The Fate of Cyanotoxins: Processes and Mechanisms

Following production and release into the environment cyanotoxins are impacted by much of the same

processes that are found to manage and mitigate cyanobacteria harmful algae blooms (cyanoHABs). These include, physical, chemical and biological.

Wayne CarmichaelProf EmeritusWright State UniversityDayton, OhioEmail: wayne.carmichael@wright.eduCurrent address: Seaside, Oregon

Goals Review bioactive compounds of cyanobacteria

A presentation of the physical, chemical and biological processes that impact degradation and fate of the higher risk cyanotoxins

Contrast those processes at work in the natural environment with those used by humans to manage and mitigate the risk from cyanotoxins

Useful Reports:

• Graham, J.L., Ziegler, A.C., Loving, B.L., and Loftin, K.A., 2012, Fate and transport of cyanobacteria and associated toxins and taste-and-odor compounds from upstream reservoir releases in the Kansas River, Kansas, September and October 2011: U.S. Geological Survey Scientific Investigations Report 2012–5129, 65 p.

• Carmichael, W. W., & Boyer, G. L. (2016). Health impacts from cyanobacteria harmful algae blooms: Implications for the North American Great Lakes. Harmful Algae, 54, 194–212.

• Wirtsbaugh, W.A., Paerl, H.P. and Dodds, W.K. (2019). Nutrients, eutrophication and harmful algal blooms along the freshwater to marine continuum. WIREs Water. 2019;6:e1373. https://doi.org/10.1002/wat2.1373.

• Toxic Cyanobacteria in Water: A Guide To Their Public Health Consequences, Monitoring and Management. 2nd

Revised Edition. 2020 in press. World Health Organization.

Cyanobacteria are ancient organisms

Great Lakes eutrophication prompted the governments of Canada and theUnited States to sign the Great Lakes Water Quality Agreement in 1972, establishing a binationalcommitment to reducenutrient loadings and clean up the lakes.

Bloom most likely the nitrogen fixing cyanobacterium genus Aphanizomenon flos-aquae (not a toxin producing species)

Harmful Algae Blooms (HABs)

• Waterblooms: Proliferation of single-celled, filamentous, colonial microalgae as well as macroalgae (seaweeds).

• Proliferation occurs when nutrients, temperature, pH and light are conducive to good growth (plus physical factors).

• Only a few of many phytoplankton species produce toxins (about 50 cyanobacteria and an equal number of marine phytoplankton).

• Three of eight algae groups (Divisions/Phyla) produce toxins.• Proliferation of nontoxic phytoplankton species can also be

harmful (depletion of oxygen). • All states in the U.S. and countries worldwide are affected by

cyanoHABs.• Waterblooms can cause water discoloration: red tides, brown

tides, blue-green (cyanobacteria) blooms.

Bioactive Metabolites of Cyanobactria

Cytotoxins—Toxins with cytotoxic (cellular) effects

“Toxins” with acute, acute-lethal, or chronic biological effects

Cytotoxic Pharmacologicals

Sampling of activities-Cytotoxic (virus, bacteria, fungi, algae, worms), Antitumor,

Cardioactive, Antiinflammatory, Antimitotic, Sunscreen

Ref: Namikoshi/Rinehart 1996. J. Indus. Microbiol. 17:373-384., Burja et al. 2001. Tetrahedron 57:9347-9377., Gerwick et al. 2001. The Alkaloids, 57:75-184., Van Wagoner et al. 2007. Adv. Appl. Microbiol. 61:89-

217.

Cyanobacteria Toxins: Health Effects

• Dermatologic effects– Lyngbyatoxins

• Neurotoxins– Anatoxins– Paralytic shellfish toxins (e.g., saxitoxin,

neosaxitoxin)– BMAA

• Hepatotoxic– Microcystins– Cylindrospermopsins

• Risk Assessment Ref: Orr and Schneider. Toxic Cyanobacteria Risk Assessment. South East Queensland Water Corp. Brisbane, Aust. Nov 2006. www.seqwater.com

CyanoHAB ToxinsRoutes of Exposure

Humans Animals Plants

Recreational Drinking water Irrigation

Drinking water Food

Food

Medical (i.e. dialysis)

Cyanotoxins—Target Organisms

LocationWater Environment

Water Users

Organisms Wild Birds & Fish Wild Invertebrates Aquacultured Fish and

Invertebrates

Domestic & Wild Animals

Humans Agriculture (Plants)

Types and Health Significance Ranking of Cyanotoxins

Microcystins (most common of cyanotoxins; widespread animal and human poisonings).

Anatoxins (worldwide animal poisonings).

Cylindrospermopsins (worldwide, animal and human poisonings).

Lyngbyatoxins (present in continental U.S.; human poisonings in South and Central Pacific).

Nodularins (sporadic worldwide animal poisonings).

Saxitoxins (sporadic worldwide animal poisonings).

beta methyl-amino (BMAA) (present worldwide; but health significance largely unknown).

USEPA 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

1) Physical: Through manipulation of the intake location and depth, aerators (destratification), mechanical mixers (long distance circulation) and barriers (sand, carbon) the numbers of cells and concentration of cyanotoxins are affected. Also light (photo oxidation), temperature and pH (degradation) will determine fate. 2) Biological: Native microbes (grazers, bacteria, fungi) in the environment and their use in water treatment processes are important to determining fate.3) Chemical controls:Phosphorus treatments (e.g. lime, aluminum sulfate, lanthanum and ferric chloride).

Used to both bind nutrients and flocculate cells.Clay particles (these are primarily used to trap cells and pull them below the photic zone). Algaecides and oxidizing compounds (e.g., copper-sulfate, hydrogen peroxide, potassiumpermanganate, herbicides and ozone).

Note: Methods for cell removal such as flocculation and physical barriers remove most cyanotoxinsalong with the cells. However, applying algaecides during a heavy bloom of toxin-producing species willcause the cells to rupture and release the cyanotoxins. The released cyanotoxins are soluble and move intothe water treatment plant or recreational area creating problems for their removal and an increased risk ofcontact with humans and animals. In addition some cyanotoxins (i.e. CYN) are released by cells during active growth.Current ref: Solutions for Managing Cyanobacterial Blooms: A scientific summary for policy makers. 2019. M.A. Burford et al. IOC/UNESCO, Paris (IOC/INF-1382).

Drinking Water Treatment Methods that Effect Fate

• Treatment to remove intracellular cells and algal toxins– Conventional treatment

• Filtration (sand, soil)• Flocculation (alum, clays, lanthanum)

• Treatment to remove extracellular algal toxins– Oxidation (ozone)– Physical removal (adsorption and absorption; carbon)– Biologically active filters

• Newer technologies– Dissolved Air Flotation (cells and intracellular toxins)– Low pressure membrane filtration– High pressure filtration– Reverse Osmosis (RO)

Micrograph of different blue-green algae in a lake water sample

Their size, unicellular/colonial morphology, prokaryotic nature with lack of sexual cycle means removing cells and cyanotoxins requires a microbial approach.

Currently about 250 analogues of the microcystins

Microcystin Biodegradation ExamplesEvent Organism Result Reference

Lake Okeechobee water samples-spiked with microcystin-LR

Bacteria: Mirobacterium, Rhizobium, Ochrobactrum

Up to 80% degradation within 20 days. HPLC detection.

Ramani et al. 2012.Biodegradation. 23:35-45.

Microcystis aeruginosa PCC7806

Fungus: Tricaptum Microcystin-LR not detected after 12 hr treatment

Jia et al. 2012. Appl. Biochem. Biotechnol. 166:987-996.

Purified microcystin-LR treated with a bacterium culture

Bacteria: Sphingomonas Linear microcystin-LR, a tetrapeptide metabolite, free ADDA

Harada et al. 2004.Toxicon. 44:107-109.

Water samples from Lake Erie, Lake Tai (China). Samples screened for mirABCD(metatranscriptomeinvolved in microcystin degradation) plus glutathione S-transferase and alkaline phosphatase (major microcystin degradation enzymes)

Microcystis aeruginosa blooms common.

MmirABCD not detected, glutathione S-transferase and alkaline phosphatase widely detected and involved in degradation

Krausfeldt et al. 2019. Frontiers in microbiology. 10: 2741.

Physical and Chemical Degradation of MicrocystinsEvent Treatment Result Reference

Reservoir water spiked with MC-LR, NOD

Treated by hydrolysis, chlorination and photodegradation

Transformation products: chlorination, UV and sunlight, but not hydrolysis (LC-QTOF MS)

Leon et al. 2019.Chemosphere. 229:538-548.

Lake mesocosm in a pond with cyanobacteria blooms

Treated with copper ethanolamine algaecide at0.6, 1.2, and 2.0 mg Cu L−1 Three densities of cyanobacteria.

MC-LR degraded with half life of 1-1.9 days for all cell densities

Kinley et al. 2018. Water Air Soil Pollut. 229:62-73.

Lab scale photocatalytic falling film reactor used to oxidize MC-LR, YR and YA.

Treated with UV (30W 90 cm long; 254 nm) and TiO2(0-5 g/L)

First order reaction kinetics. Half life of < 5 minutes.

Shepard et al. 1998. Toxicon. 26(12):1895-1901.

Mechanically collected bloom biomass applied to crop and forest lands. Lake Tai, China.

Clay/soil adsorption and unassisted degradation followed by cell/MC toxin assessment.

Nutrients, cells and cyanotoxins reduced up to 80% after 1 year. Risk noted for toxin leakage and plant accumulation.

Chen et al. 2012. Envir. Sci. Technol. 46:13370-13376.

Temperature effect on MC release from cells. Grand Lake St Marys, Ohio.

Temperature variation in lake water and temperature effect in the lab on MC release from Planktothrix.

Cell biomass increased to 18 C and then declined. Extracellular toxin max at 20-25 C. 36% MC increase at about 20C

Walls et al. 2018. Sci Total Envir. 610-611:786-795.

Anabaena (now called Dolichospermum) can produce Microcystins, Anatoxin-a, Anatoxin-a(s) and saxitoxins

Cylindrospermopsis

The anatoxins, cylindrospermopsin and saxitoxins

Now Named: Guanitoxin, re-naming a cyanobacterial organophosphate toxin. 2020.M. F. Fiorea, S. Thomaz de Limaa, W. W. Carmichael, S. M.K. McKinniec,J. R. Chekand, B. S. Moore. Harmful Algae. 92 (Feb. 101738)

ATX, CYN and STX ExamplesEvent Treatment Result ReferenceATX-a reference compound as the fumarate salt. Lab test.

UV Photolysis and UV/hydrogen peroxide degradation

Degradation followed second order rate constant. Mechanism was by hydroxyl oxidation. H2O2 enhanced the UV effect.

Afzal et al. 2009. Water Research. 44(1):278-286.

ATX-a reference compound. Lab test.

UV/H2O2 degradation NH4, NO2, NO3 nitrogen byproducts. Six degradation byproducts produced. None had residual toxicity.

Tak et al. 2018. Chem. Engineer. J. 334:1016-1022.

Manganese oxidizing bacteria from natural waters

Evaluate the removal of CYN

Pseudomonas, Comamonadaceae, Ideonella found to be effective from 3-28 days.

Martinez-Ruiz et al. 2020. Chemosphere. 238:124625.

Saxitoxin producing Anabaena circinalis

Chlorine exposure in lab tests.

STX and analogues were oxidized. Chlorination byproducts were produced (trihalomethanes, haloacetic acids, N-nitrosodimethylamine)

Zamyadi et al. 2010. EnvirSci Technol. 44:9055-9061.

Summary Toxicity Chlorinated Cyanotoxins: Merel et al. 2010. State of the art on cyanotoxins in water and their behavior toward chlorine. Toxicon. 55: 677-691.

NH2

CH2 – CH – CO2H

NHCH3

MW 118.14, m.p. 168 oC

BMAA DegradationEvent Treatment Result Reference

Reaction between BMAA and 5 drinking water oxidants. Lab tests.

Chlorine, potassium permanganate, ozone, hydrogen peroxide, hydroxyl radical

Reaction rates: OH>ozone>chlorine>>permanganate=H2O2. Higher pH=higher rate. Natural water slower than deionized water.

Chen et al. 2018. Water Research. 142:187-195.

BMAA treated with chlorine

Lab tests. Intermediates followed by MS/MS.

Four intermediates found. BMAA loss second-order reaction. Chlorinated intermediates also reacted with chlorine. A reductant was found to reduce intermediates back to BMAA.

Chen et al. 2017. Envir. Sci. Technol. 51(3): 1303-1311.

Summary Points

• Physical. Barriers to remove cells are effective ifcells are kept intact.

• Biological. Effective but hard to replicate in watertreatment systems. Common occurrence innatural waters, especially in waters with a historyof HABs.

• Chemical. All oxidation methods effective on allthe cyanotoxins. Treatment conditions must betailored to the toxin types. For examplechlorination not effective on ATX-a.

Questions?

Wayne Carmichael

Professor Emeritus

wayne.carmichael@wright.edu