The South Australian Safe Drinking Water Act: summary of the first year of operation.

The Safe Drinking Water Act 2011 was introduced in South Australia to provide clear direction to drinking water providers on how to achieve water safety. The Act requires drinking water providers to register with SA Health and develop a risk management plan (RMP) for their water supply that includes operational and verification monitoring plans and an incident notification and communication protocol. During the first year of operation, 212 drinking water providers registered under the Act, including one major water utility and a range of small to medium sized providers in regional and remote areas of the State. Information was captured on water source(s) used and water treatment. Rainwater was the most frequently reported drinking water source (66%), followed by bore water (13%), on-supply or carting of mains water (13%), mixed source (rainwater with bore water backup) (6%) and surface water (3%). The majority of providers (91%) treated the water supply, 87% used disinfection. During the first year of operation, 16 water quality incidents were formally reported to SA Health. These included both microbial and chemical incidents. Case studies presented highlight how the RMPs are assisting drinking water providers to identify incidents of potential health concern and implement corrective actions.

Novel toxic effects associated with a tropical Limnothrix/Geitlerinema-like cyanobacterium.

The presence of a toxic strain of a fine filamentous cyanobacterium belonging to the Oscillatorialean family Pseudanabaenacea was detected during a survey of cyanobacterial taxa associated with the presence of cylindrospermopsin in dams in Central Queensland (Australia). The strain, AC0243, was isolated and cultured, its genomic DNA extracted and 16S RNA gene sequenced. Phylogenetic analysis placed AC0243 with Limnothrix species, although this genus appears polyphyletic. Moreover, not all morphological characters are consistent with this genus but more closely fit the description of Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo. The potential toxic effects of AC0243 extract were assessed chemically and biologically. Cell free protein synthesis was inhibited by the extract. Exposure of Vero cells to the extract resulted in a significant reduction in cellular ATP levels following 24-72 h incubation. The presence of cylindrospermopsin was excluded based on the nature of responses obtained in cell and cell-free assays; in addition, (i) it could not be detected by HPLC, LC-MS, or immunological assay, and (ii) no genes currently associated with the production of cylindrospermopsin were found in the genome. Other known cyanobacterial toxins were not detected. The apparent novelty of this toxin is discussed.

Cytotoxicity screening for the cyanobacterial toxin cylindrospermopsin.

The cell lines C3A, HepG2, NCI-87, HCT-8, HuTu-80, Caco-2, and Vero were screened for sensitivity to the cyanobacterial toxin cylindrospermopsin (CYN), with the aim of determining the most sensitive cells to be used in cytotoxicity tests. Cell lines were chosen to be representative of the organs targeted by the toxin; liver, kidney, intestine, and were expected to have different metabolic activities and uptake capabilities. Over the range of cell lines tested, IC(50) determinations at 24 h (MTT assay) ranged fourfold, from 1.5 muM for hepatocyte-derived cell lines (C3A IC(50) = 1.5 +/- 0.54; HepG2 IC(50) = 1.5 +/- 0.87) to 6.5 +/- 3.3 micro the colon-derived Caco-2 cell line. The cell-line sensitivity seemed to decrease in cell lines derived from progressively more distal regions of the gastrointestinal tract: gastric > duodenal > ileal > colonic. The greater sensitivity of the hepatic cell lines to CYN was also apparent in 7-d exposure studies, with low toxin concentrations exerting cytotoxic effects that were not seen in other cell lines. Short-term exposure of C3A cells to CYN (1-6 h) was shown to induce cytotoxicity at 24 h despite a washout and recovery incubation, demonstrating the protracted and apparently irreversible nature of CYN's toxic effects.

Cyanotoxins: Which detection technique for an optimum risk assessment?

The presence of toxigenic cyanobacteria (blue-green algae) in drinking water reservoirs poses a risk to human and animal health worldwide. Guidelines and health alert levels have been issued in the Australian Drinking Water Guidelines for three major toxins, which are therefore the subject of routine monitoring: microcystin, cylindrospermopsin and saxitoxin. While it is agreed that these toxic compounds should be monitored closely, the routine surveillance of these bioactive chemicals can be done in various ways and deciding which technique to use can therefore be challenging. This study compared several assays available for the detection of these toxins and their producers in environmental samples: microscopy (for identification and enumeration of cyanobacteria), ELISA (Enzyme-Linked ImmunoSorbant Assay), PPIA (Protein phosphatase inhibition assay), PSI (Protein synthesis inhibition), chemical analysis and PCR (Polymerase Chain Reaction). Results showed that there was generally a good correlation between the presence of potentially toxigenic cyanobacteria and the detection of the toxin by ELISA. Nevertheless data suggest that cell numbers and toxin concentrations measured in bioassays do not necessarily correlate and that enumeration of potentially toxic cyanobacteria by microscopy, while commonly used for monitoring and risk assessment, is not the best indicator of real toxin exposure. The concentrations of saxitoxins quantified by ELISA were significantly different than those measured by LC-MS, while results were comparable in both assays for microcystin and cylindrospermopsin. The evaluation of these analytical methods led to the conclusion that there is no "gold standard" technique for the detection of the aforementioned cyanotoxins but that the choice of detection assay depends on cost, practicality, reliability and comparability of results and essentially on the question to be answered, notably on toxin exposure potential.

Cylindrospermopsin genotoxicity and cytotoxicity: role of cytochrome P-450 and oxidative stress.

Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.

Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 µg/L. mES exposed to 0-1 µg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 µg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 µg/mL, the Lowest OEC was 1 µg/mL (p < 0.001, n = 3), and the IC50 was 0.86 µg/mL after 24 h. The ADWG 1 µg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.

The growing use of recycled water in large urban centres requires comprehensive public health risk assessment and management, an important aspect of which is the assessment and management of residual trace chemical substances. Bioanalytical methods such as in vitro bioassays may be ideal screening tools that can detect a wide range of contaminants based on their biological effect. In this study, we applied thirteen in vitro assays selected explicitly for their ability to detect molecular and cellular effects relevant to potential chemical exposure via drinking water as a means of screening for chemical contaminants from recycled water at 9 Australian water reclamation plants, in parallel to more targeted direct chemical analysis of 39 priority compounds. The selected assays provided measures of primary non-specific (cytotoxicity to various cell types), specific (inhibition of acetylcholinesterase and endocrine receptor-mediated effects) and reactive toxicity (mutagenicity and genotoxicity), as well as markers of adaptive stress response (modulation of cytokine production) and xenobiotic metabolism (liver enzyme induction). Chemical and bioassay analyses were in agreement and complementary to each other: the results show that source water (treated wastewater) contained high levels of biologically active compounds, with positive results in almost all bioassays. The quality of the product water (reclaimed water) was only marginally better after ultrafiltration or dissolved air floatation/filtration, but greatly improved after reverse osmosis often reducing biological activity to below detection limit. The bioassays were able to detect activity at concentrations below current chemical method detection limits and provided a sum measure of all biologically active compounds for that bioassay, thus providing an additional degree of confidence in water quality.

Toxicity of the cyanobacterium Limnothrix AC0243 to male Balb/c mice.

A growing list of freshwater cyanobacteria are known to produce toxic agents, a fact which makes these organisms of concern to water authorities. A cultured strain of Limnothrix (AC0243) was recently shown to have toxic effects in in vitro bioassays. It did not produce any of the known cyanobacterial toxins. The intrapertoneal toxicity of aqueous extracts of the material was therefore tested in mice to determine whether the observed effects might be of public health relevance to drinking water supplies. The results indicate that Limnothrix AC0243 is acutely toxic to mice, causing widespread cellular necrosis in the liver, kidneys and gastrointestinal tract within 24 h of exposure. Sub-lethal effects lasted at least 7 d. These results suggest that Limnothrix AC0243 produces a novel toxin ("Limnothrixin") and that further work is therefore urgently required to quantify the potential public health implications.

Cylindrospermopsin, a blue-green algal toxin, inhibited human luteinised granulosa cell protein synthesis in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) inhibits protein synthesis, and CYP450 enzymes metabolise CYN to cytotoxic endproducts. Human chorionic gonadotrophin (hCG) stimulates the de novo synthesis of StAR and CYP450 aromatase. Human IVF-derived granulosa cells (GC) (n=7) were exposed to 0-5µM CYN±1IU/ml hCG for 2-24h. After 24h pre-culture GC responded to hCG by increasing estradiol 17ß (E(2)) and progesterone (P(4)) synthesis. Three micromolar of CYN±1IU/ml hCG for 24h was not cytotoxic and did not affect basal or hCG-stimulated E(2) or P(4) production, but did inhibit protein synthesis (p<0.05, n=4). hCG-stimulated steroidogenesis was not reduced by CYN, suggesting a lack of effect on StAR or CYP450 aromatase protein synthesis. hCG enhanced the effects of CYN on GC protein synthesis. Twenty four hours exposure to 0.1µM CYN did not affect GC, supporting the establishment of a 0.0024µM Guideline level for CYN in public water supplies.

Novel cytotoxicity associated with Anabaena circinalis 131C.

The production of secondary metabolites by cyanobacteria is extensively varied. Anabaena spp. have been shown to produce the toxins saxitoxin, anatoxin-a, anatoxin-a(s), cylindrospermopsin and microcystin. In this study we show cytotoxicity associated with Anabaena circinalis 131C that could not be attributed to its high saxitoxin content. Analytical, ELISA and enzymatic methods excluded the presence of other known cyanobacterial toxins leading to the suggestion of novel toxicity.

Cyanotoxins are not implicated in the etiology of coral black band disease outbreaks on Pelorus Island, Great Barrier Reef.

Cyanobacterial toxins (i.e. microcystins) produced within the microbial mat of coral black band disease (BBD) have been implicated in disease pathogenicity. This study investigated the presence of toxins within BBD lesions and other cyanobacterial patch (CP) lesions, which, in some instances ( approximately 19%), facilitated the onset of BBD, from an outbreak site at Pelorus Island on the inshore, central Great Barrier Reef (GBR). Cyanobacterial species that dominated the biomass of CP and BBD lesions were cultivated and identified, based on morphology and 16S rRNA gene sequences, as Blennothrix- and Oscillatoria-affiliated species, respectively, and identical to cyanobacterial sequences retrieved from previous molecular studies from this site. The presence of the cyanotoxins microcystin, cylindrospermopsin, saxitoxin, nodularin and anatoxin and their respective gene operons in field samples of CP and BBD lesions and their respective culture isolations was tested using genetic (PCR-based screenings), chemical (HPLC-UV, FTICR-MS and LC/MS(n)) and biochemical (PP2A) methods. Cyanotoxins and cyanotoxin synthetase genes were not detected in any of the samples. Cyanobacterial species dominant within CP and BBD lesions were phylogenetically distinct from species previously shown to produce cyanotoxins and isolated from BBD lesions. The results from this study demonstrate that cyanobacterial toxins appear to play no role in the pathogenicity of CP and BBD at this site on the GBR.

An examination of the antibiotic effects of cylindrospermopsin on common gram-positive and gram-negative bacteria and the protozoan Naegleria lovaniensis.

The importance of the toxin cylindrospermopsin to the function and fitness of the cyanobacteria that produce it remains a matter of conjecture. Given that the structure of cylindrospermopsin has commonalities with other antibacterial protein synthesis inhibitors, such as streptomycin, authors tested the possibility that the toxin might act as an antibacterial compound that can kill competing microbes. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were tested by the minimal inhibitory concentration method and significant antibacterial activity was only observed at a cylindrospermopsin concentration of 300 microg mL(-1) after exposure for 5 days. No effect on log phase growth of E. coli was observed for this same toxin concentration. Protein synthesis was inhibited by cylindrospermopsin in E. coli 70S extracts, reduced by 25% compared with controls when treated with 41.5 microg mL(-1) of the toxin; however, a much greater reduction of 97% was observed for chloramphenicol in the same experiment. Naegleria lovaniensis, a phagotrophic protozoan, was more susceptible to cylindrospermopsin, with a decrease in the number of N. lovaniensis plaques after 24-h treatment with 5-50 microg mL(-1) of toxin and an LC(50) of approximately 60 microg mL(-1). Given these results, cylindrospermopsin is clearly not antibacterial at concentrations found in environmental waters, nor will it adversely affect N. lovaniensis at these concentrations. For organisms that are able to ingest cylindrospermopsin-producing cells, the response of N. lovaniensis to the toxin suggests that only a few ingested cells would be enough to kill predatory organisms with similar susceptibility.

Cylindrospermopsin-induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes.

The toxicology of the cyanobacterial alkaloid cylindrospermopsin (CYN), a potent inhibitor of protein synthesis, appears complex and is not well understood. In exposed mice the liver is the main target for the toxic effects of CYN. In this study primary mouse hepatocyte cultures were used to investigate the mechanisms involved in CYN toxicity. The results show that 1-5 microM CYN caused significant concentration-dependent cytotoxicity (52%-82% cell death) at 18 h. Protein synthesis inhibition was a sensitive, early indicator of cellular responses to CYN. Following removal of the toxin, the inhibition of protein synthesis could not be reversed, showing behavior similar to that of the irreversible inhibitor emetine. In contrast to the LDH leakage, protein synthesis was maximally inhibited by 0.5 microM CYN. No protein synthesis occurred over 4-18 h at or above this concentration. Inhibition of cytochrome P450 (CYP450) activity with 50 microM proadifen or 50 microM ketoconazole diminished the toxicity of CYN but not the effects on protein synthesis. These findings imply a dissociation of the two events and implicate the involvement of CYP450-derived metabolites in the toxicity process, but not in the impairment of protein synthesis. Thus, the total abolition of protein synthesis may exaggerate the metabolite effects but cannot be considered a primary cause of cell death in hepatocytes over an acute time frame. In cell types deficient in CYP450 enzymes, protein synthesis inhibition may play a more crucial role in the development of cytotoxicity.