ORIGINAL ARTICLE

Impacts of CO2-induced seawater acidification on coastalMediterranean bivalves and interactions with other climaticstressors

P. Range • M. A. Chıcharo • R. Ben-Hamadou • D. Pilo • M. J. Fernandez-Reiriz •

U. Labarta • M. G. Marin • M. Bressan • V. Matozzo • A. Chinellato •

M. Munari • N. T. El Menif • M. Dellali • L. Chıcharo

Received: 5 March 2012 / Accepted: 14 May 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract The effects of seawater acidification caused by

increasing concentrations of atmospheric carbon dioxide

(CO2), combined with other climatic stressors, were stud-

ied on 3 coastal Mediterranean bivalve species: the mussel

Mytilus galloprovincialis and the clams Chamelea gallina

and Ruditapes decussatus. CO2 perturbation experiments

produced contrasting responses on growth and calcification

of juvenile shells, according to species and location. In the

Northern Adriatic (Italy), long-term exposure to reduced

pH severely damaged the shells of M. galloprovincialis and

C. gallina and reduced growth for the latter species. Sea-

water in the Ria Formosa lagoon (Portugal) was consis-

tently saturated in carbonates, which buffered the impacts

on calcification and growth. After 80 days, no shell

damage was observed in Portugal, but mussels in the

acidified treatments were less calcified. Reduced clearance,

ingestion and respiration rates and increased ammonia

excretion were observed for R. decussatus under reduced

pH. Clearance rates of juvenile mussels were significantly

reduced by acidification in Italy, but not in Portugal. Both

locations showed a consistent trend for increased ammonia

excretion with decreasing pH, suggesting increased protein

catabolism. Respiratory rates were generally not affected.

Short-term factorial experiments done in Italy revealed that

acidification caused alterations in immunological parame-

ters of adult bivalves, particularly at temperature and

salinity values far from the optimal for the species in the

Mediterranean. Overall, our results showed large variations

in the sensitivities of bivalves to climatic changes, among

different species and between local populations of the same

species. Expectations of impacts, mitigation and adaptation

strategies have to consider such local variability.

Keywords Carbon dioxide � Ocean acidification �Mollusks � Coastal waters � Gulf of Cadiz � Lagoon

of Venice

Introduction

The combustion of fossil fuels by human populations is

increasing atmospheric concentrations of carbon dioxide

(CO2) at an unprecedented rate. It is now unequivocally

accepted that this is causing global climatic changes, with

noticeable increases in global temperature, sea level rise

and changes to marine carbon chemistry (Meehl et al.

2007). Global average surface air temperature has

increased by about 0.7–0.8 �C during the last century

(Hansen et al. 2006) and further warming, ranging from 1.8

P. Range (&) � M. A. Chıcharo � R. Ben-Hamadou � D. Pilo �L. Chıcharo

CCMAR, Universidade do Algarve, Faro, Portugal

e-mail: prange@ualg.pt

Present Address:

R. Ben-Hamadou

Department of Biological and Environmental Sciences,

College of Arts and Sciences, Qatar University, Doha, Qatar

M. J. Fernandez-Reiriz � U. Labarta

CSIC, Instituto de Investigaciones Marinas, Vigo, Spain

M. G. Marin � M. Bressan � V. Matozzo � A. Chinellato �M. Munari

Department of Biology, University of Padova, Padova, Italy

N. T. El Menif � M. Dellali

Department of Biology, University of Carthage,

Carthage, Tunisia

Present Address:

L. Chıcharo

CIMA, Universidade do Algarve, Faro, Portugal

123

Reg Environ Change

DOI 10.1007/s10113-013-0478-7

to 4.0 �C, has been predicted until the end of the 21st

century. Sea surface temperature is expected to increase

between 2 and 4.5 �C during the same period (Meehl et al.

2007). Large-scale intensification of the hydrological cycle

is also expected, with increased frequency of droughts and

floods in many regions of the world. Combined with global

mean sea level rise, the modifications in freshwater runoff

will determine changes to seawater salinity, particularly in

estuarine and coastal areas (Nicholls et al. 2007). This is

worrying because, although coastal areas occupy less than

10 % of the total ocean surface (Wollast 1998), human

interactions with marine organisms mainly occur within

these areas, which account for more than 90 % of global

fisheries (Pauly et al. 2002). The uncertainty associated

with these global averages is, however, still large, and the

magnitude of change will differ markedly between regions

(Brierley and Kingsford 2009). This clearly highlights the

need for assessing the ecological impacts of global climate

change at regional and local levels (Philippart et al. 2007;

Byrne 2011; Parker et al. 2013).

Enclosed shallow seas are particularly vulnerable to

these perturbations, and the Mediterranean has been con-

sidered a ‘‘hotspot’’ for climatic change (Diffenbaugh et al.

2007). According to Rosa et al. (2012), the following

trends are expected in the Mediterranean basin during the

21st century: increasing air temperatures, of between 2.2

and 5.1 �C; decreasing rainfall of between 4 and 27 %;

longer periods of drought, related to an increased frequency

of days with temperatures above 30 �C; sea level rise of

around 35 cm; and saline intrusion. These extreme climatic

events are already perceived as having strong effects on

marine biodiversity in this region (Lejeusne et al. 2010).

The uptake of CO2 by the oceans is also increasing the

concentration of hydrogen (H?) and bicarbonate (HCO3-)

ions, while decreasing the concentration of carbonate ions

(CO32-). The saturation state of calcium carbonate

(CaCO3), which is essential to the formation of shells and

skeletons of many marine organisms, is also decreasing.

This process of ocean acidification (OA) has already

decreased the average pH of ocean surface waters by

0.1 U, from pre-industrial levels (Haugan and Drange

1996). Conservative projections based on the IPCC emis-

sions scenarios for the 21st century (SRES-A2) indicate

further declines of ocean pH ranging between 0.21 and

0.36 U (Joos et al. 2011). Under unrestricted emissions

scenarios, larger reductions, varying between 0.7 and 1.3

pH units, have been hypothesized for the more distant

future (Caldeira and Wickett 2003, 2005).

Bivalves (mussels, oysters, clams, etc.) play an important

role in the structure of aquatic ecosystems, because they

dominate the macrofauna of many estuarine and coastal

areas, link primary producers with top consumers, couple

pelagic and benthic processes, and provide habitat for many

other organisms. In a global change scenario, increasing

temperature, variations in salinity, and reduced pH may

affect physiological performance of marine bivalves, thus

reducing growth, reproductive fitness, and survival (Mato-

zzo and Marin 2011). Considering the importance of

bivalve aquaculture, commercial and artisanal fisheries in

the Mediterranean basin, any adverse effects on their via-

bility, productivity, nutritional quality, or market value can

have relevant societal implications (Cooley et al. 2011).

The ACIDBIV project ‘‘Integrated impacts of marine

acidification, temperature and precipitation changes on

bivalve biodiversity and fisheries’’ was an international

cooperation, involving partners from 4 Mediterranean

countries: Portugal, Italy, Spain, and Tunisia. The project

was aimed at investigating the cumulative effects of

changes in temperature, precipitation, and ocean acidifi-

cation, as predicted in climate changes scenarios, on three

of the most ecologically and commercially important

coastal bivalve species in the region. The Mediterranean

mussel Mytilus galloprovincialis (Lamarck 1819) is the

strongest contributor to the aquaculture sector in the

European Union (313730 tonnes in 2009, Eurostat 2011),

with its production largely concentrated in the extensive

culture system of the Galician rıas (NW Spain). The

grooved carpet clam Ruditapes decussatus (Linnaeus 1758)

is widely distributed in the Mediterranean and northeastern

Atlantic, extending from Mauritania to the English Atlantic

coast. Intensive harvesting of this species occurs mainly in

the Iberian Peninsula, France, and other Mediterranean

countries (Amaral 2008). The Venus clam Chamelea gal-

lina (Linnaeus 1758) is distributed from the Portuguese

south coast to the Mediterranean, including the Black Sea,

being particularly abundant in the Adriatic (Rufino et al.

2006). This species is commercially exploited in the

inshore waters of Italy, Turkey, and Morocco, where it has

great economic importance (Moschino et al. 2008).

Concerns about the effects of OA on marine organisms

have motivated an increasing number of CO2 forcing exper-

iments in recent years. These previous studies showed

important variability in the responses, among species, popu-

lations, and life stages (reviewed by Doney et al. 2009;

Hendriks et al. 2010; Kroeker et al. 2010; Hofmann et al. 2010;

Gattuso and Hansson 2011; Parker et al. 2013). In the present

study, juvenile bivalves were studied in long-term (up to

202 days) CO2 perturbation experiments. The long duration

of these experiments, which exceeded most previous studies

(Andersson et al. 2011), was considered necessary to ade-

quately measure the response to seawater acidification, in

terms of growth, composition, and morphology of bivalve

shells. Experiments were done simultaneously in two distinct

coastal areas, in Portugal and Italy, using similar species and

pH values, which were based on the IPCC SRES-A2 CO2

emissions scenario. This novel approach allowed us to assess

P. Range et al.

123

the generality of the observed patterns and to gain a better

insight into the underlying mechanisms for the complex

responses of bivalves to ocean acidification.

Environmental stressors can have simple (additive) or

interactive (synergistic or antagonistic) effects on marine

organisms and ecosystems. Despite meriting considerable

research effort in recent years, the biological impacts of

OA have been largely considered in isolation (Byrne 2011).

Interactive effects with other climate change stressors, such

as temperature (Metzger et al. 2007; Walther et al. 2009;

Lannig et al. 2010; Melatunan et al. 2011) or salinity

(Dickinson et al. 2012), are still poorly understood (but see

Portner 2008; Sokolova et al. 2012 and Harvey et al. 2013).

Accordingly, we used short-term controlled multifactorial

experiments to investigate the complex interactions of pH,

temperature, and salinity on the physiology of adult

bivalves. Several indicators of animal well-being were

measured at the biochemical, cellular, and organism level.

For logistical reasons, namely the difficulties in maintain-

ing large experimental designs with multiple stressors over

long periods, only acute (short-term) responses were

evaluated in these experiments.

In the ACIDBIV project, we studied the regional sen-

sitivities of bivalves exposed to seawater acidified by CO2

and interactions with other environmental stressors. Inno-

vative experimental approaches were used, to disentangle

effects within and among species and life stages, from the

same region and from different regions (Gulf of Cadiz and

Northern Adriatic). Parts of the dataset summarized here

have been used to test other hypotheses, at local scales or

species-specific effects. These previous publications are

fully listed in Table 1 and are cited when appropriate. We

hope this integrated analysis of our findings will help

managers to anticipate the impacts of global climate

change for coastal bivalve populations in the Mediterra-

nean region and contribute to mitigation and adaptation to

any adverse consequences.

Methods

Long-term CO2 perturbation experiments with juvenile

bivalves

Study sites

Two experimental rearing systems were set up for these

long-term experiments. In Portugal, an indoor system was

installed in a bivalve hatchery operated by the Instituto

Portugues do Mar e da Atmosfera (IPMA), located in the

Ria Formosa lagoon, close to Tavira (37�7017.7300N,

7�37012.1900W). The Ria Formosa is a mesotidal shallow

lagoon, with very limited freshwater input, separated from

the Atlantic ocean (Gulf of Cadiz) by a chain of five barrier

islands and two peninsulas. The physicochemical charac-

teristics of seawater were continuously monitored during

the experiments. The ranges of recorded values (daily

means) were 26–33 for salinity, 15.5–24.5 �C for temper-

ature, 7.9–8.3 for pH, 90–103 % for dissolved oxygen, and

3,252–3,934 lmol kg-1 for total alkalinity (TA-Table 1).

In Italy, an outdoor system was set up in the Hydrobio-

logical Station of Chioggia, in the lagoon of Venice, near to

the inlet connecting with the Northern Adriatic Sea

(45�13024.0700N, 12�1703.6000E). The Northern Adriatic Sea

is a relatively closed basin covering a shallow continental

shelf area, and it is surrounded by highly industrialized

regions and influenced by freshwater inflow. Po and Adige

rivers only discharge 58,0009106 m3 year-1 (Cushman-

Roisin et al. 2001). Changes in riverine inputs, depending on

season and precipitation regime, are responsible for vari-

ability in salinity values and TA loads. In recent surveys, an

average TA value of 2,658.9 ± 18.1 lmol kg-1 was found

(Luchetta et al. 2010). In Venetian coastal waters, tempera-

ture ranges from 3.6 to 28.6 �C (mean 17.3 ± 7.1 SD) and

salinity from 18 to 38 (mean 34.1 ± 2.8 SD, from 2001 to

2008 dataset provided by Servizio Acque Marino Costiere,

A.R.P.A.V.). Due to the proximity to the lagoon inlet, sea-

water parameters at the Hydrobiological Station of Chioggia

exhibit patterns of variations quite similar to those occurring

in the near coastal waters.

Rearing systems

In order to test the long-term effects of increased CO2 and

decreased pH, under natural patterns of variation for other

environmental variables, the experiments were done in

flow-through systems. This avoided the accumulation of

metabolic waste products, which might interfere with the

pH treatments. The carbonate chemistry of seawater was

manipulated by bubbling pure CO2 gas, to achieve pH

reductions of the magnitude estimated from the IPCC

SRES-A2 emission scenarios for the year 2100 and beyond

(Caldeira and Wickett 2003, 2005; Joos et al. 2011). The

effects of seawater acidification by CO2 were tested against

unmanipulated seawater, with naturally varying pH values.

In Portugal, two species were tested, the clam

R. decussatus and the mussel M. galloprovincialis, under

three levels of seawater pH: two acidified treatments and one

unmanipulated (control) level. The juvenile bivalves were

fed with a 1:1 mixture of two microalgae strains, Isochrysis

galbana and Chaetoceros calcitrans, supplied in continuous

flow. The average chlorophyll-a concentrations in the

experimental tanks ranged between 0.435 and 2.027 lg l-1.

The experiment with R. decussatus started on 07/07/2009

and lasted for 75 days. The experiment with M. gallopro-

vincialis started on 9/12/2009 and lasted for 84 days.

Impacts of CO2-induced seawater

123

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P. Range et al.

123

In Italy, two species were tested simultaneously, the

mussel M. galloprovincialis and the clam C. gallina. The

juvenile bivalves were reared during 202 days, from

October 2009 to April 2010, under two levels of seawater

pH: one acidified treatment and one unmanipulated (con-

trol) level. Microalgal food (Isochrysis galbana) was

manually supplied twice a day to each tank, with average

chlorophyll-a concentrations ranging between 0.864 and

14.848 lg l-1 (for further methodological details see

Table 1 and references therein).

Biological responses measured

In all the experiments, the juvenile bivalves were counted

and measured (shell length, width, height, and live weight),

and the dead individuals were recorded at regular intervals.

At the end of the exposure, the morphology and calcifi-

cation (i.e., total calcification minus total dissolution,

estimated by variations in shell weight) of the shells were

assessed. In Italy, the alterations in shell integrity were also

evaluated by specific damage indices (adapted from

Thomsen et al. 2010) and measurements of damaged area

and shell thickness.

The key physiological rates of the juvenile bivalves

were measured on pooled subsets of individuals.

Clearance rates (CR) were estimated using a flow-

through approach (Hildreth and Crisp 1976; Filgueira

et al. 2006). Ingestion rates (IR) were calculated as the

product of CR and food concentration, and absorption

efficiency was estimated by the method of Conover

(1966). Scope for growth (SFG) was calculated as

described by Labarta et al. (1997). Respiration rates and

ammonia excretion rates were measured in closed

chambers, from the difference in concentration between

chambers with and without animals (Solorzano 1969;

Widdows 1985).

Whole-body and pooled samples of different bivalve

tissues were collected at the end of the experiments, to be

analyzed for biochemical composition: total carbohydrates

and glycogen (Strickland and Parsons 1968), proteins

(Lowry et al. 1951) and lipids (Bligh and Dyer 1959,

modified by Fernandez-Reiriz et al. 1989). In Portugal, the

ratio of RNA to DNA was determined in tissue from the

foot (R. decussatus) or adductor muscle (M. galloprovin-

cialis), according to the methods of Caldarone et al. (2001)

and Chicharo et al. (2007).

The biological responses were tested for differences

among the distinct levels of pH using t-tests (Italy) and

single-factor analyses of variance (Portugal). The statistical

results were subsequently used for a trend analysis and the

results expressed in terms of positive, negative, nonlinear,

and neutral effects.

Short-term factorial experiments on adult bivalves

Rearing system

The flow-through system for the multifactorial experiments

on adult M. galloprovincialis and C. gallina was set up

indoors, at the Hydrobiological Station of Chioggia. Values

for seawater temperature (22 and 28 �C) were chosen,

hypothesizing a future increase, while for salinity,

increasing and decreasing values were considered (28, 34

and 40), based on possible local and seasonal variations

(dataset provided by Servizio Acque Marino Costiere,

A.R.P.A.V.). Three levels of seawater pH were tested, 8.1

(mean value recorded along the northwestern Adriatic

coast), 7.7 (-0.4 pH U), and 7.4 pH (-0.7 pH U). To

avoid spawning and possible subsequent mortality, exper-

iments were performed far from periods of sexual maturity,

i.e., in summer for mussels and in winter/spring for clams.

Since it was logistically difficult to simultaneously

manipulate pH and salinity, for each species, three exper-

iments were carried out sequentially with different levels of

salinity. In each experiment, bivalves were exposed for 7 days

to six combinations of pH and temperature (for further

methodological details see Table 1 and Matozzo et al. 2012).

Biological responses measured

Immunological biomarkers: total hemocyte count (THC),

hemocyte neutral red (NR) uptake, and hemolymph lyso-

zyme activity were evaluated, as described by Matozzo and

Marin (2010). Among the suite of biomarkers applied,

THC and neutral red uptake were chosen as representative

of important functional issues, such as the potential capa-

bilities to defend against pathogens. At each salinity,

responses obtained were compared with those of animals

maintained at 8.1 pH and 22 �C, considered as controls.

For each salinity, results were analyzed by Permutational

Analysis of Variance (PERMANOVA) using temperature

and pH as fixed factors (See Matozzo et al. 2012 for further

details).

Results and discussion

Long-term exposure of juvenile bivalves to seawater

acidified by CO2

Survival

The survival of juvenile bivalves under long-term exposure

to increased CO2 and reduced seawater pH differed

markedly between the two locations studied, even for the

Impacts of CO2-induced seawater

123

common species M. galloprovincialis. In Italy, both species

studied showed a significant increase in mortality in

acidified treatments, relative to controls (Table 2). The

magnitude of this increase was, however, much larger in

C. gallina than in M. galloprovincialis, emphasizing the

interspecific variability in the sensitivity of bivalves to OA.

Beniash et al. (2010) and Dickinson et al. (2012) have also

observed significant increases in mortality of juvenile

oysters exposed to elevated pCO2. Nevertheless, these

findings seem to contradict previous experimental evidence

indicating that, under realistic levels of CO2 increase, the

survival of adult and juvenile bivalves would not be

compromised (Berge et al. 2006; Beesley et al. 2008;

Hendriks et al. 2010). The periods of exposure used in our

study (75–212 days) and by Dickinson et al. (11 weeks) lar-

gely exceeded most previous experiments, allowing a better

appraisal of the long-term effects on survival, which may

contribute to reconcile these apparently divergent results.

In Portugal, no differences in survival were detected for

M. galloprovincialis after 84 days of exposure (average

mortality under 10 %). In contrast, after 75 days of expo-

sure, mortality of R. decussatus was significantly reduced

in the acidified treatments (average mortality 16 %), rela-

tive to controls (average mortality 36 %). This pattern,

although surprising, is not without precedent (Berge et al.

2006) and was probably associated with unexpected

spawning events in the control and intermediate acidifi-

cation treatments (Range et al. 2011). In fact, stress

exposure during spawning events can cause high mortali-

ties due to increased energy costs and thus less energy

available for stress tolerance. In a subsequent experiment

with adult mussels, Casimiro (2011) reported desynchro-

nized gametogenic development, with delayed spawning

in females, under a pH reduction of same magnitude

(-0.7 U). Bibby et al. (2008) had previously hypothesized

that exposure to acidified seawater could alter the repro-

ductive condition of bivalves, causing them to reabsorb

their gametes, as an energy saving strategy, possibly con-

tributing to increased survival. On the other hand, delayed

or desynchronized gametogenic development may reduce

the reproductive success and, ultimately, affect the abun-

dance and sustainability of bivalve populations. Further

investigation is clearly needed to test these hypotheses.

Growth

The response in terms of growth also differed markedly,

among species and locations (Table 2). In Italy, the

growth of M. galloprovincialis was unaffected by expo-

sure to increased CO2, whereas in C. gallina, the growth

parameters were severely altered. Shell length was unaf-

fected in Portugal, but interspecific and intraspecific

variations in somatic growth were particularly strong,

with both species exhibiting distinct response patterns at

different times of sampling: R. decussatus, neutral or

negative effects; M. galloprovincialis, neutral or positive

effects. The slightly different methodological approaches

used in Portugal and Italy may have contributed to

exacerbate the observed differences between the two

locations, but it seems unlikely that this could fully

explain these contrasting responses. Overall, in contrast

with previous studies (Thomsen and Melzner 2010;

Thomsen et al. 2010), CO2-induced acidification seemed

to affect somatic growth more frequently than shell

Table 2 Responses of bivalves to long-term exposure (75–202 days)

to increased CO2 and reduced pH

Type of response Ria

Formosa

North

Adriatic

RD MG MG CG

Survival

No. survivors at the end of experiments Ø ! !

Growth

Length of the shell Ø Ø Ø !Somatic tissue (weight) Ø! Ø Ø !

Calcification and morphology of the shells

Shell weight Ø ! Ø !Shell thickness ! !Shell integrity Ø Ø ! !

Feeding and digestive behavior

Absorption efficiency

Clearance rate ! Ø ! !Ingestion rate ! Ø

Scope for growth

Metabolism

Ammonia excretion Ø

Respiration ! Ø Ø Ø

O/N ratio ! Ø Ø !Biochemical composition

Carbohydrates Ø! Ø

Glycogen Ø! Ø

Proteins Ø! Ø ! !Lipids Ø Ø

Indices of condition

Somatic weight/shell weight Ø \ Ø !Somatic weight/shell length Ø !RNA/DNA ! Ø

Positive (%), negative (!), nonlinear (\), and neutral (Ø) effects;

entries with more than one symbol denote variations among experi-

ments or size classes; nonlinear responses could not be found in the

North Adriatic, since only two levels of pH were tested

RD Ruditapes decussatus, MG Mytilus galloprovincialis, CG Cham-

elea gallina

P. Range et al.

123

growth. This implies, as proposed by Findlay et al.

(2009), that calcification may not be the critical process

impacted by ocean acidification.

Calcification and morphology of the shells

In Italy, mussels and clams showed increasing erosion of

the shell during the exposure to CO2-induced acidification.

As reported in previous studies (Ries et al. 2009; Thomsen

et al. 2010; Rodolfo-Metalpa et al. 2011), external disso-

lution usually started from the umbonal region and pro-

gressed toward the margin of the shell. Dissolution was

usually associated with some degree of damage to the

periostracum and was never recorded under control pH.

The extent of the damaged area varied considerably

between the two species. A similar pattern was observed

for shell thickness, which was significantly reduced, for

both species, in the acidified treatments, relative to controls

(Table 2).

Mussels in Portugal also exhibited some erosion of the

periostracum at the umbo during the incubations, but that

type of damage was limited in frequency and extent and

unrelated to the experimental treatments. Nevertheless, the

inorganic weight of M. galloprovincialis shells was sig-

nificantly reduced in the elevated CO2 treatments and the

magnitude of this effect generally increased (up to 9 %)

with the size of the individuals. In contrast, the variations

in shell weight of R. decussatus were independent of the

pH treatments.

In a recent review, Parker et al. (2013) reported that

83 % of the 24 mollusk species studied to date showed a

negative effect of OA on calcification, while only 17 %

showed neutral or positive effects. Overall, our results

seem to corroborate this general pattern. Furthermore, the

average total alkalinity of seawater in the Ria Formosa

lagoon (&3,550 lmol kg-1) largely exceeds the typical

oceanic values (2,325 lmol kg-1; Gattuso and Lavigne

2009) and those recorded in the lagoon of Venice

(2,889–2,933 lmol kg-1) and the Northern Adriatic Sea

(Luchetta et al. 2010). This buffered seawater prevented

sub-saturation of CaCO3 and minimized the effects on net

calcification, even for the most extreme pH reductions

(-0.7 pH units, Range et al. 2011). The carbonate chem-

istry of coastal waters is known to depend on the balance

between anthropogenic CO2 emissions and watershed

processes affecting the export of nutrients, organic and

inorganic carbon, acids and carbonate alkalinity to the

ocean (Borges and Gypens 2010; Duarte et al. 2013). The

Ria Formosa basin is mainly constituted by carbonate

rocks, which contribute to the strong mineralization and

supersaturation of the groundwater with respect to car-

bonate minerals (Almeida and Silva 1987; Stigter et al.

2006). Benthic microbial processes can also increase

alkalinity in coastal ecosystems through sediment–water

fluxes, particularly in anoxic sediments (Cyronak et al.

2013; Duarte et al. 2013). Accordingly, continental inputs,

evaporation and anaerobic processes in sediments are

probably increasing TA within this coastal lagoon, at least

in areas of restricted exchange.

Interestingly, although seawater acidification caused

severe corrosion of M. galloprovincialis shells in Italy,

growth (shell and somatic) was unaffected. Similar pH

reductions (-0.7 U) also caused some loss of inorganic

shell material for mussels in Portugal, while having no

effect or even increasing organic tissue weight (Table 2).

These results suggest, as hypothesized by Melzner et al.

(2011), that under increased concentrations of CO2, mus-

sels will preferentially allocate resources to the conserva-

tion of somatic tissue, in detriment of shell integrity.

Feeding and digestive behavior

In general, seawater acidification had a negative effect on

the feeding and digestive behavior of the juvenile bivalves,

decreasing clearance and IR (Ruditapes decussatus) in

acidified treatments, relative to controls. In contrast, a

distinct response was observed for the mussel M. gallo-

provincialis in Portugal, with the pH reductions tested

having no effect the feeding behavior (clearance and

ingestion rates) and actually increasing the absorption rate

and SFG (Table 2). Again, the longer period of exposure

used in Italy (202 days) may have contributed to exacer-

bate the differences in the response of M. galloprovincialis

between the two locations. Nevertheless, the results of

feeding behavior are in agreement with the patterns

observed for survival, growth, and shell integrity, sug-

gesting that juvenile M. galloprovincialis have a high tol-

erance to CO2-induced acidification, at least in highly

alkaline coastal waters, such as the Ria Formosa lagoon.

Metabolism

Excretion of ammonia consistently increased under long-

term exposure to CO2-induced seawater acidification, for

all species and locations (Table 2). This type of response

has previously been observed in similar experiments

(Michaelidis et al. 2005; Thomsen and Melzner 2010),

although Liu and He (2012) recently reported an opposite

pattern in three species of bivalve from south China. In

contrast, respiration rates were either unaffected (C. gallina

and M. galloprovincialis) or decreased (R. decussatus) with

pH. This interspecific variability in the respiration rates of

bivalves under high CO2 has previously been observed in

other studies (Thomsen and Melzner 2010; Liu and He

2012). The ratio of oxygen consumed to nitrogen excreted

(O:N) was unaffected for the mussels, but significantly

Impacts of CO2-induced seawater

123

decreased with pH in both species of clams (Table 2).

Furthermore, the physiological responses of juvenile mus-

sels suggest a re-orientation of their metabolism from

aerobic to anaerobic mode under acidified conditions.

According to Montecinos et al. (2009), this type of

mechanism constitutes an adaptation typical of intertidal

organisms, allowing them to maintain homeostasis during

tidal cycles.

Biochemical composition

Both species studied in Italy had their protein content

significantly reduced by seawater acidification, while in

Portugal, only the larger size classes of the clam

R. decussatus were affected and not in all the sampling

periods (Table 2). This pattern is consistent with the

hypothesis of increased protein catabolism, indicated by

the smaller values of the O:N ratio observed in both species

of clams. The O:N index denotes the proportion of protein in

relation to lipids or carbohydrates catabolized for metabolic

energy requirements. Accordingly, a fast rate of protein

catabolism, relative to lipids or carbohydrates, is expressed

by a small value and is generally indicative of a stressed

condition (Widdows 1985). More recently, Thomsen and

Melzner (2010) suggested that enhanced protein metabolism

with increasing seawater pCO2 may contribute to intracel-

lular pH regulation. Again, the response of the common

species (M. galloprovincialis) differed between locations,

indicating that there were other variables interacting or

mediating the effects of seawater acidification by CO2 and

causing the effects to be site-specific. Nevertheless, the

absence of any significant effect on the biochemical com-

position of juvenile mussels in Portugal corroborates the

relative tolerance of this species to OA, at least in the

specific conditions of the Ria Formosa lagoon.

Indices of condition

The indices used to assess the physiological condition

of the juvenile bivalves seemed to corroborate the gen-

eral pattern of interspecific variability. The mussel

M. galloprovincialis was either unaffected (Italy) or

showed a nonlinear response to CO2-induced seawater

acidification (Portugal). In contrast, with the exception of

somatic weight/shell weight for R. decussatus, which was

unaffected, both species of clams generally had signifi-

cantly smaller values for the indices in acidified treatments,

relative to controls (somatic weight/shell length for

C. gallina and RNA/DNA for R. decussatus, Table 2).

According to Kroeker et al. (2010), these variations in

sensitivity among organism have important implications

for ecosystem responses. Widdicombe et al. (2011) pro-

posed that organisms living in sediments (infauna), an

environment that is frequently high in CO2, would be

inherently more tolerant to OA than organisms that live on

the sediment surface (epifauna) or in open-water. Overall,

this hypothesis is not supported by our results, since

infaunal species (the clams C. gallina and R. decussatus)

generally showed a more detrimental response to long-term

acidification than the mussel M. galloprovincialis, which is

an epifaunal organism. In order to avoid any experimental

artifacts caused by different substrates, no sediment was

used in the exposure tanks, which constitutes a common

approach in this type of CO2 forcing experiments. Never-

theless, given that the habitat conditions were unnatural for

clams, a greater level of confidence must be assigned to the

results obtained for mussels. This should be considered

when attempting to extrapolate our results to natural

populations.

Integrated effects of temperature, salinity, and pCO2

on adult bivalves

The combined effects of pH, temperature, and salinity on

the immune parameters of bivalves were evaluated for the

first time in the present study. Results showed particular

modulation patterns depending on the species and the

biological response considered.

Total hemocyte count (THC) varied markedly among

experimental conditions (Table 3). In bivalves, increases in

the THC values are generally considered as a consequence

of proliferation or movement of cells from tissues into

hemolymph, whereas decreases are likely due to cell lysis

or increased movement of cells from hemolymph to tissues

(Pipe and Coles 1995). When compared with controls (8.1

pH, 22 �C), a different pattern of THC variation was

observed in the two species considered (C. gallina and

M. galloprovincialis) for both increasing temperature and

decreasing pH at each salinity tested.

Table 3 Pattern of variation in total hemocyte count (THC) and

hemocyte neutral red (NR) uptake with increasing temperature

(22–28 �C) and decreasing pH (8.1–7.4) compared to controls (8.1

pH—22 �C), at different levels of salinity; positive (%), negative

(!), and nonlinear (\) effects

Salinity Type of response Mytilus

galloprovincialis

Chamelea gallina

Temperature pH Temperature pH

28 THC \ ! !NR uptake \ \

34 THC ! ! \NR uptake ! !

40 THC \ ! ! \NR uptake \ \ ! !

P. Range et al.

123

It is interesting to note that at salinity 34, THC generally

decreased in mussels and increased in clams at low pH and

high temperature values. At the two extreme salinities,

increased temperature and reduced pH differently affected

THC response of mussels, whereas they generally

decreased that of clams.

Differences in the degree of NR dye uptake by hemo-

cytes may reflect alterations to cell membranes (including

lysosomal membranes) and/or weakening of hemocyte

pinocytotic capability (Coles et al. 1995; Hauton et al.

1998). In clams, NR uptake was affected significantly by

pH, temperature, and their interaction at all salinities tested

(Table 3). Results obtained demonstrate that exposure of

C. gallina to reduced pH and increased temperature

resulted in a different pattern of variation in NR uptake

between clams kept at the two extreme salinities (28 and

40). In mussels, NR uptake was influenced significantly by

the pH/temperature interaction at salinity 28 and by pH and

the pH/temperature interaction at salinities 34 and 40

(Table 3). Overall, results obtained suggest that effects of

the tested experimental conditions on NR uptake were

more obvious at salinities 34 and 40, even if variation

patterns were different.

These results and those from other immunomarkers

(Matozzo et al. 2012) highlight the different immuno-

modulation mechanisms in the two species, as well as

different susceptibility to the stress conditions applied. In

bivalves, the immune system is relatively simple and its

functioning is based on both humoral and cell-mediated

responses. Circulating hemocytes play an essential role in

phagocytizing pathogens and foreign materials, as well as

in the production of hydrolytic and oxidative enzymes,

reactive oxygen species (ROS), reactive nitrogen interme-

diates, and antimicrobial peptides (Hine 1999; Cima et al.

2000; Galloway and Depledge 2001). Since climate chan-

ges can alter the geographic distribution of parasitic dis-

eases, increase abundance of parasite populations, and

promote pathogen transmission (Poulin 2006; Morgan and

Wall 2009; Martin et al. 2010), evidences of impairment in

bivalve immune defense, such as alterations in number and

functionality of circulating hemocytes, arise particular

concern.

Conclusions

The present study has shown large variations in the sen-

sitivities of bivalves to climatic changes, among different

species and between local populations of the same species.

In the lagoon of Venice (North Adriatic, Italy), the long-

term exposure (202 days) of juvenile bivalves to a pH

reduction of 0.7 U caused severe shell damage and

increased mortality of the clam C. gallina and the mussel

M. galloprovincialis and strongly reduced growth of the

former species. No such effects were apparent in the

Ria Formosa lagoon (Gulf of Cadiz, Portugal), where

M. galloprovincialis and the clam R. decussatus were able

to survive and grow normally, when exposed to similar

perturbations, although during shorter periods (84 and

75 days, respectively). Local populations of the shared

species M. galloprovincialis also differed markedly in their

responses to seawater acidification by CO2. This clearly

indicates that there were other interacting or mediating

factors, causing the effects to be locally variable and

emphasizing the danger of extrapolating results from one

region to another, even for the same species or process

(CIESM 2008). The elevated alkalinity of seawater from

the Rıa Formosa probably buffered the impacts on the

carbonate supply of calcification and growth and may

contribute to offset OA at the local scale. Multiple drivers

affect seawater carbonate chemistry in nearshore coastal

habitats, increasing its variability relative to the open

ocean. Accordingly, these coastal areas should be further

investigated to assess the generality and underlying

mechanisms determining the sensitivity of marine organ-

isms to OA and other anthropogenic perturbations of

marine pH (Duarte et al. 2013).

In Italy, exposure to elevated CO2 and reduced pH

caused alterations in immune parameters in adult mussels

and clams, particularly when maintained at extreme tem-

perature and salinity values. Similarly to juveniles, adults

of the two species showed different susceptibility to the

stress conditions applied, with the clam C. gallina being

less tolerant to environmental changes than the mussel

M. galloprovincialis. This is a clear indication that climate-

related variables can have synergistic effects, at least on

some species of bivalves. Accordingly, in order to increase

our predictive capacity about the ecological consequences

of global change, local stressors should also be investi-

gated, in an intergraded way.

Heavy fishing effort, repeated mortality events, and

recruitment failures have inflicted dramatic decreases in

population densities of C. gallina and R. decussatus in

recent years. The distinct biochemical, cellular, and

organismal responses measured in this study indicated that

climatic change may constitute an additional threat to these

clams, by further reducing the abundance of natural pop-

ulations and increasing the risk of local extinction. Given

that habitat conditions during our experiments were more

favorable to epifaunal than to infaunal organisms, greater

confidence must be assigned to the expectations of impacts

relative to mussels than to clams. Accordingly, despite the

relative tolerance exhibited by M. galloprovincialis,

changes in distribution and abundance of this species

cannot be excluded, under more extreme scenarios of cli-

mate change. Considering the economic relevance of these

Impacts of CO2-induced seawater

123

species in the Mediterranean region, the potential for sig-

nificant socioeconomic impact is also clear.

The long-term strategy to limit the impacts of climate

change on the marine environment, by imposing strong

reductions of greenhouse gas emissions, is clearly not

under the governance of local managers. Coastal marine

ecosystems are, however, subjected to multiple other

stresses, including overfishing, pollution, and loss of hab-

itat. In the current state of our knowledge, the most

effective approach to improve the resilience of these eco-

systems to climate change is probably to adopt measures to

limit these other stresses. Another important issue is the

adaptation in human activities that depend on this changing

marine environment (Caldeira 2010). In this respect, the

results of the present study emphasize the need for species-

specific and locally adapted measures for sustaining the

socioeconomic roles of bivalves in coastal Mediterranean

areas, under future scenarios of climatic change. Some of

the aspects that should be addressed, for each species and

across multiple geographic regions, are: anthropogenic

impacts on water quality (including pH and alkalinity),

design and operation of fishing gears, larval rearing and

extensive cultivation techniques.

Acknowledgments This is a contribution of the ACIDBIV project,

which is part of the CIRCLE Med network. Funding was provided by

the Foundation for Science and Technology (FCT) of Portugal (ERA-

CIRCLE/0004/2007), the Regional Ministry of Innovation and Industry

of the Galician Government, and the Italian Ministry for Environment,

Land and Sea, in the framework of Circle ERA Net project (which is

funded by the European Commission 6th Framework Programme). PR

was also supported by a post-doctoral grant from FCT (SFRH/BPD/

69959/2010). The authors would like to acknowledge the staff of the

Bivalve Production Group at IPMA-Tavira for their continuous support.

Comments by the editors of this special issue and two anonymous

referees substantially improved the original manuscript.

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