ICES m ar. Sei. Sym p., 197: 132-140. 1993

On the dark bottle in the 14C method for measuring marine phytoplankton production

Karl Banse

Banse, K. 1993. On the dark bottle in the l4C method for measuring marine phytoplankton production. - ICES mar. Sei. Symp., 197: 132-140.

In the radiocarbon method for determining phytoplankton productivity, a variety of protocols has been introduced for the dark bottle (DB) during the past one or two decades; some protocols omit the DB. A perusal of the literature does not produce evidence for processes that regularly lead to significant COj uptake in the DB but do not occur in the light bottle (LB) too. Therefore, abiotic and biotic CCK uptake in the LB that is not due to photosynthesis should continue to be corrected for by subtracting DB values. Contrary to its purpose, a zero-time uptake correction may not account fully for abiotic processes. In three tropical data sets with low to moderate photosyn­thetic rates, failure to subtract DB values from LB values leads to substantial overestimates of primary production. Such bias affects means and possibly also patterns on a world map of marine phytoplankton production.

Karl Banse: School o f Oceanography, WB-10, University o f Washington, Seattle, WA 98195, USA.

Introduction

This review of marine literature addresses the usefulness of the dark bottle (DB) in the radiocarbon (14C) method for assessing phytoplankton production. Traditionally (e.g., Steemann Nielsen, 1963), the DB was to provide the blank correction for possible particulate contami­nation of the 14C ampoules, abiotic isotope exchange, and biotic uptake of C 0 2 and was incubated for the same length of time as the light bottle (LB); therefore, the DB values were subtracted from the LB uptake (e.g., Par­sons et al., 1984). During the last one or two decades, some workers (e.g., Seki, 1968; Taguchi and Platt, 1977) separated the biotic dark uptake from the other pro­cesses by poisoning one of the paired DB immediately after adding the isotope, then incubating both. Others (e.g. Morris et al., 1971) did not subtract the measured dark uptake from the LB value in the belief that much of the dark uptake was due to the photoautotrophs and that too little was known as to whether or not the process was restricted to the DB. More recently, Legendre et al. (1983) advised against subtracting the traditional DB measurement based on additional reasoning (see be­low). Still others (e.g.. Smith et al., 1977; Chavez and Barber, 1987) followed Morris et al. (1971) and added isotope to a darkened replicate of the LB sample that was filtered immediately to determine (for subsequent subtracting) the zero-time effect due to rapid isotopic

exchange, particles in the ampoules, and artifacts like insufficient removal of label from the filters. Some (e.g ., R. W. Eppley, pers. comm.) at times did not employ DB in open-ocean studies because of the small correction expected or experienced.

Four decades after the introduction of the method by Steemann Nielsen (1952), the interpretation of the pro­cesses in the DB continue to be difficult (e.g ., Carpenter and Lively, 1980; Legendre etal., 1983; Li, 1986; Harris etal., 1989). Undisputed abiotic error sources, however, exist that may lead to high DB values, e.g., insufficient removal of label from the filter matrix, and need there­fore to be routinely checked and corrected. The dis­agreement largely concerns the non-photosynthetic bi­otic uptake that undoubtedly takes place in the dark (e.g., Harris etal., 1987). For the routine method, in my opinion, the point here is: Do processes occur regularly (or at least generally) in the DB that are absent from the LB but involve C 0 2 uptake and that are significant over the time span o f the usual14C experiment at seal Because of the absence of persuasive observations to this effect, I suggest in this paper that dark uptake (and not only zero­time fixation) should at least be regularly determined and preserved in data reports even if the originators themselves would not subtract them from the LB values. I also show that omission or inclusion of a correction for dark uptake, over and above the zero-time correction, may lead to substantial differences in results. Hence, I

IC E S m ar . Sei. S y m p . . 197 (1993) The ,4C method fo r measuring marine phytoplankton production 133

favor subtraction of DB values in routine work in the open sea.

This presentation proceeds from the re-interpretation of three published data sets to reviews of light effects on growth by heterotrophic and chemolithotrophic bac­teria, of dark uptake by photoautotrophs, and a criticism of the zero-time correction. Processes in the LB, which will not occur in the DB, especially the sinister issue of grazing on newly 14C-labelled phytoplankton, will be mentioned.

Evaluation o f som e previous data sets

Open-ocean DB values from depths near light satu­ration (i.e., at reasonably high rates of photosynthesis) can be a few percent of the LB values, for example as illustrated by Steemann Nielsen (1960) for a number of sites or in my unpublished data from the Laccadive Sea (median of [DB/LB] x 100 = 3.7% [range 1.5 to 8.0%; n = 7]; median photosynthesis [LB — DB] about 8p g C L 1 IT ‘). DB values in other data sets for the open sea and near light saturation, however, may approach LB rates (e.g., Li, 1986: fig. 14; see also following paragraph).

For two field and one laboratory data set with simi­larly high DB/LB ratios, for which raw data happened to be available, I suggest that the authors very likely dealt with high blanks (that, hence, should have been sub­tracted from the LB) rather than with phenomena of algal metabolism which might be absent in the LB. Figures 1 and 2 depict observations from the vicinity of the Costa Rica Dome used by King (1986; data in Kuntz etal., 1975), with DB rates a 0 .3/ / g C P 1 h 1 arbitrarily separated from the lower values. The DB rates neither correlate with chlorophyll (Fig. 1), in spite of the undoubted existence of dark metabolism by photoauto­trophs, nor with LB values (not illustrated; r = 0.24, p =0.14 for slope, n = 39). Further in this data set, LB = DB + f(I) [chi] (Fig. 2; here, f[I] is the chlorophyll-specific photosynthetic rate as dependent on irradiance I). Therefore, for arithmetic rather than physiological reasons, the average DB value will lead to high DB/LB ratios at low LB values and to the correlation of LB/DB with pigment depicted by King (1986, fig. 2) and pre­viously reported by Morris et al. (1971; see following paragraph). I surmise that the few high DB rates in Figure 1 might have been artifactual outliers: Enhanced bacterial l4C uptake is unlikely because the high rates were not accompanied by elevated total ATP or esti­mated non-plant ATP. (Phytoplankton ATP was esti­mated by subtracting the product of observed chloro­phyll with the carbon-to-chlorophyll ratio, C/chl, from the total ATP. A C/chl ratio by weight of 86.6 was determined by King [1986] from regression of ATP on

%Ioo 100 A 5 0

v 2 5

0.8

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0 2 - - - o — v- q------- - P ~ ^ P ~ ^ a _

-« A " t o A O

-, 02 fj.q i Chlorophyll

0 0.1 0 .3 0 .4

Figure 1. Dark uptake on chlorophyll concentration in the vicinity of the Costa Rica Dome (data from Kuntz et al. , 1975). Circles, upright triangles, and inverted triangles for 100, 50, and 25% light depths, respectively. Filled symbols for dark uptake a: 0 .3 ^g C 1” ' h _ l . Broken line from regression using all data, full-drawn line after omitting values a 0.3 fxg C l” 1 h _ l .

chi and dividing the slope by the usual C/ATP ratio of 250 .1 also lowered C/chi arbitrarily by one-third in view of my admonition [Banse, 1977] that non-algal ATP of the microbial loop is apt to covary with chi, leading to an artificially high C/ATP.) There was no correlation be­tween DB values and total ATP, estimated non-plant ATP, and ETS (electron transport system) activity (all data from Kuntz et al., 1975).

The same correlation of LB/DB with pigment (cell numbers), as well as of dark uptake with cell numbers, led Morris etal. (1971) to identify biochemical and plant- physiological processes that might not occur in the LB,

3 .75 av

o 100a 5 0

v 2 5

DB Regressions

fj.g i Chlorophyll

Figure 2. Uptake in the light on chlorophyll concentrations in the vicinity of the Costa Rica Dome, with line from linear regression through data points (r = 0.81) and two regression lines from Figure 1. Data source and symbols as in Figure 1, filled symbols corresponding to likewise designated samples in that figure.

134 K. Banse I C E S m ar . Sei. S y m p . . 197(1993)

and therefore to suggest that DB values not be sub­tracted. The laboratory data of this broadly cited paper are replotted in Figure 3 and suggest that DB = a + b [algal cell numbers]. (The intercepts of the three lines [cf. figure caption] with the ordinate are significant at p < 0.08.) In my interpretation, the reported large decline of the ratio of dark uptake/algal cell with increasing cell numbers (last column of table 2, Morris et al., 1971) resulted from the large intercepts of the regression lines rather than from any physiological cause. As the authors used unialgal cultures, bacteria might have been the principal reason for the intercepts, as well as for the large proportionality coefficients (b), because the bac­terial cell numbers (hence, dark uptake) must have been proportional to the inocula of the experimental bottles,i.e., to the algal cell numbers. Instead, the authors suggested that the proportionality reflected dark uptake by the algae. Note that my explanation (as that by Morris et al., 1971) represents a weak inference sensu Platt (1964), since bacterial and algal dark uptakes were not differentiated by the experimental design. (Like the authors, I cannot explain the break of the curves in the Dunaliella experiment: a simple repeat of the experi­ment might have clarified the matter.)

Finally, Prakash et al. (1991) reported clear geo­graphic trends of DB/LB ratios from open-sea surface samples collected on meridional sections through the Atlantic and Pacific oceans, with DB being <0.1 to >0.5 of LB in the subtropical gyres and the high southern latitudes. The replotted data (Fig. 4) do not indicate a geographic trend of the DB rates (67 of 93 values being

<3- £

Cells m l " ' £ Ë1005 0

150-

£ 100 -

5 0 -

0J

6 x 10s Cells m r ' l s i )

6 x 103 Cells )53 40 2

Figure 3. Dark uptake on cell numbers, with lines from linear regression (data from Table 2, Dark Uptake, column A, Morris et al., 1971). Circles for Phaeodactylum tricornutum, triangles for Dunaliella tertiolecta\ upright triangles for plots of all data; filled symbols and full-drawn line, experiment with a 20,000 cells ml inverted triangles, open symbols, and broken lines, experiment with s 6,750 cells mT 1 (the inverted triangles depict the same data as the open upright triangles but on another scale).

o

0.5

(3©0@ )0.0

60°S 40°S 0 ° 20°N 40°N

Figure 4. Dark uptake in surface samples on latitude in sections through the Atlantic and Pacific oceans (calculatcd from Table 1, Prakash et al., 1991). Filled symbols, Atlantic values a 0.15 /.ig L 1 h _ l . Horizontal line, 0.1C)/<g C I” 1 h - '.

< 0 .10^g C I-1 I T 1), except that most of the high values were recorded on a cruise in the North Atlantic. DB values >0.15 jug C 1~' h~ ' aside, the DB rates are not correlated with ATP (no chlorophyll observations; all data, r = 0.19, p = 0.07 for slope, n = 93; after omitting one outlier, r = 0.04, p = 0.72. For DB values >0 .15^g C l -1 h -1 , r = 0.50, p = 0.03 for slope, n = 19; remaining material, r = 0.11, p = 0.35 for slope, n = 74). As in the case of the previous two articles, I suggest high blanks (median, 0.068 / ( g C T 1 h -1) as the principal reason for the geographic trends of DB/LB ratios discussed in the paper.

Three further publications (Seki, 1968; Taguchi and Platt, 1977; Petersen, 1979) are referenced to show that coastal sites with reasonably clear water seem to be similar to the open sea in respect to the question at issue in this review (cf. Steemann Nielsen, 1960). Only during periods of allochthonous supply of organic matter, or during the winter season with low LB rates, did the DB/ LB ratios reach several 10% (or even 100%) in these coastal waters.

One conclusion from the re-interpretation of the data by King (1986), Morris et al. (1971), and Prakash et al. (1991) paraphrases earlier authors, e.g., Steemann Niel­sen (1952, 1963), ICES (1981), and Harris etal. (1989): dark bottles should be employed and the results commu­nicated. I believe that authors owe to their readers an indication of the quality of their data even though other biases of the 14C technique cannot be judged so easily, e.g., trace metal contamination and partial poisoning of the LB. Apart from handling artifacts of the water samples discussed by, e.g., Furnas (1987) and Harris et al. (1989), variable quality of DB data from differing ampoule preparations was demonstrated by Bresta et al. (1987, fig. 3); systematic differences among sets of DB

IC E S m ar . Sei. S y m p . . 197 (1993) The UC method fo r measuring marine phytoplankton production 135

values, which can be obtained even by workers of the same “school” , are illustrated by the measurements of Petersen (1979; cf. sections at 51° and 62°N with data from Godhavn and the subarctic eddy in his fig. 1). Thus, DB data should be determined. Reasons will be ad­vanced in the following sections (or repeated, as far as the early authors are concerned) that the DB values should continue to be subtracted from the LB rates after a judicious consideration of outliers.

Light effects on bacterial production

Inhibition of bacterial growth by light, acting directly on the bacteria or via a release of antibiotics by photosyn- thetizing phytoplankton, has been suspected for a long time to be a source of differences in C 0 2 uptake between LB and DB (cf. reviews by Carpenter and Lively, 1980, and Peterson, 1980; new data by Sieracki and Sieburth, 1986). If such inhibition were regular, however, bac­terial production usually should be higher during the night than during the day. New marine evidence to this effect is not strong (Sieracki and Sieburth, 1985, 1986), ambiguous (Turley and Lochte. 1986), neutral (Wil­liams and Yentsch, 1976), or shows the absence of such an effect (Ward, 1984). For example, more rod-like bacteria divided at night than during the day in deep coastal waters, whereas, in the same samples, coccoid bacteria did the opposite (Turley and Lochte, 1986). Previously, Riemann et al. (1984) recorded minor diel changes in bacterial production but more marked differ­ences in the percentage of active bacteria in a shallow coastal environment, with higher fractions, as well as larger mean cell volumes, frequently being present dur­ing the early daylight hours (note: not in darkness!). In contrast, at the same site on the same dates, with algal cells removed, Riemann and Sondergaard (1984) did not find systematic diel patterns in CO 2 uptake in the dark. Li ( 1986) noted that much of the dark uptake of l4C by marine plankton is due to the <1 /<m fraction, and that uptake at night can often approach or equal that during the daylight hours. Space constraints preclude com­ments on his calculations for anaplerotic uptake in the DB. DB rates observed by him increased after sunset, but it was not shown that the underlying process had not occurred also in the LB. Finally, in experiments directed at the issue of subtracting or not subtracting DB from LB values, Li and Dickie (1991) did not observe significant differences between the LB and DB in the rate of bacterial growth processes for the first six to twelve hours.

For nitrifying (chemolithotrophic) bacteria, newer laboratory data, cited in Ward (1987), support the previous records of light inhibition of bacterial growth. However, I note that the oxidation into nitrite of am­

monium liberated by heterotrophs cannot possibly con­tribute much dark uptake of CO 2 in the photic zone: Even if all primary production was remineralized in the photic zone and no ammonium was taken up by the photoautotrophs but all of it was nitrified, little chemo­lithotrophic C 0 2 uptake would ensue since only 0.03 to 0.06 mol C is formed per mol N oxidized (Karl et al., 1984; see also the low observed oxidation rates in Ward et al., 1982, and the ratio of assimilation/nitrification in Ward, 1987).

Thus, it has not been demonstrated that the so far studied processes of bacterial C 0 2 uptake occur often and significantly only in the DB. Can there be, however, bacterial heterotrophic processes in the well-lit part of the euphotic zone of the open sea that are high relative to photosynthesis, occur equally in LB and DB, and thus cause DB values to be high relative to the difference LB - DB? (Note that this condition “equally” excludes significant grazing on 14C-labelled phytoplankton in the LB and the resulting enhanced heterotrophic C 0 2 up­take by the microbial loop in the LB but not the DB; this situation is discussed near the end of the paper.) I suggest negative answers for the two most likely scenar­ios: Bacterial production at a deep site can be high, relative to that of phytoplankton, only (1) near and after the end of phytoplankton blooms or (2) in upwelled deep-water with few algae in the euphotic zone. In the first case, bacterial heterotrophic production might approach or exceed primary production. However, even then, the bacterial dark uptake of l4C 0 2 cannot be large since heterotrophic C 0 2 assimilation is only a few per­cent of the newly formed cell material (6% is a fre­quently used average, cf. Romanenko et al., 1972, but this number is often regarded as too high, e.g., Jordan and Likens. 1980; see also Li, 1982). I confess to being baffled by the 14C accumulation rates observed in the dark by Li and Dickie (1991), but note the caveats by Harris et al. (1989) for work in oligotrophic waters (especially the artifactual particle production followed by rapid bacterial growth that presumably proceeds equally fast in LBs and DBs). The second scenario, high bacterial production in the photic zone that is main­tained by influx of deep water, can be illuminated by considering stations subject to continued, material verti­cal diffusion and advection in King (1986). Setting out from his vertical diffusion model it can be estimated that his DB values (Fig. 2, herein) are about two orders of magnitude higher than allowed by bacterial production from vertical flux of DOC and, thus, had another cause. In conclusion, in the well-lit part of the open-sea eupho­tic zone, dark uptake values of C 0 2 from the in situ activity of heterotrophic bacteria generally will not lead to DB rates that are high relative to the difference LB - DB. If they are high, other reasons should be looked for.

136 K. Banse IC E S m ar . Sei. S y m p . . 197 (1993)

Dark uptake by photoautotrophs

Algal physiology in the light clearly differs from that in the dark. Since growth processes continue in the dark, one might expect a relation of dark uptake by carboxy­lation with growth rate as well as with biomass of phytoplankton. Does this uptake, however, matter rela­tive to other processes in the DB?

Dark uptake and photosynthesis [LB; photosynthesis does not equal growth rate!] were highly correlated in my unpublished observations from the Laccadive Sea (r = 0.93; near light saturation at seven stations), less highly correlated at nine stations, each with three sam­pling depths, in the South China Sea and the north­eastern Indian Ocean (Saijo and Takesue, 1965; r = 0.44, p = 0.02 for slope, n = 27), were variably corre­lated but significantly so in Morris et al. (1971), weakly correlated in Prakash et al. (1991; r = 0.25. p = 0.02 for slope, n = 93. For DB rates <0.15 / ( g C r 1 I T 1 [cf. Fig. 4], r = 0.09, p = 0.47 for slope, n = 74), but not correlated in King (1986). The DB rates of Saijo and Takesue (1965) were approximately 0.02jug C l_l h 1 so that any, statistically non-significant, dark uptake by photoautotrophs must have been small indeed. Dark uptake and phytoplankton biomass were correlated in Morris et al. (1971; Fig. 3 herein), but not in the data of Saijo and Takesue (1965), Petersen (1979; northern North Atlantic; from inspection of fig. 1), and King (1986; Fig. 2 herein). Finally, dark uptake in North Atlantic oligotrophic waters that was largely accounted for by heterotrophic bacteria suggests a small role of photoautotrophic uptake (Li and Dickie, 1991).

Note that a positive correlation of DB values with photosynthetic rates or biomass permits only a weak inference (Platt, 1964), as it maybe due to carboxylation in the dark by photoautotrophs only, heterotrophs only, or both, in the latter case reflecting the heterotrophic activity of a microbial loop that is more vigorous with more phytoplankton and (hence, with usually more) photosynthesis and grazing. In contrast, the absence of a correlation between DB values and chlorophyll or phytoplankton biomass is strong inference that marine phytoplankton does not regularly take up significant amounts of C 0 2 in the dark.

Some transient uptake of C 0 2 by phytoplankton, short relative to the length of routine incubations for measuring photosynthesis at sea, might occur in the DB as an after-effect of the previous illumination, as surmised by Taguchi and Platt (1977). This phenomenon was not found for axenic Skeletonema costatum (Mague et al., 1980, fig. 2), but was reported by Legendre et al. (1983) for a culture of Phaeodactylum tricornutum (not stated whether or not axenic). I wonder whether such after-effects, if they broadly occur but last only for 10-20 min (as observed by Legendre et al., 1983), would

matter in routine fieldwork where, normally, opaque collecting bottles are being used and some time passes before inoculation with l4C in the ship's laboratory. (The statement by Ignatiades et al. [1987], that dark uptake in the first 20-40 min is elevated seems simply to reflect the arithmetic effect of the high, but not corre­lated, zero-time uptake, i.e., a high blank.)

Since my paper addresses routine conditions, I neglect other transients like the enhanced dark uptake of C 0 2 by severely nitrogen-limited algae upon exposure to ammonium or nitrate (e.g., Legendre et al., 1983 from Morris et al., as cited; Elrifi and Turpin, 1987; Turpin et al., 1988): It is questionable in how many samples severely nutrient-starved phytoplankton would occur at, say, ten stations in an oligotrophic setting like that in King (1986). Moreover, the physiological adjustment to fresh nutrient supply may take place also in the light (cf. Guy et al., 1989). Finally, averaged over 6-12 h of incubation, the effect if present would be negligibly small (cf. Goldman and Dennett, 1986).

To eliminate an expected dark uptake by phyto­plankton, Legendre et al. (1983) suggested, as a stan­dard protocol, incubation of DBs to which DCMU had been added. Before this procedure is generally accepted, I suggest that the experiments be replicated since one of the two time course observations at issue shows higher 14C 0 2 uptake in the DCMU-poisoned DB than the DCMU-poisoned LB.

In conclusion, because of ambiguous evidence, I re­gard the issue of dark uptake by photoautotrophs, which would significantly affect the routine l4C method, as still open. A simple replication with axenic cultures of the experiments by Morris et al. (1971) that engendered so much discussion and, I think, confusion about the DB, might yield a pragmatic clarification of the issue. In any case, the here demonstrated absence of correlation of DB uptake with chlorophyll shows that without testing for uptake by photoautotrophs, such uptake cannot any longer be assumed to be regularly present and signifi­cant, and therefore cannot be used as an argument for not subtracting the DB value from the LB rate.

Zero-tim e determination and the

traditional dark bottle

Determinations of (a) the zero-time effect (from fil­tration of the live sample immediately after addition of isotope, without incubation) or (b) abiotic fixation (from incubation of the killed sample parallel to the LB, as done by Taguchi, 1983) differ from (c) the traditional DB (live sample treated like the LB except that it is not exposed to light). Smith et al. (1977) state that protocol (a) determines the abiotic processes so that subtracting

IC E S m ar . Sei. S y m p . , 197 ( 1993) The l4C method fo r measuring marine phytoplankton production 137

the zero-time uptake from the LB yields the photosyn­thetic C 0 2 uptake by phytoplankton. Actually, how­ever, it overestimates photosynthesis because the differ­ence, LB minus “uptake (a)” , includes the biotic C 0 2 uptake in the LB that is unrelated to light but would be measured in the DB by protocol (c). Note that all protocols underestimate the photoautotrophic forma­tion of particulate organic matter to the extent that grazing mineralizes part of the newly formed and labelled particulate material during the incubation (cf. Smith et a!., 1984, and below).

I am not aware of systematic comparisons between protocols (a) and (b) excepting some remarks that will be mentioned later. The already discussed measure­ments by Morris etal. (1971) on algal cultures, however, illuminate problems with the “zero-time blank” . Although this blank was subtracted from the “later experiments” (Morris et al., 1971, p. 855, lower right- hand), all plots of DB rates on cell concentrations yield statistically significant intercepts, i.e., apparent dark fixation in the absence of algal cells (Fig. 3). Presum­ably, therefore, at least some of the abiotic fixation takes a few minutes, i.e., longer than provided by the “zero­time” protocol, as also indicated by the results of Legendre et al. (1983) and stated (without data) by Cadée (1983). More studies addressing this issue are highly desirable.

Since the zero-time correction may fail even in respect to abiotic fixation, it should be replaced by a poisoned replicate incubated with the other bottles (protocol [b]). Furthermore, because the purpose of either protocol (a) or (b) is to provide a blank, the replicate sample should be treated as similarly to the experimental as possible so that incubation (protocol [b]) is the correct procedure a priori. There is no reason to expect that abiotic fixation of C 0 2 in the dark will differ from that in the light.

The difference between protocols [b] and [c], the biotic dark uptake by autotrophs and heterotrophs, apparently has been studied in the open sea only by Gieskes et al. (1979: table V; waters of varying trophy in the tropical Atlantic) and by Gieskes and Kraay (1980: table 2; spring bloom offshore in the northern North Sea), although Taguchi (1983) and Harris et al. (1987) also used both protocols. There is no correlation be­tween the uptakes in the first data set; the median of the fixation ratios (xlOO) of the formalin-killed sample is 9% of the live sample (i.e., of the traditional DB; mean, 16%, n = 16). Also, there is no correlation between the uptakes in the data of Gieskes and Kraay (1980), but in seven comparisons among their two sets of ten experi­ments each, protocol (b) yielded a higher uptake than (c); only in two of the 20 pairs was the DB uptake (protocol c) twice as high as that of protocol (b). No reason for the differing results is apparent. Harris et al. (1987, p. 1008) apparently also found biotic dark uptake

in 12- and 24-h incubations of oligotrophic waters to be appreciably larger than zero-time fixation.

Subtracting D B values and assessing

global phytoplankton production

Many authors, especially of studies in warm oligotrophic seas, have been disturbed by small (sometimes negative) differences of LB — DB and the concomitant high DB/ LB ratios even near light saturation and, therefore, have concluded that subtraction of DB rates is inadvisable. At least two issues need to be kept in mind, though, before reaching a false conclusion: First, negative values may arise as small differences between two relatively large numbers (large because of high blanks), both of which have experimental (including subsampling) variability attached. To illuminate the problem, consider photo­synthesis near light saturation at a chlorophyll a (chi) concentration of 0.05 pg l“ 1 as it widely occurs in the lower latitudes (e.g., Fig. 1); assume a C/chl ratio (by weight) of as high as 100 and 1 (2) cell divisions per day. The resulting net production of 5 (10) pg C l-1 d “ 1 or <0.5 (1) pg C l -1 I T 1 (12-h days) even at optimal irradiance is not much higher than the DB rates of 0.05 to 0.15 pg C l“ 1 h _l as in Figures 1 and 4. Thus, unless the blank values (DB rates) are materially reduced (as, e.g., in Steemann Nielsen, 1960; Saijo and Takesue, 1965) and LBs and DBs are replicated (cf. Harris et al. , 1989), the measurements of dark uptake may not be precise enough for a profitable discussion of the utility of the DB. Second, Harris et al. (1989) demonstrated the effect of mechanical disturbance on plankton communi­ties incarcerated in bottles, which often leads to particle production and enhanced bacterial growth such that high DB rates may go hand-in-hand with conspicuously high LB rates; the LB rates per unit of chlorophyll a, not corrected for the DB, often are far higher than is physiologically feasible. Such a handling artifact might be a reason for the seeming outliers in Figures 1 and 4, although the LB rates/chl for the high DB rates are not excessive; again, as restated recently by Harris et al. (1989), little can be surmised without replication. Other handling artifacts were studied by Furnas (1987).

An entirely different issue regarding the utility of the DB for correcting LB rates has to do with processes in the LB that are absent from the DB. The theory of the DB presumes that the biomass effecting dark uptake is about the same in DB and LB. When, however, one to two cell divisions of phytoplankton per day are quite ordinary in warm waters (Banse 1991: fig. 2), which in the bottles may or may not be accompanied by intensive grazing, and 24-h incubations are used to overcome the effect of diel periodicity of algal physiology, then the basis of the DB correction is removed. The problem of

138 K. Banse IC E S m ar . Sei. S y m p . . 197 (1993)

how to find and apply here a blank correction will as much be a question of comprehending the community ecology of the LB with its primary producers, grazers, heterotrophic bacteria, and freshly l4C-labelled dis­solved organic matter (Laws, 1984; Smith et al., 1984; Harris eta l., 1987; Marra etal., 1988), as of understand­ing the cell physiology of heterotrophic bacteria and phytoplankton in the DB vis-à-vis that in the LB. Under such conditions, one should indeed wonder whether to subtract more than the zero-time blank. In any event, because of the grazing during the incubations, the tradi­tional LB in half-day to 24-h experiments does not measure phytoplankton particulate production per se, but something less than that. In contrast, the DB assumes its traditional role, to serve as a blank, wher­ever the dilution method (Landry et al., 1984) can be applied (wherever the phytoplankton is not dependent on nutrient regeneration by grazers on an hourly basis) and the true, instead of the apparent, phytoplankton production rate is estimated. Logically, here, the DB should be subtracted from the LB.

In conclusion, it seems to me that because of the long­standing concern about the DB, many of those users of various DB protocols, who are interested in a world map of marine phytoplankton production, seem to have lost sight of the importance of correct blanks in the l4C method: Even if DB/LB x 100 is only a few percent near light saturation, the integral of DB rates through the euphotic zone may be a substantial fraction of the same integral for the LB, since the rates of dark uptake tend to be much more uniform with depth than those of photo­synthesis. For example, for the 13 stations treated by King (1986) from the vicinity of the Costa Rica Dome, the average dark uptake down to the 1% light depth of 15.5 mg C m-2 h 1, when multiplied by 11 h of daylength and extrapolated to 365 days, would translate into 62 g C m~2 y~1. For my measurements from the Laccadive Sea, the median of all 16D B of 1 .9 ^ g C l_l d -1 in the similar extrapolation to 55 m bottom depth (the photic zone reached the sea bed) would yield 38 g C n T 2 y-1 . Such figures are conspicuous relative to annual rates of photo­synthesis reported from the warm seas. Note, though, that both figures give the upper limit of bias: The hourly rates used for the estimates were based on noon-to- sunset incubations. It is inappropriate, however, to multiply the zero-time effect or a similar measure of the abiotic fixation by daylength; the dark bottle corrects only for dark fixation and uptake during the incubation time.

An example for the lower limit to the bias introduced by not subtracting DB values, for a similar ocean area, is provided by the averages of biotic dark uptake rates reported by Taguchi (1983; from traditional DB minus poisoned DB, cf. Taguchi and Platt, 1977, and noon-to- sunset incubations, El-Sayed and Taguchi, 1979). The

rates in the North Equatorial Current and the North Equatorial Counter Current of the central Pacific, ex­trapolated t o 365 days, were 4 g C rrT2 y_l (n = 8) and 15 g C m 2 y _1 (n = 7), respectively; these averages were 10 and 32( ! )% , respectively, of photosynthesis corrected for dark uptake. Formulated differently, the regression of the DB rates on the (LB—DB) values, integrated through the euphotic zone (as read from Taguchi 1983, fig. 3), indicates a biotic uptake of 20% of corrected photosynthesis (r = 0.62, n = 13; within each of the two regions, the relation between DB and [LB - DB] is not significant). Thus, subtracting or not subtracting DB values will certainly affect means and possibly even patterns on a map of primary production of the oceans.

My conclusion is a restatement of the traditional view that DB values (a) are useful as a general quality check for a data set and (b) should be subtracted from the LB values in regional work even though the processes that do or might occur in the DB are not entirely understood. While this view is not universally accepted to the west of the Atlantic Ocean (see Introduction), it seems to be the prevailing opinion in the European-dominated ICES region (ICES, 1990).

A ckn ow led gem ents

The preparation of the manuscript was partially sup­ported, and participation in the ICES symposium of 1992 at La Rochelle fully supported, by grant NAGW- 1007 from the National Aeronautics and Space Adminis­tration (NASA). J. R. Postel helped with computations, an anonymous reviewer was a stern task master regard­ing partially supported statements, and D. P. Henry made suggestions about the presentation. Contribution no. 1965 of the School of Oceanography, University of Washington.

R eferences

Banse, K. 1977. Determining the carbon-to-chlorophyll ratio of natural phytoplankton. Mar. Biol., 41: 199-212.

Banse. K. 1991. Rates of phytoplankton cell division in the field and in iron enrichment experiments. Limnol. Oceanogr., 36: 1886-1898.

Bresta, A.-M., Ursin, C., and Jensen, L. M. 1987. Intercom­parison of l4C labelled bicarbonate solutions prepared by different institutes for measurement of primary production in natural waters and monoalgal cultures. J. Plankton Res., 9: 317-325.

Cadee, G. C. 1983. Aspects of the l4C method. Dark fixation. Hydrobiol. Bull., 17: 39-40.

Carpenter, E. J., and Lively, J. S. 1980. Review of estimates of algal growth using l4C tracer techniques. In Primary produc­tivity in the sea, pp. 161-178. Ed. by P. G. Falkowski. Plenum Press, New York. 531 pp.

Chavez, F. P., and Barber, R. T. 1987. An estimate of new

I C E S m ar . Sei. S y m p . . 197 (1993) The ,4C method fo r measuring marine phytoplankton production 139

production in the equatorial Pacific. Deep-Sea Res., 34: 1229-1243.

Elrifi, I. R .. and Turpin, D. H. 1987. Short-term physiological indicators of N deficiency in phytoplankton: a unifying model. Mar. Biol., 96: 425-432.

El-Sayed, S. Z ., and Taguchi, S. 1979. Phytoplankton standing crop and primary productivity in the tropical Pacific. In Marine geology and oceanography of the Pacific manganese nodule province, pp. 241-286. Ed. by J. L. Bischoff and D. Z. Piper. Plenum Press, New York and London. 842 pp.

Furnas, J. M. 1987. Effects of prescreening on productivity of size-fractionated phytoplankton. Limnol. Oceanogr., 32: 483-491.

Gieskes, W. W., and Kraay, G. W. 1980. Primary productivity and phytoplankton pigment measurements in the northern North Sea during FLEX ‘76. ‘■Meteor” Forsch. Ergebnisse A, 22:105-112.

Gieskes, W. W., Kraay, G. W., and Baars, M. A. 1979. Current l4C methods for measuring primary production: gross underestimates in oceanic waters. Neth. J. Sea Res., 13: 58-78.

Goldman, J. C., and Dennett, M. R. 1986. D arkC O i uptake by the diatom Chaetoceros simplex in response to nitrogen pulsing. Mar. Biol., 90: 493-500.

Guy, R. D., Vanlerberghe, G. V., andTurpin, D. H. 1989. The significance of phospho-enol pyruvate carboxylase during ammonium assimilation: carbon isotope discrimination in photosynthesis and respiration by the N-limited green alga Selenastrum minutum. Plant Physiol., 89: 1150-1157.

Harris, G. P., Ganf, G. G ., and Thomas, D. P. 1987. Producti­vity, growth rates and cell size distributions of phytoplankton in the SW Tasman Sea: implications for carbon metabolism in the photic zone. J. Plankton Res., 9: 1003-1030.

Harris, G. P., Griffiths, F. B., and Thomas, D. P. 1989. Light and dark uptake and loss of l4C: methodological problems with productivity measurements in oceanic waters. Hydro- biologia, 173: 95-105.

Ignatiades L., Karydis, K., and Pagou, K. 1987. Patterns of dark 14C 0 2 incorporation by natural marine phytoplankton communities. Microb., Ecol., 13: 249-259.

ICES. 1981. Report of the Working Group on the Methodology of Primary Production, Including Guidelines for the Measurement of Primary Production in the ICES Area. Biological Oceanography Committee. ICES CM 1981/L: 46. 31 pp.

ICES. 1990. Report of the ICES C Primary Production Intercomparison Exercise, Hirtshals, Denmark, June 1987. ICES Coop. Res. Rcpt. 170. 46 pp.

Jordan, M. J., and Likens, G. E. 1980. Measurement of planktonic bacterial production in an oligotrophic lake. Lim­nol. Oceanogr., 25: 719-732.

Karl, D. M., Knauer, G. A ., Martin, J. H ., and Ward, B. B. 1984. Bacterial chemolithotrophy in the ocean is associated with sinking particles. Nature (Lond.), 309: 54-56.

King, F. D . 1986. The dependence of primary production in the mixed layer of the eastern tropical Pacific on the vertical transport of nitrate. Deep-Sea Res., 33: 733-754.

Kuntz, D., Packard, T., Devol, A., and Anderson, J. 1975. Chemical, physical and biological observations in the vicinity of the Costa Rica Dome (January-February, 1973). Univer­sity of Wash., Dept, of Ocean. Tech. Rept. No. 321. 187 pp.

Landry, M. R., Haas, L. W., and Fagerness, V. L. 1984. Dynamics of microbial plankton communities: experiments in Kaneohe Bay, Hawaii. Mar. Ecol. Progr. Ser., 16: 127— 133.

Laws, E. A. 1984. Improved estimates of phytoplankton car­

bon based on l4C incorporation into chlorophyll a. J. Thcor. Biol., 110: 425^34.

Legendre, L., Demers, S., Yentsch, C. M., and Yentsch, C. S. 1983. The l4C method: patterns of dark C 0 2 fixation and DCMU correction to replace the dark bottle. Limnol. Ocea­nogr., 28: 996-1003.

Li, W. K. W. 1982. Estimating heterotrophic bacterial produc­tivity by inorganic radiocarbon uptake: importance of estab­lishing time courses of uptake. Mar. Ecol. Prog. Ser., 8:167— 172.

Li, W. K. W. 1986. Experimental approaches to field measure­ments: methods and interpretation. In Photosynthetic pico- plankton, pp. 251-286. Ed. byT. Platt and W. K. W. Li. Can. Bull. Fish, aquat. Sei., 214. 583 pp.

Li, W. K. W., and Dickie, P. M. 1991. Light and dark l4C uptake in dimly-lit oligotrophic waters: relation to bacterial activity. J. Plankton Res., 13(Suppl.): 29-44.

Mague, T. H., Friberg, E., Hughes, D. J., and Morris, I. 1980. Extracellular release of carbon by marine phytoplankton: a physiological approach. Limnol. Oceanogr., 25: 262-279.

Marra, J., Haas, L. W., and Heinemann, K. R. 1988. Time course of C assimilation and microbial food web. J. Exp. Mar. Biol. Ecol., 115: 263-280.

Morris, I., Yentsch, C. M., and Yentsch, C. S. 1971. Relation­ship between light carbon dioxide fixation and dark carbon dioxide fixation by marine algae. Limnol. Oceanogr., 16: 854-858.

Parsons, T. R., Maita, Y., and Lalli, C. M. 1984. A manual of chemical and biological methods for seawater analysis. Per­gamon Press, Oxford. 173 pp.

Petersen, G. H. 1979. On the analysis of dark fixation in primary production computations. J. Cons. int. Explor. Mer, 38: 326-330.

Peterson, B. J. 1980. Aquatic primary productivity and the 14C - C 0 2 method: a history of the productivity problem. Ann. Rev. Ecol. Syst., 11: 359-385.

Platt. J. R. 1964. Strong inference. Science (New York), 146: 347-353.

Prakash, A ., Sheldon, R. W., and Sutcliffe, W. H ., Jr. 1991. Geographic variation of oceanic 14C dark uptake. Limnol. Oceanogr., 36: 30-39.

Riemann, B., Nielsen, P., Jeppesen, M., Marcussen, B., and Fuhrman, J. A. 1984. Diel changes in bacterial biomass and growth rates in coastal environments, determined by means of thymidine incorporation into DNA, frequency of dividing cells (FDC), and microautoradiography. Mar. Ecol. Prog. Ser., 17: 227-235.

Riemann, B., and Sondergaard, M. 1984. Measurements of diel rates of bacterial secondary production in aquatic en­vironments. Appl. Environ. Microbiol., 47: 632-638.

Romanenko, V. I., Overbeck, J., and Sorokin, Y. I. 1972. Estimation of production of heterotrophic bacteria using ,4C. In Techniques for the assessment of microbial pro­duction and decomposition in fresh waters, pp. 82-85. Ed. by Y. I. Sorokin and H. Kadota. International Biological Pro­gram, Handbook No. 23. Blackwell, Edinburgh. 112 pp.

Saijo, Y., and Takesue, K. 1965. Further studies on the size distribution of photosynthesizing phytoplankton in the Indian Ocean. J. Ocean. Soe. Japan, 20: 264-271.

Seki, H. 1968. Relation between production and mineralization of organic matter in Aburatsubo Inlet, Japan. J. Fish. Res. Bd Can., 25: 625-637.

Sieracki, M. E., and Sieburth, J. McN. 1985. Factors control­ling the periodic fluctuation in total planktonic bacterial populations in the upper ocean: comparison of nutrient, sunlight and predation effects. Mar. Microb. Food Webs, 1: 35-50.

140 K. Banse I C E S m ar . Sei. S y m p . . 197(1993)

Sieracki. M. E., and Sieburth, J. McN. 1986. Sunlight-induced growth delay of planktonic marine bacteria in filtered sea­water. Mar. Ecol. Prog. Ser., 33: 19-27.

Smith, R. E.. Gcider, R. J.. and Platt, T. 1984. Microplankton productivity in the oligotrophic ocean. Nature (Lond.), 311 : 252-254.

Smith, W. O ., Jr., Barber, R. T., and Huntsman, S. A. 1977. Primary production off the coast of northwest Africa: ex­cretion of dissolved organic matter and its heterotrophic uptake. Deep-Sea Res., 24: 3 5 ^7 .

Steemann Nielsen, E. 1952. The use of radio-active carbon (C 14) for measuring organic production in the sea. J. Cons, perm. int. Explor. Mer, 18: 117-140.

Steemann Nielsen, E. 1960. Dark fixation of C 0 2 and measure­ments of organic productivity. With remarks on chemo- synthesis. Physiol. PI., 13: 348-357.

Steemann, Nielsen, E. 1963. Productivity, definition and measurement. In The sea, vol. 2, pp. 129-164. Ed. by M. N. Hill. Interscience Publishers, New York. 554 pp.

Taguchi, S. 1983. Dark fixation of C 0 2 in the subtropical North Pacific Ocean and the Weddell Sea. Bull. Plankton Soc. Japan, 30: 115-124.

Taguchi, S., and Platt, T. 1977. Assimilation of ,4C 0 2 in the

dark compared to phytoplankton production in a small coastal inlet. Est. Coastal Mar. Sei., 5: 679-684.

Turley, C., and Lochte, K. 1986. Diel changes in the specific growth rate and mean cell volume of natural bacterial com­munities in two different water masses in the Irish Sea. Microb. Ecol., 12: 271-282.

Turpin, D. H.. Elrifi, I. R., Birch, D. G., Weger, H. G ., and Holmes, J. J. 1988. Interactions between photosynthesis, respiration, and nitrogen assimilation in microalgac. Can. J. Bot.,66: 2083-2097.

Ward. B. B. 1984. Photosynthesis and bacterial utilization of phytoplankton exudates: results from pre- and post­incubation size fractionation. Oceanol. Acta, 7: 337-343.

Ward, B. B. 1987. Nitrogen transformation in the Southern California Bight. Deep-Sea Res., 34: 785-805.

Ward, B. B., Olson, R. J., and Perry, M. J. 1982. Microbial nitrification rates in the primary nitrite maximum off southern California. Deep-Sea Res., 29: 247-255.

Williams, P. J. leB., and Yentsch, C. S. 1976. An examination of photosynthetic production, excretion of photosynthetic products and heterotrophic utilization of dissolved organic compounds with reference to results from a coastal subtropi­cal sea. Mar. Biol., 35: 31-40.