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. Principle

The 14C method, originally proposed by Steeman-Nielsen (1952), is used to estimate the uptake of dissolved inorganic carbon (DIC) by planktonic algae 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 amount of 14C-DIC added, the 14C retained in particulate organic matter (14C-POC) at the end of the incubation and the metabolic discrimination between the two isotopes of carbon (i.e., 5% discrimination against the heavier 14C isotope), then it is possible to estimate the total uptake of carbon from the following relationship:

                              DIC * 14C-POC * 1.05
                C uptake  =   --------------------
                                  14C-DIC added 

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. Cleaning

2.1. HCl (Baker Instra-Analyzed) solution (1M) is prepared with high purity hydrochloric acid and freshly-prepared glass distilled deionized water (DDW).
2.2. 500 ml polycarbonate bottles are rinsed twice with 1M HCl (Baker Instra-Analyzed) and left overnight filled with the same acid solution. The acid is removed by rinsing the bottles three times with DDW before air drying.
2.3. Go-Flo bottles, fitted with teflon-coated springs, are rinsed three times with 1M HCl and DDW before use.
2.4. Pipette tips used in the preparation of the isotope stock and in the inoculation of samples are rinsed three times with concentrated HCl (Baker Instra-Analyzed), three times with DDW and once with the sodium carbonate solution (Chapter 14, section 3.2) and stored in a clean polyethylene glove until used.

3. Isotope Stock

3.1. The preparation of the isotope stock is performed wearing polyethylene gloves. A 25 ml acid-washed teflon bottle and a 50 ml acid-washed polypropylene centifuge tube are rinsed three times with DDW.
3.2. 0.032 g of anhydrous Na2CO3 (ALDRICH 20,442-0, 99.999% purity) are dissolved in 50 ml DDW in the centrifuge tube to provide a solution of 6 mmol Na2CO3 per liter.
3.3. 3.5 ml of NaH-14CO3 (53 mCi mmol-1; Research Products Inc.) are mixed with 16.5 ml of the above prepared Na2CO3 solution in the teflon bottle.
3.4. The new stock activity is checked by counting triplicate 10 µl samples with 1 ml β-phenethylamine in 10 ml Aquasol-II.
3.5. Triplicate 10 µl stock samples are also acidified with 1 ml of 2 M HCl, mixed intermittently for 1-2 hours and counted in 10 ml Aquasol-II to confirm that there is no 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.

4. Incubation Systems

Typically we measure primary production using in situ incubation techniques.

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 polycarbonate spreader bar which is then attached to the 200 m, 1/2" polypropylene in situ line at the depths corresponding to the sample collections.
4.2. Generally eight incubation depths are selected (5-175 m, approximately).

5. Sampling

5.1. Approximately 3 hours before local sunrise, seawater samples are collected with acid- washed, 12-liter Go-Flo bottles using Kevlar line, metal-free sheave, teflon messengers and a stainless steel bottom weight. A dedicated hydrowinch is used for the primary productivity sampling procedures in a further effort to reduce/eliminate all sources of trace metal contamination.
5.2. Under low light conditions, water samples are transferred to the incubation bottles (500 ml polycarbonate bottles) and stored in the dark. Polyethylene gloves are worn during sample collection and inoculation procedures. No drawing tubes are used.

6. Isotope Addition and Sample Incubation

6.1. Three light bottles, three dark bottles and 1 time-zero control (see Chapter 14, section 8) are collected at each depth for in situ incubation. In situ dark bottles are deployed in specially- designed, double-layered cloth bags with VelcroR closures.
6.2. After all water samples have been drawn from the appropriate Go-Flo bottles, 250 µl of the 14C-sodium carbonate stock solution is added to each sample using a specially-cleaned pipette tip. 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 light conditions and begins as soon as the incubation bottles are recovered from the in situ array.
7.2. 200 µl are removed and placed into a second LSC vial containing 0.5 ml of β-phenethylamine. This sample is used for the determination of total radioactivity in each sample.
7.3. The remainder is filtered through a 25 mm diameter GF/F filters. The filters are placed into prelabelled, clean glass liquid scintillation counting vials (LSC vials) and stored at -20 °C.

8. 14C Sample Processing

8.1. One ml of 2 M HCl is added to each sample vial (under the hood). Vials are covered with their respective caps and shaken in a vortex mixer for at least 1 hour with venting at 20 minute intervals. To vent, the vials are removed from the shaker, and the cap opened (under the hood). After shaking is completed, the vials are left open to vent under the hood for an additional 24 hours.
8.2. Ten ml of Aquasol-II are added per vial (including vials for total 14C radioactivity) and the samples are counted in a liquid scintillation counter. Samples are counted again after 2 and 4 weeks, before discarding. Counts have shown a consistent increase during the first two weeks and become stable between the second and the fourth week. This is probably the result of sample hydrolysis or diffusion of radioactivity from the GF/F filter matrix, thereby reducing the extent of self-absorption. Only the 4-week count is used for 14C calculations. Counts per min (CPM) are converted to disintegration per min (DPM) using the channels ratio program supplied by the the manufacturer (Packard Instrument Co.).

9. Calculations

From the data derived above we can estimate several properties of the phytoplankton populations at Station ALOHA. Total daylight organic carbon production is calculated from the 12-hour uptake data (after corrections for 12-hour dark activities). Net daily organic carbon production is calculated from the 24-hour light/dark samples (corrected for the time-zero blank activities). Phytoplankton population respiration is taken as the difference between the 12-hour light and the 24-hour light/dark incubations. 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