A Barrick

I Barjhoux

J M Marion

A Châtel

H Perrien-Ettajani

M Mouloud

P Gillet

I Mettais

C Mouneyrac

Investigating the Impact of Remediation Efforts on Hediste diversicolor in the Seine Estuary Using Multiple Levels of Biological Organization

The Seine Estuary is historically one of the most contaminated estuaries in Europe. In 2002 the estuary underwent major redevelopment which emphasized environmental remediation to measure biological effects. The present study investigated the health status of the sentinel species Hediste diversicolor in the Seine estuary by comparing data from a sampling campaign immediately after the Seine underwent reconstruction and 10 years afterward. Both studies implemented multiple levels of biological organization ranging from enzymatic biomarkers to population density. Integrative modeling was used to establish a holistic status assessment for H. diversicolor in the Seine. H. diversicolor populations in the Seine estuary were impacted in both campaigns suggesting no significant improvements over the 10-year period. This interpretation however can be improved by increasing the number of reference sites used to establish the baseline natural variation, integrating additional environmental parameters into the model and measuring additional biological endpoints, such as reproductive status.

Introduction

The Seine Basin in France represents a catchment area of approximately 78,650 km² and encompasses 40% of the French economic activity including intensive agricultural activities, strong industrialization, and significant urbanization [START_REF] Billen | The Seine system: Introduction to a multidisciplinary approach of the functioning of a regional river system[END_REF]. Due to a long history of human activity, the Seine estuary is considered one of the most contaminated water systems in Europe with a wide variety of persistent pollutants in the environment including metals, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) [START_REF] Blanchard | Persistent toxic substance inputs to the river Seine basin (France) via atmospheric deposition and urban sludge application[END_REF][START_REF] Hamzeh | Trace Metal Mobilization from Surficial Sediments of the Seine River Estuary Trace Metal Mobilization from Surficial Sediments of the Seine River Estuary 0-15[END_REF][START_REF] Thévenot | Critical budget of metal sources and pathways in the Seine River basin (1994-2003) for Cd, Cr, Cu, Hg, Ni, Pb and Zn[END_REF]. This results in a complex mixture of anthropogenic contamination that arises in short-term and long-term impacts on ecosystem health and makes effective mitigation difficult. The Seine estuary is a major water system that is crucial for the life history of many invertebrates, fish, and birds, making it crucial to investigate the severity of anthropogenic impacts making anthropogenic contaminants an ecological concern. Starting in 2002, the estuary underwent major redevelopment and rehabilitation work (dredging to improve water circulation, reconstruction of meanders, prolongation of breakwaters, and creation of an artificial island) under the Port 2000 project which extended the harbor and closed out the northern flank of the estuary [START_REF] Tecchio | Evaluating ecosystem-level anthropogenic impacts in a stressed transitional environment : The case of the Seine estuary[END_REF]. The construction of the port fundamentally shifted hydrology and deposition of sediments into the ecosystem which may have mixed effects on organisms [START_REF] Dauvin | Recent changes in estuarine benthic and suprabenthic communities resulting from the development of harbour infrastructure[END_REF]. Sediments are a sink for numerous persistent compounds and reworking sediments could potentially reintroduce sequestered contaminants into the ecosystem and threaten both benthic and pelagic organisms [START_REF] Fisson | Qualité des eaux de l'estuaire de la Seine[END_REF]. The project to expand the port integrated ecological considerations in an attempt to mitigate anthropogenic impacts but major changes in hydrology and sedimentary characteristics of the estuary caused clear modifications in benthic communities [START_REF] Cuvilliez | Geomorphology Morphological responses of an estuarine intertidal mud fl at to constructions since 1978 to 2005[END_REF][START_REF] Dauvin | Benthic indicators : From subjectivity to objectivity -Where is the line ?[END_REF].

Investigating environmental impact of such a development plan, it is functionally and economically unrealistic to screen for every potential contaminant, and implementing biological tools may be more cost-effective [START_REF] Amiard | Les biomarqueurs dans l'évaluation de l'état écologique des milieux aquatiques[END_REF]. To assess the health status of the Seine estuary it was proposed to use biomarkers addressing multiple levels of biological organization from sub-individual endpoints to population dynamics, as biomarkers may provide early warning tools to identify stress caused by contaminants [START_REF] Amiard | Les biomarqueurs dans l'évaluation de l'état écologique des milieux aquatiques[END_REF]. The effectiveness of biomarkers in biomonitoring frameworks is recognized but their deployment is limited risk assessment strategies due the wide range of responses that can be observed and the effect this has on interpretation of impact. Concentrations of anthropogenic contaminants can be determined by chemical analysis to identify if harmful compounds are present at concentrations above the natural background making it comparatively easy, when coupled with biological response, to identify whether or not a site is contaminated and if amelioration efforts improve environmental conditions.

To assess the influence of the Port 2000 construction on the Seine estuary, the National Ecotoxicology Program (PNETOX) conducted an environmental risk assessment survey during the construction of the harbour (2002)(2003)(2004). The project implemented a multitude of biotic and abiotic endpoints and compared values to the Authie estuary. At the time of the initial study, Authie was identified to be an estuary with similar environmental parameters, equivalent latitude and comparatively lower levels of contamination [START_REF] Durou | Environmental quality assessment in estuarine ecosystems : Use of biometric measurements and fecundity of the ragworm Nereis diversicolor ( Polychaeta[END_REF]. Industrialization is also absent in the Authie estuary but low levels of metal and PAH contaminant are present and, in the context of the study, was considered an ideal comparator for assessing the quality Seine estuary [START_REF] Francescangeli | Multidisciplinary study to monitor consequences of pollution on intertidal benthic ecosystems (Hauts de France, English Channel, France): Comparison with natural areas[END_REF]. Hediste diversicolor (H. diversicolor), a sediment-dwelling polychaete worm, was selected for the study due to its status as a keystone, sentinel species with a broad geographic distribution (Durou et al., 2007a;[START_REF] Kalman | Assessment of the health status of populations of the ragworm Nereis diversicolor using biomarkers at different levels of biological organisation[END_REF][START_REF] Thit | Bioaccumulation, subcellular distribution and toxicity of sediment-associated copper in the ragworm Nereis diversicolor: The relative importance of aqueous copper, copper oxide nanoparticles and microparticles[END_REF]. The sampling survey investigated the impact at different levels of biological organization on H. diversicolor [START_REF] Amiard-Triquet | Environmental Assessment of Estuarine Ecosystems: A Case Study[END_REF]. The results of the project indicate that biomass and density of the suprabenthos declined between 2002 and 2004 (approximately 80%) and that population levels of H. diversicolor diminished too [START_REF] Gillet | Response of Nereis diversicolor population ( Polychaeta , Nereididae ) to the pollution impact e Authie and Seine estuaries[END_REF][START_REF] Tecchio | Evaluating ecosystem-level anthropogenic impacts in a stressed transitional environment : The case of the Seine estuary[END_REF]. The study also highlighted evidence linking anthropogenic activities in the Seine estuary to sub-individual effects in H. diversicolor [START_REF] Durou | Environmental quality assessment in estuarine ecosystems : Use of biometric measurements and fecundity of the ragworm Nereis diversicolor ( Polychaeta[END_REF]Durou and Mouneyrac, 2007). Previous studies have demonstrated that benthic communities show a steep decline after major alterations and that longer periods postconstruction are necessary to verify the effectiveness of remediation efforts [START_REF] Meng | Responses of macroinvertebrates and local environment to short-term commercial sand dredging practices in a flood-plain lake[END_REF]. It can also be unclear how to interpret data from multiple levels of biological organization in a clear and concise interpretation of environmental health. When considering the response of organisms however, it can be unclear how to interpret data from multiple levels of biological organization and create clear conclusions on the health status of organisms in a polluted site.

To follow up on the longevity of adverse effects of pollution in the Seine estuary, project ECOTONES (2015-2017) was initiated approximately 10 years later measuring the same biological endpoints as PNETOX. Project ECOTONES aimed to assess if the overall health of H. diversicolor in the Seine estuary improved because of remediation efforts. Interpreting organismal responses using multiple levels of biological organization can often be unclear, leading to inconsistent interpretations of health status in contaminated estuaries. Applying a method of biomarker response integration that weights biological endpoints based on the level of biological organisation can potentially be used to establish concise conclusions on health status.

One of the proposed methods to integrate biological responses at different levels of organization is based on the use of a line of evidence (LOE) for biomarkers [START_REF] Barjhoux | Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River[END_REF][START_REF] Piva | Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: A practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays[END_REF]. This LOE model compiles biological responses at multiple levels of organization into an integrated hazard quotient (HQ) associated with a hazard class ranking from absent (no observable biological impact) to severe (significant important impact), allowing for the comparison of temporal biomarker datasets through quantitative (HQ values) and qualitative (hazard classes) methods. Six biological endpoints were used in the present study: oxidative stress biomarkers (catalase (Cat), glutathione-s-transferase (GST), and thiobarbituric reactive substances (TBARs)), neurotoxicityspecific response (acetylcholinesterase activity (AChE)), energy reserves (total lipid and glycogen content) and population density. For each integrated endpoint, reference level and thresholds were derived from the baseline assessment criteria (BAC) and the environmental assessment criteria (EAC) previously defined (Barrick et al., 2018a[START_REF] Barrick | A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor[END_REF] which integrate the effects of confounding factors (salinity, weight of individuals and temperature) on the studied biomarkers into the analysis.

Materials and methods

Sample site and collection

The Seine estuary in northern France is a system influenced by high tides and the estuary is split in three spatial areas: the Northern Flank (NF), the Central Channel (CC), and the Southern Flank (SF) (Figure 1). Both PNETOX and ECOTONES sampling campaigns were carried out in the NF region of the estuary. In both projects, H. diversicolor individuals were collected in the intertidal zone at low tide in the Seine estuary (49°27′17.52″ N and 0°11′19.57″ E). Historical data for the spring, summer, and fall seasons of the 2002-2004 period in the Seine estuary were collected from previous studies (Durou et al., 2007a;Durou and Mouneyrac, 2007;[START_REF] Gillet | Response of Nereis diversicolor population ( Polychaeta , Nereididae ) to the pollution impact e Authie and Seine estuaries[END_REF].

Sampling for ECOTONES was carried out two times each year (spring and summer for 2015-2017). The temperature and the salinity of water remaining on the mudflat surface were recorded using a field thermometer (CG867) and a salinometer (WTW LF 196). Biological endpoints, time points and sampling protocols comparable to the historical data generated in PNETOX. To analyze density, four quadrats (625 cm 2 and 25 cm deep) were collected during each sampling campaign [START_REF] Ait Alla | Response of Nereis diversicolor ( Polychaeta , Nereidae ) populations to reduced wastewater discharge in the polluted estuary of Oued Souss , Bay of Agadir[END_REF][START_REF] Gillet | Response of Nereis diversicolor population ( Polychaeta , Nereididae ) to the pollution impact e Authie and Seine estuaries[END_REF]. Quadrats were then sieved to collect organisms (200µm mesh size) and organisms retained were collected and stored in 70% ethanol. For biomarker analyses, samples were collected gently by hand in parallel with the collection of samples for density. Twenty sexually immature worms of equivalent size (0.11±0.03g wet weight) were collected and individually analyzed for enzymatic biomarkers (AChE, GST, Cat, and TBARs) and twenty worms of equivalent size were collected and individually analyzed for energetic reserves (glycogen and lipids). Samples were immediately frozen in liquid nitrogen, transported to the laboratory, and stored at -80°C until analysis.

Biomarker analysis

Biomarkers in the present study were analyzed following protocols used in the PNETOX program. Briefly, enzymatic biomarker responses were assessed using the techniques described in Durou et al., 2007. Samples were individually homogenized in buffer solution (0.08 M Tris, 2 M NaCl, 1 mM of 1,4-Dithiothreitol, and cocktail protease inhibitor diluted to 1:1000 at pH 7.4 and 4 °C) in a 1:3 ratio of tissue to the solution using a mortar and pestle. The homogenates were then centrifuged for 30 minutes at 9000×g. Total protein content was calculated by following the Bradford assays [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF][START_REF] Galgani | Semi-automated colorimetric and enzymatic assays for aquatic organisms using microplate readers[END_REF]. GST activity was determined spectroscopically at 340nm by measuring the rate that 1-choloro-dinitrobenzene is conjugated with reduced glutathione [START_REF] Habig | Glutathione S-Transferases[END_REF]. Cat activity was calculated by measuring the consumption of H2O2 spectroscopically at 240 nm. Results were expressed as µmoles.min -1 .mg of protein -1 [START_REF] Aebi | Oxygen Radicals in Biological Systems[END_REF]. AChE activity was calculated by using a coupled reaction measuring the catalyzation of acetylthiocholine to produce thiocholine (which is then reduced by 5,5'-Dithiobis(2-nitrobenzoic acid) to produce thionitrobenzoate [START_REF] Bocquené | Characterisation and assay conditions for use of AChE activity from several marine species in pollution monitoring[END_REF]. The rate of the reaction was measured by colorimetric spectroscopy. Lipid peroxidation was measured by the formation of thiobarbituric reactive substances quantified by malondialdehyde absorbance at 530 nm [START_REF] Knight | Specificity of the thiobarbituric acid reaction: its use in studies of lipid peroxidation[END_REF].

Energetic reserves were analyzed following the protocol previously described in Durou et al. (2007). Individuals were ground into a powder using liquid nitrogen and then prepared as a homogenate in a citrate buffer (0.1 M, pH 5.0). Total lipid content was determined following the method defined in [START_REF] Frings | ImprovedDeterminationof Total Serum Lipids by the Sulfo-Phospho-Vanillin Reaction[END_REF]. Glycogen content was determined by partitioning the sample in two (with or without enzymatic digestion by amyloglucosidase) following the protocol described in (Carr and Neff, 1984a).

Data integration within Biomarker index and development of LOE model

To establish a baseline that accounted for natural variation, the method described in [START_REF] Barrick | A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor[END_REF] was applied. Multi-polynomial regression models were previously developed for each biomarker to integrate confounding factors (salinity, temperature, and weight of individual) into the analysis, establishing thresholds of expected natural variation using data from the Authie estuary as a reference point. Authie was used as the reference point for this study to maintain comparison with the historical data and, while the use of several reference sites is ideal, there is limited comparable data available for other estuaries. For each regression model, a baseline assessment criterion (BAC) was used to define the expected range of variation that would occur naturally in healthy individuals. A banding approach was used to define the threshold of disturbance, also called environmental assessment criterion (EAC), with twice the lower limit of the BAC used for stress-induced inhibition endpoints (AChE, energy reserve assays, and density) to indicate the biomarker is severely impacted. Formulas were previously defined for biomarkers (AChE, GST, CAT, TBARS, glycogen, and lipids) in [START_REF] Barrick | A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor[END_REF]Barrick et al. ( , 2018)). For population density, a regression model was calculated following the same method. BAC/EAC values were established for each sampling periods in the Seine estuary using confounding factors measured during sampling periods for both projects.

To characterize in situ biological responses for H. diversicolor for integrated biomarker responses an LOE model was generated following a previously defined method with some adjustments to fit data collected [START_REF] Barjhoux | Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River[END_REF][START_REF] Piva | Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: A practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays[END_REF]. The application of the LOE model requires the establishment of reference values (or control values), thresholds (Th) values indicating the range of expected natural variation, and an effect threshold above which physiological disturbances are expected to occur. Mean values from the BAC models were used as reference values and the Th value was calculated from the lower or upper limits of the BAC depending on how the biological responses change under stress conditions, i.e. the 'ecologically relevant' direction of biomarker modulation. Effect threshold values were derived from EAC values for each biological endpoint. In the LOE model, only unilateral modulations were taken into consideration. Details of the LOE parametrization and calculations are presented in the supplemental material and summarized below.

To further develop the model, measured responses were expressed as a percentage change over the respective reference value and divided by the Th value. A correction factor was applied to the resulting ratio using the following method: a 0.2-factor for values less than or equal to the Th values, a 0.5-factor for responses above the Th value but lower than the effect threshold, and a 1factor for values that exceeded the effect threshold. These calculations resulted in an 'effect' value E(i) for each biological response which was reported on an evaluation grid for hazard classification ranging from absent to severe. E(i) values were then weighted (Ew(i)) according to the biological significance of the considered biomarker following the approach described in [START_REF] Piva | Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: A practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays[END_REF]: a weight of 0.75 was applied to TBARS and AChE, a 0.5-weight was assigned to CAT and GST, a 1.2 weight was given to lipid and glycogen, and a weight of 2.8 for density measurements as an intermediate value between mortality (3.0), histopathology and reproductive failure (2.4).

Finally, a cumulative biomarker hazard quotient (BiomHQ) was generated by calculating the weighted average of the Ew(i) values of biomarkers attributed to the moderate class of hazard and summing the Ew(i) values with responses belonging to the major or the severe class of hazard. The contribution of each endpoint and class of endpoint to the BiomHQ value was also calculated as described by [START_REF] Barjhoux | Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River[END_REF]. Another global index (BiomHQ index) was also generated by adding the percentage of biomarkers divided into the different classes of hazard weighed with coefficients increasing with the attributed hazard (from 0.7 for absent to 8 for severe; according to [START_REF] Piva | Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: A practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays[END_REF]. This new index was reported on an evaluation grid for a global hazard classification (absent to severe).

Results

When assessing the health status of H. diversicolor using individual biomarker hazard rankings many of the biological endpoints demonstrated wide variations ranging from "absent" to "severe" between sampling campaigns (Table 1). Of the endpoints used in the present study TBARS, glycogen and density were the endpoints the indicated stress. Annually, hazard rankings for the PNETOX campaign suggested stress response for TBARS, AChE, GST, catalase, lipids, glycogen and density for 2002 whereas in 2003 AChE, GST, glycogen and density indidicated stress and in 2004 only GST and density demonstrated a stress response. For project ECOTONES TBARS, AChE, lipids, glycogen and density consistently suggested a stress response for 2015-2017 with catalase indicating moderate stress in 2015.

Individual biological endpoints were then weighted following the approach used in [START_REF] Piva | Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: A practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays[END_REF] and results were integrated into biomarker hazard quotients (BiomHQ) to classify stress response (Figure 2). This index represents the cumulative response profiles of all biological endpoints reletative to reference values and can be interpreted as a quantitative indicator of the intensity of deviation from expected values (ie out of the range of basal variations defined by the reference and the threshold Th values). Results from the PNETOX program showed limited variations of the BiomHQ values, with maximal values (13-15.5) observed between July 2002 and February 2003 and the lowest (0-3) between November 2003 and May 2004. On the contrary, BiomHQ values calculated with ECOTONES results proved to be generally higher and more variable with indices reaching about 20 and 35 in March 2015 and July 2015, respectively. Individual BiomHQ values are calculated based on the number of biomarker responses/ severity of response for each data set. In some cases this leads to lower values indicating higher stress response (e.g. 2002 vs 2003). The BiomHQ index, a more qualitative description of the biological disturbance observed at each sampling time, was calculated according to the weighted distribution of biomarkers within the different classes of risk attributed for each E(i) value. This second index is used to attribute the risk classification for each sampling period and the annual averages (see figure S1 in the supplemental material).

The contribution of each biomarker to the interpretation of hazard were also assessed (Figure 3). In many of the sampling campaigns for PNETOX, oxidative stress (mainly TBARS and GST), neurotoxicity (AChE), and populational (density) markers were major contributing factors to the BiomHQ values. Other responses such as energetic reserves and CAT also influenced the level of hazard in some of the PNETOX sampling campaigns but more punctually. For the ECOTONES sampling campaign, TBARs were a major contributor in defining the health status for H. diversicolor the Seine estuary. Energetic reserves contributed more to the hazard classification in the ECOTONES sampling campaign compared to the PNETOX samples. AChE, CAT, and population density also factored into the level of environmental hazard in ECOTONES but their contribution was dependent on the sample period and no clear profile could be identified.

Annually, the population density was a major indicator for 2003 and 2004 whereas TBARS, neurotoxicity, and energetic reserves (in particular glycogen) were the most contributive biological endpoints for 2002 and 2015-2017. As observed for the first PNETOX campaigns, the highest BiomHQ values are associated with more complex contribution profiles indicating that impact can be detected at different levels of response.

Discussion

Polychaete worms are ideal target organisms for monitoring ecosystem health as they are sensitive to anthropogenic activity and play a crucial role as contaminant vectors across the trophic chain [START_REF] Green Etxabe | Identifying conserved polychaete molecular markers of metal exposure: Comparative analyses using the Alitta virens (Annelida, Lophotrochozoa) transcriptome[END_REF]. The objective of the present study was to investigate if remediation efforts in the Seine estuary had improved the health status of H. diversicolor in a way that accounts for natural variatio using a multi-biomarker approach following an integrative method of analysis.

The BAC/EAC model attempts to account for effects environmental parameters (temperature, salinity and weight) on the biological endpoints used in the study by defining the range of values expected to naturally occur for each endpoint. The results of the model demonstrated large fluctuations seasonally and annually for all biological endpoints, suggesting that natural variation may not be completely accounted for and that longer time series may be necessary to characterize this relationship more appropriately. Additional research is necessary to further clarify the complex relationship between biological endpoints, natural variation and effects of anthropogenic activity [START_REF] Galgani | Semi-automated colorimetric and enzymatic assays for aquatic organisms using microplate readers[END_REF]. Measurement of temperature and salinity in the ECOTONES project were measured to conform to the how data was collected in the PNETOX campaign and measured environmental temperature and salinity at the time of sampling. Using an average temperature and salinity across several timepoints might improve the model's ability to predict how these parameters influence measured biological endpoints. Integrating additional parameters such as granulometry, available organic matter and reproductive status can also significantly improve the model as these endpoints can alter metabolic activity which, in turn, can influence subindividual endpoints [START_REF] Díaz-Jaramillo | Multibiomarker approach at different organization levels in the estuarine Perinereis gualpensis (Polychaeta; Nereididae) under chronic and acute pollution conditions[END_REF].

Annually PNETOX data indicated less stress in 2003 and 2004 when compared to the initial sampling in 2002 which would be consistent with recovery after a major environmental perturbation like the construction of the port. Data for the ECOTONES campaign was relatively consistent 2015-2017 which suggested the site was majorly impacted. This observation would indicate that despite the 10-year difference between the two projects, H. diversicolor populations in the Seine estuary were still majorly impacted and effects of contamination likely persistent and chronic (please see supplemental materials for data regarding measured contaminants present. The site used to collect H. diversicolor in the Seine estuary is an active shipping zone and has been demonstrated to have high concentrations of PAHs, PCBs and organocholorine pesticides which have been previously, and proportionally, accumulated in H. diversicolor (Durou et al., 2007a;[START_REF] Lesueur | Ecotoxicology and Environmental Safety Toxicity of sediment-bound pollutants in the Seine estuary , France , using a Eurytemora af fi nis larval bioassay[END_REF]. The Seine estuary also has high concentrations of metal in the sediment with previous studies indicating a poor correlation between metal concentrations in the environment and concentrations in H. diversicolor [START_REF] Amiard-Triquet | A comprehensive methodology for the ass essment of the health status of estuarine ecosystems[END_REF][START_REF] Berthet | Accumulation and Soluble Binding of Cadmium , Copper , and Zinc in the Polychaete Hediste diversicolor from Coastal Sites with Different Trace Metal E nvironmental C ontamination Accumulation and Soluble Binding of Cadmium , Copper , and Zinc in the Polyc[END_REF][START_REF] Saiz-Salinas | Nereis diversicolor : an Unreliable Biomonitor of Metal Contamination in the ' Ria de Bilbao ' ( Spain )[END_REF]. Nonetheless, chronic exposure of H. diversicolor to both persistent organic pollutants and metallic trace elements present in the sediment of the Seine estuary may be responsible for the effects highlighted by the LOE model.

Biological endpoints used in the study account for a wide range of responses including neurotoxicity, oxidative stress, energetic reserves, and population density. Surprisingly, endpoints that consistently suggested adverse effects were mainly TBARS, glycogen and density suggesting that H. diversicolor is impacted at multiple levels of biological organization. Results also indicated that, of the subcellular endpoints, TBARS was one of the more consistent biomarkers. One potential explanation for this observation is that TBARS are indicative of damage and suggest that defense mechanisms are consistently overwhelmed in the Seine estuary, implying chronic effects. Enzymatic biomarkers (AChE, GST and Cat) displayed seasonal patterns which is unsurprising as the previous BAC/EAC models indicated a wide range of natural variation (Barrick et al., 2018b;[START_REF] Beliaeff | Exploratory data analysis of the Mediterranean component of the BEEP programme[END_REF][START_REF] Hagger | Biomarkers and Integrated Environmental Risk Assessment : Are There More Questions Than Answers ?[END_REF]. It has also been demonstrated that enzymatic biomarkers can be influenced seasonal variation in solar exposure and adjustments to energetic budgets [START_REF] Díaz-Jaramillo | Multibiomarker approach at different organization levels in the estuarine Perinereis gualpensis (Polychaeta; Nereididae) under chronic and acute pollution conditions[END_REF]. Future adjustments may need to consider a piece-wise integrative model in order to investigate the relationship between levels of biological organization at specific time points, in particular the relationship between subcellular responses and dynamic energy budgets .

When considering the results of energetic reserves, the LOE model suggested glycogen levels are impacted in the Seine estuary, which is consistent with the BAC model previously generated [START_REF] Barrick | A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor[END_REF]. Glycogen also had a considerable contribution to the interpretation of environmental health status suggesting that it may be a useful endpoint in identifying sites adversely impacted by anthropogenic activity. With respect to lipids there was some evidence suggesting moderate stress but overall was less impacted than glycogen. This may suggest that glycogen is more responsive to environmental condition which may make it an ideal endpoint for measuring anthropogenic activity. One potential explanation for this is that glycogen is the primary energy resource (maintenance, reproduction, growth) when accounting for environmental stress (Carr and Neff, 1984b). It is also important to consider that energetic reserves are highly dependent on the reproductive status of the organisms and that contamination may alter biological clocks [START_REF] Pook | The metabolic and fitness costs associated with metal resistance in Nereis diversicolor[END_REF]. Previous work on the PNETOX data demonstrated that the sexual reproductive status of H. diversicolor in the Seine were equivalent to individuals in Authie, suggesting an allocation of energy towards reproduction rather than growth (Durou et al., 2007b). Integration of reproductive status into the model would be beneficial as it would help characterize the effects of this relationship on other biological endpoints.

Population density values consistently fell outside the range of expected natural variation, providing evidence that the Seine is still majorly impacted and potentially influencing the ecosystem. This is consistent with a previous interpretation which suggested that low population densities may be due to irrigation and sediment reworks in the Seine estuary [START_REF] Gillet | Response of Nereis diversicolor population ( Polychaeta , Nereididae ) to the pollution impact e Authie and Seine estuaries[END_REF]. It is however important to note that polychaete population densities can display significant variations across time and space [START_REF] Kristensen | Life cycle, growth and production in estuarine populations of the polychaetes Nereis virens and N. diversicolor[END_REF]. Despite Authie being considered a good comparator estuary for the Seine intersite differences, such as nuances in sediment texture and algal cover, can potentially make it an unreliable comparator site for density assessments [START_REF] Alzeny | Spatio-temporal population and reproductive responses in Hediste diversicolor (Polychaeta: Nereididae) from the Damietta Harbor Barge Canal[END_REF]. Integrating multiple references sites and accounting for these environmental parameters can potential improve the quality of the model. Perturbations to the density of H. diversicolor indicated impact during the PNETOX sampling campaign may be attributed to immediate alterations to the sediment dynamics as result of the construction process rather than the presence of contamination. The results of the model suggested 'moderate' to 'major' effects towards H. diversicolor density across the 10-year period, suggesting the site is still impacted. Previous research on macrofaunal composition in the Seine estuary are resilient to anthropogenic perturbation and one potential explanation for the interpretation of the model in the present study is that the response profile is localized [START_REF] Bacouillard | Long-term spatio-temporal changes of the muddy fine sand benthic community of the Bay of Seine ( eastern English Channel[END_REF]. Future alterations to the model should consider including multiple sampling sites in the Seine estuary.

No true reference values exist but the model attempts to account for expected environmental variation through the application of a BAC/EAC index. While this approach allows for clear, concise results for communicating environmental health to regulatory bodies, it is important to note that additional research is necessary to clarify the complex relationship between biological endpoints, natural environmental variation and effects of anthropogenic activity [START_REF] Galgani | Semi-automated colorimetric and enzymatic assays for aquatic organisms using microplate readers[END_REF]. Some of the endpoints used in the model demonstrated clear seasonal patterns indicating that longer time series may be necessary. It also necessary to look at additional endpoints linked to seasonal variation such as nutrient load, turbidity, and sediment transport.

The LOE model establishes a first step in developing a comprehensive model to provide an holistic assessment of H. diversicolor to improve the assessment of health status. Improvements can be made to the initial model by including other biological endpoints such as genotoxicity, reproductive success, or scope for growth. This was not done in the present study due to a lack of necessary data to develop relevant BAC indices for these endpoints. In addition to this, different biological endpoints have varying degrees of sensitivity to environmental disturbance and it may be necessary to clarify these relationships in future studies. Through the integration of population density into the LOE model it becomes possible to extrapolate how sublethal endpoints can lead to adverse effects on the population. Future research will need to expand on the relationship between endpoints as well as how environmental parameters may influence these interpretations.

Conclusion

"The proposed model provides a holistic assessment of the status of H. diversicolor individuals in the Seine estuary but to be effective there needs to be effective integration with other LOEs, in particular sediment type and concentrations of contaminants, to effectively establish a weight of evidence approach that characterizes environmental quality. The present study proposes a first attempt at developing an LOE model for H. diversicolor that integrates subcellular endpoints, subindividual and populational effects to establish a holistic approach to environmental health. This approach was applied to biological responses analyzed in the sentinel species H. diversicolor following two long term studies in the Seine estuary. The LOE model implemented the use of a BAC/EAC model which attempts to correct for variation due to natural variations such as temperature, salinity and weight of the organisms. The quality of this assessment can be improved upon by integrating time series data for the environmental parameters, which could potentially capture how climate change influences H. diveriscolor.

There are however major limitations with the results as the data demonstrates large seasonal variations in the interpretation of health status for the biomarkers and future research will need to investigate how to account for this. The use of only one reference site to establish the BAC/EAC index also limits the efficacy of the model and endeavors should be made establish additional data at other low impact sites. Finally, the interpretation of environmental impact is also hampered by the lack of of contaminant data and the results would be strengthened by chemistry data.

Overall, the present study demonstrates that H. diversicolor could be an effective model organisms for environmental hazard assessment. The model could however be improved by additional endpoints, such as genotoxicity and reproductive stage,can further describe the health of individuals in the Seine estuary. Results suggested that H. diversicolor in the Seine estuary are moderately to majorly impacted by environmental condition, indicating that the status of the Seine estuary has not notably improved between the two sampling campaigns. This however is only one line of evidence and other lines of evidence need to generate in order to effectively characterize the impact human activity has had on the Seine estuary, in particular coupling additional physicochemical parameters such as contamination levels in the environment and bioaccumulation measurements in key species into a weight of evidence approach."

Figure 1 :

 1 Figure 1: Location of the study area in the Seine estuary -Northern flank. Black bar indicates 1km. (Credits: Google Maps, Images @2019 Google, Data SIO, NOAA, U.S. Navyn NGA, GEBCO, Images @2019 CNES, Airbus, Landsat, Copernicus, Maxar Technologies, Données cartographique @2019).

Figure 2 :

 2 Figure 2 : Evaluation and classification of an integrated assessment of biological endpoints measured in the Seine estuary. Values are based on the biomarker hazard quotient (BiomHQ) and indicate the degree in which biological endpoints deviated from the range of expected natural variation.

Figure 3 :

 3 Figure 3: Contribution of each biomarker to the BiomHQ value calculated for each sampling campaign.N/A indicates that contributions could not be calculated due to BiomHQ value equals to 0.

Figure 4 :

 4 Figure 4: Contribution of each class of biological endpoints to the BiomHQ value calculated for each sampling campaign. N/A indicates that contributions could not be calculated due to BiomHQ value equals to 0.

Acknowledgements

This research was funded through the public interest group Seine-Aval (GIP Seine-Aval, SA5 2014-ECONTONES) under the project ECOTONES. Samples were collected for the ECOTONES data using the generation of the model were collected with assistance from the Normandy Littoral Monitoring Unit.