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A single run HPLC method utilizing ion exchange as the separation mode with a novel mobile phase system coupled to chemical postcolumn oxidation and fluorescence detection has been developed and demonstrated to be applicable to the quantitative analysis of paralytic shellfish poisons (PSPs) produced by Australian cyanobacteria (Anabaena circinalis) and other cyanobacteria. Both the cyanobacterial matrix and natural water constituents did not significantly affect the performance of this method. The daily precision of this method was adequate for it to be considered as a routine analytical tool for direct PSP analysis (prePSP concentration is not required) of cyanobacterial extracts and water bodies containing PSPs (C1, C2, GTX2, GTX3, NEO, STX) in the low parts per billion concentration range (10-70 ppb).

South Australian freshwaters from a wide variety of environments were analysed for bromide and the results correlated with both chloride and total dissolved solids (TDS) concentrations. A linear relationship was obtained which allows chloride data to be successfully used to estimate bromide concentrations. Bromide displayed a slightly better correlation with TDS indicating that an improved estimate of bromide could be made by reference to TDS data which is more easily and commonly obtained, and generally available extensively as historical data. The bromide content in relation to its ratio with both chloride and TDS contents was around seventy percent of the corresponding ratios found in seawater, a finding reported in other published data. The ability to estimate bromide concentrations is a potentially useful tool in the drinking water industry as it enables the assessment of the extent of bromate formation (predicted through the use of mathematical models and other water quality data) which is an important factor when ozonation is being considered as a treatment option, particularly as many water industry regulartory bodies have imposed stringent limits on the levels of acceptable bromate.

When treating water subject to a cyanobacterial bloom the first priority should be removal of intact cells using separation techniques such as coagulation or membrane filtration. Chlorination and ozonation are effective for the destruction of residual dissolved microcystins and cylindrospermopsin. Anatoxin-a can be effectively removed using ozone, although chlorine is relatively ineffective. Oxidation techniques do not appear to be the best method for the treatment of saxitoxins under normal treatment plant operating conditions. Powdered activated carbon can be effective for the removal of all toxins, except, perhaps, microcystin LA, provided the appropriate carbon and the correct dose is applied. Granular activated carbon filters show a limited lifetime for the adsorption of most microcontaminants, including cyanotoxins. The biodegradation of cyanotoxins across GAC filters shows great potential as a treatment process.

Changes in toxicity were studied during batch culture of a strain ofAnabaena circinalis (AWQC ANA-311F) producing paralyticshellfish toxins (PSTs). PSTs were extracted from cells and culture mediumduring the culture period of 29 days. Samples were analysed by HPLC withpost-column oxidation coupled to fluorescence detection, and tested with themouse neuroblastoma bioassay (MNB). Seven PSTs were detected, but only fivewerequantified. Based on chemical analysis of PSTs by HPLC, a‘theoretical’ saxitoxin concentration related to mouse toxicity wascalculated. Neuroblastoma cell survival was measured during the MNB andcorrelated to total toxicity of the A. circinalis samples.Comparison was made between the changes in total toxicity shown by MNB and the‘theoretical’ saxitoxin concentration shown by HPLC. There was asignificant positive correlation between the two in the culture medium, but notthe cells.

The occurrence of cyanobacteria (blue-green algae) blooms and the possibility of production of cyanotoxins (algal toxins) have become major concerns for drinking water providers worldwide. Ozone has been shown to be effective for the destruction of some classes of toxins under specific conditions, although most researchers agree that the dose and contact time required will depend on water quality. The clarification of the relative effects of water quality parameters such as dissolved organic carbon concentration and character, and alkalinity, has not been previously attempted. In this study the cyanotoxins microcystin LR and LA and anatoxin-a were ozonated at a range of ozone doses in four treated waters with very different water quality. For both the toxins, 100% destruction was related to a residual ozone concentration present after 5 min. This was, in turn, related to the water quality and indicated that a direct reaction with molecular ozone could be responsible for the destruction. The results confirmed that both the toxins would be destroyed under conditions usually utilised for ozonation prior to granular activated carbon (GAC) filtration. This will apply under a range of water quality conditions but not necessarily a range of temperatures. The saxitoxin class of compounds was very resistant to oxidation by ozone and would require further treatment such as GAC filtration.

Toxic cyanobacteria (blue-green algae) are becoming recognized as a greater water quality problem as blooms become more common in waters affected by increasing nutrient levels, and knowledge of the properties of the toxin increases. Of particular importance are the hepatotoxic cyclic peptides produced by certain strains of Microcystis species and by Nodularia spumigena. The effects of chlorine and chloramine on these hepatotoxins in both freeze-dried and intact material were investigated. The destruction of the toxins was monitored using HPLC analysis. In a number of cases mouse bioassays were used for confirmation of the destruction of the toxins and to ensure that toxic by-products, not detectable by HPLC, were not formed. Contrary to evidence reported in the literature, chlorine was effective in destroying toxins, as long as sufficient chlorine was used. Toxins were destroyed under conditions such that a chlorine residual of at least 0.5 mg/l was present after 30 min contact time. The destruction of toxins was pH dependent; chlorinating agents such as calcium and sodium hypochlorite were not as effective at high dose rates due to elevation of pH. Chloramination had little effect on toxins. Although the degradation mechanisms have yet to be established, the results show that chlorination may be an effective and practical method for the removal of cyanobacterial peptide toxins from drinking water.

Despite the growth in aquifer storage and recovery (ASR) as a technique for the provision of potable water supplies, quantitative data on the fate of disinfection byproducts that may be present in the injected water remain rare. This study evaluates the data from eight ASR sites in Australia and the United States that cover a wide range of source water compositions, hydrogeological environments, and operating conditions. Rates of attenuation and formation of trihalomethanes (THMs) in groundwater were determined using analytical techniques that took dilution effects into account. Half-lives varied by more than 2 orders of magnitude (e.g.,1 to120 days for total THMs) and were both compound- and site- specific. Chloroform was most persistent, and more highly brominated compounds tended to be less persistent, as has generally been found. For any particular THM compound, much of the variability could be explained by contrasts in geochemical conditions within the aquifer since microbial degradation is the primary mechanism for THM attenuation. As such, bounds on the half-life were defined according to the redox state of the groundwater. In situ formation of some THMs in the aquifer after injection was directly observed at a number of sites, and was predicted to have taken place at all sites. The variance in formation estimates was large between the different methods used. Formation may be more common than previously thought because of the low frequency of groundwater sampling after injection and concomitant attenuation and mixing.

Unchlorinated treated waters from two Australian reservoirs were spiked with microcystin-LA and -LR extracted from a toxic scum of Microcystis aeruginosa. The two waters had considerably different water quality and therefore ozone demands. The spiked sample waters were ozonated using the batch method of ozonation at a range of doses and the samples were analysed for toxins using high-performance liquid chromatography (HPLC). The toxin content of the samples was also determined using a protein phosphatase type 2A inhibition assay (PP2A) and toxicity via the standard mouse bioassay. The HPLC results correlated well with the PP2A results and toxicity tests for both waters. A loss of both toxins and toxicity was observed with increasing ozone dose, resulting in a complete loss of toxicity for both waters once an ozone residual had been achieved. At this ozone residual no toxin was detected using HPLC. The results indicate that microcystins are not transformed into toxic by-products.

To simplify our efforts in acquiring toxicological information on endotoxins produced by cyanobacteria, a method development study was undertaken to identify relatively hazard-free and efficient procedures for their extraction. One article sourced and two novel methods were evaluated for their ability to extract lipopolysaccharides (LPSs) or endotoxins from cyanobacteria. The Limulus polyphemus amoebocyte lysate (LAL) assay was employed to compare the performance of a novel method utilizing a 1-butanol-water (HBW) solvent system to that of Westphal's (1965) phenol-water system (HPW) for the extraction of endotoxin from various cyanobacteria. The traditional HPW method extracted from 3- to 12-fold more endotoxin from six different cyanobacterial blooms and culture materials than did the novel HBW method. In direct contrast, the novel HBW method extracted ninefold more endotoxin from a non-microcystin producing Microcystis aeruginosa culture as compared to the HPW method. A solvent system utilizing N,N'-dimethylformamide-water (HDW) was compared to both the HPW and HBW methods for the extraction of endotoxin from natural samples of Anabaena circinalis, Microcystis flos-aquae, and a 1:1 mixture of Microcystis aeruginosa/Microcystisflos-aquae. The LAL activities of these extracts showed that the novel HDW method extracted two- and threefold more endotoxin from the Anabaena sample that did the HBW and HPW methods, respectively. The HDW method also extracted approximately 1.5-fold more endotoxin from the Microcystis flos-aquae sample as compared to both the HBW and HPW methods. On the other hand, the HBW method extracted 2- and 14-fold more endotoxin from the Microcystis flos-aquae/Microcystis aeruginosa mixture than did the HPW and HDW methods, respectively. Results of this study demonstrate that significant disparities exist between the physicochemical properties of the cell wall constituents not only of different cyanobacterial species but also of different strains of the same cyanobacterial species, as showing by the varying effectiveness of the solvent systems investigated. Therefore, a sole method cannot be regarded as universal and superior for the extraction of endotoxins from cyanobacteria. Nevertheless, the ability of the novel HBW and HDW methods to utilize easily handled organic solvents that are less hazardous than phenol render them attractive alternatives to the standard HPW method.

Chlorination was investigated as a treatment option for degrading and thus removing saxitoxins (paralytic shellfish poisons, PSPs) produced by cyanobacteria (blue-green algae) from water. It was found to be effective with the order of ease of degradation of the saxitoxins being GTX5 (B1) approximately dcSTX > STX > GTX3 approximately C2 > C1 > GTX2. However the effectiveness of chlorine was pH dependent. Degradation as a function of pH was not linear with the degree of degradation increasing rapidly at around pH 7.5. At pH 9 > 90% removal was possible provided a residual of 0.5 mg l(-1) free chlorine was present after 30 min contact time. The more effective degradation at higher pH was unexpected as chlorine is known to be a weaker oxidant under these conditions. The more effective degradation, then, must be due to the toxins, which are ionisable molecules, being present in a form at higher pH which is more susceptible to oxidation. The feasibility of using chlorine to remove saxitoxins during water treatment will therefore depend strongly on the pH of the water being chlorinated. Degradation may be improved by pH adjustment but may not be a practical solution. Although saxitoxins were degraded in that the parent compounds were not detected by chemical analysis, there is no indication as to the nature of the degradation products. However, acute toxicity as determined by the mouse bioassay was eliminated.

Knowledge on the behaviour of disinfection by-products (DBPs) during aquifer storage and recovery (ASR) is limited even though this can be an important consideration where recovered waters are used for potable purposes. A reclaimed water ASR trial in an anoxic aquifer in South Australia has provided some of the first quantitative information at field-scale on the fate and transport of trihalomethanes (THMs) and haloacetic acids (HAAs). The results revealed that THM half-lives varied from

In April 2000, the water supply for Yorke Peninsula in South Australia was deemed non-potable when extracts from a proliferation of the benthic cyanobacterium Phormidium aff. formosum in Upper Paskeville Reservoir were found to be lethally toxic by intraperitoneal injection into mice (400 mg kg−1). Routine water quality monitoring had failed to detect the development of the Phormidium until complaints of musty taste and odour, attributable to the production of 2-methyl-isoborneol (MIB), were received from the consumers. The 185 ML open-balancing storage, receiving filtered and chloraminated water from the River Murray, was isolated from the drinking water supply and a health alert was issued to approximately 15,000 consumers. The identity of the toxin(s) is thus far unknown, but clinical symptoms of toxicity in mice and chemical characteristics are distinct from the known major cyanotoxins. Preliminary characterisation of this toxin indicates that it has low solubility in water and organic solvents and is strongly associated with the particulate cellular material of the filaments. Toxicity of extracts was diminished by boiling and by treatment with chlorine, but not by chloramines. Further testing of floating cyanobacterial mats in the Torrens Lake in the city of Adelaide (Phormidium aff. formosum) and Myponga Reservoir (Phormidium aff. amoenum) in 2000/2001 was also found to be toxic by mouse bioassay. Toxicity is yet to be confirmed in monospecific cultured strains and further studies are required to identify the toxin and assess its health significance. Genetic characterisation of isolates has commenced in an attempt to classify their relatedness and to assist in the rapid identification of potentially toxic strains. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 506–511, 2001

A colorimetric phosphatase inhibition assay using protein phosphatase 2A and p-nitrophenyl phosphate as substrate for determining cyanobacterial peptide hepatotoxins directly in water without sample preconcentration has been developed. The assay uses commercially available materials and is much more simple to use than similar procedures using radiolabelled substrates. It has similar sensitivity to the radiolabelled assays and, with a working range of around 0.2-1 microg/L, is able to determine these toxins at concentrations below the provisional World Health Organisation guideline of 1 microg/L for microcystin-LR. The method appears robust and not to be affected by the sample matrix apart from possibly some components of cellular material if present at very high levels in extracts of cyanobacterial material. It is not affected by the presence of low levels of methanol in sample extracts.

A colorimetric protein phosphatase inhibition assay based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1 was developed for analysis of waters for cyanobacterial hepatotoxins. The phosphate released in the assay was determined using a malachite green reagent. Good agreement with toxin concentrations determined by HPLC was obtained. The assay was capable of determining these toxins at concentrations around 1 microgram/L with high precision and without sample concentration. This is of considerable benefit as the World Health Organisation specifies a provisional guideline of 1 microgram/L for microcystin-LR. There was evidence, however, that the sample matrix might affect quantification, leading to false positive results. Thus the assay should be viewed as a screening procedure, and confirmatory analyses by an alternative procedure should be carried out for positive results. Further work is required to resolve the question of matrix interferences if phosphatase inhibition assays are used directly for measuring toxin levels in water, especially if this information is used to check compliance with water quality guidelines.

Aquifer storage and recovery is recognized in the USA as having a significant role for inter-season storage of drinking water, and in Europe and Australia also for its potential for water treatment. However a better knowledge of water quality changes during aquifer storage and recovery, along with better understanding of sustainable treatment processes in aquifers is necessary to enable water utilities to take advantage of this technique. The usefulness of aquifer storage and recovery to improve the quality of injected water is being investigated at several sites. Specific interest is on the attenuation rates of microbial pathogens and organic compounds (both natural and synthetic) in saturated groundwater at artificial recharge sites. Microbial pathogens of particular interest are enteric viruses and protozoa, while the organic chemicals being investigated include several disinfection-by-products and endocrine disruptors. The aim is to encapsulate the data obtained from this investigation into models for the prediction of changes in water quality, and which can be used by water utilities and regulators to evaluate pretreatment requirements for aquifer storage and recovery. This will also improve accuracy of information for the protection of the health of consumers and the environment.

Paralytic shellfish poisons (PSPs) are produced by freshwater cyanobacteria and pose a threat to human and animal drinking-water supplies. The wide range of toxin analogues (and the likelihood that further analogues remain to be discovered) means that chromatographic methods are not always reliable indicators of toxicity. Although the mouse bioassay remains the method of choice in the seafood industry, its use is increasingly being questioned on ethical grounds. The cell-based Neuro-2A neuroblastoma toxicity assay is an alternative bioassay validated for testing shellfish extracts, so it was of interest to determine its applicability with the different suite of toxin analogues produced by cyanobacteria. Cyanobacterial bloom samples from Australia, Brazil, and France were assayed using the neuroblastoma assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS), high-performance liquid chromatography with postcolumn derivatization and fluorescence detection, and the Jellett Rapid Test for PSP. To assess interlaboratory variability, the neuroblastoma assay was set up in laboratories in Paris (France) and Adelaide (Australia). Neuroblastoma and chromatographic methods gave comparable results except in the case of the neurotoxic Brazilian samples: LC-MS/MS did not detect the putative new PSPs contained in these samples. Inter- and intralaboratory variability of the neuroblastoma assay was typical of biological assays but no greater than that found for interassay variability between different chromatographic determinations. The batch of Jellett Rapid Tests for PSP used did not yield quantitative results. Overall, the neuroblastoma assay was useful as a screening assay for determination of toxicity caused by saxitoxin neurotoxins in freshwater cyanobacteria, having the advantage of being sensitive to unidentified toxins that currently cannot be quantified by chromatographic means.

Saxitoxin-group neurotoxins (paralytic shellfish poisons) have been identified in a cultured strain of Anabaena circinalis and in natural bloom samples in which this species was the dominant organism collected from widely distributed sites in the Murray-Darling Basin of Australia. These toxins have hitherto been isolated almost exclusively from 'red tide' dinoflagellates and contaminated shellfish. Two 'aphantoxins', which appear to be identical to two of the paralytic shellfish poisons, have been identified in a cyanobacterium from a small number of sites in New Hampshire, USA. The conclusions are supported by electrophysiological studies and by high-performance liquid chromatographic (HPLC) and fast atom bombardment-mass spectrometric (FAB-MS) analyses.

Low levels of formaldehyde in water were determined by derivatization with 2,4-dinitrophenylhydrazine at an optimized pH (1.5–2.5), solid-phase extraction with C18 adsorption cartridges and analysis by reversed-phase high-performance liquid chromatography with ultraviolet detection. A novel procedure for the removal of formaldehyde present as an impurity in blank water was responsible for lowering the detection limit to 0.1 µg l–1 for a 200 ml sample. A strong anion-exchange resin, in the hydrogen sulfite form, was used to adsorb formaldehyde and any other aldehyde impurities present in blank water. The use of C18 adsorption cartridges also minimized background effects. The recovery of C1—C3 aldehydes spiked into purified blank water was 83–93% with a relative standard deviation of 1.4–6.4%.

Pesticide residues were detected in 83% of water samples and in 100% of sediment samples from streams draining a horticultural catchment in the Mt Lofty‐Ranges of South Australia. Highest concentrations were detected during the first half of the summer growing season extending from October to December and particularly during runoff events occurring during this period. The organochlorlne insecticides DDT, lindane and endosulfan and the organophosphorus insecticide chlorpyrifos were detected in sufficiently high concentrations to adversely affect aquatic environments. However, only low concentrations of the insecticide lindane and the herbicide chlorthal‐dimethyl could be detected at the reservoir outlet and did not adversely affect water quality for human consumers according to maximum Residue limit recommendations from the National Health and Medical Research Council.

The stannous chloride reduction procedure for the colorimetric determination of total phosphorus in wastewater samples using potassium persulphate digestion was evaluated. Samples with salinities as low as 50 mg/1 gave phosphorus levels approximately 10% lower than the true levels when absorbance readings were taken after the recommended time interval of 10 minutes. With increasing salinity, the error increased to approximately 20%. This error can be overcome by the addition of sodium chloride to both standards and samples to achieve salinity levels of 2,000 mg/1 or greater, and making absorbance measurements after allowing at least 2 but not more than 3 minutes for colour development.

Les valeurs obtenues ne sont pas affectees par la presence d'autre methanes trihalogenes ni par la composition de l'eau. On peut donc utiliser les valeurs obtenues avec de l'eau distillee pour la conception d'installations d'aeration pour l'elimination du polluant

The binding of the inhibitors N-trifluoroacetyltryptophan, N- trifluoroacetylphenylalanine, N-acetyl-tryptophan and N- acetylphenylalanine to chymotrypsin has been studied by 19F N.M.R. spectroscopy at several pH values. Methods for determining the binding parameters, KI and ΔB, including a model for enzyme oligomerization and competitive inhibition from a second inhibitor, are discussed and a general non-linear least-squares method is presented. Values of KI and ΔB are recorded for D and L enantiomers of tryptophan derivatives and for D-phenylalanine derivatives. The results are discussed in terms of a model for the aromatic binding site of chymotrypsin.