THE MICROCYSTIN GROUP OF CYANOTOXINS: STRUCTURE, OCCURRENCE, MONITORING AND MANAGEMENT OPTIONS AND T

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THE MICROCYSTIN GROUP OF CYANOTOXINS STRUCTURE,
OCCURRENCE, MONITORING AND MANAGEMENT OPTIONS AND
THE DEVELOPMENT OF HUMAN RISK ASSESSMENT MODELS
Wayne W. Carmichael Dept. of Biological Sciences
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Types of Harmful Algal Blooms

• Produce dense blooms leading to oxygen stress.
• Dinoflagellates
• Cyanobacteria (prokaryotic microbes)
• Produce potent toxinsillness and death via food
  chain or biomass accumulation.
• Paralytic shellfish poisoning (PSP)
• Diarrheal shellfish poisoning (DSP)
• Neurotoxic shellfish poisoning (NSP)
• Ciguatera fishfood poisoning (CFP)
• Estuary-associated syndrome (EAS)

Dinoflagellate

• Amnesic shellfish poisoning (ASP)

• Cyanobacteria toxin poisoning (CTP)

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HAB Legislation U.S.

• Harmful Algal Bloom Eradication and Control Act
  of 1998-Subcommittee on Oceans and Fisheries-U.S.
  Senate
• Harmful Algae Bloom and Hypoxia Research
  Amendment Act of 2003-Subcommittee on
  Environment, Technology and Standards-U.S. House
  of Representatives Committee on Science (includes
  study of CyanoHABs in navigatable waters-i.e.
  Lake Erie)
• National Plan for Algal Toxins and Harmful Algal
  Blooms - March 21-25, 2004 Workshop-Charleston,
  SC-NOAA

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The Cyanotoxins

• CytotoxinsToxins with cytotoxic (cellular)
  effects
• Toxins with acute, acute-lethal, or chronic
  biological effects

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Toxins
Alkaloid Toxin Produced By
Anabaena, Aphanizomenon, and Planktothrix
(Oscillatoria) Anabaena, and Planktothrix
(Oscillatoria) Aphanizomenon, Cylindrospermopsis,
Umezakia and Raphidiopsis
Anatoxin-a Anatoxin-a(s) Cylindrospermopsi
n
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Toxins (continued)
Alkaloid Toxin Produced By

Anabaena, Aphanizomenon, Lyngbya, Cylindrospermops
is, Planktothrix and Trichodesmium
(marine) Lyngbya and Schizothrix(marine)
Paralytic Shellfish Toxins (Saxitoxins) Lyngb
yatoxin, Debromoaplysiatoxin and Aplysiatoxin
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Toxins (continued)
Cyclic Peptide Produced By
Microcystins Nodularins
Anabaena, Microcystis, Nostoc, Planktothrix
(Oscillatoria), Hapalosiphon and Synechococcus
(marine) Nodularia (brackish water)
Lipopolysaccharide Produced By (LPS)
Endotoxin
All Cyanobacteria
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CyanotoxinsMode of Action

• Neurotoxins
• Hepatotoxins
• Dermatoxins

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CyanotoxinsTarget Organisms

• Location
• Water Environment
• Water Users

• Organisms
• Wild Birds Fish
• Wild Invertebrates
• Aquacultured Fish and Invertebrates
• Domestic Wild Animals
• Humans
• Agriculture (Plants)

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Lake George-St. Johns River Mixed
phytoplankton-toxigenic cyanobacteria only 10-20
of the population
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Lyngbya wollei
Mat Forming Benthic Freshwater species - produces
Saxitoxins
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Lower Potomac River-August 2004
Photo courtesy of Richard Lacouture - ANSERC
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CyanoHAB Search A List of References to the
Toxic Cyanobacteria

• By Wayne Carmichael
• January 2004

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• CyanoHAB Search evolved from 30 years of studies
  of toxic blue-green algae, and contains 3,063
  references citing 705 journals written by 4,687
  authors and editors.

• The database is comprised of toxic cyanobacteria
  references in an electronic format (ProCite)
  that can be searched, sorted and listed by
  author, year, title, reference source or subject.

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1996 Safe Drinking Water Act Amendments

• Authorizes regulation of contaminants in drinking
  water
• Establishes procedure for making regulatory
  decisions
• Requires at least 5 contaminants to be considered
  every 5 years

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Federal Register March 1998 Drinking Water
Candidate Contaminant List
Research Priority for Cyanobacteria (blue-green
algae) and their Toxins

• Health Research
• Treatment Research
• Analytical Methods Research
• Occurrence Priorities

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CCL2 Microorganisms

• Adenoviruses
• Aeromonas
• Caliciviruses
• Coxsackieviruses
• Cyanobacteria and their toxins
• Echoviruses
• Helicobacter pylori
• Microsporidia
• Mycobacterium avium complex

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Priority Listing of Cyanotoxins for Action

• Microcystins-Analytical standards, rapid
  detection and setting of guideline values
• Cylindrospermopsins- Analytical standards, rapid
  detection and setting of guideline values
• Anatoxin-a- Analytical standards, rapid detection
  and setting of guideline values
• Saxitoxins, Nodularins, LPS-Secondary
  priority-pending further evaluation of occurrence
  in freshwaters

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Etiologic agents associated with drinking water
outbreaks, by water type United States,
1989-2000 (n 175)
Surface Water (n 44)
Ground Water (n 131)
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Cyanotoxin Analyses Methods

• Biological - Bioassay
• Small animal-mouse, invertebrate LD and LC50
• Microbial
• Biochemical-i.e. Immunological (ELISA) Enzyme
  (PPIA) Cell Receptor
• Analytical- HPLC, MS, NMR
• Genetic-PCR

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Immuno and Enzyme Assays-Microcystins, Saxitoxin
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LC/MS Analysis of Cyanotoxins
CYN
STX
ANTX-a
MCYST-RR
NODLN
MCYST-YR
MCYST-LR
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ESIMS Fragment Ions for a Typical Liver Analyses
of Microcystins
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SELDI-TOFMS Process
Target compound - capture, purification,
analysis, and processing from complex biological
mixtures directly on ProteinChip Array
surfaces.
(Protein capture and micropurification)
Detect the target compounds with TOFMS.
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MCYST-LR
SELDI-TOFMS spectrum of microcystin-LR in Great
Blue Heron Gut Content
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Genetic Studies

• Genetic - PCR
• 16S rRNA
• nifH (gene for dinitrogen reductase-N2 fixation)
• cpcBA-IGS (intergenic spacer region between two
  phycobilisome subunits)
• MCY (microcystin gene)
• Other toxin genes (CYN, Antx-a) are being studied

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Examples of Applications for Methods Developed

• Migratory Waterfowl Deaths
• Aquacultured Fish Losses
• Aquacultured Invertebrates
• Analyses of Human Serum and Tissue
• Great Lakes CyanoHABs
• Lesser Flamingos-Africa
• Dog Deaths - Nebraska

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Management and Mitigation of
Anatoxins Saxitoxins Cylindrospermopsins Microcys
tins Nodularins Lyngbyatoxins LPS ?
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• Management
• Use of a Risk-Management Framework
• Mitigation
• Choice of proper point for water abstraction
• Choice of optimum water treatment techniques
• Choice of proper algicide

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Based upon Australian National Health and
Medical Research Council (NHMRC) and the
Agricultural and Resource Management Council of
Australia and New Zealand (ARMCANZ)---2001--Amendm
ents to the Australian Drinking Water Guidelines
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Murrray-Darling River Toxic algae bloom-1993
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Australian Publications

• Australian Drinking Water Guidelines-1996-National
  Health and Medical Research Council (NHMRC)
• Evaluation of Analyt Meth Detect Quant
  Cyanotoxins in Rel to Aust Drinking Water
  Guidelines-2001-NHMRC
• Guidelines for Managing Risks in Recreational
  Water-2004-NHMRC (Draft)
• Management Strategies for Toxic Blue-Green Algae
  Literature Review. Aust. W.Q.C. House et al.
  2004.
• Water Treatment Options for Dissolved
  Cyanotoxins. Aqua. 53229-239. 2004

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Microcystins
Description

• Family of 75 cyclic heptapeptides
• Molecular weight from 909-1037
• Main one is microcystin-LR (leucine/arginine)
• Liver toxic by inhibition of protein phosphatases
• Tumor promoter

Produced by Microcystis, Anabaena, Planktothrix
(Oscillatoria), Nostoc, Anabaenopsis,
Hapalosiphon, certain cyanobacterial picoplankton
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Microcystins (continued)
Health Guidelines
MAC Cell Concentration WHO 1.0 µg/L
Australia 1.3 µg/L 6,500
cells/mL Canada 1.5 µg/L New Zealand 1 µg/L
Oregon, USA 1 µg/g Brazil 1.0 µg/L
Water Treatment Catchment and source water
managementmaintain flow, destratify, nutrient
reduction, chemicals (chlorine, ozone), removal
with carbon (GAC, PAC)
No tumor promotion safety factor algae
health food cell lysis promotes toxin
release and distribution Regulated not just a
guideline value
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WHO Provisional Guideline MCYST-LR (1998)

• Based upon two animal studies
• 1) Mouse oral NOAEL 40 µg/kg bw/day
• UF 1000 (10x for intraspecies 10x for
  interspecies 10x for limited database)
• TDI 0.04 µg/kg bw/day
• 2) Pig oral LOAEL 100 µg/kg bw/day
• UF 1500 (10x for intraspecies 3x for
  interspecies 5x for extrapolating LOAEL to
  NOAEL10x for less than lifetime exposure)
• TDI 0.067 µg/kg bw/day

(MCYST-LR equivalents)
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WHO Provisional Guideline (continued)

• Using TDI 0.04 µg/kg bw/day
• Guideline value TDI x bw x P
• L
• bw 60 kg (70 kg Australia)
• P 0.8 (0.9 Australia)
• L 2 L/day
• Results in 1.3 µg/L

0.96 µg/L
1.0 µg/L/day
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Acknowledgements

• U.S. - EPA
• James Sinclair
• Betsy Hilborn
• Florida (Funding sources)
• FMRI
• SJWMD through FMRI
• Wright State Univ.
• John Blakelock
• Laurel Carmichael
• Jeanette Frey
• Charles Friday
• Jennifer Ott
• Jerome Servaites
• Mary Stukenberg
• Moucun Yuan

• AwwaRF
• CDC-Atlanta
• CRC for Water Quality and Treatment-Adelaide
• UNC-IMS
• Hans Paerl and crew
• Canada
• E.A.D. Allen
• Univ. Illinois-NIH
• Ken Rinehart
• Robert Coates
• Queensland Health-EnTox
• Glen Shaw
• Geoff Eaglesham

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Thank You From the