12 minutes reading time (2390 words)


    B. C. Nicholson & J. Rositano
    Australian Centre for Water Quality Research
    Salisbury, South Australia

    A group of organisms currently receiving attention due to their ability to produce toxins are the widespread, and rather innocuous-looking, freshwater Algae. In the marine environment toxic algae have been a recognised danger to man for some time. Major concerns are paralytic shellfish poisoning (PSP)(1)(2)(3) and ciguatera poisoning, (2)(3) and to a lesser extent an allergic dermatitis reaction known as "swimmer's itch"(4). Numerous human fatalities have resulted from the consumption of shellfish (PSP poisoning) or fish (ciguatera poisoning) which have accumulated toxic dinoflagellates. Toxicity in freshwater algae is almost entirely restricted to the blue-green algae which are more correctly known as cyanobacteria due to their close relationship to bacteria.

    The first scientific report of a toxic blue-green algal bloom was a report(5) in in 1878 of the death of livestock which drank water from Lake Alexandrina, approximately 75 km from Adelaide, at the time of a heavy bloom of Nodularia spumigena. In the intervening years numerous incidents from various parts of the world of deaths of animals including dogs, sheep, cattle, birds and bats following consumption of water affected by heavy algal blooms have been reported in the literature. Various species of cyanobacteria have been involved.(6) Blooms of toxic cyanobacteria which are now widespread throughout the River Darling and other areas of NSW are currently attracting substantial media attention.

    The dangers of toxic algal blooms to animals are enhanced by the properties of the organisms involved. The algae are free-living but tend to float and form scums. Gentle wind and wave action concentrates these scums at the shores of lakes and rivers in bays where animals drink. Man appears to have escaped direct acute poisoning due to the rather repulsive and unpalatable appearance of the water with these thick, green scums which often have a strong odour. The scums, which often extend several metres from the shore, resemble thick, green paint.

    As many of the large rivers and man-made lakes serve as water supplies, the presence of such toxic constituents is of concern to water supply authorities. While the possibility of supplying a lethal dose through the water reticulation system is remote, there have been several reports implicating cyanobacterial toxins in gastric illnesses in communities served by water containing high numbers of algae.(3) In Australia an outbreak of hepato-enteritis in Queensland was associated with the copper sulfate treatment of a heavy bloom of algae in a water supply dam.(7) A strain of the cyanobacterium Cylindrospermopsis raciborskiisubsequently isolated from the dam was shown to be hepatotoxic.(8) Liver damage in the population of Armidale in NSW has also been correlated with high levels of the cyanobacterium Microcystis aeruginosa in the water supply reservoir.(9) This cyanobacterium has recently been shown to be a tumour promoter.(10) Contact with water containing cyanobacterial scums has recently been reported as a cause of pneumonia and other complaints. (11)

    Algal blooms are favoured by conditions which include sunlight, warm temperatures and sufficient nutrients. Increasing nutrient levels in water bodies from human activities (nitrogen from fertilisers and phosphorus from fertilisers and detergents) are a common problem worldwide and there is little doubt that algal blooms are increasing in frequency and severity both in Australia (12) and overseas (13) as a result.

    The management of algal blooms is made difficult by the existence of toxic and non-toxic strains of particular species which morphologically appear identical. Toxicity can also vary considerably over the life of a bloom and is influenced by environmental factors such as light intensity, pH etc. (14) Toxicity may also vary spatially within a bloom. Toxicity in some cases may thus be more a matter of degree rather than a change in the proportion of toxic and non-toxic strains. Some toxic algae also appear to lose their toxicity after long periods in culture(15).

    Several species of cyanobacteria can produce toxins(16). Both neurotoxins and hepatotoxins are produced (Table 1). These differ markedly in their chemistry and mode of action. Most toxins are highly toxic with LD50 values (mouse - intraperitoneal (ip)) in the low μg/kg range. In contrast the oral LD50 for potassium cyanide to rats is 10 mg/kg.

    The toxins responsible for acute toxic effects are produced within the cell and may be released to the surrounding water when the cell dies and the cell wall ruptures. These exotoxins are of greatest interest because of their high acute toxicity. Some cyanobacteria produce lipopolysaccharide (LPS) endotoxins as part of the cell wall. These are either the sole toxic product or are produced together with an exotoxin. LPS endotoxins are not acutely toxic (18) and may be responsible for human illnesses (gastric upsets and skin complaints) when cyanobacteria are present in very high numbers. This is supported by the finding that the cyanobacterium implicated in an outbreak of gastric illnesses produces LPS endotoxins but is not acutely toxic.(18) Nevertheless the acute toxicity of the exotoxins and the presence of high numbers of toxic, cyanobacteria in waters used for drinking, especially with indications of liver damage, are reasons for considerable concern. Only recently have conclusive chemical structural data for these cyanobacterial toxins been reported.

    NEUROTOXINSNeurotoxins have been identified from several species of cyanobacteria. Anatoxin-a (ANTX-A), a secondary amine, molecular weight 165 (Figure 1), is quite toxic with an LD50 (mouse -ip) of 200μg/kg and has been isolated from various species of cvanobacteria. The structure of anatoxin-a(s) (ANTX-A(S)) which has been foundin strains of A. flos-aquae has recently been elucidated. It has a molecular weight of 252 (Figure 1) and is much more toxic with an LD50 (mouse - ip) of 20μg/kg. it appears to be an anticholinesterase agent.(16) Two aphantoxins have been identified from strains of A. flos-aquae (Figure 1).

     These two toxins appear identical to toxins isolated from marine dinoflagellates responsible for paralytic shellfish poisoning. It is interesting that relatively diverse organisms such as cyanobacteria and dinoflagellates produce the same toxins. While neurotoxic species of Anabaena have often been recorded in Australia, no species of Aphanizomenon have have yet been found to be toxic. Other neurotoxins are still to be characterized.


    Several species of cyanobacteria produce highly hepatotoxic chemicals (Table 1). Those characterized to date have been shown to be small cyclic peptides. These are of particular interest as the species involved are much more common bloom organisms. These toxins cause rapid death in animals through massive enlargement of the river and engorgement with brood. The animal essentially bleeds to death into its own liver. The heptapeptides, which are produced almost entirely by species of Microcystis, are now known most commonly as microcystins. A pentapeptide isolated from Nodularia spumigena is called nodularin.(19) Nodularin appears to be the only toxin produced by N. spumigena (2O) whereas other species of cyanobacteria often produce more than one toxin at the same time. A. flos-aquae can produce both neurotoxins and hepatotoxins.

    The structure of nodularin is shown in Figure 2. It has a molecular weight of 824 and contains arginine, glutamic acid, β-methylaspartic acid, N- methyldehydrobutyrine and a unique β-amino acid given the name Adda. N. spumigena occurs commonly in Australia and was the first toxic algae described in the literature(5). Blooms still occur in Lake Alexandrina and have caused problems in recent years. They have been shown to be toxic with an LD50 (mouse - ip) of 25-50 mg freeze-dried cells/kg. Nodularin has been identiti6d as the toxic constituent. (21,22) The acute toxicity of nodularin is reported as 30- 50 μg/kg (LD50; mouse - ip).(16) During these episodes the lake could not be used for water supply and it also had to be closed to recreational use. Blooms of toxic N. spumigena also frequently occur in other more brackish environments in Australia, eg. the Gippsland Lakes in Victoria and the Peel-Harvey inlet in Western Australia.(12)

    Microcystins are cyclic heptapeptides which initially appeared to contain 5 invariant and 2 variant amino acids, the invariant amino acids being in the D- configuration (where enantiomers are possible) and the variant amino acids being the more usual L-enantiomers. Microcystins also contain the unique p- amino acid Adda found in nodularin. A 2 letter suffix (XY) is ascribed to each individual toxin to denote the two invariant amino acids. of the toxins isolated and characterized to date, X can be leucine (L), arginine (R) or tyrosine (Y); Y can be arginine (R), alanine (A) or methionine (M)(Figure 3). Molecular weights of microcystins vary from 909 to 1044 with acute toxicities varying between 50- 1000 μg/kg (mouse - ip).(16) Recently some desmethyl variants of the "invariant" amino acids plus have been reported.(23) Variants have been isolated from Nostoc sp with an acetyl rather than a 9-methoxy group of Adda (Table 2)(24).

    Both overseas and Australian studies have shown that more than one microcystin can be found in a bloom of M. aeruginosa at any one time. Up to six microcystins have been found in one Australian sample. A unique finding has been a major change in the identities of toxins found from a particular location over a relatively short period of time(21). Microcystins and nodularin can be extracted from scum material and determined by reversed phase HPLC with UV, or better still, photodiode-array detection. These toxins can be identified by their absorption of 240nm (Figure 4). Toxins from several Australian strains have been isolated by preparative HPLC and identified using FAB mass spectrometry.(21) Work is now progressing on identifying further toxins (including the use of 2-dimensional NMR), developing methods for the determination of toxins in water and the behaviour of toxins in water treatments processes. 


    Funding support from the Urban Water Research Association of Australia (UWRAA) and the SA Engineering and Water Supply Department for some of the work described above is gratefully acknowledged.REFERENCES

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    Brenton Nicholson (MRACI) gained his B.Sc.(Hons.) and Ph.D. degrees in organic chemistry from the University of Adelaide in 1969 and 1974 respectively. He followed these with a Graduate Diploma in Business Administration from the University of South Australia in 1988. Since 1975 he has been employed by the Engineering and Water Supply Department, the South Australian water authority responsible for water resources management, water supply and sewerage treatment throughout the state. Located at the State Water Laboratory, he manages the Organic Chemistry Unit which carries out organic chemical-based monitoring as well as research on organic chemicals in relation to their effects on water quality. Through the Australian Centre for Water Quality Research, a collaborative venture of the Laboratory, the School of Chemical Technology of the University of South Australia, and the Department of Soil Science, Waite Agricultural Research Institute, he is managing several projects, amongst which those dealing with algal toxins have a high priority. In particular the behaviour of toxins in water treatment processes is currently a project of very high priority. He has been a member of the State Analytical Chemistry Group of RACI since 1982 and its Secretary since 1986.

    Joanna Rositano (MRACI) gained her Honours degree in organic chemistry from the University of Adelaide in 1989. She was employed as a Research Officer in the Organic Chemistry Department of that University for two years investigating the functionalisation of amino acids for use in medical research. At the beginning of 1991 she joined the Australian Centre for Water Quality Research as a Research Officer to investigate the behaviour of algal toxins in water treatment processes. The project also involves the isolation and characterization of as yet unidentified toxins using a range of chemical techniques.



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