Juan Blanco

Biomass production and variation in the biochemical profile (total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae

Abstract Seven species of marine microalgae ( Pavlova lutheri, Isochrysis galbana, Tetraselmis suecica , Chaetoceros calcitrans, Phaeodactylum tricornutum, Rhodomonas sp. and Heterosigma akashiwo ) were harvested at three phases in the growth curve and biochemical composition (total protein, carbohydrates, RNA, lipids and fatty acids) was determined. Carbohydrate and lipid contents increased with the development of the culture, while protein levels increased in the later phases of the culture in the case of diatoms and Rhodomonas sp. and decreased in I. galbana, P. lutheri and T. suecica . Saturated fatty acids and, to a lesser extent, monoethylenic fatty acids represent between 70 and 100% of the total fatty acids. Polyunsaturated fatty acids reached their highest values in the exponential phase in Rhodomonas sp. (30.99%), in an early stationary phase in P. tricornutum (19.58%) and C. calcitrans (9.06%) and in a late stationary phase in I. galbana (12.89%), P. lutheri (4.24%) and T. suecica (18.58%). RNA levels never exceeded 2.5%. The daily production was calculated for each batch culture in three growth phases and was compared with the production of a semicontinuous culture maintained in the exponential phase. Rhodomonas sp., C. calcitrans and P. tricornutum showed a lower daily production under semicontinuous culture than in batch cultures, while I. galbana, P. lutheri, T. suecica and H. akashiwo showed the opposite. The daily production of each of the biochemical components also varied with the species.

Dinophysis Toxins: Causative Organisms, Distribution and Fate in Shellfish

Several Dinophysis species produce diarrhoetic toxins (okadaic acid and dinophysistoxins) and pectenotoxins, and cause gastointestinal illness, Diarrhetic Shellfish Poisoning (DSP), even at low cell densities (<103 cells·L−1). They are the main threat, in terms of days of harvesting bans, to aquaculture in Northern Japan, Chile, and Europe. Toxicity and toxin profiles are very variable, more between strains than species. The distribution of DSP events mirrors that of shellfish production areas that have implemented toxin regulations, otherwise misinterpreted as bacterial or viral contamination. Field observations and laboratory experiments have shown that most of the toxins produced by Dinophysis are released into the medium, raising questions about the ecological role of extracelular toxins and their potential uptake by shellfish. Shellfish contamination results from a complex balance between food selection, adsorption, species-specific enzymatic transformations, and allometric processes. Highest risk areas are those combining Dinophysis strains with high cell content of okadaates, aquaculture with predominance of mytilids (good accumulators of toxins), and consumers who frequently include mussels in their diet. Regions including pectenotoxins in their regulated phycotoxins will suffer from much longer harvesting bans and from disloyal competition with production areas where these toxins have been deregulated.

First identification of azaspiracid and spirolides in Mesodesma donacium and Mulinia edulis from Northern Chile

In an attempt to evaluate the risk for human consumption associated to the accumulation of lipophilic toxins by two commercially important bivalves: macha (Mesodesma donacium) and clam (Mulinia edulis) in Coquimbo Bay (Chile), monitoring of these species was carried out from March to September 2008. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to detect okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins and spirolides. Low levels of Azaspiracid-1 and 13-desmethyl C spirolide were found in both species. The toxins were detected at different dates throughout the monitoring period and in some cases both toxins were detected in the same sample. In all cases, the concentration of the toxins was below the limit of quantification of the technique used and therefore these detections are only indicative of a potential risk. This is the first report of the occurrence of these groups of toxins in Chile and suggests that it is necessary to monitor routinely these substances to warrant public health and shellfish exportations.

Depuration and anatomical distribution of the amnesic shellfish poisoning (ASP) toxin domoic acid in the king scallop Pecten maximus

The depuration kinetics of the domoic acid of four body fractions (digestive gland, adductor muscle, gonad+kidney and gills+mantle) of the scallop Pecten maximus was studied over 295 days. The scallops, which had acquired the toxins during a Pseudo-nitzschia australis episode that took place the week before the beginning of the experiment, were maintained in tanks with running seawater. All the body fractions, except the adductor muscle, decreased their domoic acid burden throughout the experiment. The amount of toxin in the muscle dropped sharply at the start of the experiment but increased again at the end, to levels that were higher than the initial ones. Several dynamic models of depuration kinetics, which included the depuration of each fraction (excluding the adductor muscle) and the transfers between them, were constructed, implemented and fitted to the data to obtain their parameters. The estimated depuration rates were very low, both considering and not considering the transfer of toxin between organs or the effect of weight loss. There were strong differences in the domoic acid burden of the body fractions studied but not between their depuration rates. No net transfer from the digestive gland, the tissue with highest domoic acid concentration, to the other fractions was found, as the inclusion of these processes in the models produced only a marginally better fit to the data. The depuration of domoic acid was slightly, but significantly, affected by biomass. Weight loss induced domoic acid loss, suggesting that part of the depuration may be produced by the direct loss of bivalve cells. The concentration or dilution effect, due to decreases or increases in biomass, documented for other species and toxins, has little importance in Pecten maximus.

Accumulation and transformation of DSP toxins in mussels Mytilus galloprovincialis during a toxic episode caused by Dinophysis acuminata.

The time course of several outbreaks of the diarrhetic shellfish poisoning (DSP) producer Dinophysis acuminata and the consequent kinetic of accumulation and loss of toxins in mussels Mytilus galloprovincialis feeding on them was studied. Samples of mussels and seawater were frequently (2-3 times a week) collected from a raft in the Ri;a de Vigo. DSP toxins content of mussels and water was analyzed by HPLC-FD and phytoplankton was quantified in an inverted light microscope. Only okadaic acid (OA) and some of its conjugated forms (OA CF), estimated by enzymatic hydrolysis, were found in the plankton samples obtained, comprised mainly of D. acuminata cells. The main accumulated form in mussels was OA reaching a maximum of 10.1 microg OA g(-1) in the digestive gland (d.g.) in 16 days, falling below the quarantine level (ca. 2 microg OA g(-1) d.g.) by 45 days. The low polarity conjugated forms (LPCF), estimated by hexane extraction, accounted for 6.2% of the total toxin burden of the mussels. To quantify the rates of the processes involved in the accumulation, transformation and loss of the toxins, two dynamic models, a one-compartment and a two-compartment, including OA and its conjugated forms as variables were designed and implemented. The one-compartment model provided a good fit to the OA and LPCF actual data (r(2)=0.92 and r(2)=0.94, respectively). The two-compartment model did not fit the data markedly better than its one-compartment counterpart (r(2)=0.93 and r(2)=0.95, for OA and LPCF, respectively). High hydrolysis rates were estimated for most of the OA CF, which means that these forms came largely from the ingested plankton. The low estimated acylation rates support the previous point and suggest that the formation of LPCF by direct acylation of the OA is of little importance in M. galloprovincialis. Only in cases where the intoxication period is very long, can the formed acyl-derivatives be important, because they seem to accumulate for a long time in the mussels, as suggested by the low hydrolysis and depuration rates estimated from model fitting.

The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis Lmk)

The accumulation of paralytic shellfish poisoning (PSP) toxins by bivalves is a serious threat to public health all over the world. However, very little is known about the uptake kinetics of these toxins and the environmental factors that may modify this process. We have studied the effect of mussel size, temperature, seston volume, food quality, and volume-specific toxin concentration (VOSTOC), on the uptake rate of paralytic shellfish poisoning (PSP) toxins by mussels (Mytilus galloprovincialis), by means of a second order factorial experiment. Over a 3-day period, the mussels were fed artificial diets containing Alexandrium minutum AL1V (a PSP toxin producer), Tetraselmis suecica, Ensiculifera sp1 and silt, to the levels required by each treatment. Mussel size, seston volume and VOSTOC were found to be statistically significant when the total toxin accumulated per weight of wet tissue was considered. Mussel size affected the uptake negatively and latter two positively. The interactions, mussel size-VOSTOC and mussel size-food quality were also significant. The response was not linear as shown by the significance of the quadratic term of mussel size. Notwithstanding, when the PSP toxins accumulation per mussel was analysed, only one factor, the VOSTOC and the interactions, food quality-mussel size and food quality-seston volume, were found to be significant. VOSTOC was the most important factor in the accumulation of toxins, in our opinion, probably due to toxin assimilation being mainly regulated by the probability of contact between the toxins and the cellular walls of the digestive system. The size of the bivalve is also especially important because toxin concentration is usually calculated per weight of bivalve tissue and because the weight-specific ingestion increases with mussel size. The food quality, which was directly related to the assimilation of organic matter, had an inverse effect on toxin assimilation. In our opinion, this is probably due to the effect of inorganic particles in enhancing the disruption of Alexandrium cells. Temperature had no effect on the uptake rate except for the accumulation of the gonyautoxin GTX1.

Automated on-line solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry for determination of lipophilic marine toxins in shellfish

Automated on-line solid-phase extraction (SPE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for fast determination of lipophilic marine toxins in shellfish samples. The direct coupling of an on-line SPE column to LC-MS/MS was accomplished using column switching techniques. Suitable chromatographic separation was performed on a reversed-phase C18 column under alkaline conditions (pH 11). The selected reversed-phase C18 SPE column allowed rapid and efficient on-line desalting of hydrolysed shellfish samples, avoiding signal suppression during mass spectrometry detection. Furthermore, the on-line SPE procedure allowed reducing matrix effects observed in raw and hydrolysed shellfish extracts. The proposed method was evaluated in terms of linearity, precision, accuracy and limits of detection (LODs). Quantitative recovery (97-102%) and satisfactory inter-day precision (RSD<8%) were achieved for all target compounds. LODs in the sub-μgkg-1 level (0.37-0.68μgkg-1) were obtained for all toxins except for okadaic acid, which showed a value of 2.75μgkg-1. Several mussel samples from North-western Spain were finally analysed in order to demonstrate the applicability of the method. Okadaic acid was the predominant toxin in all samples, although other lipophilic toxins were also detected.

Depuration of mussels (Mytilus galloprovincialis) contaminated with domoic acid

Abstract Domoic acid is a neurotoxin responsible for the Amnesic Shellfish Poisoning (ASP). With the aim of determining its depuration kinetics in raft mussels Mytilus galloprovincialis and the effects of body weight, salinity and temperature on it, an experiment involving these factors was carried out. Mussels which had incorporated this toxin to a concentration of 153 μg·g–1 of soft tissue during a bloom of the pennate diatom Pseudo nitzschia australis, were collected from the Galician Rias and placed at temperatures of 18 and 22 oC and salinities of 12.5 and 31. Mussel samples were taken at the start of the experiment and daily during the four subsequent days and the domoic acid contents of the soft tissues were analysed. The simple one-compartment kinetics, that is a kinetics in which all toxin depurates at the same rate, was fitted to the data, with good quantitative results. Notwithstanding, the deviations of the model from the actual data were dependent on time, suggesting that such a simple model is not enough to correctly describe the data, and that a more complex kinetics may be more adequate. A two-compartment kinetic model, in which two pools of toxins exist (compartments), each one depurating at different rate, described qualitatively better the depuration but its quantitative contribution to the fit was not statistically significant. The parameters of the model, obtained by least squares fitting, suggest the possibility of a small second compartment of very small or null depuration rate, as detected in other species. This kind of model would explain the reduced quantitative contribution of the second (slowly depurating) compartment.Low salinity was shown to reduce the depuration rate. The two other factors checked, temperature and body weight, nor any interaction had significant effect on depuration rate.

The effect of water conditioned by a PSP-producing dinoflagellate on the growth of four algal species used as food for invertebrates

Abstract The effect of the water conditioning produced by two dinoflagellate species of the tamarensis group, Alexandrium tamarense (strain not containing Paralytic Shellfish Poisoning (PSP) toxins) and Alexandrium lusitanicum (strain containing PSP toxins), on the growth of four algal species commonly used in feeding invertebrates was tested. Three of these species were very strongly affected by the water conditioned with A. lusitanicum, namely, Skeletonema costatum, Pavlova lutheri and Isochrysis galbana, and, presumably, toxins kill most of the cells. Tetraselmis suecica was only slightly affected by the toxin conditioned medium. This fact explains some of the problems encountered in culturing certain phytoplankton species when the water used has been conditioned by a bloom of PSP-producing species. On the other hand, water conditioned by A. tamarense slightly promoted the growth of all the species tested. Some possible ways of avoiding the effect of the toxins, at an industrial level, are suggested.

Amphidoma languida (Amphidomatacea, Dinophyceae) with a novel azaspiracid toxin profile identified as the cause of molluscan contamination at the Atlantic coast of southern Spain

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC-MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg-1 (reaching maximum levels of >500μg AZA-1 eq. kg-1 in Chamelea gallina and >250μg AZA-1 eq. kg-1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins.