Report on the 2nd Five-Year Review of EPA’s Recreational Water Quality Criteria

Office of Water

EPA

822R23003

May 2023

Report on the 2

Five-Year Review of

EPA’s Recreational Water Quality

Criteria

Disclaimer

While this document cites statutes and regulations that contain legally binding requirements, it

does not itself impose legally binding requirements on EPA, states, tribes, other regulatory

authorities, or the regulated community. EPA, state, tribal, and other decision makers retain the

discretion to adopt approaches on a case-by-case basis that differ from those discussed in this

document as appropriate and consistent with statutory and regulatory requirements. This

document does not confer legal rights or impose legal obligations upon any member of the

public. This document does not constitute a regulation, nor does it change or substitute for any

Clean Water Act (CWA) provision or EPA regulations. EPA can update this document as new

information becomes available. EPA and its employees do not endorse any products, services, or

enterprises. Mention of trade names or commercial products in this document does not constitute

an endorsement or recommendation for use.

Acknowledgments

This report was developed by the U.S. Environmental Protection Agency’s (EPA’s) Office of

Science and Technology (OST), Office of Water (OW). OST/OW authors are Katie Bentley

(formerly EPA Region 3, currently at OWM), John Ravenscroft, Lars Wilcut, Tracy Bone,

Shamima Akhter, Adrienne Keel, Lesley D’Anglada (formerly at OST, currently at OITA),

Betsy Behl, Sara Hisel-McCoy, Susan Euling, Shari Barash (formerly at OST, currently at

OPPT), and Menchu Martinez. EPA authors from the Office of Research and Development

(ORD) are Orin Shanks, Tim Wade, and Rick Greene. EPA contributors, including to systematic

literature review screening, and section reviewers include Kevin Oshima (ORD), Donna Hill

(ORD), Elizabeth Hilborn (ORD), Jingrang Lu (ORD), Aabir Banerji (ORD), Armah de la Cruz

(ORD), Blake Schaeffer (ORD), Toby Sanan (ORD), Dan Tettenhorst (ORD), Asja Korajkic

(ORD), Alison M. Franklin (ORD), Rich Haugland (ORD), Marika Schulhof (former AAAS

Fellow in OST), Czarina Cooper (OST), Sharon Nappier (OST), and Tom Glazer (OGC).

Literature searches and screening and technical editing was conducted by ICF International LLC

(EPA contract number: 68HERC19D0003, Task Order number: 68HERC21F0443). ICF Task

Order Managers: Audrey Ichida and Lucas Rocha Melogno. ICF Technical Contributors: Kaedra

Jones, Laura Tuhela-Reuning, Caelen Caspers, Nidhi Patel, Madison Lee, Ryan Gan, Hannah

Eglington, Afroditi Katsigiannakis, Connie Wanchen Xiong, Jaycee Mayer, Chenyang Wang,

Lauren Browning, and Sara Schwarzkopf. ICF subcontractors: Jeff Soller and Mary Schoen.

Contents

I. Introduction ............................................................................................................................. 1

II. Background .............................................................................................................................. 3

A. Brief Description of the 2012 RWQC and Key Aspects of Implementation .................... 3

1. Criteria Magnitude, Duration, and Frequency: Geometric Mean and Statistical

Threshold Value ........................................................................................................ 3

B. How the 2

Five-Year Review Was Conducted ............................................................... 5

1. Scope and Methods of the Review ............................................................................ 5

2. Inventory of Scientific Information Published Since 2016 ....................................... 5

3. Summary of Recreational Criteria Implementation Tools Developed and EPA

Outreach and Training Since 2016 ............................................................................ 5

4. Sources of Information and How Information Was Accessed .................................. 6

5. A Systematic Review of Available Peer-Reviewed Literature ................................. 6

III. Findings of the 2

Five-Year Review ..................................................................................... 9

A. New Information on Health Effects after Exposure to Recreational Waters .................... 9

1. Recreational Water Epidemiological Studies ........................................................... 9

2. Use of QMRA to Understand Risks in Recreational Water Settings ...................... 16

3. Outbreak Studies of Illnesses in Ambient Recreational Waters Associated with

Enteric Pathogens .................................................................................................... 22

4. New Information on Children’s Exposure in Recreational Waters ........................ 25

5. Summary of Major Findings from New Information on Health Effects and

Children’s Exposure ................................................................................................ 27

B. New Information on Coliphages ..................................................................................... 28

1. Research on Coliphages .......................................................................................... 29

2. New Epidemiology Studies Including Coliphages ................................................. 32

3. QMRA ..................................................................................................................... 33

4. Studies of Wastewater Treatment Efficacy ............................................................. 33

5. Summary of New Information on Coliphages ........................................................ 34

C. New Information on Cyanotoxins ................................................................................... 34

1. RWQC Guidance Values and Health Advisories for Cyanotoxins ......................... 35

2. Health Studies ......................................................................................................... 36

3. Summary of New Information on Cyanotoxins ...................................................... 43

D. New Information on Antimicrobial Resistance ............................................................... 44

1. EPA AMR Research ............................................................................................... 44

2. Summary of New Information on AMR ................................................................. 45

E. New Information on Human and Non-Human FSI ......................................................... 45

1. Human FSI Research .............................................................................................. 46

2. Non-Human FSI Advances ..................................................................................... 48

3. Community-based FSI ............................................................................................ 49

4. Summary of New Information on FSI .................................................................... 50

F. New Information on Analytical Methods for Recreational Waters ................................. 51

1. FIB .......................................................................................................................... 51

iii

2. Coliphage Methods ................................................................................................. 53

3. Cyanotoxin Methods ............................................................................................... 55

4. AMR Bacteria ......................................................................................................... 56

5. Human FSI .............................................................................................................. 57

6. Summary of New Findings on Analytical Methods ................................................ 58

G. New Implementation Tools ............................................................................................. 58

1. Implementation of the 2012 RWQC ....................................................................... 58

2. Cyanotoxins ............................................................................................................ 60

3. Predictive Modeling Research ................................................................................ 61

4. Process Modeling Research .................................................................................... 61

5. Outreach and Grants ................................................................................................ 62

6. BEACH Act Grant Funding .................................................................................... 62

IV. Summary of Major Findings and Priorities for Further Work ............................................... 64

A. Summary of Major Findings ........................................................................................... 64

1. Summary of Findings Identified in New Epidemiological Studies ........................ 64

2. Summary of New Information on Coliphages ........................................................ 65

3. Summary of New Information on Cyanotoxins ...................................................... 65

4. Summary of New Information on AMR ................................................................. 65

5. Summary of New Information on FSI .................................................................... 66

6. Summary of New Findings on Analytical Methods ................................................ 66

B. Assessment of the Need to Revise the 2012 RWQC ....................................................... 66

C. Priorities for Further Work .............................................................................................. 67

V. References ............................................................................................................................. 71

Appendix A. Literature Search and Review Strategies .............................................................. A-1

Appendix B. Summaries of Studies Reviewed by EPA .............................................................. B-1

Tables

Table 1. 2012 RWQC Recommended GM and STV Values for 36 and 32 Illnesses/1,000

Recreators (NEEAR-GI Illness [NGI]) for Marine and Fresh Waters ............................. 3

Table 2. Recreational Criteria or Swimming Advisory Recommendations for Microcystins and

Cylindrospermopsin ....................................................................................................... 36

Figures

Figure 1. Process for Identifying Literature for Some of the Five-year Review Topics: 2016–

2021 ................................................................................................................................ 7

Acronyms

AFO animal feeding operation

ALT alanine aminotransferase

AMR antimicrobial resistance

aOR adjusted odds ratio

ARG antimicrobial resistant genes

aRR adjusted risk ratio

ATX anatoxin-a

AWQC ambient water quality criteria

B. theta Bacteroides thetaiotaomicron

BASINS Better Assessment Science Integrating Point and Nonpoint Sources

BAV Beach Action Value

BEACH Beaches Environmental Assessment and Coastal Health

BMAA β-N-methylamino-L-alanine

BNR biological nutrient removal

BTB blood-testes barrier

BUN blood urea nitrogen

bw body weight

CAFO combined animal feeding operation

CAT Catellicoccus

CCE calibrator cell equivalent

CDC Centers for Disease Control and Prevention

CE cell equivalents

CFR Code of Federal Regulations

CFU colony forming unit

CI confidence interval

CSO combined sewer overflow

CWA Clean Water Act

DAL double agar layer

dcNEOSTX decarbamoyl neosaxitoxin

dhATX dihydroanatoxin-a

D-HFUF dead-end hollow-fiber ultrafiltration

DMF direct membrane filtration

DNA deoxyribonucleic acid

dPCR digital polymerase chain reaction

E. coli Escherichia coli

eDNA environmental deoxyribonucleic acid

EFSA European Food Safety Authority

EPA Environmental Protection Agency

ESBL extended spectrum beta-lactamase

EU European Union

FDA Food and Drug Administration

FIB fecal indicator bacteria

FR Federal Register

FSI fecal source identification

g grams

GC gene copies

GI gastrointestinal

GI.1 norovirus genogroup I, genotype 1

GII.4 norovirus genogroup II, genotype 4

GJs gap junctions

GM geometric mean

GTX gonyautoxin

HAB harmful algal bloom

HDA helicase-dependent amplification

HESD Health Effects Support Document

HSPF Hydrological Simulation Program-Fortran

HPyVs human polyomaviruses

HUC Hydrologic Unit Codes

IC inhibitory concentration

IEM integrated environmental monitoring

IgG Immunoglobulin G

Inv-IMS/ATP inversely coupled immunomagnetic separation/adenosine triphosphate

i.p. intraperitoneal

kg-d kilograms per day

L liter

LAMP loop-mediated isothermal amplification

lethal dose 50 percent (median lethal dose)

LDH lactate dehydrogenase

LRV log reduction value

LOAEL lowest-observed-adverse-effect level

LWTX lyngbyatoxins

M. smithii Methanobrevibacter smithii

MC microcystin

mL milliliter

MPN most probable number

MRA-IT microbial risk assessment-interface tool

MSC male-specific coliphage

MST microbial source tracking

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide

µg microgram

NEEAR National Epidemiological and Environmental Assessment of Recreational

Water

NGI NEEAR gastrointestinal

nmol/kg nanomoles per kilogram

vii

NIST National Institute of Standards and Technology

NPDES National Pollutant Discharge Elimination System

NOAEL no-observed-adverse-effect level

NORS National Outbreak Reporting System

ODH Ohio Department of Health

OHEPA Ohio Environmental Protection Agency

OR odds ratio

ORD Office of Research and Development (U.S. EPA)

PDV phocine distemper virus

PFU plaque forming unit

PI3K/AKT phosphatidylinositol 3 kinase/protein kinase B

PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses

PSP Paralytic Shellfish Poisonings

QMRA quantitative microbial risk assessment

qPCR quantitative polymerase chain reaction

R-squared Pearson's correlation coefficient squared

RBT risk-based threshold

RfD reference dose

RNA ribonucleic acid

rRNA ribosomal ribonucleic acid

ROS reactive oxygen species

RPMI Roswell Park Memorial Institute

RT-PCR reverse transcriptase-polymerase chain reaction

RT-qPCR reverse transcriptase-quantitative polymerase chain reaction

RVA Group A Rotavirus

RWQC Recreational Water Quality Criteria

SAL single-agar layer

SCCWRP Southern California Coastal Water Research Project

SDWA Safe Drinking Water Act

spp. species (plural)

SRM Standard Reference Method

STEC Shiga toxin-producing E. coli

STV statistical threshold value

STX saxitoxin

STX-Cou Fluorescent coumarin-coupled STX

STXeq saxitoxin equivalents

SWAT soil and water assessment tool

SWCNT single-walled carbon nanotubes

SWCNT-COOH carboxylated single-walled carbon nanotubes

Ti/Abs title and abstracts

TJs tight junctions

TMDL Total Maximum Daily Load

TSM Technical Support Materials

viii

TMRNEL Tetramethylrhodamine nick end labeling

UK United Kingdom

U.S. United States

UV ultraviolet

WHO World Health Organization

WHOI Woods Hole Oceanographic Institution

WOS Web of Science

WQS water quality standards

WRRF water resource recovery facility

WWTP wastewater treatment plant

Executive Summary

The Beaches Environmental Assessment and Coastal Health (BEACH) Act amendments to the

Clean Water Act (CWA) Section 304(a)(9)(B) require the United States (U.S.) Environmental

Protection Agency (EPA) to conduct a review of the current 304(a) National Recommended

pathogen and pathogen indicator Recreational Water Quality Criteria (RWQC) every 5 years,

and, as necessary, revise the RWQC. In conducting this review, EPA considered several factors,

including the availability and evaluation of the latest scientific knowledge and additional

implementation support needs. EPA conducted a systematic review and evaluation of scientific

information and collection of updated information on recreational criteria implementation tools

since the previous 2017 five-year review. Specifically, EPA evaluated health and

epidemiological studies related to indicators of fecal pollution, studies related to children’s

health, new data on coliphages as indicators of enteric viruses, cyanotoxins health studies,

antimicrobial resistance, fecal source identification, studies addressing advances in analytical

methods, and implementation materials including advances in the use of models to predict water

quality and assess risk. This report contains extensive information in these topic areas along with

priorities for further work. EPA has completed a detailed review of the latest scientific

knowledge and has determined that current science supports a revision of the 2012 RWQC and

that there are several additional implementation tools that EPA can make available to manage

recreational waters.

The available science demonstrates an increased health risk for children compared to adults

when recreationally exposed to fecal contamination. Further, studies indicate that the use of

culturable fecal indicator bacteria alone to evaluate recreational waters impacted by human

sources can result in under-protection of human health. In these waters quantitative polymerase

chain reaction (qPCR)-based criteria (i.e., based on a molecular testing method using qPCR)

offer greater protection. Since 2017, EPA has published nationally validated protocols for qPCR

methods and codeveloped (along with the National Institute of Standards and Technology

[NIST]) a standard reference material for national use that supports the use of qPCR

technologies. To address these issues, first EPA plans to develop additional criteria

recommendations for qPCR-enumerated enterococci protective of children, which would also be

protective of all recreators. Second, EPA plans to continue to develop recommendations for

coliphages to help address potential risks from human enteric viruses in ambient waters. Third,

EPA plans to explore how best to use human fecal source identifiers, such as HF183, for water

quality management. Being able to demonstrate that a waterbody has been impacted by human

fecal contamination will enable risk managers to use the appropriate tools to evaluate and

manage risks for waters impacted by human sources.

I. Introduction

United States (U.S.) Environmental Protection Agency (EPA) has conducted a five-year review

of its 2012 Recreational Water Quality Criteria (RWQC), as required by the Beaches

Environmental Assessment and Coastal Health (BEACH) Act amendments to the Clean Water

Act (CWA) Section 304(a)(9)(B). The last review was conducted in 2017. In conducting this

review, EPA considered several factors, including the availability and evaluation of the latest

scientific knowledge and additional implementation support needs. The Agency used the

information in this “state-of-the science” report to assess whether new or revised RWQC are

necessary at this time.

The development of the 2012 RWQC and five-year reviews are requirements of the BEACH Act

of 2000. Through that Act, EPA has provided grants to states, territories, and tribes to implement

water quality monitoring and notification programs for coastal recreation waters

(including the

Great Lakes) since 2002. The 2012 RWQC included development of a beach advisory threshold

for use in posting swimming advisories and the recommended ambient water quality criteria

(AWQC). Swimming advisory decisions based on water quality monitoring are intended to

reduce the risk to recreators and other users of these waters from illness associated with exposure

to human fecal contamination and provide the public with information to make decisions about

their actions. AWQC developed under CWA Section 304(a) are recommendations based on the

latest science which states and authorized tribes can adopt as part of their water quality standards

(WQS). In the case of the 2012 RWQC, EPA’s recommendations were designed to protect

primary contact recreation, in all coastal and non-coastal waters designated for recreational use.

The criteria, once adopted by states and authorized tribes and approved by EPA under CWA

section 303(c), become part of the regulatory structure of the state or authorized tribe for

protection of primary contact uses for the applicable waters. The recreational criteria values that

are part of a state or authorized tribe’s approved WQS have a direct bearing on the issuance of

National Pollutant Discharge Elimination System (NPDES) discharge permits, waterbody

assessments, the decisions regarding attainment of WQS under CWA Sections 303(d) and

305(b), and the development of targets for total maximum daily loads (TMDLs) for restoring

impaired waters.

The criteria values specified in the RWQC are for densities of culturable fecal indicator bacteria

(FIB) in water. The FIB, enterococci, and Escherichia coli (E. coli) are not pathogenic under

usual circumstances, but their presence in water above specified levels can indicate the presence

of fecal contamination potentially containing viral, bacterial, or protozoan pathogens associated

The BEACH Act of 2000 defines coastal recreation waters as follows:

The term “coastal recreation waters” means:

(i) The Great Lakes; and

(ii) Marine coastal waters (including coastal estuaries) that are designated under section 303(c) by a Stat

r use for swimming, bathing, surfing, or similar water-contact activities.

The term “coastal recreation waters” does not include:

(i) Inland waters; or

(ii) Waters upstream of the mouth of a river or stream having an unimpaired natural connection with the

open sea.

with an elevated risk of illness. Therefore, ensuring that the RWQC are consistent with the

current state of the science and are protective of human health is key to protecting the health of

users of all waters designated for primary contact recreation.

EPA identified the following objectives for this review of the 2012 RWQC:

• Identify the latest science and information available since the publication of the 2017

five-year review that may impact the 2012 criteria.

• Discuss the status of adoption of the RWQC.

• Include the latest science and information on health effects relevant to RWQC,

coliphages, cyanotoxins, antimicrobial resistance (AMR), fecal source identification, and

analytical methods that have the potential to impact recreational uses.

• Identify additional indicators and microbial methods including those that have become

more refined or feasible and include this information.

• Provide information on the implementation materials for the 2012 criteria including site-

specific tools, implementation materials for cyanotoxins, predictive and process modeling

for both fresh and marine waters, outreach, and training.

An important goal of this review is to evaluate whether revisions to the 2012 RWQC are

necessary based on the overall review of new information provided in the previous 5 years,

described later in this report.

II. Background

A. Brief Description of the 2012 RWQC and Key Aspects of Implementation

The 2012 RWQC, which use enterococci and E. coli as predictors of gastrointestinal (GI)

illnesses in recreational waters, are described below along with several other important aspects of

how the criteria can be implemented.

1. Criteria Magnitude, Duration, and Frequency: Geometric Mean and Statistical

Threshold Value

The 2012 RWQC consist of three primary components: magnitude, duration, and frequency.

Magnitude: The magnitudes of the bacterial indicators are the measured densities of the FIB

from the water quality density distribution used for the criteria, expressed both as a geometric

mean ([GM] 50th percentile value) and as a statistical threshold value ([STV] 90th percentile value).

Duration: The duration is the period over which excursions of the magnitude values are recorded

and calculated. EPA recommended a duration of 30 days in the criteria for both the GM and the STV.

Frequency: The frequency is how often the GM or the STV are exceeded. EPA recommended no

exceedances for the GM over the period of the duration.

Because the STV reflects the 90th percentile of the distribution of values used to determine the

RWQC, the RWQC allowed for a 10 percent exceedance of the STV. EPA selected the estimated

90th percentile of the water quality distribution to account for the expected variability in water

quality measurements, while limiting the amount of time allowed to exceed the STV as a

threshold of water quality impairment.

EPA was clear that “both the GM and the STV would be part of the WQS, and therefore both

targets would be used to determine whether a waterbody attains the WQS for primary contact

recreation” (U.S. EPA, 2012a).

Table 1. 2012 RWQC Recommended GM and STV Values for 36 and 32 NGI

Illnesses/1,000 Recreators for Marine and Fresh Waters

CRITERIA

ELEMENTS

Recommendation 1

Estimated Illness Rate (NGI): 36/1,000

Recommendation 2

Estimated Illness Rate (NGI): 32/1,000

Indicator

(CFU/100 mL)

STV

(CFU/100 mL)

STV

(CFU/100 mL)

Enterococci

(marine and fresh water)

35 130 30 110

E. coli (fresh water)

126 410 100 320

Duration and frequency: The waterbody GM should not be greater than the selected GM magnitude in any 30-day

interval. There should not be greater than a 10 percent excursion frequency of the selected STV magnitude in the

same 30-day interval. NEEAR = National Epidemiological and Environmental Assessment of Recreational Water;

NGI = NEEAR gastrointestinal.

EPA recommends using EPA Methods described at https://www.epa.gov/cwa-methods to measure culturable

enterococci and E. coli. Units are colony forming units (CFU) per milliliter (mL).

Criteria values were provided for culture-enumerated FIB at two illness rates, 32 and 36 NGI

illnesses per 1,000 swimmers. Based on EPA’s analysis of the available information, either set of

thresholds protects the designated use of primary contact recreation and, therefore, protects the

public from the risk of exposure to harmful levels of pathogens from fecal contamination. The

two sets of numeric concentration thresholds included in the 2012 RWQC provide states and

authorized tribes flexibility to make their own risk-management decisions. The recommendations

for criteria illness rate apply to both marine and fresh waters, regardless of the intensity of use of

the beach.

In addition to recommending criteria values, EPA also provided states and authorized tribes with

Beach Action Values (BAVs) for use in notification programs. The BAV was defined as the 75th

percentile of the water quality distribution of values of E. coli and Enterococcus species (spp.) in

the epidemiological studies used by EPA to establish a health link between gastrointestinal (GI)

illness and levels of culturable FIB. EPA’s intent was to provide the BAV for states and

authorized tribes as a precautionary tool for beach management decisions. EPA recommended

the BAVs for use by the states for issuing beach notifications/advisories in their public health

programs, but not as part of the 2012 RWQC recommendations under CWA Section 304(a).

States, territories, and authorized tribes continue to make progress adopting RWQC. Thirty-one

jurisdictions have adopted, and EPA has approved, revised RWQC for all primary contact

waters. Three additional jurisdictions have adopted, and EPA has approved, revised RWQC for

their coastal recreation (i.e., BEACH Act) waters only. One jurisdiction only includes fecal

coliform as FIB in its WQS. Seven additional jurisdictions use fecal coliform as FIB for some

but not all of their waters designated for primary contact recreation (e.g., they use enterococci as

the FIB in their marine waters and fecal coliform in their fresh waters).

Although identified in 2017 as a possible barrier to adoption of the 2012 RWQC, criteria that are

based on use intensity does not appear to be hindering continued adoption of the 2012 RWQC, as

states and authorized tribes with coastal recreation waters have continued to adopt the 2012

RWQC without the inclusion of use intensities. More than half the states have adopted the 2012

RWQC or an equivalently protective criteria in all their waters designated for primary contact

and an additional three states have adopted the 2012 RWQC for their coastal recreation waters;

all authorized tribes with coastal recreation waters have adopted the 2012 RWQC.

While the culture-enumerated FIB at two illness rates were the recommended criteria in 2012,

EPA also included quantitative polymerase chain reaction (qPCR)-based values as additional

information. EPA developed and validated a molecular testing method using qPCR as a rapid

analytical technique for the detection and quantitation of enterococci in recreational water (EPA

Method 1611, EPA 2012b). EPA included qPCR-based values for the GM, STV, and BAV for

both illness rates protective of the general population in the 2012 RWQC document. Because of

potential matrix interference issues in water types other than those studied at the NEEAR

effluent-affected beach sites, EPA’s Office of Research and Development (ORD) continued

research to characterize and refine the qPCR methodology (Haugland et al., 2016) resulting in

the publication of Method 1609.1 (EPA Method 1609.1; U.S. EPA, 2015c).

Following publication of the 2012 recreational criteria, EPA provided additional information on

tools for evaluating and managing recreational waters, such as predictive modeling and sanitary

surveys, and stressed the need for a tiered approach to developing beach monitoring plans in the

2014 National Beach Guidance and Required Performance Criteria for Grants. The Agency also

provided Technical Support Materials for developing site-specific criteria and for the use of

alternative indicators or methods at recreational beaches (see

https://www.epa.gov/wqc/recreational-water-quality-criteria-and-methods).

B. How the 2

Five-Year Review Was Conducted

1. Scope and Methods of the Review

This section describes the measures EPA has taken to assess advances in the state of the science

supporting the 2012 RWQC since 2016, the date of the last five-year review’s literature cut-off

year (published in 2018). It also addresses advances in implementation. The measures include an

inventory of the relevant scientific information published since 2016, a description of

recreational criteria implementation tools applied at recreational settings, and information on

sources of information and how information was accessed.

2. Inventory of Scientific Information Published Since 2016

A thorough inventory of scientific information published since 2016 for topics central to

recreational waters monitoring and assessment is the core of this review (Figure 1). Several

general categories of relevant information were identified:

i. Health studies, including epidemiological studies, outbreak studies, children’s health

studies, and the application of quantitative microbial risk assessment (QMRA) to water

quality data and complex settings at recreational beaches.

ii. Summary of advancements in coliphage health studies and methods since 2016.

iii. Cyanotoxins health studies, methods, and criteria and implementation since 2016.

Note that cyanotoxin recreational criteria are not subject to the BEACH Act because they

are neither pathogens nor pathogen indicators. EPA included cyanotoxins in the literature

reviews since EPA has recreational criteria for two cyanotoxins.

iv. Summary of scientific advancements in AMR since 2016.

v. Fecal source identification (also called microbial source tracking in the literature),

including human and non-human fecal source markers and tracking.

vi. Performance, implementation, and updates of microbial methods for FIB and

alternative indicators 2016 to present.

3. Summary of Recreational Criteria Implementation Tools Developed and EPA Outreach

and Training Since 2016

A further category of activities and tools related to water quality monitoring and contextual

assessment of beach settings was identified as highly relevant to the implementation of the

BEACH Act and activities related to the 2012 RWQC. This category of implementation tools

includes:

i. Site-specific implementation tools

ii. Sanitary surveys and watershed assessments

iii. Cyanotoxins implementation tools

iv. Predictive modeling

v. Process modeling

4. Sources of Information and How Information Was Accessed

The collection and analysis of information in each of these categories included accessing post-

2016 information from two broad sources:

• EPA recreational water research and publications relating to that research.

• External (non-EPA) academic research conducted by researchers at academic

institutions and government organizations that have focused on recreational water

activities and science related to the BEACH Act.

5. A Systematic Review of Available Peer-Reviewed Literature

EPA performed systematic searches of the peer-reviewed literature for articles pertaining to

health studies, including epidemiological studies of recreational water-contact activities,

outbreak studies, advances in recreational exposure descriptions for children, characterization of

children’s illness susceptibility, observed illness rates in children upon exposure, and the

application of QMRA to water quality data and complex settings at recreational beaches;

advances in human and non-human fecal source identification (microbial source tracking); and

advances in molecular methods used in recreational waters to measure indicators of fecal

contamination. Multiple sets of search terms applicable to the topic were applied to references in

Web of Science and PubMed (http://www.ncbi.nlm.nih.gov/pubmed) (see Appendix A for

detailed search terms). Searches for gray literature were also performed. Abstracts were screened

for relevance to the scope of the search. The literature search was limited to English-language,

peer-reviewed citations published between January 2016 and November 2021, with the exception

of searches pertaining to children’s health studies. Searches pertaining to advances in

recreational exposure descriptions for children, characterization of children’s illness

susceptibility, and observed illness rates in children upon exposure were limited to articles

published between January 2018 and November 2021. Following the abstract screening, the full

text of articles passing scope was reviewed for specific information related to each topic (see

Appendix A for details). Diagrams of the process and results are provided in Figure 1, below.

The steps of a systematic review as described in the Preferred Reporting Items for Systematic

Reviews and Meta-Analyses (PRISMA) protocol, which defines the minimum set of items for

reporting in systematic reviews. The search strategy, search terms, screening criteria, and

PRISMA diagrams are provided in Appendix A. Results of the systematic reviews and

summaries of studies reviewed are included in Appendix B.

Figure 1. Process for Identifying Literature for Some of the Five-year Review Topics: 2016–2021

Three literature searches (Methods Progress; Health Studies; Children’s Risk) were conducted (Appendix A) to

identify articles for four relevant topics: Microbial source tracking (MST); Molecular methods; Health studies; and

Children’s risk. The MST topic includes fecal source identification (FSI) and bacterial source tracking. Note that the

literature on three additional topics, AMR, cyanotoxins, and coliphages, that is described in those sections was

identified from additional literature searches conducted using different methods and is not shown here.

Articles were cross-tagged to other document topics/sections when applicable, resulting in some articles being

counted for more than one topic.

Pre-screening prioritization of the Title/Abstracts (Ti/Abs) for Health Studies was conducted; 1,293 Ti/Abs were

reduced to 162 based on machine learning supervised clustering using 37 seed title/abstracts (see Appendix A).

Fifty-one cross-tagged articles were added after prioritization, resulting in 213 studies.

Identification

Screening

Eligibility

Records Identified via

Database Searching

Method Progress n = 732

Health Studies

n = 213

Children’s Risk n = 495

Records Identified via

Gray Literature Searching

Method Progress n = 37

Health Studies n = 61

Children’s Risk n = 30

Total number of titles identified for Ti/Abs review

Method Progress n = 769

Health Studies n = 274

Children’s Risk n = 525

Full-text articles reviewed

Method Progress n = 107 (MST); 120 (Methods)

Health Studies n = 118

Children’s Risk n = 38

Relevant articles reviewed for 5-year review

Method Progress n = 85 (MST); 80 (Methods)

Health Studies n = 44

Children’s Risk n = 12

PubMed

n = 1,746

WOS

n = 1,490

Gray

Literature

n = 128

Databases

Total Records Identified

EPA also performed systematic searches of the peer-reviewed literature for articles pertaining to

characterizing the human health effects of cyanotoxins from all routes of exposure. The

cyanotoxins of interest for this topic included: anatoxins, cylindrospermopsin, microcystins,

nodularins, and saxitoxins. The literature searches for anatoxins, microcystins, and

cylindrospermopsin built upon work previously conducted by the Office of Water and covered

the period from January 2014 to April 2021. The literature searches for all other cyanotoxins

were conducted with no start date to April 2021. Multiple sets of search terms applicable to the

topic were applied to references in Web of Science (WOS), PubMed, and Scopus (see Appendix

A for search terms). Abstracts were screened for relevance to the scope of the search. Following

the abstract screening, the full text of articles passing scope was reviewed for specific

information related to each topic. Search strategy, search terms, screening criteria, and PRISMA

diagrams are provided in Appendix A. Results of the systematic reviews are included in

Appendix B.

III. Findings of the 2

Five-Year Review

A. New Information on Health Effects after Exposure to Recreational Waters

The systematic literature review identified 44 health studies and 12 children’s risk studies

(Figure 1) that were evaluated for the Second Five-Year Review report (Appendix B). Additional

relevant literature was identified via an ad hoc process (e.g., provided by subject matter experts).

From this ad hoc process, twelve references relevant to health study information (Teunis et al.,

2008, 2016, 2018, 2020; U.S. EPA. 2010, 2019; Boehm et al., 2015; Wade et al., 2019; Vanden

Esschert et al., 2020; Egorov et al., 2021; Goh et al., 2021; California Water Boards, 2022) and

two references relevant to children’s risk (U.S. EPA, 2011 and Dufour et al., 2017) were

identified.

Health studies of recreational water exposure broadly fall into three categories: epidemiological

studies, QMRA-based studies, and outbreak reports or studies (also referred to as surveillance

summaries).

• Epidemiological studies quantify associations between reported health symptoms (or

biomarkers of infection) in participants and a measure of water quality, such as an

indicator of fecal contamination, on the day of swimming exposure. These studies

generally provide information on the level of illness experienced by recreators, and

many have observed statistically significant associations between illness and measures

of water quality (e.g., FIB). Epidemiological studies have informed the development of

recreational water guidelines in the United States and elsewhere.

• QMRA-based studies characterize the probability of illness by focusing on the

pathogens that can cause illness and, in this context, can provide risk-based thresholds

(RBTs) for measures of water quality (including traditional and alternative surrogates

of fecal contamination) that are based on the fecal sources affecting a waterbody.

• Outbreak studies are retrospective investigations that describe the conditions and

consider possible etiologic agents resulting from clusters of reported illnesses. All three

types of studies inform human health effects that recreators experience due to fecal

contamination of recreational waters and may inform the understanding of the

waterborne pathogens responsible for causing illness. This section presents studies

identified in the published scientific literature in each of these three study categories,

followed by a summary of major findings.

1. Recreational Water Epidemiological Studies

New Epidemiological Studies Conducted in the United States: 2016–2022

Children’s Health Studies

The systematic literature search (see Figure 1 and Appendix A for method details) that identified

new literature since the first five-year review (i.e., 2016–2021) found four recreational water

epidemiology studies conducted in the United States that included enterococci data (Wade et al.,

2022; Arnold et al., 2016, 2017; Benjamin-Chung et al., 2017). Several studies evaluated the

children’s health risk from exposure to fecal contamination in the beach environment.

Wade et al. (2022) analyzed a pooled data set of over 80,000 beachgoing children, adolescents,

and adults from 13 beach sites across the United States to compare illness risks and water quality

by collecting measures of both culture- and qPCR-enumerated enterococci, exposure levels,

beach sites, and health endpoints for different age groups. Sites previously studied by EPA

included: West Beach, Huntington Beach, Washington Park Beach, Edgewater Beach, Fairhope

Beach, Goddard State Park Beach, Surfside Beach, and Boquerón Beach. California sites studied

by the University of California, Berkeley and the Southern California Coastal Water Research

Project (SCCWRP) included: Mission Bay, Doheny Beach, Malibu Beach, and Avalon Beach.

As part of the analysis, beach sites studied were categorized by the likelihood of human fecal

contamination and whether the human source was a point source. Seventy percent of all

participants had at least some contact with water and 27 percent stayed in the water 60 minutes

or more. Children 12 years of age and under comprised approximately 25 percent of the total

study population from all sites. The authors used odds ratios (ORs) among swimmers to

characterize associations between levels of Enterococcus spp., measured by culture and qPCR,

for different age groups across different exposures, beach category, and health endpoints (GI and

respiratory illness). The authors found that GI symptoms were the most sensitive health endpoint

associated with fecal contamination across all age groups, exposures, and site categories.

Children were at higher risk of swimming-associated illness compared to older adolescents and

adults with the highest mean ORs observed for GI illness among children under 10 years old at

beaches affected by effluent. Statistically significant associations were consistently observed

with Enterococcus qPCR cell equivalents (CE) across sites impacted by human fecal

contamination. The highest odds ratios (OR = 2.32, 95% confidence interval [CI]: 1.33–4.06)

were between Enterococcus qPCR and children 6 years old and under who spent at least 60

minutes in the water; ORs were similar for children in the “’10 and under” and “’12 and under”

age groups. Respiratory illness was significantly associated with Enterococcus qPCR exposures

among children 4 and under who spent 60 minutes or more in the water at sites affected by

effluent. Cough was also associated with Enterococcus CFU at sites affected by effluent among

children 4 and under, although small sample sizes resulted in imprecise estimates for these

associations. Combining data across all sites, the association between GI illness and culturable

enterococci, although positive, was not statistically significant for the general population (i.e.,

data from all participants combined). ORs for culturable enterococci and GI illness increased at

“likely human-impacted” sites, particularly among those who swallowed water (1.37, 95% CI:

1.04–1.82) and there was evidence for increasing risk with younger aged children at sites

affected by effluent. For example, among children under 6 years old at the sites affected by

treated effluent the OR was 1.82 (95% CI: 1.14–2.91), which was significantly higher compared

to adults 18 years old and over. ORs for severe GI illness

among swimmers, which accounted

for approximately 20 percent of all reported GI illness, were statistically significant for

Enterococcus qPCR (1.16, 95% CI: 1.03–1.31) and higher for children under 6 years old (1.50,

Severe GI illness was defined as a GI illness episode that lasted three of more days or resulted in a visit to the

hospital, doctor’s office, or emergency room.

95% CI: 1.13–1.99), but not for culturable enterococci. The study conclusions were that age,

source of fecal contamination, and the intensity of swimming exposure are important factors

affecting the association between Enterococcus spp. Exposure from swimming-associated

illnesses.

Arnold et al. (2016) conducted a meta-analysis of 13 beach sites and nearly 90,000 subjects. The

study combined individual level-data from studies conducted by EPA and the University of

California, Berkeley, which were coordinated to ensure similar designs and measurement

methods. Data from 13 prospective cohorts were combined and integrated into a single data set.

The beaches represented a range of recreational water conditions across the United States: four

freshwater beaches were in the Great Lakes region, four marine beaches were in Southern

California, and the remaining five marine beaches were spread along the Gulf Coast, Eastern

Seaboard, and Puerto Rico. Nine of the beaches were located near a known source of treated

sewage discharge and the remaining four beaches had more diffuse contamination from urban

runoff. The primary outcome was incident diarrhea defined as three or more loose or watery

stools in 24 hours. Swimmers were defined as those who immersed their body in water and were

further classified on the basis of water quality, that is, whether the FIB, Enterococcus, exceeded

the revised EPA guideline value of 35 CFU per 100 ml.

Compared to non-swimmers, diarrhea incidence increased with swimming exposure.

Enterococcus levels were positively associated with diarrhea risk within age strata, with the

largest absolute increases in risk among children 0 to 4 years old. Swimmers exposed to

Enterococcus above EPA guidelines had higher diarrhea incidence compared to non-swimmers

and compared to swimmers exposed to water below the guideline, but only at beaches with a

known point source of human fecal pollution. Swimmers exposed to Enterococcus levels above

the EPA guidelines had higher diarrhea incidence compared to non-swimmers as well as with

swimmers exposed below the guideline in all age groups. The finding was more pronounced in

children: 0 to 4 years old exposed to water above regulatory guidelines reported 103 episodes per

1,000 compared to 48 episodes per 1,000 among non-swimmers at beaches with a known point

source. For swimmers that recreated in waters below the guideline level, the illness rate was 65

episodes per 1,000 compared, which is significantly higher than non-swimmers (48/1,000) but

lower than the rate for swimmers who recreated in waters above the guideline level (103/1,000).

The authors noted the uncertainties about the causes of diarrhea, which include non-infectious

causes of diarrhea (e.g., swallowing excess saltwater), outcome reporting bias, or pathogens

present in recreational waters that do not covary with Enterococcus. Children 0 to 4 and 5 to 10

years old had the most water exposure, stronger associations between levels of water quality and

illness, and the largest attributable health burden. Overall, recreational water exposure was

associated with increased risk of acute gastroenteritis resulting in missed daily activities with the

highest risk and burden among young children.

Arnold et al. (2016) also performed a comparative analysis for association to health effects for

FIB and qPCR. They found that the exposure-response relationship was more consistently

monotonic for Enterococcus measured using qPCR methods versus culture methods across

different analyses, even when restricted to beaches without a known point source of pollution.

Arnold et al. (2017) conducted a longitudinal cohort study of adult surfers (18 years old and

older), a group of recreators with potentially high exposure profiles, in California waters to

evaluate health impacts associated with water quality, measured by culturable FIB, under dry and

wet weather conditions. The study collected illness information on GI illness, sinus infections,

ear infections, fever, skin rashes, and infected wounds. Under dry conditions, recreational

exposures were associated with an increased incidence of all health outcomes compared to the

nonexposed group (adjusted incidence rate ratio = 1.86; 95% CI: 1.27, 2.71). With the exception

of infected wounds, illness symptoms were not associated with increases in culturable

enterococci levels. Culturable enterococci levels were strongly associated with multiple health

outcomes after wet-weather exposure. In the wet weather flows discharging to the beaches,

human fecal contamination from urbanized areas was identified. FIB (culturable enterococci,

fecal coliforms, and total coliforms) were significantly associated with illness up to 3 days after

rainfall. The median wet weather-associated excess risk at 35 CFU enterococci per 100 mL was

16 GI illnesses per 1,000 (95% CI: 5, 27) or less than half the rate of illness associated with the

EPA’s RWQC recommendations. The significance of this study is that it identifies wet weather

as another factor that can affect loading of fecal contamination to recreational waters and can

result in increased human health risk to recreators. The results support posting of beaches after

rainstorms when human fecal sources are likely to affect recreational waters and management

actions that would reduce fecal loading in urban runoff.

Benjamin-Chung et al. (2017) conducted a pooled analysis of six prospective cohort studies at

coastal beaches in California, Alabama, and Rhode Island. Water quality was measured using

culturable enterococci and coliphages. Knowledge of contamination inputs was used to interpret

indicator monitoring. Human fecal pollution was unlikely on all study days at two of the beaches

(Mission Bay and Malibu). At Fairhope and Goddard beaches, human fecal pollution was likely

on all study days. The remaining two beaches (Doheny and Avalon) had evidence of variable

human input such as groundwater influx conveying sewage through the sand or a berm that

blocked direct discharge of contaminated water at the beach. Among all participants across the

study sites and under all conditions combined, there was no association between GI illness and

levels of culturable enterococci or coliphages. However, associations between culturable

enterococci or coliphage levels and GI illness were observed when human fecal pollution was

“likely present” and there was some evidence that the association with illness for male-specific

coliphage (MSC; also known as F+ or F-specific) was stronger than for enterococci.

Studies Assessing Antibody Response

Four health effects studies were identified that characterized seroconversion, an immune

response to infection, in recreators (Wade et al., 2018, Augustine, et al., 2020, Egorov et al.,

2018, and Egorov et al., 2021).

Wade et al. (2018) collected saliva samples as part of the U.S. EPA Boquerón Beach

epidemiological study to test for immunoglobulin G (IgG) responses to genogroup I.1 (GI.1) and

genogroup II.4 (GII.4) noroviruses. Immunoconversions for noroviruses were observed in 34

subjects, or 2.6 percent of the study population (n = 1,298). Swimmers who reported submerging

their heads while swimming had a significantly higher rate of immunoconversion compared to

non-swimmers. Immunoconversion was not statistically associated with GI symptoms. The

results of this study demonstrate the potential for transmission of noroviruses among recreators

at a marine beach. Another study by Wade et al. (2019) found similar results.

Augustine et al. (2020) demonstrated the use of a rapid salivary antibody assay to assess the

prevalence of salivary antibodies against hepatitis A virus and immunoconversions in a

population of beachgoers at Boquerón Beach, Puerto Rico. Results showed an

immunoprevalence rate of approximately 16 percent among participants, which is less than half

the overall immunoprevalence rate among U.S.-born persons 2 years old or older. Only 1.43

percent of the participants who provided all three samples were found to have hepatitis A virus

immunoconversions. There was no statistically significant association between hepatitis A

immunoconversions and any of the demographic or exposure risk factors tested. Previous health

studies conducted at this location reported low rates of GI illness (e.g., <2 NGI/1,000 recreators),

low levels of pathogens, and good water quality based on levels of enterococci below the EPA

RWQC (U.S. EPA, 2010; Soller et al., 2016). This study demonstrated the potential of rapid

assays for immunoconversions to a waterborne virus like hepatitis A virus to inform the

protection of recreational water and public health (Augustine et al., 2020).

Egorov et al. (2018) conducted a prospective cohort study recruiting local families with children

in Lawrence, Massachusetts (n = 1986) that overrepresented children (1,170 children <18 years;

58.8%). The results were that 12.5 percent of participants that immunoconverted to

Cryptosporidium, tested through salivary immunoassay, were symptomatic. Immunoconversions

by study participants to Cryptosporidium after recreational exposure to natural waterbodies or

swimming pools was associated with adjusted odds ratios (aORs) of 2.3 (0.4;15.4) and 4.9 (1.6;

15.5), respectively. No association between immunoconversion with noroviruses and swimming

in natural waterbodies (i.e., rivers, lakes, or oceans) was observed. However, only 3.6 percent of

participant responses indicated swimming in pools and 2.5 percent in natural waterbodies,

reducing the sample size included in analysis. For all oral exposure pathways analyzed,

incidence rates of Cryptosporidium immunoconversions declined steadily with age: 8.5 per 100

person-years in children between 1 and 10 years of age, 5.6 per 100 person-years for age 11 to 20

years, 4.2 per 100 person-years for age 21 to 40, and 1.5 per 100 person-years in adults ages 41

to 85 years. Norovirus genogroups I and II also showed lower rates of immunoconversions in the

group age 41 to 85 compared to ages 1 to 10. This community-level study demonstrated the

utility of the salivary antibody immunoassay methodology for the detection of infections with

Cryptosporidium and norovirus. In this study, the immunoconversions were related to potential

exposure pathways, thus providing useful information for improving public health protection.

Egorov et al. (2021) conducted a prospective salivary antibody study at a Lake Michigan beach

to study seroconversion to norovirus and Cryptosporidium. Among 872 study participants, there

were seven cases of seroconversion, including six individuals with seroconversion to noroviruses

and two to Cryptosporidium, with one study participant seroconverting to both pathogens. Those

that seroconverted to norovirus were more likely to experience vomiting within 4 days of a beach

visit (p = 0.003). This study provided further evidence that recreational water exposure can be

associated with symptomatic illness and asymptomatic waterborne infections.

New Non-U.S. Epidemiological Studies: 2016–2022

Three recreational water epidemiology studies conducted outside the United States and one

review of epidemiological data were identified (Joosten et al., 2017; Kuhn et al., 2018, and

Verhougstraete et al., 2020; Young, 2016).

Joosten et al. (2017) conducted a prospective cohort study to assess risk factors for health

complaints (GI illness, respiratory illness, and complaints associated with the skin) associated

with recreational exposure to urban waters affected by wet weather-associated sewage overflows

during canal swimming events in two Netherland cities in 2015. Prior to the events, E. coli and

enterococci monitoring data from the canals were below the European Union (EU) thresholds.

By the afternoon of the day of the event, E. coli levels greatly exceeded the EU threshold in one

of the two cities while enterococci remained below the EU threshold. Thirty-one percent of

swimmers (427 of 1,375 swimmers) reported GI illness symptoms following exposure to canal

waters affected by sewage overflows resulting in an adjusted risk ratio (aRR) of 6.3 (95% CI:

4.1–9.5). Five out of seven stool samples provided by participants tested positive for various

norovirus genotypes. One water sample from one of the canals tested positive for genogroup I

norovirus and two water samples tested positive for genogroup II norovirus. The conclusion of

this study is that the outbreak of acute GI illness in swimmers was related to the presence of

norovirus, which was possibly linked to wet weather-associated sewage overflows affecting the

canals.

Kuhn et al. (2018) conducted a prospective case-control study among Danish persons that

included 446 children ages 1 to 5 years old (from a total of 3,119 study participants ages 1 to 30

years old) to identify risk factors for campylobacteriosis. Study participants provided information

by completing an online questionnaire. Consumption of contaminated food, animal contact,

bathing in fresh waters, contact with beach sand, and bathing in a paddling pool were identified

as significant risk factors for increased risk of campylobacteriosis. The authors estimated that

4 percent of sporadic Danish campylobacteriosis cases may be caused by recreational water

contact (Kuhn et al., 2018). The authors indicated that, based on their estimates, recreational

water contact and contact with sand was likely linked to a large proportion of campylobacteriosis

in Denmark’s children and young adults.

Verhougstraete et al. (2020) developed adjusted risk difference models (excess gastrointestinal

illness per swimming event) for children (<10 years of age) and “non-children” (≥10 years of

age) across five Brazilian beaches using epidemiological data collected in the United Kingdom

(UK) and Brazil. Water quality at the Brazilian beaches exceeded the maximum fecal

streptococci levels measured in the UK studies 11.8 percent of the time. Risks associated with

the elevated indicator levels equated to 53 and 96 NGI per 1,000 recreators for non-children and

children, respectively. The study concluded that the World Health Organization (WHO)

recreational water quality guidelines, based largely on the epidemiological study performed at

UK beaches, were not appropriate as a basis for guideline development in tropical settings where

there is minimal wastewater treatment. Pathogen profiles distinct to tropical waters and point

source discharges with minimal treatment were two factors identified for supporting the

development of more regionally specific guidelines.

Young (2016) reviewed studies published before 2015 of infectious disease transmission in

marine bathing waters, sources of pathogens in marine waters, and epidemiological evidence for

the association between marine bathing and infectious disease. Numerous studies demonstrated

an increased risk of gastrointestinal illness associated with marine swimming compared to non-

swimming. However, an association between levels of FIB and illness among swimmers was not

consistently found across the studies reviewed. The authors suggest that traditional FIB may not

be predictive of human health impacts when human waste was not the predominant source of

pathogens. In one study, 71 percent of gastrointestinal episodes in Southern California were

estimated to occur when the water quality was considered safe for bathing, potentially implying

that most cases of illness associated with marine bathing could arise from the lowest risk

exposures, combined with high numbers of people exposed. The authors identified the need for

research to identify additional markers for human health risk at non-point source beaches and the

use of rapid methods to improve public health protection.

Summary of Findings Identified in New Epidemiological Studies

These studies vary in their focus, but conclusions highlight that:

• While FIB can be a useful indicator in waters impacted by raw and poorly treated

sewage, the inconsistent performance of culture-enumerated FIB related to health

outcomes was demonstrated in multiple studies, which suggests that other indicators

(e.g., Enterococcus measured by qPCR) are more predictive of risk of illness.

• qPCR-enumerated enterococci performed better than FIB as a predictor of risk in waters

dominated by human fecal sources, and there was some evidence that culture-enumerated

enterococci were associated with increased GI illness when human fecal pollution was

suspected based on knowledge of point sources.

• Some waters receiving human fecal contamination have measured viral pathogens at

levels expected to lead to health outcomes and yet also be below EPA recommended

water quality level for culturable FIB.

• Health risk in recreational waters is greatest in children 0 to 10 years old compared to

both adolescents and adults.

• The health risk from recreating during or following wet weather compared to dry weather

can be due to changes in fecal loading dynamics to a waterbody.

• Use of salivary assays to estimate seroconversion to waterborne pathogens can provide

early information on infection by specific pathogens by recreators exposed to fecal

contamination. Studies found evidence of seroconversion to norovirus and

Cryptosporidium among swimmers and also found that those who seroconverted were

more likely to have gastrointestinal symptoms compared to those who did not

seroconvert.

2. Use of QMRA to Understand Risks in Recreational Water Settings

QMRA studies published between 2016 and 2022 included studies that characterized human

health risk from recreating in waters contaminated by human and non-human fecal sources; used

QMRA to develop or evaluate risk-based water quality values for alternative indicators; and

evaluated the importance or sensitivity of specific data as input to QMRA for recreational water

purposes. The new studies described below report several important trends with respect to the

use of QMRA to study recreational water risks. As with prior QMRA studies, the risks evaluated

in these studies characterize risk of GI illness and do not include other health endpoints, such as

respiratory illness.

Characterization of Risks from Human Fecal Sources

Soller et al. (2016) conducted a QMRA that included fecal indicator and pathogen monitoring

concurrently with an epidemiological study to characterize the risk of GI illness at Boquerón

Bay, Puerto Rico. Results of the QMRA were used to improve interpretation of a recreational

water epidemiological study. Results of the water quality study component of the QMRA

demonstrated low levels of pathogens and good water quality. The QMRA findings provide a

plausible explanation for the lack of relationship between fecal indicator organism detection and

swimming-related illness in the epidemiological study. The QMRA estimated a swimming-

associated risk of less than 2 NGI per 1,000 recreation events, which was below the level of

illness that the epidemiological study was designed to detect.

Vergara et al. (2016) conducted a QMRA to estimate the GI illness risk to a population from

primary and secondary contact recreation in an urban catchment in Singapore using field

measurements of norovirus, adenovirus, and Cryptosporidium. Land use in the catchment was

mostly residential with some industrial and office areas and human contamination can be

attributed to non-point sources of pollution (Vergara et al., 2016). Norovirus was detected more

frequently and at higher levels compared to adenovirus. QMRA results showed a higher illness

risk associated from exposure to norovirus compared to adenovirus. Risks for children (<18

years), based on children-specific recreational water ingestion data, were higher compared to

adults and exceeded 36 NGI per 1,000 recreators approximately 5.6 percent of the time. The

higher prevalence and illness risk of norovirus supported the use of norovirus as a reference

pathogen for QMRA in recreational waters in Singapore.

Lapen et al. (2016) conducted a QMRA of freshwater recreation in rivers in Ontario, Canada.

Using Cryptosporidium as the reference pathogen, two river basins in Ontario were monitored

for Cryptosporidium oocysts levels and species/genotype data were collected. The QMRA

included two approaches: one assumed all observed oocysts were infectious to humans, and for

the second, risk was based on the fraction of oocysts that were Cryptosporidium hominis and/or

Cryptosporidium parvum, which are the predominant human infectious forms of the parasite.

Compared to assuming all oocysts are infective to humans, the estimated infection risk was one

order of magnitude lower when only human infectious forms were selected, and fluctuations in

risk were also observed depending on seasonality. Results of this study demonstrate the

variability in potential risk when different pollution sources can occur. Also, the availability of

pathogen data at the species/genotype level can help inform more accurate estimates of potential

human infectious risk in a QMRA.

Eregno et al. (2016) coupled discharge-based hydrodynamic modeling with QMRA to estimate

the risk of infection from swimming in marine waters following a rainfall event with combined

sewer overflow events (CSOs). Of the simulated bacteria, protozoa, and virus infection risks, the

virus risk dominated, as represented by norovirus, and exceeded the infection health benchmark

of 19 GI illnesses per 1,000 swimmers in the days after the rainfall event. This modeling result

corroborates the findings of epidemiology studies (described above) that identified wet weather

and sewage overflow as factors leading to increased pathogen levels and infection risks. The

study demonstrates the potential utility of combining discharge-based hydrodynamic modeling

with health modeling in QMRA to estimate risk under wet weather conditions to inform beach

management conditions.

Soller et al. (2017) used QMRA to predict the GI illness risk to adult surfers associated with wet

weather at marine beaches in Southern California impacted by urban stormwater. In the QMRA,

sanitary survey information, monitoring data for fecal indicators, human microbial source

markers, reference pathogens, site-specific dilution estimates, and literature-based data for

incidental ingestion, pathogen dose-response, and morbidity were considered together to

generate risk estimates associated with wet weather stream flows affecting coastal nearshore

waters. As part of a broader “Surfer Health Study,” data collection for the QMRA was conducted

in parallel with a longitudinal recreational water epidemiological study focusing on surfer

exposure (Arnold et al., 2017). Water quality monitoring showed the presence of human fecal

contamination and fecal-associated pathogens in the stormwater discharging to the coastal water

where surfers were exposed. The results of the health modeling demonstrated that enteric

viruses, as indexed by norovirus, were an important etiologic agent of GI illness among surfers.

The QMRA was bolstered by a sensitivity analysis characterizing the uncertainty associated with

using published norovirus dose-response information. QMRA risk estimates from this study

corroborated the reported epidemiological results (Arnold et al., 2017). Predicted average illness

levels were lower at higher levels of FIB when compared to the epidemiological data that

informed EPA’s national recommendations. For both the QMRA (Soller et al., 2017) and

epidemiological (Arnold et al., 2017) components of the “Surfer Health Study,” the predicted

and reported GI illness levels represent a specific short-term high-risk scenario associated with

exposure to human fecal contamination present in coastal stream discharges containing urban

stormwater. The QMRA methodology used in this study and the reported results can be useful in

developing alternative health-based water quality criteria specific to risks associated with wet

weather impacts in an urbanized setting.

Bortagaray et al. (2020) assessed the risk of infection and illness for Group A Rotavirus (RVA)

in the Santa Lucia and Uruguay watersheds in Uruguay that are affected by raw sewage from two

cities. The authors performed qPCR on surface water samples and developed a QMRA

framework for people who use surface waters from the rivers. RVA was detected at all sampling

locations in both watersheds with an approximate detection frequency of 40 percent of samples.

Detection frequency increased during the coldest month of the year. The mean level of RVA was

1.3 × 10

genomic copies per liter (L). Due to the frequency and occurrence of RVA, QMRA

results demonstrated that populations using both rivers had comparable risks of infection and

illness.

Shoults et al. (2021) conducted a reverse QMRA for a natural swimming pool relying on

biological treatment processes, including a biofilm filter, a zooplankton filter, a hydro-botanic

filter, and a submerse filter in constructed wetlands coupled with ultraviolet (UV) irradiation

prior to the treated water being returned to the pool. The authors found that of the four reference

pathogens included (norovirus, Campylobacter, Cryptosporidium, and Giardia), only norovirus

exceeded the median recreational water risk benchmark. Log reduction values (LRVs) for the

reference pathogens were developed. The authors indicated that more than 1 day of treatment

would be needed to achieve the risk benchmarks after heavy bather use. Finally, the authors

concluded that ultraviolet disinfection had little effect on reducing the treatment time required.

Abia et al. (2016) conducted a QMRA to evaluate the public health risk associated with exposure

to pathogenic bacteria in polluted river water under undisturbed conditions and conditions of

sediment resuspension in the Apies River, Gauteng, South Africa. River water was monitored for

E. coli levels and the presence or absence of three feces-associated enteric pathogens: Salmonella

spp., Shigella spp., and Vibrio cholerae. The authors assumed that 8 percent of generic E. coli

counts were pathogenic. Ingestion rates of 1 mL, 50 mL, and 100 mL were used to represent

different levels of ingestion exposure. The results indicated that risks of infections increased

during the wet season. A 2-log increase in water E. coli count following sediment disturbance led

to approximately 10 times higher probability of infection than when sediments were undisturbed.

Liao et al. (2016) linked a watershed-scale microbial fate and transport model with a stochastic

dose-response model in a QMRA to predict human health risks from different fecal sources in an

urban watershed for comparison with regulatory benchmarks in order to prioritize remediation

efforts. Results indicated that human illness risks were consistently higher than 36 illnesses per

1,000 people for the study watershed, even when the predicted FIB levels were in compliance

with the E. coli GM standard of 126 CFU per 100 mL. Sanitary sewer overflows were associated

with the greatest risk of illness, which is of particular concern, given increasing indications that

sewer leakage is ubiquitous in urban areas. Uncertainty analysis suggested the accuracy of risk

estimates would be improved by additional site-specific pathogen data. The authors recommend

integrating this QMRA with water quality management planning to provide greater clarity to

stakeholders and decision makers (Liao et al., 2016).

Characterization of Risks from Non-Human Fecal Sources and Mixed Sources

Gitter et al. (2020) used QMRA to estimate the probability of GI illness from exposure to a

freshwater creek impacted by a mixture of sources. Pathogen concentration was estimated based

on FIB MST source apportionment of human, wildlife, cattle, and domestic animals, assuming

all detected FIB were from fresh contamination. The results indicate that the risk of illness from

norovirus, representing human fecal sources, contributed the greatest risk to human health. The

most frequent sources of E. coli at the sites monitored were from non-human sources (Gitter et

al., 2020).

Lim et al. (2017) conducted a source-apportionment QMRA to assess risk of GI illness at a

Southern California beach (Baby Beach, City of Dana Point) that did not have a known source of

human fecal contamination. Dry weather inputs of enterococci included bather shedding and

birds. Wet weather inputs of enterococci included the potential presence of sewage in stormwater

outflows (up to 20% of enterococci loading), animal feces from wildlife and dogs, and

nonpathogenic sources, such as plant and soil associated sources. Historical enterococci

measurement data were used to evaluate risks in dry and wet weather scenarios. During dry

weather, the median recreational waterborne illness risk at this beach is below the EPA’s RWQC

target illness rate of 36 illness cases per 1,000 bathers regardless of the fecal source contributing

enterococci. During wet weather, the median recreational waterborne illness risk predicted by the

QMRA depends on the assumed level of human waste associated with stormwater; the risk is

below the EPA RWQC illness risk benchmark 100 percent of the time, provided that <2 percent

of the FIB in stormwater are of human origin.

Evaluating Alternative Water Quality Surrogate Measures and Derivation of Risk-Based

Thresholds

Boehm et al. (2018) investigated the risk of gastrointestinal illness associated with swimming in

surface waters with aged sewage contamination. A systematic literature search and meta-analysis

compiled decay rate constants for reference pathogens and human feces-associated source

markers, which were incorporated into the QMRA. The QMRA evaluated HF183, a human-

associated fecal source marker, as an index for sewage present and thereby provided insight into

how risk relates to HF183 concentrations in surface water. Risks were modeled for aged sewage

and sewage of unknown age. The risks from recreational exposure to fresh and aging sewage

were primarily attributed to the reference pathogen norovirus. Based on the range of decay

values (n = 52) from the literature, HF 183 was shown to decay (~0.4 log

-1

) faster than

norovirus as sewage ages. Therefore, the risk associated with a fixed number of gene copies of

HF183 increases with the age of contamination. A HF183 risk-based water quality threshold for

fresh sewage was 9,700 copies per 100 mL and 900 copies per 100 mL after sewage aged for

2.5 days.

Ahmed et al. (2018a) estimated concentrations (genome copies [GC]) of sewage-associated

markers in 100 mL of beach water sample contaminated with either fresh untreated or secondary

treated sewage that exceeded the median illness rate of 36 per 1,000 people at a single event for

reference pathogens norovirus and human adenovirus 40/41. The concentration of 3.22 × 10

of the HF183 in 100 mL of water sample corresponded to a risk above the GI illness benchmark

value when beach water was contaminated with fresh untreated sewage and roughly the same for

waters contaminated by secondary treated effluent.

Wu et al. (2020) identified RBTs (corresponding with 36 illnesses per 1,000 swimmers) within a

QMRA context in a hypothetical waterbody contaminated by a continuous loading of both

human and non-human fecal contamination. Their results indicated that a larger difference in

decay rates between pathogen and indicator leads to an increased miscalculation of

gastroenteritis risk compared to calculations that do not account for differential decay. Given the

continuous loading scenario, the difference in RBT between fresh and aged contamination was

less for human marker HF183 in the human contamination scenarios than for animal markers in

the animal contamination scenarios. The median RBT for human contamination was

approximately 3.5 to 3.7 log

gene copies per 100 HF183.

Goh et al. (2021) estimated RBT of human polyomaviruses (hPyVs), Bacteroides

thetaiotaomicron (B. theta), and Methanobrevibacter smithii (M. smithii) that correspond to the

acceptable GI illness risks associated with recreational activities in Singapore using a QMRA

approach. Among the three MST markers, hPyVs showed the highest specificity (100%) to

sewage samples, followed by M. smithii (97%) and B. theta (90%). All MST markers showed

100 percent sensitivity toward sewage contamination, with B. theta present in highest abundance

in sewage, followed by hPyVs and M. smithii. Field data showed that the MST markers at

threshold concentrations were able to classify the safe level in more than 83 percent of the

samples. This study presents the range of threshold concentrations in MST markers in freshwater

and seawater matrices by considering two scenarios (i.e., decay and dilution) under different

exposures (i.e., minimum, average, and maximum ingestion rates for swimming activity).

Brown et al. (2017) interpreted measured concentrations of Catellicoccus (CAT) genes (from

gull contamination) in recreational waters. The authors took 37 gull fecal samples to measure the

CAT gene concentrations and integrated the measurements in a QMRA framework using dose-

response functions from reference pathogens. They estimated that when the level of CAT

surpasses 4 × 10

copies per 100 mL of water, the median predicted illness exceeds three

illnesses per 100 swimmers.

Boehm and Soller (2020) incorporated updated data for microbial decay rates and aging of

mixtures of human sewage contamination to derive a risk-based water quality threshold for the

human marker HF183 in ambient waters using QMRA. The authors identified a threshold of

1–525 HF183 copies per 100 mL that corresponded with the health benchmark of 32 illnesses

per 1,000 for different contamination scenarios. This threshold was estimated for a scenario in

which human contamination in sewage of different ages co-occurs with contamination from gull

feces.

Schoen et al. (2020) used QMRA to explore the RBT of various markers for different

recreational water quality scenarios based on a series of assumptions and uncertainties. For

example, they found that fresh sewage RBT estimates were not always protective when aged

sewage was present, and aged sewage RBT estimates often fell below the marker lower limit of

quantification. Conservative RBT estimates of 9.3 × 10

and 9.1 × 10

(copies/100 mL) for

HF183/BacR287 and CPQ_056, respectively, were predicted when fresh sewage was greater (by

volume) than aged at the time of measurement. Conversely, genetic markers may not be effective

indicators when aged sewage contributes the majority of pathogens but minimal marker levels,

relative to fresh contamination. This study describes the importance of taking into account the

impact of a potentially slower decay rate and higher abundance when modeling public health

risk. The results highlight the utility of QMRA that incorporates pollutant age and mixture

scenarios, and the potential influence of site-specific factors on estimating RBT values. The

approach discussed in this study has been refined in multiple previous studies (Boehm et al.,

2015, 2018; Boehm and Soller, 2020) and supports establishing health protective RBTs for

alternative indicators.

Van Abel et al. (2017) conducted a QMRA to assess risk in surface waters used for drinking,

domestic, and recreational purposes in South Africa. Water monitoring results for three rivers

demonstrated a frequency of occurrence of norovirus genogroup I (50%) and genogroup II

(74%), with genogroup I occurring at lower levels than genogroup II. Risk estimates were based

on the occurrence of norovirus, exposure route and the dose-response model selected. Risk of

norovirus infection was sensitive to the choice of dose-response model chosen. Recreational risk

varied with the level of exposure (i.e., swimming, playing on the shore adjacent to the river, and

boating) with swimming yielding the highest risk. Depending on the dose-response model, river

waters frequently exceeded an annual recreational water illness benchmark of 0.03 (genogroup I:

46%–96%, genogroup II: 74%–96%). The risk burden was greater for individuals from

vulnerable subpopulations who may utilize untreated surface waters for multiple uses.

Sunger et al. (2019) used QMRA incorporating literature values of pathogen densities in

secondary treated wastewater effluents to simulate the risk of illness from swimming in

freshwater receiving wastewater effluent. Data from both disinfected and non-disinfected

secondary effluent were used. Enteric viruses, as indexed by norovirus, dominated the estimate

risk of GI illness.

Dose-response modeling has advanced for several waterborne pathogens that are commonly used

as reference pathogens in recreational water QMRAs. Adenovirus (Teunis et al., 2016),

norovirus (Teunis et al., 2020), and Campylobacter (Teunis et al., 2018) dose-response modeling

has been further developed by combining data sets and conducting Bayesian analyses to better

characterize infection and illness rates. Teunis et al. (2020) reported a meta-analysis of clinical

challenge studies for norovirus and norovirus outbreaks from consumption of oysters. Data

included were from 14 challenge studies using six different norovirus inocula and nine

outbreaks. The study showed how challenge studies and “natural experiments” (outbreaks) can

be combined in a multilevel, hierarchical dose-response framework. Overall, the results of the

study confirm the high infectivity of norovirus reported by earlier studies (Teunis et al., 2008).

Teunis et al. (2020) indicate that an updated dose-response model may be applied in QMRA,

with the potential benefit of differentiating between norovirus genogroups and host secretor

status.

Evaluating Parameter Selection and Sensitivity Analyses

Ballesté et al. (2018) examined the impact of decay rates of fecal source identification (FSI)

markers and FIB from different sources. The authors concluded that it is important to take into

consideration the rate of inactivation of bacteria and FSI markers in QMRA or other models used

to model pollution sources in freshwaters. Decay rates were found to be affected by changes in

temperature (seasons), sunlight, predation rates, presence of organic matter, fecal source, and

alkalinity. Intrinsic characteristics of indicators and markers for specific sources influenced

seasonal changes in decay rates.

Federigi et al. (2019) used PRISMA guidelines to review QMRAs of recreational waters

published between 2003 and 2018. PRISMA guidelines define the minimum set of items for

reporting in systematic reviews. They found research gaps included: 1) a lack of epidemiological

data on the main pathogens of concern in specific geographic areas; 2) a lack of site-specific

water quality data; 3) little consideration of recovery efficiency or infectivity of pathogens; 4)

lack of consideration of host-specific factors (like previous immunity or immunodeficient); and

5) lack of QMRA model validation. The authors conclude that the use of QMRA is “very

promising for management of recreational waters and as a mean[s] to improve the regulations,”

but additional research in the areas identified could improve the reliability of results.

Poma et al. (2019) evaluated type of methodology used for finding the best fit for pathogen data

and reported that this is a critical consideration in QMRA, especially in data sets with numerous

non-detects, because different approaches can result in very different estimations of risk. Five

different alternatives to represent censored data were evaluated. They found that the decision on

how to treat censored data, especially when they are abundant in the data set, is crucial. These

findings led the authors to suggest that, if in doubt about which approach to use, one should

adopt a more protective approach, such as using half the detection limit.

Summary of Findings from New QMRA Studies

• Additional evidence shows that waters affected by human fecal inputs can pose higher

health risks compared to non-human fecal sources.

• Several studies utilized norovirus as a primary reference pathogen to characterize risk

from human enteric viruses in waters affected by human fecal pollution.

• QMRA combined with epidemiological data can be a robust approach for

characterizing elevated health risks in acute exposure recreational scenarios.

• Children-specific exposure data can be used in a QMRA to estimate potential illness

risk to recreating children.

• Across analyses, the age of fecal contamination was identified as an important factor

affecting risk estimates and calculation of RBT values for alternative indicators.

3. Outbreak Studies of Illnesses in Ambient Recreational Waters Associated with Enteric

Pathogens

Outbreaks of illness in recreational waters are defined as the occurrence of similar illnesses in

two or more persons, epidemiologically linked by location and time of exposure to recreational

water or to pathogens, toxins, or chemicals aerosolized or volatilized from recreational water into

the surrounding air (Graciaa et al., 2018). The National Outbreak Reporting System (NORS) is a

passive reporting system through which state and local health officials voluntarily report

outbreaks to the Centers for Disease Control and Prevention (CDC). CDC reported NORS

information on leading etiologies causing outbreaks nationally in ambient recreational waters

between 2000–2014 (Graciaa et al., 2018). Vanden Esschert et al. (2020) discussed three

outbreaks in detail including reported FIB monitoring data for two of the outbreaks.

Additionally, four recreational water-related outbreaks of illness occurring outside the United

States were identified in the peer-reviewed scientific literature (Mosnier et al., 2018; Polkowska

et al., 2018; Schets et al., 2018; Sips et al., 2020). During 2000–2014, CDC reported 140

untreated recreational water-associated outbreaks resulting in at least 4,958 cases and two deaths

of which 95 outbreaks were confirmed to have a pathogen etiology, including five outbreaks

with multiple etiologies (Graciaa et al., 2018). The 95 outbreaks represented at least 3,125 cases;

enteric pathogens caused 80 (84%) resulting in 2,704 cases, with norovirus linked to 21

outbreaks (22%), pathogenic E. coli linked to 19 (20%), Shigella linked to 14 (15%), and

Cryptosporidium linked to 12 (13%). Of the eight outbreaks related to toxins or chemicals, seven

(88%) were caused by algal toxins associated with harmful algal blooms (HABs). Most

outbreaks were associated with freshwater venues (84%) and occurred during the June–August

timeframe (81%). Statistical analyses indicated the outbreak number was not significantly

different over the 15-year reporting period (Graciaa et al., 2018).

CDC recorded 119 outbreaks associated with ambient recreational water for the period 2009–

2019. In 69 of the outbreaks (58%), enteric pathogens were identified as the cause: norovirus (19

[22%]; 1,858 cases); Shiga toxin-producing E. coli (STEC) (19 [22%]; 240 cases);

Cryptosporidium (27 [19%]; 237 cases); and Shigella (14 [16%]; 713 cases) (Vanden Esschert et

al., 2020). In July 2018, 97 people reported becoming ill with GI symptoms after swimming at

Woods Pond Beach in Maine. Recreators reporting head submersion or swallowing water were

approximately three times more likely to be ill. Two stool specimens collected from four ill

people tested positive for norovirus genogroup I although the source of contamination was not

identified (Vanden Esschert et al., 2020). In August 2019, 69 cases of STEC infection were

identified in persons who reported swimming at a public lake in Minnesota. No E. coli

monitoring results from this outbreak exceeded state recreational water criteria during April–

October. No evidence of a point source of fecal contamination was identified, but investigators

did report multiple swimmers and lifeguards had contact with the lake while ill (Vanden Esschert

et al., 2020). In July 2019, 24 cases of shigellosis were identified in California following contact

with Santa Ana River water. E. coli concentrations in Santa Ana River water exceeded

acceptable levels and ranged from 350 to 1,600 most probable number (MPN) per 100 mL. Flow

in the Santa Ana River consists of discharges from multiple wastewater treatment plants

(WWTPs) and non-point sources of pathogens affect the quality of the river (California Water

Boards, 2022).

Four international outbreak investigations that demonstrated an increased risk burden among

children recreating in contaminated recreational water and/or provided specific etiological

information on the cause of the outbreak were found in the literature search. Norovirus was

identified as a cause of outbreaks at public beaches (Polkowska et al., 2018; Schets et al., 2018)

or other untreated recreational water venues (Sips et al., 2020). One study characterized a

Cryptosporidium-related outbreak among children in an isolated village with poor sanitation

(Mosnier et al., 2018).

Outbreaks of norovirus affected at least 1,093 people swimming at beaches in Finland in 2014

reported via a web-based survey (Polkowska et al., 2018) and 100 people swimming in a lake in

the Netherlands in 2012 (Schets et al., 2018); norovirus was detected in 19 of 23 (Polkowska et

al., 2018) and 4 of 5 (Schets et al., 2018) stool samples, respectively. No point sources of human

fecal contamination were identified at the lakes involved in either outbreak.

Polkowska et al. (2018) conducted a retrospective cohort study on an outbreak of gastroenteritis

in Tampere, Finland, which included children from 0 to 17 years old. Beach water monitoring

demonstrated acceptable levels of FIB (range from 0 to 5 CFU enterococci per 100 mL and 1 to

27 CFU E. coli/100 mL). The authors developed a web-based survey to identify infection sources

and risk factors. Cases in the general population included children who visited beaches at four

different lakes. Identified risk factors included exposure by getting water into the mouth while

swimming, swallowing water while swimming, submersing the head under water, and playing at

the beach. Generally, children 0 to 17 years old were more frequently affected, which the authors

related to higher exposure associated with recreating activities. Risks associated with submerging

the head under water were higher for children 0 to 4 years compared to the general population

(risk ratio 3.55 vs. 3.24). For other risk factors, the risk ratio was either lower for children

compared to the general population or not significant. The potential sources of enteric pathogens

detected (norovirus, Campylobacter jejuni, and rotavirus) were infected beachgoers.

Environmental health officers reported poor hygienic conditions at public beaches, including

children urinating or defecating on the beaches, overflowing trash receptacles, and used diapers

observed on the beach. (Polkowska et al., 2018). In both outbreaks, infected individuals may

have contaminated lake waters and contributed to secondary transmission of norovirus between

recreators. The authors suggested a need for new indicators of water quality for norovirus and

development of evidence-based recommendations regarding timing of safe reopening of

recreational water venues associated with outbreaks.

Schets et al. (2018) reported an outbreak of norovirus infection in Gelderland, the Netherlands,

among swimmers at a freshwater lake. Most cases were children with gastroenteritis symptoms,

and 82 percent of the cases had been in the water for 30–60 minutes. An epidemiological

investigation, combined with detection of norovirus genogroup I in stool samples of patients and

sand samples collected at the lake, suggested that exposure to the recreational lake resulted in the

outbreak. The most likely source of contamination was determined to be an infected human and

the authors concluded that active communication about human shedding of viruses during and

after diarrhea is needed along with guidance to refrain from swimming when contamination is

suspected.

Outbreaks of recreational water-associated GI illness reported since 2017 show that children can

be disproportionately susceptible to health effects from exposure to pathogens in recreational

waters compared to adults (Mosnier et al., 2018; Schets et al., 2018; Sips et al., 2020).

Mosnier et al. (2018) describe an increase in cryptosporidiosis reported over a 6-month period

among children living in a remote area along the Maroni River in French Guiana. Questionnaires

were distributed to collect data on demographics, food consumption, river interaction (play,

washing), symptoms, and outcome. Stool samples were collected and analyzed 3 months after

the onset of symptoms. Mosnier et al. (2018) report on a specific outbreak cluster of 14 cases, all

children, aged between 4.5 and 38 months, and all linked to Cryptosporidium hominis. The

outbreak was notable because it represented a peak in occurrence compared to previous sporadic

cases and occurred during a dry period. Human behavior and activities in the river area were

suspected to be the driver for the outbreak among the children. Numerous playgrounds on the

riverside included clothes washing, bathing, cooking/eating, playing, and defecation. Fecal

contamination and high turbidity were noted at water supply locations. Although this outbreak

likely resulted via multiple factors occurring at this location, it does provide evidence that

children, who have immature immune systems, can be at a disproportionate risk of contracting

severe infectious diseases when pathogen prevalence increases in a population.

Sips et al. (2020) described a norovirus outbreak in a natural playground in the Netherlands

where untreated water flows from a nearby river into a recreational lake. The Public Health

Service received 21 case notifications via the national online reporting system and an additional

100 cases (mostly children) were identified following an online query of the local health-related

Facebook platform. Water and fecal samples from humans and birds were tested and human

introduction of norovirus was identified as the most likely cause of the outbreak.

Summary of Findings from New Outbreak Reports

• Outbreak data document increased risk to children in recreational waters

• Outbreak surveillance in the United States and elsewhere continues to indicate the

importance of human enteric viruses as a leading etiologic agent of illness in ambient

recreational waters.

• Some outbreaks documented high levels of FIB in waters linked to the outbreak, but

some outbreaks occurred despite low densities of FIB.

4. New Information on Children’s Exposure in Recreational Waters

In 2019, EPA published Recommended Human Health Recreational Ambient Water Quality

Criteria or Swimming Advisories for Microcystins and Cylindrospermopsin, which used data

from Dufour et al. (2017) to characterize incidental ingestion for children ages 6 to 10 years old

and 7 to 11 years old compared to adults (Cyanotoxin AWQC, U.S. EPA, 2019). Section 4.2.3.1

of the Cyanotoxin AWQC includes graphical visualizations of ingestion volumes for different

age groups, information on the length of time an individual spends in the water (duration of

exposure), and a calculation of the daily incidental ingestion rate. Children ages 6 to 10 years old

have a daily incidental ingestion rate higher than any other age group. Section 7.3.2 of the

Cyanotoxin AWQC discusses exposure factors for children younger than 6 years old. Data for

ingestion by children <6 years old is more qualitative in nature and there are large uncertainties

given the lack of measured incidental ingestion data specifically for this age group, thus it is

challenging to compare this group directly to other age groups. However, using assumptions and

estimates for exposure for younger children from EPA’s 2011 Exposures Factor Handbook and

the data reported from Schets et al. (2011), the daily ingestion rate for children <6 years old was

estimated to be lower than the 6- to 10-year-old age group. Section 7.2.1 of the Cyanotoxin

AWQC presents data on duration of recreational exposures from both DeFlorio-Barker et al.

(2017) and EPA’s Exposure Factors Handbook (U.S. EPA, 2011).

DeFlorio-Barker et al. (2018) compiled self-reported swimming durations from epidemiological

study surveys from 12 beaches in which participants were asked to estimate, in minutes, the total

time they spent in the water. The study combined the data for time spent in the water with

incidental ingestion volumes reported in Dufour et al. (2018) to estimate the incidental ingestion

of water swallowed per swimming event by age group. Parents or guardians were responsible for

answering survey questions assessing exposures such as getting water in the mouth or

swallowing water, on behalf of their minor children. The study results represent 7,534 children

ages 8 to 12 years old and 5,998 children ages 4 to 7 years old recreating in fresh and marine

water. Marine recreators spent more time in the water compared to freshwater recreators. The

authors suggest that behaviors may have been influenced by the warmer water at most of the

marine sites (California and Gulf Coast) compared to the freshwater sites in the Great Lakes.

Children ages 4 to 7 and 8 to 12 years old had the highest exposures. Children 6 to 12 years old

were estimated to have the highest ingestion amounts and male children ingested more water

compared to females. This study provides additional evidence that children have higher exposure

in recreational water compared to adults and supports the results of other studies that show

children have higher risks of GI illness from recreational exposures.

The Influence of Children’s Behavior on Exposure to Recreational Waters

Recent studies of children’s behavior support other conclusions that they spend more time in

water and engage in more vigorous activity. Ferguson et al. (2019) conducted an observational

study focused on children ages 1 to 6 years old and their behavior patterns at recreational marine

beaches in Florida and Texas. Analyzing data from more than 400 parental surveys in both

locations, the authors found that over 46 percent of the study participants stay for 3 hours or

longer at the beach when going with their children, increasing their potential time of exposure

compared to adults who stay for less than 3 hours. Survey respondents also indicated that

children ages 1 to 6 years old spent 31–43 percent of the time in the water, 27–35 percent in the

intertidal zone, and 34–45 percent in dry sand. The authors found that age was the most relevant

factor in how and where children play (gender being less influential), with dry sand being the

most common zone for children 1 to 6 years old, although 6-year-old children spent the most

time in water. Hygiene practices like washing hands before eating at the beach significantly

varied by region (p < 0.001), indicating that only 37 percent of children washed their hands

before eating in Florida compared to 63 percent in Texas. Lastly, the authors observed that the

time spent digging in the sand was longer than previously published estimates. Although the data

were self-reported, they provide insights about children’s behaviors in marine coastal beaches.

Ferguson et al. (2021), used a virtual timing device to quantify real-time, sequential micro-

activity pattern data collected from videos of 120 children at four different beaches. This study is

the first to use this approach extensively for collecting data on children’s behavior at beaches.

Across all sexes, age groups, and beaches, wading was the most common activity and seawater

was the most common location where children played. For all age groups (0 to 6 years old), the

majority of time was time spent wading (activity) for both male (40.7%) and female (41.5%),

which corresponds to the seawater location where the most time was spent by both female

(45.8%) and male (47.5%) children. The fraction of time spent wading was 38.9 percent for

children 0 to 24 months, 37.6 percent for children 25 to 48 months old, and 45.1 percent for

children greater than 48 months. Although the study did not address ingestion directly, the

authors reported mouth contacts and for the majority of time, the mouth was not in contact with

any surface. When the mouth did contact a surface, the most common item was food (2%), the

next common was drinks (1.2%). The authors concluded that, based on activity patterns,

exposures for young females and males may be similar, and for dermal exposure and ingestion

exposure, the right hand is influential.

Summary of Findings from New Studies on Children’s Exposure

New studies on children’s exposure indicate that risk differences can be influenced by one or

more of the following:

• Children’s recreational exposure is greater than any other age group. In fact, studies

showed that ages 6 to 12 years old had greatest total volume of ingestion of

recreational water, independent of body size.

• Children’s greater exposure is due to behaviors such as increased time spent in water,

length of time spent playing in the sand, and more vigorous activity, which are

associated with greater health risk compared to adolescents and adults.

5. Summary of Major Findings from New Information on Health Effects and Children’s

Exposure

• Recent publications have advanced understanding and further confirm that

epidemiological and outbreak data demonstrate that children 0 to 10 years have a

higher risk of illness compared to adults when exposed to fecal contamination in

recreational waters.

• Children’s behavior, such as increased time spent in water, length of time spent playing

in the sand, and more vigorous activity, can increase their potential exposure to fecal

contamination and is associated with greater health risk compared to other life stages,

especially those 18 and over. Children’s recreational exposure is greater than

adolescents and adults because they ingest more water in proportion to their body

weight than the other age groups.

• Studies demonstrate that human fecal contamination can pose higher risks of illness in

recreators relative to some non-human fecal sources.

• While FIB can be a useful indicator where raw and poorly treated sewage dominates

water quality, multiple studies discuss the inconsistent performance of culture-

enumerated FIB related to health and point to other indicators to better represent the

potential risk of illness (e.g., Enterococcus measured by qPCR).

• Waters receiving human fecal contamination can have viral pathogens present at

health-relevant levels and yet also be below recommended water quality levels for

culturable FIB.

• The health risk of recreating during or following wet weather compared to dry weather

can be different due to changes in fecal loading dynamics to a waterbody.

• Recent QMRA studies corroborate past findings that waters affected by human fecal

inputs can pose higher health risks compared to non-human fecal sources.

• Several QMRAs utilized norovirus as a primary reference pathogen to characterize risk

from human enteric viruses in waters affected by human fecal pollution.

• Across QMRA studies, the age of fecal contamination was identified as an important

factor affecting risk estimates and calculation of RBT values for alternative indicators.

• Outbreak surveillance in the United States and elsewhere continues to indicate the

importance of human enteric viruses as a leading etiologic agent of illness in ambient

recreational waters, even where low densities of FIB occur.

• Multiple outbreak investigations demonstrate the increased health burden children

experience when exposed to fecal pollution in ambient waters.

B. New Information on Coliphages

Since the finalization of the first five-year review, new peer-reviewed scientific literature on

coliphages has been published. In particular, EPA has focused on literature assessing coliphages

as indicators of fecal contamination and their ability to act as a surrogate for enteric viruses in

human fecal contamination that impacts recreational waters. The sources of the new literature for

this section on coliphages include studies cited in the literature review performed by Nappier et

al. (2019a) and thorough discussions with experts (e.g., U.S. EPA, 2017a) and colleagues.

In 2019, a systematic literature review and meta-analysis characterizing the occurrence of

coliphages in raw wastewater and ambient waters was published (Nappier et al., 2019a). The

review identified studies published up to January 2017 of coliphages occurrence in raw

wastewater and ambient waters of sufficient quality and study design to support estimating

coliphage distributions accounting for geographic region and season. Study results reviewed

consistently found that somatic coliphages occur in significantly higher numbers compared to

MSC in both raw wastewater and ambient waters, which is consistent with other published

results. In ambient waters, somatic coliphages were more prevalent compared to MSC. Analysis

of the available quantitative data and distribution estimates showed higher densities of somatic

coliphages compared to MSC in raw wastewater, but this difference is not significant when

accounting for geographic region or season. Seasonal trends for both somatic and MSC were

noted with a winter peak for both coliphage types, which is similar to the seasonal variation

reported in the literature for noroviruses. The distributions for somatic and MSC reported in

Nappier et al. (2019a) could be used to support various risk assessment applications, such as

water reuse, shellfish, and understanding recreational water risk.

Additional studies found in the published scientific literature since 2017 have emphasized the

need for a viral surrogate to evaluate risk of human fecal contamination affecting ambient

waters. Culturable FIB are more effectively reduced by conventional WWTP processes than are

enteric viruses, such as adenovirus and norovirus, and protozoa (Dias et al., 2018; Korajkic et al.,

2022). Because FIB are disproportionately inactivated by most water treatment and

environmental degradation processes compared to viruses, Farkas et al. (2020) concluded that

FIB are “poor indicators of viral infection risk” and should not be used as the sole indicator of

human fecal contamination in water quality monitoring programs. Furthermore, Farkas et al.

(2020) pointed out that somatic coliphages and male-specific ribonucleic acid (RNA)

bacteriophages are used to assess wastewater contamination but are not always specific to human

feces. While some studies corroborated the finding that FIB are useful as a fecal contamination

indicator, the level of FIB may not be a good indicator of waterborne viral pathogens (Dias et al.,

2018 Korajkic et al., 2018).

1. Research on Coliphages

Since the first five-year review, a series of studies were published measuring coliphage

occurrence trends in recreational waters and wastewater (Wanjugi et al., 2018, Korajkic et al.,

2020a; Jones et al., 2022), environmental decay and fate and transport characteristics of

coliphages (Korajkic et al., 2019a; McMinn et al., 2019; McMinn et al., 2020; Boehm et al.,

2019), wastewater disinfection effectiveness on coliphages (Korajkic et al., 2022; Jones et al.,

2022), co-occurrence of coliphages and common host-associated fecal source markers (Li et al.,

2021), and wastewater treatment process efficacy on the removal of pathogens and indicators

(Ryu et al., 2021).

Wanjugi et al. (2018) characterized the incidence of culture-enumerated MSC and somatic

coliphages, E. coli and enterococci in recreational waters in the Great Lakes basin. FIB

concentrations were consistently higher than the concentration of MSC and somatic coliphages.

Somatic coliphage levels were consistently higher compared to MSC. Ultraviolet light absorption

and water temperature, but not FIB levels, were closely associated with coliphage concentrations

suggesting different persistence trends between FIB and coliphages in Great Lake waters. In

general, physico-chemical properties and recreational area parameters (e.g., meteorological)

were better predictors of FIB compared to coliphages at Great Lake recreational sites tested in

this study (Wanjugi et al., 2018).

Korajkic et al. (2020a) reported geospatial trends of culturable bacteriophages (MSC, somatic,

total coliphages, and GB-124 phage), and multiple fecal pollution-related genetic markers in

49 primary influent wastewater samples collected from rural and urban WWTPs across the

United States. Total coliphages were enumerated using the CB390 host. Like the pattern reported

for the Great Lakes region by Wanjugi et al. (2018), coliphages consistently occurred at levels

lower than FIB at a national scale. On average, there was a trend of higher somatic coliphage

levels compared to MSC levels, however, log

plaque forming units (PFU) per 10 mL

concentrations of somatic, MSC, and total coliphages were not significantly different from each

other (p ≥ 0.469). Notably, the indicators tested in this study exhibited a geographic stability in

their occurrence, regardless of urban or rural location designation. However, the data collected

represent a three-month monitoring effort during the winter season. This study provides a large,

paired data set consisting of multiple viral and bacterial indicators that can support new or

updated QMRA models (Korajkic et al., 2020a).

Other studies reported on decay characteristics of both coliphage types and adenoviruses in lake

water (McMinn et al., 2020), the effect of various biotic and abiotic environmental parameters on

die-off of coliphages and other fecal microbiota in ambient waters (Korajkic et al., 2019a), as

well as fate and transport of coliphages and other indicators of fecal pollution in a constructed

wetland designed to treat an impaired creek (McMinn et al., 2019).

Boehm et al. (2019) conducted a systematic review and meta-analysis of decay rates of

mammalian viruses and coliphages in surface waters. The decay rate constants of MSC and

somatic coliphages were similar to those of enterovirus, human astrovirus, and rotavirus A.

Norovirus (specifically Norwalk virus), hepatovirus A, and mastadenovirus had smaller decay

rate constants than coliphages. Decay rate constants were higher with increasing water

temperatures. The decay rate constants for coliphages were smaller in fresh water versus

estuarine and marine waters. However, this pattern was not observed for mammalian viruses,

most of which had insufficient data to make a comparison (Boehm et al., 2019).

McMinn et al. (2020) conducted an in situ mesocosm study in a freshwater lake to compare

decay characteristics of MSC and somatic coliphages to human adenovirus 2. Effects of sunlight

and indigenous protozoans on viral decay characteristics were also evaluated. The results

demonstrated that decay of coliphages and adenovirus were similar in the mesocosms and that

the greatest log reductions were observed when viruses were exposed to both sunlight and

predation. Somatic coliphages were the most affected by sunlight (McMinn et al., 2020).

Korajkic et al. (2019a) summarized the most recent published literature on the decay of fecal

microorganisms in the aquatic environment. Studies included in the review demonstrated longer

survival of coliphages in waters < 20℃, and faster decay of coliphages, FIB, and viral pathogens

(measured by culture-based techniques) in marine waters relative to freshwaters (Korajkic et al.,

2019a).

McMinn et al. (2019) monitored levels of culture-enumerated FIB, MSC, somatic coliphages,

and clostridia as well as genetic markers for Campylobacter, E. coli and enterococci, human-

associated (HF183) and bird-associated (GFD) MST markers through a constructed wetland to

evaluate its effectiveness at reducing microbial contamination from sewer overflows in stream

water fed to the wetland. Increases in fecal indicator concentrations in the wetland system were

noted. The increase could be attributed to indicator regrowth within the wetland and authors

noted elevated bird activity in areas corresponding to increases in fecal indicators. Lack of

reduction in fecal indicators due to fecal inputs by wildlife has been noted in other studies.

General fecal indicators, such as enterococci and coliphages, widely occur in warm-blooded

animals so bird deposition in the wetland can lead to false assumptions on the effectiveness of

the constructed wetland. The concentration of MSC and somatic coliphages did not significantly

change through the wetland treatment system. Somatic coliphages were correlated to culture-

enumerated E. coli levels (p = <0.0001, R

= 0.486). Some treatment efficacy in the wetland was

noted based on decreases of HF183 through the system, whereas increases in the GFD marker

were observed due to the elevated bird activity.

Korajkic et al., (2022) evaluated the effectiveness of two wastewater disinfection strategies

(chlorination and UV light exposure) on removal of coliphages, FIB, and viral pathogens.

Somatic, MSC, and total (CB-390 host strain) coliphages were measured from influent, and 20–

40 L final effluent samples concentrated using dead-end hollow-fiber ultrafiltration (D-HFUF).

Regardless of the effluent disinfection strategy, FIB were generally more sensitive to chlorine

and UV disinfection compared to coliphages. Both MSC and somatic coliphages were equally

susceptible to chlorination, but MSC were more resistant to UV light. Comparisons of coliphage

removal to that of viral pathogens was rarely feasible due to many samples with no detectable

pathogens, but removal of MSC correlated to that of adenovirus removal when chlorination was

the treatment strategy. MSC, GB-124 bacteriophage, and crAssphages were the most resistant to

UV disinfection and performed similarly to infectious enteric viruses and adenoviruses.

Li et al. (2021) evaluated paired measurements of coliphages (MSC and somatic), FIB, and FSI

genetic markers for human, ruminant, canine, and avian feces at beach and river sites in the

Great Lakes region to study the co-occurrence of fecal indicators and host-associated genetic

markers. Study findings demonstrated that general fecal indicators used for monitoring water

quality can influence the interpretation of paired FSI measurements and potentially lead to

prioritization of different pollutant sources for remediation. Routine monitoring with somatic

coliphages or MSC often led to contradictory fecal pollution source trends compared to bacterial

indicators. Both FIB and coliphages can originate from multiple animal sources. Knowledge of

the sources of contamination contributing to a waterbody would improve interpretation of the

general fecal indicator monitoring results.

Ryu et al. (2021) characterized the occurrence of MSC and somatic coliphages in primary

influent and their associated LRVs after secondary treatment and chlorine or UV disinfection.

Coliphages were detected in all influent samples (range 20 to 11,700 PFU/100 mL) but had low

detection rates in effluent samples (7% of effluent samples had detectable MSC and 14% of

effluent samples had detectable somatic coliphages). For both coliphage types, LRVs were

similar (approximately 5-log

reduction) for both chlorine and UV disinfection. LRVs for

coliphages were greater than the LRV for adenovirus, suggesting less stability than adenovirus.

Fitzmorris et al. (2022) conducted a narrative review using the One Water framework to assess

the strengths and weaknesses of using bacteriophages as viral indicators in wastewater, biosolids,

reclaimed water, recreational water, and shellfish for public health purposes. This review

indicated that scientists are reaching a consensus that bacterial indicators do not account or

represent the risk that viral pathogens pose to public health. Fitzmorris et al. (2022) suggested

that the fate and transport phenomena of bacterial pathogens is different for viral pathogens

present in surface water, groundwater, and wastewater (affected by, for example, treatment

processes, size differences, and lack of metabolic overhead for biological survival). Therefore,

monitoring for fecal indicator viruses, specifically coliphages, could support bacterial fecal

indicator monitoring as part of site-specific health-protection criteria.

2. New Epidemiology Studies Including Coliphages

Epidemiological studies evaluating the association between coliphages measured in waterbodies

and GI illness have been conducted since the first five-year review (Griffith et al., 2016;

Benjamin-Chung et al., 2017). The interest in understanding this association is because enteric

viral pathogens have been recognized as the leading causative agents of recreational water

illnesses in the United States (Graciaa et al., 2018; Vanden Esschert et al., 2020). As a result,

coliphages have also been considered in the context of QMRA to integrate monitoring efforts

with risk assessment modeling to estimate potential health impacts (Boehm, 2019).

Griffith et al. (2016) combined results across three epidemiological prospective cohort studies at

three California beaches to compare culturable MSC with culturable enterococci in relationships

with illness among swimmers. Culturable MSC measured by EPA Method 1602 had a stronger

association with GI illness than culturable enterococci enumerated by EPA Method 1600, at

Avalon and Doheny, where human fecal pollution was known to be affecting the beaches.

Human source markers performed as well or better compared to culturable enterococci at the site

with known human sewage inputs from faulty infrastructure. Overall, site-specific conditions at

each beach determined which indicator best predicted GI illness.

Benjamin-Chung et al. (2017) conducted a pooled analysis of six prospective cohort studies at

coastal beaches in California, Alabama, and Rhode Island. Water quality was measured using

enterococci and coliphages. Under all conditions combined, there was no association between GI

illness and levels of enterococci or coliphages. Associations between coliphage levels and GI

illness were observed only when human fecal pollution was likely present. Additionally, when

human fecal contamination was present, culturable enterococci only performed well when

coliphages were also detected. When enterococci levels were <35 CFU per 100 mL, coliphages

were detected in 72 percent (somatic) and 79 percent (male-specific) of samples taken, indicating

that viral pathogens may be present in waters receiving human fecal contamination even when

water quality is below the EPA criteria value. The study findings included similar associations

between somatic coliphages or enterococci with gastrointestinal illness. Additionally, in human

fecal-contaminated marine waters, there was some indication of MSC having a stronger

association with illness than enterococci.

3. QMRA

Boehm (2019) used a QMRA framework to estimate RBTs associated with the illness

benchmark of 32 NGI per 1,000 recreators for MSC and somatic coliphages in ambient waters

receiving untreated wastewater inputs. The framework considers recreational exposure to various

ages of wastewater and the associated coliphages and enteric pathogens to account for the

microbial decay over time. The effect of temperature difference on decay rates were also

considered. Exposure to fresh, unaged wastewater contamination resulted in estimated RBTs for

somatic (60 PFU/100 mL) and MSC (30 PFU/100 mL) coliphages. The estimated RBT generally

decreased as the wastewater ages because coliphages decay more quickly compared to norovirus,

which was used as a reference pathogen in the QMRA, effectively indicating the estimated risk

increases per indicator over time. Estimated RBTs decrease more quickly at higher temperatures

(25°C vs. 15°C). When the age of contamination is unknown, estimated RBTs for both

coliphages ranged between 1 and 10 PFU per 100 mL.

4. Studies of Wastewater Treatment Efficacy

The fate and persistence of norovirus, MSC, and somatic coliphages were evaluated throughout

the treatment process at nine water resource recovery facilities (WRRFs) that utilized various

types of secondary and tertiary treatment and disinfection (Worley-Morse et al., 2019).

Reduction of norovirus genogroups I and II was more similar to reduction of coliphages than FIB

(Worley-Morse et al., 2019). Reduction of MSC and somatic coliphages was variable and

dependent on the specific unit processes employed by a given WRRF (Worley-Morse et al.,

2019). Facilities with non‐biological nutrient removal (BNR) activated sludge and chlorine (de

facto chloramine) disinfection had less reduction of coliphages (1–3 log

) compared to FIB

(5 log

) (Worley-Morse et al., 2019). Greater than a 4 log

reduction was reported for both FIB

and coliphages at facilities that employed BNR processes and UV or ozone disinfection (Worley-

Morse et al., 2019). Although a 4 log

reduction was reported for FIB at a facility using

peracetic acid for disinfection, only a 2–3 log

reduction was reported for coliphages (Worley-

Morse et al., 2019). Worley-Morse et al. (2019) conclude that many WRRFs in the United States,

in particular those with disinfection steps using peracetic acid and chlorine in the presence of

ammonia (or chloramines), have greater reduction of FIB than coliphages. Thus, viral indicators

may serve as better predictors of the fate of enteric viruses, including norovirus (Mann et al.,

2019; Worley-Morse et al., 2019).

Jones et al. (2022) conducted a systematic review and meta-analysis of occurrence of coliphages

in effluent. Jones et al. found somatic coliphages and MSC density data from WWTP effluent

that had undergone primary, secondary, or tertiary treatment, or disinfection. The densities of

MSC and somatic coliphages in WWTP effluent were significantly lower than the densities in

the influent after receiving secondary biological treatment (with and without nutrient removal) or

tertiary treatment (e.g., treatment ponds or phosphate removal), or disinfection through

chlorination, UV irradiation, or both processes. This systematic review suggested that the

combination of tertiary treatment processes may result in greater viral inactivation in WWTP

effluent compared to a single effluent disinfection step. Jones et al. (2022) also concluded that

considering somatic coliphages and MSC as indicators of fecal contamination of sewage and

treated effluent continues to be useful to assess its potential impact on public health and

wastewater utilities.

5. Summary of New Information on Coliphages

• Both somatic and MSC consistently occur in raw wastewater across the United States.

Somatic coliphages are generally more numerous than MSC, and thus have potential to

be used as indicators of fecal contamination in waters affected by sewage and

wastewater treatment (particularly disinfected effluent).

• Viral indicators, such as coliphages, may perform better as surrogates of the fate of

human enteric viruses compared to culture-enumerated FIB during wastewater

treatment.

• Coliphages can exhibit a higher decay rate relative to viral pathogens in ambient

waters.

• Because coliphages have been shown to have a stronger relationship to illness than FIB

where human fecal sources were present, knowledge of the fecal sources affecting a

particular waterbody can improve interpretation of coliphage monitoring data.

C. New Information on Cyanotoxins

While cyanotoxins are not subject to the five-year review since they do not meet the criteria of

pathogens and pathogen indicators, EPA assessed the new information on health effects after

cyanotoxin exposure from literature searches conducted in 2021 by EPA (ORD and Office of

Science and Technology [OST]; see Appendix A for more information on methods). The 2021

literature search focused on six cyanotoxins: anatoxins, β-methylamino-L-alanine (BMAA),

cylindrospermopsin, microcystins, nodularins, and saxitoxins. From the relevant articles

identified in the 2021 literature search, new (defined as published since 2016) studies of health

effects after exposure to cyanotoxins are described here.

Cyanobacteria, also called blue-green algae, are naturally occurring photosynthetic bacteria

found in freshwater and brackish waters. Under certain environmental conditions, such as

elevated levels of nutrients, warmer temperatures, still water, and plentiful sunlight,

cyanobacteria can rapidly multiply to form HABs. Some species of cyanobacteria are able to

produce toxic compounds, known as cyanotoxins, which can be harmful to human and animal

health (U.S. EPA, 2019). EPA developed a public HAB web portal that contains information on

cyanobacteria and managing cyanotoxins in drinking and recreational waters

(https://www.epa.gov/cyanohabs).

U.S. exposure information indicates that cyanobacterial HABs have been reported in ambient

waters in all states and occasionally in marine waters (U.S. EPA, 2019; Woods Hole

Oceanographic Institute [WHOI], 2016). During a cyanobacterial HAB, the toxin concentration

can rapidly increase and may become elevated before a visible bloom is observed (Chorus et al.,

2000; WHO, 2021b; U.S. EPA, 2019) and persist after the bloom fades. Therefore, human

exposures can occur before and after the visible signs of a bloom. The most common

cyanotoxins found in ambient waters in the United States are microcystins, cylindrospermopsin,

anatoxin-a, and saxitoxins (U.S. EPA, 2009a; U.S. EPA, 2019). Nodularins are typically

produced by HABs in brackish waters and share similarities to microcystins in both structure and

adverse human health effects resulting from exposure. Exposure to elevated levels of

cyanobacteria and the toxins they produce during recreational activities could lead to adverse

health effects ranging from a skin rash, fever-like symptoms, body aches, respiratory irritation,

and gastrointestinal symptoms to more serious adverse health effects associated with organ

damage or neurological effects (U.S. EPA, 2019).

1. RWQC Guidance Values and Health Advisories for Cyanotoxins

In 2019, EPA published recreational water quality criteria and/or swimming advisories for two

cyanotoxins, microcystins and cylindrospermopsin, based on the best available science (U.S.

EPA, 2019). EPA recommended criteria for these two cyanotoxins include a magnitude (8 μg/L

microcystins or 15 μg/L cylindrospermopsin) and duration (not to be exceeded in more than

three 10-day assessment periods over the course of a recreational season) for freshwaters with a

recreational designated use (Table 2). The recommended magnitude represents the concentration

of microcystins or cylindrospermopsin at or below that which is not expected to result in adverse

human health effects from short-term recreational exposure to the toxins via incidental ingestion

by children while swimming in freshwaters. The recommended values are based on critical

studies identified from the available toxicity information described in the Health Effects Support

Document for the Cyanobacterial Toxin Microcystins (U.S. EPA, 2015a) and Health Effects

Support Document for the Cyanobacterial Toxin Cylindrospermopsin (U.S. EPA, 2015b). These

recommendations are intended as guidance to states, territories, and authorized tribes to consider

when developing WQS. Alternatively, these recommendations can be used as the basis of

swimming advisories for notification purposes in recreational waters to protect the public.

Table 2. Recreational Criteria or Swimming Advisory Recommendations for Microcystins

and Cylindrospermopsin

Application of

Recommended

Values

Microcystins

Cylindrospermopsin

Magnitude

(µg/L)

Duration Frequency

Magnitude

(µg/L)

Duration Frequency

Recreational

Water Quality

Criteria

One in 10-day

assessment

period across a

recreational

season

More than three

excursions in a

recreational

season, not to be

exceeded in more

than 1 year

One in 10-day

assessment

period across a

recreational

season

More than three

excursions in a

recreational

season, not to be

exceeded in more

than 1 year

Swimming

Advisory

1 day

Not to be

exceeded

1 day

Not to be

exceeded

These recommendations can apply independently within an advisory program or in water quality standards. States

can choose to apply either or both toxin recommendations when evaluating excursions within and across recreational

seasons.

An excursion is defined as a 10-day assessment period with any toxin concentration higher than the criteria

magnitude. When more than three excursions occur within a recreational season, and that pattern reoccurs in more

than 1 year, it is an indication the water quality has been or is becoming degraded and is not supporting its

recreational use. Units are micrograms (μg) per L.

The WHO Guidelines on Recreational Water Quality - Volume 1: Coastal and Fresh Waters

(WHO, 2021a) derived guideline values for four cyanotoxins: anatoxin-a and analogs (60 µg/L),

cylindrospermopsins (6 µg/L), microcystins (24 µg/L), and saxitoxins (30 µg/L). Differences

between EPA’s and WHO’s values for microcystins and cylindrospermopsin are due to

differences in input values such as children’s body weights and incidental ingestion. For

microcystins, WHO selected a different critical study compared to EPA for the basis of

developing recreational values. Additional information for these four cyanotoxins can be found

in WHO’s Toxic Cyanobacteria in Water, 2nd Ed (Chorus and Welker, 2021).

2. Health Studies

Since the last five-year review, new studies of health effects after exposure to cyanotoxins have

been published. Two single-dose, acute toxicity studies (Chernoff et al., 2020; Chernoff et al.,

2021) and three repeat-dose, chronic toxicity studies (Li et al., 2016a; Wang et al., 2016b; Zhou

et al., 2020) of health effects following oral exposure to microcystin were identified. One repeat-

dose, chronic toxicity study of health effects following oral exposure to cylindrospermopsin

(Chernoff et al., 2018) was published. Review of new data from new studies on

cylindrospermopsin and microcystins does not suggest changes to the reference doses (RfDs).

Further details of the studies for microcystins and cylindrospermopsin are described below.

Microcystins

Acute Studies

When EPA developed the Health Advisories in 2015 and the RWQC for Microcystins in 2019,

EPA derived an RfD for all microcystins using microcystin-LR (MC-LR) as a surrogate based on

MC-LR having a robust toxicological data set and relatively high potency among the microcystin

congeners (U.S. EPA, 2015a). Lethal dose 50 percent (LD

) studies of the most common

microcystin congeners identified a relationship between the congeners with hydrophobic L-

amino acids (-LA, -LR, and -YR) and the highest toxicity, and between hydrophilic amino acids,

such as microcystin-RR, and lower toxicity (Stoner et al., 1989; Gupta et al., 2003).

New studies on microcystin have been published since 2016. Chernoff et al. (2020, 2021)

characterized the acute toxicity of multiple congeners of microcystin. Chernoff et al. (2020)

reported acute toxicity for oral exposure of BALB/c mice to 10 different microcystin congeners

(MC-LR, MC-LA, MC-LF, MC-LW, MC-LY, MC-RR, [Asp3]MC-RR, [Asp3,Dhb7]MC-RR,

MC-WR, and MC-YR) by gavage as single 7 mg/kg doses. Significant differences in the single-

dose acute toxicity of microcystin congeners were observed, with MC-LR, MC-LA, MC-LY, and

MC-YR having effects on ≥ seven toxicity indicators e.g., moribundity, increases in relative liver

weight, and changes in clinical chemistry parameters. Noticeably, MC-LA and MC-LR showed

the greatest toxicity, including significant increases in moribundity. Consistent with previously

published studies, hydrophobic congeners of microcystin (e.g., MC-LR) were more toxic than

the hydrophilic congeners (e.g., MC-RR) (U.S. EPA, 2015a). Of the hydrophobic congeners

tested, MC-LR was more toxic than MC-LW, MC-LF, and MC-WR.

In a follow up study, Chernoff et al. (2021) administered single doses of several microcystin

congeners (LA, LR, LY, and RR) by gavage to BALB/c mice at levels ranging from 0.5 to

11 mg/kg, depending on the congener (Chernoff et al., 2021). All animals were euthanized

24 hours post-dosing. In this study, MC-LA was the most toxic congener among the four with

toxicity. MC-LA induced significant toxic effects in the liver that led to increases in total serum

bilirubin at 3 mg/kg, which was identified as the lowest-observed-adverse-effect level (LOAEL).

In a 28-day exposure of MC-LR, Heinze (1999) identified a LOAEL of 50 μg per kilograms per

day (μg/kg-d), 60 times lower than the acute LOAEL identified by Chernoff et al. (2021) for

MC-LA. The Heinze (1999) LOAEL for MC-LR was used for the derivation of the EPA’s

recreational criteria. Therefore, microcystin recreational criteria based on MC-LR is protective of

short-term, 1- to 28-day exposures to all four microcystin congeners. This newly identified study

(Chernoff et al., 2021) does not change the microcystin recreational criteria as the LOAEL from

an acute exposure study of MC-LR is protective of primary contact exposures to other

hydrophobic microcystins.

Chronic Studies

Three new studies of health effects after chronic exposure to microcystin were identified. Wang

et al. (2016b) administered 1, 10, or 40 μg/L of MC-LR in drinking water for 6 months to female

BALB/C mice to determine the impact of chronic, low-dose exposure to MC-LR on the lungs

(Wang et al., 2016b). The authors observed alveolar collapse and lung cell apoptosis with altered

cell junction integrity at all doses. The authors were not able to detect the presence of MC-LR in

lungs by immunoblot analysis and quantitative dose-response data were not provided.

Li et al. (2016a) characterized the chronic toxicity of MC-LR per L in adult male C57BL/6 mice

treated with 1, 5, 10, 20, and 40 µg MC-LR per L in drinking water for 12 months. Statistically

significant differences in lung/body weight ratios, caused by an increase in the level of lung

inflammatory cytokines that resulted in thickening of the alveolar septa, were observed (Li et al.,

2016a). The authors did not provide quantitative data, however, so a dose-response curve could

not be developed.

Zhou et al. (2020) evaluated the effects of chronic exposure to MC-LR on mice testes. Male mice

(age and strain not specified) were exposed to 0, 1, 10, or 100 μg/L MC-LR (purity >96%;

dissolved in 0.1% methanol) in drinking water for 90 or 180 consecutive days. Mice were noted

to be 15 to 25 grams (g), but body weights in Figure 1 presented in the study appear to be more

than 25 g on day 0. Therefore, it is not clear if the mice used were adults. Average intake was

estimated to be 0.15, 1.5, and 15 μg/kg-d by the study authors based on an assumed, rather than

measured, water intake of 1.5 mL per 10 g body weight and presumably the average biweekly

body weights. Body weight was significantly decreased in 10 and 100 μg/L groups beginning at

154 and 126 days, respectively. There are methodological issues with the lack of measured water

intake, the lack of reporting about the age of the animals, and the uncertainties with body weight

measurements at the start of the study. No significant change or trend in relative testes weight at

either 90 or 180 days was observed. However, absolute testes weights were not reported. Based

on decreases in body weights, it would be expected that there would be a change in absolute

testes weights. The lack of these data is a limitation in assessing effects on the testes.

Histological evaluation indicated an increase in abnormal seminiferous tubules in the 100 μg/L

group at both 90 and 180 days and in the 10 μg/L group at 180 days. Zhou et al. (2020) cite

methods used by Chen et al. (2011). As noted in section 7.4.2 of EPA (2015a), peer reviewers

identified concerns with the histological procedure used by Chen et al. (2011) and the issues do

not appear to have addressed in Zhou et al. (2020). Of 4,950 proteins quantified by isobaric tags

for relative and absolute quantitation (iTRAQ)-based proteomics, 20 proteins (8 upregulated and

12 downregulated) were significantly different between the 10 μg/L and control group. The

altered protein expression corresponded to 15 pathways, including renin-angiotensin system,

extracellular matrix-receptor interaction, phosphatidylinositol 3 kinase/protein kinase B

(PI3K/AKT) signaling pathway, focal adhesion, tight junction (TJs), and gap junction (GJs). The

histological analysis found that the blood-testes barrier (BTB) was significantly more permeable

in the two higher dose groups compared to the control group. The results suggest that MC-LR

causes dysfunction of the BTB through affecting TJs and GJs. Adverse effects in the Zhou et al.

(2020) study occurred at the two higher dose groups, with an estimated intake of 1.5 and 15

µg/kg-d, which are lower than the LOAEL of 50 µg/kg-d identified in the Heinze (1999) study

used to develop the EPA’s recreational criteria recommendation for microcystins. However, the

multiple methodological concerns, described above, of the Zhou et al. (2020) study precludes

EPA from considering the data to develop revised criteria.

The three chronic effects studies identified as part of EPA’s literature review assessed the

potential adverse respiratory (Li et al., 2016a; Wang et al., 2016b) and male reproductive (Zhou

et al., 2020) effects after long-term exposure to MC-LR. However, limitations associated with

the study designs and reporting prevented the quantitative use of the information and would not

support revising the RfD used to derive the 2019 recreational criteria recommendation for MC.

Cylindrospermopsin

One new oral toxicity study of cylindrospermopsin was identified. The study exposed adult male

and female mice to cylindrospermopsin (75 to 300 μg/kg-d) by gavage for 90 days (Chernoff et

al., 2018). Health effects observed were elevated organ to body weight ratios of the liver and

kidney at all dose levels, increase in serum alanine aminotransferase (ALT) activity, decreases of

blood urea nitrogen (BUN) and serum cholesterol concentrations in males, plus high monocyte

counts in both genders compared to the negative control. The study identified a LOAEL of

75 μg/kg-d based on significant effects in liver and kidney/body weight ratios, reduced BUN,

increased serum monocytes, and multiple signs of histopathology. The health effects as well as

the histopathological findings are consistent with the toxicological findings found in the earlier

studies of Humpage and Falconer (2002, 2003) that were used for the derivation of EPA’s RfD

for cylindrospermopsin. Although a comparison of no-observed-adverse-effect levels (NOAELs)

between the Chernoff et al. (2018) and Humpage and Falconer (2002, 2003) studies is not

possible, the higher LOAEL (75 μg/kg-d) reported in the Chernoff study is higher than the

60 μg/kg-d dose reported in the Humpage and Falconer study, so the new information would not

support revising the RfD used to derive EPA’s 2019 recreational criteria recommendation for

cylindrospermopsin.

Other Cyanotoxins

EPA identified new toxicity studies for anatoxin-a and saxitoxins but not for nodularins. There is

not enough information to support the derivation of RfDs for either anatoxin-a or nodularins. The

EPA literature search did identify a number of new studies, including dose-response studies, for

saxitoxins.

Anatoxin-a

One new study was identified on the lethal dose of anatoxin-a (ATX) and its congener

dihydroanatoxin-a (dhATX) (Puddick, et al., 2021). Purified anatoxin-a and dhATX from the

benthic cyanobacterium Microcoleus autumnalis were administered to female Swiss albino mice

by intraperitoneal (i.p.) injection and by oral ingestion (gavage and feeding) for 14 days to

determine the LD

. The researchers observed a difference in lethal doses for each exposure

route, with i.p. injection dhATX was less toxic than anatoxin-a (0.73 mg/kg for dhATX and

0.23 mg/kg for anatoxin-a), but dhATX was more toxic than anatoxin-a with gavage (2.5 mg/kg

for dhATX and 10.6 mg/kg for anatoxin-a) and feeding (8 mg/kg for dhATX and 25 mg/kg for

anatoxin-a). The study design focused on identifying lethality only and did not analyze for

nonlethal health effects such as neurotoxicity, histopathology, hematology, and serum chemistry,

and therefore, this study could not be used to derive a noncancer RfD for ATX or dhATX.

Zhong et al. (2020) is an in vitro study that evaluated the immunotoxicity of anatoxin-a using

Carassius auratus lymphocytes. Non-adherent lymphocytes isolated from the kidneys of C.

auratus fish (6–10 months old) were cultured in Roswell Park Memorial Institute (RPMI)-1640

medium with 5 percent fetal bovine serum, free from antibiotics, and exposed to 0, 0.01, 0.1, 1,

and 10 mg/L for 12 hours. In vitro immunotoxicity from exposure to anatoxin-a was evidenced

by increases in apoptosis of lymphocytes, which increased in a dose-dependent manner, based on

both deoxyribonucleic acid (DNA) fragmentation and flow cytometry. Flow cytometry results

indicated that the percentage of apoptotic lymphocytes exposed to 0.01, 0.1, 1, and 10 mg/L of

anatoxin-a reached 18.89, 22.89, 39.23, and 35.58 percent, respectively, with less than 15 percent

observed in controls. Ultrastructural changes associated with anatoxin-a exposure identified

using transmission electron microscopy included cytoplasmic condensation, vacuolation, and

swollen mitochondria in C. auratus lymphocytes. Oxidative stress also increased in a dose-

dependent manner as measured by increases in reactive oxygen species (ROS) and

malonaldehyde, and decreases in superoxide dismutase, glutathione, catalase, glutathione

reductase, glutathione peroxidase, and glutathione-s-transferase. In vivo studies are needed to

confirm the immunotoxicity findings of Zhong et al. (2020).

No human epidemiological data were identified that assessed health effects after exposure to

anatoxin-a. One new human case report of food poisoning following exposure to sea figs

contaminated with anatoxin-a was identified but the study did not measure or report dose

information (Biré et al., 2020).

Studies assessing adverse effects from either the inhalation or dermal route of exposure were not

identified for anatoxin-a. Anatoxin-a was detected on glass fiber filters using in field-deployed

air sampler during an active harmful algal bloom in Massachusetts in 2019 (Sutherland et al.,

2021), but the study was not designed to quantify or characterize the human health risk from

inhalation of anatoxin-a in water droplets.

For anatoxin-a, the current available information on health effects falls short for determining an

RfD. In the absence of EPA recreational water recommendations for anatoxin-a, some states

have adapted the WHO health-based reference values for acute anatoxin-a exposure (WHO,

2021b). EPA will continue to evaluate new scientific data on anatoxin-a and its effects as they

become available.

Saxitoxins

Saxitoxins are a group of 57 analogs grouped by structural similarity, including the parent

compound (STX), neosaxitoxin (neoSTX), gonyautoxins (GTXs), C-toxins,

decarbamoylsaxitoxins, and lyngbyatoxins (LWTXs). STX equivalents (STXeq) are often

reported in studies as the total concentration of STX variants, or they may represent

concentrations adjusted for toxicity (WHO, 2020). New studies on health effects after exposure

to saxitoxin have been published since 2016. EPA has initiated the development of a Health

Effects Support Document (HESD) for STXs. Health effects studies, both human studies and

toxicological studies, were identified and reviewed through EPA’s literature search. The

available human studies include case reports of morbidity and mortality resulting from paralytic

shellfish poisonings (PSPs) in adults and children, as well as epidemiologic and case-series

studies that assessed human health effects after exposure to saxitoxins. Development of this

HESD will continue in 2023.

Human Case Reports

A case of PSP poisoning in the United States was reported in 2016 when an elderly female who

ate clams harvested in Roslyn Beach in Kodiak, Alaska, reported symptoms of nausea with dry

heaving, weakness, respiratory depression, and shock (Coleman et al., 2018). PSP testing of the

clams from the suspected meal determined a saxitoxin concentration of 277 µg per 100 g, 309 µg

per 100 g of neosaxitoxin, 576 to 2,490 µg per 100 g of multiple GTX, 7.52 to 11.3 µg per 100 g

of decarbamoyl, and 10.8 to 221 µg per 100 g of C-toxins. Saxitoxins, neosaxitoxins, and GTX1-

4 were also found in the urine of the patient (64.0 µg/g-creatinine, 60 µg/g-creatinine, and 492-

4,780 µg/g-creatinine, respectively).

Knaack et al. (2016) evaluated 11 patients with suspected PSP. Of the 11 patients, four were

confirmed to have STX-PSP by urine testing (24–364 ng STX/g-creatinine) and five patients had

clinical manifestations of PSP. Results revealed that dysphagia and dysarthria appeared to be

stronger indicators of PSP than paresthesia and nausea, which are commonly used to clinically

diagnose patients with PSP. Meal remnants obtained from six presumptive PSP cases were

analyzed and all six samples tested positive for PSP toxins. The results of this report are limited

because only 4 of 11 patients were confirmed to have PSP.

Animal Studies

Diehl et al. (2016) conducted a toxicological study in rats exposed to two doses of saxitoxin

equivalents orally via drinking water for 30 days and then assessed for behavioral effects. The

results of the behavioral test battery indicate that the STX-exposed rats had decreases in learning

and memory processes compared to negative controls (Diehl et al., 2016).

Selwood et al. (2017) conducted a series of studies to determine the acute toxicity of STX and

multiple STX analogs administered by i.p. injection, as compared to oral gavage or consumption.

Exposure data were used to identify the LD

and NOAEL. Mice were first dosed a step below

the best preliminary estimates of the LD

, and subsequent animals receive a lower dose (if the

previous animal dies) or a higher dose (if the previous animal survives). In general, the LD

values for each STX analog were lower following i.p. injection than they were with oral

administration. This was attributed to slower absorption of the toxins via the oral route.

Nonlethal health effects of exploratory behavior, grip strength, changes in abdominal breathing,

and lethargy were assessed and used as the basis for the NOAEL values for oral gavage (544

nanomoles per kilogram [nmol/kg] for STX; 276 nmol/kg [neoSTX] to 25,500 nmol/kg [C3&4]

for STX analogs) and dietary exposures (4,360 nmol/kg [decarbamoyl neosaxitoxin

(dcNEOSTX)] to 17,400 nmol/kg [C1&2] for STX analogs; not determined for STX). The

symptoms of toxicity manifested by the STX analogs following the oral gavage and feeding

routes of exposure were proportional to those for i.p. injection except that the onset of toxicity,

recovery from sublethal doses, and time to death were delayed for the oral exposure routes. The

recovery and time to death results for the different exposure routes (i.p. injection < gavage <

feeding) indicate the toxin has to be delivered to systemic circulation before the effects are

manifested (Selwood et al., 2017).

In Vitro Studies

Peripheral blood mononuclear cells obtained from wild-captured harbor seals were used to study

the effects of STX on immune cell modulation and phocine distemper virus (PDV) replication

(Bogomolni et al., 2016). Exposure to 10 ppb STX led to a 78 percent increase in lymphocyte

proliferation compared to the control. In STX-exposed cultures, there was an 8-fold increase in

lymphocyte fraction PDV loads on average at day 5 post-infection, and a 2.5-fold increase in the

supernatant fraction PDV load at day 9 post-infection. Given these findings, the authors suggest

that exposure to STX could increase systemic virus dissemination upon in vivo infection in

marine mammals.

D'Mello et al. (2017) investigated the cytotoxicity of STX and other cyanotoxins on primary

human astrocytes using the lactate dehydrogenase (LDH) release assay. LDH activity was

significantly increased with 0.1 nmol STX, and inhibitory concentration (IC) IC

and IC

(concentration giving 20% or 50% of the maximum inhibitory response) values were determined

to be 0.65 and 0.95 nmol, respectively. Cell proliferation was significantly decreased with STX

exposure at both the IC

and IC

doses, with proliferation at the IC

dose at 46.4 ± 2.7 percent

of the control.

Two mammalian neuronal cell lines were used to study effects of extended, low-dose exposure

to STX on neuronal development (O'Neill et al., 2017). STX exposure prevented normal

morphological changes in neuronal morphology even with the lowest concentration tested. The

effects observed appeared to be dose dependent. The results support a conclusion that STX

exposure can cause a change in neuronal cell morphologies in both the central and autonomic

nervous system.

The interactions between pristine single-walled carbon nanotubes (SWCNTs) or carboxylated

single-walled carbon nanotubes (SWCNT-COOH) and STX were evaluated to understand the

potential effects of those interactions on cell toxicity (Ramos et al., 2017). No effects on cell

viability or cell proliferation were reported with any treatment at 30 minutes. At 24 hours,

exposure to STX with SWCNT led to a significant decrease in cell viability compared to

controls. No other statistically significant effects at 24 hours were found. These data suggest a

weak interaction between STXs and SWCNTs that could alter the effect of STX on mammalian

neuronal cells.

Abi-Khalil et al. (2017) investigated the immunotoxicity and localization of STX in hemocytes

from a species of oyster. Fluorescent coumarin-coupled STX (STX-Cou) was detected in the

cytoplasm of oyster hemocytes but did not co-localize with the mitochondria. The negative

control, Gua-Cou, showed no specific localization of fluorescent coumarin labeling, suggesting a

STX-specific accumulation in the cytoplasm of oyster hemocytes. Upon evaluation of

phosphatidylserine translocation and membrane permeability, a strong green-fluorescent signal

indicated the presence of phosphatidylserine on the cell surface in both Etoposide and STX-

treated hemocytes. Additionally, the signal depended on dose; it was detected after treatment

with 3.3 µM STX but not with 0.8 µM STX (Abi-Khalil et al., 2017).

The apoptotic potential of STX was determined as the imaging of cells exposed to 0.8 µM STX

showed marked chromatin condensation at the nucleus periphery, a hallmark of apoptosis. A

similar effect was noted in the positive control (50 µM of Etoposide) but not the sterile sea water

negative control. DNA fragmentation and apoptosis were determined in cells exposed to 0.8 µM

STX for 3.5 hours. Tetramethylrhodamine nick end labeling (TMRNEL) revealed that

hemocytes exposed to 0.8 µM STX exhibited a greater proportion of cells with fragmented

nuclear DNA compared to controls. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium

bromide (MTT) assay results showed that exposure to 3.3 µM STX resulted in a decrease (34% ±

8%) in living cells similar to that of 50 µM Etoposide (49% ± 8%), however, no effect was

observed after exposure to 0.8 µM STX. Over a 2-hour period, exposure to STX did not exhibit a

detectable effect on ROS concentrations, suggesting that cell death observed in other assays is

not due to ROS (Abi-Khalil et al., 2017).

Some state and international agencies have developed health-based values for saxitoxins in

drinking and recreational waters. The Ohio Environmental Protection Agency (OHEPA) issues

Recreational Public Health Advisories at 0.8 μg/L (OHEPA, 2022). The Ohio Department of

Health (ODH) also issues “Do Not Drink” advisories for saxitoxins in drinking water: 0.3 μg/L

for bottle-fed infants and children younger than school age, pregnant women, nursing mothers,

individuals with pre-existing liver conditions or who are immunocompromised, and individuals

receiving dialysis treatment; and 1.6 μg/L for all people of all ages as well as for pets and

livestock (ODH, 2020). WHO (WHO, 2020) estimated an acute oral RfD for saxitoxins of

0.5 µg/kg-body weight (bw) based on mild shellfish poisoning symptoms from 500 case reports

exposed to PSP, as reported by the European Food Safety Authority (EFSA, 2009). EFSA

estimated a NOAEL of 0.5 μg STXeq kg-bw based on human PSP symptoms and supported by

animal studies: based on the point of departure of the lowest acute NOAEL for neoSTX of

87 µg/kg-bw (Munday et al., 2013) from a gavage toxicology study, an acute oral RfD for

neoSTX of 0.87 µg/kg-bw (applying an uncertainty factor of 100) was derived. This value is of

the same order of magnitude as the reference values obtained with human data (WHO, 2020).

Nodularins

No new oral toxicity studies of nodularins, published since 2016, were identified in EPA’s 2021

literature search. EPA has previously reviewed the available toxicity data for nodularins and

found that the previously published (before 2016) data are inadequate to derive an RfD to use to

support developing RWQC and/or Swimming Advisories.

3. Summary of New Information on Cyanotoxins

• EPA has identified several repeat-dose, acute, and chronic toxicity studies of health

effects of cyanotoxins published in the last 5 years.

• For cylindrospermopsin and microcystins, these new studies either supported the existing

reference doses identified in EPA’s HESDs or did not provide new information to

support derivation of a revised RfDs.

• EPA evaluated several toxicity studies characterizing effects from exposure to anatoxin-

a, saxitoxins, and nodularins.

o Work is underway at EPA to evaluate data to support development of an HESD

for saxitoxins.

o The limited available toxicity information available for anatoxin-a and nodularins

does not support development of an HESD at this time.

D. New Information on Antimicrobial Resistance

To identify new literature on AMR and antimicrobial resistant genes (ARGs), EPA reviewed

three relevant review articles that were published since the last five-year review (Nappier et al.,

2020; Korajkic et al., 2020b; Franklin et al., 2021).

1. EPA AMR Research

The review article, Korajkic et al. (2020b), on AMR in Enterococcus species in marine and

estuarine environments indicates that E. faecium and E. faecalis exhibit resistance to the highest

number of antibiotics, followed by E. casseliflavus, E. hirae, and E. durans. Further, studies

found that all Enterococcus species were resistant to erythromycin. The highest proportion of

resistant enterococci were measured in the water column (>18%), with other important reservoirs

of resistant enterococci being feces and tissues of animals such as seabirds, whales, and clams,

and to a lesser degree sediments and sands. Regarding methodology, the review identified a lack

of standardized procedures to study, detect, and classify antibiotic resistant Enterococcus in the

environment and recommended the establishment a standardized framework for studying

antibiotic resistance in the environment.

The review article by Franklin et al. (2021) on the available molecular methods to analyze AMR

in surface waters was conducted to support One Health Assessments

(https://www.epa.gov/healthresearch/one-health). This article described three state-of-the-art

methodologies that can provide information about the presence, diversity, and dynamics of ARG

targets in the environment including surface waters (Franklin et al., 2021). The three techniques,

high-throughput qPCR, metagenomics, and whole-genome sequencing, are all culture-

independent and can target a high diversity of genes simultaneously. The article noted that there

is value in combining molecular information with culture-based approaches because molecular

methods can identify most, if not all, genetic elements that may confer AMR in a single

environmental isolate and/or a microbial community, while culture-based methods can provide

phenotypic confirmation of AMR in environmental isolates. Used together, the methods can

more fully characterize AMR in surface waters.

The state-of-the-science review by Nappier et al. (2020) of AMR bacteria and ARGs in

recreational waters reported that new studies found that ARGs can be acquired through

spontaneous mutations and can persist in microbial populations via selective pressure. For

example, AMR and heavy metal resistance genes often co-occur on the same mobile genetic

elements (e.g., plasmids), resulting in a co-selection and propagation of mobile genetic elements

carrying ARGs, even in the absence of antibiotics. Since resistant genes can move between

pathogenic and nonpathogenic microorganisms, a better mechanistic understanding of horizontal

gene transfer and AMR selection is warranted to improve AMR monitoring. Nappier et al.

(2020) also reported that studies indicate that untreated wastewater, treated WWTP effluent,

medical waste, pharmaceutical production, biosolids, agriculture, aquaculture, plant, and animal

agriculture (e.g., concentrated animal feeding operations [CAFOs]), and the feces of birds and

other wildlife can be sources of AMR bacteria and ARGs. Such observations are consistent with

prior studies that show differential effects of different treatment processes on AMR bacteria and

ARGs.

2. Summary of New Information on AMR

• Treatment processes commonly used at WWTPs are not typically calibrated to target

removal of AMR bacteria or ARGs.

• An increased understanding of horizontal gene transfer mechanisms and AMR

selection can be used to improve monitoring for AMR in ambient waters.

• A combination of techniques (including both molecular- and culture-based) are

necessary to fully understand AMR in surface waters.

E. New Information on Human and Non-Human FSI

FSI techniques are used to characterize fecal sources potentially present in polluted waters.

These methods rely on the detection and/or quantification of host-identifiers such as chemical or

microbial targets closely associated with a particular pollution source (Hagedorn et al., 2011).

Accurate and reliable FSI technologies have the potential to improve future water quality

management in the United States. The 2012 RWQC (U.S. EPA, 2012a) has provisions that

recommend these FSI methods for use as a sanitary characterization tool and has published

information to support alternative criteria eligibility of FSI (U.S. EPA, 2014a).

EPA has invested considerable resources to develop, validate, standardize, and implement FSI

technologies. High-priority research areas include completion and publication of standardized

EPA methods for human-associated qPCR methods, development and public release of a

companion standard reference material, further investigation of data interpretation approaches,

and science to advance virus-based human FSI method development.

Since 2017, EPA researchers have published 15 peer-reviewed studies, released two nationally

validated human-associated FSI qPCR methods, and codeveloped a standard reference material

in collaboration with the National Institute of Standards and Technology (NIST). In addition to

EPA activities, there are numerous significant contributions by other researchers that have

advanced FSI recreational water science.

The systematic literature review identified 85 relevant articles (Figure 1) discussing advances in

MST or fecal source identification techniques (Appendix B). Additionally, 22 relevant references

were identified via an ad hoc process (Blatchley et al., 2007; Shanks et al., 2009, 2010, 2011,

2014; Sinclair et al., 2009; Okoh et al., 2010; Soller et al., 2010a,b; Hagedorn et al., 2011;

Boehm et al., 2013; Ervin et al., 2013; Dutilh et al., 2014; U.S. EPA, 2014a; Qiu et al, 2015;

Stachler et al., 2017; Korajkic et al., 2020a; McKee et al, 2020; Shrestha et al., 2020; Ahmed et

al., 2021; Kralj et al., 2021; Sivaganesan et al., 2022).

1. Human FSI Research

Advances in Bacterial-Based FSI

Researchers have advanced methods for bacterial-based identification of human fecal sources in

recreational water. In particular, the Bacteroides HF183 genetic marker is well characterized

(Ahmed et al., 2016b; Holcomb and Stewart, 2020; Jiang et al., 2018; Somnark et al., 2018;

Staley and Edge, 2016; Vadde et al., 2019) and routinely used for qPCR studies due to its strong

human-host association (Devane et al., 2019; Staley and Edge, 2016; Symonds et al., 2017).

HF183 was found to be a more specific marker for combined sewage overflows than human

polyomavirus, pepper mild mottle virus, and six other human pathogens in Cobbs and Tacony

Creeks in Philadelphia (McGinnis et al., 2018). HF183 was also readily detected at Lake

Pontchartrain beach and was used to determine that rainfall events introduce large amounts of

human fecal bacteria into lake waters (Xue et al., 2018). Green et al. (2019) assessed HF183 and

ruminant-associated markers during dry weather in Onondaga Creek, New York. They found that

the upstream ruminant inputs decayed and/or were diluted out and that the high levels of urban

bacterial contamination were human associated and most likely due to failing infrastructure

and/or illicit discharges independent of rain events. In addition to HF183, other human-

associated genetic markers have been used for identifying human sources in recreational waters,

such as HumM2, HumM3, NifH, and BifHM (Devane et al., 2019; Zeki et al., 2021; Zhang et al.,

2020; Ballesté et al., 2018; Shanks et al., 2009).

Napier at al. (2017) assessed the presence of four human-associated Bacteroides markers

(HF183, BsteriF1, BuniF2, and HumM2) with self-reported gastrointestinal illness, diarrhea, and

respiratory illnesses. The analysis used data from 12,060 adult visitors who enrolled in the

NEEAR studies at six beaches from 2003 to 2007. The HF183/HumM2 detection rate at NEEAR

marine and freshwater beaches were 26 percent and 28 percent, respectively. Overall, findings

suggest inconsistent associations between illness and the detection of human-associated

Bacteroides markers. The authors concluded that quantitative measures of human fecal pollution,

rather than presence-absence, may be necessary to improve assessment of potential links

between public health risk and the occurrence of human fecal pollution.

Mattioli et al. (2020) used HF183 as a microbial tracer to evaluate potential sources of

contamination of a drinking water well linked to multiple outbreaks of norovirus documented

over a month-long period. This is a new application for human-associated FSI genetic markers,

used to mitigate exposure.

Advances in Virus-Based FSI

Enteric viruses, such as noroviruses, are reported to be the dominant etiological agents of

recreational waterborne disease (Sinclair et al., 2009; Soller et al., 2010b). These enteric viruses

can react to waste treatment and environment stressors in different ways compared to bacterial

fecal indicators (Blatchley et al., 2007; Okoh et al., 2010; Qiu et al., 2015). Viral FSI

technologies offer an alternative to current bacterial-based methods; however, many are either

not sensitive enough or lack sufficient host specificity (Boehm et al., 2013). An ideal viral FSI

method would target a virus that is both closely associated with a particular animal host and is

highly abundant. There is a growing body of evidence suggesting that some virus-based FSI

genetic targets are highly abundant in sewage, are closely associated with human waste, and

persist for longer periods of time in wastewater and environmental settings compared to many

bacterial fecal indicators.

Since 2017, there have been significant advances in the performance characterization of recently

reported crAssphage-like human-associated genetic markers (Stachler et al., 2017) identified

from a metagenome cross-assembly (Dutilh et al., 2014). EPA scientists and external partners

conducted studies demonstrating a uniform distribution of crAssphage-like sequences in

untreated wastewater across the contiguous United States (Korajkic et al., 2020a). Notable

efforts include evidence that crAssphage-like sequences are highly abundant in sewage occurring

at similar concentrations to HF183 genetic markers (Ahmed et al., 2018b), correlate with some

enteric viral pathogens (Crank et al., 2020), more closely mimic enteric virus decay rates

compared to bacterial-based FSI targets (Ahmed et al., 2021; Crank et al., 2019), and can be

recovered in multiple ambient surface water types (Petcharat et al., 2020). However, research

also suggests, like any other methodology, that standardized protocols are important for

generating consistent and reliable measurements within and between laboratories (Ahmed et al.,

2020).

Advances in Anthropogenic Chemical-Based FSI

Napier et al. (2018) used data from seven NEEAR study beaches impacted by WWTP discharges

for Enterococcus spp. measured by qPCR and 62 potential anthropogenic chemical markers. The

markers were assessed for potential associations with self-reported GI illness, diarrhea, and

respiratory illnesses. Across all beaches, no individual chemical marker or chemical category

showed strong or consistent association between Enterococcus spp. measured by qPCR and

health outcomes.

New Tools for Human-Associated FSI

EPA developed and published two methods (Method 1696.1 and 1697.1; U.S. EPA, 2022a,b)

and collaborated with NIST to develop a standard reference material (SRM 2917) that functions

with 11 qPCR FSI protocols, including human-associated HF183/BacR287 and HumM2

protocols (Kralj et al. 2021). These accomplishments represent two key advances for FSI science

since 2017 and are discussed in more detail in the methods section (Section III.F).

A series of studies were conducted to support the implementation of EPA Methods 1696.1 and

1697.1. Notable contributions included the demonstration of genetic target occurrence uniformity

in untreated wastewater across the contiguous United States (Korajkic et al., 2020a), a large-

scale field demonstration at 29 freshwater sites (Li et al., 2019), and use of QMRA modeling to

investigate links to public health risk in raw sewage (Schoen et al., 2020), as well as the use of a

new censored data analysis approach to rank recreational water sites based on human fecal

pollution levels (Cao et al., 2018a) to investigate potential links with rainfall (Shrestha et al.,

2020) and describe human fecal pollution source trends in waters routinely monitored for

bacterial and viral fecal indicators (Li et al., 2021).

2. Non-Human FSI Advances

Since 2017, there has been a growing interest in the use of non-human FSI methods to

characterize wildlife, pet, and agricultural fecal sources potentially present in recreational waters,

especially using qPCR-based methods. Non-human fecal source information can help prioritize

impaired sites, focus mitigation resources, and manage public health risk. Most research

activities focused on avian-, ruminant-, and swine-associated genetic markers.

Ruminant (i.e., cattle and deer), avian (i.e., gull and Canada goose), and domestic pets (i.e., dogs)

can contribute to elevated FIB levels in ambient surface waters (Ervin et al., 2013). In response,

EPA conducted a series of studies to explore the implementation of non-human FSI methods.

Notable contributions include the large-scale implementation of ruminant-, cattle-, avian-, swine-

and dog-associated qPCR methods at over 30 freshwater sites (Li et al., 2019, McKee et al.,

2020), development of SRM 2917 in collaboration with NIST, which functions with avian,

ruminant, dog, and swine FSI qPCR methods ( Sivaganesan et al., 2022; Willis et al., 2022), and

characterization of decay trends in fresh and marine waters (Korajkic et al., 2019b).

A variety of host-associated avian genetic markers have been investigated, including the general

avian marker GFD targeting Helicobacter spp. (Ahmed et al., 2016a; Symonds et al., 2017;

Vadde et al., 2019; Zhang et al., 2020), the poultry-associated markers LA35 and CL targeting

Brevibacterium sp. (Gibson et al., 2017), and the poultry-associated BifPL targeting

Bifidobacterium (Ballesté et al., 2018). Multiple gull-associated markers targeting Catellicoccus

marimammalium were also evaluated including CAT (Wu et al., 2017), Gull2 (Cloutier and

McLellan, 2017; Symonds et al., 2017), Gu112 (Thulsiraj et al., 2017), and qGull4 (Staley et al.,

2018a,b). Overall, findings suggest that these methods can be highly specific for avian targets,

however, most methods exhibit less than ideal sensitivity.

Multiple swine-associated markers were investigated, including Pig2Bac, targeting Bacteroidales

(Ballesté et al., 2018; Derx et al., 2021; He et al., 2016; Kongprajug et al., 2019; Liang et al.,

2021; Somnark et al., 2018; Vadde et al., 2019; Zhang et al., 2020), L.amylovorus, targeting

Lactobacillus amylovorus (He et al., 2016; Zhang et al., 2020), and PF, targeting Bacteroidales

(Symonds et al., 2017). In general, Pig2Bac exhibited a high specificity and sensitivity

suggesting that this methodology may be a reliable method for future recreational water quality

testing.

Ruminant FSI methods are generally grouped into cattle-associated and ruminant-associated

methods. Several studies explored the use of cattle-associated qPCR methods including

Bac3qPCR (Kongprajug et al., 2020) and CowM2/CowM3 (Xue et al., 2018; Xue et al., 2019).

Ruminant-associated methods can identify cattle as well as other animals that share the same

digestive physiology such as deer and elk, among others. Numerous ruminant-associated

methods were investigated in field studies targeting BacCow (Seidel et al., 2017; Symonds et al.,

2017); BifCW, targeting Bifidobacterium (Ballesté et al., 2018); and Bacteroidetes 16S

ribosomal RNA (rRNA) genes, including BoBac (Bushon et al., 2017) and BacR (Derx et al.,

2021; Kolm et al., 2019). Some ruminant sources such as cattle may under some circumstances

represent a similar health risk to humans (Soller et al., 2010a). In addition, cattle-associated

genetic marker shedding patterns are linked to animal diet (Shanks et al., 2011, Shanks et al.,

2010) and age (Shanks et al., 2014) resulting in potentially different occurrence expectations

from one monitoring site to another.

Feng and McLellan (2019) analyzed Bacteroides populations in sewage and non-human animal

hosts by targeting the V4–V5 and V6 16S rRNA gene hypervariable genome regions. Findings

suggest that the most abundant Bacteroides in untreated sewage were not human fecal associated

but sewage infrastructure derived. A qPCR assay was developed targeting an abundant sewage

infrastructure-associated Bacteroides. The authors assert that a qPCR assay that targets

organisms closely associated with the sewer pipe habitat can potentially provide source

information that is independent of the human fecal microbiome and could be useful for

identifying sewage pollution in water. Beyond microbial populations, Staley et al. (2018a)

evaluated environmental DNA (eDNA) utilizing the mitochondrial cytochrome oxidase I genetic

marker for DNA barcoding, which identifies DNA from various animal species. eDNA from cow

and chicken were only detected in creek and beach surface waters in Lake Ontario following an

extreme rain event. The authors note that caution should be used when interpreting eDNA results

because these sequences may not be of fecal origin. Advances in community-based FSI science

suggest that these approaches can complement FIB and quantitative FSI methods such as qPCR,

as well as identify new infrastructure-associated genetic targets. Additional research is warranted

to further characterize the utility of these methodologies.

3. Community-based FSI

Microbial community-based FSI methods are an active area of research for characterizing

recreational waters. Characterizing the occurrence of microbial groups utilizing sequencing for

identification can provide information about fecal sources and other microbial inputs. In addition

to genomics (nucleic acid sequencing), metabolomics (characterization of metabolites) has been

explored for characterizing recreational waters. For example, Beale et al. (2017) utilized

metagenomics sequencing (hypervariable V5 and V6 regions of the 16S rRNA gene) to report

the most abundant metabolically active bacteria in water samples from five sites along the

Brisbane River, Queensland, Australia. They also conducted gas chromatography mass

spectrophotometry analysis of water samples to characterize metabolites, such as sugars, fatty

acids, among others. Additional research is warranted to investigate the impacts of agricultural

practices, sewage treatment, and environmental endpoints using community-based approaches

(Beale et al., 2017). Another study utilized amplicon sequencing of the V3–V4 region of the

bacterial 16S rRNA gene to determine human sewage, avian, and horse fecal sources in the

Navesink River in New Jersey (Phelan et al., 2019).

4. Summary of New Information on FSI

• EPA Methods 1696.1 and 1697.1 are now available and facilitate the use of human-

associated qPCR technologies in recreational settings (see more details in Section III.F,

Analytical Methods).

• Field demonstrations have been published that use EPA Methods 1696.1 and 1697.1 and

establish their performance in wastewater and surface waters.

• Release of SRM 2917, a high-quality standard control material that functions with 11 FSI

qPCR methods indicative of human (HF183/BacR287, HumM2, CPQ_056, CPQ_064),

cattle (CowM2, CowM3), ruminant (Rum2Bac), canine (DG3, DG37), swine (Pig2Bac),

and avian (GFD) fecal sources, facilitates implementation of FSI qPCR methods.

• A new censored data analysis approach has been published that allows for quantitative

analyses of FSI qPCR data sets consisting of high proportions of non-detections and

detections below the limit of quantification.

• As the use of FSI tools for human fecal pollution characterization becomes a more

common practice, practitioners are recognizing that the contribution of non-human fecal

pollution sources plays an integral role in water quality management.

• Recent studies on microbial community-based FSI offer new strategies and tools to

characterize fecal pollution sources; however, additional studies are needed to evaluate

performance and demonstrate utility for recreational water quality monitoring.

• Understanding the decay of microorganisms associated with specific fecal pollution

sources is important for many FSI applications. Unlike culture-based FIB methods, FSI

genetic methods target nucleic acids instead of cells capable of growing and reproducing

on selective and differential media. This fundamental difference can contribute to

variable persistence behaviors in ambient surface water settings.

• Potential surrogates for enteric viral pathogens, such as enterococci qPCR, culturable

MSC and somatic coliphages, and human-associated genetic markers (HF183 and Hum

M2), have been included in health study evaluations and provide additional information

for assessing potential risk of illness (Griffith et al., 2016; Benjamin-Chung et al., 2017;

Ahmed et al., 2018a; Boehm et al., 2018; Boehm and Soller, 2020; Schoen et al., 2020;

Wade et al., 2022).

• Advances have been made in establishing potential links between public health risk and

human-associated FSI marker measurements using QMRA (see Section III.A, QMRA,

for details).

F. New Information on Analytical Methods for Recreational Waters

As discussed above, EPA has identified five types of indicators of risk in recreational waters:

FIB, coliphages, cyanotoxins, antimicrobial resistant bacteria and genes, and human and non-

human FSI genetic markers. Following is a discussion of the new research and scientific

information on the analytical methods for identifying and quantifying these potential risk

indicators.

The systematic literature search identified 80 articles (Figure 1) relevant to analytical methods

(Appendix B). Additional relevant articles were identified via an ad hoc process (U.S. EPA,

2012b, 2015c, 2020; Sivaganesan et al., 2018, 2022; Steele et al., 2018; Nshimyimana et al.,

2019; Lane et al., 2020a; Kralj et. al., 2021; Bone et al., 2022).

1. FIB

The 2012 RWQC protect human health from exposure to fecal contamination by detecting and

enumerating FIB. There are many analytical methods to enumerate E. coli and enterococci in

recreational waters. These methods either enumerate by growing the bacteria on selective and

differential media (culture methods) or by quantifying specific sequences of DNA from the FIB

(molecular methods). Currently, there are more than a dozen microbiological methods approved

for use under the CWA to monitor ambient water (see Code of Federal Regulations (CFR) 40

CFR 136.3.IH) and each method is a culture-based method.

Culture methods have been used to monitor water quality for decades. In May of 2021, EPA

approved a culture method for E. coli in ambient water that requires only 10 hours to provide a

result (May 19, 2021, Federal Register (FR) 86 FR 27235 and 40 CFR136.3.IH.3) and

https://www.epa.gov/cwa-methods). This incubation time is the shortest of the culture methods.

The length of time culture methods take to provide results means that people may have already

been exposed to fecally contaminated water, and water quality may have changed by the time the

result is available. Lag time between sampling and results may lead to unnecessary or delayed

action.

Since 2016, significant work has been conducted to update existing microbial methods, or add

methods with a faster turnaround time, applicable to the FIB E. coli and enterococci including

updated molecular-based qPCR methods and draft digital PCR (dPCR) methods. Molecular

methods can provide results faster than culture methods. EPA Method 1609.1 produces

enterococci results in about 4 hours, allowing monitoring in the morning and the ability to take

action to close a beach by noon, if necessary, and thereby reducing the number of swimmers

exposed to fecal contamination as indicated by elevated levels of FIB (EPA Method 1609.1).

This method is based on detecting specific sequences of enterococci DNA using qPCR

technology. Though faster than culture methods, molecular methods are generally more

expensive and technically more difficult to perform than culture methods. The target sequence

can be detected in viable but non-culturable, and metabolically inactive, cells and it is possible to

detect the target sequence associated with free DNA from cells that have lysed. Research in

molecular water monitoring methods is advancing rapidly and there are additional molecular

technologies under development or that are being deployed in the field in addition to qPCR.

PCR-Based Methods for FIB

EPA has made scientific advancements in qPCR methods, including the development and

publication of new methods, the development of standard control materials, the completion of

multiple laboratory performance studies, and studies characterizing linkages between FIB qPCR

and culture-based measurements in recreational waters.

A qPCR approach offers the advantage over culture methods of providing rapid results (2–6

hours versus 10–48 hours), allowing managers to make same-day decisions to protect

beachgoers. Since the initial public release of the Enterococcus qPCR method (EPA Method

1611; U.S. EPA, 2012b), EPA has made significant advances, including addressing potential

matrix interference issues in water types other than those studied at the NEEAR effluent-affected

beach sites culminating in the publication of an improved qPCR-based method for enterococci

enumeration (EPA Method 1609.1; U.S. EPA, 2015c) and the development of a draft E. coli

qPCR protocol (Haugland, et al., 2021). Despite the advantages of a qPCR water quality

monitoring approach, the use of these methods by state water quality programs has been slow

(Shrestha and Dorevitch, 2020).

Since 2017, EPA has published 18 manuscripts, developed standard control materials, and

released updated automated data analysis tools to help address research and implementation

priorities. Recent studies are summarized below.

Implementation of qPCR methods requires the generation of a standard curve to interpret results.

Quality and consistency of standard curves strongly influences the precision and reproducibility

of qPCR measurements. To address this need, EPA researchers developed a reference material

(IDTSMART-KAN_Std1_Xho), using droplet digital PCR (ddPCR) and advanced statistics, for

both the Enterococcus and E. coli qPCR methods in the Great Lakes region (Sivaganesan et al.,

2018). To help facilitate qPCR method implementation on a national scale, EPA researchers

recently collaborated with NIST to codevelop SRM 2917, which is expected to replace the initial

reference material in future recreational water analyses (Kralj et al., 2021; Sivaganesan et al.,

2022; Willis et al., 2022). EPA also reviewed qPCR methods for microbial water quality

monitoring (Nappier et al., 2019b).

To evaluate the performance of the E. coli qPCR method and establish custom data acceptance

metrics for future practitioners, EPA helped organize two ad hoc multi-laboratory studies. The

first effort consisted of 21 participating laboratories, primarily from the Great Lakes region using

a standardized protocol with the same set of reagents and consumables (Sivaganesan et al.,

2019). Findings suggested that a qPCR-based methodology can be successfully implemented

across a large network of laboratories and led to the development of custom data acceptance

metrics. In the second study, the feasibility of multiple laboratories to meet newly developed data

acceptance metrics was evaluated with most participants, both new and experienced,

demonstrating acceptable qPCR measurements (Aw et al., 2019). Given these findings, a

companion data analysis workbook was customized to incorporate new data acceptance metrics

and streamline calculations (Lane et al., 2020a).

Several studies were conducted to characterize linkages between FIB qPCR and culture-based

measurements in recreational waters. One effort investigated the temporal stability of enterococci

culture and qPCR paired measurements at freshwater recreational beaches over a 24-hour period

(Wymer et al., 2021). Findings reinforced the concept that same-day qPCR results are more

indicative of current water quality conditions compared to culture results obtained 24 hours after

sample collection. Another study generated nearly 7,000 E. coli culture and qPCR paired

measurements from 101 Lake Michigan recreational beach sites (Haugland et al., 2021). This

data set provided the State of Michigan with the scientific basis for the adoption of a statewide

Beach Notification Value for the E. coli qPCR method (Lane et al., 2020b) and could serve as a

blueprint for other states, territories, and authorities.

In 2020, EPA developed and evaluated a dPCR method using 38 recreational fresh and marine

water samples collected from 24 states across the country. The results were used to develop draft

quantitative quality control criteria that could be used to assess laboratory proficiency prior to

conducting a multi-laboratory validation study. The method incorporates elements from ORD’s

qPCR methods as well as a dPCR method being piloted in San Diego, California, recreational

waters (U.S. EPA, 2020). The results from the study indicate that the dPCR procedure is suitable

for multi-laboratory validation in both fresh and marine waters for E. coli and/or enterococci

(Bone et al., 2022).

2. Coliphage Methods

As discussed in Section III.B, coliphages are better indicators of some enteric viruses in

recreational waters compared to traditional methods measuring FIB. In 2018, EPA published

updated culture-based single-agar layer (SAL) methods to enumerate MSC and somatic

coliphages in recreational waters and wastewater (Method 1642) and in secondary wastewater

with no disinfection (Method 1643) (U.S. EPA, 2018ab). EPA Method 1642 includes

ultrafiltration to concentrate larger sample volumes (2 L), needed for recreational water

monitoring to address potential low coliphage densities in ambient waters downstream of human

fecal inputs. EPA conducted a multi-laboratory validation study (U.S. EPA, 2018c) of Method

1642 for MSC and somatic coliphages in fresh and marine recreational waters and secondary

wastewater effluents with disinfection. EPA Method 1643 reflects the results of a multi-

laboratory validation study of EPA Method 1602 for 100 mL secondary (no disinfection)

wastewater samples for MSC and somatic coliphages and is used for monitoring secondary (no

disinfection) wastewater matrices under the CWA. For both methods, the multi-laboratory

validation results allowed for the development of initial precision and recovery and ongoing

precision and recovery and matrix spike quality control acceptance criteria. Results from both

methods can be obtained in 16 to 24 hours.

EPA researchers published a study comparing performance of EPA Method 1642 to that of two

other alternative methods (modified SAL and direct membrane filtration [DMF]) capable of

processing large volumes of surface waters (1 L per coliphage type) (McMinn et al., 2018). All

methods were evaluated for the following metrics: percentage of samples with no detectable

coliphage, concentrations of coliphage, cost, and time required to process a single sample.

Method 1642 outperformed both alternative methods as it generated the least percentage of non-

detects (≤35%), the highest coliphage concentrations (0.30–1.65 log

PFU/L) and required the

least amount of time, while cost per sample was comparable to that of DMF.

An additional study evaluated performance of Method 1642 on freshwater and marine ambient

water samples collected nationwide and assessed the effect of spike titer protocols (i.e., SAL

versus double agar layer [DAL]), which are required for initial and ongoing precision recovery

tests (Korajkic et al., 2021). Study findings indicated that average percent recovery (53%–72%)

was comparable to previous reports, confirming a consistent performance of Method 1642 across

a wide variety of water types. Somatic coliphage recovery was not affected by water type, but

MSC results were significantly lower in marine waters compared to freshwater. Similarly,

somatic coliphage were not affected by spike titer protocol, but utilization of DAL significantly

underestimated MSC recovery compared to SAL.

Currently, there are E. coli host strains under evaluation that detect both MSC and somatic

coliphages. These host strains include CB-390 and C-3000 (Rose et al., 2004; Guzmán et al.,

2008; Agulló-Barceló et al., 2016; Korajkic et al., 2020a). A single host strain could simplify

Methods 1642 and 1643 by allowing the enumeration of both MSC and somatic coliphages in a

SAL assay.

Other New Studies for FIB Using PCR

Some notable studies compared EPA methods to other methods. For example, Byappanahalli et

al. (2018) compared culture methods (EPA Method 1603 for E. coli and Method 1600 for

enterococci) to qPCR-based methods (modified EPA Method 1611 qPCR for Enterococcus) at

coastal beaches and rivers. Overall, the culture-based and qPCR-based results for both indicators

were correlated in this study. While the qPCR methods would have resulted in fewer water

quality advisories or notifications, the authors concluded that the increased benefit of same-day

results provided by qPCR provides better protection overall.

Dorevitch et al., (2017) analyzed nine Chicago beaches by E. coli culture (Colilert method) and

Enterococcus spp. qPCR (EPA Methods 1609 and 1609.1). This study looked at the two pieces

of information available to beach managers on a given day: Enterococcus qPCR results of

samples collected that morning and E. coli culture results of samples collected the previous day.

The study found that if the same-day qPCR results had not been available, culture-based results

from water samples collected the day before would have triggered unnecessary, belated beach

advisories 24 percent of the time. And, on 4.7 percent of the beach-days, culture results would

have resulted in a “failure to act” (meaning, failure to trigger advisories) when advisories were

needed (based on exceedance of the same-day qPCR results). Furthermore, the 71.3 percent

concordance of beach actions between same-day qPCR and culture results from water samples

collected the day before can be explained entirely by chance. The authors concluded that there is

little scientific rationale for continued E. coli culture testing in Chicago beaches waters for the

purposes of public notification.

Digital PCR

Detection of enterococci in water samples was compared using ddPCR, qPCR (EPA Method

1609.1), and culture-based Enterolert (Crain et al., 2021). A comparison of enterococci qPCR

and ddPCR monitoring data demonstrated an Index of Agreement of 0.89 and a significant

Pearson’s correlation coefficient of r = 0.87 (p < 0.001). A subset of data used for the ddPCR and

qPCR comparison also included Enterolert data, which was evaluated using linear regression to

develop an intrinsic copy number equation that was then used to adjust the direct comparison

between ddPCR and Enterolert monitoring data. A ddPCR advisory threshold of 1,413 gene

copies per 100 mL was derived from this analysis and determined to be equivalent to the BAV of

104 MPN per 100 mL used in California (Crain et al., 2021). The ddPCR threshold value was

implemented as a BAV to monitor coastal beach water quality at San Diego County beaches in

2022 (County of San Diego Department of Environmental Health, http://www.sdbeachinfo.com).

Other FIB Method Technologies

Multiple methods for the real-time measurement of E. coli and enterococci have been evaluated,

including culture-based methods (chromogenic and fluorogenic substrates), cultivation-

independent detection methods (i.e., flow cytometry, biosensors), and microbial identification by

mass spectrometry (matrix-assisted laser desorption ionization time-of-flight, as reviewed by

Bonadonna et al., 2019). A critical review suggests that none of these technologies are currently

able to achieve real-time analysis. Additional research is warranted to develop real-time

approaches for monitoring FIB.

Kolm et al. (2017, 2019) developed helicase-dependent amplification (HDA) assays to detect

different fecal targets in surface water including fecal indicators. Only a heating block is required

for use of the assay, and the sample is applied directly to a test strip that detects and displays the

amplification products by marker-specific hybridization probes using a colorimetric reaction in 2

hours. The HDA assay yielded comparable results in sensitivity, specificity, and limits of

detection to qPCR. The authors suggest that this low-cost simple assay that requires minimal

training could be used instead of qPCR to yield comparable results (Kolm et al., 2019).

Loop-mediated isothermal amplification (LAMP) is a nucleic acid diagnostic method that is

potentially useful for testing of water samples in low-resource settings lacking sophisticated

laboratory equipment and highly trained personnel (Martzy et al., 2017; Nieuwkerk et al., 2020).

Oliveira et al. (2020) used LAMP to detect E. coli (uidA) in polluted lake and river samples.

Quantification of Enterococcus spp. and E. coli using an MPN-LAMP approach and qPCR

protocol were correlated, demonstrating that the MPN-LAMP method could be used for water

quality testing in areas that have limited resources (Fu et al., 2021).

3. Cyanotoxin Methods

As discussed in Section III.C, under certain environmental conditions cyanobacteria can rapidly

multiply and form HABs and some species may produce cyanotoxins. EPA has developed

several methods for the analysis of various cyanotoxins in ambient water, including Method 546:

Determination of Total Microcystins and Nodularins in Drinking and Ambient Waters (U.S.

EPA, 2016); Single Laboratory Validated Method for Determination of Cylindrospermopsin and

Anatoxin-a in Ambient Freshwaters (U.S. EPA, 2017b); and Single Laboratory Validated

Method for Determination of Microcystins and Nodularin in Ambient Freshwaters (U.S. EPA,

2017c).

Since 2017, EPA has published seven molecular technology-related peer-reviewed studies on

employing molecular tools for the detection of toxigenic cyanobacteria and cyanotoxins (Chen et

al., 2017; Zhu et al., 2019; Lu et al., 2019, 2020; Li et al., 2020; Wang et al., 2021; Tanvir et al.,

2021; Duan et al., 2022). EPA’s research found that there are high correlations between

microcystin and the mcy gene specific qPCR/reverse transcriptase (RT)-qPCR signals, while mcy

specific RT-qPCR can signal activities of toxin-producing genes and toxigenic species and

indicate the initiation of bloom-formation and toxin production and the subsequent subsidence of

toxin production. An early warning signal of one-week prior to microcystin production using

qPCR/RT-qPCR methods was identified (Lu et al., 2020). An additional study developed early

warning threshold values of mcy gene copy or transcript numbers corresponding to the

recommended swimming advisories for microcystins (Duan et al., 2022). The qPCR and RT-

qPCR threshold values provided here are specific for Harsha Lake, but they have been supported

by the data from lab culture experiments and current research collaborations with regions/states

(Duan et al., 2022).

In 2020, EPA developed qPCR detection methods for the detection of microcystin producers (Lu

et al., 2020), and filed a patent (U.S. Serial Number 16/142,319) on CyanoHAB qPCR assays.

This patent is for detecting genus-specific toxigenic cyanobacteria, providing a standardized

qPCR protocol, and the early warning process for cyanotoxin production. EPA is presently

developing qPCR detection methods for anatoxin, saxitoxin, and cylindrospermopsin producers

in lakes and rivers and qPCR methods for the detection of toxic/nontoxic benthic cyanobacteria.

4. AMR Bacteria

As discussed above in Section III.D, AMR bacteria can be produced by untreated wastewater and

many other sources and pose a risk to human health. No standard methods exist for measuring

AMR in recreational waters. Researchers are using a variety of different methods to study

environmental AMR with variations in the bacteria or genes targeted, the antibiotics tested, and

type of method (culture or molecular methods). This variation hinders cross comparison of data

sets. WHO has selected E. coli, specifically extended spectrum beta-lactamase (ESBL)-E. coli,

for global surveillance spanning human, animal, and environmental samples (Matheu et al.,

2017; WHO, 2018, 2019).

Human and Non-Human FSI

As described in Section III.C, the source of fecal contamination provides important information

needed to fully estimate the health risk and remediation options for any fecally contaminated

waters. Since 2017, EPA researchers have published 15 peer-reviewed studies on FSI, released

two nationally validated human-associated microbial source tracking qPCR methods, and

codeveloped a standard control material in collaboration with NIST.

5. Human FSI

EPA and numerous external partners collaborated to publish nationally validated EPA Methods

1696.1 and 1697.1 for human-associated qPCR assays HF183/BacR287 and HumM2,

respectively (U.S. EPA, 2022a,b). These methods include standardized protocols, custom data

acceptance metrics, a self-administered method proficiency testing protocol, and an automated

data analysis tool (https://www.epa.gov/cwa-methods/other-clean-water-act-test-methods-

microbiological).

EPA Method 1696.1 and 1697.1 implementation requires the use of a standard control material

to interpret results. In response, EPA designed a control material that functions with 11 qPCR

FSI protocols, including HF183/BacR287 and HumM2, and partnered with NIST to develop a

large-scale preparation for mass distribution on a national scale. The result is SRM 2617. This

material was subject to a rigorous set of certification experiments by NIST using dPCR (Kralj et

al., 2021); performance was confirmed by EPA using qPCR (Sivaganesan et al., 2022; Willis et

al., 2022) and it was made available to the public in April 2022.

Adaptation of dPCR for FSI

Since 2017, research focusing on the development and implementation of dPCR protocols for

FSI applications has increased (Pendergraph et al., 2021; Staley et al., 2018b; Steele et al., 2018;

Zimmer-Faust et al., 2021). dPCR can offer greater precision, superior trace level detection, and

is less prone to amplification inhibition compared to qPCR (Cao et al., 2016) . However, the

conversion of well-characterized qPCR protocols to a dPCR platform is not always

straightforward. For example, some researchers have observed a decrease in specificity with

dPCR protocols (Nshimyimana et al., 2019; Staley et al., 2018b), while others report the opposite

compared to a respective qPCR method (Nshimyimana et al., 2019). In addition, dPCR is ideal

for multiplex applications when the goal is to simultaneously amplify two or more targets in the

same reaction. Multiplexing can streamline laboratory testing workflow, reduce costs, and offer

increased certainty in fecal pollution characterization. Duplex dPCR-based FSI protocols have

been developed by combining the widely used HF183 16S rRNA human-associated genetic

marker with a crAssphage-like method (CPQ_056) (Ahmed et al., 2019) and an enterococci

assay (Cao et al., 2018b).

Emerging Technologies for FSI

Emerging methods are also being tested for utility in FSI. Three methods, a test strip (Kolm et

al., 2017, 2019), inversely coupled immunomagnetic separation/adenosine triphosphate (Inv-

IMS/ATP) (Zimmer-Faust et al., 2018), and LAMP (Jiang et al., 2018), are discussed in more

detail above. Briefly, a test strip was used to differentiate between spring/groundwater, surface

water, and wastewater exhibiting a high degree of agreement with paired qPCR measurements

(Kolm et al., 2017). A ruminant-associated version of the test strip correctly detected six

ruminant sources but did not react with human and 15 other non-ruminant sources (Kolm et al.,

2019). LAMP was used to identify human fecal contamination in water by detecting as few as

17 copies per mL of human-associated Bacteroides HF183 sequence with no false positives from

dog or cat feces (Jiang et al., 2018).

6. Summary of New Findings on Analytical Methods

• Analytical methods continued to advance over the previous 5 years, providing more

precise and rapid tools to assess the quality of recreational waters.

• Development of dPCR methods (e.g., Crain et al., 2021) offers the promise of rapid

results as compared to bacterial culture methods.

• Publication of EPA Methods 1642 and 1643 for culture-based coliphage methods allows

enumeration of MSC and somatic coliphage types in recreational waters.

• Development of FSI methods (1696.1 and 1697.1) and standard control material (SRM

2617) advances useful tools to mitigate and further inform the human health risk once

fecal contamination has been identified.

G. New Implementation Tools

This section discusses the development of implementation tools over the past 5 years, including

those for the 2012 RWQC and 2019 Cyanotoxins AWQC; advances in both predictive and

process modeling tools; and an update on the status of outreach and training efforts, as well as

information on BEACH Act grant funding.

1. Implementation of the 2012 RWQC

Site-Specific Alternative Recreational Criteria

As scientific advancements lead to new technologies for quantifying indicators of fecal

contamination in terms of improvements in rapidity, sensitivity, specificity, and method

performance for site-specific applications, states and authorized tribes could consider using these

methods to develop site-specific water quality criteria. Information demonstrating that these new

site-specific alternative criteria developed by states and authorized tribes are scientifically

defensible and protective of the recreational use is necessary to support new or revised water

quality standards.

To help states and authorized tribes in this effort, EPA recently developed and made public an

Excel spreadsheet tool (AltCalc Tool; U.S. EPA, 2021a,b,c) that complements EPA’s Site-

Specific Alternative Recreational Criteria Technical Support Materials for Alternative Indicators

and Methods (Alternative Indicators TSM; U.S. EPA, 2014a; available online at:

https://www.epa.gov/wqc/recreational-water-quality-criteria-and-methods). The Alternative

Indicators TSM outlines the scientific information needed before an alternative indicator/method

can replace the use of a recommended or approved method on a site-specific basis and describes

how to compare indicators after users have identified a candidate site and an alternative

indicator/method that has desirable attributes. The AltCalc Tool facilitates this comparison by

providing a user-friendly way to compute the index of agreement and Pearson's correlation

coefficient squared (R-squared) statistics for microbial water quality data sets from two different

microbial water quality methods. The index of agreement and R-squared values are used to

assess whether the alternative water quality method is appropriate for replacing an existing water

quality method using the steps in EPA’s Alternative Methods TSM (pages 16–19).

Several states have taken advantage of the information in the Alternative Indicators TSM

(available online at: https://www.epa.gov/wqc/recreational-water-quality-criteria-and-methods).

In Michigan, the state and EPA collaborated on a large-scale project to evaluate EPA Draft

Method C for enumerating E. coli with qPCR compared to previously approved EPA culture-

based methods (Best et al., 2018; Lane et al., 2020b; Haugland et al., 2021). Results from this

study were used to facilitate efforts to implement qPCR-based E. coli detection for rapid

recreational water quality monitoring in the State of Michigan. See the Outreach, Training, and

Grants section below for more information. In California, EPA Region 9 approved a pilot

program for the rapid ddPCR test to assess beach water quality to provide same-day notices in

San Diego County. (Wang et al., 2016a; California Water Boards Water Quality Coordinating

Committee, 2017; U.S. EPA, 2020; Crain et al., 2021). Other examples include Illinois

(Shrestha and Dorovitch, 2019), Hawaii (Fujioka et al., 2015; Esgaib Vaz Guimarães, 2017),

and Wisconsin (Hernandez, 2018).

Sanitary Surveys

In July 2020, EPA released its updated Sanitary Survey App for Marine and Fresh Waters with

enhanced features to help states, territories, and tribes gather sanitary survey data to identify

sources of fecal contamination and potential HAB events affecting coastal recreation waters. In

2021, EPA was able to allow increased access to the Sanitary Survey App for use by local

governments, participatory scientists, non-governmental organizations, and the public. The

surveys allow users to collect and share data on potential sources of fecal pollution and

information on potential HAB events in local surface waters, including designated recreational

waters. The data from the Sanitary Survey App can be exported for use in predictive models and

for sharing within or between groups.

The Sanitary Survey App is now a mobile web application and can be used on any device (i.e.,

phone, tablet, computer). Special features include photo storage, real-time geolocation, links to

websites such as the National Weather Service to access data, and free data storage. Additional

information is located on the Sanitary Surveys for Recreational Waters web page.

The EPA Sanitary Survey App is available for use by states, territories, tribes, local

governments, citizen science and environmental groups, and the public. EPA conducted

extensive outreach since the release of the app in July 2020. Stakeholders were notified of the

availability of the app by email, listserv announcements, social media posts, press releases, and

presentations at four national conferences, two state beach conferences, and several state and

tribal meetings. EPA also provided more than 15 virtual training webinars that included

demonstrations of the freshwater and marine routine surveys. EPA plans to continue its outreach

efforts to raise awareness and encourage use of the app by all stakeholders.

2. Cyanotoxins

EPA published materials to support implementation of the 2019 recommended recreational water

quality criteria and/or swimming advisories for microcystins and cylindrospermopsin. These

materials include the Final Technical Support Document: Implementing the 2019 Recommended

Human Health Recreational Ambient Water Quality Criteria or Swimming Advisories for

Microcystins and Cylindrospermopsin (U.S. EPA, 2021d), and guidance on EPA’s Monitoring

and Responding to Cyanobacteria and Cyanotoxins in Recreational Waters web page.

The

technical support document contains several questions and answers relating to the recommended

recreational water quality criteria and/or swimming advisories for microcystins and

cylindrospermopsin. The information in this document is primarily intended to support states,

authorized tribes, and territories interested in adopting the recommended criteria into their state

or tribal WQS or using the recommended values as the basis for swimming advisories and

related public notification purposes. The document provides recommendations on how to

monitor for cyanobacteria and two of their known cyanotoxins (microcystins and

cylindrospermopsin) in waterbodies and information on how to complete assessments, list

impaired or threatened waters, and develop TMDLs. EPA’s Monitoring and Responding to

Cyanobacteria and Cyanotoxins in Recreational Waters web page and materials found there

provide detailed information to support states and authorized tribes in their efforts to monitor and

respond to cyanobacterial blooms and cyanotoxins in recreational waters. EPA also developed

HABs infographics that can be used by federal agency, state, territorial, tribal, and local

government partners to help them educate recreators about the potential dangers of HABs in both

marine and freshwaters.

To aid in tracking the occurrence of HABs and their toxins in the nation’s freshwaters, EPA

developed the Tracking CyanoHABs Storymap. This user-friendly, interactive resource compiles

state-issued recreational waterbody and drinking water health advisories for HABs from across

the country. Each month, EPA collects available information on HAB advisories in drinking and

recreational freshwater systems that state health and/or environmental agencies publicly report

online. This information is displayed in two interactive maps that illustrate current advisories and

advisories since 2015.

In addition, EPA facilitated a series of workshops across the country to build relationships and

identify shared HABs-related goals, needs, and barriers among federal, state, and tribal CWA

and Safe Drinking Water Act (SDWA) programs. The workshops, held from 2015 to 2018 across

the country, provided information on the health outcomes associated with exposure to HABs,

strategies for the prevention and management of HABs in surface waters, and effective treatment

techniques for HABs-related cyanotoxins in drinking water. In addition, the workshops provided

a forum for state and tribal health and environmental agencies to exchange information on their

HABs programs, experiences, and needs. EPA also held a series of webinars specific to tribal

waters in 2021. Information on these and other webinars can be found on EPA Newsletter and

Collaboration and Outreach on HABs website.

https://www.epa.gov/cyanohabs/monitoring-and-responding-cyanobacteria-and-cyanotoxins-recreational-waters

3. Predictive Modeling Research

The 2012 RWQC encourage the use of predictive models to supplement water quality

monitoring to enable more timely beach notification decisions. Predictive modeling uses past

water quality data and current observed hydrometeorological measurements to estimate water

quality at a given time. Accurate and reliable predictive models can improve water quality

management.

EPA maintains an active research program focusing on priority research areas identified in the

2017 Five-Year Review of the 2012 Recreational Water Quality Criteria (U.S. EPA, 2018d)

including development of predictive models and technology transfer to stakeholders. EPA has

organized and participated in multiple workshops and training sessions on the use of Virtual

Beach across the United States, including in-person workshops in Ohio, South Carolina, and

New England. Additionally, EPA has held over a dozen online training sessions and webinars in

the last 5 years. Together, these online and in-person training opportunities reached

approximately four hundred participants.

4. Process Modeling Research

Process or deterministic models are valuable tools for the simulation of contaminant sources,

concentrations, transport, and exploration of mitigation scenarios in recreational waters. Single

models and integrated environmental modeling (IEM) are two types of approaches that can be

applied when simulating the fate and transport of microbial contaminants at the watershed scale.

IEM consists of a construct of modules and/or models that simulate individual processes where

data is exchanged seamlessly to produce an output that targets the problem of interest (Kim et al.,

2018; Whelan et al., 2018). Over the past 5 years, EPA has published multiple QMRA software

modules and tutorials. The workflow was developed to estimate risk associated with inputs of

microbial contaminants from a variety of land uses at the watershed scale (Whelan et al.,

2017a,b). Data collection has been automated for watershed analysis, allowing Hydrologic Unit

Code (HUC)-8 or Pour Point analyses (Whelan et al., 2015). The IEM automatically delineates

watersheds and sub-watersheds and the system accounts for minimum stream lengths, minimum

subwatershed sizes, and minimum land use type sizes. Local data files contain a Microbial

Properties Database that provides physico-microbial properties relevant to release, fate,

transport, exposure, and effects modeling (Whelan et al., 2017c). Precipitation data can be

entered from either monitored gauges or radar/remote sensing precipitation sources. The system

automatically estimates microbial loads on all sub-watersheds based on the number of animals

reported per land use patterns by calculating manure-based source terms and processes point and

non-point sources of fecal microbial contaminants. The information is used to prepopulate a

watershed model, the Hydrological Simulation Program-Fortran (HSPF), with microbial decay

and transport constants (Kim et al., 2017). The system is compatible with both graphical and

tabular viewers in HSPF and Better Assessment Science Integrating Point and Nonpoint Sources

(BASINS) and allows users to investigate water flows and microbial concentration time series at

multiple locations throughout the watershed. Finally, the system is designed to be linked to a

pathogen risk model, the microbial risk assessment-interface tool (MRA-IT). Supporting

information for the use, training, and application of the QMRA software can be found in the

Quantitative Microbial Risk Assessment Tutorial – Primer.

Modeling the fate and transport of MSC and somatic coliphages was another example of an IEM

application where information produced in laboratory studies contributed with critical data inputs

for the development of a hydrodynamic coliphage model in the Great Lakes. EPA evaluated the

light-induced inactivation of MSC and somatic coliphages using both laboratory and wastewater

treatment plant-isolated strains (Zepp et al., 2018; Nelson et al., 2018). Solar simulators were

used to develop biological weighing functions and ultraviolet inactivation constants that, in turn,

were used to model the inactivation of coliphages over a range of conditions in aquatic

environments. A three-dimensional model of coliphage inactivation and transport was developed

for Washington Park Beach in Lake Michigan using the organism-specific action spectra

developed in the laboratory studies (Safaie et al., 2020). Results indicated that coliphage models

outperformed a previously tested E. coli model suggesting that coliphages may serve as

conservative indicators of microbiological contamination.

EPA published a study that evaluated the importance of sediment resuspension in FIB loading

calculations in watersheds of various sizes (Bradshaw et al., 2021). While suspended transport

was the dominant mechanism for FIB movement regardless of stream size, FIB loads from

overland and bedload transport should be considered when performing modeling and microbial

assessments at the watershed scale.

EPA also modeled FIB using a high-frequency, multi-year microbial data set with the Soil and

Water Assessment Tool (SWAT) (Sowah et al., 2020). SWAT was applied to understand the

sources and parameters impacting microbial water quality in a mixed-used watershed in

southeastern United States. A key scientific advancement of this work was the development of a

criterion to evaluate the performance of bacterial models in SWAT.

5. Outreach and Grants

EPA Stakeholder Technology Transfer

An essential step in the successful implementation of recreational water quality, laboratory-based

methods is technology transfer. Molecular and newly developed coliphage culture methods can

be technically demanding, requiring specialized equipment, detailed procedures, and specialized

training making it challenging to implement on a national scale. To help address this need, EPA

ensures public access to standardized protocols and companion implementation tools, as well as

provides technical support and hands-on training opportunities. Since 2017, EPA has provided

support to 56 different federal, state, territory, or local laboratories, as well as to more than 20

academic research groups. These activities help facilitate the proper use of newly developed

recreational water quality laboratory methods.

6. BEACH Act Grant Funding

Under the BEACH Act, EPA awards grants to eligible state, territorial and tribal applicants to

help them and their local government partners monitor water quality at coastal and Great Lakes

beaches. When bacteria levels are too high for safe swimming, these agencies notify the public

by posting beach warnings or closing the beach. Since 2001, state, and local governments,

territories and tribes have been awarded nearly $206 million in EPA BEACH Act grants to

monitor beaches for FIB, maintain and operate public notification systems, and report results of

monitoring and notification activities to EPA. While beach grant funding totals allocated by

Congress were $9.2 million in 2019 and 2020, $10.1 million has been awarded in 2022. See

https://www.epa.gov/beach-tech/beach-grants for detailed information on BEACH Act

grant awards.

IV. Summary of Major Findings and Priorities for Further Work

A. Summary of Major Findings

1. Summary of Findings Identified in New Epidemiological Studies

• Recent publications have advanced understanding and further confirm that

epidemiological and outbreak data demonstrate that young children (i.e., 10 years old and

under) have a higher risk of illness compared to adults when exposed to fecal

contamination in recreational waters.

• Young children’s (i.e., 10 years old and under) behavior, such as increased time spent in

water, length of time spent playing in the sand, and more vigorous activity, can increase

their potential exposure to fecal contamination and is associated with greater health risk

compared to other age groups, especially those 18 and over.

• Children’s recreational exposure is greater than adolescents and adults because they

ingest more total volume of water, independent of body weight, than do the other age

groups.

• Studies demonstrate that human fecal contamination can pose higher risks of illness in

recreators relative to some non-human fecal sources.

• While FIB can be a useful indicator where raw and poorly treated sewage dominates

water quality, multiple studies discuss the inconsistent performance of culture-

enumerated FIB related to health and point to other indicators to better represent the

potential risk of illness (e.g., Enterococcus measured by qPCR).

• Waters receiving human fecal contamination can have viral pathogens present at health-

relevant levels and yet also be below recommended water quality levels for culturable

FIB.