RESUMO
Cyanobacterial blooms pose a risk to wild and domestic animals as well as humans due to the toxins they may produce. Humans may be subjected to cyanobacterial toxins through many routes, e.g., by consuming contaminated drinking water, fish, and crop plants or through recreational activities. In earlier studies, cyanobacterial cells have been shown to accumulate on leafy plants after spray irrigation with cyanobacteria-containing water, and microcystin (MC) has been detected in the plant root system after irrigation with MC-containing water. This paper reports a series of experiments where lysis of cyanobacteria in abstracted lake water was induced by the use of hydrogen peroxide and the fate of released MCs was followed. The hydrogen peroxide-treated water was then used for spray irrigation of cultivated spinach and possible toxin accumulation in the plants was monitored. The water abstracted from Lake Köyliönjärvi, SW Finland, contained fairly low concentrations of intracellular MC prior to the hydrogen peroxide treatment (0.04 µg L-1 in July to 2.4 µg L-1 in September 2014). Hydrogen peroxide at sufficient doses was able to lyse cyanobacteria efficiently but released MCs were still present even after the application of the highest hydrogen peroxide dose of 20 mg L-1. No traces of MC were detected in the spinach leaves. The viability of moving phytoplankton and zooplankton was also monitored after the application of hydrogen peroxide. Hydrogen peroxide at 10 mg L-1 or higher had a detrimental effect on the moving phytoplankton and zooplankton.
Assuntos
Irrigação Agrícola/métodos , Cianobactérias , Microcistinas , Eliminação de Resíduos Líquidos/métodos , Microbiologia da Água , Animais , Finlândia , Humanos , Peróxido de Hidrogênio , LagosRESUMO
The composition and abundance of cyanobacteria and their toxins, microcystins (MCs), and cylindrospermopsins (CYN) were investigated using samples collected at monthly intervals from the Amudde side of Koka Reservoir from May 2013 to April 2014. Cyanobacteria were the most abundant and persistent phytoplankton taxa with Microcystis and Cylindrospermopsis species alternately dominating the phytoplankton community of the reservoir and accounting for up to 84.3 and 11.9% of total cyanobacterial abundance, respectively. Analyses of cyanotoxins in filtered samples by HPLC-DAD and LC-MS/MS identified and quantified five variants of MCs (MC-LR, MC-YR, MC-RR, MC-dmLR, and MC-LA) in all samples, with their total concentrations ranging from 1.86 to 28.3 µg L-1 and from 1.71 to 33 µg L-1, respectively. Despite the presence and occasional abundance of Cylindrospermopsis sp., cylindrospermopsin was not detected. Redundancy analysis (RDA) showed that the environmental variables explained 82.7% of the total variance in cyanobacterial abundance and microcystin concentration. The presence of considerably high levels of MCs almost throughout the year represents a serious threat to public health and life of domestic and wild animals.
Assuntos
Cianobactérias/crescimento & desenvolvimento , Monitoramento Ambiental , Microcistinas/análise , Microbiologia da Água , Poluentes da Água/análise , Alcaloides , Toxinas Bacterianas , Toxinas de Cianobactérias , Cylindrospermopsis , Etiópia , Microcystis , Fitoplâncton , Uracila/análogos & derivadosRESUMO
A broad-spectrum noncompetitive immunoassay allowing sensitive and simple detection of a group of similar compounds would be an ideal tool for screening low-molecular weight analytes (<2000 Da) having many variants. However, the development of an essential antibody pair capable of sandwich-type recognition of the analytes' small generic core structure is a demanding task due to limited space available for simultaneous binding of two different antibodies. We report here a generic noncompetitive assay for cyanobacterial microcystins (MCs) and nodularins (Nod), a group of structurally related small cyclic peptides (â¼1000 Da) with more than 100 naturally occurring analogs. The assay is based on the unique combination of a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domain (scFv) and a monoclonal antibody capable of binding to an Adda-group (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) present in all MCs/Nod. The anti-IC scFv was isolated from a large synthetic antibody library with phage display and used to develop a single-step sandwich-type noncompetitive immunocomplex assay. The sensitive time-resolved immunofluorometry-based assay is capable of detecting all the 11 tested commonly occurring hepatotoxins (MC-LR, -dmlR, -RR, -dmRR, -LA, -LY, -LF, -LW, -YR, -WR, and Nod-R) at concentration below 0.1 µg/L in a 1 h assay. Using MC-LR, the most studied toxic and widely distributed of the toxins, the calculated detection limits (based on blank + 3SD response) are â¼0.026 µg/L in 1 h and â¼0.1 µg/L in 10 min assay time. This is by far the fastest reported immunoassay for MCs and Nod with a detection limit far below the World Health Organization's guideline limit (1 µg/L of MC-LR equivalent in drinking water). The assay was validated with spiked tap and lake water as well as with environmental surface water samples. The developed assay provides a simple, rapid, and highly sensitive tool for the quantitative detection of MCs/Nod with the additional benefit of automation and high-throughput possibilities for large scale screening of drinking and environmental surface water samples. Furthermore, the study describes the first demonstration of the assay intended for the detection of an analyte group comprising similar low-molecular weight compounds exhibiting the benefits of a reagent excess type assay.
RESUMO
This paper presents a case study of a massive fish mortality during a Cylindrospermopsis raciborskii bloom in Aleksandrovac Lake, Serbia in mid-December 2012. According to a preliminary investigation of the samples taken on November 6 before the fish mortalities and to extended analyses of samples taken on November 15, no values of significant physicochemical parameters emerged to explain the cause(s) of the fish mortality. No industrial pollutants were apparent at this location, and results excluded the likelihood of bacterial infections. Even after freezing, the dissolved oxygen concentration in the water was sufficient for fish survival. High concentrations of chlorophyll a and phaeophytin occurred in the lake, and phytoplankton bloom samples were lethal in Artemia salina bioassays. A bloom of the cyanobacterium C. raciborskii was recorded during November. Although the A. salina bioassays indicated the presence of toxic compounds in the cyanobacterial cells, the cyanotoxins, microcystins, cylindrospermopsin and saxitoxin were not detected.
Assuntos
Cylindrospermopsis/crescimento & desenvolvimento , Monitoramento Ambiental , Peixes/fisiologia , Proliferação Nociva de Algas , Lagos/microbiologia , Poluentes Químicos da Água/toxicidade , Alcaloides , Animais , Toxinas Bacterianas/toxicidade , Toxinas de Cianobactérias , Microcistinas , Uracila/análogos & derivados , Uracila/toxicidade , Microbiologia da ÁguaRESUMO
The Earth has had a permanently oxic atmosphere only since the great oxygenation event (GOE) 2.3-2.4 billion years ago but recent geochemical research has revealed short periods of oxygen in the atmosphere up to a billion years earlier before the permanent oxygenation. If these "whiffs" of oxygen truly occurred, then oxygen-evolving (proto)cyanobacteria must have existed throughout the Archaean aeon. Trapping of oxygen by ferrous iron and other reduced substances present in Archaean oceans has often been suggested to explain why the oxygen content of the atmosphere remained negligible before the GOE although cyanobacteria produced oxygen. We tested this hypothesis by growing cyanobacteria in anaerobic high-CO2 atmosphere in a medium with a high concentration of ferrous iron. Microcystins are known to chelate iron, which prompted us also to test the effects of microcystins and nodularins on iron tolerance. The results show that all tested cyanobacteria, especially nitrogen-fixing species grown in the absence of nitrate, and irrespective of the ability to produce cyanotoxins, were iron sensitive in aerobic conditions but tolerated high concentrations of iron in anaerobicity. This result suggests that current cyanobacteria would have tolerated the high-iron content of Archaean oceans. However, only 1 % of the oxygen produced by the cyanobacterial culture was trapped by iron, suggesting that large-scale cyanobacterial photosynthesis would have oxygenated the atmosphere even if cyanobacteria grew in a reducing ocean. Recent genomic analysis suggesting that ability to colonize seawater is a secondary trait in cyanobacteria may offer a partial explanation for the sustained inefficiency of cyanobacterial photosynthesis during the Archaean aeon, as fresh water has always covered a very small fraction of the Earth's surface. If oxygenic photosynthesis originated in fresh water, then the GOE marks the adaptation of cyanobacteria to seawater, and the late-Proterozoic increase in oxygen concentration of the atmosphere is caused by full oxidation of the oceans.
Assuntos
Cianobactérias/metabolismo , Compostos Ferrosos/metabolismo , Oxigênio/metabolismo , Archaea/metabolismo , Evolução Biológica , Microcystis/metabolismo , Nodularia/metabolismo , Oxirredução , FotossínteseRESUMO
Many marine dinoflagellates, including several species of the genus Alexandrium, Gymnodinium catenatum, and Pyrodinium bahamense are known for their capability to produce paralytic shellfish toxins (PST), which can cause severe, most often food-related poisoning. The recent discovery of the first PST biosynthesis genes has laid the foundation for the development of molecular detection methods for monitoring and study of PST-producing dinoflagellates. In this study, a probe-based qPCR method for the detection and quantification of the sxtA4 gene present in Alexandrium spp. and Gymnodinium catenatum was designed. The focus was on Alexandrium ostenfeldii, a species which recurrently forms dense toxic blooms in areas within the Baltic Sea. A consistent, positive correlation between the presence of sxtA4 and PST biosynthesis was observed, and the species was found to maintain PST production with an average of 6 genomic copies of sxtA4. In August 2014, A. ostenfeldii populations were studied for cell densities, PST production, as well as sxtA4 and species-specific LSU copy numbers in Föglö, Åland, Finland, where an exceptionally dense bloom, consisting of 6.3×106cellsL-1, was observed. Cell concentrations, and copy numbers of both of the target genes were positively correlated with total STX, GTX2, and GTX3 concentrations in the environment, the cell density predicting toxin concentrations with the best accuracy (Spearman's ρ=0.93, p<0.01). The results indicated that all A. ostenfeldii cells in the blooms harbored the genetic capability of PST production, making the detection of sxtA4 a good indicator of toxicity.
Assuntos
Dinoflagellida/genética , Dinoflagellida/metabolismo , Saxitoxina/genética , Saxitoxina/metabolismo , Dinoflagellida/química , Finlândia , Densidade Demográfica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.
Assuntos
Cianobactérias/química , Inibidores Enzimáticos/química , Peptídeos Cíclicos/química , Países Bálticos , Cromatografia Líquida de Alta Pressão , Humanos , Elastase Pancreática/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Água do Mar , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , Trombina/antagonistas & inibidoresRESUMO
Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⻹ to 4.92 µg·mg⻹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.
Assuntos
Alcaloides/biossíntese , Anabaena/metabolismo , Aphanizomenon/metabolismo , Toxinas Bacterianas/biossíntese , Uracila/análogos & derivados , Microbiologia da Água , Poluição Química da Água , Alcaloides/análise , Alcaloides/química , Alcaloides/isolamento & purificação , Anabaena/classificação , Anabaena/crescimento & desenvolvimento , Anabaena/isolamento & purificação , Aphanizomenon/classificação , Aphanizomenon/crescimento & desenvolvimento , Aphanizomenon/isolamento & purificação , Toxinas Bacterianas/análise , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Toxinas de Cianobactérias , Lagos/microbiologia , Tipagem Molecular , Estações do Ano , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Turquia , Uracila/análise , Uracila/biossíntese , Uracila/química , Uracila/isolamento & purificação , Qualidade da ÁguaRESUMO
The cyanobacterial cytotoxin cylindrospermopsin (CYN) has become increasingly common in fresh waters worldwide. It was originally isolated from Cylindrospermopsis raciborskii in Australia; however, in European waters, its occurrence is associated with other cyanobacterial species belonging to the genera Aphanizomenon and Anabaena. Moreover, cylindrospermopsin-producing strains of widely distributed C. raciborskii have not yet been observed in European waters. The aims of this work were to assess the occurrence of CYN in lakes of western Poland and to identify the CYN producers. The ELISA tests, high-performance liquid chromatography (HPLC)-DAD, and HPLC-mass spectrometry (MS)/MS were conducted to assess the occurrence of CYN in 36 lakes. The cyrJ, cyrA, and pks genes were amplified to identify toxigenic genotypes of cyanobacteria that are capable of producing CYN. The toxicity and toxigenicity of the C. raciborskii and Aphanizomenon gracile strains isolated from the studied lakes were examined. Overall, CYN was detected in 13 lakes using HPLC-MS/MS, and its concentrations varied from trace levels to 3.0 µg L(-1). CYN was widely observed in lakes of western Poland during the whole summer under different environmental conditions. Mineral forms of nutrients and temperature were related to CYN production. The molecular studies confirmed the presence of toxigenic cyanobacterial populations in all of the samples where CYN was detected. The toxicity and toxigenicity analyses of isolated cyanobacteria strains revealed that A. gracile was the major producer of CYN.
Assuntos
Aphanizomenon/isolamento & purificação , Lagos/microbiologia , Uracila/análogos & derivados , Poluentes da Água/análise , Alcaloides , Anabaena/genética , Anabaena/isolamento & purificação , Aphanizomenon/genética , Aphanizomenon/metabolismo , Toxinas Bacterianas , Toxinas de Cianobactérias , Cylindrospermopsis/genética , Cylindrospermopsis/isolamento & purificação , Monitoramento Ambiental , Genes Bacterianos/genética , Fitoplâncton/genética , Fitoplâncton/isolamento & purificação , Fitoplâncton/metabolismo , Polônia , Análise de Sequência de DNA , Temperatura , Uracila/análise , Uracila/biossíntese , Microbiologia da Água , Poluentes da Água/metabolismoRESUMO
Blooms of toxic cyanobacteria, associated with illness and mortality in humans and animals, are becoming increasingly common worldwide. The safe use of surface waters for drinking water production and recreation necessitates assessment of toxigenic cyanobacteria. We have developed simple and reliable sample preparation and qPCR methods to detect microcystin-producing strains of three major bloom-forming genera, Anabaena, Microcystis and Planktothrix. The mcyB second thiolation motif, previously not recognized as a potential target for qPCR, was used as a basis for primer and genus-specific probe design. Assay specificity and sensitivity was confirmed with cultured cyanobacterial strains and the effect of different sample preparation methods on quantification was investigated. Sample filtration and cell lysis reduced assay time and resulted in more efficient amplification compared to DNA extraction. Positive correlation (p<0.005) between mcyB copy numbers and microcystin concentrations was observed in environmental samples. The results encourage the use of qPCR in water risk management.
Assuntos
Monitoramento Ambiental/métodos , Reação em Cadeia da Polimerase em Tempo Real , Anabaena/classificação , Anabaena/genética , Animais , Cianobactérias/classificação , Cianobactérias/genética , DNA Bacteriano/química , Microcistinas/metabolismo , Microcystis/genética , Gestão de Riscos/métodosRESUMO
Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Microcistinas/química , Microcistinas/toxicidade , Aminoácidos/análise , Apoptose/efeitos dos fármacos , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Fosfolipídeos/análise , Fosfoproteínas Fosfatases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.
Assuntos
Proteínas de Bactérias/biossíntese , Nodularia/metabolismo , Peptídeos Cíclicos/metabolismo , Peptídeos/metabolismo , Água do Mar/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Países Bálticos , Cromatografia Líquida/métodos , Variação Genética/genética , Espectrometria de Massas/métodos , Nodularia/genética , Nodularia/isolamento & purificação , Oceanos e Mares , Peptídeos/química , Peptídeos/genética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Austrália do Sul , TurquiaRESUMO
Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography-mass spectrometry (LC-MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS-MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (+/-20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC-ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.
Assuntos
Toxinas Bacterianas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Microcistinas/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , CianobactériasRESUMO
Six modern narrow-bore (50 mm length x 2-2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 microm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1-4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 microm particle-based or monolithic reversed-phase columns for routine use in microcystin and nodularin HPLC analyses.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Cianobactérias/metabolismo , Toxinas Marinhas/isolamento & purificação , Microcistinas/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Toxinas de Cianobactérias , Toxinas Marinhas/metabolismo , Microcistinas/metabolismo , Peptídeos Cíclicos/metabolismoRESUMO
Four ultra performance liquid chromatography (UPLC) columns with different reversed-phase characteristics were tested in the chromatographic separation of 10 microcystins and three nodularins, cyanobacterial peptide toxins. The columns had been designed by the manufacturer to withstand the ultra-high pressure generated by sub-2microm stationary phase particles and the Waters ACQUITY UPLC system in ultra-fast separations. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, with three gradient rise times: 1, 1.5 and 2min. The UV detection of the toxins was performed by a photodiode array detector. The chromatographic performance was evaluated both visually and by calculating chromatographic parameters such as capacity factor, resolution, peak width at half height, selectivity and peak asymmetry. The best chromatographic performance as judged by visual inspection was given by the ACQUITY BEH Shield RP18 and ACQUITY BEH Phenyl columns. The BEH Shield RP18 column showed excellent selectivity and resolution of chosen peak pairs considered as critical. A further advantage of the UPLC system was the high sample throughput with a total analysis time of 3.12min (injection-to-injection) equalling to 461 separations per 24h.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Microcistinas/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Microcystis/química , Nodularia/químicaRESUMO
Eight reversed-phase columns intended for rapid HPLC were assessed for the separation of thirteen microcystins and nodularins, cyclic peptidic hepatotoxins. The instrumentation consisted of an Agilent Technologies 1200 Rapid Resolution high performance liquid chromatography system coupled to a mass spectrometer, Bruker Daltonics Ultra Performance High Capacity Ion Trap MS (HCT Ultra) with electrospray ionisation (RRLC-ESI-IT-MS). The columns tested were 2-2.1 mm x 50 mm in diameter and length, and contained small particles (1.8-2.7 microm), or monolithic silica supports for fast performance. The shortest total run time achieved was 3 min 15 s including equilibration and injection. Critical microcystin pairs were still resolved. Several columns showed excellent performance.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Microcistinas/análise , Microcystis/química , Peptídeos Cíclicos/análise , Propriedades de SuperfícieRESUMO
Cyanobacterial dominance in eutrophic lakes causes water quality problems due to the production of toxins harmful to humans and animals, as well as a number of odorous compounds. Cylindrospermopsin (CYN) is a potent cytotoxic cyanobacterial metabolite involved in triggering illness in humans. The occurrence of CYN has been mostly associated with tropical and subtropical cyanobacteria. We analyzed CYN concentration and phytoplankton assemblages of three lakes located in western Poland during the summers of 2006 and 2007. CYN was detected in 46% of our samples using the HPLC and LC-MS/MS methods. CYN concentrations were in the range of 0.16-1.8 microg L(-1) and exceeded the drinking water guideline value of 1 microgL(-1) in two samples. This is the first report of CYN occurrence in this part of Europe and provides further evidence that this toxin is common not only in subtropical and tropical regions. The lakes were dominated by Planktothrix agardhii but the occurrence of the CYN investigated here might be associated with the invasive species Cylindrospermopsis raciborskii and/or native Aphanizomenon gracile.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Água Doce/microbiologia , Toxinas Marinhas/isolamento & purificação , Microcistinas/isolamento & purificação , Uracila/análogos & derivados , Alcaloides , Aphanizomenon/metabolismo , Toxinas de Cianobactérias , Cylindrospermopsis/metabolismo , Monitoramento Ambiental , Água Doce/química , Fitoplâncton/metabolismo , Polônia , Uracila/isolamento & purificaçãoAssuntos
Toxinas Bacterianas/análise , Cianobactérias/patogenicidade , Toxinas Marinhas/análise , Microcistinas/análise , Alcaloides , Animais , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/toxicidade , Cianobactérias/isolamento & purificação , Toxinas de Cianobactérias , Suplementos Nutricionais/análise , Suplementos Nutricionais/toxicidade , Monitoramento Ambiental , Eutrofização , Peixes/metabolismo , Água Doce/análise , Água Doce/microbiologia , Sedimentos Geológicos/análise , Humanos , Toxinas Marinhas/farmacocinética , Toxinas Marinhas/toxicidade , Microcistinas/farmacocinética , Microcistinas/toxicidade , Peptídeos Cíclicos/análise , Plantas/química , Frutos do Mar/análise , Tropanos/análise , Uracila/análogos & derivados , Uracila/análiseRESUMO
Toxin-producing cyanobacteria are abundant in surface waters used as drinking water resources. Microcystins (MC) produced by certain cyanobacteria present acute and chronic toxicity, and their removal in drinking water treatment processes is of increasing concern. Previous studies have demonstrated that chlorine and potassium permanganate are feasible oxidants for the removal of MCs present in drinking water resources, although the oxidation might lead to toxic oxidation products. In this paper, the toxicity of the oxidation products of MC-LR and -RR has been studied using protein phosphatase 1 inhibition assay (PPIA). The HPLC and ELISA analyses correlated with the PPIA results for both toxins. The samples containing the oxidation products were fractionated by HPLC and the toxicity of the fractions was tested with PPIA. The results revealed that protein phosphatase 1 inhibition emerged only from intact MC, while the oxidation products were non-toxic. Similar results were obtained in experiments performed in natural waters: no reaction products or interactions exhibiting protein phosphatase 1 inhibition were detected.
Assuntos
Cloro/química , Microcistinas/química , Permanganato de Potássio/química , Cloro/toxicidade , Toxinas Marinhas , Microcistinas/toxicidade , Oxirredução , Permanganato de Potássio/toxicidadeRESUMO
Microcystins (MCs) are cyclic heptapeptides and protein phosphatase inhibitors produced by many species of cyanobacteria. MCs have been shown to cause adverse effects on animals as well as plants and therefore methods are needed for analysing MCs in different matrices. We assessed the effects of MC exposure on broccoli (Brassica oleracea var. italica) and mustard (Sinapis alba) by watering the seedlings with water containing 0, 1 or 10 microgMCsL(-1) (concentrations typically found in natural waters). Morphological characteristics, chlorophyll concentrations and chlorophyll fluorescence were investigated, but the only distinct difference compared to control plants was a slight (<10%) growth inhibition seen in broccoli. Afterwards the MC concentration of selected plant samples was quantitated using liquid chromatography-mass spectrometry. Among the four MC variants present in the exposure mixture, only MC-LR was clearly detectable, and the toxin was found only in the roots of broccoli and mustard. The detected MC-LR concentrations ranged from 0.9 to 2.6ng (g fresh weight)(-1).