Microcystins
(endorsed 2011)
Guideline
Based on health considerations, the concentration of total microcystins in drinking water should not exceed 1.3 mg/L expressed as microcystin-LR toxicity equivalents (TE).
General description
Microcystins are a large group of hepatotoxic peptides that are produced by a range of cyanobacteria. They were first characterised in the early 1980s and named after the cyanobacterium Microcystis aeruginosa, from which they were initially isolated. This group of cyanotoxins includes over 90 structural variants of cyclic heptapeptides (consisting of seven amino acids in a ring structure), with molecular weights in the range 800-1100 (Chorus and Bartram 1999 Chapter 3). Within this common structure there can be modifications in all seven amino acids, but the most frequent variations are substitution of L-amino acids at positions 2 and 4. The nomenclature of microcystins is based on the variable amino acids in position 2 and 4; for example, using the amino acid abbreviations for the L-amino acids, microcystin-LR possesses leucine (L) in position 2 and arginine (R) in position 4. Microcystin-LR is the best characterised and one of the most toxic variants of microcystin. Most of the structural variants are highly toxic within a narrow range, although some non-toxic variants have been identified (Chorus and Bartram 1999 Chapter 3).
Microcystins are most commonly produced by species of the genus Microcystis. They have, however, been shown to be produced by species of the planktonic genera Anabaena (Dolichospermum)¹, Planktothrix (Oscillatoria), Nostoc, Anabaenopsis and Radiocystis and also by a terrestrial (soil) species Haphalosiphon hibernicus, indicating the potential for their widespread occurrence in the environment. Within these genera and species, there can be both toxigenic (toxin-producing) and non-toxigenic genotypes. Nevertheless, the majority of human and animal microcystin-related poisonings worldwide are associated with the presence of Microcystis.
The toxicity of individual populations of M. aeruginosa is variable, and one extensive survey of the toxicity across the Murray-Darling Basin indicated that 56% of field samples tested were hepatotoxic (Baker and Humpage, 1994). A natural population may consist of a mixture of toxic and non-toxic genotypes, and this is believed to explain why population toxicity may vary over time and between samples (Chorus and Bartram 1999 Chapter 3). Environmental factors are regarded as the driving force determining these processes.
These cyanotoxins are largely water-soluble and are therefore, with a few exceptions, unable easily to penetrate biological membranes. Microcystins are thought to enter the bloodstream of mammals from the intestine, predominantly through the bile acid transport system. The absorbed toxins are then concentrated into liver cells, and cause hepatoenteritis. Microcystins are extremely stable chemically and remain potent even after boiling; however they are biodegraded by a range of aquatic bacteria found naturally in lakes and rivers. The half-lives for breakdown of microcystins in natural water have been shown to range from 5 to 20 days (Jones et al. 1994).
Australian significance
Microcystins are the most significant drinking water quality issue in relation to cyanobacterial blooms in south-eastern Australia. In Australia, they are produced predominantly by Microcystis aeruginosa, but can occasionally be produced by Anabaena (Dolichospermum) spp., although this appears to be rare.
The growth of cyanobacteria and blooms are favoured by nutrient enrichment (largely phosphorus but also nitrogen), combined with warm temperatures and calm, stable water conditions, such as those occurring in slow-flowing rivers and thermally stratified lakes.
The water supply problems associated with cyanobacteria include offensive tastes and odours and the production of toxins.
Treatment of drinking water
The first line of defence against cyanobacteria is catchment management to minimise nutrient inputs to source waters. Source water management techniques to control cyanobacterial growth include maintaining flow in regulated rivers; water mixing techniques, both to eliminate stratification and reduce nutrient release from sediments in reservoirs; and the use of algicides in dedicated water supply storages. Caution is necessary in using algicides if a bloom has developed because these agents will disrupt cells and liberate microcystins which largely occur as intracellular toxins that could otherwise be removed by cell removal as noted below. Once intracellular toxins are released they are much more difficult to manage. Microcystins will eventually be released into the water phase when a developed bloom declines and algal cells lyse, reinforcing the need to prevent blooms as far as possible. Algicide use should be in accordance with local environment and chemical registration regulations. Where multiple intakes are available, withdrawing water selectively from different depths can minimise the intake of high accumulations of cyanobacterial cells at the surface.
Water treatment processes can be highly effective in removing both cyanobacterial cells and microcystins. As with other cyanotoxins, a high proportion of microcystins remain intracellular unless cells are lysed or damaged, and can therefore be removed by coagulation and filtration in a conventional treatment plant (Chorus and Bartram 1999 Chapter 9). It should be noted that using oxidants such as chlorine or ozone to treat water containing cyanobacterial cells, while killing the cells, will also result in the release of free toxin; therefore pre-chlorination or pre-ozonation are not recommended without a subsequent step to remove dissolved toxins.
Microcystins are readily oxidised by a range of oxidants, including ozone and chlorine. Adequate contact time and pH control are needed for optimal removal of these compounds, and this will be more difficult to achieve in the presence of whole cells (Drikas et al. 2002). Microcystins are also adsorbed from solution by both granular activated carbon and, less efficiently, by powdered activated carbon. (Drikas et al. 2002). Because powdered activated carbon may be a more practical option for intermittent or emergency use, it is important to seek advice and carefully select the most appropriate type for toxin removal, as carbons vary significantly in performance for different compounds. Boiling is not effective for destruction of microcystins.
If treatment is instituted in response to the presence of toxin-producing cyanobacteria, the effectiveness of the process needs to be confirmed by testing for toxin in the product water.
Methods of identification and detection
Animal bioassays (mouse tests) have traditionally been used for detecting the entire range of cyanotoxins, including microcystins. These tests provide a definitive indication of toxicity, although they cannot be used for precise quantification of compounds in water or for determining compliance with the guideline value. A number of techniques are available for determining microcystins in water (Chorus and Bartram 1999 Chapter 13) The analytical technique selected needs to allow quantitative comparison with the guideline value in terms of toxicity equivalents. When quantitative standards are available, the most precise technique in this regard is liquid chromatography with confirmation by mass spectrometry (LC–MS/MS); although this technique still involves estimation of the concentration and therefore toxicity of some microcystins in a sample against microcystin-LR as the analytical standard, which may result in a slight overestimate of total microcystins (as microcystin-LR, toxicity equivalents).
A range of commercially available immunoassay (ELISA) kits offer a rapid technique for screening and semi-quantitative measurement of toxins in cyanobacterial cell material and in water. A potential limitation of these assays is the poor cross-reactivity of the antibodies between variants of microcystin, which can lead to underestimation of total toxicity.
Cyanobacteria are detected by light microscopy, identified using morphological characteristics, and counted per standard volume of water (Hotzel and Croome 1999). Practical keys for their identification are provided in Baker and Fabbro (2002).
Health considerations
Microcystins are primarily hepatotoxins. The mechanism of toxicity involves inhibition of protein phosphatase enzymes in eucaryotic cells. Acute exposure to high doses of microcystin administered either intravenously (i.v.) or by intraperitoneal (i.p.) injection causes severe liver damage and is characterised by a disruption of liver cell skeletal structure, a loss of sinusoidal structure, increases in liver weight due to intrahepatic haemorrhage, haemodynamic shock, heart failure and death.
There is a significant number of reports describing animal poisonings from ingesting water that contains Microcystis, with some examples confirming hepatotoxicity and the associated presence of microcystins (Ressom et al. 1994). Significant human illness has been strongly associated with exposure to microcystins in recreational waters (Turner et al. 1990). In an unfortunate incident in 1996, a large number of dialysis patients in Caruaru, Brazil, were exposed to microcystins intravenously and experienced a range of symptoms including headache, eye pain, blurred vision, nausea and vomiting (Jochimsen et al. 1998). This incident showed that human sensitivity is similar to that found in animal studies. The patients’ livers were painfully enlarged and many experienced subcutaneous, nasal or uterine bleeding. Liver samples from 52 of the 76 people who died in this incident were examined; they showed disruption of liver plates and liver cell morphology, necrosis, apoptosis, and cholestasis; and intracellular structures were also deranged. Using various assumptions, the patients who died were exposed to an estimated raw water microcystin concentration of 19.5 μg L-1 by dialysis, but much higher exposure levels would be required to cause these serious health outcomes by ingestion because of the much lower uptake of microcystin across the gut (Carmichael et al. 2001).
Microcystins are also toxic when inhaled. A study with mice showed that intranasal introduction of microcystin-LR resulted in extensive necrosis of the epithelium of the nasal mucosa of both the olfactory and respiratory zones, progressing to destruction of large areas of tissue down to levels of deep blood vessels (Fitzgeorge et al. 1994). The by this route of administration was the same as the i.p. , and dose-dependent liver lesions were observed. The same authors also demonstrated cumulative liver damage after repeated dosing.
In experimental animal studies, microcystin-LR can produce extreme acute toxicity. In mice the is in the range of 0.025 to 0.15 mg/kg bodyweight for the i.p. route, and 5 and 10.9 mg/kg bodyweight respectively for oral administration for two different strains of mice. These differences show that much higher levels of exposure are required by ingestion compared with i.p. injection. Even higher values have been demonstrated in rats (Chorus and Bartram 1999 Chapter 4).
Microcystins promote the growth of tumours in experimental animals (Falconer 1991, Nishiwaki-Matsushima et al. 1992). The significance of this for humans, who may be subject to chronic exposure via drinking water, is unclear.
Microcystins have been implicated in causing liver damage in an Australian population exposed via reticulated town water supply where the source water contained blooms of Microcystis (Falconer et al. 1983).
The International Agency for Research on Cancer (IARC) convened an expert Working Group in 2006 to assess the evidence for the carcinogenicity of microcystins. Their conclusion after considering all of the evidence but emphasising the strength of the mechanistic data, was that microcystin-LR is “possibly carcinogenic to humans” (group 2B) (Grosse et al. 2006).
Microcystins are currently regarded as non-genotoxic.
Derivation of guideline
where:
40 mg/kg body weight per day is the No Observed Adverse Effect Level (NOAEL) from a 13-week ingestion study with microcystin-LR in mice, based on liver histopathology and serum enzyme level changes (Fawell et al. 1994);
70kg is the average weight of an adult;
0.9 is the proportion of total daily intake attributed to the consumption of water;
2 L/day is the average amount of water consumed by an adult;
1000 is the safety factor derived from extrapolation of an animal study to humans (10 for interspecies variability, 10 for intraspecies variability and 10 for limitations in the database, related particularly to the lack of data on chronic toxicity and carcinogenicity).
The guideline is derived for total microcystins and expressed as microcystin-LR toxicity equivalents (TE). This is because the total microcystin concentration should be considered in relation to potential health impacts.
The World Health Organization (WHO) evaluation of the health-based information for cyanobacterial toxins (Gupta 1998, WHO 1998, Chorus and Bartram 1999 Chapter 5) concluded that there are insufficient data to allow a guideline value to be derived for any cyanobacterial toxins other than microcystin-LR. The guideline recommended by the WHO for drinking water is 1 mg/L (rounded figure) for total microcystin-LR (free plus cell-bound), based on the Fawell et al. (1994) sub-chronic study. This guideline value for microcystin-LR is provisional, as the database is regarded as limited (WHO 1998).
The approach being taken for guideline derivation here is essentially similar to that used by WHO (Chorus and Bartram 1999 Chapter 5). The same ingestion study in mice was used to calculate the NOAEL. The difference between the Australian guideline of 1.3 mg/L total microcystin (as microcystin-LR TE) and the WHO provisional guideline of 1 mg/L microcystin-LR is due to use of a different average body weight for an adult (70 kg versus 60 kg), and a different proportion of the daily microcystin intake attributed to drinking water (0.9 in the Australian guideline versus 0.8 selected by WHO). The higher figure is due to lower potential exposure in Australia from other environmental sources, such as contaminated bathing water, and via dietary supplements potentially containing microcystins.
Where M. aeruginosa occurs in drinking water supplies and toxin monitoring data are unavailable, cell numbers can provide a preliminary indication of the potential hazard to public health. For a highly toxic population of M. aeruginosa (toxin cell quota of 0.2 pg total microcystins/cell; mean cell volume of 87 ), a cell density of approximately 6,500 cells/mL (biovolume of 0.6 ) would be equivalent to the guideline of 1.3 mg/L microcystin-LR (TE) if the toxin were fully released into the water. This number is indicative only; toxin determination is required for health risk assessment.
Notification procedure
It is recommended that a notification procedure be developed by water and health authorities. A tiered framework should be considered. Initial notification to health authorities could be provided when numbers of M. aeruginosa reach 30% of the density equivalent to the guideline value of 1.3 μg/L microcystin (2,000 cells/mL; biovolume 0.2 ), while an alert could be provided when cell numbers are equivalent to the guideline value (6,500 cells/mL; biovolume 0.6 ). For microcystin-producing species other than M. aeruginosa, notifications and alerts should be based on biovolumes.
In all cases, cell numbers should be used only as preliminary signals and as triggers for toxin testing to enable assessment of potential health risks.
¹ A change of nomenclature has been proposed for Anabaena to Dolichospermum (Wacklin et al, 2009). Both names are cited due to common usage of Anabaena and recognising that references cited use the name Anabaena.
NOTE: Important general information is contained in PART II, Chapter 5
References
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Baker P, Humpage AR (1994). Toxicity associated with commonly occurring cyanobacteria in surface waters of the Murray-Darling Basin, Australia. Australian Journal of Marine and Freshwater Research, 45:773-786.
Carmichael WW, Azevedo SMFO, An JS, Molica, RJR, Jochimsen EM, Lau S, Rinehart KL, Shaw GR, Eaglesham GK (2001). Human fatalities from cyanobacteria: Chemical and biological evidence for cyanotoxins. Environmental Health Perspectives, 109:663-668.
Chorus I, Bartram J (eds) (1999). Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management. E&FN Spon, London.
Drikas M, Newcombe, G, Nicholson B (2002). Water treatment options for cyanobacteria and their toxins. In: Blue-Green Algae: Their significance and management within water supplies. CRC for Water Quality & Treatment Occasional Paper 4, pp 75-92.
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