MD Hélène Godet-Mardirossian

MD Nathalie-Sybille Goddet

MD François Dolveck

MD Michel Baer

MD Dominique Fletcher

MD Alexis Descatha
email: alexis.descatha@rpc.aphp.fr

Management of acute allergic reactions by dispatching physicians in a Medical Emergency Dispatch Centre

Background:

Acute allergic reactions often occur in out-of-hospital settings, and some of these reactions may cause death in the short term. However, initial diagnosis, management and processing of acute allergic reactions by Medical Emergency Dispatch Centres are not documented. We sought to describe acute allergic reactions and their management by a Medical Emergency Dispatch Centre.

Methods:

A prospective study was conducted from 20th August 2006 to 5th November 2006 on incoming calls for acute allergic reactions to the Medical Emergency Dispatch Centre for the Hauts de Seine (Paris West suburb, France). The agreement between initial diagnosis (made by dispatching physician) and final diagnosis (made by the physician who later examined the patient), and between initial and final severity, were evaluated using Cohen's weighted Kappa coefficient.

Results:

210 calls were included. The diagnoses made by the dispatching physician were: in 58.1% of cases urticaria, in 23.8% angioedema, in 13.3% laryngeal edema, and in 1.9% anaphylactic shock. The agreement between initial and final diagnoses was evaluated by a kappa coefficient at 0.44 (CI 95%: 0.26-0.61) and the agreement between initial and final severity was evaluated using a kappa coefficient at 0.37 (CI 95%: 0.24-0.50).

Conclusion:

We have highlighted only moderate agreement between the initial severity assessed by the dispatching physician and the final severity assessed by the physician later examining the patient. This demonstrates the need to develop a tool for assessing severity of acute allergic reactions for dispatching physicians in Medical Emergency Dispatch Centres.

INTRODUCTION

The Epidemiology of Anaphylaxis Working group of the American College of Allergy, Asthma and Immunology, summarised the findings from major international epidemiological studies and concluded that the overall frequency of episodes of anaphylaxis lies between 30 and 950 cases per 100,000 persons per year. [START_REF] Lieberman | Epidemiology of anaphylaxis: findings of the American College of Allergy, Asthma and Immunology Epidemiology of Anaphylaxis Working Group[END_REF] In France in 2005-2006, acute allergic diseases represented 0.9% to 2% of Emergency Medical System (EMS) visits with an emergency service physician on board. [START_REF]Assessment of the activity of emergency services in the Ile de France in 2004[END_REF][START_REF] Goddet | Fréquence et prise en charge des pathologies allergiques en réanimation pré-hospitalière[END_REF] Acute allergic reactions can take on very different clinical presentations and potentially involve numerous organs, including the skin and the respiratory, cardiovascular, gastrointestinal, and neurological systems. Even though acute allergic reactions can be easily distinguish by emergency physicians, many definitions of what constitutes a case exist, and no consensus can be observed. For instance, there is no universally agreed definition of anaphylaxis and there is no universally agreed criteria for diagnosis. [START_REF] Sampson | Second symposium on the definition and management of anaphylaxis: Summary report. Second national institute of allergy and infectious disease/food allergy and anaphylaxis network symposium[END_REF] As there is no consensus regarding the definition of anaphylaxis, the criteria for inclusion of patients into studies may differ from one country to another and this increases the difficulty in estimating incidence rates.

One of the main features of the French pre-hospital EMS (SAMU, Service d'Aide Médicale d'Urgence) is the participation of a physician at each stage in the organization. Actually, in the French Medical Emergency Dispatch Centres, all calls received are processed by a dispatching physician. [START_REF] Nemitz | Advantages and limitations of medical dispatching: the French view[END_REF] All calls for medical problem is received on the French pre-hospital EMS ("15", the national free medical emergency phone number) and are medically dispatched. There is no alternate EMS or screening system. The "SAMU" is a hospital department whose function is to centralise all emergency medical calls and organise an appropriate response. If a patient, a primary care physician, or everybody else face to an emergency problem and need an evaluation, triage, prehospital treatment or evacuation, he referrers to the centralized pre-hospital French EMS. Effective management of allergic emergencies by pre-hospital French EMS is dependent on the diagnosis and assessment of severity made by dispatching physicians in the Medical Emergency Dispatch Centre.

The aim of the study was to describe acute allergic events leading to prehospital EMS decisions in a circumscribed French district.

We aim to describe the characteristics of acute allergic reactions, key elements in the initial processing, and the agreement on the one hand between the initial and final diagnose and on the other hand between the initial and final severity. The study comprised two data collection stages. First, the dispatching physician filled in the "dispatch questionnaire" during the patient's call. Second, each patient included in the study was contacted on the phone by the investigating physician who filled in the "follow-up questionnaire". These questionnaires were both specifically instituted for this study.

METHODS

Study

Data collection

The following details were obtained from each patient during completion of the "dispatch questionnaire": age, gender, regular allergy medications (antihistamines, corticosteroids), symptoms (clinical features), and probable cause of allergic reaction (presumed aetiology).

The following items were also recorded in this questionnaire: initial severity evaluated by the dispatching physician using the French medical classification of emergency patients, clinical diagnosis (urticaria, angioedema, laryngeal edema, and anaphylactic shock) and response finally chosen by the dispatching physician (advice over the phone, medical consultation with a general practitioner (GP), ambulance with first-aid personnel on board and ambulance with an emergency service physician on board).

The patient was contacted for the "follow-up questionnaire" which provided information about evolution, hospitalization and diagnosis made by the physician who later examined the patient.

The "initial severity" was studied using the CCMU severity score (the French medical classification of emergency patients, CCMU, Classification Clinique des Malades des Urgences, Table 1) evaluated by the dispatching physician in two classes: "not severe" with a stable prognosis (CCMU 1 and CCMU 2) and "severe" with unstable condition, likely to worsen (CCMU 3 to 5). [START_REF] Fourestié | Classification clinique des malades des urgences. Définition et reproductibilité[END_REF] The "final severity" was considered in two classes, "severe" and "not severe" according to whether or not hospitalization occurred, whatever the unit.

Data analysis

Bivariate statistical analyses were used to describe acute allergic reactions. Relationships between qualitative variables were studied using Pearson's chi 2 test and Fisher's exact test when the conditions for applying the chi 2 test were not met. Comparisons of averages were performed using Student's T-test. The agreement between initial and final diagnosis on one hand, and between initial and final severity on the other hand, were evaluated using Cohen's weighted Kappa coefficient. The statistical analyses were conducted using the SAS software, Institute Inc. Cary, NC, USA (version 9.1). A P-value less than 0.05 was considered statistically significant.

Treatment of missing data

The reason for missing data regarding final diagnosis was that not all patient calls were followed by a medical consultation. All other variables had very little missing data (less than 1%).

Ethics

The protocol was approved by the French Commission on Individual Freedom and Data

Storage (CNIL, Commission Nationale Informatique et Liberté).

RESULTS

Two hundred and ten patients participated in the study.

Table 2 displays the characteristics of calls and patients, and the clinical features of the patients included. In more than 90%of cases, calls for acute allergic reactions came from the patient's home and the caller was the patient him/herself or a person close to him. The femalemale ratio of patients was 0.63. Ages ranged from 0.4 to 95 years with a mean age of 30.2, lower among men than among women, respectively 25.8 and 32.9 years (P<0.05). More than a half the patients had a history of allergy, and 19.5% had a regular allergy medications (H1antihistamines: 14.8%, corticosteroids: 3.3%, H2-antihistamines: 0.5%, desensitization: 1%).

The clinical symptoms the most frequently reported at the time of the call were pruritus (52.4%), facial edema (29.2%), localized erythema (24.8%), localized urticaria (superficial swelling of the skin resulting to a red raised itchy lesion) (23.3%), generalized urticaria (22.4%), generalized erythema (14.8%), paresthesia (14.8%). No significant difference was evidenced between men and women apart for conjunctive erythema and watering eyes (more frequent among men than women, P<0.05).

Diagnosis made by dispatching physician and response

The diagnoses made by the dispatching physician were: in 58.1% of cases urticaria, in 23.8% angioedema, in 13.3% laryngeal edema, and in 1.9% anaphylactic shock (table 2). No statistical significant difference in the distribution of diagnoses was observed between men and women. For the initial severity, 11.1% of allergic reactions were assessed as severe by the dispatching physician and 88.9% as not severe (table 3), without significant differences between men and women, among age groups, or among presumed allergens (P>0.05).

There was no statistical association between the response chosen by the dispatching physician and the patient's gender, but the response differed significantly with age (P<0.001). In the extreme age groups (0-2 years and 75-95 years), the response was in most cases a medical consultation with a GP. The response varied significantly with the diagnosis. The more "severe" the physician's diagnosis (laryngeal edema and anaphylactic shock), the more likely was the response to involve intervention (P<0.0001). The response also varied according to the degree of severity assessed by the dispatching physician (P<0.0001). The reactions assessed as "severe" led to sending an ambulance with emergency physicians on board in more than 75% of cases.

In the majority of cases of urticaria and angioedema, the patient stayed at home (82.6%).

More than a half of patients with laryngeal edema were hospitalized (58.3%). The reactions described by the dispatching physician as anaphylactic shock were all hospitalized (table 4).

Agreement.

The agreement between initial and final diagnoses was evaluated by a weighted kappa coefficient at 0.44 (CI 95%: 0.26-0.61). The diagnosis made by the dispatching physician was the same as the diagnosis by the physician later examining the patient in 53.98% of cases (table 5). The agreement between final and initial severity was evaluated using a kappa coefficient at 0.37 (CI 95%: 0.24-0.50). There is a significant difference between initial severity assessed by the dispatching physician and final severity assessed according to the hospitalization criterion (P<0.0001). Dispatching physicians estimated that 11.1% allergic reactions were severe. The physicians later examining patients assessed the proportion of severe allergic reactions to be 31.7%. Thus, 24.2% of the reactions classified initially as "not severe" in dispatch centre were finally classified as "severe". The severity of allergic reactions was, in almost a quarter of the cases, underestimated by dispatching physicians.

DISCUSSION

Acute allergic reactions often occur in out-of-hospital setting, their incidence is growing, and some of these reactions can cause death. However, allergic reactions leading to management by pre-hospital emergency services and their management by dispatch physicians have not been focused on previously. There is no consensus regarding management of allergic reactions, but, in order to homogenize our practices we implemented procedures about allergic reaction (with increase of consistency between physicians). There is no randomised controlled clinical trial in humans providing unequivocal evidence for their triage and treatment. Indeed, prompt medication with epinephrine is important for survival, and trying other medication would be unethical in some cases. [START_REF] Simons | First-aid treatment of anaphylaxis to food: Focus on epinephrine[END_REF][START_REF] Simons | Evidence-based management of anaphylaxis[END_REF][START_REF] Mclean-Tooke | Adrenaline in the treatment of anaphylaxis: what is the evidence?[END_REF] Only a few studies have been conducted in prehospital setting. [START_REF] Dewachter | Prise en charge d'une réaction anaphylactique en extrahospitalier et aux urgences : revue de la littérature[END_REF][START_REF] Kane | Anaphylaxis in the prehospital setting[END_REF] In USA in 2002, a study described the management of anaphylaxis in out-of-hospital setting and reported that 2.8 million runs (visit by EMS personnel) comprised between 0.34% and 0.82% runs for allergy. [START_REF] Kane | Anaphylaxis in the prehospital setting[END_REF] In 1996 Maio et al showed that allergic reactions in prehospital setting accounted for 0.4% of adult cases and 0.5% of paediatric cases. [START_REF] Maio | Emergency medical services outcomes project I (EMSOP I): prioritizing conditions for outcomes research[END_REF] The authors concluded that there was no standardization of case definitions, and management varied considerably. In France, allergic reactions leading to management by the Medical Emergency Dispatch Centres have not been described before. Therefore the comparison between initial and final severity has never been made. Most studies in dispatch centres were only interested in calls that resulted in sending an ambulance with a physician on board. [START_REF] Barrier | Les appels d'urgence au SAMU[END_REF][START_REF] Gros | Le risque allergique en réanimation pré hospitalière[END_REF] We considered all calls, regardless of the response. These elements have to our knowledge never been studied before.

Even if it is doubtful that the true etiology of an allergic reaction could be clearly designated by a dispatch physician, the presumed etiology in our study was consistent with the results of a study in the United Kingdom which found that the etiology was medication in 30% of cases, a food in 30%, and an insect bite or sting in 32% of cases. [START_REF] Peng | A Population-Based Study of the Incidence, Cause, and Severity of Anaphylaxis in the United Kingdom[END_REF] Our study showed low correlation between the initial severity assessed by the dispatching physician and the final severity. A similar finding was observed for the initial diagnosis and final diagnosis. The diagnosis relevance and the estimation of severity in dispatch centre were studied by Besnier et al. [START_REF] Besnier | Evaluation de la pertinence diagnostique et de l'estimation de la gravité en régulation médicale[END_REF] For all medical conditions combined, it was shown that the diagnosis made by the dispatching physician was the same as the diagnosis made thereafter by the physician examining the patient in 51.9% of cases. Severity was underestimated by the dispatching physician in 14.4% of cases. The same analysis performed on our study shows results of the same order. However the severity of a larger proportion of acute allergic reactions was underestimated (21.67%). This low level of agreement between the initial and the final evaluation demonstrates the difficulty in assessing the severity of these reactions in dispatch centres. The disparity between initial and final assessment could also be due to a worsening of the reaction over time or to the opposite an improvement over time, since both are possible with or without triage. We need to study the management of acute allergic reactions by dispatching physicians, because effective management is crucial for pathologies liable to lead to death in the short term.

We chose to study the initial severity as assessed by the dispatching physician using the French clinical classification of emergency patients. This classification is widely known, reproducible and adaptable to a dispatch centre. Its reliability, assessed by a measure of reproducibility, is good, with a kappa coefficient at 0.72. [START_REF] Fourestié | Classification clinique des malades des urgences. Définition et reproductibilité[END_REF] Other severity scores exist but they are complex and require physiological and biological measures, making their use impossible in a dispatch centre where physicians only have the phone interview.

For our analysis of final severity, we chose to describe as "severe" those reactions that required hospitalization. In emergency departments, Brown chose hypotension and hypoxia as severity indicators. [START_REF] Brown | Clinical features and severity grading system[END_REF] In a dispatch centre, it is not possible to obtain physiological data or even accurate clinical information for all patients. We therefore chose the variable "hospitalization or not" as a proxy to assess the final severity, since this information was available for all patients. The known major factor for hospitalization in emergency room is severity. However the terminology for severity based on the criteria for hospitalization is not necessarily accurate. Furthermore, hospitalization criteria are likely variable, and many patients are hospitalized for reactions that are not severe while other patients with anaphylaxis are not necessarily hospitalized. However, we assume that prehospital physicians will decide to hospitalize patients with severe reactions or with reactions that can worsen.

In our study, the dispatching physicians included cases of acute allergic reactions. Even though the definition is clinically defined, there is no universally agreed definition and there are no criteria for diagnosis of anaphylaxis. [START_REF] Sampson | Second symposium on the definition and management of anaphylaxis: Summary report. Second national institute of allergy and infectious disease/food allergy and anaphylaxis network symposium[END_REF][START_REF] Nemitz | Advantages and limitations of medical dispatching: the French view[END_REF] Furthermore, the second data set was acquired via information from the patient, which involves recall bias and lack of medical knowledge.

One can question the fact that patients can provide reliable information regarding the diagnosis made by the examining physician and even about the evolution. However, they could certainly indicate whether they were hospitalized.

Each urticaria assessed by the dispatching physician as a potential allergic urticaria had been included in the study. Urticaria is not a specific allergic reaction pattern, it mostly occurs in situations such as mainly viral infections, non-steroidal anti-inflammatory drugs use, and other unspecific triggers of histamine liberation without involvement of the adaptive immune system. However we had to consider each diagnosis of allergic urticaria made by dispatching physicians.

We encountered only few serious reactions. However this described the "real life" of acute allergic reactions seen by emergency physicians. Some clinical symptoms did not appear at all during our study and others were very sporadic. As many studies of acute allergic reactions our study was affected by the small size of samples of patients selected and studied. [START_REF] Neugut | Anaphylaxis in the United States: an investigation into its epidemiology[END_REF] Nevertheless, despite this relative lack of cases, our study highlights the difficulty of assessing the severity of acute allergic reactions for the dispatching physician.

The lack of demonstrated consistency between the range of dispatch physicians could represent a limitation. However, in order to homogenize our practices, we implemented a procedure seven years ago for allergic reaction for management of allergic disorders. [START_REF] Goddet | Fréquence et prise en charge des pathologies allergiques en réanimation pré-hospitalière[END_REF] We decided to contact the patient few days after the call for outcome rather than his physician, because we felt more confident about simple questions for patients than contacting a very large number of physician of our district (n>500, whereas one physician is not assigned to one patient in France).

CONCLUSION

Issues relating to allergic reactions managed by prehospital emergency dispatch centres had not previously been explored. In a relatively short time, the dispatching physician has to make a diagnosis and chose the best response for the patient, with the only information from the phone interview. Acute allergy can take the forms of a range of clinical signs and the evolution can be very fast and the outcome potentially fatal. These characteristics make it difficult to assess the severity of allergic reactions in dispatch centre. We have highlighted only moderate agreement between the initial severity assessed by the dispatching physician and the final severity assessed by the physician later examining the patient. This demonstrates the need to develop a tool for assessing severity of acute allergic reactions for dispatching physicians in medical emergency dispatch centres.

  design A prospective study was conducted from 20th August 2006 to 5th November 2006 on incoming calls to the Medical Emergencies Dispatch Centre for Hauts de Seine district, Paris West suburb. Cases of acute allergic reactions were included by dispatching physicians regardless of the patient's age, the caller and the place of call. Cases were included if the dispatching physician recorded a diagnosis of acute allergic reaction with the clinical presentation included any generalized skin, gastrointestinal, respiratory, cardiovascular, or neurological manifestations. Calls for asthma features alone were excluded from the study because the assessment of severity and criteria for emergency dispatch are established.

Table 1 -The French medical classification of emergency patients (Classification Clinique des Malades des Urgences CCMU)

 1 CCMU I: Condition injury or functional prognosis considered stable failure to act with additional diagnostic or therapeutic conduct in the emergency department. CCMU II: Condition injury or functional prognosis considered stable and necessity of complementary diagnostic or therapeutic CCMU III: Condition injury or functional prognosis may worsen but not involving life CCMU IV: Pathological situation involving life and not involving the practice of immediate resuscitation manoeuvre. CCMU V: Pathological situation involving life and necessity of immediate resuscitation manoeuvres.

Table 2 -Characteristics of calls and patients and individual clinical reaction features (n=210) Time of the day (24 hour format

 2

	)	n	%	Regular allergy medications
	0h 8h	31	14.8%	Antihistamines H1	31	14.8%
	8h 18h	84	40.0%	Corticosteroids	7	3.30%
	18h 24h	95	45.2%	Desensitization	2	1.0%
	Place of call			Antihistamines H2	1	0.5%
	Home	180 85.7%	No regular medications	167 79.5%
	School	8	3.8%	Clinical features (Symptoms)	n	%
	Workplace	9	4.3%	Pruritus	110 52.4%
	Public place	5	2.4%	Facial edema	61	29.2%
	Medical office	6	2.9%	Localized erythema	52	24.8%
	Unknown	2	1.0%	Localized urticaria	49	23.3%
	Who called?			Generalized urticaria	47	22.4%
	Patient or a person close to him	192 91.4%	Generalized erythema	31	14.8%
	Rescuer	1	0.5%	Paresthesia	31	14.8%
	Nurse	10	4.8%	Conjunctival erythema and watering eyes	16	7.6%
	Physician	7	3.3%	Mucosa oedema	14	6.7%
	Gender			Sense of choking	14	6.7%
	Men	81	38.6%	Other edema	12	5.7%
	Women	129 61.4%	Difficulty speaking	12	5.7%
	Age			Rhinitis	12	5.7%
	0-2 years	21	10.0%	Respiratory distress	11	5.2%
	3-15 years	52	24.8%	Nausea, vomiting, diarrhea	9	4.3%
	16-44 years	85	40.5%	Dysphagia	9	4.3%
	45-64 years	35	16.7%	Cough	8	3.8%
	65-74 years	3	1.4%	Dizziness	8	3.8%
	75-95 years	14	6.7%	Collapse. with or without unconsciousness	8	3.8%
	Personal allergic history			Laryngeal dyspnoea	7	3.3%
	Yes	115 54.8%	Abdominal pain	5	2.4%
	No	83	39.5%	Chest pain	3	1.4%
	Unknown	12	5.7%	Wheezing	0	0.0%
	Familial allergic history			Cardiac arrest	0	0.0%
	Yes	43	20.5%	Coma	0	0.0%
	No	75	35.7%	Sneezing	0	0.0%
	Unknown	92	43.8%
	Presumed etiology (Allergen)
	Drug	72	34.3%
	Food	51	24.3%
	Other (mostly insect bites)	68	32.4%
	Unknown	19	9.0%

Table 3 -Initial diagnosis and initial severity

 3 : diagnosis made by dispatching physician, CCMU=French medical classification of emergency patients. ** variable "initial severity": two missing data (where emergency physician unable to classify severity)

	Initial Diagnosis*	n	%
	Urticaria	122	58.1%
	Angioedema	50	23.8%
	Laryngeal edema	28	13.3%
	Anaphylactic shock	4	1.9%
	Other (collapse, conjunctivitis…)	6	2.9%
	Total	210	100.0%
	Initial severity*
	CCMU 1	137	65.9%
	CCMU 2	48	23.1%
	CCMU 3	15	7.2%
	CCMU 4	7	3.4%
	CCMU 5	1	0.5%
	Total	208**	100.0%
	*initial diagnosis

Table 4 -Orientation of patients depending on initial diagnosis

 4

		Orientation** (n=207)
		Home		Hospitalization		n (total)
	Initial diagnosis* (n=210) n	%	n	%
	Urticaria	99	82.6%	21	17.4%	120
	Angioedema	28	57.1%	21	42.9%	49
	Laryngeal edema	11	41.7%	17	58.3%	16
	Anaphylactic shock	0	0.0%	4	100.0%	4
	Other	3	50.0%	3	50.0%	6
	n (total)	140		67		207
	*initial diagnosis: diagnosis made by dispatching physician
	**variable "orientation": three missing data (1%)

Table 5 -Initial and final diagnosis

 5 : diagnosis made by dispatching physician **final diagnosis: diagnosis made by the physician who later examined the patient.

		Initial diagnosis*	Final diagnosis**
		n	%	n	%
	Urticaria	122	58,1%	53	46.9%
	Angioedema	50	23,8%	31	27.4%
	Laryngeal edema	28	13.3%	7	6.2%
	Anaphylactic shock	4	1.9%	3	2.7%
	Other	6	2.9%	19	16.8%
	n (total)	210	100.0%	113	100.0%
	*initial diagnosis


J M Thiriet

F Meriaudeau

J C Burguillo

H Fremont

H Yahoui

P De Fooz

Toward an International Curricula Network for exchanges and LifeLong Learning

The ELLEIEC ERASMUS thematic network runs from October 2008 to September 2011. This project deals with several aspects linked to LifeLong Learning in Electrical and Information Engineering in Europe. One of the actions of the project is to set some ICN (International Curricula Networks) in some disciplines (specialities in EIE) and levels (Bachelor or Master) aiming at defining the necessary learning outcomes for some curricula. It is defined and agreed at the European level by the ICNs and can be used for AEL (Accreditation of Experience Learning) for European workers who want to validate some diploma, corresponding to their actual knowledge and skills. AEL could be partial, so it is crucial to have the possibility, for workers who need it, to validate some modules; these modules can be prepared either in a classical class or remotely. The modules (or pieces of modules) which are available remotely to students, and agreed by the partners of the ICN, are called IM (International Modules).

I. INTRODUCTION

During the last years, an important work has been achieved within the framework of the European programmes in order to emphasise and allow the mobility of students, the exchange of teachers, the recognition of diploma (programmes ERASMUS, now in the LLP programme)…

These reflections have been done for classical programmes within HEIs throughout Europe and also within the programmes dedicated to vocational studies (LEONARDO, ValeurTech, EQF) to emphasise Life Long Learning and credit accumulation all around the life, with an approach based on competences [START_REF] Ward | The alignment of Generic, Specific and Language Skills within the Electrical and Information Engineering discipline, Application of the TUNING approach[END_REF].

The main purposes in these programmes can be summarized as follows:

favouring the equality among citizens in Europe, giving them the possibility to obtain their diploma in any European country and also ideally to be able to work anywhere within the European Union, -fostering Life-Long Learning in order to allow citizens to adapt themselves to the rapidly-evoluting needs of the society, all around their life, as far as jobs are concerned.

This paper presents the concept of ICN (International Curricula Network), which can act as a pedagogic committee at the European level for a particular discipline or sub-discipline.

II. PROBLEMATIC

Three challenges are now facing the European Union concerning mobility or "equal mobility" (which means equality of mobility opportunities for each European citizen):

-Mobility of students, that is partially achieved by the ERASMUS exchange programme through the European Credit Transfert System (ECTS), and the Curriculum Development programme (+ ERASMUS Mundus), for the development of joint degrees.

-Mobility of workers (recognition of diploma) which is at the moment only partial, even if an important work is done in order to adopt or to converge to a European Qualifications Framework and maybe to a European Accreditation [START_REF] Pasquet | Quality and Accreditation in Europe: implication in Electrical and Information Engineering[END_REF] System or Methodology (EUR-ACE (Accreditation of Engineering Programmes)) … -Mobility of LL citizens, which means the possibility for a citizen to accumulate credits and possibly to validate diploma during all her/his life, with a European recognition (credits, diploma) of the process she/he goes through. We can illustrate with an example, even if it is "theoretical": o to leave the university with a bachelor degree in a first country, o to work in a company of a second country, with the diploma from the 1 st country, o to pass some credits in a professional speciality during this time at the company, o to go to a third country in order to prepare some theoretical credits, o to accumulate all these credits to get a master level, for instance, in another country, o finally, to go to a fifth country with this master obtained through accumulation of credits to work in a company or at educational institutions (colleges, universities…), or to do a Ph.D.

The main purpose of this work is to enhance the employability of people by allowing them to learn and validate their knowledge all life long and to recognise these lifelong-acquired competences in the various European countries and systems.

Our reflection concerns the field of Electrical and Information Engineering (it is our speciality) and in the ERASMUS programme (ERASMUS network) since it is targeted at the moment to Universities and HEIs, because there is a challenge in introducing these concepts to the universities and to provide them with some tools in order to develop and implement in the future a quality LL process, and we think our HEIs should be the main actors in that point, within the frame of the Bologna process [START_REF] Martins | Overview of the Bologna Process: Implementation in Europe In Electrical and Information Engineering[END_REF].

III. INTERNATIONAL CURRICULA NETWORKS (ICN)

An ICN is a cluster of partners who agree all together to a common or joint curriculum in a speciality. Ideally these shared curricula should be recognised at the accreditation level, but the process can be difficult in some cases. In order to initiate the ICN, we consider two pragmatic cases: -Cross recognition of "curricula " (learning outcomes, competences, Tuning approach), which is the fact when at least two institutions agree on common modules for an ERASMUS exchange either of students or of teachers. -Shared recognition of modules, which is the case when a set of modules is developed and implemented within the frame of a programme; in particular distant modules can be used. The last role of the ICN is to act as a "pedagogical committee" to identify good approaches in the setting of existing or new LLL curricula.

IV. EXAMPLE OF AN ERASMUS-BASED ICN ON

NETWORKING AT THE BACHELOR LEVEL Within the frame of the "Final year of the Professional Bachelor's Degree In Computer Networks and Telecommunications specialising in Wireless Networks and Security"(WiNS) [START_REF] Thiriet | Terriez-Implementation of an International "Professional" Bachelor's Degree in Wireless Networks and Security in Grenoble -Colloque sur l'Enseignement des Technologies et des[END_REF] This semester with courses was recognized by the students' home institution. For the spring semester, the Belgian students went back in Liège and passed some courses and a Training project. Both of these were recognized in Grenoble and as a consequence the students obtained the two diplomas. The Spanish students stayed in Grenoble in order to get some courses and went in companies for their internship in France: these were recognized both by Grenoble and Vigo meaning that the students got also two diplomas.

Therefore, this first experiment of an ICN done in Grenoble obtained concrete results.

Another experiment concerns the case of a Slovak student, who attended the autumn semester in Grenoble, and then went in Germany (within an ERASMUS agreement between Grenoble and the German institution). So this student, registered in Kosice, spent one semester in Grenoble and one in Germany, and got both the Slovak and French bachelors.

The last scenario concerns a Finnish student, who attended only the autumn semester, and then went back to Finland for the spring semester. In his Finnish curriculum, the student followed courses but no internship. He naturally got his Finnish diploma. Concerning the French Professionnal Bachelor, an intership experience was missing, on the formal point of view, but this student got a previous professional experience, working previously in some companies in networking and security ; the final jury decided to give this student the "Licence Pro", considering his professional experience as an AEL (Accreditation of Experiential Learning).

These are some examples of an ICN, composed of several universities in Europe, around an existing curriculum, based on ERASMUS exchanges.

2.

Within the frame of staff ERASMUS exchanges Vigo and Liège are so two partner universities of Grenoble in this experiment. Juan Carlos Burguillo from Vigo and Pierre de Fooz from Liège came in Grenoble within the staff ERASMUS programme in order to give a "bit-size" module :

-"Firewall configuration and WiFi security" within the frame of the module "Security of networks" for the Spanish colleague, -"IPV6 protocol" within the "network" module for the Belgian colleague.

These "bit-size" modules were of course recognised by Grenoble, by the home institution of the students but also on an informal way by the other institutions collaborating with Grenoble within this WiNS programme: for example a student coming from AGH Krakow, following the WiNS course in Grenoble, validating the bit-size module of "Firewall configuration and WiFi security" from Vigo.

Various universities from the ELLEIEC project are partners of UJF Grenoble in this experiment: -CTU Prague (partner P13) -Technological Educational Institute of Crete (partner P32) -University of Vigo (partner P35) -Technical university of Kosice (partner P41) -AGH University of science and Technology (partner P45) -Technical University of Valencia (partner P55) -Haute-Ecole de la Province de Liège (partner P60) -Cracow University of Technology (new partner).

Another interesting experiment, not further developed in this paper, concerns the setting of 6 LLL modules, developed by a set of partners within the CD project ESPANT (http://www.advancednetworking.eu/), in the field of networking. A cross-fertilisation experiment is on the way between ELLEIEC and ESPANT. Considering these three masters, one can see the diversity of an ICN:

V. EXAMPLE OF AN ERASMUS MUNDUS-BASED ICN

At the mobility level: VIBOT is based on a rotation basis of all students at the same time (every semester) within 3 European Universities, one in France, one in Spain and one in Scotland; CIMET includes 4 European Partners (Norway, Finland, Spain and France) with students being split between the European Universities depending on their chosen specialty; EMARO relies on a consortium of 3 European Universities (Italy, Poland, France) as well as 3 non European Universities (Thailand, Japan, China). For the first year, students are split into two groups and stay in one University (France or Poland), afterwards they can go in any of the three European Universities for the third semester and in any of the 3 non European University for their research Internship.

Regarding the course structure, the three masters offer also a different approach: VIBOT has decided to have their course contents commonly defined and held every semester in each university in common with local students who follow a national degree in their university. The contents are discussed and adapted every year, based on research progress, during the Academic Board. Regarding CIMET, students may attend different courses depending on the university, and the specialty; but they can also have similar courses delivered on different campuses, taught by the same lecturer either through Intensive Modules or through Vision Conference teaching Methods. EMARO is closer to CIMET than VIBOT but the course content being highly technologically based, some modules have a slightly different content. All consortia welcome numerous guest professors from different countries sponsored by the Erasmus Mundus Program as scholars, who are teaching in some of the modules. Their lectures are fully recognized by all institutions of the consortia, leading to degrees which contents might vary from year to year but with similar acquired competencies.

Regarding, the degree awarded, all consortia recognized the modules gained at the partner institutions. They are moving to a Joint Degree but at the present time they are only delivering multiple degrees or double degrees. VIBOT is delivering a triple Degrees, EMARO is giving a double degree whereas students enrolled in CIMET can either get a double or triple degree depending on their mobility. Nonetheless, all consortia deliver a Diploma Supplement which describes the mobility of the students, explains the difference between the grading systems, and gives some details about the research topics tackled during the fourth semester.

Regarding the administrative aspects, the three consortia are following the same route: a centralized system at the Coordinating Institution, a set of quality procedures to monitor all the aspects of the EMMCs (administrative support, course content and competencies delivered, transferable skills, housing support, ….), a set of meetings for recruiting the students and discussing about course content adaptation, quality assessment and reporting.

Employability and sustainability are also of main concern for these three consortia which are developing various actions for these: conferences with industrial partners, Job fairs, interview for job and summer placements… Extension on the same basis (double or multiple degrees) with non European universities is also happening for some of these programmes. For instance, VIBOT is also proposing a similar scheme with Asian Universities in Indonesia and Malaysia, where students obtained a double degree by doing their first year in Asia and the second year in France. The course contents are elaborated jointly then validated by each university and the procedures (selection, grading system, diploma supplement…) are reported in a signed MOA.

All these masters running in parallel with national master, mobility through the usual routes (Erasmus Student Exchange, student exchanges through MOAs..) are therefore offered to students registered in the national degree.

VI. CONCLUSION

In order to enhance the mobility of citizens during their life, following the white book of the European Commission on LifeLong Learning, we should invent new strategies, using existing tools such as ERASMUS exchanges, IP, CD, ERASMUS-MUNDUS and TEMPUS. The interests are to put energy in common to develop and propose High-Quality International Modules, validated by International Curricula Networks, considered as parts of normal curricula within at least 2 partner universities in the consortium, and being proposed for Life-Long Learning, to any interested citizens.

Within this frame, the paper illustrates two approaches, an Erasmus-based ICN in networking at the bachelor level and an Erasmus-Mundus based ICN in Vision and Robotics at the master level.

  [START_REF] Thiriet | Professional" Bachelor's Degree in Wireless Networks and Security[END_REF] programme, launched in Grenoble in September 2007 as an unexpected outcome of the previous Thematic network EIE-Surveyor (www.eie-surveyor.org), the following experiments have been achieved in 2009-2010, as a first attempt to define the precepts of International Curricula Networks and International Modules.

	The first experiment concerns both student and teacher
	international exchanges.
	1.	Within the frame of the student ERASMUS
	exchanges, 4 students attended the autumn semester of the
	WiNS class:
	-	Two students from University of Vigo /
	Universidade de Vigo (partner P35 of ELLEIEC) in
	"Technical Telecommunication Engineering, speciality in
	Telecommunication Systems / Enxeñaría Técnica de
	Telecomunicación Especialidade de Sistemas de
	Telecomunicación (ETTST)"
	-	Two students from Higher Education Institution
	of the Province of Liège / Haute-Ecole de la Province de
	Liège (P60) in "Bachelor in Industrial computing, option
	network and telecommunications / Bachelier en
	informatique et systèmes finalité réseaux et
	télécommunications"

  Among the list of EMMCs, 3 are in the fields of Vision and Robotics (EMARO : European Master on Advanced Robotics, VIBOT (www.vibot.org) : Erasmus Mundus Master in VIsion and roBOTics, CIMET: Master Eramus Mundus "Color in Informatics and Media Technology) and no EMJDs selected yet.

		ndia/selected_projects_action_1_master_courses_en.php
		and	at	:
		http://eacea.ec.europa.eu/erasmus_mundus/results_compe
		ndia/selected_projects_action_1_joint_doctorates_en.php.
		ON
	VISION AND ROBOTICS AT THE MASTER LEVEL
	Erasmus Mundus Programs (Erasmus Mundus Master
	Course EMMC and Erasmus Mundus Joint Doctorate
	EMJD) are very good examples of ICN. Since the launch
	of the Program (2003) and the first round of EMMCs
	(2004), the number of ICN in the field of computer Vision
	and/or Robotics has increased and they have gained
	visibility for non European Countries and for non
	European students through actions developed within the
	Action 3 (former Action 4) of the Erasmus Mundus
	Programme. The list of all EMMC and EMJD can be
	found	at
	http://eacea.ec.europa.eu/erasmus_mundus/results_compe

ACKNOWLEDGMENT

The authors wish to thank the European Commission for the grant in the Life long Learning Programme, the Executive Agency for its help and all the partners of the project for their contribution.

Project Number: ELLEIEC 142814-LLP-1-2008-FR-ERASMUS-ENW Instrument type: ERASMUS NETWORK as well as the grant obtained for the ERASMUS MUNDUS action 3 EACOVIROE (www.eacoviroe.org) project.


E J Rochelle-Newall
email: emma.rochelle-newall@ird.fr

X Mari

O Pringault
email: olivier.pringault@ird.fr

Sticking properties of transparent exopolymeric particles (TEP) during aging and biodegradation

Keywords: TEP, Bacterial production, Bacterial respiration, DOC, residence time, carbon cycling

Although many studies have addressed the role of bacteria in the degradation of organic matter, few have examined how bacteria alter the physico-chemical properties of dissolved and colloidal organic matter in coastal systems. Here we investigate how the sticking properties of Transparent Exopolymeric Particles (TEP) varied with DOM age in batch cultures. We show that in two contrasted sites, despite different initial TEP sticking properties and bulk concentrations, after 48 hours, the sticking properties were similar and increased (i.e., TEP became stickier) with increasing DOM age. We propose that TEP occurring after 48 hours of incubation are mainly of heterotrophic origin, which is in contrast to the previously identified TEP of autotrophic origin. These results highlight the potential importance of bacterial DOM production, particularly in the aphotic zone, and further underline the potential of bacterial heterotrophs to produce biologically refractory dissolved organic matter that is physically reactive (i.e. sticky).

Introduction

Dissolved organic carbon (DOC) comprises the largest pool of bioreactive carbon in the oceans (e.g. [START_REF] Hansell | Preface[END_REF]. This large and highly diverse pool of organic molecules is made up of a wide range of carbon molecules differing dramatically in both molecular weight and bioavailability. This complexity has rendered the chemical identification of many of the carbon moieties extremely difficult and up to 80% remains unidentifiable [START_REF] Benner | Chemical composition and reactivity[END_REF]. The main result of this is that DOC concentrations are often measured as a bulk property and little consideration is given to how concentrations and properties of different fractions change over time.

Whilst DOC is the dominant form of organic carbon in aquatic systems, particulate and colloidal organic carbon (POC and COC, respectively) can represent a biologically important, though often small, percentage of the total organic carbon. Moreover, the transfer of matter between these pools is highly dynamic with colloidal particles forming a dynamic bridge between the DOC and POC pools [START_REF] Verdugo | The oceanic gel phase: a bridge in the DOM-POM continuum[END_REF]. Transparent exopolymeric particles (TEP) are COC structures, comprised of acidic polysaccharides [START_REF] Mopper | The Role of Surface-Active Carbohydrates in the Flocculation of a Diatom Bloom in a Mesocosm[END_REF][START_REF] Zhou | The role of surface-active carbohydrates in the formation of transparent exopolymer particles by bubble adsorption of seawater[END_REF]. They are ubiquitous in the aquatic environment and can represent up to 50% of the standing stock of POC in a system [START_REF] Passow | Transparent exopolymer particles (TEP) in aquatic environments[END_REF].

Bioreactivity or bioavailability describes the biological remineralisation rates of organic matter (e.g. [START_REF] Amon | Bacterial utilization of different size classes of dissolved organic matter[END_REF][START_REF] Raymond | Bacterial consumption of DOC during transport through a temperate estuary[END_REF]Rochelle-Newall et al., 2004). This remineralisation is dependent upon several factors, notably the chemical structure and molecular size of the organic matter [START_REF] Amon | Bacterial utilization of different size classes of dissolved organic matter[END_REF], the availability of organic or inorganic nutrients and the C:N ratio of the material (e.g. [START_REF] Kroer | Bacterial growth efficiency on natural dissolved organic matter[END_REF] or the degree of photolysis (e.g. [START_REF] Obernosterer | Differences in the optical and biological reactivity of the humic and nonhumic dissolved organic carbon component in two contrasting coastal marine environments[END_REF]. Although the latter of these is a physical process, the first two are related either directly or indirectly to the source and age of the organic matter. For example, it is generally considered that marine organic matter is more bioavailable than riverine or marsh derived organic matter [START_REF] Del Giorgio | Patterns in dissolved organic matter lability and consumption across aquatic ecosystems[END_REF].

Similarly, it is generally considered that organic matter released during photosynthesis, constituted mainly of carbohydrates [START_REF] Biersmith | Carbohydrates in phytoplankton and freshly produced dissolved organic matter[END_REF], is more bioavailable than the 'background' organic matter in marine systems although this bioavailability does vary with the phytoplankton species present (e.g. [START_REF] Renaud | Effects of the colonial cyanobacterium Trichodesmium spp. on bacterial activity[END_REF]. Wild et al.(2004) have also demonstrated that coral reef mucus is highly bioavailable to the surrounding bacterial communities, thus providing a potential major source of available organic matter in otherwise oligotrophic waters. This increased bioavailability of phytoplanktonic and coral reef organic matter may well be one of the reasons why we generally observe a tighter relationship between bacterial production and primary production in oligotrophic systems than what is observed in more mesotrophic systems [START_REF] Morán | Dissolved primary production and the strength of phytoplankton bacterioplankton coupling in contrasting marine regions[END_REF].

Sinking organic matter from the mixed layer of aquatic systems is a major pathway via which organic carbon is exported to the sediments. However, in order to sink, organic matter needs to have a density sufficiently high to ensure that it sinks rather than floats. Evidently, larger particles such as large diatoms will fall through the water column and it has recently been shown that even picoplankton contribute to this export [START_REF] Richardson | Small phytoplankton and carbon export from the surface ocean[END_REF], however, it is less clear what controls the export of dissolved organic matter to the deeper layers. Recent work has shown that TEP play an important role in the vertical transport of dissolved organic matter by coalescing and aggregating with dense particles to form larger, more dense aggregates that then sink (Azetsu-Scott and [START_REF] Azetsu-Scott | Ascending marine particles: Significance of transparent exopolymer particles (TEP) in the upper ocean[END_REF][START_REF] Engel | Polysaccharide aggregation as a potential sink of marine dissolved organic carbon[END_REF].

However, the factors controlling the sticking properties of TEP, the process that allows the formation of large aggregates, have not yet been clearly identified. While it is known that the presence of metals can alter the sticking properties and hence reduce the formation TEP [START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF], less is known about the impact of aging and bacterial degradation on the sticking properties of TEP.

Recently, it was proposed that residence time of organic matter plays a role in determining whether or not organic matter is exported from a system [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF]. Here we present the results from an incubation experiment aimed at testing the impact of aging on the sticking properties of TEP. The objective of this study was to determine how the sticking properties of TEP altered with age of the DOM pool at two contrasting sites subject to different flushing rates.

Material and Methods

Samples were collected from two stations in the Southwest lagoon of New Caledonia (see Mari et al., 2007 for map). The first station (Stn. Lag) is located close to the coral barrier reef system and is considered to be isolated from anthropogenic influences. The second station is located within the Grande Rade Bay (Stn. Ind). This bay is heavily influenced by both industrial and urban effluents. Furthermore, the average e-flushing times for the two stations differ, with Stn. Lag having a lower e-flushing time than that of Ind: 0.5 and 45 days, respectively [START_REF] Jouon | Calculations of hydrodynamic time parameters in a semi-opened coastal zone using a 3D hydrodynamic model[END_REF].

Sample water (40 L) was collected from each station at a depth of 3 m with a 10 L Niskin bottle. The sample was transported back to the laboratory in acid cleaned, polycarbonate 20 L carboys (Nalgene). Upon return to the laboratory, 25 L of water were pre-filtered through a 1.0 µm membrane filter (Whatman Cyclopore) in order to remove larger particles and grazers.

Two acid-washed (10% HCl) and Milli-Q water rinsed, polycarbonate (Nalgene) carboys were then filled with 20 L of 1.0 µm filtered water. Both before and after filtration, samples were collected for Chlorophyll a (Chl a), nutrient and, DOC concentration in order to determine the efficacy of the filtration and to control for any nutrient contamination arising from the manipulations. No differences were found between the before and after filtrations for nutrients and DOC (data not shown).

The two carboys were incubated in the dark and at constant temperature (24 °C) during 32 days. Samples were collected at T0 and after 1, 2, 3, 5, 8, 11, 17 and 32 days for analysis of various biological and chemical parameters, as detailed below.

Samples for bacterial abundance (1.8ml) were fixed with glutaraldehyde (final conc. 0.5%) and stored at -80°C until enumeration. Abundance was determined after staining with SYBR Green I (Molecular Probes; 1: 25,000 dilution of commercial stock) for 10 min at room temperature using a flow cytometer (FACSCalibur; Becton Dickinson) equipped with a 488nm Argon laser [START_REF] Gasol | Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities[END_REF].

Bacterial production (BP) was measured using 3 H-leucine, following the method of [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3 H-leucine[END_REF]. Briefly, triplicate 1.5 mL sub-samples plus one killed control were incubated for 1 h in the presence of 20 nM (final concentration) high specific activity 3 Hleucine (Perkin Elmer) at the incubation temperature. The coefficient of variation (CV) of the triplicate measurements was always less than 5%. Leucine uptake was converted to carbon using the conversion factor 1.55 kg C mol -1 leu [START_REF] Kirchman | Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments[END_REF].

Bacterial respiration (BR) was measured at each time point using the oxygen microelectrode technique of [START_REF] Briand | The use of oxygen microprobes to measure bacterial respiration for determining bacterioplankton growth efficiency[END_REF]. The microprobes (Unisense, Denmark) are designed with an exterior guard cathode (Revsbech 1989), which results in extremely low oxygen consumption by the electrodes (4.7-47 x 10 -7 µmol O 2 h -1 ). Probes have a response time shorter than 1 second and a precision of 0.05%. The precision of oxygen microprobe is equivalent to highly precise Winkler techniques [START_REF] Briand | The use of oxygen microprobes to measure bacterial respiration for determining bacterioplankton growth efficiency[END_REF].

Bacterial Carbon Demand (BCD) was calculated as the sum of BP and BR. Finally, bacterial growth efficiency (BGE; %) was calculated as BP/BCD * 100.

Chlorophyll a was determined fluorometrically (Turner Designs Trilogy) on 50 mL samples collected on GF/F (Whatman) glass fibre filters using the method of [START_REF] Holm-Hansen | Fluorimetric determination of chlorophyll. Rapports et procès-verbaux des réunions -Conseil international pour[END_REF]. The filters were frozen (-80 °C) until measurement which was always within 72 h and generally within 24 h after sampling.

Nitrate and nitrite concentrations (DIN) were determined according to [START_REF] Raimbault | Feasibility of using an automated colorimetric procedure for the determination of seawater nitrate in the 0 to 100 nM range: examples from field and culture[END_REF] on a Bran+Luebbe Autoanalyzer III with an average detection limit and CV of 20 nmol L -1 and 3% and 2 nmol L -1 and 8% for eutrophic and oligotrophic waters, respectively.

Phosphate concentrations (DIP) were determined on a Bran+Luebbe Autoanalyzer III with an detection limit of 10 nmol L -1 and an average CV of 6 to 11% between replicates [START_REF] Torréton | Correspondence between the distribution of hydrodynamic time parameters and the distribution of biological and chemical variables in a semienclosed coral reef lagoon[END_REF].

Particulate organic carbon (POC) and particulate nitrogen (PN) were measured on 1 L subsamples immediately filtered onto 25-mm Whatman GF/F filters pre-combusted at 550°C for 2 h. After filtration, the filters were dried at 60°C for 24 h and then frozen for later analysis. Analyses were carried out with a LECO-900 CHN analyzer calibrated with ethylenediaminetetraacetic acid standards. For the determination of organic carbon, carbonates were removed with 100 µL of 2 mol L -1 HCl and renewed until there was no longer any effervescence. Acidified samples were kept in a drying oven (60-70°C) for 24 h. Detection limits were 40 and 10 µg for carbon and nitrogen, respectively.

Total organic carbon (TOC) analyses were performed on 10 mL sub-samples collected in pre-combusted (450°C, overnight) 10 mL glass ampoules, preserved with 12 µL 85% phosphoric acid (H 3 PO 4 ) and flame sealed. Samples were stored in the dark until analysis.

TOC concentration was measured on a Shimadzu TOC VCPH analyzer, using potassium phthalate calibration standards over the measurement range 0 to 250 µmol C L -1 . Certified reference materials (CRM; Hansell Laboratory, University of Miami) were also used to assess the performance of the instrument on and between measurement days. The machine blank was between 5 and 10 µmol C L -1 for the measurement days and the CV of the measurement was always less than 2% of the mean of triplicate injections of duplicate samples. DOC was calculated by subtraction of the POC measurements from the TOC concentrations.

The sticking properties of transparent exopolymeric particles were studied largely following the method of [START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF]. In brief, after filtration through the 1 µm membranes, 2 L of the filtrate originating from each station that contained sub-micrometer TEP precursors were put inside a mixing device composed of a grid oscillating vertically inside two 2 L Plexiglas cylindrical containers, which generates small-scale turbulence inside the containers under the control of a motor controlling the oscillation frequency. Polybead® Carboxylate Red Dyed microspheres of 6-µm (Polysciences, Inc.) were added in the filtrate to yield a theoretical final concentration (C f ) of 5000 particles mL -1 . This value is determined from the manufacturers' product data sheet and the same volume of bead solution was added to each cylindrical container throughout the experiment. The formation of TEP-bead aggregates was followed during three successive 1 h periods, under increasing turbulence intensities. The turbulence kinetic energy dissipation rate, ε, was set to 0.1, 1 and 10 cm 2 s -3 , during the first, second, and third hour of the experiment, respectively. Samples were collected every 15 min in order to determine TEP size spectra and the relationship between the numbers of attached beads vs. TEP size. TEP were stained with Alcian Blue (Alldredge et al. 1993). The TEP size spectra and the relationships between TEP size and numbers of TEPattached beads were determined from 5 mL sub-samples filtered onto 0.2 µm polycarbonate filters after transfer of the retained particles onto a microscope slide (Passow and Alldredge 1994). TEP size spectra were determined microscopically for each slide by counting and sizing TEP at two successive magnifications (250x and 400x). Ten images were taken per slide and for each magnification. The equivalent spherical diameter (ESD) of individual TEP was calculated by measuring its cross-sectional area with an image-analysis system (ImagePro Plus, MediaCybernetics) and counts were combined and classified into 20 logarithmic size classes (Mari and Burd 1998). The TEP volume concentration was calculated from the TEP size spectra assuming a spherical volume for each particle. Estimates of TEP carbon (TEP-C) concentration were obtained by combining TEP size spectra with the relationship giving the carbon content of a given TEP particle according to its size (Mari 1999).

A relationship between TEP size and the number of attached beads was calculated for each sample by sizing individual TEP and enumerating its associated beads at 250x magnification using a compound light microscope. A minimum of 20 mixed aggregates of TEP-beads were studied for each slide. The number of attached beads was fitted to a linear relationship, n = ad p + b, where n is the number of associated beads in the mixed aggregate, d p (µm) is the TEP ESD, and a and b are constants for a given sample. Numbers of TEP-attached beads and TEP size were plotted in linear coordinates and the constants a and b were determined from regression analysis. The constant a, thereafter called the sticking slope, describes the ability of TEP to stick to beads (i.e. the higher a is, the higher the sticking properties are) and, thus, relates to the propensity of TEP to initiate the formation of large organic aggregates [START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF].

Results

The in situ Chl a concentrations were higher at Stn. Ind (0.44 +/-0.02 µg L -1 ; mean +/sd) than in Stn. Lag (0.24 +/-0.02; µg L -1 ), however, after filtration the concentrations were the same (0.04 µg L -1 , Table 1). In situ nutrient concentrations were also low and there was no difference between the initial concentrations and the concentrations measured immediately post filtration (data not shown).

Nutrient concentrations were low at both stations at the time of sampling (Table 1). In general, concentrations were higher at Stn. Ind, reflecting the coastal nature of this site and this general trend of higher nutrients at Stn. Ind continued throughout the 32 days experiment.

There was little difference between the initial DOC concentrations at the two sites.

Both stations had initial concentrations of around 70 µM C which decreased during the experiment. However, the amount of DOC removed during the incubation was higher in the Stn. Ind incubation than in Stn. Lag. Indeed, at Stn. Ind, 10% of the initial DOC was removed compared to only 7% at Stn. Lag.

The POC:PON molar ratios changed during the incubations. The C:N ratio in the Stn.

Ind incubation was initially higher than that of Stn. Lag (19.5 and 4.7, respectively; Table 1).

After 24 hours, the C:N ratio of Stn. Ind decreased to around 5 whilst that of Stn. Lag increased slightly to 8.6. Thereafter, the C:N molar ratio remained relatively constant for both incubations. At the end of the experiment the ratio was close to that of Redfield (7 and 6.2 for Stn. Ind and Stn. Lag, respectively). The only exceptions to this were on days 8 and 17 in Stn.

Lag, where exceptionally high values of over 30 were observed. Initial bacterial abundance was higher at Stn. Ind than Stn. Lag. In both incubation BA increased during the first 24 hrs and thereafter declined towards the end of the incubation (Fig. 1A). Initial bacterial production rates were low in the incubations (Fig. 1B). BP increased thereafter in the two incubations, with the increases in Stn. Ind being up to a factor of 5 higher than those of Stn. Lag. However, this peak in bacterial activity was transient and by day 5, the values had returned to low levels for both incubations. Respiration was initially low in Stn. Lag, peaked on day 3 and thereafter fell to near initial values (Fig. 1C). In contrast, in Stn. Ind, respiration was initially higher than in Stn. Lag and after a slight decrease on day 5, increased towards a maximum at day 32. The high bacterial production rates and relatively low bacterial respiration rates meant that bacterial growth efficiency (BGE) reached maximum values on day 1 in Stn. Ind (Fig. 1D). This was in contrast to that observed in the Stn. Lag incubation where BGE remained very low (<10%), with only a slight increase on day 1; BGE then decreased in both incubations to attain the same, low levels towards the end of the incubation. TEP-C concentration was very similar at Stn. Ind and Stn. Lag at the start of the experiment (3.1 and 2.7 µM C, respectively; Fig. 2A). The TEP-C concentration then increased rapidly in Stn. Lag on day 1 to represent 14% of total DOC concentration and thereafter decreased to levels lower than the initial concentrations of 4%. TEP-C concentration in Stn. Ind also initially increased however, the increase was much less dramatic than in Stn. Lag, reaching only 6% of total DOC as opposed to 4% at the start of the experiment. By the end of the experiment, TEP-C concentration represented only 0.1% of total DOC.

The number of beads that stick to TEP is a measure of the sticking properties of the particles (Fig. 3, Table 2). Despite similar initial TEP-C concentrations, the number of beads that were stuck to each TEP particle was 4 times higher in Stn. Lag than in Stn. Ind (Figs. 2B, 3, Table 2). However, this difference in sticking properties was transient and by day 2, there was no difference between the two stations. After day 2, the number of beads stuck to each TEP then increased towards the end of the experiment in both incubations to reach the same concentrations as was initially observed at Stn. Lag (Fig. 2B). Similarly, the sticking slope (a) which describes 'the slope of the sticking properties curve' (i.e. Fig. 3, Table 2) and is a measure of the ability of TEP to stick to the beads, was highest in Stn. Lag at the start of the experiment (Fig. 2C). By day 2, a had decreased to the same level as in Stn. Ind and both values then followed the same increasing trend towards the end of the experiment.

In order to assess the bioavailability of the organic matter and TEP-C for the two sites sampled, we examined the relationship between TEP-C concentration and BGE (Fig. 4). We observed an increasing linear trend between TEP-C concentration and BGE for Stn. Ind. The trend was less apparent for Stn. Lag. In other words, increases in TEP-C were related to higher increases in BGE in Stn. Ind than in Stn. Lag. If we consider that BGE can be used as proxy for bioavailability (Rochelle-Newall et al., 2004), the higher BGE per unit TEP observed in Stn. Ind suggests that TEP in this incubation was more bioavailable than that in Stn. Lag, despite there being a higher concentration of TEP in Stn. Lag.

Discussion

Previous work from the southwest lagoon of New Caledonia has shown that marked gradients in inorganic and organic matter concentrations exist between the outer lagoon and the inner bays [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF][START_REF] Rochelle-Newall | Phytoplanktonbacterioplankton coupling in a sub-tropical South Pacific coral reef lagoon[END_REF] and this has been shown to be related to the water residence times in this system [START_REF] Migon | Geochemical and hydrodynamic constraints on the distribution of trace metal concentrations in the lagoon of Noumea, New Caledonia[END_REF][START_REF] Torréton | Correspondence between the distribution of hydrodynamic time parameters and the distribution of biological and chemical variables in a semienclosed coral reef lagoon[END_REF].

In this work, we failed to see similar marked gradients despite large differences in the average flushing time at the two sites, 0.5 and 45 days for Stn. Lag and Stn. Ind, respectively [START_REF] Jouon | Calculations of hydrodynamic time parameters in a semi-opened coastal zone using a 3D hydrodynamic model[END_REF]. Indeed, the only bulk parameters that demonstrated a large difference were silicates, where we observed a factor of 6 difference in concentration between Stn. Ind and Stn. Lag (data not shown). The reason for the lack of difference in the other nutrients is probably due to the wind regime prior to sampling. The days preceding sampling were characterized by north westerly winds as opposed to the south easterly trade winds usually experienced in this site and such conditions generally favor an enhanced mixing of the water masses [START_REF] Pinazo | Impact of wind and freshwater inputs on phytoplankton biomass in the coral reef lagoon of New Caledonia during the summer cyclonic period: a coupled three-dimensional biogeochemical modeling approach[END_REF]. Nevertheless, even despite the relatively small differences in bulk parameters, particularly in organic carbon concentrations, differences were observed when we examined the characteristics of the organic matter in terms of sticking properties and in terms of bacterial growth efficiency, at least during the first hours of the incubation. BGE is a measure of both substrate properties and community capacity. Therefore, while it is clear that substrate quality is important, bacterial genetic functional diversity is also intrinsically linked with bioavailability [START_REF] Rochelle-Newall | Degradation of riverine dissolved organic matter by seawater bacteria[END_REF]). The differences observed in our experiments in BGE could therefore be due to available nutrient concentrations [START_REF] Kroer | Bacterial growth efficiency on natural dissolved organic matter[END_REF], the age and the origin of the organic matter [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF], or even the bacterial community composition present (Weinbauer et al., in press). Inorganic and organic nutrient limitation can have an impact on DOM bioavailability and BGE [START_REF] Kroer | Bacterial growth efficiency on natural dissolved organic matter[END_REF][START_REF] Kroer | Relationships between biovolume and carbon and nitrogen content of bacterioplankton[END_REF] particularly in sites that are nutrient limited, such as oligotrophic water.

Indeed, recently [START_REF] Jacquet | Responses of the phytoplankton communities to increased anthropogenic influences (Southwestern Lagoon, New Caledonia)[END_REF] showed that at some periods of the year it is probable that at the Lag station, nitrogen is the most limiting nutrient. However, given the small differences in inorganic nutrient concentrations observed here, it is unlikely that there was a large difference in limitation between the two sites.

Renewal rates, or flushing times, also play a role in determining the age and concentrations the DOM [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF]. The flushing time of Stn. Lag is on the order of 0.5 days and it is unlikely that large stocks of terrestrial organic matter are present in this water. Moreover, this site is adjacent to the coral reef barrier and it is probable that a proportion of the DOC measured was of coral origin. Corals are known to produce large amounts of mucus and this mucus is bioavailable to bacteria [START_REF] Moriarty | Temporal and spatial variation in bacterial production in the water column over a coral reef[END_REF][START_REF] Vacelet | Microbial utilization of coral mucus in long term in situ incubation over a coral reef[END_REF]Wild et al., 2004). [START_REF] Moriarty | Temporal and spatial variation in bacterial production in the water column over a coral reef[END_REF] found high glucosidase activities in coral mucus suggesting high carbohydrate concentrations. This may well explain the higher stickiness observed at the beginning of the incubation in the offshore station as the mucus is known to form aggregates and to enhance particle export (Wild et al., 2004). In contrast, Stn. Ind is located in a coastal bay that has a much longer flushing time (up to 45 days). The longer flushing time leads to the accumulation of older organic matter originating from a wider range of sources (terrestrial, aquatic as well as industrial and urban) than that of the lagoon site. Moreover, this inshore station is subject to industrial inputs from the nearby nickel smelt as well as from untreated urban wastewater and runoff. Recent work [START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF] has shown that the presence of metal can reduce the stickiness of TEP and it is known that in the Stn. Ind metal concentrations are higher than those of the lagoon site [START_REF] Migon | Geochemical and hydrodynamic constraints on the distribution of trace metal concentrations in the lagoon of Noumea, New Caledonia[END_REF].

Despite the initial differences in TEP stickiness, it is interesting to note that after day 2, there were no significant differences between the number of beads stuck to TEP or the sticking slopes (Figs. 2, 3 and Table 2). This seems to indicate that whatever differences existed in TEP sticking potential between the two sites disappeared rapidly and were no longer measurable after 24 hours suggesting a bacterially-mediated homogenization of the DOC composition of the available DOC fraction. Moreover, from day 2 onwards sticking properties increased in the incubations towards the end of the experiment. This seems to indicate that the sticking properties of TEP increase with the age of organic matter. This poses an interesting question as recent work from this system, based on in situ measurements has proposed that freshly produced organic matter was more sticky than older organic matter [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF]. This is in contrast to what we observed in these incubations, perhaps pointing towards an alternative source of organic matter such as bacterial recycling of organic matter. Bacteria are known to release dissolved organic compounds which can represent up to 25% of bacterial respiration in terms of carbon [START_REF] Stoderegger | Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton[END_REF]. This carbon is considered to be refractory and so it is probable that during batch incubations, such as those we performed, considerable accumulations of bacterially derived DOM occur, particularly in situations of low nutrient concentrations. Using the values of 15.2 amol C cell -1 h -1 of [START_REF] Stoderegger | Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton[END_REF], we calculated that the average rates of C release in the incubations varied between 4 and 19 pmol C h -1 and between 5 and 25 pmol C h -1 , for Stn Lag and Ind, respectively. These are just rough estimations of bacterial carbon release as our incubation set up differed from that of [START_REF] Stoderegger | Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton[END_REF], nevertheless, it gives us an estimate of the carbon transformation rates in the incubations. Over the period of the incubation, and if we consider that each C is used only once by the bacteria, we consider that approximately 5 to 6 nmol C were released by the bacteria. Considering that the final TEP-C content was 50 and 179nM C for Stn. Lag and Ind, respectively, we calculate that the potential contribution of bacterial C to TEP-C is of the order of 3 to 10 % of the total TEP-C pool.

We propose that this bacterially released DOM plays a role in the increased stickiness in the incubations. This reposes on the hypothesis of the existence of at least two types of "sticky-TEP". The first type is a by-product of primary production as proposed by [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF] and by [START_REF] Engel | The role of transparent exopolymer particles (TEP) in the increase in apparent particle stickiness (alpha) during the decline of a diatom bloom[END_REF] for in situ and algal cultures, and by Wild et al (2004) for coral reef mucus, respectively. This autotrophically produced sticky TEP is probably highly bioavailable and has a relatively short half life [START_REF] Engel | The role of transparent exopolymer particles (TEP) in the increase in apparent particle stickiness (alpha) during the decline of a diatom bloom[END_REF][START_REF] Grossart | Bacterial production and growth efficiencies: Direct measurements on riverine aggregates[END_REF]Wild et al., 2004), and as is evidenced by the rapid decrease in TEP sticking properties in Stn. Lag during the first 24 h of the incubation. This autotrophically produced sticky-TEP is subject to various factors that can alter its stickiness, such as the presence of heavy metals and changes in pH [START_REF] Mari | Does ocean acidification induce an upward flux of marine aggregates?[END_REF][START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF] and as pointed out by [START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF] this potentially explains why the sticking properties of TEP in Stn. Ind are lower than that of Stn.

Lag, despite higher slightly initial nutrient and chlorophyll a concentrations.

In contrast to the autotrophic production of sticky TEP, the second type of sticky TEP is heterotrophically produced through bacterial transformation of existing DOM. This TEP fraction accumulates within the bulk TEP pool with time and increases the overall sticking properties of the TEP pool, similar to the accumulations of bacterially produced CDOM in batch cultures [START_REF] Rochelle-Newall | Production of chromophoric dissolved organic matter fluorescence in marine and estuarine environments: an investigation into the role of phytoplankton[END_REF]. Active bacteria produce mucopolysaccharidic capsular material [START_REF] Luft | Ruthenium Red and Violet .1. Chemistry, Purification, Methods of Use for Electron Microscopy and Mechanism of Action[END_REF][START_REF] Stoderegger | Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton[END_REF] and this material forms aggregates and due to its polysaccharide content, is stained by Alcian Blue and is therefore included in the TEP pool. Moreover, even in the absence of freshly produced photosynthetic products, bacteria continue to produce this capsular material which accumulates in the water column due to its relatively recalcitrant nature [START_REF] Stoderegger | Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton[END_REF]. Thus effectively forming a shunt of semi-labile organic matter towards the refractory and hence weakly bioavailable, DOM pool.

While it is clear that we cannot rule out abiotic production of sticky TEP in our incubations, it is clear that despite differing initial DOM physico-chemical qualities, the final stickiness was similar. It is already known that metals can alter TEP stickiness [START_REF] Mari | Metal induced variations of TEP sticking properties in the southwestern lagoon of New Caledonia[END_REF] and that sticky TEP is produced by autotrophic processes (e.g. [START_REF] Engel | Polysaccharide aggregation as a potential sink of marine dissolved organic carbon[END_REF][START_REF] Mari | Water residence time: A regulatory factor of the DOM to POM transfer efficiency[END_REF]Wild et al., 2004). Here we suggest that the hypothesis of a heterotrophic source of sticky TEP clearly merits further investigation. This potential pathway of organic carbon transformation further highlights the fact that bacteria should not just be considered as remineralisers of DOM but also as transformers of DOM in aquatic systems.

The biogeochemical impacts of these transformations in terms of vertical transport of particles, particularly below the euphotic zone, away from autotrophic DOM production, need to be considered. Table 1. Organic and inorganic nutrient and dissolved organic carbon concentrations (DOC) and the C:N molar ratio of the dissolved organic fractions for both sample sites. Chla : extracted chlorophyll a; DIN : dissolved inorganic nitrogen; DON : dissolved organic nitrogen; DIP : dissolved inorganic phosphorus; DOP : Dissolved organic phosphorus; sd : standard deviation of the analytical replicates.

Stn

Figure 1 .

 1 Figure1. Bacterial abundance, respiration, production and growth efficiency in the incubations. A: bacterial abundance (BA, 10 5 cells/ml); B: bacterial production (BP, µmol C l -1 h -1 ); C: respiration (R, µmol C l -1 h -1 ) and D: bacterial growth efficiency (BGE, %). Open circles: Stn. Lag; Filled circles: Stn. Ind. Note log scale on x-axis.

Figure 2 .

 2 Figure 2. Relationship between BGE (%) and BP (µmol C l -1 h -1 ). A: Stn. Lag (open circles, r 2 = 0.961, p=0.006); B: Stn. Ind (filled circles, r 2 = 0.99, p< 0.0001. Note the log scale on the x-axis. Figure deleted

Figure 2 .

 2 Figure 2. Concentration and sticking properties of TEP (transparent exopolymeric particles). A: concentration of TEP (µmol L -1 C); B: number of beads per TEP (10 -3 µm -3 ); C: sticking slope (a). Open circles: Stn. Lag; Filled circles: Stn. Ind. Note log scale on xaxis.

Figure 3 .

 3 Figure 3. Relationship between the number of beads attached to each TEP and the equivalent spherical diameter of TEP for the duration of the experiment. Statistical details of the regressions are given in Table 2. Left side column: Stn. Ind, right side column Stn.Lag.

Figure 4 .

 4 Figure 4. Relationship between TEP-C concentration and BGE. Open circles: Stn. Lag; Filled circles: Stn. Ind.

Table 2 .

 2 Statistical parameters of the stickiness regression calculations.

	. Ind

Acknowledgements

. This research was financed by the PNETOX program of the French Ministry for Ecology and Sustainable Development (Ministère de l'Ecologie et du Développement Durable), the French National Research Agency (ANR-BLANC program: MAORY project), the Ministry of Overseas Territories (Ministère de l'Outre Mer) and the French Research Institute for Development (IRD).