Flow scintillation counting of 14 C-labeled microalgal photosynthetic pigments

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1 Journal of Plankton Research Vol.18 no.1 pp , 1996 Flow scintillation counting of 14 C-labeled microalgal photosynthetic pigments J.L.Pinckney, D.F.Millie 1, K.E.Howe, H.W.Paerl and J.P.Hurley 2 University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell Street, Morehead City, NC 28557, 'US Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, 11 Robert E. Lee Boulevard, New Orleans, LA 7124 and 2 Bureau of Research, Wisconsin Department of Natural Resources, 135 Femrite Drive, Monona, WI53716 and Water Chemistry Program, University of Wisconsin, 66 North Park Street, Madison, WI 5376, USA Abstract. Photopigment radiolabcling, a useful method for measuring the in situ carbon-specific growth rates of microalgae, is based on the determination of synthesis rates of chemosystematic (i.e. specific for microalgal phylogenetic groups) chlorophylls and carotenoids using photosynthetically assimilated I4 C as a radiotracer. The reliability of this method depends on accurate measurements of the 14 C-specific activity of individual photopigments. Typically, photopigments are separated by highperformance liquid chromatography (HPLC) with fraction collection of individual peaks, followed by further purification and standard scintillation counting. To simplify analyses, we evaluated in-line flow scintillation counting to determine its applicability and reliability for measuring the activity of radiolabeled photopigments. Incubations were conducted using both pure cultures and natural phytoplankton samples. The radiochemical purity of photopigments was determined by extract acidification (1% HC1) to transform chlorophylls into degradation products. Purity was also checked by comparing absorbance spectra with purified standards. Although 14 C-labeled colorless compounds are a common feature in radiograms, these compounds do not co-elute with photopigments using our HPLC protocol. Flow scintillation counting, coupled with a highly selective HPLC protocol, provides an efficient, reliable and feasible alternative to fraction collection/repurification methods for measuring the 14 C-specific activity of microalgal photosynthetic pigments. Introduction High-performance liquid chromatography (HPLC), which provides rapid and accurate quantification of chlorophylls and carotenoids, has become the preferred method for pigment-based chemosystematic characterization of microalgae (e.g. Gieskes and Kraay, 1986; Bidigare et al., 199; Wilhelm and Manns, 1991; Millie et ai, 1993; Pinckney et al, 1995a; Tester 1995). Photopigment radiolabeling is a useful method for measuring phytoplankton growth rates based on the synthesis rates of these chemosystematic photopigments. Under conditions of balanced growth, photopigment synthesis rates equal phytoplankton carbon-specific growth rates (Redalje and Laws, 1981; Riemann et al, 1993). The combination of HPLC methods and in situ photopigment radiolabeling can be used to determine the apparent growth rates of different phytoplankton phylogenetic groups (i.e. diatoms, chlorophytes, cyanobacteria, dinoflagellates, etc.) in natural mixed assemblages (Gieskes et al, 1993; Goericke and Welschmeyer, 1992a,b; Redalje, 1993). Detailed descriptions and validation of the photopigment radiolabeling method are provided in Redalje and Laws (1981), Redalje (1983), Riemann et al (1993) and Goericke and Welschmeyer (1992a,b). The commonly used method for measuring the 14 C-specific activity of Downloaded from by guest on 6 October 218 Oxford University Press 1867

2 J.LPinckney el at. individual photopigments involves separation and purification by HPLC, followed by fraction collection of individual pigments eluting from the column (Goericke, 1992; Redalje, 1993; Goericke and Welschmeyer, 1993; Hein and Riemann, 1995). Fraction collection is a tedious and imprecise process that requires extensive sample manipulation, collection of multiple HPLC eluant fractions for each peak, and long times (>1 h) for scintillation counting. Another disadvantage is the impracticality of fraction collection for all photopigments in natural samples (1-2 individual pigments). Because our research program required the application of the photopigment radiolabeling method to measure the taxon-specific growth rates of mixed (natural) phytoplankton assemblages as part of a long-term monitoring program, a more practical protocol for assessing the 14 C activity of chemotaxonomic pigments was needed. Flow-scintillation counting is a common technique in pharmaceutical and industrial chemistry. Radioisotope quantification is achieved using an in-line radiodetector that mixes the cocktail with the eluant downstream from the HPLC columns, pumps the mixture into a counting cell, and measures fluorescence with paired photomultiplier tubes. The small volume of the counting cell (25-15 u,l) provides reliable counts at low activities over short (seconds) time scales. Like HPLC chromatograms, radiograms are obtained in real-time and can be used to quantify the activity of individual radioisotope peaks. Although this technology has been applied in other research areas, the utility and reliability of this approach have not been evaluated for routine microalgal photopigment analyses. Previous work on photopigment radiolabeling has highlighted one particular concern when applying this new technique. Co-elution of 14 C-labeled colorless compounds with photopigments can lead to significant overestimates of the 14 C- specific activity of the pigment (Goericke, 1992; Jespersen et ai, 1992). Other studies have relied on secondary purification procedures (saponification, acidification, phase separation) to eliminate radiochemical impurities prior to radioactivity quantification (Gould and Gallagher, 199; Goericke and Welschmeyer, 1992a,b, 1993; Redalje, 1993). In this paper, we outline a simple method for quantifying the 14 C-specific activity of microalgal photosynthetic pigments using in-line flow scintillation counting. Because of potential contamination by colorless compounds, the radiochemical purity of radiogram peaks was checked by comparing absorbance spectra with pure standards and acidification of pigment extracts to alter the structure of chlorophyllous compounds. Method Hardware, software, data analysis The instrumentation consisted of a binary gradient pump (Spectra Physics SP88), autosampler (Spectra Physics AS3) with a 2 \t\ injection loop, column heater (Alltech 33), photodiode array detector (PDA; Shimadzu SPD- MlOav, 2-8 nm range) and an in-line radiodetector (Packard Radiomatic 525a flow scintillation counter) with a 5 u,l liquid counting cell. For the PDA, spectra (38-67 nm) were obtained at 2 s intervals for the duration of each run and photopigment peaks were quantified at 44 nm. Chromatograms were 1868 Downloaded from by guest on 6 October 218

3 Flow scintillation counting of 14 C-labeIed pigments Table L Gradient flow table for HPLC and radiodetector. Solvent A is 8% methanol:2% ammonium acetate (.5 M) and solvent B is 8% methanol:2% acetone Startup Run Shut-down Tune (min) %A %B HPLC flow rate (ml min" 1 ) Scintillation cocktail flow rate (ml min" 1 ) analyzed using Shimadzu EZChrom 3.2 software. Radiodetector data were acquired at 2 s intervals and converted to disintegrations per minute (d.p.m.) using software (Radiomatic Flo-One 2.) that corrects for variable flow rates, counting cell volume, scintillation cocktail mixing rates and counting efficiency (see below). HPLC columns The method used for photopigment separations was a combination of the Mantoura and Llewellyn (1983) and Van Heukelem et al (1992,1994) protocols. Multiple reverse-phase C 18 columns were connected in series. A single monomeric guard column (Rainin Microsorb,.46 X 1.5 cm, 3 \im packing) was followed by a single monomeric reverse-phase Qg column (Rainin Microsorb-MV,.46 X 1 cm, 3 u.m packing) and two polymeric reverse-phase C 18 columns (Vydac 21TP,.46 X 25 cm, 5 u-m packing). This column configuration was originally devised to enhance photopigment separations from sediment samples containing numerous (>15) photopigment and pigment degradation products. Monomeric columns provide strong retention and high efficiency, while polymeric columns select for similar compounds with minor differences in molecular structure (Van Heukelem et al, 1992). In addition to increasing the number of theoretical plates, using both monomeric and polymeric columns optimizes photopigment separations based on two different molecular properties (coarse and fine structure). Polymeric columns are temperature sensitive and necessitate the use of a column heater to maintain a constant 4 C and optimize photopigment separations (Van Heukelem et al, 1994). Downloaded from by guest on 6 October 218 Gradients A non-linear binary gradient, adapted from Van Heukelem et al (1992), was used for pigment separations (Table I). Solvent A consisted of 8% methanol:2% 1869

4 J.L.Pindtne) el at. Table IL Photopigment identification table for labeled peaks in chromatograms. Retention times are approximate Number Retention time (min) Photopigment Chlorophyllide a Chlorophyll c 2 Chlorophyll C\C2 Fucoxanthin 9' cis-neoxanthin cis-fucoxanthin Phaeophorbide a Violaxanthin Diadinoxanthin Antheraxanthin Alloxanthin Monadoxanthin Diatoxanthin Lutein Zeaxanthin Chlorophyll b Crocoxanthin Chlorophyll a allomer Chlorophyll a Phaeophytin b PT-Carotene Phaeophytin a Pe-Carotene PP-Carotene ammonium acetate (.5 M adjusted to ph 7.2) and solvent B was composed of 8% methanol:2% acetone. Solvents were degassed with helium before each run and filtered in-line (Whatman IFD L588). Low-viscosity scintillation cocktail (Packard Ultima-Flo M) was mixed automatically (in-line) with eluant downstream from the PDA at a constant 3:1 cocktail to eluant mixing ratio (based on eluant flow rate) (Table I). Quench curve As with other scintillation counters, flow scintillation counting is subject to fluor quenching that results in an underestimate of 14 C activity. Because the gradient used for photopigment separations contains acetone and water (strong quenching agents), a quench curve was constructed to correct measured counts per minute (c.p.m.) to d.p.m. using the range of solvent mixtures and flow rates encountered during runs. Gradient quench curves were constructed by mixing a known amount of 14 C with the scintillation cocktail (15. ixci in 5 ml cocktail) to achieve a constant activity level. The HPLC gradient was run, 14 C-labeled cocktail mixed with the solvent eluant (in-line with a mixing T) and the 14 C activity (cp.m.) measured at 2 s intervals. The ratio of measured c.p.m. to the known d.p.m. activity of the cocktail was used to calculate 14 C counting efficiency for the gradient (Figure 1). This gradient quench curve was used to convert measured cp.m. to d.p.m. for subsequent samples containing radiolabeled photopigments. The reduction in 187 Downloaded from by guest on 6 October 218

5 Flow scintillation counting of "C-bbeied pigments ^ 4 2 n ^ ^ ^ ^ ^ \ SB I FLOW " / _ \ \ ' y.... i.... i.. i.. i. \.. i Retention Time (min) Fig. L HPLC gradient, flow rates, (upper panel) and radiodetector quench curve (lower panel). For the quench curve, the mean (solid line) ± 1 SD (dashed lines) for three separate determinations is shown. efficiency with increasing retention time is attributed to the increasing proportion of acetone in the solvent gradient. The sharp downward spike at 41 min is due to a reduction in the flow rate of scintillation cocktail while acetone was still flowing out of the columns. No photopigments of interest elute in the 4-42 min time period. Although chlorophyllous photopigments are also strong quenching agents, the quantity of photopigment (ng) is small relative to cocktail volume (ml) and has no measurable effect on counting efficiency. Incubation and labeling Photopigment radiolabeling was accomplished by incubating phytoplankton in the presence of NaH 14 CO 3, and allowing incorporation of 14 C into chlorophylls and carotenoids Carbon fixation by photosynthesis provides 14 C-containing precursor molecules that enter photopigment precursor pools and are incorporated into photopigment molecules. To evaluate flow-scintillation counting, we performed experiments on both cultures and mixed natural phytoplankton assemblages. Pure cultures of Dunaliella tertiolecta (class Chlorophyceae, CCMP 132), Rhodomonas salina (class Cryptophyceae, CCMP 1319) and Phaeodactylum tricornutum (class Bacillariophyceae, CCMP 1327) were obtained from the Provasoli-Guillard Center for Culture of Marine Phytoplankton (Bigelow Laboratory 5.5 g Downloaded from by guest on 6 October 218

6 J.L.Pincknej el al. for Ocean Sciences, West Boothbay Harbor, ME) and maintained in f/2 growth media. These species were selected to provide a range of characteristic chemotaxonomic pigments for radiolabeling measurements. Natural phytoplankton samples were collected from two locations in the Neuse River estuary, North Carolina, USA. For 14 C labeling, 1 ml of water containing pure cultures (Rhodomonas, Dunaliella and Phaeodactylum) in exponential growth phase or mixed natural phytoplankton assemblages were placed in clear polycarbonate flasks, inoculated with 1 M-Ci of 14 C (NaH 14 CO 3 ; 54.4 mci mmoh), and sealed. Culture incubations were conducted in an environmental chamber (25 C, 1 u.e m~ 2 s" 1 ) with a 12 h light:dark cycle. Natural phytoplankton samples were incubated outside under in situ irradiances and temperatures. Typically, incubations were from dawn to dawn (-24 h) to allow sufficient turnover of precursor photopigment pools (Goericke and Welschmeyer, 1993; Redalje, 1993; Riemann et al, 1993; Mingelbier et al., 1994). Photopigment extraction Phytoplankton samples for photopigment analysis were obtained by vacuum filtration (< 3Torr) onto glass fiber filters (Whatman GF/F). Filters were placed in disposable polypropylene plastic centrifuge tubes (1 ml), a known volume (1-4 ml) of 1% acetone added, and sonicated (Fisher Sonic Dismembrator, Model 3, with microtip) for 3-6 s in an ice slurry to reduce heating. Tubes were wrapped in aluminum foil, placed in a freezer (-2 C) and extracted overnight (-12 h). After extraction, samples were centrifuged (5 min at 45 r.p.m.) and the supernate filtered through a.45 (xm PTFE filter (Gelman Acrodisc). The extract was then dispensed into amber glass autosampler vials (2. ml), sealed with Teflon-lined caps and placed in the autosampler for HPLC analysis. The injection volume for all runs was 2 jil. Experiments Radiolabeled photopigment extracts from pure cultures and natural phytoplankton samples were injected into the HPLC to obtain chromatograms for comparison with radiograms from the radiodetector. Chromatograms allow the identification and quantification of individual photopigments (chlorophylls, carotenoids and degradation products), while radiograms provide a measure of 14 C activity (d.p.m.) for each pigment peak. As a check for photopigment purity, we compared the absorbance spectra (38-67 rim) of each peak with spectra obtained from purified pigment standards. Differences between spectra were attributed to the presence of contaminants and were used as an indicator of relative photopigment purity. To verify further the radiochemical purity of peaks, chromatograms and radiograms were obtained before and after acidification (1% HC1). Extracts were neutralized using 1% NaOH before HPLC analysis Acidification results initially in the demetallation of chlorophylls to form pheophytin (Svec, 1978). Exposure to acidic conditions may result in the further breakdown of the chlorophyll molecule 1872 Downloaded from by guest on 6 October 218

7 Flow sdntfltatkni connting of l4 C-bbeled pigments 15, 12, I. 9, O 6, 3, 15, 12, J. 9, 6, 3, -B Rhodomonas Culture,,, I,, AA J,.ijy, i Retention Time (min) Acidified Fig. 2. Chromatograms (A and C) and radiograms (B and D) for the R.salina (a cryptophyte) pure culture before (A and B) and after acidification (C and D) with 1% HC1. Numbers above peaks correspond to photopigments listed in Table II. to lower-molecular-weight degradation products (pheophorbides, etc.) (Svec, 1978; Rowan, 1989; Gieskes et ai, 1991). For phytoplankton samples, acidification effects are most evident with respect to chlorophyll a (Chi a). Therefore, changes in the radiogram peak associated with Chi a following acidification should provide evidence that the measured activity is associated with the Chi a molecule rather than some other co-eluting or colorless compound. This principle was used to evaluate our ability to assess reliably the radiochemical purity of 14 C-labeled Chi a. 5 Downloaded from by guest on 6 October 218 Results and discussion Culture radiolabeling The monospecific algal cultures were incubated under nearly ideal growth conditions and exhibited rapid growth and incorporation of I4 C into photopigments 1873

8 J.L-Ptnckney el al. o. O s a. O I 3, 2,4 1,8 1,2 6 3, 2,4 1,8 1,2 6 ID L c A V,, r 1 Dunaliella Culture I l J1i r-r-t- Vt- Wvs Retention Time (min) 19 Acidified Acidified -j..a,.^a-. Fig. 3. Chromatograms (A and C) and radiograms (B and D) for the D.tertiolecta (a chlorophyte) pure culture before (A and B) and after acidification (C and D) with 1% HC1. Numbers above peaks correspond to photopigments listed in Table II. (Figures 2-4). Because the scintillation counter is located downstream from the PDA, there is a short lag time (-3 s) that results in a small shift (to the right) in radiogram peaks relative to the chromatogram. Changing flow rates during the run also contribute to small differences between radiograms and chromatograms. The radiograms have not been shifted to compensate for this time lag. However, in most cases, there is a clear relationship between pigment and 14 C activity peaks. The total activity (d.p.m.) for each pigment is determined by integrating the area under each peak in the radiogram. The Rhodomonas culture showed characteristic pigment peaks with high 14 C activity (Figure 2A and B). However, several large 14 C peaks did not correspond to colored (i.e. in the visible range, 4-7 nm) compounds. These peaks are probably associated colorless compounds (e.g. lipids, organic solubles, etc) and, if these compounds co-elute with photopigments, can result in contamination that leads to overestimates of photopigment 14 C-specific activity. Acidified (then Downloaded from by guest on 6 October 218

9 Flow scintillation counting of 14 C-bbeled pigments 2, 16, - J. 12, V 8, 4, 2, 16, 12, 8, 4, -D C IB - A Phaeodactylum Culture l*y 1 4 I 19 J 8 18 U ULi» A Retention Time (min) Acidified Acidified 24 I ^r-^ Fig. 4. Chromatograms (A and C) and radiograms (B and D) for the P.tricornutum (a diatom) pure culture before (A and B) and after acidification (C and D) with 1% HC1. Numbers above peaks correspond to photopigments listed in Table II. neutralized) pigment extracts were used to verify that the activity peaks associated with chlorophyllous compounds (primarily Chi a) were attributable to 14 C incorporated in the chlorophyll molecule (Figure 2C and D). Acidification resulted in the conversion of Chi a to pheophytin a and a shift in the 14 C activity peak previously associated with the Chi a peak. However, the pheophytin a radioactivity peak was lower than the pre-acidification Chi a peak. This difference can be attributed to further degradation of pheophytin a under the acidic conditions. Also note the reduction in 14 C peak size associated with Chi c 2. The residual activity after the removal of Chi c 2 indicates some radiochemical impurity associated with Chi c 2. Peaks associated with carotenoids (alloxanthin, monadoxanthin and crocoxanthin) and other peaks were not significantly affected by acidification, and indicate that chlorophyll degradation products (or other compounds that are altered by acidic conditions) did not contribute to these peaks Downloaded from by guest on 6 October 218

10 J.LPinckney el at. 5 4 u. a O 8 e Retention Time (min) Fig. S. Chromatogram (lower panel) and radiogram (upper panel) for a freshwater phytoplankton community collected from the Neuse River, NC. Numbers above peaks correspond to photopigments listed in Table II. Similar results were obtained for the Dunaliella and Phaeodactylum cultures (Figures 3 and 4). Several 14 C peaks were clearly associated with photopigments, but numerous other peaks, attributed to colorless compounds, were also detected. Extract acidification (Figures 3C and D and 4C and D) demonstrated the fidelity of the Chi a and Chi b radiogram peaks, and the radiochemical purity of these compounds. Carotenoid peaks were unaffected by acidification. Radiolabeling experiments were conducted on several other cultured phytoplankton species (Synechococcus elongatus, Tetraselmis sp., Skeletonema costatum, Anabaena sp. and Trichodesmium sp.) with similar results (data not shown). Natural phytoplankton radiolabeling While pure cultures provide optimal conditions for radiolabeling experiments (rapid growth, ample nutrients, healthy population, etc.), the ultimate goal of this method is to provide a useful tool for measuring 14 C incorporation into photopigments of natural phytoplankton assemblages Two examples representing a freshwater and an estuarine phytoplankton assemblage were selected to illustrate the application of flow-scintillation counting. The freshwater phytoplankton assemblage contained a range of photopigments representative of several microalgal taxonomic groups (diatoms, cryptomonads, cyanobacteria, chlorophytes) (Figure 5). Some radiogram peaks are clearly correlated with chromatogram peaks, but many radiogram peaks are associated with colorless compounds or small amounts of colored (unidentified) compounds. The noisy signal in the radiogram illustrates the effects of low 14 C activity and shows the lower limit of 1876 Downloaded from by guest on 6 October 218

11 Flow scintillation counting of I4 C-labeled pigments detection for the radiodetector (-5 d.p.m.). For the estuarine phytoplankton sample, the same major taxonomic groups were present (except cryptomonads) and most of the major photopigments showed incorporation of 14 C (Figure 6). Again, colorless compounds produced several large peaks in the radiogram. Absorbance spectra Absorbance spectra (38-67 nm) for the major photopigments in each sample were compared to spectra for pure (crystalline) photopigment standards to check for co-eluting pigments or colored contaminants and verify the identity of individual peaks. The results of the analysis for the estuarine phytoplankton sample (Figure 6) showed close agreement between sample pigments and standards (Figure 7). Spectra comparisons, even for lutein and zeaxanthin, which are usually difficult to separate, demonstrate the selectivity of the HPLC protocol for individual pigments. Similar results have been obtained consistently for phytoplankton and benthic microalgal samples collected from a wide range of freshwater, estuarine and marine habitats (Pinckney et al, 1994,1995a,b,c). Our unique HPLC protocol (i.e. using two types of Ci 8 columns) provides clean pigment peaks that are essential for reliably quantifying the 14 C-specific activity for individual photopigments. Method evaluation The combination of a highly selective HPLC photopigment separation protocol coupled with in-line flow scintillation counting provides an efficient, reliable and feasible method for assessing the 14 C-specific activity of individual photopigments. This procedure offers several advantages over fraction collection (dropcatch) methods. First, the 14 C-specific activity of all the major photopigments (chlorophylls and carotenoids) can be determined for each sample, rather than targeting a small, select group of photopigments for analysis. The presence of colorless (e.g. lipids, organic solubles, etc.) contaminants is a prominent feature in both culture and natural samples. These contaminants have been implicated as a cause of overestimates of pigment radiolabeling rates (Goericke, 1992) and resulted in the recommendation that photopigments be purified (by pre-treatment, acidification, phase separation or saponification) before measuring radioactivity (Goericke, 1992; Gieskes et al., 1993; Goericke and Welschmeyer, 1993; Redalje, 1993; Riemann et al., 1993). Radiograms provide clearly defined peaks that can be reliably integrated to obtain d.p.m. for each peak. Unlike standard scintillation counters that require long (>1 h) counting times for each sample, the small counting cell volume (5 u.1) and hardware configuration of the in-line radiodetector provide sufficient sensitivity to measure low (<1 d.p.m.) activities instantaneously during the HPLC run. Contamination of radioisotope peaks by colorless compounds and/or co-eluting carotenoids may be a problem for some of the photopigments in natural phytoplankton extracts. In mixed phytoplankton assemblages, which produce numerous carotenoids, the method does not always provide sufficient resolution to separate all radioisotope peaks. Although there are occasional instances where closely 1877 Downloaded from by guest on 6 October 218

12 J.LPinckrjey el al. a o 8 e 3, 2,5 2, 1,5 1, Retention Time (min) Fig. 6. Chromatogram (lower panel) and radiogram (upper panel) for an estuarine phytoplankton community collected from the Neuse River, NC Numbers above peaks correspond to photopigments listed in Table II. 8 8 X Fucoxanthin _.. I Lutein Zeaxanthin Chlorophyll a Downloaded from by guest on 6 October Wavelength (nm) 68 Fig. 7. Absorbance spectra for photopigments obtained from the estuarine phytoplankton sample (Figure 6) (dashed line) and spectra for pure photopigment standards (solid line). 1878

13 Flow gtintillatioii counting of 14 C-tabeled pigments eluting carotenoids result in radioisotope peak cross-contamination, most of the carotenoids in mixed pigment extracts seem to be radiochemically pure. Using the HPLC protocol described above, co-elution of radioisotope peaks is a special case rather than a general rule for carotenoids The primary emphasis of this work is the demonstration of the radiochemical purity of Chi a rather than carotenoids. Purification of Chi a by acidification is the recommended method for the fraction collection technique and we have presented an analog for this technique. Unfortunately, there is no simple method for verifying the radiochemical purity of carotenoids. However, there is some indirect evidence (i.e. pigment spectra) that the major carotenoids are radiochemically pure. Clearly, more work needs to be done in this area and we hope that this work will stimulate more research on the topic. This method has been used for routine phytoplankton analyses since July 1994 without any significant problems. However, there are a few additional aspects that should be considered by others contemplating the implementation of this method. Because of the small amount of photopigment (ng) that is actually analyzed, the amount of 14 C required for incubations is much higher than for standard productivity measurements. Large incubation volumes are needed to obtain concentrated extracts that produce significant quantities of the photopigments with sufficient 14 C activity for reliable quantification. For oligotrophic ocean waters, this requires large volumes of seawater (and associated radioactive waste). For coastal, estuarine and freshwater habitats, incubation volumes of.5-11 provide enough material for analysis. Flow scintillation counting is a useful and reliable tool for routine assessments of the 14 C-specific activity of microalgal photosynthetic pigments. The application of this method for measuring the group-specific growth rates of natural phytoplankton assemblages in situ opens the door for much-needed research on the ecological and physiological processes that determine phytoplankton community structure, function and spatio-temporal dynamics. Acknowledgements We thank CDonahue and M.Fitzpatrick for technical support, O.Schofield for enlightening discussions, L.Van Heukelem for guidance on polymeric columns and protocols, and three anonymous reviewers for their helpful comments. Funding for this study was provided by USDA Project , NC Sea Grant (NOAA) Project R/MER-3 and NSF Project OCE Support for J.L.P. was provided by an appointment to the Global Change Distinguished Postdoctoral Fellowships sponsored by the US Department of Energy, Office of Health and Environmental Research, and administered by the Oak Ridge Institute for Science and Education. References Bidigare,R.R., Marra,J., Dickey.T.D., Iturriaga.R., BakerJCS., Smith,R.G and Pak,H. (199) Evidence for phytoplankton succession and chromatic adaptation in the Sargasso Sea during spring Mar. EcoL Prog. Ser, 6, Gieskes,W.W.C and Kraay.G.W. (1986) Floristic and physiological differences between the shallow and deep nanophytoplankton community in the eutrophic zone of the open tropical Atlantic revealed by HPLC analysis of pigments. Mar. BioL, 9L, Downloaded from by guest on 6 October 218

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