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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, M ervyn Greaves3, Henry Elderfield3, Jan Peene4, Carlo H. R.
Heip4'5, Jack J. M id d elb u rg 4,6
1 C en tre N ational d e la R echerche Seientifiq u e-ln stitu t N ational d e s S ciences d e l'Univers, L ab o rato ire d 'O c é a n o g ra p h ie d e Villefranche, V illefranche-sur-M er, France,
2 U niversité P ierre e t M arie Curie-Paris 6, O b se rv ato ire O céan o lo g iq u e d e V illefranche, V illefranche-sur-M er, F rance, 3 D e p a rtm e n t o f Earth Sciences, U niversity o f
C am bridge, C am bridge, E ngland, 4 C en tre for E stuarine a n d M arine Ecology, N eth e rlan d s In stitu te o f Ecology, Y erseke, T he N eth erlan d s, 5 D e p a rtm e n t o f M arine O rganic
B iogeochem istry, Royal N etherlands In stitu te for Sea R esearch, Den Burg, T he N eth erlan d s, 6 Faculty o f G eosciences, U trech t University, U trecht, T h e N eth erlan d s
Abstract
O c e a n acidification, d u e t o a n t h r o p o g e n i c C 0 2 a b s o r p t io n by t h e o c ea n , m ay have p r o fo u n d im p a c ts o n m arine biota.
Calc areous o r g a n is m s a re e x p e c te d t o b e particularly sensitive d u e t o t h e d e c re a s in g availability o f c a r b o n a t e ions driven by
de c r e a s in g pH levels. Recently, s o m e s tu d ie s fo c u se d o n t h e early life s t a g e s of m ollusks t h a t a re s u p p o s e d l y m o r e sensitive
to e n v ir o n m e n ta l d i s t u r b a n c e s t h a n a d u lt s ta g es . A l th o u g h t h e s e s tu d ie s h a v e s h o w n d e c r e a s e d g r o w t h rates a n d increased
p r o p o r tio n s o f a b n o r m a l d e v e l o p m e n t u n d e r low pH con ditio ns, t h e y did n o t allow a ttr ib u tio n t o pH i n d u c e d c h a n g e s in
physiology o r c h a n g e s d u e t o a d e c r e a s e in a r a g o n i te sa tu r a tio n s ta te . This s t u d y aim s to asse ss t h e im p a c t of several
c a r b o n a te - s y s te m p e rtu r b a ti o n s o n t h e g r o w t h o f Pacific o y ste r (Crassostrea gigas) larvae d u rin g t h e first 3 day s of
d e v e l o p m e n t (until shelle d D-veliger larvae). S e a w a t e r with five different c h em istrie s w as o b t a i n e d by s e p a ra tely
m a n ip u la tin g pH, total alkalinity a n d a r a g o n i te s a tu ra tio n s t a te (calcium addition). Results s h o w e d t h a t t h e d e v e l o p m e n t a l
s uc c ess a n d g r o w t h ra tes w e r e n o t directly a ffe cted by c h a n g e s in pH o r a r a g o n i te sa tu r a tio n s t a te b u t w e r e highly
c orre late d w ith t h e availability o f c a r b o n a t e ions. In c o n tr a s t t o previo us studies, b o t h d e v e l o p m e n t a l su c c ess into viable Ds h a p e d larvae a n d g r o w t h rates w e r e n o t significantly a lte re d as long as c a r b o n a t e ion c o n c e n tr a t io n s w e r e a b o v e a ra g o n i te
s a tu r a tio n levels, b u t t h e y str on gly d e c r e a s e d b e lo w sa tu ra tio n levels. T h es e results s u g g e s t t h a t t h e m e c h a n i s m s u se d by
t h e s e o r g a n is m s to r e g u la te calcification rates a re n o t efficient e n o u g h t o c o m p e n s a t e for t h e low availability o f c a r b o n a t e
Ions u n d e r corrosive con ditio ns.
C ita tio n : G azeau F, G attu so J-P, G reaves M, Elderfield H, P e e n e J, e t al. (2011) Effect o f C a rb o n a te C hem istry A lteration o n th e Early Em bryonic D ev elo p m en t o f
th e Pacific O yster (Crassostrea gigas). PLoS ONE 6(8): e23010. doi:10.1 3 7 1 /jo u rn al.p o n e.0 0 2 3 0 1 0
E d ito r : Jo h n M urray R oberts, H eriot-W att U niversity, U nited Kingdom
R e c e iv e d Jan u ary 10, 2011; A c c e p te d July 11, 2011; P u b lis h e d A u g u st 10, 2011
C o p y r ig h t: © 2011 G azeau e t al. This is an o p en -ac cess article d istrib u te d u n d e r th e te rm s o f th e C reative C o m m o n s A ttrib u tio n License, w h ich perm its
un restricted use, distrib u tio n , a n d re p ro d u c tio n in an y m ed iu m , p ro v id ed th e original a u th o r a n d so u rc e a re cred ited .
F u n d in g : This research has received s u p p o rt from th e N eth erlan d s O rg an izatio n o f Scientific R esearch a n d is a co n trib u tio n to th e "E u ro p ean P ro ject o n O cean
A cidification" (EPOCA) w hich received fu n d in g from th e E uropean C o m m u n ity 's S ev en th F ram ew o rk P ro g ram m e (FP7/2007-2013) u n d e r g r a n t a g r e e m e n t n°
211384. T he fu n d e rs h ad n o role in stu d y d esig n , d a ta collection a n d analysis, decisio n to p u blish, o r p re p a ra tio n o f th e m an u scrip t.
C o m p e tin g In te re s ts : T he au th o rs h av e d ec lared th a t n o c o m p e tin g in terests exist.
* E-mail: f.gazeau@ obs-vlfr.fr
(calcite, a ragonite or high-m agnesian calcite), and, as a conse­
q uence o f ocean acidification, will significantly decrease in the
com ing decades. It m ust b e stressed th a t c arb o n a te saturation
states d e p en d n o t only o n p H b u t also o n total alkalinity levels.
T o ta l alkalinity m easures the ability o f a solution to neutralize
acids to the equivalence p o in t o f c arb o n a te or b ica rb o n ate , acting
as a n a tu ra l buffer to the inco rp o ratio n o f anthro p o g en ic C 0 2 in
the ocean. As the add itio n (or rem oval) o f C 0 2 to a solution does
n ot change its alkalinity a n d since the dissolution o f calcium
c arb o n a te m inerals in the w ater colum n a n d in the sedim ents, as
well as alkalinity inputs from co n tinental rock w eathering, are very
slow processes, they a re 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 o n the grow th o f calcifying organism s [5]. Species
th a t pro d u ce aragonite, less soluble th a n low -m agnesian calcite in
seaw ater, will be especially at risk. As am o rp h o u s calcium
c arb o n a te a n d a ragonite have b e en identified as the m ain C a C 0 3
m inerals in larval stages o f b enthic mollusks [6], there is a strong
Introduction
D u e to the a b sorption o f a nthropogenic C 0 2 by the ocean,
seaw ater p H has alread y declined by 0.1 un it c o m p a red w ith p re ­
industrial values [1] a n d is p rojected to decrease by a n o th e r 0.35
unit by the e n d o f the c entury [2]. T h is process, know n as ocean
acidification, will m ost likely have p ro fo u n d im pacts on m arine
biota. Besides the direct effect o f decreasing p H on the physiology
a n d m etabolism o f m arin e organism s th ro u g h a disruption o f in ter­
cellular tran sp o rt m echanism s [3], calcareous organism s are
p articularly sensitive due to the decreasing availability o f
c arb o n a te ions ( C 0 3~_ ) driven b y increasing p C 0 2. T h e calcium
c arb o n a te saturatio n state (Í2) is defined as:
w here K 'sp is the stoichiom etric solubility pro d u ct, a function o f
tem p eratu re, salinity, pressure a n d the m ineral phase considered
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Oyster Larvae U nder Modified C arbonate Chem istry
need to carefully assess the effects o f ocean acidification a n d the
associated alteratio n o f the c arb o n a te chem istry o n their
developm ent. Sm all changes in the a b u n d an c e a n d developm ental
success o f these larval stages control the size a n d viability o f the
b en th ic populations [7] a n d therefore could induce significant
changes in the functioning o f coastal ecosystem s. Indeed, shellfish
are ecosystem engineers governing energy a n d n u trie n t flows in
coastal ecosystem s, provide habitats for m an y b en th ic organism s
a n d form a n im p o rta n t food source for birds [8,9]. M oreover,
global shellfish a q u acu ltu re p ro d u c tio n re ac h ed 13.1 m illion tons
in 2008 (27% o f the global a q u acu ltu re yield), corresponding to a
com m ercial value o f U S$13.1 billion. T h e Pacific O yster
(Crassostrea gigas) was the m ost cultivated species in 2008 w ith a
volum e o f 6.5 m illion tons o r 9.5% o f the total w orld aqu acu ltu re
p ro d u c tio n (F IS H S T A T Plus, U niversal software for fishery
statistical tim e series, V ersion 2.3, F ood a n d A griculture
O rg a n iza tio n o f the U n ited N ations, Fisheries D e p artm en t, D a ta
a n d Statistics U nit., 2000).
Several recen t studies have focused o n 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] a n d m ost o f th em have re p o rte d
negative im pacts o f decreasing pF l levels o n the grow th a n d
developm ent o f these organism s. K u rih a ra e t al. [12,13], P arker
et al. [15] a n d G a zeau e t al. [11] have investigated the effects o f
decreasing p F l o n the early em bryonic (from fertilization to the Dveliger stage) developm ent o f com m ercially im p o rta n t bivalve
species. T his developm ental p e rio d is o f the u tm o st im portance
since the onset o f shell m ineralization occurs d u rin g the
tro ch o p h o re larval stage a n d shells are fully m ineralized w hen
larvae re ac h 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] o n Crassostrea
gigas a n d Mytilus galloprovincialis show ed a strong decrease o f
developm ental success into viable D -shaped larvae a n d grow th
rates w ith increased p C 0 2. Flow ever, a pFl (on the N ational
B u reau o f S tandards scale, h ereafter referred to as p H NBS) o f ~ 7 .4
was used (0.7 u n it low er th a n control values), a value low er th an
th a t p rojected to occur a t the e n d o f this century. M oreover, due to
low a m b ie n t total alkalinity levels, seaw ater was highly u n d e rsat­
u ra te d w ith respect to a ragonite in the low pFl conditions in these
two studies (Í2a o f 0.68 a n d 0.49, respectively). P ark er e t al. [15]
studied the early em bryonic developm ent o f the Sydney rock
oyster Saccostrea glomerata a t a m b ie n t (375 patm ), 600, 750 a n d
1000 p a tm p C 0 2 levels. T his e x p erim en t show ed a general
decrease in the percentage a n d size o f D -veliger w ith increasing
p C 0 2. Flowever, m an ip u latio n o f the c arb o n a te chem istry was
p erfo rm ed b y addition o f a strong acid (F1C1) to reduce pFl, a
technique th a t is n o t reco m m en d ed as acid add itio n also decreases
total alkalinity, w hich is n o t anticipated to occur in the com ing
decades. M oreover, as values o f total alkalinity a n d C a C 0 3
saturation state w ere n o t provided, com parisons w ith sim ilar
studies are n o t straightforw ard. G a zeau e t al. [11] re p o rte d o n the
im pacts o f decreasing pF l levels on the first 2 d developm ent o f
blue mussel (.Mytilus edulis) larvae. T h ey show ed th a t a decrease o f
P H n b s to ~ 7 .8 (control: —8.1), associated w ith a supersaturation
w ith respect to a ragonite (Í2a~ 1 .4 ), h a d no effect o n the
percentage o f em bryos developing to viable D -veliger larvae.
T h e effect o n average final shell length was lim ited (----- 5 ± 1 % ).
T h e ir results show th a t a decrease o f p FInbs to —7.6, associated
w ith a slight u n d e rsatu ra tio n w ith respect to aragonite, h a d
significant effects on the percentage o f em bryos th a t developed to
D -veliger larvae norm ally a n d m ore p ro n o u n c ed effects on
average final D -veliger shell lengths (-----1 3 ± 1 % ). Flow ever, this
study did n o t allow the discrim ination b etw een the physiological
effect o f pF l decrease, via a disruption o f inter-cellular tran sp o rt
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m echanism s, a n d the effect o f the a ragonite saturation state, on the
larval developm ent o f this species.
T h e objectives o f the p re sen t study are to investigate the effect o f
various c arb o n a te chem istry alterations, p erfo rm ed by m an ip u lat­
ing pFl, total alkalinity a n d the saturation state w ith respect to
calcium c arb o n a te separately o n the survival a n d grow th o f Pacific
oyster (Crassostrea gigas) larvae du rin g the first 3 days o f their
developm ent.
M aterials and Methods
E xperim ental se t-u p
A b a tc h o f one m illion em bryos o f the Ja p a n e se oyster
(Crassostrea gigas) was pro v id ed b y the com m ercial h a tc h ery Roem
van Terseke (Yerseke, T h e N etherlands) on 3 J u n e 2009. T his batch
was tran sp o rte d w ithin 15 m in to a tem p eratu re-reg u lated room
(19°C) a t the .Netherlands Institute o f Ecology (Yerseke, T h e N e th e r­
lands) a n d evenly distributed into 15 beakers o f 4.5 1 (larval
co n cen tratio n o f ~ 1 5 ind. m l- 1 ), c o ntaining filtered (0.2 pm)
seaw ater from the O osterschelde, the n e arb y tidal inlet. Five
treatm en ts w ere considered, each o f th em in triplicate (see Fig. 1
for the experim ental setup). O n e trea tm e n t served as a control i.e.
beakers w ere gently b u b b led w ith ex ternal a m b ie n t air. T w o
beakers w ere b u b b led w ith air a t 1000 a n d 2000 p a tm o f C 0 2 (T2
a n d T 3 , respectively). T h e fourth tre a tm e n t (T4) was b u b b led w ith
external a m b ie n t air, after total alkalinity (Tx ) was decreased to
— 1000 pm ol k g -1 by ad d in g 14 m l o f F1C1 0.1 N a n d 10.6 g o f
C a C l2-2Fl20 in o rd e r to reach saturation states w ith respect to
a ragonite a n d calcite o f 1.4 a n d 2.2, respectively. T h e last gro u p o f
beakers (T5) was b u b b led w ith 4000 p a tm C 0 2 a n d A T was
increased b y ad d in g 1.6 g o f N a H C 0 3. G as cylinders w ith
certified C 0 2 concentrations (1000, 2000 a n d 4000 patm ) w ere
supplied by W estfalen. E m bryos w ere allow ed to develop, w ithout
ad ditional feeding, until larvae re ac h ed the shelled D -veliger stage,
i.e. 72 h.
C a rb o n a te ch em istry m e a su re m e n ts
S eaw ater p H (on the total scale, h ereafter referred to as p H T)
a n d te m p e ra tu re w ere m easu red twice a day in the beakers. p H x
was m easu red using a p H m eter (M etrohm , 826 p H m obile) w ith a
glass electrode (M etrohm , electrode plus) calib rated o n the total
scale using T r is /H C l a n d 2 -a m in o p y rid in e /H C l buffer solutions
w ith a salinity o f 35.0 [20]. Sam ples for salinity a n d T x w ere taken
a t the start a n d a t the e n d o f the experim ent. Sam ples for A T w ere
filtered o n G F /F filters, poisoned w ith H g C l2 a n d stored in the
d ark p e n d in g m ea su re m e n t (within few days). Salinity was
m easured using a con d u ctim eter (R adiom eter C D M 230). T ripli­
cate poten tio m etric m easurem ents o f T x w ere p erfo rm ed using a
M e tro h m titra to r a n d a glass electrode (M etrohm , electrode plus).
M easurem ents w ere c arried o u t on 50 m l sam ples a t 25°C a n d T x
was calculated using a G ra n function. T itratio n s o f T x from
sta n d ard seaw ater p rovided by A. G. D ickson (batch 82, n = 10)
w ere o n average w ithin 0.46 pm ol kg 1 o f the n om inal value. All
p a ram ete rs o f the c a rb o n a te chem istry w ere d e term in ed from
p H x , T x , tem p e ra tu re a n d salinity using the R package seacarb
D e ­
sa m p lin g a n d m e a su re m e n ts
A t the e n d o f the incubation period, the beakers w ere em ptied.
T w o liters w ere passed th ro u g h a 30 p m sieve a n d co n ce n tra ted in
50 m l th a t was fixed in a 5% neutralized-form alin seaw ater
solution to determ in e developm ental success (% o f D-veliger
larvae) a n d D -veliger shell length a n d area. T h e developm ental
success into viable D -sh ap ed larvae was defined as the percentage
2
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Oyster Larvae U nder Modified C arbonate Chem istry
A m bient air
450 patm
CO
+ NaHCO
+ CaCI
T2 (x3
T3 (x3)
pHT ™7.72
4 t ~ 2450
Q ~ 1.5
pHT~ 7.44
~ 2450
T5 (x3)
C ontrol
Targeted values
pHT~ 8.03
Ar ~ 2450
O ~ 2.8
0
-
pHT ~ 7.7
Ar -1 0 0 0
0 - 1 .4
0.8
pHT ~ 7.58
A^ ~ 6762
O ~ 3.2
F ig u re 1. E x p e rim e n ta l s e t-u p . For each tre a tm e n t (in trip lica te; T 18.9±0.1°C), the ta rg e t AT (total alkalin ity in pm ol k g - 1 ), pHx and Q a
(saturation state o f th e seawater w ith respect to aragonite) are indicated, Ht was co n trolled by b u b b lin g a m b ie n t o r h ig h -C 0 2 air. AT was decreased
in T4 by HCI a d d itio n and increased in T5 by NaFiC03 ad d itio n . In T4, ca :ium concentrations have been increased above a m b ie n t levels by CaCI2
ad d itio n . See te x t fo r m ore details.
doi:10.1371/journal.pone.0023010.g001
o f “ n o rm a l” D -shape larvae follow ing the criteria p ro p o sed b y His
et al. [22], after observation o f at least 500 larvae p e r replicated
culture. D-veliger larvae shell length (anterior to posterior
dim ension o f the shell parallel to the hinge) a n d a rea w ere m easured
o n 100 larvae p e r replicate based o n pictures taken u n d e r a
m icroscope (20 x; 0.01 p m precision in length m easurem ent), using
the software L eica Q w in P ro version 2.4. A t the e n d o f the
experim ental period, 2 1 o f the cultures w ere filtered onto G F /F
filters for subsequent analyses o f calcium (Ca~+) concentrations.
Larvae-free seaw ater was filtered onto G F /F filters (4 x) a n d served
as blanks for Ca~+ m easurem ents. Ca~+ concentrations w ere de te r­
m ined by inductively coupled plasm a em ission spectrophotom etry
(IC P-O ES) after m ultiple rinses w ith deionised w ater to rem ove
seaw ater Ca~+. In addition to Ca~+ from the larvae, Ca~+ retained
o n the filters m ay com e from residual sea salts, from the G F /F filters
them selves a n d /o r from the analytical blank o f the p rocedure. T o
Table 1. E nvironm ental p a r a m e t e r s a n d c a r b o n a t e c h em istry fo t h e five different t r e a t m e n t s d u rin g t h e c o u rs e o f t h e e x p e r i m e n t
(m e a n ± SD).
T1 (c o n tr o l)
T2
T3
T4
T5
34.3 ± 0 .0
M e a s u r e d p a r a m e te r s
T e m p e ra tu re (°C)
18.9±0.1
Salinity
34.0± 0.1
34.1 ±0.1
34.1 ±0.1
35.4 ±0.1
pHT
8.03 ±0.01
7.72 ± 0 .0 3
7.41 ± 0 .0 3
7.67 ± 0 .0 3
7.62 ± 0 .1 2
A j (jxmol k g - 1 )
2452.7 ± 6 .6
2446.2 ± 8 .2
2443.1 ±3.1
1093.8 ± 4 .0
6726.6 ± 3 7 .6
C o m p u te d p a r a m e te r s
493.5 ± 4 2 .7
3730.4 ± 9 4 6 .5
2443.4 ± 1 0 .6
1029.4 ± 8 .9
6589.3 ± 1 3 3 .9
[HC03_ ]
2010.0 ± 13.0
2209.4± 15.1
2320.3 ± 8 .6
972.9 ± 9 .8
6238.0 ± 1 6 1 .9
[C 032~]
181.9 ± 4 .2
97.3 ± 6 .3
50.4 ± 3 .3
40.0 ± 2 .4
226.5 ± 6 0 .3
Qa
2.8± 0.1
1.5 ±0.1
oo
o
1.6 ±0.0*
3.5 ± 0 .9
2.3 ± 0 .2
1.2±0.1
2.4 ± 0 .1 *
5 .4 ± 1 .4
+l
Qc
+1
2170.5 ± 1 5 6 .9
2340.8 ± 1 2 .3
o
1019.9 ± 7 9 .9
2207.0± 10.1
©
448.7 ± 1 5 .6
'¡r
p C 0 2 (jiatm )
CT (jxmol k g - 1 )
The partial p ressu re o f C 0 2 (pC 02), dissolved inorganic ca rb o n c o n c en tratio n (CT) as well as th e satu ra tio n s ta te o f seaw ate r w ith re s p e c t to a ra g o n ite an d calcite (Qa an d
Q c respectively) w ere c o m p u te d from pHT an d to ta l alkalinity (Ap.
*: Q a an d Q c w e re increased by add itio n o f calcium (CaCI2-2H20 ; x2.6 in situ Ca2+ co n cen tratio n s).
doi:10.1371 /journal.pone.0023010.t001
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Oyster Larvae U nder Modified C arbonate Chem istry
separate the contribution to total Ca~+ from these com ponents, the
filters w ere rinsed by soaking three times in 30 m l deionised w ater,
increasing the d u ra tio n each tim e, w ith approxim ately 10 sec
allow ed for the first rinse, 25 m in for the second a n d a n h o u r for the
third. T h e w ater from each rinse was analyzed for Ca~+ a n d N a+ to
d eterm ine the seaw ater contribution, th en acidified to ~ 0 .1 M with
nitric acid a n d reanalysed to determ ine the w ater insoluble
com ponent rem oved from the filters du rin g rinsing. A fter the third
rinse, rem aining calcium was dissolved from the filters b y im m ersing
in 30 m l 0.1 M nitric acid for 30 m inutes a n d the concentrations
determ ined b y IC P -O E S . Ca~+ concentrations w ere th en corrected
for the co ncentration o f Ca~+ observed o n the blank filters following
the sam e pro ced u re a n d the total acid soluble Cia was calculated. As
only one filter p e r trea tm e n t was analysed, the stan d ard deviations
associated w ith these m easurem ents presented in the next section
correspond to those for the blank filters. C alcium concentrations
w ere norm alized by the a m o u n t o f eggs inoculated into the beakers
at the start o f the experim ent, assum ing th a t the distribution o f the
original b a tc h has been p e rform ed hom ogeneously.
Statistics
Since norm ality a n d hom oscedasticity tests could not b e used
due to the small n u m b e r o f replicates (3), differences in percentage
o f viable D -sh ap ed larvae, final shell lengths a n d areas as well as in
the a m o u n t o f calcium in co rp o ra te d betw een the different
treatm en ts w ere tested by m eans o f K ruskal-W allis tests a n d
post-hoc D u n n ’s m ultiple com parison tests (G ra p h p ad Instat
software). F or all tests, differences w ere considered significant at
p < 0 .0 5 . In the follow ing section, d a ta are presen ted as m eans ±
SD . In o rd e r to relate the different m easu red p a ram ete rs to the
c arb o n a te chem istry p a ram ete rs a t w hich the organism s w ere
exposed d u rin g the incubations, lin ear a n d n on-linear regressions
w ere p erfo rm ed a n d the significance o f these relationships was
tested using student’s t-tests.
Results and Discussion
T h e e n vironm ental (tem perature a n d salinity) a n d carb o n ate
chem istry p a ram ete rs are show n in T ab le 1 for each treatm en t.
to 3000
O
2000
6000
-
4000
4000 1
7T
2000
Calcium
added
T1
T2
T3
T4
Calcium
added
T5
T1
T2
T3
SO
T4
F ig u re 2 . C a r b o n a te c h e m is try c o n d itio n s d u rin g th e e x p e r im e n ta l p e rio d . p C 0 2: partial pressure o f C 0 2, pHT: pH on the to ta l scale, AT
to ta l alkalinity, CT: dissolved inorgan ic carbon, Q a and Q c: 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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Oyster Larvae U nder Modified C arbonate Chem istry
P aram eters o f the c arb o n a te chem istry are also presen ted in Fig. 2
for each e x perim ental beaker. T e m p e ra 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 en ts while it was slightly higher in T 5 (34.3± 0.0) due to the
ad dition o f N a H C 0 3 a n d m ore th a n 1 u n it h igher in T 4 due to
C a C l2 ad dition (35.4± 0.1). p C 0 2 values w ere close to target
values in m ost cases, except for one experim ental b eak er o f T 5 in
w hich b u b b lin g was n o t optim al a n d p C 0 2 was m u ch low er th an
the expected value (2502 vs. 4000 patm ). p H x v aried from
8 .0 3 ± 0 .0 1 in the control tre a tm e n t (T l) to 7.41 ± 0 .0 3 in the
beakers th a t w ere b u b b led w ith 2000 p a tm C 0 2 e n ric h ed air (T3).
Aj- was sim ilar in the first 3 treatm en ts while it has been
successfully decreased to -—1000 pinoi kg 1 in T 4 a n d increased
to —6800 pinoi kg-1 in T 5 . T o ta l dissolved inorganic carb o n (CT)
concentrations w ere the lowest in T 4 (1 0 2 9 ± 9 pinoi kg ) a n d the
highest in T 5 (6 5 8 9 ± 1 3 4 pinoi k g - ) w ith variable levels w ithin
the beakers due to the non-optim al C 0 2 equilibration. Seaw ater
was su p ersatu rated w ith respect to calcite in all treatm en ts w ith the
lowest value for T 3 (1.2± 0.1). U n d e rsa tu ra tio n w ith respect to
a ragonite was observed in T 3 (0.8± 0.1) w hile the o th er 4
treatm en ts show ed ñ arat! values over 1, w ith a level o f 1 .6 ± 0 .0
in T 4 due to add itio n o f C a C l2.
D evelopm ental success into viable D -sh ap ed larvae, average Dveliger shell length a n d a rea as well as the a m o u n t o f Ca~+
in co rp o ra te d p e r egg inoculated, for the five different treatm ents,
are show n in Fig. 3. Percentages o f viable D -sh ap ed larvae w ere
90% in the control trea tm e n t a n d not significantly different
betw een T l , T 2 , T 3 a n d T 5 , while significantly low er values w ere
observed for T 4 beakers (average o f 1 9 ± 1%). F inal D -veliger shell
ab
S
length a n d a rea d a ta show ed the sam e p a tte rn , w ith no
significantly different values betw een T l , T 2 , T 3 a n d T 5 a n d
significantly low er values in T 4 as c o m p a red to the control
trea tm e n t. Final D -veliger shell length a n d a re a w ere respectively
11 ± 1 % a n d 2 0 ± 2 % sm aller in T 4 as c o m p a red to control values.
Significantly less Ca~+ was in co rp o rated by the pop u latio n in T 4
th a n in the o th er treatm en ts w ith a decrease o f 45 ± 1 4 % w ith
respect to control values. Final D -veliger shell length a n d a rea as
well as the a m o u n t o f Ca~+ in co rp o ra te d are plo tted against pF lx
a n d f í a in Fig. 4. N o n e o f these p a ram ete rs w ere co rrelated w ith
pF lx o r f í a. A lthough seaw ater p F lx values in T 4, T 2 a n d T 5 w ere
sim ilar at —7.65, the larvae w ere sm aller in T 4 . Increasing the
saturation state w ith respect to a ragonite by ad d in g C a 2+ (T4) did
n ot positively affect the larval developm ent since D -veliger shell
length a n d a rea as well as the a m o u n t o f Ca~+ in co rp o ra te d w ere
low er in T 4 th a n in T 2 w hich h a d sim ilar f í a levels (i.e. —1.5). O n
one h and, no significant linear a n d /o r non -lin ear relationships
w ere found b etw een all these p a ram ete rs a n d pFl or f í a. O n the
o th er h a n d , these p a ram ete rs w ere significantly co rrelated w ith the
co n cen tratio n o f C 0 3~ ions. M ichaelis-M enten functions w ere
used to fit the d a ta (shell length: r~ = 0.90; shell area: r~ = 0.90;
calcium in corporated: r~ = 0.74). C a rb o n ate ion concentrations at
the aragonite satu ratio n level w ere estim ated for each trea tm e n t
(average o f 6 4 .4 ± 0 .2 pinoi kg ) a n d plo tted as a d o tted line on
Fig. 4. A bove the C 0 3~ saturation level, the effects o f decreasing
C 0 3~- concentrations o n shell grow th a n d Ca~+ in co rp o ra tio n as
well as o n the percen tag e o f viable D -sh ap ed larvae d u rin g these
first 72 h o f developm ent w ere n o t significant (Fig. 4). Below the
saturation level, decreasing C 0 3~ concentrations resulted in
ab
100
<n a t
F ig u re 3 . L a rv al d e v e lo p m e n ta l p a ra m e te rs a t th e e n d o f th e in c u b a tio n p e rio d in th e fiv e tr e a tm e n ts (7 2 h; T l to T 5 ). P r o p o r tio n o f
e m b r y o s t h a t d e v e l o p e d t o 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 s h e ll a r e a a n d le n g t h o f D -v e lig e r la r v 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 tiv 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 i n c o r p o r a t e d ( ± S E ; lo w e r r i g h t p lo t) a r e s h o w n . D if f e r e n t l e tte r s o n b a r s i n d i c a t e s ig n if ic a n t
d if f e r e n c e s in t h e m e d i a n v a l u e s (K ru sk a l-W allis a n d p o s t - h o c D u n n 's m u l tip l e c o m p a r i s o n te s t s , p < 0 . 0 5 ) .
d o i:1 0 .1 3 7 1 /jo u r n a l.p o n e .0 0 2 3 0 1 0 .g 0 0 3
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A ugust 2011 | Volum e 6 | Issue 8 | e23010
Oyster Larvae U nder Modified C arbonate Chem istry
Hatching rate
—
►
5000'
4500'
4000'
►I
3500'
3000'
E
3
O
®
u ^—
-a
'o
“ .S
2 o»
o S
ai.
a
o
c
7.2
7.4
7.6
7.6
8.0
.2 O
1
2
pHT
3
Ö»
4
5
6
0
100
200
300
400
[c o 32i
F ig u re 4 . R e la tio n s h ip s b e tw e e n la rv a l d e v e lo p m e n ta l p a ra m e te rs a n d c o n d itio n s o f th e c a rb o n a te c h e m is try in th e fiv e
tr e a tm e n ts . R elationships betw een th e average (±S D ) shell leng th and area o f D-veliger larvae as w ell as the a m o u n t o f calcium incorporated at the
end o f the 72 h in cub ation period, and the average (±SD ) con d itio n s o f the carbonate chem istry in th e five tre atm e nts are show n; pHT: pH on the
to ta l scale (left plots), Q a: saturation state w ith respect to aragonite (m iddle plots) and [C 032~]: carbonate ion con centration (rig h t plots). On the rig h t
plots, the d o tte d lines refer to the carbonate ion con centration at th e aragonite saturation level (see te x t fo r details).
doi:10.1371/journal.pone.0023010.g004
sm aller larvae, less Ca~+ inco rp o ratio n in the shells a n d m uch
low er developm ental success (decrease in percentage o f viable Dveliger larvae).
T h e presen t study is, to the best o f o u r know ledge, one o f the
first to investigate the effects o f c arb o n a te chem istry m odifications
o n the grow th o f a m arin e calcifier by separately assessing the
effects o f decreases in p H , c arb o n a te ion availability a n d seaw ater
saturation state w ith respect to calcium c arb o n ate. S eparating
these p otential factors is crucial as total alkalinity levels are not
constant in the ocean a n d a sim ilar decrease in p H does n o t lead to
sim ilar decline in calcium c arb o n a te saturation state. A sim ilar
study has b e en p erfo rm ed recently b y J u r y et al. [23], based on
m anipulations o f the seaw ater c arb o n a te chem istry, to d eterm ine
w hich p a ra m e te r controls coral calcification. T h ey show ed th a t the
calcification ra te o f Madracis auretenra is m ainly governed by the
b ica rb o n ate ions con cen tratio n a n d not, as expected, b y the
a ragonite saturation state. In contrast, in the presen t study, the
b ica rb o n ate ion co n cen tratio n as well as C x c o n ce n tra tio n are not
co rrelated w ith a n y o f the physiological processes m easured (data
not shown). M oreover, the presen t study shows th a t p H is not the
m ain driver o f the observed decreases in developm ental success
PLoS ONE I w w w .plosone.org
a n d grow th rates. F or instance, a decrease o f p H x to ~ 7 .6 (T5;
below the p rojected levels for the e n d o f the presen t century) h a d
no significant effect on these larvae w ith sim ilar developm ental
success a n d grow th rates as c o m p a red to control conditions. It
m ust be stressed that, for this treatm en t, A T was artificially
increased in o rd e r to m ain tain a seaw ater saturation state w ith
respect to a ragonite above 1. T his saturation state depends on the
availability o f b o th Ca~+ a n d C 0 3~_ ions. Increasing Ca~+
concentrations in o rd e r to artificially m ain tain
above 1 (T4)
h a d no beneficial effect o n the larval developm ent o f oysters. As
Ca~+ concentrations are n o t lim iting in seaw ater ( ~ 10 m m ol kg- *),
the m ain factor governing the grow th o f oyster larvae in o u r study
was C 0 3~ ion concentration. Several studies have already shown
lim ited effects o f calcium addition above 10 m m ol k g - 1 w ith coral
calcification rates reaching a p late au at these concentrations
[24,25]. T h e relationship betw een m easured param eters (develop­
m ental success, shell length a n d area a n d in co rp o rated calcium) a n d
the availability o f C 0 3~ show ed th at decreasing C 0 3~ levels only
h a d significant effects o n the larval developm ent below C 0 3~
levels corresponding to aragonite saturation. O n one h and, this is in
contrast w ith results from G a zeau et al. [11] th a t show ed that
6
A ugust 2011 I Volum e 6 | Issue 8 | e23010
Oyster Larvae U nder Modified C arbonate Chem istry
D -veliger shells o f the blue m ussel (.Mytilus edulis) w ere 5 ± 1 % sm aller
following a 0 .2 5 -0 .3 4 p H un it decrease corresponding to super­
saturated conditions w ith respect to aragonite. O n the o th er hand,
G azeau et al. [11] show ed th a t developm ental rates into viable D shaped larvae a t this p H level w ere n o t significandy altered, a finding
w hich is consistent w ith present results. Accordingly, oyster larvae
a p p ea r m ore resistant th a n blue m ussel larvae to a decrease o f p H
as long as C 0 32 - concentrations rem ain above the aragonite
saturation level. F u rth e r decreasing C 0 32 concentrations below
C 0 32 values corresponding to aragonite saturation has dram atic
consequences as only ~ 6 0 % a n d ~ 2 0 % o f the em bryos h a d
developed to viable larvae a t C 0 32 concentrations o f 5 0 .4 ± 3 .3
a n d 4 0 .0 ± 2 .4 pm ol kg- 1 , respectively, as c o m p a red to m ore th an
90% in the treatm ents exposed to C 0 32 supersaturated condi­
tions. In the field, the different pressures exerted by the e nvironm ent
a n d p red ato rs result in considerable m ortality rates, du rin g the
free-sw im m ing larval period, possibly a p p ro ac h in g 99% [26]. A n
additional decrease in developm ental success as observed in the
presen t study, u n d e r 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
con cen trate C a 2+ a n d C 0 32 ions a t the site o f calcification [27]
a n d should therefore be able to regulate calcification rates u n d e r
suboptim al concen trations o f C a 2+ a n d C 0 32 - . T h e fact that,
even u n d e r C 0 32 concentrations below aragonite saturated
conditions (T3 a n d T 4 , assum ing calcium ad dition has no effect
in T4), som e larvae w ere able to pro d u ce a shell highlights the
efficiency o f regulatory m echanism s. H ow ever, the percen tag e o f
em bryos developing to viable D -veliger larvae a n d the shell sizes o f
these viable D -veliger larvae w ere sm aller, suggesting th a t the
regulation is n o t efficient enough to com pensate for the low
C 0 32 ion availability below a ragonite satu rated conditions.
N evertheless, m an y m olluskan species are a d a p te d to a n d able to
survive u n d e r low alkalinity conditions such as those in freshw ater
ecosystem s. In the m arin e environm ent, bivalve grow th has been
re p o rte d b y T unnicliffe et al. [17] u n d e r extrem ely u n d e rsatu ra te d
conditions prevailing close to deep h y d ro th erm al sites, although
shell grow th rates w ere significandy low er th a n in non-acidified
areas. R ecendy, T h o m sen et al. [28] have show n th a t blue mussels
a re actively grow ing in a b a y o f the W estern Baltic Sea naturally
en rich ed w ith high C 0 2 w ater, a n d also juvenile recru itm en t
occurs in sum m er tim e coinciding w ith low p H levels a n d
a ragonite u n d e rsatu ra te d conditions. In the O osterschelde tidal
inlet (1998-2006, m on th ly m easurem ents, 5 stations), surface
p H NBs varied annually betw een 8.00 a n d 8.24, while A T varied
betw een 2334 a n d 2567 pm ol kg 1 (data n o t shown). T h e
organism s inhab itin g this ecosystem are therefore never exposed
to corrosive w aters a n d are even used to relatively high calcium
c arb o n a te saturation levels, especially in spring a t the tim e o f
recruitm ent. W h e th e r the organism s inhab itin g environm ents w ith
relatively high calcium c arb o n a te saturation levels will be able to
a d a p t to the an ticip ated decreases in p H a n d saturation levels in
the com ing decades rem ains a n open question. A ccording to the
presen t results, the effects o f ocean acidification o n larvae of
Crassostrea gigas from the O osterschelde estuary du rin g the first 3
days o f developm ent are n o 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 n o t
expected th a t seaw ater will becom e corrosive for aragonite
following a decrease o f 0.3 to 0.4 p H unit. H ow ever, the presen t
study only focused o n the developm ental p e rio d betw een em bryos
a n d D -veliger larvae, there is still a need to p erform experim ents on
the full larval developm ent o f this species a n d to investigate the
response o f o th er crucial physiological processes th a t have n o t b een
considered in the p re sen t study such as respiration a n d excretion.
Author Contributions
Conceived and designed the experiments: FG J-P G J P C H R H JJAT.
Perform ed the experiments: FG. Analyzed the data: FG J-P G M G H E
C H R H JJ M . C ontributed reagents/m aterials/analysis tools: F G J-P G M G
H E J P C H R H JJ M . W rote the paper: F G J-P G M G JJM .
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A ugust 2011 I Volum e 6 | Issue 8 | e23010