Céline Du Boys
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Pay out policy decisions in France A managerial perspective

Keywords: JEL classifications: G35 Dividend, Share repurchase, Managers Survey, France, Agency theory 1

Cet article s'attache avant tout à bousculer l'approche purement quantitative des politiques de dividende ou de rachat d'actions en présentant une description qualitative de ces politiques. Notre étude terrain auprès de 12 décideurs français apporte certes un éclairage hexagonal mais il permet surtout une meilleure compréhension des éléments explicatifs ou d'influence des politiques de distribution. Notre étude présente la distribution de liquidités aux actionnaires comme le résultat d'un consensus entre plusieurs variables : capacité financière de l'entreprise, influences externes et pression de l'actionnariat. Nos résultats confortent la fonction de signal de la distribution et la politique de lissage des dividendes. De nouveaux éléments venant renforcer la réflexion apparaissent : existence d'une culture de la distribution, influence des caractéristiques des managers et des concurrents. Enfin cet article remet en question le rôle de distribution des rachats d'actions et souligne la forte influence des actionnaires majoritaires.

 [START_REF] Dereeper | L'Utilisation des Programmes de Rachat d'Actions en France[END_REF]). Observation shows that investors ask for dividends whereas they are heavily taxed on it and that stock prices rise after pay out. In United States, in the last years, stock repurchases importance has been growing to the detriment of dividends' one [START_REF] Grullon | Dividends, Share Repurchases, and the Substitution Hypothesis[END_REF]).

Empirical observations don't match up with the classic theoretical frame defined by [START_REF] Modigliani | Dividend Policy, Growth and the Valuation of Shares[END_REF]: on perfect and efficient markets, firm value is only determined by investment. There is no dividend premium on stock prices and investors have no preference between dividend and capital gain.

The gap between theory and empirical studies was called the "dividend puzzle" by [START_REF] Black | The Dividend Puzzle[END_REF]. This puzzle wonders why investors like dividends whereas they are heavily taxed on it and so on why firms pay out big amounts of money every year. The puzzle has become more complex with the growing importance of share repurchases.

This paper surveys research on pay out policies focusing on the firm point of view. It sets up some answers to why firms pay out. We will not focus on explanations of share price reactions or investment strategies linked to pay out policies. We will only focus on what happens inside the firm.

To follow this path, we have chosen to classify concepts depending on who takes the decision in the firm: shareholders or managers. If there is no agency conflict, decisions will be in accordance with shareholders' wishes. Otherwise, managers will influence pay out policies.

Of course, shareholders' wishes are not homogeneous and some shareholders may be more influential than others. In French firms, the major agency conflict is not between managers and shareholders, but between controlling shareholders and minority shareholders. [START_REF] Ginglinger | Ownership Structure and Open market stock repurchases in France[END_REF]). In this case, shareholders' influence is main shareholder's one.

Why firms pay out?

2.1.

No agency conflict between shareholders and managers.

If there is no conflict between shareholders and managers or if this conflict has been resolved, managers stick to shareholders stakes. Pay out can then be justified by various theoretical explanations:

-Pay out happens because shareholders want it :

First, shareholders may ask for pay out if they need regular cash and if dividend is the cheapest way to get it.

This happens if transaction costs on capital gains are superior to dividend taxes. On a second hand, shareholders may face institutional constraints that require them to invest in paying out firms. Empirical support for those two explanations is weak: few institutional constraints exist (in particular in France) and most of the times, transaction costs are smaller than dividend taxes.

Shareholders may also ask for pay out because they like it. Behavioral finance shows that some human bias may lead to such non-rational preferences [START_REF] Shefrin | Explaining Investor Preference for Cash Dividends[END_REF], [START_REF] Baker | A Catering Theory of Dividends[END_REF]). Empirical support of that theory is growing.

-Pay out is observed because of information asymmetry between shareholders and managers.

Managers are better informed of firm's future cash flows and signal this information to shareholders through pay out. Theory tells pay out is a good signal because it is costly and because bad signals are penalized [START_REF] Albouy | Signaux et Prix des Actions[END_REF]). This signal hypothesis leads to various theoretical models where managers adjust dividend to their forecasts: if they are good, they increase dividend and vice versa. [START_REF] Bhattacharya | Imperfect Information, Dividend Policy, and "The Bird In The Hand" Fallacy[END_REF]; [START_REF] Miller | Dividend Policy Under Asymmetric Information[END_REF]).

Empirical support of the signal theory is ambiguous. On one hand, dividend changes are followed by variation of stock price, which shows that information is integrating in prices. Moreover analysts modify their forecasts after pay out. But, on the other hand, earnings vary much more than dividends, so what does it signal? Moreover, big companies, where information asymmetry is weaker because of transparence legislation, are the one paying the biggest amounts of dividends.

Existence of an agency conflict between managers and shareholders.

In case of an agency conflict between shareholders and managers, various theories justify cash pay out. First, shareholders use pay out to monitor managers. Second, private interests of managers may influence pay out policies.

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Shareholders use pay out to monitor managers

According to [START_REF] Jensen | Theory of the Firm: Managerial Behavior, Agency Costs and Ownership Structure[END_REF], public firm's managers have self interests differing from shareholders' ones. They spend firm's money to promote their own interests and not shareholders' ones. [START_REF] Jensen | Agency Costs of Free Cash Flow, Corporate Finance, and Takeovers[END_REF] shows that pay out enables to minimize cash under managers' control. This situation leads managers to regularly ask the market for money and face shareholders' monitoring [START_REF] Rozeff | Growth, Beta and Agency Costs as Determinants of Dividend Pay Out Ratios[END_REF]; [START_REF] Easterbrook | Two Agency-Cost Explanations of Dividends[END_REF]).

Allen and Michaely (2002) underlined limits of that theory: if shareholders know that managers overinvest, why would they not impeach them a different way? This leads us to question efficiency of corporate governance mechanisms.

Once more, empirical support of that theory is ambiguous, mainly because it is difficult to measure managers' overinvestment. In our opinion, proxies are the heart of the matter because of the difficulty to measure Tobin's Q, main proxy used for overinvestment. Some pinpoint the weakness of that theory [START_REF] Lang | Dividend Announcements: Cash Flow Signaling vs. Free Cash Flow Hypothesis[END_REF]; [START_REF] Yoon | Signaling, Investment Opportunities, and Dividend Announcements[END_REF]), some others support it, for example, [START_REF] Borokhovich | Dividends, Corporate Monitors and Agency Costs[END_REF] using more precise proxies to measure agency conflict than those traditionally used.

-Managers' own interests influence pay out policies Sometimes shareholders do not monitor managers because they cannot or do not want to. What consequences does it have on pay out policies?

* Managers can use pay out to consolidate their entrenchment. [START_REF] Hu | Managerial Entrenchment and Pay Out Policies[END_REF] show influence of entrenchment factors on pay out policies. [START_REF] Fuller | Flexibility and Dividends, AFA San Diego Meetings[END_REF] present a model where managers' value operating flexibility and increase it by reducing dividends and conserving cash.

Choice between stock repurchase and dividend.

As mentioned in introduction, cash pay out policies have two possible shapes: dividend and share repurchase.

Those two means being distinct, firms are often confronted with the choice of the pay out tool. Research is also interested on that point and speculates on the links between those tools. In our opinion, three main interrogations come into view: are dividend and repurchase only pay out tools? Are they complementary tools of pay out thanks to their particular features? Is a substitution of one tool by the other possible?

To the first question, we can answer that dividend is only a mean of pay out, but share repurchase has other functions. For example, it might be used as a relution tool.

Concerning the substitute or complement hypothesis, [START_REF] Grullon | Dividends, Share Repurchases, and the Substitution Hypothesis[END_REF] have supported the substitution hypothesis of dividend by share repurchase. But [START_REF] Dereeper | L'Utilisation des Programmes de Rachat d'Actions en France[END_REF] showed that in France amounts paid in dividend have not decreased since share repurchases are allowed. This supports the complementary hypothesis. Anyway, most theories make the hypothesis that dividend and share repurchase are potential substitutes.

Let's now see the main theoretical justifications to the use of one or the other tool for pay out.

No agency conflict between managers and shareholders.

If there is no conflict of interest between managers and shareholders, the firsts follow the wishes of the lasts. In consequence, the choice between share repurchase and dividend results from the potential following situations.

Dividend will be chosen if shareholders have a preference for dividend (regular need of cash or irrational preference as reported by Behavioral Finance).

Shareholders may also prefer share repurchase for its special attributes:

-In most countries, share repurchases are less taxed, so shareholders might prefer such a pay out [START_REF] Dittmar | Why do Firms Repurchase Stock ?[END_REF]). [START_REF] Barclay | Dividends and Dominant Corporate Shareholders, AFA San Diego Meetings[END_REF] do not support the tax clientele hypothesis. They find no evidence that dividends are higher in firms that have corporate blockholders, whereas corporations should have an interest in dividend paying firms. On the contrary, [START_REF] Blouin | Payout Policy: A Shareholder-Level Analysis[END_REF] demonstrate that the tax status of influential shareholders plays a significant role in firms' pay out policies.

-Repurchase can enhance shareholder value. It can be used to cancel lost due to stock options dilution.

Moreover, it helps to reevaluate stock prices [START_REF] Dittmar | Why do Firms Repurchase Stock ?[END_REF]). Repurchase also enables risk transfer from shareholders to debtholders by reducing the amount of capital [START_REF] Dittmar | Why do Firms Repurchase Stock ?[END_REF]).

-At last, stock repurchases enable non-selling shareholders to consolidate their control of the firm.

Presence of agency conflict between managers and shareholders.

In presence of agency conflict, two situations appear:

-Shareholders compel managers through mean of pay out

On one hand, dividend helps to attract some particular institutional investors better at monitoring managers than current shareholders [START_REF] Simon | A Theory of Dividends Based on Tax Clientele[END_REF]). This model is supported in France by [START_REF] Debaere | Les Investisseurs Institutionnels ont-ils une Influence sur la Politique de Dividendes?[END_REF] and is inconsistent with [START_REF] Grinstein | Institutional Holdings and Payout Policy[END_REF] study.

On the other hand, repurchase is good for corporate governance: [START_REF] Oswald | Open Market Share Reacquisitions, Surplus Cash, and Agency Problems[END_REF] show that after stocks repurchase, the quality of firm's governance improves. So shareholders may force managers to repurchase stocks to improve their control.

-Managers choose to do repurchase or dividend because it is in their interest. Each tool might have specific advantages for managers:

* Repurchase is less restricting than dividend. Repurchase does not have to be regular through time, so managers undergo fewer constraints from shareholders using this tool. Moreover, managers can use stock repurchases to protect from market control: facing a takeover that threatens their job, managers can buyback shares. This leads the takeover to be more expensive and so less likelihood. [START_REF] Dittmar | Why do Firms Repurchase Stock ?[END_REF]).

* Dividend helps to strengthen managers' entrenchment [START_REF] Hu | Managerial Entrenchment and Pay Out Policies[END_REF]).

* If managers have stock options, they have an incentive to repurchase shares. Because repurchase helps to minimize dilution effect of dividends and increases Earnings per Share, it enhances managers' stock options value. This hypothesis receives a strong empirical support [START_REF] Fenn | Corporate Payout Policies and Managerial Stock Incentives[END_REF], Weisbenner (2000), [START_REF] Kahle | When a Buyback isn't a Buyback: Open Market Repurchases and Employee Options[END_REF]).

This survey of main theories explaining why firms pay out and why there are dividends or stock repurchases has shown limits of actual research. Empirical support to those theories is either limited or ambiguous. Afterwards, it is hard to know how to move forward the resolving of the dividend puzzle.

We think the solution of the dividend puzzle may lie in a complex integrated model, with many variables.

Theories presented here might lack some important variables. Ways to find new explanatory variables have to be found. One direction researches have taken is to study practitioners' understanding of pay out policies.

Managers' point of view

One way to lighten the puzzle may be to investigate practitioners understanding of pay out policies. So, we now survey the results of those studies, and in particular Lintner's one. [START_REF] Lintner | Distribution of Incomes of Corporations Among Dividends, Retained Earnings, and Taxes[END_REF] pioneering contribution shows that the determinant factor of dividend policy is the change in dividend. Most managers suppose shareholders prefer and value stable or growing pay out ratios. Changes in dividend rate must be justified by reasons understandable to shareholders, like for example a variation in earnings. Lintner shows that managers believe that it is necessary to pay out substantial part of their earnings in dividends. Even managers less worried about shareholders or dividends are convinced that dividend must reflect firm's earnings, otherwise it can have a bad effect on some factors (such as the stability of their own position).

Surveyed firms have a target pay out ratio. This target ratio is an aim and not something to be applied immediately. Target ratio and speed of adjustment vary depending on firms, sectors and economic situations.

Results of managers surveys made in the last 20 years confirm Lintner's results, showing that pay out policies determinants have not much evolved in the last fifty years. Moreover, those surveys show stock repurchase motivations are various and hazy. On the whole, repurchase are not considered as a dividend substitution or as a mean of pay out [START_REF] Baker | Why Companies Use Open-Market Repurchases: A Managerial Perspective[END_REF]; [START_REF] Brav | Payout policy in the 21st century[END_REF]; [START_REF] Gillet | Le Rachat d'Actions: Théorie et Pratique de Gouvernement d'Entreprise[END_REF])

Those surveys underline the importance of past pay out policies and of managers' beliefs. They also question the role of stock repurchase. But their interest is tempered by their limits: they are either old or limited. Lintner's paper is 50 years old and more recent papers use only quantitative surveys, which tend to induce too much the answers.

II. Justification of a qualitative approach

Facing those researches and their limits, it is difficult to progress in the puzzle solving. Which research ways have to be followed to overcome the puzzle?

In this paper, we make the hypothesis that a better knowledge of field reality can provide a first way. Through a qualitative approach, we think we can understand better pay out policies from inside the firm. This might be an interesting step to new theories and new links between variables.

This idea is reinforced by the suspicion we have that current theories cannot apply perfectly to the French market. Those theories form a coherent frame inspired by Anglo-Saxons markets. But given the complexity of the puzzle, it seems difficult to put aside the effect of context variables such as legislation or culture (La [START_REF] Porta | Agency Problems and Dividend Policies around the World[END_REF]).

The process of this paper is to focus on pay out policies, from inside the firm, in France. We try to provide an elaborated description of decision process, of variables influencing pay out and of players' feelings about pay out policies. We propose to bring a contribution to the solving of the puzzle at a national, then global level.

To enable that description we have chosen a qualitative process. We interviewed individually important decision-makers inside French listed companies. To warrant the scientific character of our process, we now present the methodology we used for that study.

Study methodology -Presentation of the population

The quality of our study is first linked to the people we have interviewed. The following table gives marks to judge the reliability and expertise of our respondent, while respecting the anonymity we have warranted. The size of our sample has been determined thanks to the saturation criteria. From the tenth interview, we thought we had reached a saturation of information collected and stopped after the twelfth interview.

-Method and interview guide

We choose to do semi-structured interviews. This lets freedom of speech to the interviewee, while avoiding the conversation to get out of hand. We made three guides: one for the managers, one for the analyst and one for the president of the minority shareholders association. The following table shows the main themes of the guide.

1 Sector classification used is from the French economic newspaper "Les Echos".

Table 2: Presentation of main themes tackled during managers' interviews.

Theme 1 Decision Process.

⇒ Identity of people or boards involved in dividend and share repurchase decisions.

Theme 2 Factors taken into account to decide dividend and share repurchase policies.

⇒ Managers' motivations to pay out: why do they repurchase shares, why do they pay dividends?

⇒ Internal variables explaining pay out: financial factors taken into account to determine pay out. But we also tried to understand firm cultural characteristics or managers psychology.

Theme 3 External influences on pay out policies ⇒ What are the external influences? Markets, shareholders…

We ran the interviews with respect for main principles advised for qualitative surveys [START_REF] Huberman | Analyse des données qualitatives[END_REF]).

-Data analysis Analysis followed two steps. First, we did the coding of the texts, then we quantitatively analyzed them. Each interview has been entirely retranscribed to enable a rigorous study.

Coding is the action of "cutting the content of a speech or a text in analysis units and integrating them in categories chosen in accordance with research aims" [START_REF] Thiétart | Méthodes de Recherche en Management[END_REF]). Analysis unit can be defined as "the element by which the researcher will proceed to the cutting of the speech or text" [START_REF] Thiétart | Méthodes de Recherche en Management[END_REF]).

In our study as in many management studies, we chose to base coding on meaning units or themes. This choice is related to the fact that we want our analysis to result in questionings and thoughts about pay out policies.

As the analysis unit is theme, its size will vary from word to paragraph. To help us in the coding, we used the NVIVO software. When each theme had been affected with no doubt to a category and that there were enough regularities, we decided to stop the analysis. At that point, we thought we had reached saturation.

As a whole, in our 12 interviews, we coded 1367 themes and affected them to 197 categories and sub categories.

The 4 main categories are the following: Decision Process, Motivations to pay out, Explanatory factors and

Description of pay out policies

Thanks to those categories, we started content analysis. There are two types of content analysis: quantitative and qualitative ones. First one computes the frequency of a theme in a category, second one tries to estimate the importance of a theme a qualitative way. "Difference between qualitative and quantitative analysis lies in the way they understand the notion of importance of a category: "number of times" for quantitative analysis and "value of a theme" in qualitative analysis." [START_REF] Thiétart | Méthodes de Recherche en Management[END_REF]).

We thought, considering the kind of interviews we did that the best method to estimate the importance of our themes was to look at their repetition. So we chose to do a quantitative content analysis.

We counted themes in each category and computed frequency of apparition of each category inside its main category. This enables us to know the importance of a theme inside its category.

Frequency computation analyzes repetition in the whole amount of texts, leaving aside who told it. So we completed our analysis by computing the number of respondents who stated each theme.

At the end of our analysis, two pieces of information permitted to quantify the importance of a theme: the frequency of apparition of a theme inside a category and the number of respondents who stated that theme.

Our 1367 themes have been divided in 4 main categories. Explanatory factors category received 62% of the themes, 18% went to the Decision Process, 10% to Motivations to pay out and 10% to Description of pay out policies. Inside each of those 4 categories, themes have been divided in sub categories for which we also computed frequencies.

III. Light on pay out policies

In the scientific literature, pay out can have two shapes: dividend or share repurchase. Two kinds of dividends exist: ordinary dividend and special dividend. One current research issue is to determine the relation between dividends and share repurchases. Are they substitutes or complements? Is share repurchase the potential substitute of ordinary dividend or of special dividend? Before presenting our results, it is important to give respondents' definition of pay out policy and more specifically of stock repurchase.

First, in their opinion, share repurchase is not a substitute to ordinary dividend. Then, two definitions of pay out emerge:

-For a minority, share repurchase is understood as a substitute to special dividend. Pay out policy has two levels. First the payment of an ordinary dividend stable and smoothed. Then, a second payment, less regular, through repurchase or special dividend.

-For a majority of respondents, share repurchase is not a mean of pay out. It answers to others aims such as relution or stock price management. In this case, pay out policy is only dividend policy.

Now that we have made clear what is pay out policy for our sample, let's present our results. First, we will describe the decision process of dividend and repurchase policies (1). In a second section, we will expose the managers' motivation to pay out dividends and stocks repurchase (2). Last, we will present explanatory factors of pay out policies (3).

Description of the decision process

Themes about decision process helped us to understand the way the decision is taken, going beyond the legal process.

Various people or boards take part in that decision: board of directors, chairman, CEO, CFO and the shareholders' meeting.

The board of directors is undeniably the main decision-maker. Its role was underlined by 11 out of 12 respondents. 48% of the "identity of dividend decision-maker" category (so 38 themes) and 24% of "identity of repurchase decision-maker" category (8 themes) support the essential role of the board.

In France, on a legal level, shareholders' meeting is the actor that ratifies the pay out proposition made by the board. In reality its power is limited. Four respondents only (11% of themes from the "identity of dividend decision-maker" category and 24% of "identity of repurchase decision-maker") underlined its decision-maker role, whereas 8 respondents told us its role was limited. In fact, shareholders always ratify the board proposition.

Only one of our respondents told us shareholders' meeting could sometimes oppose the board. He commented on the rejection that the shareholders' meeting had done to a dividend proposal, the year before the interview.

Shareholders leaded by a few institutional investors had voted a higher dividend than the board proposal. The power of the board questions its homogeneity. Who are the directors? How much do they agree with average shareholder's point of view?

The financial management has a key role in the determination of pay out policy. It builds pay out proposals and advises decision-makers (10 respondents out of 12 underlined this role). This role of consultant is sometimes shared with bank counselors who help firms to finalize their pay out decisions. The CFO usually implements share repurchases. He might also be the initiator of repurchase programs.

Those elements summarize the decision process as our respondents presented it. They all described a quite similar situation. Yet, 7 out of 12 respondents told us their feeling of being a particular case in the way they run their pay out policy. This feeling is usually linked to a particularity of their firm; still it does not lead in our opinion to different processes.

It is also interesting to see that the interviewees described the pay out decision as simple and non-strategic. The decision seems evident and is taken quickly. The gap between the huge amount of literature about the dividend puzzle and managers' feeling that pay out is a very simple decision is striking. Here are a few verbatim to illustrate this point.

Respondent n° 6 "Those decisions are neither strategic, nor very important"

Respondent n° 7 "In our firm, the dividend policy is very, very simple"

Respondent n° 4 "It is not sophisticated, it is really not sophisticated. We are more in the emotional reasoning"

Let's now see how and why those pay out decisions are taken.

Motivations to pay out

To understand pay out policies, it is essential to know why those decisions are taken. In this section, we will present results about motivations that drive managers to pay dividends or to vote repurchase programs. Literature

shows various potential motivations, we will compare them to the ones issued from this study.

A first result of interest is the fact that respondents have more talked about their motivations to do share repurchase than about their motivations to pay dividend. For respondents, it doesn't seem necessary to talk about dividend motivations because they are obvious: it is a shared feeling that it is necessary to pay out dividends.

According to 4 respondents, it is normal to pay out dividends if the firm can. There must be no talking about it:

dividend is the normal remuneration for shareholders. For the others, payment of dividend is a way to satisfy shareholders (33% of the category "dividend motivations", that is to say 12 themes), to send a positive signal (22% or 8 themes) and to supply shareholders with cash they asked for (14% or 5 themes). Pay out is not linked to any clientele effect.

On the other hand, there are numerous motivations to vote and implement a repurchase program. Relution effect of repurchases is the first motivation brought up. Relution effect is mainly used to block dilution linked to stock options, but also convertible bonds (11 of out 12 respondents talked about this motivation, and it represents 44% of the category "repurchase motivations" or 36 themes). Stock repurchase is also a tool to manage stock price (15%) by improving volatility and liquidity. It is also used to finance external growth (10%) or to revalue the stock price (10%).

Revaluation may be a motivation, but respondents showed they were not sure of repurchase effect on stock price.

They agreed on the fact that dividend causes an increase in price but disagreed on repurchase effect.

Respondents thought its effect on stock price was either positive or negative, but mentioned the difficulty to measure repurchase consequences on stock prices. This is consistent with the results from the literature. Some studies show a positive reaction of stock prices after a repurchase. But according to Allen and Michaely (2002) those results are ambiguous.

Even if the repurchase program is not going to be implemented, respondents think it is necessary to vote it (9%):

Respondent n°6: "It is nearly an obligation to have a share repurchase program" -"I can't imagine a listed company not having a stock repurchase program" -"We have had that program for a long… long time. Above all, for comfort reasons" Respondent n°9: "But it seems weird -as there is only one shareholders' meeting a year -to not have a repurchase program voted, in case of. "

Those results do not entirely match the motivations listed by the literature. Here are a few examples:

Share repurchase and pay out: Let's note that the repurchase program does not seem motivated by paying out unlike what literature assumes. No respondent clearly displayed as a motivation for repurchasing the fact of paying out. Nevertheless, 5 respondents listed annulment of shares as a motivation (6%) and this is technically associated to pay out. This result is amazing knowing that some managers mentally associate repurchase to pay out (as we will see later on).

Takeovers: Repurchase, as a protection against takeover is neither a motivation displayed by managers. Of course, this motivation may not be the kind managers would tell first, but some of the firms interviewed have recently been threatened by a takeover, so it could have appeared as a motivation. This result is consistent with [START_REF] Brav | Payout policy in the 21st century[END_REF]. In their survey only 14% of their respondents consider stocks repurchase as a protection against takeovers.

Signal:

Contrary to what the literature says, signal doesn't come out of that study as a motivation for pay out, but more as a consequence of pay out. Our respondents has mentioned 57 times the fact that pay out sends a signal, but they only mentioned it 8 times as a motivation for their pay out policy.

Once again those results are consistent with the ones from [START_REF] Brav | Payout policy in the 21st century[END_REF]. On page 17, they sum up this fact :

"Payout policy conveys information, however it rarely is thought of as a tool to separate a company from competitors"

We found the signal sent by dividend pay out is different from the one sent by special dividend or share repurchase. This is consistent with signal theory.

On one hand, ordinary dividend sends a positive signal, indication of managers' trust in future firm's earnings.

The stability and slight growth of dividend make the signal better, whereas cutting the dividend sends a bad signal.

On the other hand, special dividend and share repurchase send negative signals. They signal an excess of cash and a lack of investment opportunities. This point is shared by half of the respondents.

This difference in signal has been theorized and tested by [START_REF] Guay | The Cash Flow Permanence and Information Content of Dividend Increases versus Repurchases[END_REF] and [START_REF] Jagannathan | Financial Flexibility and the Choice between Dividends and Share repurchases[END_REF]. They show that dividend is used to pay out regular cash, contrary to repurchase used to pay out temporary cash.

To conclude, it appears that in the French case, dividend is the normal remuneration of shareholders. Share repurchase is a financial tool, which use is motivated by relution and stock price management. The pay out motivation of repurchase program does not appear clearly, even if some respondents talked about annulment of shares.

Let's now take an interest in explanatory factors of dividend and repurchase policies.

Explanatory factors of pay out policy

Let's now present elements taken into account by decision-makers to decide on their pay out policy. Those elements can be objective or subjective and have a direct or indirect influence.

We conclude from our study that decision of pay out policy is the result of a consensus between several variables with different importance. We classified those factors in three categories: internal and financial variables (3.1), elements linked to managers and shareholding (3.2), and elements external to the firm (3.3).

We will first present those factors, and then we will comment factors explaining the choice made between dividend and repurchase (3.4).

Internal and Financial factors

Those elements are important explanatory factors if we refer to their frequency of quotation: 33% of the category "Explanatory factors" or 278 themes.

Some financial elements have an influence on pay out policies: earnings, cash, investment opportunities and debt level. These results support anterior researches.

Earnings: Level of earnings is one of the first elements firms take into account when deciding pay out (13% of category "Internal and Financial factors" or 36 themes). This is not surprising because dividend is seen as a share of benefits between shareholders.

But, 5 respondents questioned the influence of earnings. Managers smooth ordinary dividend and insofar as it is possible, they will avoid cutting it. So dividend is not going to be the exact reflect of earnings and will sometimes be totally disconnected. So pay out is more linked to average firm performance than to net earnings.

Early, influence of earnings on level of payout has been underlined by empirical literature. [START_REF] Lintner | Distribution of Incomes of Corporations Among Dividends, Retained Earnings, and Taxes[END_REF] showed that the main factor justifying change in dividend payment is change in earnings. However smoothing of dividends make the relation unclear. Later, [START_REF] Benartzi | Do Changes in Dividends Signal the Future or the Past?[END_REF] found that changes in dividends result from changes in past earnings and not futures earnings2 .

Level of debt: For 9 interviewees, debt level is an essential adjustment factor for dividend (15% or 42 themes).

Sometimes, it is the first criteria: if debt is high, firm cannot pay out too much cash in order to be able to improve its situation. At the opposite, if debt is low, firm pays out to avoid that the level of net debt becomes negative and damages firm value.

Importance of debt on pay out policy is also underlined by [START_REF] Brav | Payout policy in the 21st century[END_REF].

A survey of literature shows a substitution between debt and dividends (Calvi-Reveyron (1998)). When debt is high, pay out is low and vice versa. In France, Calvi-Reveyron (1999) found long term debt rate has a negative influence on dividend yield. This is consistent with our results.

Investment opportunities:

Respondents underlined several times the influence of investment opportunities on pay out policy (29% of category "Internal and Financial factors", 78 themes). Level of investment of the firm's sector plays on long-term level of ordinary dividend. In case of higher than average investment, ordinary dividend is not reduced but special dividends and repurchases are cancelled.

Level of ordinary dividend is fixed according to the average level of investment the firm does. Those three verbatim illustrate that point:

Respondent n° 7: "He (the CEO) said: the firm has to invest a lot, we are in an activity in which we need to do research, in which we need to invest in men […] and he considered that a third of benefits would go to shareholders and that the rest had to be reinvested in the firm"

Respondent n° 8: "We are on a long term logic, we say to ourselves the group earns 3 billions euros, we pay 1 in dividends and we keep 2 billions to finance internal or external growth"

Respondent n° 2: "The business was on a very good market segment. This segment is developing and is profitable. As a result, we could say that optimization of capital consisted in not paying dividends […] and that it is better to keep the maximum amount of money to be able to finance growth."

If there is a big investment, ordinary dividend is not cut, even if it means looking for new funding (according to two respondents). Sporadic investments influence above all the level of repurchases or special dividends. Firm will pay out this way if it hasn't any investment opportunities (15 themes and 8 respondents). In case of big investments, ordinary dividend growth is slowed down, repurchases or special dividends are cut.

Respondent n° 1: "For us, the dividend is something on what shareholders must be able to count, except accident or permanent drop in earnings. On the contrary, if we have a big investment, at this condition, of course, there will be no special dividend"

Respondent n° 4: "No, (in case of big investment), we would not decrease the dividend […], we would look for an increase in capital or we would increase debt or we would sell an other asset, but I don't think that dividend policy would be broken, for all that."

Respondent n°11: "For example, firm X (one middle size firm on French stock market): if they decided to pay out special dividend last year, it is because they judged that in the firm's strategy, there was no way to value the share, because keeping the cash would have not created development opportunities"

Cash level: Cash level independently from earnings level, has an influence on pay out, especially on special dividend and repurchase. The use of those two means of pay out seems to be highly motivated by the presence of cash inside the firm (41 themes or 15% of the category and 11 respondents).

Influence of cash level and investment opportunities on pay out policies remind us Free Cash Flow theory. [START_REF] Jensen | Agency Costs of Free Cash Flow, Corporate Finance, and Takeovers[END_REF] defined FCF as the cash remaining after having invested in good project. Level of FCF plays a role on the determination of pay out. Shareholders ask for pay out because it decreases cash under management control and though helps managers monitoring. The effect of FCF on pay out policy has been underlined in many studies (Lang and Litzenberg (1989), [START_REF] Nohel | Share Repurchases and Firm Performance: New Evidence on the Agency Costs of Free Cash Flows[END_REF] for USA, [START_REF] Stouraitis | The Impact of Ownership Structure on the Dividend Policy of Japanese Firms with Free Cash Flow Problem[END_REF] for Japan). Our survey underlines once more the importance of FCF.

Moreover, the two financial institutions interviewed underlined the influence of level of equity on pay out policy. This specificity is explained by the bank regulation.

Other internal factors:

One point little developed by the scientific literature (except by [START_REF] Frankfurter | Dividend Policy: Theory and Practice[END_REF]) has been quoted by our respondents. It is the existence of a pay out (or non-pay out) culture in some companies (10% of themes in "Internal and Financial factors" or 28 themes). This culture pushes decisionmakers to pursue past pay out policies. They will be inclined to pay out dividend if the firm has always done so and not pay out if the firm has never done so. Nine respondents mentioned the existence of pay out culture. Here are two verbatim to illustrate that point:

Respondent n°11: "The proposition that is made is function of last financial year, inevitably and then function of historical pay out policy of the firm" Respondent n°7: "Firm culture is to pay out a third of benefits if there are benefits and nothing if there are losses.

It has always been the case" -"As a conclusion, it seems to me that the dividend results of an arbitrary decision, decided a long time ago"

Elements linked to shareholding and managers.

Those elements are essential if we refer to their frequency of quotation (40% of themes of category "explanatory factors" or 348 themes).

Our respondents underlined the importance of shareholding structure, of shareholders' preferences and of chairman or CEO characteristics. Moreover, managers interviewed exposed their principles or conceptions of how pay out policies must be run. These principles strongly influence the decision.

Shareholding structure:

Our results show that two determinants of shareholding structure influence pay out decisions: the existence of a majority shareholder (35% of the category "Influence of shareholding structure" or 21 themes) and the amount of floating capital (17% or 10 themes).

-Presence of a majority shareholder:

Respondents exposed the consequences of having a majority shareholder. Depending on who is the main shareholder, we note different patrimonial interests that play on pay out (17 themes and 6 respondents). The way the shareholder financed its shares is an important determinant: if he borrowed money, he needs a steady cash flow to refund its loan and will ask for dividend. On the contrary, if he inherited his shares, he slows down pay out. Our study also shows that majority shareholders try to limit dilution of cash and power and favor pay out that limits both kind of dilution, depending on their priorities (10 themes and 4 respondents). Here are two verbatim to illustrate this influence:

Respondent n° 8: "We have a very strong demand from X (the majority shareholder) and we know that we can't

[…] cut or double a dividend"

Respondent n° 3: "At the last board meeting, the CFO of X (majority shareholder of firm 3bis) kindly remarked that the more we pay out, the more there would be flight of capital outside the group. This is fair, knowing that their main worry is to manage their funded debt" -Part of floating capital:

When floating is high, managers are less influenced by shareholders desires and are more free to decide pay out.

But, in one of the ten companies we studied, it appears that the high floating capital had given back power to the shareholders' meeting. And that it has driven to a failure of board dividend proposition.

Shareholders' preferences:

Shareholding structure gives more or less power to one shareholder, to a group of shareholders or to managers.

Influence on pay out policy will depend on the will of the ones who decide.

Respondents commented on shareholders' preferences and their influence. First, investors, mainly individuals, like dividends. Moreover, according to 5 respondents, individuals tend to wary of share repurchases, they don't understand it and are suspicious about it.

Anyway, for a majority of respondents, individual preferences and taxes (except majority shareholder's one) are not taken into account at the time of the pay out decision. The global state of the market is more influent than individual preferences. For example, at the end of 2004, firms thought about the impact of the new dividend tax system, but they did not put interest in individual taxes. Only the preferences of influent shareholders can play on the choice of pay out policy.

Our results on shareholders' influence found an echo in literature. They tend to support the agency theory and not the tax theory. Empirical evidence on the influence of taxes on pay out policies is once more weakened (Allen and Michaely (2002), [START_REF] Brav | Payout policy in the 21st century[END_REF]), except for the tax influence of main shareholders (Consistent with [START_REF] Lie | The Role of Personal Taxes in Corporate Decisions: an Empirical Analysis of Share Repurchases and Dividends[END_REF] that showed managers are more aware of their shareholders' taxes when institutionals hold large blocks).

Our first result coherent with agency theory is the influence of shareholders concentration and the non-influence of minority shareholders on pay out. The presence of a large shareholder enables less monitoring and enables less dividends. This link has been supported by [START_REF] Rozeff | Growth, Beta and Agency Costs as Determinants of Dividend Pay Out Ratios[END_REF], [START_REF] Moh | An Investigation of the Dynamic Relationship Between Agency Theory and Dividend Policy[END_REF], [START_REF] Dempsey | Effects of Agency and Transactions Costs on Dividend Payout Ratios: Further Evidence of the Agency-Transactions Cost Hypothesis[END_REF] and [START_REF] Lloyd | Agency Costs and Dividend Payout Ratio[END_REF].

Some recent papers also underlined the influence of large shareholders' identity on dividend level. Correia Da Silva et al ( 2004)'s study in Germany showed that firms which main shareholder is a bank tend to pay out less than firms with other types of main shareholder or no main shareholder. Perez -Gonzalez (2000) also showed that pay out is more impacted when the main shareholders is an individual than when it is an institutional, or when there is no main shareholder.

At last, neutrality of minority shareholders on pay out policies is supported by many empirical studies [START_REF] Crockett | Dividend Policy in Perspective: Can Theory Explain Behavior?[END_REF], [START_REF] Grinstein | Institutional Holdings and Payout Policy[END_REF], [START_REF] Jain | Institutional Investors do not Prefer Dividends: Individual Investors do[END_REF], [START_REF] Lie | The Role of Personal Taxes in Corporate Decisions: an Empirical Analysis of Share Repurchases and Dividends[END_REF])

Managers' characteristics:

Let's tackle now the influence of management's characteristics on pay out policies. The category "Influence of chairman or CEO" holds 21% of the themes of its upper category "Influence of Managers". The rest of this category is held by the category "Managers' conceptions of pay out policy"

Our results show that chairman characteristics such as its education, its personality or the fact that he is at the same time chairman and CEO or shareholder might influence pay out policies.

Managers' conceptions of how to run pay out policies

In the course of interviews, respondents discussed about their conceptions of how to run pay out policy. They mostly stated on how to run a dividend policy and on the relationship between share repurchase and dividend.

Those themes are often mentioned and undeniably influence pay out policies. Managers tend to agree on dividend smoothing, but disagree on the position share repurchase holds.

-Dividend smoothing:

Respondents agreed on the necessary stability of ordinary dividend (10 respondents said so and 36 themes have been counted). Dividend has to be stable and if possible slightly growing. Managers avoid cutting it. They will neither increase it if it has to be cut the year following. This is not a surprising result as the importance of smoothing dividend has been underlined in many studies based on managers surveys [START_REF] Lintner | Distribution of Incomes of Corporations Among Dividends, Retained Earnings, and Taxes[END_REF][START_REF] Brav | Payout policy in the 21st century[END_REF][START_REF] Baker | Factors Influencing Dividend Policy Decisions of NASDAQ Firms[END_REF]). Those results are also supported by Allen and Michaely (2002) who showed a great stability of dividend payment during the years 1972-1998.

-Positioning Stock repurchase / Dividend:

Our study helps to understand share repurchase reality as a mean of pay out.

A majority of respondent (40% of the themes on "Positioning Stock repurchase / dividend") do not consider stock repurchases as a mean of pay out. Stock repurchase is only used for relution or stock price management.

Most of those respondents understand that repurchase can be a mean of pay out, but they do not want to use it like that.

For the others, (43% of that category), pay out policy involves two parts:

-First, an ordinary dividend smoothed and stable -Then, shares repurchase program or a special dividend to pay out extra cash. This pay out is unstable.

First, those results lighten a difference between USA and France. [START_REF] Brav | Payout policy in the 21st century[END_REF] found managers do not consider dividend and share repurchase as substitutes, but they do consider repurchase as a mean of pay out. This might be linked to the fact that share repurchase has been allowed for a long time in USA whereas it is only allowed since 1998 in France. In consequence American point of view might comes to France one day.

Second, our results (for those managers who consider repurchase as a mean of pay out) echoe those from [START_REF] Guay | The Cash Flow Permanence and Information Content of Dividend Increases versus Repurchases[END_REF] and [START_REF] Jagannathan | Financial Flexibility and the Choice between Dividends and Share repurchases[END_REF] that showed that repurchase is used to pay non recurrent cash.

Existence of a conflicting agency relation

Five respondents showed that they were conscious of the existence of an agency relation between shareholders and managers. They mentioned that if this relation is conflicting, shareholders ask for pay out to avoid letting free cash in the firm. Those results fit agency theory which conclude to the influence of conflicting agency relation on pay out policies.

Elements external to the firm

Our study shows that managers also take into account elements from outside the firm when they decide pay out policies. Those elements collected 20% of themes from our category "explanatory factors" (183 themes).

What is said or done outside the firm influences pay out. The main influence comes from financial markets (65% of themes from « External influence » or 119 themes) and then from competitors (14%, or 26 themes).

Financial markets' influence

This influence materializes mainly through analysts and portfolio managers' opinions (23 themes). Press and foreign markets also influence firms in their pay out policies.

Those actors create a kind of "market opinion", which is listened at very carefully by firm managers. Those actors judge firms, but above all they raise rules that have to be followed. Respondents mentioned (17 themes)

the strong markets' influence on good corporate governance principles, information and transparence requirements, accepted level of risk….

More precisely, several points of influence concerning pay out policies comes out of our study:

-Dividend culture:

There is a strong dividend culture on markets. It is good to pay dividends and firms must do it. Some interviewees think markets would not understand that repurchases take the place of dividends. At the end of the interviews, I asked some respondents if they could imagine a substitution of dividends by share repurchases. The following verbatim shows we are far from it in France:

Respondent n°7: "Where I am very surprised, it's because it didn't come to us, or I am wide of the mark, or the company for which I work is totally wide of the mark, but this issue of pay out policy with dividend and share repurchase has not come to us at all."

-Shares repurchase fashion:

3 respondents out of 12 mentioned that share repurchases is a fashion that has to be followed. Besides, most respondents think a share repurchase program has to be voted even if it is not planned to implement it. Not having such a program can be bad for the firm's image on markets.

Respondent n°5: "There was a little fashion effect in stock repurchase and at a time share repurchase was a way to be in good books, it was very in fashion, all companies at the forefront were doing it. So, we thought about the issue and we said after all why not us."

-Yield share and Growth share:

Markets distinguish Yield shares and Growth shares. Firms with high yield, called "yield shares" pay out big money. Firms with high growth, called "growth shares" have a good growth of their stock price. This distinction influences firms at the time of their pay out decision. If a firm is ranked in yield shares, the dividend paid out is very important and pay out policy will be looked at very carefully. Whereas, growth shares thanks to their stock price potential don't have pay out obligations.

Competitors influence

Six respondents mentioned their main competitors as a model for their pay out policies. The influence of the sector obtained ambiguous support from the literature. In our study, concerning pay out, firms try to be the less distinguishable from their competitors they can.

Factors explaining choice between dividend and share repurchase.

As seen before, some respondents assimilate repurchases to pay out. The study of those interviews has permitted to find out a few explanatory factors of the choice made between the two means of pay out. As said previously, this choice is done mainly between special dividends and repurchases.

The main determinant of that choice is the difference in the shape of the two tools (61% of themes from category "Explanatory factors of choice between dividend and repurchase" or 20 themes). The way the cash is received is different. Some respondents have the feeling that share repurchase does not pay out every shareholders. Even more, they feel they pay only the leaving shareholders. Some others have a different feeling about that. They think share repurchase lets more freedom to shareholders to choose or not to receive cash.

The flexibility of share repurchase compared to ordinary dividend is also a mentioned argument. This might influence the choice between the tools (21% of themes of the category, 7 themes). Respondents talked very little about the fact that signals sent by each tool or taxes are different. This is yet something important in theory.

The flexibility of share repurchase has been underlined by Allen and Michaely (2002) : in the USA during 1972-1988 period, stocks repurchases were much more volatile than dividends. [START_REF] Guay | The Cash Flow Permanence and Information Content of Dividend Increases versus Repurchases[END_REF] and [START_REF] Jagannathan | Financial Flexibility and the Choice between Dividends and Share repurchases[END_REF] also insisted on the flexibility and less restricting aspect of shares repurchase.

Other studies based on surveys also contradict influence of taxes on choice between dividend and shares repurchases. [START_REF] Brav | Payout policy in the 21st century[END_REF] and [START_REF] Baker | Why Companies Use Open-Market Repurchases: A Managerial Perspective[END_REF])

Conclusion

This paper does not intend to resolve the complexity of pay out policy issues. It makes the assumption that the dividend puzzle is still not resolved, because of a lack of explanatory variables. To move forward in its resolution and find new theories or links between variables, a better knowledge of the reality of pay out policies is necessary. To reach that aim, we proceeded to a qualitative survey of dividend and share repurchase decisionmakers.

The results of this study are limited statistically by the size of our sample. However this research enables to point out and stress the main variables that interfere in pay out decision in France. It is only the beginning of a larger research; it lightens potential variables that need to be studied.

So what are the contributions of this paper?

First, it shows pay out decision is the result of a consensus between various elements: financial capacity of the firm, external influences, and shareholding pressure.

It supports elements from the literature: influence of financial variables, signal function of pay out or smoothing of dividend policy. It introduces new elements that may lead to interesting questioning: existence of pay out culture in firms, influence of managers' characteristics or competitors' behavior.

This paper also underlines divergences with the literature. Those differences might be linked to the French cultural context, but they might also question the validity of some theories.

  Respondent n°8: "We look at what our friends are doing, […], we look at each others and we check that we are not disconnecting us from what the market is doing." Respondent n°9: "And, of course, we also look at what our big competitors are doing, to avoid, if there are differences, to have arbitrages against us."

Table 1 :

 1 Presentation of the population of respondents

	RESP. POSITION

See Allen andMichaely (2002) for a literature review on links between dividend and future earnings.

For example, the role of share repurchase is questioned. We show in this paper that stock repurchase is not fully considered as pay out neither by firms, nor by markets. This point might be even more striking among all listed firms because we studied big companies that tend to repurchase more. Share repurchase can be used as pay out, but it is more often used as relution tool. It also seems important to note the difference between the vote of a repurchase program and its implementation. Voting a program does not mean the firm is going to repurchase. Is all this a cultural difference or a backward state?

Secondly, this paper contradicts the literature that emphasizes the role of taxes in pay out decisions. Our results show that shareholders' tax is not a meaningful variable, at the firm level.

Last, we found that majority shareholders have a strong influence on pay out. It may be linked to French and European specificity of firms' capital holding. 3 Managers' and main shareholders' power on pay out decision leads to question the necessity for pay out. Are pay out policies decided in the firm's interest, in shareholders'one or in the private interest of some influent people? Isn't it done to the detriment of minority shareholders or stakeholders? This reinforces the idea to lighten pay out mechanisms by a deeper study on corporate governance systems of the firm.

The ramification of our study could then take the way of understanding effect of governance variables on shape of pay out policies. It seems essential to check the efficiency of corporate governance structure to monitor firms' decision: do corporate governance mechanisms permit monitoring on pay out policies? Is pay out policy a mean of governance?

3 See Correia da Siva et al. (2004) for a cross cultural survey on differences between shareholding structures.


M Gimenes

E Lambert

Colin M Macleod
email: cmacleod@uwaterloo.ca

Nigel Gopie

Kathleen L Hourihan

Karen R Neary

Jason D Ozubko

Keehan Bailey

Emily Bryntwick

Samantha Chaves

Domenica De Pasquale

Nicole Deckert

Grace Hsiao

Jingjing Ji

Marita Partanen

Molly Pottruff

Is spelling memory improved by reading aloud ?

Keywords: memory, encoding, production, generation, distinctiveness

In 8 recognition experiments, we investigated the production effect-the fact that producing a word aloud during study, relative to simply reading a word silently, improves explicit memory. Experiments 1, 2, and 3 showed the effect to be restricted to within-subject, mixed-list designs in which some individual words are spoken aloud at study. Because the effect was not evident when the same repeated manual or vocal overt response was made to some words (Experiment 4), producing a subset of studied words appears to provide additional unique and discriminative information for those words-they become distinctive. This interpretation is supported by observing a production effect in Experiment 5, in which some words were mouthed (i.e., articulated without speaking); in Experiment 6, in which the materials were pronounceable nonwords; and even in Experiment 7, in which the already robust generation effect was incremented by production. Experiment 8 incorporated a semantic judgment and showed that the production effect was not due to "lazy reading" of the words studied silently. The distinctiveness that accrues to the records of produced items at the time of study is useful at the time of test for discriminating these produced items from other items. The production effect represents a simple but quite powerful mechanism for improving memory for selected information.

In 1978 Slamecka and Graf reported a thorough set of experiments demonstrating that producing a word from a cue (e.g., producing fast from the cue rapid-f) leads to considerably better memory for that word than does simply reading the word. The phenomenon that they called the "generation effect" has subsequently become one of the most widely used manipulations in memory research, leading to their article becoming a citation classic (see Slamecka, 1992), now having been cited more than 500 times and having spawned hundreds of directly related articles (for a meta-analysis, see Bertsch, Pesta, Wiscott, & McDaniel, 2007).

The generation effect is a member of a very select club: manipulations that have consistent, reliable effects on retention of a stimulus that was presented only once. Other such manipulations include imagery (Paivio, 1971) and elaboration (Craik & Lockhart, 1972). All three of these involve some recoding of the stimulus, and all produce quite substantial memory benefits, often an improvement of 10% or even more relative to a "standard" baseline of simply reading the word. Enriching the encoding of the stimulus definitely enhances memory for it, in line with what we know generally about mnemonic techniques (see Higbee, 1988).

Improving Memory by Saying a Word Aloud

In the same period, another factor was first reported to benefit memory but, unlike the others, it failed to attract subsequent research attention. In the study in question, Hopkins and Edwards (1972) tested a key assumption of frequency theory (Ekstrand, Wallace, & Underwood, 1966)-that recognition should be better for pronounced than for unpronounced words because pronunciation would increment the item's frequency (see Hopkins, Boylan, & Lincoln, 1972, for evidence that pronunciation does increase judged frequency). To test this prediction, Hopkins and Edwards used two recognition tests: two-alternative forced choice (Experiment 1) and yes/no (Experiment 2). In both experiments, three groups of subjects studied 100-word lists. There were two pure-list groups-one read all 100 words aloud, and one read all 100 words silently-and one mixed-list group, which read 50 of the words aloud and 50 silently.

In comparing the two pure-list conditions, Hopkins and Edwards (1972) found no between-subjects benefit to reading words aloud. But in the mixed-list condition, words read aloud were better recognized than those read silently. This pattern held for both types of recognition test, with the within-subject benefit of reading aloud being approximately 10%. Hopkins and Edwards suggested that the effect was at encoding. Overall, the results showed that the production effect is mainly based on phonological information but it can be observed even when only orthographic information is available

  The production effect(MacLeod et al., 2010) : reading a word aloud allows for better memory performance than simply reading it

  MacLeod, C. M., Gopie, N., Hourihan, K. L., Neary, K. R., & Ozubko, J. D. (2010). The production effect: Delineation of a phenomenon. Jour nal of E x p e r i m e n t a l P s y c h o l o g y : Learning, Memory, Cognition, 36, 671-685. doi: 10.1037/ a0018785 CONTACT manuel.gimenes@univ-poitiers.fr CREDITS Vectors graphics designed by Freepik

This research was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant A7459.


Simon Pouil

Rachel Clausing

Dr Marc Metian
email: m.metian@iaea.org

Paco Bustamante

Marie Yasmine Dechraoui-Bottein

Use of radiotracer techniques to study the influence of brevetoxin exposure on trace element bioaccumulation in the blue mussel Mytilus edulis

Keywords: Biomonitoring, Bivalves, Brevetoxins, Metals, Radioligand receptor

Marine organisms are exposed to and affected by a multitude of chemicals present in seawater and can accumulate in their tissues a wide range of contaminants as well as natural biotoxins associated with harmful algal blooms (HABs). Trace elements and biotoxins may modify physiological functions in exposed organisms, and studies have been conducted to better understand their respective kinetics and effects in marine species. Despite the increasing concern of concurrent toxic HABs and pollution events due to anthropogenic pressures and global change, very little information is available on their combined effects. Chemical interactions between biotoxins and trace elements have been reported, and exposure to certain biotoxins is known to modify ion transport pathways, suggesting that biotoxins have the potential to alter trace element uptake. Using specific and sensitive radiotracer techniques (radioligand receptor binding assay and γ-spectrometry), this laboratory study examined the influence of pre-exposure to the brevetoxins (PbTxs)-producing microalgae Karenia brevis on the bioaccumulation of selected non-essential (Cd) and essential (Co, Mn and Zn) trace elements in the blue mussel Mytilus edulis. PbTxs are a group of neurotoxins known to accumulate in bivalves but also to have lethal effects on a number of marine organisms including fish and mammals. We found that, over 23 days exposure to the radiotracers, the bioaccumulation of the dissolved essential trace elements Co, Mn and Zn in M. edulis was not significantly affected by pre-exposure to toxic K. brevis. In contrast, the uptake rate constant ku of Cd was significantly higher in the pre-exposed group (p < 0.05), likely caused by a decrease in mussel clearance rates after K. brevis exposure. These results suggest that the effects of algal toxin exposure on bioaccumulation of trace elements in mussels may be trace elementdependent.

Introduction

In coastal marine environments, dissolved organic and inorganic elements coming from both natural or anthropogenic sources are prevalent and can play an important role in the physiology of inhabiting organisms. Their concentrations and distributions in the marine environment have been increasing over the last decades, mostly due to anthropogenic activities such as industrialization, urbanization and agriculture (e.g. Förstner and Wittman, 2012). Some trace elements, required for basic metabolic processes such as Co, Mn, and Zn, are considered essential to marine organisms; components of various enzyme functional groups, they play a structural role in respiratory pigments and metalloenzymes, and can activate co-factors for various proteins [START_REF] Simkiss | Metal ions in cells[END_REF][START_REF] Williams | The bakerian lecture, 1981: natural selection of the chemical elements[END_REF]. Other trace elements such as Cd, having no known biological function for organisms, are considered as non-essential. Nevertheless, all trace elements, essentials and non-essentials, may cause adverse effects on organismal physiology if their concentrations exceed thresholds beyond which homeostatic regulation processes are saturated (Förstner and Wittman, 2012). In addition to those chemicals resulting from geological weathering processes [START_REF] Morel | The biogeochemical cycles of trace metals in the oceans[END_REF], naturally occuring chemicals such as biotoxins associated with harmful algal blooms (HABs) accumulate in marine marine organism, where they may affect physiological functions.

The frequency and intensity of toxin-producing HABs has been increasing over the last decades [START_REF] Hallegraeff | A review of harmful algal blooms and their apparent global increase[END_REF][START_REF] Smayda | Novel and nuisance phytoplankton blooms in the sea: evidence for a global epidemic[END_REF][START_REF] Sournia | Red tide and toxic marine phytoplankton of the world ocean: an inquiry into biodiversity[END_REF][START_REF] Van Dolah | Marine algal toxins: Origins, health effects, and their increased occurrence[END_REF], increasing the likelihood of filtering organisms, planktivorous organisms, as well as upper trophic level organisms to be exposed to biotoxins. Bivalve molluscs, in particular, have been found to be capable of accumulating biotoxins, including azaspiracid, okadaic acid, saxitoxins, domoic acid and brevetoxins (PbTxs) [START_REF] Fao | Assessment and management of biotoxin risks in bivalve molluscs[END_REF]. Toxic to humans, those biotoxins may also cause severe physiological and behavioral changes in bivalve species [START_REF] Manfrin | Physiological and molecular responses of bivalves to toxic dinoflagellates[END_REF]. Modification in valve closure and on feeding and filtration activities have been reported in various bivalve species after exposure to different toxin-producing microalgae [START_REF] Contreras | Effects of short-term exposure to paralytic shellfish toxins on clearance rates and toxin uptake in five species of New Zealand bivalve[END_REF][START_REF] Echevarria | Effects of Karenia brevis on clearance rates and bioaccumulation of brevetoxins in benthic suspension feeding invertebrates[END_REF][START_REF] Shumway | Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species[END_REF][START_REF] Shumway | The effects of the toxic dinoflagellate Protogonyaulax tamarensis on the feeding and behaviour of bivalve molluscs[END_REF]. For example, reduction in filtration activity, as estimated by the measurement of clearance rates, has been demonstrated in four bivalve species exposed to PbTx (e.g. [START_REF] Leverone | Comparative effects of the toxic dinoflagellate Karenia brevis on clearance rates in juveniles of four bivalve molluscs from Florida, USA[END_REF].

Nevertheless, such effects may be variable or even conflicting, where reduced filtration rates have been observed in the soft shell clam Mya arenaria and the ribbed mussel Geukensia demissa while the oyster Ostrea edulis increased rates of filtration under identical experimental conditions [START_REF] Shumway | The effects of the toxic dinoflagellate Protogonyaulax tamarensis on the feeding and behaviour of bivalve molluscs[END_REF].

Changes in filtration activity as observed in the presence of biotoxins could affect the ability of bivalves to bioaccumulate trace elements from the dissolved pathway [START_REF] Wang | Comparison of metal uptake rate and absorption efficiency in marine bivalves[END_REF]. Thus, it can be postulated that trace element bioaccumulation in bivalves may be altered by exposure to toxin-producing harmful algae. Moreover, trace element bioavailability and potential transfer in aquatic food chains may be affected as they have the potential to complex with algal toxin molecules which often contain functional groups such as amino, carboxyl, phenol, sulfhydryl and hydroxyl groups, [START_REF] Humble | Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography[END_REF][START_REF] Rue | Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudo-nitzschia[END_REF]. For example, Cu bioavailability has been shown to be affected by the formation of a domoic acid-Cu complex [START_REF] Rue | Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudo-nitzschia[END_REF]. In addition, changes in membrane potential that occur with exposure to algal neurotoxins such as PbTxs may impact cellular ion transport [START_REF] Berman | Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca 2+ influx[END_REF][START_REF] Liberona | Sodiumdependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca 2+ release in rat skeletal myotubes[END_REF][START_REF] Tian | Proteomic modification in gills and brains of medaka fish (Oryzias melastigma) after exposure to a sodium channel activator neurotoxin, brevetoxin-1[END_REF] and consequently may affect distribution of trace elements to target tissues and cells, and hence their accumulation in bivalves.

This study assessed the effects of pre-exposure to Karenia brevis culture (containing K. brevis cells and solubilized PbTxs) on bioaccumulation of non-essential (i.e. Cd) and essential (i.e.

Co, Mn and Zn) trace elements in the blue mussel Mytilus edulis. Higher specific nuclear techniques were used (1) to quantify PbTx concentrations accumulated in mussels using a radioligand receptor binding assay (RBA) and (2) to determine the uptake kinetics of studied metals using γ-radiotracers ( 109 Cd, 57 Co, 54 Mn and 65 Zn). Two levels of biological organization were considered in this study, including both individual tissues and the entire blue mussel, in order to evaluate, after exposure to PbTxs, the uptake kinetics of trace element in the who body and the dynamics of trace element transfer among compartments.

Materials and Methods

Origin and maintenance of organisms

One hundred blue mussels M. edulis, a species widely used as a bioindicator for monitoring of trace elements, were purchased from a seafood seller (Les Halles du Midi, Monaco). They were transported to International Atomic Energy Agency (IAEA-EL) premises in Monaco, and acclimated to laboratory conditions for 1 month (continuously aerated, open-circuit 300-L plastic tank; flux: 150 L h -1 ; salinity: 38; temperature: 20 ± 1 °C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) prior to experimentation. During this period, mussels were fed a daily algal diet of Isochrysis galbana. They were confirmed to be PbTx-free by RBA analysis, K. brevis culture (NOAA-1 strain isolated from Charlotte Harbor, FL, USA) was grown in a 20-L plastic container containing 0.45 µm filtered aged seawater (dark-incubated for 1 month minimum; salinity: 38; temperature: 20 ± 1°C; pH: 8.0 ± 0.1; bilateral luminosity 66-80 μE m -2 s -1 ; light/dark: 12h/12h) spiked with an atypical f10k medium (Holmes et al., 1991). K. brevis were harvested at the stationary phase (i.e. approx. 10000 cells mL -1 ) for use in the experiment.

Experimental Procedures

2.2.1. Exposure to K. brevis culture Mussels M. edulis (33.1 ± 5.1 g wet wt) were evenly distributed at the bottom of three 20-L aquaria (n = 40; same environmental conditions as described in section 2.1.). Prior to distribution, byssal threads were cut with a scissors to limit mussel movement and hence maintain a homogeneous distribution of the mussels throughout the tank during the whole experiment. An experimental treatment was randomly assigned to each aquarium. Mussels from aquaria 1 and 2 were exposed to K. brevis culture (concentration of 980  20 cells mL -1 in the exposure tank) while aquarium 3 served as a control without algal exposure. K. brevis culture was added to aquaria 1 and 2 for 1 hour daily (i.e. time required for the complete filtration of K. brevis cells by the mussels, data not shown) in closed circuit, over a period of 4 days. Culture was added to the aquaria with extreme care to prevent cell lysis. Cell abundance was checked manually by optic microscopy in 3 replicate counts. After the 1-h exposure period, seawater was changed and mussels fed briefly (30 min with I. galbana) in K brevis-free conditions. After the last exposure to K. brevis (day 4), eight mussels from aquaria 1 and 2 were collected, sacrificed and dissected to quantify the PbTx concentrations in combined soft tissues for some mussels and in the following isolated body compartments for others: (1) gills, ( 2) digestive gland and (3) remaining soft-parts. To follow PbTx depuration, mussels from the first K. brevis-exposed aquarium (aquarium 1) were kept in K. brevis-free conditions (open-circuit; conditions as described in section 2.1) for 23 days, after which eight mussels were sacrificed and dissected as described above. The remaining mussels of the second K. brevis-exposed aquarium (aquarium 2) were exposed to dissolved radiotracers for 23 days as described in section 2.2.2.

Mussels from the third 20-L aquarium (aquarium 3) were maintained in the same conditions as the mussels in aquarium 1 but were not exposed to cultures of K. brevis. All handlings (water changes, feeding) were repeated on this aquarium to ensure potential levels of stress and numbers of individuals were identical between all the aquaria throughout the experiment.

Exposure to dissolved radionuclides

Remaining mussels (n=32) in both aquarium 2 (K. brevis-exposed) and aquarium 3 (control), were exposed in the same 20-L aquaria to a mixture of dissolved γ-radiotracers (aquaria conditions as previously described in the section 2.2.1) over 23 days (nominal activities of 0.5 Bq mL -1 for 54 Mn and 57 Co and 1 Bq mL -1 for 65 Zn and 109 Cd). The duration of exposure was chosen to allow accurate determination of kinetic parameters. To keep the environmental radioactivity constant, spiked seawater was renewed regularly (i.e. at the time of each γcounting as detailed below), with a maximum of 3 days between two renewals of seawater (following [START_REF] Metian | Delineation of Pb contamination pathways in two Pectinidae: The variegated scallop Chlamys varia and the king scallop Pecten maximus[END_REF]. Seawater radioactivity in the two radiotracer-exposed aquaria (2 and 3) was checked before and after each spike renewal by γ-counting 150-ml water samples (time-integrated activities available in Table 1). After each water renewal and before radiotracer spiking, mussels were fed 30 min with I. galbana. Eight tag-identified mussels from each aquarium were collected using forceps at different time intervals (after 1, 3, 6, 8, 10, 13, 15, 17, 20 and 23 days of exposure), rinsed three times in clean seawater, weighed and then radioanalyzed (γ-counted) alive to quantify whole-body radiotracer concentration. After counting, mussels were returned to their respective aquarium. At 3, 9 and 23 d of exposure, mussels were collected (n=4), sacrificed, and dissected into 4 compartments: (1) gills, ( 2) digestive gland, (3) remaining soft-parts and (4) shell. Samples were γ-counted in order to assess the radionuclide body distribution.

2.2.3. PbTx quantification using -radioligand receptor binding assay (RBA) PbTx from whole K. brevis culture were extracted using 500 mg C-18 SPE cartridges (recovery: 99-125%, [START_REF] Twiner | Extraction and analysis of lipophilic brevetoxins from the red tide dinoflagellate Karenia brevis[END_REF]. Solvent extraction for PbTx from mussel tissues was performed as previously described [START_REF] Poli | Neurotoxic shellfish poisoning and brevetoxin metabolites: A case study from Florida1[END_REF][START_REF] Pouil | The role of marine biotoxins on the trophic transfer of Mn and Zn in fish[END_REF], tested (recovery: 90-108%, [START_REF] Dickey | Monitoring brevetoxins during a Gymnodinium breve red tide: Comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts[END_REF]. Briefly, samples of each tissue type (n=4) were homogenized (T25 Ultra-Turrax Basic, IKA) in acetone, sonicated, and centrifuged at 3000 g for 10 min. Each supernatant was collected, and the process was repeated two more times. Combined supernatants were evaporated under a stream of nitrogen in a water bath at 40 °C. Dried samples were resuspended in 80% methanol, mixed by inversion after the addition of equal volumes of n-hexane, and centrifuged at 3000 g for 1 min to facilitate phase separation. The methanol phase was collected in glass tubes and evaporated under a stream of nitrogen. All dry extracts (from K. brevis culture and mussel tissues) were resuspended in 100% methanol and stored at -18°C until analysis. The activity-based RBA was used to detect and quantify composite PbTx concentrations in the different tissue extracts (according to [START_REF] Bottein | Bioassay methods for detection of Npalmitoylbrevetoxin-B2 (BTX-B4)[END_REF][START_REF] Dechraoui Bottein | Receptor binding assay for the analysis of marine toxins: detection and mode of action[END_REF], with some modification) using PbTx-3 as the standard. Samples were analysed at three dilutions in triplicate in a 96-well filterplate format (MSFB N6B 50 MultiscreenHTS), and radioactivity was counted in a liquid scintillation -counter (MicroBeta2 Microplate Counter, PerkinElmer) after a 1-h period of dark incubation. Toxin quantification of samples was calculated from PbTx-3 standard curves (4-parameter logistic regressions) determined using GraphPad Prism software version 6.0 (San Diego, USA). Since γ-emitters (here 109 Cd, 57 Co, 54 Mn and 65 Zn) can cause inference in β-detection with liquid scintillation counting (here tritiated PbTx: [ 3 H]PbTx-3), quantification of PbTx concentrations cannot be performed on radiotracer-exposed samples.

Radiotracers and γ-spectrometry measurements

Uptake kinetics of radionuclides in mussels were determined using high specific activity γradiotracers ( 109 CdCl2 in 0.5M HCl, T1⁄2: 464 days, 57 CoCl2 in 0.1M HCl, T1⁄ 2: 271 days, 54 MnCl2 in 0.5M HCl, T1⁄ 2: 312 days, and 65 ZnCl2 in 0.1M HCl, T1⁄2: 244 days) purchased from Polatom, Poland. Radioanalyses were carried out using a γ-spectrometer system composed of 4 Germanium -N or P type -detectors (EGNC 33-195-R, Canberra® and Eurysis®) connected to a multi-channel analyser and information was treated using a computer equipped with a spectra analysis software (Interwinner 6, Intertechnique®). The radioactivity in living mussels and samples was determined by comparison with standards of appropriate geometry spiked with known activity [START_REF] Cresswell | Aquatic live animal radiotracing studies for ecotoxicological applications: Addressing fundamental methodological deficiencies[END_REF]. Briefly, calibration was conducted with a mussel "phantoms" created by filling empty shells with paper towels soaked with known quantities of radiotracers in 2M HCl. For samples of seawater and soft tissues, the calibration was done by using similar container filled with same volume of a 2M HCl solution spiked with known concentrations of radiotracers. Corrections were made for background and physical radioactive decay [START_REF] Rodriguez Y Baena | Experimental evidence for 234 Th bioaccumulation in three Antarctic crustaceans: Potential implications for particle flux studies[END_REF].

Data treatment and statistical analyses

Uptake kinetics of radiotracers were expressed in terms of change in concentration factor (CF) over time, where the CF is the ratio between whole-body activity (Bq g -1 wet wt) and timeintegrated activity of radiotracers in seawater (Bq g -1 ). Radiotracer uptake kinetics were described using a simple linear regression model (Eq. ( 1)), or by a saturation exponential kinetic model (Eq. ( 2)) if the observed kinetics tended to reach a steady-state equilibrium:

CF t = k u t
(1)

CF t = CF ss (1 -e -k e t ) (2)
with CF ss = 𝑘 𝑢 𝑘 𝑒

where CFt and CFss are the concentration factors at time t in days (CFt) and at steady-state (CFss), respectively, ku is the uptake rate constant (d -1 ) and ke is the depuration rate constant (d - 1 ; e.g. [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: Sea water and food exposures[END_REF][START_REF] Whicker | Radioecology: Nuclear energy and the environment[END_REF].

In order to capture the influence of K. brevis pre-exposure on radionuclide uptake capacities in mussels, statistical comparisons were conducted using individual uptake kinetics: individual parameters (ku and CFss) were obtained from the best-fit model at the global scale (Eq. 1) for each individual. Differences between these parameters were then tested using two-tailed Wilcoxon-Mann-Whitney non-parametric tests. For each body compartment, the same statistical test was used to compare trace element distribution during the uptake period between control mussels and those pre-exposed to K. brevis culture. The same procedure was used to compare PbTx concentrations in the body compartments of the mussels from the exposed and the depurated mussels. The level of significance for statistical analyses was always set at α = 0.05. All the statistical analyses were performed using R software 3.0.1 (R Development Core

Team, 2014).

Results and Discussion

We found that blue mussels accumulated PbTxs in non-negligible quantities (i.e. 0.49 ± 0.22 µg g -1 wet wt in whole-soft tissues after the last exposure to the K. brevis, Fig. 1). Moreover, concentrations of PbTx were found to be more than 4-fold higher in the digestive gland (up to 2.80 ± 1.00 µg g -1 wet wt, Fig. 1) compared to other tissues, suggesting that mussels accumulated PbTx mainly by ingestion of K. brevis cells. As PbTxs were also found in the gills, this suggests that PbTx may have been accumulated in part from dissolved PbTxs in the water during exposure to K. brevis culture (i.e. K. brevis intact cells and solubilized PbTx). After the 23-d depuration period, PbTx were still strongly retained in mussels' tissues (78% of initial values: 0.38 ± 0.14 µg g -1 wet wt in the whole-soft tissues after three weeks of depuration, n = 4). [START_REF] Mcfarland | Uptake and elimination of brevetoxin in the invasive green mussel, Perna viridis, during natural Karenia brevis blooms in southwest Florida[END_REF] showed that another mytilid mussel, the green-lipped mussel Perna viridis, retained high levels of PbTxs after a K. brevis bloom, where tissue concentrations remained above the regulatory limit for human consumption for 4-5 months. This timeline is significantly longer than the depuration time of 2-8 weeks that has been found for oyster and clam species [START_REF] Mcfarland | Uptake and elimination of brevetoxin in the invasive green mussel, Perna viridis, during natural Karenia brevis blooms in southwest Florida[END_REF]. These findings demonstrated that PbTx were present in the tissues of the mussel throughout the trace element exposure period and suggested that depuration and detoxification processes operate on a longer timescale than that of this experiment (three weeks) in the K. brevis exposed mussels.

After 4 exposures to K. brevis culture (both cells and solubilized PbTx) at ecologically relevant cell concentrations (~1000 cells mL -1 ; [START_REF] Gannon | Effects of Karenia brevis harmful algal blooms on nearshore fish communities in southwest Florida[END_REF][START_REF] Stumpf | Monitoring Karenia brevis blooms in the Gulf of Mexico using satellite ocean color imagery and other data[END_REF], mussels were exposed to dissolved trace elements for 23 days. Our results showed that whole-body uptake kinetics of all essential trace elements (Co, Mn and Zn) followed saturation kinetics regardless of pre-exposure to PbTx (R 2 : 0.75-0.91, n=8; Fig. 2 andTable 2). Uptake kinetics of the nonessential trace element Cd were best described by a linear model (Fig. 2 and Table 2).

Comparisons among the rates at which elements were taken up in the whole organisms (ku) and the estimated CFss indicated that the essential elements (Co, Mn and Zn) were bioaccumulated similarly (p>0.05, Fig. 2 and Table 2) between unexposed M. edulis and those exposed to K. brevis cells. In contrast, a statistically significant higher uptake rate constant ku was found for Cd in K. brevis-exposed mussels compared to controls (p=0.049, Fig. 2) suggesting PbTx preexposure increased Cd uptake.

An important driver of variation in rates of bioaccumulation among bivalve species is the clearance rate. A negative relationship between trace element absorption efficiency (from the dissolved pathway) and clearance rates have been demonstrated for three marine bivalves (P. 2007) found a species-dependent reduction in clearance rate (79% for the bay scallop Argopecten irradians, or 38% for the green mussel P. viridis) in the presence of 1000 cells mL -1 of K. brevis culture. Although the clearance rate was not measured in our study, a preliminary experiment showed that ingestion rate (i.e. phytoplankton cells ingested by individual h -1 ) decreased in mussels pre-exposed to K. brevis (data not shown). Thus, we suggest that pre-exposure to K. brevis may also have induced a decrease in the clearance rate in mussels, leading to a higher Cd uptake rate constant ku. Although it is a non-essential element, Cd was mainly distributed in the mussel soft tissues (82 % after 9 days of waterborne exposure, Fig. 3). In contrast, the other elements were present predominantly in the shell, likely through adsorptive process (28-70 % after 9 days, Fig. 3). This finding further suggests that an active mechanism such as a decrease of the clearance rate may be responsible for the effects on Cd bioaccumulation in the mussel tissues.

As previously indicated, the distribution of trace elements among the mussel body compartments (i.e. digestive gland, gills and remaining tissues and shell) were determined throughout the exposure period. Distribution of all trace elements in each tissue was not significantly affected by pre-exposure to K. brevis (p>0.05, Fig. 3), indicating that both storage mechanisms of such elements remain constant despite the accumulation of PbTx in mussels.

The current study presents the first findings on the effects of pre-exposure to toxin-producing microalgae on the bioaccumulation of dissolved trace elements in a bivalve species. Care needs to be taken in how to expand on these results. Some of the functional groups generally present in algal toxin molecules, such as amino, carboxyl, phenol, sulfhydryl and hydroxyl groups, have the potential to complex with metal ions. Such complexes could affect the bioavailability of metals throughout aquatic food chains, as has been demonstrated in the case of cyanobacterial microcystins and domoic acid produced by the diatom Pseudo-nitzschia spp. [START_REF] Humble | Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography[END_REF][START_REF] Rue | Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudo-nitzschia[END_REF]. Thus, further investigations should be conducted to investigate the influence of concomitant exposure of metals and algal toxins in bivalves.

Conclusion

This study revealed statistically significant differences in the dissolved Cd whole-body uptake kinetics in common mussels pre-exposed to simulated blooms of PbTx-producing K. brevis in comparison to control mussels. This difference is likely related to decrease of the clearance rate of the K. brevis-exposed mussels. Nevertheless, no difference was observed in the uptake of the studied essential trace elements (Co, Mn, Zn). These findings indicate that the occurrence of PbTx in the environment at concentrations of ~1000 cells mL -1 appear to have a limited effect on the subsequent bioaccumulation of trace elements by this species of bivalves. More research is certainly needed to confirm observed patterns at higher PbTx dosing or more chronic exposures. In addition, as harmful algae can produce a variety of toxins that can potentially interact with trace elements and have diverse modes of action which can vary between bivalve species, further investigations are needed to study the influence of (1) a concomitant exposure to PbTx and trace elements (2) other biotoxins on trace element bioaccumulation in mussels (3) concomitant exposure using other species of bivalves. pre-exposed (white) or not (black) to K. brevis expressed as Concentration Factors (means ± SD). Parameters of uptake kinetics and their statistics are given in Table 1. Letter denotes significant difference (p<0.05) between ku of the mussels from the two experimental conditions.

  viridis, Septifer virgatus, and Ruditapes philippinarum;[START_REF] Wang | Comparison of metal uptake rate and absorption efficiency in marine bivalves[END_REF], indicating that individuals filtering a larger quantity of water display lower trace element absorption efficiency. Harmful algae have also been shown to alter clearance rates in bivalves, although both the magnitude and direction of effects depend on the species.[START_REF] Hegaret | Diverse feeding responses of five species of bivalve mollusc when exposed to three species of harmful algae[END_REF] demonstrated in the common mussel that the exposure to three harmful algal species (Prorocentrum minimum, Alexandrium fundyense, and Heterosigma akashiwo) increases the clearance rates, whileLeverone et al. (

Figure 1 .

 1 Figure 1. Concentration of PbTx-3 in four mussel compartments (digestive gland, gills and remaining soft-parts and whole-soft parts, n=4) after four simulated blooms of K. brevis (black) and then, after three weeks of depuration (white). Data are Means + SD.

Figure 2 .

 2 Figure 2. Whole-body uptake of 54 Mn, 57 Co, 65 Zn and 109 Cd in blue mussel (M. edulis, n = 8)

Figure 3 .

 3 Figure 3. Distribution of54 Mn,57 Co,65 Zn and 109 Cd among three body compartments (digestive gland, gills, remaining soft parts and shell) of blue mussel (M. edulis) pre-exposed (white) or not (black) to K. brevis over the radiotracer uptake period. At each time four mussels were dissected. All the values are expressed as percentage of the whole-body activity (Means + SD). For all the radiotracers, no significant difference was found at each time between preexposed mussels and those from the control group.

Table 1 .

 1 Time-integrated activities measured during the 23-day exposure to the dissolved radiotracers for mussels pre-exposed to K. brevis culture (aquarium 2) and for non-exposed mussels (aquarium 3, i.e. control group). Data are Means ± SD.

	Radiotracer	Aquarium 2 (Pre-exposed to K. brevis, Bq mL -1 )	Aquarium 3 (Control group, Bq mL -1 )
	54 Mn	0.39 ± 0.17	0.41 ± 0.16
	57 Co	0.43 ± 0.11	0.43 ± 0.12
	65 Zn	0.80 ± 0.39	0.78 ± 0.40
	109 Cd	0.93 ± 0.17	1.00 ± 0.12

Acknowledgments

The IAEA is grateful for the support provided to its Environment Laboratories by the Government of the Principality of Monaco. MM is Nippon foundation Nereus program Alumni.

The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a senior Member.

for 23 d to waterborne radionuclide (n=8) 1) after a pre-exposure to toxic K. brevis and 2) in the control group (non-exposed). ASE: asymptotic standard error; R 2 : determination coefficient of the uptake kinetics.

Radiotracer

Probability of the model adjustment: a p < 0.001.