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# Oxygen production and carbon fixation in oligotrophic coastal bays and the relationship with gross and net primary production

Natalia Gonzalez, Jean-Pierre Gattuso, Jack J. Middelburg

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Natalia Gonzalez, Jean-Pierre Gattuso, Jack J. Middelburg. Oxygen production and carbon fixation in oligotrophic coastal bays and the relationship with gross and net primary production. Aquatic Microbial Ecology, 2008, 52 (2), pp.119-130. 10.3354/ame01208. hal-03494356

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# Oxygen production and carbon fixation in oligotrophic coastal bays and the relationship with gross and net primary production

Natalia González<fnref n="1" />,<fnref n="3" />,*, Jean-Pierre Gattuso<fnref n="1" />, Jack J. Middelburg<fnref n="2" />

**ABSTRACT:** Planktonic primary production and respiration in 2 coastal oligotrophic sites of the Northwest Mediterranean Sea were examined. Primary production was quantified using 3 methods (light and dark changes in dissolved O₂, ¹⁸O-labeling and ¹⁴C uptake technique) using *in situ* bottle incubations. Gross primary production (GPP) based on the O₂ light-dark technique was not significantly different from that using the ¹⁸O-labeling technique, indicating that the former technique provides accurate estimates of GPP in these environments. Respiration in the dark was not significantly different from respiration in the light. Total ¹⁴C uptake (including the dissolved and particulate organic carbon fractions) during the whole duration of the light period approached GPP and consequently overestimated net primary production.

**KEY WORDS:** Primary production · Oligotrophic coastal bays · Light-dark O₂ changes · ¹⁸O-labeling ·

¹⁴C uptake · Respiration in the light

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Oceanic primary production (PP) accounts for about 50% of total carbon fixation in the biosphere (Field et al. 1998). Accurate quantification of marine PP is therefore fundamental in understanding and characterizing global carbon cycling. To avoid confusion, it is important to distinguish between some of the terms related to PP. PP can be quantified at the initial stage of the photosynthetic process by following changes in dissolved oxygen or at a later stage by measuring carbon fixation. Gross primary production (GPP) is the photosynthetic carbon uptake by phytoplankton which can be estimated using the O₂ light-dark and the ¹⁸O-labeling methods. Net community production (NCP) is GPP less the organic carbon lost through community respiration and can be measured using the O₂ light–dark method. Net primary production (NPP) is the GPP minus autotrophic respiration and can be

measured using the ¹⁴C method when the ¹⁴C and ¹²C carbon pools are in equilibrium.

Traditionally, PP is estimated from either oxygen changes during light/dark incubations or the transfer of ¹⁴C from bicarbonate to the particulate organic matter pool produced, thus ignoring the dissolved organic matter fraction (see references in Barber & Hilting 2002). Subsequently, the ¹⁸O-labeling technique for gross oxygen production, and bottle incubation independent techniques such as the triple oxygen isotope method (¹⁸O₂, ¹⁷O₂, and ¹⁶O₂), fast repetition rate fluorometry (FRRF) and indirect methods (O₂ and CO₂ changes, net transports of allochthonous organic carbon) have been used (e.g. Grande et al. 1989, Luz & Barkan 2000, Juranek & Quay 2005, Sarma et al. 2005, Pringault et al. 2007). However, each technique has its strengths and weaknesses (e.g. Robinson & Williams 2005).

Unfortunately, most of these methods involve incubation over a period of time and this may induce a

<footnote n="1">¹CNRS, Laboratoire d'océanographie de Villefranche, BP 28, 06234 Villefranche-sur-Mer Cedex, France; Université Pierre et Marie Curie–Paris 6, Laboratoire d'océanographie de Villefranche, BP 24, 06230 Villefranche-sur-Mer, France</footnote>

<footnote n="2">²Netherlands Institute of Ecology (NIOO-KNAW), Postbus 140, 4400 AC Yerseke, The Netherlands</footnote>

<footnote n="3">³Present address: Área de Biodiversidad y Conservación, ESCET, Universidad Rey Juan Carlos, Tulipán s/n., Móstoles 28933 Madrid, Spain</footnote>
# Oxygen production and carbon fixation in oligotrophic coastal bays and the relationship with gross and net primary production

Natalia González<fnref n="1" />,<fnref n="3" />,*, Jean-Pierre Gattuso<fnref n="1" />, Jack J. Middelburg<fnref n="2" />

**ABSTRACT:** Planktonic primary production and respiration in 2 coastal oligotrophic sites of the Northwest Mediterranean Sea were examined. Primary production was quantified using 3 methods (light and dark changes in dissolved O₂, ¹⁸O-labeling and ¹⁴C uptake technique) using *in situ* bottle incubations. Gross primary production (GPP) based on the O₂ light-dark technique was not significantly different from that using the ¹⁸O-labeling technique, indicating that the former technique provides accurate estimates of GPP in these environments. Respiration in the dark was not significantly different from respiration in the light. Total ¹⁴C uptake (including the dissolved and particulate organic carbon fractions) during the whole duration of the light period approached GPP and consequently overestimated net primary production.

**KEY WORDS:** Primary production · Oligotrophic coastal bays · Light-dark O₂ changes · ¹⁸O-labeling · ¹⁴C uptake · Respiration in the light

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Oceanic primary production (PP) accounts for about 50% of total carbon fixation in the biosphere (Field et al. 1998). Accurate quantification of marine PP is therefore fundamental in understanding and characterizing global carbon cycling. To avoid confusion, it is important to distinguish between some of the terms related to PP. PP can be quantified at the initial stage of the photosynthetic process by following changes in dissolved oxygen or at a later stage by measuring carbon fixation. Gross primary production (GPP) is the photosynthetic carbon uptake by phytoplankton which can be estimated using the O₂ light-dark and the ¹⁸O-labeling methods. Net community production (NCP) is GPP less the organic carbon lost through community respiration and can be measured using the O₂ light–dark method. Net primary production (NPP) is the GPP minus autotrophic respiration and can be

measured using the ¹⁴C method when the ¹⁴C and ¹²C carbon pools are in equilibrium.

Traditionally, PP is estimated from either oxygen changes during light/dark incubations or the transfer of ¹⁴C from bicarbonate to the particulate organic matter pool produced, thus ignoring the dissolved organic matter fraction (see references in Barber & Hilting 2002). Subsequently, the ¹⁸O-labeling technique for gross oxygen production, and bottle incubation independent techniques such as the triple oxygen isotope method (¹⁸O₂, ¹⁷O₂, and ¹⁶O₂), fast repetition rate fluorometry (FRRF) and indirect methods (O₂ and CO₂ changes, net transports of allochthonous organic carbon) have been used (e.g. Grande et al. 1989, Luz & Barkan 2000, Juranek & Quay 2005, Sarma et al. 2005, Pringault et al. 2007). However, each technique has its strengths and weaknesses (e.g. Robinson & Williams 2005).

Unfortunately, most of these methods involve incubation over a period of time and this may induce a

<footnote n="1">¹CNRS, Laboratoire d'océanographie de Villefranche, BP 28, 06234 Villefranche-sur-Mer Cedex, France; Université Pierre et Marie Curie–Paris 6, Laboratoire d'océanographie de Villefranche, BP 24, 06230 Villefranche-sur-Mer, France</footnote>

<footnote n="2">²Netherlands Institute of Ecology (NIOO-KNAW), Postbus 140, 4400 AC Yerseke, The Netherlands</footnote>

<footnote n="3">³Present address: Área de Biodiversidad y Conservación, ESCET, Universidad Rey Juan Carlos, Tulipán s/n., Móstoles 28933 Madrid, Spain</footnote>
bottle effect, which can result in changes in biomass and community structure (e.g. Gattuso et al. 2002). For instance, incubations are often carried out on the deck of a ship or in an incubator in which temperature, and in particular light conditions, deviate from those in *situ*. Therefore, there is a critical need to develop alternative techniques with no incubation. A number of incubation-free approaches have been developed, e.g. FRRF and the measurement of the expression of the gene coding for RUBISCO (Corredor et al. 2004), but these do not yet have the required precision. Air-sea gas exchange of oxygen provides another direct method to estimate PP (e.g. Bates et al. 1998), but the uncertainty of the air-sea gas exchange estimates is rather high (Juranek & Quay 2005). Recent studies (e.g. Luz & Barkan 2000) consider the 3 oxygen isotopes method as one of the more accurate methods to estimate GPP. Nevertheless, it relies on uncertain parameterization of the air-sea gas exchange which may lead to relatively large errors of GPP (Juranek & Quay 2005).

Several studies have compared methods to derive PP in the open ocean (e.g. Williams et al. 1983, Bender et al. 1987, Grande et al. 1989, Juranek & Quay 2005, Sarma et al. 2005) and have identified a number of difficulties. First, a photosynthetic quotient (PQ = moles of O₂ released/moles of CO₂ fixed) is required to compare PP based on carbon fixation and oxygen release methods. This PQ can vary widely although it generally ranges between 1.2 and 1.8 (Laws 1991). Second, interpreting ¹⁴C uptake is not as straightforward as the significance of recycling, i.e. the release of fixed C as CO₂, varies depending on the duration of the incubation and community structure. According to some authors (see references in Langdon et al. 1995), ¹⁴C uptake should theoretically approximate NPP or GPP depending on how fast the pool of organic carbon reaches isotopic equilibrium with the dissolved inorganic carbon pool. Although there is no consensus, ¹⁴C fixation is thought to approach NPP when 24 h incubations are used; when shorter incubations are used, it approximates GPP (e.g. Weger et al. 1989). Marra (2002) has argued that ¹⁴C fixation approximates NPP otherwise it would require an unrealistic PQ. However, others (e.g. Langdon et al. 1995) consider that ¹⁴C fixation gives a good estimation of GPP or something between GPP and NPP (e.g. Laws et al. 2000). Third, the ¹⁴C uptake rates reported in the literature often concern only particulate ¹⁴C fixation. If dissolved organic carbon (DO¹⁴C) is produced but not measured, the ¹⁴C fixation rate will consequently underestimate total PP (Karl et al. 1998). Fourth, light respiration may be higher than dark respiration as a result of photoenhancement of mitochondrial respiration, photorespiration and photoreduction of O₂ (e.g. Bender et al.

1987, Dickson & Orchard 2001, Dickson et al. 2001, Pringault et al. 2007). The differences in respiration in the light and dark are another contentious issue of direct relevance to the accuracy of the light-dark technique which assumes that these rates are similar.

The current debate on the measurement of PP remains unresolved because of the lack of *in situ* assessments, artefacts associated with incubations, the difficult distinction between NPP and GPP, the rapid release and consumption of photosynthesis derived dissolved organic matter, the impact of metabolic processes not directly related to photosynthesis and the critical assumption of equal community respiration in light and darkness. In addition, the present debate is largely limited to open ocean communities (Grande et al. 1989, Laws et al. 2000, Juranek & Quay 2005) and fresh-water ecosystems (e.g. Carignan et al. 2000), while methods inter-comparisons in coastal ecosystems are rather limited (Sarma et al. 2005, Gazeau et al. 2007).

The main goal of the present work was to provide a critical appraisal of GPP measurements. Such an evaluation is essential to further our understanding of global carbon cycling. We present PP rates from Mediterranean oligotrophic coastal waters, based on 3 methods (light and dark changes in dissolved O₂, ¹⁸O-labeling and ¹⁴C-uptake technique) using *in situ* bottle incubations. These data will be used to revisit the controversial topics of whether respiration rates in the light and dark are equal and whether ¹⁴C fixation approximates GPP or NPP.

## MATERIALS AND METHODS

**Study site and sampling.** Experiments were carried out in the Bay of Palma (BP) and at Point B, Bay of Villefranche-sur-mer (BV) (43° 41' N, 7° 19' E) (Table 1). Both embayments are oligotrophic systems in the NW Mediterranean Sea with extensive seagrass (*Posidonia oceanica*) meadows and negligible freshwater inputs. The bottom depth of the stations varied between 13 m and 33 m in the BP and was 80 m in the BV. The study at the BP was conducted on board the RV 'Mytilus' during June 2002 and the experiments were performed at 4 stations: Posidonia (Pos), Enderrocat (End), Bahía (B) and Stn 4 (S4) (Table 1). The study in the BV was carried out using the Sagitta at the Point B time series station (Table 1) in October 2002, December 2002, March 2003 and July 2003. Further details about the BP and BV study sites are provided in Gazeau et al. (2005) and Gomez & Gorsky (2003), respectively.

**Hydrography, irradiance and nutrients.** Vertical CTD profiles were carried out at each station using a CTD SeaBird SBE19 (BP) or SBE25 (BV). Profiles of
bottle effect, which can result in changes in biomass and community structure (e.g. Gattuso et al. 2002). For instance, incubations are often carried out on the deck of a ship or in an incubator in which temperature, and in particular light conditions, deviate from those *in situ*. Therefore, there is a critical need to develop alternative techniques with no incubation. A number of incubation-free approaches have been developed, e.g. FRRF and the measurement of the expression of the gene coding for RUBISCO (Corredor et al. 2004), but these do not yet have the required precision. Air-sea gas exchange of oxygen provides another direct method to estimate PP (e.g. Bates et al. 1998), but the uncertainty of the air-sea gas exchange estimates is rather high (Juranek & Quay 2005). Recent studies (e.g. Luz & Barkan 2000) consider the 3 oxygen isotopes method as one of the more accurate methods to estimate GPP. Nevertheless, it relies on uncertain parameterization of the air-sea gas exchange which may lead to relatively large errors of GPP (Juranek & Quay 2005).

Several studies have compared methods to derive PP in the open ocean (e.g. Williams et al. 1983, Bender et al. 1987, Grande et al. 1989, Juranek & Quay 2005, Sarma et al. 2005) and have identified a number of difficulties. First, a photosynthetic quotient (PQ = moles of O₂ released/moles of CO₂ fixed) is required to compare PP based on carbon fixation and oxygen release methods. This PQ can vary widely although it generally ranges between 1.2 and 1.8 (Laws 1991). Second, interpreting ¹⁴C uptake is not as straightforward as the significance of recycling, i.e. the release of fixed C as CO₂, varies depending on the duration of the incubation and community structure. According to some authors (see references in Langdon et al. 1995), ¹⁴C uptake should theoretically approximate NPP or GPP depending on how fast the pool of organic carbon reaches isotopic equilibrium with the dissolved inorganic carbon pool. Although there is no consensus, ¹⁴C fixation is thought to approach NPP when 24 h incubations are used; when shorter incubations are used, it approximates GPP (e.g. Weger et al. 1989). Marra (2002) has argued that ¹⁴C fixation approximates NPP otherwise it would require an unrealistic PQ. However, others (e.g. Langdon et al. 1995) consider that ¹⁴C fixation gives a good estimation of GPP or something between GPP and NPP (e.g. Laws et al. 2000). Third, the ¹⁴C uptake rates reported in the literature often concern only particulate ¹⁴C fixation. If dissolved organic carbon (DO¹⁴C) is produced but not measured, the ¹⁴C fixation rate will consequently underestimate total PP (Karl et al. 1998). Fourth, light respiration may be higher than dark respiration as a result of photoenhancement of mitochondrial respiration, photorespiration and photoreduction of O₂ (e.g. Bender et al.

1987, Dickson & Orchard 2001, Dickson et al. 2001, Pringault et al. 2007). The differences in respiration in the light and dark are another contentious issue of direct relevance to the accuracy of the light-dark technique which assumes that these rates are similar.

The current debate on the measurement of PP remains unresolved because of the lack of *in situ* assessments, artefacts associated with incubations, the difficult distinction between NPP and GPP, the rapid release and consumption of photosynthesis derived dissolved organic matter, the impact of metabolic processes not directly related to photosynthesis and the critical assumption of equal community respiration in light and darkness. In addition, the present debate is largely limited to open ocean communities (Grande et al. 1989, Laws et al. 2000, Juranek & Quay 2005) and fresh-water ecosystems (e.g. Carignan et al. 2000), while methods inter-comparisons in coastal ecosystems are rather limited (Sarma et al. 2005, Gazeau et al. 2007).

The main goal of the present work was to provide a critical appraisal of GPP measurements. Such an evaluation is essential to further our understanding of global carbon cycling. We present PP rates from Mediterranean oligotrophic coastal waters, based on 3 methods (light and dark changes in dissolved O₂, ¹⁸O-labeling and ¹⁴C-uptake technique) using *in situ* bottle incubations. These data will be used to revisit the controversial topics of whether respiration rates in the light and dark are equal and whether ¹⁴C fixation approximates GPP or NPP.

## MATERIALS AND METHODS

**Study site and sampling.** Experiments were carried out in the Bay of Palma (BP) and at Point B, Bay of Villefranche-sur-mer (BV) (43° 41' N, 7° 19' E) (Table 1). Both embayments are oligotrophic systems in the NW Mediterranean Sea with extensive seagrass (*Posidonia oceanica*) meadows and negligible freshwater inputs. The bottom depth of the stations varied between 13 m and 33 m in the BP and was 80 m in the BV. The study at the BP was conducted on board the RV 'Mytilus' during June 2002 and the experiments were performed at 4 stations: Posidonia (Pos), Enderrocat (End), Bahía (B) and Stn 4 (S4) (Table 1). The study in the BV was carried out using the Sagitta at the Point B time series station (Table 1) in October 2002, December 2002, March 2003 and July 2003. Further details about the BP and BV study sites are provided in Gazeau et al. (2005) and Gomez & Gorsky (2003), respectively.

**Hydrography, irradiance and nutrients.** Vertical CTD profiles were carried out at each station using a CTD SeaBird SBE19 (BP) or SBE25 (BV). Profiles of
<table><caption>Table 1. Summary of selected variables within photic layer at stations sampled at Bay of Villefranche (BV) and Bay of Palma (BP). Also shown are averages ± SE of each vertical depth, BV and BP. %cyano: % cyanobacteria; PER: percent of extracellular release TOC: total organic carbon; Pos: *Posidonia*; B: Bahía; S4: Stn 4; End: Enderrocat</caption><thead><tr><th>Location</th><th>Depth (m)</th><th>Sunrise-sunset (h)</th><th>Irradiance (µE m<sup>-2</sup> s<sup>-1</sup>)</th><th>NO<sub>3</sub> (µmol l<sup>-1</sup>)</th><th>Chl a (µgl<sup>-1</sup>)</th><th>%cyano (%)</th><th>PER (%)</th><th>TOC ± SE (µmol l<sup>-1</sup> d<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="4">Oct02 (BV)</td><td>0.5</td><td>11.43</td><td>1287</td><td>0.47</td><td>0.15</td><td>43.86</td><td>69.21</td><td>0.56 ± 0.07</td></tr><tr><td>10</td><td>11.43</td><td>300</td><td>0.24</td><td>0.15</td><td>41.39</td><td>50.41</td><td>0.32 ± 0.07</td></tr><tr><td>20</td><td>11.43</td><td>153</td><td>0.75</td><td>0.15</td><td>17.13</td><td>65.68</td><td>0.40 ± 0.03</td></tr><tr><td>30</td><td>11.43</td><td>110</td><td>2.21</td><td>0.36</td><td>28.03</td><td>37.82</td><td>0.21 ± 0.11</td></tr><tr><td rowspan="4">Dec02 (BV)</td><td>0.5</td><td>9.09</td><td>739</td><td>1.14</td><td>0.31</td><td>13.8</td><td>5.01</td><td>0.25 ± 0.02</td></tr><tr><td>10</td><td>9.09</td><td>219</td><td>0.69</td><td>0.29</td><td>6.66</td><td>19.46</td><td>0.20 ± 0.03</td></tr><tr><td>20</td><td>9.09</td><td>69</td><td>0.44</td><td>0.29</td><td>5.69</td><td>19.37</td><td>0.13 ± 0.01</td></tr><tr><td>30</td><td>9.09</td><td>20</td><td>0.68</td><td>0.17</td><td>4.45</td><td>37.74</td><td>0.05 ± 0.01</td></tr><tr><td rowspan="4">Mar03 (BV)</td><td>0.5</td><td>11.21</td><td>374</td><td>2.16</td><td>1.09</td><td>1.39</td><td>37.25</td><td>2.19 ± 0.20</td></tr><tr><td>10</td><td>11.21</td><td>215</td><td>2.95</td><td>0.66</td><td>2.14</td><td>31.20</td><td>2.86 ± 0.09</td></tr><tr><td>20</td><td>11.21</td><td>100</td><td>3.75</td><td>0.84</td><td>1.15</td><td>64.26</td><td>1.88 ± 0.07</td></tr><tr><td>30</td><td>11.21</td><td>48</td><td>3.01</td><td>0.40</td><td>2.21</td><td>86.59</td><td>1.03 ± 0.31</td></tr><tr><td rowspan="4">Jul03 (BV)</td><td>0.5</td><td>15.21</td><td>1598</td><td>0.64</td><td>0.13</td><td>30.13</td><td>21.41</td><td>0.71 ± 0.04</td></tr><tr><td>10</td><td>15.21</td><td>593</td><td>0.30</td><td>0.12</td><td>47.40</td><td>16.96</td><td>0.65 ± 0.10</td></tr><tr><td>20</td><td>15.21</td><td>288</td><td>0.54</td><td>0.11</td><td>36.71</td><td>44.76</td><td>0.33 ± 0.04</td></tr><tr><td>30</td><td>15.21</td><td>221</td><td>1.20</td><td>0.21</td><td>35.80</td><td>43.19</td><td>0.58 ± 0.03</td></tr><tr><td rowspan="4">Pos (BP)</td><td>0.5</td><td>15.00</td><td>998</td><td>0.30</td><td>0.42</td><td>4.86</td><td>16.44</td><td>1.92 ± 0.31</td></tr><tr><td>3</td><td>15.00</td><td>677</td><td>0.32</td><td>0.44</td><td>0.14</td><td>45.09</td><td>3.74 ± 0.35</td></tr><tr><td>6</td><td>15.00</td><td>424</td><td>0.55</td><td>0.44</td><td>3.48</td><td>47.86</td><td>2.57 ± 0.15</td></tr><tr><td>10</td><td>15.00</td><td>228</td><td>0.32</td><td>0.39</td><td>6.76</td><td>36.82</td><td>2.27 ± 0.95</td></tr><tr><td rowspan="4">B (BP)</td><td>0.5</td><td>15.00</td><td>974</td><td>0.26</td><td>0.29</td><td>12.38</td><td>40.80</td><td>1.00 ± 0.38</td></tr><tr><td>5</td><td>15.00</td><td>659</td><td>0.22</td><td>0.26</td><td>14.00</td><td>31.78</td><td>1.34 ± 0.30</td></tr><tr><td>15</td><td>15.00</td><td>276</td><td>0.30</td><td>0.49</td><td>5.42</td><td>37.29</td><td>1.04 ± 0.42</td></tr><tr><td>30</td><td>15.00</td><td>75</td><td>0.27</td><td>0.26</td><td>11.75</td><td>36.62</td><td>1.13 ± 0.00</td></tr><tr><td rowspan="4">S4 (BP)</td><td>0.5</td><td>15.00</td><td>921</td><td>0.32</td><td>0.14</td><td>7.93</td><td>61.57</td><td>1.13 ± 0.23</td></tr><tr><td>5</td><td>15.00</td><td>635</td><td>0.32</td><td>0.08</td><td>13.24</td><td>55.13</td><td>0.60 ± 0.13</td></tr><tr><td>16</td><td>15.00</td><td>277</td><td>0.40</td><td>0.07</td><td>28.70</td><td>67.54</td><td>0.41 ± 0.27</td></tr><tr><td>30</td><td>15.00</td><td>80</td><td>0.51</td><td>0.12</td><td>32.36</td><td>60.69</td><td>0.24 ± 0.01</td></tr><tr><td rowspan="4">End (BP)</td><td>0.5</td><td>15.00</td><td>1060</td><td>0.40</td><td>0.21</td><td>7.64</td><td>68.86</td><td>0.30 ± 0.02</td></tr><tr><td>5</td><td>15.00</td><td>699</td><td>0.30</td><td>0.18</td><td>11.09</td><td>80.36</td><td>0.29 ± 0.07</td></tr><tr><td>10</td><td>15.00</td><td>441</td><td>0.30</td><td>0.13</td><td>14.18</td><td>80.63</td><td>0.20 ± 0.02</td></tr><tr><td>15</td><td>15.00</td><td>278</td><td>0.36</td><td>0.12</td><td>19.14</td><td>83.28</td><td>0.35 ± 0.20</td></tr><tr><td>Average ± SE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Depth 1</td><td></td><td></td><td></td><td>0.71 ± 0.23</td><td>0.34 ± 0.11</td><td>15.2 ± 5.1</td><td>40.0 ± 8.7</td><td>1.01 ± 0.25</td></tr><tr><td>Depth 2</td><td></td><td></td><td></td><td>0.67 ± 0.33</td><td>0.27 ± 0.07</td><td>17.0 ± 6.2</td><td>41.3 ± 7.4</td><td>1.25 ± 0.47</td></tr><tr><td>Depth 3</td><td></td><td></td><td></td><td>0.88 ± 0.41</td><td>0.32 ± 0.09</td><td>14.1 ± 4.5</td><td>53.4 ± 6.9</td><td>0.87 ± 0.32</td></tr><tr><td>Depth 4</td><td></td><td></td><td></td><td>1.07 ± 0.36</td><td>0.25 ± 0.04</td><td>17.6 ± 4.7</td><td>52.8 ± 7.5</td><td>0.73 ± 0.26</td></tr><tr><td>BV</td><td></td><td></td><td></td><td>1.32 ± 0.28</td><td>0.34 ± 0.07</td><td>19.9 ± 4.3</td><td>40.7 ± 5.5</td><td>0.77 ± 0.21</td></tr><tr><td>BP</td><td></td><td></td><td></td><td>0.34 ± 0.02</td><td>0.25 ± 0.04</td><td>12.1 ± 2.2</td><td>53.1 ± 4.9</td><td>1.16 ± 0.25</td></tr></tbody></table>

light penetration were performed with a spherical sensor (LI-193SA, LI-COR Biosciences) connected to a LI-1400 data-logger. Seawater was collected at 4 depths within the photic layer at each station (Table 1) using single 12 l Niskin bottles. Dissolved inorganic nutrients were measured using an automated colorimetric method and chlorophyll a (chl a) was measured by HPLC. In order to convert chl a to biomass (total autotrophic biomass), we used the ratio of carbon/chl a = 120 (Marañón 2005). The *Prochlorococcus* spp., and *Synnechococcus* spp. biomass were estimated following the methodology and conversion factors described in González et al. (2003). The percent of *Prochlorococcus* spp. and *Synnechococcus* spp. to total autotrophic biomass (% cyanobacteria) was estimated as the ratio of *Prochlorococcus* spp. and *Synnechococcus* spp. biomass to total autotrophic biomass. Samples for measurement of PP and respiration were incubated *in situ* on a vertical line from sunrise to sunset or for 24 h. The processes measured are summarized in Table 2.

**¹⁴C technique.** The incorporation of carbon into the dissolved and particulate fractions was measured using the ¹⁴C technique (Steeman Nielsen 1952). Water samples (30 ml) were transferred to transparent glass bottles at each station and depth. The samples were incubated from sunrise to sunset in 1 dark
<table><caption>Table 1. Summary of selected variables within photic layer at stations sampled at Bay of Villefranche (BV) and Bay of Palma (BP). Also shown are averages ± SE of each vertical depth, BV and BP. %cyano: % cyanobacteria; PER: percent of extracellular release TOC: total organic carbon; Pos: *Posidonia*; B: Bahía; S4: Stn 4; End: Enderrocat</caption><thead><tr><th>Location</th><th>Depth (m)</th><th>Sunrise-sunset (h)</th><th>Irradiance (µE m<sup>-2</sup> s<sup>-1</sup>)</th><th>NO<sub>3</sub> (µmol l<sup>-1</sup>)</th><th>Chl a (µgl<sup>-1</sup>)</th><th>%cyano (%)</th><th>PER (%)</th><th>TOC ± SE (µmol l<sup>-1</sup> d<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="4">Oct02 (BV)</td><td>0.5</td><td>11.43</td><td>1287</td><td>0.47</td><td>0.15</td><td>43.86</td><td>69.21</td><td>0.56 ± 0.07</td></tr><tr><td>10</td><td>11.43</td><td>300</td><td>0.24</td><td>0.15</td><td>41.39</td><td>50.41</td><td>0.32 ± 0.07</td></tr><tr><td>20</td><td>11.43</td><td>153</td><td>0.75</td><td>0.15</td><td>17.13</td><td>65.68</td><td>0.40 ± 0.03</td></tr><tr><td>30</td><td>11.43</td><td>110</td><td>2.21</td><td>0.36</td><td>28.03</td><td>37.82</td><td>0.21 ± 0.11</td></tr><tr><td rowspan="4">Dec02 (BV)</td><td>0.5</td><td>9.09</td><td>739</td><td>1.14</td><td>0.31</td><td>13.8</td><td>5.01</td><td>0.25 ± 0.02</td></tr><tr><td>10</td><td>9.09</td><td>219</td><td>0.69</td><td>0.29</td><td>6.66</td><td>19.46</td><td>0.20 ± 0.03</td></tr><tr><td>20</td><td>9.09</td><td>69</td><td>0.44</td><td>0.29</td><td>5.69</td><td>19.37</td><td>0.13 ± 0.01</td></tr><tr><td>30</td><td>9.09</td><td>20</td><td>0.68</td><td>0.17</td><td>4.45</td><td>37.74</td><td>0.05 ± 0.01</td></tr><tr><td rowspan="4">Mar03 (BV)</td><td>0.5</td><td>11.21</td><td>374</td><td>2.16</td><td>1.09</td><td>1.39</td><td>37.25</td><td>2.19 ± 0.20</td></tr><tr><td>10</td><td>11.21</td><td>215</td><td>2.95</td><td>0.66</td><td>2.14</td><td>31.20</td><td>2.86 ± 0.09</td></tr><tr><td>20</td><td>11.21</td><td>100</td><td>3.75</td><td>0.84</td><td>1.15</td><td>64.26</td><td>1.88 ± 0.07</td></tr><tr><td>30</td><td>11.21</td><td>48</td><td>3.01</td><td>0.40</td><td>2.21</td><td>86.59</td><td>1.03 ± 0.31</td></tr><tr><td rowspan="4">Jul03 (BV)</td><td>0.5</td><td>15.21</td><td>1598</td><td>0.64</td><td>0.13</td><td>30.13</td><td>21.41</td><td>0.71 ± 0.04</td></tr><tr><td>10</td><td>15.21</td><td>593</td><td>0.30</td><td>0.12</td><td>47.40</td><td>16.96</td><td>0.65 ± 0.10</td></tr><tr><td>20</td><td>15.21</td><td>288</td><td>0.54</td><td>0.11</td><td>36.71</td><td>44.76</td><td>0.33 ± 0.04</td></tr><tr><td>30</td><td>15.21</td><td>221</td><td>1.20</td><td>0.21</td><td>35.80</td><td>43.19</td><td>0.58 ± 0.03</td></tr><tr><td rowspan="4">Pos (BP)</td><td>0.5</td><td>15.00</td><td>998</td><td>0.30</td><td>0.42</td><td>4.86</td><td>16.44</td><td>1.92 ± 0.31</td></tr><tr><td>3</td><td>15.00</td><td>677</td><td>0.32</td><td>0.44</td><td>0.14</td><td>45.09</td><td>3.74 ± 0.35</td></tr><tr><td>6</td><td>15.00</td><td>424</td><td>0.55</td><td>0.44</td><td>3.48</td><td>47.86</td><td>2.57 ± 0.15</td></tr><tr><td>10</td><td>15.00</td><td>228</td><td>0.32</td><td>0.39</td><td>6.76</td><td>36.82</td><td>2.27 ± 0.95</td></tr><tr><td rowspan="4">B (BP)</td><td>0.5</td><td>15.00</td><td>974</td><td>0.26</td><td>0.29</td><td>12.38</td><td>40.80</td><td>1.00 ± 0.38</td></tr><tr><td>5</td><td>15.00</td><td>659</td><td>0.22</td><td>0.26</td><td>14.00</td><td>31.78</td><td>1.34 ± 0.30</td></tr><tr><td>15</td><td>15.00</td><td>276</td><td>0.30</td><td>0.49</td><td>5.42</td><td>37.29</td><td>1.04 ± 0.42</td></tr><tr><td>30</td><td>15.00</td><td>75</td><td>0.27</td><td>0.26</td><td>11.75</td><td>36.62</td><td>1.13 ± 0.00</td></tr><tr><td rowspan="4">S4 (BP)</td><td>0.5</td><td>15.00</td><td>921</td><td>0.32</td><td>0.14</td><td>7.93</td><td>61.57</td><td>1.13 ± 0.23</td></tr><tr><td>5</td><td>15.00</td><td>635</td><td>0.32</td><td>0.08</td><td>13.24</td><td>55.13</td><td>0.60 ± 0.13</td></tr><tr><td>16</td><td>15.00</td><td>277</td><td>0.40</td><td>0.07</td><td>28.70</td><td>67.54</td><td>0.41 ± 0.27</td></tr><tr><td>30</td><td>15.00</td><td>80</td><td>0.51</td><td>0.12</td><td>32.36</td><td>60.69</td><td>0.24 ± 0.01</td></tr><tr><td rowspan="4">End (BP)</td><td>0.5</td><td>15.00</td><td>1060</td><td>0.40</td><td>0.21</td><td>7.64</td><td>68.86</td><td>0.30 ± 0.02</td></tr><tr><td>5</td><td>15.00</td><td>699</td><td>0.30</td><td>0.18</td><td>11.09</td><td>80.36</td><td>0.29 ± 0.07</td></tr><tr><td>10</td><td>15.00</td><td>441</td><td>0.30</td><td>0.13</td><td>14.18</td><td>80.63</td><td>0.20 ± 0.02</td></tr><tr><td>15</td><td>15.00</td><td>278</td><td>0.36</td><td>0.12</td><td>19.14</td><td>83.28</td><td>0.35 ± 0.20</td></tr><tr><td>Average ± SE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Depth 1</td><td></td><td></td><td></td><td>0.71 ± 0.23</td><td>0.34 ± 0.11</td><td>15.2 ± 5.1</td><td>40.0 ± 8.7</td><td>1.01 ± 0.25</td></tr><tr><td>Depth 2</td><td></td><td></td><td></td><td>0.67 ± 0.33</td><td>0.27 ± 0.07</td><td>17.0 ± 6.2</td><td>41.3 ± 7.4</td><td>1.25 ± 0.47</td></tr><tr><td>Depth 3</td><td></td><td></td><td></td><td>0.88 ± 0.41</td><td>0.32 ± 0.09</td><td>14.1 ± 4.5</td><td>53.4 ± 6.9</td><td>0.87 ± 0.32</td></tr><tr><td>Depth 4</td><td></td><td></td><td></td><td>1.07 ± 0.36</td><td>0.25 ± 0.04</td><td>17.6 ± 4.7</td><td>52.8 ± 7.5</td><td>0.73 ± 0.26</td></tr><tr><td>BV</td><td></td><td></td><td></td><td>1.32 ± 0.28</td><td>0.34 ± 0.07</td><td>19.9 ± 4.3</td><td>40.7 ± 5.5</td><td>0.77 ± 0.21</td></tr><tr><td>BP</td><td></td><td></td><td></td><td>0.34 ± 0.02</td><td>0.25 ± 0.04</td><td>12.1 ± 2.2</td><td>53.1 ± 4.9</td><td>1.16 ± 0.25</td></tr></tbody></table>

light penetration were performed with a spherical sensor (LI-193SA, LI-COR Biosciences) connected to a LI-1400 data-logger. Seawater was collected at 4 depths within the photic layer at each station (Table 1) using single 12 l Niskin bottles. Dissolved inorganic nutrients were measured using an automated colorimetric method and chlorophyll a (chl a) was measured by HPLC. In order to convert chl a to biomass (total autotrophic biomass), we used the ratio of carbon/chl a = 120 (Marañón 2005). The *Prochlorococcus* spp., and *Synnechococcus* spp. biomass were estimated following the methodology and conversion factors described in González et al. (2003). The percent of *Prochlorococcus* spp. and *Synnechococcus* spp. to total autotrophic biomass (% cyanobacteria) was estimated as the ratio of *Prochlorococcus* spp. and *Synnechococcus* spp. biomass to total autotrophic biomass. Samples for measurement of PP and respiration were incubated *in situ* on a vertical line from sunrise to sunset or for 24 h. The processes measured are summarized in Table 2.

**$^{14}$C technique.** The incorporation of carbon into the dissolved and particulate fractions was measured using the $^{14}$C technique (Steeman Nielsen 1952). Water samples (30 ml) were transferred to transparent glass bottles at each station and depth. The samples were incubated from sunrise to sunset in 1 dark
<table><caption>Table 2. Summary of different processes and how they were determined</caption><thead><tr><th>Acronym</th><th>Definition</th><th>Comments</th></tr></thead><tbody><tr><td>GPP-<sup>18</sup>O</td><td>Photosynthetic release of <sup>18</sup>O from H<sub>2</sub> <sup>18</sup>O during daytime</td><td>There is no <sup>18</sup>O release at night-time</td></tr><tr><td>NCP<sub>12h</sub></td><td>Change in O<sub>2</sub> during daytime</td><td>GPP less community respiration during daytime: GPP – CR<sub>light</sub></td></tr><tr><td>NCP</td><td>Change in O<sub>2</sub> over 24 h</td><td>GPP less community respiration over 24 h: GPP – CR</td></tr><tr><td>CR</td><td>O<sub>2</sub> decrease in the dark bottles during 24 hours</td><td>Community respiration over 24 h in the dark. It is assumed that light and dark respirations are equal</td></tr><tr><td>CR<sub>light</sub></td><td>GPP-<sup>18</sup>O<sub>2</sub> – NCP<sub>12h</sub></td><td>Community respiration during daytime</td></tr><tr><td>CR<sub>dark</sub></td><td>NCP<sub>12h</sub> – NCP<sub>24h</sub></td><td>Community respiration during night-time</td></tr><tr><td>GPP-O<sub>2</sub></td><td>CR + NCP</td><td>GPP, assuming that light and dark respiration are equal</td></tr><tr><td>GPP<sub>light</sub></td><td>CR<sub>light</sub> + CR<sub>dark</sub> + NCP</td><td>GPP estimates take into account light respiration</td></tr><tr><td>DO<sup>14</sup>C</td><td>Photosynthetic production of dissolved organic <sup>14</sup>Carbon</td><td>From sunrise to sunset</td></tr><tr><td>PO<sup>14</sup>C</td><td>Photosynthetic production of particulate organic <sup>14</sup>Carbon</td><td>From sunrise to sunset</td></tr><tr><td>TO<sup>14</sup>C</td><td>Photosynthetic production of total organic <sup>14</sup>Carbon (DO<sup>14</sup>C + PO<sup>14</sup>C)</td><td>NCP ≤ TO<sup>14</sup>C ≤ GPP</td></tr></tbody></table>

and 3 transparent biological oxygen demand bottles, after the addition of 30 µCi (1110 kBq) of sodium bicarbonate (NaH<sup>14</sup>CO<sub>3</sub>). Upon completion of the incubation, two 5 ml aliquots of each sample were placed into 20 ml scintillation vials for determination of total labeled organic carbon (TO<sup>14</sup>C). Two more 5 ml aliquots of each sample were filtered over 0.22 µm of membrane filter for determination of total particulate organic carbon (PO<sup>14</sup>C) and the filtrate collected for determination of total dissolved organic carbon (DO<sup>14</sup>C). Filters were placed into vials and exposed to concentrated hydrochloric acid fumes for a minimum of 12 h before the addition of 3.5 ml of Ultima Gold liquid scintillation cocktail (Packard Instruments). Liquid samples (TO<sup>14</sup>C and DO<sup>14</sup>C) were acidified with 100 µl of hydrochloric acid (5 N) and bubbled with air for 2 h before adding 15 ml of Ultima Gold XR scintillation cocktail. Radioactivity (dpm) was measured in a Tri-Carb 4000 scintillation counter (Packard Instruments). Counts from the dark bottles were subtracted from counts measured in the light bottles to correct for non-photosynthetic <sup>14</sup>C-incorporation.

**GPP-<sup>18</sup>O technique.** Gross oxygen production was determined by spiking seawater with H<sub>2</sub> <sup>18</sup>O and measuring the quantity of <sup>18</sup>O produced during the incubation. At each water depth, 8 headspace vials of 27 ml were filled carefully to prevent trapping of air bubbles. Four vials were sealed immediately, fixed with saturated mercuric chloride (6.5%) and stored upside down in the dark pending the isotope analysis. The other 4 vials were spiked with H<sub>2</sub> <sup>18</sup>O to a final isotopic composition (δ<sup>18</sup>O-H<sub>2</sub>O) of 300 to 500‰. This isotopic composition is defined as:

$$ \delta^{18}\text{O} = \left[ \frac{(^{18}\text{O}/^{16}\text{O})_{\text{sample}}}{(^{18}\text{O}/^{16}\text{O})_{\text{std}}} - 1 \right] \cdot 1000 $$

where std corresponds to V-SMOW (Vienna Standard Mean Ocean Water).

Samples were deployed *in situ* from sunrise to sunset alongside the samples used for measurement of <sup>14</sup>C uptake and fixed with saturated mercuric chloride upon completion of the incubation. In the laboratory, 500 µl of headspace was created with helium and allowed to equilibrate for 24 h (<sup>18</sup>O-O<sub>2</sub> measurements). The extracted water was injected into helium-flushed vials (<sup>18</sup>O-H<sub>2</sub>O). Pure CO<sub>2</sub> (100 µl) was then added and samples were allowed to equilibrate for 24 h. δ<sup>18</sup>O-H<sub>2</sub>O was therefore measured as δ<sup>18</sup>O-CO<sub>2</sub>. Determinations of δ<sup>18</sup>O-O<sub>2</sub> and δ<sup>18</sup>O-CO<sub>2</sub> were accomplished using an elemental analyzer interfaced Finnigan Deltaplus Isotope Ratio Mass Spectrometer. An overflow technique was used to limit air contamination of the needle.

Gross oxygen production (GPP-<sup>18</sup>O) was calculated from the isotopic composition of the dissolved oxygen in the initial and incubated samples following the equation of Dickson et al. (2001). Standard deviation is based on propagation of error assuming that the errors of the measurements were independent.

**GPP-O<sub>2</sub> technique.** For estimating production and consumption of dissolved oxygen, seawater samples (20 replicates) were transferred into dark and transparent BOD bottles (60 ml) and incubated at sunrise *in situ* alongside the samples used with the GPP-<sup>18</sup>O and <sup>14</sup>C techniques. At each station and depth, 5 bottles were immediately fixed (time 0) with Winkler reagents (Knap et al. 1996), 5 dark bottles were fixed after 24 h, 5 transparent bottles were fixed at sunset (about 12 h incubation).
<table><caption>Table 2. Summary of different processes and how they were determined</caption><thead><tr><th>Acronym</th><th>Definition</th><th>Comments</th></tr></thead><tbody><tr><td>GPP-<sup>18</sup>O</td><td>Photosynthetic release of <sup>18</sup>O from H<sub>2</sub><sup>18</sup>O during daytime</td><td>There is no <sup>18</sup>O release at night-time</td></tr><tr><td>NCP<sub>12h</sub></td><td>Change in O<sub>2</sub> during daytime</td><td>GPP less community respiration during daytime: GPP – CR<sub>light</sub></td></tr><tr><td>NCP</td><td>Change in O<sub>2</sub> over 24 h</td><td>GPP less community respiration over 24 h: GPP – CR</td></tr><tr><td>CR</td><td>O<sub>2</sub> decrease in the dark bottles during 24 hours</td><td>Community respiration over 24 h in the dark. It is assumed that light and dark respirations are equal</td></tr><tr><td>CR<sub>light</sub></td><td>GPP-<sup>18</sup>O<sub>2</sub> – NCP<sub>12h</sub></td><td>Community respiration during daytime</td></tr><tr><td>CR<sub>dark</sub></td><td>NCP<sub>12h</sub> – NCP<sub>24h</sub></td><td>Community respiration during night-time</td></tr><tr><td>GPP-O<sub>2</sub></td><td>CR + NCP</td><td>GPP, assuming that light and dark respiration are equal</td></tr><tr><td>GPP<sub>light</sub></td><td>CR<sub>light</sub> + CR<sub>dark</sub> + NCP</td><td>GPP estimates take into account light respiration</td></tr><tr><td>DO<sup>14</sup>C</td><td>Photosynthetic production of dissolved organic <sup>14</sup>Carbon</td><td>From sunrise to sunset</td></tr><tr><td>PO<sup>14</sup>C</td><td>Photosynthetic production of particulate organic <sup>14</sup>Carbon</td><td>From sunrise to sunset</td></tr><tr><td>TO<sup>14</sup>C</td><td>Photosynthetic production of total organic <sup>14</sup>Carbon (DO<sup>14</sup>C + PO<sup>14</sup>C)</td><td>NCP ≤ TO<sup>14</sup>C ≤ GPP</td></tr></tbody></table>

and 3 transparent biological oxygen demand bottles, after the addition of 30 µCi (1110 kBq) of sodium bicarbonate (NaH<sup>14</sup>CO<sub>3</sub>). Upon completion of the incubation, two 5 ml aliquots of each sample were placed into 20 ml scintillation vials for determination of total labeled organic carbon (TO<sup>14</sup>C). Two more 5 ml aliquots of each sample were filtered over 0.22 µm of membrane filter for determination of total particulate organic carbon (PO<sup>14</sup>C) and the filtrate collected for determination of total dissolved organic carbon (DO<sup>14</sup>C). Filters were placed into vials and exposed to concentrated hydrochloric acid fumes for a minimum of 12 h before the addition of 3.5 ml of Ultima Gold liquid scintillation cocktail (Packard Instruments). Liquid samples (TO<sup>14</sup>C and DO<sup>14</sup>C) were acidified with 100 µl of hydrochloric acid (5 N) and bubbled with air for 2 h before adding 15 ml of Ultima Gold XR scintillation cocktail. Radioactivity (dpm) was measured in a Tri-Carb 4000 scintillation counter (Packard Instruments). Counts from the dark bottles were subtracted from counts measured in the light bottles to correct for non-photosynthetic <sup>14</sup>C-incorporation.

**GPP-<sup>18</sup>O technique.** Gross oxygen production was determined by spiking seawater with H<sub>2</sub><sup>18</sup>O and measuring the quantity of <sup>18</sup>O produced during the incubation. At each water depth, 8 headspace vials of 27 ml were filled carefully to prevent trapping of air bubbles. Four vials were sealed immediately, fixed with saturated mercuric chloride (6.5%) and stored upside down in the dark pending the isotope analysis. The other 4 vials were spiked with H<sub>2</sub><sup>18</sup>O to a final isotopic composition (δ<sup>18</sup>O-H<sub>2</sub>O) of 300 to 500‰. This isotopic composition is defined as:

$$ \delta^{18}\text{O} = \left[ \frac{(^{18}\text{O}/^{16}\text{O})_{\text{sample}}}{(^{18}\text{O}/^{16}\text{O})_{\text{std}}} - 1 \right] \cdot 1000 $$

where std corresponds to V-SMOW (Vienna Standard Mean Ocean Water).

Samples were deployed *in situ* from sunrise to sunset alongside the samples used for measurement of <sup>14</sup>C uptake and fixed with saturated mercuric chloride upon completion of the incubation. In the laboratory, 500 µl of headspace was created with helium and allowed to equilibrate for 24 h (<sup>18</sup>O-O<sub>2</sub> measurements). The extracted water was injected into helium-flushed vials (<sup>18</sup>O-H<sub>2</sub>O). Pure CO<sub>2</sub> (100 µl) was then added and samples were allowed to equilibrate for 24 h. δ<sup>18</sup>O-H<sub>2</sub>O was therefore measured as δ<sup>18</sup>O-CO<sub>2</sub>. Determinations of δ<sup>18</sup>O-O<sub>2</sub> and δ<sup>18</sup>O-CO<sub>2</sub> were accomplished using an elemental analyzer interfaced Finnigan Deltaplus Isotope Ratio Mass Spectrometer. An overflow technique was used to limit air contamination of the needle.

Gross oxygen production (GPP-<sup>18</sup>O) was calculated from the isotopic composition of the dissolved oxygen in the initial and incubated samples following the equation of Dickson et al. (2001). Standard deviation is based on propagation of error assuming that the errors of the measurements were independent.

**GPP-O<sub>2</sub> technique.** For estimating production and consumption of dissolved oxygen, seawater samples (20 replicates) were transferred into dark and transparent BOD bottles (60 ml) and incubated at sunrise *in situ* alongside the samples used with the GPP-<sup>18</sup>O and <sup>14</sup>C techniques. At each station and depth, 5 bottles were immediately fixed (time 0) with Winkler reagents (Knap et al. 1996), 5 dark bottles were fixed after 24 h, 5 transparent bottles were fixed at sunset (about 12 h incubation).
tion) and the remaining 5 bottles at the following sunrise (24 h incubation). In the BP, 10 extra dark bottles at each depth and station were incubated and fixed after 12 and 48 h, to compare the effect of the incubation time on the respiration rates. The dissolved oxygen concentration was determined by the Winkler titration technique with potentiometric end-point detection. Analyses were performed with a redox electrode (9778-SC, Orion) and a custom built titrator and standardizations were similar to those described by (Knap et al. 1996). The planktonic community respiration (CR) and net community production during the light period (NCP<sub>12h</sub>) and 24 h (NCP) were respectively estimated by regressing O<sub>2</sub> in the dark and transparent bottles against time. Daily GPP-O<sub>2</sub> was estimated as the difference between NCP and CR (Table 2). We obtained a pooled variation coefficient of 0.19 ± 0.03% (mean ± SE, n = 176) for NCP and CR. Dark community respiration during night time (CR<sub>dark</sub>) was estimated as the difference between NCP<sub>12h</sub> and NCP (Table 2). Community respiration in the light (CR<sub>light</sub>) was estimated as the difference between GPP-<sup>18</sup>O and NCP<sub>12h</sub>. This avoids the use of CR estimated in bottles incubated for 24 h in the dark, which assumes that dark respiration is equivalent to light respiration. Consequently, we could distinguish between GPP-O<sub>2</sub> and GPP<sub>light</sub>, which takes into account light respiration (Table 2). We filtered the data for occasions when CR<sub>dark</sub> and CR<sub>light</sub> values were anomalously negative. These values are considered impossible when the data are less

than twice its standard error (Williams 2000). However, we do not have standard errors for CR<sub>light</sub> and CR<sub>dark</sub>. Therefore, we compared NCP<sub>12h</sub> and NCP and GPP-<sup>18</sup>O and NCP<sub>12h</sub>, respectively, using paired t-tests. A few pairs that were significantly different and gave negative values of CR<sub>dark</sub> and CR<sub>light</sub>, were then excluded from the analyses. In order to compare PP in oxygen and carbon units, molar flux ratios (ΔO<sub>2</sub>/ΔCO<sub>2</sub>, apparent PQ) were estimated (Williams et al. 1983) as the ratio between GPP-O<sub>2</sub> or GPP-<sup>18</sup>O (µmol O<sub>2</sub> l<sup>-1</sup> h<sup>-1</sup>) and TO<sup>14</sup>C (µmol C l<sup>-1</sup> h<sup>-1</sup>).

**Statistical analysis.** A series of random complete block designs (RCB; Quinn & Keough 2002) were performed to evaluate differences among depths and between bays, taking the different stations within the BP and BV as blocks. When the sphericity was not met we used the Greenhouse-Geisser adjustment (Quinn & Keough 2002). Variance homogeneity was tested with Cochran's analysis.

We used reduced major axis regressions (RMA, Model II regression) because it is more appropriate than standard ordinary least squares regression (OLS, Model I regression) when both variables are subject to measurement error (Sokal & Rohlf 1995). However, results obtained with both analyses are reported, in order to enable comparison with previous studies. The regression lines were compared using Student's t-tests (Zar 1999) because the analysis of covariance assumes a Model I regression. Data are reported as mean ± standard error of the mean (SE).

## RESULTS

### Irradiance, nutrients and chlorophyll

The profiles of irradiance were measured at approximately mid-day at each station (Table 1). Obviously, the highest irradiances were measured at surface and during summer. The highest nitrate (NO<sub>3</sub>) values were observed during the most productive time, in March 2003 (see Table 1). NO<sub>3</sub> concentration did not show any significant vertical variability but tended to be higher at the BV (Table 3) because all stations in the BP were measured during summer (June 2002). Concentrations of chl a were below 1.1 µg l<sup>-1</sup>, and did not exhibit any vertical variability or difference across the study sites (Table 3).

<table><caption>Table 3. Random complete block design (RCB). Bay of Villefranche (BV) and Bay of Palma (BP)</caption><thead><tr><th>Parameter</th><th>Source</th><th>df</th><th>F-statistic</th><th>p-value</th><th>A posteriori<br>comparison (Simple)</th></tr></thead><tbody><tr><td rowspan="3">Nitrate (log)</td><td>Bay</td><td>1</td><td>5.01</td><td>0.06</td><td rowspan="12"></td></tr><tr><td>Depth</td><td>3</td><td>1.98</td><td>0.15</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.43</td><td>0.73</td></tr><tr><td rowspan="3">Chl a</td><td>Bay</td><td>1</td><td>0.30</td><td>0.59</td></tr><tr><td>Depth</td><td>3</td><td>0.77</td><td>0.52</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.24</td><td>0.86</td></tr><tr><td rowspan="3">TO<sup>14</sup>C</td><td>Bay</td><td>1</td><td>0.33</td><td>0.58</td></tr><tr><td>Depth</td><td>3</td><td>2.3</td><td>0.10</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.3</td><td>0.80</td></tr><tr><td rowspan="3">NCP<sup>a</sup></td><td>Bay</td><td>1</td><td>0.64</td><td>0.45</td></tr><tr><td>Depth</td><td>1.2</td><td>1.04</td><td>0.35</td></tr><tr><td>Bay × Depth</td><td>1.2</td><td>0.33</td><td>0.61</td></tr><tr><td rowspan="3">CR</td><td>Bay</td><td>1</td><td>39.3</td><td>0.001</td><td rowspan="3">BV&lt;BP</td></tr><tr><td>Depth</td><td>3</td><td>0.32</td><td>0.80</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.43</td><td>0.73</td></tr><tr><td rowspan="3">GPP-O<sub>2</sub></td><td>Bay</td><td>1</td><td>2.32</td><td>0.17</td><td rowspan="3">1=2&gt;3=4<sup>b</sup></td></tr><tr><td>Depth</td><td>3</td><td>7.69</td><td>0.002</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.22</td><td>0.88</td></tr><tr><td rowspan="3">GPP-<sup>18</sup>O</td><td>Bay</td><td>1</td><td>5.05</td><td>0.06</td><td rowspan="3">1&gt;4<sup>b</sup></td></tr><tr><td>Depth</td><td>3</td><td>7.70</td><td>0.002</td></tr><tr><td>Bay × Depth</td><td>3</td><td>1.7</td><td>0.18</td></tr></tbody></table><p><sup>a</sup>Sphericity was not met so the Greenhouse-Geisser adjustment was used</p><p><sup>b</sup>The numbers correspond to each depth</p>
tion) and the remaining 5 bottles at the following sunrise (24 h incubation). In the BP, 10 extra dark bottles at each depth and station were incubated and fixed after 12 and 48 h, to compare the effect of the incubation time on the respiration rates. The dissolved oxygen concentration was determined by the Winkler titration technique with potentiometric end-point detection. Analyses were performed with a redox electrode (9778-SC, Orion) and a custom built titrator and standardizations were similar to those described by (Knap et al. 1996). The planktonic community respiration (CR) and net community production during the light period (NCP<sub>12h</sub>) and 24 h (NCP) were respectively estimated by regressing O<sub>2</sub> in the dark and transparent bottles against time. Daily GPP-O<sub>2</sub> was estimated as the difference between NCP and CR (Table 2). We obtained a pooled variation coefficient of 0.19 ± 0.03% (mean ± SE, n = 176) for NCP and CR. Dark community respiration during night time (CR<sub>dark</sub>) was estimated as the difference between NCP<sub>12h</sub> and NCP (Table 2). Community respiration in the light (CR<sub>light</sub>) was estimated as the difference between GPP-<sup>18</sup>O and NCP<sub>12h</sub>. This avoids the use of CR estimated in bottles incubated for 24 h in the dark, which assumes that dark respiration is equivalent to light respiration. Consequently, we could distinguish between GPP-O<sub>2</sub> and GPP<sub>light</sub>, which takes into account light respiration (Table 2). We filtered the data for occasions when CR<sub>dark</sub> and CR<sub>light</sub> values were anomalously negative. These values are considered impossible when the data are less

than twice its standard error (Williams 2000). However, we do not have standard errors for CR<sub>light</sub> and CR<sub>dark</sub>. Therefore, we compared NCP<sub>12h</sub> and NCP and GPP-<sup>18</sup>O and NCP<sub>12h</sub>, respectively, using paired t-tests. A few pairs that were significantly different and gave negative values of CR<sub>dark</sub> and CR<sub>light</sub>, were then excluded from the analyses. In order to compare PP in oxygen and carbon units, molar flux ratios (ΔO<sub>2</sub>/ΔCO<sub>2</sub>, apparent PQ) were estimated (Williams et al. 1983) as the ratio between GPP-O<sub>2</sub> or GPP-<sup>18</sup>O (µmol O<sub>2</sub> l<sup>-1</sup> h<sup>-1</sup>) and TO<sup>14</sup>C (µmol C l<sup>-1</sup> h<sup>-1</sup>).

**Statistical analysis.** A series of random complete block designs (RCB; Quinn & Keough 2002) were performed to evaluate differences among depths and between bays, taking the different stations within the BP and BV as blocks. When the sphericity was not met we used the Greenhouse-Geisser adjustment (Quinn & Keough 2002). Variance homogeneity was tested with Cochran's analysis.

We used reduced major axis regressions (RMA, Model II regression) because it is more appropriate than standard ordinary least squares regression (OLS, Model I regression) when both variables are subject to measurement error (Sokal & Rohlf 1995). However, results obtained with both analyses are reported, in order to enable comparison with previous studies. The regression lines were compared using Student's t-tests (Zar 1999) because the analysis of covariance assumes a Model I regression. Data are reported as mean ± standard error of the mean (SE).

## RESULTS

### Irradiance, nutrients and chlorophyll

The profiles of irradiance were measured at approximately mid-day at each station (Table 1). Obviously, the highest irradiances were measured at surface and during summer. The highest nitrate (NO<sub>3</sub>) values were observed during the most productive time, in March 2003 (see Table 1). NO<sub>3</sub> concentration did not show any significant vertical variability but tended to be higher at the BV (Table 3) because all stations in the BP were measured during summer (June 2002). Concentrations of chl a were below 1.1 µg l<sup>-1</sup>, and did not exhibit any vertical variability or difference across the study sites (Table 3).

<table><caption>Table 3. Random complete block design (RCB). Bay of Villefranche (BV) and Bay of Palma (BP)</caption><thead><tr><th>Parameter</th><th>Source</th><th>df</th><th>F-statistic</th><th>p-value</th><th>A posteriori<br>comparison (Simple)</th></tr></thead><tbody><tr><td rowspan="3">Nitrate (log)</td><td>Bay</td><td>1</td><td>5.01</td><td>0.06</td><td rowspan="12"></td></tr><tr><td>Depth</td><td>3</td><td>1.98</td><td>0.15</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.43</td><td>0.73</td></tr><tr><td rowspan="3">Chl a</td><td>Bay</td><td>1</td><td>0.30</td><td>0.59</td></tr><tr><td>Depth</td><td>3</td><td>0.77</td><td>0.52</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.24</td><td>0.86</td></tr><tr><td rowspan="3">TO<sup>14</sup>C</td><td>Bay</td><td>1</td><td>0.33</td><td>0.58</td></tr><tr><td>Depth</td><td>3</td><td>2.3</td><td>0.10</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.3</td><td>0.80</td></tr><tr><td rowspan="3">NCP<sup>a</sup></td><td>Bay</td><td>1</td><td>0.64</td><td>0.45</td></tr><tr><td>Depth</td><td>1.2</td><td>1.04</td><td>0.35</td></tr><tr><td>Bay × Depth</td><td>1.2</td><td>0.33</td><td>0.61</td></tr><tr><td rowspan="3">CR</td><td>Bay</td><td>1</td><td>39.3</td><td>0.001</td><td rowspan="3">BV&lt;BP</td></tr><tr><td>Depth</td><td>3</td><td>0.32</td><td>0.80</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.43</td><td>0.73</td></tr><tr><td rowspan="3">GPP-O<sub>2</sub></td><td>Bay</td><td>1</td><td>2.32</td><td>0.17</td><td rowspan="3">1=2&gt;3=4<sup>b</sup></td></tr><tr><td>Depth</td><td>3</td><td>7.69</td><td>0.002</td></tr><tr><td>Bay × Depth</td><td>3</td><td>0.22</td><td>0.88</td></tr><tr><td rowspan="3">GPP-<sup>18</sup>O</td><td>Bay</td><td>1</td><td>5.05</td><td>0.06</td><td rowspan="3">1&gt;4<sup>b</sup></td></tr><tr><td>Depth</td><td>3</td><td>7.70</td><td>0.002</td></tr><tr><td>Bay × Depth</td><td>3</td><td>1.7</td><td>0.18</td></tr></tbody></table><p><sup>a</sup>Sphericity was not met so the Greenhouse-Geisser adjustment was used</p><p><sup>b</sup>The numbers correspond to each depth</p>
Carbon fixation, oxygen production and
consumption

TO<sup>14</sup>C production ranged from 0.05 to 3.74 µmol l<sup>-1</sup>
d<sup>-1</sup> (Table 1). The highest TO<sup>14</sup>C values were observed
in March at Point B station whereas the *Posidonia* sta-
tion in summer was the most productive of the stations
studied in the BP. TO<sup>14</sup>C did not show a difference
across study sites (Table 3) but showed a marginally
vertical variability (i.e. a decrease) as a function of
depth (Table 1). We observed a wide range of variabil-
ity in the relative contribution of DOC production to

total PP (% DOC) with an average of 40 and 54%
(Table 1) for the BV and BP, respectively.

Estimates of NCP and NCP<sub>12h</sub> ranged from -6.01 to
5.25 and -3.99 to 6.39 µmol l<sup>-1</sup> d<sup>-1</sup>, respectively. Esti-
mates of NCP (µmol l<sup>-1</sup>h<sup>-1</sup>) were significantly higher
than NCP<sub>12h</sub> (t-test, t<sub>31</sub> = 4.5, n = 32, p < 0.0001). NCP
showed a tendency to decrease with depth (Table 4)
but this was not significant (Table 3). Differences be-
tween areas in NCP were not significant (Table 3). Es-
timates of CR ranged from 0.25 to 7.0 µmol l<sup>-1</sup> d<sup>-1</sup> and
did not exhibit significant vertical variability but were
significantly higher in the BP than in the BV (Table 3).

<table><caption>Table 4. Summary of selected biological variables within the photic layer (4 depths, see Table 1) at the stations sampled in BV and BP. dt = daytime (from sunrise to sunset) nt = night-time; nd = no data. Also shown are averages (± SE) of each vertical depth, BV and BP. See Table 1 for location abbreviations</caption>
  <thead>
    <tr>
      <th>Location</th>
      <th>NCP<sub>12h</sub> ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>NCP ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>R ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>GPP-O<sub>2</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>GPP-<sup>18</sup>O ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>CR<sub>light</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> dt<sup>-1</sup>)</th>
      <th>CR<sub>dark</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> nt<sup>-1</sup>)</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td rowspan="4">Oct02</td>
      <td>-0.24 ± 0.12</td>
      <td>-0.83 ± 0.15</td>
      <td>2.64 ± 0.21</td>
      <td>1.82</td>
      <td>0.76 ± 0.12</td>
      <td>1.00</td>
      <td>0.58</td>
    </tr>
    <tr>
      <td>0.00 ± 0.06</td>
      <td>-0.47 ± 0.16</td>
      <td>1.84 ± 0.15</td>
      <td>1.37</td>
      <td>1.20 ± 0.33</td>
      <td>1.19</td>
      <td>0.47</td>
    </tr>
    <tr>
      <td>0.43 ± 0.14</td>
      <td>-0.34 ± 0.25</td>
      <td>0.89 ± 0.21</td>
      <td>0.55</td>
      <td>0.76 ± 0.21</td>
      <td>0.33</td>
      <td>0.77</td>
    </tr>
    <tr>
      <td>0.51 ± 0.21</td>
      <td>-0.16 ± 0.26</td>
      <td>0.49 ± 0.25</td>
      <td>0.32</td>
      <td>0.43 ± 0.17</td>
      <td>-0.08</td>
      <td>0.67</td>
    </tr>
    <tr>
      <td rowspan="4">Dec02</td>
      <td>1.02 ± 0.38</td>
      <td>1.38 ± 0.26</td>
      <td>0.76 ± 0.26</td>
      <td>2.14</td>
      <td>0.50 ± 0.06</td>
      <td>-0.52<sup>a</sup></td>
      <td>-0.36</td>
    </tr>
    <tr>
      <td>1.04 ± 0.39</td>
      <td>0.30 ± 0.35</td>
      <td>0.25 ± 0.26</td>
      <td>0.55</td>
      <td>0.33 ± 0.10</td>
      <td>-0.71<sup>a</sup></td>
      <td>0.74</td>
    </tr>
    <tr>
      <td>-0.27 ± 0.26</td>
      <td>-0.29 ± 0.31</td>
      <td>1.03 ± 0.30</td>
      <td>0.74</td>
      <td>0.12 ± 0.07</td>
      <td>0.39</td>
      <td>0.02</td>
    </tr>
    <tr>
      <td>nd nd</td>
      <td>1.01 ± 0.24</td>
      <td>0.40 ± 0.28</td>
      <td>1.41</td>
      <td>0.47 ± 0.09</td>
      <td>nd</td>
      <td>5.38</td>
    </tr>
    <tr>
      <td rowspan="4">Mar03</td>
      <td>5.07 ± 0.99</td>
      <td>5.25 ± 0.77</td>
      <td>0.61 ± 1.69</td>
      <td>5.86</td>
      <td>3.45 ± 0.38</td>
      <td>-1.62<sup>a</sup></td>
      <td>-0.18</td>
    </tr>
    <tr>
      <td>4.55 ± 0.40</td>
      <td>2.66 ± 0.55</td>
      <td>0.73 ± 0.30</td>
      <td>3.39</td>
      <td>4.39 ± 0.40</td>
      <td>-0.16</td>
      <td>1.89</td>
    </tr>
    <tr>
      <td>1.77 ± 0.92</td>
      <td>2.02 ± 0.77</td>
      <td>0.96 ± 0.81</td>
      <td>2.99</td>
      <td>1.57 ± 0.19</td>
      <td>-0.19<sup>a</sup></td>
      <td>-0.26</td>
    </tr>
    <tr>
      <td>-0.47 ± 0.41</td>
      <td>0.73 ± 0.45</td>
      <td>0.64 ± 0.25</td>
      <td>1.37</td>
      <td>0.37 ± 0.18</td>
      <td>0.84</td>
      <td>-1.20<sup>a</sup></td>
    </tr>
    <tr>
      <td rowspan="4">Jul03</td>
      <td>0.62 ± 0.48</td>
      <td>-0.31 ± 0.11</td>
      <td>1.38 ± 0.04</td>
      <td>1.07</td>
      <td>0.52 ± 0.73</td>
      <td>-0.10</td>
      <td>0.92</td>
    </tr>
    <tr>
      <td>0.35 ± 0.24</td>
      <td>-0.57 ± 0.18</td>
      <td>1.99 ± 0.22</td>
      <td>1.42</td>
      <td>1.50 ± 0.45</td>
      <td>1.15</td>
      <td>0.92</td>
    </tr>
    <tr>
      <td>-0.07 ± 0.24</td>
      <td>-0.26 ± 0.24</td>
      <td>0.28 ± 0.23</td>
      <td>0.02</td>
      <td>0.50 ± 0.13</td>
      <td>0.57</td>
      <td>0.19</td>
    </tr>
    <tr>
      <td>0.12 ± 0.26</td>
      <td>0.19 ± 0.30</td>
      <td>0.27 ± 0.27</td>
      <td>0.46</td>
      <td>0.45 ± 0.12</td>
      <td>0.32</td>
      <td>-0.07</td>
    </tr>
    <tr>
      <td rowspan="5">Pos</td>
      <td>3.82 ± 0.90</td>
      <td>0.38 ± 0.64</td>
      <td>4.44 ± 0.98</td>
      <td>4.83</td>
      <td>6.33 ± 0.69</td>
      <td>2.51</td>
      <td>3.44</td>
    </tr>
    <tr>
      <td>4.14 ± 0.67</td>
      <td>0.44 ± 1.08</td>
      <td>5.34 ± 0.51</td>
      <td>5.77</td>
      <td>5.55 ± 1.65</td>
      <td>1.41</td>
      <td>3.71</td>
    </tr>
    <tr>
      <td>3.35 ± 0.31</td>
      <td>0.84 ± 0.50</td>
      <td>4.97 ± 0.45</td>
      <td>5.81</td>
      <td>5.87 ± 0.62</td>
      <td>2.52</td>
      <td>2.51</td>
    </tr>
    <tr>
      <td>6.18 ± 0.48</td>
      <td>4.41 ± 0.77</td>
      <td>1.01 ± 0.77</td>
      <td>5.42</td>
      <td>5.03 ± 1.07</td>
      <td>-1.15<sup>a</sup></td>
      <td>1.77</td>
    </tr>
    <tr>
      <td>2.15 ± 0.43</td>
      <td>0.83 ± 0.68</td>
      <td>3.63 ± 0.41</td>
      <td>4.45</td>
      <td>5.43 ± 1.03</td>
      <td>3.29</td>
      <td>1.32</td>
    </tr>
    <tr>
      <td rowspan="3">B</td>
      <td>2.60 ± 0.56</td>
      <td>1.10 ± 0.89</td>
      <td>2.02 ± 0.48</td>
      <td>3.12</td>
      <td>4.88 ± 0.95</td>
      <td>2.28</td>
      <td>1.50</td>
    </tr>
    <tr>
      <td>1.49 ± 0.67</td>
      <td>-0.28 ± 1.08</td>
      <td>2.60 ± 0.73</td>
      <td>2.31</td>
      <td>3.47 ± 0.22</td>
      <td>1.98</td>
      <td>1.77</td>
    </tr>
    <tr>
      <td>0.45 ± 0.24</td>
      <td>0.41 ± 0.38</td>
      <td>1.68 ± 0.30</td>
      <td>2.08</td>
      <td>2.48 ± 0.31</td>
      <td>2.03</td>
      <td>0.04</td>
    </tr>
    <tr>
      <td rowspan="4">S4</td>
      <td>2.03 ± 0.49</td>
      <td>0.68 ± 0.79</td>
      <td>nd 0.98</td>
      <td>nd</td>
      <td>1.87 ± 0.57</td>
      <td>-0.16<sup>a</sup></td>
      <td>1.35</td>
    </tr>
    <tr>
      <td>1.32 ± 0.32</td>
      <td>0.90 ± 0.51</td>
      <td>1.65 ± 0.81</td>
      <td>2.55</td>
      <td>1.40 ± 0.33</td>
      <td>0.08</td>
      <td>0.42</td>
    </tr>
    <tr>
      <td>0.30 ± 0.41</td>
      <td>-0.94 ± 0.66</td>
      <td>1.50 ± 0.48</td>
      <td>0.55</td>
      <td>1.11 ± 0.27</td>
      <td>0.81</td>
      <td>1.24</td>
    </tr>
    <tr>
      <td>-1.67 ± 0.64</td>
      <td>-4.49 ± 1.02</td>
      <td>5.79 ± 0.30</td>
      <td>1.30</td>
      <td>0.48 ± 0.24</td>
      <td>2.14</td>
      <td>2.82</td>
    </tr>
    <tr>
      <td rowspan="4">End</td>
      <td>2.30 ± 0.27</td>
      <td>0.84 ± 0.16</td>
      <td>2.48 ± 0.41</td>
      <td>3.31</td>
      <td>4.67 ± 0.19</td>
      <td>2.37</td>
      <td>1.46</td>
    </tr>
    <tr>
      <td>0.72 ± 0.16</td>
      <td>-1.12 ± 0.25</td>
      <td>4.03 ± 0.22</td>
      <td>2.91</td>
      <td>2.52 ± 0.58</td>
      <td>1.80</td>
      <td>1.84</td>
    </tr>
    <tr>
      <td>1.56 ± 0.24</td>
      <td>-0.77 ± 0.38</td>
      <td>1.22 ± 0.25</td>
      <td>0.45</td>
      <td>2.25 ± 0.30</td>
      <td>0.69</td>
      <td>2.33</td>
    </tr>
    <tr>
      <td>-3.99 ± 0.28</td>
      <td>-6.01 ± 0.44</td>
      <td>7.10 ± 0.08</td>
      <td>1.09</td>
      <td>1.81 ± 0.24</td>
      <td>5.80</td>
      <td>2.02</td>
    </tr>
    <tr>
      <td colspan="8">Average ± SE</td>
    </tr>
    <tr>
      <td>Depth 1</td>
      <td>2.10 ± 0.61</td>
      <td>1.03 ± 0.65</td>
      <td>2.28 ± 1.45</td>
      <td>3.35 ± 1.77</td>
      <td>2.94 ± 0.83</td>
      <td>1.81 ± 0.60</td>
      <td>1.07 ± 0.42</td>
    </tr>
    <tr>
      <td>Depth 2</td>
      <td>1.84 ± 0.61</td>
      <td>0.40 ± 0.42</td>
      <td>2.23 ± 1.68</td>
      <td>2.64 ± 1.61</td>
      <td>2.72 ± 0.69</td>
      <td>1.11 ± 0.33</td>
      <td>1.44 ± 0.38</td>
    </tr>
    <tr>
      <td>Depth 3</td>
      <td>1.07 ± 0.43</td>
      <td>0.00 ± 0.34</td>
      <td>1.68 ± 1.49</td>
      <td>1.68 ± 1.96</td>
      <td>1.96 ± 0.67</td>
      <td>1.04 ± 0.32</td>
      <td>1.07 ± 0.38</td>
    </tr>
    <tr>
      <td>Depth 4</td>
      <td>0.94 ± 1.28</td>
      <td>-0.49 ± 1.16</td>
      <td>2.17 ± 2.70</td>
      <td>1.68 ± 1.61</td>
      <td>1.44 ± 0.58</td>
      <td>1.84 ± 0.87</td>
      <td>1.80 ± 0.72</td>
    </tr>
    <tr>
      <td>BV</td>
      <td>1.30 ± 0.53</td>
      <td>0.64 ± 0.39</td>
      <td>0.95 ± 0.17</td>
      <td>1.59 ± 0.37</td>
      <td>1.08 ± 0.30</td>
      <td>0.50 ± 0.15</td>
      <td>0.77 ± 0.36</td>
    </tr>
    <tr>
      <td>BP</td>
      <td>1.67 ± 0.59</td>
      <td>-0.17 ± 0.59</td>
      <td>3.30 ± 0.49</td>
      <td>3.06 ± 0.48</td>
      <td>3.45 ± 0.48</td>
      <td>2.12 ± 0.36</td>
      <td>1.84 ± 0.24</td>
    </tr>
  </tbody>
</table>

<footnote><sup>a</sup>Values anomalously negative; these are not included in the analyses</footnote>
Carbon fixation, oxygen production and
consumption

TO<sup>14</sup>C production ranged from 0.05 to 3.74 µmol l<sup>-1</sup>
d<sup>-1</sup> (Table 1). The highest TO<sup>14</sup>C values were observed
in March at Point B station whereas the *Posidonia* sta-
tion in summer was the most productive of the stations
studied in the BP. TO<sup>14</sup>C did not show a difference
across study sites (Table 3) but showed a marginally
vertical variability (i.e. a decrease) as a function of
depth (Table 1). We observed a wide range of variabil-
ity in the relative contribution of DOC production to

total PP (% DOC) with an average of 40 and 54% (Table 1) for the BV and BP, respectively.

Estimates of NCP and NCP<sub>12h</sub> ranged from -6.01 to
5.25 and -3.99 to 6.39 µmol l<sup>-1</sup> d<sup>-1</sup>, respectively. Esti-
mates of NCP (µmol l<sup>-1</sup>h<sup>-1</sup>) were significantly higher
than NCP<sub>12h</sub> (t-test, t<sub>31</sub> = 4.5, n = 32, p < 0.0001). NCP
showed a tendency to decrease with depth (Table 4)
but this was not significant (Table 3). Differences be-
tween areas in NCP were not significant (Table 3). Es-
timates of CR ranged from 0.25 to 7.0 µmol l<sup>-1</sup> d<sup>-1</sup> and
did not exhibit significant vertical variability but were
significantly higher in the BP than in the BV (Table 3).

<table><caption>Table 4. Summary of selected biological variables within the photic layer (4 depths, see Table 1) at the stations sampled in BV and BP. dt = daytime (from sunrise to sunset) nt = night-time; nd = no data. Also shown are averages (± SE) of each vertical depth, BV and BP. See Table 1 for location abbreviations</caption>
  <thead>
    <tr>
      <th>Location</th>
      <th>NCP<sub>12h</sub> ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>NCP ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>R ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>GPP-O<sub>2</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>GPP-<sup>18</sup>O ± SE<br>(µmol O<sub>2</sub> l<sup>-1</sup> d<sup>-1</sup>)</th>
      <th>CR<sub>light</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> dt<sup>-1</sup>)</th>
      <th>CR<sub>dark</sub><br>(µmol O<sub>2</sub> l<sup>-1</sup> nt<sup>-1</sup>)</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td rowspan="4">Oct02</td>
      <td>-0.24 ± 0.12</td>
      <td>-0.83 ± 0.15</td>
      <td>2.64 ± 0.21</td>
      <td>1.82</td>
      <td>0.76 ± 0.12</td>
      <td>1.00</td>
      <td>0.58</td>
    </tr>
    <tr>
      <td>0.00 ± 0.06</td>
      <td>-0.47 ± 0.16</td>
      <td>1.84 ± 0.15</td>
      <td>1.37</td>
      <td>1.20 ± 0.33</td>
      <td>1.19</td>
      <td>0.47</td>
    </tr>
    <tr>
      <td>0.43 ± 0.14</td>
      <td>-0.34 ± 0.25</td>
      <td>0.89 ± 0.21</td>
      <td>0.55</td>
      <td>0.76 ± 0.21</td>
      <td>0.33</td>
      <td>0.77</td>
    </tr>
    <tr>
      <td>0.51 ± 0.21</td>
      <td>-0.16 ± 0.26</td>
      <td>0.49 ± 0.25</td>
      <td>0.32</td>
      <td>0.43 ± 0.17</td>
      <td>-0.08</td>
      <td>0.67</td>
    </tr>
    <tr>
      <td rowspan="4">Dec02</td>
      <td>1.02 ± 0.38</td>
      <td>1.38 ± 0.26</td>
      <td>0.76 ± 0.26</td>
      <td>2.14</td>
      <td>0.50 ± 0.06</td>
      <td>-0.52<sup>a</sup></td>
      <td>-0.36</td>
    </tr>
    <tr>
      <td>1.04 ± 0.39</td>
      <td>0.30 ± 0.35</td>
      <td>0.25 ± 0.26</td>
      <td>0.55</td>
      <td>0.33 ± 0.10</td>
      <td>-0.71<sup>a</sup></td>
      <td>0.74</td>
    </tr>
    <tr>
      <td>-0.27 ± 0.26</td>
      <td>-0.29 ± 0.31</td>
      <td>1.03 ± 0.30</td>
      <td>0.74</td>
      <td>0.12 ± 0.07</td>
      <td>0.39</td>
      <td>0.02</td>
    </tr>
    <tr>
      <td>nd nd</td>
      <td>1.01 ± 0.24</td>
      <td>0.40 ± 0.28</td>
      <td>1.41</td>
      <td>0.47 ± 0.09</td>
      <td>nd</td>
      <td>5.38</td>
    </tr>
    <tr>
      <td rowspan="4">Mar03</td>
      <td>5.07 ± 0.99</td>
      <td>5.25 ± 0.77</td>
      <td>0.61 ± 1.69</td>
      <td>5.86</td>
      <td>3.45 ± 0.38</td>
      <td>-1.62<sup>a</sup></td>
      <td>-0.18</td>
    </tr>
    <tr>
      <td>4.55 ± 0.40</td>
      <td>2.66 ± 0.55</td>
      <td>0.73 ± 0.30</td>
      <td>3.39</td>
      <td>4.39 ± 0.40</td>
      <td>-0.16</td>
      <td>1.89</td>
    </tr>
    <tr>
      <td>1.77 ± 0.92</td>
      <td>2.02 ± 0.77</td>
      <td>0.96 ± 0.81</td>
      <td>2.99</td>
      <td>1.57 ± 0.19</td>
      <td>-0.19<sup>a</sup></td>
      <td>-0.26</td>
    </tr>
    <tr>
      <td>-0.47 ± 0.41</td>
      <td>0.73 ± 0.45</td>
      <td>0.64 ± 0.25</td>
      <td>1.37</td>
      <td>0.37 ± 0.18</td>
      <td>0.84</td>
      <td>-1.20<sup>a</sup></td>
    </tr>
    <tr>
      <td rowspan="4">Jul03</td>
      <td>0.62 ± 0.48</td>
      <td>-0.31 ± 0.11</td>
      <td>1.38 ± 0.04</td>
      <td>1.07</td>
      <td>0.52 ± 0.73</td>
      <td>-0.10</td>
      <td>0.92</td>
    </tr>
    <tr>
      <td>0.35 ± 0.24</td>
      <td>-0.57 ± 0.18</td>
      <td>1.99 ± 0.22</td>
      <td>1.42</td>
      <td>1.50 ± 0.45</td>
      <td>1.15</td>
      <td>0.92</td>
    </tr>
    <tr>
      <td>-0.07 ± 0.24</td>
      <td>-0.26 ± 0.24</td>
      <td>0.28 ± 0.23</td>
      <td>0.02</td>
      <td>0.50 ± 0.13</td>
      <td>0.57</td>
      <td>0.19</td>
    </tr>
    <tr>
      <td>0.12 ± 0.26</td>
      <td>0.19 ± 0.30</td>
      <td>0.27 ± 0.27</td>
      <td>0.46</td>
      <td>0.45 ± 0.12</td>
      <td>0.32</td>
      <td>-0.07</td>
    </tr>
    <tr>
      <td rowspan="5">Pos</td>
      <td>3.82 ± 0.90</td>
      <td>0.38 ± 0.64</td>
      <td>4.44 ± 0.98</td>
      <td>4.83</td>
      <td>6.33 ± 0.69</td>
      <td>2.51</td>
      <td>3.44</td>
    </tr>
    <tr>
      <td>4.14 ± 0.67</td>
      <td>0.44 ± 1.08</td>
      <td>5.34 ± 0.51</td>
      <td>5.77</td>
      <td>5.55 ± 1.65</td>
      <td>1.41</td>
      <td>3.71</td>
    </tr>
    <tr>
      <td>3.35 ± 0.31</td>
      <td>0.84 ± 0.50</td>
      <td>4.97 ± 0.45</td>
      <td>5.81</td>
      <td>5.87 ± 0.62</td>
      <td>2.52</td>
      <td>2.51</td>
    </tr>
    <tr>
      <td>6.18 ± 0.48</td>
      <td>4.41 ± 0.77</td>
      <td>1.01 ± 0.77</td>
      <td>5.42</td>
      <td>5.03 ± 1.07</td>
      <td>-1.15<sup>a</sup></td>
      <td>1.77</td>
    </tr>
    <tr>
      <td>2.15 ± 0.43</td>
      <td>0.83 ± 0.68</td>
      <td>3.63 ± 0.41</td>
      <td>4.45</td>
      <td>5.43 ± 1.03</td>
      <td>3.29</td>
      <td>1.32</td>
    </tr>
    <tr>
      <td rowspan="3">B</td>
      <td>2.60 ± 0.56</td>
      <td>1.10 ± 0.89</td>
      <td>2.02 ± 0.48</td>
      <td>3.12</td>
      <td>4.88 ± 0.95</td>
      <td>2.28</td>
      <td>1.50</td>
    </tr>
    <tr>
      <td>1.49 ± 0.67</td>
      <td>-0.28 ± 1.08</td>
      <td>2.60 ± 0.73</td>
      <td>2.31</td>
      <td>3.47 ± 0.22</td>
      <td>1.98</td>
      <td>1.77</td>
    </tr>
    <tr>
      <td>0.45 ± 0.24</td>
      <td>0.41 ± 0.38</td>
      <td>1.68 ± 0.30</td>
      <td>2.08</td>
      <td>2.48 ± 0.31</td>
      <td>2.03</td>
      <td>0.04</td>
    </tr>
    <tr>
      <td rowspan="4">S4</td>
      <td>2.03 ± 0.49</td>
      <td>0.68 ± 0.79</td>
      <td>nd 0.98</td>
      <td>nd</td>
      <td>1.87 ± 0.57</td>
      <td>-0.16<sup>a</sup></td>
      <td>1.35</td>
    </tr>
    <tr>
      <td>1.32 ± 0.32</td>
      <td>0.90 ± 0.51</td>
      <td>1.65 ± 0.81</td>
      <td>2.55</td>
      <td>1.40 ± 0.33</td>
      <td>0.08</td>
      <td>0.42</td>
    </tr>
    <tr>
      <td>0.30 ± 0.41</td>
      <td>-0.94 ± 0.66</td>
      <td>1.50 ± 0.48</td>
      <td>0.55</td>
      <td>1.11 ± 0.27</td>
      <td>0.81</td>
      <td>1.24</td>
    </tr>
    <tr>
      <td>-1.67 ± 0.64</td>
      <td>-4.49 ± 1.02</td>
      <td>5.79 ± 0.30</td>
      <td>1.30</td>
      <td>0.48 ± 0.24</td>
      <td>2.14</td>
      <td>2.82</td>
    </tr>
    <tr>
      <td rowspan="4">End</td>
      <td>2.30 ± 0.27</td>
      <td>0.84 ± 0.16</td>
      <td>2.48 ± 0.41</td>
      <td>3.31</td>
      <td>4.67 ± 0.19</td>
      <td>2.37</td>
      <td>1.46</td>
    </tr>
    <tr>
      <td>0.72 ± 0.16</td>
      <td>-1.12 ± 0.25</td>
      <td>4.03 ± 0.22</td>
      <td>2.91</td>
      <td>2.52 ± 0.58</td>
      <td>1.80</td>
      <td>1.84</td>
    </tr>
    <tr>
      <td>1.56 ± 0.24</td>
      <td>-0.77 ± 0.38</td>
      <td>1.22 ± 0.25</td>
      <td>0.45</td>
      <td>2.25 ± 0.30</td>
      <td>0.69</td>
      <td>2.33</td>
    </tr>
    <tr>
      <td>-3.99 ± 0.28</td>
      <td>-6.01 ± 0.44</td>
      <td>7.10 ± 0.08</td>
      <td>1.09</td>
      <td>1.81 ± 0.24</td>
      <td>5.80</td>
      <td>2.02</td>
    </tr>
    <tr>
      <td colspan="8">Average ± SE</td>
    </tr>
    <tr>
      <td>Depth 1</td>
      <td>2.10 ± 0.61</td>
      <td>1.03 ± 0.65</td>
      <td>2.28 ± 1.45</td>
      <td>3.35 ± 1.77</td>
      <td>2.94 ± 0.83</td>
      <td>1.81 ± 0.60</td>
      <td>1.07 ± 0.42</td>
    </tr>
    <tr>
      <td>Depth 2</td>
      <td>1.84 ± 0.61</td>
      <td>0.40 ± 0.42</td>
      <td>2.23 ± 1.68</td>
      <td>2.64 ± 1.61</td>
      <td>2.72 ± 0.69</td>
      <td>1.11 ± 0.33</td>
      <td>1.44 ± 0.38</td>
    </tr>
    <tr>
      <td>Depth 3</td>
      <td>1.07 ± 0.43</td>
      <td>0.00 ± 0.34</td>
      <td>1.68 ± 1.49</td>
      <td>1.68 ± 1.96</td>
      <td>1.96 ± 0.67</td>
      <td>1.04 ± 0.32</td>
      <td>1.07 ± 0.38</td>
    </tr>
    <tr>
      <td>Depth 4</td>
      <td>0.94 ± 1.28</td>
      <td>-0.49 ± 1.16</td>
      <td>2.17 ± 2.70</td>
      <td>1.68 ± 1.61</td>
      <td>1.44 ± 0.58</td>
      <td>1.84 ± 0.87</td>
      <td>1.80 ± 0.72</td>
    </tr>
    <tr>
      <td>BV</td>
      <td>1.30 ± 0.53</td>
      <td>0.64 ± 0.39</td>
      <td>0.95 ± 0.17</td>
      <td>1.59 ± 0.37</td>
      <td>1.08 ± 0.30</td>
      <td>0.50 ± 0.15</td>
      <td>0.77 ± 0.36</td>
    </tr>
    <tr>
      <td>BP</td>
      <td>1.67 ± 0.59</td>
      <td>-0.17 ± 0.59</td>
      <td>3.30 ± 0.49</td>
      <td>3.06 ± 0.48</td>
      <td>3.45 ± 0.48</td>
      <td>2.12 ± 0.36</td>
      <td>1.84 ± 0.24</td>
    </tr>
  </tbody>
</table>

<footnote><sup>a</sup>Values anomalously negative; these are not included in the analyses</footnote>
GPP-O₂ estimates were below 5.8 µmol l⁻¹ d⁻¹ and showed significantly higher values at the surface decreasing with depth (Table 3) but did not differ between the 2 bays. The highest values of PP (GPP-O₂, NCP₁₂h, NCP) were found at the Posidonia station in the BP and in March 2003 in the BV. More than half (55%) of the GPP/CR ratios were higher than 1 (net autotrophy) with an average of 1.71 ± 0.35. However, the average respiration rate was not significantly different from that of average GPP (t-test, t₃₀ = 0.60, n = 31, p = 0.55).

Estimates of CR based on incubations lasting 12 h (0.08 ± 0.02 µmol O₂ l⁻¹ h⁻¹), 24 h (0.10 ± 0.02 µmol O₂ l⁻¹ h⁻¹) or 48 h (0.12 ± 0.02 µmol O₂ l⁻¹ h⁻¹) were not significantly different (F₁, 21 = 1.09, p = 0.35).

GPP-¹⁸O was below 6.3 µmol l⁻¹ d⁻¹ and similar to GPP-O₂ (Table 4). The highest values of GPP-¹⁸O coincided with the highest concentrations of chl *a* and the highest values of the other metabolic rates (GPP-O₂, NCP₁₂h, NCP). GPP-¹⁸O showed significant depth variability with the highest values in the surface waters (Table 3) but no differences between areas were evident.

Estimates of CR<sub>light</sub> were not significantly different from those of CR<sub>dark</sub> (t-test t<sub>22</sub> = 0.45, n = 23, p = 0.65). However, incubation times for CR<sub>light</sub> and CR<sub>dark</sub> were different and for that reason we normalized these estimates by time. We observed that CR<sub>dark</sub> h⁻¹ was not significantly different from CR<sub>light</sub> (t-test, t<sub>22</sub> = 1.89, n = 23, p = 0.07). Consequently, we observed no differences between CR<sub>dark</sub> + CR<sub>light</sub> and CR (paired t-test, t<sub>25</sub> = 0.36, n = 26, p = 0.71), but they were strongly correlated (r² = 0.82, p < 0.001, Fig. 1) with a slope<sub>RMA</sub> not significantly different from 1 (Table 5). In addition, GPP-O₂ was not significantly different from GPP<sub>light</sub> (paired t-test, t<sub>23</sub> = 1.70, n = 24, p = 0.10). In order to precisely estimate the effect of light on the rates of oxygen consumption, we examined the relation between irradiance (µE m⁻² s⁻¹)

<figure><img src="image_1.png" /><figcaption>Fig. 1. Relationship between community respiration (CR<sub>dark</sub> + CR<sub>light</sub> [CR<sub>d+1</sub>]) estimated as the sum of dark and light respiration rates and community respiration (CR) estimated from the light–dark O₂ method. Dashed line = reduced major axis (RMA) regression</figcaption></figure>

and CR<sub>light</sub>/CR<sub>dark</sub> but the correlation was not significant (r<sub>s</sub> = 0.22, n = 23, p = 0.32). Also, there was no significant correlation between percentage of cyanobacteria and CR<sub>light</sub>/CR<sub>dark</sub> (r<sub>s</sub> = 0.1, n = 23, p = 0.65). Finally, we compared GPP-O₂ vs. GPP<sub>light</sub> as a function of the water metabolic conditions. We found that GPP-O₂ was not significantly different from GPP<sub>light</sub> in autotrophic (t-test, t<sub>9</sub> = 1.97, n = 10, p = 0.07) or in heterotrophic (t-test, t<sub>13</sub> = 0.49, n = 14, p = 0.63) waters.

### Comparison of oxygen production and carbon fixation rates

GPP-O₂ closely correlated with GPP-¹⁸O (r² = 0.74, n = 31, p < 0.001, Fig. 2). The slope<sub>RMA</sub> (0.89 ± 0.03) was not significantly different from 1 and the intercept

<table><caption>Table 5. Parameters of the regressions (RMA: reduced major axis; OLS: ordinary least squares) and Student's t-tests for the slope and intercept of regression lines</caption><thead><tr><th></th><th>r<sup>2</sup></th><th>n</th><th>df</th><th>RMA</th><th>OLS</th></tr></thead><tbody><tr><td>CR<sub>light</sub> + CR<sub>dark</sub> vs. CR</td><td>0.82</td><td>24</td><td>22</td><td>t<sub>slope</sub> = 0.94, p > 0.05<br>t<sub>intercept</sub> = 0.27, p > 0.05</td><td></td></tr><tr><td>GPP-O<sub>2</sub> vs. GPP-<sup>18</sup>O</td><td>0.74</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 1.27, p > 0.05<br>t<sub>intercept</sub> = 1.07, p > 0.05</td><td>t<sub>slope</sub> = 2.74, p < 0.01<br>t<sub>intercept</sub> = 2.18, p < 0.05</td></tr><tr><td>GPP-O<sub>2</sub> vs. TO<sup>14</sup>C (PQ = 1)</td><td>0.66</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 4.41, p < 0.001<br>t<sub>intercept</sub> = 1.74, p > 0.05</td><td>t<sub>slope</sub> = 2.69, p < 0.01<br>t<sub>intercept</sub> = 2.97, p < 0.01</td></tr><tr><td>GPP-O<sub>2</sub> vs. TO<sup>14</sup>C (PQ = 1.5)</td><td>0.66</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 1.97, p > 0.05<br>t<sub>intercept</sub> = 1.73, p > 0.05</td><td>t<sub>slope</sub> = 0.26, p > 0.05<br>t<sub>intercept</sub> = 2.97, p < 0.01</td></tr><tr><td>NCP vs. TO<sup>14</sup>C (PQ = 1)</td><td>0.47</td><td>29</td><td>27</td><td>t<sub>slope</sub> = 3.25, p < 0.001<br>t<sub>intercept</sub> = 3.46, p < 0.001</td><td>t<sub>slope</sub> = 1.01, p > 0.05<br>t<sub>intercept</sub> = 1.77, p > 0.05</td></tr><tr><td>NCP vs. TO<sup>14</sup>C (PQ = 1.5)</td><td>0.47</td><td>29</td><td>27</td><td>t<sub>slope</sub> = 1.29, p > 0.05<br>t<sub>intercept</sub> = 3.46, p < 0.001</td><td>t<sub>slope</sub> = 0.93, p > 0.05<br>t<sub>intercept</sub> = 1.77, p > 0.05</td></tr></tbody></table>
GPP-O₂ estimates were below 5.8 µmol l⁻¹ d⁻¹ and showed significantly higher values at the surface decreasing with depth (Table 3) but did not differ between the 2 bays. The highest values of PP (GPP-O₂, NCP₁₂h, NCP) were found at the Posidonia station in the BP and in March 2003 in the BV. More than half (55%) of the GPP/CR ratios were higher than 1 (net autotrophy) with an average of 1.71 ± 0.35. However, the average respiration rate was not significantly different from that of average GPP (t-test, t₃₀ = 0.60, n = 31, p = 0.55).

Estimates of CR based on incubations lasting 12 h (0.08 ± 0.02 µmol O₂ l⁻¹ h⁻¹), 24 h (0.10 ± 0.02 µmol O₂ l⁻¹ h⁻¹) or 48 h (0.12 ± 0.02 µmol O₂ l⁻¹ h⁻¹) were not significantly different (F₁, 21 = 1.09, p = 0.35).

GPP-¹⁸O was below 6.3 µmol l⁻¹ d⁻¹ and similar to GPP-O₂ (Table 4). The highest values of GPP-¹⁸O coincided with the highest concentrations of chl *a* and the highest values of the other metabolic rates (GPP-O₂, NCP₁₂h, NCP). GPP-¹⁸O showed significant depth variability with the highest values in the surface waters (Table 3) but no differences between areas were evident.

Estimates of CR<sub>light</sub> were not significantly different from those of CR<sub>dark</sub> (t-test t<sub>22</sub> = 0.45, n = 23, p = 0.65). However, incubation times for CR<sub>light</sub> and CR<sub>dark</sub> were different and for that reason we normalized these estimates by time. We observed that CR<sub>dark</sub> h⁻¹ was not significantly different from CR<sub>light</sub> (t-test, t<sub>22</sub> = 1.89, n = 23, p = 0.07). Consequently, we observed no differences between CR<sub>dark</sub> + CR<sub>light</sub> and CR (paired t-test, t<sub>25</sub> = 0.36, n = 26, p = 0.71), but they were strongly correlated (r² = 0.82, p < 0.001, Fig. 1) with a slope<sub>RMA</sub> not significantly different from 1 (Table 5). In addition, GPP-O₂ was not significantly different from GPP<sub>light</sub> (paired t-test, t<sub>23</sub> = 1.70, n = 24, p = 0.10). In order to precisely estimate the effect of light on the rates of oxygen consumption, we examined the relation between irradiance (µE m⁻² s⁻¹)

<figure><img src="image_3.png" /><figcaption>Fig. 1. Relationship between community respiration (CR<sub>dark</sub> + CR<sub>light</sub> [CR<sub>d+1</sub>]) estimated as the sum of dark and light respiration rates and community respiration (CR) estimated from the light–dark O₂ method. Dashed line = reduced major axis (RMA) regression</figcaption></figure>

and CR<sub>light</sub>/CR<sub>dark</sub> but the correlation was not significant (r<sub>s</sub> = 0.22, n = 23, p = 0.32). Also, there was no significant correlation between percentage of cyanobacteria and CR<sub>light</sub>/CR<sub>dark</sub> (r<sub>s</sub> = 0.1, n = 23, p = 0.65). Finally, we compared GPP-O₂ vs. GPP<sub>light</sub> as a function of the water metabolic conditions. We found that GPP-O₂ was not significantly different from GPP<sub>light</sub> in autotrophic (t-test, t<sub>9</sub> = 1.97, n = 10, p = 0.07) or in heterotrophic (t-test, t<sub>13</sub> = 0.49, n = 14, p = 0.63) waters.

### Comparison of oxygen production and carbon fixation rates

GPP-O₂ closely correlated with GPP-¹⁸O (r² = 0.74, n = 31, p < 0.001, Fig. 2). The slope<sub>RMA</sub> (0.89 ± 0.03) was not significantly different from 1 and the intercept

<table><caption>Table 5. Parameters of the regressions (RMA: reduced major axis; OLS: ordinary least squares) and Student's t-tests for the slope and intercept of regression lines</caption><thead><tr><th></th><th>r<sup>2</sup></th><th>n</th><th>df</th><th>RMA</th><th>OLS</th></tr></thead><tbody><tr><td>CR<sub>light</sub> + CR<sub>dark</sub> vs. CR</td><td>0.82</td><td>24</td><td>22</td><td>t<sub>slope</sub> = 0.94, p > 0.05<br>t<sub>intercept</sub> = 0.27, p > 0.05</td><td></td></tr><tr><td>GPP-O<sub>2</sub> vs. GPP-<sup>18</sup>O</td><td>0.74</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 1.27, p > 0.05<br>t<sub>intercept</sub> = 1.07, p > 0.05</td><td>t<sub>slope</sub> = 2.74, p < 0.01<br>t<sub>intercept</sub> = 2.18, p < 0.05</td></tr><tr><td>GPP-O<sub>2</sub> vs. TO<sup>14</sup>C (PQ = 1)</td><td>0.66</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 4.41, p < 0.001<br>t<sub>intercept</sub> = 1.74, p > 0.05</td><td>t<sub>slope</sub> = 2.69, p < 0.01<br>t<sub>intercept</sub> = 2.97, p < 0.01</td></tr><tr><td>GPP-O<sub>2</sub> vs. TO<sup>14</sup>C (PQ = 1.5)</td><td>0.66</td><td>31</td><td>29</td><td>t<sub>slope</sub> = 1.97, p > 0.05<br>t<sub>intercept</sub> = 1.73, p > 0.05</td><td>t<sub>slope</sub> = 0.26, p > 0.05<br>t<sub>intercept</sub> = 2.97, p < 0.01</td></tr><tr><td>NCP vs. TO<sup>14</sup>C (PQ = 1)</td><td>0.47</td><td>29</td><td>27</td><td>t<sub>slope</sub> = 3.25, p < 0.001<br>t<sub>intercept</sub> = 3.46, p < 0.001</td><td>t<sub>slope</sub> = 1.01, p > 0.05<br>t<sub>intercept</sub> = 1.77, p > 0.05</td></tr><tr><td>NCP vs. TO<sup>14</sup>C (PQ = 1.5)</td><td>0.47</td><td>29</td><td>27</td><td>t<sub>slope</sub> = 1.29, p > 0.05<br>t<sub>intercept</sub> = 3.46, p < 0.001</td><td>t<sub>slope</sub> = 0.93, p > 0.05<br>t<sub>intercept</sub> = 1.77, p > 0.05</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>Fig. 2. Relationship between gross primary production measured by the light-dark O₂ method (GPP-O₂) and gross primary production measured by the ¹⁸O technique (GPP-¹⁸O). Dashed line = reduced major axis (RMA) regression</figcaption></figure>

(0.27 ± 0.52) was not significantly different from 0 (Table 5). Note that the slope<sub>OLS</sub> was significantly different from 1 and the intercept was significantly different from 0 (Table 5).

GPP-O₂ was correlated with TO<sup>14</sup>C (Fig. 3A) with a slope<sub>RMA</sub> (1.90 ± 0.04) significantly higher than 1 (Table 5). TO<sup>14</sup>C and NCP were poorly correlated (r<sup>2</sup> =

0.26. p < 0.05). However, 3 outliers were identified and removed from the analysis (Zar 1999), improving the correlation (r<sup>2</sup> = 0.47, p < 0.001, Fig. 3B). The slope<sub>RMA</sub> (1.83 ± 0.08) was also significantly higher than 1 (Table 4). The average of the molar flux ratio was 1.5 ± 0.17 (GPP-O<sub>2</sub>/<sup>14</sup>C) and 2.1 ± 0.23 (GPP<sup>18</sup>O/<sup>14</sup>C), if we did not consider the outlier data with molar flux ratios > 6. The latter average was considered high and out of the general range (1.2 to 1.8). Therefore, the <sup>14</sup>C uptake rates were adjusted for a factor of 1.5 to compare <sup>14</sup>C uptake vs. GPP-O<sub>2</sub> and NCP. The new regression line between TO<sup>14</sup>C and GPP-O<sub>2</sub> (Fig. 3C) had a slope<sub>RMA</sub> (1.19 ± 0.04) and an intercept (0.47 ± 0.55) which were not significantly different from 1 and 0, respectively (Table 5). The new comparison between TO<sup>14</sup>C and NCP (Fig. 3C) showed a slope<sub>RMA</sub> (1.14 ± 0.08) not significantly different from 1, but the intercept (−1.07 ± 0.64) was significantly different from 0 (Table 5).

DISCUSSION

Oxygen production and consumption

The rates of GPP-O₂ (2.43 to 0.34 µmol l⁻¹ d⁻¹) measured in the bays of BV and BP are similar to those results measured in other oligotrophic coastal systems

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. (A) Relationship between gross primary production measured by the light-dark O₂ method (GPP-O₂) and daily ¹⁴C uptake. (B) Relationship between net community production (NCP) measured by the light-dark O₂ method and daily ¹⁴C uptake. Dashed lines = reduced major axis (RMA) regressions. (C) Relationship between TO¹⁴C and NCP (dashed line and circles; 1.14 ± 0.08 × TO¹⁴C – 1.07 ± 0.64), and GPP-O₂ (solid line and squares; 1.19 ± 0.04 × TO¹⁴C + 0.47 ± 0.55), assuming a photosynthetic quotient of 1.5</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 2. Relationship between gross primary production measured by the light-dark O₂ method (GPP-O₂) and gross primary production measured by the ¹⁸O technique (GPP-¹⁸O). Dashed line = reduced major axis (RMA) regression</figcaption></figure>

(0.27 ± 0.52) was not significantly different from 0 (Table 5). Note that the slope<sub>OLS</sub> was significantly different from 1 and the intercept was significantly different from 0 (Table 5).

GPP-O₂ was correlated with TO<sup>14</sup>C (Fig. 3A) with a slope<sub>RMA</sub> (1.90 ± 0.04) significantly higher than 1 (Table 5). TO<sup>14</sup>C and NCP were poorly correlated (r<sup>2</sup> =

0.26. p < 0.05). However, 3 outliers were identified and removed from the analysis (Zar 1999), improving the correlation (r<sup>2</sup> = 0.47, p < 0.001, Fig. 3B). The slope<sub>RMA</sub> (1.83 ± 0.08) was also significantly higher than 1 (Table 4). The average of the molar flux ratio was 1.5 ± 0.17 (GPP-O<sub>2</sub>/<sup>14</sup>C) and 2.1 ± 0.23 (GPP<sup>18</sup>O/<sup>14</sup>C), if we did not consider the outlier data with molar flux ratios > 6. The latter average was considered high and out of the general range (1.2 to 1.8). Therefore, the <sup>14</sup>C uptake rates were adjusted for a factor of 1.5 to compare <sup>14</sup>C uptake vs. GPP-O<sub>2</sub> and NCP. The new regression line between TO<sup>14</sup>C and GPP-O<sub>2</sub> (Fig. 3C) had a slope<sub>RMA</sub> (1.19 ± 0.04) and an intercept (0.47 ± 0.55) which were not significantly different from 1 and 0, respectively (Table 5). The new comparison between TO<sup>14</sup>C and NCP (Fig. 3C) showed a slope<sub>RMA</sub> (1.14 ± 0.08) not significantly different from 1, but the intercept (−1.07 ± 0.64) was significantly different from 0 (Table 5).

DISCUSSION

Oxygen production and consumption

The rates of GPP-O₂ (2.43 to 0.34 µmol l⁻¹ d⁻¹) measured in the bays of BV and BP are similar to those results measured in other oligotrophic coastal systems

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

<figure><img src="image_6.png" /><figcaption>Fig. 3. (A) Relationship between gross primary production measured by the light-dark O₂ method (GPP-O₂) and daily ¹⁴C uptake. (B) Relationship between net community production (NCP) measured by the light-dark O₂ method and daily ¹⁴C uptake. Dashed lines = reduced major axis (RMA) regressions. (C) Relationship between TO¹⁴C and NCP (dashed line and circles; 1.14 ± 0.08 × TO¹⁴C – 1.07 ± 0.64), and GPP-O₂ (solid line and squares; 1.19 ± 0.04 × TO¹⁴C + 0.47 ± 0.55), assuming a photosynthetic quotient of 1.5</figcaption></figure>
(0.1 to 16.9 µmol l⁻¹ d⁻¹; Duarte & Agustí 2004, Gazeau et al. 2005, Hashimoto et al. 2005). The rates of GPP¹⁸O measured during the present study (2.27 ± 0.34 µmol l⁻¹ d⁻¹) are higher than the rates reported for open ocean samples incubated *in situ* (0.71 to 0.09 µmol l⁻¹ d⁻¹; Grande et al. 1989) and *in vitro* (0.96 µmol l⁻¹ d⁻¹; Juranek & Quay 2005), similar to those reported for eutrophic estuaries (3.1 ± 0.6 µmol l⁻¹ d⁻¹; Gazeau et al. 2007), but lower than those measured in laboratory incubations of samples from coastal mesocosms (43.5 to 1.2 µmol l⁻¹ d⁻¹; Bender et al. 1987).

Dickson & Orchardo (2001) suggested that the H₂¹⁸O spike method should be preferred because it does not require the assumption that respiration in the light equals respiration in the dark, as does the oxygen light–dark method. However, in agreement with Bender et al. (1987) our results showed that GPP based on ¹⁸O-labeling and the light-dark method, were not significantly different (Fig. 2). The H₂¹⁸O spike and O₂ light-dark method both involve bottle incubation. Sarma et al. (2005) observed that estimates of GPP based on *in situ* FRRF, which does not involve incubations, were similar to those obtained with the GPP-O₂ technique in the Bay of Sagami (Japan). Obviously, the bottle incubation procedure does not accurately mimic all aspects of *in situ* conditions. The light climate and hydrodynamics within the bottle may be different to that outside, and communities and biomass may change during incubation. However, our results revealed no significant differences in dark respiration rates when incubated for short (12 h) and long hours (24 and 48 h). Consequently, although GPP rates may be influenced by bottle incubations, we believe this artefact to be minor and our estimations robust.

## Respiration in the light and dark

Most studies lack simultaneous determination of light and dark respiration, because of methodological difficulties, complicating comparison of our results with previous studies. The mean values of dark and light respiration in the BP and BV were higher than the values reported by Grande et al. (1989) for offshore oligotrophic waters (<0.5 µmol l⁻¹ per 12 h). Oxygen consumption rates under light conditions were not significantly different from those in the dark, in agreement with Marra & Barber (2004). These results confirm that (daily) CR assuming CR<sub>light</sub> = CR<sub>dark</sub> is not significantly different from CR<sub>light</sub> + CR<sub>dark</sub> (Table 5). Consequently, GPP-O₂ based on dark respiration was not significantly different from GPP<sub>light</sub> that takes CR<sub>light</sub> into account. However, we did not observe a higher degree of underestimation of GPP-O₂ in net heterotrophic conditions, as observed in some other

studies (e.g. Pringault et al. 2007). In an attempt to explain this difference, we note that most CR<sub>light</sub> estimates from previous studies (e.g. Pringault et al. 2007) were obtained from *in vitro* conditions, under constant and saturating light (1000 µmol photons m⁻² s⁻¹), while 90% of our samples received *in situ* irradiances much lower than saturating light, falling to values as low as 20 µmol photons m⁻² s⁻¹ at bottom depths in winter. Our data did not reveal evidence for respiration photo-enhancement within our range of light intensities (20 to approximately 2000 µE m⁻² s⁻¹). This is consistent with the findings of Lewitus & Kana (1995) showing limited differences between light and dark respiration under balanced growth conditions (i.e. respiration at the growth irradiance and not saturating light). Moreover, the lack of relationship between respiration in the light (CR<sub>light</sub> or CR<sub>light</sub>/CR<sub>dark</sub>) and percentage of cyanobacteria is also consistent with literature reports (see references in Lewitus & Kana 1995), which showed important light stimulation of oxygen uptake by different phytoplankton species (e.g. *Synnechococcus* spp., diatoms) but only at high irradiances (light energy in excess). Our values for CR<sub>light</sub> and CR<sub>dark</sub> were estimated by difference and some negative values (see ‘Materials and methods’) were obtained. These negative values were retained and are presented because Williams (2000) argued that only the negative values derived from experimental anomalies (see ‘Materials and methods’) should be removed, otherwise it would bias the data set. However, this is not a common procedure (e.g. Grande et al. 1989, Pringault et al. 2007).

The similarity of rates of light and dark respiration that we found requires discussion in the context of existing knowledge (Williams & Lefèvre 2007). The enhanced O₂ consumption during the light period reported by some authors (e.g. Bender et al. 1987, Dickson & Orchardo 2001, Dickson et al. 2001) has been attributed to photorespiration, the Mehler reaction, and mitochondrial respiration or its prokaryotic equivalent. However, photorespiration in phytoplankton is usually low due to the existence of a CO₂ concentrating mechanism (CCM) (Weger et al. 1989). Additionally, although the Mehler reaction can decrease real gross photosynthesis in cultures by 10% (Laws et al. 2000), its contribution to O₂ consumption in the light is minor (Weger et al. 1988). The alternative explanation for enhanced respiration in the light is increased mitochondrial respiration. However, although it can be inhibited (e.g. Bate et al. 1988) or stimulated (e.g. Weger et al. 1989), the current consensus is that it is similar in the dark and light (Geider 1992). Besides these physiological mechanisms, there is also a community level mechanism for enhanced respiration in the light: immediate release of fixed carbon by auto-
(0.1 to 16.9 µmol l⁻¹ d⁻¹; Duarte & Agustí 2004, Gazeau et al. 2005, Hashimoto et al. 2005). The rates of GPP¹⁸O measured during the present study (2.27 ± 0.34 µmol l⁻¹ d⁻¹) are higher than the rates reported for open ocean samples incubated *in situ* (0.71 to 0.09 µmol l⁻¹ d⁻¹; Grande et al. 1989) and *in vitro* (0.96 µmol l⁻¹ d⁻¹; Juranek & Quay 2005), similar to those reported for eutrophic estuaries (3.1 ± 0.6 µmol l⁻¹ d⁻¹; Gazeau et al. 2007), but lower than those measured in laboratory incubations of samples from coastal mesocosms (43.5 to 1.2 µmol l⁻¹ d⁻¹; Bender et al. 1987).

Dickson & Orchardo (2001) suggested that the H₂¹⁸O spike method should be preferred because it does not require the assumption that respiration in the light equals respiration in the dark, as does the oxygen light–dark method. However, in agreement with Bender et al. (1987) our results showed that GPP based on ¹⁸O-labeling and the light-dark method, were not significantly different (Fig. 2). The H₂¹⁸O spike and O₂ light-dark method both involve bottle incubation. Sarma et al. (2005) observed that estimates of GPP based on *in situ* FRRF, which does not involve incubations, were similar to those obtained with the GPP-O₂ technique in the Bay of Sagami (Japan). Obviously, the bottle incubation procedure does not accurately mimic all aspects of *in situ* conditions. The light climate and hydrodynamics within the bottle may be different to that outside, and communities and biomass may change during incubation. However, our results revealed no significant differences in dark respiration rates when incubated for short (12 h) and long hours (24 and 48 h). Consequently, although GPP rates may be influenced by bottle incubations, we believe this artefact to be minor and our estimations robust.

## Respiration in the light and dark

Most studies lack simultaneous determination of light and dark respiration, because of methodological difficulties, complicating comparison of our results with previous studies. The mean values of dark and light respiration in the BP and BV were higher than the values reported by Grande et al. (1989) for offshore oligotrophic waters (<0.5 µmol l⁻¹ per 12 h). Oxygen consumption rates under light conditions were not significantly different from those in the dark, in agreement with Marra & Barber (2004). These results confirm that (daily) CR assuming CR<sub>light</sub> = CR<sub>dark</sub> is not significantly different from CR<sub>light</sub> + CR<sub>dark</sub> (Table 5). Consequently, GPP-O₂ based on dark respiration was not significantly different from GPP<sub>light</sub> that takes CR<sub>light</sub> into account. However, we did not observe a higher degree of underestimation of GPP-O₂ in net heterotrophic conditions, as observed in some other

studies (e.g. Pringault et al. 2007). In an attempt to explain this difference, we note that most CR<sub>light</sub> estimates from previous studies (e.g. Pringault et al. 2007) were obtained from *in vitro* conditions, under constant and saturating light (1000 µmol photons m⁻² s⁻¹), while 90% of our samples received *in situ* irradiances much lower than saturating light, falling to values as low as 20 µmol photons m⁻² s⁻¹ at bottom depths in winter. Our data did not reveal evidence for respiration photo-enhancement within our range of light intensities (20 to approximately 2000 µE m⁻² s⁻¹). This is consistent with the findings of Lewitus & Kana (1995) showing limited differences between light and dark respiration under balanced growth conditions (i.e. respiration at the growth irradiance and not saturating light). Moreover, the lack of relationship between respiration in the light (CR<sub>light</sub> or CR<sub>light</sub>/CR<sub>dark</sub>) and percentage of cyanobacteria is also consistent with literature reports (see references in Lewitus & Kana 1995), which showed important light stimulation of oxygen uptake by different phytoplankton species (e.g. *Synnechococcus* spp., diatoms) but only at high irradiances (light energy in excess). Our values for CR<sub>light</sub> and CR<sub>dark</sub> were estimated by difference and some negative values (see ‘Materials and methods’) were obtained. These negative values were retained and are presented because Williams (2000) argued that only the negative values derived from experimental anomalies (see ‘Materials and methods’) should be removed, otherwise it would bias the data set. However, this is not a common procedure (e.g. Grande et al. 1989, Pringault et al. 2007).

The similarity of rates of light and dark respiration that we found requires discussion in the context of existing knowledge (Williams & Lefèvre 2007). The enhanced O₂ consumption during the light period reported by some authors (e.g. Bender et al. 1987, Dickson & Orchardo 2001, Dickson et al. 2001) has been attributed to photorespiration, the Mehler reaction, and mitochondrial respiration or its prokaryotic equivalent. However, photorespiration in phytoplankton is usually low due to the existence of a CO₂ concentrating mechanism (CCM) (Weger et al. 1989). Additionally, although the Mehler reaction can decrease real gross photosynthesis in cultures by 10% (Laws et al. 2000), its contribution to O₂ consumption in the light is minor (Weger et al. 1988). The alternative explanation for enhanced respiration in the light is increased mitochondrial respiration. However, although it can be inhibited (e.g. Bate et al. 1988) or stimulated (e.g. Weger et al. 1989), the current consensus is that it is similar in the dark and light (Geider 1992). Besides these physiological mechanisms, there is also a community level mechanism for enhanced respiration in the light: immediate release of fixed carbon by auto-
trophs and uptake and respiration by heterotrophic consumers. Although a major fraction (~47%) of the fixed carbon was excreted in our study, the rate of respiration was rather constant; there was not a difference among incubations lasting 12, 24 or 48 h. Hence, bacteria do not directly depend on current algal production of labile carbon. In oligotrophic ecosystems where PP is low and in coastal sites, bacteria carbon demand tends to be higher than algal DOC release (Morán et al. 2002). This implies the existence of other autochthonous (e.g. cellular lysis, grazer activity) or allochthonous dissolved organic sources in addition to algal release, making it possible that phytoplankton release did not significantly enhance respiration in the light.

The present work shows robust evidence that under natural conditions respiration in the light is not significantly different to respiration in the dark. Consequently, the light-dark method is an accurate and unambiguous method to measure marine PP.

## O₂ vs. ¹⁴C rates

¹⁴C fixation data including DOC fraction are scarce, especially in oligotrophic coastal systems. The rates (from 0.96 to 0.16 µmol l⁻¹ d⁻¹) were higher than those obtained in the open ocean with or without accounting for the DO-¹⁴C fraction (< 1.5 µmol l⁻¹ d⁻¹, Grande et al. 1989, Juranek & Quay 2005), and within (0.1 to 9.4 µmol l⁻¹ d⁻¹, Hashimoto et al. 2005) or lower than the range of coastal environments (e.g. 4 to 60 µmol l⁻¹ d⁻¹, Morán et al. 2002; >60 µmol l⁻¹ d⁻¹, Marañón et al. 2004). ¹⁴C uptake is lower than GPP if part of the ¹⁴C pool in the dissolved organic form is not collected and measured or if some photosynthetically fixed ¹⁴C is re-mineralized during the incubation. It is therefore important to consider the incubation time. Weger et al. (1989) suggested that the ¹⁴C technique estimates GPP when short incubations (1 to 3 h) are used because respiratory losses can be ignored. In agreement with earlier works (e.g. Raine 1983, Langdon et al. 1995) our results showed that ¹⁴C uptake in incubations of up to 6 to 12 h, overestimate NPP and are closer to GPP. There are also uncertainties regarding the magnitude of extracellular release of recently fixed carbon. Most previous papers that have concluded that ¹⁴C uptake is closer to NPP, did not account for the DOC fraction (e.g. Marra 2002, Juranek & Quay 2005). However, the contribution of dissolved PP can be important, especially in oligotrophic systems where productivity tends to be low (Moran et al. 2002). Therefore, PP can be significantly underestimated if the DO-¹⁴C fraction is not taken into account (e.g. Marañón et al. 2004). Dissolved organic carbon release accounted for about 47% of total carbon fixation in the present study. We

have no evidence that the high DOC production rates were an artefact of improper handling of samples. For instance, care was taken to ensure that the phytoplankton assemblage was not light shocked during sampling (samples were collected before sunrise and were incubated *in situ*). However, incubation times were rather long (9 to 15 h) depending on the day light length and some ¹⁴C may have been recycled. Dickson et al. (2001) and Hansell & Carlson (1998) employed shorter incubations (4 to 6 h) and nevertheless also obtained high percentages of DOC production (e.g. 79 ± 5% and 59 to 70%, respectively). Our results clearly highlight the need to include the dissolved organic fraction in oligotrophic coastal waters such as those found in the Mediterranean.

When the oxygen and ¹⁴C fluxes are compared, careful attention needs to be paid to the PQ. It can vary widely (from 0.5 to 5) but generally ranges between 1.2 and 1.8 (Laws 1991). We obtained an apparent PQ of 1.5 (molar flux ratio O₂/¹⁴C), which is within the PQ range, and was derived from natural assemblages and not from non-specific phytoplankton blooms or cultures. According to Rabinowitch (1945), PQ is different from the conventional value of 1.0, mainly due to the synthesis of lipids (PQ > 1) or organic acids (PQ < 1) rather than the carbohydrates. In contrast, Burris (1981) observed that PQs higher than 1 were more likely as a consequence of the nitrogen substrate used by phytoplankton. When nitrate as a replacement for ammonia is assimilated by phytoplankton PQ tends to increase. PQ rises as a result of O₂ release during the reduction of nitrate to ammonium, in the range of 1.5 to 1.8 (Williams et al. 1979) or up to 2 (Raine 1983). Laws (1991) used alternative methods to estimate PQ based on balanced chemical equations and reported PQ estimates of 1.4 for new production (nitrate as the main source of nitrogen) and 1.1 for recycled production (ammonium as the main source of nitrogen). In our study area (Northwest Mediterranean), we observed abundances of cyanobacteria of about 2 × 10⁵ cells ml⁻¹ (data not shown). This abundance is even higher than that reported by Duarte & Agustí (2004) in the Bay of Blanes (10⁴ cells ml⁻¹), considering that cyanobacteria largely dominate the autotrophic biomass in summer. According to Bergman et al. (1997), aerobic N₂ fixation by cyanobacteria entails O₂ consumption as part of the nitrogenase autoprotection mechanism. Therefore, our data confirm that PQ values higher than the commonly used value of 1.25 are not unusual (e.g. Williams et al. 1979). In any case, and using more conservative PQs (<1.5), our results confirmed that PP based on ¹⁴C method is closer to gross than NPP (Table 5).

Finally, it should be noted that these conclusions were obtained from comparing TO-¹⁴C vs. GPP-O₂ and NCP rates using model II reduced major axis regression
trophs and uptake and respiration by heterotrophic consumers. Although a major fraction (~47%) of the fixed carbon was excreted in our study, the rate of respiration was rather constant; there was not a difference among incubations lasting 12, 24 or 48 h. Hence, bacteria do not directly depend on current algal production of labile carbon. In oligotrophic ecosystems where PP is low and in coastal sites, bacteria carbon demand tends to be higher than algal DOC release (Morán et al. 2002). This implies the existence of other autochthonous (e.g. cellular lysis, grazer activity) or allochthonous dissolved organic sources in addition to algal release, making it possible that phytoplankton release did not significantly enhance respiration in the light.

The present work shows robust evidence that under natural conditions respiration in the light is not significantly different to respiration in the dark. Consequently, the light-dark method is an accurate and unambiguous method to measure marine PP.

## O₂ vs. ¹⁴C rates

¹⁴C fixation data including DOC fraction are scarce, especially in oligotrophic coastal systems. The rates (from 0.96 to 0.16 µmol l⁻¹ d⁻¹) were higher than those obtained in the open ocean with or without accounting for the DO-¹⁴C fraction (< 1.5 µmol l⁻¹ d⁻¹, Grande et al. 1989, Juranek & Quay 2005), and within (0.1 to 9.4 µmol l⁻¹ d⁻¹, Hashimoto et al. 2005) or lower than the range of coastal environments (e.g. 4 to 60 µmol l⁻¹ d⁻¹, Morán et al. 2002; >60 µmol l⁻¹ d⁻¹, Marañón et al. 2004). ¹⁴C uptake is lower than GPP if part of the ¹⁴C pool in the dissolved organic form is not collected and measured or if some photosynthetically fixed ¹⁴C is re-mineralized during the incubation. It is therefore important to consider the incubation time. Weger et al. (1989) suggested that the ¹⁴C technique estimates GPP when short incubations (1 to 3 h) are used because respiratory losses can be ignored. In agreement with earlier works (e.g. Raine 1983, Langdon et al. 1995) our results showed that ¹⁴C uptake in incubations of up to 6 to 12 h, overestimate NPP and are closer to GPP. There are also uncertainties regarding the magnitude of extracellular release of recently fixed carbon. Most previous papers that have concluded that ¹⁴C uptake is closer to NPP, did not account for the DOC fraction (e.g. Marra 2002, Juranek & Quay 2005). However, the contribution of dissolved PP can be important, especially in oligotrophic systems where productivity tends to be low (Moran et al. 2002). Therefore, PP can be significantly underestimated if the DO-¹⁴C fraction is not taken into account (e.g. Marañón et al. 2004). Dissolved organic carbon release accounted for about 47% of total carbon fixation in the present study. We

have no evidence that the high DOC production rates were an artefact of improper handling of samples. For instance, care was taken to ensure that the phytoplankton assemblage was not light shocked during sampling (samples were collected before sunrise and were incubated *in situ*). However, incubation times were rather long (9 to 15 h) depending on the day light length and some ¹⁴C may have been recycled. Dickson et al. (2001) and Hansell & Carlson (1998) employed shorter incubations (4 to 6 h) and nevertheless also obtained high percentages of DOC production (e.g. 79 ± 5% and 59 to 70%, respectively). Our results clearly highlight the need to include the dissolved organic fraction in oligotrophic coastal waters such as those found in the Mediterranean.

When the oxygen and ¹⁴C fluxes are compared, careful attention needs to be paid to the PQ. It can vary widely (from 0.5 to 5) but generally ranges between 1.2 and 1.8 (Laws 1991). We obtained an apparent PQ of 1.5 (molar flux ratio O₂/¹⁴C), which is within the PQ range, and was derived from natural assemblages and not from non-specific phytoplankton blooms or cultures. According to Rabinowitch (1945), PQ is different from the conventional value of 1.0, mainly due to the synthesis of lipids (PQ > 1) or organic acids (PQ < 1) rather than the carbohydrates. In contrast, Burris (1981) observed that PQs higher than 1 were more likely as a consequence of the nitrogen substrate used by phytoplankton. When nitrate as a replacement for ammonia is assimilated by phytoplankton PQ tends to increase. PQ rises as a result of O₂ release during the reduction of nitrate to ammonium, in the range of 1.5 to 1.8 (Williams et al. 1979) or up to 2 (Raine 1983). Laws (1991) used alternative methods to estimate PQ based on balanced chemical equations and reported PQ estimates of 1.4 for new production (nitrate as the main source of nitrogen) and 1.1 for recycled production (ammonium as the main source of nitrogen). In our study area (Northwest Mediterranean), we observed abundances of cyanobacteria of about 2 × 10⁵ cells ml⁻¹ (data not shown). This abundance is even higher than that reported by Duarte & Agustí (2004) in the Bay of Blanes (10⁴ cells ml⁻¹), considering that cyanobacteria largely dominate the autotrophic biomass in summer. According to Bergman et al. (1997), aerobic N₂ fixation by cyanobacteria entails O₂ consumption as part of the nitrogenase autoprotection mechanism. Therefore, our data confirm that PQ values higher than the commonly used value of 1.25 are not unusual (e.g. Williams et al. 1979). In any case, and using more conservative PQs (<1.5), our results confirmed that PP based on ¹⁴C method is closer to gross than NPP (Table 5).

Finally, it should be noted that these conclusions were obtained from comparing TO-¹⁴C vs. GPP-O₂ and NCP rates using model II reduced major axis regression
sion (RMA) instead of the ordinary least squares regression (OLS) model I (see Table 5). According to McArdle (1987), when the coefficient of determination is sufficiently large ($r^2$ > 0.9) it does not matter which model is used. However, when the coefficient of determination is low and the X variable is subject to significant error, it is recommended to carry out both regressions (model I and II), selecting always the RMA if there is any difference. In this respect, our paper differs from others that rely on model I regression (e.g. Marra 2002, Marra & Barber 2004, Juranek & Quay 2005).

*Acknowledgements*. Many individuals contributed to the successful completion of the field work, in particular J. Nieuwenhuize, M.-D. Pizay and the skippers of the research boats. The CTD data were kindly provided by C. Heyndrickx and the SOMLIT program. We thank an anonymous reviewer and Prof. Williams for constructive feedback and editor P. del Giorgio for his patience. This research was supported by the EU in the framework of the EUROTROPH project (contract # EVK3-CT-2000-00040) and by an M.C. individual fellowship (MCFI-2001-01425). Additional support was provided by NWO (Dutch-French co-operation on sea research program).

## LITERATURE CITED

Barber RT, Hilting AK (2002) History of the study of plankton productivity. In: Williams PJLeB, Thomas DN, Reynolds CS (eds) Phytoplankton productivity: carbon assimilation in marine and freshwater ecosystems. Blackwell Science, Oxford, p 16–43

► Bate GC, Sueltemeyer DF, Fock HP (1988) ¹⁶O₂/¹⁸O₂ analysis of oxygen exchange in *Dunaliella tertioleta*. Evidence for the inhibition of mitochondrial respiration in the light. Photosynth Res 16:219–231

► Bates NR, Hansell DA, Carlson CA (1998) Distribution of CO₂ species, estimates of net community production, and air-sea CO₂ exchange in the Ross Sea polynya. J Geophys Res 103:2883–2896

Bender ML, Grande K, Johnson K (1987) A comparison of four methods for determining planktonic community production. Limnol Oceanogr 32:1085–1098

Bergman B, Gallon JR, Rai AN, Stal LJ (1997) N₂ fixation by non-heterocystous cyanobacteria. FEMS Microbiol Rev 19:139–185

► Burris JE (1981) Effects of oxygen and inorganic carbon concentrations on the photosynthetic quotients of marine algae. Mar Biol 65:215–219

Carignan R, Planas D, Vis C (2000) Planktonic production and respiration in oligotrophic shield lakes. Limnol Oceanogr 45:189–199

► Corredor JE, Wawrik B, Paul JH, Tran H and others (2004) Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs. Appl Environ Microbiol 70:5459–5468

► Dickson ML, Orchardo J (2001) Oxygen production and respiration in the Antarctic Polar Front region during the annual spring and summer. Deep-Sea Res II 48:4101–4126

► Dickson ML, Orchardo J, Barber RT, Marra J, McCarthy JJ, Sambrotto RN (2001) Production and respiration rates in the Arabian Sea during the 1995 Northeast and southwest Monsoons. Deep-Sea Res II 48:1199–1230

Duarte CM, Agustí S (2004) Controls on planktonic metabolism in the Bay of Blanes, northwestern Mediterranean littoral. Limnol Oceanogr 49:2162–2170

► Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240

► Gattuso JP, Peduzzi S, Pizay MD, Tonolla M (2002) Changes of bacterial community composition during measurements of community and microbial respiration. J Plankton Res 24:1197–1206

Gazeau F, Duarte CM, Gattuso JP, Barrón C and others (2005) Whole-system metabolism and CO₂ fluxes in a Mediterranean Bay dominated by seagrass beds (Palma Bay, NW Mediterranean). Biogeosciences 2:43–60

► Gazeau F, Middelburg JJ, Loijens M, Vanderborght JP, Pizay MD, Gattuso JP (2007) Planktonic primary production in estuaries: comparison of ¹⁴C, O₂, and ¹⁸O methods. Aquat Microb Ecol 46:95–106

Geider RJ (1992) Respiration: Taxation without representation? In: Falkowski PG, Woodhead AD (eds) Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, p 333–360

► Gomez F, Gorsky G (2003) Annual microplankton cycles in Villefranche Bay, Ligurian Sea, NW Mediterranean. J Plankton Res 25:323–339

► González N, Anadón R, Viesca L (2003) Carbon flux through the microbial community in a temperate sea during summer: role of bacterial metabolism. Aquat Microb Ecol 33: 117–126

► Grande KD, Williams PJLeB, Marra J, Purdie DA, Heinemann K, Eppley RW, Bender ML (1989) Primary production in the North Pacific gyre: a comparison of rates determined by the ¹⁴C, O₂ concentration and ¹⁸O methods. Deep-Sea Res A 36:1621–1634

► Hansell DA, Carlson CA (1998) Net community production of dissolved organic carbon. Global Biogeochem Cycles 12: 443–453

Hashimoto N, Yamaguchi Y, Ishimaru T (2005) Relationship between net and gross primary production in the Sagami Bay, Japan. Limnol Oceanogr 50:1830–1835

► Juranek LW, Quay PD (2005) In vitro and in situ gross primary and net community production in the North Pacific Subtropical Gyre using labeled and natural abundance isotopes of dissolved O₂. Global Biogeochem Cycles 19: GB3009

Karl DM, Hebel DV, Bjökman K, Letelier RM (1998) The role of dissolved organic matter release in the productivity of the oligotrophic North Pacific Ocean. Limnol Oceanogr 43:1270–1286

Knap AH, Michaels AE, Close A, Ducklow HW, Dickson AG (1996) Protocols for the joint global ocean flux study (JGOFS) core measurements. JGOFS Report 19:170. Reprint of the IOC manuals and gudies No. 29, UNESCO 1994.

► Langdon C, Marra J, Knudson C (1995) Measurements of net and gross O₂ production, dark O₂ respiration, and ¹⁴C assimilation at the Marine Light-Mixed Layers site (59°N, 21°W) in the northeast Atlantic Ocean. J Geophys Res 100:6645–6653

► Laws EA (1991) Photosynthetic quotients, new production and net community production in the open ocean. Deep-Sea Res A 38:143–167

► Laws EA, Landry MR, Barber RT, Campbell L, Dickson ML, Marra J (2000) Carbon cycling in primary production bottle incubations: inferences from grazing experiments and photosynthetic studies using ¹⁴C and ¹⁸O in the Arabian Sea. Deep-Sea Res II 47:1339–1352
sion (RMA) instead of the ordinary least squares regression (OLS) model I (see Table 5). According to McArdle (1987), when the coefficient of determination is sufficiently large ($r^2 > 0.9$) it does not matter which model is used. However, when the coefficient of determination is low and the X variable is subject to significant error, it is recommended to carry out both regressions (model I and II), selecting always the RMA if there is any difference. In this respect, our paper differs from others that rely on model I regression (e.g. Marra 2002, Marra & Barber 2004, Juranek & Quay 2005).

*Acknowledgements*. Many individuals contributed to the successful completion of the field work, in particular J. Nieuwenhuize, M.-D. Pizay and the skippers of the research boats. The CTD data were kindly provided by C. Heyndrickx and the SOMLIT program. We thank an anonymous reviewer and Prof. Williams for constructive feedback and editor P. del Giorgio for his patience. This research was supported by the EU in the framework of the EUROTROPH project (contract # EVK3-CT-2000-00040) and by an M.C. individual fellowship (MCFI-2001-01425). Additional support was provided by NWO (Dutch-French co-operation on sea research program).

## LITERATURE CITED

Barber RT, Hilting AK (2002) History of the study of plankton productivity. In: Williams PJLeB, Thomas DN, Reynolds CS (eds) Phytoplankton productivity: carbon assimilation in marine and freshwater ecosystems. Blackwell Science, Oxford, p 16–43

► Bate GC, Sueltemeyer DF, Fock HP (1988) ¹⁶O₂/¹⁸O₂ analysis of oxygen exchange in *Dunaliella tertioleta*. Evidence for the inhibition of mitochondrial respiration in the light. Photosynth Res 16:219–231

► Bates NR, Hansell DA, Carlson CA (1998) Distribution of CO₂ species, estimates of net community production, and air-sea CO₂ exchange in the Ross Sea polynya. J Geophys Res 103:2883–2896

Bender ML, Grande K, Johnson K (1987) A comparison of four methods for determining planktonic community production. Limnol Oceanogr 32:1085–1098

Bergman B, Gallon JR, Rai AN, Stal LJ (1997) N₂ fixation by non-heterocystous cyanobacteria. FEMS Microbiol Rev 19:139–185

► Burris JE (1981) Effects of oxygen and inorganic carbon concentrations on the photosynthetic quotients of marine algae. Mar Biol 65:215–219

Carignan R, Planas D, Vis C (2000) Planktonic production and respiration in oligotrophic shield lakes. Limnol Oceanogr 45:189–199

► Corredor JE, Wawrik B, Paul JH, Tran H and others (2004) Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs. Appl Environ Microbiol 70:5459–5468

► Dickson ML, Orchardo J (2001) Oxygen production and respiration in the Antarctic Polar Front region during the annual spring and summer. Deep-Sea Res II 48:4101–4126

► Dickson ML, Orchardo J, Barber RT, Marra J, McCarthy JJ, Sambrotto RN (2001) Production and respiration rates in the Arabian Sea during the 1995 Northeast and southwest Monsoons. Deep-Sea Res II 48:1199–1230

Duarte CM, Agustí S (2004) Controls on planktonic metabolism in the Bay of Blanes, northwestern Mediterranean littoral. Limnol Oceanogr 49:2162–2170

► Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240

► Gattuso JP, Peduzzi S, Pizay MD, Tonolla M (2002) Changes of bacterial community composition during measurements of community and microbial respiration. J Plankton Res 24:1197–1206

Gazeau F, Duarte CM, Gattuso JP, Barrón C and others (2005) Whole-system metabolism and CO₂ fluxes in a Mediterranean Bay dominated by seagrass beds (Palma Bay, NW Mediterranean). Biogeosciences 2:43–60

► Gazeau F, Middelburg JJ, Loijens M, Vanderborght JP, Pizay MD, Gattuso JP (2007) Planktonic primary production in estuaries: comparison of ¹⁴C, O₂, and ¹⁸O methods. Aquat Microb Ecol 46:95–106

Geider RJ (1992) Respiration: Taxation without representation? In: Falkowski PG, Woodhead AD (eds) Primary productivity and biogeochemical cycles in the sea. Plenum Press, New York, p 333–360

► Gomez F, Gorsky G (2003) Annual microplankton cycles in Villefranche Bay, Ligurian Sea, NW Mediterranean. J Plankton Res 25:323–339

► González N, Anadón R, Viesca L (2003) Carbon flux through the microbial community in a temperate sea during summer: role of bacterial metabolism. Aquat Microb Ecol 33: 117–126

► Grande KD, Williams PJLeB, Marra J, Purdie DA, Heinemann K, Eppley RW, Bender ML (1989) Primary production in the North Pacific gyre: a comparison of rates determined by the ¹⁴C, O₂ concentration and ¹⁸O methods. Deep-Sea Res A 36:1621–1634

► Hansell DA, Carlson CA (1998) Net community production of dissolved organic carbon. Global Biogeochem Cycles 12: 443–453

Hashimoto N, Yamaguchi Y, Ishimaru T (2005) Relationship between net and gross primary production in the Sagami Bay, Japan. Limnol Oceanogr 50:1830–1835

► Juranek LW, Quay PD (2005) In vitro and in situ gross primary and net community production in the North Pacific Subtropical Gyre using labeled and natural abundance isotopes of dissolved O₂. Global Biogeochem Cycles 19: GB3009

Karl DM, Hebel DV, Bjökman K, Letelier RM (1998) The role of dissolved organic matter release in the productivity of the oligotrophic North Pacific Ocean. Limnol Oceanogr 43:1270–1286

Knap AH, Michaels AE, Close A, Ducklow HW, Dickson AG (1996) Protocols for the joint global ocean flux study (JGOFS) core measurements. JGOFS Report 19:170. Reprint of the IOC manuals and gudies No. 29, UNESCO 1994.

► Langdon C, Marra J, Knudson C (1995) Measurements of net and gross O₂ production, dark O₂ respiration, and ¹⁴C assimilation at the Marine Light-Mixed Layers site (59°N, 21°W) in the northeast Atlantic Ocean. J Geophys Res 100:6645–6653

► Laws EA (1991) Photosynthetic quotients, new production and net community production in the open ocean. Deep-Sea Res A 38:143–167

► Laws EA, Landry MR, Barber RT, Campbell L, Dickson ML, Marra J (2000) Carbon cycling in primary production bottle incubations: inferences from grazing experiments and photosynthetic studies using ¹⁴C and ¹⁸O in the Arabian Sea. Deep-Sea Res II 47:1339–1352
<figure><img src="image_5.png" /></figure>

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<figure><img src="image_6.png" /></figure>

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<figure><img src="image_7.png" /></figure>

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

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<figure><img src="image_7.png" /></figure>

Lewitus AJ, Kana TM (1995) Light respiration in six estuarine phytoplankton species: contrasts under photoautotrophic and mixotrophic growth conditions. *J Phycol* 31:754–761

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

Luz B, Barkan E (2000) Assessment of oceanic productivity with the triple-isotope composition of dissolved oxygen. *Science* 288:2028–2031

Marañón E (2005) Phytoplankton growth rates in the Atlantic subtropical gyres. *Limnol Oceanogr* 50:299–310

Marañón E, Cermeño P, Fernández E (2004) Significance and mechanism of photosynthetic production of dissolved organic carbon in a coastal eutrophic ecosystem. *Limnol Oceanogr* 49:1652–1666

Marra J (2002) Approaches to the measurements of plankton production. In Willimas PJLeB, Thomas DN, Reynolds CS (eds) Phytoplankton productivity: carbon assimilation in marine and freshwater ecosystems. Blackwell, Oxford, p 78–108

<figure><img src="image_9.png" /></figure>

Marra J, Barber RT (2004) Phytoplankton and heterotrophic respiration in the surface layer of the ocean. *Geophys Res Lett* 31:L09314

McArdle BH (1987) The structural relationship: regression in biology. *Can J Zool* 66:2329–2339

<figure><img src="image_10.png" /></figure>

Móran XAG, Estrada M, Gasol JM, Pedrós-Alió C (2002) Dis-
solved primary production and the strength of phyto-
plankton-bacterioplankton coupling in contrasting marine
regions. *Microb Ecol* 44:217–223

Pringault O, Tassas V, Rochelle-Newall E (2007) Consequences of respiration in the light on the determination of production in pelagic systems. *Biogeosciences* 4:105–114

Quinn GP, Keough MJ (2002) Experimental design and data analysis for biologists. Cambridge University Press, Cambridge

Rabinowitch EI (ed) (1945) Photosynthesis and related
processes, Vol I. Interscience Publishers, New York

Raine RCT (1983) The effect of nitrogen supply on the photo-
synthetic quotient of natural phytoplankton assemblages.
Bot Mar 26:417–423

Robinson C, Williams PJLeB (2005) Respiration and its mea-

surement in the surface waters. In: del Giorgio PA, Williams PJLeB (eds) Respiration in aquatic ecosystems. Oxford University Press, New York, p 147–180

Sarma VVSS, Abe O, Hashimoto S, Hinuma S, Saino T (2005) Seasonal variations in triple oxygen isotopes and gross oxygen production in the Sagami Bay, central Japan. *Limnol Oceanogr* 50:544–552

Sokal RR, Rohlf FJ (1995) Biometry, the principles and prac-
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