On the occasion of the workshop of the group on aquatic primary productivity (GAP) held in Konstanz (1982), 14C-uptake rates were determined by two widely used, well-established procedures. In order to avoid any variation in the results caused by manipulation of the samples, subsamples for both determinations were withdrawn from the same bottles. The acid-bubbling method (ABM) yielded results which exceeded those of the filtration method by about 30%. Excretion of 14C labelled dissolved organic matter was negligibly small and therefore cannot account for the observed differences. Based on available information also other possible explanations discussed can Likely be dismissed. Hence additional effort is needed to identify and eliminate possible shortcomings in either metho

Gächter, René

Mares, Antonin

tilzer, Max M.

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Journal of Plankton Research Volume 6 Number 2 1984Determination of phytoplankton production by the radiocarbonmethod: a comparison between the acidification and bubblingmethod (ABM) and the filtration technique*Rene Gachter, Antonin Mares and Max M. Tilzer1Swiss Federal Institute for Water Resources and Water Pollution Control(EAWAG), CH-8600 DUbendorf, Switzerland, and lLimnological Institute,University of Constance, P.O. Box 5560, Konstanz, FRG(Received May 1983; accepted November 1983)Abstract. On the occasion of the workshop of the group on aquatic primary productivity (GAP)held in Konstanz (1982), "C-uptake rates were determined by two widely used, well-established pro-cedures. In order to avoid any variation in the results caused by manipulation of the samples, sub-samples for both determinations were withdrawn from the same bottles. The acid-bubbling method(ABM) yielded results which exceeded those of the filtration method by about 30%. Excretion of "Clabelled dissolved organic matter was negligibly small and therefore cannot account for the observeddifferences. Based on available information also other possible explanations discussed can likely bedismissed. Hence additional effort is needed to identify and eliminate possible shortcomings in eithermethod.IntroductionArthur and Rigler (1967) first showed that the specific activity of the labeledmaterial retained on the filters (as cpm ml"1 filtered) may decrease with theamount of sample filtered. This was attributed to cell leakage with increasingfiltering times and pressure. Algae release dissolved organic material which canbe utilised by heterotrophic microorganisms. Therefore it is to be considered asprimary production but is not measured by the filtration technique (Watt, 1966;Fogg, 1971; Berman, 1976; Mague et al., 1980; Larsson and Hagstrom, 1982).Sharp (1977) attributed what previously had been termed 'extracellular release' toexperimental artifacts such as cell leakage, organic contamination and release bycultural shock. Regardless of whether loss of I4C labeled dissolved organic carbon(DOC) is due to artifacts or release by viable cells, primary production is under-estimated if labeled DOC is not measured.The acidification and bubbling technique (ABM) introduced by Schindler et al.(1972), offers several advantages over the conventional filtration technique, (i)Fragile organisms are not destroyed by filtration and photosynthates are not lost,(ii) It avoids contamination of the sample with dissolved (McMahon, 1973) orpaniculate inorganic l*C which might occur at high pH values, when CaCO3precipitates, (iii) Activities of added inorganic 14C and assimilated 1*C aremeasured with equal efficiencies and therefore no quench corrections are needed.Acidification has been shown not to interfere with 14C counting efficiency, (iv)Because the liquid scintillation counting technique does not discriminate betweenThis paper is the result of a study made at the Group for Aquatic Primary Productivity (GAP) FirstInternational Workshop held at the LJmnological Institute, University of Konstanz, in April 1982.© IRL Press Limited. Oxford, England 359R.G3chUr, A.Marb and M.M.TUzerhomogeneously suspended 14C labeled algal cells and dissolved "C, thephenomenon of self-absorption which might occur when algae are concentratedon Filters (Pugh, 1973; Gargas, 1975) is negligible in the ABM (Gachter et al.,1979).The main shortcoming of the ABM as compared to the filtration technique isits lower sensitivity because only relatively small fractions of the entire sample (inour case 6%) are used for the determination. Therefore, relatively 'hot' labels arerequired which in turn may raise blanks. By careful handling moreover, several ofthe above mentioned shortcomings of the 14C filtration technique can be avoidedor at least minimised.The objectives of our experiments were: (i) To compare the results of the ABMtechnique with those of the filtration technique routinely applied at the Limno-logical Institute at Konstanz. In order to avoid any variation in the results causedby the manipulation of the samples, subsamples for both determinations of the14C-uptake rate were withdrawn from the same bottles, (ii) To estimate the label-ing rate of DOC. Our experiments were carried out in two systems that differedmarkedly with regard to total phytoplankton biomass and species composition(Lake Constance and experimental pond at the Limnological Institute, respec-tively). For details see Sakamoto et al., 1984).MethodsTwo light bottles and one dark bottle of -120 ml volume were used for the in-cubations. In situ exposure lasted for 4 h. The activity of the 14C bicarbonatesolution added to each bottle was 8 /tCi. Dissolved inorganic carbon (DIQ wasdetermined from alkalinity titrations (0.1 n HC1 and methyl-orange-bromecresole-green indicator) and pH-measurements. The concentration of DIC wascalculated by the following equation derived from Stumm and Morgan (1981):= Alk.(Meq I"1) 12,0002K710-PHwhere A:, (= 109M) and K2(= 10"10M) are the dissociation constants of the car-bonic acid at 20°C.After incubation two 7 ml samples were withdrawn from each flask for theABM technique and 50 ml for the filtration technique. The samples were process-ed as follows:Acidification and bubbling method (ABM)Determination of 14C available and 14C assimilated. Two subsamples werepipetted into glass scintillation vials. In order to determine the activity of the add-ed 14C (14C available) one subsample was mixed with 10 ml of Luma-Gel® , aready for use scintillator. At this water to fiuor ratio a stable monophasic gel isproduced, keeping 14C labeled particles in suspension. The other subsample wasacidified with 0.1 ml of 0.1 N HC1 and bubbled with air for 1 h as shown inFigure 1 prior to addition of Luma-Gel® . This procedure removes inorganic 14C360Comparison of radiocarbon techniquesN2iFig. 1. Apparatus for bubbling acidified samples. (1) Plexiglaj tubing with attached silicone rubberstoppers, each of which is bored through with a hypodermic needle; (2) liquid scintillation countingvials; (3) CO, absorber; and (4) line to vacuum pump.quantitatively and the remaining activity ("C assimilated) is due to labeledorganic 14C in either particulate or dissolved form.Determination of1AC activity in the filtrate P*C filtrate). To estimate the pro-duction of "C labeled dissolved organic matter, particles were removed by lowvacuum filtration (Sartorius membrane filters, pore size 0.2 /im). Then 7 ml ali-quots were acidified and treated as described above.Determination of background activity ^*C background). To consider possibletrace impurities of organic 14C in the 1*C stock solution, lake water was passedthrough a Sartorius membrane filter (pore size 0.2 /tm) to remove algae andbacteria. Then 8 fiC "C/100 ml were added. Ten 7 ml aliquots were acidified im-mediately after labeling, bubbled for 1 h and mixed with 10 ml Luma-Gel® asdescribed above. These samples yielded an activity of 40.1 ±3 .0 cpm.Calculation of results. Total primary production (PP) and the production of14C labeled DOM ("C-DOM) were calculated as follows:pp _ l*C assimilated - background"C available - background"C-DOM = 14C filtrate - background14C available - backgroundDICwhere DIC is the concentration of dissolved inorganic carbon. No correction forisotope discrimination in carbon uptake rates was considered. "C-DOM mayunderestimate the true rates of "C-DOC excretion because during the 4 h ex-posure period the specific activity of excreted DOC is not constant and not equalto that of the available DIC, but increases from zero at the beginning to anunknown value at the end of the experiment. However, observations of Mague etal. (1980) indicate, that 14C labeling of the pool supplying "C-DOM may occurvery quickly after incubation with H"CO3~. Excretion of DOC may further beunderestimated due to uptake and partial mineralisation of labeled exudates byheterotrophic organisms.361R.Gachter, A.Marts and M.M.TDzerLAKE CONSTANCEmgC m h1 2 3 4 5 6 715 JmPONDmgC in" h1 2 3 4 5 6 7 80.15-0.30-050-¥tACi0)Fig. 2. Comparison of primary production measurements in (a) Lake Constance and (b) experimen-tal pond. ( • ) light and (O) dark bottles ABM. ( • ) and (A) dark bottles filtration technique.Filtration techniqueDetermination of 1*C added and l*C assimilated. To determine the activity ofthe added 14C solution, after incubation, 1 ml was withdrawn from each flaskwith a precision pipette, and injected into a scintillation vial that contained onedrop of 2 N NaOH to prevent escaping of 14CO2. Thereafter 2.5 ml of unisolvegel (Zinsser, Frankfurt/M.) were added. Filtration onto 0.8 /an cellulose acetatemembranes (Sartorius, Gottingen) was performed at dim light. The vacuum was- 35 mm Hg. By using two 6-place manifolds the filtration time was minimised.The filters were rinsed with distilled water three times and thereafter dissolved in3.0 ml of dioxane cocktail (Riedel de Haen No. 24224). No fuming with HC1 asproposed by Berman (1973) was applied because of consistently low blanks.Counting was performed in a Beckman LS 7500 liquid scintillation counter. Thecounting efficiencies determined by using toluene standards were 80% in the 1-mlwater samples in unisolve gel and 90% for the dissolved filters in Dioxanecocktail.Calculation of results. Results were calculated by using the following equation:Qs, = DIC(mgCm-') • <?"* fl~l *<*cpmw f2"1 50Where cpmp are the counts of the filters, cpmw counts of the wateT samples andfx = 0.90 and f2 = 0.80 are the counting efficiencies of the dissolved filters indioxane and of 1 ml of 14C solution in Unisolve cocktail, respectively, 1.06 is theisotope discrimination factor and 50 is a factor to relate the activity of the added14C to the volume that has been filtered. Calculations are routinely performed ona HP 9825 computer by using a program developed by Dr. H. Kausch, Hamburg.362Comparison of radiocarbon technlqnesPhotosynthetic rates are given as hourly incubation averages by subtracting thedark value at each depth from the values obtained in the light bottles.Results and DiscussionBoth in the lake and in the pond experiment the acid bubbling method resultedin consistently higher photosynthetic rates than the filtration technique (Figure2). The results obtained by the filtration technique in the lake were 70 db 13% andin the pond 74 ± 10% of the results of the acid bubbling method (mean valuesand s.d. of the measurements at different depths of the profile). In the lake therelative discrepancy was highest at near-optimum light-levels. In the pond no con-sistent vertical trend was apparent.This is in sharp contrast with results obtained by Theodorsson (1975) whofound that results obtained by the two methods deviated <5%. Gieskes andKraay (1980) found excellent correspondence between the two methods on oneoccasion. On two other occasions however, they observed in near surface layers,in agreement with our findings, deviations of up to 40%.Since in our case no additional experiments were performed we can onlyspeculate about the reasons of this discrepancy. Possible explanations include thefollowing: (i) Incomplete removal of inorganic "C-label from the sample by theacid bubbling procedure which could lead to an overestimation of photosynthesisby this method, (ii) Extracellular release and/or leakage of labeled organicmaterial during filtration by cell rupture or osmotic-shock. (The activity in thefiltrate of the Lake Constance samples was 40.3 ± 7 cpm and thus was identicalto the background. This indicates that stripping of "CO2 after acidification wascomplete and that release of 14C-DOM was undetectably small. In the pond,extracellular release and/or lysis of DOM was only 1-2% of total primary pro-duction. Therefore excretion cannot account for the observed differences.) (iii)Filtration errors caused by passing of small algal cells through the filters. (Filtra-tion errors are not likely since the smallest diameter of phytoplankton species inLake Constance exceeded 1 ftin.) (iv) Incorrect quenching corrections in one orboth scintillation cocktails used in the filtration technique. (Quench correctionfactors f j and f2 have frequently been estimated to be in the order of 0.9 and 0.8,respectively. Thus it is very unlikely that inaccurate quench correction could ac-count for the observed discrepancy.)Filtration differed in the two methods in so far as in the ABM pore size offilters was 0.2 /un and in the filtration technique it was 0.8 /im. In addition filterswere rinsed three times with distilled water in the filtration technique.Experiments performed on other occasions using either lake water or distilledwater for rinsing yielded equal results so that 14C losses due to osmotic shock arehighly unlikely. However, we cannot exclude the possibility that cell membranesbecame partly destroyed when sucking the filters three times to dryness.Peterson (1978) emphasised that organic carbon budgets for phytoplanktonwill be subject to large uncertainties as long as we do not better understand what14C uptake measures. This experiment has shown that production of particulatecarbon determined from identical samples by two widely used methods can differfar beyond the standard error within either method. This demonstrates that dif-363R.Gicnter, A.Maris and M.M.TDzerficulties in the interpretation of 14C measurements already begin at a purelytechnical level. The sobering fact that the reasons responsible for the observeddiscrepancy could not be clearly determined demonstrates that additional ex-perimental effort is needed to identify and eliminate possible short-comings ineither method. Only if all basic methodological problems are solved can we ad-dress the question which processes are actually measured by the "C method.ReferencesArthur.C.R. and Rigkr.F.M.: 1967,, 'A possible source of error in the "C method of measuringprimary productivity*, Limnol. Oceanogr., 12, 121-126.Berman.T.: 1973,' Modification in filtration methods for the measurement of inorganic "C uptake byphotosynthesizing algae', / . Phycol., 9, 327-330.Berman.T.: 1976, 'Release of dissolved organic matter by photosynthesizing algae in Lake Kinneret,Israel', Freshwat. Biol., 6, 13-18.Fogg.G.E.: 1971, 'Extracellular products of algae in freshwater', Arch. Hydrobiol. Bah. Ergebn.Limnol., 5, 1-25.Gfichter.R. and Marcs,A.: 1979, 'Comments on the acidification and bubbling method for determin-ing phytoplankton production', Oikos, 33, 69-73.Gargas.E. (ed.): 1975, 'A Manual for Primary Production Studies in the Baltic', Baltic MarineBiologists, Publication 2.Gieskes.W.W.C. and Kraay.G.W.: 1980, 'Primary productivity and phytoplankton pigment measure-ments in the northern North Sea during FLEX' 76', 'Meteor' Forsch.-Ergebnisse, A 22, 105-112.Larsson.U. and HagjtrOm, A: 1982, 'Fractionated phytoplankton primary production, exudaterelease and bacterial production in a Baltic eutrophication gradient', Mar. Biol., 67, 57-70.Mague.T.H., Friberg.E., Hughes,D.J. and Morris,I.: 1980, 'Extracellular release of carbon bymarine phytoplankton; a physiological approach', Limnol. Oceanogr., 25, 262-279.McMahon.J.W.: 1973, 'Membrane filter retention — a source of error in the "C method ofmeasuring primary production', Limnol. Oceanogr., 18, 319-324.Peterson,B.J.: 1978, 'Radicarbon uptake: its relation to net paniculate carbon production', Limnol.Oceanogr., 23, 179-184.Pugh.P.R.: 1973, 'An evaluation of liquid irintillarinn counting techniques for use in aquatic primaryproduction studies', Limnol. Oceanogr., 18, 310-319.Sakamoto et at.: 1984, 'Joint field experiments for comparisons of measuring methods of photo-synthetic production', / . Plankton Res., 6, 365-383.Schindler.O.W., Schmidt,R.V. and Reich,R.A.: 1972, 'Acidification and bubbling as an alternativeto filtration in determining phytoplankton production by the 14C method', J. Fish. Res. Bd. Can.,29, 1627-1631.Sharp.J.H.: 1977, 'Excretion of organic matter by marine phytoplankton. Do healthy cells do it?',Limnol. Oceanogr., 22, 381-399.Stumm.W., Morgan.J.T.: 1981, Aquatic Chemistry, Wiley Interscience, New York, 780 pp.Theodorsson.P. and Bjarnason.J.O.: 1975, 'The acid-bubbling method for primary productivitymeasurements modified and tested', Limnol. Oceanogr., 20, 1018-1019.Watt.W.D.: 1966, 'Release of dissolved organic material from the cells of phytoplankton popula-tions', Proc. R. Soc. Lond. B., 164, 521-551.364

Determination of phytoplankton production by the radiocarbon method: a comparison between the acidification and bubbling method (ABM) and the filtration techn1que

Determination of phytoplankton production by the radiocarbon method: a comparison between the acidification and bubbling method (ABM) and the filtration techn1que

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