Evaluation of the threat of marine CO2 leakage-associated acidification on the toxicity of sediment metals to juvenile bivalves

Aquatic Toxicology 166 (2015) 63–71

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Evaluation of the threat of marine CO2 leakage-associated acidification on the toxicity of sediment metals to juvenile bivalves M. Dolores Basallote a,∗,1 , Araceli Rodríguez-Romero b , Manoela R. De Orte a,1 , T. Ángel Del Valls a , Inmaculada Riba a a Cátedra UNESCO/UNITWIN WiCop, Departamento de Química-Física, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Polígono Río San Pedro s/n, 11510 Puerto Real, Cádiz, Spain b Departamento de Ecología y Gestión Costera, Instituto de Ciencias Marinas de Andalucía (CSIC), Campus Río San Pedro, 11510 Puerto Real, Cádiz, Spain

a r t i c l e

i n f o

Article history: Received 11 November 2014 Received in revised form 12 June 2015 Accepted 8 July 2015 Available online 17 July 2015 Keywords: CO2 leakage R. philippinarum Sediment elutriate Acute effects Metal toxicity

a b s t r a c t The effects of the acidification associated with CO2 leakage from sub-seabed geological storage was studied by the evaluation of the short-term effects of CO2 -induced acidification on juveniles of the bivalve Ruditapes philippinarum. Laboratory scale experiments were performed using a CO2 -bubbling system designed to conduct ecotoxicological assays. The organisms were exposed for 10 days to elutriates of sediments collected in different littoral areas that were subjected to various pH treatments (pH 7.1, 6.6, 6.1). The acute pH-associated effects on the bivalves were observed, and the dissolved metals in the elutriates were measured. The median toxic effect pH was calculated, which ranged from 6.33 to 6.45. The amount of dissolved Zn in the sediment elutriates increased in parallel with the pH reductions and was correlated with the proton concentrations. The pH, the pCO2 and the dissolved metal concentrations (Zn and Fe) were linked with the mortality of the exposed bivalves. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The storage of CO2 in sub-seabed geological formations to reduce the anthropogenic CO2 atmospheric emissions has been legally allowed since 2007 (LondonProtocol, 2007; OSPAR, 2007; Reguera et al., 2009). Globally, almost forty million tons of CO2 have been captured per annum from the 21 “active” large scale Carbon Capture and Storage (CCS) projects (GlobalCCSInstitute, 2014). Nevertheless, it has been estimated that 0.2% of the total gas storage would escape, which is the equivalent of a hundred thousand tons of CO2 per year if a storage capacity approximately 100 gt of CO2 is considered (Bellerby and Golmen, 2013). The escape of CO2 has been proposed from two main sources: the transport facilities and the storage areas (Leung et al., 2014). Abandoned wells, geological discontinuities or operational accidents have been described as such mechanisms that facilitate leakage (Blackford et al., 2014). The release of CO2 through the sediments to the water column will cause a reduction of

∗ Corresponding author. Tel.: +34 956016040; fax: +34 956016040. E-mail address: [email protected] (M.D. Basallote). 1 Present address: Instituto do Mar, Campus Baixada Santista, Universidade Federal de São Paulo, Av. Alm. Sandanha da Gama, 89-Ponta da Praia/SP CEP, 11030-400, Santos, SP, Brazil. http://dx.doi.org/10.1016/j.aquatox.2015.07.004 0166-445X/© 2015 Elsevier B.V. All rights reserved.

the pH value. Factors, such as the leakage rate, the sediment buffering capacity, and the transport and dispersions processes would determine the effects on a marine ecosystem exposed to the potential leaks. Recent studies have postulated that marine organisms could tolerate or adapt to few tenths of units of reduction of ocean seawater pH (up to 0.5) (Hofmann and Todgham, 2010; Melzner et al., 2009; Pistevos et al., 2011; Sunday et al., 2011), including the changes expected for the near-future ocean acidification (OA) due to the oceans’ CO2 capture from the atmosphere (Caldeira and Wickett, 2005). However, a sharp and sudden decrease in pH because of a possible CO2 leakage would not permit the time necessary for the marine biota to acclimate (Basallote et al., 2012). Furthermore, the responses to increases in the dissolved CO2 are species-specific (Basallote et al., 2014; Byrne et al., 2010; Halsband and Kurihara, 2013; Ishimatsu et al., 2005). The chemical characteristics of the area, such as elevated alkalinity values, could contribute to buffer increased acid release (Range et al., 2011). Thus, the responses of organisms to pCO2 vary within and between populations (Parker et al., 2011). For example, species living in waters near hydrothermal vents will show an ability to buffer ion exchanges or CO2 transport (Seibel and Walsh, 2001). However, estuarine and marine organism during embryonic and larval stages are expected to be more susceptible to environmental changes than mature organisms (Pörtner, 2008).

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Human actions, such as fossil fuel combustion and industrial activity cause a continuous influx of pollutants into marine ecosystems. Metals are among the most common contaminants in both water and sediments (Doney et al., 2009; Zeng et al., 2015). The pH is one of the main environmental factors that determine the organic and inorganic speciation of the metals present in marine ecosystems (Millero et al., 2009). Recent studies have postulated that the acidification processes would affect the stability of metals trapped on marine sediments (Ardelan et al., 2009; De Orte et al., 2014b; Payan et al., 2012; Roberts et al., 2013), leading to increases in the concentrations of metals dissolved in the overlying water (Basallote et al., 2014; De Orte et al., 2014a), which could increase the toxic effects on the surrounding biota. The sediment quality is expected to be affected by pH decreases, leading to increases in the toxicity of sediments that would not produce toxic effects at the natural environmental pH. Bivalve mollusks are among the species most susceptible to ocean acidification, and these organisms are widely used in laboratory experiments. The bivalve Ruditapes philippinarum has been identified as a suitable organism to study toxicity in coastal systems (Casado-Martínez et al., 2006a; Riba et al., 2003). This species has a wide geographical distribution, and the organisms are easily manipulated under laboratory conditions (Rodríguez-Romero et al., 2014b). The effects of OA on bivalve mollusks have been widely studied, and negative effects on the morphology, physiology, growth, and behavior of bivalves associated with pH decrease have been demonstrated (Gazeau et al., 2013; Ishimatsu and Dissanayake, 2010). Moreover, juveniles and small bivalves seem to be more susceptible than higher size classes to the dissolutioninduced mortality associated with CO2 acidification (Green et al., 2004). Previous studies have postulated increased mortality of bivalves exposed to elevated pCO2 , comparable to the levels expected to occur due to CO2 leakages (>1500 ␮atm) (Basallote et al., 2012; Rodríguez-Romero et al., 2014b). This study attempted to identify the short-term effects of CO2 induced acidification on the juvenile bivalves R. philippinarum by performing laboratory scale experiments with a CO2 -bubbling system designed to conduct ecotoxicological assays. The organisms were exposed for 10 days to sediment elutriates subjected to various pH treatments. The dissolved metal concentrations in the sediment elutriates were measured to assess possible interactions among the pH, dissolved metals and toxicity. The exposures were selected according to several scenarios related to CO2 leakages from sub-seabed storage formations. To this end, the expected local pH decrease of more than 0.5 pH units as a consequence of CO2 escape from its storage site was compared to the present-day pH of a coastal estuarine area. Because there is little information about the actual CO2 leaks from commercial CCS projects, the magnitude of the potential leaks are unpredictable (Dewar et al., 2013). Thus, the lowest pH treatments presented in this work were selected based on the pH measured in natural CO2 vents, such as the natural CO2 vent recently discovered above a natural salt dome in the Southern North Sea (pH 6.8 ± 0.2) (McGinnis et al., 2011), the Volcanic Ischia Island in Italy (lowest pH registered 6.57) (Hall-Spencer et al., 2008), or the submarine volcanic eruption of El Hierro, at the Canary Island (pH < 6.0) (Santana-Casiano et al., 2013).

2. Material and methods 2.1. Sampling Sediment samples were collected from two different littoral areas in the Gulf of Cádiz at the southwestern part of the Iberian Peninsula (see Supplementary information (SI), Fig. S1). The Río San Pedro (RSP) sediment was collected from the Bay of Cádiz,

which is a relatively protected area connected to the Atlantic Ocean through intertidal channels and salt marshes. The sampling site was a shallow, tidally controlled creek area, mainly influenced by marine aquaculture, shipbuilding industry, and urban discharges among other anthropogenic activities (DelValls et al., 1998; Ligero et al., 2002; Silva et al., 2012). The other sediment samples, Mazagón (MAZ) and Muelle de Levante (ML), were collected from the Ría of Huelva, which is known as one of the most metal-contaminated estuaries in the world (Sainz et al., 2004). The Ría of Huelva is a complex system of drainage streams (tidally controlled) that receives inputs from the Odiel and Tinto Rivers, which are affected by chronic metal contamination within their basins due to mining activities since the beginning of human occupation. Additionally, this area is highly affected by industrial activity and urban sewage discharges (Blasco et al., 2010; Riba et al., 2003). The test sediments were selected on the basis of the best available information to represent presumably low and high levels of metal contamination in the sediments. The RSP sediment was considered to represent sediment from a relatively unpolluted reference site (Casado-Martínez et al., 2006b; Riba et al., 2010). Surface sediments (the top layer from 0 to 10 cm depth) were collected and transported to the laboratory (Marine and Environmental Science Faculty, University of Cádiz) in 25 L acid-washed polypropylene containers. The containers were hermetically closed and stored at 4 ◦ C in darkness for no longer than 15 days until the toxicity tests were performed. The sediments were sub-sampled for physical and chemical characterization. For the grain size distribution measurements, duplicate portions of approximately 100 g with their water content were separated. More than 6 g of sieved wet sediment were sub-sampled in triplicate for subsequent determination of organic matter (OM). For the metal and organic carbon (TOC) assessments, sediment samples were collected and frozen prior to the sample drying procedure. The seawater used in the experiments (RSPsw ) was collected from the water surface (1 m depth) during high tide at the same site as the RSP sediment. After collection, the seawater was transported to the laboratory and kept in a 400-L tank. Once in the tank, it was continuously filtered through a high-power external filter TM (CristalProfi-e900) containing nitrate-removal stones (Denitrate , Seachem). The seawater (0.45 ␮m filtered) was used for preparing the sediment elutriates. 2.2. Elutriate preparation and experimental set-up A laboratory-scale CO2 injection system (Basallote et al., 2012; De Orte et al., 2014b) was adapted to work with sediment elutriates (2 L test chambers), employing a range of pH treatments. Modification of the USEPA method (1998) was used for the sediment elutriation procedure. Thus, the sediment elutriates were obtained by mixing (60 rpm for 30 min) the sediment and filtered seawater (1:4 v/v). Before agitation, the sediment–seawater mixture was acidified by the injection of CO2 bubbles until the target pH was reached. The “AT Control System” from Aqua Medic GmbH (Bissendorf, Germany) was used to manipulate and control the pH in each test chamber independently. This system monitors and controls the pH in each test vessel using electrodes (NBS scale) placed inside the chamber and connected to the computer system. The pH is regulated by adding CO2 gas through a solenoid valve that opens when the pH increases 0.01 U above the predetermined pH values and closes after the target pH is reached. After agitation, the containers were sealed and the mixtures were left to settle in darkness overnight. The remaining supernatant liquid was carefully siphoned off in a new clean test vessel without disturbing the settled material. Various pH treatments ranging from 8.0 to 6.0 (two replicates per pH) were selected for each of the sediment samples. Four pH treatments were selected to compare the

M.D. Basallote et al. / Aquatic Toxicology 166 (2015) 63–71

reduced pH treatments based on the predicted pH reduction due to CO2 leaks (nominally pH 7.1, 6.6, 6.1) (SI Table SI) with a control un-manipulated pH sediment elutriate (nominally pH 8.0).

2.3. Toxicity testing Juveniles of R. philippinarum (weight 0.21 ± 0.04 g, major axis 10.37 ± 1.05 mm) were obtained from an aquaculture farm (Haliotis S.L., Cádiz, Spain) and kept in filtered clean seawater (pH 7.9 ± 0.1; salinity (S) 34; dissolved oxygen (DO) >81%; temperature (T) 18 ± 1 ◦ C) to acclimate to the laboratory conditions for 15 days. The clams were fed with a mixture of marine microalgae (Tetraselmis chuii, Isochrysis galbana, and Chaetoceros gracilis). The organisms (n = 80) were exposed to the pH-treated sediment elutriates in duplicate for 10 days. The water was continuously aerated to maintain normoxic conditions (䊐60%). To avoid a sudden change in the pH, the elutriate pH were gradually reduced by 0.5 pH unit per day during the exposures of the organisms. No food was provided to the organisms during the exposure period. The mortality was evaluated during the exposure time (Riba et al., 2004, 2010). The percent survival at each exposure pH treatment was selected as endpoint.

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2.5. Statistical analysis The mortality was calculated based on survival percentages measured after the exposure time. The variation in mortality rates between pH treatments was examined using a Kruskal Wallis test because a normal distribution and homogeneity of variances was not achieved. The statistical software SPSS 15.0 was used to determine significant differences (p < 0.05) among the different pH treatments. Thus, for the sediment elutriates tested, significant differences between the pH 7.1, 6.6, and 6.1 and the sediment elutriates at their environmental pHs, not manipulated by CO2 injection (nominal pH 8.0), were studied. The Spearman rank correlation method was used to determine the correlation between the sediment elutriate toxicity and pH as well as between the dissolved metals and the pH. The mortality data were used to calculate the median effect pH (LpH50 ), which was defined as the pH that causes lethal effects in 50% of the population exposed. This parameter was estimated using the pH that caused mortality in 50% of the population exposed to the different pH treatments and was calculated by the linear interpolation method for lethal toxicity. A factor analysis was conducted to explain the relationship observed between the initial set of variables and a smaller number of factors using principal component analysis (PCA) performed with the SPSS 15.0 software.

2.4. Analytical procedure

3. Results

The analytical procedure has been previously reported by Basallote et al. (2014). Briefly, the grain size distribution was determined according to ASTMD422-63 (2007) and Gee and Or (2002). The sediment textural classification was performed as described by Flemming (2000), using the percentage of sand and fines. TOC was measured using the technique described by Gaudette et al. (1974), as modified by El-Rayis (1985). OM in the sediment was determined through loss of ignition (LOI) in a muffle furnace at 450 ◦ C. The metal concentrations (Fe, Cr, Cu, Ni, Co, Zn, Pb, Cd, and As) in the sediment were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS) (Thermo Elemental X7 Series, Thermo Electron, Winsford, England) after microwave digestion (Speedwave of Berghof). The dissolved metal concentrations in the sediment elutriates were measured in filtered (0.45 ␮m) subsamples acidified to pH < 2 with ultrapure HNO3 at the beginning (day 1) and at the end (day 10) of the exposure time. The concentrations of metals were determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Thermo Elemental Series-X). Polyatomic salt water interferences were controlled using the coalition-reaction cell. Elutriate subsamples were collected (50 mL) on days 1, 5, and 10 for measurements of total alkalinity (TA). The samples were collected avoiding any head space and preserved in darkness for no longer than 24 h. The TA was measured by an automatic titration method (Mettler Toledo, T50) using a combined glass electrode (Mettler Toledo, DGi115-SC) calibrated on the NBS scale. The AT control system measured pH, and the TA values were used to calculate the carbonate system speciation using the CO2SYS program (Pierrot et al., 2006), with the dissociation constant from Mehrbach et al., (1973), refitted by Dickson and Millero (1987). During the experimental period, T (18 ± 1 ◦ C), S (34 ± 1) and DO (>60%) were monitored and controlled daily. The pHNBS recorded by the AT control system in each aquarium was verified regularly against the values obtained using a portable pH meter (model: Phenomenal 1000H; accuracy ±0.005 pH) calibrated using pH buffer solutions of 4.00 and 7.00. The oxygen saturation was recorded using an Oxy 4000H meter (accuracy ±0.5% of the measured value), and salinity was measured using a conductivity EC 30 m (VWR, accuracy ±1% of the measured value).

3.1. Sediment and seawater physicochemical parameters 3.1.1. Sediment characterization The proportions of TOC and OM were similar for the RSP and MAZ samples. Slightly higher TOC and OM values were observed for the ML sediment site compared with the other sediments (Table 1). Likewise, the percentage of fine particles (<0.63 ␮m) and of sand (>0.63 ␮m) were similar for RSP and MAZ, and both sediments were classified as muddy sand. ML was classified as sandy mud according to the USDA classification. As expected, the sediment samples from the Ría of Huelva (ML and MAZ) contained higher metal concentrations than the sediment from the Bay of Cádiz (RSP). 3.1.2. Water chemistry The RSPsw that was used to prepare the sediment elutriates (pH 7.9 ± 0.1, S 34 ± 1, alkalinity 2709.52 ␮M) exhibited concentrations below the detection limit of the equipment for all metals with the exception of Fe, which showed a concentration of 4.97 ␮g L−1 . The mean values for the carbonate system speciation in the sediment elutriates were calculated based on the pHNBS registered by the AT control system, T (25 ◦ C), S (34) and TA (SI Table S1). The non-CO2 -manipulated elutriates (pH 8.0) showed partial pressures of CO2 (pCO2 ) ranging between 365.28 ± 16.40 ␮atm for the RSPelut and 442.88 ± 0.79 ␮atm for the MLelut . As expected, the highest pCO2 was observed at the lowest pH treatments, but the differences among the concentrations were suggested to be related to the different sediment buffer capacities. 3.2. Toxicity test The mortality of the bivalves increased with the reduction in the elutriate pH values (Fig. 1). After 10 days of exposure, similar survival rates (95–100%) were recorded for the control pH, the 7.1 pH treatment, and the 6.6 pH treatment for the three studied sediments. Significant differences (p < 0.05) in the survival rate were observed at the lowest pH treatment (6.1) relative to the control (pH 8.0) for all the sediment elutriates. The bivalve mortality values after 10 days of exposure were used to calculate the LpH50

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Table 1 The summarized results of the physicochemical parameters of the sediments from the Río San Pedro (RSP), Mazagón (MAZ), and Muelle de Levante (ML) sites used in the experiments, including the percentage of sand (0.063–2 mm), percentage of fine particles; fines (<0.063 mm), organic carbon content (TOC), and organic matter (OM). The AL1 and AL2 refer to the Spanish Action levels for dredged material management (CEDEX, 1994). RSP Sand (%) Fines (%) TOC (%) OM (%) Fe As Pb Cd Cr Cu Ni Zn Co  Metals Natural pH

MAZ

56.12 43.89 1.06 5.97 15,239.00 7.14 22.3 0.12 72.7 18 14 49 5.8 15,428.07 7.3 (±0.2)

53.13 46.87 1.34 7.47 42,436.00 121 229 0.54 58.1 754 26.4 872 18.5 44,515.54 7.3 (±0.1)

120

Mortality %

100

ML 33.57 66.43 2.21 8.15 60,591.00 203 253 1.11 89 981 31 1023 17.2 63,189.31 6.8 (±0.2)

AL1 – – – – – 80 120 1 200 100 100 500 – – –

AL2 – – – – – 200 600 5 1000 400 400 3000 – – –

* **

RSP MAZ ML

80 60 40 20 0 8.0

7.1

6.6

6.1

pH Fig. 1. Averages and standard deviations of the R. philippinarum mortality percentages after 10 days of exposure to the sediment elutriates (RSP, MAZ, and ML) subjected to various pH treatments. The pH 8.0 refers to the control treatment, i.e., the sediment elutriate at the natural pH without any CO2 manipulation. The asterisks indicate significant differences (*p < 0.05) between the sediment elutriate at the different pH treatments (6.1, 6.6, 7.1) and the control pH treatment (8.0). The absence of a bar for the pH treatments pH 8.0 (RSP, ML), pH 7.1 (MAZ), and pH 6.6 (MAZ, ML) indicates that no mortality (0%) was observed for the exposure to that elutriate.

(Fig. 2). Thus, the calculated pH values that caused 50% mortality of the exposed populations were 6.33, 6.45, and 6.43 for the RSPelut , MAZ elutriate (MAZelut ), and ML elutriate (MLelut ), respectively. All of the studied sediments exhibited mortality rates that significantly correlated with the pH (SI Table S2). 3.3. Metal behavior The results for dissolved Fe, Zn, As, and Pb in the sediment elutriates subjected to the various pH treatments for experimental days 1 and 10 are plotted in Fig. 3. Among the dissolved elements measured, only Fe, Zn, As, and Pb exhibited detectable concentrations in the sediment elutriates. Differences in the dissolved metal concentrations were observed between the beginning (day 1) and the end (day 10) of the experiments. The dissolved Fe concentration decreased as the pH was reduced for the day 1 for MAZelut and MLelut samples. However, the concentration of this metal increased as the pH decreased on day 1 for the RSPelut sample. For the three sediments tested, the concentration of the dissolved Fe was increased at the lowest pH treatment on day 10 of the experiment. The dissolved concentration of the metalloid As for the three sediments tested on day 1 was reduced as the pH was reduced. Although the As concentrations at pH 8.0 (control treatments) were different depending on the station (RSPelut 6026 ␮g L−1 ; MAZelut

Fig. 2. Mean LpH50 values calculated for the juvenile R. philippinarum organisms exposed to elutriates that were prepared with sediment from three different littoral sites (RSP, MAZ, and ML) and were adjusted to several pH values by CO2 injection.

14,635 ␮g L−1 ; and MLelut 10,225 ␮g L−1 ), the As levels were similar in the 3 studied sediments for the pH 6.1 condition on day 1 of experiment (ranging between 4.7 and 7.5 ␮g L−1 ). On day 10, decreases in the concentrations of dissolved As as a function of the pH reduction were also measured in the RSP and MAZ sediment elutriates. However, for the MLelut , a higher concentration of dissolved As was measured at pH 6.1 compared with the pH 7.1 and 6.6 treatments. Regarding the dissolved Zn, increases asso-

M.D. Basallote et al. / Aquatic Toxicology 166 (2015) 63–71 60

RSP Fe

day 1 day 10

50

60

Fe d1 Fe d10

µg L-1

30

40

30

30

20

20

20

10

10

10

0

0 8.0

7.1

6.6

0

8.0

6.1

7.1

6.6

6.1

8.0

7.1

6.6

pH

pH 10

day 1 day 10

ML Fe

50

40

40 µg L-1

MAZ Fe

50

µg L-1

60

67

RSP As

20

day 1 day 10

8

6.1

pH 16 day 1 day 10 14

MAZ As

ML As

As d1 As d10

15

4

µg L-1

µg L-1

µg L-1

12 6

10

10 8

5 2

6 0

0 8.0

7.1

6.6

4

6.1

8.0

7.1

60 day 1 day 10 50

RSP Zn

100

MAZ Zn

day 1 day 10

40

6.6

6.1

30

ML Zn

day 1 day 10

80 60 40

20 20

10 0 8.0

7.1

6.6

0 8.0

6.1

7.1

6.6

8.0

6.1

7.1

0,5

RSP Pb

day 1 day 10

2,0

6.6

6.1

pH

pH

pH 2,5

7.1 pH

µg L-1

18 16 14 12 10 8 6 4 2 0 -2 -4

8.0

6.1

pH

µg L-1

µg L-1

pH

6.6

1,4

MAZ Pb

day 1 day 10

0,4

ML Pb

day 1 day 10

1,2

1,0

0,3

µg L-1

µg L-1

µg L-1

1,0 1,5

0,2

0,8 0,6 0,4

0,5

0,1

0,0

0,0

0,2 Control

7.1

6.6

6.1

0,0 Control

7.1

pH

6.6 pH

6.1

Control

7.1

6.6

6.1

pH

Fig. 3. Dissolved concentrations of Zn, Fe, As, and Pb on day 1 and 10 of the experiment for each sediment elutriate pH treatments from the three study sites (RSP, MAZ, and ML). The error bars represent the standard error for duplicate treatments.

ciated with the decreased pH were found for all of the studied sediments. In addition, the highest Zn concentrations were measured on day 10 at the lowest pH level (6.1) for all elutriate types. For lead, no specific pattern with respect to the pH decrease was observed. Although the RSP and MAZ samples demonstrated an increase in the Pb concentration at the intermediate pHs (7.1, 6.6) on day 1 of the experiment, a reduction in concentration between pH 6.6 and 6.1 is observed. For the ML site, the Pb concentration decreased with the reduced pHs. On the last day of the experiment, the Pb concentrations showed slight variations with pH for all of the sediments tested, with values under 0.6 ␮g L−1 . Among the metals measured, changes in the dissolved Zn and As among the pH treatments were relatively more pronounced in sediment elutriates from MAZ and ML sites.

Significant correlations were observed between the pH and the dissolved Zn concentrations on days 1 and 10 of the experiment for all the sediments tested and for As for the RSP and MAZ sediment samples (SI Table S2). For the ML sediment sample, the As was correlated with pH only on day 1 of the exposure time. On day 1 of the experiment, the Fe and Pb were also correlated with the pH as well as on day 10 for the RSP sample. A principal component analysis (PCA) was performed with 11 variables including: elutriate pH and pCO2 , bivalve biological responses (mortality) and dissolved metal concentrations (Fe, Zn, As, and Pb) on days 1 and 10 of the exposure time to link the metals, pH and lethal responses (Table 2). The analysis indicated that the original variables could be represented by 4 components that explain almost 90% of the total variance of the data set. The first principal factor linked the pH and pCO2 with the mortality as well

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Table 2 Sorted rotated component matrix with component loadings of the total variables on the 5 principal components. Only loadings 䊐0.3 are shown in the table. Variables

R. philippinarum mortality pCO2 pH Dissolved Fe day 1 Dissolved Fe day 10 Dissolved Zn day 1 Dissolved Zn day 10 Dissolved As day 10 Dissolved As day 1 Dissolved Pb day 1 Dissolved Pb day 10

Components 1 (31%)

2 (26%)

3 (21%)

4 (12%)

0.901 0.864 −0.594 – 0.96 0.355 0.522 – – – –

– – 0.563 0.91 – −0.817 −0.756 – – 0.34 0.45

– −0.325 0.441 – – – – 0.972 0.939 – –

– – – – – – – – – 0.845 −0.692

as the dissolved metals Fe and Zn on day 10 of the experiment and the Zn on day 1. Factor 2 combines the pH and the metals Zn and Pb at the beginning and at the end of the exposure time and the Fe on day 1. Factor 3 links the dissolved metal As with the pH and the pCO2 . Finally, factor 4 links the dissolved Pb on days 1 and 10 of the experiment. The represented weights for the factors that define the sediment elutriate pH treatments (SI Fig. S2) show that factor 1, which links the pH, pCO2 , mortality of the clams, and the dissolved metal (Fe on day 10 and Zn on days 1 and 10), is relevant only at pH 6.1 for the three sediments studied. 4. Discussion Potential CO2 leakage could mobilize and re-suspend high quantities of sediments. Hence, tests of elutriates are further justified in the context of CO2 escape and could mimic sediment re-suspension scenarios as a consequence of flows of CO2 through the sediment during leak events (Payan et al., 2012). To the best of our knowledge, accidental CO2 leakage from storage sites in sub-seabed formation has not been reported. Controlled CO2 leaks have been experimentally applied to simulate the behavior of CO2 plumes under escape events (Blackford et al., 2014; Blackford and Kita, 2013; Denchik et al., 2014; Jones et al., 2014). The shallow CO2 injection experiment (1.67 t of CO2 , 6 days duration) conducted at Svelvik (Norway) through a well at 20 m depth below the ground water (300–400 m seawater depth), demonstrated a complex CO2 plume migration path, with gas escaping outside the monitoring area. Less than 5% of the injected CO2 escaped to the atmosphere; consequently, a significant proportion of CO2 was dissolved into the water, leading to a decrease in pH of approximately 2 units, which is consistent with the lowest pH treatment (pH 6.1) used in our experiments. It is postulated that these geochemical changes would impact the dissolution of minerals, thereby releasing chemical elements and contaminants into the seawater and affecting the water quality (Denchik et al., 2014; Harvey et al., 2012). Marine calcifying organisms are among the species most susceptible to being impacted by CO2 acidification because their calcification rate could be altered. In addition, bivalves living in the upper sediment layer would be directly affected by the sediment–seawater interface acidification process. In the current study, strongly reduced survival rates were observed after the short-term exposure to the sediment elutriates at the lowest pH values, proving that a dramatic reduction of the seawater ecosystem’s pH would have lethal effects on juvenile bivalve organisms. Similar effects on marine organism mortality as a consequence of pH reduction after a short period of exposure have been reported previously, especially on marine calcifying organisms. After 5 days of exposure to pH 6.1, 100% mortality was observed in the crab

Necora puber (Spicer et al., 2007). The sea urchin Psammechinus miliaris also showed 100% mortality after 7 days exposure to pH 6.16 (Miles et al., 2007). Similarly, 100% mortality on adults of R. philippinarum exposed to seawater and whole sediment–seawater experiments at pH 6.1 or below were found by Rodríguez-Romero et al. (2014b) and Basallote et al. (2012). The exposure route (i.e., whole sediment, sediment elutriate, or filtered seawater) does not seem to influence the negative effects of the acidification process (Basallote et al., 2012). On the contrary, the sediment composition and properties as well as the life stages of the organisms are among the factors that influence the effects of the acidification on the bivalves. The median effect pH (LpH50 ) calculated for the juvenile clams R. philippinarum exposed to sediment elutriates prepared from three sediments with different levels of metal contamination ranged between 6.33 and 6.43 pH units, while the LpH50 calculated for adult organisms exposed to the whole sediment from the RSP and RSP seawater was 6.26 pH units (Basallote et al., 2012). Hence, it is suggested that adult bivalves could tolerate a lower pH than juveniles of the same species under similar exposure conditions. Mortality in adult bivalves at high pCO2 (䊐10 000 ␮atm) has been reported in the literature, whereas, in the case of larvae of juveniles, mortality was observed at lower pCO2 values (1000–5000 ␮atm) (Gazeau et al., 2013). The undersaturation of carbonate is one consequence of an increase in CO2 and reduction of pH. Although CO2 does not modify the TA in a constant total inorganic carbon (TIC) environment, as CO2 is being bubbled in to manipulate the pH, the balance in carbon species is modified, leading to an increase in the bicarbonate ions and consequently, the TA values (Millero and Sohn, 1992; Riebesell et al., 2010). Dissolution related to undersaturation represents a significant source of mortality for juvenile bivalves (Green et al., 2009). Although the organisms in the present study were not exposed for prolonged time intervals, it was observed that the outermost organic cover that protects the shell of the clams, the periostracum (Eyster, 1986), was lost in all of the individuals exposed to the lowest pH treatments. Small bivalves have been reported by Green et al. (2004) to be more susceptible to shell dissolution mortality than higher size classes. Furthermore, even moderate increase in the pCO2 (1500 ␮atm) caused shell corrosion in Cerastoderma edule exposed for 3 months to acidified seawater. Metal mobilization associated with acidification has been previously reported for marine and estuarine sediments (Ardelan and Steinnes, 2010; De Orte et al., 2014c; Roberts et al., 2013; Rodríguez-Romero et al., 2014a; Simpson et al., 2004). The form in which the metals are associated with the sediments and their concentrations are some of the factors controlling the mobility and availability of metals in marine ecosystems (Ure and Davidson, 2008). Furthermore, the toxicity of environmental metals depends on their speciation, mobility and bioavailability (Cappuyns and Swennen, 2008). The behavior of Zn was clearly related to the pH decrease in our experiments, with increases in the dissolved metal concentration observed at the reduced pH values. Correlations between the dissolved Zn and the pH were measured on days 1 and 10 of the experiments. The first component of the PCA linked the pH and pCO2 as well as the dissolved Zn on day 1 and 10 of the experiment with the survival of the bivalves. The second component also combined the pH and the Zn at the beginning and at the end of the exposure time. A similar behavior of increased Zn as the pH is decreased by CO2 -induced acidification has been previously shown (Basallote et al., 2014; De Orte et al., 2014b,c; RodríguezRomero et al., 2014a). The Zn results from our study confirm that acidification by addition of CO2 affects the mobility of the metal present in marine sediments. Thus, at a normal marine ecosystem pH, this metal is bound to the sediments and not available to the organisms. However, it could become available and subsequently

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potentially toxic to the exposed biota if the pH were reduced as result of CO2 leakage. Fe did not follow any clear pattern related to acidification. The Fe concentrations were lower on day 10 of exposure than on day 1, with the exception of the lowest pH treatment, 6.1, where a higher Fe concentration was observed on the last day of experiment for MAZelut and MLelut . The first factor of the PCA results linked the pH and pCO2 with the survival and the dissolved Fe on day 10. Low Fe solubility in the surface seawater has been described. Furthermore, the rapid oxidation rate of this metal could be responsible for Fe precipitation during the experimental time, and the precipitation of Fe might remove other metals by adsorption. Additionally, a disturbance in the Fe–Mn shuttle in the sediment could lead to increased concentrations of toxic metals (Ardelan and Steinnes, 2010). With respect to As, the total dissolved concentration decreased with acidification. This behavior has been previously reported in experiments conducted with CO2 . These results indicate that the total As in seawater would be less available to the marine biota under acidic conditions. Factor 3 shown by the calculated PCA linked the dissolved metal As with the pH and the pCO2 . A lower solubility of arsenate compared to the arsenite species under acidic condition has been postulated by Morin and Calas (2006). Consequently, an increase in toxicity associated with an increase in the proportion of the most harmful species of As, As(III), could occur. De Orte et al. (2014b) reported an increase in the proportion of the most harmful species of arsenic, As(III), and thus, increased toxicity as a consequence of a decrease in the pH. However, the results from the present study did not show a relationship between As and the increase in toxicity. The concentrations of Pb correlated with pH for RSPelut and MAZelut on day 1 of the experiment and for MLelut and RSPelut on days 1 and 10 of the experiment. Millero et al. (2009) described the behavior of lead during a pH reduction as causing a large increase in the chloride complexation, with only a 10% of the increase being its free from. The presented results showed that the dissolved Pb amounts were always below those expected to produce chronic effects according to the USEPA criteria (8.1 ␮g L−1 ) in all of the sediment elutriates tested. Additionally, the dissolved Pb was not associated with an increase in the toxicity to the exposed bivalves. In summary, the metals bound to sediments would be affected by pH decreases associated with CO2 leak events. The Zn, which is originally non-available and non-toxic at natural pH, would become available and subsequently toxic if the pH is reduced. Injection of CO2 through the sediment layer will also alter the stability of minerals such as pyrite and ferrihydrite (De Orte et al., 2014b). Therefore, the sediment characteristics play important roles in the metal behavior under CO2 -associated acidification. Nevertheless, others metals or pollutants not evaluated here could contribute to the lethal toxic effects presented in this work. In addition to metals, other contaminants including oil hydrocarbons, persistent organic pollutants, pesticides, and eutrophication nutrient elements enter into marine ecosystems due to human activities. Considering the coexistence of OA and pollution in many coastal regions, these environmental stressors may have combined effects on marine ecosystems that need to be addressed (Zeng et al., 2015). Because benthic bivalves are filter-suspension-feeding organisms that live directly in contact with sediments, the influx of a metal into the organism is expected to be proportional to the metal concentration in the water (Bryan et al., 1979; Rodríguez-Romero et al., 2014b). Therefore, bivalves will filter particles in suspensions that were previously affected by acid extraction from sediments (Riba et al., 2010), leading to the possibility of metal accumulation. The complexity of predicting the migration pathways of CO2 leakage events even when the leaks are from a known and controlled injection point has been previously described. Moreover, the

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seepage could occur at locations not necessarily close to the injection well (Blackford et al., 2014; Jones et al., 2014). These types of uncertainties related to the monitoring measures make it difficult to determine the implication of the escapes of CO2 in the existing marine biota. Thus, it would be helpful to compile the most possible information about the effects of potential CO2 leakage from the sub seabed storage sites on marine ecosystems. 5. Conclusions Unconsolidated sediment re-suspension as a consequence of flows of CO2 through the sub-seabed associated with acidification (pH reductions) would affect the geochemical composition of sediment–seawater. According to our results, reductions below pH 6.5 will cause lethal effects to the juveniles of the bivalves, R. philippinarum, exposed to all the studied sediments. The dissolved metal Zn was strongly correlated with the decrease in the pH and was associated with increased toxicity of the sediment to the exposed organisms. Nevertheless, although the acidification processes would influence the mobility of the other dissolved elements (Fe, As, and Pb), they were not associated with an increase in toxic effects on the exposed organisms. Other stressors of marine ecosystems such as rising temperature, salinity variations, and deoxygenation of the oceans are expected to co-occur during the current century. Thus, marine ecosystems will be increasingly subjected to multiple stressors simultaneously. Therefore, it is strongly recommended that the effects of marine acidification processes be addressed in the context of the effects of other climate change-associated environmental stressors in future work. Acknowledgements Research presented in this document was supported partially by the Spanish Ministerio de Economía y Competitividad, under Grant Reference CTM2011-28437-C02-02/TECNO and CTM2012-36476C02-01/TECNO. The authors are also grateful to international Grant from Bank Santander/UNESCO Chair UNITWIN/WiCop for funding this work. The authors would like to thank to the anonymous reviewers for their valuable comments on this manuscript. M.R. de Orte thanks FAPESP for the postdoctoral fellowship granted under process 2014/22273-1. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2015.07. 004 References Ardelan, M.V., Steinnes, E., 2010. Changes in mobility and solubility of the redox sensitive metals Fe, Mn and Co at the seawater–sediment interface following CO2 seepage. Biogeosciences 7, 569–583 http://www.biogeosciences.net/567/ 569/2010/ Ardelan, M.V., Steinnes, E., Lierhagen, S., Linde, S.O., 2009. Effects of experimental CO2 leakage on solubility and transport of seven trace metals in seawater and sediment. Sci. Total Environ. 407, 6255–6266 http://www.sciencedirect.com/ science/article/pii/S0048969709008481 ASTMD422-63, 2007. Standard test method for particle-size analysis of soils. Basallote, M., Rodríguez-Romero, A., Blasco, J., DelValls, A., Riba, I., 2012. Lethal effects on different marine organisms, associated with sediment–seawater acidification deriving from CO2 leakage. Environ. Sci. Pollut. Res. 19, 2550–2560, http://dx.doi.org/2510.1007/s11356-11012-10899-11358 Basallote, M.D., De Orte, M.R., DelValls, T.Á., Riba, I., 2014. Studying the effect of CO2 -induced acidification on sediment toxicity using acute amphipod toxicity test. Environ. Sci. Technol. 48, 8864–8872, http://dx.doi.org/8810.1021/ es5015373

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