Ανάπτυξη μεθόδων προσδιορισμού κυανοτοξινών και οργανικών ρύπων σε νερά με υγροχρωματογραφία-συζευγμένη φασματομετρία μαζών

Τα κυανοβακτήρια αναπτύσσονται στο υδάτινο περιβάλλον και κάτω από ευνοϊκές συνθήκες πολλαπλασιάζονται σχηματίζοντας έντονες ανθίσεις (blooms) ελευθερώνοντας συχνά στο νερό τοξικούς δευτερογενείς μεταβολίτες, τις κυανοτοξίνες που αποτελούν σημαντικό κίνδυνο για τα υδάτινα οικοσυστήματα, τους ταμιευτήρες πόσιμου νερού και την δημόσια υγεία. Οι κυανοτοξίνες είναι μία μεγάλη ομάδα ενώσεων με ποικίλες φυσικοχημικές ιδιότητες, χημικές δομές και τοξική δράση (ηπατοτοξικές, νευροτοξικές, δερματοτοξικές, κυτταροτοξικές). Οι κυανοτοξίνες κατηγοριοποιούνται κυρίως με βάση τη δομή τους σε κυκλικά πεπτίδια (όπως Microcystins, Nodularins), αλκαλοειδή (όπως Cylindrospermopsin, Anatoxin), λιποπολυσακχαρίτες και αμινοξέα. Οι περισσότερες μελέτες για την ανάλυση των κυανοτοξινών εστιάζουν στον προσδιορισμό μίας κατηγορίας κυανοτοξινών. Στην παρούσα εργασία αναπτύχθηκε μία γρήγορη, απλή και ευαίσθητη αναλυτική μέθοδος για τον ταυτόχρονο προσδιορισμό διαφορετικών κατηγοριών κυανοτοξινών (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 και MC-LF), καθώς και των πολύ διαδεδομένων θαλάσσιων φυκοτοξινών Okadaic acid και Domoic acid στο νερό με μία μόνο ανάλυση. Ο προσδιορισμός των ενώσεων έγινε με υγροχρωματογραφία – συζευγμένη φασματομετρία μαζών (LC-MS/MS). Για την προκατεργασία του δείγματος και την αποτελεσματική εκχύλιση όλων των αναλυτών από το νερό αναπτύχθηκε μέθοδος εκχύλισης στερεάς φάσης (SPE), με διάταξη διπλού φυσιγγίου αποτελούμενη από Oasis HLB και Hyper PGC. Επιπλέον, αναπτύχθηκε μέθοδος για την ταυτόχρονη εκχύλιση διαφορετικών κατηγοριών κυανοτοξινών (Cylindrospermopsin, Anatoxin-a and Microcystins) από λυοφιλοποιημένη κυανοβακτηριακή βιομάζα, όπου μελετήθηκε η επίδραση διάφορων παραγόντων στην απόδοση της εκχύλισης. Οι αναπτυχθείσες μέθοδοι εφαρμόστηκαν αποτελεσματικά στην ανάλυση δειγμάτων από λίμνες της Ελλάδας, όπου ανιχνεύθηκε και ταυτοποιήθηκε για πρώτη φορά στον ελλαδικό χώρο η παρουσία των κυανοτοξινών Cylindrospermopsin, Anatoxin-a και ποικίλων Microcystins. Οι οργανικοί ρύποι του νερού, εκτός από φυσικής προέλευσης, είναι και επικίνδυνες ουσίες από ανθρωπογενείς δραστηριότητες. Στα πλαίσια της προστασίας του υδάτινου περιβάλλοντος και της δημόσιας υγείας εκδόθηκε η πιο πρόσφατη ευρωπαϊκή οδηγία (2013/39/ΕΕ) για τις ουσίες προτεραιότητας με αναθεωρημένα και αυστηρότερα πρότυπα ποιότητας περιβάλλοντος (ΠΠΠ) και με την απόφαση 2015/495/ΕΕ δημιουργήθηκε ο πρώτος κατάλογος επιτήρησης με αναδυόμενους ρύπους του νερού. Οι οδηγίες αυτές προτείνουν νέες στρατηγικές για την αντιμετώπιση της ρύπανσης του νερού και την ανάπτυξη νέων μεθόδων για την συστηματική παρακολούθηση των ρύπων του νερού. Στην παρούσα εργασία αναπτύχθηκε μία πολυδύναμη μέθοδος για τον προσδιορισμό 29 οργανικών ρύπων του νερού (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2,4,6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine και Triazophos) με LC-MS/MS, εκ των οποίων οι 13 περιλαμβάνονται είτε στις ουσίες προτεραιότητας είτε στον κατάλογο επιτήρησης και οι υπόλοιπες 16 είναι ευρέως χρησιμοποιηθέντα φυτοφάρμακα, πολλά εκ των οποίων έχουν απαγορευθεί. Για τον καθαρισμό του δείγματος και την προσυγκέντρωση των αναλυτών από το νερό, αναπτύχτηκε μέθοδος SPE επιτυγχάνοντας υψηλή ευαισθησία με LODs της τάξης των ng/L.Cyanobacteria, also known as blue-green algae, are present in surface waters and under favorable environmental conditions can form extensive algal blooms and release hazardous toxic compounds (cyanotoxins), posing a significant risk to aquatic ecosystems, drinking water sources and human health. Cyanotoxins comprise a large group of organic compounds, with a variety of physicochemical properties, chemical structures and toxic activity (hepatotoxicity, neurotoxicity, cytotoxicity, dermatotoxicity). Cyanotoxins are classified based on their structure on cyclic peptides (i.e. Microcystins, Nodularins), alkaloids (i.e. Cylindrospermopsin, Anatoxin), lipopolysaccharides and amino acids. Most of the studies on the occurrence of cyanotoxins have been targeted to specific compounds or to variants within a class of cyanotoxins. In this thesis, a fast, simple and sensitive analytical method was developed for the simultaneous determination of multi-class cyanobacterial and algal toxins in water in one run. 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) in MRM mode. A dual Solid Phase Extraction (SPE) cartridge assembly (two tandem SPE cartridges, Oasis HLB and Hyper PGC) was applied for the efficient extraction of target compounds from water. Moreover, a method was developed for the simultaneous extraction of several cyanotoxins (Cylindrospermopsin, Anatoxin-a and Microcystins) from lyophilized cyanobacterial biomass. During method development several extraction parameters were evaluated. Detection and identification of cyanotoxins Cylindrospermopsin, Anatoxin-a and a wide range of Microcystins in Greek lakes for the first time have demonstrated the applicability and efficiency of the proposed methods. Apart from the natural contaminants of water there are also anthropogenic contaminants with emerging concern. The latest EU Directive on Environmental Quality Standards (EQS Directive 2013/39/EU) has proposed new strategies against water pollution and novel monitoring methods for priority substances. Additionally, the EU Decision 2015/495/EE has established a watch list of emerging contaminants for Union-wide monitoring in the field of water policy. The above directives and decisions implements revised and stricter EQS and making necessary the development and validation of novel multi-residue methods for the determination of micropollutants of water. In this thesis, the development of a novel method for the simultaneous determination of various organic pollutants (i.e. pesticides, endocrine disruptors, pharmaceuticals) in water is presented. The 29 target compounds (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2.4.6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine and Triazophos), 13 of which are either considered as priority substances or included in watch list and the rest 16 were widely used pesticides many of them have been banned, were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in MRM mode. SPE method, utilizing Oasis HLB cartridges, was developed for sample clean-up and analytes preconcentration from water, enabling high sensitivity with LODs at ng/L level

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.</p

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.</p

Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.Peer reviewe

Method development for the determination of cyanotoxins and organic pollutants in water with liquid chromatography – tandem mass spectrometry

Cyanobacteria, also known as blue-green algae, are present in surface waters and under favorable environmental conditions can form extensive algal blooms and release hazardous toxic compounds (cyanotoxins), posing a significant risk to aquatic ecosystems, drinking water sources and human health. Cyanotoxins comprise a large group of organic compounds, with a variety of physicochemical properties, chemical structures and toxic activity (hepatotoxicity, neurotoxicity, cytotoxicity, dermatotoxicity). Cyanotoxins are classified based on their structure on cyclic peptides (i.e. Microcystins, Nodularins), alkaloids (i.e. Cylindrospermopsin, Anatoxin), lipopolysaccharides and amino acids. Most of the studies on the occurrence of cyanotoxins have been targeted to specific compounds or to variants within a class of cyanotoxins. In this thesis, a fast, simple and sensitive analytical method was developed for the simultaneous determination of multi-class cyanobacterial and algal toxins in water in one run. 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) in MRM mode. A dual Solid Phase Extraction (SPE) cartridge assembly (two tandem SPE cartridges, Oasis HLB and Hyper PGC) was applied for the efficient extraction of target compounds from water. Moreover, a method was developed for the simultaneous extraction of several cyanotoxins (Cylindrospermopsin, Anatoxin-a and Microcystins) from lyophilized cyanobacterial biomass. During method development several extraction parameters were evaluated. Detection and identification of cyanotoxins Cylindrospermopsin, Anatoxin-a and a wide range of Microcystins in Greek lakes for the first time have demonstrated the applicability and efficiency of the proposed methods.Apart from the natural contaminants of water there are also anthropogenic contaminants with emerging concern. The latest EU Directive on Environmental Quality Standards (EQS Directive 2013/39/EU) has proposed new strategies against water pollution and novel monitoring methods for priority substances. Additionally, the EU Decision 2015/495/EE has established a watch list of emerging contaminants for Union-wide monitoring in the field of water policy. The above directives and decisions implements revised and stricter EQS and making necessary the development and validation of novel multi-residue methods for the determination of micropollutants of water. In this thesis, the development of a novel method for the simultaneous determination of various organic pollutants (i.e. pesticides, endocrine disruptors, pharmaceuticals) in water is presented. The 29 target compounds (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2.4.6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine and Triazophos), 13 of which are either considered as priority substances or included in watch list and the rest 16 were widely used pesticides many of them have been banned, were determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in MRM mode. SPE method, utilizing Oasis HLB cartridges, was developed for sample clean-up and analytes preconcentration from water, enabling high sensitivity with LODs at ng/L level.Τα κυανοβακτήρια αναπτύσσονται στο υδάτινο περιβάλλον και κάτω από ευνοϊκές συνθήκες πολλαπλασιάζονται σχηματίζοντας έντονες ανθίσεις (blooms) ελευθερώνοντας συχνά στο νερό τοξικούς δευτερογενείς μεταβολίτες, τις κυανοτοξίνες που αποτελούν σημαντικό κίνδυνο για τα υδάτινα οικοσυστήματα, τους ταμιευτήρες πόσιμου νερού και την δημόσια υγεία. Οι κυανοτοξίνες είναι μία μεγάλη ομάδα ενώσεων με ποικίλες φυσικοχημικές ιδιότητες, χημικές δομές και τοξική δράση (ηπατοτοξικές, νευροτοξικές, δερματοτοξικές, κυτταροτοξικές). Οι κυανοτοξίνες κατηγοριοποιούνται κυρίως με βάση τη δομή τους σε κυκλικά πεπτίδια (όπως Microcystins, Nodularins), αλκαλοειδή (όπως Cylindrospermopsin, Anatoxin), λιποπολυσακχαρίτες και αμινοξέα. Οι περισσότερες μελέτες για την ανάλυση των κυανοτοξινών εστιάζουν στον προσδιορισμό μίας κατηγορίας κυανοτοξινών. Στην παρούσα εργασία αναπτύχθηκε μία γρήγορη, απλή και ευαίσθητη αναλυτική μέθοδος για τον ταυτόχρονο προσδιορισμό διαφορετικών κατηγοριών κυανοτοξινών (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 και MC-LF), καθώς και των πολύ διαδεδομένων θαλάσσιων φυκοτοξινών Okadaic acid και Domoic acid στο νερό με μία μόνο ανάλυση. Ο προσδιορισμός των ενώσεων έγινε με υγροχρωματογραφία – συζευγμένη φασματομετρία μαζών (LC-MS/MS). Για την προκατεργασία του δείγματος και την αποτελεσματική εκχύλιση όλων των αναλυτών από το νερό αναπτύχθηκε μέθοδος εκχύλισης στερεάς φάσης (SPE), με διάταξη διπλού φυσιγγίου αποτελούμενη από Oasis HLB και Hyper PGC. Επιπλέον, αναπτύχθηκε μέθοδος για την ταυτόχρονη εκχύλιση διαφορετικών κατηγοριών κυανοτοξινών (Cylindrospermopsin, Anatoxin-a and Microcystins) από λυοφιλοποιημένη κυανοβακτηριακή βιομάζα, όπου μελετήθηκε η επίδραση διάφορων παραγόντων στην απόδοση της εκχύλισης. Οι αναπτυχθείσες μέθοδοι εφαρμόστηκαν αποτελεσματικά στην ανάλυση δειγμάτων από λίμνες της Ελλάδας, όπου ανιχνεύθηκε και ταυτοποιήθηκε για πρώτη φορά στον ελλαδικό χώρο η παρουσία των κυανοτοξινών Cylindrospermopsin, Anatoxin-a και ποικίλων Microcystins. Οι οργανικοί ρύποι του νερού, εκτός από φυσικής προέλευσης, είναι και επικίνδυνες ουσίες από ανθρωπογενείς δραστηριότητες. Στα πλαίσια της προστασίας του υδάτινου περιβάλλοντος και της δημόσιας υγείας εκδόθηκε η πιο πρόσφατη ευρωπαϊκή οδηγία (2013/39/ΕΕ) για τις ουσίες προτεραιότητας με αναθεωρημένα και αυστηρότερα πρότυπα ποιότητας περιβάλλοντος (ΠΠΠ) και με την απόφαση 2015/495/ΕΕ δημιουργήθηκε ο πρώτος κατάλογος επιτήρησης με αναδυόμενους ρύπους του νερού. Οι οδηγίες αυτές προτείνουν νέες στρατηγικές για την αντιμετώπιση της ρύπανσης του νερού και την ανάπτυξη νέων μεθόδων για την συστηματική παρακολούθηση των ρύπων του νερού. Στην παρούσα εργασία αναπτύχθηκε μία πολυδύναμη μέθοδος για τον προσδιορισμό 29 οργανικών ρύπων του νερού (17α-Ethinylestradiol, β-Estradiol, Diclofenac, Alachlor, Atrazine, Chlorfenvinphos, Chlorpyriphos, Diuron, Isoproturon, Simazine, Trifluralin, Pentachlorophenol, 2,4,6-Trichlorophenol, Azinphos-ethyl, Azinphos-methyl, Chlorotoluron, Cyanazine, Diazinon, Dimethoate, Ethion, Fenthion, Linuron, Malathion, Methidathion, Metribuzine, Monuron, Phosalone, Propazine και Triazophos) με LC-MS/MS, εκ των οποίων οι 13 περιλαμβάνονται είτε στις ουσίες προτεραιότητας είτε στον κατάλογο επιτήρησης και οι υπόλοιπες 16 είναι ευρέως χρησιμοποιηθέντα φυτοφάρμακα, πολλά εκ των οποίων έχουν απαγορευθεί. Για τον καθαρισμό του δείγματος και την προσυγκέντρωση των αναλυτών από το νερό, αναπτύχτηκε μέθοδος SPE επιτυγχάνοντας υψηλή ευαισθησία με LODs της τάξης των ng/L

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

Analytical workflow integrating LC-HRMS untargeted analysis and CyanoMetDB for fast and extensive detection of cyanobacterial metabolites

Cyanobacteria produce a large number of secondary metabolites including cyanotoxins and a variety of bioactive peptides with great structural diversity. Identification of these metabolites (cyanometabolites) is a great analytical challenge due to the extremely limited availability of analytical standards and lack of a well-documented fragmentation mass spectra database. In the present study, an analytical workflow was developed for the detection of the cyanometabolites in bloom samples from Greek lakes. Samples were extracted [1,2] and analyzed by LC-HRMS (Orbitrap Fusion Lumos Tribrid MS) in data depended acquisition (DDA) mode. Fragmentation spectra of compounds were obtained with collision-induced dissociation (CID) and higher-energy C-trap dissociation (HCD) modes. Acquired data were processed with Compound Discoverer software in combination to the recently published CyanoMetDB mass list [3] and other related tools for the annotation and structural elucidation of cyanometabolites. Verification of proposed structures was performed based on in silico fragmentation and fragment ion search (FISh) scoring. Application of the workflow revealed the presence of numerous congeners belonging to the cyanotoxins class microcystins and to the understudied cyanopeptides classes of cyanopeptolines, microginins, aeruginosins, anabaenopeptins and aeruginosamides. Furthermore, new congeners were annotated clearly demonstrating the suitability of the approach for the characterization of cyanobacterial chemodiversity. [1] C. Christophoridis, S.-K. Zervou, K. Manolidi, M. Katsiapi, M. Moustaka-Gouni, T. Kaloudis, T. M. Triantis and A. Hiskia, Scientific Reports 8 (2018) 17877. [2] S.-K. Zervou, K. Moschandreou, A. Paraskevopoulou, C. Christophoridis, E. Grigoriadou, T. Kaloudis, T. M. Triantis, V. Tsiaoussi and A. Hiskia, Toxins 13 (2021) 394. [3] M. R. Jones, E. Pinto, M. A. Torres, F. Dörr, H. Mazur-Marzec, K. Szubert, L. Tartaglione, C. Dell’Aversano, C. O. Miles, D. G. Beach, P. McCarron, K. Sivonen, D. P. Fewer, J. Jokela and E. M.-L. Janssen, Water Research 196 (2021) 117017

A Greek Raphidiopsis raciborskii strain and Microcystins: A toxic relationship

The cyanobacterium Raphidiopsis raciborskii is extensively studied for its toxicity and invasive behavior, which is presumably enhanced by global warming. The widening of its geographic distribution and the isolation of strains showing high optimum growth temperature underline its ecological heterogeneity, suggesting the existence of different ecotypes. In this study, we investigate the ecotoxicology of Raphidiopsis raciborskii TAU-MAC 1414 strain, isolated from Lake Karla, Greece. The effect of different growth conditions (temperature, light intensity, phosphorus concentration, co-culture with toxic and non-toxic Microcystis spp. extracts) on microcystin production by R. raciborskii was examined. MC-LR and MC-HilR were detected with LC-MS/MS, mainly during the cultivation of R. raciborskii with toxic or non-toxic Microcystis spp. strains’ extracts. Further, the subcellular phytotoxic effects of R. raciborskii on Oryza sativa (rice) are discussed. Our research demonstrated unambiguously for the first time that R. raciborskii is able to produce microcystins under certain conditions, shedding new light in the ecotoxicology of the species, whereas the triggering of microcystin production remains to be further investigated

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

Membrane Lipidome Reorganization and Accumulation of Tissue DNA Lesions in Tumor-Bearing Mice: An Exploratory Study

Increased rates of reactive oxygen/nitrogen species (ROS/RNS) are involved in almost all cancer types, associated with tumor development and progression, causing damage to biomolecules such as proteins, nucleic acids and membrane lipids, in different biological compartments. We used a human tumor xenograft mouse model to evaluate for the first time in parallel the remodeling of fatty acid moieties in erythrocyte membrane phospholipids and the level of ROS-induced DNA lesions in liver and kidney tissues. Using liquid chromatography tandem mass spectrometry the 5&#8242;R and 5&#8242;S diastereoisomers of 5&#8242;,8-cyclo-2&#8242;-deoxyadenosine and 5&#8242;,8-cyclo-2&#8242;-deoxyguanosine, together with 8-oxo-7,8-dihydro-2&#8242;-deoxyadenosine, were determined in mice at young (4- and 5-weeks) and old (17-weeks) ages and compared with control SCID mice without tumor implantation. Tumor-bearing mice showed a higher level of ROS-damaged nucleosides in genomic DNA as the age and tumor progress, compared to controls (1.07&#8211;1.53-fold in liver and 1.1&#8211;1.4-fold in kidney, respectively). The parallel fatty acid profile of erythrocyte membranes showed a profound lipid remodeling during tumor and age progression consisting of PUFA consumption and SFA enrichment (ca 28% and 58%, respectively, in late stage tumor-bearing mice), markers of enhanced oxidative and proliferative processes, respectively. Membrane lipid remodeling and ROS-induced DNA lesions may be combined to afford an integrated scenario of cancer progression and ageing, reinforcing a holistic vision among molecular markers rather than the biomarker identification in a single compartment