Massive fish death associated with the toxic cyanobacterial Planktothrix sp. bloom in the Béni-Haroun Reservoir (Algeria).

In July 2017, a massive bloom of the potentially toxic cyanobacterial species Planktothrix sp. was observed in the Béni-Haroun Reservoir (Algeria), which was followed by a massive fish death. Many questions were raised in association with the role of cyanotoxins and the fish massive mortality. The objective of this paper is twofold: (1) to investigate the variability of physicochemical and cyanobacterial parameters (chlorophyll-a, phycocyanin, allophycocyanin, and microcystins) throughout the period of July 2017 to June 2018; and (2) to determine the free and total MC levels in viscera and muscle tissues of the common carp (Cyprinus carpio), which are found dead in the considered reservoir in October 2017. Our results showed microcystin (MC) concentrations in water samples (by the protein phosphatase PP2A assay) had reached 651.2 ng MC-LR equiv./L. Total MC levels (free + bound) in the viscera and muscle tissues of sampled dead fish were at 960.24 and 438.54 µg MC-LR equiv./kg dw, respectively. It is assumed that high concentrations of MC observed in the tissues of common carp induced a strong degradation of the visceral contents resulting in the complete lysis of the hepatopancreas, and presumably the massive fish death.

Cyanobacteria, Cyanotoxins, and Neurodegenerative Diseases: Dangerous Liaisons.

The prevalence of neurodegenerative disease (ND) is increasing, partly owing to extensions in lifespan, with a larger percentage of members living to an older age, but the ND aetiology and pathogenesis are not fully understood, and effective treatments are still lacking. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are generally thought to progress as a consequence of genetic susceptibility and environmental influences. Up to now, several environmental triggers have been associated with NDs, and recent studies suggest that some cyanotoxins, produced by cyanobacteria and acting through a variety of molecular mechanisms, are highly neurotoxic, although their roles in neuropathy and particularly in NDs are still controversial. In this review, we summarize the most relevant and recent evidence that points at cyanotoxins as environmental triggers in NDs development.

Are Bacterio- and Phytoplankton Community Compositions Related in Lakes Differing in Their Cyanobacteria Contribution and Physico-Chemical Properties?

Bacterioplankton community composition has become the center of research attention in recent years. Bacteria associated with toxic cyanobacteria blooms have attracted considerable interest. However, little is known about the environmental factors driving the bacteria community, including the impact of invasive cyanobacteria. Therefore, our aim has been to determine the relationships between heterotrophic bacteria and phytoplankton community composition across 24 Polish lakes with different contributions of cyanobacteria including the invasive species Raphidiopsis raciborskii. This analysis revealed that cyanobacteria were present in 16 lakes, while R. raciborskii occurred in 14 lakes. Our results show that bacteria communities differed between lakes dominated by cyanobacteria and lakes with minor contributions of cyanobacteria but did not differ between lakes with R. raciborskii and other lakes. Physical factors, including water and Secchi depth, were the major drivers of bacteria and phytoplankton community composition. However, in lakes dominated by cyanobacteria, bacterial community composition was also influenced by biotic factors such as the amount of R. raciborskii, chlorophyll-a and total phytoplankton biomass. Thus, our study provides novel evidence on the influence of environmental factors and R. raciborskii on lake bacteria communities.

Reversed evolution of grazer resistance to cyanobacteria.

Exploring the capability of organisms to cope with human-caused environmental change is crucial for assessing the risk of extinction and biodiversity loss. We study the consequences of changing nutrient pollution for the freshwater keystone grazer, Daphnia, in a large lake with a well-documented history of eutrophication and oligotrophication. Experiments using decades-old genotypes resurrected from the sediment egg bank revealed that nutrient enrichment in the middle of the 20th century, resulting in the proliferation of harmful cyanobacteria, led to the rapid evolution of grazer resistance to cyanobacteria. We show here that the subsequent reduction in nutrient input, accompanied by a decrease in cyanobacteria, resulted in the re-emergence of highly susceptible Daphnia genotypes. Expression and subsequent loss of grazer resistance occurred at high evolutionary rates, suggesting opposing selection and that maintaining resistance was costly. We provide a rare example of reversed evolution of a fitness-relevant trait in response to relaxed selection.

DNA damages induced by both endotoxin and exotoxin produced by cyanobacteria.

Cyanobacteria are prokaryotes involved in the contamination of aquatic environments since they release toxins that are highly potent and dangerous for living organisms. Prokaryotes produce endo and exotoxins, among others. Exotoxins are highly toxic, while endotoxins have milder toxic effects. The present study evaluated the cytotoxicogenetic potency of both toxins studying them in different concentrations of cyanobacterial biomasses (1 µg/L, 1.5 µg/L, 2 µg/L), to assess the amount of exotoxin present in the cultured medium in which the cyanobacteria were grown. For this evaluation, we used an extract taken from the medium in a concentration of 10%. Our results showed that genotoxic and mutagenic changes in Allium cepa could be observed in all of the varying concentrations of biomass (endotoxin action) and also in the medium induced with exotoxin. Even at low concentrations, these toxins were highly effective at triggering changes in the DNA molecules of organisms exposed to them. This information is highly significant when considering environmental contamination caused by cyanobacteria blooms, since the results of this study show that these toxins may not only kill organisms when found in high concentrations, but also induce mutations when found in low concentrations. Since these mutations are expressed later on in the organisms, it is impossible to associate the observed effect with the event that induced the damage.

Analytical Technique Optimization on the Detection of ß-cyclocitral in Microcystis Species.

ß-Cyclocitral, specifically produced by Microcystis, is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that ß-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. ß-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for ß-cyclocitral have already been reported, ß-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why ß-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of ß-cyclocitral. Among these, heating (60 °C) was critical for the ß-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that ß-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of ß-cyclocitral in an aquatic environment.

Adverse effect of cylindrospermopsin on embryonic development in zebrafish (Danio rerio).

Eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is receiving great interest due to its increasing presence in waterbodies. However, the toxic effects of CYN on zebrafish development are poorly understood, especially the toxicological mechanism, which is still unclear. In this study, we examined the adverse effects of CYN on embryonic development in zebrafish. CYN (2-2000 nM) exposure decreased embryos survival rate, hatching rate, body length and eye size in a concentration-dependent manner and caused abnormalities in embryo morphology, including pericardial edema, spinal curvature, tail deformity, uninflated swim bladder, cardiac and vascular defects. CYN at concentrations of 20 nM or higher significantly increased ROS level and promoted cell apoptosis in zebrafish embryos. To preliminarily elucidate the potential mechanism of zebrafish developmental toxicity caused by CYN, we examined the expression of oxidative stress- and apoptotic-related genes. CYN could promote the expression of oxidative stress-related genes (SOD1, CAT and GPx1) and induce changes in transcriptional levels of apoptotic-related genes (p53, Bax and Bcl-2). Taken together, CYN induced adverse effects on zebrafish embryos development, which may associate with oxidative stress and apoptosis. These outcomes will advance our understanding of CYN toxicity, environmental problems and health hazards caused by climate changes and eutrophication.

Field cyanobacterial blooms producing retinoid compounds cause teratogenicity in zebrafish embryos.

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.

Diagnóstico do tratamento de esgotos no Rio Grande do Sul e sua associação aos riscos com a saúde / Diagnosis of sewage treatment in Rio Grande do Sul and its association with health risks

O presente trabalho tem como objetivo estimar o volume de esgoto gerado no RS em Demanda Bioquímica de Oxigênio (DBO kg/dia), e o volume de esgoto (DBO kg/dia) que chega até as estações de tratamento de esgoto ETEs; diagnosticar a eficiência das estações de tratamento de esgotoem relação ao percentual de remoção de (DBO kg/dia); correlacionando com a presença de cianobactérias próximas aos pontos de captação de água superficial para consumo humano; analisar se há inativação ou remoção de protozoários no lançamento do efluente ao retornar ao manancial. Como resultado obteve-se o volume total de matéria orgânica lançada nos corpos hídricos por dia estimado no Rio Grande do Sul é 511.769,00 DBO kg/dia no RS. As Estações de Tratamento de Esgoto captam 390.285,45 DBO kg/dia apresentaram resultados de remoção de 145.500,50 DBO kg/dia e de 22.963,21 DBO kg/dia residual; O volume ocioso das ETEs fica em torno de 5.049,89 L/s. O processo de eliminar ou inativar grande quantidade de protozoários presentes no afluente se dá através de etapas terciárias avançadas nas ETEs. Não encontramos no RS no ano 2019 ETEs com esses tipos avançados de tratamento. Sugere-se então que haja um melhor aproveitamento destas estações de tratamento no que se refere à quantidade e qualidade de volume de esgoto tratado e que sejam ampliados estudos para viabilidade de projetos de execução de novas ETEs para os demais municípios que despejam seus esgotos in natura nos mananciais impactando-os.

This work aims to estimate the volume of sewage generated in RS in Biochemical Oxygen Demand (BOD kg/day), and the volume of sewage (BOD kg/day) that reaches the sewage treatment stations (STS)s; diagnose the efficiency of sewage treatment station in relation to the percentage of removal (BOD kg/day); correlating with the presence of cyanobacteria close to the surface water intake points for human consumption; analyze if there is inactivation or removal of protozoa in the discharge of the effluent when returning to the source. As a result, the total volume of organic matter released into water bodies per day estimated in Rio Grande do Sul is 511,769.00 BOD kg/day in RS. The Sewage Treatment Plants capture 390,285.45 BOD kg/day showed removal results of 145,500.50 BOD kg/day and 22,963.21 BOD kg/day residual; The idle volume of ETEs is around 5,049.89 L/s. The process of eliminating or inactivating a large amount of protozoa present in the tributary takes place through advanced tertiary stages in the STSs. We did not find in RS in the year 2019 STSs with these advanced types of treatment. It is therefore suggested that there is a better use of these treatment plants with regard to the quantity and quality of the volume of treated sewage and that studies for the feasibility of projects for the execution of new STSs be expanded to the other municipalities that dump their raw sewage in water sources impacting them.

Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats.

Benthic cyanobacterial proliferations in rivers are have been reported with increasing frequency worldwide. In the Eel and Russian rivers of California, more than a dozen dog deaths have been attributed to cyanotoxin toxicosis since 2000. Periphyton proliferations in these rivers comprise multiple cyanobacterial taxa capable of cyanotoxin production, hence there is uncertainty regarding which taxa are producing toxins. In this study, periphyton samples dominated by the cyanobacterial genera Anabaena spp. and Microcoleus spp. and the green alga Cladophora glomerata were collected from four sites in the Eel River catchment and one site in the Russian River. Samples were analysed for potential cyanotoxin producers using polymerase chain reaction (PCR) in concert with Sanger sequencing. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry, and anatoxin quota (the amount of cyanobacterial anatoxins per toxigenic cell) determined using droplet digital PCR. Sequencing indicated Microcoleus sp. and Nodularia sp. were the putative producers of cyanobacterial anatoxins and nodularins, respectively, regardless of the dominant taxa in the mat. Anatoxin concentrations in the mat samples varied from 0.1 to 18.6 µg g-1 and were significantly different among sites (p < 0.01, Wilcoxon test); however, anatoxin quotas were less variable (< 5-fold). Dihydroanatoxin-a was generally the most abundant variant in samples comprising 38% to 71% of the total anatoxins measured. Mats dominated by the green alga C. glomerata contained both anatoxins and nodularin-R at concentrations similar to those of cyanobacteria-dominated mats. This highlights that even when cyanobacteria are not the dominant taxa in periphyton, these mats may still pose a serious health risk and indicates that more widespread monitoring of all mats in a river are necessary.

Functional characterization of aminotransferase involved in serine and aspartate metabolism in a halotolerant cyanobacterium, Aphanothece halophytica.

Aminotransferases catalyze the reversible pyridoxal phosphate-dependent transfer of amino groups from amino acids to oxo acids and play important roles for the balance between carbon and nitrogen metabolism. In this report, four aminotransferases (Ap1-Ap4) from a halotolerant cyanobacterium Aphanothece halophytica were examined. The results revealed that Ap1 and Ap2 exhibited the aspartate:2-oxoglutarate aminotransferase (AspAT) activity whereas Ap2 catalyzed further aminotransferase activities with alanine (AlaAT) and LL-diaminopimelate (an intermediate for the synthesis of Lys/peptidoglycan) as amino donors. Ap4 exhibited bifunctional aminotransferase with phosphoserine (PSAT) and glycine (GGAT) as amino donors. No activity was observed for Ap3. We identified third gene encoding phosphoserine phosphatase (PSP) in phosphorylate serine biosynthetic pathway. The levels of mRNA for Ap2 and ApMurE encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase were increased after salt stress. These results suggest the link among photorespiratory metabolite (serine, glycine, glyoxylate), phosphorylate serine biosynthetic pathway and aspartate metabolism via aminotransferases for the synthesis of peptidoglycan and betaine under salt stress conditions.

Fungicides at environmentally relevant concentrations can promote the proliferation of toxic bloom-forming cyanobacteria by inhibiting natural fungal parasite epidemics.

Fungal parasites of the phylum Chytridiomycota (chytrids) are increasingly recognized as potent control agents of phytoplankton, including toxic bloom-forming cyanobacteria. We experimentally tested whether agricultural fungicides can interfere with natural epidemics caused by parasitic chytrid fungi and thereby favor cyanobacterial bloom formation. Specifically, we exposed the toxic bloom-forming cyanobacterium Planktothrix and its chytrid parasite Rhizophydium megarrhizum to different concentrations of the widely used agricultural fungicides tebuconazole and azoxystrobin, as well as the medical fungicide itraconazole (the latter was included to test its potential to suppress infection in vitro). Environmentally relevant concentrations of tebuconazole (20-200 µg/L) and azoxystrobin (1-30 µg/L) significantly decreased infection prevalence over a timespan of seven days, while not affecting the growth of uninfected cyanobacteria. Itraconazole suppressed infection completely. Our findings demonstrate that agricultural fungicide run-off has the potential to inhibit natural chytrid epidemics and, thereby, to promote the proliferation of toxic cyanobacteria.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, ß-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and ß-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 µg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of ß-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Transfer of a cyanobacterial neurotoxin, ß-methylamino-l-alanine from soil to crop and its bioaccumulation in Chinese cabbage.

Most cyanobacteria can synthesize the notorious neurotoxin ß-methylamino-l-alanine (BMAA) that is transferred and bioaccumulated through natural food webs of aquatic ecosystems and ultimately arises the potential human health risks by the consumption of BMAA-contaminated aquatic products. Fertilization of cyanobacterial composts in farmlands may also lead to BMAA contamination in soil and its possible transfer and bioaccumulation within major crops, thereby threatening human health. In this study, we first detected a high level of BMAA (1.8-16.3 µg g-1) in cyanobacterial composts. In order to assess the health risks from cyanobacterial composts, we planted Chinese cabbage, a favourite vegetable in China, in BMAA-contaminated soil (4.0 µg BMAA/g soil) and detected the levels of free and protein-associated BMAA in soil and crop organs during the whole growth cycle by HPLC-MS/MS, respectively. Our results demonstrated that BMAA indeed transferred from soil to root, stem and leaf of Chinese cabbage during the growth cycle. The BMAA level finally accumulated in the edible portions was much higher than the initial level in soil, including 13.82 µg g-1 in leaf and 4.71 µg g-1 in stem. The discovery of the neurotoxin BMAA in this vegetable, an ending in human consumption, not only provides a BMAA transfer pathway in farmland ecosystems, but also is alarming and requires attention due to the potential risk of cyanobacterial composts to human health.

Assessment of the cytotoxicity, antioxidant activity and chemical composition of extracts from the cyanobacterium Fischerella major Gomont.

Cyanoprokaryotes (Cyanobacteria/Cyanophyta) are ancient photosynthetic prokaryotic organisms with cosmopolitan distribution. They are producers of a number of biologically active substances with antitumor and antifungal activity, vitamins, antibiotics, algaecides, insecticides, repellents, hormones, immunosuppressants and toxins. So far, the cyanobacterium Fischerella major Gomont has not been studied regarding its impact on the environment and human health. In this study, the cytotoxic, antioxidant and antitumor activities of four extracts prepared from Fischerella major were evaluated in vitro. In addition, the total phenolic content and the potential for production of cyanotoxins were also analyzed. The conducted GC/MS analysis identified 45 compounds with different chemical nature and biological activity. Presence of microcystins and saxitoxins was detected in all Fischerella major extracts. In vitro testing on cell cultures showed a significant concentration- and time-dependent cytotoxic effect on all cell lines (HeLa, SK-Hep-1 and FL) treated at three exposure times (24, 48 and 72 h) with four extracts. A selective antitumor effect was not observed. This is the first study demonstrating biological activity of extracts from Fischerella major, which makes it an interesting subject for further research, including environmental risk assessments (as producer of cyanotoxins) or as a potential source of pharmaceuticals.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Sensitive detection of oxidative DNA damage in cyanobacterial cells using supercoiling-sensitive quantitative PCR.

Supercoiling-sensitive quantitative PCR (ss-qPCR) is a sensitive technique to detect DNA damage in cultured animal cells and cultured/clinical human cells in vitro. In this study, we investigated whether the ss-qPCR method can be applied as a sensitive means to detect oxidative DNA damage in unicellular organisms. We used the model cyanobacterium Synechococcus elongatus PCC 7942 as a test organism and H2O2 as an exogenetic oxidative toxicant. Results showed that a significant increase in the plasmid DNA damage of S. elongatus PCC 7942 was induced by H2O2 in a dose- and time-dependent manner. The sensitivity of ss-qPCR in detecting DNA damage of the cyanobacterium was higher than the cell inhibition method (up to 255 times) as calculated from the slopes of fitted curves in the tested sub-toxic concentration range of 1-5 mM H2O2. Ss-qPCR also detected repairable low-intensity DNA damage in the cyanobacterium when DNA repair inhibitors were used. The detection limit of modified ss-qPCR was one tenth of that of previous methods. We also observed that ss-qPCR can be used to detect genomic DNA conformation change of cyanobacterium exposed to H2O2. Thus, this method will provide a powerful technical support for investigating the mechanisms of cyanobacterial DNA damage by environmental factors, especially intracellular reactive oxygen species enhancement-related factors.

Determination of the Settling Rate of Clay/Cyanobacterial Floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

Nitrogen limitation, toxin synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event.

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0-25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 µg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

Widespread anatoxin-a detection in benthic cyanobacterial mats throughout a river network.

Benthic algae fuel summer food webs in many sunlit rivers, and are hotspots for primary and secondary production and biogeochemical cycling. Concerningly, riverine benthic algal assemblages can become dominated by toxic cyanobacteria, threatening water quality and public health. In the Eel River in Northern California, over a dozen dog deaths have been attributed to cyanotoxin poisonings since 2000. During the summers of 2013-2015, we documented spatial and temporal patterns of cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a in benthic cyanobacterial mats. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 Anabaena-dominated or Phormidium-dominated cyanobacterial mat samples were collected from 27 locations to measure intracellular anatoxin-a (ATX) and microcystins (MCY). Anatoxin-a levels were higher than microcystin for both SPATT (mean MCY = 0.8 and ATX = 4.8 ng g resin-1 day-1) and cyanobacterial mat samples (mean MCY = 0.074 and ATX = 1.89 µg g-1 DW). Of the benthic mats sampled, 58.9% had detectable anatoxin-a (max = 70.93 µg g-1 DW), while 37.6% had detectable microcystins (max = 2.29 µg g-1 DW). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence in benthic cyanobacterial mats in a North American watershed.