Estimation of N or C uptake rates by phytoplankton using

Transcription

1 Journal of lankton Research Vol. 19 no. 2 pp , 1996 SHORT COMMUNICATION Estimation of N or C uptake rates by phytoplankton using 15 N or 13 C: revisiting the usual computation formulae Louis Legendre and Michel Gosselin 1 Department de biologie, Universite Laval, Quebec, QC G1K 74 and ' Department d'oce'anographie, Universite du Quebec a Rimouski, 310 Allee des Ursulines, Rimouski, QC G5L 3A1, Canada Abstract. The uptake of N by phytoplankton is generally estimated using the I5 N technique and, under some circumstances, the uptake of C is estimated using 13 C. Rigorous examination of formulae for computing net transport rates leads to several interesting and even unexpected conclusions. These are that the I5 N or B C technique formula for computing net transport rates (p) is identical to that of the 14 C technique, in spite of apparent dissimilarities which reflect differences in equipment used for determining non-radioactive and radioactive isotopes; the so-called specific uptake rates (V) should not be used with natural samples, except as a step in the calculation of transport rates (p); estimation of p is unaffected by the presence/absence of non-phytoplanktonic paniculate organic matter (OM) in the incubated sample; the practice of adding the concentration of tracer to the denominator of expression representing the concentration of tracer in the dissolved phase at the beginning of incubation should be discontinued; and the concentration of OM should be determined from the inoculated sample at the end of incubation (or, alternatively, from a sample incubated in parallel) and not from a water sample taken at the beginning of the incubation. Thirty-five years ago, Dugdale et al. (1961) introduced to biological oceanography the I5 N technique to determine the uptake rates of nitrogen by phytoplankton. More than 15 years later, Slawyk et al. (1977, 1979) conducted the first dual tracer measurements using the stable isotopes 13 C and I5 N, to simultaneously estimate the uptake rates of dissolved inorganic carbon and nitrogen. A number of different formulae have been used to calculate the uptake rates of nitrogen (e.g. Neess et al., 1962; Dugdale and Goering, 1967; Eppley et al., 1977) and carbon (e.g. Slawyk et al., 1977; Hama et al., 1983). These were reviewed and compared by Collos and Slawyk (1985) and Collos (1987). Dugdale and Wilkerson (1986) and Collos (1987) recommend two different equations to calculate N transport (also called absolute uptake) rates. One is to be used when the concentration of particulate organic nitrogen (ON) is measured at the end of incubation, and the other, for situations when ON is determined at the beginning. The two equations provide equivalent results when phytoplankton use only one source of nitrogen. When algae use several sources of nitrogen, as is generally the case in natural populations, the only valid equation for calculating transport rates is the first (i.e. ON measured at the end of incubation). The two equations can also be used for computing C transport rates ( 13 C method; Collos and Slawyk, 1985). In addition, Lund (1987) developed equations to calculate N transport rates when the initial concentration of ON is known instead of the final, and when there is simultaneous uptake of several N sources. Recent papers (Bronk et al., 1994; Slawyk and Raimbault, 1995) have shown that part of the nitrogen taken up by phytoplankton may be lost during Oxford University ress 263

2 L.Legendre and M.Gosselin incubation, so that one must distinguish between gross and net transport rates. Here, the two equations generally used for computing net transport rates of dissolved inorganic carbon and nitrogen in the stable isotope ( I5 N and/or I3 C) technique are critically examined. The exercise leads to several interesting, and sometimes unexpected, conclusions. The two usual equations for computing net transport rates (p: mass volume" 1 time"') are both derived from the following general equation (equation 3 in Collos, 1987): A + A* _ %/>«(/>, + />*) - %f g(fo + S) At %D*At All symbols are denned in Table I. The sums (o + J) an d (A + *) correspond to the concentrations of particulate organic matter (OM) in the sample before and after incubation, respectively. Given that: it follows from equation 1 that: A + A* = ( t + *) - ( o + * o ) (2) pa* = ( t + *) - (o + J) (3) Using equation 3, it is easy to derive two forms of equation 1: %*(t +?) - %g[( t + \) - p At] %D*At _ %*[p At + (p + S)] ~ %* Q (o + j), %D\At K) Equations (4) and (5) can be developed to isolate p on the left-hand side. For equation 4, this gives: p(%d* - %*)At = (%* - %o")(, + J) l ( ' ' which provides the formula for calculating p when ( t + *) is known: (%p* - %S) ( t + p (%D* - %*) At K ' In a similar way, equation 5 provides the formula for cases when (o + *,) is used instead of ( t + *): (%r - %$) (p + S) (% >? - %t) At l m ' The two equations are both derived from equation 1, so that they are mathematically equivalent. The net transport rate of N (or C) is generally computed using equation 6 (which corresponds to equation 2 in Dugdale and Wilkerson, 1986, and equation 4 in Collos, 1987). A slightly different form of this equation is: 264

3 Formulae for N and C uptake by phytoplankton Table I. Definitions of symbols in the text and the equations Concentrations and changes in concentrations (mass volume' 1 ) D* Concentration of tracer (heavy isotope) added to the sample at beginning of incubation. Do Concentration of light isotope in the dissolved phase before incubation (natural concentration). Dp Concentration of heavy isotope in the dissolved phase before incubation (nat. cone.), Concentration of light isotope in the particulate phase after incubation. /** Concentration of heavy isotope in the particulate phase after incubation, ^o Concentration of light isotope in the particulate phase before incubation (nat. cone.) Q Concentration of heavy isotope in the particulate phase before incubation (nat. cone.) A Increase of light isotope in the particulate phase during incubation. A* Increase of heavy isotope in the particulate phase during incubation. A* noc Increase of heavy isotope in the particulate phase during incubation for the inoculated sample A/'Jj, Increase of heavy isotope in the particulate phase during incubation for a sample incubated in parallel (without tracer, i.e. natural water). Concentrations (atom %) %D* Concentration of heavy isotope in the dissolved phase of the sample at beginning of incubation (i.e. after addition of the tracer). %DQ Concentration of heavy isotope in the dissolved phase in the natural environment. %* Concentration of heavy isotope in the particulate phase after incubation. %*, Concentration of heavy isotope in the particulate phase before incubation. Time and rates At Incubation time. V Specific uptake rate (time" 1 ). p Net transport rate (mass volume" 1 time"'). = (%p* - %pg) (ft + f) m (/oi/j /OUQ) where %/"5 in the denominator is replaced by %DQ. This is based on the assumption that %D* Q = %Q (%D*, is not determined in field applications). According to Collos and Slawyk (1985), Dugdale and Wilkerson (1986), and Collos (1987), equations 6 or 8 yield unbiased results even when more than one N or C source is taken up by phytoplankton. There are two alternative ways of computing %D*: AI %D*=(D*+D* 0 )/(D 0 + D* 0 ) (9) %D* = (D* + D* 0 )/(D 0 + D* 0 + D*) (10) The two equations are equivalent when D* is small relative to (Do + DJ). Using the first definition of %D* (equation 9), equation 8 can be developed as follows: / * n T "T r n, + l + A+A* p + y (p + * o + A + A*) 265

4 L.Legendre and M.Gosselin If equation 10 is used instead of equation 9, as is generally the case in the literature, p is then computed as: / * Q + A* * o \ _ \Q \» + * O u + A + A* O» +» *J/ (o + o + A*) ~~ D*+D* o D* o \ D 0 + D* 0 +D* D 0 + D*J The consequences of using equations 11 or 12 will be examined below. Equation 11 can be further developed as follows: A, pa* -* O A Expanding the numerator of equation 13 gives: D* At D o +D* o In natural waters, the concentrations of heavy isotopes I5 N or 13 C in the OM are much smaller than those of the corresponding light isotopes 14 N or 12 C. Values for isotopic ratios in the literature are expressed as 5 15 N and 5 13 C: in the sample X I5 N/ I4 N in the standard -) _ / I3 C/ I2 C in the sample I3 C/ I2 -ljxlooo (16) C in the standard ~ V For 5 I5 N, the standard is atmospheric N 2, in which 15 N/ 14 N = ; the values for isotopic ratios in marine seston generally range between 8 15 N = +2 and +10 (e.g. Voss et al., 1996). For 5 13 C, the standard is ee Dee Belemnite, whose 13 C/ I2 C = ; the values for marine seston are 5 I3 C = approx. 25 (e.g. Voss et al., 1996). It follows from equations 15 and 16 that, in most phytoplankton samples, 15 N/ I4 N = and 13 C/ I2 C = This allows the computation of values for two expressions in the numerator of equation 14: forn ' *= 0 " 6 and ^ r forc ' T^T/T and - 266

5 Formulae for N and C uptake by phytoplankton Identical results (to the third decimal) would be obtained for 8 I5 N ranging between -21 and +224 and 5 I3 C between -48 and +32. It follows from the two equations that, for both N and C: -%1 and **0 (19) Given equation 19, equation 14 becomes: = ^(D 0 + D* 0 )-L (20) It is interesting to note that, even if the concentration of OM (/\ + *) is an explicit term in equation 8, it is cancelled out by other terms, so that it does not appear in the final form (equation 20). When calculating the net transport of N and C using the I5 N or I3 C tracers, equation 20 can be rewritten as: = 4^Xt [N-nutrient] 0 (21) = 7 ^ ick (22) where [N-nutrient]o is the ambient concentration, at the beginning of incubation, of the dissolved N-nutrient whose net transport is being estimated, and [DIC]o is the concentration of dissolved inorganic carbon in sea water. It must be noted that equation 22 is identical to the formula used to determine primary productivity with the I4 C radioactive tracer (refs. in eterson, 1980): A I4 C rimary productivity = -^-^ [DIC] 0 (23) = Equations 20 to 22 show that equation 8 estimates: fraction of tracer taken by phytoplankton x concentration of substrate in water incubation time The form of equation 8 compared with that of equation 23 is dictated by the fact that a mass spectrometer provides ratios of isotopic abundances of the light and heavy isotopes in particulate matter, whereas a liquid scintillation counter determines the activities of radioactive isotopes. A basic assumption of methods using either stable or radioactive isotopes is that isotope discrimination by phytoplankton is negligible. This leads to two important conclusions. Firstly, one finds in the literature, in addition to the net transport rate (p), the so-called specific uptake rate (V). The latter is the transport rate divided by the concentration of OM: V = p/om (24) 267

6 L.Legendre and M.Gosselin Several authors first compute V and use it to calculate p: p = V x OM As long as most of the measured OM is phytoplankton, equation 24 provides a valid normalization. However, when a significant proportion of OM is not phytoplankton, as is often the case in natural waters, dividing phytoplankton N or C net transport rates (equations 21 and 22) by ON or OC concentrations (in which there are large non-phytoplanktonic components) does not provide a valid normalization. This is why primary productivity, when determined on natural phytoplankton using the 14 C technique (equation 23), is never normalized to OC but always to chlorophyll a (chl a). There is no reason why the same rule should not apply to p. This is consistent with the recent practice (e.g. Levasseur el ai, 1990; Dickson and Wheeler, 1995) of normalizing nitrogen transport rates to chl a (p/chl a). However, since there is no constant ratio of chl a to phytoplankton carbon, nitrogen, or cell volume, normalization to chl a is by no means ideal but chl a is, at least, specific to phytoplankton. It follows that computed V values are generally not true specific rates, so that V should not be used with natural samples, except as a step in the calculation of p. Secondly, the fact that OM is not in the final form of the equation used to compute p (equation 20) means that p is unaffected by the presence or not of non-phytoplanktonic OM in the incubated sample. The same conclusion had already been reached by Dugdale and Goering (1967) and Dugdale and Wilkerson (1986), based on a different reasoning. The above discussion was based on equation 9. However, the definition of % >* in equation 10 is presently more frequently used than that in equation 9, and the following development examines the consequence of using equation 10 instead of equation 9. Developing equation 12, which was derived from equation 10, gives: p = ^l(d 0 + D* 0 ) (\+ D{} ' "' ), where I ^-J-^ I should be «1 (25) Because, in equation 25, the additional term relative to equation 20 should be quite small, the difference in calculated p values would also be small. However, since equation 20 clearly shows that equation 11 estimates net transport rates, there seems to be no reason to use the definition of %D* in equation 10 instead of that in equation 9. Hence, when D* is small relative to Do, the practice of adding D* to the denominator of %D* should be discontinued. When D* is not small relative to Do, there is no way of computing unbiased p, as shown in the next paragraph. There are situations when the amount of heavy isotope added to the sample is high relative to the natural concentration, e.g. when N transport is estimated in oligotrophic waters. In such cases, the term (DJ -f D*)/Do is not negligible (it can even be >1) so that, as shown by equation 25, equation 11 would overestimate p. In fact, when the amount of tracer is high relative to the natural concentration and is thus an enrichment, no equation can correct for the fact 268

7 Formulae for N and C uptake by phytoplankton that N transport in the incubated sample does not represent transport under the natural, lower N concentration. The problem does not occur when determining C transport by marine phytoplankton because, in oceans, DIC concentrations are high relative to the amount of added I3 C tracer. The situation may be different in fresh waters, where DIC concentrations are sometimes relatively low. There is no absolute rule for deciding whether D* is small or large relative to DQ. In waters with high concentrations of nitrogenous nutrients (i.e. A> + Z>o >0.5uM), >? should not exceed 10% of the ambient value (Dugdale and Goering, 1967; Dugdale and Wilkerson, 1986). Hence, (D* o + D*)/D o <0.1 (equation 25). In waters with low concentrations of nitrogenous nutrients (i.e. DO + DQ <0.5UM), /)* must not exceed the limit of detection of the nutrient (McCarthy, 1980), which is ~0.05uM for dissolved inorganic nitrogen. In oligotrophic waters, (DQ + D*)/D$ may thus lie between 0.5 and 1.0, which would overestimate p as discussed in the previous paragraph. This may nevertheless be acceptable for some purposes. The final case to be examined is the computation of p using the concentration of OM in a water sample taken at the beginning of incubation, instead of that in the sample at the end of incubation. As shown above, p is then calculated with equation 7 (Collos and Slawyk, 1985, their equation 4; Dugdale and Wilkerson, 1986, their equation 7; Collos, 1987, his equation 5). According to Collos and Slawyk (1985) and Collos (1987), equation 7 can be used only when algae take a single source of nitrogen (or carbon). Since this is generally not the case for natural populations, equation 7 should not be used and the concentration of OM should thus be determined on the inoculated sample at the end of incubation (or, alternatively, on a sample incubated in parallel) and not on a water sample taken at the beginning of incubation. Determination of OM on the inoculated sample or, alternatively, on a sample incubated in parallel depends on whether the available equipment measures, on a single filter, the relative concentrations of light and heavy isotopes and the absolute concentration of OM. If so, isotope and OM concentrations are determined simultaneously on the incubated sample. If not, isotope and OM concentrations must be determined independently, on either two samples incubated in parallel (with and without added tracer, respectively) or two fractions of a large-volume inoculated sample. In cases when the concentration of OM is determined on a sample (without added tracer) incubated in parallel to the inoculated sample (end of previous paragraph), equation 11 becomes: n \ + ll. f inoc O + V (Q + *Q + A+, /n* i n* n* Using equation 19, development of equation 26 gives: 269

8 L.Legendre and M.Gosselin A*._.. 1 = D* u h = 1 (27) However, because A* noc and A* at are small relative to o + Q + A, equation 27 shows that using OM measured on a sample incubated in parallel to the inoculated sample would only slightly underestimate p (equation 20). Lund (1987) proposed a set of equations to calculate p when only the initial (and not the final) ON is known and when there is simultaneous uptake of other (unlabelled) N sources. In order to use these equations, the uptake of each N source must be determined separately. The same set of equations could perhaps also be used, when only the initial ON or OC are known, to compute C and N uptake (two tracers) or, as discussed by Collos and Slawyk (1985), to assess the simultaneous uptake of DIC and dissolved organic carbon. In several instances, determining the net transport of all N and/or C sources taken up by natural phytoplankton may be quite demanding. In summary, the above results show that: (i) the 15 N or I3 C technique formula for computing net transport rates (p) is identical to that of the I4 C technique, apparent dissimilarities reflecting differences in equipment used for determining non-radioactive and radioactive isotopes; (ii) the so-called specific uptake rates (V) should not be used with natural samples, except as a step in the calculation of transport rates (p); (iii) estimation of p is unaffected by the presence or absence of non-phytoplanktonic OM in the incubated sample; (iv) the practice of adding the concentration of tracer to the denominator of expression representing the concentration of tracer in the dissolved phase at the beginning of incubation should be discontinued; and (v) the concentration of OM should be determined on the inoculated sample at the end of incubation (or, alternatively, on a sample incubated in parallel) and not on a water sample taken at the beginning of incubation. Acknowledgements Contribution to the programmes of CIROQ (Groupe interuniversitaire de recherches oceanographiques du Quebec) and GREC (Groupe de recherche en environnement cotier). The authors thank the two reviewers and Drs. Jacques Dionne, Bert Klein and Warwick F. Vincent for useful suggestions. Research grants from the Natural Sciences and Engineering Research Council of Canada were instrumental in completion of the work. References Bronk.DA., Glibert..M. and Ward.B.B. (1994) Nitrogen uptake, dissolved organic nitrogen release, and new production. Science, 265, Collos,Y. (1987) Calculations of I5 N uptake rates by phytoplankton assimilating one or several nitrogen sources. Appl. Radial, hot., 38,

9 Formulae for N and C uptake by phytoplankton Co!los,Y. and Slawyk,G. (1985) On the compatibility of carbon uptake rates calculated from stable and radioactive isotope data: implications for the design of experimental protocols in aquatic primary productivity. J. lankton Res., 7, Dickson,M.L. and Wheeler..A. (1995) Nitrate uptake rates in a coastal upwelling regime: A comparison of N-specific, absolute, and Chi a-specific rates. Limnol. Oceanogr., 40, Dugdale.R.C. and Goering,J.J. (1967) Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol. Oceanogr., 12, Dugdale.R.C, Menzel,D.W. and Ryther,J.H. (1961) Nitrogen fixation in the Sargasso Sea. Deep-Sea Res., 7, Dugdale,R.C. and Wilkerson.F.. (1986) The use of I5 N to measure nitrogen uptake in eutrophic oceans; experimental considerations. Limnol. Oceanogr., 31, Eppley,R.W., Sharp.J.H., Renger.E.H., erry,m.j. and Harrison,W.G. (1977) Nitrogen assimilation by phytoplankton and other microorganisms in the surface waters of the Central North acific Ocean. Mar. Bioi, 39, Hama,T., Miyasaki,T., Ogawa,Y., Iwakuma,T., Takahashi,M., Otsuki,A. and Ichimura,S. (1983) Measurement of photosynthetic production of a marine phytoplankton population using a 13 C stable isotope. Mar. Biol, 73, Levasseur,M., Harrison,.J., Heimdal,B.R. and Therriault.J.C. (1990) Simultaneous nitrogen and silicate deficiency of a phytoplankton community in a coastal jet-front. Mar. Bioi, 104, Lund,B.Aa. (1987) Mutual interference of ammonium, nitrate, and urea on uptake of I5 N sources by the marine diatom Skelelonema costalum (Grev.) Cleve. J. Exp. Mar. Biol. Ecol., 113, McCarthy,J.J. (1980) Nitrogen. In Morris,I. (ed.) The physiological ecology of phytoplankton. Blackwell, Oxford, pp Neess.J.C, Dugdale,R.C, Dugdale,V.A. and Goering,J.J. (1962) Nitrogen metabolism in lakes. I. Measurement of nitrogen fixation with N 15. Limnol. Oceanogr., 7, eterson,b.j. (1980) Aquatic primary productivity and the I4 C-CO2 method: a history of the productivity problem. Annu. Rev. Ecol. Syst., 11, Slawyk,G., Collos.Y. and Auclair,J.C. (1977) The use of the I3 C and I5 N isotopes for the simultaneous measurements of carbon and nitrogen turnover rates in marine phytoplankton. Limnol. Oceanogr., 22, Slawyk,G. and Raimbault.. (1995) Simple procedure for simultaneous recovery of dissolved inorganic and organic nitrogen in l5 N-tracer experiments and improving the isotopic mass balance. Mar. Ecol. rog. Ser., 124, Slawyk,G., Collos,Y. and Auclair,J.C. (1979) Reply to comment by Fishen et al. Limnol. Oceanogr., 24, Voss,M., Altabet,M. and von Bodunge.B. (1996) Stable isotopes in sedimenting particles as indicator of euphotic zone processes. Deep-Sea Res., 43, Received on April 2, 1996; accepted on September 30,