EPA #823‐R1‐0002

Applicability of Great Lakes NEEAR Dataset to Inland 

Recreational Water Criteria: Summary of Key Studies 

U.S. Environmental Protection Agency 

November 2010 

Inland 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 flowing, inland impounded, and Great Lakes coastal 

settings ................................................................................................................................ 7

1.3.1. Qualitative comparison of indicator processes in inland and coastal waters............. 7

1.3.2. Brief review of epidemiology studies 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 Critical Research and Science 

Needs for the Development of Recreational 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 (Dorevitch et al. 2010)............................................................ 27

2.2.3. Literature Review of Assessment of the 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 Criteria to Inland Waters 

(compilation of findings from multiple reports) ............................................................... 38

References..................................................................................................................................... 43

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Inland Waters Summary Report November 2010 

Tables 

Table 1. Key areas that the reviewed reports and 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 Workshop on Critical Research and Science Needs for the 

Development of Recreational Water Quality Criteria for Inland Waters (WERF 

2009) ..............................................................................................................................12

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).......................................................................................14

Table 4. Study purpose, methodology, findings and limitations for Literature Review of 

Assessment of the Applicability of Existing Epidemiology Data to Inland Waters

(USEPA 2010a) .............................................................................................................15

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) ...........................................................................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 findings, and limitations of Methodology 

Performance and Relevance to Applicability 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 research 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 fecal 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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Inland Waters Summary Report November 2010 

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 Environmental 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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Inland Waters Summary Report November 2010 

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Inland Waters Summary Report November 2010 

Executive Summary 

This report evaluates the applicability of NEEAR Great Lakes data to inland waters and assesses 

the similarities and differences between coastal freshwaters and inland freshwaters to establish if 

there are significant differences to justify additional studies to support applicability 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 several finding categories in the table below. The 

reports and articles provide (1) overall assessments on whether criteria developed on the basis of 

studies of coastal waters (marine/estuarine and Great Lakes waters) are 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 differences in implementing 

new or revised criteria to inland and coastal waters, 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 suggestions provided by each report’s authors. Major 

findings as reported in the reports for each of the categories are summarized below (not in order 

of importance). Note that the table presents the major findings as proposed by the study authors. 

The findings differ in the degree to which they are 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. physical and biological processes). This 

observation is consistent with findings from quantitative microbial risk 

assessment (QMRA) studies and the limited 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 the 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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Inland Waters Summary Report November 2010 

Finding category Conclusions and observations 

Experts participating in the 2009 Water Environment Research Foundation 

(WERF) inland waters workshop speculate 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 are 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 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. 

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 typical of coastal sites because of differences 

in age of fecal pollution, presence and concentration 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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Inland Waters Summary Report November 2010 

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 effects observed in epidemiology studies of coastal 

waters. A decision to extend or not extend those criteria to inland waters must be supported by 

analysis of the science of inland and coastal waters as well as analysis of practical concerns, such 

as likely differences in use and sampling of inland 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 necessary to inform extrapolation 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 conclusions 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 reports 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 literature, 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 generally relate to perceptions 

or findings that indicator organisms 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 water and (2) the fate and transport of indicators 

relative to pathogens and loads from specific fecal pollution sources. Practical concerns relate to 

the development of monitoring schemes and interpretation of sample results for inland waters. 

Paramount among those concerns is the interpretation for all waters of indicator densities from 

samples collected during and immediately after precipitation (especially rain) events (NRC 

2004). 

To develop context for this report, PowerPoint presentations from stakeholder meetings and 

correspondence between EPA and stakeholders were reviewed. Questions drawn from those 

communications are presented below. The questions 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 the performance of indicators in inland and 

coastal waters? What are alternative criteria appropriate for fecal pollution sources other 

than publicly owned treatment works (POTW) effluent? 

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Inland Waters Summary Report November 2010 

 Does indicator performance differ in flowing waters, impounded inland and coastal 

waters? 

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

 If E. coli is a better indicator of fecal pollution in freshwaters, could Enterococcus criteria 

be equally protective of human health for inland and coastal waters? 

Questions related to implementing 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 relaxed during extreme-flow events? 

 How will culture methods be integrated with new or revised criteria? Is a rapid method 

needed for inland waters, particularly those with low use? 

1.2. Methodology 

1.2.1. Documents reviewed 

Eight studies were originally proposed 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 below). The condensed version of the workshop 

proceedings was reviewed separately because it has 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 Workshop on Critical Research and Science Needs 

for the Development of Recreational Water 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 Variability (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 Sources of Microbial 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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Inland Waters Summary Report November 2010 

8. Results of the Single-Laboratory Validation of EPA Method A for Enterococci and 

Method B for Bacteroidales in Waters by TaqMan® Quantitative Polymerase Chain 

Reaction (qPCR) Assay (USEPA 2010d) 

In addition, the following five key articles were selected and reviewed 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 Culturable Enterococci Densities with 

Hydrometeorological Conditions” (Byappanahalli et al. 2010) 

13. “A Cross Comparison of QPCR to Agar-Based 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 five published research articles (#9 to 13), are 

discussed together in Section 2.3 to condense 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 divided into topics that 

inform the performance of indicators and topics related to differences in implementing criteria in 

inland and coastal waters. 

Topics related to the performance of indicators in inland and coastal waters include findings on 

the intrinsic physical, biological, and hydrologic differences between 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 coastal 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 coastal waters entails review of epidemiology, 

hydrodynamics, fecal pollution sources, use patterns, 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 specific factors pertinent to extending coastal water 

criteria to inland waters reviewed in each study are presented in Table 1. 

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Inland Waters Summary Report November 2010 

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 quality and environmental factors on 

FIB DNA persistence in marine settings 

“Discrimination of Viable and Dead Fecal 

Bacteroidales Bacteria 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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Inland Waters Summary Report November 2010 

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 flowing, inland impounded, and Great Lakes 

coastal settings 

Inland waters are waterbodies that are not coastal recreational waters as defined by the 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) waterbodies, but not the Great Lakes, which are defined as 

coastal waters under the Beaches Environmental Assessment and Coastal 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 readers understand and interpret findings of the 

studies reviewed. Two types of comparisons are provided. In Section 1.3.1, the physical, 

hydrological, and biological differences in coastal and inland sites are described. Such 

differences influence indicator fate and transport and the association of indicators 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 inland 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 inland and coastal waters are diverse, generalizations about these 

waters are provided in this section as an introduction to the findings of the studies reviewed for 

this report. Intrinsic physical differences between inland and coastal settings include the 

following: 

 the ways in which the sites are loaded with fecal pollution; 

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

 hydrograph and indicator organism density responses to rain events; 

 the locations and mechanisms important in resuspension of sediment indicators; 

 the dilution of fecal pollution loads; and 

 average insolation (average incident solar radiation per water surface area). 

Those differences are illustrated in Figure 1 and Figure 2 and are described below. Other 

important differences between recreational sites include the predominant fecal pollution 

source(s) and the frequency of use for recreation. Those differences are not intrinsic to inland 

and coastal waters, and no data sources providing means for quantitative assessment of their 

importance were reviewed for this report. 

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Inland Waters Summary Report November 2010 

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 Waters Summary Report November 2010 

Inland and coastal waters receive fecal indicator bacteria (FIB) from point sources, diffuse 

sources including nonpoint sources, direct deposition (e.g., gulls or cattle in a stream), and 

resuspension of FIB in sediments or overbanks. It is likely that the overall predominant sources 

in inland waters differ from those in coastal waters. However, for a specific stream compared to 

a specific coastal water, the sources might be the 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 exposure necessary to observe a given response (e.g., 

adverse health effect) in the exposed population also differs. 

FIB loading and hydrodynamics in POTW-impacted inland and coastal waters are generally 

similar. POTW discharges to both inland and coastal 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 for inland and coastal sites. Non-POTW fecal 

pollution is loaded to receiving waters primarily 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 receiving fecal pollution are lower than those of 

coastal waters, fecal pollution in inland waters is expected to be generally less dilute than that in 

coastal waters. Because dose-response functions for individual pathogens are not linear, 

differences in dilution in coastal and inland waters might result in differences in incidence of 

illness typical of the two sites. Differences in incidence 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 and sediments, the ratio of the sediment area to 

the water volume, and the mechanisms most responsible for resuspension of sediment-associated 

indicator organisms. Together, those factors can cause differences in the abundance of 

resuspended indicator organisms in the two settings. 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 quality from indicator 

density measurements. None of the studies reviewed for this report sought to associate inland or 

coastal waters with specific soil and sediment types or to compare and contrast the extent to 

which growth is likely in either setting, although one report (WERF 2009) asserts that growth is 

more likely in the soils and sediments of inland waters and at the water-sediment interface. In 

inland waters, resuspension is by means of turbulent shear at the stream bottom, while in coastal 

waters, resuspension is primarily due to wave action. The net resuspended indicator load at a 

given site is a function of both the abundance of sediment-associated indicators and the processes 

by which free or particle-associated organisms are drawn from the sediments. Although a 

quantitative comparison of resuspension in inland and coastal waters is not possible, it is likely 

that resuspension is different in inland and coastal waters because of the different sediments 

typical of the sites and the very different mechanisms that cause resuspension in the two settings. 

No information in the documents reviewed indicates whether the differences would affect the 

relationship between indicator density and adverse health outcomes in exposed persons. 

1.3.2. Brief review of epidemiology studies conducted for inland waters 

At present, epidemiology studies provide the best 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 and health risks via modeling. Recently published 

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Inland Waters Summary Report November 2010 

QMRA studies indicate the potential for connecting indicator densities and health effects via 

modeling (Schoen and Ashbolt 2010; Soller et el. 2010a, 2010b). 

As context for the studies reviewed in this document, a brief review of epidemiology studies 

conducted at inland sites is presented below. These studies were conducted for different purposes 

and with different methodologies, and these differences 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 association of the incidence of 

health endpoints with the level (density) of indicators to which swimmers were exposed. In some 

cases, no statistically significant association is observed. That lack of association could relate to 

a lack of association between the indicator and a specific fecal pollution source or could relate to 

the epidemiology study design. In a limited number 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 have 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 Commission [EC] 2009a, 2009b [referred to as Epibathe 

studies]; Marion et al. 2010). They are grouped below as follows: 

 investigation of (presumably) POTW-impacted inland sites (Dufour 1984; Weidenmann 

et al. 2006; EC 2009a, 2009b; Marion et al. 2010); 

 investigation of untreated domestic sewage affected inland flowing waters (Ferley et al. 

1989) 

 investigation of unspecified inland waters1

 with unspecified fecal sources (Seyfried et al., 

1985a, 1985b) 

 investigation of flowing and impounded inland waters (Dufour 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 (Calderon 1991; Dufour 1984; Marion et al. 2010; 

Stevenson et al. 1953; Seyfried et al. 1985a, 1985b); and 

 use of retrospective cohort study design (Ferley et al. 1989). 

In all of those epidemiology studies, statistically significant differences 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 incidence 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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Inland Waters Summary Report November 2010 

1984; Seyfried et al. 1985b; Marion et al. 2010), and one study related indicator density to GI 

illness risk via estimation of a no adverse effect level of indicator organism density 

(Wiedenmann et al. 2006). No such relationships have been proposed for livestock and wildlifeaffected inland waters, although two studies (Seyfried et al. 1985a, 1985b; Wiedenmann et al. 

2006) pooled illness rates and indicator densities without regard to fecal pollution source. 

Calderon (1991) found no association between indicator level and incidence of GI illness in 

recreational waters with exclusively 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 beaches 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 control trial (RCT) study design. At 

present, results from PC and RCT study designs cannot be used interchangeably, and no 

techniques currently exist for converting their statistical outputs to allow meaningful quantitative 

comparisons across study designs. 

Based on these studies, E. coli (as measured by the culture-based 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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Inland Waters Summary Report November 2010 

2. Documents review 

This section is comprised of a summary of findings (Tables 2 through 7) and a detailed review of 

the documents (Section 2.2). Five out of eight reports (#1 to 4 and 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 Section 2.3 to compile aspects of 

methodology performance with respect to applicability of criteria 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 coastal 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 science 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 presence 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 inactivation) 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 imperative 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 position 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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Inland Waters Summary Report November 2010 

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 water 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) the 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 pollution 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 treatment facilities than coastal waters. 

Implementation realities group findings

 Approaches that have been used or could 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 analysis approaches and statistical 

measures) for assessing water quality; and 

o using temporary or permanent 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 based 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 specific data. The major limitation/data gap 

identified in the WERF report was the scarcity of epidemiology studies of inland 

waters. 

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Inland 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 diseases 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 variability 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 sanitary 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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Inland Waters Summary Report November 2010 

Table 4. Study purpose, methodology, findings and limitations for Literature Review of 

Assessment of the Applicability of Existing 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 pathogens 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, can be applied to inland waters that are 

also affected primarily by POTW effluents. 

 For inland waters that are affected predominantly by sources other than POTW 

effluent, the available science is not sufficient to support the extension of the 

relationships developed in the Great Lakes 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, insolation, 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 literature 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 culture-based membrane filtration. 

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Inland Waters Summary Report November 2010 

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, respectively. 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 monitoring are important components in the 

development of beach monitoring plans. These 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 develop 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 correlation 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 survey and limited to the studies reviewed. 

 Most of the studies reviewed reported results 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 characterization 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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Inland Waters Summary Report November 2010 

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 discharges 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 measurements for a variety of dischargesof-concern to recreational waters across 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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Inland Waters Summary Report November 2010 

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 influence 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-based methods with qPCR assays. This 

leads to the following: 

o qPCR cell equivalent counts are consistently 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), prompting the design of live-only qPCR assays, 

such as PMA-qPCR; 

o accounting for different persistence 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 reviewed 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 strongly reduced (2-5 orders of magnitude) 

through wastewater treatment processes, especially by secondary treatment and 

disinfection; qPCR inventories experience 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 recreational waters receive greater fluxes 

per volume of solar radiation (due to higher surface area-to-volume ratio 

compared with streams), afternoon differences 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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Inland Waters Summary Report November 2010 

Limitations  Data and experience with qPCR are limited; no single epidemiological study 

reviewed 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 need 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 recreational water research can be 

extrapolated to apply to inland waters. 

2. Identify additional near- and long-term research that could aid in the development of 

water quality criteria applicable to inland waters. 

The workshop was organized around the following five thematic areas: 

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

 health risks: epidemiology and risk assessment; 

 water matrix: hydrology, chemistry, 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 differences in water quality 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 have smaller volumes and less dilution potential 

than coastal waters. 

 The banks of inland waters are more favorable for tree growth, which could result in 

greater shading of inland waters than coastal waters. Such shading is significant because 

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Inland Waters Summary Report November 2010 

sunlight inactivation is a significant factor in the extra-enteric persistence 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 report question that assertion. Flows in inland waters are 

characterized by higher velocities than those observed in coastal waters. Those higher 

velocities can keep particles and particle-associated bacteria suspended. 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 physical conditions that are 

more conducive to FIB growth than the soils 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 growth in coastal sediments and sands has been 

documented in many studies, and no studies definitively support a greater propensity for 

FIB growth in the inland environment 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 quality criteria based on Great Lakes epidemiology 

studies for inland waters. That group’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 and inland waters likely face similar risks but 

that the presence of non-fecal indicator sources at sites could result 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 either fresh and marine sites when culture-based 

methods are used for measuring indicator organism 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 with 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 were 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 relatively small number of 

studies. In general, the group members hypothesized that 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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Inland Waters Summary Report November 2010 

Weighing the findings from the epidemiology studies 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 underprotective of public 

health. 

 Position 2—applying the results from the Great Lakes studies to inland flowing waters is 

a reasonable step based on supporting information in the literature 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 generally 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 public health at inland waters.” 

Water matrix

The group noted the importance of fecal pollution 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 indicators 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 interface area to water volume 

for inland waters, (2) finer sediment sizes typical in inland waters, (3) greater presence of 

wetting and drying areas (per volume of water), and (4) relatively high 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 used for systematic evaluation of differences 

between inland and coastal sites: 

 regression models, 

 mechanistic models, and 

 QMRA models. 

Each of those models is associated with limitations. Regression models have, to date, been 

formulated on the basis of relatively limited sets of water quality and physical condition data. 

That limits the applicability of the models for sites 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 waters, mechanistic models should include the 

following: 

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Inland Waters Summary Report November 2010 

 three-dimensional models of indicator transport for coastal sites, inclusive of wind-driven 

flows; 

 accurate modeling of dilution, turbidity, and shading; 

 association of inland sites with finer particle sizes and greater association of 

microorganisms with particles; and 

 higher organic content and potential for regrowth for inland sites. 

Although the Water Matrix Group did 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 generalization of results of 

individual simulations. 

The group noted that QMRA modeling would have to overcome great uncertainty 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 suited, including exploration of relative 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 livestock) the relationship between indicator level 

and health effects for different fecal pollution sources 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, and the persistence of pathogens in the fecal 

pollution source relative to the persistence of indicator organisms. The prevalence and 

abundance of both FIB and pathogens varies widely between fecal pollution sources and within 

each fecal pollution source. 

The Sources Group stated that because the prevalence 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 fecal pollution sources are more associated with 

inland waters than coastal waters. Note that septic systems differ widely in their design and 

operation and likely result in fecal pollution loads that vary widely. Additionally, runoff from 

urbanized areas with high proportions of impervious 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 sources to receiving waters, differences in pathogen and indicator 

density and prevalence in source materials, differences in loading during rain events, and 

differences in land use. The group hypothesized that coastal waters are generally more associated 

with urbanized land use, higher impervious surface 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 association of coastal and inland waters with specific sources of fecal 

pollution and characteristics are hypotheses. 

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Inland Waters Summary Report November 2010 

The group concluded by stating that knowledge gaps—particularly for the abundance, 

prevalence, and pathogenicity of pathogens in livestock and wildlife 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 Lakes/coastal studies could be extrapolated to 

inland waters. 

Implementation realities 

The group focused on flexibilities that are present in non-U.S. beach programs and might be 

considered for implementation with new or revised 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 studies. Additionally, states can designate specific 

classes of waterbodies or specific circumstances for different, scientifically defensible water 

quality standards. Such a designation could be made for waters known to be affected primarily 

by animal sources or for temporary changes in microbial water quality criteria following rain 

events. 

Flexibility is provided in World Health Organization (2003) standards by using sanitary survey 

findings in selecting water quality criteria appropriate for a specific site. Recreational sites 

without human fecal pollution sources and low bather density are considered to have good water 

quality at indicator densities higher than those for sites with known human fecal impacts or high 

bather densities. 

European Union bathing water quality standards (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 could be disregarded for classification purposes 

because of short-term pollution during the last assessment period. 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 beach sampling and water quality assessment are 

conducted in the context of other regulatory programs, including NPDES permitting, 

303(d)/TMDL use attainment assessments, and BEACH Act monitoring. Monitoring needs and 

realities differ for these programs and techniques and methods for assessing water quality also 

differ. Flexibility in the overall regulatory context might be provided with the issuance of water 

quality criteria on the basis 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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Inland Waters Summary Report November 2010 

Research priorities 

After assessing the state of the science and knowledge gaps in each topic area, the expert groups 

developed prioritized lists of suggested research activities intended to provide improved 

information for development or extension of criteria for inland waters. The priority short- and 

longer-term research activities proposed by the workshop participants 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 tools for implementation of new AWQC 

Longer-term Characterize fate and transport of animal pathogens in relation to indicators 

Conduct epidemiology studies in inland waters 

Table 9. Comprehensive list of proposed research 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. Include 

the 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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Inland Waters Summary Report November 2010 

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 its 

applicability 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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Inland Waters Summary Report November 2010 

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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Inland Waters Summary Report November 2010 

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 method 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 and 

exposure 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 publication (Dorevitch et al. 2010) describing the 

workshop findings and knowledge gaps related to the extension of criteria developed for coastal 

to inland waters. In the following section, the report findings are summarized. 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 

27 
Inland 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 interpreted and prioritized elements from the full study, the peerreviewed version of the report summary is also reviewed in this report. 

Differences between inland and coastal waters 

Dorevitch et al. (2010) identifies the following three 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 pathogens of similar infectivity and virulence. 

2. Hydrogeochemical differences among inland lakes, rivers, and coastal waters have 

nondifferential effects on the transport and fate of indicators and pathogens. 

3. The criteria derived from the studies conducted at sewage-affected coastal beaches 

protect against illness in inland settings, where 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 that inland waters are 

predominantly in rural areas and have a greater likelihood than coastal waters of being affected 

by agricultural or wildlife fecal pollution sources. Further, pathogens present 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 importance of source and etiology of illnesses arising 

from recreation, it does not quantify the extent to which coastal and inland waters are affected by 

different fecal pollution sources. Though many river miles 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 counties of the Great Lakes 

states support large populations of dairy cattle, while coastal counties on the Delaware and 

Chesapeake bays are locations of intensive chicken 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 and that inland water hydraulics generates greater 

suspended indicator loads than coastal hydraulics. Inland water sediments are believed to 

generate larger FIB loads because the extra-enteric 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 higher than that for coastal sites. The study also 

asserts that indicator organisms harbored or growing in sediments are more likely to be 

resuspended into the water column due to more favorable hydraulics for resuspension in inland 

waters because boundary layers (indicative 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 arising from non-fecal sources than the 

proportion in coastal waters. Thus, for inland and coastal sites with the same fecal pollution 

source, an indicator density observed in inland water is likely to correspond to a different 

28 
Inland Waters Summary Report November 2010 

presence of fecal pollution and a different risk than for the coastal water at which the same 

indicator density is observed. Such a decoupling of indicator organisms from fecal pollution 

sources represents a significant difference in indicator performance for inland and coastal waters. 

Hydrogeological differences might influence indicator performance in inland and coastal waters. 

First, fecal pollution sources are often 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 associated 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, higher indicator densities are associated 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 settings. Inland waters are subject to highly 

variable volumetric flow rates, bottom shear stresses, and turbulence. Those parameters are less 

variable for coastal waters. Advection dominates fecal pollution transport in flowing inland 

waters, whereas advection via longshore currents, other large-scale currents, 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 variation 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 described above, turbulence can promote decoupling of 

indicator density from fecal pollution sources. 

Critical research questions and a research agenda 

The report proposes four areas encompassing the research that should be conducted to improve 

the understanding and estimation of health risks arising from recreation in inland waters. The 

four areas are presented below, along with suggested short- and long-term research activities for 

closing data gaps and improving understanding. 

1. Microbial indicators as predictors of risk 

The spatiotemporal variability and determinants of FIB need to be characterized for 

hydrologically diverse settings. Although not detailed in the report, these diverse settings include 

small rural streams, small urban streams, large flowing waters, and impounded waters. Shortand long-term research suggested include meta-analysis of epidemiological relationships 

developed for waters affected by different fecal 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 sources 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 studies, optimization and anchoring of QMRA by 

means of data and relationships from epidemiology studies, and field sampling of feces from 

farm animals and wildlife to quantify the occurrence and dynamics of pathogens of human 

29 
Inland Waters Summary Report November 2010 

concern. In the long term, the report suggests that epidemiology studies should be conducted at 

inland sites with different dominant fecal pollution sources. Epidemiological studies should 

differentiate between pathogens posing different hazards, particularly 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 optimized. A database describing the relationships 

between molecular- and culture-based determinations 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 ranging from small inland waters to large inland 

waters to coastal sites. The persistence of molecular method targets 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. Quantitative PCR methods, particularly for source-specific 

markers, should be optimized, standardized, and applied in epidemiology studies. 

4. Other approaches for predicting inland water recreation health risks 

The other approaches suggested by Dorevitch and colleagues for predicting risks include 

predictive modeling (such as Nowcast forecasting) 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 activities suggested include evaluation of the viability of regression 

models and mechanistic models for risk management 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. Monitoring and forecasting systems are in use for 

managing risks to drinking water systems with surface sources. Numerous well-tested 

contaminant transport models are available for mechanistic modeling of FIB or pathogens in 

streams; given the hydraulics and geometry of streams, these models are much less complex than 

flow models of coastal sites and have the potential for use in real time. Challenges to use of 

regression and mechanistic models 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 used to relate indicator and pathogen fate and 

transport to risk. When used in tandem with epidemiological analyses, QMRA can add context to 

epidemiology study findings. In the absence of epidemiology studies (as is currently the case for 

inland waters), QMRA can be used to develop estimates of human health 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 scientific community and must provide sufficient data to allow realistic 

risk estimates specific to the geography, biology, and hydrology of inland waters. The report 

advocates anchoring QMRA (retrospective studies) as a short-term research need. Such an 

30 
Inland Waters Summary Report November 2010 

exercise would necessitate generating improved 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 inland 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 clear understanding of the proportion 

of indicators that is directly related to a specific fecal pollution source and the proportion not 

related to fecal pollution sources and thus indicative of a different and likely lower risk. 

The authors reason that applying criteria derived on the basis of epidemiology studies of coastal 

sites to inland waters should 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 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 coastal 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 above (Dorevitch et al. 2010; 

WERF 2009), background material was assembled 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 expand upon sections pertaining to epidemiological data and health 

effects. The document (USEPA 2010a) underwent 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 compare the performance of indicators in inland and 

coastal waters. The authors compare the performance of indicators in coastal and inland waters 

on the basis of the following features: 

 demonstrated correlation with health risk; 

 similar or greater survival time 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 features 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 the health effects associated with recreation in 

surface waters; (2) the occurrence and variability of indicators in coastal and inland settings; and 

(3) the persistence and growth of indicators and the association of indicators with pathogens. 

Absent from the report are discussion of differences in resuspension for typical inland and 

coastal waters, discussion of differences in typical fecal pollution sources for different settings, 

31 
Inland Waters Summary Report November 2010 

discussion of suites of pathogens characteristic of different fecal pollution sources, and 

evaluation of the role of dilution in indicator performance. 

Epidemiology and modeling studies 

To date, epidemiology studies have demonstrated correlations of health risk with indicator level 

only for waters primarily affected by POTW discharge. 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 studies, 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 cautiously, because the health effects relations in the Dufour study are 

based on seasonal averages of indicator densities 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 effects observed in the two studies 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 for 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. However, in the absence of epidemiology studies, 

modeling studies could provide a means for evaluating 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 occurrence of pathogens for non-POTW sources—including cattle manure, 

swine manure, chicken manure, and gull droppings. Those studies rely on pathogen and indicator 

occurrence and abundance as reported in the literature for estimating the pathogen suite and 

resulting illnesses rates associated with ingestion of runoff from livestock operations or from 

beaches contaminated with gull droppings. Those models are stochastic and account for the 

variability in the occurrence and abundance of pathogens and indicators in fecal sources. The 

QMRA models indicate that, at a given indicator level, recreation in waters affected by cattle 

runoff poses a similar risk to recreation in waters affected by human fecal pollution sources, 

whereas recreation in swine-, chicken-, and gull-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 finding that different fecal pollution sources have 

different risks at the same indicator level does not consider inland and coastal sites to be 

substantially different. Rather, it is presumed that the source, not the setting, is the critical feature 

of a site. 

Watershed modeling is a related but alternative modeling technique that can be used as an 

alternative to epidemiology studies for assessing health risks association with recreation in 

surface waters. Like QMRA models, watershed models rely on data collected from external 

sources as input for models that predict net pathogen loads watersheds discharge to receiving 

32 
Inland 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 comparing 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 spread and of skew in distribution of occurrence, 

could not be used. Moreover, studies were limited to relatively few waterbodies and each study 

had different designs and objectives, so how representative their data are with respect to the 

overall distribution of FIB among inland and coastal waters is uncertain. Thus, comparisons of 

the occurrence of indicators in coastal 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 allow 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 waters 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 concludes that the occurrence of indicators in inland 

waters and in coastal waters is not substantially different. It is important to note that none of the 

studies used in developing the occurrence ranges 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, representing 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 sediments, 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 sufficiently 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 reported growth of enterococci 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 indicators or whether there are fewer studies on enterococci. The presence 

33 
Inland Waters Summary Report November 2010 

of sediments is a primary factor in determining whether growth 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 result in a different proportion of indicators in 

inland waters arising from sediments than the proportion in coastal waters. The proportion of 

indicators attributable to resuspension from sediments is determined by the density of indicators 

in the sediments and the resuspension of the indicators via turbulence arising either from shear 

stress at an inland water streambed or from wave action or tidal 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 water types, indicator decay rates depend on the same 

physical factors and are widely variable. Contrary to expectations, reported inactivation rates for 

marine and freshwaters overlapped. Studies have established decreased persistence of E. coli

with salinity, so the overlapping ranges indicate that the combination of factors determining 

survival are such that their net effect is inactivation rates spanning the same range. Fewer studies 

were found providing Enterococcus inactivation rates. The studies reviewed in USEPA (2010a) 

indicate that the same factors govern the persistence 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 enumeration in inland waters and there are no available 

studies that have established health effects relationships for inland waters based on qPCR. 

Therefore, the health effects associated with inland waters and coastal waters must be deduced 

either from a direct association 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 inland 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 clear 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 indicators and pathogens via ultraviolet (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 inland 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 occur, 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 

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Inland Waters Summary Report November 2010 

Sampling and Consideration of Variability (Temporal and Spatial) for Monitoring of 

Recreational Waters (USEPA 2010b). That report describes the temporal and spatial variations 

in indicator densities for both inland and coastal waters and at all 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 review of the literature produced 

the following findings: 

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

 Temporal variability, from greatest to least, is as follows: Event variability (rain events) > 

diurnal variability > tidal-time-scale variability (coastal sites only) > monthly/seasonal 

variability (considering only the recreational use season) > short-time-scale variability 

(for samples taken at knee depth and greater). 

 For coastal sites, sources of spatial variability, from greatest to least, are as follows: 

variation with depth of sample collection > variation with site features such as point 

sources or features inhibiting mixing > along-shore variation > 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 > variation with depth below the water surface where sample is 

collected > cross-stream variation (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 anticipated at that site. Thus, data should be collected before monitoring 

scheme development to quantify spatial and temporal variability. Two vehicles suggested for 

collecting those data are sanitary surveys and pilot monitoring activities. Perhaps most important, 

sanitary surveys include collection of data on the fecal pollution sources with the potential to 

affect a site and the route by which fecal pollution could be delivered to the site. For coastal 

waters, transport modeling can be complex, given variability in currents, wave-generated 

turbulence, and such. For inland waters, identification 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 densities during rain events. As noted in that report, extremely high 

indicator densities can arise in both inland and coastal waters during 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 acceptable 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 should be selected based on the ability of a small number of 

samples to adequately describe water quality at the site and should target areas of beaches in 

35 
Inland Waters Summary Report November 2010 

closer proximity to fecal pollution sources (portions of the beach with significantly different 

mixing should be sampled separately). In addition, 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 provides 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 indicators 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 waters 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 relative contributions of different fecal 

pollution sources can be determined for a particular site. Knowing the contribution of the 

sources will allow improved risk estimates for 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, the general features of indicator and pathogen 

dynamics are known for inland and coastal sites, but quantitative data for relating 

different processes affecting risk are not available. 

3. QMRA models for recreational exposure are becoming increasingly 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 QMRA 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 collection on pathogen and indicator occurrence and 

abundance in diverse fecal pollution sources (literature survey and field study); development and 

characterization of the performance 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 fecal pollution sources to health risks. 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 abundance in specific fecal pollution 

sources, and the development of source-specific QMRA models. Each of these areas is reviewed 

below, with findings most closely related to differences in inland 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, and rotaviruses) and indicator organisms (e.g., 

36 
Inland Waters Summary Report November 2010 

Bacteroidales, Enterococcus, and E. coli) in treated and untreated sewage, urban runoff, runoff 

from undeveloped sites, livestock feces, wildlife feces, and companion animal feces. 

Field studies involved sampling wastewater treatment plant, CSO, and stormwater effluents prior 

to mixing with receiving waters. Because the drainages contributing to the 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 used to enumerate pathogens and indicators (to 

the extent that both methods are possible for a given microorganism). Novel or particularly 

relevant findings from the field 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 pollution 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 prevalences 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 specific settings, because risks due to multiple 

pathogens are additive. 

Spiking experiments were conducted such that filtration 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 determined that using holding media stabilized 

bacterial populations without adversely effecting protozoa. The authors also noted that from raw 

data that, in some cases, recoveries above 100 percent were realized, 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 QMRA studies. Correctly 

characterizing pathogen densities is very important given the low 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 pollution source. The model incorporated data on 

the prevalence and abundance of all the priority 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 sources among coastal and inland waters. 

In general, viruses were consistently found to be the risk drivers for all sources, with norovirus 

producing the dominant health risk, even for agricultural runoff. The authors do not comment on 

the degree to which animal noroviruses are host-adapted 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 

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Inland Waters Summary Report November 2010 

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

compared with inland waters. 

The authors discuss methods by which QMRA might be used for generating new recreational 

water criteria or in implementation of those criteria. An important precursor to such uses is 

calibration of the models based on findings of epidemiology studies and development of models 

with specific components that reflect site characteristics ascertained 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 scenarios for sites, whether they are inland or 

coastal. 

Quantitative MST 

Along with QMRA model development, the authors developed a quantitative MST methodology 

based on quantification of universal, 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 comparison of risks between sites (including inland 

and coastal sites). The performance of the quantitative 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 findings 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 coastal waters. The emphasis of this section is 

method performance and relative differences in qPCR and culture targets, rather than the 

association of indicators (as measured 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 the plant is discharging to an inland 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 indicator bacteria in environmental 

waters and the factors that may impact qPCR performance. 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 extent to which those factors differ in inland 

and coastal settings is yet to be established. The section concludes with a review of several 

studies directly comparing culture and qPCR method performance in inland and coastal waters. 

38 
Inland Waters Summary Report November 2010 

Factors influencing method performance 

Generally, FIB densities derived from qPCR have been consistently reported higher by several 

orders of magnitude than those derived from culture-based assays in both coastal and inland 

water settings, 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 assay. All DNA present in a sample is amplified 

equally, regardless of the viability status of its host. This total DNA inventory is comprised of 

free and dead cell DNA (constituting ambient background DNA), viable but not culturable 

bacterial (VBNC) DNA, which is predominant with respect to the last culturable and viable 

fraction (corresponding to the cells enumerated by culture-based methods). 

In report # 7 (Final report: Comparative Evaluation of Molecular and Culture Methods for Fecal 

Indicator Bacteria for use in Inland Recreational Waters (WERF 2010b)) the study authors view, 

based on the results of their study, is that qPCR methods, as currently optimized, cannot be 

applied universally across all inland water bodies. Differences 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, unresolved inhibition, between-lab method variability 

(e.g., difference in extraction efficiency or development of calibration curves), and the relative 

contribution of DNA from viable v. non-viable cells. All of these factors vary among 

waterbodies and laboratories and are 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 comparing 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 (qPCR counts) for samples taken on a given day were 

nearly always one order of magnitude and frequently two orders of magnitude greater than those 

for the MF method. Haugland and colleagues suggested 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 distinguishing live and dead cells, 

below). This finding is significant to the difference in inland and coastal waters in that the 

persistence of indicators and relative abundance of live culturable 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 beaches 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 samples collected at 3 PM. These plots demonstrate 

the importance of sunlight inactivation on culture counts and the relative insensitivity 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 culturable 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, this correlation is lost at low 

39 
Inland Waters Summary Report November 2010 

culture-based densities due to high variability of the qPCR in this range and predominance of 

VBNC over culturable bacteria and possibility due to the high uncertainty of qPCR assays when 

fewer than 100 cells (or qPCR targets) are present (WERF 2010b). These findings may be of 

significance in the comparison of inland and coastal 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 culturable 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 propidium monoazide (PMA) to remove DNA from non-intact cells 

from the PCR reaction, resulting in better correlations with culture-based methods. For samples 

from the wastewater plant effluent, gene copies from qPCR with PMA were only 30 percent of 

those from qPCR without PMA. The difference between qPCR with and without PMA was 

greater than two orders of magnitude for samples of wastewater plant effluent. If optimized, 

these new techniques have the potential for making the analysis of coastal and inland waters 

more consistent. 

40 
Inland Waters Summary Report November 2010 

POTW effluent 

Strong reductions of cultivable FIB (2-5 orders of magnitude) have been observed throughout 

wastewater treatment, especially during secondary treatment and disinfection. Although this 

illustrates the ability of such treatments to inhibit cultivability, it does not necessarily 

demonstrate that the treatment trains have caused 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 criteria design but also from the standpoint 

of public health, highlighting the potential for a large proportion 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 could be greater than that for coastal waters. 

Some water matrices may have a strong influence on the comparison of qPCR cell equivalents 

and MF CFUs. For example, PCR amplification 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 unchlorinated 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 enumerations part of the time and below culture 

enumerations for other samples. Two plausible 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 measure different features of bacteria and those 

features vary differently between samples. In the case of chlorinated secondary effluent, 

relatively high enumerations by qPCR potentially indicate the presence of dead cells or extracellular DNA. For samples from a marine environment (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 relative abundances of enterococci as measured by 

cultural methods and qPCR differed for samples taken 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 significantly different from 

that of the samples enumerated via cultural methods 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 the lake water as the cause for significantly higher qPCR counts 

than culture counts because samples were analyzed after a 1:5 dilution employed to prevent 

inhibition. Further, the authors report that the qPCR Enterococcus counts in the tributary and the 

lake water were not significantly greater than those 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 other loading of qPCR targets to the beach sites from sources other 

than the tributary. 

Telech et al. (2009) developed a series of regression models using several rapidly-measured 

environmental variables (e.g., rainfall, turbidity, number of bathers, 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 

41 
Inland Waters Summary Report November 2010 

using either type of analytical detection method (i.e., qPCR or membrane filtration), the variables 

exhibiting the strongest relationship with Enterococcus densities in the models were consistent 

within the analytical detection method. The authors noted that meteorological, physical, water, 

and beach characteristics explained more variability 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 their correlation with culture counts. 

Although empirical, their methodology relies upon a condition-specific correction and as such is 

related to the theoretical 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 discordance 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 increased densities of background DNA (not associated 

with viable culturable cells). To improve correlation between culture and qPCR densities, the 

authors proposed use of a correction factor for conditions associated with high background DNA 

densities. In the resulting model, if specific rainfall and wave heights 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 factors and the conditions under which they are applied would be 

substantially different for inland and coastal waters. 

Finally, report # 7 (Final report: Comparative Evaluation of Molecular and Culture Methods for 

Fecal Indicator Bacteria for use in Inland Recreational Waters (WERF 2010b)) reviewed for this 

study reports stronger associations of culture indicator density 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 modeling 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 also noted that for the 3 qPCR methods used, low 

levels of qPCR targets were hard to analyze for cell numbers below 100 cells. This shortcoming 

of qPCR may influence the correlation 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 among all sites, with distinct 

differences observed among inland and coastal sites when the two site types were compared 

directly. These differences may not be the result of intrinsic differences in inland and coastal 

sites but rather the result of site-specific differences in water quality or source characteristics for 

the sites studied. 

42 
Inland Waters Summary Report November 2010 

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