Evaluation of Analytical Methods for Detection and Quantification of Cyanotoxins in Relation to Australian Drinking Water Guidelines

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2 Evaluation of Analytical Methods for Detection and Quantification of Cyanotoxins in Relation to Australian Drinking Water Guidelines Brenton C. Nicholson Michael D. Burch Cooperative Research Centre for Water Quality and Treatment Endorsed October 2001 A report prepared for the National Health and Medical Research Council of Australia, the Water Services Association of Australia, and the Cooperative Research Centre for Water Quality and Treatment i

3 Commonwealth of Australia 2001 ISBN Print: Online: This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from AusInfo. Requests and enquiries concerning reproduction and rights should be addressed to the Manager, Legislative Services, AusInfo, GPO Box 1920, Canberra ACT address: The strategic intent of the NHMRC is to provide leadership and work with other relevant organisations to improve the health of all Australians by: fostering and supporting a high quality and internationally recognised research base; providing evidence based advice; applying research evidence to health issues thus translating research into better health practice and outcomes; and promoting informed debate on health and medical research, health ethics and related issues. NHMRC web address: Cover picture: Courtesy Mike Burch, CRCWQT This document is sold through AusInfo Government Info Bookshops at a price which covers the cost of printing and distribution only. For publication purchases please contact AusInfo on their toll-free number , or through their internet address: ii

4 C O N T E N T S E X E C U T I V E S U M M A RY A C R O N Y M S A N D A B B R E V I AT I O N S V X I 1. I N T R O D U C T I O N Toxic cyanobacteria Cyanobacterial toxins (cyanotoxins) Peptide hepatotoxins (microcystins and nodularin) Neurotoxins Cylindrospermopsin Lipopolysaccharide (LPS) endotoxins 7 2. S C O P E O F T H I S R E V I E W General Terms of Reference 9 3. T OX I N A N A LY S I S G E N E R A L C O N S I D E R AT I O N S Toxin extraction Concentration/cleanup procedures Sample preservation General toxicity-based assays D E T E R M I N AT I O N O F M I C R O C Y S T I N S A N D N O D U L A R I N General Extraction procedures Concentration/cleanup procedures Sample preservation Determination of microcystins and nodularin Toxicity-based assays Instrumental analyses High performance liquid chromatography (HPLC) Capillary electrophoresis (CE) Thin layer chromatograph (TLC) MMPB method Enzyme linked immunosorbent assays (ELISAs) Phosphatase inhibition assays Other assays 24 iii

5 5. D E T E R M I N AT I O N O F N E U R OTOX I N S ( S A X I T OX I N S ) Extraction procedures Concentration/cleanup procedures Sample preservation Determination of saxitoxins Toxicity-based assays Instrumental analyses (HPLC, CE) Enzyme linked immunosorbent assays (ELISAs) Other assays D E T E R M I N AT I O N O F C Y L I N D R O S P E R M O P S I N S U M M A RY A N D C O N C L U S I O N S Peptide hepatotoxins (microcystins and nodularin) Saxitoxins Cylindrospermopsin 33 8 A C K N OW L E D G M E N T S R E F E R E N C E S 4 1 L I S T O F F I G U R E S Figure 1.1: Structures of peptide hepatotoxins 2 Figure 1.2: Structures of the anatoxins 4 Figure 1.3: Structures of the saxitoxins 5 Figure 1.4: Structure of cylindrospermopsin 6 L I S T O F TA B L E S Table 5.1 Table 7.1 Table 7.2 Concentration of saxitoxins equivalent to a guideline of 3 µg/l saxitoxin equivalents and detection limits without sample concentration 26 Summary of applicability of analytical methods for determining microcystins in relation to the Terms of Reference 34 Summary of applicability of analytical methods for determining saxitoxins in relation to the Terms of Reference 37 iv

6 E X E C U T I V E S U M M A RY P U R P O S E O F T H I S R E V IE W The Australian Drinking Water Guidelines (ADWG) have been undergoing a process of rolling revision by NHMRC/ARMCANZ since The NHMRC/ARMCANZ Drinking Water Review Coordinating Group identified that guidelines for cyanobacteria and their toxins would be developed as part of the review for 1999/2000. A working party completed a review of information on cyanobacteria and their toxins in relation to drinking water and public health in April This has resulted in the production of four Fact Sheets for individual classes of toxins: microcystins, nodularin, saxitoxins, and cylindrospermopsin (Fact Sheets 17a 17d). The outcome of the review and subsequent consultation process was that a guideline value was recommended for total microcystins (Fact Sheet 17a), and that no guideline values could be set for concentrations of nodularin, saxitoxins or cylindrospermopsin due to the lack of adequate data. These findings are essentially similar to the position taken by the World Health Organization in their recent review of cyanotoxins as part of the continuing process of updating the second edition of the WHO Guidelines for Drinking Water Quality (GDWQ) (WHO, 1998). The WHO position is that there are insufficient data to allow a guideline value to be derived for any cyanobacterial toxins other than microcystin-lr. The WHO guideline is 1 µgl -1 for total microcystin-lr (free plus intracellular). In addition, the WHO assessment indicated that the guideline value of 1 µgl -1 for microcystin-lr is provisional, as the database is limited, new data for the toxicity of cyanobacterial toxins are being generated, and the guideline value covers only microcystin-lr. The various WHO working groups have indicated that at the time of their deliberations (1997/98) there was insufficient information available to derive guidelines for either neurotoxins or cylindrospermopsin. A significant difference between the provisional WHO guideline and the NHMRC/ARMCANZ guideline is that the Australian guideline gives advice in relation to the concentration of total microcystins whereas WHO restricts its advice to the single compound microcystin-lr. Microcystin-LR is the most widespread variant in geographic terms and best characterised toxicologically of this class of hepatotoxins, of which there are in excess of 75 structural types. The Fact Sheet for microcystins (17a) in the ADWG states that the Australian guideline for Total Microcystins is 1.3 µgl -1 expressed as toxicity equivalents of microcystin-lr. The rationale for the Australian guideline covering total microcystins is that blooms of Microcystis aeruginosa which is the most common toxin producing cyanobacterium in Australia, generally contain a range of variants of microcystin in varying amounts. Experience indicates that the number of variants in an individual sample can range from a few to up to more than 20 in some cases. It is the cumulative toxicity of the microcystins in total that represents the potential hazard to human health from ingestion via drinking water. Therefore the unit recommended for the quantitative expression of this cumulative toxicity in the guideline is total microcystins expressed as toxicity equivalents of microcystin-lr. A consequence of the recommendation of such a guideline for microcystins is the potential requirement for identifying and quantifying specific toxin variants where contamination is suspected in a water supply. The process of guideline setting in general by NHMRC considers the requirements and current limitations of the measurement technology. In the case of analysis for cyanobacterial toxins the basic principles of techniques available for measuring compounds of a particular class vary considerably. Given the above background it was proposed that the revision of the NHMRC guidelines for toxins should consider the status of analytical methods. Therefore this review of toxin analytical methods was undertaken separately under the direction of the Drinking Water v

7 Review Coordinating Group for the ADWG Revision, with support from NHMRC, the Water Services Association of Australia and the Cooperative Research Centre for Water Quality and Treatment. This review is intended to provide guidance in the selection of appropriate methods for measuring cyanotoxins to assist water supply authorities and health authorities in the interpretation and application of new guidelines. T E R M S O F R E FE R EN C E O F T H I S RE V IE W The review involved an assessment of the current range of analytical methods for cyanotoxins. This included methods that have been used for microcystins, saxitoxins and cylindrospermopsin. The assessment of each method (for microcystins and saxitoxins) addressed the following points: Principle of the technique; Detection limit and precision; Current usage and reliability, ie, is the current usage a research application only, and is it amenable to routine use in a commercial laboratory? Degree of documentation of the method in the literature, ie, are published standard protocols available? Level of expertise required by the operator/analyst; Economics of the method, ie, relative cost of set-up and application; and Assessment of whether the method is immediately amenable for compliance with the guideline (for microcystins only). S U M M ARY O F F I N DI N GS General A number of techniques are available for determining cyanotoxins in water. For microcystins these range from immunological or biochemical screening techniques based on ELISA and enzyme activity (protein phosphatase inhibition) assays respectively, to quantitative chromatographic techniques based on high performance liquid chromatography (HPLC) and more sophisticated (and expensive) liquid chromatography-mass spectrometry (LC-MS). Less common analytical techniques of capillary electrophoresis (CE) and MALDI-TOF mass spectrometry have also been evaluated in some laboratories. Analytical techniques based on either HPLC or LC-MS can also be used for determining saxitoxins or cylindrospermopsin in water. Animal bioassays (mouse tests), and in some cases assays based on isolated cell lines, are also available for screening the entire range of toxins. Appropriate and careful handling of samples both prior to and during analysis is extremely important to ensure an accurate determination of toxin concentration. Microcystin toxins are readily degraded both photochemically (ie, in light) and microbiologically. Samples should be kept refrigerated and in the dark prior to analysis, and should not be exposed to strong light during the preparation and analytical procedures. Saxitoxins may interconvert unless extracts are prepared in dilute acetic acid and kept refrigerated. Toxin extraction To determine the concentration of total toxin in any water sample it may be necessary to first release the toxins that are present within the cell (ie, intracellular). This is because current analytical procedures only determine toxins in the dissolved or free state. This vi

8 process can be carried out in such a way that the total toxin content is measured, or the cellular material containing toxins can be separated from the water phase for individual determination of intracellular and extracellular toxin. This approach should be included in the analysis of unfiltered water supply samples where intracellular toxins may be present. In the case of filtered water, this step is unnecessary, as all toxins will be in solution and total toxin is represented by the concentration of dissolved toxin. The most appropriate technique for liberating intracellular toxin is freeze/thawing in the presence of a solvent appropriate for the particular toxin. The requirement to ensure toxin release from cells prior to analysis applies to all toxic cyanobacteria and classes of toxins. Mouse bioassays Mouse bioassays have in the past always provided a definitive indication of mammalian toxicity for cyanobacterial material, although they cannot be used for precise quantification of cyanotoxins at low concentrations in water. While they may indicate the class of toxin, and could potentially be calibrated in terms of a specific toxin, they do not have sufficient sensitivity for application to water samples without an impractical degree of preconcentration. Microcystins Amongst the range of techniques evaluated in this review for the determination of microcystins in water, there is no single technique that provides an accurate measure of the toxin concentration in microcystin LR toxicity equivalents where complex mixtures of microcystins occur in a water sample. Each technique generates a value that requires assumptions in order to derive a result in toxicity equivalents. Irrespective of the detection method, there are a number of criteria which must be satisfied if a toxin concentration in terms of microcystin-lr toxicity equivalents is to be obtained for comparison with the proposed guideline level. These are: The toxin must be able to be unequivocally identified; An analytical standard must be available for the specific toxin so that the concentration can be accurately determined; and An acute toxicity value (LD 50 ) must be available for that specific toxin so that a concentration in microcystin-lr toxicity equivalents can be calculated. Given these criteria, all methods have limitations. However, in relation to the terms of reference for this review, the technique which is currently most suitable to monitor in relation to the guideline is the instrumental technique of HPLC with photo diode array (PDA) or mass spectral detection. The following is a summary assessment of the strengths and weaknesses of the techniques that were evaluated: High performance liquid chromatography (HPLC): This technique can generally separate most microcystins, and PDA detection is relatively reliable for identification of chromatographic peaks as microcystins based on their spectra. However, the identification of specific microcystins based solely on retention times can be prone to error. Quantification is achieved by comparison of analyte responses to those of commercially available standards. There are a limited number of microcystin standards available commercially and these are not certified quantitative analytical standards. The availability of certified standards is currently a limitation for precise quantification of microcystins for all methods currently used, however this is likely to improve as commercial suppliers respond to the world wide demand for monitoring these toxic compounds in water supplies. The technique of HPLC- PDA fulfils other practical criteria for use in routine monitoring of microcystin in having acceptable detection limits in relation to the guideline, good documentation with a degree of standardisation, and a long history of use both within Australia and overseas in commercial analytical laboratories. vii

9 In situations where standards are unavailable for particular toxins, or the identity of the microcystin is unknown, it is necessary to estimate the concentration of these microcystins against microcystin-lr as the analytical standard based on the assumption that individual variants have similar responses with PDA detection as work to date has shown this to be so. In terms of toxicity, it must also be assumed that this is also the same as microcystin-lr. In this case a slight overestimation of total microcystins (as microcystin-lr toxicity equivalents) may result as microcystin-lr is the most acutely toxic of the microcystin variants with other variants having similar or lower toxicities. The result is a conservative estimate of the hazard from total microcystins in drinking water for these samples which is desirable for the protection of public health. Liquid chromatography-mass spectrometry (LC-MS): Mass spectrometry as a detection technique following liquid chromatographic separation offers a superior means of identifying separated components as microcystins, and in the case of MS/MS detection, it may be possible to identify specific compounds for which published spectra are not available. Currently however these techniques are not widely used or offered in routine analytical laboratories due to methods not being well developed and the cost of instrumentation. This situation may change relatively quickly, with the likelihood of a combination of improved method development, and the availability of suitable instrumentation at lower cost in major commercial analytical laboratories. In this case the technique of LC/MS may become the method of choice for the routine determination of these toxins in the near future. As with PDA detection, standards must be available in order to accurately quantify specific toxins. Although with PDA detection, the responses of the various microcystins are similar and a reasonable estimate of concentration can be made with reference to a microcystin-lr standard, the same is not true with MS detection, ie, the total ion current response depends markedly on the particular microcystin. Hence concentrations of microcystins for which standards are not available cannot be estimated from this analysis. Enzyme linked immunosorbent assays (ELISA): Analytical techniques based on enzyme linked immunosorbent assays (ELISA) have high sensitivity, however the cross-reactivity of the various microcystins and nodularin depends upon the similarity in chemical structure to the microcystin against which antibodies have been raised (generally microcystin-lr) and not toxicity. Therefore, an issue to be considered in the application of these antibodies to a sample with an unknown or complex mixture of microcystins is the potential for poor reaction with some components. Depending on both the cross reactivity and the toxicity of a microcystin, ELISAs can over- or underestimate the toxin concentration in terms of microcystin-lr toxicity equivalents. In cases where the mixture of toxins is well characterised, for example given a water body with ongoing or regular contamination with toxic Microcystis with a consistent toxin profile, the use of ELISA for ongoing monitoring is quite acceptable. However, as ELISA techniques can greatly over- or underestimate the concentration with some variants, they cannot be relied on as quantitative assays. They are, however, useful screening tools. Protein phosphatase inhibition assays: Protein phosphatase inhibition assays are also sensitive procedures for determining this class of toxins. While the response and measurement of toxins in this assay is related to the mode of toxic action of microcystins, there is not a direct relationship between measurement of enzyme inhibition and mammalian toxicity. The data to date suggest that such assays provide an overestimation of toxin concentration in terms of microcystin-lr toxicity equivalents which may be acceptable as the result is conservative and therefore provides for added protection of public health. However, response data with a much larger range of microcystin variants needs to be obtained in order to determine whether there is an underestimation of toxin concentration with some variants. At this stage, then, these assays are also restricted to use as screening tools. In addition, for direct monitoring of waters at low toxin concentrations, the effects of sample matrix on the results obtained requires further evaluation. viii

10 Both ELISA and phosphatase inhibition assays are currently not widely used and documented for routine water supply monitoring in this country. However as these methods are further evaluated, their acceptance will increase if they can be shown to be reliable. The techniques may then become low-cost methods for toxin screening, with the advantage of very low detection limits relative to the guideline value. Positive results can then be followed up with more definitive analyses such as HPLC. Saxitoxins The analytical technique of high performance liquid chromatography (HPLC) with postcolumn derivatisation and fluorescence detection can be used to quantify a range of saxitoxins in both water and cellular material where appropriate standards are available. These procedures require analysis under three different sets of conditions to individually determine the range of saxitoxins produced by cyanobacteria. They are highly sensitive and specific, but the possibility of coeluting interferences must be kept in mind. This information can then be used to derive an estimate of total toxins in terms of saxitoxin equivalents (STX-eq) using a conversion based on specific mouse toxicities. It is the method of choice for determining these compounds at present. Instrumental methods based on capillary electrophoresis or MS detection currently require further development. Commercially available ELISAs do not appear to be applicable to toxins from Australian cyanobacteria with their particular profiles, as these assays have been developed to respond to saxitoxin itself and the toxins predominating in Australian cyanobacteria, the C-toxins, do not cross react. The neuroblastoma or saxiphilin assays may have some potential for monitoring waters contaminated with saxitoxins but require further development, mainly to improve sensitivity. Cylindrospermopsin Cylindrospermopsin can be directly determined in water samples using LC/MS/MS with a detection limit of around 1µg/L. This is the method of choice, as it has not been demonstrated that conventional HPLC with UV or PDA detection has adequate specificity to be applied to water samples containing low levels of cylindrospermopsin. ix

11 K E Y F IN D I N GS FOR M O N ITO R I N G FOR M I C RO C YS T I N S I N R ELAT I O N TO T H E AUSTRAL I AN DRINKING WAT E R GUIDELI N E The techniques which provide the most reliable measurement for compliance with the ADWG for microcystins in water are is high performance liquid chromatography (HPLC) with photo diode array (PDA) detection.). Liquid chromatography with mass spectral confirmation of toxin identity and quantification is suitable if standards for the toxins present are available. For compliance monitoring in relation to the guideline, the concentrations of individual microcystins are determined by calibration against standards. The relative toxicity of microcystins other than microcystin LR are then converted to microcystin-lr toxicity equivalents based on the ratio of their published LD 50 (mouse, i.p.) relative to that of microcystin-lr. A comprehensive list of relative toxicities of microcystin variants is provided by Sivonen and Jones (1999). In situations where standards are unavailable for particular toxins in a sample it is necessary to use HPLC with PDA detection for analysis and to estimate the concentration and therefore toxicity of these microcystins against microcystin-lr as the analytical standard. In this case a slight overestimate of total microcystins (as microcystin-lr, toxicity equivalents) may result. The technique of ELISA is useful for routine screening of water for toxin contamination. In situations where the sample is well characterised in terms of toxin composition, and results are cross-calibrated initially and at periodic intervals against other techniques (HPLC-PDA) it can be regarded as a reliable measure of total microcystins in microcystin-lr concentration equivalents. For samples containing toxins of unknown identities, it cannot provide a reliable quantitative result. Phosphatase inhibition assays show promise for monitoring microcystins in relation to the ADWG but require further development. Mouse bioassays are not suitable for determining microcystins at low concentration in water. These assays do not have sufficient sensitivity for application to water samples without impractical levels of preconcentration. Mouse bioassays are useful for initial screening of highly concentrated cyanobacterial samples (eg scums ) of unknown toxicity. The results of a quantitative assay will indicate acute toxic effects and may also indicate the class of toxin (eg neurotoxin, hepatotoxin) from reactions and pathology in test animals. This can then allow for further quantitative testing by an alternative analytical method. Appropriate and careful handling of samples both prior to and during analysis is extremely important to ensure an accurate determination of toxin concentration. Microcystin toxins are readily degraded both photochemically (ie in light) and microbiologically. Samples should be kept refrigerated and in the dark prior to analysis, and should not be exposed to strong light during the preparation and analytical procedures. x

12 AC RO N Y M S A N D A B B R E V I AT I O N S Adda ADWG ARMCANZ ATP CE ELISA GTX HPLC MALDI-TOF LC MMPB MS NeoSTX NHMRC NOM MS PDA PSPs STX TLC UV WHO 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid Australian Drinking Water Guidelines Agriculture and Resource Management Council of Australia and New Zealand Adenosine triphosphate Capillary electrophoresis Enzyme linked immunosorbent assay Gonyautoxin High performance liquid chromatography Matrix assisted laser desorption ionisation time-of-flight Liquid chromatography 3-methoxy-2-methyl-4-phenylbutryric acid Mass spectrometry Neosaxitoxin National Health and Medical Research Council Naturally occurring organic matter Mass spectrometry/mass spectrometric Photo-diode array Paralytic shellfish poisons Saxitoxin Thin layer chromatography Ultraviolet World Health Organization xi

13 1. I N T RO D U C T I O N 1. 1 T OX I C C YANOBAC T E R IA The increasing prevalence of algal blooms in freshwaters arising through increasing eutrophication is placing greater pressures on the uses of water for both drinking and recreational purposes. Blue-green algae, more correctly known as cyanobacteria, are well known for their ability to produce potent toxins which have been responsible for numerous animal deaths (Schwimmer and Schwimmer, 1968; Carmichael et al., 1985; Beasley et al., 1989; Kuiper-Goodman et al., 1999). While the unpalatable appearance of freshwater affected by heavy planktonic algal blooms has probably prevented significant human consumption with consequent fatalities, there is increasing evidence from both Australia and overseas that low-level exposure may have chronic health effects in man. Cyanobacteria have been implicated in episodes of human illnesses in Australia (Bourke et al., 1983; Falconer et al., 1983), North America (Tisdale, 1931a,b; Veldee, 1931; Dillenberg and Dehnel, 1960; Lippy and Erb, 1976; Billings, 1981), the United Kingdom (Turner et al., 1990), and Africa (Zilberg, 1966). Recently deaths of dialysis patients in Brazil from water contaminated with cyanotoxins were reported (Jochimsen et al., 1998). There is also epidemiological evidence from China of a link, in part, between cyanobacteria and cancer (Yu, 1994; Ueno et al., 1996a). Toxic cyanobacteria are cosmopolitan; they have been recorded from every continent including Antarctica (Carmichael and Falconer, 1993; Sivonen and Jones, 1999; Hitzfeld et al., 2000). Of the cyanobacterial blooms tested to date, per cent have been toxic (Codd, 1995). However not all blooms of a particular species may be toxic. In fact toxicities of blooms of the same species can vary markedly both geographically and with time (Carmichael and Gorham, 1981). While initially toxicity appeared to be restricted to planktonic cyanobacteria, benthic forms which form mats in water bodies have also been shown to be toxic (Edwards et al., 1992; Carmichael et al., 1997; Mez et al., 1997) C YANOBAC T E R IAL TOX I N S (C YANOTOX I N S ) Cyanobacteria produce several toxins, including the neurotoxins, (anatoxins and saxitoxins), hepatotoxins, cylindrospermopsin and lipopolysaccharide (LPS) endotoxins (Carmichael and Falconer, 1993; Carmichael, 1997). A summary of the toxins produced by the various species of cyanobacteria is given by Sivonen and Jones (1999) Peptide hepatotoxins (microcystins and nodularin) The hepatotoxins are cyclic peptides with the most frequently encountered compounds being the microcystins, cyclic heptapeptides produced most commonly by Microcystis aeruginosa but also by other species of Microcystis and other genera such as Planktothrix (Oscillatoria), Anabaena, Nostoc, Anabaenopsis, and Hapalosiphon (Sivonen and Jones, 1999). A similar cyclic pentapeptide, nodularin, which is equally as toxic as the most toxic microcystins (Rinehart et al., 1988) is commonly produced by Nodularia spumigena which is normally a brackish water cyanobacterium. Other similar cyclic pentapeptide toxins have also been characterised, eg, motuporin isolated from a marine sponge (de Silva et al., 1992) and [L-Har 2 ]-nodularin (Beattie et al., 2000; Saito et al., 2001). The structures of the peptide hepatotoxins are shown in Figure

14 (1) (2) (3) Figure 1.1: Structures of peptide hepatotoxins (1) General structure of microcystins, (2) microcystin-lr, (3) nodularin 2

15 Microcystins initially appeared to contain 5 invariant and 2 variant amino acids. One of the invariant amino acids is a unique b-amino acid called Adda. A 2-letter suffix (XY) is ascribed to each individual toxin to denote the 2 variant amino acids (Carmichael et al., 1988). X is commonly leucine, arginine or tyrosine, and Y, arginine, alanine and methionine. Variants of all the invariant amino acids have now been reported, eg, desmethyl amino acids and/or replacement of the 9-methoxy group of Adda by an acetyl moiety. Currently there are in excess of 60 variants of microcystin which have been characterised (Rinehart et al., 1994: Sivonen and Jones, 1999). Of these 60 compounds, microcystin-lr would appear to be the microcystin most commonly found in cyanobacteria. It is also common for more than one microcystin to be found in a particular strain of cyanobacterium (Namikoshi et al., 1992; Lawton et al., 1995). The microcystin variants may also differ in toxicity (Carmichael, 1992). The literature indicates that hepatotoxic blooms of M. aeruginosa containing microcystins occur commonly worldwide (Sivonen and Jones, 1999). The cyclic pentapeptide nodularin contains amino acids similar or identical to those found in microcystins, namely arginine, glutamic acid, b-methylaspartic acid, N-methyldehydrobutyrine and also Adda (Rinehart et al., 1988). Motuporin has arginine replaced by valine (de Silva et al., 1992) and in [L-Har 2 ]-nodularin, arginine is replaced by homoarginine (Beattie et al., 2000; Saito et al., 2001) Neurotoxins Anatoxins Toxins in this class identified to date are the neurotoxic alkaloids anatoxin-a, homoanatoxina and anatoxin-a(s) (Figure 1.2). Anatoxins have only been identified in cyanobacteria from the Northern Hemisphere; they have not yet been found in neurotoxic cyanobacteria in Australia. To date the only neurotoxic cyanobacterium encountered in Australia would appear to be Anabaena circinalis. Of all samples of A. circinalis analysed from the Murray- Darling basin, none contained anatoxin-a. Symptoms of neurotoxicity precluded the presence of anatoxin-a(s) (Baker and Humpage, 1994; Baker et al., 1993; Velzeboer et al., 2000). Saxitoxins (paralytic shellfish poisons (PSPs)) The neurotoxic saxitoxins or paralytic shellfish poisons (PSPs) are one of a number of groups of toxins produced by dinoflagellates in the marine environment (Figure 1.3). Shellfish feeding on toxic dinoflagellates can themselves become toxic and hazardous if consumed, even causing human fatalities (Kao, 1993). Poisoning incidents usually coincide with the sudden proliferation of these organisms to produce visible blooms, the so-called red tides (Hallegraeff, 1993; Anderson, 1994). Saxitoxins have now also been found to the responsible for neurotoxicity in three cyanobacterial species overseas; Aphanizomenon flos-aquae (Ikawa et al., 1985; Mahmood and Carmichael, 1986; Ferreira et al., 2001), Lyngbya wollei (Carmichael et al., 1997) and Cylindrospermopsis raciborskii (Lagos et al., 1999). Saxitoxins in Danish lakes appear to be produced by Anabaena lemmermannii (Kaas and Henriksen, 2000). Toxin profiles are complex and variable, similar to those that have now been found in dinoflagellates and contaminated shellfish. 3

16 (1) (2) (3) Figure 1.2: Structures of the anatoxins (1) anatoxin-a, (2) homoanatoxin-a, (3) anatoxin-a(s) 4

17 O R 4 H R 1 N NH NH H 2 N N NH OH OH R 2 R3 R4=CONH 2 (carbamate toxins) R1 R2 R3 Net Relative charge toxicity STX H H H +2 1 neostx OH H H GTX1 OH H OSO GTX2 H H OSO GTX3 H OSO 3 - H GTX4 OH OSO 3 - H R4 = CONHSO 3 - (n-sulfocarbamoyl (sulfamate) toxins) GTX5 (B1) H H H GTX6 (B2) OH H H +1 - C1 (epigtx8) H H OSO C2 (GTX8) H OSO 3 - H C3 OH H OSO C4 OH OSO 3 - H R4=H (decarbamoyl toxins) dcstx H H H dcneostx OH H H +2 - dcgtx1 OH H OSO dcgtx2 H H OSO dcgtx3 H OSO 3 - H dcgtx4 OH OSO 3 - H +1 - Figure 1.3: Structures of the saxitoxins (paralytic shellfish poisons (PSPs)). Toxicity data from Oshima et al. (1995b) 5

18 Saxitoxins are also the neurotoxins present in A. circinalis, the only cyanobacterium yet found to be neurotoxic, in Australia (Baker and Humpage, 1994; Humpage et al., 1994; Negri et al., 1995, 1997; Velzeboer et al., 2000). The widespread occurrence of saxitoxins, especially in Australian neurotoxic A. circinalis, makes them a very important class of cyanobacterial toxins, at least in this country. In relation to A. circinalis in Australia, toxin profiles appear to be relatively constant and dominated by the C toxins (Negri et al., 1997; Velzeboer et al., 2000). There is also some limited evidence that this cyanobacterium can produce both neurotoxins and hepatotoxins (Bowling and Baker, 1996), a phenomenon which has been reported overseas with A. flos-aquae (Al-Layl et al., 1988; Sivonen et al., 1989). The saxitoxins are a relatively complex class of 18 compounds with widely differing toxicities which can be divided into three groups as shown in Figure 1.3. They can also be divided into three groups based on the net charge of the molecule under acidic conditions (Shimizu, 1988; Hall et al., 1990) (Figure 1.3). This grouping comprises the saxitoxins (saxitoxin (STX), neosaxitoxin (neostx) and decarbamoyl derivatives) (charge +2;), the gonyautoxins (GTXs) including decarbamoyl derivatives (charge +1) and C toxins (charge 0). These properties form the basis of analytical methods involving high performance liquid chromatography (HPLC) Cylindrospermopsin In 1979 at Palm Island, Queensland, Australia there was a severe outbreak of hepatoenteritis in the population supplied with drinking water from a dam which had been treated with copper sulphate to kill a heavy bloom of algae (Byth, 1980). Subsequent research on Cylindrospermopsis raciborskii from this source showed it to produce toxicological effects in animals consistent with the symptoms observed at Palm Island. On this basis it was subsequently suggested that the 1979 outbreak was caused by toxic C. raciborskii (Hawkins et al., 1985). This species has also been responsible for cattle deaths in Queensland (Saker et al., 1999). C. raciborskii is predominantly a tropical species although monitoring of the Murray-Darling Basin has indicated that it is present in the more southern areas of the basin (Baker and Humpage, 1994; Baker et al., 1993). An hepatotoxic alkaloid toxin was isolated from C. raciborskii and named cylindrospermopsin (Figure 1.4) (Ohtani et al., 1992). It has also subsequently been isolated from the cyanobacterium Umezakia natans in Japan (Harada et al., 1994) and Aphanizomenon ovalisporum in both Australia (Shaw et al., 1999) and Israel (Banker et al., 1997). Cylindrospermopsin can be classified as an hepatotoxic alkaloid but toxicological studies have shown that, while the principal organ affected is the liver, other organs such as the kidney are also affected (Hawkins et al., 1985; Terao et al., 1994; Falconer et al., 1999). Recently a toxic minor component of a strain of Aph. ovalisporum from Israel, 7-epicylindrospermopsin, has been characterised (Banker et al., 2000) indicating that toxins other than cylindrospermopsin itself need to be considered when dealing with these cyanobacteria. Figure 1.4: Structure of cylindrospermopsin 6

19 1.2.4 Lipopolysaccharide (LPS) endotoxins The LPS endotoxins are perhaps the least understood of the toxins produced by cyanobacteria. These toxins are constituents of the outer wall of both cyanobacteria and heterotrophic gram-negative bacteria (Sykora et al., 1980; Keleti and Sykora, 1982). LPS endotoxins produced by cyanobacteria are less toxic than those produced by bacteria; however they may be responsible for illnesses such as gastroenteritis in human populations exposed to cyanobacteria (Keleti et al., 1979; Codd et al., 1989). Consequently the involvement of LPS endotoxins in episodes of human toxicity warrants further attention. 7

20 2. S C O P E O F T H I S R E V I E W 2.1 GE N E R AL The concern over the possible health effects associate with consumption of water containing cyanobacterial toxins has led to consideration of drinking water guidelines for these compounds (WHO, 1998; NHMRC/ARMCANZ, 2001). In order to determine exposures to toxins, and to monitor compliance with guidelines, it is necessary to have reliable and effective methods of analysis for these compounds. This document is a review of existing analytical methodologies for cyanotoxins. This review will to some extent expand on existing reviews of analytical methods, focussing on the terms of reference, and toxins found in Australian cyanobacteria. Reviews of toxin analysis are given by Harada et al. (1999), Harada (1996a,b; microcystins), Falconer (1993), Meriluoto (1997; microcystins, especially their chromatography) and Meriluoto et al. (1996, 2000; microcystins). Reviews on determining saxitoxins in seafood and the marine environment have some relevance to determining these compounds in Anabaena and water (Sullivan, 1993; Hallegraeff et al., 1995; Botana et al., 1997; Quilliam, 1996) T E R M S OF R E FE R E N C E The terms of reference specified for this review were to consider the status of current cyanotoxin analytical methods and critically assess: Principle of each technique; What it measures; Detection limit and precision; Current usage and reliability, ie, is the current usage a research application only and is it amenable to routine use in a commercial laboratory? Degree of documentation of the method in the literature, ie, are published standard protocols available? Level of expertise required by the operator/analyst; Economics of the method, ie, relative cost of set-up and application; and Assessment of whether the method is immediately amenable for compliance with the guideline ( for microcystins only) In terms of the various classes of toxins, individual classes will be treated independently as there is no scope for determining more than one class at a time except for some toxicitybased assays such as the mouse bioassay. The review will cover the current range of monitoring and analytical methods for microcystins, saxitoxins and cylindrospermopsin, and will focus on methods applied to, or with the potential for application to, water samples. It will not cover procedures applicable for the elucidation of toxin structures, unless they are applicable to water samples on a routine basis. The review will not include the neurotoxic anatoxins as this class of toxins has yet to be found in Australian cyanobacteria. Methods will encompass instrumental analytical procedures, bioassays, enzyme linked immunosorbent assays (ELISAs) and activity assays, in particular protein phosphatase inhibition assays for microcystins. Analyses must take into account the stability of toxins in samples once sampled, ie, their potential degradation by chemical, photochemical or microbiological processes, and possible interconversion. Lysing of cells will affect distribution of intracellular and extracellular toxins, but this is not important if total toxin concentration only is required. 9

21 3. TOX I N A N A LY S E S G E N E R A L C O N S I D E R AT I O N S 3. 1 T OX I N EX T R AC T I ON Toxins in water bodies at the time of a bloom will be present in both the water (free, dissolved or extracellular toxins) and the cyanobacterial cells (intracellular toxins). If this water is consumed without any treatment, especially treatment that will remove cellular material intact and therefore remove intracellular toxin, exposure will be to the total toxin content. Analytical methods must therefore be able to determine both the intracellular and extracellular toxins. The extraction step of the analytical determination is generally independent of the detection component of the method and can therefore be considered independently. However procedures for optimum extraction of toxins can be compound specific. Current analytical procedures only determine toxins in the dissolved or free state. Therefore the basis of any analytical procedure where toxins are contained in cellular material must include a step to extract or release intracellular toxin. This can be carried out in such a way that the total toxin content is measured, or the cellular material containing toxins can be separated for a separate determination of intracellular and extracellular toxin. In the absence of cellular material, eg, in the case of filtered water, this step is unnecessary, and the extracellular toxin is the total toxin. This extraction of intracellular toxins is absolutely necessary as in healthy blooms containing peptide hepatotoxins at least, the major proportion of the toxin present is contained within cells (see section on these compounds). It would appear that only when a bloom collapses, or is treated with an algicide such as copper sulphate, and cells die, that toxins are released to the surrounding water in significant concentrations. In contrast, healthy blooms of C. raciborskii may have a significant proportion of the toxin cylindrospermopsin in the water phase (Chiswell et al., 1999). Extraction of intracellular toxins can be regarded as a two-stage process. Firstly the cell wall must be ruptured or lysed to provide access of the extracting solvent to the toxin in the cell. Secondly the toxin within the cell is dissolved in the solvent. Generally these two processes are studied as one. It is not possible to determine absolute recoveries from intact cells as it is not possible to spike the cells themselves. The procedure which gives maximum recovery with acceptable precision is considered to be the optimum. Cell lysis can be achieved by freeze-drying, often followed by sonication in solution, or by repeated freeze-thawing and/or sonication of whole cell material. Extraction is effected with water, organic solvents such as methanol or dilute acid, depending on the toxins C O N C EN T R AT I ON / C LE ANUP PRO C E DU R E S At low concentrations, direct determination of toxins may not be feasible due to the inadequate sensitivity of the detection procedure. This necessitates a sample preconcentration step to increase the concentration of toxin in solution. This may also serve as a cleanup step by allowing removal of co-extracted material which may interfere in the analysis. These procedures are compound specific, and may also only have relevance to one detection protocol. For example, the separation and detection/quantification of toxins using high performance liquid chromatography (HPLC) requires separation of toxins using liquid chromatography and detection/quantification of the separated toxins using various techniques. Concentration of toxins can be carried out using solid-phase extraction cartridges. The sample is passed through a cartridge which retains the toxins by adsorption. Toxins are eluted from these cartridges using solvents such as methanol. These extracts may be reduced in volume by 11

22 evaporation, thereby resulting in toxins being concentrated relative to the original sample. These cartridges also extract the naturally occurring organic matter (NOM) present in water, and following elution and concentration of toxins from the cartridge, the NOM is also isolated and concentrated. In some analyses, co-extracted NOM can interfere in the chromatographic separation by producing peaks which may obscure the toxin peaks. This masking of toxin peaks can therefore make quantification imprecise. In the case of microcystins, procedures involving selective elution of toxins versus co-extracted interferences such as NOM have been developed to minimise this problem (Tsuji et al., 1994b). Thus solid phase extraction can be used not only to concentrate toxins present in a sample but also to effect a cleanup by selectively eliminating interferences. The details of cleanup/concentration procedures are discussed under the individual toxin classes S AMPLE P R ES E RVAT I ON The stability of toxins in water samples has received little attention, but is critical to the accurate determination of toxin concentrations. Lysing of intact cells and release of toxins during storage can alter the proportion of intracellular and extracellular toxins. In the absence of other processes, this will not affect the total toxin concentration. In terms of water quality guidelines where the total toxin concentration is the issue, such a situation will not be important. However, the degradation of dissolved toxins during storage can lead to an underestimate of total toxin in the sample. Another issue with saxitoxins is their possible interconversion, a phenomenon which can significantly affect the concentration in terms of toxicity equivalents. The stability of toxins once extracted from samples, ie, in sample extracts, must also be considered if the analysis is to accurately determine the concentration in the original water sample GE N E R AL TOX I C I TY- B ASED ASSAYS The traditional approach to determine the toxicity of cyanobacterial samples has been the mouse bioassay, where toxin extracts are administered by intraperitoneal injections (Falconer, 1993). This has been used primarily to determine the toxicity of bloom material, and, from the toxic response, the identity of the class of toxin can be inferred. It has generally been used in a qualitative manner to determine a bloom as toxic or non-toxic. However, as dose determines the toxic effect, a cut-off point in terms of toxicity should be defined. Everything is toxic if the dose is sufficient, even the salts that may be present in a sample highly concentrated by evaporation. In terms of symptoms, the peptide hepatotoxins (microcystins and nodularin) generally cause death within 4 hours with symptoms of a liver engorged with blood. The neurotoxins can cause death much more quickly (within 15 minutes) with neuromuscular symptoms and typically with no evidence of tissue damage on post-mortem (Falconer, 1993). This assay, although it can potentially be calibrated against a specific toxin such as microcystin-lr, and therefore produce a result in terms of microcystin-lr toxicity equivalents, does not have the sensitivity or precision to be applicable to water samples. It is not practicable to use for water samples with concentrations around 1 2 µg/l, the approximate range of the guideline for microcystins, as a significant concentration factor is required. For microcystin-lr with an LD 50 of 50 µg/kg, the lethal dose is around 1 µg for a 20 gm mouse which requires a 1 L sample to be concentrated to 1 ml for intraperitoneal injection to produce a lethal response. It is also impracticable and unacceptable from the point of view of number of mice required to obtain a quantitative result when a number of samples require analysis. The ethics of animal toxicity testing, together with the practical issues associated with the mouse bioassay has led to toxicity assays with other organisms. These assays may be toxin specific, and are discussed under the specific toxins. Hiripi et al. (1998) reported an assay using the African locust. All classes of toxins elicited a toxic response. As it was not possible to discriminate between the classes of toxins, this assay could not then be used as a monitoring tool for specific toxins. In contrast, another assay using the desert locust responded only to saxitoxins (McElhiney et al., 1998). 12

23 4. D E T E R M I N AT I O N O F M I C R O C YS T I N S A N D N O D U L A R I N 4. 1 GE N E R AL A significant difference between the provisional WHO drinking water guideline for microcystin-lr (WHO, 1998) and the NHMRC/ARMCANZ guideline is that the latter considers advice in relation to the concentration of total microcystins whereas WHO restricts its advice to microcystin-lr alone. Microcystin-LR appears to be the most widespread and bestcharacterised variant of this class of toxins, of which over 60 variants have been characterised (Rinehart et al., 1994; Sivonen and Jones, 1999). NHMRC/ARMCANZ also stipulates that the Australian guideline for total microcystins be expressed as toxicity equivalents of microcystin-lr. The rationale for this approach is that populations of Microcystis that are toxic usually contain a range of microcystins in varying amounts (Namikoshi et al., 1992; Fastner et al., 1999). These microcystins may vary in toxicity (Rinehart et al., 1994; Sivonen and Jones, 1999), and it is the cumulative toxicity, not the mass amount of toxins, which represents the potential hazard to human health from ingestion via drinking water. This requirement for determining toxin content in terms of toxicity equivalents of microcystin-lr may substantially limit the application of some existing analytical procedures E X T R AC T I ON P RO C E DU R E S As already discussed, the effective extraction of intracellular toxins is necessary as in healthy blooms containing peptide hepatotoxins, the major proportion of the toxin present is contained within cells (Park et al., 1998a,b; Jones and Orr, 1994; Tsuji et al., 1996; Lindholm and Meriluoto, 1991; Heresztyn and Nicholson, 1997; Lam et al., 1995b, Ueno et al., 1996b). It is only when a bloom collapses, or is treated with an algicide such as copper sulphate, and cells die that toxins are released to the surrounding water in significant concentrations (Jones and Orr, 1994; Kenefick et al., 1993; Lam et al., 1995b). There is also some evidence that small amounts of some toxins released to the surrounding water during the relatively healthy stages of a bloom are rapidly degraded microbiologically as organisms capable of degrading toxins become established during the bloom (Heresztyn and Nicholson, 1997). Some lysis of cyanobacterial cells occurs during the freeze-drying process employed when isolating cyanobacterial material for investigation, and this assists in subsequent extraction into solution. Freeze-thawing also achieves this end, and has been utilised in a number of analytical procedures for the peptide hepatotoxins (Lawton et al., 1994), as has methanol extraction of cells dried at 55 C (Coyle and Lawton, 1996) and sonication (Rositano and Nicholson, 1994; Meriluoto and Eriksson, 1988). Extraction of microcystins from lysed cells has been carried out using a number of solvent systems. Acetic acid/water mixtures (Harada et al., 1988b) or alcohol/water mixtures (Siegelman et al., 1984; Krishnamurthy et al., 1986; Brooks and Codd, 1986; Gjolme and Utkilen, 1994; Meriluoto and Eriksson, 1988) were initially used as extractants. Water, in conjunction with sonication has also been shown to be applicable to live cells (Rositano and Nicholson, 1994; van Hoof et al., 1994). More recent studies have investigated these extraction procedures, especially those using methanol/water mixtures, in some detail. With the more hydrophobic microcystins, it was found that methanol was slightly more effective than methanol/water mixtures, and also effective with the more hydrophilic toxins (Lawton et al., 1994). However methanol has been reported to be less effective than 5 per cent acetic acid or 75 per cent methanol for the extraction of the hydrophilic microcystin-rr (Gjolme and Utkilen, 1996; Fastner et al., 1998; Wirsing et al., 1998). Ward et al. (1997) also found that decreasing the methanol content decreased the recovery of the more hydrophobic 13