Participants: Survey No. 2: William M. Balch, Katherine Kilpatrick
Measurements, protocols and QA/QC:
C stock preparation: The C-bicarbonate stock solution will be cleaned by acidification with sulfuric acid to drive off the CO into the headspace of an enclosed glass vessel and the headspace gas recirculated through 1 ml of 1 N NaOH to collect the . The 1 ml of NaOH will be subsequently diluted with Milli-Q water to a final activity of about 1 mCi per ml, 0.2 mm filter sterilized and stored in acid cleaned, sterile, glass vials with teflon screw caps. The stock will be removed with sterile, acid-cleaned, plastic micropipetters (not metal syringe needles). Addition of this alkaline stock to seawater at the levels we will be using changes the pH of the seawater by less than 0.01 pH units.
Bottle preparation: All incubations will be done in 250 ml polycarbonate bottles. The bottles will have been soaked for 5 days in warm Alconox detergent solution (to avoid the ammonium contamination found in Micro detergent), rinsed 3 times and soaked over night with deionized water, rinsed once with Milli-Q water, then soaked 3 days in 20 % HCl solution, and rinsed 5 times in Milli-Q water. At sea, used bottles will be rinsed with isotope free filtered sea water, rinsed once with Milli-Q water and 90 ethanol to kill any bacterial/algal films adhering to the bottle walls, followed by a 10 % HCl rinse, then 3 rinses with Milli-Q water.
Sampling protocol: Samples will be drawn from Niskin bottles before sunrise, and transferred to the 250 polycarbonate bottles (and kept in darkness). 20 mCi of C-bicarbonate stock will be added to each bottle from the 10 depth cast, and the bottles will be either placed in the deck incubator or put in large mesh bags to be attached to the in situ arrays, then deployed. Samples will be incubated for 24 hours, after which the contents of each bottle will be split into two 100 ml aliquots, and filtered onto 0.4 mm nuclepore filters (these filters are required to maintain low interstitial water content, hence low background C activity). Each replicate filter will be fumed for 3 minutes in a desicator containing concentrated HCl to drive off activity due to calcite. Filters will then be placed in a scintillation cocktail, and radioactivity measured in a liquid scintillation counter on board the ship. Calcification rates are calculated based on the difference between the unfumed filter counts and the fumed filter counts.
QA/QC: We will check our photosynthesis numbers against R. T. Barber's for internal consistancy, especially since different types of filters must be used. We will also check by filtering our own samples with the various filter types. Trace metal contamination will be checked by adding EDTA to a separate C incubation bottle and comparing this to the untreated control. A check on loss of organic material following fuming has been performed by incubating non-calcifying coccolithophore clones with C, and fuming as described above. When the filter activities were subtracted, the difference was zero, not a negative value, which demonstrates that no organic carbon is lost in the fuming procedure.
Calcite standing stock measurements: The abundance of calcium carbonate coccoliths can be estimated continuously using an optical technique. Water is diverted from the autoanalyzer flow stream and run through a glass cuvette in front of a laser beam (660 nm light). Light scatter at 90 is measured with a photodiode, and this value will be recorded using a data logging system. Every 5 minutes, the stream passing the laser will be diluted with glacial acetic acid to drop the pH to 6, and dissolve calcium carbonate, and the 90 scatter will be measured again. The difference in the scatter values represents the 90 light scatter due to the acid labile fraction. This has been shown to be highly correlated to the standing stock of coccoliths (see Balch et al., 1991; Limnology and Oceanography; 36: 629--643). These measurements will be run during transects and vertical profiles.
QA/QC: The standing stock estimates will be calibrated using microscope counts made on the ship, as well as counts from samples preserved with Lugols solution. The microscope we use has polarization optics which allow the birefringent coccoliths to be easily counted (and any other calcium carbonate particles for that matter).