Effect of increased &amp;lt;i&amp;gt;p&amp;lt;/i&amp;gt;CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; on the planktonic metabolic balance during a mesocosm experiment in an Arctic fjord

<jats:p>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 (t4 to t13); phase 2 – nutrient addition until the second chlorophyll a minimum (t14 to t21); phase 3 – the second chlorophyll a minimum until the end of this study (t22 to t28). 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 stoichiometric 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.                     </jats:p>

section_content: 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 remineralized 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. 

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 (CO 2 ) in the atmosphere leads to an increase of the partial pressure of CO 2 (pCO 2 ) 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 pCO 2 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 14 C fixation or production of particulate organic carbon (POC) at elevated pCO 2 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 pCO 2 mostly enhances primary production. Measurements of primary production based on 14 C 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 pCO 2 levels. The oxygen-based NCP measurement has shown a significant decrease in NCP of Emiliania huxleyi at elevated pCO 2 in a N-limited chemostat culture (Sciandra et al., 2003), and insignificant changes in NCP of semi-natural plankton community at different pCO 2 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 pCO 2 treatments plus two replicated controls (no CO 2 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).
section_name: Introduction

section_name: Materials and methods

section_content: 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 m 3 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 CO 2 manipulation was carried out between 6 and 11 June (t -1 to t4), establishing seven enhanced pCO 2 treatments and two replicated controls (no pCO 2 treatment). pH and approximate pCO 2 levels in all mesocosms on t8/9 and t26/27 are shown in Table 1 (see also Bellerby et al., 2012). While pH and pCO 2 changed in all mesocosms because of air/sea gas exchange and biological carbon uptake to different degrees, the gradients of pH and pCO 2 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 (NO 3 ), 0. 31 µM of phosphate (PO 4 ), 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 CO 2 (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). 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 (t30). Sampling for NCP and CR determination ended on 5 July (t28).
section_name: Experimental setup and sampling

section_content: 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 × 10 4 -2 × 10 4 individuals m -3 ) in the mesocosms between t -2 and t11 (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 t7 and between t12 and t28. 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 (t6). For the CR measurement, dark BOD bottles were incubated at the in situ mooring site until 18 June (t11), and clear BOD bottles were incubated in a dark laboratory incubator from 19 June (t12) 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 O 2 L -1 d -1 ). GCP was calculated as the difference between NCP and CR. The combined uncertainty of GCP (SE GCP ) was calculated using the standard error of NCP (SE NCP ) and CR (SE CR ) according to

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 1end of CO 2 manipulation until nutrient addition (t4 to t13);

(2) phase 2 -nutrient addition until the second Chl a minimum (t14 to t21); (3) phase 3 -the second Chl a minimum until the end of this study (t22 to t28); (4) phase 2 + 3 (t14 to t28); and (5) whole period (t4 to t28). 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.
section_name: Net community production, community respiration, and gross community production

section_content: Linear regression was used to analyze the significance of responses of NCP, CR, GCP, and ratios of cumulative NCP to cumulative consumption of NO 3 and PO 4 to increased pCO 2 levels. When pCO 2 -dependent NCP, CR, and GCP were analyzed on a certain day, the pCO 2 values measured on that

Mean ± 2. 9 ± 0. 02 ± 0. 00 ± 0. 59 ± 0. 05 ± 0. 13 ± 11. 3 ± 4. 7 ± 2. 5 ± 0. 21 ± SD 0. 1 0. 01 0. 00 0. 05 0. 01 0. 02 1. 4 0. 9 0. 6 0. 02 (n = 9) 1 The data on temperature were based on the mean of 0-12 m in each mesocosm (Schulz et al., 2012). 2 The data on Chl a are based on the HPLC method (Schulz et al., 2012). day were used. For the regression analysis of the cumulative parameters, the mean pCO 2 during the corresponding period was used. All statistical analyses were performed with R (R Development Core Team, 2008).
section_name: Statistical analysis

section_content: The Arctic coastal water used to fill the mesocosms had low concentrations of dissolved inorganic nutrients and Chl a. The concentrations of NO 3 and PO 4 but not NH 4 were close to the detection limit of the conventional nutrient analysis, and the mean Chl a concentration determined by the highperformance liquid chromatography method was 0. 21 µg L -1 on t0 (Table 2 ; Schulz et al., 2012). The mean ratios of dissolved inorganic nitrogen (DIN: sum of NH 4, NO 3, and NO 2 ) to PO 4, DIN to Si, and Si to PO 4 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 t13 (Schulz et al., 2012). After the addition of nutrients, the net consumption rate of NO 3 and PO 4 was statistically higher in higher pCO 2 mesocosms from t17 to t22, 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 -1 (t0 to t30) 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 (t8, range: 0. 5-0. 9 µg L -1 ) and twice after the nutrient addition (t19 and t28, range: 0. 3-1. 0 µg L -1 and 0. 6-1. 8 µg L -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 pCO 2 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 O 2 L -1 d -1 between t -1 and t28 (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 (t24 or t28). There were a few exceptions: the smallest GCP was observed after the nutrient addition (t16) in M6, and the highest value was observed before the nutrient addition (t12) 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 pCO 2 on t24 (-0. 008 ± 0. 002 µmol O 2 L -1 d -1 µatm -1, P < 0. 05). 

The extent of temporal variation of CR (-5. 6 to 0. 25 µmol O 2 L -1 d -1 ) was about half of that of GCP during the experiment (Fig. 1 ). The positive CR measured in M2 on t7 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 pCO 2 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 t7, M5 and M7 on t12, M6 on t16, and M6 on t24). 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 (t26 or t28), respectively. There were a few exceptions: the lowest NCP was observed after the nutrient addition (t16) in M6, and the highest was observed before the nutrient addition (t12) in M9. NCP in all mesocosms but M9 tended to increase 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 pCO 2 level. Linear regression analysis detected a significant decrease of NCP with increase of pCO 2 level on t24 and t26 (slope ± se: -0. 008 ± 0. 003 and -0. 008 ± 0. 001 µmol O 2 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 CO 2 perturbation (i. e., no CO 2 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 pCO 2 -dependent decrease of cumulative NCP during phase 3 (slope ± se: P = 0. 001) and during phase 2 + 3 (slope ± se:

-0. 04 ± 0. 01 µmol O 2 L -1 µatm -1, n = 9, F-test, P = 0. 005). The cumulative NCP slightly increased with increasing pCO 2 during phases 1 and 2, although the regression slope was not significant (P > 0. 05). The relationship between cumulative NCP and pCO 2 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 pCO 2 was always insignificant (F-test, P > 0. 05). The cumulative GCP significantly decreased with increasing pCO 2 during phase 3 (slope ± se: -0. 05 ± 0. 01 µmol O 2 L -1 µatm -1, n = 9, F-test, P < 0. 001) and during phase 2 + 3 (slope ± se: -0. 04 ± 0. 01 µmol O 2 L -1 µatm -1, n = 9, F-test, P = 0. 019). 

The net consumption rate of NO 3 and PO 4 with increasing pCO 2, 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 NO 3 and PO 4 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 NO 3 to PO 4 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 NO 3 and PO 4 (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 pCO 2 -dependent decrease in ratio of cumulative NCP to cumulative consumption of NO 3 and PO 4 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).
section_name: Results

section_content: This experiment was set up as a gradient of pCO 2 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 NO 3, PO 4, Si, and Chl a, and the nutrient stoichiometry suggests a depletion of dissolved inorganic nitrogen and Si compared to PO 4 (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 pCO 2 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 postbloom plankton community in a high-latitude fjord estimated using the oxygen technique significantly decreased with increasing pCO 2 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 ) to cumulative consumption of NO 3 (µmol N L -1 ) and PO 4 (µmol P L -1 ) vs. the mean pCO 2 (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). 

experiment in all mesocosms, with no consistent pattern found in response to changes in pCO 2. As a result, the cumulative gross community production (the difference between NCP and CR) significantly decreased with increasing pCO 2 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 NO 3 and PO 4 significantly decreased with increasing pCO 2 during phase 3, suggesting alterations of the stoichiometric C:N and C:P uptake by the plankton community in response to increasing pCO 2. 

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 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). Conceptually, these methods are considered to measure the process of NCP. However, there are some differences in practice (see Czerny et al., 2012 ; 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. 

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 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. (Fig. 3; Silyakova et al., 2012).
section_name: Discussion

section_content: We speculate that these differences were due to (1) less frequent measurement of O 2 -NCP, (2) less representative measurement of O 2 -NCP, and/or (3) possible modification of C and O 2 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 O 2 -NCP. It should be noted that the correlation between C T -NCP and 13 C-NCP was insignificant during phase 1 (Table 3 ). During phases 2 + 3, O 2 -NCP was measured every second day, while C T -NCP and 13 C-NCP were measured every day (except on t26 and t28 for 13 C-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 O 2 -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. O 2 -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 pCO 2 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 pCO 2 compared to the ambient pCO 2. This demonstrates the confounding effects of the synergistic and antagonistic interactions of pCO 2 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 O 2 -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 pCO 2, and pCO 2related 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 NO 3 and PO 4 (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 pCO 2 treatments may add further uncertainty to the comparison of O 2 -NCP with the carbon-based NCP, although to our knowledge change of photosynthetic quotient in response to increasing pCO 2 remains to be clarified. A significant increase of C T -NCP and an insignificant response of O 2 -NCP with increasing pCO 2 have been reported from a pCO 2 -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 pCO 2 and concludes that natural phytoplankton assemblages consistently show a relatively modest increase in carbon fixation at elevated pCO 2 (Hendriks et al., 2010). Yoshimura et al. (2010) reported that increasing pCO 2 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 CO 2 -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 pCO 2. 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 pCO 2 for pelagic plankton communities (Fig. 2 ; de Kluijver et al., 2012; Silyakova et al., 2012). 

The net consumption rate of NO 3 and PO 4 was higher at the higher levels of pCO 2 during phase 2 and at the lower pCO 2 levels during phase 3 (Schulz et al., 2012). During phase 2, a statistically significant, positive correlation with increasing pCO 2 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 NO 3 at higher pCO 2 levels observed in the same study (Schulz et al., 2012) is in contrast with the findings of Riebesell et al. (2007) who reported higher NO 3 consumption at lower pCO 2 levels at the beginning of the experiment. While the cumulative consumption of NO 3 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 pCO 2 levels and about 6 at the higher pCO 2 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 pCO 2 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 pCO 2 mesocosms (Czerny et al., 2012). The mechanism(s) that caused these stoichiometric responses remains to be elucidated. Yet it is evident that increasing pCO 2 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 pCO 2 levels showed insignificant response of NCP during phases 1 and 2 and a significant decrease of NCP as a function of increasing pCO 2 during phase 3. CR was relatively stable throughout the experiment in all mesocosms. As a result, the cumulative GCP significantly decreased with increasing pCO 2 only during phase 3. Similarly, the ratios of cumulative NCP to cumulative consumption of NO 3 and PO 4 showed insignificant response during phase 2 but significant decrease during phase 3 with increasing pCO 2. The results suggest that elevated pCO 2 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 O 2 -NCP vs. C T -NCP and 13 C-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 provide 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

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