Cyanotoxins: producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation.

Cyanobacteria were present on the earth 3.5 billion years ago; since then they have colonized almost all terrestrial and aquatic ecosystems. They produce a high number of bioactive molecules, among which some are cyanotoxins. Cyanobacterial growth at high densities, forming blooms, is increasing in extension and frequency, following anthropogenic activities and climate changes, giving rise to some concern for human health and animal life exposed to cyanotoxins. Numerous cases of lethal poisonings have been associated with cyanotoxins ingestion in wild animal and livestock. In humans few episodes of lethal or severe human poisonings have been recorded after acute or short-term exposure, but the repeated/chronic exposure to low cyanotoxin levels remains a critical issue. The properties of the most frequently detected cyanotoxins (namely, microcystins, nodularins, cylindrospermopsin and neurotoxins) are here critically reviewed, describing for each toxin the available information on producing organisms, biosynthesis/genetic and occurrence, with a focus on the toxicological profile (including kinetics, acute systemic toxicity, mechanism and mode of action, local effects, repeated toxicity, genotoxicity, carcinogenicity, reproductive toxicity; human health effects and epidemiological studies; animal poisoning) with the derivation of health-based values and considerations on the risks for human health.

Risk to human health associated with the environmental occurrence of cyanobacterial neurotoxic alkaloids anatoxins and saxitoxins.

Cyanobacteria are ubiquitous photosynthetic micro-organisms forming blooms and scums in surface water; among them some species can produce cyanotoxins giving rise to some concern for human health and animal life. To date, more than 65 cyanobacterial neurotoxins have been described, of which the most studied are the groups of anatoxins and saxitoxins (STXs), comprising many different variants. In freshwaters, the hepatotoxic microcystins represent the most frequently detected cyanotoxin: on this basis, it could appear that neurotoxins are less relevant, but the low frequency of detection may partially reflect an a priori choice of target analytes, the low method sensitivity and the lack of certified standards. Cyanobacterial neurotoxins target cholinergic synapses or voltage-gated ion channels, blocking skeletal and respiratory muscles, thus leading to death by respiratory failure. This review reports and analyzes the available literature data on environmental occurrence of cyanobacterial neurotoxic alkaloids, namely anatoxins and STXs, their biosynthesis, toxicology and epidemiology, derivation of guidance values and action limits. These data are used as the basis to assess the risk posed to human health, identify critical exposure scenarios and highlight the major data gaps and research needs.

Human glutathione transferases catalyzing the conjugation of the hepatoxin microcystin-LR.

Many cyanobacterial species are able to produce cyanotoxins as secondary metabolites. Among them, microcystins (MC) are a group of around 80 congeners of toxic cyclic heptapeptides. MC-LR is the most studied MC congener, in view of its high acute hepatotoxicity and tumor promoting activity. Humans may be exposed to cyanotoxins through several routes, the oral one being the most important. The accepted pathway for MC-LR detoxication and excretion in the urine is GSH conjugation. The GSH adduct (GS-MCLR) formation has been shown to occur spontaneously and enzymatically, catalyzed by glutathione transferases (GSTs). The enzymatic reaction has been reported but not characterized both in vitro and in vivo in animal and plant species. No data are available on humans. In the present work, the MC-LR conjugation with GSH catalyzed by five recombinant human GSTs (A1-1, A3-3, M1-1, P1-1, and T1-1) has been characterized for the first time. All GSTs are able to catalyze the reaction; kinetic parameters K(m), k(cat), and their relative specific activities to form GS-MCLR were derived (T1-1 > A1-1 > M1-1 > A3-3 ≫ P1-1). In the range of MC tested concentrations used (0.25-50 µM) GSTT1-1 and A1-1 showed a typical saturation curve with similar affinity for MC-LR (≈80 µM; k(cat) values 0.18 and 0.10 min(-1), respectively), A3-3 and M1-1 were linear, whereas GSTP1-1 showed a temperature-dependent sigmoidal allosteric curve with a k(cat) = 0.11 min(-1). The enzymes mainly expressed in the liver and gastrointestinal tract, GSTA1-1, T1-1, and M1-1, seemed to be mainly involved in the MC-LR detoxification after oral exposure, whereas P1-1 kinetics and location in the skin suggest a role related to dermal exposure. Considering the high frequency of some GST polymorphism, especially M1 and T1 gene deletion, with complete loss in activity, this information could be the first step to identify groups of individual at higher risk associated with MC exposure.

Is Chronic Exposure to Raw Water a Possible Risk Factor for Amyotrophic Lateral Sclerosis? A Pilot Case-Control Study.

BACKGROUND: The etiopathogenesis of amyotrophic lateral sclerosis (ALS) is still largely unknown. METHODS: We performed a case-control study (33 cases and 35 controls) in Umbria, Italy. We investigated associations between common lifestyle, clinical factors, as well as environmental exposures potentially implicated with ALS onset. Face-to-face interviews were carried out. All cases were recruited and diagnosed according to El Escorial criteria. Case-control comparisons were made for educational and residential status, occupational exposures, and clinical and lifestyle factors prior to cases' dates of diagnosis. RESULTS: Our results showed an increased risk of ALS for subjects chronically exposed to raw water use (odds ratio (OR) = 6.55, 95% confidence interval (CI): 2.24-19.12). Garden activities showed a tight association with ALS as well, very likely as a consequence of chronic raw water exposure. Indeed, we could exclude an impact for pesticides, as no significant differences were observed in pesticide exposure in the two groups interviewed. However, cases were more often exposed to fertilizers. After adjustment for age, sex, and heavy physical activities, exposure to raw water was still associated with increased ALS risk (OR = 4.74, 95% CI: 1.33-16.85). DISCUSSION: These findings suggest an association between ALS and exposure to raw water, which should be further investigated for the presence of chemicals interfering with nervous system functionality.

Water-related diseases outbreaks reported in Italy.

Water related disease outbreak (WRDO) statistics in Italy from 1998 to 2005 have been discussed in this paper. The true incidence of WRDO is not reflected in the National Surveillance System (NSS), although this study has provided information on pathogens associated to different water sources, incidence in Regions and inadequacy of regulations. 192 outbreaks and 2546 cases of WRD were reported to the NSS, an average of 318 cases per year. Cases were associated to shellfish (58.79%), drinking water (39.94%) and agricultural products (1.25%). WRDs have been detected in 76% of Regions: central and southern Regions showed lower percentage of cases (35.4%) due to under-reporting. Most of WRD cases in the North were related to drinking water; WRDs in marine coastal Regions were mostly related to shellfish. 49% of Districts (Province) notified WRDs, including only 101 Municipalities. Pathogenic microorganisms were identified in a few cases from clinical investigations. They included enteric viruses, Norwalk viruses, Salmonella, Shigella, Giardia and Campylobacter. There is the need to improve the existing NSS in relation to WRDs. An adequate WRDs Surveillance System should be based on connection between health and environmental authorities, priority pathogens and critical areas identification, response capability and contingency plans.

Alkylphenols and their ethoxylates in seafood from the Tyrrhenian Sea.

The present study reports the results of an investigation on occurrence of octylphenols (OPs), nonylphenols (NPs) and their respective ethoxylates (with 1-6 ethoxylic group) in aquatic species of commercial interest from the Tyrrhenian Sea. Samples were collected at Livorno, Fiumicino and Salerno commercial harbors, during September-October 2003. The data obtained showed that almost all of the tested compounds were found in all the samples. NP was generally detected at the highest concentrations. Among the tested species common to the three sampling sites, mullets and seabreams generally showed high values of total alkylphenols (APs) and alkylphenol ethoxylates (APEOs) (44-55 ng g(-1) and 27-525 ng g(-1) fw), yet, the maximum concentrations were found in shrimps from Fiumicino (1255 ng g(-1) fw). On the contrary, the lowest concentrations were observed in hakes and anchovies (34-36 ng g(-1) and 6-37 ng g(-1) fw). Tuna, exhibited very high concentrations of total alkylphenolic compounds (APEs) (889 ng g(-1) fw). Seafood from Fiumicino fishing area, which is under the influence of the River Tiber, showed a degree of contamination of at least one order of magnitude higher than the other two sites. On the basis of the results of this study, the corresponding daily intakes of NPs are much lower than the TDI proposed by the Danish Environmental Agency.

Cyanobacteria blooms in water: Italian guidelines to assess and manage the risk associated to bathing and recreational activities.

Cyanobacteria thrive in many aquatic environments, where they can produce cyanotoxins with different toxicological profile. Anthropic pressure and climate changes are causing the expansion in terms of time and space of their blooms, increasing the concerns for human health in several exposure scenarios. Here the update of the Italian guidelines for the management of cyanobacterial blooms in bathing water is presented. A risk-based approach has been developed according to the current scientific knowledge on cyanobacteria distribution in the Italian Lakes and on chemical, toxicological and epidemiological aspects of different cyanotoxins, summarized in the first part of the paper. Oral, dermal and inhalation exposure to cyanotoxins, during recreational activities, are individually examined, to develop a framework of thresholds and actions aimed at preventing harmful effects for bathers. Guidelines, also by comparing international guidance values and/or guidelines, provide criteria to plan environmental monitoring activities, health surveillance and public communication systems. Finally the still important scientific gaps and research needs are highlighted.

Leaching potential of some phenylureas and their main metabolites through laboratory studies.

BACKGROUND, AIMS AND SCOPE: Laboratory studies were conducted with the aim of defining the leaching potential of some phenylureas and their metabolites. A first study was performed for calculating their leaching index (as GUS) on the base of intrinsic properties: persistence (as DT50) and mobility (as Koc) in soil. Another study consisted of aged column leaching experiments whose meaning was to semi-quantify the occurrence of the tested compounds in the leachates, so simulating in field conditions. METHODS: The tested compounds were: diuron, linuron and monolinuron (parents); 3,4-dichloroaniline (DCA), 4-chloroaniline (CLA), 1-(3,4-dichlorophenyl)-3-methylurea (DCPMU), 1-(3,4-dichlorophenyl)urea (DCPU), 1-(4-chlorophenyl)urea (CPU) and monuron, this latter considered both as a metabolite and parent compound. The Koc values of the examined substances were determined by the HPLC screening methods, according to the OECD TG 121. DT50 determinations and aged column leaching experiments were carried out according to SETAC procedures. RESULTS AND DISCUSSION: The examined compounds showed a rather wide range of persistence in soil, with DT50 values less than 2 days for DCA and CLA, close to 8 days for DCPU and CPU and from 16 (diuron) up to 24.8 (DCPMU) days for the others. Their mobility was generally high, based on their Koc values, which ranged from 33 (CPU) to 406 (linuron). The GUS indices indicated that monuron has a clear potential to contaminate groundwater (> 2.8); DCPMU, monolinuron, CPU and diuron are intermediate contaminants (1.8-2.8). Linuron, DCPU, CLA and DCA exhibited a non-leaching behaviour (< 1.8). The aged leaching column experiments showed that parents were found in the leachates at very high percentages respect to the doses applied. The metabolites reached much less percentages, the highest values were observed for monuron from diuron (5.7), CPU (7.2) and DCPMU (8.2%). CONCLUSION: Diuron, Monuron, CPU and DCPMU on the basis of their intrinsic properties, formation from their parents and occurrence in leachates from aged column leaching studies, seem to possess the characteristics of groundwater contaminants. The methodological approach of this study is relatively easy and rapid, hence it can represent a tool for a first screening of compounds such as pesticide metabolites (generally available only in small quantities and for which a field study is not conceivable) or other compounds for which not adequate environmental data are available.

Cyanobacteria biennal dynamic in a volcanic mesotrophic lake in central Italy: Strategies to prevent dangerous human exposures to cyanotoxins.

Vico Lake, a volcanic meso-eutrophic lake in Central Italy, whose water is used for drinking and recreational activities, experienced the presence of the microcystins (MC) producing cyanobacterium Planktothrix rubescens. In order to assess the human health risks and to provide the local health authorities with a scientific basis for planning tailored monitoring activities, we studied P. rubescens ecology and toxicity for two years. P. rubescens generally dominated the phytoplankton community, alternating with Limnothrix redekei, potentially toxic. P. rubescens was distributed throughout the water column during winter; in summer it produced intense blooms where drinking water is collected (-20 m); here MC were detected all year round (0.5-5 µg/L), with implications for drinking water quality. In surface waters, MC posed no risk for recreational activities in summer, while in winter surface blooms and foams (containing up to 56 µg MC/L) can represent a risk for people and children practicing water sports and for animals consuming raw water. Total phosphorus, phosphate and inorganic nitrogen were not relevant to predict densities nor toxicity; however, a strong correlation between P. rubescens density and aminopeptidase ectoenzymatic activity, an enzyme involved in protein degradation, suggested a role of organic nitrogen for this species. The fraction of potentially toxic population, determined both as mcyB(+)/16SrDNA (10-100%) and as the MC/mcyB(+) cells (0.03-0.79 pg MC/cell), was much more variable than usually observed for P. rubescens. Differently from other Italian and European lakes, the correlation between cell density or the mcyB(+) cells and MC explained only ∼50 and 30% of MC variability, respectively: for Vico Lake, monitoring only cell or the mcyB(+) cell density is not sufficient to predict MC concentrations, and consequently to protect population health. Finally, during a winter bloom one site has been sampled weekly, showing that monthly sampling during such a phase could greatly underestimate the 'hazard'. Our results highlight the need to adopt a stepwise monitoring activity, considering the lake and the cyanobacteria specific features. This activity should be complemented with communication to the public and involvement of stakeholders.

Alkylphenols and alkylphenol ethoxylates contamination of crustaceans and fishes from the Adriatic Sea (Italy).

This paper presents the results of an investigation on the occurrence of alkylphenols (APs) and their ethoxylates (APEs) in 8 edible marine species from the Adriatic Sea and tries to estimate the corresponding intake for the Italian population. Two crustaceans, Nephrops norvegicus (Norway lobster) and Squilla mantis (spottail mantis shrimp), plus six fish species, Engraulis enchrascicolus (anchovy), Scomber scombrus (Atlantic mackerel), Merluccius merluccius (European hake), Mullus barbatus (red mullet), Solea vulgaris (common sole) and Lophius piscatorius (angler) were analyzed for their content of nonylphenol (NP), octylphenol (OP) and octylphenol polyethoxylates (OPEs). These compounds were found in all analysed samples. NP was detected at the highest concentrations: 118-399 and 9.5-1431 ng g(-1) fresh weight (fw) respectively in crustaceans and fish. OP was found at respective levels of 2.7-4.7 and 0.3-3.8 ng g(-1) fw in crustaceans and fish, whereas OPE was determined at respective concentrations of 1.2-16.8 and 0.2-21.1 ng g(-1) fw in the same species. These results, together with those from a previous study on 4 edible mollusc, allow to estimate respective daily intakes for NP, OP, and OPE of about 12, 0.1, and 0.1 microg day(-1) for an Italian adult living along the Adriatic Coast. In relation to NP and OP, these intakes are much lower than the doses associated with toxic effects in laboratory animals (9 mg kg(-1) bw for rats). Nevertheless, data of exposure from other sources to these chemicals and others with similar biological characteristics are needed.

[Mucilage in the Adriatic Sea: role of possible causal agents and environmental factors]. / Le mucillagini nel Mar Adriatico: ruolo dei possibili agenti causali e dei fattori ambientali.

The massive accumulation of gelatinous material at and below the seawater surface occurred in Northern Adriatic, at a frequency and with such an extension to cause serious environmental and economic damages. The present review describes the updated knowledge and considers the main hypotheses on mucilage formation. They focus, in turn, on organisms that produce mucilage, on the particular hydrodynamic of the North Adriatic basin, on the role of nutrients, but the trigger mechanism has not yet been really understood. Finally, the theoretical framework recently proposed by Azam et al. is presented. In the organic matter continuum view, organisms of the microbial loop just represent a part of the whole picture from which specific mechanisms of interactions are examined and few important pathways are outlined for future research.

[Risk to human health associated with marine toxic algae]. / Il rischio sanitario associato alle tossine di alghe marine.

Among the about 5000 of marine algal species, some 75 produce algal toxins. These latter mainly belong to the taxa first of dynoflagellates and then diatomes. The so far known human intoxications have been associated with mollusc consumption. The main poisoning syndromes have been termed, on the basis of the observed symptoms, as paralytic, diarrhetic, neurotoxic and amnesic and shorthened as: PSP (paralytic shellfish poisoning), DSP (diarrhetic shellfish poisoning), NSP (neurotoxic shellfish poisoning), ASP (amnesic shellfish poisoning), respectively. This paper is a review of the problem of human health implications associated with marine toxic algae, with particular reference to the situation of the Mediterranean and the Italian coastal areas.

[Accidents in recreational waters]. / Incidenti nelle aree di balneazione.

Several countries fail to systematically collect statistical data on accidents in bathing waters of recreational areas. Yet, drowning, near-drowning, head trauma and spinal chord injuries are important causes of mortality and morbidity throughout the world. In Italy, the available statistical data are circumscribed to drowning, and show that between 1969 and 1997 the mortality dropped from some 1,200 to some 500 cases/year, and its rate from 21 to 7.5 deaths per million resident/year. The mortality for drowning is much higher in males than in females, but in the period examined it halved in both the sexes. From 1969 to 1997, the mortality rates considerably decreased in all the age subgroups, but particularly in that of young people. The analysis of these data indicates that some general factors have played an important role, like a better education and information, a greater surveillance by adults and a better organisation of summer resorts. A national strategy aimed at preventing recreational accidents would provide much better results than those obtained without any planning.

Survival, growth and toxicity of Microcystis aeruginosa PCC 7806 in experimental conditions mimicking some features of the human gastro-intestinal environment.

Cyanotoxins (CTX) are widely produced by several cyanobacteria (CB), increasingly spreading in most water bodies and terrestrial habitats, and represent a risk for human health. CB are prokaryotes, and although mostly autotrophic, several examples of heterotrophy in symbiotic relationship with different organisms have been described. In addition to the known routes of exposure, it has been hypothesized that CB might 'colonize' human intestine with relevant implications for human health. Colonization is a complex process and requires specific features of the possible invaders. Still, a short-term persistence as living and toxin-producing organisms within the intestinal lumen of the host could represent an 'internal' source of exposure to CTX. In this work we ran microcosm experiments (4-18days), looking at Microcystis aeruginosa PCC7806 resistance and cyanotoxin-producing capabilities in darkness, 37°C, pH 2, and subsequent recovery in a rich medium, in darkness, 37°C, in the presence of enteric bacteria, mimicking few important features of the gastrointestinal environment. We measured cyanobacterial populations and growth, microcystin (MC) production and the presence of mcyB gene. M. aeruginosa could grow in the dark at 37°C up to 17days, and survive at pH 2 at a rate between 30% and 70%, depending on the age and toxicity of the starting culture. Cell lysis resulted in a substantial amounts of MC released, not degraded at gastric pH. Following the acidic passage, still in the dark at 37°C, M. aeruginosa restarted to grow within 24h for the next 3-4days, independently on the presence of intestinal bacteria, maintaining the MC cell quota and mcyB gene. Our results show new features of CB: a significant resistance of M. aeruginosa in conditions far from its optimal one, that is an environment mimicking some of the important characteristics of human gastrointestinal tract, suggesting the possibility of an internal source of exposure to CTX, with implications for the risk assessment.

The conjugation of microcystin-RR by human recombinant GSTs and hepatic cytosol.

Many cyanobacterial species can produce cyanotoxins, among which mycrocistins (MC) are a group of ≈100 congeners of hepatotoxic cyclic heptapeptides. MC-RR differs from MC-LR, the most studied congener only for one residue (arginine vs leucine), resulting in a ten-fold difference in the acute toxicity in mice. Although humans may be exposed to MC through several routes and kinetics appeared to be the major factor affecting congener-specific toxicity, little is known on MC metabolism. The accepted pathway for MC detoxication is GSH conjugation: here the MC-RR conjugation with GSH catalyzed by 5 recombinant human GSTs and human liver cytosol (HLC) has been characterized and appeared to be more efficient than MC-LR conjugation. The catalytic efficiency score is T1-1>A1-1≈P1-1>M1-1>A3-3 (0.161-0.056pmol GSMC-RR (µgproteinminµM)(-1)). In HLC the spontaneous reaction is favored vs the enzymatic one (ratio 3:1) at physiological GSH content. However, at low MC-RR concentrations, representative of repeated oral exposure, and low GSH content (down to 0.05mM), possibly associated to exposure to drugs or in patients affected by several pathologies, the relevance of the enzymatic reaction progressively increases, providing the predominant contribution to MC-RR detoxication.

Impact of climate change on waterborne diseases.

Change in climate and water cycle will challenge water availability but it will also increase the exposure to unsafe water. Floods, droughts, heavy storms, changes in rain pattern, increase of temperature and sea level, they all show an increasing trend worldwide and will affect biological, physical and chemical components of water through different paths thus enhancing the risk of waterborne diseases. This paper is intended, through reviewing the available literature, to highlight environmental changes and critical situations caused by floods, drought and warmer temperature that will lead to an increase of exposure to water related pathogens, chemical hazards and cyanotoxins. The final aim is provide knowledge-based elements for more focused adaptation measures.

Contamination by Microcystis and microcystins of blue-green algae food supplements (BGAS) on the Italian market and possible risk for the exposed population.

Blue green algae supplements (BGAS) are generally proposed as health-promoting natural products for their purported beneficial effects. Spirulina spp. and Aphanizomenon flos aquae are mainly used in BGAS production. They are usually collected from the natural environment, where other potentially toxic cyanobacteria can be present, making possible BGAS contamination by cyanotoxins, with potential risk for human health. In this work we apply a combined approach, by using chemical and molecular techniques, on BGAS of 17 brands available in Italy. Samples containing Spirulina-only were free of contamination. The Aphanizomenon flos aquae-based samples were contaminated by highly variable levels of microcystins (MC-LR and MC-LA congeners), up to 5.2 µg MC-LR equivalents per gram product. The highest variability (up to 50 fold) was among batches of the same brand, although intra-batch differences were also evidenced. PCR analyses were positive only for the presence of Microcystis sp., identified as the toxin-producing species responsible for contamination. At the maximum contamination levels found, a risk for consumers can be expected following chronic or sub-chronic exposure to a reasonable daily BGAS consumption of 4 g. The need for a strict monitoring by producers and Health Authority to assure an adequate protection for consumers is underscored.

Emerging health issues of cyanobacterial blooms.

This paper describes emerging issue related to cyanobacterial dynamics and toxicity and human health risks. Data show an increasing cyanobacteria expansion and dominance in many environments. However there are still few information on the toxic species fitness, or on the effects of specific drivers on toxin production. Open research fields are related to new exposure scenario (cyanotoxins in water used for haemodialysis and in food supplements); to new patterns of co-exposure between cyanotoxins and algal toxins and/or anthropogenic chemicals; to dynamics affecting toxicity and production of different cyanotoxin variants under environmental stress; to the accumulation of cyanotoxins in the food web. In addition, many data gaps exist in the characterization of the toxicological profiles, especially about long term effects.

Alkylphenols in adipose tissues of Italian population.

Alkylphenols (APs) and AP ethoxylated compounds (APEs) were screened in human subcutaneous adipose tissue samples from Italy. The samples were collected during bariatric surgery from 16 subjects (three men and 13 women) and a total of seven alkylphenol compounds (APs) was detected. Nonylphenol (NP) was the compound found at the highest level (mean 122 ng g⁻¹ fresh weight; range 10-266 ng g⁻¹ fw). Several nonylphenol ethoxylates (NPEOs) were found in all the sample analysed though the frequency of detection decreased with the increasing number of ethoxylic groups. NP4EO was found only in four patients ranging from trace amounts to 41.3 ng g⁻¹ fw. Total nonylphenols (NPEs) ranged between 45 and 1131 ng g⁻¹ fw, whereas the concentration of total octylphenols (OPEs) was at least 10 times lower (range 6-80 ng g⁻¹ fw). Our findings show that the average concentration of NP is about two times higher than that found in women from Southern Spain and up to three times that of people from Switzerland. Similarly, OP mean level is two times that reported in Finland and Spain populations. This is the first study that reports the presence of alkylphenols in the Italian population adipose tissue and it draws a baseline for further researches in order to depict a trend in human exposure to these compounds and to investigate possible consequences for human health.