Dataset: HOT Primary Production

Photosynthetic production of organic matter was measured by the ¹⁴C tracer method. All incubations from 1990 through mid-2000 were conducted in situ at eight depths (5, 25, 45, 75, 100, 125, 150 and 175m) over one daylight period using a free-drifting array as described by Winn et al. (1991). Starting October 2000 (HOT-119), samples were collected from only the upper six depths while the lower two depths were modeled based on the monthly climatology. During 2015, all incubations were conducted in situ on a free floating, surface tethered array. Integrated carbon assimilation rates were calculated using the trapezoid rule with the shallowest value extended to 0 meters and the deepest extrapolated to a value of zero at 200 meters.

A summary of methodology is listed below. Full details can be found at the HOT Field & Laboratory Protocols page.   (http://hahana.soest.hawaii.edu/hot/protocols/protocols.html#) or below in Related Publications section (Karl et al.)

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 

2. Cleaning
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. HCl (Baker Instra-Analyzed) solution (1M) is prepared with high purity hydrochloric acid and freshly-prepared glass distilled deionized water (DDW). 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. Go-Flo bottles, fitted with teflon-coated springs, are rinsed three times with 1M HCl and DDW before use. 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
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. 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. 3.5 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. The new stock activity is checked by counting triplicate 10 µl samples with 1 ml β-phenethylamine in 10 ml Aquasol-II. 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 primary production is measured using in situ incubation techniques. 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. Generally eight incubation depths are selected (5-175 m, approximately).

5. Sampling
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. 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
Three light bottles, three dark bottles and 1 time-zero control (see HOT Protocols 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 Velcro closures. 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. 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
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. 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.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
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. Ten milliliters 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. Therefore, 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 manufacturer (Packard Instrument Co.)

---------
Analysis History for HOT program

• HOT-1 to HOT-7: on deck incubations only
• HOT-8 to HOT-17: on deck and in situ incubations
• HOT-18 to present: in situ incubations only
• HOT 97 to present: sampled from CTD rosette mounted PVC bottles only. Previously used Go-Flo bottles with Kevlar line and Teflon messengers.
• HOT 119 to present: six incubation depths (5-125 meters), light bottles only. Previously had eight depths (5-175 m) with both light and dark incubations
• HOT 178: began using Ultima Gold LLT scintillation cocktail. Switched from Aquasol II that was used previously.