Vol. 520: 21–34, 2015 MARINE ECOLOGY PROGRESS SERIES Published February 3 doi: 10.3354/meps11123 Mar Ecol Prog Ser

Effect of acidification on an Arctic community from Disko Bay, West Greenland

Christina Thoisen1,2,*, Karen Riisgaard3, Nina Lundholm4, Torkel Gissel Nielsen3,5, Per Juel Hansen2

1Department of Environmental, Social and Spatial Change, Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark 2Centre for Ocean Life, Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark 3National Institute of Aquatic Resources, DTU Aqua, Section for Ocean Ecology and Climate, Technical University of Denmark, Kavalergården 6, 2920 Charlottenlund, Denmark 4Natural History Museum of Denmark, University of Copenhagen, Sølvgade 83S, 1307 Copenhagen K, Denmark 5Greenland Climate Research Centre, Greenland Institute of Natural Resources, Kivioq 2, PO Box 570, 3900 Nuuk, Greenland

ABSTRACT: Long-term measurements (i.e. months) of in situ pH have not previously been reported from the Arctic; this study shows fluctuations between pH 7.5 and 8.3 during the 2012 in a coastal area of Disko Bay, West Greenland. The effect of acidification on phyto- from this area was studied at both the community and species level in experimental pH treatments within (pH 8.0, 7.7 and 7.4) and outside (pH 7.1) in situ pH. The growth rate of the phytoplankton community decreased during the experimental acidification from 0.50 ± 0.01 d−1 (SD) at pH 8.0 to 0.22 ± 0.01 d−1 at pH 7.1. Nevertheless, the response to acidification was species- specific and divided into 4 categories: I, least affected; II, affected only at pH 7.1; III, gradually affected and IV, highly affected. In addition, the colony size and chain length of selected species were affected by the acidification. Our findings show that coastal phytoplankton from Disko Bay is naturally exposed to pH fluctuations exceeding the experimental pH range used in most studies. We emphasize that studies on ocean acidification should include in situ pH before assumptions on the effect of acidification on marine organisms can be made.

KEY WORDS: Ocean acidification · Coastal · Arctic phytoplankton · Growth rate · pH · CO2 · DIC

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INTRODUCTION 18th century to ~7.8 by the end of the 21st century (Feely et al. 2009). When pH of seawater decreases, 2− The impacts of ongoing ocean acidification on mar- the concentration of CO3 (carbonate ion) also − ine organisms are a highly debated topic within the decreases, while HCO3 (bicarbonate ion) remains in scientific community. Anthropogenic emissions are vast concentrations (for details on seawater carbon- expected to increase the level of atmospheric CO2 ate chemistry see Zeebe & Wolf-Gladrow 2003). from ~280 ppm in the mid-18th century to ~700 ppm The majority of phytoplankton examined in the liter- st − by the end of the 21 century (Riebesell et al. 2009). ature utilize both HCO3 and CO2 for photosynthesis, − Approximately 25% of the emitted atmospheric CO2 while some use either HCO3 or CO2 (e.g. Giordano is absorbed into the oceans (Riebesell et al. 2010) et al. 2005). Increased CO2 could potentially increase where chemical reactions alter the composition of primary production, especially for species relying on dissolved inorganic carbon (DIC) while lowering pH diffuse uptake of CO2 since the energy usage for the (acidification). The average pH of ocean surface uptake of inorganic carbon through carbon concen- waters is expected to decrease from ~8.2 in the mid- trating mechanisms (CCMs) is likely to be reduced

*Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 22 Mar Ecol Prog Ser 520: 21–34, 2015

(Rost et al. 2008). However, extracellular pH (pHe) cation has been conducted in the pH range 7.7 to 8.2, may influence the intracellular pH (pHi) of unicellu- despite the fact that natural fluctuations of pH are lar organisms and affect physiological processes such much greater in coastal areas and often decrease to as ion transport, enzyme activity, protein function <7.7 (e.g. Hansen 2002, Hofmann et al. 2011). To and nutrient uptake (Gattuso & Hansson 2011 and obtain compelling results regarding effects from references therein, see also Nimer et al. 1994). Thus, ocean acidification, measurements of natural pH increased CO2 may not result in enhanced primary fluctuations in a given area are needed to experi- production if other physiological processes are mentally expose organisms to pH values beyond affected negatively by acidification. present-day naturally occurring levels. The formation of chains and colonies in marine The Arctic Ocean is presumed to be very sensitive phytoplankton is well known. For , it is pre- to ocean acidification due to the high solubility of sumed that this reduces sinking velocity and preda- CO2 in cold water and the decreasing sea ice cover tion, and increases sexual reproduction (Smayda & (Slagstad et al. 2011). However, only short-term Bolelyn 1966, Takabayashi et al. 2006, Kooistra et al. measurements (over days) of pH levels in the polar 2007, Bergkvist et al. 2012). Likewise, the prymnesio- open ocean and in the brine of sea ice have been con- phyte Phaeocystis globosa forms different sized ducted (e.g. Gleitz et al. 1995, Hofmann et al. 2011). colonies depending on the type of grazer present Short-term measurements do not provide thorough (Jakobsen & Tang 2002). The effects of acidification insight into the pH fluctuations that organisms are on the formation of chains and colonies have, to our exposed to over a longer time scale. It is hereby cru- knowledge, not been studied until now. Considering cial to collect in situ pH data in the Arctic region dur- that acidification affects physiological processes, it is ing long-term measurements (over months). This will likely that these formations are affected. allow for a more qualified study on the effects of acid- Ocean pH is considered stable on seasonal and ification on Arctic organisms, and contribute to a bet- diurnal scales due to the buffering capacity of seawa- ter estimate of the impact of ocean acidification in the ter, with an average open ocean surface pH of ~8.2 Arctic region. (Feely et al. 2009). This stability in pH is particularly The aim of the present study was to monitor the true for open oceans where algal biomass is usually long-term fluctuations of in situ pH levels in a coastal low. In contrast, pH in coastal ecosystems can exhibit water column during the Arctic phytoplankton large seasonal and diurnal fluctuations shaped by spring bloom while examining the effect of acidifica- factors such as photosynthesis, respiration, up - tion on phytoplankton growth via incubations with welling, CO2 venting, trophic state and water resi- natural phytoplankton assemblages exposed to dif- dence time (e.g. Feely et al. 2008, Wootton et al. 2008, ferent pH levels. Hofmann et al. 2011, Duarte et al. 2013). Seasonal variations in pH levels in temperate coastal ecosys- tems correlate with productivity, and the fluctuation MATERIALS AND METHODS decreases with distance from the shore (Provoost et al. 2010). In shallow and temperate coastal areas, the Study site pH can vary from 7.4 to 9.2 on a diurnal basis (Mid- delboe & Hansen 2007), and in the Danish eutrophic The experiment was conducted in spring 2012 at Mariager Fjord, the minimum and maximum pH dur- Arctic Station (University of Copenhagen, Denmark) ing a 10 yr period was ~7.1 and ~9.7, respectively in Qeqertarsuaq on Disko Island, West Greenland (Hansen 2002). Smaller fluctuations in the range of (Fig. 1). A few days after the breakup of sea ice (April 0.1 to ~0.6 pH units were measured in inner Danish 14) the plankton community in Disko Bay was col- waters over 6 decades (Duarte et al. 2013), and yearly lected from the R/V ‘Porsild’ at a 300 m deep monitor- fluctuations of pH in the western Gulf of Finland reg- ing station (69° 13 N, 53° 22 W) outside of Qeqertar- ularly spanned 1 pH unit (Brutemark et al. 2011). suaq. Depth distribution of salinity and temperature Studies on natural fluctuations of pH have focused on was recorded with a Seabird CTD every 3rd day and temperate coastal waters, and studies on the toler- samples for pH measurements were collected at ance of phytoplankton to acidification have primarily depths of 1, 40, 50, 75, 100, 150, 200 and 250 m with a dealt with temperate phytoplankton (e.g. Lundholm Niskin bottle. The plankton community was collected & Hansen 2004, Kim & Lee 2006, Berge et al. 2010, using a 10 l Niskin bottle from the depth of the chloro- Lohbeck et al. 2012, McCarthy et al. 2012). Unfortu- phyll maximum (5 m depth) and the sample was re- nately, the majority of studies on the effect of acidifi- verse-filtered into a 25 l container via a silicon tube Thoisen et al.: Effect of acidification on Arctic phytoplankton 23

4 treatments (pH 8.0, 7.7, 7.4 and 7.1) 50 km were placed at 3 ± 2°C on a flat turning 100 m 200 m plankton wheel (1 rpm) in front of a 300 m light source (100 µE m−2 s−1, 12 h GREENLAND V light:12 h dark cycle). The phytoplank- A I G 70°E AT ton community exposed to pH 7.7, 7.4 DISKO and 7.1 was lowered in steps of 0.5 pH units per 12 h by adding strongly acidi- DISKO fied seawater with a pH of 4.9 ± 0.1 rcle ic Ci Qeqertarsuaq (Fig. 2; Day 0). After 24 h, all incubation Arct Godhavn IlulissatIlulissat bottles had reached their respective pH JacobshavnJacobshavn set points. To produce acidic seawater, DISKO BAY 69°E 2 l of 0.45 µm filtered seawater were bubbled strongly with CO2 (ca. 5 min) and then with O2 (Hede Nielsen) (ca. AasiaatAasiaat 5 s) to increase the concentration of O2 Fig. 1. Location of EgedesmindeEgedesminde Qeqer tarsuaq on >100%, which was measured with a Disko Island, West WTW Oxi 3210 oxygen meter using a Greenland, where WTW DurOx oxygen probe. During the Arctic Station (×) is remaining part of the experiment, the situated 68°E 56°W 54°W 52°W acidic seawater was added to bottles of 2 l 0.45 µm filtered seawater to obtain seawater for dilution with the specific through a cylinder with a 250 µm nylon mesh to re- experimental pH values; 7.7, 7.4 and 7.1 (Fig. 2; Day move mesozooplankton. Immediately after filtration, 1 to end of experiment). Before each sampling, the the plankton assemblage was carefully mixed and acidic seawater and the pH-specific seawater for transferred to 12 incubation bottles via a silicon tube. dilution were produced as described. The phyto- plankton community at pH 8.0 was used as a control, resembling the pH in Disko Bay at the time of sam- Seawater for dilution pling, and diluted with 0.45 µm filtered seawater without adjustment of pH. The treatment at pH 8.0 Seawater used for diluting the treatments during was terminated on Day 11, while pH 7.7 and 7.4 were the experiment was collected from the sampling sta- terminated on Day 17, and pH 7.1 on Day 16. tion below the pycnocline (150 to 200 m) to ensure nutrient-rich water. The water was filtered through a 0.45 µm Whatman® polycap filter and stored dark Sampling and dilution and cold (3 ± 2°C) in 25 l containers. Average pH, temperature and salinity of the seawater were 7.9 ± The incubation bottles were removed in triplicates 0.1, 3.3 ± 0.1 °C and 34.3 ± 0.0, respectively. AU val- from the plankton wheel and kept cold in a cooling ues are ± SD. box filled with snow during sampling (~20 min). The phytoplankton community was diluted throughout sampling and additional dilutions were done to avoid

Experimental setup and CO2 manipulation high biomasses that would cause large fluctuations in pH and nutrient limitation. First, pH was measured The experiment was run in triplicated 1 l Nalgene® and samples were withdrawn. Then, the incubation polycarbonate bottles and pH was adjusted by apply- bottles were refilled with pH-specific seawater (dilu- ing gaseous CO2 (UN 1013 Carbon dioxide, Class 2, tion). If dilution was insufficient to obtain the experi- 2A, ADR; Air Liquide Denmark). The incubation bot- mental pH, a few drops of acidic seawater were tles were filled to capacity (1.23 l), allowing no head- added. The phytoplankton community was always space in order to avoid fluctuations of seawater diluted to a concentration of ~5 µg chl a l−1. The chemistry as well as avoiding negative effects of air intensity of sampling depended on the growth rate of bubbles on protists; the top was then sealed with the phytoplankton community (using chl a as a parafilm before applying the lid. Triplicate bottles of proxy) and due to the extraction time of chl a the 24 Mar Ecol Prog Ser 520: 21–34, 2015

Day 0 buffers of pH 7 and 10 (WTW Technical Buffer, NIST 8.0 scale). Prior to use (≥10 min), the pH electrode was immersed in filtrated seawater to stabilize.

CO2 7.7 (g) DIC and nutrients

pH 7.4 Samples for DIC were fixed with 100 µl Hg2Cl2 4.9 (mercury(I)chloride) in airtight glass vials (12 ml)

without headspace to avoid CO2 leaking out of the Acidic seawater 7.1 water phase. Samples were stored in dark and cold conditions until measurements 1 mo later at Marine pH treatment Biological Laboratorium in Helsingør, Denmark. Trip- licate measurements were conducted on an IRGA (in- − Day 1 to end of experiment frared gas analyzer) by comparing with a HCO3 (bi- carbonate) standard of 2.0 mM (for procedure see 8.0 − Nielsen et al. 2007). The carbon speciation (HCO3 , 2− Seawater CO3 and CO2* [*includes H2CO3 and CO2]) was cal- culated using the program CO2SYS developed for CO system calculations (Lewis & Wallace 1998). CO2 7.7 7.7 2 (g) Samples for measurements of the inorganic nutri- 3− − ents phosphate (PO4 ), nitrate (NO3 ) and silicate pH 7.4 7.4 (Si(OH)4) were transferred into plastic bottles (35 ml) 4.9 and frozen immediately. The samples were analyzed at the Institute for Bioscience, University of Aarhus, Acidic seawater 7.1 7.1 following the procedures of Grasshoff (1976) and Valderrama (1981). pH-specific seawater pH treatment for dilution

Fig. 2. Experimental setup. Day 0: Acidic seawater was used Chl a to reduce the pH treatments of the phytoplankton commu- nity to the respective pH levels, except the control, which A 50 to 100 ml sample of the phytoplankton com- remained at pH 8.0. Day 1 to end of experiment: pH-specific munity was filtrated through a 0.7 µm GF/F filter and seawater for dilution of the phytoplankton in the pH treat- ments was lowered to the respective pH levels with acidic a 10 µm Nitex® plankton gauze, respectively. Imme- seawater. The control treatment (pH 8.0) was diluted with diately afterwards, the filters were extracted in 5 ml filtered seawater without adjustment of pH 96% ethanol in glass vials and stored in the dark at room temperature. The next day, chl a was measured on a TD-700 Fluorometer (Turner Designs) following dilution to ~5 µg chl a l−1 was an approximate esti- Jespersen & Christoffersen (1987). The growth rate mate. The control treatment at pH 8.0 was sampled of the phytoplankton community (chl a) was calcu- nearly every day, while pH 7.7, 7.4 and 7.1 were sam- lated from the cumulative growth due to the dilution pled every 2nd or 3rd day. On Day 5, the desired con- technique. centration of 5 µg chl a l−1 was reached in all pH treatments and Day 0 to 5 was set as the period of acclimation (not included in the results). Enumeration and identification of phytoplankton species

pH Samples for enumeration of phytoplankton taxa (120 to 250 ml) were transferred to brown glass bot- pH was measured with a WTW pH 3210 pH meter tles (250 to 300 ml) containing acidic Lugol’s iodine equipped with a WTW SenTix 41 electrode. The elec- (2% final concentration) and stored in the dark at trode was calibrated using a 2-point calibration and room temperature. Cells of dominating species were regularly checked for correct measurements in counted on an inverted microscope (Olympus CK40) Thoisen et al.: Effect of acidification on Arctic phytoplankton 25

Table 1. The dominant phytoplankton species from Disko Bay in this study. Enumeration of single cells, colonies or chains of the given species is indicated by ‘X’. The presence of single cells of species which were not enumerated is indicated by ‘–’. Blanks indicate that colonies or chains are not formed by the species

Species Single cells Colonies/chains

Diatoms Thalassiosira spp. X X spp. (incl. C. socialis)XX Navicula vanhoeffenii XX Navicula cf. granii –X Navicula spp. (incl. N. transitans var. derasa f. delicatula)X Prymnesiophyte Phaeocystis pouchetii –X Prasinophyte Pyramimonas sp. X

using 25 or 50 ml sedimentation chambers (Hydro- Dilution of the phytoplankton community (chl a) Bios) with 24 h settling time. A minimum of 400 cells was calculated as: or 6 transects were enumerated. Cells in colonies or C2 chains, single cells, or both were enumerated (Table 1). volr = 1−⋅ volt (1) ()C The growth rate was calculated from the cumulative 1 growth due to the dilution technique. Species were where volr and volt is the volume replaced with pH- identified based on light microscopic characters specific seawater and total volume of the incubation using Tomas (1997), except for Navicula cf. granii bottle, respectively, and C is the chl a concentration which was identified using a JEOL-1010 transmis- before (C1) and after (C2) dilution. sion electron microscope (TEM; Jeol). Prior to identi- Growth rate of the phytoplankton community fication by TEM, a sample of the phytoplankton com- (chl a) and species was calculated assuming expo- munity at pH 8.0 was rinsed following Lundholm et nential growth: N al. (2002). ln t 2 ()N μ = t1 (2) tt21− Colony, chain and cell size where μ is the growth rate and N is chl a or cell con-

centration at time t2 and t1. The cumulative concen- The diameter of 25 random colonies of the prymne- tration (μ’) of chl a and cells were calculated using μ siophyte Phaeocystis pouchetii was measured on an due to the dilution technique: inverted microscope (Olympus CK40). The number ’eN μ⋅t22()tt − 1 of cells in the chains of the species Thalas- μ = t1 ⋅ (3) siosira spp., Navicula vanhoeffenii, Chaetoceros spp. where N is chl a or cell concentration and μ is the and Navicula cf. granii were enumerated in 25 ran- growth rate at time t2. dom chains. Single cells and chains ≥2 cells were The growth rate for a pH treatment was calculated included for Thalassiosira spp., Navicula vanhoef- as the average of the triplicate samples’ linear fenii and Chaetoceros spp. Only chains ≥2 cells were regressions of ln(cumulative chl a concentration) or included for Navicula cf. granii due to difficulty iden- ln(cumulative cell concentration) after acclimation tifying single cells on the inverted microscope. with the slopes equalling to the growth rates.

Calculations Statistics

− 2− DIC speciation (HCO3 , CO3 and CO2*) was cal- Significant differences were analyzed using culated with the program CO2SYS and the following ANOVA (1-way) followed by pairwise multiple com- available inputs: Set of constants: K1, K2 from parisons with the Holm-Sidak method. The null

Mehrbach et al. (1973) refit by Dickson & Millero hypothesis (H0) was that there was no difference (1987); KHSO4: Dickson; pH scale: Seawater (SW) between pH treatments, and H0 was rejected when scale (mol kg−1 SW). For further information about p < 0.05. The significance level was set to 0.05 in all CO2SYS see Lewis & Wallace (1998). analyses. 26 Mar Ecol Prog Ser 520: 21–34, 2015

RESULTS

Hydrography of sampling station in Disko Bay

On the day of sampling (14 April 2012), the temperature and salinity of the surface water were −1.6°C and 33.2, respectively. At a depth of 300 m, this increased to 3.6°C and 34.4, respectively. A pycnocline at 160 m depth kept the spring bloom in the upper water column with a maximum chl a concentration of 1.4 µg l−1 at 5 m depth (data not shown). The phyto- plankton community was sampled from the maximum chl a where temperature, salinity and pH were −1.4°C, 33.2 and 7.9, respectively. The vertical distribution of in situ pH at a water column depth of 250 m was measured during the spring of 2011 and 2012 (Fig. 3). In 2011, pH was not measured throughout the spring bloom but varied between 7.9 and 8.5 from the end of April to the end of May. In 2012, pH var- ied between 7.5 and 8.3 from the end of March to the end of May. In March 2012, pH in the water column was 7.8 for approx. 2 wk and increased during April. However, there was a decrease to pH 7.5 in early April and mid-May. At the end of May, pH decreased for a longer period to 7.8, with a small increase to pH 8.0 in the upper part of the water column.

Experimental pH, DIC and nutrients

pH in the experimental treatments fluctuated minimally from the designated pH levels of 8.0, 7.7, 7.4 and 7.1 throughout the experimental period (Table 2). The concentration of DIC in the triplicate of each pH treatment was not sig- Fig. 3. In situ pH of Disko Bay. Vertical distribution of pH at the nificantly different between the midway point coastal sampling station at a depth of 250 m during (a) spring 2011 and (b) 2012 and the end of the experiment (Holm-Sidak, p > 0.05) (data not shown). DIC increased from 2222.2 ± 2.3 µmol kg−1 at pH 8.0 to 2522.6 ± Table 2. Average pH in the experimental treatments from Day 1 to 35.9 µmol kg−1 at pH 7.1 (Table 2). The DIC the end of the experiment (±SD, n = 3). The concentration of dis- solved organic carbon (DIC) is the average of 2 sampling times concentration at pH 7.1 was significantly dif- (midway and at the end of the experiment). The carbon speciation ferent from the other pH treatments (Holm- of DIC is given in percent. *includes H2CO3 and CO2 Sidak, p < 0.05). The carbon speciation of DIC in the pH treatments was dominated by bicar- − 2− pH DIC HCO3 CO3 CO2* − −1 bonate ion (HCO3 ) at >90%, while the con- (µmol kg ) (%) (%) (%) 2− centration of carbonate ion (CO3 ) decreased 8.01 ± 0.04 2222.2 ± 2.3 94.3 ± 0.2 4.7 ± 0.3 1.0 ± 0.1 and carbon dioxide (CO2*) increased with low- 2− 7.71 ± 0.04 2286.4 ± 8.8 95.5 ± 0.0 2.3 ± 0.0 2.1 ± 0.0 ered pH. The concentration of CO3 and CO2* 7.44 ± 0.04 2341.7 ± 40.0 95.1 ± 0.1 1.4 ± 0.1 3.5 ± 0.2 was significantly different between all pH 7.15 ± 0.05 2522.6 ± 35.9 92.1 ± 0.2 0.6 ± 0.0 7.3 ± 0.2 treatments (Holm-Sidak, p < 0.05). Thoisen et al.: Effect of acidification on Arctic phytoplankton 27

− 3− In situ concentrations of the nutrients NO3 , PO4 and Si(OH)4 at the depth of the chlorophyll maximum were 9.27 ± 0.1 µM, 0.77 ± 0.01 µM and 7.51 ± 0.12 µM, respectively, and fitted the Redfield ratios of 16N:1P and 15Si:16N. The nutrient concentrations in the pH treatments were not significantly different from the in situ concentrations throughout the exper- imental period (ANOVA, p > 0.05).

Growth of the phytoplankton community

The growth rate of the total phytoplankton commu- nity (i.e. chl a > 0.7 µm) was 0.50 ± 0.01 d−1 at pH 8.0 and decreased to 0.22 ± 0.01 d−1 at pH 7.1 (Fig. 4). The growth rate of the phytoplankton community Fig. 5. Temporal size composition of the fraction of the phyto- >10 µm (i.e. chl a >10 µm) was affected to the same plankton community >10 µm at pH 8.0, 7.7, 7.4 and 7.1. Data points are means ± SD (n = 3) degree but had a significantly higher growth rate at pH 8.0 (0.58 ± 0.02 d−1), 7.7 and 7.4. There was no sig- nificant difference between the growth rates of the species (Fig. 6). At pH 8.0, the growth rates of dia- total phytoplankton community and the phytoplank- toms were the highest among the species (~0.4 to ton community >10 µm at pH 7.1 (Holm-Sidak, p < ~0.6 d−1), whereas the prymnesiophyte Phaeocystis 0.05). pouchetii and the prasinophyte Pyramimonas sp. had The phytoplankton community >10 µm was the lower growth rates (~0.3 d−1). For all species, the dominating size fraction after Day 10 at pH 8.0, 7.7 growth rates at pH 7.1 were significantly lower and 7.4 (Fig. 5). In contrast, at pH 7.1 the size fraction (>50% compared to pH 8.0), with few species not >10 µm did not exceed 40% during the experiment. showing any growth at this pH. The effect of acidification on the growth rate was divided into 4 categories (Fig. 6): Category I, the spe- Growth of the phytoplankton species cies least affected. This was the prasinophyte Pyra- mimonas sp, and although there was a significant In general, the highest growth rates of the phyto- difference in growth rate between some of the pH plankton species were obtained at pH 8.0 and the treatments (Holm-Sidak, p < 0.05), a 32% reduction lowest at pH 7.1, but the growth rates differed among in growth rate from pH 8.0 to 7.1 was the smallest compared to the other species. Category II, species not affected by acidification in the pH range 8.0 to 0.7 7.4. These comprised the diatom Navicula spp. and the prymnesiophyte P. pouchetii. At pH 7.1 neither 0.6 species could sustain growth (Holm-Sidak, p < 0.05). Category III, species gradually affected by acidifica- ) 0.5 -1 tion. These were the chain-forming diatoms Thalas- 0.4 siosira spp., Navicula vanhoeffenii and Chaetoceros spp. with similar growth rates and a similar reduction 0.3 in growth rate reaching ~0.2 d−1 at pH 7.1. The growth rate for each species was not significantly dif- Growth rate (d rate Growth 0.2 ferent between pH 7.7 and 7.4 (Holm-Sidak, p >

0.1 Total chl a 0.05). Category IV, the species highly affected by Chl a >10 µm acidification. This was the chain-forming diatom 0.0 Navivula cf. granii, which had a significantly differ- 7.1 7.4 7.7 8.0 ent growth rate for each pH treatment (Holm-Sidak, pH p > 0.05). This species had one of the highest growth Fig. 4. Growth rate of the phytoplankton community at pH rates at pH 8.0 but was also the most sensitive species 8.0, 7.7, 7.4 and 7.1. Data points are means ± SD (n = 3) and could not sustain growth at pH 7.1. 28 Mar Ecol Prog Ser 520: 21–34, 2015

0.8 0.8 Pyramimonas sp. I Navicula spp. II Phaeocystis pouchetii ) 0.6 0.6 -1

0.4 0.4

0.2 0.2

Growth rate, µ (d 0.0 0.0 +

-0.2 -0.2 7.1 7.4 7.7 8.0 7.1 7.4 7.7 8.0

0.8 0.8 Thalassiosira spp. III Navicula cf. granii IV

) Chaetoceros spp.

-1 0.6 Navicula vanhoeffenii 0.6

0.4 0.4

0.2 0.2 Growth rate, µ (d 0.0 0.0

–0.2 -0.2 7.1 7.4 7.7 8.0 7.1 7.4 7.7 8.0 pH pH Fig. 6. Growth rates of phytoplankton species. The species were divided into categories based on their tolerance to acidifica- tion: least affected (I); affected only at pH 7.1 (II); gradually affected (III); and highly affected (IV). (+) indicates presence of Phaeocystis pouchetii colonies until Day 10 at pH 7.1. Data points are means ± SD (n = 3)

Effect of acidification on colony size Day 11, compared to 4 cells at pH 7.1. A similar effect and chain length on chain length was observed for N. vanhoeffenii and Chaetoceros spp., while the chain length of N. cf. The colony size of Phaeocystis pouchetii was granii did not appear to be affected (data not shown). reduced during acidification (Fig. 7). On Day 5, the frequency of P. pouchetii colonies with a diameter of up to 280 µm was similar at pH 8.0, 7.7 and 7.4, with DISCUSSION a slight decrease in size with increasing acidification (only data for pH 8.0 is shown). However, at pH 7.1 a Fluctuations of pH in Disko Bay during spring decrease in colony size was observed on Day 5 with the majority of colonies measuring up to 80 µm. Few data are available on fluctuations of pH in Microscopical observations of P. pouchetii colonies at Arctic marine ecosystems and these mostly report the pH 7.1 revealed that the cells were unorganized and fluctuations in open surface waters. Data on pH from clustered, making it impossible to enumerate the coastal regions of the Arctic have so far been com- cells in the colonies after Day 5. After 10 d no P. pletely lacking, but here we document prominent pouchetii colonies were present at pH 7.1 (Fig. 6). fluctuations from pH 7.5 to 8.3 in a 250 m water col- Also, the chain length (i.e. no. of cells) of Thalassio - umn in Disko Bay during the spring bloom of 2012. sira spp. decreased with acidification (Fig. 8). At pH These fluctuations resemble observations in other 8.0, the majority of chains contained up to 8 cells on coastal areas, e.g. pH at 2 m depth in the upwelling Thoisen et al.: Effect of acidification on Arctic phytoplankton 29

70 80 pH 8.0 pH 8.0 60

50 60 Day 1 Day 1 Day 11 40 Day 5 40 30

Frequency (%) 20 Frequency (%) Frequency 20 10

0 -40 -80 20 60 00 40 80 0 1 41 1-1 1-1 1-2 1-2 1-2 8 12 16 20 24 0246810121416 60 80 pH 7.1 pH 7.1 50 60 40

30 40

20 Frequency (%) Frequency (%) 20 10

0 0 -40 -80 20 60 00 40 80 1 41 1-1 1-1 1-2 1-2 1-2 8 12 16 20 24 0 2 4 6 8 10 12 14 16 Colony diameter (µm) Chain length (no. of cells)

Fig. 7. Colony size. Frequency of the colony diameter of the Fig. 8. Chain length. Frequency of the chain length (i.e. no. prymnesiophyte Phaeocystis pouchetii at pH 8.0 and 7.1 of cells) of the diatom Thalassiosira spp. at pH 8.0 and 7.1 on Days 1 and 5 on Days 1 and 11 area of Point Ano Nuevo, USA, varied from 7.7 to 8.1 2012 due to an earlier breakup of sea ice which over 30 d from mid-May (Hofmann et al. 2011) and allowed for a prolonged spring bloom (~6 wk) with a pH in Narragansett Bay, USA, fluctuated from ~7.6 to longer period for photosynthesis to raise pH in the ~8.5 over 1 yr at 5 m depth (Hinga 1992). water column (T. G. Nielsen pers. obs.). On the day of The observed pH fluctuations in Disko Bay were sampling (14 April 2012), the salinity in the water col- caused by several factors. Before the spring bloom, umn increased from the surface and downwards due i.e. during the Arctic polar night and sea ice cover, to the melting of sea ice and a halocline was present with continuous lack of solar irradiance, there was a at a depth of ~160 m, maintaining the photosynthetic − 3− low pH at the sampling station due to the dominance plankton above this depth. Nutrients (NO3 , PO4 of respiration processes. As the solar irradiance and Si(OH)4) in the chlorophyll maximum (5 m) were increased during the transition to polar day, factors plentiful at the time of sampling because the phyto- such as chl a, photosynthesis and pH increased in the plankton spring bloom had not yet peaked. water column. Advection of sea ice into the bay A pH below 7.5 was not measured in Disko Bay reduced the solar irradiance into the water column during spring but it is not unlikely that pH may and resulted in a decrease in pH twice during 2012 decrease to values near 7.1 during the next centuries (early and mid-May) due to decreased photosynthe- with ongoing ocean acidification. For instance, Woot- sis and increased respiration (Fig. 3). pH in the water ton et al. (2008) found a significant decrease of pH in column was generally higher in 2011 compared to coastal waters on the Washington Shelf over a period 30 Mar Ecol Prog Ser 520: 21–34, 2015

of 8 yr in association with increasing atmospheric Effect of acidification on the growth rate of

CO2, and a similar acidification could likely be occur- phytoplankton species ring in Disko Bay at present time. The phytoplankton community in Disko Bay was dominated by diatoms, haptophytes and prasino- Nutrients, DIC and pH phytes. The investigated species (Table 1) have pre- viously been reported in Disko Bay (Nielsen & − The nutrient concentrations of Si(OH)4, NO3 and Hansen 1999, DATMAPD marine protist database at 4− PO3 in the experimental treatments exceeded those www2.bio.ku.dk/disko/ [accessed 11.11.2012]) with shown to be limiting for phytoplankton, including the exception of Navicula cf. granii, in spite of this cold water species (e.g. Egge & Aksnes 1992, Nelson being a typical Arctic species (Guillard & Kilham & Tréguer 1992). This excluded nutrient limitation as 1977). The ciliate species Strombidium sp. and a cause for the decreasing growth rates during the Lohmaniella oviformis were also present in the pH experiment. The concentration of DIC and carbon treatments but due to a very low cell concentration speciation in the treatment at pH 8.0 correlate well the grazing by these ciliates on the phytoplankton is with literature data on ocean surface waters (e.g. assumed to have been insignificant. The tolerance of Feely et al. 2009). At increased experimental acidifi- marine protists to pH has previously been found to be cation, the inorganic carbon availability, which could species-specific (Tortell et al. 2002, Kim & Lee 2006, potentially benefit the autotrophic phytoplankton, Berge et al. 2010) and the present study supports this, was changed, theoretically, for the better with a vast as the phytoplankton species could be divided into 4 − concentration of HCO3 and increased CO2. How- well-defined categories (Fig. 6). Studies show that ever, a benefit from this inorganic carbon availability prasinophytes are quite tolerant to acidification and was not reflected in the phytoplankton growth rate. actually increase in abundance, possibly due to a

The pH of the treatments fluctuated minimally (≤0.05 higher exploitation of the elevated CO2 levels units) throughout the experiment providing a valid (Meakin & Wyman 2011, Newbold et al. 2012). The response of the phytoplankton community to the prasinophyte Pyramimonas sp. in the present study experimental pH values. did not attain a higher growth rate during acidifica- tion. However, it was the species least affected and our study supports the proposal that prasinophytes Effect of acidification on phytoplankton community may be fairly tolerant to acidification. Also the growth rate diatom Navicula spp. and the haptophyte Phaeocys- tis pouchetii were quite tolerant to acidification and The growth rate of the phytoplankton community were only affected at pH 7.1. P. pouchetii is very from Disko Bay was affected negatively by the exper- common in Arctic waters with colonies up to 2 mm in imental acidification (Fig. 4), even for those commu- diameter (Schoemann et al. 2005) and the broad tol- nities grown within the range of in situ pH measured erance to acidification could be a contributing factor in Disko Bay in 2012 (pH 7.5 to 8.3). The effect was to the formation of the well-known large Phaeocystis observed as a gradual decrease in community blooms. However, the colony size of P. pouchetii growth rate with decreasing pH from 8.0 to 7.1. Many was severely reduced at pH 7.1 (Fig. 7) and colonies studies have explored the effect of acidification on disappeared after 10 d. Normally, the cells in P. phytoplankton, but they tend to study the growth pouchetii colonies are embedded in groups of 4 but response within a narrow pH limit of 8.2 to 7.8 (e.g. at pH 7.1 the cells were clustered. It is possible that Wang et al. 2010, Yang & Gao 2012), probably low pH hampered the uptake or utilization of certain because this reduction has been predicted to occur cations necessary for the mucus gelling in these for open oceans by the end of the 21st century. How- colonies (van Boekel 1992). Only cells in colonies of ever, it is problematic to use such a narrow range of P. pouchetii were enumerated so the present study pH to study the tolerance of coastal phytoplankton to did not clarify if single cells (4 to 7 µm; Schoemann et acidification because pH in coastal waters fluctuates al. 2005) were either unable to tolerate a pH of 7.1 or within a larger range (as shown in this study). Fur- if they were present but simply unable to form thermore, it is necessary to expose organisms to a pH colonies. lower than that measured in situ to evaluate the sus- Solitary species of the diatom genus Navicula are ceptibility of marine organisms to the ongoing ocean primarily benthic forms and common in sea ice from acidification. which they are released into the water column when Thoisen et al.: Effect of acidification on Arctic phytoplankton 31

the sea ice melts (Tomas 1997). In contrast to our 2011 and references therein, see also Nimer et al. study, where Navicula spp. could not sustain growth 1994). at pH 7.1, Wang et al. (1998) found a tolerance to The phytoplankton species in the present study acidification of benthic diatoms as growth rates have been reported to also be present in the brine decreased by merely ~20% from pH 8.0 to 7.0. How- channels of Arctic sea ice (Abelmann 1992, Ikävalko ever, a comparison of the tolerance of species be- & Gradinger 1997, Werner et al. 2007, Niemi et al. tween different experiments is not optimal due to dif- 2011) where pH can vary from the surrounding pH of ferences in experimental conditions and strains. seawater up to pH 9.9 (Gleitz et al. 1995). Thus, the

Diatoms incorporate silicic acid (Si(OH)4) into frus- phytoplankton is subjected to a pH as high as 9.9 in tules during growth and this process is known to be the brine of sea ice and as low as 7.5 in the water col- negatively affected by acidification (Hervé et al. umn (Fig. 3). This seasonal exposure to a broad pH 2012). This could be a reason for the decreasing range up to 2.4 pH units could indicate that some growth rates and chain lengths found in the present Arctic phytoplankton species may be currently study. However, the single-celled diatom Navicula exposed to their potential maximum physiological spp. was more tolerant to acidification than the range. This could explain why some species could chain-forming diatoms (Thalassiosira spp., Chaeto- not grow at pH 7.1, a pH exceeding the natural fluc- ceros spp., N. vanhoeffenii and N. cf. granii), which tuations in Disko Bay during spring. In the present suggests that chain-forming diatoms possess similar study, the phytoplankton appears to be more or less physiological mechanisms affected by acidification adapted to the natural fluctuations of pH, leading to a (Fig. 8), especially regarding chain elongation. Con- broad tolerance to the experimental acidification trary to this, the chain length of N. cf. granii did not between pH 8.0 and 7.4. The species-specific toler- differ between pH 8.0 and 7.1 although the growth ances to pH may contribute to the dominance of dif- rate was highly affected. The effect of acidification ferent species throughout the year as pH fluctuates. on the chain length of diatoms found in this study is Three of 7 phytoplankton species were not able to unclear. The shorter chain lengths at low pH could grow at pH 7.1, and if the pH in Disko Bay reaches simply be a result of lower growth rates of the pH 7.1 by the end of this century, a shift in the phyto- diatoms obtained at pH 7.1 as shown by Takabayashi plankton community structure may occur according et al. (2006), where changes in temperature and to our results. Such a shift in species composition, nutrient availability caused a lower growth rate and colony size and chain length could affect the Arctic shorter chain lengths of the diatom Skeletonema food web. Phaeocystis sp. is an important component costatum. However, all diatoms in the present study of the phytoplankton in Arctic marine ecosystems, tolerated a pH range from 8.0 to 7.4 with either stable with a variety of grazers grazing on different sizes of or reduced growth rates, and similar results were Phaeocystis colonies (van Boekel 1992 and refer- obtained from natural enclosures at Rhode Island, ences therein, see also Tang 2003, Schoemann et al. USA, where the frequency of diatoms was highest at 2005). In addition, the success of Phaeocystis blooms the pH range ~8.0 to ~7.4 and decreased with acidifi- has been attributed to the ability to form large gelat- cation (Hinga 1992). inous colonies, and this study shows that the colonies It is well known that voltage-gated H+ channels in were severely affected at pH 7.1. Hence, if colonies the plasma membrane of phytoplankton are ex- of P. pouchetii are inhibited by the ongoing acidifica- tremely sensitive to changes in external pH (pHe) tion, they could become absent as an important food (e.g. Taylor et al. 2012). The predicted drop in the source for certain grazers, should the pH decrease to average ocean surface water pH to ~7.8 by the end of values near pH 7.1. this century (Feely et al. 2009) is thought to reverse the proton motive force, thereby impairing the pas- sive efflux of H+ out of the cell which regulates intra- Is pH tolerance size-dependent? cellular pH (pHi). Changes in pHi studied by Hervé et al. (2012) with the diatom Thalassiosira weissflogii A size-dependent pH tolerance has been proposed found that a lowering of pHe from 8.5 to 6.4 led to a for and diatoms, with small species drop in pHi from 7.6 to 6.7. This decrease in pHi may suggested to be able to tolerate a higher pH com- have severe physiological consequences, e.g. ion pared to larger species (Lundholm & Hansen 2004, transport, enzyme activity and protein function, Søderberg & Hansen 2007). The large surface:vol- which will be reflected in reduced growth rates as ume area in small species is thought to provide a bet- observed in the present study (Gattuso & Hansson ter regulation of pHi during increased pHe and the 32 Mar Ecol Prog Ser 520: 21–34, 2015

same could apply for the tolerance to low pH. How- 2000). Thus, if the present study had been conducted ever, considerable variations among species of simi- later in the season, it is likely that species composi- lar sizes were also reported and a simulation study by tion and pH tolerance would have differed from the Flynn et al. (2012) showed that large phytoplankton results obtained by the present study conducted in was better adapted to variable pH conditions com- spring. pared to smaller sizes. In the present study, a size- dependent pH tolerance was found for the temporal composition of the phytoplankton community where CONCLUSIONS large cells (>10 µm) dominated at pH 8.0, 7.7 and 7.4 and small cells (≤10 µm) dominated at pH 7.1 (Fig. 5). In situ pH in Disko Bay varied from 7.5 to 8.3 dur- Similar results were obtained by Nielsen et al. (2010) ing the spring bloom in 2012, and the present study for a coastal plankton community from Øresund shows that the growth rate of the phytoplankton Strait, Denmark, where large cells (>15 µm) domi- community decreased with pH within the in situ pH nated at pH 8.0, 7.8 and 7.6 and small cells (10 to range. The phytoplankton community, as well as the 15 µm) dominated at pH 6.0. Thus, small phytoplank- investigated phytoplankton species, did not benefit ton cells could be more tolerant to low pH (ca ≤7.1) from acidification despite abundant nutrients and compared to larger cells, but further research is inorganic carbon for growth and photosynthesis. A needed to clarify the contrasting findings. likely explanation could be that the phytoplankton growth is not limited by inorganic carbon at pH 8.0 and/or that negative effects of lowered pH on physi- Population genetic adaption and experimental ological processes exceed the potentially beneficial

acidification effect of increased CO2. The study also reveals that a further decrease to pH 7.1 may have severe effects The present study was conducted over a short time on the productivity of the phytoplankton community. span (17 d), which did not allow sufficient time for The response to acidification was species-specific, population genetic adaptation to the changing envi- with some species being quite sensitive and others ronment. The pH in treatments was adjusted from more tolerant. If future ocean acidification causes a the in situ pH of Disko Bay (pH 7.9) to the experimen- decrease in pH in Disko Bay to ~7.1, a shift in the tal values of 7.7, 7.4 and 7.1 within 24 h and does not community structure and production may occur reflect the rate of ocean acidification occurring grad- unless phytoplankton populations genetically adapt ually over centuries. However, the present study to the changing conditions over time. shows the response of phytoplankton to the natural Studies on the effect of ocean acidification should variations of pH in Disko Bay (pH 7.5 to 8.3), includ- include the natural fluctuations of in situ pH in order ing some degree of tolerance when exposed to a low to expose organisms from a specific site to an experi- pH of 7.1. Several strains of a species are present mental pH range exceeding the natural fluctuations within a natural assemblage, and population genetic of pH. In addition, information on genetic diversity adaption could potentially lead to the evolution of the within species, and how this potentially affects the strains which are more tolerant of low pH. Thus, the species’ response to acidification, is required. effect of ocean acidification on phytoplankton might not be as significant as could be expected. However, Acknowledgements. The authors thank the helpful crew at such predictions are difficult to confirm, and studies Arctic Station in Qeqertarsuaq and the scientific crew of adaption are time-consuming. Few studies on involved in collecting data and analysing samples for this adaption have been conducted; studies on the cocco- study. The authors are also grateful to the anonymous lithophores and Calcidiscus lepto- reviewers for their constructive critique. The project was porus suggest that adaption to acidification may in funded by the European Commission FP7 EURO-BASIN (Grant Agreement: 264 933), a Freja stipend from the Fac- fact occur over time (Langer et al. 2006, Lohbeck et ulty of Science, the Greenland Climate Research Centre al. 2012). (Project 6505), and the VKR Centre for Ocean Life. Studies on the effect of acidification, using natural phytoplankton assemblages, need to take the time of year into consideration because seasonal distribution LITERATURE CITED and diversity of species change over time due to vari- Abelmann A (1992) Diatom assemblages in Arctic sea ice — ations of environmental conditions such as presence indicator for ice drift pathways. Deep-Sea Res II 39: of sea ice, temperature, pH and salinity (von Quillfeldt S525−S538 Thoisen et al.: Effect of acidification on Arctic phytoplankton 33

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Editorial responsibility: Steven Lohrenz, Submitted: August 12, 2013; Accepted: November 14, 2014 New Bedford, Massachusetts, USA Proofs received from author(s): January 14, 2015