SEPA
         United States   Office of Water    EPA- 820R15101
         Environmental   Mail Code 4304T   June 2015
         Protection Agency
Drinking Water Health Advisory
 for the Cyanobacterial Toxin
      Cylindrospermopsin

-------
         Drinking Water Health Advisory
for the Cyanobacterial Toxin Cylindrospermopsin
                      Prepared by:

            U.S. Environmental Protection Agency
                 Office of Water (4304T)
            Health and Ecological Criteria Division
                 Washington, DC 20460
             EPA Document Number: 820R15101
                   Date: June 15, 2015

-------
                              ACKNOWLEDGMENTS

This document was prepared by U.S. EPA Scientists Lesley V. D'Anglada, Dr.P.H. (lead) and
Jamie Strong, Ph.D. Health and Ecological Criteria Division, Office of Science and Technology,
Office of Water. EPA gratefully acknowledges the valuable contributions from Health Canada's
Water  and  Air  Quality Bureau,  in  developing the  Analytical Methods and  Treatment
Technologies information included in this document.

This Health Advisory was provided for review and comments were received from staff in the
following U.S. EPA Program Offices:
      U.S. EPA Office of Ground Water and Drinking Water
      U.S. EPA Office of Science and Technology
      U.S. EPA Office of Research and Development
      U.S. EPA Office of Children's Health Protection
      U.S. EPA Office of General Counsel

This Health Advisory was provided for review and comments were received from the following
other federal and health agencies:
      Health Canada
      U.S. Department of Health and Human Services, Centers for Disease Control and
      Prevention
       Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
                              TABLE OF CONTENTS

ACKNOWLEDGMENTS	I
TABLE OF CONTENTS	II
LIST OF TABLES	IV
LIST OF FIGURES	IV
ABBREVIATIONS AND ACRONYMS	V
EXECUTIVE SUMMARY	1
1.0    INTRODUCTION AND BACKGROUND	3
   1.1    Current Criteria, Guidance and Standards	3
2.0    PROBLEM FORMULATION	5
  2.1    Cyanobacteria and Production of Cylindrospermopsin	5
  2.2    Physical and Chemical Properties	5
  2.3    Sources and Occurrence	7
       2.3.1   Occurrence in Surface Water	8
       2.3.2  Occurrence in Drinking Water	8
  2.4    Environmental Fate	9
       2.4.1   Persistence	9
       2.4.2  Mobility	9
  2.5    Nature of the Cylindrospermopsin Toxin	9
       2.5.1   Toxicokinetics	9
       2.5.2  Noncancer Health Effects Data	10
       2.5.3   Mode of Action for Noncancer Health Effects	11
       2.5.4  Carcinogenicity Data	12
  2.6    Conceptual Model	12
       2.6.1   Conceptual Model Diagram	15
       2.6.2  Factors Considered in the Conceptual Model for Cylindrospermopsin	15
  2.7    Analysis Plan	16
3.0    HEALTH EFFECTS ASSESSMENT	19
  3.1    Dose-Response	19
       3.1.1   Critical Study Selected	19
       3.1.2  Endpoint Selection	22
  3.2    Ten-Day Health Advisory	23
       3.2.1   Bottle-fed Infants and Young Children ofPre-school Age	23
       3.2.2  School-age Children through Adults	24
       3.2.3   Uncertainty Factor Application	24
4.0    RISK CHARACTERIZATION	26
  4.1    Studies Supporting Determination of Critical Study	26
  4.2    Study Duration	26
  4.3    Allometric Scaling Approach	
27
      Drinking Water Health Advisory for Cylindrospermopsin - June 2015          ii

-------
  4.4   Uncertainty and Variability	27
  4.5   Susceptibility	28
  4.6   Distribution of Body Weight and Drinking Water Intake by Age	28
  4.7   Distribution of Potential Health Advisory Values by Age	29
5.0    ANALYTICAL METHODS	31
6.0    TREATMENT TECHNOLOGIES	32
  6.1   Management and Mitigation  of Cyanobacterial Blooms in Source Water	32
  6.2   Drinking Water Treatment	33
      6.2.1   Conventional Treatment for Cylindrospermopsin	33
      6.2.2   Chemical Oxidation	34
      6.2.3   Ultraviolet Irradiation	35
  6.3   Point-of-Use (POU) Drinking Water Treatment Units	35
7.0    REFERENCES	36
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015      iii

-------
                                  LIST OF TABLES

Table 1-1. International Guideline Values for Cylindrospermopsin 	4
Table 1-2. State Guideline Values for Cylindrospermopsin	4
Table 2-1. Chemical and Physical Properties of Cylindrospermopsin	7
Table 3-1. Kidney Weight Data from Oral Toxicity Study of Cylindrospermopsin Administered
    Daily over Eleven Weeks (Humpage and Falconer, 2002, 2003)	21
Table 3-2. Selected Clinical Chemistry, Hematology and Urinalysis Findings (Humpage and
    Falconer, 2002, 2003)	21
                                 LIST OF FIGURES

Figure 2-1. Structure of Cylindrospermopsin (de la Cruz etal., 2013)	6
Figure 2-2. Structurally Related Cylindrospermopsins (de la Cruz et al., 2013)	6
Figure 2-3. Conceptual Model of Exposure Pathways to Cylindrospermopsin in Drinking Water
     	14
Figure 4-1. 90th Percentile Drinking Water Ingestion Rates by Age Group	29
Figure 4-2. Ten-day Health Advisories for Cylindrospermopsin by Age Group	30
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       iv

-------
                       ABBREVIATIONS AND ACRONYMS
BMD
BMDL
BW
CAS
CCL
CWA
CYP450
DAF
DBF
DMSO
DNA
DWI
ELISA
EPA
g
GAC
GFR
HA
HAB
HESD
HPLC
ICR
i.p.
kg
KQC
KOW
L
LC
LCAT
LC-ESI/MS

LCMRL
LC-MS/MS
LOAEL
MCH
US
MNBNC
mg
mL
mmol
MOA
MWCO
N
Benchmark Dose
Benchmark Dose Level
Body Weight
Chemical Abstracts Service
Contaminant Candidate List
Clean Water Act
Cytochrome P450
Dissolved Air Flotation
Disinfection By-Products
Dimethylsulfoxide
Deoxyribonucleic Acid
Drinking Water Intake
Enzyme Linked Immunosorbent Assay
U.S. Environmental Protection Agency
Gram
Granular Activated Carbon
Glomerular Filtration Rate
Health Advisory
Harmful Algal Bloom
Health Effects Support Document
High Performance Liquid Chromatography
Institute for Cancer Research
Intraperitoneal
Kilogram
Organic Carbon:Water Partition Coefficient
Octanol:Water Partition Coefficient
Liter
Liquid Chromatography
Lecithin Cholesterol Acyl Transferase
Liquid Chromatography Tandem Electrospray lonization Mass
Spectrometry
Lowest Concentration Method Reporting Limit
Liquid Chromatography Tandem Mass Spectrometry
Lowest-Ob served-Adverse-Effect Level
Mean Corpuscular Hemoglobin
Microgram
Micromole
Micronucleated Binucleated Cells
Milligram
Milliliter
Millimole
Mode of Action
Molecular Weight Cut-off
Nitrogen
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
N/A
NARS
ng
NHANES
NLA
NOAEL
NOM
OECD
P
PAC
PDA
Pg
POU
RBC
RfD
SDWA
SHE
SPE
TOC
TOXLINE
UF
USAGE
USGS
UV
Not Applicable
National Aquatic Resource Surveys
Nanogram
National Health and Nutrition Examination Survey
National Lake Assessment
No-Observed-Adverse-Effect Level
Natural Organic Material
Organization for Economic Cooperation and Development
Phosphorus
Powdered Activated Carbon
Photodiode Array
Picogram
Point-of-Use
Red Blood Cell
Reference Dose
Safe Drinking Water Act
Syrian Hamster Embryo
Solid Phase Extraction
Total Organic Carbon
Toxicology Literature Online
Uncertainty Factor
U.S. Army Corps of Engineers
U.S. Geological Survey
Ultraviolet
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       vi

-------
                               EXECUTIVE SUMMARY

       Cylindrospermopsin is a toxin produced by a variety of cyanobacteria including:
Cylindrospermopsis raciborskii (C. raciborskii), Aphanizomenonflos-aquae, Aphanizomenon
gracile, Aphanizomenon ovalisporum, Umezakia natans, Anabaena bergii, Anabaena lapponica,
Anabaena planctonica, Lyngbya wollei, Rhaphidiopsis curvata, and Rhaphidiopsis mediterranea.

       Many environmental factors such as the ratio of nitrogen to phosphorus, temperature,
organic matter availability, light attenuation and pH play an important role in the development of
Cylindrospermopsin blooms, both in fresh and marine water systems. These species do not tend
to form visible surface scums and the highest concentrations of cells occurs below the water
surface. Cylindrospermopsin may be retained within the cell, but most of the time it is found in
the water (extracellular) or attached to particulates present in the water.

       This Health Advisory (HA) for the cyanobacterial toxin Cylindrospermopsin is focused
on drinking water as the primary source of exposure. Exposure to cyanobacteria and their toxins
may also occur by ingestion of toxin-contaminated food, including consumption offish, and by
inhalation and dermal contact during bathing or showering and during recreational activities in
waterbodies with the toxins. While these types of exposures cannot be quantified at this time,
they are assumed to contribute less to the total cyanotoxin exposures than ingestion of drinking
water. Due to the  seasonality of cyanobacterial blooms, exposures are not expected to be chronic.

       Limited animal studies demonstrate absorption of Cylindrospermopsin from the  intestinal
tract primarily in the liver, but also in the kidney and spleen. Limited data are available on the
metabolism of Cylindrospermopsin, but evidence indicates that metabolism and toxicity are
mediated by the hepatic cytochrome P450  (CYP450) enzyme system. The periacinar region of
the liver, an area where substantial CYP450-mediated xenobiotic metabolism occurs, appears to
be the main target of Cylindrospermopsin toxicity and where Cylindrospermopsin and its
metabolites bind to proteins. The few studies evaluating elimination suggest that
Cylindrospermopsin is rapidly eliminated primarily in the urine, but also in feces.

       The main source of information on the toxicity of Cylindrospermopsin in humans is from
qualitative reports of a hepatoenteritis-like illness attributed to acute or short-term consumption
of drinking water  containing C. raciborskii. Symptoms reported include fever, headache,
vomiting, bloody  diarrhea, hepatomegaly, and kidney damage with loss of water, electrolytes and
protein. No reliable data are available on the exposure levels of Cylindrospermopsin that induced
these effects.

       Based on oral and intraperitoneal (i.p.) studies in mice treated with purified
Cylindrospermopsin or extracts of C.  raciborskii cells, the liver and kidneys appear to be the
primary target organs for Cylindrospermopsin toxicity.

       The U.S. Environmental Protection Agency (EPA) identified a study by Humpage and
Falconer (2002, 2003) conducted on mice as the critical study used in the derivation of the
reference dose (RfD) for Cylindrospermopsin. The critical effects identified in the study
areincreased kidney weight and decreased urinary protein. The NOAEL (No Observed Adverse
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
Effect Level) was determined to be 30 ug/kg/day based on kidney toxicity. The total uncertainty
factor (UF) applied to the NOAEL was 300. This was based on a UF of 10 for intraspecies
variability, a UF of 10 for interspecies variability, and a UF of 3 (10'/2) to account for deficiencies
in the database.

       EPA is issuing a Ten-day HA for cylindrospermopsin based on the Humpage and
Falconer (2002, 2003) 11-week study. Studies of a duration of 7 days up to 30 days are typically
used to derive Ten-day HAs. In this case, a subchronic study was determined to be suitable for
the derivation of the HA. Although the duration of the Humpage and Falconer (2002, 2003)
study is longer (77 days) than the studies typically used for the derivation of a Ten-day HA, the
short-term studies available for cylindrospermopsin (Shaw et al., 2001; Reisner et al., 2004) are
not suitable for quantification; 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 short-term HA is consistent with the available data and most appropriately matches
human exposure scenarios for cyanobacterial blooms in drinking water. Cyanobacterial blooms
are usually seasonal, typically occurring from May through October. In the presence of algal cell
pigments, photochemical degradation of cylindrospermopsin can occur rapidly, with reported
half-lives of 1.5 to 3 hours. In the absence of pigments, however, there is little degradation. The
biodegradation of cylindrospermopsin in natural water bodies is a complex process that can be
influenced by many environmental factors, including concentration, water temperature and the
presence of bacteria. Half-lives of 11 to 15 days and up to 8 weeks have been reported for
cylindrospermopsin in surface waters.  In addition, concentrations in finished drinking water can
be reduced by drinking water treatment and management measures.

       The Ten-day HA value for bottle-fed infants  and young children of pre-school age is 0.7
|ig/L and for school-age children through adults is 3  |ig/L for cylindrospermopsin.  The two
advisory values use the same toxicity data (RfD) and represent differences in drinking water
intake and body weight for different human life stages. The first advisory value is based on the
summation of the time-weighted drinking water intake/body weight ratios for birth to < 12
months of age (U.S. EPA's Exposure Factors Handbook, 201 la). The second advisory value is
based on the mean body weight and the 90th percentile drinking water consumption rate for
adults age 21 and over (U.S. EPA's Exposure Factors Handbook, 201 la), which is similar to that
of school-aged children. Populations such as pregnant women and nursing mothers, the elderly,
and immune-compromised individuals or those receiving dialysis treatment may be more
susceptible than the general adult population to the health effects of cylindrospermopsin. As a
precautionary measure, individuals that fall into these susceptible groups may want to consider
following the recommendations for children pre-school age and younger. This HA is not a
regulation;  it is not legally enforceable; and it does not confer legal rights or impose legal
obligations on any party.

       No epidemiological studies of the association of cylindrospermopsin and cancer are
available. Also, no  chronic cancer bioassays of purified cylindrospermopsin in animals were
identified. Therefore, under the U.S. EPA's (2005) Guidelines for Carcinogen Risk Assessment,
there is inadequate information to assess carcinogenic potential of cylindrospermopsin.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
1.0  INTRODUCTION AND BACKGROUND

       EPA developed the non-regulatory Health Advisory (HA) Program in 1978 to provide
information for public health officials or other interested groups on pollutants associated with
short-term contamination incidents or spills for contaminants that can affect drinking water
quality, but are not regulated under the Safe Drinking Water Act (SDWA). At present, EPA lists
HAs for 213 contaminants  (http://water.epa.gov/drink/standards/hascience.cfm).

       HAs identify the concentration of a contaminant in drinking water at which adverse
health effects are not anticipated to occur over specific exposure durations (e.g., one-day, ten-
days, and a lifetime). HAs  serve as informal technical guidance to assist Federal, State and local
officials, and managers of public or community water systems in protecting public health when
emergency spills or contamination situations occur. An HA provides information on the
environmental properties, health effects, analytical methodology, and treatment technologies for
removal of drinking water contaminants.

       The Health Effects Support Document for Cylindrospermopsin (U.S. EPA, 2015a) is the
peer-reviewed, effects assessment that supports this HA.  This document is available at
http://www2.epa.gov/nutrient-policy-data/health-and-ecological-effects. The HAs are not
legally enforceable Federal standards and are subject to change as new information becomes
available. The structure of this Health Advisory is consistent with EPA's Framework for Human
Health Risk Assessment to Inform Decision Making (U. S .EP A, 2014).

       EPA is releasing the Recommendations for Public Water Systems to Manage Cyanotoxins
in Drinking Water (U.S. EPA, 2015b) as a companion to the HAs for microcystins and
Cylindrospermopsin. The document is intended 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. It is designed to provide information
and a framework that PWSs and others as appropriate may consider to inform their decisions on
managing the risks from cyanotoxins in drinking water.
1.1     Current Criteria, Guidance and Standards

       Currently there are no U.S. federal water quality criteria, or regulations for cyanobacteria
or cyanotoxins in drinking water under the SDWA or in ambient waters under the Clean Water
Act (CWA). The Safe Drinking Water Act (SDWA), as amended in 1996, requires the EPA to
publish a list of unregulated contaminants every five years that are not subject to any proposed or
promulgated national primary drinking water regulations, which are known or anticipated to
occur in public water systems, and which may require regulation. This list is known as the
Contaminant Candidate List (CCL). The EPA's Office of Water included cyanobacteria and
cyanotoxins on the first and second CCL (CCL 1, 1998; CCL 2, 2005). EPA included
cyanotoxins, including anatoxin-a, Cylindrospermopsin, and microcystin-LR, on CCL 3 (2009)
and the draft CCL 4 (April 2015 for consideration).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
       SDWA requires the Agency to make regulatory determinations on at least five CCL
contaminants every five years. When making a positive regulatory determination, EPA
determines whether a contaminant meets three criteria:

       •  The contaminant may have an adverse effect on the health of persons,
       •  The contaminant is known to occur or there is substantial likelihood the contaminant
          will occur in public water systems with a frequency and at levels of concern, and
       •  In the sole judgment of the Administrator, regulating the contaminant presents a
          meaningful opportunity for health risk reductions.

       To make these determinations, the Agency uses data to analyze occurrence (prevalence
and magnitude) and health effects. EPA continues gathering this information to inform future
regulatory determinations for cyanotoxins under the SDWA. The SDWA also provides the
authority for EPA to publish non-regulatory HAs or take other appropriate actions for
contaminants not subject to any national primary drinking water regulation. EPA is providing
this HA and the HA for microcystins to assist State and local officials in evaluating risks from
these contaminants in drinking water.

       Internationally, three countries and two U.S. states have developed drinking water
guidelines for cylindrospermopsin, as shown in Table 1-1 and Table 1-2, respectively.
Table 1-1. International Guideline Values for Cylindrospermopsin
Country
Australia
New Zealand
Brazil
Guideline Value
lUg/L
lUg/L
15ug/L
(recommended)
Source
Australian Drinking Water Guidelines 6
(NHMRC, NRMMC, 201 1)
Drinking-water Standards for New Zealand 2005
(Ministry of Health, 2008)
Guidelines for Drinking Water Quality, Official LA
Report's, Regulation MS N 518/2004 (Brasil, 2009)
Table 1-2. State Guideline Values for Cylindrospermopsin
State
Ohio
Oregon
Guideline Value
lUg/L
l^g/L
Source
State of Ohio Public Water System Harmful Algal
Bloom Response Strategy (OHEPA, 2014)
Public Health Advisory Guidelines, Harmful Algae
Blooms in Freshwater Bodies. (OHA, 2015)
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
2.0  PROBLEM FORMULATION

       The development of the HA begins with problem formulation, which provides a strategic
framework by focusing on the most relevant cyanotoxin properties and endpoints identified in
the Health Effects Support Document for Cylindrospermopsin (U.S. EPA, 2015 a).
2.1     Cyanobacteria and Production of Cylindrospermopsin

       Cyanobacteria, formerly known as blue-green algae (Cyanophyceae), are a group of
bacteria with chlorophyll-a capable of photosynthesis (light and dark phases) (Castenholz and
Waterbury, 1989). Most Cyanobacteria are aerobic photoautotrophs, requiring only water, carbon
dioxide, inorganic nutrients and light for survival, while others have heterotrophic properties and
can survive long periods in complete darkness (Fay, 1965). Some species are capable of nitrogen
fixation (diazotrophs) (Duy et al., 2000), producing inorganic nitrogen compounds for the
synthesis of nucleic acids and proteins. Cyanobacteria can form symbiotic associations with
animals and plants, such as fungi, bryophytes, pteriodophytes, gymnosperms and angiosperms
(Rai, 1990), supporting their growth and reproduction (Sarma, 2013; Hudnell, 2008; Hudnell,
2010).

       Under the right conditions of pH, nutrient availability, light, and temperature,
Cyanobacteria can reproduce quickly, forming a bloom. Although studies of the impact of
environmental factors on cyanotoxin production are ongoing, nutrient (nitrogen, phosphorus and
trace metals) supply rates, light, temperature, oxidative stressors, interactions with other biota
(viruses, bacteria and animal grazers) and, most likely, the combined effects of these factors are
all involved (Paerl and Otten 2013a, 2013b). Fulvic and humic acids reportedly encourage
Cyanobacteria growth (Kosakowska et al., 2007).

       Cylindrospermopsin is a toxin produced by a variety of Cyanobacteria including:
Cylindrospermopsis raciborskii (C. raciborskii), Aphanizomenonflos-aquae, Aphanizomenon
gracile, Aphanizomenon ovalisporum, Umezakia natans, Anabaena bergii, Anabaena lapponica,
Anabaena planctonica,  Lyngbya wollei, Rhaphidiopsis curvata, and Rhaphidiopsis mediterranea.
2.2     Physical and Chemical Properties

       The cyanotoxin Cylindrospermopsin is a tricyclic alkaloid with the following molecular
formula CisF^iNsOyS (Ohtani et al., 1992) and a molecular weight of 415.43 g/mole. It is
zwitterionic (i.e., a dipolar ion with localized positive and negative charges) (Ohtani et al., 1992)
and is believed to be derived from a polyketide that uses an amino acid starter unit such as
glycocyamine or 4-guanidino-3-oxybutyric acid (Duy et al., 2000). The chemical structure of
Cylindrospermopsin is presented in Figure 2-1. Two naturally occurring congeners of
Cylindrospermopsin have been identified (Figure 2-2): 7-epicylindro-spermopsin (the epimer of
cylindrospremopsin) and 7-deoxyCylindrospermopsin (Norris et al., 1999; de la Cruz et al.,
2013). Recently, Wimmer et al. (2014) identified two new analogs, 7-deoxy-desulfo-
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
           Figure 2-1. Structure of Cylindrospermopsin (de la Cruz et al., 2013)
                                                 H  OH
                  03SO
                   me1
                                    H
      Figure 2-2. Structurally Related Cylindrospermopsins (de la Cruz et al., 2013)
          7-epi-cylindrospermopsin
7-deoxy Cylindrospermopsin
                          HO H
             R)     N      NH    HN      NH
      H,C'
                                                 H3C'
Cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin, from the Thai strain of
C. raciborskii. However, it is not clear if these are Cylindrospermopsin congeners, precursors or
degradation products. Chlorination of water containing Cylindrospermopsin can produce 5-
chlorocylindrospermopsin and cylindrospermic acid.

       The physical and chemical properties of Cylindrospermopsin are presented in Table 2-1.
Cylindrospermopsin generally exists in a zwitterionic state (with both positive and negative ions)
and is highly soluble in water (Moore et al., 1998, Chiswell et al., 1999). Cylindrospermopsin is
isolated for commercial use mostly from C. raciborskii. Other physicochemical properties of
Cylindrospermopsin in the environment such as vapor pressure, boiling and melting points, soil
organic carbon-water partition coefficient (Koc), octanol-water partition coefficient (Kow), and
vapor pressure and Henry's Law constant are unknown. Available information on the chemical
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
Table 2-1. Chemical and Physical Properties of Cylindrospermopsin
Property
Chemical Abstracts Service (CAS)
Registry #
Chemical Formula
Molecular Weight
Color/Physical State
Boiling Point
Melting Point
Density
Vapor Pressure at 25 °C
Henry' s Law Constant
KQW
KQC
Solubility in Water
Other Solvents
Cylindrospermopsin
143545-90-8
Ci5H21N507S
415.43 g/mole
white powder
N/A
N/A
2.03g/cm3
N/A
N/A
N/A
N/A
Highly
Dimethylsulfoxide (DMSO) and methanol
       Sources: Chemical Book, 2012; TOXLINE, 2012
breakdown, biodegradation and mobility of Cylindrospermopsin in the environment is discussed
in the Environmental Fate section.
2.3
Sources and Occurrence
       Many environmental factors such as the ratio of nitrogen to phosphorus, temperature,
organic matter availability, light attenuation and pH play an important role in the development of
Cylindrospermopsin blooms (Paerl and Huisman, 2008; Paerl and Often, 2013). Although
cylindrospermopsin-producing cyanobacteria (such as C. raciborskii) occur mostly in tropical or
subtropical regions, they have also been found in warmer temperate regions, both in fresh and
marine water systems. These species do not tend to form visible surface scums and the highest
concentrations of cells occurs below the water surface (Falconer 2005). Cylindrospermopsin may
be retained within the cell, but most of the time it is found in the water (extracellular) or attached
to particulates present in the water (Chiswell et al., 2001).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
2.3.1       Occurrence in Surface Water

       EPA's National Aquatic Resource Surveys (NARS) generate national estimates of
pollutant occurrence every 5 years. In 2007, the National Lakes Assessment (NLA) conducted
the first-ever national probability-based survey of algal toxins, but did not include
cylindrospermopsin. The United States Geological Survey (USGS) subsequently analyzed the
stored samples collected during the NLA and reported that cylindrospermopsin was present in
5% of the samples; however, concentrations of cylindrospermopsin were not reported (Loftin
and Graham, 2014). Future NARS plan to include other algal toxins, including
cylindrospermopsin.

       Cylindrospermopsin  was also detected in 9% of the blooms sampled during a 2006 USGS
survey of 23 lakes in the Midwestern U.S. (Graham et al., 2010). The low concentrations of
cylindrospermopsin detected (0.12 to 0.14 |ig/L) in the study occurred in bloom communities
dominated by Aphanizomenon orAnabaena andMicrocystis.

       Many states monitor for harmful algal blooms (HABs). State monitoring efforts are
expanding with greater awareness of the toxic effects of HABs. These monitoring efforts tend to
focus on priority waters used for recreation or drinking water. Sampling is seasonal or on
occasions when blooms are observed.

       Cylindrospermopsin  has been detected in lakes throughout multiple states. In a 1999
study, cylindrospermopsin was detected in 40% of 167 water samples taken from 87 water
bodies in Florida during the  months of June and November (Burns, 2008). However,  the actual
cylindrospermopsin concentrations were not reported. In 2005, the U.S. Army Corps of
Engineers (USAGE) detected cylindrospermopsin at a maximum concentration of 1.6 |ig/L in
lake water samples from Oklahoma (Lynch and Clyde, 2009). In Grand Lake St. Marys, Ohio,
cylindrospermopsin concentrations as high as 9  |ig/L were reported in 2010 (OHEPA, 2012).
2.3.2       Occurrence in Drinking Water

       The occurrence of cyanotoxins in finished drinking water depends on their levels in the
raw source water and the effectiveness of the treatment methods used for removing
cyanobacteria and cyanotoxins during the production of drinking water. Currently there is no
federal or state program in place that requires monitoring for cyanotoxins at U.S.  drinking water
treatment plants. Therefore, data on the presence or absence of cyanotoxins in finished drinking
water are limited.

       EPA used information from the published literature to evaluate the potential  occurrence
of cylindrospermopsin in public water systems. In the single publication identified, the results of
a 2000 survey of toxins in drinking water treatment plants in Florida were reported (Burns,
2008). In this survey, cylindrospermopsin was detected at concentrations ranging from 8 |ig/L to
97 |ig/L in nine finished drinking water samples.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
2.4     Environmental Fate

       Different physical and chemical processes are involved in the persistence, breakdown,
and movement of cylindrospermopsin in aquatic systems.
2.4.1       Persistence

       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 cell pigments,
cylindrospermopsin tends to be relatively stable in sunlight, with a half-life of 11 to 15 days in
surface waters (Funari and Testai, 2008). Cylindrospermopsin remains a potent toxin even after
boiling for 15 minutes (Chiswell et al., 1999).

       Degradation of cylindrospermopsin increases in the presence of cell pigments such as
chlorophyll-a and phycocyanin. When exposed to both sunlight and cell pigments,
cylindrospermopsin breaks down rapidly, more than 90% within 2 to 3 days (Chiswell  et al.,
1999). Cylindrospermopsin has been shown to be decomposed by bacteria in laboratory studies;
the biodegradation is influenced by the toxin concentration, temperature and pH. Mohamed and
Alamri (2012) reported that cylindrospermopsin was degraded by Bacillus bacteria and
degradation occurred in 6 days at the highest toxin concentration (300 ug/L) and in 7 or 8 days at
lower concentrations (10 and 100 ug/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.
2.4.2       Mobility

       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.
2.5     Nature of the Cylindrospermopsin Toxin

2.5.1       Toxico kinetics

       Animal studies show that cylindrospermopsin is absorbed from the gastrointestinal tract
(Humpage and Falconer, 2003; Shaw et al., 2000, 2001) and that the tissue distribution occurs
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
primarily to the liver, but also to the kidneys and spleen after intraperitoneal (i.p.) exposure
(Norrisetal.,2001).

       The metabolism and toxicity of cylindrospermopsin is mediated by the hepatic
cytochrome P450 (CYP450) enzyme system. The periacinar region of the liver, an area where
substantial CYP450-mediated xenobiotic metabolism occurs, appears to be the main target of
cylindrospermopsin toxicity and where cylindrospermopsin and its metabolites bind to proteins
(Runnegar et al. 1995; Shaw et al. 2000, 2001; Norris et al., 2001).

       Animal studies evaluating the elimination of cylindrospermopsin in urine and feces after
i.p. exposures found a continued urinary and fecal excretion over the monitoring period (24
hours) and a mean total recovery  from the urine and feces of 76.9% of the administered dose
after 24 hours (Norris et al., 2001). Urinary excretion accounted for 68.4% of the 24-hour total
and fecal excretion for 8.5%. There was considerable interanimal variability in this study.
2.5.2       Noncancer Health Effects Data

2.5.2.1        Human Studies

       Human data on oral toxicity of cylindrospermopsin are limited, but suggest that liver and
kidney are potential target organs for toxicity. 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 C. 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, 2006). Exposed individuals showed mild irritation, but
no statistically significant 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 C. raciborskii
(Stewart et al., 2006).
2.5.2.2        Animal Studies

       Most of the information on the noncancer effects of cylindrospermopsin in animals is
from oral and i.p. administration studies in mice exposed to purified compound or extracts of C.
raciborskii cells. Studies conducted with purified toxin are preferred because extracts may
contain other toxins or compounds with similar chemical physical properties that co-elute with
the toxin. Effects on the liver and kidney, including changes in organ weights and
histopathological lesions, along with increases in the hematocrit level in serum and deformation
of red blood cell  are observed following short-term and subchronic oral exposure to
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       10

-------
cylindrospermopsin (Humpage and Falconer, 2002, 2003; Reisner at al., 2004; Sukenik et al.,
2006). Oral and i.p. acute toxicity studies in mice also report histopathological effects in both
liver and kidney. No chronic toxicity studies evaluating cylindrospermopsin are available.

      No oral reproductive or developmental studies are available for cylindrospermopsin.
Developmental toxicity studies following i.p. administration of cylindrospermopsin provide
some evidence for maternal toxicity and decreased postnatal pup survival and body weight
(Rogers et al., 2007; Chernoff et al., 2011). Sibaldo de Almeida et al. (2013) did not find any
visceral  or skeletal malformations in the offspring of pregnant rats receiving an oral dose of 3
mg/kg/day purified cylindrospermopsin during gestation (GD 1-20).
2.5.3       Mode of Action for Noncancer Health Effects

2.5.3.1        Liver

       The occurrence of toxicity in the liver suggests a protein-synthesis inhibition mechanism
of action for cylindrospermopsin. In vitro and in vivo studies have been conducted to
demonstrate the ability of cylindrospermopsin to inhibit hepatic protein synthesis, which could
impact mouse urinary protein production leading to decreased urinary excretion of these proteins
(Froscio et al., 2008, 2009; Terao et al., 1994). Available evidence indicates that protein
synthesis inhibition is not decreased by broad-spectrum CYP450 inhibitors, but they do reduce
cytotoxicity (Froscio et al., 2003; Bazin et al., 2010). Hepatotoxicity appears to be CYP450-
dependent, which indicates a possible involvement of oxidized and/or fragmented metabolites
and mechanisms other than protein  synthesis inhibition (Froscio et al., 2003;  Humpage et al.,
2005; Norris et al., 2001, 2002). Despite the number of studies that have been published, the
mechanisms for liver and kidney toxicity by cylindrospermopsin are not completely
characterized.
2.5.3.2        Red Blood Cells

       There was evidence of effects on red blood cells (RBCs) in the Reisner et al. (2004) and
Humpage and Falconer (2002) studies of purified cylindrospermopsin. In the Reisner et al.
(2004) report, microscopic examination of blood samples snowed the presence of RBCs 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 RBC
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 (LCAT), an enzyme associated with high-density lipoproteins and
the esterifi cation of plasma cholesterol. Effects on the cholesterol content of the RBC membrane
can occur with inhibition of the enzyme increasing membrane fluidity and mean corpuscular
volume. Associated effects were observed in the Reisner et al. (2004) and Humpage and
Falconer (2002) studies. Removal of the abnormal blood cells by the spleen increases both
spleen weight and serum bilirubin as well as stimulates hematopoiesis. Additional research is
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015        11

-------
needed to examine the LCAT enzyme inhibition hypothesis in order to confirm whether it
accounts for the effects on the RBC as a result of cylindrospermopsin exposure.
2.5.3.3       Kidney

       No mode of action information for kidney effects was observed in the available studies of
cylindrospermopsin. Since all the studies were conducted in mice, a species that excretes low
molecular weight proteins in urine, there is a need to conduct a study of cylindrospermopsin in a
laboratory species that does not excrete protein in the urine in order to determine whether there
are comparable effects on kidney weight, protein excretion and renal cellular damage. Kidney
necrosis and a decreased renal failure index at the high cylindrospermopsin doses provide
support for the effects on the kidney.
2.5.4       Carcinogenicity Data

       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 initiated tumors in mice (Falconer and Humpage, 2001). Cell
transformation in Syrian hamster embryo (SHE) cells was observed using purified
cylindrospermopsin (Marie et al., 2010). Transformation frequency increased at the lowest
concentrations (from 1 x 10"2 to IxlO"7 ng/mL) but not at the highest concentrations (1 or IxlO"1
ng/mL).

       Mutagenicity studies (e.g., the Ames Assay) have not observed mutagenic activity of
cylindrospermopsin (Sieroslawska, 2013). A few i.p. studies investigating the in vivo
genotoxicity (DNA damage) from exposure to cylindrospermopsin showed DNA strand breakage
in the liver of Balb/c mice (Shen et al., 2002) and covalent binding between DNA and
cylindrospermopsin, or a metabolite, in Quackenbush mouse liver (Shaw et al., 2000). In vitro
mutagenic and genotoxic cell assays have shown potential damage to DNA expressed as an
increase in micrenucleated binucleate cells (MNBNC) in the colon adenocarcinoma line and the
human hepatoma line (Bazin et al., 2010), in the human lymphoblastoid cell line (Humpage et
al., 2000), in HepG2 cells (Straser et al.,  2011), and in isolated human peripheral lymphocytes
(Zegura et al., 2011). DNA breaks also have been observed in primary hepatocytes by comet
assay (Humpage et al., 2005).
2.6     Conceptual Model

       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 HA values. The conceptual model demonstrates the relationship
between exposure to cylindrospermopsin in drinking water and adverse health effects in the
populations at risk.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       12

-------
       HAs describe non-regulatory concentrations of drinking water contaminants at which
adverse health effects are not anticipated to occur over specific exposure durations (e.g., one-
day, ten-days, and a lifetime). HAs also contain a margin of safety to protect sensitive members
of the population. They serve as informal technical guidance to assist federal, state and local
officials, as well as managers of public or community water systems, in protecting public health.
They are not to be construed as legally enforceable federal standards.

       Assessment endpoints for HAs can be developed for both short-term (one-day and ten-
day) and lifetime exposures periods using information on the non-carcinogenic and carcinogenic
toxicological endpoints of concern. Where data are available, endpoints will reflect susceptible
and/or more highly exposed populations.

    •   A One-day HA is typically calculated for an infant (0-12 months or 10kg child),
       assuming a single acute exposure to the chemical and is generally derived from a study of
       less than seven days' duration.

    •   A Ten-day HA is typically calculated for an infant (0-12  months or 10kg child), assuming
       a limited period of exposure of one to two weeks, and is generally derived  from a study
       of 7 to 30-days duration.

    •   A Lifetime HA is derived for an adult (>21  years or 80kg adult), and assumes an
       exposure period over a lifetime (approximately 70 years). It is usually derived from a
       chronic study of two years duration, but subchronic studies may be used by adjusting the
       uncertainty factor employed in the calculation. For carcinogens, the HA documents
       typically provide the concentrations in drinking water associated with risks for one excess
       cancer case per ten thousand persons exposed up to one excess cancer case per million
       exposed for Group A and B carcinogens and those classified as known or likely
       carcinogens (U.S. EPA, 1986, 2005). Cancer risks are not provided for Group C
       carcinogens or those classified as "suggestive", unless the cancer risk has been
       quantified.

       For each assessment endpoint EPA uses one or more measures of effect (also referred to
as a point of departure), which describe the change in the attribute of the assessment endpoint in
response to chemical exposure, to develop acute, short-term, longer term (subchronic) or chronic
reference values when the data are available. The measures of effect selected represent impacts
on survival,  growth, system function, reproduction and development.

       This conceptual model provides useful information to characterize and communicate the
potential health risks related to exposure to cyanotoxins in drinking water. The sources of
cyanotoxins in drinking water, the route of exposure for biological receptors of concern (e.g., via
various human activities such as drinking, food preparation and consumption) and the potential
assessment endpoints (i.e., effects such as kidney and liver toxicity, and reproductive  and
developmental effects) due to exposure to cylindrospermopsin are depicted in the conceptual
diagram below (Figure 2-3).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015

-------
Figure 2-3. Conceptual Model of Exposure Pathways to Cylindrospermopsin in Drinking
Water
   STRESSOR
   SOURCES
  EXPOSURE ROUTE
  RECEPTORS
  ENDPOINTS
                                           Cylindrospermopsin
                                Lakes, Reservoirs and Rivers
                            Shallow ground water under the
                            direct influence of surface water
                                              Finished Drinking Water
Dermal

Showering.
Bathing

Washing
dishes



Outside activities
- (eardenins. car
Inhalation


Incidental
— inhalation while
showering

Incidental
inhalation while
washing dishes

                                               washing, etc.)
                              Incidental ingestion
                             while outside activities
                               (gardening, car
                               washing, etc.)
Kidney
Damage
 Liver
Damage
Hematological
  Damage
Developmental
   Effects
Cancer
                                                                           Legend
Quantitative
Data Available

Incomplete or
Quantitative Data
Not Available
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015
                                                             14

-------
2.6.1       Conceptual Model Diagram

       Cyanobacteria are a common part of freshwater and marine ecosystems. An increase in
water column stability, high water temperatures, elevated concentrations of nutrients and low
light intensity have been associated with an increase and/or dominance of cylindrospermopsin-
producing cyanobacteria in surface waters (or aquatic ecosystems). The presence of detectable
concentrations of cyanotoxins in the environment is closely associated with these blooms. Winds
and water currents can potentially transport cyanobacterial blooms to areas within the proximity
of water intakes for drinking water treatment plants. If not managed in source waters, or removed
during drinking water treatment, cyanobacteria and cyanotoxins may result in exposure that
could potentially affect human health.
2.6.2      Factors Considered in the Conceptual Model for Cylindrospermopsin

Stressors: For this HA, the stressor is Cylindrospermopsin concentrations in finished drinking
water.

Sources: Sources of Cylindrospermopsin include potential sources of drinking water such as
rivers, reservoirs and lakes in the U.S. where blooms producing Cylindrospermopsin occur.
Shallow private wells under the direct influence of surface water (in hydraulic connection to a
surface water body) can also be impacted by cylindrospermopsin-producing blooms if the toxins
are drawn into the well along with the water from the surface water. There is substantially less
information on exposure from this source.

Routes of exposure: Exposure to cyanotoxins from contaminated drinking water sources may
occur via oral exposure (drinking water, cooking with water, and incidental ingesting from
showering); dermal exposure (contact of exposed parts of the body with water containing toxins
during bathing or showering, washing dishes or outside activities); inhalation exposure (during
bathing or showering); or intravenous exposure (e.g., via dialysis). Toxicity data are available for
the oral route of exposure from drinking water, but are not available to quantify dose response
for other exposure routes (inhalation, dermal, dietary and intravenous exposures).

Receptors: The general population (adults and children) could be exposed to cyanotoxins
through dermal contact, inhalation and/or ingestion. Infants and pre-school age children can be at
greater risk to Cylindrospermopsin because they consume more water per unit body weight than
adults. Other individuals of potential sensitivity include persons with kidney and/or liver disease
due to the compromised detoxification mechanisms in the liver and impaired excretory
mechanisms in the kidney. There are no human data to quantify risk to pregnant woman or to
evaluate the transfer of cyanotoxins across the placenta. Data are also not available on the
transfer of cyanotoxins through the milk from nursing mothers or on the risk to the elderly.
Given this lack of information, pregnant women, nursing mothers, and the elderly may also be
potentially sensitive populations. Data from the episode in a dialysis clinic in Caruaru, Brazil
where microcystins, and possibly Cylindrospermopsin, were not removed by treatment of dialysis
water (Carmichael  et al., 2011), identify dialysis patients as a population of potential concern in
cases where the drinking water source for the clinic is contaminated with cyanotoxins. EPA has
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       15

-------
data to quantify risk to infants, children, and adults based on variability in potential exposure
(body weight and drinking water intake rate). However, data are not available to quantify risk to
pregnant woman, nursing mothers, persons with liver or kidney disease, or dialysis patients. Data
are not available to derive a one-day HA for children because  studies with single oral dosing do
not provide dose-response information. A lifetime HA for cylindrospermopsin is not
recommended as the types of exposures being considered are short-term and episodic in nature.
Although the majority of the cyanobacterial blooms in the U.S. occur seasonally, usually during
late summer, some toxin-producing strains can occur early in the season and can last for days or
weeks.

Endpoints: Human data on oral toxicity of cylindrospermopsin are limited, but have shown
effects on the liver following potential exposure to cylindrospermopsin. Acute, short-term and
subchronic studies in animals show effects on the liver, RBC and kidney. In addition, some
studies suggest that cylindrospermopsin may lead to reproductive and developmental  effects;
however, these data are limited. In vitro mutagenic and genotoxic cell assays with
cylindrospermopsin have shown varied results with some indications of potential damage to
DNA. However, these data are limited, and there has been no long term bioassay of purified
cylindrospermopsin. Thus, available data are inadequate to assess the carcinogenic potential of
cylindrospermopsin at this time. Available toxicity data are described in the Health Effects
Support Document (HESD)for Cylindrospermopsin (U.S. EPA, 2015 a). Kidney effects were
selected as the endpoint on which to base the measure of effect. Liver and hematological effects
were not as sensitive as the reported kidney effects.
2.7     Analysis Plan

       The Health Effects Support Document (HESD)for Cylindrospermopsin (U.S. EPA,
2015a) provides the health effects basis for development of the HA, including the science-based
decisions providing the basis for estimating the point of departure. To develop the HESD for
cylindrospermopsin, a comprehensive literature search was conducted from January 2013 to May
2014 using Toxicology Literature Online (TOXLINE), PubMed component and Google Scholar
to ensure the most recent published information on cylindrospermopsin was included. The
literature search included the following terms: cylindrospermopsin, human toxicity, animal
toxicity, in vitro toxicity, in vivo toxicity, occurrence, environmental fate, mobility and
persistence. EPA assembled available information on occurrence, environmental fate,
mechanisms of toxicity, acute, short-term, subchronic and chronic toxicity and cancer in humans
and animals, toxicokinetics, and exposure. Additionally, EPA considered information from the
following risk assessments during the development of the cylindrospermopsin health risk
assessment:

   •   Health Canada (2012) Toxicity Profile for Cyanobacterial Toxins

   •   Enzo Funari and Emanuela Testai (2008) Human Health Risk Assessment Related to
       Cyanotoxins Exposure

   •   Tai Nguyen Duy, Paul Lam, Glen Shaw and Des Connell (2000) Toxicology and Risk
       Assessment of Freshwater Cyanobacterial (Blue-Green Algal) Toxins in Water
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       16

-------
   •   ILS (2000) Cylindrospermopsin [CASRN143545-90-8] Review of Toxicological
       Literature

       The toxicity data available for an individual pollutant vary significantly. An evaluation of
available data was performed by EPA to determine data acceptability. The following study
quality considerations from U.S. EPA's (2002) A Review of the Reference Dose and Reference
Concentration Processes were used in selection of the studies for inclusion in the HESD and
development of the HA.

   •   Clearly defined and stated hypothesis.

   •   Adequate description of the study protocol, methods and statistical analyses.
   •   Evaluation of appropriate endpoints. Toxicity depends on the amount, duration, timing
       and pattern of exposure, and may range from frank effects (e.g., mortality) to more subtle
       biochemical, physiological, pathological or functional changes in multiple organs and
       tissues.

   •   Application of the appropriate statistical procedures to determine an effect.
   •   Establishment of dose-response relationship (i.e., no observed adverse effect level
       (NOAEL) and/or lowest observed adverse effect level (LOAEL) or data amenable to
       modeling of the dose-response in order to identify a point of departure for a change in the
       effect considered to be adverse (out of the range of normal biological viability). The
       NOAEL is the highest exposure level at which there are no biologically significant
       increases in the frequency or severity of adverse effect between the exposed population
       and its appropriate  control. The LOAEL is the lowest exposure level at which there are
       biologically significant increases in frequency or severity of adverse effects between the
       exposed population and its appropriate control group.

       After the available  studies were evaluated for inclusion in the HESD and HA, the critical
study was selected based on consideration of factors including exposure duration (comparable to
the duration of the HA being derived), route of exposure (oral exposure via drinking water,
gavage, or diet is preferred), species sensitivity, comparison of the point of departure with other
available studies demonstrating an effect, and confidence in the study (U.S. EPA, 1999). Once, a
point of departure is chosen for quantification, uncertainty factors appropriate for the study
selected are then applied to the point of departure to account for variability and uncertainty in the
available data.

       For Cylindrospermopsin, toxicity and exposure data are available to develop a Ten-day
HA. EPA used measures of effect and estimates of exposure to derive the Ten-day HAs using the
following equation:

                           _   NOAEL or  LOAEL or BMDL
                     JTL/Y  —  	
                                        UF x DWI/BW
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015        17

-------
Where:
NOAEL or  =  No- or Lowest-Observed-Adverse-Effect Level (mg/kg bw/day) from a study
   LOAEL     of an appropriate duration (up to 7 days and 7-30 days for the One-day and
               Ten-day HAs, respectively).

    BMDL  =  When the data available are adequate, benchmark dose (BMD) modeling can
               be performed to determine the point of departure for the calculation of HAs.
               The benchmark dose (BMD) approach involves dose-response modeling to
               obtain dose levels corresponding to a specific response level near the low end
               of the observable range of the data (U.S.EPA, 2012). The lower 95%
               confidence limit is termed the benchmark dose level (BMDL).

       UF  =  Uncertainty factors (UF) account for: (1) intraspecies variability (variation in
               susceptibility across individuals); (2) interspecies variability (uncertainty in
               extrapolating animal  data to humans; (3) uncertainty in extrapolating from a
               LOAEL to a NOAEL; and (4) uncertainty associated with extrapolation when
               the database is incomplete. These are described in U.S. EPA, 1999 and U.S.
               EPA, 2002.

  DWI/BW  =  For children, a normalized ratio of drinking water ingestion to body weight
               (DWI/BW) was calculated using data for infants (birth to <12 months). The
               estimated drinking water intake body weight ratio (L/kg/day) used for birth to
               < 12 months of age are the 90th percentile values of the consumers only
               estimates of direct and indirect water ingestion based on 1994-1996, 1998
               CSFII (Continuing Survey of Food Intakes by Individuals) (community water,
               mL/kg/day) in Table 3-19 in the U.S. EPA (201 la) Exposure Factors
               Handbook. The time  weighted average of DWI/BW ratios values was derived
               from multiplication of age-specific DWI/BW ratios (birth to <1 month, 1 to <
               3 months, 3 to < 6 months, and 6 to <12 months) by the age-specific fraction
               of infant exposures for these time periods.

               For adults (>21 years of age), EPA updated the default BW assumption to 80
               kg based on National Health and Nutrition Examination Survey (NHANES)
               data from 1999 to 2006 as reported in Table 8.1 of EPA's Exposure Factors
               Handbook (U.S. EPA, 201 la). The updated BW represents the mean weight
               for adults ages 21 and older.

               EPA updated the default DWI to 2.5 L/d, rounded from 2.546 L/d, based on
               NHANES data from 2003 to 2006 as reported in EPA's Exposure Factors
               Handbook (U.S. EPA 201 la, Table 3-33). This rate represents the consumer's
               only estimate of combined direct and indirect community water ingestion at
               the 90th percentile for adults ages 21 and older.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015
18

-------
3.0  HEALTH EFFECTS ASSESSMENT

       The health effects assessment provides the characterization of adverse effects and
includes the hazard identification and dose-response assessment. The hazard identification
includes consideration of avail able information on toxicokinetics; identification, synthesis and
evaluation of studies describing the health effects of cylindrospermopsin; and the potential Mode
of Action (MO As), or toxicity pathways related to the health effects identified.
3.1     Dose-Response
3.1.1       Critical Study Selected

       The critical study selected for the derivation of the reference dose (RfD) for
cylindrospermopsin is Humpage and Falconer (2002, 2003). Humpage and Falconer (2002,
2003) is a comprehensive toxicity study in which male mice were exposed by gavage to purified
cylindrospermopsin from cell extract for 11 weeks. The study authors used four dose groups,
adequate numbers of animals per dose group (10) and evaluated a variety of endpoints.
Statistically significant, dose-related effects on the kidney, liver and serum chemistry were
observed. The kidney was the most sensitive target of toxicity. The Humpage and Falconer
(2002) data are supported by the short-term Reisner et al. (2004) results showing exposure-
duration-related increased kidney weights, liver weights and testes weights, and hematological
effects (acanthocytes or abnormal red blood cells (RBCs) and changes in hematocrit) following a
21-day exposure.

       Purified cylindrospermopsin in water was administered by gavage in doses of 0, 30, 60,
120 or 240 ug/kg/day to groups of male Swiss albino mice (6 to 10 mice per dose group) for 11
weeks (Humpage and Falconer, 2002, 2003). The cylindrospermopsin was from an extract of
freeze-dried C. raciborskii cells Woloszynska  (AWT 205) purified using sephadex size-
exclusion gel  (G-10). The individual sephadex fractions were assayed using high-performance
liquid chromatography (HPLC) and concentrated to a sample that was 47% cylindrospermopsin
by dry  weight and 53% phenylalanine. Food and water consumption, and body weight were
examined throughout the study. After 9 weeks of exposure, the study authors report conducting  a
clinical examination to detect physiological and behavioral signs of toxicity but do not specify
the parameters evaluated. Hematology evaluations (4 to 5 per dose group, except the high dose),
serum chemistry (4 to 6 per dose group), and urinalysis (6 or 10 per dose group) were conducted.
All the evaluations were conducted either near or at the end of the treatment period.

       Postmortem examinations were done on the following organs: liver, spleen, kidneys,
adrenal glands, heart, testes, epididymis and brain, including measurement of organ weights.
Comprehensive histological evaluations were conducted in accordance with the
recommendations from the Organization for Economic Cooperation and Development (OECD).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015        19

-------
       No deaths or visual clinical signs of toxicity were reported in mice exposed to purified
cylindrospermopsin under the study conditions. The mean final body weight was 7-15% higher
in all dose groups compared to controls, but was not dose-related and was only statistically
significant at 30 and 60 ug/kg/day (Humpage and Falconer, 2003). No significant changes were
observed in food consumption. In all dose groups, the water intake was significantly reduced;
water consumption was 53% of the control level at 30 ug/kg/day and the higher dose groups
were 68-72% of the control levels.

       Relative kidney weight was significantly increased in a dose-related manner at >60
ug/kg/day (12-23% greater than controls; see Table 3-1). Relative liver weight was significantly
increased (13% greater than controls) only at the highest dose (240 ug/kg/day). Relative spleen,
adrenal and testes weights were increased  for doses >60 ug/kg/day, but the differences from
control were not statistically  significant (Humpage and Falconer, 2002).

       Selected serum chemistry (n= 4-6), hematology (n=4-5) and urinalysis (n=6-10) results
are shown in Table 3-2. The hematology and serum chemistry evaluations showed no dose-
related, statistically significant changes, although serum albumin, total bilirubin and cholesterol
were increased compared to controls at all doses (Humpage and Falconer,  2002). The increases
in cholesterol were significant for the 30 and 60 ug/kg/day groups, but not at the higher doses.
The serum urea concentration was slightly decreased at the two highest doses. A nonsignificant
increase in red cell polychromasia (high number of RBCs) was indicated for all  doses, but
quantitative data were not presented. Packed red cell volume  was slightly increased and mean
corpuscular hemoglobin was slightly decreased (Table 3-2) when compared to controls, although
the changes were not dose related. When combined with the bilirubin results and the increased
relative spleen weight, the hematological data suggest the possibility of minor RBC effects. One
of the limitations in the serum chemistry and hematology data is the small number of samples
evaluated, a factor that impacts the determination of statistical significance (Humpage and
Falconer, 2002).

       There was a significant decrease in the urine protein-creatinine ratio (g/mmol creatinine)
at 120 and 240 ug/kg/day compared to the controls (51% and 37% of controls, respectively; both
p<0.001) (Humpage and Falconer, 2002).  Also, a significant decrease in urine specific gravity
normalized for creatinine was seen at 240  ug/kg/day compared to the control (p<0.001). The
renal glomerular filtration rate (GFR) was decreased compared to controls at all doses, but the
differences were not dose dependent or statistically significantly different from controls. The
renal failure index1 was decreased slightly at > 120 ug/kg/day; the differences from control  were
not statistically significant (Humpage and  Falconer, 2002). Tubular retention of low molecular
weight urinary proteins could account for the decreased urinary protein and possibly the
increased kidney weight. 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
functional, highly-polymorphic, low-molecular-weight urinary proteins that play important roles
in social recognition and mate assessment  (Cheetham et al., 2009). The relevance of the urinary
protein findings in mice to humans is unknown.
1 Renal failure index= (urinary sodium concentration x plasma creatinine concentration) / urinary creatinine
concentration
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       20

-------
Table 3-1. Kidney Weight Data from Oral Toxicity Study of Cylindrospermopsin
Administered Daily over Eleven Weeks (Humpage and Falconer, 2002, 2003)
        Dose
     (ug/kg/day)
Number
             Relative Kidney
                 Weight
           Control

          g/lOOg BW
         Exposed
         g/lOOg BW
                    % Difference
                         Significance
          0
       (Control)
   10
1.48
          30
   10
1.48
1.57
+6
Not significant
          60
             1.48
           1.66
             + 12
             p<0.001
         120
             1.48
           1.82
             +23
             p<0.001
         240
             1.48
           1.78
             +20
            PO.001
Table 3-2. Selected Clinical Chemistry, Hematology and Urinalysis Findings (Humpage
and Falconer, 2002, 2003)
Endpoint
N
Dose (ug/kg/day)
0
30
60
120
240
Clinical Chemistry
Urea (mmol/L)
Albumin (g/L)
Cholesterol (mmol/L)
Bilirubin (mmol/L)
4-6
4-6
4-6
4-6
9.24
23.8
3.26
2.62
9.22
26.6
4.60**
2.72
8.55
26.0
4.65**
2.88
7.51
26.0
3.68
3.06
7.92
25.8
4.08
3.07
Hematology
Packed Cell volume (L/L)
Mean Corpuscular
Hemoglobin (MCH, pg/L)
4-5
4-5
0.38
16.8
0.39
15.7
0.39
16.4
0.39
16.4
ND
ND
Urinalysis
Volume (mL)
Creatinine (mmol/L)
Specific gravity/creatinine
Protein/creatinine (g/mmol)
Renal Failure Index
(mmol/L)
6-10
6-10
6-10
6-10
4-6
9.85
0.57
1.79
4.3
4.3
11.18
0.49
2.04
3.6
4.3
10.38
0.54
1.91
3.3
4.5
11.74
0.51
1.99
2 2**
3.6
6.74
0.72**
1.44*
1.6**
3.6
ND = not determined
Significantly different from control: *p<0.05; **p<0.01.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015
                                                                21

-------
       Serum albumin and total serum protein were not decreased in the Humpage and Falconer
studies (2002, 2003). The most sensitive effects observed by Humpage and Falconer (2002,
2003) were dose-related decreases in the urinary protein: creatinine ratio at >120 ug/kg/day and
increased relative kidney weight at >60 ug/kg/day. The noted decrease in urinary protein
excretion could reflect an impact on excretion of mouse urinary proteins given the fact that total
serum protein was not significantly increased compared to controls for all dose groups. Mouse
urinary proteins are synthesized in the liver (Clissold and Bishop, 1982) and transported to the
kidney for excretion. If cylindrospermopsin did  reduce liver protein synthesis, a  decrease in total
serum protein would be expected. However, this was not the case, suggesting a lack of an effect
on synthesis of the urinary proteins in the liver.

       The Humpage and Falconer (2002, 2003) postmortem tissue examinations showed
histopathological damage to the liver based on scores assigned for necrosis, inflammatory foci
and bile duct changes at >120 ug/kg/day. The percent of animals with liver lesions in the 120 and
240 |ig/kg/day dose groups was 60% and 90%, respectively, when compared to 10%, 10% and
20% for the 0, 30, and 60 |ig/kg/day dose groups, respectively. Severity scores were not given,
and the liver lesions were not further described.  There was proximal renal tubular damage in
kidney sections from two mice in the 240 ug/kg/day dose group (Humpage and Falconer, 2002,
2003).

       The 11-week study by Humpage and Falconer (2002, 2003) provides a NOAEL (30
|ig/kg/day) and a LOAEL (60 jig/kg/day) for dose-related, statistically significant increases in
kidney weights along with indicators of reduced renal function effects at higher doses. Because
of the similarity in the type of effects observed and the LOAELs from the Humpage and
Falconer (2002, 2003) and Reisner et al. (2004)  studies, the selection of the NOAEL from
Humpage and Falconer was determined to be the most appropriate point of departure for ten-day
exposures in infants, children and adults despite its longer exposure duration.
3.1.2       Endpoint Selection

       Upon considering all effects observed by Humpage and Falconer (2002, 2003), increased
relative kidney weight was considered the most appropriate basis for quantitation. Adverse
effects on the kidneys were manifested by decreases in urinary protein concentration and
increased relative kidney weight. The study authors reported significantly increased relative
kidney weight at >60 ug/kg/day, decreased urinary protein and liver lesions at > 120 ug/kg/day
and renal tubular lesions at 240 ug/kg/day (Humpage and Falconer, 2002, 2003). Relative kidney
weight increased significantly in a dose-related manner beginning at 60 ug/kg/day (12-23%
greater than controls), and relative liver weight was significantly increased at 120 ug/kg/day (12-
23% greater than controls)  and at the high dose of 240  ug/kg/day (13% greater than controls).
Relative spleen, adrenal and testes weights were increased for doses >60 jig/kg/day, but the
differences from control, although dose-related, were not statistically significant. Humpage and
Falconer (2002, 2003) identified the LOAEL as 60 |ig/kg/day and the NOAEL as 30 |ig/kg/day
based on the dose-related and statistically significant increase in relative kidney weight.  These
adverse effects are potential indicators of suppressed hepatic protein synthesis that was not
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       22

-------
reflected in the measurement of total serum protein and/or increased retention of low molecular
weight mouse urinary proteins by the kidney because of damage to the renal tubules.

       In the single dose drinking water study by Reisner et al. (2004), hematological effects
(acanthocytes, increased hematocrit) and increased organ weights (liver, testicular and kidney) in
young (4 week) male Institute for Cancer Research (ICR) mice were observed following a three
week exposure to purified cylindrospermopsin. The 66 |ig/kg/day LOAEL is comparable to that
from the Humpage and Falconer  (2002, 2003) study (60|ig/kg/day). Humpage and Falconer
(2002, 2003) evaluated 5 different doses using 6 to 10 mice per dose group; Reisner et al. (2004)
used one dose with 8 male mice.  Reisner et al. (2004) demonstrated effects in comparable
parameters to those impacted in Humpage and Falconer at a dose of 66 jig/kg/day with a three
week exposure. They also demonstrated a trend for effects on kidney weight and hematocrit
across the three-week duration of exposure. Because the renal  effects reported in Humpage and
Falconer (2002, 2003) did not occur at 11 weeks for the 30 |ig/kg dose, the point of departure
from the Humpage and Falconer  study was determined to be the most appropriate for the
quantitative assessment. Thus, the quantification from the Humpage and Falconer NOAEL based
on kidney weight changes provides the best point of departure for ten-day exposures in children
and adults despite its longer exposure duration.
3.2     Ten-Day Health Advisory

The Ten-day HA is considered protective of non-carcinogenic adverse health effects over a ten-
day exposure to cylindrospermopsin in drinking water.

3.2.1       Bottle-fed Infants and Young Children of Pre-school Age

The Ten-day HA for bottle-fed infants and young children of pre-school age is calculated as
follows:


                     Ten-day HA  =     3°^g/kg/day     = 0.7 ug/L
                                    300 x 0.15 L/kg/day
Where:
     30 |ig/kg/day     =   The NOAEL for kidney effects in mice exposed to
                         cylindrospermopsin in water for 11 weeks (Humpage and
                         Falconer, 2002, 2003).
     300             =   The composite uncertainty factor (UF) including a 10 for
                         intraspecies variability (UFH), a 10 for interspecies differences
                         (UFA), and a 3 for uncertainties in the database (UFo).
     0.15 L/kg/day    =   Normalized drinking water intakes per unit body weight over
                         the first year of life based on the 90th percentile of drinking
                         water consumption and the mean body weight (U.S. EPA,
                         201 la).
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       23

-------
The Ten-day HA of 0.7 |ig/L is considered protective of non-carcinogenic adverse health effects
for bottle-fed infants and young children of pre-school age over a ten-day exposure to
cylindrospermopsin in drinking water.
3.2.2       School-age Children through Adults

The Ten-day HA for school-age children through adults is calculated as follows:

                      rr    .   TTA        30ug/kg/day
                      Ten-day HA  =	      —	 = 3 ug/L
                                      300 x 0.03L/kg/day

Where:
        30 |ig/kg/day      =  The NOAEL for kidney effects in mice exposed to
                            cylindrospermopsin in water for 11 weeks (Humpage and
                            Falconer, 2002, 2003).
        300              =  The composite UF including a 10 for intraspecies variability
                            (UFn),  a 10 for interspecies differences (UFA), and a 3 for
                            uncertainties in the database (UFD).
        0.03 L/kg/day     =  Drinking water intake per unit body weight based on adult default
                            values  of 2.5 L/day and 80 kg (U.S. EPA, 201 la).

The Ten-day HA of 3 jig/L is considered protective of non-carcinogenic adverse health effects
for children of school age through adults over a ten-day exposure to cylindrospermopsin in
drinking water.


3.2.3       Uncertainty Factor Application

   •   UFH - A Ten-fold value is applied to account for variability in the human population. No
       information was available to characterize interindividual and age-related variability in the
       toxicokinetics or toxicodynamics among humans. Individuals with a low RBC count as a
       result of genetic or nutritional factors could be more sensitive to cylindrospermopsin
       exposures than the general population. Individuals with pre-existing kidney/liver
       problems may also be more sensitive. Pregnant woman, nursing mothers, and the elderly
       could also be sensitive to cylindrospermopsin exposures.

   •   UFA - A Ten-fold value is applied to account for uncertainty in extrapolating from
       laboratory animals to humans (i.e., interspecies variability). Information to quantitatively
       assess toxicokinetic or toxicodynamic differences between animals and humans is
       unavailable for cylindrospermopsin. Allometric scaling is not applied in the development
       of the Ten-Day HA values for  cylindrospermopsin. The allometric scaling approach is
       derived from the relationship between body surface area and basal metabolic rate in
       adults (U.S. EPA, 201 Ib). This approach is not appropriate for infants and children due to
       the comparatively slower clearance during these ages and the limited toxicokinetic data
       available to assess the appropriateness of body weight scaling in early life.


               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       24

-------
           - An uncertainty factor of 3 (10°5 = 3.16) is selected to account for deficiencies in
       the database for cylindrospermopsin. The database for cylindrospermopsin includes
       limited human studies. The database for studies in laboratory animals includes oral
       exposure acute,  short-term and subchronic studies, but many of them lacked a
       comprehensive evaluation of a wide spectrum of effects. The database lacks chronic
       toxicity and multi-generation reproductive and developmental toxicity studies using the
       oral route of exposure. There is a lack of data on neurological and immunological
       endpoints. The RBC parameters evaluated differed between the Humpage and Falconer
       (2002, 2003) and Reisner et al. (2004) studies.

       The default factors typically used cover a single order of magnitude (i.e., 101). By
convention, in the Agency, a value of 3 is used in place of one-half power (i.e.,  10'/2) when
appropriate (U.S. EPA,  2002).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       25

-------
4.0  RISK CHARACTERIZATION

     The following topics describe important conclusions used in the derivation of the health
advisory. This section characterizes each topic and its impact on the health advisory.
4.1     Studies Supporting Determination of Critical Study

       Increases in kidney weight and hematological effects are detected in all three studies
(Humpage and Falconer, 2002, 2003; Reisner et al., 2004; and Sukenik et al., 2006). However,
the type of hematological effects varied among studies as did the statistical significance of the
observed effects. Humpage and Falconer (2002, 2003) found signs indicative of hemolysis
(increased bilirubin, spleen weight and polychromasia (high number of RBCs with low
hemoglobin)), while Reisner et al. (2004) and Sukenik et al. (2006) found acanthocytes
(abnormal RBCs). Increases in kidney weight were significant for Humpage and Falconer (2002,
2003) and Sukenik et al. (2006), but not statistically significant for Reisner et al. (2004).
Humpage and Falconer (2002, 2003) and Reisner et al. (2004) used purified cylindrospermopsin,
while Sukenik et al. (2006) used an extract in spent medium. Of these three studies, Humpage
and Falconer (2002, 2003) provides a NOAEL (30 |ig/kg/day) and a LOAEL (60 |ig/kg/day) for
dose-related statistically significant increases in kidney weights and indications of renal function
effects at higher doses. Although the percent change in kidney weight is the same  for Reisner et
al. (2004), only the change observed by Humpage and Falconer (2002, 2003) was  statistically
significant.
4.2     Study Duration

       The short-term studies with appropriate durations (typically 7 days up to 30 days)
available for cylindrospermopsin (Shaw et al., 2001; Reisner et al., 2004), are not suitable for
quantification, as described below. However, the Reisner study does support the use of the
Humpage and Falconer (2002, 2003) study for the derivation of the Ten-day HA, despite the
longer duration of the study.

       The Shaw et al (2001) study reported the results from multiple experiments. These
experiments each have limitations including use of extract, lack of adequate numbers of animals
and monitored endpoints, and the limited number of doses tested that preclude their use in
quantification. The oral data for purified extract from Shaw et al. (2001) identified fatty liver as
an adverse effect in mice following a 14 day gavage exposure to 0.05 mg/kg/day. However, the
only effects mentioned in the published paper are the liver effects and an absence of
lymphophagocytosis in the spleen.

       Reisner et al. (2004) conducted a 21 day study in mice and showed significant increases
in hematocrit, acanthocytes (abnormal RBCs), and liver and testes weights effects at a 66
|ig/kg/day dose and a duration-related nonsignificant increase in and kidney weight. This study
was not selected for development of the Ten-day HA because this study used a single dose;
however, the effects to that dose after 3-weeks were comparable to the effects seen in the
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       26

-------
Humpage and Falconer (2002, 2003) study at a slightly lower 60 mg/kg/day dose after 11 weeks.
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 3-weeks in the shorter duration study.
4.3     Allometric Scaling Approach

       Allometric scaling was not applied in the development of the RfD for
cylindrospermopsin. In the development of short-term advisory values (One-day and Ten-day),
parameters are used that reflect exposures and effects for infants up to one year of age, rather
than for adults. The body weight scaling approach is derived from the relationship between body
surface area and basal  metabolic rate in adults. Infants/children surface area and basal metabolic
rates are very different than adults with a slower metabolic rate. In addition, limited toxicokinetic
data are available to assess the appropriateness of body weight scaling in early life. The body
weight scaling procedure has typically been applied in the derivation of chronic oral RfDs and
cancer assessments, both of which are concerned with lifetime repeated exposure scenarios (U.S.
EPA, 2012). Thus,  given the development of a Ten-Day HA value, and the application of the
Ten-Day HA to infants and pre-school age children, the application of the body weight scaling
procedure is not appropriate for this scenario.

       In addition, for short-term advisories (one-day and ten-day  duration), EPA assumes all
exposure is derived from drinking water and, therefore, no Relative Source Contribution (RSC)
term is applied. For lifetime health advisory values, EPA does include an RSC that reduces the
advisory value to account for other potential sources.
4.4     Uncertainty and Variability

       Uncertainty factors were applied in several areas to adjust for incomplete information.
Human data on the toxic effects of cylindrospermopsin are limited. Quantification for the
absorption, distribution and elimination of cylindrospermopsin in humans following oral,
inhalation or dermal exposure is not well understood. The clinical significance in humans for
biological changes observed in experimental animals such as increased kidney weight, decreased
urinary protein levels, decrease in renal failure index and the formation of acanthocytes
(abnormal RBCs) is not known. In animal studies with cylindrospermopsin, adverse effects
(RBC effects)  observed have not been fully characterized. No  data are available to quantify the
differences between humans and animals for the critical health endpoints. There is uncertainty
regarding susceptibility and variability characterized in the human population following
exposure to cylindrospermopsin. Additional information is needed on the potential health risks
from mixtures of cylindrospermopsin with other cyanotoxins, bioactive molecules with an effect
on living organisms and chemical stressors present in ambient water and/or drinking water
supplies. The critical study was conducted only in male mice and therefore, any gender-specific
effects of cylindrospermopsin are not understood.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       27

-------
4.5     Susceptibility

       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. Based on
the results from animal studies, individuals with liver and/or kidney disease might be more
susceptible than the general population because of compromised detoxification mechanisms in
the liver and impaired excretory mechanisms in the kidney. 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 used by a clinic for hemodialysis is
contaminated with cyanotoxins.

       The data on RBC acanthocytes 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 (rare autosomal recessive disorder that interferes with the
normal absorption of fat and fat-soluble vitamins from food) and hypobetalipoproteinemia are
associated with abnormal RBC 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.

       Based on the currently available science, evidence is lacking to assess differences in
susceptibility between infants, children and adults. There are, however, significant differences in
exposure between these life-stages that impact risk.
4.6     Distribution of Body Weight and Drinking Water Intake by Age

       Both body weight and drinking water intake distributions vary with age. EPA has
developed two health advisory values, a Ten-day HA of 0.7 |ig/L based on exposure to infants
over the first year of life, and a Ten-day HA of 3 |ig/L based on exposure to adults, over 21 years
of age. Section 4.7 discusses how EPA recommends application of these values to other age
groups.
       The U.S. EPA (201 la) Exposure Factors Handbook provides values for drinking water
ingestion rate and corresponding body weight. The estimated 90th percentile of community water
ingestion for the general population (males and females of all ages) has been used as the default
value for water ingestion. EPA plotted the 90th percentile of drinking water intake using Table 3-
19 for ages < 3 years, and Table 3-38 for ages >3 years due to sample size in  the respective
studies. Age groups < 3 months in Table 3-19 were combined due to insufficient sample sizes.
Figure 4.1 represents the 90th percentile drinking water ingestion rates (L/kg/day) for each age
group (located on top of the columns). Bottle-fed ages are shown in red (first three columns on
the left).
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       28

-------
         Figure 4-1. 90* Percentile Drinking Water Ingestion Rates by Age Group
               0.25 -i
     _
     1
     a
     a
     •c
     Q
     o
     ON
       Adapted from U.S. EPA 2011 Exposure Factors Handbook (U.S.EPA, 201 la).
       Based on the drinking water intake rates for children <12 months (0.15 L/kg-day), the
exposure of children is over 4 times higher than that of adults >21  years old on a body weight
basis (0.034 L/kg-day). Infants from birth to 3 months may be exclusively bottle-fed and
therefore, have a higher ingestion rate. After 3 months of age, typically around 4 to 6 months of
age, other food and liquids are introduced into the infant diet, lowering the ingestion rate of
drinking water. Drinking water contributes the highest risk of the total cyanotoxins intake for
infants to one-year-olds fed exclusively with powdered formula prepared with tap water
containing cyanotoxins. At the age of 6, children's intake of drinking water relative to their body
weight is approximately the same as those of an adult (>21 years). Data evaluating the transfer of
cylindrospermopsin through breast milk are not available for humans.
4.7     Distribution of Potential Health Advisory Values by Age

       Using the ingestion rates for each age group (from Figure 4-1), EPA estimated Ten-day
HA values for cylindrospermopsin for each age group (plotted in Figure 4-2) to demonstrate the
variability due to body weight and drinking water intake by age.

       EPA decided to apply the Ten-day HA value calculated for infants over the first year of
life (0.7 |ig/L) to all bottle-fed infants and young children of pre-school age because these age
groups have higher intake per body weight relative to adults. As Figure 4-2 demonstrates, when
the Ten-day HA is estimated by age group, the calculated HA value for infants from birth to 3
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015
29

-------
       Figure 4-2. Ten-day Health Advisories for Cylindrospermopsin by Age Group
    4.0
    E.5
    = !i
    3.7
    l.f,
    3.5
    3.4
    13
    3.2
    3.1
    3.0
    ;.5
    2.8
    2.7
 — Z4
    ?2.3
    :.:
 3 I

 I"
 "* L7
    1.1
    1.0
    0.5
    O.i
    0.7
    O.S
    as
    0.4
    0.;
    0.1
    0.1
    0 0
                                School-age children and
                                   adults 3.0 ug/L
           Bottle fed infants up to
         school age children 0.7 ug/L
birth to
  <3
months

               3 to <6
               months
6 to < 12
 mouths
lto<2
years
2to<3
years
 3to<6
  years

Age Range
6 to < 11 11 to <1616 to <1818 to <21  > 21
 years   years    years    years   years
months old is 0.4 |ig/L, slightly below the infant health advisory value of 0.7 |ig/L. EPA believes
that infants from birth to 3 months old are not at a disproportionate risk at a 0.7 |ig/L advisory
value because a safety factor of 30 is built into this calculation to account for human variability
and deficiencies in the database. The estimated Ten-Day HA values for infants from 3 months
old through pre-school age groups (less than 6 years old), are at or above the advisory value of
0.7 |ig/L. Therefore, children within these age groups are adequately protected by the advisory
value for bottle-fed infants and young  children of pre-school age. EPA decided to apply the adult
Ten-Day HA value of 3 |ig/L to school age children (children older than or equal to 6 years)
through adulthood because children's intake of drinking water relative to body weight in this age
group is almost the same as those of an adult (>2l years).
                 Drinking Water Health Advisory for Cylindrospermopsin - June 2015
                                                                                   30

-------
5.0  ANALYTICAL METHODS

       The primary methods used for the analysis of cylindrospermopsin are liquid
chromatography (LC) and enzyme linked immunosorbent assay (ELISA).  Several detection
modes are generally coupled with LC including single channel ultraviolet (UV)/visible and
multi-channel UV photodiode array (PDA), electrospray ionization mass spectrometry (LC-
ESI/MS), and electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Due to the
limited selectivity of UV-based detectors, the use of mass spectrometric detection is becoming
more commonplace. Commercial ELISA test kits are also available for cylindrospermopsin
detection. These kits are available in both semiquantitative and quantitative formats and are
easily adapted to field or "screening" measurements.

       EPA has recently released Method 545 (U.S. EPA, 2015c) which is a LC-ESI/MS/MS
method for the determination of cylindrospermopsin and anatoxin-a in drinking water. This
method requires the operation of the mass spectrometer in MS/MS mode to enhance
selectivity. In this method, samples are preserved with ascorbic acid (dechlorinating agent) and
sodium bisulfate (microbial inhibitor). In the laboratory, aliquots (1 mL) of sample are taken for
analysis, and internal  standards are added. An aliquot of the sample is injected into an LC
equipped with an analytical column that is interfaced to the mass spectrometer. The analytes are
separated and then identified by comparing the acquired mass spectra and retention times to
reference spectra and  retention times  for calibration standards acquired under identical liquid
chromatography tandem mass spectrometry (LC-MS/MS) conditions. The concentration of each
analyte is determined using the integrated peak area and internal standard technique. A single
laboratory lowest concentration method reporting limit (LCMRL) of 0.063 |ig/L was  determined
for cylindrospermopsin along with an average value of 0.083 |ig/L for all participants of a multi-
lab evaluation (n=4) (Winslow et al.,  2006). Method 545: Determination of Cylindrospermopsin
and Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem
Mass Spectrometry (LC-ESI/MS/MS) is available at
http://water.epa.gov/scitech/drinkingwater/labcert/analyticalmethods_ogwdw.cfm.

       Other LC methods have generally used UV spectroscopic detection for
cylindrospermopsin analysis. These methods have often incorporated solid phase extraction
(SPE) to preconcentrate the target analyte, reduce matrix interferences or both. Quantitation
limits have ranged from 4 |ig/L to < 0.1 |ig/L based on the instrumental setup and the use of
preconcentration steps (Papageorgiou et al., 2012).

       Commercial ELISA kits for the detection of cylindrospermopsin are available from
several vendors. These kits claim a working concentration range of 0.05 and 2  |ig/L.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       31

-------
6.0  TREATMENT TECHNOLOGIES

       The information below is adapted from the draft Health Canada Guidelines for
Cyanobacteria Toxins in Drinking Water, available later in 2015.

       Detailed information on the operational considerations of a variety of treatment methods
can be found in the International Guidance Manual for the Management of Toxic Cyanobacteria
(GWRC, 2009) and Management Strategies for Cyanobacteria (Blue-Green Algae): A Guide for
Water Utilities (Newcombe et al., 2010) available at: http://www.waterra.com.au/cyanobacteria-
manual/PDF/GWRCGuidanceManualLevel 1 .pdf and
http://www.researchgate.net/profile/Lionel_Ho/publication/242740698_Management_Strategies
_for_Cyanobacteria_(Blue-
Green_Algae)_A_Guide_for_Water_Utilities/links/02e7e52d62273e8f70000000.pdf.
       For additional information on treatment strategies commonly used or being considered by
water systems vulnerable to cyanotoxins, please see Recommendations for Public Water Systems
to Manage Cyanotoxins in Drinking Water (U.S. EPA, 2015b).
6.1     Management and Mitigation of Cyanobacterial Blooms in Source Water

       Algaecides can be applied to lakes and reservoirs to mitigate algal blooms, including
Cyanobacteria. In most cases, depending on the Cyanobacteria species present, the application of
algaecides has the potential to compromise cell integrity releasing cyanotoxins into the source
waters. Chemical treatment to control blooms in drinking water sources in the early stages of the
bloom when cyanobacterial  concentrations are still relatively low (usually from 5,000 to 15,000
cells/mL) (WHO,  1999), are less likely to release significant cyanotoxin concentrations upon cell
lysis and may mitigate or prevent a cyanobacterial bloom from proliferating as the season
progresses. If a Cyanobacteria bloom does occur, utilities may investigate alternative raw water
sources, change intake locations or levels to withdraw raw water with minimal cyanotoxin
concentrations, or investigate methods of destratification in the water source. Purchasing water
from a neighboring interconnected water system that is unaffected by the bloom may also be an
option for some systems.

       Clays and commercial products such as aluminum sulfate (alum) have been used for the
management of blooms in source waters. Alum treatment efficiency depends on the alum dose
and the type of flocculant. Aeration and destratification have also been used to treat
cyanobacterial blooms, usually in smaller water bodies (from one acre to several tens of
acres). Active mixing devices, diffuse air bubblers, and other means of reducing stratification
have proven to be effective in controlling outbreaks and persistence of blooms in relatively small
shallow impoundments (around < 20 feet). These strategies can be applied to the entire source
water body or to just a portion of the lake depending on the need, size and depth of the water
body relative to the source water intake(s).

       The use of ultrasonic sound waves, or soni cation, to disrupt cyanobacterial cells has also
been investigated as a potential  source water treatment option (Rajasekhar et al., 2012).
Drawbacks include that application frequencies are difficult to calculate and are system-specific;


               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       32

-------
and that applications on large scale require more powerful, and therefore, more expensive
equipment. Sonication shows potential for use in cyanobacterial bloom management, but further
study to determine effective operating procedures is needed before it can be considered as a
feasible approach (Rajasekhar et al., 2012).

       Excess nutrients are thought to be a primary driver of cyanobacterial blooms. Long-term
prevention of cyanobacterial blooms likely requires reductions in nutrient pollution. Excess
nitrogen and phosphorus in aquatic systems can stimulate blooms and create conditions under
which harmful cyanobacteria thrive. Thus, managing nutrient pollution sources within a
watershed in addition to waterbody-specific physical controls (in systems that are amenable to
those controls) tends to be the most effective strategy. Nutrient pollution can be from urban,
agricultural, and atmospheric sources, and therefore, reductions can be achieved through a
variety of source control technologies and best management practices.
6.2     Drinking Water Treatment

       Effective treatment of cyanotoxins in drinking water includes the evaluation and selection
of appropriate treatment methods. The water treatment methods need to be tailored to the type(s)
of cyanobacteria present, the site-specific water quality (e.g., pH, temperature, turbidity,
presence of natural organic material (NOM)), the treatment processes already in place and
multiple treatment goals (e.g., turbidity and total organic carbon (TOC) removal, disinfection
requirements, control of disinfection by-products (DBF) formation). Utilities need to have an
understanding of the type and concentration of cyanotoxins present in the source water and
should conduct  site-specific evaluations such as jar testings and piloting in order to determine the
most effective treatment strategy. Potential target parameters include:  chlorophyll-a, turbidity,
cyanobacterial cells and extracellular and intracelluar toxins. Care should be taken to avoid cell
lysis. A multi-barrier approach consists of conventional filtration for intracellular
cylindrospermopsin removal and additional processes such as activated carbon, biodegradation,
advanced oxidation, and small-pore membrane processes (e.g. nanofiltration and reverse
osmosis), for the removal or oxidation of extracellular cylindrospermopsin. The most effective
way to deal with cyanobacteria cells and their toxins, is to remove the cells intact, without
damaging them, to prevent the release of additional extracellular toxins into the water.

       When released from the cell, cylindrospermopsin can be found dissolved or attached to
other materials such as particulate or soluble substances. Powdered activated carbon (PAC) has
proven to be effective for removal of extracellular cylindrospermopsin. Limited information is
available on the adsorption of cylindrospermopsin onto granular activated carbon (GAC).
6.2.1       Conventional Treatment for Cylindrospermopsin

       In the absence of cell damage, conventional treatment employing coagulation,
flocculation, clarification (sedimentation or dissolved air flotation) and rapid granular filtration
can be effective at removing intact cells and the majority of intracellular toxins (cell bound)
(Chow et al.,  1998; Newcombe et al., 2015). However, if toxins are released into solution, a
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       33

-------
combination of conventional treatment processes with oxidation, adsorption and/or advanced
treatment needs to be considered to treat both intracellular and extracellular cyanotoxins. Rapid
sand filtration without pre-treatment (i.e., direct filtration, without coagulation/clarification) is
not effective for cyanobacterial cell removal.

       Conventional water treatment (coagulation, flocculation, sedimentation or dissolved air
flotation (DAF), and filtration) is considered effective for removal of intracellular toxins but
ineffective for dissolved cyanotoxins such as cylindrospermopsin, which is partially dissolved in
water under normal growth conditions (Chow et al., 1999; Rapala et al., 2006; Carriere et al.,
2010).  Application of a multiple barrier approach has the potential to be effective (Newcombe et
al., 2015). Ho et al.  (2008, 2011) conducted bench-scale studies and modeling on the use of
PAC for the adsorption of cylindrospermopsin. The results demonstrated that a PAC dose of 25
mg/L and a contact time of 60 minutes would be required to reduce 5 |ig/L of
cylindrospermopsin to less than  1 |ig/L. When concentrations of cylindrospermopsin are 1-2
ug/L or 3-4 ug/L, the recommended doses of PAC are 10-20  mg/L and 20-30 mg/L, respectively
(Newcombe et al., 2010).

       Dixon et al. (201 Ib) also conducted laboratory-scale testing of integrated membrane
systems for cyanotoxin removal. The results showed that an ultrafiltration system with pre-
treatment using 2.2 mg/L of alum and 20 mg/L of PAC resulted in 97% removal of intra- and
extra-cellular cylindrospermopsin to achieve a treated water concentration of less than 0.1 ug/L
(Dixon et al.,  201 la). Nanofiltration and reverse osmosis would likely be effective in removing
dissolved toxins, but only a few  studies have been conducted. Dixon et al. (201 la) studied the
removal of cyanobacterial toxins by nanofitration and found that average removals between 90-
100% could be achieved for cylindrospermopsin using membranes with a low molecular weight
cut-off (MWCO) (< 300 Daltons).

       In practice, full-scale treatment plants use a combination of treatment technologies (i.e.,
conventional  filtration and chemical oxidation) in order to remove both intracellular and
extracellular cyanotoxins. Extracellular cylindrospermopsin may be removed by many treatment
plants using existing treatments such as chlorination or by the addition  of PAC (Carriere et al.,
2010).  Although it is possible to remove both intracellular and extracellular toxins effectively
using a combination of treatment processes, the removal efficiency can vary considerably.
Utilities need to ensure that they are using their existing treatment processes to their fullest
capacity for removal of both cyanobacterial cells and extracellular toxins, and that the
appropriate monitoring is being conducted to ensure that adequate removal is occurring at each
step in the treatment process.
6.2.2       Chemical Oxidation

       Chemical oxidation using chlorine or ozonation can be effective at oxidizing
cylindrospermopsin, but can also cause the cells to lyse, resulting in an increase in concentrations
of extracellular toxins in drinking water. By applying conventional filtration (or another filtration
process) first to remove the  majority of intact cells, the extracellular cylindrospermopsin is less
likely to increase due to cell lysis when water is treated with oxidants. In cases where pre-
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       34

-------
oxidation (oxidant applied anywhere along the treatment process prior the filter influent) is
practiced, it may need to be discontinued during an algal bloom or adjustments to the oxidant
type and doses may be needed to minimize cell rupture prior to filtration (Newcombe et al.,
2015).

       Different cyanotoxins react differently to oxidants depending on the individual
characteristics of the source water such as TOC, temperature and pH (Westrick  et al., 2010).
While chlorination is an effective treatment for oxidizing cylindrospermopsin, its effectiveness is
dependent on pH. Rodriguez et al. (2007) found that at a pH of 7 and an initial chlorine dose of 1
mg/L, oxidation of cylindrospermopsin is fast, with almost complete reaction after 30 minutes.
Other chlorinated oxidants such as chloramines and chlorine dioxide have little  impact on
cylindrospermopsin due to a slow reaction rates. For example, the reaction of chlorine dioxide
with cylindrospermopsin is relatively slow with a second-order rate constant of 0.9 M'V1 at pH
8. The rate  constant is pH-dependent and decreases significantly under mildly acidic conditions.
Chlorine dioxide may be used to inactivate C. raciborskii, however, in typical drinking  water
treatment applications, it does not appear to be practical for oxidizing cylindrospermopsin given
its slow reaction rate (de la Cruz et al., 2013). Oxidation by potassium permanganate is
temperature dependent and has not been shown to be effective in oxidizing cylindrospermopsin.
Water treatment utilities that use chloramines or chlorine dioxide as disinfectants to  reduce the
formation of regulated disinfection by-products may want to reconsider oxidation efficacy for
cyanotoxin inactivation during periods when algal toxins are present in source waters, while
balancing these other treatment objectives. Ozone has been  shown to effectively oxidize
cylindrospermopsin in laboratory-scale studies (de la Cruz et al., 2013). At pH 8, approximately
95% of cylindrospermopsin (initial concentration of 415 |ig/L) was oxidized using 0.38 mg/L Os.
6.2.3       Ultraviolet Irradiation

       Studies have indicated that ultraviolet (UV) irradiation may be effective for the oxidation
of cylindrospermopsis cells (Westrick et al., 2010). However, exposure times and/or UV doses
tested in the bench-scale experiments were greater than those typically applied in drinking water
treatment.
6.3     Point-of-Use (POU) Drinking Water Treatment Units

       Limited information is available on residential treatment units for the removal of
cyanobacteria cells and cyanotoxins. At this time, no units have been evaluated for removal of
cylindrospermopsin. Further studies need to be conducted to assess the efficacy of home
filtration devices for various cyanotoxins, including cylindrospermopsin, and for other filtering
conditions such as increased toxin load and the presence of other contaminants in drinking water.
Third-party organizations are currently developing certification standards to test POU devices to
evaluate how well they remove cyanotoxins from drinking water treatment units. Those
standards are expected in the near future.

More information about treatment units and the contaminants they can remove can be found at
http ://www. nsf org/C ertifi ed/DWTU/.


                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       35

-------
7.0  REFERENCES

Bazin, E., Mourot, A., Humpage, A. R., etal. (2010). Genotoxicity of a Freshwater Cyanotoxin,
       Cylindrospermopsin, in Two Human Cell Lines: Caco-2 and HepaRG. Environmental
       andMolecular Mutagenesis, 51 (3): 251-259.

Brasil. (2009). Ministerio da Saude. Secretaria de Vigilanica em Saude. Coordena9ao-Geral de
       Vigilancia em Saude Ambiental. Portaria MS n.° 518/2004 /Ministerio da Saude,
       Secretaria de Vigilancia em Saude, Coordena9ao-Geral de Vigilancia em Saude
       Ambiental - Brasilia: Editora do Ministerio da Saude, 32 p. - (Serie E. Legisla9ao de
       Saude) ISBN 85-334-0935-4,
       http://bvsms.saude.gov.br/bvs/publi cacoes/portaria_ms_n518_2004.pdf

Byth, S. (1980). Palm Island mystery disease. MedicalJournal of Australia, 2(1): 40-42.

Burns, J. (2008). Toxic cyanobacteria in Florida waters. In: Hudnell, H.K. (Ed.), Cyanobacterial
       Harmful Algal Blooms: State of the Science and Research Needs (Vol. 619, pp. 127-137).
       Springer Press, New York, NY.

Carmichael, W.W., S.M.F.O. Azevedo, J.S. An et al. (2001). Human fatalities from
       cyanobacteria: Chemical and biological evidence for cyanotoxins. Environmental Health
       Perspective 109(7):663-668.

Carriere, A, Prevost, M., Zamyadi, A., et al. (2010). Vulnerability of Quebec drinking water
       treatment plants to cyanotoxins in a climate change context. Journal of Water and
       Health, 5(3): 455-465.

Castenholz, R.W. and Waterbury, J.B.  (1989). In: J.T.  Staley, M.P. Bryant, N. Pfennig and J.G.
       Holt Eds. Bergey's Manual of Systematic Bacteriology. Vol. 3, Williams & Wilkins,
       Baltimore, 1710-1727. (Cited in WHO 1999)

Cheetham, S.A., Smith, AL. et al. (2009). Limited cariation in major urinary proteins of
       laboratory mice. Physiology and Behavior, 95(2): pp.253-361.

Chemical Book. (2012). CAS Index. Retrieved September 25, 2012 from the World Wide Web:
       http://www.chemicalbook.com/Search_EN.aspx?keyword

Chernoff, N., E. H. Rogers, et al. (2011). Toxicity and recovery in the pregnant mouse after
       gestational exposure to the Cyanobacterial toxin, Cylindrospermopsin. Journal of Applied
       Toxicology, 31(3): 242-254.

Chiswell, R., Shaw, K., Eaglesham, G. R., etal. (1999). Stability of Cylindrospermopsin, the
       toxin from the cyanobacterium Cylindrospermopsis raciborskii, effect of pH,
       temperature, and sunlight on decomposition. Environmental Toxicology, 14(1): 155-161.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       36

-------
Chow, C.W.K.., et al. (1998). The effect of ferric chloride flocculation on cyanobacterial cells.
       Water Research, 32(3):808-814.

Chow, C.W. K., et al. (1999). The impact of conventional water treatment processes on cells of
       the cyanobacterium microcystis aeruginosa. Water Research, 33(15):3253-3262.

de la Cruz, A. A.,  Hiskia, A.,  Kaloudis, T., et al. (2013). A review on cylindrospermopsin:  the
       global occurrence, detection, toxicity and degradation of a potent cyanotoxin.
       Environmental Science: Processes and Impacts, 15(11): 1979-2003.

Dixon, M., Falconet C, Ho L., et al. (201 la). Removal of cyanobacterial metabolites by
       nanofiltration from two treated waters. Journal Hazardous Materials, 188(1-3): 288-295.

Dixon, M., Richard, Y., Ho, L., Chow, C., O'Neill, B. andNewcombe, G. (201 Ib). A
       coagulation-powdered activated carbon ultrafiltration, multiple barrier approach for
       removing toxins from two Australian cyanobacterial blooms. Journal of Hazardous
       Materials,  186(2-3): 1553-1559.

Duy, T.N., Lam, P.K.S., Shaw, G.R., and Connell, D.W. (2000). Toxicology and risk assessment
       of freshwater cyanobacterial (blue-green algal) toxins in water. Reviews of Environmental
       Contaminants Toxicology. 163:113-186.

Falconer, I.R. (2005) Cyanobacterial Toxins of Drinking Water Supplies: Cylindrospermopsins
       and Mcrocystins. CRC Press Boca Raton, Florida. 263p

Falconer, I.R. and A.R. Humpage. (2001). Preliminary evidence for in vivo tumour initiation by
       oral administration of extracts of the blue-green alga Cylindrospermopsis raciborskii
       containing the toxin cylindrospermopsin. Environ. Toxicol. 16(2): 192-195.

Fay, P. (1965). Heterotrophy and nitrogen fixation in Chlorogloeafritschii. J. Gen. Microbiol.,
       39:11-20. (Cited in WHO 1999)

Froscio, S. M., E.  Cannon, et al. (2009). Limited uptake of the cyanobacterial toxin
       cylindrospermopsin by Vero cells. Toxicon 54(6): 862-868.

Froscio, S. M., Humpage, A. R., Burcham, P. C., etal. (2003). Cylindrospermopsin-induced
       protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes.
       Environmental Toxicology, 18(4): 243-251.

Froscio, S. M., Humpage, A. R., Wickramasinghe, W., et al.  (2008). Interaction of the
       cyanobacterial toxin cylindrospermopsin with the eukaryotic protein synthesis system.
       Toxicon, 51(2): 191-198.

Funari, E. and Testai, E. (2008). Human Health Risk Assessment Related to Cyanotoxins
       Exposure.  Critical Reviews in Toxicology, 38(2): 97-125.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       37

-------
Global Water Research Coalition (GWRC) (2009). International Guidance Manual for the
      Management of Toxic Cyanobacteria. Ed, G. Newcombe. Global Water Research
      Coalition and Water Quality Research Australia. London, United Kingdom.

Graham, J., Loftin, K., Meyer, M., and Ziegler, A. (2010). Cyanotoxin mixtures and taste-and-
      odor-compounds in cyanobacterial blooms from the midwestern United States.
      Environmental Science and Technology, 44(19): 7361-7368.

Griffiths, D. J. and Saker, M. L. (2003). The Palm Island mystery disease 20 years on: A review
      of research on the cyanotoxin cylindrospermopsin. Environmental Toxicology, 18(2): 78-
      93.

Health Canada. (2012). Toxicity Profile for Cyanobacterial Toxins. Prepared for Water Quality
      and Science Division of Health Canada by MTE GlobalTox. MTE File No.: 36348-100.
      January 27, 2012. 48 pages.

Ho, L., Lambling, P., Bustamante, H, et al. (2008). Biological filtration processes for the
      removal of algal metabolites. The Cooperative Research Center for Water Quality and
      Treatment. Research Report 64. Salisbury, Australia.

Ho, L., McDowall, B., Wijesundara, S., et al. (2011). Application of powdered activated carbon
      for the adsorption of cylindrospermopsin and microcystin toxins from drinking water
      supplies. Water Research, 45(9): 2954-2964.

Hudnell, H.K. (ed.) (2008). Cyanobacterial Harmful Algae Blooms, State of the Science and
      Research Needs. Proceedings of the Interagency, International Symposium on
      Cyanobacterial Harmful Algal Blooms. RTF North Carolina, Sept. 2005. Advances in
      Experimental Medicine & Biology. Springer Science. Vol. 619, 948 pp.

Hudnell, H.K. (2010).The state of U.S. freshwater harmful algal blooms assessments policy and
      legislation. Toxicon 55, pp!024-1034.

Humpage, A. R. and Falconer, I. R. (2002). Oral Toxicity of Cylindrospermopsin: No Observed
      Adverse  Effect Level Determination in Male Swiss Albino Mice.   The  Cooperative
      Research Centre for Water  Quality and Treatment, Salisbury, South Australia.  Research
      Report No. 13.(93 pages).

Humpage, A. R. and Falconer, I. R. (2003). Oral toxicity of the cyanobacterial toxin
      cylindrospermopsin in male Swiss albino mice: Determination of no observed adverse
      effect level for deriving a drinking water guideline value. Environmental Toxicology,
      18(2): 94-103.

Humpage, A. R., Fenech, M, Thomas, P. and Falconer, I. R.  (2000). Micronucleus induction and
      chromosome loss in transformed human white cells indicate clastogenic and aneugenic
      action of the  cyanobacterial toxin, cylindrospermopsin. Mutation Research/Genetic
      Toxicology andEnvironemntalMutagenesis, 472(1-2): 155-161.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       38

-------
Humpage, A. R., Fontaine, F., Froscio, S., Burcham, P., and Falconer, I. R. (2005).
       Cylindrospermopsin genotoxicity and cytotoxicity: Role of cytochrome P-450 and
       oxidative stress. Journal of Toxicology and Environmental Health, Part A., 68(9): 739-
       753.

ILS (Integrated Laboratory Systems) (2000). Cylindrospermopsin: Review of Toxicological
       Literature.  Prepared by Integrated Laboratory Systems for National Toxicology Program,
       NIEHS, USEPA. December 2000. (37 pages.)

Kane, J. P. and Havel, R. J. (1989). Disorders of the biogenesis and secretion of lipoproteins
       containing the B apolipoproteins. In Screiver, C.R., Beaudet, A. L., Sly, W. S., Valle, D.:
       The Metabolic Basis of InhertedDisease. New York, NY: McGraw-Hill (6th ed.) p.
       1154-1155.

Klitzke, S, Beusch, C, and Fastner, J. (2011). Sorption of the cyanobacterial toxins
       Cylindrospermopsin and anatoxin-a to sediments, Water Research, 45(3):1338-46

Klitzke, S., and J. Fastner. (2012). Cylindrospermopsin degradation in sediments—the role of
       temperature, redox conditions, and dissolved organic carbon. Water
       Research,\;46(5): 1549-5 5

Kosakowska, A., Nedzi, M. and Pempkowiak, J. (2007). Responses of the toxic cyanobacterium
       Microcystis aeruginosa to iron and humic substances. Plant Physiology and
       Biochemistry, 45, pp. 365-370.

Loftin, K. and Graham, J. (2014). Occurrence ofCyanobacteria and Associated Toxins in
       Surface Water, Relative Risk, and Potential Changes Due to Environmental Factors in
       the United States. USGS presentation, November 2014. Retrieved on May 30th, 2015
       from the World Wide Web:
       http://ky.water.usgs.gov/proj ects/HABS/Presentations/USGS%20HAB%20Occurrence%
       20and%20Environmental%20Factors%2011_3_2014 .pdf

Lynch, R. and Clyde, T. (2009). A Survey of Cyanobacterial Toxins in Oklahoma Reservoirs.
       Paper presented at the 18th Annual Oklahoma cela Lakes and Wartersheds Association
       Conference on April 1 -3, 2009. Retrieved on September 29, 2012 from the World Wide
       Web: http://www.oclwa.org/pdf/2006%20Presentation%20PDFs/040506_6_lynch.pdf

Marie, M. A., Bazin E., Fessard, V., et al. (2010). Morphological cell transformation of Syrian
       hamster embryo (SHE) cells by the cyanotoxin Cylindrospermopsin. Toxicon, 55(7):
       1317-1322.

Matthijs, H.C.P. et al. (2012). Selective suppression of harmful cyanobacteria in an entire lake
       with hydrogen peroxide. Water Research. 46, 1460-1472.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       39

-------
Ministry of Health. (2008). Drinking-water Standards for New Zealand 2005 (Revised 2008).
       Wellington: Ministry of Health. Retreived from the World Wide Web:
       http://www.health.govt.nz/system/files/documents/publications/drinking-water-standards-
       2008-junl4.pdf

Mohamed, Z. A. and Alamri, S. A. (2012). Biodegradation of cylindrospermopsin toxin by
       microcystin-degrading bacteria isolated from cyanobacterial blooms. Toxicon, 60(8):
       1390-1395.

Moore, M. R., Seawright, A. A., Chiswell, R. R., et al. (1998). The cyanobacterial toxin
       cylindrospermopsin: Its chemical properties and toxicology. Proceedings of the British
       Toxicology Annual Congress, Guilford, England, UK, April 19-22, 1998. Human and
       Experimental Toxicology,  17(9): 469-534.

NHMRC, NRMMC (2011). Australian Drinking Water Guidelines Paper 6 National Water
       Quality Management Strategy. National Health and Medical Research Council, National
       Resource Management Ministerial Council, Commonwealth of Australia, Canberra.
       http://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/eh52_australian_drinki
       ng_water_guidelines_l 50413 .pdf

Newcombe, G., House, J., Ho, L., et al. (2010). Management Strategies for Cyanobacteria (blue-
       green algae): A Guide for Water Utilities. Water Quality Research Australia. Research
       Report 74. Adelaide, Australia. (112 pages).

Newcombe, G., Dreyfus, J., Monrolin, Y., Pestana, C., et al. (2015). Optimizing Conventional
       Treatment for the Removal of Cyanobacteria and Toxins. Water Research Foundation,
       Denver, CO.

Norris, R. L.G., Eaglesham, G. K., Pierens, G., et al. (1999). Deoxycylindrospermopsin, an
       analog of cylindrospermopsin from Cylindrospermopsisrac/'&or.s'A://. Environmental
       Toxicology, 14(1): 163-165

Norris, R. L. G., Seawright, A. A., Shaw, G. R., et al. (2001). Distribution of 14C
       cylindrospermopsin in vivo in the mouse. Environmental Toxicology, 16(6): 498-505.

Norris, R. L. G., Seawright, A. A. Shaw, G. R., et al. (2002). Hepatic xenobiotic metabolism of
       cylindrospermopsin in vivo in the mouse. Toxicon, 40(4): 471-476.

OH EPA (Ohio Environmental Protection Agency) (2012). 2011 Grand Lake St. Marys Algal
       Toxin Sampling Data. Retrieved September 25, 2012 from the World Wide Web:
       http://www. epa. state. oh.us/dsw/HAB.aspx

OH EPA (Ohio Environmental Protection Agency) (2014). Public Water System Harmful Algal
       Bloom Response Strategy. Retrieved April 25, 2014 from the World Wide Web:
       http://epa.ohio.gov/Portal s/28/documents/HABs/PWS_HAB_Response_Strategy_2014.p
       df
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       40

-------
Ohtani, I, R.E. Moore and M.T.C. Runnegar. (1992). Cylindrospermopsin: A potent hepatotoxin
       from the blue-green algae Cylindrospermopsis raciborskii. Journal of American
       Chemical Society. 114(20):7941-7942.

OHA (Oregon Health Authority) (2015). Public Health Advisory Guidelines, Harmful Algae
       Blooms in Freshwater Bodies. Harmful Algae Bloom Surveillance (HABS) Program,
       Public Health Division, Center for Health Protection, Retrieved April 25, 2014 from the
       World Wide Web:
       http://public.health.oregon.gov/HealthyEnvironments/Recreation/HarmfulAlgaeBlooms/
       Documents/HABPublicHealthAdvisoryGuidelines%2020150424x.pdf

Paerl H. W. andHuisman, J. (2008). Blooms like it hot. Science, 320(5872): 57-58

Paerl, H.W. and Otten, T.G. (2013a). Blooms Bite the Hand That Feeds Them. Science. 342, 25,
       433-434.

Paerl, H.W. and Otten, T.G. (2013b). Harmful Cyanobacterial Blooms: Causes, Consequences,
       and Controls. MicrobialEcology. 65: 995-1010.

Papageorgiou, J., Nicholson, B., Wickramasinghe, et al. (2012). Criteria for Quality Control
       Protocols for Various Algal Toxin Methods. Water Research Foundation. Web Report
       #2942.

Pilotto, L., Hobson, P., Burch, M. D., et al. (2004). Acute skin irritant effects of cyanobacteria
       (blue-green algae) in healthy volunteers. Australian and New Zealand Journal of Public
       Health, 28(3): 220-224.

Rai, A.N. (1990). CRC Handbook of Symbiotic Cyanobacteria. CRC Press, Boca Raton, 253 pp.
       (Cited in WHO 1999)

Rajasekhar, P., et al. (2012). A review of the use of soni cation to control cyanobacterial blooms.
       Water Research. 46, 4319-4329.

Rapala, J., M. Niemela, K. Berg, L. Lepisto, and K. Lahti. (2006). Removal of cyanobacteria,
       cyanotoxins, heterotrophic bacteria and endotoxins at an operating surface water
       treatment plant. Water, Science and Technology. 54:3:23.

Reisner, M., Carmeli, S., Werman, M., and Sukenik, A. (2004). The cyanobacterial toxin
       Cylindrospermopsin inhibits pyrimidine nucleotide synthesis and alters cholesterol
       distribution in mice. Toxicological Sciences, 82(2): 620-627.

Rodriguez, E., Onstad, G. D., Kull, T. P. J., et al. (2007). Oxidative elimination of cyanotoxins:
       comparison of ozone, chlorine, chlorine dioxide and permanganate. Water Research,
       41(15): 3381-3393.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       41

-------
Rogers, E. H., Zehr, R. D., Gage, M. I, et al. (2007). The cyanobacterial toxin,
       cylindrospermopsin, induces fetal toxicity in the mouse after exposure late in gestation.
       Toxicon, 49(6): 855-864.

Runnegar, M. T., Kong, S. M., Zhong, Y. Z., and Lu, S. C. (1995). Inhibition of reduced
       glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat
       hepatocytes. Biochemical Pharmacology, 49(2): 219-225.

Sarma, T.A. (2013). Cyanobacterial Toxins in Handbook ofCyanobacteria. CRC Press. Taylor
       and Francis Group, pp. 487-606.

Shaw, G. R., Seawright, A. A., Moore, M. R., and Lam, P. K. (2000). Cylindrospermopsin, a
       cyanobacterial alkaloid: Evaluation of its toxicologic activity.  Therapeutic Drug
       Monitoring, 22(1): 89-92.

Shaw, G. R., Seawright, A. A., and Moore, M. R. (2001). Toxicology and human health
       implications of the cyanobacterial toxin cylindrospermopsin. In: Dekoe, W. J., Samson,
       R. A., van Egmond, H. P., et al. (Eds), Mycotoxins andPhycotoxins in Perspective at the
       Turn of the Millennium. IUPAC & AOAC International, Brazil, pp. 435-443.

Shen, X., Lam, P. K. S., ShawG. R., and Wickramasinghe, W. (2002). Genotoxicity
       investigation of a cyanobacterial toxin, cylindrospermopsin. Toxicon, 40(10): 1499-1501.

Sibaldo de Almeida, C., Costa de Arruda, A. C., Caldas de Queiroz, E., et al. 2013. Oral
       exposure to cylindrospermopsin in pregnant rats: reproduction and foetal toxicity studies.
       Toxicon, 74: pp. 127-129

Sieroslawska, A. (2013). Assessment of the mutagenic  potential of cyanobacterial extracts and
       pure cyanotoxins. Toxicon, 74(0): 76-82.

Stewart, I,  Robertson, I. M., Webb, P. M., et al. (2006). Cutaneous hypersensitivity reactions to
       freshwater cyanobacteria - human volunteer studies. BMC Dermatology, 6:6.

Straser, A., Filipic, M., and Zegura, B. (2011). Genotoxic effects of the cyanobacterial
       hepatotoxin cylindrospermopsin in the HepG2 cell line. Archives of toxicology, 85(12):
       1617-1626.

Sukenik,  A, Reisner, M., Carmeli, S., and Werman, M. (2006). Oral Toxicity of the
       Cyanobacterial Toxin Cylindrospermopsin in Mice: Long-Term Exposure to Low Doses.
       Environmental Toxicology, 21(6): 575-582.

Terao, K., S. Ohmori, K. Igarashi  et al. (1994). Electron microscopic studies on experimental
       poisoning in mice induced by cylindrospermopsin isolated from blue-green alga
       Umezakia natans. Toxicon. 32(7): 833-843.
                Drinking Water Health Advisory for Cylindrospermopsin - June 2015       42

-------
Toxicology Literature Online (TOXLINE) (2012). Toxicology Data Network, National Institute
       of Health. Retrieved on September 25, 2012 from the World Wide Web:
       http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen7TOXLINE

U.S. EPA (United States Environmental Protection Agency). (1986). Guidelines for
       Mutagenicity Risk Assessment. Fed. Reg. 51(185):34006-34012. Available from:
       http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=23160

U.S. EPA (United States Environmental Protection Agency) (1999). Drinking Water Health
       Advisories: Pesticides. Lewis Publishers. ISBN: 978-0-87371-235-4.

U.S. EPA (United States Environmental Protection Agency) (2002). A Review of the Reference
       Dose and Reference Concentration Processes. Risk Assessment Forum, Washington, DC;
       EPA/630/P-02/0002F. Available from: http://www.epa.gov/iris/backgrd.html

U.S. EPA (United States Environmental Protection Agency). (2005). Guidelines for Carcinogen
       Risk Assessment. Risk Assessment Forum, Washington, DC; EPA/630/P-03/001B.
       Available from: http://www.epa.gov/iris/backgrd.html

U.S. EPA (United States Environmental Protection Agency) (2009). National Lakes Assessment:
       A Collaborative  Survey of the Nation's Lakes. EPA 841-R-09-001. Available from:
       http: //www. epa. gov/owow/L AKES/1 akes survey/pdf/nla_report_l ow_r e s. pdf

U.S. EPA (United States Environmental Protection Agency). (201 la). Exposure Factors
       Handbook: 2011 edition. Office of Research and Development, National Center for
       Environmental Assessment, Washington, DC. EPA/600/R-090/052F. 1436pp.
       http://www.epa.gov/ncea/efh/pdfs/efh-complete.pdf

U.S. EPA (United States Environmental Protection Agency). (201 Ib). Recommended Use of
       Body Weight 3/4 as the Default Method in Derivation of the Oral Reference Dose.
       EPA/100/R11/0001.  http://www2.epa.gov/sites/production/files/2013-
       09/documents/recommended-u se-of-bw34.pdf

U.S. EPA (United States Environmental Protection Agency). (2012). Benchmark dose technical
       guidance document [external review draft]. EPA/630/R-00/001. Available from:
       http://www.epa.gov/iris/backgrd.html

U.S. EPA (United States Environmental Protection Agency). (2014). Framework for Human
       Health Risk Assessment to Inform Decision Making. Office of the Science Advisor, Risk
       Assessment Forum, Washington, DC; EPA/100/R-14/001. Available from:
       http://www2.epa.gov/programs-office-science-advisor-osa/framework-human-health-
       risk-assessment-inform-deci si on-making

U.S. EPA (United States Environmental Protection Agency). (2015a). Health Effects  Support
       Document for the Cyanobacterial Toxin Cylindrospermopsin. EPA 820R15101,
       Washington, DC; June, 2015. Available  from:
       http://www2.epa.gov/nutrient-policy-data/health-and-ecological-effects
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       43

-------
U.S. EPA (United States Environmental Protection Agency). (2015b). Recommendations for
       Public Water Systems to Manage Cyanotoxins in Drinking Water. EPA-815R15010,
       Washington, DC; 2015. Available from: http://www2.epa.gov/nutrient-policy-data/
       guidelines-and-recommendations

U.S. EPA (United States Environmental Protection Agency). (2015c) Method 545.
       Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by Liquid
       Chromatography Electrospray ionization Tandem Mass Spectrometry (LC/ESI-MS/MS).
       EPA-815-R-15-009, Cincinnati, OH. Available at:
       http://water. epa.gov/scitech/drinkingwater/labcert/upload/epa815rl 5009.pdf

Wang, Z. et al. (2012). An integrated method for removal of harmful cyanobacterial blooms in
       eutrophic lakes. Environmental Pollution., 160, 34-41.

Westrick J., Szlag, D.,  Southwell, B. and Sinclair, J. (2010). A review of cyanobacteria and
       cyanotoxins removal/inactivation in drinking water treatment. Analytical Bioanalytical
       Chemistry, 391'(5): 1705-1714.

Wimmer, K. M., Strangman, W. K., and Wright, J. L. C. (2014). 7-Deoxy-desulfo-
       cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin: Two new
       Cylindrospermopsin analogs isolated from a Thai strain of Cylindrospermopsis
       raciborskii. Harmful Algae, 37(0): 203-206.

Winslow, S. D., Pepich, B. V., Martin, J. J.,  et al. (2006). Statistical Procedures for
       Determination and Verification of Minimum Reporting Levels for Drinking Water
       Methods. Environmental Science & Technology, 40(1): 281-288.

WHO (World Health Organization). 1999. Toxic Cyanobacteria in Water: A Guide to their
       Public Health Consequences, Monitoring, and Management, I. Chorus and J. Bartram,
       Eds. E&FN Spon, London, UK.

Zegura, B., Gajski, G., Straser, A. and Garaj-Vrhovac, V. (2011).  Cylindrospermopsin induced
       DNA damage and alteration in the expression of genes involved in the response to DNA
       damage, apoptosis and oxidative stress. Toxicon, 58(6-7): 471-479.
               Drinking Water Health Advisory for Cylindrospermopsin - June 2015       44

-------