N-nitrosodimethylamine and trihalomethane formation and minimisation in Southeast Queensland drinking water.

This study assesses the prevalence of disinfection by-product (DBP) precursors in some Southeast Queensland drinking water sources by conducting formation potential experiments for the four regulated trihalomethanes (THMs), and the potent carcinogen, N-nitrosodimethylamine (NDMA). NDMA formation potentials were consistently low (<5-21 ng/L), and total THM (tTHM) formation potentials were consistently below the Australian Drinking Water Guideline (250 µg/L). NDMA concentration of finished drinking waters was also monitored and found to be <5 ng/L in all cases. The effect of coagulation and advanced oxidation on the formation of NDMA and THMs is also reported. UV/H(2)O(2) pre-treatment was effective in producing water with very low THMs concentrations, and UV irradiation was an effective method for NDMA degradation. H(2)O(2) was not required for the observed NDMA degradation to occur. Coagulation using alum, ferric chloride or poly(diallyldimethylammonium chloride) (polyDADMAC) was ineffective in removing DBPs precursors from the source water studied, irrespective of the low dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) attained. Rather, coagulation with polyDADMAC caused an increase in NDMA formation potential upon chloramination, and all coagulants led to an increased tTHM formation potential upon chlorination due to the high bromide concentration of the source water studied.

Occupational and environmental hazard assessments for the isolation, purification and toxicity testing of cyanobacterial toxins.

Cyanobacteria can produce groups of structurally and functionally unrelated but highly potent toxins. Cyanotoxins are used in multiple research endeavours, either for direct investigation of their toxicologic properties, or as functional analogues for various biochemical and physiological processes. This paper presents occupational safety guidelines and recommendations for personnel working in field, laboratory or industrial settings to produce and use purified cyanotoxins and toxic cyanobacteria, from bulk harvesting of bloom material, mass culture of laboratory isolates, through routine extraction, isolation and purification. Oral, inhalational, dermal and parenteral routes are all potential occupational exposure pathways during the various stages of cyanotoxin production and application. Investigation of toxicologic or pharmacologic properties using in vivo models may present specific risks if radiolabelled cyanotoxins are employed, and the potential for occupational exposure via the dermal route is heightened with the use of organic solvents as vehicles. Inter- and intra-national transport of living cyanobacteria for research purposes risks establishing feral microalgal populations, so disinfection of culture equipment and destruction of cells by autoclaving, incineration and/or chlorination is recommended in order to prevent viable cyanobacteria from escaping research or production facilities.

Dermatitis associated with exposure to a marine cyanobacterium during recreational water exposure.

BACKGROUND: Anecdotal evidence reported an outbreak of symptoms on Fraser Island during the late 1990 s similar to those expected from exposure to dermotoxins found in the cyanobacterium L. majuscula. This coincided with the presence of a bloom of L. majuscula. METHODS: Records from the Fraser Island National Parks First aid station were examined. Information on cyanobacterial blooms at Fraser Island were obtained from Queensland National Parks rangers. RESULTS: Examination of first aid records from Fraser Island revealed an outbreak of symptoms predominantly in January and February 1998. CONCLUSION: During a bloom of L. majuscula there were numerous reports of symptoms that could be attributed to dermotoxins found in L. majuscula. The other four years examined had no L. majuscula blooms and the number of L. majuscula symptoms was much reduced. These cases comprised a high percentage of the cases treated at the first aid station.

Cyanobacterial poisoning in livestock, wild mammals and birds--an overview.

Poisoning of livestock by toxic cyanobacteria was first reported in the 19th century, and throughout the 20th century cyanobacteria-related poisonings of livestock and wildlife in all continents have been described. Some mass mortality events involving unrelated fauna in prehistoric times have also been attributed to cyanotoxin poisoning; if correct, this serves as a reminder that toxic cyanobacteria blooms predate anthropogenic manipulation of the environment, though there is probably general agreement that human intervention has led to increases in the frequency and extent of cyanobacteria blooms. Many of the early reports of cyanobacteria poisoning were anecdotal and circumstantial, albeit with good descriptions of the appearance and behaviour of cyanobacteria blooms that preceded or coincided with illness and death in exposed animals. Early necropsy findings of hepatotoxicity were subsequently confirmed by experimental investigations. More recent reports supplement clinical and post-mortem findings with investigative chemistry techniques to identify cyanotoxins in stomach contents and tissue fluids.

Brine shrimp bioassay: importance of correct taxonomic identification of Artemia (Anostraca) species.

Despite the common use of the brine shrimp bioassay in toxicology, there is confusion in the literature regarding citation of the correct taxonomic identity of the Artemia species used. The genus Artemia, once thought to be represented by a single species Artemia salina, is now known to be composed of several bisexual species as well as parthenogenetic populations. Artemia franciscana is the best studied of the Artemia species and is considered to represent the vast majority of studies in which Artemia is used as an experimental test organism. We found that in studies referring to the use of A. salina, the zoogeography of the cyst harvest site indicated that the species used was actually A. franciscana. Those performing bioassays with Artemia need to exercise diligence in assigning correct species identification, as the identity of the test organism is an important parameter in assuring the validity of the results of the assay.

Studies of the comparative in vitro toxicology of the cyanobacterial metabolite deoxycylindrospermopsin.

Cyanobacteria are capable of producing metabolites that are in some cases toxic to humans and other animals. Of these metabolites, the toxin cylindrospermopsin (CYN) is produced by a number of species of cyanobacteria including Cylindrospermopsis raciborskii, and its toxicity has been documented. The CYN analog deoxycylindrospermopsin (deoxyCYN) is commonly produced in varying proportions by the cyanobacteria that produce CYN. The toxicological profile of CYN suggests that it is primarily a hepatotoxin, but with the capacity to damage other organs and tissues. Limited in vivo information is available on the toxicity of deoxyCYN and suggests it to be of low potency. The aim of this research was to determine the comparative toxicology of deoxyCYN using in vitro systems. Using cell viability assays, it was shown that deoxyCYN had inhibitory effects on cell viability and proliferation of a similar magnitude to that of CYN. Morphological changes in deoxyCYN-treated cells were similar to those of CYN. Investigation of protein synthesis inhibition demonstrated that deoxyCYN was of similar potency to CYN. Inhibition of protein synthesis is an acknowledged mechanism of toxicity for CYN, and the results produced here suggest that deoxyCYN operates by similar toxicological mechanisms to CYN and that in vivo animal testing should be undertaken to clarify the potential for risk to humans from this toxin.

Induction of p53-regulated gene expression in human cell lines exposed to the cyanobacterial toxin cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that induces a range of genotoxic indicators in a variety of models. The possible involvement of the tumor suppressor protein p53 in cylindrospermopsin-induced gene expression was examined in cultured human dermal fibroblasts and the human hepatocellular carcinoma cell line HepG2. After 6 h of exposure to CYN, concentration-dependent increases in mRNA levels were observed for the p53 target genes CDKN1A, GADD45alpha, BAX, and MDM2, indicating an early activation of p53. After 24 h, relative mRNA levels for these genes remained elevated. Accumulation of p53 protein occurred after longer exposures in the HepG2-derived cell line C3A. Data suggest that cylindrospermopsin induces stress responses that result in the activation of the p53 transcription factor.

Occurrence and seasonal variations of algal toxins in water, phytoplankton and shellfish from North Stradbroke Island, Queensland, Australia.

A number of marine microalgae are known to produce toxins that can accumulate in shellfish and when eaten, lead to toxic and potentially fatal reactions in humans. This paper reports on the occurrence and seasonal variations of algal toxins in the waters, phytoplankton and shellfish of Southeast Queensland, Australia. These algal toxins include okadaic acid (OA), domoic acid (DA), gymnodimine (GD), pectenotoxin-2 (PTX-2) and pectenotoxin-2-seco acid (PTX-2-SA), which were detected in the sampled shellfish and phytoplankton, via HPLC-MS/MS. Dissolved OA, PTX-2 and GD were also detected in the samples collected from the water column. This was the first occasion that DA and GD have been reported in shellfish, phytoplankton and the water column in Queensland waters. Phytoplankton tows contained both the toxic Dinophysis and Pseudo-nitzschia algae species, and are suspected of being the most likely producers of the OA, PTX-2s and DA found in shellfish of this area. The number of cells, however, did not correlate with the amount of toxins present in either shellfish or phytoplankton. This indicates that toxin production by algae varies with time and the species present and that number of cells alone cannot be used as an indicator for the presence of toxins. The presence of OA and PTX-2s were more frequently seen in the summer, while DA and GD were detected throughout the year and without any obvious seasonal patterns.

Cyanobacterial lipopolysaccharides and human health - a review.

Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae. We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans. There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation.

Recreational and occupational field exposure to freshwater cyanobacteria--a review of anecdotal and case reports, epidemiological studies and the challenges for epidemiologic assessment.

Cyanobacteria are common inhabitants of freshwater lakes and reservoirs throughout the world. Under favourable conditions, certain cyanobacteria can dominate the phytoplankton within a waterbody and form nuisance blooms. Case reports and anecdotal references dating from 1949 describe a range of illnesses associated with recreational exposure to cyanobacteria: hay fever-like symptoms, pruritic skin rashes and gastro-intestinal symptoms are most frequently reported. Some papers give convincing descriptions of allergic reactions while others describe more serious acute illnesses, with symptoms such as severe headache, pneumonia, fever, myalgia, vertigo and blistering in the mouth. A coroner in the United States found that a teenage boy died as a result of accidentally ingesting a neurotoxic cyanotoxin from a golf course pond. This death is the first recorded human fatality attributed to recreational exposure to cyanobacteria, although uncertainties surround the forensic identification of the suspected cyanotoxin in this case. We systematically reviewed the literature on recreational exposure to freshwater cyanobacteria. Epidemiological data are limited, with six studies conducted since 1990. Statistically significant increases in symptoms were reported in individuals exposed to cyanobacteria compared to unexposed counterparts in two Australian cohort studies, though minor morbidity appeared to be the main finding. The four other small studies (three from the UK, one Australian) did not report any significant association. However, the potential for serious injury or death remains, as freshwater cyanobacteria under bloom conditions are capable of producing potent toxins that cause specific and severe dysfunction to hepatic or central nervous systems. The exposure route for these toxins is oral, from ingestion of recreational water, and possibly by inhalation.A range of freshwater microbial agents may cause acute conditions that present with features that resemble illnesses attributed to contact with cyanobacteria and, conversely, acute illness resulting from exposure to cyanobacteria or cyanotoxins in recreational waters could be misdiagnosed. Accurately assessing exposure to cyanobacteria in recreational waters is difficult and unreliable at present, as specific biomarkers are unavailable. However, diagnosis of cyanobacteria-related illness should be considered for individuals presenting with acute illness following freshwater contact if a description is given of a waterbody visibly affected by planktonic mass development.

Epidemiology of recreational exposure to freshwater cyanobacteria--an international prospective cohort study.

BACKGROUND: Case studies and anecdotal reports have documented a range of acute illnesses associated with exposure to cyanobacteria and their toxins in recreational waters. The epidemiological data to date are limited; we sought to improve on the design of some previously conducted studies in order to facilitate revision and refinement of guidelines for exposure to cyanobacteria in recreational waters. METHODS: A prospective cohort study was conducted to investigate the incidence of acute symptoms in individuals exposed, through recreational activities, to low (cell surface area < 2.4 mm2/mL), medium (2.4-12.0 mm2/mL) and high (> 12.0 mm2/mL) levels of cyanobacteria in lakes and rivers in southeast Queensland, the central coast area of New South Wales, and northeast and central Florida. Multivariable logistic regression analyses were employed; models adjusted for region, age, smoking, prior history of asthma, hay fever or skin disease (eczema or dermatitis) and clustering by household. RESULTS: Of individuals approached, 3,595 met the eligibility criteria, 3,193 (89%) agreed to participate and 1,331 (37%) completed both the questionnaire and follow-up interview. Respiratory symptoms were 2.1 (95%CI: 1.1-4.0) times more likely to be reported by subjects exposed to high levels of cyanobacteria than by those exposed to low levels. Similarly, when grouping all reported symptoms, individuals exposed to high levels of cyanobacteria were 1.7 (95%CI: 1.0-2.8) times more likely to report symptoms than their low-level cyanobacteria-exposed counterparts. CONCLUSION: A significant increase in reporting of minor self-limiting symptoms, particularly respiratory symptoms, was associated with exposure to higher levels of cyanobacteria of mixed genera. We suggest that exposure to cyanobacteria based on total cell surface area above 12 mm2/mL could result in increased incidence of symptoms. The potential for severe, life-threatening cyanobacteria-related illness is likely to be greater in recreational waters that have significant levels of cyanobacterial toxins, so future epidemiological investigations should be directed towards recreational exposure to cyanotoxins.

Primary irritant and delayed-contact hypersensitivity reactions to the freshwater cyanobacterium Cylindrospermopsis raciborskii and its associated toxin cylindrospermopsin.

BACKGROUND: Freshwater cyanobacteria are common inhabitants of recreational waterbodies throughout the world; some cyanobacteria can dominate the phytoplankton and form blooms, many of which are toxic. Numerous reports in the literature describe pruritic skin rashes after recreational or occupational exposure to cyanobacteria, but there has been little research conducted on the cutaneous effects of cyanobacteria. Using the mouse ear swelling test (MEST), we sought to determine whether three toxin-producing cyanobacteria isolates and the purified cyanotoxin cylindrospermopsin produced delayed-contact hypersensitivity reactions. METHODS: Between 8 and 10 female Balb/c mice in each experiment had test material applied to depilated abdominal skin during the induction phase and 10 or 11 control mice had vehicle only applied to abdominal skin. For challenge (day 10) and rechallenge (day 17), test material was applied to a randomly-allocated test ear; vehicle was applied to the other ear as a control. Ear thickness in anaesthetised mice was measured with a micrometer gauge at 24 and 48 hours after challenge and rechallenge. Ear swelling greater than 20% in one or more test mice is considered a positive response. Histopathology examination of ear tissues was conducted by independent examiners. RESULTS: Purified cylindrospermopsin (2 of 9 test mice vs. 0 of 5 control mice; p = 0.51) and the cylindrospermopsin-producing cyanobacterium C. raciborskii (8 of 10 test mice vs. 0 of 10 control mice; p = 0.001) were both shown to produce hypersensitivity reactions. Irritant reactions were seen on abdominal skin at induction. Two other toxic cyanobacteria (Microcystis aeruginosa and Anabaena circinalis) did not generate any responses using this model. Histopathology examinations to determine positive and negative reactions in ear tissues showed excellent agreement beyond chance between both examiners (kappa = 0.83). CONCLUSION: The irritant properties and cutaneous sensitising potential of cylindrospermopsin indicate that these toxicological endpoints should be considered by public health advisors and reservoir managers when setting guidelines for recreational exposure to cyanobacteria.

Cutaneous hypersensitivity reactions to freshwater cyanobacteria--human volunteer studies.

BACKGROUND: Pruritic skin rashes associated with exposure to freshwater cyanobacteria are infrequently reported in the medical and scientific literature, mostly as anecdotal and case reports. Diagnostic dermatological investigations in humans are also infrequently described. We sought to conduct a pilot volunteer study to explore the potential for cyanobacteria to elicit hypersensitivity reactions. METHODS: A consecutive series of adult patients presenting for diagnostic skin patch testing at a hospital outpatient clinic were invited to participate. A convenience sample of volunteers matched for age and sex was also enrolled. Patches containing aqueous suspensions of various cyanobacteria at three concentrations were applied for 48 hours; dermatological assessment was made 48 hours and 96 hours after application. RESULTS: 20 outpatients and 19 reference subjects were recruited into the study. A single outpatient produced unequivocal reactions to several cyanobacteria suspensions; this subject was also the only one of the outpatient group with a diagnosis of atopic dermatitis. No subjects in the reference group developed clinically detectable skin reactions to cyanobacteria. CONCLUSION: This preliminary clinical study demonstrates that hypersensitivity reactions to cyanobacteria appear to be infrequent in both the general and dermatological outpatient populations. As cyanobacteria are widely distributed in aquatic environments, a better appreciation of risk factors, particularly with respect to allergic predisposition, may help to refine health advice given to people engaging in recreational activities where nuisance cyanobacteria are a problem.

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants.

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 microg/l drinking water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check, the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 microg/l were found for MCs, PSPs and CYN, respectively. However, only traces (<1.0 microg/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety.

Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil.

In February 1996, an outbreak of illness occurred at a hemodialysis clinic in Caruaru, Pernambuco State-Brazil. At this clinic 116 (89%) of 131 patients experienced visual disturbances, nausea, vomiting, and muscle weakness, following routine haemodialysis treatment. Subsequently, 100 patients developed acute liver failure. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called 'Caruaru Syndrome'. Examination of previous years' phytoplankton counts showed that cyanobacteria were dominant in the water supply reservoir since 1990. Analyses of carbon and other resins from the clinic's water treatment system plus serum and liver tissue of patients led to the identification of two groups of hepatotoxic cyanotoxins: microcystins (cyclic heptapeptides) in all of these samples and cylindrospermopsin (alkaloid hepatotoxic) in the carbon and resins. Comparison of victims symptoms and pathology with animal studies on these two cyanotoxins, leads us to conclude that the major contributing factor to death of the dialysis patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR and -AR. In 2000, a review of the Brazilian regulation for drinking water quality, promoted by Brazilian Health Ministry with collaboration of PAHO, incorporated cyanobacteria and cyanotoxins into this new regulation as parameters that must to be monitored for water quality control.

Trace organic compounds in the marine environment.

Trace organic chemicals include a range of compounds which, due to a combination of their physico-chemical properties and toxicological implications, have been described as a serious threat to the biotic environment. A global treaty to regulate the manufacture and release of some of the most persistent trace chemicals has been promulgated and signed. The marine environment is an important sink for many trace chemicals, some of which accumulate in the marine food chain and in particular in marine mammals. With respect to the global distribution of trace organic chemicals, the levels of organohalogen compounds in the Southern Hemisphere are comparatively lower for a given environmental compartment and latitude compared to the Northern Hemisphere. A debate is currently evolving about the toxicity of alternative halogen substitutions such as bromine instead of chlorine and also of mixed halogen substitution. Recently a series of potentially natural bioaccumulative and persistent organohalogen chemicals have been found in marine mammals and turtles at levels in excess of those of anthropogenic trace organochlorines including PCBs and DDE. Little is known about the sources, behaviour and effects of natural trace organic chemicals. This manuscript presents an overview on the occurrence of trace organic chemicals in different compartments of the aquatic environment. Important knowledge gaps with regards to trace chemicals in the marine environment are presented.

Health risk assessment for cyanobacterial toxins in seafood.

Cyanobacteria (blue-green algae) are abundant in fresh, brackish and marine waters worldwide. When toxins produced by cyanobacteria are present in the aquatic environment, seafood harvested from these waters may present a health hazard to consumers. Toxicity hazards from seafood have been internationally recognised when the source is from marine algae (dinoflagellates and diatoms), but to date few risk assessments for cyanobacterial toxins in seafood have been presented. This paper estimates risk from seafood contaminated by cyanobacterial toxins, and provides guidelines for safe human consumption.

Establishing a public health analytical service based on chemical methods for detecting and quantifying Pacific ciguatoxin in fish samples.

A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 microg/kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 microg/kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p<0.001, Fishers Exact Test).

Human fatality associated with Pacific ciguatoxin contaminated fish.

Ciguatera is a food poisoning identified as the principal risk factor in the consumption of tropical fish in Oceania. The syndrome, which follows ingestion of ciguatoxin-contaminated ciguateric fishes, is characterised by an array of gastrointestinal and neurological features. In this report we examine forensic samples associated with a human fatality using a (3)H-brevetoxin binding assay and reversed-phase HPLC/MS and HPLC/MS/MS. Three Pacific ciguatoxins (P-CTX) were detected in the implicated fish flesh sample by LC-MS/MS, implicating multiple P-CTXs in the fatal case. Additionally, ciguatoxin was identified in a liver sample obtained at post-mortem. The level of ciguatoxin detected (0.14 ppb P-CTX-1 equivalents by binding assay) indicated that at least 10% of the ingested P-CTX-1 remained in the human liver 6 days after the toxic fish was consumed. This study confirms the potential of tropical reef fish to accumulate sufficient P-CTX to be lethal to humans, especially if the liver and viscera are consumed as part of the meal.