Assessment of rapid bioassays for detecting cyanobacterial toxicity

Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato-and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3–17 mg l^-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2–14 mg l^-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.     

Water-blooming and toxin-producing cyanobacteria in Swedish fresh and bracish waters, 1981–1995

During the period 1981–1995 331 phytoplankton samples from 153stations in different parts of Sweden were analysed regardingspecies composition and possible toxicity. Toxins were indicated in156 samples which gives a percentage of 47. The toxicity wasconfirmed by mouse bioassay. All samples were collected fromwater-blooming lakes, mainly containing taxa belonging to thecyanobacterial genera Anabaena, Aphanizomenon, Microcystis and Planktothrix. In addition to informationabout occurrence of neuro- or hepatotoxins, knowledge about thedistribution of water-blooming lakes in the country was obtained aswell as about their predominating taxa. Microcystisaeruginosa, Anabaena flos-aquae and Aphanizomenonflos-aquae were the most frequently occurring taxa in the lakes.Among the taxa observed, nine different cyanobacterial species withcertainty produce toxins. Microcystis spp. most frequentlyproduced toxic blooms (only hepatotoxins). Anabaena spp.,however, caused the majority of waterblooms. The Anabaenaflos-aquae-group produced neurotoxins [incl. anatoxin-a(s)],neurotoxins + hepatotoxins or only hepatotoxins. Aphanizomenon flos-aquae was only recorded as toxin-producing inthree Swedish lakes, which may be compared with the conditions inFinland, where it was toxic in 36 bloom samples. Aspects are givenon toxicity versus lake acidification and trophic conditions, aswell as on health effects on animals and humanbeings.     

Effects of salinity and source of inocula on germination of Anabaena akinetes from a tidally influenced lake

1. Sedimentary akinetes (resting stages) may represent significant potential inocula for nuisance blooms of cyanobacteria. We studied the effects of salinity and sediment source on the germination and subsequent growth of Anabaena flos‐aquae akinetes from a shallow, tidally influenced lake. 2. Surface sediments collected from littoral and open‐water sites were used as inocula to culture A. flos‐aquae akinetes in four salinities (0.1, 2.2, 4.4 and 6.5) over 22 days. Akinete germination and development was followed by counting developmental stages every second day. 3. Filament growth, but not akinete germination, was inhibited by salinity and there were significantly fewer filaments at 6.5 than at 0.1 and 2.2. Cultures inoculated with littoral sediment had more akinetes, germlings and filaments than those inoculated with open‐water sediment. 4. Sediment is a potential source of inocula for Anabaena blooms in the lake, which potentially could develop solely from this source because germination and subsequent filament growth do not depend on the existence of an initial pelagic Anabaena population.     

The rise of potentially toxin producing cyanobacteria in Lake Naivasha,Great African Rift Valley,Kenya

Lake Naivasha, an important inland water ecosystem and a crucial freshwater resource in the Great African Rift Valley, has displayed clear signals of degradation in recent decades. We studied the phytoplankton composition and biomass levels in the period 2001–2013 and noted a progressive increase in the occurrence of potentially toxic cyanobacteria. Analyses for the presence of cyanotoxins such as microcystins (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a) were carried out on samples collected in 2008–2013. Among the cyanotoxins tested, low concentrations of MC were detected in the lake. This is the first record of the occurrence of MC in Lake Naivasha. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Lake Naivasha were carried out to establish the taxa of the dominant species. Amplification of the aminotrasferase (AMT) domain responsible for cyanotoxin production confirmed the presence of the mcyE gene belonging to the microcystin synthesis gene cluster in field samples containing Microcystis and Planktothrix species. These findings suggest that toxin producing cyanobacteria could become a threat to users of this over-exploited tropical lake in the near future.     

Toxin-Producing Anabaena flos-aquae Induces Settling of Chlamydomonas reinhardtii,a Competing Motile Alga

Toxin production is an adaptation that allows cyanobacteria in resource-limiting environments to ameliorate the effects of herbivory and competition with other phototrophs. We demonstrate that the cyanobacterial toxins anatoxin-a and microcystin-LR paralyze the motile green alga Chlamydomonas reinhardtii. In addition, both purified toxins and cyanobacterial extracellular products containing these toxins cause the alga to settle faster than in nontoxic media. In microcosm experiments, the presence of either the cyanobacterium or its extracellular products induce settling in the alga, similar to the response observed with the addition of both anatoxin-a and microcystin-LR. The cyanobacterial production of paralyzing toxins represents a novel mechanism for phytoplankton settling. This prokaryotic/eukaryotic chemical interaction may create a competitor-free zone for cyanobacteria in lake environments, predicating optimal conditions for a toxic cyanobacterial bloom.     

Hindcasting cyanobacterial communities in Lake Okaro with germination experiments and genetic analyses

Cyanobacterial blooms are becoming increasingly prevalent worldwide. Sparse historic phytoplankton records often result in uncertainty as to whether bloom-forming species have always been present and are proliferating in response to eutrophication or climate change, or if there has been a succession of new arrivals through recent history. This study evaluated the relative efficacies of germination experiments and automated rRNA intergenic spacer analysis (ARISA) assays in identifying cyanobacteria in a sediment core and thus reconstructing the historical composition of cyanobacterial communities. A core (360 mm in depth) was taken in the central, undisturbed basin of Lake Okaro, New Zealand, a lake with a rapid advance of eutrophication and increasing cyanobacteria populations. The core incorporated a tephra from an 1886 volcanic eruption that served to delineate recent sediment deposition. ARISA and germination experiments successfully detected akinete-forming nostocaleans in sediment dating 120 bp and showed little change in Nostocales species structure over this time scale. Species that had not previously been documented in the lake were identified including Aphanizomenon issatschenkoi , a potent anatoxin-a producer. The historic composition of Chrococcales and Oscillatoriales was more difficult to reconstruct, potentially due to the relatively rapid degradation of vegetative cells within sediment.     

Growth and survival of Bordetella bronchiseptica in natural waters and in buffered saline without added nutrients

Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.     

Growth and survival of Bordetella bronchiseptica in natural waters and in buffered saline without added nutrients.

Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.     

Molecular detection of potentially toxic cyanobacteria and their associated bacteria in lake water column and sediment

We investigated the molecular diversity of cyanobacteria and bacteria during a water bloom in a lake with a long history of toxic cyanobacterial blooms (Lake Kastoria, Greece). We also tested the hypothesis whether bloom-forming cyanobacteria are preserved in the lake’s sediment 2 years after the bloom. The dominant cyanobacteria during the bloom included the potentially toxin-producing Microcystis aeruginosa and several other Chroococcales forms closely related to the genus Microcystis. This suggests that the use of cyanobacterial-specific primers seems to be very informative in describing the cyanobacteria during the water blooms. The bacterial community showed high diversity, consisting mostly of singleton and doubleton phylotypes. The majority of the phylotypes were typical lake bacteria including some potential pathogens and toxin metabolising bacteria, suggesting that the dominant toxic cyanobacteria did not have any significant effect on the bacterial community structure. In the sediment, 2 years after the water bloom, no bloom-forming cyanobacteria were retrieved, suggesting that they cannot be preserved in the sediment. Similar to the water column, sediment bacterial diversity was also high, consisting mostly of yet-uncultured bacteria that are related to environments where organic matter degradation takes place.     

Occurrence of anatoxin-a(s) during a bloom of <Emphasis Type="Italic">Anabaena crassa</Emphasis> in a water-supply reservoir in southern Brazil

Cyanobacterial blooms and the accompanying production of cyanotoxins are a serious global problem. Toxic blooms of Anabaena species are common in lagoons and reservoirs of southern Brazil. Worldwide, species of the genus Anabaena produce the majority of the known hepatotoxins (microcystins) and neurotoxins [anatoxin-a, anatoxin-a(s), and saxitoxins]. This report links a bloom of Anabaena crassa in the Faxinal Reservoir, the main water supply for the city of Caxias do Sul (400,000 inhabitants) in southern Brazil, to the occurrence of anatoxin-a(s) in the water. During the bloom period, the reservoir was strongly stratified, with higher temperatures and a deep anoxic hypolimnion. Two methods for sample concentration (direct and complete extraction) were tested, and direct extraction of samples proved to be more efficient. Water samples collected during the bloom showed 9% acetylcholinesterase inhibition at 50 mg mL⁻¹, corresponding to 0.61 μg of anatoxin-a(s) per gram of lyophilized powder. At these concentrations, symptoms of neurotoxicity and mortality were not observed in tests with Swiss albino mice. Although the concentrations of anatoxin-a(s) in the Faxinal Reservoir were low, these results are important because this is the first record of the toxin for A. crassa. Furthermore, this cyanotoxin is not yet included in Brazilian legislation for drinking-water monitoring, because of the lack of information about toxicity levels and risk calculation for oral doses. The data presented here contribute to the basis for the future inclusion of this toxin in Brazilian legislation for drinking-water quality control, and for the development of analytical methods for this toxin.     

An assessment of the influence of recruitment from the sediment on the development of planktonic populations of cyanobacteria in a temperate mesotrophic lake

1. A number of planktonic cyanobacteria species form resting stages that survive in the sediments of lakes. The significance of this life history strategy to the ecology of new planktonic populations was investigated in Esthwaite Water, a mesotrophic lake in the English Lake District.
2. A simple trapping technique was used to quantify vertical movements of five species of buoyant gas-vacuolate cyanobacteria from close to the sediments, along a depth transect.
3. 'Recruitment' from the sediments was found to be widespread amongst the cyanobacteria species associated with the summer phytoplankton community.
4. Estimates of the vertical upward fluxes of cyanobacteria based upon trap catches could not account for observed increases in the planktonic populations suggesting that 'recruitment' was not a significant source of biomass.
5. Vertical upward movements of Anabaena solitaria were recorded prior to this species becoming established in the plankton suggesting that benthic populations might be a source of cells for initial pelagic growth of populations of this species.
6. Low numbers of vegetative filaments of Anabaena flos-aquae , Aphanizomenon flos-aquae and Oscillatoria agardhii were observed in the plankton through the winter. These small overwintering populations appeared to be the primary source of inocula for the large summer populations of these species.     

Different Genotypes of Anatoxin-Producing Cyanobacteria Coexist in the Tarn River, France

Repeated dog deaths occurred in 2002, 2003, and 2005 after the animals drank water from the shoreline of the Tarn River in southern France. Signs of intoxication indicated acute poisoning due to a neurotoxin. Floating scum and biofilms covering pebbles were collected in the summers of 2005 and 2006 from six different sites along 30 km from the border of this river. The cyanobacterial neurotoxic alkaloid anatoxin-a and/or its methyl homolog, homoanatoxin-a, was detected in the extracts of most samples examined by gas chromatography-mass spectrometry. Fifteen filamentous cyanobacteria of the order Oscillatoriales were isolated and displayed four distinct phenotypes based on morphological characteristics and pigmentation. Three of the phenotypes can be assigned to the genus Oscillatoria or Phormidium, depending on the taxonomic treatises (bacteriological/botanical) employed for identification. The fourth phenotype is typical of the genus Geitlerinema Anagnostidis 1989. Eight strains rendered axenic were analyzed for production of anatoxin-a and homoanatoxin-a, and all strains of Oscillatoria/Phormidium proved to be neurotoxic. The genetic relatedness of the new isolates was evaluated by comparison of the intergenic transcribed spacer sequences with those of six oscillatorian strains from the Pasteur Culture Collection of Cyanobacteria. These analyses showed that the neurotoxic representatives are composed of five different genotypes, three of which correspond to phenotypes isolated in this study. Our findings prove that neurotoxic oscillatorian cyanobacteria exist in the Tarn River and thus were most likely implicated in the reported dog poisonings. Furthermore, they reemphasize the importance of monitoring benthic cyanobacteria in aquatic environments to fully assess the health risks associated with these organisms.     

Characterization of akinetes from cyanobacterial strains and lake sediment: A study of their resistance and toxic potential

Nostocalean cyanobacteria are known to proliferate abundantly in eutrophic aquatic ecosystems, and to produce several cyanotoxins, including anatoxin-a. In this study, we investigated both the resistance and toxic potential of the akinetes (resistant cells), using cyanobacterial cultures and akinetes extracted from the sediment of Lake Aydat (France) sampled in the winter and spring. Intact and lysed akinetes were differentiated using a double control based on the autofluorescence of akinetes and SYTOX-green staining. The percentage of resistant akinetes found in several different abiotic stress conditions was highly variable, depending on the species and also on the sampling season. Thus, the resistance of akinetes and their ability to germinate seems to follow a species-specific process, and akinetes can undergo physiologic changes during the sedimentary phase of the Nostocale life cycle. This study also revealed the first evidence of anatoxin-a genes in akinetes, with anaC and anaF genes detected in akinetes from all cyanobacterial producer cultures. The low number of anaC genes, almost exclusively detected using nested PCR, in the sediment at Lake Aydat suggests a limited but existent past population of toxic Nostocales in this lake. Given the key role of akinetes in the annual cycle and subsequent summer proliferation, it can be interesting to integrate the surveillance of akinetes in the management of lakes exposed to recurrent cyanobacterial blooms.     

Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”

Increasingly, harmful algal blooms (HABs) are being reported worldwide due to several factors, primarily eutrophication, climate change and more scientific monitoring. All but cyanobacteria toxin poisonings (CTPs) are mainly a marine occurrence. CTPs occur in fresh (lakes, ponds, rivers and reservoirs) and brackish (seas, estuaries, and lakes) waters throughout the world. Organisms responsible include an estimated 40 genera but the main ones are Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, and Oscillatoria (Planktothrix). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins with biotoxins being responsible for acute lethal, acute, chronic and sub-chronic poisonings of wild/domestic animals and humans. The biotoxins include the neurotoxins; ana-toxin-a, anatoxin-a(s) and saxitoxins plus the hepatotoxins; microcystins, nodularins and cylindrospermopsins. Confirmations of human deaths from cyanotoxins are limited to exposure through renal dialysis at a haemodialysis center in Caruaru, Brazil, in 1996. A major effort to compile all available information on toxic cyanobacteria including issues of human health, safe water practices, management, prevention and remediation have been published by the World Health Organization. This paper will review our current understanding of CTP's including their risk to human health.     

Anatoxin-A(S), a naturally occurring organophosphate,is an irreversible active site-directed inhibitor of acetylcholinesterase (EC 3.1.1.7)

Anatoxin-a(s) is a guanidine methyl phosphate ester (unprotonated molecular ion equals 252 daltons) isolated from the freshwater cyanobac-terium (blue-green alga) Anabaena flos-aquae strain NRC 525–17. Previous work has shown anatoxin-a(s) to be a potent irreversible inhibitor of electric eel ace-tylcholinesterase (EC 3.1.1.7, AChE). In the present study the interaction of anatoxin-a(s) with AChE was investigated by protection studies and since similarities have been noted between anatoxin-a(s) and the synthetic organophosphate anticholinesterases, the ability of reactivators to reactivate the inhibited enzyme was investigated. Treatments directed toward eliminating poisoning symptoms and in vivo protection from anatoxin-a(s) poisonings were investigated using oxime reactivators and atropine or pretreatment with a carbamate and atropine. Anatoxin-a(s) was shown to be an active site-directed inhibitor of acetyl-cholinesterase which is resistant to oxime reactivation due to the structure of its enzyme adduct. In vivo pretreatment with physostigmine and high concentrations of 2-PAM were the only effective antagonists against a lethal dose of anatoxin-a(s).     

蓝藻毒素对底栖动物的毒理学研究进展

近年,由于人类活动加剧,大量氮磷等营养物质流入湖泊等缓流水体,导致水体富营养化。而由此引起有害蓝藻水华的频繁爆发,使生态环境和人类健康受到严重威胁。相关研究表明,蓝藻水华的爆发不仅能够使水体水质恶化,其中一些产毒藻类还会产生大量蓝藻毒素,对水生生物产生重要影响。底栖动物作为水体生态系统的重要组成部分,在食物网中有重要作用,同时其中的许多种类又与人类息息相关,因此关于水华蓝藻毒素对淡水底栖动物的毒理学研究具有重要意义。在介绍蓝藻毒素概况的基础上,综述了蓝藻毒素的致毒机理和对底栖动物的影响,展望了研究方向。     

洱海蓝藻爆发的时空特征及影响因子

湖泊生态环境对区域气候变化和流域人类活动十分敏感。随着流域的持续开发,洱海作为云贵高原第二大淡水湖泊面临着严重的生态与环境问题,主要包括水质恶化和生态功能的衰退,其中蓝藻水华问题尤为突出。针对湖泊现代监测数据存在时间序列较短、连续监测记录缺乏、监测位点不完全一致等问题,应用沉积物记录开展色素等多指标分析和环境变化重建研究,并对洱海湖区南、中、北3个湖盆的沉积物记录进行对比分析,从而探讨洱海富营养化与蓝藻爆发的历史与变化特征,并识别藻类响应模式的空间异同。沉积物色素记录结果表明,洱海蓝藻生物量变化具有明显的时空差异性,呈现由南至北,先后增加、最后呈现蓝藻水华全湖性持续爆发的模式。进一步的简约模型方差分解结果表明气候变暖和营养盐富集是洱海蓝藻生物量变化的主要驱动因子,此外相对较浅的南部湖盆还受到水位波动、水动力减弱、水生植物演化的综合影响。因此,在气候变暖的背景下,控制水体营养盐输入、合理调控湖泊水位、提高水体透明度并恢复水生植物是控制洱海蓝藻水华爆发和进行生态恢复的重要措施。     

晋阳湖丝状蓝藻的形态学分析与分子鉴定北大核心CSCD

蓝藻是地球上最古老的生物之一,其形态结构较为简单,为产氧型光合作用的原核生物。山西省晋阳湖为华北地区最大的人工湖,该研究以采自晋阳湖水体及岸边附着的蓝藻为材料,采用经典毛细管法分离纯化出5株丝状蓝藻,利用光学显微镜观察其形态结构特征(如细胞形状、藻丝体宽度、是否有鞘)和显微结构,并采用16S rRNA序列分析其系统发育关系,以明确晋阳湖的蓝藻种类,为预防湖泊蓝藻水华的发生、维护水资源环境稳定与生态平衡提供理论数据。结果显示:(1)所分离纯化的5株丝状蓝藻依形态特征归属于3个科,其中2株(JYH005和JYH012)为细鞘丝藻亚科(Leptolyngbyaceae),2株(JYH008和JYH022)为伪鱼腥藻科(Pseudanabaenaceae),1株(JYH010)为沙丝藻科(Desertifilaceae)。(2)基于16S rRNA序列构建的系统发育树显示,5株丝状蓝藻中JYH005为结丝藻属(Nodosilinea)的一种;JYH008可归为Arthronema,该株蓝藻在培养条件下观察到不同的形态特征,可能为新物种;JYH010为沙丝藻属(Desertifilum)的一种;JYH012可归为细鞘丝藻属(Leptolyngbya);JYH022与伪鱼腥藻科聚为一支,由于与该科其他藻相似度低于90%,且不能聚为一支,因此只能归为伪鱼腥藻科。研究表明,基于16S rRNA序列系统发育分析与形态学鉴定结果相一致。该研究结果丰富了山西省晋阳湖丝状蓝藻的多样性,为该湖的资源利用和环境保护提供了一定的科学依据。     

微囊藻毒素在滇池鱼体内的积累水平及分布特征

为了解富营养化水体中鱼体内微囊藻毒素(MC)的积累水平及其分布特征,2003年4月和9月份两次在滇池试验区采集了鲢、鳙和草鱼等鱼种,用ELISA方法对鱼体中肝、肾、空肠、胆、肌肉等不同组织中MC的含量进行了检测。结果表明,MC在所有样品中均能检测到,且主要分布在鱼体的肝肾脏和消化道等器官,而肌肉和非消化道器官中毒素含量相对较低。不同鱼种不同组织对MC的富集程度也明显不同,鲢鳙中肝脏和肾脏这两个主要的靶器官对MC的蓄积能力就远高于草鱼。同时,不同季节MC在鱼体内的积累水平也明显不同,4月份鱼样中MC的含量普遍低于9月份鱼样中MC的含量。最后按照WHO生活饮用水安全标准的建议进行推算,所有鱼肉中的MC均没有超过其推荐的人体每日可允许摄入量(≤0.04μg/kg人体重),初步推断鱼肉中MC暂时还未危及到人体健康,但仍具有潜在的风险性。     

Genotoxicity of microcystic cyanobacteria extract of a water source in China.

The water pollution of toxic cyanobacteria (blue-green algae) is a worldwide problem and worsens with industrialization. Microcystins are potent cyclic heptapeptidic hepatotoxins produced mainly by Microcystis aeruginosa, and their hepatotoxicity has been well-documented. In contrast, information on the genotoxic effects of microcystins is relatively scarce. In our present study, the genotoxicity of microcystic cyanobacteria extract (MCE) of a water source in China was studied using Salmonella typhimurium assay (Ames test), comet assay (Single cell gel electrophoresis) and mouse micronucleus test. Results from Ames test indicated that MCE had strong mutagenicity regardless of the presence of S9. Moreover, MCE was able to induce DNA damage in primary cultured rat hepatocytes examined by comet assay. In addition, MCE also enhanced bone marrow micronucleated polychromatic erythrocytes in mice. The analysis of HPLC showed that the main component of MCE was microcystin-LR. The understanding of the potent genotoxicity of MCE will help to establish the possible link between water cyanobacteria contamination and high risk of primary liver cancer found in some endemic areas.