Effects of CO2 perturbation on phosphorus pool sizes and uptake in a mesocosm experiment during a low productive summer season in the northern Baltic Sea

Abstract

Studies investigating the effect of increasing CO₂ levels on the phosphorus cycle in natural waters are lacking although phosphorus often controls phytoplankton development in many aquatic systems. The aim of our study was to analyse effects of elevated CO₂ levels on phosphorus pool sizes and uptake. The phosphorus dynamic was followed in a CO₂-manipulation mesocosm experiment in the Storfjärden (western Gulf of Finland, Baltic Sea) in summer 2012 and was also studied in the surrounding fjord water. In all mesocosms as well as in surface waters of Storfjärden, dissolved organic phosphorus (DOP) concentrations of 0.26 ± 0.03 and 0.23 ± 0.04 µmol L^-1, respectively, formed the main fraction of the total P-pool (TP), whereas phosphate (PO₄) constituted the lowest fraction with mean concentration of 0.15 ± 0.02 in the mesocosms and 0.17 ± 0.07 µmol L^-1 in the fjord. Transformation of PO₄ into DOP appeared to be the main pathway of PO₄ turnover. About 82 % of PO₄ was converted into DOP whereby only 18 % of PO₄ was transformed into particulate phosphorus (PP). PO₄ uptake rates measured in the mesocosms ranged between 0.6 and 3.9 nmol L^-1 h^-1. About 86 % of them was realized by the size fraction < 3 µm. Adenosine triphosphate (ATP) uptake revealed that additional P was supplied from organic compounds accounting for 25-27 % of P provided by PO₄ only. CO₂ additions did not cause significant changes in phosphorus (P) pool sizes, DOP composition, and uptake of PO₄ and ATP when the whole study period was taken into account. However, significant short-term effects were observed for PO₄ and PP pool sizes in CO₂ treatments > 1000 µatm during periods when phytoplankton biomass increased. In addition, we found significant relationships (e.g., between PP and Chl <i>a</i>) in the untreated mesocosms which were not observed under high <i>f</i>CO₂ conditions. Consequently, it can be hypothesized that the relationship between PP formation and phytoplankton growth changed with CO₂ elevation. It can be deduced from the results, that visible effects of CO₂ on P pools are coupled to phytoplankton growth when the transformation of PO₄ into POP was stimulated. The transformation of PO₄ into DOP on the other hand does not seem to be affected. Additionally, there were some indications that cellular mechanisms of P regulation might be modified under CO₂ elevation changing the relationship between cellular constituents.

Biogeosciences, 13, 30353050, 2016 www.biogeosciences.net/13/3035/2016/ doi:10.5194/bg-13-3035-2016 Author(s) 2016. CC Attribution 3.0 License.

Monika Nausch1, Lennart Thomas Bach2, Jan Czerny2, Josephine Goldstein1,a, Hans-Peter Grossart4,5, Dana Hellemann2,b, Thomas Hornick4, Eric Pieter Achterberg2,3, Kai-Georg Schulz2,c, and Ulf Riebesell2

1Leibniz Institute for Baltic Sea Research, Seestrasse 15, 18119 Rostock, Germany

2GEOMAR Helmholtz Centre for Ocean Research Kiel, Dsternbrooker Weg 20, 24105 Kiel, Germany

3Ocean and Earth Science, University of Southampton National Oceanography Centre Southampton, Southampton SO14 3ZH, UK

4Leibniz-Institute for Freshwater Ecology and Inland Fisheries, Zur alten Fischerhtte 2, 16775 Stechlin, Germany

5Potsdam University, Institute for Biochemistry and Biology, Maulbeerallee 2, 14469 Potsdam, Germany

anow at: Max-Planck Odense Center on the Biodemography of Aging & Department of Biology, Campusvej 55, 5230 Odense M, Denmark

bnow at: Department of Environmental Sciences, University of Helsinki, PL 65 00014 Helsinki, Finland

cnow at: Centre for Coastal Biogeochemistry, School of Environment, Science and Engineering, Southern Cross University, Lismore, Australia

Correspondence to: Monika Nausch ([email protected])

Received: 25 September 2015 Published in Biogeosciences Discuss.: 30 October 2015 Revised: 12 April 2016 Accepted: 25 April 2016 Published: 24 May 2016

Abstract. Studies investigating the effect of increasing CO2 levels on the phosphorus cycle in natural waters are lacking although phosphorus often controls phytoplankton development in many aquatic systems. The aim of our study was to analyse effects of elevated CO2 levels on phosphorus pool sizes and uptake. The phosphorus dynamic was followed in a CO2-manipulation mesocosm experiment in the

Storfjrden (western Gulf of Finland, Baltic Sea) in summer 2012 and was also studied in the surrounding fjord water. In all mesocosms as well as in surface waters of Storfjrden, dissolved organic phosphorus (DOP) concentrations of 0.26 0.03 and 0.23 0.04 mol L1, respec

tively, formed the main fraction of the total P-pool (TP), whereas phosphate (PO4) constituted the lowest fraction with mean concentration of 0.15 0.02 in the mesocosms and

0.17 0.07 mol L1 in the fjord. Transformation of PO4

into DOP appeared to be the main pathway of PO4 turnover.

About 82 % of PO4 was converted into DOP whereby only 18 % of PO4 was transformed into particulate phosphorus (PP). PO4 uptake rates measured in the mesocosms ranged between 0.6 and 3.9 nmol L1 h1. About 86 % of them was

realized by the size fraction < 3 m. Adenosine triphosphate (ATP) uptake revealed that additional P was supplied from organic compounds accounting for 2527 % of P provided by PO4 only. CO2 additions did not cause signicant changes in phosphorus (P) pool sizes, DOP composition, and uptake of PO4 and ATP when the whole study period was taken into account. However, signicant short-term effects were observed for PO4 and PP pool sizes in CO2 treatments > 1000 atm during periods when phytoplankton biomass increased. In addition, we found signicant relationships (e.g., between PP and Chl a) in the untreated mesocosms which were not observed under high f CO2 conditions. Consequently, it can be hypothesized that the relationship between PP formation and phytoplankton growth changed with CO2 elevation. It can be deduced from the results, that visible effects of CO2 on P pools are coupled to phytoplankton growth when the transformation of PO4 into POP was stimulated. The transformation of PO4 into DOP on the other hand does not seem to be affected. Additionally, there were some indications that cellular mechanisms of P regulation might be modied un-

Published by Copernicus Publications on behalf of the European Geosciences Union.

Effects of CO2 perturbation on phosphorus pool sizes and uptake in a mesocosm experiment during a low productive summer season in the northern Baltic Sea

3036 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

der CO2 elevation changing the relationship between cellular constituents.

1 Introduction

Increasing emissions of anthropogenic CO2 into the atmosphere and subsequent acidication of the ocean can potentially affect the diversity of organisms and the functioning of marine ecosystems (Eisler, 2012). The rise in atmospheric CO2 concentrations was accelerated from 3.4 0.2 in the

1980s to 4.0 0.2 PgC yr1 in the 2000s leading to increases

of CO2 in ocean surface waters at a similar rate (IPCC, 2013).

Atmospheric CO2 is projected to rise to 7501000 ppm and higher in 2100 (IPCC, 2001) corresponding with a decrease in open ocean pH by 0.30.5 units (Caldeira and Wickett, 2005) from the present level of 8.1. Although this process

is of global signicance and all parts of the oceans are at risk, there will be regional differences in the degree of acid-ication (Borges et al., 2005). Thus, to determine the CO2-related changes in the oceans, multiple studies in different regions are required. Semi-enclosed coastal regions, such as the Baltic Sea, react with higher changes in pH to CO2 elevation than open ocean waters due to high freshwater inputs resulting in a reduced buffer capacity (Orr, 2011).

In the Baltic Sea, several studies of CO2 effects have been undertaken on the organism level of sh (Frommel et al., 2013), zooplankton (Pansch et al., 2012; Vehmaa et al., 2012), macrophytes (Pajusalu et al., 2013), benthic species (Hiebenthal et al., 2013; Stemmer et al., 2013), and lamentous cyanobacteria (Czerny et al., 2009; Eichner et al., 2014; Wannicke et al., 2012). Studies on the impacts of elevated CO2 at the ecosystem level, however, have thus far been limited to Kiel Bight in the western Baltic Sea (Engel et al., 2014; Rossoll et al., 2013; Schulz and Riebesell, 2013), which may fundamentally differ from other parts of the Baltic Sea.

Next to nitrogen, phosphorus (P) controls the productivity of phytoplankton in the ocean (Karl, 2000; Sanudo-Wilhelmy et al., 2001; Tyrrell, 1999) and is a limiting factor in some regions (Ammerman et al., 2003). The total phosphorus (TP) pool comprises phosphate (PO4), dissolved organic phosphorus (DOP), and particulate organic (POP) and inorganic (PIP) phosphorus. There is a continuous transformation of phosphorus between these P species due to their uptake, conversion, and release by organisms as well as by interaction with minerals. While PO4 is the preferred P-species of phyto- and bacterioplankton, DOP can become an important P source when PO4 is depleted (Llebot et al., 2010;

Lomas et al., 2010). DOP includes nucleic acids, phospholipids, and adenosine triphosphate (ATP; Karl and Bjrkman, 2002) which are structural and functional components of all living cells, but, can also be released into the surrounding water.

Figure 1. The Baltic Sea and the location near the peninsula Hanko in the western Gulf of Finland where the mesocosms were deployed.

In general, there is little knowledge on how the P cycle is affected by ocean acidication and how related changes in P availability inuence the response of organisms to CO2 elevation. In CO2 manipulation experiments, particulate phosphorus dynamics were studied to determine effects on C : P stoichiometry of phytoplankton (Riebesell and Tortell, 2011;Sugie and Yoshimura, 2013) and PO4 concentration dynamics to estimate its utilization (Bellerby et al., 2008). CO2 effects on phosphorus pool sizes and PO4 uptake have so far been studied by Tanaka et al. (2008) in the Raunefjorden, Norway and by Unger et al. (2013) and Endres et al. (2013) in laboratory experiments with cultures of Nodularia spumigena. In order to reduce the gap of knowledge, we studied the impact of elevated CO2 on phosphorus pool sizes, the DOP composition, and PO4 uptake of a northern Baltic Sea plankton community. These measurements provide important information on potential changes in P cycling under increasing CO2 levels and contribute to a better understanding of the P cycle in brackish water ecosystems.

2 Material and methods

2.1 Experimental design and CO2 manipulation

The study was conducted in the northwestern Gulf of Finland, in the proximity of the Tvrminne Zoological Station (TZS; Fig. 1), between 17 June and 4 August 2012, using the KOSMOS mesocosm system (Riebesell et al., 2013).Nine mesocosms (M1M9) were moored in the open waters of the Storfjrden (5951.5 N, 2315.5 E) at a water depth

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M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3037

of 30 m. Only six of them were included throughout the

whole study period since leakages in the remaining three rendered them unusable. Equipment and deployment procedures are described in detail by Paul et al. (2015b). Briey, polyurethane enclosure bags of 2 m in diameter and 18.5 m in length were mounted in oating frames and lowered in such a way that 17 m of each bag was immersed in the wa

ter column and 1.5 m remained above the water surface.

Large organisms were excluded from the mesocosms by a 3 mm mesh installed at the top and bottom of the bags before closure. The mesocosms were deployed 10 days prior to CO2 manipulation to rinse the bags and for full water exchange.Sediment traps were mounted on the lower ends to close them water tight, while the upper ends were raised above the water surface to prevent water entry during wave action. The mesocosms were covered with a dome-shaped roof to prevent nutrient input by birds and potentially signicant fresh water input by rain. Salinity gradients were removed by bubbling the mesocosms with compressed air for 3.5 min, so that 5 days before the start of the experiment (day 5) the water

body was fully homogeneous.

CO2 was injected at day 0 and the subsequent 4 days by pumping various quantities of 50 m-ltered and CO2-enriched fjord water into seven of the mesocosms as described by Riebesell et al. (2013). The intended CO2 and pH gradients were reached after the last treatment on day4. Details are described in Paul et al. (2015b). For the two untreated (control) mesocosms, only ltered fjord water was added to adjust the water volume to that of the treated mesocosms. To compensate for outgassing, the CO2 manipulation was similarly repeated in the upper 7 m layer of the mesocosms on day 16.

2.2 Sampling

Daily sample collection started 3 days before the rst CO2 injection (day 3). Parallel samples were taken from the

surrounding fjord. Sampling over the entire 17 m depth was carried out using an integrating water sampler (IWS HYDROBIOS-KIEL) that was lowered slowly on a cable by hand. The sampling frequency differed depending on the parameter to be observed as shown in the overview by Paul et al. (2015b).

Phosphorus pool parameters and uptake rates were determined every second day, except for dissolved organic phosphorus (DOP) components, which were measured every 4 days. Termination of the measurements varied due to logistical constrains. Thus, total phosphorus (TP) and DOP were sampled only until day 29 whereas other parameters were sampled until day 43.

The collected water was lled in HCl-cleaned polyethylene canisters that had been pre-rinsed with sample water. All containers were stored in the dark. Back on land, subsamples were processed immediately for each P-analysis. The other analyses were carried out within a few hours of sample col-

lection and sample storage in a climate room at in situ temperature.

2.3 Analytical methods

2.3.1 Temperature, salinity, and carbonate chemistry

Measurements in the fjord and in each mesocosm were conducted using a CTD60M memory probe (Sea and sun technology, Trappenkamp, Germany) lowered from the surface to a depth of 17 m at about 0.3 m s1 in the early afternoon (01:3002:30 p.m.). For these parameters, depth-integrated mean values are presented here.

The carbonate system is described in detail in Paul et al. (2015b). The pHT (total scale) was determined using a spectrophotometric method (Dickson et al., 2007) on a Cary 100 (Varian) and the dye m-cresol as indicator. Extinction was measured at 578 (E1) and 434 nm (E2) in a 10 cm cuvette. The pH was calculated from the ratio of E1 and E2 (Clayton and Byrne, 1993).

DIC was measured using a coulometric AIRICA system (MARIANDA, Kiel) measuring the infrared absorption after N2 purging of the sample and calibration with certied reference material (CRM; A. Dickson, University of California, San Diego).

The f CO2 was calculated from DIC, pHT, salinity and using the stoichiometric equilibrium constant for carbonic acid of Mehrbach et al. (1973) as retted by Lueker et al. (2000).

2.3.2 Chlorophyll and inorganic nutrients

Subsamples of 500 mL were ltered onto GF/F-lters. Chl a was extracted in acetone (90 %) in plastic vials by homogenisation of the lters for 5 min in a cell mill using glass beads.After centrifugation (10 min, 800 g, 4 C) the supernatant

was analysed on a uorometer (TURNER 10-AU) at an excitation of 450 nm and an emission of 670 nm to determine Chl a concentrations (Jeffrey and Welschmeyer, 1997).

A segmented continuous-ow analyzer coupled with a liquid-waveguide capillary ow-cell (LWCC) of 2 m length was used to determine phosphate (PO4) and the sum of nitrite and nitrate (NO2 + NO3) at nanomolar precision (Patey

et al., 2008). The PO4 determination was based on the molybdenum blue method of Murphy and Riley (1962), and NO3+NO2 on the method of Morris and Riley (1963). PO4

concentrations from the same subsample were also measured manually using a 5 cm cuvette (Grasshoff et al., 1983). In most of the samplings PO4 data obtained from both methods did not differ signicantly (paired t test: p = 0.262,

t = 1.127, n = 109).

2.3.3 Dissolved organic phosphorus (DOP)

For the determination of DOP, duplicate 40 mL subsamples were ltered through pre-combusted (6 h, 450 C) glass ber lters (Whatman GF/F) and stored in 50 mL vials (Falkon) at

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Dissolved phospholipids

The phosphate content of the dissolved phospholipids (PL-P) was analysed using a modied method of Suzumura and In-gall (2001, 2004). Briey, 400 mL subsamples of the ltrate were stored at 20 C until further processing. The sam

ples were then thawed in a water bath at 30 C and extracted twice with 100 mL of chloroform. The chloroform phase was collected, concentrated to 5 mL in a rotary evaporator (Heidolph Instruments, Schwabach, Germany), and then transferred into microwave tubes. The chloroform was completely evaporated by incubating the tubes in a 60 C water bath overnight. After the addition of 20 mL of deionized water (Milli-Q, Millipore), the samples were digested with potassium peroxydisulfate in alkaline medium and microwaved as described for the DOP analysis. Six standard concentrations of phospholipids, ranging from 0 to 125 g L1, were prepared by adding the respective amounts of a stock solution containing 5 mg of l-phosphatidyl-dl-glycerol sodium salt (PG, Sigma Aldrich, P8318) mL1 to the aged seawater. The detection limit was 0.8 nmol L1. The blanks contained only chloroform and were processed as for the samples.

Dissolved DNA and RNA

Dissolved DNA and RNA (dDNA and dRNA) concentrations were determined according to Karl and Bailiff (1989) and as described by Unger et al. (2013). For each sample, 200 mL of the ltrate was gently mixed with the same volume of ethylene-diamine-tetracetic acid (EDTA, 0.1 M, pH9.3, Merck, 1.08454) and 4 mL of cetyltrimethyl-ammonium bromide (CTAB, Sigma-Aldrich, H5882) and stored frozen at 20 C for at least 24 h. After thawing the samples, the

precipitate was collected onto combusted (450 C, 6 h) glass ber lters (25 mm, GF/F Whatman), placed into annealed vials, and stored frozen at 80 C until further analysis.

DNA concentrations were measured using a uorescence-spectrophotometer (Hitachi F 2000), and RNA concentrations using a dual-beam UV/VIS-spectrophotometer U3010 (Hitachi).

Coupled standards (DNA + RNA) containing 110 g

DNA (Sigma Aldrich, D3779) L1 and 20120 g RNA (Sigma Aldrich, R1753) L1 were prepared in aged seawater as described above. A reagent blank served as the reference and aged seawater as the background control. The P-contents of the DNA and RNA were calculated by multiplying the measured values by a factor of 2.06 nmol P per g dDNA and2.55 nmol P per g dRNA. The latter values were determined by the microwave digestion of standard substrates.

2.3.5 Particulate organic phosphorus, carbon, and nitrogen

Particulate phosphorus (PP) was analysed using two methods in parallel. In the aqueous method, 40 mL of unl-

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3038 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

20 C until further processing. The thawed samples were oxidized in a microwave (MARSXpress, CEM, Matthews, USA; Johnes and Heathwaite, 1992) after the addition of potassium peroxydisulfate in an alkaline medium (Bhaya et al., 2000). The P concentration, measured as PO4 in a 10 cm cuvette, represents the total dissolved phosphorus (DP) concentration. DOP was calculated as the difference between the DP concentrations in the ltered and digested samples and the corresponding PO4 concentration analysed as described above.

2.3.4 Dissolved organic phosphorus compounds

For all analysed components, subsamples were pre-ltered through pre-combusted (6 h, 450 C) lters (Whatman GF/F)

to remove larger particles followed by ltration through0.2 m cellulose acetate lters to remove picoplankton. Sub-samples were prepared for storage according to the specic method used for each compound. After the analyses, the phosphorus content of measured DOP compounds was summed and the amount subtracted from the total DOP concentration. The difference is dened as the uncharacterized DOP.

Dissolved ATP

The method of (Bjrkman and Karl, 2001) adapted to Baltic Sea conditions (Unger et al., 2013) was used to determine dissolved adenosine triphosphate (dATP). An Mg(OH)2 precipitate, including the co-precipitated nucleotides, was obtained by treating 200 mL of the ltrate with 2 mL of 1 M NaOH (1 % v/v). The precipitate was allowed to settle overnight and then centrifuged at 1000 g for 15 min. The supernatant was discarded and the precipitate was transferred into 50 mL Falcon tubes, centrifuged again (1.5 h, 1680 g).

The resulting pellet was dissolved by drop-wise addition of5 M HCl. The samples were frozen at 20 C until further

processing. The pH of the thawed samples was adjusted to7.2 by the addition of TRIS buffer (pH 7.4, 20 mM). The nal volume was recorded. The dATP concentrations were measured in triplicate using the rey bioluminescence assay and a Sirius luminometer (Berthold Detection Systems Pforzheim, Germany), as described by Unger et al. (2013).Standard concentrations were prepared as described above, using aged Baltic Sea water and six ATP concentrations (adenosine 50-triphosphate disodium salt hydrate, Sigma-Aldrich, A2383) ranging from 1 to 20 nmol L1. The detection limit of the bioluminescence assay was 2.5 nmol L1.

The uorescence slope of the standard concentrations was used to calculate dATP concentrations, correcting for the nal sample volume. The P-content of the dATP (dATP-P) was calculated by assuming that 1 mol of ATP is equivalent to 3 mol P.

M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3039

tered subsamples were frozen at 20 C and analysed as

described for DOP using the potassium peroxydisulfate digestion (Grasshoff et al., 1983). The measured PO4 concentration represents total phosphorus (TP). PP is the difference between the TP concentration in the unltered digested sample and the sum of DOP+PO4. In the lter-method,

500 mL subsamples were ltered onto pre-combusted GF/F-lters that were then placed into Schott bottles containing 40 mL of deionised water. PP was digested to PO4 by the addition of oxidizing decomposition reagent (Oxisolv, Merck) followed by heating in a pressure cooker for 30 min. The PO4 concentrations of the cooled samples were determined spectrophotometrically according to Grasshoff et al. (1983). Paired t test revealed signicant differences between two methods; however, the difference between the means of the lter method and of the aqueous method (0.19 0.03 mmol L1 and 0.1 0.04 mol L1, re

spectively) were near the detection limit (0.02 mol L1) of the methods. Thus, solely the mean values obtained from both measurements are used in the following.

Particulate carbon (PC) and nitrogen (PN) were analysed by ltering 500 mL samples onto pre-combusted (450 C, 6 h) glass ber lters (Whatman GF/F), which were then stored frozen at 20 C. PC and PN concentrations were

measured by ash combustion of the dried (60 C) lters using a EuroEA elemental analyser coupled with a Cono II interface to a Finnigan DeltaPlus mass spectrometer and included organic and inorganic matter.

2.3.6 Phosphate and ATP uptake

PO4 uptake was measured by addition of radioactively labelled phosphate [33P]PO4 (specic activity of 111 TBq mmol1, Hartmann Analytic GmbH) at concentrations of 50 pmol L1 to 50 mL subsamples, which were then incubated under laboratory light and the in situ temperatures for 2 h. For each mesocosm, three parallel samples and a

blank were prepared. The blank was obtained by the addition of formaldehyde (1 % nal concentration) 10 min before radiotracer addition, in order to poison the samples. At dened time intervals within the incubation, 5 mL subsamples were taken from each of the parallel samples and ltered onto polycarbonate (PC) lters pre-soaked with a cold 20 mM PO4 solution to prevent non-specic [33P]PO4 binding. The lters were rinsed with 5 times 1 mL of particle-free bay water and placed in 6 mL scintillation vials. Scintillation liquid (4 mL IrgaSafe; Perkin Elmer) was added and the contents of the vials were mixed using a vortex mixer. After allowing the samples to stand for at least 2 h, the radioactivity on the lters was counted in a Perkin Elmer scintillation counter. PC-lters of 0.2 m and 3 m pore sizes (What-man and Millipore, respectively) were used to determine up-take by the whole plankton community and the size fraction > 3 m, respectively. Picoplankton uptake was calculated as the difference between the activity on the 0.2 and 3 m lters.

[ 33P]ATP (specic activity of 111 TBq mmol1, Hart-mann Analytic GmbH) was added to triplicate 10 mL samples and a blank, each in a 20 mL vial, at a concentration of 50 pmol L1. The samples were incubated in the dark at the in situ temperature for 1 h. The uptake was stopped by addition of 200 L of a cold 20 mM ATP solution to the samples, which were then ltered and processed as described for the PO4 uptake measurements.

2.3.7 Bacterial production (BPP)

Rates of bacterial protein production (BPP) were determined by incorporation of 14[C]-leucine (14C-Leu, Simon and Azam, 1989) according to Grossart et al. (2006). Trip-licates and a formalin-killed control were incubated with

14C-Leu (7.9 GBq mmol1; Hartmann Analytic GmbH, Germany) at a nal concentration of 165 nmol L1, which ensured saturation of uptake systems of both free and particle-associated bacteria. Incubation was performed in the dark at in situ temperature (between 7.8 and 15.8 C) for 1.5 h.

After xation with 2 % formalin, samples were ltered onto5.0 m (attached) nitrocellulose lters (Sartorius, Germany) and extracted with ice-cold 5 % trichloroacetic acid (TCA) for 5 min. Thereafter, lters were rinsed twice with ice-cold 5 % TCA, once with ethanol (96 % v/v), and dissolved with ethylacetate for measurement by liquid scintillation counting. Afterwards the collected ltrate was ltered on 0.2 m (free-living) nitrocellulose lters (Sartorius, Germany) and processed in the same way as the 5.0 m lters. Standard deviation of triplicate measurements was usually < 15 %. The sum of both fractions (free-living bacteria and attached bacteria) is referred to total BPP. The amount of incorporated

14C-Leu was converted into BPP by using an intracellular isotope dilution factor of 2 (Simon and Rosenstock, 1992).A conversion factor of 0.86 was used to convert the protein produced into carbon (Simon and Azam, 1989).

2.4 Statistical analyses

The Grubbs test, done online (http://graphpad.com/quickcalcs/Grubbs1.cfm

Web End =graphpad.com/quickcalcs/

http://graphpad.com/quickcalcs/Grubbs1.cfm

Web End =Grubbs1.cfm ) was applied to identify outliers in all data sets.The outliers were removed from further statistical analyses.

Spearman Rank correlations were carried out to describe the relationship between the development of the parameters over time in the mesocosms and in the fjord using Statistica 6 software.

Short-term CO2 effects on PP concentrations at days 02 and 2343 between the CO2 treatments were veried with an ANCOVA analysis using the SPSS software. The days were treated as a covariate interacting with the treatments.Paired t test was applied to check the differences in PO4 concentrations between the treatments.

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3040 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

Figure 2. (a) f CO2 values in the mesocosms and in the fjord throughout the experiment. Small black dots show the f CO2 in the ambient fjord water. Treatment of the mesocosms with CO2 saturated fjord water at the beginning of the experiment (days 04)

created different f CO2 levels in the mesocosms: blue symbols represent the untreated mesocosms, grey the intermediate, and red the high CO2 treated mesocosms. The treatment was repeated at day16. (b) Corresponding pH ranges in the mesocosms during the four phases. Despite decreasing trends over time, a gradient between the mesocosms was kept over the whole period.

3 Results

3.1 Development in the mesocosms

3.1.1 CO2, pH, temperature and salinity

The different mesocosms were characterized based on their averaged f CO2 and pH values from day 1 until day 43 (Fig. 2a, b):

M1 365 atm f CO2, pH 8.08 M5 368 atmf CO2 pH 8.07 M7 497 atm f CO2, pH 7.95

M6 821 atm f CO2, pH 7.74 M3 1007 atm f CO2, pH 7.66

M8 1231 atm f CO2, pH 7.58M1 and M5 were the untreated mesocosms and served as controls.

Temperature development in the mesocosms closely followed that in the fjord ranging from 7.82 to 15.86 C. Based

Table 1. Minimum, maximum and mean values of hydrographical parameters and f CO2 for the different phases in the fjord. Temperatures in the mesocosms were identical with those in surrounding fjord water.

phase min max mean

water temperature ( C)

0 7.82 8.71 8.20 I 9.66 15.86 12.27 II 7.89 14.79 11.68 III 8.35 12.61 10.83

salinity

0 5.72 5.85 5.78 I 5.46 5.85 5.65 II 5.67 6.04 5.82 III 5.9 6.05 5.98

pH

0 8.09 8.23 8.16 I 8.11 8.30 8.17 II 7.81 8.30 8.00 III 7.75 7.93 7.83

f CO2 (atm)

0 250 347 298 I 207 336 283 II 208 679 465 III 521 800 668

on this (compare Paul et al., 2015b for details), the experiment was divided into four phases (Fig. 3): phase 0: day

3 to day 0; phase I: days 116, phase II: days 1730 and phase III: day 31 until the end of the measurements. Temperature dropped from 8.71 to 7.82 C in phase 0 and rose from8.07 C at the start of phase I to the maximum of 15.86 C by the end of this phase. During phase II, the temperature decreased to 7.89 C interrupted by a short reversal on days 22 and 23. During phase III, the temperature increased to12.61 C (Table 1).

Salinity (5.69 0.01) remained relatively stable in all

mesocosms throughout the entire experimental period (Fig. 3).

3.1.2 Phytoplankton biomass

Chlorophyll a (Chl a) reached maximum concentrations of2.062.48 g L1 at day 5 (Fig. 4). Average concentrations of1.94 0.23 g L1 in phase I exceeded those in phases II and

III when Chl a decreased to a mean of 1.08 0.16 g L1.

The increase in Chl a in the high CO2 mesocosms by0.27 g L1 in phase III was marginal for Baltic Sea summer conditions. According to Paul et al. (2015b), this represents an increase of 24 % which is a signicant difference compared to the controls.

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M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3041

on day 21. Thereafter, the mean TP remained constant at0.54 0.03 mol L1 until the end of the measurements.

Thus, the loss of phosphorus (116 34 nmol L1) from the

17 m layer during the 29-day measurement period was calculated to be 4.0 nmol L1 day1. The decline in TP can be explained by loss through sedimentation of PP (Paul et al., 2015b).

Particulate phosphorus (PP) concentrations varied from0.10 to 0.23 mol L1 in all CO2 treatments (Figs. 5b, 6). We expected that the decrease in TP was reected in PP. However, parallel changes occurred only periodically. PP concentrations increased during the rst 5 days after the bags were closed. This increase was stimulated by CO2 additions from day 0 to day 2 (ANCOVA: p = 0.004, F = 20.811; Fig. 7a).

Subsequently, PP declined in parallel with TP until day 21, albeit with a lower amount. Averaged over all mesocosms, TP decreased by 0.12 0.03 mol L1, whereas PP declined

only by 0.06 0.01 mol L1 during this period. From day

23 until the end of the measurements, PP remained at relatively constant concentrations; however, PP concentrations in the high CO2 treated mesocosms exceeded those in the other mesocosms signicantly (ANCOVA: p < 0.0001, F = 11.99;

Figs. 5b, 7). PP developed in parallel with PC. The two parameters were positively correlated in the untreated and the intermediate CO2 treatments, but not in the high CO2 treatments (Table 2). Figures 3 and 6b show that the increase in Chl a was delayed by 23 days compared to the increase in PP during the rst growth event. A correlation between PP and Chl a was detected only for the untreated mesocosms (Table 2).

Dissolved organic phosphorus (DOP) concentrations in the mesocosms ranged between 0.18 and 0.36 mol L1 constituting 3271 % of the TP pool (Fig. 5c). DOP did not change signicantly in response to the CO2 perturbations, and were similar to the concentrations in fjord water. Concentrations of 0.3 mol L1 were measured on days 6 and

7 (phase I) and on day 23 (phase II); the high DOP value in the intermediate CO2 treatment at day 19 was identied as an outlier according to Grubbs test (Fig. 5c).

In phase I, DOP initially increased in parallel with Chl a and BPP but reached its maximum 12 days later, after which it decreased only marginally until the end of this phase, independent of changes in BPP and Chl a (Fig. 8c, d). In phase II, the peak conformed to that of BPP. DOP correlated with temperature only in the high f CO2 mesocosms (Table 2). In addition, the composition of DOP did not change with increasing CO2 (Fig. 10). The sum of RNA ( 47 %) plus the

unidentied fraction constituted 9899 % of the DOP pool whereas the other measured compounds contributed only 1 2 % (Table 3).

Phosphate (PO4) concentrations ranged between 0.06 and0.21 mol L1, with deviations between the mesocosms only in nanomolar range. The mean contribution of PO4 to TP was 25 6 %, which was the lowest among all TP frac

tions (Fig. 6). From the start of the measurements to day

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Figure 3. Temperature and salinity averaged over the 17 m surface layer of the mesocosms and the fjord. The data were obtained from daily CTD casts. Large symbols represent temperature and the small symbols salinity. Fjord water is shown as black dots with broken line while blue symbols denote untreated, grey intermediate and red high f CO2 levels in the mesocosms. According to the temperature regime, the experimental period can be divided into four phases (phases 0, I, II and III).

Figure 4. Chl a concentrations in fjord water and in the mesocosms with different f CO2 conditions. The development over time can be divided into three phases as well. Blue represents untreated, grey intermediate, and red highly treated f CO2 levels. Black dots with dotted line are the Chl a concentrations in the fjord water.

We observed a signicant relationship between Chl a and PO4 in the untreated and intermediate treated mesocosms that diminished with increasing f CO2 as indicated by lower p values. The statistical signicance was lost in the highest f CO2 mesocosms (Table 2).

3.1.3 Phosphorus pools

Total phosphorus (TP) concentrations in the mesocosms ranged between 0.49 and 0.68 mol L1 (Fig. 5a) during the experiment without statistically signicant differences between the CO2 treatments. Shortly after the bags were closed, the decline in TP concentrations began and continued until the beginning of phase II. On average, TP concentrations decreased from 0.63 0.02 on day 3 to 0.51 0.01 mol L1

3042 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

Table 2. Mesocosms in which the Spearman Rank correlation between P-pools or uptake rates and other parameters was signicant. The relationship of PP with TP and Chl a was signicant only in the untreated mesocosms while the correlation to PC was also signicant in the mesocosms with intermediate CO2 levels. DOP was related to temperature only in the high CO2 treatments. Under high f CO2 conditions, the PO4 uptake in the size fraction > 3 m correlated with Chl a and the P content of phytoplankton.

Relationship between f CO2 Signicant responses

(atm) r p n

PPTP 365 0.599 0.008 18 368 0.515 0.029 18

PPChl a 365 0.479 0.0130 25 368 0.584 0.0022 25

365 0.832 < 0.0001 21

PO4Chl a 368 0.756 0.0011 20

497 0.674 0.0008 21

821 0.524 0.0147 21

1007 0.634 0.0027 20

365 0.542 0.0061 24 PPPC 368 0.625 0.0011 24

497 0.404 0.0490 24 821 0.551 0.0052 24

DOPtemperature 1007 0.488 0.0470 17 1231 0.525 0.0310 17

497 0.743 0.0056 12 PO4 uptake > 3 mChl a 821 0.674 0.0081 14

1231 0.476 0.0310 14

497 0.601 0.0380 12

PO4 uptake > 3 mPOP/Chl a 821 0.631 0.0160 14

1231 0.626 0.0165 14

Table 3. Contribution of different phosphorus components to DOP in the mesocosms and in the fjord.

f CO2 Contribution to DOP (%)

(atm) ATP-P PL-P DNA-P RNA-P sum unidentied P

Fjord 0.7 0.7 0.04 69.4 70.84 29.16 365 0.7 0.5 0.03 44.1 45.33 54.67 368 0.6 0.5 0.03 46.9 48.03 51.97 497 0.6 0.4 0.04 49.5 50.54 49.46 821 0.6 0.4 0.03 41.8 42.83 57.17 1003 0.8 0.4 0.04 60.1 61.34 38.66 1231 0.5 0.4 0.03 48.6 49.53 50.47

namics of PP and Chl a concentrations, which were signicantly elevated in the high CO2 treatments. Thus, the transformation of PO4 to POP via stimulated biomass formation may have been promoted under high CO2 conditions in phaseIII.

Since PO4 was never fully exhausted, phosphorus limita

tion of phyto- and bacterioplankton can be excluded. This interpretation is supported by the PC : PP ratios, which varied between 84.4 and 161.1 in all treatments (Paul et al., 2015b) deviating only slightly from the Redeld ratio.

3.1.4 Uptake of PO4 and ATP

PO4 turnover times of 1.58.4 days (mean 4.0 1.2 days,

n = 112) in all mesocosms indicated no dependency

on the CO2 treatment (Fig. 9a). Gross PO4 uptake rates were in the range of 0.63.9 nmol L1 h1 (mean1.7 0.6 nmol L1 h1, n = 112), or 14.394.4 nmol L1

day1 (mean 41.3 13.8 nmol L1 day1; Fig. 9b, Table 4).

The rates were highest on days 4 and 9 (phase I) and decreased thereafter until day 15, followed by an increase to a mean maximum rate of 2.3 0. 5 nmol L1 h1 (n = 6) at

day 27. The size fraction < 3 m was responsible for 59.1 to

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13, PO4 declined by 0.06 mol L1 (or 3.5 nmol L1 day1) from initial values of 0.16 0.01 mol L1 (Fig. 5d). Sub

sequently, concentrations increased again, by an average of2.6 nmol L1 day1, until the end of the experiment. There were no signicant differences between CO2 treatments until day 23, when high CO2 concentrations led to slightly lower PO4 concentrations (Fig. 5d). Afterwards, PO4 concentrations in the high f CO2 mesocosms were signicantly lower than those in the untreated mesocosms (t = 6.51,

p = 0.0003). This observation is in accordance with the dy-

M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3043

Table 4. PO4- and ATP uptake rates in the fjord and in the mesocosms. Minimum, maximum and mean values as well as the contribution of the size fraction < 3 m to the total activity are given for the whole period of investigation (each: n = 16 for PO4 and n = 6 for ATP uptake).

Total PO4 uptake Total ATP-P uptakef CO2 (n moll1 h1) portion (%) (n moll1 h1) portion (%)

min max mean < 3 m min max mean < 3 m

Fjord 0.87 2.81 1.63 0.58 76 15 0.04 0.51 0.26 0.15 92 5

365 0.82 3.89 1.67 0.82 81 11 0.14 1.08 0.43 0.33 96 2

368 0.65 2.74 1.61 0.58 86 7 0.16 0.97 0.47 0.27 96 2

497 0.61 3.03 1.52 0.59 86 6 0.20 1.07 0.54 0.28 96 2

821 0.91 2.83 1.60 0.59 88 8 0.14 0.71 0.36 0.21 97 2

1003 0.67 3.79 1.73 0.85 86 6 0.22 0.69 0.39 0.15 97 1

1231 0.87 2.23 1.53 0.43 87 6 0.17 0.67 0.44 0.17 97 2

98.4 % of the total PO4 uptake (mean 86.5 7.6 %) whereas

the size fraction > 3 m accounted for only 1.640.9 % (mean13.5 7.4 %). Thus, PO4 was taken up mainly by picoplank

ton. However, only the uptake rate by the size fraction > 3 m was positively related to Chl a and inversely related to the P content of the biomass (Table 2). Thus, the PO4 uptake was obviously stimulated when the phytoplankton biomass increased and the cellular P decreased simultaneously. The relationship between PO4 uptake by this fraction and Chl a became evident only in the CO2-amended conditions indicating that the interaction between P uptake, cellular P-content and growth of phytoplankton was stimulated under elevated CO2 conditions.

ATP turnover times of 0.2 to 3.6 days (mean0.94 0.74 days, n = 90) were much shorter than the

PO4 turnover times and did not vary between the treatments (Fig. 9c). Between 0.05 and 0.36 nmol ATP L1 h1 (mean0.14 0.08 nmol L1 h1, n = 36) were hydrolysed, cor

responding to a P supply of 0.14 and 1.08 nmol L1 h1 (mean 0.44 0.25 nmol L1 h1, n = 36). Thus, phosphorus

additionally supplied from ATP accounted for 25 %

of that provided by PO4. The picoplankton size fraction (< 3 m) was responsible for 9099 % of ATP uptake, with only a marginal portion (1.69.5 %) attributable to the phytoplankton fraction > 3 m (Table 4).

3.2 Hydrography and pool sizes in the fjord

Large variations in f CO2 and pH occurred in fjord water during the period of investigation (Table 1). The relationship of f CO2 with temperature and salinity indicated that the CO2 conditions were inuenced predominantly by changes in the water masses, specically by upwelling which affected both the relationship of f CO2 with PO4 and probably the correlation of f CO2 with Chl a and PC (Table 2). f CO2 ranged from 207 atm (Fig. 2a) at days 12-16 when temperatures were highest to 800 atm at day 33 when deep water input occurred which was indicated by pH below 7.75.

Chl a concentrations were between 1.12 and 5.46 g L1 (mean 2.29 1.11 g L1; n = 38), with distinct phases cor-

relating with temperature, salinity and pH. However, the Chl a maximum occurred at the beginning of phase II, which was 12 days after the maximum temperature. Shortly thereafter, Chl a decreased to its lowest level before it increased again, albeit only marginally to 1.93 g L1 during phase III (Fig. 4).

TP concentrations from day 3 until day 29 ranged be

tween 0.54 and 0.70 mol L1 (mean 0.61 0.04 mol L1;

n = 19; Figs. 5a, 6). With a general decreasing tendency, TP

undulated with a frequency of about 10 days in the period of phases 0 to the rst half of phase I and of 6 days in the second half of phase I to II. For the period under investigation, the TP fractions had the following characteristics.

PP concentrations varied from 0.13 to 0.30 mol L1 (mean 0.20 0.04 mol L1; n = 29), thus accounting for

23.451.8 % (mean 34.7 7.9 %; n = 19) of the TP pool.

The development of PP over time did not follow that of TP (Fig. 5b). PP concentrations were highest between days 8 and 19, when the accumulation of PP in the biomass was reected in declining C : P ratios from 180 to 107 (Paul et al., 2015b) and thereafter remained at the low ratio until the end of the measurements. The PP increase in phase III occurred in parallel to Chl a and to the PO4 decrease (Fig. 6). Thus PO4 was transformed into PP via biomass production. The calculated P content of phytoplankton was 0.050.15 (mean 0.1) mol PP (g Chl a)1.

DOP substantially contributed (2645 %) to the TP pool (Fig. 6). Concentrations ranged between 0.19 and0.29 mol L1 (mean 0.24 0.03 mol L1; n = 17), with

high concentrations occurring in parallel to those of TP

in phases I and II (Fig. 5c). The very low DOP value of0.11 mol L1, on day 29, was an outlier (Grubbs test). For the whole study period, DOP concentrations correlated positively with PP (p = 0.034, n = 17) and inversely with PO4

concentrations (p = 0.005, n = 17). A similar behaviour be

tween DOP and Chl a was restricted to phases 0 and I, whereas the relationship was inverse in phase II (Fig. 8b). As shown in Figure 8a, the DOP and BPP levels alternated with the same rhythm, but inversely, in phases 0 and I and changed to a parallel development in phase II. Statistical analysis was

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3044 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

Figure 6. Contribution of the individual P-fractions to TP in fjord water and in the respective mesocosms. The data are averaged for the period when TP measurements were done (day 3 to day 29).

(Fig. 6). With a few exceptions, PO4 concentrations declined from the beginning of the study period until the end of phase I and increased during phase II and the beginning of phaseIII. These changes were caused by upwelling of PO4 enriched deep water of higher salinity and lower temperatures.The subsequent decline in PO4 between days 33 and 40 was caused by the stimulation of phytoplankton production, as indicated by the increase in Chl a concentration (Fig. 4).

4 Discussion

An increase in CO2 in marine waters and the associated acid-ication may potentially have multiple effects on organisms and biogeochemical element cycling (Gattuso and Hansson, 2011). Reported ndings indicate wide-ranging responses, probably depending on the investigated species and growth conditions. For example, CO2 stimulation as well as lack of stimulation were found for primary production and carbon xation (Beardall et al., 2009; Boettjer et al., 2014), DOC release (Engel et al., 2014; MacGilchrist et al., 2014) and phytoplankton growth (Riebesell and Tortell, 2011). An interaction of CO2 effects with phosphorus and iron avail-

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Figure 5. (ad) Development of total phosphorus (TP) and the three measured P-fractions in fjord water (black dots with dotted line) and in the mesocosms over time. Blue represents untreated, grey intermediate and red high f CO2 treatment levels.

not feasible because DOP and BPP were not always sampled

on the same day and only very few data pairs were available. PO4 concentrations ranged between 0.06 and0.41 mol L1 (mean 0.21 0.09 mol L1, n = 21),

thus comprising 24.3 11.2 % (n = 21) of the TP pool

M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3045

Figure 7. PP concentration in the mesocosms during the initial phase from day 0 to day 2 (a) and from day 23 until the end (b) of experiment.

ability has been found by Sun et al. (2011) for a diatom Pseudo-nitzschia multiseries and by Yoshimura et al. (2014) for a diatom-dominated subarctic plankton community. Thus, responses of organisms and ecosystems to enhanced CO2 concentrations are complex and still poorly understood. The present study is the rst to determine the effects of increased CO2 levels on phosphorus cycling in a brackish water ecosystem.

4.1 Response of P-pools and P-uptake to enhanced CO2 in the mesocosms

The Finnish coast off the Gulf of Finland is one of the most important upwelling regions in the Baltic Sea. During our investigation in 2012, surface temperatures, obtained from the NOAA satellite (Siegel and Gerth, 2013), showed that upwelling persisted during the whole study period but with varying intensity. The intensity of upwelling shaped the pattern of temperature not only in the fjord but also in the mesocosms varying from 7.8 to 15.9 C. Such variations in temperature inuence the phosphorus transformation and interleave with CO2 effects.

While nutrients were added in previous CO2 enrichment experiments (Riebesell et al., 2008, 2013; Schulz et al., 2008), no amendments were undertaken in this study in order to be close to natural conditions. Initial PO4 concentrations of only 0.17 0.01 mol L1 were measured, however, PO4

was never exhausted (Figs. 5, 6). Cellular C : P and N : P ratios were close to the Redeld ratio. Therefore, phosphorus limitation unlikely occurred in this experiment. Simultane-

ous low nitrate and ammonium concentrations (Paul et al., 2015b) formed nutrient conditions that benet the growth of diazotrophic cyanobacteria. However, a cyanobacteria bloom failed to appear, despite the low-level presence of Aphanizomenon sp. and Dolichospermum sp. (Paul et al., 2015a) as potential seed stock. For Baltic Sea summer conditions, the phytoplankton development with maximum Chl a concentrations of 2.22.5 g L1 remained relatively low with the highest contribution of cryptophytes and chlorophytes in phase I and at the beginning of phase II. Picoplankton was mostly the dominating size fraction, amounting to 20

70 % of Chl a in phase I and up to 85 % in phase III (Paul

et al., 2015b). However, a positive correlation of f CO2 with Chl a was observed only for the size fraction > 20 m. The abundance of diatoms that could be a part of this fraction increased from day 23 to day 30 and might have an inuence

on this relationship.

Against this background, the CO2 perturbation did not cause signicant changes in phosphorus pool sizes, DOP composition, and P-uptake rates from PO4 and ATP when the whole study period was considered. However, small yet signicant short-term effects on PO4 and PP pool sizes were ob-served in phases I, III and partially in phase II (Fig. 7). CO2 elevation stimulated the formation of PP until day 3 (Fig. 5b) when chlorophytes, cyanobacteria, prasinophytes and the pico-cyanobacteria started to grow (Paul et al., 2015b).

The effects of CO2 addition on PO4 and PP pool sizes were evident from day 23 onwards (Figs. 5b, 7). PO4 concentrations were slightly, but signicantly lower in the high CO2 treatment than in the untreated mesocosms, accompanied by signicantly elevated PP concentrations. This indicates that the transformation of PO4 into PP was likely stimulated under high CO2 conditions. Since Chl a was also elevated at similar PP : Chl a ratios, the PO4 taken up was used for new biomass formation. However, the elevated transformation of PO4 into PP was not reected in the PO4 uptake rates which can be seen as gross uptake rates. But, an increase of PP, caused by biomass formation, while the PO4 uptake remained unchanged can only occur when the P release from organisms is reduced. Thus, it is likely the net uptake was modied under CO2 elevation and not the gross uptake.

While in phases II and III, high CO2 levels caused a change in the PP and PO4 pools for about 22 days, changes lasting only 2 days have been observed at the beginning of phase I (Fig. 7a), but, shorter effects cannot be excluded.Uptake and release are assumed to be continuous processes and can alter the P pool sizes on timescales shorter than 1 day. Thus, variations and differences in the treatments can be overseen at daily sampling. Unger et al. (2013) demonstrated that an accelerated PO4 uptake by the cyanobacterium

Nodularia spumigena under elevated CO2 incubations could only be observed during the rst hours. Thereafter, the differences were balanced and the same level of radiotracer labelling was reached in all treatments. An acceleration in the formation of particulate P under CO2 elevation without any

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3046 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

Figure 8. Development of DOP in relation to bacterial production (BPP) and phytoplankton biomass (Chl a) in the fjord (a, b) and in the mesocosms (c, d). For mesocosms, mean values averaged over all treatments are given.

Figure 9. Turnover times of PO4 (a) and ATP (c) in the mesocosms as well as the respective uptake rates (b, d).

changes of PO4 turnover times was also observed by Tanaka et al. (2008). They observed an increase of the PP amount and an earlier appearance of the PP maximum under CO2 elevation.

Correlations calculated by using the Spearman rank test between P pools or uptake rates and other parameters for each mesocosm are presented in Table 2. The relationships

between PP and TP with Chl a disappeared at elevated f CO2, whereas correlations developed between PP and PC as well as between the PO4 uptake by phytoplankton in the > 3 m size class and the PP : Chl a ratio (Table 2). These shifts could be caused by changes in the phytoplankton composition deduced from CO2 effects on the pigment composition (Paul et al., 2015b).

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M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes 3047

However, the PO4 decline ( 34.5 nmol L1) was stronger

than that of the total dissolved P pool since DOP increased in parallel by 26.5 nmol L1. Thus, about 77 % of the PO4 re

duction was retrieved as DOP and remained in the dissolved P-pool as the main pathway of PO4 transformation.

4.2 Phosphorus dynamics in the Storfjrden

Nutrients in upwelled water during our study were depleted in dissolved inorganic nitrogen and enriched in PO4, as reported for other upwelling areas of the Baltic Sea (Lass et al., 2010). Thus, ammonium and NO2+NO3 concentrations

in the surface water were only in the nanomolar range (Paul et al., 2015b). PO4 increased in parallel with the increase in salinity and decrease in temperature. Maximum PO4 concentrations of 0.33 and 0.42 mol L1 (Figs. 5, 6) were observed at the end of the upwelling events in phases 0 and II, respectively. The correlation with Chl a and PP indicated that PO4 was utilized during plankton growth in the subsequent relaxation phases I and III. Due to PO4 input into surface water, the phytoplankton community was unlikely P-limited indicated by PC : PP ratios of 86189 (mean 125, n = 23; Paul

et al., 2015b). However, the PO4 availability might be not the only reason for the good P-nutritional status of the plankton. It can be deduced from the long PO4 turnover times in the mesocosms, where external input was excluded, that the P demand of the plankton community might be low. The P content deduced from PP : Chl a ratios of 0.050.15 mol P ( g Chl a)1 was somewhat lower than those observed during an upwelling event along the east coast of Gotland, where ratios between 0.1 and 0.2 mol P ( g Chl a)1 (Nausch et al., 2009) were estimated.

PP concentrations of 0.130.3 mol L1 were in the range typically observed in the Baltic Proper (Nausch et al., 2009, 2012). However, PP concentrations in the Gulf of Finland may reach higher values, as was the case in the summer of 2008, when the observed PP concentration was0.35 0.07 mol L1 (Nausch and Nausch, 2011).

DOP concentration of 0.27 0.02 mol L1 during our

study was similar to that detected in the Gulf of Finland in the summer of 2008 (Nausch and Nausch, 2011). In the Baltic Sea, DOP exhibits vertical gradients with maximum concentrations in the euphotic surface layer and lower than0.1 mol L1 at depths below 25 m. Thus, the observed DOP dynamics in surface water during our study can be assumed to be the result of release, consumption and mineralization by organisms or input from land. The relationship of DOP with Chl a and BPP (Fig. 8) indicated that the increased DOP concentrations in phase I may be due to release by phytoplankton supplemented by bacterial release. DOP can be accumulated in water only when the release exceeded the consumption or degradation. During phase II, phytoplankton biomass was low and DOP release should thus be minor. Since the small mesozooplankton increased in the fjord similar to those reported for the mesocosms in phases II and III (Paul et al.,

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Figure 10. Development of DOP compounds in the mesocosms and in the fjord from day 0 to day 27.

Independent of the CO2 treatment, TP decreased by2.6 nmol L1 day1 in all mesocosms over the course of the experiment, in agreement with the measured sedimentation rates (Paul et al., 2015b). The strongest decrease ( 3.2 nmol L1 day1) occurred during phase I. Of the

total TP removal during this phase (48 nmol L1), 84 % ( 40.5 nmol L1) could be explained by the decrease in PP

and 16 % ( 8 nmol L1) by changes in the dissolved pool.

3048 M. Nausch et al.: Effects of CO2 perturbation on phosphorus pool sizes

2015b) DOP could be released during grazing combined with the observed temporal offset of BPP and DOP maxima. Thus, the observed DOP variations may be the result of processes in the surface water.

5 Conclusions

Surface water in Storfjrden showed highly variable f CO2 conditions and reached levels of up to 800 atm, which is similar to that expected in ca. 100 years from now. Deduced from the high frequency of upwelling events, organisms experience elevated f CO2 more or less regularly. Thus, a general impact of f CO2 on P pools and P uptake rates in the mesocosms could not be identied for the overall period of investigation. However, short-term responses to f CO2 elevation lasting only few days were observed for the transformation of PO4 into PP that was linked with stimulation of phytoplankton growth. Although statistically signicant, it is dif-cult to assess if the differences between the treatments are of ecological relevance. Such short-term variations are possible in the phosphorus dynamics since the pools size can be transformed within hours and there changes are in the nanomolar concentration range. There are also indications that relationships of P pool sizes or uptake with Chl a and PC can change as f CO2 increases, but the underlying mechanisms are still unclear. The transformation of PO4 into DOP was not affected by CO2 elevation. It may be the major pathway of phosphorus cycling under hydrographical and phytoplankton growth conditions as occurred in our experiment.

Acknowledgements. We are grateful to the KOSMOS team for their invaluable help with the logistics and maintenance of the mesocosms throughout the experiment. In particular, we sincerely thank Andrea Ludwig for organizing and coordinating the campaign and for the daily CTD measurements. We appreciate the assistance of Jehane Ouriqua in the nutrient analysis and that of many other participants who carried out the samplings. We also appreciate the collegial atmosphere during the work and thank everyone who contributed to it. We would also like to acknowledge the staff of the Tvrminne Zoological Station for their hospitality and support, for allowing us to use the experimental facilities, and for providing CTD data for the summers of 200820011. Finally, we thank Jana Woelk for analysing the phosphorus samples in the IOW. This study was funded by the BMBF project BIOACID II (FKZ 03F06550).

Edited by: C. P. D. Brussaard

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