Arnaud A Gbetchedji

MD Gilles D Houndetoungan

PhD Hubert C Hounsossou

PhD Neige M Y Journy

PhD Nadia Haddy

Carolé Rubino

MD Olivier Biaou

PhD Daton Medenou

Kuassi M Amoussou-Guenou

PhD Florent De Vathaire

MS Arnaud A Gbetchedji

MD Carole Rubino

MD Kuassi M Amoussou- Guenou

PhD Rodrigue S Allodji
email: rodrigue.allodji@gustaveroussy.fr

Orcid Ids

Alfred Arnaud Gbetchedji

A systematic review of occupational radiation individual dose monitoring among healthcare workers exposed in Africa

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

Word Count: 3547 of 4000; Abstract: 291 of 300 Short running title: Review of radiation dose monitoring among radiology healthcare workers in Africa KEYWORDS: dose monitoring, occupational exposure, diagnostic radiology, Africa, healthcare workers, radiography, diagnostic X-Ray, medical imaging, review. ABSTRACT Dosimetric monitoring is useful to requested limit exposures to ionizing radiation in medical occupational settings, and reduce subsequent health risks. Scientific literatures, such as UNSCEAR report, 2017 and International Atomic Energy Agency Report, 2014, updated informations on this subject, whereas, such a few African works were found. That's the reasons why we undertake this study, which consists in summarizing existing informations on monitoring external radiation exposure doses for whole body, from medical workers on this continent.

Using standard terms and combine the different keywords research for radiation dose monitoring among radiology healthcare workers in Africa, from the titles, abstracts, and full texts, we found 3139 articles in PubMed/MEDLINE, Google Scholar and INIS databases. Two reviewers screened the retrieved publications based on predefined eligibility criteria to identify relevant studies, extract key information from each, and summarize data in table .   A total of twenty potentially relevant articles were identified. Among these twenty articles, fifteen reported the overall average annual effective dose. Studies included in this systematic review represent an inventory of the radiation protection of medical workers in various countries of Africa, with a focus on the monitoring of occupational radiation exposure. The size of studied populations ranged between 81 and 5152 Occupational Exposed Workers. The mean annual effective doses ranged from 0.44 to 8.20 mSv in all specialities of Medical sectors, while diagnostic radiology ranged from 0.07 to 4.37 mSv. For the nuclear medicine and radiotherapy from medical groups, the mean annual effective dose varied between 0.56 and 6.30 mSv. Industrial and Research/Teaching sectors's data which are presented in Table 2b for comparison, varied between 0.38 to 19.40 mSv.

In conclusion, more studies implemented on dosimetric monitoring in Africa are needed to get a real picture of occupational exposure in this continent. KEYWORDS: Dose monitoring, occupational exposures, diagnostic radiology, Africa, healthcare, workers radiography, diagnostic X-Ray, medical imaging.

INTRODUCTION

Occupational exposure to ionizing radiation occurs in many professions, including medicine, research/teaching and industry workers [START_REF]UNSCEAR 2016 Report of the United Nations Scientific Committee on the Effects of Atomic Radiation[END_REF]. Diagnostic investigations using radiation have become a critical feature of medical practice. As such, it raises concern about the potential risk that these advancements may pose for both patients and professionals [START_REF] Dauda | Medical doctors' awareness of radiation exposure in diagnostic radiology investigations in a South African academic institution South[END_REF].

The cancer and non-cancer diseases caused by effects of radiations exposure on humans, has major implications to public health and radiation standard setting [START_REF] Wang | Cancer incidence and risk estimation among medical x-ray workers in China[END_REF].

Public interest in the long-term effects of radiation on humans has then increased and has been focused on carcinogenic effects from protracted exposure to low doses.

The role of individual monitoring in the nuclear industry is undisputed, with the need for good record keeping and regular review [START_REF]The information system on occupational exposure in medicine, industry and research[END_REF]. Individual monitoring in medical sector is also important for the Occupational Exposed Workers (OEW). The monitoring of medical workers chronically exposed to ionizing radiation is common practice in many countries but remains limited on the African continent. This is evidenced in the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) 2017 report [START_REF]UNSCEAR 2016 Report of the United Nations Scientific Committee on the Effects of Atomic Radiation[END_REF]. It states that literature dealing with medical exposure in Africa, Asia and Latin America is limited [START_REF]UNSCEAR 2016 Report of the United Nations Scientific Committee on the Effects of Atomic Radiation[END_REF]. Our study will focus on Africa, because, according to the literature [START_REF]The Information System on Occupational Exposure in Medicine, Industry[END_REF], it always remains the continent with the lowest participation rate in international surveys on radiation protection. The updating of information on this subject through scientific literature, such as UNSCEAR report, 2017 and International Atomic Energy Agency, 2014b, led us to undertake this study. That consists in making to summarize existing information on monitoring of doses external radiation exposure for whole body of medical workers on this continent. This will also highlight the improvement in dosimetric monitoring between the different countries, after analysis of the annual effective mean doses between different practices.

Methods

Search strategy and study selection

We searched the PubMed/MEDLINE, Google Scholar and INIS databases using a combination of the following keywords with Booleans operators 'OR' 'AND': dose monitoring, occupational exposure, diagnostic radiology OR radiography OR diagnostic X-Ray OR medical imaging Africa AND medical workers, in the titles, abstracts, AND full texts. The reference lists of eligible articles were also reviewed to identify studies that we might have missed by searching terms in the titles and abstracts. We included articles, published in English or French up to the 31st of August 2019, with an update on 20th of May 2020, which reported results regarding the dose monitoring of occupational radiation exposure from IR. We excluded studies that only provided descriptions of the system of radiation protection or did not report information on the dose monitoring of occupational radiation exposure from ionizing radiation for whole body. Similarly, studies focused, only on the radiation protection of patients or monitoring doses of the hand in interventional radiology, were excluded. In this review, inclusion criteria were (i) article fully available in English or French languages (ii) articles which provided mean annual effective doses related to different medical, industrial and research specialities or teacher. The results of the identification and selection process are displayed in a flow diagram (Fig. 1), as requested in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [START_REF] Moher | Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement[END_REF].

Data extraction

Information on country, study design, source population, sample size and period were extracted from the articles using a predefined data extraction form (Table 1). Data extraction was performed independently by two reviewers (AG, RA), who crosschecked their reports to validate the information extracted from the original articles and reach a consensus data synthesis.

Results

Study Selection and Characteristics

Using the defined key words, three thousand one hundred and thirty-nine (3139) articles were identified (Fig. 1). At the end, the twenty selected studies have been published between at 1986 to 2018. These articles provides the dosimetric monitoring of workers exposed to ionizing radiation whole body and others described dosimetric monitoring practices. Among these twenty articles (Table 1) fifteen reported the overall mean annual effective dose (Tables 2a and2b). Five studies not reported in Table 2a and Table 2b addressed issues of whether or not dosimetric monitoring exists in the facilities concerned or the assessment of compliance to international radiation protection standards; without reporting the individual radiation doses. From twenty studies, 70% and 15% studies were cross-sectional (reporting exposures during a oneyear period) and retrospective (reporting mean annual effective doses during a period of 16 years on average), respectively. The largest size of the source populations was 5152 provided in the field of diagnostic radiology by Ghana between 2000 and 2009.

It was followed by Tanzania with 757 diagnostic radiology workers on 1000, while 2a). For the Nuclear medicine and radiotherapy specialities of medical sector, the mean annual effective dose varied between 0.56 and 6.30 mSv (Table 2a).

Discussion

The updating of information on this subject through scientific literature, such as UNSCEAR report, 2017 and International Atomic Energy Agency, 2014b, where African countries were very little represented, led us to undertake this study which consists in making to summarize existing information on monitoring of doses external radiation exposure for whole body of by medical workers on this continent. Studies included in this systematic review represent, across various countries, an update of dosimetric's doses monitoring of medical workers in Africa, with a focus on the monitoring of occupational radiation exposure in diagnostic radiology sector. Because a wide numbers of medical workers are exposed to lower doses which may not be without consequences. We were able to find articles only from 11 countries out of 54 in Africa. This number is not representative as monitoring programs are available in some other countries, but they did not published papers on occupational radiation doses. The annual mean effective doses were provided by a little less than half of the studies selected for several exposed groups (medical, industry and research/teaching groups). The scarcity of dose monitoring data in African countries may be explained in part by the low participation rates of these countries in International Atomic Energy Agency (IAEA) technical studies. The results showed that, out of 55 countries that participated in the study from IAEA on individual dosimetry's monitoring of Interventional Cardiology from medical sector [START_REF]The Information System on Occupational Exposure in Medicine, Industry[END_REF], only 2 were in Africa, and it had the lowest participation rate of 6% (2 countries among 32 representing the total number of participating countries). No specific study produced by the IAEA, for the field of diagnostic radiology and nuclear medicine was found.

In addition, several African countries (Benin, Burundi, Green cap, Central African republic, Comoros, Republic of Congo, Djibouti, Equatorial Guinea, Eritrea, Guinea, Guinea-Bissau, Lesotho, Liberia, Malawi, Rwanda, Chad, Togo) [START_REF]IAEA 2016 The Role of Dosimetry in Protecting Workers Exposed to Ionizing Radiation (VIENNE: IAEA)[END_REF] do not yet have a dosimetry service functional in their country, despite the steps taken by the Mission Radiation Protection Advisory Team, IAEA, since 1984 [START_REF][END_REF]. However, the countries that have some may not publish the results of their surveillance. During the IAEA meeting in 2017 in Africa as part of Strengthening National Capabilities on Occupational Radiation Protection, 21 out of 28 participating countries had a dosimetric monitoring program [START_REF]IAEA 2016 The Role of Dosimetry in Protecting Workers Exposed to Ionizing Radiation (VIENNE: IAEA)[END_REF]. The IAEA would have declared that only 17

countries participated in an intercomparison study [START_REF]IAEA 2016 The Role of Dosimetry in Protecting Workers Exposed to Ionizing Radiation (VIENNE: IAEA)[END_REF]. This would confirm the thesis of the UNSCEAR 2017 report from the low participation of African countries in the studies.

The requested dose limit for effective dose is 20 mSv per year on average over 5 consecutive years (100 mSv in 5 years) and 50 mSv in one year [START_REF]Radiation protection and safety of radiation sources: Basic international safety standards[END_REF]. A wide range of data has been produced by the different articles that have been selected. But as part of our study, we also compared the mean annual effective doses between medical practices on the one hand and between disciplines on the other hand. In medical sector's workers in study carried out by Farai and Obed, the OEW have received a mean annual effective dose above the 1/3 of 20 mSv, annual dose limit requested.

However, in three articles some extreme values are above the annual requested dose limit [START_REF] Yakoro | First assessment of individual monitoring of medical workers occupationally exposed to ionizing radiation in Burkina Faso 7[END_REF][START_REF] Osei | Occupational Radiation Exposure in Ghana[END_REF][START_REF] Muhogora | Occupational dose trends in Tanzania 7[END_REF]. That is the case of one OEW from study carried out in Burkina Faso in 1990 by Yakoro et al. [START_REF] Yakoro | First assessment of individual monitoring of medical workers occupationally exposed to ionizing radiation in Burkina Faso 7[END_REF] where the value is 42.84 mSv in two months monitoring. The reason advanced by authors is the improper location of the said dosimeter. That is the same reason provided by others authors.

In all height articles (Table 2a), the diagnostic radiology group had the lowest values and for seven articles in Table 2b, the medical sectors presents the lowest values of mean annual effective doses, except for the study by Farai and Obed [START_REF] Farai | Occupational Radiation Protection Dosimetry in Nigeria[END_REF]. We also found, when a comparison was made between the mean of the annual effective doses inside the medical specialities (diagnostic radiology, nuclear medicine and radiotherapy), diagnostic radiology was favoured, presenting the low annual effective dose means. However, we have observed in this study between different countries, the mean annual effective doses ranged from 0,07 to 4.37 mSv (Table 2a), between 2011 and 2007, at Burkina Faso and Nigeria respectively, for the diagnostic radiology group. This difference observed from one country to another can be explained by the fact that organizational characteristics of medical fields can be different. Improvement of practices and devices over time can influence exposure time. That is the reason why the mean annual effective doses has decreased between period from 2007 to 2011 between Burkina Faso [START_REF] Tapsoba | État du suivi dosimétrique des travailleurs dans les services de radiologie de la ville de Ouagadougou[END_REF] and Nigeria [START_REF] Nzotta | Occupational Radiation Dose to X-ray workers in Radiological units in South Eastern Nigeria[END_REF]. However, we observe that in the years 2007, at the level of the diagnostic radiology sector, several countries presented the highest annual dose means values [START_REF] Hasford | Assessment of annual whole-body occupational radiation exposure in medical practice in Ghana (2000-09)[END_REF][START_REF] Korir | Estimation of annual occupational effective doses from external ionising radiation at medical institutions in Kenya South[END_REF][START_REF] Nzotta | Occupational Radiation Dose to X-ray workers in Radiological units in South Eastern Nigeria[END_REF]. That is for the same reason that between 1990 to 1999 and 2008 to 2009 for studies carried out in Nigeria [START_REF] Farai | Occupational Radiation Protection Dosimetry in Nigeria[END_REF] and Ghana [START_REF] Adjei | Personal dose analysis of TLD glow curve data from individual monitoring records[END_REF] respectively, there is a decreasing considerable of mean annual effective doses. The same trends are observed through the studies performed by Bayou et al.

[10] at Ethiopia and Gordon et al. [START_REF] Gordon | Optimization of radiation protection for the control of occupational exposure in Ghana[END_REF] at Ghana, where the mean annual effective doses decrease from 4.51 to 0.57 mSv between 1988 and 2011, respectively.

However, we note the heterogeneity of mean annual effective doses for the same year where the measures carried out between different countries, in medical sectors. The This situation is certainly due to a growth in radiological procedures performed on the same devices in recent years.

Conclusion

This study showed that few countries in Africa publish the results of their dosimetric monitoring. It also shows that the doses recorded are sometimes relatively high, hence the need to encourage systematic dosimetric monitoring of all exposed workers for better monitoring of their state of health.

The paucity of international publications of dosimetric monitoring data in African countries does not reveal the manifold efforts made by them to optimize the protection of workers and the public. However, several countries have shown, through published studies, the improvement of radiation protection occupational.

Fig. 1 :

 1 Fig. 1: PRISMA diagram selection process s for paper publishedOn Pubmed: Monitoring of occupational exposures in diagnostic radiology radiation workers; on others sources: Monitoring, occupational exposures, diagnostic radiology; Dosimeter radiation workers medical imaging, medical X-Ray healthcare; Screening process: included the words "Africa". Criteria of eligibility: articles which report the individual dose's radiation about medical workers. Reasons for exclusion: * articles which report the dose's radiation about patients; **did not match inclusion criteria (i.e., radiation protection studies investigating the doses monitoring for whole body of medical workers).

  types of device (high or low dose rate) or radiation protection systems takes in place can explain this heterogeneity. The same conclusion on improvement of practices, can be drawn regarding to the comparative study intra-countries of the annual effective mean doses for the medical sector between Gordon and Adjei's studies where the doses decrease from 0.57 to 0.44 mSv between 2002 and 2009 in Ghana. In Nigeria, Farai and Ogundare's studies show a decrease in the annual mean effective doses from 8.20 to 3.70 mSv between 1990 and 2001. In contrast to these two countries, the studies carried out in Burkina Faso show an opposite trend, from 2007 to 2011, the dose increased of < 0.10 to 0.47 mSv according to studies by Yakoro and Tapsoba.

Table 1 :

 1 Tapsoba et al.[START_REF] Tapsoba | État du suivi dosimétrique des travailleurs dans les services de radiologie de la ville de Ouagadougou[END_REF] reported only 81 medical workers on 157, in Ouagadougou at Burkina-Faso. The different studies reported data on workers exposed to ionizing radiation in all fields using the ionizing radiation, but more particularly in different specialities from medical sectors (Table2a). All medical sectors, industrial and research/teaching's data are presented in Table2bfor comparison. Summary of twenty selected studies

					Provided mean annual	Provided collective
	Authors	Country	Type of studies	Source population (sample size)	individual doses	annual doses	Period
					(Yes/No)	(Yes/No)
	[8]	Burkina Faso	Longitudinal	OEW of Medical (3 to 121)	Yes	No	2007-2010
	[7]	Burkina-Faso	Cross-sectional	OEW in diagnostic radiology department (81)	Yes	No	2010-2011
	[9]	Egypt	Retrospective	OEW (radioactive source) (27)	No	No	2002-2012
	[10]	Ethiopia	-	OEW (100 to 450)	Yes	Yes	1977-1988
	[11]	Ghana	Cross-sectional	OEW of medicine, industrial, research and education and teaching (650)	Yes	Yes	2002-2007
	[12]	Ghana	Retrospective	OEW of diagnostic radiology (5152), radiotherapy (747), nuclear medicine (87)	Yes	Yes	2000-2009
				OEW of medicine, industrial and
	[13]	Ghana	Cross-sectional	research/teaching: radiotherapy (8),	Yes	Yes	2008-2009
				diagnostics (556), nuclear medicine (4)
	[14]	Ghana	Cross-sectional	OEW in medicine, industrial, research and education and teaching (254 to 761)	Yes	Yes	1988-1995
	[15]	Kenya	Cross-sectional	OEW in diagnostic radiology department (367)	Yes	Yes	2007
	[16]	Madagascar	Cross-sectional	OEW in diagnostic radiology department (260)	Yes	No	1990 -2000
	[17]	Malawi	Cross-sectional	OEW in diagnostic radiology department (Number of workers not defined)	No	No	2018
	[18]	Nigeria	Longitudinal	OEW of medicine, industrial, and research (640)	Yes	Yes	1990 -1999
	[19]	Nigeria	Cross-sectional	OEW of Medical (30 to 192)	Yes	Yes	1999-2001
	[20]	Nigeria	Cross-sectional	Radiographs (500)	No	No	2009
	[21]	Nigeria	Cross-sectional	OEW (Number of workers not defined)	No	No	2011
	[22]	Nigeria	Cross-sectional	OEW of medicine and industrial (500)	Yes	Yes	2000-2001
	[23]	Nigeria	Cross-sectional	OEW of medicine, radiographers and radiologists (59)	Yes	No	2005-2007
	[24]	Sudan	Cross-sectional	OEW in cardiology department (Number of workers not defined)	No	No	2010
	[25]	Tanzania	Retrospective	OEW in medicine, industrial, research and education and teaching (730)	Yes	Yes	1996-2010
	[26]	Tanzania	Cross-sectional	OEW in medicine, industrial, research and education and teaching (1000)	Yes	Yes	1986-1997
	OEW: Occupational Exposed Worker.

Table 2a :

 2a Summary of height articles from the fifteen included studies reporting mean annual individual doses for different specialities of medical sectors

TLD = Thermoluminescent Dosimeter.

Table 2b :

 2b Summary of seven articles from the fifteen included studies reporting mean annual individual doses for all specialities of medical sectors and others sectors (Industrial/ Research/Teaching)

	Authors	Type of dosimeter	Mean annual individual doses (in mSv) Diagnostic Radiology Nuclear Medicine Radiotherapy
	[12]	TLD	2.94	6.30	5.24
	[25]	TLD	1.50	1.50	-
	[23]	TLD	4.37	-	-
	[14]	TLD	0.80	0.56	1.12
	[7]	TLD	0.07-0.47	-	-
	[15]	TLD	2.52	-	-
	[8]	TLD	In mean 85.22 % of doses < 0.10	-	-
	[16]	TLD	0.90-2.00	-	-
			Mean annual individual doses (in mSv)
	Authors	Type of dosimeter	Medical sectors	Industrial	Others sectors Research/Teaching group
	[18]	TLD	8.20	2.34	4.39
	[10]	TLD	4.51	-	-
	[11]	TLD	0.57	0.54	-
	[13]	TLD	0.44	0.54	0.38
	[19]	TLD	3.20-3.70	4.70-19.40	-
	[26]	TLD	1.68	0.93	0.92
	[22]	TLD	2.7	16.25	-
	TLD = Thermoluminescent Dosimeter.

Acknowledgments

This work was supported by the Division for Africa, Department of Technical Cooperation of International Atomic Energy Agency (IAEA). We especially thanks to Catherine Luccioni and Olivier Laurent for their real involvements in the reading and revising the manuscript. We are also grateful to Imène Mansouri and Françoise Terrier for their unfailing support and advice.

Abbreviations

ALARA

Conflicts of Interest

The authors declare that there is no conflict of interest.


P Ory

V Hamani

P.-E Bodet

L Murillo

Marianne Graber
email: mgraber@univ-lr.fr

The variegated scallop, Mimachlamys varia, undergoes alterations in several of its metabolic pathways under short-term zinc exposure

Keywords: Zinc, UHPLC/QToF mass spectrometry, metabolomics, Mimachlamys varia

The variegated scallop (Mimachlamys varia) is a filter feeder bivalve encountered in marine regions of the Atlantic coast. In particular, it is present in the La Rochelle marina (France), where it is used for the biomonitoring of marine pollution, due to its ability to strongly bioaccumulate pollutants. In this semi-closed environment, contamination generated by port activities leads to an accumulation of both organic and metal pollutants. Zinc is one of these pollutants, present at a dose of up to 150 to µg.L -1 . This study investigated the effects of 48 h zinc exposure upon the metabolic profiles of Mimachlamys varia using UHPLC/QToF (ultrahigh performance liquid chromatography-quadrupole time-of-flight) tandem mass spectrometry metabolomics. After acclimation in mesocosms recreating in situ conditions, both controls and exposed with Zn 2+ (150 μg.L -1) bivalves were dissected to recover the gills after 48 h and stored at -80°C before metabolites extraction. UHPLC/QToF tandem mass spectrometry was performed to study metabolite composition of samples. Statistical analysis of results using multivariate techniques showed a good classification between control and exposed groups. Eleven identified metabolites were found to be downmodulated in exposed scallops. These variations could reflect potential zinc effects on several of the biological processes, such as energy metabolism, osmoregulation and defense against oxidative stress.

Among the eleven metabolites highlighted, four were reported for the first time in an aquatic organism exposed to Zn. This study demonstrates once again the diversity of interactions between bivalves and metals and the complexity of the physiological response of marine bivalves to pollutants.

Introduction

Marine ecosystems are the final receptacle of contaminants released to the environment, especially in seaports and other industrialized or strongly anthropised coastal areas. Boat harbours, for instance, may be hotspots of contaminant accumulation [START_REF] Bighiu | Metal contamination in harbours impacts life-history traits and metallothionein levels in snails[END_REF][START_REF] Johnston | Bearing the burden of boat harbours: Heavy contaminant and fouling loads in a native habitat-forming alga[END_REF]. Indeed, they are likely to receive both a cumulative load of pollutants from upstream point and nonpoint sources from watershed, and many pollutants generated at the marina itself. In addition, the construction of a marina generally creates a condition of reduced water circulation and protection from wave action. The result is an area with limited natural water circulation and exchange. The marina of La Rochelle, located in the south-west of France, perfectly illustrates this phenomenon with its complex geometry and infrastructure, especially since its expansion in 2014 [START_REF] Huguet | Characterisation of the water renewal in a macro-tidal marina using several transport timescales[END_REF]. Over time, this reduced circulation and increased pollutant generation can increase pollutant concentrations in the water column, sediments, and aquatic organisms. This often results in greater impacts in the area concerned on ecological communities [START_REF] Sim | Sediment Contaminants and Infauna Associated with Recreational Boating Structures in a Multi-Use Marine Park[END_REF]. Because of their environmental persistence, heavy metals are one of the major harmful toxic pollutants for the marine environment [START_REF] Stankovic | Biota as toxic metal indicators[END_REF] and especially in harbours and marina [START_REF] Bighiu | Metal contamination in harbours impacts life-history traits and metallothionein levels in snails[END_REF][START_REF] Galkus | Heavy Metal Contamination of Harbor Bottom Sediments[END_REF]. The major proportion of these trace elements is trapped in coastal sediments where they can be continuously released into the water column and incorporated into food webs. The availability of trace elements to aquatic species is of prime concern both in terms of the prediction of the effects of metal pollution on ecosystems and in terms of public health when bioaccumulated in edible animals.

In the following study, we focused attention on Zn pollution, at a concentration such as the maximum concentration that can be reached in the marina of La Rochelle. Zinc is found at 157 µg.L -1 in the water of the Marillac area, which is one of the four docks that make up the marina of les Minimes [START_REF] Creocean | Suivi des rejets des activités de carénage -Port de plaisance des Minimes[END_REF]. It may arise from nonpoint sources from watershed : rainwater runoff sources (outdoor zinc or galvanised surfaces, tire wear debris, etc.), zinc-containing paints, anti-corrosion additives for watercontaining equipment, fireworks, motor oil/lubricating oils additives, industrial air emissions, industrial runoff, zinc minerals or wastes in construction materials, fertiliser and agricultural micronutrient products (California Stormwater Quality Association [CASQA] 2015). It also has its origin in the boat components and their maintenance: zinc anodes used to deter corrosion of metal hulls and engine parts, zinc constituent of motor oil and tires, antifouling paints [START_REF] Eklund | Pleasure Boatyard Soils are Often Highly Contaminated[END_REF].

Among the marine organisms most affected by metal pollution are bivalve mollusks, given their filterfeeding attitude, which leads to a strong accumulation of the pollutants. In order to investigate the unknown toxicological effects and mechanisms of metals on bivalves, environmental metabolomics is commonly applied for several years now (for reviews see: [START_REF] Booth | Metabolomics and its application to studying metal toxicity[END_REF][START_REF] Viant | Mass spectrometry based environmental metabolomics: a primer and review[END_REF][START_REF] Lankadurai | Environmental metabolomics: an emerging approach to study organism responses to environmental stressors[END_REF]. As examples of recent metabolomics application in the case of a metal contamination of bivalves, [START_REF] Aru | Metabolic responses of clams, Ruditapes decussatus and Ruditapes philippinarum, to short-term exposure to lead and zinc[END_REF] investigated the effects of short-term lead and zinc exposure upon the metabolic profiles of clams Ruditapes decussatus and Ruditapes philippinarum. [START_REF] Nguyen | Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes[END_REF] applied an integrated approach that combines flow cytometry and metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in mussel (Perna canaliculus). [START_REF] Wu | Differential metabolic responses in three life stages of mussels Mytilus galloprovincialis exposed to cadmium[END_REF] found that mussel response to cadmium was dependent on life cycle stage. Many other current applications of metabolomics to study the effects of metals or other water contaminants on marine organisms have been reported in many studies (for a review see Pomfret et al., 2019).

Here, we used the marine bivalve Mimachlamys varia, in the family Pectinidae, commonly known as the variegated scallop, as a model organism. Due to its relative abundance along the Atlantic coasts, its sedentary nature and also because this species has been shown to have a very elevated pollutants incorporation and retention capacity, it has been proposed as a potential indicator species for the marine biomonitoring (Breitwieser et al., 2018a;Metian et al., 2009aMetian et al., , 2009b;;[START_REF] Milinkovitch | In situ evaluation of oxidative stress and immunological parameters as ecotoxicological biomarkers in a novel sentinel species (Mimachlamys varia)[END_REF]. In particular, it is relatively easy to sample in the marina of La Rochelle, where it has been used for ecotoxicological studies (Breitwieser et al., 2018b). Furthermore, it is a commercially harvested species also captured for leisure activities, directly on the shore or by snorkeling and it represents therefore a definite economic value.

Previously, we performed a first metabolomics study on M. varia that highlighted a complex metabolic reprogramming that occurs during tidal cycles after an emersion period of 2 h and upon re-immersion [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF]. It appeared that metabolites depend on the moment of the tidal cycle as well as on the ambient environment (oxygenation, salinity and temperature conditions, seasonality, etc.). In order to minimise the effects of all these important interplaying environmental factors and understand the underlying mechanisms affecting M. varia exposed to zinc, we grew scallops in aquaria under controlled and continuously immersed conditions and we exposed the animals to zinc concentration equal to that found in La Rochelle marina. A comparative metabolomics study was performed in gills samples, using UHPLC/QToF mass spectrometry, in order to provide an overview of the relationship between zinc exposure and metabolome changes. Gills were preferred over other tissues, as they have been shown to be, along with the mantle and digestive gland, the main storage organs for metals in oysters [START_REF] Wang | Trace metals in oysters: molecular and cellular mechanisms and ecotoxicological impacts[END_REF]. In addition, gills are generally considered as the main entry of dissolved metals in marine filter-feeder organisms [START_REF] Marigómez | Cellular and subcellular distribution of metals in molluscs[END_REF].

Materials and Methods

Animals and experimental design

Scallops, Mimachlamys varia, (shell length from 3 to 5 cm) were collected from La Pointe du Grouin to Loix en Ré (Ré island, France) and acclimated one month in the laboratory in filtered through a 50 µm nylon filter and sterilised seawater. The physicochemical parameters of the water were controlled every two days and were kept constant by a biweekly water renewal (pH 8.0, temperature 10°C, salinity 33 g.kg -1 seawater, nitrite, nitrate, and ammonia <0.025 mg.L -1 ). Food as liquid algal culture (Shellfish Diet 1800®, Reed Mariculture, Campbell, CA, USA) was distributed three times per week, at a rate of 0.02 mL per individual.

Mortality of less than 3% was observed in acclimation tanks.

For this experiment, 36 scallops were randomly distributed in six 18-litres aquaria. Three randomly selected aquaria were contaminated with a ZnCl2 solution (nominal zinc concentration 150 µg.L -1 ). The other three aquaria received the same amount of seawater.

After 48 hours of exposure, the six scallops remaining in each aquarium were sampled. The gill tissues of each individual were dissected, drained on absorbent paper and put in cryovial on ice. Gill tissues from three individuals were grouped, snap-frozen in liquid nitrogen and stored in a freezer at -80°C. From this sampling, six exposed samples and six controls were obtained. These control samples were used as a reference for the analyses.

Sample preparation for metabolomics study

Sample preparation was performed according to a previously described method [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF], inspired by a metabolomics study in Phycotoxines laboratory (Ifremer, Nantes, France) [START_REF] Mondeguer | Pinnatoxines en lien avec l'espèce Vulcanodinium rugosum (II)[END_REF]. In summary, samples were thawed, homogenized by manual grinding and then crushed with a homogenizer. These three last operations were performed on ice. They were then adjusted to 1 g and stored at -80°C before extraction procedure. This last protocol included a triple solvent extraction by successively using acetone twice and methanol. The three pooled supernatants were recovered and dried with a stream of nitrogen, before resuspension with 2 mL methanol/water 20/80 (v/v), storage at -80°C, filtration with 0.2 µm filters and LC/MS analysis.

UHPLC/QToF MS analysis of metabolites

The analysis of metabolites in gills was performed by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry, according to a previously described method [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF], with the same analytical equipment. 5 μL of the samples were injected in a column "Acquity UPLC HSST3" (Waters) (2.1 × 150 mm, 1.7 μm), and the products were eluted at a flow rate of 300 μL.min -1 using a gradient composed of solvents A (water/formic acid 100/0.001 (v/v)) and B (acetonitrile/ formic acid 100/0.001 (v/v)). The gradient used was the same as described previously [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF]. The analyses were performed in positive and negative ionisation mode with MS E function in a centroid mode. The MS parameters applied in the ESI source for the two ionisation modes were the same as described previously [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF], except that desolvation gas flow-rate was 300 L.h -1 and capillary voltage was 3 kV (+) and 2.5 kV (-) and sampling cone 35 V. The instrument was adjusted for the acquisition on a 50-2100 m/z interval, with a scan time of 0.5 s. A 20 to 40 V ramp was used as collision energy for the high energy mode of MS E .

The mass spectrometer was calibrated before analysis using 0.5 mM sodium formiate solution and the Leucine Enkephalin (M = 555.62 Da, 1 ng.μL -1 ) was used as a lock-mass.

Compound identification was performed as explained below.

Quality Control

A pool sample was prepared by combining 100 μL of each gills tissue extract. This mixture was divided into ten vials that were used as quality-control samples (QCs). They were regularly distributed and injected throughout the sample list to ensure analytical repeatability. Samples of gill tissue extracts were measured in randomised order. Blanks were prepared with the last extraction solvent and injected at the beginning and at the end of the sample sequence, to allow the subsequent subtraction of contaminants or components coming from the extraction solvent.

Chemicals

Acetonitrile, Methanol, Acetone with HPLC grade purity were from Carlo Erba. Water was prepared using a Milli-Q reagent water system. Succinate and L-phenylalanine were purchased from Sigma-Aldrich (now part of Merck).

Statistical analysis

Metabolites data were obtained and treated as ion peak intensity as previously described [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF].

Data were processed on the online and freely available Workflow4Metabolomics (W4M) platform (http://workflow4metabolomics.org) for pretreatments and multivariate analyses processing as previously described [START_REF] Ory | Metabolomics based on UHPLC-QToF-and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence[END_REF]. Pretreatments included batch correction and removing metabolites with coefficients of variation (CV) > 0.4 in the QC samples. Quality metrics tool (supplied by W4M Core Development Team) provided visualising the data matrices, batch correction efficiency and supplied a rapid highlight of potential sample outliers.

Log-transformation normalised the data and Pareto scaling was applied before the multivariate analysis process. The data were analysed with unsupervised (Principal Component Analysis, PCA) and supervised (Partial Least Square Discriminant Analysis, PLS-DA) methods. This last method is a multivariate regression and prediction based on the separation between two classes: reference and Zn-exposed samples after a 48 h exposure. It allowed the selection of metabolites involved in class separation in order to identify metabolites affected by Zn exposure. Metabolites responsible for classification between groups were selected according to their contribution as variable in the component prediction in PLS-DA model, described as Variable Importance in Projection parameter (VIP), considering only variables with VIP values higher than 1 indicative of significant differences between groups.

Predictability performance was evaluated for each built PLS-DA model. To assess the statistical significance, t-tests were performed on each metabolite intensities difference between reference and Zn exposed samples, after normality and homoscedasticity were previously checked. Otherwise, Mann-Whitney tests were performed.

Identification and relative level of metabolites

The identification of metabolites was performed by using the human metabolome database (http://www.hmdb.ca) and the METLIN database (https://metlin.scripps.edu/landing_page.php?pgcontent=mainPage) and also for lipids and lipid-derivatives with the online lipid database: (http://www.lipidmaps.org/tools/ms/LMSD_search_mass_options.php). A research was performed with the mass value of metabolites and a limit with a mass error lower 5 ppm.

Moreover, for succinate and L-phenylalanine, analytical standards were used with the LC/MS method to verify this identification. Metabolites were finally expressed in relative abundance, which was calculated for each metabolite by dividing the abundance in each sample by the median abundance across all the samples.

Results

Data processing

Mass spectrometry untreated analyses detected 20 946 m/z features for negative ionisation mode results in LC/MS and 44 256 for positive ionisation mode. These large sets of data obtained for metabolomics profiling were preprocessed applying a Lowess regression model fitting with the pool values as batch correction [START_REF] Van Der Kloet | Analytical error reduction using single point calibration for accurate and precise metabolomic phenotyping[END_REF].

After preprocessing and removal of metabolites with CV>0.4 in QC samples, the database of metabolites counted for 1556 and 3581 compounds detected with negative and positive ionisation modes in LC/MS respectively.

PCA Analysis

PCA was performed as a first method to confirm differences in metabolite levels between reference animals and animals exposed to zinc and detect eventual outlier samples. The score plots of PCA analysis are shown in Figure 1. Each symbol represented an individual sample, and the dot scattering indicated similarities or differences in metabolic compositions between samples. PCA performed with data sets from both reference and exposed individuals showed that two groups were well separated according to Zn exposure for the negative mode but fairly well separated for the positive mode, due to a high dispersion of Zn-exposed samples along the 2 nd axis. Thus, the intensities of the compounds, considered as variables, contributed to the separate structure observed between reference and exposed individuals. The two first axes accounted for 31%-12% and 24%-14% of the total variability among samples for negative and positive data sets respectively. Reference samples and samples exposed to Zn were grouped and distinguished mainly along the second axis (t2) for both ionisation conditions. The model fitted the data well. However, too many variables were considered to highlight a specific contribution to the total variability within both data sets.

Hotelling's T² statistic tested the presence of outliers measuring the variation within the PCA model.

Samples with large Hotelling T² had an unusual variation inside the model. In this case, they were not representative of the modeled data and can be considered as outliers if p-value < 0.05. The PCA results showed one outlier sample in the negative mode of data sets (Zn48-2). This outlier sample was removed from sample matrix data set to perform PLS-DA.

PLS-DA analyses

PLS-DA analyses were then performed, to identify metabolites whose abundance was related to exposure to Zn (Figure 1). The first PLS-DA models were built to force the separation between the two populations in the two datasets. (negative and positive modes). The performances of the model were proven, as the results of both analyses in negative and positive modes showed a relative data consistency with R 2 (cumulative) = 0.99 and 0.99, respectively. Moreover, the prediction performance of the models reached Q 2 (cumulative) = 0.88 and 0.57 for negative and positive modes respectively, which corresponds to good prediction capability.

However, the permutation test was not significant (p-value > 0.05) for these first models due to the very large number of variables. In order to improve the prediction performance of the model and to select the variables with the largest contribution, successive PLS-DA models were built by selecting each time only the variables with VIPs > 1. This process was stopped when model predictive performance stopped to increase.

In the negative mode data set, a total of three successive models selecting 465, 155 and 59 metabolites with VIPs > 1 were performed reaching Q 2 = 0.931. In the positive mode data set, three successive models selecting 360, 137 and 40 metabolites with VIPs > 1 were performed reaching Q 2 = 0.959. All these selected metabolites showed a significant difference between the reference samples and the samples exposed to Zn (t-test or Mann-Whitney test, p-value < 0.05).

Metabolite modulation

Mass-matching analysis using the different online metabolome databases mentioned above or comparison with standards allowed putative annotation of eleven metabolites showing a significant difference between reference and Zn exposed samples (Table 1).

Interestingly, all of them were down-modulated when going to references to Zn exposed samples. The relative abundance of all these metabolites decreased under zinc exposure by more than 30% (Figure 2) : Lphenylalanine (-39.02%, p = 0.05), L-kynurenine (-70.31%, p = 0.0038), picolinic acid (-61.93%, p = 0.013), neuroprostane 1 (-58.61%, p = 0.027), neuroprostane 2 (identified as 14-hydroperoxy-H4-neuroprostane or 11-hydroperoxy-H4-neuroprostane or 10-hydroperoxy-H4-neuroprostane) (-40.22%, p = 0.05 ), neuroprostane 3 (256 candidates corresponding to neuroprostanes on 266 entries on lipid maps databank for this m/z) (-52.28%, p = 0.05), succinate (-42%, p = 0.05) , S-(PGA2)-glutathione or S-(PGJ2)-glutathione (-64.07%, p = 0.012), xanthine (-46.65%, p = 0.018), hexanoylcarnitine (-36.81%, p = 0.044), dodecenoylcarnitine (-49.12%, p = 0.0005).

Discussion

Scallops exposed for 48 h to a nominal zinc concentration of 150 µg.L -1 in laboratory conditions undergo alterations of their metabolism. These alterations correspond to metabolism of two amino acids (e.g. Lphenylalanine and L-tryptophan), bioactive lipid compounds (glutathione conjugated prostaglandin and neuroprostanes), purine metabolism (e.g. xanthine), tricarboxylic acid (TCA) cycle intermediates (succinate) and metabolism of fatty acids (hexanoylcarnitine and dodecenoylcarnitine). These variations could reflect potential effects on several of the biological processes discussed below, such as energy metabolism, including means to face increased energy demand and potential mitochondrial dysfunction, osmoregulation, oxidative stress defense and purine and tryptophan metabolism as discussed below.

It should be mentioned that the decrease in phenylalanine could also result from disturbed food intake under stress induction.

Energy and fatty acid metabolism

In the present study the decrease of four metabolites (L-phenylalanine, hexanoylcarnitine, trans-2dodecenoylcarnitine and succinate) for Zn exposed scallops, may be related with energy metabolism.

The decrease of phenylalanine may reflect its consumption as substrate for energy metabolism, in place of carbohydrates or lipids, which are used in normal circumstances. Indeed, phenylalanine is an amino acid being both glucogenic and ketogenic and it can be consumed under energy deficient conditions in aquatic invertebrates [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. In case of Zn exposure, this energy deficiency may arise from an elevated basal metabolic demand due to the need to develop protective mechanisms for the cells, such as overproduction of metallothioneins, glutathione, molecular chaperones, antioxidants pathways [START_REF] Cherkasov | Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica: linking cellular and mitochondrial responses[END_REF][START_REF] Ivanina | Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin[END_REF][START_REF] Sokolova | Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change[END_REF]. Moreover, toxic metals such as cadmium, copper, zinc and mercury have been shown to reduce mitochondrial efficiency and coupling and elevated proton leak in marine organisms [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. In the same vein, after 9 days of exposure with nominal Zn concentration of 30 μg.L -1 , oysters gills were analysed by proteomic analysis and it was concluded that Zn affected the TCA cycle by influencing the expressions of Fe-containing proteins and disrupted the electron transport chain by inhibiting complex I-IV related protein expressions [START_REF] Meng | Respiration disruption and detoxification at the protein expression levels in the Pacific oyster (Crassostrea gigas) under zinc exposure[END_REF]. It should be mentioned that other stress factors such as hypoxia, the presence of other pollutants or high temperature could concomitantly modify the effect of zinc on the metabolism of bivalves. Recently [START_REF] Belivermis | Effects of variable deoxygenation on trace element bioaccumulation and resulting metabolome profiles in the blue mussel (Mytilus edulis)[END_REF] showed that hypoxia diminished the Ag and Zn uptake in mussels and induced significantly greater abundance of several amino acids, amino sulfonic acids, dicarboxylic acids, carbohydrates and other metabolites compared to Zn exposed mussels in normoxia. [START_REF] Sokolov | Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas[END_REF] observed a decrease of protein synthesis during hypoxia in intertidal oysters Crassostrea gigas and interpreted this as an energysaving mechanism.

Two different acylcarnitine, hexanoylcarnitine and trans-2-dodecenoylcarnitine, were found to be downregulated in presence of zinc. The first derivative of carnitine (hexanoylcarnitine) is a medium-chain acylcarnitine, whereas the second one (trans-2-dodecenoylcarnitine) is a long-chain acylcarnitine. Long chain fatty acids have to be combined with carnitine to enter the mitochondria, where they undergo betaoxidation for energy production. In contrast, the medium-chain fatty acids can enter the mitochondrial membrane directly for beta-oxidation without the need of carnitine-mediated transport. Therefore, in our case, the defect of trans-2-dodecenoylcarnitine mainly affects the oxidation of the corresponding long-chain fatty acids. However, the metabolism of acylcarnitines is not only related to the transport of fatty acids, but also plays a key role in maintaining the homeostasis of the mitochondrial acyl-CoA/CoA ratio [START_REF] Connor | High-resolution analysis of metabolic cycles in intertidal mussel Mytilus californianus[END_REF][START_REF] Gracey | Transcriptional and metabolomic characterization of spontaneous metabolic cycles in Mytilus californianus under subtidal conditions[END_REF] and in other physiological processes such as peroxidation of fatty acids. Therefore, the observed change in acylcarnitine profile in presence of zinc may be related more generally with potential mitochondrial dysfunction and abnormal fatty acid metabolism.

Lastly, succinate was found to decrease upon Zn exposure. It is a metabolite intermediate of the TCA cycle, which is oxidized into fumarate by the iron-sulfur enzyme succinate dehydrogenase (SDH). Zn affects the TCA cycle by influencing the expressions of Fe-containing proteins [START_REF] Pagani | Disruption of iron homeostasis in Saccharomyces cerevisiae by high zinc levels: a genome-wide study[END_REF] and it is therefore so not surprising to observe that succinate is impacted by Zn in our study. What is more surprising is that the succinate appears in the present study to be down-regulated after Zn exposure. Indeed, in several previous studies, an accumulation of succinate was observed after Zn exposure : in the gills of Pacific oyster (Crassostrea gigas) exposed to excess Zn solution (30 μg.L -1 ) for 9 days [START_REF] Meng | Respiration disruption and detoxification at the protein expression levels in the Pacific oyster (Crassostrea gigas) under zinc exposure[END_REF], in the digestive gland of Ruditapes philippinarum exposed to ZnCl2 (nominal concentrations: 20, 50, 100 and 150 μg.L -1 ) for 48 h [START_REF] Aru | Metabolic responses of clams, Ruditapes decussatus and Ruditapes philippinarum, to short-term exposure to lead and zinc[END_REF] and in adductor muscles of the pedigree White clam of Ruditapes philippinarum after exposure with Zn 2+ (50 μg.L -1 ) for 48 h [START_REF] Wu | NMR-Based Metabolomic Investigations on the Differential Responses in Adductor Muscles from Two Pedigrees of Manila Clam Ruditapes philippinarum to Cadmium and Zinc[END_REF]. This accumulation of succinate was interpreted as molecular sign of the shift in cellular metabolism to anaerobiosis. It was explained in details by proteomic analysis, by a decrease in SDH expression and a simultaneously increase in enzyme succinyl-CoA synthetase (SCS) expression, which allows succinate biosynthesis in the TCA cycle, under Zn exposure of oysters [START_REF] Meng | Respiration disruption and detoxification at the protein expression levels in the Pacific oyster (Crassostrea gigas) under zinc exposure[END_REF]. Nevertheless, in the same study, fumarase (FUM), another Fe-S enzyme that converts fumarate to L-malate in the TCA cycle, showed significantly increased levels of expression at the same time.

This was interpreted as a compensation mechanism for ensuring that the TCA cycle remains active when exposed to metals [START_REF] Meng | Respiration disruption and detoxification at the protein expression levels in the Pacific oyster (Crassostrea gigas) under zinc exposure[END_REF]. This indicates that Fe-S enzymes of the TCA cycles can be either upor down-regulated upon Zn exposure and that defense strategy against Zn toxicity exists in bivalves, as it is the case with bacteria exposed to metals such as Al that interfere with Fe metabolism [START_REF] Chenier | Involvement of Fumarase C and NADH Oxidase in Metabolic Adaptation of Pseudomonas fluorescens Cells Evoked by Aluminum and Gallium Toxicity[END_REF].

In another study, two pedigrees (White and Zebra) of clam Ruditapes philippinarum were exposed with Zn 2+ (50 µg.L -1 ) during 48 h and the metabolomics results showed differences in digestive gland between White and Zebra clam samples in succinate levels: in Zebra clam, succinate was increased, whereas it was decreased in White clam after Zn exposure [START_REF] Ji | Toxicological evaluation of two pedigrees of clam Ruditapes philippinarum as bioindicators of heavy metal contaminants using metabolomics[END_REF]. In conclusion, it seems clear that Zn affects the TCA cycle metabolites, through its effect on Fe-S enzymes involved in the cycle. Concerning the level of succinate, it may be increased or decreased upon Zn exposure, depending on the species, with respect to over-or under-expression of the different Fe-S enzymes of the TCA cycle.

However, succinate has multiple roles in cell metabolism and its decrease may be linked among others with formation and elimination of reactive oxygen species, as discussed below.

Osmoregulation

Down-modulation of L-phenylalanine on the one hand and of prostaglandine-glutathione and neuroprostanes on the other hand, in Zn exposed scallops may be related to osmoregulation phenomena. Indeed, L-phenylalanine is a catecholamine precursor and its lower level may lead to a decrease in dopamine. Dopamine and serotonin are neurotransmitters involved in the modulation of Na + and K + transport in gills of crustaceans [START_REF] Mo | Dopamine as a modulator of ionic transport and Na+/K+-ATPase activity in the gills of the chinese crab Eriocheir sinensis[END_REF] and in lateral cilia in gills of bivalves [START_REF] Carroll | The nervous system control of lateral ciliary activity of the gill of the bivalve mollusc, Crassostrea virginica[END_REF].

The down-modulation of dopamine may thus have a repercussion on osmoregulation of cells submitted to Zn exposure. A similar relationship between L-phenylalanine decrease and osmoregulation was previously suggested [START_REF] Aru | Metabolic responses of clams, Ruditapes decussatus and Ruditapes philippinarum, to short-term exposure to lead and zinc[END_REF]. These authors observed a down-modulation of phenylalanine in presence of ZnCl2 (20, 50, 100 and 150 μg.L -1 ) under laboratory conditions after 48 h exposure for the clam Ruditapes decussatus, associated with a significant increment in organic osmolyte production (hypotaurine and homarine) and a decrease of the content of free amino acid.

Prostaglandins (PGs) are an important group of bioactive lipid compounds in living organisms; they are involved in mammals in a great variety of physiopathological processes like for instance inflammation, signaling or reproduction. These molecules play similar roles in marine invertebrates, being involved in the control of gametogenesis, ion transport, and defense. These molecules also represent very important lipid mediators in marine invertebrates [START_REF] Di Costanzo | Prostaglandins in Marine Organisms: A Review[END_REF]. PGs biosynthesis starts with arachidonic acid, a polyunsaturated fatty acid (PUFA), that is oxidised by enzymes such as cyclooxygenase or lipoxygenase to give precursors, which are then further transformed by a large number of possible enzymes, giving rise to the formation of different families of PGs, like PG J2 and PGA2. These last PGs may undergo addition reactions with glutathione (GSH) to give new reactive compounds in humans [START_REF] Murphy | Glutathione adducts of oxyeicosanoids[END_REF]. In the present study, among the metabolites showing a down regulation after 48 h Zn exposure, a compound putatively identified as S-(PGA2)-glutathione or S-(PGJ2)-glutathione was obtained. This would indicate that these types of conjugates are also present in bivalves and are impacted by exposure to Zn.

Isoprostanes (IsoPs) and the IsoP-like molecules are PG-like compounds formed in vivo via the nonenzymatic free radical-initiated peroxidation of a number of different PUFAs. Among them, neuroprostanes (MPs) are generated from the PUFA docosahexaenoic acid (DHA) [START_REF] Milne | Isoprostane Generation and Function[END_REF]. PGs, IsoPs and IsoP-like molecules have been identified recently in molluscs [START_REF] Bonnefille | Metabolomics assessment of the effects of diclofenac exposure on Mytilus galloprovincialis: Potential effects on osmoregulation and reproduction[END_REF][START_REF] Chakraborty | Response of pro-inflammatory prostaglandin contents in anti-inflammatory supplements from green mussel Perna viridis L. in a time-dependent accelerated shelf-life study[END_REF], which is not surprising given that PUFAs are essential molecules for the survival of bivalves [START_REF] Da Costa | Essential Fatty Acid Assimilation and Synthesis in Larvae of the Bivalve Crassostrea gigas[END_REF] and that they are highly susceptible to oxidation because of the large number of their double bonds.

The role of PGs, IsoPs and Iso-P like molecules in invertebrates has not yet been fully elucidated. Some studies show that they have a role in osmoregulation : PGE2 was shown to be involved in Na transport regulation in gills of the freshwater mussel Ligumia subrostata and also in clams [START_REF] Saintsing | Modification of sodium transport in freshwater mussels by prostaglandins, cyclic AMP and 5-hydroxytryptamine: effects of inhibitors of prostaglandin synthesis[END_REF] and it was shown that hyposmotic stress significantly increased prostaglandin synthesis in the marine bivalve Modiolus demissus [START_REF] Freas | Ioninc and osmotic influences on prostaglandin release from the gill tissue of a marine bivalve, Modiolus demissus[END_REF]. In the present case, a down-modulation of three compounds identified as neuroprostanes was obtained after Zn exposure and this may be linked also with osmoregulation. In fish, prostaglandins were shown to be involved in Cl -secretion [START_REF] Avella | Regulation of Clsecretion in seawater fish (Dicentrarchus labrax) gill respiratory cells in primary culture[END_REF] and effluent exposure resulted also in widespread reduction (between 50% and 90%) in PG profiles obtained through metabolomics in fish tissues and plasma [START_REF] David | Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics[END_REF].

Oxidative stress defense

Besides their possible role in the above mentioned osmoregulation, IsoPs and Iso-P like molecules have become biomarkers of choice in human for assessing endogeneous oxidative stress linked with many pathological disorders, because they are chemically stable [START_REF] Milne | Human Biochemistry of the Isoprostane Pathway[END_REF]. Oxidative stress is characterised by an imbalance between cellular antioxidant defenses and overproduction of free radicals, particularly reactive species of oxygen (ROS). Lipids are readily attacked by free radicals, resulting in the formation of a number of peroxidation products. In bivalves, like in other animals, there is a close relationship between environmental stress and the rate of cellular ROS generation and consequently lipid peroxidation reactions [START_REF] Regoli | Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms[END_REF]. In our case, the modification of the Iso-P like molecules profile could therefore be linked to an increased oxidative stress in the presence of Zn.

Another down-modulated metabolite under Zn exposure that may be linked with oxidative stress defense is succinate. The roles of succinate in mitochondrial ROS formation and elimination are numerous and complex [START_REF] Tretter | Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis[END_REF]. It was shown that succinate exerted an inhibitory effect on lipid peroxidation in mitochondria. This protective role of succinate against oxidation was attributed to the maintenance of a pool of reduced coenzyme Q. Succinate was oxidised by the membrane-bound succinate dehydrogenase and the enzyme activity was only moderately sensitive to oxidants, therefore it was more efficient in maintaining reduced coenzyme Q, thus maintaining membrane integrity.

Other metals and in particular copper are able to induce oxidative stress in bivalves. Cu 2+ -exposed haemocytes showed a remarkable increase in ROS production, which induced oxidative stress in mussel (Perna canaliculus) [START_REF] Nguyen | Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes[END_REF]. This was accompanied by a decrease in GSH. This compound is an important antioxidant, able to react with electrophilic oxidants (e.g., H2O2) resulting in conversion of two GSH molecules into its oxidized form (GSSG). Two other molecules decrease simultaneously upon Cu 2+ exposure: cysteine, which is involved in the GSH metabolic pathway and methionine, which takes place in the transsulfuration pathway, a source of cysteine for GSH. These concomitant phenomena were identified as important signatures of oxidative stress [START_REF] Nguyen | Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes[END_REF].

Purine metabolism

A lower level of xanthine in presence of Zn suggests a disordered nucleotide metabolism. Indeed, in the catabolism of purine nucleotides, adenosine monophosphate (AMP) and guanosine monophosphate (GMP) both lead to xanthine [START_REF] Van Waarde | Operation of the purine nucleotide cycle in animal tissues[END_REF]. AMP is degraded to inosine and then by phosphorolysis to hypoxanthine. In presence of xanthine oxidase (XOD), xanthine is formed from oxidation of hypoxanthine.

GMP is degraded to guanosine and then by phosphorolysis to guanine, which is transformed to xanthine by guanine deaminase. This makes the metabolic interrelationships between nucleotides and purine base nucleosides, and finally xanthine, very strong [START_REF] Harkness | Hypoxanthine, xanthine and uridine in body fluids, indicators of ATP depletion[END_REF]. Therefore, we can consider that xanthine is at the center of the interplay between ATP and GTP metabolism. Since the nucleotides ATP and GTP are the energy carriers molecules in cells, it can be hypothesised that zinc would induce an increased demand for energy by the cells.

Previously, a decrease of inosine (between -30 and -80%) and hypoxanthine (between -40 and -50%) was observed in whole body of zebrafish, after exposure to ZnCl2 at different concentrations (10, 100, 500 and 1000 µg.L -1 for 72 h). It was interpreted as an impairment of the purine metabolism caused by exposure to Zn. As it was combined with an ATP level increased, it was hypothesised that Zn would induce an increased energy demand to fulfil essential maintenance costs and ensure organism survival (e.g. cellular protective mechanisms, detoxification) [START_REF] Kim | Metabolic responses in zebrafish (Danio rerio) exposed to zinc and cadmium by nuclear magnetic resonance -based metabolomics[END_REF][START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF].

Tryptophan metabolism

In humans, L-tryptophan is metabolised by two main pathways : the serotonin pathway (around 1% of Ltryptophan metabolism) and the kynurenine pathway (around 95% of L-tryptophan metabolism). In the kynurenine pathway, L-tryptophan gives one of three main end products: kynurenic acid, picolinic acid and NAD. This pathway begins with the oxidative cleavage of L-tryptophan by either one of the two enzymes indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase also called tryptophan pyrolase (TDO) to produce L-formylkynurenine, which is then converted to L-kynurenine by a formamidase. L-kynurenine is a branch point and can be converted to the following: 1) kynurenic acid by kynurenine aminotransferases; 2) 3-hydroxykyruninine by kynurenine 3-monooxygenase; or 3) anthranilic acid. 3-hydroxykyruninine further undergoes enzymatic or chemical transformations to give either picolinic acid or NAD [START_REF] Grant | The Physiological Action of Picolinic Acid in the Human Brain[END_REF].

The kynurenine metabolism was shown to be highly conserved throughout evolution and the enzyme IDO was discovered in mammals, lower vertebrates, several invertebrates, fungi and a number of bacterial species [START_REF] Cervenka | Kynurenines: Tryptophan's metabolites in exercise, inflammation, and mental health[END_REF].

In the present study, a down-regulation of both picolinic acid and kynurenine was observed after Zn exposure, showing an impact of this metal on tryptophan metabolism. This trend was also observed in another study with the marine bivalve Mytilus galloprovincialis, when exposed to a wastewater treatment plants effluent extract: both L-formylkynurenine and L-kynurenine were found to be down-regulated (respectively -55%, p ≤ 0.05 and -63%, p ≤ 0.05) compared to non-exposed animals thanks to a non-targeted metabolomics study [START_REF] Dumas | Metabolomics approach reveals disruption of metabolic pathways in the marine bivalve Mytilus galloprovincialis exposed to a WWTP effluent extract[END_REF].

In another study using proteomic analysis with the pacific oyster Crassostrea gigas the enzyme kynurenine formamidase was found to be down-regulated under aerial exposure [START_REF] Zhang | Proteomic Basis of Stress Responses in the Gills of the Pacific Oyster Crassostrea gigas[END_REF], which again corresponds to an impairment of the tryptophan metabolism. This was accompanied by the suppression of many physiological activities and this was interpreted as a means of redistributing available resources to fuel stress responses [START_REF] Zhang | Proteomic Basis of Stress Responses in the Gills of the Pacific Oyster Crassostrea gigas[END_REF].

Conclusion

In the present study, we chose the variegated scallop Mimachlamys varia, for assessing zinc contamination effects on their metabolism, at a concentration such as the one the metal can reach in La Rochelle marina. An untargeted UHPLC/QToF tandem mass spectrometry metabolomics approach was chosen, as it offers a high level of sensitivity to highlight impacts due to short-time metal exposure. Metabolite profiling of gills tissues could clearly separate scallops with no metal inputs and those exposed to 48 h zinc exposure, under laboratory conditions. Among the high quantity of modulated metabolites, only a few of them were identified, because of the current lack of knowledge on scallop metabolism. They include amino acids or their metabolites, bioactive lipid compounds, including four metabolites never before identified in bivalves, metabolites of purine or the TCA cycle and carnitine conjugates of fatty acids. These molecules reflect potential zinc effects on several of the biological processes, such as energy metabolism, osmoregulation, defense against oxidative stress, metabolism of purine and tryptophan.

This study demonstrates once again that metabolomics approach, when used in untargeted mode is able to reveal novel and unanticipated physiological perturbations, without a priori knowledge of the metabolome. This is valuable in the present case where a poorly studied animal model is used. The limitations of the technique come from the fact that there are a large number of unknown metabolites that remain unannotated in the databases, so that some metabolite features cannot be attributed to a molecular structure. The development of new methods dedicated to the identification of metabolites on the one hand and to the search for the connectivity of metabolic pathways on the other hand, combined with the enrichment of databases, will make it possible in the future to carry out more and more advanced impact analyses.

Moreover, further study is needed to assess the effect of other trace elements present in the marine environment and better understand the combined effects of pollutants. La Rochelle marina sets up multiple actions to ensure a sustainable development of coastal activities while preserving the aquatic environment. These actions, framed by the "Ports Propres" AFAQ certification (AFNOR : Association française de normalisation), are based on criteria approved at the European level by the CWA 16987 agreement of the "Clean Harbour Guidelines". Among them, some aim to develop approaches to highlight the physiological modifications of marine organisms impacted by environmental variation. Our results show that metabolomics is a powerful method that can brings out small environmental variations, such as zinc concentration, through metabolic changes in scallops.

FIGURE CAPTIONS

Figure 1: Score plots of PCA and PLS-DA for negative mode (LC-Negative), positive mode (LC-Positive) of liquid chromatography. Ellipses score plots represented the confidence limit (95%) of Hotelling's T2 statistic (Green ellipse for reference samples and red ellipse for Zn-Exposed samples). Figure 2: Box plots of the relative abundance of the 11 identified metabolites in Mimachlamys varia gills showing a significant difference between reference and Zn-exposed samples. Green plots represent the reference samples, red plots represent the zinc exposed samples. The mean value is given by the dark diamond-shaped symbol.

  Figure 1

Acknowledgements

We gratefully thank E. Dubillot (from laboratory "Littoral Environnement et Sociétés" (LIENSs), UMR 7266, CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, F-17042 La Rochelle Cedex 01, France) for technical assistance. We also thank the Marina of La Rochelle for their assistance in particularly for the data transmission and the "Musée Mer Marine Bordeaux" for the involvement in the V. HAMANI PhD.

Funding sources

Funding for this work came from the following separate sources: (i) Contrat de Plan Etat-Région FEDER and the CNRS ECONAT Axe 1 -Ressources Marines Littorales: qualité et éco-valorisation and Axe 2 -Gestion Intégrée des Zones Littorales et Portuaires (ii) French Ministry of Higher Education, Research and Innovation for doctoral grant of V. Hamani (Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation, MESRI).