v/EPA
United States
Environmental
Protection Agency
Office of Water
Mail Code 4304T
EPA 822-R-19-001
May 2019
Recommended Human Health Recreational
Ambient Water Quality Criteria or
Swimming Advisories for Microcystins
and Cylindrospermopsin

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Recommended Human Health Recreational
Ambient Water Quality Criteria or Swimming Advisories
for Microcystins and Cylindrospermopsin
Prepared by:
U.S. Environmental Protection Agency
Office of Water (4304T)
Health and Ecological Criteria Division
Washington, DC
EPA Document Number: 822-R-19-001
Date: May 2019

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NOTICES
This document has been drafted and approved for publication by the Health and Ecological Criteria
Division, Office of Science and Technology, United States Environmental Protection Agency, and is
approved for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Recommended Human Health Recreational Ambient Water Quality Criteria or
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FOREWORD
Section 304(a) of the Clean Water Act (CWA) requires the Administrator of the U.S. Environmental
Protection Agency (EPA) to publish water quality criteria that accurately reflect the latest scientific
knowledge on the kind and extent of all identifiable effects on health and welfare that might be expected
from the presence of pollutants in any body of water, including ground water.
The EPA is publishing these recommended values under CWA 304(a) for states to consider as the basis
for swimming advisories for notification purposes in recreational waters to protect the public. The EPA
envisions that if states decide to use the values as swimming advisory values they might do so in a
manner similar to their current recreational water advisory programs. Alternatively, states may consider
using these same values when adopting new or revised water quality standards (WQS). If adopted by
states as WQS and approved by the EPA under CWA 303(c), the WQS could be used for all CWA
purposes. States may also wish to consider using these values as both swimming advisory values and
WQS.
This document has undergone an EPA intra-agency peer-review process. The Health and Ecological
Criteria Division, Office of Science and Technology, Office of Water, U.S. Environmental Protection
Agency has completed the final review and the document is approved for publication. The values were
derived using the existing peer-reviewed and published science on the adverse human health effects of
the toxins including previous EPA analysis, such as the EPA's Health Effects Support Document for the
Cyanobacterial Toxin Microcystins and Health Effects Support Document for the Cyanobacterial Toxin
Cylindrospermopsin (HESDs), and the EPA's Drinking Water Health Advisory for the Cyanobacterial
Microcystin Toxins and Drinking Water Health Advisory for the Cyanobacterial Toxin
Cylindrospermopsin (Drinking Water Health Advisories) (U.S. EPA 2015a, 2015b, 2015c, 2015d). The
EPA used established criteria methodologies (U.S. EPA 2000) and recreation-specific exposure
parameters from the EPA's Exposure Factors Handbook (EFH) (U.S. EPA 2011) to derive these values.
Detailed information that can be found in the EPA's HESDs and Drinking Water Health Advisories is
summarized in this document.
The term "water quality criteria" is used in two sections of the CWA section 304(a)(1) and section
303(c)(2). The term has a different legal meaning in each section. In section 304, the term represents a
non-regulatory, scientific assessment of effects on human health or aquatic life. The criteria
recommendations presented in this document are such a scientific assessment. If the state or authorized
tribe adopts water quality criteria associated with specific designated uses as WQS under section 303,
and approved by the EPA, they become applicable CWA WQS in ambient waters within that state or
tribe. Water quality criteria adopted in state or tribal WQS could have the same numerical values as
criteria developed by the EPA under section 304. Alternatively, states and authorized tribes may derive
numeric criteria based on other scientifically defensible methods, but the criteria must be protective of
designated uses. States and tribes can adopt criteria into their standards. When approved by the EPA, the
criteria become Clean Water Act-applicable WQS. Guidelines to assist in modifying the criteria
recommendations presented in this document are contained in the Water Quality Standards Handbook
(U.S. EPA 2012).
This document provides recommendations only. It does not establish or affect legal rights or obligations.
It does not establish a binding norm and cannot be finally determinative of the issues addressed. Agency
decisions in any particular situation will be made by applying the CWA and EPA regulations on the
basis of specific facts presented and scientific information then available.
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ACKNOWLEDGMENTS
The development of this document was made possible through an effort led by John Ravenscroft, EPA
Project Manager, Health and Ecological Criteria Division, Office of Science and Technology, Office of
Water. The following Office of Science and Technology staff provided valuable contributions to the
development and review of this document: Tracy Bone, Dr. Lesley D'Anglada, Dr. Joyce Donohue, Lars
Wilcut, Oakridge Institute for Science and Education (ORISE) fellow Meghann Niesen, and summer
intern Ana-Maria Murphy-Teixidor.
The EPA gratefully acknowledges the valuable contributions of the EPA internal technical reviewers
who reviewed this document. Staff from the following EPA program and regional offices completed a
formal review of these Human Health Recreational Ambient Water Quality Criteria (AWQC) or
Swimming Advisories for Microcystins and Cylindrospermopsin.
U.S. EPA Office of Children's Health Protection
U.S. EPA Office of General Counsel
U.S. EPA Office of Policy
U.S. EPA Office of Research and Development
U.S. EPA Office of Water
Office of Ground Water and Drinking Water
Office of Science and Technology
Office of Wastewater Management
Office of Wetlands, Oceans, and Watersheds
U.S. EPA Regional Offices
Region 1
Region 4
Region 5
Region 7
Region 8
Technical support was provided by ICF and its subcontractor Bigelow Laboratory for Ocean Sciences
under EPA Contract No. EP-C-16-011. The EPA acknowledges important input received from the states,
tribes, local governmental agencies, individual citizens, and stakeholder groups, such as the Association
of Clean Water Administrators, and other nongovernmental organizations, who submitted comments on
the draft.
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TABLE OF CONTENTS
NOTICES	3
FOREWORD	4
ACKNOWLEDGMENTS	5
TABLE OF CONTENTS	6
LIST OF TABLES	9
LIST OF FIGURES	11
ACRONYMS AND ABBREVIATIONS	 12
1.0 EXECUTIVE SUMMARY	15
2.0 INTRODUCTION AND BACKGROUND	18
International and State Guidelines	18
3.0 NATURE OF THE STRESSORS	26
3.1	Cyanobacteria and Cyanobacterial Blooms	26
3.1.1 Environmental Factors Influencing Occurrence of Cyanobacteria and
Cyanotoxins	28
3.2	Cyanotoxins	35
3.2.1	Chemical and Physical Properties	35
3.2.2	Sources and Occurrence in Surface Waters	38
3.2.3	Estuarine and Marine Waters	44
3.2.4	Other Sources of Microcystins and Cylindrospermopsin	45
3.3	Environmental Fate	45
3.3.1	Mobility	45
3.3.2	Persistence	46
3.4	Toxicokinetics	48
4.0 PROBLEM FORMULATION	49
4.1	Conceptual Model	49
4.1.1	Conceptual Model Diagram for Recreational Exposure	49
4.1.2	Factors Considered in the Conceptual Model for Microcystins and
Cylindrospermopsin	50
4.2	Analysis Plan	51
4.2.1	Approach for Recreational AWQC and Swimming Advisory Derivation	52
4.2.2	Measures of Effect	52
4.2.3	Measures of Exposure	53
4.2.4	Relative Source Contribution (RSC)	58
5.0 EFFECTS ASSESSMENT	59
5.1	Hazard Identification	59
5.1.1	Noncancer Health Effects	59
5.1.2	Cancer	70
5.2	Dose-response Assessment	71
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6.0 RECOMMENDED RECREATIONAL CRITERIA AND SWIMMING ADVISORY
DERIVATION	72
6.1	Microcystins Magnitude	72
6.2	Cylindrospermopsin Magnitude	72
6.3	Frequency and Duration for Recreational Criteria	73
6.4	Frequency and Duration for Swimming Advisory	75
6.5	Recommended Recreational Criteria and Swimming Advisory for Mi crocystins and
Cylindrospermopsin	75
7.0 EFFECTS CHARACTERIZATION	77
7.1	Enhanced Susceptibility	77
7.2	Recreational Exposure Duration	77
7.2.1 Comparison of Duration of Exposure Distributions	79
7.3	Evaluation of Health Protective Values for Different Lifestages	82
7.3.1	Consideration of Multiple Lifestages	82
7.3.2	Exposure Factors for Children Younger Than Six Years Old	86
7.4	Other Recreational Exposure Pathways	87
7.4.1	Inhalation of Cyanotoxins	87
7.4.2	Dermal Absorption	91
7.5	Cyanobacterial Cells	94
7.5.1	Health Effects Associated with Cyanobacterial Cells and Uncertainties	94
7.5.2	Cyanobacteria Biomass Measurements as Indicators of Hazard	96
7.5.3	Use of Cyanobacteria Cell Densities in Guidelines	101
7.6	Other Sources of Mi crocystins and Cylindrospermopsin	108
7.6.1	Drinking Water	108
7.6.2	Groundwater	109
7.6.3	Fish and Shellfish	109
7.6.4	Dietary Supplements	109
7.6.5	Ambient Air	110
7.6.6	Soils and Sediments	110
7.7	Tribal Considerations	110
7.8	Livestock and Pet Concerns	110
7.8.1 States and Animal HAB Guidelines	112
8.0 REFERENCES	113
APPENDIX A. INTERNATIONAL RECREATIONAL WATER GUIDELINES FOR
CYANOTOXINS AND CYANOBACTERIA	A-l
APPENDIX B. STATE RECREATIONAL WATER GUIDELINES FOR
CYANOTOXINS AND CYANOBACTERIA	B-l
APPENDIX C. LITERATURE SEARCH DOCUMENTATION	C-l
APPENDIX D. REVIEW OF THE STATE OF THE SCIENCE ON
CYANOBACTERIAL CELLS HEALTH EFFECTS	D-l
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APPENDIX E. INCIDENTAL INGESTION EXPOSURE FACTOR COMBINED
DISTRIBUTION ANALYSIS	E-l
APPENDIX F. INGESTION STUDIES	F-l
APPENDIX G. INFORMATION ON CELLULAR CYANOTOXIN AMOUNTS	G-1
APPENDIX H. TABLES OF STATE-ISSUED GUIDELINES SPECIFIC TO ANIMAL
CYANOTOXIN POISONING	II-l
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LIST OF TABLES
Table 2-1. WHO (2003a) Recreational Guidance/Action Levels for Cyanobacteria,
Chlorophyll a, and Estimated Corresponding Microcystin Level	19
Table 2-2. International Recreational Water Guideline or Action Levels for Cyanobacteria
and Microcystins	20
Table 2-3. State Guideline or Action Levels for Microcystins, Cylindrospermopsin, and
Cyanobacterial Cells in Recreational Water	23
Table 3-1. Abbreviations for Selected Microcystins (Yuan et al. 1999)	 36
Table 3-2. Chemical and Physical Properites of Microcystin-LR	37
Table 3-3. Chemical and Physical Properties of Cylindrospermopsin	38
Table 3-4. States Surveyed as Part of the 2007 NLA with Water Body Microcystin
Concentrations above 10 [j,g/L (U.S. EPA 2009)	41
Table 4-1. Results of the Combined Distribution Analysis	58
Table 5-1. Liver Effects in Animals Exposed to Microcystins in Selected Acute and Short-
term Studies as Discusssed in the EPA's Health Effects Support Document for
the Cyanobacterial Toxin Microcystins (U.S. EPA 2015d)	60
Table 5-2. Kidney and Liver Effects in Animals Exposed to Cylindrospermopsin (Purified) in
Acute and Key Short-term Studies Health Effects Support Document for the
Cyanobacterial Toxin Cylindrospermopsin (U.S. EPA 2015c)	63
Table 6-1. Recreational Criteria or Swimming Advisory Recommendations for Microcystins
and Cylindrospermopsin51	76
Table 7-la. Durations of Recreational Exposures in Minutes per Day	78
Table 7-lb. Durations of Recreational Exposures in Minutes per Swimming Eventa	78
Table 7-2. Parameters Used to Fit Recreation Duration Distributions in Freshwater	80
Table 7-3. Calculated Daily Incidental Ingestion Rates Based on EFH and DFB Datasets	82
Table 7-4. Mean Body Weight by Age Group Based on U.S. EPA (2011)	84
Table 7-5. Inputs for Calculation of Protective Values for Microcystins and
Cylindrospermopsin	85
Table 7-6. Microcystins Magnitude Comparison Between Children Six to 10 and Children
One to Less Than Six Years Old	87
Table 7-7. Ingestion Parameters and Estimated Ingestion Dose for Screening-level
Comparative Inhalation Exposure Analysis	89
Table 7-8. Inhalation Exposure Parameters and Estimated Inhaled Dose	89
Table 7-9. Results of Screening Analysis Comparing Ingestion and Inhalation Doses	90
Table 7-10. Ingestion Parameters and Estimated Ingestion Dose for Screening-level
Comparative Dermal Absorption Exposure Analysis	92
Table 7-11. Parameters Used to Estimate Skin Permeability of Microcystins	92
Table 7-12. Dermal Absorption Exposure Parameters and Estimated Dermal Absorbed Dose... 93
Table 7-13. Results of Screening Analysis Comparing Ingestion and Dermal Absorbed Doses. 94
Table 7-14. Aggregated Cell Quota Summary Data for Selected Microcystin and
Cylindrospermopsin-producing Genera	105
Table B-l. Summary Counts of State Recreational Water Guidelines for Cyanotoxins and
Cyanobacteria by Type and Scope of Guidelines	B-l
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Table B-2. State Recreational Water Quality Guideline for Cyanotoxins and Cyanobacteria
Sorted by Type	B-2
Table C-l. Internet URL Domains Searched for Research Question 4	C-6
Table C-2. Number of Journal Articles Returned by Three Search Strategies for Research
Question 5	C-8
Table D-l. Cyanobacteria Epidemiological Studies Summary	D-4
Table E-l. Parameters Used to Fit Ingestion Distributions	E-l
Table E-2. Parameters Used to Fit Recreation Duration Distributions	E-2
Table F-l. Studies of Incidental Ingestion Volumes While Recreating	F-6
Table G-l. Summary of Cyanotoxin Cell Quota Data Literature Search Results	G-l
Table G-2. Summary of Study Prioritization	G-4
Table G-3. Cell Quota Data for Microcystin and Cylindrospermopsin-Producing Genera	G-l
Table G-4. Cell Quota Appendix Summary Data for Microcystin and Cylindrospermopsin-
producing Genera	G-l8
Table H-l. California Environmental Protection Agency (2012) Action levels for Selected
Pet and Livestock Scenarios	H-l
Table H-2. California Environmental Protection Agency (2012) Reference Doses and Acute
and Subchronic Action Levels for Canine Exposure to Cyanotoxins in Drinking
Water	H-l
Table H-3. Oregon Dog-specific Guideline Values for Cyanotoxins in Recreational Waters
(Mg/1.)	H-2
Table H-4. Grayson County, Texas Microcystin Guidelines for Dogs	H-2
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LIST OF FIGURES
Figure 2-1. State Guidelines for Cyanotoxins and Cyanobacteria in Recreational Water by
Type and Scope of Guidelines	22
Figure 3-1. Environmental Factors Influencing Total Cyanobacterial Blooms, Reproduced
from Paerl and Otten (2013b)	29
Figure 3-2. Structure of Microcystin (Kondo et al. 1992)	 36
Figure 3-3. Structure of Cylindrospermopsin and Structurally Related Cylindrospermopsins
(de la Cruz et al. 2013)	37
Figure 3-4. Generalized Distribution of Cyanobacterial HABs in the United States and
Territories	39
Figure 3-5. State-reported HAB Notices by EPA Region, June 2 to August 1, 2017	39
Figure 4-1. Conceptual Model of Exposure Pathways to the Cyanotoxins, Microcystins and
Cylindrospermopsin, and Cyanobacteria in Surface Waters While Recreating	49
Figure 4-2. Combined Distributions for Age Groups	54
Figure 4-3. Incidential Ingestion for Age Groups Based on Appendix E Dufour Data	55
Figure 4-4. Direct Contact Recreational Exposure Duration by Age Group, Based on Table
16-20 in U.S. EPA (2011)	57
Figure 7-1 a and b. Comparison of Children's Duration of Time Spent Recreating	81
Figure 7-2. Incidental Ingestion During Recreational Activity Based on Age (Appendix E)	83
Figure 7-3. Comparison of Children and Adults Incidental Ingestion Rate During
Recreational Activity Adjusted for Body Weight	84
Figure 7-4. Comparison of Calculated Recreational Health Protective Values for
Microcystins and Cylindrospermopsin for Children, Older Children, and Adults ... 85
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ACRONYMS AND ABBREVIATIONS
ACC
ambient cyanotoxin concentration
AWQC
ambient water quality criteria
AWWARF
American Water Works Association Research Foundation
BAX
BCL2 Associated X, Apoptosis Regulator
Bel-2
BCL2 Apoptosis Regulator
BGAS
blue-green algae supplements
BID
BH3 interacting domain death agonist
BW
body weight
C.
Cylindrospermopsis
CalEPA
California Environmental Protection Agency
CAS
Chemical Abstracts Service
CAWS
Chicago Area Waterway System
CCD
cyanobacterial cell density
CDC
U.S. Centers for Disease Control and Prevention
CDEEP
Connecticut Energy and Environmental Protection
CDPH
Connecticut Department of Public Health
CFU
colony forming unit
CI
confidence interval
cm
centimeter
CTA
cell toxin amount
CWA
Clean Water Act
CyAN
Cyanobacteria Assessment Network
CYP450
Cytochrome P450
DFB
DeFlorio-Barker et al. (2017)
DIN
dissolved inorganic nitrogen
DIP
dissolved inorganic phosphorus
DON
dissolved organic nitrogen
dw
dry weight
E.
Escherichia
EFH
Exposure Factors Handbook
ELISA
Enzyme Linked Immunosorbent Assay
EPA
U.S. Environmental Protection Agency
FAQs
frequently asked questions
Fe
iron
fg
femtogram
g
grams
GI
gastrointestinal
GI2
more severe gastrointestinal symptom index
GM
geometric mean
GSD
geometric standard deviation
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HAB	harmful algal bloom
HAB1SS	Harmful Algal Bloom-related Illness Surveillance System
HESD	Health Effects Support Document
HPLC	high performance liquid chromatography
IDEQ	Idaho Department of Environmental Quality
IQR	interquartile range
IR	ingestion rate
kg	kilograms
km	kilometer
Koc	soil organic carbon-water partition coefficient
Kow	octanol-water partition coefficient
L	liter
LA	leucine, alanine
LC/MS/MS	liquid chromatography with tandem mass spectrometry
LF	leucine, phenylalanine
lethal concentration causing the death of 50 percent of a group of test
LC50	animals
LD511	lethal dose causing the death of 50 percent of a group of test animals
LOAEL	lowest-observed-adverse-effect-level
LOD	level of detection
LPS	lipopoly saccharide
LR	leucine, arginine
LW	leucine, tryptophan
LY	leucine, tyrosine
M.	Microcystis
"3
m	cubic meter
nicy	three-letter nomenclature for genes that produce microcystins
mg	milligram
mL	milliliter
MW	molecular weight
MS	mass spectroscopy
n	sample size
N	nitrogen
N/A	not available
NASA	National Aeronautics and Space Administration
ng	nanogram
NHMRC	National Health and Medical Research Council
NLA	National Lakes Assessment
NOAA	National Oceanic and Atmospheric Administration
NOAEL	no-observed-adverse-effect-level
NORS	National Outbreak Reporting System
NYSDOH	New York State Department of Health
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OATP	organic anion transporting polypeptide
OHHABS	One Health Harmful Algal Bloom System
OPP	EPA Office of Pesticide Programs
OR	odds ratio
ORSANCO	Ohio River Valley Water Sanitation Commission
P	phosphorus
PCR	polymerase chain reaction
pg	picogram
pH	potential of hydrogen
ppb	parts per billion
PWS	public drinking water system
qPCR	quantitative polymerase chain reaction
rDNA	ribosomal deoxyribonucleic acid
RfD	reference dose
ROS	reactive oxygen species
RR	relative risk or when microcystin-RR it means arginine, arginine
RSC	relative source contribution
SDWA	Safe Drinking Water Act
SWIMODEL	Swimmers Exposure Assessment Model
t	event duration
TBD	to be determined
TDI	tolerable daily intake
TN:TP	total nitrogen ratio to total phosphorus
TOXLINE	Toxicology Literature Online
U.S.	United States of America
UF	uncertainty factor
URL	Uniform Resource Locator
|.ig	microgram
"3
(.mi	cubic micrometer
USGS	U.S. Geological Survey
WHO	World Health Organization
WHOl	Woods Hole Oceanographic Institute
WoS	Web of Science
WQS	water quality standards
WSDE	Washington State Department of Ecology
YR	tyrosine, arginine
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1.0 EXECUTIVE SUMMARY
Cyanobacteria, also commonly referred to as blue-green algae, are photosynthetic bacteria that are
ubiquitous in nature and are found in surface waters. Environmental conditions that promote excessive
growth of cyanobacteria in surface waters can lead to situations in which cyanobacterial cell density is
high, known as blooms. Nitrogen and phosphorus levels, the ratio of nitrogen to phosphorus, water
temperature, organic matter availability, light attenuation, pH, and water column stratification are
environmental factors that play an important role in the development of cyanobacterial blooms and their
production of cyanotoxins. Some cyanobacteria, but not all, have the ability to produce toxins. The
toxin-producing cyanobacteria contain genes that confer the ability to produce toxins and are referred to
as toxigenic cells. The abundance of toxigenic cyanobacteria can vary within the overall cyanobacteria
population, between waterbody to waterbody, and over time within a single waterbody.
Microcystins can be produced by a variety of toxigenic cyanobacteria genera, including Microcystis,
Anabaena, Dolichospermum, Nostoc, Oscillatoria, Fischerella, Planktothrix, and Gloeotrichia. Some of
these species can be distributed through the water column, concentrate in the upper layers, or form
surface scums depending on environmental conditions. More than 100 microcystin congeners exist,
which vary based on amino acid composition. The majority of toxicological data on the effects of
microcystins are available for microcystin featuring leucine and arginine (microcystin-LR), which is
also a frequently monitored congener. Microcystins are water soluble and tend to remain contained
within the toxigenic cyanobacterial cell until the cell breaks and they are released into the water.
Microcystins typically have a half-life of four to 14 days in surface waters or may persist longer,
depending on factors such as photodegradation, bacteria, and the presence of organic matter.
Microcystins can persist even after a toxigenic cyanobacterial bloom is no longer visible.
Cylindrospermopsin can be produced by a variety of toxigenic cyanobacteria species, including
Cylindrospermopsis raciborskii, Aphanizomenon, Anabaena, Lyngbya wollei, and Raphidiopsis. Some
of these species tend not to form visible surface scums, and the highest concentrations of total
cyanobacterial cells typically occur below the water surface. Two congeners of cylindrospermopsin, as
well as two structural analogs, have been identified. Cylindrospermopsin can be retained within the cell
or released into the water. The biodegradation of cylindrospermopsin in natural water bodies is a
complex process that can be influenced by many environmental factors, including toxin concentration,
water temperature, sunlight, and the presence of cell pigments and bacteria. Half-lives of 11 to 15 days
and up to eight weeks have been reported for cylindrospermopsin in surface waters.
This document for microcystins and cylindrospermopsin focuses on the human health risks associated
with incidental ingestion while recreating in freshwaters containing these harmful cyanotoxins. The
recommended cyanotoxin values apply to freshwaters with the recreational designated use. The toxins
that are produced by cyanobacteria growing in freshwaters can enter estuarine and marine waters as
waters containing the toxins flow downstream. The EPA recognizes that there may be circumstances
where harmful cyanobacterial blooms (also known as harmful algal blooms or HABs) can impact
downstream marine and estuarine waters. This document provides information on occurrence and
incidental ingestion in estuarine and marine waters for states to consider but does not provide
recommendations for those waters. Exposure to cyanobacteria and their toxins can also occur through
non-recreational pathways such as consumption of cyanotoxin-contaminated drinking water and food
(including fish), and during bathing or showering. This document does not address or provide
recommendations for non-recreational exposures.
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The EPA is publishing these recommended values for microcystins1 and cylindrospermopsin under the
Clean Water Act (CWA) section 304(a) for states to consider as the basis for swimming advisories for
notification purposes to protect public health in recreational waters. The EPA envisions that if states
decide to use the values as swimming advisory values, they would do so in a manner similar to their
current recreational water advisory programs. Alternatively, states may consider using these same values
when adopting new or revised water quality standards (WQS). If adopted as WQS and approved by the
EPA under the CWA section 303(c), the WQS could be used for all CWA purposes. States may also
wish to consider using these values as both swimming advisory values and WQS.
The recommended values in this document leverage the information that the EPA collected and
evaluated in its Health Effects Support Document for the Cyanobacterial Toxin Microcystins and Health
Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin (HESDs), and the EPA's
Drinking Water Health Advisory for the Cyanobacterial Microcystin Toxins and Drinking Water Health
Advisory for the Cyanobacterial Toxin Cylindrospermopsin (Drinking Water Health Advisories) (U.S.
EPA 2015a, 2015b, 2015c, 2015d).
The EPA evaluated the health effects of microcystins and derived a reference dose (RfD) in its 2015
Health Effects Support Document for the Cyanobacterial Toxin Microcystins (U.S. EPA 2015d).
Exposure to elevated levels of microcystins can potentially lead to liver damage. The critical study for
the derivation of the microcystins RfD was conducted by Heinze (1999) based on rat exposure to
microcystin-LR in drinking water. The critical effect from this study was slight to moderate liver lesions
with necrosis and increased liver weight and enzymes associated with tissue damage. The EPA
established the RfD based on microcystin-LR and used it as a surrogate for other microcystin congeners.
Monitoring and toxicity studies suggest that the microcystin-LR is the most frequently occurring
congener and is more toxic than other congeners of microcystin evaluated (Loftin et al. 2016b; U.S.
EPA 2015d; Ito et al. 2002; Rinehart et al. 1994; Vesterkvist and Meriluoto 2003; WHO 1999). The
EPA used the RfD to derive its previously published Drinking Water Health Advisories for microcystins
(U.S. EPA 2015a) and the recommended values in this document. The dose and critical effects that the
EPA used from Heinze (1999) to establish the RfD are supported by a Guzman and Solter (1999) study,
also conducted in rats.
The EPA evaluated the health effects of cylindrospermopsin and derived an RfD in its 2015 Health
Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin (U.S. EPA 2015 c). The
kidneys and liver appear to be the primary target organs for cylindrospermopsin toxicity. The critical
study that the EPA used to derive the cylindrospermopsin RfD was conducted by Humpage and
Falconer (2002, 2003) based on drinking water exposure to mice. Adverse effects on the kidneys were
manifested by decreases in urinary protein concentration and increased relative kidney weight. Upon
considering all effects observed by Humpage and Falconer (2002, 2003), increased relative kidney
weight was considered the most appropriate basis for quantitation (U.S. EPA 2015c). The EPA used the
RfD to derive its previously published Drinking Water Health Advisories for cylindrospermopsin (U.S.
EPA 2015b).
Based on available noncancer health effects information, the EPA is recommending values protective of
primary contact recreation as follows:
1 Microcystins comprise a class of over 100 congeners and unless specified otherwise, "microcystins" refers to total
microcystins.
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•	For microcystins, the recommended recreational value is 8 micrograms ((_ig)/liter (L).
•	For cylindrospermopsin, the recommended recreational value is 15 (J,g/L.
These values are based on the exposure experienced by recreating children due to their higher exposures
compared with other age groups. Given that toxigenic cyanobacterial blooms typically are seasonal
events, recreational exposures are likely to be episodic, and may be short term in nature. The EPA
recommends that if used as a swimming advisory to protect swimmers at a beach, these values not be
exceeded on any single day. If used as a water quality criterion for assessment and listing purposes, the
EPA recommends a maximum of three excursions across a recreational season and observation of that
pattern across multiple years to reflect seasonal dynamics and occurrence patterns of HABs.
At this time, available data are insufficient to develop quantitative recreational values for total
cyanobacterial cell density related to inflammatory health endpoints. The reported epidemiological
relationships between cell density exposure and specific health outcomes (e.g., dermal symptoms,
eye/ear irritation, fever, gastrointestinal (GI) illness, and respiratory symptoms) are not consistent. The
uncertainties related to the epidemiological study differences, such as study size, species and strains of
cyanobacteria present, and the total cyanobacterial cell densities associated with significant health
effects, do not provide sufficient information to determine a consistent association between total
cyanobacterial densities associated with adverse inflammatory health effects. The EPA recognizes that
some states have included total cyanobacterial cell density values as an important part of their FLAB
management strategy. Available information on health endpoints, cell density, and guidelines developed
by other authorities on total cyanobacteria cells is described in the Effects Characterization section of
the document (section 7.5) and in Appendix D.
Because the EPA's recommendations in this document are cyanotoxin concentrations, it can be helpful
for risk-management purposes to understand how this relates to toxigenic cyanobacteria in the
waterbody, as the abundance of toxigenic cells in a water body affects the amount of cyanotoxin
produced. The number of toxigenic cyanobacteria relative to the number of total cyanobacteria can vary
in time and space. Quantifying the abundance of toxigenic cyanobacteria is a better predictor of potential
toxin production compared to quantifying total cyanobacteria. The EPA presents a toxigenic cell number
based on the number of toxigenic cells that could produce microcystins equivalent to the recommended
magnitude. The Effects Characterization section also describes gene-based detection methods (i.e.,
quantitative polymerase chain reaction (qPCR)) that can target and quantify the toxigenic subpopulation
of cyanobacteria that are present in a waterbody.
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2.0 INTRODUCTION AND BACKGROUND
Section 304(a) of the CWA requires the Administrator of the EPA to publish water quality criteria that
accurately reflect the latest scientific knowledge on the kind and extent of all identifiable effects on
health and welfare that might be expected from the presence of pollutants in any body of water.
Currently there are no U.S. federal water quality criteria or regulations for cyanobacteria or cyanotoxins
in drinking water under the Safe Drinking Water Act (SDWA) or in ambient waters under the CWA. No
cyanotoxins are included on EPA's priority pollutant list.2 In 2015, the EPA published non-regulatory
Drinking Water Health Advisories (U.S. EPA 2015a, 2015b) to provide information for public health
officials or other interested groups on two cyanotoxins (microcystins and cylindrospermopsin) that can
affect drinking water quality but are not regulated under SDWA.
The EPA is publishing these recommended values for microcystins and cylindrospermopsin under the
CWA section 304(a) for states to consider as the basis for swimming advisories for notification purposes
to protect public health in recreational waters. The EPA envisions that if states decide to use the values
as swimming advisory values, they would do so in a manner similar to their current recreational water
advisory programs. Alternatively, states may consider using these same values when adopting new or
revised WQS. If adopted as WQS and approved by the EPA under the CWA section 303(c), the WQS
could be used for all CWA purposes. States may also wish to consider using these values as both
swimming advisory values and WQS.
The EPA-recommended values in this document leverage the information that the EPA collected and
evaluated in its Health Effects Support Document for the Cyanobacterial Toxin Microcystins and Health
Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin (HESDs), and its Drinking
Water Health Advisory for the Cyanobacterial Microcystin Toxins and Drinking Water Health Advisory
for the Cyanobacterial Toxin Cylindrospermopsin (Drinking Water Health Advisories) (U.S. EPA
2015a, 2015b, 2015c, 2015d).
This document for microcystins and cylindrospermopsin focuses on the human health risks associated
with incidental ingestion while recreating in freshwaters containing these harmful cyanotoxins. The
recommended cyanotoxin values apply to freshwaters with the recreational designated use. The toxins
that are produced by cyanobacteria growing in freshwaters can enter estuarine and marine waters as
waters containing the toxins flow downstream. The EPA recognizes that there may be circumstances
where harmful cyanobacterial blooms (also known as HABs) can impact downstream marine and
estuarine waters. This document provides information on occurrence and incidental ingestion in
estuarine and marine waters for states to consider but does not provide recommendations for those
waters. Exposure to cyanobacteria and their toxins can also occur through non-recreational pathways
such as consumption of cyanotoxin-contaminated drinking water and food (including fish), and during
bathing or showering. This document does not address or provide recommendations for non-recreational
exposures.
International and State Guidelines
The World Health Organization (WHO 2003a) published a series of guideline values for recreational
exposure to cyanobacteria associated with incremental severity and probability of health effects at
increasing densities of total cyanobacteria and corresponding concentrations of chlorophyll a (if
2 https://www.epa.gov/sites/prodiiction/files/2015-09/doaHnents/prioritv-poHutant-list-epa.pdf
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cyanobacteria dominate) (Table 2-1). The WHO also considered the potential for liver damage by
microcystins in deriving the recommended total cyanobacterial cell densities. Potential concentrations of
microcystins that could be associated with each guidance level are discussed in the WHO document.
However, it should be noted that actual microcystin concentrations at each WHO action level could vary
depending on the composition of toxigenic strains in the cyanobacterial community present and the
dominant species of microcystin producer present in a bloom. For example, at a total cyanobacterial cell
density of 100,000 cells/milliliter (mL), an estimated microcystin concentration of 20 [j,g/L could occur
assuming all cells present are toxin-producing Microcystis species and the average cellular toxin content
was 0.2 picogram (pg) microcystin per cell (WHO 2003a). Microcystin concentrations could range from
50 to 100 (J,g/L, or higher, if another toxin-producing species, such as Planktothrix, is present at the same
cell density.
Table 2-1. WHO (2003a) Recreational Guidance/Action Levels for Cyanobacteria, Chlorophyll a,
and Estimated Corresponding Microcystin Level
Relative Probability of
Acute Health Effects
Cyanobacteria (cells/mL)
Chlorophyll a (ng/L)
Estimated Corresponding
Microcystin Levels (jtg/L)
Low
< 20,000
< 10
< 10a
Moderate
> 20,000-100,000
>10-50
2-4 to 20a b
High
> 100,000
>50
>20
a WHO estimated that 2 to 4 |ig microcystins/L may be expected, with 10 |ig/L possible, at a cell density of 20,000 cells/mL
if microcystin-producing cyanobacteria are dominant.
b At 100,000 cyanobacterial cells/mL, a concentration of 20 |ig microcystins/L is likely if the bloom consists of Microcystis
and has an average toxin content of 0.2 pg/cell.
For these guidelines, the WHO recommended values that included the potential health effects from
exposure to total cyanobacteria because it was "unclear whether all important cyanotoxins had been
identified and that the health outcomes observed after recreational exposure could be related to
cyanobacterial substances other than the well-known cyanotoxins" (WHO 2003a). The different
guideline levels were an effort to distinguish between irritative or inflammatory-response symptoms
associated with total cyanobacterial cells and the more severe hazard of exposure to elevated
concentrations of cyanotoxins, particularly microcystins. The cell-associated inflammatory responses are
represented by the low probability of adverse health effects category of < 20,000 cells/mL,
corresponding to < 10 [j,g/L chlorophyll a if cyanobacteria dominate. According to the WHO, as the
density of cyanobacteria increase above that level, the probability of inflammatory responses increases,
and the potential for more severe adverse health effects associated with exposure to the cyanotoxins also
increases. The WHO high-risk category includes both > 100,000 cells/mL, corresponding to 50 [j,g/L of
chlorophyll a, if cyanobacteria dominate, and > 20 [j,g/L microcystin levels. Health effects at this level
are expected to be primarily due to the toxic effects of microcystins. Very high densities of cells
occurring in scums—for example, >10 million cells/mL or > 5,000 chlorophyll a—can be associated
with very high concentrations of toxin, for example 2,000 [j,g/L of microcystins in the top 4 cm of a
water body (WHO 2003a). Scums that accumulate along the shoreline due to wind can be associated
with a thousand-fold higher density of cells (WHO 2003a).
The WHO guideline value development was informed by results from a review conducted by Chorus
and Bartram (1999). A primary study identified in this review was a prospective epidemiology study by
Pilotto et al. (1997), which evaluated health effects after recreational exposure to total cyanobacteria and
reported associations between total cyanobacterial cell densities and health. Pilotto et al. (1997) found a
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significant association among recreators exposed to greater than 5,000 cells/mL. The WHO chose a
guideline level of 20,000 cells/mL to represent the upper bound of the "low probability of adverse health
effects" category (WHO 2003a). While the association among recreators exposed to greater than 5,000
cells/mL for more than one hour and one or more symptoms reported in Pilotto et al. (1997) was
statistically significant, the WHO states that they represented less than 30 percent of the individuals
exposed (Chorus and Bartram 1999). Therefore, the level of health effect and the small number of
people affected at 5,000 cells/mL were not considered by the WHO to be a basis to justify action
(WHO 2003a).
The WHO pointed out that the potential concentration of microcystins could vary depending on the
composition of toxigenic strains within the overall cyanobacterial community present and the dominant
species of microcystin producer present in a bloom. The WHO states that, at the same cyanobacterial
cell density, cyanotoxin levels could approximately double if Planktothrix agardhii were the dominant
member of the community.
Many countries have adopted the multiple parameters that the WHO discusses for recreational waters
including cell density, biovolume, and cyanotoxin concentration (see Table 2-2). Some international
authorities have multiple action levels. For brevity, Table 2-2 presents the guideline reflecting the lowest
concentration of microcystins or density of cyanobacterial cells or narrative guidelines that
recommended or triggered a health protective action for countries that have adopted action levels. For a
more complete list of guideline or action levels and recommended actions for international jurisdictions,
see Appendix A. The EPA did not identify any recreational guideline levels for cylindrospermopsin
established by other international regulatory authorities.
Table 2-2. International Recreational Water Guideline or Action Levels for Cyanobacteria and
Microcystins
Jurisdiction
Lowest Recreational Water Guideline/Action Lever'
Reference
Australia13
microcystins (total): >10 |ig/L
or Microcystis aeruginosa (total): > 500 to < 5,000 cells/mL
or cyanobacteria (total): > 0.4 to < 4 mm3/L (where a known toxin
producer is dominant in the total biovolume)
or total biovolume of all cyanobacterial material >10 mm3/L (where
known toxins are not present)
Australian Government
National Health and Medical
Research Council (2008)
Canada
microcystins (total): > 20 |ig/L (expressed as microcystin-LR)
or cyanobacteria (total): > 100,000 cells/mL
Health Canada (2012)
Cuba
cyanobacteria: > 1 of the species known as potentially toxic
or phytoplankton cells: > 20,000 - to < 100,000 cells/mL,
> 50 percent of cells cyanobacteria
German Federal Environment
Agency (2012)°
Czech
Republic
cells: > 20,000 cells/mL
German Federal Environment
Agency (2012)°
Denmark
chlorophyll a: > 50 |ig/L. dominated by cyanobacteria
or visible surface scum
German Federal Environment
Agency (2012)°
European
Union
appropriate monitoring must be implemented if there is a risk of
proliferation of algae. Member state authorities responsible must
take management measures and provide information immediately if
a proliferation of cyanobacteria (or blue algae) occurs.
European Parliament and the
Council of the European Union
(2006)
Finland
algae (includes cyanobacteria): detected
German Federal Environment
Agency (2012)°
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Jurisdiction
Lowest Recreational Water Guideline/Action Lever'
Reference
Franceb
microcystins: > 25 |ig/L
or cyanobacteria: > 20,000 to < 100,000 cells/mL (± 20 percent)
German Federal Environment
Agency (2012)°
Germany
Secchi Disk reading > 1 m and (microcystins): >10 |ig/L
or chlorophyll a (with dominance by cyanobacteria): > 40 |ig/L
or biovolume: > 1 mm3/L)
German Federal Environment
Agency (2012)°
Hungary
microcystins: > 4 to < 10 |ig/L
or cell count: > 20,000 to < 50,000 cells/mL
or chlorophyll a (with dominance by cyanobacteria): > 10 to
< 25 (ig/L
German Federal Environment
Agency (2012)°
Italyb
microcystin-LR: > 20 |ig/L equivalents
or cyanobacterial cell count for cyanotoxin-producing species other
than those that produce microcystins (e.g., cylindrospermopsin) >
100,000 cells/mL (± 20 percent)
or transparency < 1 m and total phosphorus > 20 |ig/L and total
cyanobacterial cell count > 2,000 to < 20,000 cells/mL
(± 20 percent)
or transparency > 1 m and total phosphorus > 20 |ig/L and total
cyanobacterial cell count < 2,000 cells/mL
Funari et al. (2017)
Netherlands
chlorophyll a: > 12.5 to < 75 |ig/L
or biovolume (cyanobacterial cell count): >_2.5 to < 15 mm3/L
German Federal Environment
Agency (2012)°
New Zealand13
microcystins (total): >12 |ig/L
or cyanobacteria (benthic): 20-50 percent coverage of potentially
toxigenic cyanobacteria attached to substrate
or cyanobacteria (total): > 0.5 to < 1.8 mm3/L (biovolume
equivalent of potentially toxic cyanobacteria)
or cyanobacteria (total): > 0.5 to < 10 mm3/L (biovolume
equivalent of the combined total of all cyanobacteria)
Wood et al. (2008)
Poland
visible blooms
German Federal Environment
Agency (2012)°
Scotland13
chlorophyll a: > 10 |ig/L with dominance of cyanobacteria
or cyanobacteria: > 20,000 cells/mL
Scottish Government Health
and Social Care Directorates
Blue-Green Algae Working
Group (2012)
Spain
cyanobacteria proliferation potential (low)
German Federal Environment
Agency (2012)°
Turkey
microcystin-LR: > 25 |ig/L equivalents
or cells: > 20,000 to 100,000 cells/mL
German Federal Environment
Agency (2012)°
World Health
Organization
(WHO)
cyanobacteria: 20,000 cells/mL
or chlorophyll a: 10 |ig/L (approximately 2-4 |ig microcystins/L,
assuming cyanobacteria dominance)
Chorus and Bartram (1999);
WHO (2003a)
a More details are provided in Appendix A.
b The lowest guideline values for each quantitative parameter (i.e., cyanotoxin concentration, cyanobacterial cell density,
biovolume) are not associated with the same action level. For example, for Australia, the lowest cyanobacterial cell density
and biovolume criteria trigger the green level surveillance mode, and the lowest cyanotoxin concentration triggered the red
level action mode.
0 Following the Vlllth International Conference on Toxic Cyanobacteria, the German Federal Environmental Agency
compiled and published in 2012 regulatory approaches to the assessment and management of cyanotoxin risks based on
contributions by member countries.
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As of March 2018, approximately 35 U.S. states have implemented cyanobacterial HAB guidelines for
recreational waterways. As graphically shown in Figure 2-1, five states have quantitative or qualitative
cyanotoxin guidelines only, and 20 states have quantitative guidelines for cyanotoxins, as well as either
quantitative or qualitative guidelines for total cyanobacterial cell density. Qualitative guidelines for cell
density use visual inspection rather than quantitative detection methods. In addition, 10 states had
quantitative guidelines for cyanobacterial cell density only or had qualitative guidelines for
cyanobacteria only. Seven states have guideline levels that address toxin-producing cyanobacteria as a
proportion of the total cyanobacterial population or include a toxin-specific cyanobacteria cell density
(California, Idaho, Maryland, New York, New Hampshire, Oregon, and Virginia). The Karuk Tribe,
located in California, developed cell-based values for posting cyanotoxin public health warnings for the
tribe's recreational waters (Kann 2014). Its values were based on the site-specific relationships between
the cell densities of Microcystis and the level of microcystes observed in Karuk waters. For example, in
the Klamath River, at 20,000 cells Microcystis/mL, the probability of exceeding 4 pg/L microcystins
was 55 percent, while at 5,000 cells/mL there were no exceedances. Because the probability of
exceeding the microcystins benchmark rapidly increased at cell densities above 5,000 Microcystis/mL,
the Karuk Tribe uses that value to inform decision-making for health warnings (Kann 2014).
Figure 2-1. State Guidelines for Cyanotoxins and Cyanobacteria in Recreational Water by Type
and Scope of Guidelines
Cyanotoxin and cyanobacteria guidelines* ~
Cyanobacteria guidelines
b'C	d
Cyanotoxin guidelines only
No cyanobacteria or cyanotoxin
a Includes states with quantitative cyanotoxin guidelines as well as either quantitative or qualitative cyanobacteria
guidelines.
b Includes states that either have quantitative cyanobacteria guidelines only or qualitative guidelines only.
c The EPA found that Texas and North Carolina published guidelines in the past, but the guidelines were no longer on their
websites.
d Missouri has presence/absence testing for cyanotoxins and quantitative thresholds.
For brevity, Table 2-3 lists the lowest recreational water guideline or narrative guidelines or action
levels for microcystins, cylindrospermopsin, or total cyanobacteria that trigger or recommend a health
protective action for U.S. states. For a more complete list of state guideline or action levels see
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Appendix B. Parameters and values used as the basis for guidelines varied across states, as did the
methodologies for developing the values.
Table 2-3. State Guideline or Action Levels for Microcystins, Cylindrospermopsin, and
Cyanobacterial Cells in Recreational Water
State
Lowest Recreational Water Guideline
or Action Level3
Reference
Arizona
Blue-green algae (mean value based on a minimum
of two sample events within one peak season):
20,000 cells/mL
and
Chlorophyll a result (mean value based on a
minimum of two sample events within one peak
season) in target range
Arizona Department of Environmental Oualitv
(2008)
California
Microcystins: 0.8 |ig/L
Butler et al. (2012); Cvanobacteria Harmful Alsal
Bloom Network (2016a. 2016b)
Cylindrospermopsin: 1 |ig/L
Toxin-producing cyanobacteria: 4,000 cells/mL
Site-specific indicators of cyanobacteria (e.g.,
blooms, scums, mats)
Colorado
Microcystin-LR: > 10 |ig/L and < 20 |ig/L
Colorado Derailment of Public Health and
Environment (2016)
Cylindrospermopsin: > 7 |ig/L
Potentially toxic algae are visible
Connecticut13
Combination of visual inspection, cell counts:
Visual rank category 2:
Blue-green algae cells > 20,000 cells/mL and
< 100,000 cells/mL
Connecticut Department of Public Health (CDPH)
and Connecticut Energy and Environmental
Protection (CDEEP) (CDPH and CDEEP 2017;
CDEEP 2017)
Delaware
Thick green white, or red scum on surface of pond
Delaware Derailment of Natural Resources and
Environmental Control: Division of Water (2016)
Florida
Cyanobacteria bloom
Florida Derailment of Environmental Protection
(2019)
Idaho
Microcystis or Planktothrix: > 40,000 cells/mL
IDEO (2015)
Sum of all potentially toxigenic taxa: > 100,000
cells/mL
Illinois
Microcystin-LR: > 10 |ig/L
Illinois Environmental Protection Asencv (2018);
Illinois Environmental Protection Asencv (2013)
Indiana
Blue-green algae: 100,000 cells/mL
Indiana Department of Environmental
Management (2018)
Microcystin-LR: 4 |ig/L
Cylindrospermopsin: 8 |ig/L
Iowa
Microcystin: > 20 |ig/L
Iowa Environmental Council (2018)
Kansas
Cyanobacteria: > 80,000 and < 250,000 cells/mL
Kansas Department of Health and Environment
(2015a): Kansas Department of Health and
Environment (2015b)
Microcystin: > 4 and < 20 |ig/L
Kentucky
Blue-green algae: > 100,000 cells/mL
Kentuckv Department for Environmental
Protection (2014)
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State
Lowest Recreational Water Guideline
or Action Level3
Reference

Microcystins: > 20 |ig/L
Commonwealth of Kentucky Department for
Environmental Protection Division of Water
(2015)
Maine
Secclii disk reading < 2 meters caused by algae
Maine Department of Environmental Protection
(2013)
Maryland
Microcystis aeruginosa or other potential
microcystin-producing blue-green algae > 40,000
cells/mL, and samples contain microcystins: > 10
PPb
Wazniak personal communication (2016);
Maryland Department of Natural Resources (2014'

Massachusetts
Blue-green algae: > 50,000 cells/mL
Massachusetts Bureau of Environmental Health
(2015); Massachusetts Department of Public
Health (2008)
Microcystins: > 14 |ig/L
Michigan
Microcystin: > 20 |ig/L
Michiean Department of Environmental Oualitv
(2018); Kohlhepp G (2015)
Chlorophyll a: > 30 |ig/L and visible surface
accumulations/scum are present, or cells are visible
throughout the water column
Missouri
Microcystins: presence (test strip range 0 to
10 ng/mL)
Missouri Department of Natural Resources (2017)
Cylindrospermopsin: presence (test strip range 0 to
10 ng/mL)
Montana
Reservoirs that seem stagnated and harbor large
quantities of algae
State of Montana Newsroom (2015)
Nebraska
Microcystin: > 20 |ig/L
Nebraska Department of Environmental Oualitv
and Nebraska Department of Health and Human
Services: Division of Public Health (2018)
New Hampshire
Cyanobacteria: > 50 percent of total cell counts from
toxigenic cyanobacteria
New Hampshire Department of Environmental
Services (2014)
New Jersey
Microcystins (as total including -LR and other
detectable congeners): 3 |ig/L
New Jersev Department of Environmental
Protection (2017)
Cylindrospermopsin: 8 |ig/L
Cyanobacterial cell count: > 20,000 cells/mL
New York
Bloom: credible report or digital imagery of a bloom
determined as likely to be potentially toxic
cyanobacteria by DEC or DOH staff
New York State Department of Environmental
Conservation (2017)
Blue-green chlorophyll a: > 25 |ig/L
Microcystin-LR: > 4 |ig/L
North Carolina
Visible discoloration or surface scum
North Carolina Health and Human Services:
Division of Public Health (2014)
North Dakota
Blue-green algae bloom is present over a significant
portion of the lake AND
microcystin-LR: > 10 |ig/L
North Dakota Department of Health: Division of
Water Oualitv (2016)
Ohio
Microcystins: 6 |ig/L
Ohio EPA (2016)
Cylindrospermopsin: 5 |ig/L
Oklahoma
Cyanobacteria: 100,000 cell/mL
Oklahoma Legislature (2012)
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State
Lowest Recreational Water Guideline
or Action Level3
Reference

Microcystin: > 20 |ig/L

Oregon
Cylindrospermopsin: > 8 |ig/L
Oregon Health Authority (2018)
Microcystin: > 4 |ig/L
Microcystis'. > 40,000 cells/mL
Planktothrix: > 40,000 cells/mL
Toxigenic species: > 100,000 cells/mL
Visible scum with documentation and testing
Pennsylvania
Microcystin: > 6 |ig/L
Pennsvlvania Department of Environmental
Protection (2014)
Cylindrospermopsin: > 5 |ig/L
HAB verified by visual observation
Rhode Island
Cyanobacteria: > 70,000 cells/mL
Rhode Island Department of Environmental
Management and Rhode Island Derartment of
Health (2013)
Microcystin-LR: > 14 |ig/L
Visible cyanobacteria scum or mat
Utah
Cyanobacteria: 20,000-10,000,000 cells/mL
Utah Derailment of Environmental Oualitv and
Derartment of Health (2017)
Microcystin: 4-2,000 |ig/L
Vermont
Cylindrospermopsin: > 10 |ig/L
Vermont Derartment of Health (2015)
Microcystin-LR (equivalents): > 6 |ig/L
Visible known blue-green algae bloom/scum or an
unknown potentially blue-green algae (i.e., not
pollen), bloom/scum
Virginia
Blue-green algal "scum" or "mats" on water surface
Virginia Derartment of Health (2012)
Microcystin: > 6 |ig/L
Microcystis: 5,000 to < 20,000 cells/mL
Washington
Bloom is forming or a bloom scum is visible (toxic
algae may be present); cyanotoxin levels do not
exceed thresholds
Hardv and Washington State Derartment of Healtl
(2008); Hardv and Washington State Derartment
of Health (2011)
Microcystins: 6 |ig/L
Cylindrospermopsin: 4.5 |ig/L
West Virginia
Blue-green algal blooms observed and monitored
West Virginia Derartment of Health and Human
Resources (2015)
Wisconsin
Cyanobacteria: > 100,000 cells/mL
Wisconsin Derartment of Natural Resources
(2012); Wisconsin Derartment of Health Services
(2016)
Visible scum layer
Werner and Masnado (2014); Wisconsin
Derartment of Health Services (2016)
a More details are provided in Appendix B.
b Connecticut states "based on US EPA's draft recreational criterion CT DPH suggests a cyanotoxin threshold of 4 |ig/L
inicrocystin."
0 The EPA found that Texas published guidelines in the past, but the guidelines were no longer on its website.
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3.0 NATURE OF I II I STRESSORS
This section describes cyanobacteria and cyanobacterial blooms that have the potential to produce
microcystins and cylindrospermopsin. It also describes the chemical and physical properties, sources and
occurrence information in different media, environmental fate, and toxicokinetics for the cyanotoxins.
The information in this section is based on information the EPA presented in its HESDs and Drinking
Water Health Advisories (U.S. EPA 2015a, 2015b, 2015c, 2015d). The EPA conducted supplemental
literature searches in September 2015 to capture new references related to the following topics:
•	Levels of human exposure to cylindrospermopsin or microcystins through recreational water
activities.
•	Health effects for humans or animals exposed to cylindrospermopsin or microcystins.
•	State and international safety levels or criteria for microcystins or cylindrospermopsin.
•	Recreational exposure ingestion rates for children's age groups.
•	Incidents of pet or livestock adverse health effects, including mortality, due to exposure to
cyanotoxins.
For detailed information on these supplemental literature searches and the five research questions that
correspond to the bullets above, see Appendix C.
Cyanobacteria are a group of microorganisms that naturally occur in freshwater and marine
environments and can be found at higher densities in eutrophic or nutrient-enriched water bodies. Many
cyanobacteria are capable of producing toxins, referred to as cyanotoxins, which can adversely affect
human health. Under the right conditions of water temperature, light, pH, nutrient availability, and other
factors, cyanobacteria can reproduce rapidly, forming what are commonly referred to as cyanobacterial
HABs. Other microorganisms can form HABs, but for the purpose of this document the usage of
"HABs" refers to cyanobacterial HABs unless otherwise specified.
3.1 Cyanobacteria and Cyanobacterial Blooms
Cyanobacteria are photosynthetic prokaryotes (Seckbach and Oren 2007) and are ubiquitous in the
environment. Cyanobacteria smaller than 2.0 [j,m are known as picocyanobacteria (Jakubowska and
Szel^g-Wasielewska 2015). The chloroplast, found in photosynthetic eukaryotes like algae and plants,
evolved from an endosymbiotic relationship with cyanobacteria (Kutschera and Niklas 2005). Ecologists
historically grouped cyanobacteria, often referred to as "blue-green algae," with eukaryotic algae
because they contain chlorophyll a and can perform oxygenic photosynthesis. However, cyanobacteria
are prokaryotes (i.e., no discrete membrane-bound nucleus or membrane-bound subcellular organelles)
and are genetically related to other bacteria in the eubacteria domain. Taxonomically, they are classified
in the phylum Cyanobacteria or Cyanophyceae (Carmichael 2008; O'Neil et al. 2012).
Cyanobacteria, including picocyanobacteria, can produce bioactive compounds including toxins, which
can be harmful. These biomolecules include hepatotoxic, neurotoxic, and cytotoxic compounds and
compounds that can result in allergic reactions (Burkholder and Glibert 2006; Carmichael 1994; Jaiswal
et al. 2008; Jakubowska and Szel^g-Wasielewska 2015; Sliwinska-Wilczewska et al. 2018; Yolk and
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Mundt 2007). Studies have shown that exposure to cyanobacterial cells can cause health effects that are
independent of the cyanotoxins; this information is detailed in Appendix D.
Under certain conditions, cyanobacteria possessing the toxin synthesis genes, also referred to as
toxigenic cyanobacteria, begin producing cyanotoxins. Numerous biotic and abiotic factors can
influence not only the dominance of cyanobacteria within the overall phytoplankton community, but
also the proportion of toxigenic cyanobacteria relative to non-toxin-producing cyanobacteria (Davis et
al. 2009; Hyenstrand et al. 1998; McCarthy et al. 2009; Neilan et al. 2013; Gobler et al. 2016). Multiple
species of cyanobacteria are capable of producing the same toxin, such as the microcystins, which can
pose a risk to human and animal health (Crawford et al. 2017). Although scientists have observed a
generalized relationship between cyanobacteria density or chlorophyll a and cyanotoxin concentration,
these relationships are affected by the dominance of the toxin-producing cyanobacteria within the
overall cyanobacterial community (Zhang et al. 2014; Loftin et al. 2016b).
Members of the genera Microcystis, Dolichospermum (Anabaena), Nostoc, Fischer ella, Planktothrix
(formerly Oscillatoria), and Gloeotrichia can produce microcystins (Carey et al. 2012b; Codd et al.
2005; Duy et al. 2000; Stewart et al. 2006c). Microcystis aeruginosa occurs mostly at the surface with
higher light intensities and in shallow lakes. Kosten et al. (2012) surveyed 143 shallow lakes along a
latitudinal gradient (between 5-55°S and 38-68°N) from subarctic Europe to southern South America.
Microcystis have been documented to occur in blooms on all continents except Antarctica and often
dominate phytoplankton assemblages in the summer (O'Neil et al. 2012). Microcystis have been
documented throughout the United States (Carmichael 2001; Jacoby et al. 2000). Species of
cyanobacteria, like Microcystis, that occur at or near the surface due to buoyancy and wind, can
accumulate on shores and bays where they can form scums (Australian Government National Health and
Medical Research Council 2008; WHO 2003b).
Cylindrospermopsin can be produced by a number of cyanobacterial species including Raphidiopsis
raciborskii (formerly Cylindrospermopsis raciborskii),3 Aphanizomenon flos-aquae, Aphanizomenon
gracile, Aphanizomenon ovalisporum, Umezakia natans, Anabaena bergii, Anabaena lapponica,
Anabaena planctonica, Lyngbya wollei, Raphidiopsis curvata, and Raphidiopsis mediterranea (B-Beres
et al. 2015; Kokocinski et al. 2013; McGregor et al. 2011; Moreira et al. 2013). These species do not
tend to form visible surface scums and the highest concentrations of cyanobacterial cells occurs below
the water surface (Falconer 2005).
Cylindrospermopsin-producing cyanobacteria occur in tropical or subtropical regions, as well as warmer
temperate regions. For example, Cylindrospermopsis raciborskii occurs in freshwater ponds, rivers,
reservoirs, and eutrophic lakes and has been found in Australia, Asia, Europe, Africa, and South,
Central, and North America (Fuentes et al. 2010). According to a survey conducted in Florida in 1999
from June to November, the most frequently observed toxigenic cyanobacteria were Microcystis
(43.1 percent), Cylindrospermopsis (39.5 percent), and Anabaena (28.7 percent) (Burns 2008).
Research indicates that cyanotoxins can confer competitive advantage for survival and replication and
are associated with physiological functions of cyanobacterial cell signaling, nutrient uptake, iron
scavenging, maintenance of homeostasis, and protection against oxidative stress (Holland and Kinnear
2013). Cylindrospermopsin production provides a competitive advantage to cyanobacteria when
phosphorus becomes scarce. Bar-Yosef et al. (2010) observed that when phosphorus is scarce, the
3 Cyanobacteria taxonomy is continuously being revised. The genus Cylindrospermopsis has been renamed to Raphidiopsis.
This document mostly maintains the genus name of Cylindrospermopsis.
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cyanobacterium Aphanizomenon ovalisporum releases cylindrospermopsin, which causes other
microorganisms to release alkaline phosphatase, a compound that will increase available free
phosphorus. Subsequently, Aphanizomenon can gain access to phosphorus made available by other
microorganisms while simultaneously conserving the energy and resources required to express and
excrete alkaline phosphatase (Bar-Yosef et al. 2010). The precise ecological function of microcystins
has not been determined conclusively (Zurawell et al. 2005). Studies comparing wild-types and mutants
of a microcystin-producing species, examining the genes involved in microcystin biosynthesis, and
evaluating Microcystis colony size have suggested that microcystins play important physiological roles
in cyanobacteria, including colony formation (Kaplan et al. 2012; Zurawell et al. 2005). Gobler et al.
(2007) observed decreased zooplankton grazing when toxigenic Microcystis were actively producing
microcystin. Although cyanotoxins can negatively affect humans and other animals, research suggests
that the primary functions of cyanotoxins are in cyanobacterial physiology and microbial ecology.
Cyanobacteria can regulate their buoyancy; thus, they can actively seek water depths with optimal
growth conditions and will enlarge their gas vesicles to adapt to turbulent conditions. When weather
conditions shift from turbulent to strongly stratified, excessively buoyant cells may accumulate at the
surface because the regulation of buoyancy takes a few days (Australian Government National Health
and Medical Research Council 2008, WHO 2003b). When the rate of cyanobacterial cell growth exceeds
the loss rate for a population, positively buoyant, floating cyanobacterial cells can also accumulate at the
surface (Falconer 1998). This accumulation can form a visibly colored scum on the water surface, which
can contain more than 10,000 cells/mL (Falconer 1998). Scums can pose an elevated health risk to
recreational users. The floating scum can be concentrated by prevailing winds in certain surface water
areas, especially at the shore as is the case for Microcystis. Scums have frequently been reported to
accumulate cells and cyanotoxin concentration by a factor of 1,000 or more, with million-fold
accumulations resulting in pea soup consistency (Australian Government National Health and Medical
Research Council 2008; WHO 2003b).
The microbial community can be complex and variable. It can consist of multiple different species and
strains of cyanobacteria and other microbes. Microbial interactions can occur within blooms, such as
competition and adaptation between toxic and nontoxic cyanobacterial strains, as well as impacts from
viruses and zooplankton grazers like Daphnia (large generalist grazers), copepods, and cladocerans (Ger
et al. 2014). Each of these microbial-related factors can cause fluctuations in bloom development and
composition.
3.1.1 Environmental Factors Influencing Occurrence of Cyanobacteria and Cyanotoxins
A variety of physical, chemical, and environmental factors can influence both cyanobacteria
proliferation and toxin production, including nutrient (e.g., nitrogen and phosphorus) concentrations,
water temperature, light levels, and pH. Other factors include water turbulence, mixing, and flushing,
oxidative stressors, and interactions with other biota (e.g., viruses, bacteria, and animal grazers), as well
as their combined effects (Paerl and Otten 2013a, 2013b). See Figure 3-1.
Total cyanobacterial density in a bloom and cyanotoxin concentration are not always closely related.
Cyanotoxin concentrations depend on the dominance and diversity of species and strains within the
bloom along with environmental and ecosystem influences on bloom dynamics (Chorus et al. 2000;
Hitzfeld et al. 2000; WHO 1999). Cyanotoxin production can vary among strains and clones of a single
species (Carmichael 1994; Utkilen and Gj0lme 1992) and within and between blooms (Codd and Bell
1985). Growth phase also can influence cyanotoxin production (Jaiswal et al. 2008). Biomass and toxin
production do not necessarily coincide (section 7.5.2.3). Francy et al. (2016) modeled the relationship of
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environmental variables compared to cyanotoxin levels. They demonstrated that some environmental
factors such as measures of the algal community (e.g., phycocyanin, cyanobacterial biovolume, and
cyanobacterial gene concentrations) and pH are strongly correlated with microcystin concentrations.
Figure 3-1. Environmental Factors Influencing Total Cyanobacterial Blooms, Reproduced from
Paerl and Otten (2013b)
Positive
•	High P (High N for some)
•	Low N (DIN, DON) (only
applies to N2 fixers)
•	Low N:P Ratios
•	Low turbulence
•	Low water flushing-Long
water residence time
•	High (adequate) light
•	Warm temperatures
•	High dissolved organic
matter
•	Sufficient Fe (+ trace
metals)
•	Low grazing rates
w
CD
•*—>
(0
cc
Cyanos
CO
CD
¦4—'
CO
CC
Diversity
Modulating factors
Strong biogeochemical gradients (e.g.
persistent stratification, stable benthos)
Heterogeneous and diverse habitats (e.g.
reefs, seagrasses, marshes, sediments,
aggregates)
Selective grazing
"Toxin" production
Negative
High DIN/total N (only
applies to N2 fixers)
Low P (DIP)
High N:P ratios
High turbulence & vertical
mixing
High water flushing-Short
water residence time
Low light (for most taxa)
Cool temperatures
Low dissolved organic
matter
Low Fe (+ trace metals)
High grazing rates
Viruses (cyanophages)
Predatory bacteria
Cyanotoxins can be found inside the cell (i.e., intracellular) or external to the cell in the water (i.e.,
extracellular). The proportion of intracellular versus extracellular cyanotoxin can vary. Extracellular
microcystins (either dissolved in water or bound to other materials) typically are less than 30 percent of
the total microcystin concentration in source water (Graham et al. 2010). Most of the microcystins are
intracellular and released into the water when the toxigenic cyanobacterial cells rupture or die.
Cylindrospermopsin can be retained within the cyanobacterial cell or released. The ratio of intracellular
to extracellular cyanotoxin can change depending on the growth phase with as much as 50 percent of
cylindrospermopsin produced by Cylindrospermopsis raciborskii released extracellularly (Griffiths and
Saker 2003).
A complex interplay of environmental factors dictates the spatial and temporal changes in the
concentration of cyanobacterial cells and their toxins with respect to the dominant species. Factors such
as the amount and timing of nutrient supply (i.e., nutrient concentration and nutrient loading), the
relative proportions of nutrients (i.e., nitrogen to phosphorus ratio), dissolved organic matter availability,
temperature, and light attenuation, as well as other physico-chemical processes, can play a role in
shaping cyanobacterial bloom composition and cyanotoxin production (Paerl and Huisman 2008; Paerl
and Otten 2013b).
Some cyanobacteria possess toxin genes that enable them to produce toxins, while other cyanobacteria
do not contain toxin genes and therefore cannot produce toxins. For example, cyanobacteria that can
produce microcystins contain a collection of genes, called iLmcy" genes, that when expressed produce
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microcystins. Multiple species of cyanobacteria can contain this set of genes. Together these species
comprise microcystin-producing toxigenic cyanobacteria. Ten genes are in the microcystin gene cluster,
mcyA through may.I (Tillett et al. 2000). Different researchers have studied the occurrence and
prevalence of these genes within cyanobacteria populations.
Environmental factors can provide competitive advantages to Microcystis relative to other
phytoplankton (Jacoby et al. 2000; Marmen et al. 2016). Evidence suggests that these environmental
factors also affect the relative abundance of microcystin-producing strains and non-microcystin-
producing strains (Marmen et al. 2016). Microcystis thrive in warmer temperatures, with optimal growth
and photosynthesis occurring above 25°C (O'Neil et al. 2012). A Japanese study between May and
November 2006 found that the toxin-producing species, Microcystis aeruginosa, dominated in months
with relatively higher water temperatures, while the non-toxin-producing species, M. wesenbergii,
dominated in months with lower water temperatures (Imai et al. 2009). The genetic composition of the
bloom can also influence the degree of toxicity associated with an algal bloom. Lee et al. (2015) found
that Microcystis typically comprised less than one percent of the total cyanobacterial abundance in
Vancouver Lake, Washington, but the majority of the Microcystis cells contained the toxin-producing
gene. Despite comprising a small percentage of the total cyanobacterial community in this lake,
Microcystis were the sole microcystin-producing cyanobacteria and were responsible for microcystin
concentrations that exceeded the WHO guidelines several times throughout the sampling period. In
addition, increases in phosphate concentrations were associated with increases in both toxigenic and
non-toxigenic Microcystis and with toxin production. The authors note that quantifying Microcystis
mcyE gene (one of the genes responsible for toxin production) copy number, rather than relying solely
on visual cell counts, might be a better estimate of overall cyanotoxin concentration (Lee et al. 2015).
Zhang et al. (2015) observed that low flow conditions favored total cyanobacteria and higher flow
conditions favored green algae. Loftin et al. (2016a) suggest that low stream flow, shallow depth, and
high water-column light penetration in Piedmont streams favored periphyton occurrence (mixture of
algae, cyanobacteria, heterotrophic bacteria, and detritus).
Phytoplankton competition and food web interactions occur as blooms develop, persist, and decline,
thereby impacting cyanotoxin concentrations in surface waters. In addition, potential warming of surface
waters and changes in precipitation could result in changes in ecosystem dynamics that lead to more
frequent formation of cyanobacteria blooms and their associated toxins (Paerl et al. 2011; Paerl and
Huisman 2008; Paerl and Otten 2013b).
3.1.1.1 Nutrients
Nutrients, particularly nutrient over-enrichment, are key environmental drivers that influence the
proportion of cyanobacteria in the phytoplankton community, the cyanobacterial biovolume, cyanotoxin
production, and the impact that cyanobacteria may have on ecosystem function and water quality (Yang
et al. 2016a; Beaulieu et al. 2013; Paerl et al. 2011). Cyanobacteria have been shown to dominate the
phytoplankton communities in eutrophic lakes (Downing et al. 2001; Monchamp et al. 2014).
Phosphorus loading has been linked to the proliferation of cyanobacteria and the shift toward
cyanobacterial dominance of the phytoplankton community (O'Neil et al. 2012). However, it is
important to consider both phosphorus and nitrogen when considering the occurrence of toxigenic
cyanobacterial blooms. Cyanobacterial toxin concentrations are also associated with nutrient levels
(Wang et al. 2002); however, different cyanobacteria species use organic and inorganic nutrient forms
differently. Dolman et al. (2012) found that total cyanobacterial biomass was higher in lakes with above-
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average nitrogen and phosphorus concentrations and that concentrations of all cyanotoxin groups were
higher in lakes with higher total nitrogen and total phosphorus concentrations.
Paerl (2008) demonstrated that nitrogen and phosphorus additions, both independently and together, can
stimulate primary productivity and Cylindrospermopsis raciborskii biomass. Elevated nitrogen and
phosphorus loading can enhance the growth and cyanotoxin levels of Microcystis blooms and
microcystin synthetase gene expression (Gobler et al. 2007; O'Neil et al. 2012; Marmen et al. 2016).
Gobler et al. (2007) found that Microcystis dominance and toxin production was stimulated by elevated
nitrogen and suppressed by nitrogen limiting conditions. Toxin production may cause the inhibition of
grazing by mesozooplankton and further accumulation of cyanobacterial cells. Willis et al. (2015) found
the highest growth rates for environmental isolates of Cylindrospermopsis raciborskii were observed
with the addition of nitrogen.
The relative abundance of nitrogen and phosphorus can be an important consideration in regards to
toxigenic cyanobacterial blooms. Loadings of nitrogen, or phosphorus, or both, to water bodies from
agricultural, industrial, and urban sources influences the development of total cyanobacterial blooms and
are associated with cyanotoxin production (Paerl et al. 2011). Smith (1983) was the first to describe a
strong relationship between the relative amounts of nitrogen and phosphorus in surface waters and
toxigenic cyanobacterial blooms. Smith proposed that cyanobacteria should be superior competitors
under conditions of nitrogen limitation because of their unique capacity for nitrogen fixation, although
many cyanobacteria like Microcystis that produce toxins do not fix nitrogen. Many toxigenic
cyanobacterial blooms are comprised of non-nitrogen-fixing genera and in the presence of elevated
phosphorus, nitrogen can be a limiting factor for biomass proliferation and microcystin production
(Gobler et al. 2007). Schindler et al. (2008) demonstrated that lower nitrogen inputs relative to
phosphorus loadings can lead to dominance of nitrogen-fixing cyanobacteria in mesocosm- and
ecosystem-scale experiments in prairie and boreal lakes. Otten et al. (2012) reported higher average
microcystin concentrations and a higher prevalence of toxigenic Microcystin biomass at sites that had
narrower TN:TP ratios (< 20) in Lake Taihu, China. Fortin et al. (2015) demonstrated that the
dominance of Microcystis depended on the ratio of nitrogen to phosphorus, with a (mass) ratio 11:1
resulting in the highest abundance of Microcystis, whereas the concentrations of each nutrient were
significant factors affecting the amount of biomass that could be generated.
Cyanotoxin concentration can be related to cyanobacterial cell abundance, which is facilitated by
nutrient availability (Welker 2008), so nutrient concentration can be correlated to cyanotoxin
concentration. Yuan et al. (2014; 2015) developed nutrient thresholds related to microcystin
concentrations, cyanobacterial biovolume, and chlorophyll a. Nutrient availability, environmental
conditions, and ecosystem interactions can affect the production and amount of toxins that cells produce
and release (Bar-Yosef et al. 2010; Dolman et al. 2012; Graham et al. 2004; Paerl et al. 2001). For
example, both nitrogen and phosphorus have been shown to promote the production of microcystins
during bloom events (Davis et al. 2009; Gobler et al. 2016; Ha et al. 2009). Horst et al. (2014) found a
significant positive relationship between cellular microcystin amounts and nitrate concentration with
nitrogen limitation related to lower cell quotas of microcystin. Ha et al. (2009) found that microcystin
concentrations were highly associated with mcyA gene copies and that high concentrations of nitrates
and ammonium increased microcystin production by promoting the growth of toxigenic Microcystis.
Elevated phosphorus has been shown to favor toxigenic strains over non-toxin strains coupled with
higher intracellular toxin concentrations (Boopathi and Ki 2014; Burford et al. 2016).
Soluble phosphates and nitrates may also result in the increased production of microcystins (ILS 2000;
O'Neil et al. 2012; Paerl and Scott 2010; Wang et al. 2010). Davis et al. (2009) found that growth rates
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of toxigenic Microcystis were higher than nontoxic strains as temperature increased in the presence of
elevated soluble phosphorus and that toxigenic cells contained more copies of the gene mcyD under
these conditions. The authors conclude that lakes experiencing this combination of factors could
experience more toxic blooms (Davis et al. 2009). In the Sacramento-San Joaquin delta in California
nitrogen and phosphorus are available in non-limiting amounts and facilitate persistence of total
cyanobacterial blooms (Berg and Sutula 2015). A study by Lehman et al. (2015) characterizes nitrogen
sources of a Microcystis bloom in the San Francisco Estuary using stable isotopes. They reported that
ammonium from the Sacramento River was the likely sole source of the nitrogen for most of the bloom,
overriding nitrate contributions from the San Joaquin River.
Jacoby et al. (2000) characterized multiple physical and chemical environmental factors associated with
blooms in the summer of 1994 and 1995 at Steilacoom Lake, Washington. The dominance of
Microcystis aeruginosa in the lake was associated with low nitrogen-to-phosphorus ratios and low
nitrate-nitrogen with sufficient ammonium-nitrogen. Microcystin concentrations were positively
correlated with increasing soluble reactive phosphorus concentrations with the highest microcystin
concentrations associated with a low ratio of soluble nitrogen to soluble reactive phosphorus (less than
five). The authors reported that microcystin production per gram cyanobacterial biomass was not
consistent, thus no relationship was found between Microcystis aeruginosa abundance and microcystin
concentration. A significant positive relationship between total phosphorus concentrations and total
cyanobacteria densities was observed in both years of the study (Jacoby et al. 2000).
During bloom events, nutrients on a local scale are incorporated into the production of biomass and
decrease in the water column within the bloom, even in eutrophic water bodies. Kuniyoshi et al. (2013)
showed that phosphate deficiency resulting from exponential biomass production can result in
approximately seven-fold increase in microcystin synthesis. Bar-Yosef et al. (2010) reported that
cylindrospermopsin-producing Aphanizomenon excrete cylindrospermopsin when phosphorus-limiting
conditions occur within the bloom, to induce other cells to produce and excrete alkaline phosphatase,
thus increasing availability of extracellular inorganic phosphate. Cylindrospermopsin is energetically
cheaper for the cell to produce relative to alkaline phosphatase (Raven 2010) and coupled with a high-
affinity phosphorus uptake protein also found in these cells, allows Aphanizomenon to increase rapidly,
outcompeting other cyanobacteria and dominate a bloom (Bar-Yousef et al. 2010). PreuBel et al. (2014)
observed that cylindrospermopsin is actively released from Aphanizomenon ovalisporum cells subjected
to phosphorus limitation, a condition that occurs during the exponential biomass production in a bloom
event.
Eutrophic systems already subject to bloom events are prone to further expansion of these blooms due to
additional nitrogen inputs, especially if these nutrients are available from internal sources. As the trophic
state increases, aquatic systems absorb higher concentrations of nitrogen (Paerl and Huisman 2008;
Paerl and Otten 2013b). Recent surveys of cyanobacterial and algal productivity in response to nutrient
pollution across geographically diverse eutrophic lakes, reservoirs, estuarine and coastal waters, and in
different experimental enclosures of varying sizes demonstrate that greater stimulation is routinely
observed in response to both nitrogen and phosphorus additions. Further, this evidence suggests that
nutrient co-limitation is widespread (Elser et al. 2007; Lewis et al. 2011; Paerl et al. 2011). These results
suggest that reductions in nutrient concentration would reduce eutrophication and cyanobacterial bloom
expansion. For example, analysis of observational data collected at high spatial scales support the idea
that controlling total phosphorus and total nitrogen could reduce the frequency of high microcystin
contamination events by reducing the biomass of total cyanobacteria in the system (Orihel et al. 2012;
Scott et al. 2013; Yuan et al. 2014). In addition, reduction of phosphorus in the absence of concurrent
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reductions in nitrogen loading may not effectively control the growth, toxicity, or both of cyanobacteria
such as Microcystis (Gobler et al. 2016). Study authors concluded that reduction of specific nutrient
species, such as soluble forms of nitrogen and phosphorus, could reduce the dominance of toxigenic
cyanobacteria in the lake microbial community, which could, in turn, decrease the incidences of elevated
toxin levels (Davis et al., 2010; Gobler et al. 2016).
3.1.1.2 Temperature
Cyanobacterial blooms commonly occur from spring to early fall in various regions of the United States
(Wynne and Stumpf 2015). Conditions such as elevated water temperatures and increased vertical
stratification in lakes and reservoirs can support proliferation of total cyanobacteria (Paerl and Huisman
2008). The increasing body of laboratory and field data (Carey et al. 2012a; De Senerpont Domis et al.
2007; Huisman et al. 2005; Jeppesen et al. 2009; Kosten et al. 2012; Reynolds 2006; Wagner and Adrian
2009; Weyhenmeyer 2001) suggest that an increase in temperature may influence cyanobacterial
dominance in phytoplankton communities. Some cyanobacteria have higher optimal growth
temperatures compared with other phytoplankton and can proliferate at higher water temperatures by
outcompeting these other phytoplankton groups (Elliott 2010; Paerl et al. 2011). Warmer water
temperatures favor surface bloom-forming cyanobacterial genera because they are heat-adapted, and
their maximal growth rates occur at relatively high temperatures, with optimum growth temperatures
ranging from 30 to 35°C and optimum microcystin production ranging from 20 to 25°C (Giannuzzi
2018; Reynolds 2006; Robarts and Zohary 1987; WHO 2003b). As the growth rates of the eukaryotic
taxa decline in response to warming water temperature, cyanobacterial growth rates reach their optima.
Davis et al. (2009) found in four U.S. lakes that concurrent increases in temperature and phosphorus
concentrations yielded the highest growth rates of toxic Microcystis cells, which led them to conclude
that eutrophication and warm temperatures may promote the growth of toxic, rather than nontoxic,
populations of Microcystis leading to blooms with higher microcystin content.
Cyanobacteria are typically known to proliferate in warm water environments such as tropical and
temperate lakes and rivers, but they can also proliferate in cooler water environments under mesophilic
and psychrophilic conditions (Seckback and Oren 2007). Cyanobacteria are also found in Antarctic
habitats where they play a significant role in microbial ecosystem dynamics by providing fixed carbon
via photosynthesis (Singh and Elster 2007). Cyanobacteria can grow in these extreme environments
because they can adapt to survive freeze/thaw cycles and they can metabolize at near 0°C (Singh and
Elster 2007).
The increase in water column stability associated with higher temperatures, less flow, and shallower
water can also favor total cyanobacteria growth (Carey et al. 2012a; Wagner and Adrian 2009). In a
study of 143 shallow lakes sampled along a latitudinal transect ranging from subarctic Europe to
southern South America, Kosten et al. (2012) reported the percentage of cyanobacteria relative to total
phytoplankton biovolume increased steeply with temperature in the lakes. The series of conditions most
likely to result in cyanobacterial dominance begin with elevated winter-spring rainfall and runoff,
followed by protracted periods of summer drought where temperatures, vertical stratification, and water
residence times all increase simultaneously (Paerl and Otten 2013b).
Indirectly, warming can increase nutrient concentrations by enhancing mineralization (Gudasz et al.
2010; Kosten et al. 2009; Kosten et al. 2010) by temperature- or anoxia-mediated sediment phosphorus
release (Jensen and Andersen 1992; S0ndergaard et al. 2003). Thus, increases in temperature can
indirectly increase cyanobacterial biomass through its effect on nutrient concentrations. Others have
suggested that warmer conditions may raise total phytoplankton biomass through an alteration of top-
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down regulation by selective grazing that favors larger size phytoplankton species and cyanobacterial
blooms (Jeppesen et al. 2009; Jeppesen et al. 2010; Teixeira-de Mello et al. 2009). The relationship
between temperature and cyanobacterial dominance can be explained not only through a temperature-
related effect on the competitive advantage of cyanobacteria, but also by factors such as the percent area
covered and the volume of the lake taken up by submerged macrophytes (Carey et al. 2012a; Kosten et
al. 2012).
Cylindrospermopsis raciborskii was first identified in the tropics but has also been increasingly found in
temperate regions since it was first found in North America in 1955 (Hong et al. 2006).
Cylindrospermopsis raciborskii blooms are most likely to occur between the temperatures of 25 to 32°C
but can sustain biomass at temperatures as low as 11°C (Antunes et al. 2015). In Florida, C. raciborskii
was found to be the dominant cyanobacteria species in one lake all year round (Burns 2008). In 2006, C.
raciborskii was detected in lakes in southern Louisiana (Fuentes et al. 2010). Conditions promoting its
growth were shallow, warm surface water (over 30°C) and low light intensities. The highest densities of
C. raciborskii were observed from June through August with densities ranging from 37,000 cells/mL to
more than 160,000 cells/mL. In a study of two lakes directly connected to Lake Michigan, Hong et al.
(2006) found low levels of C. raciborskii only in the late summer, and these were associated with
elevated bottom water temperatures and phosphorus concentrations.
3.1.1.3	Sunlight
Sunlight availability and turbidity can have a strong influence on the cyanobacteria species that
predominate, as well as the depth at which they occur (Carey et al. 2012a; Falconer 2005). The authors
(Carey et al. and Falconer) found a greater proportion of the total phytoplankton biovolume attributable
to cyanobacteria in lakes with high rates of light absorption. They could not establish cause and effect
from their field data, but other controlled experiments and field data have demonstrated that light
availability can affect the competitive balance among a large group of shade-tolerant species of
cyanobacteria, primarily Oscillatoriales and other phytoplankton species (Scheffer et al. 1997; Smith
1986).
3.1.1.4	pH Levels
Total cyanobacterial blooms intensify and persist at pH levels between six and nine (Caraco and Miller
1998; WHO 2003a). Kosten et al. (2012) noted that pH affected cyanobacteria abundance in lakes along
a latitudinal transect from Europe to southern South America. The percentage of cyanobacteria in the
143 shallow lakes sampled highly correlated with pH, increasing as the pH increased. Shapiro (1984)
hypothesized that cyanobacteria have a competitive advantage over other phytoplankton species because
they are efficient users of carbon dioxide in water. When dissolved carbon dioxide is high (low pH),
conditions favor growth and replication of the green algal colonies over the blue-green cyanobacteria
(Caraco and Miller 1998; Shapiro 1984). At alkaline pH levels, inorganic carbon is present as carbonate
anion rather than as carbon dioxide, carbonic acid, or bicarbonate anion. This situation favors the growth
of cyanobacteria because they can carry out photosynthesis when the levels of dissolved carbon dioxide
are very low (high pH). The blue-green algae have a much higher photosynthetic demand for the
dissolved carbon dioxide allowing them to out compete the green algae for the limited supply (Caraco
and Miller 1998; Shapiro 1984). Thus, a higher water column pH can correlate with a higher proportion
of cyanobacteria in an algal bloom.
The Caraco and Miller (1998) study suggests that pH and dissolved carbon dioxide, although chemically
linked, are also independent factors in bloom dynamics because, even when dissolved carbon dioxide in
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water is mechanically enriched, an alkaline pH still favors growth of the cyanobacteria over the green
algae if nutrient inputs are constant.
3.2 Cyanotoxins
Much of the information and the studies summarized in this section for microcystins and
cylindrospermopsin are described in detail in the EPA's HESDs and Drinking Water Health Advisories
for microcystins and cylindrospermopsin (U.S. EPA 2015a, 2015b, 2015c, 2015d). The EPA's HESDs
established the scientific basis for the EPA Drinking Water Health Advisories and also informed the
EPA in developing these ambient water quality criteria (AWQC) or swimming advisories. This section
summarizes the information that is provided in more detail in the EPA's HESDs. Additional information
can be found in the EPA's HESDs for microcystins and cylindrospermopsin (U.S. EPA 2015c, 2015d).
3.2.1 Chemical and Physical Properties
Structurally, microcystins are monocyclic heptapeptides that contain seven amino acids joined end to
end and then head to tail to form cyclic compounds that are comparatively large; molecular weights
range from approximately 800 to 1,100 g/mole for the different congeners (e.g., microcystin-LR is
995.17 g/mole). The cyclic peptides include more than 100 congeners of microcystins (Niedermeyer
2014). Figure 3-2 provides the structure of microcystin where X and Y represent variable amino acids.
Although substitutions mostly occur in positions X and Y, other modifications have been reported for all
the amino acids (Puddick et al. 2015).
The microcystin congeners are named based on their two variable amino acids (Carmichael et al. 1988).
For example, microcystin-LR, the most common congener (Carmichael 1992). The letters used to
identify the variable amino acids are the standard single letter abbreviations for the amino acids found in
proteins. The variable amino acids are usually the L-amino acids as found in proteins. In Figure 3-2,
which shows the structure of microcystin-LR, leucine is in the X position and arginine is in the Y
position. Table 3-1 lists the most common microcystin congeners, including the amino acids in the X
and Y positions.
There are other variants of microcystins besides those that arise because of the two interchangeable
amino acids on the microcystin ring. For example, demethylated congeners have been observed in
Europe; Wejnerowski et al. (2018) identified demethylated forms of microcystin-RR and microcystin-
LR in a toxigenic cyanobacterial bloom in Poland. Observations of demethylated microcystins suggest
that more than 200 microcystin congeners are possible.
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Figure 3-2. Structure of Microcystin (Kondo et al. 1992)
Table 3-1. Abbreviations for Selected Microcystins (Yuan et al. 1999)
Microcystin Congeners
Amino Acid in X
Amino Acid in Y
Microcystin-LR
Leucine
Arginine
Microcystin-RR
Arginine
Arginine
Microcystin-YR
Tyrosine
Arginine
Microcystin-LA
Leucine
Alanine
Microcystin-LY
Leucine
Tyrosine
Microcystin-LF
Leucine
Phenylalanine
Microcystin-LW
Leucine
Tryptophan
The preponderance of toxicological data on the effects of microcystins result from tests using the
microcystin-LR congener. Toxicity data suggest that microcystin-LR is as potent as or more potent than
other studied microcystins and that the most toxic microcystins are those with the more hydrophobic L-
amino acids (e.g., -LA, -LR, and -YR); the least toxic are those with hydrophilic amino acids, such as
microcystin-RR (U.S. EPA 2015d; Ito et al. 2002; Rinehart et al. 1994; Vesterkvist and Meriluoto 2003;
WHO 1999). Data on the -RR, -YR, and -LA congeners, however, are limited, and toxicity values
cannot be derived for them. Therefore, values developed from data specific to microcystin-LR can
represent other present microcystin congeners.
Table 3-2 provides chemical and physical properties of microcystin-LR. Microcystins are water soluble.
In aquatic environments, the cyclic peptides tend to remain contained within the cyanobacterial cell and
are released in substantial amounts only when the cell walls are broken down (cell lysis).
Cylindrospermopsin is a tricyclic alkaloid with the molecular formula of C15H21N5O7S (Ohtani et al.
1992) and a molecular weight of 415.43 g/mole. It is a dipolar ion with localized positive and negative
charges (Ohtani et al. 1992). The chemical structure of cylindrospermopsin is presented in Figure 3-3(a).
Two naturally occurring congeners of cylindrospermopsin have been identified, 7-epi-
cylindrospermopsin (the epimer of cylindrospermopsin) and 7-deoxycylindrospermopsin; see Figure 3-
3(b) and (c) (de la Cruz et al. 2013; Norris et al. 1999). Recently, Wimmer et al. (2014) identified two
new analogs, 7-deoxy-desulfo-cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin,
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from the Thai strain of Cylindrospermopsis rciciborskii. However, it is not clear if these are
cylindrospermopsin congeners, precursors, or degradation products.
Table 3-2. Chemical and Physical Properites of Microcystin-LR
Property
Microcystin-LR
Chemical Abstracts Registry (CAS) Number
101043-37-2
Chemical Formula
C49H74N10O12
Molecular Weight
995.17 g/mole
Color/Physical State
Solid
Boiling Point
Not available (N/A)
Melting Point
N/A
Density
1.29 g/cm3
Vapor Pressure at 25°C
N/A
Henry's Law Constant
N/A
Log Octanol-Water Partition Coefficient (Kow)
2.16; -1.41 to 1.67 as pH decreases
Soil Organic Carbon-Water Partition Coefficient (Koc)
N/A
Solubility in Water
Highly*
Other Solvents
Ethanol and methanol
Sources: Chemical Book (2012); TOXLINE (2012); Ward and Codd (1999) and McCord et al. (2018) for log Kow.
* Microcystin congeners vary in their relative solubility in water.
Figure 3-3. Structure of Cylindrospermopsin and Structurally Related Cylindrospermopsins (de
la Cruz et al. 2013)
(a) Cylindrospermopsin
H OH
(b) 7-epi-cylindrospermopsin (the epimer of cylindrospermopsin)
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(c) 7-deoxycylindrospermopsin
©
The physical and chemical properties of cylindrospermopsin are presented in Table 3-3.
Cylindrospermopsin is highly soluble in water (Chiswell et al. 1999; Moore et al. 1998). It is isolated for
commercial use mostly from Cylindrospermopsis raciborskii. Some relevant physico-chemical
properties of cylindrospermopsin could not be identified, and no physico-chemical properties were
found for the structurally related cylindrospermopsins.
Table 3-3. Chemical and Physical Properties of Cylindrospermopsin
Property
Cylindrospermopsin
CAS Registry Number
143545-90-8
Chemical Formula
C15H21N5O7S
Molecular Weight
415.43 g/mole
Color/Physical State
White powder
Boiling Point
N/A
Melting Point
N/A
Density
2.03 g/cm3
Vapor Pressure at 25°C
N/A
Henry's Law Constant
N/A
K-ow
N/A
Koc
N/A
Solubility in Water
Highly
Other Solvents
Dimethyl sulfoxide and methanol
Sources: Chemical Book (2012); TOXLINE (2012).
3.2.2 Sources and Occurrence in Surface Waters
Because they are a natural part of algal communities, cyanobacteria are commonly observed in
freshwater systems. The occurrence of HABs has been documented in surface waters of all 50 states as
well as U.S. territories between 2006 and 2015 as shown in Figure 3-4 (Richlen 2016; WHOI2016).
Figure 3-4 also identifies areas where more widespread HAB problems have occurred (e.g., parts of the
Great Lakes).
Figure 3-5 shows the number of 2017 freshwater HAB recreational notices states publicly reported,
organized by the EPA region between June 2 and August 1, 2017. To develop this regional summary
map, the EPA researched and compiled publicly available reports posted on states' websites between
these dates. During that time, states reported at least 281 notices for freshwater HABs with reported
microcystin concentrations ranging from not detected (i.e., below the limit of detection) to 382 pg/L.
These notices included cautions, warnings, public health advisories, and public health warnings due to
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the presence of total cyanobacteria, cyanotoxins, or both. These notices can last for multiple days. The
review was not exhaustive and might not reflect all the monitoring, beach, or general health advisories
(e.g., some advisories at local or county-level may not be posted on the state website). Thus, the number
of actual ITAB notices during this time might be higher. In addition, many states have only recently
begun to monitor HABs, so monitoring may be limited.
2015. It reflects input fromHAB experts with broad experience in FIAB events and reports to the U.S. National Office for
Harmful Algal Blooms (Richlen 2016; WHOI2016). Each state that has experienced one or more cyanobacterial HAB is
indicated with a single green dot. Larger green ovals mark areas where more widespread cyanobacterial HAB problems
occurred.
Figure 3-5. State-reported HAB Notices by EPA Region, June 2 to August 1, 2017
41
19
31


29


i
2
B
17
37
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3.2.2.1 Microcystins
Microcystins are the most common cyanotoxins found worldwide and have been reported in surface
waters in most of the states in the United States (Funari and Testai 2008; Loftin et al. 2016b; U.S. EPA
2009). Dry-weight concentrations of microcystins in surface freshwater toxigenic cyanobacterial blooms
or surface freshwater samples reported worldwide between 1985 and 1996 ranged from 1 to 7,300 jag/g.
Water concentrations of extracellular plus intracellular microcystins ranged from 0.04 to 25,000 (J,g/L.
The remainder of this section provides examples of microcystin concentrations reported in ambient
waters in the United States.
The EPA (U.S. EPA 2009) reported on the 2007 National Lakes Assessment (NLA), a national
probability-based survey of the nation's lakes, ponds, and reservoirs. The NLA provided estimates of the
condition of natural and man-made freshwater lakes, ponds, and reservoirs greater than 4 hectares
(10 acres) and at least one meter deep. The 2007 NLA study surveyed 1,028 inland lakes and included
measured microcystin concentrations, total cyanobacterial cell counts, and chlorophyll a concentrations.
Microcystins were quantified using enzyme-linked immunosorbent assays (ELISA) with a detection
limit of 0.1 [j,g/L (Loftin et al. 2016b). At each lake site, crews collected samples at a single station
located at the deepest point in the lake and at ten stations around the lake perimeter. Due to the design of
the survey, samples were taken at random and not necessarily where a bloom was occurring.
The 2007 NLA found that total cyanobacteria were detected in 98 percent of samples and were the
dominant member of the phytoplankton community in 76 percent of samples (Loftin et al. 2016b; U.S.
EPA 2009). Subsequent analysis indicated that potential microcystin-producing species occurred in
95 percent of samples (Loftin et al. 2016b). Microcystins were the most commonly detected class of
cyanotoxins found in 32 percent of lakes in the contiguous United States (Loftin et al. 2016b; U.S. EPA
2009) and 39 percent of streams in the southeastern United States (Loftin et al. 2016a). Microcystins
present in lakes ranged from the limit of detection (0.1 (J,g/L) to 225 [j,g/L with a mean concentration of
3.0 [j,g/L (detections only). Approximately 1.1 percent of lake samples exceeded 10 [j,g/L microcystins,
and approximately 27 percent and 44 percent of lakes exceeded the WHO low-risk threshold for
cyanobacterial abundance and chlorophyll a, respectively (Loftin et al. 2016b).
Lakes in states with microcystins levels > 10 [j,g/L reported in the 2007 NLA are shown in Table 3-4.
The 2007 NLA data show two states (North Dakota and Nebraska) had nine percent of samples above
10 (J,g/L. Other states including Iowa, Texas, South Dakota, and Utah also had samples that exceeded
10 (J,g/L, but the frequency of detection was lower. Several of the 2007 NLA samples in North Dakota,
Nebraska, and Ohio exceeded 20 [j,g/L (192, 225, and 78 (J,g/L, respectively).
In 2012, the EPA expanded on the 2007 NLA to include smaller water bodies in this statistically
designed survey. Results represent the population of natural lakes, ponds, and reservoirs across the
lower 48 states (not including the Great Lakes or the Great Salt Lake). To be included, in the survey
lakes had to be larger than 2.47 acres (1 hectare), at least 3.3 feet (1 meter) deep, with a minimum
quarter acre (0.1 hectare) of open water (U.S. EPA 2016). Data were collected from 1,038 lakes selected
from a stratified random sample based on ecoregion, state, and surface area in the larger inference
population (the set of 111,818 lakes). The NLA used thresholds established by the WHO to determine
risk of exposure to cyanotoxins. Microcystins were detected in 39 percent of lakes monitored, but less
than one percent exceeded the WHO estimates for microcystins at moderate or high risk of exposure.
Less than one percent of lakes are in the most and moderately disturbed condition (i.e., have a high or
moderate risk of exposure), and 99 percent are either least disturbed or show no detection of
microcystins. Between 2007 and 2012, the percentage of lakes categorized as most disturbed for
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microcystins did not change (U.S. EPA 2016), even though there was a significant increase in the
detection of microcystins (+9.5 percent).
Table 3-4. States Surveyed as Part of the 2007 NLA with Water Body Microcystin Concentrations
above 10 jig/L (U.S. EPA 2009)
State
Number
of Sites
Sampled
Percentage of Samples with
Detection of Microcystins
> 10 jig/L
Maximum Detection
of Microcystins
North Dakota
38
9.1 percent
192 (ig/L
Nebraska
42
9.1 percent
225 (ig/L
South Dakota
40
4.9 percent
33 (ig/L
Ohio
21
4.5 percent
78 (ig/L*
Iowa
20
4.5 percent
38 (ig/L*
Utah
26
3.6 percent
15 (ig/L*
Texas
51
1.8 percent
28 (ig/L*
* Single sample.
The NLA used total cyanobacterial cell counts as an indicator of water quality impacts of microcystins;
15 percent of lakes were classified in the most disturbed condition, 23 percent were classified as
moderately disturbed, and 61 percent were classified as least disturbed. Between 2007 and 2012, there
was a statistically significant increase (+8.3 percent) in the number of lakes in the most disturbed
category for cyanobacterial cell counts. Lakes that were considered most disturbed exceeded the WHO
recreational levels of concern (20 jag of microcystins/L).
A survey conducted during the spring and summer of both 1999 and 2000 in more than 50 lakes in New
Hampshire found measurable microcystin concentrations in all samples (Haney and Ikawa 2000).
Microcystins were analyzed by ELISA and were found in all the lakes sampled with a mean
concentration of 0.1 pg/L.
A survey conducted in Florida in 1999 found potential microcystin-producing genera in water samples,
including, Microcystis (43.1 percent), Anabcienci (28.7 percent), Plcmktothrix (13.8 percent),
Aphcmizomenon (7.2 percent), and Coelosphaerium (3.6 percent) (Burns 2008). Although Plcmktothrix
and Aphcmizomenon were found less frequently than were the other genera, at times they accounted for a
significant portion of the cyanobacterial community present. Microcystins were the most commonly
found toxins in Florida waters, occurring in all samples analyzed containing cyanotoxins (Burns 2008).
In 2002, the Monitoring and Event Response to Harmful Algal Blooms in the Lower Great Lakes project
evaluated the occurrence and distribution of cyanotoxins in the lower Great Lakes region (Boyer 2007).
Analysis for total microcystins was performed using protein phosphatase inhibition assay. Microcystins
were detected in at least 65 percent of the samples, mostly in Lake Erie, Lake Ontario, and Lake
Champlain.
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A 2004 study of the Great Lakes found high levels of cyanotoxins during the month of August
(Makarewicz et al. 2006). Microcystin-LR was analyzed by protein phosphatase inhibition assay (limit
of detection of 0.003 (J,g/L) and was detected at levels of 0.008 [j,g/L in the nearshore and 0.076 [j,g/L in
the bays and rivers. This study reported higher levels of microcystin-LR (1.6 to 10.7 (J,g/L) in smaller
lakes in the Lake Ontario watershed.
In 2005, Washington State Department of Ecology developed the Ecology Freshwater Algae Program to
focus on the monitoring and management of cyanobacteria in Washington lakes, ponds, and streams
(WSDE 2012). Microcystin levels ranged from the detection limit (0.05 (J,g/L) to 4,620 [j,g/L in 2008, to
18,700 ng/L in 2009, to 853 ^g/L in 2010, and to 26,400 ^g/L in 2011 (Hamel 2009, 2011, 2012).
In 2006, the U.S. Geological Survey (USGS) conducted a study of 23 lakes in the midwestern United
States in which total cyanobacterial blooms were sampled to determine the co-occurrence of
cyanotoxins in cyanobacterial blooms (Graham et al. 2010). This study reported that microcystins were
detected in 91 percent of the lakes sampled with 17 percent of microcystin-positive samples exceeding
20 (J,g/L. The researchers also found that cylindrospermopsin co-occurred with microcystins in
nine percent of samples (Graham et al. 2010). Mixtures of all the microcystin congeners measured (-LA,
-LF, -LR, -LW, -LY, -RR, and -YR) were common. Microcystin-LR and -RR were the dominant
congeners detected with mean concentrations of 104 and 910 (J,g/L, respectively.
The Ohio EPA (2012) has been monitoring inland lakes since 2007 for cyanotoxins. Of the Ohio lakes
sampled during the 2007 NLA, 36 percent had detectable levels of microcystins. In 2010, the Ohio EPA
sampled Grand Lake St. Marys for cylindrospermopsin, microcystins, and other cyanotoxins.
Microcystin levels ranged from below the detection limit (<0.15 (J,g/L) to more than 2,000 (J,g/L.
Follow-up samples taken in 2011 for microcystins indicated concentrations exceeded 50 [j,g/L in August.
During the same month, sampling in Lake Erie found microcystin levels exceeding 100 (J,g/L.
The USGS monitored Lake Houston in Texas from 2006 to 2008 and found microcystins in 16 percent
of samples and at concentrations less than or equal to 0.2 [j,g/L (Beussink and Graham 2011). The USGS
also did a study in the Upper Klamath Lake in Oregon in 2007 and detected total microcystin
concentrations between 1 [j,g/L and 17 [j,g/L (VanderKooi et al. 2010). In 2011, the USGS conducted a
study on the upstream reservoirs of the Kansas River to characterize the transport of cyanobacteria and
associated compounds (Graham et al. 2012). Concentrations of total microcystins were low in the
majority of the tributaries with the exception of Milford Lake, which had higher total microcystin
concentrations, some exceeding the Kansas recreational guideline level of 20 (J,g/L. Upstream from
Milford Lake, a cyanobacterial bloom was observed with a total microcystin concentration of
150,000 (J,g/L. When sampled a week later, total microcystin concentrations were less than 1 (J,g/L. The
study authors indicated that this might be due to dispersion of microcystins through the water column or
to other areas, or by degradation of microcystins via abiotic and biological processes. Samples taken
during the same time from outflow waters contained total microcystin concentrations of 6.2 (J,g/L.
In 2008, the National Oceanic and Atmospheric Administration (NOAA) began monitoring for total
cyanobacterial blooms in Lake Erie using high temporal resolution satellite imagery. Using the Great
Lakes Coastal Forecast System, forecasts of bloom transport are created to estimate the trajectory of the
bloom, which are distributed as bulletins to local managers, health departments, researchers, and other
stakeholders. To evaluate bloom toxicity, the Great Lakes Environmental Research Laboratory collected
samples at six to eight stations each week for 24 weeks, measuring cyanotoxin concentrations as well as
chlorophyll biomass and an additional 18 parameters (e.g., nutrients) to improve future forecasts of these
blooms. Microcystins were separated into particulate (cell-bound) and dissolved (extracellular) phases
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(Graham and Jones 2007; Zastepa et al. 2014). In 2014, particulate microcystin concentrations ranged
from below detection to 36.7 (J,g/L. Samples taken in 2015 and 2016 showed particulate microcystin
concentration ranges from below detection to 9.19 [j,g/L and from below detection to 21.26 (J,g/L,
respectively. Particulate microcystin concentrations peaked in August 2014 at all sites. Dissolved
microcystin concentrations were also collected at each site in 2014 from September until the end of the
sampling period in November, as well as during the field sampling seasons in 2015 and 2016. During
the final months of sampling in 2014 (October to November), dissolved microcystin concentrations were
detected with peak concentrations of 0.8 [j,g/L (mean: 0.28 +/- 0.2 (J,g/L) whereas particulate microcystin
concentrations were below detection limits on many dates, indicating that a majority of the microcystins
(mean: 72 percent +/- 37 percent) were in the dissolved form, as the bloom declined in intensity.
Measured dissolved microcystin concentrations in the following two years ranged from levels below
detection to peaks of 0.69 [j,g/L in September 2015 and 1.76 [j,g/L in July 2016 (NOAA 2014).
A 2014 survey of southeastern U.S. streams detected microcystins in 39 percent of the samples (29 of 75
sites) (Loftin et al. 2016a). The stream sample concentrations ranged from the minimum reporting limit
of 0.1 [j,g/L to 3.2 (J,g/L. In some cases, the source of the cyanobacteria in flowing water bodies was
traced to an upstream water body such as a lake or reservoir.
From August to October 2015, a bloom identified as Microcystis aeruginosa occurred on the Ohio River
(ORSANCO 2017). Patches of the bloom covered 636 miles of the river and peaked in late September.
The Ohio River Valley Water Sanitation Commission (ORSANCO) collected over 150 river samples,
which were analyzed for microcystins. Of the samples collected by ORSANCO, 15 (10 percent) were
greater than 6 (J,g/L. The highest microcystin concentration was 1900 [j,g/L from a sample collected at
river mile 468.8 (Cincinnati, Ohio). No toxins were detected in finished drinking water (tested by
utilities and state agencies). Ohio, West Virginia, Kentucky, and Indiana issued recreation notices for the
Ohio River as the bloom extended into their areas. Illinois issued a precautionary statement concerning
recreation in the river due to concern that the bloom would reach their border. These recreation
advisories were lifted after the bloom ended (ORSANCO 2017).
From July 14 to September 14, 2016, an extensive cyanobacterial bloom covering 100 square miles
occurred in Utah Lake, Jordan River, and nearby canals. Microcystin-LR concentrations ranged from
below the detection limit to 0.23 (J,g/L, and the highest total microcystin concentration reported was
176 [j,g/L (Utah Department of Environmental Quality 2016). Both maximum values were from samples
collected at the surface near an accumulation of cyanobacteria. Cyanobacteria composition observed
during the 2016 bloom varied in both time and space, but was primarily dominated by Aphanizomenon
or Dolichospermum. Other taxa including Geitlerinema, Pseudanabaena, and Phormidium were also
observed in significant densities in a few samples (Utah Department of Environmental Quality 2016).
Lake Okeechobee, located north of the Everglades, is the largest freshwater lake in Florida. It is subject
to agricultural runoff from adjacent cattle farms and sugar cane fields, which contribute to the formation
of massive cyanobacterial blooms (Parker 2016). Water may be pumped out of the lake to the coast
through the St. Lucie River and the Caloosahatchee River to prevent the lake level from rising too high
after periods of heavy rain (Parker 2016). In July 2016, a 239-square mile cyanobacterial bloom in Lake
Okeechobee was discharged and flowed through canals, rivers, and estuaries to the ocean. As a result of
the microcystin levels in the river and at the coast, and the visible cyanobacterial scum in the lake and
river, a state of emergency was declared in the counties of Martin, St. Lucie, Palm Beach, and Lee. From
May 4 to August 4, 2016, the Florida Department of Environmental Protection took approximately 200
water samples from the St. Lucie River and estuary, Caloosahatchee River and estuary, Lake
Okeechobee, Indian River Lagoon, and other nearshore marine locations (Florida Department of
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Environmental Protection 2016). The microcystin concentrations in freshwater were reported in Lake
Okeechobee (from not detected to 382.3 (J,g/L). Elevated levels were also reported in the St. Lucie River
and the St. Lucie Canal (from not detected to 80.3 (J,g/L). Among the cyanobacteria species identified
were Microcystis aeruginosa, Scrippsiella trochoidea, Planktolyngbya limnetica, Dolichospermum
circinalis, and Plectonemawollei (Florida Department of Environmental Protection 2016).
3.2.2.2 Cylindrospermopsin
In general, fewer surface water occurrence data were available for cylindrospermopsin compared with
microcystins. During blooms, testing for microcystins is much more common than is testing for
cylindrospermopsin.
In a 1999 study, Cylindrospermopsis was detected in 40 percent of 167 water samples taken from 87
water bodies in Florida (Burns 2008). The actual cylindrospermopsin concentrations were not reported,
but all samples containing the organism Cylindrospermopsis were positive for the toxin
cylindrospermopsin.
In 2005, the U.S. Army Corps of Engineers detected cylindrospermopsin at a maximum concentration of
1.6 [j,g/L in lake water samples from Oklahoma (Lynch and Clyde 2009).
The USGS detected cylindrospermopsin in nine percent of blooms sampled during a 2006 USGS survey
of 23 lakes in the midwestern United States (Graham et al. 2010). The low concentrations of
cylindrospermopsin detected (0.12 to 0.14 (J,g/L) in the study occurred in bloom communities dominated
by the genera Aphanizomenon or Anabaena and Microcystis.
The USGS analyzed the stored samples collected during the 2007 EPA NLA (U.S. EPA 2009) and
detected cylindrospermopsin in four percent of samples, with a mean concentration 0.56 [j,g/L and a
range from the limit of detection, 0.01 (J,g/L, to a maximum of 4.4 [j,g/L (Loftin et al. 2016b). Potential
cylindrospermopsin-producing species (Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, and
Raphidiopsis) occurred in 67 percent of samples (Loftin et al. 2016b). Cylindrospermopsins occurred
most frequently in the midwestern and south-central United States and parts of Florida.
In Grand Lake in St. Marys, Ohio, cylindrospermopsin concentrations as high as 9 [j,g/L were reported in
2010 (Ohio EPA 2012).
3.2.3 Estuarine and Marine Waters
In Japan, the Isahaya Reservoir discharges water into Isahaya Bay. The reservoir experiences algal
blooms seasonally, with species including nontoxic cyanobacteria as well a microcystin-producing
Microcystis aeruginosa (Umehara et al. 2012). Water from the reservoir is discharged to the bay after
rainfall events, even during periods of Microcystis aeruginosa blooms. Between November 2008 and
November 2009, Umehara et al. (2012) estimated that 64.5 kilograms (kg) of microcystins were
discharged to the bay, of which only 0.7 kg deposited on the floor. The authors speculated that because
the majority of microcystins remain in the water, it is likely that they are washed out to other coastal
areas with strong tides (Umehara et al. 2012).
In 2007, Miller et al. (2010) confirmed the presence of Microcystis and microcystins in Lake Pinto's
downstream tributaries within 1 kilometer (km) of Monterey Bay in California after a large
cyanobacterial bloom in the lake, and detected microcystins in nearshore marine waters following the
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rainy season. The same researchers observed sea otters dying from consuming microcystin-contaminated
clams, mussels, and oysters near ocean outflows of freshwater systems (Miller et al. 2010). A follow-up
study was designed by Gibble and Kudela (2014) to identify the potential pathways leading to
microcystin contamination in coastal ecosystems in and around Monterey Bay. They surveyed 21 sites at
the land-sea interface in 2010-2011 followed by a survey of four watersheds in 2011-2013. In the first
year of a three-year study, microcystins were detected in 15 of 21 freshwater, estuarine, and marine
locations. In the two subsequent years, monitoring focused on four major watersheds that feed into
Monterey Bay. The authors observed high microcystin concentrations in both autumn and spring seasons
and concluded that microcystins are likely present throughout the year and transfer to the coastal
environment, with the potential to be a persistent issue in the Monterey Bay area. The authors also
correlated anthropogenic nutrient loadings with microcystins. Concentrations ranged from undetectable
up to 20 ng/g resin, which translates to approximately 20 parts per billion (ppb) microcystins in the
water column.
Otten et al. (2015) used microbial source tracking techniques to trace the source of a toxic Microcystis
bloom in the Klamath River in Oregon to a single upstream reservoir. The use of assays targeting gene
sequences for phycocyanin and microcystin synthase allowed the quantification of total and toxigenic
Microcystis. Their results showed that large quantities of cyanobacterial cells could withstand passage
through hydroelectric installations and transport over 300 km. Microcystin concentrations ranged from
165 [j,g/L in a reservoir upstream to 3.6 [j,g/L within the lower estuary less than 1 km from the Pacific
Ocean (Otten et al. 2015).
The large cyanobacterial bloom in Lake Okeechobee, Florida, in 2016 (described above) flowed
downstream and impacted estuarine and marine waters, resulting in beach closures along the Atlantic
(Chaney 2016; Florida Department of Environmental Protection 2016). From May 4 to August 4, 2016,
the Florida Department of Environmental Protection sampled freshwater, estuarine waters, and
nearshore marine waters. The highest concentration reported (414.3 (J,g/L) was collected in Martin
County at Bathtub Reef, a beach along the Atlantic Ocean. Sampling efforts in estuarine water, for
example at a marina in the St. Lucie River, reported a concentration of 78 (J,g/L. The majority of marine
waters sampled had low levels of microcystins (not detected or approximately 1 (J,g/L).
3.2.4 Other Sources of Microcystins and Cylindrospermopsin
Cyanotoxins have the potential to occur in drinking water, ground water, fish, shellfish, dietary
supplements, air, soil, and sediments. These potential sources of cyanotoxins are discussed briefly in
section 7.6. Exposure to these toxins in finished drinking water is also characterized in the Drinking
Water Health Advisories (U.S. EPA 2015a, 2015b).
3.3 Environmental Fate
Different physical and chemical processes are involved in the persistence, breakdown, and movement of
microcystins and cylindrospermopsin in aquatic systems as described below.
3.3.1 Mobility
Microcystins may adsorb onto naturally suspended solids and dried crusts of cyanobacteria. They can
precipitate out of the water column and reside in sediments for months (Falconer 1998; Han et al. 2012).
A study conducted by the USGS and the University of Central Florida determined that microcystin-LR
and cylindrospermopsin did not sorb in sandy aquifers and were transported along with ground water
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(O'Reilly et al. 2011). The authors suggested that the removal of microcystin-LR was due to
biodegradation.
Cyanotoxins that are produced by cyanobacteria growing in freshwaters can enter estuarine and marine
waters as waters containing the toxins flow downstream. Studies have demonstrated that toxigenic
cyanobacteria can travel long distances in freshwater and can reach estuarine and marine waters from
coastal lakes, reservoirs, and rivers (Preece et al. 2017).
In sediments, cylindrospermopsin exhibits some adsorption to organic carbon, with little adsorption
observed on sandy and silt sediments (Klitzke et al. 2011). The low adsorption of cylindrospermopsin
reduces its residence time in sediments, thus reducing the opportunity for microbial degradation.
3.3.2 Persistence
3.3.2.1 Microcystins
Microcystins are relatively stable and resistant to chemical hydrolysis or oxidation at or near neutral pH.
Elevated or low pH or temperatures above 30°C may cause slow hydrolysis. Microcystins have been
observed to persist for 21 days to two to three months in solution and up to six months in dry scum
(Funari and Testai 2008; Rapala et al. 2006). Environmental conditions such as temperature, pH,
presence of light, salinity, and presence of certain aquatic bacteria can influence the rate of microcystin
degradation (Schmidt et al. 2014). Microcystins can persist even after a cyanobacterial bloom is no
longer visible (Lahti et al. 1997b; Zastepa et al. 2014). In a study by Zastepa et al. (2014), dissolved
microcystin-LA was present at a concentration of 20 [j,g/L or greater for 9.5 weeks even though the
Microcystis bloom was not visible after five weeks.
In the presence of full sunlight, microcystins undergo photochemical breakdown, but this varies by
microcystin congener (Chorus et al. 2000; WHO 1999). Zastepa et al. (2014) suggest that microcystin-
LA degrades at a slower rate than microcystin-LR, -RR, and -YR congeners. The presence of water-
soluble cyanobacterial cell pigments, in particular phycobiliproteins, enhances this breakdown.
Breakdown can occur in as few as two weeks to longer than six weeks, depending on the concentration
of pigment and the intensity of the light (Tsuji et al. 1994, 1995).
Several other factors, including pH, wavelength of light (Schmidt et al. 2014), and whether microcystins
are dissolved or present in particulate matter (Lahti et al. 1997b) can affect the rate of transformation or
photodegradation. According to Tsuji et al. (1994, 1995), microcystin-LR was photodegraded with a
half-life of about five days in the presence of 5 mg/L of extractable cyanobacterial pigment. Humic
substances can act as photosensitizers and can increase the rate of microcystins breakdown in sunlight.
Others have found that high concentrations of humic acids can slow the rate of microcystins
transformation by sunlight (Schmidt et al. 2014). In deeper or turbid water, the breakdown rate is slower.
Welker and Steinberg (2000) estimated the maximum rate of microcystin-LR degradation in the
presence of humic substance photosensitizers. Extrapolating results from their small experimental tubes
to a water column of 1 meter, Schmidt et al. (2014) estimated the half-life of microcystin-LR to be 90 to
120 days per meter of water depth in surface waters. The researchers demonstrated that the wavelength
of light can also affect degradation rates; complete microcystins degradation was observed within one
hour when exposed to 254-nm light and within five days using 365-nm light. According to Lahti et al.
(1997b), microcystin-LR follows first-order decay kinetics, with a decimal reduction time of 30 days for
dissolved microcystins compared with 15 days for microcystins found in particulate matter. Zastepa et
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al. (2014) also found that dissolved microcystin-LA persists longer than microcystin-LA in particulates,
with in situ half-lives of 15.8 days and 6.5 days, respectively.
Microcystins are susceptible to biodegradation by aquatic bacteria found naturally in surface waters
(Jones et al. 1994). Bacteria isolates of Arthrobacter, Brevibacterium, Rhodococcus, Paucibacter, and
various strains of the genus Sphingomonas (Pseudomonas) have been reported to be capable of
degrading microcystin-LR (de la Cruz et al. 2011; Han et al. 2012). These degradative bacteria have also
been found in sewage effluent (Lam et al. 1995), lake water (Cousins et al. 1996; Jones et al. 1994; Lahti
et al. 1997b), and lake sediment (Lahti et al. 1997a; Rapala et al. 1994; U.S. EPA 2015a). Lam et al.
(1995)	reported that the biotransformation of microcystin-LR followed a first-order decay with a half-
life of 0.2 to 3.6 days. In a study conducted by Jones et al. (1994) with microcystin-LR in different
natural surface waters, microcystin-LR persisted for three days to three weeks; however, more than 95
percent loss occurred within three to four days. A study by Christoffersen et al. (2002) measured half-
lives in the laboratory and in the field of approximately one day, driven largely by bacterial aerobic
metabolism. These researchers found that approximately 90 percent of the initial amount of microcystins
disappeared from the water phase within five days, irrespective of the starting concentration. Other
researchers (Edwards et al. 2008) have reported half-lives of four to 14 days, with longer half-lives
associated with a flowing stream and shorter half-lives associated with lakes. Microcystin-LR
degradation by Sphingopyxis species was observed with an optimal degradation rate at pH values
between 6.5 and 8.5 (Schmidt et al. 2014). Several studies have demonstrated bacterial degradation of
microcystin-LR, but other congeners, such as microcystin-LF or -LA, were not significantly degraded
(Zastepa et al. 2014). During periods of high toxigenic cyanobacterial densities, the composition of other
bacteria in the community may shift in response. In a study of the San Juan reservoir in Spain, Lezcano
et al. (2017) found that several classes, orders, and families of known biodegrading bacteria, such as the
Spirobacillales order, increased by more than a factor of 1.5 during the peak of a cyanobacterial bloom.
The increase in relative abundance suggests that these biodegraders may play a role in microcystins
degradation in the environment. Although microcystin-degrading bacteria might be present, initial
degradation rates could be slow because the bacteria need time to begin using the toxins as carbon or
energy sources (Hyenstrand et al. 2003). Microcystins can accumulate in the water column if these
biodegrading bacteria are not present at the time of a toxic bloom (Schmidt et al. 2014). Cousins et al.
(1996)	demonstrated that microcystin experimentally added into reservoir water has a half-life of three
to four days, whereas microcystin spiked into the same matrix but sterilized (so biodegrading bacteria
are dead) had no significant change in the 12 days of the experiment. The authors concluded that
biodegradation was the primary mechanism of microcystin reductions in the raw reservoir water.
Where rivers discharge to the ocean, freshwater cyanobacteria, cyanotoxins, or both can enter the marine
environment (Andersen et al. 1993; Miller et al. 2010). Miller et al. (2010) confirmed the transfer of
freshwater microcystins to the marine environment; the researchers found that after introducing
Microcystis cyanobacteria to a saline environment, cyanobacteria can survive for 48 hours before lysing
and releasing microcystins. Microcystin concentrations in these experiments decreased in the range of
44 to 71 percent after one hour in the saline environment, but continued to be detected in the seawater
for at least 21 days, based on a detection limit of 0.02 [j,g/L (Miller et al. 2010).
3.3.2.2 Cylindrospermopsin
Cylindrospermopsin is relatively stable in the dark and at temperatures from 4°C to 50°C for up to five
weeks (ILS 2000). Cylindrospermopsin is also resistant to changes in pH and remains stable for up to
eight weeks at pH 4, 7, and 10. In the absence of cyanobacterial cell pigments, cylindrospermopsin tends
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to be relatively stable in sunlight, with a half-life of 11 to 15 days in surface waters (Funari and Testai
2008).
Like microcystins, degradation of cylindrospermopsin increases in the presence of cell pigments such as
chlorophyll a and phycocyanin, a blue photosynthetic pigment found in cyanobacteria. When exposed to
both sunlight and cell pigments, cylindrospermopsin breaks down rapidly—more than 90 percent within
two to three days (Chi swell et al. 1999).
Bacteria have been shown to decompose cylindrospermopsin in laboratory studies; the biodegradation is
influenced by the cyanotoxin concentration, temperature, and pH. Mohamed and Alamri (2012) reported
that Bacillus bacteria degraded cylindrospermopsin and that degradation occurred in six days at the
highest toxin concentration (300 (J,g/L) and in seven or eight days at lower concentrations (10 and
100 (J,g/L, respectively). The biodegradation rate was also reported to depend on temperature and pH,
with the highest rates occurring in warm waters (25 and 30°C) and neutral to slightly alkaline conditions
(pH 7 and 8). Klitzke and Fastner (2012) confirmed the observations of Mohamed and Alamri (2012),
noting that a decrease in temperature from 20 to 10°C slowed down degradation by a factor of 10. They
also found that degradation slowed significantly under anaerobic conditions, with half-lives of 2.4 days
under aerobic conditions and 23.6 days under anaerobic conditions.
3.4 Toxicokinetics
Limited data are available regarding the toxicokinetics of microcystins in environmental exposure
conditions (U.S. EPA 2015d). Available intestinal data indicate that the organic anion transporting
polypeptide (OATP) family transporters facilitate the absorption of microcystins from the intestinal tract
into liver, brain, and other tissues, as well as their export out of organs and tissues (Cheng et al. 2005;
Fischer et al. 2005; Svoboda et al. 2011). However, bile acids and other physiologically relevant
substrates compete with microcystins for transporter uptake by the liver (Thompson and Pace 1992);
reduction or elimination of liver toxicity has been observed during in vivo or in vitro exposures when
microcystin uptake by OATP transporters is limited or inhibited (Hermansky et al. 1990a, 1990b;
Runnegar et al. 1995; Runnegar and Falconer 1982; Runnegar et al. 1981). Both in vivo and in vitro
studies have shown biliary excretion of microcystins (Falconer et al. 1986; Pace et al. 1991; Robinson et
al. 1991), possibly via conjugation with cysteine and glutathione (Kondo et al. 1996). Additional details
of microcystin toxicokinetics can be found in the EPA's Drinking Water Health Advisory and HESD for
microcystins (U.S. EPA 2015a, 2015d).
Limited toxicokinetic data for cylindrospermopsin are available and are derived from mice
intraperitoneal studies and in vivo studies that do not necessarily reflect environmental exposure
conditions (U.S. EPA 2015c; Pichardo et al. 2017). Cylindrospermopsin is absorbed from the GI tract
(Humpage and Falconer 2003; Shaw et al. 2001; Shaw et al. 2000) and is distributed primarily to the
liver but also to the kidneys and spleen (Norris et al. 2001). The metabolism and toxicity of
cylindrospermopsin is mediated by hepatic cytochrome P450 (CYP450) enzymes, and the periacinar
region of the liver appears to be the main target of toxicity where cylindrospermopsin and its metabolites
bind to proteins (Norris et al. 2001; Runnegar et al. 1995; Shaw et al. 2001; Shaw et al. 2000).
Elimination of cylindrospermopsin was continuous over a monitoring period of 24 hours, with a large
mean total recovery primarily from urine, and to a smaller extent, feces, after 24 hours (Norris et al.
2001). Additional details of cylindrospermopsin toxicokinetics can be found in the EPA's Drinking
Water Health Advisory and HESD for cylindrospermopsin (U.S. EPA 2015b, 2015c).
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4.0 PROBLEM FORMULATION
4.1 Conceptual Model
This conceptual model provides useful information that characterizes and communicates the potential
health risks related to exposure to microcystins and cylindrospermopsin in recreational waters. The
model depicts the sources of the cyanotoxins in these waters, the recreational routes of exposure for
sensitive biological receptors of concern, and the potential assessment endpoints (e.g., effects such as
kidney and liver toxicity) (Figure 4-1).
Figure 4-1. Conceptual Model of Exposure Pathways to the Cyanotoxins, Microcystins and
Cylindrospermopsin, and Cyanobacteria in Surface Waters While Recreating
4.1.1 Conceptual Model Diagram for Recreational Exposure
The conceptual model is intended to explore potential links of exposure to a contaminant or stressor with
the adverse effects and toxicological endpoints important for management goals, including the
development of recreational AWQC. Boxes that are shaded darker green indicate pathways that the EPA
considered quantitatively in estimating the advisory level, whereas boxes shaded lighter green indicate
data were sufficient for qualitative use and the white boxes did not have sufficient data for the EPA to
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evaluate quantitatively or qualitatively. The solid lines are for the cyanotoxins and the dotted lines are
for the cyanobacterial cells.
4.1.2 Factors Considered in the Conceptual Model for Microcystins and Cylindrospermopsin
Stressors
The stressors are microcystins and cylindrospermopsin concentrations in water. These toxins can be
produced by cyanobacteria occurring in freshwater. The EPA concluded that although statistically
significant associations with adverse health effects occur across a wide range of cyanobacterial cell
densities, criteria cannot be derived based on cyanobacterial cell density at this time. Effects related to
cyanobacterial cells are discussed in section 7.5.1 and Appendix D.
Sources
Cyanobacteria occur naturally in surface waters, such as lakes, ponds, rivers, estuaries, bays, lagoons,
and oceans in or surrounding the United States. Some genera of the cyanobacteria, including
Microcystis, Cylindrospermopsis, Anabaena, Planktothrix, and Nostoc, can produce the cyanotoxins
microcystins and cylindrospermopsin. This assessment focuses on cyanotoxins produced by these
cyanobacteria in freshwater. These toxins have the potential to affect downstream waters, including
coastal areas where surface water containing the toxins discharges into estuarine and marine waters.
Routes of Exposure
Exposure to cyanotoxins from recreational water sources can occur via oral exposure (incidental
ingestion while recreating); dermal exposure (contact of exposed parts of the body with water containing
cyanotoxins during recreational activities such as swimming, wading, or water skiing); and inhalation
exposure to contaminated aerosols (while recreating). The route of exposure considered quantitatively in
this assessment is the oral exposure to microcystins and cylindrospermopsin via incidental ingestion
while swimming. Inhalation can occur from exposures from personal watercraft and boat spray. Dermal
exposure can occur through recreational water contact; however, significant dermal absorption of
microcystins and cylindrospermopsin is not expected due to the large size and charged nature of these
molecules and the lack of dermal receptor sites capable of uptake (Butler et al. 2012; U.S. EPA 2004;
U.S. EPA 2007). Sufficient data to quantify toxicity via the inhalation and dermal exposure routes were
not available. The dermal and inhalation routes of exposure are discussed further in the Effects
Characterization section (7.4).
Receptors
Anyone who recreates in a water body where cyanotoxins are present could be exposed to cyanotoxins
through ingestion, dermal contact, and inhalation of aerosols while recreating in contaminated surface
waters. Recreating children can be at greater risk from exposure to microcystins or cylindrospermopsin
because they have smaller body mass compared to adults, they spend more time in contact with the
water compared to adults, and they incidentally ingest more water than adults while recreating.
Therefore, the EPA has determined that childhood is the most vulnerable lifestage due to potential
increased exposure while recreating when compared with adults. The EPA evaluates and discusses
differences between lifestages in the Effects Characterization section (7.3).
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While there are examples in the literature and reports of animal poisonings and death due to exposure of
cyanotoxins, values protective of animals such as dogs and livestock are not generated in this document.
However, section 7.8 discusses some animal-specific issues, including a summary of guidelines that
several states have developed for animals.
Endpoints
Available microcystin toxicity data indicate that the primary target organ for microcystins is the liver as
described in the EPA's HESD for microcystins (U.S. EPA 2015d).
Available cylindrospermopsin toxicity data are described in the EPA's HESD for cylindrospermopsin
(U.S. EPA 2015c). For cylindrospermopsin, the EPA selected kidney effects as the endpoint on which to
quantify the measure of effect. However, in both the critical study and the supporting studies there is
evidence that cylindrospermopsin can also alter the shape of red blood cells.
Clinical, epidemiological, and outbreak study results (see Appendix D) suggest a link between an
increase in adverse inflammatory symptoms among recreators and elevated cyanobacterial cell densities.
However, there is considerable uncertainty and variability associated with the epidemiological results,
which did not identify consistent effects at similar cyanobacterial densities. Specifically, significant
associations occur across a wide range of cell densities; associations vary with different self-reported
health endpoints or combined symptom categories. Potential inflammatory health effects related to
exposure to total cyanobacterial cells are described in the Effects Characterization section (7.5.1) and in
Appendix D, both of which include a discussion of the uncertainties related to associations with
cyanobacterial cells.
4.2 Analysis Plan
The EPA's 2000 Methodology for Deriving Ambient Water Quality Criteria for the Protection of Human
Health (2000 Human Health Methodology) outlines the Agency's process for deriving AWQC and
guides the development of these recreational criteria and swimming advisory recommendations (U.S.
EPA 2000).
The 2000 Human Health Methodology includes identifying the population subgroup that should be
protected and evaluating cancer and non-cancer endpoints, measures of effect, measures of exposure,
and relative source contribution (RSC). In this analysis plan, the EPA describes: (1) the RfD previously
derived for microcystins and cylindrospermopsin (measure of effect); (2) the calculation for the
recreational criteria; (3) incidental ingestion exposure in terms of volume ingested, duration of exposure,
and body weight (measure of exposure) described in the EPA's Exposure Factors Handbook (EFH) and
data reported in the peer-reviewed scientific literature; and (4) discusses the RSC. These criteria focus
on human exposure as a result of primary contact recreation activities, such as swimming, during which
immersion and incidental ingestion of ambient water are likely.
The EPA's HESD for microcystins and HESD for cylindrospermopsin (U.S. EPA 2015c, 2015d)
provide the health effects basis for the development of the Drinking Water Health Advisories for
microcystins and cylindrospermopsin (U.S. EPA 2015a, 2015b), including the basis for estimating the
point of departure. To develop its HESDs for microcystins and cylindrospermopsin, the EPA assembled
available information on toxicokinetics, acute, short-term, subchronic, and chronic toxicity along with
developmental and reproductive toxicity, neurotoxicity, immunotoxicity, genotoxicity, and cancer in
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humans and animals. For detailed descriptions of the literature search strategies, see the EPA's HESDs
for microcystins and cylindrospermopsin (U.S. EPA 2015c, 2015d).
The EPA's HESDs were subject to rigorous internal and external peer review before being finalized in
2015. The information evaluated for these documents also supports the development of the recreational
criteria and swimming advisories for microcystins and cylindrospermopsin, which evaluate exposure via
recreational water ingestion. The EPA conducted supplemental literature searches to capture new
references, including effects related to recreational exposure to cells. For detailed information on the
search terms, see Appendix C.
4.2.1 Approach for Recreational AWQC and Swimming Advisory Derivation
The recreational AWQC and swimming advisory recommendations for microcystins and
cylindrospermopsin are calculated as described in the 2000 Human Health Methodology and presented
in the equation below:
BW
Recreational AWQC (|_ig/L) = RfD X ———
1R
Where:
RfD = reference dose (ng/kg body weight/day)
BW = mean body weight (kg)
IR = ingestion rate (L/day) (discussed in section 4.2.3.1)
4.2.1.1 Magnitude, Duration, and Frequency
Recreational criteria, like other 304(a) criteria, consist of a magnitude, duration, and frequency.
Magnitude is the numeric expression of the maximum amount of the contaminant that may be present in
a water body that supports the designated use. Duration is the period over which the magnitude is
calculated. Frequency of excursion describes the number of times the contaminant may be present above
the magnitude over the specified period (duration). A criterion is derived such that the combination of
magnitude, duration, and frequency protect the designated use (e.g., primary contact recreation).
4.2.2 Measures of Effect
The EPA's HESDs for microcystins and cylindrospermopsin (U.S. EPA 2015c, 2015d), provide the
health effects basis for development of an oral toxicity value or the RfD, including the selection of the
critical study and critical endpoints and application of uncertainty factors (UFs). In derivation of the
recreational criteria and swimming advisory recommendations, the EPA uses these toxicity values as the
measure of effect for oral exposure through incidental ingestion while recreating. The RfDs described in
the EPA's HESDs are based on short-term and subchronic studies and therefore are an estimate (with
uncertainties spanning perhaps an order of magnitude) of the daily exposure to the human population
(including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects
during a short-term exposure period.
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4.2.3 Measures of Exposure
The EPA selected incidental ingestion during primary contact activities (such as swimming) in
derivation of the recreational criteria and swimming advisories because data suggest that incidental
ingestion can be considered the highest potential exposure pathway for cyanotoxins while recreating.
Dorevitch et al. (2011) studied the volume of water ingested during a range of recreational activities in
the Chicago Area Waterway System (CAWS) and at a public outdoor swimming pool. Study
participants took part in one of the following activities on the CAWS: canoeing, fishing, kayaking,
motor boating, or rowing. In the swimming pool, participants took part in canoeing, fishing, kayaking,
swimming, or wading/splashing. The results indicate that the odds of ingesting a teaspoon or more of
water are significantly higher among swimmers than among those who just immersed their head in a
swimming pool or those who participated in the other, more limited contact activities on surface waters.
Therefore, the EPA determined that using a swimmer scenario for exposure as the basis for the criteria is
protective of these other aquatic activities.
Inhalation exposure occurs during swimming; however, data are not sufficient to quantify health effects
resulting from inhalation exposure to cyanotoxins at this time. See section 7.4.1 for a characterization of
potential effects from inhalation exposure.
Dermal exposure happens during swimming; however, significant dermal absorption of the toxins
microcystins and cylindrospermopsin is not expected due to the large size and charged nature of these
molecules (Butler et al. 2012; U.S. EPA 2004; U.S. EPA 2007). Because available data are not
sufficient, the EPA is not quantifying effects resulting from dermal exposure to cyanotoxins. See section
7.4.2 for a characterization of dermal exposure to these cyanotoxins.
Dermal exposure to cyanobacterial cells can also result in adverse health effects, such skin rashes, eye
irritation, and ear irritation. Because adequate effects data are not available, the EPA is not quantifying
effects resulting from exposure to cells at this time; effects are described qualitatively. Available
epidemiological study results do not provide consistent associations between cell densities and the
inflammatory health endpoints. See section 7.5.1 for a characterization of potential effects from
recreational exposure to cyanobacterial cells.
All recreational exposure studies that included both children and adults found that age tended to
influence incidental ingestion exposure while recreating. More specifically, children tend to ingest more
water and spend more time in the water compared with adults (Dufour et al. 2017; Dufour et al. 2006;
Schets et al. 2011; U.S. EPA 2011). Data supporting the selected exposure factors are described in the
sections that follow.
The measure of exposure is the 90th percentile of the daily incidental ingestion rate (volume of surface
water incidentally ingested per day) and body weight (kg). Both body weight and incidental ingestion
while recreating are parameters that vary with age. The EPA's 2000 Human Health Methodology (2000)
outlines EPA's process for deriving AWQC and guides the development of these recreational criteria
and swimming advisory recommendations.
4.2.3.1 Incidental Ingestion
To calculate the recreational incidental ingestion rate in units of volume per day, the EPA combined a
distribution of incidental ingestion volumes (volume per event normalized to volume per hour) and a
distribution of exposure durations (hours per day). The EPA uses the 90th percentile of the combined
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distribution of ingestion rate and exposure duration to represent incidental ingestion per day, consistent
with the EPA's Human Health Methodology (U.S. EPA 2000). Probability density plots of the combined
distributions are shown in Figure 4-2. The ingestion data demonstrate that the mean ingestion rate for
children six to 10 years is higher than for older children and adults. These data are discussed in the
following sections.
Figure 4-2. Combined Distributions for Age Groups
1
0.75
>¦
to
c
cu
O
>- 0.5
15
ro
_Q
o
Q_
0.25
0
0.001	0.1
Volume Ingested (L/d)
Ingestion Volume Studies
The EPA evaluated seven studies on ingestion and selected the dataset collected and analyzed by Dufour
et al. (2017) for development of these AWQC or swimming advisory recommendations. This study used
the same methodology as an earlier study (Dufour et al. 2006) but included 10 times more participants.
Both studies used cyanuric acid as an indicator of amount of pool water ingested while swimming in an
outdoor pool. Pool water samples were collected before the start of swimming activities, and
participants' urine was collected for 24 hours after the swimming event ended; pool water and urine
samples were analyzed for cyanuric acid. The dataset collected by Dufour et al. (2017) included age
information for each particpant ages six to 81 years, whereas the 2006 study classified individuals as
over or under 18 years old. Both studies did not include children younger than six years old. The 2017
6 to 10 years
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study recorded time spent in the water for each participant. The 2017 study results highlighted that
younger children tested ingested more than older children or adults. The EPA selected the Dufour et al.
(2017) dataset to calculate incidental ingestion volume because of the larger number of particpants, the
inclusion of additional age groups, and recording of the duration exposure of each participant. The raw
data collected and analyzed by Dufour et al. (2017) was provided by the study authors (U.S. EPA
2018a). The EPA adjusted (i.e., normalized) the volume ingested by each participant to one hour based
on the length of time that participant reported being in the water. The summary statistics the EPA
calculated using this dataset are shown in Appendix E (Table E-l). Figure 4-3 shows the raw data
density plots for the Appendix E Dufour data separately grouped as age groups six to 10, 11 to 17, and
18 years and over. The density plots show the volume of incidental ingestion (mL) per recreational event
on a log scale. To develop the distribution, each participant's volume ingested was adjusted to one hour
based on the length of time that participant reported being in the water. Incidental ingestion was
recorded for 66 individuals in the six- to 10-year category.
Figure 4-3. Incidential Ingestion for Age Groups Based on Appendix E Dufour Data
1.2-
0.9 -
>~
'1/1
c
QJ
Q
.-K °"6 "
15
ro
-Q
O
L_
Q.
0.3-
0.0 -
1	10	100
Volume Ingestion (mL/event)
Appendix F describes seven studies that reported incidental ingestion while recreating, but only three
others reported ingestion estimates for children (Dufour et al. 2006; Schets et al. 2011; Suppes et al.
2014). These other studies reported children's ingestion volumes similar to Dufour et al. (2017).
Although these other studies corroborate the Dufour et al. (2017) findings, they were not selected for
deriving the ingestion rate. Dufour et al. (2006) had fewer age groups (i.e., six to 17 and 18+ years),
smaller sample size, and did not record time spent in water for each participant, making it a less robust
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study than Dufour et al. (2017). Schets et al. (2011) collected data in the Netherlands, which may not be
representative of the United States due to different behavioral trends in the resident population,
including effects of temperature on recreating patterns. In addition, Schets et al. (2011) ingestion
volumes are based on self-reported estimates; parents estimated volumes for children five and younger.
Self- and parent-reported estimates are more uncertain than the methods used by Dufour et al. (2017).
Suppes et al. (2014) used video and urine analysis to estimate ingestion volume. In Suppes et al. (2014)
quantitative data were available for 35 participants, which is much lower than the sample size for
Dufour et al. (2017). In addition, Suppes et al. (2014) only reported two age groups, children (five to 17
years) and adults (18+ years), which does not allow for the finer discernment of exposure patterns that is
possible with the Appendix E and U.S. EPA (2018a) data.
Appendix F also describes the methodology used by the EPA's Office of Pesticide Programs (OPP) to
calculate exposures to pool chemicals during swimming to support registration decisions. The
Swimmers Exposure Assessment Model (SWIMODEL) (U.S. EPA 2003) uses incidental ingestion
values for children that are twice the values used for noncompetitive adult swimmers. The model
assumes an incidental ingestion rate of 0.050 L/hour for children ages seven to 10 years and 11 to 14
years while swimming noncompetitively. Incidental ingestion rates among adults while swimming
competitively and noncompetitively are 0.0125 L/hour and 0.025 L/hour, respectively.
Duration of Recreational Exposure
Duration of recreational exposure quantifies the length of time people might be exposed to cyanotoxins
during their primary contact recreational use. Duration is needed to convert recreational ingestion rates
in units of volume per hour to an amount incidentally ingested per day, which is the exposure parameter
needed to derive the recommended cyanotoxin values.
The EPA selected recreational exposure data from the EFH (U.S. EPA 2011) for the development of
these criteria/swimming advisories. The EPA's EFH (2011) lists time spent per 24 hours in an outdoor
spa or pool for different age groups. The data are based on analysis of the National Human Activity
Pattern Survey (U.S. EPA 1996). Figure 4-4 compares point estimates for the recreational duration data
for different age groups and shows that recreators ages five to 11 years (n = 15) tend to spend more time
in the water than other child age groups and adults. A duration was not provided for children younger
than age one year.
The EPA investigated available exposure parameters for children younger than six years old, but they
have large uncertainties given the lack of measured incidental ingestion data for this age group (see
section 7.3.2). See section 7.2 (Recreational Exposure Duration) for further discussion of the available
data for recreational exposure duration. The EPA used the distribution of exposure durations for children
ages five to 11 years (n = 15; units are hour/day) as described below to calculate incidental ingestion
per day.
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Figure 4-4. Direct Contact Recreational Exposure Duration by Age Group, Based on Table
16-20 in U.S. EPA (201 l)a
6.0
5.0
4.0
>
CD
o
I2 3.0
o
2.0
1.0
0.0
i
1-4 years
1



1






5-11 years
C Mean E Median H 90th percentile
a This figure shows a comparison of point estimates. The EPA used the whole distribution for ages five to 11 years in
deriving the AWQC and swimming advisory magnitudes.
Determination of Incidental Ingestion per Day
The incidental ingestion volume per day the EPA used to calculate the AWQC or swimming advisories
is the product of the distribution of children's incidental ingestion rate for children ages six to 10 years
(Appendix E; U.S. EPA 2018a) and the distribution of exposure durations for children ages five to 11
years (U.S. EPA 2011). The lifestage grouping for the duration data include children one year older and
one year younger than the lifestage group for the incidental ingestion data.
The individual ingestion rate data points (adjusted to L/hour) were used to calculate a mean and standard
deviation of the log-normal transformed dataset. This distribution was combined with the distribution of
hours of recreation per day (ages five to 11 years) from the 2011 EFH (Table 16-20 Time Spent
(minutes day) in Selected Outdoor Locations, Doers Only, At Home in the Outdoor Pool or Spa). The
mathematical relationship between the two variables and the daily incidental ingestion rate is shown in
this equation:
Ingestion Volume (L/hour) x Recreation Duration (hour/day)
= Daily Incidental Ingestion Rate (L/day)
The EPA used probabilistic (Monte Carlo) simulation to develop the combined distribution of these
variables as follows:
•	Estimated statistical distributions for hourly ingestion rate and recreation duration for different
age groups.
•	Sampled randomly one value from each of these distributions.
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• Multiplied the two sampled values.
•	Repeated a large number of times (i.e., 100,000 times) to populate the distribution for daily
ingestion rate (L/day) or the combined distribution.
•	Reported results as summary statistics of the combined distribution.
The distribution shape that best fit the datasets was log-normal for both ingestion volume and exposure
duration. Table 4-1 presents summary statistics for different age groups based on the combined
distribution analysis. As per the EPA's 2000 Human Health Methodology (U.S. EPA 2000), the 90th
percentile of exposure, represented by this combined distribution (0.21 L/day) was used as a point
estimate for deriving the AWQC or swimming advisories. Details and the R code for this analysis are
shown in Appendix E. Appendix E also includes the mean, median, and standard deviation for the
distributions for ages six to 10, 11 to 17, and 18 years and older.
Table 4-1. Results of the Combined Distribution Analysis
Age Group
Summary Statistics for Ingestion Rate (L/day)
Median
Mean
90th Percentile
6 to 10 years
0.063
0.094
0.21
11 to 17 years
0.038
0.058
0.13
18+ years
0.015
0.04
0.10
4.2.3.2 Body Weight
Table 8-1 in the EPA's EFH (U.S. EPA 2011) reported body weight statistics based on the National
Health and Nutrition Examination Survey, including for a range of age groups. The EPA selected
children aged six to 10 years because it reflected the age group with higher ingestion volumes
(Appendix E; U.S. EPA 2018a; U.S. EPA 2011) and exposure duration (U.S. EPA 2011).4 As per the
EPA's 2000 Human Health Methodology (U.S. EPA 2000), mean body weight (31.8 kg) was used for
deriving the AWQC or swimming advisories. Section 7.3.2 provides a discussion of younger children's
exposure factors.
4.2.4 Relative Source Contribution (RSC)
The RSC component of the AWQC calculation allows a percentage of the exposure to a contaminant to
include other potential exposure sources. The RSC describes the portion of the RfD available for
AWQC-related sources (U.S. EPA 2000); the remainder of the RfD is allocated to other sources of the
contaminant. The EPA focused on recreational exposures to microcystins and cylindrospermopsin in
ambient freshwaters. To derive recommendations protective of the recreational designated use, the EPA
assumes all cyanotoxin exposure is from incidental ingestion of water while recreating; therefore, no
RSC term is applied.
4 The age group six to 10 years includes 10-year-old children. The EPA's Exposure Factors Handbook labels this age group
as six to < 11 years.
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5.0 EFFECTS ASSESSMENT
The health effects studies summarized below for microcystins and cylindrospermopsin are described in
detail in the EPA's HESDs and Drinking Water Health Advisories for these two cyanotoxins (U.S. EPA
2015a, 2015b, 2015c, 2015d).
5.1 Hazard Identification
5.1.1 Noncancer Health Effects
5.1.1.1 Animal Toxicity Studies
Microcystins
The preponderance of animal toxicity data on the noncancer effects of microcystins is restricted to the
microcystin-LR congener. Available data on the RR, YR, and LA congeners do not provide dose-
response information sufficient for quantification. The EPA is using data on effects of microcystin-LR
to represent other microcystin congeners (U.S. EPA 2015d). Observed effects in animals exposed orally
or via intraperitoneal infusion to microcystin-LR include liver, reproductive, developmental, kidney, and
GI effects (Chernoff et al. 2002; Falconer et al. 1998; Fawell et al. 1999; Fitzgeorge et al. 1994; Guzman
and Solter 1999, 2002; Heinze 1999; Ito et al. 1997a, 1997b; Yoshida et al. 1997). Most oral and
injection studies in laboratory animals have demonstrated that the liver is a primary target organ for
microcystin toxicity. Liver effects, as well as kidney effects, have been reported in acute, short-term, and
subchronic oral studies in laboratory animals exposed to microcystin-LR, in addition to reproductive
effects following short-term and subchronic oral exposures. Studies evaluating the chronic toxicity of
microcystins have not shown clinical signs of toxicity and are limited by study design and by the lack of
quantitative data. For individual study details see the EPA's HESD for microcystins (U.S. EPA 2015d).
Available animal data on the acute oral toxicity of microcystin-LR provide evidence of hepatotoxicity.
Liver effects described in the above studies are summarized in Table 5-1. A single oral dose of 500 jag
microcystin-LR/kg resulted in diffuse hemorrhage in the liver of mice and rats; more pronounced liver
damage occurred at higher doses (Ito et al. 1997a; Fawell et al. 1999; Yoshida et al. 1997). Studies that
utilized parenteral administration of microcystin-LR show a steep dose-response with rapid onset of
liver damage.
The findings in acute and subchronic studies support the liver as a target organ for microcystin-LR
toxicity. The EPA identified a 28-day short-term study by Heinze (1999) as the critical study for
derivation of an RfD. Male hybrid rats (10/group) were administered microcystin-LR in drinking water
at doses of 0, 50, or 150 (J,g/kg body weight (Heinze 1999). Liver effects included increased liver weight,
and slight to moderate liver necrosis lesions with or without hemorrhages at the low dose and with dose-
related increases in necrotic severity. The necrosis was accompanied by changes in serum enzymes
indicative of liver damage. All rats in each dose group had liver necrosis. Data were not collected prior
to the end of the study so it is not known when during the 28-day study period these effects were
manifested.
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Table 5-1. Liver Effects in Animals Exposed to Microcystins in Selected Acute and Short-term
Studies as Discusssed in the EPA's Health Effects Support Document for the Cyanobacterial Toxin
Microcystins (U.S. EPA 2015d)
Species
Exposure
Route
Dosing Regimen
Micro-
cystin
Congener
Description of
Liver Effects
Study
Female
BALB/c mice
(n = 7)
Gavage
Single dose of 0, 8,000,
10,000, or 12,500 (ig/kg
Examination at 24 hours
after treatment
LR
Centrilobular
hemorrhage,
hepatocyte
degeneration
Yoshida et al.
(1997)
Male ICR
mice aged
(n = 29 age 32
weeks) and
young (n = 12
age 5 weeks)
Gavage
Single dose of 500 (ig/kg
Animals sacrificed at 2, 5,
and 19 hours after treatment
LR
Bleeding and
disappearance of
hepatocytes in the
whole liver or in
centrilobular region,
friable tissue,
necrosis, or
eosinophilic changes
in the centrilobular
region
Ito et al.
(1997a)
CR1:CD-
1(ICR)BR(VA
F plus) mice
and
CR1:CD(SD)B
R(VAF plus)
rats (5 males
and 5 females
per group)
Gavage
Single dose of 500, 1,500,
or 5,000 (ig/kg (no control)
Animals sacrificed at day 14
post treatment
LR
Darkly discolored
and distended livers;
moderate or marked
centrilobular
hemorrhage of liver;
diffuse hemorrhage
in the liver
Fawell et al.
(1999)
Male ICR
mice (n = 5 per
group)
Gavage
Repeated doses of 0, 4.6,
23, 46, 93, or 186 (ig/kg/day
for 7 days
Animals sacrificed at day 7
RR
Dose-dependent
increase in apoptosis
Huang et al.
(2011)
Male hybrid
rats (Fi
generation of
female
WELS/Fohm
x male BDIX)
(10 per group)
Drinking water
Repeated doses of 0, 50, or
150 (ig/kg/day for 28 days
LR
Hepatocyte
degeneration
hemorrhage, and
necrosis; increase in
periodic acid-Schiff-
positive substances
(indicating cell
damage), Kupffer cell
activation
Heinze
(1999)
Male Sprague-
Dawley rats
(3 per group)
Intraperitoneal
infusion
Repeated doses of 0, 16, 32,
or 48 (ig/kg/day for 28 days
LR
Fibrous tissue, cell
death, necrosis, lipid
vacuoles, Kupffer
cell activation (+2
and +3 severity
rating)
Guzman and
Solter (1999)
The liver effects in the Heinze (1999) study were supported by additional data from a study by Guzman
and Solter (1999). Rats exposed via intraperitoneal infusion displayed histological evidence of liver
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damage (i.e., inflammation, fibrous tissue, necrosis, and apoptosis). The study authors identified a no-
observed-adverse-effect-level (NOAEL) of 16 [j,g/kg/day and a lowest-observed-adverse-effect-level
(LOAEL) of 32 [j,g/kg/day. Microcystin-LR was delivered directly to the livers of the animals in the
study by implanted osmotic pumps and this may account for the liver effects observed at lower doses
compared to Heinze (1999). Guzman and Solter (1999) only included three rats per group exposed to
doses of 0, 16, 32, or 48 [j,g/kg/day of microcystin for 28 days, which is a limitation of the study design.
Although adverse liver effects were observed, the limited numbers of animals per dose group (n = 3) and
the exposure route, which bypassed intestinal barriers to absorption, resulted in greater uncertainty than
Heinze (1999). Thus, Guzman and Solter (1999) was not used to derive the RfD.
Some studies observed other kinds of effects following short-term or subchronic oral or intraperitoneal
exposures. These studies, including limitations, are discussed in the EPA's HESD for microcystins (U.S.
EPA 2015d). Potential effects included reproductive toxicity in males (Chen et al. 2011), maternal
mortality (Fawell et al. 1999; Chernoff et al. 2002), and fetal body weight changes (i.e., at 2,000 (J,g/kg,
administered orally during gestational days six to 15, at which significant maternal mortality was
observed) (Fawell et al. 1999). Chernoff et al. (2002) did not report adverse effects on fetal or pup
weights in two separate intraperitoneal studies.
Cylindrospermopsin
The available acute, short-term, and subchronic studies for cylindrospermopsin (Bazin et al. 2012;
Humpage and Falconer 2002; 2003; Reisner et al. 2004; Terao et al. 1994; Shaw et al. 2001) support the
liver and kidneys as the primary targets for cylindrospermopsin toxicity (summarized in Table 5-2), with
effects on red blood cells also evident. These effects were observed in mice given single or repeated
doses of purified cylindrospermopsin via oral administration or intraperitoneal injection (Bazin et al.
2012; Humpage and Falconer 2002, 2003; Reisner et al. 2004; Terao et al. 1994). The EPA did not find
health effects information for other cylindrospermopsin congeners or analogs.
No oral reproductive or developmental studies are available for cylindrospermopsin. Developmental
toxicity studies following intraperitoneal administration of cylindrospermopsin provide some evidence
for maternal toxicity and decreased postnatal pup survival and body weight (Chernoff et al. 2011;
Rogers et al. 2007). For individual study details, see the EPA's HESD for cylindrospermopsin (U.S.
EPA 2015c).
The RfD for cylindrospermopsin was derived from the 11-week critical study by Humpage and Falconer
(2002, 2003). This study was an 11-week study in mice, and the critical effect identified was kidney
toxicity. The short-term studies available for cylindrospermopsin (Shaw et al. 2001; Reisner et al. 2004),
were also evaluated and are considered supportive of the critical study; however, the EPA concluded
that they were not suitable for quantification based on limitations including the use of extract, lack of
adequate numbers of animals, monitored endpoints, the limited number of doses tested and endpoints
monitored.
Humpage and Falconer (2002, 2003) identified a NOAEL of 30 [j,g/kg/day and a LOAEL of
60 [j,g/kg/day for increases in relative kidney weight in mice treated with purified cylindrospermopsin by
gavage for 11 weeks. There were indications of reduced renal function effects, decreased urinary
protein, and red blood cell effects (including increased bilirubin, spleen weight and polychromasia,
indicative of hemolysis) at doses above the LOAEL. Although effects on kidney weight and urine
protein levels were observed in male mice, the biological relevance of the latter effect and whether it
would also occur in female mice needs further investigation. Mice are known to excrete a group of
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highly polymorphic, low-molecular-weight urinary proteins that play important roles in social
recognition and mate assessment. The relevance of the urinary protein findings in mice to humans is
unknown. Humpage and Falconer (2002, 2003) found signs indicative of hemolysis (e.g., increased
bilirubin, spleen weight and polychromasia), however these changes were not statistically significant.
Results from Reisner et al. (2004) corroborate Humpage and Falconer (2002, 2003) with comparable
effects observed in mice during a three-week study. The kidney and red blood cell effects observed by
Reisner et al. (2004) occurred at a LOAEL of 66 [j,g/kg/day in drinking water. The study authors
demonstrated significant increases in hematocrit, acanthocytes (abnormal red blood cells), and liver and
testes weights in exposed animals and a duration-related nonsignificant increase in kidney weight. The
red blood cell effects were seen as early as the end of the first week of dosing and were present in each
of the three weekly blood samples collected. Sukenik et al. (2006) observed similar effects on red blood
cells (increases in hematocrit from week 16 to 32 accompanied by increased numbers of acanthocytes up
to week 42) in male and female mice exposed to gradually increasing concentrations of
cylindrospermopsin (i.e., from 100 to 550 (J,g/L) in drinking water for 42 weeks. Mice were given
cylindrospermopsin in the form of spent medium on which cultures of Aphanizomenon ovalisporum had
been grown; other medium components were not characterized. The authors proposed a LOAEL of
20 [j,g/kg/day (equivalent to 200 (J,g/L) for male and female mice based on changes in hematocrit at
16 weeks (Sukenik et al. 2006). This study was not selected as a critical study because this study used a
single dose; however, the kidney and red blood cell effects at that dose after three weeks were
comparable to the effects seen in the Humpage and Falconer (2002, 2003) study at a slightly lower
60 mg/kg/day dose after 11 weeks.
The short-term study by Shaw et al. (2001) was also considered in the development of the RfD for
cylindrospermopsin. Shaw et al. (2001) reported liver effects (fatty infiltration) in mice given 50 (J,g/kg
purified cylindrospermopsin by gavage for 14 days; this dose is lower than the NOAEL identified in the
key study by Humpage and Falconer (2002, 2003). However, the EPA concluded that the Shaw et al.
(2001) study was not suitable for quantification based on the limited number of doses tested.
A 90-day oral toxicity study by Chernoff et al. (2018) demonstrated signs of hepatic and renal injury in
mice at all dose levels (0, 75, 150, and 300 (j,g/kg/day). Liver toxicity effects were noted by elevated
absolute and relative liver weights, increases in serum alanine aminotransferase activity, reduced serum
blood urea nitrogen and cholesterol levels, and increased incidence of hepatocellular hypertrophy and
cord disruption. Renal toxicity effects were demonstrated in elevated absolute and relative kidney
weights and renal cellular hypertrophy, tubule dilation, and cortical tubule lesions. Males showed more
susceptibility to toxic effects; liver and kidney/body weight ratios, reduced cholesterol levels, cellular
signs of inflammation, and degree and extent of renal histopathological damage were all observed to be
more prominent in males. A NOAEL was not determined for any dose level based on significant liver
and kidney effects exhibited in the 75 (J,g/kg group. The LOAEL of 75 (J,g/kg observed by Chernoff et al.
(2018) is higher than the Humpage and Falconer (2002, 2003) NOAEL of 30 (J,g/kg.
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Table 5-2. Kidney and Liver Effects in Animals Exposed to Cylindrospermopsin (Purified) in
Acute and Key Short-term Studies in the Health Effects Support Document for the Cyanobacterial
Toxin Cylindrospermopsin (U.S. EPA 2015c)
Species
Exposure
Route
Dosing Regimen
Description of Kidney
and Liver Effects
Study
Male Swiss albino mice
(10 mice per group,
except the highest dose
group, which included
6 mice)
Gavage
Repeated doses of 0, 30,
60, 120, or 240 (ig/kg/day
for 11 weeks
Kidney: dose-related
increases in relative kidney
weight, proximal renal
tubular damage, decreased
urinary protein
Liver: necrosis,
inflammatory foci, and bile
duct changes
Humpage and
Falconer
(2002, 2003)
CD-I (Swiss-Webster)
mice
(18 to 20 per group)
Gavage
Repeated doses of 0, 75,
150, or 300 (ig/kg/day for
90 days
Kidney: elevated absolute
and relative kidney
weights, renal cellular
hypertrophy, tubule
dilation cortical tubule
lesions
Liver: elevated absolute
and relative liver weights,
increases in serum alanine
aminotransferase activity,
reduced serum blood urea
nitrogen and cholesterol
levels, increased incidence
of hepatocellular
hypertrophy and cord
disruption
Chernoff et
al. (2018)
Male Swiss mice
(3 per group)
Gavage
Single dose of 1,000,
2,000, or 4,000 (ig/kg
Examination at 24 hours
after treatment
Liver: dark red liver,
apoptosis in the liver and
the kidneys
Bazin et al.
(2012)
Male ICR mice
(n = 24, single group)
Intraperitoneal
injection
Single dose of 200 (ig/kg
Three animals sacrificed at
8 time points,
16-100 hours after
treatment
Kidney: proliferation of the
endoplasmic reticulum and
fat droplet accumulation in
cells along the brush
borders of the tubules plus
limited single cell necrosis
Liver: necrosis in the
centrilobular region
Terao et al.
(1994)
Male ICR mice
(4 per group)
Drinking water
Repeated doses of 0 or 0.6
mg/L (estimated at
66 (ig/kg/day) for 3 weeks
Kidney: duration-related
nonsignificant increase in
kidney weight
Liver: increases in relative
weight
Reisner et al.
(2004)
Quackenbush mice
(4 per group)
Intraperitoneal
injection
Single dose of 200 (ig/kg
Liver: fatty infiltration and
cell necrosis
Shaw et al.
(2001)
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Species
Exposure
Route
Dosing Regimen
Description of Kidney
and Liver Effects
Study
Quackenbush mice
(4 per group)
Gavage or
intraperitoneal
injection
0 to 300 (ig/kg/day (oral)
or 0 to 25 (ig/kg/day
(intraperitoneal injection)
for 14 days
Liver: fatty infiltration
(oral), foamy
hepatocellular cytoplasm
(intraperitoneal injection)
Shaw et al.
(2001)
The Humpage and Falconer (2002, 2003) study was determined to be the most appropriate for the
quantitative assessment because the LOAEL at 11 weeks would be protective for the effects seen at
three weeks in the shorter duration study. For these reasons, this RfD was deemed suitable for
development of the short-term drinking water health advisory and for use in recreational exposure
scenarios. The EPA's HESD and Health Advisory documents for cylindrospermopsin describe the
selection of the critical study and effect in detail and provide the rationale for applicability of the longer-
term duration study (U.S. EPA 2015c).
5.1.1.2 Human Studies
Microcystins
The EPA identified the available epidemiological, outbreak, and case study reports on adverse health
effects from oral exposures to microcystins. Limited human studies examining microcystin effects on
humans exposed via drinking water are available, and no dose response data from oral exposure to
microcystins in ambient water were identified. The scant human data on the oral toxicity of microcystin-
LR are limited by the potential co-exposure to other pathogens, cyanotoxins, and microorganisms; by
the lack of quantitative information; and by the failure to control for confounding factors. Available
human studies evidence is supportive of the liver as a target organ for toxicity (Carmichael 2001;
Falconer et al. 1983; Giannuzzi et al. 2011; Hilborn et al. 2013; Jochimsen et al. 1998; Li et al. 201 lb).
The EPA identified four epidemiological studies, three case reports, and two outbreak summaries that
evaluated human health effects associated with recreational exposures to cyanobacteria and
microcystins. This human health effects information is summarized in the paragraphs that follow.
Backer et al. (2008) characterized microcystin concentrations in blood and reported symptoms in people
recreating in a lake with a Microcystis aeruginosa bloom to those of people recreating in a nearby
bloom-free lake. Low levels of total microcystins (detection limit = 0.08 ng/m3) were detected in air
samples collected above a lake bloom. Phytoplankton counts ranged from 175,000 to 688,000 cells
per mL with > 95 percent of those cells being cyanobacteria. Cell densities of potentially toxigenic
cyanobacteria ranged from approximately 54,000 to 144,000 cells/mL. Although a visible bloom was
present and contained cyanobacterial species capable of producing microcystin, microcystin
concentrations in water during the study were low and ranged from 2 to 5 |_ig/L. Recreational users of
the lake at the time of the bloom had no detectable microcystins in their blood and did not report an
increase in GI, dermal, respiratory, or neurological symptoms after spending time on the lake.
Adenoviruses (level of detection (LOD) = 1,250 gene copy equivalents) and enteroviruses (LOD = 200
plaque forming units/10 L) were not detected in any water sample. This study was limited in the number
of participants (n = 104) and included a limited number of exposure days in the analysis (three days).
The study demonstrated that people recreating on or in a water body can be exposed to aerosolized
microcystins. However, given the limited number of participants and exposure days, and the low levels
of microcystins present in the water and as aerosols, there were no reported increases in self-reported
symptoms following recreational exposures. Other symptoms consistent with microcystin intoxication
(e.g., liver toxicity) were not included in the study.
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Backer et al. (2010) applied the same experimental approach at three lakes in California. Two of the
lakes experienced blooms producing much higher microcystin concentrations compared with the lakes
studied in the Backer et al. (2008) study, and the third lake did not contain a toxin-producing bloom.
Eighty-one people, aged 12 and older, participated in the study and engaged in waterskiing, using
personal watercraft, swimming, or wading. Total microcystins present in the lake containing toxic
blooms ranged from <10 [j,g/L to > 500 (J,g/L. Measured microcystin concentrations from personal air
samples ranged from the limit of detection (0.1 ng/m3) to 2.89 ng/m3; the mean air concentration was
0.4 ng/m3. Similarly, nasal swabs ranged from below the limit of detection to 5 ng, and all blood
samples were below the limit of detection. Recreators had a significantly higher amount of microcystins
present in nasal swabs after exposure. No statistically significant differences were noted in the frequency
of reported GI, dermal, or respiratory symptoms between participants immediately after they engaged in
direct- or indirect-contact recreational activities in the lake with a cyanobacterial bloom and those in a
lake without a cyanobacterial bloom. Other symptoms consistent with microcystin intoxication (e.g.,
liver toxicity) were not included in the study. Adenoviruses or enteroviruses were not detected at the
study locations. The authors concluded that it is possible for microcystins to become aerosolized, which
in turn represents a potential route of exposure to recreators. They recommended additional research
studying larger populations and sensitive subgroups.
Levesque et al. (2014) conducted a prospective study of residents living in proximity to three lakes in
Canada affected by cyanobacteria and microcystins to investigate the relationship between recreational
exposure, specifying full contact and limited contact with lake water, and the incidence of GI, dermal,
respiratory, and other symptoms (e.g., ear pain, muscle pain). Full contact included swimming,
waterskiing, windsurfing, use of watercraft involving launching, accidental falls, and similar activities,
and limited contact included fishing, use of watercraft not involving launching, and other activities. The
authors reported a dose-effect relationship (p-trend = 0.001) between total cyanobacterial cell counts and
severe GI illness with a significant increase in reported symptoms starting at 20,000 cells/mL and above.
The study reported a relative risk value of 3.28 (95 percent confidence interval (CI): 1.69-6.37) for the
more severe GI symptom index (i.e., GI2, defined as diarrhea or vomiting or (nausea and fever) or
(abdominal cramps and fever)) for exposures by full or limited contact to concentrations higher than
100,000 cells/mL (Levesque et al. 2014). Adjusted relative risks of GI illness were significantly high for
limited contact, but no relationship was found between GI symptoms and full contact. The authors
explained that study participants avoided full contact with lake waters when high densities of
cyanobacteria were visible, but continued to have limited contact. No significant fecal contamination
measured by Escherichia coli (E. coli) was observed with geometric means in the lakes ranging from
8 to 145 colony forming units (CFU)/100 mL.5 No associations were observed between any symptoms
and recreational exposures to microcystins. Overall, the microcystin concentrations were low during the
study, and the reported lower bound of the upper tertile was 0.2456 (J,g/L. The maximum microcystin
concentrations for which recreational-related GI symptoms were reported was 7.65 g/L; however,
microcystins occurred at much higher concentrations (e.g., maximum reported microcystin
concentrations of 108 [j,g/L and 773 [j,g/L at two of the study locations), but there was no significant
trend of increasing illness symptoms with elevated toxin concentrations. The study did not characterize
the primary endpoint of concern for exposure to microcystins (i.e., liver toxicity) and did not conduct the
necessary medical testing to determine liver function impairment.
Levesque et al. (2016) provided additional analysis of the prospective study reported previously.
Because GI illness was significantly associated with increasing cyanobacterial cell densities and GI
5 Current Canadian recreational water guidelines for E. coir, geometric mean < 200 E. coli/100 mL and single-sample
maximum < 400 E. coli/100 mL (Health Canada 2012).
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symptoms can be related to cellular constituents, also termed endotoxins in the literature, the authors
characterized the relationship between endotoxin exposure and illness in the study participants.
Endotoxins include cell wall-associated lipopolysaccharides present in cyanobacteria and Gram negative
bacteria. Frozen filters collected during the study were analyzed for endotoxins. The authors found a
weak correlation between endotoxin levels and cyanobacteria cell density and reported a significant
trend of increasing GI illness with increasing endotoxin concentrations. They also suggest that endotoxin
concentrations could be a surrogate for another stressor. They cite other researchers that have suggested
the endotoxins could be contributed by other members of the microbial community or the reported
symptoms could be related to another stressor (Berg et al. 2008; Blahova et al. 2013; Rapala et al. 2002;
Stewart 2006d).
In a recent case report by Vidal et al. (2017), a 20-month-old child and three adults reported GI
symptoms several hours after engaging in bathing and other recreational activities at beaches in
Montevideo, Uruguay, during January 2015. At that time, a cyanobacterial bloom of mainly Microcystis
occurred in the River de la Plata. While the GI symptoms in the adults (i.e., diarrhea) rapidly resolved,
the child's symptoms (i.e., diarrhea and vomiting) persisted. The child developed fatigue and jaundice,
and five days after the exposure, she was admitted to hospital. Tests showed significant increases in
bilirubin and serum liver enzymes, and a diagnosis of acute liver failure was given. The child was
recommended for, and received, a liver transplant. The city government's beach monitoring program
from April 2014 to March 2015 reported mean and maximum microcystin concentrations of 2.9 (J,g/L of
56 (J,g/L, respectively. These levels were reported in water samples from the beaches the family used
with cyanobacteria presence but without cyanobacterial foam. Mean and maximum microcystin
concentrations of 2,900 [j,g/L and 8,200 (J,g/L, respectively, were reported in water samples with
cyanobacterial foam. The monitoring program also reported geometric means of fecal coliform values
below the limit of 1,000 CFU/100 mL. After the child received a liver transplant, histological analysis of
the explanted liver revealed liver damage characterized by hemorrhagic necrosis, intracytoplasmic
cholestasis, large and multinucleated hepatocytes, proliferation, and nodular regeneration. The
pathological findings and detection of microcystin-LR in the liver (2.4 ng microcystin-LR/g and 75.4 ng
(D-Leul) microcystin-LR/g liver) led to a diagnosis of acute liver failure related to exposure to
microcystin-LR and cyanobacteria.
In another case report, acute intoxication with microcystin-producing cyanobacterial blooms in
recreational water was reported in Argentina in 2007 (Giannuzzi et al. 2011). A male Jet Skier was
exposed to a Microcystis bloom containing 33,680 and 35,740 cells/mL. A level of 48.6 (J,g/L of
microcystin-LR concentrations was detected in water samples associated with the bloom. The subject
was immersed for two hours as a result of an accident that required him to swim to the shoreline towing
the Jet Ski. Four hours later the subject reported experiencing nausea and abdominal pain. Three days
later the subject sought medical assistance because of respiratory distress requiring his hospitalization.
One week after the exposure, the patient developed a hepatotoxicosis with a significant increase of
serum alanine aminotransferase, aspartate aminotransferase, and y-glutamyltransferase. With treatment,
the patient recovered within 20 days.
An outbreak among army recruits undergoing canoe exercises who had consumed reservoir water
containing a bloom of Microcystis aeruginosa reported symptoms of headache, sore throat, vomiting
and nausea, stomach pain, dry cough, diarrhea, blistering around the mouth, and pneumonia (Turner et
al. 1990). Microcystins, including microcystin-LR, were present in bloom samples. However, high
levels of E. coli were also found in reservoir water after two weeks. The authors suggested that exposure
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to microcystins may have had a role in some of the clinical symptoms; however, this case report
information is insufficient to establish cause and effect.
Dziuban et al. (2006) and Hilborn et al. (2014) reported 10 outbreaks associated with recreational
exposure to cyanobacteria in which microcystins were detected. Hilborn et al. (2014) reported that eight
of these investigations evaluated the presence of cyanotoxins; eight detected microcystins; and two
detected cylindrospermopsin. In four of the outbreaks, microcystin concentrations ranged from 0.2 [j,g/L
to > 2,000 (J,g/L. Four outbreaks had microcystin concentrations > 20 (J,g/L. Cylindrospermopsin and
anatoxin-a also were detected in three of the outbreaks. In one outbreak, 20.8 [j,g/L microcystins was
measured, and other cyanotoxins were either not detected or measured. The nine persons reporting
illness for this outbreak had symptoms that included abdominal cramps (3 people), diarrhea (3), nausea
(3) vomiting (2), fever (2), headache (2), rash (8), eye irritation (1), ear ache (1), neurologic symptoms
(2), tingling (2), confusion (1), and respiratory symptoms (1) (Hilborn et al. 2014). Dziuban et al. (2006)
reported on two 2004 cyanobacteria-associated outbreaks in which 22 cases of illness were associated
with elevated levels of microcystins in Nebraska lakes. The predominant illnesses in both outbreaks
included dermatitis and gastroenteritis, and individuals who sought medical care showed a combination
of rashes, diarrhea, cramps, nausea, vomiting, and fevers. Walker et al. (2008) also reported about a
Nebraska outbreak. Levels of total microcystins at the east swimming beach of Pawnee Lake exceeded
15 ppb on July 12, 2004, and a health alert was issued. However, heavy public use of Pawnee Lake
occurred that weekend and more than 50 calls were received from the public, complaining about
symptoms such as skin rashes, lesions, blisters, vomiting, headaches, and diarrhea after swimming or
water skiing in Pawnee Lake (Walker et al. 2008). The outbreak reports data are not sufficient to
establish cause and effects for microcystins because of weaknesses in the nature of the data reported and
the many potential confounding variables. The researchers concluded that the disease outbreak data
suggest that the time to onset of effects might be rapid, that children might be at higher risk for illness,
and that these types of outbreaks occur during the warmer months. Hilborn et al. (2014) noted that
HAB-associated illness from recreational exposure might be underreported due to multiple possible
exposure routes and the non-specific nature of potential health effects.
Graham et al. (2009) counted 36 states with anecdotal reports of acute cyanotoxin poisonings of
animals, humans, or both as reported in journal articles and newspaper articles (Chorus and Bartram
1999; Hilborn et al. 2014; Huisman et al. 2005; Yoo et al. 1995).
Information on the human health effects of microcystins based on epidemiological studies related to
drinking water exposures to microcystins are discussed in detail in the EPA's HESD for microcystins
(U.S. EPA 2015d). These studies are summarized in the paragraphs that follow.
An epidemiology study done in Australia compared the hepatic enzyme levels from patients served by a
public water supply contaminated with a Microcystis aeruginosa bloom with enzyme levels from
patients living in areas served by water supplies uncontaminated by cyanobacteria (Falconer et al. 1983).
Although the authors observed significant variability in enzyme levels between the two groups, the
findings were attributed by the authors to the imprecise method of study participant selection and
confounding factors such as alcoholism and chronic kidney disease among some of the participants.
A cross-sectional study conducted in China assessed the relationship between the consumption of
drinking water and aquatic food (carp and duck) contaminated with microcystins and liver damage in
children (Li et al. 201 lb). The authors found that mean serum levels of microcystins ranged from below
detection to 1.3 jag microcystin-LR equivalents/L. According to the authors, hepatitis B infection was a
greater risk for liver damage among these children than the microcystins exposure.
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An outbreak of acute liver failure occurred in a dialysis clinic in 1996 in Caruaru, Brazil, where dialysis
water was contaminated with microcystins, and possibly cylindrospermopsin. Of the 130 patients who
received their routine hemodialysis treatment (intravenously) at that time, 116 reported symptoms of
headache, eye pain, blurred vision, nausea, and vomiting. Subsequently, 100 of the affected patients
developed acute liver failure and, of these, 76 died (Carmichael et al. 2001; Jochimsen et al. 1998).
Analyses of blood, sera, and liver samples from the patients revealed microcystins.
In another contamination event at a dialysis center in Rio de Janeiro, Brazil, in 2001, 44 dialysis patients
were potentially exposed to microcystin concentrations of 0.32 (J,g/L, detected in the activated carbon
filter used in an intermediate step for treating drinking water to prepare dialysate (Soares et al. 2005).
Concentrations of 0.4 [j,g/L microcystin-LR were detected in the drinking water. Serum samples were
collected from 13 dialysis patients 31 to 38 days after the detections in water samples, and patients were
monitored for eight weeks. Concentrations of microcystin-LR in the serum ranged from 0.46 to
0.96 ng/mL. Although the biochemical outcomes varied among the patients, markers of hepatic cellular
injury and of chlolestasis (elevations of AST, ALT bilirubin, ALP, and GGT) in serum during weeks
one to eight after treatment frequently exceeded normal values (Hilborn et al. 2013). Because
microcystin-LR was not detected in the dialysate during weekly monitoring after the first detection, the
authors suggested that the patients were not continuously exposed to the toxin and that the toxin detected
in the serum after eight weeks may have been present in the form of bound toxin in the liver (Soares et
al. 2005). Results were consistent with a mild to moderate mixed liver injury (Hilborn et al. 2013).
Although the patients in the study had pre-existing diseases, the direct intravenous exposure to dialysate
prepared from surface drinking water supplies put them at risk for cyanotoxin exposure and resultant
adverse effects (Hilborn et al. 2013).
Cylindrospermopsin
No epidemiological studies were identified for recreational exposure to cylindrospermopsin.
Hilborn et al. (2014) reported two outbreaks associated with recreational exposure to HABs in which
cylindrospermopsin was detected between 2009 and 2010. However, cyanobacteria, microcystins, and
other cyanotoxins were also present. As mentioned earlier, the results reported from the outbreaks
should not be interpreted as cause and effect.
Human data on oral toxicity of cylindrospermopsin are limited, but results indicate that kidney and liver
exhibit adverse effects due to cylindrospermopsin exposures. Information on the human health effects of
cylindrospermopsin based on epidemiological studies related to drinking water are discussed in detail in
the EPA's HESD for cylindrospermopsin (U.S. EPA 2015c). This information is summarized in the
paragraphs that follow.
Reports of a hepatoenteritis-like outbreak (mostly in children) in Palm Island, Australia, in 1979 were
attributed to consumption of drinking water with a bloom of Cylindrospermopsis raciborskii, a
cyanobacteria that can produce cylindrospermopsin. No data are available on exposure levels or
potential co-exposures to other cyanobacterial toxins and microorganisms. The majority of the cases,
mostly children, required hospitalization. The clinical picture included fever, headache, vomiting,
bloody diarrhea, hepatomegaly, and kidney damage with loss of water, electrolytes, and protein (Byth
1980; Griffiths and Saker 2003).
Dermal exposure to cylindrospermopsin was evaluated using skin-patch testing in humans (Pilotto et al.
2004; Stewart et al. 2006a). Exposed individuals showed mild irritation, but no statistically significant
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dose-response relationship or reaction rates were found between skin reactions and increasing cell
concentrations for either whole or lysed cells (Pilotto et al. 2004). No detectable skin reactions were
observed in individuals exposed to lyophilized Cylindrospermopsis raciborskii (Stewart et al. 2006a).
5.1.1.3 Mode of Action for Noncancer Health Effects
Microcystins
Mechanistic studies have shown the importance of membrane transporters for systemic uptake and tissue
distribution of microcystins by all exposure routes (Feurstein et al. 2010; Fischer et al. 2005). The
importance of the membrane transporters to systemic uptake and tissue access is demonstrated by
studies where there was either no liver damage or reduced damage when the hepatic organic anion
transporting polypeptide (OATP) receptors were inhibited (Hermansky et al. 1990a, 1990b; Thompson
and Pace 1992). OATPs are a transporter family that controls uptake of microcystins by the liver
(Fischer et al. 2005).
The uptake of microcystins causes protein phosphatase inhibition and a loss of coordination between
cytoskeletal protein phosphorylation by kinases and dephosphorylation by phosphatases. This event
initiates altered cell function followed by cellular apoptosis and necrosis (Barford et al. 1998). Both
cellular kinases and phosphatases keep the balance between phosphorylation and dephosphorylation of
key cellular proteins controlling organization of the cytoskeleton, metabolic processes, gene regulation,
cell cycle control, transport and secretory processes, and cell adhesion. Each of the microcystin
congeners evaluated (LR, LA, and LL) interacts with catalytic subunits of protein phosphatases PP1 and
PP2A, inhibiting their functions (Craig et al. 1996).
As a consequence of the microcystin-induced changes in cytoskeleton proteins, an increase in cellular
reactive oxygen species (ROS) leads to cellular apoptosis. In both in vitro and in vivo studies, cellular
pro-apoptotic Bax and Bid proteins increased whereas anti-apoptotic Bcl-2 decreased (Fu et al. 2005;
Huang et al. 2011; Li et al. 201 la; Takumi et al. 2010; Weng et al. 2007; Xing et al. 2008).
Mitochondrial membrane potential and permeability transition pore changes (Ding and Nam Ong 2003;
Zhou et al. 2012) lead to membrane loss of cytochrome c, a biomarker for apoptotic events. Wei et al.
(2008) identified a time-dependent increase in ROS production and lipid peroxidation in mice after
exposure to microcystin-LR. Following intraperitoneal injection of 55 (J,g/kg of body weight
microcystin-LR, the levels of hepatic ROS increased within 0.5 hours of treatment and continued to
accumulate for up to 12 hours in a time-dependent manner.
Cylindrospermopsin
Despite the number of studies that have been published, the mechanisms for liver and kidney toxicity by
cylindrospermopsin are not completely characterized.
In vitro and in vivo studies showed that cylindrospermopsin can inhibit hepatic protein synthesis
(Froscio et al. 2003; Froscio et al. 2008; Terao et al. 1994), which could impact mouse urinary protein
production leading to decreased urinary excretion of these proteins. Available evidence indicates that
protein synthesis inhibition is not decreased by broad-spectrum CYP450 inhibitors, but they do reduce
cytotoxicity (Bazin et al. 2010; Froscio et al. 2003). Hepatotoxicity appears to be CYP450-dependent,
which indicates a possible involvement of oxidized or fragmented metabolites and mechanisms other
than protein synthesis inhibition (Froscio et al. 2003; Humpage et al. 2005; Norris et al. 2002; Norris et
al. 2001).
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In the Reisner et al. (2004) and Sukenik et al. (2006) reports, microscopic examination of blood samples
showed the presence of red blood cells with spiked surfaces rather than their normal biconcave-disc
shape. The authors attributed the acanthocyte formation to an increase in the cholesterol to phospholipid
ratio of the red blood cell membrane. Phospholipids constitute the matrix material of cell membranes.
The authors hypothesized that this change was the consequence of decreased activity of plasma lecithin
cholesterol acyl transferase, an enzyme associated with high-density lipoproteins and the esterification
of plasma cholesterol. Effects on the cholesterol content of the red blood cell membrane can occur with
inhibition of the enzyme increasing membrane fluidity and mean corpuscular volume. Removal of the
abnormal blood cells by the spleen increases both spleen weight and serum bilirubin as well as
stimulates hematopoiesis. Additional research is needed to examine the lecithin cholesterol acyl
transferase enzyme inhibition hypothesis to confirm whether it accounts for the effects on the red blood
cell as a result of cylindrospermopsin exposure.
Kidney necrosis and a decreased renal failure index at the high cylindrospermopsin doses in Humpage
and Falconer (2002, 2003) are also indicative of an effect on the kidney. Numerous signs of renal
damage including proteinuria, glycosuria, and hematuria were also observed in humans after a
hepatoenteritis-like outbreak in Palm Island, Australia, in 1979 (Byth 1980). The outbreak was attributed
to consumption of drinking water from source waters with a bloom of Cylindrospermopsis raciborskii.
These effects have been shown to be related to impaired kidney function (Byth 1980); however, no
mode of action information for kidney effects was observed in the available animal or human studies of
cylindrospermopsin. Because all the studies were conducted in mice, a species that excretes low-
molecular-weight proteins in urine, a study is needed of cylindrospermopsin in a laboratory species that
does not excrete protein in the urine to determine whether there are comparable effects on kidney
weight, protein excretion, and renal cellular damage.
5.1.2 Cancer
5.1.2.1 Weight of Evidence Classification
While there is evidence of an association between liver and colorectal cancers in humans and
microcystins exposure and some evidence that microcystin-LR is a tumor promoter in mechanistic
studies, there is "inadequate information to assess carcinogenic potential" of microcystins in humans
(U.S. EPA 2005b). The human studies are limited by lack of exposure information and the uncertainty
regarding whether these studies adequately controlled for confounding factors such as hepatitis B
infection. No chronic cancer bioassays for microcystins in animals are available. The EPA (U.S. EPA
2005a) states that the descriptor of" inadequate information to assess carcinogenic potentiaF is
appropriate when available data are judged inadequate for applying one of the other descriptors or for
situations where there is little or no pertinent information or conflicting information. The guidelines also
state that (p. 2-52) "Descriptors can be selected for an agent that has not been tested in a cancer bioassay
if sufficient other information, e.g., toxicokinetic and mode of action information, is available to make a
strong, convincing, and logical case through scientific inference." In the case of microcystins, the data
suggest that microcystin-LR may be a tumor promoter but not an initiator. Without strong
epidemiological data and a chronic bioassay of purified microcystin-LR, the data do not support
classifying microcystin-LR as a carcinogen.
No chronic cancer bioassays of cylindrospermopsin were located in the literature. Limited data from an
in vivo study showed no indication that the cyanobacterial extract containing cylindrospermopsin in the
presence of a tumor promotor indicated preneoplastic changes consistent with its having tumorigenic
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activity in mice (Falconer and Humpage 2001). Following the EPA guidelines (U.S. EPA 2005a), there
is inadequate information to assess carcinogenic potential of cylindrospermopsin.
5.2 Dose-response Assessment
The RfD value for microcystins used to derive this recreational AWQC or swimming advisory is
described in the EPA's HESD for microcystins (U.S. EPA 2015d). The EPA identified a 28-day study in
male hybrid rats by Heinze (1999) as the critical study (described in section 5.1.1). A LOAEL of
50 [j,g/kg/day was identified based on increased liver weight, slight to moderate liver necrosis (necrotic
severity was dose-related) with hemorrhages, and increased enzyme levels, which was used to derive an
RfD of 0.05 [j,g/kg/day. The EPA selected the study by Heinze (1999) based on the appropriateness of
the study duration, the use of multiple doses, dose-related toxicological responses, and histopathological
evaluations of toxicity. After 28 days of exposure, rat organ weights (liver, kidneys, adrenals, thymus,
and spleen) were measured, and hematology, serum biochemistry, and histopathology of liver and
kidneys were evaluated. The critical effect in the Heinze (1999) study was supported by additional acute
and subchronic data as described in the EPA's HESD for microcystins and summarized in section
5.1.1.1. The EPA's selection of uncertainty factors and derivation of the RfD are documented in its
HESD for microcystins (U.S. EPA 2015d).
The RfD value for cylindrospermopsin used to derive the AWQC and swimming advisory is described
in the EPA's HESD for cylindrospermopsin (U.S. EPA 2015c). The EPA identified an 11-week study in
mice by Humpage and Falconer (2002, 2003) as the critical study for development of the RfD. The
NOAEL was 30 [j,g/kg/day dose for increases in relative kidney weight seen at the LOAEL of
60 [j,g/kg/day. Increased relative kidney weights was the critical effect on which to base the point of
departure. The EPA's selection of UFs and derivation of the RfD are documented in its HESD for
cylindrospermopsin (U.S. EPA 2015c).
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6.0	RECOMMENDED RECREATIONAL CRITERIA AND SWIMMING ADVISORY
DERIVATION
This section summarizes the inputs and shows the calculation for the recommended recreational criteria
and swimming advisories for microcystins and cylindrospermopsin.
6.1	Microcystins Magnitude
The magnitude of the recommended recreational criteria and swimming advisory for microcystin toxins
is calculated as follows:
BW
Recreational value (|_ig/L) = RfD x ———
1R
Where:
RfD (ng/kg/day) = 0.05 ng/kg/day (U.S. EPA 2015d)
BW (kg)	= 31.8 kg (mean body weight of children six to 10 years; U.S. EPA
2011)
IR (L/day)	= 0.21 L/day (90th percentile daily recreational water incidental
ingestion rate for children age six to 10 years; Appendix E; U.S.
EPA 2018a; U.S. EPA 2011; see section 4.2.3.1)
31.8 kg
Microcystins recommended recreational value = 0.05 |j,g/kg/day x 		— = 8 |j,g/L
0.21 L/day
6.2	Cylindrospermopsin Magnitude
The magnitude of the recommended recreational criteria and swimming advisory values for
cylindrospermopsin is calculated as follows:
BW
Recreational value (|_ig/L) = RfD x
IR
Where:
RfD (ng/kg/day) = 0.1 ng/kg/day (U.S. EPA 2015c)
BW (kg)	= 31.8 kg (mean body weight of children six to 10 years; U.S. EPA
2011)
IR (L/day)	= 0.21 L/day (90th percentile daily recreational water incidental
ingestion rate for children age six to 10 years; Appendix E; U.S.
EPA 2018a; U.S. EPA 2011; see section 4.2.3.1)
31.8 kg
Cylindrospermopsin recommended recreational value = 0.1 |j,g/kg/day x	— = 15 |j,g/L
0.21 L/day
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6.3 Frequency and Duration for Recreational Criteria
The frequency and duration components of a criterion describe how often and for how long a water
body's conditions can exceed the magnitude and be protective of the designated use (U.S. EPA 2005c).
HABs can occur naturally, but can be an uncommon event due to a convergence of climatic and other
environmental factors that result in a single short-term bloom lasting days or a couple of weeks. In some
cases, multiple HABs can occur in a single year. Alternatively, longer-term HABs can occur regularly in
some waters lasting for a few weeks, months, or possibly all year. HABs can occur while conditions
conducive to cyanobacterial proliferation exist and limit the use of the water body for primary
recreation. Water bodies where a toxic HAB has occurred in the past may experience repeat occurrences
of elevated toxins when bloom-promoting conditions reoccur. In some circumstances, anthropogenic
inputs are identified and controlled, and the conditions that cause the bloom can be mitigated.
The EPA recognizes that a single sample above the cyanotoxin criteria magnitude does not necessarily
indicate that the designated recreational use is not attained. However, when cyanotoxin concentrations
exceed the criteria magnitude either in multiple short-term blooms within a year or from a single bloom
that persists for an extended period within a year, and when these patterns occur in more than one year,
the designated recreational use may not be attained. The frequency and duration components discussed
in this section support the identification of a trend or pattern of cyanotoxin excursions that state decision
makers can use to inform the evaluation of a water body. The EPA recommends that decisions on
whether the designated recreational use is attained should be flexible enough to address both types of
exposure patterns when patterns reoccur in more than one year (short-term blooms that occur frequently
in a recreational season, or blooms that persist for an extended period during a recreational season).
States may want to evaluate the pattern of bloom occurrence and toxin concentrations within and across
years to determine if there is a trend toward degradation of the water quality.
The EPA's recommended criteria duration rely on the underlying toxicity data used to derive the criteria.
For both toxins, animal toxicological studies consistently demonstrate adverse health effects at various
dosages and relevant timeframes. See Tables 5-1 and 5-2. For microcystins, the key study (Heinze 1999)
shows adverse liver effects from repeated microcystin exposures (50 and 150 (J,g/kg body weight) during
a study duration of 28 days. Another supporting study showed similar effects (Guzman and Solter 1999).
For cylindrospermopsin, the key study (Humpage and Falconer 2002, 2003) had a duration of 11 weeks.
The shorter-term studies available for cylindrospermopsin (Shaw et al., 2001; Reisner et al., 2004) were
not suitable for quantification due to study limitations; however, effects observed in these studies are the
same or similar to the Humpage and Falconer study (2002, 2003) and occur at similar doses. The
LOAEL derived from Humpage and Falconer (2002, 2003) was determined to be protective for the
adverse effects observed in the shorter duration studies. For both key studies, adverse health effects were
noted at the end of the study period and it is not known if those effects occurred earlier.
The criteria are based on the same science used to develop the EPA's Drinking Water Health Advisories
for microcystins and cylindrospermopsin, which are 10-day advisories (U.S. EPA 2015a, 2015b). The
10-day drinking water health advisory values represent concentrations of cyanotoxins in finished
drinking water below which adverse noncarcinogenic effects are not expected to result from ingestion of
drinking water over a 10-day period. Following the detection and confirmation of microcystins or
cylindrospermopsin in finished drinking water above the health advisory values, the EPA recommends
that drinking water utilities initiate actions to reduce exposure to consumers including determining when
to notify drinking water consumers who may be more susceptible to adverse outcomes (U.S. EPA
2015c). If the advisory level continues to be exceeded after 10 days, additional public health measures
can be taken, including a do-not-drink and do-not-boil water advisory. Recreational water managers
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have fewer options to reduce exposure to toxins in recreational waters than do drinking water treatment
operators, as recreational water does not go through a treatment process.
The EPA recommends states use 10-day assessment periods over the course of a recreation season to
evaluate ambient water body condition and recreational use attainment. The 10-day period links the
water body assessment to the adverse health effects from ingestion of the toxins over short-term
exposures, consistent with the EPA's Drinking Water Health Advisory (described in greater detail in
section 5.1). Also, Cordell (2012) discussed decade-long trends in outdoor recreation activities showing
a significant proportion (43 percent) of Americans visited a beach in 2005-2009, up almost 21 percent
over the previous decade. Over the same timeframe, participation in swimming in lakes and streams
(42 percent of the population) increased by 14 percent (Cordell 2012). Beach visitation surveys have
shown that nearly half (47 percent) of the local population are regular beach users with five or more
visits in a recreation season (Caldwell et al. 2013). The recommended assessment period is reasonable
considering beach visitation rates for recreators living in proximity to a beach or vacationing at a beach
for a week or two with daily beach visits expected. Exposure to recreational waters containing
microcystins or cylindrospermopsin at or below the recommended magnitude concentrations over the
short-term 10-day duration would not be expected to result in the adverse health effects discussed in
section 5.
The EPA recommends that if toxin concentrations are higher than the criterion magnitude in a sample
collected during a 10-day assessment period, that period should be considered an excursion from the
recreational criteria. Elevated toxin concentrations can occur over hours, days, or a couple of weeks and
are counted as excursions in a recreational season. A short-term HAB that does not reoccur can result in
a small number of excursions of the criteria but is not expected to result in impairment of the
recreational use. Such algal blooms may result from conditions that occur naturally (e.g., as a result of
unusually hot conditions), but not frequently. Following an excursion (an exceedance during the 10-day
assessment period), the EPA recommends increasing the monitoring frequency to better understand the
temporal and spatial nature of cyanotoxin occurrence in the affected waterbody.
In some waterbodies, longer-term HABs can persist for many weeks to months with conditions
conducive to cyanobacterial proliferation. This can result in many excursions of the recommended toxin
values during a recreation season. The EPA recommends that when more than three excursions (an
exceedance during the 10-day assessment period) occur within a recreational season and that pattern
reoccurs in more than one year, it is an indication the water quality is or is becoming degraded such that
the water body no longer supports the recreational use. Recreational freshwaters at lower latitudes can
have longer recreational seasons compared with those waters found at higher latitudes. For those waters
in more temperate areas with a recreational season of approximately 100 days (i.e., from Memorial Day
to Labor Day), three excursions could translate into a maximum of 30 percent of the recreational season
not supporting the designated recreational use. Surface waters in areas with longer recreational seasons
can also experience conditions that can support HAB proliferation and cyanotoxin occurrence for a
longer period of the year. A maximum of three excursions across a recreational season reflects seasonal
dynamics and occurrence patterns of HABs within years and the potential for adverse health effects over
a short-term duration of exposure (i.e., approximately 30 days).
The EPA recognizes that multiple environmental factors can cause variability in bloom formation and
toxin production, and that some years may produce HABs that occur for long periods, or HABs of
shorter duration that occur repeatedly throughout a single recreational season, but such events may not
occur every year. Therefore, the EPA concludes that it is appropriate to consider a pattern of multiple
excursions within a recreational season as well as in multiple years (i.e., more than one year) when
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determining whether the use is attained. It is important to note that the years with multiple excursions do
not have to be consecutive to indicate a water quality problem. The upper-bound frequency (e.g., one
year out of three years) is a risk-management decision that states need to determine when developing
their water quality standards (WQS). States should include in their WQS the maximum number of years
a pattern of cyanotoxin excursions can occur for the recreational use to remain supported.
The EPA does not recommend using a 10-day average concentration or a rolling average to determine an
excursion, consistent with available toxicity information. States have flexibility in applying the 10-day
assessment period. Some may choose to use pre-defined 10-day assessment periods for water bodies
with a documented history of HAB occurrence or detection of elevated levels of cyanotoxins. Another
approach is to begin the 10-day assessment period upon observation of a visible bloom. However, only
considering the presence of visible blooms can miss episodes of elevated toxins (Raymond 2016). States
are encouraged to consider the application of the frequency and duration components to capture elevated
toxin concentrations, which may or may not coincide with the general proliferation of total
cyanobacteria at high densities. More information on implementation of these values as criteria is
provided in technical support materials.
6.4	Frequency and Duration for Swimming Advisory
Local and state governments can use swimming advisories to provide information to recreators on their
potential exposure to cyanobacteria and their toxins. Some local and state governments currently post
notifications for swimmers, in the form of advisories or warnings, when a cyanobacterial bloom is
reported in recreational waters or when cyanotoxin levels exceed advisory thresholds. Table B-2 in
Appendix B summarizes currently available information on state cyanotoxin-related guidelines and
associated actions, including the issuance of swimming advisories.
The EPA recommends that the magnitude of the swimming advisory value not be exceeded on any
single day, to provide timely information for people visiting beaches. The EPA also recommends that
any exceedance of the recommended magnitude result in a swimming advisory being issued until the
toxin concentration falls below the recommended magnitude. By increasing the monitoring frequency at
a site where a swimming advisory is issued, water resources managers may get a clearer understanding
of the temporal and spatial nature of water quality that can be useful in making decisions that protect the
recreational use. Increased monitoring can also help water managers decide when to remove an
advisory. The EPA has published materials for recreational water body managers that describe
communicating risk to the public about cyanotoxins in recreational water bodies, monitoring, and
responding to HABs (U.S. EPA 2017).
6.5	Recommended Recreational Criteria and Swimming Advisory for Microcystins and
Cylindrospermopsin
The magnitude, duration, and frequency are summarized in Table 6-1.
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Table 6-1. Recreational Criteria or Swimming Advisory Recommendations for Microcystins and
Cylindrospermopsin3
Application of
Recommended
Values
Microcystins
Cylindrospermopsin
Magnitude
(Hg/L)
Duration
Frequency
Magnitude
(Hg/L)
Duration
Frequency
Recreational
Water Quality
Criteria
8
1 in 10-day
assessment
period across a
recreational
season
More than 3
excursions in a
recreational season,
not to be exceeded in
more than one yearb
15
1 in 10-day
assessment
period across a
recreational
season
More than 3
excursions in a
recreational season,
not to be exceeded
in more than one
yearb
Swimming
Advisory
One day
Not to be exceeded
One day
Not to be exceeded
a These recommendations can apply independently within an advisory program or in WQS. States can choose to apply
either or both toxin recommendations when evaluating excursions within and across recreational seasons.
b 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 one year, it is an
indication the water quality has been or is becoming degraded and is not supporting its recreational use. As a risk-
management decision, states should include in their WQS an upper-bound frequency stating the number of years
that pattern can reoccur and still support its recreational use.
The recommended magnitude represents the concentration of microcystins or cylindrospermopsin that is
not expected to result in adverse human health effects from short-term recreational exposure to the
toxins via incidental ingestion while swimming, based on exposure to young children. The adverse
health effects include liver toxicity (for microcystins) and kidney toxicity (for cylindrospermopsin) and
could result from exposures to waters containing elevated levels of these toxins.
The water quality criteria developed by the EPA describe the magnitude, duration, and the frequency
of occurrence of pollutants. HABs may be caused or exacerbated by human activities and elevated
nutrient concentrations, but cyanotoxins differ from other pollutants as they are not typically discharged
into a water body. The EPA developed recommended criteria for these cyanotoxins that provide a
magnitude (8 pg/L microcystins or 15 pg/L cylindrospermopsin) and duration (not to be exceeded in
more than three 10-day assessment periods over the course of a recreational season). The EPA expects
states to make an explicit risk management decision regarding the frequency (i.e., the number of years
this pattern of exceedances can occur in the waterbody) and still support its recreational use.
As a basis for issuing a swimming advisory, the EPA recommends a concentration of 8 pg/L
microcystins or 15 pg/L cylindrospermopsin not be exceeded on a single day. This is consistent with the
goal of a swimming advisory to provide prompt information to people who wish to use the water body
for recreation.
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7.0 EFFECTS CHARACTERIZATION
7.1	Enhanced Susceptibility
Based on the available studies in animals, individuals with liver or kidney disease may be more
susceptible to health effects than the general population as the detoxification mechanisms in the liver
and impaired excretory mechanisms in the kidney may be compromised. Data from an episode in a
dialysis clinic in Caruaru, Brazil, where microcystins (and possibly cylindrospermopsin) were not
removed by treatment of dialysis water, identify dialysis patients as a population of potential concern in
cases where the drinking water source was contaminated with cyanotoxins.
The data on red blood cell acanthocytes observed in animal studies of cylindrospermopsin suggest that
individuals that suffer from anemia (e.g., hemolytic or iron-deficiency) might be a potentially sensitive
population. Several rare genetic defects such as abetalipoproteinemia (i.e., a rare autosomal recessive
disorder that interferes with the normal absorption of fat and fat-soluble vitamins from food) and
hypobetalipoproteinemia are associated with abnormal red blood cell acanthocytes, which appears to
result from a defect in expression of hepatic apoprotein B-100, a component of serum low-density
lipoprotein complexes (Kane and Havel 1989). Individuals with either condition might be sensitive to
exposure to cylindrospermopsin.
Available animal data are not sufficient to determine if there is a definitive difference in the response of
males versus females following oral exposure to microcystins. Fawell et al. (1999) observed a slight
difference between male and female mice in body weight and serum proteins, but no sex-related
differences in liver pathology. Available animal data are not sufficient to determine if there is a
definitive difference in the response of males versus females following oral exposure to
cylindrospermopsin.
7.2	Recreational Exposure Duration
Recreational exposure data available in the literature are expressed in two primary ways: 1) the volume
of water incidentally ingested during recreation (e.g., L/hr), and 2) the duration of the recreational
activity (e.g., minutes of recreation per day). A daily incidental ingestion rate distribution was developed
by combining these two distributions (for more information see Appendix E). The 90th percentile of the
daily incidental ingestion rate distribution for children (see section 7.3) was selected for the derivation of
the criteria and swimming advisories, consistent with the 2000 Human Health Methodology.
The EPA identified the following sources of data on the duration of the recreational activity: the EPA's
EFH (2011); Schets et al. (2011); and DeFlorio-Barker et al. (2017) (DFB study). See Table 7-la and
Table 7-lb for summary overviews of these studies. One major difference between the studies is in the
unit of exposure, reported in minutes per day in one study and minutes per swimming event in the two
other studies.
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Table 7-la. Durations of Recreational Exposures in Minutes per Day
Reference
Recreational
Environment
Age Group
(Years Old)
Sample Size
Mean
Units
U.S. EPA
Exposure
Factors
Handbook
(2011)
In Outdoor Pool or
Spa
1 to 4
9
85.6
minutes per day
5 to 11
15
164.2

12 to 17
5
97.0

18 to 64
44
117.6

>64
10
78.9

Table 7-lb. Durations of Recreational Exposures in Minutes per Swimming Event3
Reference
Recreational
Environment
Age Group
(Years Old)
Sample Size
Mean
Units
Schets et al.
(2011)
Freshwater
< 15 Years
1,689
79.0
minutes per event
16+
4,123
54.0
Swimming Pool
< 15
1,689
81.0
minutes per event
16+
4,123
67.5
DeFlorio-
Barker et al.
(2017)
Freshwater
< 1
171
56
minutes per event
1 to 3
1,061
66.7
4 to 7
l,738b
88.5
8 to 12
2,136°
92.9
13 to 18
1,855
64
19 to 34
5,478
45.4
35+
8,058
47
Marine
< 1
350
60.5
1 to 3
2,687
79.1
4 to 7
4,260
107.8
8 to 12
5,398
121.4
13 to 18
4,021
102
19 to 34
10,786
68.2
35+
19,745
66.9
a Additional information is needed to translate minutes per event to minutes per day.
b Number of children ages 4-7 reported to have contact with water: 1,562.
0 Number of children ages 8-12 reported to have contact with water: 1,901.
The EPA considered these three studies and selected the EFH for use in deriving the criteria and
swimming advisories primarily because the EFH dataset represents exposures in minutes per day. Other
datasets measured the duration of recreational exposure on an event basis, which require assumptions
about how many recreational events occur per day to create the relevant distribution. The EPA
conducted analyses comparing these datasets, as described below to evaluate the differences in the
distributions given differences in sample size, and evaluated differences given different assumptions of
number of events per day.
The EFH (U.S. EPA 2011) lists time spent per 24 hours in an outdoor spa or pool for different age
groups (including children five to 11 years old). The EPA acknowledges that the reported sample size
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for this study is small (n = 15) for the five-to-11-year-old group. Schets et al. (2011) demonstrate that
time spent in swimming pools is similar to time spent in freshwater and therefore EPA concluded that
these data are representative of recreational exposure in freshwater. The EFH also presents data for
minutes spent "outdoors at a pool/river/lake." The EPA did not select these data as it is uncertain if this
is time spent in the water, or total time "at" the location.
Schets et al. (2011) investigated swimming durations in freshwater, marine water, and pools. They
surveyed 8,000 adults, 1,924 of whom also provided estimates for their eldest child (<15 years of age)
and found that children spend, on average, 25 minutes longer swimming in freshwater compared to
adults. Schets et al. (2011) reported similar mean duration times between swimming pools and
freshwater locations for children less than 15 years old (average of 81 and 79 minutes per event,
respectively; upper 95 percent CI: 200 and 270 minutes per event, respectively).
The DFB study (DeFlorio-Barker et al., 2017) 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. 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 2,136 children ages eight to 12 years and 1,738 children ages
four to seven recreating in freshwater. Marine recreators spent more time in the water compared with
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 with the freshwater sites in the
Great Lakes.
Although not represented in Table 7-1 a or b, the EPA's OPP uses a different approach to estimate
chemical exposures for children during pool swimming, for use in its SWIMODEL (U.S. EPA 2003).
This model simulates short-term exposure using a high-end estimate of exposure-time per event to
represent a maximum, one-time exposure. It also simulates intermediate/long-term exposure using a
shorter event duration to represent an average of maximum and minimum exposures over time. Among
competitive children swimmers, the short-term exposure duration used by the SWIMODEL is one hour
per day for children ages six to 10 and two hours per day for children ages 11 to 15 years based on a
survey of swim coaches (U.S. EPA 2003). The competitive swimming scenario (e.g., children
swimming laps) is appropriate for conducting risk assessments of exposure to swimming pool
chemicals. However, it is less relevant to children's recreational activities in lakes or rivers and therefore
was not used in this assessment.
7.2.1 Comparison of Duration of Exposure Distributions
Because the DFB study has a much larger sample size compared to the study results reported in EPA's
EFH, the EPA conducted a statistical analysis to compare the distributions of duration of exposure.
Because the DFB study age groupings and the EFH age groupings do not exactly align, the EPA
compared the four-to-seven and the eight-to-12 age groups from the DFB study with the five-to-11 age
group presented in the EFH. Both studies include self-reported data, which are prone to recall bias.
Adult recollection of their children's time spent in the water is also uncertain. However, there is no
reason to believe there would be differential recall bias between the studies.
Table 7-2 shows the parameters used to create distributions for EFH and DFB studies. The EPA used
assumptions of one swimming event per day and two events per day to translate the DFB duration from
minutes per event to minutes per day for two different age groups. The EPA assumed the underlying
distributions of exposure durations were log-normal. The observed mean and standard deviations in
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Table 7-2 defined the parameters of the underlying log-normal distributions. The standard deviations
take into consideration the numbers of samples, and therefore address differences in numbers of
participants in the EFH and DFB studies. A large number (1 million) of samples were drawn from each
log-normal distribution defined using these parameters. The distributions were truncated to reflect the
observed maximum and minimum values in the EFH and DFB studies for the age groups of interest.
Figures 7-1 a and b show the five resulting distributions: the EFH distribution and the DFB distributions
assuming one (Figure 7-la) and two (Figure 7-lb) events per day.
Table 7-2. Parameters Used to Fit Recreation Duration Distributions in Freshwater
Parameter Source
Age Group
(sample size)
Mean
(min/day)
Standard
deviation
Minimum
(min/day)
Maximum
(min/day)
EPA 2011 EFH
(minutes/day)
5 to 11 years
(n=15)
164.2
103.97
25
450
DFB 2017
(minutes/day, assuming
one event/day)
4 to 7 years
(n= 1,562)
88.5
(1 event)
62.8
2
300
DFB 2017
(minutes/day, assuming
two events/day)
177
(2 events)
125.6
4
600
DFB 2017
minutes/day, assuming one
event/day)
8 to 12 years
(n= 1,901)
92.9
(1 event)
64.7
2
360
DFB 2017
minutes/day, assuming two
event/day)
185.8
(2 events)
129.4
4
720
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Figure 7-1 a and b. Comparison of Children's Duration of Time Spent Recreating
Figure 7-la.
Figure 7-lb.
1000
1000
>-
TJ
3
c
aj
4—1
c
aj
Q.
l/l
100
10
100
10
EFH	DFB	DFB
5 to 11 years 4 to 7 years 8 to 12 years	5 to 11 years 4 to 7 years 8 to 12 years
1 event/day 1 event/day	2 events/day 2 events/day
Comparison of children's time spent in water between EP A's 2011 Exposure Factors Handbook (five to 11 years old)
(EFH; U.S. EPA 2011) and the DeFlorio-Barker study (DFB) (four to seven and eight to 12 years old) (DeFlorio-Barker et
al. 2017) assuming one swimming event per day (Figure 7-la) or two swimming events per day (Figure 7-lb) for the DFB
data. The range of each distribution is represented by the vertical solid line, the short horizontal line indicates the median,
and blue diamonds represent the mean. Letters beside the means denote significant differences of the means.
The EPA conducted two statistical tests to compare these distributions; one based on the means of the
distributions and the other based on the full distributions. The full duration distribution, not the mean, in
combination with the distribution of volume ingested per hour, was used to calculate the daily incidental
ingestion rate. The EPA also explored how these comparisons change when one assumes that children
engage in one or two swimming events per day (e.g., those who swam, took a break, and then re-entered
the water at a later point in the day). The changes in the parameters are shown in Table 7-2.
For the comparison of the means, the EPA used a two-tailed t-test with unequal variances. The mean of
the EFH is statistically different from both the DFB age group means (p-value < 0.001) for both one and
two events per day. The means of the two DFB age groups are not statistically different from each other
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(p-value = 0.08) assuming both one event and two events per day. Statistical differences between the
means are denoted by letters (a and b) in Figure 7-1. Assuming two events per day for the DFB studies,
the means for both DFB study age groups are significantly higher (p < 0.001) than the EFH mean. The
larger sample size available in the DFB study results in a narrower confidence interval around the mean
time spent in water, compared to the 95 percent CI for the mean used in the EFH.
For the comparison of the distributions, the EPA used the Kruskal-Wallis test. Results show that the
EFH distribution is not statistically significantly different from either DFB age group distributions (p-
value = 0.499, assuming one event per day; p-value = 0.498, assuming two events per day).
The EPA concluded that because the EFH and DFB distributions are not significantly different, the EFH
dataset is the most appropriate for deriving criteria and swimming advisory values as it does not require
additional assumptions about the number of swimming events that occur per day. The 90th percentile
incidental ingestion rates are shown in Table 7-3 below for the EFH distribution and for the DFB
distributions. The resulting 90th percentiles of daily incidental ingestion rate are also shown. The 90th
percentile of daily ingestion rate based on the EFH distribution most closely corresponds to the 90th
percentile of daily ingestion rate using the DFB dataset when two swimming events per day are
assumed.
Table 7-3. Calculated Daily Incidental Ingestion Rates Based on EFH and DFB Datasets
Volume per Hour
Data Source
Event Duration Data
Source
Age Group (years)
Events per Day
(if assumed)
90th Percentile
Daily Ingestion
Rate (L/day)
Recreational
AWQC Appendix E
Ml dataset (L/lir)
EPA Exposure Factors
Handbook (201 l)a
(lir/day)
5 to 11
not needed
0.21
DeFlorio-Barker et al.
(2017) (DFB)
(hr/event)
4 to 7
1
0.11
2
0.23
8 to 12
1
0.12
2
0.24
a This distribution was used in the derivation of the criteria and recreational swimming advisories.
7.3 Evaluation of Health Protective Values for Different Lifestages
The EPA compiled and evaluated available information for various life stages before selecting children
ages six to 10 years as the basis for the recreational criteria values or swimming advisory. This section
discusses potential health protective values for children and adults (section 7.3.1) and focuses on
exposures of younger children (less than six years) (section 7.3.2).
7.3.1 Consideration of Multiple Lifestages
The EPA used the Appendix E and the Dufour et al. (2017) dataset provided in U.S. EPA (2018a) to
generate the box and whisker plots shown in Figure 7-2 for three life stages (children six to 10 years,
children 11 to 17 years, and adults 18 years or older). The Appendix E Dufour data for volume ingested
per swimming event was normalized to one hour. Each participant's volume ingested was adjusted to
one hour based on the length of time that participant reported being in the water. The EPA converted
volume of water ingested from L/event to L/hour, then used the swimming duration per day from the
EPA's 2011 EFH (hours/day). The distributions were assumed to be log-normal and the plot is
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visualized in log space. The EPA used the Appendix E Dufour data on ingestion rate (shown in
Figure 7-2) and the body weight estimates from the EPA's (2011) EFH (kg) to calculate the ingestion
normalized by body weight (L/kg/day) shown in Figure 7-3.
Figure 7-2. Incidental Ingestion During Recreational Activity Based on Age (Appendix E)
0.1
T3
01
4—1
to
01
00
c
01
E
_2
o
>
0.001
6 to 10	11 to 17 18 and over
years	years
In this box plot, the horizontal line the middle of the box is the median (Q2). The length of the box is the interquartile range
(IQR) or the 25th percentile to the 50th percentile. The upper whisker vertical line extends to the greatest value less than or
equal to Q3+1.5*IQR; the lower whisker extends to the smaller value less than or equal to Q1-1.5*IQR. The dots represent
extreme values that are either greater than the upper whisker or lower than the lower whisker.
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Figure 7-3. Comparison of Children and Adults Incidental Ingestion Rate During Recreational
Activity Adjusted for Body Weight
0.0070
ro
cc
c
o
CD
CuO
c
c
CD
U
c
>-
"O
CuO
c
(D
L)
(D
Q_
O
(T)
0.0060
0.0050
0.0040
0.0030
0.0020
0.0010
0.0000
6 to 10 years
11 to 17 years
18+ years
Body weight varies by age. Table 8-1 in the EPA's EFH (U.S. EPA 2011) reported recommended
statistics based on the 1999-2006 National Health and Nutrition Examination Survey. Table 7-4 shows
the mean body weight for the age groups compared in this section (U.S. EPA 2011).
Table 7-4. Mean Body Weight by Age Group Based on U.S. EPA (2011)
Age Group
Body Weight (kg)
Children 6 to 10 years
31.8
Children 11 to 17 years
56.8
Adults 18 to 64
80
The EPA estimated recreational health protective values for these three different age groups for
microcystins and cylindrospermopsin to demonstrate the variability due to body weight, recreational
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water incidental ingestion, and exposure duration by lifestage. Inputs for these calculations are in
Table 7-5.
Table 7-5. Inputs for Calculation of Protective Values for Microcystins and Cylindrospermopsin
Age Group
Ingestion Ratea (L/day)
Body Weight b (kg)
Children 6 to 10 years
0.21
31.8
Children 11 to 17 years
0.13
56.8
Adults 18+ years
0.10
80.0
a Value is 90th percentile of the combined distribution (i.e., ingestion and duration data combined); see Appendix E.
b For children age 6 to 10 years, the mean body weight for the 6-to-10-year age group (31.8 kg) was used. For 11 to
17 years, the mean body weight for the 11- to 15-year-old age group (56.8 kg) was used because it was the closest age
group available from the EPA's Exposure Factors Handbook (U.S. EPA 2011). For adults 18+ the mean body weight
for the 21+ year age group (80 kg) was used (U.S. EPA 2011).
As illustrated in Figure 7-4, the AWQC and swimming advisories the EPA calculated to be protective of
children ages six to 10 years are also protective of older children and adults.
Figure 7-4. Comparison of Calculated Recreational Health Protective Values for Microcystins and
Cylindrospermopsin for Children, Older Children, and Adults
Q0
a>
ro
>

¦4—1
U

-------
7.3.2 Exposure Factors for Children Younger Than Six Years Old
In the calculation of the cyanotoxin values reported in section 6, the EPA utilized exposure parameters
reported in the EFH (U.S. EPA 2011) and peer-reviewed study data (study design presented in Dufour et
al. 2017; data analyzed in Appendix E; U.S. EPA 2018a). The available incidental ingestion volume and
exposure duration values from the Appendix E and the EPA's EFH (U.S. EPA 2011), respectively, were
limited to specific age ranges. For incidental ingestion, the data reported were limited to children
six years old and older because the Dufour et al. (2017) study design did not include children younger
than six years. The EPA's EFH (U.S. EPA 2011) provided a mean recreational exposure duration for
children ages one to four years (1.4 hour/day). This duration is shorter than the mean duration for
children ages five to less than 11 years (2.7 hour/day). Values for exposure duration were not available
for children younger than one year.
The EPA found one other study that characterized incidental ingestion for children. Schets et al. (2011)
reported incidental ingestion volumes and durations of recreational events for children ages zero to < 15
years. However, the study did not further divide this cohort into younger children and older children.
The incidental ingestion data for children <15 years represent parental estimates of volumes of
freshwater incidentally ingested by their children, which is a different methodological approach
compared to the more quantitative approach used by Dufour et al. (2017). The exposure durations were
also parental estimates.
The EPA calculated the 90th percentile incidental ingestion rate per day for children younger than six
years old in order to compare the daily ingestion rate (L/day) between children six to 10 years and those
younger than six years. The daily ingestion rate (0.21 L/day) used to derive the recreational criteria was
calculated by combining the distributions for incidental ingestion and exposure duration via a
probabilistic (Monte Carlo) analysis (described in section 4.2.3.1). The daily ingestion rate for children
younger than six years old (0.11 L/day) was a mixed-age estimate calculated by dividing the 90th
percentile for incidental ingestion for children age six to 10 years (0.077 L/hour; see Appendix E) by the
mean exposure duration for children one to four years (1.4 hour/day; U.S. EPA 2011). The daily
ingestion rate for children younger than six years old is lower than for children six to 10 years old. This
calculation was also performed using data from Schets et al. (2011) and resulted in a daily ingestion rate
of 0.1 L/day.6 The EPA evaluated the effect of using parameter values for children younger than
six years by including an age-specific body weight and the mixed-age estimate for the daily ingestion
rate (L/day) parameters. Table 7-6 shows a comparison of the microcystins magnitude for the two
different age groups, children ages six to 10 years and children ages one to less than six years.
The estimates for children younger than six years have large uncertainties given the lack of measured
incidental ingestion data specifically for this age group. Information on exposure durations for children
less than one-year-old is also lacking. Because exposure durations are greatest for five- to 11-year-olds,
the EPA concluded that calculating the ingestion rate using a higher duration was protective of children
younger than six years old. Research designed to fill this data gap could be helpful for characterizing the
risks to children younger than six years old. Specifically, data to better characterize the volume of water
ingested during recreational events would enhance EPA's confidence that the criteria values are
protective of children younger than six years old.
6 This number was calculated as follows: 0.07 L/hour (90th percentile ingestion volume for age zero to less than 15 years
from Schets et al. (2011)) divided by 1.4 hr/d (mean for children one to four years from U.S. EPA 2011).
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Table 7-6. Microcystins Magnitude Comparison Between Children Six to 10 and Children
One to Less Than Six Years Old
Age Group
RfD (jig/kg/dav)
Body Weight (kg)
Ingestion
Rate (L/dav)
Magnitude (jig/L)
Magnitude (jug/L)
Rounded
6 to 10 years
0.05
31.8
0.21
7.57
8
1 to < 6 years
0.05
15.6 a
0.11
7.09
7
a This value is the weighted mean of the age groups one to less than two years, two to less than three years, three to less
than six years (U.S. EPA 2011).
7.4 Other Recreational Exposure Pathways
The EPA selected primary contact activities and incidental ingestion of water as the primary exposure
pathway for derivation of the recreational criteria and swimming advisories. Inhalation and dermal
toxicity data were not available; however, there are limited available data to estimate inhalation and
dermal exposure. The EPA conducted analyses to compare inhalation and dermal exposure to incidental
ingestion of the cyanotoxins while recreating. Section 7.4.1 compares recreational ingestion and
inhalation exposures to microcystins. Similarly, section 7.4.2 compares recreational ingestion and
dermal exposure. Section 7.7 briefly discusses tribal considerations. Further research is needed to better
understand the toxicity from inhalation and dermal exposure to cyanotoxins. The EPA describes the
screening analyses in this section because sufficient data to quantify toxicity via these routes were
not available.
7.4.1 Inhalation of Cyanotoxins
Volatilization of microcystins and cylindrospermopsin from water to air is not expected due to their size
and charges. Both cyanotoxins are rather large molecules compared to volatile chemicals. Microcystin-
associated acid groups are charged at the pH of normal surface waters. Cylindrospermopsin features
both negative and positive changes and, like other zwitterions, do not volatilize significantly into the air
from water (Butler et al. 2012).
According to Wood and Dietrich (2011), waterborne cyanotoxins can be aerosolized through a bubble-
bursting process, in which the cyanobacteria and cyanotoxins are ejected and carried into the air where
they can bind to particulate matter. Microcystins that are free or bound to particulate matter in air can be
deposited into the deepest bronchiolar or alveolar cavities; air borne cyanobacterial cells from
aerosolized water droplets would likely be deposited in the upper respiratory tract (Wood and
Dietrich 2011).
The EPA identified field studies that measured recreators' exposure levels to aerosols containing
microcystins from lakes with blooms containing microcystin-producing Microcystis aeruginosa. The
studies found low inhalation exposures. In one study, Backer et al. (2008) used personal air samplers in
a three-day study of recreational activities in a lake with a cyanobacterial bloom, either carried by the
study participant or placed on the participant's boat. The microcystin concentrations in air ranged from
below the limit of detection (0.0037 ng/m3) to 0.456 ng/m3. Backer et al. (2010) also detected
microcystins in ambient air for one day, at one lake, and only from the shoreline sampler. The average
air concentration was 0.052 ng/m3. Backer et al. (2010) also collected 44 personal air samples, which
ranged from the limit of detection (0.1 ng/m3) to 0.4 ng/m3. The study identified no associations between
health effects and microcystin concentrations from inhalation exposure from activities that included
swimming, water skiing, Jet Skiing, or boating. The authors noted that the daily mean microcystin
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concentrations in personal air samples did not correlate with the concentrations of Microcystis
aeruginosa cells, dissolved microcystins, or total microcystins in the sampled lake water.
In another study by Backer et al. (2010), the lakes had a wider range of concentrations of microcystins
(< 10 to > 500 (J,g/L). The study authors measured microcystins exposure via personal air samplers, nasal
swabs, and blood samples for individuals whose activities included swimming, boating,
tubing/wakeboarding, riding watercraft, wading, and fishing at the lakes. They found low microcystin
levels in personal air samplers below the limit of detection (0.1 ng/m3) to 2.89 ng/m3 and also in nasal
swabs below the limit of detection (0.1 ng) to 5 ng. The average aerosolized microcystin concentration
was approximately 0.3 ng/m3. Based on the nasal swab data, the investigators estimated on average that
the adults inhaled 0.8 ng of microcystins. Microcystin concentration in the water-soluble plasma fraction
of the study subjects was also below the limit of detection (1 (J,g/L). The investigators cautioned that
microcystin might be bound to a protein component in the blood or sequestered in liver tissue.
Wood and Dietrich (2011) studied Lake Rotorua (New Zealand) when it was experiencing a dense
bloom of microcystin-producing Microcystis. The authors measured a maximum microcystin
concentration in the water of 2,140 (J,g/L and air concentrations from 0.0003 to 0.0018 ng/m3.
Cheng et al. (2007) used high volume and personal air samplers to measure microcystins in the air at a
lake with a cyanobacterial bloom. The authors measured low microcystin concentrations in the water
(approximately 1 (J,g/L) and air concentrations ranging from below the detection limit (0.02 ng/m3) to
0.08 ng/m3.
The EPA performed a screening analysis to characterize potential relative exposures. The EPA analyzed
the relative potential dose of the cyanotoxins via inhalation exposure compared to oral ingestion to
evaluate if recreational criteria values or swimming advisories based on ingestion could be protective of
the other exposure routes. Although the recreational use is primary contact recreation, such as
swimming, data are available for secondary contact activities such as Jet Skiing or boating and white-
capped wave, bubble-bursting action, which can result in cyanotoxins becoming aerosols (microscopic
liquid or solid particles suspended in air).
Using the information from Cheng et al. (2007) and inhalation exposure parameters provided in the
EPA's EFH (2011), the EPA compared the estimated microcystin ingested dose to the inhaled dose. The
first step in this comparative screening analysis was to calculate the incidental ingestion dose using the
following equation:
Ingestion dose (ng/day) = Ingestion rate x Concentrationwater
Where:
Ingestion rate = 90th percentile incidental ingestion rate based on combined distributions
of incidental ingestion (Appendix E) and recreational duration
(U.S. EPA 2011) (L/day)
Concentrationwater = assumed concentration in water (1,000 ng/L from Cheng et al. (2007))
(ng/L)
The parameters used in the calculation of the estimated ingestion dose for each age group are presented
in Table 7-7.
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Table 7-7. Ingestion Parameters and Estimated Ingestion Dose for Screening-level Comparative
Inhalation Exposure Analysis
Age Group
Ingestion Rate (L/day)a
Concentration in Water
(ng/L) b
Ingestion Dose (ng/day)
Children
0.21
1000
210
Adults
0.10
1000
100
a Daily recreational incidental ingestion rate calculated in combined distribution analysis for children and adults as
described in section 4.2.3.1.
b Cheng et al. (2007) measured 0.08 ng/m3 in air near surface waters with a concentration of 1 |ig/L microcystins. This
concentration in water was assumed as part of this analysis because Cheng et al. (2007) provided aerosolized levels given
a specific concentration in water.
The second step in the comparative screening analysis was to estimate the inhaled dose using the
following equation:
Inhalation dose (ng/day) = Inhalation rate x Inhalation duration x Concentration^
Where:
Inhalation rate = inhalation rate from the EPA's EFH (U.S. EPA 2011; Table 6-2)
(m3/min)
Inhalation duration = inhalation exposure duration from the EPA's EFH (U.S. EPA 2011;
Table 16-20) (minutes/day)
Concentrationair = concentration in air (0.08 ng/m3) assumed from Cheng et al. (2007)
(ng/m3)
The inhalation exposure parameters the EPA used in this equation and the resulting estimated inhaled
dose are listed in Table 7-8. The EPA selected inhalation rates for children and adults from the EPA's
EFH (U.S. EPA 2011). For this conservative comparative analysis, the EPA selected the highest 95th
percentile short-term, moderate intensity activity level inhalation rate—the volume of air inhaled per
minute (m3/minute)—listed for children and adults in EPA's EFH Table 6-2 "Recommended Short-
Table 7-8. Inhalation Exposure Parameters and Estimated Inhaled Dose
Age
Group
Inhalation
Rate (m3/min)a
Duration of Inhalation
Exposure per Day
(minutes/day)b
Daily Inhalation Rate
Adjusted for Duration
of Exposure (m3/day)
Concentration
in Air
(ng/m3)c
Estimated
Inhalation Dose
(ng/day)
Children
0.037
560
21
0.08
1.7
Adults
0.04
511
20
0.08
1.6
a The EPA's Exposure Factors Handbook (EFH; U.S. EPA 2011) did not report recommended short-term, moderate
intensity activity level inhalation rate values for children or adults in aggregate; used highest inhalation rate listed for
children and adult age groups for this conservative screen. For children, it was the age group 16 to < 21 years, and for
adults, it was 51 to < 61 years.
b Values are the longest 90th percentile duration reported for child and adult age groups in the EPA's EFH (U.S. EPA 2011)
from Table 16-20 "Time Spent (minutes/day) in Selected Outdoor Locations, Doers Only, Outdoors at a Pool/River/Lake."
The child and adult age groups with the longest durations spent near or in the water were children 1 to 4 years old and adults
18 to 64 years old.
0 Cheng et al. (2007) measured 0.08 ng/m3 in air near surface waters with a concentration of 1 mg/L microcystins.
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Term Exposure Values for Inhalation (males and females combined)" The child and adult age groups
with the highest of these inhalation rates were 16 to < 21 years and 51 to < 61 years, respectively.
To estimate the amount inhaled in a day, the EPA multiplied the inhalation rates for children and adults
by an estimated daily inhalation exposure duration for each of these age groups. The EPA estimated
daily inhalation exposure duration using a different dataset from the set it used for the incidental
ingestion analysis (described in section 4.2.3.1). This was because people do not need to enter the water
to be exposed via inhalation, they only need to be mar or at the water. In contrast, recreators who
incidentally ingest water while swimming must be in the water.
The EPA's EFH (U.S. EPA 2011) provides in Table 16-20 the time spent (in minutes/day) outdoors at a
pool/river/lake. The EPA estimated inhalation exposure duration using the number of minutes per day
spent outdoors at a pool/river/lake (U.S. EPA 2011). The EPA selected the longest 90th percentile
duration values reported for child and adult age groups. The child and adult age groups with the longest
times spent outdoors at a pool/river/lake were children one to four years old and adults 18 to 64 years old.
A comparison of the EPA's EFH data provided for time spent outdoors at a pool/river/lake and time
spent in the water indicates that all age groups spent more time at a pool/river/lake than they spend in a
pool/spa (U.S. EPA 2011). Consistent with the trend that children have longer durations of recreation in
water than adults, children's time spent near recreational waters was greater than adults. The children's
age group exposure patterns differed between the datasets. The data suggest younger children (one to
four years) spend more time at recreational waters compared to school-aged children (five years and
older), but children five to 11 years old spend more time in the water compared to other children (U.S.
EPA 2011).
It is reasonable that younger children spend more time engaged in activities at a pool/river/lake
compared to time spent recreating in recreational waters. The EPA selected this dataset to characterize
inhalation exposure because younger children can spend more time playing on a beach, where they can
be exposed to aerosolized cyanotoxins, than in the water where incidental ingestion can be the primary
route of exposure.
The final step for this comparative screening analysis was to compare the ingestion and inhalation doses.
The results are presented in Table 7-9. Using conservative assumptions for inhalation rates and
inhalation exposure duration and comparing with daily incidental ingestion rates, the ingested dose is
estimated to be higher than the estimated inhaled dose for children and adults. This analysis is for
screening only and is highly uncertain. Further research is needed to better understand the toxicity from
inhalation exposure to cyanotoxins.
Table 7-9. Results of Screening Analysis Comparing Ingestion and Inhalation Doses
Age Group
Ingestion Dose (ng/day)
Inhalation Dose (ng/day)
Children
210
1.7
Adults
100
1.6
a Calculations used unrounded parameters; results slightly differ with rounded values.
This analysis supports the conclusion that the inhaled dose can be much less than the incidental
ingestion dose while recreating. The studies conducted by Backer et al. (2008, 2010) found low
microcystin levels in aerosols above lakes with low or high microcystin concentrations and did not
detect microcystin levels in the blood of study participants. In an animal study, no clinical signs or
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effects on body or organ weights were observed after exposure to microcystin-LR aerosol (Benson et al.
2005). The EPA did not conduct a similar analysis for cylindrospermopsin because published measured
air concentration data for this cyanotoxin were not available.
The California Environmental Protection Agency (CalEPA) came to a similar conclusion for water
skiers (Butler et al. 2012). They cited Cheng et al. (2007) and noted that their results showed that a liter
of water contains 700,000 to 800,000 times the amount of cyanotoxins as in a cubic meter of air.
CalEPA calculated that this concentration is equivalent to 1.3 to 1.4 [j,L aerosolized microcystins/m3.
Compared to the ingestion assumptions used for swimmers in the calculation of their recreational
guideline (i.e., 50 mL/hour), CalEPA calculated that a water skier would have to inhale at least 35,000
m3/hour while skiing to achieve a dose equal to the swimmer, which is 17,000 times the inhalation rate
of a marathon runner. CalEPA concluded that a water skier would not inhale enough aerosol to receive a
dose similar to what a swimmer gets from ingestion.
Another comparison considers spray exposures from personal watercraft and boat spray. Sinclair et al.
(2016) modeled a water-spray exposure scenario and observed much lower exposures than those
resulting from swimming or limited contact recreational activities reported in the previous study. Thus,
the EPA expects that the comparison above based on exposure from secondary contact recreation is
protective of primary contact recreation. Sinclair et al. (2016) also measured urinary concentrations of
cyanuric acid after 26 participants' exposure to spray in a simulated 10-minute car wash situation.
Subjects wore a protective coverall with hood, vinyl gloves, waterproof footwear, and safety glasses to
ensure that only their face and mouths were exposed. The estimated median and 90th percentile
ingestion volumes were 0.18 and 1.89 mL, respectively. Converted to a duration of one hour, the
amounts would be 1.08 mL and 11.3 mL, which are much lower than the incidental ingestion intakes per
hour.
7.4.2 Dermal Absorption
The EPA did not find any peer-reviewed measured data for microcystins or cylindrospermopsin dermal
absorption. The EPA's Dermal Exposure Assessment: A Summary of EPA Approaches (U.S. EPA 2007)
states that to get through the skin, a chemical must dissolve into the stratum corneum, which is a
stabilized lipid barrier; therefore, lipid solubility is required initially (U.S. EPA 2007).
The EPA performed a comparative screening analysis to estimate the potential dermal absorbed dose of
microcystins and compare it to the incidentally ingested dose. The first step in this comparative
screening analysis was to calculate the incidental ingestion dose using the following equation:
Ingestion dose = Ingestion rate x Concentrationwater
Where:
Ingestion rate	= 90th percentile incidental ingestion rate based on combined
distributions of incidental ingestion (Appendix E) and recreational
duration (EFH; U.S. EPA 2011) (L/day)
Concentrationwater = concentration in water assumed as the health protective value the EPA
derived in this document for microcystins (mg/L)
The parameters used in the calculation of the estimated ingestion dose are presented in Table 7-10.
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Table 7-10. Ingestion Parameters and Estimated Ingestion Dose for Screening-level Comparative
Dermal Absorption Exposure Analysis
Ingestion Rate (L/day)a
Chemical Concentration in Water
(mg/L)b
Ingestion Dose (mg/day)
0.21
0.008
0.002
a Daily recreational incidental ingestion rate calculated in combined distribution analysis for children and adults as
described in section 4.2.3.1.
b Concentration in water assumed to be the health protective value for microcystins the EPA derived in this document,
converted to mg/L.
To estimate the potential dermal absorbed dose, the EPA used exposure equations in its Risk Assessment
Guidance for Saperfand (U.S. EPA 2004). The first step was to use chemical-specific octanol-water
partition coefficient and molecular weight values to estimate dermal permeability, a parameter needed
for the equation to estimate dermally absorbed dose. Octanol-water partition coefficients are available
for four microcystins, including microcystin-LR. Ward and Codd (1999) estimated the log octanol-water
partition coefficients of microcystin-LR, -LY, -LW and -LF using high performance liquid
chromatography (HPLC) as 2.16, 2.92, 3.46, and 3.56, respectively. The EPA could not estimate
cylindrospermopsin dermal absorption due to the lack of these lipophilicity parameters.
The equation to estimate skin permeability coefficient from U.S. EPA (2004) is:
Log Kp= -2.80 + 0.66 x log KOW-0.0056 x MW
Where:
Kp	= dermal permeability coefficient of compound in water
(cm/hour)
Kow = octanol-water partition coefficient from Ward and Codd (1999)
(dimensionless)
MW = molecular weight (g/mole)
The chemical-specific dermal exposure parameters used to estimate skin permeability are listed in Table
7-11.
Table 7-11. Parameters Used to Estimate Skin Permeability of Microcystins
Microcystin Congener
Log Kowa
Molecular
Weight (g/mole)
Skin Permeability
Coefficient (Log KP)
Skin Permeability
Coefficient (KP)
(cm/hour)
Microcystin-LR
2.16
995.17
-6.95
1.1 x 10"7
Microcystin-LY
2.92
1002.16
-6.48
3.3 x 10"7
Microcystin-LW
3.46
1025.2
-6.26
5.5 x 10"7
Microcystin-LF
3.56
986.16
-5.97
1.1 x 10"6
a Ward and Codd (1999)
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The equation to estimate dermal absorbed dose for highly ionized organic chemicals from U.S. EPA
(2004) is:
Dermal absorbed dose = Kp x Concentrationwater x t
Where:
Dermal absorbed dose = dermal absorbed dose per event (mg/cm2-event)
Kp	= dermal permeability coefficient of compound in water
(cm/hour)
Concentrationwater	= chemical concentration in water (mg/cm3)
t	= event duration (hour/event)
The exposure parameters and estimated microcystins absorbed dose based on these calculations are
presented in Table 7-12.
Table 7-12. Dermal Absorption Exposure Parameters and Estimated Dermal Absorbed Dose
Microcystin
Congener
Chemical Cone, in
Water (mg/cm3)a
Event Durationb
(hour/event)
(mean for 5- to
11-year-olds)
Dermal
Absorbed Dose
per Event
(mg/cm2-event)
Total Body
Surface Area
(cm2)0
Dermal Absorbed
Dose per Event
(mg/event)
Microcystin-LR
8 x 10"6
2.7
2.4 x 10"12
14,800
3.6 x 10"8
Microcystin-LY
7.1 x 10"12
1.0 x 10"7
Microcystin-LW
1.2 x 10"11
1.8 x 10"7
Microcystin-LF
2.3 x 10"11
3.4 x 10"7
a Concentration in water assumed to be the health protective value for microcystins the EPA derived in this document,
converted to mg/cm3.
b Event duration is defined as time spent per day in outdoor pool or spa at home as reported in the EPA's EFH (U.S. EPA
2011).
0 Value is 95th percentile Children 6 to 10 years from U.S. EPA (2011), converted to cm2.
The final step for this comparative screening analysis was to compare the ingestion and dermal absorbed
doses. The results are presented in Table 7-13. The estimated ingested dose is higher than the estimated
dermal absorbed dose for children. This assessment is highly uncertain. Further research is needed to
better understand the toxicity from dermal exposure to cyanotoxins.
CalEPA also concluded dermal absorption of microcystins and cylindrospermopsin while swimming is
not expected to be significant due to the large size and charged nature of these molecules (Butler et al.
2012). CalEPA eliminated the dermal absorption pathway from its risk assessment of microcystins and
cylindrospermopsin citing evidence that similarly large molecules such as antibiotics have not been able
to be formulated in a way to penetrate the skin (Butler et al. 2012). A U.S. Army-contracted in vitro
study by Kemppainen et al. (1990) measured microcystin dermal penetration in 48 hours through
excised human abdominal skin and found 0.9 (± 0.3) percent of the total dose in water penetrated
through the skin; however, this study has not been peer reviewed.
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Table 7-13. Results of Screening Analysis Comparing Ingestion and Dermal
Absorbed Doses
Microcystin Congener
Ingestion Dose (mg/day)
Dermal Absorbed Dose3
(mg/event)
Microcystin-LR
0.002
3.6 x 10"8
Microcystin-LY
0.002
1.0 x 10"7
Microcystin-LW
0.002
1.8 x 10"7
Microcystin-LF
0.002
3.4 x 10"7
a Calculations used unrounded parameters; results slightly differ with rounded values.
7.5 Cyanobacterial Cells
Cyanobacteria are associated with two distinct types of stressors, as described in the conceptual model,
section 4.1. The first type of stressor are the toxins (microcystins and cylindrospermopsin) produced by
the cyanobacteria. Section 3 of this document discusses the nature of these stressors and section 5
discusses related health effects endpoints. These stressors are the basis of the recreational criteria and
swimming advisories. The second type of stressor is cyanobacterial cells. At this time, available data are
insufficient to develop quantitative recreational values for total cyanobacterial cell density related to
inflammatory health endpoints. However, various state and international agencies use total
cyanobacterial cell densities in decision-making to determine water quality and to post recreational
warnings to the public.
Exposure to cyanobacteria cells in ambient waters is associated with numerous inflammatory health
endpoints, including: rashes, respiratory and GI distress, and ear and eye irritation. These effects can be
the result of direct contact with bioactive compounds in the cyanobacteria (also referred to as
"endotoxins"), or by contact with cyanobacteria-associated microbial commensals via dermal, oral, or
inhalation exposure routes (Eiler and Bertilsson 2004; Gademann and Portmann 2008). Section 7.5.1
and Appendix D provide more information about the health effects associated with exposure to
cyanobacteria cells based on the scientific literature and related uncertainties. Section 7.5.2 presents
information about the use of total cyanobacteria, or other biomass metrics, as an indicator of potential
hazard associated with cells or cyanotoxins. Gene-based enumeration methods, satellite remote sensing
and uncertainties related to use of cells as indicators are also described. Section 7.5.3 discusses
guidelines that use total cyanobacterial cell density as an indicator for toxin presence, quantification of
toxigenic cells, and an approach providing cell density estimates related to the recommended 304(a)
cyanotoxin criteria.
7.5.1 Health Effects Associated with Cyanobacterial Cells and Uncertainties
Various health studies, described in more detail in Appendix D, relate recreational exposure to
increasing densities of cyanobacterial cells with increased incidence of specific health endpoints that can
be described as acute inflammatory or allergenic reactions. The EPA identified epidemiological studies,
clinical studies, and recreational water outbreak reports in searches of the publicly available and peer-
reviewed scientific literature that characterize the human health effects associated with recreating in
surface waters where cyanobacteria were present (see Appendix D).
The epidemiological studies provide evidence for statistically significant associations between
cyanobacterial cell densities and possible inflammatory or allergenic health endpoints:
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•	Pilotto et al. (1997) reported a significant association with the occurrence of one or more
symptoms, such as skin rashes, eye irritation, ear irritation, gastrointestinal distress, fever and
respiratory symptoms, and exposure to greater than 5,000 cells/mL for more than one hour. In
discussing the significance of the trend of increasing symptom occurrence and with the 5,000
cells/mL cut point, Pilotto et al. (1997) specifically suggested that the 20,000 cell/mL threshold
might be too high to be adequately protective of recreators.
•	Stewart et al. (2006d) found a significant increase in the inflammatory health effects associated
with recreators exposed to > 100,000 total cyanobacteria/mL or a total cyanobacterial surface
area >12 mm2/mL.
•	Levesque et al. (2014) observed a significant increase in GI symptoms associated with
recreational contact. The increase in GI symptoms was significant in the > 20,000-cells/mL and
> 100,000-cells/mL categories, and the positive trend for increasing illness with increased total
cyanobacterial cell densities also was significant at p-value = 0.001.
•	Lin et al. (2015) reported significant associations between respiratory symptoms and exposure to
the 25th to 75th percentile range of cyanobacterial cells excluding picocyanobacteria (range 37-
237 cells/mL) and between reported respiratory, rash, and earache symptoms and exposure to the
highest quartile (range 237-1,461 cells/mL). The 1,461-cells/mL value was the highest cell
density observed in that study (Lin et al. 2015).
•	Levesque et al. (2016) reported a significant trend of increasing of GI illness in recreators
associated with exposure to the concentration of endotoxins. The authors noted a positive
correlation between endotoxin concentrations and total cyanobacterial counts. Relative risks for
GI illness were higher for families that also received drinking water from the lakes studied or
from wells under the influence of surface water contamination. There was no relationship
between GI illness and exposure to E. coli. Relative risks also increased for recreators engaged in
full (e.g., swimming, water skiing, diving, etc.) or limited (e.g., fishing, use of watercraft)
contact recreation and adjustment for the level of exposure did not alter the health relationship.
The variability in the reported epidemiological associations in these studies in both the range of
cyanobacterial cell densities reported and specific symptomologies characterized limited identification
of a discrete cyanobacterial cell density value associated with a consistent level of effect. Some
researchers have suggested that the lack of a described dose-response characterizing cell-related
inflammatory health effects could suggest a "threshold" rather than a specific dose-response relationship
(Cochrane et al. 2015; Stewart et al. 2006b). Allergy is an example of a threshold mechanism, meaning
that there is a level of exposure (i.e., a threshold value) below which the development of sensitization
and the elicitation of an allergic reaction will not occur. Defining accurate numerical values for
threshold exposure levels is difficult due to lack of validated methods and uncertainties about the
mechanism of sensitization (Cochrane et al. 2015).
Scientists investigating the health effects posed by cyanobacteria have pointed out factors that contribute
to the epidemiological variability observed and uncertainties in determining what level of cyanobacterial
cells result in a specific level of inflammatory responses. For example:
•	There are differing cyanobacterial community composition and proportions of the more
allergenic, non-cyanotoxin-producing strains relative to the cyanotoxin-producing strains at each
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site. Researchers have reported non-toxin-producing strains can be more allergenic compared to
toxin-producing strains (Torokne et al. 2001).
•	There is variability in sensitivity in the study populations.
•	There are differences among the specific sites studied.
•	The limited size of some studies could have affected the ability to detect significantly increased
rates of illness in individual symptom categories (Pilotto et al. 1997; Stewart et al. 2006b). Small
sample size diminishes the statistical power of the study and the ability to detect an association if
one exists (Rothman et al. 2008).
•	The incomplete characterization or consideration of frank or opportunistic pathogens that could
co-occur with cyanobacteria in ambient waters also complicates conclusions related to the
etiologic agent of the reported symptoms (Levesque et al. 2014; Lin et al. 2015; Pilotto et al.
1997; Stewart et al. 2006b).
The number of cells in freshwater reported to be statistically-associated with a significant increase in
inflammatory endpoints ranged from 5,000 to 100,000 cells per mL. The EPA concluded that, although
significant associations with adverse health effects occur across a wide range of cyanobacterial cell
densities, the EPA cannot derive the CWA section 304(a) criteria based on total cyanobacterial cell
density at this time. There is considerable uncertainty and variability associated with the epidemiological
results that did not identify consistent effects at similar cell densities and available data do not support a
consistent quantitative dose-response relationship.
Additional research is needed to better describe the health effects associated with exposure to
cyanobacteria with more precision using consistent health symptomologies in context with the
community of cyanobacteria present (e.g., population of toxigenic versus non-toxin-producing
cyanobacteria, shifts in community profile during the study, etc.) and other factors that influence the
proliferation of cyanobacteria. Based on currently available science, inflammatory illnesses are
significantly increased at values above 100,000 cyanobacterial cells per mL. Guideline values currently
in use (see sections 2.1 and 7.5.3) that are within the 5,000 to 100,000 cell density range can find
supporting scientific evidence in the peer-reviewed literature described above and in Appendix D.
7.5.2 Cyanobacteria Biomass Measurements as Indicators of Hazard
Under certain conditions, cyanobacteria possessing the toxin synthesis genes, also referred to as
toxigenic cyanobacteria, begin producing cyanotoxins. Toxigenic cyanobacteria are a functional
subgroup of the total cyanobacterial population that may be present in a water body and the proportion
of toxigenic cells present can vary geographically and over time. Numerous biotic and abiotic factors
can influence not only the dominance of cyanobacteria within the overall phytoplankton community, but
also the proportion of toxigenic cyanobacteria relative to non-toxin-producing cyanobacteria (Davis et
al. 2009; Hyenstrand et al. 1998; McCarthy et al. 2009; Neilan et al. 2013; Gobler et al. 2016). Multiple
species of cyanobacteria are capable of producing the same toxin, such as the microcystins, which can
pose a risk to human and animal health (Crawford et al. 2017). Although scientists have observed a
generalized relationship between total cyanobacteria density or chlorophyll a and cyanotoxin
concentration, these relationships are affected by the dominance of the toxin-producing cyanobacteria
within the overall cyanobacterial community (Zhang et al. 2014; Loftin et al. 2016b).
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Total cyanobacterial cell biomass, described by cell densities or other metrics, such as chlorophyll a, can
function as a measure of the ecological health of a water body and as an indicator of potential public
health hazards, such as inflammatory reactions from exposure to cells and adverse health effects
associated with the presence of cyanotoxins. The extent, frequency, persistence, and severity of
cyanobacteria proliferation can indicate the eutrophic status of a water body (Yuan and Pollard 2015).
Surface water enrichment with nitrogen, notably reduced forms of nitrogen, and phosphorus have been
linked to cyanobacteria becoming the dominant phytoplankton (Beaulieu et al. 2013; Glibert et al. 2016;
Paerl 2008; Watson et al. 1997). Proliferating cyanobacterial biomass can result in an increased potential
for toxins being produced (Pearl et al. 2001; Otten et al. 2012).
Although there can be large variation in the number of toxigenic cyanobacteria present relative to non-
toxigenic cyanobacteria in any given body of water, measures of the total cyanobacterial biomass, such
as cell counts, chlorophyll, or even visual assessments, can be used effectively in decision-making as
early warnings of potential HAB-associated hazards (Loftin et al. 2016b). Pacheco et al. (2016) stated
that these measurements can be good indicators of the potential risk of cyanotoxin exposure and useful
when access to more sophisticated approaches, resources, or expertise may be limiting. Measurements of
total cyanobacteria may also be particularly useful in waters with a history of HAB occurrence and the
presence of elevated cyanotoxins.
7.5.2.1 Remote Sensing Techniques for Estimating Cyanotoxins
New and innovative methods, such as remote sensing techniques using satellite imagery, coupled with
quantitative analysis to identify cyanobacterial blooms are of increasing interest to states. To date, these
techniques cannot yet detect cyanotoxins, but they can quantify cyanobacterial densities in water bodies,
an indicator of potential for cyanotoxin presence. Satellite measures of chlorophyll a, phycocyanin, or
both are used to estimate cyanobacterial cell density based on validated algorithms that quantify
relationships between these parameters and in situ measurements of cell density. For example, Stumpf
(2014) and Wynne et al. (2010) readily detected by satellite areas of high Microcystis densities in larger
freshwater bodies, such as Lake Erie.
U.S. EPA has collaborated since 2015 with the National Aeronautics and Space Administration
(NASA), NOAA, and the USGS on the Cyanobacteria Assessment Network (CyAN) project. This
project is developing the capability to detect and quantify total cyanobacterial blooms and related water
quality of U.S. lakes and estuaries using satellite data records (U.S. EPA 2018b). This includes
improving interpretation of satellite data and refining algorithms across satellite platforms. CyAN
defined an approach for identifying lakes that can be spatially resolved (i.e., visually separated) with
satellite imagery given differences in pixel resolutions, a method to quantify frequency of bloom
occurrence in recreational freshwater sites, and a method for evaluating changes in the spatial extent of
cyanobacterial blooms over time to support state-level assessments (Clark et al. 2017; Urquhart et al.
2017). CyAN has developed a mobile application that makes its processed satellite data more widely
available. In 2017, the application was made available to state agencies for beta testing (U.S. EPA
2018b). A CyAN project that compares satellite-based estimations of total cyanobacterial cell density
data from monitoring programs in eight states in the eastern United States found that satellite
information provided robust estimates for freshwater lakes greater than 100 hectares when the cell
densities less than 109,000 cells/mL and above 1 million cells/mL (Lunetta et al. 2015). The estimates
were less on target for intermediate densities (i.e., between 110,000 and 1 million cells). The authors
attributed this lower performance to the gap in taxonomic information needed to facilitate conversions
between cell count and cell volumes (Lunetta et al. 2015).
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Challenges remain for using remote sensing for cyanotoxin detection and mapping and Stumpf et al.
(2016) identify these and a strategy for resolving them. The challenges they note include the lack of a
steady relationship between the indicator pigments (i.e., chlorophyll a or phycocyanin) and cyanotoxins.
These relationships may be valid for several weeks but start to vary over longer time periods due to
changes in the amount of cyanotoxin produced as a function of cyanobacterial biomass. Strategic
collection of pigment and toxin measurements will improve the application of remote sensing and
associated models. The Ocean Land Colour Imager on the Sentinel-3 satellite, launched in 2016, will
help address this need and improve data availability for most medium to large lakes around the world.
Given the inherent spatial uncertainty in the distribution of blooms and the potential issues with use of
the appropriate satellite product, more attention should be given to the use of field measurements of
reflectance to parameterize derivative-based pigment models (Tomlinson et al. 2016). This approach
will help standardize processing of the satellite data to consistent reflectance-based products.
Standardization is a factor in pigment and cyanotoxin measurement that will also require closer scrutiny.
Propagation of known measurement error and uncertainty into the models will establish confidence
levels for a variety of applications besides toxin maps. Improving strategies for collecting pigment
measurement with toxin measurement will allow a better understanding and use of remote sensing to
inform monitoring of toxins in lakes.
7.5.2.2 Molecular Methods for Estimating Cyanotoxins
Scientists have applied newer methods of quantifying microbes in environmental matrices, which
increases understanding of bloom dynamics and functional subgroups of cyanobacteria, such as the
toxigenic cells (Davis et al. 2009). The use of gene-based enumeration methods allows the quantification
of cyanobacteria that contain specific gene sequences for toxin synthesis—without which a cell cannot
produce the toxin. When toxigenic cyanobacteria are characterized with these tools, they have been
shown to be better predictors of subsequent increases in toxin concentrations than with other traditional
enumeration methods.
More recently, the use of gene-based quantification methods has helped to shed light on the community
dynamics within a bloom, understand some of the factors that trigger toxic blooms, and provide faster
and less expensive measurements of potential bloom toxicity compared to ELISA- and LC/MS/MS-
based methodologies. Researchers have shown that microcystins and cylindrospermopsin are produced
by non-ribosome-associated peptide synthetases (Dittmann et al. 1997; Moreira et al. 2013). The
microcystin synthetase complex is encoded by 10 mcy genes (mcyA to mcyj) (Neilan et al. 2013).
Studies have characterized the abundance of various mcy genes in ambient waters (Pacheco et al. 2016;
Qiu et al. 2013). The cylindrospermopsin synthetase gene cluster, cyr, is not as well characterized, but
has been studied in multiple cylindrospermopsin-producing cyanobacteria (Neilan et al. 2013). Other
researchers have used qPCR methods to characterize the relative abundance of total cyanobacteria,
Cylindrospermopsis raciborskii and cylindrospermopsin synthase in lake water (Moreira et al. 2011).
Selected examples of monitoring studies using gene-based approaches are described below.
• Davis et al. (2009) characterized toxic and nontoxic strains of Microcystis by quantifying the
mcyD (toxigenic strains) and the 16S rDNA genes (all Microcystis) in four lakes in the
northeastern United States over a two-year period. At all sites, toxigenic Microcystis were a
better predictor of microcystin concentrations compared to total cyanobacteria, total Microcystis,
chlorophyll a, or other environmental factors. Gene copies of mcyD were significantly correlated
with microcystin concentrations in every lake studied (Davis et al. 2009).
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•	HABs in lakes and reservoirs are prevalent in Alberta, Canada, and are affected predominantly
by elevated microcystins (Alberta Health 2014). Multiple Canadian governmental departments
and public health laboratories in Alberta conducted a monitoring and advisory program for
cyanobacteria at beaches. Among the findings were: microcystin-producing cyanobacteria
species were dominant in most lakes with blooms peaking in late August to September,
microcystin concentrations exceeding Canadian guidelines were not consistently associated with
elevated total cyanobacterial cell densities in most cases, and the mcyE gene measured by qPCR
was a good predictor for cyanobacterial blooms in some lakes (Alberta Health 2014).
•	In response to the 2014 Lake Erie HAB event that contaminated the drinking water of Toledo,
Ohio, the EPA revised the monitoring requirements for Ohio public water systems. Included in
those requirements are testing for the mycE gene. If > 5 mycE genes/[j,L are detected in raw water
samples, public water systems must monitor for microcystins (Ohio EPA 2017). Ohio is
currently testing qPCR methods for total cyanobacteria (16s rDNA) and toxigenic cyanobacteria
such as microcystin (mcyE gene) and saxitoxin (sxlA gene) producers. Ohio's HAB response
strategy for recreational waters (Ohio EPA 2017) includes qPCR assessment for cyanotoxin-
production genes as an option for cyanobacterial screening. If the qPCR testing indicates an
abundance of toxigenic cyanobacteria, additional analysis for the toxin is recommended (Ohio
EPA 2017).
•	In Lake Champlain, in the northeastern United States, Fortin et al. (2015) applied qPCR-based
methods and high-throughput sequencing to evaluate the effect of physico-chemical parameters
and nutrients on the dynamics of cyanobacterial community. The researchers observed that total
cyanobacteria were correlated with microcystin concentrations (Fortin et al. 2015). They also
showed a significant correlation between the microcystin concentrations, the abundance of the
mcyD gene, and the abundance of Microcystis 16S rDNA gene copies. Previous work had shown
that Microcystis were the predominant microcystin producer present in the same water body
(Ngwa et al. 2014).
•	Pacheco et al. (2016) reviewed studies examining relationships between the prevalence of
microcystin synthetase genes and microcystin concentration, and between chlorophyll a or cell
density and microcystin concentration. While many studies included in the review did show a
correlation for both comparisons, some did not. A lack of correlation between the synthetase
genes and microcystin concentration was reported in studies that: (1) extracted the particulate-
associated microcystins only; (2) included waters with very low concentrations of total
microcystins (e.g., < 0.5 (J,g/L); or (3) in one study, monitored lakes at a single fixed point in the
pelagic zone at the deepest site in each lake using depth-integrated water samples representing
the entire photic zone (Beversdorf et al. 2015a; Pacheco et al. 2016). For studies not reporting a
correlation between chlorophyll a or cell density and toxin concentration, only particulate-
associated microcystin was analyzed or a very low concentration (e.g., < 0.05 (J,g/L) of total
microcystins was observed (Pacheco et al. 2016). Zhang et al. (2014), one of the studies included
in the Pacheco et al. (2016) review, characterized cylindrospermopsin- and microcystin-
producing genotypes in the Macau reservoir, China, and found high cylindrospermopsin
concentrations correlated to the prevalence of the pks gene (r2 = 0.95, p-value < 0.01) and that
Cylindrospermopsis dominated the cyanobacterial population in the reservoir studied.
•	Crawford et al. (2017) applied an integrated monitoring approach including microscopic
cyanobacteria identification, multiplex qPCR for toxin genes, and toxin analysis to assess
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potential risks and inform bloom management decisions in a HAB event on the Murray River,
Australia, in 2016. The qPCR results showed that cylindrospermopsin and saxitoxin genes were
present, but were below the level of quantification. No microcystin genes were detected. The
qPCR results were corroborated with the lack of detection of any cylindrospermopsin,
microcystin, or saxitoxin (Crawford et al. 2017).
7.5.2.3 Uncertainties in Using Cyanobacterial Cells as Indicators
While cell density and pigment measurements can be useful for early detection of cyanobacterial
proliferation and informative for bloom monitoring, these approaches may not be sufficiently accurate to
predict risk from cyanotoxins (Pacheco et al. 2016). Uncertainties related to the use of total
cyanobacteria in decision-making related to toxin concentrations should be considered.
1.	Toxigenic cell densities can be a better indicator of the potential of a bloom to produce cyanotoxins
compared to measures of total cyanobacterial biomass.
The amount of toxin produced by a toxigenic cyanobacterial cell and the relative abundance of toxigenic
strains relative to non-toxigenic ones can vary considerably and be affected by environmental factors
(Gobler et al. 2016). Gene-based quantification of toxigenic cyanobacteria can be beneficial for
decision-making for HAB management approaches (Lee et al. 2015; Crawford et al. 2017). Davis et al.
(2009) observed that quantifying toxigenic Microcystis was a better predictor of in situ microcystin
levels than other surrogates, such as total cyanobacteria and chlorophyll a. The use of qPCR to
characterize temporal and spatial variations in the abundance of toxigenic strains can identify the
capability of a bloom to produce toxins, and hence the potential for recreator exposure to toxins,
including perhaps prior to the hazardous condition occurring (Pacheco et al. 2016).
The importance of the toxigenic cyanobacterial cells has been recognized by the WHO and previously
discussed in section 2.1. Based on toxigenic Microcystis, approximately 20 jag microcystins per L could
be expected, but other species, such as Planktothrix, can contain higher microcystin concentrations in a
cell compared to Microcystis (Fastner et al. 1999). Thus, the WHO commented that microcystin
concentrations could be much higher (e.g., 50-100 (J,g/L) if species with high microcystin content
dominate a bloom (WHO 2003a).
2.	Total cyanobacteria can be informative as an indicator for the presence of toxins if toxigenic species
are abundant or the dominant members of the cyanobacterial community.
Evidence from prior monitoring may demonstrate toxigenic strains tend to dominate blooms in a water
body or that a prior bloom had increased densities of toxigenic species occurring in conjunction with
elevated toxins. Studies showing good correlation between increased cell densities or other parameters
linked to cell proliferation and elevated toxin concentrations can also show the bloom is dominated by
toxin-producing species (Rinta-Kanto et al. 2009; Zhang et al. 2014; Pacheco et al. 2016). In one study
on Lake Erie over multiple seasons, Rinta-Kanto et al. (2009) observed a positive correlation between
the abundance of cyanobacterial and Microcystis gene copies and the number of microcystin synthetase
genes. Microcystis were a strong contributor to the concentration of microcystins in Lake Erie and the
relative abundance of Microcystis cells was correlated with microcystin concentrations (Rinta-Kanto et
al. 2009). Lack of correlation can occur when toxigenic cell density is low or undetectable (Crawford et
al. 2017) or low concentrations of toxin are recorded (Rinta-Kanto et al. 2009) and in such cases
measures of total cyanobacteria are not good indicators of toxins.
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3. The proliferation of toxigenic cells and the timing of the presence of elevated toxin concentrations
may or may not coincide with the visible proliferation of a HAB.
Decisions to issue recreational water warnings/advisories, or initiate monitoring for cyanotoxins based
on total cyanobacteria once a bloom is observed (i.e., green, discolored water, or scum
formation/accumulation associated with high densities of cells) may overlook situations where
extracellular toxins are present. Cells may accumulate in locations different from where the bloom
originated (e.g., by wind or wave action, or both, or be transported downstream). A cell density of
40,000 cells/mL is lower than what might be typically associated with a visible bloom (WHO 2003a).
Decision points contingent on visually confirmed blooms may miss or delay the identification of the
hazardous condition associated with exposure to elevated cyanotoxins, especially in water bodies with a
previous history of HAB events or toxin detections and the downstream waters potentially affected by
the HAB.
Davis et al. (2009, 2010) observed bloom dominance shift between toxigenic strains and non-toxigenic
strains over the course of a summer. Spatial and temporal dynamics in cyanobacterial population
succession is noted in other seasonal studies (Sabart et al. 2010; Otten et al. 2012, Beversdorf et al.
2015b; Fortin et al. 2015; Chen et al. 2017). Ha et al. (2009) observed similar seasonal variations in both
the gene copies of microcystin synthetase genes and for total cyanobacteria gene copies, although the
cyanobacterial community was consistently dominated by microcystin-producing cells throughout
the study.
7.5.3 Use of Cyanobacteria Cell Densities in Guidelines
7.5.3.1 Cyanobacteria Cell Guidelines
A number of states and international agencies include both total cyanobacteria and toxigenic
cyanobacteria density guidelines to account for both inflammatory- and toxin-associated health
endpoints. Cyanobacterial cell densities used by states and local health departments to provide guidance
to recreators on water quality are presented elsewhere in this document (see Table 2-3 for a list of states
with cyanobacterial cell density guidelines; see Appendix B for state guidelines and associated actions).
As discussed in section 2.1, the 35 states that currently have HAB-related guidelines include different
approaches and guideline levels (see Table 2-3). Seven states have guideline levels that address toxin-
producing cyanobacteria as a proportion of the total cyanobacterial population or include a toxin-
specific cyanobacteria cell density (California, Idaho, Maryland, New York, New Hampshire, Oregon,
and Virginia). The Karuk Tribe, located in California, developed cell-based values for posting
cyanotoxin public health warnings for the tribe's recreational waters (Kann 2014).
As described in section 2.1 of this document, the WHO (2003a) guideline value development was
informed by results from a review conducted by Chorus and Bartram (1999) and a prospective
epidemiology study by Pilotto et al. (1997), which evaluated health effects after recreational exposure to
cyanobacteria and reported associations between cyanobacterial cell densities and health. The WHO
recommended three tiers of guideline values describing an increasing scale of potential adverse health
effects and "between the chiefly irritative symptoms caused by unknown cyanobacterial substances and
the potentially more severe hazard of exposure to high concentrations of cyanotoxins, particularly
microcystins."
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•	The lowest tier of guideline values (< 20,000 cyanobacterial cells per ml; <10 [j,g/L
chlorophyll a) was mainly associated with a significant increase in irritative or allergenic effects
(the inflammatory health endpoints). The WHO, using conservative assumptions, also estimated
that microcystin concentrations of 2 to 4 (J,g/L, and possibly up to 10 (J,g/L, may be expected at a
cell density of 20,000 cells/mL where microcystin producers dominate.
•	The second tier (20,000 to 100,000 cyanobacterial cells per ml; 10 to 50 [j,g/L chlorophyll a),
describing a moderate probability of adverse health effects from cyanotoxins was informed by
(1) modifying the value for the WHO drinking water guideline for microcystin-LR for a
recreational exposure scenario and (2) translating microcystin concentrations to cell densities
based on the average microcystin content of Microcystis cells. The WHO, using conservative
assumptions, also estimated that 100,000 cyanobacterial cells/mL could correspond to 20 jag
microcystins/L if a bloom consists of Microcystis and has an average microcystin content of 0.2
pg/cell.
•	At the third tier (> 100,000 cells per mL; > 50 (J,g/L chlorophyll a) "there is the potential for
some frequently occurring species (i.e., Microcystis) to form scums," which can "increase risks
for bathers and others involved in body-contact water sports." The high probability of adverse
health effects category is associated with the elevated potential for exposure to cyanotoxins and
the potential for severe health outcomes. "The presence of cyanobacterial scum in swimming
areas represents the highest risk of adverse health effects due to abundant evidence for
potentially severe health outcomes associated with these scums."
•	Very high densities of cells occurring in scums (e.g., >10 million cells/mL or > 5,000 [j,g/L
chlorophyll a) can be associated with very high concentrations of toxin.
The Australian National Health and Medical Research Council (NHMRC) published a two-tiered
guideline for managing cyanobacteria in recreational water (NHMRC 2008). Tier one includes numeric
targets for microcystins based on children's recreational exposures and a toxigenic cell density for
Microcystis aeruginosa. The NHMRC recommends a secondary guideline for the protection from health
hazards associated with high densities of non-toxigenic cyanobacteria consistent with the WHO
cyanobacterial cell density recommendations for the moderate probability of health effects. NHMRC
used the epidemiological results published by Stewart et al. (2006b) to inform the derivation of the
Australian total cyanobacteria guideline number. Stewart et al. (2006b) found a significant increase in
the inflammatory health effects associated with recreators exposed to >100,000 total cyanobacteria/mL
or a total cyanobacterial surface area >12 mm2/mL. Because different cyanobacteria species can have
different sizes, the surface area estimate of biomass can take those size differences into account (e.g.,
1,000 very big cells versus 1,000 very small cells). NHMRC converted the cell surface reported by
Stewart et al. (2006b) to an equivalent biovolume and rounded that value to 10 mm3/L. This biovolume
guideline value applies when toxigenic cyanobacteria are absent in a bloom (NHMRC 2008)
NHMRC calculated a child-based total microcystin concentration of 9.4 (J,g/L, rounded to 10 [j,g/L
(NHMRC 2008). The authors then converted the toxin concentration to an equivalent toxigenic cell
density (50,000 Microcystis aeruginosa!mL) using the microcystin cell quota value (0.2 pg/cell). To
account for the potential hazard posed by other microcystin-producing cyanobacteria, the cell density
was converted into a biovolume equivalent (4 mm3/L). Other species have different cell sizes, so the
biovolume measurement allows comparisons with the other known toxin-producing cyanobacteria that
may be present. The biovolume equivalent applies to the total of all cyanobacteria where a known toxin
producer is dominant (NHMRC 2008).
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7.5.3.2 Amount of Toxin per Cell
Toxigenic cyanobacteria produce cyanotoxins that can accumulate inside the cells or be released to the
water column. The amounts of toxin produced by a toxigenic cyanobacterium is also referred to as "cell
quota." There is variability in the estimate of cyanotoxin concentrations associated with cell density, in
part because a bloom can contain both the toxigenic and non-toxin-producing strains of the same species
and cyanobacterial community differences between locations could affect the level of cyanotoxin that is
present. Thus, it is important to understand the abundance of toxigenic cyanobacteria in a water body.
As discussed above, characterizing the abundance of toxin genes can be a better predictor of toxin
produced than can calculations based on a toxin cell quota. The WHO's microcystin estimates at the
different risk levels were based on converting the recommended total cyanobacterial cell density using a
Microcystis cell quota value for microcystins (0.2 pg/cell) derived from a laboratory study conducted by
Mole et al. (1997) reporting an average microcystin cell quota in laboratory cultures of 0.2 pg/cell
(range: 0.07-0.3 pg/cell) (Fitzgerald et al. 1999), but other species and strains of microcystin producers
could result in much higher water-column microcystin concentrations given the same cell density
(WHO 2003a).
The EPA searched the published peer-reviewed scientific literature for information on the amount of
microcystin and cylindrospermopsin produced by or contained in a cell to inform the development of
toxigenic cell densities equivalent to the recommended criteria concentrations. Appendix G presents the
details related to the search strategy, reference prioritization and search results. The search resulted in
the collation of multiple studies reporting cell quotas for microcystin and cylindrospermopsin in
multiple genera of cyanobacteria. Laboratory-based culture studies with numerous clones of Microcystis
aeruginosa, Cylindrospermopsis raciborskii, Planktothrix agardhii, and Planktothrix rubescens were
also found. Many of these references also included either biomass-toxin conversions or graphic data that
would support conversion factors from cyanobacterial cell density (expressed in a variety of units
including: cells/L, biovolume (|j,m3/L), and chlorophyll alL) to toxin concentrations for these species.
Aggregated data are presented in Table 7-12. Table G-3 in Appendix G provides additional detail on the
studies identified containing cell quota information.
To facilitate a comparison of this information with the value used by the WHO, the EPA organized the
reported cell quota information by toxin and by genus (Table 7-14). Within each row, the study type,
quantification method, reported means and ranges, and references to the original study are included. Not
every study reported a mean, median, maximum, or minimum, so each row represents a collation of the
values reported. Ranges of reported cell quotas were large. For example, for all microcystin-producing
genera, reported cell quotas ranged from 0 to 4.3 pg/cell and the reported range of the means were 0.015
to 0.58 pg/cell. For Microcystis, the mean of the means, for seven studies published between 2008 and
2013, was 0.15 pg/cell. This value is similar to the 0.2 pg/cell value used by the WHO and provides
additional evidence that this conversion factor is supported by multiple scientific studies. For the genus
Planktothrix, the studies identified by the EPA do not suggest that this genus produces much higher
amount of microcystin compared to Microcystis. However, the EPA's literature search focused on more
recently published data and the Planktothrix values in the summary table come from only two recent
studies that may have not characterized toxin production under optimal conditions. Based on the data
presented in Table 7-14, the EPA concluded that the microcystin cell quota used by the WHO is
supported. The caveat expressed by the WHO (i.e., cell quota values can be variable within and between
species of microcystin-producing cyanobacteria) is also substantiated by the EPA's literature search
results. The EPA included the 0.2 pg/cell value in the calculation of a toxigenic cell density for
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microcystin-producing cyanobacteria equivalent to the recommended toxin magnitude (see section
7.5.3.3).
The EPA also collated similar information for cylindrospermopsin cell quotas. As with other aspects of
cylindrospermopsin, less information was available, but multiple field and laboratory studies reporting
the mass of toxin per cell were identified. The range of cylindrospermopsin cell quotas (0.0028-
14.6 pg/cell in Cylindrospermopsis) was larger than for microcystins, as was the range of reported
means (0.0028-0.17 pg/cell). The highest value (14.6 pg/cell) was reported from a field study (see Table
G-l). The highest value reported in a laboratory study was 0.17 pg/cell. The mean value for all studies
was 0.047 pg/cell (n = 10) and for field studies (n = 2) was 0.023 pg/cell. Given the few number of field
studies, large uncertainties exist with how representative the mean is of the central tendency of the
range. Less information was identified for Aphanizomenon, another well-known cylindrospermopsin
producer. To have a similar confidence level in the cylindrospermopsin cell quota data compared to
microcystins, additional data and an improved sense of the central tendency within the reported ranges is
needed. At present, the EPA is not sufficiently confident in the cylindrospermopsin cell quota database
to estimate a toxigenic cell density specific for cylindrospermopsin.
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Table 7-14. Aggregated Cell Quota Summary Data for Selected Microcystin and Cylindrospermopsin-producing Genera
Toxin
Genus
Quantification
Method3; Study
Typeb
Range of
Means0
Meancd
Median of
Means0
Minimum;
Maximum00
References
Microcystins
All microcystin-
producing genera
Mass per cell;
Field and lab
0.015 pg/cell
- 0.58 pg/cell
0.11 pg/cell
0.091 pg/cell
0 pg/cell -
4.3 pg/cell
Orr and Jones (1998); Jahnichen et al. (2001);
Wiedner et al. (2003); Akcaalan et al. (2006);
Jahnichen et al. (2007); Briand et al. (2008);
Fahnenstiel et al. (2008); Vasconcelos et al.
(2011); Sitoki et al. (2012); Tao et al. (2012);
Wood et al. (2012); Cires et al. (2013); Sabart
et al. (2013); Wang et al. (2013); Pineda-
Mendoza et al. (2014); Cilia et al. (2016); Wei
et al. (2016)
Microcystis
Mass per cell;
Field and lab
0.015 pg/cell
- 0.58 pg/cell
0.11 pg/cell
0.072 pg/cell
0 pg/cell -
4.3 pg/cell
Orr and Jones (1998); Jahnichen et al. (2001);
Wiedner et al. (2003); Jahnichen et al. (2007);
Fahnenstiel et al. (2008); Vasconcelos et al.
(2011); Sitoki et al. (2012); Tao et al. (2012);
Wood et al. (2012); Cires et al. (2013); Sabart
et al. (2013); Wang et al. (2013); Pineda-
Mendoza et al. (2014); Cilia et al. (2016); Wei
et al. (2016)
Mass per cell;
Field
0.015 pg/cell
- 0.58 pg/cell
0.15 pg/cell
0.075 pg/cell
0 pg/cell;
4.19 pg/cell
Fahnenstiel et al. (2008); Vasconcelos et al.
(2011); Sitoki et al. (2012); Tao et al. (2012);
Cires et al. (2013); Sabart et al. (2013); Wang
et al. (2013)
Planktothrix
Mass per cell;
Field and lab
0.076 pg/cell
- 0.24 pg/cell
0.12 pg/cell
0.10 pg/cell
0.076 pg/cell;
0.24 pg/celle
Akcaalan et al. (2006); Briand et al. (2008);
Mass per cell;
Field
0.091 pg/cell
- 0.24 pg/cell
0.16 pg/cell
0.16 pg/cell
0.091 pg/cell;
0.24 pg/celle
Akcaalan et al. (2006); Briand et al. (2008)
Fisherella
Mass per
biomass; Lab
N/A
N/A
N/A
43 (ig/g
Cires et al. (2014)
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Toxin
Genus
Quantification
Method3; Study
Typeb
Range of
Means0
Meancd
Median of
Means0
Minimum;
Maximum00
References
Cylindrospermopsin
Aphanizomenon
Mass per
biomass; Field
and lab
N/A
N/A
N/A
7,390 (ig/g;
9,330 (ig/g
Yilmaz et al. (2008)
Cylindrospermopsis
Mass per cell;
Field and lab
0.0028 pg/cell
- 0.17 pg/cell
0.047
pg/cell
0.027 pg/cell
0.0028
pg/celle; 14.6
pg/cell
Hawkins et al. (2001); Orr et al. (2010);
Carneiro et al. (2013); Mohamed and Al-
Shehri (2013); Davis et al. (2014);
Pierangelini et al. (2015); Willis et al. (2015);
Willis et al. (2016); Yang et al. (2016a)

Mass per cell;
Field
0.023 pg/cell
0.023
pg/cell
N/A
0.006 pg/cell;
14.6 pg/cell
Orr et al. (2010); Mohamed and Al-Shehri
(2013)

Mass per cell;
Lab
0.0028 pg/cell
- 0.17 pg/cell
0.052
pg/cell
0.031 pg/cell
0.0028
pg/celle;
0.17 pg/celle
Hawkins et al. (2001); Carneiro et al. (2013);
Davis et al. (2014); Pierangelini et al. (2015);
Willis et al. (2015); Willis et al. (2016); Yang
et al. (2016a)

Mass per
biovolume; Lab
N/A
N/A
N/A
416 fg/(im3;
447 fg/(im3
Pierangelini et al. (2015)
fg = femtogram; pg = picogram; |ig = microgram; N/A = not available.
a Various methods were used to quantify toxin quotas and quota values were presented in different forms, including toxin mass per cyanobacterial cell and toxin
mass per cyanobacterial biomass.
b Studies were conducted in two different settings: the field (i.e., enviromnental) or a laboratory.
0 Study authors reported data using multiple measurement units. When possible, the EPA converted data to the standard units of pg per cell. The EPA did not
identify appropriate conversion factors that would allow genus-specific conversion of quotas described in mass per biomass to mass per cell.
d Shows single reported mean if only one study was available or average of reported means.
e If reported toxin quota means from one study were the lowest or highest toxin quotas reported within a genus, then these values were listed as the minimum or
maximum values, respectively, to better reflect the range of toxin quota values.
f Cylindrospermopsis is now known as Raphidiopsis.
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Challenges with collating this information include the variable conditions under which the studies
characterized toxin quotas and the various ways the toxin quota data were reported. Conditions under
which the toxin quotas were studied include laboratory and field conditions, different environmental and
collection-based strains included in the study, and the different environmental conditions existing at the
various locations where the field studies were conducted. For the latter, information on some of the
external factors affecting toxin production is summarized above to help demonstrate the complex
interactions that affect not just if the toxin is produced, but also how much toxin can be produced. The
various ways that toxin cell quotas were reported include: toxin mass per cell, toxin mass per unit
biomass, and toxin mass per unit biovolume. When possible, the EPA converted the cell quota
information into pg per cell to enable a straightforward comparison to the WHO value.
7.5.3.3 Toxigenic Cyanobacteria Value Associated with Recommended Microcystins
Criteria/Swimming Advisory
As discussed in section 7.5.3.2 the abundance of toxigenic cells in a water body affects the amount of
cyanotoxin produced. The number of toxigenic cyanobacteria relative to the number of total
cyanobacteria can vary in time and space. Quantifying the abundance of toxigenic cyanobacteria is a
better predictor of potential toxin production compared to total cyanobacteria. Below, the EPA presents
a similar approach to that used by the WHO to calculate a cyanobacterial cell density corresponding to
recommended criteria/ swimming advisory value for microcystins. Because more data are available for
microcystins compared to cylindrospermopsin, this calculation is based on microcystins only.
Cyanobacterial cell density (CCD) =
Ambient cyanotoxin concentration (ACC)
Cell toxin amount (CTA)
Where:
CCD
calculated toxigenic cell density associated with a specific toxin
concentration
ACC
specific toxin concentration target in ambient water (e.g., AWQC
value)
CTA
amount of toxin produced in a cyanobacterial cell
For the microcystins-producing cyanobacteria (e.g., Microcystis)'.
ACC
CTA
8 (J,g/L; recommended recreational criteria value for microcystins
0.2 pg/cell; reported mean concentration of microcystin in a cell of
microcystin-producing cyanobacteria
Adding in the conversion factors to convert units, the equation is:
CCD = ACC (ng/L) x 106 pg/ng
CTA (0.2 pg/cell)
X
L
1000 mL
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Adding in the values,
8 ng/L x 106 pg/|Lig
0.2 pg/cell
X
1000 mL
1 L
= 40,000 cells/mL
Thus, a toxigenic microcystin-producing cell density of 40,000 cells/mL has the potential to result in a
microcystin concentration of 8 (J,g/L.
7.6 Other Sources of Microcystins and Cylindrospermopsin
Although the EPA is not including other sources of cyanotoxins in this recreational exposure scenario,
the Agency has included summary information on potential sources of cyanotoxins, such as drinking
water, ground water, fish, shellfish, dietary supplements, air, soil, and sediments. Exposure to
cyanotoxins in finished drinking water is characterized in the Drinking Water Health Advisories
(U.S. EPA 2015a, 2015b). States may wish to consider these other sources of cyanotoxins in their public
health approach.
7.6.1 Drinking Water
The occurrence of cyanotoxins in drinking water depends on their levels in the raw source water and the
effectiveness of treatment methods for removing cyanobacteria and cyanotoxins during the production
of drinking water. The EPA has provided Recommendations for Public Water Systems to Manage
Cyanotoxins in Drinking Water to assist public drinking water systems (PWSs) that choose to develop
system-specific plans for evaluating their source waters for vulnerability to contamination by
microcystins and cylindrospermopsin (U.S. EPA 2015e). Cyanotoxin management plan templates, water
treatment optimization, and a communications tool box are also available on the EPA's Cyanotoxins in
Drinking Water website (U.S. EPA 2015e).
The American Water Works Association Research Foundation (AWWARF) conducted a study on the
occurrence of cyanobacterial toxins in source and treated drinking waters from 24 public water systems
in the United States and Canada in 1996-1998 (AWWARF 2001). Of 677 samples tested, microcystins
were found in 80 percent (539) of the waters sampled, including source and treated waters. Only two
samples of finished drinking water were above 1 (J,g/L. A survey conducted in 2000 in Florida (Burns
2008) reported that microcystins were the most commonly found toxin in pre- and post-treated drinking
water. Finished water concentrations ranged from below detection levels to 12.5 (J,g/L.
During the summer of 2003, a survey was conducted to test for microcystins in 33 U.S. drinking water
treatment plants in the northeastern and midwestern United States (Haddix et al. 2007). Microcystins
were detected at low levels ranging from undetectable (< 0.15 (J,g/L) to 0.36 [j,g/L in all 77 finished water
samples.
In August 2014, the city of Toledo, Ohio, issued a do-not-drink or -boil advisory to nearly 500,000
customers in response to the presence of total microcystins in the city's finished drinking water at levels
up to 2.50 (J,g/L. The presence of the toxins was due to a cyanobacterial bloom near Toledo's drinking
water intake located on Lake Erie. The advisory was lifted two days later, after treatment adjustments
led to the reduction of the cyanotoxin concentrations to concentrations below the WHO guideline value
of 1 [j,g/L in all samples from the treatment plant and distribution system.
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During the late spring and early summer of 2018, both microcystins and cylindrospermopsin were found
in the finished drinking water of Salem, Oregon (Novak Consulting Group 2018). Salem's finished
drinking water source is the North Santiam River, which is fed by Detroit Lake, a reservoir located
southeast of the city. In late May 2018, the State of Oregon issued a recreational advisory for
cyanotoxins for Detroit Lake. Less than a week later, the City of Salem issued a do not drink advisory
due to the presence of levels of microcystins and cylindrospermopsin in drinking water exceeding health
advisories. The drinking water advisory was lifted in the beginning of July based on many consecutive
days of finished water results being below health advisory levels.
The EPA has published Drinking Water Health Advisories to address microcystins and
cylindrospermopsin in drinking water (U.S. EPA 2015a, 2015b).
7.6.2	Ground Water
Only very limited data are available on microcystins in ground water and no monitoring data were
identified for cylindrospermopsin. A study reported microcystins in ground water from a well located
near the shore of Lake Chaohu, in China (also known as Chao Lake), which contained high microcystin
concentrations (Yang et al. 2016b). Therefore, under certain conditions, ground water hydraulically
connected to surface water has the potential to be contaminated by cyanotoxins.
7.6.3	Fish and Shellfish
Fish and shellfish living in waters affected by a cyanobacterial bloom may accumulate cyanotoxins in
their muscle tissue and internal organs (Gibble et al. 2016; Kinnear 2010). Some authors have found that
microcystins accumulate less in the edible parts of aquatic organisms, such as muscle (Deblois et al.
2011; Gutierrez-Praena et al. 2013; Song et al. 2009; Vareli et al. 2012; Wilson et al. 2008; Xie et al.
2005; Zimba et al. 2006). Cylindrospermopsin has also been found in fish and shellfish exposed for
longer periods of time to a cyanobacterial bloom (Funari and Testai 2008; Ibelings and Chorus 2007;
Kinnear 2010; Saker and Eaglesham 1999). For additional information on occurrence of microcystins
and cylindrospermopsin in fish and shellfish, please see the Health Advisory document published (U.S.
EPA 2015a, 2015b).
7.6.4	Dietary Supplements
Extracts from Arthrospira (Spirulina) and Aphanizomenon flos-aquae have been used as dietary blue-
green algae supplements (BGAS) (Funari and Testai 2008). These supplements are reported to have
beneficial health effects including supporting weight loss, and increasing alertness, energy and mood
elevation for people suffering from depression (Jensen et al. 2001). A study suggested that BGAS could
be contaminated with microcystins ranging from 1 j_ig/g up to 35 j_ig/g (Dietrich and Hoeger 2005). In
two separate studies, Heussner et al. (2012) and Roy-Lachapelle et al. (2017) both analyzed 18 different
commercially available BGAS for the presence of cyanotoxins. Heussner et al. (2012) reported that all
products containing Dolichospermum flos-aquae (formerly Aphanizomenon flos-aquae) tested positive
for microcystins at levels < 1 (J-g microcystin-LR equivalents/g dry weight. Cylindrospermopsin was not
found in any of the supplements. Roy-Lachapelle et al. (2017) reported that of the 14 products
containing Spirulina, three contained total microcystins at levels < 1 (_ig/g. All four products containing
Dolichospermum flos-aquae tested positive for total microcystins ranging from 0.8 [j,g/g to 8.2 [j,g/g
using the Adda oxidation method and from 0.52 j_ig/g to 5.8 j_ig/g using the sums of microcystins
standards. Cylindrospermopsin was not found in any of the supplements.
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7.6.5	Ambient Air
Four studies provide air concentration data for cyanotoxins indicating that recreational surface waters
with toxigenic cyanobacterial blooms can result in aerosolized cyanotoxins (Backer et al. 2008, 2010;
Wood and Dietrich 2011; Cheng et al. 2007). These studies are summarized in section 7.4.1.
7.6.6	Soils and Sediments
Microcystins can adsorb onto naturally suspended solids and dried crusts of cyanobacteria. Cyanotoxins
can precipitate out of the water column and reside in sediments for months (Falconer 1998; Han et al.
2012; Wu et al. 2012). In sediments, cylindrospermopsin adsorbs to organic carbon, with little
adsorption observed in sandy and silt sediments (Klitzke et al. 2011). The low adsorption of
cylindrospermopsin in sediments/silts with low levels of organic carbon reduces the opportunity for
microbial degradation.
Maghsoudi et al. (2015) tested adsorption of cyanotoxins onto three fractionated sediment particles,
clay-silt (< 75 (j,m), fine sand (75-315 um) and coarse sand (315-2000 (j,m) and found that adsorption
capacity of coarse sand fraction for all the tested cyanotoxins was less than four percent of the clay-silt
fraction. They found that highest adsorption for cylindrospermopsin, microcystin-LW, and microcystin-
LF were 73, 57, and 55 percent, respectively, and occurred within two hours. Desorption experiments
demonstrated that less than nine percent of cyanotoxins desorbed from sediment within 96 hours.
Song et al. (2015) found that a statistically significant part of the variability of the microcystin
concentration in the sediments could be explained by a combination of variables in the water column,
such as total microcystins in the water, cyanobacterial biomass in water, pH, and temperature.
7.7	Tribal Considerations
The EPA considered alternative exposure scenarios tribal communities might have, given their cultural
practices. Native American food foraging customs or cultural or religious ceremonies can put them into
primary or secondary contact with cyanotoxins. Primary contact ceremonial use may include the use of a
surface water body for religious or traditional purposes by members of a tribe, involving immersion and
intentional or incidental ingestion of water (Eastman 2007).
It is uncertain whether these activities would lead to cyanotoxin exposures higher than the primary
recreational contact assumptions for incidental ingestion and exposure duration used in this assessment.
7.8	Livestock and Pet Concerns
The earliest observations of adverse effects of cyanobacterial exposure to animals include the rapid
death of stock animals in Australia in 1878 (Francis 1878). Since then, numerous cases of mammal and
bird deaths have been documented (Backer et al. 2015; Hilborn and Beasley 2015). These cases were
reported throughout the 20th century on all continents except Antarctica (Stewart et al. 2008). The
impacts of cyanotoxins on domestic and companion animals are likely under-recognized because many
cases are misdiagnosed, few cases are biochemically confirmed, and even fewer are reported in the
scientific literature or to animal health systems (Zaias et al. 2010).
Livestock and pets potentially can be exposed to higher concentrations of cyanotoxins, or have increased
exposure to cyanotoxins than humans because they are known to consume cyanobacterial scum and mats
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and drink cyanobacteria-contaminated water (Backer et al. 2013). Dogs are also at risk, as they may lick
cyanobacterial cells from their fur after swimming in a water body with an ongoing bloom (CDC
2017a). Mats and scums can represent thousand-fold to million-fold concentrations of cyanobacterial
cell populations, and published microcystin concentrations have ranged up to 24 mg microcystins/L
from scum material (Chorus and Bartram 1999). Common signs of HAB cyanotoxin poisonings in pets
include repeated vomiting, diarrhea, loss of appetite, abdominal swelling, stumbling, seizures,
convulsions, disorientation, inactivity, or skin rashes and hives, and in extreme cases collapse and
sudden death (CDC 2017a; New York Sea Grant 2014; Trevino-Garrison et al. 2015). Although reports
of livestock deaths are uncommon, in extreme cases, death can occur minutes after drinking from a
contaminated water source. Acute symptoms of cyanotoxin poisoning can include loss of appetite,
weakness, staggering, or inflammation of the muzzle, ear, or udder. Higher levels of cyanotoxins can
lead to severe liver damage, the development of jaundice, and severe photosensitization. Often livestock
or pets that recover from these ailments can then suffer from chronic failure to thrive (Australia
Department of Economic Development Jobs Transport and Resources 2013; Robinson and Alex 1987).
The Centers for Disease Control and Prevention (CDC) provides multiple resources, such as frequently
asked questions (FAQs), Veterinarian Cards, and Animal Safety Alerts, to help educate the public of the
dangers associated with cyanotoxin exposure to pets (CDC 2017a, 2017b, 2017c). The CDC suggests
that pet owners prevent their animals from playing in or drinking scummy water. If a dog has been
swimming in scummy water, the CDC recommends rinsing them off immediately to prevent the dog
from licking cyanobacteria off their fur (CDC 2017b).
The CDC recommends that pet owners contact a veterinarian if their animal shows the following
symptoms of cyanotoxin poisoning: loss of appetite, loss of energy, vomiting, stumbling and falling,
foaming at the mouth, diarrhea, convulsions, excessive drooling, tremors and seizures or any other
unexplained sickness after being contacted with water (CDC 2017c). While there have been no HAB-
associated human deaths in the United States, there have been many pet deaths (especially dogs) due to
cyanotoxin exposure via swimming and ingesting contaminated waters. Overall, CDC encourages the
public to follow the phrase "when in doubt, its best to keep out" (CDC 2017a).
The One Health Concept acknowledges a connection between human, animal, and environmental health,
suggesting that HAB-associated animal illnesses and deaths could serve as predictors of potential HAB-
associated risks in humans (CDC 2017d). Following this concept, the CDC created a voluntary reporting
system called the One Health Harmful Algal Bloom System (OHHABS) (CDC 2017d). While there are
other reporting systems that capture aggregate information on human illnesses or outbreaks, such as the
National Outbreak Reporting System (NORS), OHHABS expands reporting to include HAB-associated
environmental data, animal case data, and human case data (CDC 2017d). By collecting this
information, the goal of OHHABS is to better understand HABs and HAB-associated illnesses.
Members of the public can report HABs and cases of HAB-related human or animal illness by
contacting local or state public health agencies (CDC 2017d).
The New York State Department of Health (NYSDOH) applied the One Health approach to implement a
pilot surveillance system of HAB-related illnesses in 2015. During this pilot period, three dogs were
reported to have GI symptoms after exposure to HABs in recreational water; one of these cases was also
associated with a human case (Figgatt et al. 2017).
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7.8.1 States and Animal HAB Guidelines
A few states have guideline levels specific to the protection of animals from cyanotoxin poisoning
(Appendix H). California calculated cattle and dog action levels for the cyanotoxins microcystin and
cylindrospermopsin (Butler et al. 2012). California first calculated an RfD (mg/kg body weight/day) for
domestic animals for each of the cyanotoxins, based on laboratory studies. For both dogs and cattle,
California estimated drinking water ingestion rates (L/kg body weight/day) based on two publications by
the National Research Council, Nutrient Requirements for Beef Cattle and Nutrient Requirements for
Dogs and Cats, and applied an UF of three to account for preferential consumption of cyanobacteria. To
determine action levels (acute action level of 100 [j,g/L for microcystins and 200 [j,g/L for
cylindrospermopsin), California divided the domestic animal RfD for each cyanotoxin by the final water
and cyanobacterial biomass intake exposure levels calculated for cattle and dogs, and performed a unit
conversion, providing a cyanotoxin concentration that would result in exposure at the RfD level or
below. The state performed these calculations for an acute (lethal) and a subchronic scenario.
Oregon followed a similar approach to California's to calculate dog-specific guideline values for the
cyanotoxins cylindrospermopsin, microcystin, anatoxin-a, and saxitoxin (Oregon Health Authority
2018). Oregon estimated tolerable daily intake (TDI) values for humans ((J-g/kg body weight/day) for
each of the cyanotoxins, and applied these values to dogs (Farrer et al. 2015). Using California's dog-
specific exposure estimate (L/kg body weight/day), Oregon divided the human TDI by the dog-specific
ingestion rate to determine its guideline values (0.2 [j,g/L for microcystin and 0.4 [j,g/L for
cylindrospermopsin).
Grayson County in Texas estimated the quantity of water that would result in a potentially lethal dose of
microcystin and cylindrospermopsin for small and large dogs. Using advisory levels of 20 ppb for
microcystin and cylindrospermopsin, the county calculated the volume of water that would result in a
lethal or near-lethal dose of cyanotoxin by extrapolating the results of mouse studies to 10- and
80-pound dogs. This estimate does not include additional dose amounts that could be ingested by a dog
while self-grooming cyanobacteria scum off its fur (Lillis et al. 2012).
At Presque Isle State Park in Pennsylvania, a HABs task force (a partnership of six agencies and
organizations) monitors for microcystin and cylindrospermopsin at multiple locations on Lake Erie
within the park. Some of the locations monitored include designated dog beaches. Warning signs are
posted specifically for dog owners when microcystin levels are detected above 0.2 [j,g/L (Schnars
personal communication 2017; Best personal communication 2017).
Other states mention animal poisoning in their guideline documents but do not give guideline values
specific to livestock or companion animals. For example, Utah and Washington report that animal
illness or death can be reason to issue or accelerate a HAB advisory warning (Hardy and Washington
State Department of Health 2008; Utah Department of Environmental Quality and Department of Health
2017). Ohio includes pets in their public health advisory at threshold levels of 6 [j,g/L for microcystin
and 5 [j,g/L for cylindrospermopsin; however, Ohio issues the disclaimer that thresholds used are
protective of human exposure and may or may not be protective of animals such as dogs or livestock
(Ohio EPA 2016). Several other states including Connecticut, Idaho, Kansas, Massachusetts, Nebraska,
Vermont, and Virginia provide information via pamphlets and state websites warning about harm to pets
or other animals or post about harm to animals in their beach warnings and advisory signage (CDPH
2017; CDEEP 2017; IDEQ 2015; Kansas Department of Health and Environment 2016; Massachusetts
Bureau of Environmental Health 2015; Nebraska Department of Environmental Quality and Nebraska
Department of Health and Human Services: Division of Public Health 2018; Vermont Department of
Health 2015; Virginia Department of Health 2012).
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APPENDIX A. INTERNATIONAL RECREATIONAL WATER GUIDELINES FOR CYANOTOXINS AND CYANOBACTERIA
Jurisdiction
Recreational Water Guideline Level
Recommended Action
Australia3
Cyanobacteria (total): >10 mm3/L
(where known toxins are not present)
Red level action mode; level 2 guideline:
•	Immediately notify health authorities for advice on health risk.
•	Make toxicity assessment or toxin measurement of water if this lias not already been done.
•	Health authorities warn of risk to public health (i.e., the authorities make a health risk
assessment considering toxin monitoring data, sample type and variability).

Cyanobacteria (total): > 4 mm3/L (where
a known toxin producer is dominant in
the total biovolume)
Red level action mode; level 1 guideline:
•	Immediately notify health authorities for advice on health risk.
•	Make toxicity assessment or toxin measurement of water if this has not already been done.
•	Health authorities warn of risk to public health (i.e., the authorities make a health risk
assessment considering toxin monitoring data, sample type and variability).

Cyanobacteria (total): > 0.4 to < 10
mm3/L (where known toxin producers
are not present)
Amber level alert mode:
•	Increase sampling frequency to twice weekly where toxigenic species are dominant within the
alert level definition (i.e., total biovolume).
•	Monitor weekly or fortnightly where other types are dominant.
•	Make regular visual inspections of water surface for scums.
•	Decide on requirement for toxicity assessment or toxin monitoring.

Cyanobacteria (total): > 0.4 to <4 mm3/L
(where a known toxin producer is
dominant in the total biovolume)
Amber level alert mode:
•	Increase sampling frequency to twice weekly where toxigenic species are dominant within the
alert level definition (i.e., total biovolume).
•	Monitor weekly or fortnightly where other types are dominant.
•	Make regular visual inspections of water surface for scums.
•	Decide on requirement for toxicity assessment or toxin monitoring.

Cyanobacteria (total): > 0.04 to <0.4
mm3/L
Green level surveillance mode:
•	Weekly sampling and cell counts at representative locations in the water body where known
toxigenic species are present; or
•	Fortnightly for other types including regular visual inspection of water surface for scums.

Cyanobacterial scums consistently
present
Red level action mode; level 2 guideline:
•	Immediately notify health authorities for advice on health risk.
•	Make toxicity assessment or toxin measurement of water if this has not already been done.
•	Health authorities warn of risk to public health (i.e., the authorities make a health risk
assessment considering toxin monitoring data, sample type and variability).
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-l

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Jurisdiction
Recreational Water Guideline Level
Recommended Action

Microcystins (total): > 10 |ig/L
Red level action mode; level 1 guideline:
•	Immediately notify health authorities for advice on health risk.
•	Make toxicity assessment or toxin measurement of water if this has not already been done.
•	Health authorities warn of risk to public health (i.e., the authorities make a health risk
assessment considering toxin monitoring data, sample type and variability).

Microcystis aeruginosa (total): > 50,000
cells/ml
Red level action mode; level 1 guideline:
•	Immediately notify health authorities for advice on health risk.
•	Make toxicity assessment or toxin measurement of water if this has not already been done.
•	Health authorities warn of risk to public health (i.e., the authorities make a health risk
assessment considering toxin monitoring data, sample type and variability).

Microcystis aeruginosa (total): > 5,000
to < 50,000 cells/ml
Amber level alert mode:
•	Increase sampling frequency to twice weekly where toxigenic species are dominant within the
alert level definition (i.e., total biovolume).
•	Monitor weekly or fortnightly where other types are dominant.
•	Make regular visual inspections of water surface for scums.
•	Decide on requirement for toxicity assessment or toxin monitoring

Microcystis aeruginosa (total): > 500 to
< 5,000 cells/ml
Green level surveillance mode:
•	Weekly sampling and cell counts at representative locations in the water body where known
toxigenic species are present; or
•	Fortnightly for other types including regular visual inspection of water surface for scums.
Canadad
Cyanobacteria (total): > 100,000 cells/ml
Issue swimming advisory.

Detection of a cyanobacterial bloom
Issue beach closure.

Microcystins (total): > 20 |ig/L
(expressed as microcystin-LR)
Issue swimming advisory.
Cuba0
Any report of toxic effect in humans or
animals
Action (in red): as for "Alert," but with increased actions for public communication.

Benthic mats: < 40 percent coverage of
surfaces with any cyanobacteria;
> 20 percent with toxicogenic
cyanobacteria; > 50 percent with
potentially toxicogenic cyanobacteria
Alert: increased sampling (weekly and more sites); daily inspection; notification to public health
unit and local managers; report to local government; warning of the public.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Jurisdiction
Recreational Water Guideline Level
Recommended Action

(particularly where they are visibly
detaching and accumulating in scum)

Cyanobacteria: < 500 cells/ml
Monthly visual inspection.
Cyanobacteria: > 1 of the species known
as potentially toxic
Alert: increased sampling (weekly and more sites); daily inspection; notification to public health
unit and local managers; report to local government; warning of the public.
Phytoplankton cells: >_20.000 to <
100,000 cells/ml, > 50 percent of cells
cyanobacteria
Alert: increased sampling (weekly and more sites); daily inspection; notification to public health
unit and local managers; report to local government; warning of the public.
Phytoplankton: > 0 to < 1,500 cells/ml
Monthly visual inspection and sampling at least four months per year.
Scum consistently present; confirmed
bloom persistence
Action (in red): as for "Alert," but with increased actions for public communication.
Czech Republic0
Cells: > 100,000 cells/ml
Second warning level: closure for public recreation.
Cells: > 20,000 cells/ml
First warning level (not otherwise specified).
Denmark0
Chlorophyll a: > 50 |_ig/L. dominated by
cyanobacteria
Relevant authorities are informed and decide when and how the public should be informed;
warnings include signs, media, and contact to local user groups such as kindergartens, scouts,
water sports clubs.
Visible surface scum
Relevant authorities are informed and decide when and how the public should be informed;
warnings include signs, media, and contact to local user groups such as kindergartens, scouts,
water sports clubs.
European Unionf
Cyanobacterial proliferation
(occurrence)
When cyanobacterial proliferation occurs and a health risk has been identified or presumed,
adequate management measures shall be taken immediately to prevent exposure, including
information to the public.
Cyanobacterial proliferation (potential
for)
Appropriate monitoring shall be carried out to enable timely identification of health risks.
Finland0
Algae (includes cyanobacteria): detected
Level 1: Possibly microscopic examination and even toxin analysis if there is a specific cause
such as very popular beach or reports of adverse health effects or animal deaths.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Jurisdiction
Recreational Water Guideline Level
Recommended Action

Algae (includes cyanobacteria): high
amount
Level 2: Preferably microscopical examination; toxin analysis; warning of the public is
compulsory.
Algae (includes cyanobacteria): very
high amount
Level 3: Preferably microscopical examination; toxin analysis; warning of the public is
compulsory.
France0
Bloom, scum change in water color
Microscopy examination. If cyanobacteria are absent: no further action. If present: counting and
genus identification.
Cyanobacteria: < 20,000 cells/ml
(± 20 percent)
Active daily monitoring. Counting at least on a weekly basis. Normal recreational activity at the
site.
Cyanobacteria: > 100,000 cells/ml
(± 20 percent)
Bathing and recreational activities are restricted. Public is informed.
Cyanobacteria: >_20.000 to
< 100,000 cells/ml (± 20 percent)
Active daily monitoring. Counting on a weekly basis. Recreational activities are still allowed; the
public is informed by posters on site.
Microcystins: 25 |ig/L
(± 5 percent)
If microcystins < 25 |ig/L bathing and recreational activities are restricted. If microcystins >
25 |ig/L bathing is banned and recreational activities are restricted. In either case, public is
informed.
Visible scum or foam in recreational or
bathing area
All water activities in this area are prohibited. Restrictions do not necessarily apply to the whole
recreational site. Other areas without scum may still be open.
Germany0
Secclii Disk reading > 1 m AND
biovolume: < 1 mm3/L
Monitor further cyanobacterial development.
Secclii Disk reading > 1 m AND
biovolume: > 1 mm3/L
Publish warnings, discourage bathing, consider temporary closure.
Secclii Disk reading > 1 m AND
chlorophyll a (with dominance by
cyanobacteria): < 40 |ig/L
Monitor further cyanobacterial development.
Secclii Disk reading > 1 m AND
chlorophyll a (with dominance by
cyanobacteria): > 40 |ig/L
Publish warnings, discourage bathing, consider temporary closure.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-4

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Jurisdiction
Recreational Water Guideline Level
Recommended Action

Secclii Disk reading > 1 m AND
microcystins: <10 |ig/L
Monitor further cyanobacterial development.
Secclii Disk reading > 1 m AND
microcystins: >10 |ig/L
Publish warnings, discourage batliing, consider temporary closure.
Visible heavy scums and/or
microcystins: >100 |ig/L
Publish warnings, discourage batliing, temporary closure is recommended.
Hungary0
Cell count: > 50,000 to < 100,000
cells/ml
No recommended actions listed, water body classification: Acceptable.
Cell count: < 20,000 cells/ml
No recommended actions listed, water body classification: Excellent.
Cell count: > 20,000 to < 50,000 cells/ml
No recommended actions listed, water body classification: Good.
Cell count: > 100,000 cells/ml
No recommended actions listed, water body classification: Unacceptable.
Chlorophyll a (with dominance by
cyanobacteria): <10 (ig/L
No recommended actions listed, water body classification: Excellent.
Clilorophyll a (with dominance by
cyanobacteria): > 10 to < 25 |ig/L
No recommended actions listed, water body classification: Good.
Clilorophyll a (with dominance by
cyanobacteria): > 25 to < 50 |ig/L
No recommended actions listed, water body classification: Acceptable.
Clilorophyll a (with dominance by
cyanobacteria): > 50 |ig/L
No recommended actions listed, water body classification: Unacceptable.
Microcystins: > 4 to < 10 (ig/L
No recommended actions listed, water body classification: Good.
Microcystins: > 10 to < 20 (ig/L
No recommended actions listed, water body classification: Acceptable.
Microcystins: < 4 |ig/L
No recommended actions listed, water body classification: Excellent.
Microcystins: > 20 (ig/L
No recommended actions listed, water body classification: Unacceptable.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-5

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Jurisdiction
Recreational Water Guideline Level
Recommended Action
Italy'
Cyanobacterial cell count for
cyanotoxin-producing species other than
microcystins (e.g., cylindrospermopsin,
anatoxin-a) > 100,000 cells/ml (± 20
percent)
Emergency phase: weekly sampling and intensified visual inspection; quantification of all
identified cyanotoxins; health surveillance; temporary bans on bathing and removal of scums from
water and shoreline in addition to alert phase management measure.

Cylindrospermopsin and anatoxin-a >
20 (ig/L
Emergency phase: weekly sampling and intensified visual inspection; quantification of all
identified cyanotoxins; health surveillance; temporary bans on bathing and removal of scums from
water and shoreline in addition to alert phase management measures.

Microcystin-LR: > 20 |ig/L equivalents
Emergency phase: weekly sampling and intensified visual inspection; quantification of all
identified cyanotoxins; health surveillance; temporary bans on bathing and removal of scums from
water and shoreline in addition to alert phase management measures.

Total cyanobacterial cell count > 20,000
cells/ml (± 20 percent) AND
microcystin-LR < 20 |ig/L equivalents
Alert phase: weekly sampling and visual inspection every 2 days; assessment of bloom extent and
stretches of coastline affected; identify presence of cyanotoxins other than microcystins (when
relevant); management measures put in place to inform citizens and prevent hazardous exposures
using informative and warning panels/signs at waterfront and/or at beach access points,
newsletters, brochures, publications on regional and national websites, local information systems,
social network, and a Ministry toll-free number.

Transparency > 1 m AND total
phosphorus < 20 |ig/L
Routine phase 1: monthly sampling.

Transparency > 1 m AND total
phosphorus > 20 |ig/L AND total
cyanobacterial cell count < 2,000
cells/ml
Routine phase 2: monthly sampling and weekly visual inspection.

Transparency < 1 m AND total
phosphorus > 20 |ig/L AND total
cyanobacterial cell count > 2,000 to
< 20,000 cells/ml (± 20 percent)
Routine phase 3: fortnightly sampling and weekly visual inspection.

Visible surface scum
Emergency phase: weekly sampling and intensified visual inspection; quantification of all
identified cyanotoxins; health surveillance; temporary bans on bathing and removal of scums from
water and shoreline in addition to alert phase management measures.
Netherlands0
Biovolume (cyanobacterial cell count):
> 0 to < 2.5 mm3/L
Surveillance level: continue fortnightly monitoring
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-6

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Jurisdiction
Recreational Water Guideline Level
Recommended Action

Biovolume (cyanobacterial cell count):
>15 mm3/L (if 80 percent dominance of
microcystin producers and microcystins
< 20 |ig/L. revert to Alert Level 1).
Alert level 2: weekly monitoring and advice against bathing (by public authority): "You are
advised not to bathe in this water;" prohibition by local authority is possible.

Biovolume (cyanobacterial cell count):
>_2.5 to < 15 mm3/L
Alert level 1: weekly monitoring and issue warning (by site operator) for duration of that week:
"Toxic blue-green algae. Risk of skin irritation or intestinal problems." In case of daily site
inspection, reevaluate the warning on a daily basis.

Chlorophyll a: > 0 to < 12.5 (ig/L
Surveillance level: continue fortnightly monitoring.

Clilorophyll a: >75 |ig/L
Alert level 2: weekly monitoring and advice against bathing (by public authority): "You are
advised not to bathe in this water;" prohibition by local authority is possible.

Clilorophyll a: >_ 12,5 to £ 75 (ig/L
Alert level 1: weekly monitoring and issue warning (by site operator) for duration of that week:
"Toxic blue-green algae. Risk of skin irritation or intestinal problems." In case of daily site
inspection, reevaluate the warning on a daily basis.

Surface scum: category 1
Surveillance level: continue fortnightly monitoring.

Surface scum: category 2
Alert level 1: weekly monitoring and issue warning (by site operator) for duration of that week:
"Toxic blue-green algae. Risk of skin irritation or intestinal problems." In case of daily site
inspection, reevaluate the warning on a daily basis.

Surface scum: category 3
Alert level 2: weekly monitoring and advice against bathing (by public authority): "You are
advised not to bathe in this water"; prohibition by local authority is possible.
New Zealand11
Cyanobacteria (benthic): 20-50 percent
coverage of potentially toxigenic
cyanobacteria attached to substrate
Alert (amber mode):
•	Notify the public health unit.
•	Increase sampling to weekly.
•	Recommend erecting an information sign.
•	Consider increasing the number of survey sites.
•	If toxigenic cyanobacteria dominate the samples, testing for cyanotoxins is advised. If
cyanotoxins are detected in mats or water samples, consult the testing laboratory to determine if
levels are hazardous.

Cyanobacteria (benthic): greater than
50 percent coverage of potentially
toxigenic cyanobacteria attached to
substrate
Action (red mode) situation 1:
•	Immediately notify the public health unit.
•	If potentially toxic taxa are present (see Table 2) then consider testing samples for cyanotoxins.
•	Notify the public of the potential risk to health.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Jurisdiction
Recreational Water Guideline Level
Recommended Action

Cyanobacteria (benthic): Up to
20 percent coverage of potentially
toxigenic cyanobacteria attached to
substrate
Surveillance (green mode):
• Undertake fortnightly surveys between spring and autumn at representative locations in the
water body where known mat proliferations occur and where there is recreational use.

Cyanobacteria (benthic): up to
50 percent where potentially toxigenic
cyanobacteria are visibly detaching from
the substrate, accumulating as scums
along the river's edge or becoming
exposed on the river's edge as the river
level drops.
Action (red mode) situation 2:
•	Immediately notify the public health unit.
•	If potentially toxic taxa are present (see Table 2) then consider testing samples for cyanotoxins.
•	Notify the public of the potential risk to health.

Cyanobacteria (total): <0.5 mm3/L
(biovolume equivalent of the combined
total of all cyanobacteria)
Surveillance (green mode):
• Undertake weekly or fortnightly visual inspection and sampling of water bodies where
cyanobacteria are known to proliferate between spring and autumn.

Cyanobacteria (total): < 500 cells/ml
Surveillance (green mode):
• Undertake weekly or fortnightly visual inspection and sampling of water bodies where
cyanobacteria are known to proliferate between spring and autumn.

Cyanobacteria (total): >1.8 mm3/L
(biovolume equivalent of potentially
toxic cyanobacteria)
Action (red mode) situation 1:
•	Continue monitoring as for alert (amber mode).
•	If potentially toxic taxa are present (see Table 1), then consider testing samples for cyanotoxins
•	Notify the public of a potential risk to health.

Cyanobacteria (total): >0.5 to <
1.8 mm3/L (biovolume equivalent of
potentially toxic cyanobacteria)
Alert (amber mode):
•	Increase sampling frequency to at least weekly.
•	Notify the public health unit.
•	Multiple sites should be inspected and sampled.

Cyanobacteria (total): > 0.5 to <
10 mm3/L (total biovolume of all
cyanobacterial material where the
cyanobacterial population lias been
tested and shown not to contain known
toxins)
Alert (amber mode):
•	Increase sampling frequency to at least weekly.
•	Notify the public health unit.
•	Multiple sites should be inspected and sampled.

Cyanobacteria (total): >_10 mm3/L (total
biovolume of all cyanobacterial material
Action (red mode) situation 2:
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Jurisdiction
Recreational Water Guideline Level
Recommended Action

where the cyanobacterial population has
been tested and shown not to contain
known toxins)
•	Continue monitoring as for alert (amber mode).
•	If potentially toxic taxa are present (see Table 1), then consider testing samples for cyanotoxins.
•	Notify the public of a potential risk to health.

Cyanobacterial scums consistently
present for more than several days in a
row
Action (red mode) situation 3:
•	Continue monitoring as for alert (amber mode).
•	If potentially toxic taxa are present (see Table 1), then consider testing samples for cyanotoxins.
•	Notify the public of a potential risk to health.

Microcystins (total): > 12 |ig/L
Action (red mode) situation 1:
•	Continue monitoring as for alert (amber mode).
•	If potentially toxic taxa are present (see Table 1), then consider testing samples for cyanotoxins.
•	Notify the public of a potential risk to health.
Poland0
Visible blooms
Sampling of bathing sites not less than 4 times per season (the interval between sampling does not
exceed one month), including responses to cyanobacteria if blooms are observed.
Scotland6
Chlorophyll a: > 10 |ig/L with
dominance of cyanobacteria
1.	Watch for scum or conditions conducive to scums.
2.	Discourage bathing and further investigate hazard.
3.	Post on-site risk advisory signs.
4.	Inform relevant authorities.

Cyanobacteria: > 20,000 cells /ml
1.	Watch for scum or conditions conducive to scums.
2.	Discourage bathing and further investigate hazard.
3.	Post on-site risk advisory signs.
4.	Inform relevant authorities.

Cyanobacterial scum
formation in bathing areas
1.	Immediate action to control contact with scums; possible prohibition of swimming and other
water contact activities.
2.	Public health follow-up investigation.
3.	Inform public and relevant authorities.
Singapore0
Chlorophyll a: < 50 |ig/L (of 95 percent
of a 3-year rolling period)
Status of the sites reviewed annually. If the assessment is that the water body is unsuitable for
primary water contact activities, the public is notified.
Spain0
Cyanobacteria proliferation potential
(High Medium Low)
Criteria for assessment of health risk and response are set locally; some health authorities use
WHO scheme, others include further risk parameters (such as number of users, type of use);
temporary closure has occasionally occurred based on the abundance of cyanobacteria.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Jurisdiction
Recreational Water Guideline Level
Recommended Action
Turkey0
Cells: < 20,000 cells/ml
Level 1: recreational activities are allowed to continue and users are informed by posters on site.
Monitoring (sampling, counting and species identification) should be done fortnightly.
Cells: 20,000-100,000 cells/ml
Level 2: At > 20 000 cells/mL, microcystins are analyzed. If microcystin-LR equivalents
>25 |ig/L. immediate action to inform relevant authorities and public. Discourage users from
swimming and other water contact activities by advisory signs on site.
Chlorophyll a (if dominated by
cyanobacteria): <10 |ig/L
Level 1: recreational activities are allowed to continue and users are informed by posters on site.
Monitoring (sampling, counting and species identification) should be done fortnightly.
Microcystin-LR: < 10 |ig/L equivalents
Level 1: recreational activities are allowed to continue and users are informed by posters on site.
Monitoring (sampling, counting and species identification) should be done fortnightly.
Microcystin-LR: > 25 |ig/L equivalents
Level 2: At > 20,000 cells/mL, microcystins are analyzed. If microcystin-LR equivalents
>25 |ig/L. immediate action to inform relevant authorities and public. Discourage users from
swimming and other water contact activities by advisory signs on site.
Visible scum in bathing area
Level 3: all activities in the water may be prohibited.
World Health
Organization (WHO)b g
Chlorophyll a: 10 |ig/L with dominance
of cyanobacteria
Low risk: post on-site advisory signs, inform relevant authorities.
Chlorophyll a: 50 |ig/L with dominance
of cyanobacteria
Moderate risk: watch for scums or conditions conducive to scums, discourages swimming and
further investigate hazard, post on-site risk advisory signs, inform relevant authorities.
Cyanobacteria: 100,000 cells/ml
Moderate risk: watch for scums or conditions conducive to scums, discourages swimming and
further investigate hazard, post on-site risk advisory signs, inform relevant authorities.
Cyanobacteria: 20,000 cells/ml
Low risk: post on-site advisory signs, inform relevant authorities.
Cyanobacterial scum formation in areas
where whole-body contact and/or risk of
ingestions/aspiration occur
High risk: immediate action to control contact with scums, possible prohibition of swimming and
other water contact activities, public health follow-up investigation, inform public and relevant
authorities.
a Australian Government National Health and Medical Research Council (2008). Guidelines for Managing Risk in Recreational Water.
b Chorus, I. and Bartram, J. (eds.) (1999). Toxic cyanobacteria in water: A guide to public health significance, monitoring and management. E. and F.N. Spon Chapman
and Hall, London United Kingdom.
c Federal Enviromnent Agency (Germany) (2012). Current approaches to Cyanotoxin risk assessment, risk management and regulations in different countries.
d Health Canada (2012). Guidelines for Canadian Recreational Water Quality, Third Edition. Water, Air and Climate Change Bureau, Healthy Enviromnents and
Consumer Safety Branch Health Canada, Ottawa, Ontario. (Catalogue No H129-15/2012E).
e Scottish Government Health and Social Care Directorates Blue-Green Algae Working Group (2012). Cyanobacteria (Blue-Green Algae) in Inland and Inshore Waters:
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-10

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Assessment and Minimization of Risks to Public Health.
'European Parliament and the Council of the European Union (2006). Directive 2006/7/EC of the European Parliament and of the Council of 15 February 2006
concerning the management of bathing water quality and repealing Directive 76/160/EEC.
g WHO (World Health Organization) (2003). Guidelines for Safe Recreational Water Environments: Volume 1: Coastal and Fresh Waters. World Health Organization.
h Wood, S; Hamilton, D; Safi, K; Williamson, W. (2008). New Zealand Guidelines for Cyanobacteria in Recreational Fresh Waters: Interim Guidelines. New Zealand
Ministry for the Environment and Ministry of Health.
Funari. W; Manganelli, M; Buratti, FM; Testai, E. (2017). Cyanobacteria blooms in water: Italian guidelines to assess and manage the risk associated to bathing and
recreational activities. Science of the Total Environment, 598, 867-880.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
A-ll

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Recommended Human Health Recreational Ambient Water Quality Criteria or
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APPENDIX B. STATE RECREATIONAL WATER GUIDELINES FOR CYANOTOXINS
AND CYANOBACTERIA
EPA compiled the information presented in this appendix based on searches of state websites for
publicly available information regarding guidelines or action levels for cyanotoxins and cyanobacteria.
Online searches for state guidance were conducted in 2015, 2016, and 2018. Direct personal
communication of state guidelines and state public comments on the draft AWQC revealed some
updated information.
Table B-l. Summary Counts of State Recreational Water Guidelines for Cyanotoxins and
Cyanobacteria by Type and Scope of Guidelines
Recreational Water
Guideline Type
and Scope
Number of States and
List of States
Additional Information
Quantitative guidelines for
cyanobacteria only
5 states:
Arizona, Idaho, Maine, New
Hampshire, Wisconsin
Measurements for these criteria include
cyanobacterial cell densities, proportion of
toxigenic cyanobacteria, chlorophyll concentration
and Secchi disk depth measurements.
Quantitative guidelines for
cyanotoxins only
4 states:
Illinois, Iowa, Nebraska, Ohio
State guidelines address four cyanotoxins in order
from most to least common:
microcystins (24 states)
anatoxin-a (11 states)
cylindrospermopsin (9 states)
saxitoxin (5 states)
Quantitative guidelines for
cyanotoxins and either
quantitative or qualitative
guidelines for cyanobacteria
20 states:
California, Colorado,
Connecticut, Indiana, Kansas,
Kentucky, Maryland,
Massachusetts, Michigan New
Jersey, New York, North Dakota,
Oklahoma, Oregon,
Pennsylvania, Rhode Island,
Utah, Vermont, Virginia,
Washington
Qualitative guidelines only
6 states:
Delaware, Florida, Missouri,
Montana, North Carolina, West
Virginia
Examples include:
presence of surface scum
visible discoloration
presence of potentially toxic algae
presence/absence test for microcystins
Guidelines under development
4 states:
Arkansas, Georgia, Minnesota,
Wyoming

Note: The EPA found that Texas and North Carolina published guidelines in the past, but the guidelines are no longer
found on their websites. Missouri is in the process of developing quantitative thresholds.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-l

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Table B-2. State Recreational Water Quality Guideline for Cyanotoxins and Cyanobacteria Sorted by Type
State
Recreational Water Guideline Level
Recommended Action
Reference
States with Guidelines Based on Cyanobacteria Only
Arizona
Blue-green algae (mean value based on a
minimum of two sample events within one
peak season): 20,000 cells/ml
and
Chlorophyll a result (mean value based on a
minimum of two sample events within one
peak season) in target range
Violation of the Narrative Nutrient Standard.
Arizona Department of Environmental
Quality (2008). Narrative Nutrient
Standard Implementation Procedures for
Lakes and Reservoirs,
litto ://www. azdea. eov/environ/water/stand
ards/download/draft nutrient.odf. Last
Accessed: 11/27/2018.
Idaho
Microcystis or Planktothrix: >40,000 cells/ml
Public health advisory posting by Public Health
District in conjunction with water body operator.
IDEQ (Idaho Department of
Environmental Quality) (2015). Blue-
Green Algae Bloom Response Plan: Final,
litto ://www. healthandwelfare .idaho. eov/Po
rtals/O/Health/EnvironmentalHealth/Idaho
%20Blue-
Grccn%20Alaac%20RcsDonsc%20PlanFin
al.ndf. Last Accessed: 11/27/2018.
Sum of all potentially toxigenic taxa: >
100,000 cells/ml
Public health advisory posting by Public Health
District in conjunction with water body operator.
Visible surface scum that is associated with
toxigenic species
Public health advisory posting by Public Health
District in conjunction with water body management
agency.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-2

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State
Recreational Water Guideline Level
Recommended Action
Reference
Maine
Secclii disk reading < 2 meters caused by
algae
Body of water considered impaired, but still safe to
swim.
Maine Department of Environmental
Protection (2013). Reports of Algal
Blooms.
litto ://www. maine. aoy/dco/watcr/lakcs/rcD
bloom.html. Last Accessed: 11/27/2018.
New Hampshire
Cyanobacteria: > 50 percent of total cell
counts from toxigenic cyanobacteria OR the
cyanobacteria cell count is greater than 70,000
cells per ml of water
Post beach advisory.
New Hampshire Department of
Enviromnental Services (2014). Beach
Advisories.
htft>://des.nh.eov/oreanization/divisions/wa
ter/wmb/beaches/advisories.htm. Last
Accessed: 11/27/2018.
Wisconsin
Cyanobacteria: > 100,000 cells/ml
Post health advisory and possible beach closure.
Wisconsin Department of Natural
Resources (2012). Draft Blue-Green Algae
Section of 303 (d) Report - 7/3/2012:
Harmful Algal Blooms.
htft>://dnr.wi.eov/lakes/blueereenaleae/doc
uments/HarmfulAlealBloomsvs2.t>df. Last
Accessed: 11/27/2018.
Wisconsin Department of Health Services
(2016). Harmful Algal Blooms Toolkit: A
Planning Guide for Public Health and
Emergency Response Professionals.
htft>s://www.dhs.wisconsin.eov/i3ublication
s/D0/D00853.Ddf. Last Accessed:
11/27/2018.

Visible scum layer
Post health advisory and possible beach closure.
Werner M, and Masnado R (2014).
Guidance for Local Health Departments:
Cyanobacteria and Human Health,
litto ://citv. milwaukee. sov/ImaseLibrarv/Gr
o ii d s/hca 11 h A ii t ho rs/ D C P/P D F s/C v a no b a c t
criaLHD.Ddf. Last Accessed: 11/27/2018.
Wisconsin Department of Health Services
(2016). Harmful Algal Blooms Toolkit: A
Planning Guide for Public Health and
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-3

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State
Recreational Water Guideline Level
Recommended Action
Reference



Emergency Response Professionals.
htft>s://www.dhs.wisconsin.eov/i3ublication
s/D0/D00853.Ddf. Last Accessed:
11/27/2018.
States with Guidelines Based on Cyanotoxin(s) Only
Illinois
Microcystin-LR: > 10 |ig/L
Appropriate lake management personnel and Illinois
EPA staff will be notified; follow-up monitoring by
the Illinois EPA may occur as professional judgment
dictates and staff laboratory, and financial resources
allow.
Illinois Environmental Protection Agency
(2013). 2013 Statewide Harmful Algal
Bloom Program.
htft>s://www2.illinois.eov/et>a/tomcs/water-
aualitv/monitoring/aleal-
bloom/Paees/2013-oroeram.ast>x. Last
Accessed: 11/27/2018.
Illinois Environmental Protection Agency
(2018). Blue-Green Algae and Harmful
Algal Blooms.
htft>s://www2.illinois.eov/et>a/tomcs/water-
aualitv/monitorine/aleal-
bloom/Paees/default.asox. Last Accessed:
12/5/2018.
Iowa
Microcystin: > 20 |ig/L
Warnings are posted at state park beaches.
Iowa Environmental Council (2018). Toxic
Blue-Green Algae: A Threat to Iowa
Beachgoers.
litto ://www. iaenvironment. ore/our-
work/clean-water-and-land-
stewardshio/swimmine-advisories. Last
Accessed: 11/27/2018.
Nebraska
Microcystin: > 20 |ig/L
Health alert; signs posted advising public to use
caution; affected swimming beaches will be closed;
boating and other recreational activities will be
allowed, but public advised to use caution and avoid
prolonged exposure to the water.
Nebraska Department of Environmental
Quality and Nebraska Department of
Health and Human Services: Division of
Public Health (2018). Fact Sheet:
Precautions and facts regarding toxic algae
at Nebraska Lakes.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-4

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State
Recreational Water Guideline Level
Recommended Action
Reference



htft>://dea.ne.eov/NDEOProe.nsf/OnWeb/
ENV042607. Last Accessed: 5/10/2019.
Ohio
Anatoxin-a: 300 |ig/L
Issue no contact advisory.
Ohio EPA (2016). State of Ohio Harmful
Algal Bloom Response Strategy For
Recreational Waters.
litto ://coa. ohio. sov/nortals/3 5/hab/H ABRe
snonseStratesv.ndf. Last Accessed:
11/27/2018.
Anatoxin-a: 80 |ig/L
Issue recreational public health advisory.
Cylindrospermopsin: 20 (ig/L
Issue no contact advisory.
Cylindrospermopsin: 5 (ig/L
Issue recreational public health advisory.
Microcystins: 20 |ig/L
Issue no contact advisory.
Microcystins: 6 (ig/L
Issue recreational public health advisory.
Saxitoxin: 0.8 (ig/L
Issue recreational public health advisory.
Saxitoxin: 3 (ig/L
Issue no contact advisory.
States with Guidelines Based on Cyanobacteria and Cyanotoxin(s)
California
Anatoxin-a: detection using an analytical
method that detects <1 |ig/L
Caution trigger level: increase monitoring and post
caution sign warning people to stay away from scum
and warning people to keep pets and livestock away
from water and scum.
Butler N, Carlisle J, Kaley KB, and
Linville R (2012). Toxicological Summary
and Suggested Action Levels to Reduce
Potential Adverse Health Effects of Six
Cyanotoxins.
htto://www.waterboards.ca. sov/water issu
es/nrosrams/neer review/docs/calif cvano
toxins/cvanotoxins053112.odf. Last
Accessed: 11/27/2018.
Cyanobacteria Harmful Algal Bloom
Network (2016a). Appendix to the
CCHAB Preliminary Changes to the
Statewide Voluntary Guidance of
CyanoHABs in Recreational Waters.
Anatoxin-a: 20 |ig/L
Warning tier 1: post warning sign stating that
swimming is not recommended and that pets and
livestock should be kept away from the water.
Anatoxin-a: 90 (ig/L
Danger tier 2: post sign stating that there is a present
danger and that people, pets and livestock should
stay out of the water and away from water spray.
Cylindrospermopsin: 1 (ig/L
Caution trigger level: increase monitoring and post
caution sign warning people to stay away from scum
and warning people to keep pets and livestock away
from water and scum.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-5

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State
Recreational Water Guideline Level
Recommended Action
Reference

Cylindrospermopsin: 4 |ig/L
Warning tier 1: post warning sign stating that
swimming is not recommended and that pets and
livestock should be kept away from the water.
htft>://www.mvwateraualitv.ca.eov/monitor
ins council/cvanohab nctwork/docs/aDDcn
dix a.odf. Last Accessed: 11/27/2018.
Cyanobacteria Harmful Algal Bloom
Network (2016b). Table 1: CyanoHAB
trigger levels for human health.
htft>://www.mvwateraualitv.ca.eov/monitor
ins council/cvanohab network/docs/trieee
rs.odf. Last Accessed: 11/27/2018.

Cylindrospermopsin: 17 |ig/L
Danger tier 2: post sign stating that
there is a present danger and that people, pets and
livestock should stay out of the
water and away from water spray.

Microcystins: 0.8 |ig/L
Caution trigger level: increase monitoring and post
caution sign warning people to stay away from scum
and warning people to keep pets and livestock away
from water and scum.

Microcystins: 6 (ig/L
Warning tier 1: post warning sign stating that
swimming is not recommended and that pets and
livestock should be kept away from the water.


Microcystins: 20 (ig/L
Danger tier 2: post sign stating that
there is a present danger and that people, pets and
livestock should stay out of the
water and away from water spray.


Site-specific indicators of cyanobacteria (e.g.,
blooms, scums, mats)
Caution trigger level: increase monitoring and post
caution sign warning people to stay away from scum
and warning people to keep pets and livestock away
from water and scum.


Toxin-producing cyanobacteria: 4,000
cells/ml
Caution trigger level: increase monitoring and post
caution sign warning people to stay away from scum
and warning people to keep pets and livestock away
from water and scum.

Colorado
Anatoxin-a: > 7 (ig/L
Issue toxic algae caution:
a.	post sign with "caution" language.
b.	perform routine testing for toxin levels.
bi. if test results are below caution thresholds, test at
Colorado Department of Public Health and
Environment. Algae bloom risk-
management toolkit for recreational
waters.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-6

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State
Recreational Water Guideline Level
Recommended Action
Reference


least once per week until algae visually subsides,
bii. if test results are above caution thresholds, test at
least twice per week until toxin levels are below
caution thresholds for two consecutive tests.
c.	notify drinking water providers and county health
department if toxin levels exceed the caution
thresholds.
d.	toxic algae caution ends when there is no visual
evidence of algae and toxin levels are non-detectable
for two consecutive weeks.
di. notify drinking water providers and county health
department that bloom lias ended,
dii. remove "caution" sign.
httDs://\vww.Colorado. aov/Daciric/cdDhc/ha
rmful-aleae-blooms. Last Accessed:
11/27/2018

Cylindrospermopsin: > 7 |ig/L
Issue toxic algae caution:
a.	post sign with "caution" language.
b.	perform routine testing for toxin levels.
bi. if test results are below caution thresholds, test at
least once per week until algae visually subsides,
bii. if test results are above caution thresholds, test at
least twice per week until toxin levels are below
caution thresholds for two consecutive tests.
c.	notify drinking water providers and county health
department if toxin levels exceed the caution
thresholds.
d.	toxic algae caution ends when there is no visual
evidence of algae and toxin levels are non-detectable
for two consecutive weeks.
di. notify drinking water providers and county health
department that bloom lias ended,
dii. remove "caution" sign.


Microcystin-LR: > 10 (ig/L and < 20 |ig/L
Issue toxic algae caution:
a.	post sign with "caution" language.
b.	perform routine testing for toxin levels.
bi. if test results are below caution thresholds, test at
least once per week until algae visually subsides.

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-7

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State
Recreational Water Guideline Level
Recommended Action
Reference


bii. if test results are above caution thresholds, test at
least twice per week until toxin levels are below
caution thresholds for two consecutive tests.
c.	notify drinking water providers and county health
department if toxin levels exceed the caution
thresholds.
d.	toxic algae caution ends when there is no visual
evidence of algae and toxin levels are non-detectable
for two consecutive weeks.
di. notify drinking water providers and county health
department that bloom lias ended,
dii. remove "caution" sign.


Microcystin-LR: > 20 |ig/L
Issue toxic algae warning:
a.	immediately post sign with "warning" language.
b.	take necessary steps to prevent contact with water
in affected area for humans and pets.
c.	notify drinking water providers and county health
department if toxin levels exceed warning thresholds.
d.	test at least twice per week until toxin levels are
below warning thresholds for two consecutive tests.
e.	posting can be reduced to "caution" language
when microcystin test results drop below the warning
threshold and no new human illness or pet deaths
have been reported for two consecutive weeks.


Saxitoxin: > 4 (ig/L
Issue toxic algae caution:
a.	post sign with "caution" language.
b.	perform routine testing for toxin levels.
bi. if test results are below caution thresholds, test at
least once per week until algae visually subsides,
bii. if test results are above caution thresholds, test at
least twice per week until toxin levels are below
caution thresholds for two consecutive tests.
c.	notify drinking water providers and county health
department if toxin levels exceed the caution
thresholds.

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-8

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State
Recreational Water Guideline Level
Recommended Action
Reference


d. toxic algae caution ends when there is no visual
evidence of algae and toxin levels are non-detectable
for two consecutive weeks.
di. notify drinking water providers and county health
department that bloom lias ended,
dii. remove "caution" sign.


Potentially toxic algae are visible
Issue toxic algae caution:
a.	post sign with "caution" language.
b.	perform routine testing for toxin levels.
bi. if test results are below caution thresholds, test at
least once per week until algae visually subsides,
bii. if test results are above caution thresholds, test at
least twice per week until toxin levels are below
caution thresholds for two consecutive tests.
c.	notify drinking water providers and county health
department if toxin levels exceed the caution
thresholds.
d.	toxic algae caution ends when there is no visual
evidence of algae and toxin levels are non-detectable
for two consecutive weeks.
di. notify drinking water providers and county health
department that bloom lias ended,
dii. remove "caution" sign.

Connecticut
Visual rank category 2: cyanobacteria present
in low numbers; there are visible small
accumulations but water is generally clear;
OR blue-green algae cells > 20,000 cells/ml
and < 100,000 cells/ml
Notify Connecticut Department of Public Health (CT
DPH), Connecticut Department of Energy and
Environmental Protection (CT DEEP); increase
regular visual surveillance until conditions change;
consider cautionary postings at public access points.
Connecticut Department of Public Health
and Connecticut Department of Energy and
Environmental Protection (CDPH and
CDEEP) (2017). Guidance to Local Health
Departments for Blue-Green Algae
Blooms in Recreational Freshwaters.
litto ://www. ct. ao v/dc c d/ 1 i b/dc c d/wa tc r/wa t
er aualitv manaucmcnt/monitori nuDubs/bl
ueereenaleaeblooms euidanceforlhds 201
7version.t>df. Last Accessed: 11/27/2018.

Visual rank category 3: cyanobacteria present
in high numbers; scums may or may not be
present; water is discolored throughout; large
areas affected; color assists to rule out
sediment and other algae; OR blue-green
algae cells > 100,000 cells/ml
Update/inform CTDPH and CTDEEP and expand
risk communication efforts; collect samples for
analysis and/or increase frequency of visual
assessment; POSTED BEACH CLOSURE: if public
lias beach access, alert water users that a blue-green
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-9

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State
Recreational Water Guideline Level
Recommended Action
Reference


algae bloom is present; POSTED ADVISORY: at
other impacted access points.
Connecticut Department of Energy and
Environmental Protection (CDEEP).
(2017). Comment Letter Regarding Human
Health Recreational Ambient Water
Quality Criteria and/or Swimming
Advisories for Microcystins and
Cylindrospermopsin. March 20, 2017.
Docket No. EPA-HQ-OW-2016-0715.
httDs://\vww. regulations. aov/dockct'.'D=EP
A-HO-OW-2016-0715. Last accessed:
11/27/2018.
Anatoxin-a: 80 |ig/L
Issue recreation advisory.
Indiana
Blue-green algae: 100,000 cells/ml
Issue recreation advisory.
Indiana Department of Environmental
Management (2018). Blue-Green Algae:
Indiana Reservoir and Lake Update.
htft>://www.in. eov/idem/aleae/. Last
Accessed: 11/27/2018.
Cylindrospermopsin: 8 |ig/L
Issue recreation advisory.
Microcystin-LR: 20 (ig/L
Close beaches.
Microcystin-LR: 4 |ig/L
Issue recreation advisory.
Cyanobacteria: > 10,000,000 cells/ml
Recommended that all in-lake recreation cease and
that picnic, camping and other public land activities
adjacent to affected waters be closed.
Kansas
Cyanobacteria: > 250,000 cells/ml
Issue public health warning.
Kansas Department of Health and
Environment (2015). Guidelines for
Addressing Harmful Algal Blooms in
Kansas Recreational Waters.
htft>://www.kdheks. eov/aleae-
illness/download/HAB Dolicv.Ddf. Last
Accessed: 11/27/2018.
Kansas Department of Health and
Environment (2015). Harmful Algal
Blooms (HABs): KDHE Agency Response
Plan. htft>://www.kdheks.eov/aleae-
illness/download/HAB response nlan.ndf.
Cyanobacteria: > 80,000 and < 250,000
cells/ml
Issue public health watch.
Microcystin: > 2,000 |ig/L
Recommended that all in-lake recreation cease and
that picnic, camping and other public land activities
adjacent to affected waters be closed.
Microcystin: > 20 (ig/L
Issue public health warning.
Microcystin: > 4 and < 20 |ig/L
Issue public health watch.
Blue-green algae: > 100,000 cells/ml
Issue anHAB advisory.
Last Accessed: 11/27/2018.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-10

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State
Recreational Water Guideline Level
Recommended Action
Reference




Kentucky
Microcystins: > 20 |ig/L
Issue recreational use advisory.
Kentucky Department for Environmental
Protection (2014). Harmful Algal Blooms:
Background.
htft>://water.kv.eov/wateraualitv/Document
s/HAB FACTs/HAB%20Backeround%20
Fact%20Sheet.t>df. Last Accessed:
11/27/2018.

Microcystis aeruginosa or other potential
microcystin-producing blue-green algae
> 40,000 cells/ml, and samples contain
microcystins: >10 ppb
Put up signs advising public of health risk, notify
local press (through joint DHMH, DNR, MDE press
release) and coordinate with local health department,
place advisory information on DNR web site (Eyes
on the Bay), Maryland Healthy Beaches web site if a
swimming beach is affected, or other local web site.
MDE will initiate emergency closure to shellfish
harvesting if warranted, and coordinate with DNR
Natural Resource Police.
Commonwealth of Kentucky: Department
for Enviromnental Protection Division of
Water (2015). Harmful Algal Blooms.
httt>://water.kv.eov/wateraualitv/t>aees/HA
BS.asD.x. Last Accessed: 11/27/2018.
Maryland
Presence of potentially toxic algae
Issue algae bloom beach alert.
Wazniak C personal communication.
(2016). Regarding Maryland Department
of Natural Resources Harmful Algal
Bloom (HAB) Monitoring and
Management SOP. Sent via email
correspondence from Catherine Wazniak,
Program Manager at the MD DNR, on
February 22, 2016, to John Ravenscroft,
U.S. EPA.
Maryland Department of Natural
Resources (2014). Harmful Algal Bloom
Management in the Chesapeake and
Coastal Bays.
litto ://dnr. marvland. eov/waters/bav/Docum
ents/HAB Management.ndf. Last
Accessed: 11/27/2018.

Blue-green algae: > 50,000 cells/ml
Toxin testing of lysed cells should be done to ensure
that guideline of 14 ppb is not exceeded.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-ll

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State
Recreational Water Guideline Level
Recommended Action
Reference
Massachusetts
Blue-green algae: > 70,000 cells/ml
Post an advisory against contact with the water.
Massachusetts Bureau of Environmental
Health (2015). MDPH Guidelines for
Cyanobacteria in Freshwater Recreational
Water Bodies in Massachusetts. Boston
Massachusetts.

Microcystins: > 14 |ig/L
Post an advisory against contact with the water.
httt>://www.mass.eov/eohhs/docs/dr>h/envir
oniric ntal/cxDOSiirc/Drotocol-
cvanobacteria.ndf. Last Accessed:
11/27/2018.

Visible cyanobacteria scum or mat is evident
MDPH recommends an immediate posting by the
local health department, state agency, or relevant
authority to advise against contact with the water
body.
Massachusetts Department of Public
Health (2008). MDPH guidelines for
cyanobacteria in freshwater recreational
water bodies in Massachusetts.
httt>://www.mass.eov/eohhs/docs/dr>h/envir
oniric ntal/cxDOSiirc/Drotocol-
cvanobacteria.ndf. Last Accessed:
11/27/2018.

Microcystin: >20 micrograms per liter (|ig/L)
Not reported.
Michigan
Other algal toxins are at or above appropriate
guidelines that have been reviewed by
MDEQ-WRD
Not reported.
Post advisory.
Michigan Department of Environmental
Quality (2018). Algae (Harmful Algal
Blooms) website
httt>://www.michiean.eov/dea/0.4561.7-
135-3313 3681 3686 3728-383630-
.00.html. Last Accessed: 11/27/2018.
Kohlhepp (2015) Harmful Algal Bloom

Chlorophyll a: >30 |ig/L and visible surface
accumulations/scum are present, or cells are
visible throughout the water column


Microcystins (as total including -LR and
other detectable congeners): 3 |ig/L

Monitoring and Assessment in Michigan
Waters. Michigan Department of
Environmental Quality Water Resources
Division. MI/DEQ/WRD-15/013.
httt>://www.michieaneov/documents/dea/
wrd-swas-aleae-
HABsummarv 551207 7.t>df. Last
Accessed: 03/6/2018.
New Jersey
Cylindrospermopsin: 8 |ig/L
Post advisory.

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-12

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State
Recreational Water Guideline Level
Recommended Action
Reference

Anatoxin-a: 27 |ig/L
Post advisory.
New Jersey Department of Environmental
Protection (2017). Cyanobacterial Harmful
Algal Bloom (HAB) Freshwater
Recreational Response Strategy,
litto ://www. state, ni. u s/dc d A v m s/b ft) m/N J H
AESRcsdoriseStratcav.ixlf. Last Accessed:
11/27/2018
Cyanobacterial cell count: > 20,000 cells/ml
Post advisory.
Visual indication of a bloom - receipt of a
bloom report or digital photograph
Suspicious Bloom: DEC HABs Program staff
determine if a bloom is Suspicious and whether
collection of a sample is feasible or warranted.
New York
Blue-green chlorophyll levels: > 25 (ig/L; OR
Microscopic confirmation that majority of
sample is cyanobacteria and present in bloom-
like densities; OR only in absence of the
previous criteria being met: microcystin > 4
|ig/L but less than 20 |ig/L and accompanied
by ancillary evidence of the presence or recent
history of a bloom
Confirmed Bloom: Signs have been developed by
NY State Department of Health for use at regulated
swimming beaches when Local Health Department
personnel or beach operators close beaches.
Online summer notification provides weekly update
on the number of HABs locations in New York is
included inMakingWaves, the DEC email
subscription.
New York State Department of
Environmental Conservation (2017).
Harmful Algal Blooms (HABs) Program
Guide.
litto://www.dec.nv. sov/docs/water odf/hab
snrosramsuide.ndf. Last Accessed:
11/27/2018.
Microcystin > 20 |ig/L (shoreline samples
only); OR microcystin > 10 |ig/L (open water
samples only); OR known risk of exposure to
anatoxin or another cyanotoxin, based on
consult between DEC HABS Program and
NYSDOH staff
Confirmed with High Toxins Bloom: Signs have
been developed by NY State Department of Health
for use at regulated swimming beaches when Local
Health Department personnel or beach operators
close beaches.
Online summer notification provides weekly update
on the number of HABs locations in New York is
included inMakingWaves, the DEC email
subscription.
Blue-green algae bloom is present AND
microcystin-LR: < 10 |ig/L
Issue advisory.
North Dakota
Blue-green algae bloom is present over a
significant portion of the lake AND
microcystin-LR: > 10 |ig/L
Issue warning.
North Dakota Department of Health:
Division of Water Quality (2016). Blue-
green algae advisories and warnings.
Cyanobacteria: 100,000 cell/ml
Issue advisory.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-13

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State
Recreational Water Guideline Level
Recommended Action
Reference



htft>://www.ndhealth.eov/wa/sw/habs/defa
ulthabs.htm. Last Accessed: 11/27/2018.
Oklahoma
Microcystin: > 20 |ig/L
Issue advisory.
Oklahoma Legislature (2012). SB 259 Bill
Summary.
httD://\vcbscrvcrl .lsb.state.ok.us/CF/2011-
12%20SUPPORT%20DOCUMENTS/BIL
LSUM/House/SB259%20cci%20a%20bill
sunxdoc. Last Accessed: 11/27/2018.
Anatoxin-a: > 20 |ig/L
Issue public health advisory.
Oregon
Cylindrospermopsin: > 20 (ig/L
Issue public health advisory.
Oregon Health Authority (2018). Oregon
Harmful Algae Bloom Surveillance
(HABS) Program Public Health Advisory
Guidelines: Harmful Algae Blooms in
Freshwater Bodies.
htft>s://www.oreeon.eov/oha/i3h/HealthvEn
vironments/Recreation/HarmfulAleaeBloo
ms/Documents/HABPublicHealtliAdvisorv
Guidelines.pdf. Last Accessed:
Microcystin: > 10 (ig/L
Issue public health advisory.
Microcystis'. > 40,000 cells/ml
Issue public health advisory.
Planktothrix: > 40,000 cells/ml
Issue public health advisory.
Saxitoxin: > 10 |ig/L
Issue public health advisory.
Toxigenic species: > 100,000 cells/ml
Issue public health advisory.
11/27/2018.
Visible scum with documentation and testing
Issue public health advisory.
Microcystin: > 6 |ig/L
Recreational Public Health Advisory.
Pennsylvania
Microcystin: > 20 |ig/L
Recreational No Contact Advisory.
Pennsylvania Department of
Enviromnental Protection (2014). Lake
Erie Harmful Algal Bloom Monitoring and
Response Strategy for Recreational
Waters.
httr)s://seasrant.i3su.edu/sites/default/files/P
A%20Lake%20Erie%20Harmfol%20Aleal
Cylindrospermopsin: > 5 (ig/L
Recreational Public Health Advisory.
Cylindrospermopsin: > 20 (ig/L
Recreational No Contact Advisory.
Anatoxin-a: > 80 |ig/L
Recreational Public Health Advisory.
Anatoxin-a: > 300 (ig/L
Recreational No Contact Advisory.
%20Bloom%20RcsDonsc%20Stratcav%20
For%20Recreational%20Waters%20-
Saxitoxin: > 0.8 |ig/L
Recreational Public Health Advisory.
%202nd%20Draft.odf. Last Accessed:
11/27/2018.
Saxitoxin: > 3 (ig/L
Recreational No Contact Advisory.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-14

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State
Recreational Water Guideline Level
Recommended Action
Reference

HAB verified by visual observation
Recreational no contact advisory.

Cyanobacteria: > 70,000 cells/ml
Issue health advisory.
Rhode Island
Microcystin-LR: > 14 |ig/L
Issue health advisory.
Rhode Island Department of
Environmental Management, and Rhode
Island Department of Health (2013).
Cyanobacteria Related Public Health
Advisories in Rhode Island.
lUtD://\vww. health. ri.aov/Diiblications/datar
coorts/2013 CvanobacteriaBloomsInRhodel
sland.ndf. Last Accessed: 11/27/2018.
Visible cyanobacteria scum or mat
Issue health advisory.
Anatoxin-a: detection 90 |ig/L
Tier 2: Warning: Issue WARNING advisory. Post
WARNING signs, sampling recommended weekly.
Utah
Anatoxin-a: > 90 (ig/L
Tier 3: Danger: Issue DANGER advisory. Post
DANGER signs, consider CLOSURE, sampling
recommended at least weekly.
Utah Department of Environmental
Quality and Department of Health (2017).
Utah HAB Guidance Summary.
lUtD://hcalthutahaoY/cnvirocDi/aDDlctrcc/
HAB/HAB Guidance Summary 2017.t>df
. Last Accessed: 11/27/2018.
Cyanobacteria: 20,000 - 10,000,000 cells/ml
Tier 2: Warning: Issue WARNING advisory. Post
WARNING signs, sampling recommended weekly.
Cyanobacteria: >10,000,000 cells/ml
Tier 3: Danger: Issue DANGER advisory. Post
DANGER signs, consider CLOSURE, sampling
recommended at least weekly.
Microcystin: 4 - 2,000 |ig/L
Tier 2: Warning: Issue WARNING advisory. Post
WARNING signs, sampling recommended weekly.
Microcystin: > 2,000 |ig/L
Tier 3: Danger: Issue DANGER advisory. Post
DANGER signs, consider CLOSURE, sampling
recommended at least weekly.
Cylindrospermopsin: > 8 |ig/L
Tier 2 or 3: Consult with Utah Department of
Environmental Quality and Utah Department of
Health as needed on this issue.
Reports of animal illnesses or death
Tier 2: Warning: Issue WARNING advisory. Post
WARNING signs, sampling recommended weekly.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-15

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State
Recreational Water Guideline Level
Recommended Action
Reference

Reports of human illness
Tier 3: Danger: Issue DANGER advisory. Post
DANGER signs, consider CLOSURE, sampling
recommended at least weekly.

Anatoxin-a: > 10 |ig/L
Close recreational beaches.
Vermont
Cylindrospermopsin: > 10 (ig/L
Close recreational beaches.
Vermont Department of Health (2015).
Cyanobacteria (Blue-green Algae)
Guidance for Vermont Communities.
htft>://www.healthvermont.eov/sites/defaul
t/files/documents/2016/12/ENV RW Cva
nobacteriaGuidance.odf. Last Accessed:
11/27/2018.
Microcystin-LR (equivalents): > 6 (ig/L
Close recreational beaches.
Visible known blue-green algae bloom/scum
or an unknown, potentially blue-green algae
(i.e., not pollen), bloom/scum
Close recreational beaches.
Blue-green algal "scum" or "mats" on water
surface
Immediate public notification to avoid all
recreational water contact where bloom is present;
continue weekly sampling.
Virginia
Microcystin: > 6 |ig/L
Immediate public notification to avoid all
recreational water contact where bloom is present;
continue weekly sampling.
Virginia Department of Health (Division of
Environmental Epidemiology) (2012).
Virginia Recreational Water Guidance for
Microcystin wAMicrocystis Blooms:
Provisional Guidance,
litto ://www. vdhvirsinia. eov/content/uoloa
ds/sites/12/2016/02/VDHMicrocvstisGuida
nce.odf. Last Accessed: 11/27/2018.
Microcystis: > 100,000 cells /ml
Immediate public notification to avoid all
recreational water contact where bloom is present;
continue weekly sampling.
Microcystis: 20,000 to 100,000 cells/ml
Notify public through press release and/or signage;
advise people and pet owners that harmful algae are
present; initiate weekly water sampling.
Microcystis: 5,000 to < 20,000 cells/ml
Local agency notification; initiate bi-weekly water
sampling.
Anatoxin-a: 1 |ig/L
Tier 2: local health posts WARNING sign; local
health takes additional site-specific steps; minimum
weekly sampling. In addition, if history of high
toxicity, or reports of illness, pet death than tier 3:
local health posts DANGER sign; lake closed.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-16

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State
Recreational Water Guideline Level
Recommended Action
Reference
Washington
Bloom is forming or a bloom scum is visible
(toxic algae may be present); toxin levels do
not exceed thresholds
Tier 1: local health posts CAUTION sign; samples
taken and sent for toxicity tests; weekly sampling
until bloom dissipates.
Hardy J, and Washington State Department
of Health (2008). Washington State
Recreational Guidance for Microcystins
(Provisional) and Anatoxin-a
(Interim/Provisional).
htft>://www.doh.wa.eov/Portals/l/Docume
nts/4400/334-177-receuide.t>df. Last
Accessed: 11/27/2018.

Cylindrospermopsin: 4.5 |ig/L
Tier 2: local health posts WARNING sign; local
health takes additional site-specific steps; minimum
weekly sampling. In addition, if history of high
toxicity, or reports of illness, pet death than tier 3:
local health posts DANGER sign; lake closed.
Hardy J, and Washington State Department
of Health (2011). Washington State
Provisional Recreational Guidance for
Cylindrospermopsin and Saxitoxin.
htft>://www.doh.wa.eov/t>ortals/l/documen
ts/4400/332-118-
cvlindrosa\%20rcDort.Ddf Last Accessed:
11/27/2018.

Microcystins: 6 |ig/L
Tier 2: local health posts WARNING sign; local
health takes additional site-specific steps; minimum
weekly sampling. In addition, if history of high
toxicity, or reports of illness, pet death than tier 3:
local health posts DANGER sign; lake closed.


Saxitoxin: 75 |ig/L
Tier 2: local health posts WARNING sign; local
health takes additional site-specific steps; minimum
weekly sampling. In addition if history of high
toxicity, or reports of illness, pet death than tier 3:
local health posts DANGER sign; lake closed.
Hardy J, and Washington State Department
of Health (2008). Washington State
Recreational Guidance for Microcystins
(Provisional) and Anatoxin-a
(Interim/Provisional).
htft>://www.doh.wa.eov/Portals/l/Docume
nts/4400/334-177-receuide.t>df. Last
Accessed: 11/27/2018.

Saxitoxin: 75 |ig/L
Tier 2: local health posts WARNING sign; local
health takes additional site-specific steps; minimum
weekly sampling. In addition, if history of high
Hardy J, and Washington State Department
of Health (2011). Washington State
Provisional Recreational Guidance for
Cylindrospermopsin and Saxitoxin.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-17

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State
Recreational Water Guideline Level
Recommended Action
Reference

toxicity, or reports of illness, pet death than tier 3:
local health posts DANGER sign; lake closed.
htft>://www.doh.wa.eov/i3ortals/l/documen
ts/4400/332-118-
cvlindrosa\%20rcDort.Ddr. Last Accessed:
11/27/2018.
States with Qualitative Guidelines Only
Delaware
Thick green, white, or red scum on surface of
pond
Post water advisory signs.
Delaware Department of Natural
Resources and Environmental Control:
Division of Water. Blue-Green Algae in
Delaware. (2016).
litto ://www. dnrec. delaware. eov/wr/INF OR
MATION/OTHERINFO/Pases/Blue-
GreenAleae.asox. Last Accessed:
11/27/2018.
Florida
Cyanobacteria bloom
Issue health advisory; post warning signs.
Florida Department of Environmental
Protection (2019). Freshwater Algal
Blooms: Frequently Asked Questions.
httos://floridadeaeov/sites/default/files/fre
shwater-aleal-bloom-faas 2019.odf Last
Accessed: 5/10/2019.
Missouri
Microcystins: presence (test strip range 0 to
10 ng/ml)
Missouri has a multi-agency proactive approach to
address events which can result in the decision to
temporary close swim beaches and post notices
regarding the bloom around the lake to protect the
citizens of Missouri from the health risk posed by
exposure to a HAB. Information is also released to
through the news media and social media to quickly
share the possible health risk with the largest
audience possible.
Missouri Department of Natural Resources
(2017)	Qualitative screening of algal toxins
in drinking water and recreational waters
using strip test by Abraxas, Inc.
httos ://dnr. mo. so v/c n v/do c s/ md n rc s d 360. d
df. Last Accessed: 11/27/2018.
Missouri Department of Natural Resources
(2018)	Harmful Algal Blooms and Blue-
Green Algae. Website
httos ://dnr. mo. eov/env/cvanobacteria. htm.
and
htft>://et>htn.dhss.mo.eov/EPHTN Data Po
rtal/odf/success-stories/MO-Blue-Green-
Cylindrospermopsin: presence (test strip range
0 to 10 ng/ml)
Anatoxin-a: presence (test strip range 0 to
2.5 ng/ml)
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-18

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State
Recreational Water Guideline Level
Recommended Action
Reference



Alsae-Task-Force-Establislunent.ndf Last
Accessed: 11/27/2018.
Montana
Reservoirs that seem stagnated and harbor
large quantities of algae
The Montana Department of Environmental Quality
advises people to avoid swimming in ponds, lakes, or
reservoirs.
State of Montana Newsroom (2015). DEQ
Issues Advisory on Blue-Green Algae
Blooms: Ponds, Lakes, and Reservoirs
Most Often Affected.
littD://news. mt. aov/Homc/ArtlVI ID/24469/
ArticleID/1564/DEO-Issues-Advisorv-on-
Blue-Green-Alsae-Blooms. Last Accessed:
11/27/2018.
North Carolina
Visible discoloration or surface scum
Microcystin testing.
North Carolina Health and Human
Services: Division of Public Health (2014).
Occupational and Environmental
Epidemiology: Cyanobacteria (Blue-green
Algae).
lUtD://cDi.Diiblichcalthnc.aov/occ/a z/alsa
e.html. Last Accessed: 11/27/2018.
West Virginia
Blue-green algal blooms observed and
monitored
Issue public health advisory.
West Virginia Department of Health and
Human Resources (2015). DHHR
Continuing to Monitor Blue-Green Algal
Blooms on the Ohio River: Residents
Advised to Adhere to Public Health
Advisory.
htft>://www.dhhr.wv. sov/News/2015/Pases
/DHHR-Continuine-to-Monitor-Blue-
Grccn-Alaal-Blooms-o n-the-Ohio-
River%3B-Residents-Advised-to-Adhere-
to-Public-Health-Advisorv.ast>x. Last
Accessed: 11/27/2018.
States with Guidelines Under Development
Arkansas
TBD
TBD
Arkansas Beautiful Buffalo River Action
Committee (2018).
httos ://bbrac .arkansas. sov/ndfs/201701205
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-19

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State
Recreational Water Guideline Level
Recommended Action
Reference



-arkansas-harmful-aleal-bloom-(habs)-
worksroun.ndf. Last Accessed:
11/27/2018.
Georgia
TBD
TBD
Georgia Department of Public Health
(2018). httt>s://www.eachd.org/t>roerams-
services/environmental-health-
2/beach water testine/. Last Accessed:
03/6/2018.
Minnesota
TBD
TBD
Minnesota Department of Health (2015).
Toxicological Summary for: Microcystin-
LR.
litto ://www. health, state. mn.us/divs/eh/risk/
euidance/ew/microcvstin.t>df. Last
Accessed: 11/27/2018.
Wyoming
TBD
TBD
Wyoming Department of Environmental
Quality (2018). Harmful Algal Bloom
Website.
httr>://dea. wvomine. eov/wad/nutrient-
Dollution/resources/harmful-aleal-blooms/.
Last Accessed: 11/27/2018.
Note: Alabama, Alaska, Hawaii, Louisiana, Mississippi, Nevada, New Mexico, South Carolina, South Dakota, Tennessee, and Texas did not have guidelines available
online. Missouri is in the process of developing quantitative thresholds.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
B-20

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APPENDIX C. LITERATURE SEARCH DOCUMENTATION
The recreational ambient water quality criteria (AWQC) or swimming advisories document for
microcystins and cylindrospermopsin relied significantly on information identified, reviewed, and
synthesized in the EPA's Health Effects Support Document for the Cyanobacterial Toxin Microcystins,
Heath Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin, Drinking Water
Health Advisory for the Cyanobacterial Microcystin Toxins, and Drinking Water Health Advisory for the
Cyanobacterial Toxin Cylindrospermopsin (U.S. EPA 2015c, 2015d, 2015a, 2015b). The EPA
conducted supplemental literature searches to answer additional questions related to recreational
exposures, exposure factors, and to identify new health data.
For the Health Effects Support Documents (HESDs), the EPA conducted a comprehensive literature
search from January 2013 to May 2014 using Toxicology Literature Online (TOXLINE), PubMed, and
Google Scholar. The EPA assembled available information on occurrence; environmental fate;
mechanisms of toxicity; acute, short-term, subchronic, and chronic toxicity and cancer in humans and
animals; and toxicokinetics and exposure. For a detailed description of the literature review search and
strategy, see the HESDs for microcystins and cylindrospermopsin (U.S. EPA 2015c, 2015d).
The EPA conducted supplemental literature searches in September 2015 to capture references published
since the completion of the HESDs' literature searches and to account for the recreational exposure
scenario. The specific questions investigated include:
1. What levels of anatoxin-a, cylindrospermopsin, or microcystins are humans—of all ages,
including children—exposed to through recreational use (activities) in freshwaters or marine
waters from incidental ingestion, inhalation, and dermal exposure routes?
2 What health effects information for humans or animals exposed to cylindrospermopsin or
microcystins (through ingestion, inhalation, and dermal exposure routes) has been published
since the health effects literature searches were conducted for the EPA's 2015 HESDs for
cylindrospermopsin and microcystins?
3.	What recreational water use safety levels or criteria have been set for microcystins or
cylindrospermopsin by states or international governments, and how did they derive them?
4.	What new information, if any, is available regarding how aquatic recreational exposure
ingestion rates in children differ among age groups between zero and 18 years?
5.	What incidents of companion animal (e.g., dogs, horses) or livestock poisonings, including
mortality or adverse health effects, due to exposure to cyanotoxins in freshwaters, marine
waters, or beaches have occurred in the past 15 years? Specifically, when and where did
these incidents occur, to which cyanotoxin were the animals exposed, how were they
exposed, and what were the weights and breeds of the affected animal(s)?
The EPA implemented a unique literature search strategy to address each research question. Trial
searches were conducted, and results were evaluated to refine the search strategies (e.g., to reduce
retrieval of citations unrelated to the research questions). The search strings were refined to improve the
relevancy of the results. The literature search strategies implemented for each research question are
subsequently detailed.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-l

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Research Question 1: What levels of anatoxin-a, cylindrospermopsin, or microcystins are humans—of
all ages, including children—exposed to through recreational use (activities) in freshwaters or marine
waters, from incidental ingestion, inhalation, and dermal exposure routes?
The EPA searched the bibliographic databases, PubMed and Web of Science (WoS), to identify
candidate journal article literature relevant to human exposure to anatoxin-a, cylindrospermopsin, or
microcystins through recreational activities. PubMed and WoS contain peer-reviewed journal abstracts
and articles on various biological, medical, public health, and chemical topics. The WoS search string
differs slightly from the PubMed search string due to how the search engines treat search terms with
more than one word. Both search strings are presented below.
Results
The searches returned 321 journal articles after removing duplicates between PubMed and WoS results.
Based on a screening review of each article's title and abstract, the EPA retrieved nine articles that
appeared to be studies that measured, reviewed, or estimated human recreational exposure to
cyanotoxins.
PubMed Search:
("A lemmermannii Raphidiopsis mediterranean OR Anabaenaflos-aquae ORflos-aquae OR anatoxin-a
OR Aphanizomenon OR cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR
Cylindrospermopsis OR Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum
OR "M aeruginosa" OK Microcystis OR microcystin OR microcystins OR Oscillatoria OR
Planktothrix OR Phormidium OR Tychonema OR Woronichinia)
AND
("boogie board" OR "boogie boarding" OR "jet ski" OR "jet skier" OR "jet skiers" OR "jet skiing" OR
"water ski" OR "water skier" OR "water skiers" OR "water skiing" OR aerosol OR boat OR boating OR
boats OR bodyboard OR bodyboarding OR canoe OR canoeing OR canoes OR capsize OR capsized OR
dermal OR inhalation OR inhale OR kayak OR kayaker OR kayaking OR kayaks OR kneeboard OR
kneeboarding OR paddle OR paddling OR raft OR rafting OR rafts OR recreation OR recreational OR
rowing OR skin OR surf OR surfer OR surfing OR swim OR swimmer OR swimmers OR swimming
OR tubing OR wading OR wakeboarding OR wakeboard)
AND
("marine water" OR "surface water" OR beach OR beaches OR estuaries OR estuarine OR estuary OR
"fresh water" OR freshwater OR lake OR lakes OR ocean OR oceans OR pond OR ponds OR reservoir
OR reservoirs OR river OR rivers OR sea OR stream OR streams OR water)
Filters: English
Date search was conducted: 10/9/2015
Publication dates searched: 1/1/1995 - 10/9/2015
Web of Science Search:
^lemmermannii Raphidiopsis mediterranean OK Anabaena flos-aquae OR flos-aquae OR anatoxin OR
Aphanizomenon OR cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis
OR Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa"
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-2

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OK Microcystis OR microcystin OR microcystins OR Oscillatoria OK Planktothrix OR Phormidium
OR Tychonema OR Woronichinia)
AND
("boogie board" OR "boogie boarding" OR "jet ski" OR "jet skier" OR "jet skiers" OR "jet skiing" OR
"water ski" OR "water skier" OR "water skiers" OR "water skiing" OR aerosol OR boat OR boating OR
boats OR bodyboard OR bodyboarding OR canoe OR canoeing OR canoes OR capsize OR capsized OR
dermal OR inhalation OR inhale OR kayak OR kayaker OR kayaking OR kayaks OR kneeboard OR
kneeboarding OR paddle OR paddling OR raft OR rafting OR rafts OR recreation OR recreational OR
rowing OR skin OR surf OR surfer OR surfing OR swim OR swimmer OR swimmers OR swimming
OR tubing OR wading OR wakeboarding OR wakeboard)
AND
("marine water" OR "surface water" OR beach OR Beaches OR estuaries OR estuarine OR estuary OR
"fresh water" OR freshwater OR lake OR lakes OR ocean OR oceans OR pond OR ponds OR reservoir
OR reservoirs OR river OR rivers OR sea OR stream OR streams OR water)
Filters: English
Date search was conducted: 10/9/2015
Publication dates searched: 1/1/1995-10/9/2015
C.l Research Question 2: What health effects information for humans or animals exposed to
microcystins, cylindrospermopsin, or anatoxin-a (through ingestion, inhalation, and dermal
exposure routes) has been published since the health effects literature searches were conducted for
the EPA's 2015 HESDs for Cylindrospermopsin and Microcystins?
The EPA searched PubMed and WoS to identify candidate journal article literature relevant to health
effects associated with exposure to anatoxin-a, cylindrospermopsin, or microcystins. The WoS search
string differs slightly from the PubMed search string due to how the search engines treat search terms
with more than one word. Both search strings are presented below.
Results
The searches returned 1,000 journal articles after removing duplicates between PubMed and WoS
results. Based on a screening review of each article's title and abstract, the EPA retrieved 40 articles that
appeared to be prospective human epidemiological studies (n = 1), ecological human epidemiologic
studies (n = 2), reviews of human health effects (n = 4), in vivo animal studies (n = 30), or reviews of in
vivo animal studies (n = 3).
PubMed Search:
("A lemmermannii Raphidiopsis mediterranean OR Anabaenaflos-aquae ORflos-aquae OR anatoxin-a
OR Aphanizomenon OR cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR
Cylindrospermopsis OR Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum
OR "M aeruginosa" OK Microcystis OR microcystin OR microcystins OR Oscillatoria OR
Planktothrix OR Phormidium OR Tychonema OR Woronichinia)
AND
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-3

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("non cancer" OR "blurred vision" OR "cell damage" OR "cellular damage" OR "health effect" OR
"health endpoint" OR "health outcome" OR "health risk" OR "loss of protein" OR "loss of water" OR
"micronucleated binucleate cell" OR abdominal pain OR ache OR acute OR alanine aminotransferase
OR allergic OR allergies OR allergy OR aspartate aminotransferase OR blister OR blistered OR blisters
OR cancer OR carcinogen OR carcinogenic OR carcinogens OR chronic OR clinical OR cough OR
dermal OR detoxification OR detoxify OR develop OR development OR developmental OR dialysis OR
diarrhea OR disease OR DNA OR dyspnea OR electrolyte OR emergency room OR enzyme OR
enzymes OR epidemiologic OR epidemiological OR epidemiology OR epilepsy OR epileptic OR
epithelium OR eye OR failure OR fever OR gastrointestinal OR genetox OR genotoxic OR
glutamyltransferase OR head OR hematologic OR hematological OR hepatic OR histopathologic OR
histopathological OR histpathology OR hospital OR hospitalizations OR hospitals OR hospitalization
OR ill OR illness OR illnesses OR intoxicate OR intoxicated OR irritate OR irritated OR kidney OR
larynx OR lesion OR lesions OR liver OR lung OR lymph OR lymph nodes OR lymphatic OR
metabolic OR metabolism OR mucosa OR mutate OR mutated OR mutation OR mutations OR nausea
OR necrosis OR neonatal OR neonate OR neonates OR neoplasm OR neurologic OR neurological OR
noncancer OR oral OR organ OR pain OR placenta OR pneumonia OR polymorphism OR
polymorphisms OR prenatal OR red blood cell OR renal OR reproduction OR respiratory OR seizure
OR sick OR sickness OR skin OR stomach OR subacute OR subchronic OR symptom OR symptoms
OR teratogen OR teratogenic OR teratogens OR throat OR toxic OR toxicity OR trachea OR tumor OR
tumors OR urinary OR urine OR vomit OR vomiting OR conjugate OR conjugated OR diagnose OR
diagnosis OR diagnosed OR diagnoses)
Filters: English
Date search was conducted: 10/9/2015
Publication dates searched: 1/1/2014-10/9/2015
Web of Science Search:
^lemmermannii Raphidiopsis mediterranean OR Anabaena flos-aquae OR flos-aquae OR anatoxin OR
Aphanizomenon OR cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis
OR Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa"
OK Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium
OR Tychonema OR Woronichinia)
AND
("non cancer" OR "blurred vision" OR "cell damage" OR "cellular damage" OR "health effect" OR
"health endpoint" OR "health outcome" OR "health risk" OR "micronucleated binucleate cell" OR
abdominal pain OR ache OR acute OR alanine aminotransferase OR allergic OR allergies OR allergy
OR aspartate aminotransferase OR blister OR blistered OR blisters OR cancer OR carcinogen OR
carcinogenic OR carcinogens OR chronic OR clinical OR cough OR dermal OR detoxification OR
detoxify OR develop OR development OR developmental OR dialysis OR diarrhea OR disease OR
DNA OR dyspnea OR electrolyte OR emergency room OR enzyme OR enzymes OR epidemiologic OR
epidemiological OR epidemiology OR epilepsy OR epileptic OR epithelium OR eye OR failure OR
fever OR gastrointestinal OR genetox OR genotoxic OR glutamyltransferase OR head OR hematologic
OR hematological OR hepatic OR histopathologic OR histopathological OR histpathology OR hospital
OR hospitalizations OR hospitals OR hospitalization OR ill OR illness OR illnesses OR intoxicate OR
intoxicated OR irritate OR irritated OR kidney OR larynx OR lesion OR lesions OR liver OR lung OR
lymph OR lymph nodes OR lymphatic OR metabolic OR metabolism OR mucosa OR mutate OR
mutated OR mutation OR mutations OR nausea OR necrosis OR neonatal OR neonate OR neonates OR
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-4

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neoplasm OR neurologic OR neurological OR noncancer OR oral OR organ OR pain OR placenta OR
pneumonia OR polymorphism OR polymorphisms OR prenatal OR red blood cell OR renal OR
reproduction OR respiratory OR seizure OR sick OR sickness OR skin OR stomach OR subacute OR
subchronic OR symptom OR symptoms OR teratogen OR teratogenic OR teratogens OR throat OR
toxic OR toxicity OR trachea OR tumor OR tumors OR urinary OR urine OR vomit OR vomiting OR
conjugate OR conjugated OR diagnose OR diagnosis OR diagnosed OR diagnoses)
Filters: English
Date search was conducted: 10/9/2015
Publication dates searched: 1/1/2014-10/9/2015
WoS research areas searched: Environmental Sciences Ecology OR Marine Freshwater Biology OR
Toxicology OR Pharmacology Pharmacy OR Public Environmental Occupational Health OR
Microbiology OR Immunology OR Biotechnology Applied Microbiology OR Biochemistry Molecular
Biology OR Research Experimental Medicine OR Water Resources OR Infectious Disease OR Science
Technology Other Topics OR Life Sciences Biomedicine Other Topics OR Gastroenterology
Hepatology OR Pediatrics.
C.2 Research Question 3: What recreational water use safety levels or criteria have been set for
microcystins or cylindrospermopsin by states or international governments and how did they
derive them?
To identify state-level recreational guidelines for cyanobacteria and cyanotoxins, the EPA searched the
websites of state-level departments of public health, environmental health, and natural resources for all
50 U.S. states. If relevant recreational guidelines were not found by searching state-level websites, the
EPA conducted Google searches of the internet using state names, key terms for cyanobacteria and
cyanotoxins (e.g., harmful algal bloom, blue-green algae, microcystin, cylindrospermopsin), and key
terms for guidelines (e.g., advisory, guidance, guideline, standard, regulation). For international
governments, the EPA used the 2012 report, Current Approaches to Cyanotoxin Risk Assessment, Risk
Management and Regulations in Different Countries, by Dr. Ingrid Chorus, Federal Environment
Agency, Germany, to identify international government recreational safety levels for cyanobacteria and
cyanotoxins. In addition, the EPA implemented the same search strategy as used for U.S. states to
identify updated international recreational guidelines or guideline levels not featured in the 2012 report
by Dr. Ingrid Chorus.
C.3 Research Question 4: What new information, if any, is available regarding how aquatic
recreational exposure ingestion rates in children differ among age groups between zero and 18
years?
Search of Bibliographic Databases
The EPA searched PubMed, WoS, and Google Scholar to identify literature that has cited, or is similar
(based on terms identified in the titles and abstracts) to, the studies that provide water ingestion data for
swimmers or during water recreational activities in the EPA's (2011) Exposure Factors Handbook
(EFH) (i.e., Dorevitch et al. 2011; Dufour et al. (2006); Schets et al. 2011). The PubMed and WoS
searches were conducted on 10/9/2015, the publication dates searched were 1/1/2011 to 10/9/2015, and
an English filter was applied. The Google Scholar search was conducted on 10/9/2015 and could not be
limited by year or language.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-5

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Results
Together all three searches returned 341 journal articles. Duplicates were removed between PubMed and
WoS, but this total might include duplicates between Google Scholar results and WoS/PubMed results.
Based on a screening review of each article's title and abstract, the EPA retrieved five articles, four of
which were published between 2013 and 2015 and appeared to measure or estimate incidental water
ingestion. The EPA also retrieved one 2012 study that assessed duration of non-swimming recreational
water exposure by using novel time lapse photography technology.
Google Search of Internet
In addition, the EPA conducted a Google search of the internet focused on specified URL domains
(listed in Table C-l) to identify candidate gray literature (e.g., state, federal, or international government
reports or guidance). The Google search string is presented below. The Google search of the internet
could not be limited by year or language.
Table C-l. Internet URL Domains Searched for Research Question 4
Organization
URL Domain
U.S. Government
•gov
.us
All U.S. States
Google Custom Search Engine
Centers for Disease Control and Prevention, including Agency
for Toxic Substances and Disease Registry
cdc.gov
Australia, including Australian Department of Health
gov.au
Canada, including Health Canada
gc.ca
European Union, including
•	European Chemicals Agency
•	European Commissions on Environment, Public Health,
Food, and Health and Consumers
europa.eu
Public Health England
hpa.org.uk
United Kingdom
gov.uk
Germany
.de
Education websites
.edu
HERA (Human and Environmental Risk Assessment) Project
heraproject.com
World Health Organization
who.int
Results
The Google search returned 390 results after removing duplicates. Based on a preliminary screen of each
result, the EPA retrieved two documents which appeared to either derive or cited an incidental ingestion
rate while recreating which had not previously been identified during the literature search process.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-6

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Google Search of Internet (conducted separately for each URL domain listed in Table C-l)
(pool OR swim OR swimmer OR swimmers OR swimming OR recreation OR recreational)
AND
(adolescents OR boys OR child OR children OR girls OR kids OR teenagers)
AND
("activity-related ingestion" OR "incidental ingestion" OR "activity-related ingestion" OR "ingestion of
water" OR "water ingestion")
AND
rate
AND
inurl:.
Filters: None
Date search was conducted: 10/9/2015
Dates searched: Not specified
Web browser: Internet Explorer
C.4 Research Question 5: What incidents of companion animal (e.g., dogs, horses) or livestock
poisonings, including mortality or adverse health effects, due to exposure to cyanotoxins in
freshwaters, marine water, or beaches have occurred in the past 15 years? Specifically, when and
where did these incidents occur, to which cyanotoxin were the animals exposed, how were they
exposed, and what were the weights and breeds of the affected animal(s)?
The EPA searched PubMed, WoS, and Agricola to identify candidate journal article literature relevant to
companion animal or livestock poisoning due to exposures to cyanobacterial cells, anatoxin-a,
cylindrospermopsin, or microcystins. The EPA first searched PubMed and WoS with a focus on dogs.
The EPA conducted two additional searches in PubMed, WoS, and Agricola focused on livestock, and
on cats and birds. The search strings for each search iteration are presented below.
Results
The number of journal articles returned by the three searches is provided in Table C-2. Based on a
screening review of the article's title and abstract, the EPA retrieved five of the 35 journal articles
retrieved during the search focused on dogs. These five articles appeared to provide information about
an incident of cyanotoxin exposure to an animal where the authors confirm that the animal was exposed
to a cyanotoxin by either measuring the concentration of cyanotoxin found in the animal or by sampling
the body of water to which the animal had contact.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-l

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Table C-2. Number of Journal Articles Returned by Three Search Strategies for Research
Question 5
Search Strategy Focus
Number of Results Returned from PubMed, WoS, and
Agricola Searches
Dogs
35a
Livestock
100
Cats and birds
169b
a Search conducted in PubMed and WoS only.
b Duplicates between PubMed/WoS results and Agricola results were not removed. Therefore, the cats and birds search
might include duplicates between Agricola results and PubMed/WoS results.
C.4.1 Search Strategy Focused on Dogs
PubMed Search
("A lemmermcmnii Raphidiopsis mediterranean ORflos-aquae OR anatoxin-a OR Aphanizomenon OR
cylindrospermopsin OR "C. raciborskiF OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskiF OR Dolichospermum OR "M aeruginosa" OR
Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(canine OR canines OR dog OR dogs OR "Canis lupus familiaris" OR "Canis familiaris")
Filters: English
Date search was conducted: 10/5/2015
Publication dates searched: 1/1/2012-10/5/2015
Web of Science Search
(" lemmermannii Raphidiopsis mediterranean OR flos-aquae OR anatoxin OK Aphanizomenon OR
cylindrospermopsin OR "C. raciborskiF OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskiF OR Dolichospermum OR "M aeruginosa" OR
Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-8

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("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(canine OR canines OR dog OR dogs OR "Canis lupus familiaris" OR "Canis familiaris")
Filters: English
Date search was conducted: 10/5/2015
Publication dates searched: 1/1/2012-10/5/2015
C.4.2 Search Strategy Focused on Livestock
PubMed and Agricola Searches
("A lemmermannii Raphidiopsis mediterranean OR flos-aquae OR anatoxin-a OR Aphanizomenon OR
cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa" OR
Microcystis OR microcystin OR microcystins OR Oscillator^ OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(alpaca OR alpacas OR bronco OR broncos OR buffalo OR bull OR bulls OR cattle OR colt OR colts
OR cow OR cows OR bovine OR bison OR oxen OR donkey OR donkeys OR duck OR ducks OR
equine OR ewe OR ewes OR fillies OR filly OR foal OR foals OR gelding OR geldings OR heifer OR
heifers OR horse OR horses OR lamb OR lambs OR livestock OR llama OR llamas OR mare OR mares
OR mule OR mules OR mustang OR mustangs OR ponies OR pony OR ram OR rams OR sheep OR
stallion OR stallions OR steer OR pig OR pigs OR piglet OR piglets)
Filters: English
Date search was conducted: 11/25/2015
Publication dates searched: 1/1/2012-11/25/2015
Web of Science Search:
^lemmermannii Raphidiopsis mediterranean OR flos-aquae OR anatoxin OK Aphanizomenon OR
cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa" OR
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-9

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Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(alpaca OR alpacas OR bronco OR broncos OR buffalo OR bull OR bulls OR cattle OR colt OR colts
OR cow OR cows OR bovine OR bison OR oxen OR donkey OR donkeys OR duck OR ducks OR
equine OR ewe OR ewes OR fillies OR filly OR foal OR foals OR gelding OR geldings OR heifer OR
heifers OR horse OR horses OR lamb OR lambs OR livestock OR llama OR llamas OR mare OR mares
OR mule OR mules OR mustang OR mustangs OR ponies OR pony OR ram OR rams OR sheep OR
stallion OR stallions OR steer OR pig OR pigs OR piglet OR piglets)
Filters: English
Date search was conducted: 11/25/2015
Publication dates searched: 1/1/2012-11/25/2015
C.4.3 Search Strategy Focused on Cats and Birds
PubMed and Agricola Searches
("A lemmermannii Raphidiopsis mediterranean OR flos-aquae OR anatoxin-a OR Aphanizomenon OR
cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa" OR
Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(feline OR felines OR cat OR cats OR kitten OR kittens OR Catus" OR "Felis Catus" OR bird OR
birds OR avian OR waterfowl)
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-10

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Filters: English
Date search was conducted: 2/1/2016
Publication dates searched: 1/1/2012-2/1/2016
Web of Science Search
^lemmermannii Raphidiopsis mediterranean OR flos-aquae OR anatoxin OR Aphanizomenon OR
cylindrospermopsin OR "C. raciborskii" OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermum OR "Cylindrospermopsis raciborskii" OR Dolichospermum OR "M aeruginosa" OR
Microcystis OR microcystin OR microcystins OR Oscillatoria OR Planktothrix OR Phormidium OR
Tychonema OR Woronichinia OR Cyanobacteria OR cyanotoxin OR Cyanotoxins OR "harmful algae"
OR "harmful algal bloom" OR blue green algae)
AND
("health effect" OR "health endpoint" OR "health outcome" OR dead OR death OR deaths OR died OR
disease OR diseased OR diseases OR exposed OR exposure OR ill OR illness OR illnesses OR infect
OR infected OR infection OR infections OR morbidity OR mortality OR poison OR poisoned OR
poisoning OR poisonings OR sick OR sickness OR toxic OR toxicity OR diagnose OR diagnosis OR
diagnosed OR diagnoses)
AND
(feline OR felines OR cat OR cats OR kitten OR kittens OR Catus" OR "Felis Catus" OR bird OR
birds OR avian OR waterfowl)
Filters: English
Date search was conducted: 2/1/2016
Publication dates searched: 1/1/2012-2/1/2016
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
C-ll

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Recommended Human Health Recreational Ambient Water Quality Criteria or	C-12
Swimming Advisories for Microcystins and Cylindrospermopsin

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APPENDIX D. REVIEW OF THE STATE OF THE SCIENCE ON CYANOBACTERIAL
CELLS HEALTH EFFECTS
D.l Introduction
This appendix provides information gathered and reviewed to determine the state of the science on
health effects from cyanobacterial cells. The EPA conducted literature searches to identify studies
relevant to the health effects from cyanobacterial cells. Detailed information on the design and
implementation of these searchers is provided in Appendix C. Results from these literature searches
were reviewed for relevance to cyanobacterial cell exposures and health effects.
D. 1.1 Animal Studies
Cyanobacterial cells cause allergenicity and irritation in animals, independent of whether the
cyanobacterial cells produce toxin. Three animal studies (Shirai et al. 1986; Stewart et al. 2006c;
Torokne et al. 2001) demonstrated hypersensitivity reactions and dermal and eye irritation in several
species that did not correlate with microcystin content. Although the number of studies is limited and
different species were evaluated in each study, these studies provide evidence to support hypersensitivity
reactions in animals from exposure to cyanobacteria when cyanotoxins are not present (Shirai et al.
1986; Torokne et al. 2001) and when they are (Stewart et al. 2006c).
Cyanobacteria bloom samples collected from five different lakes or ponds were tested for allergenic and
irritative effects in guinea pigs and rabbits, respectively (Torokne et al. 2001). The microcystin content
(presumed to be total LR, RR, and YR) ranged from not detected to 2.21 mg/g. To determine
sensitization, guinea pigs were initiated with an intradermal injection of freeze-dried cyanobacteria
followed seven days later by topical application at the injection site. Sensitization was moderate to
strong in 30-67 percent of guinea pigs and did not correlate with microcystin content. The
Aphanizomenon ovalisporum sample (a non-toxin-producing strain) sensitized 91 percent of the animals
and was the strongest allergen. Skin irritation tests in albino rabbits showed slight or negligible
irritation, except for Aphanizomenon ovalisporum, which showed moderate irritation. The eye irritation
evaluation in rabbits was positive for four of the five samples containing Microcystis.
Shirai et al. (1986) reported that C3H/HeJ mice, immunized intraperitoneal with either sonicated or live
cells from a Microcystis water bloom, developed delayed-type hypersensitivity when challenged two
weeks later with a subcutaneous injection sonicated Microcystis cells. A positive reaction, as assessed by
footpad swelling, was seen in mice immunized with either live cells or sonicated cells. Both toxic and
nontoxic Microcystis cells induced delayed-type hypersensitivity in this mouse study. Because this strain
of mouse is unresponsive to lipopolysaccharide (LPS), the footpad delayed-type hypersensitivity was
not related to LPS, thus, the antigenic component of the sonicated cyanobacterial cells is not known.
Stewart et al. (2006c) conducted a mouse ear swelling test in which cylindrospermopsin and
Cylindrospermopsis raciborskii solutions generated irritation of the abdominal skin exposed during
induction (two percent w/v lysed cell solution containing 73 [j,g/mL cylindrospermopsin). Subsequent
dermal exposures to the Cylindrospermopsis raciborskii solution produced hypersensitivity reactions
(p = 0.001). The cyanobacteria Microcystis aeruginosa and Anabaena circinalis elicited no responses in
this test.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
D-l

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Two of the cyanobacterial cell studies in animals found that rodents became sensitized after exposure
and subsequent challenge to non-toxin strains (Shirai et al. 1986; Torokne et al. 2001). Torokne et al.
(2001) found that a nontoxic strain was more sensitizing and irritating than the toxic strains evaluated.
These experiments support the conclusion that there is no relationship between the cyanotoxin content
and the allergenic effect of cyanobacteria.
D. 1.2 Clinical and Laboratory Human Studies
Several types of studies and reports provide information on associations between cyanobacteria
exposure and health effects. Clinical and in vitro studies (Bernstein et al. 2011; Geh et al. 2015; Pilotto
et al. 2004; Stewart et al. 2006a) have been able to assess associations between cyanobacteria exposure
and human health effects including dermal and allergenic reactions. Three clinical studies assessed
dermal exposure to cyanobacterial cells using skin-patch or skin-prick testing in humans (Bernstein et al.
2011; Pilotto et al. 2004; Stewart et al. 2006a). Some of the exposed individuals showed mild irritation
or allergenicity. No statistically significant dose-response relationships were found between skin
irritation and increasing cyanobacterial cell concentrations. The allergenicity study suggests that
cyanobacteria are allergenic, particularly among people with chronic rhinitis (Bernstein et al. 2011).
Skin-patch testing in humans was performed by Pilotto et al. (2004) with laboratory-grown
cylindrospermopsin-producing Cylindrospermopsis raciborskii cells, both whole and lysed, which were
applied using adhesive patches at concentrations ranging from < 5,000 to 200,000 cells/mL to the skin of
50 adult volunteers. After 24 hours, patches were removed and evaluation of the erythematous reactions
were graded. Analysis of participants' reactions to patches treated with whole cells showed an odds ratio
(OR) of 2.13 and a 95 percent confidence interval (CI) of 1.79-4.21 (p < 0.001). Lysed cells patch
analysis showed an OR of 3.41 and a 95 percent CI of 2.00-5.84 (p < 0.001). No statistically significant
increase or dose-response between skin reactions and increasing cell concentrations for either patches
(whole or lysed) was observed. Subjects had skin reactions to the cylindrospermopsin, and positive
control patches more frequently than to the negative control patches. The mean percentage of subjects
with a reaction was 20 percent (95 percent CI: 15-31 percent). When subjects reacting to negative
controls (39) were excluded, the mean percentage was 11 percent (95 percent CI: 6-18 percent).
Evaluation of erythematous reactions showed that mild irritations (grade 2) were resolved in all cases
within 24 to 72 hours.
Stewart et al. (2006a) conducted a skin-patch test with 39 volunteers (20 dermatology outpatients; 19
controls) who were exposed to six cyanobacterial suspensions, including toxigenic species, nontoxigenic
species, mixed suspensions, and two cyanobacterial LPS extracts. All cyanobacterial suspensions of
lyophilized cells were tested at three concentrations, 0.25 percent w/v, 0.05 percent w/v, 0.005 percent
w/v, and the estimated doses of cyanotoxins were 2.4 ng/kg cylindrospermopsin and 2.6 ng/kg
microcystins. Only one subject showed significant responses to cyanobacterial suspensions, specifically
to two suspensions of cyanobacterial cells: Cylindrospermopsis raciborskii and mixed Microcystis
aeruginosa and Cylindrospermopsis raciborskii, both of which contained one or more cyanotoxins. This
subject showed no evidence of any dose-response effect in the dermal reactions. None of the participants
reacted to the cyanobacterial LPS extracts, which ranged from 260 ppb to 31 ppm. This small clinical
study demonstrated that dermal hypersensitivity reactions to cyanobacteria exposure occur infrequently,
and further research into risk factors for predisposition to this type reaction could be beneficial.
Bernstein et al. (2011) studied skin sensitization to nontoxic extracts of Microcystis aeruginosa in 259
patients with chronic rhinitis over two years. Patients were evaluated with aeroallergen skin testing and
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
D-2

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skin-prick testing. The authors found that 86 percent of the subjects had positive skin-prick tests to
Microcystis aeruginosa, and that patients with existing allergic rhinitis were more likely to have
reactions and sensitization to cyanobacteria than the controls (non-atopic health subjects). This study
indicated that cyanobacterial allergenicity is associated with the nontoxic portion of the cyanobacteria.
Geh et al. (2015) studied the immunogenicity of extracts of toxic and nontoxic strains of Microcystis
aeruginosa in patient sera (18 patients with chronic rhinitis and three non-atopic healthy subjects as
documented in Bernstein et al. 2011). Enzyme Linked Immunosorbent Assay (ELISA) test was used to
test IgE-specific reactivity, and gel electrophoresis, followed by immunoblot and mass spectrometry,
was done to identify the relevant sensitizing peptides. The authors found an increase in specific IgE in
those patients tested with the nontoxic Microcystis aeruginosa extract than the extract from the toxic
strain. After pre-incubation of the nontoxic extract with various concentrations of microcystin, the
authors found that phycocyanin and the core-membrane linker peptide were responsible for the release
of P-hexosaminidase in rat basophil leukemia cells. The authors concluded that non-toxin-producing
strains of cyanobacteria are more allergenic than toxin-producing strains in allergic patients, and that the
toxin may have an inhibitory effect on the allergenicity of the cyanobacterial cells.
Facciponte et al. (2018) used polymerase chain reaction (PCR) to detect aerosolized cyanobacteria
inhaled into the human respiratory tract. They found cyanobacteria at high frequencies in the upper
respiratory tract (92.2 percent) and central airway (79.3 percent) of the study subjects (n = 77). The
findings suggests that humans inhale aerosolized cyanobacteria, which can remain in the nostrils and the
lungs.
D.1.3 Epidemiological Studies, Case Reports, and Outbreaks
Among the epidemiological studies discussed here, some identified significant associations between
cyanobacteria exposure and a range of health outcomes including dermal, eye/ear, gastrointestinal (GI),
and respiratory effects. Several of these studies also measured one or more cyanotoxins and found no
association between cyanotoxin occurrence or exposure and health effects. Additional evidence from
outbreak and case reports provides support for health effects associated with cyanobacteria exposure.
The studies vary in study design, methods used, size of study population, cyanobacterial species
evaluated, health effects identified, and cyanobacterial cell densities associated with human health
effects. Therefore, substantial uncertainty remains regarding the associations between cyanobacterial
cell exposure and human health effects. Overall, these studies provide evidence of statistically
significant associations between cyanobacterial cell exposure and human health effects even in the
absence of cyanotoxins. However, the reported associations between cyanobacterial cell densities and
health outcomes are not consistent.
Eight epidemiological studies evaluated short-term health effects associated with recreational exposure
to cyanobacterial blooms (El Saadi et al. 1995; Levesque et al. 2014; Lin et al. 2015; Philipp 1992;
Philipp and Bates 1992; Philipp et al. 1992; Pilotto et al. 1997; Stewart et al. 2006d). See Table D-l for
a summary list of these studies. The health outcomes evaluated included dermal, GI, respiratory, and
other acute effects, such as eye or ear symptoms. Seven studies evaluated recreational exposure to
freshwater cyanobacteria, and one evaluated exposure to marine water cyanobacteria (Lin et al. 2015).
Two studies included field sites in the continental United States or Canada (Levesque et al. 2014;
Stewart et al. 2006d), three occurred in the United Kingdom (Philipp 1992; Philipp and Bates 1992;
Philipp et al. 1992), and three were conducted in subtropical and tropical regions in Australia (El Saadi
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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et al. 1995; Pilotto et al. 1997) and Puerto Rico (Lin et al. 2015). These epidemiological studies are
discussed below in chronological order.
Table D-l. Cyanobacteria Epidemiological Studies Summary
Reference
Study Design, n,
and
Location
Cyanobacteria
Identified
Cyanotoxins
Measured
Health
Association3
Lowest Significant
Cyanobacterial Cell
Density (cells/mL)
Philipp (1992)
Cross-sectional
n = 246
United Kingdom
(Hampshire)
Microcystis sp.,
Gleotrichia sp.

No statistically
significant health
associations
No quantitative
cyanobacterial cell
densities provided
Philipp and Bates
(1992)
Cross-sectional
n = 382
United Kingdom
(Somerset)
Microcystis sp.,
Gleotrichia sp.

No statistically
significant health
associations
No quantitative
cyanobacterial cell
densities provided
Philipp et al. (1992)
Cross-sectional
n = 246
United Kingdom
(Lincolnshire, South
Yorkshire)
Oscillatoria sp.,
Aphanizomenon sp.,
Aphanothece sp.,
Merismopedia sp.

No statistically
significant health
associations
No quantitative
cyanobacterial cell
densities provided
El Saadi et al.
(1995)
Case-control
n cases =102 GI, 86
dermatological
n controls = 132
Australia (South
Australia)
Anabaena sp.,
Aphanizomenon sp.,
Planktothrix sp.,
Anabaena circinalis,
Microcystis aeruginosa

No statistically
significant health
associations
No quantitative
cyanobacterial cell
densities provided
Pilotto et al. (1997)
Cross-sectional
n = 295 exposed
n = 43 unexposed
Australia (South
Australia, New South
Wales, Victoria)
Microcystis aeruginosa,
Microcystis sp.,
Anabaena sp.,
Aphanizomenon sp.,
Nodularia spumigena
Hepatotoxins
detected by
mouse
bioassay
Significant positive
association between
combined symptoms
(GI, dermal,
respiratory, fever,
eye or ear irritation)
and cyanobacteria
> 5,000
Stewart et al.
(2006d)
Cohort (prospective)
n= 1,331
Australia
(Queensland, New
South Wales) and
Florida
Cyanobacteria
identified, species not
specified
Microcystins
detected by
HPLC with
photodiode
array detection
or ELISA;
cylindro-
spermopsin
and anatoxin-a
detected by
HPLC-
MS/MS;
saxitoxins not
detected by
HPLC with
fluorescence
detection
Significant positive
association between
respiratory
symptoms and
cyanobacteria
Significant positive
association between
combined symptoms
(GI, dermal,
respiratory, fever,
eye or ear irritation)
and cyanobacteria
> 100,000b
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Reference
Study Design, n,
and
Location
Cyanobacteria
Identified
Cyanotoxins
Measured
Health
Association3
Lowest Significant
Cyanobacterial Cell
Density (cells/mL)
Levesque et al.
(2014)
Cohort (prospective)
n = 466
Canada (Quebec)
Cyanobacteria
identified, species not
specified
Microcystins
detected by
ELISA
Significant positive
association between
GI symptoms with
fever and
cyanobacteria
20,000-100,000
Liiiet al. (2015)c
Cohort (prospective)
n= 15,726
Puerto Rico
(Boqueron)
Cyanophyte filament,
Pseitdanabaena sp.,
Picocyanophyte,
Synechococcits sp.,
Synechocystis sp.,
Cyanophyte cell pair,
Phormidium sp.,
Lyngbya sp.,
Trichodesmium sp.,
Aphanothece sp.,
Johannesbaptistia sp.,
Komvophoron sp.,
Cyanophyte colony,
Cyanophyte unicell
sphere
Lyngbyatoxin-
a and debromo-
aplysiatoxin
measured but
not detected by
HPLC-MS
Significant positive
association between
respiratory illness
and cyanobacteria
other than
picocyanobacteria
36.7-237.4



significant positive
association between
rash and
cyanobacteria other
than
picocyanobacteria
>237.4
sp. = unspecified species of the genus; HPLC = high performance liquid chromatography; MS = mass spectrometry;
MS/MS = tandem mass spectroscopy
a Includes only significant associations between recreational cyanobacteria exposure and health effects.
b Values were converted from cyanobacterial cell surface area (> 12.0 mm2/mL) to cyanobacterial cell density (> 100,000
cells/mL) using conversions in NHMRC (2008). Relationship between biomass and cyanobacterial cell density can vary by
species and cell size (Lawton et al. 1999; Stewart et al. 2006d).
°Linet al. (2015) evaluated picocyanobacteria and cyanobacteria other than picocyanobacteria separately.
Three cross-sectional studies were conducted by Philipp et al. (Philipp 1992; Philipp and Bates 1992;
Philipp et al. 1992) to evaluate health effects related to exposure to cyanobacteria from recreational
activities including sailing, windsurfing, and fishing in water bodies in the United Kingdom.
Questionnaires were administered to participants who visited one of six inland lakes to evaluate
exposure and morbidity (including dermal, eye/ear, GI, and respiratory symptoms). Several species of
cyanobacteria were identified and, in some cases, cyanobacterial levels exceeded the National Rivers
Authority threshold for "potential to cause harm." Only minor morbidity was identified among
recreators, and no statistically significant associations between cyanobacteria exposure and morbidity
were identified.
El Saadi et al. (1995) conducted a case-control study in Australia to evaluate exposure to river water
with detectable levels of cyanobacteria and GI and dermatological symptoms evaluated by a medical
practitioner. This river was used as a source for drinking water, domestic water, and recreational water.
The authors found no significant association between recreational exposure to river water with
cyanobacteria and GI or dermatological symptoms. Cyanotoxins were not measured, but species of
cyanobacteria were present that were capable of producing cyanotoxins.
These four studies (El Saadi et al. 1995; Philipp 1992; Philipp and Bates 1992; Philipp et al. 1992)
provided no quantitative data on cyanobacterial cell densities. Therefore, they could not help inform
determination of a quantitative level associated (or not associated) with health effects.
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Four more recent epidemiological studies assessed the association between exposure to recreational
waters containing cyanobacteria and human health and provide quantitative density data for
cyanobacterial cells (Levesque et al. 2014; Lin et al. 2015; Pilotto et al. 1997; Stewart et al. 2006d).
These studies reported at least one statistically significant association between exposure to cyanobacteria
and human health outcomes, including GI illness (Levesque et al. 2014), respiratory symptoms (Lin et
al. 2015; Stewart et al. 2006d), dermal symptoms (Lin et al. 2015), or combined symptomology (GI,
dermal, respiratory, and other symptoms) (Pilotto et al. 1997; Stewart et al. 2006d). These associations
were linked to a range of densities of cyanobacterial cells from as low as > 5,000 cells/mL (Pilotto et al.
1997) to as high as 100,000 cells/mL (analogous to > 12 mm2/mL (NHMRC 2008; Stewart et al. 2006d).
In contrast to the studies that examined all cyanobacteria, Lin et al. (2015) evaluated picocyanobacteria,
larger cyanobacterial cells, and total phytoplankton, and reported health effects associated with 37-
1,461 cells/mL for cyanobacteria other than picocyanobacteria.
Pilotto et al. (1997) investigated the health effects from recreational exposures (including jet-skiing,
water skiing, swimming, and windsurfing) to cyanobacteria in Australia. The study included 852
participants, 777 who had water contact and were considered exposed, and 75 not exposed. There were
338 recreators (295 exposed, 43 not exposed) after exclusion of those who experienced symptoms or had
recreational exposure in the five days prior to the initial interview at the water recreation site (the after
exclusion study group). Health outcomes evaluated included diarrhea, vomiting, flu-like symptoms (e.g.,
cough), skin rashes, mouth ulcers, fevers, or eye or ear infections. Water samples were collected for
evaluation of cyanobacterial cell counts, hepatotoxins, and neurotoxins.
In the after exclusion study group, when all symptoms were combined, the authors found a significant
trend of increasing symptom occurrence with duration of exposure at seven days post-exposure (p-value
for trend =0.03). Similarly, in the after exclusion study group there was a significant trend of increasing
symptom occurrence with increasing cyanobacterial cell count (p-value for trend = 0.04). To account for
the combined effect of duration of exposure and cyanobacterial cell density, unexposed participants
were compared with those exposed for up to 60 minutes and for more than 60 minutes to water with up
to 5,000 cells/mL and to water with more than 5,000 cells/mL. For the after exclusion study group, a
significant trend of increasing symptom occurrence with increasing levels of exposure was identified (p-
value for trend = 0.004). In addition, participants with recreational exposure for more than 60 minutes to
cyanobacterial densities above 5,000 cells/mL had a significantly higher symptom occurrence rate at
seven days post-exposure than unexposed participants (OR = 3.44, CI: 1.09-10.82). In this study, the
significant trends observed in the after exclusion study group were not observed when all participants
were included.
Pilotto et al. (1997) reported toxicity data collected by the Australia Water Quality Center. Presence or
absence of particulate (intracellular) hepatotoxins in concentrated surface water phytoplankton samples
was measured by mouse bioassay. The authors reported that hepatotoxins were identified at one site on
two separate interview days and at three sites for one day each. No evidence of neurotoxins was
detected. They reported that no significant association was found between the presence of hepatotoxins
and symptom occurrence at two and seven days after exposure. Data and analysis methods were not
provided. The authors point out that trends were observed at seven days and not at two days after
exposure and this might suggest a delayed rather than an immediate allergic response. The authors also
stated they could not rule out other causative factors, such as other microorganisms, that could co-occur
with cyanobacteria. The results from this study informed the recommendations made by WHO in
Guidelines for Safe Recreational Water Environments (WHO 2003).
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Stewart et al. (2006d) conducted a prospective cohort study to investigate the incidence of acute
symptoms in individuals exposed to cyanobacteria via recreational activities in lakes and rivers in
Australia and Florida. This study included 1311 recreators with any water contact-related activity (e.g.,
swimming, boat entry/egress). Cyanobacterial cell densities were characterized in terms of cell surface
area rather than cell counts (to normalize for cell size differences among different species). Authors
evaluated incidence of acute symptoms in recreators exposed to low, medium and high levels of
cyanobacteria.
Study subjects were asked to complete a self-administered questionnaire before leaving for the day after
enrollment and to submit to a telephone follow-up interview. The questionnaire and follow-up interview
forms gathered information on various acute illnesses, their onset and severity. Respiratory symptoms
among study participants in the high recreational exposure group (total cyanobacterial cell surface area >
12 mm2/mL on day of recreation) were significantly greater compared to participants in the low
recreational exposure group (< 2.4 mm2/mL) (adjusted OR = 2.1, 95 percent CI: 1.1-4.0). Respiratory
symptoms were defined as difficulty breathing, dry cough, productive cough, runny nose, unusual
sneezing, sore throat, or wheezy breathing. Reports of any symptom among study participants in the
high exposure group were significantly greater compared to reports among study participants in the low
recreational exposure group (adjusted OR = 1.7, 95 percent CI: 1.0-2.9). However, when subjects with
recent prior recreational water exposure were excluded the result remained positive but not significant
(adjusted OR = 1.6, 95 percent CI: 0.8-3.2). A dose-response relationship between increased
cyanobacterial biomass and increased symptom reporting was not identified. The authors speculated that
the pattern in their data could be due to a threshold effect. No other significant associations with health
effects were identified.
For water samples that contained potentially toxic cyanobacteria, Stewart et al. (2006d) measured
cyanotoxins including microcystins, saxitoxins, cylindrospermopsin and anatoxin-a by HPLC or HPLC-
MS/MS methods. Cyanotoxins were infrequently identified and only at low levels. Microcystins were
detected on two occasions (1 and 12 (J,g/L). Cylindrospermopsin was found on seven occasions (ranging
from 1 to 2 (J,g/L). Anatoxin-a was identified on a single recruitment day at a concentration of 1 (J,g/L. A
statistically significant increase in symptom reporting was found to be associated with anatoxin-a
exposure, but the number of exposed subjects was very low (n =18). No relationship between fecal
indicator bacteria (fecal coliforms) and symptoms was identified.
Levesque et al. (2014) conducted a prospective study of health effects including GI, respiratory, dermal,
eye/ear, and other symptoms associated with cyanobacteria and microcystin exposure at three lakes in
Canada (Quebec), one of which was a local supply of drinking water. The study evaluated acute
symptoms in humans (466 subjects included in analysis) living in proximity to lakes affected by blooms
and analyzed recreational exposure (full and limited contact) and drinking water exposure scenarios for
both cyanobacterial cells and microcystins.
More severe GI symptoms, defined as diarrhea, vomiting, nausea and fever, or abdominal cramps and
fever, were associated with recreational contact (full and limited) and cyanobacteria. For the more
severe GI symptoms, the adjusted relative risk (RR) increased with cyanobacterial cell counts providing
evidence of a dose-response relationship (p-value for trend = 0.001, < 20,000 cells/mL: RR = 1.52,
95 percent CI: 0.65-3.51; 20,000-100,000 cells/mL: RR = 2.71, 95 percent CI: 1.02-7.16;
> 100,000 cells/mL: RR = 3.28, 95 percent CI: 1.69-6.37). No evidence of a dose-response relationship
for cyanobacterial cell counts and the less severe GI symptoms was found. No relationship was observed
between duration of contact or head immersion and risk of GI symptoms. A significant increase for both
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the less and the more severe GI symptoms was found with contact in the more highly impacted lakes
(median cell densities 20,001-21,485 cells/mL), but not in the less impacted lake (median 1,032
cells/mL). No relationship was observed between microcystin concentrations and risk of GI symptoms.
No significant associations between recreational exposures to cyanobacteria and health effects other than
GI effects were identified.
To evaluate possible co-exposures, authors measured microcystin concentrations and E. coli as a fecal
indicator. Levesque et al. (2014) measured particulate (intracellular) and dissolved microcystins by
ELISA and found that microcystin concentrations varied by lake and by sample location (littoral versus
limnetic). Microcystins were detected in all three lakes. At Lake William the median values were below
the limit of detection at littoral and limnetic stations, with maximum values of 0.63 [j,g/L and 0.02 (J,g/L,
respectively. At Lake Roxton littoral stations, the median concentration was 0.23 [j,g/L (range:
0.008 [j,g/L-108.8 (J,g/L) and at limnetic stations the median was 0.12 [j,g/L (range: 0.04 (J,g/L-
1.12 (J,g/L). The Mallets Bay littoral stations had a median of 0.70 [j,g/L (range: under limit of detection -
773 (J,g/L) and the limnetic stations had a median of 0.35 [j,g/L (range: 0.001 [j.g/L-125 (J,g/L).
Levesque et al. (2014) reported that as a whole the microcystin concentrations during contact were
relatively low (first tertile: < 0.0012 (J,g/L; second tertile: 0.0012-0.2456 (J,g/L; third tertile: >
0.2456 (J,g/L). Symptoms were examined in relation to recreational and drinking water exposure to
cyanobacteria and microcystins. Only GI symptoms were associated with recreational contact. The
highest microcystin concentration at which an episode of GI symptoms was reported was 7.65 (J,g/L.
There was no significant increase in adjusted RR of GI symptoms with recreational exposure to more
than 1 [j,g/L microcystins. Adjusted RR (adjusted for gender, gastrointestinal (GI) symptoms reported in
the two weeks prior to data collection, residence's source of drinking water) for GI illness without fever
and GI illness with fever were 1.06 (95 percent CI=0.32-3.52) and 1.48 (95 percent CI = 0.41-5.23),
respectively. There were significant increases in adjusted RR of several symptoms in participants who
received their drinking water from a source contaminated by cyanobacteria (muscle pain, GI illness,
skin, and ear symptoms).
Levesque et al. (2014) found that the geometric mean of E. coli at the three lakes ranged from 0 to 145
CFU per 100 mL, and there was no association between GI illness and E. coli levels. The authors noted
that GI symptoms could have other causes, such as Aeromonas infections; however, the symptoms were
not related to fecal contamination as measured by culturable E. coli. They also noted that people avoided
full recreational contact during blooms and more people engaged in limited contact recreation at higher
cell counts. This observation explains the counterintuitive finding that participants with limited contact
exposure (fishing, watercraft without direct water contact) had higher likelihood of symptom reporting
compared to participants with full contact.
A follow-up analysis (Levesque et al. 2016) characterized the same health data as Levesque et al. (2014)
to evaluate the relationship of bacterial endotoxin (e.g., LPS) concentration to GI symptoms. Endotoxin
concentrations were slightly correlated with cyanobacterial counts (polychoric correlation
coefficient = 0.57). The highest tertile of endotoxin concentration (> 48 endotoxin units/mL) was
significantly associated with GI illness both with and without fever (GI illness without fever RR = 2.87,
CI: 1.62-5.08; GI illness with fever RR = 3.11, CI: 1.56-6.22). Adjustment to the level of cyanobacteria
did not alter the relationship between endotoxin and GI illness and authors hypothesize that other Gram
negative bacteria might play a role in the relationship between endotoxin levels and GI illness as has
been suggested in a previous study (Berg et al. 2011). Authors note that they stored filtered water
samples at -80 °C for several months prior to conducting endotoxin testing and that another study
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(O'Toole et al. 2009) showed a 44 percent mean decline in the concentration of endotoxins in samples
stored at -80 °C for several weeks compared to samples stored at 4 °C for 24 hours. Levesque et al.
(2016) caution that concentrations reported could be underestimated and should be interpreted on an
ordinal basis. Two other studies conducting endotoxin testing on frozen samples found concentrations of
a similar magnitude as this study (Berg et al. 2011; Rapala et al. (2002).
Lin et al. (2015) conducted a prospective study based on data collected in 2009 at Boqueron, Puerto
Rico for 26 study days involving 15,726 enrollees to examine the association between phytoplankton
cell counts and illness among beachgoers. Three categories of phytoplankton were evaluated:
picocyanobacteria, cyanobacteria other than picocyanobacteria, and total phytoplankton. The analysis
compared people exposed at phytoplankton cell count levels > 25th percentile (e.g., 25th to 75th
percentile, > 75th percentile) to people exposed at levels < 25th percentile (range of cyanobacteria other
than picocyanobacteria: < 37 to 1461 cells/mL).
The Lin et al. (2015) study reported significant associations between recreational exposure to
cyanobacteria other than picocyanobacteria and respiratory symptoms, rash, and earache. For the other
symptoms measured, including eye irritation, no significant associations were observed. More
specifically, cyanobacterial (other than picocyanobacterial) densities of 37 to 237 cells/mL (> 25th to
< 75th percentile) and densities > 237 cells/mL (> 75th percentile) were associated with increased
respiratory symptoms (> 25th to < 75th percentile, OR = 1.30, 95 percent CI: 1.08-1.56; > 75th
percentile, OR = 1.37, 95 percent CI: 1.12-1.67) in study participants who reported body immersion.
Respiratory symptom occurrence was defined as any two of the following: sore throat, cough, runny
nose, cold, or fever. Cyanobacterial (other than picocyanobacterial) densities >237 cells/mL were
associated with rash (OR = 1.32, 95 percent CI = 1.05-1.66) and earache (OR = 1.75, 95 percent
CI: 1.09-2.82). Study participants who reported head submersion or swallowing of water showed no
relationship between recreational exposures to cyanobacteria (other than picocyanobacteria) and
respiratory symptoms. There was no association between recreational exposures to cyanobacteria (other
than picocyanobacteria) and respiratory symptoms in study participants who reported head submersion
or swallowing of water. A statistically significant association between cyanobacterial cell exposure
(other than picocyanobacterial cell exposure) and all health effects combined was also observed.
Lin et al. (2015) measured the dermatoxins, debromoaplysiatoxin, and lyngbyatoxin, using HPLC-mass
spectrometry and did not detect levels above the limit of detection of 1.0 ppb. Authors reported that
debromoaplysiatoxin and lyngbyatoxin-a are photolabile and are unlikely to persist in the water column
(Moikeha and Chu 1971). They noted that the health effects identified in this study were consistent with
previous blooms of Lyngbya majuscula, which can produce these toxins, though Lyngbya only
comprised three percent of total planktonic cyanobacteria (other than picocyanobacteria). It is also
possible that the cyanobacterial cells or associated contaminants could be having direct health effects as
cyanotoxins levels were below the limit of detection.
To evaluate possible co-exposures, some studies measured cyanotoxins and fecal indicators. Lin et al.
(2015), Levesque et al. (2014), Pilotto et al. (1997), and Stewart et al. (2006d) measured one or more
cyanotoxins or total hepatotoxins. In some cases, cyanotoxin levels were below the limit of detection. To
determine if study participants possibly were exposed to fecal contamination, three of the studies
(Levesque et al. 2014; Lin et al. 2015; Stewart et al. 2006d) measured bacterial fecal indicators at some
study locations and times. Of the studies that measured bacterial fecal indicators, none found an
association between bacterial fecal indicators and health effects. Of these studies, the only one with data
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available for viral fecal indicators or concentrations of waterborne pathogens was Lin et al. (2015)
provided in Wade et al. (2010) and Soller et al. (2016).
In summary, although four studies identified significant associations between cyanobacteria exposure
and health effects, the type of health effect identified varied. One study reported a significant association
between GI illness and exposure to cyanobacteria (Levesque et al. 2014). Stewart et al. (2006d) and Lin
et al. (2015) identified statistically significant associations between cyanobacterial cell exposure and
respiratory effects. Lin et al. (2015) also found a statistically significant association between earache and
cyanobacterial densities. Both Pilotto et al. (1997) and Stewart et al. (2006d) found statistically
significant associations between cyanobacterial cell exposure and all symptoms combined. The three
cross-sectional studies conducted in the United Kingdom in 1990 found no statistically significant
associations, although some minor elevated morbidity was observed in exposed individuals (Philipp
1992; Philipp and Bates 1992; Philipp et al. 1992). Another 1992 case-control epidemiological study in
Australia found no statistically significant symptoms for exposed recreators (El Saadi et al. 1995).
The Centers for Disease Control and Prevention (CDC) has collected information on illness outbreaks
associated with HABs, which commonly involve cyanobacteria. This information includes human health
effects and water-sampling results voluntarily reported to the Waterborne Disease Outbreak Surveillance
System via the National Outbreak Reporting System and the Harmful Algal Bloom-related Illness
Surveillance System. CDC published summary information on HAB-associated outbreaks from
recreational exposures focusing on 2009-2010 with limited additional information available for
outbreaks that occurred in 2001, 2004, and 2011-2012 (Dziuban et al. 2006; Hilborn et al. 2014; Hlavsa
et al. 2014; Yoder et al. 2004). CDC defines a recreational water-associated outbreak as the occurrence
of similar illnesses in two or more persons, epidemiologically linked by location and time of exposure to
recreational water or recreational water-associated chemicals volatilized into the air surrounding the
water.
The 2009-2010 reporting cycle was notable, as almost half (46 percent) the recreational water outbreaks
reported to CDC were associated with HABs (Hilborn et al. 2014). Three of the outbreaks confirmed the
presence of cyanobacteria, and four confirmed the presence of microcystins at levels greater than
20 (J,g/L. GI and dermatologic symptoms were the most commonly reported symptom categories
associated with HAB-related outbreaks in freshwater (Dziuban et al. 2006; Hilborn et al. 2014; Hlavsa et
al. 2014; Yoder et al. 2004). For the cyanobacteria-associated outbreaks with reported symptom counts,
the most common symptoms reported were GI related, including vomiting, diarrhea, and nausea
(estimated to be > 40 percent). The second most frequent outbreak symptom reported was skin rash (>
27 percent cases reported). Fever, earache, skin irritation, and headache were the next most frequently
reported symptoms (11 percent, nine percent, and nine percent of cases reported, respectively).
During 2009 and 2010 in the United States, 11 outbreaks of illness associated with HABs were reported
to CDC, all occurring in freshwater lakes and reported via the National Outbreak Reporting System
(NORS) and the Harmful Algal Bloom-related Illness Surveillance System (HABISS). Hilborn et al.
(2014) analyzed the HAB outbreak data from 2009-2010 and found the 11 outbreaks affected at least
61 persons, resulting in two hospitalizations, and included GI, dermatologic, respiratory, neurologic, and
other symptoms. Sixty-six percent of case patients were individuals aged one to 19 years (n = 38 of 58
total) and 35 percent were aged nine years or younger (n = 20). In addition, in a cyanobacteria-
associated outbreak in 2001, 42 children were affected. Outbreak data are typically limited in scope and
thought to represent an underreporting of the "true" occurrence of illness in a population, but available
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information suggests that children may share a disproportional share of the health burden associated
with recreational exposures to cyanobacterial HABs.
Dziuban et al. (2006) and Walker et al. (2008) reported on outbreaks in Nebraska. Dziuban et al. (2006)
described two 2004 cyanobacteria-associated outbreaks in which 22 cases of illness were reported from
exposure to Nebraska lakes. The predominant illnesses in both outbreaks included dermatitis and
gastroenteritis, and individuals who sought medical care showed a combination of rashes, diarrhea,
cramps, nausea, vomiting, and fevers. Walker et al. (2008) also reported about a Nebraska outbreak.
Levels of total microcystins at the east swimming beach of Pawnee Lake exceeded 15 ppb on July 12,
2004, and a health alert was issued. However, heavy public use of Pawnee Lake occurred that weekend
and more than 50 calls were received from the public, complaining about symptoms such as skin rashes,
lesions, blisters, vomiting, headaches, and diarrhea after swimming or water skiing in Pawnee Lake
(Walker et al., 2008).
D.2 Mode of Action
Few mechanistic investigations have been completed on how exposure to cyanobacterial cells might
lead to inflammatory response. Torokne et al. (2001) evaluated the sensitization and irritation potential
of Microcystis, Anabaena, Cylindrospermopsis, and Aphanizomenon bloom and strain samples and
found no correlation between the cyanotoxin content and allergenicity. For example, the nontoxic
Aphanizomenon was the most allergenic sample, more allergenic than the most toxic cyanobacterial cells
they studied, Microcystis aeruginosa. Stewart et al. (2006e) concluded that cutaneous effects strongly
suggest allergic reactions, and symptoms such as rhinitis, conjunctivitis, asthma, and urticaria (or hives)
also indicate immediate hypersensitivity responses, which are probably explained by a cascade action of
pro-inflammatory cytokines.
Bernstein et al. (2011) suggested that the allergenic structure of cyanobacteria might be associated with
a non-toxin-producing part of the organism. Building on this conclusion, Geh et al. (2015) conducted a
series of experiments to identify the cyanobacteria allergen(s) responsible for sensitization. Study
participants were given skin-prick tests with extracts from nontoxic Microcystis aeruginosa strains.
Serum from these individuals was collected from a subset of 15 patients who elicited strong skin test
responses to Microcystis aeruginosa and from three healthy control subjects. The lysate from nontoxic
Microcystis aeruginosa strains was significantly (p < 0.01) more immunoreactive than the lysate from
the toxin-producing strains, which suggests that the nontoxic strain was more allergenic than the toxic
strain. They found, however, that IgE binds to Microcystis aeruginosa peptides present in lysates of both
the toxic and nontoxic strains. Geh et al. (2015) also performed a P-hexosaminidase release assay, as a
surrogate assay for measuring histamine release, to identify functional activity of the Microcystis
aeruginosa extracts using rat basophil leukemia cells. The authors concluded that the same allergen is
present in toxic and nontoxic Microcystis aeruginosa lysates, but suggest the toxic Microcystis
aeruginosa lysate might contain an endogenous inhibitor that prevents IgE from effectively binding to
the specific allergen. The further analysis by Geh et al. (2015) of the sera of individuals exposed to
nontoxic Microcystis aeruginosa lysate indicated that either linker core-membrane peptide or
phycocyanin, or both, are potentially responsible for Microcystis aeruginosa allergenicity.
Epidemiological studies and case reports suggest respiratory effects that could be consistent with an
allergic or hay fever type reaction (Giannuzzi et al. 2011; Stewart et al. 2006e). Inhalation exposure to
bacterial endotoxins (i.e., a toxin that is part of the cyanobacterial cell as opposed to exotoxins such as
microcystins and cylindrospermopsin) has been found to be associated with pulmonary disease,
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including asthma, chronic obstructive airway disease, and emphysema (Stewart et al. 2006b). A recent
review of the structure and effects of cyanobacterial LPS suggested that it could act as an antagonist of
the TLR4 receptor and inhibit the inflammatory-response pathway (Durai et al. 2015).
Stewart et al. (2006e) also noted that, although symptoms and time to onset can be disparate, several
reports described:
"a collective group of symptoms resembling immediate or Type-I hypersensitivity reactions.
Immediate hypersensitivity reactions are commonly associated with atopy, which is the familial
tendency to react to naturally occurring antigens, mostly proteins, through an IgE-mediated
process. Atopy frequently manifests as a spectrum of diseases, e.g., seasonal rhinitis,
conjunctivitis, asthma, and urticaria."
Documentation of this type of respiratory response is consistent with results from Geh et al. (2015) and
further supports that immune system response follows exposure to cyanobacteria.
In older literature, cyanobacterial LPS was suspected as being a cause of inflammatory response because
this cell structure, also found in many Gram negative bacterial species, has been observed to initiate
acute inflammatory responses in mammals that are typical of a host reaction to tissue injury or infection
(Stewart et al. 2006b). The Stewart et al. (2006e) review, however, found evidence to support this
mechanism lacking. Although all cyanobacteria contain the pigment phycocyanin, not all species of
cyanobacteria have shown dermal reactions. Also, some species of cyanobacteria produce toxins that are
known dermal irritants (e.g., lyngbyatoxin-a). Pilotto et al. (2004), however, found that 20-24 percent of
the study participants exposed to cyanobacterial cells via skin patches for 24 hours showed dermal
reactions to cyanobacteria species, both whole and lysed cells.
Stewart et al. (2006b) noted that the effects of microcystin- and cylindrospermopsin-producing bacteria
on the GI tract could suggest that cyanotoxins and LPS from the cyanobacteria or other bacteria residing
in the gut might cross a gut mucosal barrier that has been disrupted and enhance the adverse effects of
cyanotoxins.
An aquatic invertebrate study using brine shrimp (Artemia salina, Daphnia magna, and Daphnia
galeata) to determine the toxicity of microcystin and cylindrospermopsin in combination with
cyanobacterial LPS found that pre-exposure to LPS increased the lethal concentration (LCso )of
cylindrospermopsin eight-fold (Lindsay et al. 2006). The authors concluded that the decrease in
susceptibility to cylindrospermopsin was due to the effects of LPS on detoxification enzyme pathways;
LPS decreased toxic metabolites of cylindrospermopsin by suppressing the invertebrate cytochrome
P450 system, thus decreasing toxicity.
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D.3 References
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Brisson G, Greer C, and Bird D (2014). Prospective study of acute health effects in relation to
exposure to cyanobacteria. Sci Total Environ, 466-467, 397-403.
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Levesque B, Gervais MC, Chevalier P, Gauvin D, Anassour-Laouan-Sidi E, Gingras S, Fortin N,
Brisson G, Greer C, and Bird D (2016). Exposure to cyanobacteria: acute health effects
associated with endotoxins. Public Health, 134, 98-101.
Lin CJ, Wade TJ, Sams EA, Dufour AP, Chapman AD, and Hilborn ED (2015). A prospective study of
marine phytoplankton and reported illness among recreational beachgoers in Puerto Rico,
2009. Environ Health Per sped, 124(4), 477-483.
Lindsay J, Metcalf JS, and Codd GA (2006). Protection against the toxicity of microcystin-LR and
cylindrospermopsin in Artemia salina and Daphnia spp. by pre-treatment with cyanobacterial
lipopolysaccharide (LPS). Toxicon, 48(8), 995-1001.
Moikeha SN, and Chu GW (1971). Dermatitis-producing alga Lynbya majuscula gomont in Hawaii. II.
Biological properties of the toxic factor 1,2. Journal of Phycology, 7(1), 8-13.
NHMRC (National Health and Medical Research Council) (2008). Guidelines for Managing Risks in
Recreational Water, https://nhmrc.gov.au/about-us/publications/guidelines-managing-risks-
recreational-water. Last Accessed: 12/5/2018.
O'Toole J, Sinclair M, Jeavons T, and Leder K (2009). Influence of sample preservation on endotoxin
measurement in water. Water Sci Technol, 60(6), 1615-1619.
Philipp R (1992). Health risks associated with recreational exposure to blue-green algae (cyanobacteria)
when dinghy sailing. Health Hyg, 13, 110-114.
Philipp R and Bates AJ (1992). Health-risks Assessment of dinghy sailing in Avon and exposure to
cyanobacteria (blue-breen algae). Water and Environment Journal, 6(6), 613-617.
Philipp R, Brown M, Bell R, and Francis F (1992). Health risks associated with recreational exposure to
blue-green algae (cyanobacteria) when windsurfing and fishing. Health Hyg, 13, 115-119.
Pilotto L, Hobson P, Burch MD, Ranmuthugala G, Attewell R, and Weightman W (2004). Acute skin
irritant effects of cyanobacteria (blue-green algae) in healthy volunteers. Aust N Z J Public
Health, 28(3), 220-224.
Pilotto LS, Douglas RM, Burch MD, Cameron S, Beers M, Rouch GJ, Robinson P, Kirk M, Cowie CT,
Hardiman S, Moore C, and Attewell RG (1997). Health effects of exposure to cyanobacteria
(blue-green algae) during recreational water-related activities. Aust N Z J Public Health, 21(6),
562-566.
Rapala J, Lahti K, Rasanen LA, Esala AL, Niemela SI, and Sivonen K (2002). Endotoxins associated
with cyanobacteria and their removal during drinking water treatment. Water Res, 36(10), 2621-
2635.
Shirai M, Takamura Y, Sakuma H, Kojima M, and Nakano M (1986). Toxicity and delayed type
hypersensitivity caused by Microcystis blooms from Lake Kasumigaura. Microbiol Immunol,
30(1), 731-735.
Soller JA, Eftim S, Wade TJ, Ichida AM, Clancy JL, Johnson TB, Schwab K, Ramirez-Toro G, Nappier
S, and Ravenscroft JE (2016). Use of quantitative microbial risk assessment to improve
interpretation of a recreational water epidemiological study. Microbial Risk Analysis, 7, 2-11.
Stewart I, Robertson IM, Webb PM, Schluter PJ, and Shaw GR (2006a). Cutaneous hypersensitivity
reactions to freshwater cyanobacteria - human volunteer studies. BMC Dermatol, 6, 6.
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hypersensitivity reactions to the freshwater cyanobacterium Cylindrospermopsis raciborskii and
its associated toxin cylindrospermopsin. BMC Dermatol, 6, 5.
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Stewart I, Webb PM, Schluter PJ, Fleming LE, Burns JW, Jr., Gantar M, Backer LC, and Shaw GR
(2006d). Epidemiology of recreational exposure to freshwater cyanobacteria - an international
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Stewart I, Webb PM, Schluter PJ, and Shaw GR (2006e). Recreational and occupational field exposure
to freshwater cyanobacteria - a review of anecdotal and case reports, epidemiological studies and
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of freshwater cyanobacteria - experimental evidence. Environ Toxicol, 16(6), 512-516.
Wade TJ, Sams E, Haugland R, Brenner KP, Li Q, Wymer L, Molina M, Oshima K, and Dufour AP
(2010). Report on 2009 National Epidemiologic and Environmental Assessment of 61
Recreational Water Epidemiology Studies (EPA/600/R-10/168).
https://archive.epa.eov/neear/web/pdf/report2009v5 508comp.pdf. Last Accessed: 12/5/2018.
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APPENDIX E. INCIDENTAL INGESTION EXPOSURE FACTOR COMBINED
DISTRIBUTION ANALYSIS
This appendix describes in detail the approach used to derive the value for ingestion rate in units of liters
per day. The ingestion rate is used in the derivation of the recommended cyanotoxin values in this
document.
To arrive at liters of ingestion per day, the EPA combined data on liters of ingestion per hour and the
number of hours spent in the water per day. Both of these parameters were represented as log-normal
distributions. The sources of the data were:
•	Recreational water ingestion per hour - The lead author of Dufour et al. (2017) provided the
EPA's Office of Water, Health and Ecological Criteria Division with the raw data collected and
analyzed in the study, which included mL of water ingested during a swimming event. Each
participant in the study also reported the length of time they spent in the water. The ingestion per
event was normalized to one hour for each participant and converted to liters to arrive at liters
ingested per hour. The mean and standard deviation were calculated for different age groups (6
to 10, 11 to 17, 18 years and up, and all ages). See Table E-l below for summary statistics for
this parameter. Subsequent to the EPA's analysis, Dufour et al. posted their raw dataset on
data.gov (U.S. EPA 2018). There are few minor variations in the dataset analyzed here and the
posted dataset (i.e., the posted dataset included an additional adult participant's results, specified
time spent in the water as 45 minutes for two participants, rounded ingestion volumes of 0.5 up
to 1, and indicated a higher ingestion volume for one adult woman). The EPA performed a
sensitivity analysis to see if these differences impacted the results and found no significant
effect. The very slight differences were within the rounding to the third decimal. No differences
were observed between the datasets for the results of the combined distribution analysis for the
six- to 10-year age group.
•	Duration of swimming per day - the EPA's 2011 Exposure Factors Handbook (EFH; Table 16-
20). Time Spent (minutes/day) in Selected Outdoor Locations, Doers Only, At Home in the
Outdoor Pool or Spa). Table E-2 below shows the summary statistics provided by the EPA's
EFH.
Table E-l. Parameters Used to Fit Ingestion Distributions
Ingestion Rate (L/hour)
Age Group (sample size)
Mean"
Standard deviation
Minimum
Maximum
6 to 10 (child) (n = 66)
0.03745
0.03355
0.00033
0.20000
11 to 17 (child) (n= 170)
0.03996
0.04377
0.00067
0.26800
18+ (adult) (n= 312)
0.02811
0.04960
0.00012
0.36800
All (6 to 50+) (n = 549)
0.03290
0.04643
0.00012
0.36800
a Arithmetic mean based on raw data provided by the Dufour et al. (2017) study authors. The ingestion rates for age groups
children (6 to 10), teens (11 to 15), and adults (16 and over) were reported as geometric means in Dufour et al. (2017).
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Table E-2. Parameters Used to Fit Recreation Duration Distributions
EPA 20.11 EFH (Excerpt from Tabic 16-20) (minutes/day)
Age Group (sample size)
Mean
Standard
deviation
Median (50th
percentile)
Minimum
Maximum
1 to 4 (n = 9)
85.6
86.3
60
15
255
5 to 11 (n= 15)
164.2
103.97
140
25
450
12 to 17 (n = 5)
97
53.8
100
40
180
18 to 64 (n = 44)
117.6
112.7
83
4
450
> 64 (n = 10)
78.9
85.3
53
1
258
R (open source programming language) was used to perform the calculations described in this appendix.
The annotated R code is shown below, following a summary of what calculations were performed and
assumptions.
The water ingestion rate per hour data from Dufour were used to compute an arithmetic mean and
standard deviation, which are in turn used to compute the log geometric mean (GM) and log geometric
standard deviation (GSD) using a mathematical conversion formula. The log GM and log GSD are used
as distributional parameters to generate 10,000 random samples representing water ingestion rates per
hour of recreational activity (L/hour).
The mean and standard deviation of the number of recreational hours spent in the water per day are
reported as summary statistics in the EFH 2011, and are used to compute the log GM and log GSD using
a mathematical conversion formula. The log GM and log GSD are used as distributional parameters to
generate 10,000 random samples representing water ingestion rates per hour of recreational activity
(hour/day).
The two component distributions are assumed to be statistically independent of each other and are
multiplied to generate a combined distribution with 10,000 values for the ingestion rate of water per day
of recreational activity in L/day. Summary statistics, including the mean, standard deviation, and point
estimates of various percentiles, are then computed from the combined distribution. The EPA chose the
90th percentile point estimate for children six to 10 (0.21 L/day) to calculate the recommended
cyanotoxin values.
References
Dufour AP, Behymer TD, Cantu R, Magnuson M, and Wymer LJ (2017). Ingestion of swimming pool
water by recreational swimmers. Journal of Water and Health, 75(3), 429-437.
U.S. EPA (United States Environmental Protection Agency) (2011). Exposure Factors Handbook 2011
Edition (Final). Washington, DC. EPA/600/R-09/052F.
https://cfpub.epa.gov/ncea/risk/recordisplav.cfm?deid=236252. Last Accessed: 11/27/2018.
U.S. EPA (United States Environmental Protection Agency) (2018). Ingestion of swimming pool water
by recreational. Swimming pool water ingestion data. Dataset associated with Dufour et al.
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(2017). Data.gov Data Catalog, https://catalog.data.gov/dataset/ingestion-of-swimming-pool-
water-bv-recreational. Last Accessed: 11/27/2018.
R Code
#Cyanotoxin recAWQC WA
#	This script is to combine distributions for water ingestion rate (L/hr) and recreational exposure
duration (hr/day) to develop a distribution for ingestion/day (L/day) and to generate a histogram of this
combined distribution
#	The first distribution is the incidental ingestion rate per hour from the Dufour dataset
#	The second distribution is the recreational exposure duration (hr/day) from the EPA 2011 Exposure
Factors Handbook Table 16-20. Time Spent (minutes/day) in Selected Outdoor Locations, Doers Only,
At Home in the Outdoor Pool or Spa
#	Both distributions are assumed to be log-normal
#####Read required libraries and set simulation sample size #################
rm(list=ls()) # Remove all current R objects from memory
library(truncnorm) #import library for truncated normal distribution
nsamp = 1000000 # specify number of samples in monte-carlo analysis
set.seed(1984756) # set seed for analysis replicability
mmmmmmmmmmmmmmmmmmmmmmmmmm
# The combined distribution function (cdist) assumes a log-normal distribution for ingestion rate
(L/hour) and a log-normal distribution for exposure duration (hr/d)
#.... using the mean and sd as parameter inputs. This function is called in later sections of the code for
each age group analysis.
cdist<-function(nsamp,mean_dur,sd_dur,min_dur,max_dur,mean_ing,sd_ing,min_ing,max_ing){
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n<-nsamp # number of samples to be drawn
transform mean and sd of duration
sd_dur_ln<-sqrt(log((sd_dur/mean_dur)A2+l)) # standard deviation of duration in log space
mean_dur_ln<-log(mean_dur)-((sd_dur_lnA2)/2) # mean of duration in log space
min_dur_ln<-log(min_dur) # minimum duration in log space
max_dur_ln<-log(max_dur)
transform mean and sd of ingestion rate
sd_ing_ln<-sqrt(log((sd_ing/mean_ing)A2+1))
mean_ing_ln<-log(mean_ing)-((sd_ing_lnA2)/2)
min_ing_ln<- -10A10
max_ing_ln<-log(max_ing)
#	draw n samples from the truncated ingestion rate distribution in L/hr
ingperhr_ln_trunc<-exp(rtruncnorm(n=n, a=min_ing_ln, b=max_ing_ln, mean=mean_ing_ln,
sd=sd_ing_ln)) #truncated log normal distribution
#	draw n samples from the truncated duration distribution (hr/d)
duration_hr_ln_trunc<-exp(rtruncnorm(n=n, a=min_dur_ln, b=max_dur_ln, mean=mean_dur_ln,
sd=sd_dur_ln))
#	compute n samples for the combined ingestion rate per day distribution (L/d)
ingperday<-ingperhr_ln_trunc*duration_hr_ln_trunc #combine distributions
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print(summary(ingperday)) # print summary statistics of the combined distribution
print(quantile(ingperday, probs=0.90)) # print 90th percentile of the combined distribution
#Generate histogram
hist(ingperday,xlab="Ingestion rate (L/day)",ylab="Probability", main - 'Truncated hybrid distribution
fit", xlim=c(0, 2.0), ylim=c(0, 1))
h=hist(ingperday)
h$density=h$counts/sum(h$counts)
plot(h,xlab="Ingestion rate (L/day)",ylab="Probability", main ="Log-normal distribution fit",
xlim=c(0, 1), ylim=c(0, 0.99), xaxp=c(0,1.5,15), freq=FALSE)
}
mmmmmmmmmmmmmmmmmmmmmmmmmm
#1. Analysis for 6 to 10 age group
#	These values are from 2011 EFH table 16-20 for ages 5 to 11.
mean_dur_min= 164.2
sd_dur_min= 103.97
min_dur_min=25
max_dur_min=450
#	Convert exposure data from the EPA's EFH from min/day to hr/day
mean_dur<-mean_dur_min/60 #mean exposure duration hr/day
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sd_dur<-sd_dur_min/60 #sd exposure duration hr/day
min_dur<-min_dur_min/60 #minimum exposure duration hr/day
max_dur<-max_dur_min/60 #maximum exposure duration hr/day
#	These ingestion rate values are computed from the Dufour dataset
mean_ing<- 0.03745 # mean ingestion rate in L/hr
sd_ing<-0.03355 # sd ingestion rate in L/hr
min_ing<-0.00033 # minimum ingestion rate in L/hr
max_ing<-0.20000 # maximum ingestion rate in L/hr
cdist(nsamp,mean_dur,sd_dur,min_dur,max_dur,mean_ing,sd_ing,min_ing,max_ing) # call combined
distribution function
mmmmmmmmmmmmmmmmmmmmmmmmmm
#11. Analysis for 11 to 17 age group
#	These values are from 2011 EFH table 16-20 for age 12 to 17
mean_dur_min=97
sd_dur_min=5 3.81
med_dur_min= 100
min_dur_min=40
max_dur_min= 180
#	Convert exposure data from the EPA's EFH from min/day to hr/day
mean_dur<-mean_dur_min/60 #mean exposure duration hr/day
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sd_dur<-sd_dur_min/60 #sd exposure duration hr/day
med_dur<-med_dur_min/60 #median exposure duration hr/day
min_dur<-min_dur_min/60 #minimum exposure duration hr/day
max_dur<-max_dur_min/60 #maximum exposure duration hr/day
# These ingestion rate values are computed from the Dufour dataset
mean_ing<-0.03996 # mean ingestion rate in L/hr
sd_ing<-0.04377 # sd ingestion rate in L/hr
min_ing<-0.00067 # minimum ingestion rate in L/hr
max_ing<-0.26800 # maximum ingestion rate in L/hr
cdist(nsamp,mean_dur,sd_dur,min_dur,max_dur,mean_ing,sd_ing,min_ing,max_ing) # call combined
distribution function
mmmmmmmmmmmmmmmmmmmmmmmmmm
#111. Analysis for 18+ age group
#	Combine exposure duration data for 18 to 64 and for >64 age groups from 2011 EFH table 16-20.
mean_dur_min=(l 17.61+78.9)/2
sd_dur_min=sqrt(( 112.7 2A2+8 5.3 2A2)/2)
min_dur_min=l
max_dur_min=450
#	Convert exposure data from the EPA's EFH from min/day to hr/day
mean_dur<-mean_dur_min/60 #mean exposure duration hr/day
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sd_dur<-sd_dur_min/60 #sd exposure duration hr/day
min_dur<-min_dur_min/60 #minimum exposure duration hr/day
max_dur<-max_dur_min/60 #maximum exposure duration hr/day
# These ingestion rate values are computed from the Dufour dataset
mean_ing<-0.02811 # mean ingestion rate in L/hr
sd_ing<-0.04960 # sd ingestion rate in L/hr
min_ing<-0.00012 # minimum ingestion rate in L/hr
max_ing<-0.36800 # maximum ingestion rate in L/hr
cdist(nsamp,mean_dur,sd_dur,min_dur,max_dur,mean_ing,sd_ing,min_ing,max_ing) # call combined
distribution function
mmmmmmmmmmmmmmmmmmmmmmmmmm
#	IV. Analysis for all age groups (including 1-4 yo)
#	Combine exposure duration data for all age groups (1 to 4, 5 to 11, 12 to 17, 18 to 64, >64) from 2011
EFH table 16-20.
mean_dur_min=(85.56+164.2+97+117.61+78.9)/5
sd_dur_min=103.71 # SD reported in EFH for all ages
min_dur_min=l
max_dur_min=450
#	Convert exposure duration data from min/day to hr/day
mean_dur<-mean_dur_min/60 #mean exposure duration hr/day
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sd_dur<-sd_dur_min/60 #sd exposure duration hr/day
min_dur<-min_dur_min/60 #minimum exposure duration hr/day
max_dur<-max_dur_min/60 #maximum exposure duration hr/day
# These ingestion rate values are computed from the Dufour dataset
mean_ing<- 0.03290 # mean ingestion rate in L/hr
sd_ing<- 0.04643 # sd ingestion rate in L/hr
min_ing<-0.00012 # minimum ingestion rate in L/hr
max_ing<-0.36800 # maximum ingestion rate in L/hr
cdist(nsamp,mean_dur,sd_dur,min_dur,max_dur,mean_ing,sd_ing,min_ing,max_ing) # call combined
distribution function
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APPENDIX F. INGESTION STUDIES
The EPA reviewed seven studies to evaluate recreation-associated incidental ingestion (DeFlorio-Barker
et al. 2017; Dorevitch et al. 2011; Dufour et al. 2006, 2017; Schets et al. 2011; Schijven and de Roda
Husman 2006; Suppes et al. 2014). Evans et al. 2006 was also reviewed, but is the same dataset as
Dufour et al. (2017), so it is not included in the table. The EPA's approach for estimating incidental
exposure while swimming used by the EPA's Office of Pesticide Programs (OPP) is also summarized
below.
F.l DeFlorio-Barker et al. (2017)
DeFlorio-Barker et al. (2017) combined ingestion data from Dufour et al. (2017) and time spent in the
water data from 12 cohorts of epidemiological studies to estimate the volume of water ingested per
swimming event. They calculated the ingested volume per minute (mL/minute) for each Dufour et al.
(2017) study participant, using the mL ingested and the self-reported time spent in the water for each
participant. The National Epidemiological and Environmental Assessment of Recreational Water Study
and Southern California Coastal Water Research Project epidemiological studies included 68,685
recreators at four freshwater and eight marine beaches. The participants in these studies estimated how
much time they spent in the water. DeFlorio-Barker et al. (2017) combined the mL/minute ingestion rate
from Dufour et al. (2017) and the self-reported time spent in the water for the epidemiological study
participants to calculate the volume of water ingested per event. The results of this study corroborate
other studies that demonstrate that, on average, children have higher incidental ingestion than adults
when recreating.
F.2 Dorevitch et al. (2011)
Dorevitch et al. (2011) evaluated incidental ingestion associated with multiple types of water contact
activities in both surface water (canoeing, fishing, kayaking, motor boating, and rowing) and in pools
(canoeing, fishing, kayaking, swimming, wading/splashing, and walking around the pool as a control).
The surface water activities did not include swimming because the water body was designated for
secondary contact recreation only. Volume of ingestion was self-reported via interviews
(3,367 participants: 2,705 individuals recreating in the Chicago Area Waterway System (CAWS, surface
water) and 662 individuals recreating at a public outdoor swimming pool). At the end of their exposure,
participants self-reported whether they ingested water, and how much, during their recreational
experience. The authors used a subset of the pool exposures to assess cyanuric acid in urine to determine
the accuracy of the self-reported ingestion volumes. There was strong agreement between self-reported
results and cyanuric acid measurement (none = 0.0014±0.008 L; drop to teaspoon = 0.0094±0.011 L;
mouthful = 0.026±0.037 L).
The results indicate that the odds of ingesting a teaspoon or more of water are significantly higher
among swimmers than among those who just immersed their head in a swimming pool or those who
participated in the other, more limited contact activities on surface waters. More specifically, rowing,
motor boating, fishing, wading/splashing, and non-capsizing kayaking and canoeing were found to be
low-ingestion activities, resulting in 95 percent upper confidence limit ingestion volumes between 0.01
and 0.012 L/hour. Less than five percent of limited contact recreators on surface waters reported
swallowing any water. The study authors considered those who capsized during canoeing or kayaking a
"middle ingestion category," with mean incidental ingestions of 0.006 to 0.005 L/hour. Swimmers were
the highest ingestion category, with a mean of 0.01 L/hour. Swimmers in a pool were more than
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50 times as likely to report swallowing a teaspoon of water compared to people who canoed or kayaked
in surface waters.
In surface water, participants ages six years and above incidentally ingested the most water while
canoeing and capsizing compared to any other activity assessed (median = 0.0036 L; mean = 0.006 L;
Upper 95 percent CI: 0.0199 L). Kayaking and capsizing in surface water resulted in nearly as high
incidental ingestion (mean = 0.005 L; Upper 95 percent CI: 0.0165). In swimming pool water,
participants ages six and above incidentally ingested the most water while swimming compared to any
other activity assessed (median = 0.006 L; mean = 0.01 L; Upper 95 percent CI: 0.0348 L). Duration of
activities was not reported, so the ingestion volumes are on a per event basis.
F.3 Dufour et al. (2006)
The EPA's Exposure Factors Handbook (EFH) (2011) presents values for incidental ingestion while
recreating values citing Dufour et al. (2006). Dufour et al. (2006) measured the incidental ingestion of
water while participants were swimming in a pool and found that children under the age of 18 ingested
higher volumes of water while swimming than adults. The 2006 study design instructed participants to
swim for at least 45 minutes, so the time the participants spent in the water is probably not
representative of preferred or regular patterns for recreation duration and the actual duration was not
recorded. Both studies reported higher ingestion among children compared to adults. The values
presented in the EFH adjusted the Dufour et al. (2006) data from a per event basis to an hourly ingestion
rate. The EFH recommends using the 97th percentile ingestion rate for children and the maximum
reported value for adults because the dataset is limited (U.S. EPA 2011).
F.4 Evans et al. (2006)
Evans et al. (2006) presented results from an observational study of incidental water ingestion during
recreational swimming activities using the same methodology as the Dufour et al. (2006) pilot study.
This study characterized ingestion volumes for younger children verses older children and adults. Evans
et al. (2006) reported higher ingestion volumes for younger children. Although study results were
presented at a conference, they were not published, so the EPA did not cite this publication in the
derivation of the recommended cyanotoxin values. However, Dufour et al. (2017) includes the data
reported by Evans et al. (2006).
F.5 Schets et al. (2011)
A study in the Netherlands by Schets et al. (2011) used questionnaires to collect estimates of water
swallowed while swimming/bathing in freshwater, marine water, and swimming pools. Of the 8,000
adults who completed the questionnaire, 1,924 also provided estimates for their eldest child (<15 years
of age). The participants estimated the amount of water they or their children swallowed while
swimming. Participants chose between four categories of water volumes: (1) no water or only a few
drops; (2) one to two mouthfuls (a shot glass); (3) three to five mouthfuls (coffee cup); and (4) six to
eight mouthfuls (soda glass). Schets et al. (2011) also conducted a series of experiments to measure the
amount of water that corresponded to a mouthful of water and converted the data in the four response
categories to volumes of water ingested per event. Adult men swallowed, on average 0.030 L/hour and
women swallowed 0.020 L/hour, with somewhat greater ingestion in marine waters than in freshwater or
a swimming pool. In fresh and marine waters children swallowed about the same as adults, and in
swimming pools they ingested more than adults, on average, 0.038 L/hour compared with 0.030 and
0.021 for males and females, respectively (Schets et al. 2011). The EPA made the assumption that
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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exposure in a swimming pool is roughly equivalent to exposure in fresh and marine waters. Schets
(2011) supports that assumption, although it is a somewhat more conservative assumption for children.
However, when bodyweight is taken into account the greater exposure to children versus adults becomes
clear. Additional research would be helpful to clarify uncertainty in differences in ingestion from
different types of waters.
F.6 Schijven and de Roda Husman (2006)
Schijven and de Roda Husman (2006) studied sport and occupational diver incidental ingestion. The
types of water studied for occupational divers (n = 37 divers) were open sea and coastal marine water,
and freshwater. For sport divers (n = 483 divers), the types of water considered were open sea and
coastal marine water, fresh recreational water, canals and rivers, city canals, and swimming pools. The
divers were asked to estimate how much water they swallowed in terms of: none, few drops, shot glass,
coffee cup, or soda glass. The authors translated the description of volumes from the questionnaires into
average volumes. Occupational divers reported incidentally ingesting more water per dive in marine
water (mean: 0.0098 L/dive; maximum: 0.1 L/dive) compared to freshwater (mean: 0.0057 L/dive;
maximum: 0.025 L/dive). Sports divers wearing an ordinary diving mask reported incidentally ingesting
the most water per dive in swimming pools (mean: 0.02 L/dive; maximum: 0.19 L/dive), followed by
recreational freshwater (mean: 0.013 L/dive; maximum: 0.19 L/dive) and coastal marine water (mean:
0.0099 L/dive; maximum: 0.19 L/dive). Sports divers wearing a full face mask reported incidentally
ingesting less water than sports divers wearing an ordinary diving mask. The mean ingestion rates in
freshwater ranged from 0.0015 to 0.019 L/hour, with the highest mean being for adult recreational divers
wearing an ordinary diving mask and the lowest mean for adult recreational divers wearing a full face
mask. The mean ingestion rates in marine water ranged from 0.0005 to 0.014 L/hour, with the highest
mean being for adult recreational divers wearing an ordinary diving mask and the lowest mean for adult
recreational divers wearing a full face mask. The age of the divers was not included in the study report.
Occupational divers dived on average 60-95 minutes and sport divers dived on average 42-52 minutes
per dive.
F.7 Suppes et al. (2014)
Suppes et al. (2014) used a similar measurement method as Dufour et al. (2006, 2017), (i.e., using
cyanuric acid as an indicator of pool water ingestion) to evaluate the rate of water ingested by 16
children ages five to 17 years. They and found that children on average ingested pool water at a higher
rate than adult participants. Total time in water, quantified by viewing videos, was used to adjust pool
water ingestion volumes to obtain rates. After adjustments for false-positive measurements were applied,
the mean rate at which adults ingested water was 0.0035 L/hour with range 0-0.051 L. The mean rate at
which children ingested water was 0.026 L/hour with range 0.0009-0.106 L/hour.
F.8 U.S. EPA (2003)
Additional estimates of incidental water ingestion rates while swimming in pools have been identified
by the EPA's OPP. OPP calculated people's exposures to pool chemicals while they swim using its
Swimmers Exposure Assessment Model (SWIMODEL) (U.S. EPA 2003). SWIMODEL uses incidental
ingestion values for children that are twice the values used for adults. Incidental ingestion rates among
adults while swimming competitively and noncompetitively are 0.0125 L/hour and 0.025 L/hour,
respectively. The model assumes an incidental ingestion rate of 0.050 L/hour for children ages seven to
10 years and 11 to 14 years while swimming noncompetitively. The 0.050 L/hour value is the value used
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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in the EPA OPP's Standard Operating Procedures (U.S. EPA 2000) and is based on recommendations
from EPA's Risk Assessment Guidance for Superfund, Part A (U.S. EPA 1989, 2000, 2003).
F.9 Summary
Although these studies used different methodologies and have limitations with respect to reporting
information for different age group categories, their results show a similar pattern compared to Dufour et
al. (2006, 2017): children ingest water at a higher rate while swimming than adults. Dufour et al. (2017)
and Dufour et al. (2006) identified mean ingestion rates for children of 0.037-0.040 and 0.049 L/hour,
respectively, and adult rates of 0.028 and 0.021 L/hour, respectively. Depending on water type, Schets et
al. (2011) found a mean ingestion volume for children aged zero to 14 years of 0.028-0.038L/hour for
children and 0.020-0.036 L/hr for males and females. The most pronounced differences were for
swimming pools, where children ingested at a higher rate (0.038 L/hour) than adults (males: 0.030
L/hour; females: 0.021 L/hour). Dorevitch et al. (2011) reported ingestion rates while swimming for all
ages of O.OlOL/hour. Suppes et al. (2014) reported an adjusted mean ingestion rate of 0.026 L/hour for
children and a rate of 0.0035 L/hour for adults.
Table F-l includes: sample size, measurement methodology, the maximum values or the upper
confidence intervals (CI) for the mean ingestion per event, time spent in the water (mean or range), and
the mean ingestion volume normalized to one hour (or range if a range of durations were reported). This
information supports comparison of the studies and help with understanding the range of different
recreational exposures from activities.
The column with normalized ingestion (mL/hour) was populated using the following methods:
•	Dufour et al. (2017) - The EPA used the individual data points from this dataset. Each
participant's volume ingested was adjusted to one hour based on the length of time that
participant reported being in the water.
•	Dufour et al. (2006) - The EPA assumed that all swimming events were 45 minutes in duration.
The values reported in Table F-l are the same as the values in EPA's EFH (2011).
•	DeFlorio-Barker et al. (2017) - Normalized data are not included in Table F-l because the
authors used the Dufour et al. (2017) rate in their modeling, so including the normalized data
would be duplicative of Dufour et al. (2017).
•	Dorevitch et al. (2011) - Study authors included normalized values in the study publication.
•	Schets et al. (2011) - The EPA used the mean duration values provided in the publication to
calculate the normalized value for each age and activity category.
•	Suppes et al. (2014) - Study authors reported volume per hour.
•	Schijven and de Roda Husman (2006) - The EPA used the range of duration values provided in
the publication to calculate the normalized value for each activity category.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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F.10 References
DeFlorio-Barker S, Arnold BF, Sams EA, Dufour AP, Colford JM Jr, Weisberg SB, Schiff KC, Wade
TJ (2017) Child environmental exposures to water and sand at the beach: Findings from studies
of over 68,000 subjects at 12 beaches. J Expo Sci Environ Epidemiol. 28(2):93-100.
Dorevitch S, Panthi S, Huang Y, Li H, Michalek AM, Pratap P, Wroblewski M, Liu L, Scheff PA, and
Li A (2011). Water ingestion during water recreation. Water Res, 45(5), 2020-2028.
http://www.ncbi.nlm.nih.eov/pubmed/21227479.
Dufour AP, Evans O, Behymer TD, and Cantu R (2006). Water ingestion during swimming activities in
a pool: A pilot study. Journal of Water Health, 4, 425-430.
Dufour AP, Behymer TD, Cantu R, Magnuson M, and Wymer LJ (2017). Ingestion of swimming pool
water by recreational swimmers. Journal of Water and Health, 15(3), 429-437.
Evans OM, Wymer LJ, Behymer TD, and Dufour AP (2006). An Observational Study: Determination of
the Volume of Water Ingested During Recreational Swimming Activities. Paper presented at the
National Beaches Conference, Niagara Falls, NY.
Schets FM, Schijven JF, and de Roda Husman AM (2011). Exposure assessment for swimmers in
bathing waters and swimming pools. Water Res, 45(7), 2392-2400.
http://www.ncbi.nlm.nih.eov/pubmec	L
Schijven J, and de Roda Husman AM (2006). A survey of diving behaviour and accidental water
ingestion among Dutch occupational and sport divers to assess the risk of infection with
waterborne pathogenic microorganisms. Environ Health Perspect, 114(5), 712-717.
http://www.ncbi.nlm.nih.eov/pubmed/16675425.
Suppes LM, Abrell L, Dufour AP, and Reynolds KA (2014). Assessment of swimmer behaviors on pool
water ingestion. J Water Health, 12(2), 269-279.
http://www.ncbi.nlm.nih.eov/pubmed/24937221.
U.S. EPA (United States Environmental Protection Agency) (1989). Risk Assessment Guidance for
Superfund. Volume I: Human Health Evaluation Manual (Part A).
U.S. EPA (United States Environmental Protection Agency) (2000). Standard Operating Procedures for
Residential Exposure Assessments. Residential Exposure Assessment Work Group (Draft).
https://www.epa.eov/sites/production/files/2015~08/documents/usepa~opp~
hed residential sops oct2012.pdf. Last Accessed: 11/27/2018.
U.S. EPA (United States Environmental Protection Agency) (2003). User's Manual: Swimmer Exposure
Assessment Model (SWIMMODEL) Version 3.0.
https://www.epa.eov/sites/prodiiction/files/2015-09/dociiments/swimodel-users~eiiide.pdf. Last
Accessed: 11/27/2018.
U.S. EPA (United States Environmental Protection Agency) (2011). Exposure Factors Handbook 2011
Edition (Final). Washington, DC. EPA/600/R-09/052F.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Table F-l. Studies of Incidental Ingestion Volumes While Recreating
Reference
Study
Sample
Size
Measurement Methodology
Water Type,
Recreational Activity
Age Group"
(Years Old)
Mean Ingestion
(Maximum Value)
(mL/event)
Mean Duration
of Event
(minutes)
Normalized Ingestion
(mL/hour)
Dataset from
Dufour (data
collection
>500
Cyanuric acid was measured in
pool water and urine samples,
and ingestion rate was
Swimming pool,
Swimming
6 to 10
59.8 (245)
each participant
reported a
duration0
37
methods
reported in
Dufour et al.
(2017))b

calculated based on duration of
swimming event

11 to 17
35.6 (267)
each participant
reported a
duration0
40



18+
23.7(279)
each participant
reported a
duration0
28




All ages (6+)
31.7(279)
each participant
reported a
duration0
33
Dufour et al.
53
Cyanuric acid was measured in
Swimming pool,
6 to < 18
37 (NR)
>45
49
(2006)

pool water and urine samples
Swimming
18+
16 (NR)
>45
21




All ages (6+)
32 (NR)
>45
43
DeFlorio-
12
Estimates of amount of water
Freshwater
6 to 10
58.9 (142)d
(NR)
-
Barker et al.
(2017)
cohorts
totaling
68,685
swallowed were self-reported

11 to 17
55.5 (140)d
(NR)
-


18+
21.9 (46.7)d
(NR)
-


Marine Water
6 to 10
74.4 (180)d
(NR)
-




11 to 17
75.6 (186.7)d
(NR)
-




18+
32.4 (72)d
(NR)
-
Dorevitch et al.
(2011)
3,367
Estimates of amount of water
swallowed were self-reported
Surface water,
Canoeing/capsizing
All ages (6+)
6 (19.9)e
No duration
constraints
-



Surface water,
Kayaking/capsizing
All ages (6+)
5 (16.5)e
No duration
constraints
-


Estimates of amount of water
swallowed were self-reported;
cyanuric acid was measured in
urine in a subset of participants
Swimming pool,
Swimming
All ages (6+)
10 (34.8)e
60
10


Swimming pool,
Canoeing/capsizing
All ages (6+)
6.6 (22.4)e
60
6.6



Swimming pool,
Kayaking/capsizing
All ages (6+)
7.9 (7.9)e
60
7.9
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Reference
Study
Sample
Size
Measurement Methodology
Water Type,
Recreational Activity
Age Group;l
(Years Old)
Mean Ingestion
(Maximum Value)
(mL/event)
Mean Duration
of Event
(minutes)
Normalized Ingestion
(mL/hour)
Schets et al.
(2011)
9,924
(1,924 of
which
were
children)
Descriptive estimates of the
amount of water swallowed
were self-reported by
participants or parents of
participants, and estimates
were converted to volumes
Freshwater,
Swimming
Oto 14
37 (170)e
79
28
15+, males
27 (140)e
54
30
15+, females
18 (86)e
54
20
Marine water,
Swimming
Oto 14
31 (140)e
65
29
15+, males
27 (140)e
45
36
15+, females
18 (90)e
41
26
Swimming pool,
Swimming
Oto 14
51 (200)e
81
38
15+, males
34 (170)e
68
30
15+, females
23 (110)e
67
21
Suppes et al.
(2014)
38
Cyanuric acid was measured
and total time in water was
quantified using videos to
adjust ingestion volumes to
rates; authors adjusted
ingestion volumes to correct
for potential false-positive
measurements from cyanuric
acid carry-over between
sample injections
Swimming pool,
Swimming (adjusted)
5 to 17
26(106)
60f
26
18+
4(51)
60f
3.5
All ages (5+)
14(106)
60f
14
Swimming pool,
Swimming (unadjusted)
5-17
59 (225)
60f
59
18+
9 (NR)
60f
9
All ages (5+)
32 (NR)
60f
32
Schijven and de
Roda Husman
(2006)
517
Descriptive estimates of the
amount of water swallowed
were self-reported, and
estimates were converted to
volumes
Freshwater,
Recreational diving
w/ordinary diving mask
Adults
13(190)
42 to 52
15 to 19
Freshwater,
Recreational diving
w/full face mask
Adults
1.3(15)
42 to 52
1.5 to 1.9
Freshwater,
Occupational diving
Adults
5.7(25)
60 to 95
4 to 6
Marine Water (coastal),
Recreational diving
w/ordinary diving mask
Adults
9.9(190)
42 to 52
11 to 14
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Reference
Study
Sample
Size
Measurement Methodology
Water Type,
Recreational Activity
Age Group;l
(Years Old)
Mean Ingestion
(Maximum Value)
(mL/event)
Mean Duration
of Event
(minutes)
Normalized Ingestion
(mL/hour)



Marine water (coastal),
Recreational diving
w/full face mask
Adults
1.3(15)
42 to 52
1.5 to 1.9



Marine Water (open sea),
Recreational diving
w/ordinary diving mask
Adults
7.7(100)
42 to 52
9 to 11



Marine water (open sea),
Recreational diving
w/full face mask
Adults
0.43 (2.8)
42 to 52
0.5 to 0.6



Marine Water (coastal and
open sea combined),
Recreational diving
w/ordinary diving mask
Adults
9.0(190)
42 to 52
10 to 13



Marine water (coastal and
open sea combined),
Occupational diving
Adults
9.8(100)
60 to 95
6 to 10



Swimming pool,
Recreational diving
w/ordinary diving mask
Adults
20(190)
42 to 52
23 to 29



Swimming pool,
Recreational diving
w/full face mask
Adults
13(190)
42 to 52
15 to 19
a Age group ranges reflect the age groupings reported in the study. In some cases the authors did not separate data by different age groups among children or between adults and children.
b The values shown are arithmetic means calculated from the Dufour dataset. The Dufour et al. (2017) publication reported ingestion volumes as geometric means for children (6 to 10
years), teens (11 to 15 years), and adults (16 years and over).
c Each participant's volume ingested was adjusted to one hour based on the length of time that participant reported being in the water.
dNo maximum values are reported in the study; 90th provided percentile in parentheses.
e No maximum values are reported in the study; upper limit of the CI is provided.
f Swimming duration was reported as > 45 minutes, however authors derived and reported only hourly ingestion per event.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
F-8

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APPENDIX G. INFORMATION ON CELLULAR CYANOTOXIN AMOUNTS
The information in the tables in this appendix was generated from a brief survey of the peer-reviewed
and published scientific literature. This survey was not a formal systematic literature search and was
conducted to evaluate the availability of data needed to calculate a cyanobacterial cell density potentially
associated with a specific cyanotoxin concentration.
The information in Tables G-3 and G-4 was generated from both a brief survey and a standardized
search of the peer-reviewed and published scientific literature. The purpose of these searches was to
evaluate the availability of cyanotoxin quota data (i.e., cyanotoxin content per cyanobacterial cell or per
unit biomass, for microcystins and cylindrospermopsin) needed to calculate a cyanobacterial cell density
potentially associated with a specific cyanotoxin concentration.
The EPA conducted a brief initial survey of the available peer-reviewed and published scientific
literature in December 2016 and identified 29 studies with data on cellular toxin amounts. After
reviewing the available data, a formal literature search was conducted. The purpose of this literature
search and screening was to identify literature relevant to answering the following research question:
What cyanotoxin cell quota data (i.e., cyanotoxin content per cyanobacterial cell or per unit biomass, for
microcystins and cylindrospermopsin) are available in the peer-reviewed literature?
Search terms were identified with support from a subject matter expert and library science professionals
and included genera of known microcystins or cylindrospermopsin producers, names of the toxins of
interest, and keywords that could indicate that quota data were reported. The search was conducted in
PubMed and results were limited to articles published in English from 1987 to March 2017. A summary
of the literature search results is provided in Table G-l.
Table G-l. Summary of Cyanotoxin Cell Quota Data Literature Search Results
Database
Results
Notes/Limits
PubMed
253
1987 to present; English
Web of Science
472
1987 to present; English
Total Unique
485

The EPA developed search strategies for each database. Both search strategies included the same set of
keywords but varied in how these keywords were strung together. The Web of Science search strategy
also included limits, a feature not characteristic of a search strategy conducted using PubMed. The
search strategies are provided below.
PubMed
Date of Search: 3/01/2017
Date Limit: 1987 to present
Language = English
Set
PubMed Seareh Strategy
1
(Anabaena[tiab] OR Anabaena[mh] OR Anabaena-flos-aquae[tiab] OR Anabaenopsis[tiab] OR
Aphanizomenon[tiab] OR Aphanizomenon[mh] OR C.-raciborskii[tiab] OR Chrysosporum-
ovalisporum[tiab] OR Cuspidothrix[tiab] OR Cylindrospermopsis[tiab] OR
Cylindrospermopsis[mh] OR Cylindrospermopsis-raciborskii[tiab] OR Cylindrospermum[tiab]
OR Dolichospcrmum|tiab| OR Fischcrclla| tiab| OR Glocotrichialtiab| OR Lyngbyaftiabl OR
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-l

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Set
PublMcd Search Strategy

M.-aeruginosa[tiab] OR Microcystis[tiab] OR Microcystis[mh] OR Microcystis-aeruginosa[tiab]
OR Nostoc[tiab] OR Nostoc[mh] OR Oscillatoria[tiab] OR Oscillatoria[mh] OR
Phormidium[tiab] OR Planktothrix[tiab] OR Sphaerospermopsis[tiab] OR Synechococcus[tiab]
OR Synechococcus[mh])
2
AND (microcystin[tiab] OR microcystins [tiab] OR microcystins[mh] OR
cylindrospermopsin[tiab] OR cylindrospermopsin[Supplementary Concept])
3
AND (quota[tiab] OR cell-content[tiab] OR cellular-concentration[tiab] OR cyanotoxin-
content[tiab] OR intracellular-content[tiab] OR intracellular-concentration[tiab] OR toxin-
content[tiab] OR microcystin-content[tiab] OR microcystin-LR-content[tiab] OR MC-
content[tiab] ORMCYST-content[tiab] OR MC-LR-content[tiab] OR intracellular-
microcystin[tiab] OR intracellular-MC[tiab] OR microcystin-production[tiab] OR microcystin-
LR-production[tiab] OR microcystins-production[tiab] OR MC-production[tiab] ORMCYST-
production[tiab] OR MC-LR-production[tiab] OR CYN-content[tiab] OR particulate-CYN[tiab]
OR cylindrospermopsin-production[tiab])
Web of Science
Date of Search: 3/01/2017
Date Limit: 1987 to present
Language = English
All terms searched in Topic (Title, Abstract, and Keywords)
Set
Web of Science Search Strategy
1
(Anabaena OR Anabaena-flos-aquae OR Anabaenopsis OR Aphanizomenon OR C.-raciborskii
OR Chrysosporum-ovalisporum OR Cuspidothrix OR Cylindrospermopsis OR
Cylindrospermopsis-raciborskii OR Cylindrospermum OR Dolichospermum OR Fischerella
OR Gloeotrichia OR Lyngbya OR M.-aeruginosa OR Microcystis OR Microcystis-aeruginosa
OR Nostoc OR Oscillatoria OR Phormidium OR Planktothrix OR Sphaerospermopsis OR
Synechococcus)
2
AND (microcystin OR microcystins OR cylindrospermopsin)
3
AND (microcystin-RR-content OR MC-RR-content OR particulate-microcystin OR
particulate-MC OR cylindrospermopsin-content OR intracellular-CYN OR quota OR cell-
content OR cellular-concentration OR cyanotoxin-content OR intracellular-content OR
intracellular-concentration OR toxin-content OR microcystin-content OR microcystin-LR-
content OR MC-content OR MCYST-content OR MC-LR-content OR intracellular-
microcystin OR intracellular-MC OR microcystin-production OR microcystin-LR-production
OR microcystins-production OR MC-production OR MCYST-production OR MC-LR-
production OR CYN-content OR particulate-CYN OR cylindrospermopsin-production)
Limits
AND
Research Areas: (AGRICULTURE OR OCEANOGRAPHY OR ENVIRONMENTAL
SCIENCES ECOLOGY OR PHARMACOLOGY PHARMACY OR EVOLUTIONARY
BIOLOGY OR BIOCHEMISTRY MOLECULAR BIOLOGY OR FISHERIES OR PLANT
SCIENCES OR BIODIVERSITY CONSERVATION OR PUBLIC ENVIRONMENTAL
OCCUPATIONAL HEALTH OR RESEARCH EXPERIMENTAL MEDICINE OR
BIOTECHNOLOGY APPLIED MICROBIOLOGY OR SCIENCE TECHNOLOGY OTHER
TOPICS OR CELL BIOLOGY OR CHEMISTRY OR LIFE SCIENCES BIOMEDICINE
OTHER TOPICS OR TOXICOLOGY OR MARINE FRESHWATER BIOLOGY OR
WATER RESOURCES OR METEOROLOGY ATMOSPHERIC SCIENCES OR ZOOLOGY
OR MICROBIOLOGY)
The EPA conducted title and abstract screening of the 253 search results (generated from both database
searches) and classified them as "relevant," "maybe relevant," or "not relevant." Titles were considered
"relevant" if the title or abstract included mention of cell quota data for microcystins or
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-2

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cylindrospermopsin or if the title or abstract indicated that the study had quantitative information on
cyanobacterial cell density and microcystins or cylindrospermopsin concentration and therefore may
contain sufficient data to calculate a quota. Titles were considered "maybe relevant" if the title or
abstract indicated the article might have information relevant to the research question. Title and abstract
did not specifically include the term "quota" but indicated that it may have had quantitative information
on cyanobacterial cell density and microcystins or cylindrospermopsin concentration or if cyanobacterial
cells were only quantified by molecular methods such as PCR and toxin concentrations were measured.
Titles were considered "not relevant" if the title/abstract did not appear to have information about
microcystins or cylindrospermopsin quotas or densities/concentrations, if the study was a spiked
cyanotoxin experiment (meaning cyanotoxins were added, not produced by cyanobacteria present), or if
the study was not a peer-reviewed article, book, or government document.
The EPA prioritized the studies to facilitate the review. Prioritization yielded a high number of studies
classified as "relevant" or "maybe relevant." Relevant studies were further prioritized for each
cyanotoxin of interest based on date of publication. The approach for prioritization is presented in Table
G-2. A full text review was conducted on Priority 1 studies only.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-3

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Table G-2. Summary of Study Prioritization
Toxin
Priority 1 Classification
Criteria
Priority 2 Classification Criteria
Priority 3 Classification
Criteria
Microcystins
Classified as relevant based
on title/abstract screening;
Did not use PCR
quantification or evaluate
benthic cyanobacteria;
Identified predominant
species without statistical
analysis;
Published in last 5 years; and
Field study or study with
both field and laboratory
component.
Studies that use only PCR for
quantification of cyanobacteria; and
All laboratory studies (internal or
external forcing, mitigation studies,
studies evaluating non-nutrient
pollutants).
Methods studies; and
Studies on benthic
cyanobacteria.
Cylindrospermopsin
Classified as relevant based
on title/abstract screening;
Did not use PCR
quantification or evaluate
benthic cyanobacteria;
Identified predominant
species without statistical
analysis;
Published in last 10 years;
and
Field study or laboratory
study.
Studies that use only PCR for
quantification of cyanobacteria.
Methods studies; and
Studies on benthic
cyanobacteria.
Extracted data from studies meeting the criteria for "Priority 1" are presented below in Table G-3 and
are further summarized in Table G-4. Relevant quota data were extracted from both the text and figures
in "Priority 1" studies. All figures were digitized using GraphPad Digitizer software, as appropriate. All
extracted data from text and figures underwent primary and secondary review for quality assurance
purposes.
The EPA's primary interest when reviewing the data was to identify the amount of toxin per
cyanobacterial cell when toxin was present in a sample. In the environment, it is possible for
cyanobacterial cells to be present with no toxin being produced (e.g., the cyanobacteria are a non-toxin-
producing strain or environmental conditions do not support toxin production). The EPA only included
quota data where toxin was detected.
The studies included in Table G-3 vary in methods used, conditions evaluated, and presentation of data.
Typically, complete, raw data were unavailable. The EPA made choices regarding selection,
presentation, aggregation, and conversion of data to develop the necessary standardization required for
comparing and analyzing these data. Specifically, if quota values were from the same sample at a single
location, the average and range were recorded; results from different sampling locations were recorded
separately; and multiple mean quota values within the same study were recorded separately (note that
separate mean values could be reported for different sampling sites or species within the same genera).
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-4

-------
The EPA found that study authors report toxin quota data in various forms, including but not limited to
toxin mass per cyanobacterial cell, toxin mass per cyanobacterial biomass, and toxin mass per
cyanobacterial biovolume. Scientific measurement units vary among studies. The EPA presents the cell
quota data in Table G-3 in the units reported by the study authors (i.e., without conversion to standard
units). However, when possible, the EPA converted data to a standard set of units, picograms (pg) per
cell, in Table G-4 so that data could be summarized and compared. The EPA did not identify appropriate
conversion factors that would allow genus-specific conversion of quotas described in mass per
biovolume to mass per cell or mass per biomass to mass per cell. The EPA considered converting
biovolume quotas using methods cited in the Australian national guidelines (Australian Government
National Health and Medical Research Council, 2008) and Ackaalan (2006), but ultimately decided that
the number of uncertainties associated with these methods were too great. Thus, data with unique units
are summarized separately in Table G-3, Table G-4, and Table 7-14.
Within Table G-3 and Table G-4, the EPA categorized studies as either "field" or "lab." Field studies
include studies where environmental samples were collected and analyzed for cell quota data without
additional manipulation of growing conditions. In some studies, environmental samples were taken to a
laboratory where growing conditions were optimized or manipulated to determine cyanotoxin cell quota.
These studies were categorized as laboratory studies. Other laboratory studies analyzed cell quota in
laboratory strains that were not collected in the environment for the purpose of the analysis. For
laboratory studies, only control data were extracted. In laboratory studies where there was no true
control the conditions closest to ambient conditions were selected (e.g., multiple conditions were tested
and none was the clear control, all data were included).
While the traditional definition of toxin quota refers to the intracellular amount of toxin, some studies
presented the total toxin present normalized by the cell density or the extracellular toxin normalized by
cell density as a quota. In other cases, methods for calculation of the quota were not very clear. If a
quota value was presented (i.e., intracellular toxin per cell) this was recorded. If this value was not
available or was not clearly described, was recorded as presented by the study authors and assumed to be
intracellular or the total amount of toxin per cell. Extracellular toxin per cell was not recorded. The EPA
recognizes that the exclusion of extracellular toxin data could lead to an underestimation of the amount
of toxin per cell, in particular for cylindrospermopsin as Cylindrospermopsis has been shown to
constitutively produce the toxin, which can stay inside the cells during log phase growth and accumulate
externally upon entering the stationary phase (Davis et al. 2014; Burford et al. 2016). Researchers have
also demonstrated that cylindrospermopsin production can be excreted in response to phosphorus
limitation and induce other cells to excrete alkaline phosphatase to the water body resulting in a
phosphorus scavenging effect (Bar-Yosef et al. 2010).
Some field studies identified the presence of cyanotoxins and multiple cyanobacterial genera including
more than one potential toxin producer with no clear predominant toxin-producing species. Table G-3
only includes cell quota values from field studies where there was a clear predominant toxin-producing
genera. In these instances, the study was grouped with the predominant toxin-producing genera. In
mixed samples with multiple cyanobacteria and no predominant toxin-producing species, quota data
were not included. The EPA recognizes that this approach presents a possible limitation to conclusions
on toxin quota as studies conducted under non-bloom conditions were excluded. Predominant species
are easier to identify when there is a bloom, however, traditional microscopic identification of
cyanobacteria does not distinguish between toxigenic and non-toxigenic strains. The proportion of
toxigenic cells within a cyanobacterial community and the copy number of the mcyD gene per cell can
vary significantly, both affected by environmental parameters (Davis et al. 2009).
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-5

-------
Table G-3 includes cell quota data for microcystin and cylindrospermopsin-producing genera. For each
study, data are provided, where available, on the genus and species of the cyanobacteria, the site where
the sample was collected or the clone used to estimate cellular toxin for, the type of study (i.e., field or
laboratory), and the reported toxin quota data. Notes relevant to each study are reported in the final
column of the table, when appropriate.
Relevant toxin data include the mean toxin quota per cell, the median toxin quota per cell, the minimum
toxin quota per cell, or the maximum toxin quota per cell. These data are reported where available and
not all data points were reported in each study. Data are presented using the units of measure reported by
the study authors.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-6

-------
Table G-3. Cell Quota Data for Microcystin and Cylindrospermopsin-Producing Genera
Toxin
Genus/Species"
Site/Clone
Study Tvpcb
Toxin Quota Data'
Reference
Notes
Microcystin
Microcystis spp.
Grangent Reservoir,
France
Field
Mean: 0.576 pg/cell
Min: 0.042 pg/cell
Max: 4.19 pg/cell
Sabart et al. (2013)
Data digitized from Figure
6b; The authors report cell
quotas for different size
ranges of Microcystis
aeruginosa cells and these
values represent the
minimum and maximum for
all sizes; Mean calculated
using all cell quota data
reported at all time points for
all sizes; Study provides
highest reported mean for
Microcystis spp. mass per
cell, field and field and lab
combined

Microcystis spp.
Lake Victoria, Kenya
Field
Mean: 17 fg/cell
Median: 553 fg/cell
Sitoki et al. (2012)
Sixteen Microcystis strains
identified

Microcystis spp.
Lake Taihu, China
Field
Mean: 0.015 pg/cell
Min: 0 pg/cell
Max: 0.159 pg/cell
Wang et al. (2013)
Data digitized from Figure
4a,b; Mean calculated using
all cell quota data reported at
all time points for all colony
sizes; Study provides
minimum cell quota value
and lowest reported mean
for Microcystis spp. mass
per cell, field, and field and
lab combined

Microcystis spp.
Dapugang River,
Lake Taihu, China
Field
Cell quota data not
presented
Xue et al. (2016)


Microcystis spp.
Umia River, Galicia,
Spain
Field
Max: 570 jxg/g
biomass
Alvarez et al. (2016)
Mixed bloom: Microcystis
aeruginosa, Scenedesmus
spp., Kirchneriella spp.;
unclear which is
predominant

Microcystis spp.
Lake Taihu, China
Field
Mean: 640.59 |ig/g
biomass
Wei et al. (2016)
Data digitized from Figure
4a,b; Only microcystin-L-R
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-7

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes




Min: 13.21 |ig/g
biomass
Max: 1389.13 jxg/g
biomass

congener reported; Mean
calculated using cell quota
data for all time points;
Study provides mean,
minimum, and maximum
cell quota values for
Microcystis spp. mass per
biomass, field

Microcystis spp.
FACHB-905
Lab
Mean: 20.25 fg/cell
Min: 17.05 fg/cell
Max: 28.47 fg/cell
Wei etal. (2016)
Data digitized from Figure
ID and Figure 2D; Mean
calculated using cell quota
data for all time points

Microcystis
aeruginosa
Lake Huron, United
States
Field
Mean: 140 fg/cell
Mix: 10 fg/cell
Max: 350 fg/cell
Fahnenstiel et al.
(2008)
Study provides highest
reported mean, maximum,
and minimum cell quota
values for Microcystis
aeruginosa mass per cell,
field, and field and lab
combined

Microcystis
aeruginosa
Aguieira reservoir,
Portugal
Field
Mean: 0.12 fg/cell
Mix: 0.07 fg/cell
Max: 0.22 fg/cell
Vasconcelos et al.
(2011)
Data digitized from Figure
5; Microcystis aeruginosa
was dominant microcystins
producer; Mean calculated
using all cell quota data for
all yearly time points

Microcystis
aeruginosa
Lake Erie, United
States
Field
Mean: 3.34 (ig/mg
biomass
Min: 1.37 (ig/mg
biomass
Horst et al. (2014)
Data digitized from Figure 3
and Figure 6; Study provides
mean and maximum cell
quota value for Microcystis
aeruginosa mass per
biomass, field

Microcystis
aeruginosa
Hartbeespoort Dam,
South Africa
Field
Min: 0.14 |ig/g
biomass
Max: 268 jxg/g
biomass
Mbukwa and Mamba
(2012)
Study provides minimum
cell quota value for
Microcystis aeruginosa mass
per biomass, field

Microcystis
aeruginosa
BCCUSP232
Lab
Mean: 18.84 fg/cell
Min: 15.07 fg/cell
Chia et al. (2016)
Data digitized from Figure
4b; Study provides lowest
reported mean for
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-8

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes




Max: 22.61 fg/cell

Microcystis aeruginosa mass
per cell, lab, field and lab
combined and the minimum
cell quota value for mass per
cell, lab

Microcystis
aeruginosa
Model was used to
simulate
cyanobacteria
Lab
Mean: 91.5 fg/cell
Jahnichen et al.
(2001)
Model used cell quota data
reported by Long et al.
(2001), Orr and Jones
(1998), Jahnichen et al.
(2001), and Watanabe et al.
(1989); Study provides
highest reported mean for
Microcystis aeruginosa mass
per cell, lab

Microcystis
aeruginosa
Model was used to
simulate
cyanobacteria
Lab
Min: 18 fg/cell
Max: 23.7 fg/cell
Jahnichen et al.
(2007)
Microcystins cell quota data
reported in the presence of
sodium and potassium,
respectively; Study provides
minimum cell quota value
for Microcystis aeruginosa
mass per cell, lab

Microcystis
aeruginosa
MASH01 non-axenic
Lab
Mean: 84.7 fg/cell
Min: 41.53 fg/cell
Max: 165.89 fg/cell
Orr and Jones (1998)
Data digitized from Figure
5; Mean calculated using
quota data presented for
each treatment

Microcystis
aeruginosa
MASH01-A19
Lab
Mean: 93.92 fg/cell
Min: 46.58 fg/cell
Max: 138.47 fg/cell
Orr and Jones (1998)
Data digitized from Figure
5; Mean calculated using
quota data presented for
each treatment; Study
provides highest reported
mean and maximum cell
quota value for Microcystis
aeruginosa mass per cell, lab

Microcystis
aeruginosa
PCC 7806
Lab
Min: 34.5 fg/cell
Max: 81.4 fg/cell
Wiedner et al. (2003)
Mean quota value not
reported, however data could
be digitized from Figure IB
to calculate a mean
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-9

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes

Microcystis
aeruginosa
Lake Rotura, New
Zealand
Lab
Mean: 0.064 pg/cell
Min: 0.017 pg/cell
Max: 0.134 pg/cell
Wood et al. (2012)
Data digitized from Figure
IB; Mean calculated using
cell quota data from all time
points; Study provides
minimum cell quota value
for Microcystis aeruginosa
mass per cell, lab

Microcystis
aeruginosa
Ontario, Canada
Lab
Min: 40.3 fg/cell
Max: 62.4 fg/cell
Pineda-Mendoza et
al. (2014)
The range of quota data
presented was assumed to be
the minimum and maximum
values

Microcystis
aeruginosa
New Mexico, United
States
Lab
Min: 34.5 fg/cell
Max: 136.3 fg/cell
Pineda-Mendoza et
al. (2014)
The range of quota data
presented was assumed to be
the minimum and maximum
values

Microcystis
aeruginosa
Umia River, Galicia,
Spain
Lab
Mean: 11 |ig/g
biomass
Alvarez et al. (2016)
Study provides lowest mean
and minimum cell quota
value fox Microcystis spp.
mass per cell, field, and field
and lab combined

Microcystis
aeruginosa
Dayet Afourgah lake,
Morocco
Lab
Max: 688.4 (ig/g
biomass
Douma et al. (2017)
Maximum reported as total
microcystins content

Microcystis
aeruginosa
Aguelmam Azigza
lake, Morocco
Lab
Max: 699 jxg/g
biomass
Douma et al. (2017)
Maximum reported as total
microcystins content

Microcystis
aeruginosa
Aguelmam Azigza
lake, Morocco
Lab
Max: 859.6 (ig/g
biomass
Douma et al. (2017)
Maximum reported as total
microcystins content

Microcystis
aeruginosa
Lake Erie, United
States
Lab
Mean: 2.44 (ig/mg
biomass
Horst et al. (2014)
Data digitized from Figure
5; Study provides highest
mean and maximum cell
quota value for Microcystis
aeruginosa mass per
biomass, lab

Microcystis
aeruginosa, M. flos-
aquae, M. novacekii
Cogotas, Spain
Field
Min: 1.2 pg/cell
Max: 4.3 pg/cell
Cires et al. (2013)
Data digitized from Figure
1; Study provides maximum
cell quota value for
Microcystis spp. Mass per
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-10

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes






cell, field, and field and lab
combined

Microcystis
aeruginosa, M. flos-
aquae, M. novacekii
Valmayor, Spain
Field
Min: 3.4 pg/cell
Max: 4.1 pg/cell
Cires et al. (2013)
Data digitized from Figure
1; Study provides maximum
cell quota value for
Microcystis spp. mass per
cell, field, and field and lab
combined

Microcystis
aeruginosa, M. flos-
aquae, M. viridis, M.
wesenbergii
Lake Taihu, China
Field
Mean: 0.027 pg/cell
Min: 0.001 pg/cell
Max: 0.087 pg/cell
Tao et al. (2012)
Data digitized from Figure
2c; Mean calculated using
all cell quota data for all
time points

Fisherella
NQAIF311 from
Queensland,
Australia
Lab
Max: 43 jxg/g
biomass
Cires et al. (2014)
Data digitized from Figure 1

Geitlerinema
Florida, United
States
Field
Min: 0.02 |ig/g
biomass
Max: 0.10 (ig/g
biomass
Gantar et al. (2009)


Geitlerinema
Florida, United
States
Lab
Mean: 0.40 jxg/g
biomass
Min: 0.15 |ig/g
biomass
Max: 0.30 (ig/g
biomass
Gantar et al. (2009)


Leptolyngbya
Florida, United
States
Field
Min: 0 jxg/g biomass
Max: 0.08 (ig/g
biomass
Gantar et al. (2009)


Leptolyngbya
FLKBBD1; Florida,
United States
Lab
Mean: 0.10 jxg/g
biomass
Min: 0.06 |ig/g
biomass
Max: 0.20 (ig/g
biomass
Gantar et al. (2009)


Phormidium
Florida, United
States
Field
Mean: 0.026 jxg/g
biomass
Gantar et al. (2009)

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-ll

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes

Planktothrix spp.
Occhito, Italy
Field
Median:
3.82 (ig/mm3
biovolume
Min: 1.27 (ig/mm3
biovolume
Max: 6.28 (ig/mm3
biovolume
Salmaso et al. (2014)
Data on minimum and
maximum digitized from
Figure 4a

Planktothrix spp.
Pusiano, Italy
Field
Median:
0.59 (ig/mm3
biovolume
Min: 0.37 (ig/mm3
biovolume
Max: 0.87 (ig/mm3
biovolume
Salmaso et al. (2014)
Data on minimum and
maximum digitized from
Figure 4a

Planktothrix spp.
Ledro, Italy
Field
Mean: 0.45 (ig/mm3
biovolume
Min: 0.12 (ig/mm3
biovolume
Max: 0.84 (ig/mm3
biovolume
Salmaso et al. (2014)
Data on minimum and
maximum digitized from
Figure 4a

Planktothrix spp.
Garda, Italy
Field
Mean: 0.31 (ig/mm3
biovolume
Min: 0 (ig/mm3
biovolume
Max: 0.32 (ig/mm3
biovolume
Salmaso et al. (2014)
Data on minimum and
maximum digitized from
Figure 4a

Planktothrix
agardhii
Bassenwaithe Lake,
England
Field
Mean: 91.2 fg/cell
Akcaalan et al. (2006)


Planktothrix
agardhii
NIES 595
Lab
Mean: 75.6 fg/cell
Akcaalan et al. (2006)


Planktothrix
rubescencs
Iznik Lake, Turkey
Field
Mean: 235.6 fg/cell
Akcaalan et al. (2006)
Study provides mean and
maximum cell quota value
for Planktothrix rubescencs
mass per cell, field

Planktothrix
rubescencs
France
Field
Min: 0.13 pg/cell
Max: 0.16 pg/cell
Briand et al. (2008)
Study provides maximum
cell quota value for
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-12

-------
Toxin Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes





Planktothrix rubescencs





mass per cell, field, and lab





and the minimum cell quota





value for mass per cell, field
Planktothrix
SL 03; Turkey
Lab
Mean: 103.9 fg/cell
Akcaalan et al. (2006)
Study provides lowest mean
rubescencs




and minimum cell quota
value for Planktothrix
rubescencs mass per cell,
lab, and field and lab
combined
Planktothrix
rubescencs
Sapanca Lake,
Turkey
Lab
Mean: 108.2 fg/cell
Akcaalan et al. (2006)

Pseudanabaena
Florida, United
States
Field
Min: 0.02 |ig/g
biomass
Max: 0.04 (ig/g
biomass
Gantar et al. (2009)

Spirulina
Florida, United
States
Field
Mean: 0.12 jxg/g
biomass
Gantar et al. (2009)

Synechococcus
Florida, United
States
Field
Min: 0.08 |ig/g
biomass
Max: 0.27 (ig/g
biomass
Gantar et al. (2009)

Multiple genera
including
Microcystis
Kiwah Island pond,
South Carolina
Field

Greenfield et al.
(2014)
Data available but were not
digitized
aeruginosa,
Anabaenopsis





Multiple genera
Lake Victoria,
Field

Mbonde and
Data available but were not
including
Microcystis spp.,
Anabaena spp., and
Tanzania


Kurmayer (2015)
digitized
Planktolyngbya spp.





Microcystis,
Aphanomenizon, and
others
Quebec lakes,
Canada
Field

Monchamp et al.
(2014)
Data available but were not
digitized
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-13

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes

Multiple genera
including
Microcystis and
Anabaena
Anzali wetland, Iran
Field

Rezaitabar et al.
(2017)
Data available but were not
digitized

Multiple genera
including
Microcystis and
Anabaena
Anzali wetland, Iran
Field

Rezaitabar et al.
(2017)
Data available but were not
digitized

Multiple genera
including
Microcystis,
Dolichospermum,
others
Lake Chaohu, China
Field

Shang et al. (2015)
Data available but were not
digitized
Cylindrospermopsin
Aphanizomenon
ovalisporum
Florida, United
States
Lab
Min: 7.39 (ig/mg
biomass
Max: 9.33 (ig/mg
biomass
Yilmaz et al. (2008)


Cylindrospermopsis
raciborskiid
Gazam Dam Lake,
Saudi Arabia
Field
Min: 0.6 pg/cell
Max: 14.6 pg/cell
Mohamed and Al-
Shehri (2013)
Study provides maximum
value for
Cylindrospermopsis
raciborskii mass per cell,
field, and field and lab
combined

Cylindrospermopsis
raciborskii
Queensland,
Australia
Field
Mean: 23.12 fg/cell
Median: 20.5 fg/cell
Min: 5.9 fg/cell
Max: 55.8 fg/cell
Orr et al. (2010)
Study provides minimum
value for
Cylindrospermopsis
raciborskii mass per cell,
field, and field and lab
combined

Cylindrospermopsis
raciborskii
Queensland,
Australia
Field
Median: 20.3 fg/cell
Min: 10 fg/cell
Max: 49.4 fg/cell
Orr et al. (2010)


Cylindrospermopsis
raciborskii
CYP 030A; Australia
Lab
Min: 3.2 ng/106 cell
Max: 5.7 ng/106 cell
Carneiro et al. (2013)


Cylindrospermopsis
raciborskii
CYP 01 IK; Australia
Lab
Min: 12.1 ng/106
cell
Carneiro et al. (2013)

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-14

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data'
Reference
Notes




Max: 24.7 ng/106
cell



Cylindrospermopsis
raciborskii
Queensland,
Australia
Lab
Min: 13.4 fg/cell
Max: 14.9 fg/cell
Davis et al. (2014)


Cylindrospermopsis
raciborskii
New South Wales,
Australia
Lab
Mean: 31 fg/cell
Min: 12 fg/cell
Max: 52 fg/cell
Hawkins et al. (2001)


Cylindrospermopsis
raciborskii
Queensland,
Australia
Lab
Min: 19 fg/cell
Max: 26 fg/cell
Pierangelini et al.
(2015)


Cylindrospermopsis
raciborskii
CS-506; Queensland,
Australia
Lab
Mean: 0.0028
pg/cell
Willis et al. (2015)
Study provides lowest mean
value for
Cylindrospermopsis
raciborskii mass per cell,
lab, and field and lab
combined and minimum cell
quota value for mass per
cell, lab

Cylindrospermopsis
raciborskii
CS-506; Queensland,
Australia
Lab
Mean: 0.018 pg/cell
Willis et al. (2015)


Cylindrospermopsis
raciborskii
Lake Wivenhoe,
Australia
Lab
Mean: 165.75 fg/cell
Willis et al. (2016)
Calculated mean based on
data in Table 1; Study
provides highest mean value
for Cylindrospermopsis
raciborskii mass per cell,
lab, and field and lab
combined and maximum cell
quota value for mass per
cell, lab

Cylindrospermopsis
raciborskii
CHAB3438, China
Lab
Mean: 43.76 fg/cell
Min: 35.89 fg/cell
Max: 52 fg/cell
Yang et al. (2016)
Data digitized from Figure
2; Mean calculated using
quota data presented for
each time point

Cylindrospermopsis
raciborskii
Queensland,
Australia
Lab
Min: 416 fg/|im3
biovolume
Max: 447 fg/(im3
biovolume
Pierangelini et al.
(2015)

Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-15

-------
Toxin
Genus/Species"
Site/Clone
Study Type1*
Toxin Quota Data' Reference
Notes

Multiple genera
including
Aphanizomenon,
Anabaena,
Nostocales, and
Cylindrospermopsis
Germany
Field
Riicker et al. (2007)
Data available but were not
digitized

Multiple genera
including
Aphanizomenon
Langer See,
Germany
Field
Wiedner et al. (2008)
Data available but were not
digitized
Abbreviations: M. = Microcystis', spp. = multiple species in the genus
aBoth the genus and species are reported where available. In some studies, the genus was reported but the species was not reported. In other studies, multiple species
were analyzed within a specific genus but the specific species were not identified. In both instances, studies were categorized as the genus name (e.g., Microcystis) spp.
Separately, in some studies multiple genera were considered. In these studies, available toxin quota data were not digitized as they could not be used for comparison
purposes. Only information about the studies are presented in this table with a note that data are available but were not digitized.
b Studies were conducted in two different settings: the field (i.e., environmental) or a laboratory. In some instances, field samples were subjected to optimized growth
conditions in the laboratory. These studies were classified as laboratory; not as field studies.
0 Toxin cell quota data were not converted and are reported in the measurement units used by the study authors. Significant figures were not normalized among the data
points.
d The genus Cylindrospermopsis has been renamed to Raphidiopsis.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-16

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Table G-4 provides the first step in summarizing and grouping cell quota data for microcystin and
cylindrospermopsin-producing genera. Studies presented in Table G-3 were grouped by genus and
species when possible. Studies that looked at more than one species within a specific genus or that did
not specify which species were considered within that genus were placed in a single group (e.g.,
Microcystis spp., Planktothrix spp.). Within each genus/species group, studies were further grouped
based on their study type and the quantification method used in that study. For each study type and
quantification method group, data were aggregated on the mean, minimum, and maximum cell quota
values presented in each study included in that group. In Table G-4, the range of the means, arithmetic
mean (of the means), median of the means, minimum cell quota value, and maximum cell quota value
are reported for the studies included in that group. Note that studies were not identified in the literature
search for all quantification methods and study types for all genus/species groups. The EPA converted
data to a standard set of units, pg per cell, when possible. No other conversions were attempted.
Additional information about the approach used to summarize the available cell quota data is provided
in the footnotes accompanying the table.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
17

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Table G-4. Cell Quota Appendix Summary Data for Microcystin and Cylindrospermopsin-producing Genera
Toxin
Genus, Species
Quantification
Method"; Study
Type1*
Ranjje of
Meanscd
Meancd
Median of Means' '
Minimum;
Maximum''1
Refcrcnees
Microcystin
Microcystis spp.
Mass per cell;
Field and lab
0.015-0.576
pg/cell
0.13 pg/cell
0.017 pg/cell
0 pg/cell;
4.30 pg/cell
Sitoki et al. (2012);
Tao et al. (2012);
Cires et al. (2013);
Sabart et al. (2013);
Wang et al. (2013);
Wei et al. (2016)
Mass per cell;
Field
0.015-0.576
pg/cell
0.16 pg/cell
0.022 pg/cell
0 pg/cell;
4.30 pg/cell
Sitoki et al. (2012);
Tao et al. (2012);
Cires et al. (2013);
Sabart et al. (2013);
Wang et al. (2013)
Mass per cell; Lab
0.020 pg/cell
0.02 pg/cell
N/A
0.017 pg/cell;
0.028 pg/cell
Wei et al. (2016)
Mass per biomass;
Field
640.59 jxg/g
biomass
640.59 (ig/g
biomass
N/A
13.21 jxg/g;
1389.13 (ig/g
biomass
Alvarez et al.
(2016); Wei etal.
(2016)
Microcystis
aeruginosa
Mass per cell;
Field and lab
0.02-0.14
pg/cell
0.09 pg/cell
0.09 pg/cell
0.01 pg/cell;
0.35 pg/cell
Orr and Jones
(1998); Jahnichenet
al. (2001); Wiedner
et al. (2003);
Jahnichen et al.
(2007); Fahnenstiel
et al. (2008);
Vasconcelos et al.
(2011);	Wood etal.
(2012);	Pineda-
Mendoza et al.
(2014); Chia et al.
(2016)
Mass per cell;
Field
0.12-0.14
pg/cell
0.13 pg/cell
0.13 pg/cell
0.01 pg/cell;
0.35 pg/cell
Fahnenstiel et al.
(2008); Vasconcelos
etal. (2011)
Mass per cell; Lab
0.02-0.09
pg/cell
0.07 pg/cell
0.08 pg/cell
0.02 pg/cell;
0.17 pg/cell
Orr and Jones
(1998); Jahnichenet
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-18

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Toxin
Genus, Species
Quantification
Method"; Study
Type1*
Ranjjc of
Meansc,d
Meancd
Median of Means' '
Minimum;
Maximum'-1
References







al. (2001); Wiedner
et al. (2003);
Jahnichen et al.
(2007); Wood et al.
(2012); Pineda-
Mendoza et al.
(2014); Chia et al.
(2016)
Mass per biomass;
Field
3,340 jxg/g
biomass
3,340 (ig/g
biomass
N/A
0.14 jxg/g
biomass;
3,340 jxg/g
biomass
Mbukwa and
Mamba (2012);
Horst et al. (2014)
Mass per biomass;
Lab
11-2,440
(ig/g biomass
1225.5 (ig/g
biomass
1225.5 jxg/g biomass
ii ng/g;
2,440 jxg/g
biomass
Horst et al. (2014);
Alvarez et al.
(2016);	Douma et al.
(2017)
Fisherella
Mass per biomass;
Lab
N/A
N/A
N/A
43 jxg/g
biomass
Gantar et al. (2009)
Geitlerinema
Mass per biomass;
Field
N/A
N/A
N/A
0.02 jxg/g;
0.10 (ig/g
biomass
Gantar et al. (2009)
Mass per biomass;
Lab
0.40 jxg/g
biomass
0.40 (ig/g biomass
N/A
0.15 jxg/g;
0.40 (ig/g
biomass
Gantar et al. (2009)
Leptolyngbya
Mass per biomass;
Field
N/A
N/A
N/A
o ng/g;
0.08 jxg/g
biomass
Gantar et al. (2009)
Mass per biomass;
Lab
0.10 jxg/g
biomass
0.10 (ig/g biomass
N/A
0.06 jxg/g;
0.20 jxg/g
biomass
Gantar et al. (2009)
Phormidium
Mass per biomass;
Lab
0.026 jxg/g
biomass
0.026 (ig/g
biomass
N/A
0.026 jxg/g
biomass
Gantar et al. (2009)
Planktothrix spp.
Mass per
biovolume; Field
N/A
N/A
N/A
0 (ig/mm3;
6.28 (ig/mm3
biomass
Salmaso et al.
(2014)
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-19

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Toxin
Genus, Species
Quantification
Method"; Study
Type1*
Ranjjc of
Meansc,d
Meancd
Median of Means' '
Minimum;
Maximum'-1
References

Planktothrix
agardhii
Mass per cell;
Field and lab
0.076-0.091
pg/cell
0.083 pg/cell
0.083 pg/cell
0.076 pg/cell;
0.091 pg/cell
Akcaalan et al.
(2006)


Mass per cell;
Field
0.091 pg/cell
0.091 pg/cell
N/A
0.091 pg/cell
Akcaalan et al.
(2006)


Mass per cell; Lab
0.076 pg/cell
0.076 pg/cell
N/A
0.076 pg/cell
Akcaalan et al.
(2006)

Planktothrix
rubescencs
Mass per cell;
Field and lab
0.104-0.236
pg/cell
0.149 pg/cell
.108 pg/cell
0.104 pg/cell;
0.16 pg/cell
Akcaalan et al.
(2006); Briand et al.
(2008)


Mass per cell;
Field
0.236 pg/cell
0.236 pg/cell
N/A
0.13 pg/cell;
0.236 pg/cell
Akcaalan et al.
(2006); Briand et al.
(2008)


Mass per cell; Lab
0.104-0.108
pg/cell
0.106 pg/cell
0.106 pg/cell
0.104 pg/cell;
0.108 pg/cell
Akcaalan et al.
(2006)

Pseudanabaena
Mass per biomass;
Field
N/A
N/A
N/A
0.02 jxg/g;
0.04 (ig/g
biomass
Gantar et al. (2009)

Spirulina
Mass per biomass;
Field
0.12 jxg/g
biomass
0.12 (ig/g biomass
N/A
0.12 jxg/g
biomass
Gantar et al. (2009)

Synechococcus
Mass per biomass;
Field
N/A
N/A
N/A
0.08 jxg/g;
0.27 jxg/g
biomass
Gantar et al. (2009)
Cylindrospermopsin
Aphanizomenon
ovalisporum
Mass per biomass;
Field and lab
N/A
N/A
N/A
7.39 jxg/g;
9.33 (ig/mg
biomass
Yilmaz et al. (2008)

Cylindrospermop
sis raciborskiig
Mass per cell;
Field and lab
0.0028-0.17
pg/cell
0.05 pg/cell
0.03 pg/cell
0.006 pg/cell;
14.6 pg/cell
Orr et al. (2010);
Mohamed and Al-
Shehri (2013);
Pierangelini et al.
(2015); Willis etal.
(2015); Yang et al.
(2016a)
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-20

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Toxin
Genus, Species
Quantification
Method"; Study
Type1*
Ranjjc of
Meansc,d
Meancd
Median of Means' '
Minimum;
Maximum'-1
References


Mass per cell;
Field
0.023 pg/cell
0.023 pg/cell
N/A
0.006 pg/cell;
14.6 pg/cell
Orretal. (2010);
Mohamed and Al-
Shehri (2013)


Mass per cell; Lab
0.0028-0.17
pg/cell
0.057 pg/cell
0.031
0.0028
pg/cell; 0.17
pg/cell
Hawkins et al.
(2001); Carneiro et
al. (2013); Davis et
al. (2014);
Pierangelini et al.
(2015); Willis etal.
(2015);	Willis etal.
(2016);	Yang et al.
(2016)


Mass per
biovolume; Lab
N/A
N/A
N/A
416 fg/(im3;
447 fg/(im3
Pierangelini et al.
(2015)
Acronyms and Abbreviations: fg = femtogram; pg = picogram; |ig = microgram; N/A = not applicable.
a Various methods were used to quantify toxin quotas and quota values were presented in different forms, including toxin mass per cyanobacterial cell and toxin mass
per cyanobacterial biomass.
b Studies were conducted in two different settings: the field (i.e., environmental) or a laboratory. In some instances, field samples were subjected to optimized growth
conditions in the laboratory. These studies were classified as laboratory; not field.
0 Study authors reported data using multiple measurement units. When possible, the EPA converted data to the standard units of pg per cell. The EPA did not identify
appropriate conversion factors that would allow genus-specific conversion of quotas described in mass per biomass to mass per cell.
d Shows single reported mean if only one study was available or average of reported means.
e Median of means not calculated if only one mean value was available or if only minimum and/or maximum cell quota values were available.
f If reported toxin quota means from one study were the lowest or highest toxin quotas reported within a genus, then these values were listed as the minimum or
maximum values, respectively, to better reflect the range of toxin quota values.
g The genus Gylindrospermopsis has recently been renamed to Raphidiopsis.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-21

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Appendix G References
Akcaalan R, Young FM, Metcalf JS, Morrison LF, Albay M, and Codd GA (2006). Microcystin analysis
in single filaments of Planktothrix spp. in laboratory cultures and environmental blooms. Water
Res, 40(S), 1583-1590.
Alvarez X, Valero E, Cancela A, and Sanchez A (2016). Freshwater algae competition and correlation
between their growth and microcystin production. Environ Sci Pollut Res Int, 23(21), 21577-
21583.
Australian Government National Health and Medical Research Council (2008). Guidelines for
Managing Risk in Recreational Water, https://nhmrc.gov.au/about-us/publications/guidelines-
m an agin g~ri sks-recreati on al - water. Last Accessed: 11/27/2018.
Bar-Yosef Y, Sukenik A, Hadas O, Viner-Mozzini Y, and Kaplan A (2010). Enslavement in the water
body by toxic Aphanizomenon ovalisporum, inducing alkaline phosphatase in phytoplanktons.
CurrBiol, 20(17), 1557-1561. http://www.ncbi.nlm.nih.eov/piibmed/20705465.
Briand E, Gugger M, Francis J-C, Bernard C, Humbert J-F, and Quiblier C (2008). Temporal variations
in the dynamics of potentially microcystin-producing strains in a bloom-forming Planktothrix
agardhii (cyanobacterium) population. ApplEnviron Microbiol, 74(12), 3839-3848.
Burford MA, Beardall J, Willis A, Orr PT, Magalhaes VF, Rangel LM, Azevedo SMFOE, Neilan BA
(2016). Understanding the winning strategies used by the bloom-forming cyanobacterium
Cylindrospermopsis raciborskii. Harmful Algae, 54, 44-53.
Carneiro RL, Pereira Ribeiro da Silva A, and Freitas de Magalhaes V (2013). Use of the cell quota and
chlorophyll content for normalization of cylindrospermopsin produced by two
Cylindrospermopsis raciborskii strains grown under different light intensities. Ecotoxicol
Environ Contam, 5(1), 93-100.
Chia MA, Cordeiro-Araujo MK, Lorenzi AS, and Bittencourt-Oliveira MDC (2016). Does anatoxin-a
influence the physiology of Microcystis aeruginosa and Acutodesmus acuminatus under different
light and nitrogen conditions. Environ Sci Pollut Res Int, 23(22), 23092-23102.
Cires S, Wormer L, Carrasco D, and Quesada A (2013). Sedimentation patterns of toxin-producing
Microcystis morphospecies in freshwater reservoirs. Toxins, 5(5), 939-957.
Cires S, Alvarez-Roa C, Wood SA, Puddick J, Loza V, and Heimann K (2014). First report of
microcystin-producing Fischerella sp. (Stigonematales, Cyanobacteria) in tropical Australia.
Toxicon, 88, 62-66.
Davis TW, Berry DL, Boyer GL, and Gobler CJ (2009). The effects of temperature and nutrients on the
growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms.
Harmful Algae, 8, 715-725.
Davis TW, Orr PT, Boyer GL, Burford MA (2014). Investigating the production and release of
cylindrospermopsin and deoxy-cylindrospermopsin by Cylindrospermopsis raciborskii over a
natural growth cycle. Harmful Algae, 31, 18-25.
Douma M, Ouahid Y, Loudiki M, Del Campo FF, and Oudra B (2017). The first detection of potentially
toxic Microcystis strains in two Middle Atlas Mountains natural lakes (Morocco). Environ Monit
Assess, 189(1), 39.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
G-22

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Fahnenstiel GL, Millie DF, Dyble J, Litaker RW, Tester PA, McCormick MJ, Rediske R, and Klarer D
(2008). Microcystin concentrations and cell quotas in Saginaw Bay, Lake Huron. Aquat Ecosyst
Health Manag, 11(2), 190-195.
Gantar M, Sekar R, and Richardson LL (2009). Cyanotoxins from black band disease of corals and from
other coral reef environments. Microb Ecol, 55(4), 856-864.
Greenfield DI, Duquette A, Goodson A, Keppler CJ, Williams SH, Brock LM, Stackley KD, White D,
and Wilde SB (2014). The effects of three chemical algaecides on cell numbers and toxin content
of the cyanobacteri a Microcystis aeruginosa and Anabaenopsis sp. Environ Manage, 54(5),
1110-1120.
Hawkins PR, Putt E, Falconer I, and Humpage A (2001). Phenotypical variation in a toxic strain of the
phytoplankter, Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) during batch culture.
Environ Toxicol, 16, 460-467.
Horst GP, Sarnelle O, White JD, Hamilton SK, Kaul RB, and Bressie JD (2014). Nitrogen availability
increases the toxin quota of a harmful cyanobacterium, Microcystis aeruginosa. Water Res, 54,
188-198.
Jahnichen S, Ihle T, Petzoldt T, and Benndorf J (2007). Impact of inorganic carbon availability on
microcystin production by Microcystis aeruginosa PCC 7806. Appl Environ Microbiol, 73(21),
6994-7002.
Jahnichen S, Petzoldt T, and Benndorf J (2001). Evidence for control of microcystin dynamics in
Bautzen Reservoir (Germany) by cyanobacterial population growth rates and dissolved inorganic
carbon. Arch Hydrobiol, 150(2), 177-196.
Mbonde AS, Sitoki L, and Kurmayer R (2015). Phytoplankton composition and microcystin
concentrations in open and closed bays of Lake Victoria, Tanzania. Aquat Ecosyst Health
Manag, 18(2), 212-220.
Mbukwa EA, M TA, and Mamba BB (2012). Quantitative variations of intracellular microcystin-LR, -
RR and -YR in samples collected from four locations in Hartbeespoort Dam in North West
Province (South Africa) during the 2010/2011 summer season. Int J Environ Res Public Health,
9, 3484-3505.
Mohamed ZA and Al-Shehri AM (2013). Assessment of cylindrospermopsin toxin in an arid Saudi lake
containing dense cyanobacterial bloom. Envion Monit Assess, 185, 2157-2166.
Monchamp ME, Pick FR, Beisner BE, and Maranger R (2014). Nitrogen forms influence microcystin
concentration and composition via changes in cyanobacterial community structure. PloS One,
9(1), e85573.
Orr PT, Rasmussen JP, Burford MA, and Eaglesham GK (2010). Evaluation of quantitative real-time
PCRto characterise spatial and temporal variations in cyanobacteria, Cylindrospermopsis
raciborskii (Woloszynska) Seenaya et Subba Raju and cylindrospermopsin concentrations in
three subtropical Australian reservoirs. Harmful Algae, 9, 243-254.
Orr PT and Jones GJ (1998). Relationship between microcystin production and cell division rates in
nitrogen-limited Microcystis aeruginosa cultures. Limno! Oceanogr, 43(1), 1604-1614.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
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Pierangelini M, Sinha R, Willis A, Burford MA, Orr PT, Beardall J, and Neilan BA (2015). Constitutive
cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2
partial pressure conditions. Appl Environ Microbiol, 81(9), 3069-3076.
Pineda-Mendoza RM, Zuniga G, and Martinez-Jeronimo F (2014). Microcystin production in
Microcystis aeruginosa: Effect of type of strain, environmental factors, nutrient concentrations,
and N:P ratio on mcyA gene expression. Aquat Ecol, 50(1), 103-119.
Rezaitabar S, Esmaili SA, BahramifarN, and RamezanpourZ (2017). Transfer, tissue distribution and
bioaccumulation of microcystin LR in the phytoplanktivorous and carnivorous fish in Anzali
wetland, with potential health risks to humans. Sci Total Environ, 575, 1130-1138.
Riicker Jl, Stiiken A, Nixdorf B, Fastner J, Chorus I, Wiedner C. (2007). Concentrations of particulate
and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes.
Toxicon. 50(6), 800-809.
Sabart M, Misson B, Descroix A, Duffaud E, Combourieu B, Salencon MJ, and Latour D (2013). The
importance of small colonies in sustaining Microcystis population exposed to mixing conditions:
An exploration through colony size, genotypic composition and toxic potential. Environ
Microbiol Rep, 5(5), 747-756.
Salmaso N, Copetti D, Cerasino L, Shams S, Capelli C, Boscaini A, Valsecchi L, Pozzoni F, and
Guzzella L (2014). Variability of microcystin cell quota in metapopulations of Planktothrix
rubescens: Causes and implications for water management. Toxicon, 90, 82-96.
Shang L, Feng M, Liu F, Xu X, Ke F, Chen X, and Li W (2015). The establishment of preliminary
safety threshold values for cyanobacteria based on periodic variations in different microcystin
congeners in Lake Chaohu, China. Environ Sci Process Impacts, 17(4), 728-739.
Sitoki L, Kurmayer R, and Rott E (2012). Spatial variation of phytoplankton composition, biovolume,
and resulting microcystin concentrations in the Nyanza Gulf (Lake Victoria, Kenya).
Hydrobiologia, 691(1), 109-122.
Tao M, Xie P, Chen J, Qin B, Zhang D, Niu Y, Zhang M, Wang Q, and Wu L (2012). Use of a
generalized additive model to investigate key abiotic factors affecting microcystin cellular quotas
in heavy bloom areas of Lake Taihu. PloS One, 7, e32020.
Vasconcelos V, Morais J, and Vale M (2011). Microcystins and cyanobacteria trends in a 14 year
monitoring of a temperate eutrophic reservoir (Aguieira, Portugal). J Environ Monit, 13(3), 668-
672.
Wang L, Liu L, and Zheng B (2013). Eutrophication development and its key regulating factors in a
water-supply reservoir in North China. J Environ Sci (China), 25(5), 962-970.
Wei N, Hu L, Song L, and Gan N (2016). Microcystin-Bound Protein Patterns in Different Cultures of
Microcystis aeruginosa and Field Samples. Toxins, 5(10), 293.
Wiedner C, Visser PM, Fastner J, Metcalf JS, Codd GA, and Mur LR (2003). Effects of light on the
microcystin content of Microcystis strain PCC 7806. Appl Environ Microbiol, 69(3), 1475-1481.
Willis A, Adams MP, Chuang AW, Orr PT, O'Brien KR, and Burford MA (2015). Constitutive toxin
production under various nitrogen and phosphorus regimes of three ecotypes of
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Swimming Advisories for Microcystins and Cylindrospermopsin
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Cylindrospermopsis raciborskii (Wotoszynska) Seenayya et SubbaRaju). Harmful Algae, 47,
27-34.
Willis A, Chuang, AW, Woodhouse JN, Neilan BA, and Burford MA (2016). Intraspecific variation in
growth, morphology and toxin quotas for the cyanobacterium, Cylindrospermopsis raciborskii.
Toxicon, 119, 307-310.
Wood SA, Dietrich DR, Cary SC, and Hamilton DP (2012). Increasing Microcystis cell density
enhances microcystin synthesis: A mesocosm study. Inland Waters, 2(1), 17-22.
Xue Q, Steinman AD, Su X, Zhao Y, and Xie L (2016). Temporal dynamics of microcystins in
Limnodrilus hoffmeisteri, a dominant oligochaete of hypereutrophic Lake Taihu, China. Environ
Pollut, 213, 585-593.
Yang Y, Jiang Y, Li X, Li H, Chen Y, Xie J, Fangfang C, and Li R (2016a). Variations of growth and
toxin yield in Cylindrospermopsis raciborskii under different phosphorus concentrations. Toxins,
9(1), 13.
Yilmaz M, Philips EJ, Szabo NJ, and Badylak S (2008). A comparative study of Florida strains of
Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production. Toxicon,
57(1), 130-139.
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Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin

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APPENDIX H. TABLES OF STATE-ISSUED GUIDELINES SPECIFIC TO ANIMAL
CYANOTOXIN POISONING
H.l California
Table H-l. California Environmental Protection Agency (2012) Action levels for Selected Pet and
Livestock Scenarios

Microcystins"
Cylindrospermopsin
Media (units)
Subchronic water intake, dog1'
2
10
water (|ig/L)
Subchronic crust and mat intake, dog
0.01
0.04
crusts and mats (mg/kg dw)°
Acute water intake, dog1'
100
200
water (|ig/L)
Acute crust and mat intake, dog
0.5
0.5
crusts and mats (mg/kg dw)°
Subchronic water intake, cattle"
0.9
5
water (|ig/L)
Subchronic crust and mat intake,
cattle"
0.1
0.4
crusts and mats (mg/kg dw)°
Acute water intake, cattle"
50
60
water (|ig/L)
Acute crusl and mat intake, cattle"
5
5
crusts and mats (mg/kg dw)°
aMicrocystins LA, LR, RR, and YR all had the same RfD so the action levels are the same.
b Subchronic refers to exposures over multiple days.
0 Based on sample dry weight (dw).
d Acute refers to exposures in a single day.
e Based on small breed dairy cows because their potential exposure to cyanotoxins is greatest.
Table H-2. California Environmental Protection Agency (2012) Reference Doses and
Acute and Subchronic Action Levels for Canine Exposure to Cyanotoxins in Drinking
Water

Microcystin
Cylindrospermopsin
Water consumption L/kg-day
0.085
0.085
Uncertainly factor (unitlcss)
3
3
Acute RfD'1 mg/kg/dav
0.037
0.04
Acute action level jig/L
100
200
Subchronic RfD mg/kg/dav
0.00064
0.0033
Subchronic action level jig/L
2
10
Reference:
Butler N, Carlisle J, Kaley KB, and Linville R (2012). Toxicological Summary and Suggested Action
Levels to Reduce Potential Adverse Health Effects of Six Cyanotoxins.
http://www.waterboards.ca.eov/water issues/programs/peer review/docs/califcvanotoxins/cvan
otoximsO	. Last Accessed: 11/27/2018.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
H-l

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H.2 Indiana
Indiana has adopted guidance for cyanotoxins for dog exposures:
"A warning to dog owners using the Fort Harrison State Park Dog Park Lake will occur whenever any
cyanotoxins are detected, and dogs will be prohibited from swimming at the values of 0.8 [j,g/L
microcystin, any anatoxin-a detection, and 1.0 [j,g/L of cylindrospermopsin."
Reference:
Indiana Department of Environmental Management (2018). Blue-Green Algae: Indiana Reservoir and
Lake Update.http://www.in.gov/idem/algae/. Last Accessed: 02/27/2018.
H.3 Oregon
Table H-3. Oregon Dog-specific Guideline Values for Cyanotoxins in
Recreational Waters (jig/L)

Microcystin
Cylindrospermopsin
Dog Guidance Value
0.2
0.4
Note: All dog-specific guideline values have been changed in this revision because California EPA's estimate of the
amount of water an exercising dog consumes per kilogram body weight was updated in 2012 (from 0.168 to 0.255 L/kg-
day). Current dog-specific guideline values are now consistent with the California EPA update. The dog-specific value for
saxitoxins was further modified by application of an uncertainty factor to the dog-specific TDI for interspecies differences
in sensitivity between humans (the species in the critical study) and dogs.
Reference:
Oregon Health Authority (2018). Oregon Harmful Algae Bloom Surveillance (HABS) Program Public
Health Advisory Guidelines: Harmful Algae Blooms in Freshwater Bodies.
https://www.oregon.gov/oha/ph/HealthvEnvironments/Recreation/HarmfulAlgaeBlooms/Docum
etrts/ iblicHealthAdvisorvGuidelines.pdf.
H.4 Grayson County, Texas
Table H-4. Grayson County, Texas Microcystin Guidelines for Dogs
Quantity of Lake Water Ingested to Receive a Potentially Lethal Dose of Microcystin, Assuming that
Mouse and Dog Toxic Responses are Equivalent

Gallons of Water
Pounds of Water
10-pound dog
2.70
22.50
80-pound dog
21.57
180.00
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
H-2

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Quantity of Lake Water Ingested to Receive a Potentially Lethal Dose of Microcystin, Assuming that
Mouse and Dog Toxic Responses are Equivalent (at actual concentrations found in Grand Lake,
Oklahoma, in June 2011) Highest measured microcystin concentration was 358 ppb.

Gallons of Water
Pou nds of Water
10-pound dog
0.15 (19.3 ounces)
1.26
80-pound dog
1.21
10.06
*This is not including additional dose amounts that could be ingested from a dog self-grooming algae scum off its fur.
**LD5o for microcystin-mouse used in calculations = 45 ng/kg
***20 ppb microcystin is algal toxin threshold for BGA Warning (condition red)
Quantity of Lake Water Ingested to Receive a Potentially Lethal Dose of Cylindrospermopsin,
Assuming that Mouse and Dog Toxic Responses are Equivalent 20 ppb Cylindrospermopsin in Lake
Water

Gallons of Water
Pounds of Water
10-pound dog
263
2,200
80-pound dog
2,109
17,601
*This is not including additional dose amounts that could be ingested from a dog self-grooming algae scum off its fur.
**LD5o for cylindrospermopsin-mouse used in calculations = 4400 |ig/kg
***20 ppb cylindrospermopsin is algal toxin threshold for BGA Warning (condition red)
Reference:
Lillis J, Ortez A, and Teel JH (2012). Blue-Green Algae Response Strategy. Sherman, Texas.
http://www.co.erayson.tx.iis/iipload/paee/0206/docs/Bliie-GTei ae Response Strateev.pdf.
Last Accessed: 12/5/2018.
Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin
H-3

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