# Effects of increased pCO2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis

T. Lacoue-Labarthe, S. Martin, F. Oberhänsli, J.-L. Teyssié, S. Markich, J.

Ross, R. Jeffree, Paco Bustamante

► To cite this version:

T. Lacoue-Labarthe, S. Martin, F. Oberhänsli, J.-L. Teyssié, S. Markich, et al., Effects of increased pCO2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis. Biogeosciences, 2009, 6 (11), pp.2561-2573. 10.5194/bg-6-2561-2009.hal-04110653v2

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# Effects of increased pCO₂ and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, *Sepia officinalis*

T. Lacoue-Labarthe<sup>1,2,*</sup>, S. Martin<sup>1</sup>, F. Oberhänsli<sup>2</sup>, J.-L. Teyssié<sup>1</sup>, S. Markich<sup>3</sup>, J. Ross<sup>1</sup>, and P. Bustamante<sup>2</sup>

Received: 1 April 2009 – Published in Biogeosciences Discuss.: 11 May 2009

Revised: 22 October 2009 – Accepted: 29 October 2009 – Published: 16 November 2009

**Abstract.** Cephalopods play a key role in many marine trophic networks and constitute alternative fisheries resources, especially given the ongoing decline in finfish stocks. Along the European coast, the eggs of the cuttlefish *Sepia officinalis* are characterized by an increasing permeability of the eggshell during development, which leads to selective accumulation of essential and non-essential elements in the embryo. Temperature and pH are two critical factors that affect the metabolism of marine organisms in the coastal shallow waters. In this study, we investigated the effects of pH and temperature through a crossed (3 × 2; pH 8.1 (pCO₂, 400 ppm), 7.85 (900 ppm) and 7.6 (1400 ppm) at 16 and 19°C, respectively) laboratory experiment. Seawater pH showed a strong effect on the egg weight and non-significant impact on the weight of hatchlings at the end of development implying an egg swelling process and embryo growth disturbances. The lower the seawater pH, the more ¹¹⁰mAg was accumulated in the tissues of hatchlings. The ¹⁰⁹Cd concentration factor (CF) decreased with decreasing pH and ⁶⁵Zn CF reached maximal values pH 7.85, independently of temperature. Our results suggest that pH and temperature affected both the permeability properties of the eggshell and embryonic metabolism. To the best of our knowledge, this is one of the first studies on the consequences of ocean acidification and ocean warming on metal uptake in marine organisms,

and our results indicate the need to further evaluate the likely ecotoxicological impact of the global change on the early-life stages of the cuttlefish.

## 1 Introduction

Atmospheric carbon dioxide (CO₂) concentration has increased from 280 parts per million (ppm) prior to the beginning of the industrial revolution to a current value of 380 ppm due to human activities (Solomon et al., 2007). It is now rising at a rate of ca. 3.3% year⁻<fnref n="1" /> (Canadell et al., 2007) that will give a concentration of 700 ppm by the end of this century, according to the Intergovernmental Panel on Climate Change (IPCC) business-as-usual CO₂ emission scenario (Solomon et al., 2007). Increasing atmospheric CO₂ may have important consequences for the Earth's climate, leading to an average warming of 3°C at the Earth's surface over the course of this century (Solomon et al., 2007). Similar trends are expected for surface ocean temperature due to the warming of the surface mixed layer (Levitus et al., 2005). Surface ocean CO₂ partial pressure (pCO₂) is also expected to increase in proportion to the atmospheric CO₂ increase due to the oceanic uptake of anthropogenic CO₂ (Sabine et al., 2004). Increasing pCO₂ in the surface ocean causes major shifts in seawater carbonate chemistry and is likely to reduce pH by 0.2–0.4 units over the course of this century (Caldeira and Wickett, 2005). Such acidification of surface

<figure><img src="image_3.png" /></figure>

Correspondence to: T. Lacoue-Labarthe (tlacouel@gmail.com)

<footnote n="1"><sup>1</sup>Littoral Environnement et Sociétés (LIENSs), UMR 6250, CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, 17042 La Rochelle Cedex 01, France</footnote>

<footnote n="2"><sup>2</sup>International Atomic Energy Agency – Marine Environment Laboratories, 4 Quai Antoine Ier, M98000 Principality of Monaco</footnote>

<footnote n="3"><sup>3</sup>Aquatic Solutions International, Level 1, 467 Miller St, Cammeray, NSW 2062, Australia</footnote>

<footnote>*now at: LOV, UMR 7093 CNRS, BP28, 06234, Villefranche-sur-mer Cedex, France</footnote>
waters could affect marine organisms and in particular those
having carbonate skeleton such as corals, coralline algae,
foraminifera and coccolithophores for which calcification
rates may decrease by 0–56% (see review by Kleypas et al.,
2006). In addition to these biological effects, new data are
emerging on the disturbances of physiological process such
as growth, development, metabolism, ionoregulation and
acid-base balance under elevated temperature and pCO₂ (e.g.
Fabry et al., 2008; Widdicombe and Spicer, 2008; Pörtner et
al., 2004; Pörtner, 2008). Moreover, it is widely accepted
that early life stages may be the more sensitive to high pCO₂
(Pörtner and Farell, 2008) especially in invertebrates (Kuri-
hara, 2008; Dupont and Thorndyke, 2009). Among the lat-
ter, cephalopods play a key role in many marine trophic
foodwebs and constitute alternative fishery resources in the
context of the ongoing decline in finfish stocks. In physi-
ological terms, they are complex organisms with an active
lifestyle and high levels of performance, e.g. high metabolic
and growth rate (Pörtner et al., 1994). Recent studies have fo-
cused on the responses of these organisms to increasing tem-
perature and pCO₂ (Melzner et al., 2007; Rosa and Seibel,
2009) and reported that their low oxygen-carrying blood pro-
tein was a target of their expected vulnerability to global
warming and ocean acidification. Indeed, oxygen affinity of
their haemocyanins decreased with decreasing pH (Bridges,
1994) and increasing temperature (Zielinski et al., 2001),
subsequently reducing their metabolic scope. Nonetheless,
data on the potential impact of both these variables on the
cephalopod early life stages are relatively scarce. On the one
hand, the temperature-dependence of development time in
the cephalopod egg is well described (Boletzky, 1974), viz.
as temperature decreases the development time increases.
Moreover, temperature affects the use of the energy bud-
get supplied by the yolk, increasing respiration of the cut-
tlefish embryo (Wolf et al., 1985) and reducing its growth
rate (Bouchaud and Daguzan, 1989). On the other hand,
D'Aniello et al. (1989) reported that squid eggs developing
in acidified seawater showed reduced survival of the larvae.
More globally, in the Coleoid common cuttlefish, Sepia of-
ficinalis, pH could interact with the egg development in two
ways: first, the eggshell hardens once the egg is laid and be-
comes thicker due to pH-induced seawater polymerization of
the mucopolysaccharidic components of the nidamental se-
cretions (Gomi et al., 1986). The eggshell therefore aims at
protecting the embryo against the external environment, e.g.
microbial attack and predation (Boletzky, 1986) but also lim-
its gas diffusion during the first developmental stages (Wolf
et al., 1985). Secondly, later in the development the cuttlefish
embryo experiences low pH in its surrounding medium, i.e.
the perivitelline fluid, because of the rising level of CO₂ as
a product of the embryo respiration (Gutowska and Melzner,
2009). In this context, increasing pCO₂ in seawater could
impact both the egg structure and the embryonic develop-
ment.

Finally, when cuttlefish Sepia officinalis migrate during
the breeding season into shallow waters to spawn (Boucaud
and Boismery, 1991), the eggs laid here are thus subject to
acute and/or chronic exposure to the various contaminants
such as metals which are released from the human activ-
ities in the marine environment. Exposed to various dis-
solved trace elements, the cuttlefish eggshell is likely to act
as a protective barrier that limits or hinders the incorpora-
tion of metals into the embryo during the first developmental
stages, with a permeability that is element-specific (Busta-
mante 2002, 2004, 2006; Lacoue-Labarthe 2008a). The sub-
sequent incorporation of water into the perivitelline fluid as
the egg swells appears to be a key process in metal pen-
tration. Thus, we hypothesized that, in coastal shallow wa-
ters, ocean acidification and warming could affect embryonic
metabolism and the shielding properties of the eggshell com-
ponents, and could lead to shifts in a) the accumulation of
essential element (Zn) and b) the capacity of the eggshell to
protect against the penetration of non-essential or toxic ele-
ments, such as Ag and Cd, known for their contrasting up-
take behaviours (Lacoue-Labarthe et al., 2008a). These three
trace elements are known to be very toxic to early develop-
ment stages of marine invertebrate (Calabrese et al., 1974).
They are also of specific interest due to their high concen-
trations in polluted spawning areas of the cuttlefish along the
French coasts such as the Seine Bay and the Gironde Estuary
(Boutier et al., 2000; Michel et al., 2000; Roux et al., 2001).

**2 Materials and methods**

**2.1 Organisms, radiotracer and experimental procedures**

Eight adult cuttlefish were collected by net-fishing off the
Principality of Monaco in April and May 2008. Male and
female cuttlefish were acclimated and maintained in open-
circuit tanks in the IAEA-MEL premises. After mating, the
fertilized eggs that were laid by each female were immedi-
ately separated to optimise their oxygenation.

The eggs (n = 300) were randomly assigned in six 5-
L plastic bottles (one bottle per treatment) filled with fil-
tered (0.45 µm) and UV sterilized Mediterranean seawater
that was pumped from 30 m depth and adjacent to Monaco
Bay. In each experimental bottle (closed system), seawater
was constantly aerated. The light/dark cycle was 12 h/12 h.
Eggs were maintained during the full development time in
controlled conditions of temperature and pH in a crossed
(2 temperatures×3 pH levels) experimental design. Seawa-
ter was renewed daily with sterilized and filtered Mediter-
ranean seawater during the first week and then every second
day to maintain good water quality. Bottles were changed
and cleaned at each seawater renewal to prevent any "bott-
tle" effect due to the development of different biomasses or
to the accumulation of detritus such as fragments of external
<table><caption>**Table 1.** *Sepia officinalis*. Two-way ANOVA parameters testing the effects of three pH (7.60, 7.85 and 8.10) and two temperatures (16 and 19°C) on the weight of the eggs and hatchlings, and on the concentration factor (CF) of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn in the hatchlings at the end of the embryonic development (see Figs. 1 and 2).</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="3">pH</th><th colspan="3">Temp</th><th colspan="3">pH X Temp</th></tr><tr><th>df</th><th>MS</th><th>F</th><th>df</th><th>MS</th><th>F</th><th>df</th><th>MS</th><th>F</th></tr></thead><tbody><tr><td>Egg Weight</td><td>2</td><td>0.587</td><td>8.8***</td><td>1</td><td>1.486</td><td>22.2***</td><td>2</td><td>0.018</td><td>0.3<sup>ns</sup></td></tr><tr><td>Hatchling Weight</td><td>2</td><td>0.0005</td><td>2.6<sup>†</sup></td><td>1</td><td>0.013</td><td>67.3***</td><td>2</td><td>3 e<sup>-5</sup></td><td>0.2<sup>ns</sup></td></tr><tr><td>¹¹⁰mAg CF</td><td>2</td><td>14 429 994</td><td>81.5***</td><td>1</td><td>579 843</td><td>3.3<sup>†</sup></td><td>2</td><td>1 023 733</td><td>5.8<sup>**</sup></td></tr><tr><td>¹⁰⁹Cd CF</td><td>2</td><td>5 080</td><td>13.2***</td><td>1</td><td>432</td><td>1.1<sup>ns</sup></td><td>2</td><td>392</td><td>1.02<sup>ns</sup></td></tr><tr><td>⁶⁵Zn CF</td><td>2</td><td>239 017</td><td>32.1***</td><td>1</td><td>26 490</td><td>3.6<sup>†</sup></td><td>2</td><td>4 535</td><td>0.6<sup>ns</sup></td></tr></tbody></table>

df = degree of freedom; MS = mean squares. Probability levels for significant effects: *p* < 0.001(***), *p* < 0.01 (**), *p* < 0.05 (*), *p* < 0.1 (†); ns = non significant.

eggshell layers, or bacterial proliferation, which could affect the metabolism of eggs or the bioavailability of chemicals. Three bottles were kept in a bath that was maintained at 16°C (ambient temperature) and three others in a bath at 19°C (elevated temperature). Temperature was controlled in each bath to within ±0.5°C using temperature controllers connected to 300 W submersible heaters. Within each temperature condition, one bottle was maintained at ambient pH (8.10) while the two others were maintained at lowered pH (7.85 and 7.60). The values of lowered pH were consistent with those that are the most realistic modelled scenarios of ocean pH to occur by the end of this century: 7.85 (*pCO₂* = 900 ppm), 7.60 (*pCO₂* = 1400 ppm), as derived from various IPCC models on trajectories of carbon emissions to the year 2100 (Orr et al., 2005). The pH was controlled in each bottle to within ±0.05 pH unit with a continuous pH-stat system (IKS, Karlsbad) that bubbled pure CO₂ into the bottles that were continuously aerated with CO₂-free air. The pH values of the pH-stat system were adjusted every two days from measurements of pH on the total scale. The pH was measured in each bottle using a pH meter (Metrohm, 826 pH mobile) with a glass electrode (Metrohm, electrode plus) calibrated on the total scale using Tris/HCl and 2-aminopyridine/HCl buffer solutions with a salinity of 38 and prepared according to Dickson et al. (2007). Total alkalinity (TA) shifts between two seawater renewals were assessed in a control bottle containing 45 eggs and maintained at ambient pH (ca. 8.1) and at a temperature of ca. 20°C. TA was measured on seawater samples filtered through 0.45 µm membranes, immediately poisoned with mercuric chloride and stored in a cool dark place pending analyses. TA was determined potentiometrically using a home-made titration system with an Orion 8103SC pH electrode calibrated on the National Bureau of Standards scale and a computer-driven Metrohm 665 Dossimat titrator. TA was calculated using a Gran function applied to pH values ranging from 3.5 to 3.0 as described by Dickson et al. (2007). The *pCO₂* was determined from pH and total alkalinity using the R package seacarb (Proye and Gattuso, 2003).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** *Sepia officinalis*. Weight (g) (A) of the eggs at the end of development (n = 10) and (B) of the hatchlings (n = 10) reared at different treatments different pH – pH 8.10, pH 7.85, pH 7.60 – for two temperatures, i.e. 16°C (grey) and 19°C (white). Results of the statistical analysis were reported on the Table 1.</figcaption></figure>
Uptake of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn in the vitellus and the embryo during the development time were expressed as changes in total activity/concentration ratio (Load concentration ratio; LCR; g; ratio between radiotracer content in the vitellus or the embryo – Bq – and time-integrated activity in seawater – Bq g⁻¹) over time (Lacoue-Labarthe et al., 2008a). Although this ratio is a rather unusual way to express metal accumulation, whole radioactivity content in the vitellus and the embryo was preferred over the concentration factor in order to overcome the problem of dramatic weight variations of these egg compartments that tends to mask the actual accumulation of metals in the whole egg.

Constants for the best fitting uptake and depuration kinetic equations (decision based on ANOVA tables for two fitted model objects) as well as their statistics of variability were estimated by iterative adjustment of the models using the nls curve-fitting routine in R freeware. The level of significance for statistical analyses was always set at $\alpha = 0.05$.

## 2.3 Chemical speciation modelling

To ensure a consistently adequate quality all seawater was carbon-filtered prior to delivery into the tanks that were used during the acclimation period and the experimental exposure. To provide additional information on whether decreasing pH (7.60 and 7.85 from a baseline of 8.10) influenced metal(loid) bioavailability in the tested waters, the speciation of Ag, Cd and Zn was calculated using the HARPHRQ geochemical speciation code (Brown et al., 1991). The input parameters were based on measured physicochemical data (salinity, 38, temperature, 16 or 19°C, dissolved organic carbon <1 mg l⁻¹; see Jeffree et al., 2006 for more details).

# 3 Results

## 3.1 Culture conditions

The pH was maintained at a mean (±SD) of 7.61±0.11, 7.84±0.04, and 8.09±0.04, at ambient temperature (16.0±0.1°C), and of 7.61±0.08, 7.84±0.04, and 8.09±0.09, at elevated temperature (18.9±0.3°C), corresponding to $pCO_2$ of 1399, 781, and 404 ppm at ambient temperature and 1440, 799, and 399 ppm at elevated temperature, respectively. Mean TA of renewed seawater was 2.597±0.012 mmol kg⁻¹. It changed by 0.010 to 0.030 mmol kg⁻¹ between two seawater renewals.

## 3.2 Chemical speciation

The results of the speciation modelling indicated that the decrease in pH (a 3-fold increase in H⁺) from 8.10 to 7.60 had only a very minor influence on the speciation of Ag, Cd or Zn. In terms of the free metal ion concentration, generally considered to represent the bioavailable form of Ag, Cd, Zn, the concentration of Ag⁺ increased from 0.6 to 1.0%, $Cd^{2+}$

increased from 2.7 to 2.8% and $Zn^{2+}$ increased from 46 to 56%, as the pH decreased from 8.10 to 7.60. Consequently, any observed influence of pH on the accumulation of these three trace elements by cuttlefish eggs can be regarded as being predominantly due to responses of the exposed biological tissues and/or competition with elevated H⁺ at the cell surface binding sites.

## 3.3 Biological results

Decreasing pH resulted in higher egg weight at the end of development at both temperatures ($p < 0.05$), with maximal values at pH 7.85 (1.60±0.21 g and 1.83±0.12 g at 16°C and 19°C, respectively). Increasing temperature led to an increase of the egg weight but no interactive effect of both pH and temperature was observed ($p > 0.05$).

Seawater pH had no significant impact on the juvenile weight at hatching time ($p = 0.08$) for both temperature, but hatchlings were smaller when they developed at 16°C than at 19°C ($p < 0.05$).

The lower the pH of the incubation seawater of the eggs, the more ¹¹⁰mAg was accumulated in the tissues of hatchlings. Moreover, this effect was amplified at low temperature, i.e. ¹¹⁰mAg CF was 2.5 and 1.6 fold higher at pH 7.60 than at normal pH, when eggs developed at 16 and 19°C, respectively. In contrast to Ag, the ¹⁰⁹Cd CF decreased with increasing $pCO_2$ ($p < 0.05$), whereas differences in temperature had no effect. Finally, ⁶⁵Zn CF showed the maximal values in the juveniles hatched at the intermediate pH 7.85, independent of temperature, and the CF at 7.60 was lower than at pH 8.10.

## 3.4 Uptake kinetics in the eggshell and the embryo

The uptake kinetics of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn in the shell of the eggs that were exposed at the three pH levels during their development are shown in Fig. 3. For the eggs reared at 19°C, ¹¹⁰mAg uptake in their eggshell displayed a linear pattern during the first 17 days with the uptake rate at normal pH being higher than at the lower pH values, i.e. 102±3 vs. 81±3 and 77±2 d⁻¹ at pH 8.10 vs. 7.85 and 7.60, respectively. Following the first 17 days of development, the CFs decreased according to a single exponential equation indicating that the tracer no longer accumulated in the eggshell, but was only depurated from it. Finally, CF values reached at the end of development were 2- and 4-fold lower at lower pHs than at normal pH, i.e. 557±97 and 317±30 vs. 1258±212 at pH 7.85 and 7.60 vs. 8.10, respectively. These results suggest that low pH limited the Ag retention in the eggshell. Similar patterns were observed at 16°C although ¹¹⁰mAg CF reached a steady-state equilibrium at pH 8.10 and the elimination rate at pH 7.85 and 7.60 were lower than those determined at 19°C (Table 2; 0.043 vs. 0.006 and 0.058 vs. 0.022 d⁻¹ at pH 7.85 and 7.60, respectively).
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** *Sepia officinalis*. $^{110m}$Ag, $^{109}$Cd and $^{65}$Zn uptake kinetics (CF; mean±SD; *n* = 3) in the eggshell from eggs exposed at three different pH – pH 8.10 (●), pH 7.85 (□), pH 7.60 (▲) – for two temperatures, i.e. 16°C (left side) and 19°C (right side).</figcaption></figure>

The $^{109}$Cd uptake kinetics in the eggshell (Fig. 3) were best described by a saturation equation during the first 15 days, and then decreased dramatically following an exponential model (Table 2), reaching the lowest CF values 20 and 10 days before the time of hatching, at 16°C and 19°C, respectively. At pH 7.60, the pattern of $^{109}$Cd accumulation changed after only 7 days of development, at both temperatures. The pH and the temperature showed a combined effect on the maximal $^{109}$Cd CF values in the eggshell, with $^{109}$Cd CF$_{7.85}$>CF$_{8.10}$>CF$_{7.60}$ and CF$_{8.10}$>CF$_{7.85}$>CF$_{7.60}$, at 16°C and 19°C, respectively; and with CF$_{7.85}$ 3.5-fold higher at 16°C than at 19°C, i.e. 930±150 and 265±50, respectively.

The $^{65}$Zn uptake kinetics (Fig. 3) increased linearly during the first 20–22 days and 17–20 days at 16°C and 19°C respectively, independent of the rearing conditions. Then, CF slightly decreased until the end of the egg development following a linear equation (Table 2). For both temperatures, $^{65}$Zn accumulation in the eggshell was lower at pH 7.60.

During the period of development, the radiotracer distribution was determined in the internal egg compartments, i.e. the vitellus and the embryo. In Table 3 is reported the $^{110m}$Ag, $^{109}$Cd and $^{65}$Zn uptake expressed in terms of metal content

(load/concentration ratio; LCR; g) to take into account the vitellus reduction and the embryo growth. The radiotracers' patterns of accumulation were determined for; i), the pooled vitellus and the developing embryo, from the day 1 to 21 and from the day 1 to 14 at 16°C and 19°C and then, ii) the embryonic tissues as soon as the embryo could be separated from the vitellus (>8 mg; stages 21–22 according to Lemaire, 1970), i.e. at day 27 and 17 at 16°C and 19°C, respectively. A significant accumulation (*p* < 0.05) of $^{65}$Zn and $^{110m}$Ag in the pooled vitellus and embryo were determined at 6 and 4 days earlier (day 15 vs. 21 and 10 vs. 14 at 16°C and 19°C; Table 3) for pH 7.60 than at the other pH values, suggesting that the low pH induced an earlier change in the eggshell permeability. During the whole period of embryonic growth, $^{110m}$Ag and $^{65}$Zn were more efficiently accumulated at pH 7.60 and 7.85, respectively, as also shown above for the hatchlings. This result implies that seawater pH had an impact on the metal accumulation capacities of the embryo during its total development. Finally, the $^{109}$Cd distribution revealed that this element was only significantly taken up from the seawater during the last week of development, at both temperatures.
<table><caption>**Table 2.** *Sepia officinalis*. Parameters of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn uptake kinetics in the eggshell of cuttlefish eggs exposed for the whole development time to radiotracers dissolved in seawater (CF; mean±SD, *n* = 3) in three *pCO₂* level treatments and two different temperatures (see Fig. 3).</caption><thead><tr><th rowspan="2">Metal</th><th rowspan="2">Temp</th><th rowspan="2">pH</th><th rowspan="2">Model</th><th colspan="4">First uptake phase</th><th colspan="4">Second uptake phase</th></tr><tr><th>k<sub>u</sub> (d<sup>-1</sup>)</th><th>CF<sub>ss</sub>±SE</th><th>k<sub>e</sub> (d<sup>-1</sup>)</th><th>R<sup>2</sup></th><th>Model</th><th>CF<sub>0</sub>±SE</th><th>k<sub>e</sub> (d<sup>-1</sup>)</th><th>CF<sub>ss</sub>±SE</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td rowspan="6">(a) ¹¹⁰mAg</td><td>16</td><td>7.60</td><td>L</td><td>70.4***</td><td>—</td><td>—</td><td>0.752</td><td>E</td><td>1169±66</td><td>0.022***</td><td>—</td><td>0.727</td></tr><tr><td>16</td><td>7.85</td><td>L</td><td>69.4***</td><td>—</td><td>—</td><td>0.870</td><td>E</td><td>1193±70</td><td>0.006*</td><td>—</td><td>0.245</td></tr><tr><td>16</td><td>8.10</td><td>L</td><td>57.5***</td><td>—</td><td>—</td><td>0.930</td><td>L</td><td>—</td><td>—</td><td>1283±52</td><td>—</td></tr><tr><td>19</td><td>7.60</td><td>L</td><td>76.6***</td><td>—</td><td>—</td><td>0.954</td><td>E</td><td>1220±36</td><td>0.058***</td><td>—</td><td>0.943</td></tr><tr><td>19</td><td>7.85</td><td>L</td><td>80.6***</td><td>—</td><td>—</td><td>0.928</td><td>E</td><td>1261±67</td><td>0.043***</td><td>—</td><td>0.767</td></tr><tr><td>19</td><td>8.10</td><td>L</td><td>101.6***</td><td>—</td><td>—</td><td>0.943</td><td>E</td><td>1674±92</td><td>0.028***</td><td>—</td><td>0.625</td></tr><tr><td rowspan="6">(b) ¹⁰⁹Cd</td><td>16</td><td>7.60</td><td>E</td><td>—</td><td>113±7</td><td>1.177**</td><td>0.914</td><td>E</td><td>85±15</td><td>0.118*</td><td>42±7</td><td>0.557</td></tr><tr><td>16</td><td>7.85</td><td>E</td><td>—</td><td>1087±150</td><td>0.131**</td><td>0.924</td><td>E</td><td>879±63</td><td>0.128***</td><td>57±32</td><td>0.899</td></tr><tr><td>16</td><td>8.10</td><td>E</td><td>—</td><td>950±423</td><td>0.061<sup>ns</sup></td><td>0.864</td><td>E</td><td>571±34</td><td>0.092***</td><td>46±22</td><td>0.927</td></tr><tr><td>19</td><td>7.60</td><td>E</td><td>—</td><td>192±21</td><td>0.549*</td><td>0.869</td><td>E</td><td>149±18</td><td>0.121**</td><td>22±12</td><td>0.694</td></tr><tr><td>19</td><td>7.85</td><td>E</td><td>—</td><td>287±21</td><td>0.413**</td><td>0.921</td><td>E</td><td>234±27</td><td>0.132**</td><td>32±21</td><td>0.780</td></tr><tr><td>19</td><td>8.10</td><td>E</td><td>—</td><td>619±58</td><td>0.161***</td><td>0.975</td><td>E</td><td>550±87</td><td>0.090*</td><td>5±90</td><td>0.724</td></tr><tr><td rowspan="6">(c) ⁶⁵Zn</td><td>16</td><td>7.60</td><td>L</td><td>33.7***</td><td>—</td><td>—</td><td>0.783</td><td>L</td><td>618±49</td><td>-2.4<sup>ns</sup></td><td>—</td><td>0.078</td></tr><tr><td>16</td><td>7.85</td><td>L</td><td>63.3***</td><td>—</td><td>—</td><td>0.887</td><td>L</td><td>1257±71</td><td>-6.9*</td><td>—</td><td>0.242</td></tr><tr><td>16</td><td>8.10</td><td>L</td><td>63.5***</td><td>—</td><td>—</td><td>0.977</td><td>L</td><td>1410±71</td><td>-13.1***</td><td>—</td><td>0.537</td></tr><tr><td>19</td><td>7.60</td><td>L</td><td>45.7***</td><td>—</td><td>—</td><td>0.934</td><td>L</td><td>770±26</td><td>-13.2***</td><td>—</td><td>0.726</td></tr><tr><td>19</td><td>7.85</td><td>L</td><td>46.4***</td><td>—</td><td>—</td><td>0.911</td><td>L</td><td>895±45</td><td>-4.8<sup>ns</sup></td><td>—</td><td>0.106</td></tr><tr><td>19</td><td>8.10</td><td>L</td><td>57.7***</td><td>—</td><td>—</td><td>0.943</td><td>L</td><td>1127±66</td><td>-7.2<sup>ns</sup></td><td>—</td><td>0.105</td></tr></tbody></table>

L and E: linear and exponential models, respectively; CF<sub>ss</sub>: concentration factor at steady-state, *k*<sub>u</sub> and *k*<sub>e</sub>: uptake and elimination rate, respectively; SE: standard error; R<sup>2</sup>: determination coefficient; p-values: <0.001 (***), <0.01 (**), <0.05 (*), >0.05 (ns).

## 3.5 Radiotracers distribution during the egg development

The distribution of the different radiotracers between the eggshell, the perivitelline fluid, the vitellus and the embryo was also determined in this experiment (Table 4). The perivitelline fluid could be considered as an intermediate compartment between the seawater and the embryo. We therefore calculated the CF between seawater and the perivitelline fluid and between the perivitelline fluid and the embryo for Ag, Cd and Zn on the last developmental day, i.e. day 63 and 42 at 16°C and 19°C, respectively (Table 4). The results highlighted that; i) Ag was efficiently concentrated in the perivitelline fluid compared to the other metals (CF<sub>Ag</sub> > 100 ≫ CF<sub>Zn</sub> ≈ 3 > CF<sub>Cd</sub> < 2) and ii) that ¹¹⁰mAg CF in the perivitelline fluid did not vary with the pH for either temperature, except at normal pH compared to the lower pHs in the 19°C-incubated group. In this experimental condition, CF values for ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn were affected by the fact that most of the eggs sampled at this time were already hatched, leading to the loss of at least some of their perivitelline content. The ¹¹⁰mAg was more effectively taken up from the perivitelline fluid with decreasing pH, with the highest CF values being attained in the embryo reared under acidified conditions. No significant effect of pH was observed on

the ¹⁰⁹Cd CF in the perivitelline fluid whereas the Cd accumulation from the perivitelline fluid to the embryo increased with decreasing pH, at 16°C. Concerning ⁶⁵Zn, the CF<sub>PVF/sw</sub> perivitelline fluid decreased with decreasing pH, whereas the CF<sub>emb/PVF</sub> were maximal at pH 7.85 leading to the highest Zn accumulation in the hatchlings as described above.

## 4 Discussion

In this study, one of the major results observed was that lowered pH had increased the egg weight by the end of the development, with an increase in the perivitelline fluid volume (results not shown). This suggests that the seawater *pCO₂* disturbed the swelling process that occurs during the last two thirds of the whole developmental period. The water intake occurred progressively with the organogenesis and enhanced the perivitelline space that the embryo requires for its growth. The mechanistic understanding of this phenomenon is not well known in cephalopod eggs, although it was observed that the water follows an osmotic gradient that is maintained by the embryo himself (Boletzky, 1986; De Leersnyder et al., 1972). It has also been suggested that the oviducal substances from the eggshell play a key role in egg swelling, and that organic compounds cross the chorion and consequently increase the osmotic pressure of the perivitelline fluid (Gomi
<table><caption>**Table 3.** *Sepia officinalis*. Load concentration ratios (LCR; g; mean±SD, *n* = 3) of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn, at different developmental time, in the pooled vitellus and embryo and in the separated embryo of eggs exposed to dissolved radiotracers in three *pCO₂* level treatments and two different temperatures.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">T °C</th><th rowspan="2">pH</th><th colspan="3">Vitellus+Embryo</th><th colspan="3">Embryo</th></tr><tr><th>Day 10</th><th>Day 15</th><th>Day 21</th><th>Day 27</th><th>Day 41</th><th>Day 48</th><th>Day 63</th></tr></thead><tbody><tr><td rowspan="3">¹¹⁰mAg</td><td>16</td><td>7.60</td><td>&lt;1</td><td>6±3</td><td>24±11</td><td>43±4</td><td>155±8</td><td>230±24</td><td>355±44</td></tr><tr><td>16</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>11±10</td><td>11±6</td><td>110±16</td><td>173±6</td><td>220±7</td></tr><tr><td>16</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>13±8</td><td>25±10</td><td>77±4</td><td>112±11</td><td>175±22</td></tr><tr><td rowspan="3">¹⁰⁹Cd</td><td>16</td><td>7.60</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>6±2</td></tr><tr><td>16</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>9±2</td></tr><tr><td>16</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>9±3</td></tr><tr><td rowspan="3">⁶⁵Zn</td><td>16</td><td>7.60</td><td>&lt;1</td><td>1.6±0.2</td><td>4±1</td><td>4±1</td><td>16±1</td><td>28±4</td><td>59±3</td></tr><tr><td>16</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>3±2</td><td>9±2</td><td>29±5</td><td>47±5</td><td>84±11</td></tr><tr><td>16</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>4±1</td><td>6±1</td><td>22±1</td><td>34±3</td><td>72±7</td></tr></tbody><thead><tr><th rowspan="2"></th><th rowspan="2">T °C</th><th rowspan="2">pH</th><th colspan="3">Vitellus+Embryo</th><th colspan="3">Embryo</th></tr><tr><th>Day 7</th><th>Day 10</th><th>Day 14</th><th>Day 17</th><th>Day 20</th><th>Day 32</th><th>Day 42</th></tr></thead><tbody><tr><td rowspan="3">¹¹⁰mAg</td><td>19</td><td>7.60</td><td>&lt;1</td><td>&lt;1</td><td>6±2</td><td>31±2</td><td>45±5</td><td>247±9</td><td>438±17</td></tr><tr><td>19</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>3±1</td><td>11±1</td><td>29±15</td><td>210±18</td><td>305±42</td></tr><tr><td>19</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>19±5</td><td>154±17</td><td>286±15</td></tr><tr><td rowspan="3">¹⁰⁹Cd</td><td>19</td><td>7.60</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>5±2</td></tr><tr><td>19</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>8±2</td></tr><tr><td>19</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;1</td><td>&lt;2</td><td>&lt;2</td><td>13±2</td></tr><tr><td rowspan="3">⁶⁵Zn</td><td>19</td><td>7.60</td><td>&lt;1</td><td>1.1±0.3</td><td>2.3±0.4</td><td>4±1</td><td>7±1</td><td>34±3</td><td>74±4</td></tr><tr><td>19</td><td>7.85</td><td>&lt;1</td><td>&lt;1</td><td>5±1</td><td>4±1</td><td>8±3</td><td>55±5</td><td>95±7</td></tr><tr><td>19</td><td>8.10</td><td>&lt;1</td><td>&lt;1</td><td>3±1</td><td>3±2</td><td>8±1</td><td>37±6</td><td>86±8</td></tr></tbody></table>

et al., 1986; Ikeda et al., 1993). Hence there are two possible explanations for our experimental results, viz: i) lowered pH disturbs the maintenance of the osmotic gradient in the perivitelline fluid by the embryo, and/or, ii) the components of the eggshell and its permeability were affected by the seawater *pCO₂*.

For eggs reared at two different temperatures, the observed course of embryonic development was fully consistent with the previous observations reported for the common cuttlefish (Boletzky, 1986); this tends to confirm that the temperature effect was homogeneous between the three pH conditions at 16 and 19°C. It is also worth noting that reduced temperature, i.e. 16°C, decreased egg swelling compared to 19°C. Although the development time was 20 days longer at 16°C than at 19°C, the incorporation of water was still limited. This effect has also been confirmed in a subsequent experiment (unpublished data). It is known that temperature influences metabolic rate (Melzner et al., 2006), and as the temperature decreases metabolism would slow down. This suggests that the egg swelling observed depended on

the embryonic metabolic level and the subsequent capacity of the embryo to maintain the osmotic gradient in the perivitelline fluid. However, our results revealed also that the egg weight increased with acidified conditions. It is also noteworthy that increasing *pCO₂* in seawater leads to metabolic depression in marine organisms due to changes in their acid-base balance (e.g. Pörtner et al., 2004; Pörtner, 2008). Indeed, due to the high Bohr coefficient of their hemocyanins, cephalopods showed reduced aerobic scope under elevated *pCO₂* in seawater and also showed a high sensitivity to hypercapnia (Pörtner et al., 2004; Melzner et al., 2007). It follows that the reduced metabolic rates (Rosa et al., 2009) of eggs under acidified conditions limited embryonic growth. Additionally, a recent study has demonstrated that at the end of the egg development, the cuttlefish embryo was surrounded by 10-fold higher *pCO₂* values in the perivitelline fluid (i.e., ≈pH 7.4) than those in seawater because of the embryo's respiration (Gutowska and Melzner, 2009). This result highlights that the embryo naturally experiences hypercapnia and still develops normally under such conditions. In
<table><caption>**Table 4.** *Sepia officinalis*. Uptake of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn expressed as CF in between the peri-vitelline fluid (PVF) and seawater and between PVF and the embryo at the end of development following three different *pCO₂* levels at two different temperatures.</caption><thead><tr><th>Experiment</th><th>7.6</th><th>16°C<br>7.85</th><th>8.1</th><th>7.6</th><th>19°C<br>7.85</th><th>8.1</th></tr></thead><tbody><tr><td>(a) ¹¹⁰mAg</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CF<sub>emb/sw</sub></td><td>3270±440<sup>a</sup></td><td>1730±100<sup>b</sup></td><td>1450±240<sup>b</sup></td><td>2910±50<sup>a</sup></td><td>2320±340<sup>b</sup></td><td>2020±120<sup>b</sup></td></tr><tr><td>CF<sub>emb/PVF</sub></td><td>36±18<sup>a</sup></td><td>17±2<sup>ab</sup></td><td>14±2<sup>b</sup></td><td>27±3<sup>a</sup></td><td>14±4<sup>b</sup></td><td>49±19<sup>a</sup></td></tr><tr><td>CF<sub>PVF/sw</sub></td><td>110±40<sup>a</sup></td><td>100±3<sup>a</sup></td><td>100±10<sup>a</sup></td><td>110±10<sup>a</sup></td><td>150±10<sup>ab</sup></td><td>46±17<sup>b</sup></td></tr><tr><td>(a) ¹⁰⁹Cd</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CF<sub>emb/sw</sub></td><td>52±14<sup>a</sup></td><td>69±13<sup>a</sup></td><td>72±26<sup>a</sup></td><td>40±30<sup>a</sup></td><td>60±21<sup>a</sup></td><td>89±11<sup>a</sup></td></tr><tr><td>CF<sub>emb/PVF</sub></td><td>62±11<sup>a</sup></td><td>47±40<sup>ab</sup></td><td>28±2<sup>b</sup></td><td>34±32<sup>a</sup></td><td>46±20<sup>a</sup></td><td>38±18<sup>a</sup></td></tr><tr><td>CF<sub>PVF/sw</sub></td><td>&lt;2<sup>a</sup></td><td>&lt;2<sup>a</sup></td><td>&lt;3<sup>a</sup></td><td>&lt;2<sup>a</sup></td><td>&lt;2<sup>a</sup></td><td>&lt;3<sup>a</sup></td></tr><tr><td>(a) ⁶⁵Zn</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CF<sub>emb/sw</sub></td><td>540±50<sup>a</sup></td><td>660±100<sup>a</sup></td><td>600±60<sup>a</sup></td><td>490±20<sup>a</sup></td><td>720±60<sup>b</sup></td><td>610±50<sup>b</sup></td></tr><tr><td>CF<sub>emb/PVF</sub></td><td>200±30<sup>ab</sup></td><td>240±80<sup>a</sup></td><td>140±10<sup>b</sup></td><td>160±30<sup>a</sup></td><td>180±10<sup>ab</sup></td><td>290±20<sup>b</sup></td></tr><tr><td>CF<sub>PVF/sw</sub></td><td>2.7±0.5<sup>a</sup></td><td>3.1±1.0<sup>a</sup></td><td>4.4±0.6<sup>b</sup></td><td>3.2±0.6<sup>a</sup></td><td>3.9±0.3<sup>ab</sup></td><td>2.1±0.3<sup>b</sup></td></tr></tbody></table>

Different letters denote statistically significant differences (Kruskall-Wallis test; *p*<0.05) between the sample pH for each temperature. CF values in *italic* form were calculated on eggs hatched for a part.

the study reported here, the reduced size of juveniles exposed at pH 7.60 suggests that the *pCO₂* of the perivitelline fluid could reach a threshold value above which the embryo does not develop normally. Consequently, it could be valuable to determine the pH/*pCO₂* levels achieved in the perivitelline fluid when eggs develop under increasing *pCO₂* conditions and assess their impact on the embryo's acid-base regulation and metabolism.

During the embryonic development of cuttlefish, the greatest amounts of the metals, such as Ag, ²⁴¹Am, Cd, Co, Hg, Mn, Pb and Zn, remain associated with the eggshell (Bustamante et al., 2002, 2004, 2006; Lacoue-Labarthe et al 2008a, 2009a, b). Indeed, the eggshell contains a high proportion of mucin proteins that also have a high content of sulphydryl-groups (Boletzky, 1986) for which Ag, Cd and Zn have a high affinity (e.g., Wedemeyer, 1968; Temara et al., 1997; Bell and Kramer, 1999). As previously described (Lacoue-Labarthe et al., 2008a), Ag and Zn accumulated linearly during the first two weeks of development, suggesting that the binding sites were not saturated during this period, in contrast to Cd. During prolonged exposure to metals (> 20 days), the accumulation of ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn decreased while the eggs were under exposure conditions, consistent with changes in the binding properties of the eggshell. This shift occurred at similar times (17 and 20 days at 19°C and 16°C, respectively) for both temperatures and consequently at different developmental stages, suggesting that the polymerization of the eggshell mucopolysaccharides due to seawater pH (Boletzky, 1986, 1998) was the main factor influencing the metal bioaccumulation in the eggshell. In this

way, the low pH (7.60) could affect eggshell polymerization and reduce the metals binding sites, as shown for the ¹¹⁰mAg and ⁶⁵Cd (Fig. 3), possibly through the competitive inhibition with H⁺ ions. This suggests that the protective role of the eggshell in hindering the incorporation of these metals into the embryo could be adversely affected.

Concerning Cd interaction with the eggshell, two characteristics were noteworthy: firstly, Cd uptake reached a steady-state equilibrium and shifted after only 7 days at pH 7.60, strongly suggesting that the mechanisms involved in the Cd accumulation were different from the other metals. Moreover, the combined effect of pH and temperature on the maximal CF values was surprising considering that both these factors affected the chemical properties of the eggshell. Therefore, it could be proposed that the metal uptake process could be driven by the accumulation capacity of the symbiotic bacteria embedded in the eggshell nidamental layers (Bloodgood et al., 1977; Barbieri et al., 1996). As the microorganism respiration influences the oxygen diffusion through the eggshell (Cronin and Seymour, 2000), their metabolism could also affect the accumulation and the retention of metals in this egg compartment. However, to the best of our knowledge, no study has assessed the effect of temperature and pH on the metabolism of these bacteria and the consequences for their population levels in the eggshell. Finally, the metal accumulation among the internal compartments (Table 3) revealed that the permeability of the eggshell seemed to be influenced by the pH with ¹¹⁰mAg and ⁶⁵Zn penetrating earlier into the pooled vitellus and embryo incubated at low pH than in those reared at normal pH. All
these results highlight the role of seawater pH in reducing
the shielding properties of the eggshell against the accumu-
lation of dissolved metals.

Regarding the ¹¹⁰mAg, ¹⁰⁹Cd and ⁶⁵Zn activities in the
hatchlings, it appeared that ¹¹⁰mAg and ¹⁰⁹Cd uptake showed
a linear relationship with the increasing pH, whereas ⁶⁵Zn
was optimally accumulated in the embryo at the interme-
diate pH. ¹¹⁰mAg was efficiently accumulated in the cuttle-
fish embryo, as previously demonstrated (Bustamante et al.,
2004; Lacoue-Labarthe et al., 2008a), presumably from the
time when the water permeability of the eggshell changed
and the perivitelline fluid started to increase in volume. A
few hours before hatching, the higher ¹¹⁰mAg CF values
recorded in the perivitelline fluid compared to the other el-
ements highlighted the capacity of Ag to concentrate in the
perivitelline space. Consistently, in the hypertonic periv-
itelline fluid, the monovalent ions such as Cl⁻, Na⁺ and
K⁺ are slightly more concentrated than divalent ions such as
Ca²⁺ and Mg²⁺ (De Leersnyder et Lemaire, 1972). More-
over, Ag could be bound to the large molecules dissolved in
the perivitelline fluid, such as the natural tranquilizer pep-
tides (Weischer and Marthy, 1983) or the organic matter ac-
cumulated from the oviducal jelly (Gomi et al., 1986; Bo-
letzky, 1986). It is noteworthy that Ag was more efficiently
taken up with decreasing seawater pH in the juveniles. This
could be explained by; i) a higher metal translocation from
the eggshell to the embryo (Lacoue-Labarthe et al., 2008a)
being linked with the reduced Ag retention capacity of the
eggshell at lower pH and by ii) greater transfer of Ag from the
perivitelline fluid to the embryo under acidified conditions in
seawater. This latter mechanism may arise for two reasons:
a) as mentioned above, increasing seawater pCO₂ could dis-
turb the low pH/high pCO₂ conditions in the perivitelline
fluid. This may subsequently modify the chemical speciation
of the metal in the embryo's surrounding medium, thus en-
hancing the Ag free ionic forms which are more bioavailable;
and/or b) increasing Ag uptake in the embryo could reflect
disturbances of ionic regulation (Wood et al., 1999) which
is highly challenged by the acid-base balance (e.g. Pörtner
et al., 2004). Therefore it seems that the embryo metabolic
rate controls the Ag uptake processes into its tissues. This
was further confirmed by the fact that low temperature lim-
ited the Ag uptake in the hatchlings at normal pH as it de-
creased the respiration rate (Wolf et al., 1985). Finally, it
was noteworthy that high Ag CF was correlated with a low
level of egg swelling and small-sized hatchlings at the end of
development at low pH. Are both these observations the re-
sults of the metabolic disturbance under acidified condition,
or are these morphological impacts the first consequences of
the presumably toxicity of the highly accumulated Ag?

Regarding ¹⁰⁹Cd and ⁶⁵Zn, the lower CF<sub>PVF/sw</sub> deter-
mined at the end of development suggested that both metal
concentrations in the perivitelline fluid were close to the
equilibrium with those in seawater (CF≈1–4). Cadmium
passed through the eggshell during the last days of devel-

opment and accumulated in the embryonic tissues during the
last developmental stages (Lacoue-Labarthe et al., 2008a).
Considering that Cd mimics Ca (Bustamante et al., 2002;
Bridges and Zalups, 2005), the decreasing Cd accumula-
tion with increasing pCO₂ whatever the temperature was,
could reflect a possible decreasing calcification rate of the
embryo under hypercapnic conditions (e.g. Gazeau et al.,
2007). However, it has been recently demonstrated that the
cuttlebone calcification was enhanced in sub-adult cuttlefish
reared at 6000 ppm CO₂ (Gutowska et al., 2008). Further
studies are now warranted to determine the impact of acidi-
fied conditions on the calcification of the cuttlebone during
the embryonic development.

Zn is an essential element required for the synthesis of nu-
merous cell constituents such as proteins and enzymes (e.g.,
Vallee and Auld, 1990). It has been demonstrated that it
is maternally transferred (Lacoue-Labarthe et al., 2008b) by
incorporation in the vitellus during oogenesis and that dis-
solved Zn in seawater accumulated in the embryo during egg
development (Bustamante et al., 2002; Lacoue-Labarthe et
al., 2009b). Then, after hatching, young cuttlefish continue
to bioaccumulate Zn very efficiently both from both seawater
and food (Bustamante et al., 2002; Miramand et al., 2006).
These facts suggest that during the embryonic development,
the high embryonic requirements for Zn are not fully cov-
ered by the maternal pool. In this study, temperature had no
effect on the recorded ⁶⁵Zn CF in the hatchlings, implying
that a longer exposure time at 16°C did not lead to the higher
metal accumulation and therefore that the Zn content in the
embryo may be regulated as a function of the metabolic rate
according to the developmental stages. Then, ⁶⁵Zn activi-
ties in the hatchlings and the embryos clearly showed that
the metal accumulation was higher at pH 7.85 during the full
developmental period and that this higher uptake was associ-
ated with a greater rate of growth of both egg and embryo.
These findings give rise to the following two hypotheses:
1) the metabolic performances of the embryo increased at
pH 7.85 enhancing the protein synthesis and subsequently
the requirements for Zn, and/or 2) the chemical speciation
of Zn in the perivitelline fluid enhanced the bioavailable
ionic species for the embryo, consequently stimulating the
metabolism and growth of the embryo.

In summary, this first study showed the strong and con-
trasting effects of pH and temperature on the bioaccumula-
tion of several metals in the cuttlefish eggs. In the context
of the ocean acidification, it appears that decreasing pH until
7.85 should lead to some possibly beneficial effects, such as
a larger egg and presumably hatchling size and a better incor-
poration of the essential element such as Zn in the embryonic
tissue. This may improve the survival the newly hatched ju-
veniles. Moreover, the incorporation of a toxic metal such
as Cd (unpublished data) in the embryonic tissue was re-
duced with increasing pCO₂ whereas the accumulation of
Ag was strongly enhanced under acidified conditions. Ac-
cording to these first results, further work is now warranted
to further assess the ecotoxicological consequences of com-
bined global change effects with a greater range of anthro-
pogenic coastal pollutants on cuttlefish egg development and
the recruitment success of juveniles into their populations.

*Acknowledgements.* We are grateful to Eleonor Hajek and Mathieu Petel (IAEA-MEL) for skilful assistance during the experiments. TL-L was supported by a post-doctoral grant of the French Conseil Général de Charente Maritime (17). The IAEA is grateful for the support provided by the Government of the Principality of Monaco to its Marine Environment Laboratories. This work was supported by the IAEA and LIENSs. This work is a contribution to the "European Project on Ocean Acidification" (EPOCA) which received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 211384.

Edited by: H.-O. Pörtner

<figure><img src="image_6.png" /></figure>

INSU
Institut national des sciences de l'Univers

The publication of this article is financed by CNRS-INSU.

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