No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
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 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.
... They are considered hepatotoxins associated with many human and animal intoxications, mostly when an algal supplement has been cross-contaminated. Some representatives of this family have been detected [85][86][87][88]. ...
... Although there are specific detection methods for this group, their presence in algal supplements has not been reported [99]. Just toxins from the groups of microcystin and anatoxin-a were demonstrated to be present in samples containing spirulina or Aphanizomenon flos-aquae [85][86][87][95][96][97]. ...
... Some other dietary supplements contain mixtures of species on purpose. Those in which Chlorella sp. or Arthrospira sp. were mixed with Aphanizomenon flos-aquae or were Aphanizomenon flos-aquae-based showed cytotoxicity and/or presence of microcystins [85,87]. ...
With the increase in global population, getting new sources of food is essential. One of the solutions can be found in the oceans due to algae. Microalgae are aquatic photosynthetic organisms largely used due to their variety of bioactive compounds. The consumption of microalgae has been carried out for centuries and is recommended by organizations such as OMS and FAO due to its nutritional value and its properties. Based on the existing literature, there is substantial evidence of the nutritional quality of the algae as well as their functional elements. However, much quantification is still necessary as well as studying possible adverse effects. The present review describes the compounds of alimentary interest present in these algae as well as different extraction techniques assisted by different energetic mechanisms (such as heat, supercritical-fluid, microwave, ultrasound, enzymes, electric field, high hydrostatic pressure, among others). The most challenging and crucial issues are reducing microalgae growth cost and optimizing extraction techniques. This review aimed a better understanding of the uses of microalgae for new researches in nutrition. Because of the use of microalgae is still a field in which there is much to discover, it is likely that more benefits will be found in its consumption.
... The consumption of FS of marine origin has also increased over the past decades due to their perceived health benefits [68,89]. Previous studies have detected cyanotoxins (i.e. ...
... Previous studies have detected cyanotoxins (i.e. toxins produced by certain species of cyanobacteria), arising from the contamination of marine raw materials used in these FS [89,90]. ...
... For example, Aphanizomenon flos-aquae is a cyanobacterium commonly used in blue-green algae (BGA) supplements. One relevant source of this BGA is the Upper Klamath Lake (Oregon, USA), where the toxinogenic cyanobacterium Microcystis aeruginosa occurs regularly, allowing the contamination of BGA products during harvesting [89]. The culture conditions commonly used to produce the BGA Spirulina and the green algae Chlorella make these raw materials less likely to be contaminated with unwanted cyanobacterial species [89]. ...
Food supplements (FS) are often consumed as one of the strategies to fight ageing-associated pathologies, especially in the case of oxidative stress-related diseases. Despite the popularity of FS, some concerns about their quality and safety have been raised, especially regarding the presence of contaminants. This paper reviews and discusses the occurrence of contaminants in marketed samples of FS in the last two decades, considering both scientific literature and notifications registered on RASFF portal. The most relevant classes of contaminants were included namely metals, toxins, pesticides, dioxins and PCBs, as well as pharmacologically active ingredients. Variable amounts of contaminants were reported in a significant number of commercially available FS. Although the presence of contaminants does not necessarily mean that their levels exceed the regulatory limits or that the FS intake constitutes a risk to human health, it alerts for the need to further monitor FS safety. The evaluation of the risk associated to the consumption of FS, especially in the elderly population, is particularly challenging due to the frequent exposure to multiple toxicants and to different exposure sources, as well as due to possible pre-existing diseases and respective therapeutics. Therefore, improved quality control procedures and monitoring programs should be pursued in order to avoid undesirable products and assure the safety of FS.
... In 2012, Heussner et al. analyzed 13 commercially available BGAS in Germany for the presence of cyanotoxins. All samples were analyzed and confirmed by PCR for the presence of the mcyE gene, a part of the MC and nodularin gene cluster [11]. Of all products tested, ten consisted of AFA, five of Spirulina platensis, and three of Chlorella pyrenoidosa [59]. ...
... Spirulina spp. are generally regarded as safe within the BGAS industry as of this time [11]. Only AFA BGAS products tested positive for MCs as well as the presence of the mcyE gene [11]. ...
... are generally regarded as safe within the BGAS industry as of this time [11]. Only AFA BGAS products tested positive for MCs as well as the presence of the mcyE gene [11]. In 2015, a confirmed case of MC poisoning in a dog was documented following the consumption of a BGAS containing organic AFA daily for just 3 weeks [9]. ...
Previous studies of recreational waters and blue-green algae supplements (BGAS) demonstrated co-occurrence of Aphanizomenon flos-aquae (AFA) and cyanotoxins, presenting exposure risk. The authors conducted a systematic literature review using a GRADE PRISMA-p 27-item checklist to assess the evidence for toxigenicity of AFA in both fresh waters and BGAS. Studies have shown AFA can produce significant levels of cylindrospermopsin and saxitoxin in fresh waters. Toxicity studies evaluating AFA-based BGAS found some products carried the mcyE gene and tested positive for microcystins at levels ≤ 1 μg microcystin (MC)-LR equivalents/g dry weight. Further analysis discovered BGAS samples had cyanotoxins levels exceeding tolerable daily intake values. There is evidence that Aphanizomenon spp. are toxin producers and AFA has toxigenic genes such as mcyE that could lead to the production of MC under the right environmental conditions. Regardless of this ability, AFA commonly co-occur with known MC producers, which may contaminate BGAS. Toxin production by cyanobacteria is a health concern for both recreational water users and BGAS consumers. Recommendations include: limit harvesting of AFA to months when toxicity is lowest, include AFA in cell counts during visible blooms, and properly identify cyanobacteria species using 16S rRNA methods when toxicity levels are higher than advisory levels.
... Due to this potential issue of MC contamination in AFA raw materials from UKL, the State of Oregon's Health Division estimated a safe concentration of 1 mg MC per g dry weight for BGA products consumed by adults, which the Oregon Department of Agriculture adopted as a regulatory standard for products sold in Oregon in 1997 (Gilroy et al., 2000). Despite this limit, researchers in Canada (Lawrence et al., 2001), Germany (Heussner et al., 2012), and Italy (Vichi et al., 2012) reported commercially available BGA products, containing AFA from UKL, with MC at or above the 1 mg/g limit. The primary objective of this study was to evaluate the presence and concentrations of MC in AFA-containing BGA dietary supplements currently available to U.S. consumers. ...
... Numerous methods have been employed for screening or confirmation of MC contamination in BGA-containing dietary supplement products including enzyme-linked immunosorbent assays (ELISAs) typically targeting the conserved Adda group, antibody-based surface plasmon resonance (SPR) biosensors, DNA-based polymerase chain reaction (PCR) assays screening for the presence of MC producing genes (i.e., screening for the potential for MC production), and chemical confirmatory methods such as Liquid Chromatography with UV detection (LC-UV) and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) (Lawrence et al., 2001;Heussner et al., 2012;Vichi et al., 2012;Parker et al., 2015;Yakes et al., 2015). The mechanism of hepatotoxicity of MC to mammals involves the potent inhibition of protein phosphatases comprising PP1, PP2A, and related enzymes, which dephosphorylate serine, threonine, and histidine residues in proteins and are critical to the control of several biological processes (MacKintosh et al., 1995). ...
... This bioactivity has been exploited to develop in-vitro assays based on protein phosphatase inhibition measuring the inhibitory effects of MC on either PP1 or PP2A towards p-nitrophenyl phosphate (An and Carmichael, 1994;Heresztyn and Nicholson, 2001). These Protein Phosphatase Inhibition Assays (PPIAs) have also been used successfully to monitor BGA dietary supplements for MC contamination (Heussner et al., 2012), but to date the lack of a verified commercially available PPIA test kit has limited the standardization required for the routine use of PPIA assays as a regulatory monitoring tool. Furthermore, most of the existing rapid monitoring tools for the detection of MC, including PPIAs, were designed for the testing of drinking and source waters with calibration at either the World Health Organization (WHO) provisional guideline value of 1 mg/L MC in drinking water (WHO, 1998), or at the recreational water exposure response guidelines that have been adopted by several U.S. states [range 4e20 mg/L] (https://www.epa.gov/nutrientpolicy-data/guidelines-and-recommendations) ...
The cyanobacterium Aphanizomenon flos-aquae (AFA), from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA) dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 μg/g microcystin (MC) for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP) regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 μg/g. The objectives of this study were (1) identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2) use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA), based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51) distinct AFA-BGA products had MC ≥0.25 μg/g (the detection limit of the kit), 10 products had MC concentrations between 0.5 and 1.0 μg/g, and 4 products exceeded the limit (1.1–2.8 μg/g). LC-MS/MS confirmed PPIA results ≥0.5 μg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 μg/g MC, most were at or below 1.0 μg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC contamination found.
... 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.
... Additionally, in the recent years, consumption of food supplements produced from dried cyanobacterial bloom material (BGAS, blue-green algal supplements) increased. Unfortunately, these are often contaminated with toxin-producing cyanobacterial species (Bautista et al. 2015;Gilroy et al. 2000;Heussner et al. 2012;Vichi et al. 2012). This is due to the manufacturing process, which involves harvesting bloom material of commonly Aphanizomenon flos-aqua (AFA), drying and then pressing into tablets without further analysis of co-occurring species or toxins. ...
... Blue-green algal supplements (BGAS) can contain MCs (Gilroy et al. 2000;Heussner et al. 2012;Lyon-Colbert et al. 2018;Roy-Lachapelle et al. 2017;Vichi et al. 2012), sometimes above the guidance value of 1 µg/g dw (dry weight) given by the Oregon Health department (Gilroy et al. 2000) or the 10 µg/g dw given by Dietrich and Hoeger (2005). Thus, the consumption of BGAS can pose a threat to humans, especially as they are marketed for many purposes, to all kinds of customers including children and manufacturers encourage taking many grams per day. ...
... The meat substitute Quorn™, which is derived from the filamentous fungus Fusarium venenatum, has been commercially available in the United Kingdom since 1985 and is now sold in many countries world-wide (Finnigan et al. 2017). Spirulina is marketed as a nutritional supplement rather than a food product because of concerns regarding the introduction of biotic and abiotic contaminants during its production (Heussner et al. 2012). ...
... Photosynthetic microorganisms such as cyanobacteria and eukaryotic microalgae require access to light and are therefore often cultivated in open ponds. As a consequence, such open systems are more vulnerable to contamination from particulate matter, other undesirable (and potentially toxin-producing) photosynthetic microorganisms (Heussner et al. 2012) in addition to algal parasites such as viruses as well as predatory zooplankton (Wang et al. 2013). Closed cultivation systems for photosynthetic microorganisms can counter these problems but result in increased capital costs. ...
Edible microbial biomass derived from bacteria, yeasts, filamentous fungi or microalgae is a promising alternative to conventional sources of food and feed. Microorganisms are a good source of protein, vitamins and, in some cases, also contain beneficial lipids. The ability of microorganisms to use simple organic substrates for growth permits industrial-scale cultivation of edible microbial biomass in geographical locations that would not compete with agricultural production. Only a handful of microbial products are currently available for human consumption. The use of microbial biomass for animal feed is limited by access to low-cost growth substrates and competition from conventional feed sources such as soy and fishmeal. At a time when the global food production system is threatened by the effects of climate change, the production of edible microorganisms has the potential to circumvent many of the current environmental boundaries of food production as well as reducing its environmental impact. Photosynthetic microorganisms such as cyanobacteria and microalgae can be cultivated for food and feed independently of arable land. In addition, recent technological developments in atmospheric carbon dioxide (CO 2) capture, extraction and catalytic conversion into simple organic compounds can be used for cultivation of edible microbial biomass for food and feed in a manner that is wholly independent of photosynthesis. The future possibilities, challenges and risks of scaled-up production of edible microbial biomass in relation to the global food system and the environment are discussed.
... efficiency, and results in higher detection limits [16]. Alternatively, other multi-toxin screening methods obtain successful retention and separation of the polar cyanotoxins, ANA, CLD, and SAX using hydrophilic interaction liquid chromatography (HILIC) [16,[19][20][21][22]. The advantage of HILIC includes functionality similar to traditional normal phase chromatography with the compatibility of solvents suitable for RPLC, allowing the same mobile phases to be used for both separation techniques [23]. ...
... Initial development of a HILIC separation method was performed on a TSKgel amide-80 due to previous reports and used for ANA, ANA-13 C, CLD, CLD-15 N, and SAX [16, 19-23, 39, 40]. Complete separation of ANA and ANA-13 C, DPA-d5, CLD and CLD-15 N, and SAX was achieved using HILIC ( Figure S2), solving the issue of ANA and DPA coelution [13,18], and providing excellent retention of the polar cyanotoxins, similar to previous results [16,[19][20][21][22]. Further, in agreement with previous work, the current study found that target analyte peak shape and retention when using HILIC were greatly affected by mobile phase buffer conditions [16]; thus, a previously reported optimized buffer with 5mM NH 4 OOCH and 3.6 mM HCOOH (pH 3.7) was utilized [19]. ...
Cyanobacteria can form dense blooms under specific environmental conditions, and some species produce secondary metabolites known as cyanotoxins, which present significant risks to public health and the environment. Identifying toxins produced by cyanobacteria present in surface water and fish is critical to ensuring high quality food and water for consumption, and protectionn of recreational uses. Current analytical screening methods typically focus on one class of cyanotoxins in a single matrix and rarely include saxitoxin. Thus, a cross-class screening method for microcystins, nodularin, anatoxin-a, cylindrospermopsin, and saxitoxin was developed to examine target analytes in environmental water and fish tissue. This was done, due to the broad range of cyanotoxin physicochemical properties, by pairing two extraction and separation techniques to improve isolation and detection. For the first time a zwitterionic hydrophilic interaction liquid chromatography column was evaluated to separate anatoxin-a, cylindrospermopsin, and saxitoxin, demonstrating greater sensitivity for all three compounds over previous techniques. Further, the method for microcystins, nodularin, anatoxin-a, and cylindrospermopsin were validated using isotopically labeled internal standards, again for the first time, resulting in improved compensation for recovery bias and matrix suppression. Optimized extractions for water and fish tissue can be extended to other congeners in the future. These improved separation and isotope dilution techniques are a launching point for more complex, non-targeted analyses, with preliminary targeted screening.
... Yet, every so often appears an article that raises concerns about the possible toxicity of microalgae, and particularly of the cyanobacterium Aphanizomenon flos aquae (AFA) from Klamath Lake, Oregon, USA, due to microcystins contamination. In 2012, for example, 2 articles were published, one from researchers of the Italian Istituto Superiore di Sanità [1], and a second one from a German University team [2]. Both of them report that some Klamath algae products tested were above the limit of 1 μg/g of microcystins, the safety limit established by the Government of Oregon for the cyanobacterial supplements, and this is supposed to constitute some kind of danger, even though the authors never tested the actual toxicity of such cyanobacterial supplements, and only took for granted the 1 μg/L of the WHO Guideline, and its automatic application to microalgae at 1 μg/g. ...
... At this level, microcystins would have remained a small toxicological problem, with a significantly reduced alarm and public expenditure in this area, as the current level of microcystins in water was found to be "…from below 1.0 μg MC-LR equiv./L to more than 8.0 μg/L in raw water…" [16]. Instead, still today there are those who push towards standards even lower than 1 μg/L, while proposing new and more expensive microcystins tests [17,2]. ...
Microcystins have been the subject of increasingly alarming popular and scientific articles, which have taken as their unquestionable foundation the provisional Guideline of 1 μg/L established by the WHO Panel on microcystins levels in water, and mechanically translated by the Oregon government as 1 μg/g of Klamath Aphanizomenon flos aquae microalgae. This article underlines the significant limitations and ultimately scientific untenability of the WHO Guideline on microcystins in water, for being based on testing methodologies which may lead to a significant overestimation of the toxicity of microcystins. I propose criteria for the realization of new experimental studies on the toxicity of microcystins, based on the essential understanding that drinking water is contaminated by whole cyanobacterial microalgae rather than purified microcystins, while it is important to differentiate between water and cyanobacterial supplements. It is indeed a mistake to automatically apply standards that are proper for water to cyanobacterial supplements, as they have different concentrations of the antioxidant substances that inactivate or significantly reduce the toxicity of microcystins, a fact that also require that each cyanobacterial supplement be tested individually and through realistic testing methodologies.
... ADS are natural products that come in the form of capsules, pills or powders, marketed internationally, easily available and advertised as beneficial for health. ADS can help elevate mood, weight reduction, boost energy, increase alertness, detoxification and can be used at the pharmacological therapies as well (Roy-Lachapelle et al., 2017;Heussner et al., 2012). Due to these health benefits derived, ADS products are sold with limited quality control, assumed to be safe and may be consumed on daily basis without medical prescription. ...
... Researchers showed that ADS can impact negative health effects on consumers due to the detected MCs trace observed. The Spirulina fusiformis, Nostoc, and Aphanizomenon flos-aquae used in manufacturing ADS products have been identified as a potent producer of MCs at low concentrations (Roy-Lachapelle et al., 2017;Heussner et al., 2012;Gilroy et al., 2000). This implies people who consume ADS products are at a higher risk of being exposed to MCs. ...
... MC and Nod have also been reported to accumulate in species such as fish and bivalves, resulting in a potential risk to human health following consumption of contaminated fishery products and animals feeding on toxic filter feeders such as bivalve molluscs [15][16][17][18][19][20]. The popularity of food supplements prepared from blue-green algae, also raises the possibility of regular human consumption of products containing naturally occurring cyanotoxins [21,22]. ...
... Optimisation experiments demonstrated that excellent recoveries for all analytes in all four matrices could be achieved through use of 80% aqueous MeOH as the extraction solvent, thereby showing similar results to those determined previously for extraction of cell material using 70% aqueous methanolic extraction [8], 75% aqueous MeOH [42] and other solvent extraction approaches [44] without the need to use more complex extraction techniques such as SPE and immunoaffinity columns [39,[43][44][45][46][55][56][57]. For extraction of algal supplement tablet powder and shellfish tissue, a wide range of solvents and extraction techniques were tested, but again the single step dispersive extraction using 80% aqueous MeOH was found to provide optimum extraction of toxins from both matrix types prior to LC-MS/MS analysis, eliminating the need for more complex and time-consuming extraction and clean-up protocols including prolonged ultrasonication, solvent evaporation steps and SPE [21,28]. The use of 80% aqueous MeOH as the optimum extraction solvent was therefore in direct agreement with the solvent used for extraction of other tissue samples, including carp larvae [57] as well as the 75% MeOH solvent methods used for extraction of mussels and fish liver [36] and other fish tissues [38] and 90% MeOH for fish tissues, mussels and oysters [34]. ...
A simple, rapid UHPLC-MS/MS method has been developed and optimised for the quantitation of microcystins and nodularin in wide variety of sample matrices. Microcystin analogues targeted were MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, LC-LW, MC-YR, MC-WR, [Asp3] MC-LR, [Dha7] MC-LR, MC-HilR and MC-HtyR. Optimisation studies were conducted to develop a simple, quick and efficient extraction protocol without the need for complex pre-analysis concentration procedures, together with a rapid sub 5min chromatographic separation of toxins in shellfish and algal supplement tablet powders, as well as water and cyanobacterial bloom samples. Validation studies were undertaken on each matrix-analyte combination to the full method performance characteristics following international guidelines. The method was found to be specific and linear over the full calibration range. Method sensitivity in terms of limits of detection, quantitation and reporting were found to be significantly improved in comparison to LC-UV methods and applicable to the analysis of each of the four matrices. Overall, acceptable recoveries were determined for each of the matrices studied, with associated precision and within-laboratory reproducibility well within expected guidance limits. Results from the formalised ruggedness analysis of all available cyanotoxins, showed that the method was robust for all parameters investigated. The results presented here show that the optimised LC-MS/MS method for cyanotoxins is fit for the purpose of detection and quantitation of a range of microcystins and nodularin in shellfish, algal supplement tablet powder, water and cyanobacteria. The method provides a valuable early warning tool for the rapid, routine extraction and analysis of natural waters, cyanobacterial blooms, algal powders, food supplements and shellfish tissues, enabling monitoring labs to supplement traditional microscopy techniques and report toxicity results within a short timeframe of sample receipt. The new method, now accredited to ISO17025 standard, is simple, quick, applicable to multiple matrices and is highly suitable for use as a routine, high-throughout, fast turnaround regulatory monitoring tool.
... 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.
... 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.
... In some countries, a few cases of acute MC intoxication from drinking contaminated water (Funari and Testai, 2008) and through recreational activities (Hilborn et al., 2014) has been reported. Humans eat fish and other seafood (Testai et al., 2016), agricultural produce (Peuthert et al., 2007), and health supplements (Heussner et al., 2012) possibly contaminated with MCs daily. Remarkably, no reported fatalities due to MC exposure via an oral route has been reported. ...
The incidence of cyanobacterial blooms is increasing due to numerous factors, especially anthropogenic influences such as eutrophication and global warming. As cyanobacteria produce toxins that are hazardous to several organisms, the ecosystem, and also humans, there is a need to accrue and evaluate the current data to assess the risk posed to us and our environment. By understanding how and why these toxins are produced, we can attempt to eliminate the threat by either managing toxin production or to eradicate these hazards from surface waters and thus limiting exposure. This chapter specifically addresses the most commonly occurring group of cyanobacterial toxins, the microcystins. We discuss the adverse implication for the environment and the human exposure risk. We conclude the chapter by discussing possible countermeasures, focusing on the phytoremediation method called the Green Liver System.
... This indicates that these fractions possessed cytotoxic potency. The cytotoxicity of extracts of Chlorella and Spirulina food supplements were previously demonstrated in vitro in A549 cells, although none of the tested products contained detectable levels of cyanotoxins, such as microcystins, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin [48]. Case reports of hepatotoxicity associated with the use of food supplements based on Chlorella and Spirulina were also reported [11,49,50]. ...
Selected species of cyanobacteria and green algae have been reported to produce lipophilic polymethoxy-1-alkenes (PMAs) which were shown to exhibit in vivo teratogenicity. Considering that information on PMAs in Arthospira sp. (known commercially as Spirulina) and Chlorella sp. cultivated for food supplement production was essentially lacking, the present study screened Chlorella (n = 10) and Spirulina (n = 13) food supplements registered in the European Union. Mass spectrometry analysis of column fractionated extracts was performed. None of the four variants previously reported in some cyanobacteria and green algae, nor any potentially related structures were detected in the studied samples. Since the isolated lipophilic fractions contained various compounds, they were further screened for in vivo teratogenicity in Danio rerio embryo, and for the potential to induce oxidative stress and genotoxicity in the liver and neurotoxicity in the brain of adult zebrafish. None of the tested food supplements had detectable levels of PMAs or any potentially related structures. No teratogenicity was revealed except for spinal curvature induced by fractions obtained from two Chlorella products. Selected fractions revealed cytotoxicity as indicated by an increased level of reactive oxygen species, catalase activity, lipid peroxidation and increased frequency of DNA strand breaks in hepatic tissue. The majority (60%) of Chlorella fractions induced an increase in cholinesterase activity in zebrafish brain homogenate while exposure to 61.5% of Spirulina fractions was associated with its decrease. The present study confirms that Chlorella and Spirulina food supplements are free of teratogenic PMAs, although the observed in vivo toxicities raise questions regarding the quality of selected products.
... Arthrospira has a long history of human consumption, and has been thoroughly studied in toxicological models, with no results prompting concern for safety (de la Jara et al., 2018;Heussner, Mazija, Fastner, & Dietrich, 2012;Koru, 2012;Marles et al., 2011). It has received the Generally Recognised As Safe (GRAS) status for human consumption by the US Food & Drug Administration (GRAS Notice 000391; GRAS Notice 000394). ...
... Consumer oriented applications, such as biomedical, pharmaceutical, or food applications, are highly demanding in terms of the cytotoxic evaluation for new compounds [32]. Cytotoxic evaluation of seaweed-based compounds [33][34][35] is under-exploited in the literature, and given their significance, this study is of key importance. The anti-tumorigenic effects of compounds extracted from seaweeds were reported [36,37], but it is also highly important to study the effects of these ingredients on normal mammalian cells, an added-value of this study. ...
Seaweeds, which have been widely used for human consumption, are considered a potential source of biological compounds, where enzyme-assisted extraction can be an efficient method to obtain multifunctional extracts. Chemical characterization of Sargassum muticum and Osmundea pinnatifida extracts obtained by Alcalase and Viscozyme assisted extraction, respectively, showed an increment of macro/micro elements in comparison to the corresponding dry seaweeds, while the ratio of Na/K decreased in both extracts. Galactose, mannose, xylose, fucose, and glucuronic acid were the main monosaccharides (3.2-27.3 mg/glyophilized extract) present in variable molar ratios, whereas low free amino acids content and diversity (1.4-2.7 g/100gprotein) characterized both extracts. FTIR-ATR and 1H NMR spectra confirmed the presence of important polysaccharide structures in the extracts, namely fucoidans from S. muticum or agarans as sulfated polysaccharides from O. pinnatifida. No cytotoxicity against normal mammalian cells was observed from 0 to 4 mglyophilized extract/mL for both extracts. The comprehensive characterization of the composition and safety of these two extracts fulfils an important step towards their authorized application for nutritional and/or nutraceutical purposes.
... It was concluded that the quantity of MCs in these brands exceeded the recommended tolerable daily intake (TDI) for adults specified by the World Health Organization (exceeding TDI up to 683% for total MCs) (Roy-Lachapelle et al., 2017;WHO, 2011). The amino acid phenylalanine in spirulina causes brain damage -especially to people suffering from phenylketonuria (Habib, Parvin, Huntington, & Hasan, 2008;Heussner, Mazija, Fastner, & Dietrich, 2012;Robb-Nicholson, 2006). These toxins could be minimized if organic fertilizers are utilized in cultivating these algae. ...
A functional food is any food that has (a) specific nutrient(s) added to it for a specific functional purpose. Selenium (Se) is a metalloid that belongs to group 16 of the periodic table. It may be obtained from myriad sources like soil, water, and living organisms. Se is required to sustain proper health in both animals and humans due to its linkage with various biological functions in the immune system. Nature has made it impossible for us to obtain sufficient Se from the diet since some regions across the globe have been designated as Se-deficient while others are becoming Se-toxic. Se deficiency is associated with a compromised immune system and increased susceptibility to various diseases. Therefore, designing Se functional foods and supplements is the way forward in curbing the menace mentioned above, since geographical location will not have any effect on the Se content of these foods. Brewing yeast has the necessary enzymes to biotransform inorganic Se (Na 2 SeO 3) to its bioactive organic form, which is incorporated in the aged selenized beer. S. cerevisiae was found to have better biotransformation efficiency than other yeast species. A traditional Slavic beverage, selenized kvass, was brewed using rye grains soaked and germinated in solutions of Na 2 SeO 3. Fruit yeasts (Lesaffre) and Evitalia were utilized as the starter cultures. A Se enriched solution was extracted from Se-biofortified pak-choi cabbage and incorporated into the wort before fermentation. Selenized yeast and algae can be taken as supplements or as food additives. There have been some reports about microcystins (MCs) in Spirulina. Therefore, the safety of Se-algae is not guaranteed. Pasteurized dried selenized supplements (yeast and algae) were proposed in formulating Selenized enriched Tom-brown. This review seeks to propose some possible foods that could be enriched with Se. A large portion of the population adequately consumes these proposed foods on a regular basis hence the target goal would be a success. Abbreviations. FOSHU-foods for specified health use; FUFOSE-functional food science in Europe; SHF-specific health promoting food; TDI-tolerable daily intake; MCs-microcystins; SeCys-selenocysteine; SeMeSeCys-selenomethyl-selenocysteine; SeMet-selenomethionine; NaHSeO 3-sodium hydroselenite; kDa-Kilo Dalton; Se-yeast-selenized-yeast; Se-algae-selenized algae; Se-Selenium; ICP-MS-liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer Selenium; MHLW-Ministry of health, labor and welfare; SOD-superoxide dismutase; CAT-catalase; GSH-Px-glutathione peroxidase; ICP-MS-inductively coupled plasma mass spectrometry; HPLC-High Performance Liquid Chromatography; MMeSe-monomethyl form of Se; DMeSe-dimethyl form of Se; TMeSe-trimethyl form of Se; SDS-Sodium dodecyl sulfate.
... such as Arthrospira maxima and Arthrospira platensis are the microalgae most commonly used for the production of dietary food supplements as they are regarded as a rich source of high quality protein, vitamins (in particular vitamin B12 and provitamin b-carotene), phenolic acids, minerals, essential amino acids, pigments, and essential fatty acids (Joventino et al., 2012;Karkos et al., 2011;Kulshreshtha et al., 2010;Vichi et al., 2012). The algal food products derived from spirulina are advertised for weight loss, as abundant sources of energy, mood improvers, and also as detoxifier (Heussner et al., 2012). Researchers have studied their numerous pharmacological functions such as anti-nephrotoxic properties (Belay et al., 1993), anticancer properties (Mathew et al., 1995), blood glucose level control (Parikh et al., 2001), hypocholesterolemic activity (Samuels et al., 2002), activation of natural killer cell (Akao et al., 2009), and hypolipidemic activity (Mazokopakis et al., 2014). ...
Spirulina has emerged as the next-generation dietary supplement owing to its health benefits. Despite the advantages, there have been reports of contamination by cyanotoxins such as microcystins that can adversely affect human health. Hence, there is a need to develop a robust, efficient, and cost-effective method to detect microcystin-producing cyanobacteria in these food supplements. In this study, we have demonstrated a multiplex polymerase chain reaction (PCR) method for identification of microcystin-contamination in spirulina dietary supplements. This method involves simultaneous amplification of phycocyanin and microcystin B encoding genes (pcb, mcyB). The sensitivity of the multiplex PCR was assessed, and the limit of detecting mcyB along with pcb was found to be 250 fg/µL. The presence of microcystin was detected in five out of seven fish food supplements indicating poor culture conditions. Hence, rigorous quality control is required for monitoring the spirulina food supplements.
... Recent concerns have been raised as to the quality and safety of selected FS following reports of their contamination or the presence of substances whose distribution is strictly regulated. For example, some microalgae-based products were found to cause significant side effects, exert cytotoxic effects in human cells, and were contaminated with significant levels of lead, aluminium (Rzymski et al. 2015a or cyanotoxins (Heussner et al. 2012) while bodybuilding FS targeted at sportsman contained prohibited stimulant compounds (Geyer et al. 2008) or anabolic androgenic steroids (Abbate et al. 2015). Moreover, as found for selenium FS distributed in Poland, the declared nutritional values often differed significantly from actual content . ...
The market of food supplements (FS) in European Union (EU) is on the rise. The present study investigated the content of essential (Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Zn) and toxic elements (Cd, Cr(VI), Ni and Pb) in 168 FS produced in the EU and designed to support various health aspects (nutrition, neurological function, reproduction, weight loss, cosmetic appearance). The majority of FS did not exceed a dissolution time of 60 min in hydrochloric acid (89.8%) and phosphate buffer (85.7%). Cd and Cr(VI) were not detected in any FS, Ni and Pb were present sporadically (in 10.1 and 6.5%, respectively) at mean concentrations of 0.06 and 0.07 mg/single unit dose, respectively. All FS containing detectable Pb levels had exceeded a maximum allowance level (3.0 mg kg−1) set by the European Commission. Generally, the determined concentrations of essential elements fall greatly below recommended safety upper limits, except Fe in selected FS (mainly for pregnant women). Compared to the value declared on the product label, the majority of tested FS displayed mineral content below the accepted margin (70–130%). In general, the majority of the studied FS can be considered safe although discrepancies between declared and determined mineral content are worrisome. These findings are important in view of FS consumer safety.
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.
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.
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.
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.
Microcystins (MCs) are hepatotoxic heptapeptides produced by cyanobacteria and are potent inhibitors of protein phosphatases in eukaryotic cells. Algae for dietary supplements are harvested from outdoor environments and can be contaminated with MCs. Monitoring of MCs in these products is necessary, but is complicated by their structural diversity (> 250 congeners). We used a combination of protein phosphatase inhibition assay (PPIA), ELISA, LC–MS/MS and nontargeted LC–high resolution MS (LC–HRMS) with thiol derivatization to characterize the total MCs in eighteen algal dietary supplements. LC-MS/MS revealed some products contained >40 times the maximum acceptable concentration (MAC) of 1 μg/g MCs, but ELISA and PPIA showed up to 50–60 times the MAC. LC–HRMS identified all congeners targeted by LC–MS/MS plus MC-(H4)YR contributing up to 18% of total MCs, along with numerous minor MCs. Recommended dosages of the products with >MAC would result in 2.6–75 times the tolerable daily intake, presenting a risk to consumers. This study confirms the need for monitoring these products, and presents strategies to fully describe the total MC pool in environmental samples and algal products.
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.
