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Toxin content and cytotoxicity of algal dietary supplements
Abstract
Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC-MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤1μg MC-LR equivalents g(-1) dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable.
... In response to a Microcystis aeruginosa bloom in Klamath Lake which could have led to the contamination of the harvest, the Oregon Health division decreed a regulatory limit of 1000 ng microcystins per gram of supplement in 1997 [18]. Several researchers have nevertheless shown that some products exceed this limit, which may be the result of insufficient quality monitoring [18,19,25]. The chronic exposure to toxins contained by these products may pose a health risk for the consumers, as most supplements are meant to be consumed daily and the long-term exposure to small doses of cyanotoxins is yet to be fully understood. ...
... Fewer reports are available regarding the analysis of other types of cyanotoxins such as other cyanopeptides of emerging concern (e.g., anabaenopeptins (AP-a and AP-b), alkaloids (ANA, saxitoxin (SXT) and derivatives), and non-proteinogenic amino acids (β-N-methylamino-Lalanine (BMAA) and isomers) [17,25,26] in algal dietary supplements. Alkaloid cyanotoxins may cause gastrointestinal symptoms and lifethreatening paralysis which can be fatal if it reaches the heart or lungs [27]; BMAA is suspected to be linked to neurodegenerative diseases including amyotrophic lateral sclerosis, dementia and Parkinson [28,29]. ...
... Therefore, analysis of total MCs is relevant to avoid underestimating the degree of contamination of supplements. Lower maximum concentrations of 500 ng g − 1 [44] and 6000 ng g − 1 [25] can also be found in the literature. ...
... In fact, BGA-based dietary supplements have become well known around the world as a result of the increase in health awareness and disease prevention and also due to the demand for a natural alternative to pharmaceutical products [7]. Most of the BGA dietary supplements derive from two filamentous genera of cyanobacteria and one microalgae: Spirulina including S. platensis and S. maxima, Aphanizomenon flos-aquae and Chlorella pyrenoidosa [8]. Spirulina is usually produced under cultured conditions and in open tank systems. ...
... Alkaloids such as cylindrospermopsin, and anatoxina (ANA) and its metabolites have also been studied separately using mainly RPLC-FL, although UV and mass spectrometry (MS) detectors have also been employed [24][25][26][27][28]. Regarding multi-toxin analysis, some studies cover the presence of diverse congeners and cyanotoxin families in dietary supplements, especially MCs, cylindrospermopsin, saxitoxin, anatoxins and some of them also included BMAA. These methods apply different extraction procedures, depending on the family and also the chromatographic method is developed separately for lipophilic and hydrophilic toxins [8,[29][30][31]. In those cases, sample treatment approaches are generally based on solid-liquid extraction (SLE) using aqueous mixtures of about 75% methanol, acidified methanol or acidified water, depending on whether cyclic peptides, alkaloids or NPAs are extracted. ...
... Firstly, mixtures of MeOH, MeCN and ethanol (EtOH) with water (80:20, v/v) were tested as extraction solvents in BGA samples. This ratio was chosen according to the literature, where around 75-90% methanolic-water mixtures are usually employed to extract MCs and NOD with optimal recoveries from dietary supplements [8,11,20,30]. With this purpose, 50 mg of organic spirulina spiked with a mixture of cyanotoxins were extracted with 4 mL of the extraction mixtures, stirred 10 min and placed in an ultrasonic bath for 15 min. ...
In recent years, the consumption of blue-green algae (BGA) dietary supplements is increasing because of their health benefits. However, cyanobacteria can produce cyanotoxins, which present serious health risks. In this work we propose hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) to determine cyanotoxins in BGA dietary supplements. Target toxins, including microcystin-leucine-arginine (MC-LR) and microcystin-arginine-arginine (MC-RR), nodularin, anatoxin-a and three non-protein amino acids, β-N-methylamino-L-alanine (BMAA), 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG), were separated using a SeQuant ZIC-HILIC column. Cyanotoxin extraction was based on solid–liquid extraction (SLE) followed by a tandem-solid phase extraction (SPE) procedure using Strata-X and mixed-mode cation-exchange (MCX) cartridges. The method was validated for BGA dietary supplements obtaining quantification limits from 60 to 300 µg·kg−1. Nine different commercial supplements were analyzed, and DAB, AEG, and MCs were found in some samples, highlighting the relevance of monitoring these substances as precaution measures for the safe consumption of these products.
... Multiple groups have developed and validated methods capable of accurately detecting MCs. The amount of different congeners that they were able to detect, was primarily dependent on the availability of standards [12,13,17,20,21,23,48]. ...
... Based on the data from the literature cited in this study, it is clear that a ± 80% MeOH extraction is the most appropriate way to extract the MCs and NOD, with the addition of a mechanical extraction and/or sonication [12,17,20,21,48]. Moreover, no additional purification seems to be needed for BGAS samples for MS/MS analysis, which significantly reduces material cost and analysis duration. ...
... They were exclusively observed in products containing Aphanizomenon flos-aquae. Our concentrations fit perfectly with the earlier reported toxin concentrations ranging between 0 and 60 µg g −1 in BGAS using different methods (e.g., MS/MS, MS, ELISA, PPIA) [12,14,17,19,20,54,55]. Roy-Lachappelle et al. ...
Food supplements are gaining popularity worldwide. However, harmful natural compounds can contaminate these products. In the case of algae-based products, the presence of toxin-producing cyanobacteria may cause health risks. However, data about the prevalence of algal food supplements on the Belgian market and possible contaminations with cyanotoxins are scarce. Therefore, we optimized and validated a method based on Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry to quantify eight microcystin congeners and nodularin in algal food supplements. Our analytical method was successfully validated and applied on 35 food supplement samples. Nine out of these samples contained microcystin congeners, of which three exceeded 1 µg g−1, a previously proposed guideline value. Additionally, the mcyE gene was amplified and sequenced in ten products to identify the taxon responsible for the toxin production. For seven out of these ten samples, the mcyE gene could be amplified and associated to Microcystis sp. EFSA and posology consumption data for algal-based food supplements were both combined with our toxin prevalence data to establish different toxin exposure scenarios to assess health risks and propose new guideline values.
... The Spirulina products are advertised as detoxifiers, energy sources, mood improvers, and also as supplements for weight loss (Heussner et al. 2012). They are claimed to possess numerous pharmacological functions such as hypocholesterolemic activity (Samuels et al. 2002), blood glucose level control (Parikh et al. 2001), hypolipidaemic activity (Mazokopakis et al. 2014), natural killer cell activation (Akao et al. 2009), reducing nephrotoxicity (Belay et al. 1993), and anticancer properties (Mathew et al. 1995). ...
... Several methods have been employed to detect the presence of microcystin including multiplex PCR ( Barón-Sola et al. 2012, Ngwa et al. 2014, loop-mediated isothermal amplification PCR (LAMP) (Zhu et al. 2014), phosphatase inhibition assay (PPIA) (Heussner et al. 2012), ELISA (Preece et al. 2015), cell-based bioassay (RCA) (Sorichetti et al. 2014), liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Parker et al. 2015), reverse-phase high-performance liquid chromatography (Sangolkar et al. 2009), surface plasmon resonance biosensor (Yakes et al. 2015), ultra-highpressure liquid chromatography-tandem mass spectrometry (Pekar et al. 2016), and MALDI-TOF (Valério et al. 2016). However, they all have a major drawback concerning the sensitivity to detect minute traces of the toxin. ...
... A study reported the presence of MC-LR and MC-LA variants of microcystin in A. flosquae-based food supplements (Vichi et al. 2012). Similarly, the presence of MC-LR, MC-LA, and [Asp 3 ]-MC-LR in A. flosquae-based food supplements was detected using LC-MS (Heussner et al. 2012). LC-HRMS is a state-ofthe-art technique which is highly useful in detecting peptides. ...
... A chromatogram for all toxins spiked in a plant-based FS matrix is depicted in Supporting Fig 1S. Variants of different extraction/purification procedures were evaluated: (i) in-house methodologies (based on the extraction with H 2 SO 4 acidified solvent or with an H 2 O/ACN mixture followed by MgSO 4 drying and hexane defatting steps), (ii) the QuEChERS procedure [28], and (iii) a generic "dilute-and-shoot" approach [29]. For each of these, varying extraction mixtures of water and organic solvent (ACN or MeOH, whether or not acidified/alkalinized) were tested, both for single versus 2-fold extraction. ...
... As for the application of the QuEChERS protocol, the procedure described by Zeng et al. [28] but with the application of a 2-fold extraction (first extraction with 5 ml H 2 O + 1 % FA, second extraction with 100 % ACN) and omission of (d)SPE gave good results. Variations of the dilute-and-shoot approach described by Heussner et al. [29] but without the inclusion of SPE indicated that extraction with MeOH/H 2 O 75/25 (v/v) and ultra-sonication led to the best results. Finally, a MeOH-based extraction solvent was chosen, as ACN-based ones induce clot formation when arabic gum is present in the formulation. ...
... In conclusion, the final extraction procedure concerns a variant of the dilute-and-shoot methodology described by Heussner et al. [29] without applying SPE and adjusted extraction solvent. ...
The presence of plant toxins and/or cyanotoxins in food supplements implies consumer health risks. Therefore, a targeted ultra-high performance liquid chromatographic-tandem mass spectrometric method to detect/quantify 25 toxins simultaneously in food supplement formulations was developed and validated. Full validation for tablets/powders and secondary validation for a liquid and soft gel capsule indicated that most compounds were efficiently extracted (≥ 75%), while others were only partly extracted (18 - 61%). Trueness was fulfilled (70 - 120%), with some exceptions (mostly at the lowest validation level). Intralaboratory repeatability, intra- and interlaboratory reproducibility values of ≤ 20%, ≤ 25%, and ≤ 25% were obtained for most, respectively. Matrix effects were found to be significant for most compounds. Good sensitivity (µg/kg level) was observed for galegin(e), lycopsamine, lycorine, rubiadin, skimmiamine, and vascin(e), in contrast to helveticoside, lucidin, lucidin-3-primveroside, plumbagin(e), and thujone, which were detected at the mg/kg level. The other compounds were characterized by a sensitivity between 10 to 1000 µg/kg. The validated methodology was applied for 52 food supplements (tablets, capsules, liquids/syrup, etc.) purchased from the Belgian market. In more than 25% of the samples, one or more toxins were detected (concentrations determined using standard addition). Lycopsamine, microcystin LR, solamargine, thujone, and vasicin(e) were the most frequently detected toxins. A clear link between the toxins detected and the plant species on the food supplement ingredient list could not always be established. This generic "dilute-and-shoot" procedure can be used for further research on toxins in food supplements and by extension other plant/algae-based food/feed commodities (herbs, edible flowers, etc.).
... water, biomass) and BGAS healthcare products. They include high-performance liquid chromatography with pulsed amperometric detector (HPLC-PAD) for lake water samples [11], HPLC-ELISA (enzyme-linked immunosorbent assay) for BGAS [12], Adda-ELISA (Adda moiety is present in all MC congeners) [13], DNA extraction and PCR analyses of mcyE gene in BGAS [13], colorimetric protein phosphatase inhibition assay (cPPIA) for water samples or BGAS [14], and liquid chromatography tandem mass spectrometry (LC-MS/MS) for BGAS [13,15]. ...
... water, biomass) and BGAS healthcare products. They include high-performance liquid chromatography with pulsed amperometric detector (HPLC-PAD) for lake water samples [11], HPLC-ELISA (enzyme-linked immunosorbent assay) for BGAS [12], Adda-ELISA (Adda moiety is present in all MC congeners) [13], DNA extraction and PCR analyses of mcyE gene in BGAS [13], colorimetric protein phosphatase inhibition assay (cPPIA) for water samples or BGAS [14], and liquid chromatography tandem mass spectrometry (LC-MS/MS) for BGAS [13,15]. ...
... water, biomass) and BGAS healthcare products. They include high-performance liquid chromatography with pulsed amperometric detector (HPLC-PAD) for lake water samples [11], HPLC-ELISA (enzyme-linked immunosorbent assay) for BGAS [12], Adda-ELISA (Adda moiety is present in all MC congeners) [13], DNA extraction and PCR analyses of mcyE gene in BGAS [13], colorimetric protein phosphatase inhibition assay (cPPIA) for water samples or BGAS [14], and liquid chromatography tandem mass spectrometry (LC-MS/MS) for BGAS [13,15]. ...
Algal blooms present naturally in marine and fresh water ecosystems are harvested as ingredients in health supplement products. Blue-green algal supplements (BGAS) such as Aphanizomenon flos-aquae, can be contaminated with highly potent toxins, most common being microcystin-LR (MC-LR). Recently, the Pharmacovigilance Section of NPRA has received adverse drug reaction (ADR) reports including liver and kidney toxicities suspected to be related to products containing Aphanizomenon flos-aquae in the local market. The objectives of this study were: (1) to develop and validate a MC-LR quantification test method and (2) to use quality control test results as a complement to managing product safety issues. During investigation on ADR reports concerning Aphanizomenon flos-aquae products, samples obtained through ADR reporting and market surveillance were used to develop and validate a test based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Testing results have been applied to supplement other investigational activities which together support follow-up regulatory actions on the ADR reports received by NPRA. This research report illustrates an example in which a test method developed in-house can serve to supplement other regulatory components in handling product safety issues.
... Toxin can accumulate in microalgae, particularly when the environment is contaminated with toxin-producing cyanobacteria, such as Microcystis aeruginosa. Two studies reported the contamination of microalgal dietary supplements (Aphanizomenon flos-aquae) with cyanobacterial hepatoxins-namely, microcystins, as confirmed by toxin detection (cPPIA, Adda-ELISA or LC/MS assays) and also the presence of microcystin-producing genes mcyE or mcyB (PCR analysis) [156,157]. Indeed, A. flos-aquae has the capability of producing cyanotoxins, such as anatoxin-a, cylindrospermopsin, microcystins and saxitoxins, and thus the presence of these cyanotoxins in A. flos-aquae products may be the result of direct toxin production by A. flos-aquae or cross-contamination with other toxinproducing cyanobacteria [158]. Along with microcystins, anatoxin-a and β-methylamino-L-alanine were detected in eight food products derived from Arthrospira or A. flos-aquae [159]. ...
... Interestingly, it has been reported that while toxins (microcystins or polymethoxy-1-alkenes) were absent in several dietary supplements derived from Arthrospira spp. and Chlorella spp., these products still exhibited cytotoxicity in human A549 cells [156] and adult zebrafish (Danio rerio) [160], although the reason for this phenomenon was unknown [156,160]. ...
... Interestingly, it has been reported that while toxins (microcystins or polymethoxy-1-alkenes) were absent in several dietary supplements derived from Arthrospira spp. and Chlorella spp., these products still exhibited cytotoxicity in human A549 cells [156] and adult zebrafish (Danio rerio) [160], although the reason for this phenomenon was unknown [156,160]. ...
Food security and environmental issues have become global crises that need transforma-tive solutions. As livestock production is becoming less sustainable, alternative sources of proteins are urgently required. These include cultured meat, plant-based meat, insect protein and single-cell protein. Here, we describe the food safety aspects of these novel protein sources, in terms of their technological backgrounds, environmental impacts and the necessary regulatory framework for future mass-scale production. Briefly, cultured meat grown in fetal bovine serum-based media can be exposed to viruses or infectious prion, in addition to other safety risks associated with the use of genetic engineering. Plant-based meat may contain allergens, anti-nutrients and thermally induced carcinogens. Microbiological risks and allergens are the primary concerns associated with insect protein. Single-cell protein sources are divided into microalgae, fungi and bacteria, all of which have specific food safety risks that include toxins, allergens and high ribonucleic acid (RNA) contents. The environmental impacts of these alternative proteins can mainly be attributed to the production of growth substrates or during cultivation. Legislations related to novel food or genetic modification are the relevant regulatory framework to ensure the safety of alternative proteins. Lastly, additional studies on the food safety aspects of alternative proteins are urgently needed for providing relevant food governing authorities with sufficient data to oversee that the technological progress in this area is balanced with robust safety standards.
... In Italy, Vichi et al. (2012) found that Aphanizomenon flosaquae-based samples were contaminated by highly variable levels of MCYSTs, up to 5.2 μg MCYST-LR equivalents per gram product. In Germany, Heussner et al. (2012), detected MCYSTs in food supplements containing "Spirulina" (0.2 μg g −1 MCYST-LR equivalents), nevertheless their samples were negative for the presence of nodularins, STXs, anatoxin-a (ANA-a) and CYNs. Heussner et al. (2012) examined different "Spirulina" products than those examined in our study and consequently no comparisons could be done between results of the two studies. ...
... In Germany, Heussner et al. (2012), detected MCYSTs in food supplements containing "Spirulina" (0.2 μg g −1 MCYST-LR equivalents), nevertheless their samples were negative for the presence of nodularins, STXs, anatoxin-a (ANA-a) and CYNs. Heussner et al. (2012) examined different "Spirulina" products than those examined in our study and consequently no comparisons could be done between results of the two studies. Roy-Lachapelle et al. (2017) investigated the presence of MCYSTs, ANA-a, two of ANA-a transformation products (dihydroanatoxin-a and epoxyanatoxin-a), CYNs, STXs and β-methylaminol-alanine (BMAA) in 18 different commercially available algae products in Canada. ...
... The presence of cyanotoxins in "Spirulina" supplements can be explained by the presence of cyanotoxin-producing cyanobacteria in these supplements. Consequently, molecular methods have been used by many researchers in order to detect toxic cyanobacteria and to corroborate cyanotoxin contamination of supplements (Heussner et al. 2012;Lyon-Colbert et al. 2018;Vichi et al. 2012). According to our results, all samples were contaminated with MCYSTs and two samples were contaminated with CYNs. ...
Arthrospira (“Spirulina”) is the most used cyanobacterium in food supplementation. “Spirulina” products are considered as superfood due to their excellent nutritional content, although several studies have shown the toxic effects of short and long term “Spirulina” consumption. In this work, a total of 31 “Spirulina” products from different brands, available in Greece, were investigated for their cyanotoxin contamination. All samples were positive for microcystins (MCYSTs) (35.7–583.5 ng g−1 dry weight [dw]). Two samples were tested positive for cylindrospermopsins (CYNs) (221.4–351.8 ng g−1 dw). No saxitoxins (STXs) were found. According to guidelines, the MCYSTs concentrations did not exceed the tolerable daily intake (TDI) set for adults, but six products exceeded the TDI set for children and 23 exceeded the TDI set for infants. The CYNs did not surpass the TDI for adults, but both samples exceeded the TDI for children and infants. This study showed that the low cyanobacterial toxin concentrations occurring in commercial “Spirulina” supplements, do not pose obvious risks for adults’ consumption, however they pose health risks for children and infants after daily consumption. To protect consumers, regular monitoring of these toxins at all stages of “Spirulina” production is recommended.
... The presence of microcystin in microalgae-based food supplements has been previously documented, especially when they contain Aphanizomenon [6,21,27,28,33,35,45,46]. The more commercialized Aphanizomenon comes from Klamath Lake (USA), and several reports about the toxicity of this product are found in literature [28][29][30]46]. Anatoxin-a has been also detected in food supplements previously [47,48]. ...
... When microalgae production takes place outdoors, it is almost impossible to avoid the presence of other species, and some can produce toxins regardless of the growing algae being cyanophytes or not. According to our results, both MC-LR and anatoxin-a were detected in a Chlorella product, as formally indicated in [30] and [49]. ...
Food supplements with microalgae are becoming increasingly abundant and can be easily found anywhere. The most popular products are based on cyanophytes, such as Aphanizomenon flos-aquae, Arthrospira platensis and Limnospira maxima, or on chlorophytes, such as Chlorella or Haematoccus. Although they are all advertised as being very beneficial for health, these products might be harmful because they may contain cyanotoxins and other contaminants, and no information on production methods or strain origins is usually provided. While legislation on the presence of microcystins in waters for different uses is clear, toxicological analyses are not compulsory for food supplements, nor for analyzing anatoxins. Given the potential risk of eating contaminated food, cyanotoxins, heavy metals and the presence of other contaminant organisms were analyzed in 10 microalgae food supplements. Microcystin-LR and anatoxin-a were detected in three analyzed products, and in both cyanophyte- and chlorophyte-based products. The light microscope study revealed the presence of different potentially harmful microbial contaminants. The ICP (OES) analyses detected high concentrations of some heavy metals, especially Pb. The results emphasize the need to promote the better control of food products containing microalgae, and to develop standard methodologies to analyze cyanotoxins and potential toxic compounds to protect consumer health.
... These effects can be mitigated through proper processing. Thermal processing of APs can lead to the formation of carcinogenic compounds like polycyclic aromatic hydrocarbons and nitrosamines [93]. Edible insects and microalgae can accumulate heavy metals depending on their growing conditions, potentially exceeding safe consumption levels. ...
To ensure food security amid dwindling natural resources, alternative proteins (APs) have been suggested as a sustainable solution. Yet, the adoption and consumption of APs remain limited. This review aims to delve into the latest progress (following PRISMA guidelines) concerning the utilization of proteins from alternative sources, particularly focusing on their effective incorporation into food products. Our findings reveal that insect proteins can improve amino acid profiles in bakery products. However, consumer acceptance remains low due to cultural biases, with optimal sensory results being achieved at lower substitution levels (5-10%). Mushroom proteins, when incorporated into meat analogs and bakery items, enhance nutritional value and offer favorable sensory properties, making them viable replacements in meat products. Plant-based proteins, such as pea and soy proteins, increase fiber and antioxidants and improve texture in meat alternatives, although formulation adjustments are necessary to meet consumer expectations for taste and overall experience. Microalgae offer unique benefits for bakery, confectionery, and dairy products by boosting protein, fatty acids, and probiotic growth while maintaining sensory acceptability. In conclusion, this study highlights that the effective incorporation of APs into food products can help in the development of healthier, more sustainable diets. That said, the success of AP acceptance will depend on continued innovations in formulation and consumer education.
... У доступних літературних джерелах повідомляється що в дієтичних добавках, отриманих з Chlorella vulgaris токсин (мікроцистин) був відсутній, однак отримані продукти виявили цитотоксичність у клітин А 549 у людини [16] і дорослих особин рибок danio (Danio rerio) [17]. Автори відмічають, що причина цього явища невідома. ...
Green algae Chlorella is positioned as a biologically active feed additive that includes proteins, polysaccharides, vitamins, minerals, glycoproteins and ß-glucans. Adding a small amount of them to the diet has a positive effect on the health and welfare of animals and poultry. However, their use in poultry farming requires an analysis of the quality and safety of the obtained products for the consumer. The purpose of the study is to assess the safety and quality of quail meat after drinking a suspension of Chlorella vulgaris microalgae. The object of study were quails of the Pharaoh breed, at the age of one day they were divided into two groups: experimental and control, 30 heads in each. The birds were kept in cages with free access to food and water. The quails of the research group were given a chlorella suspension (DSTU EK ISO 8692:2022 EN) in their drinking water. For drinking, the prepared chlorella suspension was diluted with drinking water to a concentration of 2*106 cells/ml grown in a glass fermenter. Weighing of quails was carried out weekly starting from day-old age. Drinking the suspension of Chlorella vulgaris increased the live weight of quail by 13.2 (p<0.05) compared to the control. Pre-slaughter examination of quails of both groups revealed a satisfactory clinical condition of the bird. An examination of 20 quail carcasses showed that they can be attributed to the first grade. According to organoleptic indicators, quail meat in the veterinary and sanitary sense belongs to benign quality. The conducted microbiological studies of quail meat show that there is no effect of the microalgae Chlorella sorokiniana in the applied doses on its bacterial contamination. The chemical parameters of the meat (pH, amino ammonia nitrogen, volatile fatty acids) during storage in the refrigerator (5 days, t= 4–5 °C) tended to increase and were within the normal range for a fresh product. During microscopy, m ulcer tissue and its disintegration were not detected in quails of both groups. The biological value of quail meat of both groups was identical, and there was no toxicity. The tasting evaluation of the broth and meat showed that drinking the microalgae Chlorella sorokiniana does not affect the studied taste indicators. Therefore, the meat of quails that received the microalgae Chlorella sorokiniana feed additive with water is of good quality according to the veterinary and sanitary examination, which allows it to be used for human consumption without restrictions. Key words: poultry, microalgae, organoleptic evaluation, microbial contamination, biological value, tasting evaluation.> <0.05) compared to the control. Pre-slaughter examination of quails of both groups revealed a satisfactory clinical condition of the bird. An examination of 20 quail carcasses showed that they can be attributed to the first grade. According to organoleptic indicators, quail meat in the veterinary and sanitary sense belongs to benign quality. The conducted microbiological studies of quail meat show that there is no effect of the microalgae Chlorella sorokiniana in the applied doses on its bacterial contamination. The chemical parameters of the meat (pH, amino ammonia nitrogen, volatile fatty acids) during storage in the refrigerator (5 days, t= 4–5 °C) tended to increase and were within the normal range for a fresh product. During microscopy, mulcer tissue and its disintegration were not detected in quails of both groups. The biological value of quail meat of both groups was identical, and there was no toxicity. The tasting evaluation of the broth and meat showed that drinking the microalgae Chlorella sorokiniana does not affect the studied taste indicators. Therefore, the meat of quails that received the microalgae Chlorella sorokiniana feed additive with water is of good quality according to the veterinary and sanitary examination, which allows it to be used for human consumption without restrictions. Key words: poultry, microalgae, organoleptic evaluation, microbial contamination, biological value, tasting evaluation.
... It is also an important cyanobacterium in numerous ecological niches, and culture breakdown has been implicated in the decline in certain bird populations that rely on it as a food source [23]. Despite this widespread use by humans and generally favourable reputation, concerns regarding its effectiveness [24,25], toxicity [26,27], and contamination with heavy metals persist [4]. ...
Commercially produced cyanobacteria preparations sold under the name spirulina are widely consumed, due to their traditional use as a nutrient-rich foodstuff and subsequent marketing as a superfood. Despite their popularity, the microbial composition of ponds used to cultivate these bacteria is understudied. A total of 19 pond samples were obtained from small-scale spirulina farms and subjected to metagenome and/or virome sequencing, and the results were analysed. A remarkable level of prokaryotic and viral diversity was found to be present in the ponds, with Limnospira sp. and Arthrospira sp. sometimes being notably scarce. A detailed breakdown of prokaryotic and viral components of 15 samples is presented. Twenty putative Limnospira sp.-infecting bacteriophage contigs were identified, though no correlation between the performance of these cultures and the presence of phages was found. The high diversity of these samples prevented the identification of clear trends in sample performance over time, between ponds or when comparing successful and failed fermentations.
... Food safety issues of using microalgae need attention. The algal biomass used as raw material needs to be of food-grade quality otherwise it poses risks of contamination from heavy metals (Rzymski et al., 2015), algal toxins (Heussner et al., 2012;Vichi et al., 2012), and other microbial toxins (Hadi & Brightwell, 2021). The major threat to food safety posed by mycoproteins is from allergens. ...
Concerns about the environmental effects of livestock production and the health effects of consuming red and processed meat are driving up demand for meat substitutes. To produce meat analogs with appropriate nutritional and sensory qualities, two types of materials can be used viz., microalgae and fungus. High concentrations of proteins, omega-3 fatty acids, and other necessary nutrients can be found in microalgae. They can be grown using a variety of techniques and processed into powders or pastes that can be added to meat substitutes. Fungi, on the other hand, are a good source of protein, fiber, and flavorings. They are capable of being cultivated on a variety of substrates and can be converted into protein concentrates or used as mycelia. Incorporating microalgae and fungus into meat substitutes can enhance their overall health profile and provide a variety of nutritional advantages in addition to offering distinctive aromas and textures that closely resemble those of meat. Overall, they use less land, water, and other resources and are more sustainable than traditional meat production. To fully realize their potential and ensure the creation of premium microbial meat products, additional study is required. This chapter serves to give an insight into what is known in the usage of fungi and microalgae in producing meat alternatives, their nutritional overview, and their potential impact on health, nutrition, and the environment.
... In the case of cultured meat, the major issues are the concerns regarding the use of genetic engineering [116], as well as the risks associated with culturing meat like exposure to infectious prions or viruses in growth media [117]. Microalgae, fungus, and bacteria are the three main single-cell protein sources, and each of them has distinct food safety issues, such as toxins [118], allergens, and high RNA levels [9]. All these issues should be properly addressed when developing new regulations and policies by governing bodies. ...
The rise in the global population has increased the demand for dietary food protein. Strategies to maximize agricultural and livestock outputs could strain land and freshwater supply and contribute to substantial negative environmental impacts. Consequently, there has been an emphasis on identifying alternative sources of edible proteins that are more sustainable, sustainable, ethical, and healthy. This review provides a critical report on future food protein sources including: plant, cultured meat, insect, and microbial, as alternative sources to traditional animal-based sources. The technical challenges associated with the production process of alternative protein sources are discussed. The most important quality parameters of alternative proteins, such as: protein composition and digestibility, allergenicity, functional and sensory attributes, and safety regulations have been documented. Lastly, future direction and conclusion have been made on future protein trends. However, further regulatory norms need to develop for safe consumption and distribution around the world.
... Notably, these studies have illuminated significant variations in microcystin content, particularly in products containing Aphanizomenon flos-aquae species. These findings underscore the elevated susceptibility of A. flos-aquae to contamination compared to other algae, such as Spirulina [64,65,67]. Conversely, earlier studies primarily grappled with the issue of crosscontamination of Arthrospira cultures with other microalgae ( Table 2). ...
... The quality of these supplements has been often questionable among the scientific community [41][42][43][44][45][46]. Several reports have called attention to potential hazardous components in Cyanobacteria-and/or Chlorella-containing food supplements sold in the European market coming from non-EU countries [11,[47][48][49]. ...
The interest in microalgae as food in Europe is growing due to its remarkable features that can foster a sustainable economy. The lack of tradition on their use among Europeans is changing and a demand for more sustainable products is increasing. The legal framework from the microalgae stakeholders' point of view has been consistently identified as a bottleneck, regardless of its nutritional value and potential to provide added-value metabolites. Microalgae-based products have been mostly consumed as food supplements, which are characterized by some general uncertainty with regards to food security of products sourced from non-European countries. The novel foods regulation is a landmark in Europe's food law defining the conditions in which a new type of food can be commercialized. Currently, a more simplified and centralized version is in place, and around eleven microalgae-based products are on the market; however, more than half are represented by Schizochytrium sp. derived products (DHA-rich oil). Microalgae have immense potential as a sustainable food source; nonetheless, there is limited experience in assessing the safety of these microorganisms, considering the uncertainty around undesirable substances present in the way they are produced and their diverse metabolites. Here, we overview the regulatory use of microalgae as food in Europe with a focus on market introduction, highlighting the administrative procedures and scientific requirements to assess food safety. We also discuss the implications of the Transparency regulation related to microalgae as novel foods and provide considerations for a more solid interaction between academia and industry.
... Moreover, AP has been considered a safe food by the European Food Safety Authority (EFSA) (Chacón-Lee and González-Mariño 2010). Interestingly, AP grows at pH values between 9-10, a pH range at which no toxic accompanying flora was found (Heussner et al. 2012). By contrast, cyanotoxin contamination might be of grave concern, especially in cyanobacterial strains grown in uncontrolled and unmonitored environments, such as outdoor ponds or lakes (Grosshagauer et al. 2020). ...
There is an urgent need to develop technologies for removing CO 2 from the atmosphere to combat climate change. Microalgae and cyanobacteria, such as Arthrospira platensis (AP), have shown promise due to their high photoautotrophic biomass production. Conventional AP culture media are supplemented with high concentrations of NaHCO 3 since AP utilizes HCO 3 - as a carbon source. These culture conditions result in significant amounts of CO 2 escaping into the atmosphere, instead of being sequestered during cultivation. Here, we investigated whether ambient air (0.042% CO 2 ) can be used for growing AP in a culture medium lacking a fossil-based carbon source. AP was cultured in 2 L glass bioreactors containing: (1) Zarrouk medium with 16.8 g/L NaHCO 3 and aeration with 0.236 vvm air with 2% CO 2 (“NaHCO 3 /CO 2 -based”) to compensate carbon loss due to CO 2 outgassing, and (2) Zarrouk medium without NaHCO 3 and a gas flow with ambient air (0.926 vvm) as the only carbon source (“air-based”). The air-based production resulted in the biofixation of 3.78 g CO2 /L during the linear growth phase. With NaHCO 3 /CO 2 -based production, a comparable amount of 3.42 g CO2 /L was obtained while 659.12 g of CO 2 was released into the atmosphere. Total protein, phycocyanin, chlorophyll-a, and carotenoids were present in similar or increased amounts in AP produced by the air-based method. We concluded that cultivation of AP with Zarrouk medium lacking NaHCO 3 but using ambient air with atmospheric CO 2 as the only carbon source is possible without reducing productivity. These results improve our understanding of how atmospheric CO 2 can be reduced by culturing AP.
... Even anaphylaxis was reported in a 14-year-old adolescent six hr. after consuming five Spirulina tablets [142]. There are also reports of severe neonatal hypercalcemia related to excessive maternal exposure to Spirulina platensis. ...
Spirulina or Arthrospira, a Cyanobacterium from the class Cyanophyceae, with a wide range of properties, has been applied for over 400 years. The present study aimed to review available investigations surrounding the clinical and pharmacological properties of Spirulina that have been carried out so far. Databases including Scopus, PubMed, Google Scholar, and Web of Science were searched for relevant literature using the keywords: (Spirulina), (pharmacology), and (clinical). About 130 papers that studied the pharmacological characteristics of Spirulina in animal models, as well as clinical trials, were selected from the beginning to 29 July 2021. According to this review, antioxidative, anti-inflammatory, anti-neoplastic, hypolipidemic, antiviral, immunomodulatory, antimicrobial, anti-atherogenic, anti-diabetic, and radio-protective functions are attributed to Spirulina. Moreover, Spirulina's positive influence on several organs, including hair, skin, liver, CNS, lung, and genitourinary tract, are ascribed to different components of various species of Spirulina such as Spirulina platensis, Spirulina fusiformis, and Spirulina maxima. Although so many studies have been accomplished on every aspect of Spirulina in recent years, the lack of a comprehensive investigation surrounding this microalga encouraged us to prepare this paper. Therefore, the present study could be considered an up-to-date overview of the clinical, pharmacological, and molecular aspects of Spirulina, resulting in more occupational research on this valuable organism.
... S. U. Zafar et al. advantages such as heart-health benefits due to their capacity to block enzymes in the renin-angiotensin-aldosterone system (RAAS) and lower blood pressure (Neklyudov et al. 2000). Algal proteins are approved for human consumption by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO); however, caution must be taken due to reports of toxins in microalgae (Heussner et al. 2012). ...
Carotenoids are pigments having a proven role as food colorants, antioxidants, health-promoting substances, food additives, feed additives, vitamins, pharmaceuticals, etc. After experiencing the hazard of synthetic entities in human life, people are again trying to “go natural.” Being natural and part of a healthy ecosystem, microalgae may have immense potential to provide many such entities. In the present scenario, microalgal systems are among the top-ranked bioresources to meet the demands of the fast-growing world population. In addition, being grown in natural water resources provides opportunities to socially backward classes to manage their lifestyle for economic upliftment and nutritional well-being. Carotenoids may be divided into primary and secondary groups. The secondary carotenoids are present in the lipid vesicles in the cytosol or plastids. Also, phycobiliproteins, phycocyanin, phycoerythrins, β-carotenes, lutins, and astaxanthins are the pigments which are commonly produced by microalgae. Many microalgal systems have been investigated so far to produce different pigments, for instance, diatoms and members of Phaeophyceae for fucoxanthins, dinoflagellates for peridinin, cryptophytes for alloxanthins, Porphyridium spp. for β-carotenes, Tetraselmis spp. for lutein, and so on. This chapter aims to provide an overview of the potential of microalgal systems to generate valuable carotenoids and pigments.
... Microalgae cultivated in open ponds are more vulnerable to contamination (Heussner et al., 2012), while enclosed cultivation systems can reduce contamination risk but can also be capital-intensive (Linder, 2019). Hybrid options like open ponds within greenhouses offer scope to balance these considerations by reducing contamination risk cost-effectively (Lu et al., 2011). ...
In 2021 the Intergovernmental Panel on Climate Change (IPCC) issued the first volume of its latest authoritative report on climate change. Underlining the seriousness of the situation, the United Nations Secretary-General branded its findings a ‘code red for humanity’. The need for climate action is now evident, but finding viable pathways forward can be elusive. Microalgae have been attracting attention as a category of ‘future food’, with species like Arthrospira platensis (spirulina) and Chlorella vulgaris (chlorella) seeing growing uptake by consumers while research interest continues to expand. One timely but neglected question is whether microalgae might offer options for promising climate actions via their agri-food applications. Specifically, might they offer scope to help secure food supplies, while also providing climate resilient livelihood pathways for vulnerable farmers already grappling with food insecurity and environmental degradation? This paper reports on a review of the academic literature on microalgae as an agri-food technology, notably their uses as a food, feed, biofertiliser, biostimulant and biochar. This family of applications was found to offer promising climate actions vis-à-vis both mitigating and adapting to climate change. Aspects pertinent to adaptation include growing rapidly under controlled conditions, reusing water, providing potent nutrition for humans and animals, and supporting resilient crop production. Agri-food applications of microalgae also provide opportunities to mitigate climate change that could be explored. The paper concludes by flagging possible risks and obstacles as well as research and policy priorities to elaborate and harness this potential.
... LC-MS is an intricate analytic technique used to detect MC variants in water. MC structure has been detected via LC-MS in other matrices such as blue-green algae dietary supplements and vegetables (Heussner et al., 2012;Parker et al., 2015;Qian et al., 2017). LC-MS detectors are limited by equipment cost, sample processing time, and technical operation (Ortiz et al., 2017;Foss et al., 2019). ...
Clean, fresh, and safe drinking water is essential to human health and well-being. Occasionally, biological contaminants taint surface water quality used for human consumption and recreation. Microcystins (MCs) and nodularins (NODs) are toxic monocyclic peptides produced by cyanobacteria in fresh and brackish water. These secondary metabolites can reach hazardous concentrations, impairing surface drinking water supplies and thereby increasing the risk of exposure to consumers. Inconsistent screening of MCs and NODs is not uncommon in drinking water systems and no provisional guidance value has been established in Florida to protect community and public health. We performed a case study of two Florida lakes supplying drinking water to adjacent communities for the purpose of monitoring MCs and NODs over the potential peak algae bloom season (June-August). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was employed to analyze concentrations of total MCs and NODs in environmental water samples from Lake Manatee and Lake Washington. Toxin concentrations were similar in each lake, averaging 0.19 μg/L. The highest toxin concentration (0.46 μg/L) was reported in Lake Manatee at Site 15, a location where other toxin concentrations demonstrated statistical significance with toxins detected at Sites 6 (p = 0.014) and 8 (p = 0.011). Inferential analyses from the Kruskal-Wallis H Test revealed a statistically significance difference in toxin concentrations by sampling month within the two Floridian drinking water systems (p < 0.001). Furthermore, phosphate and nitrite concentrations strongly correlated with total MCs and NODs in each lake (p < 0.01). Although results indicate a low probable health risk from cyanotoxins, more research is needed to understand the intrinsic nature of MCs and NODs by examining their prevalence, distribution, and dynamics in surface drinking water supplies serving nearby communities.
... The commercial dried algae (Chlorella and Spirulina) showed high nutrient value (being rich in vitamins B12, C and D2) and is sold as dietary supplements, as sources of proteins and carbohydrates, rather than a food product because of concerns regarding the introduction of biotic and abiotic contaminants during its production. [192] The extracted products from Chlorella and Spirulina can be added to food and feed to improve their nutritional value, for containing high-value compounds as pigments (e.g., astaxanthin), antioxidants (e.g., ß-carotene), proteins (e.g., phycocyanin) and fatty acids (e.g., omega-3, docosahexaenoic acid -DHA and eicosapentaenoic acid -EPA). Due to its high protein content, NASA has used Spirulina as a dietary supplement for astronauts during space missions. ...
There has been a growing demand towards alternative protein sources due to population growth and increasing consumer awareness on sustainability and environmental issues. Proteins from various plant, marine, insect and microbial sources are considered as excellent alternatives to substitute traditional animal-based proteins due their relatively low cost, sustainable production and nutritional value. This work is an overview of the physicochemical properties, functionality and nutritional quality of novel protein sources. Current information on the proximate composition, amino acid profile, digestibility, physicochemical
and functional properties is presented. Effects of extraction method
applied for obtaining protein ingredients from novel sources on protein
composition, functionality and nutritional quality are discussed. Findings of some of the recent studies focusing on modification of protein structure and improvement of functionality are reviewed. Potential end product applications and challenges related to the production and use of protein ingredients obtained from novel sources are discussed. Furthermore, future research recommendations are presented.
... MC have been shown to be toxic to cattle [3][4][5], dogs [6,7] and other mammals [8][9][10]. Human exposure is frequent by accidental or deliberate ingestion of contaminated water, food [11][12][13], and food supplements [14,15], but also due to inhalation of aerosolised toxins [16]. Most notably, parenteral exposure of dialysis patients to contaminated water has highlighted the toxic potential of MC [17][18][19]. ...
Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [β-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.
... RP-C18 columns have been used as well in combination with polar columns like amide [24] and about all with hydrophilic interaction liquid chromatography (HILIC) columns [25][26][27][28][29][30][31] for multiclass cyanotoxin analysis purposes. ...
The presence of cyanobacteria and cyanotoxins in all water bodies, including ocean water and fresh water sources, represents a risk for human health as eutrophication and climate change are enhancing their level of proliferation. For risk assessment and studies on occurrence, the development of reliable and sensitive analytical approaches able to cover a wide range of cyanotoxins is essential. This work describes the development of an HILIC-MS/MS multiclass method for the simultaneous analysis of eight cyanotoxins in reservoir water samples belonging to three different classes according to their chemical structure: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin, anatoxin-a) and three non-protein amino acids isomers such as β-methylamino-L-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). A SeQuant ZIC-HILIC column was employed to achieve the chromatographic separation in less than 12 min. Previously, a novel sample treatment based on a tandem solid-phase extraction (SPE) system using mixed cation exchange (MCX) and Strata-X cartridges was investigated with the aim of extracting and preconcentrating this chemically diverse group of cyanotoxins. The Strata-X cartridge, which was configured first in the line of sample flow, retained the non-polar compounds and the MCX cartridge, which was at the bottom of the dual system, retained mainly the non-protein amino acids. The optimization procedure highlighted the importance of sample ion content for the recoveries of some analytes such as microcystin-RR and the non-protein amino acid isomers. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and precision in terms of repeatability and intermediate precision. This work represents the first analytical method for the simultaneous analysis of these multiclass cyanotoxins in reservoir water samples, achieving LOQs in the very low range of 7·10⁻³ – 0.1 μg L⁻¹. Despite high recoveries obtained at the LOQ concentration levels (92.8–70.9%), relative standard deviations lower than 15.3% were achieved.
... Common routes of MC/NOD exposure include the uptake of contaminated drinking water, algal food supplements, and food (aquatic animals from contaminated water bodies or vegetables and fruits watered with contaminated water). 1,2 MCs are a group of cyclic heptapeptide cyanotoxins with high toxicity and broad distribution. They are secondary metabolites of different species of freshwater cyanobacteria (formerly known as bluegreen algae), such as Anabaena, Microcystis, Nostoc, and Planktothrix, 3,4 and released in water when algal cells are lysed under stress or age. ...
... In their study, they only found microcystins, which were present in products of Aphanizomenon flos-aquae. Extracts from all the tested samples showed cytotoxic effects, which leads to the conclusion that additional components should be present that have the potential to induce adverse effects in consumers [72]. ...
Microalgal products are an emerging class of food, feed, and nutraceuticals. They include dewatered or dried biomass, isolated pigments, and extracted fat. The oil, protein, and antioxidant-rich microalgal biomass is used as a feed and food supplement formulated as pastes, powders, tablets, capsules, or flakes designed for daily use. Pigments such as astaxanthin (red), lutein (yellow), chlorophyll (green), or phycocyanin (bright blue) are natural food dyes used as isolated pigments or pigment-rich biomass. Algal fat extracted from certain marine microalgae represents a vegetarian source of n-3-fatty acids (eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), γ-linolenic acid (GLA)). Gaining an overview of the production of microalgal products is a time-consuming task. Here, requirements and options of microalgae cultivation are summarized in a concise manner, including light and nutrient requirements, growth conditions, and cultivation systems. The rentability of microalgal products remains the major obstacle in industrial application. Key challenges are the high costs of commercial-scale cultivation, harvesting (and dewatering), and product quality assurance (toxin analysis). High-value food ingredients are commonly regarded as profitable despite significant capital expenditures and energy inputs. Improvements in capital and operational costs shall enable economic production of low-value food products going down to fishmeal replacement in the future economy.
... Due to the public concerns regarding the side effects of BGAS, more cautionary statements have been amended regarding the certification of such products in markets. In USA, the tolerable daily intake (TDI) of BGAS is at the level of 1 μg MCeq g −1 and Switzerland allows the daily consumption of 2 μg MC L −1 for adults and subsequent lower amounts for infants and children's [101]. Based on the average body weight i.e., infants (5 kg), children (20 kg) and adults (50 kg) can tolerate a maximum consumption of 0.2. ...
Microalgae are unicellular photosynthetic organisms that have been recently attracted potential interests and have applications in food, nutraceuticals, pharmaceuticals, animal feed, cosmetics, and biofertilizers industry. Microalgae are rich in a variety of high-value bioactive compounds which have potential benefits on human health and can be used for the prevention and curing of many disease conditions. But scale-up and safety issues remain a major challenge in the commercialization of microalgal products in a cost-effective manner. However, techniques have been developed to overcome these challenges and successfully selling the products derived from microalgae as food, cosmetics and pharmaceutical industries. Microalgae are rich in many nutrients and can be used for the production of functional food and nutraceuticals, safety and regulatory issues are major concerns and extensive research is still needed to make microalgae a commercial success in the future. Many practical difficulties are involved in making the microalgal food industry commercially viable. The present review focuses on the industrial applications of microalgae and the challenges faced during commercial production.Graphic abstract
... A number of ubiquitously present cyanobacteria genera, e.g. Microcystis, Planktothrix, Anabaena, Dolichospermum, have been demonstrated to produce MC(Svirčev et al. 2019).The main exposure route for humans is drinking water, albeit excessive exposure can occur when cyanobacterial supplements are voluntarily consumed(Heussner et al. 2012). The World Health Organization (WHO) suggested a safe value of 1 µg/l (1 nM) for MC-LR into their guidance on cyanobacteria in drinking water (World Health Organization.Water et al. 2004). ...
... We have found no evidence that any of the known cyanometabolites is produced by Spirulina analyzed in this work. In the case of this genus, a confusing thing is that many studies still use the incorrect name Spirulina platensis instead of Arthrospira platensis [93,[96][97][98]. ...
With the improved understanding of the cyanobacterial diversity, the polyphasic approach has become a widely accepted way to classify the organisms. The aim of the study was to describe and compare phenotypic and molecular traits of two strains of filamentous, helically coiled cyanobacteria CCNP1310 and 06S082, originating from different habitats of the Baltic Sea. Genotypic and phylogenetic analyses were focused on 16S rDNA, cpcBA-IGS and mcyE target sequences, while the phenotypic characterization included morphology, ultrastructures visualized with transmission electron microscopy, pigment and other metabolite profiles, determined with high-performance liquid chromatography and liquid chromatography-mass spectrometry, respectively. Results indicated that both tested strains belong to the Spirulinales order. 16S rDNA phylogeny confirmed that in the case of the analyzed microorganisms, cryptic diversity may occur. The final reclassification of this order requires analysis of a larger group of strains. Despite the high morphological similarity, the strains showed significant differences in the produced metabolites. The analysis of photosynthetic pigments indicated that in the cyanobacteria of the order Spirulinales the model of response to changing light regime might be preserved even after a long time of growth in culture. Cyanometabolite profiles determined spectrometrically showed that the characterized organisms differ in terms of the produced metabolites. The presented, comprehensive characterization of the strains can also be useful in further studies on the diversity, taxonomy, and metabolic potential of cyanobacteria of the order Spirulinales.
... Extracts from products consisting of Spirulina sp., Aphanizomenon flos-aquae and Chlorella sp. appear to be contaminated with microcystins and noludarin toxins (Heussner et al., 2012). Moreover, another safety aspect is the exposure to biological and non-biological contamination when algae such as Spirulina and Chlorella are cultivated in open systems. ...
The coexistence of algae and bacteria in nature dates back to the very early stages when life came into existence. The interaction between algae and bacteria plays an important role in the planet ecology, cycling nutrients, and feeding higher trophic levels, and have been evolving ever since. The emerging concept of algal-bacterial consortia is gaining attention, much towards environmental management and protection. Studies have shown that algal-bacterial synergy does not only promote carbon capture in wastewater bioremediation but also consequently produces biofuels from algal-bacterial biomass. This review has evaluated the optimistic prospects of algal-bacterial consortia in environmental remediation, biorefinery, carbon sequestration as well as its contribution to the production of high-value compounds. In addition, algal-bacterial consortia offer great potential in bloom control, dye removal, agricultural biofertilizers, and bioplastics production. This work also emphasizes the advancement of algal-bacterial biotechnology in environmental management through the incorporation of Industry Revolution 4.0 technologies. The challenges include its pathway to greener industry, competition with other food additive sources, societal acceptance, cost feasibility, environmental trade-off, safety and compatibility. Thus, there is a need for further in-depth research to ensure the environmental sustainability and feasibility of algal-bacterial consortia to meet numerous current and future needs of society in the long run.
... Although cyanobacteria have the potential to produce toxins, increasingly genera such as Aphanizomenon and Spirulina (potentially including Arthrospira) are being promoted as health food supplements (Bishop and Zubeck 2012). Toxin analysis of such supplements has indicated that they are capable of producing a range of toxins, including microcystins, anatoxin-a and β-N-methylamino-L-alanine (BMAA) (Rawn et al. 2007;Rellán et al. 2009;Heussner et al. 2012;Glover et al. 2015;Roy-LaChapelle et al. 2017). The presence of toxins in health food supplements is a potential risk to consumers, such that in the State of Oregon, commercial products of Aphanizomenon flos-aquae are legislated to not contain microcystins at a concentration of greater than 1 μg/g (Marsan et al. 2017). ...
In some parts of the world, cyanobacteria are used as a food in the human diet, due to their ready availability. Lake Chad, has long been a traditional site for the collection of Arthrospira fusiformis which is dried and processed at the lake into thin wafers called Dihé for later consumption or is transported to market for sale. However, Dihé purchased from markets in Chad has not been analyzed for known cyanobacterial toxins or assessed for total amino acid content. Since BMAA in traditional foodstuffs of the indigenous Chamorro people of Guam causes neurodegenerative illness, it is important that Dihé from Chad be analyzed for this neurotoxin. BMAA and its isomer AEG were not detected in our analyses, but a further isomer DAB was detected as both a free and bound amino acid, with an increase in the free concentration after acid hydrolysis of this fraction. Microcystins were present in 6 samples at up to 20 μg/g according to UPLC-PDA, although their presence could not be confirmed using PCR for known microcystin synthetic genes. Amino acid analysis of the cyanobacterial material from Chad showed the presence of large amounts of canonical amino acids, suggesting that this may supplement indigenous people on low protein diets, although regular monitoring of the foodstuffs for the presence of cyanotoxins should be performed.
This chapter discusses the biotechnology of marine green organisms, with major focus on their derived bioactive compounds and their use for medicinal purposes. It focuses on water being a basic source of life as the earth composes about 70% extant water bodies which are inhabitants to diverse forms of living organisms. It examined the attention of science and technology into marine plants (algae) which gives rise to marine green biotechnology. Bioactive compounds that are derivable from marine organisms either as natural products or as metabolites were looked into as well as their numerous health benefits for man. Furthermore, the chapter discusses various advances of these marine-derived bioactive compound and their applications or utilization for medical purposes, importantly in the management of chronic diseases including diabetes, cardiovascular diseases (CVDs), Inflammation and cancer. In conclusion, Marine green environment is portrayed a unique habitat, harbouring diverse organisms with great potentials to produce bioactive substances of distinctive biological and physicochemical properties of importance for human health.
Aphanizomenon flos-aquae (AFA) is a Gram-negative nitrogen-fixing freshwater filamentous cyanobacterium with a rich nutritional profile that is approved for human consumption by both the Food and Drug Administration and the European Food and Safety Authorities. It has a high protein content (60-70%) and contains numerous vitamins, minerals and trace elements together with several high-value compounds with nutraceutical properties, such as C-phycocyanin and β-phenylethylamine. 500-1000 t of AFA dry biomass are currently wild harvested from natural blooms that occur seasonally in Klamath Lake, Oregon, USA, and distributed as a nutritional supplement worldwide. The requirements and unreliability of wild harvesting, owing to the dependence of AFA growth on environmental conditions and potential contamination by microcystin toxins, threaten the availability of biomass supply and restricts commercial expansion. In this review we propose AFA cultivation in open ponds or closed photobioreactors to obtain a reliable production of unialgal biomass to resolve the supply issue and enhance AFA as a feedstock for specific high-value by-products. Furthermore, we explore potential strategies for maximising overall yield and seasonal robustness by using a synergistic combination of cultivation and wild harvesting. Controlled AFA cultivation would also facilitate the use of genetic manipulation to generate bespoke strains with improved commercial applications, such as increasing the cyanobacterium’s nitrogen-fixation rate to enhance its value as a biofertiliser. Ultimately, realising the untapped biotechnological potential of AFA requires a better understanding of its fundamental biology, robust methodologies for laboratory and large-scale cultivation, and the development of AFA-specific genetic engineering techniques.
The intricate nature of cyanotoxin exposure through food reveals a complex web of risks and uncertainties in our dietary choices. With the aim of starting to unravel this intricate nexus, a comprehensive review of 111 papers from the past two decades investigating cyanotoxin contamination in food was undertaken. It revealed a widespread occurrence of cyanotoxins in diverse food sources across 31 countries. Notably, 68% of the studies reported microcystin concentrations exceeding established Tolerable Daily Intake levels. Cyanotoxins were detected in muscles of many fish species, and while herbivorous fish exhibited the highest recorded concentration, omnivorous species displayed a higher propensity for cyanotoxin accumulation, exemplified by Oreochromis niloticus. Beyond fish, crustaceans and bivalves emerged as potent cyanotoxin accumulators. Gaps persist regarding contamination of terrestrial and exotic animals and their products, necessitating further exploration. Plant contamination under natural conditions remains underreported, yet evidence underscores irrigation-driven cyanotoxin accumulation, particularly affecting leafy vegetables. Finally, cyanobacterial-based food supplements often harbored cyanotoxins (57 % of samples were positive) warranting heightened scrutiny, especially for Aphanizomenon flos-aquae-based products. Uncertainties surround precise concentrations due to methodological variations (chemical and biochemical) and extraction limitations, along with the enigmatic fate of toxins during storage, processing, and digestion. Nonetheless, potential health consequences of cyanotoxin exposure via contaminated food include gastrointestinal and neurological disorders, organ damage (e.g. liver, kidneys, muscles), and even elevated cancer risks. While microcystins received significant attention, knowledge gaps persist regarding other cyanotoxins' accumulation, exposure, and effects, as well as combined exposure via multiple pathways. Intriguing and complex, cyanotoxin exposure through food beckons further research for our safer and healthier diets.
Alternative proteins have gained popularity as consumers look for foods that are healthy, nutritious, and sustainable. Plant proteins, precision fermentation-derived proteins, cell-cultured proteins, algal proteins, and mycoproteins are the major types of alternative proteins that have emerged in recent years. This review addresses the major alternative-protein categories and reviews their definitions, current market statuses, production methods, and regulations in different countries, safety assessments, nutrition statuses, functionalities and applications, and, finally, sensory properties and consumer perception. Knowledge relative to traditional dairy proteins is also addressed. Opportunities and challenges associated with these proteins are also discussed. Future research directions are proposed to better understand these technologies and to develop consumer-acceptable final products.
A significant factor that contributes to the majority of neurodegenerative disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), and multiple sclerosis (MS), is the deterioration of the brain's anti-inflammatory and antioxidant protective mechanisms that can occur in a number of situations. Consequently, the brain becomes susceptible to the harmful impacts of free radicals. To treat neurodegenerative and neurological disorders, researchers have concentrated on the creation of novel medications, particularly natural bioactive compounds that can correct imbalance in redox system and the ensuing harm to the cells without producing toxicity to the neurones. In addition to promoting a healthy immune response and mitigating the negative impacts of an overactive immune system, marine green spirulina is a rich source of anti-inflammatory as well as antioxidant compounds that has ability to enhance the physiologic brain activities, and the growth and development of the nervous system. The most recent research on marine green spirulina's modulatory and neuroprotective properties, as well as its beneficial impacts on activation of glial cells and therapeutic application in neurodegenerative disorders, particularly MS, PD, and AS, is compiled in this study.
Keywords: Marine green spirulina; Antioxidant; Multiple sclerosis; Alzheimer’s disease; Parkinson’s disease.
“Marine greens” refers to the vast and diverse ecosystem of marine algae or seaweeds that exist in oceans, seas, and other bodies of saltwater. Algae are a type of plant that can grow both in the water and on land. However, marine green algae are only found in saltwater. They play a significant role in marine ecology, as they serve as the primary food source for many aquatic organisms and provide important nutrients to the oceanic food chain. Marine green algae play a vital role in maintaining the health and balance of marine ecosystems. They serve as a primary food source for many aquatic animals, including fish, crustaceans, and other invertebrates, and are also consumed by larger marine mammals such as manatees and sea otters. Additionally, they produce significant amounts of oxygen through photosynthesis, which helps to maintain the oxygen balance in marine environments. The algae also absorb carbon dioxide from the atmosphere and store it, thereby reducing the amount of carbon in the atmosphere and mitigating the effects of climate change. Marine green algae are an essential component of marine ecosystems, providing crucial nutrients, oxygen, and ecological balance. The biodiversity and variety of these algae are crucial to the wellbeing of numerous marine species and help mitigate the effects of climate change by reducing carbon dioxide in the atmosphere. The preservation and conservation of these species are crucial to maintaining the health of our oceans and by extension, the planet. Therefore, this review proposes to look at the ability of marine green to remediate contaminant
The increased consumption of blue-green algae (BGA)-based dietary supplements has raised concern about their food safety, especially about cyanotoxin presence. The hyphenation of liquid chromatography with ion mobility mass spectrometry represents a relevant tool to screen several compounds in a large variety of food matrices. In this work, ultrahigh-performance liquid chromatography coupled to traveling wave ion mobility spectrometry/quadrupole time-of-flight mass spectrometry (UHPLC-TWIMS-QTOF) was employed to establish the first comprehensive TWIMS-derived collision cross section database (TWCCSN2) for phycotoxins. The database included 20 cyanotoxins and 1 marine toxin. Accurate m/z, retention times, and TWCCSN2 values were obtained for 81 adducts in positive and negative electrospray (ESI+/ESI-) modes. Reproducibility and robustness of the TWCCSN2 measurements were determined to be independent of the matrix. A screening was carried out on 19 commercial BGA dietary supplements of different composition. Cyanotoxins were confidently identified in five samples based on retention time, m/z, and TWCCSN2.
Citation: Pinchart, P.-E.; Leruste, A.; Pasqualini, V.; Mastroleo, F. Microcystins and Cyanobacterial Contaminants in the French Small-Scale Productions of Spirulina (Limnospira sp.). Toxins 2023, 15, 354. Abstract: Spirulina is consumed worldwide, in the form of food or dietary supplements, for its nutritional value and health potential. However, these products may contain cyanotoxins, including hepatotoxic microcystins (MCs), produced by cyanobacterial contaminants. The French spirulina market has the particularity of being supplied half-locally by approximately 180 small-scale spir-ulina production farms. Data about this particular production and possible contaminations with other cyanobacteria and MCs are scarce. Thus, we collected the results of MC analyses and total cyanobacteria counts, carried out between 2013 and 2021, from 95 French spirulina producers who agreed to share their data. These data consisted of MC concentrations determined with an enzyme-linked immunosorbent assay (ELISA) using 623 dry spirulina samples and 105 samples of spirulina cultures. In addition, potentially unsafe samples of dry spirulina were further investigated through mass spectrometry, as duplicate analysis. We confirmed that the situation of the French spirulina production stayed within the safe regulatory level in terms of MC levels. On the other hand, the inventory of cyanobacterial contaminants, based on 539 count results, included 14 taxa. We present their prevalence, interannual evolution and geographical distribution. We also suggested improvements in cultivation practices to limit their propagation. Key Contribution: This first analysis of a large dataset on cyanobacteria and microcystin contamination provided an understanding of the situation of the French spirulina production and an insight into the cyanobacteria population and distribution.
As fossil fuels are declining, global economies need sustainable energy sources. Biofuel, the most promising candidate for the energy crisis, is still not competitive as compared to fossil fuels. Thus, higher yields of biofuel and simultaneous production of high-value commodities (HVCs) to compensate for the production cost are a plausible strategy. Microalgae in this context are a successful candidate, due to their ability to produce various nutraceuticals, pharmaceuticals, and many other HVCs alongside biofuels. Higher sequestration of greenhouse gases (GHGs) to biomass and the ability to recycle nutrient-rich cosmopolitan waste with a biorefinery-based approach give microalgae an advantage over other photoautotrophs. This biofuel and HVC-rich biomass can be further enhanced by regulating some specific targets like pathway genes, transcription factors, enhancers, and repressors. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered regulatory interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) are some technologies used for this purpose. The combination of these synthetic biology approaches with bioprocess improvement can help in increasing biofuel and HVC production. However, the unavailability of microalgal genomes is a bottleneck in their genetic engineering for a sustainable biorefinery. The advances in multi-omics technologies give possible solutions for effectively understanding the microalgal genomes. Thus, in this chapter, we discuss microalgal physiology in light of synthetic biology and aforementioned genome editing techniques in combination with major bottlenecks and their solutions.KeywordsMicroalgaeBiorefinerySynthetic biologyGenetic engineeringBiofuels
Dietary supplements are widely consumed in the EU and the USA. Based on their similarity to pharmaceuticals, consumers mistakenly believe that dietary supplements have also been approved for safety and efficacy. However, in the absence of mandatory testing, data on supplement quality is scarce. Thus, we applied liquid chromatography coupled with tandem mass spectrometry to analyse the quality of dietary supplements containing tryptophan (Trp). We examined 22 supplements in tablets or capsules, produced in the USA, Great Britain, Germany, France, Czech Republic, and Poland. Trp release, crucial for bioavailability and efficiency, was assessed. Additionally, we performed a qualitative analysis of the main ingredient and screened for contaminants. Among the contaminants, we detected Trp’s metabolites, condensation products of Trp and carbonyl compounds, Trp degradation products, degradation products of kynurenine, and other contaminants such as glucosamine and melatonin. The main ingredient content was in the range of 55–100% in capsules and 69–87% in tablets. Surprisingly, almost no Trp release was noted from some supplements. Our study confirms the need to advance research on supplements. We believe that the high-quality analysis of supplements based on reliable analytical techniques will be an important contribution to the discussion on the regulatory framework of these products.
Microalgae represent a valuable source of protein, increasingly used as food and feed supplements in aquaculture, animal, and human nutrition. In addition to producing high-quality protein from simply light, carbon dioxide, water, and minimal nutrients, microalgae are capable of producing a vast array of bioactive compounds which have been exploited in many applications such as pharmaceutical, nutraceutical, functional food, fertilizer, bioenergy, and bioplastics. These versatile cell factories hold the solutions to many societal problems without compromising the use of agricultural land. This chapter describes the biological activity of important chemical groups such as fatty acids, pigments, and vitamins which all contribute to good health and well-being as well as the deadly natural toxins produced by some microalgae which have been detected in commercial dietary supplements and must be considered in the sustainable production of algal biomass and products.
Clean, fresh, and safe drinking water is essential to human health and well-being. Occasionally, chemical pollutants taint surface water quality used for consumption. Microcystins (MCs) are toxic heptapeptides produced by freshwater cyanobacteria. These secondary metabolites can reach hazardous concentrations, impairing surface drinking water supplies. Inconsistent screening of MCs is not uncommon in Florida waters as no provisional guidance value is established to protect public health. The occurrence of MCs in Lake Manatee and Lake Washington was monitored over the potential peak algae bloom season (June-August). An indirect competitive enzyme-linked immunosorbent assay (icELISA) quantified total MCs in two drinking water systems. Varied concentrations occurred between June and July, whereas concentrations peaked in August. Overall, MC prevalence was higher in Lake Manatee than Lake Washington. Colorimetric assays measured phosphate and nitrite in environmental water samples. Phosphate and nitrite concentrations strongly correlated with total MCs (p < 0.01). The results indicate the intrinsic nature of environmental MCs in surface drinking water supplies and the need to examine hepatotoxin dynamics to preserve drinking water quality in community served areas.
Marine organisms are vital sources of staple and functional food but are also the major dietary route of human exposure to total arsenic. We surveyed the total arsenic content and the mass fractions of hydrophilic arsenic species from five different marine food types cutting across the food chain from microalgae, macroalgae, bivalve clam, crustaceans and finfish. Total arsenic was determined using inductively coupled plasma-mass spectrometry (ICP-MS) while arsenic speciation analysis was performed using high-performance liquid chromatography (HPLC) coupled to ICP-MS as the detector. The total arsenic contents ranged from 133 ± 11 ng/g to 26,630 ± 520 ng/g. The mass fractions of inorganic arsenic (iAs), arsenobetaine (AsB), dimethylarsinic acid (DMA), and the four commonly occurring arsenosugars (AsSugars) are reported. Extractable hydrophilic arsenic species accounted for 10 % (aquacultured shrimp) to 95 % (kelp) of the total arsenic. DMA was established to be a byproduct of the decomposition of AsSugars in acid extracts of samples known to contain these species.
Cyanobacterial harmful algal blooms (CyanoHABs) produce microcystins (MCs) which are associated with animal and human hepatotoxicity. Over 270 variants of MC exist. MCs have been continually studied due of their toxic consequences. Monitoring water quality to assess the presence of MCs is of utmost importance although it is often difficult because CyanoHABs may generate multiple MC variants, and their low concentration in water. To effectively manage and control these toxins and prevent their health risks, sensitive, fast, and reliable methods capable of detecting MCs are required. This paper aims to review the three main analytical methods used to detect MCs ranging from biological (mouse bioassay), biochemical (protein phosphatase inhibition assay and enzyme linked immunosorbent assay), and chemical (high performance liquid chromatography, liquid chromatography-mass spectrometry, high performance capillary electrophoresis, and gas chromatography), as well as the newly emerging biosensor methods. In addition, the current state of these methods regarding their novel development and usage, as well as merits and limitations are presented. Finally, this paper also provides recommendations and future research directions towards method application and improvement.
this book aims to give background information as well as practical guidance. Chapters 2 and 3 provide the
background for understanding the behaviour of cyanobacteria and their toxin production
in given environmental conditions. Chapter 4 reviews the evidence regarding health
impacts, primarily for public health experts establishing national guidelines or academics
identifying and addressing current research needs. Chapters 5-7 provide guidance on
safe practices in the planning and management of drinking water supplies and
recreational resorts. Readers who access the book with specific questions regarding
prevention of cyanobacterial growth or their removal in drinking water treatment will find
Chapters 8 and 9 of direct relevance. Guidance on the design and implementation of
monitoring programmes is given in Chapter 10, and Chapters 11-13 provide field and
laboratory methods for monitoring cyanobacteria, their toxins and the conditions which
lead to their excessive growth.
Algae preparations are commonly used in alternative medicine. We examined the effects of algae extracts on normal hematopoietic cells and leukemia cells. Ethanol extracts were prepared of Dunaliella salina (Dun), Astaxanthin (Ast), Spirulina platensis (Spir), and Aphanizomenon flos-aquae (AFA). Cell viability effects were completed by Annexin staining. Ast and AFA inhibited HL-60 and MV-4-11 whereas Dun and Spir had no effect. Primary AML blasts demonstrated increased apoptosis in AFA. Primary CLL cells showed apoptosis at 24 hours after exposure to Dun, Ast, Spir, and AFA. High AFA concentrations decreased viability of normal marrow cells. Normal CD34+ viability was inhibited by Dun. Dun and AFA inhibited BFU-E, but all extracts inhibited CFU-GM. Cell-cycle analysis of AML cell lines showed G0/G1 arrest in the presence of AFA. These data suggest that algae extracts may inhibit AML cell lines and leukemia blasts, but they may also have potential inhibitory effects on normal hematopoiesis.
In western cultures, certain cyanobacteria have beenan accepted source of microalgal biomass for food forabout 30 years, in particular Spirulina(Arthrospira) platensis and S. maxima. Beginning in the early 1980s, another species, Aphanizomenon flos-aquae was adopted for similaruses. This is harvested from Upper Klamath Lake, thelargest freshwater lake system in Oregon. In 1998 theannual commercial production of Aphanizomenonflos-aquae was about 1 106 kg. Since thisspecies is not cultured like Spirulina inoutdoor ponds or raceways, it requires very differentprocedures for harvesting and processing. These arereviewed here and include extensive off-lake screensor on-lake barges, which dewater and concentrate thecells. Other procedures, such as those for removal ofdetritus and mineral materials, and those formonitoring and reducing the amounts of certaincontaminant cyanobacteria, which can producecyanotoxins, have also become important in qualitycontrol and marketing.
Cyanobacteria (blue-green algae) are abundant in fresh, brackish and marine waters worldwide. When toxins produced by cyanobacteria are present in the aquatic environment, seafood harvested from these waters may present a health hazard to consumers. Toxicity hazards from seafood have been internationally recognised when the source is from marine algae (dinoflagellates and diatoms), but to date few risk assessments for cyanobacterial toxins in seafood have been presented. This paper estimates risk from seafood contaminated by cyanobacterial toxins, and provides guidelines for safe human consumption.
The cyanobacteria or "blue-green algae", as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites.
Cyanobacterial microcystins (MCs) represent a toxin group with > 100 variants, requiring active uptake into cells via organic anion-transporting polypeptides, in order to irreversibly inhibit serine/threonine-specific protein phosphatases. MCs are a human health hazard with repeated occurrences of severe poisonings. In the well-known human MC intoxication in Caruaru, Brazil (1996), patients developed signs of acute neurotoxicity, e.g., deafness, tinnitus, and intermittent blindness, as well as subsequent hepatotoxicity. The latter data, in conjunction with some animal studies, suggest that MCs are potent neurotoxins. However, there is little data to date demonstrating MC neuron-specific toxicity. MC exposure-induced cytotoxicity, caspase activity, chromatin condensation, and microtubule-associated Tau protein hyperphosphorylation (epitopes serine199/202 and serine396) were determined. Neurite degeneration was analyzed with confocal microscopy and neurite length determined using image analysis. MC-induced apoptosis was significantly increased by MC-LF and MC-LW, however, only at high concentrations (≥ 3μM), whereas significant neurite degeneration was already observed at 0.5μM MC-LF. Moreover, sustained hyperphosphorylation of Tau was observed with all MC congeners. The concentration- and congener-dependent mechanisms observed suggest that low concentrations of MC-LF and MC-LW can induce subtle neurodegenerative effects, reminiscent of Alzheimer's disease type human tauopathies, and thus should be taken more seriously with regard to potential human health effects than the apical cytotoxicity (apoptosis or necrosis) demonstrated at high MC concentrations.
The Dietary Supplements Information Expert Committee (DSI-EC) of the United States Pharmacopeial Convention (USP) reviews the safety of dietary supplements and dietary supplement ingredients for the purpose of determining whether they should be admitted as quality monographs into the United States Pharmacopeia and National Formulary (USP-NF). The United States Food and Drug Administration (FDA) has enforcement authority to pursue a misbranding action in those instances where a dietary supplement product indicates that it conforms to USP standards but fails to so conform. Recently DSI-EC undertook a safety evaluation of spirulina, a widely used dietary ingredient. DSI-EC reviewed information from human clinical trials, animal studies, and regulatory and pharmacopeial sources and analyzed 31 adverse event reports regarding spirulina to assess potential health concerns. At the conclusion of this review, DSI-EC assigned a Class A safety rating for Spirulina maxima and S. platensis, thereby permitting the admission of quality monographs for these dietary supplement ingredients in USP-NF. DSI-EC continually monitors reports concerning the safety of dietary supplements and dietary supplement ingredients for which USP dietary supplement monographs are developed. The DSI-EC may revisit the safety classification of spirulina as new information on this dietary ingredient becomes available.
Cylindrospermopsin (CYN), a potent hepatoxin, occurs in freshwaters worldwide. Several cyanobacterial species produce the toxin, but the producing species vary between geographical regions. Aphanizomenon flos-aquae, a common algae species in temperate fresh and brackish waters, is one of the three well-documented CYN producers in European waters. So far, no genetic information on the CYN genes of this species has been available. Here, we describe the complete CYN gene cluster, including flanking regions from the German Aphanizomenon sp. strain 10E6 using a full genome sequencing approach by 454 pyrosequencing and bioinformatic identification of the gene cluster. In addition, we have sequenced a approximately 7 kb fragment covering the genes cyrC (partially), cyrA and cyrB (partially) of the same gene cluster in the CYN-producing Aphanizomenon sp. strains 10E9 and 22D11. Comparisons with the orthologous gene clusters of the Australian Cylindrospermopsis raciborskii strains AWT205 and CS505 and the partial gene cluster of the Israeli Aphanizomenon ovalisporum strain ILC-146 revealed a high gene sequence similarity, but also extensive rearrangements of gene order. The high sequence similarity (generally higher than that of 16S rRNA gene fragments from the same strains), atypical GC-content and signs of transposase activities support the suggestion that the CYN genes have been horizontally transferred.
Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been
detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean
genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN
concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating
and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically
classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked
immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by
ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX],
saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake
Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.
Certain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the two L-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathione S-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.
The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or Leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to protein serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning.
The presence of paralytic shellfish poisoning (PSP) toxins in cultures of Aphanizomenon flos-aquae, isolated from the Crestuma-Lever reservoir, was found by reversed phase high performance liquid chromatography employing two isocratic elution systems for the separation of PSP toxins. With the first isocratic elution protocol, the presence of apolar toxins as saxitoxin, decarbamoyl saxitoxin and neosaxitoxin not detected. On the other hand, GTX4, GTX1 and GTX3 as well as Cs toxins were present either in the Aphanizomenon flos-aquae cells collected directly from the bloom or in the other toxic isolates priorly cultivated in laboratory conditions.
Cyanobacteria can generate molecules hazardous to human health, but production of the known cyanotoxins is taxonomically sporadic. For example, members of a few genera produce hepatotoxic microcystins, whereas production of hepatotoxic nodularins appears to be limited to a single genus. Production of known neurotoxins has also been considered phylogenetically unpredictable. We report here that a single neurotoxin, beta-N-methylamino-L-alanine, may be produced by all known groups of cyanobacteria, including cyanobacterial symbionts and free-living cyanobacteria. The ubiquity of cyanobacteria in terrestrial, as well as freshwater, brackish, and marine environments, suggests a potential for wide-spread human exposure.
The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.
The combination of hydrophilic interaction liquid chromatography with electrospray mass spectrometry (HILIC–MS) has been investigated as a tool for the analysis of assorted toxins produced by cyanobacteria. Toxins examined included saxitoxin and its various analogues (1–18), anatoxin-a (ATX-a, 19), cylindrospermopsin (CYN, 20), deoxycylindrospermopsin (doCYN, 21), and microcystins-LR (22) and -RR (23). The saxitoxins could be unequivocally detected in one isocratic analysis using a TSK gel Amide-80 column eluted with 65% B, where eluent A is water and B is a 95% acetonitrile/water solution, both containing 2.0 mM ammonium formate and 3.6 mM formic acid. The analysis of ATX-a, CYN and doCYN required 75% B isocratic. Simultaneous determination of 1–21 was also possible by using gradient elution. HILIC proved to be suitable for the analysis of microcystins, but peak shape was not symmetric and it was concluded that these compounds are best analysed using existing reversed-phase methods. The HILIC–MS method was applied to the analysis of field and cultured samples of Anabaena circinalis and Cylindrospermopsis raciborskii. In general, the method proved quite robust with similar results obtained in two different laboratories using different instrumentation.
Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood–brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent Km values amounted to 7 ± 3 μM for OATP1B1, 9 ± 3 μM for OATP1B3, and 20 ± 8 μM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood–brain barrier.
Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N2 fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.
This review provides an in-depth survey of the recorded incidences in aquatic organisms of mortality and disease events suspected or known to be caused by microalgal or ciliate blooms, their biotoxins, or their harmful mechanisms. Some 200 species of dinoflagellates, diatoms, raphidophytes, prymnesiophytes, silicoflagellates, cilliates, and cyanobacteria are currently known to be, suspected to be, or have the potential to be toxic or harmful to a wide spectrum of organisms. This review summarizes the current information on toxic or harmful microalgal species that affect aquatic organisms (and, when relevant, those that affect terrestrial organisms, including humans), provides an updated list of such species, cites pertinent case histories, and includes relevant information on harmful or toxic species from freshwater, brackish, and marine ecosystems. It is hoped that this review will provide documentation and reference material suitable for researchers, students, managers, and resource and health professionals alike and that it will stimulate future research questions critical to our understanding of harmful algal bloom (HAB) species and the significant effects they have upon aquatic systems.
Blue green algae supplements (BGAS) are generally proposed as health-promoting natural products for their purported beneficial effects. Spirulina spp. and Aphanizomenon flos aquae are mainly used in BGAS production. They are usually collected from the natural environment, where other potentially toxic cyanobacteria can be present, making possible BGAS contamination by cyanotoxins, with potential risk for human health. In this work we apply a combined approach, by using chemical and molecular techniques, on BGAS of 17 brands available in Italy. Samples containing Spirulina-only were free of contamination. The Aphanizomenon flos aquae-based samples were contaminated by highly variable levels of microcystins (MC-LR and MC-LA congeners), up to 5.2μg MC-LR equivalents per gram product. The highest variability (up to 50 fold) was among batches of the same brand, although intra-batch differences were also evidenced. PCR analyses were positive only for the presence of Microcystis sp., identified as the toxin-producing species responsible for contamination. At the maximum contamination levels found, a risk for consumers can be expected following chronic or sub-chronic exposure to a reasonable daily BGAS consumption of 4g. The need for a strict monitoring by producers and Health Authority to assure an adequate protection for consumers is underscored.
There is mounting evidence that the cyanobacterial toxins, the microcystins, can act as tumor promoters. However, due to their requirement for active uptake by the cell, there have been few in vitro studies of the mechanism by which this might occur. Most of our understanding of this process has been deduced from experiments using the cell permeant okadaic acid, despite differences in the effects of these toxins. A cell culture system was developed in which nonmitogenically stimulated proliferating primary mouse hepatocytes could be exposed to microcystin-LR and the effects on various cell cycle parameters could be determined. It was found that cytokinesis was stimulated and the rate of apoptosis reduced by picomolar concentrations of microcystin-LR, whereas at higher (nanomolar) concentrations, cytokinesis appeared to be inhibited and cell death was induced. Cell killing was selective at these higher concentrations, favoring retention of a proliferatively active cohort of cells. The differences in effect between okadaic acid and microcystin-LR found by other researchers were confirmed, although in this system the effective concentrations of the toxins were approximately 100-fold lower than those reported previously. The mechanistic implications of these findings with regard to tumor promotion are discussed. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 61–75, 1999
Main Berlin waterways are lowland rivers Spree, Dahme, and Havel, which are connected to some shallow lakes. High nutrient concentrations support heavy blooms of cyanobacteria which produce toxic substances (“cyanotoxins”). Concentrations of microcystins (MC), anatoxin-a, chlorophyll-a, and cyanobacterial biomass were analyzed in surface water. Two methods [enzyme linked immunosorbert assay (ELISA) and high-pressure liquid chromatography (HPLC)] were used for MC analysis. Different preparation techniques were carried out determining dissolved and cell-bound microcystins. Furthermore, the health implication for people using lakes for swimming and recreation was assessed as a basis of a proposal to health and water authorities for further regulations. Concentrations of total (cell-bound and dissolved) microcystin (with ELISA) varied between 0.14 and 119 μg/L; 95 of 128 samples showed values above 1 μg/L (nearly 74%), 49 above 10 μg/L (nearly 38%), and 2 above 100 μg/L (nearly 2%). With wide variability, concentrations of microcystins measured with HPLC were 65% (median) of values obtained using the immunologic method. Compared to total microcystin concentrations (dissolved and cell-bound toxin) proportion of dissolved toxin varied from 0.3 to 12% (n=46; mean: 4%). Using animal toxicity data for risk assessment we assume that for children a single intake of 25 μg MC-LR has no adverse effects. Under conservative uptake assumption for contaminated water a concentration of 100 μg MC/L in surface water is safe. On this basis, regulations (as provisional orientation) are proposed to health and water authorities. Levels of anatoxin-a measured in 14 surface samples of two Berlin lakes (Unterhavel and Müggelsee) were under detection limit of 0.01 μg/L. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 120–130, 2000
A must-have health companion for herbalists, naturopaths, complementary medicine practitioners and students
"Recommended evidence-based reference on Complementary Medicines" National Pharmacy Board 2010
Herbs and Natural Supplements, 3rd Edition: An evidence-based guide presents evidence-based information on the 130 most popular herbs, nutrients and food supplements used across Australia and New Zealand.
This exhaustive textbook is organised alphabetically by each herb or nutrient's common name. Herbs and nutrients are then accompanied by critical information such as daily intake, main actions and indications, adverse reactions, contraindications and precautions, safety in pregnancy and more.
This new edition of Herbs and Natural Supplements has been expanded with new chapters on pregnancy and wellness.
It also features 10 new monographs for Arginine, Dunaliella, Elde, Goji, Pelargonium, Prebiotics, Red Yeast Rice, Rhodioloa, Shatavari and Taurine.
Spirulina is a microscopic filamentous alga that is rich in proteins, vitamins, essential amino acids, minerals and essential fatty
acids like γ-linolenic acid (GLA). It is produced commercially and sold as a food supplement in health food stores around
the world. Up to very recently, the interest in Spirulina was mainly in its nutritive value. Currently, however, numerous people are looking into the possible therapeutic effects
of Spirulina. Many pre-clinical studies and a few clinical studies suggest several therapeutic effects ranging from reduction of cholesterol
and cancer to enhancing the immune system, increasing intestinal lactobacilli, reducing nephrotoxicity by heavy metals and
drugs and radiation protection. This paper presents a critical review of some published and unpublished data on therapeutic
effects of Spirulina.
Cyanobacteria-derived microcystin-leucine-arginine (MCLR), commonly characterized as a hepatotoxin, has recently been found to show neurotoxicity, but the exact mechanism is still unknown. To further our understanding of the neurotoxic effects of MCLR and the mechanisms behind it, we used two-dimensional gel electrophoresis and mass spectrometry analysis to identify global protein profiles associated with MCLR-induced neurotoxicity. MCLR-treated hippocampi showed alterations in proteins involved in cytoskeleton, neurodegenerative disease, oxidative stress, apoptosis, and energy metabolism. After validation by Western blot and quantitative real-time PCR, the expressions of three proteins related to neurodegenerative disease, septin 5, α-internexin, and α-synuclein, were identified to be altered by MCLR exposure. Based on our proteomic analysis that MCLR toxicity might be linked to neurodegeneration, we examined the activity of serine/threonine-specific protein phosphatases (PPs), which are markers of neurodegenerative disease. MCLR was found to induce inhibition of PPs and abnormal hyperphosphorylation of the neuronal microtubule-associated protein tau. This was found to lead to impairment of learning and memory, accompanied by severe histological damage and neuronal apoptosis in the hippocampal CA1 regions of rats. Our results support the hypothesis that MCLR could induce neurotoxic effects, the reason for which could be attributed to the disruption of the cytoskeleton, oxidative stress, and inhibition of PPs in the hippocampus. Moreover, MCLR was found to induce tau hyperphosphorylation, spatial memory impairment, neuronal degenerative changes, and apoptosis, suggesting that this cyanotoxin may contribute to Alzheimer's disease in humans.
Aphanizomenon flos-aquae (A. flos-aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). The LD(50) of the aphantoxin preparation was determined to be 11.33 μg/kg (7.75 μg saxitoxin equivalents (STXeq) per kg) following intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos-aquae DC-1 toxins 7.73 and 9.28 μg /kg (5.3 and 6.4 μg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT-PCR at different timepoints postinjection. Low-dose aphantoxin exposure was associated with chromatin condensation, cell-membrane blebbing, and the appearance of apoptotic bodies. High-dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and necrosis. At later timepoints apoptosis appeared to predominate in the low-dose group, whereas necrosis predominated in the high-dose group. RT-PCR revealed that mRNA levels of the apoptosis-related genes encoding p53, Bax, caspase-3, and c-Jun were upregulated after aphantoxin exposure, but there was no evidence of DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing necrosis. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2011.
A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg(-1). The detection capability (CCβ) of the assay was determined to be ≤ 0.85 mg kg(-1) for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg(-1). The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.
Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ∼70-80% when HepG2 cells were incubated with 0-500 μg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0%, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects.
Cyanobacterial bloom was observed in a highly eutrophic dam reservoir, Zemborzycki, near Lublin (SE Poland) over a warm period in the year 2007. The water bloom consisted of several cyanobacterial taxa: Anabaena circinalis, Anabaena spiroides, Anabaena flos-aquae, Planktothrix agardhii, Aphanizomenon flos-aquae, Aphanizomenon gracile, and Microcystis flos-aquae. Anabaena spp., and Aphanizomenon spp., potential producers of neurotoxic anatoxin-a, quantitatively predominated in the studied bloom. High-performance liquid chromatography (HPLC) analysis of surface scum sampled during Anabaena circinalis domination revealed the presence of anatoxin-a at a high concentration (1,035.59 microg per liter of surface scum). At the same time, neither gas chromatography/mass spectrometry (GC/MS) nor microcystin enzyme-linked immunosorbent assay (ELISA) test showed the presence of other frequently found cyanotoxins, microcystins. Toxicity of cyanobacterial bloom was assessed by the crustacean acute toxicity test Daphtoxkit F pulex using Daphnia pulex, and by the chronic toxicity test Protoxkit F with a ciliate protozoan Tetrahymena thermophila. The crude extract of cyanobacterial scum showed high toxicity for Daphnia pulex, with 24-h median effective concentration (EC50) value of 90.3 microg/L of anatoxin-a, which corresponded to the cyanobacterial density in the scum of 1.01 g dry weight/L. For Tetrahymena thermophila, 24-h EC50 was lower, evaluated to be 60.48 microg/L of anatoxin-a, which corresponded to a cyanobacterial density of 0.68 g dry weight/L of the scum. On the basis of evaluated toxicity units, the cyanobacterial extract was classified at class IV toxicity, which means high toxic hazard.
The neurotoxin anatoxin-a (ATX), has been detected in several northeast German lakes during the last two decades, but no ATX producers have been identified in German water bodies so far. In 2007 and 2008, we analyzed phytoplankton composition and ATX concentration in Lake Stolpsee (NE Germany) in order to identify ATX producers. Sixty-one Aphanizomenon spp. strains were isolated, morphologically and phylogenetically characterized, and tested for ATX production potential by liquid chromatography-tandem mass spectrometry (LC-MS/MS). New primers were specifically designed to identify a fragment of a polyketide synthase gene putatively involved in ATX synthesis and tested on all 61 Aphanizomenon spp. strains from L. Stolpsee and 92 non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from German lakes Langersee, Melangsee and Scharmützelsee. As demonstrated by LC-MS/MS, ATX concentrations in L. Stolpsee were undetectable in 2007 and ranged from 0.01 to 0.12 microg l(-1) in 2008. Fifty-nine of the 61 strains isolated were classified as Aphanizomenon gracile and two as Aphanizomenon issatschenkoi. One A. issatschenkoi strain was found to produce ATX at concentrations of 2354+/-273 microg g(-1) fresh weight, whereas the other A. issatschenkoi strain and A. gracile strains tested negative. The polyketide synthase gene putatively involved in ATX biosynthesis was found in the ATX-producing A. issatschenkoi strain from L. Stolpsee but not in the non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from lakes Stolpsee, Langersee, Melangsee, and Scharmützelsee. This study is the first confirming A. issatschenkoi as an ATX producer in German water bodies.
Wasserverunreinigungen durch toxische Cyanobakterien sind ein weltweit wachsendes Problem, das ein ernstes Risiko für Menschen darstellt. Am wahrscheinlichsten sind humane Vergiftungen möglich durch chronische Vergiftungen mit niedrigen Toxinkonzentrationen, zum Beispiel durch Kontaminationen von Trinkwasser, Nahrung (Fisch, Garnelen, Nahrungsergänzungsmittel auf Blau-Grün Algen Basis) oder durch Freizeitaktivitäten in Gewässern, die ein Massenvorkommen von toxischen Cyanobakterien aufweisen. Microcystine (MC) repräsentieren die am häufigsten vorkommende Gruppe von zyklischen Heptapeptiden mit mehr als 80 verschiedenen strukturellen Varianten (Kongenere). Es konnte gezeigt werden, dass MC-LR im Vergleich zu anderen Kongeneren sowohl in vitro als auch in vivo toxisch ist, wobei momentan vermutet wird, dass der Mechanismus der Toxizität hauptsächlich von der spezifischen und irreversiblen Inhibition von Serin/Threonin Protein Phosphatasen (PP) herrührt. Durch ihre chemische Struktur und Größe ist es MC nicht möglich, durch Zellmembranen hindurch zu diffundieren, sondern sie benötigen hierfür Transporter, sogenannte organic anion transporting polypeptides (Nager Oatp / Mensch OATP). Des Weiteren besitzen nicht alle Oatps/OATPs dieselbe Affinität und Kapazität für all diejenigen MC Kongenere, die während einer Cyanoblüte auftreten können. Deshalb ist die Verteilung, d.h. die Toxikokinetik von einzelnen bzw. mehreren Kongeneren, absolut abhängig von der spezifischen Expression von Oatps/OATPs im Gewebe und in dessen Zellen. Erste Anhaltspunkte nach humanen und tierischen (Wild- und Haustiere) MC Intoxikationen deuten auf eine Hepato-, Nephro- und Neurotoxizität hin. Tatsächlich findet man Oatps/OATPs in Hepatozyten und kortikalen Epithelzellen der Niere, die auch einzelne MC Kongenere transportieren können. Um eine Neurotoxizität induzieren zu können, sollten daher Oatps/OATPs nicht nur in der Blut-Hirn-Schranke, sondern auch in neuronalen Membranen exprimiert werden. Ziel dieser Arbeit war es zu untersuchen, ob MC potente Neurotoxine darstellen. Hauptaugenmerk wurde auf eine Neuronen spezifische sowie MC Kongener abhängige Toxizität gelegt. In ersten in vitro Versuchen mit primären murinen Whole Brain Cells (mWBCs) sollten Anhaltspunkte gesammelt werden, die auf eine generelle MC Kongener abhängige Neurotoxizität hindeuten. Für eine genauere Abschätzung sollte in weiteren Versuchen mit primären murinen Cerebellar Granule Neurons (mCGNs), d.h. primäre Neurone, die Präsenz von mOatps, ein MC Kongener abhängiger Transport, PP Inhibition sowie eine in Folge auftretende Neuritendegradation und Cytotoxizität (Nekrose/Apoptose) untersucht werden. Abschließend sollte ein erster in vivo Versuch mit Mäusen die beobachteten in vitro Effekte bestätigen. Zusammengefaßt zeigen die Daten eine mOatp abhängige Verteilung von MCs in mWBCs und mCGNs sowie einen MC Kongener anhängigen Cytotoxizitäts-Mechanismus, der MC-LF als den potentesten neurotoxischen MC Kongener darstellt. Da einzelne MC Kongenere sehr stark in ihrem neurotoxischen Potential variierten, sollte die momentane Risikobewertung, die nur auf MC-LR beruht, neu überarbeitet werden. Dies ist von höchster Wichtigkeit, da MC-LF häufig in Oberflächengewässern einer auftretenden Cyanoblüte zu finden ist. Folglich zeigen die Daten dieser Studie das Vorkommen eines neuen bis jetzt noch nicht in Betracht gezogenen Risikos durch MC für Mensch und Tier. Contamination of natural waters by toxic cyanobacteria is a growing worldwide problem, representing serious risks to public health. Human poisonings have been associated with chronic exposure to low toxin concentrations via drinking water, contaminated food (e.g. fish, prawns, Blue-Green Algae Supplements) or as a result of recreational water activities in surface waters with toxic cyanobacteria blooms. Microcystins (MCs) are the most commonly found group of cyclic heptapeptide cyanotoxins with more than 80 structural variants (congeners). MC-LR, in comparison to other MC congeners, was demonstrated to be toxic in vitro and in vivo, whereby the mechanism of toxicity (toxicodynamics) is currently assumed to primarily stem from the specific and irreversible inhibition of serine/threonine protein phosphatases (PPs). However, due to their structure and size, MCs cannot penetrate cell membranes by simple diffusion but rather require organic anion transporting polypeptides (rodent Oatp / human OATP) for an active uptake. Moreover, not all Oatps/OATPs have an identical affinity and capacity for MC congeners present in an actual cyanobacterial bloom situation. Therefore, the distribution i.e. toxicokinetics of an individual MC congener(s) appear to entirely depend on the tissue and cell-type inherent expression of specific Oatp/OATP transporters. Initial evidence from human and animal (wild and domestic) MC intoxications suggests that MCs can elicit hepato-, nephro- as well as neurotoxicity. Indeed, the presence of Oatp/OATP in hepatocytes and renal cortical epithelial cells have been demonstrated and also shown to transport some of the MC congeners. Consequently, OATPs/Oatps should be present not only at the blood-brain barrier, but also within the neuronal membrane in order that MCs can induce the assumed neurotoxicity. The overall goal of this work was to investigate whether or not MCs represent potent neurotoxins in vitro and in vivo with a focus on neuron-specific toxicity induced by three different MC congeners. During initial in vitro experiments using primary murine Whole Brain Cells (mWBCs) preliminary evidence for single MC congener dependent general neurotoxicity should be assessed. For a more refined assessment of neurotoxicity, primary murine Cerebellar Granule Neurons (mCGNs), i.e. primary neurons, should allow determination of the presence of mOatps, MC congener- dependent uptake, and ensuing PP inhibition, neurite degeneration and cytotoxicity (necrosis/apoptosis). Finally, a first in vivo experiment with mice should allow confirmation of the effects observed in vitro. In summary the data suggest a mOatp dependent distribution of MCs in mWBCs and mCGNs as well as a MC congener dependent mechanism of cytotoxicity, pointing to MC-LF as being the most potent MC for potential neurotoxic effects. As individual MC congeners differ strongly in their potential neurotoxicity, the current risk assessment, based solely on MC-LR, may need a revision. The latter is of utmost importance, as the most potent potential neurotoxic MC, MC-LF, occurs with regularly in cyanobacterial blooms of surface waters. Thus, the results of these studies suggest the presence of a new and not yet carefully considered hazard for humans and animals.
Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.
In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.14.72 μg g−1 (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria.
A single filament clonal isolate of Aphanizomenon flos-aquae was made from a water bloom sample taken at a small pond near Durham, New Hampshire, in 1980. When batch cultured the strain was toxic to mice and had an i.p. LD50 of about 5.0 mg/kg. Using an extraction procedure originally designed for paralytic shellfish poisons and other neurotoxins of freshwater cyanobacteria, a purification method was developed. The procedure involved acidified water/ethanol extraction of the cells followed by ultrafiltration, gel filtration, use of C18 cartridges to remove pigments, ion-exchange and high performance liquid chromatography using u.v. detection at 220 or 240 nm. Thin-layer chromatography and high performance liquid chromatography results indicate that Aphanizomenon flos-aquae NH-5 may produce paralytic shellfish poisons, mainly neo-saxitoxin and saxitoxin. Three labile toxins were also detected which were not similar to any of the known paralytic shellfish poisons.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Seafood poisoning has been recognized as a problem in both coastal and inland populations for millennia. Many types of sea creatures from shellfish to the largest fish have been implicated. Severe cases of many different types of seafood poisonings can result in fatalities. While the pathophysiology of the toxins is well known in some cases, others, like ciguatera, remain somewhat confusing. As a result, the treatment of these conditions remains controversial, although supportive care continues to be the mainstay of therapy. In this manuscript, we review the pathophysiology, clinical presentation, and treatment of some of the most common and toxic varieties of seafood poisoning resulting from toxins.
The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.
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Four different cell models were chosen for comparison of OTA and OTB toxicity: primary porcine (PKC), rat (RPTC) and human renal proximal epithelial cells (HKC) from both sexes and a porcine renal cell line: LLC-PK1. Culture conditions were tested and optimized for each respective cell type (species/sex and origin). All cell types were characterized for epithelial origin and growth patterns and following optimization of dosing strategies and assay procedures, a strict study design was implemented to avoid systemic variations. Due to possible sensitivity differences, three simple endpoints were chosen to provide basic data for interspecies comparison: neutral red uptake, MTT reduction and cell number. Of the endpoints tested neutral red appeared the most sensitive, although all three parameters yielded comparable EC50's. Sex-differences were observed between male and female HKC cells following 96 h exposure to OTA, with HKC(m) being more sensitive than HKC(f). No sex-difference was observed in PKC cells, however, the PKC were approximately 3 and 10 times more sensitive than HKC(m) and HKC(f), respectively, to OTA and OTB. Interestingly, the CI95 of the EC50 values obtained for OTA (15.5-16.5 microM) and OTB (17.0-2 1.0 microM) were comparable in the PKC cells. In contrast, OTB had lower cytotoxicity than OTA in HKC and LLC-PK1 (approx. 2-fold) and no effects in RPTC. Overall, HKC(m) were nearly as sensitive as PKC towards OTA, followed by RPTC, LLC-PK1 and HKC(f), thus suggesting a sex specific sensitivity in humans towards OTA induced cytotoxicity.
A colorimetric phosphatase inhibition assay using protein phosphatase 2A and p-nitrophenyl phosphate as substrate for determining cyanobacterial peptide hepatotoxins directly in water without sample preconcentration has been developed. The assay uses commercially available materials and is much more simple to use than similar procedures using radiolabelled substrates. It has similar sensitivity to the radiolabelled assays and, with a working range of around 0.2-1 microg/L, is able to determine these toxins at concentrations below the provisional World Health Organisation guideline of 1 microg/L for microcystin-LR. The method appears robust and not to be affected by the sample matrix apart from possibly some components of cellular material if present at very high levels in extracts of cyanobacterial material. It is not affected by the presence of low levels of methanol in sample extracts.
Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).
Water samples taken from 93 freshwater and brackish water locations in Aland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography-mass spectrometry (LC-MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC-UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS-MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 microg l(-1) were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 microg l(-1). The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC-UV were generally in good agreement with the LC-MS SIR results.
This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcystins and compares this to the guidance values proposed for microcystins in water by the World Health Organization, and for blue-green algal food supplements by the Oregon State Department of Health. The basis of the risk assessment underlying these guidance values is viewed as being critical due to overt deficiencies in the data used for its generation: (i) use of one microcystin congener only (microcystin-LR), while the other presently known nearly 80 congeners are largely disregarded, (ii) new knowledge regarding potential neuro and renal toxicity of microcystins in humans and (iii) the inadequacies of assessing realistic microcystin exposures in humans and especially in children via blue-green algal food supplements. In reiterating the state-of-the-art toxicology database on microcystins and in the light of new data on the high degree of toxin contamination of algal food supplements, this review clearly demonstrates the need for improved kinetic data of microcystins in humans and for discussion concerning uncertainty factors, which may result in a lowering of the present guidance values and an increased routine control of water bodies and food supplements for toxin contamination. Similar to the approach taken previously by authorities for dioxin or PCB risk assessment, the use of a toxin equivalent approach to the risk assessment of microcystins is proposed.
Three single-filament isolates of Aphanizomenon flos-aquae from two German lakes were found to produce remarkable amounts of the cyanobacterial hepatotoxin cylindrospermopsin (CYN). CYN-synthesis of the strains were evidenced both by LC-MS/MS analysis and detection of PCR products of gene fragments which are implicated in the biosynthesis of the toxin. The strains contain CYN in the range of 2.3-6.6 mg g(-1) of cellular dry weight. To our knowledge this is the first report of CYN in A. flos-aquae.
The frequent occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in the (sub)tropics has been largely associated with cyanobacteria of the order Nostocales of tropical origin, in particular Cylindrospermopsis raciborskii. C. raciborskii is currently observed to spread northwards into temperate climatic zones. In addition, further cyanobacteria of the order Nostocales typically inhabiting water bodies in temperate regions are being identified as CYN-producers. Therefore, data on the distribution of CYN in temperate regions are necessary for a first assessment of potential risks due to CYN in water used for drinking and recreation. A total of 127 lakes situated in the north-eastern part of Germany were investigated in 2004 for the presence of the toxin CYN and the phytoplankton composition. The toxin could be detected in half of the lakes (n = 63) and in half of 165 samples (n = 88). Concentrations reached up to 73.2 microg CYN/g DW. CYN thus proved more widely distributed than previously demonstrated. The analyses of phytoplankton data suggest Aphanizomenon sp. and Anabaena sp. as important CYN producers in Germany, and confirm recent findings of Aphanizomenon flos-aquae as CYN-producing species frequently inhabiting water bodies in temperate climatic regions. The data shown here suggest that CYN may be an important cyanobacterial toxin in German water bodies and that further data are needed to assess this.
Cyanobacterial blooms in New Zealand surface water resources have been surveyed and, in response to strict new standards for drinking water, more intensive monitoring for cyanotoxins has been initiated. Aphanizomenon issatschenkoi was recently identified in a New Zealand lake and was found to produce the potent neurotoxin anatoxin-a (ATX). A strain of Aph. issatschenkoi (CAWBG02) was cultured for ATX production and a novel derivative of ATX was found to account for a high proportion of the toxin content in the Aph. issatschenkoi cells. Spectroscopic data (LC-UV, liquid chromatography with ultraviolet absorption detection; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-HRMS, liquid chromatography with high resolution mass spectrometry) identified this derivative as 11-carboxyl anatoxin-a. Although precursors with a carboxyl group on C11 have been postulated in the biosynthetic pathway for ATX from amino acids and acetate, this is the first identification of a specific intermediate. The production of ATX and the intermediate by Aph. issatschenkoi was studied under different growth conditions. Concentrations of ATX and the intermediate increased in the aerated culture to 170 microg/L and 330 microg/L, respectively, at 21 days (18 x 10(9) cells/L). Cell concentrations did not markedly increase during subsequent growth to 37 days. ATX concentrations decreased, and 11-carboxyl ATX concentrations continued to increase during this period. Toxin production by Aph. issatschenkoi cells was maximal at 6 days of growth (0.08-0.09 pg/cell each; 2.3 x 10(8) cells/L). Other ATX analogues and metabolites were not detected in the cultures. Freeze-thawing of cultures resulted in complete conversion of the intermediate to ATX with a half-life of 5 min, and this conversion was inhibited by acidification, heating of the culture to 100 degrees C, or addition of methanol. The implications of the findings for mechanisms of biosynthesis of anatoxins by cyanobacteria and for monitoring of water bodies for cyanotoxins are discussed.