Deriving safe short-term chemical exposure values (STEV) for drinking water.

Small and brief exceedances of chemicals above their guideline values in drinking water are unlikely to cause an appreciable increased risk to human health. As a result, short-term exposure values (STEV) can be derived to help decide whether drinking water can still be supplied to consumers without adverse health risks. In this study, three approaches were applied to calculate and compare STEV for pesticides. The three approaches included basing a STEV on the acute reference dose (ARfD) (Approach 1), removing conventional attribution rates and uncertainty factors from current guideline values (Approach 2) and extrapolating 1 d and 7 d no observed adverse effect levels (NOAEL) from existing toxicity data using a log-linear regression (Approach 3). Despite being very different methods, the three approaches produced comparable STEV generally within an order of magnitude, which often overlapped with other existing short-term exposure values such as short-term no adverse response levels (SNARL) and health advisories (HA). The results show that adjusting the current guideline value using standard extrapolation factors (Approach 2) often produced the most conservative values. Approach 2 was then applied to two other chemical classes, disinfection by-products (DBPs) and cyanotoxins, demonstrating the wider applicability of the approach.

Effect of increasing bromide concentration on toxicity in treated drinking water.

Research is increasingly indicating the potential chronic health effects of brominated disinfection by-products (DBPs). This is likely to increase with elevated bromide concentrations resulting from the impacts of climate change, projected to include extended periods of drought and the sudden onset of water quality changes. This will demand more rigorous monitoring throughout distribution systems and improved water quality management at water treatment plants (WTPs). In this work the impact of increased bromide concentration on formation of DBPs following conventional treatment and chlorination was assessed for two water sources. Bioanalytical tests were utilised to determine cytotoxicity of the water post disinfection. Coagulation was shown to significantly reduce the cytotoxicity of the water, indicating that removal of natural organic matter DBP precursors continues to be an important factor in drinking water treatment. Most toxic species appear to form within the first half hour following disinfectant addition. Increasing bromide concentration across the two waters was shown to increase the formation of trihalomethanes and shifted the haloacetic acid species distribution from chlorinated to those with greater bromine substitution. This correlated with increasing cytotoxicity. This work demonstrates the challenges faced by WTPs and the possible effects increasing levels of bromide in source waters could have on public health.

Evaluating Evidence for Association of Human Bladder Cancer with Drinking-Water Chlorination Disinfection By-Products.

Exposure to chlorination disinfection by-products (CxDBPs) is prevalent in populations using chlorination-based methods to disinfect public water supplies. Multifaceted research has been directed for decades to identify, characterize, and understand the toxicology of these compounds, control and minimize their formation, and conduct epidemiologic studies related to exposure. Urinary bladder cancer has been the health risk most consistently associated with CxDBPs in epidemiologic studies. An international workshop was held to (1) discuss the qualitative strengths and limitations that inform the association between bladder cancer and CxDBPs in the context of possible causation, (2) identify knowledge gaps for this topic in relation to chlorine/chloramine-based disinfection practice(s) in the United States, and (3) assess the evidence for informing risk management. Epidemiological evidence linking exposures to CxDBPs in drinking water to human bladder cancer risk provides insight into causality. However, because of imprecise, inaccurate, or incomplete estimation of CxDBPs levels in epidemiologic studies, translation from hazard identification directly to risk management and regulatory policy for CxDBPs can be challenging. Quantitative risk estimates derived from toxicological risk assessment for CxDBPs currently cannot be reconciled with those from epidemiologic studies, notwithstanding the complexities involved, making regulatory interpretation difficult. Evidence presented here has both strengths and limitations that require additional studies to resolve and improve the understanding of exposure response relationships. Replication of epidemiologic findings in independent populations with further elaboration of exposure assessment is needed to strengthen the knowledge base needed to better inform effective regulatory approaches.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Community composition, toxigenicity, and environmental conditions during a cyanobacterial bloom occurring along 1,100 kilometers of the Murray River.

A cyanobacterial bloom impacted over 1,100 km of the Murray River, Australia, and its tributaries in 2009. Physicochemical conditions in the river were optimal to support a bloom at the time. The data suggest that at least three blooms occurred concurrently in different sections of the river, with each having a different community composition and associated cyanotoxin profile. Microscopic and genetic analyses suggested the presence of potentially toxic Anabaena circinalis, Microcystis flos-aquae, and Cylindrospermopsis raciborskii at many locations. Low concentrations of saxitoxins and cylindrospermopsin were detected in Anabaena and Cylindrospermopsis populations. A multiplex quantitative PCR was used, employing novel oligonucleotide primers and fluorescent TaqMan probes, to examine bloom toxigenicity. This single reaction method identified the presence of the major cyanotoxin-producing species present in these environmental samples and also quantified the various toxin biosynthesis genes. A large number of cells present throughout the bloom were not potential toxin producers or were present in numbers below the limit of detection of the assay and therefore not an immediate health risk. Potential toxin-producing cells, possessing the cylindrospermopsin biosynthesis gene (cyrA), predominated early in the bloom, while those possessing the saxitoxin biosynthesis gene (sxtA) were more common toward its decline. In this study, the concentrations of cyanotoxins measured via enzyme-linked immunosorbent assay (ELISA) correlated positively with the respective toxin gene copy numbers, indicating that the molecular method may be used as a proxy for bloom risk assessment.

Cytotoxic and genotoxic effects of cylindrospermopsin in mice treated by gavage or intraperitoneal injection.

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN-induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 µg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty-four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 µg/kg CYN (P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration.

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.

Toolbox for the sampling and monitoring of benthic cyanobacteria.

Benthic cyanobacteria are a nuisance because they produce highly potent toxins and taste and odour compounds. Despite this, benthic cyanobacteria remain far less studied than their planktonic counterparts. For example, little is known about their growth or the seasonality of their secondary metabolite production. Moreover, sampling and monitoring techniques commonly used for the survey of planktonic species are not necessarily applicable to benthic forms. This study aimed to develop and validate a new sampling device for the routine monitoring of benthic mats. Molecular monitoring techniques were established and validated on environmental samples collected in a South Australian reservoir (SA-L2). A total of eight qPCR assays were applied to samples in order to track seasonal variations in cyanobacteria concentrations and associated secondary metabolite production. Next Generation Sequencing was utilised to conduct a microbial community composition analysis and to select the most appropriate substrate material for the sampling of benthic cyanobacteria. The concentration of the secondary metabolites geosmin and 2-methyl-isoborneol were quantified using High-Performance Liquid Chromatography, and concentrations of key nutrients (N, P) were quantified in water samples. The sampling device designed proved efficient and easy to use in the field. The qPCR assay designed for the amplification of the cyanobacterial MIB synthase had a high efficiency with a minimum limit of quantification of 4 cell-equivalents per reaction and identified a potential source of MIB in SA-L2 Reservoir. The peak season for benthic growth and secondary metabolite production was observed in spring. Proportionally, 35% of the variability in water geosmin concentrations can be explained by benthic actinobacterial and cyanobacterial activity, showing that freshwater benthic mats represent a significant source of taste and odour compounds.

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.

Transformation of endocrine disrupting chemicals, pharmaceutical and personal care products during drinking water disinfection.

Pharmaceuticals and personal care products (PPCPs) and endocrine disrupting compounds (EDCs) are frequently detected in drinking water sources. This raises concerns about the formation of potentially more toxic transformation products (TPs) after drinking water disinfection. This study applied a combination of computational and experimental methods to investigate the biological activity of eight EDCs and PPCPs commonly detected in source waters (acetaminophen, bisphenol A, carbamazepine, estrone, 17α-ethinylestradiol, gemfibrozil, naproxen and triclosan) before and after disinfection. Using a Stepped Forced Molecular Dynamics (SFMD) method, we detected 911 unique TPs, 36% of which have been previously reported in the scientific literature. We calculated the likelihood that TPs would cause damage to biomolecules or DNA relative to the parent compound based on lipophilicity and the occurrence of structural alerts, and applied two Quantitative Structure-Activity Relationship (QSAR) tools to predict toxicity via receptor-mediated effects. In parallel, batch experiments were performed with three disinfectants, chlorine, chlorine dioxide and chloramine. After solid-phase extraction, the resulting TP mixtures were analyzed by chemical analysis and a battery of eleven in vitro bioassays covering a variety of endpoints. The laboratory results were in good agreement with the predictions. Overall, the combination of computational and experimental chemistry and toxicity methods used in this study suggest that disinfection of the studied EDCs and PPCPs will produce a large number of TPs, which are unlikely to increase specific toxicity (e.g., endocrine activity), but may result in increased reactive and non-specific toxicity.

Effects of blue-green algal toxin cylindrospermopsin (CYN) on human granulosa cells in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) occurs in public water supplies. CYN was hepatotoxic when administered orally to mice, and cytotoxic and genotoxic to human cell lines. To determine the effects of CYN on primary human IVF-derived granulosa cells, 0-1 microg/ml CYN was added to cells for 2, 4 or 6h+/-hCG (n=6), or for 24, 48 and 72 h (n=6). Cytotoxicity was evaluated by MTT assay, and secreted progesterone or estrogen quantified by radioimmunoassay. 24h exposure to 1 microg/ml CYN was cytotoxic (p<0.05), whereas 0.0625 microg/ml CYN did not cause cytotoxicity or affect estrogen production, but did inhibit basal progesterone production (p<0.01). Similarly, 6h exposure to 1 microg/ml CYN did not affect cytotoxicity or hCG-stimulated estrogen production, but did inhibit hCG-stimulated progesterone production (p<0.01). In this in vitro assay, CYN inhibited progesterone production and therefore has the potential to be an endocrine disrupter by changing the progesterone:estrogen ratio in women.

Bacterial degradation of microcystin toxins in drinking water eliminates their toxicity.

Microcystin-LR and -LA were readily biodegraded by a bacterium, Sphingpoyxis sp. LH21, in a treated reservoir water. Detection of the microcystins was conducted using high-performance liquid chromatography (HPLC), protein phosphatase 2A (PP2A) inhibition assay and a cell-based cytotoxicity assay. The HPLC results correlated well with the two assays. The decrease in cytotoxicity, coupled with the associated decrease in microcystin concentrations, indicated that no cytotoxic by-products were being generated, highlighting the applicability of biodegradation as a feasible treatment option for effective microcystin removal.

Application of the neuroblastoma assay for paralytic shellfish poisons to neurotoxic freshwater cyanobacteria: interlaboratory calibration and comparison with other methods of analysis.

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.

Extended Low-Dose Exposure to Saxitoxin Inhibits Neurite Outgrowth in Model Neuronal Cells.

The potent neurotoxin saxitoxin (STX) belongs to a group of structurally related analogues produced by both marine and freshwater phytoplankton. The toxins act by blocking voltage-gated sodium channels stopping the inflow of sodium ions and the generation of action potentials. Exposure from marine sources occurs as a result of consuming shellfish which have concentrated the toxins, and freshwater exposure can occur from drinking water although there have been no acute poisonings from the latter source to date. Previously, the majority of research into this group of toxins, collectively known as the paralytic shellfish toxins, has focused on acute exposure resulting in paralytic shellfish poisoning. While acute exposure guidelines exist for both sources, there are no chronic exposure guidelines and there has been minimal research into this pattern of exposure despite the known role of electrical activity in neurogenesis. We aimed to investigate this pattern of exposure and its potential effects on neurodevelopment using model neuronal cells. PC12 and SH-SY5Y cells were exposed to STX (0.25-3 µg/l) for 7 days, after which time they were stained with TRITC-Phalloidin, to observe adverse morphological effects. Cells exposed to STX had a significant decrease (18-85%) in long axonlike projections, instead exhibiting a significant increase in shorter projections classified as filopodia (p < 0.05). The results suggest that extended low-dose exposure to STX can inhibit proper neurite outgrowth at concentrations well below guideline levels for both sources of exposure making it a potential public health concern.

Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs.

Cyanobacteria represent a health hazard worldwide due to their production of a range of highly potent toxins in diverse aquatic environments. While planktonic species have been the subject of many investigations in terms of risk assessment, little is known about benthic forms and their impact on water quality or human and animal health. This study aimed to purify isolates from environmental benthic biofilms sampled from three different drinking water reservoirs and to assess their toxin production by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR). Microscopic observation of the isolates allowed the identification of various filamentous cyanobacterial genera: Anabaena (benthic form), Calothrix and Nostoc from the Nostocales and Geitlerinema, Leptolyngbya, Limnothrix, Lyngbya, Oxynema, Phormidium and Pseudanabaena representing non-heterocystous filamentous cyanobacteria. The Phormidium ambiguum strain AWQC-PHO021 was found to produce 739 ng/mg of dry weight (d/w) of cylindrospermopsin and 107 ng/mg (d/w) of deoxy-cylindrospermopsin. The Nostoc linckia strain AWQC-NOS001 produced 400 ng/mg (d/w) of a microcystin analogue. This is the first report of hepatotoxin production by benthic cyanobacteria in temperate Australian drinking water reservoirs. These findings indicate that water quality monitoring programs need to consider benthic cyanobacteria as a potential source of toxins.

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.

Hypothetical scenario exercises to improve planning and readiness for drinking water quality management during extreme weather events.

Two hypothetical scenario exercises were designed and conducted to reflect the increasingly extreme weather-related challenges faced by water utilities as the global climate changes. The first event was based on an extreme flood scenario. The second scenario involved a combination of weather events, including a wild forest fire ('bushfire') followed by runoff due to significant rainfall. For each scenario, a panel of diverse personnel from water utilities and relevant agencies (e.g. health departments) formed a hypothetical water utility and associated regulatory body to manage water quality following the simulated extreme weather event. A larger audience participated by asking questions and contributing key insights. Participants were confronted with unanticipated developments as the simulated scenarios unfolded, introduced by a facilitator. Participants were presented with information that may have challenged their conventional experiences regarding operational procedures in order to identify limitations in current procedures, assumptions, and readily available information. The process worked toward the identification of a list of specific key lessons for each event. At the conclusion of each simulation a facilitated discussion was used to establish key lessons of value to water utilities in preparing them for similar future extreme events.

Low dose extended exposure to saxitoxin and its potential neurodevelopmental effects: A review.

Saxitoxin (STX) and its analogs, the paralytic shellfish toxins (PSTs), are a group of potent neurotoxins well known for their role in acute paralytic poisoning by preventing the generation of action potentials in neuronal cells. They are found in both marine and freshwater environments globally and although acute exposure from the former has previously received more attention, low dose extended exposure from both sources is possible and to date has not been investigated. Given the known role of cellular electrical activity in neurodevelopment this pattern of exposure may be a significant public health concern. Additionally, the presence of PSTs is likely to be an ongoing and possibly increasing problem in the future. This review examines the neurodevelopmental toxicity of STX, the risk of extended or repeated exposure to doses with neurodevelopmental effects, the potential implications of this exposure and briefly, the steps taken and difficulties faced in preventing exposure.

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.

Health risk assessment of cyanobacterial (blue-green algal) toxins in drinking water.

Cyanobacterial toxins have caused human poisoning in the Americas, Europe and Australia. There is accumulating evidence that they are present in treated drinking water supplies when cyanobacterial blooms occur in source waters. With increased population pressure and depleted groundwater reserves, surface water is becoming more used as a raw water source, both from rivers and lakes/reservoirs. Additional nutrients in water which arise from sewage discharge, agricultural run-off or storm water result in overabundance of cyanobacteria, described as a 'water bloom'. The majority of cyanobacterial water-blooms are of toxic species, producing a diversity of toxins. The most important toxins presenting a risk to the human population are the neurotoxic alkaloids (anatoxins and paralytic shellfish poisons), the cyclic peptide hepatotoxins (microcystins) and the cytotoxic alkaloids (cylindrospermopsins). At the present time the only cyanobacteral toxin family that have been internationally assessed for health risk by the WHO are the microcystins, which cause acute liver injury and are active tumour promoters. Based on sub-chronic studies in rodents and pigs, a provisional Guideline Level for drinking water of 1 microg/L of microcystin-LR has been determined. This has been adopted in legislation in countries in Europe, South America and Australasia. This may be revised in the light of future teratogenicity, reproductive toxicity and carcinogenicity studies. The other cyanobacterial toxin which has been proposed for detailed health risk assessment is cylindrospermopsin, a cytotoxic compound which has marked genotoxicity, probable mutagenicity, and is a potential carcinogen. This toxin has caused human poisoning from drinking water, and occurs in water supplies in the USA, Europe, Asia, Australia and South America. An initial health risk assessment is presented with a proposed drinking water Guideline Level of 1 microg/L. There is a need for both increased monitoring data for toxins in drinking water and epidemiological studies on adverse health effects in exposed populations to clarify the extent of the health risk.