Annual variations of total and fractionated chlorophyll and phytoplankton groups in the Gulf of Cadiz.

Chlorophyll and phytoplankton distribution and concentration in the Gulf of Cadiz were studied during 2014 and 2015. In both years the highest chlorophyll concentrations are found at coastal stations during spring. Pico- and nanophytoplankton are the main contributors to total chlorophyll, with highest concentrations at the outer limit of the continental shelf. Microphytoplankton is responsible for most of the inshore chlorophyll. Picophytoplankton was analyzed to determine density, biomass and distribution. Prochlorococcus and Synechococcus show differences in distribution depending on distance from the coast and depth. Our results suggest temperature and consequent water stratification seem to be the main factors determining deep fluorescence maxima (DFM), mainly formed by picophytoplankton, especially Prochlorococcus. Pigment identification assisted by CHEMTAX analysis was carried out to analyze relative concentrations of larger phytoplankton.

Effects of TiO2 nanoparticles and sunscreens on coastal marine microalgae: Ultraviolet radiation is key variable for toxicity assessment.

Given the large numbers of sunbathers on beaches, sunscreen compounds are being released into the coastal aquatic environment in significant amounts. Until now the effect of these potential pollutants on microbiota has been not well-known. Phytoplankton is a key component of the microbiota community. It forms the basis of the aquatic trophic networks, and any change in the natural population of phytoplankton can affect the structure of aquatic biota. This paper describes an experiment performed outdoors (in natural sunlight conditions including ultraviolet radiation (UVR) and with UVR blocked) on mixed microalgae populations (four species from different key marine taxonomic groups, Nannochloropsis gaditana, Chaetoceros gracilis, Pleurochrysis roscoffensis and Amphidinium carterae), for three days, exposed to a range of concentrations of three commercial sunscreens (with variable TiO2 concentrations: highest concentration for sunscreen C, followed by sunscreen A; and sunscreen B did not contain TiO2 in its composition). With regard to UVR effect, in the absence of sunscreens, the most sensitive species is the centric diatom, Chaetoceros gracilis, and the least is Nannochloropsis gaditana; this last species presented the same behavior in the absence of UVR and with high sunscreen concentrations. The toxicity gradient obtained for sunscreens and nanoparticles under UVR is: TiO2 NPs>Sunscreen C>Sunscreen A>Sunscreen B. The differential sensitivity of microalgae to sunscreens and TiO2 NPs can produce a change in the dynamics of phytoplankton populations and provoke undesirable ecological effects (such as giving dinoflagellates more prominence). The effects of UVR, commonly neglected in bioassays, could alter the results in important ways and should be considered when performing environmentally-relevant bioassays. The toxicity mediated by hydrogen peroxide production associated with the concentration of TiO2 NPs cannot be considered the only factor responsible for the toxicity: the organic compounds in the sunscreens must also be taken into account.

Toxicity of copper in natural marine picoplankton populations.

Standard microalgae toxicity tests should be able to establish responses in real ecosystems. Natural marine picoplankton samples collected during the months of March, June, August, October 2007 and January 2008, where exposed to 72 h copper toxicity tests. Results analysed by flow cytometry distinguished two groups, with different cytometric characteristics that can match with two of Synechococcus populations. EC(50) values for these two populations resulted low, ranging from 0.62 to 26.28 microg L(-1), this converts copper in a very powerful contaminant and Synechococcus in one of the most sensitive groups of phytoplankton. Differences in EC(50) values for a same population can be related to the month of collection including different initial cellular densities and oceanographic parameters that can affect the picoplankton's tolerance and distribution.

Comparison of supercritical fluid and ultrasound-assisted extraction of carotenoids and chlorophyll a from Dunaliella salina.

In the work described here the extraction processes of carotenoids and chlorophylls were analysed using two extraction techniques, namely ultrasound-assisted extraction and supercritical fluid extraction, and the results are compared. The solvents used for the ultrasound-assisted extraction were N,N'-dimethylformamide and methanol and for the supercritical fluid extraction, carbon dioxide. The raw material studied was Dunaliella salina, a microalgae characterized by the high levels of carotenoids present in its cellular structure. The results indicate that the supercritical fluid extraction process is comparable to the ultrasound-assisted extraction when methanol is used as solvent. In addition, the supercritical extraction process is more selective for the recovery of carotenoids than the conventional technique since it leads to higher values for the ratio carotenoids/chlorophylls. Finally, the effects of pressure and temperature on the extraction yields of the supercritical fluid extraction process were studied.

Effect of linear alkylbenzene sulfonate (LAS) and atrazine on marine microalgae.

Five marine microalgae (Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana), in the same biovolume quantity, were exposed to 72h growth-inhibition tests with atrazine and LAS. In all cases, the inhibition effect of atrazine was higher than that of LAS up to two orders of magnitude higher in the case of T. chuii. In a second part of the study, initial cellular densities for each microalga strain and fixed organic compound concentration were varied, and results show density has a clear influence in growth inhibition tests. Finally, the sum of all data obtained in the study was expressed in terms of "toxic cellular quota" (mass of chemical substance per cell). The result was a sigmoid curve with a good fit, including the two main factors in toxicity tests, initial cellular density and contaminant concentration. This toxic cellular quota exhibits a general tendency to increase with cell volume/size.

Sediment toxicity tests involving immobilized microalgae (Phaeodactylum tricornutum Bohlin).

Populations of calcium-alginate immobilized marine diatom Phaeodactylum tricornutum were exposed to two sediments containing different levels of surfactant (LAS). Toxic responses were compared for free and immobilized algae. Although there is a direct relation between LAS content in sediment and inhibition, immobilized algae suffered less inhibition than free cells, over all when fluorescence is chosen as a biomarker for biomass. When cells are counted from dissolved beads, inhibition of growth is closer to the values found for free cells. Immobilization can be useful for in situ experiments but protection of cells inside the alginate beads against toxic capacity of xenobiotics must be taken into account.

Calcium alginate immobilized marine microalgae: experiments on growth and short-term heavy metal accumulation.

Growth of 11 calcium alginate immobilized marine microalgal species belonging to eight taxonomical groups has been checked in the present work. Cellular densities inside the calcium alginate beads were monitored during 17 days. Good growth and maintenance of the structure of the beads were both found for some of the assayed species. One of those species (Tetraselmis chui, Prasinophyceae) was selected in order to perform a short term (up to 24 h) heavy metal accumulation experiment. Beads of calcium alginate containing (or not) cells of T. chui were exposed to 820 microg L(-1) Cu and 870 microg L(-1) Cd separately during a 24 h period, and accumulation of heavy metals in the beads was measured after this time and compared. Concentration of each metal in the supernatants was monitored at 5, 10, 60 min and 24 h from the beginning of the experiment. After 24 h, practically all Cu was removed by the beads. Beads with immobilized algae removed around 20% of total Cd, while beads without algae removed half of that percentage.

Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae).

A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media.

Acute toxicity of LAS homologues in marine microalgae: esterase activity and inhibition growth as endpoints of toxicity.

The toxicity of two linear alkylbenzene sulfonate (LAS) homologues (C(10) and C(13)) was evaluated in four marine microalgae (Nannochloropsis gaditana, Tetraselmis suecica, Rhodomonas salina, and Isocrysis galbana), using growth inhibition rate and esterase activity (measured by flow cytometry) as endpoints. The inhibitor effect was higher for the C(13) LAS homologue than for C(11), in both responses analyzed. When both endpoints were compared, the growth inhibition rate was between 2 and 5 times more sensitive than esterase activity. Among microalgae species, R. salina exhibited the highest sensitivity.

Marine microalgae toxicity test for linear alkylbenzene sulfonate (LAS) and alkylphenol ethoxylate (APEO).

Different microalgal species have been used in growth-inhibition tests to determine the toxic concentrations of anionic and non-ionic surfactants to phytoplankton. The species used were selected from different taxonomic groups, all of considerable ecological relevance to marine environments. The toxicity of the C13 LAS homologue to the microalgal species selected was usually one order of magnitude greater than that of the C11 homologue. The toxicity of a commercial LAS mixture to different microalgal species was also checked. For this material and C. gracilis, cellular counting by means of a Neubauer chamber and by use of a flow cytometer were compared; differences between the two methods were insignificant. The toxicity of decaethoxylated nonylphenol non-ionic surfactant to C. gracilis was also checked; the EC50 value for this compound was 1.0 mg L(-1).

Influence of cellular density on determination of EC(50) in microalgal growth inhibition tests.

Growth inhibition tests for copper were carried out on four marine microalgal species: Chlorella autotrophyca, Nannochloris atomus (Chlorophyceae), Phaeodactylum tricornutum (Bacillariophyceae), and Isochrysis aff. galbana (Primnesiophyceae). The test initial cellular densities were reduced to 50 and 10% from the recommended initial cellular density in most of standardized assays. OECD test protocol (originally described for freshwater) was adapted for seawater. The EC(50) values were reduced when initial cellular density decreased. The green algae used in this study exhibited lower sensitivity than P. tricornutum and quite lower than I. aff. galbana. The latter species was found to be very sensitive to copper. The concept of cellular toxic quote (amount of toxic per cell) is defined in order to improve the results of toxicity tests.

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana.

Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry. Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport. Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3-. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium.

In vitro populations of rotifer Brachionus plicatilis Müller demonstrate inhibition when fed with copper-preaccumulating microalgae.

Four marine microalgal species (Chlorella autotrophyca, Nannochloropsis gaditana, Tetraiselmis chuii, and Isochrysis aff. galbana) were exposed for 24 h to 1 mg L(-1) dissolved copper and then transferred to fresh medium. After that, a group of 10 neonate rotifers were fed with these four microalgal species. The levels of accumulated copper in cellular concentrations of the microalgae were checked, with the result of around 40% of original concentration, with the exception of I. aff. galbana (25% of original concentration). In all cases, cells with preaccumulated metal caused a delay of 1 or 2 days in populational development of rotifers (increase in "lag phase"). The microalgae that were not fed to rotifers (disposed in parallel series) did not significantly transfer metal to the medium after the first day.