Types and Distribution of Bioactive Polyunsaturated Aldehydes in a Gradient from Mesotrophic to Oligotrophic Waters in the Alborán Sea (Western Mediterranean).

Polyunsaturated aldehydes (PUAs) are bioactive molecules suggested as chemical defenses and infochemicals. In marine coastal habitats, diatoms reach high PUA production levels during bloom episodes. Two fractions of PUA can usually be analyzed: pPUA obtained via artificial breakage of collected phytoplankton cells and dissolved PUA already released to the environment (dPUA). In nature, resource supply arises as a main environmental controlling factor of PUA production. In this work, we monitored the vertical distribution and daily variation of pPUA associated with large-size phytoplankton and dPUA, at three sites located in the Alborán Sea from mesotrophic to oligotrophic waters. The results corroborate the presence of large-size PUA producers in oligotrophic and mesotrophic waters with a significant (58%-85%) diatom biomass. In addition to diatoms, significant correlations between pPUA production and dinoflagellate and silicoflagellate abundance were observed. 2E,4E/Z-Heptadienal was the most abundant aldehyde at the three sites with higher values (17.1 fg·cell-1) at the most oligotrophic site. 2E,4E/Z-Decadienal was the least abundant aldehyde, decreasing toward the oligotrophic site. For the first time, we describe the daily fluctuation of pPUA attributable to cellular physiological state and not exclusively to taxonomical composition. Our results demonstrate the persistence of threshold levels of dPUA deep in the water column, as well as the different chromatographic profiles of dPUA compared with pPUA. We propose different isomerization processes that alter the chemical structure of the released PUAs with unknown effects on their stability, biological function, and potential bioactivity.

Changes in membrane lipids and carotenoids during light acclimation in a marine cyanobacterium Synechococcus sp.

Time course of carotenoid and membrane lipid variation during high light (HL) acclimation (about 85 meu mol m-2 s-1), after transfer from low light (LL) (5-10 meu mol m-2 s-1), was determined in a marine Synechococcus strain. Highperformance liquid chromatography (HPLC) coupled to diode array detector (DAD) or electrospray ionization mass spectrometry (ESI-MS) was used for compound separation and detection. Myxoxanthophyll rose within a time interval of 8 h to 24 h after the onset of exposure to HL. Beta -carotene content started to decrease after 4 h of the onset of exposure to HL. Zeaxanthin content rose with exposure to HL, but it was only significant after 24 h of exposure. Carotenoid changes are in agreement with a coordinated activity of the enzymes of the myxoxanthophyll biosynthetic pathway, with no rate-limiting intermediate steps. Lipid analysis showed all species with a C18:3/C16:0 composition increased their content, the changes of PG (18:3/16:0) and MGDG(18:3/16:0) being primarily significant. Major lipid changes were also found to occur within 24 h. These changes might suggest reduction and reorganization of the thylakoid membrane structure. Hypotheses are also drawn on the role played by lipid molecule shape and their possible effect in membrane fluidity and protein accommodation.

Ranking sediment samples from three Spanish estuaries in relation to its toxicity for two benthic species: the microalga Cylindrotheca closterium and the copepod Tisbe battagliai.

The present study assesses the sediment toxicity levels of three Spanish estuaries, as well as the suitability of two microorganisms, the benthic microalga Cylindrotheca closterium and the harpacticoid copepod Tisbe battagliai, as test organisms in whole-sediment toxicity assays. The sensitivity of both species to potentially polluted sediments was compared. Three sites at the southwest of the Iberian Peninsula were chosen: the Ría of Huelva, the Guadalquivir Estuary, and the Bay of Algeciras. Inhibition data were based on growth for C. closterium and fecundity for T. battagliai. No toxicity was recorded for the microalga in the Guadalquivir Estuary and the Bay of Algeciras. However, for T. battagliai, inhibition of fecundity was approximately 50% in those zones, indicating higher sensitivity. Samples from stations in the Ría of Huelva were the most toxic of all those assayed; inhibition values higher than 90% were obtained for both organisms. The highest values for total metal concentrations such as arsenic (As), cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), tin (Sn), and zinc (Zn) were found in the Ría of Huelva, which can be classified as severely impacted. The Guadalquivir Estuary and the Bay of Algeciras can be considered moderately impacted. In general, both methodologies are suitable for application in ecotoxicological studies.

Sensitivity of Cylindrotheca closterium to copper: influence of three test endpoints and two test methods.

The present study checked the suitability of Cylindrotheca closterium (Bacillariophyceae) as a test species for ecotoxicology studies. To date, only limited use has been made of microphytobenthos in ecotoxicology, and C. closterium has been employed as a target organism in this study because the biological group is considered to be very relevant ecologically. The main objective was to assess the response of C. closterium to a contaminant-type (copper) using three different test endpoints (esterase activity, chlorophyll fluorescence and population growth) and two different test methods (Erlenmeyer flasks and microplates), to evaluate which combination of test conditions would provide the most sensitive approach for assessment of effects. Regardless of the endpoints, the response of C. closterium to copper was very similar; however lower sensitivity (EC50 of 27.8 +/- 0.7 microg Cu L(-1) was observed when tests were carried out in microplates. Chlorophyll fluorescence measured by flow cytometry as total FL3 was slightly more sensitive (EC50 of 4.7 +/- 0.1 microg Cu L(-1)) than the other parameters measured, probably because it takes into account the effect on chlorophyll fluorescence and cell density simultaneously. The test method (Erlenmeyer flask or microplate) was the determining factor for the observed differences in sensitivity. These differences found for the two methods are explained by the higher metal adsorption capacity of microplate vessel walls (more than 40%), which decreases the available copper. C. closterium was demonstrated to be a suitable organism for adoption in ecotoxicological studies, given the reliability of the three endpoints and also of the two test methods evaluated here.

Ring test for whole-sediment toxicity assay with -a- benthic marine diatom.

This work presents the results of an interlaboratory proficiency exercise for whole-sediment toxicity assays with the benthic marine diatom Cylindrotheca closterium. An assay protocol was established and followed by all participating laboratories. Cell growth after 72 h exposure was the endpoint used. Four sediment samples of unknown toxicity were assayed. The main problem encountered during this exercise was the differences in the cell growth of algae exposed to reference sediment. Those differences may be associated with changes in the physiological status of the initial culture due to temperature changes during transport to the other laboratories. In general, the method proposed presented good replicability (precision between replicates) and reproducibility (interlaboratory precision). Around 80% (17 out of 21) of results obtained were classified as satisfactory (Z-scores <2). The whole-sediment assay with C. closterium presented here can be considered sufficiently successful for possible use as a standard toxicity test. The assay is simple to perform, the proposed species is ecologically relevant as an integral component of microphytobenthos, and is widely distributed around the world. These positive factors suggest that the whole-sediment assay with the benthic marine diatom C. closterium can be used as a reliable tool in marine sediment quality assessment.

Toxicity and bioaccumulation of copper and lead in five marine microalgae.

On five marine microalgae with the same biovolume quantity (Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana) 72-h exposure toxicity tests with copper and lead were performed. For both metals, 72-h EC50s showed T. chuii as the most tolerant and R. salina as one of the most sensitive. Besides copper and lead EC(50) concentrations, metal concentrations in solution and accumulated on/in the cell where also analysed. T. chuii, the most tolerant species accumulated high copper concentrations (EC(50(Cu))=330 microgL(-1); EC(50(Pb))=2600 microgL(-)1), and R. salina the most sensitive to copper, accumulated the highest amount of this metal (EC(50(Cu))=50 microgL(-1)). Results of this study show that there is no specific relationship between cell tolerance and accumulated metal on/in the cell. On the other hand, due to an established evidence of the influence of cellular density in microalgae toxicity tests, this effect was also studied. Results showed reduced EC(50) values when initial cellular densities decreased. In this study, the term "toxic cellular quota" was used to express all data. This allowed, in a single expression, the combination of two parameters that clearly influence growth, cellular density and toxic concentration.

Sediment integrative assessment of the Bay of Cádiz (Spain): an ecotoxicological and chemical approach.

This study consisted of the sediment toxicity assessment of the Bay of Cádiz based on two endpoints: growth inhibition for Cylindrotheca closterium (benthic microalgae) and fecundity inhibition for Tisbe battagliai (harpacticoid copepod). A new methodology to eliminate (but not as storage technique) the autochthonous biota present in the sediment samples by immersing them in liquid nitrogen (-196 degrees C) was also assessed. Sediment toxicity data showed different toxicity levels for both organisms. In general, T. battagliai was more sensitive; however a good correlation (r=0.75; p<0.05) between sediment toxicity results for both species was found. Data in pore water (pH, redox potential, and toxicity for microalgae and copepod) and sediment (pH, redox potential, organic carbon, and metal concentrations) demonstrated that ultra-freezing did not alter sample characteristics; thus, this technique can be adopted as a pre-treatment in whole-sediment toxicity tests in order to avoid misleading results due to presence of autochthonous biota. Multivariate statistical analysis such as cluster and principal component analysis using chemical and ecotoxicological data were employed. Silt and organic matter percentage and lead concentration were found to be the factors that explain about 77% of sediment toxicity in the Bay of Cádiz. Assay methodology determined in this study for both assayed species is considered adequate to be used in sediment toxicity monitoring programs. Results obtained using both species show that the Bay of Cádiz can be considered a moderately polluted zone.

Effects of cold-dark storage on growth of Cylindrotheca closterium and its sensitivity to copper.

Cylindrotheca closterium cells were maintained at low temperature (4+/-1 degrees C) and dark conditions up to 21 weeks to assess the effect on survival and physiological status. From a control culture under standard conditions, three densities were prepared: (A) 2 x 10(4), (B) 10 x 10(4), and (C) 25 x 10(4) cells ml(-1). Weekly, inoculums of each stored density were exposed to continuous light and at 20+/-1 degrees C. Sensitivity to copper for microalgal cultures was evaluated in order to assess possible changes in cells sensitivity due to storage. Concurrently, assays with a control culture were carried out in order to assess the sensitivity of C. closterium to copper and to be able to generate a standard sensitivity control chart with a mean value of EC50-72 h+/-2SD (standard deviation). Density-C presented higher cell yield values, between 40% and 80% relative to control culture. Cell density showed to be important feature that may be taken into account in cell storage experiments. There was an increase in sensitivity of cells submitted to storage; however results always kept in the range established as standard sensitivity with no statistically significant difference with regards to control culture. EC50-72 h mean value for the control culture was 29+/-10 mug Cul(-1), while for densities-A, B and C were 22+/-7; 23+/-9 and 23+/-8 microg Cul(-1), respectively. In spite of drastic changes in the environmental conditions due to storage, it is concluded that C. closterium cells stored during 5 months remained metabolically active and with no significant change in its sensitivity.

Extraction of carotenoids and chlorophyll from microalgae with supercritical carbon dioxide and ethanol as cosolvent.

The extraction of carotenoids and chlorophylls using carbon dioxide modified with ethanol as a cosolvent is an alternative to solvent extraction because it provides a high-speed extraction process. In the study described here, carotenoid and chlorophyll extraction with supercritical CO(2 )+ ethanol was explored using freeze-dried powders of three microalgae (Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina) as the raw materials. The operation conditions were as follows: pressures of 200, 300, 400 and 500 bar, temperatures of 40, 50 and 60 degrees C. Analysis of the extracts was performed by measuring the absorbance and by using empirical correlations. The results demonstrate that it is necessary to work at a temperature of 50-60 degrees C and a pressure range of 300-500 bar, depending on the type of microalgae, in order to obtain the highest yield of pigments. The best carotenoid/chlorophyll ratios were obtained by using supercritical fluid extraction + cosolvent instead of using conventional extraction. The higher selectivity of the former process should facilitate the separation and purification of the two extracted pigments.

Supercritical CO2 extraction of beta-carotene from a marine strain of the cyanobacterium Synechococcus species.

Dynamic extraction of carotenoids from a marine strain of Synechococcus sp. (Cyanophyceae) with supercritical CO2 (SC-CO2) was investigated with regard to operation pressure and temperature effects on extraction efficiency. Extraction yield (milligrams of pigment per gram of dry weight) for SC-CO2) was compared with the extraction yield for dimethylformamide (DMF). Carotenoids extracted with SC-CO2 were beta-carotene (Ct), zeaxanthin (Z), beta-cryptoxanthin (Cr), and equinenone; chlorophyll a was poorly extracted, whereas myxoxanthophyll, another major carotenoid, was not extracted under any experimental condition. The highest relative yield, which is defined here as y(r) = [(mg of pigment(SC-CO2)/mg of pigment(DMF))] x 100, was 76.1 +/- 8.6% for Ct, but it rose to 87.0 +/- 3.4% when 15% ethanol was used as cosolvent. The pressure effect on y(r) was found to be significant (p < 0.05) for both Cr and Z, along with total carotenoids, whereas the effect of square T (TT) was significant for only Ct. From empirical correlations, pairwise pressure (bar) and temperature (degrees C), respectively, for optimal extraction were determined to be (358, 50) for Ct, (454, 59) for Cr, and (500, 60) for Z. Cell disruption by sonication or detergent treatment of the biomass did not improve the extraction efficiency. Matrix structure together with material state could explain the low carotenoid extraction yield obtained with SC-CO2 as compared to DMF in Synechococcus sp. However, the process can be applied to selective extraction of different carotenoids.

Interaction of UV radiation and inorganic carbon supply in the inhibition of photosynthesis: spectral and temporal responses of two marine picoplankters.

The effect of ultraviolet radiation (UVR) on inhibition of photosynthesis was studied in two species of marine picoplankton with different carbon concentration mechanisms: Nannochloropsis gaditana Lubian possesses a bicarbonate uptake system and Nannochloris atomus Butcher a CO2 active transport system. Biological weighting functions (BWFs) for inhibition of photosynthesis by UVR and photosynthesis vs irradiance (PI) curves for photosynthetically active radiation (PAR) were estimated for both species grown with an enriched CO2 supply (high dissolved inorganic carbon [DIC]: 1% CO2 in air) and in atmospheric CO2 levels (low DIC: 0.03% CO2). The response to UVR and PAR exposures was different in each species depending on the DIC treatment. Under PAR exposure, rates of maximum photosynthesis were similar between treatments in N. gaditana. However, the cultures growing in high DIC had lower sensitivity to UVR than the low DIC cultures. In contrast, N. atomus had higher rates of photosynthesis under PAR exposure with high DIC, but the BWFs were not significantly different between treatments. The results suggest that one or more processes in N. gaditana associated with HCO3- transport are target(s) for UV photodamage because there was relatively less UV inhibition of the high DIC-grown cultures in which inorganic carbon fixation is supplied by passive CO2 diffusion. Time courses of photochemical efficiency in PAR, during UV exposure and during subsequent recovery in PAR, were determined using a pulse amplitude modulated fluorometer. The results were consistent with the BWFs. In all time courses, a steady state was obtained after an initial decrease, consistent with a dynamic balance between damage and repair as found for other phytoplankton. However, the relationship of response to exposure showed a steep decline in activity that is consistent with a constant rate of repair. A novel feature of a model developed from a constant repair rate is an explicit threshold for photosynthetic response to UV.

Mycosporine-like amino acid (MAAs) production by Heterocapasa sp. (Dinophyceae) in indoor cultures.

The possibility of using mycosporine-like amino acids (MAAs), with an apparent sunscreen function in nature, as ultraviolet radiation (UVR) blockers to prevent skin injury has been raised by diverse authors. Production of MAAs by the dinoflagellate Heterocapsa sp. (Dinophyceae) is shown here. Three major peaks with absorption maxima at 330.8, 332.0 and 333.2 nm were detected by high performance liquid chromatography (HPLC) analysis of methanolic extracts in all tested conditions. Analysis of crude extract by mass spectroscopy with electrospray ionization (MS-EI) showed a set of molecular ions ([M+H](+)) with main peaks being at m/z 242.4, 288.4, 303.3 and 333.3 u.m.a. According to these data, along with retention times, the MAA profile of Heterocapsa sp. is assumed to be composed of shinorine (lambda(max)=334 nm), mycosporine-2-glycine (lambda(max)=331 nm) and palythinol (lambda(max)=332 nm). A constitutive MAA content of about 4 microg (10(6) cells)(-1) was measured under exposure to PAR only. A maximal accumulation of MAA per culture volume of 1.1 mg l(-1) was obtained after 72 h of exposure to PAR+UVA, while the highest production rate (0.025 mg l(-1) h(-1)) was computed after 24 h of exposure to PAR+UVA+UVB.