15TH INTERNATIONAL SYMPOSIUM ON TOXICITYASSESSMENT

Lethal effects on different marine organisms, associatedwith sediment–seawater acidification derivingfrom CO2 leakage

M. D. Basallote & A. Rodríguez-Romero & J. Blasco &

A. DelValls & I. Riba

Received: 9 December 2011 /Accepted: 27 March 2012# Springer-Verlag 2012

Abstract CO2 leakages during carbon capture and storagein sub-seabed geological structures could produce potentialimpacts on the marine environment. To study lethal effectson marine organisms attributable to CO2 seawater acidifica-tion, a bubbling CO2 system was designed enabling a bat-tery of different tests to be conducted, under laboratoryconditions, employing various pH treatments (8.0, 7.5, 7.0,6.5, 6.0, and 5.5). Assays were performed of three exposureroutes (seawater, whole sediment, and sediment elutriate).Individuals of the clam (Ruditapes philippinarum) and early-life stages of the gilthead seabream, Sparus aurata, wereexposed for 10 days and 72 h, respectively, to acidified cleanseawater. S. aurata larvae were also exposed to acidifiedelutriate samples, and polychaete organisms of the specieHediste diversicolor and clams R. philippinarum were alsoexposed for 10 days to estuarine whole sediment. In the fishlarvae elutriate test, 100 % mortality was recorded at pH 6.0,after 48 h of exposure. Similar results were obtained in the clam

sediment exposure test. In the other organisms, significantmortality (p<0.05) was observed at pH values lower than 6.0.Very high lethal effects (calculating L[H+]50, defined as the H+

concentration that causes lethal effects in 50 % of the popula-tion exposed) were detected in association with the lowest pHtreatment for all the species. The implication of these results isthat a severe decrease of seawater pH would cause high mor-tality in marine organisms of several different kinds and lifestages. The study addresses the potential risks incurred due toCO2 leakages in marine environments.

Keywords Carbon capture and storage (CCS) . pH .Toxicitytest . Seawater . Sediment . Sediment elutriates

Introduction

The long-term sequestration of carbon in sub-seabed geolog-ical structure is one of the CO2 mitigation strategies consid-ered in numerous conventions and directives such as theLondon Protocol (1996, 1997), London Convention (2006,2007), OSPAR Convention 2007, and the EC Directive,European Parliament on 17 December 2008. Amendmentsof the London Protocol were adopted in 2006 with the objectof regulating this activity by including the “Risk Assessmentand Management Framework for CO2 Sequestration in Sub-seabed Geological Structures” and the “Specific Guidelinesfor Assessment of Carbon Dioxide Streams for Disposal intoSub-seabed Geological Formations.”

At present, several countries are interested in the devel-opment of carbon capture and storage (CCS) technologies.Furthermore, the EU is promoting and contributing to prog-ress in CCS technologies by financially supporting newprojects. Thus, since numerous projects on CO2 storage insub-seabed geological structures are currently in hand, studies

Communicated by Philippe Garrigues

M. D. Basallote (*) :A. DelValls : I. RibaDepartamento de Química-Física, Facultad de Ciencias del Mar yAmbientales, Universidad de Cádiz. UNESCO/UNITWIN Wicop.Campus de Excelencia Internacional del Mar (CEIMAR),Polígono Río San Pedro s/n,Puerto Real 11510 Cádiz, Spaine-mail: dolores.basallote@uca.es

A. Rodríguez-Romero : J. BlascoDepartamento de Ecología y Gestión Costera, Instituto de CienciasMarinas de Andalucía (CSIC),Campus Río San Pedro,11510 Puerto Real, Cádiz, Spain

J. BlascoUnidad Asociada Oceanografía Interdisciplinar UCA-CSIC,Campus Río San Pedro,11510 Puerto Real, Cádiz, Spain

Environ Sci Pollut Res (2012) 19:2550–2560DOI 10.1007/s11356-012-0899-8

are urgently needed to obtain reliable research-based informa-tion for correct risk assessment and to plan monitoring net-works (Keating et al. 2011).

Despite the potential contribution of CCS to the reductionof the atmospheric concentration of CO2, one of the risksassociated with this technology lies in possible CO2 migra-tion or leakages from the storage site. Different scenarios ofCO2 leaks are possible, such as gas migration throughcompromised boreholes or gas-permeable natural faults(Annunziatellis et al. 2008), and gas seepage could occurduring the injection process.

One of the main impacts expected in CO2 leakage sce-narios is the acidification of the environment. A decrease ofpH associated with the increase of CO2 could producechemical changes in the sediment–seawater interface, lead-ing to biogeochemical alteration in marine ecosystems. Inaddition, acidic conditions due to decreased pH will enhancethe mobility of trace metals or other contaminants that couldbe present in marine sediment, making these substancesmore available in the overlaying water column (Ardelan etal. 2009; Riba et al. 2010).

The bivalve mollusks are organisms widely used in labora-tory experiments, and they tolerate a wide range of salinity andtemperature values (Casado-Martínez et al. 2006; Riba et al.2004b). Marine calcifying organisms are among the speciesmost susceptible to impact by CO2 acidification, since theircalcification rate could be altered. These organisms play es-sential functional roles in coastal ecosystems and also consti-tute commercially important marine products (Ishimatsu et al.2004). Consequently, studies of these kinds of organism and oforganisms with calcareous skeletons or shells must be consid-ered when assessing the possible effects of CO2 acidification.

Fish larvae have been used previously to test the toxicity ofseveral chemicals or pollutants present in marine environ-ments (DelValls et al. 1998a, b; Hampel and Blasco 2002;Oliva et al. 2008). According toMcKim (1977) and Ishimatsuet al. (2004), the early developmental stages of marine organ-isms are generally more susceptible to environmental stressorsthan adults. Thus, negative impacts of elevated CO2 environ-ments appear to be particularly strong during early develop-mental stages. Even if adult organisms may be adaptable tohigher CO2 levels, evaluations of the effects of CO2 acidifi-cation events need to consider not only susceptible marinespecies but also the life stages of those species when they areespecially vulnerable.

Polychaetes have been utilized frequently as indicator spe-cies of environmental conditions. The exposure of this kind oforganism has been shown to be a useful tool for assessingenvironmental quality through toxicology tests. Because oftheir relative immobility and intimate contact with the sedi-ment, they are exposed to any harmful material present notonly in the sediments but also in the pore water and watercolumn (Dean 2008; Kalman 2009; Ramos-Gómez 2011).

The purpose of this study is to test the impact on marineorganisms of CO2-induced acidification, due to leakagesfrom marine storage in geological formations, by studyinglethal effects on several marine species. This work addressesseveral ecologically relevant CCS scenarios, by comparingthe present-day pH of a coastal-estuarine area (pH 8.0), thepredicted decrease of ocean surface pH by 0.3–0.5 U (topH 7.5) by 2100 (Caldeira and Wickett 2005), and thepredicted pH reduction in particular marine environmentsdue to leakages from CO2 storage sites (Payan et al. 2012),i.e., possible pH variation of more than 0.5 U (to pH 7.0,6.5, 6.0, and 5.5). A bubbling CO2 system has beendesigned to conduct a battery of short-term toxicity testsunder laboratory conditions (non-pressurized) employingdiverse pH treatments. The effects of elevated CO2 levelsin three marine organisms (clams of the specie Ruditapesphilippinarum, fish larvae of the specie Sparus aurata, andthe ragworm Hediste diversicolor) have been studied usingstandardized toxicity test guidelines, since toxicity tests arean important means of assessing the toxicity of marineenvironments.

Materials and methods

CO2 system

A non-pressurized laboratory-scale CO2 injection systemhas been designed to expose marine organisms to differentpH treatments (Fig. 1). The “AT Control System” fromAqua Medic of North America (Loveland, CO, USA) is acomputer-controlled system for measuring and controllingpH levels electronically in aquaria. It was used in this studyto modify, control, and maintain different pH levels, inde-pendently, in several test chambers. The pH in each aquar-ium was measured by pH electrodes (NBS scale), connectedto the computer system. The pH was monitored continuous-ly, and pH values were adjusted by injecting pure CO2 gasthrough a solenoid valve. The valve delivers the gas in eachaquarium when pH rises above the set value, in 0.01 U, untilthe required pH value is reached. The CO2 injection systemwas adapted to work with three routes of exposure: cleanseawater, whole sediment, and sediment elutriates. Twodifferent sizes of test chamber were used depending on theorganism exposed: One is a fish box with dimensions 35×27×29cm (capacity 25 L, approximately), and the other is a2-L glass beaker. The whole sediment toxicity test wasconducted applying a water-to-sediment ratio of 1:3 (v/v),adapted from Riba et al. (2004b). Sediment elutriate wasobtained by mixing sediment and filtered seawater (ratio 1:4v/v). The mix was maintained under agitation (60 rpm) for30 min, followed by a settling down process for at least 12 h(DelValls et al. 1998a, b; Beiras 2002). Then the supernatant

Environ Sci Pollut Res (2012) 19:2550–2560 2551

fraction was carefully extracted and placed in a new glassvessel. The pH of each elutriate was adjusted before andafter mixing, by injection of CO2. The pH in each aquariumwas verified daily using a portable pH meter (model:Phenomenal 1000 H; accuracy±0.005 pH), calibrated usingpH buffer solutions of 4.00 and 7.00. The oxygen saturationwas recorded using an Oxy 4000 H meter (accuracy±0.5 %of measured value). Salinity was measured by a Conductivity3000 H meter (accuracy±1 % of measured value). Totalalkalinity (TA) was determined by automatic titration(Metrohm 794) using a combined glass electrode (Metrohm,ref. 6.0210.100) calibrated on a total scale. The pH and TAvalues were measured in a separate laboratory system andused to determine the seawater carbonate system speciation.

Water and sediment samples

Water and sediment used were collected from the Río SanPedro shallow tidal creek area, located at the Bay of Cádiz(SW Spain) (36°31′52.90″N; 6°12′48.43″W) (Fig. 2). Thewater used in all experiments was collected from the surface(1 m depth), during high tide (salinity 31±1), transported tothe laboratory (Marine and Environmental Science Faculty,University of Cádiz), and placed in a 400-L tank and filteredusing a high-power external filter with 1 mm pore size. Thesediment sampling procedure was according to Riba et al.(2004a). Sub-samples were collected for chemical quantifi-cation of organic carbon, organic matter content, and metals.Sediments were sieved through a 2-mm mesh and stored in acooler (4°C) for 2 weeks in darkness until the toxicity testswere carried out. Before use, sediment was homogenizedwith a Teflon spoon until no color or textural differences

were detected. All beakers were thoroughly cleaned withacid (10% HNO3) and rinsed in double de-ionized (Milli-Q)water prior to sampling and storage. Organic matter contentin the sediment was obtained through calcinations (LOI) in amuffle furnace at 450°C. Organic carbon content was deter-mined using the technique described by Gaudette et al.(1974), modified by El-Rayis (1985). Metal concentrationin sediment was determined using the method of aqua regiaextraction (ISO11466 1995). Trace metal concentrations(Al, As, Co, Cr, Cu, Fe, Ni, Pb, and Zn) were determinedby inductively coupled plasma optical emission spectrome-try (ICP-OES). For mercury, a LECO AMA 254 analyzerwas used. The results were checked using MESS-1 NRCreference material.

Toxicity test

Toxicity tests were carried out to determine acute effectsassociated with pH changes by increases in CO2 concentra-tion. Three different marine species were used in this study.Temperature (degree Celsius), oxygen saturation (percent),salinity, and pH (NBS scale) were measured daily inside theexposure vessels (see Table 1). The pH values of the treat-ments ranged between 8.0 and 5.5 in the toxicity testsemploying exposure to liquid phase (clear seawater andsediment elutriates). However, in the toxicity tests in whichwhole sediment was used, the pH values did not fall belowthe value of 6.0. In the experiments, the pH value wasdecreased slowly, by 0.5 U/day, until the selected pH valuewas reached, in order to avoid a sudden change of pH in theexperiment chambers. Survival (percent) for the time ofexposure was measured as the endpoint. Aliquot samples

CO2 bottle

Solenoid valve

Automatic CO2 central

AT control System

sediment layer

SeawaterDuplicate exposure aquaria

Whole sediment Duplicate exposure aquaria

pH sensor

(a)

(b)

Sediment elutriate experiment adaptationTriplicate exposure 25 mL vessel

(c)

Fig. 1 Schematic design of theCO2 injection system used todevelop the toxicity testscarried out in this study. Thissystem intends to adjust,maintain, and control the pH inthe aquarium automatically byCO2 bubbles injection

2552 Environ Sci Pollut Res (2012) 19:2550–2560

were taken at the different pH treatments vessels to measureTA.

Fish larvae

The experimental design of the fish larvae toxicity test wasadapted from DelValls et al. (1998a, b), OECD (1992,1998), and USEPA (2002) to obtain normalized results forfish early-life stage toxicity test. Although the CO2 injectionsystem was designed to work with very large aquaria, it wasadapted to work with small organisms such as fish larvae. Inthat case, for both seawater and elutriates test, the pH treat-ments were prepared in 2-L glass vessels. One-day-old fishlarvae of gilthead seabream (S. aurata) were acquired fromthe Marine Culture Laboratory (University of Cádiz). The

larvae were maintained for 2 days in filtered seawater withlight aeration and temperature 19°C±1 for acclimatization.Three samples of aliquot were taken from each pH treatmentand placed in 25-mL polypropylene vessels. After the accli-matization time, fish larvae (n010) were placed in each sam-ple solution for 72 h. The pH values in the treatments were 8.0(±0.1), 7.5 (±0.1), 7.0 (±0.1), 6.5 (±0.1), 6.0 (±0.1), and 5.5(±0.1). In order to maintain the pH in the 25-mL vessels, thesolution was replaced three times each day. The number ofsurviving organisms was counted after the exposure time.

Bivalves

Clams (R. philippinarum) were obtained from an aquacul-ture farm (Haliotis S. L., Cádiz, Spain) and preserved in the

Bay of Cádiz

Iberian Peninsula

Río San Pedro

Puerto Real

Atlantic Ocean

San Fernando

Cádiz

Sample site

N

Fig. 2 Map of the location ofwater and sediment collectionin the southwestern of theIberian Peninsula

Table 1 Summarized description of the different toxicity tests and different marine organisms tested in this study (organism type, exposure route,pH range, experiment physicochemical parameters: T, S, and DO, duration and endpoint evaluated)

Organism Exposure route pH range T (°C) S DO (%) Duration Endpoint

S. aurata larvae Seawater 8.0−5.5 19±1 31±1 >80 72 h Mortality

S. aurata larvae Sediment elutriate 8.0−5.5 19±1 31±1 >80 72 h Mortality

R. philippinarum Seawater 8.0−5.5 20±1 31±1 >80 10 days Mortality

R. philippinarum Whole sediment 8.0−6.0 20±1 31±1 >80 10 days Mortality

H. diversicolor Whole sediment 8.0−6.3 20±1 31±1 >80 10 days Mortality

T temperature, S salinity, DO oxygen saturation

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laboratory to acclimatize for 1 week. Clams were fed with amixture of marine microalgae (Tetraselmis chuii, Isocrhysisgalbana, and Chaetoceros gracilis). Salinity (31±1), temper-ature (20°C±1), and dissolved oxygen saturation (>80 %)were monitored daily both during the acclimatization periodand during the exposure time. Each pH treatment was carriedout in duplicate. In clean seawater experiments, organisms(n015) were exposed for 10 days in aquaria of 25-L capacity.The pH values of the treatments were from 8.0 (±0.1) to 5.5(±0.1). In another experiment, clams (n018) were exposed tovarious pH treatments of whole sediment–seawater interface(ratio 1:3v/v; 6.25 L of sediment, and 18.75 L of water,approximately), with a pH range between 8.0 (±0.1) and 6.0(±0.1). Water was replaced every 3 days in both experiments.Mortality was checked at the beginning and at the end of theexposure time (Riba et al. 2004b, 2010).

Polychaetes

Polychaete organisms (H. diversicolor) were collected fromthe intertidal zone of the Río San Pedro intertidal creek area,taken to the laboratory, and preserved there for 1 week foracclimatization, until the beginning of the experiment. Salinity(31±1), temperature (20°C±1), and dissolved oxygen satura-tion (>80 %) were monitored daily both during the acclimati-zation period and during the exposure time. Each pHtreatment was carried out in duplicate. A total of 12 organisms(n012) were placed in 25 L aquaria (ratio 1:3v/v; 6.25 L ofsediment and 18.75 L of water, approximately) containingsediment–seawater. The pH values of treatments were 8.0(±0.1), 7.3 (±0.1), and 6.3 (±0.3). The water was changedevery 4 days. Mortality was checked at the beginning and atthe end of the exposure period (Kalman 2009).

Data calculation

Data of total inorganic carbon (TIC), bicarbonate (HCO3−),

carbonate (CO32−), the partial pressure of CO2 (pCO2), and

saturation states for aragonite and calcite were calculatedusing the CO2SYS program (Pierrot et al. 2006) with disso-ciation constant from Mehrbach et al. (1973) with refit byDickson and Millero (1987) and KSO4 using Dickson (1990).

Mortality was calculated based on survival percentagemeasured after exposure for 10 days (clams and ragworms)

and for 72 h (fish larvae). The data were used to calculatethe toxic parameter LC50 associated with H+ concentration.The L[H+]50 was defined as the H+ concentration thatcauses lethal effects in 50 % of the population exposed.This parameter was estimated through calculated mortalityassociated with the different pH treatments, using the pHthat provokes mortality in 50 % of the population exposed.It was calculated by the linear interpolation method forlethal toxicity. Variation in mortality rates of each organismbetween pH treatments was examined, using analysis ofvariance, followed by Dunnett’s test, by use of SPSS 17.0statistical software, to determine significant differences (p<0.05) between the results obtained in each pH treatment andthe control pH treatment.

Results

The results of metal concentration measured in the sedimentused in the experiments (Al, As, Cd, Co, Cr, Cu, Fe, Ni, Pb,Zn, and Hg) are given in Table 2. The highest metal concen-tration was found for aluminum, followed by zinc, copper,chrome, nickel, iron, lead, arsenic, cobalt, and mercury, in thatorder; cadmium concentration was below the ICP-OES detec-tion limit. Organic carbon content is 1.06±0.13 and organicmatter content 6.83±0.07. Mean values for carbonate systemspeciation are represented in Table 3. Table 3 refers to thecarbon parameters calculated in the clear seawater treatmentsat different pH values. The salinity parameter used was 31 andtemperature was 20°C. The pH range is between 7.88 and 5.92(total scale). The pCO2 value calculated was 436.01 μatm atthe highest pH and 67,763.03 μatm at lowest pH value. It alsorefers to the carbon parameters calculated in sediment elutriatetreatments at different pH. The salinity parameter usedwas 31.The pH is between 7.77 and 6.54 (total scale), and in compar-ison with the seawater results, an extremely high pCO2 valueof 119,459.10 can be observed at the lowest pH value.

Figure 3 represents mortality associated with pH value forthe fish larvae exposed to acidified seawater and sedimentelutriate. The pH value of 8.0 indicates the control sample inboth exposure routes. Significant mortality (p<0.05) com-pared to control pH was recorded in the fish larvae seawatertoxicity test at pH values of 6.0 and 5.5, representing mortalityrates of 60 and 100 %, respectively. Significant mortality (p<

Table 2 Summary of the physicochemical characteristics for the sediment used in the toxicity tests

Parameters Al As Cd Co Cr Cu Fe Ni Pb Zn Hg C org OM

Data 2.74 3.37 n.d. 2.48 6.30 10.75 5.66 5.67 5.53 20.99 0.10 1.06±0.13 6.83±0.07

Organic carbon content, grain size, and metals concentration data are given. All concentration are expressed as milligrams per kilogram dry weight,except the organic carbon content, organic matter content, Fe, and Al are expressed in percent

C org organic carbon, OM organic matter, n.d. not detected

2554 Environ Sci Pollut Res (2012) 19:2550–2560

0.05) was observed in sediment elutriate at pH values of 7.0 orless. Non-significant mortality (p<0.05) was observed in therest of the pH treatments. Mortality recorded in the clamtoxicity tests (water and whole sediment) is shown in Fig. 4.

A mortality rate of 100 % was calculated in both pH treat-ments after 10 days of exposure to acidified seawater.Significant mortality (p<0.05) was observed at pH values of6.0 and 5.5. The whole sediment exposure test resulted insignificant mortality (p<0.05) at pH of 6.0, since mortality of100 % was calculated at the end of the exposure time. Non-

Table 3 Summary of carbonate system speciation in seawater pH treatments and sediment elutriate pH treatments

pHtot pHNBS TA TIC HCO3− CO3

2− CO2 pCO2 ΩArag ΩCal

Seawater pH treatments

7.88 (±0.00) 8.15 2,604.56 2,489.68 2,362.34 99.34 28.00 436.01 1.51 2.43

7.60 (±0.07) 7.84 2,747.22 2,733.28 2,616.23 54.12 62.93 981.55 0.82 1.32

7.22 (±0.00) 7.42 2,754.98 2,895.17 2,703.34 21.33 170.50 2,659.30 0.32 0.52

7.03 (±0.02) 7.22 2,864.74 3,127.45 2,830.82 14.11 282.52 4,406.42 0.21 0.34

6.35 (±0.01) 6.53 2,917.63 4,330.10 2,910.80 3.00 1,416.30 22,089.70 0.05 0.07

5.92 (±0.00) 6.10 3,315.74 7,659.30 3,313.60 1.30 4,344.70 67,763.03 0.02 0.03

Sediment elutriate pH treatments

7.77 (±0.02) 8.03 2,668.80 2,594.35 2,475.35 79.60 39.40 598.40 1.21 1.94

7.78 (±0.00) 8.04 3,444.74 3,353.85 3,200.40 104.80 48.65 758.70 1.59 2.56

7.55 (±0.00) 7.79 4,366.06 4,383.36 4,192.55 76.66 114.15 1,780.10 1.17 1.87

7.43 (±0.01) 7.65 16,509.16 16,876.10 16,063.90 215.15 597.05 9,311.70 3.27 5.25

6.54 (±0.02) 6.73 24,913.22 32,530.75 24,831.45 40.10 7,659.20 119,459.10 0.61 0.95

The carbon parameters were calculated based on total pH, temperature (20°C), salinity (31), and total alkalinity. pH units are moles per kilogramSW for the pH total scale and moles per kilogram H2O for the pH NBS scale. TA is expressed in micromolars and TIC, HCO3

− , CO32− , and CO2

are expressed in micromoles per kilogram. pCO2 is expressed in micro-atmospheres

pHtot total pH, TA total alkalinity, pHNBS pH NBS scale, ΩArag saturation state of aragonite, ΩArag saturation state of calcite

0

20

40

60

80

100

120

8.0 7.5 7.0 6.5 6.0 5.5

Mo

rtal

ity

(%)

pH

(a) S. aurata larvae -seawater

0

20

40

60

80

100

120

8.0 7.7 7.5 7.0 6.5 6.0 5.5

Mo

rtal

ity

(%)

pH

(b) S. aurata -sediment elutriate

Fig. 3 Results of mortality of the fish larvae S. aurata exposed toacidified seawater (a) and sediment elutriate (b). pH 7.7 is referring tothe elutriate pH got from the mixing between seawater and estuarinesediment without any CO2 injection

0

20

40

60

80

100

120

8.0 7.5 7.0 6.5 6.0 5.5

Mo

rtal

ity

(%)

pH

(a) R. philippinarum - seawater

0.00

20.00

40.00

60.00

80.00

100.00

120.00

8.0 7.0 6.5 6.0

Mo

rtal

ity

(%)

pH

(b) R. philippinarum - whole sediment

Fig. 4 Results of mortality associated with pH for the clam R. philip-pinarum exposed to acidified seawater (a) and whole sediment (b)

Environ Sci Pollut Res (2012) 19:2550–2560 2555

significant mortality (p<0.05) was observed in the other pHtreatments. A general trend of the highest mortality at thelowest pH values was observed in clams and fish larvaetoxicity tests. Non-significant mortality (p<0.05) was detectedin ragworm H. diversicolor exposed to whole sediment atdifferent pH values (Fig. 5).

The L[H+]50 values obtained for each organism in thedifferent exposure routes are summarized in Table 4. The unitsfor concentrations of protons ([H+]) are moles per kilogramH2O for the NBS scale. The pH value that provokes mortalityin 50 % of the fish larvae population exposed to clean seawa-ter and sediment elutriate is 6.08±0.07 and 6.31±0.03, re-spectively. From those values, the L[H+]50 value estimated is8.25×10−7 mol kg−1 H2O in the seawater exposure route, and4.90×10−7 mol kg−1 H2O in the sediment elutriate exposureroute. In both the toxicity tests with clams, similar L[H+]50values were calculated. The pH value at which 50 % ofindividual clams exposed to seawater died was 6.26±0.00,and the equivalent pH value in the clams exposed to wholesediment was also 6.26±0.00. Therefore, the L[H+]50 valueestimated is 5.51×10−77 mol kg−1 H2O for seawater and5.50×10−7 mol kg−1 H2O for whole sediment toxicity tests.The L[H+]50 value could not be calculated for polychaeteorganisms because no mortality higher than 16 % wasdetected in any of the pH treatments.

Discussion

To mimic environmental conditions in laboratory experimentsis a goodway to assess the possible effects of CO2 leakages onmarine ecosystems. Bubbling CO2 into experimental vesselshas been contemplated as one of the techniques useful in thecontext of studying the consequences of ocean acidification,according to the “Guide to best practices for ocean acidifica-tion research and data reporting” (Riebesell et al. 2010). Thismethodology is considered an efficient way to manipulate pHvalues (and carbonate chemistry) in laboratory experiments

(Gattuso et al. 2010) and appropriate for the studies requiredfor working on this topic.

It has been demonstrated that sudden changes in pH couldhave drastic adverse effects on marine species, but it is alsoimportant to consider the acclimatization of the organisms tochanges in the acidity of their media. Thus, the speed at whichthe experimental pH treatments are imposed could have asignificant impact on the results obtained from tests. In thiswork, the pH values were lowered slowly, in each experimen-tal vessel (by 0.5 U/day, until the pH values selected werereached), in order to avoid rapid changes of pH that wouldprobably cause the direct death of many of the organisms.Although recent studies have shown that marine animalsmight will adapted to changes in pH levels and to climatechanges in general (Sunday et al. 2011; Hofmann andTodgham 2010; Melzner et al. 2009; Pistevos et al. 2011),sudden changes such as those likely to be provoked due toCO2 leakages from sub-seabed storages sites would not givetime for the acclimatization of certain species. According toHaedrich (1996), “any change that takes place too quickly toallow for a compensating adaptive change within the geneticpotential of finely-adapted deep-water organisms is likely tobe harmful” (Seibel and Walsh 2001).

In addition, many other experimental factors, such astemperature and salinity, oxygen depletion, and ammonialevel, must be controlled in toxicity test assays, especially ifseveral parameters, such as pH, are being modified andwhen sediments are used. However, the results of this workare not linked to the alteration of these kinds of factor.Parameters such as temperature, salinity, and oxygen satu-ration have been controlled to comply with the optimallevels for the organisms in toxicity test standard guidelines.Considering that sediment has been used in some of theexperiments conducted in this work, the NH3/NH4

+ levelshould also be controlled. Nevertheless, of the two forms inwhich ammonia is present in aquatic ecosystems, the onlytoxic form is the non-ionized form, NH3. Thus, since it isknown that ammonia is less toxic at lower pH levels (below7.0, all ammonia is in the form of ammonium), it is notexpected that the mortality of organisms measured in moreacid pH treatments was a consequence of NH3/NH4

+ levels.Furthermore, NH3/NH4

+ levels were below 1.3 mg/L indifferent toxicity tests, in which sediment from the RíoSan Pedro intertidal creek area was used.

Another important factor that should be taken into ac-count in laboratory experiments is whether food has beenprovided during the period of exposure, not only thechanges in CO2 concentration and lower pH values. In arecent study in which the effects of food algae concentrationand pCO2 on Mytilus edulis were studied, Melzner et al.(2011) show that elevated seawater pCO2 coupled with foodlimitation increase negative effects on this organism by en-hanced shell dissolution and corrosion. Furthermore, Parker et

0102030405060708090

100

8.0 7.3 6.3

Mo

rtal

ity

(%)

pH

H. diversicolor - whole sediment

Fig. 5 Results of mortality for H. diversicolor exposed to wholesediment at three different pH

2556 Environ Sci Pollut Res (2012) 19:2550–2560

al. (2011) show that organisms with no naturally abundantfood source are probably better able to support the energyrequirements imposed by increased CO2 acidification. In theexperiments carried out in this study, only the clams were feedad libitum. No food was provided to fish larvae and poly-chaete organisms; it was assumed that test sediments wouldprovide a food source during the exposure of organism towhole sediment (ASTM 2007). The fish larvae fed from theirown yolk sac because they were still young enough to havethat source (OECD 1992).

Coastal and estuarine areas are likely to be susceptible toimpact by CO2 leaks because, depending on the siting ofstorage places and the type of leakage occurring and on oceancurrents and environmental conditions, the CO2 releasedcould reach the coast. It is known that metals are among themost common contaminants existing in estuarine sedimentsand aquatic systems (Luoma and Davis 1983; Riba et al.2004b). A decrease in pH reflects an increase in hydrogenion concentrations. Consequently, the hydrogen ions will pro-tonate the aquatic ligands and replace the trace metals, causingan increase in the free metals ion activity; this is consideredthe most bioavailable form of dissolved metals (Kalman2009). However, the subsequent toxicity in aquatic organismswill depend not only on the concentration and form of themetal in the sediment but also on the physicochemical con-ditions such as texture, organic content, and pH (DelValls etal. 1998b). Focusing on this work, the sediment used presentslow values of concentrations in metals. All metal concentra-tions measured in the Río San Pedro sediment were below themetal concentration levels associated with toxic effects, con-sidering various Sediment Quality Guidelines (SQGs) such asthe Spanish Action Levels for Dredged Material management(CEDEX 1994) based on sediment geochemical considera-tions, SQGs in the Atlantic Coast of Spain (Riba et al. 2004a),and the site-specific SQG for the Gulf of Cádiz (Choueri et al.2009). According to Riba et al. (2003), the mobility of metalsfrom the sediment to the water column increases when the pHvalue decreases; consequently, metals would become morebioavailable, especially those metals more easily mobilizedfrom sediments to water. Similarly, observations have beenverified by Ardelan et al. (2009), who determined that

possible CO2 seepages from submarine geological storageformations into marine ecosystems are expected to causerelease of metals into the sediment–seawater interface, result-ing in an enhanced metal flux to the water column. The keyrole of pH on the bioavailability of metals is highlighted in thiswork because it is well-known that metals are one of the mainpollutants associated with sediments and coastal ecosystems,and the relationship between the acidification of the media andthe availability of metals is important. However, correlatingthe mobility of metals with the different pH levels tested is notamong the specific objectives of this study.

This work has examined the acute effects of reduced pH ondifferent kinds and life stages of marine organisms, in order tocalculate L[H+]50 values, meaning the pH or [H+] values thatcause mortality in 50 % of the population exposed. In the fishlarvae toxicity test, the calculated [H+] value that provokesthis mortality rate was higher in the acidified seawater toxicitytest than in the sediment elutriates. That result means that, in atoxicity test under the same conditions, a pH value of 7.0would cause this mortality rate if fish larvae are exposed tosediment seawater elutriate; however, it would be necessaryfor the pH value to fall to 6.0 to detect significant (p<0.05)mortality in organisms exposed to clean seawater. Althoughthis organism appears to be more sensitive to exposure to adrop in pH in sediment elutriates than in acidified seawater,that result cannot be attributed to metal availability since, aspreviously explained, the metal concentrations present in thesediment used were below the values associated with toxiceffects, according to several different SQGs. Similar resultswere found by Ishimatsu et al. (2004) for fish larvae exposedto seawater, where 100 % mortality was observed in larvae ofthe fish Pagrus major exposed to seawater at pH of 5.9 and60 % mortality at pH of 6.2.

Comparing the results of our toxicity tests in which clamorganisms were used, a similar L[H+]50 value was detectedfor both routes of exposure (seawater and whole sediment);thus, it was found that lethal effects in this specie do dependon pH or [H+] but do not depend on the particular exposureroute. The sublethal effects of CO2-induced acidification inbivalve mollusks have been widely studied. These studiescorroborate that exposure to a small reduction in the pH of the

Table 4 Summary of LC50 defined as the [H+] that provokes 50 % mortality in the population exposed, calculated for eachorganism and exposure route

Organism Exposure route LpH50 L[H+]50

S. aurata (fish larvae) Seawater 6.08±0.07 8.25×10−7

S. aurata Sediment elutriate 6.31±0.03 4.90×10−7

R. philippinarum (adult clam) Seawater 6.26±0.00 5.51×10−7

R. philippinarum Whole sediment 6.26±0.00 5.50×10−7

H. diversicolor (adult polychaete) Whole sediment No effects –

Units for [H+ ] are moles per kilogram H2O for the NBS scale (now the National Institute of Standards and Technology)

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media results in adverse effects on morphology, physiology,growth, and behavior of the organisms (Ishimatsu andDissanayake 2010). In addition, Green et al. (2004) found thatjuveniles of the bivalveMercenaria mercenaria were suscep-tible to dissolution-inducedmortality when theywere exposedto surface sediment under-saturated with respect to aragonite(Ω aragonite≈0.3), which corresponds to pH values of around7.0–7.2. Further, Green et al. (2004) demonstrated that smallbivalves are more susceptible to dissolution mortality thanhigher size classes, concluding that any physical, biological,or anthropogenic mechanism that disrupts the sediment satu-ration state, such as a decline in pH value, may impact bivalvesurvival and benthic community structure present in that area.A further summary of mortality percent of bivalves as afunction of pH can be found in Green et al. (2004) andAuerbach et al. (1997).

In the ragworm whole sediment exposure toxicity test, onthe other hand, non-significant (p<0.05) lethal effects weredetected (no mortality rate higher than 16 % was observedafter 10 days’ exposure). That result confirms the hightolerance of this specie to environmental processes of acid-ification. Beside that inconclusive results, similar answerswere found by Fleeger et al. (2006), who reported that thenematode did not respond to CO2 exposure by differentialrecruitment, mortality, or migration. Furthermore, for thepolychaete Nereis virens, tolerance to pH values below 6.5was demonstrated by Widdicombe and Needham (2007).

When assessing the impact of increased CO2 concentra-tions in aquatic ecosystems, it is important to select speciesthat play a key role in the structure and functioning of theecosystem. However, the response of organisms exposed toelevated pCO2 values could vary within and between pop-ulations, as shown by Parker et al. (2011). For example,deep-living animals would be highly susceptible to the CO2

release and consequent changes in pH values likely toaccompany deep-sea CO2 sequestration. On the other hand,species well-adapted to elevated pCO2 can be found, such asthose living in waters near to hydrothermal vents or instagnant tide pools, which show greater ability for bufferingion exchange and transport of CO2 (Seibel and Walsh 2001).Furthermore, it is during the critical embryonic and larvalstages that estuarine and marine organisms are perhaps mostsusceptible to environmental change because they are stilldeveloping their physiological capacities (Pörtner 2008). Inaddition, the various possible mechanisms of leakage shouldbe taken into account, by differentiating between the variousways that CO2 may be released, such as through continuousdiffuse seepages, temporary point-source leaks, and contin-uous point-source leaks (Blackford et al. 2009). Finally,because different effects have been demonstrated in studiesusing CO2 injection or some other process, such as acidadditions, to produce the required acidification, new meth-ods for studying CO2 leakage into ecosystems should be

focused on CO2-induced acidification in both laboratory andin situ experiments.

Conclusions

The potential increase of CO2 concentration in marine eco-systems associated with leakages from CCS systems couldincrease the acidification of the surrounding environmentcausingmortality in the biota (S. aurata larvae,R. philippinarum)living there. In this study, the highest rates of mortality wereobserved at pH values lower than 6.5 in each pH test treat-ment, except in the case of the H. diversicolor toxicity test, inwhich inconclusive results (no significant mortality (p<0.05))were observed. A range of pH values, between 8.0 and6.0, that two different marine species (S. aurata and R. phil-ippinarum) could support has been established. From thisinformation, we can select the interval of pH values for futuretoxicity tests to study influences of CO2 leakages in a con-taminated ecosystem matrix and to determine sublethal effectson marine organisms.

In addition to the direct impact that a dramatic CO2 leakagecould provoke in the surrounding ecosystem, some otherindirect effects such as increased bioavailability of certaincontaminants already present in the areas affected by theincrease of CO2 concentration need to be considered. Thus,new methods linking toxicity studies with in-laboratory acid-ifying treatments by injecting CO2 gaseous should help in theunderstanding of the risk associated with CO2 leakages fromsub-seabed geological formations.

It is an acknowledged reality that the ocean acidificationpredicted for the next century could cause damage in marineecosystems. To date, numerous studies have been conductedto assess the effects of ocean acidification due to increasedatmospheric CO2 and the decline in pH predicted by the year2100 has been estimated at ≈0.4 U (Caldeira and Wickett2003; Blackford and Gilbert 2007; Havenhand and Schlegel2009). However, effects on ecosystems associated with asharp and sudden decrease in pH because of possible CO2

leakages from CCS systems are largely unknown and need tobe investigated. For that reason, it remains necessary to ad-dress in more detail the influence of acidification associatedwith possible CO2 leakage events on the behavior, bioavail-ability, toxicity, and risk of contaminants linked to the sedi-ment–seawater matrix. Therefore, further studies to investigatethe mobility and bioavailability of metals present in aquaticecosystems at different acidification levels are recommended.

Acknowledgments The work described was supported partially bygrants from the Spanish Ministry of Science and Innovation(CTM2008-06344-C03-02/ TECNO, CTM2008-06344-C03-03/TECNO, and CTM2011-28437-C02-02) and by grant P08-556 RNM3924 from the Regional Government of Andalusia (Junta de Andalu-cía). We thank the following for their contributions: M. De Orte for her

2558 Environ Sci Pollut Res (2012) 19:2550–2560

valuable collaboration in the setup of the CO2 system and performanceof the toxicity tests and the Marine Aquaculture Working Group(Marine and Environmental Science Faculty, University of Cádiz).MDB also thanks Prof. Dr. Jesús Forja for his personal clarifications.This is the CEIMAR journal publication 5.

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