United States Office of Water EPA- 820R15100
Environmental Mail Code 4304T June 2015
Protection Agency
Drinking Water Health Advisory
for the Cyanobacterial
Microcystin Toxins
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Drinking Water Health Advisory
for the Cyanobacterial Microcystin Toxins
Prepared by:
U.S. Environmental Protection Agency
Office of Water (43 04T)
Health and Ecological Criteria Division
Washington, DC 20460
EPA Document Number: 820R15100
Date: June 15,2015
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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 Microcystins-June 2015
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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 6
2.1 Cyanobacteria and Production of Microcystins 6
2.2 Physical and Chemical Properties 6
2.3 Sources and Occurrence 9
2.3.1 Occurrence in Surface Water 10
2.3.2 Occurrence in Drinking Water 13
2.4 Environmental Fate 14
2.4.1 Persistence 14
2.4.2 Mobility 15
2.5 Nature of the Stressor-Characteristics of the Microcystin Toxins 15
2.5.1 Toxicokinetics 15
2.5.2 Noncancer Health Effects Data 16
2.5.2.1 Human Studies 16
2.5.2.2 Animal Studies 17
2.5.3 Mode of Action for Noncancer Health Effects 18
2.5.4 Carcinogenicity Data 18
2.6 Conceptual Model for Microcystins 19
2.6.1 Conceptual Model Diagram 20
2.6.2 Factors Considered in the Conceptual Model for Microcystins 22
2.7 Analysis Plan 23
3.0 HEALTH EFFECTS ASSESSMENT 26
3.1 Dose-Response 26
3.1.1 Study Selection 26
3.1.2 Endpoint Selection 28
3.2 Ten-day Health Advisory 28
3.2.1 Bottle-fed Infants and Young Children of Pre-school Age 28
3.2.2 School-age Children through Adults 29
3.2.3 Uncertainty Factor Application 29
4.0 RISK CHARACTERIZATION 31
4.1 Use of microcystin-LR as a surrogate for total microcystins 31
Drinking Water Health Advisory for Microcystins-June 2015
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4.2 Consideration of Study Duration 31
4.3 Consideration of Reproductive Effects asEndpoint 32
4.4 Allometric Scaling Approach 33
4.5 Benchmark Dose (BMD) Modeling Analysis 33
4.6 Carcinogenicity Evaluation 33
4.7 Uncertainty and Variability 34
4.8 Susceptibility 35
4.9 Distribution of Body Weight and Drinking Water Intake by Age 35
4.10 Distribution of Potential Health Advisory Values by Age 36
5.0 ANALYTICAL METHODS 38
6.0 TREATMENT TECHNOLOGIES 41
6.1 Management and Mitigation of Cyanobactedal Blooms in Source Water 41
6.2 Drinking Water Treatment 42
6.2.1 Conventional Treatment for Microcystins 43
6.2.2 Adsorption 44
6.2.3 Chemical Oxidation 45
6.2.4 Other Filtration Technologies 46
6.2.5 Combined Treatment Technologies 47
6.3 Point-of-Use (POU) Drinking Water Treatment Units 48
7.0 REFERENCES 49
Drinking Water Health Advisory for Microcystins-June 2015 iii
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LIST OF TABLES
Table 1-1. International Guideline Values for Microcystins 4
Table 1-2. State Guideline Values for Microcystins 5
Table 2-1. Abbreviations for Microcystins (Yuan et al., 1999) 8
Table 2-2. Chemical and Physical Properties of Microcystin-LR 9
Table 2-3. States surveyed as part of the 2007 National Lakes Assessment with water body
Microcystin concentrations above the WHO advisory guideline level for recreational water
of 10|ig/L(U.S. EPA, 2009) 11
Table 3-1. Liver Weights and Serum Enzyme Levels in Rats Ingesting Microcystin-LR in
Drinking Water (Heinze, 1999) 27
Table 3-2. Histological Evaluation of the Rat Livers after Ingesting Microcystin-LR in Drinking
Water (Heinze, 1999) 27
LIST OF FIGURES
Figure 2-1. Structure of Microcystin (Kondoetal., 1992) 7
Figure 2-2. Structure of the amino acids Adda and Mdha (Harada et al., 1991) 8
Figure 2-3. Conceptual Model of Exposure Pathways to Microcystins in Drinking Water 21
Figure 4-1. 90th Percentile Drinking Water Ingestion Rates by Age Group 35
Figure 4-2. Ten-day Health Advisories for Microcystins by Age Group 37
Drinking Water Health Advisory for Microcystins-June 2015 iv
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ABBREVIATIONS AND ACRONYMS
rgt
A
Adda
ALT
ALP
AST
AWWARF
BMD
BMDL
BW
C102
CAS
CASA
CCL
CWA
DBF
DL
EBCT
ELISA
EPA
FSH
g
GC/MS
GAC
GLERL
H202
HA
HAB
HESD
HPLC
IARC
i.p.
Kg
L
LC/MS
LDH
LD50
LH
LOAEL
MC-LA
MC-LR
MC-RR
MC-YR
MC-YM
F-Glutamyltransferase
Alanine
3-Amino-9-Methoxy-2,-6,-8-,Trimethyl-10-Phenyldeca-4,-6-Dienoic Acid
Alanine Aminotransferase
Alkaline Phosphatase
Aspartate Aminotransferase
American Water Works Association Research Foundation
Benchmark Dose
Benchmark Dose Level
Body Weight
Chlorine Dioxide
Chemical Abstracts Service
Computer-Assisted Sperm Analysis
Contaminant Candidate List
Clean Water Act
Disinfection By-Products
Detection Limit
Empty Bed Contact Time
Enzyme-Linked Immunosorbent Assay
U.S. Environmental Protection Agency
Follicle Stimulating Hormone
Gram
Gas Chromatograph/Mass Spectrometry
Granular Activated Carbon
Great Lakes Environmental Research Laboratory
Hydrogen Peroxide
Health Advisory
Harmful Algal Bloom
Health Effects Support Document
High-Performance Liquid Chromatography
International Agency for Research on Cancer
Intraperitoneal
Kilogram
Leucine
Liquid Chromatography/Mass Spectrometry
Lactate Dehydrogenase
Lethal Dose to 50% of Organisms
Luteinizing Hormone
Lowest-Observed-Adverse-Effect Level
Microcystin-LA
Microcystin-LR
Microcystin-RR
Microcystin-YR
Microcystin-YM
Drinking Water Health Advisory for Microcystins-June 2015
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Mdha
MERHAB-LGL
US
LOQ
Mdls
mg
ml
MMPB
MOA
MF
MWCO
NDEA
NF
NLA
NOAA
NOAEL
NOD
NOM
OATp
PAC
PAS
PBS
PDA
P-GST
POD
POU
PP2
PP1
PPIA
RfD
RO
ROS
SDWA
SPE
TEF
TOC
TOXLINE
TUNEL
UF
UF
USGS
UV
WHO
Methyl dehy droal anine
Monitoring and Event Response to Harmful Algal Blooms in the Lower
Great Lakes
Microgram
Micromole
Level of Quantification
Method Detection Limit
Milligram
Milliliter
2-methyl-3-methoxy-4-phenylbutyric acid
Mode of Action
Microfiltration
Molecular Weight Cut-Off
N-Nitrosodiethylamine
Nanofiltration
National Lakes Assessment
National Oceanic and Atmospheric Administration
No-Observed-Adverse-Effect Level
Nodularin
Natural Organic Material
Organic Acid Transporter Polypeptides
Powdered Activated Carbon
Periodic Acid-Schiff
Phosphate-Buffered Saline
Photodiode Array Detector
glutathione S-transferase placental form-positive
Point of Departure
Point-of-Use
Protein Phosphatase 2A
Protein Phosphatase 1
Protein Phosphatase Inhibition Assays
Reference Dose
Reverse Osmosis
Reactive Oxygen Species
Safe Drinking Water Act
Solid-Phase Extraction
Toxicity Equivalency Factors
Total Organic Carbon
Toxicology Literature Online
Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End-
Labeling Assay
Uncertainty Factor
Ultrafiltration
United States Geological Survey
Ultraviolet
World Health Organization
Drinking Water Health Advisory for Microcystins-June 2015
VI
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EXECUTIVE SUMMARY
Microcystins are toxins produced by a number of cyanobacteria species, including
members ofMicrocystis, Anabaena, Nodularia, Nostoc, Oscillatoria, Fischerella, Planktothrix,
and Gloeotrichia. Approximately 100 microcystin congeners exist, which vary based on amino
acid composition. Microcystin-LR is one of the most potent congeners and the majority of
toxicological data on the effects of microcystins are available for this congener.
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
microcystin blooms, both in fresh and marine water systems and could encourage toxin
production. Microcystins are water soluble and tend to remain contained within the cyanobacterial
cell (intracellular), until the cell breaks and they are released into the water (extracellular).
This Health Advisory (HA) for microcystins 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 data in humans and animals demonstrate the absorption of microcystins from the
intestinal tract and distribution to the liver, brain, and other tissues. Elimination from the body
requires facilitated transport using receptors belonging to the Organic Acid Transporter
polypeptide (OATp) family. Data for humans and other mammals suggest that the liver is a
primary site for binding these proteins (i.e., increased liver weight in laboratory animals and
increased serum enzymes in laboratory animals and humans). Once inside the cell, these toxins
covalently bind to cytosolic proteins (PP1 and PP2) resulting in their retention in the liver.
Limited data are available on the metabolism of microcystins, but most of the studies indicate that
microcystins can be conjugated with glutathione and cysteine to increase their solubility and
facilitate excretion.
The main source of human health effects data for microcystins is from acute recreational
exposure to cyanobacterial blooms. Symptoms include headache, sore throat, vomiting and
nausea, stomach pain, dry cough, diarrhea, blistering around the mouth, and pneumonia.
However, human data on the oral toxicity of microcystins are limited and confounded by:
potential co-exposure to other contaminants; a lack of quantitative information; and other
confounding factors. Reports of human intravenous exposure to dialysate prepared with
microcystin-contaminated water indicated acute liver failure and death in a large number of the
exposed patients.
Studies in laboratory animals demonstrate liver, kidney, and reproductive effects
following short-term and subchronic oral exposures to microcystin-LR. Studies evaluating the
chronic toxicity of microcystins have not shown clinical signs of toxicity and are limited by study
design and by the lack of quantitative data.
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The U.S. Environmental Protection Agency (EPA) identified a study by Heinze (1999)
conducted on rats as the critical study used in the derivation of the reference dose (RfD) for
microcystins. The critical effects identified in the study are increased liver weight, slight to
moderate liver lesions with hemorrhages, and increased enzyme levels as a result of exposure to
microcystin-LR. The lowest-observed-adverse-effect level (LOAEL) was determined to be 50
ug/kg/day, based on these effects. The drinking water route of exposure matches potential
drinking water exposure scenarios in humans. The total uncertainty factor (UF) applied to the
LOAEL was 1000. This was based on a UF of 10 for intraspecies variability, a UF of 10 for
interspecies variability, a UF of 3 (101/2) for extrapolation from a LOAEL to no-observed-adverse-
effect level (NOAEL), and a UF of 3 (101/2) to account for deficiencies in the database. EPA is
using microcystin-LR as a surrogate for other microcystin congeners. Therefore, the HA based on
this critical study applies to total microcystins.
EPA is issuing a Ten-day HA for microcystins based on the Heinze (1999) short-term, 28-
day study. Studies of a duration of 7 to 30 days are typically used to derive Ten-day HAs. The HA
is consistent with this duration and appropriately matches human exposure scenarios for
microcystins in drinking water. Cyanobacterial blooms are usually seasonal, typically occurring
from May through October. Microcystins typically have a half-life of 4 days to 14 days in surface
waters, (depending on the degree of sunlight, natural organic matter, and the presence of bacteria)
and can be diluted via transport. 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 0.3
|ig/L and for school-age children through adults is 1.6 |ig/L for microcystins. The two advisory
values use the same toxicity data (RfD) and represent differences in drinking water intake and
body weight for different 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. The
second advisory value is based on the mean body weight and 90th percentile drinking water
consumption rates 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 population to the health effects of microcystins. 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.
Applying the U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment, there is
inadequate information to assess carcinogenic potential of microcystins. The few available
epidemiological studies are limited by their study design, poor measures of exposure, potential
co-exposure to other contaminants, and the lack of control for confounding factors. No long term
animal studies were available to evaluate dose-response for the tumorigenicity of microcystins
following lifetime exposures. Other studies evaluating the tumor promotion potential of
microcystin found an increase in the number and/or size of GST-P positive foci observed. In two
promotion studies, microcystin-LR alone showed no initiating activity.
Drinking Water Health Advisory for Microcystins-June 2015
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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 Microcystim (U.S.EPA, 2015 a) 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 EP A's Framework for Human Health
Risk Assessment to Inform Decision Making (U.S.EPA, 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).
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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 cylindrospermopsin to assist State and local officials in evaluating risks from
these contaminants in drinking water.
Internationally, eighteen countries and three U.S. states have developed drinking water
guidelines for microcystins, as shown in Table 1.1 and Table 1.2, respectively, based on lifetime
exposures.
Table 1-1. International Guideline Values for Microcystins
Country
Guideline Value
Source
Brazil, China, Czech Republic,
Denmark, Finland, France, Germany,
Italy, Japan, Korea, Netherlands,
Norway, New Zealand, Poland,
South Africa, and Spain
1.0 ug/L
microcystin-LR
Based on the World Health
Organization (WHO) Provisional
Guideline Value of lug/L for
drinking water
(WHO, 1999; 2003)
Australia
1.3 ug/L microcystin-
LR (toxicity
equivalents)
Australian Drinking Water
Guidelines 6
(NHMRC, NRMMC, 2011)
Canada
1.5 ug/L
microcystin-LR
Guidelines for Canadian
Drinking Water Quality:
Supporting Documentation
Cyanobacterial Toxins-
Microcystin-LR
(Health Canada, 2002)
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Table 1-2. State Guideline Values for Microcystins
State
Minnesota
Ohio
Oregon
Guideline Value
0.04 |ig/L Microcystin-LR
1 ng/L Microcystin
1 |ig/L Microcystin-LR
Source
Minnesota Department of Health (MDH, 2012)
Public Water System Harmful Algal Bloom
Response Strategy (Ohio EPA, 2014)
Public Health Advisory Guidelines, Harmful Algae
Blooms in Freshwater Bodies. (OHA, 2015)
For drinking water, the provisional WHO Guideline value for microcystin-LR of 1 ug/L
(or the underlying Tolerable Daily Intake (TDI) of 0.04 ug/kg) has been widely used as the basis
for national standards or guideline values (WHO, 1999, 2003). Following the release of the WHO
provisional guideline, drinking-water standards or national guideline values were adopted in 16
countries. Australia and Canada have used the TDI, but have adapted other factors in the
calculation to reflect their national circumstances (e.g. body weight or amounts of water
consumed), thus reaching somewhat higher guidance values or standards (Chorus, 2012). A few
countries have issued guideline values specifically for microcystin-LR while others use
microcystin-LR as a surrogate for all microcystin congeners (i.e. toxicity equivalents). Values are
similar across all countries, ranging between 1.0 and 1.5 ug/L based on lifetime exposures.
Drinking Water Health Advisory for Microcystins-June 2015
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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 Microcystim (U.S. EPA, 2015 a).
2.1 Cyanobacteria and Production of Microcystins
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 (N, P 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).
Microcystins are produced by several cyanob acted al species, including Anabaena,
Fischerella, Gloeotrichia, Nodularia, Nostoc, Oscillatoria., members ofMicrocystis, and
Planktothrix (Duy et al., 2000; Codd et al., 2005; Stewart et al., 2006a; Carey et al., 2012).
2.2 Physical and Chemical Properties
The cyclic peptides include around 100 congeners of microcystins. Table 2-1 lists only the
most common microcystins congeners. Figure 2-1 provides the structure of microcystin where X
and Y represent variable amino acids. Although substitutions mostly occur in positions X and Y,
other modifications have been reported for all of the amino acids (Puddick et al., 2015). The
amino acids are joined end-to-end and then head to tail to form cyclic compounds that are
comparatively large (molecular weights ranging from -800 to 1,100 g/mole).
Drinking Water Health Advisory for Microcystins-June 2015
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Figure 2-1. Structure of Microcystin (Kondo et al., 1992)
C02H
Microcystin congeners vary based on their amino acid composition and through
methylation or demethylation at selected sites within the cyclic peptide (Duy et al., 2000). The
variations in composition and methylation account for the large number of toxin congeners. The
microcystins are named based on their variable amino acids, although they have had many other
names (Carmichael et al., 1988). For example, microcystin-LR, the most common congener,
contains leucine (L) and arginine (R) (Carmichael, 1992). The letters used to identify the variable
amino acids are the standard single letter abbreviations for the amino acids found in proteins. The
variable amino acids are usually the L-amino acids as found in proteins. In this HA, the term
microcystin may be followed by the abbreviations for the variable amino acids. For example,
microcystin-LR is for the microcystin with leucine in the X position of Figure 2-1 and arginine in
the Y position. Most research has concentrated on microcystin-LR, with lesser amounts of data
available for the other amino acid combinations. For the purpose of this HA, microcystin-LR is
used as the surrogate for total microcystins.
Structurally, the microcystins are monocyclic heptapeptides that contain seven amino
acids: two variable L-amino acids, three common D-amino acids or their derivatives, and two
novel D-amino acids. These two D-amino acids are: 3S-amino-9S-methoxy-2,6,8S,-trimethyl-10-
phenyldeca-4,6-dienoic acid (Adda) and methyldehydroalanine (Mdha). Adda is characteristic of
all toxic microcystin structural congeners and is essential for their biological activity (Rao et al.,
2002; Funari and Testai, 2008). Mdha plays an important role in the ability of the microcystins to
inhibit protein phosphatases. Figure 2-2 illustrates the structures of these two unique amino acid
microcystin components.
Microcystins are water soluble. In aquatic environments, the cyclic peptides tend to
remain contained within the cyanobacterial cell and are released in substantial amounts only upon
cell lysis. The microcystins are most frequently found in cyanobacterial blooms in fresh and
brackish waters (WHO, 1999). Table 2-2 provides chemical and physical properties of
microcystin-LR.
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Table 2-1. Abbreviations for Microcystins (Yuan et al., 1999)
Microcystin Congeners
Microcystin-LR
Microcystin-RR
Microcystin-YR
Microcystin-LA
Microcystin-LY
Microcystin-LF
Microcystin-LW
Amino Acid in X
Leucine
Arginine
Tyrosine
Leucine
Leucine
Leucine
Leucine
Amino Acid in Y
Arginine
Arginine
Arginine
Alanine
Tyrosine
Phenylalanine
Tryptophan
Figure 2-2. Structure of the amino acids Adda and Mdha (Harada et al., 1991).
20
CH 0
H2C
3
Adda
Mdha
Drinking Water Health Advisory for Microcystins-June 2015
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Table 2-2. Chemical and Physical Properties of Microcystin-LR
Property
Chemical Abstracts Registry (CAS) #
Chemical Formula
Molecular Weight
Color/Physical State
Boiling Point
Melting Point
Density
Vapor Pressure at 25°C
Henry's Law Constant
J^-OW
Koc
Solubility in Water
Other Solvents
Microcystin-LR
101043-37-2
C49H?4NloOl2
995.17g/mole
Solid
N/A
N/A
1.29g/cm3
N/A
N/A
N/A
N/A
Highly
Ethanol and methanol
Sources: Chemical Book, 2012; TOXLINE, 2012
2.3
Sources and Occurrence
Cyanotoxin production is strongly influenced by the environmental conditions that
promote growth of particular cyanobactedal species and strain. Nutrient concentrations, light
intensity, water turbidity, temperature, competing bacteria and phytoplankton, pH, turbulence,
and salinity are all factors that affect cyanobacterial growth and change in cyanobacteria
population dynamics. Although environmental conditions affect the formation of blooms, the
numbers of cyanobacteria and toxin concentrations produced are not always closely related.
Cyanotoxin concentrations depend on the dominance and diversity of strains within the bloom
along with environmental and ecosystem influences on bloom dynamics (Hitzfeld et al., 2000;
Chorus et al., 2000; WHO, 1999). Extracellular microcystins (either dissolved in water or bound
to other materials) typically make up less than 30% of the total microcystin concentration in
source water (Graham et al., 2010). Most of the toxin is intracellular, and released into the water
when the cells rupture or die. Both intracellular and extracellular microcystins may also be
present in treated water, depending on the type of treatment processes in place.
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2.3.1 Occurrence in Surface Water
Microcystins are the most common cyanotoxin found worldwide and have been reported
in surface waters in most of the U.S. and Europe (Funari and Testai, 2008). Dry-weight
concentrations of microcystins in surface freshwater cyanobactedal blooms or surface freshwater
samples reported worldwide between 1985 and 1996 ranged from 1 to 7,300 |ig/g. Water
concentrations of extracellular plus intracellular microcystins ranged from 0.04 to 25,000 |ig/L.
The concentration of extracellular microcystins ranged from 0.02 to a high of 1,800 |ig/L reported
following treatment of a large cyanobacteria bloom with algaecide (WHO, 1999) and the U.S.
Geological Survey (USGS) reported a concentration of 150,000 |ig/L total microcystins, in a lake
in Kansas (Graham et al., 2012).
According to a survey conducted in Florida in 1999 between the months of June and
November, the most frequently observed cyanobacteria wereMicrocystis (43.1%),
Cylindrospermopsis (39.5%), andAnabaena spp (28.7%) (Burns, 2008). Of 167 surface water
samples taken from 75 waterbodies, 88 samples were positive for cyanotoxins. Microcystin was
the most commonly found cyanotoxin in water samples collected, occurring in 87 water samples.
In 2002, the Monitoring and Event Response to Harmful Algal Blooms in the Lower Great
Lakes (MERHAB-LGL) project evaluated the occurrence and distribution of cyanobacterial
toxins in the lower Great Lakes region (Boyer, 2007). Analysis for total microcystins was
performed using Protein Phosphatase Inhibition Assay (PPIA). Microcystins were detected in at
least 65% of the samples, mostly in Lake Erie, Lake Ontario, and Lake Champlain. The National
Oceanic and Atmospheric Administration (NOAA) Center of Excellence for Great Lakes and
Human Health (CEGLHH) continues to monitor the Great Lakes and regularly samples algal
blooms for microcystin in response to bloom events.
A 2004 study of the Great Lakes found high levels of cyanobacteria during the month of
August (Makarewicz et al., 2006). Microcystin-LR was analyzed by PPIA (limit of detection of
0.003 |ig/L) and was detected at levels of 0.084 ug/L in the nearshore and 0.076 ug/L in the bays
and rivers. This study reported higher levels of microcystin-LR (1.6 to 10.7ug/L) in smaller lakes
in the Lake Ontario watershed.
In 2006, the USGS conducted a study of 23 lakes in the Midwestern U.S. in which
cyanobacterial blooms were sampled to determine the co-occurrence of toxins in cyanobacterial
blooms (Graham et al., 2010). This study reported that microcystins were detected in 91% of the
lakes sampled. Mixtures of all the microcystin congeners measured (LA, LF, LR, LW, LY, RR,
and YR) were common and all the congeners were present in association with the blooms.
Microcystin—LR and -RR were the dominant congeners detected with mean concentrations of
104 and 910 ug/L, respectively.
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 the nation's lakes, ponds and reservoirs (U.S.EPA,
2009). This baseline study of the condition of the nation's lakes provided estimates of the
condition of natural and man-made freshwater lakes, ponds, and reservoirs greater than 10 acres
and at least one meter deep. A total of 1,028 lakes were sampled in the NLA during the summer
Drinking Water Health Advisory for Microcystins-June 2015 10
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of 2007. The NLA measured microcystins using Enzyme Linked Immunosorbent Assays (ELISA)
with a detection limit of 0.1 jig/L as well as cyanob acted al cell counts and chlorophyll-a
concentrations, which were indicators of the presence of cyanob acted al toxins. Samples were
collected in open water at mid-lake. Due to the design of the survey, no samples were taken
nearshore or in other areas where scums were present.
A total of 48 states were sampled in the NLA, and states with lakes reporting microcystins
levels above the WHO's moderate risk1 threshold in recreational water (>10 |ig/L) are shown in
Table 2-3. Microcystins were present in 30% of the lakes sampled nationally, with sample
concentrations that ranged from the limit of detection (0. 1 |ig/L) to 225 |ig/L. Two states (North
Dakota and Nebraska), had 9% of samples above 10 |ig/L. Other states including Iowa, Texas,
South Dakota, and Utah also had samples that exceeded 10 |ig/L. Several samples in North
Dakota, Nebraska, and Ohio exceeded the WHO high risk threshold value for recreational waters
of 20 |ig/L (192 and 225 |ig/L, respectively). EPA completed a second survey of lakes in 2012,
but data have not yet been published.
Microcystins have been detected in most of the states of the U.S., and over the years many
studies have been done to determine their occurrence in surface water. USGS, for example, did a
study in the Upper Klamath Lake in Oregon in 2007 and detected total microcystin concentrations
between 1 |ig/L and 17 |ig/L (VanderKooi et al., 2010). USGS also monitored Lake Houston in
Texas from 2006 to 2008, and found microcystins in 16% of samples with concentrations less
than or equal to 0.2 |ig/L (Beussink and Graham, 201 1). In 201 1, USGS conducted a study on the
upstream reservoirs of the Kansas River, a primary source of drinking water for residents in
northeastern Kansas, to characterize the transport of cyanobacteria and associated compounds
(Graham et al., 2012). Concentrations of total microcystin were low in the majority of the
tributaries with the exception of Milford Lake, which had higher total microcystin concentrations,
some exceeding the Kansas recreational guidance level of 20 |ig/L. Upstream from Milford Lake,
a cyanob acted al bloom was observed with a total microcystin concentration of 150,000 |ig/L.
When sampled a week later, total microcystin concentrations were less than 1 |ig/L. The study
authors indicated that this may be due to dispersion of microcystins through the water column or
to other areas, or by degradation of microcystins via abiotic and biological processes. Samples
taken during the same time from outflow waters contained total microcystin concentrations of 6.2
In 2005, Washington State Department of Ecology developed the Ecology Freshwater
Algae Program to focus on the monitoring and management of cyanobacteria in Washington
lakes, ponds, and streams (WSDE, 2012). The data collected have been summarized in a series of
reports for the Washington State Legislature (Hamel, 2009, 2012). Microcystin levels ranged
from the detection limit (0.05 |ig/L) to 4,620 |ig/L in 2008, 18,700 |ig/L in 2009, 853 |ig/L in
20 1 0, and 26,400 |ig/L in 20 1 1 .
1 The WHO established guideline values for recreational exposure to cyanobacteria using a three-tier approach: low
risk (<20,000 cyanobacterial cells/ml corresponding to <10 ug/L of MC-LR); moderate risk (20,000-100,000
cyanobacterial cells/ml corresponding to 10-20 ug/L of MC-LR); and high risk (>100,000 cyanobacterial cells/ml
corresponding to >20 ug/L for MC-LR).
Drinking Water Health Advisory for Microcystins-June 2015 11
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Table 2-3. States Surveyed as Part of the 2007 National Lakes Assessment with Water Body
Microcystins Concentrations above the WHO Advisory Guideline Level for Recreational
Water of 10 ug/L (U.S. EPA, 2009)
State
North Dakota
Nebraska
South Dakota
Ohio
Iowa
Utah
Texas
Number
of Sites
Sampled
38
42
40
21
20
26
51
Percentage of Samples
with Detection of
Microcystins >10 ug/L
9.1%
9.1%
4.9%
4.5%
4.5%
3.6%
1.8%
Maximum Detection
of
Microcystins
192 |ig/L
225 |ig/L
33,ig/L
78 |ig/L*
38 |ig/L*
15 |ig/L*
28 |ig/L *
Single Sample
Other surveys and studies have been conducted to determine the occurrence of
microcystin in lakes in the United States. A survey conducted during the spring and summer of
1999 and 2000 in more than 50 lakes in New Hampshire found measureable microcystin
concentrations in all samples (Haney and Ikawa, 2000). Microcystins were analyzed by ELISA
and were found in all of the lakes sampled with a mean concentration of 0.1 |ig/L. In 2005 and
2006, a study conducted in New York, including Lake Ontario, found variability in microcystin-
LR concentrations within the Lake Ontario ecosystem (Makarewicz et al., 2009). Of the samples
taken in Lake Ontario coastal waters, only 0.3% of the samples exceeded the WHO provisional
guideline value for drinking water of 1 |ig/L. However, 20.4% of the samples taken at upland
lakes and ponds within the Lake Ontario watershed, some of them sources of drinking water,
exceeded 1 |ig/L. During 2008 and 2009, a study was conducted in Kabetogama Lake, Minnesota
which detected microcystin concentrations in association with algal blooms (Christensen et al.,
2011). Microcystin concentrations were detected in 78% of bloom samples. Of these, 50% were
above 1 ug/L, and two samples were above the high risk WHO recreational level of 20 ug/L.
A study from 2002 evaluated water quality including chlorophyll-a concentration,
cyanobacterial assemblages, and microcystin concentrations in 11 potable water supply reservoirs
within the North Carolina Piedmont during dry summer growing seasons (Touchette et al., 2007).
Microcystins concentrations were assessed using ELISA. The study found that cyanobacteria
were the dominant phytoplankton community, averaging 65-95% of the total phytoplankton cells.
Although microcystin concentrations were detected in nearly all source water samples,
concentrations were <0.8 ug/L.
Since 2007, Ohio EPA (OHEPA, 2012) has been monitoring inland lakes for cyanotoxins.
Of the 19 lakes in Ohio sampled during the NLA, 36% had detectable levels of microcystins. In
Drinking Water Health Advisory for Microcystins-June 2015
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2010, OHEPA sampled Grand Lake St. Marys for anatoxin-a, cylindrospermopsin, microcystins,
and saxitoxin. Toxin levels ranged from below the detection limit (<0.15 ug/L) to more than
2,000 ug/L for microcystins. Follow-up samples taken in 2011 for microcystins indicated
concentrations exceeding 50 ug/L in August. During the same month, sampling in Lake Erie
found microcystins levels exceeding 100 ug/L.
In 2008, NOAA began monitoring for cyanobactedal blooms in Lake Erie using high
temporal resolution satellite imagery. Between 2008 and 2010, Microcystis cyanobacterial blooms
were detected associated with water temperatures above 18°C (Wynne et al., 2013). Using the
Great Lakes Coastal Forecast System (GLCFS), forecasts of bloom transport are created to
estimate the trajectory of the bloom, and these are distributed as bulletins to local managers,
health departments, researchers and other stakeholders. To evaluate bloom toxicity, the Great
Lakes Environmental Research Laboratory (GLERL) collected samples at six stations each week
for 24 weeks, measuring toxin concentrations as well as chlorophyll biomass and an additional 18
parameters (e.g., nutrients) to improve future forecasts of these blooms. In 2014, particulate toxin
concentrations, collected from 1 meter depth, ranged from below detection to 36.7
ug/L. Particulate toxin concentrations peaked in August, 2014 at all sites, with the Maumee Bay
site yielding the highest toxin concentration of the entire sampling period. Dissolved toxin
concentrations were collected at each site from September until November when the field season
ended. During the final months of sampling (October-November), dissolved toxin concentrations
were detected with peak concentrations of 0.8 ug/L (mean: 0.28 +/- 0.2 ug/L) whereas particulate
toxin concentrations were below detection limits on many dates, indicating that a majority of the
toxins (mean: 72% +/- 37%) were in the dissolved pool as the bloom declined in intensity.
Concentrations of microcystins were detected during sampling in 2005 and 2006 in lakes
and ponds used as a source of drinking water within the Lake Ontario watershed (Makarewicz et
al., 2009). A microcystin-LR concentration of 5.07 ug/L was found in Conesus Lake, a source of
public water supply that provides drinking water to approximately 15,000 people. Microcystin-LR
was also detected at 10.716 ug/L in Silver Lake, a public drinking water supply for four
municipalities.
2.3.2 Occurrence in Drinking Water
The occurrence of cyanotoxins in drinking water depends on their levels in the raw source
water and the effectiveness of treatment methods for removing cyanobacteria and cyanotoxins
during the production of drinking water. Currently, there is no program in place to monitor for the
occurrence of cyanotoxins at surface-water treatment plants for drinking water in the U.S.
Therefore, data on the presence or absence of cyanotoxins in finished drinking water are limited.
The American Water Works Association Research Foundation (AWWARF) conducted a
study on the occurrence of cyanobacterial toxins in source and treated drinking waters from 24
public water systems in the United States and Canada in 1996-1998 (AWWARF, 2001). Of 677
samples tested, microcystin was found in 80% (539) of the waters sampled, including source and
treated waters. Only two samples of finished drinking water were above 1 ug/L. A survey
conducted in 2000 in Florida (Burns, 2008) reported that microcystins were the most commonly
Drinking Water Health Advisory for Microcystins-June 2015 13
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found toxin in pre- and post-treated drinking water. Finished water concentrations ranged from
below detection levels to 12.5 |ig/L.
During the summer of 2003, a survey was conducted to test for microcystins in 33 U.S.
drinking water treatment plants in the northeastern and Midwestern U.S. (Haddix et al., 2007).
Microcystins were detected at low levels ranging from undetectable (<0.15 |ig/L) to 0.36 jig/L in
all 77 finished water samples.
In August 2014, the city of Toledo, Ohio issued a "do not drink or boil advisory" to nearly
500,000 customers in response to the presence of total microcystins in the city's finished drinking
water at levels up to 2.50 |ig/L. The presence of the toxins was due to a cyanob acted al bloom
near Toledo's drinking water intake located on Lake Erie. The advisory was lifted two days later,
after treatment adjustments led to the reduction of the cyanotoxin concentrations to concentrations
below the WHO guideline value of 1 |ig/L in all samples from the treatment plant and distribution
system.
2.4 Environmental Fate
Different physical and chemical processes are involved in the persistence, breakdown, and
movement of microcystins in aquatic systems as described below.
2.4.1 Persistence
Microcystins are relatively stable and resistant to chemical hydrolysis or oxidation at or
near neutral pH. Elevated or low pH or temperatures above 30°C may cause slow
hydrolysis. Microcystin is not destroyed by boiling (Rao et al., 2002). In natural waters kept in the
dark, microcystins have been observed to persist for 21 days to 2-3 months in solution and up to 6
months in dry scum (Rapala et al., 2006; Funari and Testai, 2008).
In the presence of full sunlight, microcystins undergo photochemical breakdown, but this
varies by microcystin congener (WHO, 1999; Chorus et al., 2000). The presence of water-soluble
cell pigments, in particular phycobiliproteins, enhances this breakdown. Breakdown can occur in
as few as two weeks to longer than six weeks, depending on the concentration of pigment and the
intensity of the light (Tsuji et al., 1993; 1995). According to Tsuji et al, microcystin-LR was
photodegraded with a half-life (time it takes half of the toxin to degrade) of about 5 days in the
presence of 5 mg/L of extractable cyanobacterial pigment. Humic substances can also act as
photosensitizers and can increase the rate of microcystin breakdown in sunlight. In deeper or
turbid water, the breakdown rate is slower.
Microcystins are susceptible to degradation by aquatic bacteria found naturally in rivers
and reservoirs (Jones et al., 1994). Bacteria isolates ofArthrobacter, Brevibacterium,
Rhodococcus, Paucibacter, and various strains of the genus Sphingomonas (Pseudomonas) have
been reported to be capable of degrading microcystin-LR (de la Cruz et al., 2011; Han et al.,
2012). These degradative bacteria have also been found in sewage effluent (Lam et al., 1995),
lake water (Jones et al., 1994; Cousins et al., 1996; Lahti et al., 1997a), and lake sediment (Rapala
Drinking Water Health Advisory for Microcystins-June 2015 14
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et al., 1994; Lahti et al., 1997b). Lam et al., in 1995 reported that the biotransformation of
microcystin-LR followed a first-order decay with a half-life of 0.2 to 3.6 days (Lam et al., 1995).
In a study done by Jones et al. (1994) with microcystin-LR in different natural surface waters,
microcystin-LR persisted for 3 days to 3 weeks; however, more than 95% loss occurred within 3
to 4 days. A study by Christoffersen et al., 2002, measured half-lives in the laboratory and in the
field of approximately 1 day, driven largely by bacterial aerobic metabolism. These researchers
found that approximately 90% of the initial amount of microcystin disappeared from the water
phase within 5 days, irrespective of the starting concentration. Other researchers (Edwards et al.,
2008) have reported half-lives of 4 to 14 days, with longer half-lives associated with a flowing
stream and shorter half-lives associated with lakes.
2.4.2 Mobility
Microcystins may adsorb onto naturally suspended solids and dried crusts of
cyanobacteria. They can precipitate out of the water column and reside in sediments for months
(Han et al., 2012: Falconer, 1998). Ground water is generally not expected to be at risk of
cyanotoxin contamination, however, ground water under the direct influence of surface water can
be vulnerable. A study conducted by the USGS and the University of Central Florida determined
that microcystin and cylindrospermopsin did not sorb in sandy aquifers and were transported
along with ground water (O'Reilly et al., 2011). The authors suggested that the removal of
microcystin was due to biodegradation.
2.5 Nature of the Stressor-Characteristics of the Microcystin Toxins
2.5.1 Toxicokinetics
No data were available that quantified the intestinal, respiratory or dermal absorption of
microcystins. Available data indicate that the Organic Acid Transporter polypeptide (OATp)
receptors facilitate the absorption of toxins from the intestinal tract into liver, brain, and other
tissues. The OATp family transporters facilitate the cellular, sodium-independent uptake and
export of amphipathic compounds such as bile salts, steroids, drugs, peptides and toxins (Cheng et
al., 2005; Fischer et al., 2005; Svoboda et al., 2011). This facilitated transport is necessary for
both uptake of microcystins into organs and tissues as well as for their export. Microcystins
compete with bile acids for uptake by the liver and is limited in the presence of bile acids and
other physiologically-relevant substrates for the transporter (Thompson and Pace, 1992). Other
studies following in vitro or in vivo exposures have shown that inhibition of microcystin uptake
by its OATp transporter reduces or eliminates the liver toxicity observed (Runnegar et al., 1981,
1995; Runnegar and Falconer, 1982; Hermansky et al., 1990a, b).
Limited information is available on the metabolism of microcystins. Some studies have
found that metabolism of microcystin-LR in mice occurs in the liver (Robinson et al., 1991; Pace
et al., 1991). Most of the available studies show minimal if any catabolism (process of breaking
down molecules into smaller units to release energy). Microcystins can be conjugated with
cysteine and glutathione to increase their solubility and facilitate excretion (Kondo et al., 1996).
Drinking Water Health Advisory for Microcystins-June 2015 15
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However, it is not clear whether hepatic cytochromes, such as cytochrome P450-facilitated
oxidation, precedes conjugation (Cote et al., 1986; Brooks and Codd, 1987). Both in vivo and in
vitro studies have shown biliary excretion (Falconer et al., 1986; Pace et al., 1991; Robinson et
al., 1991).
2.5.2 Noncancer Health Effects Data
2.5.2.1 Human Studies
The human data on the oral toxicity of microcystin-LR are limited by the potential co-
exposure to other pathogens and toxins, by the lack of quantitative information, and by the failure
to control for confounding factors.
Only a few epidemiological and case studies are available on the toxicity of microcystins
in humans. An outbreak among army recruits who had consumed reservoir water with a
cyanobacteria bloom withM aeruginosa reported symptoms of headache, sore throat, vomiting
and nausea, stomach pain, dry cough, diarrhea, blistering around the mouth, and pneumonia
(Turner et al., 1990). Microcystins, including microcystin-LR, were present in bloom samples.
However, high levels of Escherichia coli were also found in reservoir water after two weeks. The
authors suggested that exposure to microcystins may have had a role in some of the clinical
symptoms.
An epidemiology study done in Australia compared the hepatic enzyme levels from
patients served by a public water supply contaminated with aM aeruginosa bloom with enzyme
levels from patients living in areas served by water supplies uncontaminated by cyanobacteria
(Falconer et al., 1983). Although the authors observed significant variability in enzyme levels
between the two groups, the findings were attributed by the authors to the imprecise method of
study participant selection and confounding factors such as alcoholism and chronic kidney disease
among some of the participants.
A cross-sectional study done in China assessed the relationship between the consumption
of water and food (carp and duck) contaminated with microcystins and liver damage in children
(Li et al., 201 la). The authors found that mean serum levels of microcystins ranged from below
detection to 1.3 ug microcystin-LR equivalents/L. According to the authors, hepatitis B infection
was a greater risk for liver damage among these children than the microcystin exposure.
Acute intoxication with microcystin-producing cyanobacteria blooms in recreational water
was reported in Argentina in 2007 (Giannuzzi et al., 2011). A single person was immersed in a
Microcystis bloom with concentrations of 48.6 |ig/L. After four hours of exposure, the patient
exhibited fever, nausea, and abdominal pain, and three days later, presented dyspnea and
respiratory distress and was diagnosed with an atypical pneumonia. A week after the exposure,
the patient developed a hepatotoxicosis with a significant increase of alanine aminotransferase
(ALT), aspartate aminotransferase (AST) and y-glutamyltransferase (yGT). The patient
completely recovered within 20 days.
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An outbreak of acute liver failure occurred in a dialysis clinic in 1996 in Caruaru, Brazil
where dialysis water was contaminated with microcystins, and possibly cylindrospermopsin. Of
the 130 patients who received their routine hemodialysis treatment (intravenously) at that time,
116 reported symptoms of headache, eye pain, blurred vision, nausea and vomiting. Subsequently,
100 of the affected patients developed acute liver failure and, of these, 76 died (Carmichael et al.,
2001; Jochimsen et al., 1998). Analyses of blood, sera, and liver samples from the patients
revealed microcystins. Although the patients in the study had pre-existing diseases, the direct
intravenous exposure to dialysate prepared from surface drinking water supplies put them at risk
for cyanotoxin exposure and resultant adverse effects (Hilborn et al., 2013).
In another contamination event at a dialysis center in Rio de Janeiro, Brazil in 2001, 44
dialysis patients were potentially exposed to microcystin concentrations of 0.32 |ig/L, detected in
the activated carbon filter used in an intermediate step for treating drinking water to prepare
dialysate (Scares et al., 2005). Concentrations of 0.4 jig/L microcystin-LR were detected in the
drinking water. Serum samples were collected from 13 dialysis patients 31 to 38 days after the
detections in water samples, and patients were monitored for eight weeks. Concentrations of
microcystin-LR in the serum ranged from 0.46 to 0.96 ng/mL. Although the biochemical
outcomes varied among the patients, markers of hepatic cellular injury chlolestasis (elevations of
AST, ALT bilirubin, ALP and GOT) in serum during weeks one to eight after treatment
frequently exceeded normal values. Since microcystin-LR was not detected during weekly
monitoring after the first detection, the authors suggested that the patients were not continuously
exposed to the toxin and that the toxin detected in the serum after eight weeks may have been
present in the form of bound toxin in the liver (Scares et al., 2005). Results were consistent with a
mild to moderate mixed liver injury.
2.5.2.2 Animal Studies
Most of the information on the noncancer effects of microcystins in animals is from oral
and intraperitoneal (i.p.) administration studies in mice and rats exposed to purified microcystin-
LR. Liver effects are observed following acute oral exposure to microcystin-LR (Yoshida et al.,
1997; Ito et al., 1997b; Fawell et al., 1999). Effects on the liver, kidney, and male reproductive
system (testicular function and sperm quality), including changes in organ weights and
histopathological lesions, are observed following short-term and subchronic oral exposure to
microcystin-LR (Heinze, 1999; Fawell et al., 1999; Huang et al., 2011; Chen et al., 2011). Oral
and i.p. developmental toxicity studies in mice provide some evidence for fetal body weight
changes and maternal mortality (Fawell et al., 1999; Chernoff et al., 2002).
According to the authors, no clinical signs of toxicity were observed in a chronic study
done in mice for 18 months by Ueno et al. (1999). Although histopathology from a 280 day study
in mice revealed infiltrating lymphocytes and fatty degeneration in the livers, no quantitative data
were provided in the study (Zhang et al., 2012).
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2.5.3 Mode of Action for Noncancer Health Effects
Mechanistic studies have shown the importance of membrane transporters for systemic
uptake and tissue distribution of microcystin by all exposure routes (Fischer et al. 2005; Feurstein
et al., 2010). The importance of the membrane transporters to tissue access is demonstrated when
a reduction in, or lack of, liver damage happens following OATp inhibition (Hermansky et al.,
1990 a,b; Thompson and Pace, 1992).
The uptake of microcystins causes protein phosphatase inhibition and a loss of
coordination between kinase phosphorylation and phosphatase dephosphorylation, which results
in the destabilization of the cytoskeleton. This event initiates altered cell function followed by
cellular apoptosis and necrosis (Barford et al., 1998). Both cellular kinases and phosphatases keep
the balance between phosphorylation and dephosphorylation of key cellular proteins controlling
metabolic processes, gene regulation, cell cycle control, transport and secretory processes,
organization of the cytoskeleton and cell adhesion. Each of the microcystin congeners evaluated
(LR, LA, and LL) interacts with catalytic subunits of protein phosphatases PP1 and PP2A,
inhibiting their functions (Craig et al., 1996).
As a consequence of the microcystin-induced changes in cytoskeleton, increases in
apoptosis and reactive oxygen species (ROS) occur. In both in vitro and in vivo studies, cellular
pro-apoptotic Bax and Bid proteins increased while anti-apoptotic Bcl-2 decreased (Fu et al.,
2005; Weng et al., 2007; Xing et al., 2008; Takumi et al., 2010; Huang et al., 2011; Li et al.,
201 Ib). Mitochondrial membrane potential and permeability transition pore changes (Ding and
Ong, 2003; Zhou et al., 2012) lead to membrane loss of cytochrome c, a biomarker for apoptotic
events. Wei et al., (2008) found a time-dependent increase in ROS production and lipid
peroxidation in mice after exposure to microcystin-LR. After receiving a 55 |ig/kg of body weight
i.p. injection of microcystin-LR, the levels of hepatic ROS increased rapidly within 0.5 hours and
continued to accumulate for up to 12 hours in a time-dependent manner.
2.5.4 Carcinogenicity Data
Several human epidemiological studies from China have reported an association between
liver or colon cancer and consumption of drinking water from surface waters containing
cyanobacteria and microcystins (Ueno et al., 1996; Zhou et al., 2002). In these studies, a
concentrations measured in a surface drinking water supply were used as a surrogate for exposure
to microcystins. Individual exposure to microcystins was not estimated, and there was no
examination of numerous possible confounding factors, such as co-occurring chemical
contaminants or hepatitis infections in the population.
A study done by Flemming et al. (2002, 2004) in Florida failed to find a significant
association for primary liver cancer between populations living in areas receiving their drinking
water from a surface water treatment plant (with the potential for microcystin exposures), and the
Florida general population, or those receiving their water from ground-water sources. The one
significant association observed was between those people in the surface water service areas,
versus those in their surrounding areas described as buffer zones. However, the nature of the
water supply for the buffer zones were not identified.
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The only longer-term oral animal study of purified microcystin-LR was conducted by Ito
et al. (1997b). Ito et al. (1997b) administered 80 jig microcystin-LR/kg/day by gavage to mice for
80 or 100 days over 28 weeks (7 months). This single dose failed to induce neoplastic nodules of
the liver. The lack of hyperplastic nodules at 7 months suggests that microcystins are not a
mutagenic initiator of tumors, however, the fairly short duration may have been a limiting factor.
Several studies suggest that microcystin-LR is a tumor promoter. In these studies, animals
were first exposed to substances known to be tumor initiators (e.g. N-methyl-N-nitroso urea or
NDEA) alone, or in combination with microcystin-LR at i.p. doses known to have no significant
impact on liver weight. The combination of the initiator and the microcystin-LR significantly
increased the number and area of glutathione ^-transferase placental form-positive (GST-P) foci
when compared to treatment with the initiator alone. The same was true for situations where the
initiator treatment was combined with a partial hepatectomy (to stimulate tissue repair) and then
exposed to microcystins i.p. (Nishiwaki-Matsushima et al., 1992; Ohta et al., 1994). GST-P foci
are regarded as indicators for potential tumors formation. The results from these studies support
the classification of microcystin as a tumor promoter.
2.6 Conceptual Model for Microcystins
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 microcystins in drinking water and adverse health effects in the populations
at risk.
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 exposure 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.
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• 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 microcystins are depicted in the conceptual diagram
below (Figure 2-3).
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 microcystin-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.
Drinking Water Health Advisory for Microcystins-June 2015 20
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Figure 2-3. Conceptual Model of Exposure Pathways to Microcystins in Drinking Water
STRESSOR
SOURCES
EXPOSURE ROUTE
RECEPTORS
Microcystins
Lakes. Reservoirs and Rivers
Shallow ground water under the
direct influence of surface water
Finished Drinking Water
Oral
-
Drinking
water
Cooking with
water
Incidental
ingestion while
showering
Incidental ingestion
while outside activities
(gardening, car
washing, etc.)
Dermal
Inhalation
Showering/
Bathing
Incidental
— i inhalation while
showering
Washing
dishes
Outside
activities
(gardening, car
washing, etc.)
Incidental
— inhalation while
washing dishes
Intravenous
1
' Dialysis
EXDPOINTS
Liver
Damage
Kidney
Damage
Reproductive
Effects
Developmental
Effects
Cancer
Quantitative
Data Available
Incomplete or
Quantitative Data
Not Available
Drinking Water Health Advisory for Microcystins-June 2015
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2.6.2 Factors Considered in the Conceptual Model for Microcystins
Stressors: For this HA, the stressor is microcystins concentrations in finished drinking water.
Sources: Sources of microcystins include potential sources of drinking water such as rivers,
reservoirs, and lakes in the U.S. where blooms producing microcystins occur. Shallow private
wells under the direct influence of surface water (in hydraulic connection to a surface water body)
can also be impacted by microcystins-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 during bathing or showering,
washing dishes, or outside activities); inhalation exposure (during bathing, showering or washing
dishes,); 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 microcystins because they consume more water per body weight than do adults. Other
individuals of potential sensitivity are 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 regarding 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 were not removed
by treatment of dialysis water, identify dialysis patients as a population of potential concern in
cases where the drinking water source for the clinic is contaminated with cyanotoxins. 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 microcystins 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 microcystins are limited, but suggest the liver as the
primary target organ. Acute, short-term, and subchronic studies in animals also demonstrate that
the liver and kidney are target organs. In addition, some studies suggest that microcystins may
lead to reproductive and developmental effects. Studies have suggested that microcystins have
tumor promotion potential if there has been co-exposure to a carcinogen or cellular organ damage.
However, these data are limited, and there has been no long term bioassay in animals to evaluate
cancer. Available toxicity data are described in the Health Effects Support Document (HESD)for
Microcystins (U.S. EPA, 2015a), and indicate that the primary target organ for microcystins is the
liver. Kidney and reproductive effects in male mice were also observed, but were either not as
Drinking Water Health Advisory for Microcystins-June 2015 22
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sensitive as the liver or lack confirmation from more than one laboratory. Data are inadequate to
assess the carcinogenic potential of microcystins at this time.
2.7 Analysis Plan
The Health Effects Support Documentfor Microcystins (HESD, U.S. EPA, 2015 a),
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
microcystins, 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 microcystins was included. Some of the search
terms included in the literature search were microcystin, microcystin congeners, 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 microcystins 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
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
Drinking Water Health Advisory for Microcystins-June 2015 23
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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 microcystins, 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:
HA =
NOAEL or LOAEL or BMDL
UF x DWI/BW
Where:
NOAEL or = No- or Lowest-Observed-Adverse-Effect Level (mg/kg bw/day) from a study
LOAEL of an appropriate duration (7 to 30 days).
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 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
Drinking Water Health Advisory for Microcystins-June 2015
24
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<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 Microcystins-June 2015 25
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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 available information on toxicokinetics; identification, synthesis and evaluation
of studies describing the health effects of microcystins; and the potential modes of action
(MOAs), or toxicity pathways related to the health effects identified.
3.1 Dose-Response
3.1.1 Study Selection
The critical study chosen for determining the guideline value is a short-term study by
Heinze (1999) in which 11-week-old male hybrid rats (Fl generation of female WELS/Fohm x
male BDIX) were administered microcystin-LR via drinking water for 28 days at concentrations
of 0 (n=10), 50 (n=10) or 150 (n=10) ug/kg body weight (Heinze, 1999). Water consumption was
measured daily, and rats were weighed at weekly intervals. The dose estimates provided by the
authors were not adjusted to account for incomplete drinking water consumption (3-7% of
supplied water was not consumed over the 28-day period). Rats were sacrificed by exsanguination
under ether anesthesia after 28 days of exposure, and evaluation of hematology, serum
biochemistry plus histopathology of liver and kidneys, and measurement of organ weights (liver,
kidneys, adrenals, spleen and thymus) was performed.
Hematological evaluation showed an increase of 38% in the number of leukocytes at the
highest dose group (150 ug/kg body weight). Serum biochemistry showed a significant increase in
both treatment groups in mean levels of alkaline phosphatase (ALP) and lactate dehydrogenase
(LDH); 84 and 100% increase in LDH, and 34 and 33% increase in ALP, in the low and high dose
groups respectively. No changes were observed in mean levels of AST (aspartate
aminotransferase), and ALT (alanine aminotransferase). An increase in relative liver weights was
observed in a dose-dependent manner; 17% at 50 ug/kg body weight, and 26% at 150 ug/kg body
weight. Mean enzyme levels and relative liver weights are shown in Table 3-1.
A dose-dependent increase in absolute liver weight was also reported, and data on the liver
weights were provided by the author in a personal communication. A dose-dependent increase in
the average absolute liver weights was also observed in all groups: 8.8 grams at the control group,
9.70 grams at the lower dose and 10.51 grams at the high dose (Table 3-1). No statistically
significant changes in other organ weights or body weights were reported, and no effects on the
kidneys were observed. Table 3-2 summarizes the histological observations of liver lesions. Liver
lesions were considered toxic and spread diffusely throughout the parenchyma indicating cell
damage expressed by an increase in cell volume, an increase in mitochondria, cell necrosis, the
activation of Kupffer cells, and an increase in the amounts of periodic acid-Schiff (PAS)-positive
substances. Liver lesions were observed in both treatment groups. No kidney effects were
observed in either dose groups. The LOAEL was determined to be 50 ug/kg/day. The selection of
Heinze (1999) as the critical study was based on the appropriateness of the study duration, the use
Drinking Water Health Advisory for Microcystins-June 2015 26
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Table 3-1. Liver Weights and Serum Enzyme Levels in Rats Ingesting Microcystin-LR in
Drinking Water (Heinze, 1999)
Control
(Mean ± SD)
50 ug/kg
(Mean ± SD)
150 ug/kg
(Mean ± SD)
Serum Enzymes
Alkaline phosphatase (ALP)
(microkatals/L)
Lactate dehydrogenase (LDH)
(microkatals/L)
9.67 ±2.20
16.64 ±4.48
13.00±3.81*
30.64 ±5. 05*
12.86 ± 1.85*
33.58± 1.16*
Liver Weight
Relative (g/100 g body weight)
Absolute (g)**
2.75 ± 0.29
8.28 ± 1.37
3.22 ±0.34*
9.70 ± 1.32
3.47 ±0.49*
10.51 ± 1.02
* p<0.05 when compared with control; katal=conversion rate of 1 mole of substrate per second.
* information provided by the author through a personal communication.
Table 3-2. Histological Evaluation of the Rat Livers after Ingesting Microcystin-LR in
Drinking Water (Heinze, 1999)
•
Activation of
Kupffer Cells
Degenerative
and Necrotic
Hepatocytes
with
Hemorrhage
Degenerative
and Necrotic
Hepatocytes
without
Hemorrhage
PAS-positive
Material
Control
Slight
Moderate
Intensive damage
0
0
0
0
0
0
0
0
0
1
0
0
50 jig/kg
Slight
Moderate
Intensive damage
0
10
0
4
6
0
0
0
0
5
5
0
150 ug/kg
Slight
Moderate
Intensive damage
0
10
0
0
6
3
0
1
0
0
8
2
Drinking Water Health Advisory for Microcystins-June 2015
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of multiple doses, dose-related toxicological responses, and histopathological evaluations of
toxicity.
3.1.2 Endpoint Selection
The point of departure selected from the Heinze (1999) study is the LOAEL (50
ug/kg/day) for liver effects (increased liver weight, slight to moderate liver necrosis lesions, with
or without hemorrhages at the low dose and increased severity at the high dose, and changes in
serum enzymes indicative of liver damage). For the lesions, incidence increases from one animal
impacted in the control group to ten animals impacted in the dosed groups. This dose-response is
more dramatic than the difference in liver weight between the control and low dose (1.17 fold)
and the differences in the ALP and LDH levels between the control and low dose group (1.34 and
1.84-fold, respectively). Therefore, the liver lesions are identified as the endpoint of greatest
concern. These differences also advise against application of benchmark dose modeling for these
effects. The male and female mice in the Fawell et al (1999) study displayed liver lesions, but the
difference between controls and the low dose group (40 ug/kg/day) was less than two-fold. In an
i.p. infusion study by Guzman and Solter (1999) with a more direct delivery of dose to the liver,
necrosis was observed at doses of 32 and 48 ug/kg/day, but not at a dose of 16 ug/kg/day, thus
providing support for the critical effect and dose.
3.2 Ten-day Health Advisory
This Ten-day HA is applied to total microcystins using microcystin-LR as a surrogate.
The Ten-day HA is considered protective of non-carcinogenic adverse health effects over a ten-
day exposure to microcystins 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:
„ , TTA 50 jug/kg/day
Ten-day HA = ^ — =0.3 ug/L
1000 x 0.15L/kg/day
Where:
50 ug/kg/day = The LOAEL for liver effects in 11-week-old male hybrid rats
exposed to microcystin-LR in drinking water for 28 days
(Heinze, 1999).
1000 = The composite UF including a 10 for intraspecies variability
(UFn), a 10 for interspecies differences (UFA), a 3 for LOAEL to
NOAEL extrapolation (UFi,), and a 3 for uncertainties in the
database (UFo).
0.15 L/kg/day = Normalized drinking water intake 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 Microcystins-June 2015 28
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The Ten-day HA of 0.3 |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
microcystins 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:
T A UA 50
Ten-day HA = - — - - =1.6 ug/L
1000 x 0.03 L/kg/day
50 |ig/kg/day = The LOAEL for liver effects in 1 1 -week-old male hybrid
rats exposed to microcystin-LR in drinking water for 28
days (Heinze, 1999).
1000 = The composite UF including a 10 for intraspecies
variability (UFn), a 10 for interspecies differences (UFA), a
3 for LOAEL to NOAEL extrapolation (UFi,), and a 3 for
uncertainties in the database (UFo).
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 1.6 jig/L is considered protective of non-carcinogenic adverse health effects
for children of school age through adults over a ten-day exposure to microcystins in drinking
water.
3.2.3 Uncertainty Factor Application
• UFn - 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 pre-existing liver
problems could be more sensitive to microcystins exposures than the general population.
Pregnant woman, nursing mothers, and the elderly could also be sensitive to microcystins
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 microcystins. Allometric scaling is not applied in the development of the
Ten-day HA values for microcystins. 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 Microcystins-June 2015 29
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- An uncertainty factor of 3 (10°5 = 3.16) is selected to account for the extrapolation
from a LOAEL to a NOAEL. The threefold factor is justified based on the evidence
suggesting that the uptake of microcystins by tissues requires membrane transporters.
Uptake from the intestines involves both apical and basolateral transporters, uptake by the
microvilli capillaries and portal transport to the liver. Transporters are again necessary for
hepatic uptake. When there is slow infusion into the peritoneum and into the portal
intraperitoneal capillaries, uptake is described as rapid because of the rich blood supply
and large surface area of the peritoneal cavity (Klassen, 1996). Delivery of the
microcystins to the intraperitoneum increases the amount of the dose that reaches the liver
for three additional reasons: 1) the apical and basolateral intestinal barriers to uptake are
eliminated with the i.p. infusion; 2) there is no dilution of dose by the gastric plus
intestinal fluids as when food residues are in the gastrointestinal track; and 3) there is no
delay in reaching the site of absorption because of gastric emptying time (Klassen, 1996).
In addition, facilitated transporter kinetics are similar to Michaelis Menton enzyme
kinetics in that there are Km and Vmax components that are defined by the affinity of the
transported substance for the transporter.
In the Guzman and Solter (1999) intraperitoneal infusion study in rats, the NOAEL is 16
|ig/kg/day and the LOAEL is 32 jig/kg/day, a two-fold difference. There is no reason to
believe that the less direct delivery from the intestines to the liver following oral
exposures through drinking water (as was used in Heinze, 1999) would have a more than
3-fold separation between a NOAEL and LOAEL had there been one in the Heinze (1999)
study.
• UFo - An uncertainty factor of 3 (1005 = 3.16) is selected to account for deficiencies in the
database for microcystins. The database includes limited human data, including studies
evaluating the association between microcystin exposure and cancers in liver and colon,
and systemic effects including liver endpoints such as elevated liver enzymes. Oral and
i.p. acute and short-term studies on mice and rats, and subchronic studies done in mice are
available. Chronic data are also available for microcystin, however, are limited by the lack
of quantitative data provided in the study. Additionally, there are limited neurotoxicity
studies (including a recent publication on developmental neurotoxicity) and several i.p.
reproductive and developmental toxicity studies. The database lacks a multi-generation
reproductive toxicity study.
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., 101/2) when appropriate (U.S.
EPA, 2002).
Drinking Water Health Advisory for Microcystins-June 2015 30
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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 Use of microcystin-LR as a surrogate for total microcystins
Among the approximately 100 different congeners of microcystins known to exist,
microcystin-LR is the most common. The difference in toxicity of microcystin congeners depends
on the amino acid composition (Falconer, 2005). Stoner et al. (1989) administered by
intraperitoneal (i.p.) purified microcystin congeners (-LR, -LA, -LY and -RR) into ten or more
adult male and female Swiss albino mice. Necropsies were performed to confirm the presence of
the pathognomonic hemorrhagic livers. The authors reported 50% lethal doses (LDso) of 36 ng/g-
bw for -LR, 39 ng/g-bw for -LA, 91 ng/g- bw for -LY and 111 ng/g-bw for -RR. Similarly, Gupta
et al., (2003) determined LDso for the microcystin congeners LR, RR and YR in female mice
using DNA fragmentation assay and histopathology examinations of the liver and lung. The acute
LDso determination showed that the most toxic congener was microcystin-LR (43.0 |ig/kg),
followed by microcystin-YR (110.6 |ig/kg) and microcystin-RR (235.4 jig/kg). The most toxic
microcystins are those with the more hydrophobic L-amino acids (-LA, -LR, -and -YM), and the
least toxic are those with hydrophilic amino acids, such as microcystin-RR.
Wolf and Frank (2002) proposed toxicity equivalency factors (TEFs) for the four major
microcystin congeners based on LDso values obtained after i.p. administration. The proposed
TEFs, using microcystin-LR as the index compound (TEF=1.0) were 1.0 for microcystin-LA and
microcystin-YR and 0.1 for microcystin-RR. The application of TEFs based on i.p. LDso values to
assessment of risk from oral or dermal exposure is questionable given that differences in
lipophilicity and polarity of the congeners may lead to variable absorption by non-injection routes
of exposure.
The potential health risks from exposure to mixtures of microcystin congeners is
unknown, and since microcystin-LR is one of the most potent congeners and has the majority of
toxicological data on adverse health effects, microcystin-LR is used as a surrogate for all
microcystins in the health advisory.
4.2 Consideration of Study Duration
EPA used a 28-day study conducted by Heinze (1999) to derive the Ten-day HA for
microcystins. It is standard to use studies that are 7 to 30 days in duration to derive a 10-day
advisory value. In the study conducted by Heinze (1999), rats were dosed daily via drinking water
with microcystin and sacrificed at the conclusion of the study. No interim sacrifices were
performed to evaluate effects at 10 days or any other time less than the full 28 days. At the
conclusion of the 28-day study, adverse effects observed in the liver included increases in liver
weight, slight to moderate liver necrosis lesions accompanying hemorrhages at the low dose with
increased severity at the high dose, and changes in serum enzymes indicative of liver damage.
Given the lack of interim effects data, it is not known when during the 28-day study these effects
were manifested.
Drinking Water Health Advisory for Microcystins-June 2015 31
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4.3 Consideration of Reproductive Effects as Endpoint
Upon consideration of all available studies, liver effects were considered the most
appropriate basis for quantitation as it was a common finding among oral toxicology studies
(Falconer et al., 1994; Fawell et al., 1999; Ito et al., 1997b). However, while the liver is the
primary target of microcystin toxicity, there have been reports of effects of microcystin-LR on the
male reproductive system and sperm development following oral exposures (Chen et al., 2011).
In a study conducted by Chen et al. (2011), oral exposures to low concentrations of
microcystin-LR for 3 to 6 months showed reproductive toxicity including decreased sperm counts
and sperm motility, as well as an increase in sperm abnormalities, decreased serum testosterone
and increased serum luteinizing hormone (LH) levels. Because these effects were observed at
doses lower (0.79 mg/kg/day) than those observed for liver effects in Heinze (1999), EPA
evaluated Chen et al. (2011) and the lesions in the testes and effects on sperm motility as the
potential critical study and points of departure for the derivation of the RfD for microcystins.
The Chen et al., 2011 study has several limitations in the experimental design and
reporting. There was a lack of data reported on testis weights and sperm motility. The authors
reported "no significant differences in testis weights," but no information was provided on the
weights of the testis or whether there was a trend toward decreasing weights that failed to be
statistically significant. Also, no information was given on the methodology used for sperm
motility evaluation. No information was provided on how samples were handled and what
measurements were made to determine the percentage of sperm motility. Although body weight
and amount of water consumed were measured, these data were not presented, and doses to the
animals were not calculated by the study authors. In addition, the purity of microcystin-LR and
the species and age of the mouse used were not reported. Male sperm characteristics such as
volume, motility, and structure of sperm differ developmentally by age. Therefore, not knowing
the age of the mice in the study introduces uncertainty in the quantification of the reproductive
effects.
The fixation and staining of the testes used for microscopic examination
(paraformaldehyde in phosphate-buffered saline (PBS) and paraffin), could result in the
generation of artifacts, such as disruption of the testicular tubes. Cytoplasmic shrinkage and
chromatin aggregations were observed in both control and experimental groups. In order to
preserve the micro structure of the testis, dual fixation such as Davidson's or Bouin's fixation
followed by PAS staining should have been done. In addition, the histopathology analysis of the
testes reported by the authors did not provide sufficient detail to adequately assess the degree of
damage.
The quality of the medium used for the sperm analysis, and the lack of additional data
from the sperm analysis measurements carried out through the computer-assisted sperm analysis
(CAS A) are additional limitations in experimental design for this study. Very few details of the
serum hormone assay protocol and the quantitative parameters of sperm motility from the CASA
analysis were provided. Therefore, the calculation for the motility of the sperm was unclear and
could not be verified.
Drinking Water Health Advisory for Microcystins-June 2015 32
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Based on the limitations in study design, report and methods used by Chen et al. (2011),
EPA concluded that the quantitative data on decreased sperm counts and sperm motility were not
appropriate for determining the point of departure for the derivation of the RfD for microcystin.
4.4 Allometric Scaling Approach
Allometric scaling was not applied in the development of the short term RfD for
microcystins. 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 short term duration of the critical study and the development of a
short term RfD for determination 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.
4.5 Benchmark Dose (BMD) Modeling Analysis
The data set reported by Heinze (1999) was evaluated for BMD modeling. Heinze (1999)
demonstrated dose-related liver changes and statistically significant effects at the lowest dose (50
Hg/kg/day). Histological changes were also observed in all the animals (ten) in each dose group
(Table 3-2). Although differences in the degree of necrosis were observed with or without
hemorrhage related to dose, all the histological effects including Kupffer cell activation and PAS
staining showed no dose-response since all ten animals at the low and high doses displayed liver
damage associated with each effect. Therefore, the dose-response for the sum of the incidence
categories (slight, moderate, and intensive damage), are not amenable to BMD modeling. As a
result, the LOAEL of 50 jig/kg/day described by Heinze (1999) was used as the POD for
development of the HA.
4.6 Carcinogenicity Evaluation
While there is evidence of an association between liver and colorectal cancers in humans
and microcystins exposure and some evidence that microcystin-LR is a tumor promoter in
mechanistic studies, there is inadequate information to assess carcinogenic potential of
microcystins in humans (U.S. EPA, 2005). The human studies are limited by lack of exposure
Drinking Water Health Advisory for Microcystins-June 2015 33
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information and the uncertainty regarding whether or not these studies adequately controlled for
confounding factors such as Hepatitis B infection. No chronic cancer bioassays for microcystins
in animals are available.
The only oral study that examined the tumorgenicity of microcystin-LR failed to find
preneoplastic nodules in the livers of groups of 22 mice receiving up to 100 doses of 0 or 80
ug/kg/day over 7 months. Some studies suggest that microcystin-LR is a tumor promoter. Given
the potential impact on the cell cytoskeleton, necrotic effects on liver cell generation of reactive
oxygen species (ROS), and other biochemical changes, this finding is not surprising. The work by
Nishiwaki-Matsushima et al., 1992 that compares glutathione S-transferase placental form-
positive (P-GST) foci from 10 |ig/L microcystin-LR to that from the phenobarbital (0.05% in the
diet) as a positive control suggests that it is at best a weak promoter. The results from the second
part of the same study that compare P-GST foci following initiation with DEN followed by
microcystin-LR (10 |ig/kg), both before and after a partial hepatectomy, support this conclusion.
The International Agency for Research on Cancer (IARC) classified microcystin-LR as a
Group 2B (possibly carcinogenic to humans) based on the conclusion that there was strong
evidence supporting a plausible tumor promoter mechanism for these liver toxins. U.S. EPA's
Cancer Guidelines (2005) state that the descriptor of "inadequate information to assess
carcinogenic potential" is appropriate when available data are judged inadequate for applying one
of the other descriptors or for situations where there is little or no pertinent information or
conflicting information. The guidelines also state that (p. 2-52) "Descriptors can be selected for
an agent that has not been tested in a cancer bioassay if sufficient other information, e.g.,
toxicokinetic and mode of action information, is available to make a strong, convincing, and
logical case through scientific inference". In the case of microcystins, the data suggest that
microcystin-LR may be a tumor promoter but not an initiator. Without stronger epidemiology
data and a chronic bioassay of purified microcystin-LR, the data do not support classifying
microcystin-LR as a carcinogen.
4.7 Uncertainty and Variability
Several uncertainty factors were applied in several areas to account for incomplete
information. Human data on the toxic effects of microcystins are limited. Quantification of the
absorption, distribution, and elimination of microcystins 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 decreased sperm count and motility, and
microscopic lesions in the testes needs further analysis. In animal studies with oral exposures to
microcystins, some adverse effects in males such as reduced testosterone levels, as well as
toxicity to the female reproductive tissues and those of offspring 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 in the
human population following exposure to microcystins and the relative toxicity of other
microcystins congeners when compared to microcystin-LR. Additional information is needed on
the potential health risks from mixtures of microcystins with other cyanotoxins, as well as
biological and chemical stressors present in source water and drinking water supplies.
Drinking Water Health Advisory for Microcystins-June 2015 34
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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 exposure to other potential sources.
4.8 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 microcystins. Fawell et al. (1999)
observed a slight difference between male and female mice in body weight and serum proteins
(ALT and AST), but no sex-related differences in liver pathology.
Studies in laboratory rodents suggest that the acute effects of microcystin-LR may be
more pronounced in adult or aged animals than in juvenile animals (Adams et al., 1985; Ito et al.,
1997a; Rao et al., 2005). In these studies, young animals showed little or no effect at microcystin-
LR doses found to be lethal to adult animals. Age-dependent differences in toxicity were observed
after both oral and i.p. exposure, suggesting that differences in gastrointestinal uptake were not
entirely responsible for the effect of age. The relevance of these age-related differences to acute
toxicity in humans is unknown. However, for infants to one-year olds fed exclusively with
powdered formula prepared with tap water, drinking water is the dominant route of exposure to
cyanotoxins. There are significant differences in exposure between these life-stages that impact
risk.
Based on the available studies in animals, individuals with liver and/or kidney disease
may be more susceptible than the general population since the detoxification mechanisms in the
liver and impaired excretory mechanisms in the kidney may be compromised. Data from an
episode in a dialysis clinic in Caruaru, Brazil where microcystins were not removed by treatment
of dialysis water, identify dialysis patients as a population of potential concern in cases where the
drinking water source for the clinic used to prepare the dialysate is contaminated with
cyanotoxins. Other potentially sensitive individuals include pregnant woman, nursing mothers,
and the elderly.
4.9 Distribution of Body Weight and Drinking Water Intake by Age
Both body weight and drinking water intake are distributions that vary with age. EPA has
developed two health advisory values, a Ten-day HA of 0.3 jig/L based on exposure to infants
over the first year of life, and a Ten-day HA of 1.6 |ig/L based on exposure to adults, over 21
years of age. Section 4.10 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
Drinking Water Health Advisory for Microcystins-June 2015 35
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Figure 4-1. 90th Percentile Drinking Water Ingestion Rates by Age Group
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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).
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 cyanotoxin 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
microcystins through breast milk are not available for humans.
4.10 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 microcystins for each age group (plotted on Figure 4-2) to demonstrate the
variability due to body weight and drinking water intake by age.
Drinking Water Health Advisory for Microcystins-June 2015
36
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Figure 4-2. Ten-day Health Advisories for Microcystins by Age Group
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<3 months months years years years years years years years years
months Age Range
EPA decided to apply the Ten-day HA value calculated for infants over the first year of
life (0.3 |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
months old is 0.2 |ig/L, slightly below the infant health advisory value of 0.3 |ig/L. EPA believes
that infants from birth to 3 months old are not at a disproportionate risk at a 0.3 |ig/L advisory
value because a 30-fold safety factor 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 0.3
|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 1.6 |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 (>21 years).
Drinking Water Health Advisory for Microcystins-June 2015
37
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5.0 ANALYTICAL METHODS
This Health Advisory (HA) for the Cyanobacterial Microcystin Toxins is applied to total
microcystins which should include all of the measureable microcystin congeners within the
cyanobacterial cells (intracellular) and outside the cell (extracellular).
Extracellular microcystins (either dissolved in water or bound to other materials) typically
make up less than 30% of the total microcystin concentration in source water (Graham et al.,
2010). Most of the toxin is intracellular, and released into the water when the cells rupture or die.
Both intracellular and extracellular microcystins may also be present in treated water, depending
on the type of treatment processes in place. Therefore, it is important to note that analysis for
microcystins should account for both intracellular and extracellular toxins in samples when intact
cells may be present. Release of intracellular microcystins is achieved by rupturing or lysing the
cell walls in order to expose the intracellular microcystins. Cell lysis can be achieved by a variety
of methods including sequential freeze-thawing, freeze drying, and mechanical or sonic
homogenization. Following cell lysis, microcystins may need to be extracted for some analytical
methods. At low concentrations, the direct determination of microcystins may not be feasible, and
a preconcentration step may be required. Typically samples are filtered and/or centrifuged after
cell lysis to remove cell fragments and particulates. This may be followed by freeze-drying or
solid-phase extraction (SPE). Typical elution solvents are dilute acid, methanol, acidified
methanol/water mixtures, and butanol/methanol/water mixtures.
Preconcentration is generally needed when techniques such as liquid chromatography are
used in order to achieve limits of detection in the low-|ig/L and ng/L range. Extraction efficiency
has been shown to vary depending on the type of solvent, the hydrophobicity of the congener, the
water content of the cells (freeze-dried versus frozen) and differences between field samples and
laboratory cultures. Variations in extraction efficiency may impact the accurate quantitation of
microcystins so the use of a surrogate compound to monitor the extraction efficiency is strongly
recommended. Responsible authorities should ensure that the appropriate methods and
preparation techniques (extraction, concentration and separation) are being used in the laboratory
depending on the type of sample and the analytical method selected.
Analytical methods available for the detection of microcystins in drinking water include
reversed phase high performance liquid chromatography (HPLC) coupled with mass
spectrometric (MS, MS/MS) or ultraviolet/photodiode array detectors (UV/PDA), Enzyme Linked
Immunosorbent Assays (ELISA), and Protein Phosphatase Inhibition Assays (PPIA).
EPA has developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS)
method for microcystins and nodularin (combined intracellular and extracellular) in drinking
water (Method 544; U.S. EPA, 2015). Accuracy and precision data have been generated in
reagent water, and finished ground and surface waters for the following compounds: microcystin-
LA (microcystin-LA), -LF (microcystin-LF), -LR (microcystin-LR), -LY (microcystin-LY), -RR
(microcystin-RR), -YR (microcystin-YR), and nodularin-R (NOD). This method is intended for
use by analysts skilled in solid phase extractions, operation of LC/MS/MS instruments, and the
interpretation of associated data. The single laboratory lowest concentration minimum reporting
levels (LCMRLs) for this method range from 2.9 to 22 ng/L (0.0029-0.022 |ig/L). The Detection
Limit (DL) for analytes in this method range from 1.2 to 4.6 ng/L. In this method, a 500 mL water
Drinking Water Health Advisory for Microcystins-June 2015 38
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sample (fortified with an extraction surrogate) is filtered, and both the filtrate and the filter are
collected. The filter is placed in a solution of methanol containing 20% reagent water and held for
at least one hour at -20 °C to release the intracellular toxins from cyanobacteria cells captured on
the filter. The liquid is drawn off the filter and added back to the 500-mL aqueous filtrate. The
500-mL sample (plus the intracellular toxin solution) is passed through a SPE cartridge to extract
the method analytes and surrogate. Analytes are eluted from the solid phase with a small amount
of methanol containing 10% reagent water. The extract is concentrated to dryness by evaporation
with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with methanol
containing 10% reagent water. A 10-uL injection is made into an LC equipped with a C8 column
that is interfaced to an MS/MS. Analytes are separated and identified by comparing the acquired
mass spectra and retention times to reference spectra and retention times for calibration standards
acquired under identical LC/MS/MS conditions. The concentration of each analyte is determined
by external standard calibration. To download Method 544 Determination of Microcystins and
Nodularin in Drinking Water by Solid Phase Extraction and Liquid Chromatography/tandem
Mass Spectrometry (LC/MS/MS), please go to: http://www.epa.gov/nericwww/ordmeth.htm
High performance liquid chromatography (HPLC) is widely used to separate microcystin
congeners. A variety of stationary phases have been used including reversed-phase Cig columns,
amide Cig columns, internal surface reversed-phase columns or ion exchange columns.
Optimization of chromatographic parameters is needed to ensure a good resolution of analytes. In
addition to mass spectrometry, ultraviolet/visible absorbance is a commonly used detection
techniques with HPLC. Most microcystin congeners have similar absorption profiles between 200
and 300 nm. The wavelength of the UV/visible detector can be set at these values to record the
responses of microcystins in sample extracts separated by the HPLC. The retention time, UV
spectra and peak area of commercially available or laboratory standards is the basis of
identification and quantification of microcystins using HPLC-UV/visible detection. However, due
to the limited number of commercially available standards, the toxins are often quantified by
comparison to an microcystin-LR standard and reported in terms of microcystin-LR equivalence.
HPLC-UV/visible is susceptible to interferences from natural organic materials (NOMs).
Detection limits will depend partially on the sample volume extracted, the concentration of the
toxins, and the presence of interfering contaminants.
A variety of antibodies have been isolated against microcystin-LR and microcystin-RR, as
well as recombinant antibody fragments and antibodies against the amino acid ADDA.
Commercial ELISA kits that contain all of the reagents needed for analysis have also been
developed and typically provide a cross reactivity chart for some of the congeners (i.e.,
microcystin-LR, -RR, YR, nodularin) that are commonly found in water. These range from 50-
85% for microcystin-RR, 35-181% for microcystin-YR and 10-124% for microcystin-LA.
Detection of the total microcystins will be expressed as the sum of the congeners provided from
ADDA ELISA. The methods detection limit (MDLs) of several commercial laboratory ELISA
kits have been reported to range from 0.04 to 0.2 |ig/L for microcystin-LR. Commercial ELISA
kits generally have quantitation ranges from 0.2 (LOQ) to an upper limit of 5 |ig/L. Two high
sensitivity ELISA plate kits have become commercially available with MDLs ranging from 0.04
to 0.05 |ig/L.
Drinking Water Health Advisory for Microcystins-June 2015 39
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PPIAs are used with a variety of detection methods and substrates including radioactive
detection assays using 32P-radiolabelled substrates and colorimetric assays using p-nitrophenol
phosphate as the substrate. The method has also been adopted for fluorescence measurements
using the substrates methylumbelliferyl phosphate. The detection limit of total microcystins,
reported as microcystin-LR equivalents (microcystin-LRequiv) using radiometric protein
phosphatase assays is approximately 0.1 |ig/L or less, and using colorimetric PP1 inhibition
assays range between 10 to 20 ng/mL (0.01 to 0.02 |ig/L).
Rapid tests for the identification of the presence of microcystins in water have been
developed for use in the field. Field test kits can be used as a presence/absence tool for
determining if a bloom is toxic or if treatment plant operations need to be adjusted during a bloom
event but do not currently have sufficient sensitivity at microcystin concentrations below 1 |ig/L
to be used for treated water analyses. Commercially-available test kits use a variety of methods
including immunochromatography (test strips), ELISA, and phosphatase inhibition to estimate the
level of microcystins in a water sample. In general, the results of field test kits should be
considered qualitative and should only be used to conduct a preliminary assessment of
microcystin levels. The applicability of test kits is between 1 and 5 |ig/L of microcystins with a
detection limit of approximately 0.5 |ig/L. Several field test kits do not include a lysing agent and,
therefore, only determine the presence of extracellular microcystins. When using these field test
kits, users should consult the manufacturer regarding an appropriate lysing technique if the
detection of both intracellular and extracellular microcystins is required.
A new approach using laser diode thermal desorption-atmospheric pressure chemical
ionization interface coupled to tandem mass spectrometry (LDTD-APCI-MS/MS) has been
developed for the analysis of total microcystins in complex environmental matrices. The method
is based on oxidation of the MCs in a sample using potassium permanganate under alkaline
conditions to produce 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB). MMPB is then
extracted and directly injected (no chromatographic separation) into the LDTD-APCI-MS/MS
system. This approach results in ultra-fast sample analysis with simple sample preparation,
reducing time and material costs associated with chromatographic separation. This method does
not require individual MC standards, but similar to ELISA and PPIA, the results do not provide
information on the identity of the individual MC congeners. The MDL and LOQ are 0.2 and 0.9
|ig/L, respectively (Roy-Lachapelle et al., 2014).
Drinking Water Health Advisory for Microcystins-June 2015 40
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6.0 TREATMENT TECHNOLOGIES
The information below is adapted from the 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 EP A Drinking Water Treatability Database for Microcystins (U.S. EPA,
2007); the International Guidance Manual for the Management of Toxic Cyanobacteria (GWRC,
2009) available at http://www.waterra.com.au/cyanobacteria-
manual/PDF/GWRCGuidanceManualLevell.pdf, and Management Strategies for Cyanobacteria
(Blue-Green Algae): A Guide for Water Utilities (Newcombe et al., 2010) available at
http://www.researchgate.net/profile/Lionel_Ho/publication/242740698_Management_Strategies_for_Cyan
obacteria_(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 cyanob acted al concentrations are still relatively low (usually under 5,000 to 15,000
cells/mL) (WHO, 1999), are less likely to release significant cyanotoxin concentrations upon cell
lysis and is able to mitigate or prevent a cyanob acted al bloom from proliferating as the season
progresses. If harmful cyanobacterial blooms occur, utilities may take action to investigate
alternative source water sources, change intake locations or levels to withdraw source 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 deep). These strategies can be applied to the entire
source water body or to just a portion of the lake depending on the need, and size and depth of the
water body relative to the source water intake(s).
Hydrogen peroxide (H2O2) has been used as an algaecide in source water because of a
rapid reaction time (90% of bloom collapsed in 3 days and 99% in 10 days), and environmentally
safe reaction products (oxygen and water) (Wang et al., 2012; Matthjis et al; 2012). The
Drinking Water Health Advisory for Microcystins-June 2015 41
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drawbacks (aside from cell lysis) are that oxidant breakdown is so rapid that repeated applications
are needed. Further understanding of this technique is needed (Matthjis et al., 2012).
The use of ultrasonic sound waves to disrupt cyanobacterial cells has also been
investigated as a potential source water treatment option (Rajasekhar et al., 2012). It is
environmentally friendly compared to chemical treatment strategies. The technique has also been
reported to be capable of degrading microcystin-LR (Song et al., 2005). Drawbacks include that
application frequencies are difficult to calculate and are system-specific; 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. Any variation in treatment methods aimed at reducing toxins
concentrations need to be tailored to the type(s) of cyanobacteria present and the site-specific
water quality (e.g. pH, temperature, turbidity, presence of natural organic material (NOM), etc.),
the treatment processes already in place, and the utility's multiple treatment goals (e.g., turbidity
and total organic carbon (TOC) removal, disinfection requirements, control of disinfection by-
products (DBF) formation, etc.). 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 lysisTo remove both
intracellular and extracellular toxins from drinking water, a multi-barrier approach is required,
which may consist 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.
Drinking Water Health Advisory for Microcystins-June 2015 42
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6.2.1 Conventional Treatment for Microcystins
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
combination of conventional treatment processes with oxidation, adsorption, and/or advanced
treatment needs to be considered to treat both intracellular and extracellular cyanotoxins.
The efficiency of the conventional treatment processes to remove cyanob acted al cells and
intracellular microcystins has been shown to vary from 60 to 99.9%. Factors that impact removal
include the cyanob acted al species and cell density, coagulant type and dose, pH, NOM, and
operational parameters such as flocculation time, frequency of filter backwashing and clarifier
sludge removal (Vlaski et al., 1996; Hoeger et al., 2004; Jurczak et al., 2005; Zamyadi et al.,
2012a, 2013c; Newcombe et al., 2015). Typically, 60 to 95% of cells and intracellular
microcystins can be removed during sedimentation with as much as 99.9% removal achieved
through filtration (Lepisto et al., 1994; Drikas et al., 2001; Hoeger et al., 2004; Newcombe et al.,
2015). The efficiency of coagulation and clarification for cell removal is dependent on pH,
coagulant type and dose and the morphological characteristics of the cyanobacteria. Rapid sand
filtration without pre-treatment (i.e., direct filtration, without coagulation/clarification) is not
effective for cyanob acted al cell removal.
If operated properly, conventional treatment (coagulation, flocculation, clarification and
filtration), does not cause cell lysis or increases in the extracellular microcystin concentrations of
treated water (Chow et al., 1998, 1999; Drikas, 2001; Sun et al., 2012). Drinking water treatment
plants utilizing conventional treatment followed by oxidation or activated carbon may remove
both intracellular and extracellular microcystins up to 99.99% of total microcystins to achieve
concentrations below 0.1 |ig/L in treated water (Karner et al., 2001; Lahti et al., 2001; Hoeger et
al., 2005; Jurczak et al., 2005; Rapala et al., 2006; Zamyadi et al., 2013a). Conventional treatment
is generally considered to have limited effectiveness for the removal of the extracellular
microcystins. Therefore, additional processes such as adsorption, chemical oxidation,
biodegradation or reverse osmosis, and nanofiltration are required to remove extracellular
microcystins.
Although microfiltration and ultrafiltration membranes can remove both cyanobacterial
cells and intracellular microcystins, removal of extracellular microcystins using ultrafiltration is
variable (35 to 70%) and microfiltration is not effective (Gijsbertsen-Abrahamse et al., 2006;
Dixon et al., 201 la, b). Nanofiltration and reverse osmosis membranes can achieve high removals
of intracellular and extracellular microcystins, from 82% to complete removal (Westrick et al.,
2010; Dixon et al., 2010). Pore size, among others, is an important factor in removal efficiency
for these processes.
Successful removal of cyanobacterial cells and intracellular microcystins will depend on
proper operations of the conventional treatment processes (Hoeger et al., 2004; Dugan and
Williams, 2006; Ho et al., 2013; Zamyadi et al., 2012a, 2013c). Operational considerations for
removing cyanobacterial cells using coagulation, flocculation and clarification are similar to
Drinking Water Health Advisory for Microcystins-June 2015 43
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considerations for achieving effective particle removal. The appropriate coagulant and
coagulation pH should be determined through jar-testing to maximize cell removal. In jar-testing,
the NOM, chlorophyll-a, or cyanobactedal cell count can be used to optimize the coagulation
conditions for cyanobacterial cell removal (Sklenar et al., 2014; Newcombe et al., 2015).
Sufficient mixing should be provided at the point of chemical addition to ensure rapid and
uniform contact, and an appropriate mixing speed should be determined to optimize the
flocculation process (GWRC, 2009). It is important to minimize the potential for the
accumulation of cyanobacterial cells as scums at the surface of sedimentation basins and filters
(Zamyadi et al., 2012a, 2013c).
Effective sludge removal from sedimentation/clarification processes is important to
minimize the release of intracellular and extracellular microcystins into the surrounding waters, as
significant cell numbers can accumulate within the sludge, and cells contained within the sludge
can lyse rapidly (Drikas et al., 2001; Ho et al., 2013; Zamyadi et al. 2012a). It has been reported
that accumulation of cyanobacterial cells and microcystins in clarifiers can lead to their
breakthrough into filter effluent. In addition, cell lysis can occur in the clarifier sludge, increasing
the extracellular concentration of microcystins in the treatment plant. Therefore sludge
management (decreased sludge age) in clarifiers and increased frequency of backwashing of
filters is important because settled/filtered cells can remain viable and possibly multiply over a
period of at least 2 to 3 weeks. Within 1 day, some cells in the sludge can lyse and release NOM
and taste and odor compounds, in addition to cyanotoxins (Newcombe et al., 2015). Additionally,
backwash water from the filters may contain cyanobacterial cells and/or extracellular
microcystins; hence, care needs to be taken if spent backwash water is recirculated to the
beginning of the treatment process to prevent the reintroduction of cells and toxins into the
treatment train. Although longer filter run-times are typically desirable between backwashing,
during periods of high algal concentrations, cells can accumulate in the filter, which can
potentially lead to a significant amount of extracellular microcystins released into the filtered
water. The optimum balance between maximizing water production and minimizing the risk of
toxin breakthrough will be plant-specific.
6.2.2 Adsorption
Adsorption processes, such as granular activated carbon (GAC) or powdered activated
carbon (PAC), are effective at removing extracellular microcystins but are not capable of
removing intact cells and intracellular toxins (Lambert et al., 1996; Newcombe, 2002; Newcombe
et al., 2003). Removal through adsorption depends on many factors including the type of activated
carbon used, the microcystin congener and water quality conditions. In general, mesoporous
carbons (such as chemically-activated wood-based carbons) are the most effective for the removal
of microcystins (Newcombe et al., 2010). Other factors such as the type of microcystin congener
present, the raw water quality (i.e., NOM and pH) and contact time affect microcystins removal
efficiency when using activated carbon processes. In addition, shortened filter run times or filter
overload may happen during cyanobacteria blooms. Therefore, water treatment plants should
conduct jar-testing to determine the most effective activated carbon dose, type, and feed point
prior to the application without affecting other water quality parameters and treatment processes
(Skienaretal., 2014).
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The performance of GAC filtration for extracellular microcystin removal depends upon
the empty bed contact time (EBCT), carbon age, carbon pore size, and raw water quality
characteristics such as NOM and pH, as well as the microcystin variant (Newcombe, 2002;
Newcombe et al., 2003; Ho and Newcombe, 2007; Wang et al., 2007). Solution chemistry can
also affect microcystin-LR adsorption onto GAC. Enhanced removal of microcystin-LR has been
observed at lower pH (2.5 versus 6.5) due to either precipitation or reduced solvency effect
(Pendleton et al., 2001).
Removal of extracellular microcystins by PAC can be highly effective (up to 95%)
depending on the microcystin congener and concentration, the PAC type and dose, the contact
time and the water quality characteristics such as TOC (Newcombe et al., 2003; Cook and
Newcombe; 2008; Ho et al., 2011). According to Newcombe et al. (2010), a PAC dose of 20
mg/L and a contact time of at least 45 minutes should be considered for removal of most
extracellular microcystins (with the exception of microcystin-LA).
6.2.3 Chemical Oxidation
Chemical oxidation using chlorine, potassium permanganate, or ozonation can be effective
at oxidizing extracellular microcystins, but can also impaired cell integrity, resulting in an
increase in concentrations of extracellular microcystins in drinking water. By applying
conventional filtration (or other filtration process) first to remove the majority of intact cells, the
extracellular microcystin concentration is less likely to increase due to cell lysis when water water
is treated with oxidants. In cases where pre-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).
The effectiveness of chemical oxidation of microcystins depends on the type of oxidant,
dose, contact time, microcystin congener and water quality characteristics such as pH and
dissolved organic carbon (DOC) (GWRC, 2009; Sharma et al., 2012). Laboratory-scale
experiments have demonstrated that the general trend for the effectiveness of cyanobacterial cell
and extracellular microcystin oxidation to be: ozone>permanganate>chlorine>chlorine-based
oxidants (Acero et al., 2005; Rodriguez et al., 2007a, b; Ding et al., 2010; Sharma et al., 2012;).
However, selection of the most appropriate oxidant for microcystins should be based on the
characteristics of each water source, the disinfection requirements, and potential formation of
disinfection by-products (DBFs) (Sharma et al., 2012).
It is also important to recognize that the use of oxidants may result in the formation of
DBFs and should be considered when selecting a strategy for oxidizing microcystins (Merel et al.,
2010; Zamyadi et al., 2012b; Wert et al., 2013). For example, ozone and chlorine dioxide can
result in the formation of inorganic DBFs, such as bromate and chlorite/chlorate, respectively.
Additionally, modifying pre-oxidation practices may compromise other treatment objectives (e.g.,
turbidity removal), and should be considered.
Drinking Water Health Advisory for Microcystins-June 2015 45
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The oxidation of microcystins by chlorine has been found to be highly effective (>90%
removal) under experimental conditions (Ho et al., 2006a; Acero et al., 2008; Merel et al., 2009;
Sorlini and Collivignarelli, 2011). However, the effectiveness of chlorination on the oxidation of
microcystins depends upon the chlorine dose, contact time, pH, temperature, and other water
quality characteristics (Sharma et al., 2012). Several studies have found that microcystins are
efficiently oxidized if pH is maintained below 8, the chlorine dose is greater than 3 mg/L and 0.5
to 1.5 mg/L of free chlorine residual is present after 30 minutes of contact time (Nicholson et al.,
1994; Acero et al., 2005; Ho et al., 2006a; Xagoraraki et al., 2006; Newcombe et al., 2010).
However, much higher chlorine doses (2 to 10 mg/L) are required to lyse the cyanobacterial cell
and then oxidize the previously cell-bound microcystins (Zamyadi et al., 2013b).
The oxidation of microcystins in water by permanganate is one of the more effective
processes for oxidizing extracellular microcystins in water (Sharma et al., 2012). Rodriguez et al.
(2007a) exhibited a 90% oxidation of microcystin-LR at a dose of 1.0 mg/L, a contact time of 60
minutes, a pH of 8, and a temperature of 20°C. Complete oxidation occurred at a dose of 1.5
mg/L (Rodriguez et al., 2007a). Treatment plants considering potassium permanganate for
oxidation of microcystins should be aware that permanganate can discolor water when it is
present in excess of 0.05 mg/L. Therefore, dosage control and process monitoring (e.g., visual
inspection of the basin effluent color, measuring permanganate residual) is important in avoiding
consumer complaints (MWH, 2012).
The oxidation of microcystins in water by ozone has been shown to be highly effective
(greater than 90% oxidation) in laboratory-scale studies (Rositano et al., 2001; Shawwa and
Smith, 2001; Brooke et al., 2006). The efficacy depends on temperature, pH, ozone dose, contact
time, and other water quality characteristics such as DOC and alkalinity (Sharma et al., 2012).
Utilities should also be aware that the use of ozone may result in the formation of bromate and
other DBFs.
Monochloramine is a weaker oxidant than chlorine and is not an effective treatment
barrier for microcystins (Westrick et al., 2010).
Most laboratory studies have found that chlorine dioxide (CICh) is not effective for
oxidizing extracellular microcystins (Kull et al., 2004, 2006; Ding et al., 2010; Sorlini and
Collivignarelli, 2011) or cyanobacterial cells and intracellular microcystins (Ding et al., 2010;
Wert et al., 2014) at dosages (1-2 mg/L) and contact times typically applied to drinking water.
6.2.4 Other Filtration Technologies
Biological filtration, using either biologically-active sand or activated carbon, has been
shown to be effective for the removal of extracellular microcystins in bench- and pilot-scale
studies (Keijola et al., 1998; Bourne et al., 2006; Ho et al., 2006b, 2008, 2012) and in limited full-
scale studies (Grutzmacher et al., 2002, Rapala et al., 2006). The removal of intact cyanobacterial
cells and their associated intracellular toxins through physical straining in slow sand filters has
also been documented (Grutzmacher et al., 2002; Pereira et al., 2012). Biological filters also have
the capability to remove particulate including intact cyanobacterial cells. Bank filtration may also
be effective for the removal of microcystins (Lahti et al., 1998; Schijven et al., 2002). A detailed
Drinking Water Health Advisory for Microcystins-June 2015 46
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review of biological treatment options for cyanotoxin removal conducted by Ho et al., (2012b)
identified the type and concentration of microcystin-degrading bacteria, concentration of
microcystins, and temperature as key factors that influence the efficiency of biological filtration
for the removal of microcystins. In addition, the concentration of other organic matter within the
source water may inhibit biodegradation, as microcystins may be a secondary substrate in the
presence of NOM. Particle size, chemical composition and roughness or topography of the
surface of the media used for filtration have also been identified as important factors for biofilm
growth and ultimately the biodegradation of microcystins (Wang et al., 2007, Ho et al., 2012).
Membrane filtration including microfiltration (MF) and ultrafiltration (UF) can achieve
greater than 98% removal of cyanobacterial cells and intracellular microcystins (Chow et al.,
1997; Gijsbertsen-Abrahamse et al., 2006; Campinas and Rosa, 2010: Sorlini et al., 2013).
Nanofiltration (NF), reverse osmosis (RO) and, to a lesser extent UF, can be used for both
intracellular and extracellular microcystin removal (Neumann and Weckesser, 1998; Lee and
Walker, 2008; Dixon et al., 201 la,b). The performance of membrane filtration for microcystin
removal depends on characteristics of the membrane such as molecular weight cut-off (MWCO)
and hydrophobicity, initial concentration, size and molecular weight of the microcystins, and
operating parameters such as flux, recoveries and degree of fouling. It is recommended that
cyanobacterial cells are removed prior to reverse osmosis to prevent membrane clogging and
fouling.
Laboratory and pilot-scale studies have demonstrated that MF and UF can remove greater
than 98% of cyanobacterial cells (Chow et al., 1997, Gijsbertsen-Abrahamse et al., 2006;
Campinas and Rosa, 2010; Sorlini et al., 2013), and ultrafiltration can be moderately effective
(35-70%) for removal of extracellular microcystins (Lee and Walker, 2008). Several studies have
also demonstrated that the release of intracellular microcystins from the shear stress on
cyanobacterial cells during MF and UF is possible, although it generally results in permeate
microcystin concentration increases of less than 12 percent (Gijsbertsen-Abrahamse et al., 2006;
Campinas and Rosa, 2010).
The removal of extracellular microcystins by NF and RO is very effective (greater than
90%) and depends on the MWCO, as the filtration of microcystins occurs via size exclusion
(Gijsbertsen-Abrahamse et al., 2006).
6.2.5 Combined Treatment Technologies
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 microcystins. Data indicate that utilities can effectively remove both intracellular and
extracellular microcystins to achieve concentrations below 0.1 |ig/L (Lahti et al. 2001; Boyd and
Clevenger, 2002; Zurawell, 2002; Hoeger et al., 2005, Jurczak et al., 2005; Rapala et al., 2006;
Haddix et al., 2007; Nasri et al., 2007; Zamyadi et al., 2013c). However, some studies have
shown that the presence of high concentrations of cells (i.e., 105 cells/mL) and/or microcystins in
raw water (100 |ig/L) may be challenging for treatment plants to reduce concentrations to below
0.1 |ig/L (Tarczyriska et al., 2001; Zamyadi et al., 2012a).
Drinking Water Health Advisory for Microcystins-June 2015 47
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In most cases, utilities will be able to effectively remove intracellular microcystins with
processes that are already in place (e.g., conventional treatment) when they are operated with a
focus on cyanobacteria cell or NOM removal. Extracellular microcystins may also be removed in
many treatment plants by using existing treatment such as chlorination after filtration or by the
addition of PAC following conventional treatment (Carriere et al., 2010). Although it is possible
to remove both intracellular and extracellular microcystins effectively using a combination of
treatment processes, the removal efficiency can vary considerably. Utilities need to ensure that
they are utilizing their existing treatment processes to their fullest capacity for removal of both
cyanobacterial cells and extracellular microcystins and that appropriate monitoring is being
conducted to ensure that adequate removal is occurring at each step in the treatment process.
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 microcystins. A study using common water filtration and purification
systems found that the efficacy of POU filtration devices to remove microcystin (LR) varies
considerably with the type of device being used (Pawlowicz et al., 2006). Microcystin-LR was
successfully removed using carbon filters allowing only 0.05 to 0.3% of the toxin load to pass
through the filter. However, more than 90% of microcystin-LR passed through string-wound
filters and pleated paper. According to the authors, the use of carbon home filter devices tested in
this study may provide additional protection beyond that provided by the drinking water treatment
plant against human exposure to microcystin-LR. Additional studies are recommended to assess
the efficacy of POU drinking water treatment units for other cyanotoxins and under other
conditions. 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/Certified/DWTU/.
Drinking Water Health Advisory for Microcystins-June 2015 48
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7.0 REFERENCES
Acero, J. L., Rodriguez, E., Meriluoto, J. (2005). Kinetics of reactions between chlorine and the
cyanobacterial toxins microcystins. Water Research, 39(8): 1628-1638.
Acero, J. L., Rodriguez, E., Majado, et al. (2008). Oxidation of microcystin with chlorine and
permanganate during drinking water treatment. Journal of Water Supply: Research and
Technology, 57(6): 371-380.
Adams, W. H., Stoner, R. D., Adams, D. G., et al. (1985). Pathophysiologic effects of atoxic
peptide fromMicrocystis aeruginosa. Toxicon. 23(3): 441-447.
AWWA Research Foundation. (2001). Assessment of Blue-Green Algal Toxins in Raw and
Finished Drinking Water. Final report #256. Prepared by Carmichael, W. W. AWWA
Research Foundation and American Water Works Association. Denver, CO.
Barford, D., Das, A. K., and Egloff, M. (1998). The structure and mechanism of protein
phosphatases: Insights into catalysis and regulation. Annual Review of Biophysics and
Biomolecular Structure, 27: 133-164.
Beussink, A. M., and Graham, J. L. (2011). Relations between hydrology, water quality, and
taste-and-odor causing organisms and compounds in Lake Houston, Texas, April 2006-
September 2008. U.S. Geological Survey Scientific Investigations Report 2011-5121,
pp.27. Reston, VA.
Bourne, D. G., Blakeley, R. L., Riddles, P., and Jones, G. J. (2006). Biodegradation of the
cyanobacterial toxin microcystin-LR in natural water and biologically active slow sand
filters. Water Research, 40(6): 1294-1302.
Boyd, E. and Clevenger, T. (2002). Removal of cyanobacterial toxins in Midwestern States. In:
Proceedings of the Water Quality and Technology Conference. American Water Works
Association, Denver, CO.
Boyer, G. L. (2007). Cyanobacterial toxins in New York and the Lower Great Lakes ecosystems.
In: H. K. Hudnell, (Ed.), Proceedings of the Interagency, International Symposium on
Cyanobacterial Harmful Algal Blooms (ISOC-HAB): State of the Science and Research
Needs, Advances in Experimental Medicine and Biology. Springer Press, New York, NY.
pp 151-163.
Brooke, S., Newcombe, G., Nicholson, B., and Klass, G. (2006). Decrease in toxicity of
microcystins LA and LR in drinking water by ozonation. Toxicon, 48(8): 1054-1059.
Brooks, W. P. and Codd, G. A. (1987). Distribution of Microcystis aeruginosa peptide toxin and
interactions with hepatic microsomes in mice. Pharmacology and Toxicology, 60(3): 187-
191. (Cited in WHO 1999).
Drinking Water Health Advisory for Microcystins-June 2015 49
-------
Burns, J., (2008). Toxic cyanobacteria in Florida waters. In: In: H. K. Hudnell, (Ed.), Proceedings
of the Inter agency, International Symposium on Cyanobacterial Harmful Algal Blooms
(ISOC-HAB): State of the Science and Research Needs, Advances in Experimental
Medicine and Biology. Chapter 5. Springer Press, New York, NY. pp.139-152.
Campinas, M. and Rosa, M. J. (2010). Evaluation of cyanobacterial cells removal and lysis by
ultrafiltration. Separation and Purification Technology, 70(3): 345-352.
Carey, C. C., Ibelings, B. W., Hoffmann, E. P., et al. (2012). Eco-physiological adaptations that
favour freshwater cyanobacteria in a changing climate. Water Research, 46(5): 1394-
1407.
Carmichael, W. W. (1992). A Status Report onPlanktonic Cyanobacteria (Blue Green Algae) and
their Toxins. EPA/600/R-92/079, Environmental Monitoring Systems Laboratory, Office
of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH.
(Cited in WHO 1999).
Carmichael, W. W., Beasley, V., Bunner, D. L., et al. (1988). Naming of cyclic hepatapeptide
toxins of cyanobacteria (blue-green algae). Toxicon 26(11): 971-973.
Carmichael W. W., Azevedo S. M., An J. S., et al. (2001). Human fatalities from cyanobacteria:
chemical and biological evidence for cyanotoxins. Environmental Health Perspectives
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,
8(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, MD. pp. 1710-1727. (Cited in WHO 1999).
Chen, Y., Xu, J., Li, Y., and Han, X. (2011). Decline of sperm quality and testicular function in
male mice during chronic low-dose exposure to microcystin-LR. Reproductive
Toxicology, 31(4): 551-557.
Cheng, X., Maher, J., Dieter, M. Z., and Klaassen, C. D. (2005). Regulation of mouse organic
anion-transporting polypeptides (OATPs) in liver by prototypical microsomal enzyme
inducers that activate transcription factor pathways. Drug Metabolism Disposition, 33(9):
1276-1282.
Chernoff, N., Hunter, E. S., Ill, Hall, L. L, et al. (2002). Lack of teratogenicity of microcystin-LR
in the mouse and toad. Journal of Applied Toxicology, 22(1): 13-17.
Christensen, V. G., Maki, R. P., and Kiesling, R. L. (2011). Relation of Nutrient Concentrations,
Nutrient Loading, and Algal Production to Changes in Water Levels in Kabetogama Lake,
Drinking Water Health Advisory for Microcystins-June 2015 50
-------
Voyageurs National Park, Northern Minnesota, 2008-09. U.S. Geological Survey Scientific
Investigations Report 2011-5096, 50 p.
Christoffersen, K., Lyck, S., and Winding, A. (2002). Microbial activity and bacterial community
structure during degradation of microcystins, Aquatic Microbial Ecology, 27: 125-136.
Chow, C. W. K., Panglisch, S., House, J., et al. (1997). A study of membrane filtration for the
removal of cyanob acted al cells. Journal of Water Supply: Research and Technology,
46(6): 324-334.
Chow, C., Drikas, M. and Ho, J. (1999). The impact of conventional water treatment processes on
cells of the cyanobacterium Microcystis aeruginosa. Water Research, 33(15): 3253-3262.
Chow, C. W. K., House, J., Velzeboer, R. M. A., et al. (1998). The effect of ferric chloride
flocculation on cyanobacterial cells. Water Research, 32(3): 808-814.
Chorus, I (Ed.). (2012). Current Approaches to Cyanotoxin Risk Assessment, Risk Management
and Regulations in Different Countries. Federal Environment Agency. Dessau-Ro|31au,
Germany. Online publication: www.uba.de/uba-info-medien-e/4390.html
Chorus, I, Falconer, I, Salas, H. S., and Bartram, J. (2000). Health risks caused by freshwater
cyanobacteria in recreational waters. Journal of Toxicology and Environmental Health-
Part B, Critical Reviews, 3(4): 323-347.
Codd, G. A., Morrison, L. F., and Metcalf, J. S. (2005). Cyanobacterial toxins: risk management
for health protection. Toxicology and Applied Pharmacology, 203(3): 264-272.
Cook, D. andNewcombe, G. (2008). Comparison and modeling of the adsorption of two
microcystin analogues onto powdered activated carbon. Environmental Technology, 29(5):
525-534.
Cote, L-M., Lovell, R. A., Jeffrey, E. H., et al. (1986). Failure of blue-green algae (Microcystis
aeruginosa) hepatotoxin to alter in vitro mouse liver enzymatic activity. Journal of
Toxicology Toxin Reviews, 52(2): 256
Cousins, I. T., Dealing, D. J., James, H. A., and Sutton, A. (1996). Biodegradation of microcystin-
LRby indigenous mixed bacterial populations. Water Research, 30(2): 481-485. (Cited in
WHO 1999).
Craig, M., Luu, H. A., McCready, T. L., et al. (1996). Molecular mechanisms underlying the
interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases.
Biochem. Cell Biology, 74(4): 569-578.
de la Cruz, A., Antoniou, M., Hiskia, A., et al. (2011). Can We Effectively Degrade
Microcystins? - Implications on Human Health. Anti-Cancer Agents in Medicinal
Chemistry, 11:19-37.
Drinking Water Health Advisory for Microcystins-June 2015 51
-------
Ding, J. Shi, H., Timmons, T., et al. (2010). Release and removal of microcystins from
Microcystis during oxidative, physical and UV-based disinfection. Journal of
Environmental Engineering, 136(1): 2-11.
Ding, W. X. and Ong, C. N.(2003). Role of oxidative stress and mitochondrial changes in
cyanobacteria-induced apoptosis and hepatotoxicity. FEMSMicrobiology Letters, 220(1):
1-7.
Dixon, M. B., Falconet, C., Ho, L., et al. (2010). Nanofiltration for the removal of algal
metabolites and the effects of fouling. Water Science Technology 61(5): 1189-99.
Dixon, M. B., Falconet C, Ho L., et al.(201 la). Removal of cyanobacterial metabolites by
nanofiltration from two treated waters. Journal of Hazardous Materials, 188(1-3): 288-95.
Dixon, M. B., Richard, Y., Ho, L., et al. (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): 553-1559.
Drikas, M., Chow, C., Christopher, W. K., et al. (2001). Using coagulation, and settling to remove
toxic cyanobacteria. Journal American Water Works Association, 93(2): 100-111.
Dugan, N. R. and Williams, D. J. (2006). Cyanobacteria passage through drinking water filters
during perturbation episodes as a function of cell morphology, coagulant and initial filter
loading rate. Harmful algae, 5: 26-35.
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
Envrionemental Contamination and Toxicology, 163: 113-186.
Edwards C., Graham, D., Fowler, N., and Lawton, L. A. (2008). Biodegradation of microcystins
and nodularin in freshwaters. Chemosphere, 73(8):1315-1321.
Falconer, I. R. (1998). Algal toxins and human health. In: J. Hubec (Ed.), Handbook of
Environmental Chemistry, Vol. 5, Part C, Quality and Treatment of Drinking Water, pp.
53-82.
Falconer, I. R., Beresford, A. M., and Runnegar, M. T. C. (1983). Evidence of liver damage by
toxin from a bloom of the blue-green alga, Microcystis aeruginosa. Medical Journal of
Australia, 1(11): 511-514.
Falconer, I. R., Buckley, T., and Runnegar, M. T. C. (1986). Biological half-life organ
distribution and excretion of 125I-labeled toxic peptide from the blue-green alga
Microcystis aeruginosa. Australian journal of biological sciences, 39(1): 17-21.
Drinking Water Health Advisory for Microcystins-June 2015 52
-------
Falconer, I. R., Burch, M. D., Steffense, M., et al. (1994). Toxicity of the blue-green alga
(Cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal
model for human injury and risk assessment. Environmental Toxicology and Water
Quality, 9(2): 131-139.
Falconer, I.R. (2005). Cyanobacterial Toxins of Drinking Water Supplies: Cylindrospermopsins
and Microcystins. CRC Press Boca Raton, Florida. 263p
Fawell, J. K., Mitchell, R. E., Everett, D. I, and Hill, R. E. (1999). The toxicity of Cyanobacterial
toxins in the mouse. 1. Microcystin-LR. Human and Experimental Toxicology, 18(3): 162-
167.
Fay, P. (1965). Heterotrophy and nitrogen fixation in Chlorogloea fritschii. Journal of General
Microbiology, 39: 11-20. (Cited in WHO 1999).
Feurstein, D., Kleinteich, J., Heussner, A. H., et al. (2010). Investigation of Microcystin
Congener-Dependent Uptake into Primary Murine Neurons. Environmental Health
Perspectives, 118(10): 1370-1375.
Fischer, W. J., Altheimer, S., Cattori, V., et al.(2005). Organic anion transporting polypeptides
expressed in liver and brain mediate uptake of microcystin. Toxicology and Applied
Pharmacology, 203(3): 257-263.
Fleming, L. E., Rivero, C., Burns, J., et al. (2002). Blue green algal (Cyanobacterial) toxins,
surface drinking water and liver cancer in Florida. Harmful Algae, 1(2): 157-168.
Fleming, L. E., Rivero, C., Burns, J., et al. (2004). Cyanobacteria exposure, drinking water and
colorectal cancer. In: K. A. Steidinger, J. H. Landsberg, C. R. Tomas and G. A.Vargo,
(Eds.), Harmful Algae 2002. Proceedings of the Xth International Conference on Harmful
Algae. Florida Fish and Wildlife Conservation Commission and Intergovernmental
Oceanographic Commission of UNESCO, Tallahassee, FL. p. 470-472.
Fu, W. Y., Chen, J. P., Wang, X. M. and Xu, L. H. (2005). Altered expression of p53, Bel-2 and
Bax induced by microcystin-LR in vivo and in vitro. Toxicon, 46(2): 171-177.
Funari, E. and Testai, E. (2008). Human Health Risk Assessment Related to Cyanotoxins
Exposure. Critical Reviews in Toxicology, 38: 97-125.
Giannuzzi, L., Sedan, D., Echenique R., and Andrinolo, D. (2011). An Acute Case of Intoxication
with Cyanobacteria and Cyanotoxins in Recreational Water in Salto Grande Dam,
Argentina. Marine Drugs, 2011(9): 2164-2175.
Gijsbertsen-Abrahamse, A. J., Schmidt, W., Chorus, L, and Heijman, S. G. J. (2006). Removal of
Cyanotoxins by ultrafiltration and nanofiltration. Journal of Membrane Science, 276: 252-
259.
Drinking Water Health Advisory for Microcystins-June 2015 53
-------
Graham, J. L., Ziegler, A. C., Loving, B. L., and Loftin, K. A.(2012). Fate and Transport of
Cyanobacteria and Associated Toxins and Taste-and-Odor Compounds from Upstream
Reservoir Releases in the Kansas River, Kansas, September and October 2011. U.S.
Geological Survey Scientific Investigations Report 2012-5129, pp. 65.
Graham, J., Loftin, K., Meyer, M., and Ziegler, A. (2010). Cyanotoxin mixtures and taste-and-
odor-compounds in cyanobactedal blooms from the midwestern United States.
Environmental Science and Technology, 44: 7361-7368.
Grutzmacher, G. Bottcher, G., Chorus, I, and Bartel, H. (2002). Removal of microcystins by slow
sand filtration. Environmental Toxicology, 17(4): 386-394.
Global Water Research Coalition (GWRC). (2009). International Guidance Manual for the
Management of Toxic Cyanobacteria. G. Newcombe (Ed.). Global Water Research
Coalition and Water Quality Research Australia. London, United Kingdom.
Gupta, Nidhi, et al. (2003). Comparative toxicity evaluation of cyanobactedal cyclic peptide toxin
microcystin congeners (LR, RR, YR) in mice. Toxicology 188; 2: 285-296.
Guzman, R. E., Solter, P. F. (1999). Hepatic oxidative stress following prolonged sublethal
Microcystin LR exposure. Toxicologic Pathology, 27: 582-588.
Haddix, P. L., Hughley, C. J., and Lechevallier, M. W. (2007). Occurrence of microcystins in 33
US water supplies. Journal American Water Works Association, 99(9): 118-125.
Han, J., B. Jeon, and H. Park. 2012. Cyanobacteria cell damage and cyanotoxin release in
response to alum treatment Water Science & Technology: Water Supply, 12.5, pp. 549-
555.
Haney, J. and Ikawa, M. (2000). A Survey of 50 NH Lakes for Microcystin (MCs). Final Report
Prepared for N.H. Department of Environmental Services by University of New
Hampshire. Retrieved September 25, 2012 from the World Wide Web:
http://water.usgs.gov/wrri/AnnualReports/2000/NHfy2000_annual_report.pdf
Harada K., Ogawa, K., Kimura, Y., et al. (1991). Microcystins from Anabaena flos-aquae NRC
525-17. Chemical Research in Toxicology, 4(5): 535-540.
Health Canada. (2002). Guidelines for Canadian Drinking Water Quality: Supporting
Documentation - Cyanobacterial Toxins-Microcystin-LR. Water Quality and Health
Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa,
Ontario. Available at http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/doc_sup-
appui/index_e. html
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. pp. 48.
Drinking Water Health Advisory for Microcystins-June 2015 54
-------
Health Canada. (2015). Guidelines for Canadian Drinking Water Quality: Guideline technical
document for public comment - Cyanobacterial toxins. Water and Air Quality Bureau,
Healthy Environments and Consumer Safety Branch, Ottawa, Ontario. Available in 2015.
Heinze, R. (1999). Toxicity of the Cyanobacterial toxin microcystin-LR to rats after 28 days
intake with the drinking water. Environmental Toxicology., 14(1): 57-60.
Hermansky, S. J., Casey, P. J., and Stohs, S. J. (1990a). Cyclosporin A - a chemoprotectant
against microcystin-LR toxicity. Toxicology Letters, 54(2-3): 279-285.
Hermansky S. J., Wolff, S. N., and Stohs, S. J. (1990b). Use of rifampin as an effective
chemoprotectant and antidote against microcystin-LR toxicity. Pharmacology 41(4): 231-
236.
Hilborn E. D., Scares R. M., Servaites J. C., et al. (2013). Sublethal Microcystin Exposure and
Biochemical Outcomes among Hemodialysis Patients. PLoS ONE, 8(7): e69518
Hitzfeld, B. S., Hoeger, J., and Dietrich, D. R. (2000). Cyanobacterial Toxins: Removal during
Drinking Water Treatment, and Human Risk Assessment. Environmental Health
Perspectives, 108: 113-122.
Ho, L., Sawade, E., and Newcombe, G. (2012b). Biological treatment options for cyanobacteria
metabolite removal-a review. Water Research, 46: 1536-1548.
Ho, L. and Newcombe, G.(2007). Evaluating the adsorption of microcystin toxins using granular
activated carbon (GACI Journal of Water Supply: Research and Technology-Aqua, 56(5):
281-291.
Ho, L., Dreyfus, J., Boyer, J., et al. (2012a). Fate of cyanobacteria and their metabolites during
water treatment sludge management processes. Science of the Total Environment,
424:232-238.
Ho, L., Barbero, A., Dreyfus, J., et al. (2013). Behaviour of Cyanobacterial bloom material
following coagulation and/or sedimentation. Journal of Water Supply: Research and
Technology-Aqua, 62(2): 350-358.
Ho, L., Onstand, G., von Gunten, U., et al. (2006a). Differences in the chlorine reactivity of four
microcystin analogues. Water Research, 40(6): 1200-1209.
Ho, L., Meyn, T., Keegan, A., et al. (2006b). Bacterial degradation of microcystin toxins within a
biologically active sand filter. Water Research, 40(4): 768-774.
Ho, L., Wijesundara, S., Shaw, G., 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.
Drinking Water Health Advisory for Microcystins-June 2015 55
-------
Ho, L., Lambling, P., Bustamante, H., 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.
Hoeger, S. J., Shaw, G., Hitzfeld, B. C., Dietrich, D. R. (2004). Occurrence and elimination of
cyanobacterial toxins in two Australian drinking water treatment plants. Toxicon, 43: 639-
649.
Hoeger, S. J., Hitzfeld, B. C., Dietrich, D. R. (2005). Occurrence and elimination of
cyanobacterial toxins in drinking water treatment plants. Toxicology and Applied
Pharmacology, 203: 231-242.
Huang, W. J., Cheng, B. L. and Cheng, Y. L. (2007). Adsorption of microcystin-LR by three
types of activated carbon. Journal of Hazardous Materials., 141: 115-122.
Huang, P., Zheng, Q., and Xu, L-H. (2011). The apoptotic effect of oral administration of
microcystin-RR on mice liver. Environmental Toxicology, 26(5): 443-452.
Hudnell, H. K. (Ed.) (2008). Cyanobacterial Harmful Algae Blooms, State of the Science and
Research Needs. Proceedings of the Inter agency, 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: 1024-1034.
Ito, E., Kondo, F., and Harada, K-I. (1997a). Hepatic necrosis in aged mice by oral administration
of microcystin-LR. Toxicon, 35(2): 231-239.
Ito, E., Kondo, F., Terao, K., and Harada, K-I. (1997b). Neoplastic nodular formation in mouse
liver induced by repeated i.p. injections of microcystin-LR. Toxicon, 35(9): 1453-1457.
Jochimsen, E. M., Carmichael W. W., An J. S., et al. (1998). Liver failure and death after
exposure to microcystins at a hemodialysis center in Brazil. New England Journal of
Medicine, 338(13): 873-8.
Jones, G. J., Blackburn, S. I, and Parker, N. S. (1994). A toxic bloom oiNodularia spumigena
Mertens in Orielton Lagoon, Tasmania. Australian Journal of Marine and Freshwater
Research, 45: 787-800. (Cited in WHO 1999).
Jurczak, T., Tarczynska, M., Izydorczyk, K., et al. (2005). Elimination of microcystins by water
treatment processes-examples from SulejowResevoir, Poland. Water Research, 39(3):
2394-2406.
Drinking Water Health Advisory for Microcystins-June 2015 56
-------
Karner, D. A., Standridge, J. H., Harrington, G. W., and Barnum, R. P. (2001). Microcystin algal
toxins in source and finished drinking water. Journal American Water Works Association.,
93(8): 72-81.
Keijola, A., Himber, K, Esala, A. L., et al. (1998). Removal of cyanobactedal toxins in water
treatment processes: laboratory and pilot plant experiments. Toxicity Assessment, 3(5):
643-656.
Klassen, C. D. and Aleksunes, L. M. (1996). Xenobiotic bile acid and cholesterol transporters:
function and regulation. Pharmacological Reviews, 62: 1-96. (As cited in Zhou et al.,
2012).
Kondo, F., Ikai, Y, Oka, H., et al. (1992). Formation, characterization, and toxicity of the
glutathione and cysteine conjugates of toxic heptapeptide microcystins. Chemical
Research in Toxicology, 5(5): 591-596.
Kondo, F., Matsumoto, H, Yamada, S., et al. (1996). Detection and identification of metabolites
of microcystins in mouse and rat liver. Chemical Research in Toxicology, 9: 1355-1359.
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: 365-370.
Kull, T. P., Sjovall, O. T., Tammenkoski, M. K., et al. (2006). Oxidation of the cyanobacterial
hepatotoxin microcystin-LR by chlorine dioxie: influence of natural organic matter.
Environmental Science and Technology, 40(5): 1504-1510.
Kull, T. P., Backlund, P. H., Karlsson, K. M., and Meriluoto, J. A. (2004). Oxidation of the
cyanobacterial hepatotoxin microcystin-LR by chlorine dioxide: reaction kinetics,
characterization, and toxicity of reaction products. Environmental Science and
Technology, 38(22): 6025-6031.
Lahti, K., Niemi, M. R., Rapala, J., and Sivonen, K. (1997a). Biodegradation of cyanobacterial
hepatotoxins - characterization of toxin degrading bacteria. Proceedings of the VII
International Conference on Harmful Algae. (Cited in WHO 1999).
Lahti, K., Rapala, J., Fardig, M., Niemela, M., and Sivonen, K. (1997b). Persistence of
cyanobacterial hepatotoxin, microcystin-LR, in particulate material and dissolved in lake
water. Water Research, 31(5): 1005-1012. (Cited in WHO 1999).
Lahti, K., Rapala, J., Kivimaki, A. L., et al. (2001). Occurrence of microcystins in raw water
sources and treated drinking water of Finnish waterworks. Water Science and
Technology, 43(1): 225-228.
Drinking Water Health Advisory for Microcystins-June 2015 57
-------
Lahti, KJ. et al. (1998). Fate of cyanobactedal hepatotoxins in artificial recharge of groundwater
and in bank filtration. In: J. H. Peters, et al. (Eds), Artifical recharge of groundwater. A.S.
Balkema, Rotterdam, The Netherlands.
Lam, A. K., Fedorak, P. M, and Prepas, E. E. (1995). Biotransformation of the cyanobacterial
hepatotoxin microcystin-LR, as determined by HPLC and protein phosphatase bioassay.
Environmental Science and Technology, 29: 242-246.
Lambert, T. W., Holmes, C. F. B., and Hrudey, S. E. (1996). Adsorption of microcystin-LR by
activated carbon and removal in full scale water treatment. Water Research, 30(6): 1411-
1422.
Lee, J. and Walker, H. W. (2008). Mechanisms and factors influencing the removal of
microcystin-LR by ultrafiltration membranes. Journal of Membrane Science, 320: 240-
247.
Lepisto, L., Lahti, K., Niemi, J., and Fardig, M. (1994). Removal of cyanobacteria and other
phytoplankton in four Finish waterworks. Algological Studies, 75: 167-181.
Li, Y., Chen, J. A., Zhao, Q., et al. (201 la). A Cross-Sectional Investigation of Chronic Exposure
to Microcystin in Relationship to Childhood Liver Damage in the Three Gorges Reservoir
Region, China. Environmental Health Perspectives. 119(10): 1483-1488.
Li, T., Huang, P., Liang, J., et al. (201 Ib). Microcystin-LR (MCLR) induces a compensation of
PP2A activity mediated by alpha4 protein in HEK293 cells. InternationalJournal of
Biological Sciences, 7(6): 740-52.
MWH. (2012). Water treatment principles and design. 3rd edition. John Wiley & Sons. New
York, NY.
Makarewicz, J., Boyer, G., Guenther, W., et al. (2006). The Occurrence of Cyanotoxins in the
Nearshore and Coastal Embayments of Lake Ontario. Great Lakes Research Review, 7:
25-29.
Makarewicz, J., Boyer, G. Lewis, T., et al. (2009). Spatial and temporal distribution of the
cyanotoxin microcystin-LR in the Lake Ontario ecosystem: Coastal embayments, rivers,
nearshore and offshore, and upland lakes. Journal of Great Lakes Research, 35: 83-89.
Matthijs, H. C., Visser, P. M., Reeze, B., et al. (2012). Selective suppression of harmful
cyanobacteria in an entire lake with hydrogen peroxide. Water Research, 46(5): 1460-
1472.
Merel, S., LeBot, B., Clement, M., et al. (2009). MS identification of microcystin-LR chlorination
by-products. Chemosphere, 74(6): 832-839.
Drinking Water Health Advisory for Microcystins-June 2015 58
-------
Merel, S., Clement, M., and Thomas, O. (2010). State of the art on cyanotoxins in water and their
behaviour towards chlorine. Toxicon, 55: 677-691.
Minnesota Department of Health (MDH) (2012). Health Based Value for Groundwater. Health
Risk Assessment Unit, Environmental Health Division, No.651-201-4899. Retrieved April
25, 2014 from the World Wide Web:
http://www.health.state.mn.us/divs/eh/risk/guidance/gw/microcystin.pdf
Nasri, H., Bouaicha, N, and Harche, M. K. (2007). A new morphospecies of Microcystis sp.
forming bloom in the Cheffia Dam (Algeria): seasonal variation of microcystin
concentrations in raw water and their removal in a full-scale treatment plant.
Environmental Toxicology, 22(4): 347-356.
Neumann, U., and Weckesser, J. (1998). Elimination of microcystin peptide toxins from water by
reverse osmosis. Environmental Toxicology, 13: 143-148.
Newcombe, G. (2002). Removal of Algal Toxins from Drinking Water Using Ozone andGAC.
American Water Works Association Reseach Foundation and American Water Works
Association. Denver, CO.
Newcombe, G., Cook, D., Brooke, S., et al. (2003). Treatment options for microcystin toxins:
similarities and differences between variants. Environmental Toxicology, 24: 299-308.
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.
Newcombe, G., Dreyfus, J., Monrolin, Y., et al. (2015). Optimizing Conventional Treatment for
the Removal of Cyanobacteria and Toxins. Water Research Foundation, Denver, CO.
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_drinkin
g_water_guidelines_l 50413 .pdf
Nicholson, B. C., Rositano, J., and Burch, M. D. (1994). Destruction of cyanobacterial peptide
hepatotoxins by chlorine and chloramine. Water Research, 28(6): 1297-1303.
Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S. et al. (1992). Liver tumor promotion by the
cyanobacterial cyclic peptide toxin microcystin-LR. Journal of Cancer Research and
Clinical Oncology, 118(6): 420-424.
O'Reilly, A., Wanielista, M., Loftin, K., and Chang, N. (2011). Laboratory simulated transport of
microcystin-LR and cylindrospermopsin in groundwater under the influence of stormwater
ponds: implications for harvesting of infiltrated stormwater. GQ10: Groundwater Quality
Management in a Rapidly Changing World (Proc. 7th International Groundwater Quality
Drinking Water Health Advisory for Microcystins-June 2015 59
-------
Conference held in Zurich, Switzerland, 13-18 June 2010). IAHS Publ 342, 2011, 107-
111.
Ohio EPA (OHEPA) (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
Ohio EPA (OHEPA). (2014). Public Water System Harmful Algal Bloom Response Strategy.
Retrieved April 25, 2014 from the World Wide Web:
http://epa.ohio.gov/Portals/28/documents/HABs/PWS_HAB_Response_Strategy_2014.pd
f
Ohta, T., Sueoka, E., lida, N., et al. (1994). Nodularin, a potent inhibitor of protein phosphatases
1 and 2A, is a new environmental carcinogen in male F344 rat liver. Cancer Research,
54(24): 6402-6406.
Oregon Health Authority (OHA) (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/D
ocuments/HABPublicHealthAdvisoryGuidelines%2020150424x.pdf
Pace, J. G., Robinson, N. A., Miura, G. A., et al. (1991). Toxicity and kinetics of 3H microcystin-
LR in isolated perfused rat livers. Toxicolgy and Applied Pharmacology, 107(3): 391-401.
Paerl, H. W. and Often, T. G. (2013a). Blooms Bite the Hand That Feeds Them. Science, 342(25):
433-434.
Paerl, H. W. and Often, T. G. (2013b). Harmful Cyanobacterial Blooms: Causes, Consequences,
and Controls. MicrobialEcology, 65: 995-1010.
Pawlowicz, M. B., Evans, J. E., Johnson, D. R., and Brooks, R. G. (2006). A study of the efficacy
of various home filtration substrates in the removal of microcysin-LR from drinking
water. Journal of Water and Health, 4(1): 99-107.
Pendleton, P., Schumann, R. and Wong, S. H. (2001). Microcystin-LR adsorption by activated
carbon. Journal of Colloid and Interface Science, 240: 1-8.
Pereira, S. P., Martins, F. C., Gomes, L. N. L., et al. (2012). Removal of cyanobacteria by slow
sand filtration for drinking water. Journal of Water, Sanitation, and Hygiene for
Development, 2(3): 133-145.
Puddick, J., Prinsep, M., Wood, S., et al. (2015). Further Characterization of Glycine-Containing
Microcystins from the McMurdo Dry Valleys of Antarctica. Toxins, 7: 493-515.
Drinking Water Health Advisory for Microcystins-June 2015 60
-------
Rai, A. N. (1990). CRC Handbook of Symbiotic Cyanobacteria. CRC Press, Boca Raton, FL. pp.
253. (Cited in WHO 1999).
Rajasekhar, P., Fan, L., Nguyen, T., and Roddick, F. A. (2012). A review of the use of sonication
to control cyanobactedal blooms. Water Research, 46: 4319-4329.
Rao, P. V. L., Gupta, N., Bhaskar, A. S. B., and Jayaraj, R. (2002). Toxins and bioactive
compounds from cyanobacteria and their implications on human health. Journal of
Environmental Biology, 3: 215-224.
Rao, P. V. L., Gupta, N., Jayaraj, R., et al. (2005). Age-dependent effects on biochemical
variables and toxicity induced by cyclic peptide toxin microcystin-LR in mice.
Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology,
140(1): 11-19.
Rapala, J., Lahti, K., Sivonen, K., and Niemeld, S. (1994). Biodegradability and adsorption on
lake sediments of cyanob acted al hepatotoxins and anatoxin-a. Letters in Applied
Microbiology, 19: 423-428. (Cited in WHO 1999).
Rapala, J., Niemela, M., Berg, K., et al.(2006). Removal of cyanobacteria, cyanotoxins,
heterotrophic bacteria and endotoxins at an operating surface water treatment plant. Water
Science and Technology, 54(3): 23-28.
Robinson, N. A., Pace, J. G., Matson, C. F., et al. (1991). Tissue distribution, excretion and
hepatic biotransformation of microcystin-LR in mice. Journal of Pharmacology and
Experimental Therapeutics, 256(1): 176-182.
Rodriguez, E., Onstad, G. D., Kull, T. P. J., et al. (2007a). Oxidative elimination of cyanotoxins:
comparison of ozone, chlorine, chlorine dioxide and permanganate. Water Research,
41(15): 3381-3393.
Rodriguez, E., Majado, M. E., Meriluoto, J., et al. (2007b). Oxidation of microcystins by
permanganate: reaction kinetics and implications for water treatment. Water Research,
41(1): 102-110.
Rositano, J., Nicholson, B. C., and Pieronne, P. (1998). Destruction of cyanobacterial toxins by
ozone . Ozone: Science and Engineering: The Journal of the International Ozone
Association, 20(3): 223-238.
Rositano, J., Newcombe, G., Nicholson, B., and Sztanjnbok. (2001). Ozonation of NOM and algal
toxins in four treated waters. Water Research, 35(1): 23-32.
Roy-Lachapelle, A., Fayad, P. B., Sinotte, M., et al. (2014). Total microcystin analysis in water
using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem
mass spectrometry. Analytical Chimica Ada, 820: 76-83.
Drinking Water Health Advisory for Microcystins-June 2015 61
-------
Runnegar, M. T. C. and Falconer, I. R. (1982). The in vivo and in vitro biological effects of the
peptide hepatotoxin from the blue-green alga Microcystis aeruginosa. South African
Journal of Science., 78: 363-366.
Runnegar, M., Berndt, N., Kong, S. M., et al. (1995). In vivo and in vitro binding of microcystin
to protein phosphatases 1 and 2 A. Biochemical and Biophysical Research
Communications, 216(1): 162-169.
Runnegar, M. T. C., Falconer, I. R., and Silver, J. (1981). Deformation of isolated rat hepatocytes
by a peptide hepatoxin from the blue-green alga Microcystis aeruginosa. Naunyn-
Schmiedeberg's Archives of Pharmacology., 317(3): 268-272.
Sarma, T.A. (2013). Cyanobacterial Toxins in Handbook of Cyanobacteria. CRC Press. Taylor
and Francis Group, pp. 487-606.
Schijven, J., Berger, P, and Miettinen, I. (2002). Removal of pathogens, surrogates, indicators and
toxins using riverbank filtration. In: C. Ray (Ed), Riverbank Filtration: Improving Source-
Water Quality. Kluwer Academic Publishers, Netherlands, pp. 73-116.
Sharma, V. K., Triantis, T. M., Antoniou, M. G., et al. (2012). Destruction of microcystins by
conventional and advanced oxidation processes: a review. Separation and Purification
Technology, 91: 3-17.
Shawwa, A. R. and Smith, D. W. (2001). Kinetics of microcystin-LR oxidation by ozone. Ozone:
Science and Engineering: The Journal of the International Ozone Association, 23: 161-
170.
Sklenar, K., Westrick, J., and Szlag, D.(2014). Managing and Mitigating Cyanotoxins in Water
Supplies. Water Research Foundation Webcast. August 28, 2014.
Scares, R.M. Yuan, M., Servaites, C. Delgado, A., V.F. Magalhaes, V., Hilborn, E., Carmichael,
W., and Azevedo, S. (2005). Sublethal exposure from microcystins to renal insufficiency
patients in Rio de Janeiro, Brazil. Environmental Toxicology, 21(2): 95-103
Song, W., Teshiba, T., Rein, K., and O'Shea, K. E. (2005). Ultrasonically induced degradation
and detoxification of microcystin-LR (cyanobacterial toxin). Environmental Science and
Technology, 39(16): 6300-6305.
Sorlini, S. and Collivignarelli, C.(2011). Microcystin-LR removal from drinking water supplies
by chemical oxidation and activated carbon adsorption. Journal of Water Supply Research
and Technology, 60(7): 403-411.
Sorlini, S., Gialdini, F., and Collivignarelli, C. (2013). Removal of cyanobacterial cells and
microcystin-LR from drinking water using a hollow fiber microfiltration pilot plant.
Desalination, 309: 106-112.
Drinking Water Health Advisory for Microcystins-June 2015 62
-------
Stewart, I, Schluter, P., and Shaw, G. (2006a). Cyanobacterial lipopolysaccharides and human
health - a review. Environmental Health., 5(1): 1-23.
Stoner, R. D.; Adams, W. H.; Slatkin, D. N.; and Siegelman, H. W. (1989). The effects of single
L-amino acid substitutions on the lethal potencies of microcystins. Toxicon, 27(7): 825-
828.
Sun, F., Pei, H.-Y., Hu, W.-R., and Ma, C.-X. (2012). The lysis of Microcystis aeruginosa in
AlCb coagulation and sedimentation processes. Chemical Engineering Journal, 193-194:
196-202.
Svoboda, M., Riha, J., Wlcek, K., et al. (2011). Organic Anion Transporting Polypeptides
(OATPs): regulation of expression and function. Current Drug Metabolism, 12(2): 139-
153.
Takumi, S., Komatsu, M., Furukawa, T., et al.(2010). p53 plays an important role in cell fate
determination after exposure to microcystin-LR. Environmental Health Perspectives,
118(9): 1292-1298.
Tarczyriska, M., Romanowska -Duda, Z., Jurczak, T., and Zalewski, M. (2001). Toxic
Cyanobacterial blooms in drinking water reservoir, causes, consequences, and
management strategy. Water Science and Technology: Water Supply, 1(2): 237-246.
Thompson, W. L. and Pace, J. G. (1992). Substances that protect cultured hepatocytes from the
toxic effects of microcystin-LR. Toxicology In vitro, 6(6): 579-587.
Touchette, B. W., Burkholder, J. M., Allen, E. H., et al. (2007). Eutrophication and cyanobacteria
blooms in run-of-river impoundments in North Carolina, U.S.A. Lake and Reservoir
Management, 23(2): 179-192.
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
Tsuji, K., Naito, S., Kondo, F., et al. (1993). Stability of microcystins from cyanobacteria: Effect
of light on decomposition and isomerization. Environmental Science and Technology, 28:
173-177. (Cited in WHO 1999).
Tsuji, K., Naito, S., Kondo, F., et al. (1995). Stability of microcystins from cyanobacteria-II.
Effect of UV light on decomposition and isomerization. Toxicon, 33(12): 1619-1631.
Turner, P. C., Gammie, A. J., Hollinrake K., and Codd, G. A. (1990). Pneumonia associated with
contact with cyanobacteria. British Medical Journal, 300(6737): 1440-1441.
Drinking Water Health Advisory for Microcystins-June 2015 63
-------
Ueno, Y., Makita, Y., Nagata, S., et al. (1999). No chronic oral toxicity of a low-dose of
microcystin-LR, a cyanobacterial hepatoxin, in female Balb/C mice. Environmental
Toxicology, 14(1): 45-55.
Ueno, Y., Nagata, S., Tsutsumi, T., et al. (1996). Detection of microcystins, a blue-green algal
hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary
liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 17(6): 1317-
1321.
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). (2007). Drinking Water Treatability
Database: Microcystins. Available from:
http://iaspub.epa.gov/tdb/pages/contaminant/contaminantOveiview.do7contaminantId—
1336577584
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/lakessurvey/pdf/nla_report_l ow_res.pdf
U.S. EPA (United States Environmental Protection Agency). (201 la). Exposure Factors
Handbook 2011 Edition (Final). Washington, DC, EPA/600/R-09/052F. Available from:
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-use-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
Drinking Water Health Advisory for Microcystins-June 2015 64
-------
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-decisi on-making
U.S. EPA (United States Environmental Protection Agency). (2015a). Health Effects Support
Document for the Cyanobacterial Toxin Microcystin. EPA-820R1502, Washington,
DC; 2015. Available from: http://www2.epa.gov/nutrient-policy-data/health-and-
ecological-effects
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 544. Determination
of Microcystins and Nodularin in Drinking Water by Solid Phase Extraction and Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS). Version 1.0, EPA/600/R-
14/474, Cincinnati, OH. Available from:
http ://www. epa.gov/nerl cwww/docum ents/Method544_Final .pdf
VanderKooi, S., Burdinck, Echols, K., et al. (2010). Algal Toxin in Upper Klamath Lake, Oregon:
Linking Water Quality to Juvenile Sucker Health. U.S. Geological Survey Fact Sheet
2009-3111, pp. 2.
Vlaski, A. van Breemen, A. N., Alaerts, G. J., et al. (1996). Optimisation of coagulation
conditions for the removal of cyanobacteria by dissolved air flotation or sedimentation.
Journal of Water Supply: Research and Technology, 45(5): 253-261.
Wang, H., Ho., L., Lewis, D. M., et al. (2007). Discriminating and assessing adsorption and
biodegradation removal mechanisms during granular activated carbon filtration of
microcystin toxins. Water Research, 41(18): 4262-4270.
Wang, Z. Li., D, Qin, H., and Li, Y. (2012). An integrated method for removal of harmful
Cyanobacterial blooms in eutrophic lakes. Environmental Pollution, 160: 34-41.
WSDE (Washington State Department of Ecology). (2012). Freshwater Algae Control Program.
Accessed December 12,
2012; http://www.ecy.wa.gov/programs/wq/plants/algae/index.html
Wei, Y., Weng, D., Li, F., et al. (2008). Involvement of INK regulation in oxidative stress-
mediated murine liver injury by microcystin-LR. Apoptosis, 13(8): 1031-1042.
Weng, D., Y. Lu, Wei, Y., et al. (2007). The role of ROS in microcystin-LR-induced hepatocyte
apoptosis and liver injury in mice. Toxicology, 232(1-2): 15-23.
Drinking Water Health Advisory for Microcystins-June 2015 65
-------
Wert, E. C., and Rosario-Ortiz, F. L. (2013). Intracellular organic matter from cyanobacteria as a
precursor for carbonaceous and nitrogenous disinfection byproducts. Environmental
Science and Technology, 47: 6332-6340.
Wert, B.C., Dong, M. M., Rosario-Ortiz, F. L., and Korak, J. (2014). Release of Intracellular
Metabolites from Cyanobacteria During Oxidation Processes. Water Research
Foundation, Denver, CO.
Westrick, J. A., Szlag, D. C., Southwell, B. J., and Sinclair, J. (2010). A review of cyanobacteria
and cyanotoxins removal/inactivation in drinking water treatment. Analytical and
Bioanalytical Chemistry, 397(5): 1705-1714.
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.
WHO (World Health Organization). (2003). Cyanobacterial Toxins: Microcystin-LR in Drinking-
Water. Background document for development of WHO Guidelines for Drinking-water
Quality, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland.
Wolf, H.-U. and Frank, C. (2002). Toxicity assessment of Cyanobacterial toxin mixtures.
Environmental Toxicology, 17(4): 395-399.
Wynne, T., Stumpf, R., Tomlinson, M. et al. (2013). Evolution of a Cyanobacterial bloom forecast
system in western Lake Erie: Development and initial evaluation. Journal of Great Lakes
Research, Supplement 39(9): 90-99.
Xagoraraki, I, Harrington, G., Zulliger, K., et al. (2006). Inactivation kinetics of the
Cyanobacterial toxin microcystin-LR by free chlorine. Journal of Environmental
Engineering, 132(7): 818-823.
Xing, M. L., Wang, X. F., and Xu, L. H. (2008). Alteration of proteins expression in apoptotic FL
cells induced by MCLR. Environmental Toxicology, 23(4): 451-458.
Yuan, M., Namikoshi, M., Otsuki, A., et al. (1999). Electrospray lonization Mass Spectrometric
Analysis of Microcystins, Cyclic Heptapeptide Hepatotoxins: Modulation of Charge
States and [M 1 H]l to [M 1 Na]l Ratio. Journal of the Amereican Society for Mass
Spectrometry, 10: 1138-1151.
Yoshida, T., Makita, Y, Nagata, S., et al. (1997). Acute oral toxicity of microcystin-LR, a
Cyanobacterial hepatotoxin in mice. Natural Toxins, 5(3): 91-95.
Zamyadi, A., MacLeod, S. L., Fan, Y., et al. (2012a). Toxic Cyanobacterial breakthrough and
accumulation in a drinking water plant: A monitoring and treatment challenge. Water
Research, 46(5): 1511-1523.
Drinking Water Health Advisory for Microcystins-June 2015 66
-------
Zamyadi, A., Ho, L., Newcombe, G., et al. (2012b). Fate of toxic cyanobactedal cells and
disinfection by-products formation after chlorination. Water Research., 46(5): 1524-1535.
Zamyadi, A., Dorner, S., Sauve, S., et al. (2013a). Species dependence of cyanobacteria removal
efficiency by different drinking water treatment processes. Water Research, 47(8): 2689-
2700.
Zamyadi, A., Dorner, S., Ndong, M., et al. (2013b). Low-risk cyanob acted al bloom sources: cell
accumulation within full-scale treatment plants. Journal American Water Works
Association, 105(11): 651-663
Zamyadi, A., Fan, Y, Daly, R. I, Prevost, M. (2013c). Chlorination of Microcystis aeruginosa:
toxin release and oxidation, cellular chlorine demand and disinfection by-products
formation. Water Research, 47(3): 1080-1090.
Zhang, Z., Zhang, X. X., Qin, W., et al. (2012). Effects of microcystin-LR exposure on matrix
metalloproteinase-2/-9 expression and cancer cell migration. Ecotoxicology and
Environmental Safety, 77: 88-93.
Zhou, L., Yu, H., and Chen, K. (2002). Relationship between microcystin in drinking water and
colorectal cancer. Biomedical and Environmental Sciences, 15(2): 166-171.
Zhou, Y., Yuan, J., Wu, J., and Han, X. (2012). The toxic effects of microcystin-LR of rat
spermatogonia in vitro. Toxicology Letters, 212(1): 48-56.
Zurawell, R. (2002). An Initial Assessment of Microcystin in Raw and Treated Municipal
Drinking Water Derived from Eutrophic Surface Waters in Alberta. Alberta Environment,
Edmonton, AB. ISBN No. 0-7785-2417-5.
Drinking Water Health Advisory for Microcystins-June 2015 67
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