Effect of Carbonate Chemistry Alteration on the Early

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Effect of Carbonate Chemistry Alteration on the Early Embryonic Development of the Pacific Oyster(Crassostrea gigas)Frédéric Gazeau1'2*, Jean-Pierre Gattuso1'2, Mervyn Greaves3, Henry Elderfield3, Jan Peene4, Carlo H. R. Heip4'5, Jack J. M iddelburg4,61 C entre National d e la R echerche Seientifique-lnstitu t National d es Sciences d e l'Univers, Laboratoire d 'O céanog raph ie d e Villefranche, Villefranche-sur-M er, France,2 Université Pierre e t Marie Curie-Paris 6, O bservato ire O céano log ique d e Villefranche, Villefranche-sur-M er, France, 3 D epartm en t o f Earth Sciences, University o f Cam bridge, C am bridge, England, 4 C entre for Estuarine and M arine Ecology, N etherlands Institu te o f Ecology, Yerseke, The N etherlands, 5 D epartm en t o f M arine Organic Biogeochem istry, Royal N etherlands Institu te for Sea Research, Den Burg, The N etherlands, 6 Faculty o f G eosciences, U trecht University, U trecht, The N etherlands

AbstractOcean acidification, d u e to a n th ro p o g e n ic C 0 2 a b so rp t ion by th e ocean, may have profound impacts on marine biota. Calcareous o rgan ism s are ex p ec ted to be particularly sensitive d u e to th e decreas ing availability of c a rb o n a te ions driven by decreas ing pH levels. Recently, so m e s tudies focused on th e early life s tages of mollusks th a t are su p p o sed ly m ore sensitive to env ironm enta l d is tu rbances th an adu lt s tages . A l though th e se s tud ies have sh o w n d e creased g ro w th rates an d increased proport ions of a bnorm a l d e v e lo p m e n t u n d e r low pH conditions, th ey did n o t allow attr ibu tion to pH induced c h an g e s in physiology o r c h an g es d u e to a d ecrease in a ragon i te sa turat ion s ta te . This s tudy aims to assess th e impact of several carbona te -sys tem pertu rba tions on th e g ro w th of Pacific oyster (Crassostrea gigas) larvae during th e first 3 days of d e v e lo p m e n t (until shelled D-veliger larvae). Seaw ate r with five different chemistr ies was o b ta in e d by separately m anipulat ing pH, total alkalinity and a rag o n i te sa tu rat ion s ta te (calcium addition). Results sh o w ed th a t th e d ev e lo p m en ta l success and g ro w th ra tes w e re n o t directly affected by c h an g es in pH o r a ragon i te sa turat ion s ta te b u t w ere highly corre lated with th e availability of c a rb o n a te ions. In con tra s t to previous studies, b o th d ev e lo p m en ta l success into viable D- sh a p e d larvae and g ro w th rates w ere no t significantly a ltered as long as c a rb o n a te ion concen tra t ions w ere a b o v e aragon i te sa turat ion levels, b u t th ey s trongly decreased b e low sa turat ion levels. These results su g g e s t th a t th e m echan ism s used by th ese o rgan ism s to regu la te calcification rates are no t efficient e n o u g h to c o m p e n sa te for th e low availability o f ca rb o n a te Ions un d e r corrosive conditions.

C ita tio n : G azeau F, G attuso J-P, G reaves M, Elderfield H, P eene J, e t al. (2011) Effect o f C arbona te Chem istry A lteration o n th e Early Em bryonic D evelopm ent o f th e Pacific O yster (Crassostrea gigas). PLoS ONE 6(8): e23010. doi:10.1371/journal.pone.0023010

E d ito r: John Murray Roberts, Heriot-W att University, U nited Kingdom

R eceived January 10, 2011; A c cep ted July 11, 2011; P u b lish ed A ugust 10, 2011

C o p y rig h t: © 2011 G azeau e t al. This is an open-access article d istribu ted u n d er th e te rm s o f th e Creative C om m ons A ttribution License, w hich perm its unrestricted use, distribution , and reproduction in any m edium , provided th e original au th o r and source are credited .

F u n d in g : This research has received su p p o rt from th e N etherlands O rganization o f Scientific Research and is a con tribu tion to th e "E uropean Project on O cean Acidification" (EPOCA) w hich received fund ing from th e European C om m unity 's S eventh Fram ew ork P rogram m e (FP7/2007-2013) u n d er g ran t a g ree m en t n° 211384. The funders had no role in study design , d a ta collection and analysis, decision to publish, o r p repara tion o f th e m anuscript.

C o m p e tin g In te rests : The au tho rs have declared th a t no com peting in terests exist.

* E-mail: f.gazeau@ obs-vlfr.fr

Introduction

D ue to the absorption of anthropogenic C 0 2 by the ocean, seawater p H has already declined by 0.1 unit com pared with p re industrial values [1] an d is projected to decrease by ano ther 0.35 unit by the end of the century [2]. T his process, know n as ocean acidification, will m ost likely have profound im pacts on m arine biota. Besides the direct effect o f decreasing p H on the physiology and m etabolism o f m arine organism s th rough a disruption o f in tercellular transport m echanism s [3], calcareous organism s are particularly sensitive due to the decreasing availability o f carbonate ions ( C 0 3~_ ) driven by increasing p C 0 2. T h e calcium carbonate saturation state (Í2) is defined as:

w here K 'sp is the stoichiom etric solubility product, a function o f tem perature, salinity, pressure and the m ineral phase considered

(calcite, aragonite or high-m agnesian calcite), and, as a consequence o f ocean acidification, will significantly decrease in the com ing decades. It m ust be stressed that carbonate saturation states depend no t only on p H bu t also on total alkalinity levels. T o tal alkalinity m easures the ability o f a solution to neutralize acids to the equivalence po in t o f carbonate or b icarbonate , acting as a natu ral buffer to the incorporation o f anthropogenic C 0 2 in the ocean. As the addition (or removal) o f C 0 2 to a solution does not change its alkalinity and since the dissolution o f calcium carbonate m inerals in the w ater colum n an d in the sediments, as well as alkalinity inputs from continental rock w eathering, are very slow processes, they are not expected to significantly buffer ocean acidification in the com ing decades [4].

Several experim ental studies have investigated the effect o f a p C 0 2 increase on the grow th o f calcifying organism s [5]. Species that produce aragonite, less soluble th an low-m agnesian calcite in seawater, will be especially at risk. As am orphous calcium carbonate an d aragonite have been identified as the m ain C a C 0 3 m inerals in larval stages o f benthic mollusks [6], there is a strong

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Oyster Larvae Under Modified Carbonate Chemistry

need to carefully assess the effects o f ocean acidification an d the associated alteration of the carbonate chem istry on their developm ent. Small changes in the abundance an d developm ental success o f these larval stages control the size and viability o f the benthic populations [7] and therefore could induce significant changes in the functioning of coastal ecosystems. Indeed, shellfish are ecosystem engineers governing energy and nu trien t flows in coastal ecosystems, provide habitats for m any benthic organisms and form an im portan t food source for birds [8,9]. M oreover, global shellfish aquaculture p roduction reached 13.1 m illion tons in 2008 (27% o f the global aquaculture yield), corresponding to a com m ercial value o f US$13.1 billion. T h e Pacific O yster (Crassostrea gigas) was the m ost cultivated species in 2008 w ith a volum e of 6.5 m illion tons o r 9.5% of the total w orld aquaculture p roduction (FISH ST A T Plus, Universal software for fishery statistical tim e series, V ersion 2.3, Food and Agriculture O rganization o f the U nited Nations, Fisheries D epartm ent, D ata and Statistics U nit., 2000).

Several recent studies have focused on the effect o f ocean acidification on the early developm ent o f m olluscan species [10,11,12,13,14,15,16,17,18] an d m ost o f them have reported negative im pacts o f decreasing pFl levels on the grow th and developm ent o f these organisms. K u rih a ra e t al. [12,13], Parker et al. [15] and G azeau e t al. [11] have investigated the effects o f decreasing pFl on the early em bryonic (from fertilization to the D- veliger stage) developm ent o f com m ercially im portan t bivalve species. This developm ental period is o f the u tm ost im portance since the onset o f shell m ineralization occurs during the trochophore larval stage and shells are fully m ineralized when larvae reach the D-veliger stage, a t the second or th ird day after fertilization [19]. Studies o f K u rih a ra et al. [12,13] on Crassostrea gigas and Mytilus galloprovincialis showed a strong decrease o f developm ental success into viable D -shaped larvae and growth rates with increased p C 0 2. Flowever, a pFl (on the N ational B ureau of S tandards scale, hereafter referred to as p H NBS) o f ~ 7 .4 was used (0.7 un it lower th an control values), a value lower than th a t projected to occur a t the end o f this century. M oreover, due to low am bien t total alkalinity levels, seawater was highly undersatu ra ted w ith respect to aragonite in the low pFl conditions in these two studies (Í2a o f 0.68 and 0.49, respectively). Parker e t al. [15] studied the early em bryonic developm ent o f the Sydney rock oyster Saccostrea glomerata a t am bien t (375 patm ), 600, 750 and 1000 pa tm p C 0 2 levels. This experim ent showed a general decrease in the percentage and size o f D-veliger with increasing p C 0 2. Flowever, m anipulation o f the carbonate chem istry was perform ed by addition o f a strong acid (F1C1) to reduce pFl, a technique th a t is no t recom m ended as acid addition also decreases total alkalinity, w hich is no t anticipated to occur in the com ing decades. M oreover, as values o f total alkalinity an d C a C 0 3 saturation state were no t provided, com parisons with similar studies are no t straightforward. G azeau e t al. [11] reported on the im pacts o f decreasing pFl levels on the first 2 d developm ent o f blue mussel (.Mytilus edulis) larvae. T hey showed th a t a decrease o f PH nbs to ~ 7 .8 (control: —8.1), associated with a supersaturation with respect to aragonite (Í2a~1 .4 ), had no effect on the percentage o f em bryos developing to viable D-veliger larvae.T h e effect on average final shell length was lim ited (----- 5± 1% ).T heir results show th a t a decrease o f p FInbs to — 7.6, associated with a slight undersaturation with respect to aragonite, had significant effects on the percentage o f em bryos th a t developed to D -veliger larvae norm ally and m ore p ronounced effects onaverage final D-veliger shell lengths (-----13±1% ). Flowever, thisstudy did no t allow the discrim ination betw een the physiological effect o f pFl decrease, via a disruption o f inter-cellular transport

m echanism s, and the effect o f the aragonite saturation state, on the larval developm ent o f this species.

T h e objectives o f the p resent study are to investigate the effect o f various carbonate chem istry alterations, perform ed by m anipulating pFl, total alkalinity and the saturation state w ith respect to calcium carbonate separately on the survival an d grow th of Pacific oyster (Crassostrea gigas) larvae during the first 3 days o f their developm ent.

Materials and Methods

Experim ental se t-upA batch o f one m illion em bryos o f the Japanese oyster

(Crassostrea gigas) was provided by the com m ercial hatchery Roem van Terseke (Yerseke, T h e N etherlands) on 3 Ju n e 2009. This batch was transported w ithin 15 m in to a tem perature-regulated room (19°C) a t the .Netherlands Institute o f Ecology (Yerseke, T h e N etherlands) and evenly distributed into 15 beakers o f 4.5 1 (larval concentration of ~ 1 5 ind. m l-1 ), containing filtered (0.2 pm) seawater from the O osterschelde, the nearby tidal inlet. Five treatm ents were considered, each o f them in triplicate (see Fig. 1 for the experim ental setup). O n e trea tm en t served as a control i.e. beakers were gently bubbled w ith external am bient air. Two beakers were bubbled w ith air a t 1000 and 2000 pa tm o f C 0 2 (T2 and T 3, respectively). T h e fourth trea tm en t (T4) was bubbled with external am bient air, after total alkalinity (Tx) was decreased to — 1000 pm ol kg-1 by adding 14 ml o f F1C1 0.1 N an d 10.6 g o f C aC l2-2Fl20 in o rder to reach saturation states with respect to a ragonite an d calcite o f 1.4 and 2.2, respectively. T h e last group of beakers (T5) was bubbled with 4000 pa tm C 0 2 an d A T was increased by add ing 1.6 g o f N a H C 0 3. Gas cylinders with certified C 0 2 concentrations (1000, 2000 and 4000 patm ) were supplied by W estfalen. Em bryos w ere allowed to develop, w ithout additional feeding, until larvae reached the shelled D-veliger stage, i.e. 72 h.

C arbonate chem istry m easu rem en tsSeaw ater p H (on the total scale, hereafter referred to as p H T)

and tem peratu re w ere m easured twice a day in the beakers. p H x was m easured using a p H m eter (M etrohm , 826 p H mobile) w ith a glass electrode (M etrohm , electrode plus) calibrated on the total scale using T r is /H C l an d 2-am inopyrid ine /H C l buffer solutions with a salinity o f 35.0 [20]. Samples for salinity and T x were taken a t the start an d a t the end o f the experim ent. Samples for A T were filtered on G F /F filters, poisoned with H gC l2 and stored in the dark pending m easurem ent (within few days). Salinity was m easured using a conductim eter (R adiom eter CDM 230). T riplicate potentiom etric m easurem ents o f T x were perform ed using a M etrohm titra to r and a glass electrode (M etrohm , electrode plus). M easurem ents were carried ou t on 50 ml samples a t 25°C a n d T x was calculated using a G ran function. T itrations o f T x from standard seawater provided by A. G. Dickson (batch 82, n = 10) were on average w ithin 0.46 pm ol kg 1 o f the nom inal value. All param eters o f the carbonate chem istry were determ ined from p H x , T x , tem perature and salinity using the R package seacarb

D e

sam pling an d m easu rem en tsA t the end of the incubation period, the beakers were em ptied.

Tw o liters were passed th rough a 30 pm sieve and concentra ted in 50 ml th a t was fixed in a 5% neutralized-form alin seawater solution to determ ine developm ental success (% o f D-veliger larvae) and D-veliger shell length an d area. T h e developm ental success into viable D -shaped larvae was defined as the percentage




Ambient air 450 patm

CO

+ NaHCO

+ CaCI

T2 (x3 T3 (x3)

Targeted values

ControlpHT~ 8.03 Ar ~ 2450 O ~ 2.8

pHT ™ 7.72 4 t ~ 2450 Q ~ 1.5

pHT ~ 7.44 ~ 2450

0 - 0.8

pHT ~ 7.7 A r -1 0 0 0 0 - 1 . 4

T5 (x3)

pHT ~ 7.58 A^ ~ 6762 O ~ 3.2

F igure 1. E xperim en ta l se t-u p . For each treatm ent (in trip licate; T (saturation state o f the seawater w ith respect to aragonite) are indicated, in T4 by HCI add ition and increased in T5 by NaFiC03 addition. In T4, ca addition. See text fo r more details. doi:10.1371/journal.pone.0023010.g001

o f “ n orm al” D -shape larvae following the criteria proposed by His et al. [22], after observation o f at least 500 larvae pe r replicated culture. D-veliger larvae shell length (anterior to posterior dim ension of the shell parallel to the hinge) an d a rea were m easured on 100 larvae pe r replicate based on pictures taken under a microscope (20 x; 0.01 pm precision in length m easurem ent), using the software Leica Q w in Pro version 2.4. A t the end o f the experim ental period, 2 1 o f the cultures were filtered onto G F /F

Table 1. Environmental p a ram ete rs and c a rb o n a te chem istry fo(m ean ± SD).

18.9±0.1°C), the target AT (total alkalin ity in pm ol kg-1 ), pHx and Qa Ht was controlled by bubbling am bient or h igh-C 02 air. AT was decreased :ium concentrations have been increased above am bient levels by CaCI2

filters for subsequent analyses o f calcium (Ca~+) concentrations. Larvae-free seawater was filtered onto G F /F filters (4 x) and served as blanks for Ca~+ m easurem ents. Ca~+ concentrations were determ ined by inductively coupled plasm a emission spectrophotom etry (ICP-OES) after multiple rinses with deionised w ater to remove seawater Ca~+. In addition to Ca~+ from the larvae, Ca~+ retained on the filters m ay come from residual sea salts, from the G F /F filters themselves a n d /o r from the analytical blank o f the procedure. T o

th e five different t r ea tm e n ts during th e course o f th e exper im en t

T1 (c o n tro l) T2 T 3 T 4 T5

M e asu red p ara m e te rs

T em peratu re (°C) 18.9±0.1

Salinity 34.0±0.1 34.1 ±0.1 34.1 ±0.1 35.4 ±0.1 34.3 ± 0 .0

pHT 8.03 ±0.01 7.72 ±0.03 7.41 ±0.03 7.67 ±0.03 7.62 ±0.12

A j (jxmol kg -1 ) 2452.7 ± 6 .6 2446.2 ± 8 .2 2443.1 ±3.1 1093.8 ± 4 .0 6726.6 ± 37 .6

C o m p u te d p a ra m e te rs

p C 0 2 (jiatm) 448.7 ± 15 .6 1019.9 ± 79 .9 2170.5 ±156 .9 493.5 ± 42 .7 3730.4 ±946.5

CT (jxmol kg -1 ) 2207.0±10.1 2340.8 ±12.3 2443.4 ± 10 .6 1029.4 ± 8 .9 6589.3 ±133 .9

[HC03_ ] 2010.0 ± 13.0 2209.4±15.1 2320.3 ± 8 .6 972.9 ± 9 .8 6238.0 ±161 .9

[C032~] 181.9± 4 .2 97.3 ± 6 .3 50.4 ± 3 .3 40.0 ± 2.4 226.5 ±60.3

Qa 2.8±0.1 1.5 ±0.1

o+1ooo

1.6 ±0.0* 3.5 ± 0 .9

Qc

©+l'¡r 2.3 ± 0 .2 1.2±0.1 2.4±0.1* 5 .4 ± 1 .4

The partial pressu re o f C 0 2 (pC 02), dissolved inorganic carbon concen tration (CT) as well as th e satu ra tion s ta te o f seaw ate r w ith respect to a ragon ite and calcite (Qa and Qc respectively) w ere com pu ted from pHT and to ta l alkalinity (Ap.*: Qa and Qc w ere increased by addition o f calcium (CaCI2-2H20 ; x2.6 in situ Ca2+ concentrations). doi:10.1371 /journal.pone.0023010.t001

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separate the contribution to total Ca~+ from these com ponents, the filters were rinsed by soaking three times in 30 ml deionised water, increasing the duration each time, with approxim ately 10 sec allowed for the first rinse, 25 m in for the second and an hour for the third. T he w ater from each rinse was analyzed for Ca~+ an d N a+ to determ ine the seawater contribution, then acidified to ~ 0 .1 M with nitric acid and reanalysed to determ ine the w ater insoluble com ponent rem oved from the filters during rinsing. After the third rinse, rem aining calcium was dissolved from the filters by im m ersing in 30 ml 0.1 M nitric acid for 30 m inutes and the concentrations determ ined by IC P-O E S . Ca~+ concentrations were then corrected for the concentration o f Ca~+ observed on the blank filters following the same procedure and the total acid soluble Cia was calculated. As only one filter per treatm ent was analysed, the standard deviations associated with these m easurem ents presented in the next section correspond to those for the blank filters. Calcium concentrations were norm alized by the am ount o f eggs inoculated into the beakers at the start o f the experim ent, assum ing that the distribution o f the original batch has been perform ed homogeneously.

StatisticsSince norm ality an d hom oscedasticity tests could not be used

due to the small num ber o f replicates (3), differences in percentage o f viable D -shaped larvae, final shell lengths and areas as well as in the am ount o f calcium incorporated betw een the different treatm ents were tested by m eans o f Kruskal-W allis tests and post-hoc D u n n ’s m ultiple com parison tests (G raphpad Instat software). For all tests, differences were considered significant at p < 0 .0 5 . In the following section, da ta are presented as m eans ± SD. In o rder to relate the different m easured param eters to the carbonate chem istry param eters a t w hich the organism s were exposed during the incubations, linear an d non-linear regressions were perform ed an d the significance o f these relationships was tested using student’s t-tests.

Results and Discussion

T h e environm ental (tem perature and salinity) and carbonate chem istry param eters are shown in T ab le 1 for each treatm ent.

to 3000

O 2000

- 4000

Calciumadded

Calciumadded

6000

4000 1

7T

2000

SO

T1 T2 T3 T4 T5 T1 T2 T3 T4

F igure 2. C arb o n ate ch em istry cond itions d u rin g th e ex p erim en ta l pe rio d . pC 02: partial pressure o f C 02, pHT: pH on the tota l scale, AT to ta l alkalinity, CT: dissolved inorganic carbon, Qa and Qc: saturation state o f the seawater w ith respect to, respectively, aragonite and calcite. doi:10.1371/journal.pone.0023010.g002

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Param eters o f the carbonate chem istry are also presented in Fig. 2 for each experim ental beaker. T em pera tu re was constant in the beakers a t 1 8 .9 ± 0 .T C . Salinity was 3 4 ± 0 .1 in the first three treatm ents while it was slightly higher in T 5 (34.3±0.0) due to the addition of N a H C 0 3 and m ore th an 1 unit h igher in T 4 due to C aC l2 addition (35.4±0.1). p C 0 2 values were close to target values in m ost cases, except for one experim ental beaker o f T 5 in w hich bubbling was no t optim al and p C 0 2 was m uch lower than the expected value (2502 vs. 4000 patm ). p H x varied from 8 .03±0 .01 in the control trea tm en t (T l) to 7.41 ± 0 .0 3 in the beakers that were bubbled with 2000 pa tm C 0 2 enriched air (T3). Aj- was similar in the first 3 treatm ents while it has been successfully decreased to -—1000 pinoi kg 1 in T 4 and increased to —6800 pinoi kg-1 in T 5. T o tal dissolved inorganic carbon (CT) concentrations were the lowest in T 4 (1029± 9 pinoi kg ) and the highest in T 5 (6589±134 pinoi kg- ) w ith variable levels within the beakers due to the non-optim al C 0 2 equilibration. Seawater was supersaturated with respect to calcite in all treatm ents with the lowest value for T 3 (1.2±0.1). U ndersaturation with respect to aragonite was observed in T 3 (0.8±0.1) while the o ther 4 treatm ents showed ñ arat! values over 1, w ith a level o f 1 .6± 0 .0 in T 4 due to addition o f C aC l2.

D evelopm ental success into viable D -shaped larvae, average D- veliger shell length and area as well as the am oun t o f Ca~+ incorporated pe r egg inoculated, for the five different treatm ents, are shown in Fig. 3. Percentages o f viable D -shaped larvae were 90% in the control treatm ent and not significantly different betw een T l , T 2, T 3 and T 5, while significantly lower values were observed for T 4 beakers (average of 1 9 ± 1%). Final D-veliger shell

length and area da ta showed the same pattern , w ith no significantly different values betw een T l , T 2, T 3 an d T 5 and significantly lower values in T 4 as com pared to the control treatm ent. Final D-veliger shell length and a rea were respectively 11 ± 1 % and 2 0 ± 2 % smaller in T 4 as com pared to control values. Significantly less Ca~+ was incorporated by the population in T 4 th an in the o ther treatm ents with a decrease o f 45 ± 1 4 % with respect to control values. Final D-veliger shell length and area as well as the am ount o f Ca~+ incorporated are plo tted against pFlx and f í a in Fig. 4. N one o f these param eters were correlated with pFlx o r f í a. A lthough seawater pF lx values in T4, T 2 an d T 5 were similar at —7.65, the larvae were smaller in T 4. Increasing the saturation state with respect to aragonite by adding C a2+ (T4) did not positively affect the larval developm ent since D-veliger shell length an d area as well as the am ount o f Ca~+ incorporated were lower in T 4 than in T 2 w hich h ad similar f í a levels (i.e. —1.5). O n one hand, no significant linear a n d /o r non-linear relationships were found betw een all these param eters an d pFl or f í a. O n the o ther hand , these param eters were significantly correlated with the concentration o f C 0 3~ ions. M ichaelis-M enten functions were used to fit the da ta (shell length: r~ = 0.90; shell area: r~ = 0.90; calcium incorporated: r~ = 0.74). C arbonate ion concentrations at the aragonite saturation level were estim ated for each treatm ent (average o f 6 4 .4 ± 0 .2 pinoi kg ) and plo tted as a dotted line on Fig. 4. Above the C 0 3~ saturation level, the effects o f decreasing C 0 3~- concentrations on shell grow th and Ca~+ incorporation as well as on the percentage o f viable D -shaped larvae during these first 72 h o f developm ent were no t significant (Fig. 4). Below the saturation level, decreasing C 0 3~ concentrations resulted in

ab abS 100

<n a t

F igure 3 . Larval d e v e lo p m en ta l pa ram eters a t th e end o f th e in cu b atio n perio d in th e fiv e trea tm e n ts (72 h; T l to T 5 ). P r o p o r t io n o f e m b r y o s t h a t d e v e lo p e d to v ia b le D -v e lig e r (± S D ; u p p e r le ft p lo t) , a v e r a g e sh e ll a r e a a n d le n g th o f D -v e lig e r la rv a e (± S D ; u p p e r r ig h t a n d lo w e r le ft p lo t , r e s p e c t iv e ly ) a s w e ll a s t h e a m o u n t o f c a lc iu m in c o r p o r a te d (± S E ; lo w e r r ig h t p lo t) a re s h o w n . D iffe re n t le t te r s o n b a r s in d ic a te s ig n if ic a n t d if f e re n c e s in t h e m e d ia n v a lu e s (K ruskal-W allis a n d p o s t - h o c D u n n 's m u l tip le c o m p a r is o n te s t s , p < 0 .0 5 ) . d o i:1 0 .1 3 7 1 /jo u rn a l .p o n e .0 0 2 3 0 1 0 .g 0 0 3

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5000'

4500'

4000'► I

3500'

3000'

E3O® — u ^

-a ' o“ .S 2 o» o S ai .aoc

7.2 7.4 7.6 7.6 8.0 .2 O 1 2 3 4 5 6 0 100 200 300 400p H T Ö» [c o 32i

F igure 4 . R elationships b e tw ee n larva l d e ve lo p m en ta l p a ram eters and cond itions o f th e ca rb o n a te ch em istry in th e fiv e trea tm e n ts . Relationships between the average (±SD) shell length and area o f D-veliger larvae as well as the am ount o f calcium incorporated at the end o f the 72 h incubation period, and the average (±SD) conditions o f the carbonate chem istry in the five treatm ents are shown; pHT: pH on the tota l scale (left plots), Qa: saturation state w ith respect to aragonite (m iddle plots) and [C032~]: carbonate ion concentration (right plots). On the right plots, the do tted lines refer to the carbonate ion concentration at the aragonite saturation level (see text fo r details). doi:10.1371/journal.pone.0023010.g004

smaller larvae, less Ca~+ incorporation in the shells an d m uch lower developm ental success (decrease in percentage o f viable D- veliger larvae).

T h e present study is, to the best o f our knowledge, one o f the first to investigate the effects o f carbonate chem istry modifications on the grow th o f a m arine calcifier by separately assessing the effects o f decreases in pH , carbonate ion availability an d seawater saturation state w ith respect to calcium carbonate. Separating these potential factors is crucial as total alkalinity levels are not constant in the ocean and a similar decrease in p H does no t lead to similar decline in calcium carbonate saturation state. A similar study has been perform ed recently by Ju ry et al. [23], based on m anipulations o f the seawater carbonate chemistry, to determ ine w hich p a ram ete r controls coral calcification. T hey showed that the calcification ra te o f Madracis auretenra is m ainly governed by the b icarbonate ions concentration an d not, as expected, by the aragonite saturation state. In contrast, in the present study, the b icarbonate ion concentration as well as C x concentra tion are not correlated with any o f the physiological processes m easured (data not shown). M oreover, the present study shows that p H is not the m ain driver o f the observed decreases in developm ental success

and grow th rates. For instance, a decrease o f p H x to ~ 7 .6 (T5; below the projected levels for the end of the present century) had no significant effect on these larvae w ith similar developm ental success and grow th rates as com pared to control conditions. It m ust be stressed that, for this treatm ent, A T was artificially increased in o rder to m aintain a seawater saturation state with respect to aragonite above 1. This saturation state depends on the availability o f bo th Ca~+ and C 0 3~_ ions. Increasing Ca~+ concentrations in o rder to artificially m aintain above 1 (T4) had no beneficial effect on the larval developm ent o f oysters. As Ca~+ concentrations are no t lim iting in seawater (~ 10 m m ol kg- *), the m ain factor governing the growth o f oyster larvae in our study was C 0 3~ ion concentration. Several studies have already shown lim ited effects o f calcium addition above 10 m m ol kg- 1 with coral calcification rates reaching a p lateau at these concentrations [24,25]. T he relationship betw een m easured param eters (developm ental success, shell length and area and incorporated calcium) and the availability o f C 0 3~ showed that decreasing C 0 3~ levels only had significant effects on the larval developm ent below C 0 3~ levels corresponding to aragonite saturation. O n one hand, this is in contrast w ith results from G azeau et al. [11] that showed that


Hatching rate

— ►




D -veliger shells o f the blue mussel (.Mytilus edulis) were 5 ± 1 % smaller following a 0 .25-0 .34 p H unit decrease corresponding to supersaturated conditions with respect to aragonite. O n the o ther hand, G azeau et al. [11] showed th a t developm ental rates into viable D- shaped larvae a t this p H level were no t significandy altered, a finding which is consistent with present results. Accordingly, oyster larvae appear m ore resistant th an blue mussel larvae to a decrease o f p H as long as C 0 32 - concentrations rem ain above the aragonite saturation level. Further decreasing C 0 32 concentrations below C 0 32 values corresponding to aragonite saturation has dram atic consequences as only ~ 6 0 % and ~ 2 0 % o f the embryos had developed to viable larvae a t C 0 32 concentrations o f 5 0 .4 ± 3 .3 and 4 0 .0 ± 2 .4 pm ol kg- 1 , respectively, as com pared to m ore than 90% in the treatm ents exposed to C 0 32 supersaturated conditions. In the field, the different pressures exerted by the environm ent and predators result in considerable m ortality rates, during the free-swimming larval period, possibly approaching 99% [26]. An additional decrease in developm ental success as observed in the present study, under C 0 32 concentrations below aragonite saturated conditions, could therefore com prom ise the survival of the populations.

M ost calcifying species, including mollusks, are able to concentrate C a 2+ and C 0 32 ions a t the site o f calcification [27] and should therefore be able to regulate calcification rates under suboptim al concentrations o f C a2+ an d C 0 32 - . T h e fact that, even under C 0 32 concentrations below aragonite saturated conditions (T3 and T 4, assum ing calcium addition has no effect in T4), some larvae were able to produce a shell highlights the efficiency o f regulatory m echanism s. How ever, the percentage of em bryos developing to viable D-veliger larvae and the shell sizes o f these viable D-veliger larvae w ere smaller, suggesting th a t the regulation is no t efficient enough to com pensate for the low C 0 32 ion availability below aragonite saturated conditions. Nevertheless, m any m olluskan species are adap ted to and able to survive under low alkalinity conditions such as those in freshwater ecosystems. In the m arine environm ent, bivalve grow th has been reported by Tunnicliffe et al. [17] under extrem ely undersaturated

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conditions prevailing close to deep hydrotherm al sites, although shell grow th rates w ere significandy lower th an in non-acidified areas. Recendy, T hom sen et al. [28] have shown th a t blue mussels are actively growing in a bay o f the W estern Baltic Sea naturally enriched with high C 0 2 w ater, an d also juvenile recruitm ent occurs in sum m er tim e coinciding with low p H levels and aragonite undersaturated conditions. In the O osterschelde tidal inlet (1998-2006, m onthly m easurem ents, 5 stations), surface p H NBs varied annually betw een 8.00 an d 8.24, while A T varied betw een 2334 and 2567 pm ol kg 1 (data no t shown). T he organism s inhabiting this ecosystem are therefore never exposed to corrosive waters and are even used to relatively high calcium carbonate saturation levels, especially in spring a t the tim e of recruitm ent. W hether the organism s inhabiting environm ents with relatively high calcium carbonate saturation levels will be able to ad ap t to the anticipated decreases in p H and saturation levels in the com ing decades rem ains an open question. A ccording to the present results, the effects o f ocean acidification on larvae of Crassostrea gigas from the Oosterschelde estuary during the first 3 days o f developm ent are no t significant as long as C 0 3 concentrations rem ains above aragonite saturated conditions. D ue to relatively high levels o f total alkalinity in this area, it is no t expected th a t seawater will becom e corrosive for aragonite following a decrease o f 0.3 to 0.4 p H unit. However, the present study only focused on the developm ental period betw een embryos and D-veliger larvae, there is still a need to perform experim ents on the full larval developm ent o f this species an d to investigate the response o f o ther crucial physiological processes that have no t been considered in the p resent study such as respiration and excretion.

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August 2011 I Volume 6 | Issue 8 | e23010


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Effect of Carbonate Chemistry Alteration on the Early