Drinking Water Health Advisory 

for the Cyanobacterial 

Microcystin Toxins 

United States 

Environmental 

Protection Agency 

Office of Water

Mail Code 4304T

EPA- 820R15100

June 2015
Drinking Water Health Advisory

for the Cyanobacterial Microcystin Toxins 

Prepared by:

U.S. Environmental Protection Agency 

Office of Water (4304T)

Health and Ecological Criteria Division

Washington, DC 20460 

EPA Document Number: 820R15100 

Date: June 15, 2015 
ACKNOWLEDGMENTS

This document was prepared by U.S. EPA Scientists Lesley V. D’Anglada, Dr.P.H. (lead) and 

Jamie Strong, Ph.D. Health and Ecological Criteria Division, Office of Science and Technology, 

Office of Water. EPA gratefully acknowledges the valuable contributions from Health Canada’s 

Water and Air Quality Bureau, in developing the Analytical Methods and Treatment Technologies 

information included in this document. 

This Health Advisory was provided for review and comments were received from staff in the 

following U.S. EPA Program Offices: 

U.S. EPA Office of Ground Water and Drinking Water 

U.S. EPA Office of Science and Technology 

U.S. EPA Office of Research and Development 

U.S. EPA Office of Children’s Health Protection

U.S. EPA Office of General Counsel

This Health Advisory was provided for review and comments were received from the following 

other federal and health agencies:

Health Canada

U.S. Department of Health and Human Services, Centers for Disease Control and 

Prevention

Drinking Water Health Advisory for Microcystins-June 2015 i 
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 ii
4.2 Consideration of Study Duration .................................................................................... 31

4.3 Consideration of Reproductive Effects as Endpoint ....................................................... 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 Cyanobacterial 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
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 μg/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 (Kondo et al., 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
ABBREVIATIONS AND ACRONYMS

Γgt Γ-Glutamyltransferase

A Alanine 

Adda 3-Amino-9-Methoxy-2,-6,-8-,Trimethyl-10-Phenyldeca-4,-6-Dienoic Acid

ALT Alanine Aminotransferase 

ALP Alkaline Phosphatase 

AST Aspartate Aminotransferase

AWWARF American Water Works Association Research Foundation 

BMD Benchmark Dose

BMDL Benchmark Dose Level

BW Body Weight

Clo2 Chlorine Dioxide

CAS Chemical Abstracts Service

CASA Computer-Assisted Sperm Analysis 

CCL Contaminant Candidate List 

CWA Clean Water Act

DBP Disinfection By-Products 

DL Detection Limit

EBCT Empty Bed Contact Time 

ELISA Enzyme-Linked Immunosorbent Assay

EPA U.S. Environmental Protection Agency 

FSH Follicle Stimulating Hormone

g Gram

GC/MS Gas Chromatograph/Mass Spectrometry 

GAC Granular Activated Carbon

GLERL Great Lakes Environmental Research Laboratory 

H2O2 Hydrogen Peroxide 

HA Health Advisory

HAB Harmful Algal Bloom

HESD Health Effects Support Document 

HPLC High-Performance Liquid Chromatography 

IARC International Agency for Research on Cancer

i.p. Intraperitoneal 

Kg Kilogram 

L Leucine

LC/MS Liquid Chromatography/Mass Spectrometry 

LDH Lactate Dehydrogenase

LD50 Lethal Dose to 50% of Organisms 

LH Luteinizing Hormone

LOAEL Lowest-Observed-Adverse-Effect Level

MC-LA Microcystin-LA

MC-LR Microcystin-LR

MC-RR Microcystin-RR

MC-YR Microcystin-YR

MC-YM Microcystin-YM

Drinking Water Health Advisory for Microcystins-June 2015 v 
Mdha Methyldehydroalanine

MERHAB-LGL Monitoring and Event Response to Harmful Algal Blooms in the Lower 

 Great Lakes

μg Microgram

μm Micromole

LOQ Level of Quantification

Mdls Method Detection Limit

mg Milligram

ml Milliliter

MMPB 2-methyl-3-methoxy-4-phenylbutyric acid

MOA Mode of Action 

MF Microfiltration

MWCO Molecular Weight Cut-Off 

NDEA N-Nitrosodiethylamine 

NF Nanofiltration

NLA National Lakes Assessment

NOAA National Oceanic and Atmospheric Administration

NOAEL No-Observed-Adverse-Effect Level

NOD Nodularin 

NOM Natural Organic Material

OATp Organic Acid Transporter Polypeptides 

PAC Powdered Activated Carbon

PAS Periodic Acid-Schiff 

PBS Phosphate-Buffered Saline 

PDA Photodiode Array Detector 

P-GST glutathione S-transferase placental form-positive

POD Point of Departure

POU Point-of-Use

PP2 Protein Phosphatase 2A 

PP1 Protein Phosphatase 1 

PPIA Protein Phosphatase Inhibition Assays

RfD Reference Dose

RO Reverse Osmosis 

ROS Reactive Oxygen Species

SDWA Safe Drinking Water Act 

SPE Solid-Phase Extraction 

TEF Toxicity Equivalency Factors 

TOC Total Organic Carbon 

TOXLINE Toxicology Literature Online

TUNEL Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick EndLabeling Assay

UF Uncertainty Factor

UF Ultrafiltration

USGS United States Geological Survey

UV Ultraviolet

WHO World Health Organization

Drinking Water Health Advisory for Microcystins-June 2015 vi 
EXECUTIVE SUMMARY 

Microcystins are toxins produced by a number of cyanobacteria species, including 

members of Microcystis, 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 of fish, 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. 

Drinking Water Health Advisory for Microcystins-June 2015 1 
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 

μg/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-adverseeffect 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 

μg/L and for school-age children through adults is 1.6 μg/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 (2011a)), 

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 2 
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 Microcystins (U.S.EPA, 2015a) is the peerreviewed, effects assessment that supports this HA. This document is available at 

http://www2.epa.gov/nutrient-policy-data/health-and-ecological-effects. The HAs are not legally 

enforceable Federal standards and are subject to change as new information becomes available. 

The structure of this Health Advisory is consistent with EPA’s Framework for Human Health 

Risk Assessment to Inform Decision Making (U.S.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). 

Drinking Water Health Advisory for Microcystins-June 2015 3 
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 μg/L 

microcystin-LR

Based on the World Health 

Organization (WHO) Provisional 

Guideline Value of 1ug/L for 

drinking water 

(WHO, 1999; 2003)

Australia

1.3 μg/L microcystinLR (toxicity 

equivalents)

Australian Drinking Water 

Guidelines 6 

(NHMRC, NRMMC, 2011)

Canada 1.5 μg/L 

microcystin-LR

Guidelines for Canadian 

Drinking Water Quality: 

Supporting Documentation 

Cyanobacterial ToxinsMicrocystin-LR 

(Health Canada, 2002)

Drinking Water Health Advisory for Microcystins-June 2015 4 
Table 1-2. State Guideline Values for Microcystins 

State Guideline Value Source

Minnesota 0.04 μg/L Microcystin-LR Minnesota Department of Health (MDH, 2012) 

Ohio 1 μg/L Microcystin Public Water System Harmful Algal Bloom 

Response Strategy (Ohio EPA, 2014)

Oregon 1 μg/L Microcystin-LR 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 μg/L 

(or the underlying Tolerable Daily Intake (TDI) of 0.04 μg/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 μg/L based on lifetime exposures. 

Drinking Water Health Advisory for Microcystins-June 2015 5 
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 Microcystins (U.S. EPA, 2015a). 

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 cyanobacterial species, including Anabaena, 

Fischerella, Gloeotrichia, Nodularia, Nostoc, Oscillatoria, members of Microcystis, 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 6 
Figure 2-1. Structure of Microcystin (Kondo et al., 1992) 

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.

Drinking Water Health Advisory for Microcystins-June 2015 7 
Table 2-1. Abbreviations for Microcystins (Yuan et al., 1999) 

Microcystin Congeners Amino Acid in X Amino Acid in Y

 Microcystin-LR Leucine Arginine 

Microcystin-RR Arginine Arginine

Microcystin-YR Tyrosine Arginine

Microcystin-LA Leucine Alanine

 Microcystin-LY Leucine Tyrosine 

 Microcystin-LF Leucine Phenylalanine

Microcystin-LW Leucine Tryptophan

 

 

Figure 2-2. Structure of the amino acids Adda and Mdha (Harada et al., 1991). 

Adda Mdha

Drinking Water Health Advisory for Microcystins-June 2015 8 
Table 2-2. Chemical and Physical Properties of Microcystin-LR

Property Microcystin-LR

Chemical Abstracts Registry (CAS) # 101043-37-2 

Chemical Formula C49H74N10O12

Molecular Weight 995.17 g/mole 

Color/Physical State Solid

Boiling Point N/A

Melting Point N/A

Density 1.29 g/cm3

Vapor Pressure at 25°C N/A

Henry’s Law Constant N/A

Kow N/A

Koc N/A

Solubility in Water Highly

Other Solvents 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 cyanobacterial 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.

Drinking Water Health Advisory for Microcystins-June 2015 9 
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 cyanobacterial blooms or surface freshwater 

samples reported worldwide between 1985 and 1996 ranged from 1 to 7,300 μg/g. Water 

concentrations of extracellular plus intracellular microcystins ranged from 0.04 to 25,000 μg/L. 

The concentration of extracellular microcystins ranged from 0.02 to a high of 1,800 μg/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 μg/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 were Microcystis (43.1%),

Cylindrospermopsis (39.5%), and Anabaena 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 μg/L) and was detected at levels of 0.084 μg/L in the nearshore and 0.076 μg/L in the bays 

and rivers. This study reported higher levels of microcystin-LR (1.6 to 10.7μg/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 μg/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 
of 2007. The NLA measured microcystins using Enzyme Linked Immunosorbent Assays (ELISA) 

with a detection limit of 0.1 μg/L as well as cyanobacterial cell counts and chlorophyll-a 

concentrations, which were indicators of the presence of cyanobacterial 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 μg/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 μg/L) to 225 μg/L. Two states (North 

Dakota and Nebraska), had 9% of samples above 10 μg/L. Other states including Iowa, Texas, 

South Dakota, and Utah also had samples that exceeded 10 μg/L. Several samples in North

Dakota, Nebraska, and Ohio exceeded the WHO high risk threshold value for recreational waters 

of 20 μg/L (192 and 225 μg/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 μg/L and 17 μg/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 μg/L (Beussink and Graham, 2011). In 2011, 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 μg/L. Upstream from Milford Lake, 

a cyanobacterial bloom was observed with a total microcystin concentration of 150,000 μg/L. 

When sampled a week later, total microcystin concentrations were less than 1 μg/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 

μg/L. 

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 μg/L) to 4,620 μg/L in 2008, 18,700 μg/L in 2009, 853 μg/L in 

2010, and 26,400 μg/L in 2011. 

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 μg/L of MC-LR); moderate risk (20,000-100,000 

cyanobacterial cells/ml corresponding to 10-20 μg/L of MC-LR); and high risk (>100,000 cyanobacterial cells/ml 

corresponding to >20 μg/L for MC-LR).

Drinking Water Health Advisory for Microcystins-June 2015 11 

 
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 μg/L (U.S. EPA, 2009)

State

Number 

of Sites 

Sampled

Percentage of Samples 

with Detection of 

Microcystins >10 μg/L 

Maximum Detection 

of 

Microcystins 

North Dakota 38 9.1% 192 μg/L

Nebraska 42 9.1% 225 μg/L

South Dakota 40 4.9% 33 μg/L

Ohio 21 4.5% 78 μg/L*

Iowa 20 4.5% 38 μg/L*

Utah 26 3.6% 15 μg/L*

Texas 51 1.8% 28μg/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 μg/L. In 2005 and 

2006, a study conducted in New York, including Lake Ontario, found variability in microcystinLR 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 μg/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 μg/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 μg/L, and two samples were above the high risk WHO recreational level of 20 μg/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 μg/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 12 
2010, OHEPA sampled Grand Lake St. Marys for anatoxin-a, cylindrospermopsin, microcystins, 

and saxitoxin. Toxin levels ranged from below the detection limit (<0.15 μg/L) to more than 

2,000 μg/L for microcystins. Follow-up samples taken in 2011 for microcystins indicated 

concentrations exceeding 50 μg/L in August. During the same month, sampling in Lake Erie 

found microcystins levels exceeding 100 μg/L. 

In 2008, NOAA began monitoring for cyanobacterial 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 

μg/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 μg/L (mean: 0.28 +/- 0.2 μg/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 μg/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 μg/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 μg/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 
found toxin in pre- and post-treated drinking water. Finished water concentrations ranged from

below detection levels to 12.5 μg/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 μg/L) to 0.36 μg/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 μg/L. The presence of the toxins was due to a cyanobacterial bloom 

near Toledo’s drinking water intake located on Lake Erie. The advisory was lifted two days later, 

after treatment adjustments led to the reduction of the cyanotoxin concentrations to concentrations 

below the WHO guideline value of 1 μg/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 of Arthrobacter, Brevibacterium, 

Rhodococcus, Paucibacter, and various strains of the genus Sphingomonas (Pseudomonas) have 

been reported to be capable of degrading microcystin-LR (de la Cruz et al., 2011; Han et al., 

2012). These degradative bacteria have also been found in sewage effluent (Lam et al., 1995), 

lake water (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 
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 
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 coexposure 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 with M. 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 a M. 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., 2011a). The authors found that mean serum levels of microcystins ranged from below 

detection to 1.3 μg 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 μg/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 γ-glutamyltransferase (γGT). The patient 

completely recovered within 20 days. 

Drinking Water Health Advisory for Microcystins-June 2015 16 
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 μg/L, detected in 

the activated carbon filter used in an intermediate step for treating drinking water to prepare 

dialysate (Soares et al., 2005). Concentrations of 0.4 μg/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 GGT) 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 (Soares 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 microcystinLR. 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). 

Drinking Water Health Advisory for Microcystins-June 2015 17 
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., 

2011b). 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 μg/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. 

Drinking Water Health Advisory for Microcystins-June 2015 18 
The only longer-term oral animal study of purified microcystin-LR was conducted by Ito 

et al. (1997b). Ito et al. (1997b) administered 80 μg 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 S-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 tenday) 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. 

Drinking Water Health Advisory for Microcystins-June 2015 19 
• 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 
Figure 2-3. Conceptual Model of Exposure Pathways to Microcystins in Drinking Water 

Drinking Water Health Advisory for Microcystins-June 2015 21 
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 
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 Document for Microcystins (HESD, U.S. EPA, 2015a), 

provides the health effects basis for development of the HA, including the science-based 

decisions providing the basis for estimating the point of departure. To develop the HESD for 

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 
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: 

UF DWI/BW

NOAEL or LOAELor BMDL HA × = 

Where:

NOAEL or 

LOAEL

BMDL

=

=

No- or Lowest-Observed-Adverse-Effect Level (mg/kg bw/day) from a study 

of an appropriate duration (7 to 30 days). 

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 
<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 (2011a) 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, 2011a). 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 2011a, 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 
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 (F1 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) μg/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 μg/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 μg/kg body weight, and 26% at 150 μg/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 μg/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 
Table 3-1. Liver Weights and Serum Enzyme Levels in Rats Ingesting Microcystin-LR in

Drinking Water (Heinze, 1999) 

* 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) 

Control

(Mean ± SD)

50 μg/kg 

(Mean ± SD)

150 μg/kg 

(Mean ± SD)

Serum Enzymes 

Alkaline phosphatase (ALP)

(microkatals/L) 9.67 ± 2.20 13.00 ± 3.81* 12.86 ± 1.85* 

Lactate dehydrogenase (LDH)

(microkatals/L) 16.64 ± 4.48 30.64 ± 5.05* 33.58 ± 1.16* 

Liver Weight

Relative (g/100 g body weight) 2.75 ± 0.29 3.22 ± 0.34* 3.47 ± 0.49* 

Absolute (g)** 8.28 ± 1.37 9.70 ± 1.32 10.51 ± 1.02 

Activation of 

Kupffer Cells

Degenerative 

and Necrotic 

Hepatocytes 

with 

Hemorrhage

Degenerative 

and Necrotic 

Hepatocytes 

without 

Hemorrhage

PAS-positive 

Material

Control

Slight 0 0 0 1 

Moderate 0 0 0 0 

Intensive damage 0 0 0 0 

50 μg/kg

Slight 0 4 0 5 

Moderate 10 6 0 5 

Intensive damage 0 0 0 0 

150 μg/kg

Slight 0 0 0 0 

Moderate 10 6 1 8 

Intensive damage 0 3 0 2 

Drinking Water Health Advisory for Microcystins-June 2015 27 
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 

μg/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 μg/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 μg/kg/day, but not at a dose of 16 μg/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 tenday 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: 

 0.3 μg/L 0.15 1000 L/kg/day

 50 μg/kg/day Ten day - HA = × =

Where: 

50 μg/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 

(UFH), a 10 for interspecies differences (UFA), a 3 for LOAEL to 

NOAEL extrapolation (UFL), and a 3 for uncertainties in the 

database (UFD).

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, 2011a).

Drinking Water Health Advisory for Microcystins-June 2015 28 
The Ten-day HA of 0.3 μg/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:

Where: 

50 μg/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 (UFH), a 10 for interspecies differences (UFA), a 

3 for LOAEL to NOAEL extrapolation (UFL), and a 3 for 

uncertainties in the database (UFD).

0.03 L/kg/day = Drinking water intake per unit body weight based on adult 

default values of 2.5 L/day and 80 kg (U.S. EPA, 2011a).

The Ten-day HA of 1.6 μg/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

• UFH - A Ten-fold value is applied to account for variability in the human population. No 

information was available to characterize interindividual and age-related variability in the 

toxicokinetics or toxicodynamics among humans. Individuals with 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, 

2011b). 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.

 1.6 μg/L L/kg/day 0.03 1000

 50 μg/kg/day Ten day - HA = × =

Drinking Water Health Advisory for Microcystins-June 2015 29 
• UFL - An uncertainty factor of 3 (100.5 = 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 

μg/kg/day and the LOAEL is 32 μg/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. 

• UFD - An uncertainty factor of 3 (100.5 = 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 
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 (LD50) of 36 ng/gbw 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 LD50 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 

LD50 determination showed that the most toxic congener was microcystin-LR (43.0 μg/kg), 

followed by microcystin-YR (110.6 μg/kg) and microcystin-RR (235.4 μg/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 LD50 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. LD50 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 
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 μg/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 microstructure 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 

(CASA) 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 
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 

μg/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 μg/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 
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 

μg/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 formpositive (P-GST) foci from 10 μg/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 μg/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 
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 microcystinLR 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 μg/L based on exposure to infants 

over the first year of life, and a Ten-day HA of 1.6 μg/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 (2011a) 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 
Figure 4-1. 90th Percentile Drinking Water Ingestion Rates by Age Group

Adapted from U.S. EPA 2011 Exposure Factors Handbook (U.S.EPA, 2011a). 

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. 

0.23

0.148

0.112

0.056 0.052 0.043 0.035 0.026 0.023 0.026 0.034

0

0.05

0.1

0.15

0.2

0.25

90%tile Drinking Water Ingestion Rate (L/kg/day)

Drinking Water Health Advisory for Microcystins-June 2015 36 
Figure 4-2. Ten-day Health Advisories for Microcystins by Age Group

 

 EPA decided to apply the Ten-day HA value calculated for infants over the first year of 

life (0.3 μg/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 μg/L, slightly below the infant health advisory value of 0.3 μg/L. EPA believes 

that infants from birth to 3 months old are not at a disproportionate risk at a 0.3 μg/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 

μg/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 Tenday HA value of 1.6 μg/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 
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-μg/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: microcystinLA (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 μg/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 
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 oC 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-μL 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/nerlcwww/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 C18 columns, 

amide C18 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 μg/L for microcystin-LR. Commercial ELISA 

kits generally have quantitation ranges from 0.2 (LOQ) to an upper limit of 5 μg/L. Two high 

sensitivity ELISA plate kits have become commercially available with MDLs ranging from 0.04 

to 0.05 μg/L. 

Drinking Water Health Advisory for Microcystins-June 2015 39 
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 μg/L or less, and using colorimetric PP1 inhibition 

assays range between 10 to 20 ng/mL (0.01 to 0.02 μg/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 μg/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 μg/L of microcystins with a 

detection limit of approximately 0.5 μg/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 

μg/L, respectively (Roy-Lachapelle et al., 2014). 

Drinking Water Health Advisory for Microcystins-June 2015 40 
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 EPA 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/cyanobacteriamanual/PDF/GWRCGuidanceManualLevel1.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 cyanobacterial 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 cyanobacterial 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 
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 byproducts (DBP) 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 
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 cyanobacterial cells and 

intracellular microcystins has been shown to vary from 60 to 99.9%. Factors that impact removal 

include the cyanobacterial 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 cyanobacterial 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 μg/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., 2011a, 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 
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 cyanobacterial 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 

(Sklenar et al., 2014). 

Drinking Water Health Advisory for Microcystins-June 2015 44 
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 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 (DBPs) (Sharma et al., 2012). 

It is also important to recognize that the use of oxidants may result in the formation of 

DBPs 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 DBPs, 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 
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 DBPs. 

 

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 (ClO2) 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 fullscale 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 
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., 2011a,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 μg/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 μg/L) may be challenging for treatment plants to reduce concentrations to below 

0.1 μg/L (Tarczyriska et al., 2001; Zamyadi et al., 2012a). 

Drinking Water Health Advisory for Microcystins-June 2015 47 
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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