Surface alterations and compound release from aligner attachments in vitro.

AIM: The aim of the present study was to assess the alterations in morphology, roughness, and composition of the surfaces of a conventional and a flowable composite attachment engaged with aligners, and to evaluate the release of resin monomers and their derivatives in an aqueous environment. METHODS: Zirconia tooth-arch frames (n = 20) and corresponding thermoformed PET-G aligners with bonded attachments comprising two composite materials (universal-C and flowable-F) were fabricated. The morphological features (stereomicroscopy), roughness (optical profilometry), and surface composition (ATR-FTIR) of the attachments were examined before and after immersion in water. To simulate intraoral use, the aligners were removed and re-seated to the frames four times per day for a 7-day immersion period. After testing, the eluents were analyzed by LC-MS/MS targeting the compounds Bis-GMA, UDMA, 2-HEMA, TEGDMA and BPA and by LC-HRMS for suspect screening of the leached dental material compounds and their degradation products. RESULTS: After testing, abrasion-induced defects were found on attachment surfaces such as scratches, marginal cracks, loss of surface texturing, and fractures. The morphological changes and debonding rate were greater in F. Comparisons (before-after testing) revealed a significantly lower Sc roughness parameter in F. The surface composition of the aligners after testing showed minor changes from the control, with insignificant differences in the degree of C = C conversion, except for few cases with strong evidence of hydrolytic degradation. Targeted analysis results revealed a significant difference in the compounds released between Days 1 and 7 in both materials. Insignificant differences were found when C was compared with F in both timeframes. Several degradation products were detected on Day 7, with a strong reduction in the concentration of the targeted compounds. CONCLUSIONS: The use of aligners affects the surface characteristics and degradation rate of composite attachments in an aqueous environment, releasing monomers, and monomer hydrolysates within 1-week simulated use.

Leaching from a 3D-printed aligner resin.

AIM: To quantitatively assess the degree of conversion and the water-leaching targeted compound from 3D-printed aligners. MATERIALS AND METHODS: 3D-printed aligners were made of photopolymerized resin (Tera Harz TC85A). The molecular structure and degree of conversion of the set resin were investigated by ATR-FTIR spectroscopy (n = 5). The aligners (n = 10) were immersed in double distilled water for 1 week at 37°C and the eluents were analysed using liquid chromatography/mass spectrometry methods (LC-ESI-MS/MS for urethane dimethacrylate [UDMA] and LC-APCI-MS/MS for bispenol-A [BPA]). RESULTS: The resin was composed of aliphatic vinyl ester-urethane monomers, with acrylate and/or methacrylate functionalization. The degree of conversion was estimated as to 83%. There was no detection of BPA in any of the assessed samples (0.25 µg/l). Quantifiable amounts of UDMA were detected in all the exposed samples, ranging from 29 to 96 µg/l. CONCLUSIONS: Although efficiently polymerized and BPA free, the great variability in the amount of UDMA monomer leached from the examined samples may raise concerns on potential health hazards after repeated intraoral exposure, which is indicated for this class of materials.

Leptothoe, a new genus of marine cyanobacteria (Synechococcales) and three new species associated with sponges from the Aegean Sea.

Cyanobacterial diversity associated with sponges remains underestimated, though it is of great scientific interest in order to understand the ecology and evolutionary history of the symbiotic relationships between the two groups. Of the filamentous cyanobacteria, the genus Leptolyngbya is the most frequently found in association with sponges as well as the largest and obviously polyphyletic group. In this study, five Leptolyngbya-like sponge-associated isolates were investigated using a combination of molecular, chemical, and morphological approach and revealed a novel marine genus herein designated Leptothoe gen. nov. In addition, three new species of Leptothoe, Le. sithoniana, Le. kymatousa, and Le. spongobia, are described based on a suite of distinct characters compared to other marine Leptolyngbyaceae species/strains. The three new species, hosted by four sponge species, showed different degrees of host specificity. Leptothoe sithoniana and Le. kymatousa hosted by the sponges Petrosia ficiformis and Chondrilla nucula, respectively, seem to be more specialized than Le. spongobia, which was hosted by the sponges Dysidea avara and Acanthella acuta. All three species contained nitrogen-fixing genes and may contribute to the nitrogen budget of sponges. Leptothoe spongobia TAU-MAC 1115 isolated from Acanthella acuta was shown to produce microcystin-RR indicating that microcystin production among marine cyanobacteria could be more widespread than previously determined.

Multi-electron reduction of Wells-Dawson polyoxometalate films onto metallic, semiconducting and dielectric substrates.

The investigation of conditions allowing multi-electron reduction and reoxidation of polyoxometalate (POM) films onto solid substrates is considered an issue of critical importance for their successful incorporation in electronic devices, different types of sensors and catalytic systems. In the present paper, the rich multi-electron redox chemistry of films of Wells-Dawson ammonium salts, namely (NH4)6P2Mo18O62 and (NH4)6P2W18O62, on top of metallic (Al), semiconducting (ITO) and dielectric (SiO2) substrates under ambient conditions is investigated. The respective Keggin heteropolyacids, H3PMo12O40 and H3PW12O40, are also investigated for comparison. On Al substrates, the Wells-Dawson ammonium salts are found to be significantly more reduced (4-6e-) compared to the respective Keggin heteropolyacids (∼2e-), in accordance with their deeper lying lowest unoccupied molecular orbital (LUMO) level. Subsequent thermal treatment in air results in reoxidation of the initially highly reduced POM films. Similar behavior is found on ITO substrates, but in initially less reduced (2-4e-) Wells-Dawson POM films. On the other hand, on SiO2 substrates, the thermal reduction of (NH4)6P2Mo18O62 film is observed and attributed to the thermal oxidation of ammonium counterions by [P2Mo18O62]6- anions. Overall, the multi-electron reduction of Wells-Dawson ammonium salts onto metallic and semiconducting substrates (Al, ITO) is determined by the relative position of the LUMO level of POMs in relation to the Fermi level of the substrate (i.e. substrate work function) and affected in a synergistic way by the presence of ammonium counterions. In contrast, on dielectric substrates (SiO2) the reduction of Wells-Dawson POMs ((NH4)6P2Mo18O62) is attributed only to the oxidation of ammonium counterions.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

Exposure to ß-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Volatile Profiling of Spirulina Food Supplements.

Spirulina, a cyanobacterium widely used as a food supplement due to its high nutrient value, contains volatile organic compounds (VOCs). It is crucial to assess the presence of VOCs in commercial spirulina products, as they could influence sensory quality, various processes, and technological aspects. In this study, the volatile profiles of seventeen commercial spirulina food supplements were determined using headspace solid-phase microextraction (HS-SPME), coupled with gas chromatography-mass spectrometry (GC-MS). The identification of volatile compounds was achieved using a workflow that combined data processing with software tools and reference databases, as well as retention indices (RI) and elution order data. A total of 128 VOCs were identified as belonging to chemical groups of alkanes (47.2%), ketones (25.7%), aldehydes (10.9%), alcohols (8.4%), furans (3.7%), alkenes (1.8%), esters (1.1%), pyrazines (0.8%), and other compounds (0.4%). Major volatiles among all samples were hydrocarbons, especially heptadecane and heptadec-8-ene, followed by ketones (i.e., 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one, ß-ionone, 2,2,6-trimethylcyclohexan-1-one), aldehydes (i.e., hexanal), and the alcohol oct-1-en-3-ol. Several volatiles were found in spirulina dietary supplements for the first time, including 6,10-dimethylundeca-5,9-dien-2-one (geranylacetone), 6,10,14-trimethylpentadecan-2-one, hept-2-enal, octanal, nonanal, oct-2-en-1-ol, heptan-1-ol, nonan-1-ol, tetradec-9-en-1-ol, 4,4-dimethylcyclohex-2-en-1-ol, 2,6-diethylpyrazine, and 1-(2,5-dimethylfuran-3-yl) ethanone. The methodology used for VOC analysis ensured high accuracy, reliability, and confidence in compound identification. Results reveal a wide variety of volatiles in commercial spirulina products, with numerous newly discovered compounds, prompting further research on sensory quality and production methods.

Spirulina-based carbon materials as adsorbents for drinking water taste and odor control: Removal efficiency and assessment of cyto-genotoxic effects.

The sensory quality of drinking water, and particularly its taste and odor (T&O) is a key determinant of consumer acceptability, as consumers evaluate water by their senses. Some of the conventional treatment processes to control compounds which impart unpleasant T&O have limitations because of their low efficiency and/or high costs. Therefore, there is a great need to develop an effective process for removing T&O compounds without secondary concerns. The primary objective of this study was to assess for the first time the effectiveness of spirulina-based carbon materials in removing geosmin (GSM) and 2-methylisoborneol (2-MIB) from water, two commonly occurring natural T&O compounds. The efficiency of the materials to remove environmentally relevant concentrations of GSM and 2-MIB (ng L-1) from ultrapure and raw water was investigated using a sensitive headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC/MS) method. Moreover, the genotoxic and cytotoxic effects of the spirulina-based materials were assessed for the first time to evaluate their safety and their potential in the treatment of water for human consumption. Based on the results, spirulina-based materials were found to be promising for drinking water treatment applications, as they did not exert geno-cytotoxic effects on human cells, while presenting high efficiency in removing GSM and 2-MIB from water.

Salivary levels of eluents during Invisalign™ treatment with attachments: an in vivo investigation.

BACKGROUND: The aim of the present study was to investigate qualitatively and quantitatively the elution of substances from polyester-urethane (Invisalign™) aligners and resin composite attachments (Tetric EvoFlow) in vivo. METHODS: Patients (n = 11) treated with the aligners and attachments (16 per patient, without other composite restorations) for an average of 20 months, who were planned for attachment removed were enrolled in the study. Patients were instructed to rinse with 50 mL of distilled water upon entry and the rinsing solution was collected (before removal). Then, the attachments were removed with low-speed tungsten carbide burs for adhesive residue removal, a thorough water rinsing was performed immediately after the grinding process to discard grinding particle residues, and subsequently, after a second water-rinsing the solution was collected for analysis (after removal). The rinsing solutions were analyzed for targeted (LC-MS/MS: Bis-GMA, DCDMA, UDMA, BPA) and untargeted (LC-HRMS: screening of leached species and their degradation products) compounds. RESULTS: Targeted analysis revealed a significant reduction in BPA after attachment removal (4 times lower). Bis-GMA, DCDMA, UDMA were below the detection limit before removal but were all detectable after removal with Bis-GMA and UDMA at quantifiable levels. Untargeted analysis reviled the presence of mono-methacrylate transformation products of Bis-GMA (Bis-GMA-M1) and UDMA (UDMA-M1), UDMA without methacrylate moieties (UDMA-M2), and 4-(dimethylamino) benzoic acid (DMAB), the degradation product of the photo-initiator ethyl-4-(dimethylamino) benzoate (EDMAB), all after attachment removal. Several amino acids and endogenous metabolites were also found both before and after removal. CONCLUSIONS: Elevated levels of BPA were traced instantaneously in patients treated with Invisalign™ and flowable resin composite attachments for the testing period. BPA was reduced after attachment removal, but residual monomers and resin degradation products were found after removal. Alternative resin formulations and attachment materials may be utilized to reduce eluents.

Shifts in phytoplankton and zooplankton communities in three cyanobacteria-dominated lakes after treatment with hydrogen peroxide.

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L-1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L-1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.

Detection of secondary cyanobacterial metabolites using LC-HRMS in Lake Karaoun.

Harmful algal blooms events have been reported worldwide and during the last decades are occurred with increasing frequency and intensity due to the climate change and the high inputs of nutrients in freshwaters from anthropogenic activities. During blooms cyanobacteria release in water their toxic secondary metabolites, known as cyanotoxins, along with other bioactive metabolites. Due to the negative impacts of these compounds on aquatic ecosystems and public health, there is an urgent need to detect and identify known and unknown cyanobacterial metabolites in surface waters. In the frame of the present study, a method based on liquid chromatography - high resolution mass spectrometry (LC-HRMS) was developed to investigate the presence of cyanometabolites in bloom samples from Lake Karaoun, Lebanon. Data analysis was performed using Compound Discoverer software with related tools and databases in combination to the CyanoMetDB mass list for detection, identification and structural elucidation of the cyanobacterial metabolites. In the course of this study, 92 cyanometabolites were annotated including 51 cyanotoxins belonging to microcystins, 15 microginins, 10 aeruginosins, 6 cyclamides, 5 anabaenopeptins, a cyanopeptolin, the dipeptides radiosumin B and dehydroradiosumin, the planktoncyclin and a mycosporine-like amino acid. Out of them, 7 new cyanobacterial metabolites, the chlorinated MC-ClYR, [epoxyAdda5]MC-YR, MC-LI, aeruginosin 638, aeruginosin 588, microginin 755C and microginin 727 were discovered. Moreover, the presence of anthropogenic contaminants was recorded indicating the pollution of the lake and emphasizing the need for assessment of the co-occurrence of cyanotoxins, other cyanobacterial metabolites and other compounds hazardous to the environment. Overall, results prove the suitability of the proposed approach for the detection of cyanobacterial metabolites in environmental samples but also highlight the necessity of spectral libraries for these compounds, considering the absence of their reference standards.

Untargeted and targeted LC-MS and data processing workflow for the comprehensive analysis of oligopeptides from cyanobacteria.

Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography-mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8-13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.

Photocatalytic degradation of organic micropollutants under UV-A and visible light irradiation by exfoliated g-C3N4 catalysts.

In the present study, the photocatalytic performance of exfoliated graphitic carbon nitride (g-C3N4) catalysts, with enhanced properties and response in UV and visible light irradiation, was evaluated for the removal of selected contaminants i.e., diuron, bisphenol A and ethyl paraben. Commercial TiO2 Degussa P25 was also used as a reference photocatalyst. The g-C3N4 catalysts demonstrated good photocatalytic activity which in some cases is comparable to TiO2 Degussa P25 leading to high removal percentages of the studied micropollutants under UV-A light irradiation. In contrast to TiO2 Degussa P25, g-C3N4 catalysts were also able to degrade the studied micropollutants under visible light irradiation. For all the studied g-C3N4 catalysts under both UV-A and visible light irradiation, the overall degradation rate decreases in the order of bisphenol A > diuron > ethyl paraben. Among the studied g-C3N4, the chemically exfoliated catalyst (g-C3N4-CHEM) showed superior photocatalytic activity under UV-A light irradiation due to its enhanced characteristics, such as pore volume and specific surface area and ~ 82.0 % in 6 min, ~75.7 % in 15 min and ~ 96.3 % in 40 min removals were achieved for BPA, DIU and EP, respectively. Under visible light irradiation, the thermally exfoliated catalyst (g-C3N4-THERM) demonstrated the best photocatalytic performance and the degradation ranged from ~29.5 to 59.4 % after 120 min. EPR data revealed that the three g-C3N4 semiconductors generate mainly O2•-, whereas TiO2 Degussa P25 generates both HO• and O2•-, the latter only under UV-A light irradiation. Nevertheless, the indirect formation of HO• in the case of g-C3N4 should also be considered. Hydroxylation, oxidation, dealkylation, dechlorination and ring opening were the main degradation pathways. The process proceeded without significant alterations in toxicity levels. Based on the results, heterogeneous photocatalysis using g-C3N4 catalysts is a promising method for the removal of organic micropollutants without the formation of harmful transformation products.

Cyanotoxins in Bloom: Ever-Increasing Occurrence and Global Distribution of Freshwater Cyanotoxins from Planktic and Benthic Cyanobacteria.

Toxic cyanobacteria in freshwater bodies constitute a major threat to public health and aquatic ecosystems [...].

Sublethal exposure to Microcystis aeruginosa extracts during embryonic development reduces aerobic swimming capacity in juvenile zebrafish.

In the last decades, cyanobacterial harmful algal blooms (CyanoHABs) pose an intensifying ecological threat. Microcystis aeruginosa is a common CyanoHAB species in freshwater ecosystems, with severe toxic effects in a wide range of organisms. In the present paper we examined whether transient and short (48 h) exposure of fish embryos to sublethal levels of M. aeruginosa crude extract (200 mg biomass dw L-1) affects swimming performance at later life stages (end of metamorphosis, ca 12 mm TL, 22,23 days post-fertilization). Pre-exposed metamorphosing larvae presented a significant decrease in swimming performance (9.7 ± 1.6 vs 11.4 ± 1.7 TL s-1 in the control group, p < 0.01), and a significant decrease in the ventricle length-to-depth ratio (1.23 ± 0.15 vs 1.42 ± 0.15 in control fish, p < 0.05). In addition, extract-exposed fish presented significantly elevated rates of vertebral abnormalities (82 ± 13% vs 7 ± 4% in the control group), mainly consisting of the presence of extra neural and haemal processes. No significant differences between groups were detected in survival and growth rates. Results are discussed in respect to the mechanisms that might mediate the detected cyanobacterial effects. This is the first evidence of a direct link between sublethal exposure to M. aeruginosa during the embryonic period and swimming performance at later life-stages. Decreased swimming performance, altered cardiac shape, and elevated vertebral abnormalities in response to early exposure to M. aeruginosa could have significant effects on fish populations in the wild.

Anabaenopeptins from Cyanobacteria in Freshwater Bodies of Greece.

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

Cyanobacterial Toxins and Peptides in Lake Vegoritis, Greece.

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018-2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 µg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 µg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.

Investigation of the Occurrence of Cyanotoxins in Lake Karaoun (Lebanon) by Mass Spectrometry, Bioassays and Molecular Methods.

Lake Karaoun is the largest artificial lake in Lebanon and serves multiple purposes. Recently, intensive cyanobacterial blooms have been reported in the lake, raising safety and aesthetic concerns related to the presence of cyanotoxins and cyanobacterial taste and odor (T&O) compounds, respectively. Here, we communicate for the first time results from a recent investigation by LC-MS/MS covering multiple cyanotoxins (microcystins (MCs), anatoxin-a, cylindrospermopsin, nodularin) in water and fish collected between 2019 and 2020. Eleven MCs were identified reaching concentrations of 211 and 199 µg/L for MC-LR and MC-YR, respectively. Cylindrospermopsin, anatoxin-a and nodularin were not detected. The determination of the total MCs was also carried out by ELISA and Protein Phosphatase Inhibition Assay yielding comparable results. Molecular detection of cyanobacteria (16S rRNA) and biosynthetic genes of toxins were carried out by qPCR. Untargeted screening analysis by GC-MS showed the presence of T&O compounds, such as ß-cyclocitral, ß-ionone, nonanal and dimethylsulfides that contribute to unpleasant odors in water. The determination of volatile organic compounds (VOCs) showed the presence of anthropogenic pollutants, mostly dichloromethane and toluene. The findings are important to develop future monitoring schemes in order to assess the risks from cyanobacterial blooms with regard to the lake's ecosystem and its uses.

Development of a fast and selective method for the sensitive determination of anatoxin-a in lake waters using liquid chromatography-tandem mass spectrometry and phenylalanine-d5 as internal standard.

Anatoxin-a is a potent alkaloid neurotoxin produced by a number of cyanobacterial species and released in freshwaters during cyanobacterial blooms. Its high toxicity is responsible for several incidents of lethal intoxications of birds and mammals around the world; therefore anatoxin-a has to be regarded as a health risk and its concentration in lakes and water reservoirs should be monitored. Phenylalanine is a natural amino acid, also present in freshwaters, isobaric to anatoxin-a, with a very similar fragmentation pattern and LC retention. Since misidentification of phenylalanine as anatoxin-a has been reported in forensic investigations, special care must be taken in order to selectively determine traces of anatoxin-a in the presence of naturally occurring phenylalanine. A fast LC tandem MS method was developed by using a 1.8 microm 50 x 2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d(5) was employed as internal standard to compensate for electrospray ion suppression and sample preconcentration losses. Both compounds were preconcentrated 1,000-fold on a porous graphitic carbon solid-phase extraction (SPE) cartridge after adjustment of sample pH to 10.5. The method was validated by using lake water spiked at four different levels from 0.01 to 1 microg L(-1). Anatoxin-a recovery ranged from 73 to 97%, intra-day precision (RSD%) ranged from 4.2 to 5.9, while inter-day precision (RSD%) ranged from 4.2 to 9.1%. Limits of detection and quantification were 0.65 and 1.96 ng L(-1) respectively. The method was successfully applied for the detection of anatoxin-a in Greek lakes at concentrations ranging from less than 0.6 to 9.1 ng L(-1).

Fragmentation mass spectra dataset of linear cyanopeptides - microginins.

Microginins are the less common class of bioactive linear cyanobacterial peptides. Recently, an investigation for their presence in cyanobacteria from Greek freshwaters and strain cultures was carried out. The present dataset is related to the research article "New microginins from cyanobacteria of Greek freshwaters" [1]. Cyanobacterial biomass from bloom samples and cultured strains were extracted with aqueous methanol. Extracts were analysed by liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (LC-qTRAP MS/MS) in information dependent acquisition (IDA) mode. Enhanced ion product (EIP) mode was applied for the collection of ion fragmentation spectra. Identification of microginins was based on the characteristic fragment ions of the unique microginin amino acid 3-amino-2-hydroxy-decanoic acid (Ahda) and its modified forms. The analysis of fragmentation spectra revealed 51 microginin structures, including 36 new variants. This article provides the dataset of fragmentation mass spectra of the microginins detected in cyanobacteria from Greek freshwaters. As this class of cyanopeptides is produced by cyanobacteria from different geographical regions, the aim of this dataset is to enable the identification of microginins in future studies and therefore to contribute to a better evaluation of their presence in freshwater bodies worldwide.

ß-Ν-Methylamino-L-alanine interferes with nitrogen assimilation in the cyanobacterium, non-BMAA producer, Synechococcus sp. TAU-MAC 0499.

The production of ß-Ν-methylamino-L-alanine (BMAA) in cyanobacteria is triggered by nitrogen-starvation conditions and its biological role, albeit unknown, is associated with nitrogen assimilation. In the present study, the effect of BMAA (773 µg L-1) on nitrogen metabolism and physiology of the non-diazotrophic cyanobacterium and non-BMAA producer, Synechococcus sp. TAU-MAC 0499, was investigated. In order to study the combined effect of nitrogen availability and BMAA, nitrogen-starvation conditions were induced by transferring cells in nitrogen-free medium and subsequently exposing the cultures to BMAA. After short-term treatment (180 min) and in the presence of nitrogen, BMAA inhibited glutamine synthetase, which resulted in low concentration of glutamine. In the absence of nitrogen, although there was no effect on glutamine synthetase, a possible perturbation in nitrogen assimilation is reflected on the significant decrease in glutamate levels. During the long-term exposure (24-96 h), growth, photosynthetic pigments and total protein were not affected by BMAA exposure, except for an increase in protein and phycocyanin levels at 48 h in nitrogen replete conditions. Results suggest that BMAA interferes with nitrogen assimilation, in a different way, depending on the presence or absence of combined nitrogen, providing novel data on the potential biological role of BMAA.