# Presentation: 283273

## THE MICROCYSTIN GROUP OF CYANOTOXINS: STRUCTURE, OCCURRENCE, MONITORING AND MANAGEMENT OPTIONS AND THE DEVELOPMENT OF HUMAN RISK ASSESSMENT MODELS

- Dept. of Biological Sciences

**THE MICROCYSTIN GROUP OF CYANOTOXINS: STRUCTURE, OCCURRENCE, MONITORING AND MANAGEMENT OPTIONS AND THE DEVELOPMENT OF HUMAN RISK ASSESSMENT MODELS**

## Types of Harmful Algal Blooms

- Produce dense blooms leading to oxygen stress.
- 	Dinoflagellates
- 	Cyanobacteria (prokaryotic microbes)
**Produce potent toxins—illness 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)

- 	**Cyanobacteria toxin poisoning (CTP)**

- 	Amnesic shellfish poisoning (ASP)

- Dinoflagellate

## 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

## Slide 4

## The Cyanotoxins

- Cytotoxins—Toxins with cytotoxic (cellular) effects
- “Toxins” with acute, acute-lethal, or chronic biological effects

## Toxins

- Anatoxin-a(s)
- Cylindrospermopsin

***Anabaena,***
***Aphanizomenon,***
- and*** Planktothrix (Oscillatoria)***
***Anabaena,***
- and*** Planktothrix (Oscillatoria)***
***Aphanizomenon,***
***Cylindrospermopsis,***
***Umezakia and Raphidiopsis***

** ****Alkaloid Toxin**** ****	****       ****Produced By**

**Toxins**

## Toxins (continued)

- Toxins (Saxitoxins)
- Lyngbyatoxin,
- Debromoaplysiatoxin
- and Aplysiatoxin

***Anabaena,***
***Aphanizomenon,***
***Lyngbya,***
***Cylindrospermopsis,***
***Planktothrix and***
***Trichodesmium (marine)***
***Lyngbya ***and ***Schizothrix***(marine)

**Toxins (continued)**

** ****Alkaloid**** ****Toxin**** ****	****       ****Produced By                 **

## Toxins (continued)

- Nodularins

***Anabaena, Microcystis, ***
***Nostoc, Planktothrix (Oscillatoria),***
***Hapalosiphon**** and ****Synechococcus (marine)***
***Nodularia ***(brackish water)

**Toxins (continued)**

** ****Cyclic Peptide ****	****           Produced By**

** ****Lipopolysaccharide         Produced By****	****(LPS)**

- Endotoxin

-             All Cyanobacteria

## Cyanotoxins—Mode of Action

-  Neurotoxins
-  Hepatotoxins
-  Dermatoxins

## Cyanotoxins—Target Organisms

**    ****Location**
- Water Environment
- Water Users

**     ****Organisms**
- Wild  Birds & Fish
- Wild  Invertebrates
- Aquacultured Fish and Invertebrates
- Domestic & Wild Animals
- Humans
- Agriculture (Plants)

## Lake George-St. Johns River

**Lake George-St. Johns River**
**Mixed phytoplankton-toxigenic**** ****cyanobacteria**** only 10-20%**** of the**** population**

## Lyngbya wollei

**Mat Forming Benthic Freshwater species - produces Saxitoxins**

## Lower Potomac River-August 2004

- Photo courtesy of: Richard Lacouture - ANSERC

**CyanoHAB Search: A List of References to the **
**Toxic Cyanobacteria**

**By Wayne Carmichael**
**January 2004**

- 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.

## Slide 16

## 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

## 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

## CCL2 Microorganisms

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

## 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**

## Etiologic agents associated with drinking water outbreaks, by water type – United States, 1989-2000 (n = 175)

**Surface Water (n = 44)**

**Ground Water (n = 131)**

## Cyanotoxin Analyses Methods

**Biological - Bioassay**
**Small animal-mouse, invertebrate LD and LC****50**** **
**Microbial**
**Biochemical-i.e. Immunological (ELISA); Enzyme (PPIA); Cell Receptor**
**Analytical- HPLC, MS, NMR**
**Genetic-PCR**

## Immuno and Enzyme Assays-Microcystins, Saxitoxin

## Slide 24

## LC/MS Analysis of Cyanotoxins

- CYN

- STX

- ANTX-a

- MCYST-RR

- NODLN

- MCYST-YR

- MCYST-LR

## ESIMS Fragment Ions for a Typical Liver Analyses of Microcystins

## SELDI-TOFMS Process

_**SELDI-TOFMS Process**_

**Target compound - capture, purification, analysis, and processing from complex biological **
**      ****mixtures directly on ProteinChip Array surfaces****.**

**Detect the target compounds with TOFMS****.**

**(Protein capture and micropurification)**

## SELDI-TOFMS spectrum of microcystin-LR

**MCYST-LR**

_**SELDI-TOFMS spectrum of microcystin-LR**_
_** **__**in Great Blue Heron Gut Content**_

## 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

## 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**

## Management and Mitigation of:

*Anatoxins*
*Saxitoxins *
*Cylindrospermopsins*
*Microcystins*
*Nodularins*
*Lyngbyatoxins*
*LPS  ?*

## 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**

## Based upon: Australian National Health and Medical Research Council (NHMRC) and the Agricultural and Resource Management Council of Australia and New Zealand (ARMCANZ)---2001--Amendments to the Australian Drinking Water Guidelines

**Based upon: Australian National Health and Medical Research Council ****(****NHMRC) and the Agricultural and Resource Management Council of Australia and New Zealand (ARMCANZ)---2001--Amendments to the Australian Drinking Water Guidelines**

## Murrray-Darling River

- Toxic algae bloom-1993

## 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. 53:229-239. 2004

## Microcystins

- 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

**Description:**

**Produced by:**	***Microcystis, Anabaena, Planktothrix (Oscillatoria), Nostoc, Anabaenopsis, Hapalosiphon,**** *certain cyanobacterial picoplankton

## Microcystins (continued)

- 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

**Microcystins (continued)**

- * No  tumor promotion safety factor
- ** algae health food
- **	* cell lysis promotes toxin release and distribution
- Regulated not just a guideline value

**Health Guidelines:**

**Water Treatment***:** Catchment and source water management=maintain flow, destratify, nutrient reduction, chemicals* (chlorine, ozone), removal with carbon (GAC, PAC)

## 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 L_x0008_OAEL to NOAEL;10x for less than lifetime exposure)
- TDI = 0.067 μg/kg bw/day

-  *(MCYST-LR equivalents)

## 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

## 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**

## Thank You 

**Thank You**** **
**From the—**