1. Phytoplankton carbon content and Chlorophyll

3. At least 20 specimens of each species are measured for cell volume calculations. When fewer than 20 specimens are present, those present are measured as they occur. The measurements required are those which are necessary for the volume calculation of a solid which best approximates the shape of any particular organism. For most organisms the measurements are taken from out-side wall to outside wall.

4. Those forms which are loricate (Diatoms, Chlorophyta, Chrysophyta) must have the active portion, i.e., protoplast measured. Filamentous and colonial forms require measurements of the individual components.

5. If cell walls are not readily visible in the filament, the length of the entire colony is measured and the number of cells is determined by dividing by the average cell length which is measured when possible.

7. From biovolume to C content: Diatoms - log 10 C (pg)= 0.758(log 10 V)- 0.422 Others - log 10 C (pg)= 0.866 (log 10 V)- 0.46 C = C content in pg V cell volume in m 3 (Strathmann, 1967) Or Diatoms - log 10 C (pg)= 0.811 (log 10 V)- 0.541 Others - log 10 C (pg)= 0.939 (log 10 V)- 0.665 Menden-Duer & Lessard (2000)

9. Phytoplankton C content (by species) in the Gulf of Trieste over a year

11. Chlorophyll measuraments Chlorophyll is a easy to detect proxy of the phytoplankton biomass, but the relationship between phytoplankton C content and chlorophyll varies from < 20 up to 80 depending on the species present in the water and their physiological state. Multiprobes usually can detect chlorophyll along the water column as fuorescence both natural and excited. It is also possible to detect chlorophyll concentration by remote sensing More frequently chl is detected by chemical analyses

13. December

14. March

15. July

16. October

20. Chlorophyll measurements: This method is chiefly employed to estimate phytoplankton biomass. The most useful chemical method for determining the total quantity of phytoplankton in seawater is to estimate the amount of chlorophyll usually as chlorophyll a. This is a rapid method for determining phytoplankton density in a sample and involves the extraction and measurement of chlorophyll concentrations. The amount of pigments as chlorophyll a, b, c and phaeophytin is considered as a measure of phytoplankton biomass.

21. Water samples are collected from Niskin bottles into clean polyethylene bottles. Samples are immediately filtered through 47 mm GF/F filters using polycarbonate in-line filters (Gelman) or through Millipore (Pore size 0.45 μm) filters and a vacuum of less than 100 mm Hg and is pumped to dryness. All steps should be carried out in the dark to avoid pigment breakdown. Filters are folded in half twice and wrapped in aluminum foil, labeled, and stored in liquid nitrogen (to avoid formation of degradation products) until shore analysis. Alternatively, filters can be placed immediately in acetone for pigment extraction if analysis is to be carried out onboard ship.

22. Some recommends addition of 1 ml of a 1% MgCO 3 suspension onto the filter to form a thin bed, which will serve as a precaution against the development of any acidity and subsequent degradation of pigment in the extract. In oligotrophic waters, for this measurement coupled with HPLC determined pigments, 4 liters are filtered. For fluorometric analysis alone, a smaller volume (0.5 - 1.0 l) may be sufficient. In coastal regions, a volume of 0.1-0.5 l may be adequate. In this case, use of 25 mm GF/F filters may be appropriate.

23. After removal from liquid nitrogen or freezer, the filter is brought to room temperature. The pigments are extracted by placing the filters in 5.0 ml 100% acetone. Work quickly and ensure all containers with acetone are capped, as it is very volatile. Work with acetone in the hood only and use gloves. The samples are covered with Parafilm to reduce evaporation, sonicated (0°C, subdued light) and allowed to extract for 24 hours in the dark at -20°C. Following extraction, samples are vortexed, filters are pressed to the bottom of the tube with a stainless steel spatula and spun down in a centrifuge at 5000 rpm for 5 minutes to remove cellular debris and the supernatant solution is considered for the determination of optical density, or transmission percentage which is mainly obtained with the aid of a fluorometer.

24. Algal pigments, particularly chlorophyll a, fluoresce in the red wavelengths after extraction in acetone when they are excited by blue wavelengths of light. The fluorometer excites the extracted sample with a broadband blue light and the resulting fluorescence in the red is detected by a photomultiplier. The significant fluorescence by phaeopigments is corrected for by acidifying the sample which converts all of the chlorophyll a to phaeopigments. By applying a measured conversion for the relative strength of chlorophyll and phaeopigment fluorescence, the two values can be used to calculate both the chlorophyll a and phaeopigment concentrations.

25. The fluorometer is allowed to warm up and stabilize for 30 minutes prior to use. The fluorometer is zeroed with 90% acetone. 1.0 ml of pigment extract is mixed with 4.0 ml 90% acetone in a cuvette and read on the appropriate door to give a reading between 30 and 100. The sample is then acidified with 2 drops of 1.2 M HCl. Further dilutions may be necessary for higher chlorophyll a concentrations. For laboratory use, the fluorometer is calibrated every 6 months with a commercially available chlorophyll a standard (Anacystis nidulans, Sigma Chemical Company). If the fluorometer is taken to sea, it is recommended that the fluorometer be calibrated before and after each cruise.

29. Fig. 1.8.1 - Boxplots degli andamenti mensili delle concentrazioni di clorofilla a. Sono evidenziati: il valore mediano mensile (  ); il valore medio mensile ( ▫ ), il riquadro comprendente il 50% dei dati (  ), le due barre che racchiudono il 75% dei dati, gli out layers (ж).

30. Fig. 1.8.2 - Boxplots degli andamenti mensili delle concentrazioni di feopigmenti. Sono evidenziati: il valore mediano mensile (  ); il valore medio mensile ( ▫ ), il riquadro comprendente il 50% dei dati (), le due barre che racchiudono il 75% dei dati, gli out layers (ж).

31. Fig. - 1.8.3 Boxplots degli andamenti mensili del rapporto Feopigmenti/Clorofilla a. Sono evidenziati: il valore mediano mensile (  ); il valore medio mensile ( ▫ ), il riquadro comprendente il 50% dei dati (), le due barre che racchiudono il 75% dei dati, gli out layers (ж).