Is the Cyanobacterial Bloom Composition Shifting Due to Climate Forcing or Nutrient Changes? Example of a Shallow Eutrophic Reservoir.

Cyanobacterial blooms in eutrophic freshwater is a global threat to the functioning of ecosystems, human health and the economy. Parties responsible for the ecosystems and human health increasingly demand reliable predictions of cyanobacterial development to support necessary decisions. Long-term data series help with identifying environmental drivers of cyanobacterial developments in the context of climatic and anthropogenic pressure. Here, we analyzed 13 years of eutrophication and climatic data of a shallow temperate reservoir showing a high interannual variability of cyanobacterial development and composition, which is a less occurring and/or less described phenomenon compared to recurrant monospecific blooms. While between 2007-2012 Planktothrix agardhii dominated the cyanobacterial community, it shifted towards Microcystis sp. and then Dolichospermum sp. afterwards (2013-2019). The shift to Microcystis sp. dominance was mainly influenced by generally calmer and warmer conditions. The later shift to Dolichospermum sp. was driven by droughts influencing, amongst others, the N-load, as P remained unchanged over the time period. Both, climatic pressure and N-limitation contributed to the high variability of cyanobacterial blooms and may lead to a new equilibrium. The further reduction of P-load in parallel to the decreasing N-load is important to suppress cyanobacterial blooms and ameliorate ecosystem health.

Demonstrated transfer of cyanobacteria and cyanotoxins along a freshwater-marine continuum in France.

The frequency of cyanobacterial proliferations in fresh waters is increasing worldwide and the presence of associated cyanotoxins represent a threat for ecosystems and human health. While the occurrence of microcystin (MC), the most widespread cyanotoxin, is well documented in freshwaters, only few studies have examined its occurrence in estuarine waters. In this study we evaluated the transfer of cyanobacteria and cyanotoxins along a river continuum from a freshwater reservoir through an interconnecting estuary to the coastal area in Brittany, France. We sampled regularly over 2 years at 5 stations along the river continuum and analysed for phytoplankton and cyanotoxins, together with physico-chemical parameters. Results show that cyanobacteria dominated the phytoplanktonic community with high densities (up to 2 × 106 cells mL-1) at the freshwater sites during the summer and autumn periods of both years, with a cell transfer to estuarine (up to 105 cells mL-1) and marine (2 × 103 cells mL-1) sites. While the temporal variation in cyanobacterial densities was mainly associated with temperature, spatial variation was due to salinity while nutrients were non-limiting for cyanobacterial growth. Cyanobacterial biomass was dominated by several species of Microcystis that survived intermediate salinities. Intracellular MCs were detected in all the freshwater samples with concentrations up to 60 µg L-1, and more intermittently with concentrations up to 1.15 µg L-1, at the most upstream estuarine site. Intracellular MC was only sporadically detected and in low concentration at the most downstream estuarine site and at the marine outlet (respectively <0.14 µg L-1 and <0.03 µg L-1). Different MC variants were detected with dominance of MC-LR, RR and YR and that dominance was conserved along the salinity gradient. Extracellular MC contribution to total MC was higher at the downstream sites in accordance with the lysing of the cells at elevated salinities. No nodularin (NOD) was detected in the particulate samples or in the filtrates.

Management of toxic cyanobacteria for drinking water production of Ain Zada Dam.

Blooms of toxic cyanobacteria in Algerian reservoirs represent a potential health problem, mainly from drinking water that supplies the local population of Ain Zada (Bordj Bou Arreridj). The objective of this study is to monitor, detect, and identify the existence of cyanobacteria and microcystins during blooming times. Samples were taken in 2013 from eight stations. The results show that three potentially toxic cyanobacterial genera with the species Planktothrix agardhii were dominant. Cyanobacterial biomass, phycocyanin (PC) concentrations, and microcystin (MC) concentrations were high in the surface layer and at 14 m depth; these values were also high in the treated water. On 11 May 2013, MC concentrations were 6.3 µg/L in MC-LR equivalent in the drinking water. This study shows for the first time the presence of cyanotoxins in raw and treated waters, highlighting that regular monitoring of cyanobacteria and cyanotoxins must be undertaken to avoid potential health problems.

Beta-N-methylamino-L-alanine: LC-MS/MS optimization, screening of cyanobacterial strains and occurrence in shellfish from Thau, a French Mediterranean lagoon.

ß-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 µg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 µg/g DW), while only several samples contained quantifiable free BMAA.

Searching for a link between the L-BMAA neurotoxin and amyotrophic lateral sclerosis: a study protocol of the French BMAALS programme.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is the most common motor neurone disease. It occurs in two forms: (1) familial cases, for which several genes have been identified and (2) sporadic cases, for which various hypotheses have been formulated. Notably, the ß-N-methylamino-L-alanine (L-BMAA) toxin has been postulated to be involved in the occurrence of sporadic ALS. The objective of the French BMAALS programme is to study the putative link between L-BMAA and ALS. METHODS AND ANALYSIS: The programme covers the period from 1 January 2003 to 31 December 2011. Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this period in the area under study (10 counties spread over three French regions) were collected. First, the standardised incidence ratio will be calculated for each municipality under concern. Then, by applying spatial clustering techniques, overincidence and underincidence zones of ALS will be sought. A case-control study, in the subpopulation living in the identified areas, will gather information about patients' occupations, leisure activities and lifestyle habits in order to assess potential risk factors to which they are or have been exposed. Specimens of drinking water, food and biological material (brain tissue) will be examined to assess the presence of L-BMAA in the environment and tissues of ALS cases and controls. ETHICS AND DISSEMINATION: The study has been reviewed and approved by the French ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-Mer IV). The results will be published in peer-reviewed journals and presented at national and international conferences.

Cylindrospermopsin accumulation and release by the benthic cyanobacterium Oscillatoria sp. PCC 6506 under different light conditions and growth phases.

We have studied the dynamics of cylindrospermopsin concentration (CYN) of a benthic cyanobacterium of the genus Oscillatoria under various light conditions over the different growth phases. The present study is the first one reporting on the effect of abiotic factors on the CYN accumulation and release by a benthic species. In particular we have measured the concentrations of both intracellular and extracellular CYN. We found that the total CYN content is highest during the exponential growth phase at intermediate light level (10 µE m(-2) s(-1)) and during the stationary growth phase at more extreme lower and higher light levels. Our results also indicate that the amount of the extracellular form varied between 56 % and 96 % of the total CYN concentrations. We found no relationship between CYN content and growth rates. These results suggest many similarities with planktonic species but also highlight some differences.

Dietary BMAA exposure in an amyotrophic lateral sclerosis cluster from southern France.

BACKGROUND: Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described. OBJECTIVES: We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA. METHODS: A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations. RESULTS: We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded. CONCLUSIONS: While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.

Development of a combined isotopic and mass-balance approach to determine dissolved organic carbon sources in eutrophic reservoirs.

A combined mass-balance and stable isotope approach was set up to identify and quantify dissolved organic carbon (DOC) sources in a DOC-rich (9mgL(-1)) eutrophic reservoir located in Western France and used for drinking water supply (so-called Rophemel reservoir). The mass-balance approach consisted in measuring the flux of allochthonous DOC on a daily basis, and in comparing it with the effective (measured) DOC concentration of the reservoir. The isotopic approach consisted, for its part, in measuring the carbon isotope ratios (δ(13)C values) of both allochthonous and autochthonous DOC sources, and comparing these values with the δ(13)C values of the reservoir DOC. Results from both approaches were consistent pointing out for a DOC of 100% allochthonous origin. In particular, the δ(13)C values of the DOC recovered in the reservoir (-28.5±0.2‰; n=22) during the algal bloom season (May-September) showed no trace of an autochthonous contribution (δ(13)C in algae=-30.1±0.3‰; n=2) being indistinguishable from the δ(13)C values of allochthonous DOC from inflowing rivers (-28.6±0.1‰; n=8). These results demonstrate that eutrophication is not responsible for the high DOC concentrations observed in the Rophemel reservoir and that limiting eutrophication of this reservoir will not reduce the potential formation of disinfection by-products during water treatment. The methodology developed in this study based on a complementary isotopic and mass-balance approach provides a powerful tool, suitable to identify and quantify DOC sources in eutrophic, DOC-contaminated reservoirs.

Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta.

Hepatotoxic microcystins (MCs) produced by cyanobacteria are known to accumulate in gastropods following grazing of toxic cyanobacteria and/or absorption of MCs dissolved in water, with adverse effects on life history traits demonstrated in the laboratory. In the field, such effects may vary depending on species, according to their relative sensitivity and ecology. The aims of this study were to i) establish how various intensities of MC-producing cyanobacteria proliferations alter the structure of gastropod community and ii) compare MC tissue concentration in gastropods in the field with those obtained in our previous laboratory experiments on the prosobranch Potamopyrgus antipodarum and the pulmonate Lymnaea stagnalis. We explored these questions through a one-year field study at three stations at Grand-Lieu Lake (France) affected by different intensities of cyanobacteria proliferations. A survey of the community structure and MC content of both cyanobacteria and gastropods was associated with a caging experiment involving P. antipodarum and L. stagnalis. In total, 2592 gastropods belonging to 7 prosobranch and 16 pulmonate species were collected. However, distribution among the stations was unequal with 62% vs 2% of gastropods sampled respectively at the stations with the lowest vs highest concentrations of MC. Irrespective of the station, pulmonates were always more diverse, more abundant and occurred at higher frequencies than prosobranchs. Only the pulmonate Physa acuta occurred at all stations, with abundance and MC tissue concentration (< or = 4.32 microg g DW(-1)) depending on the degrees of MC-producing cyanobacteria proliferations in the stations; therefore, P. acuta is proposed as a potential sentinel species. The caging experiment demonstrated a higher MC accumulation in L. stagnalis (< or = 0.36 microg g DW(-1) for 71% of individuals) than in P. antipodarum (< or = 0.02 microg g DW(-1) for 12%), corroborating previous laboratory observations. Results are discussed in terms of differential gastropod sensitivity and MC transfer through the food web.

First occurrence of cylindrospermopsin in freshwater in France.

Eleven waterbodies in Western France dominated by cyanobacteria of the genera Aphanizomenon and Anabaena were analyzed in September 2006 for microcystins (MC) and cylindrospermopsin (CYN). CYN was detected for the first time in France in four of them in the presence of Aphanizomenon flos-aquae and in the presence of Anabaena planctonica in the other. The intracellular concentrations of CYN measured by LC-MS/MS ranged between 1.55 and 1.95 microg/L. The occurrence of CYN represents an additional health hazard to MC especially because Aphanizomenon flos-aquae is the third most common species in freshwaters in France.

Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs.

Community structure and microcystin accumulation of freshwater molluscs were studied before and after cyanobacterial proliferations, in order to assess the impact of toxic blooms on molluscs and the risk of microcystin transfer in food web. Observed decrease in mollusc abundance and changes in species richness in highly contaminated waters were not significant; however, relative abundances of taxa (prosobranchs, pulmonates, bivalves) were significantly different before and after cyanobacterial bloom. Pulmonates constituted the dominant taxon, and bivalves never occurred after bloom. Microcystin accumulation was significantly higher in molluscs from highly (versus lowly) contaminated waters, in adults (versus juveniles) and in pulmonates (versus prosobranchs and bivalves). Results are discussed according to the ecology of molluscs, their sensitivity and their ability to detoxify.

A phycocyanin probe as a tool for monitoring cyanobacteria in freshwater bodies.

In many countries, the presence of cyanobacteria in freshwater bodies used for both drinking water and recreational purposes is under increasing public health attention. Water managers are considering how to implement monitoring that leads to a minimization of the risks incurred by the users of potentially contaminated sites. To address this question, this study involved assessing the performance of a submersible probe for measuring phycocyanin-specific fluorescence as a function of cyanobacterial biomass, with the aim of applying it as a tool for surveillance management. Its advantages and limits compared to more traditional analyses are discussed. The monitoring of cyanobacteria in the water bodies of western France was carried out using a minifluorimeter specific to the fluorescence of phycocyanin, a pigment specific to cyanobacteria. The results are compared with the analyses recommended by the World Health Organisation (chlorophyll a and cell counting). This study based on nearly 800 samples shows a significant correlation between the phycocyanin content and the cyanobacterial biomass, expressed as the number of cells per mL (R2 = 0.73). This submersible probe is simple and rapid to use, making it possible to take into account horizontal and vertical heterogeneities in the proliferation growth. In this way, we are able to detect at an early stage the conditions that could potentially lead to a risk, in order to start sampling. Due to its sensitivity, this tool proves suitable for monitoring aimed at reducing the risks incurred by the users of contaminated sites and launching preventative actions. The use of the phycocyanin probe provides an effective tool to complement traditional analyses of cyanobacterial presence. It is suggested that a surveillance protocol based on phycocyanin concentration can significantly improved the accuracy of the extent of cyanobacterial bloom development in the light of spatial and temporal variabilities associated with these occurrences.

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.

Interactions between cyanobacteria and gastropods II. Impact of toxic Planktothrix agardhii on the life-history traits of Lymnaea stagnalis.

Hepatotoxins are frequently produced by many cyanobacterial species. Microcystins (MCs) are the most frequent and widely studied hepatotoxins, with potentially hazardous repercussions on aquatic organisms. As a ubiquitous herbivore living in eutrophic freshwaters, the snail Lymnaea stagnalis (Gastropoda: Pulmonata) is particularly exposed to cyanobacteria. The toxic filamentous Planktothrix agardhii is common in temperate lakes and is therefore, a potential food resource for gastropods. In the first part of this study, we demonstrated the ingestion of toxic P. agardhii by L. stagnalis during a 5 weeks exposure, with concomitant accumulation of, on average, 60% of total MCs ingested. After 3 weeks of non-toxic food (lettuce), approximately 90% of MCs were eliminated from tissues. Here, we investigate the impact of toxic P. agardhii consumption on the life-history traits (survival, growth and fecundity), locomotion and the structure of digestive and genital glands of juvenile and adult L. stagnalis. We observed a decrease of growth regardless of age, although this was more marked in juveniles, and a reduction of fecundity in adults. Survival and locomotion were not affected. Reduction of growth and fecundity continued to be observed even after feeding of non-toxic food for 3 weeks. The structure of the digestive gland was altered during the intoxication period but not irreversibly as cells tended to recover a normal status after the 3-week detoxification period. No histopathological changes occurred in the genital gland and oocytes, and spermatozoids were present in the gonadic acini. The density of cyanobacterial suspensions used in this study was comparable to those regularly observed in lakes, particularly in eutrophic waters. These results are discussed in terms of the negative impact of toxic cyanobacteria on natural communities of freshwater gastropods, and potential cascading effects on the equilibrium and functioning of the ecosystem.

Interactions between cyanobacteria and gastropods I. Ingestion of toxic Planktothrix agardhii by Lymnaea stagnalis and the kinetics of microcystin bioaccumulation and detoxification.

The last two decades have been marked by an increasing occurrence of toxic cyanobacterial blooms in aquatic ecosystems. These pose an expanding threat to the environment and to human health. Among the intracellular toxins produced by cyanobacteria, microcystins (hepatotoxins) are the most frequent and widely studied. As an ubiquitous herbivore living in eutrophic freshwaters, the freshwater snail Lymnaea stagnalis (Gastropoda: Pulmonata) is particularly exposed to cyanobacteria. The toxic filamentous Planktothrix agardhii is common in temperate lakes and is, therefore, a potential food resource for gastropods. We have studied the consumption of P. agardhii by L. stagnalis juveniles and adults in the presence or absence of non-toxic food (lettuce) over a 5-weeks period. Intoxication was followed by a 3-week detoxification period when snails were fed only on lettuce. The kinetics of microcystin accumulation and detoxification in the gastropods were established using the ELISA analytical method. The results showed an ingestion of toxic P. agardhii by L. stagnalis, even in the presence of lettuce, and the absence of food selection regardless of the age of the snails. Juveniles and adults consumed the same number of cells per millilitre and consumption was proportional to food availability. On average, 63% of cyanobacteria available were taken up during the first 24h. After 5 weeks of intoxication, 61% of the toxins present in the ingested cyanobacterial cells had accumulated in snail tissues (95% in the digestive-genital gland complex) with a concentration up to 80.4+/-4.9microggDW(-1). Toxin accumulation was greater in the gastropods fed on P. agardhii alone than those fed on the mixed diet, and was also greater in juveniles than in adults. After the removal of toxic cyanobacteria, detoxification was rapid: 64% of the toxins disappeared from snail tissues during the first week, but microcystins were still detected after 3 weeks (on average, 3.5+/-0.9microggDW(-1)). These results are discussed in terms of potential contamination to the food web.

Variation in the response of juvenile and adult gastropods (Lymnaea stagnalis) to cyanobacterial toxin (microcystin-LR).

Owing to the increasing public health problem related to the proliferation of toxin-producing cyanobacteria in aquatic ecosystems, we have investigated the response of the pond snail Lymnaea stagnalis exposed to 33 microg/L microcystin-LR for 6 weeks, through its life traits (survival, growth, fecundity) and locomotion; uptake of microcystin-LR was also quantified in the snail body tissues. To study the potential plasticity of the response related to the development stage, snails were exposed to the toxin as sexually immature and mature. According to our results, microcystin-LR accumulated in snail tissues at a higher level in juveniles (7.96 ng/g fresh weight) versus adults (2.17 ng/g fresh weight). Whatever the age, survival, growth, and locomotion were not affected by the toxin, and fecundity of polluted adults was reduced by half. These results are discussed in terms of negative effects of aqueous microcystin-LR occurrence on the dynamics of natural populations of gastropods.