SUMMARY: The 14C-radiotracer method is used to measure the assimilation of dissolved inorganic carbon (DIC) by phytoplankton as an estimate of the rate of photosynthetic production of organic matter in the euphotic zone.

1. Rationale and Assay Principle

The 14C method, originally devised by Steeman-Nielsen (1952), is used to estimate the uptake of DIC by photoautotrophic microorganisms in the water column. The method is based on the fact that the biological uptake of 14C-labeled DIC is proportional to the biological uptake of 12C-DIC. If one knows the initial concentration of DIC in a water sample, the DIC pool specific radioactivity (14C/12C), the 14C retained in particulate matter (14C-POC) at the end of the incubation and the metabolic discrimination between the two isotopes of carbon (i.e., 6% discrimination against the heavier 14C isotope), then it is possible to estimate the total uptake of carbon from the following relationship:

                            DIC * 14C-POC * 1.06
                Cuptake  =   --------------------
                                14C-DIC added 

2. Precautions

Due to the potentially toxic effects of trace metals on phytoplankton metabolism in oligotrophic waters, the following procedure is used to minimize the contact between water samples and possible sources of contamination.

2.1. Trace metal grade HCl (Fisher for example) solution (1M) is prepared with DIW.
2.2. Polycarbonate (PC) bottles (500 ml) are rinsed twice with trace metal grade 1M HCl and left overnight filled with the same acid solution. The acid is discarded and the bottles are rinse at least 3 times with DIW before air drying in a clean environment.

3. Isotope Stock

3.1. A 60 ml acid-washed Teflon bottle and a 100 ml acid-washed Teflon bottle are rinsed 3 times with DIW.
3.2. A 6 mM Na2CO3 solution is made up of anhydrous Na2CO3 in DIW (Sigma-Aldrich; 99.999% purity; 0.064 g:100 ml DIW) is made up in the Teflon bottle.
3.3. NaH-14CO3 (MPBiomedicals # 17441H, 2 mCi ml-1) is mixed with the above-prepared Na2CO3 solution in the Teflon bottle to yield a working stock solution of 200 µCi ml-1. The working stock solution is made up to the volume required for each cruise.
3.4. The activity is measured by counting triplicate 10 µl samples with 1 ml beta-phenethylamine (Sigma-Aldrich) in 10 ml Ultima Gold LLT (Perkin-Elmer).
3.5. Triplicate 10 µl stock samples are also acidified with 1 ml of 2 M HCl, vented for 24 hours and counted in 10 ml Ultima Gold LLT to assess the level of 14C-organic carbon contamination. The acidification is done under the hood. The acidified dpm should be <0.001% of the total dpm of the 14C preparation or else the working stock 14C solution should not be used. These last two steps are performed periodically, and always when receiving new batches from the manufacturer.

4. Incubation Systems

Typically we measure primary production using in situ incubation techniques, but both in situ and on-deck procedures have been used in the HOT program.

4.1. A free-floating array equipped with VHF radio and strobe light is used for the in situ incubations. Incubation bottles are attached to a horizontal PC spreader bar which is then attached to the 200 m, 1/2" POLYPRO in situ line at the depths corresponding to the sample collections.
4.2. Generally six to eight incubation depths are used with the top 175 m.

5. Sampling

5.1. Approximately 3 hr before local sunrise, seawater samples are collected using the CTD rosette mounted PVC bottles.
5.2. Under low-light conditions, water samples are collected into the sample rinsed incubation bottles (500 ml clear PC bottles) directly from the spigot, filled to the brim, capped and stored in the dark. Powder-free vinyl gloves are worn during sample collection and inoculation procedures.

6. Isotope Addition and Sample Incubation

6.1. Three field replicate bottles are collected at each depth for in situ incubation.
6.2. After all water samples have been drawn from the appropriate bottles, 200 µl of the 14C-NaHCO3 stock solution is added to each sample using a clean repipettor tip (final 14C-activity ~ 80-100 µCi l-1). The bottles are capped tightly. The samples are deployed before dawn on a free-floating, drifter buoy array.
6.3. At local sunset, the free-floating array is recovered and all in situ bottles are immediately placed in the dark and processed as soon as possible. The time of recovery is recorded.

7. Filtration

7.1. Filtration of the samples is done under low or red light conditions and begins as soon as the incubation bottles are recovered from the in situ array.
7.2. From each incubation bottle 250 µl is removed and placed into 20 ml glass, liquid scintillation counting vials (LSC vials) containing 0.5 ml of phenethylamine. This sample is used for the determination of total radioactivity in each sample and to calculate the specific activity of 14C-inorganic carbon in the samples.
7.3. The remainder of the sample is filtered through a 25 mm diameter GF/F filter. The filter is placed into a second clean glass LSC vial and stored at -20°.

8. 14C Sample Processing

8.1. One ml of 2 M HCl is added to each sample vial containing a filter (in a fume hood). Vials are left open to vent under the hood for 24 hr.
8.2. After the samples have vented 10 ml of Ultima Gold LLT scintillation cocktail is added per vial including the vials for total 14C radioactivity. The samples are counted in a liquid scintillation counter. Samples are counted again after one month. Only the last count is used for primary production calculations. Counts per min (cpm) are converted to disintegration per min (dpm) by using the instrument’s tSIE protocol (transformed Spectral Index of the External standard) and quench curve (Perkin-Elmer TR 2800).

9. Calculations

From the data derived above we can estimate several properties of the phytoplankton populations at Sta. ALOHA. Total daylight organic carbon production is calculated from the 12-hr uptake data. Net primary production is used as the estimate of phytoplankton carbon production for the purposes of comparison to other ecosystem-level processes (e.g., standing stock assessments, vertical C-flux, etc.).

10. Equipment/Supplies

11. Reagents

12. References

13. HOT Program Analytical Summary