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Pelagic nitrogen dynamics in the Vietnamese upwelling area according to stable nitrogen and carbon isotope data
ARTICLE IN PRESS
Deep-Sea Research I 54 (2007) 596–607 www.elsevier.com/locate/dsri
Pelagic nitrogen dynamics in the Vietnamese upwelling area according to stable nitrogen and carbon isotope data Natalie Loicka,, Joachim Dippnera, Hai Nhu Doanb, Iris Liskowa, Maren Vossa a
Department of Marine Biology, Baltic Sea Research Institute, FR Germany, Seestr. 15, 18119 Rostock, FR Germany b Institute of Oceanography Nha Trang, Cau Da 01, Nha Trang, Khan Hoa, SR Vietnam Received 12 September 2005; received in revised form 13 December 2006; accepted 16 December 2006 Available online 8 January 2007
Abstract Upwelling and nitrogen (N) fixation provide new N for primary production off southern central Vietnam. Here we evaluate the roles of both N sources for zooplankton nutrition by comparing d15N and d13C values in nitrate, particulate organic matter (POM), and six net-plankton size fractions from monsoon and intermonsoon seasons. The d13C values in POM and the net-plankton size fractions differed by 2–4% at any time. We assume that plankton from the POM filters was dominated by nano-and picoplankton as opposed to micro- and mesoplankton in the net-samples. The implications of this are discussed in terms of size differential pathways of C and N in the planktonic food web. We used d15N to estimate the differences in N nutrition between the actual upwelling region and the oligotrophic area further offshore. The d15N values of the net-plankton size fractions were depleted in d15N by ca. 2% outside compared to inside the upwelling area during the monsoon season. We attribute these patterns to the additional utilization of N derived from N fixation. The concomitant findings of high N fixation rates reported earlier and low subthermocline nitrate (nitratesub) values of 2.9–3.6% support this conclusion. Net-plankton d15N values increased with size, pointing to the dominance of higher trophic levels in the larger size fractions. According to a two source mixing model N fixation may have provided up to 13% of the N demand in higher trophic levels. r 2007 Elsevier Ltd. All rights reserved. Keywords: Upwelling; Nitrogen isotopes; Carbon isotopes; Trophic relationships; Zooplankton; Nitrogen fixation; Vietnam; South China Sea
1. Introduction The upwelling area off southern central Vietnam is the most productive one of three in the South Corresponding author. Tel.: +49 381 5197 267;
fax: +49 381 5197 440. E-mail addresses: [email protected] (N. Loick), [email protected] (J. Dippner), [email protected] (H.N. Doan), iris.liskow@io-warnemuende. de (I. Liskow), [email protected] (M. Voss). 0967-0637/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr.2006.12.009
China Sea (Liu et al., 2002). However, it is also the least well-studied one, with most biogeochemical work relying on remote sensing data without ground truth validation (Liu et al., 2002). During summer monsoon from June to September upwelling nitrate regularly fertilizes the otherwise nitrogen (N)-limited area within a 40 km long strip along the coast (Dippner et al., 2006; Pham et al., 2002). This results in chlorophyll a values of up to 0.9 mg m3 according to CZCS and SeaWiFS data (Liu et al.,
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2002). Besides upwelling, the Mekong River influences the sea area off southern central Vietnam. However, no additional nitrate or phosphate is carried further offshore into the sea area off southern central Vietnam, where the riverine influence is still detectable by salinities below approximately 33.2 (Dippner et al., 2006). Voss et al. (2006) found high N fixation rates in the low-salinity waters, which accounted for ca. 10% of the new production. The trophic transfer of N has not yet been studied in this region, although different phytoplankton communities are expected to support different heterotrophs; e.g., only a few species directly feed on cyanobacteria (Sellner, 1997). Indirect transfer of fixed N via the microbial loop has also been considered and may support heterotrophs (Montoya et al., 2002). In this study we evaluated the roles of the different N sources for zooplankton nutrition by means of stable N isotopes in subthermocline nitrate (nitratesub) and by carbon (C) and N stable isotopes in particulate organic matter (POM) and six net-plankton size fractions. d15N in nitratesub will be used as tracer for the source nitrate. In the upper water column fractionation processes have to be considered to correctly interpret stable N isotope data (Needoba et al., 2003; Altabet, 1996). The unilateral excretion of light ammonium seems to be the principal cause for enrichment in the d15N of consumers by 2–3.5% compared to the diet (Peterson and Fry, 1987). This fact is widely used as a trophic level indicator in ecological studies (Peterson and Fry, 1987). d13C can be used as a food source indicator, because diet and consumers in marine systems generally differ by less than 1% (Peterson and Fry, 1987). Combined measurements of stable N and C isotopes in nitratesub, POM and the different plankton size fractions therefore can serve as indicators of the food web structure and the ultimate N and C sources for secondary production. A two-source mixing model will be used to estimate the fraction of N in nitrate and plankton derived from N fixation during non-upwelling spring intermonsoon (SpIM) and upwelling southwest monsoon (SWM) seasons.
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southwest monsoon 2004 (8–26 July, Fig. 1). Altogether 46 stations were sampled with a seabird CTD system with attached 10-l water bottles. Fourty-five stations were analysed for nutrients and chlorophyll a, 43 for particulate organic matter (POM), and 33 for d15N-nitrate from 5–6 depth during the three sampling periods. Additionally, 41 stations were sampled for net-plankton, separated into 6 size fractions. Samples for POM, nutrients and chlorophyll a were taken from standard depths covering surface waters (0 or 5 m), 20, 40, 50, 70, 100, and 150 m depth. Additionally, samples were taken from the chlorophyll maximum and from maximum 800 m depth at few stations. Nitrate was analysed after Grasshoff et al. (1983) with a precision of 70.1 mmol l1. Chlorophyll a was analysed after Jeffrey and Humphrey (1975). GF/ F-filtered seawater for isotopic analysis of nitrate was collected when nitrate concentration exceeded 1.5 mmol l1. Samples were transferred to polyethylene bottles and preserved with concentrated HCL to a final pH of 1–2. Nitrate was collected for isotopic analysis by reduction to ammonia, which was then extracted from solution by diffusion and trapping on an acified GF/ F-filter (Sigman et al., 1997).
2. Material and methods The Vietnamese upwelling area was sampled during three cruises on board R.V. Nghien Cuu Bien during southwest monsoon 2003 (18–28 July), spring intermonsoon 2004 (21 April–2 May), and
Fig. 1. Station map during the three sampling periods in southwest monsoon (SWM) and spring intermonsoon seasons.
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drying at 60 1C and storage in aluminium foil, all plankton size fractions were qualitatively inspected under a stereo microscope (50 magnification). At some stations not enough material for isotopic analysis in all plankton size fractions could be sampled. In the following, the terms net-plankton size fractions will be used for plankton from the nethauls, whereas POM will be used for plankton filtered on GF/ F filters. For isotope, PON and POC concentration analysis, dry acified POM and nitrate filters were transferred to a tin capsule; net-plankton size fractions were ground to fine powder and weighed in tin capsules. All natural abundance measure-
POM was collected by vacuum filtration (o400 mm Hg vacuum) of 1–5 l of seawater through precombusted (450 1C for 2 h) 25 mm GF/ F-filters. Visible organisms on the filters were removed. Filters were dried at 60 1C and stored dry for analysis in the laboratory onshore. Net-plankton was collected with a 45 and a 200 mm mesh size JUDAY-net (opening diameter 0.37 m) through the upper 100 m or the entire water column in the case of shallow stations. Plankton from the 45 mm net was separated into two size fractions by passing it through 166 and 55 mm Nitex sieves. Plankton caught with the 200 mm net was separated into 4 size fractions by passing it through 2500, 1500, 1000, and 330 mm Nitex sieves. Prior to
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Fig. 3. Horizontal nitrate concentrations in mmol l from surface waters (0 or 5 m), 20, and 40 m depth during southwest monsoon (SWM) and spring intermonsoon (SpIM) seasons. Black dots show the sampling stations. Note: CI differ between plots as follows: (a–e) 0.1 mmol l1 and (f–e) 0.5 mmol l1.
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ments were made by continuous-flow isotope mass spectrometer (Finnigan Delta S or Delta Plus) via a Conflow II open split interface. Calibration for the total particulate organic carbon (POC) and nitrogen (PON) determination was done daily with an acetanilide standard. All isotope abundances are expressed in d notation as follows: dX (%) ¼ [(Rsample/Rstandard)1)] 103, where X is 13C or 15N, and R is the 13C: 12C or 15N: 14N ratio. IAEA-N3 (KNO3) and IAEA-CH-6 (sucrose) were used as the standards for N and C, respectively. A laboratory internal standard (Peptone, Merck) was run for every 6th sample for net-plankton and every 4th sample for POM filters. The peptone standard indicated an analytical error associated with the isotope measurements of less than 0.2% for both isotopes. Two replicates were analysed from each sample of net-plankton. For comparability of POM and net-plankton values, the vertically integrated concentrationweighted d15N-PON was calculated for the upper 100 m or the entire water column in the case of shallow stations according to Eq. (1) after Montoya et al. (1992): P DZ i d15 NPONi ½PONi 15 P d NPON0max : 100 m ¼ , DZ i ½PONi (1) where DZ is the thickness of the individual layers between sampled depths, i denotes the depth layers between the surface and 100 m depth or the entire water column in the case of a shallow station, [PON] is the concentration in mM and d15N-PON is the isotope ratio in %. The same was done for the d13C-POC. Vertically integrated concentration weighted d15N from nitratesub was also calculated according to Eq. (1) for the depths range between the mixed layer and the maximum depth of 150 m. This depth interval includes the starting horizon of the upwelling velocity at approximately 125 m depth (Vo, 1997). The mixed layer depths were estimated from the maximum in the buoyancy frequency of the water column. Vertically integrated chlorophyll a and POC concentrations were calculated from surface to 100 m depth after the trapezoidal rule.
3. Results 3.1. Sea surface temperature (SST) and sea surface salinity (SSS) During SWM 2003 a weak gradient in SST off the coast of Vietnam was observed (Fig. 2). This reflected the weak upwelling conditions for the post-ENSO year 2003 as described in detail by Dippner et al. (2006). Compared to 2003 SST was 2 1C cooler during SWM 2004 and reflected a stronger upwelling intensity. During SpIM no SST gradient was found; therefore no coastal upwelling occurred. Similar to temperature, SSS showed strong coastal gradients during both SWM periods (Fig. 2). No gradient was found during SpIM. During both SWM periods low salinity waters of less than 33.2 entered the study area from the south. These waters have been described as an extension of the Mekong River plume that turns northwards during SWM by Dippner et al. (2006). During SpIM no low salinity waters were found and SSS was evenly high between 33.9 and 34.0. 3.2. Nitrate concentrations Coastal upwelling of nitrate was indicated during the SWM, when nitrate concentrations increased from zero at the surface to 4 and 8 mmol l1 at 40 m depth (Fig. 3). The nutricline was situated at approximately 20 m within the upwelling area and at 40–60 m offshore during SWM. Upwelling nitrate reached surface waters only at station 22 during SWM 2003 (Fig. 3a) and at station 52 during SWM 2004 (Fig. 3c). During SpIM the nutricline generally was found at 60 m depth, whereas no horizontal gradient was apparent. Elevated values of up to 5 mmol l1 at 40 m depth indicated an eddy formation at station 54 (Fig. 3h). Furthermore enhanced surface concentrations of up to 0.9 mmol l1 were found at the southernmost coastal stations during SWM 2004 (Fig. 3c) and of up to 1.6 mmol l1 at 20 m depth during SpIM 2004 (Fig. 3e), possibly due to a riverine influence. However, transport of riverine nitrate into the investigation area was restricted to the southern, shallow sites, whereas
Fig. 4. d15N values of NO 3 (open symbols) and PON (closed symbols) from inshore sites, shown on the upper panel, and offshore sites, shown on the lower panel including one northern and one southern station during southwest monsoon (SWM) and spring inter-monsoon (SpIM) seasons.
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the rest of the upwelling area remained unaffected by riverine nitrate. Nitrate concentrations in the water column were similar to values previously reported by Pham et al. (2002). 3.3. Stable isotope data d15N values in nitrate were most variable in the upper 150 m (Fig. 4). During SWM 2003 values ranged from 2.6% to 4.9%, during SpIM from 0.8% to 5.3%, and during SWM 2004 from 2.2% to 7.2%. Below 150 m to a maximum deth of 800 m d15N-nitrate was similar between seasons with an average value of 4.070.6% (n ¼ 15). Vertical profiles of d15N in nitrate were rather similar in and outside the upwelling area during SWM seasons (Fig. 4). Only during SpIM were lower values found at coastal stations compared to stations 442 km offshore. The concentration weighted average values of nitratesub were 3.570.3 (n ¼ 11 stations, SWM 2003), 2.970.9 (n ¼ 19 stations, SpIM 2004), and 3.7%70.6 (n ¼ 15 stations, SWM 2004, Fig. 5). During SWM 2003 and SpIM 2004 surface d15N in particulate organic matter (POM) varied around 2% whereas during SWM 2004 values were ca. 2% enriched (Fig. 4). d15N-POM increased with depth by 2–4%, a distribution generally attributed to isotopic fractionation in the course of remineralization of POM (Montoya et al., 1992). The vertical profiles were similar between stations in and outside the upwelling area during all sampled seasons (Fig. 4). The integrated values d15 NPON0max : 100 m for the upper water column to a maximum of 100 m had average values of 2.9% (70.5, n ¼ 9, SWM 2003), 2.8% (71.0, n ¼ 24, SpIM 2004), and 4.2% (70.8, n ¼ 40, SWM 2004). No clear in-offshore trend was found in d15N-PON (Fig. 5). Average d13 CPOC0max : 100 m values were 24.1% (70.3, n ¼ 9, SWM 2003), 23.5% (70.3, n ¼ 24, SpIM 2004), and 23.1% (70.8, n ¼ 40, SWM 2004, Fig. 6). POM was in 81% of all cases enriched over the nitrate isotopic signature by 2.171.2% (n ¼ 69) and only in 16 samples depleted by 1.771.5% during SWM seasons. During SpIM season 65% of all POM samples were enriched over the nitrate isotopic signature by 2.271.4% (n ¼ 30) and in 16 cases depleted by 2.471.9%. The d15N values in the six net-plankton size fractions generally increased from the smallest (55–166 mm) to the largest (42500 mm) size fraction
(Fig. 5). Lowest d15N values of 1.4% and 0.6% were found during SWM 2003 and 2004, respectively. Highest d15N values were also found during SWM seasons (8.0% and 7.8% in 2003 and 2004, respectively). During SpIM 2004 values ranged from 3.0% to 6.8% and no spatial differences were found between in- and offshore stations (440 km offshore, Fig. 5). During monsoon season d15N values in all net-plankton size fractions were on average 1.6% (SWM 2003) and 2.3% (SWM 2004) depleted outside compared to inside the upwelling area. Fig. 6 shows the d13C values of the smallest netplankton size fraction 55–166 mm, the combined values for the other 5 net-plankton size fractions (166 to 42500 mm) and for the POM-filters (40.7 mm). The 6 net-plankton size fractions were statistically not different from each other and had mean values of 20.9% (70.5, n ¼ 69, SWM 2003), 21.3% (70.6, n ¼ 137, SpIM 2004), and 21.2% (70.8, n ¼ 104, SWM 2004, Fig. 6). In contrast, d13C values in POM were significantly lower than in the net-plankton size fractions. 4. Discussion 4.1. Differences between POM and net-plankton C cycling in marine planktonic food webs is generally separated in two different pathways, the microbial and the herbivore pathway (Legendre and Le Fe`vre, 1991; Pesant et al., 2000). We found two different phytoplankton communities in our study area. The first is represented by the POM-filter samples. Particulate organic C concentrations correlate highly significantly with chlorophyll a concentrations in the upper 100 m of the water column (Fig. 7). This indicates that POM was dominated by phytoplankton rather than by detritus. The second phytoplankton community is represented by the size fraction 55–166 mm. The dominance of phytoplankton in this size fraction is indicated by an average C:N ratio of 7.4 (70.8, n ¼ 44). Furthermore, microscopic inspection on board identified diatoms like Rhizosolenia spp., Chaetoceros spp., Coscinodiscus spp., Thalassiosira spp., Thalasionema spp., Hemiaulus spp., Pseudonizschia spp. or Stephanopyxis spp., cyanobacteria like Trichodesmium spp., and dinoflagellates like Protoperidinium spp., Ceratium spp., Amphisolenia spp., or Pyrocystis spp. in this size fraction. The size fractions 4166 mm were characterized by increasing d15N values with size (Fig. 5). According to Fry and Quinones (1994) this
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Fig. 5. d N patterns of particulate organic matter (POM 4 0.7 mm) and six net-plankton size fractions (55–42500 mm) during (a) southwest monsoon 2003, (b) spring intermonsoon 2004, and (c) southwest monsoon 2004. The continuous lines separate inshore (o42 km off the coast) and offshore (442 km off the coast) stations. Dotted lines give the integrated concentration weighted d15N value of subthermocline nitrate for each season. Note: Inshore during SWM seasons means within the upwelling area.
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Fig. 6. Average d13C values of POM, net-plankton size fraction 55–166 mm, and net-plankton size fraction 166 to 42500 mm. Numbers of samples are given above the x-axis for each respective season and plankton size fraction for size fraction 166 to 42500 mm (top), size fraction 55–166 mm (middle), and POM (bottom). Dotted area indicates the 71% interval for potential food source of plankton in size fractions 166 to 42500 mm according to Peterson and Fry (1987).
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reflects an increase in trophic levels with size due to different zooplankton groups. In order to identify the potential food source of the zooplankton the stable C isotope data of POM and size fraction 55–166 mm were compared to those of the zooplankton size fractions 4166 mm. According to Peterson and Fry (1987) food for zooplankton must have d13C values that are within a71% interval of the zooplankton value. It was found that the stable C isotope values (d13C) in POM (40.7 mm) were 2–3% lower than the d13C values in the zooplankton size fractions 4166 mm (Fig. 6). In contrast the phytoplankton size fraction 55–166 mm differed less than 1% from the zooplankton size fractions 4166 mm (Fig. 6). We therefore conclude that phytoplankton in size fraction 55–166 mm is the potential food source for zooplankton 4166 mm, whereas POM may be excluded as food source for zooplankton 4166 mm. The d13C values in our POM samples are similar to the d13C values of pico- and nanoplankton covering a size range of o3–20 mm from a study in the Mediterranean Sea by Rau et al. (1990). The d13C values from
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our net-plankton are similar to the micro-and mesoplankton values from Rau et al. (1990) that covered a size range of 20 to 4150 mm. This indicates that the filter plankton may belong to the microbial, whereas the net-plankton may belong to the herbivore food web. This interpretation is consistent with observed co-existences of pico-and nanoplankton populations and larger-sized phytoplankton supporting micro- and mesozooplankton populations in the oceans as reviewed by Kiørboe (1993). In contrast to the probably limited C transfer between POM and larger plankton we found indications for intensive transfer of N from the POM size fraction into the larger plankton size fractions at sites of high N fixation. Voss et al. (2006) measured high N fixation rates in the POM size fraction exclusively at the offshore sites during SWM. Our results show that at these sites the d15N values of the net-plankton size fractions were lowered by ca. 2% compared to the sites of low N fixation (Fig. 5). We interpret this as transfer of N from N fixation in the microbial loop into the herbivore food web. N from N fixation by cyanobacteria has a theoretical d15N value of 1% (Montoya et al., 2002). It may be an additional new N source in oligotrophic regions, and its incorporation can be traced by a lowering in the d15N values of the pelagic food web as shown, e.g., by Fry and Quinones (1994). However, it is not clear in which inorganic or organic form this N is transferred. We found d15N values in POM below the d15N values of subthermocline nitrate despite low N2 fixation rates, especially in the upwelling area during SWM 2003 (Fig. 5a). Besides N2 fixation only the uptake of ammonium may cause d15N values of surface particles below the d15N values of subthermocline nitrate (Sigman and Casciotti, 2001). Regenerated ammonium as N source therefore may be an additional N source especially for nano-and picoplankton as found by Wafar et al. (2004). In contrast larger phytoplankton cells are considered to depend on alternative N sources, especially on nitrate when it is available in high concentrations, because of a strong diffusion limitation for ammonium (Stolte et al., 1994). 4.2. d15N patterns in net-plankton in relation to d15N in nitrate The d15N values in the net-plankton size fractions (55Z2500 mm) were higher than the average d15N values in subthermocline nitrate (nitratesub) through-
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out the study area during SpIM and within the upwelling area during SWM (Fig. 5). In contrast the lower trophic levels and the assumed zooplankton food source (size fraction 55–166 mm) had d15N values below the nitratesub values at offshore stations during SWM. These differences can be explained by the spatially different utilization of new N sources. Whereas N from N fixation was an additional new N source for the net-plankton community outside the upwelling area during SWM, nitrate from below the nutricline was the only new N source in the upwelling area and during SpIM. This is consistent with the N fixation rates and new production rates measured by Voss et al. (2006) and the identification of nitratesub from Maximum Salinity Water as the sole nitrate source for primary production by Dippner et al. (2006). Utilization of nitratesub leads to d15N values of the assimilating organism, e.g., diatoms, that may be up to 12% enriched compared with the source nitrate (Needoba et al., 2003; Altabet, 1996). Important factors affecting isotope fractionation in phytoplankton are species composition, swimming abilities, light regime and the assimilatory activity of nitrate reductase (Granger et al., 2004; Needoba and Harrison, 2004). d15N values in the net-plankton size fractions higher than the nitratesub values therefore possibly reflect the fractionation effect during nitrate uptake. In contrast, the concomitant finding of high N2 fixation rates strongly supports the theory that the incorporation of N from N fixation rather than ammonium caused the observed lowering in the d15N values in the net plankton outside the upwelling area during SWM. A strong influence of N2 fixation on the N cycle throughout the area is furthermore indicated by d15N values of nitratesub between 2.9% and 3.7% since only N2 fixation can lead to d15N values in nitratesub below the global range of 4.5–6% (Liu and Kaplan, 1989). We generally observed an increase in d15N with plankton size (Fig. 5). Enrichments in d15N with increasing plankton size are attributed to trophic fractionation effects (Rolff, 2000; Fry and Quinones, 1994). However, the observed increases in d15N between the consecutive size fractions of on average 0.2% (70.6, n ¼ 263) were much smaller than the reported 2–3.5% increase between consumer and diet (Peterson and Fry, 1987). One reason may be the insufficient resolution of trophic levels and the overlap within one size fraction. Furthermore d15N in bulk plankton may be altered by a variety of factors such as species composition (Montoya et al., 1992), food quality or
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quantity (Adams and Sterner, 2000), N turnover (Montoya et al., 1991), or amino acid composition (McClelland and Montoya, 2002). For example, the salp Salpa aspera can feed effectively on a broad range of particle sizes since it uses a mucus net to filter suspended particles out of the sea water (Montoya et al., 1992). The copepod Pareuchaeta norvegica selectively feeds on copepods and other small zooplankton and has higher d15N values than the salp. The d15N in different zooplankton species therefore varies depending on their feeding habits (Montoya et al., 1992). Nutritional stress can cause increases in d15N of up to 0.4% in Daphnia magna, which are significantly correlated with the C:N ratio of the food (Adams and Sterner, 2000). As the N content of the food source decreases from a C:N of 6–25, the corresponding d15N values of the daphnids feeding on the algae increased from 1% to 4% (Adams and Sterner, 2000). However, we found no correlation between C:N ratios and d15N of the netplankton samples. The natural abundance of 15N in various components of an ecosystem can change markedly on a time scale of days (Montoya et al., 1991). Differences in N turnover will lead to different kinetics in the isotope responses of plankton species to changes in d15N of the food source (Montoya et al., 2002). For example, the turnover time of Acartia tonsa was estimated to be 7.670.6 days, whereas the ctenophore Mnemiopsis leidyi was assumed to have much slower turnover times, as indicated by less variable d15N values (Montoya et al., 1991). Furthermore d15N in bulk plankton may alter by variations in d15N of amino acids attributed to internal processing or variations in the acquired food source (McClelland and Montoya, 2002). However, although amino acids like glutamic acid, alanine, aspartic acid, isoleucine, leucine, proline, and valine are up to 7% enriched in the consumer compared to the diet, they always seem to reflect the trophic increase in bulk d15N (McClelland and Montoya, 2002). Despite these factors only trophic fractionation can cause the general increase in d15N with size and is confirmed by other studies (e.g. Montoya et al., 2002; Fry and Quinones, 1994).
zooplankton food source is a mixture of the d15N values of the 2 new N sources nitrate and N from N fixation. For this estimation the d15N value of the zooplankton food source, the d15N of the source nitrate and the d15N of N from N fixation have to be known. We assume that size fraction 55–166 mm is the zooplankton food source. The mean d15N value of this size fraction at sites of high N fixation was 2.9% during SWM 2003 and 3.1% during SWM 2004. Furthermore, we use the theoretical d15N value of 1% as value for regenerated N from N fixation from Montoya et al. (2002). Nitratesub was found to be the only nitrate source and had d15N values of 3.5% and 3.7% during SWM 2003 and 2004, respectively. Based on these values N fixation contributed 9% and 13% of N of the zooplankton food source during SWM 2003 and 2004, respectively. The d15N patterns of the larger size fractions prove that this N is also transferred to higher trophic levels. This contribution is much less than in the oligotrophic North Atlantic, where N2 fixation was estimated to contribute on average 51% to the organic N in particles in the upper 100 m (Montoya et al., 2002). Our estimation from the natural abundance stable N isotope measurements closely match the rate estimations by Voss et al. (2006) that N2 fixation may contribute approximately 10% of the new production. This shows that N2 that is fixed by pico- and nanoplankton is transferred in large part into the herbivore food web. Acknowledgments The German-Vietnamese cooperation has been financed by the German Research Foundation (DFG), the Federal Ministry for Economic Cooperation and Development (BMZ) and the Vietnamese Ministry of Science and Technology (MOST), which is greatly acknowledged. We thank Lam Ngoc Nguyen, Bui Hong Long, and Rolf Peinert for their organisation before and during the cruises. The authors are indebted to Barbara Deutsch and Joseph Montoya for helpful comments and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG/Bo-768).
4.3. Contribution of N fixation The proportion of N from N fixation for higher trophic levels can be estimated according to the 2-source mixing model of Montoya et al. (2002). For this model it is assumed that the d15N value of the
References Adams, T.S., Sterner, R.W., 2000. The effect of dietary nitrogen content on trophic level 15N enrichment. Limnology and Oceanography 45 (3), 601–607.
ARTICLE IN PRESS N. Loick et al. / Deep-Sea Research I 54 (2007) 596–607 Altabet, M.A., 1996. Isotopic fractionation during biochemical reactions. In: Ittekot, S., Honjo, D. (Eds.), Scope: Particle Flux in the Ocean. Wiley, Chichester, pp. 156–183. Dippner, J., Nguyen, K.V., Hein, H., Ohde, T., Loick, N., 2006. Monsoon Induced Upwelling off the Vietnamese Coast. Ocean Dynamics (in press). Fry, B., Quinones, R.B., 1994. Biomass spectra and stable isotope indicators of trophic level in zooplankton of the Northwest Atlantic. Marine Ecology Progress Series 112 (1–2), 201–204. Granger, J., Sigman, D.M., Needoba, J.A., Harrison, P.J., 2004. Coupled nitrogen and oxygen isotope fractionation of nitrate during assimilation by cultures of marine phytoplankton. Limnology and Oceanography 49 (5), 1763–1773. Grasshoff, K., Ehrhardt, M., Kremling, K., 1983. Methods of Seawater Analysis. Verlag Chemie, Weinheim, p. 419. Jeffrey, S.W., Humphrey, G.F., 1975. New spectrophotometric equation for determining chlorophyll a, b, c1 and c2. Biochemie und Physiologie der Pflanzen 167, 194–204. Kiørboe, T., 1993. Turbulence, phytoplankton cell size, and the structure of pelagic food webs. Advances in Marine Biology 29, 1–72. Legendre, L., Le Fe`vre, J., 1991. From individual plankton cells to pelagic ecosystems and to global biogeochemical cycles. In: Demers, S. (Ed.), Particles Analysis in Oceanography. Springer, Berlin, pp. 261–300. Liu, K.K., Chao, S.Y., Shaw, P.T., Gong, G.C., Chen, C.C., Tang, T.Y., 2002. Monsoon-forced chlorophyll distribution and primary production in the South China Sea: observations and a numerical study. Deep-Sea Research I 49 (8), 1387–1412. Liu, K.K., Kaplan, I.R., 1989. The eastern tropical Pacific as a source of 15N-enriched nitrate in seawater off southern California. Limnology and Oceanography 34 (5), 820–830. McClelland, J.W., Montoya, J.P., 2002. Trophic relationships and the nitrogen isotopic composition of amino acids. Plankton Ecology 83 (8), 2173–2180. Montoya, J.P., Horrigan, S.G., McCarthy, J.J., 1991. Rapid, storm-induced changes in the natural abundance of 15N in a planktonic ecosystem, Chesapeake Bay, USA. Geochimica et Cosmochimica Acta 55, 3627–3638. Montoya, J.P., Wiebe, P.H., McCarthy, J.J., 1992. Natural abundance of 15N in particulate nitrogen and zooplankton in the Gulf Stream region and warm-core ring 86A. Deep-Sea Research Part A (Oceanographic Research Papers) 39 (1A suppl.), S363–S392. Montoya, J.P., Carpenter, E.J., Capone, D.G., 2002. Nitrogen fixation and nitrogen isotope abundances in zooplankton of the oligotrophic North Atlantic. Limnology and Oceanography 47 (6), 1617–1628. Needoba, J.A., Harrison, P.J., 2004. Influence of low light and A light: Dark cycle on NO 3 uptake, intracellular NO3 , and
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nitrogen isotope fractionation by marine phytoplankton. Journal of Phycology 40 (3), 505–516. Needoba, J.A., Waser, N.A., Harrison, P.J., Calvert, S.E., 2003. Nitrogen isotope fractionation in 12 species of marine phytoplankton during growth on nitrate. Marine Ecology Progress Series 255, 81–91. Pesant, S., Legendre, L., Gosselin, M., Bjornsen, P.K., Fortier, L., Michaud, J., Nielsen, T.G., 2000. Pathways of carbon cycling in marine surface waters: the fate of small-sized phytoplankton in the Northeast Water Polynya. Journal of Plankton Research 22 (4), 779–801. Peterson, B.J., Fry, B., 1987. Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18, 293–320. Pham, V.T., Nguyen, T.A., Hoang, T.D., 2002. Some remarks on the distribution of nutrients along the transect Nha TrangLuzon (Vietnamese-Philippines cooperative investigation, JOMSRE II, May 2000). In: Nguyen, T.A. (Ed.), Collection of Marine Research Works XII, 91–102. Rau, G.H., Teyssie, J.L., Rassoulzadegan, F., Fowler, S.W., 1990. 13C/12C and 15N/14N variations among size-fractionated marine particles: Implications for their origin and trophic relationships. Marine Ecology Progress Series 59 (1–2), 33–38. Rolff, C., 2000. Seasonal variation in d13C and d15N of sizefractioned plankton at a coastal station in the northern Baltic proper. Marine Ecology Progress Series 203, 47–65. Sellner, K.G., 1997. Physiology, ecology, and toxic properties of marine cyanobacteria blooms. Limnology and Oceanography 42 (5pt. 2), 1089–1104. Sigman, D.M., Casciotti, K.L., 2001. Nitrogen isotopes in the ocean. In: Thorpe, S., Threbion, K. (Eds.), Encyclopedia of Ocean Sciences. Elservier Verlag, New York, pp. 1884–1894. Sigman, D.M., Altabet, M.A., Michener, R., McCorkle, D.C., Fry, B., Holmes, R.M., 1997. Natural abundance-level measurement of the nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonia diffusion method. Marine Chemistry 57 (3–4), 227–242. Stolte, W., Mc Collio, T., Noordeloos, A.A.M., Reigman, R., 1994. Effect of N source on the size distribution within marine phytoplankton populations. Journal of Experimental Marine Biology and Ecology 184, 83–97. Vo, V.L., 1997. Atlas of natural condition, ecology and bioproduction of strong upwelling sea region in southern central Vietnam. In: Vo, V.L., Nguyen, T.A. (Eds.). Science and Technics Publishing House, Nha Trang, p. 2. Voss, M., Bombar, D., Loick, N., Dippner, J.W., 2006. Riverine influence on nitrogen fixation in the upwelling region off Vietnam, South China Sea. Geophysical Research Letters 33, L07604, doi:10.1029/2005GL025569. Wafar, M., L0 Helguen, S., Raikar, V., Maguer, J-F., Le Corre, P., 2004. Nitrogen uptake by size-fractionated plankton in a permanently well-mixed temperate coastal waters. Journal of Plankton Research 26 (19), 1207–1218.
Deep-Sea Research I 54 (2007) 596–607 www.elsevier.com/locate/dsri
Pelagic nitrogen dynamics in the Vietnamese upwelling area according to stable nitrogen and carbon isotope data Natalie Loicka,, Joachim Dippnera, Hai Nhu Doanb, Iris Liskowa, Maren Vossa a
Department of Marine Biology, Baltic Sea Research Institute, FR Germany, Seestr. 15, 18119 Rostock, FR Germany b Institute of Oceanography Nha Trang, Cau Da 01, Nha Trang, Khan Hoa, SR Vietnam Received 12 September 2005; received in revised form 13 December 2006; accepted 16 December 2006 Available online 8 January 2007
Abstract Upwelling and nitrogen (N) fixation provide new N for primary production off southern central Vietnam. Here we evaluate the roles of both N sources for zooplankton nutrition by comparing d15N and d13C values in nitrate, particulate organic matter (POM), and six net-plankton size fractions from monsoon and intermonsoon seasons. The d13C values in POM and the net-plankton size fractions differed by 2–4% at any time. We assume that plankton from the POM filters was dominated by nano-and picoplankton as opposed to micro- and mesoplankton in the net-samples. The implications of this are discussed in terms of size differential pathways of C and N in the planktonic food web. We used d15N to estimate the differences in N nutrition between the actual upwelling region and the oligotrophic area further offshore. The d15N values of the net-plankton size fractions were depleted in d15N by ca. 2% outside compared to inside the upwelling area during the monsoon season. We attribute these patterns to the additional utilization of N derived from N fixation. The concomitant findings of high N fixation rates reported earlier and low subthermocline nitrate (nitratesub) values of 2.9–3.6% support this conclusion. Net-plankton d15N values increased with size, pointing to the dominance of higher trophic levels in the larger size fractions. According to a two source mixing model N fixation may have provided up to 13% of the N demand in higher trophic levels. r 2007 Elsevier Ltd. All rights reserved. Keywords: Upwelling; Nitrogen isotopes; Carbon isotopes; Trophic relationships; Zooplankton; Nitrogen fixation; Vietnam; South China Sea
1. Introduction The upwelling area off southern central Vietnam is the most productive one of three in the South Corresponding author. Tel.: +49 381 5197 267;
fax: +49 381 5197 440. E-mail addresses: [email protected] (N. Loick), [email protected] (J. Dippner), [email protected] (H.N. Doan), iris.liskow@io-warnemuende. de (I. Liskow), [email protected] (M. Voss). 0967-0637/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr.2006.12.009
China Sea (Liu et al., 2002). However, it is also the least well-studied one, with most biogeochemical work relying on remote sensing data without ground truth validation (Liu et al., 2002). During summer monsoon from June to September upwelling nitrate regularly fertilizes the otherwise nitrogen (N)-limited area within a 40 km long strip along the coast (Dippner et al., 2006; Pham et al., 2002). This results in chlorophyll a values of up to 0.9 mg m3 according to CZCS and SeaWiFS data (Liu et al.,
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2002). Besides upwelling, the Mekong River influences the sea area off southern central Vietnam. However, no additional nitrate or phosphate is carried further offshore into the sea area off southern central Vietnam, where the riverine influence is still detectable by salinities below approximately 33.2 (Dippner et al., 2006). Voss et al. (2006) found high N fixation rates in the low-salinity waters, which accounted for ca. 10% of the new production. The trophic transfer of N has not yet been studied in this region, although different phytoplankton communities are expected to support different heterotrophs; e.g., only a few species directly feed on cyanobacteria (Sellner, 1997). Indirect transfer of fixed N via the microbial loop has also been considered and may support heterotrophs (Montoya et al., 2002). In this study we evaluated the roles of the different N sources for zooplankton nutrition by means of stable N isotopes in subthermocline nitrate (nitratesub) and by carbon (C) and N stable isotopes in particulate organic matter (POM) and six net-plankton size fractions. d15N in nitratesub will be used as tracer for the source nitrate. In the upper water column fractionation processes have to be considered to correctly interpret stable N isotope data (Needoba et al., 2003; Altabet, 1996). The unilateral excretion of light ammonium seems to be the principal cause for enrichment in the d15N of consumers by 2–3.5% compared to the diet (Peterson and Fry, 1987). This fact is widely used as a trophic level indicator in ecological studies (Peterson and Fry, 1987). d13C can be used as a food source indicator, because diet and consumers in marine systems generally differ by less than 1% (Peterson and Fry, 1987). Combined measurements of stable N and C isotopes in nitratesub, POM and the different plankton size fractions therefore can serve as indicators of the food web structure and the ultimate N and C sources for secondary production. A two-source mixing model will be used to estimate the fraction of N in nitrate and plankton derived from N fixation during non-upwelling spring intermonsoon (SpIM) and upwelling southwest monsoon (SWM) seasons.
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southwest monsoon 2004 (8–26 July, Fig. 1). Altogether 46 stations were sampled with a seabird CTD system with attached 10-l water bottles. Fourty-five stations were analysed for nutrients and chlorophyll a, 43 for particulate organic matter (POM), and 33 for d15N-nitrate from 5–6 depth during the three sampling periods. Additionally, 41 stations were sampled for net-plankton, separated into 6 size fractions. Samples for POM, nutrients and chlorophyll a were taken from standard depths covering surface waters (0 or 5 m), 20, 40, 50, 70, 100, and 150 m depth. Additionally, samples were taken from the chlorophyll maximum and from maximum 800 m depth at few stations. Nitrate was analysed after Grasshoff et al. (1983) with a precision of 70.1 mmol l1. Chlorophyll a was analysed after Jeffrey and Humphrey (1975). GF/ F-filtered seawater for isotopic analysis of nitrate was collected when nitrate concentration exceeded 1.5 mmol l1. Samples were transferred to polyethylene bottles and preserved with concentrated HCL to a final pH of 1–2. Nitrate was collected for isotopic analysis by reduction to ammonia, which was then extracted from solution by diffusion and trapping on an acified GF/ F-filter (Sigman et al., 1997).
2. Material and methods The Vietnamese upwelling area was sampled during three cruises on board R.V. Nghien Cuu Bien during southwest monsoon 2003 (18–28 July), spring intermonsoon 2004 (21 April–2 May), and
Fig. 1. Station map during the three sampling periods in southwest monsoon (SWM) and spring intermonsoon seasons.
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drying at 60 1C and storage in aluminium foil, all plankton size fractions were qualitatively inspected under a stereo microscope (50 magnification). At some stations not enough material for isotopic analysis in all plankton size fractions could be sampled. In the following, the terms net-plankton size fractions will be used for plankton from the nethauls, whereas POM will be used for plankton filtered on GF/ F filters. For isotope, PON and POC concentration analysis, dry acified POM and nitrate filters were transferred to a tin capsule; net-plankton size fractions were ground to fine powder and weighed in tin capsules. All natural abundance measure-
POM was collected by vacuum filtration (o400 mm Hg vacuum) of 1–5 l of seawater through precombusted (450 1C for 2 h) 25 mm GF/ F-filters. Visible organisms on the filters were removed. Filters were dried at 60 1C and stored dry for analysis in the laboratory onshore. Net-plankton was collected with a 45 and a 200 mm mesh size JUDAY-net (opening diameter 0.37 m) through the upper 100 m or the entire water column in the case of shallow stations. Plankton from the 45 mm net was separated into two size fractions by passing it through 166 and 55 mm Nitex sieves. Plankton caught with the 200 mm net was separated into 4 size fractions by passing it through 2500, 1500, 1000, and 330 mm Nitex sieves. Prior to
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ments were made by continuous-flow isotope mass spectrometer (Finnigan Delta S or Delta Plus) via a Conflow II open split interface. Calibration for the total particulate organic carbon (POC) and nitrogen (PON) determination was done daily with an acetanilide standard. All isotope abundances are expressed in d notation as follows: dX (%) ¼ [(Rsample/Rstandard)1)] 103, where X is 13C or 15N, and R is the 13C: 12C or 15N: 14N ratio. IAEA-N3 (KNO3) and IAEA-CH-6 (sucrose) were used as the standards for N and C, respectively. A laboratory internal standard (Peptone, Merck) was run for every 6th sample for net-plankton and every 4th sample for POM filters. The peptone standard indicated an analytical error associated with the isotope measurements of less than 0.2% for both isotopes. Two replicates were analysed from each sample of net-plankton. For comparability of POM and net-plankton values, the vertically integrated concentrationweighted d15N-PON was calculated for the upper 100 m or the entire water column in the case of shallow stations according to Eq. (1) after Montoya et al. (1992): P DZ i d15 NPONi ½PONi 15 P d NPON0max : 100 m ¼ , DZ i ½PONi (1) where DZ is the thickness of the individual layers between sampled depths, i denotes the depth layers between the surface and 100 m depth or the entire water column in the case of a shallow station, [PON] is the concentration in mM and d15N-PON is the isotope ratio in %. The same was done for the d13C-POC. Vertically integrated concentration weighted d15N from nitratesub was also calculated according to Eq. (1) for the depths range between the mixed layer and the maximum depth of 150 m. This depth interval includes the starting horizon of the upwelling velocity at approximately 125 m depth (Vo, 1997). The mixed layer depths were estimated from the maximum in the buoyancy frequency of the water column. Vertically integrated chlorophyll a and POC concentrations were calculated from surface to 100 m depth after the trapezoidal rule.
3. Results 3.1. Sea surface temperature (SST) and sea surface salinity (SSS) During SWM 2003 a weak gradient in SST off the coast of Vietnam was observed (Fig. 2). This reflected the weak upwelling conditions for the post-ENSO year 2003 as described in detail by Dippner et al. (2006). Compared to 2003 SST was 2 1C cooler during SWM 2004 and reflected a stronger upwelling intensity. During SpIM no SST gradient was found; therefore no coastal upwelling occurred. Similar to temperature, SSS showed strong coastal gradients during both SWM periods (Fig. 2). No gradient was found during SpIM. During both SWM periods low salinity waters of less than 33.2 entered the study area from the south. These waters have been described as an extension of the Mekong River plume that turns northwards during SWM by Dippner et al. (2006). During SpIM no low salinity waters were found and SSS was evenly high between 33.9 and 34.0. 3.2. Nitrate concentrations Coastal upwelling of nitrate was indicated during the SWM, when nitrate concentrations increased from zero at the surface to 4 and 8 mmol l1 at 40 m depth (Fig. 3). The nutricline was situated at approximately 20 m within the upwelling area and at 40–60 m offshore during SWM. Upwelling nitrate reached surface waters only at station 22 during SWM 2003 (Fig. 3a) and at station 52 during SWM 2004 (Fig. 3c). During SpIM the nutricline generally was found at 60 m depth, whereas no horizontal gradient was apparent. Elevated values of up to 5 mmol l1 at 40 m depth indicated an eddy formation at station 54 (Fig. 3h). Furthermore enhanced surface concentrations of up to 0.9 mmol l1 were found at the southernmost coastal stations during SWM 2004 (Fig. 3c) and of up to 1.6 mmol l1 at 20 m depth during SpIM 2004 (Fig. 3e), possibly due to a riverine influence. However, transport of riverine nitrate into the investigation area was restricted to the southern, shallow sites, whereas
Fig. 4. d15N values of NO 3 (open symbols) and PON (closed symbols) from inshore sites, shown on the upper panel, and offshore sites, shown on the lower panel including one northern and one southern station during southwest monsoon (SWM) and spring inter-monsoon (SpIM) seasons.
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the rest of the upwelling area remained unaffected by riverine nitrate. Nitrate concentrations in the water column were similar to values previously reported by Pham et al. (2002). 3.3. Stable isotope data d15N values in nitrate were most variable in the upper 150 m (Fig. 4). During SWM 2003 values ranged from 2.6% to 4.9%, during SpIM from 0.8% to 5.3%, and during SWM 2004 from 2.2% to 7.2%. Below 150 m to a maximum deth of 800 m d15N-nitrate was similar between seasons with an average value of 4.070.6% (n ¼ 15). Vertical profiles of d15N in nitrate were rather similar in and outside the upwelling area during SWM seasons (Fig. 4). Only during SpIM were lower values found at coastal stations compared to stations 442 km offshore. The concentration weighted average values of nitratesub were 3.570.3 (n ¼ 11 stations, SWM 2003), 2.970.9 (n ¼ 19 stations, SpIM 2004), and 3.7%70.6 (n ¼ 15 stations, SWM 2004, Fig. 5). During SWM 2003 and SpIM 2004 surface d15N in particulate organic matter (POM) varied around 2% whereas during SWM 2004 values were ca. 2% enriched (Fig. 4). d15N-POM increased with depth by 2–4%, a distribution generally attributed to isotopic fractionation in the course of remineralization of POM (Montoya et al., 1992). The vertical profiles were similar between stations in and outside the upwelling area during all sampled seasons (Fig. 4). The integrated values d15 NPON0max : 100 m for the upper water column to a maximum of 100 m had average values of 2.9% (70.5, n ¼ 9, SWM 2003), 2.8% (71.0, n ¼ 24, SpIM 2004), and 4.2% (70.8, n ¼ 40, SWM 2004). No clear in-offshore trend was found in d15N-PON (Fig. 5). Average d13 CPOC0max : 100 m values were 24.1% (70.3, n ¼ 9, SWM 2003), 23.5% (70.3, n ¼ 24, SpIM 2004), and 23.1% (70.8, n ¼ 40, SWM 2004, Fig. 6). POM was in 81% of all cases enriched over the nitrate isotopic signature by 2.171.2% (n ¼ 69) and only in 16 samples depleted by 1.771.5% during SWM seasons. During SpIM season 65% of all POM samples were enriched over the nitrate isotopic signature by 2.271.4% (n ¼ 30) and in 16 cases depleted by 2.471.9%. The d15N values in the six net-plankton size fractions generally increased from the smallest (55–166 mm) to the largest (42500 mm) size fraction
(Fig. 5). Lowest d15N values of 1.4% and 0.6% were found during SWM 2003 and 2004, respectively. Highest d15N values were also found during SWM seasons (8.0% and 7.8% in 2003 and 2004, respectively). During SpIM 2004 values ranged from 3.0% to 6.8% and no spatial differences were found between in- and offshore stations (440 km offshore, Fig. 5). During monsoon season d15N values in all net-plankton size fractions were on average 1.6% (SWM 2003) and 2.3% (SWM 2004) depleted outside compared to inside the upwelling area. Fig. 6 shows the d13C values of the smallest netplankton size fraction 55–166 mm, the combined values for the other 5 net-plankton size fractions (166 to 42500 mm) and for the POM-filters (40.7 mm). The 6 net-plankton size fractions were statistically not different from each other and had mean values of 20.9% (70.5, n ¼ 69, SWM 2003), 21.3% (70.6, n ¼ 137, SpIM 2004), and 21.2% (70.8, n ¼ 104, SWM 2004, Fig. 6). In contrast, d13C values in POM were significantly lower than in the net-plankton size fractions. 4. Discussion 4.1. Differences between POM and net-plankton C cycling in marine planktonic food webs is generally separated in two different pathways, the microbial and the herbivore pathway (Legendre and Le Fe`vre, 1991; Pesant et al., 2000). We found two different phytoplankton communities in our study area. The first is represented by the POM-filter samples. Particulate organic C concentrations correlate highly significantly with chlorophyll a concentrations in the upper 100 m of the water column (Fig. 7). This indicates that POM was dominated by phytoplankton rather than by detritus. The second phytoplankton community is represented by the size fraction 55–166 mm. The dominance of phytoplankton in this size fraction is indicated by an average C:N ratio of 7.4 (70.8, n ¼ 44). Furthermore, microscopic inspection on board identified diatoms like Rhizosolenia spp., Chaetoceros spp., Coscinodiscus spp., Thalassiosira spp., Thalasionema spp., Hemiaulus spp., Pseudonizschia spp. or Stephanopyxis spp., cyanobacteria like Trichodesmium spp., and dinoflagellates like Protoperidinium spp., Ceratium spp., Amphisolenia spp., or Pyrocystis spp. in this size fraction. The size fractions 4166 mm were characterized by increasing d15N values with size (Fig. 5). According to Fry and Quinones (1994) this
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Fig. 5. d N patterns of particulate organic matter (POM 4 0.7 mm) and six net-plankton size fractions (55–42500 mm) during (a) southwest monsoon 2003, (b) spring intermonsoon 2004, and (c) southwest monsoon 2004. The continuous lines separate inshore (o42 km off the coast) and offshore (442 km off the coast) stations. Dotted lines give the integrated concentration weighted d15N value of subthermocline nitrate for each season. Note: Inshore during SWM seasons means within the upwelling area.
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Fig. 6. Average d13C values of POM, net-plankton size fraction 55–166 mm, and net-plankton size fraction 166 to 42500 mm. Numbers of samples are given above the x-axis for each respective season and plankton size fraction for size fraction 166 to 42500 mm (top), size fraction 55–166 mm (middle), and POM (bottom). Dotted area indicates the 71% interval for potential food source of plankton in size fractions 166 to 42500 mm according to Peterson and Fry (1987).
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reflects an increase in trophic levels with size due to different zooplankton groups. In order to identify the potential food source of the zooplankton the stable C isotope data of POM and size fraction 55–166 mm were compared to those of the zooplankton size fractions 4166 mm. According to Peterson and Fry (1987) food for zooplankton must have d13C values that are within a71% interval of the zooplankton value. It was found that the stable C isotope values (d13C) in POM (40.7 mm) were 2–3% lower than the d13C values in the zooplankton size fractions 4166 mm (Fig. 6). In contrast the phytoplankton size fraction 55–166 mm differed less than 1% from the zooplankton size fractions 4166 mm (Fig. 6). We therefore conclude that phytoplankton in size fraction 55–166 mm is the potential food source for zooplankton 4166 mm, whereas POM may be excluded as food source for zooplankton 4166 mm. The d13C values in our POM samples are similar to the d13C values of pico- and nanoplankton covering a size range of o3–20 mm from a study in the Mediterranean Sea by Rau et al. (1990). The d13C values from
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our net-plankton are similar to the micro-and mesoplankton values from Rau et al. (1990) that covered a size range of 20 to 4150 mm. This indicates that the filter plankton may belong to the microbial, whereas the net-plankton may belong to the herbivore food web. This interpretation is consistent with observed co-existences of pico-and nanoplankton populations and larger-sized phytoplankton supporting micro- and mesozooplankton populations in the oceans as reviewed by Kiørboe (1993). In contrast to the probably limited C transfer between POM and larger plankton we found indications for intensive transfer of N from the POM size fraction into the larger plankton size fractions at sites of high N fixation. Voss et al. (2006) measured high N fixation rates in the POM size fraction exclusively at the offshore sites during SWM. Our results show that at these sites the d15N values of the net-plankton size fractions were lowered by ca. 2% compared to the sites of low N fixation (Fig. 5). We interpret this as transfer of N from N fixation in the microbial loop into the herbivore food web. N from N fixation by cyanobacteria has a theoretical d15N value of 1% (Montoya et al., 2002). It may be an additional new N source in oligotrophic regions, and its incorporation can be traced by a lowering in the d15N values of the pelagic food web as shown, e.g., by Fry and Quinones (1994). However, it is not clear in which inorganic or organic form this N is transferred. We found d15N values in POM below the d15N values of subthermocline nitrate despite low N2 fixation rates, especially in the upwelling area during SWM 2003 (Fig. 5a). Besides N2 fixation only the uptake of ammonium may cause d15N values of surface particles below the d15N values of subthermocline nitrate (Sigman and Casciotti, 2001). Regenerated ammonium as N source therefore may be an additional N source especially for nano-and picoplankton as found by Wafar et al. (2004). In contrast larger phytoplankton cells are considered to depend on alternative N sources, especially on nitrate when it is available in high concentrations, because of a strong diffusion limitation for ammonium (Stolte et al., 1994). 4.2. d15N patterns in net-plankton in relation to d15N in nitrate The d15N values in the net-plankton size fractions (55Z2500 mm) were higher than the average d15N values in subthermocline nitrate (nitratesub) through-
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out the study area during SpIM and within the upwelling area during SWM (Fig. 5). In contrast the lower trophic levels and the assumed zooplankton food source (size fraction 55–166 mm) had d15N values below the nitratesub values at offshore stations during SWM. These differences can be explained by the spatially different utilization of new N sources. Whereas N from N fixation was an additional new N source for the net-plankton community outside the upwelling area during SWM, nitrate from below the nutricline was the only new N source in the upwelling area and during SpIM. This is consistent with the N fixation rates and new production rates measured by Voss et al. (2006) and the identification of nitratesub from Maximum Salinity Water as the sole nitrate source for primary production by Dippner et al. (2006). Utilization of nitratesub leads to d15N values of the assimilating organism, e.g., diatoms, that may be up to 12% enriched compared with the source nitrate (Needoba et al., 2003; Altabet, 1996). Important factors affecting isotope fractionation in phytoplankton are species composition, swimming abilities, light regime and the assimilatory activity of nitrate reductase (Granger et al., 2004; Needoba and Harrison, 2004). d15N values in the net-plankton size fractions higher than the nitratesub values therefore possibly reflect the fractionation effect during nitrate uptake. In contrast, the concomitant finding of high N2 fixation rates strongly supports the theory that the incorporation of N from N fixation rather than ammonium caused the observed lowering in the d15N values in the net plankton outside the upwelling area during SWM. A strong influence of N2 fixation on the N cycle throughout the area is furthermore indicated by d15N values of nitratesub between 2.9% and 3.7% since only N2 fixation can lead to d15N values in nitratesub below the global range of 4.5–6% (Liu and Kaplan, 1989). We generally observed an increase in d15N with plankton size (Fig. 5). Enrichments in d15N with increasing plankton size are attributed to trophic fractionation effects (Rolff, 2000; Fry and Quinones, 1994). However, the observed increases in d15N between the consecutive size fractions of on average 0.2% (70.6, n ¼ 263) were much smaller than the reported 2–3.5% increase between consumer and diet (Peterson and Fry, 1987). One reason may be the insufficient resolution of trophic levels and the overlap within one size fraction. Furthermore d15N in bulk plankton may be altered by a variety of factors such as species composition (Montoya et al., 1992), food quality or
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quantity (Adams and Sterner, 2000), N turnover (Montoya et al., 1991), or amino acid composition (McClelland and Montoya, 2002). For example, the salp Salpa aspera can feed effectively on a broad range of particle sizes since it uses a mucus net to filter suspended particles out of the sea water (Montoya et al., 1992). The copepod Pareuchaeta norvegica selectively feeds on copepods and other small zooplankton and has higher d15N values than the salp. The d15N in different zooplankton species therefore varies depending on their feeding habits (Montoya et al., 1992). Nutritional stress can cause increases in d15N of up to 0.4% in Daphnia magna, which are significantly correlated with the C:N ratio of the food (Adams and Sterner, 2000). As the N content of the food source decreases from a C:N of 6–25, the corresponding d15N values of the daphnids feeding on the algae increased from 1% to 4% (Adams and Sterner, 2000). However, we found no correlation between C:N ratios and d15N of the netplankton samples. The natural abundance of 15N in various components of an ecosystem can change markedly on a time scale of days (Montoya et al., 1991). Differences in N turnover will lead to different kinetics in the isotope responses of plankton species to changes in d15N of the food source (Montoya et al., 2002). For example, the turnover time of Acartia tonsa was estimated to be 7.670.6 days, whereas the ctenophore Mnemiopsis leidyi was assumed to have much slower turnover times, as indicated by less variable d15N values (Montoya et al., 1991). Furthermore d15N in bulk plankton may alter by variations in d15N of amino acids attributed to internal processing or variations in the acquired food source (McClelland and Montoya, 2002). However, although amino acids like glutamic acid, alanine, aspartic acid, isoleucine, leucine, proline, and valine are up to 7% enriched in the consumer compared to the diet, they always seem to reflect the trophic increase in bulk d15N (McClelland and Montoya, 2002). Despite these factors only trophic fractionation can cause the general increase in d15N with size and is confirmed by other studies (e.g. Montoya et al., 2002; Fry and Quinones, 1994).
zooplankton food source is a mixture of the d15N values of the 2 new N sources nitrate and N from N fixation. For this estimation the d15N value of the zooplankton food source, the d15N of the source nitrate and the d15N of N from N fixation have to be known. We assume that size fraction 55–166 mm is the zooplankton food source. The mean d15N value of this size fraction at sites of high N fixation was 2.9% during SWM 2003 and 3.1% during SWM 2004. Furthermore, we use the theoretical d15N value of 1% as value for regenerated N from N fixation from Montoya et al. (2002). Nitratesub was found to be the only nitrate source and had d15N values of 3.5% and 3.7% during SWM 2003 and 2004, respectively. Based on these values N fixation contributed 9% and 13% of N of the zooplankton food source during SWM 2003 and 2004, respectively. The d15N patterns of the larger size fractions prove that this N is also transferred to higher trophic levels. This contribution is much less than in the oligotrophic North Atlantic, where N2 fixation was estimated to contribute on average 51% to the organic N in particles in the upper 100 m (Montoya et al., 2002). Our estimation from the natural abundance stable N isotope measurements closely match the rate estimations by Voss et al. (2006) that N2 fixation may contribute approximately 10% of the new production. This shows that N2 that is fixed by pico- and nanoplankton is transferred in large part into the herbivore food web. Acknowledgments The German-Vietnamese cooperation has been financed by the German Research Foundation (DFG), the Federal Ministry for Economic Cooperation and Development (BMZ) and the Vietnamese Ministry of Science and Technology (MOST), which is greatly acknowledged. We thank Lam Ngoc Nguyen, Bui Hong Long, and Rolf Peinert for their organisation before and during the cruises. The authors are indebted to Barbara Deutsch and Joseph Montoya for helpful comments and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG/Bo-768).
4.3. Contribution of N fixation The proportion of N from N fixation for higher trophic levels can be estimated according to the 2-source mixing model of Montoya et al. (2002). For this model it is assumed that the d15N value of the
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