following five thematic areas:

 indicators and pathogens: biology, ecology, and methods;

 health risks: epidemiology and risk assessment;

 water matrix: hydrology, chem istry, geology, and modeling;

 sources: human vs. nonhuman and point vs. nonpoint; and

 implementation realities. Workshop participants were assigned to one of the five topic areas, and each topic area group produced a separate section for inclusion in the WERF report summarizing the workshop. Findings in each topic area are described below. Indicators and pathogens The indicators and pathogens group explored diff erences in water qualit y at inland and coastal sites and based their recommendations on those differences and the differences noted in epidemiology studies. The water quality differences noted by the group are summarized below.

 Coastal and inland water indicator dynamics are different. Coastal indicator dynamics are more stable and less variable than those of inland waters. That is because inland waters are more diverse than coastal waters and ha ve smaller volumes and less dilution potential than coastal waters.

 The banks of inland waters are more favorab le for tree growth, which could result in greater shading of inland wate rs than coastal waters. Such shading is significant because

19Inland Waters Summary Report November 2010

sunlight inactivation is a signifi cant factor in the extra-enteric pers istence of indicator organisms.

 Inland waters are generally more shallow than coastal waters. The group relates the difference to greater particle settling in inland waters than coastal waters. [Note that the authors of this summary re port question that assertion. Flows in inland waters are characterized by higher veloci ties than those observed in coastal waters. Those higher velocities can keep particles and particle-asso ciated bacteria suspe nded. When tributaries enter coastal waters, influent plumes disperse , velocities decrease, and particles tend to settle.]

 Inland waters are believed to be associated with soils and physica l conditions that are more conducive to FIB growth than the soil s and physical conditions associated with coastal waters. [Note that although the authors of this report find that assertion to be reasonable, it is important to note that grow th in coastal sediments and sands has been documented in many studies, and no studies de finitively support a greater propensity for FIB growth in the inland environm ent than the coastal environment.] After reviewing water quality and the dynamics of indicators in inland and coastal settings, the group directly addressed the use of water qual ity criteria based on Great Lakes epidemiology studies for inland waters. That gr oup’s assessment is that insufficient evidence exists for direct extrapolation of criteria from Great Lakes studies for use in inland waters. The group speculated that swimmers in POTW-impacted Great Lakes a nd inland waters likely face similar risks but that the presence of non-fecal indi cator sources at sites could resu lt in differences in the meaning of indicator levels at inland and coastal sites.
Health risks The group contrasted the results of epidemiological investigations conducted for the following types (setting) of water:

 fresh and marine waters,

 flowing and non-flowing waters, and

 waters with different fecal pollution sources. Comparisons of epidemiology studies for fresh and marine sites showed that indicator organism levels do not relate to the same levels at ei ther fresh and marine sites when culture-based methods are used for measuring indicator organi sm density. The comparison also identified fecal pollution source as more important than water type (marine or fresh) as a determinant of whether the indicator level is associated w ith observed adverse health effects. The group was unable to contrast studies for flowing and non-flowing waters because limitations in study designs or differences between studies we re too great to allow meaningful comparisons. Comparisons of epidemiology studies conducted for waters with different fecal pollutions sources were also hampered by differences in study designs and a rela tively small number of studies. In general, the group members hypothesized th at differences in fecal pollution source are likely more important than those in water quality and other features that differentiate inland and coastal sites.

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Weighing the findings from the epidemiology st udies described above, the group developed the following two positions:

 Position 1—applying the results from the Great Lakes studies to inland flowing waters is not supported by the scientific literature because directly comparable studies have not been conducted. It is unknown whether such application is under protective of public health.

 Position 2—applying the results from the Great Lakes studies to inland flowing waters is a reasonable step based on s upporting information in the liter ature and approaches taken worldwide and is unlikely to result in the underprotection of public health. The group recognized the need for new criteria to be developed and the low likelihood that additional epidemiology studies will be conducted in time for use in developing new or revised criteria. Under such circumstances, the group gene rally supported position 2, stating, “It is the opinion of the Health Risks Group that water quality criteria derived from Great Lakes studies would likely be protective of pub lic health at inland waters.”

Water matrix The group noted the importance of fecal pollu tion source, water physical and chemical properties, microbial ecology, and hydraulics in the association of indicator organisms with health effects. Considering those factors, the group focused on the potential for settling, regrowth, and resuspension of indi cators in inland waters, and the use of predictive models for evaluating differences between inland and coastal sites. Inland water conditions that appear to favor indicator occurrence, growth, and resuspension from sediments were thought to be (1) higher ratio of sediment-water inte rface area to water volume for inland waters, (2) finer sedi ment sizes typical in inland wa ters, (3) greater presence of wetting and drying areas (per volume of water), and (4) relatively hi gh velocities and higher potential for resuspension at sediment-water interfaces. The group acknowledged that additional research is required before the effects of those features on the loads of non-fecal indicators is known. The group discussed the use of predictive models for evaluating differences between coastal and inland waters. Three types of models can be us ed for systematic eval uation of differences between inland and coastal sites:

 regression models,

 mechanistic models, and

 QMRA models.
Each of those models is associated with lim itations. Regression models have, to date, been formulated on the basis of relatively limited se ts of water quality and physical condition data. That limits the applicability of the models for s ites outside those used for developing the model. Debate remains regarding the most appropriate independent variables, and it is possible that those variables differ between sites, further reducing the generality of regression models. Mechanistic models can be developed at many scales and including/excluding many processes. To allow comparison of inland and coastal wa ters, mechanistic models should include the following:

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 three-dimensional models of indicator transpor t fo r coastal sites, inclusive of wind-driven flows;

 accurate modeling of dilution, turbidity, and shading;

 association of inland sites with finer pa rticle sizes and grea ter association of microorganisms with particles; and

 higher organic content and potential for regrowth for inland sites. Although the Water Matrix Group di d not explicitly note it, the development of such models would require large amounts of data, additional data collection, and the simulation of multiple scenarios or development of techniques that would allow the genera lization of results of individual simulations. The group noted that QMRA modeling would have to overcome great uncerta inty in parameters of the risk models. To lend credibility to QMRA modeling activities, the group strongly recommended studies in which QMRA models are anchored to epidemiology study results. Once anchored, QMRA would provide an avenue for the investigation of phenomena for which other modeling activities are less suite d, including exploration of relativ e risks during events and of worst-case scenarios.
Sources The group noted that even within a particular fecal pollution source (i.e ., human treated, human nonpoint, livestock, companion animals, and livesto ck) the relationship between indicator level and health effects for different fecal pollution so urces differs with level of treatment of the waste—including the proximity of the waste to the receiving water, the prevalence and abundance of pathogens in the fecal pollution, an d the persistence of pa thogens in the fecal pollution source relative to the persistence of indicator organisms. The prevalence and abundance of both FIB and pathogens varies wide ly between fecal polluti on sources and within each fecal pollution source. The Sources Group stated that because the preval ence of on-site wastewater treatment (septic) systems, which they considered to be nonpoint sources of pollution, is much greater for inland waters than for coastal waters, these human f ecal pollution sources are more associated with inland waters than coastal waters. Note that septic systems differ wide ly in their design and operation and likely result in feca l pollution loads that vary wi dely. Additionally, runoff from urbanized areas with high proportions of imperv ious surface area differs from agricultural or rural areas, although the explicit implications of the differences between rural and urban runoff were not stated. The group related differences between inland and coastal water settings to differences in proximity of fecal pollution sour ces to receiving waters, differe nces in pathogen and indicator density and prevalence in source materials, di fferences in loading during rain events, and differences in land use. The group hypothesized that coastal waters are gene rally more associated with urbanized land use, higher impervious surf ace areas, and point and diffuse human pollution sources than inland waters. However, the group acknowledged that inland waters comprise a diverse group and that the associ ation of coastal and inland waters with specific sources of fecal pollution and characteri stics are hypotheses.

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The group concluded by stating that knowle dge gaps—particularly for the abundance, prevalence, and pathogenicity of pathogens in livestock and w ildlife wastes, are so profound and the inherent variability in nonpoint source-affected systems is so great that it could not assess whether water quality criteria based on Great La kes/coastal studies could be extrapolated to inland waters. Implementation realities The group focused on flexibilities that are pres ent in non-U.S. beach programs and might be considered for implementation with new or revi sed AWQC. Specific needs for flexibility should recognize the following factors/elements:

 water quality and risk vary dramatically with rain events;

 different fecal pollution sources pose different hazards; and

 the proportion of non-fecal, resuspended organisms varies with site type. The current AWQC (USEPA 1986) implementation provides flexibility via use of different monitoring schemes for assessing water at beaches with different levels of usage, and through provision of an off-ramp by which site-specific water quality criteria could be established on the basis of sanitary surveys and epidemiology stud ies. Additionally, states can designate specific classes of waterbodies or specific circumstances for different, scientifically defensible water quality standards. Such a designa tion could be made for waters known to be affected primarily by animal sources or for temporary changes in mi crobial water quality criteria following rain events. Flexibility is provided in Worl d Health Organization (2003) sta ndards by using sanitary survey findings in selecting water quality criteria a ppropriate for a specific site. Recreational sites without human fecal pollution sources and low bath er density are considered to have good water quality at indicator densities hi gher than those for sites with known human fecal impacts or high bather densities. European Union bathing water quality standard s (EP/CEU 2006) provide flexibility through discounting of samples collected during short-term pollution events. Within this framework, up to 15 percent of the total samples at a site c ould be disregarded for classification purposes because of short-term pollution during the last assessment peri od. The Implementation Realities Group notes that inland waters are most influenced by wet weather events because they are more closely associated with urbanized areas. Note that this assessment is somewhat at odds with that of the Sources Group. The Implementation Realities Group noted that b each sampling and water quality assessment are conducted in the context of other regulat ory programs, including NPDES permitting, 303(d)/TMDL use attainment assessments, and BEACH Act monitoring. Monitoring needs and realities differ for these program s and techniques and methods fo r assessing water quality also differ. Flexibility in the overall regulatory cont ext might be provided with the issuance of water quality criteria on the ba sis of multiple methods for counting indicator organisms, or through the provision of multiple statistical approaches for developing monitoring plans and assessing results of microbial water quality sampling.

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Research priorities After assessing the state of the science and knowledge gaps in each topic area, the expert groups developed prioritized lists of suggested rese arch activities intended to provide improved inform ation for development or extension of crite ria for inland waters. Th e priority short- and longer-term research activiti es proposed by the workshop part icipants is presented in Table 8
, and a comprehensive list is presented in Table 9

.

Table 8. Priority research activities from the 2009 Inland Waters Workshop (WERF 2009)

Time frame Research Identify and quantify human pathogens in animal feces Examine relationships between qPCR and culture-based FIB Short-term

Optimize and anchor QMRA models to observed health effects data obtained from epidemiology studies and develop QMRA t ools for implementation of new AWQC Characterize fate and transport of animal pathogens in relation to indicators Longer-term Conduct epidemiology studies in inland waters

Table 9. Comprehensive list of proposed resear ch activities from the 2009 Inland Waters Workshop (WERF 2009)
Group Short-term research priorities Longer-term research priorities

Indicators and Pathogens  Examine relationships between qPCR- and culture-based FIB and develop a database of results to date of other reliable potential new monitoring methods  Conduct studies on watershed assessment information to be used as input for site-specific water quality criteria

 Investigate the potential for speciation of enterococci to identify fecal-specific strains (preferably human) from environmental strains, and then apply results to future epidemiology studies

 Conduct epidemiology studies that take into account urban runoff and nonpoint sources of fecal contamination. Includethe use of culture-based and molecularbased analytical methods. Include sensitive populations (particularly children).

 Conduct epidemiology studies incorporating the measurement of pathogens of interest along with indicators to determine the correlations of these organisms and to better understand their associations with diseases at downstream recreational locations

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Group Short-term research priorities Longer-term research priorities
Health Effects  Optimize and anchor QMRA models to epidemiology studies

 Incorporate source characterization methods into ongoing epidemiology studies

 Optimize and standardize qPCR methods and enhance their interpretation

 Conduct meta-analysis of epidemiology studies by source  Conduct further epidemiology research to identify how much uncertainty exists in using the results of epidemiology studies conducted in Great Lakes and coastal settings to establish criteria for inland flowing waters

Water Matrix  Apply model-driven nowcasting or forecasting (or both) in current epidemiologic studies to determine a relationship of model variables to health outcome

 Further develop and test regression models in inland waters (at rivers with different morphologies from those in Ohio, Georgia, and Kansas)

 Focus on prior epidemiology studies to build models after the fact and compare the results from such retrospective models to the observed human health effects

 Include an in-depth evaluation of target microbe/pathogen sources and environmental conditions in epidemiology studies to evaluate the sources and parameters needed for coupling health outcomes with model output

 Evaluate reverse QMRA for itsapplicability in developing sitespecific criteria

 Apply data mining activities to identify data gaps that limit application of models and identify longer-term research goals  Use modeling approaches to characterize source impacts as an integral part of the design of all recreational water epidemiology studies

 Further develop and test regression models in all types of inland waters to clarify their applicability, limits, and future research needs

 Focus on developing the fundamental understanding and knowledge needed for establishing mechanistic models

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Group Short-term research priorities Longer-term research priorities
Sources  Identify and quantify human pathogens in animal feces

 Identify and quantify human pathogens in various types of on-site systems, especially during wet weather and including disinfection efficacy of systems and their rate of failure  Identify fate and transport of important human pathogens identified in animal feces

 In future and ongoing epidemiology studies, include agricultural, nonpoint source runoff, measure human pathogens identified in animal feces

 In characterization of land-use and sanitary surveys, define consistent template and quantify important inputs

 In developing water quality safety plans and developing best management practices (BMPs), determine recreational component of water safety plans involving EPA, utilities, scientists, and community collaboration

 In developing water quality safety plans and developing BMPs, undertake research to quantify effectiveness of various BMPs and their applicability to various watershed scenarios

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Group Short-term research priorities Longer-term research priorities Implementation Realities  Perform analyses of all existing inland water epidemiological data (domestic and international), including multiple sub-analyses (e.g., flowing versus quiescent, source type, relationships between indicators, relationships of indicators to human health risk)

 Develop more science to understand regrowth or resuspension of enterococci in sediment of inland flowing waters

 Research the effects of prolonged holding times on microbiological analytical results

 Develop early communication on aspects or options of implementation guidance and national level expectations in terms of adoption for inland waters

 Use longer averaging periods for assessment purposes to deemphasize short-term excursions

 Consider modification or use suspensions during defined high-flow conditions

 Consider developing site-specific criteria based on QMRA or other methods

 Designate a new designated use (or uses) on the basis of physical waterbody characteristics or the types of activities that a waterbody is used for (shallow water use or secondary contact use)

 Develop a guidance document to facilitate criteria adoption by states  Develop data to quantify risk in waters affected by nonhuman sources

 Develop a flowing water sample design on the basis of stream characteristics

 Develop a translation between current criteria and any new criteria

 Standardize methods including an evaluation of met hod robustness and a certification program for regulated laboratories

 Conduct studies evaluating the impact of sediment-borne resuspension events especially during high-flow periods

 Conduct studies to determine appropriate indicators for each CWA purpose and a method to bridge current assessment criteria and methodologies to new ones

 Develop data on pathogens/infectivity andexposure information for input to sitespecific QMRA models

 Use QMRA for estimating health risks and developing appropriate and detailed exposure pathways for inland waters

2.2.2. Meeting Report: Knowledge and Gaps in Developing Microbial Criteria for Inland Recreational Waters (Dorevitch et al. 2010) As described in Section 2.2.1, in February 2009, WERF with support from EPA planned and conducted a workshop to explore similarities and differences between inland and coastal waters. That workshop resulted in a peer-reviewed pub lication (Dorevitch et al . 2010) describing the workshop findings and knowledge ga ps related to the extension of criteria developed for coastal to inland waters. In the following section, the report findings are su mmarized. Differences between inland and coastal waters with the potential to necessitate different criteria are summarized first. Then research questions and a research agenda are presented. Note that the

27Inland Waters Summary Report November 2010

study is a synthesis of the full report (WERF 2009) from the workshop (Section 2.2.1). Because the authors of the study interp reted and prioritized elements from the full study, the peerreviewed version of the report summary is also reviewed in this report. Differences between in land and coastal waters Dorevitch et al. (2010) identifies the following th ree assumptions that must be made to extend AWQC developed for sewage-affected coastal sites to inland sites:
1. Similar densities of FIB reflect a similar risk in inland and coastal settings, presumably because they reflect a similar exposure to path ogens of similar infec tivity and virulence. 2. Hydrogeochemical differences among inland la kes, rivers, and coastal waters have nondifferential effects on the transport a nd fate of indicators and pathogens. 3. The criteria derived from the studies c onducted at sewage-affected coastal beaches protect against illness in inland settings, wh ere the predominant source could be wildlife or agricultural animals. Foremost, the report notes that the fecal pollution source is the primary site feature determining the risk of illness from recreational exposure. The authors assert th at inland waters are predominantly in rural areas and have a greater likeli hood than coastal waters of being affected by agricultural or wildlife fecal pollution sources . Further, pathogens pr esent in livestock and wildlife fecal wastes differ in both abundance and human health effects from those present in sewage. Zoonotic agents can differ from pathogens of human origin either in their ability to initiate infection or in the hazard they pose. The authors of this summary report note that although the reviewed report notes the importan ce of source and etiology of illnesses arising from recreation, it does not quantif y the extent to which coastal and inland waters are affected by different fecal pollution sources. Though many river m iles are in rural areas, rivers are loaded by POTWs, combined sewer overflows (CSOs), septic systems, and other human sources. For some streams during dry periods, POTW effluent can constitute a significant portion of flow. Likewise, animal sources affect coastal sites. For example, coastal count ies of the Great Lakes states support large populations of dairy cattle, while coasta l counties on the Delaware and Chesapeake bays are locations of intensive ch icken production (NASS 2010). Shorebirds and dogs are also considered important fecal pollution sources for coastal waters. Dorevitch et al. (2010) identifies indicator growth and mobilization in sediments as a significant potential difference between inland and coastal water indicator performance. Specifically, the report implies that the inland water sediment and soil environment is more favorable to indicator organism growth than the coastal environment a nd that inland water hydraulics generates greater suspended indicator loads than coastal hydraul ics. Inland water sediments are believed to generate larger FIB loads because the extra-ente ric growth conditions for FIB are thought to be most favorable at the water-sediment boundary, and the ratio of the water-sediment boundary length to the stream cross-sectional area is highe r than that for coastal sites. The study also asserts that indicator organisms harbored or growing in sedi ments are more likely to be resuspended into the water column due to more favorable hydraulics for resuspension in inland waters because boundary layers (i ndicative of the shear stress on bottom sediments) occupy a larger fraction of inland water volume than of coastal water volume. Together, those phenomena result in a larger proportion of indicators in inland waters aris i ng from non-fecal sources than the proportion in coastal waters. Thus, for inland an d coastal sites with the same fecal pollution source, an indicator density obs erved in inland water is likely to correspond to a different

28Inland Waters Summary Report November 2010

presence of fecal pollution and a different risk than for the coastal water at which the sam e indicator density is observed. Such a decoupli ng of indicator organisms from fecal pollution sources represents a significant difference in i ndicator performance for inland and coastal waters. Hydrogeological differences might influence indicator performance in inland and coastal waters. First, fecal pollution sources are of ten closer to receiving waters for inland water than for coastal waters. Given the smaller water volumes typical of inland waters, less dilution of the fecal pollution is expected for inland waters than for coastal waters. This finding does not pose a difficulty in use of indicators for inland and coastal waters; the higher indicator densities observed in inland waters are related to higher fecal pollution densities and higher public health risk. Increased risk is expected to be associat ed with proximity to the fecal pollution source. Decreased risk is expected to be associated with exposure to more dilute fecal pollution. Indicators shed by the bathers can be diluted less in inland waters than in coastal waters. All other factors being equal, highe r indicator densities are associ ated with higher fecal pollution concentration and increased risk. An additional difference in hydrogeology for inland and coastal waters with significance to indicator performance is the flow typical of the setti ngs. Inland waters are subject to highly variable volumetric flow rates, bottom shear stre sses, and turbulence. Those parameters are less variable for coastal waters. Advection domina tes fecal pollution transport in flowing inland waters, whereas advection via longshore currents , other large-scale curr ents, or wind-driven water flows are the drivers behind most transport of fecal pollution into and out of coastal sites. This difference results in more extreme variatio n in indicator densities during storm events for inland waters than for coastal waters, as observed by Nevers et al. (2007) for coastal streams near Great Lakes beaches. Furthermore, as describe d above, turbulence can promote decoupling of indicator density from fecal pollution sources. Critical research questions and a research agenda The report proposes four areas en compassing the resear ch that should be conducted to improve the understanding and estimation of health risks arising from r ecreation in inland waters. The four areas are presented below, along with suggested short- and long-term research activities for closing data gaps a nd improving understanding.
1. Microbial indicators as predictors of risk The spatiotemporal variability and determinan ts of FIB need to be characterized for hydrologically diverse settings. Alth ough not detailed in the report, these diverse settings include small rural streams, small urban streams, larg e flowing waters, and impounded waters. Shortand long-term research suggest ed include meta-analysis of epidemiological relationships developed for waters affected by different feca l pollution sources (short-term), and mechanistic fate and transport modeling of FIBs for the range of water types expected to be important as recreational waters.
2. Fecal pollution sources as predictors of pathogen exposure and health risk In the topic of fecal pollution s ources as predictors of pathogen exposure and health risk, shortterm research activities suggested in the report are development of a sanitary survey tool for use in inland water epidemiology and QMRA studi es, optimization and anchoring of QMRA by means of data and relationships from epidemio logy studies, and field sa mpling of feces from farm animals and wildlife to quantify the o ccurrence and dynamics of pathogens of human

29Inland Waters Summary Report November 2010

concern. In the long term, the re port suggests that epidemiology studies should be conducted at inland sites with different dom inant fecal pollution sources. Ep idemiological studies should differentiate between pathogens pos ing different hazards, pa rticularly those associated with very serious health outcomes (e.g., zoonotic E. coli O157:H7).
3. Molecular methods for water quality testing
Given the likelihood that molecular methods will be employed in new AWQC, molecular methods should be better understood and optimi zed. A database describing the relationships between molecular- and culture-based determinati ons of indicator densities should be developed on the basis of findings reported in the technical literature. Given the differences in transport and fecal pollution loading for inland and coastal waters, that database would likely include relationships for a wide variety of settings ra nging from small inland waters to large inland waters to coastal sites. The pe rsistence of molecular method targ ets should be established. This step is critical given that one performance criterion of indicator organisms is persistence similar to that of the pathogens they indicate. Quantita tive PCR methods, particular ly for source-specific markers, should be optimized, standardi zed, and applied in epidemiology studies.
4. Other approaches for predicting in land water recreation health risks The other approaches suggested by Dorevitch and colleagues for pred icting risks include predictive modeling (such as Nowcast forecastin g) and QMRA. Regression models have been demonstrated to be more predictive of whether an AWQC will be exceeded than simple use of a prior day’s indicator density for coastal sites. Flow models can be used to improve regression models, though real-time multidimensional modeling of coastal sites has not been demonstrated to date. Modeling research activit ies suggested include evaluation of the viability of regression models and mechanistic models for risk manage ment and as supplements to FIB monitoring for inland waters. There are several indications that regression and mechanistic models are feasible for use in inland water risk management. Moni toring and forecasting systems are in use for managing risks to drinking water systems w ith surface sources. Numerous well-tested contaminant transport models are available for mechanistic modeling of FIB or pathogens in streams; given the hydraulics and ge ometry of streams, these mode ls are much less complex than flow models of coastal sites a nd have the potential for use in r eal time. Challenges to use of regression and mechanistic m odels include the following:

 sporadic nature of fecal indicator and pathogen loads to streams;

 knowledge gaps regarding FIB and pathogen growth in sediments and resuspension;

 incomplete knowledge of the persistence of the microorganisms in diverse inland water settings; and

 need for sensors and protocols for collecting real-time data for use in predictive models. The QMRA framework is flexible and can be us ed to relate indicator and pathogen fate and transport to risk. When used in tandem with ep idemiological analyses, QMRA can add context to epidemiology study findings. In the absence of epid emiology studies (as is currently the case for inland waters), QMRA can be used to develop estimates of human hea lth risk that can be compared to those observed for coastal waters. To be used in either of those modes, QMRA must have credibility with the scientif ic community a nd must provide sufficient data to allow realistic risk estimates specific to th e geography, biology, and hydrology of inland waters. The report advocates anchoring QMRA (retrospective studies) as a short-term research need. Such an

30Inland Waters Summary Report November 2010

exercise would necessitate generating improve d models and can be used to advance the credibility of QMRA with the scientific and policy communities. Report conclusions Dorevitch et al. (2010) conclude that the distinction between inla nd and coastal waters is less important than differences in the fundamental processes and variables of the systems. The processes and variables include the scale of the waterbody, the fecal pollution source(s), indicator and pathogen dynamics in sediments, and other factors related to the transport and fate of indicators and pathogens. Of particular importance is a clea r understanding of the proportion of indicators that is directly related to a specific fecal polluti on source and the proportion not related to fecal pollution sources and thus i ndicative of a different and likely lower risk. The authors reason that applying cr iteria derived on the basis of epidemiology studies of coastal sites to inland waters should result in sporad ic mild illnesses at rates no higher and possibly lower than those experienced in coastal waters. However, coastal and inland waters might pose very different risks of severe diseases such as hemolytic uremic syndrome arising from exposure to E. coli O157:H7 and other pathogenic E. coli strains. The origin of those different risks is the difference in the dilution capacity of typical inland waters and co astal waters, as well as the difference in pathogenic organisms in sources typical of coastal and inland waters.

2.2.3. Literature Review of Assessment of the Applicability of Existing Epidemiology Data to Inland Waters (USEPA 2010a) Before the 2009 WERF workshop whose findings are described a bove (Dorevitch et al. 2010; WERF 2009), background material was assemble d describing and comparing the occurrence, fate and transport of indicator organisms in inland and coastal waters. After the workshop, the draft report was revised to expa nd upon sections pertaining to ep idemiological data and health effects. The document (USEPA 2010a) underwen t a second round of revisions in August 2010. That version of the report is reviewed below. The purpose of the report is to assess and compar e the performance of indicators in inland and coastal waters. The authors compare the performa nce of indicators in coastal and inland waters on the basis of the following features:

 demonstrated correlation with health risk;

 similar or greater survival ti me than the target pathogen;

 similar or greater transport than the target pathogen;

 presence in greater numbers than the pathogen; and

 specificity to a fecal source or an identifiable source of origin.
Indicators demonstrate all those featur es for both inland and coastal waters. The report presents data and analysis from the peer-reviewed literature grouped into three sections (1) epidemiology and modeling studies of th e health effects associat ed with recreation in surface waters; (2) the occurrence an d variability of indicators in coastal and inland settings; and (3) the persistence and growth of indicators a nd the association of i ndicators with pathogens. Absent from the report are discussion of di fferences in resuspension for typical inland and coastal waters, discussion of differences in typi cal fecal pollution sources for different settings,

31Inland Waters Summary Report November 2010

discussion of suites of pathogens characterist ic of different fecal pollutio n sources, and evaluation of the role of dilu tion in indicator performance. Epidemiology and modeling studies To date, epidemiology studies have demonstrated co rrelations of health risk with indicator level only for waters primarily affected by POTW disc harge. For freshwater sites, studies have demonstrated association of health risk with indicator level for E. coli enumerated by cultural methods (Dufour 1984; EC 2009a, 2009b; Marion et al. 2010) and for Enterococcus enumerated by qPCR (Wade et al. 2006, 2008). Among those st udies, the only two that are potentially directly comparable are the Dufour and Marion studies, because both used the prospective cohort (PC) study design and the same illness definition . Direct comparison of the results of those studies should be done cautiousl y, because the health effects re lations in the Dufour study are based on seasonal averages of indicator densitie s whereas in the Marion study they are based on individual days’ samples taken at an inland reservoir. In general, the results of both epidemiology studies indicate that health effect s observed in the two st udies are comparable, despite the studies being conducted more than two decades apart (the GI definition was the same in both studies). Other epidemiology studies will require analyses and conversion of data prior to comparison. For example, both health effects and water quality data from randomized control trial (RCT) epidemiology studies could require conversion fo r comparison with PC epidemiological health effects data. Modeling provides an alternative to epidemiology studies for assessing health risks associated with recreational use of surface waters. As noted above, epidemiology studies provide the firmest basis on which to develop criteria. Howe ver, in the absence of epidemiology studies, modeling studies could provide a means for evalua ting health risks. Modeling studies described in the USEPA (2010a) include QMRA and watershed modeling approaches. Recently published QMRA studies (Schoen and Ashbolt 2010; Soller et al. 2010a,b) have linked indicator level to the occurrenc e of pathogens for non-POTW s ources—including cattle manure, swine manure, chicken manure, and gull droppings. Those studies rely on pathogen and indicator occurrence and abundance as repo rted in the literature for es timating the pathogen suite and resulting illnesses rates associated with ingesti on of runoff from livestock operations or from beaches contaminated with gull droppings. Thos e models are stochastic and account for the variability in the occurrence and abundance of pa thogens and indicators in fecal sources. The QMRA models indicate that, at a given indicator level, recreati on in waters affected by cattle runoff poses a similar risk to recreation in wa ters affected by human fecal pollution sources, whereas recreation in swine-, chicken-, and gul l-affected waters poses a significantly lower health risk. The results will be refined in future studies to account more accurately for fate and transport processes and manure treatment. The findi ng that different fecal pollution sources have different risks at the same indi cator level does not consider inland and coastal sites to be substantially different. Rather, it is presumed that the source, not th e setting, is the critical feature of a site. Watershed modeling is a related but alternative modeli ng technique that can be used as an alternative to epidemiology studies for assessi ng health risks associa tion with recreation in surface waters. Like QMRA models, watershed mode ls rely on data collected from external sources as input for models that predict net pathogen loads wa tersheds discharge to receiving

32Inland Waters Summary Report November 2010

waters. The watershed models can be used in developing sampling schemes, in assessing differences of risk in different portions of a watershed, or in com par ing the contribution of different fecal pollution sources to the pathogen loads to receiving waters. Because the models are site-specific, they might have a greater role in implementing new or revised criteria than in developing the criteria.
FIB occurrence in inland and coastal waters Nearly all available data on the occurrence of FIB are presented as ranges, not as statistical distributions. Accordingly, more meaningful comparison of ranges of indicator densities with metrics such as geometric means, measures of sp read and of skew in di stribution of occurrence, could not be used. Moreover, studies were limite d to relatively few waterbodies and each study had different designs and objectiv es, so how representative their data are with respect to the overall distribution of FIB among inland and coasta l waters is uncertain. Thus, comparisons of the occurrence of indicators in coasta l and inland waters were considered screening level and were made on the basis of reported ranges. The report presented indicator occurrence density ranges for E. coli , enterococci, fecal coliforms, total coliforms, and fecal streptococci. To allo w comparison across setting types, ranges for the following setting types were determined:

 coastal freshwaters;

 coastal marine waters;

 estuarine;

 inland flowing, main;

 inland flowing, small; and

 inland non-flowing.
Coastal marine sites exhibited the highest variation in both E. coli and Enterococcus densities, somewhat contrary to expectations. Coastal wate rs are subject to much greater dilution than inland waters and are farther from fecal pollution sources than typical inland waters. Given the limitations of the occurrence data, the report concl udes that the occurrence of indicators in inland waters and in coastal waters is not substantially different. It is im portant to note that none of the studies used in developing the occurrence rang es attempted to ascertain the source of the indicator organisms. Growth and persistence of FIB Indicator survival curves typically exhibit shoulder behavior (the shoulder being the initial curve before the exponential portion, re presenting that damage has to accumulate to a certain level before cells begin to die), followed by first- order decay. The factors most important in determining the decay rate are presence of sedi ments, insolation, and the presence/absence of predators. Less significant factors are temperature and salinity. Nutrient availability and pH also influence persistence, but they do not vary su fficiently among waters to result in significant differences in persistence among sites. Fecal indicator bacteria growth has been observed in coastal and inland settings and in both large and small streams. Fewer studies have re ported growth of en terococci than of E. coli and fecal coliforms; however, it is not certain whether this is because enterococci growth is less prevalent than that of the other indicator s or whether there are fewer stud ies on enterococci. The presence

33Inland Waters Summary Report November 2010

of sediments is a primary factor in determini ng whether g rowth is observed. Because inland and coastal waters differ in the sediments present and the ratio of the sediment-water interface area to the water column volume, that difference could re sult in a different propo rtion of indicators in inland waters arising from sediments than the proportion in coastal wa ters. The proportion of indicators attributable to resuspension from se diments is determined by the density of indicators in the sediments and the resuspension of the i ndicators via turbulence ar ising either from shear stress at an inland water stream bed or from wave action or tida l processes in coastal sites. Reported E. coli inactivation rates do not differentiate indicator performance in Great Lakes waters from that in inland waters. In both wate r types, indicator decay rates depend on the same physical factors and are widely va riable. Contrary to expectations , reported inactivation rates for marine and freshwaters overlapped. Studies have established decr eased persistence of E. coli with salinity, so the overlappi ng ranges indicate that the comb ination of factors determining survival are such that their net effect is inac tivation rates spanning the same range. Fewer studies were found providing Enterococcus inactivation rates. The studi es reviewed in USEPA (2010a) indicate that the same factors govern the persiste nce of enterococci in inland and coastal waters and that persistence is comparable in the two settings. Co-occurrence of FIB and pathogens At the time that report was completed, only two epidemiology studies have yielded health effects relationships for culture-based indicator enumerat ion in inland waters a nd there are no available studies that have established health effects relationships fo r inland waters based on qPCR. Therefore, the health effects as sociated with inland waters and coastal waters must be deduced either from a direct asso ciation of pathogens and indicators or from an association of indicators and pathogens with fecal pollution sources. In USEPA (2010a), studies attempting to correlate pathogen and indicator occurrence were reviewed. The relevant studies included studies of bacterial, protozoan, and viral pathogens for inla nd and coastal waters. In short, co-occurrence and correlation between pathogens and E. coli and Enterococcus were not observed for any setting. Though not reported, it is cl ear that pathogen variability differs from that of indicators, even for a given fecal pollution source. Other processes, including the following, can cause pathogen-to-indicator ratios to vary:

 different removal rates of indicators and pathogens via settling in flowing and nonflowing settings;

 different removal rates of i ndicators and pathogens via ul traviolet (UV) inactivation (assuming the incident UV radiation is different in coastal and inland settings because of shading or other features); and

 different sources or loading rates associated with different settings.

2.2.4. Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b) As noted in Section 1.1.2, stakeholders have expressed concerns over both science and the application of new or revised AWQC to inla nd waters. Concerns repeatedly expressed by stakeholders are (1) sampling and interpretation of results for inland waters at which there is the potential for recreation but recreation does not occu r, and (2) concerns that samples taken during or shortly after rain events, when indicator densities are extremely high, will unduly influence water quality assessment. Data and interpretations germane to those concerns are presented in

34Inland Waters Summary Report November 2010

Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of Recreational Waters (USEPA 2010b). That report describes the tem poral and spatial variations in indicator densities for both in land and coastal waters and at al l relevant spatial and temporal scales. Indicator variability in inland and coastal waters A full review of the findings regarding indicator spatial and temporal variability provided in USEPA (2010b) is outside the scope of this report. In brief, a re view of the literature produced the following findings:

 Regardless of site type, the grea test variations in indicator density arise from rain events.

 Temporal variability, from greates t to least, is as follows: Ev ent variability (rain events) > diurnal variability > tidal-tim e-scale variability (coastal sites only) > monthly/seasonal variability (considering only the recreational use season) > short-tim e-scale variability (for samples taken at knee depth and greater).

 For coastal sites, sources of spatial variab ility, from greatest to least, are as follows: variation with depth of sample collection > va riation with site feat ures such as point sources or features inhibi ting mixing > along-shore varia tion > variation with depth below the water surface where sample is collected.

 For inland sites, sources of spatial variability, from greatest to least, are as follows: along-stream variation > varia tion with depth below the water surface where sample is collected > cross-stream va riation (i.e., downstream of the mixing zone for point sources). Developing monitoring plans Monitoring plans chosen for specific sites should be designed on the basis of the variability in indicator density anticip ated at that site. Thus, data s hould be collected before monitoring scheme development to quantify spatial and tem poral variability. Two ve hicles suggested for collecting those data are sanitary surveys and pilot monitoring activities. Perhaps most important, sanitary surveys include collection of data on th e fecal pollution sources with the potential to affect a site and the route by which fecal polluti on could be delivered to the site. For coastal waters, transport modeling can be complex, give n variability in currents, wave-generated turbulence, and such. For inland waters, identific ation of sources can be complex because many inland water fecal pollution sources are diffuse. None of the studies reviewed in USEPA (2010b) directly addressed the consideration of extremely high indicator densitie s during rain events. As noted in that report, extremely high indicator densities can arise in both inland and coastal waters dur ing rain events, although given the lower dilution and proximity to sources of inland waters, the impact on inland waters is expected to be greater than that on coastal waters. Inland sites can require different monitoring strategies because of their accessibility, length, and frequency of use. Ideally, the number of samples is chosen using a variation of power analysis, with the detectable difference related to the accept able range in risk or a range of risks that is measurable within the overall population. Because of cost or logistical considerations, beach managers might not be able to sample with the density or frequency suggested by power analysis. Sampling locations shoul d be selected based on the ab ility of a small number of samples to adequately describe water quality at the site and should target areas of beaches in

35Inland Waters Summary Report November 2010

closer proximity to fecal pollution sources (po rti ons of the beach with significantly different mixing should be sampled separately). In additio n, collection of samples in the morning appears to offer the best balance between practicality and generation of data that are conservative estimators of human health effects. It also pr ovides the best correlation between culture-based and qPCR results.

2.2.5. Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters (WERF 2010a) The overall objectives of this study are (1) to identify and address data gaps pertaining to loadings and concentrations of waterborne pathogens and indicator s in discharges to recreational waters that are affected by fecal pollution; and (2 ) to compile, analyze and synthesize the data in QMRA models and waterborne risk management frameworks. These objectives are related to the comparison of inland and coastal wa ters because of the following:
1. Pathogen densities typical of specific fecal pollutions sources are characterized. These data can be used to compare the relative risks posed by inland and coastal sites if it is found that inland and coastal sites are impacted predominantly by different fecal pollution sources. 2. The study develops a method by which the re lative contributions of different fecal pollution sources can be determined for a part icular site. Knowing the contribution of the sources will allow improved risk estimates fo r both inland and coastal sites and will provide information regarding the how the dynamics of indicators and pathogens differ for inland and coastal sites. At present, th e general features of indicator and pathogen dynamics are known for inland and coastal si tes, but quantitative data for relating different processes affecti ng risk are not available. 3. QMRA models for recreationa l exposure are becoming in creasingly available. New methodologies and data for use in QMRA are provided in this study. Although the new data and methodologies will be useful if QMRA is used for comparing the relative risks of inland and coastal sites, because the QM RA model developed in this study does not consider the characteristics of the waters receiving fecal pollution, the results do not directly address differences in indicator performance for inland and coastal sites. Elements of this study were: data collect ion on pathogen and indi cator occurrence and abundance in diverse fecal polluti on sources (literature survey a nd field study); development and characterization of the performan ce of microbial methods for all of the relevant pathogens and indicators; development of a quantitative microbial source tracking (MST) procedure for estimating the contributions of various sources to the density of indicators at a specific site; and QMRA and modeling to relate feca l pollution sources to health risk s. In a generic sense, all of these activities are pertinent to comparison of indicator performance in inland and coastal sites. The activities with the most direct relevance to differences between these settings are the quantification of pathogens and indicator prevalence and abunda nce in specific fecal pollution sources, and the development of source-specific QM RA models. Each of these areas is reviewed below, with findings most closel y related to differences in inla nd and coastal waters highlighted.
Data collection
Literature searches and field studies were used to quantify the prevalence and abundance of several waterborne pathogens (e.g., Salmonella
, Campylobacter
, Cryptosporidium
, Giardia
, adenoviruses, enteroviruses, noroviruses, a nd rotaviruses) and indicator organisms (e.g.,

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Bacteroidales
, Enterococcus , and E. coli ) in treated and untreated sewage, urban runoff, runoff from undeveloped sites, livestoc k feces, wild life feces, and companion animal feces. Field studies involved sampling wast ewater treatment plant, CSO, and stormwater effluents prior to mixing with receiving waters. Because the drai nages contributing to th e discharges are wellcharacterized, they might allow generalization of the findings to other drainages with similar characteristics. Sampling was conducted during (precipitation) event and non-event conditions. Both culture- and molecular-based methods were us ed to enumerate pathoge ns and indicators (to the extent that both methods are possible for a given microorganism). Novel or particularly relevant findings from the fiel d studies include the following:

 Cryptosporidium density in runoff from forested land was much higher than that in runoff from lands with other uses;

 Salmonella occurred far more frequently in all fecal pollution sources than the other bacterial pathogens; and

 the dominant viruses differed by fecal polluti on source, with enterovirus and norovirus most prevalent in runoff from residential and commercial/ light industrial drainages, rotavirus more plentiful in discharges from agricultural operations, and different adenovirus types occurring with different pr evalences among the fecal pollution sources. The occurrence of some pathogens was found to be correlated with other pathogens. This finding might be significant in assessing risk related sp ecific settings, because risks due to multiple pathogens are additive. Spiking experiments were conducted such that f iltration efficiencies, detection limits, and the effect of hold times could be established for the bacterial and protozoan organisms chosen for the study. From those experiments, the authors dete rmined that using holdi ng media stabilized bacterial populations without adve rsely effecting protozoa. The aut hors also noted that from raw data that, in some cases, rec overies above 100 percent were r ealized, and in many cases there were declines in recovery during the 72-hour hold times. The results of the spiking studies do not contribute directly to the comparison of inland and coastal waters. However, they do provide information with which pathogen abundance might be characterized in QM RA studies. Correctly characterizing pathogen densities is very important given the lo w densities at which pathogens normally occur and at which some pathogens can initiate infection and illness in humans.
QMRA A QMRA model was developed for each fecal pollu tion source. The model incorporated data on the prevalence and abundance of all the priori ty pathogens in each fecal pollution source and used two exposure scenarios—direct exposure to effluent/runoff and exposure to diluted effluent/runoff. As noted above, it is difficult to relate the QMR results directly to differences between inland and coastal sites because there are no quantitative data describing and comparing the distribution of fecal pollution sour ces among coastal and inland waters. In general, viruses were consiste ntly found to be the risk drivers for all sources, with norovirus producing the dominant health risk, even for ag ricultural runoff. The authors do not comment on the degree to which animal noroviruses are host-ada pted and their potential to be infectious to exposed persons. It was also found that dilution (as simulated in the QMRA) was not associated with significant risk reduction. This finding is significant, in that one significant difference between inland and coastal suggested by participants in the WERF experts workshop (Dorevitch

37Inland Waters Summary Report November 2010

et al. 2010; WERF 2009) was the much higher diluti on of fecal pollution in coastal waters as

com pared with inland waters. The authors discuss methods by which QMRA mi ght be used for generating new recreational water criteria or in implementation of those crit eria. An important precursor to such uses is calibration of the models based on findings of ep idemiology studies and development of models with specific components that reflect site char acteristics ascertai ned through sanitary surveys or other data collection. Underlying the discussion of QMRA is the need to formulate the model for consistency with the pathogens and exposure s cenarios for sites, whether they are inland or coastal. Quantitative MST Along with QMRA model development, the au thors developed a quantitative MST methodology based on quantification of uni versal, human, cow, and dog Bacteroidales , and that was capable of determining the extent to which different fecal pollution sources (human, dog, and cow) contributed fecal indicators in a fecal pollution sample. Given the assumption that fecal pollution source is the most important determinant of risk for a given receiving water, this tool, along with sanitary surveys, has the potential to allow co mparison of risks between sites (including inland and coastal sites). The performance of the quant itative MST method was found to be sensitive to the selection of the different host-specific indicators included in the methodology, and on the measurement error associated with each of the indicators. Two illustrations of model performance showed that the model performs well under some circumstances and might be improved such that it could also perform well when fecal pollution from species other than humans, dogs, or cows is present.

2.3. Methodology Performance and Relevance to Applicability of Criteria to Inland Waters (compilation of findi ngs from multiple reports )
This section contains a review of the findings from reports #5, 7, and 8, and peer-reviewed publications 9 through 13 (Section 1.2.1) that are relevant to the difference in performance of indicator-method combinations in inland and coas tal waters. The emphasis of this section is method performance and relative differences in qPCR and culture targets, rather than the association of indicators (as meas ured by different methods) with health effects. Some of the factors reviewed in this section are common to both inland and coastal waters. For example, chlorinated POTW effluent typically has much higher indicator counts via qPCR than culture methods, regardless of whether th e plant is discharging to an in land water or a coastal water. Other features reviewed herein differentiate inland from coastal waters, such as the degree of qPCR inhibition exhibited in specific water types. The section begins with a review of qPCR enumeration of indicat or bacteria in environmental waters and the factors that may impact qPCR perf ormance. Those factors, which include solar radiation, chlorination, die-off and predation of qPCR and culture targets, and inhibition, may differ among inland and coastal waters, though the ex tent to which those f actors differ in inland and coastal settings is yet to be established. The section concludes with a review of several studies directly comparing cultu re and qPCR method performance in inland and coastal waters.

38Inland Waters Summary Report November 2010

Factors influencing method performance Generally, FIB densities derived fr om qPCR have been consistent ly reported higher by several orders of magnitude than those derived from culture-based assays in both coastal and inland water settin gs, and under a variety of conditions (e.g., throughout wastewater treatment, at POTW effluent, different times of day). This is mainly due to the comprehensive or unspecific nature of DNA amplification in the qPCR assa y. All DNA present in a sample is amplified equally, regardless of the viability status of its host. This total DNA inve ntory is comprised of free and dead cell DNA (constituting ambient background DNA), viable but not culturable bacterial (VBNC) DNA, which is predominant w ith respect to the last culturable and viable fraction (corresponding to the cells enum erated by culture-based methods). In report # 7 (Final report: Comparative Evaluati on of Molecular and Culture Methods for Fecal Indicator Bacteria for use in Inland Recreational Waters (W ERF 2010b)) the study authors view, based on the results of their study, is that qP CR methods, as currently optimized, cannot be applied universally across a ll inland water bodies. Differe nces among inland sites (and presumably among coastal sites as well) that may limit the performance of a particular qPCR assay are the fecal contamination source, unres olved inhibition, between-lab method variability (e.g., difference in extraction efficiency or deve lopment of calibration cu rves), and the relative contribution of DNA from viab le v. non-viable cells. All of these factors vary among waterbodies and laboratories and ar e not intrinsic to inland waters per se. None-the-less, this finding implies that method performance can differ from site to site and should be considered when interpreting indicator levels for a given site or compari ng indicator levels among sites. In a study comparing qPCR and culture counts of Enterococci, Haugland et al. (2005) noted that geometric mean values of cell equivalents (qP CR counts) for samples taken on a given day were nearly always one order of magn itude and frequently two orders of magnitude greater than those for the MF method. Haugland and colleagues suggest ed that the much higher densities indicated by the qPCR assays are a result of the inability of the qPCR technique used to distinguish between DNA from live and dead cells (see the discussion on distinguishi ng live and dead cells, below). This finding is significan t to the difference in inland and coastal waters in that the persistence of indicators and re lative abundance of live culturab le cells and qPCR targets may differ for inland and coastal sites. A discrepancy in correlation between qPCR and culture-based results for morning and afternoon samples was consistently observed for samples taken at Great Lakes b eaches during the NEARR epidemiology studies (USEPA 2010c; report 5 reviewed in this summary). Plots of qPCR counts of Enterococcus against culture counts of Enterococcus for one of the beaches for data collected at 8 AM and 3 PM are shown in Figure 3 and 4. Slopes of linear regression models of the logtransformed densities of the two data sets are significantly different, with the 8 AM samples exhibiting a slope much closer to 1 than the sam p les collected at 3 PM. These plots demonstrate the importance of sunlight inactivation on culture counts and th e relative insens itivity of qPCR targets to solar radiation. In dark conditions culture and qPCR targets have shown different persistences, with persistence of naked DNA in seawater mesocosms on the order of three times longer than that of viable cultu rable enterococci (Walters et al., 2009). The insensitivity of qPCR targets to solar radiation and the much slower decay of qPCR targets underscore the necessity to account for ambient background DNA at recreational sites when using molecularbased monitoring techniques. Culture-based and qPCR results are well correlated at high FIB densities (viable and culturable fraction predominant). However, th is correlation is lost at low

39Inland Waters Summary Report November 2010

culture-based densities du e to high variability of the qPCR in this range and predominance of VBNC over culturable bacteria and possibility due to the high uncer tainty of qPCR assays when fewer than 100 cells (or qPCR targets) are pr esent (WERF 2010b). These findings may be of significance in the comparison of inland and coasta l waters if inland waters are generally more shaded than coastal waters, if beaches on inland waters tend to be impacted by “fresher” fecal pollution, or if the microbial ecology of inland and coastal sites are sufficiently different to impact the persistence of culturabl e cells and DNA from non-intact cells.

Figure 3. Paired qPCR and Culture Enterococcus Data: Huntington Beach: 8 AM Samples Only

Figure 4. Paired qPCR and Culture Enterococcus Data: Huntington Beach: 3 PM Samples Only

Alternative molecular methods or refinements to qPCR may improve the ability of molecular methods to distinguish between live and dead cells. For example, Bae and Wuertz (2009) developed a modified qPCR propi dium monoazide (PMA) to rem ove DNA from non-intact cells from the PCR reaction, resulting in better correlat ions with culture-based methods. For samples from the wastewater plant efflue nt, gene copies from qPCR with PMA were only 30 percent of those from qPCR without PMA. The differe nce between qPCR with and without PMA was greater than two orders of magn itude for samples of wastewater plant effluent. If optimized, these new techniques have the potential for maki ng the analysis of coastal and inland waters more consistent.

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POTW effluent Strong reductions of cultivable FIB (2-5 orders of magnitude) have been observed throughout wastewater treatm ent, especially during seconda ry treatment and disi nfection. Although this illustrates the ability of such treatments to inhibit cultivability, it does not necessarily demonstrate that the treatment trains have cau sed cell death. This is because qPCR counterpart inventories experience only small reductions or remain unchanged. Such dichotomy between method outputs is critical both from the standpoint of cr iteria design but also from the standpoint of public health, highlighting the potential for a large proport ion of VBNC bacteria being released to recreational waters. In addition, because inland waters generally receive less-dilute chlorinated POTW effluent than coastal waters, the difference in qPCR and culture densities for inland waters impacted by POTW effluent coul d be greater than that for coastal waters. Some water matrices may have a strong influe nce on the comparison of qPCR cell equivalents and MF CFUs. For example, PCR amplificatio n efficiency may be lower in high turbidity waters than low turbidity waters. He and Jiang (2005) developed a qPCR assay for Enterococcus and evaluated the assay against MF for unchlor inated primary and secondary POTW effluent, chlorinated secondary effluent, and marine samples from multiple sites. For unchlorinated sewage, the difference between qPCR and culture enumerations of Enterococcus varies widely, with qPCR enumerations exceeding culture enumer ations part of the time and below culture enumerations for other samples. Two plausibl e explanations for this finding, in addition to matrix-related effects, are that (1) qPCR results are uncertain and vary significantly between samples, or (2) qPCR and culture methods measur e different features of bacteria and those features vary differently between samples. In the case of chlorinated secondary effluent, relatively high enumerations by qPCR potentially i ndicate the presence of dead cells or extracellular DNA. For samples from a marine enviro nment (Table 4), qPCR results are consistently higher than those of culture methods. Impact of Setting (Inland v. Coastal) on the Relative Abundance of qPCR and Culture Targets Byappanahalli et al. (2010) found that the rela tive abundances of enterococci as measured by cultural methods and qPCR differed for samples ta ken at beach sites and for samples taken from a tributary discharging to Lake Michigan in the vicinity of the beaches. The authors found that the mean of the samples enumerated via cultural methods was not signif icantly different from that of the samples enumerated via cultural me thods in the tributary. For the two beaches monitored in the study, the mean of the qPCR counts were 1.6 and 2.1 times the mean CFU counts and the mean cultural and qPCR counts were significantly different for both beaches. The authors discount inhibition in th e lake water as the cause fo r significantly higher qPCR counts than culture counts because samples were anal yzed after a 1:5 dilution employed to prevent inhibition. Further, the au thors report that the qPCR Enterococcus counts in the tributary and the lake water were not significantly greater than t hose on the beach. These findings support slower removal of qPCR targets than culturable indicator bacteria as discharge from the tributary is advected to the beaches or othe r loading of qPCR targ ets to the beach sites from sources other than the tributary. Telech et al. (2009) developed a series of re gression models using several rapidly-measured environmental variables (e.g., rainfa ll, turbidity, number of bather s, wind speed and direction) for predicting qPCR and culturable (membrane filtration) Enterococcus counts at four Great Lakes beaches. Although the explanatory variables differed by beach for regression models

41Inland Waters Summary Report November 2010

using either type of analytical detection method (i.e., qPCR or me mb rane filtration), the variables exhibiting the strongest relationshi p with Enterococcus de nsities in the models were consistent within the analytical detection method. The aut hors noted that meteorologi cal, physical, water, and beach characteristics explained more variab ility in FIB densities measured by membrane filtration than by qPCR. Lavender and Kinzelman (2009) developed an empirical model for correcting qPCR Enterococcus and E. coli densities (as CE) and improving thei r correlation with culture counts. Although empirical, their methodology relies upon a condition-specific correc tion and as such is related to the theoreti cal model described above. The study entailed collection of samples at several locations and analysis of the samples for Enterococcus and E. coli via membrane filtration and qPCR. Analysis of data found di scordance between qPCR and culture data during rain events or when wave height was above a certain threshold. The authors attributed poor correlation under those conditions to increase d densities of background DNA (not associated with viable culturable cells). To improve correlation betwee n culture and qPCR densities, the authors proposed use of a corr ection factor for conditions as sociated with high background DNA densities. In the resulting model, if specifi c rainfall and wave hei ghts are observed, the qPCR indicator density is reduced by a correction factor associated with those conditions and estimated from the data. The correction factors proposed by the authors were different for different sites. It is likely that correction f actors and the conditions under whic h they are applied would be substantially different for inland and coastal waters. Finally, report # 7 (Final repor t: Comparative Evaluation of Molecular and Culture Methods for Fecal Indicator Bacteria for use in Inland Recrea tional Waters (WERF 2010b)) reviewed for this study reports stronger associations of culture indicator dens ity with qPCR indicator density of Great Lakes waters than for an effluent dominated river or an inland lake. This finding was based on logistic regression m odeling performed on paired culture and qPCR analyses from Great Lakes waters, rivers, inland lakes, effluent dominated waters , and waters not dominated by POTW effluents. The authors of that study al so noted that for the 3 qPCR methods used, low levels of qPCR targets were hard to analyze fo r cell numbers below 100 cells. This shortcoming of qPCR may influence the corr elation between culture and qPCR cell densities if consistently low indicator densities are typical of an inland or a coastal site. Summarizing, correlations between qPCR and culture counts differ amo ng all sites, with distinct differences observed among inland and coastal site s when the two site types were compared directly. These differences may not be the resu lt of intrinsic differences in inland and coastal sites but rather the result of site-specific differen ces in water quality or source characteristics for the sites studied.

42Inland Waters Summary Report November 2010



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