Microbiota and Cyanotoxin Content of Retail Spirulina Supplements and Spirulina Supplemented Foods

[EN]Cyanobacterial biomass such as spirulina (Arthrospira spp.) is widely available as a food supplement and can also be added to foods as a nutritionally beneficial ingredient. Spirulina is often produced in open ponds, which are vulnerable to contamination by various microorganisms, including some toxin-producing cyanobacteria. This study examined the microbial population of commercially available spirulina products including for the presence of cyanobacterial toxins. Five products (two supplements, three foods) were examined. The microbial populations were determined by culture methods, followed by identification of isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), and by 16S rRNA amplicon sequencing of the products themselves and of the total growth on the enumeration plates. Toxin analysis was carried out by enzyme-linked immunosorbent assay (ELISA). Several potentially pathogenic bacteria were detected in the products, including Bacillus cereus and Klebsiella pneumoniae. Microcystin toxins were detected in all the products at levels that could lead to consumers exceeding their recommended daily limits. Substantial differences were observed in the identifications obtained using amplicon sequencing and MALDI-TOF, particularly between closely related Bacillus spp. The study showed that there are microbiological safety issues associated with commercial spirulina products that should be addressed, and these are most likely associated with the normal means of production in open ponds.S

Implementation of the Water Framework Directive: Lessons Learned and Future Perspectives for an Ecologically Meaningful Classification Based on Phytoplankton of the Status of Greek Lakes, Mediterranean Region

The enactment of the Water Framework Directive (WFD) initiated scientific efforts to develop reliable methods for comparing prevailing lake conditions against reference (or nonimpaired) states, using the state of a set biological elements. Drawing a distinction between impaired and natural conditions can be a challenging exercise. Another important aspect is to ensure that water quality assessment is comparable among the different Member States. In this context, the present paper offers a constructive critique of the practices followed during the WFD implementation in Greece by pinpointing methodological weaknesses and knowledge gaps that undermine our ability to classify the ecological quality of Greek lakes. One of the pillars of WDF is a valid lake typology that sets ecological standards transcending geographic regions and national boundaries. The national typology of Greek lakes has failed to take into account essential components. WFD compliance assessments based on the descriptions of phytoplankton communities are oversimplified and as such should be revisited. Exclusion of most chroococcal species from the analysis of cyanobacteria biovolume in Greek lakes/reservoirs and most reservoirs in Spain, Portugal, and Cyprus is not consistent with the distribution of those taxa in lakes. Similarly, the total biovolume reference values and the indices used in classification schemes reflect misunderstandings of WFD core principles. This hampers the comparability of ecological status across Europe and leads to quality standards that are too relaxed to provide an efficient target for the protection of Greek/transboundary lakes such as the ancient Lake Megali Prespa

Differential Effect of Hydroxen Peroxide οn Toxic Cyanobacteria of Hypertrophic Mediterranean Waterbodies

Cyanobacterial blooms have been known since ancient times; however, they are currently increasing globally. Human and ecological health risks posed by harmful cyanobacterial blooms have been recorded around the world. These risks are mainly associated with their ability to affect the ecosystem chain by different mechanisms like the production of cyanotoxins, especially microcystins. Their expansion and their harmful effects have led many researchers to seek techniques and strategies to control them. Among them, hydrogen peroxide could be a promising tool against cyanobacteria and cyanotoxins and it is well-established as an environmentally friendly oxidizing agent because of its rapid decomposition into oxygen and water. The aim of the present study was to evaluate the effect of hydrogen peroxide on phytoplankton from two hypertrophic waterbodies in Greece. The effect of hydrogen peroxide on concentration of microcystins found in the waterbodies was also studied. Treatment with 4 mg/L hydrogen peroxide was applied to water samples originated from the waterbodies and Cyanobacterial composition and biomass, phycocyanin, chlorophyll-a, and intra-cellular and total microcystin concentrations were studied. Cyanobacterial biomass and phycocyanin was reduced significantly after the application of 4 mg/L hydrogen peroxide in water treatment experiments while chlorophytes and extra-cellular microcystin concentrations were increased. Raphidiopsis (Cylindrospermopsis) raciborskii was the most affected cyanobacterial species after treatment of the water of the Karla Reservoir in comparison to Aphanizomenon favaloroi, Planktolyngbya limnetica, and Chroococcus sp. Furthermore, Microcystis aeruginosa was more resistant to the treatment of Pamvotis lake water in comparison with Microcystis wesenbergii and Microcystis panniformis. Our study showed that hydrogen peroxide differentially impacts the members of the phytoplankton community, affecting, thus, its overall efficacy. Different effects of hydrogen peroxide treatment were observed among cyanobacerial genera as well as among cyanobacterial species of the same genus. Different effects could be the result of the different resistance mechanisms of each genus or species to hydrogen peroxide. Hydrogen peroxide could be used as a treatment for the mitigation of cyanobacterial blooms in a waterbody; however, the biotic and abiotic characteristics of the waterbody should be considered

Effects of microcystin concentrations on aquatic animals

Cyanobacteria have been known as natural components of ecosystems since 3 billion years. Nevertheless, cyanobacterial toxicity has been known to humans since 2000 years. Freshwaters of different trophic conditions are the main environments of cyanobacteria. The increased eutrophication and the cyanobacterial blooming can disturb the balance of a lake through many ways, like cyanotoxin production. Cyanotoxins can introduce animal and human health hazards. The most frequent cyanotoxins are the hepatotoxins microcystins and nodularins. The main toxicity mechanism of microcystins is the inhibition of protein phosphates 1 and 2A. The bioaccumulation of microcystins has been detected in aquatic and terrestrial animals. The aim of the present study is the enrichment of information, related to the presence of microcystin’s concentrations in Greek freshwaters and the effect of microcystins on aquatic organisms. The approach of the aim was conducted by a) the detection of microcystin concentrations in different lake waters, b) the detection of microcystin concentrations in tissues of fish species Carassius gibelio from different freshwaters, c) the assessment of the environmental parameters, which affect yearly microcystin concentrations in Lake Pamvotis, d) the assessment of different susceptibility in microcystins, among different length classes of fish species Rutilus panosi, e) the determination of seasonally distribution of microcystins in aquatic organisms of Lake Pamvotis, f) the assessment of microcystin biomagnification, g) the investigation of natural cyanobacterial extracts toxicity from Lake Pamvotis, h) the investigation of human health hazards from the presence of microcystins in Greek freshwaters. Microcystin concentrations were detected in all the studied lakes. The highest values of microcystins were detected in Lake Koronia concerning both concentrations in surface scum (15896 ng MC-LR eq /l) and in water (3748.6 ng MC-LR eq /l). The neighbouring Lake Volvi had the lowest concentrations in surface scum (1086 ng MC-LR eq /l) and in water (209.56 ng MC-LR eq /l) respectively. Significant concentrations were found in the remaining lakes. The concentrations of microcystins found in Greek freshwaters are comparable of those in other Mediterranean lakes. High microcystin concentrations were detected in Carassius gibelio’s tissues of Lakes: Koronia, Kastoria, Pamvotis, Doirani and Mikri Prespa. Microcystin concentrations were detected in liver (124.4 ±23.4 ng MC-LR eq /g), kidneys (63.3±12.2 ng MC-LR eq /g), brain (43.8±10.2 ng MC-LR eq /g), intestine (35.3±15.7 ng MC-LR eq /g), gonads (9.27±5.2 ng MC-LR eq /g) and muscle (7.1±2.5 ng MC-LR eq /g). High microcystin concentration was detected in brain, for the first time, supporting the possible neurotoxicity of microcystins. Microcystis, Anabaena and Aphanizomenon were the dominant cyanobacterial genus in Lake Pamvotis. Aqueous microcystin concentrations (0.10μg MC-LR eq /l - 8.5μg MC-LR eq /l) in Lake Pamvotis, during the year 2008 were lower than cell-bound microcystins (0.87μg MC-LR eq /l -11.54μg MC-LR eq /l). Microcystin concentrations in the littoral zone of Lake Pamvotis were higher than those in pelagic zone. Strong correlations were detected between microcystin concentrations and orthophosphate concentrations, chl-a concentrations, phycocyanin concentrations and water temperature. Microcystin concentrations were detected in liver (288.46 ng MC-LR eq /g-561.13 ng MC-LR eq /g), kidneys (194.32 ng MC-LR eq /g -542.20 ng MC-LR eq /g), brain (206.57 ng MC-LR eq /g -416.08ng MC-LR eq /g) and muscle (17.30 ng MC-LR eq /g - 20.15ng MC-LR eq /g) of Rutilus panosi. Microcystin concentrations were significant different between the different length classes of Rutilus panosi. Bigger-sized fishes accumulated lower microcystin concentrations in comparison to smaller-sized fishes.Τα κυανοβακτήρια αποτελούν φυσικό κομμάτι του γήινου οικοσυστήματος, εδώ και 3 δισεκατομμύρια χρόνια, και η τοξικότητα τους ήταν ήδη γνωστή στον ανθρώπινο πληθυσμό εδώ και δύο χιλιάδες χρόνια. Εσωτερικά ύδατα με ποικίλα τροφικά επίπεδα αποτελούν τα συνηθέστερα ενδιαιτήματα των κυανοβακτηρίων. Ο ευτροφισμός των λιμνών και κατά συνέπεια η άνθηση των κυανοβακτηρίων είναι υπεύθυνες για μια σειρά δυσάρεστων γεγονότων που διαταράσσουν την ισορροπία των λιμναίων οικοσυστημάτων, ανάμεσα σε αυτά και η παραγωγή κυανοτοξινών. Οι κυανοτοξίνες είναι επικίνδυνες για τα ζώα και τον άνθρωπο, καθώς αυτές μπορούν να προκαλέσουν ασθένεια ακόμα και θνησιμότητα σε συγκεντρώσεις που απαντώνται στο φυσικό περιβάλλον. Από τις κυανοτοξίνες μεγαλύτερη εξάπλωση και αφθονία παρουσιάζουν οι ηπατοτοξίνες, όπως οι μικροκυστίνες και οι νοντουλαρίνες. Ο κύριος μηχανισμός τοξικότητας των μικροκυστινών είναι η μη αναστρέψιμη αναστολή των πρωτεϊνικών φωσφατασών 1 και 2Α. Η βιοσυσσώρευση των μικροκυστινών έχει αποδειχθεί σε μια πληθώρα υδρόβιων και χερσαίων οργανισμών. Σκοπός της παρούσας εργασίας είναι η κάλυψη του κενού που υπάρχει στη διεθνή και ελληνική βιβλιογραφία σχετικά με την παρουσία των μικροκυστινών στα εσωτερικά ύδατα και την επίδραση των παραπάνω τοξινών στους υδρόβιους ζωϊκούς οργανισμούς. Η προσέγγιση του στόχου έγινε με α) τον προσδιορισμό της συγκέντρωσης των μικροκυστινών (ενδοκυττάριων και εξωκυττάριων) στο νερό εσωτερικών υδάτινων οικοσυστημάτων της Ελλάδος, β) τον προσδιορισμό της συγκέντρωσης των μικροκυστινών στους ιστούς τους ιχθύος Carassius gibeliο, των ελληνικών εσωτερικών υδάτινων οικοσυστημάτων, γ) την εκτίμηση των παραμέτρων της ποιότητας του νερού της λίμνης Παμβώτιδας, που συμβάλλουν στην ετήσια δυναμική των μικροκυστινών, δ) την εκτίμηση της διαφορετικής ευαισθησίας στις μικροκυστίνες, που εμφανίζουν οι ιχθύες του είδους Rutilus panosi, ανάλογα με το μέγεθος τους, ε) τον προσδιορισμό της εποχιακής κατανομής των μικροκυστινών σε υδρόβιους οργανισμούς της Λίμνης Παμβώτιδας, στ) την εκτίμηση της πιθανής βιομεγένθυσης των μικροκυστινών διαμέσου της τροφικής αλυσίδας, ζ) τον έλεγχο της τοξικότητας των κυανοβακτηριακών εκχυλισμάτων της Λίμνης Παμβώτιδας με τη χρήση βιοδοκιμών σε επίμυες και σε ιχθύες του είδους Danio rerio και η) τη διερεύνηση της πιθανής απειλής της δημόσιας υγείας από την παρουσία των μικροκυστινών στα ελληνικά εσωτερικά υδάτινα οικοσυστήματα. Σύμφωνα με τα αποτελέσματα της παρούσας μελέτης, ανιχνεύθηκαν σημαντικές ποσότητες μικροκυστινών στα εσωτερικά υδάτινα οικοσυστήματα που εξετάστηκαν (με τη χρήση της ενζυμοσυνδεόμενης ανοσοπροσροφητικής τεχνικής [ELISA]). Οι συγκεντρώσεις των μικροκυστινών διέφεραν σημαντικά ανάμεσα στα οικοσυστήματα. Στη Λίμνη Κορώνεια εντοπίστηκαν οι υψηλότερες συγκεντρώσεις εξωκυττάριων (3748.6 ng MC-LR eq /l) και ενδοκυττάριων μικροκυστινών (15896 ng MC-LR eq /l), σε σχέση με τα υπόλοιπα υδάτινα συστήματα. Αντιθέτως, στη Λίμνη Βόλβη εντοπίστηκαν οι χαμηλότερες συγκεντρώσεις εξωκυττάριων (209.56 ng MC-LR eq /l) και ενδοκυττάριων μικροκυστινών (1086 ngMC-LReq/l). Στα υπόλοιπα υδάτινα οικοσυστήματα, εντοπίστηκαν ενδιάμεσες συγκεντρώσεις εξωκυττάριων και ενδοκυττάριων μικροκυστινών. Από αυτά, υψηλές συγκεντρώσεις μικροκυστινών βρέθηκαν στις Λίμνες Καστοριά (3426 ng MC-LReq/l και 15842 ngMC-LReq/l για τις εξωκυττάριες και ενδοκυττάριες μικροκυστίνες, αντιστοίχως) και Παμβώτιδα (1791 ng MC-LReq/l και 11286 ng MC-LR eq /l για τις εξωκυττάριες και ενδοκυττάριες μικροκυστίνες, αντιστοίχως). Χαμηλότερες συγκεντρώσεις εντοπίστηκαν στις Λίμνες: Τριχωνίδα (230 ng MC-LReq/l και 1226 ngMC-LReq/l για τις εξωκυττάριες και ενδοκυττάριες μικροκυστίνες, αντιστοίχως) και Βεγορίτιδα (523 ng MC-LReq /l και 1346 ng MC-LReq /l για τις εξωκυττάριες και ενδοκυττάριες μικροκυστίνες, αντιστοίχως)

Microbiological Confinement of Two Adjacent Water Wells in Lake Karla Basin, Greece

We analyzed the bacterial and archaeal community structure of two adjacent irrigation well waters of the Lake Karla Basin, Central Greece, in order to elucidate their connectivity or confinement by using 454 tag pyrosequencing of the 16S rRNA genes. Although considerable overlap was found at the phylum/high taxonomic level, and also at the operational taxonomic units (OTU) level, the dominant, and most likely active, prokaryotes represented by these OTUs were very different between the two wells. As expected, we found higher bacterial species richness compared to that of archaeal, and this renders Bacteria better for the study of connectivity or confinement of water wells. Some of the taxonomic groups found are amongst those found typically in the terrestrial subsurface and also those that have been recently described, enhancing the importance of the subsurface for expanding our knowledge on microbial diversity. The majority of the archaeal and several of the bacterial OTUs, including the most dominant ones in each well, were related to marine or saline environments, indicating the previously suggested persistence of fertilizer residuals in the basin’s soils

Assessment of microcystin distribution and biomagnification in tissues of aquatic food web compartments from a shallow lake and evaluation of potential risks to public health

The objectives of this study were: (1) to examine the distribution and bioaccumulation of microcystins in the main components of the food web (phytoplankton, zooplankton, crayfish, shrimp, mussel, snail, fish, frog) of Lake Pamvotis (NW Greece), (2) to investigate the possibility of microcystin biomagnification and (3) to evaluate the potential threat of the contaminated aquatic organisms to human health. Significant microcystin concentrations were detected in all the aquatic organisms during two different periods, with the higher concentrations observed in phytoplankton and the lower in fish species and frogs. This is the first study reporting microcystin accumulation in the body of the freshwater shrimp Atyaephyra desmsaresti, in the brain of the fish species common carp (Cyprinus carpio) and in the skin of the frog Rana epirotica. Although there was no evidence for microcystin biomagnification, the fact that microcystins were found in lake water and in the tissues of aquatic organisms, suggests that serious risks to animal and public health are possible to occur. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Pamvotis due to the high-concentrations of accumulated microcystins