Comparative evaluation of sea-urchin larval stage sensitivity to ocean acidification

Chemosphere 184 (2017) 224e234

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Comparative evaluation of sea-urchin larval stage sensitivity to ocean acidification M.C. Passarelli a, *, A. Cesar b, c, I. Riba a, T.A. DelValls a
diz, Spain UNESCO/UNITWIN WiCop, Physico Chemical Department, Faculty of Marine and Environmental Studies, CEIMAR, University of Ca ~o Paulo (UNIFESP), Santos, Sa ~o Paulo, Brazil Department of Ocean Sciences, Federal University of Sa c ~o Paulo, Brazil Department of Ecotoxicology, Santa Cecília University (UNISANTA), Santos, Sa a

b

h i g h l i g h t s
CO2-induced acidification changes the metal mobility from Brazilian and Spanish sediments.
The pH reduction causes effects on embryo-larval development of sea urchins.
The tropical sea urchin Lytechinus variegatus shows to be more tolerant to ocean acidification than Paracentrutos lividus.
The ICpH50 for the embryo-larval development was ranged from pH 7.30 to 6.79.
The As dissolved in the elutriate sediment was correlated with the pH reduction and toxicity of the sediment.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 March 2017 Received in revised form 16 May 2017 Accepted 1 June 2017

Changes in the marine carbonate system may affect various calcifying organisms. This study is aimed to compare the sensitivity of embryo-larval development of two species of sea urchins (Paracentrutos lividus and Lytechinus variegatus) collected and exposed to samples from different coastal zone (Spain and Brazil) to ocean acidification. The results showed that the larval stages are very sensitive to small changes in the seawater's pH. The larvae from P. lividus species showed to be more sensitive to acidified elutriate sediments than larvae from L. variegatus sea urchin. Furthermore, this study has demonstrated that the CO2 enrichment in aquatic ecosystems cause changes on the mobility of the metals: Zn, Cu, Fe, Al and As, which was presented different behavior among them. Although an increase on the mobility of metals was found, the results using the principal component analysis showed that the pH reduction show the highest correlations with the toxicity and is the main cause of embryo-larval development inhibition. In this comparative study it is demonstrated that both species are able to assess potential effects of the ocean acidification related to CO2 enrichment by both near future scenarios and the risk associated with CO2 leakages in the Carbon Capture and Storage (CCS) process, and the importance of comparative studies in different zones to improve the understanding of the impacts caused by ocean acidification. © 2017 Published by Elsevier Ltd.

Handling Editor: Jim Lazorchak Keywords: CO2 enrichment Paracentrutos lividus Lytechinus variegatus Toxicity tests Metal mobility

1. Introduction Changes in the marine carbonate system may affect various calcifying organisms. As CO2 is added to seawater, there are increases in Hþ and bicarbonate [HCO 3 ] and simultaneous decreases in water's pH and carbonate ion concentration [CO2 3 ] (Orr, 2011). This imbalance in the chemistry of seawater can cause many impacts on the marine ecosystem, especially to the organisms that

* Corresponding author. E-mail address: [email protected] (M.C. Passarelli). http://dx.doi.org/10.1016/j.chemosphere.2017.06.001 0045-6535/© 2017 Published by Elsevier Ltd.

have calcareous skeletal structures. CO2 increases in the ocean may occur both by the capacity of CO2 exchanges with its dissolved form between the atmosphere and surface seawater as well by CO2 leaks during the carbon capture and storage (CCS) process. Previous studies have showed a 7.5% increases in greenhouse gases from 2005 to 2011, with carbon dioxide contributing 80% of this amount (IPCC, 2013). The seawater surface pH is predicted to have a reduction around 0.4 units by 2100 and 0.77 units by 2300 (Caldeira and Wickett, 2005; IPCC, 2007). Briefly, this reaction is because there is a natural equilibrium between the ocean and the atmosphere. When there is an excess of atmosphere CO2 concentrations due to the anthropogenic

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activities, the gas is absorbed by the ocean as a sink (Sabine et al., 2004), reacting with the seawater and consequently the pH decrease (Millero, 1995). On other hand, the CCS could contribute 15e55% of the cumulative mitigation effort worldwide by 2100 (IPCC, 2005). This technique consists of the separation of carbon dioxide from industrial and energy-related sources, transport to an offshore geological formation, and long-term isolation from the atmosphere. However, CO2 leakage may occur during this process, which may contribute with the imbalance of seawater's chemistry. In this context, many studies have been performed in order to analyze the impacts from changes in the marine carbonate system as well as pH reduction to the organisms (Moulin et al., 2011; De Orte et al., 2014a; Basallote et al., 2012, 2014, 2015; Rodriguez-Romero et al., 2014a, 2014b; Bautista-Chamizo et al., 2016; Wang et al., 2016). According to Barry et al. (2011), for taxa affected by ocean acidification, individual physiological stress can lead to reduced growth, size, reproductive output, and survival. Reduced calcification rates have been also well established, particularly to corals and mollusks (Gazeau et al., 2007; Doney et al., 2009). Echinoderms appear less tolerant of low pH waters than many groups, as indicated by their conspicuous absence from habitats with naturally high CO2 levels such as hydrothermal vents (Grassle, 1986) and they are highly variable in response, mainly due to the great variability in degree of calcification within the phylum (Wood et al., 2008). As calcifying organisms tend to be more vulnerable to carbonate seawater alteration, early life stages, such as fertilization, embryogenesis, and larval development, are expected to be particularly sensitive to changes in environmental conditions (Bryrne, 2011; Havenhand and Schlegel, 2009; Havenhand et al., 2008; Raven et al., 2005; Ross et al., 2011). Studies have showed a decrease of fertilization and early development stages in sea urchins in low pH conditions (Kurihara et al., 2007; Havenhand et al., 2008; Moulin et al., 2011). Thus, it highlights the importance of studying the impact of pH decrease on early life stages once the developmental success of a population level depends on the survival of the embryos and larvae. Another important point that should be considered is the habit of marine organisms. Coastal organisms are regularly experiencing hypercapnic conditions (elevated pCO2 levels). This suggests that they might be pre-adapted to relatively high ambient pCO2 levels (Portner et al., 2011). Nonetheless, it has been reported that since infauna organisms live in an environment that is often high in CO2, they will be inherently more immune to ocean acidification than organisms that live on sediment surface (epifauna) (Widdicombe et al., 2011). Therefore, the region where organisms live and their habit are strongly correlated with the effect on changes in carbonate seawater to marine organisms. Recently there has been a debate on whether the tropics or temperate zones are more vulnerable to climate warming (Ghalambor et al., 2006; Tewksbury et al., 2008) and the acclimation capacity of species that live in these zones (Vinagre et al., 2016). Considering that the ocean acidification would be one of the causes from the global warming, it is also very important to study its impacts on the organisms from different zones. Thus, comparative studies to analyze the impact from seawater pH changes and consequently ocean acidification to different species are highlighted. This study aimed to compare the sensitivity of embryo-larval development of two sea urchins species (Paracentrutos lividus and Lytechinus variegatus) to ocean acidification. The species Paracentrutos lividus and Lytechinus variegatus are used in samples collected in Spain and Brazil, respectively. Both of these areas have projects with operating (in Rio de Janeiro and Bahia states, Brazil) and dormant (Ponferrada, Spain) status of CCS system, and can be

225

impacted by the use of this technology (SCCS, 2017). Also, there is
diz that could be used in approved a storage area in the Gulf of Ca the future for CCS (BOE, 2008). Experiments were performed in laboratory scale with a CO2-bubbling system designed to conduct ecotoxicological assays. The pluteus larval stage was analyzed for 24 h (L. variegates) and 48 h (P. lividus) to sediment elutriates subjected to various pH treatments. The exposition time established for each species was related to time for them get themselves to the pluteus stage. Sediment samples were collected different areas located in Spain and in Brazil. The dissolved metal concentrations in the sediment elutriates were measured to assess possible interactions among the pH, dissolved metals and toxicity. 2. Materials and methods 2.1. Sampling Sediment samples were collected from four sites in two different littoral areas. Two sampling sites are located in Santos Estuary and Bay, S~ ao Paulo eBrazil (Fig. 1), and the others two in Bay of Cadiz, Spain (Fig. 2). The Santos Estuarine System is located on the central coast of ~o Paulo, in southeastern Brazil. This region is of the state of Sa economic importance due to the industrial complex in the city of ~o, the Port of Santos, the potential for tourism, and the Cubata fisheries and natural resources provided by mangroves that occur within the estuary. The establishment of the sampling site in the Santos Estuary (CPI) is based on previous studies (Lamparelli et al., 2001; Abessa et al., 2005; Cesar et al., 2006, 2007; Torres et al., 2015) which show contamination gradients from the inner portions of the estuary to the external areas. In contrast, the site from Santos bay (PAI) is considered relatively clean when compared with the local guideline which establish values for dredged sediment (CONAMA 454, 2012), and has been used as a reference site in this area (Szalaj et al., 2016; Goulding et al., 2017). The sampling sites in Spain are located in San Pedro River (RSP)
diz. The sites are relatively and Trocadero (TRO) both in Bay of Ca protected areas connected to the Atlantic Ocean through intertidal channels and salt marshes. These areas are influenced by marine aquaculture, shipbuilding industry, and urban discharges among other anthropogenic activities (DelValls et al., 1998; Silva et al., 2012). The RSP shows low metal concentration, while in TRO is considered an area with intermediate metal concentration (Basallote et al., 2014). These values were also compared with the local guidelines which establish the concentrations of metals for dredged sediments considered to be no dangerous (CEDEX, 2015) (Table 2). Collection procedures and transport of sediment samples

Fig. 1. Map showing the sediment sampling sites located in Piaçaguera Channel (CPI) and Palmas Island (PAI), Santos, Brazil. The triangle reflects the organisms collect site.

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modifications of the USEPA method (1998). The sediment-seawater (ratio 1:4 v/v) was firstly acidified by the injection of CO2 gas until the pH target was reached. Then, sediment elutriate was obtained by mixing acidified sediment and seawater. The mix was maintained under agitation (60 rpm) for 30 min, followed by a settling down process for at least 12 h as previously reported by Basallote et al. (2015). After this procedure, the supernatant fraction was carefully extracted and placed in a new glass vessel (100 ml of sample per replicate). Elutriates were again acidified by the injection of CO2 bubbles to maintain the target pH treatments during all the experiment. The pH values of the treatments ranged between 8.0 and 6.0 (pHs: 8.0 ± 1, 7.5 ± 1, 7.0 ± 1, 6.5 ± 1 and 6.0 ± 1) with 3 replicates. Fig. 2. Map showing the sediment sampling sites located in Río San Pedro (RSP) and Trocadero (TRO), Cadíz, Spain.

followed the USEPA (2001). After collection, all sediment samples were taken to the laboratory and sieved through a 2 mm plastic meshed to remove the gravel fraction, and homogenized. The samples were stored at 4  C in darkness until their use (no more than 2 weeks). Sub-samples were collected for chemical quantification of organic carbon, organic matter content, and metals. Scuba divers in areas located in the Bay of Cadiz and Bay of Santos, respectively, collected the sea urchins Paracentrutos lividus and Lytechinus variegatus. These test species have been recommended in standardized protocols of environmental quality to use their embryos in chronic toxicity tests (ABNT NBR 15350 2012; CONAMA 452 2012; CEDEX, 2015). 2.2. Experimental set-up The laboratory-scale injection system (Fig. 3) employed in this study is described by De Orte et al. (2014a). In brief: the CO2 injection system proposed seeks to provide a laboratory-based simulation of the acidification process in the marine environment caused by CO2 leaks during the carbon dioxide capture and storage process in stable underwater structures. The AT Control System from Aqua Medic (Bissendorf, Germany) was used to independently manipulate and control the pH in each test chamber. 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. Adding CO2 gas through a solenoid valve that opens when the pH increases 0.01 units above the predetermined pH values and closes after the target pH is reached regulates the pH. The sediment elutriate procedure was made according to

Fig. 3. Schematic design of the CO2 injection system used in this study adapted from De Orte et al., (2014a). a) Sediment elutriate with pH adjusted. b) Supernatant fraction exposed to testing; triplicate exposure in 100 ml of elutriates solution.

2.3. Sediment e water characterization Sediment sub-samples were collected for the chemical quantification of organic matter content and metals. More than 6 g of sieved wet sediment were sub-sampled in triplicate for subsequent determination of organic matter (OM). Metals dissolved in elutriate seawater were measured in filtered (0.45 mm) subsamples acidified to pH < 2 with ultrapure HNO3 in order to perform the metals analyses. Trace metal (Al, As, Co, Cr, Cu, Fe, Ni, Pb, and Zn) in the sediment and elutriate were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES). This method uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. These analyses were performed in the Scientific Instrumentation Center of the University of Granada. Overlying seawater (50 mL) was collected for the total alkalinity (TA) analysis. TA was determined by automatic titration (Mettler Toledo, T50) using a combined glass electrode (Mettler Toledo, DGi115-SC) calibrated on the NBS scale. Both TA and the pH of the CO2 system were used to determine the speciation of the seawaterecarbonate system. The pH (from the registered data at the AT Control System) and TA were used to calculate the parameters of the seawater carbonate system using the CO2SYS program (Pierrot et al., 2006) with the dissociation constant from Mehrbach et al. (1973) refitted by Dickson (1990) and Millero et al. (1987) and KSO4 using Dickson (1990). 2.4. Embryos-larval development assay Specimens of Paracentrutos lividus and Lytechinus variegatus
diz, were collected, from a rocky intertidal platform in the Gulf of Ca located in a protected area (Southwestern Spain) and from Palm Island in Bay of Santos e Brazil, respectively. After the collection, the sea urchins were transported in cool boxes to the laboratory and placed in aquaria with aerated seawater before testing. The gametes were obtained from different ways, following the standards applied in each laboratory. The procedure used for the or
diz was by dissection and direct extractions ganisms from Bay of Ca from the gonads and KCl (0,5 M) injection was the method used to obtain the gametes from the organisms collected in Bay of Santos, following ABNT NBR 15350 (2012). As soon as the organisms started releasing the gametes, they were targeted for the fertilization procedure. The sperm were transferred to glass backers using a Pasteur pipette and cooled by ice cubes, while the eggs were transferred to backers with filtered seawater. The fertilization was attained by adding 1 mL of sperm solution to the 200 mL of ovules solution. Density of fertilized eggs was estimated, about 600 embryos were transferred to glass tubes containing 100 ml of elutriate solution in different pH concentrations for a period of 48 h at a temperature of 18  C to Paracentrutos lividus, and of 24 h at 25  C to Lytechinus variegates. The exposition

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time established for each species was related to time for them get themselves to the pluteus stage, following ABNT NBR 15350 (2012) and CEDEX (2015) protocols. After the exposure time, the assay was finished adding 40% formaldehyde and observed under inverted microscope to measure embryogenesis success. Table 1 shows a summarized description of toxicity test and different sea urchin used in this study. The first 100 larvae from each replicate were analyzed when the larvae developed to the pluteus stage was considered as an endpoint for the embryo-larval toxicity test (ABNT NBR 15350, 2012). 2.5. Statistical analysis The linear interpolation method (Norberg-King, 1988) was used to calculate the set of the half maximal of Inhibitory Concentration IC50 (48 h and 24 h) for the embryo-larval development assays. ANOVA followed by the Dunnett's test were used to identify the pH concentrations significantly different from the control for each assay. For all assays, significant differences were determined when p < 0.05. Statistical analysis was performed employing TOXSTAT 3.5. 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. The analysis was performed on the matrix (varimax-normalized rotation) and a component loading cutoff of 0.40 was used in selecting variables for inclusion into factors. The variables were autoscaled (standardized) to be treated with equal importance (DelValls and Chapman, 1998; DelValls et al., 2002). 3. Results 3.1. Sediment and seawater characterization The concentration of Fe, Cu, Co, Zn, Cd, Pb, As, and Cr in the sediments samples used in this study are given in Table 2. The highest metal concentration was found for Fe (1474 mg kg1 sediment dry) in CPI sample site. The metal zinc presented higher concentrations in TRO (156 mg kg1 of sediment dry) and CPI (133 mg kg1 of sediment dry) sites. The PAI site showed the lowest metal concentrations followed by RSP site. In the TRO site it also highlights the concentrations of Pb and Fe, respectively. Regarding to the sites located in Brazil, besides the Fe and Zn concentrations, the concentration of Cr (30.9 mg kg1 of sediment dry) in the CPI sediment site was also highlighted. The seawater used in the experiments in Spain as well as in Brazil exhibited concentrations below the detection limit of the equipment for many of metals measured which are shown in Table 2. Both areas have the seawater pH average of 8.0 ± 1. Furthermore, low values for alkalinity were found in these waters, which were estimated are 2859 mM and 2230 mM for water used in the experiments performed in Spain and Brazil, respectively. 3.1.1. Metal mobility In Fig. 4 it is shows the metal mobility (As, Zn, Cu, Al and Fe) in the elutriates treatment exposed at different pH levels using all

Table 1 Summarized description of the toxicity tests and different sea urchins tested in this study. Organisms

pH range T ( C)

L. variegatus 8.0e6.0 P. lividus 8.0e6.0

S (psu) DO (%) Duration (h) Endpoint

25 ± 1 35 ± 1 18 ± 1 31 ± 1

>80 >80

T temperature; S salinity; DO oxygen saturation.

24 h 48 h

Pluteus stage Pluteus stage

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sediments samples (RSP, TRO, PAI and CPI). In the case of Al and Fe, their concentrations were measured only in the elutriate sediments from Spain. The others metals measured presented concentrations below of detection limit of the equipment or low mobility. The mobility of the metals Al and Fe among the pH levels were observed only in the sediment samples from Spain (RSP and TRO). The mobility of Fe from sediment to water increased substantially from the control (50 mg L1) to pH 6.0 (1070 mg L1) for TRO sediment, while a little mobility of Fe was observed in the elutriate of RSP sediment. The concentration of Al increased from 43 mg L1to 96 mg L1at pH 6.5 and 6.0 in the elutriate sediment of RSP. Cu concentrations increased from 27 mg L1 to 61 mg L1 at pH 6.5 in the elutriate of sediment from RSP. In the case of the TRO sediment, these concentrations increased to 85 mg L1 pH 6.5 in the elutriate sediment. However, there was little mobility of Cu concentrations among the pH levels in elutriates of sediments from Brazil. Zn concentrations decreased at pH 7.0 and pH 6.5 and increased again at pH 6.0 in sediment elutriate from both the RSP and the TRO. However, its concentrations increased from 40 mg L1 to 91 mg L1 in the elutriate from the control to pH 7.5 using the sediment from CPI. The concentrations of As showed increases strongly to 84 mg L1, 142 mg L1 and 95 mg L1 at pH 6.0 in the elutriates of TRO, PAI and CPI, respectively. 3.2. Toxicity tests For the highest pH treatments (pH control and 7.5), similar embryo-larval development success (>90%) was recorded for the sediment elutriates tested, except at pH 7.5 from RSP and TRO sediment (80% of embryo-larval development success). Figs. 5 and 6 show the results from larval development success of P. lividus and L. variegatus, respectively, to sediment elutriates samples. The larval development success presented significant differences (p < 0.05) at pH 7.5 at 7.0 for sediment from RSP, TRO and PAI. Regarding the elutriates treatment using sediment from Brazil, a significant statistical difference was found in the larvae of L. variegatus exposed starting at pH 6.5. For all sediment tested, a significant difference (p < 0.05) was shown in lowers pH (pH 6.5 and 6.0). The ICpH50 was defined as the median inhibition pH values that cause adverse responses in 50% of the embryo-larval development. The results were estimated based on abnormal larval development for the larvae exposed to the elutriate sediment subjected to the pH treatments. The IC50 was ranged from pH 7.30 using RSP elutriate sediment to 6.79 using CPI elutriate sediment (Table 3). These results show that there is lower concentration of protons in the stations from Spain needed to produce toxicity than those related to Brazil in a gradient: RSP ¼ TRO > PAI > CPI. The latter need more acidification to produce toxicity than the other stations. 3.3. Linking contamination, toxicity and acidification A principal component analysis (PCA) was performed in order to compare the results from two different littoral areas in relation to the effects of ocean acidification on the embryo-larval development of sea-urchins. For it, results from four sediment sampling sites at different pH levels were treated using a multivariate analysis. The application of PCA to the variables indicates that the 10 variables can be described by 3 factors or principal components (Table 4). These explain 88.6% of the variance in the original data set. The criterion selected to interpret a variable associated with a particular factor was loading of 0.4 or higher. This approximates Comreys (1973) cutoff of 0.55 for a good association between an original variable and a component, and also takes into account discontinuities in the magnitudes of the loadings of the original variables.

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Table 2 Summary of the sediment characterization from the sites used in the toxicity tests (RSP and TRO at Spain and PAI and CPI at Brazil) compared with the local guidelines for dredged sediments (CEDEX, 2015 to compare the sites from Spain and CONAMA 454, 2012 to compared the sites from Brazil), and the metals concentrations in the seawater (SW) used in the experiment in each country. Sediment mg kg1

As

Cd

Cr

Cu

Fe

Ni

Pb

Zn

OM (%)

Fines (%)

RSP TRO PAI CPI CEDEX CONAMA SWSpain SWBrazil

7.14 16.1 3.16 2.83 1000 19 <0.05a <0.05a

0.12 0.29 0.06 0.25 72 1.2 <0.02a <0.02a

20.3 33.2 27.6 30.9 1000 81 <0.006a <0.006a

18 42.1 7.2 11 2500 34 0.071 0.017

15.2 35.8 548 1474 e e 0.038 0.041

14 34.7 13.7 5.92 1000 20.9 <0.007a <0.007a

22.3 62.6 18 11.8 1000 46.7 <0.015a <0.015a

49 156 123 133 2500 150 0.014 0.041

5.97 20.2 1.11 13.1 e e No applied

48.9 73.7 26.8 34.6 e e

a

Values refer to the detection limit for each metal on the equipment (mg L1).

Fig. 4. Dissolved concentrations of Cu, Zn, As, Al and Fe using sediment elutriates from all sediment samples used in this study at the different pH levels.

Each factor is described according to the dominant group of variables.

The first principal factor (47.9% of variance) linked with positive values the sediment parameters, such as metal concentrations, the

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Table 4 Sorted rotated component matrix with component loadings of the total variables on the three principal components. Only loadings >0.4 are shown in the table. Variables

Larval Development Inhibition (%) pH Reduction Zn Elu (mg L1) Cu Elu (mg L1) As Elu (mg L1) Zn Sediment (mg Kg1) Cu Sediment (mg Kg1) As Sediment (mg Kg1) Fines (%) OMa (%) Variance (%) Cumulative (%) a

Fig. 5. Results from larval development success of Paracentrutos lividus to pH reduction using the sediment elutriate treatments from Spain (RSP and TRO). *means significant difference to the control (p < 0,05).

Components Factor 1

Factor 2

Factor 3

e e e 0.90 e e 0.99 0.97 0.97 0.86 47.9 47.9

0.94 0.97 e e 0.44 e e e e e 21.4 69.3

e e 0.74 e 0.66 0.80 e e e 0.40 19.3 88.6

OM reflects Organic Matter.

biological effect. In order to confirm these factor descriptions and to establish the relationship between components associated with the embryolarval development inhibition and the acidification at each of the studied stations, it was proposed a representation of estimated factor scores from each case (stations and pH levels) to the centroid of all cases for the original data (Fig. 7). The positive values of factor 2 which linked the main cause of larval development inhibition in the sea urchins to acidification and mobility of As has shown positive values in all sediment samples sites used in this study at different values of pH. In general, it could be observed that the Factor Score for Factor 2 increases when the pH decreases being positive in different stations at different pH values. This positive value was associated at pH 7.0 for all sampling sites, except for CPI sample site that showed a positive factor 2 only starting at pH 6.5. Also, it is observed a strong correlation between the factor 1 and the TRO sediment site in all pH levels tested. 4. Discussion

Fig. 6. Results from larval development success of Lytechinus variegatus to pH reduction using the sediment elutriate treatments from Brazil (PAI and CPI). *means significant difference to the control (p < 0,05).

Table 3 Results of median inhibition pH (ICpH50) using tested elutriate sediment. *SD means Standard Deviation. Sediment elutriates

RSP

TRO

PAI

CPI

ICpH50 SD*

7.30 0.011

7.29 0.014

7.18 0.12

6.79 0.009

percentage of fines, and the amount of organic matter. In addition to the sediment parameters, the first factor also linked the concentrations of Cu in elutriate treatments. The second factor (21.4% of variance) linked with positive values the embryo-larval development inhibition, concentrations of As in the elutriate treatment and the pH reduction. The third factor (19.3% of variance) linked with positive values the concentrations of Zn and As in the elutriate treatments and the concentrations of Zn and OM in the sediment. Only F2 link toxicity endpoints with acidification and concentration of As, the other two factors were not associated with adverse

Our study assessed the effect from pH changes in seawater to embryo-larval development of Paracentrutos lividus and Lytechinus variegatus sea urchins, using different elutriates sediment samples. The effects from ocean acidification to marine organisms have been widely studied in the last years (De Orte et al., 2014a; RodriguezRomero et al., 2014a; 2014b; Almagro-Pastor et al., 2015). Release of carbon dioxide (CO2) from human activities is reported as a significant contributor to observed climate changes and ocean acidification (IPCC, 2014). The ranged pH (7.5e6.0) chosen in this study was to mimic the effect from future scenarios of CO2 in seawater, which as mentioned above is estimated to reduction around 0.4 units by 2100 and 0.77 units by 2300 (Caldeira and Wickett, 2005; IPCC, 2007) as well as possible CO2 leakages during the storage process of CSS technique. Elutriate treatment is already standardized and have been applied as a tool to mimic sediment re-suspension events (USEPA, 1998; Casado-Martínez et al., 2007), providing an important response in relation to contamination as well toxicity from sediment. It is well known that when sediment is mixed several contaminants become more toxic than in whole sediment (Thompson et al., 1999; McDonald, 2005; Casado-Martínez et al., 2007). Sediment geochemical properties, organic matter and the sediment grain size are extremely important when studying sediment contamination. Moreover, factors such pH and salinity may influence the bioavailability of chemical compounds in sediment. Ocean acidification has been shown to change in the speciation of metal

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Fig. 7. Estimated factor scores prevalence for each of the four sediment sampling sites used in this study in relation to the pH levels.

ions in seawater (e.g., increase in free form) transforming them thus more toxic to the biota (Zeng et al., 2015). Potential CO2 leakage events could mobilize and re-suspend high quantities of sediments. In this sense, it highlights the importance of studying the effects of ocean acidification to marine organisms, using elutriate assays. The observed results from metals concentrations in the sediment sampling sites used in this study corroborate to those reported in previous studies (Basallote et al., 2012; 2014; De Orte et al., 2014a; Cesar et al., 2007; Choueri et al., 2009; Torres et al., 2015). As expected the lower metal concentrations were found for the control sediment site in each country studied (RSP and PAI), being among them the lowest metals concentrations related to sediment from PAI. Additionally, studies have been performed in the last years in TRO and CPI sites which indicate these areas as an intermediate contaminated site (Basallote et al., 2012; 2014; De Orte et al., 2014a; Cesar et al., 2007; Choueri et al., 2009; Torres et al., 2015). Sediment from TRO presents higher concentrations of all metals tested than sediment from RSP. The highest concentrations were observed related to Pb (62.6 mg/kg) and Zn (156 mg/kg) in TRO sample site. However, these results are below the limit allowed by CEDEX (2015) as associated with biological effects or risk to the environment. Regarding the sediment samples from Brazil, highest metal concentrations were found in the CPI site mainly for Fe and Zn, which concentrations were 1474 mg kg1 and 133 mg kg1, respectively. However, these values are also below the limit defined by CONAMA 454 (2012) which classified the levels of metal concentrations in dredged sediment. The decrease in seawater pH will result in a reduction in the concentration of both hydroxide and carbonate (OH and CO2 3 ).

This reduction will change the speciation of a number of metals that form strong complexes with OH and CO2 3 , consequently will have a higher fraction in their free forms at lower pH (Millero et al., 2009). As mentioned above, it was observed a higher mobility from certain metals (Cu and Fe, for instance) to elutriate sediments in relation to pH reduction. These results are expected once these metals form carbonate complexes, which as previously explained are strongly affected by change in pH, resulting in an increase in their free ionic forms (Millero et al., 2009). The concentration of Cu were found to be above the seawater quality criteria based in the chronic value for Cu (3.1 mg L1) in the elutriate sediment from RSP and TRO samples, even in the non-acidified treatment (control, no CO2-added). Table 5 shows the values recommended by USEPA for acute and chronic toxicity in saltwater as well the values found in this study that reach the concentrations up above to those

Table 5 Summarized of metal concentrations in elutriate sediment (current study), compared with the values recommended by USEPA (1995) for acute and chronic toxicity in saltwater as well the pH level that these metals reach their concentrations up above those recommended by the national protocol. Values are expressed in mg L1. Metals

Usepa Acute

As Cu Zn

69 4.8 90

Current Study Chronic

36 3.1 81

Spain

pH Brazil

RSP

TRO

PAI

CPI

e 26 e

78 73 e

100 e e

60 5 91

6.5 7.5 7.5

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recommended by this national protocol. A similar behavior from metal mobility associated with low pH by CO2 induced acidification has been previously shown (Basallote et al., 2014, 2015; De Orte et al., 2014b; Rodriguez-Romero et al., 2014a). In De Orte et al. (2014b) also showed results associating the increase of Zn mobility with low pH levels in overlying water. These authors analyzed the mobility from metal in sediment to water in two steps, 1 and 10 days of exposure time, which showed higher mobility from Fe for 1 day and from Cu and Zn from day 1 until day 10 in relation to pH reduction. The national recommended aquatic life criteria indicate a limit of zinc concentrations of 81 mg L1 to chronic toxicity in saltwater (USEPA, 1995). The results obtained in this study suggest that the concentrations of Zn could thus reach the concentrations up above (91 mg L1 at pH 7.5 in the CPI sediment elutriate) those recommended by the national recommended aquatic life criteria indicate as a limit to chronic toxicity in saltwater (Table 5). In the case of the metal As, its concentrations increased considerably among the pH levels in all sediment tested, except in the RSP sediment that its concentration was below of the detection limit of the equipment in all pHs used in this study. The speciation form of this metalloid can explain these results. It has been reported that arsenite has a higher bioavailability than arsenate and is considered more toxic to marine organisms (Neff, 1997). The USEPA (1995) recommending the values of 69 mg L1 and 36 mg L1 to acute and chronic toxicity in saltwater. This way, it highlights the concentrations of As in the PAI sediment that increased from 40 mg L1 in the control (no CO2 added) to 142 mg L1 at pH 6.0. Furthermore, the results of PCA indicated an association between the embryo-larval development inhibition and the concentrations of As in the elutriate sediment in relation to pH reduction. This result confirms that the metal As could become more bioavailable and toxic to marine organisms in situations of disturbance causes by CO2 leakages events. There were no recommendation values for the metals Fe and Al in saltwater in the water quality criteria established by Usepa. However, the concentrations of these metals showed an increased at pH 6.5 and 6.0 in the RSP and TRO sediment elutriates. According to Millero et al. (1987), the lifetime of dissolved iron depends on its oxidation rate, which is strongly associated with pH. Previous studies have reported similar Fe behavior in RSP sediment elutriates and overlying water (Basallote et al., 2014, 2015). Additionally, De Orte et al. (2014b) also reported substantial increases in Fe concentrations at pH 6.5 (around 0.15 mg L1) due to acidification after 24 h of exposure to CO2 leakages. However, after 10 days of experimentation, no significant (p < 0.05) Fe mobility was observed in their results. According the study reported by Martín-Torre et al. (2016), this fact occurs as a consequence of the Fe(II)-Fe(III) oxidation and precipitation of Fe(III). Many works have reported effects from ocean acidification on fertilization (Szalaj et al., 2016; Barros et al., 2013) larval mortality (Basallote et al., 2012; Barros et al., 2013) and larval development (Gianguzza et al., 2014; Szalaj et al., 2016) to marine organisms. According to Barry et al. (2011), taxa that live a part of their lives (often life-history phases) in open waters, are expected to be particularly sensitive to ocean acidification and this vulnerability may have large effects on population level. The larvae of P. lividus showed to be more sensitive to acidified elutriate sediments than larvae of L. variegatus sea urchin. Significant effects (p < 0.05) were found from the larval development success of P. lividus at pH 7.0 in all sediment tested, except for CPI, which significant effects (p < 0.05) were found only starting at pH 6.5 for the larvae of L. variegatus. These results agree with the study reported by Szalaj et al. (2016), which found significant effects (p < 0.05) on the

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development D-shaped larval from the mussels Perna perna exposed to both contaminated and non-contaminated elutriate sediments at pH 7.6. Furthermore, these authors found values of median effect concentration EpH50 for larval development of pH 7.80 and 7.86, which indicates a strong sensitivity from the mussel larvae even in small changes in the seawater pH. Effects from ocean acidification to Lytechinus variegatus sea urchin was already observed in previous studies. Albright et al. (2012) found in their study a difference of 28% in the weight of juvenile L. variegatus exposed for 3 months to seawater with 800 matm compared to the control (380 matm). According to Moulin et al. (2011), larvae from intertidal sea urchin were more resistant to acidification than those of species collected from subtidal sites. These authors suggested that organisms living in the stressful intertidal zone may be adapted or acclimatized to pH stress. However, both species used in this study are from intertidal zone, which indicates that the difference between the results obtained should be not related to the organism habitat, in this case. In a research reported by Gianguzza et al. (2014) using the Arbacia lixula sea urchin related that the pH effect of development rate was dependent on the temperature. According to these authors, even at pH 8.2 development to pluteus stage was faster in warmer temperature. Ecotoxicology assays under laboratorial conditions have been very useful and indicate for many types of biomonitoring programs (Environment Canada, 1992; Chapman, 1995; Cesar et al., 2007), which provide specific information from the effects of contaminants to aquatic organisms. The advantage of this type of assays is that the physico-chemical parameters are adjusted according to the optimal conditions for each test-specie. Thus, the temperature not should be associated as a potential cause of effect to the larval success development in this study. It may be suggested in this study that the sensitivity observed in the sea urchin L. lividus could be associated to sediment contamination in the sites from Spain, as these sediments present higher concentrations of metals than the sediments samples from Brazil. Also, It may be suggested that P. lividus is more sensitive than L. variegatus to ocean acidification since this species living in a semi-enclosed sea which is particularly vulnerable to ocean warming due to their cold-water habitats and limited dispersal ability (Merzouk and Johnson, 2011; Zhang et al., 2017). The use of integrated methods have been very applied to the characterization of environmental quality (DelValls et al., 2002; Cesar et al., 2007; Pereira et al., 2011; 2014; Baruarem et al., 2013; Torres et al., 2015). According to Cesar et al. (2014), such analysis allows a deeper understanding of the relationships between variables in the dataset with minimal loss of information, providing an objective procedure to achieve the final decision about the environmental risks assessment. The majority of studies have used this approach in recent years in order to identify the relationship between biological effects and metal toxicity (Baruarem et al., 2013; Cesar et al., 2014; Pereira et al., 2014) but this approach has been also used to understand the effects from ocean acidification (Riba et al., 2010; Basallote et al., 2014, 2015; De Orte et al., 2014a; Rodriguez-Romero et al., 2014a; Szalaj et al., 2016; Goulding et al., 2017). The results of PCA obtained in this study indicate that the inhibition on the larval development of sea urchin was strongly correlated with the pH reduction and the concentrations of As in the elutriate treatments. These results agree with the results reported by Szalaj et al. (2016) that identified pH reduction as a main cause of adverse effects on the mussels Perna perna. Nevertheless, studies have demonstrated a greater correlation between biological effects and toxicity caused by the increase of dissolved Zn in related to low pH values. The increase of mobility of dissolved Zn contributed with the mortality

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of adult and juveniles bivalves (Riba et al., 2010; Basallote et al., 2015), and amphipods (Basallote et al., 2014). Moreover, it has also been reported an association between other metals and adverse effects such as the increase on the mobility of Cu, As and Ni related to the pH reduction. In Rodriguez-Romero et al. (2014a) was reported a correlation between the increase of Cu, Zn and As with the mortality of Hediste diversicolor polichaeta. Furthermore, the metals Ni and As have been demonstrated to be associated with the mortality of amphipod Hyale youngi from Brazil (Goulding et al., 2017). This study reported a comparative approach between the results from tropics and temperate zones (Brazil and Spain) in relation to the effects of ocean acidification caused by CO2 enrichment on the embryo-larval development of L. variegatus and L. lividus sea urchin. The results obtained of metal mobility (Fig. 4) suggest that if the data were treated separately, the metal As could be also associated with the larval development inhibition on the sea urchin L. variegatus, and the metal Cu and Al to be associated with the larval development inhibition on the sea urchin L. lividus. However, it is important to note that all of these related studies before have been strongly associated adverse effects with the pH reduction even if the data are treated on separately way, which indicates this factor as a potential toxic to the marine organisms. Furthermore, it is observed similar results for all the sediment sampling sites and the prevalence of the factor 2 (e.g., related to embryo-larval development inhibition) that inform about the toxicity related to low values of pHs (Fig. 6). In the case of the TRO sediment, it was found a higher positive correlation also with the factor 1 (e.g., related to sedimentary matrix). These results suggest that the larval development inhibition was also correlated with sediment parameters such as metal concentrations, percentage of fines and organic matter, and certain dissolved metals. These results are similar to previous studies that have reported strong correlations between sediment parameters and mortality rates (Nipper, 1998; Krull et al., 2014). Additionally, when assessing the mobility of metals in a sediment-water environment, it should also be considered that the mobility of the metals present in marine sediments depends on their association with the solid phases to which they are bound (Ure and Davidson, 2001), and that the acidification of the samples tends to release the metals that are less strongly associated with sediments increasing their potential bioavailability (Riba et al., 2004). The sediment samples from Spain have showed to be thinner than the sediment samples from Brazil. This way, it is suggested that the higher mobility in the elutriate sediments from Spain in related with the form which the metals are present in the sediment as well the size of these sediment. There is a clear trend for all the stations about the prevalence of Factor 2 that increase its value when the pH values decrease. As mentioned before, the positive correlation of factor 2 starting at pH 7.0 for RSP, TRO and PAI stations and at pH 6.5 for CPI station. It also may be suggested in this study that there were no influence of metals concentrations in the RSP and PAI sampling sites (e.g., related to the cleaner sampling sites in each country); that it was found no correlation with the factor 1 among the pH levels in both sites (RSP and PAI). In the case of the CPI sediment site, it was showed a higher correlation in the factor score associated with Factor 3 in all pH values used in this study; which it is suggests to be related with the concentrations of Zn in both sediment and elutriate sediment treatments. Besides larvae from the tropical sea urchin (L. variegatus) showed to be more tolerant to pH changes in the seawater than larvae from the temperate sea urchin (P. lividus). It is highlighted in this study that the main cause associated with the larval development inhibition was the pH reduction, which it may be change according to the contamination level of the

sediment sample. These findings contribute with the studies of the risk characterization in relation to CO2 enrichment in the ocean and to improve the knowledge of the effects from the ocean acidification phenomena to marine organisms living in different zones. It is also suggest the urgent need to perform experiments on a wide range with different species from different zones, in order to understand and predict the best way to control these effects and support a sustainable development. 5. Conclusion The present study provides information on the consequences of changes in the seawater pH and ocean acidification on the embryolarval development of sea-urchins from different coastal zone (Spain and Brazil). The larvae from Paracentrutos lividus specie showed to be more sensitive to acidified elutriate sediments than larvae from Lytechinus variegatus sea urchin. However, it was suggested that these sensitivities could be associated to sediment contaminations; the sediments tested from Spain have presented higher metal concentrations than the sediments used in the Brazilian samples. It has been demonstrated that the CO2 enrichment in aquatic ecosystems cause changes to the mobility of the next metals: Zn, Cu, As, Fe and Al, and reach concentrations up above those recommended by Environmental Protection Agency for chronic toxicity in saltwater. Furthermore, the principal component analysis indicates that the main cause of the larval development inhibition was associated with low values of pH (at pH 7.0 in the Río San Pedro, Trocadero and Palma Island elutriates sediments, and at pH 6.5 in the Piaçaguera channel elutriate sediment). It can also be associated with the concentrations of As in the elutriate sediments. Moreover, the results using the factor scores also showed a strong correlation of the factor 2 (related with embryo-larval inhibition) with the pH reduction in all stations tested in this study. From these results, it can be established from this comparative study that both species are able to assess potential effects of the ocean acidification related to CO2 enrichment by both near future scenarios and the risk associated with CO2 leakages in the carbon capture and storage process. Additionally, the increase on the mobility of metals in both zones could indicate the importance of comparative studies in different types of environment and improve the understood of the risks associated with the ocean acidification. Acknowledgements The authors are grateful to international Grant from Santander Bank/UNESCO Chair UNITWIN/WiCop for funding this study. The first author would like to thank the Erasmus Mundus Program for the doctoral fellowship (2014-0693/001-001-EMJD). A. Cesar would like to thank the Brazilian National Council for Scientific and Technological Development (CNPq) for the productivity fellowship (No. 305869/2013-2). T.A. DelValls would like to thank the Ministry of Education in Spain for supporting his participation in this work through the Movilidad de Profesores en el Extranjero program under grant (No. PRX16/00074). Thanks are due to Prof. Ionan Marigomez and Prof. Manu Soto from PIE at University of Basque Country for their input to improve the quality of this paper. Also thanks are due to Dr. Fernando S. Cortez and Fabio H. Pusceddu at Santa Cecília University for the support in the data analsysis. The authors would also like to thank Eric Russell for the English revision. References SCCS Scottish Carbon Capture & Storage. http://www.sccs.org.uk/expertise/map.

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