Seawater carbonate chemistry and biological processes during experiments with clownfish Amphiprion percula, 2009

The persistence of most coastal marine species depends on larvae finding suitable adult habitat at the end of an offshore dispersive stage that can last weeks or months. We tested the effects that ocean acidification from elevated levels of atmospheric carbon dioxide (CO2) could have on the ability of larvae to detect olfactory cues from adult habitats. Larval clownfish reared in control seawater (pH 8.15) discriminated between a range of cues that could help them locate reef habitat and suitable settlement sites. This discriminatory ability was disrupted when larvae were reared in conditions simulating CO2-induced ocean acidification. Larvae became strongly attracted to olfactory stimuli they normally avoided when reared at levels of ocean pH that could occur ca. 2100 (pH 7.8) and they no longer responded to any olfactory cues when reared at pH levels (pH 7.6) that might be attained later next century on a business-as-usual carbon-dioxide emissions trajectory. If acidification continues unabated, the impairment of sensory ability will reduce population sustainability of many marine species, with potentially profound consequences for marine diversity.

Seawater carbonate chemistry and biological processes during experiments with barnacle Semibalanus balanoides, 2010

The Arctic Ocean and its associated ecosystems face numerous challenges over the coming century. Increasing atmospheric CO2 is causing increasing warming and ice melting as well as a concomitant change in ocean chemistry ("ocean acidification"). As temperature increases it is expected that many temperate species will expand their geographic distribution northwards to follow this thermal shift; however with the addition of ocean acidification this transition may not be so straightforward. Here we investigate the potential impacts of ocean acidification and climate change on populations of an intertidal species, in this case the barnacle Semibalanus balanoides, at the northern edge of its range. Growth and development of metamorphosing post-larvae were negatively impacted at lower pH (pH 7.7) compared to the control (pH 8.1) but were not affected by elevated temperature (+4 °C). The mineral composition of the shells did not alter under any of the treatments. The combination of reduced growth and maintained mineral content suggests that there may have been a change in the energetic balance of the exposed animals. In undersaturated conditions more mineral is expected to dissolve from the shell and hence more energy would be required to maintain the mineral integrity. Any energy that would normally be invested into growth could be reallocated and hence organisms growing in lowered pH grow slower and end up smaller than individuals grown in higher pH conditions. The idea of reallocation of resources under different conditions of pH requires further investigation. However, there could be long-term implications on the fitness of these barnacles, which in turn may prevent them from successfully colonising new areas.

Seawater carbonate chemistry, calcification and shell size of hard clam Mercenaria mercenaria during experiments, 2011

Increasing atmospheric carbon dioxide threatens to decrease pH in the world's oceans. Coastal and estuarine calcifying organisms of significant ecological and economical importance are at risk; however, several biogeochemical processes drive pH in these habitats. In particular, coastal and estuarine sediments are frequently undersaturated with respect to calcium carbonate due to high rates of organic matter remineralization, even when overlying waters are saturated. As a result, the post-larval stages of infaunal marine bivalves must be able to deposit new shell material in conditions that are corrosive to shell. We measured calcification rates on the hard clam, Mercenaria spp.,in 5 post-larval size classes (0.39, 0.56, 0.78, 0.98, and 2.90 mm shell height) using the alkalinity anomaly method. Acidity of experimental water was controlled by bubbling with air-CO2 blends to obtain pH values of 8.02, 7.64, and 7.41, corresponding to pCO2 values of 424, 1120, and 1950 µatm. These pH values are typical of those found in many near-shore terrigenous marine sediments. Our results show that calcification rate decreased with lower pH in all 5 size classes measured. We also found a significant effect of size on calcification rate, with the smaller post-larval sizes unable to overcome dissolution pressure. Increased calcification rate with size allowed the larger sizes to overcome dissolution pressure and deposit new shell material under corrosive conditions. Size dependency of pH effects on calcification is likely due to organogenesis and developmental shifts in shell mineralogy occurring through the post-larval stage. Furthermore, we found significantly different calcification rates between the 2 sources of hard clams we used for these experiments, most likely due to genotypic differences. Our findings confirm the susceptibility of the early life stages of this important bivalve to decreasing pH and reveal mechanisms behind the increased mortality in post-larval juvenile hard clams related to dissolution pressure, that has been found in previous studies.

Seawater carbonate chemistry, mass, length and gene expression of Sepia officinalis during experiments, 2011

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immuno histochemical methods, we demonstrate that Na+/K+-ATPase (soNKA), a V-type H+-ATPase (soV-HA), and Na+/HCO3- cotransporter (soNBC) are co-localized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater pCO2 (0.16 and 0.35 kPa) over a time-course of six weeks in different ontogenetic stages. The applied CO2 concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII and COX. In contrast, no hypercapnia induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However a transiently increased demand of ion regulatory demand was evident during the initial acclimation reaction to elevated seawater pCO2. Gill Na+/K+-ATPase activity and protein concentration were increased by approximately 15% in during short (2-11 day), but not long term (42 day) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the down regulation of ion-regulatory and metabolic genes in late stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater pCO2.

Seawater carbonate chemistry and Strongylocentrotus purpuratus body length and gene expression pattern changes during experiments, 2011

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO2 41 Pa e.g. 399 µatm) and CO2 acidified seawater with pH of 7.7 (pCO2 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10 % reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (Two way ANOSIM: Global R = 1) while acidification effects were less pronounced (Global R = 0.518). Significant differences in gene expression patterns (ANOSIM R = 0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23 and 36% in msp130, SM30B, SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

Seawater carbonate chemistry and trace element accumulation during experiments with common cuttlefish, Sepia officinalis, 2009

Cephalopods play a key role in many marine trophic networks and constitute alternative fisheries resources, especially given the ongoing decline in finfish stocks. Along the European coast, the eggs of the cuttlefish Sepia officinalis are characterized by an increasing permeability of the eggshell during development, which leads to selective accumulation of essential and non-essential elements in the embryo. Temperature and pH are two critical factors that affect the metabolism of marine organisms in the coastal shallow waters. In this study, we investigated the effects of pH and temperature through a crossed (3?2; pH 8.1 (pCO2, 400 ppm), 7.85 (900 ppm) and 7.6 (1400 ppm) at 16 and 19°C, respectively) laboratory experiment. Seawater pH showed a strong effect on the egg weight and non-significant impact on the weight of hatchlings at the end of development implying an egg swelling process and embryo growth disturbances. The lower the seawater pH, the more 110 mAg was accumulated in the tissues of hatchlings. The 109Cd concentration factor (CF) decreased with decreasing pH and 65Zn CF reached maximal values pH 7.85, independently of temperature. Our results suggest that pH and temperature affected both the permeability properties of the eggshell and embryonic metabolism. To the best of our knowledge, this is one of the first studies on the consequences of ocean acidification and ocean warming on metal uptake in marine organisms, and our results indicate the need to further evaluate the likely ecotoxicological impact of the global change on the early-life stages of the cuttlefish.

Seawater carbonate chemistry and its effects on properties and sinking velocity of aggregates of the coccolithophore Emiliania huxleyi, 2010

Coccolithophores play an important role in organic matter export due to their production of the mineral calcite that can act as ballast. Recent studies indicated that calcification in coccolithophores may be affected by changes in seawater carbonate chemistry. We investigated the influence of CO2 on the aggregation and sinking behaviour of the coccolithophore Emiliania huxleyi (PML B92/11) during a laboratory experiment. The coccolithophores were grown under low (~180 µatm), medium (~380 µatm), and high (~750 µatm) CO2 conditions. Aggregation of the cells was promoted using roller tables. Size and settling velocity of aggregates were determined during the incubation using video image analysis. Our results indicate that aggregate properties are sensitive to changes in the degree of ballasting, as evoked by ocean acidification. Average sinking velocity was highest for low CO2 aggregates (~1292 m d-1) that also had the highest particulate inorganic to particulate organic carbon (PIC/POC) ratio. Lowest PIC/POC ratios and lowest sinking velocity (~366 m d-1) at comparable sizes were observed for aggregates of the high CO2 treatment. Aggregates of the high CO2 treatment showed a 4-fold lower excess density (~4.2*10**-4 g cm**-3) when compared to aggregates from the medium and low CO2 treatments (~1.7 g*10**-3 cm**-3). We also observed that more aggregates formed in the high CO2 treatment, and that those aggregates contained more bacteria than aggregates in the medium and low CO2 treatment. If applicable to the future ocean, our findings suggest that a CO2 induced reduction of the calcite content of aggregates could weaken the deep export of organic matter in the ocean, particularly in areas dominated by coccolithophores.

Seawater carbonate chemistry and echinoderm Asterias rubens biological processes during experiments, 2011

We compared effects of exposure to predicted near-future (2100) ocean acidification (OA; pH 7.7) and normal seawater (Control; pH 8.1) on immune and stress responses in the adult sea star Asterias rubens. Analyses were made after one week and after six months of continuous exposure. Following one week exposure to acidified water, the pH of coelomic fluid was significantly reduced. Levels of the chaperon Hsp70 were elevated while key cellular players in immunity, coelomocytes, were reduced by approximately 50%. Following long-term exposure (six months) levels of Hsp70 returned to control values, whereas immunity was further impaired, evidenced by the reduced phagocytic capacity of coelomocytes and inhibited activation of p38 MAP-kinase. Such impacts of reduced seawater pH may have serious consequences for resistance to pathogens in a future acidified ocean.

Pore water chemistry of samples near the sediment-water interface

Rhizon samplers were originally designed as micro-tensiometers for soil science to sample seepage water in the unsaturated zone. This study shows applications of Rhizons for porewater sampling from sediments in aquatic systems and presents a newly developed Rhizon in situ sampler (RISS). With the inexpensive Rhizon sampling technique, porewater profiles can be sampled with minimum disturbance of both the sediment structure and possible flow fields. Field experiments, tracer studies, and numerical modeling were combined to assess the suitability of Rhizons for porewater sampling. It is shown that the low effort and simple application makes Rhizons a powerful tool for porewater sampling and an alternative to classical methods. Our investigations show that Rhizons are well suited for sampling porewater on board a ship, in a laboratory, and also for in situ sampling. The results revealed that horizontally aligned Rhizons can sample porewater with a vertical resolution of 1 cm. Combined with an in situ benthic chamber system, the RISS allows studies of benthic fluxes and porewater profiles at the same location on the seafloor with negligible effect on the incubated sediment water interface. Results derived by porewater sampling of sediment cores from the Southern Ocean (Atlantic sector) and by in situ sampling of tidal flat sediments of the Wadden Sea (Sahlenburg/Cuxhaven, Germany) are presented.

Seawater carbonate chemistry, chlorophyll a and phosphate during experiments with Trichodesmium erythraeum IMS101 (CCMP1985), 2010

In this laboratory study, we monitored the buildup of biomass and concomitant shift in seawater carbonate chemistry over the course of a Trichodesmium bloom under different phosphorus (P) availability. During exponential growth, dissolved inorganic carbon (DIC) decreased, while pH increased until maximum cell densities were reached. Once P became depleted, DIC decreased even further and total alkalinity (TA) dropped, accompanied by precipitation of aragonite. Under P-replete conditions, DIC increased and TA remained constant in the postbloom phase. A diffusion-reaction model was employed to estimate changes in carbonate chemistry of the diffusive boundary layer. This study demonstrates that Trichodesmium can induce precipitation of aragonite from seawater and further provides possible explanations about underlying mechanisms.

Seawater carbonate chemistry and crab Necora puber size and elements in tissue during experiments, 2010

Ocean acidification (OA) is predicted to play a major role in shaping species biogeography and marine biodiversity over the next century. We tested the effect of medium-term exposure to OA (pH 8.00, 7.30 and 6.70 for 30 d) on acid-base balance in the decapod crab Necora puber-a species that is known to possess good extracellular buffering ability during short-term exposure to hypercapnic conditions. To determine if crabs undergo physiological trade-offs in order to buffer their haemolymph, we characterised a number of fundamental physiological functions, i.e. metabolic rate, tolerance to heat, carapace and chelae [Ca2+] and [Mg2+], haemolymph [Ca2+] and [Mg2+], and immune response in terms of lipid peroxidation. Necora puber was able to buffer changes to extracellular pH over 30 d exposure to hypercapnic water, with no evidence of net shell dissolution, thus demonstrating that HCO3- is actively taken up from the surrounding water. In addition, tolerance to heat, carapace mineralization, and aspects of immune response were not affected by hypercapnic conditions. In contrast, whole-animal O2uptake significantly decreased with hypercapnia, while significant increases in haemolymph [Ca2+] and [Mg2+] and chelae [Mg2+] were observed with hypercapnia. Our results confirm that most physiological functions in N. puber are resistant to low pH/hypercapnia over a longer period than previously investigated, although such resistance comes at the expenses of metabolic rates, haemolymph chemistry and chelae mineralization.

Seawater carbonate chemistry and calcification rate of colonies of Stylophora pistillata, 1998

The carbonate chemistry of seawater is usually not considered to be an important factor influencing calcium-carbonate-precipitation by corals because surface seawater is supersaturated with respect to aragonite. Recent reports, however, suggest that it could play a major role in the evolution and biogeography of recent corals. We investigated the calcification rates of five colonies of the zooxanthellate coral Stylophora pistillata in synthetic seawater using the alkalinity anomaly technique. Changes in aragonite saturation from 98% to 585% were obtained by manipulating the calcium concentration. The results show a nonlinear increase in calcification rate as a function of aragonite saturation level. Calcification increases nearly 3-fold when aragonite saturation increases from 98% to 390%, i.e., close to the typical present saturation state of tropical seawater. There is no further increase of calcification at saturation values above this threshold. Preliminary data suggest that another coral species, Acropora sp., displays a similar behaviour. These experimental results suggest: (1) that the rate of calcification does not change significantly within the range of saturation levels corresponding to the last glacial-interglacial cycle, and (2) that it may decrease significantly in the future as a result of the decrease in the saturation level due to anthropogenic release of CO2 into the atmosphere. Experimental studies that control environmental conditions and seawater composition provide unique opportunities to unravel the response of corals to global environmental changes.

Seawater carbonate chemistry and clownfish Amphiprion percula size and otholith development during experiments, 2011

Calcification in many invertebrate species is predicted to decline due to ocean acidification. The potential effects of elevated CO2 and reduced carbonate saturation state on other species, such as fish, are less well understood. Fish otoliths (earbones) are composed of aragonite, and thus, might be susceptible to either the reduced availability of carbonate ions in seawater at low pH, or to changes in extracellular concentrations of bicarbonate and carbonate ions caused by acid-base regulation in fish exposed to high pCO2. We reared larvae of the clownfish Amphiprion percula from hatching to settlement at three pHNBS and pCO2 levels (control: ~pH 8.15 and 404 µatm CO2; intermediate: pH 7.8 and 1050 µatm CO2; extreme: pH 7.6 and 1721 µatm CO2) to test the possible effects of ocean acidification on otolith development. There was no effect of the intermediate treatment (pH 7.8 and 1050 µatm CO2) on otolith size, shape, symmetry between left and right otoliths, or otolith elemental chemistry, compared with controls. However, in the more extreme treatment (pH 7.6 and 1721 µatm CO2) otolith area and maximum length were larger than controls, although no other traits were significantly affected. Our results support the hypothesis that pH regulation in the otolith endolymph can lead to increased precipitation of CaCO3 in otoliths of larval fish exposed to elevated CO2, as proposed by an earlier study, however, our results also show that sensitivity varies considerably among species. Importantly, our results suggest that otolith development in clownfishes is robust to even the more pessimistic changes in ocean chemistry predicted to occur by 2100.

Seawater carbonate chemistry and biological processes during experiments with Patella vulgata, 2010

The effect of short-term (5 days) exposure to CO2-acidified seawater (year 2100 predicted values, ocean pH = 7.6) on key aspects of the function of the intertidal common limpet Patella vulgata (Gastropoda: Patellidae) was investigated. Changes in extracellular acid-base balance were almost completely compensated by an increase in bicarbonate ions. A concomitant increase in haemolymph Ca2+ and visible shell dissolution implicated passive shell dissolution as the bicarbonate source. Analysis of the radula using SEM revealed that individuals from the hypercapnic treatment showed an increase in the number of damaged teeth and the extent to which such teeth were damaged compared with controls. As radula teeth are composed mainly of chitin, acid dissolution seems unlikely, and so the proximate cause of damage is unknown. There was no hypercapnia-related change in metabolism (O2 uptake) or feeding rate, also discounting the possibility that teeth damage was a result of a CO2-related increase in grazing. We conclude that although the limpet appears to have the physiological capacity to maintain its extracellular acid-base balance, metabolism and feeding rate over a 5 days exposure to acidified seawater, radular damage somehow incurred during this time could still compromise feeding in the longer term, in turn decreasing the top-down ecosystem control that P. vulgata exerts over rocky shore environments.

Seawater carbonate chemistry and biological processes of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicus ssp. braarudii) during experiments, 2011

All species of coccolithophore appear to respond to perturbations of carbonate chemistry in a different way. Here, we show that the degree of malformation, growth rate and stable isotopic composition of organic matter and carbonate produced by two contrasting species of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicus ssp. braarudii) are indicative of differences between their photosynthetic and calcification response to changing DIC levels (ranging from ~1100 to ~7800 µmol/kg) at constant pH (8.13 ± 0.02). Gephyrocapsa oceanica thrived under all conditions of DIC, showing evidence of increased growth rates at higher DIC, but C. braarudii was detrimentally affected at high DIC showing signs of malformation, and decreased growth rates. The carbon isotopic fractionation into organic matter and the coccoliths suggests that C. braarudii utilises a common internal pool of carbon for calcification and photosynthesis but G. oceanica relies on independent supplies for each process. All coccolithophores appear to utilize bicarbonate as their ultimate source of carbon for calcification resulting in the release of a proton. But, we suggest that this proton can be harnessed to enhance the supply of CO2(aq) for photosynthesis either from a large internal HCO3- pool which acts as a pH buffer (C. braarudii), or pumped externally to aid the diffusive supply of CO2 across the membrane from the abundant HCO3- (G. oceanica), likely mediated by an internal and external carbonic anhydrase respectively. Our simplified hypothetical spectrum of physiologies may provide a context to understand different species response to changing pH and DIC, the species-specific delta p and calcite "vital effects", as well as accounting for geological trends in coccolithophore cell size.