New Zealand Guidelines for cyanobacteria in recreational fresh waters: Interim Guidelines

This document is divided into four main sections, plus 14 appendices. Section 1. Introduction provides an overview of the purpose and status of the document as well as advice on who should use it. Section 2. Framework provides a background to the overall guidelines approach, recommendations on agency roles and responsibilities, and information on the condition of use of this document. Section 3. Guidelines describes the recommended three-tier monitoring and action sequence for planktonic and benthic cyanobacteria. Section 4. Sampling provides advice on sampling planktonic and benthic cyanobacteria. The appendices give further background information and include templates for data collection and reporting, including: • background information on known cyanotoxins and their distribution in New Zealand • information on the derivation of guideline values • photographs of typical bloom events • a list of biovolumes for common New Zealand cyanobacteria • templates for field assessments • suggested media releases and warning sign templates. A glossary provides definitions for abbreviations and terms used in these guidelines

Les toxines des cyanobactéries : revue de synthèse

La toxicité du phytoplancton est un problème dont l'importance est grandissante en France comme en Europe. Cette toxicité se manifeste surtout lors de l'ingestion de cyanobactéries formant des fleurs d'eau superficielles liées à l'eutrophisation. Microcystis aeruginosa est l'espèce la plus fréquemment incriminée, mais 75 % des souches de cyanobactéries d'eau douce seraient des toxiques potentielles. Lorsque des clones sont isolés d'un plan d'eau dans lequel des manifestations de toxicité ont été observées, des souches toxiques et non toxiques sont obtenues. La connaissance des conditions d'expression de la toxicité représente un sujet important actuellement peu étudié. Un mammifère peut mourir s'il passe dans son sang 0,07 mg de toxine de Microcystis par kg de son poids. En pratique, de nombreux cas de morts de bétail ont été recensés. Les toxines qui causent des accidents sont des endotoxines non rejetées par les cellules vivantes, mais libéré au cours de leur lyse. Ceci explique que des cas d'intoxication humaine par l'intermédiaire de l'eau de boisson ont pu être observés. On distingue principalement : des microcystines, hépatotoxines produites par diverses cyanobactéries dont Microcystis et Oscillatoria, et des anatoxines, neurotoxines produites par des Anabaena. Certaines cyanobactéries produiraient un mélange des deux formes. En sus du test de toxicité classique sur la souris, plusieurs autres tests existent. L'analyse est généralement réalisée par chromatographie liquide à haute pression. Des produits actifs synthétisés par des cyanobactéries possèdent une fonction antibiotique et sont susceptibles de jouer un rôle dans le comportement des espèces et leur dominance ou de les protéger contre le broutage. Ces produits seraient des exotoxines différentes des précédentes.The aim of the paper is to present in the French language the international knowledge related to freshwater cyanobacterial toxins, a problem of great significance in our European countries but largely unknown by people in France. An analytical review of a selection of works chosen from the extensive existing literature is presented. At the present time, the mains works come from : U.S.A., with CARMICHAEL and coworkers, continuing the researchs from CORHAM; Scotland, with CODD; Scandinavia with ERIKSSON, LINDHOLM; and Japan with WATANABE, HARADA, but also many others scientists.In freshwater, poisoning is typically associated with the ingestion of the cyanobacteria appearing in large amount, at the surface of some water bodies, and called water blooms. Many cases of livestock death (concerning sheeps, calves but also adult oxen and horses), associated with the consumption of such water blooms are reported, also, deaths of wild mammals (muskrats, and hogs), birds (ducks, geese), fishes, invertebrate, and human illness following bathing are known. But, because toxins are not destroyed by conventional sand filtration treatment, human illness may also arise from drinking water taken out from an impoundment with a cyanobacterial bloom. Cases are known from United States, Scandinavia and Sardinia. This tap water problem is serious because probabilities of long term diseases, such as tumors promotion, are now considered high.Seventy-five percent of fresh water cyanobacterial strains are potentially toxic, but, on the whole, only some clones from a single species, simultaneously isolated out of an unique body of water, are toxic. However, there are no evidence that the nontoxic strains could never become toxic.Cyanobacteria are also known as blue-green algae, Myxophyceae or Cyanophyta, and are typically microscopic prokaryotes, but with chlorophyll a. Toxic clones belong to : a) the Chroococcales, single coccoid cells embedded in a gelatinous matrix, represented by species of the genera Coelosphaerium, Gomphosphaeria, and Microcystis whose the species M. aeruginosa is the most frequently quotted toxic Cyanobacteria. b) the Nostocales, filamentous forms, some of them with exocellular sheath. Many are nitrogen fixing species belonging to the genera Nodularia, Anabaena, Aphanizomenon and Nostoc, others belong to the genera Oscillatoria, and are sot known as nitrogen fixers.Most often the mice toxicity test is used to identify toxic water blooms, it allows to define the lethal doses of the toxins or of the toxic organisms. But some others tests have been applied or suggested, they use others animals : fishes, zooplankton or microorganisms, and isolated organs or cells cultures, many are not specific. Currently the modern immunological tests are not yet adapted to identify fresh-water cyanobacterial toxins.The main toxins that can be distinguished are the microcystins and the anatoxins. The microcystins are hepatotoxins from various cyanobacteria belonging principally to the genera Microcystis and Oscilatoria; they promote liver haemorrhages. The anatoxins are neurotoxins from Anabaena and death occur by breath arrest. Some cyanobacteria simultaneously produce the two forms. A mammal may be killed by a blood level content of 0.07 mg of Microcystis toxin per kg of body weight. Cyanobacterial toxins are endotoxins which diffuse during cell lysis. This explain why toxins can be found in water from impoundments with cyanobacterial blooms. In this case, the toxins can possibly originate in a thick metalimnic plankton layer, not seen from surface.Toxin analysis is usually performed using high pressure liquid chromatography but also thin layer chromatography, and particularly the high performance modem technique. Molecular structures are eluciated using, fast atoms bombardment spectrometry, mass spectrometry and nuclear magnetic resonance. The microcystins are cyclic heptapeptides of low molecular weight, they differed in amino-acids composition; a characteristic one, built up with 20 carbons, is called ADDA. Microcystins toxicity ensue as they act as strong inhibitors upon phosphatase activities. The anatoxins-a are alkaloids, others anatoxins are peptides or currently unknown.The causes of the expression of the toxicity remain to be elucidated. In the years to come, much progress can be achieved by using new genetic tools. Nevertheless, as the largest problems occur always associated with water-blooms, and rise under high sunlight in hot periods, toxicity appears in the whole, associated with eutrophication, and as many toxic species are nitrogen fixers which do not need inorganic nitrogen to grow, problems follow generally a plentiful phosphorous load of water bodies due to human operations. As toxins accumulate in the cells, they could be actives either only after cells consumption, or after toxins discharge during cell lysis. Cyanobacterial toxicity is not due to bacteria associated with the cyanobacteria and can appear in pure axeniccultures. Toxicity is not associated with the presence of cell plasmids. It was shows that optimal conditions for growth did sot coincide with those for toxin production and vary with the growth phase. On the whole, the optimum temperatures, for toxin maximum production, were at about 25 °C for different cultures. Light intensity would be the primary important factor for the production of the toxin, but, in cyanobacteria cultures, this production can occur at relatively low light intensity.Some related cyanobacterial products are not true toxins but are exotoxins acting as antibiotics and can affect species behaviour or dominance and help deter grazers. Since LEFEVRE and coworkers studies, and after a long quiescent period, ecologists take these products anew into account when studying plankton ecology and successions. Only some phytoplankton responds to cyanobacterial extracellular products. Among zooplankton there are species avoiding actively the cyanobacteria and insensitive ones. Chemists have already started search for antitoxins chemicals and found promising curative results. On the other band, biotechnology could take advantage of all these various cyanobacterial products to obtain new drags having pharmacological or agronomical uses. Some true toxins could be used as anti-neoplastics, and products, involved in allelopathic reactions, have antibiotic and antivirus activities. The use of toxins as commercial algicide for chlorophycean waterbloom control had been suggested, but the action spectra of the toxins must be precisely known before extensive implementation. From another point of view, « microalgal » by-products, used as food additives have to be carefully checked for possible toxins.As SKULBERG et al. could rite in 1984, toxic blue-green algal blooms is a growing problem in Europe. Scientists involved in health supervision had to be watchful to it in a way to prevent people from possible major accidents

Oceanus.

v. 26, no. 2 (1983

Microalgal toxin(s): characteristics and importance

Prokaryotic and eukaryotic microalgae produce a wide array of compounds with biological activities. These include antibiotics, algicides, toxins, pharmaceutically active compounds and plant growth regulators. Toxic microalgae, in this sense, are common only among the cyanobacteria and dinoflagellates. The microalgal toxins is either important as material for useful drugs or one of the great mysteries in the world of biotoxicology. The aquatic poisons have long remained one of the great mysteries in the world of biotoxicology. There is evidence that these toxic organisms are on the increase, perhaps as a result of increased global pollution. The ability of cyanobacterial populations to produce potent toxins and annual examples of associated human and animal health problems have raised the position of cyanobacteria in the priorities for the management and protection of water quality in countries where health problems associated with the toxins have been perceived. The purpose of this review is to discuss the present understanding of microalgal toxins from microalgae in a manner that will stimulate interdisciplinary research with these microorganisms. Key Words: Toxin, cyanobacteria, microalgae, dinoflagellate. African Journal of Biotechnology Vol.3(12) 2004: 667-67

Saxitoxins: role of prokaryotes

Thesis (M.S.) University of Alaska Fairbanks, 2001Saxitoxins, the toxins associated with paralytic shellfish poisoning (PSP), are synthesized by dinoflagellates, cyanobacteria, and possibly bacteria. The specific objectives of this study were to determine growth conditions that promote high and low levels of toxin accumulation in Aphanizomenon flos-aquae (cyanobacterium) and Pseudomonas stutzeri (bacterium). Putative saxitoxins of P. stutzeri identified by HPLC-FLD in this study, and previously by other laboratories, were determined to be 'imposters' based on their chemical and physical properties, suggesting that this bacterium may not synthesize PSP toxins. In the cyanobacterium, toxin production was enhanced under higher light intensities and temperatures. Toxin accumulation reached maximal levels when cellular nitrogen was from either (NO₃-+NH₄)-N or N₂-N, while urea-N drastically reduced toxin levels. These data will be used in future studies aimed at identifying the genes involved in saxitoxin synthesis via molecular technologies that rely upon expression of the 'saxitoxin genes' under different growth conditions

Florações de Cianobactérias tóxicas no Reservatório do Funil: dinâmica sazonal e consequências para o zooplâncton

revista vol 13 nº 2.indd The Funil water reservoir, located in the Paraíba do Sul River Valley in the municipality of Resende (Rio de Janeiro State, Brazil), has become eutrophic during the last two decades and undergone recurrent blooms of cyanobacteria. This study presents temporal series of physical, chemical and biological data from the reservoir encompassing an overall period of four years (from June/02 to March/06). Monthly, measurements of conductivity, transparency, temperature, pH, dissolved oxygen, and water samples for the analyses of nutrients (N and P), chlorophyll-a, phytoplankton and zooplankton composition and cyanotoxins in seston and in net plankton were performed. Toxicity tests with native and temperate cladocerans species were also performed. The results showed that the high input of N and P favored the persistent dominance of cyanobacteria. A temporal pattern was observed mainly related to changes in water temperature, characterizing two distinct periods: a warm-wet period with cyanobacterial bloom, and a cold-dry period with general reduced biomass. Cyanobacteria included potential hepatotoxins (microcystins) producers like Microcystis spp., and potential neurotoxins (saxitoxins) producers like Anabaena circinalis and Cylindrospermopsis raciborskii. In fact, elevated concentrations of microcystins and saxitoxins were found in the phytoplankton, and high levels of microcystins in the zooplankton, suggesting that these toxins may be transferred through the food chain. The toxicity tests revealed that the cyanobacterial blooms had toxic effects on cladocerans, causing death, reduction in the rate of population increase (r), and paralysis, in agreement with the mechanism of action of the cyanotoxins present.revista vol 13 nº 2.indd El Embalse de Funil, localizado en el valle del Río Paraíba do Sul, municipio de Resende (RJ), se convirtió en un ambiente eutrófico a lo largo de las últimas dos décadas, con floraciones recurrentes de cianobacterias. Este estudio presenta una serie temporal de datos físicos, químicos y biológicos del embalse abarcando un período de cuatro años (Junio/02 a Marzo/06). Durante dicho período, se tomaron mensualmente medidas de conductividad eléctrica, transparencia de la columna de agua, temperatura, pH, oxigeno disolvido, muestras de agua para análisis de nutrientes (N y P), clorofila-a, de la composición de la comunidad fitoplanctônica y zooplanctónica y cianotoxinas en el seston y colectas en red de plancton. También fueron realizados ensayos de toxicidad con cladóceros nativos así como también con una especie de zonas templada. Los resultados encontrados evidencian que el elevado y constante aporte de N y P favorece la ocurrencia de cianobacterias durante todo el año. Sin embargo, los patrones temporales están principalmente relacionados con el cambio en la temperatura, caracterizando dos períodos diferentes: cálido-húmedo, con floraciones de cianobacterias, y frío-seco, con una biomasa reducida. Entre las cianobacterias presentes están las especies potencialmente productoras de hepatotoxinas (microcistinas), como Microcystis spp., y de neurotoxinas (saxitoxinas), como Anabaena circinalis e Cylindrospermopsis raciborskii. Fueron encontradas concentraciones elevadas de microcistinas y saxitoxinas en el fitoplancton y microcistinas en el zooplancton, sugiriendo que puede existir transferencia trófica de dichas toxinas en la cadena alimenticia. Los ensayos de toxicidad revelaron que las floraciones de cianobacterias ejercieron efectos tóxicos sobre los cladóceros, como alta mortalidad, reducción de la tasa de crecimiento poblacional (r) y parálisis de los movimientos natatorios, que parecen estar relacionados con el mecanismo de acción de las cianotoxinas presentes.O Reservatório do Funil, situado no vale do Rio Paraíba do Sul, município de Resende (RJ), tornou-se ao longo das últimas duas décadas um ambiente eutrófico, com florações recorrentes de cianobactérias. Este estudo apresenta uma série temporal de dados físicos, químicos e biológicos do reservatório, abrangendo um período amostral de quatro anos (junho/02 a março/06). Mensalmente, foram realizadas medidas de condutividade elétrica, transparência da coluna d'água, temperatura, pH, oxigênio dissolvido, e coletas de água para análise de nutrientes (N e P), clorofila-a, cianotoxinas do seston e do plâncton de rede, e da composição da comunidade fitoplanctônica e zooplanctônica. Foram também realizados testes de toxicidade com cladóceros nativos e de origem temperada. Os resultados mostraram que o elevado e constante aporte de N e P favorece a ocorrência de cianobactérias durante todo ano. Entretanto, a variabilidade temporal está principalmente relacionada às alterações de temperatura, caracterizando dois períodos distintos: quente-chuvoso, com floração de cianobactérias e frio-seco, com reduzidas biomassas. Entre as cianobactérias presentes estão espécies potencialmente produtoras de hepatotoxinas (microcistinas), como Microcystis spp., e de neurotoxinas (saxitoxinas), como Anabaena circinalis e Cylindrospermopsis raciborskii. Foram encontradas concentrações elevadas de microcistinas e saxitoxinas no fitoplâncton e microcistinas no zooplâncton, sugerindo que pode haver transferência trófica destas toxinas na cadeia alimentar. Os testes de toxicidade revelaram que as florações de cianobactérias exerceram efeitos tóxicos para os cladóceros, como alta mortalidade, redução da taxa de crescimento populacional (r) e paralisia dos movimentos natatórios, que parecem estar relacionados ao mecanismo de ação das cianotoxinas presentes

シジョウ ランソウ Oscillatoria agardhii ノ セイリ カッセイ ペプチド ニ カンスル ケンキュウ

藍藻類は光合成を行う原核生物で、淡水湖沼、汽水域、海洋、土壌など様々な環境下で生育しており、生育環境によってユニークな化学構造や生理活性を示す物質を生産することが知られている。特に、海洋生物に共生する藍藻から有用な化合物が単離されている。藍藻は培養も比較的容易なため、医薬品リード化合物の新たな天然物資源として注目されている。 淡水産藍藻の有毒物質については精力的な研究が行われ、肝臓毒や神経毒が構造決定されている。しかし、淡水産藍藻の毒物以外の生理活性物質についてはあまり研究が行われておらず、特に、有毒種が多い糸状藍藻Oscillatoria属についてはほとんど化学成分研究が行われていない。 そこで、本研究では、Oscillatoria属の中でも自然界で優占種となることが多いOscillatoria agardhiiに注目し、その生理活性物質の検索を行うことを計画した。その結果、糸状藍藻Oscillatoria agardhijは化学構造的に非常に興味深い生理活性ペプチドを生産していることを見出した。これらの生理活性ペプチドについて詳細に研究を行い、以下のような知見を得た。 1) O. agardhiiが生産する肝臓毒microcystinを単離し、その構造を解析した結果、MSとアミノ酸分析では既存の3-desmethylmicrocystinと区別がつかないDhb-microcystin-RR，HtyR，およびLRの構造をNMRを用いて明らかにした。このDhb-microcystinの構造を決定する際に、HMBC法よりも微弱な結合を測定可能なdecoupled-HMBC法が有効であった。Dhb-microcystinの細胞毒性はmicrocystin-LRよりやや強い値を示した。 2) トリプトファンの分子内環化反応とイソプレンユニットの付加反応によって生合成されたと考えられる新規異常アミノ酸(3a-cis)-1，2，3，3a，8，8a-hexahydro-3a-(3-methyl-2-butenyl)-pyrrolo[2，3-b]indol-2-carboxylic acid (oscillatoric acid)ユニットを含む環状ペプチドOscillatorinを単離し、その構造を決定した。Oscillatorinはキモトリプシンを強力に阻害し、そのIC50は8x10-7 Mであった。 3) 3-Amino-6-hydroxy-2-piperidone (Ahp)残基を含む新規環状ペプチドOscillapeptinA～C、およびGの構造を決定した。OscillapeptinBはトリプシンの活性を強力に阻害し、そのIC50は7x10-9Mであった。OscillapeptinA、C、およびGはキモトリプシンを強力に阻害し、そのIC50はそれぞれ3x10-9 M， 7x10-8 M，4x10-9 Mであった。また、その構造と阻害酵素特異性に関して新たな知見を得た。 4)ウレイド結合を有し、homotyrosineおよびN-Meアミノ酸を含む環状ペプチドOscillamideA～C，H，およびYを単離した。OscillamideBおよびYはキモトリプシンを阻害した。 5)淡水産の藍藻から塩素で置換された3-amino-10-chloro-2-hydroxydecanoic acid (CIAhda)を含む新規鎖状ペプチドOscillagininA およびその脱塩素体OscillagininBを単離し、その構造を決定した。OscillagininAおよびBはキモトリプシンを阻害した。 本研究でOscillatoria agardhiiから得られたペプチドは、環状となったものが多く、さらに、Ahpやoscillatoric acidなどの環状アミノ酸やβ-アミノ酸などの異常アミノ酸を多く含んでいた。これら環状ペプチドは、デプシペプチドとなったものやウレイド結合を含むものもあり、構造的に興味深い。さらに、これら環状ペプチドはアミノ酸組成も多様性に富み、医薬品のリード化合物となることが期待される。 本研究で用いたOscillatona agardhiiは環状ペプチドの宝庫ともいえるもので、Dhbmicrocystin以外は同じ構造の化合物が見つからないほど、株によって生産しているペプチドの構造がそれぞれ異なり、医薬品リード化合物の資源としても有用であることが確認された。doctoral thesi