Joana Osswald

First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria

Anatoxin-a is a potent neurotoxin produced by several species of cyanobacteria. This alkaloid may cause fatal intoxication to exposed organisms and this has raised concerns over the increasing popularity of food supplements containing cyanobacteria. These are being marketed with alleged health properties for animal and human consumption. These supplements most commonly contain the genera Spirulina (Arthrospira) and Aphanizomenon and their consumption represent a potential route for anatoxin-a exposure in cases where adequate quality control is not undertaken. In this work, several dietary supplements containing cyanobacteria from different commercial suppliers were evaluated for the presence of anatoxin-a by high performance liquid chromatography with fluorescence detection. Additionally, the presence of the previously derivatized anatoxin-a was confirmed by using Gas chromatography-mass spectrometry. A total of 39 samples were analysed in the study. Results showed that three of the samples (7.7%) contained anatoxin-a, at concentrations ranging from 2.50 to 33 microg g(-1). Quality control of cyanobacterial food supplements is required to avoid potential health effects in humans and animals.

Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp.

This study compares the effects of pure anatoxin-a and cyanobacterial extracts of an anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.

Uptake and depuration of anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions

Cyanobacterial blooms tend to be more common in warm and nutrient-enriched waters and are increasing in many aquatic water bodies due to eutrophication. The aim of this work is to study the accumulation and depuration of anatoxin-a by Mytilus galloprovincialis a widespread distributed mussel living in estuarine and coastal waters and recognized worldwide as a bioindicator (e.g. Mussel Watch programs). Research on the distribution and biological effects of anatoxin-a in M. galloprovincialis is important. Nevertheless, the risk of human intoxication due to the consumption of contaminated bivalves should also be considered. A toxic bloom was simulated in an aquarium with 5 x 10(5) cell ml(-1) of Anabaena sp. (ANA 37), an anatoxin-a producing strain. Mussels were exposed to Anabaena for 15 days and then 15 days of depuration followed. Three or more animals were sampled every 24h for total toxin quantification and distribution in soft tissues (edible parts). Water samples were also taken every 24h in order to calculate total dissolved and particulate anatoxin-a concentrations. Anatoxin-a was quantified by HPLC with fluorescence detection. No deaths occurred during accumulation and depuration periods. One day after the beginning of depuration, the toxin could not be detected in the animals. Anatoxin-a is distributed in the digestive tract, muscles and foot and is probably actively detoxified.

Toxic effects of pure anatoxin-a on biomarkers of rainbow trout, Oncorhynchus mykiss.

Anatoxin-a is a neurotoxin produced by various bloom-forming cyanobacteria. Although it shows widespread occurrence and is highly toxic to rodents, its mechanisms of action and biotransformation, and effects in fish species are still poorly understood. The main aim of this study was, thus, to investigate sub-lethal effects of anatoxin-a on selected biochemical markers in rainbow trout fry in order to get information about the mechanisms of toxicity and biotransformation of this toxin in fish. Trout fry were administered sub-lethal doses of anatoxin-a (0.08-0.31 μg g⁻¹) intraperitoneally. Livers and muscle tissue were collected 72 h later for quantification of key enzyme activities as biochemical markers. Enzymes assessed in muscle tissues were related to cholinergic transmission (acetylcholinesterase [AChE]), energy metabolism (lactate dehydrogenase [LDH] and NADP⁺-dependent isocitrate dehydrogenase [IDH]). Enzymes assessed in the liver were involved in biotransformation (ethoxyresorufin-O-deethylase [EROD] and glutathione S-transferases [GST]). The results indicated a significant increasing trend for AChE activity with the dose of anatoxin-a, possibly representing an attempt to cope with overstimulation of muscle activity by the toxin, which competes with acetylcholine for nicotinic receptors binding. Anatoxin-a was also found to significantly induce the activities of liver EROD and GST, indicating the involvement of phase I and II biotransformation in its detoxification. Likewise, lactate dehydrogenase activity recorded in fry muscle increased significantly with the dose of anatoxin-a, suggesting an induction of the anaerobic pathway of energy production to deal with toxic stress induced by the exposure. Altogether, the results suggest that under continued exposure in the wild fish may experience motor difficulties, possibly becoming vulnerable to predators, and be at increased metabolic demand to cope with energetic requirements imposed by anatoxin-a biotransformation mechanisms.

Development and Validation of an SPE-HPLC-FL Method for the Determination of Anatoxin-a in Water and Trout (Oncorhincus mykiss)

Anatoxin-a is a neurotoxic alkaloid which is produced by various cyanobacteria genera and it is highly toxic to animals. Anatoxin-a risk evaluation is not fully determined yet. Therefore, it is imperative to implement detection and quantification methods in the various possible matrices. The recent worldwide shortage of acetonitrile, as well as environmental reasons, demand an urgent alternative to this high-performance liquid chromatography (HPLC) mobile phase solvent. This work describes an internal study that validated the measuring method of anatoxin-a in water and lyophilized trout matter by means of HPLC with fluorimetric detection (FLD) utilizing methanol as mobile phase.

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.