Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO2 and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO2 levels (from 380 to 1120 µatm) indicate no affect of elevated pCO2 on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO2 levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO2 related bias.
Citation: Stemmer K, Nehrke G, Brey T (2013) Elevated CO2 Levels do not Affect the Shell Structure of the Bivalve Arctica islandica from the Western Baltic. PLoS ONE 8(7): e70106. https://doi.org/10.1371/journal.pone.0070106
Editor: Anna Stepanova, Texas A&M University, United States of America
Received: December 26, 2012; Accepted: June 17, 2013; Published: July 29, 2013
Copyright: © 2013 Stemmer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the bmbf (Bundesministerium für Bildung und Forschung/Federal Ministry of Education and Research) in the framework of the project BIOACID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Marine biogenic carbonates such as bivalve shells represent complex composites of organic and inorganic phases , , . Fossil and recent shells are valuable bioarchives for paleo-climate reconstructions , ,  and environmental monitoring purposes . Furthermore, bivalve shells provide information on environmental conditions at times of shell formation in the form of structural and biogeochemical properties , , . However, in bivalve shells, some of the “classic” proxy systems (e.g. trace elements) developed for paleo-temperature, salinity and food availability have been shown to be affected by growth patterns, crystal fabric structure , the organic and the mineral phase of the biogenic carbonate (calcite, the more soluble aragonite or both) , , .
Increased seawater pCO2 and therefore decreased pH leads to a reduced saturation level for calcium carbonates  and therefore hampers shell or skeleton formation , . Apparently, several species of marine calcifiers can cope with such conditions , , ,  albeit this adaptation may coincide with changes in shell microstructure and chemistry , , , . Raising atmospheric CO2 and the corresponding decrease in ocean pH represents a challenge for marine calcifiers on a global scale (e.g. ).
A number of studies evaluated the impact of high pCO2 and low pH on marine bivalve shells (e.g. , , , ) but just a few of these took a closer look on shell growth in height or thickness and on internal shell crystal fabrics , , . In the North Atlantic bivalve A. islandica, Hiebenthal et al.  found shell stability, shell growth and tissue lipofuscin accumulation (indicating stress levels) to be unaffected by high pCO2 (up to 1700 µatm), indicating that this species may be less vulnerable to ocean acidification.
Due to its longevity (up to several centuries, , , ), its distinct internal growth band pattern , , its wide distribution in the northern Atlantic , , and its long fossil record A. islandica represents a valuable bioarchive , , . Wanamaker et al.  e.g. used shell-derived temperature proxies (δ18Oc) of A. islandica to reconstruct ocean temperature variability over the last millennium.
The mineral phase present within the shell of A. islandica is aragonite with an outer shell layer (OSL) comprising the outer shell margin and forming the distinct shell increments and growth checks, and an inner shell layer (ISL) extending from the oldest part of the shell, the umbo, to the pallial line , , . Both layers are separated by a thin myostracum. In addition, a protective organic layer, the periostracum, covers the outer shell. The shell is formed at the inner shell surface (growth in thickness) and the shell margin (growth in height), i.e. at two separate locations of precipitation divided by the attachment of the mantle tissue at the pallial line: The inner extrapallial fluid (EPF) is in contact with the ISL that is not yet formed outward the pallial line where the outer EPF is in contact with the OSL. It is suggested that shell precipitates directly from the EPF situated in space between secretory mantle tissue and shell surface , , . However, to what extent the EPF is involved in shell formation is not clear and subject of current research. Outer and inner shell layer are composed of distinct crystal morphotypes that can be differentiated in shell cross-sections, with different affinities to the uptake of elements , . Irregular simple prisms, irregular complex crossed lamellar and crossed acicular-crossed lamellar microstructures have been described by Ropes et al.  and are also observed in A. islandica shells from the North Sea , whereas the shells from the Kiel Bight (Western Baltic Sea) mostly display homogeneous crystals in the outer shell layer and simple crossed lamellar structures in the inner shell layer .
There is substantial evidence that element and isotope signatures of biogenic carbonates used as proxy data are affected by the crystal fabric structure of the biogenic carbonate to some extent , . Crystal growth rate, size and crystal fabric structure within bivalve shells can strongly influence trace element concentrations, as shown by, e.g., Carré et al. ; Freitas et al. . Regarding A. islandica, Schoene et al.  recommends to restrict sampling for geochemical analysis to one type of shell crystal fabric to avoid structure related bias.
In Kiel Bight, Arctica islandica lives below the thermohaline pycnocline (>15 m), and is thus exposed to strong environmental fluctuations, i.e. low and variable salinity (18–23), periods of low oxygen availability during summer stratification and correspondingly, fluctuating pCO2 levels with peaks over 1000 µatm (Boknis Eck Time Series Station, . Shell growth and shell crystal fabric structure represent an integrated response of the physiological and biochemical activities in the organism to the surrounding environmental conditions . Compared to A. islandica from fully marine environments, the life span of Kiel Bight animals is distinctly shorter, the shells are generally thinner  and smaller and show a less organized microstructure  than A. islandica from the North Sea or Iceland. Nevertheless, A. islandica is a prominent and abundant key species in Western Baltic benthic communities .
The aim of this study is to investigate the impact of pCO2 on the shell microstructure of A. islandica from the Kiel Bight, in order to evaluate the possible impact such changes would have on shell based proxies.
Materials and Methods
Field sampling did not require specific permissions but was in accordance with general governmental regulations. No endangered or protected species were involved.
Specimens of Arctica islandica were collected in February 2010 from the “Süderfahrt” location (N 54°31′–32′ E 10°41′–48′) in Kiel Bight, Western Baltic. Samples were dredged from the seafloor in 20 m water depth. Quahogs were transported to the AWI Wadden Sea Station Sylt and kept in an aerated flow-through tank with natural sediment for an acclimation time of 3 months. A. islandica is a high saline species and shows optimum growth at between 6 and 10°C , , . Therefore, salinity and temperature of experimental seawater were slowly increased to experimental starting conditions with salinity of 29 and a temperature of 10°C. Small animals of 15 to 25 mm height were chosen for this study. The ontogenetic age of A. islandica from Kiel Bight in that size range (15–25 mm) is about 4–5 years , . According to the A. islandica growth model of Begum et al. , shell production for the size range 15–25 mm amounts to 0.18 to 0.46 g calcium carbonate (CaCO3) y−1. This seems to be a comparatively narrow range compared to lifetime range in shell production (up to 1.27 g y−1). Shell sizes were distributed randomly over treatments. Initial shell size did not differ significantly between treatments (ANOVA, p = 0.288).
During the experiments, artificial calcium carbonate free sediment (Vitakraft® quarz gravel 1−2 mm grain size) was used to avoid pH buffering. Food supply (DT’s Premium Blend, T’s Plankton Farm, Sycamore, IL, containing Nannochloropsis oculata, Phaeodactylum tricorutum and Chrorella sp.), 3 ml was added every two days.
To mark the start of the experiment the animals were immersed for 4½ hours in a calcein solution (100 mg/l seawater) followed by two washing steps as described in Riascos et al. . Calcein is a fluorescent dye with an excitation and emission wavelength of 495/515 nm respectively. It is incorporated in biogenic calcium carbonate at the actual location of carbonate growth , i.e. the outer shell margin in bivalves  and was shown not to alter the element signature of the carbonate . All clams were pumping water (shell open and siphons visible) during the staining period and were therefore exposed to the fluorochrome. No animal died during the staining. The perturbation experiment started immediately after the staining procedure.
CO2 Perturbation Experiment
A. islandica were kept at three different pCO2 - gas-levels for 90 days: The control group at 380 µatm (ambient atmospheric CO2 level) and experimental groups at 760 µatm (2x actual pCO2) and 1120 µatm (4x preindustrial pCO2), respectively. CO2 concentrations of the experimental water were maintained by a gas mixing system (HTK, Hamburg, Germany). Experimental temperature was set to 10°C but showed slight fluctuations over the 90 days owing to North Sea warming as well as slight differences between incubations related to technical conditions (Table 1).
The experimental setup is shown in Figure 1.
The temperature controlled room was supplied with one filter-tank (sediment filter and UV-sterilization), one 250 l perturbation tank and one 30 l perturbed header tank per pCO2-level. From the header tanks the CO2-enriched water and the control water flowed down to triplicates of 4 l incubation tanks. In each tank 10 animals were incubated, i.e. a total of 30 clams per pCO2-level. A plastic lid sealed all tanks to prevent gas exchange with the atmosphere. Seawater-flow rate from header to incubation tank was 150 ml/min.
Water Chemistry and Calculations
pH, salinity and temperature of the treatment tanks were measured once a day. To determine the total alkalinity, water samples (25 ml) (AT, determined by means of potentiometric titration using the Gran method) were collected once a week. The pH electrode (WTW 3310 pH meter with SenTix Mic electrode, Weilheim, Germany) was calibrated with NBS buffers before each measurement. Carbonate chemistry was calculated using the program CO2SYS  with the input of pH (NBS scale), AT and the constants of Mehrbach et al. . Measured and calculated water parameters from our controlled perturbation experiment under different pCO2 conditions are listed in Table 1.
After 90 days experimental exposition, the quahogs were chucked and the soft tissues removed. Shells were carefully cleaned by hand and air-dried. From each treatment, 15 shells (5 per replicate) were randomly chosen for growth analysis. The staining with the fluorochrome calcein marked the start of the pCO2 incubation and allowed to identify shell material grown under experimental conditions.
Shells were submerged in NaOCl (13%) solution for 1 h to remove the organic layer (periostracum) and subsequently rinsed twice with de-ionisized water. Shells were checked for calcein marks using a fluorescence stereoscope (Olympus SZX12, Figure 2). In most shells, the calcein mark was not found along the whole shell edge, but only intermittently. If the mark was detectable at the line of strongest growth (LSG, Fig. 1), the shell was cross-sectioned along the LSG, if not, along a line through the calcein mark closest to the LSG. To prevent shell damage during cutting, metal epoxy (Toolcraft) was applied to the marked shell area one day before sectioning. Cross-sections were ground using grinding paper (Buehler) with grit sizes of P1200/P2400/P4000 followed by a polishing step with Buehler diamond polycrystalline suspensions (3 µm) and a final polish with aluminum oxide suspension (1 µm). The samples were then carefully rinsed using de-ionized water and air-dried.
New grown shell was measured at the line of strongest growth (LSG). Arrows indicate the direction of growth (dog).
Shell growth in height, i.e. addition of incremental growth, was measured from the end of the calcein mark (start of pCO2 incubation) to the outer shell margin (end of pCO2 incubation). If growth could not be measured directly on the LSG trajectory, the measurement was transformed to growth at LSG assuming isometric shell growth in all directions. Shell growth in thickness (i.e. the distance between inner and outer valve surface) was measured at the end of the calcein mark perpendicular to the direction of growth (Figure 3). All measurements were performed under a fluorescence stereoscope (Olympus SZX12) using the program ANALYSIS.
(A) Sketch of shell-cross-section with major shell structures. (B) Magnification of outer shell margin from the cross-section showing calcein mark and additional shell growth in height and thickness.
Differences in shell growth in height and thickness between treatments were analyzed by one-way ANOVA and subsequent TUKEY HSD post-hoc tests (significance level alpha = 0.05).
Shell microstructures were studied by scanning electron microscopy (XL30 ESEM, Philips) of shell cross-sections. Polished samples were coated with gold and scanned with an accelerating Voltage of 10 kV and a beam current of 1.7 nA. Shell-layers chosen for analysis are shown in Figure 4 A, B, C.
(A) Transition zone of shell material grown under normal and pCO2 perturbated conditions in the outer shell layer (OSL) (green line = calcein mark). (B) Inner shell layer (ISL). (C) Last precipitated shell material at the very tip of the shell.
The calcein mark used to mark the start of the experiment could be detected in 80% of the shells (Table 2) etched with NaOCl. Within most shells the calcein mark did not appear along the whole shell margin but only in the fastest growing segments (Figure 2). This indicates asynchronous shell growth of Arctica islandica during short time periods (4½ h calcein immersion). However, since this finding is outside the scope of this study we will not pursue it further here.
pCO2 level had no significant effect on shell growth in height and thickness (one-way ANOVA, height: F = 0.503, p = 0.609; thickness: F = 1.227, p = 0.306). Growth varied between 0.96 µm/day and 9.14 µm/day in height and between 0.70 µm/day and 2.88 µm/day in thickness (Figure 5A, B respectively; Table 2).
Shell crystal fabrics of A. islandica formed during the pCO2 incubations did not differ between exposures to pCO2 levels ranging from 380 to 1120 µatm (Figure 6). In the cross-sections, the outer and inner shell layers (OSL/ISL) were clearly distinguishable by their crystal fabrics: The shell region of the OSL is characterized by a homogeneous distribution of irregular shaped crystals with an average diameter of 1.5 µm (Figure 6A). The inner shell layer was build from distinct crossed-lamellar crystal fabrics ,  (Figure 4B; Figure 6B). The tips of the very recently formed shell, i.e. the latest formed crystal fabrics are also similar in all experimental animals: They consist of homogeneously distributed but irregularly shaped crystals with an average diameter of 5 µm (Figure 4C; Figure 6C).
Crystal fabrics of new shell material grown under different pCO2 levels. (A) The growth line (gl) stained with calcein at starter point of the pCO2 incubation is visible due to smaller crystals. HOM = homogeneous crystal fabric. (B) Inner shell layer (ISL) with distinct crossed-acicular crystal fabric. (C) Latest formed crystal fabric.
Our study indicates that shell growth and crystal microstructure of Arctica islandica from Kiel Bight are not altered by pCO2 in the range of 380–1120 µatm.
Individual shell growth rates varied over a wide range (0.96–9.14 µm/day in height and 0.70–2.88 µm/day in thickness) but were not affected by the pCO2 treatment. Each shell formed new shell material with distinct and specific crystal fabrics in the outer and inner shell layers. The widely accepted concept of extracellular matrix mediated mineralization in bivalves ,  suggests that mineral formation requires a microenvironment that provides and maintains a sufficient supersaturation for nucleation and growth of the mineral phase. Our results indicate that A. islandica possesses a high physiological control over the chemical composition at the site of calcification, even when exposed to elevated proton concentrations, i.e. low pH.
Current implications suggest that elevated pCO2 and lowered pH can have various effects on bivalve species and other marine calcifiers, apparently depending on species and experimental conditions , , , , . Accordingly, we are still far from a consistent picture of the cause-and-effect mechanisms involved.
To our knowledge there are few studies of pCO2 effects on newly grown bivalve shell material. Hahn et al.  report changes in shell ultrastructure of the Mediterranean Mytilus galloprovincialis that were transplanted in the field from normal to a high pCO2 level site with a pH of 8.1 and 7.3 respectively. However, it remains unclear to which extent other (uncontrolled) environmental factors may have also affected shell properties. Melzner et al.  observed dissolution of the internal aragonite (nacre) layer in the blue mussel Mytilus edulis from Kiel Fjord under exposition to pCO2>2000 µatm. These mussels experience seasonal pCO2 peaks of >4000 µatm and corresponding pH values as low as 7.1 and thus are presumed to be adapted to such conditions , . However, a direct comparison with our findings is difficult because (i) the shell of M. edulis consists of two calcium carbonate polymorphs, calcite on the outside and aragonite on the inside and (ii) pCO2 impact on newly grown shell material in shell regions comparable to those investigated in our study was not analyzed.
What makes Arctica Islandica so Special?
The findings that elevated pCO2 levels neither affect shell growth rate (, this study) nor shell microstructure (this study) indicate that A. islandica is in full physiological and chemical control of the shell formation process, including carbonate precipitation. This tolerance can have two possible explanations: (a) pre-adaptation through species-specific lifestyle; (b) pre-adaptation to regularly enhanced pCO2 levels in Kiel Bight.
- A. islandica is unique among bivalves as the deliberate exposition to high pCO2 and low pH conditions is part of its life strategy: A. islandica can perform extreme “metabolic rate depression” (MDR), i.e. animals may stop water pumping and bury deeper into the sediment for several days, while reducing metabolic activity to very low levels , . These sediments are often hypercapnic (physiological effects of elevated pCO2) and can be undersaturated with respect to aragonite , whereas body fluids, i.e. haemolymph, mantle water, and extrapallial fluid are naturally acidified and may become even more acidic under anaerobic conditions , . Our findings support the hypothesis of Hiebenthal et al.  that the specific lifestyle of A. islandica may serve as a pre-adaptation to forthcoming elevated ocean pCO2. This feature may have also added to the long-term success of A. islandica. A. islandica is the only remnant of an ancient genus of the once diverse Arcticidae  and apparently was able to survive major past climatic oscillations, showing a high abundance through geological times and a wide distribution in the Northern Atlantic. Presence of the genus Arctica during the high CO2 Cretaceous epoch (3.7 to 14.7 times the modern pre-industrial value of 285 ppm) ,  indicates that this species may be pre-adapted to high pCO2 levels in general and not only in a Baltic sub-population.
- An alternative explanation would be that A. islandica from Kiel Bight are well adapted to the strongly fluctuating conditions (salinity, temperature, oxygen availability, pCO2) at this locality. As a consequence this population can tolerate also elevated pCO2 levels. Adaption to fluctuating and increased pCO2 may add to the general expression of pronounced stress response at the expense of lifespan  with that of Kiel Bight animals of ∼40 yrs  compared to A. islandica from Iceland populations living up to 400 yrs in fully marine environment , . The robustness towards changing conditions of Kiel Bight animals is also reflected in our experiment where A. islandica was not affected by fully saline North Sea water used during the experiment.
Future research on A. islandica from different localities (Iceland, Kattegat, White Sea, ect.), possibly in combination with genetic approaches (e.g. transcriptomics)  will show whether or not the observed pCO2 tolerance of this bivalve is unique for the Kiel Bight population or if represents a species-specific feature.
However, synergistic effects of pCO2 and other parameters such as temperature, food availability and salinity have not been considered yet. Furthermore, we still lack a detailed understanding of the mechanisms and controls of shell formation, which are a matter of ongoing and future research. We need to uncover the processes involved in biomineralization and before resolving this interdisciplinary enigma we can only report species-specific responses and hypothesize the processes behind it.
Our study shows that shell growth and shell microstructure of young Arctica islandica from Kiel Bight are not affected by the pCO2 up to 1120 µatm. Correspondingly, isotope and element-based proxies derived from A. islandica shells are unbiased regarding changes in shell crystal fabric structure caused by varying environmental pCO2 levels. Whether or not this robustness applies to all A. islandica populations or just to the one from the Western Baltic remains to be seen.
Conceived and designed the experiments: KS TB GN. Performed the experiments: KS. Analyzed the data: KS TB GN. Contributed reagents/materials/analysis tools: TB GN. Wrote the paper: KS TB GN.
- 1. Addadi L, Joester D, Nudelman F, Weiner S (2006) Mollusk shell formation: A source of new concepts for understanding biomineralization processes. Chemistry-a European Journal 12: 981–987.
- 2. Cuif JP, Dauphin Y, Sorauf JE (2011) Biominerals and fossils through time: Cambridge University Press.
- 3. Nudelman F, Gotliv BA, Addadi L, Weiner S (2006) Mollusk shell formation: Mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre. Journal of Structural Biology 153: 176–187.
- 4. Marchitto TM, Jones GA, Goodfriend GA, Weidman CR (2000) Precise temporal correlation of holocene mollusk shells using sclerochronology. Quaternary Research 53: 236–246.
- 5. Schoene BR, Kroencke I, Houk SD, Castro ADF, Oschmann W (2003) The cornucopia of chilly winters: Ocean quahog (Arctica islandica, L., Mollusca) master chronology reveals bottom water nutrient enrichment during colder winters (North Sea). Senckenbergiana Maritima 32: 165–175.
- 6. Wanamaker AD Jr, Kreutz KJ, Schoene BR, Maasch KA, Pershing AJ, et al. (2009) A late Holocene paleo-productivity record in the western Gulf of Maine, USA, inferred from growth histories of the long-lived ocean quahog (Arctica islandica). International Journal of Earth Sciences 98: 19–29.
- 7. Krause-Nehring J, Brey T, Thorrold SR (2012) Centennial records of lead contamination in northern Atlantic bivalves (Arctica islandica). Marine Pollution Bulletin 64: 233–240.
- 8. Epstein S, Buchsbaum R, Lowenstam HA, Urey HC (1953) Revised carbonate-water isotopic temperature scale Geological Society of America Bulletin. 64: 1315–1325.
- 9. Hickson JA, Johnson ALA, Heaton THE, Balson PS (1999) The shell of the Queen Scallop Aequipecten opercularisis (L.) as a promising tool for palaeoenvironmental reconstruction: evidence and reasons for equilibrium stable-isotope incorporation. Palaeogeography Palaeoclimatology Palaeoecology 154: 325–337.
- 10. Schone BR, Castro ADF, Fiebig J, Houk SD, Oschmann W, et al. (2004) Sea surface water temperatures over the period 1884–1983 reconstructed from oxygen isotope ratios of a bivalve mollusk shell (Arctica islandica, southern North Sea). Palaeogeography Palaeoclimatology Palaeoecology 212: 215–232.
- 11. Schoene BR, Radermacher P, Zhang Z, Jacob DE (2011) Crystal fabrics and element impurities (Sr/Ca, Mg/Ca, and Ba/Ca) in shells of Arctica islandica - implications for paleoclimate reconstructions. Palaeogeography Palaeoclimatology Palaeoecology DOI:https://doi.org/10.1016/j.palaeo.2011.05.013.
- 12. Addadi L, Raz S, Weiner S (2003) Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization. Advanced Materials 15: 959–970.
- 13. Strasser CA, Mullineaux LS, Walther BD (2008) Growth rate and age effects on Mya arenaria shell chemistry: Implications for biogeochemical studies. Journal of Experimental Marine Biology and Ecology 355: 153–163.
- 14. Feely RA, Sabine CL, Lee K, Berelson W, Kleypas J, et al. (2004) Impact of anthropogenic CO2 on the CaCO3 system in the oceans. Science 305: 362–366.
- 15. Gazeau F, Quiblier C, Jansen JM, Gattuso J-P, Middelburg JJ, et al.. (2007) Impact of elevated CO2 on shellfish calcification. Geophysical Research Letters 34. doi: https://doi.org/10.1029/2006GL028554.
- 16. Ries JB, Cohen AL, McCorkle DC (2009) Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology 37: 1131–1134.
- 17. Langer G, Nehrke G, Probert I, Ly J, Ziveri P (2009) Strain-specific responses of Emiliania huxleyi to changing seawater carbonate chemistry. Biogeosciences 6: 2637–2646.
- 18. Thomsen J, Gutowska MA, Saphoerster J, Heinemann A, Truebenbach K, et al. (2010) Calcifying invertebrates succeed in a naturally CO2-rich coastal habitat but are threatened by high levels of future acidification. Biogeosciences 7: 3879–3891.
- 19. Tunnicliffe V, Davies KTA, Butterfield DA, Embley RW, Rose JM, et al. (2009) Survival of mussels in extremely acidic waters on a submarine volcano. Nature Geoscience. 2: 344–348.
- 20. Hahn S, Rodolfo-Metalpa R, Griesshaber E, Schmahl WW, Buhl D, et al. (2012) Marine bivalve shell geochemistry and ultrastructure from modern low pH environments: environmental effect versus experimental bias. Biogeosciences 9: 1897–1914.
- 21. Melzner F, Stange P, Truebenbach K, Thomsen J, Casties I, et al. (2011) Food Supply and Seawater pCO(2) Impact Calcification and Internal Shell Dissolution in the Blue Mussel Mytilus edulis. Plos One 6(9): e24223
- 22. Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean Acidification: the other CO2 Problem. Annual Review of Marine Science 1: 169–192
- 23. Michaelidis B, Ouzounis C, Paleras A, Portner HO (2005) Effects of long-term moderate hypercapnia on acid-base balance and growth rate in marine mussels Mytilus galloprovincialis. Marine Ecology-Progress Series 293: 109–118.
- 24. Rodolfo-Metalpa R, Houlbreque F, Tambutte E, Boisson F, Baggini C, et al. (2011) Coral and mollusc resistance to ocean acidification adversely affected by warming. Nature Climate Change 1: 308–312.
- 25. Hiebenthal C, Philipp E, Eisenhauer A, Wahl M (2012) Effects of seawater pCO2 and temperature on shell growth, shell stability, condition and cellular stress of Western Baltic Sea Mytilus edulis (L.) and Arctica islandica (L.). Marine Biology doi 10.1007/s00227-012-2080-9.
- 26. Abele D, Brey T, Philipp E (2009) Bivalve models of aging and the determination of molluscan lifespans. Experimental Gerontology 44: 307–315.
- 27. Strahl J, Philipp E, Brey T, Broeg K, Abele D (2007) Physiological aging in the Icelandic population of the ocean quahog Arctica islandica. Aquatic Biology 1: 77–83.
- 28. Wanamaker AD Jr, Heinemeier J, Scourse JD, Richardson CA, Butler PG, et al. (2008) Very long-lived mollusks confirm 17th century ad tephra-based radiocarbon reservoir ages for north Islandic shelf-waters Radiocarbon. 50: 399–412.
- 29. Epple VM, Brey T, Witbaard R, Kuhnert H, Paetzold J (2006) Sclerochronological records of Arctica islandica from the inner German Bight. Holocene 16: 763–769.
- 30. Ropes JW (1984) Procedures for preparing acetate peels and evidence validating the annual periodicity of growth lines formed in the shells of the ocean quahogs, Arctica islandica Marine Fisheries Review. 46: 27–35.
- 31. Dahlgren TG, Weinberg JR, Halanych KM (2000) Phylogeography of the ocean quahog (Arctica islandica): influences of paleoclimate on genetic diversity and species range. Marine Biology 137: 487–495.
- 32. Nicol D (1951) Recent species of the veneroid pelecypod Arctica. Journal of Washington Academic Science 41: 102–106.
- 33. Schoene BR, Hickson J, Oschmann W (2005) Reconstruction of subseasonal environmental conditions using bivalve mollusk shells-A graphical model. Mora,Germán (Hrsg.). Isotopic and Elemental Tracers of Cenozoic Climate Change. Boulder, Colo.: Geological Soc. of America. 2005. S. 21–31. doi: https://doi.org/10.1130/0-8137-2395-7.21.
- 34. Schone BR, Fiebig J, Pfeiffer M, Gless R, Hickson J, et al. (2005) Climate records from a bivalved Methuselah (Arctica islandica, Mollusca; Iceland). Palaeogeography Palaeoclimatology Palaeoecology 228: 130–148.
- 35. Weidman CR, Jones GA (1994) Kyger (1994) The long-lived mollusk Arctica islandica - a new paleoceanographic tool for the reconstruction of bottom temperatures for the continental shelves of the northern North-Atlantic ocean. Journal of Geophysical Research-Oceans 99: 18305–18314.
- 36. Wanamaker AD Jr, Kreutz KJ, Schoene BR, Pettigrew N, Borns HW, et al. (2008) Coupled North Atlantic slope water forcing on Gulf of Maine temperatures over the past millennium. Climate Dynamics 31: 183–194.
- 37. Dunca E, Mutvei H, Goransson P, Morth C-M, Schoene BR, et al. (2009) Using ocean quahog (Arctica islandica) shells to reconstruct palaeoenvironment in A-resund, Kattegat and Skagerrak, Sweden. International Journal of Earth Sciences 98: 3–17.
- 38. Morton B (2011) The biology and functional morphology of Arctica islandica (Bivalvia: Arcticidae): A gerontophilic living fossil. Marine Biology Research 7: 540–553.
- 39. Ropes JW, Jones DS, Murawski SA, Serchuk FM, Jearld A (1984) Documentation of annual growth lines in ocean quahogs, Arctica islandica L.. Fishery Bulletin 82: 1–19.
- 40. Crenshaw MA (1980) Mechanisms of shell formation and dissolution. Topics in Geobiology 1: 115–132.
- 41. Wheeler AP (1992) Mechanisms of molluscan shell formation. In: Calcification in Biological Systems. ed. E. Bonucci, CRC Press, Boca Raton, 179–215.
- 42. Wilbur KM (1983) Shell formation. The Mollusca Volume 4 Physiology Part 1: 253–287.
- 43. Dauphin Y, Guzman N, Denis A, Cuif JP, Ortlieb L (2003) Microstructure, nanostructure and composition of the shell of Concholepas concholepas (Gastropoda, Muricidae). Aquatic Living Resources 16: 95–103.
- 44. Meibom A, Cuif JP, Hillion FO, Constantz BR, Juillet-Leclerc A, et al.. (2004) Distribution of magnesium in coral skeleton. Geophysical Research Letters 31.
- 45. Carre M, Bentaleb I, Bruguier O, Ordinola E, Barrett NT, et al. (2006) Calcification rate influence on trace element concentrations in aragonitic bivalve shells: Evidences and mechanisms. Geochimica Et Cosmochimica Acta 70: 4906–4920.
- 46. Freitas PS, Clarke LJ, Kennedy H, Richardson CA (2009) Ion microprobe assessment of the heterogeneity of Mg/Ca, Sr/Ca and Mn/Ca ratios in Pecten maximus and Mytilus edulis (bivalvia) shell calcite precipitated at constant temperature. Biogeosciences 6: 1209–1227.
- 47. Hansen HP, Giesenhagen HC, Behrends G (1999) Seasonal and long-term control of bottom-water oxygen deficiency in a stratified shallow-water coastal system. Ices Journal of Marine Science 56: 65–71.
- 48. Riisgard HU, Randlov A (1981) Energy budgets, growth and filtration-rates in Mytilus edulis at different algal concentrations Marine Biology. 61: 227–234.
- 49. Brey T, Arntz WE, Pauly D, Rumohr H (1990) Arctica (Cyprina) islandica in Kiel Bay (western Baltic) - growth, production and ecological significance Journal of Experimental Marine Biology and Ecology. 136: 217–235.
- 50. Hiebenthal C, Philipp EER, Eisenhauer A, Wahl M (2012) Interactive effects of temperature and salinity on shell formation and general condition in Baltic Sea Mytilus edulis and Arctica islandica. Aquatic Biology 14: 289–298.
- 51. Mann R (1982) The seasonal cycle of gonadal development in Arctica islandica from the southern New England shelf. U S Fish and Wildlife Service Fishery Bulletin 80: 315–326.
- 52. Begum S, Basova L, Heilmayer O, Philipp EER, Abele D, et al. (2010) Growth and energy budget models of the bivalve Arctica islandica at six different sites in the Northeast Atlantic realm Journal of Shellfish Research. 29: 107–115.
- 53. Riascos J, Guzman N, Laudien J, Heilmayer O, Oliva M (2007) Suitability of three stains to mark shells of Concholepas concholepas (Gastropoda) and Mesodesma donacium (Bivalvia). Journal of Shellfish Research 26: 43–49.
- 54. Moran AL (2000) Calcein as a marker in experimental studies newly-hatched gastropods. Marine Biology 137: 893–898.
- 55. Dissard D, Nehrke G, Reichart GJ, Nouet J, Bijma J (2009) Effect of the fluorescent indicator calcein on Mg and Sr incorporation into foraminiferal calcite. Geochemistry Geophysics Geosystems 10 (1). doi: https://doi.org/10.1029/2009GC002417.
- 56. Lewis Wallace (1998) Basic program for CO2 system in seawater. Oak Ridge National Laboratory.
- 57. Mehrbach C, Culberso.Ch, Hawley JE (1973) Pytkowic.Rm (1973) Measurement of apparent dissociation-constants of carbonic-acid in seawater at atmospheric pressure Limnology and Oceanography. 18: 897–907.
- 58. Weiner S, Dove P (2003) An Overview on Biomineralization Processes and the Problem of the Vital Effect. In Biomineralization. Eds. P.M. Dove, S. Weiner and J.J. De Yoreo. Mineralogical Society of America, Washington, D.C., v. 54, p. 1–31.
- 59. Berge JA, Bjerkeng B, Pettersen O, Schaanning MT, Oxnevad S (2006) Effects of increased sea water concentrations of CO2 on growth of the bivalve Mytilus edulis L. Chemosphere. 62: 681–687.
- 60. Strahl J, Brey T, Philipp EER, Thorarinsdottir G, Fischer N, et al. (2011) Physiological responses to self-induced burrowing and metabolic rate depression in the ocean quahog Arctica islandica. Journal of Experimental Biology 214: 4223–4233.
- 61. Green MA, Jones ME, Boudreau CL, Moore RL, Westman BA (2004) Dissolution mortality of juvenile bivalves in coastal marine deposits. Limnology and Oceanography 49: 727–734.
- 62. Crenshaw MA (1972) Inorganic composition of molluskan extrapallial fluid. Biological Bulletin 143: 506–512.
- 63. Melzner F, Thomsen J, Koeve W, Oschlies A, Gutowska MA, et al.. (2012) Future ocean acidification will be amplified by hypoxia in coastal habitats. Marine Biology: doi: https://doi.org/10.1007/s00227-00012-01954-00221.
- 64. Lutz RA, Mann R, Goodsell JG, Castagna M (1982) Larval and early post-larval development of Arctica islandica. Journal of the Marine Biological Association of the United Kingdom 62: 745–769.
- 65. Caldeira K, Rampino MR (1991) The Mid-Cretaceous Super Plume, carbon dioxide, and global warming. Geophysical Research Letters 18: 987–990.
- 66. Philipp EER, Wessels W, Gruber H, Strahl J, Wagner AE, et al. (2012) Gene Expression and Physiological Changes of Different Populations of the Long-Lived Bivalve Arctica islandica under Low Oxygen Conditions. Plos One 7: e44621
- 67. Begum S, Basova L, Strahl J, Sukhotin A, Heilmayer O, et al. (2009) A metabolic model for the ocean quahog Arctica islandica - effects of animal mass and age, temperature, salinity, and geography on respiration rate. Journal of Shellfish Research 28: 533–539.