Sarah Alami

Laurène Courouve
email: laurene.courouve@cemka.fr

Guila Lancman

Pierrette Gomis

Gisele Al-Hamoud

Corinne Laurelli

Hélène Pasche

Gilles Chatellier

Grégoire Mercier

François Roubille

Gisele Al- Hamoud

Cécile Delval

Isabelle Durand-Zaleski

Organisational Impact of a Remote Patient Monitoring System for Heart Failure Management: The Experience of 29 Cardiology Departments in France

Keywords: heart failure, remote patient monitoring, organisational impact, health technology assessment, ambulatory, emergency department

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Chronic heart failure (CHF) is a global pandemic that currently affects the lives of 64 million people worldwide [START_REF] Gbd | Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2017[END_REF] and 2.3% of the French population [START_REF] De Peretti | Prévalences et statut fonctionnel des cardiopathies ischémiques et de l'insuffisance cardiaque dans la population adulte en France: Apports des enquêtes déclaratives Handicap-Santé[END_REF]. The condition is characterised by a high mortality rate in the post-discharge period and a high likelihood of hospital readmission for acute, decompensated heart failure [START_REF] Gabet | National trends in rate of patients hospitalized for heart failure and heart failure mortality in France, 2000-2012[END_REF][START_REF] Tuppin | Two-year outcome of patients after a first hospitalization for heart failure: A national observational study[END_REF]. Remote patient monitoring (RPM) systems (such as Chronic Care ConnectTM (CCC TM ) e-health solution) are now emerging as additional tools for CHF care management.

In France, a national scheme for promoting and funding RPM (Expérimentations de Télémédecine pour l'Amélioration des Parcours en Santé (ETAPES)) was launched in 2014. For inclusion in ETAPES, RPM systems must meet strict specifications and must combine a digital device, medical tele-monitoring and therapeutic support [START_REF]Direction Générale de L'offre de Soins. Étapes Télémédecine, Rapport au Parlement, Evaluation des Expérimentations de Télésurveillance du Programme National Etapes[END_REF]. Although an evaluation of the device's clinical benefit, impact on quality of life, and economic impact is obligatory, other consequences must also be explored-notably with regard to access to care, quality of care, and care organisation [START_REF]Intégration de la Cartographie Relative à L'impact Organisationnel Pour L'évaluation des Technologies de Santé[END_REF]. Until now, these evaluations were mainly focused on assessing the medical and economic impacts [START_REF] Farnia | Evaluation Criteria of Noninvasive Telemonitoring for Patients With Heart Failure: Systematic Review[END_REF]. The assessment of a device's organisational impact has lagged behind the other assessments because of the lack of a specific methodology and guidelines [START_REF]Health Technology Assessment Handbook[END_REF]-even though this is essential for fully appraising the influence of medical technologies [START_REF] Roussel | Impact organisationnel: Définition et méthodes d'évaluation pour dispositifs médicaux[END_REF].

In order to tackle this problem and include the organisational impact in the assessment process, the French National Authority for Health (Haute Autorité de Santé (HAS)) published a guide in December 2020 [START_REF]Cartographie des Impacts Organisationnels Pour L'évaluation des Technologies de Santé[END_REF]. The guide gave a definition of organisational impact ("an effect or consequence of the health technology on the characteristics and functioning of an organisation involved in the care process or the user's life pathway") and suggested criteria for measuring and justifying the RPM device's effects in this respect. The organisational impact has now been included in the criteria evaluated by the French National Medical Device and Health Technology Evaluation Committee (Commission Nationale d'Evaluation des Dispositifs Médicaux et Technologies de Santé (CNEDiMTS)) [START_REF]Intégration de la Cartograhie Relative à L'impact Organisationnel dans les Critères D'évaluation CNEDiMTS-Note de Cadrage[END_REF].

The organisational impact can be documented from various perspectives (that of the patient, the healthcare professional, the healthcare system, the hospitals, etc.) and by using various methods and data sources. The objective of the present study was to describe the organisational impact of CCC TM on CHF management from the perspective of healthcare professionals using the device.

Methods

Survey Design

An online survey was conducted among 31 French cardiology departments (CDs) known to use CCCTM for CHF management. The questionnaire was sent to all 31 public or privatesector institutions known to have monitored at least 20 patients with CCCTM between 2018 and 2020.

Data were collected between 16 April and 26 April 2021 using an online questionnaire. In each CD, only one questionnaire was filled; the survey was designed to be completed by the team as a whole.

Chronic Care Connect TM

Chronic Care Connect TM is a remote patient monitoring (RPM) solution for heart failure (HF) management composed of a class IIA medical device and non-medical human assistance. A connected scale allows daily weight collection, and a mobile application allows patients recording of HF symptoms. One of the CCC TM 's unique features is the integration of this new, non-hospital-based stakeholder: a monitoring centre whose nurses are specially trained in remote monitoring and initially screen the alerts received. If an alert is judged to be relevant, it is sent to the patient's CD.

Study Measurements and Outcomes

The entire questionnaire was based on the organisational impact map for health technology assessment when the standards were applicable [START_REF]Cartographie des Impacts Organisationnels Pour L'évaluation des Technologies de Santé[END_REF]. This mapping process is a structured way of identifying and quantifying organisational impacts, in which three macro-criteria are divided into several sub-criteria.

For macro-criterion 1, the various sub-criteria covered time consumption, the speed or duration of care processes, the equipment and infrastructures used in the RPM process, and the skills needed so that the stakeholders could implement the care process. For macro criterion 2, the sub-criteria were related to stakeholder training and skill transfers. Lastly, for macro-criterion 3, the sub-criteria covered impacts related to communication, society, and the environment.

For each sub-criterion, an assessment of the potential impact of CCC TM was performed (Table 1). When the sub-criterion was not relevant for CCC TM , "not applicable" was stated in the questionnaire. To assess the organisational impact of each sub-criterion, several data sources and methods are used, including this survey among health professionals. All the results only concern the organisational impact of CCC TM .

Statistical Analysis

Most of the statistical analyses were descriptive. All statistical analyses were performed with SAS® software (version 9.4, SAS® Institute Inc., Cary, NC, USA).

Results

Participants

A link to the online survey was sent to the 31 CDs equipped with the CCC TM device and which had sufficient experience of its use (i.e., RPM with at least 20 patients). Of the 31 CDs contacted, 29 (94%) completed the study questionnaire. In most CDs, several different healthcare professionals answered the survey: most were cardiologists (27 out of 29) and nurses (18 out of 29). The response rates per question were 100% unless otherwise stated.

Characteristics of the Participating Cardiology Departments

From the system's date of deployment in France (1 February 2018) to the survey closure date (26 April 2021), the 29 participating CDs monitored 63% of the patients remotely monitored by CCC TM over the same time frame. On average, 122 patients per CD were monitored (Table 2). Twenty-four of the CDs (83%) were in public-sector institutions, and five (17%) were in private-sector institutions. The CDs had used the device for an average of 23 months; 14 (48%) had used it for more than 24 months, and 7 (24%) had used it for less than 12 months.

Impacts of the Health Technology on the Care Process (Macro-Criterion 1)

Impact on the Initiation of the Care Process

Healthcare professionals were asked about the time interval between cardiac decompensation and the initiation of medical care. Most of the respondents declared that RPM for patients with CHF was associated with a shorter time interval: 21 of the 29 (72%) CDs "absolutely agreed" and 7 (24%) "mostly agreed".

Impact on the Pace or Duration of the Process

The use of RPM might modify the process pace and overall duration of the care process as it includes alert management, which necessitates additional time. Alert management includes several phases, acknowledging the alert, making a diagnosis, responding to the alert, and triggering the intervention (Table 3). The survey results showed that nurses performed most of the tasks in alert management: nurses were primarily involved in overseeing the first phase (acknowledgement of the alert) in 23 of the 24 (96%) CDs, whereas cardiologists were primarily involved to a lesser extent (in 16 of the 29 CDs (55%)).

Twenty-two of the twenty-nine CDs (76%) chose to deal with the alerts Monday to Friday, during office hours. In the seven other CDs, the various management procedures were explained by the low number of remotely monitored patients. Depending on the number of patients and the type of healthcare professional involved, alert management may require dedicated time. In CDs with less than 50 patients being remotely monitored, the average time spent on this task was 4.1 h per week for nurses and 1.3 h per week for cardiologists. In CDs with more than 50 patients being remotely monitored, the average time spent was 14.3 h per week for nurses and 1.3 h per week for cardiologists.

Regarding the overall duration of the care process, RPM was initially prescribed for six months. In 28 of the 29 CDs (97%), this prescription was renewed. In 12 CDs (41%), the proportion of patients with a renewed prescription was over 80%. Renewal of the prescription was prompted by unstable disease (in 23 of the 29 CDs (79%)) or a patient request (again in 23 of the 29 (79%)).

Impact on Process Timing or Content

The survey results demonstrated that all participating CDs had specifically changed their organisational structure in response to alert management and thus changed the care process content (Figure 1). Sixteen of the twenty-nine CDs (55%) had specifically allotted time for outpatient consultations after an emergency alert. Fourteen out of sixteen (88%) had started the consultations at the same time as (or shortly after) the introduction of RPM. Eighteen of the CDs (62%) had created an organisational structure dedicated to CHF medication titration [START_REF] Greene | Titration of Medical Therapy for Heart Failure with Reduced Ejection Fraction[END_REF]; only a third of the CDs had implemented it before RPM deployment. In 16 of the 18 CDs (88%), the medication was titrated during a face-to-face consultation. However, six of the 18 had the option to do it over the phone.

To avoid admission to the emergency department (ED), 25 of the 29 CDs (86%) set up a procedure for direct emergency admission to the CD. In 21 of the 25 CDs (84%), this system had been implemented at the same time as (or shortly after) the introduction of RPM.

Figure 1. Specific organisational structures and procedures implemented in cardiology departments for alert management after decompensation of HF. * Titration of the dose of a drug in order to maximise its effectiveness without increasing the risk of adverse drug events, as recommended for patients with HF [START_REF]Intégration de la Cartograhie Relative à L'impact Organisationnel dans les Critères D'évaluation CNEDiMTS-Note de Cadrage[END_REF][START_REF] Greene | Titration of Medical Therapy for Heart Failure with Reduced Ejection Fraction[END_REF].

Impact on the Organisation of Human Resources

The implementation of RPM for patients with CHF requires qualified, trained human resources. In 24 of the 29 CDs (83%), a dedicated RPM team had been set up. Two CDs out of five (40%) stated that they were not monitoring enough patients for a dedicated team, and two others (40%) declared that they lacked funding.

Whether or not a team was assigned, in all questioned CDs, at least one cardiologist was involved in RPM, and at least one nurse was involved in 24 of the 29 CDs (83%). On average, 1.3 healthcare professionals (full-time equivalents (FTEs), and regardless of their role) per CD were involved in RPM (median: 1). For the period from June 2019 to June 2020, the mean number of remotely monitored patients per FTE healthcare professional was 74 (median: 38; range: 11-340).

Impact on the Allocation of Materials and Equipment

Along with human resources, the survey also included questions on equipment. According to the healthcare professionals, the use of RPM did not require any additional equipment because CCC TM relied on the CD's existing computers (according to 18 of the 24 (75%) healthcare professionals who answered this item), and the CD had implemented a dedicated telephone line (according to 16 of the healthcare professionals). Only four CDs (14%) had funded the creation of a dedicated remote monitoring room.

Impact on the Continuity of Care

The healthcare professionals were also asked about the impact of RPM on the continuity of care and mainly reported difficulties when team members were on leave. Twenty of the 29 (76%) respondents declared difficulties in ensuring care continuity: 16 (55%) encountered some difficulties and 4 (14%) encountered many difficulties).

Impacts of the Health Technology on the Abilities and Skills Required of Stakeholders to Implement the Care Process (Macro-Criterion 2)

Impact on the Skills Required of Stakeholders

To manage RPM in a CHF setting, for both healthcare professionals and patients training was required [START_REF] Mcdonagh | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: Developed by the Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) With the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF]. Patients were mainly trained to use the device (i.e., the smart scale and the tablet computer) but also had access to a disease management program supervised by a nurse to help them manage their disease daily.

According to the survey respondents, this training took an average of 4 h per patient over a 12-month period. Thus, training in disease management for 50 patients would take a total of 6 weeks per FTE healthcare professional.

Most of this training was delivered by nurses (in 22 of the 29 CDs (76%)). Disease management training was sometimes delivered by the device supplier (11 out of 29 (38%)), but this was mainly due to a lack of human resources in the CDs (mentioned by 10 out of 11 respondents (91%)).

In 19 of the 29 CDs (66%), some or all of the healthcare professionals involved in RPM received specific training. In some cases, this training was delivered as part of continuing professional development on CHF or disease management.

Impact on Physician-to-Nurse Delegation of Duties

In 15 of the 29 CDs (52%), the organisational structure implemented for RPM allowed cardiologists to delegate certain medical procedures to nurses. In 6 CDs (21%), task delegation was part of a collaborative agreement with the health authorities: for example, nurses were allowed to manage alerts, refer patients, and perform follow-up consultations for medication titration in the absence of a cardiologist. In the other 9 CDs (31%), task delegation was mostly limited to alert screening (mentioned five times), the prescription of standard laboratory tests (mentioned three times) and changes in treatment supervised by a cardiologist (mentioned three times).

The device also demanded skills transfers: the nurses contributed significantly to the administrative work and the coding of medical procedures.

Impact on the Coordination between the Stakeholders

A New, Non-Hospital-Based Stakeholder

One of CCC TM 's specific features is the introduction of a new, non-hospital-based stakeholder into the care process. Twenty-six of the twenty-nine (90%) survey participants stated that the existence of a monitoring centre with a dedicated team of nurses was the main reason for choosing CCC TM . The respondents considered that the alerts screened by the monitoring centre were relevant (mean relevancy score: 8.2 out of 10).

The equipment provided and the manufacturer's experience in the field of RPM were also mentioned as factors that facilitated the implementation of this new organisational structure (according to 22 (76%) and 17 (59%) CDs, respectively).

Coordination between Ambulatory Care and Hospital Care

RPM requires coordination between hospital stakeholders and those in ambulatory care, including general practitioners (GPs). The respondents noted that GPs did not have direct access to the RPM software. The GPs were sent updates about the patients' follow-up by the hospital staff-usually by e-mail (according to 22 of the 28 respondents (79%)) or by phone (22 out of 28 (79%)). The respondents stated that the implementation of RPM helped to improve coordination with primary care: 8 of the 29 (28%) agreed absolutely with this statement, and 11(38%) agreed somewhat.

Impact on Healthcare Professionals' Working Conditions and the Patients' Quality of Life

Fourteen of the twenty-nine (48%) respondents agreed that RPM could also impact the healthcare professionals' working conditions. All fourteen considered that the organisational structure implemented for RPM helped to improve the healthcare professional patient relationship, gave the healthcare professionals more independence, and increased their level of satisfaction. However, twelve of the fourteen (86%) respondents also reported a decrease in the speed and quantity of work to some extent.

According to the surveyed healthcare professionals, RPM had a positive impact on the patient's quality of life and level of satisfaction: 16 of the 29 (55%) respondents considered that the quality of life was very much better, and 11 (38%) considered that quality of life was somewhat better.

Discussion

The present survey of hospital-based healthcare professionals' opinions provided a description of the organisational implementation of CCC TM for RPM for CHF management. This survey stands out because it used a standardised method to directly address all the organisational impacts (whether positive or negative) perceived by a representative panel of professionals with significant expertise in CHF management and RPM. Furthermore, it should be possible to apply this method to other health technologies in other contexts. Whereas, in previous studies, the organisational impact was less explored and poorly documented, partially due to the lack of a standardised method. Most studies on the impact of health technologies have only referred to certain organisational impacts, such as the effects on human resources and organisational structures [START_REF] De Vries | Health professionals' expectations versus experiences of internetbased telemonitoring: Survey among heart failure clinics[END_REF][START_REF] Seto | Attitudes of heart failure patients and health care providers towards mobile phone-based remote monitoring[END_REF].

The results show that the CDs restructured their working practices over time. Most had implemented a new organisational structure upon or shortly after the implementation of RPM for decompensations of CHF. Twenty-four of the CDs (83%) had created a dedicated RPM team. More than half of the CDs had created specific outpatient consultations for patients with an emergency alert; in fourteen of the sixteen (88%), this initiative was taken upon or shortly after the introduction of RPM. Eighteen of the twenty-nine CDs (62%) had introduced specific procedures for medication titration; twelve of the eighteen (67%) did this after RPM had been implemented. Twenty-five of the twenty-nine CDs (86%) admitted patients directly and thus avoided an ED admission. Even though the CDs had changed their organisational structure over time, the survey results highlighted differences from one department to another. These differences were mainly related to each team's level of familiarity with RPM and the number of remotely monitored patients. Healthcare professionals were generally satisfied with the introduction of a new stakeholder (a monitoring centre with a dedicated team of nurses) for some care management tasks. Moreover, RPM does not demand a substantial investment in equipment; only computers and a phone line are needed to manage the patients' alerts. However, in the short term, RPM requires dedicated staff time to be set aside, and this can differ from one healthcare professional to another. Cardiologists spent an average of 2 h a week on alert management, whereas nurses spent an average of 10 h a week on the same task. Nevertheless, the literature data show that in the long term, RPM with an appropriate organisational structure is associated with shorter lengths of stay in the ED and in hospitals in general, which might free up time for patients and hospital staff [START_REF] Johnson | Remote monitoring for heart failure: Assessing the risks of readmission and mortality[END_REF]. Our present survey also demonstrated that RPM led to more training and more skill transfer for the participating healthcare professionals. Lastly, the healthcare professionals stated that the changes in organisational structure improved the quality of patient care. Most of the respondents reported that care provision took less time, with a smoother care pathway and better quality of life for the patients.

The main limitation of the present study is embedded in its design; the results reflected the opinions of hospital-based healthcare professionals of a relatively small sample of French CDs. Indeed, for this study, only CDs already using CCCTM were targeted, which is already limiting the survey pool. Among these CDs using the device, only those with at least 20 patients were included. Although it was the most relevant choice in this situation, the results cannot be extrapolated to RPM devices in general. Moreover, as there is no scoring system in the questionnaire, it is impossible to have a hierarchy of the mentioned organisational impacts. Secondly, some of the HAS' criteria could not be adapted for the assessment of CCC TM . The "mapping" technique is a robust tool for identifying organisational impacts but cannot be used directly to assess health technology devices. Lastly, impacts that were not thought to be relevant to CCC TM were not assessed (i.e., macro-criterion 3).

Given the great variety of organisational structures highlighted by our survey, it will now be necessary to assess the corresponding clinical and economic impacts on patients with CHF and to disseminate the best-performing practices. To this end, a quantitative study is currently performed using the French national healthcare database (Système National des Données de Santé), which should complement the qualitative data on healthcare professionals' perceptions collected in the present study. The quantitative study will enable the assessment of other organisational impacts (such as the hospital readmission rate and the lengths of stay in the ED or in the hospital, as the clinical and economic impacts) as a function of the CD's organisational structure and human resources.

Conclusions

The present survey is the first to have assessed the organisational impact of the implementation of the CCC TM RPM device for CHF management. The results highlighted the variety of organisational structures, which tends to structure with the use of the device.

Acknowledgments:

The authors thank Max Rene and Pierre-Emmanuel Augustin (CDM eHealth, Air Liquide Santé International (Bagneux, France)) for their assistance with this project. We would also like to thank all the cardiologists and nurses who completed the survey.

Funding:

This research was funded by Air Liquide Santé International (Bagneux, France).

Informed Consent Statement:

Not applicable.

Data Availability Statement:

Data is unavailable due to privacy or ethical restrictions.


Lory-Anne Baker
email: l-anne.baker@doctorant.uca.fr

Aude Beauger

Sofia Kolovi

Olivier Voldoire

Elisabeth Allain

Vincent Breton

Patrick Chardon

Didier Miallier

Céline Bailly

Gilles Montavon

Agnès Bouchez

Frédéric Rimet

Cécile Chardon

Valentin Vasselon

Luc Ector

Carlos E Wetzel

David G Biron

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities

Keywords: diatom diversity, mineral springs, naturally occurring radioactivity, rbcL, metabarcoding, genetic variants

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities

Lory-Anne Baker, Aude Beauger, Sofia Kolovi, Olivier Voldoire, Elisabeth Allain, Vincent Breton, Patrick Chardon, Didier Miallier, Céline Bailly, Gilles F Montavon, et al.

To cite this version:

Lory-Anne Baker, Aude Beauger, Sofia Kolovi, Olivier Voldoire, Elisabeth Allain, et al.. Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities. Science of the Total Environment, 2023Environment, , 873, pp.162270. 10.1016Environment, /j.scitotenv.2023.162270.162270. hal-04033005

Introduction

Since the formation of the Earth some 4.5 billion years ago, background radiation levels have varied spatially and temporally in the habitats of the planet [START_REF] Karam | Changes in terrestrial natural radiation levels over the history of life[END_REF]. For at least 3.5 billion years, life has been evolving and adapting in ecosystems with different levels of natural radioactivity [START_REF] Martell | Radionuclide-induced evolution of DNA and the origin of life[END_REF][START_REF] Garzón | Radioactivity as a significant energy source in prebiotic synthesis[END_REF]. Currently, there are regions on the Earth where natural radioactivity can reach very high levels, such as India (Mohanty, 2004), Iran (Khodashenas, 2012) and Brazil [START_REF] Oliveira | Natural radioactivity in mineral spring waters of a highly radioactive region of Brazil and consequent population doses[END_REF]. In Europe, median concentration of uranium in soils is estimated to be around 2 mg.kg -1 , with a range of variation from less than 0.1 to more than 50 mg.kg -1 (De Vos et al., 2006). In France, the highest concentrations are found in the Massif Central (a mountain range in the middle of France), where it can reach hundreds of mg.kg -1 in granitic soil (Garnier-Laplace, 2010).

In the Massif Central, several mineral springs show significant levels of natural radioactivity [START_REF] Jacquet | Contribution à l'étude de la radioactivité des eaux minérales et du magnétisme des roches volcaniques du département du Puy-de-Dôme [Contribution to the study of mineral waters and magnetism of volcanic rocks in the department of Puy-de-Dôme[END_REF][START_REF] Boineau | Les sources minérales du Massif Central français et leur cadre géologique[END_REF][START_REF] Risler | Description et classification géologique des sources minérales et thermales du Massif Central [Description and geological classification of the mineral and thermal springs of the Massif Central[END_REF][START_REF] Ielsch | Estimation and mapping of uranium content of geological units in France[END_REF]. This natural radioactivity comes from 40 K and the decay chains of three primordial radioelements 235 U, 238 U and 232 Th that are naturally present in the Earth's crust, especially in granitic rocks [START_REF] Métévier | L'uranium de l'environnement à l'homme[END_REF]. Because of its longer half-life (4,47 billion years), 238 U and its nuclear decay products ( 226 Ra, 222 Rn, 210 Po) are today the most dominant radionuclides in mineral springs [START_REF] Erőss | Uranium in groundwater-The importance of hydraulic regime and groundwater flow system's understanding[END_REF][START_REF] Rihs | Long-term behaviour of continental hydrothermal systems: U-series study of hydrothermal carbonates from the French Massif Central (Allier Valley)[END_REF][START_REF] Morais | Use of Biomphalaria glabrata as a bioindicator of groundwater quality under the influence of NORM[END_REF]. They are α-particle emitters, the most mutagen ionising radiation involved in radioactive stress mainly related to internal irradiation in cells. Thus, mineral springs in the French Massif Central are not only extreme environments due to their significant mineralisation levels, but also their high level of natural radioactivity.

Living organisms that inhabit these extreme environments have been evolving and adapting to these conditions. Such mineral springs are therefore interesting 'natural laboratories' in which to study the influence of mineralisation and natural radioactivity on biodiversity. These ecosystems are characterised by short food webs which could be interesting to study the effects of natural radioactivity on primary producers. Diatoms are part of the phytobenthos, and one of the main groups of primary producers in aquatic ecosystems, including mineral springs. Algae are known to be good indicator for radionuclides in aquatic environments and show differences in radionuclides tolerances between species due to their ecological variations (Pradhan, 2019;[START_REF] Banerjee | Bioremediation of uranium from waste effluents using novel biosorbents: a review[END_REF].

The influence of radioactivity on diatoms can be studied at three ecological organizational levels, viz. individual, population and community levels [START_REF] Stevenson | Ecological assessments with algae: a review and synthesis[END_REF][START_REF] Pandey | The use of diatoms in ecotoxicology and bioassessment: Insights, advances and challenges[END_REF].

Gross and Koczy, (1946) (cited in [START_REF] Driver | Ecotoxicity literature review of selected Hanford Site contaminants[END_REF] reported a severe reduction of diatom survival in a radioactive and experimental medium. In addition, some surveys have already been conducted to study the impact of radioactivity on the communities of diatoms in tailing waters (Ruggles, 1979cited in Driver, 1994), brackish waters [START_REF] Snoeijs | Benthic diatoms as monitoring organisms for radionuclides in a brackish-water coastal environment[END_REF], and in rivers [START_REF] Herlory | Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge 25 on a stream: case study of the Ritord watershed (Center-West France)[END_REF] and mineral springs (Millan, 2019). High diatom species richness (>50) was found in springs with significant levels of radioactivity by Millan et al. (2019). This study also showed that high levels of 222 Rn activities in mineral springs are correlated with higher rates of benthic diatom frustule deformations. All of these studies suggest that diatoms can resist high radioactivity levels but did not provide any information on the effect of radioactivity on the structure of diatom communities themselves.

In the present study, and in order to bring new insights at both population and community levels, DNA metabarcoding was employed to assess the effect of enhanced natural radioactivity of water and sediments on benthic diatom communities in several mineral springs of the Massif Central. To this end, a 312 bp region of the chloroplast gene rbcL (encoding for the Ribulose Bisphosphate Carboxylase Large) was used as a barcode for diatom species identification [START_REF] Vasselon | Application of highthroughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter ?[END_REF], a convenient marker for such discriminations [START_REF] Mann | DNA barcoding for species identification and discovery in diatoms[END_REF][START_REF] Kermarrec | A next-generation sequencing approach to river biomonitoring using benthic diatoms[END_REF][START_REF] Bailet | Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization[END_REF]. Following on from earlier published results of Millan et al. (2019), where species richness was high even in radioactive mineral springs, our main working hypothesis was that radioactivity is not a major factor that influences diatom genetic richness. Furthrmore, because microbial species have different levels of resistance to ionising radiation [START_REF] Shuryak | Review of microbial resistance to chronic ionizing radiation exposure under environmental conditions[END_REF], our second hypothesis was that radioactivity can influence diatom community structure because ionising radiation can shape the microbial community. In this light, a series of studies were performed in order to test these ideas empirically.

Material and methods

Study sites and sampling

The sampling campaign took place in autumn from the 15 th to the 30 th October 2019, where diatom samples were collected from a total of 16 mineral springs distributed in the Auvergne region of the French Massif Central (Fig. 1). The French Massif Central (over 85,000 km 2 ) is influenced by oceanic, continental, and Mediterranean climates. It is composed of mountain reliefs, plateaus, and volcanoes (quiescent), where the Hercynian platform appears at the surface, surrounded by different sedimentary basins. Classically, the many faults in the massif allow the circulation of water in the rocks and the emergence of this having travelled some distance across the earth's crust. The study sites (mineral springs) present a diversity of physical, chemical, and radiological characteristics including a three orders of magnitude gradient of natural radioactivity.

Sampling of diatom communities

Benthic diatom samples were collected in the 16 mineral springs (Fig. 1). Since it was earlier demonstrated that the substrate has little influence on benthic diatom composition in mineral springs [START_REF] Baker | The substrate, a key factor or not, to explain the species diversity of diatom communities in mineral springs[END_REF], the biofilm was recovered and considered to be the dominant substrate, representative of each of the springs sampled. In some springs, biofilm was collected by scrubbing the upper surface of rocks over which water flowed with a clean toothbrush. In others, biofilm was sampled by scraping the first millimetre or so of sediments from the spring basin.

For each sample, the suspension in the was fixed with using ≥70% ethanol as required by

European protocols for diatom metabarcoding procedures [START_REF] Cen | Water quality -CEN/TR 17245 -Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses[END_REF].

Physical and chemical analyses

At the 16 spring sampling sites, water temperature (°C), conductivity (µS.cm -1 ) and pH (pH unit -Sorenson scale) were measured with a multi-parameter WTW probe FC 340i (VWR International, Radnor, PA, USA) by submerging the probe in the water. The percentage of dissolved oxygen (DO) was obtained with an Ysi ProODO oxygen probe (Yellow Springs Instruments, OH, USA). For chemical analyses, 200 mL of water was initially filtered with Whatman GF/C filters. For cation and anion analysis, a Thermo Scientific Dionex ICS1100 and a Thermo Scientific Dionex Aquion systems were used, respectively, to measure concentrations of the following ions (expressed as mg.L -1 ): lithium (Li + ), sodium (Na + ), ammonium (NH4 + ), potassium (K + ), magnesium (Mg 2+ ), calcium (Ca 2+ ), fluoride (F -), chloride (Cl -), nitrite (NO2 -), nitrate (NO3 -) and phosphate (PO4 3-).

Radiological and trace elements characterization

The activity of dissolved 222 Rn in water samples collected using 1 L Marinelli beakers was measured on a well-shaped Germanium detector (GCW3523, Canberra, 35% relative efficiency) operated at Clermont Physics Laboratory (University Clermont Auvergne, Aubière, France) upon returning from the field. For other radiological analyses, water, and sediment samples were collected in clean vials from the water-column either near the emergence or at its outlet. Samples were transported to the laboratory under cold conditions in refrigerated boxes protected from light and heat.

Analyses were conducted at Subatech (Nantes, France) and ICN (Nice, France) laboratories.

After PTFE filtration (0.45µm) and acidification of water samples (HNO3 or HCl for Po analysis), uranium was analysed by ICP-QMS (Xseries 2, Thermo Electron). Ultra-trace concentrations of 226 Ra were measured without chemical purification by HR-ICP-MS in lowresolution mode (Element XR, Thermo Scientific) with an APEX-Q high sensitivity desolvating sample solution introduction system. In order to remove major polyatomic interferences from the spectrum of 226 Ra in low-resolution mode thereby potentially leading to inaccurate assessment of concentrations of this the commonest isotope of this highly radioactive element, preliminary water sample solution analyses were also performed in medium resolution mode where major interferences can be resolved. The observed consistency of 226 Ra activity measured in both low and middle resolution modes showed no significant evidence of the production of major interferences [START_REF] Verlinde | A new rapid protocol for 226 Ra separation and preconcentration in natural water samples using molecular recognition technology for ICP-MS analysis[END_REF]. Preference for use of the low-resolution mode was characterised by a high sensitivity (4.7×105 CPS/Bq. mL -1 ) and a low instrumental detection limit (0.8 mBq.L -1 ) for 226 Ra. Pb and Bi-based polyatomic interferences unresolved in low and medium resolution modes were not considered in the present study due to low signal intensities measured for Pb (< 60,000 CPS) and Bi (< 1 000 CPS). Lastly, 210 Po was measured after spontaneous deposition onto a silver disc, by alpha spectrometry using dual alpha spectrometers EG&G Ortec 576A equipped with boron-implanted silicon detectors [START_REF] Le | 210 Po sequential extraction applied to wetland soils at uranium mining sites[END_REF].

Sediment samples were sieved to 2mm and dried at 105°C. 226 Ra and 228 Ac were analysed using a γ-Spectrometer with coaxial hyperpure Ge detector (GX4519, Mirion, >45% relative efficiency at 1.33 MeV between 10 keV to 10 MeV) according to NF EN ISO 18589-3.

Measurements were conducted at least four weeks after the sealing of the sample container to ensure the secular equilibrium between 226 with concentrated HNO3, centrifugation at 2650g during 10 min, then PTFE filtration (0.45µm).

DNA extraction and PCR amplification for amplicons library preparation

For each mineral spring, two sub-samples (duplicates) of 2 mL of biofilm were taken for DNA extraction. After homogenization by manually shaking of the raw material, samples were centrifuged at 13 000 rpm for 30 min. at 4°C to remove the supernatant (ethanol). Total genomic DNA was extracted from the pellet with the commercial DNA extraction kit Nucleospin for Soil (Macherey-Nagel, Düren, Germany). DNA quality of the samples was assessed by spectrophotometry using the NanoDrop ND-1000 (Nanodrop Technologies, Wilmington, Delaware). From each extraction duplicate, PCR amplifications targeting a 312 bp fragment of the marker gene rbcL were done on two groups of triplicates. Amplification was performed using the primers as described in [START_REF] Vasselon | Application of highthroughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter ?[END_REF]. Each PCR product group was pooled to provide a PCR duplicate per extraction replicate. In summary, four replicates per mineral spring were obtained. Library pools were sequenced on four Illumina MiSeq runs (2x250 bp paired-end V3) by GenoToul GeT-PlaGe platform (INRAE Genomics, Toulouse, France).

Analysis of diatom communities

The sequencing platform performed the demultiplexing and provided two fastq files per mineral spring, one corresponding to forward reads (R1) and reverse reads (R2), respectively.

The R package DADA2 was used for the amplicon sequence variant (ASV) from the demultiplexed Miseq following the method described by [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF]. An average of 132,420 sequences per sample were obtained before using DADA2 to filter the sequences.

Primers were removed from the R1 and R2 reads using CutAdap [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF]. Based on their quality profile, R1 and R2 reads were truncated to 200 and 170 bp, respectively. Reads with ambiguities and errors (maxEE) higher than 2 were eliminated. Then, chimeric sequences were removed from the samples, resulting in an average of 120,364 sequences per sample. Amplicon sequence variants (ASV) detected were assigned to taxa using version 10 of the curated reference database for diatoms Diat.barcode (Rimet, 2021) available at https://doi.org/10.15454/D7FO2T. ASV's not classified as Bacillariophyta were removed from the analysis. A total of 565 ASVs was detected and assigned to the phylum of Bacillariophyta.

Several species and genera were not assigned taxonomically to any known sequence, as is frequently the case in metabarcoding analyses (e.g. [START_REF] Rivera | Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software[END_REF]). Thus, an internal database of GEOLAB (that will be added to Diat.barcode) allowed the identification of some of the most abundant species belonging to the genus Navicula in springs, such as N.

sanctamargaritae Beauger, using light microscopy.

The diversity data of all samples were exploited using the 'Phyloseq' package [START_REF] Mcmurdie | phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data[END_REF] [START_REF] Hill | Diversity and evenness: a unifying notation and its consequences[END_REF] diversity index) were calculated for each replicate of the biological samples using the function "estimate_richness" (Fig. 2).

Replicates in metabarcoding allow better extrapolation of diatom diversity in mineral springs. To reduce the number of files to be analysed, replicates of the same spring were merged using the function "merge_sample." This function allows all ASVs present in the different replicates to be combined as effectively representative of one sample per spring. Relative abundances of species were calculated in each sample, and only species with an abundance >> 1% and representative of all the studied springs, were retained. The plot of the species composition for all samples was built with the ggplot2 package (Fig. 3). The ecology of the diatom species was determined using several diatom atlases, research papers and books [START_REF] Cleve-Euler | Die Diatomeen von Schweden und Finnland. Part III. Monoraphideae, Biraphideae 1[END_REF][START_REF] Carter | A taxonomic study of diatoms from standing freshwaters in Shetland[END_REF][START_REF] Hartley | A Check-List of the Freshwater, Brackish and Marine Diatoms of the British Isles and Adjoining Coastal Waters[END_REF]Krammer et al, 1997aKrammer et al, , 1997bKrammer et al, , 2000aKrammer et al, , 2000b[START_REF] Krammer | Bacillariophyceae. 4. Teil: Achnanthaceae, kritische Ergänzungen zu Navicula (Lineoatae) und Gomphonema[END_REF][START_REF] Krammer | Diatoms of Europe[END_REF][START_REF] Krammer | Diatoms of Europe[END_REF]Potatova and Hamilton, 2007;[START_REF] Lange-Bertalot | Toleranzgrenzen und Populationsdynamik benthischer Diatomeen bei unterschiedlich starker Abwasserbelastung[END_REF], 2001;[START_REF] Lange-Bertalot | Diatoms of Europe: Diatoms of the European Inland Waters and Comparable Habitats[END_REF], 2017;Bey andEctor, 2013a, 2013b;[START_REF] Beauger | Two new Navicula species (Bacillariophyceae) from Western Europe[END_REF][START_REF] Levkov | Diatoms of the European Inland Waters and Comparable Habitats[END_REF][START_REF] Li | The morphology and molecular phylogenetics of some marine diatom taxa within the Fragilariaceae, including twenty undescribed species and their relationship to Nanofrustulum, Opephora and Pseudostaurosira[END_REF].

Statistical analysis

Prior to performing statistical analyses, radiological measures were modified in order to obtain the contribution of the spring. The limits of detection (LD) are very low and below the environmental background noise. For the statistical study, samples below the LD were set to "0". LD values were subtracted from all results.

Different statistical analyses were perfomed to test the effect of physical, chemical and radiological variables on the biotic data. As abiotic data (table S1, S2) did not follow a normal distribution, they were standardised using z-score normalisation ((x-mean)/standard deviation).

This transformation also allows comparison of variables that initially had different units. Fortynine (49) abiotic variables were available from measures in water and sediments of the mineral springs monitored (Table S1, S2). To increase statistical robustness, collinearity tests were performed in parallel with principal component analyses (PCA), and thus the final dataset was reduced to 16 variables. These analyses were performed on the concentrations of major ions, trace elements and radionuclides. Variables with a cosine of less than 0.4 on both axes of the PCA were not retained. When two variables had a correlation coefficient >> 0.9, the variable with the higher cosine was retained as presented in Table 1. Thus, in the springs, some radiologic parameters were more important and showed more variation in the spring water or the sediment. Dissolved oxygen, conductivity, pH, and water temperature were added to the dataset due to their ecological importance for diatoms [START_REF] Patrick | Ecology of freshwater diatoms and diatom communities[END_REF]. A final PCA was performed on the main environmental parameters selected in each spring.

The correlation between ASV richness and physico-chemical and radiological variables was tested by calculating Pearson correlation coefficient with XLSTAT 2020 version 5.1, to determine if these elements measured in our study influenced the number of diatom species in mineral springs (Table 2).

In order to extract further information from our analyses, the different diatom communities of mineral springs were compared. A matrix of dissimilarities based on Jaccard dissimilarity index [START_REF] Jaccard | Étude comparative de la distribution florale dans une portion des Alpes et des Jura[END_REF]) was generated with the function vegedist of the R package "Vegan" (Oksanen et al., 2013, version 2.5-7). This index only takes into account the absence/presence of species, so a second matrix with the abundance-based Jaccard dissimilarity index "ab.Jaccard" (or chao jaccard) described in [START_REF] Chao | Abundance-based similarity indices and their estimation when there are unseen species in samples[END_REF] was also generated. The function "dist.ldc" of the package "Adespatial" version 0.3-14 [START_REF] Dray | Community ecology in the age of multivariate multiscale spatial analysis[END_REF]) was used to generate a dissimilarity matrix with this second index. The individual influence of five environmental and radiological factors such as "Geographical location", " 238 U", " 222 Ra", " 210 Po", " 228 Ac" activities level on diatom community diversity in springs were determined (Table S3) with a non-parametric MANOVA analysis [i.e. ADONIS (Permutational MANOVA)] based on Jaccard and "ab. Jaccard" dissimilarity indices using the function Adonis of the vegan R package. The "geographical location" represents not only springs that belong to the same geographical area but also to similar hydrogeological contexts linked to the fault system and the geological layers associated. The radioactivity classes for four radionuclide variables (i.e. 222 Rn, 238 U, 226 Ra, 210 Po level, 228 Ac) were determined from the orders of magnitude observed for these variables in the sediments of the sampled springs (Table 1). For 238 U and 226 Ra activities, three classes were determined by: Low: activity < 0.01 Bq.L -1 ; Medium: 0.01 Bq.L -1 ≤ activity ≤ 0.1 Bq.L -1 ; High: activity > 0.1 Bq.L -1 and Low: activity < 0.1 Bq.g -1 ; Medium: 0.1 Bq.g -1 ≤ activity ≤ 1 Bq.g -1 ; High: activity > 1 Bq.g -1 respectively.

Similarly, 210 Po activity levels were Low: activity < 0.00001 Bq.L -1 ; Medium: 0.00001 Bq.L -1

≤ activity ≤ 0.0001 Bq.L -1 ; High: activity > 0.0001 Bq.L -1 . For 228 Ac activity levels, the classes were: Low: activity < 1 Bq.g -1 ; Medium: 1 Bq.g -1 ≤ activity ≤ 10 Bq.g -1 ; High: activity > 10 Bq.g -1 .

Further correlations between the 14 most abundant species and physico-chemical and radiological variables were computed with XLSTAT 2020 version 5.1. The difference of ASV structure between radioactivity classes (Table S3) was estimated using the Multi-Response Permutation Procedure (MRPP) [START_REF] Zimmerman | Use of an improved statistical method for group comparisons to study effects of prairie fire[END_REF][START_REF] Mielke | The application of multivariate permutation methods based on distance functions in the earth sciences[END_REF]. MRPP is non-parametric, multivariate approach that tests the null hypothesis that two or more predefined groups are not different in species composition (here, predefined groups are geographical location and 238 U, 226 Ra, 210 Po, 228 Ac activities). The computed MRPP statistic delta (δ) represents the overall weighted mean of within-group means of pairwise dissimilarities among plots. In addition, the chance-corrected within-group agreement A statistic was computed (A = 0 means that in-group and out-group heterogeneities are equal; A = 1 means that all members of each group are identical and different from the members of other groups). This analysis was complemented with an Indicator Species Analysis (ISA) [START_REF] Dufrêne | Species assemblages and indicator species: the need for a flexible asymmetrical approach[END_REF][START_REF] Mcgeoch | Scaling up the value of bioindicators[END_REF] again using the vegan R package.

Results

Environmental variables in mineral springs

The physical, chemical, and radiological characteristics of the studied mineral spring sites are reported in Table 1. Conductivity of the mineral springs varied between 3090 and 9680 µS.cm - 1 (mean of 5682.56 ± 1693.30 µS.cm -1 ). pH did not display significant variations among the mineral springs (mean ± SD: 6.9 ± 0.3). The mean water temperature of the studied mineral springs was 16.6 ± 3.6 °C. Mineral springs were not well oxygenated with a mean value of 22.3 ± 3.6 %. Concentrations of Na + and HCO3 -are high in mineral springs with an average of 939± 361 mg.L -1 and 2318± 516 mg.L -1 respectively. In the water, 238 U activity varied between detection limit (2.5 mBq.L -1 ) and 0.193 Bq.L -1 and 210 Po varied between 9 and 392 µBq.L -1 . In the sediments, 226 Ra activity variation extended on almost two orders of magnitude, from 0.56 to 52.1 Bq.g -1 .Lastly, , considering all the springs, 228 Ac varied between detection limit (45 mBq.L -1 ) and 28.8 Bq.g -1 .

Analysis of the data sets using PCA (Fig. 2) shows the distribution of springs based on their physical, chemical and radiological parameters. Axis 1 and 2 explained, respectively, 41.53 % and 24.34 % of the data variability. Overall, PCA allows identification of four groups.

Thus, in the top quadrant, the springs for which the 226 Ra activity is significant, include Daguillon, Graviers, Ours, Tennis and Tête de lion. Also, Chemin, Graviers, Ours, Salut, Saulcée, Tennis and Tête de lion are represented by higher 228 Ac activity. Bard, Saladis, Giraudon and Tennis springs show higher Ca 2+ , HCO3 -and K + concentrations. In the lower left quadrant, Estreys, Sail, Salins, Saulcée and Trois sauts show high 210 Po, 238 U activities and Mn 2+ and PO4 3-concentrations. In the lower right quadrant, Giraudon, Rocs bleus, Saladis are associated with higher temperature, conductivity, pH and DO and also with high Br -and Na + concentrations.

Influence of physical, chemical and radiological factors on ASV richness

A total of 565 ASV were detected. Figure 3 shows the number of ASVs observed and Shannon's diversity index obtained for each mineral spring using the four technical replicates. The number of ASVs ranged from 6 and 74 with a mean 29 ± 17. The mineral spring with the most of ASVs was "Salut" (i.e. 74 ± 7) and the lowest "Salins" (i.e. 8 ± 1). In terms of Shannon's diversity index, the richest spring was "Chemin" (i.e. 2.38 ± 0.04), the poorest "Tennis" (i.e. 0.18 ± 0.09).

There was no significant correlation between ASV richness and physico-chemical or radiological ( 238 U, 226 Ra, 210 Po, 228 Ac activities) parameters.

Influence of environmental and radiological factors on diatom communities' structure

The contribution of several environmental factors, including radiological parameters, to the variability of diatom communities in mineral springs was determined using an Adonis test (Table 3). These contributions were calculated by taking into account the ASVs shared between samples (Jaccard) and the abundance of these ASVs (ab.Jaccard). The geographical location of the mineral springs explains, respectively, 47.0 % (Jaccard) and 53.5% (ab.Jaccard) of the ASV variability. For the radiological parameters, 238 U activities in the spring water explains 17.2 % (Jaccard) and 38.6 % * (ab.Jaccard) of the variability while its descendant 226 Ra activity in the spring sediments explains 10.7 % (Jaccard) and 7.7 % (ab.Jaccard). 210 Po activities in the springs' water contributes to 5.6 % (Jaccard) and 0 % (ab.Jaccard) of the variability while 228 Ac activities in the springs sediments explains 13.3 % (Jaccard) and 3.5 % (ab.Jaccard) of this variability.

Taxonomic composition of diatom communities in mineral springs

Sixty percent of the ASVs were assigned to a diatom genus, forty-two percent to a species.

Fifty-two genera were determined of which only 22 were associated to ASVs with read abundance > 10 % in all samples. The dominant genera were Achnanthidium, Amphora, Anomoeoneis, Caloneis, Craticula, Cyclotella, Diatoma, Diploneis, Epithemia, Fallacia, Fistulifera, Fragilaria, Gedaniella, Gomphonema, Halamphora, Mayamaea, Navicula, Nitzschia, Pinnularia, Planothidium, Sellaphora, Surirella. The most dominant ASV (relative abundance above 1% in at least on sample) were assigned to 32 diatom species (Figure 4): 69%

of the diatom species are freshwater species, 17% found in both freshwater and marine ecosystems, 7% in brackish water, and 7% in marine habitats. Among the 20 dominant ASVs, some could not be assigned to a species and belong to the genera Gomphonema, Navicula, Pinnularia and Surirella. The other dominant assigned species are Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea. Different ASVs were assigned to the same diatom species suggesting the presence of genetic variants.

Pearson correlations between the abundance of the 14 dominant ASVs and the physical, chemical and radiological parameters were determined (Table S4). All significant correlations were positive. Na + was correlated with Pinnularia sp2 (R²=0.60), which also correlated with several parameters as PO4

Discussion

This study provides the first analysis of diatom communities living in mineral springs using DNA metabarcoding to assess the influence of enhanced natural radioactivity on these assemblages. These extreme aquatic environments in effect comprise a field laboratory in which to assess the contribution of natural radioactivity through radionuclides' activity on diatom community structure. From this, two hypotheses were posited: (1) that radioactivity is not a factor that particularly influences diatom genetic richness; and (2), that radioactivity is a factor that can influence diatom community structure.

Natural radioactivity did not impact ASV richness

A first parameter to describe the α-diversity of communities with metabarcoding is ASV richness. Generally, diatom ASV richness was high in the springs monitored and varied greatly between mineral springs with a factor of about 11. No significant correlations were observed between ASV richness and the various radiological parameters, viz. 238 U, 226 Ra, 210 Po, 228 Ac activity. These results validate the first hypothesis: radioactivity does not influence diatom community genetic ASV richness. In the literature and to our knowledge, no other study besides the present one has yet been conducted on the effect of radioactivity on ASV richness of microbial communities. Even so, a negative impact of radioactivity on the species richness of macroorganisms in terrestrial ecosystems are a reduction of species richness, for instance forest birds in an area with high radiation doses around Chernobyl [START_REF] Moller | Species richness and abundance of forest birds in relation to radiation at Chernobyl[END_REF]Mousseau 2016).

For microorganisms, [START_REF] Shuryak | Modeling species richness and abundance of phytoplankton and zooplankton in radioactively contaminated water bodies[END_REF] suggest that diatom richness in some radioactive waterbodies in Russia can be greatly reduced by high radiation levels: in this particular study, a negative correlation of -0.81 was measured between diatoms species richness and the level of radioactive contamination. The result obtained in our study is significantly higher than that observed in the monitored mineral springs because radioactive levels are elevated by a factor of eight in the radioactive reservoirs in Russia. In another study, comparison of bacterial communities in geographically-separated soils affected by caesium releases showed that species richness was lower in the most contaminated soils [START_REF] Ihara | Direct comparison of bacterial communities in soils contaminated with different levels of radioactive cesium from the first Fukushima nuclear power plant accident[END_REF]. However, in aquatic environments, different results are reported: for example, [START_REF] Herlory | Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge 25 on a stream: case study of the Ritord watershed (Center-West France)[END_REF], revealed that diatom species richness was not affected by the treated uranium mining effluents; [START_REF] Videvall | Impact of ionizing radiation on the environmental microbiomes of Chernobyl wetlands[END_REF] that radioactivity had no effect on prokaryote richness in Chernobyl wetlands, whilst [START_REF] Murphy | Abundance and diversity of aquatic macroinvertebrate communities in lakes exposed to Chernobyl-derived ionising radiation[END_REF] also showed that radioactivity had no effect on macroinvertebrate communities from lakes exposed to Chernobyl-derived ionising radiation. The latter results

were obtained in the context of anthropogenic contamination by the addition of radionuclides to the environment, which implies stress on the surrounding organisms due to the introduction of these elements. In the case of radioactive mineral springs, organisms grow in the presence of radioactivity for long periods. Thus, we assume that organisms are adapted to radioactivity in mineral springs where the radioactive level is higher.

Uranium activity explained part of diatom community structure

Geographical location was found to be the main factor explaining diatom community structure in mineral springs considering ASV occurrence. This result is similar to those of [START_REF] Potapova | Benthic diatoms in USA rivers: distributions along spatial and environmental gradients[END_REF], [START_REF] Virtanen | The roles of environment and space in shaping stream diatom communities[END_REF], [START_REF] Soininen | Global patterns and drivers of species and trait composition in diatoms[END_REF] and Baker et al. (2021) that studied benthic diatom assemblages in rivers, streams and springs. 238 U activity was the second parameter that explained the variability of the diatom communities, whereas the variability of diatom community structure was more poorly explained by 226 Ra, 210 Po and 228 Ac activities.

In the literature, study of the effects of uranium on communities of various macro-and microorganisms is quite common in the context of anthropogenic contamination or laboratory experiments. Several studies have demonstrated that uranium in water courses and static bodies of water influences the taxonomic composition of microbial communities [START_REF] Reardon | Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer[END_REF][START_REF] Islam | Molecular assessment on impact of uranium ore contamination in soil bacterial diversity[END_REF][START_REF] Brzoska | The long-term effect of uranium and pH on the community composition of an artificial consortium[END_REF]Sutcliff, 2017;[START_REF] Suriya | Assessment of bacterial community composition in response to uranium levels in sediment samples of sacred Cauvery River[END_REF][START_REF] Zeng | Assessment of Bacterial Community Composition of Anaerobic Granular Sludge in Response to Short-Term Uranium Exposure[END_REF][START_REF] Chuiyun | The microbial community composition and population change during bioremediation of uranium tailings[END_REF][START_REF] Videvall | Impact of ionizing radiation on the environmental microbiomes of Chernobyl wetlands[END_REF]. For example, [START_REF] Suriya | Assessment of bacterial community composition in response to uranium levels in sediment samples of sacred Cauvery River[END_REF] showed that increased concentration of uranium in sediments of the Cauvery River in India favour the presence of several Proteobacteria and Gammaproteobacteria species. Exposure of the bacterial community found in anaerobic granular sludge to uranium leads to the modification of the composition of the community [START_REF] Zeng | Assessment of Bacterial Community Composition of Anaerobic Granular Sludge in Response to Short-Term Uranium Exposure[END_REF]. However, there do not seem to be any studies on the effect of naturally occurring uranium in the environment. This study is therefore a pioneer study on diatom community structure in natural radioactive areas. Moreover, uranium contamination (from mining, nuclear accidents, or the release of nuclear waste into the environment) is a relatively recent event, as it is linked to the growing demand for uranium by the domestic and military industries since the Second World War. In the context of mineral springs, microorganisms have been naturally

exposed to uranium in a continuous and prolonged manner, and uranium activity seems to have a significant influence on the structure of the communities, especially considering the genetic variants present.

This last view validates the second hypothesis: radioactivity is a factor that can influence diatom community structure. Furthermore, bioaccumulation of 226 Ra by several organisms such as plants, algae, bacteria, plankton and zooplankton is also documented to occur [START_REF] Iyengar | The natural distribution of radium[END_REF][START_REF] Zakeri | Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress[END_REF][START_REF] Carvalho | The environmental behaviour of radium: revised edition[END_REF][START_REF] Heidari | Isolation of an efficient biosorbent of radionuclides ( 226 Ra, 238 U): green algae from high-background radiation areas in Iran[END_REF]. 226 Ra accumulates in a similar way to calcium in human and animal body tissues, by accumulating on the surface of bones, whilst also leading to the cancers of various kinds [START_REF] Gad | Encyclopedia of Toxicology[END_REF][START_REF] Gad | Encyclopedia of Toxicology[END_REF]. Diatoms can also absorb and use calcium in their metabolic processes calcium and therefore it is also likely that 226 Ra is accumulated by diatoms and thereby involved in their metabolism too. 210 Po bioaccumulation is well documented in marine environments [START_REF] Bennett | Concentrations and temporal variations of 210 Po, 210 Pb, and Al in the surf zone ecosystem of Copalis Beach, Washington[END_REF][START_REF] Carvalho | Radionuclide concentration processes in marine organisms: A comprehensive review[END_REF][START_REF] Uddin | 210 Po concentration in selected diatoms and dinoflagellates in the northern Arabian Gulf[END_REF], marine phytoplankton [START_REF] Horowitz | 210 Po and 210 Pb distributions during a phytoplankton bloom in the North Atlantic: implications for POC export[END_REF], and invertebrates from the Baltic sea (Skwarze and Falkowski, 1988). All these radionuclides can be accumulated in cells of living organisms. [START_REF] Brown | Radiation doses to aquatic organisms from natural radionuclides[END_REF] observed that 210 Po and 226 Ra are the main contributors of absorbed dose rates when they measured the internal concentration of several radionuclides within marine and freshwater organisms (e.g., phytoplankton, macrophytes). However, in our study, 226 Ra and 210 Po are the two radionuclides that contribute the least to explanation of the community structure of diatoms frequenting natural bodies of water.

Taxonomic composition of diatom communities

The main challenges and limitations of the metabarcoding method as here described are the reference barcoding libraries themselves. Indeed, these reference libraries are often incomplete, and difficult to produce and maintain (e.g. [START_REF] Weigand | DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work[END_REF]. Morphological discrimination under a powerful microscope is still found to be necessary for species identification and can in turn be complemented by metabarcoding. The lack of genus and/or species identification could also be explained by the presence of new cryptic taxa due to the insularity of mineral springs.

Indeed, springs are biotopes that can be marked by insularity via physical or physico-chemical barriers. Such barriers are characterised by high levels of physical stress related for example to temperature water availability and radioactivity [START_REF] Cantonati | Crenic habitats, hotspots for freshwater biodiversity conservation: toward an understanding of their ecology[END_REF][START_REF] Cartwright | Ecological islands: conserving biodiversity hotspots in a changing climate[END_REF]. Thus, they present specific biotopes distinct from surrounding ecosystems and as such, may contain novel species due to potential allopatric speciation events.

A large proportion of the taxa herein observed by us could not be identified down to species level, but the general ecology of those that could be so identified demonstrated that most of those colonizing springs are present in various types of environments (freshwater, marine species and brackish water). Furthermore, their distributions are related to some chemical similarities between mineral springs and marine or brackish environment such as high conductivity, high ions contents among others. Also, some diatoms species have large ecological range and inhabited environments with different ecological statue like freshwater, springs and brackish water. Only Navicula sanctamargaritae is typical to mineral springs [START_REF] Beauger | Biodiversity and ecology of diatoms in mineral springs of the area of Sainte Marguerite (Saint-Maurice-ès-Allier, Massif central, France)[END_REF]. Thus, the observations made in the mineral springs show that some species are well distributed in the mineral springs, but their abundance varies according to the local environmental conditions.

The correlations between the species associated to the 14 most abundant ASVs and physical, chemical and radiological parameters of the springs monitored clearly revealed that one genetic variant of Planothidium frequentissimum is particularly abundant in mineral springs with high natural concentrations of the isotope 238 U. This result was confirmed with MRPP and ISA, which revealed interestingly that this species is associated with high 238 U activity. Hence, this finding means that there are clearly genetic variants of the same diatom species that do not have the same distribution and ecological tolerances. This finding agrees with those of Pérez-Burillo et al. ( 2021), working on the distribution and ecological preferences of four diatom species, who concluded that ecological tolerance of some diatom species resulted from overlapping preferences among genetic variants. These genetic variants individually showed much more restricted preferences and distributions. In a previous study, a higher frequency of deformations in the most radioactive springs had been observed for P. frequentissimum (Millan, 2019).

Mineral springs with high levels of radioactivity (i.e. 238 U activity) are habitats in which environmental pressure is more important than springs with low levels of radioactivity. If so, it questions the ability of this species to tolerate intracellular accumulation of radionuclides.

Indeed, bioaccumulation of radionuclides depends on several environmental factors including external concentrations of these radionuclides in the water. In this context, [START_REF] Snoeijs | Benthic diatoms as monitoring organisms for radionuclides in a brackish-water coastal environment[END_REF], demonstrated that external concentrations of radionuclides and benthic diatom concentrations are related: the higher the radionuclides concentrations, the higher the intracellular concentrations in the diatoms. This thus strongly suggests that in environments with high concentrations of radionuclides, diatom species are able to tolerate high radionuclide concentrations. If this in turn is so, the genetic variant A of P. frequentissimum is a potentially a good bio-indicator species for high natural uranium levels in spring water. Lastly, diatoms are the main producers in aquatic environments and the possible high accumulation of radionuclides within the cells implies -importantlythat diatoms are themselves potential vectors of radionuclides in the high levels of the food webs.

Conclusions

Mineral springs are extreme environments that may have naturally high levels of radioactivity.

Such environments are good field 'laboratories' to observe and understand the adaptation of organisms in the face of higher levels of natural and chronic background radioactivity. Our present study, the first on diatom community structure in natural radioactive areas, reveals that DNA metabarcoding proves that diatom species are very abundant in the mineral springs of the Massif Central. Radioactivity in the springs has no apparent effect on the diatom ASV richness in these mineral springs. Geographical location is the first factor explaining the structure of diatom communities in the mineral springs, and 238 U the second factor. This radionuclide is known to influence microbial community composition. Very interestingly, one of the genetic variants of Planothidium frequentissimum was detected as a bio-indicator of high level of uranium in natural environment (i.e., mineral springs). Lastly, our results show that some diatom species are well distributed in the mineral springs, but their abundance varies according to the environmental conditions found locally. Thus, some species are particularly abundant in mineral springs with a high enhanced level of natural radioactivity. This exemplifies the fact that diatoms are organisms that have a great capacity for tolerance to radionuclides.

Figure 1 :

 1 Figure 1: Location map of the study sites. (A) Map of Europe, (B) Map of the Massif Central in France, (C) Map of the 16 mineral springs monitored in the Massif Central; photographs of some mineral springs (D, E, F).monitored.

Figure 2 :

 2 Figure 2: Principal component analysis (PCA) shows the springs' distribution based on physical, chemical and radiological variables measured in water (w) or sediments (s).

Figure 3 :

 3 Figure 3: Boxplot showing the mean of (A) ASV richness corresponding to benthic diatom species richness and (B) Shannon's diversity for each technical replicate (N=4) of the mineral springs monitored in the Massif Central (France).

Figure 4 :

 4 Figure 4: Taxonomic composition of diatom communities for the mineral springs monitored in the Massif Central based on DNA metabarcoding results for the rbcL chloroplast gene. Only species with reads relative abundances above 1% are represented.

  Ra, 222 Rn,214 Pb and 214 Bi. The data were then processed via LabSOCS™ Calibration Software. 238 U,232 Th and trace elements were measured by ICP-QMS (Xseries 2, Thermo Electron) after microwave digestion (Ethos Easy Milestone)

Table 1 :

 1 Physical, chemical and radiological variables measured at the mineral springs monitored in the Massif Central (France) after the selection of variables with stepwise followed by the subtraction of detection limits (DL). Cond: conductivity; DO: dissolved oxygen; Temp: Temperature. 238 U; 210 Po 226 Ra; 228 Ac expressed radionuclides activity in spring water (w) or sediments (s).

	Springs	Na + (mg.L -1 )	K + (mg.L -1 )	Ca 2+ (mg.L -1 )	Br -(mg.L -1 )	PO4 3-(mg.L -1 )	HCO3 -(mg.L -1 )	Cond (µS.cm -1 )	pH	DO (%)	Temp (°C)	238 U (w) (Bq.L -1 )	226 Ra (s) (Bq.g -1 )	210 Po (w) (µBq.L -1 )	228 Ac (s) (Bq.g -1 )	Mn 2+ (s) (mg.g -1 )
	Bard	1,028.88	185.88	233.76	1.88	0.12	3,012	6,380	6.81	3.1	14.7	0	2.2		0.6	0.33
	Chemin	990.42	179.01	202.55	1.72	0.09	2,515	4,860	6.41	15.3	14.2	0.002	10.8		26.1	0.24
	Daguillon	503.16	112.22	297.06	0.56	0.13	2,142	4,030	6.6	29	13.9	0	23.0		4.6	0.30
	Estreys	504.61	45.10	128.21	1.65	0.11	1,185	3,480	6.38	16.4	16	0.012	1.9	17	0.0	0.40
	Giraudon 1,787.50	189.50	151.28	4.91	0.70	2,814	9,201	7.51	54	32.1	0	4.2		5.9	0.24
	Graviers	823.10	159.59	381.87	1.66	0.13	2,730	5,430	6.69	19.5	14.4	0	26.7		17.9	0.39
	Ours	713.30	146.24	313.27	1.20	0.16	2,648	5,490	6.72	13.8	13.7	0	52.1		28.7	0.11
	Rocs bleus 1,396.77	144.23	255.14	3.68	0.06	2,696	8,150	7	9.3	19.2	0	0.6		0.1	0.16
	Sail	686.62	83.46	85.67	1.13	0.54	1,150	5,390	7.02	9.2	16.6	0.004	0.8		0.0	0.32
	Saladis	1,813.91	197.52	112.55	4.09	0.05	2,928	9,680	7.82	88.6	16.1	0	1.2		0.1	0.17
	Salins	291.27	53.52	204.16	0.23	0.53	1,948	3,090	6.83	20.1	13.3	0.193	0.8	382	0.4	9.32
	Salut	1,041.30	189.46	179.92	1.67	0.09	2,740	5,850	6.46	11.7	12.1	0	9.5		12.8	0.10
	Saulcée	464.48	67.75	173.14	0.95	0.05	1,412	3,420	6.66	26	14.4	0.082	19.3	158	11.7	5.35
	Tennis	1,408.88	154.92	281.91	3.34	0.09	2,778	8,380	7.05	8.9	27.1	0	21.6		11.9	0.11
	Tête de lion	768.30	147.04	239.54	1.62	0.08	2,454	4,200	6.99	18.9	14.4	0	15.9		12.9	0.41
	Trois sauts	803.89	69.23	135.35	0.89	0.06	1,940	3,890	6.66	12.8	13.1	0.004	2.1		1.1	0.53

Table 2 :

 2 Pearson correlation coefficient (R) between ASV richness of benthic diatom communities and the physical, chemical and radiological variables measured in studied mineral springs of the Massif Central (N=16). ASV: Amplicon sequence variant richness, Cond: Conductivity, DO: Dissolved oxygen, Temp: Temperature, 238 U; 210 Po 226 Ra;228 Ac expressed radionuclides activity in spring water (w) or sediments (s). Values in bold are significant p-value (α = 0.05).

		ASV	Na +	K +	Ca 2+	Br -	PO4 3-HCO3 -	Cond	pH	DO	Temp	238 U	226 Ra	210 Po	228 Ac	Mn 2+
													(w)	(s)	(w)	(s)	(s)
	ASV	1	-0.01	0.29	-0.32	-0.15	-0.23	0.01	-0.11	-0.26	-0.15	-0.36	-0.25	-0.15	-0.25	0.18	-0.21
	Na +	-0.01	1	0.75	-0.13	0.95	0.05	0.68	0.96	0.73	0.53	0.66	-0.49	-0.23	-0.48	-0.08	-0.50
	K +	0.29	0.75	1	0.27	0.63	-0.12	0.90	0.73	0.40	0.30	0.29	-0.54	0.13	-0.52	0.35	-0.55
	Ca 2+	-0.32	-0.13	0.27	1	-0.12	-0.29	0.48	-0.03	-0.25	-0.32	-0.07	-0.10	0.70	-0.08	0.53	-0.09
	Br -	-0.15	0.95	0.63	-0.12	1	0.13	0.56	0.93	0.73	0.52	0.78	-0.42	-0.24	-0.41	-0.14	-0.43
	PO4 3-	-0.23	0.05	-0.12	-0.29	0.13	1	-0.17	0.11	0.35	0.15	0.47	0.35	-0.25	0.34	-0.24	0.30
	HCO3 -	0.01	0.68	0.90	0.48	0.56	-0.17	1	0.67	0.37	0.22	0.26	-0.35	0.19	-0.33	0.30	-0.36
	Cond	-0.11	0.96	0.75	-0.03	0.93	0.11	0.67	1	0.77	0.48	0.68	-0.45	-0.13	-0.43	-0.11	-0.46
	pH	-0.26	0.73	0.40	-0.25	0.73	0.35	0.37	0.77	1	0.73	0.56	-0.09	-0.24	-0.08	-0.34	-0.09
	DO	-0.15	0.53	0.30	-0.32	0.52	0.15	0.22	0.48	0.73	1	0.26	-0.02	-0.16	-0.01	-0.19	-0.01
	Temp	-0.36	0.66	0.29	-0.07	0.78	0.47	0.26	0.68	0.56	0.26	1	-0.20	-0.12	-0.20	-0.13	-0.21

Table 3 :

 3 Non-parametric Multivariate Analysis of Variances (NPMANOVA or Adonis) results for unifactorial analysis of location, 238 U, 226 Ra, 210 Po and 228 Ac activity levels in spring water (w) or sediments (s), made with Jaccard (J) and ab.Jaccard (ab.J) distances performed on ASV matrix with relative abundances. The matrix contains only ASV with a relative abundance of more than 2% in at least one sample of the mineral springs monitored in the Massif Central (N=16). Significant p-value is indicated with (*).

	Factors	R² (J)	R² (ab.J)
	Location	47.01 %	53.53 %

Table 4 :

 4 Multiple Response Permutation Procedure (MRPP) results for analysis of differences of community structure between sample group of location, 238 U, 226 Ra, 210 Po and 228 Ac activity levels in spring water (w) or sediments (s) made with Jaccard (J) and ab.Jaccard (ab.J) distances performed on ASV matrix with relative abundances. The matrix contains only ASV with a relative abundance of more than 2% in at least one sample of the mineral springs monitored in the Massif Central (N=16). MRPP is based on 999 permutations. ASV: Amplicon sequence variant richness, Cond: Conductivity, DO: Dissolved oxygen, Temp: Temperature.

	Distance index	Geographic

location 238 U Activity 226 Ra Activity 210 Po Activity 228 Ac Activity

	Supplementary
	(J)	Chance-corrected within-group A	0.146	0.026	-0.049	-0.010	0.005
		Observed δ	0.674	0.768	0.455	0.797	0.785
		Expected δ	0.789	0.789	0.434	0.789	0.789
		Significance of δ	p=0.002	p>0.05	p>0.05	p>0.05	p>0.05
	(ab.J)	Chance-corrected within-group A	0.368	0.230	-0.011	-0.040	0.005
		Observed δ	0.274	0.334	0.797	0.451	0.785
		Expected δ	0.434	0.434	0.789	0.434	0.789
		Significance of δ	p=0.11	p=0.004	p>0.05	p>0.05	p>0.05

Table 3 :

 3 Qualitative variables of the mineral springs monitored in the Massif Central indicating geographical location, and activity levels of 238 U, 210 Po 226 Ra and 228 Ac in water (w) or in sediment (s).

	Springs	Geographical location 238 U level (w) 226 Ra level (s)	210 Po level (w) 228 Ac level (s)
	Bard	Boudes	low	medium	low	low
	Chemin	Ternant les eaux	medium	high	low	high
	Daguillon	Joze	low	high	low	medium
	Estreys	Polignac	low	medium	medium	low
	Giraudon	Saint-Nectaire	low	medium	low	medium
	Graviers	Joze	low	high	low	high
	Ours	Joze	low	high	low	high
	Rocs bleus	Martes-de-Vevre	low	low	low	low
	Sail	Mirefleurs	low	low	low	low
	Saladis	Martes-de-Vevre	low	medium	low	low
	Salins	Apcher	high	low	high	low
	Salut	Ternant les eaux	low	medium	low	high
	Saulcée	Coudes	medium	high	high	high
	Tennis	Sainte-Marguerite	low	high	low	high
	Tête de lion Saint-Floret	low	high	low	high
	Trois sauts	Leyvaux	low	medium	low	medium

U activity level (w) 17.18 % 38.59 % * 226 Ra activity level (s) 10.69 % 7.74 % 210 Po activity level (w) 5.64 % 0 228 Ac activity level (s) 13.34 % 3.45 %

Acknowledgements

We thank François Keck for his expertise and advices in terms of analysis of the metabarcoding data, Hugh D. Loxdale for his English editing, and the two anonymous reviewers for their valuable comments, which have greatly improved the manuscript.

Funding sources: This study was funded by a grant from the Centre National de la Recherche Scientifique (NEEDS, project Acronym: DISCOVER)) including a CNRS doctoral grant for L.A. BAKER. We acknowledge financial support from CNRS-INEE within the context of the Zone Atelier Territoires Uranifères and the Clermont Auvergne Metropole.

Conflicts of Interest:

The authors declare no conflict of interest. S1: Physical and chemical variables measured in the mineral springs monitored in the Massif Central (France). N=16

Supplementary Table

Springs

Na + (mg.L -1 ) K + (mg.L -1 ) Ca 2+ (mg.L -1 ) Br - (mg.L -1 ) PO4


Christian Mougin

Véronique Gouy

Vincent Bretagnolle

Julie Berthou

Context and objectives of Recotox

 For many years, the scientific community underlines the lack of long-term and wide scale monitoring of chemical effects on ecosystems.

 Ecotoxicological studies are mostly supported by protocols often far away from realistic environmental conditions.

 Hence, our potential to understand and foresee the ecotoxicological effects remains limited.

 In this respect, the aims of RECOTOX are to:

 Promote a transverse and integrated research to follow, understand and mitigate the environmental (and health) impacts of pesticides,  Analyse the chain "pressures-exposures-impacts" by coordinating and integrating the in natura observation and experiment in spatial ecotoxicology and toxicology,  Share between its sites a common culture in ecotoxicology.

Why RECOTOX?

 Needs expressed by the scientific community



  Scientists: controlled conditions > in natura, realism, long-term  Site managers: opening and évolution  Needs expressed by programming organisations  2013 : IFRES report by the Group inter Alliances AllEnvi, AvieSan et Athena  2014 : Workshop INRA-AllEnvi : Pesticides, ecotoxicology and environmental exposure  2015 : CNRS-INSU and Ecodyn program  2017 : Workshop GT9 AllEnvi : new scientific challenges near risks and catastrophes  Ecophyto program …  Needs expressed by other stakeholders  ANSES, phyto-pharmacovigilance  Public society  Open data and open science What is RECOTOX? 22 / 11 / 2017  A network of sites providing various and additional agro-pedo-climatic conditions to monitor, understand and mitigate the impacts of pesticides (synthetic, metals, biopesticides)  A scientific and operating project  To consider:  the chain pressure-exposure-impact  the factors controlling the vulnerability of the agro-ecosystems, their ability of résilience  the exposure to multi-substances, the multiple stress and their long-term effects  the various compartments of agro-ecosystems  In realistic in natura conditions  To be the French focal point, partner for discussion with stakeholders (risk assessors and managers, pharmacovigilance...), provider of open data… of intensification and pesticide use • Various contexts of contamination (type, amount, temporality and spatialisation) • Types of soils (continental, tropical…) small (10 km²) large (100 à 1000 km²) catchment  Disciplines, approaches and knowledge • Soil science, agronomy, hydrology, chemistry, biology, ecotoxicology… • Survey, field monitoring and experiment, analytical methods, modeling • Field and laboratory pre-existing instrumentation • Pre-existing data (including long-term sequences) • Existing sample collections (soils, DNAs…), passive samplers… • Links with operational partners The project, since 2017  WP0 : animation and management of the network  WP1: promoting the network for an integrated and transversal research in ecotoxicology Incubation for innovative projects and production of position papers  WP2: up-scaling approaches in ecotoxicology, from plot to landscape, considering several compartments (soil, water, air, biota) Support for increasing ecological relevance, up-scaling  WP3: from ecotoxicology to toxicology Characterisation of exposure, and (human) exposome  WP4: harmonizing analytical methods and measurements, sharing equipments, documentary resources, technological intelligence within the sites Methods, skills and equipment sharing, quality management (reliability, traceability)  WP5: developing a focal point to acquire, store, diffuse and archive data for end users at the local and national scale Metadata standardization, geo-referenced sites, data management plan  WP6: communication, exploitation and dissemination of results Scientific and technical publication, training, workshops But also challenges (common and shared culture, progress, interoperability…)

/ 11 / 2017

• Cu is present at higher levels at Montmay and Pizay stations than in the upstream station Chatillon • Diuron levels are similar within the 3 stations tested

Thanks for your attention !

We are grateful to:

-Site managers -Alliance AllEnvi

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