PHYCOB  - Assessment of phycotoxins and their producing species in the Black Sea. The main scientific objectives for the proposed research cruise are: 1) To assess the presence of toxic microalgae in the plankton assemblage of the Western Black Sea. 2) To quantitatively determine the spatial distribution of toxic phytoplankton species and their corresponding toxins in the plankton. 3) To quantitatively describe the spatial distribution of toxic phytoplankton resting stages in surface sediments and determine hotspots of recruitment and bloom initiation. 4) To define correlations among hydrographic and/or meteorological conditions and occurrence of HAB species. 5) To characterize the plankton communities accompanying HAB species together with toxin analysis of size fractionated samples. 6) To identify heterotrophic dinoflagellates, ciliates, or other small zooplankton as possible toxin vectors. 7) To isolate toxic Black Sea microalgal species and establish monoclonal cultures for characterization of Black Sea strains. 8) To perform an interseasonal comparison of toxigenic plankton species by combining the data sets collected during the R/V Akademik cruise that was performed in May/June 2019 with our data from summer. 9) To compare the data set from the Western Black Sea to the Southern (North-) Western North Sea, Baltic Sea and West Greenland obtained during earlier expeditions for site comparisons. The cruise started on 11th September from Istanbul and ended on 17th September back to Istanbul on board of the R/V Tübitak Marmara. Lab work after each station consisted of processing the bottle water and net tows, whereas sediment samples were only transferred into centrifugation tubes and stored for posterior resting stage analysis and germination at IOW in the fridge at 4 °C. Bottle water was taken for nutrient analysis, dissolved oxygen, pH, dissolved organic matter (DOM), particulate organic carbon (POC) and nitrogen (PON), cobalamins (vitamin B12), plankton species determination and quantification, DNA, live sample inspection by microscopy, cell isolation and phycotoxin determination of smaller dinoflagellates. In contrast, phytoplankton net samples from 30 m depth to surface were used for species determination and quantification, DNA, live sample inspection by microscopy, cell isolation and phycotoxin determination of bigger dinoflagellates. Nutrients, dissolved oxygen, pH:  - Nitrite and nitrate as nitrogen (NO3+NO2), ammonium as nitrogen (NH4+), ortho-phosphate as phosphorus (o-PO4) and silica (SiO2) analyzes were measured by a QuAAtro39 Continuous Segmented Flow Analyzer consisting  of an autosampler, a peristaltic pump, a chemistry manifold, a detector and data acquisition software. This protocol is in accordance with standard methods for the examination of water and wastewater.  - Dissolved oxygen (DO) was measured by using the Winkler method, in which a divalent manganese solution and strong alkali are added sequentially to the samples. Potentiometric measurements are performed with Metrohm 905 Titrando automatic titrator system. Dissolved oxygen samples are taken and analyzed in duplicate. The relative percent difference (RPD) of duplicates did not exceed 4.3% (Average is 0.62%). All results were also compared with the CTD dissolved oxygen data.  - pH measurements of sea waters were measured in lab by Mettler Toledo SevenMulti pH meter. The pH meter was calibrated every two days with the calibration standards at the values of 7.00 and 9.21. During the measurements, the pH values were recorded together with the current sample temperature values. - DOM: water samples from the CTD-rosette were taken at various depth (usually 3 m, 10 m, thermocline/Chl-max and 2nd Chl-max. Additionally, at station 1, 4, 10, 15, and 23 deep CTD profiles were taken with additional samples at 100 m, 250 m, 500 m and 1000 m) depth. These samples and filtered through GFF-syringe filters to remove the particulate fraction (<0.7 μm). Chl-a, POC, PON, B12, flow cytometry: - Samples for size fractionated chlorophyll a and particulate organic carbon and nitrogen (POC/PON) were collected from the surface, pycnocline and the deep chlorophyll a maximum. For POC/PON water was filtered onto pre-combusted glass fiber filters, and stored at -20 °C until subsequent analysis on an elemental analyzer in the home lab. Chl-a samples were collected on GFF and on polycarbonate filters with a 2 mm pore size. At each depth 5 mL of seawater was collected and preserved with buffered formalin (1% final concentration) for flowcytometric analysis. - Vitamin B12: Samples were filtered through a 0.7/0.2 mm filter cartridge. After particles were removed, the pH was adjusted to 6.2-6.6 with 1.2 N HCl and the water was slowly (<1.5 mL min-1) pulled through C18 containing columns using a microperistaltic pump for preconcentration prior to analysis.. After all of the sample was processed in this way, the columns were rinsed with 10 mL of deinonized water, removing any salts. Vitamins are eluted from the resin with a small volume of solvent resulting in a 15-20,000 fold concentration factor. Vitamins are analysed by UHPLC-MS/MS. Species quantification: At each station, lugol fixed subsamples of rosette water samples were taken and stored for later quantitative cell counts. At selected stations, samples were fixed with formaldehyde for later taxonomic studies using electron microscopy. Furthermore, 1 L sea water samples from surface (3m), thermocline and chlorophyll maximum layers were taken for the investigation of phytoplankton communities (taxonomic structure, numerical abundance, and biomass) from surface (3m), thermocline and chlorophyll maximum layers. The samples were fixed with formaldehyde 37%. Species identification and cell counting of these samples are done post-cruise under light microscope (Olympus). In addition, on-board analyses by microscopy of live net tow and rosette water samples was performed to characterise the plankton communities, and to document the dinophyte species composition by photo- and video recording. In order to identify and isolate potentially toxin-producing species of dinophytes, rosette water samples were screened by 200 µm gauze, concentrated on 5 µm polycarbonate filters and inspected under the microscope. In addition, various pre-cultures were started by isolating single cells of species of interest. After transportation back to AWI Germany, these samples have been inspected for the growth of algal species of interest, especially for cultures of toxigenic species of Alexandrium, Lingulodinium, Protoceratium, or Azadinium. DNA: An 1 L aliquot of pooled water from surface water, 10 m depth and the thermocline was filtered through 1 μm pore-size polycarbonate filters. In addition, an aliquot (50 mL) of the net tow concentrate was fixed with formaldehyde for analysis by light microscopy and another aliquot (150 mL) was filtered for DNA. DNA from the filters was extracted with Chelex buffer (5%) and stored for analysis. Phytotoxins: An 8 L aliquot of pooled water from surface water, 10 m depth and the thermocline was filtered through 5 µm polycarbonate filters for azaspiracid and karlotoxin determination. The filters were extracted by repeated rinsing with methanol, the methanolic extracts were transferred to centrifugation filters and filtered at a cutt-off of 0.45 µm by centrifugation. Filtrates were transferred into analytical glass vials for posterior LC-MS/MS determination at AWI. The remaining aliquot of the net tow concentrate was filtered through a filter array of 200, 50, and 20 µm meshes. The residue of each mesh was rinsed with filtrated seawater into a centrifugation tube, homogenized by shaking, and divided into two aliquots. Both aliquots of each size fraction were centrifuged at maximum speed for 15 min. After centrifugation, supernatants were decanted and the remaining cell pellets resuspended with a small volume of filtrated seawater and transferred to cryovials containing ceramic beads. The samples were centrifuged again and supernatants carefully removed with a pipette. To one of the two size fraction aliquots 500 µL methanol was added and the same volume of dilute acetic acid to the other. All samples were homogenized with FastPrep instrument by reciprocal shaking at maximum speed and homogenated were centrifuged. After centrifugation, the toxin containing extracts were removed and transferred to centrifugation filters. The extracts were filtered by centrifugation and the filtered extracts were transferred into analytical glass vials for LC-MS/MS and LC-FLD determination.