Milena Bruno

Seasonal succession of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum blooms with cylindrospermopsin occurrence in the volcanic Lake Albano, Central Italy

The cyanobacterial toxin cylindrospermopsin is rapidly spreading in the European temperate Countries. Cylindrospermopsin was detected for the first time in Italy in 2004; in this study, the presence of this toxin in Albano Lake (Central Italy) has been correlated to the cyanobacterial species Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum and their population dynamics. In 2004, these two species succeeded in the lake during spring, summer, and early autumn without overlapping, causing superficial blooms. Cylindrospermopsin was detected in lake samples by LC‐MS/MS and ELISA immunoassay, showing extracellular superficial values ranging from 2.6 to 126 μg/L, and water column values ranging from 0.41 to 18.4 μg/L. Twenty‐six of 30 positive water samples (86%) exceeded the recommended limit of 1 μg/L. Intracellular values up to 42.3 μg/g were measured. Moreover, cylindrospermopsin was detected in tissues from two Salmo trutta trouts (up to 2.7 ng/g) and in a well for drinking water supply (1.6 μg/L). For the first time, two cyanobacterial species producing cylindrospermopsin were detected in the same lake in Italy.

Anatoxin-a and a previously unknown toxin in Anabaena planctonica from blooms found in Lake Mulargia (Italy)

Algal extracts of Anabaena planctonica from Lake Mulargia in Italy were tested for toxins by mouse bioassay, the Microtox system, and GC-MS and HPLC chromatography. Anatoxin-a was identified by GC-MS after derivatization with pentafluorobenzyl bromide. Hepatotoxins were also present and are perhaps related to the microcystins present in other species of Anabaena.

Liquid chromatography coupled to quadruple time-of-flight tandem mass spectrometry for microcystin analysis in freshwaters: method performances and characterisation of a novel variant of microcystin-RR.

Cyanobacteria, also called blue-green algae, occur worldwide within water blooms in eutrophic lakes and drinking water reservoirs, producing several biotoxins (cyanotoxins). Among these, microcystins (MCs) are a group of cyclic heptapeptides showing potent hepatotoxicity and activity as tumour promoters. So far, at least 89 MCs from different cyanobacteria genera have been characterised. Herein, ion trap, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) and quadruple time-of-flight (Q-ToF) mass spectrometry (MS)-based methods were tested and compared for analysing MCs in freshwaters. Method performances in terms of limit of detection, limit of quantification, mean recoveries, repeatability, and specificity were evaluated. In particular, a liquid chromatography/electrospray ionisation (LC/ESI)-Q-ToF-MS/MS method was firstly described to analyse MCs in freshwaters; this technique is highly selective and sensitive, and allowed us to characterise the molecular structure of an unknown compound. Indeed, the full structural characterisation of a novel microcystin variant from a bloom of Planktothrix rubescens in the Lake Averno, near Naples, was attained by the study of the fragmentation pattern. The new cyanotoxin was identified as the 9-acetyl-Adda variant of microcystin-RR.

Determination of cylindrospermopsin in freshwaters and fish tissue by liquid chromatography coupled to electrospray ion trap mass spectrometry

Cylindrospermopsin (CYN) is a toxic alkaloid-like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI-MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI-MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in southern Italy.

Microcystin-like toxins in different freshwater species of Oscillatoria

In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tenuis and Oscillatoria mougetii were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms.

Chemical composition and biological origin of ‘dirty sea’ mucilages

Abstract The carbohydrate composition of the extracellular exudates of the native algae cells floating in the Adriatic sea during the summer have been determined. These results were then compared with similar analyses performed on the excreted organic matter produced by in vitro cultures of Amphora coffeaeformis and Cylindrotheca fusiformis , both of which have been isolated in the past in the same areas of the Adriatic sea. Separation of the jelly-like polymeric components of the mucilages is described. The insoluble fraction presents a monomer distribution similar to that of C. fusiformis , while the unprecipitable fraction is similar to that of A. coffeaeformis . Native mucilages appear to be an intimate mixture of the extracellular exudates produced by the in vivo metabolism of the two diatoms.

A peptidomic approach for monitoring and characterising peptide cyanotoxins produced in Italian lakes by matrix‐assisted laser desorption/ionisation and quadrupole time‐of‐flight mass spectrometry

In recent years, the occurrence of cyanobacterial blooms in eutrophic freshwaters has been described all over the world, including most European countries. Blooms of cyanobacteria may produce mixtures of toxic secondary metabolites, called cyanotoxins. Among these, the most studied are microcystins, a group of cyclic heptapeptides, because of their potent hepatotoxicity and activity as tumour promoters. Other peptide cyanotoxins have been described whose structure and toxicity have not been thoroughly studied. Herein we present a peptidomic approach aimed to characterise and quantify the peptide cyanotoxins produced in two Italian lakes, Averno and Albano. The procedure was based on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry mass spectrometry (MALDI-TOF-MS) analysis for rapid detection and profiling of the peptide mixture complexity, combined with liquid chromatography/electrospray ionisation quadrupole time-of- flight tandem mass spectrometry (LC/ESI-Q-TOF-MS/MS) which provided unambiguous structural identification of the main compounds, as well as accurate quantitative analysis of microcystins. In the case of Lake Averno, a novel variant of microcystin-RR and two novel anabaenopeptin variants (Anabaenopeptins B(1) and Anabaenopeptin F(1)), presenting homoarginine in place of the commonly found arginine, were detected and characterised. In Lake Albano, the peculiar peptide patterns in different years were compared, as an example of the potentiality of the peptidomic approach for fast screening analysis, prior to fine structural analysis and determination of cyanotoxins, which included six novel aeruginosin variants. This approach allows for wide range monitoring of cyanobacteria blooms, and to collect data for evaluating possible health risks to consumers, through the panel of the compounds produced along different years.

Dynamics of Dinophysis sensu lato species (Dinophyceae) in a coastal Mediterranean environment (Adriatic Sea)

Species of Dinophysis Ehrenberg sensu lato were collected opportunistically along the southern Adriatic coasts (Apulia, Italy) during a survey of phytoplankton community structure and dynamics carried out from 1995 to 1997. Most previous reports of Dinophysis spp. in the Adriatic Sea concentrated on northern populations, with few data available on phytoplankton inhabiting the southern coastal areas. Thus, the present study is the first on Dinophysis sensu lato species composition and spatio-temporal distribution in these essentially oligotrophic waters. Twelve Dinophysis species were identified; some of which can be assigned to the genus Phalacroma. Dinophysis sacculus, D. fortii, D. caudata, Phalacroma rotundatum and P. mitra are potentially producers of diarrhetic shellfish poisoning. Seasonal changes in Dinophysis and Phalacroma populations are presented, as well as their relationships with hydrographic and biotic factors. These dinoflagellates were associated with mixing conditions, low water temperatures and high nutrient inputs. Furthermore, the statistical analysis evidenced significant correlations of Dinophysis species to chlorophyll a and phytoplankton. Ecological implications arising from the data are discussed

Diarrhoeic shellfish toxins in Adriatic Sea mussels evaluated by an ELISA method

A competitive enzyme immunoassay (ELISA) was used to determine the presence of okadaic acid (OA) and/or dinophysistoxin-1 (DTX-1) in mussels. The accuracy and sensitivity of the ELISA method has been checked. The sensitivity of the method (100 ng/g of hepatopancreas) makes it possible to determine OA concentrations ten times lower than the tolerance limits established by the Health Authorities of many countries. For the first time, OA and/or DTX-1 were detected in mussels (Mytilus galloprovincialis) collected in different stations along the Adriatic Sea coasts.

ELISA and LC-MS/MS methods for determining cyanobacterial toxins in blue-green algae food supplements

The use of natural products as a diet supplement is increasing worldwide but sometimes is not followed by adequate sanitary controls and analyses. Twenty samples of pills and capsules of lyophilised cyanobacteria (blue-green algae), commercialised in Italy as dietary supplements, were found positive at the Vibrio fischeri bioassay. Further analyses with ELISA and LC-MS/MS methods revealed the presence of four microcystin (MC) analogues, MC-LR, -YR, -LA, -RR and two demethylated forms of MC-RR. The highest total microcystin content was 4.5 and 1.4 µg g−1 in pills and capsules, respectively. The ELISA measurements, compared to the LC-MS/MS analyses, showed significantly lower concentrations of microcystins in pills, this confirming a possible ELISA underestimate of mixed microcystins, due to different sensitivities for some toxic analogues.