Photosynthetic production of organic matter was measured by\u00a0the 14C tracer method. Beginning July 1989 (HOT-008), all incubations were conducted\u00a0in situ\u00a0on a free-floating, surface tethered array. All incubations from 1990 through mid-2000 were conducted in situ at eight depths (5, 25, 45, 75, 100, 125, 150 and 175 m) over one daylight period using a free-drifting array as described by Winn et al. (1991). Starting October 2000 (HOT-119), samples were\u00a0collected\u00a0from only the upper six depths while the lower two depths were modeled based on the monthly climatology. 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.
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A summary of methodology is listed below.\u00a0Full details can be found at the HOT\u00a0Field & Laboratory Protocols page.\u00a0 \u00a0(http://hahana.soest.hawaii.edu/hot/protocols/protocols.html#) or below in Related Publications section (Karl et al.)
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1. Principle
\nThe 14C method, originally proposed by Steeman-Nielsen (1952), is used to estimate the uptake of dissolved inorganic carbon (DIC) by photoautotrophic microorganisms\u00a0in the water column. The method is based on the fact that the biological uptake of\u00a014C-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:
\n\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 DIC * 14C-POC * 1.06
\n\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 C uptake\u00a0 =\u00a0 \u00a0--------------------
\n\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 14C-DIC added\u00a0
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2. Precautions
\nDue 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. Trace metal grade HCl (i.e. FisherScientific)\u00a0solution (1M) is prepared with high purity hydrochloric acid and deionized water (DIW). Polycarbonate bottles (500 ml) are\u00a0rinsed twice with trace metal grade 1M HCl and left overnight filled with the same acid solution. The acid is discarded and the bottles are rinsed at least three times with DIW before air drying in a clean environment.
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3. Isotope Stock
\nFor the preparation of the isotope stock, a\u00a060 ml acid-washed Teflon bottle and a 100 ml acid-washed Teflon bottle are rinsed three times with DIW. A six\u00a0millimolar (6 mM) sodium carbonate solution containing anhydrous Na2CO3 in DIW (Sigma-Aldrich; 99.999% purity; 0.064 g:100 ml DIW) is made up in the Teflon bottle. 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 microCuries per milliliter (\u00b5Ci ml-1). The working stock solution is made up to the volume required for each cruise. The radiocarbon activity is measured by counting triplicate 10 \u00b5l samples with 1 ml beta-phenethylamine (Sigma-Aldrich) in 10 ml Ultima Gold LLT (Perkin-Elmer). Triplicate 10 \u00b5l 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\u00a0preparation 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.
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4. Incubation Systems
\nTypically primary production is measured using in situ incubation techniques, but both in situ and on-deck procedures have been used in the HOT program. 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 six to eight incubation depths are used with the top 175 meters.\u00a0
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5. Sampling
\nApproximately 3 hours before local sunrise, seawater samples are collected using the PVC bottles mounted on the CTD rosette.\u00a0Under 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.
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6. Isotope Addition and Sample Incubation
\nThree field replicate bottles are collected at each depth for in situ incubation. After all water samples have been drawn from the appropriate bottles, 200 \u00b5l of the 14C-NaHCO3 stock solution is added to each sample using a clean re-pipettor tip (final 14C-activity ~ 80-100 \u00b5Ci l-1). The bottles are capped tightly. 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.
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7. Filtration
\nFiltration of the samples is done under low light conditions and begins as soon as the incubation bottles are recovered from the in situ array. From each incubation bottle 250 \u00b5l are removed and placed into 20 mL glass liquid scintillation counting (LSC) vials. This sample is used for the\u00a0determination of total radioactivity in each sample and to calculate the specific activity of 14C-inorganic carbon in the samples. The remainder of the sample is filtered through a 25 mm diameter GF/F filter. The filter is placed into a second\u00a0clean glass LSC vial\u00a0and stored at -20 \u00b0C.
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8. 14C Sample Processing
\nOne 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 hours. 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\u2019s tSIE protocol (transformed Spectral Index of the External standard) and quench curve (Perkin-Elmer TR 2800).
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\nAnalysis History for HOT program
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\n- HOT-1 to HOT-7: on deck incubations only
\n- HOT-8 to HOT-17: on deck and\u00a0in situ\u00a0incubations
\n- HOT-18 to present:\u00a0in situ\u00a0incubations only
\n- HOT 97 to present: sampled from CTD rosette mounted PVC bottles only. Previously used\u00a0Go-Flo bottles with Kevlar line and Teflon messengers.
\n- HOT 119 to present: six incubation depths (5 to 125 meters) with light bottles only. Previously had eight depths (5 to 175 meters) with both light and dark incubations
\n- HOT 178: began using Ultima Gold LLT scintillation cocktail. Switched from Aquasol II that was used previously.\u00a0
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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.
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From the data derived here we can estimate several properties of the phytoplankton populations at Station ALOHA:
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\n- \u00a0Total daylight organic carbon production is calculated from the 12-hour uptake data (after corrections for 12-hour dark activities).
\n- 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.).
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Quality Flags
\nQuality Flags were assigned for the bottle, chlorophyll, phaeopigments, light incubation, dark incubation, salinity & bacteria values respectively.
\n1: not quality controlled
\n2: good data
\n3: suspect (questionable) data
\n4: bad data
\n5: missing value
\n\u200b9: variable not measured during this cast