The use of the 14C - method for measurement of primary production in aquatic ecosystem was first outlined by Steemann Nielsen (1952). The principle of the 14C - technique is: additions of 14CO2 in the form of NaH14CO3 to the water sample, where during photosynthesis the algae incorporate the tracer into organic matter. If the total content of CO2 in the experimental water is known, and if the known amount of 14CO2 is added, the rate of primary production can be calculated.
The daily plankton algae production can be estimated by the use of the different methods:
1. In situ method
2. Incubator methods
a. Simulated in situ methods (deck incubator)
b. Other incubator methods (artificial light incubator).
The only difference between the above listed methods is the incubation process.
Except of this the procedure of our measurements was the same.
Procedure:
Preparation of 14C solution:
The 5% w/v sodium chloride solution was prepared from analytical quality salt and distilled water. To each litre of this solution 0.3 g of anhydrous sodium carbonate, Na2CO3 and 1 pellet (ca. 0.2 g) of sodium hydroxide were added. The "carrier free" solution of Na2CO3 with an activity of about 18.5 MBq to 37 MBq per mililitre was obtained. The pH of the solution was adjusted to 9.5-10.0.
Standarisation of 14C solution in the ampoules:
Concentrations corresponding to a 1cm-3 14C solution with the radioactivity of 37-148 kBq cm-3 per 100 cm3 sample is applicable for production studies in the Baltic Sea. The radioactivity of the NaH14CO3 solution and the filters with sestone were measured by the same technique to allow the direct comparison of the two activities. Since May 1991 the liquid scintillation counting has been used.
Sampling:
Usually we chose the following depths for our measurements: 0, 1, 2, 3, 5, 7, 10, 15, 20, 30 meters. The levels closer to the surface are more frequent than the recommended ones. Such a choice of the levels enables a better observation of the maximum of primary production profile and photoinhibition process in the Baltic Sea. Most often the samples were taken between 08 and 10 a.m. except of the experiments with multiplied incubation during the day. The non-toxic bathometer was used.
Incubation:
For the incubation the 100 ml bottles, with standard laboratory grinding and glass stoppers were used. The bottles were filled immediately after sampling. The 0.2 ml of 14C solution was added to obtain 185 kBq per 100 ml sample. The treatment of all the samples, both before and after the incubation, took place in dimmed light, the bottles were kept in darkness. We used to add two "dark bottles" at each station to measure the dark fixation of carbon, one from the surface and second from the deepest level. At the end of experiment the stoppers were removed from the bottles and 1 ml of neutral formaldehyde solution was added to kill organisms and to stop the photosynthesis.
The differences between in situ, simulated and artificial light incubation methods are given at the end of this file.
Filtration:
Water samples were filtered immediately after the production experiment had been stopped. Filtration was carried out in dimmed light. We used the Sartorius filters with pore size 0.45 micrometer. The suction did not exceed 0.4 atm. To remove possible 14C precipitates extracellularly the filters were transferred to the desiccator and exposed to vapours of fuming HCl for 5 min.
Determination of radioactivity:
The Geiger counter with efficiency 6% was used till 1990. (Ocassionally the measurements were carried out with various scintillation counters - in these instances an information is added to data file.) Since May 1991 the Beckman LS 6000 IC liquid scintillation counter has been used.
Total CO2 concentration:
The total carbon can be expressed:
sigmaCO2 = Carbonate alkalinity * F
These values were estimated according to Strickland and Parsons (1972) and Gowinska (1975).
Production calculations:
The total carbon uptake, Pt, during the time t is calculated for every sample according to the following equation:
Pt=dpm(a) totalCO2(c) 12(d) 1.05(e) 1.06(f) k1 k2 k3/dpm(b)
where:
Pt - carbon uptake, [mg C m-3 h-1].
(a) - sample dpm - background dpm - net dpm/sample.
(b) - the activity of the added 14C solution, dpm.
(c) - concentration of total CO2 in the experimental water, [mM/dm3].
(d) - 12: the atomic weight of carbon , converts [mM/dm3] to [mg/dm3].
(e) - a correction for the effect of 14C discrimination, the uptake of the 14C-isotope is 5% slower than that of the 12C-isotope.
(f) - a correction for the respiration of organic matter produced during the experiment. This has empirically been found to represent 6% at optimal photosynthesis. The production rate is thus corrected to represent the rate of gross production.
k1 - a correction factor for subsampling, e.g. 15 cm3 were filtered from a sample of 27 cm3 and 1 cm3 NaH14CO3 was added, then k1=28/15=1.87.
k2 - a time correction factor, e.g. when converting production per 2 hours to production per hour, then k2=0.5.
k3 - a unit conversion factor, e.g. when converting [mg C dm-3] to [mg C m-3], then k3=103.
Incubation methods:
In situ measurements
The in situ technique for measuring production is today the most available approximation of the production in space and time. So we used it most often. In this technique samples are resuspended at the standard depths after 14C incubation. In doing this, special care must be taken to avoid the light excess
because the phytoplankton from greater depths could be "light-shocked". In various experiments the incubation ranged from 2 hours to a "half-day exposition" (from sunrise to midday, or from midday to sunset), most often for 4 hours.
Simulated in situ measurements
Using this technique the samples were placed in an incubator cooled by the surface sea water and exposed to daylight. In order to eliminate UV radiation the samples were kept under a sufficient thick water layer. This method was used only for surface samples during joined Polish-Russian cruises.
Artificial light incubator measurements
In the incubator the experimental bottles were agitated to avoid sedimentation.
The incubator was flooded with overboard water. Irradiances inside the incubator, in the distance of the sample bottles from the light source, are given in data files.
The description of all procedure is given above.
References:
Gowinska A., Ochocki S., Popowska B., Renk H., Torbicki H., 1975, Comparative studies on methods of inorganic carbon determination in research works on primary production., Stud. Mater. MIR 15, (in Polish).
Nielsen G., Bresta A., [Eds.], 1984, Guidlines for the measurement of phytoplankton primary production, BMB 1, 2nd. ed.
Steemann Nielsen E., 1952, The use of radio-active carbon 14C for measuring organic production in the sea., J. Con. Perm. int. Explor. Mer 18(2), 117-140.
Strickland J., D., H., Parsons T., R., 1960, A manual of sea water analysis, Fish. Res. Board Canada, Ottawa.