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.

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.

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.

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 [...].

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.

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.

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.

Kinetic and mechanistic investigation of water taste and odor compound 2-isopropyl-3-methoxy pyrazine degradation using UV-A/Chlorine process.

The present study was launched as a continuation of global efforts to tackle problems associated with two important aesthetic characteristics, taste and odor (T&O), of drinking water. The UV-A/Chlorine process, a promising advanced oxidation process (AOP), was evaluated for the first time for the removal of 2-isopropyl-3-methoxy pyrazine (IPMP), a widely reported compound in the literature that causes unpleasant taste and odor when present in water at or below the ng L-1 level. It was found that the studied process was efficient for the removal of IPMP in both ultrapure and drinking water. The initial chlorine dosage influenced significantly the degradation efficiency under initial neutral pH values. Degradation efficiency of IPMP was slightly inhibited by using drinking water as matrix. Scavenging experiments highlighted the significant role of various reactive species (e.g. HO, ClO, Cl, Cl2-) generated during the process that have not been studied comprehensively until now. In addition, the significant role of HO was further verified by Electron paramagnetic resonance spectroscopy (EPR) experiments. Overall, the formation of diverse radicals during the UV-A/Chlorine treatment enhanced the degradation of IPMP, promoting mainly the formation of hydroxy, hydroperoxy and dealkylated derivatives. In contrast, chlorinated by-products were only identified in traces.

Diversity, Cyanotoxin Production, and Bioactivities of Cyanobacteria Isolated from Freshwaters of Greece.

Cyanobacteria are a diverse group of photosynthetic Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against a broad spectrum of organisms and cell lines. In this study, 29 strains isolated from freshwaters in Greece were classified using a polyphasic approach and assigned to Chroococcales, Synechococcales, and Nostocales, representing 11 genera and 17 taxa. There were good agreements between 16S ribosomal RNA (rRNA)-cpcBA-internal genetic spacer (IGS) characterization and morphological features, except for the Jaaginema-Limnothrix group which appears intermixed and needs further elucidation. Methanol extracts of the strains were analyzed for cyanotoxin production and tested against pathogenic bacteria species and several cancer cell lines. We report for the first time a Nostoc oryzae strain isolated from rice fields capable of producing microcystins (MCs) and a Chlorogloeopsis fritschii strain isolated from the plankton of a lake, suggesting that this species may also occur in freshwater temperate habitats. Strains with very high or identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activities. Extracts from Synechococcus cf. nidulans showed the most potent antibacterial activity against Staphylococcus aureus, whereas Jaaginema sp. strains exhibited potent cytotoxic activities against human colorectal adenocarcinoma and hepatocellular carcinoma cells. Jaaginema Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) 0110 and 0210 strains caused pronounced changes in the actin network and triggered the formation of numerous lipid droplets in hepatocellular carcinoma and green monkey kidney cells, suggesting oxidative stress and/or mitochondrial damage leading to apoptosis.

Neurotoxin BMAA and its isomeric amino acids in cyanobacteria and cyanobacteria-based food supplements.

Cyanobacteria are photosynthetic microorganisms distributed globally in aquatic and terrestrial environments. They are also industrially cultivated to be used as dietary supplements, as they have a high nutritional value; however, they are also known to produce a wide range of toxic secondary metabolites, called cyanotoxins. BMAA (ß-methylamino-l-alanine) and its most common structural isomers, DAB (2,4-diaminobutyric acid) and AEG (N-2-aminoethylglycine) produced by cyanobacteria, are non-proteinogenic amino acids that have been associated with neurodegenerative diseases. A possible route of exposure to those amino acids is through consumption of food supplements based on cyanobacteria. The review critically discusses existing reports regarding the occurrence of BMAA, DAB and AEG in cyanobacteria and cyanobacteria-based food supplements. It is shown that inconsistencies in reported results could be attributed to performance of different methods of extraction and analysis applied and in ambiguities regarding determination of soluble and bound fractions of the compounds. The critical aspect of this review aims to grow awareness of human intake of neurotoxic amino acids, while results presented in literature concerning dietary supplements aim to promote further research, quality control as well as development of guidelines for cyanotoxins in food products.

Occurrence and diversity of cyanotoxins in Greek lakes.

Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm3MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754 ng mg-1 dw, followed by MC-LR (458 ng mg-1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria).

A European Multi Lake Survey dataset of environmental variables, phytoplankton pigments and cyanotoxins.

Under ongoing climate change and increasing anthropogenic activity, which continuously challenge ecosystem resilience, an in-depth understanding of ecological processes is urgently needed. Lakes, as providers of numerous ecosystem services, face multiple stressors that threaten their functioning. Harmful cyanobacterial blooms are a persistent problem resulting from nutrient pollution and climate-change induced stressors, like poor transparency, increased water temperature and enhanced stratification. Consistency in data collection and analysis methods is necessary to achieve fully comparable datasets and for statistical validity, avoiding issues linked to disparate data sources. The European Multi Lake Survey (EMLS) in summer 2015 was an initiative among scientists from 27 countries to collect and analyse lake physical, chemical and biological variables in a fully standardized manner. This database includes in-situ lake variables along with nutrient, pigment and cyanotoxin data of 369 lakes in Europe, which were centrally analysed in dedicated laboratories. Publishing the EMLS methods and dataset might inspire similar initiatives to study across large geographic areas that will contribute to better understanding lake responses in a changing environment.

New SPE-LC-MS/MS method for simultaneous determination of multi-class cyanobacterial and algal toxins.

Cyanobacterial and algal toxins comprise a large group of harmful metabolites, belonging to different chemical classes, with a variety of chemical structures, physicochemical properties and toxic activities. In this study, a fast, simple and sensitive analytical method was developed for the simultaneous determination of multi-class cyanobacterial and algal toxins in water. The target compounds were: Cylindrospermopsin, Anatoxin-a, Nodularin, 12 Microcystins ([D-Asp3]MC-RR, MC-RR, MC-YR, MC-HtyR, [D-Asp3]MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), Okadaic acid and Domoic acid. Analytes were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A dual Solid Phase Extraction (SPE) cartridge assembly was applied for the extraction of target compounds from water. Optimized SPE parameters included cartridge material, initial sample pH, sequence of the cartridges in the SPE assembly as well as composition and volume of the elution solvent. The method was validated, providing acceptable mean recoveries and reproducibility for most analytes. Limits of detection were at the ngL-1 level. The method was successfully applied in real lake water samples from Greece, where a wide range of Microcystins were detected for the first time, at concentrations ranging from 0.034 to 63µgL-1.

Assessment of the roles of reactive oxygen species in the UV and visible light photocatalytic degradation of cyanotoxins and water taste and odor compounds using C-TiO2.

Visible light (VIS) photocatalysis has large potential as a sustainable water treatment process, however the reaction pathways and degradation processes of organic pollutants are not yet clearly defined. The presence of cyanobacteria cause water quality problems since several genera can produce potent cyanotoxins, harmful to human health. In addition, cyanobacteria produce taste and odor compounds, which pose serious aesthetic problems in drinking water. Although photocatalytic degradation of cyanotoxins and taste and odor compounds have been reported under UV-A light in the presence of TiO2, limited studies have been reported on their degradation pathways by VIS photocatalysis of these problematic compounds. The main objectives of this work were to study the VIS photocatalytic degradation process, define the reactive oxygen species (ROS) involved and elucidate the reaction mechanisms. We report carbon doped TiO2 (C-TiO2) under VIS leads to the slow degradation of cyanotoxins, microcystin-LR (MC-LR) and cylindrospermopsin (CYN), while taste and odor compounds, geosmin and 2-methylisoborneol, were not appreciably degraded. Further studies were carried-out employing several specific radical scavengers (potassium bromide, isopropyl alcohol, sodium azide, superoxide dismutase and catalase) and probes (coumarin) to assess the role of different ROS (hydroxyl radical OH, singlet oxygen (1)O2, superoxide radical anion [Formula: see text] ) in the degradation processes. Reaction pathways of MC-LR and CYN were defined through identification and monitoring of intermediates using liquid chromatography tandem mass spectrometry (LC-MS/MS) for VIS in comparison with UV-A photocatalytic treatment. The effects of scavengers and probes on the degradation process under VIS, as well as the differences in product distributions under VIS and UV-A, suggested that the main species in VIS photocatalysis is [Formula: see text] , with OH and (1)O2 playing minor roles in the degradation.

Photocatalytic degradation of cylindrospermopsin under UV-A, solar and visible light using TiO2. Mineralization and intermediate products.

Cyanobacteria (blue-green algae) are considered an important water quality problem, since several genera can produce toxins, called cyanotoxins that are harmful to human health. Cylindrospermopsin (CYN) is an alkaloid-like potent cyanotoxin that has been reported in water reservoirs and lakes worldwide. In this paper the removal of CYN from water by UV-A, solar and visible light photocatalysis was investigated. Two different commercially available TiO2 photocatalysts were used, i.e., Degussa P25 and Kronos-vlp7000. Complete degradation of CYN was achieved with both photocatalysts in 15 and 40 min under UV-A and 40 and 120 min under solar light irradiation, for Degussa P25 and Kronos vlp-7000 respectively. Experiments in the absence of photocatalysts showed that direct photolysis was negligible. Under visible light irradiation only the Kronos vlp-7000 which is a visible light activated catalyst was able to degrade CYN. A number of intermediates were identified and a complete degradation pathway is proposed, leading to the conclusion that hydroxyl radical attack is the main mechanism followed. TOC and inorganic ions (NO2-, NO3-, SO4(2-) and NH4+) determinations suggested that complete mineralization of CYN was achieved under UV-A in the presence of Degussa P25.

Determination of microcystins and nodularin (cyanobacterial toxins) in water by LC-MS/MS. Monitoring of Lake Marathonas, a water reservoir of Athens, Greece.

A method for the determination of the hepatotoxic cyanotoxins microcystins (MCs, i.e. MC-LR, MC-RR, MC-YR, MC-LA) and nodularin (NOD) in water was developed using liquid chromatography with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) after solid phase extraction (SPE). New patterns of fragmentation of MC-LA were observed under the experimental conditions used. The method was fully validated to meet accreditation criteria. Mean recoveries at three concentration levels (0.006, 0.1 and 1 µg L(-1)) ranged between 70 and 114% with %RSD values generally below 20%. Detection limits were 2 ng L(-1) for all hepatotoxins. The method was applied to study the occurrence of MCs and NOD in Lake Marathonas, a water reservoir of Athens, over a period from July 2007 to December 2010. The protein phosphatase inhibition assay (PPIA) was additionally used for fast screening of samples. MC-YR, MC-LR and MC-RR were detected and found to vary seasonally with consistent peaks during early autumn, having maximum concentrations of 717, 451 and 174 ng L(-1), respectively. The results of this study constitute the first report on the presence, concentration levels and seasonal variations of MCs in Lake Marathonas. None of the target cyanotoxins were detected in treated drinking water samples during the period of the study.

Efficient removal of microcystin-LR by UV-C/H2O2 in synthetic and natural water samples.

The destruction of the commonly found cyanobacterial toxin, microcystin-LR (MC-LR), in surface waters by UV-C/H(2)O(2) advanced oxidation process (AOP) was studied. Experiments were carried out in a bench scale photochemical apparatus with low pressure mercury vapor germicidal lamps emitting at 253.7 nm. The degradation of MC-LR was a function of UV fluence. A 93.9% removal with an initial MC-LR concentration of 1 µM was achieved with a UV fluence of 80 mJ/cm(2) and an initial H(2)O(2) concentration of 882 µM. When increasing the concentration of MC-LR only, the UV fluence-based pseudo-first order reaction rate constant generally decreased, which was probably due to the competition between by-products and MC-LR for hydroxyl radicals. An increase in H(2)O(2) concentration led to higher removal efficiency; however, the effect of HO scavenging by H(2)O(2) became significant for high H(2)O(2) concentrations. The impact of water quality parameters, such as pH, alkalinity and the presence of natural organic matter (NOM), was also studied. Field water samples from Lake Erie, Michigan and St. Johns River, Florida were employed to evaluate the potential application of this process for the degradation of MC-LR. Results showed that the presence of both alkalinity (as 89.6-117.8 mg CaCO(3)/L) and NOM (as ∼2 to ∼9.5 mg/L TOC) contributed to a significant decrease in the destruction rate of MC-LR. However, a final concentration of MC-LR bellow the guideline value of 1 µg/L was still achievable under current experimental conditions when an initial MC-LR concentration of 2.5 µg/L was spiked into those real water samples.

Development of an integrated laboratory system for the monitoring of cyanotoxins in surface and drinking waters.

A system of analytical processes has been developed in order to serve as a cost-effective scheme for the monitoring of cyanobacterial toxins on a quantitative basis, in surface and drinking waters. Five cyclic peptide hepatotoxins, microcystin-LR, -RR, -YR, -LA and nodularin were chosen as the target compounds. Two different enzyme-linked immunosorbent assays (ELISA) were validated in order to serve as primary quantitative screening tools. Validation results showed that the ELISA methods are sufficiently specific and sensitive with limits of detection (LODs) around 0.1 microg/L, however, matrix effects should be considered, especially with surface water samples or bacterial mass methanolic extracts. A colorimetric protein phosphatase inhibition assay (PPIA) utilizing protein phosphatase 2A and p-nitrophenyl phosphate as substrate, was applied in microplate format in order to serve as a quantitative screening method for the detection of the toxic activity associated with cyclic peptide hepatotoxins, at concentration levels >0.2 microg/L of MC-LR equivalents. A fast HPLC/PDA method has been developed for the determination of microcystins, by using a short, 50mm C18 column, with 1.8 microm particle size. Using this method a 10-fold reduction of sample run time was achieved and sufficient separation of microcystins was accomplished in less than 3 min. Finally, the analytical system includes an LC/MS/MS method that was developed for the determination of the 5 target compounds after SPE extraction. The method achieves extremely low limits of detection (<0.02 microg/L), in both surface and drinking waters and it is used for identification and verification purposes as well as for determinations at the ppt level. An analytical protocol that includes the above methods has been designed and validated through the analysis of a number of real samples.