Cyanobacterial Toxins of Drinking Water Supplies: Cylindrospermopsins and Microcystins - Chapter 10 pps

Thus, foraccurate measurement of a cyanobacterial toxin in a drinking water supply, it isimportant to measure total toxin in the water — that is, toxin in cells plus free toxin in the wa

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Cylindrospermopsins and Microcystins

To assure public safety of drinking water supplies, harmful organisms and toxiccontaminants must be reduced to harmless levels In order to be able to provide thisassurance when toxic cyanobacterial water blooms occur on supply reservoirs, ana-lytical techniques are required of sufficient sensitivity to characterize any hazard.The early approach to assessing potential hazard from a cyanobacterial bloomwas the toxicity testing of scum or concentrate samples by injection into mice(Falconer 1993) This method has limitations through lack of sensitivity and speci-ficity and ethical difficulties due to subjecting animals to potentially painful treat-ment In the last decade, a series of alternative approaches have been developed,including microbiotests using invertebrates, enzyme inhibition assays, enzyme-linked immunosorbent assays (ELISAs), and a range of chemical analytical tech-niques This chapter considers these methods and also evaluates the remaining rolefor in vivo mouse assays

Cyanobacterial toxins are synthesized within the cells of the organisms andlargely remain within the cells during growth However, when the cells senesce orare killed, there may be high concentrations of toxin that are free in the water.Blooms senesce and lyse (die) naturally, so that a water body with a Microcystis or

Planktothrix bloom that is forming a decaying scum will have appreciable quantities

of free toxin in the water Copper treatment of reservoirs kills the cyanobacterialcells and releases toxins into the water During water treatment, the early addition

of chlorine will lyse the cells, similarly releasing toxin into the water Thus, foraccurate measurement of a cyanobacterial toxin in a drinking water supply, it isimportant to measure total toxin in the water — that is, toxin in cells plus free toxin

in the water — for a reliable assessment of potential hazard

The majority of bioassay and analytical techniques are insufficiently sensitive

to directly measure the low concentrations of microcystins, nodularins, and drospermopsins that occur in the bulk water in reservoirs To provide sufficientconcentration of toxins, several methods have been developed that will selectivelyconcentrate toxin for analysis These are described in this chapter

cylin-It has recently become possible to measure the total toxins in water without aconcentration step, and these methods are currently being validated As the toxinlevels are frequently in the submicrogram-per-liter range, great sensitivity is required.With the World Health Organization’s (WHO) determination of a provisional

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186 Cyanobacterial Toxins of Drinking Water Supplies

Guideline Value of 1 µg/L for microcystin-LR in drinking water and a similarconcentration recommended for cylindrospermopsin, analytical techniques for tapwater must be accurate down to concentrations of 0.1 µg/L (approximately 0.1 nMfor microcystins and 0.2 nM for cylindrospermopsin) Immunoassays and analyticaltechniques of suitable sensitivity are becoming available and others are under devel-opment Several based on ELISAs for microcystins and nodularin are availablecommercially as kits

10.1 TOXIN CONCENTRATION

There are two quite different approaches to concentrating cyanobacterial toxinspresent in a water bloom in a reservoir so that an effective analysis can be undertaken.Which approach is selected depends on the genus of toxic cyanobacterium and thegrowth phase of the water bloom The microcystin- and nodularin-containing genera,such as Microcystis, Planktothrix, and Nodularia, retain the toxin within the cells

in growing, healthy colonies and filaments (Welker, Steinberg et al 2001) Thesimplest method of concentrating toxin for analysis in water blooms of these organ-isms is to concentrate the cells, as minimal amounts of free toxin will be present inthe water This approach is discussed later in Section 10.7

The other approach applies when a water bloom is naturally lysing, has beendosed with copper sulfate, or is of a genus that “leaks” toxin into the free water

Cylindrospermopsis is such a genus, with a considerable quantity of toxin in thewater even in healthy, growing cultures and natural blooms (Hawkins, Putt et al.2001) In these cases a deceptive underestimate of total toxin in the water will resultfrom measuring cell toxicity only

To ensure that all the toxin content of a sample of water containing cyanobacteria

is available for concentration and assay, it is essential to lyse the intact cyanobacterialcells Freeze-thawing of samples is effective for some cyanobacterial species, but

Microcystis is particularly difficult to disrupt Repeated cycles of sonication andfreeze-thawing may be required Freeze-drying followed by resuspension and son-ication are also effective The cell debris can be removed by filtration or centrifu-gation (Falconer 1993; Lawton, Beattie et al 1994)

Concentration of toxin from lysed cyanobacteria can be undertaken by twomethods One is to freeze-dry, or evaporate off, the bulk of the water prior to assay.The resulting solution may require pH adjustment The other (preferable) approachrelies on adsorption of toxins onto a solid phase in an appropriate cartridge As thetechnique differs between toxins, it is dealt with separately in Sections 10.3 and 10.7

10.2 IN VIVO RODENT TOXICITY ASSAYS

The basis of toxicology is the adverse effect of the toxic compound on mammals,which provides evidence for potential toxicity to people The identification of toxicity

in cyanobacteria followed poisonings of domestic animals and of people and wasinitially investigated in rodents and domestic animals Concentrated scum sampleswere the preferred material for toxicity investigation, as sufficient concentration of

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Detection and Analysis of Cylindrospermopsins and Microcystins 187

toxin for observable pathological changes was required Direct measurement of lowconcentrations of toxins in water was not feasible with the in vivo rodent assays, astheir low sensitivity required about 3 µg of toxin per mouse for lethality

To provide effective concentrations of toxin for rodent assays, most samplesrequire cell or toxin concentration; for fresh bloom or cell-culture samples, this hasbeen done by cell concentration followed by in vivo toxicity measurement Thismethod has provided the basic data for the investigations of the toxic species, themechanisms of toxicity, and the development of more sensitive assays

The use of whole-animal assays is currently opposed on ethical grounds and isbeing replaced by the variety of microbiotests and in vitro test systems available.Only in the case of investigation of a possible public health risk from a newlydiscovered toxic cyanobacterial species or verification of the cause of livestockpoisoning by an uncharacterized cyanobacterial bloom is it essential to use liveanimals When a cyanobacterial species that has not been investigated for toxicityappears in a drinking water supply, especially in the posttreatment distributionsystem, it is still necessary to undertake mouse toxicity tests In a recent case of thisproblem, Phormidium, a normally benthic cyanobacterium, detached from the sed-iment of a distribution reservoir and entered the public water supply Immediatetoxicity testing using mice identified the organism as poisonous, and the users ofthe supply were alerted and provided with alternative drinking water (Baker et al.2001) In this case the toxin has not been identified and is not any of the presentlydescribed toxins For investigation of the toxicity of cyanobacterial blooms of generaknown to produce particular toxins, microbiotests or in vitro tests are appropriateand sufficiently rapid

10.2.1 M ETHODS FOR M OUSE T ESTS — I NTACT C ELLS

Toxicity testing, using rodents, of an uncharacterized cyanobacterial bloom posing

a health risk can be undertaken by the following procedure It is first necessary tocollect a sample of as dense bloom material as possible in order to obtain sufficienttoxin This can be done by collecting from a scum concentration along the edge ofthe water or by use of a plankton net The cells can be processed to remove water

by filtration at the lakeside or transported to the laboratory with minimal heating orshaking This material can be concentrated by allowing the sample to stand overnightand collecting the buoyant cells, or by centrifuging a sample at sufficient speed tocollapse the gas vacuoles and sediment the cells, or by filtering the sample through

a paper, glass-fiber, or membrane filter The cell paste can then be used directly,stored refrigerated, or freeze- or air-dried for more extended storage

For in vivo toxicity testing, resuspension of the cells is done in physiological orphosphate-buffered saline (pH 7.5, 0.05 M), at 200-mg dry weight in 10 mL ofsaline Concentrated suspensions of fresh cyanobacterial cells can also be useddirectly, without drying, using about 1 g of cell paste in 10 mL of saline In thiscase a dry-weight determination is required for the suspension Cell rupture of fresh

or dried cells is essential for the rodent assay and can be performed by sonicationand freeze-thawing of the suspension (Falconer 1993; Lawton, Beattie et al 1994)

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The assay is carried out by intraperitoneal injection into test animals Thisrequires bacteriologically sterile solution to avoid infection The suspension can befiltered through a bacterial filter prior to injection or, if the toxicity is anticipated to

be due to peptide or other heat-stable toxins, the suspension can be held in a boilingwater bath for 10 min and then filtered through a sterile filter for injection On thebasis of the dry weight of the solids in the suspension, doses from 50 to 500 mg/kgbody weight can be used to determine lethal dose, with doses of 0.1 to 1.0 mLinjected To minimize the number of animals used, four dose rates (say 50, 100,

250, and 500 mg/kg) administered to pairs of animals will provide basic information

If no pathological changes are seen at the highest dose, it is unlikely that the bloom

is of significance as a health risk Any animals showing distress should be euthanizedimmediately, and all animals should be euthanized at 24 h after dosing Clinicalobservation should include respiratory rate, motor activity, piloerection, salivationand lacrymation, and blanching of extremities

Postmortem examination of internal organs is informative, as hepatotoxins, such

as microcystins and nodularins, and cytotoxins, such as cylindrospermopsins, willcause changes in liver weight and appearance and show hepatocyte damage onhistopathological examination Microcystins and nodularins at acutely toxic doseswill cause swelling and darkening of the liver without substantial damage to otherorgans (Falconer, Jackson et al 1981)

Cylindrospermopsins will cause damage to several organs, including lymphocytenecrosis in the spleen and damage to the proximal tubule in the kidney, which can

be seen on histopathological examination (Falconer, Hardy et al 1999; Seawright,Nolan et al 1999) Neurotoxins cause neurological symptoms and death in theabsence of visually observable postmortem changes, and hence can be differentiatedfrom other toxins on postmortem examination (Falconer 1993)

An assessment of relative toxicity, given by Harada, Kondo et al (1999) as LD50

in milligrams of dry cyanobacterial cells per kilogram of mouse body weight (thedose at which 50% of the mice are killed within 24 h by toxin), is as follows:

Greater than 1000 mg/kg body weight: Nontoxic

500 to 1000 mg/kg: Low toxicity

100 to 500 mg/kg: Medium toxicity

Less than 100 mg/kg: High toxicity

The same scale of toxicity can be applied to the minimal lethal dose (the lowestdose at which death occurred), which can be expected to be below but close to the

LD50

10.2.2 S ENESCENT OR L YSED S AMPLES

The method described above assesses in vivo toxicity of cell-bound toxins and isapplicable only to healthy bloom material collected and handled without cell lysis

If cell lysis has occurred in the bloom or after collection, then total lysis of thebloom sample should be undertaken by freeze-thawing and sonication The extent

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of lysis in collected bloom material depends substantially on the genus of bacterium as well as handling conditions Microcystis is very resistant to lysis,whereas the filamentous cyanobacteria are much more sensitive, especially Ana- baena. After lysis, the suspension should be centrifuged and filtered to obtain aparticle-free solution of toxins Toxin concentration from lysed samples can beundertaken as described later for the individual types of toxin

cyano-For injection into mice, the toxin extract is redissolved in physiological saline

or phosphate-buffered saline for intraperitoneal injection Dose rate can be imated by assuming a lethal dose of (say) 100 µg/kg and administering doses of 50,

approx-100, 250, and 500 µg/kg Clinical observation, euthanasia, and postmortem nation follow the procedures described above unless cylindrospermopsin is sus-pected In this case, if no mortality is seen at 24 h, it will be necessary to extendthe time of observation to 7 days, as this toxin is slow acting All animals shouldthen be euthanized at 7 days for postmortem examination and histopathology

exami-10.2.3 E THICS P ERMISSION

In most countries permission is required from an ethics committee within the tution prior to any toxicity testing in mammals Some jurisdictions require theexperimenter to also have a personal license, which certifies the individual as beingcompetent to carry out the tests specified by the license In general, standard LD50determination is not permitted unless rigorous evaluation of a potential pharmaceu-tical product or pesticide is being undertaken The aim of this restriction is tominimize the number of animals used and reduce the potential suffering of lethaltoxicity testing For assessment of toxicity of water or scum samples, an approximateminimal lethal doseis a satisfactory substitute for an LD50 determination This can

insti-be carried out with four doses of cyanobacterial extract administered over a 10-foldconcentration range to pairs of Swiss albino mice weighing 25 to 30 g

10.3 CYLINDROSPERMOPSIN BIOASSAY AND

ANALYSIS

A Cylindrospermopsis bloom was responsible for the human poisoning at PalmIsland, Australia, described in Chapter 5 The cyanobacterium was isolated and itstoxicity investigated, initially using mice (Hawkins, Runnegar et al 1985) Later,after isolation and identification of the toxin cylindrospermopsin (Ohtani, Moore

et al 1992), it became possible to apply cell-based, biochemical, and chemicalassays It was also possible to calibrate microbiotests for cylindrospermopsin tox-icity, providing a less technological method for assay suitable for laboratories with-out sophisticated chemical analytical equipment

C raciborskii appears to release toxin into the water during growth, unlike

Microcystis, which retains toxin in the cells until cell death (Chiswell, Shaw et al.1999) Therefore measurement of only the cell content of toxin may substantiallyunderestimate the overall toxin content of the water Hence both the toxin in thecells and in the free water phase require measurement The most direct method ofassuring that total toxin is measured is to lyse/rupture the cells in the water sample

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by freeze-thawing and/or sonication of the bulk sample Freeze-drying the bulksample is also effective, with subsequent extraction of toxin in 5% concentration ofacetic acid in water (Hawkins, Chandrasena et al 1997) Dissolving cylindrosperm-opsin in deionized water is also possible, but methanol extraction of the dried samplemay lose cylindrospermopsin while bringing into solution a range of other potentiallybioactive compounds (Hiripi, Nagy et al 1998)

If a water bloom of cyanobacteria is likely to contain cylindrospermopsins orother alkaloid cyanobacterial toxins, solid-phase adsorption cartridges can be usedfor toxin concentration Polygraphite cartridges — for example, Carbograph ExtractClean — are conditioned before use by washing with methanol (5 mL) followed byhigh-purity water (5 mL) After the water (pH 6 to 8) containing filtered cyanobac-terial lysate is run through the column, elution by 5% formic acid in methanol, 2 × 5

mL, will recover alkaloid toxins (Norris, Eaglesham et al 2001) The concentratedsample that is eluted from the cartridge can be dried with nitrogen and weighedprior to assay

Cylindrospermopsin adsorbs strongly to polyethylene, so only glass containersshould be used for its extraction and analysis It is stable in the dark between pH 4and 10 at room temperatures but breaks down in sunlight in the presence of celldebris It is stable to boiling at neutral pH for 15 min (Chiswell, Shaw et al 1999)

10.3.1 B IOASSAYS FOR C YLINDROSPERMOPSIN

In vivo rodent assay, which has been extensively undertaken for cylindrospermopsin,describes the features of toxicity (Hawkins, Runnegar et al 1985; Hawkins, Chan-drasena et al 1997; Falconer, Hardy et al 1999; Seawright, Nolan et al 1999) Themouse assay technique used is described earlier in this chapter and is not nowgenerally required for cylindrospermopsin analysis, as a range of alternatives areavailable

During recent years, the use of insects and zooplankton as assay organisms forenvironmental toxins has become more common, and a number of standardized kitsare available for this purpose (Persoone, Janssen et al 2000) These test systems usedehydrated cysts, eggs, or the equivalent as sources of the test organisms and astandardized protocol for measuring toxicity Evaluation of these organisms formonitoring cyanobacterial toxins, in place of mouse tests, has shown promisingresults (Tarczynska, Nalecz-Jawecki et al 2001) In a similar manner to the toxicitytests using rodents, these organisms respond to neurotoxins, hepatotoxins, and alka-loid toxins, with dose–response curves of varying sensitivity They thus provide ageneral test for toxicity even when the nature of the toxin is unknown A range ofprotozoa, crustacea, insecta, and eukaryotic algae have been explored for sensitivity

in toxicity tests (Persoone, Janssen et al 2000)

Larger insects have also been tried as assay systems for cyanobacterial toxins,particularly neurotoxins, and the African locust has also been shown to be sensitive

to toxicity from C raciborskii and Microcystis aeruginosa (Hiripi, Nagy et al.1998)

The most promising organism that provides sufficient sensitivity to cyanobacterialtoxins for test use is the small freshwater crustacean Thamnocephalus platyurus This

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organism has been demonstrated to be effective for the assay of cylindrospermopsincarried out on samples cultured from a toxic water bloom of C raciborskii in LakeBalaton, Hungary (Torokne 1997) The lethal concentration to 50% of the testorganism (LC50) for T platyurus was 0.61 mg of freeze-dried cells per milliliter ofassay solution, and the LD50 of the same samplefor mice was 550 mg/kg

On the basis of published data for C raciborskii containing cylindrospermopsin,the mouse toxicity of the Lake Balaton material is equivalent to a toxin content ofapproximately 0.5 mg cylindrospermopsin per gram of dried cells (Hawkins, Chan-drasena et al 1997) Therefore the T platyurus assay provided an LC50 (calculated

as pure toxin) of about 0.3 µg cylindrospermopsin per milliliter of solution Thissensitivity is adequate for testing the toxicity of freeze-dried scums and cell con-centrates or concentrated extracts of bulk water samples eluted from solid-phaseadsorption cartridges The LC50 is 300 times higher than the concentration of theproposed Guideline Value for cylindrospermopsin in drinking water of 1 µg/L(Humpage and Falconer 2003); hence this microbiotest is not suitable for the directanalysis of the toxin in water supplies

Another organism, more widely used in toxicity tests, is the brine shrimp Artemia salina This can be readily obtained in the form of eggs, which are then hatched foruse The quoted LC50 for purified cylindrospermopsin was 8.1 µg/mL at 24 h and0.71 µg/mL at 72 h (Metcalf, Lindsay et al 2002) This increased sensitivity withextended time of observation is similar to the reduction in experimental LD50 inmice, when the time is extended from 24 h to 7 days (see Chapter 6) It probablyrelates to two independent toxic mechanisms, the earlier lethal response with lowersensitivity followed by a later, more sensitive response through inhibition of proteinsynthesis

The use of these microbiotests in place of the mouse bioassay has led to costsavings while also avoiding mammalian testing These tests have similar technicaladvantages and disadvantages when compared to mouse tests, as they provide ageneral toxicity screen with some indication of toxin type Their sensitivity issatisfactory for concentrated material but insufficient for the direct monitoring oftoxin content in bulk water Cylindrospermopsin has, however, been successfullyconcentrated from lake water by the use of C-18 and polygraphite cartridges inseries, with 100% recovery (Metcalf, Beattie et al 2002) The method for use ofCarbograph solid-phase cartridges for the concentration of cylindrospermopsin fromdilute solution in water is described earlier in this chapter

Cylindrospermopsin is also toxic to plants, and inhibition of the growth ofetiolated seedlings of Sinapis alba (mustard) has been used as an assay (Vasas,Gaspar et al 2002)

10.4 CELL-BASED AND CELL-FREE TOXICITY

MEASUREMENT OF CYLINDROSPERMOPSIN

Cylindrospermopsin is toxic to a wide variety of cells, though the sensitivity ofdifferent cell types to toxin is likely to vary widely through differences in xenobioticmetabolizing capability of the cells Damage to a human lymphocyte cell line has

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been shown, at a concentration of 1 µg/mL (approximately 2 µM) (Humpage, Fenech

et al 2000) Primary mouse hepatocytes appear to be more sensitive, with toxicityand inhibition of protein synthesis shown at 0.25 µg/mL (approximately 0.5 µM)(Froscio, Humpage et al 2003) These concentrations, which are toxic to isolatedcells in culture, are comparable to those shown to be toxic in the microbiotests withcrustaceans The cell-culture systems do not present any substantial advantages forassay of cylindrospermopsin, due to cost, the time involved, and the low sensitivity.The cell-free protein synthesis inhibition assay, which uses a rabbit reticulocytelysate as a source of protein-synthesizing capacity, is appreciably more sensitive,with a detection limit of 50 nM (0.025 µg/mL) in the assay solution (Froscio,Humpage et al 2001) This assay provided accurate quantitation of cylindrosperm-opsin concentrations in water of 0.2 to 1.2 µg/mL (0.5 to 3.0 µM) Water sampleconcentration or concentration of toxin by solid-phase adsorption is required prior

to use of this assay for field samples

Both methods of preparation require the covalent linkage of the toxin to a carrierprotein, and bovine serum albumin, ovalbumin, and polylysine have been used Forantiserum production, the conjugated toxin is then administered intradermally torabbits or other animals, together with an adjuvant to stimulate antibody formation.Repeat injections are given to boost antibody titer For the production of polyvalentantibodies, rabbits or larger animals are used and blood samples collected for theseparation of antibodies from serum (Chu, Huang et al 1989)

For monoclonal antibody formation, mice are used, and repeat injections of theconjugated toxin are administered intraperitoneally Spleen cells are collected andhybridized in vitro with a transformed cell line to form a series of clones of hybridcells Screening is done in culture plates using the original toxin coated onto theplate to identify cell clones secreting antibodies A color-producing enzyme linked

to an antimouse immunoglobulin is used to visualize the antibody-secreting clones.Selected clones are then multiplied in culture or injected intraperitoneally into mice

to generate ascites fluid, and the antitoxin antibodies are purified (Kfir, Johannsen

et al 1986a,b)

The monoclonal or polyvalent (polyclonal) antitoxin antibodies are then used in

a competitive binding assay, which can be carried out in multiwell plates Two assaymethods can be used The direct competition assay employs competition between

an unknown toxin concentration and toxin linked to an enzyme These compete foravailable binding sites on the antibody, which is coated onto the plate surface Thecolor development arises from the enzyme linked to the toxin, which is bound on

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the plate As the test toxin concentration rises, less toxin linked to enzyme is bound

to the plate and the color development decreases (Figure 10.1) (Chu, Huang et al

1990)

The alternative approach is an indirect competition assay in which the plate is

coated with toxin linked to a protein and there is competition between toxin in

solution and toxin on the plate for binding sites on antitoxin antibody in solution

The result is visualized by use of an anti-immunoglobulin linked to an enzyme,

which will quantitatively bind to the antitoxin antibodies adhering to the toxin on

the plate (Chu, Huang et al 1989) As before, increased toxin in the test material

will decrease color development The β-galactosidase and horseradish peroxidase

enzymes are often used with suitable substrates that provide the color reaction

quantitating the enzyme bound to the plate

At the time of writing, several laboratories are developing enzyme-linked

immu-noassays for detecting cylindrospermopsin in water supplies These immuimmu-noassays

have potential for laboratory-based quantitative assays using multiwell plates or to

be developed into a “dipstick” that can be used on site at a lake for an approximate

measure of toxin presence Adequate sensitivity for direct estimation of

concentra-tions in the range of 0.2 to 20 µg/L in bulk water will be needed in these assays for

use by water supply agencies

10.6 INSTRUMENT-BASED TECHNIQUES FOR

CYLINDROSPERMOPSIN 10.6.1 H IGH -P ERFORMANCE L IQUID C HROMATOGRAPHY (HPLC)

Pure cylindrospermopsin has a UV absorbance maximum at 262 nm, which can be

observed in a photodiode array detector When coupled to an HPLC system fitted

with an ODS column (Cosmosil 5C18-AR), a mobile phase of 5% methanol will

provide separation from other cyanobacterial cell constituents (Harada, Ohtani et al

FIGURE 10.1 Standard curve for the direct competitive immunoassay of microcystin-LR

using a polyclonal antibody coated onto a plate Color development by horseradish peroxidase

coupled to microcystin-LR (From Chu, Huang et al 1990 With permission.)

Log toxin conc (ng/mL)

0 20 40 60 80 100

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1994) A modification of this was used by Hawkins, Chandrasena et al (1997),

employing a linear gradient of 0 to 5% methanol followed by isocratic 5% methanol

with a Spherisorb ODS-2 packed column to separate cylindrospermopsin from

extracts of toxic C raciborskii

Use of this approach with environmental samples has also shown advantages in

gradient elution from the column A more extensive gradient from 0 to 50% methanol

containing 0.05% trifluoracetic acid was used with a sensitivity of detection from 1

to 300 ng cylindrospermopsin on the column Some interference from peaks eluting

close to cylindrospermopsin was observed (Welker, Fastner et al 2002) Caution is

required if high concentrations of organic solvents are used, as above 50% methanol

or 30% acetonitrile a marked decrease in measured toxin content was observed,

which may be due to self-association of the cylindrospermopsin molecule (Metcalf,

Beattie et al 2002) The majority of environmental samples will require

preconcen-tration before application to HPLC

Capillary electrophoresis has been used to analyze cylindrospermopsin from

Aphanizomenon ovalisporum (Vasas, Gaspar et al 2002), a technique that may

have future potential

The definitive analytical technique at present is HPLC followed by tandem

mass spectrometry (MS/MS) The initial HPLC separation used a C-18 column at

40ºC, with a gradient of 1 to 60% methanol buffered with 5 mM ammonium acetate

for 6 min, followed by 60% methanol for 1 min The injection volume was 110

µL Effluent splitting supplied 20% of the flow to the MS/MS interface The

original M+H ion at 416 m/z was fragmented to 194 m/z, which was measured

for quantitation

The determination was linear between 1 and 600 µg/L cylindrospermopsin in

water, showing great sensitivity (Eaglesham, Norris et al 1999) The accuracy of

the assay at a concentration of 5.2 µg cylindrospermopsin per liter was 93.5% This

method is very costly for the equipment and requires highly skilled operators It is

likely to continue as the reference technique, as it has very high sensitivity, specificity

and accuracy Less costly methods will be required for routine water analysis

10.7 MICROCYSTINS AND NODULARINS: BIOASSAY

AND ANALYSIS

Microcystis blooms have caused worldwide deaths of livestock, and there are early

reports of “water bloom” as a cause of poisoning of domestic animals in the U.S

(Fitch, Bishop et al 1934); these were followed by studies of the pathology of

toxicity in rats (Ashworth and Mason 1946) Many whole-animal studies followed,

using laboratory and domestic animals (see Carmichael and Falconer 1993) Studies

of the effects of microcystin on isolated hepatocytes (Runnegar, Falconer et al 1981)

preceded the final structural characterization of the toxins (Botes, Tuinman et al

1984) Since that time a range of assays of varying approach have been developed

for microcystins, the most widely used being ELISA, protein phosphatase inhibition

assay (PPI), HPLC, and assays based on mass spectroscopy (MS)

As a consequence of the WHO’s determination of a provisional Guideline

Value for microcystin-LR in drinking water of 1.0 µg/L, there has been worldwide

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activity in refining and validating methods of analysis Sensitivity and precision

are improving, and novel approaches for online flow techniques for microcystin

measurement are under development

Nodularia first came to attention as a consequence of a large-scale poisoning of

domestic livestock (Francis 1978) and later poisonings of dogs (Lundberg, Edler

et al 1983) Its toxicity was examined in mice (Runnegar, Jackson et al 1988) and

the toxin nodularin was structurally identified (Rinehart, Harada et al 1988) The

similarity of the toxic action and chemical structure of nodularin to that of

micro-cystin has allowed modification of the assays for micromicro-cystin to be applied to

nodularin assay Nodularin has occurred in drinking water and in seafood (Sipia,

Kankaanpaa et al 2001; Sipia, Kankaanpaa et al 2002) and has the same toxic

potency as microcystin-LR It is therefore necessary to have assays that will measure

the concentrations in water and food There are general assays for both microcystins

and nodularins; a specific ELISA assay for nodularin is available (Mikhailov,

Har-mala-Brasken et al 2001)

10.8 SAMPLE COLLECTION AND HANDLING FOR

MICROCYSTINS

Fresh samples of reservoir water containing cyanobacteria and their toxins are

subject to bacterial degradation and therefore best stored for short periods at reduced

temperatures prior to concentration or analysis Microcystins and nodularins are,

however, very stable compounds, which are resistant to boiling at neutral pH; they

can be stored as dried water samples or dried cell concentrates at room temperature

for weeks prior to analysis An early study used boiling to concentrate microcystins

from 20-L samples collected during water treatment (Falconer, Runnegar et al

1983) Drying in a convection oven at moderate temperature (40 to 50ºC) can be

used to evaporate water from cyanobacterial cell samples on filters for later extraction

and analysis

Because microcystins remain within the cyanobacterial cells until cell death, the

toxicity of healthy bloom samples containing microcystins can be measured simply

by collecting cells from a known volume of water for analysis Cells can be

con-centrated by filtration, centrifugation, or flotation or by evaporation of water from

the sample by freeze- or air-drying The dried cells can be extracted by 75% methanol

in water to quantitatively dissolve microcystins and nodularins (Fastner, Flieger et al

1998) Wet cell concentrates can also be extracted with methanol, using 2.5 mL of

cells plus 7.5 mL of methanol In both cases a second extraction with 75% methanol

will maximize toxin recovery Filtration or centrifugation will remove particulate

residue These methanolic samples can be dried with nitrogen or by evaporation at

45ºC under reduced pressure For bioassay, they are redissolved in water,

physio-logical saline, or culture medium; for instrumental analysis, they are redissolved in

appropriate solvents Sonication of the cell suspension, which is required for aqueous

extraction of toxin, may also assist in organic solvent extraction of fresh cells or

dried material

When total toxin content is required — for example, in senescent blooms, after

copper treatment of the reservoir, or in samples collected during or after water

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treatment — bulk water samples for microcystin or nodularin measurement are

handled differently from cell concentrates If intact cells are present in the sample,

lysis is necessary to obtain all the toxin in the aqueous phase, which can be done

by freeze-thawing and sonication

Concentration of microcystins and nodularins from water and removal of

inter-fering inorganic and organic compounds can be carried out using solid-phase

adsorp-tion cartridges For blooms expected to contain peptide toxins, reversed-phase

octa-decyl (C-18) silanized silica gel (ODS) cartridges have been extensively used There

are a variety of commercially available types with different characteristics (Meriluoto

1997) SepPak, Bond Elut, and Baker cartridges, for example, have been used The

cartridge is activated by washing with 5 mL of methanol followed by 5 mL of water

The filtered cyanobacterial solution is passed through the cartridge, which is then

washed with water Elution of microcystins and nodularins can be done by 70%

methanol solution, 2 × 5 mL, and the eluate dried under a stream of dry nitrogen

and weighed (Lawton, Edwards et al 1994) The product can then be dissolved in

phosphate-buffered saline for intraperitoneal injection or in high-purity water or

methanol/water for use in other assays

An alternative method of concentration of microcystins and nodularin from dilute

solution in water samples is the use of immunoaffinity columns These employ a

cartridge containing either Sepharose beads coated with antimicrocystin antibodies

or silica beads coated with similar antibodies The filtered water sample is passed

through the affinity column, which selectively attaches microcystins and nodularins

After washing the cartridge with 25% methanol/water, the toxins are eluted with

4% acetic acid in 80% methanol/water (Aranda-Rodriguez, Kubwabo et al 2003)

The use of immunoaffinity columns to concentrate microcystins in lake water in

Japan has shown that they have potential in environmental monitoring and a

possi-bility of reuse, which minimizes cost (Kondo, Ito et al 2002)

The most sensitive of the presently available assay techniques can be undertaken

on water samples without concentration, though the effects of interfering ions and

organic material on precision may be considerable Concentration/cleanup

proce-dures reduce these possibilities of interference in the assay results

10.9 BIOASSAYS FOR MICROCYSTINS AND

NODULARINS

Rodent assays for determination of microcystin or nodularin were of vital importance

in the initial characterization of these hepatotoxins but are now not permitted in

many countries, and alternatives are available The method for rodent assay of

cyanobacterial concentrates is described earlier (in Section 10.2) A range of

crus-tacean and insect assays have been evaluated (Harada, Kondo et al 1999), with the

A salina (brine shrimp) assay being among the most easily performed (Kiviranta,

Sivonen et al 1991; Campbell, Lawton et al 1994) This assay may be carried out

with commercially available eggs (sold by aquarium retailers or biological supply

companies) or by purchase of a prepared kit The protocol is described in Harada,

Kondo et al (1999) The sensitivity is moderate, with LC50 values of about 4 µg/mL

(4mg/L) for microcystin-LR (Delaney and Wilkins 1995; Lahti, Ahtiainen et al

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1995) This is adequate for toxicity testing of scums or cell concentrates or centrated eluates from solid-phase adsorption cartridges (see earlier in this chapter)

con-It is not sufficiently sensitive for direct testing of bulk water samples, for which asensitivity of 0.1 to 0.5 µg/L is necessary, to meet the WHO’s Guideline Value formicrocystin-LR of 1 µg/L

10.9.1 C ELL -B ASED A SSAYS FOR M ICROCYSTINS

Freshly isolated rat hepatocytes in suspension culture were shown to be sensitive tomicrocystins by dose-related deformation Thirty nanograms of microcystin wereshown to cause deformation of approximately 60% of 106 hepatocytes in 1 mL ofincubation mixture (Runnegar, Falconer et al 1981) This method was further devel-oped as an assay tool for detection of microcystins in cyanobacterial blooms (Auneand Berg 1986) Examination of the relative potencies of microcystins-LR, -YR,

and -RR to reduce hepatocyte viability showed responses that followed the in vivo

toxicity of the three microcystins The LD50 for hepatocytes after 20 h of incubationwith microcystin-LR was 50 ng/mL (Heinze 1996) This is appreciably more sen-sitive than whole organism assays but still inadequate for unconcentrated samples.Salmon hepatocytes were found to be two- to fivefold less sensitive than rat hepat-ocytes to cell death in suspension culture when incubated with a range microcystinand nodularin concentrations (Fladmark, Serres et al 1998)

10.9.2 B ACTERIAL L UMINESCENCE A SSAYS

Toxicity tests based on bacterial luminescence measurement have proved generallyunsatisfactory for cyanobacterial toxins, as they respond to nontoxic components ofcyanobacteria and not to the toxins of concern (Lawton, Beattie et al 1994) More

recently a Vibrio fisheri bioluminescence assay has been reevaluated for nodularin

measurement, with more promising results (Dahlmann, Ruhl et al 2001)

10.10 ELISA FOR MICROCYSTINS AND NODULARINS

10.10.1 P OLYCLONAL A NTIBODIES

The early ELISA methods in which antibodies to microcystin-LR were raised inrabbits proved successful and have formed the basis of commercially available kits(Chu, Huang et al 1989, 1990) The background to this method was described indiscussing ELISA techniques for cylindrospermopsin earlier in this chapter Thedirect competitive assay in which the antimicrocystin antibody was coated onto theplate was used Microcystin linked to peroxidase enzyme was used as the competitorwith the standard or unknown microcystin concentration Color development in thewells of the plate measured the amount of peroxidase activity on the washed plate

As the unknown concentration of toxin increased, so the enzyme-linked microcystinbound to the antibodies on the plate decreased (Figure 10.1) The sensitivity rangewas 0.5 to 10.0 ng/mL, with a detection limit of 10 pg in 50-µL volume per assay.

This detection limit (of 0.2 µg/L) was sufficient to detect microcystin in trated lake water

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