Cyanobacteria Toxicity

It is clear that cyanobacteria are toxin-producing organisms that can be found almost anywhere on Earth.  What is interesting is that prior to the last decade or so, they seldom provoke public health concerns.  In their normal low populations, they are usually unnoticed.  However, under the proper conditions, these organisms can and do make a significant impact on public health.  In response, public health and environmental professionals use the phrase harmful algal blooms or HABs.  It is perhaps an acknowledgement that the problem is more serious, widespread and older than originally thought by the general public.  There are published reports of livestock/ human sickness and death linked to HABs from across the world and dating back a thousand years [Chorus­ and Bartram, 1999].  Most reported outbreaks come from China, India, Australia, United States and Brazil.  It can only be speculated how many outbreaks go unrecognized or unreported.

Cases of human exposure to cyanotoxins can be divided into two categories: acute and chronic.  This division is based on how quickly and severely symptoms begin.  Acute poisoning symptoms occur very soon after exposure, often with great severity.  They are often described as flu-like symptoms: severe nausea, vomiting, diarrhea, cramps and fever.  Acute cyanotoxin poisoning is infrequently fatal to humans, but there are fatalities.  Because of the obvious cause (ingestion) and effect (symptoms), these cases are more thoroughly investigated.  Most acute fatalities are pets and livestock.

Chronic cyanotoxin exposure is the ingestion of lower concentrations over longer periods of time.  It is far more common than acute exposure in humans.  But the problem is that chronic cases are less obvious.  Sometimes the connection between ingestion of toxin and onset of symptoms is missed.  One of the purposes of this publication is to educate readers to know the risks and protect themselves.

1.) Measuring Toxicity

One way to measure the toxicity of chemicals is a chemical analysis called the Lethal Dose, 50% test, or LD₅₀.  This method measures the amount of a chemical that kills 50% of the test subjects after a specific period of time.  The subjects for these tests are usually rats.  The amount of chemical is measured in milligrams and the body weight of the test subject is measured in kilograms: so LD₅₀ values are expressed in units of mg/kg.  This is equivalent to parts per million or ppm.  The lower the LD₅₀ value indicates the more toxic the chemical. 

While it is important to be able to identify the cyanobacteria that are potential sources of cyanotoxin, it is also important to directly detect or measure the cyanotoxin in the water.  This sometimes requires access to some analytical methods and instrumentation.  The three most commonly used methods are test strips, enzyme-linked immunosorbant assay (ELISA) and liquid chromatography coupled with mass spectrometry (LC/MS). 

A.) Test strips to detect cyanotoxins are the most recently developed of the three methods.  They are specifically designed to be easily used in the field.  They are similar in principle to a home pregnancy test.  The kit contains an antibody specific to the toxin and a color-producing chemical reaction embedded on a paper strip.  When the antibody binds to some of the toxin, color is produced.  The amount of color produced is proportional to the amount of toxin present.  The results from test strips are described as semi-quantitative within a narrow concentration range (10-fold).  The detection limits for the test strips are set up to be appropriate for drinking and recreational water testing [Humpage, et al., 2012].

B.) The ELISA (Enzyme Linked Immunosorbant Assay), like the test strip, utilizes an antibody that binds specifically to the toxin and the chemicals needed for a color-producing reaction.  The ELISA is more accurate and has a broader concentration range than the test strips, but also has serious drawbacks.  The ELISA must be done in the lab.  There are several ELISA validation procedures [Triantis, et al., 2010].   A major problem with an ELISA, for example, is that it cannot tell the difference among the 80 different forms of microcystins.  Given the differences in toxicity among the different forms of microcystins, this could lead to an overestimation of the true toxicity of a water sample. 

C.) Liquid chromatography with mass spectrometry (LC/MS) is generally considered the best method to detect and measure cyanotoxins because of its superior sensitivity, precision and efficiency [Oehrle and Westrick, 2003].   The liquid chromatography portion separates or resolves the toxins from all of the other chemicals in the water sample based on their chemical properties.  The mass spectrometer identifies and quantifies the toxins based on the mass of the molecule and how they break apart when ionized.  Given the correct instrument and method, all of the cyanotoxins can be clearly identified and measured from lake water in less than eight minutes [Oehrle, et al., 2010].

Cyanotoxin Classification

There are two ways to classify cyanotoxins: by their target and by their chemical properties.  The target method looks at the organs/systems that the toxin damages.  Neurotoxins damage the brain and nervous system, hepatotoxins damage the liver, cytotoxins damage cells and dermatoxins damage skin and mucous membranes.   The chemical method classifies the toxin by its chemical properties.  Based on their chemistry there are four groups of cyanotoxins: cyclic peptides, alkaloids, lipopolysaccharides (LPS) and neurotoxic amino acids.

1.) Cyclic Peptide Cyanotoxins

Cyclic peptide cyanotoxins are a chain of five to seven amino acids that folds back on itself to form a circle.  This is why they are cyclic (circular) and peptide (a chain of amino acids held together by peptide bonds).   Most cyanobacterial poisonings are caused by these cyclic peptide cyanotoxins.  The two most common cyclic peptide cyanotoxins are the microcystins and the nodularins [Carmichael, 1997]. 

A.) Microcystins were named after the organism in which they were first discovered: the cyanobacteria Microcystis [Carmichael, et al., 1988].  Other cyanobacteria produce microcystins, but Microcystis is most frequently linked with incidences of algal poisoning in freshwater [Carmichael, 2001]. 

a.) Physical properties Microcystin is made up of seven amino acids that form a ring.  Of the seven amino acids, five remain constant but two are variable [Carmichael, 1997].  This variation produces 80 different forms of microcystin, each with its own properties and toxicities [Bourne, et al., 1996].  The most toxic form, microcystin-LR, is about twice as toxic as the less toxic forms.  Because of this variability, the molecular weight of microcystin is listed as a range from 800 to 1100 daltons [Chorus and Bartram, 1999].  Some of the amino acids used to synthesize microcystins (and nodularins) are unusual, non-essential amino acids not generally used by animals to make proteins.  Also, microcystins are water soluble and do not easily move across lipid cell membranes.  These properties make microcystins difficult to break down by animals, so they are usually sent to the liver where they accumulate.  Though stable in animals, aquatic microorganisms can break down microcystins in 1-2 days [Cousins, et al., 1996; Welker and Steinberg, 1999; Surono, et al., 2008; Somdee, et al., 2013].  In water treatment plants, chlorination and/or ozonation quickly oxidize microcystins.

b.) Mode of action Because microcystins do not easily cross cell membranes, they are not released into the environment until the algal cell ruptures [Orr and Jones, 1998].  As previously stated, ingested microcystins are transported to the liver and accumulate there.  As they build up there, they interfere with the function of liver enzymes.  This causes damage to the liver cells and an increase in cancer risk [Nishiwaki-Matsushima, et al., 1991; Mankiewicz, et al., 2003, Campos and Vasconcelos  2010].  The LD₅₀ of the most toxic form (microcystin-LR) is 0.025-0.150 mg/Kg [Carmichael, 1997].

c.) Acute microcystin poisoning in humans is rare because of the large volume of contaminated water needed to deliver enough microcystin.  However, in February 1996, in the city of Caruaru (Northeast Brazil) at a hemodialysis clinic, 131 patients had intravenous exposure to microcystin [Azevedo, et al., 2002].  It was in the water used for the dialysis procedures.  Immediately after treatment, 116 patients had visual disturbances, nausea, vomiting and muscle weakness.  In the following months, 100 of these patients suffered liver failure.  By December 1996, 52 of these patients were dead [Jochimsen, et al., 1998; Kuiper-Goodman, et al., 1999].  This is the worst documented case of acute human cyanotoxin poisoning, but certainly not the only one.  Acute poisonings are much more common in fish, birds and pets due to their smaller body mass [Teneva, et al., 2012].  Symptoms of acute poisoning are whitening of the mucous membranes, vomiting, cold extremities and diarrhea [Carmichael, 1992]. 

d.) Chronic exposure is more common in larger animals and is indicated by gastrointestinal upset, diarrhea, vomiting and potential liver cancer. Of particular concern to health professionals is chronic cyanotoxin exposure from drinking water. A study in Australia reported elevated liver enzyme levels in blood samples (an indication of liver damage) from residents who drank treated water from a reservoir which had annual HABs [Falconer, et al., 1983].  A study in China reported a significantly higher incidence of liver cancer among individuals who drank water from ditches containing HABs in comparison with those from the same area who drank groundwater [Yu, 1989].  Lesser exposure routes are recreation on contaminated lakes [D'Anglada, 2014], consumption of shellfish and crustaceans (these organisms accumulate microcystin, which is not broken down by cooking) [IARC, 2010] and blue-green algae dietary supplements [Carmichael, et al., 2000; Trout, 2004].  The following genera are reported to produce microcystins under bloom conditions: Anabaena, Anabaenopsis, Arthrospira, Gloeotrichia, Hapalosiphon, Microcystis, Nostoc, Oscillatoria, Phormidium, Plectonema, Planktothrix and Rivularia.

B.) Nodularins, the other cyclic peptide toxin, were named after the Nodularia genus in which they were discovered.  It was the first cyanobacteria reported to poison animals [Francis 1878] but nodularin was isolated much later [Rinehart, et al., 1988] possibly because many Nodularia are marine organisms.  Their HABs form mostly in the Baltic Sea and around New Zealand and Australia [Sivonen, et al., 1989; Laamanen, et al., 2001]. 

a.) Physical properties of nodularins are similar to microcystins, including stability, target organ, mode of action and symptoms [Sivonen, et al., 1989].  They are a chain of five amino acids that fold to form a circle.  Because it is a smaller molecule than most microcystins (824 daltons), it is thought to enter the liver cells more easily and do more cellular damage.  The LD₅₀ of nodularins is 0.06 mg/kg [Chen, et al., 2013].  The genera reported to produce nodularins in HABs are: Aphanizomenon, Nodularia and Nostoc.

2.) Alkaloid Cyanotoxins

Alkaloids are a very diverse category of chemicals, normally made by plants, which have a chemical structure that contains a system of rings with a nitrogen atom within.  Like ammonia, another nitrogen compound, it has an alkaline pH.  Other alkaloids include cocaine, quinine, strychnine, nicotine, morphine and many others.

Alkaloid cyanotoxins fit into this alkaloid category because of their chemical structure.  They are small chemical compounds with a wide variety of structures, stabilities and toxic properties [Sivonen and Jones, 1999; Hoehn and Long, 2008].  Because this is such a diverse category of chemicals, it had to be subdivided into more manageable groups.  The first subdivision is based on their target organs and systems.  One group attacks the nervous system: they are the Neurotoxic Alkaloid Cyanotoxins.  There are two groups of neurotoxic alkaloids cyanotoxins: Anatoxins and Saxitoxins.  The other group of alkaloid cyanotoxins attack cells: they are the Cytotoxic Alkaloid Cyanotoxins.  They will be discussed later.

Alkaloid Cyanotoxin, Neurotoxins:  Type 1: Anatoxins

There are three types of anatoxins: anatoxin-a, homoanatoxin-a and anatoxin-a(S) (now know as guanitoxin [Fiore, et al., 2020]).

A.) Anatoxin-a was first isolated from the genus Anabaena in 1972.  However, it was originally observed by Paul Gorham in 1964.  He named it Very Fast Death Factor because cattle would die in seven minutes after drinking contaminated water [Carmichael, et al., 1977]. 

a.) Physical properties of anatoxin-a: they are small molecules (165 daltons) that are stable in water only in the dark [Chorus and Bartram, 1999].  It is readily broken down by UV light (especially at alkaline pH) and by microorganisms [Osswald, et al., 2007].

b.) Mode of action: Gorham documented instances of cattle displaying symptoms of muscle spasms, convulsions, paralysis and suffering respiratory arrest within seven minutes of ingestion [Carmichael, et al., 1977].  These symptoms gave researchers some insight into how and where this toxin worked.  In the body, the place where nerves and muscles physically meet and chemically communicate is called the neuromuscular junction.  There, the muscle cell has a chemical ‘switch’, which when turned to the ‘ON’ position, tells the muscle cell to contract.  The nerve cell fires to release a chemical, called a neurotransmitter, which turns the muscle cell’s switch to the ‘ON’ position, so that it contracts under the control of the nervous system.  However, the muscle cell also has an internal mechanism which turns the switch back to the ‘OFF’ position, to allow the muscle cell to stop contracting.  Anatoxin-a acts as a neurotoxin by imitating the neurotransmitter and then turning the muscle cell’s switch to the ‘ON’ position permanently.  This makes the muscle cell try to contract even after it becomes exhausted, which leads to the symptoms described above.  The LD₅₀ of anatoxin-a is reported to be 0.375 mg/kg [Fawell, et al., 1999].  There are only reported cases of acute anatoxin-a exposure because it is so toxic; no chronic exposure has been knowingly observed.  The cyanobacteria genera reported to produce anatoxin-a are: Anabaena, Aphanizomenon, Cylindrospermum, Microcystis, Oscillatoria, Phormidium, Planktothrix and Raphidiopsis.

B.) Homoanatoxin-a was first isolated from the cyanobacteria Phormidium [Skulberg, et al., 1992].  There is limited information on this compound.  The structure, toxicity and stability data for homoanatoxin-a show it is very similar to anatoxin-a.  It is also a small molecule: 179 daltons.  The LD₅₀ is reported as 0.29-0.58 mg/kg [Lilleheil, et al., 1997].  Like anatoxin-a, homoanatoxin is also broken down by UV light and in an alkaline pH.  The same genera that produce anatoxin-a also produce homoanatoxin-a.

C.) Anatoxin-a(S) [guanitoxin] was first isolated from Anabaena [Mahmood & Carmichael, 1986a; 1987], but which has since been reclassified as Dolichospermum.

a.) Physical properties of guanitoxin are very different from those of anatoxin-a.  Guanitoxin has a molecular weight of 252 daltons.  It resembles the chemical and toxicological properties of organophosphate insecticides (Malathion and parathion) and the chemical weapon sarin.  Studies of subtoxic exposure to sarin show some similarities in symptoms [Scremin, et al., 2003]. Guanitoxin is the only known naturally-occurring organophosphate toxin.

b.) Mode of Action Guanitoxin acts as a neurotoxin by making the nerve cell continuously fire and release neurotransmitter.  This makes the muscle cell continue to contract [Metcalf and Codd, 2014].  Because of the high toxicity of guanitoxin it is regulated as a potential chemical warfare agent [Patocka, et al., 2011]. 

c.) Acute exposure symptoms are muscle spasms, gasping, convulsions, profuse salivation and death by respiratory collapse.  The (S) in anatoxin-a(S) means “salivation factor” [Carmichael, 1992; 2001].  If a cow drinks a few liters of contaminated water it can die in a matter of seconds [Sivonen and Jones, 1999].  The LD₅₀ is reported as 0.02 mg/kg [Mahmood and Carmichael, 1986a].  Guanitoxin breaks down rapidly under elevated temperatures (>40°C) and alkaline conditions, but is fairly stable at a lower pH [Carmichael 1992].   Anabaena (Dolichospermum) is the only genus reported to produce guanitoxin.

Alkaloid Cyanotoxins, Neurotoxins: Type 2. Saxitoxins

A.) Saxitoxins are also known as Paralytic Shellfish Poisons (PSPs), even though shellfish do not produce the poison.  Most saxitoxins are produced by another type of algae, the dinoflagellates.  They are the toxic agent of red tides.

a.) Physical properties: there are 20+ forms of saxitoxin [Sivonen and Jones, 1999].  All forms are water soluble and are stable for 1-10 weeks at an acidic pH.  Chlorination and alkaline pH readily break down saxitoxins.  This can be a problem because the least toxic saxitoxin forms (C-toxins) can break down into the most toxic forms (dc-GTXs) [Negri, et al., 1997].  This form is the most toxic non-protein known: 8000 times more toxic than cyanide [Pearson, et al., 1990].  The LD₅₀ reported for this saxitoxin form is 0.01 mg/kg [Pearson, et al., 2010].

b.) Mode of Action Saxitoxins act as a neurotoxin by degrading the ability of nerve cells to transmit a signal [Metcalf and Codd, 2014].  This leads to a tingling sensation in the mouth, dizziness, weakness and paralysis; then nausea, vomiting and tachycardia.  These symptoms can begin within 5 minutes of exposure; death normally occurs within 2-12 hours.  In non-fatal cases, the symptoms usually pass within 1-6 days [Metcalf and Codd, 2014]. 

c.) Exposure to saxitoxins comes from drinking contaminated water or eating contaminated shellfish, lobster or fin fish.  Shellfish concentrate saxitoxins in their flesh in a process called bioaccumulation.  Cooking does not degrade the toxin [Doyle, 2011].  Saxitoxins are rapidly removed by the kidneys and excreted in the urine [Metcalf and Codd, 2014].  The freshwater cyanobacteria that produce saxitoxins are Aphanizomenon, Anabaena and Lyngbya [Mur, et al., 1999].

Alkaloid Cyanotoxins, Cytotoxins

A.) Cylindrospermopsin is the primary cytotoxic alkaloid.  It was isolated in 1992 after extensive investigation of a 1979 incident known as the “Palm Island Mystery Disease” [Kinnear, 2010].  On this small island off the northeastern coast of Australia, 140 children and 10 adults exhibited symptoms of vomiting and headache.  This was followed by liver enlargement, bloody diarrhea and dehydration [Kuiper-Goodman, et al., 1999].  None of the 150 died, but most required extensive therapy.  Analysis revealed all those affected drank treated water from the same reservoir.  Prior to the outbreak, a bloom of Cylindrospermum occurred in that reservoir.  It was treated with a copper sulfate algaecide which caused the Cylindrospermum cells to rupture and release cylindrospermopsin into the water [Bourke, et al., 1983; Kuiper-Goodman, et al., 1999].  This is why treating algal blooms must be done carefully, if at all.

a.) Physical properties Cylindrospermopsin is an alkaloid with a larger and more complex structure than the other alkaloids discussed earlier.  It has a multiple ring structure and a molecular weight of 415 daltons [Adamski, et al., 2014].  It is highly water-soluble and is not broken down by heat, sunlight or pH [Adamski, et al., 2014] but is degraded by chlorination [Kinnear, 2010].  Studies of biodegradation of cylindrospermopsin in aquatic systems show that it is not rapidly broken down [Adamski, et al., 2014].  These finding indicate the possibility of bioaccumulation in animal tissues.  It is a stable molecule.

b.) Mode of Action Cylindrospermopsin produces widespread cell and tissue damage in multiple organs, particularly the liver and kidneys [Kuiper-Goodman, et al., 1999].  The poison inhibits protein synthesis [Bazin, et al., 2009] and damages the strands of DNA [Shen, et al., 2002].  It is considered a potent carcinogen.  The LD₅₀ reported for cylindrospermopsin is 0.30 mg/kg [Ohtani, et al., 1992].  The freshwater cyanobacteria that are reported to produce cylindrospermopsins are: Anabaena, Aphanizomenon, Cylindrospermopsis, Cylindrospermum, Lyngbya and Raphidiopsis.

3.) Lipopolysaccharides

A.) Lipopolysaccharides (LPS) are also called endotoxins [Sivonen and Jones, 1999].

a.) Physical Properties LPS are structural components of the outer cell wall membrane of Gram-negative bacteria (which include cyanobacteria) [Chorus and Bartram, 1999].  These are large, multi-component molecules with molecular weights beginning at 10,000 daltons and up.

b.) Mode of Action The toxins can cause vomiting, diarrhea, fever and allergic reactions in humans who come in contact with them.  The chief health concern is eliciting anaphylactic shock in sensitized individuals.  The main impact is from direct contact to skin or respiratory passages rather than ingestion [Sivonen and Jones, 1999].

LPS was first isolated from the genus Anacystis [Weise, et al., 1970], but have since been found in almost all cyanobacteria (the genus Synechococcus in particular).     

4.) Neurotoxic Amino Acids

A.) BMAA, or β-N-methylamino-L-alanine, was discovered in 1960 in roots of tropical tree fern (cycad) genus Gunnera.  The cyanobacteria Nostoc lives inside the roots in a symbiotic relationship and was the source of BMAA [Doyle, 2011].  This was found in the process of investigating the high incidence of amyotrophic lateral sclerosis (ALS) and ALS-like conditions, referred to as ALS/parkinsonism dementia complex, among the indigenous people of Guam. 

a.) Physical properties Neurotoxic amino acids are chemically and structurally different than the 20 standard amino acids normally used to build proteins.  Unusual amino acids are not normally incorporated into proteins, but some are known to have significant physiological effects.  BMAA is water soluble and has a molecular weight of 118 daltons [Krüger, et al., 2012].  BMAA is similar to the standard amino acid glutamic acid and is readily broken down by microorganisms.  Chlorination, ozonation and carbon filtration have little effect on BMAA. 

b.) Symptoms In 1954, the ALS/PDC rate in Guam was almost 100 times the worldwide rate [Banack, et al., 2010].  Chronic symptoms are reduction in muscle mass, loss of coordination, paralysis and death.  It was found that BMAA is a neurotoxin, but the mode of action is not clear [Weiss, et al., 1989].  There is evidence that BMAA can act on the nerve cell’s receptors and cause over activation, leading to nerve cell death [Lobner, 2009].  Another study found that proteins containing BMAA are not able to assume their correct three dimensional structure – they do not fold correctly.  These misshapen proteins could lead to the plaques and neurofibrillary tangles associated with Alzheimer’s disease [Dunlop, et al., 2013].  Other research suggests links between HABs, BMAA exposure and areas that have increased rates of neurodegenerative diseases [Banack, et al., 2010; Masseret, et al, 2013].  More study is needed. 

BMAA is present in a wide range of cyanobacteria.  Of the cyanobacteria tested, 95% of the genera and 97% of the strains produced BMAA [Cox, et al., 2005].  Many cyanobacteria produce BMAA in the lab.  It is in HABs, but it has not been found in open waters.

5.) Other Cyanotoxins

There are other toxic compounds from cyanobacteria that do not fit into the above categories.

A.) Aplysiatoxin is a compound that found in protective secretions.  It functions primarily as a powerful irritant/fish repellant.  It is a relatively large (671.6 daltons) and has a complex, multi-ring structure.  It is thought to be carcinogenic [Sivonen and Jones, 1999; Metcalf and Codd, 2014].  It is found in the genera: Lyngbya, Oscillatoria and Planktothrix. 

B.) Lyngbyatoxin-a is a relatively complex compound that is a potent blistering agent.  It is a medium to large molecule (437.6 daltons) and has a complex, multi-ring structure.  It acts as a fish repellant and is thought to be carcinogenic [Sivonen and Jones, 1999; Metcalf and Codd, 2014].  It is found in the genera Lyngbya, Oscillatoria and Planktothrix.

Summary

To summarize: cyanobacteria produce a wide variety of compounds, some of which are toxic to animals.  These toxins are generally not public health issues under most circumstances.  But, under bloom conditions, they can be a genuine problem.  Part of the purpose of the information compiled here is to help individuals learn to recognize these problem conditions and take appropriate steps to protect themselves and their families, pets and livestock.

Cyanotoxin Classification

References

[ 1] Carmichael, 1997           

[ 2] Sivonen, et al., 1989       

[ 3] Chen, et al., 2013

[ 4] Carmichael, et al., 1977

[ 5] Fawell, et al., 1999

[ 6] Skulberg, et al., 1992

[ 7] Lilleheil, et al., 1997

[ 8] Metcalf and Codd, 2014

[ 9] Mahmood and Carmichael, 1986a

[10] Pearson, et al., 2010

[11] Kuiper-Goodman, et al., 1999

[12] Ohtani, et al., 1992

[13] Sivonen and Jones, 1999

[14] Weiss, et al., 1989

[15] Fiorea, et al, 2020