Accepted Manuscript Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles? M. Bressan, A. Chinellato, M. Munari, V. Matozzo, A. Manci, T. Marčeta, L. Finos, I. Moro, P. Pastore, D. Badocco, M.G. Marin PII:

S0141-1136(14)00081-6

DOI:

10.1016/j.marenvres.2014.04.009

Reference:

MERE 3886

To appear in:

Marine Environmental Research

Received Date: 21 October 2013 Revised Date:

8 April 2014

Accepted Date: 18 April 2014

Please cite this article as: Bressan, M., Chinellato, A., Munari, M., Matozzo, V., Manci, A., Marčeta, T., Finos, L., Moro, I., Pastore, P., Badocco, D., Marin, M.G., Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles?, Marine Environmental Research (2014), doi: 10.1016/ j.marenvres.2014.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles?

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Bressan M.a, *, Chinellato A.a, Munari M.a, Matozzo V.a, Manci A.a, Marčeta T.a, Finos L.b,

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Moro I.a, Pastore P.c, Badocco D.c, Marin M.G.a

5 a Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy

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b Department of Statistical Sciences, University of Padova, Via C. Battisti 241, 35121 Padova, Italy

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c Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy

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Corresponding author: email: [email protected] (M. Bressan); tel: +390498276242; fax:

+390498276230

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ACCEPTED MANUSCRIPT Abstract

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Anthropogenic emissions of carbon dioxide are leading to decreases in pH and changes in the

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carbonate chemistry of seawater. Ocean acidification may negatively affect the ability of marine

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organisms to produce calcareous structures while also influencing their physiological responses and

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growth. The aim of this study was to evaluate the effects of reduced pH on the survival, growth and

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shell integrity of juveniles of two marine bivalves from the Northern Adriatic sea: the

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Mediterranean mussel Mytilus galloprovincialis and the striped venus clam Chamelea gallina. An

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outdoor flow-through plant was set up and two pH levels (natural seawater pH as a control, pH 7.4

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as the treatment) were tested in long-term experiments. Mortality was low throughout the first

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experiment for both mussels and clams, but a significant increase, which was sensibly higher in

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clams, was observed at the end of the experiment (6 months). Significant decreases in the live

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weight (-26%) and, surprisingly, in the shell length (-5%) were observed in treated clams, but not in

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mussels. In the controls of both species, no shell damage was ever recorded; in the treated mussels

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and clams, damage proceeded via different modes and to different extents. The severity of shell

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injuries was maximal in the mussels after just 3 months of exposure to a reduced pH, whereas it

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progressively increased in clams until the end of the experiment. In shells of both species, the

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damaged area increased throughout the experiment, peaking at 35% in mussels and 11% in clams.

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The shell thickness of the treated and control animals significantly decreased after 3 months in

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clams and after 6 months in mussels. In the second experiment (3 months), only juvenile mussels

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were exposed to a reduced pH. After 3 months, the mussels at a natural pH level or pH 7.4 did not

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differ in their survival, shell length or live weight. Conversely, shell damage was clearly visible in

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the treated mussels from the 1st month onward. Monitoring the chemistry of seawater carbonates

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always showed aragonite undersaturation at 7.4 pH, whereas calcite undersaturation occurred in

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only 37% of the measurements. The present study highlighted the contrasting effects of

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acidification in two bivalve species living in the same region, although not exactly in the same

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habitat.

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Keywords:

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Mussel, Clam, Acidification, Carbon dioxide, pH, Calcium carbonate, Shell damage, Northern

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Adriatic

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1. Introduction

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Due mainly to anthropogenic activities, carbon dioxide in the atmosphere has been increasing from

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pre-industrial levels, in the range of 172-300 ppmv (Lüthi et al., 2008), to the present concentration

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of 395 ppmv (NOAA, 2013). Emissions increased at a rate of 1.0% yr−1 in the 1990s and peaked at

ACCEPTED MANUSCRIPT 3.4% yr−1 between 2000 and 2008 (Le Quéré et al., 2009). The oceans have taken up approximately

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25% of the CO2 released by human activities and this value is expected to increase up to 90% at

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millennial timescales (Kleypas et al., 2006; Sabine et al., 2004). These increasing amounts of CO2

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dissolved in the ocean have resulted in decreased pH levels and changes in seawater carbonate

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chemistry, with a reduction of the saturation states of aragonite and calcite, which are the most

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prevalent shell-forming carbonates (Kleypass et al., 2006; Orr et al., 2005).

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It has been calculated that the pH of the ocean surface was 0.1 units higher in pre-industrial times,

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which corresponds to a 30% increase in the concentration of hydrogen ions (Caldeira and Wickett,

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2003; Raven et al., 2005). Under unrestricted emissions, the simulated atmospheric CO2 levels are

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predicted to exceed 1,900 ppm by approximately 2300, and the maximum predicted reduction in the

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ocean surface pH is 0.77 units (Caldeira and Wickett, 2003, 2005; Raven et al., 2005). Following

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ocean acidification, the 20% decrease in the saturation state of calcium carbonate calculated for the

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period 1766-2007 is projected to further decline by approximately 40% by 2100 (Gattuso and

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Hansson, 2011).

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Because of natural and anthropogenic factors, estuarine and coastal environments are characterised

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by a large spatial and temporal variability in physico-chemical and biological features, including

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seawater carbonate levels (Range et al., 2012). The onset of ocean acidification may exacerbate the

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current patterns of background variability in these habitats, which experience seawater CO2

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concentrations that are often significantly higher than expected based on equilibrium with the

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atmosphere (Andersson and Mackenzie, 2011; Frankignoulle et al., 1998).

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Although reductions in pH may negatively affect the survival, growth, reproduction and

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physiological performance of all marine organisms, variations in carbonate chemistry have

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detrimental effects mostly on marine calcifiers, which are hindered in producing calcareous skeletal

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structures (Fabry et al., 2008). Among marine calcifiers, bivalves play an important role in coastal

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ecosystems that involves linking primary producers with top consumers, coupling pelagic and

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benthic processes and acting as essential ecosystem engineers for many other species. In addition,

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bivalves are often important resources with a high market value for both fisheries and the

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aquaculture industry. Quantitative and qualitative variations in bivalve production may entail

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negative societal consequences, mainly in countries with low adaptability and high nutritional or

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economic dependence on molluscs (Cooley et al., 2011).

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Different life stages of bivalves are known to respond in different ways to variations in

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environmental parameters, including acidification (Kroeker et al., 2010; Ross et al., 2011). At the

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population level, larval tolerance influences survival throughout the pre-settlement and settlement

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phases, whereas the success of juveniles affects recruitment, which in turn plays a part in

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ACCEPTED MANUSCRIPT determining adult abundance. Due to their high growth rates, juveniles are particularly suitable for

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evaluating the effects of exposure to acidification. To obtain better insight into these effects, a long

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acclimation period should be considered in experimental designs. However, few studies have been

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carried out on time spans of more than a few months and no study has covered a full annual cycle

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(Gazeau et al., 2012). A long-term experimental approach was applied in the present study with the

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aim of investigating how two species from the Northern Adriatic Sea with a different ecological

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tolerance to environmental variables, but similar economical relevance – the Mediterranean mussel

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Mytilus galloprovincialis Lmk and the striped venus clam Chamelea gallina (L) – may cope with

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reduced seawater pH levels, as predicted in future global change scenarios. In adults of M.

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galloprovincialis and C. gallina from the Northern Adriatic Sea, previous studies have investigated

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immunological and biochemical parameters, demonstrating a strong interaction between pH and

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temperature (Matozzo et al., 2012, 2013).

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The Mediterranean mussel, M. galloprovincialis, is the most widely distributed of the three sibling

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Mytilus species (Wonham, 2004) and is considered one of the 100 "World's Worst" invaders by the

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ISSG of the IUCN (Lowe, 2004). It inhabits hard substrates in harbours, estuaries and open coastal

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areas, ranging from the intertidal zone to a depth of 40 m. Due to its distribution in areas with tidal

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changes in abiotic factors, M. galloprovincialis is considered a very tolerant species, particularly to

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temperature, salinity and pH. However, Anestis et al. (2007) found that it cannot survive at

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seawater temperatures of 26°C or higher for extended periods of time (Anestis et al., 2007). Among

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Mytilus species, M. galloprovincialis is the major contributor to the mussel aquaculture industry,

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with an economic value of more than 10.6 billion USD in 2010 and showing a global production of

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107,488 tonnes (FAO, 2012). In 2011, the Northern Adriatic regions produced approximately

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40,000 tonnes of cultured mussels (Osservatorio Socio Economico della Pesca e dell’Acquacoltura

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– Veneto Agricoltura, 2012 a).

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The striped venus clam, C. gallina, is an infaunal bivalve species distributed throughout the

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Mediterranean and Black Sea and along the Algarve coast (Portugal) (Gaspar et al., 2004) in well-

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sorted fine sand biocenoses (Pérès and Picard, 1964). In the Northern Adriatic sea, C. gallina lives

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buried in subtidal sands at depths up to approximately 10 m. C. gallina shows relatively low

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tolerance to temperature (Monari et al., 2007a; Moschino and Marin, 2006) and salinity (Matozzo et

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al., 2007; Monari et al., 2007b) variations. Indeed, optimal habitat for this species exhibits limited

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variations not only in temperature and salinity, but also in sediment characteristics such as grain

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size, oxygenation and redox potential (always > 300 mV) (Barillari et al., 1978; Brooks et al.,

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1991). Clam distribution appeared not related with pH values in sediments (Barillari et al., 1978).

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Due to its typical euryoxic habit, C. gallina demonstrated the lowest tolerance to anoxia among

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ACCEPTED MANUSCRIPT various Adriatic bivalve species (Brooks et al., 1991; de Zwaan et al., 1991). Furthermore, C.

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gallina displays a slow growth rate (Keller et al., 2002), mostly above 27 °C (Ramón and

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Richardson, 1992).

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Along the Italian coasts of this region, a large clam fishery has been present since the 1970s, though

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a collapse to one-sixth of the previous production level was recorded in recent years (Romanelli et

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al., 2009); in 2011, the production was more than 3,000 tonnes (Osservatorio Socio Economico

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della Pesca e dell’Acquacoltura – Veneto Agricoltura, 2012 b). In comparison with other bivalve

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species, C. gallina shows relatively low tolerance to temperature (Monari et al., 2007a; Moschino

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and Marin, 2006) and salinity (Matozzo et al., 2007; Monari et al., 2007b) variations and displays a

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slow growth rate (Keller et al., 2002).

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The two studied species belong to the Mytilidae and Veneridae families, which differ markedly in

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their shell structure and composition. The mollusc shell is a composite biomaterial: the mineral

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phase (CaCO3) accounts for 95 to 99% of its weight, with the remaining 1 to 5% consisting of an

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organic matrix (Marin and Luquet, 2004). In bivalves, the shell consists of an outer proteinaceous

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layer, known as the periostracum, and the calcified shell, which is a mixture of protein and CaCO3,

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either in the form of aragonite alone or as aragonite and calcite together (Kennedy et al., 1969),

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deposited in different (generally 2 or 3) superimposed layers (Marin and Luquet, 2004).

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The periostracum protects the calcified shell, provides the first support for CaCO3 crystals, seals the

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extrapallial space to achieve supersaturation conditions and provides the site of nucleation for

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CaCO3 (Hahn et al., 2012; Marin and Luquet, 2004). Mytilidae and Veneridae shells differ in the

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extent and thickness of the periostracum and CaCO3 layers. In M. galloprovincialis individuals with

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lengths of 15 to 60 mm, the shells exhibit a consistently high (>90%) periostracum cover (Scardino

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et al., 2003), whereas clam shells remain largely exposed to ambient seawater after deposition, with

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the periostracum being very thin (Alemany, 1986-1987; Ries et al., 2009).

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In mussels, the calcified part of the shell presents two layers: the outer carbonate shell layer, which

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is composed of calcite prisms, and the inner layer, which consists of nacreous aragonite (Marin and

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Luquet, 2004; Hahn et al., 2012). In M. galloprovincialis from Ischia port, the thickness of the

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calcite and aragonite layers varies significantly during the life span of individuals, whereas the

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thickness of the periostracum remains more constant (Hahn et al., 2012).

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The shell of C. gallina, similar to all Veneridae Chioninae (Jones, 1979; Mikkelsen et al., 2006),

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consists principally of 2 aragonite layers: an outer shell layer subdivided into 2 sublayers, i.e., the

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outer prismatic layer (OPL) and the middle crossed lamellar layer (MCLL), and an inner

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homogeneous layer (Alemany, 1986-1987).

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ACCEPTED MANUSCRIPT In addition, the CaCO3 polymorphs are qualitatively different in mussels and clams: in mussels,

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low-Mg calcite (4 mol% MgCO3) (Ries et al., 2009). As a consequence, clams may be more

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threatened by elevated pCO2 levels because they use the most soluble forms of CaCO3 for the

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deposition of their shells (Morse et al., 2007).

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Based on previous reports on bivalve juveniles reared under conditions of increased CO2 and

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reduced pH levels, we hypothesised that there would be at least three effects of acidification: 1.

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reduced survival; 2. reduced growth in both size and weight; and 3. shell injuries. Due to the well-

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known differences between the two studied species, we further hypothesise that 4. juvenile mussels

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and clams may exhibit different sensitivities. In the present study, these hypotheses were tested by

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measuring various parameters, including mortality, shell and soft tissue growth, shell thickness

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variations and shell damage, throughout long-term exposure to low pH seawater.

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164 2.1 Animals

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Because the spat of M. galloprovincialis and C. gallina is available only in autumn in the Northern

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Adriatic Sea, 0+ cohort juveniles were sampled in that season: mussels were collected from an

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offshore culture park and clams were harvested using commercial fishing gear.

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After collection, epibionts were gently scraped from the shells. The bivalves were counted and their

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morphometric and morphological parameters were measured.

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2.2 Experiments

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The experimental plant was built outdoor at the Hydrobiological Station of Chioggia (Northern

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Adriatic, Italy, University of Padova), using a large tank (3.5 m3) supplied with seawater flowing

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from the lagoon of Venice (southern basin). Within this large tank, six smaller tanks (60 litres each)

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were almost completely submerged and were supplied with seawater at a continuous flow rate of 80

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ml/min (Fig. 1). Three of the smaller tanks acted as controls (named controls from here on) with

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naturally varying pH values, as in the lagoon water, whereas the other three tanks were maintained

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at a constant pH of 7.4 (treated) by bubbling CO2 using an automatic control system (Aquarium

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Controller Evolution, mod. ACQ110, Aquatronica, Italy) connected to pH electrodes (ACQ310N-

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PH, Aquatronica, Italy). The low pH value was chosen according to the maximum pH reduction

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predicted in ocean surface by 2300 by (Caldeira and Wickett, 2003, 2005).

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ACCEPTED MANUSCRIPT Two experiments were conducted. The first began in October 2009 and lasted until April 2010 (202

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days). At the beginning of the experiment, 400 juvenile mussels (mean length 1.15 cm) and 600

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juvenile clams (mean length 0.76 cm), laid upon a rigid plastic grid, were suspended in each tank.

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In the second experiment, which was initiated in October 2010 and lasted until January 2011 (94

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days), only juvenile mussels were used (500 individuals per tank, mean length 1.81 cm).

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Throughout the latter experiment, the carbonate chemistry of the seawater was also studied. Under

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each experimental condition, two replicate tanks contained animals, whereas the third tank did not

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contain animals to evaluate any possible effects of the bivalves on seawater chemistry.

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In both experiments, the juveniles fed on phytoplankton and organic matter suspended in the

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continuously flowing lagoon water and were additionally provided with Isochrysis galbana twice a

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day, increasing the algae concentration in each experimental tank by 54 x 103 cells ml-1. In both

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experiments, the seawater temperature and salinity were checked daily and chlorophyll-a was

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measured before and after supplying I. galbana, according to the spectrophotometric method

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described in Parsons et al. (1984).

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2.3 Morphometric and morphological parameters

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All bivalves were counted monthly and dead individuals were recorded. The shell lengths of at least

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200 individuals were measured monthly using a Vernier calliper accurate to 0.1 mm. In clams, the

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length corresponds to the anterior/posterior axis and is measured perpendicularly from the height

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line, matching the dorsal/ventral axis. Conversely, in mussels, length is defined as the distance from

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the umbo to the opposite shell margin.

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To avoid stress to the animals and minimise errors during weighing, the bivalves were dried as

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much as possible using blotting paper and their total live weight was estimated in pools of 40

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specimens using an analytical balance (0.01 g). The number of pools that were weighed monthly in

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each tank varied according to the abundance of the surviving juveniles, ranging from 10 to 8 for

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mussels and from 14 to 8 for clams.

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After 98 (3rd month) and 202 (6th month) days in the first experiment and after 31 (1st month) and 98

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days (3rd month) in the second experiment, further measurements were made. Shell length was

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measured in 30 mussels and 30 clams per tank and shell alterations were evaluated both

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qualitatively, using a specific damage index, and quantitatively, by measuring the percentage of

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damaged area, mostly occurring at the umbo level. In the same individuals, the dry weights (after 48

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h at 60°C) of the shell and soft tissues and shell thickness were also evaluated, but only in the first

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experiment.

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ACCEPTED MANUSCRIPT In both species, the shell damage index (DI) was graded according to the type of alterations found,

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regardless of the extent of the damaged area. For M. galloprovincialis, the DI was graded as follows

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by examining the injuries of the periostracum and underlying prismatic layer of calcite: 0 = no

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damage; 1 = periostracum discolouration; 2 = breakage and lifting of the periostracum, prismatic

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layer visible; or 3 = dissolution of the prismatic layer, inner aragonitic nacreous layer visible. For C.

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gallina, the DI was expressed as the sum of the damage level observed in the umbonal area plus the

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damage level on the remaining outer shell surface. In the umbonal area, damage was graded as

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follows: 0 = no damage; 1 = partial dissolution of the outer prismatic layer (OPL), i.e., OPL

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partially maintained with some visible traces of concentric ribs; or 2 = total dissolution of the OPL,

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i.e., middle crossed lamellar layer visible and ribs completely lacking. On the remaining external

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shell surface, damage was graded as follows: 0 = no damage; 1 = partial damage of the OPL, i.e.,

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loss of shine, discoloured areas on some concentric ribs; or 2 = total dissolution of the OPL, i.e.,

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colour loss, many concentric ribs flattened.

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The damaged umbonal area was measured and was recorded as a percentage of the outer surface of

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the left valve using an image analysis system (Leica Application Suite v3) connected to a stereo

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microscope (Leica S8APO). Finally, to measure the shell thickness of both species, the right valves

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of 30 shells per tank were sectioned along the growth axis, following embedding in epoxy resin

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(Araldite2020, Huntsman) and grinding. Shell thickness was measured at various specific points

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(the umbonal area and shell margin and three points in-between) in these sections using a stereo

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microscope and the above-mentioned image analysis system. In C. gallina, at intermediate points in

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the sectioned shells, thickness was measured at both the rib and ridge levels (Fig. 2). In the

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sectioned valves from both the control and treated bivalves, the mean thickness was calculated for

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each specific point on the shell in the 3rd and 6th month.

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For scanning electron microscopy (SEM) analysis, after embedding the bivalve shells in epoxy

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resin, they were cut, fixed on aluminium stubs and gold coated using an Edwards S 150B Sputter

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Coater. The specimens were observed with a Stereoscan 260 (Cambridge Instruments) scanning

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electron microscope, operating at 12 kV.

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ACCEPTED MANUSCRIPT 2.4 Seawater chemistry

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Seawater samples were collected weekly from 4 November 2010 to 12 January 2011. Total

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alkalinity (TA) was determined via potentiometric titration. Seawater hardness was measured using

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two independent methods: 1) complexometric titration with EDTA and Eriochrome Black T as an

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end-point indicator (APHA 2340C) (APHA., 1995), and 2) ion chromatographic determination of

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calcium and magnesium.

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Thermodynamic constants were computed according to Millero (1995). The pH values refers to sea

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water scale (SWS).The dissolved inorganic carbon (DIC) content and CO2 partial pressure (pCO2)

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in seawater were computed at the sampling temperature using the TA values. Application of the

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error propagation law allowed the computation of the standard deviations of the DIC and pCO2

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values. These values were 10 µmol/Kg and 15 µatm, respectively. The saturation states of calcite

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(Ωcal) and aragonite (Ωara) were computed based on the solubility products reported in Millero

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(1995) and Millero et al. (2006). The carbonate concentration values were obtained from TA

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measurements.

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2.5 Statistical analyses

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Statistical comparisons between juveniles maintained in control and reduced pH conditions were

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performed through a mixed model approach. In each tank, the evolution over time of mortality,

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length and weight parameters was summarized using trend statistics. To perform these analyses, the

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estimated slopes were used for length and weight, while the mean counts per month, averaged

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among the three control or three treatment tanks, were used for mortality. The treatment tanks were

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then compared with the control ones by a t-test, which can be used safely since for each tank we can

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invoke the central limit theorem. Since the number of measures in each tank was balanced, this

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method can be used to safely analyse mixed models (Finos and Basso, 2013).

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For damage index and percent damaged area parameters, the control samples had constant values

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(=0), thus an interaction analysis can be misleading. In this case, we performed over-time analyses

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(i.e. testing if treated or control samples had changed over time, not comparing treatment with

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control samples).

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Finally, for thickness data, parametric ANOVA mixed models were performed using treatment

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(acidified vs natural conditions) as fixed effect nested in time (3rd vs 6th month) and tank as random

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effect. For each pH condition, the effect was further tested over time by a parametric ANOVA

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mixed model, setting the time as fixed effect and the tank as random effect.

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The software R (R Core Team 2013) and STATISTICA 9 were used to perform the above

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mentioned analyses, whereas the PRIMER-6 PERMANOVA plus software packages were used to

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perform principal components analysis (PCA) on the whole data set.

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ACCEPTED MANUSCRIPT 3. Results

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3.1 First experiment

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During the first experiment, the temperature ranged from 3.6°C in January to 22.1°C in April

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(average 11.9 ± 4.84°C) and the salinity ranged from 27.95 to 35.52 psu (average 33.06 ± 1.45 psu).

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The pH was nearly constant under both the natural (average 8.21 ± 0.10, min 8.09, max 8.43) and

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acidified (average 7.41 ± 0.06, min 7.33, max 7.56) conditions. For the trend of pH values under

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natural and acidified conditions see Supplementary figure 1. In the experimental tanks, the average

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chlorophyll-a concentration ranged from 0.864 ± 0.327 µg l-1 to 14.848 ± 3.463 µg l-1.

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Mortality was low until the 5th month for both species, with similar values being recorded for the

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control and treated bivalves: 5.6% in control (natural pH conditions) and 5.5% in treated (pH 7.4)

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mussels, 1.1% in control and 1.7% in treated clams. A large increase in mortality was only observed

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at the end of the experiment, mostly in treated animals. Cumulative mortality was significantly

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higher in treated bivalves with respect to controls, with a sensibly higher difference being observed

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in clams (39%) than in mussels (11%) (Fig. 3).

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At the end of the experiment, the control and treated mussels both showed significantly increased

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shell lengths and live weights (p=0.000) (Figs 4, 5). In the clams, significant increases were only

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observed in shell length and live weight (p=0.023) in the control tanks, whereas in treated

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individuals, significant (p=0.000) decreases in weight (-26%) and even in length (-5%) were

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observed (Figs 4, 5).

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Throughout the experiment, no significant difference in length or live weight was found between

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the control and treated mussels. Conversely, length and weight were affected in the treated clams,

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with both parameters progressively decreasing with respect to the values measured in the 1st month

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(Table 1). A significant difference in live weight arose between the control and treated juvenile

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clams early during the experiment, from the 3rd month onward (Table 1). Although a steady

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decrease in length was observed in the treated clams throughout the experiment, the difference with

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respect to the controls was significant only in the 6th month (Table 1).

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In contrast to what was observed in the mussels, in the 6th month, the dry weights of both the shells

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and soft tissues of the treated clams differed significantly from those of the controls (Tables 2, 3).

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During the experiment, the treated mussels and clams also showed increasing shell damage,

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beginning in the umbonal region, extending toward the shell margin. In the controls of both species,

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no damage was ever recorded (Figs 6, 7). The alterations observed in the umbonal areas of the clam

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shells ranged from the corrosion of concentric ribs, which appeared to be thinner in the treated

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specimens, to the complete degradation at the end of experiment (Figs 7 T6 and b). Furthermore,

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modifications of the hinge were clearly visible (Fig. 7 b).

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ACCEPTED MANUSCRIPT In all of the treated mussels, the maximum (DI = 3) was observed after 3 months, although the

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extent of the damaged area was quite variable (average 22%) (Tables 2, 3). From the 3rd to the 6th

315

months, the damaged area showed a significant peak at 35% (Tables 2, 3). The shell thickness of the

316

treated and control mussels differed significantly only after 6 months (Table 3). Significant

317

differences in shell thickness were also observed between the 3rd and 6th month in treated mussels

318

(Table 3). The differences in mean shell thickness between the control and treated mussels are

319

shown in Figure 8 a. The reported values refer to the measurements made in the 3rd and 6th months

320

at six specific points in the sectioned valves. Shell corrosion was higher at the umbo level (-0.085

321

mm), mostly in the 6th month, but it progressively decreased towards the margin.

322

In the treated specimens of C. gallina, shell damage increased significantly from the 3rd to the 6th

323

month, with the DI increasing from 2.20 to 3.12 and the percentage of damaged area increasing

324

from 6% to 11% (Tables 2, 3). Similarly, clam shell thickness significantly decreased from the 3rd

325

to the 6th month (Tables 2, 3). As shown in Figure 8 b, the differences in mean shell thickness

326

observed between the control and treated clams reached their highest values in the 6th month, with a

327

maximum detected at the umbonal level (-0.181 mm). The minimum difference was recorded at the

328

marginal level in the 3rd month (-0.044 mm). Different corrosion patterns across the sections were

329

detected at the two measurement times: in the 3rd month, from the umbo toward the margin,

330

intermediate points showed an increasing level of corrosion; and in the 6th month, intermediate

331

points and the margin exhibited similar levels of corrosion, without clear differences between ribs

332

and ridges (Fig. 8 b).

333

In addition, in the 6th month alone, all of the treated clams displayed a loss of shine of the inner

334

surface of the shell, extending from the umbonal area to the pallial line (Fig. 9).

335

The multivariate statistical analyses performed on all of the parameters measured in the juvenile

336

mussels and clams at the end of experiment revealed the different behaviours of the two species

337

(Fig. 10). The first two PCA axes accounted for the majority of the total observed variation (86.7%

338

for mussels, 97.5% for clams) and clearly divided the tanks at a natural pH, on the right side of the

339

plot, from treatment tanks, on the left (Fig. 10). In the clams, both the control and treated samples

340

appeared more closely clustered than in mussels.

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341 342

3.2 Second experiment

343

During this experiment, the temperature varied from 5.0 to 15.3°C (average 10.8 ± 3.6°C) and the

344

salinity from 27.55 to 34.51 psu (average 30.93 ± 1.93 psu). In the experimental tanks, the average

345

chlorophyll-a concentration ranged from 1.357 ± 0.964 mg m-3 to 16.775 ± 4.531 mg m-3. Under

346

both the natural (8.22 pH) and acidified pCO2 (7.38 pH) conditions, no significant differences were

ACCEPTED MANUSCRIPT found between the tanks with and without mussels (Table 4). Although the TA values under natural

348

pH conditions did not significantly differ from those at pH 7.4, under seawater acidification, DIC

349

and pCO2 significantly increased (p 4). These saturation levels are higher than in Northern Adriatic basin, though

375

contrasting processes, such as the seawater temperature patterns controlling pCO2 and primary

376

production, may show a different balance, thus influencing calcium carbonate saturation states at

377

both spatial and temporal scales (Cantoni et al., 2012; Luchetta et al., 2010).

378

Under acidified conditions, the mean Ωc value remained slightly higher than 1, whereas Ωa

379

indicated undersaturation, resulting in an increased risk of shell corrosion and/or failure of shell

380

deposition, mainly in bivalves with aragonitic shells, such as Veneridae Chioninae species.

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ACCEPTED MANUSCRIPT 381 4.2 Effects on bivalve juveniles

383

Because the effects of acidification on shell growth and integrity could be affected by the

384

experimental duration, as demonstrated by Bamber (1990) and Yamada and Ikeda (2000), in

385

mussels and zooplankton, respectively, we must consider the days of exposure when comparing our

386

results to those from other studies. Medium- or long-term effects of acidification on juveniles have

387

been reported for several bivalve species. The duration of exposure in these experiments ranged

388

from 44 to 140 days, whereas our results refer to a considerably longer exposure time (202 days).

389

The longest experiments on juvenile mussels reported in the scientific literature are listed in Table

390

6A. Although some reports are available on the medium- or long-term exposure to acidification in

391

juveniles of Veneridae species (Table 6B), the effects of acidification in C. gallina were evaluated

392

for the first time in the present study. Further information is available from similar experiments on

393

juveniles of other bivalve species (Table 6C).

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4.2.1 Hypothesis 1: reduced survival

396

In both species, our results confirmed our first hypothesis of reduced survival in acidified

397

conditions.

398

In the first experiment, our results showed that there was a significant increase in mortality in both

399

mussels and clams maintained at pH 7.4 for 202 days, although clams exhibited a more marked

400

drop in survival (-39% with respect to controls). It is important to stress that under acidified

401

conditions, mortality increased greatly for both species at the end of experiment, whereas it was

402

very low during the previous five months. Interestingly, the same low mortality rate was found after

403

3 months in the second experiment on mussels. It must be noted that the mortality observed at the

404

end of the first experiment coincided with the highest seawater temperature values recorded during

405

the study period (22°C in April). Detrimental synergistic relationships between increases in

406

temperature and pCO2 have arisen in various studies on bivalve juveniles (e.g., Berge et al., 2006;

407

Hiebenthal et al., 2012; Talmage and Gobler, 2011). Increased temperatures, as long as they are

408

within the thermal range of tolerance of a species, increase physiological rates and may hide the

409

negative effects of acidification. Conversely, increased temperatures outside the range of tolerance

410

are detrimental and could exacerbate any stress caused by acidification (Pörtner, 2008). Although

411

22°C is not outside the range of thermal tolerance of either of the examined species from the

412

Northern Adriatic Sea, the increased temperatures in spring, together with the long period of

413

maintenance under the experimental conditions, may have compounded the responses of the

414

bivalves to the reduced pH.

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ACCEPTED MANUSCRIPT In M. galloprovincialis juveniles reared for 84-90 days at approximately pH 7.3, no mortality was

416

observed (Michaelidis et al., 2005; Range et al., 2012). Furthermore, in Mytilus chilensis, CO2

417

values ranging from 390 to 1,000 ppm (8.0-7.6 pH) were not found to affect survival in juveniles

418

(Duarte et al., 2014). In M. edulis spat, significant mortality has been reported only by Berge et al.

419

(2006), after 23 days of exposure at pH ≤ 6.7 and after 37 days at a higher experimental pH (up to

420

pH 8.1), when an increase in water temperature (up to 24°C) occurred. Hiebenthal et al. (2012)

421

observed that M. edulis juveniles were largely insensitive to a seawater pCO2 of almost 1,700 µatm

422

(approximately 7.7 pH), whereas their mortality sharply increased between 20 and 25°C,

423

irrespective of the tested pCO2 level (pH range: 8.01- 7.63).

424

After 75 days of rearing at 7.7 or 7.4 pH, R. decussatus juveniles exhibited lower mortality relative

425

to control individuals (Range et al., 2011), whereas at pH values lower than 6.5, mortality increased

426

within a few days (Bamber, 1987). In sediments undersaturated with respect to aragonite (Ωaragonite

427

0.3- 0.4), Green et al. (2004; 2009) observed significantly higher mortality rates and shell

428

dissolution in Mercenaria mercenaria juveniles within only few weeks. Conversely, in the same

429

species, a reduced seawater pH (7.6) combined with a high temperature (28°C) significantly

430

increased larval mortality up to 82%, although juvenile survival was not affected (Talmage and

431

Gobler, 2011).

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434

Despite what we observed for survival, the second hypothesis of reduced growth in acidified

435

conditions was confirmed only for C. gallina.

436

During the two experiments, positive growth was recorded in terms of both length and live weight

437

in the mussels, with no significant differences being detected between the control and treated

438

animals. The dry weight of the soft tissues and shells also did not differ in the control and treated

439

specimens. Similarly, Range et al. (2012) did not observe any reduction in the size or live weight of

440

M. galloprovincialis juveniles maintained at pH 7.6 or 7.3 for 84 days. Conversely, our results

441

demonstrated that after 6 months, the pH 7.4-treated mussels displayed shells that were significantly

442

thinner compared to both the controls and the treated individuals measured in the 3rd month.

443

As stated by Gazeau et al., 2012, shell thickness may indicate the effects of acidification more

444

accurately than areal measurements. In the present study, the obtained shell thickness values

445

indicated that shell dissolution occurred in the mussels under acidified conditions, but only after

446

more than 3 months. This dissolution took place mostly in the umbonal region, which is the oldest

447

part of the shell. In Littorina littorea, shell thickness has also been found to significantly decline at

448

a low pH (∼ 6.5 pH) (Bibby et al., 2007), though this was observed under experimental acidified

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ACCEPTED MANUSCRIPT conditions that were more severe than those used in the present work. Thomsen et al. (2010) did not

450

detect any decrease in the thickness of the calcite and aragonite layers of the shells of M. edulis

451

juveniles maintained under acidified conditions that were even more severe than those applied here

452

(∼pH 7.2). However, in their study, thickness measurements were performed only on shell parts that

453

were newly formed during exposure to elevated pCO2, thus evaluating shell deposition rather than

454

corrosion.

455

In the present study, although a reduced shell thickness was observed in the treated mussels, the

456

lack of a difference in shell size and weight with respect to controls suggest that a different

457

chemical composition of shells may occur under reduced pH. In this regard, it is of note that in M.

458

galloprovincialis juveniles, Range et al. (2012) found that the ashed shell weight (shell inorganic

459

component) significantly decreased at pH 7.6-7.3 with respect to controls (8 pH).

460

In the literature, there are many studies focused on mussel growth in acidified conditions (Gazeau et

461

al., 2012). In M. galloprovincialis juveniles, shell length is significantly and positively correlated

462

with pH levels below 7.5, and this value has been suggested to be harmful for shelled molluscs

463

(Michaelidis et al., 2005). Berge et al. (2006) found that in M. edulis juveniles, a detrimental effect

464

on growth appeared to be evident between 7.4 and 7.1 pH, whereas a strong significant decrease in

465

growth arose below 7.1 pH. In summer and winter trials, a high seawater pCO2 (∼ 7.2 pH) was

466

shown to significantly reduce growth in terms of shell length in M. edulis from Kiel Fjord, whereas

467

it did not affect growth in terms of somatic mass (Thomsen et al., 2010; Melzner et al., 2011). In

468

these experiments, Thomsen et al., 2010 also found significant effects of acidification on shell mass

469

growth. An additive effect of a high pCO2 and food limitation was observed by Melzner et al., 2011.

470

In M. chilensis juveniles, the net rate of calcium deposition and total weight were found to be

471

negatively affected by CO2 levels (minimum pH of ∼7.6), but no effect of temperature was revealed

472

(Duarte et al., 2014). In their study, no interaction between the pCO2 and temperature was found,

473

whereas in M. edulis, growth arrest was observed at 7.7 pH, but only at high temperatures (25°C)

474

(Hiebenthal et al., 2012).

475

Assuming that the reduced shell thickness recorded in this study may represent a first signal of the

476

impact of acidification on growth, a threshold value for the appearance of negative effects on

477

juvenile mussels may be set at 7.5-7.4 pH, in agreement with the findings of Berge et al. (2006) and

478

Michaelidis et al., (2005), provided that the environmental temperature remains lower than 25°C

479

(Hiebenthal et al., 2012).

480

In the present study, after 6 months, treated clams showed significantly lower shell lengths, live

481

weights and shell and soft tissue dry weights than did the controls. Interestingly, under acidified

482

conditions the clams did not grow, but their live weight decreased greatly and a slight reduction in

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ACCEPTED MANUSCRIPT shell length was even observed. Conversely, the clams maintained under natural pH conditions

484

showed increased shell lengths and live weights at the end of the experiment. The results obtained

485

in the controls indicated that rearing outside the sediment did not suppress clam growth and thus

486

that the differences recorded at a decreased pH could result from acidification. The highly

487

significant decrease in shell length highlighted that corrosion occurred not only on the outer or inner

488

shell surface but also along the shell margin. To our knowledge, this effect of seawater acidification

489

is reported for the first time in bivalves here. A detrimental effect on growth was confirmed at the

490

end of the first experiment based on the reduction of shell thickness. This effect, maximum at umbo

491

level, was consistent across the remaining extent of the sectioned valve, from the first intermediate

492

point up to the margin.

493

The experiments performed on juveniles of other Veneridae species have been shorter in duration

494

and have not found any significant difference in growth, measured in terms of size, weight and net

495

calcification (Range et al., 2011; Talmage and Gobler, 2011). However, these results were obtained

496

at pH values (7.8 pH) higher than ours (Range et al., 2011; Talmage and Gobler, 2011) or at the

497

same pH value (pH 7.4) but in seawater that was consistently supersaturated with respect to CaCO3,

498

thus attenuating any detrimental effects of acidification on carbonate availability, even under pCO2

499

values exceeding 4000 µatm (Range et al., 2011). In shorter experiments conducted at lower pH

500

values (< 7.0), V. decussata juveniles ceased feeding activity and growth in terms of both shell size

501

and, more pronouncedly, flesh weight (Bamber, 1987). Nevertheless, it must be noted that Bamber

502

(1987) performed his experiment by acidifying seawater via the addition of sulphuric acid;

503

compared to gas bubbling, this type of pH manipulation may result in responses of a different

504

magnitude (Kikkawa et al., 2004).

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505

4.2.3 Hypothesis 3: occurrence of shell injuries

507

This hypothesis was fully confirmed. Indeed, exposure to acidification resulted in injuries on the

508

outer shell surface of all mussels and clams. In both species, damage was initiated in the umbonal

509

area within a short period, being observed after 1 month in mussels in the second experiment.

510

Although there were different characteristics of the damage observed depending on the species, the

511

injuries were propagated from the umbo towards the ventral margin, as described for decreasing

512

shell thickness.

513

In previous studies conducted in naturally acidic seawater of the Mediterranean Sea and Kiel Fjord

514

(Rodolfo-Metalpa et al., 2011; Thomsen et al., 2010), dissolution of the external shell surface of

515

mussels (M. edulis) was also found to begin in the umbo region and to entail damage to the

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ACCEPTED MANUSCRIPT periostracum. Conversely, in laboratory trials, no response to elevated pCO2 was found in blue

517

mussels by Ries et al. (2009) or in Mediterranean mussels by Range et al. (2012).

518

In our juvenile mussels, the dissolution of the prismatic layer, revealing the inner aragonitic

519

nacreous layer, which corresponded to the maximum damage level found, was observed after 3

520

months. In contrast, during the second experiment, shell damage became visible as early as the end

521

of the 1st month. From the 3rd to the 6th month, the severity of damage did not increase, but the

522

damaged area progressively extended over time, covering up to 35% of the external valve surface.

523

The statistical significance of the comparison between the mean extent of damage recorded in the

524

3rd and 6th months stresses how much shell corrosion increased in mussels after the 3rd month, as

525

noted for thickness.

526

In both hard clams (M. mercenaria) and soft clams (Mya arenaria), net dissolution of the shell was

527

previously observed under the highest pCO2 treatment applied (7.45 pH), where the experimental

528

seawater was undersaturated with respect to aragonite and high-Mg calcite (Ries et al., 2009).

529

Conversely, Range et al. (2011) did not detect any shell damage in R. decussatus after 75 days of

530

exposure to a pH of 7.4. In contrast to the carbonate concentration measured by Ries et al. (2009),

531

undersaturated conditions for both aragonite and calcite were never observed by Range et al.

532

(2011).

533

In the clams subjected to the low pH treatment in the present work, the shell injuries continued to

534

worsen, extending to complete discoloration and flattening of the concentric ribs not only in the

535

umbo area but also over the entire outer shell surface. In contrast to what was observed in mussels,

536

in the 6th month, early signals of damage were also detected on the inner shell surface of all treated

537

clams, in the form of a loss of shine. Internal shell corrosion has previously been reported by

538

Melzner et al. (2011) in M. edulis juveniles from Kiel Fjord, mainly when pCO2 levels were high

539

and coupled with food limitation. This kind of damage which was attributed to partial dissolution

540

of the nacre for energy recovery and somatic mass maintenance in stress conditions. The authors

541

demonstrated that the observed change in the colour of the corroded inner shell surface was caused

542

by the dissolution of nacre tablets and the resulting changes in light refraction due to the remaining

543

organic material. The loss of shine observed on the inner shell surface in our clams may also reflect

544

the dissolution of the nacreous aragonitic tablets, which are more exposed to extrapallial fluid.

545

Shell carbon in aquatic molluscs is derived mainly from ambient DIC (McConnaughey and Gillikin,

546

2008). Nevertheless, most calcifying species, including freshwater and marine molluscs, are well

547

adapted to acidic conditions resulting in carbonate undersaturation. Indeed, bivalves are able to

548

concentrate Ca2+ and CO32- at the site of calcification and regulate calcification rates under

549

suboptimal concentrations of these ions (Gazeau et al., 2011; McConnaughey and Gillikin, 2008).

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ACCEPTED MANUSCRIPT Thomsen et al. (2010) demonstrated that M. edulis shows active recruitment and growth in the

551

waters of Kiel Fjord, which are naturally enriched with high CO2 levels and undersaturated for both

552

aragonite and calcite. Bathymodiolus brevior exhibits growth under the extremely undersaturated

553

conditions of deep hydrothermal vents, revealing an incomplete dependence on environmental

554

conditions (Tunnicliffe et al., 2009). Hiebenthal et al. (2012) found strong evidence that shell

555

growth in mussels from Kiel Fjord is independent of the seawater CaCO3 saturation state and pCO2

556

levels between 308 and 1,655 µatm (8.0 - 7.6 pH). Range et al (2012) demonstrated that, when

557

reared under acidified (7.6–7.3 pH) conditions in which oversaturation of both aragonite and calcite

558

occurred, juvenile M. galloprovincialis could continue to exhibit calcification and growth in coastal

559

lagoon waters of Southern Portugal.

560

However, contrasting results have been obtained in a number of studies reporting evidence of the

561

dependence of bivalve shell calcification and growth on the saturation of seawater carbonates (see

562

Gazeau et al., 2012, for a review), as demonstrated by our results. In Venice lagoon water acidified

563

to a pH of 7.4, undersaturation was consistently recorded for aragonite, but only occasionally for

564

calcite (37% of measurements). These conditions caused a more severe impact on the aragonitic

565

shells of clams, even leading to a reduction with respect to the length values measured in the 1st

566

month.

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4.2.4 Hypothesis 4: different sensitivities of juvenile M. galloprovincialis and C. gallina

569

In agreement with previous data on the biological features of the two studied species, our

570

hypothesis of different sensitivities of juvenile mussels and clams to acidification was fully

571

confirmed. Indeed, all of the parameters measured – i.e., mortality, length, live weight, shell and

572

soft tissue dry weight, shell damage and thickness – highlighted a greater impact of the reduced pH

573

in C. gallina than in M. galloprovincialis, with the former showing increases in detrimental effects

574

throughout the experiment. In particular, the increased temperature recorded at the end of the

575

experiment (April) exacerbated the effect of pH reduction to a greater extent in clams than in

576

mussels; this difference was mostly indicated by the final mortality values obtained in the two

577

species.

578

PCA was carried out on all of the parameters measured, and the high values of total variance

579

accounted for by the first two axes highlighted how definitively this statistical analysis

580

discriminated the relationships linking the measured variables to the experimental conditions tested.

581

For both mussels and clams, some interesting associations between specific parameters and control

582

or treatment conditions were stressed. Indeed, in clams, parameters for which high values

583

corresponded to detrimental effects (mortality, qualitative and quantitative shell damage) were

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ACCEPTED MANUSCRIPT clearly linked to acidified conditions, whereas variables indicating positive growth and the general

585

well-being of bivalves were linked to the controls. Conversely in mussels, shell thickness grouped

586

with two control replicates, while mortality and shell damage grouped with two treatment

587

replicates, and all of the other variables measured were located between one control (N1) and one

588

treatment (A3) replicate (fig. 10 A).

589

The most dramatic effect observed in clams was the reduction of shell length, which has not

590

previously been reported for any bivalve species and may be a consequence of limited tolerance to

591

environmental variations as well as of slow growth rates (Keller et al., 2002; Matozzo et al., 2007;

592

Monari et al., 2007a; Moschino and Marin, 2006). Although severe injuries were detected in

593

juvenile mussel shells under acidified conditions, the same amount of growth was measured in the

594

control and treated individuals. In addition to the different tolerances of the mussels and clams to

595

the environmental changes, the shell damage and growth observed in the two species may be related

596

to their different shell composition: clam shells are exclusively aragonitic, whereas mussel shells

597

are calcitic-aragonitic and almost entirely covered by the periostracum. For this reason, clams may

598

be more affected by the aragonite undersaturation recorded in the reduced pH tanks, as reported

599

above in section 4.1.

600

The occurrence of corrosion on the inner shell surface observed in clams, but not in mussels, may

601

be explained by the occurrence of increased stress conditions due to a reduced energy input at low

602

pH, as demonstrated by the reduction of clearance rates (Marin, unpublished results). Interestingly,

603

similar shell damage was described by Melzner et al. (2011) in M. edulis juveniles at low food and

604

high pCO2 levels. In our experiment, the growth of control clams and both treated and control

605

mussels suggested that the food supply was sufficient. Due to their higher sensitivity, the clams

606

reared at 7.4 pH needed to recover extra energy to cope with acidification stress. This condition

607

resulted not only in partial shell dissolution but also in a decreased soft tissue dry weight, which

608

was detected as early as the 3rd month.

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610

5. Conclusions

611

Various studies on the effects of acidification on bivalve adults and juveniles stress the relevance of

612

interspecific differences (this study), intraspecific mechanisms of acclimation or genetic adaptation

613

in local populations (Parker et al., 2011), different sensitivities of various life-history stages

614

(Talmage and Gobler, 2011), and local variability in seawater chemistry (Range et al., 2011, 2012).

615

Under seawater acidification, these constraints influence bivalve responses, which in turn can be

616

differently modulated by the synergistic effects of other stressors (e.g., temperature, salinity, food

617

concentration, environmental contaminants) in both laboratory and field conditions. In this regard,

ACCEPTED MANUSCRIPT the impact of acidification on even single species may result in overall effects at food-web and

619

ecosystem levels.

620

Many studies aimed at evaluating the effects of acidification on marine organisms have highlighted

621

difficulties in extrapolating general proxies from a single species or population. The present study

622

corroborates this conclusion, showing contrasting effects in two bivalve species living in the same

623

region, although not exactly in the same habitat. To assess future risks to bivalves due to ocean

624

acidification, further research should be carried out on multiple species from differing geographic

625

locations, accounting for potential carryover effects on subsequent generations in long-term

626

experiments and exposure to multiple stressors, whose impacts may increase under predicted

627

climate change scenarios.

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628 Acknowledgements

630

This study is a contribution to the project “The integrated impacts of marine acidification,

631

temperature and precipitation changes on bivalve coastal biodiversity and fisheries: how to adapt

632

(ACIDBIV).” This project is part of the CIRCLE Med projects, which are funded by the Italian

633

Ministry for Environment, Land and Sea (IMELS) (DEC/RAS/649/2008), the Foundation for

634

Science and Technology of Portugal, and the Regional Ministry of Innovation and Industry of the

635

Galician Government, in the framework of the CIRCLE ERA Net project (which is funded by the

636

European Commission 6th Framework Programme).

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ACCEPTED MANUSCRIPT 6. References

639 640 641 642

Alemany, J.A., 1986-1987. Comparaison morphologique de la structure de la coquille et de l'enroulement en spirale chez Chamelea gallina (Mörch, 1853) et chez Venus verrucosa L. 1758 (Mollusca: Bivalvia). Archives d’Anatomie Microscopique et de Morphologie Expérimentale 75 (1), 61-74.

643 644

Amaral, V., Cabral, H.N., Bishop, M.J., 2012. Moderate acidification affects growth but not survival of 6-month-old oysters. Aquatic Ecology 46, 119–127.

645 646

Andersson, A.J., Mackenzie, F.T., 2011. Ocean acidification: setting the record straight. Biogeosciences Discussions 8, 6161–6190.

647 648 649 650

Anestis A., Lazou A., Pörtner H.O., Michaelidis B., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 293, R911–R921.

651 652 653

Bamber, RN., 1987. The effects of acidic sea water on young carpet-shell clams Venerupis decussata (L.) (Mollusca: Veneracea). Journal of Experimental Marine Biology and Ecology 108, 241-260.

654 655

Bamber, R.N., 1990, The effects of acidic sea water on three species of lamellibranch molluscs. Journal of Experimental Marine Biology and Ecology 143, 181-190.

656 657 658

Barillari, A., Boldrin, A., Mozzi, C., Rabitti, S., 1978. Alcune relazioni tra natura dei sedimenti e presenza della vongola Chamelea gallina, nell’Alto Adriatico, presso Venezia. Atti Istituto Veneto Scienze Lettere Arti Classe Sci Fi Mat Nat 137, 19–34.

659 660 661

Beniash, E., Ivanina, A., Lieb, N.S., Kurochkin, I., Sokolova, I.M., 2010. Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica. Marine Ecology Progress Series 419, 95–108.

662 663 664

Berge, J.A., Bjerkeng, B., Pettersen, O., Schaanning, M.T., Øxnevad, S., 2006. Effects of increased sea water concentrations of CO2 on growth of the bivalve Mytilus edulis L. Chemosphere 62, 681– 687.

665 666 667

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Fig. 1 - Outdoor experimental plant at the Hydrobiological Station of Chioggia (Northern Adriatic,

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Italy).

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Fig. 2 – Mussel (A) and clam (B) shells sectioned along the growth axis. Yellow bars indicate

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points of thickness measurement; arrows indicate measurement at rib (black) and ridge (white)

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level.

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Figure 3 - First experiment: percent cumulative mortality in juvenile M. galloprovincialis (A) and

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C. gallina (B) throughout the experiment under natural (Nc) and acidified (Ac) conditions. Bars are

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for standard deviation (replicate tanks: n = 3). Asterisks are for t-test significance: * = p