Narimane Dorey
email: narimane.dorey@gmail.com

Sophie Martin

François Oberhänsli

Jean-Louis Teyssié

Ross Jeffree

Thomas Lacoue-Labarthe

Ocean acidification modulates the incorporation of radio-labeled heavy metals in the larvae of the Mediterranean sea urchin Paracentrotus lividus

The marine organisms which inhabit the coastline are exposed to a number of anthropogenic pressures that may interact. For instance, the accumulation of toxic metals present in coastal waters is expected to be modified by ocean acidification through e.g. changes in physiological performance and/or elements availability. Changes in bioaccumulation due to lowering pH are likely to be differently affected depending on the nature (essential vs. non-essential) and speciation of each element. The Mediterranean is of high concern for possible cumulative effects due to strong human influences on the coastline.

The aim of this study was to determine the effect of ocean acidification (from pH 8.1 down to -1.0 pH units) on the incorporation kinetics of six trace metals (Mn, Co, Zn, Se, Ag, Cd, Cs) and one radionuclide ( 241 Am) in the larvae of an economically-and ecologically-relevant sea urchin of the Mediterranean coastline: Paracentrotus lividus. The radiolabelled metals and radionuclides added in trace concentrations allowed precise tracing of their incorporation in larvae during the first 74 hours of their development.

Independently of the expected indirect effect of pH on larval size/developmental rates, Paracentrotus lividus larvae exposed to decreasing pHs incorporated significantly more Mn and Ag and slightly less Cd. The incorporation of Co, Cs and 241 Am was unchanged, and Zn and Se exhibited complex incorporation behaviors. Studies such as this are necessary prerequisites to the implementation of metal toxicity mitigation policies for the future ocean.

+ GRAPHICAL ABSTRACT

+ HIGHLIGHTS -The radionuclide method allowed accurate tracing of the metal incorporation within this microscopic planktonic species.

-Metal incorporation in this sea urchin larvae is strongly correlated with the indirect delaying effect of seawater acidification on larval size.

-Independently of the size effect, acidification additionally directly affects the incorporation behavior of four metals (Mn, Ag, Se, Zn).

-The nature of the modulation is specific to each metallic element (see graphical abstract).

-The relationship between speciation, bioaccumulation and toxicity in the context of changing seawater pH requires more research.

+ KEYWORDS: Developmental biology, Bioaccumulation kinetics, Metals, Radionuclide, Pollution, CO 2 , Ocean acidification

INTRODUCTION

The marine organisms which inhabit the coastline are exposed to a number of anthropogenic pressures. On a local scale, metallic trace elements -originating from activity occurring along river basins (e.g. mine discharge, agriculture, wastewater treatment) -are harmful contaminants of the coastal marine environments and of concern to humans for ocean exploitation [START_REF] Förstner | Metal pollution in the aquatic environment[END_REF]. The French Mediterranean coastline, which is strongly influenced by the discharges of the Rhône river that drains industrialized and urban basins, is particularly exposed to metal pollution (UNEP/MAP, 2013). Metals can be highly toxic at low levels, especially to early development stages of marine invertebrates, and they have long been shown to alter (for example) sea urchin gametogenesis, larval growth, morphological development (e.g. Cu: [START_REF] Bougis | Effet du cuivre sur la croissance du pluteus d'oursin (Paracentrotus lividus)[END_REF]Cd: Pagano et al., 1982 and[START_REF] Filosto | Environmentally relevant cadmium concentrations affect development and induce apoptosis of Paracentrotus lividus larvae cultured in vitro[END_REF]Mn: Pinsino et al., 2010) and biomineralization (reviewed by [START_REF] Matranga | Chapter 8: Echinoderms as blueprints for biocalcification: Regulation of skeletogenic genes and matrices[END_REF].

In parallel, the ocean is facing global changes, a consequence of the increase in anthropogenic carbon dioxide (CO 2 ) in the atmosphere (280 to 400 ppm between 1850 and 2013, Etheridge et al., 1996 and Scripps CO 2 Program). On top of a warming (average + 0.1°C per decade between 1970(average + 0.1°C per decade between and 2010(average + 0.1°C per decade between , IPCC, 2013)), the surface ocean waters also experience an acidification (average -0.1 pH units -i.e. 30% increase in acidity -between pre-industrial levels and 2000: Caldeira and Wickett, 2008, -0.0044 units per year in the northwestern Mediterranean: [START_REF] Flecha | Trends of pH decrease in the Mediterranean Sea through high frequency observational data: indication of ocean acidification in the basin[END_REF]. This ocean acidification is projected to decrease the current average pH of the global oceans (≈ 8.1) by a further 0.3 to 0.4 pH units by the end of the 21 st century (IPCC, 2013). Ocean acidification has been demonstrated to be a major threat for marine organisms, impacting their physiology (e.g. sea urchin larvae disrupted in their acid-base regulation: [START_REF] Stumpp | CO 2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay[END_REF]and feeding: Stumpp et al., 2013, increased metabolism: Dorey et al., 2013 and modulated gene expression, in functions such as immunity: [START_REF] Runcie | Genomic characterization of the evolutionary potential of the sea urchin Strongylocentrotus droebachiensis facing ocean acidification[END_REF] as well as their fitness (e.g. negative effect on sea urchin larval growth: [START_REF] Dorey | Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH[END_REF][START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF][START_REF] Stumpp | CO 2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay[END_REF] and sea urchin gonadal growth: [START_REF] Siikavuopio | Effects of carbon dioxide exposure on feed intake and gonad growth in green sea urchin, Strongylocentrotus droebachiensis[END_REF]Stumpp et al., 2012b: exposure time≤56 days, but see [START_REF] Dupont | Long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis[END_REF] no difference after 16 months).

Ocean acidification has also been demonstrated to modify larval behavior (e.g. settlement behavior of Bugula neritina: [START_REF] Pecquet | Ocean acidification increases larval swimming speed and has limited effects on spawning and settlement of a robust fouling bryozoan, Bugula neritina[END_REF] and cognitive performances (e.g. sensory capacities and spatial navigation in larvae of fish: [START_REF] Munday | Ocean acidification impairs olfactory discrimination and homing ability of a marine fish[END_REF] and more largely interactions within communities (e.g. [START_REF] Asnaghi | Cascading effects of ocean acidification in a rocky subtidal community[END_REF]. Early life stages, often considered as bottlenecks for population dynamics, have however been shown to be more resistant than previously thought [START_REF] Kroeker | Impacts of ocean acidification on marine organisms : quantifying sensitivities and interaction with warming[END_REF]. For instance, sea urchin larvae are resistant to a seawater acidification down to 7.1, albeit with larval growth being generally reduced when pH is lowered (Paracentrotus lividus: [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF], Strongylocentrotus droebachiensis: Dorey et al., 2013). Overall, studies show that responses are however species-and stage-specific [START_REF] Kroeker | Impacts of ocean acidification on marine organisms : quantifying sensitivities and interaction with warming[END_REF], with for instance the larvae of another echinoderm, Ophiotrix fragilis, experiencing 100% mortality at pH NBS 7.9 (vs. 70% in control pH: [START_REF] Dupont | Near-future level of CO 2driven ocean acidification radically affects larval survival and development in the brittlestar Ophiothrix fragilis[END_REF].

The resulting interactions of multiple stressors in the future ocean could lead to non-independent effects (e.g. [START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF]. The Mediterranean Sea is of high concern for possible cumulative effects (global change, over-fishing, pollution; UNEP/MAP, 2013). Although an important body of works demonstrated that increasing temperature arises metal toxicity (see review by [START_REF] Sokolova | Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change[END_REF] together with an elevation of the organism's metabolism and a modification of its physiology (e.g. feeding, osmoregulation), few studies focused on the effects of ocean acidification on the bioaccumulation and toxicity of metallic contaminants (see review Table 1). Yet, the changes of pH and seawater chemistry caused by increased CO 2 can modify the speciation of metals, because element's speciation is largely dependent on physicochemical parameters (salinity, pH, redox potential: Millero et al., 2009;[START_REF] Stockdale | Effect of ocean acidification on organic and inorganic speciation of trace metals[END_REF], and therefore their bioavailability for organisms. CO 2 -driven acidification has been shown to modulate trace metal accumulation in both cephalopods early-life stages (Lacoue-Labarthe et al., 2011a, 2009) and the mantle cells of clams (Ivanina et al., 2013). A change in bioaccumulation efficiencies could lead to a change of metal toxicity.

Additionally, pH can impact the organism's abilities to deal with metal bioaccumulation and toxicity, as both are likely to interfere with metabolism and more specifically ionoregulation activities [START_REF] Pörtner | Biological impact of elevated ocean CO 2 concentrations: lessons from animal physiology and earth history[END_REF]Stumpp et al., 2012a). While sea urchin larvae have been shown to be relatively tolerant to lowered seawater pH -even for values well below the average projections for 2100 -, the effects are strong at the sub-lethal level. Decreasing pH resulted in larval growth delay (but conserved calcification in the form of calcium accumulation and relative arm length: [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF], increased metabolism (e.g. respiration rates increased by 9% per 0.1 pH unit decrease in [START_REF] Dorey | Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH[END_REF] and changes in the expression of traits relating to cell division, metabolism and immune activity (pH 7.7: [START_REF] Runcie | Genomic characterization of the evolutionary potential of the sea urchin Strongylocentrotus droebachiensis facing ocean acidification[END_REF]. These resulting sub-lethal changes could possibly be linked to the increase in energy demand [START_REF] Stumpp | CO 2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay[END_REF] in order to maintain intracellular medium, via the use of energy-demanding membrane pumps (Stumpp et al., 2012a). Within these processes, ionic pumps such as -ATPase pumps play a key role by creating electrochemical gradients used by secondary active transporters [START_REF] Stumpp | pH regulation and excretion in Echinoderms[END_REF]).

Yet, in marine organisms, these ion channels represent a major pathway for the entrance of metallic positively-charged ions (e.g. fish: [START_REF] Webb | Bioaccumulation and distribution of silver in four marine teleosts and two marine elasmobranchs: influence of exposure duration, concentration, and salinity[END_REF]. Ocean acidification is therefore likely to increase metal pollutant bioaccumulation in organisms possessing acid-base regulation systems such as the sea urchin larvae. More information on the interactions between pH and metal toxicity is thus required to improve predictive mathematical models and coastal management [START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF].

The aim of this study was to determine the effect of ocean acidification on the incorporation kinetics of six trace metals and one radionuclide (Mn, Co, Zn, Se, Ag, Cd, Cs, 241 Am) in an economically-and ecologically-relevant sea urchin of the Mediterranean coastline: P. lividus. These elements were chosen for their essential (e.g. Mn, Co, Se and Zn function as catalyst or activators of enzymatic reactions) and non-essential characters (e.g. Ag, Cd and 241 Am), their toxicity ("very toxic and relatively accessible" for Co, Se, Zn, Ag and Cd according to [START_REF] Förstner | Metal pollution in the aquatic environment[END_REF] and their occurrence in the coastal areas (e.g. 241 Am is a toxic and radioactive isotope associated with nuclear waste). The radiolabelled metals and radionuclide added in trace concentrations allowed precise tracing of their incorporation in larvae exposed to six different pH conditions (from pH T 8.1 down to -1.0 pH units) during the first 74 hours of development. We expected to observe decreased accumulation of radionuclides when pH was lowered due to the impaired larval developmental rates and we wished to detangle the more direct effects of pH on bioaccumulation due to e.g. changes in metal speciation, the larval physiology/metabolism and/or impairment of detoxification processes. We also expected differences in how metals would react to pH changes to be based on the nature of the metals (essential vs. non-essential) and their major form in the seawater (e.g. chloride-dominated vs. free form) and hence their expected speciation changes.

MATERIAL AND METHODS

Biological material, spawning and experimental conditions

In March and April 2009, specimens of adult sea urchin (P. lividus) were collected by divers from subtidal rocky shores (≈10m deep) in the Bay of Villefranche-sur-mer (NW Mediterranean coastline, France). This period corresponds to a few months prior to natural spawning in this area (mid-May to July, [START_REF] Boudouresque | Ecology of Paracentrotus lividus[END_REF]. The adults were maintained in a flow of unfiltered natural seawater (depth=10m, salinity=38) in the Laboratoire d'Océanographie de Villefranche and fed with Posidonia oceanica until fertilization (end of April). Three females and one male were opened around the peristome to retrieve gametes from the ripe gonads. Eggs were rinsed three times with filtered seawater (FSW; 0.22 μm, salinity=38) and pooled together. Sperm was collected dry using a Pasteur pipette and stored at 4°C in a small tube before use. Within an hour after collection the gametes were transferred to the Radioecology Laboratory (Environment Laboratories of the International Atomic Energy Agency, Monaco) premises for fertilization. A concentrated egg suspension (≈ 4 000 000 eggs) was transferred to six 1-l beakers filled with FSW (20°C) and diluted sperm (1:50 ratio in FSW) was added to the egg suspension. Eggs were gently agitated for 15 min to optimize fertilization and then distributed to six replicates 4.5-l glass beakers per pH level (N=36 beakers) filled with radiolabeled FSW (seven radionucleids, detailed below). Embryos (start density of ca. 130 per ml) were grown in aerated FSW at their respective pH levels (pH T : 7.1, 7.3, 7.5, 7.7, 7.9, 8.1;

Table 2).

Natural variability in pH has been described to be broad in certain coastal areas (e.g. [START_REF] Thomsen | Calcifying invertebrates succeed in a naturally CO2-rich coastal habitat but are threatened by high levels of future acidification[END_REF] and the shift of the pH range as a whole in the future (e.g. [START_REF] Hauri | Spatiotemporal variability and long-term trends of ocean acidification in the California Current System[END_REF] is highlighted as an important reason to investigate of wide ranges of pH (ΔpH down to -1.0 unit; e.g. [START_REF] Dorey | Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH[END_REF]. Yearly surface pH variations are much tamer near our site, with extreme pH values recorded ranging from 7.95 to 8.20 (DYFAMED station in the Ligurian Sea, 50 km from the coast, monthly data from 1995-2011: Geri et al., 2014, and SOMLIT Villefranche-sur-Mer Station, 500 m from the shore, April-December 2016, n=37: http://somlitdb.epoc.u-bordeaux1.fr/bdd.php) as well as in general in the northwestern Mediterranean Sea (e.g. pH T = 7.893 ± 0.007 near the Strait of Gibraltar in Aug 2012-June 2015, n=15937, [START_REF] Flecha | Trends of pH decrease in the Mediterranean Sea through high frequency observational data: indication of ocean acidification in the basin[END_REF]. This small pH seasonal variation window has been consistently shifting at a rate of -0.0044 units per year [START_REF] Flecha | Trends of pH decrease in the Mediterranean Sea through high frequency observational data: indication of ocean acidification in the basin[END_REF]. Nevertheless, a wide pH range approach also allows for a more precise understanding of the shape and intensity of the biological response to ocean acidification, with a focus in this study on the metal bioaccumulation efficiency in sea urchin early life stages.

Experimental set-up and carbonate chemistry

Temperature (≈ 20 °C) was controlled to within ± 0.5°C in the 300-l bath surrounding the culture beakers (300W submersible heater coupled to a temperature controller: IKS, Karlsbad, Germany). Seawater pH was maintained to the desired levels within each culture beaker within ±0.05 pH units using a continuous pH-stat system (IKS) that controlled the addition of CO 2 into the beakers (see Martin et al., 2011 for detailed description). The pH values of the pH-stat system were adjusted from the measurements of pH on the total scale (pH T ) in each beaker using a pHmeter (Metrohm 826 pH mobile, Metrohm, Herisau, Switzerland) calibrated using TRIS (Tris/HCl) and AMP (2-aminopyridine/HCl) buffer solutions with a salinity of 38. In order to avoid contamination of the lab equipment used for alkalinity determination with radioactive agents, total alkalinity (TA) was assessed potentiometrically in seawater from radiotracer-free culture beakers maintained in the same pH/temperature conditions and done in parallel in spring 2009 (see below in 2.4; detailed description in [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF]). The carbonate system parameters (pCO 2 , Ωa and Ωc) were calculated from these measurements using the R package seacarb [START_REF] Lavigne | seacarb: seawater carbonate chemistry with R. R package version 2[END_REF] with the dissociation constants from [START_REF] Mehrbach | Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure[END_REF] as refitted by [START_REF] Dickson | A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media[END_REF].

Radiolabelling and radioactivity analyses

Radiotracers were purchased from Amersham, UK ( 109 Cd), Polatom, Poland ( 110m Ag, 134 Cs, 75 Se), Cyclotron, Russia ( 65 Zn), and Isotope Product Laboratory, USA ( 241 Am, 60 Co, 54 Mn).

For determination of larval metal uptake, eggs were incubated in seawater labeled with dissolved 54 Mn (1 kBq l -1 as 54 MnCl 2 ), 60 Co (1 kBq l -1 as 60 CoCl 2 ), 65 Zn (1 kBq l -1 as 65 ZnCl 2 ), 75 Se (1 kBq l -1 as Na 2 75 SeO3), 109 Cd (1 kBq l -1 as 109 CdCl 2 ), 110m Ag (1 kBq l -1 as 110m AgNO 3 ), 134 Cs (1 kBq l -1 as 134 CsCl), and 241 Am (0.35 kBq l -1 as 241 AmNO 3 ) by spiking typically 5 to 10 µl of stock solutions without affecting the tank seawater pH. These concentrations correspond to the addition of 21, 3, 133, 5, 6, and 0.7 ng l -1 of Ag, Am, Cs, Co, Se and Zn stable element respectively.

While the specific activities of 109 Cd and 54 Mn sources were not available from the providers, the use of these radionuclides usually results in the addition of ng l -1 of metal or less (see Lacoue-Labarthe et al., 2011b), which correspond to environmentally-realistic trace metal concentrations [START_REF] Bruland | Controls of Trace Metals in Seawater[END_REF][START_REF] Yoon | Dissolved trace metals in the Western Mediterranean Sea: total concentration and fraction isolated by C18 Sep-Pak technique[END_REF]. This allows us to add simultaneously the eight radiotracers in each experimental microcosm, while being able to consider that the metals do not interact in this mixture at these concentrations.

At 28, 52 and 74 hpf (hours post-fertilization; i.e. until four-arm pluteus stage), culture samples of 300 to 1000 ml (≈10300 to 65500 individuals depending on the culture density) were collected in triplicates in each pH treatment for radioactivity counting. Larvae were collected on 20-µm polycarbonate membranes, re-suspended and rinsed three times in radiotracer-free FSW.

The filters with embryos or larvae were then radio-counted (γ-spectrometry system consisting of Germanium detectors; see e.g. [START_REF] Lacoue-Labarthe | Temperature and pCO 2 effect on the bioaccumulation of radionuclides and trace elements in the eggs of the common cuttlefish, Sepia officinalis[END_REF]. Sub-samples of 150 ml of the remaining filtered seawater were sampled and radio-counted in order to measure the radiotracer concentrations in the seawater at sampling time. In the culture beakers, the sampled seawater volume was replaced with pH-equilibrated FSW and spiked with adjusted concentrations of radiotracers to maintain activities as constant as possible. Measured concentrations of radiotracers in the seawater varied by 5.9 ± 11.5 % of the desired value all along the experiment (N=30 for each metal).

During the experiment, larval density was followed every day for the first 74 hpf (count at the binocular microscope of six replicates of 1 or 2 ml -depending on larval density -of seawater for every pH treatment at 22, 47 and 74 hpf). Larval density decreased from 100-130 ind.ml -1 at 22 hpf to 10-25 ind.ml -1 at 74 hpf. Total larval mortality (%) was calculated from the difference between initial (22 hpf) and final (74 hpf) larval density corrected for consecutive seawater dilutions (Table 3).

Data treatment and statistical analyses

The accumulation of metals and radionuclide in sea urchin larvae was expressed as change of concentration factor (CF; Bq larvae -1 ), here defined as the radiotracer activities measured in the larval sample divided by the number of larvae per sample (estimated from larval density) and normalized by the time-integrated activity in the seawater over time. In order to limit total radiation exposure for experimental workers and waste management cost, only one 4.5-l tank was dedicated to each pH condition. Hence, triplicates of sampled larvae are considered as technical replicates according to [START_REF] Hurlbert | Pseudoreplication and the design of ecological field experiments[END_REF] and the bioaccumulation of radiotracers in larvae has been tested considering the measured pH T as a continuous covariate instead [START_REF] Havenhand | Designing ocean acidification experiments to maximise inference[END_REF].

Larval body lengths (L, μm) at each sampling times (Table 3) were back-calculated from the growth rates measured in the same pH conditions during a parallel experiment (without radionuclides; spring 2009: [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF]. This experiment was running at the same time as the present experiment and was identical (set-up: N=36 4.5-l glass beakers, identical pool of embryos, pH, temperature and seawater origin) to the exception of the sampling times (±1h) and the initial density that was ca. 4 times lower at the start (32-35 embryos per ml vs. 130 per ml here). Although we are aware of the possible confounding effect this could have on growth rates, this difference was quickly reduced due to the large sampling required in the radio-labeled experiment (10 individuals per ml at 73h vs. 10-25 individuals per ml at 74h in the experiment presented here). To confirm the relationships between metal accumulation and larval size, we tested the fit of linear (CF = a x L + b) and logarithmic models (CF = a x log(L) + b). The model comparison confirmed linear model to consistently have higher AIC (Akaike Information Criterion). Because of the well-documented developmental delay which sea urchin larvae encounter in acidified conditions [START_REF] Dorey | Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH[END_REF][START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF][START_REF] Stumpp | CO 2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay[END_REF], larval body length was used instead of time during the analyses in order to avoid any growthdependent effect. Since CF and larval size (L) were significantly linearly correlated, we therefore performed statistical analyses on the CF normalized by larval size (CF/L: Bq larvae -1 μm -1 ).

In order to assess the effect of pH on the bioaccumulation of metals, we tested the effect of two continuous variables (mean pH T and Time) on the CF normalized by larval size (CF/L; Bq larvae -1 μm -1 ) for each metal using generalized linear model analyses. We then fitted generalized additive models (gam, R package mgcv) to the CF/L at the final time-point (74 hpf) to unravel the different shapes of the relationships between each metal accumulation and pH. The basis dimensions for smooths were chosen visually and using the available function gam.check in the mgcv package. All the statistical analyses were performed using the R software (R Core Team, 2009), with a significance level of α=0.05. Results are expressed as mean ± SD.

RESULTS

Culture conditions

The mean pH T values, as well as the calculated carbonate chemistry parameters, are shown in Table 2. Mean temperature was 20.13 ± 0.34 °C (N=282). The pH T values in every treatment were significantly different from each other (1-way ANOVA with Welch correction: p<0.0001; F=30946 and df=5, followed by pairwise t-test with Holm adjustment: p<0.0001).

Because it was not possible to measure TA in radiolabelled samples, TA was measured in a similar experiment where the mean TA averaged between 2550 and 2565 μmol.kg -1 depending on the culture conditions (changes of 5 to 35μmol.kg -1 from 0 to 73 hpf in each treatment, see [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF]. The mean TA (≈2558 mol.kg -1 ) was therefore used for the carbonate chemistry calculations presented here. pCO 2 values ranged from 335 ± 20 μatm in the 8.1 pH condition to 4537 ± 438 μatm in the lowest-pH condition. Seawater was under-saturated in the lowest-pH treatment (pH T =7.12) for both calcite and aragonite and only in the pH T =7.32 treatment with respect to aragonite.

Larval densities were followed every day in each beaker (n=6 replicates per day) and decreased with time due to seawater dilution (Table 3: 130 to 10 larvae.ml -1 ). Larval mortality (%) was calculated from the difference between the expected larval density at 74 hpf taking into account dilutions (i.e. 28.8 larvae.ml -1 ) and the measured density in each pH treatment at 74 hpf.

Larval mortality was generally low (12-22%), except for pH T =7.12 where it reached 64% mortality in three days (Table 3).

Metal incorporation in larvae

Concentration factors (CF) increased with time for all the metals except Cs (Fig. 1A), as metals accumulated in the body of larvae in the first 74 hpf. Accordingly, the presence of Cs in the larval bodies was always relatively small (CF= 6.8 10 -6 ± 6.5 10 -6 Bq larvae -1 ). Between 28 and 74 hpf, the CF increased on average by ~10-fold for Mn, Se and Am (respectively by 9.1, 13.1 and 12.2 times) and by ~5-fold for the other four metals (4.8 times for Co, 5.3 times for Zn, 6.4 times for Cd and 4.8 times for Ag).

Seventy-four hours after fertilization, the larvae in the lowest pH treatment were ~40% smaller than the larvae grown at pH T 8.1 (Table 2, also see [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF] for complete description of the results). Because the CF was strongly correlated with larval size (L) for all metals but Cs (Fig. 1B: CF=a x L + b), we analyzed the effect of time and average pH T on the CF standardized by larval body length (CF/L, Bq larvae -1 μm -1 ) using generalized linear models (Table 4). Taking into account the three time points, the pH T significantly affected the CF/L of the following three metals: Se, Cd and Ag. This analysis also confirmed that time had a significant effect on the standardized accumulation of all metals except for Cs (Table 4).

In order to understand how average pH T shaped the incorporation of metals in the larvae, we fitted generalized additive models to the CF/L data on 74 hpf (Fig. 2; the statistical results are detailed in Table 5). At this time-point, neither Co, Cs nor Am accumulations were significantly affected by pH T . However, pH T had a significant effect on the accumulation Se, Cd and Ag (the same metals found significantly-affected in the generalized linear models analysis: Table 4 and5), and two other additional metals: Mn and Zn (Table 5). The accumulation (CF/L) significantly decreased when pH T was lowered for three metals: Zn, Se and Cd, while it was significantly increased in Mn and Ag (Table 5). These trends were also observed at 24 and 58 hpf (data not shown).

The accumulation of Cd in larval tissues decreased linearly with lowering pH T (deviance explained by the model: D=25%; Fig. 2 andTable 5): the larval content by size-unit was halved in the 7.1 pH treatment compared to pH 8.1 (CF/L=0.66.10 -6 ± 0.58.10 -6 vs. 1.2.10 -6 ± 0.06.10 -6 Bq larvae -1 μm -1 respectively). The larval content of Zn linearly decreased from pH 7.9 to 7.1 (D=73%: CF/L= 5.0.10 -6 ± 0.2.10 -6 to 3.8.10 -6 ± 0.6.10 -6 Bq larvae -1 μm -1 respectively, i.e. -24%; Fig. 2 andTable 5) but the larval content at the 7.1 pH treatment was comparable to that of the pH 8.1 (CF/L=3.7.10 -6 ± 0.2.10 -6 Bq larvae -1 μm -1 ). Similarly, the larval content of Se at the 7.1 pH treatment was comparable to that of pH 8.1 (CF/L=7.5.10 -7 ± 2.8.10 -7 and 8.8.10 -7 ± 1.6.10 -7

Bq larvae -1 μm -1 respectively) while the larval content linearly decreased by more than twice compared to pH 8.1 for the 7.3 pH treatment (CF/L= 2.6.10 -7 ± 0.8.10 -7 Bq larvae -1 μm -1 ; D=66%; Fig. 2 andTable 5). The differences observed could not be attributable to a change in larval size as the analyses were made on size-standardized CF.

The accumulation of Ag in larval tissues significantly (p=0.037; Fig. 2 andTable 5) and gradually increased with lowering pH T (D=67%): the larval content (CF/L) was more than 3.5 times higher in the 7.1 pH treatment than at pH 8.1 (CF/L=6.5.10 -6 ± 1.9.10 -6 vs. 1.8.10 -6 ± 0.3.10 -6 Bq larvae -1 μm -1 respectively). The accumulation of Mn in larval tissues significantly (p=0.033; Fig. 2 andTable 5) increased with lowering pH T (D=67%) although the main difference was visible for the 7.1 pH treatment where the larval content was ~2 times higher than in the rest of the treatments (CF/L=4.3.10 -6 ± 1.7.10 -6 for 7.1 while other ranged from 1.5.10 -6 ± 0.4.10 -6 to 1.9.10 -6 ± 0.3.10 -6 Bq larvae -1 μm -1 ).

Discussion

Ocean acidification modulates the incorporation of radio-labeled heavy metals in sea urchin larvae

In this study, we observed that a decrease in seawater pH can alter the incorporation of essential and non-essential elements. A known effect of pH is the slowing down of developmental rates in sea urchin larvae, and here we standardized incorporation by size in order to better focus on the direct effects of pH on metal incorporation. Paracentrotus lividus larvae exposed to decreasing pHs from 8.1 to 7.1 for 74 hpf incorporated significantly more Mn (2.5 fold increase in concentration at 7.1 compared to the other pHs), significantly more Ag (3.7 fold increase in concentration for a pH unit decrease) and slightly less Cd (1.9 fold decrease for a pH unit decrease to non-significant if one zero data point is taken out). Zn and Se exhibited complex incorporation behavior with pH: an initial increase for Zn (1.35 fold from pH 8.1 to 7.9) followed by a gradual decrease back to the initial (pH 8.1) values at pH 7.1, while Se concentration gradually decreased by 3.4 fold until pH 7.3 to sharply rise back nearly to the initial (pH 8.1) concentrations at pH 7.1. The incorporation of Co, Cs and 241 Am was unchanged, partly owing to low concentration detected in the larvae for e.g. Cs.

Remarkably, there was no obvious trend linked to the nature of the elements (essential vs. non-essential): lowering pH significantly increased the concentration of Ag and Mn in sea urchin larvae but slightly decreased Cd when Zn and Se concentration had opposite behaviors. There was no evident impact on the other elements tested (Co, 241 Am, Cs). The bioaccumulation behaviors observed did also not appear obviously linked to the type of the element form (whether chloride-dominated for Cd and Ag, oxidized state for Se or mostly found as free form for Cs, Co, Zn, Mn) and the consequent speciation changes. For instance, Ag and Cd have similar speciation changes with lowering pH, resulting in a very little increase in the toxic form due to pH (Cd 2+ represents 20.1% of the Cd forms at pH 8.1 and 20.2% at pH 7.4 in the model by [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF], but end up having different bioaccumulation reactions to pH. It is thus unlikely that the observed disturbances in elemental bioaccumulation with pH are only due to speciation changes or to increased metabolism (increased incorporation) and these disturbances must be specific to each metal and their entrance pathways and mechanisms.

The nature of the modulation is specific to each metallic element

Silver is toxic at low levels for sea urchin larvae (EC 50 of 40-85 μg l -1 for P. lividus, [START_REF] Warnau | Spermiotoxicity and embryotoxicity of heavy metals in the echinoid Paracentrotus lividus[END_REF]. Here, 110m Ag was taken up at higher concentrations when pH was lowered in the tissues of the sea urchin larvae. This observation is consistent with previous observations of increased Ag accumulation with lowering pH in whole eggs and newly hatched juveniles of cephalopods (Lacoue-Labarthe et al., 2011a, 2009). Although reasons specific to the embryonic development of the studied cephalopods had been discussed (peri-vitellin fluid properties affecting the specificity of Ag speciation and availability and higher metal translocation from the eggshell to the embryo), this study shows that increased Ag incorporation with lower pH was also visible in animals with a different life history. Silver is thought to enter marine invertebrates via Na + /K + -ATPase pumps (e.g. shrimp, sea hare, urchin: [START_REF] Bianchini | Mechanism of acute silver toxicity in marine invertebrates[END_REF]. These pumps are likely to be more active in the sea urchin larvae exposed to acidified seawater as a way to maintain internal stasis [START_REF] Stumpp | pH regulation and excretion in Echinoderms[END_REF]. Therefore, this strengthen the hypothesis that the increase in Ag incorporation at low pH is driven by increased activities in ionic regulatory mechanisms (Lacoue-Labarthe et al., 2011a): an increased metabolism could consequently increase the transport of Ag into the cells.

Cadmium has no known biological role, it is highly toxic to sea urchin larvae and has been effectively used as an inducer of skeleton malformation [START_REF] Matranga | Chapter 8: Echinoderms as blueprints for biocalcification: Regulation of skeletogenic genes and matrices[END_REF]. It also induces the expression of stress proteins [START_REF] Roccheri | Cadmium induces the expression of specific stress proteins in sea urchin embryos[END_REF], methallothionein genes [START_REF] Russo | Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos[END_REF] and even apoptosis [START_REF] Filosto | Environmentally relevant cadmium concentrations affect development and induce apoptosis of Paracentrotus lividus larvae cultured in vitro[END_REF]. Even 15 hours after Cd removal from the culture, negative effects can persist [START_REF] Roccheri | Cadmium induces the expression of specific stress proteins in sea urchin embryos[END_REF]. In our study, 109 Cd uptake did not seem to be strongly affected by pH (except from one point at 7.1), which is in accordance with the absence of strong speciation changes for this element. Cadmium is thought to enter the cell membrane in the form of Cd 2+ passively via calcium channels [START_REF] Rainbow | Cadmium, zinc and the uptake of calcium by two crabs, Carcinus maenas and Eriocheir sinensis[END_REF], and to mimic Ca 2+ behavior. Accordingly to the behavior of Cd observed in our study (no effect of pH), [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF] found no effect of pH on the accumulation of 45 Ca in the larvae. However, Cd uptake has been shown to be either decreased with lowering pH (isolated clam cells: Ivanina et al., 2013;cephalopods: Lacoue-Labarthe et al., 2011, 2009;phyoplankton: Xu et al., 2012), increased (bivalves: Shi et al., 2016) or to remain unaffected by pH (bivalves: [START_REF] Götze | Interactive effects of CO 2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria[END_REF].

The hypothesis that the lowered Cd accumulation in acidified water is only due to reduced metabolism in both clam cells and squid embryos does not corroborate our observations. However, one plausible mechanism for limited Cd incorporation at lower pH, even with the increased metabolic rates for sea urchin larvae, could be that there is a strong competition for binding sites with increased H + ion concentration in low pH as suggested by [START_REF] Pascal | The toxicological interaction between ocean acidity and metals in coastal meiobenthic copepods[END_REF]. Ivanina et al. (2013) also discuss possibly strong interactions between Cd accumulation in the cells and other essential metals (Zn), which may increase toxicity by replacement on the binding sites in case Cd pollution is present in the extra-cellular medium.

Zinc is an essential element to normal cellular function, as it acts as an enzyme co-factor for e.g. DNA and RNA-polymerase or carbonic anhydrase. However, zinc contamination of the environment from anthropogenic sources (e.g. discharges of mining products and wastes, use of fertilizers and wood preservatives: ATSDR, 2013), has been associated with several toxic effects.

In sea urchin larvae, this metal has been shown to induce strong morphological abnormalities with delayed/blocked development at the blastula stage, exogastrula, spaceship Apollo-like gastrula, radicalized pluteus [START_REF] Kobayashi | Effects of heavy metals on sea urchin embryo development. 1. Tracing the cause by the effects[END_REF][START_REF] Radenac | Bioaccumulation and toxicity of four dissolved metals in Paracentrotus lividus sea-urchin embryo[END_REF]. Ocean acidification is expected to increase the relative concentration of dissolved Zn 2+ from 80% at pH T of 8.1 to 95% at pH T 7.4. Zn bioaccumulation was lowered by ~1.5 fold in coral tissues when pH decreased (pH T 7.5 and 7.8 vs. 8.1) and in coral skeleton (pH T 7.5 vs. 7.8 and 8.1; [START_REF] Houlbrèque | Effects of increased pCO 2 on zinc uptake and calcification in the tropical coral Stylophora pistillata[END_REF]. On the contrary, here we observed an initial increase (x1.3) for Zn in the first 0.2 pH unit decrease followed by a gradual decrease back to the initial (pH 8.1) values at pH 7.1. A similar bioaccumulation behavior (maximum CF found at pH T 7.85 vs. pH T 8.1 and 7.60) was found for cuttlefish newly hatched juveniles [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF] and squid paralarvae (Lacoue-Labarthe et al., 2011a). As previously proposed, this bell-like behavior may be linked to the complex interactions between the requirement to maintain acid-base balance and the function of Zn as a co-factor of carbonic anhydrase, an enzyme involved in the skeletogenesis but also in the regulation of acid-base balance (see further details in [START_REF] Karakostis | Characterization of an alpha type carbonic anhydrase from Paracentrotus lividus sea urchin embryos[END_REF] and, in particular, for its putative role in proton excretion in the stomach of sea urchin larvae [START_REF] Stumpp | pH regulation and excretion in Echinoderms[END_REF]. Seawater acidification might therefore enhance carbonic anhydrase synthesis and consequently, Zn requirement in the tissues in the first acidification step. The decrease in Zn accumulation at even lower pHs (≤7.7) might be due to an increased competition with protons for the cells binding sites, which might limit Zn cation incorporation.

Selenium displayed the same kind of trends with a decreased accumulation with lowered pH, except at the extreme value of pH 7.1, suggesting again a competition between protons and Se free form ion. To date, little information on the mechanisms involved in dissolved Se uptake in marine animals is available to link physiological responses of sea urchin larvae to Se bioaccumulation processes. It is noteworthy that the accumulation values observed for Se compared to the other trace elements are relatively low which is congruent with the fact that Se is known to be mainly accumulated from the trophic route [START_REF] Sandholm | Uptake of selenium by aquatic organisms[END_REF]. Selenium plays an essential role as a co-factor for the reduction of anti-oxidant enzymes, especially in marine organisms. It is mostly found in the water under its oxidized state (SeO 4 2-or SeO 3 -), which presumes that it is little affected by CO 2 -driven seawater acidification [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF]. It enters the water from surface runoff, drainage, coal mining, treatment plants and historically oil refineries with concentrations in polluted effluents that can reach 0.25 mg l -1 (average seawater concentration: 0.09 µg l -1 ; ATSDR, 2003). Selenium toxicity to sea urchin has been seldom studied, to our knowledge; only one study found a significant effect of Se on larval abnormality (EC 50 of 26 μg l -1 in plutei of Diadema antillarum; [START_REF] Bielmyer | The effects of metals on embryo-larval and adult life stages of the sea urchin, Diadema antillarum[END_REF] and only a single study investigated the effect of pH on its bioaccumulation (undetected in cuttlefish newly hatched juveniles; [START_REF] Lacoue-Labarthe | Temperature and pCO 2 effect on the bioaccumulation of radionuclides and trace elements in the eggs of the common cuttlefish, Sepia officinalis[END_REF].

Finally, manganese is an essential element which is involved in enzyme activation and several regulatory functions such as e.g. bone mineralization, energetic metabolism or immunological and nervous system activities (see review by [START_REF] Pinsino | Manganese : A new emerging contaminant in the environment[END_REF]. It is however an emerging pollutant in the marine environment which is present in several human-made waste (e.g. metallic and chemical products, sewage) and it is a known neurotoxin in humans. In water, it is principally found as Mn 2+ with a slight increase in the toxic form presence with decreasing pH (97.3% at pH 8.1 to 99.05% at pH 7.4: [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF]. Extensive work on P. lividus larvae [START_REF] Pinsino | Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development[END_REF][START_REF] Pinsino | Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton[END_REF][START_REF] Pinsino | Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects[END_REF] demonstrated that, although high levels of Mn did not create lethal effects, Mn accumulated in the cells with time and led to strong sub-lethal effects. For instance, the authors observed that Mn exposure disturbed calcium uptake and increased morphological abnormalities (at a concentration of 61.6 mg l -1 , Mn prevented skeletal growth and modified gene expression linked to skeletogenesis), and also increased heat-shock protein expression. In this study, we observed that lowering pH increased Mn bioaccumulation in tissues, but this was mostly due to an increase at pH T 7.1. Until this extreme pH value, the absence of any change in Mn bioaccumulation remain consistent with the quasi-stable concentration of the free form ion, suggesting that responses of sea urchin larvae to decreasing pH do not affect its Mn metabolism. At pH T 7.1, it is difficult to interpret the sudden increase since larvae are usually highly abnormal in their morphology (pers. data: 46 ± 40% abnormality vs. 1.5 ± 3.2% for S. droebachiensis at pH T 7.1: [START_REF] Dorey | Assessing physiological tipping point of sea urchin larvae exposed to a broad range of pH[END_REF].

Conclusions

In conclusion, we have shown that CO 2 -induced pH changes can modify the bioaccumulation rates of metals in sea urchin larvae. The variance in accumulation was mostly explained by the indirect effect of pH on larval growth but effects independent of the size were also significant for five metals. The relationship between the changes in speciation, bioaccumulation and toxicity are not straightforward and urgently requires more research. In particular, studies combining toxicity with environmentally-relevant levels of metals and bioaccumulation data are vital to implement the necessary policies for water and fisheries management. Combined pH/metal toxicity studies have been carried out mainly on Cd and Cu [START_REF] Campbell | Ocean acidification increases copper toxicity to the early life history stages of the polychaete Arenicola marina in artificial seawater[END_REF]Ivanina et al., 2015, 2014, Ivanina and Sokolova, 2015, 2013;[START_REF] Lewis | Metal contamination increases the sensitivity of larvae but not gametes to ocean acidification in the polychaete Pomatoceros lamarckii (Quatrefages)[END_REF] and tend to show an increase of metal toxicity with lowering pH. In our study, we show that the responses in terms of bioaccumulation are specific to each metal and this highlights the current need to understand the mechanisms which operate in seawater with lowered pH (e.g. passive or active regulation of metal uptake, metal uptake as by-products of metabolic rates or acid-base balance regulation), which will in turn be essential to be able to implement more accurate water management policies.

Finally, this study did not consider the processes of detoxification (e.g. [START_REF] Pinsino | Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton[END_REF] and the reversibility of trace metal effects (e.g. [START_REF] Morroni | Reversibility of trace metals effects on sea urchin embryonic development[END_REF], which could play an important role in determining the sensitivity of sea urchin larvae to a future ocean where seawater acidification and metal pollution co-exist, and represents a challenging gap of knowledge. [START_REF] Basallote | Studying the effect of CO 2induced acidification on sediment toxicity using acute amphipod toxicity test[END_REF] nor nano-metals (e.g. [START_REF] Hu | CO 2 -induced pH reduction increases physiological toxicity of nano-TiO 2 in the mussel Mytilus coruscus[END_REF]. For a review by metals, see also Ivanina & Sokolova (2015) for each pH treatment), temperature (20.13 ± 0.34 °C, n=282, measured every 15 min in one tank), salinity (38) and a mean total alkalinity (TA) of 2558 μmol.kg -1 using the R package seacarb. Data are expressed as mean ± sd. Larval body length (L; μm) was calculated for each sampling time (hpf) and pH treatment used in the experiment (lengths calculated from [START_REF] Martin | Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO 2 -driven acidification[END_REF]. 119.8 ± 10.0 54.5 ± 6.5 25.2 ± 5.4 12.5 7.9

103.8 ± 6.9 53.5 ± 4.9 23.9 ± 4.0 16.8 8.1 126.0 ± 13.7 75.7 ± 7.9 24.7 ± 4.1 14.2 FIGURE 1: Concentration factor (CF±SD; 10 -4 Bq larvae -1 ) of metals in the different pH treatments: diamond (7.1), triangle (7.3), circles: hollow (7.5), light-grey (7.7), grey (7.9), black (8.1); as a function of A. time (three time points: 28, 52 and 74 hpf) and B. body length (μm). The results of the linear regression between CF and body length for each metal are presented in grey (R 2 and p for the p-value). 5.

Fitzer↓

  pH ↓ Cd and ↑ Cu accumulationIvanina et al., 2013

FIGURE 2 :

 2 FIGURE 2: Larvae metal content (CF/L) at 74 hpf changes with A. pH and B. calculated H +

TABLE 1 .

 1 Literature review of the effects of CO 2 -induced acidification on metal bioaccumulation and toxicity in marine invertebrates 687 using lab-based experiments. Article search was performed in Web of Science using the keywords 'ocean acidification' and 'metal' in 688 July 2017. This review does not include publications on CSS-leakage experiments, field or sediment toxicity studies (but see e.g.

	689

.

  Stage: S= 690 sperm, F=fertilization, E=embryos, L=larvae, J=juveniles, A=adults, gen=generations. pCO 2 is indicated when it was measured, or an additional 691 carbonate chemistry parameter was measured (e.g. TA, DIC).

	Type of study
	Group
	Species
	Stage
	concentration)	(nominal	elements	Chemical

Measured pH (pCO 2 ) treatments Conclusions Reference

							and uptake	experiment	Toxicity														(radiotracers)	Bioaccumulation
	Clam	Oyster,		Scallop		Mussel		Bivalves Mussel			Echinoderms Urchin				Bivalves Oyster		Cnidarians Coral		Squid					Cephalopods Cuttlefish		Crustaceans Copepod		Clams	Mussel,
	Adults			Adults		Adults		Adults			Larvae	Embryos,			Adults		Adults		Juveniles	Embryos,		Embryos		Embryos		2 gen.	A, E, L, J,	Adults	Adults	Adults
	(50 μg l -1 )	Cu or Cd		Cd (40 μg l -1 )		Cu (0.1 μM)		Cd (20 μg l -1 )			(trace)	Ag, Cd, Cs, 241 Am	Mn, Co, Zn, Se,		Cd, 241 Am (trace)	Mn, Co, Zn, Ag,	Zn (trace)		Mn, Zn (trace)	Ag, Cd, Co, Hg,		Se (trace)	Am, Cs, Co, Mn,	Ag, Cd, Zn (trace)		Cu (20 μg l -1 )		Cd (50 μg l -1 )	(50 μg l -1 )	Cd or Cu	Cd (50 μg l -1 )
	(860), 7.76 (1330)	pH NBS =8.11 (531), 7.93		8.05, 7.60		(1420)	pH NBS =8.11 (485), 7.68	(2980)	pH NBS =8.17 (430), 7.42		(4540)	(1700), 7.3 (2840), 7.1	(625), 7.7 (1020), 7.5	pH T =8.1 (335), 7.9	(900), 7.5 (2040)	pH T =8.1 (380), 7.8	7.78 (900), 7.46 (2040)	pH T =8.09 (ambient),	(850), 7.60 (1500)	pH T =8.1 (380), 7.85		(800), 7.61 (1400)	pH T =8.04 (400), 7.84	(800), 7.61 (1400)	pH T =8.09 (400), 7.84	7.82 (400), 7.67 (590)	8.06 (210), 7.95 (285),	8.07, 7.79, 7.42	(970), 7.52 (2090)	pH NBS =8.14 (415), 7.83	(1100), 7.60 (2590)	pH NBS =8.18 (580), 7.93
	gene expression	accumulation, interactive effects on	Intermediate pH ↑ Cu	or Cd toxicity	No/little effect of pH on Cd uptake	acidosis and ↑ Cu toxicity	↓ pH enhanced extracellular	genotoxicity	accumulation, ↓ pH ↑ Cd	No effect of pH on Cd	modulations of Mn, Zn and Se	↓ pH ↑ Ag accumulation, non-linear			bioaccumulation	No effect of pH on metals'	and skeleton (7.5)	↓ Zn accumulation in tissue (7.8)	accumulation in Embryos	accumulation, modulates Hg	↓ pH ↑ Ag andZn, ↓ Cd and Mn	↓ pH ↓ Co and Mn accumulation		modulates Zn accumulation	↓ pH ↑ Ag and ↓ Cd accumulation,	toxicity	↓ pH ↑Cu accumulation and	↓ pH ↑ Cd accumulation	↓ pH ↑ Cu and Cd accumulation	immunology	accumulation, ↓ pH ↑ Cd effects on	No effect of pH on Cd
	Götze et al., 2014			Benedetti et al., 2016		Lewis et al., 2016		Nardi et al., 2017			Present study				Belivermis et al., 2016		Houlbrèque et al., 2011		2011	Lacoue-Labarthe et al.,		2012	Lacoue-Labarthe et al.,	2009	Lacoue-Labarthe et al.,			Shi et al., 2016	Ivanina et al., 2015	Ivanina et al., 2014

TABLE 2 .

 2 Experimental conditions: Measured pH T and calculated carbonate chemistry in each of the pH treatments. Measurements in italic (pCO 2 and saturation states: Omega calcite and aragonite) are calculated from pH T (measurements taken every 15 min from 0 to 74 hpf, N=282

TABLE 3 . Larval densities and mortality:

 3 Larval density (larvae.ml -1 ; mean ± SD) in each of the six pH treatments and three time-points (N=6 replicates of 1 to 2 ml of seawater per pH treatment and time point) and total mortality (%

	larvae) after 74 hpf.
		Density (larvae.ml -1 )		Mortality
	pH treatment	22 hpf	47 hpf	74 hpf	at 74 hpf (% larvae)
	7.1	119.7 ± 18.0 42.8 ± 5.5 10.3 ± 1.3	64.1
	7.3	122.0 ± 5.9 49.8 ± 2.9 23.8 ± 4.3	17.4
	7.5	131.0 ± 5.0 50.3 ± 6.8 23.6 ± 1.7	21.5
	7.7

Acknowledgements:

The IAEA is grateful for the support provided to its Marine Environment Laboratory by the Government of the Principality of Monaco. We are grateful to D. Luquet, L. Gilletta and J.-Y. Carval for sea urchin sampling in the field. 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.

 TABLE 4. Generalized linear models results testing the effect of two continuous variables (mean pH T and Time) on the CF/L (Bq larvae -1 μm -1 ), for each metal. The best model was always without interaction term (pHT: Time) and so it is the one presented here. The variables that had a significant effect in CF/L are shown in bold for each metal.

Metal