# Effect of increased pCO(2) on the planktonic metabolic balance during a mesocosm experiment in an Arctic fjord

T. Tanaka, S. Alliouane, R. G. B. Bellerby, J. Czerny, A. De Kluijver, U.

Riebesell, K. G. Schulz, A. Silyakova, J. -P. Gattuso

► To cite this version:

T. Tanaka, S. Alliouane, R. G. B. Bellerby, J. Czerny, A. De Kluijver, et al.. Effect of increased pCO(2) on the planktonic metabolic balance during a mesocosm experiment in an Arctic fjord. Biogeosciences, 2013, 10 (1), pp.315-325. 10.5194/bg-10-315-2013 . hal-03502650

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# Effect of increased pCO₂ on the planktonic metabolic balance during a mesocosm experiment in an Arctic fjord

T. Tanaka<fnref n="1" />,<fnref n="2" />, S. Alliouane<fnref n="1" />,<fnref n="2" />, R. G. B. Bellerby<fnref n="3" />,<fnref n="4" />,<fnref n="5" />, J. Czerny<fnref n="6" />, A. de Kluijver<fnref n="7" />, U. Riebesell<fnref n="6" />, K. G. Schulz<fnref n="6" />, A. Silyakova<fnref n="3" />,<fnref n="4" />, and J.-P. Gattuso<fnref n="1" />,<fnref n="2" />

*Correspondence to:* T. Tanaka (tsuneo.tanaka@obs-vlfr.fr)

Received: 24 July 2012 – Published in Biogeosciences Discuss.: 17 August 2012

Revised: 20 November 2012 – Accepted: 10 December 2012 – Published: 23 January 2013

**Abstract.** The effect of ocean acidification on the balance between gross community production (GCP) and community respiration (CR) (i.e., net community production, NCP) of plankton communities was investigated in summer 2010 in Kongsfjorden, west of Svalbard. Surface water, which was characterized by low concentrations of dissolved inorganic nutrients and chlorophyll *a* (a proxy of phytoplankton biomass), was enclosed in nine mesocosms and subjected to eight *pCO₂* levels (two replicated controls and seven enhanced *pCO₂* treatments) for one month. Nutrients were added to all mesocosms on day 13 of the experiment, and thereafter increase of chlorophyll *a* was provoked in all mesocosms. No clear trend in response to increasing *pCO₂* was found in the daily values of NCP, CR, and GCP. For further analysis, these parameters were cumulated for the following three periods: phase 1 – end of CO₂ manipulation until nutrient addition (*t*4 to *t*13); phase 2 – nutrient addition until the second chlorophyll *a* minimum (*t*14 to *t*21); phase 3 – the second chlorophyll *a* minimum until the end of this study (*t*22 to *t*28). A significant response was detected as a decrease of NCP with increasing *pCO₂* during phase 3. CR was relatively stable throughout the experiment in all mesocosms. As a result, the cumulative GCP significantly decreased with increasing *pCO₂* during phase 3. After the nutrient addition, the ratios of cumulative NCP to cumulative

consumption of NO₃ and PO₄ showed a significant decrease during phase 3 with increasing *pCO₂*. The results suggest that elevated *pCO₂* influenced cumulative NCP and stoichio-metric C and nutrient coupling of the plankton community in a high-latitude fjord only for a limited period. However provided that there were some differences or weak correlations between NCP data based on different methods in the same experiment, this conclusion should be taken with caution.

## 1 Introduction

The balance between photosynthetic carbon production and consumption of organic carbon in the ocean's surface layer is of importance in understanding the ocean's role in the global carbon cycle. Marine phytoplankton play an important role in the carbon cycle, being responsible for about half of the global primary production (Field et al., 1998). A large portion of organic carbon produced by photosynthesis is mineralized by respiration (del Giorgio and Duarte, 2002). Heterotrophic prokaryotes (hereafter "bacteria") can consume a significant fraction of primary production in pelagic systems (Cole et al., 1988; Ducklow and Carlson, 1992). Mineral nutrients (e.g., N, P) can be a limiting factor of growth or organic carbon production by phytoplankton and bacteria.

<footnote n="1">¹INSU-CNRS, Laboratoire d'Océanographie de Villefranche, UMR7093 BP 28, 06234 Villefranche sur Mer cedex, France</footnote>

<footnote n="2">²Université Pierre et Marie Curie-Paris 6, Observatoire Océanologie de Villefranche, 06230 Villefranche sur Mer cedex, France</footnote>

<footnote n="3">³Uni Bjerknes Centre, Allégaten 55, 5007 Bergen, Norway</footnote>

<footnote n="4">⁴Bjerknes Center for Climate Research, Allégaten 55, 5007 Bergen, Norway</footnote>

<footnote n="5">⁵Geophysical Institute, University of Bergen, Allégaten 70, 5007 Bergen, Norway</footnote>

<footnote n="6">⁶Helmholtz Centre for Ocean Research Kiel (GEOMAR), Düsternbrooker Weg 20, 24105 Kiel, Germany</footnote>

<footnote n="7">⁷Department of Ecosystems Studies, Royal Netherlands Institute of Sea Research (NIOZ), The Netherlands</footnote>
Because of the stoichiometric constraint on an organism's elemental composition, changes in the stoichiometric coupling between organic carbon and mineral nutrients in the lower part of the pelagic food web in response to environmental change may have consequences for the carbon cycle and/or the nutrient cycle in the ocean (Thingstad et al., 2008).

The increasing concentration of carbon dioxide (CO₂) in the atmosphere leads to an increase of the partial pressure of CO₂ (pCO₂) in seawater, and related changes in the chemistry of the carbonate system, such as a reduced pH (Orr, 2011). These changes could lead to changes in carbon production and consumption (Riebesell and Tortell, 2011) and, therefore, to changes in oxygen production and consumption. Net community production (NCP) is defined as the balance between gross community production (GCP) and community respiration (CR). NCP thus describes the net metabolism of the ecosystem. A positive NCP indicates that more organic carbon is produced than respired, so-called net autotrophy, while negative NCP indicates respiration exceeds primary production, net heterotrophy.

The effects of increasing pCO₂ on production and respiration of pelagic plankton have been studied on single-species in laboratory cultures up to semi-natural communities in field mesocosms (reviewed by Riebesell and Tortell, 2011). Primary production measured by ¹⁴C fixation or production of particulate organic carbon (POC) at elevated pCO₂ is enhanced (Hein and Sand-Jensen, 1997; Riebesell et al., 2000; Zondervan et al., 2001; Schippers et al., 2004; Leonardos and Geider, 2005; Egge et al., 2009; Borchard et al., 2011), decreased (Sciandra et al., 2003), or shows no significant difference compared to the control (Tortell et al., 2002; Delille et al., 2005). It thus has been shown that increasing pCO₂ mostly enhances primary production. Measurements of primary production based on ¹⁴C fixation or POC production are relatively numerous, but few studies have examined the metabolic balance (i.e., NCP, CR, and GCP) of planktonic communities based on changes of dissolved oxygen (DO) concentration at different pCO₂ levels. The oxygen-based NCP measurement has shown a significant decrease in NCP of *Emiliania huxleyi* at elevated pCO₂ in a N-limited chemo-stat culture (Sciandra et al., 2003), and insignificant changes in NCP of semi-natural plankton community at different pCO₂ levels in mesocosm experiments (Delille et al., 2005; Egge et al., 2009).

The objective of the present study was to investigate the effect of ocean acidification on the balance between GCP and CR (i.e., NCP) of a plankton community in a northern high-latitude fjord. The Arctic Ocean is particularly affected by ocean acidification: undersaturation for aragonite already occurs seasonally and will spread the whole surface of the Arctic Ocean in 2100 (Steinacher et al., 2009). In summer 2010, a multidisciplinary experiment was conducted for about one month using free-floating mesocosms deployed at Ny-Ålesund, Spitsbergen, as part of the EPOCA (European Project on Ocean Acidification) project. Seven

enhanced pCO₂ treatments plus two replicated controls (no CO₂ enrichment) were established for post-bloom plankton community at the start of the experiment. Nutrients were added to all mesocosms in the middle of the experiment in order to stimulate phytoplankton growth. A series of chemical, biogeochemical, biological, and physiological parameters were measured during this experiment. We have analyzed NCP, CR, and GCP based on changing concentrations of dissolved oxygen in incubation bottles together with other related chemical and biological parameters. We also compare NCP estimated by different methods based on dissolved oxygen (this study), stable carbon isotope (de Kluijver et al., 2012), and total carbon (Silyakova et al., 2012).

## 2 Materials and methods

### 2.1 Experimental setup and sampling

The mesocosm experiment was conducted in Kongsfjorden, northern Spitsbergen (78°56.2′N, 11°53.6′E), in June and July 2010 as part of the EPOCA Svalbard experiment (see Czerny et al., 2012; Riebesell et al., 2012; Schulz et al., 2012 for details). In Kongsfjorden, the spring phytoplankton bloom occurs in April, which results in low concentration of dissolved inorganic nutrients (Rokkan Iversen and Seuthe, 2011; Hodal et al., 2012). Nine Kiel off-shore mesocosms (KOSMOS: thermoplastic polyurethane 0.5 to 1 mm thick, 17 m long, and 2 m in diameter, approximately 50 m³ volume) were deployed on 31 May 2010 (t-7). The site of the mesocosm mooring was ice-free during the experiment except for a few occasions when ice floats needed to be pushed out from the site (Riebesell et al., 2012). All mesocosms were filled with nutrient-poor, post-bloom, and sieved (3 mm mesh) fjord water. The CO₂ manipulation was carried out between 6 and 11 June (t-1 to t4), establishing seven enhanced pCO₂ treatments and two replicated controls (no pCO₂ treatment). pH and approximate pCO₂ levels in all mesocosms on t8/9 and t26/27 are shown in Table 1 (see also Bellerby et al., 2012). While pH and pCO₂ changed in all mesocosms because of air/sea gas exchange and biological carbon uptake to different degrees, the gradients of pH and pCO₂ between the treatments remained until the end of the experiment (Bellerby et al., 2012; Schulz et al., 2012; Silyakova et al., 2012). To induce the development of a phytoplankton bloom, 5 µM of nitrate (NO₃), 0.31 µM of phosphate (PO₄), and 2.5 µM of silicate (Si) were added early in the morning on 20 June (t13) (i.e., before the routine sampling) to all mesocosms. The nutrient concentrations were chosen to simulate an upwelling event (Schulz et al., 2012). The additions of CO₂ (t-1 to t4) and inorganic nutrients (t13) were performed in the upper 13 m of the mesocosms using the dispersal device in order to assure an even distribution in the water column (Riebesell et al., 2012; Schulz et al., 2012).
<table><caption>**Table 1.** Average pH<sub>T</sub> and approximate pCO₂ levels (µatm) on t8/9 and t26/27 during the experiment (Bellerby et al., 2012).</caption><thead><tr><th rowspan="2">Mesocosm</th><th colspan="2">t8/9</th><th colspan="2">t26/27</th></tr><tr><th>pH<sub>T</sub></th><th>pCO<sub>2</sub></th><th>pH<sub>T</sub></th><th>pCO<sub>2</sub></th></tr></thead><tbody><tr><td>3*</td><td>8.32</td><td>185</td><td>8.36</td><td>165</td></tr><tr><td>7*</td><td>8.31</td><td>185</td><td>8.37</td><td>160</td></tr><tr><td>2</td><td>8.18</td><td>270</td><td>8.25</td><td>220</td></tr><tr><td>4</td><td>8.05</td><td>375</td><td>8.15</td><td>290</td></tr><tr><td>8</td><td>7.96</td><td>480</td><td>8.07</td><td>365</td></tr><tr><td>1</td><td>7.81</td><td>685</td><td>7.94</td><td>500</td></tr><tr><td>6</td><td>7.74</td><td>820</td><td>7.90</td><td>555</td></tr><tr><td>5</td><td>7.64</td><td>1050</td><td>7.80</td><td>715</td></tr><tr><td>9</td><td>7.51</td><td>1420</td><td>7.73</td><td>855</td></tr></tbody></table>

Depth-integrated water samples (0–12 m) were collected in each mesocosm using Hydro-Bios integrated water samplers (5 L volume). Samples for nutrients and chlorophyll *a* (Chl *a*) were collected in the morning (09:00–11:00), whereas those for measurement of community metabolism were collected in the afternoon (13:00–15:00). Such a separate sampling program was used because of logistical constraints. The experiment ended on 7 July (*t*30). Sampling for NCP and CR determination ended on 5 July (*t*28).

## 2.2 Net community production, community respiration, and gross community production

Water samples from each mesocosm were distributed into 12 biological oxygen demand (BOD) bottles (60 mL) by overflowing by 4–5 times the bottle volume, as soon as the water samples were brought back to shore. Four bottles were immediately fixed with Winkler reagents to determine the initial concentration of dissolved oxygen (DO), and served as a control. Then, two quadruple sets of bottles served for determination of NCP and CR. Prior to filling, the BOD bottles had been washed with HCl (5 %) and rinsed thoroughly with Milli-Q water.

Meroplankton larvae (*Cirripedia nauplii*) were abundant ($1 \times 10^4 - 2 \times 10^4$ individuals m$^{-3}$) in the mesocosms between *t* − 2 and *t*11 (Niehoff et al., 2012), and several individuals of this species and mesozooplankton were sometimes distributed in quadruple sets of BOD bottles (Tanaka et al., unpublished). The heterogeneous inclusion of large organisms can to an extent contribute to the variation in concentration of DO between replicated samples during the first half of the experiment. However since we wanted to minimize the perturbation, water samples collected from the mesocosms were poured into BOD bottles without any pre-treatment.

NCP and CR were measured every 2 and every 4 days, respectively, between *t* − 1 and *t*7 and between *t*12 and *t*28. That is, the incubations of both NCP and CR bottles and

that of NCP bottles alone were done alternatively during the experiment. BOD bottles for NCP measurement were incubated for 24 h at a mooring site, which was located about 300 m from the mesocosms. The BOD bottles were incubated at 4 m depth, at which the irradiance corresponded to the average irradiance of the water column sampled in the mesocosms (0 to 12 m). As the summer season progressed, the mooring site sometimes became influenced by the plume of a nearby stream. Therefore, the mooring was moved closer (about 100 m) to the mesocosms on 13 June (*t*6). For the CR measurement, dark BOD bottles were incubated at the in situ mooring site until 18 June (*t*11), and clear BOD bottles were incubated in a dark laboratory incubator from 19 June (*t*12) onwards due to logistical constraints. The temperature in the laboratory incubator was adjusted to the mean water temperature in the top 12 m on the day of sampling (2 to 4 °C). Preliminary measurements of CR with Kongsfjorden samples did not detect a statistically significant decrease in DO during the first 24 h of incubation in the dark. The CR samples were therefore incubated for 48 h. Upon completion of the incubation, the bottles were fixed with Winkler reagents as described by Knap et al. (1996).

DO concentrations were determined with an automated Winkler titration method using a potentiometric end-point detection (Titrando888). Reagents and standardizations were similar to those described by Knap (1996). Rates of NCP and CR were determined by linear regression of DO against time (slope ± standard error: µmol O₂ L⁻¹ d⁻¹). GCP was calculated as the difference between NCP and CR. The combined uncertainty of GCP (SE<sub>GCP</sub>) was calculated using the standard error of NCP (SE<sub>NCP</sub>) and CR (SE<sub>CR</sub>) according to

$$SE_{GCP} = \sqrt{SE_{NCP}^2 + SE_{CR}^2}. \qquad (1)$$

The cumulative values of NCP, CR, and GCP were calculated for different periods, which were defined based on the timing of manipulations and the temporal changes of phytoplankton biomass (see Riebesell et al., 2012): (1) phase 1 – end of CO₂ manipulation until nutrient addition (*t*4 to *t*13); (2) phase 2 – nutrient addition until the second Chl *a* minimum (*t*14 to *t*21); (3) phase 3 – the second Chl *a* minimum until the end of this study (*t*22 to *t*28); (4) phase 2 + 3 (*t*14 to *t*28); and (5) whole period (*t*4 to *t*28). Because NCP and CR were measured every 2 and 4 days, respectively during most of the experimental period, the data on the days when the measurement was not done were estimated by the linear interpolation. The cumulative values were then summed up for the corresponding period.

## 2.3 Statistical analysis

Linear regression was used to analyze the significance of responses of NCP, CR, GCP, and ratios of cumulative NCP to cumulative consumption of NO₃ and PO₄ to increased pCO₂ levels. When pCO₂-dependent NCP, CR, and GCP were analyzed on a certain day, the pCO₂ values measured on that

<footnote>* Mesocosms 3 and 7 received no pCO₂ manipulation (i.e., control).</footnote>
<table><caption>**Table 2.** Initial condition in the nine mesocosms. Data on *t*0 are shown as a reference (Schulz et al., 2012). DIN (dissolved inorganic nitrogen) is the sum of NO₃, NO₂, and NH₄.</caption><thead><tr><th>Parameter</th><th>Temperature (°C)<sup>1</sup></th><th>NO<sub>3</sub> (µmol·N L<sup>-1</sup>)</th><th>NO<sub>2</sub> (µmol·N L<sup>-1</sup>)</th><th>NH<sub>4</sub> (µmol·N L<sup>-1</sup>)</th><th>PO<sub>4</sub> (µmol·P L<sup>-1</sup>)</th><th>Si (µmol·Si L<sup>-1</sup>)</th><th>DIN: PO<sub>4</sub></th><th>DIN: Si</th><th>Si: PO<sub>4</sub></th><th>Chl a (µg L<sup>-1</sup>)<sup>2</sup></th></tr></thead><tbody><tr><td>Mean ± SD<br>(n = 9)</td><td>2.9 ± 0.1</td><td>0.02 ± 0.01</td><td>0.00 ± 0.00</td><td>0.59 ± 0.05</td><td>0.05 ± 0.01</td><td>0.13 ± 0.02</td><td>11.3 ± 1.4</td><td>4.7 ± 0.9</td><td>2.5 ± 0.6</td><td>0.21 ± 0.02</td></tr></tbody></table>

day were used. For the regression analysis of the cumulative parameters, the mean pCO₂ during the corresponding period was used. All statistical analyses were performed with R (R Development Core Team, 2008).

## 3 Results

The Arctic coastal water used to fill the mesocosms had low concentrations of dissolved inorganic nutrients and Chl *a*. The concentrations of NO₃ and PO₄ but not NH₄ were close to the detection limit of the conventional nutrient analysis, and the mean Chl *a* concentration determined by the high-performance liquid chromatography method was 0.21 µg L⁻<fnref n="1" /> on *t*0 (Table 2; Schulz et al., 2012). The mean ratios of dissolved inorganic nitrogen (DIN: sum of NH₄, NO₃, and NO₂) to PO₄, DIN to Si, and Si to PO₄ were 11.3, 4.7, and 2.5, respectively, in all mesocosms (Table 2; Schulz et al., 2012). The water temperature was homogeneous in the water column of the mesocosms at the beginning, and increased gradually, especially in the upper 5 to 10 m, until the end of the experiment (~2.7 to ~5.5 °C), while it was always similar in all mesocosms (coefficient of variation: 0 to 6%; see Schulz et al., 2012).

The concentrations of dissolved inorganic nutrients remained low in all mesocosms until the nutrient addition performed on *t*13 (Schulz et al., 2012). After the addition of nutrients, the net consumption rate of NO₃ and PO₄ was statistically higher in higher pCO₂ mesocosms from *t*17 to *t*22, while the cumulative nutrient consumption became similar in all mesocosms toward the end of the experiment (Schulz et al., 2012). The concentration of Chl *a* ranged from 0.1 to 2.6 µg L⁻<fnref n="1" /> (*t*0 to *t*30) and increased during the experiment (Fig. 1, see also Schulz et al., 2012). Peaks of Chl *a* were observed three times in all mesocosms: once before the nutrient addition (*t*8, range: 0.5–0.9 µg L⁻<fnref n="1" />) and twice after the nutrient addition (*t*19 and *t*28, range: 0.3–1.0 µg L⁻<fnref n="1" /> and 0.6–1.8 µg L⁻<fnref n="1" />, respectively). The first Chl *a* peak during phase 1 was largely dominated by haptophytes, while, after the nutrient enrichment, the second was due to prasinophytes, dinoflagellates, and cryptophytes, and the third was due to haptophytes, prasinophytes, dinoflagellates,

and chlorophytes (Schulz et al., 2012). Top-down control on nanophytoplankton by microzooplankton grazing and viral lysis was important especially during phase 1 (Brussaard et al., 2012). The Chl *a* concentration at elevated pCO₂ was statistically higher during phase 2, but lower during phase 3 (Schulz et al., 2012).

GCP ranged from −0.5 ± 1.0 to 11.2 ± 2.3 µmol O₂ L⁻<fnref n="1" /> d⁻<fnref n="1" /> between *t* − 1 and *t*28 (Fig. 1). Note that negative values were not statistically different from 0 (F-test, *P* > 0.05). The lowest and the highest GCPs were mostly observed, respectively, before the nutrient addition and towards the end of the experiment (*t*24 or *t*28). There were a few exceptions: the smallest GCP was observed after the nutrient addition (*t*16) in M6, and the highest value was observed before the nutrient addition (*t*12) in M9. After the nutrient addition, while GCP showed two peaks in M1 and a reduction in M9, it increased towards the end of the experiment in the other mesocosms. Linear regression analysis detected a significant decrease of GCP as a function of increasing pCO₂ on *t*24 (−0.008 ± 0.002 µmol O₂ L⁻<fnref n="1" /> d⁻<fnref n="1" /> µatm⁻<fnref n="1" />, *P* < 0.05).

The extent of temporal variation of CR (−5.6 to 0.25 µmol O₂ L⁻<fnref n="1" /> d⁻<fnref n="1" />) was about half of that of GCP during the experiment (Fig. 1). The positive CR measured in M2 on *t*7 was not statistically different from zero (F-test, *P* = 0.89). The highest CR was observed before the nutrient addition in M1, M2, M3, M4, and M7, and after the nutrient addition in M5, M6, M8, and M9. Linear regression analysis detected no significant relationship between CR and pCO₂ levels on any day (F-test, *P* > 0.05).

NCP was mostly close to zero or positive during the experiment (Fig. 1). Statistically significant negative NCP values were detected seven times (M2 and M8 on *t* − 1, M7 on *t*7, M5 and M7 on *t*12, M6 on *t*16, and M6 on *t*24). Similar to GCP, the smallest and the highest NCP in each mesocosm were mostly observed before the nutrient addition and towards the end of the experiment (*t*26 or *t*28), respectively. There were a few exceptions: the lowest NCP was observed after the nutrient addition (*t*16) in M6, and the highest was observed before the nutrient addition (*t*12) in M9. NCP in all mesocosms but M9 tended to increase

<footnote n="1">¹ The data on temperature were based on the mean of 0–12 m in each mesocosm (Schulz et al., 2012).</footnote>

<footnote n="2">² The data on Chl *a* are based on the HPLC method (Schulz et al., 2012).</footnote>
<figure><img src="image_4.png" /></figure>

**Fig. 1.** Temporal changes of gross community production (GCP: open upside triangle), net community production (NCP: filled diamond), and community respiration (CR: open downside triangle) based on changes of dissolved oxygen during incubation. Values are mean ± standard error (µmol O₂ L⁻¹ d⁻¹). The color code is as follows: blue for low pCO₂ treatments (M3, M7, and M2), grey for intermediate pCO₂ treatments (M4, M8, and M1) and red for high pCO₂ treatments (M6, M5, and M9). Black solid lines are HPLC-based chlorophyll a concentration (µg L⁻¹: Schulz et al., 2012). Vertical dotted lines separate phases 1, 2, and 3 during the experiment (see also the text). pCO₂ values (µatm) denote the mean during the experiment (Bellerby et al., 2012).

after the nutrient addition towards the end of the experiment. The temporal variation of NCP in each mesocosm during the experiment tended to decrease with increasing pCO₂ level. Linear regression analysis detected a significant decrease of NCP with increase of pCO₂ level on t24 and t26 (slope ± se: -0.008 ± 0.003 and -0.008 ± 0.001 µmol O₂ L⁻¹ d⁻¹ µatm⁻¹, respectively on t24 and t26, F-test, *P* < 0.05 for both cases).

The cumulative NCP revealed that it was negative in only one mesocosm (M7) before the nutrient addition (phase 1) (Fig. 2). It should be noted that M3 and M7 were treated as the control in the same way with regard to the CO₂ perturbation (i.e., no CO₂ enrichment). During phase 3, only the cumulative NCP in M9 was negative. The proportion of the cumulative NCP during the whole period was highest during phase 3 in all mesocosms except M9. The cumulative CR in all mesocosms tended to be similar between different phases. The proportion of the cumulative GCP was highest during phase 3 in all mesocosms except M1, 6, and 9.

A linear regression describes the pCO₂-dependent decrease of cumulative NCP during phase 3 (slope ± se: -0.05 ± 0.01 µmol O₂ L⁻¹ µatm⁻¹, *n* = 9, F-test,

<figure><img src="image_5.png" /></figure>

**Fig. 2.** Relationship between cumulative GCP (open upside triangle), NCP (filled diamond), and CR (open downside triangle) (µmol O₂ L⁻¹) vs. the mean pCO₂ (µatm) during (a) phase 1 (end of CO₂ manipulation until nutrient addition: t4 to t13), (b) phase 2 (nutrient addition until the second chlorophyll minimum: t14 to t21), (c) phase 3 (the second chlorophyll minimum until the end of this study: t22 to t28), (d) phase 2 + 3 (after nutrient addition: t14 to t28), and (e) the whole period (t4 to t28). The lines indicate statistically significant relationship (F-test, *P* < 0.05).

$P = 0.001$) and during phase 2 + 3 (slope ± se: -0.04 ± 0.01 µmol O₂ L⁻¹ µatm⁻¹, *n* = 9, F-test, *P* = 0.005). The cumulative NCP slightly increased with increasing pCO₂ during phases 1 and 2, although the regression slope was not significant (*P* > 0.05). The relationship between cumulative NCP and pCO₂ during the whole period was statistically negative (F-test, *P* = 0.02) only when the data from M7 were not included in the analysis. The relationship between CR and pCO₂ was always insignificant (F-test, *P* > 0.05). The cumulative GCP significantly decreased with increasing pCO₂ during phase 3 (slope ± se: -0.05 ± 0.01 µmol O₂ L⁻¹ µatm⁻¹, *n* = 9, F-test, *P* < 0.001) and during phase 2 + 3 (slope ± se: -0.04 ± 0.01 µmol O₂ L⁻¹ µatm⁻¹, *n* = 9, F-test, *P* = 0.019).

The net consumption rate of NO₃ and PO₄ with increasing pCO₂, both significantly increased during phase 2 and decreased during phase 3 (F-test, *P* < 0.05; Schulz et al., 2012). During the period between t14 and t28 (phase 2 + 3), the cumulative consumption of NO₃ and PO₄ was respectively similar between nine mesocosms
(5.3–5.5 µmol N L⁻¹, 0.32–0.38 µmol P L⁻¹), and thus the ratios of cumulative consumption of NO₃ to PO₄ were similar in all mesocosms (range: 14–17, n = 9; Schulz et al., 2012), which is close to Redfield ratio of 16. Assuming a photosynthetic quotient of 1.25 (Williams, et al., 1979), the ratios of cumulative NCP to cumulative consumption of NO₃ and PO₄ (i.e., C:N and C:P ratios) in all mesocosms were 0.1 to 5.2 and 1 to 76, respectively, during phase 2 and -1.6 to 12 and -24 to 210, respectively, during phase 3 (Fig. 3). The negative ratios were due to the negative cumulative NCP in M9 during phase 3. The pCO₂-dependent decrease in ratio of cumulative NCP to cumulative consumption of NO₃ and PO₄ was detected during phase 3 (slope ± se: -0.014 ± 0.003 µmol C µmol N⁻¹ µatm⁻¹, n = 9, F-test, P = 0.003; slope ± se: -0.22 ± 0.05 µmol C µmol P⁻¹ µatm⁻¹, n = 9, F-test, P = 0.003, respectively) and during phase 2 + 3 (slope ± se: -0.005 ± 0.001 µmol C µmol N⁻¹ µatm⁻¹, n = 9, F-test, P = 0.011; slope ± se: -0.07 ± 0.02 µmol C µmol P⁻¹ µatm⁻¹, n = 9, F-test, P = 0.019, respectively).

## 4 Discussion

This experiment was set up as a gradient of pCO₂ levels with a range of 185 to 1420 µatm on t8/9 (Table 1; Bellerby et al., 2012). The water used to fill the mesocosms had low concentrations of NO₃, PO₄, Si, and Chl *a*, and the nutrient stoichiometry suggests a depletion of dissolved inorganic nitrogen and Si compared to PO₄ (Table 2; Schulz et al., 2012). The water temperature increased gradually, especially in the upper 5 to 10 m, in all mesocosms during the experiment (2.7 to 5.5 °C; Schulz et al., 2012). The growth rate of planktonic organisms is sensitive to increasing water temperature (Eppley, 1972; Rose and Caron, 2007; Kirchman et al., 2009). Significant effects of temperature and/or nutrient supply on microbial metabolism are observed in cold waters (e.g., Pomeroy et al., 1991), but CR remained relatively stable in our study. Since our experimental design did not include a control mesocosm with regard to water temperature and nutrients, it is not possible to separate the effect of temperature and availability of nutrients from that of ocean acidification on the responses of NCP, CR, and GCP values reported in the present study. In other words, the responses of the metabolic balance in each mesocosm can be regarded as an effect of a given pCO₂ set up at the start of the experiment, overridden with a gradual increase of water temperature throughout the experiment and a nutrient enrichment on t13 to all mesocosms.

The cumulative net community production of the post-bloom plankton community in a high-latitude fjord estimated using the oxygen technique significantly decreased with increasing pCO₂ after nutrient addition during the period of the second Chl *a* minimum until the end of this study (phase 3). Community respiration was relatively stable throughout the

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Relationships between ratios of NCP (µmol C L⁻¹ d⁻¹) to cumulative consumption of NO₃ (µmol N L⁻¹) and PO₄ (µmol P L⁻¹) vs. the mean pCO₂ (µatm) during phase 2 (nutrient addition until the second chlorophyll minimum: t14 to t21) (circle), phase 3 (the second chlorophyll minimum until the end of this study: t22 to t28) (triangle), and phase 2 + 3 (after nutrient addition: t14 to t28) (square). NCP values based on changes of dissolved oxygen concentration were converted to carbon unit under an assumption of photosynthetic quotient of 1.25 (Williams et al., 1979). The solid and dotted lines indicate statistically significant relationship during phase 3 and phase 2 + 3, respectively (F-test, P < 0.05).</figcaption></figure>

experiment in all mesocosms, with no consistent pattern found in response to changes in pCO₂. As a result, the cumulative gross community production (the difference between NCP and CR) significantly decreased with increasing pCO₂ level during phase 3. While the nutrient addition on t13 induced an increase of phytoplankton biomass in all mesocosms, the ratio of cumulative NCP to cumulative consumption of NO₃ and PO₄ significantly decreased with increasing pCO₂ during phase 3, suggesting alterations of the stoichiometric C:N and C:P uptake by the plankton community in response to increasing pCO₂.

In this experiment NCP of whole plankton community was determined based on three different methods: temporal changes of total carbon (C_T) concentration (hereafter, C_T-NCP) (Czerny et al., 2012; Silyakova et al., 2012), net ¹³C-POC production (hereafter, ¹³C-NCP) (de Kluijver et al., 2012), and net oxygen production in incubation bottles (hereafter, O₂-NCP) (this study). Conceptually, these methods are considered to measure the process of NCP. However, there are some differences in practice (see Czerny et al., 2012;
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Comparison of NCP measured with three different methods: temporal changes of total carbon concentration (hereafter, $C_T$-NCP) (Silyakova et al., 2012), net $^{13}$C-POC production (hereafter, $^{13}$C-NCP) (de Kluijver et al., 2012), and net oxygen production in incubation bottles (hereafter, $O_2$-NCP) (this study). The data were cumulated for each period: phase 1 ($t8$ to $t13$; circle), phase 2 ($t14$ to $t21$; upward triangle), phase 3 ($t22$ to $t27$; downward triangle), phase 2 + 3 ($t14$ to $t22$; square), and the whole period ($t8$ to $t27$; cross). When NCP was not measured every day, the data were linearly interpolated before cumulation. See also Table 3. The solid lines indicate the 1 : 1 relationship.</figcaption></figure>

de Kluijver et al., 2012; Silyakova et al., 2012). $C_T$-NCP is based on daily measurements of $C_T$ in integrated samples (no incubation) and on a correction for gas exchange. $^{13}$C-NCP is a measure of mesocosm-scale accumulation of $^{13}$C-POC in the water column and sediment material, and also does not require incubation (addition of $^{13}$C-bicarbonate to the mesocosms on $t-4$). $O_2$-NCP is based on changes in the concentration of DO during 24 h incubation of the integrated samples at a fixed depth outside the mesocosms.

Cumulative $O_2$-NCP was generally similar to cumulative $C_T$-NCP and higher than cumulative $^{13}$C-NCP (Fig. 4). Cumulative $O_2$-NCP positively correlated with both cumulative $C_T$- and $^{13}$C-NCP during phases 3 and 2 + 3 ($P < 0.05$) (Table 3). However, the correlation between $O_2$-NCP and the other NCPs was low during the other phases. Cumulative $C_T$- and $^{13}$C-NCP always showed the same trend and were highly correlated ($r = 0.785-0.976, P < 0.05$) except during phase 1 (Table 3), while cumulative $^{13}$C-NCP tended to be smaller than cumulative $C_T$-NCP (Fig. 4). Production rate of POC and DOC for the < 200 µm community measured using $^{14}$C in the same experiment, which is somewhat between net and gross primary production, was always higher than these three NCPs (see Engel et al., 2012). Hence, the responses of cumulative NCP with increasing $pCO_2$ were somewhat different between the three measurements: (1) insignificant responses of $O_2$-NCP with increasing $pCO_2$ during phases 1 and 2, but significant positive responses of $C_T$-NCP during phase 1 and $^{13}$C-NCP during phase 2; (2) insignificant responses of $O_2$-NCP with increasing $pCO_2$ during the whole period, but significant negative response of $^{13}$C-NCP (Fig. 2; de Kluijver et al., 2012; Silyakova et al., 2012). The cumulative $O_2$-NCP during phases 1 and 2 in response to increasing $pCO_2$ tended to be positive but was statistically insignificant. Moreover the ratio of cumulative $O_2$-NCP to $NO_3$ and $PO_4$ with increasing $pCO_2$ was significant during phase 3, but the

<table><caption>**Table 3.** Correlation analysis of NCP measured with three different methods: temporal changes of total carbon concentration ($C_T$-NCP) (Silyakova et al., 2012), net $^{13}$C-POC production ($^{13}$C-NCP) (de Kluijver et al., 2012), and net oxygen production in incubation bottles ($O_2$-NCP) (this study). The data were cumulated for each period: phase 1 ($t8$ to $t13$), phase 2 ($t14$ to $t21$), phase 3 ($t22$ to $t27$), phase 2 + 3 ($t14$ to $t22$), and the whole period ($t8$ to $t27$). When NCP was not measured every day, the data were linearly interpolated before cumulation. Significant coefficients of correlation are shown in bold with * ($P < 0.05$), ** ($P < 0.01$), and *** ($P < 0.001$) ($n = 9$ for all cases). See also Fig. 4.</caption><thead><tr><th>Period</th><th>Parameter</th><th><i>C</i><sub>T</sub>-based</th><th><sup>13</sup>C-based</th></tr></thead><tbody><tr><td>phase 1</td><td></td><td>0.369</td><td>-0.086</td></tr><tr><td>phase 2</td><td></td><td>-0.108</td><td>0.377</td></tr><tr><td>phase 3</td><td>O<sub>2</sub>-based</td><td><b>0.927</b><sup>***</sup></td><td><b>0.913</b><sup>***</sup></td></tr><tr><td>phase 2 + 3</td><td></td><td><b>0.792</b><sup>*</sup></td><td><b>0.751</b><sup>*</sup></td></tr><tr><td>whole period</td><td></td><td>0.113</td><td>0.128</td></tr><tr><td>phase 1</td><td></td><td></td><td>0.553</td></tr><tr><td>phase 2</td><td></td><td></td><td><b>0.785</b><sup>*</sup></td></tr><tr><td>phase 3</td><td><i>C</i><sub>T</sub>-based</td><td>-</td><td><b>0.937</b><sup>***</sup></td></tr><tr><td>phase 2 + 3</td><td></td><td></td><td><b>0.976</b><sup>***</sup></td></tr><tr><td>whole period</td><td></td><td></td><td><b>0.838</b><sup>**</sup></td></tr></tbody></table>

ratio of cumulative $C_T$-NCP to $NO_3$ and $PO_4$ was insignificant with increasing $pCO_2$ (Fig. 3; Silyakova et al., 2012).
We speculate that these differences were due to (1) less frequent measurement of O₂-NCP, (2) less representative measurement of O₂-NCP, and/or (3) possible modification of C and O₂ coupling of the plankton community. Since the cumulative calculation was based on only three data points during phase 1 (Fig. 1), the less frequent measurement could contribute to a relatively large uncertainty for the cumulative O₂-NCP. It should be noted that the correlation between C_T-NCP and ¹³C-NCP was insignificant during phase 1 (Table 3). During phases 2 + 3, O₂-NCP was measured every second day, while C_T-NCP and ¹³C-NCP were measured every day (except on t26 and t28 for ¹³C-NCP). Unless skewed increases of NCP happened on daily scale, it is difficult to explain why a similar response was detected for all three NCP datasets during phase 3 but the response of O₂-NCP was different from the others only during phase 2 (marginally positive for C_T-NCP during phase 2, *P* = 0.08). As mentioned above, there were some important differences in measurement and incubation of samples between the three methods. O₂-NCP was measured by incubating the integrated samples for 24 h at a fixed depth at the mooring site. Although we chose the incubation depth of 4 m at which the irradiance corresponded to the average irradiance of the water column sampled in the mesocosms (0 to 12 m), the mooring site was occasionally influenced by the plume of a nearby stream, resulting in reduced water transparency and irradiance. In the mesocosms, photosynthetically active radiation at 14.5 m and 4.2 m depth varied in a range of 2–15 % and 10–30 %, respectively, in comparison to the surface layer (0.1 to 0.2 m), which was likely because of temporal changes of phytoplankton biomass (Schulz et al., 2012). Gao et al. (2012) recently reported that the growth rate of three species of diatoms subjected to elevated pCO₂ is inversely related to light at irradiance levels above 22 to 36 % of surface irradiance in the South China Sea, and the threshold of photoinhibition occurs at lower irradiance in elevated pCO₂ compared to the ambient pCO₂. This demonstrates the confounding effects of the synergistic and antagonistic interactions of pCO₂ and irradiance conditions on the response of phytoplankton (e.g., Boyd et al., 2010). In this study, irradiance was not measured at the mooring site during the experiment, preventing the comparison of the light condition between the mesocosm site and the mooring site. It has been reported that long incubation (24 h for NCP and 48 h for CR in this study) in bottles can result in important changes in the abundance, activity, and composition of the community, leading in turn to significant changes in the planktonic metabolism (Pomeroy et al., 1994; Calvo-Díaz et al., 2011). However, unless the irradiance was significantly different between the mesocosm site and the mooring site and/or the artifact of bottle incubation was only significant during phase 2, it is difficult to explain why a similar response was detected for all three NCP datasets during phase 3 but only the response of O₂-NCP was different from the others during phase 2. Interestingly, Schulz et al. (2012) report that numerous parameters

of standing stock and phytoplankton composition positively or negatively correlated with increasing pCO₂, and pCO₂-related differences in phytoplankton pigment composition, phytoplankton carbon biomass, and organic matter became increasingly significant as the experiment progressed. While the photosynthetic quotient was assumed to be 1.25 in this study, it generally varies in a range of 1.2 to 1.8 (Laws, 1991). A study in the Canadian high Arctic reports an apparent photosynthetic quotient in the range of 1.3 to 1.8 (Platt et al., 1987). Assuming a photosynthetic quotient of 1.8, the ratios of cumulative NCP to cumulative consumption of NO₃ and PO₄ (i.e., C:N and C:P ratios) in all mesocosms would be proportionally reduced (C:N = 0.05 to 3.6 and C:P = 1 to 52 during phase 2 and C:N = -1.1 to 8.2 and C:P = -17 to 146 during phase 3) but the significant relationships would remain unchanged (data not shown). It is possible that the photosynthetic quotient is a function of the nutrient concentration (Williams et al., 1979). In addition, an application of a constant photosynthetic quotient to all pCO₂ treatments may add further uncertainty to the comparison of O₂-NCP with the carbon-based NCP, although to our knowledge change of photosynthetic quotient in response to increasing pCO₂ remains to be clarified. A significant increase of C_T-NCP and an insignificant response of O₂-NCP with increasing pCO₂ have been reported from a pCO₂-manipulated mesocosm experiment performed using relatively temperate coastal water in Norway (60.6° N, 5.2° E) (Riebesell et al., 2007; Bellerby et al., 2008; Egge et al., 2009).

Ocean acidification leads to both negative and positive effects on biological processes (reviewed by Hendriks et al., 2010; Kroeker et al., 2010; Liu et al., 2010). A meta-analysis suggests that photosynthetic organisms show higher growth rates with increasing pCO₂ and concludes that natural phytoplankton assemblages consistently show a relatively modest increase in carbon fixation at elevated pCO₂ (Hendriks et al., 2010). Yoshimura et al. (2010) reported that increasing pCO₂ treatment resulted in a significantly smaller accumulation of dissolved organic carbon with a reduced contribution of fucoxanthin-containing phytoplankton such as diatoms in the phytoplankton community in a CO₂-manipulated experiment in the Sea of Okhotsk. They did not find any significant effect on Chl *a* and POC, although phytoplankton growth or production was not measured. Their results may hint at reduced NCP with increasing pCO₂. In the present study, even though phase 3 seems relatively short (one-third of the whole experimental period), the cumulative NCP based on all three measurements showed significantly negative effect of increasing pCO₂ for pelagic plankton communities (Fig. 2; de Kluijver et al., 2012; Silyakova et al., 2012).

The net consumption rate of NO₃ and PO₄ was higher at the higher levels of pCO₂ during phase 2 and at the lower pCO₂ levels during phase 3 (Schulz et al., 2012). During phase 2, a statistically significant, positive correlation with increasing pCO₂ was found for the concentrations of DOC, POC, PON, and POP, but not for any stoichiometric ratio
(Schulz et al., 2012). The higher consumption rate of NO₃ at higher pCO₂ levels observed in the same study (Schulz et al., 2012) is in contrast with the findings of Riebesell et al. (2007) who reported higher NO₃ consumption at lower pCO₂ levels at the beginning of the experiment. While the cumulative consumption of NO₃ was similar between the mesocosms during phase 2+3 in the present study, the ratio of POC to PON in the mesocosm water column was about 8 at the lower pCO₂ levels and about 6 at the higher pCO₂ levels at the end of the experiment (Schulz et al., 2012). This trend was amplified in the ratio of POC to PON in the sediment materials of mesocosms (Czerny et al., 2012). The abundance of diatoms increased faster in the lower pCO₂ treatments from t20 onward in the same experiment (Aberle et al., 2012; Schulz et al., 2012), and higher amounts of diatom-derived material were collected in the sediment traps of the lower pCO₂ mesocosms (Czerny et al., 2012). The mechanism(s) that caused these stoichiometric responses remains to be elucidated. Yet it is evident that increasing pCO₂ resulted in alteration of stoichiometric coupling of C and nutrients in the present study, which may change the nutritional value for higher trophic levels.

In conclusion, the metabolic parameters (NCP, CR, and GCP) of planktonic communities based on changes of DO concentration at different pCO₂ levels showed insignificant response of NCP during phases 1 and 2 and a significant decrease of NCP as a function of increasing pCO₂ during phase 3. CR was relatively stable throughout the experiment in all mesocosms. As a result, the cumulative GCP significantly decreased with increasing pCO₂ only during phase 3. Similarly, the ratios of cumulative NCP to cumulative consumption of NO₃ and PO₄ showed insignificant response during phase 2 but significant decrease during phase 3 with increasing pCO₂. The results suggest that elevated pCO₂ influenced cumulative NCP and stoichiometric C and nutrient coupling of the plankton community in a high-latitude fjord only for a limited period. Since there were some differences or weak correlations between O₂-NCP vs. C_T-NCP and ¹³C-NCP during phases 1 and 2, this conclusion should be taken with caution.

**Acknowledgements.** This work is a contribution to the "European Project on Ocean Acidification" (EPOCA) which received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 211384. Additional financial support was provided by IPEV, the French Polar Institute. We gratefully acknowledge the logistical support of Greenpeace International for its assistance with the transport of the mesocosm facility from Kiel to Ny-Ålesund and back to Kiel. We also thank the captains and crews of M/V ESPERANZA of Greenpeace and R/V Viking Explorer of the University Centre in Svalbard (UNIS) for assistance during mesocosm transport and during deployment and recovery in Kongsfjorden. We thank the staff of the French-German Arctic Research Base at Ny-Ålesund (AWIPEV), in particular Marcus Schumacher, for on-site lo-

gistical support. This study could have been performed without the kind support of all members of the EPOCA 2010 scientific party.

Edited by: T. F. Thingstad

<figure><img src="image_8.png" /></figure>

INSU

Institut national des sciences de l'Univers

The publication of this article is financed by CNRS-INSU.

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