Le Luherne

Le Pape Olivier

A Paumier
email: alexis.paumier@irstea.fr

Impacts of green tides on estuarine fish assemblages

Keywords: Fish community, Estuarine ecology, Green tides, Macroalgae, Nursery

All around the world, an increasing proportion of estuarine systems are facing massive proliferations of green macroalgae, called green tides, in response to nutrient enrichment. The consequences of this perturbation for ichthyofauna that use estuarine systems as essential fish habitats remain understudied. To estimate these consequences, we combined outputs of both macroalgae proliferation and fish community surveys conducted for the European Water Framework Directive in thirteen estuaries in northwestern France, a region where green tides are of great concern. The approach revealed the influence of green tides on estuarine fish communities. The response of each community to the green tides differed according to their functional guild composition. Benthic and marine juvenile guilds were negatively impacted, while demersal and pelagic fish guilds appeared to be more resilient. Green tides, which significantly affect the suitability of fish habitat, change the composition of the fish community and may hinder the future recruitment of marine fish species that rely on estuaries during the juvenile stage.

trophic webs [START_REF] Raffaelli | Ecological impact of green macroalgal blooms[END_REF]. However, only a few studies have considered the fish community, especially in estuarine systems [START_REF] Raffaelli | Ecological impact of green macroalgal blooms[END_REF][START_REF] Lyons | Macroalgal blooms alter community structure and primary productivity in marine ecosystems[END_REF]. During recent decades, the intensity of GTs and the number of impacted sites have increased in northwestern France, with high levels of proliferation observed from mid spring to the end of summer [START_REF] Ménesguen | Nitrogen loadings and macroalgal (Ulva sp.) Mass Accumulation in Brittany (France)[END_REF]. Locally, a small-scale analysis of shallow intertidal beaches and estuarine mudflats revealed differences in the fish communities between control and impacted sites during green algae proliferation. Negative impacts for benthic and marine juvenile fish species begin at a low proliferation rates, and green tides significantly decrease fish species diversity and overall fish density until the complete disappearance of fish at a high level of GT (Le Luherne et al., 2016). Proliferations of green macroalgae and fish communities are both monitored in this region to assess the ecological quality of estuarine systems and to achieve a status that is compliant with the goals of the European Water Framework Directive (WFD). Complementing the previous local small-scale approach [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF], this study provides a quantitative evaluation of the effects of contrasting levels of GT proliferation on fish communities in estuaries over a much larger geographic area. The effects of GTs on fish communities were examined through the analysis of fish density and species richness collected from thirteen estuaries to explore the following questions:

-Is there a significant difference between the fish communities in affected and non-affected estuaries? -At what scale can we detect GT impacts on estuarine fish communities? -Do functional groups of fish respond differently to GTs?

Materials and methods 2.1 General approach

To study the effects of green tides on estuarine fish, a generalized linear model (GLM) approach was applied (2.5.). We used several survey-based (2.2.) descriptors of the fish community (2.3). The "natural" environmental variability of fish in estuaries was integrated by using four major environmental variables that control fish assemblage (i.e., ecoregion, season, depth, and salinity; [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF]2.3.). Then, we tested separately the GT effect using four descriptors, from a large scale to a local scale (2.4.).

Study area and fish sampling

We investigated the effect of GT in thirteen estuaries located in northwestern France (Fig. 1). The selection of these study sites allowed the coupling of fish survey and green tide indicator data. Furthermore, the pressure represented by GTs differed among these estuaries (e.g., from non-affected to moderately affected. These estuaries qualities were evaluated by ELFI, which is an indicator of estuarine quality based on the fish metrics (Table 1). From 2008 to 2014, fish were sampled in thirteen estuaries as part of a monitoring program that evaluates the ecological status of transitional waters in relation to the WFD [START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. Standardized fish surveys were conducted in spring (between April and June) and autumn (between September and November) for each site and sampling year [START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. Each survey used a beam trawl with an opening of 1.5 m wide and 0.5 m high and with a 8-mm stretched mesh in the cod-end [START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. The beam trawl was hauled in a counter-current direction for 15 min at a standard speed of 1.5-3 knots [START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. An average bottom surface of 1100 m² was swept during each haul. Salinity and depth were recorded for each beam trawl haul, the depth ranged between 0.75 and 23 m, and the salinity ranged between 2. 5 and 35.1. From 20085 and 35.1. From to 20145 and 35.1. From , 1348 beam trawl hauls have been conducted, and 85 fish species and 79,125 individuals have been sampled.

Fish community metrics and environmental variables

Fish density and species richness were selected as indicators of the fish community status because they are assumed to decrease with an increase in habitat disturbances [START_REF] Gibson | Impact of habitat quality and quantity on the recruitment of juvenile flatfishes[END_REF][START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. Species richness was estimated based on the number of species captured in each trawl haul, and the total density was defined as the number of individuals captured per hectare. In addition, to evaluate the impact of GTs on the use of fish habitat, each species was classified into functional guilds, including: 3 vertical distribution guilds (i.e., pelagic, demersal and benthic) and 2 ecological guilds (i.e., marine juvenile and resident) [START_REF] Franco | Life strategies of fishes in European estuaries: the functional guild approach[END_REF]Potter et al., 2015). Vertical distribution guilds describe the spatial distribution of fish in the water column and illustrate the dependence of the organism on the substratum (Table 2). Ecological guilds describe the use of estuaries during the species' life cycle (Table 2). Other ecological fish guilds, such as amphidromous or marine adventive species [START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF], were not well sampled in the beam trawl surveys; thus, our approach did not account for these guilds. The fish densities for each of these five guilds were calculated for each trawl haul. Because estuaries are exposed to strong environmental gradients, it was necessary to consider the natural source of environmental variability [START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF][START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF][START_REF] Nicolas | Functional diversity in European estuaries: Relating the composition of fish assemblages to the abiotic environment[END_REF] before providing a reliable assessment of the green tide impacts on estuarine fish communities. Four main environmental variables were considered: the sampling season of the survey, the ecoregion where the estuary is located, the salinity and the depth during the trawl haul. Preliminary tests have shown that these four sources of environmental variability were not correlated. -Fish communities change dramatically between spring and autumn [START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF]. The most marked shift was observed for marine juveniles, whose density decreased drastically from spring to autumn, revealing a high mortality rate after settlement [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF][START_REF] Pape | The food limitation hypothesis for juvenile marine fish[END_REF]. These density variations and the seasonality of GT proliferations led us to separate spring and autumn analyses.

-A biogeographic classification based on the Marine Ecoregions of the World [START_REF] Spalding | Marine Ecoregions of the World: A Bioregionalization of Coastal and Shelf Areas[END_REF] was used to separate the thirteen estuaries into two ecoregions: "North Brittany" and "South Brittany" (Fig. 1). -Salinity and depth, which are among the main drivers structuring fish communities in estuaries, were [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF]) also considered.

Green tide monitoring in WFD and GT index computation

To quantify the intensity of the GTs, we used four indicators provided by the Center for Study and Promotion of Algae (CEVA, France) and the WFD (Table 3). Three of these indicators (GT1, GT2 and GT3) were based on quantitative maps of GT, and the last indicators (GT4) was based on a WFD indicators (Table 3). The quantitative maps were obtained by combination of orthophotographies and field monitoring. Orthophotographies were obtained from an aircraft during low tide, and field monitoring was performed to determine the associated density of macroalgae. The quantitative maps represented the macroalgal proliferation as a percentage of algal cover. The first three indicators of GTs were derived from these maps at different spatio-temporal scales (Table 3): -At a large scale (i.e., the estuary scale), the ecological quality ratios (EQRs) were used (GT1). The EQR index is a validated indicator of estuarine quality in terms of macroalgal proliferation that is applied in the WFD [START_REF] Wilkes | Intercalibration of opportunistic macroalgae in coastal and transitional waters v4. UE Water Framework Directive intercalibration technical report[END_REF]. The EQR index is composed of four scores: poor, moderate, good and high. Inside each of the thirteen estuaries, a low variability of the EQR was observed over the period of 2008-2014 (Supp. Fig. 1.1). Among the thirteen estuaries, some were almost clear of green macroalgae (high), while others were impacted by GTs (moderate, Table 1). No poor EQR were recorded within the studied estuarine.

-At a local scale (i.e., the trawl haul scale), we used two indicators based on the same data (Table 3): (i) the macroalgal mat surface ratio within the trawl hauls (GT2), and (ii) the macroalgal mat surface ratio near the trawl hauls (GT3). The macroalgal mat surface ratio within the trawl hauls (GT2) was computed using QGis software by combining the quantitative maps of macroalgal proliferation with the trawling location from 2008 to 2014 (which was the same period as the fish trawl surveys). Preliminary analysis and expert knowledge validated the inter-annual steadiness of this distribution. The macroalgal mat surface ratio near the trawl samples (GT3) was also computed by combining the quantitative maps of macroalgal proliferation with the trawl haul location. To achieve this, a surface buffer was calculated around each beam trawl haul (Supp. Fig. 2.1). Several surface buffers were calculated to compute the index of the macroalgal surface ratio near the trawl hauls with different buffer distances (i.e., from 100 to 1000 m). Inside each buffer, the proportion of sea surface (excluding land cover) covered by green macroalgae was calculated up to a certain threshold (i.e., >25%, >50% and >75%) (Supp. Fig. 2.1). The influence of the buffer distances and thresholds was assessed in preliminary analysis before the GT3 was integrated as explaining covariates in the models of fish metrics. We screened the correlation levels between the GT index in the vicinity of trawl hauls according to the different buffer distances that were defined around the beam trawl hauls (ranging from 100 to 1000 m) and the different algal cover thresholds ( > 25%; > 50% and > 75%). In addition, at a local scale, we tested a fourth GT descriptor on a restricted dataset. The Ulva spp. density per beam trawl (GT4) was estimated from an additional protocol added to the WFD trawl surveys in 2013 and 2014 (Table 3). These data were available for 227 beam trawl hauls and were collected using a WFD standardized protocol [START_REF] Scanlan | The monitoring of opportunistic macroalgal blooms for the water framework directive[END_REF] The influence of each of these four indicators on fish metrics was tested separately to determine the most appropriate scale for assessing the effect of GTs on estuarine fish.

Modelling the effect of GTs on the fish community

The multi-scalar approach used for the GT indicators allowed us to explore the appropriate scale for detecting the impacts of GTs on fish communities. Because there is no optimal scale to describe an ecological phenomenon [START_REF] Levin | The problem of pattern and scale in ecology[END_REF][START_REF] Wheatley | Factors limiting our understanding of ecological scale[END_REF], we chose to compare the response of the fish community to these four GT indexes in separate analyses. The modelling approach integrated the response of fish to the natural variability and the four GT indexes used in four separate models (Eq. 1):

Fish metrics ~ environmental variables + GT* (Eq. 1) where the environmental variables represent the four natural drivers (i.e. ecoregion + salinity + depth), and "GT*" corresponds to one of the four GT metrics (Table 3).

2.5.1 Species richness The species richness was modelled using GLM with a Poisson distribution (Eq. 2). This distribution is most commonly used for analysing count data in trawl surveys [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF].

Species richness ~ (environmental variables) + (GT*) + ε (link function = log) (Eq. 2) where the environmental variables represent the four natural drivers, and "GT*" corresponds to one of the four GT metrics (Table 3). 2.5.2 Fish density The fish survey data were characterized by a large proportion of zeros (Supp. Fig. 1.2.a). According to this zero-inflated distribution, we used a delta distribution [START_REF] Aitchison | The lognormal distribution[END_REF]. This approach is appropriate for the analysis of fish survey data [START_REF] Stefansson | Analysis of groundfish survey abundance data: combining the GLM and delta approaches[END_REF][START_REF] Pape | Quantitative description of habitat suitability for the juvenile common sole (Solea solea, L.) in the Bay of Biscay (France) and the contribution of different habitats to the adult population[END_REF]. The delta model [START_REF] Aitchison | On the Distribution of a Positive Random Variable Having a Discrete Probability Mass at the Origin[END_REF][START_REF] Pennington | Efficient Estimators of Abundance, for Fish and Plankton Surveys[END_REF] combines two distinct GLMs: one sub-model for fish presence and absence (Eq. 3), and one sub-model for positive density (Eq. 4).

-Binomial sub-models for fish presence Y (1/0) ~ (environmental variables) + (GT*) + ε (link function = logistic) (Eq. 3) where Y (1/0) represents the presence or absence of fish (i.e., 1 or 0). The area under the curve (ROC) was used as a criterion to validate the goodness-of-fit of each sub-model [START_REF] Manel | Evaluating presence-absence models in ecology: the need to account for prevalence: Presence-absence modelling[END_REF][START_REF] Vasconcelos | Predicting estuarine use patterns of juvenile fish with Generalized Linear Models[END_REF].

-Sub-models for positive fish density Log (Y (>0)) ~ (environmental variables) + (GT*) + ε (link function = identity) (Eq. 4) where Y (>0) is the density of fish when at least one fish was caught. The log-transformed positive densities satisfy the conditions of a linear distribution and the homogeneity of variance for these sub-models (Supp. Fig. 3.1.). The value of the actual data vs. the predicted relationship was used as a criterion to validate the goodness-of-fit of these sub-models [START_REF] Manel | Evaluating presence-absence models in ecology: the need to account for prevalence: Presence-absence modelling[END_REF][START_REF] Vasconcelos | Predicting estuarine use patterns of juvenile fish with Generalized Linear Models[END_REF].

-Coupling The two sub-models (Eqs. 3 and 4) were coupled (Eq. 5) to estimate fish density [START_REF] Stefansson | Analysis of groundfish survey abundance data: combining the GLM and delta approaches[END_REF]. A correction was applied to the positive sub-models to obtain unbiased estimations from log-transformed data (Laurent, 1963). (Eq. 5) where Ŷ is the fish density estimated by the delta model; Y (1/0) is the probability of the presence of fish provided by the binomial model; Y (>0) is the logarithmic density of fish provided by the log-normal model; and α²(ln(Y (>0)) is the standard error associated with the log-normal model. To quantify the uncertainty that accounts for the combination of the two sub-models' errors, we used a random sampling approach. We predicted the presence of fish on 5,000 subsamples that were randomly generated with the binomial model, and we log-transformed the densities of 5,000 subsamples that were generated with the GLM model using positive density values. Then, these predictions were coupled, and we computed the 10%, 50% and 90% quantiles of the 5,000 predictions [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF].

Preliminary analysis and modelling options

There was a single exception to the use of the delta model, and it concerned the pelagic fish guild. The proportion of non-null observations of pelagic fish was too low to allow for fitting a positive sub-model. As a result, a single binomial model (Eq. 3) was developed for this guild (Table 4). The GLM approach requires linearity in the relations between the response variable and the covariates. This assumption was preliminarily tested before the integration of the 2 environmental variables (e.g., depth and salinity, and ecoregion was a class factor) as linear factors in the GLM. For depths down to 16 m, we observed atypical observations and non-linear effects on fish metrics. Thus, to account for the linear effect of depth, we removed trawl samples that were deeper than 16 m. By doing so, we narrowed the data set by less than 1% of the survey data. In a few other cases where we found non-linear patterns, we integrated the environmental variable as a class factor in the GLMs (see pelagic fish in Table 4). The models were run using R software (R Core Team 2016). For both sub-models, the selected level of statistical significance of the four environmental variables and the GT indexes of the GLM was 5% (i.e., only the environmental variables that were significant at the 5% level were retained, and the same was applied to GT*). Both the explained percentage of deviance and the Akaike information criterion (AIC) were used to assess the effects of the environmental variables and the GTs on the fish metrics.

Results

Fish community and its environmental variables

The majority of fish caught belonged to the demersal (71.5%) and benthic (26.6%) guilds, while a minority of fish were pelagic (1.9%). For the ecological guilds, the residents were the most frequently caught species (70%), followed by the marine juveniles (13%). Other ecological guilds were poorly represented and were, thus, not included. Globally, the statistical significance of depth and ecoregion were high in the models, and the salinity appeared to be a structuring factor for the density of the benthic and marine juvenile guilds in spring (Table 4). Selected models had satisfying goodness-of-fit values (Supp. Table 3.1; Supp. Fig. 3.1).

GT index computation

The study revealed that few beam trawls were conducted within the algal mat: 54 trawl hauls occurred in spring and 71 hauls occurred in autumn (i.e., 9% of the fish community data). Furthermore, the macroalgal mat surface ratio (GT2) was very low at these 125 beam trawl hauls. Accordingly, the green macroalgae density accumulated per beam trawl haul, which was available in 2013 and 2014 (GT4), was very low (mean biomass = 0.0035 kg/m 2 , Supp. Fig. 1.2.b). The macroalgal mat surface ratio near the beam trawl haul (GT3) differed among the threshold and buffer combinations. For identical buffer distances, the different algal cover thresholds were correlated (more than 80%; Supp. Fig. 2.2.). In the radius range of 300-800 m, the levels of correlation were also high between the GT indexes for the different buffer sizes and the algal cover thresholds (Supp. Fig. 2.2.). We selected a single combination of buffer distance and algal cover to compute the macroalgal mat surface ratio near the beam trawl hauls (GT3): the 25% threshold of algal cover within the 500 m buffer.

Large-scale effect of GTs

Accounting for the statistical significance of the environmental variables (Table 4), the significant effects of the EQR (GT1) were detected for 13 of the 14 models in spring and for 11 of the 14 models in autumn (Table 4). However, a single fish metricthe density of benthic fish in springhad a monotonous relationship with GT1 (i.e., a continually decreasing density associated with a decreasing gradient of the EQR). Moreover, the deviance explained by GT1 for the density of benthic fish was the highest (32% for the presenceabsence model and 2% for the positive density model). Furthermore, the confidence bands around the prediction associated with the average and high EQR scores did not overlap, and there was a distinguishable difference in density (Fig. 2). GTs negatively impacted the benthic guild in spring. The deviance explained for the other fish metrics was less than 10%. According to this low signal at the estuarine scale, there was no significant correlation between GT1 and ELFI. 3.4 Local-scale effect of GT Accounting for the statistical significance of the environmental variables, the two indexes of proliferation at beam trawl locations (GT2 and GT4) were not statistically significant for either the spring or autumn surveys. Conversely, statistically significant effects (Table 4) were observed for the algal cover near the beam trawl haul (GT3). In spring, the probability of the presence of the resident guild responded to GTs positively. In autumn, statistical significance was detected for 6 of the 12 models. Positive effects of the GTs were observed on total fish density, demersal fish density, and resident fish density. In contrast, GTs had a negative effect on the benthic and marine juvenile fish densities. The density of marine juveniles was halved when 20% of the surface at an area less than 500 m from a beam trawl haul had a green macroalgae cover greater than 25% (Fig. 3).

Discussion

This study provided evidence on the effects of GTs on estuarine fish communities, and it focused on thirteen estuarine systems with contrasting levels of GTs. It appeared that GTs had both negative and positive effects on the fish communities. Benthic fish were especially sensitive and negatively impacted, and marine juveniles appeared to be dramatically more sensitive than were resident fish. This confirmed previous findings from coastal areas [START_REF] Howell | Juvenile winter flounder distribution by habitat type[END_REF][START_REF] Jokinen | Decline of flounder (Platichthys flesus (L.)) at the margin of the species' distribution range[END_REF][START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF], and enabled us to extend these conclusions to estuaries.

Can we consider correlations to GT indexes as causal links to GTs?

Fish that inhabit estuaries have adapted to strong environmental gradients (e.g., "estuarine quality paradox" theory; [START_REF] Elliott | The Estuarine Quality Paradox, Environmental Homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF]. Thus, it was critical to consider the environmental constraints before providing a reliable assessment of the impacts of GTs on the estuarine fish communities [START_REF] Nicolas | Functional diversity in European estuaries: Relating the composition of fish assemblages to the abiotic environment[END_REF]. Accounting for these "natural" factors that structure fish communities at different scales (time: seasonal patterns; space: ecoregion at a mesoscale, depth and salinity at a local scale) allowed us to account for a part of the "natural" variability and autocorrelations in the fish survey data, which enabled a reliable assessment of the effects of GTs. In addition, the potential effects of the other anthropogenic pressures potentially combined with the effects of GTs were not considered. The lack of an exhaustive assessment of the ecological status of these thirteen estuaries in the WFD, e.g., with regard to the concentration of xenobiotics, prevents the present approach from accounting for them. These potential confounding factors smooth the strength of the conclusions based on analysis at the estuarine scale. However, the use of local-scale GT indexes, especially GT3, allows for a thin-scale analysis of the link between GTs and fish metrics, with a dramatically less probable influence of potential confounding factors.

Large-scale impacts of GT

At the large scale, a single negative effect of GTs was revealed on the benthic guild. It was previously demonstrated that this guild was the most sensitive to green macroalgae proliferation [START_REF] Bowen | The ecological effects of urbanization of coastal watersheds: historical increases in nitrogen loads and eutrophication of Waquoit Bay estuaries[END_REF][START_REF] Bricker | Effects of nutrient enrichment in the nation's estuaries: A decade of change[END_REF][START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF][START_REF] Le Luherne | Influence of Green Tides in Coastal Nursery Grounds on the Habitat Selection and Individual Performance of Juvenile Fish[END_REF]. However, there was no further signal of change in the fish community in relation to GT1. The relatively low intensity of GTs in these estuaries, the minimum score of EQR encountered in this survey was a moderate status of estuarine quality in terms of macroalgal proliferation, prevents dramatic changes in the fish communities at the global scale of estuaries. 4.3 Local-scale impacts of GT First, we analysed the effects of GTs directly at the trawling location using two indicators (i.e., macroalgal mat surface ratio on beam trawl hauls, GT2, and Ulva spp. density per beam trawl haul, GT4). From previous restricted small-scale analysis, these two indicators of the local intensities of GTs were expected to have significant effects on the fish community. However, none of the fish metrics presented a significant response to the GT descriptors at the trawling locations. This lack of response was explained by the fish sampling protocol. Indeed, during the WFD survey, trawl hauls were conducted beside the algal mats to avoid the clogging of the net. Consequently, the surfaces and biomasses of algae were low at the trawling locations. The maximum Ulva spp. density per beam trawl haul recorded was 0.05 kg/m² in spring and 0.18 kg/m² in autumn. These low algal densities explain the absence of significant effects on fish metrics. Indeed, Le Luherne et al. ( 2016) observed a minimum algal density threshold of 0.30 kg/m² from which the fish community was significantly impacted by expanded blade green macroalgae. The under-sampling of the algal mats during the surveys explained the inability to provide a clear-cut assessment of the impacts of GTs on fish at the trawling positions. To improve this assessment, the impacts of the GTs should be analysed using a dedicated protocol that has been fitted for a before-after control-impact analysis [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF]. To cope with the under-sampling of the algal mats during the fish surveys, we computed the macroalgal mat surface ratio near the beam trawl hauls to test the effect of the spread of the GTs beyond the algal mats. This local descriptor succeeded in detecting the effect of GTs on estuarine fish. A single significant and positive effect of GTs on resident fish was observed in spring based on the GT index near the beam trawl hauls. This moderate effect may be caused by the very limited biomass and cover of algae before the proliferation season begins [START_REF] Adams | Assessing cause and effect of multiple stressors on marine systems[END_REF][START_REF] Andrades | The influence of allochthonous macroalgae on the fish communities of tropical sandy beaches[END_REF]. Proliferation occurs between May and September [START_REF] Merceron | In situ growth potential of the subtidal part of green tide forming Ulva spp[END_REF]. As a result, a potential scale mismatch between the GT indicator and the fish surveys may have occurred during the spring campaigns of certain years. Conversely, half of the functional guilds presented a significant response to GT3 in autumn. Resident and demersal fish responded positively to moderate algal cover in their vicinity [START_REF] Adams | Assessing cause and effect of multiple stressors on marine systems[END_REF][START_REF] Andrades | The influence of allochthonous macroalgae on the fish communities of tropical sandy beaches[END_REF]. However, benthic and marine juvenile fish were negatively impacted. This sensitivity of benthic and marine juvenile fish was already observed during GTs, with a dramatic decrease in their density even during moderate proliferation [START_REF] Pihl | Relating plaice (Pleuronectes platessa) recruitment to deteriorating habitat quality: effects of macroalgal blooms in coastal nursery grounds[END_REF][START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF], as well as a decrease in individual performance [START_REF] Le Luherne | Influence of Green Tides in Coastal Nursery Grounds on the Habitat Selection and Individual Performance of Juvenile Fish[END_REF].

Generalization on the impact of GTs on fish

The density of benthic fish responded negatively to the GT1 and GT3 indicators of proliferation, with strong evidence supporting the sensitivity of this guild to GTs. This sensitivity may be explained by the GT pattern of proliferation. GTs generate physical, chemical and trophic perturbations that affect the fish community within a small area (Nordström et al., 2007;[START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF]. Physical stress is linked to the degradation of the physical structure of the habitat. GTs first colonize the estuarine floor, modifying the structure of the substratum [START_REF] Solidoro | Long term simulations of population dynamics of Ulva r. in the lagoon of Venice[END_REF], and then the water column is colonized [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF]. This modification of the bottom habitat by GTs mainly affects macrobenthic communities [START_REF] Quillien | Effects of macroalgal accumulations on the variability in zoobenthos of high-energy macrotidal sandy beaches[END_REF], including benthic fish [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF][START_REF] Le Luherne | Influence of Green Tides in Coastal Nursery Grounds on the Habitat Selection and Individual Performance of Juvenile Fish[END_REF]. Trophic perturbations are linked to a decrease in fish foraging efficiency within the mats of Ulva spp (Nordström et al., 2007;[START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF]. Finally, chemical perturbations could occur with anoxic events within the sediment or at the sediment-water interface below the mats of Ulva spp. [START_REF] Baden | Eutrophication on Benthic Communities Including Fish: Swedish West Coast[END_REF][START_REF] Sundbäck | Response of benthic microbial mats to drifting green algal mats[END_REF]. Therefore, the benthic fish are the first to be impacted due to the degradation of their habitat [START_REF] Wennhage | Vulnerability of newly settled plaice (Pleuronectes platessa L.) to predation: effects of habitat structure and predator functional response[END_REF][START_REF] Wennhage | From flatfish to sticklebacks: assemblage structure of epibenthic fauna in relation to macroalgal blooms[END_REF]. Following the rapid impact on benthic fish, shifts from a benthic to a pelagic community could be observed after GT events [START_REF] Bowen | The ecological effects of urbanization of coastal watersheds: historical increases in nitrogen loads and eutrophication of Waquoit Bay estuaries[END_REF]. In the Baltic Sea, a massive change in fish composition was associated with a decrease in the foraging efficiency of predatory fish, and this change was induced by a GT [START_REF] Pihl | Relating plaice (Pleuronectes platessa) recruitment to deteriorating habitat quality: effects of macroalgal blooms in coastal nursery grounds[END_REF][START_REF] Österblom | Human-induced Trophic Cascades and Ecological Regime Shifts in the Baltic Sea[END_REF]. In fish survey data from the thirteen studied estuaries, the proportion of pelagic fish was low (<2%) with regard to previous estimations of European estuarine fish assemblages (25%; [START_REF] Elliott | The structure and components of European estuarine fish assemblages[END_REF]. This result revealed that the fish community was not fully represented in the French WFD surveys. Indeed, the beam trawl is well adapted to sample the benthic and demersal communities, but this method is inadequate for sampling pelagic fish [START_REF] Courrat | Anthropogenic disturbance on nursery function of estuarine areas for marine species[END_REF][START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF]. GTs mainly disturb benthic and demersal species [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF], and the present study); thus, the sampling bias did not compromise the validity of the present approach for these fish guilds. Moreover, the effects of GTs on pelagic fish occurred at a high level of GT proliferation, which was not observed in the present data set in the sampled areas. However, potential shifts from a benthic to a pelagic community after GT events [START_REF] Bowen | The ecological effects of urbanization of coastal watersheds: historical increases in nitrogen loads and eutrophication of Waquoit Bay estuaries[END_REF] could not be assessed with this sampling protocol. This type of assessment requires a dedicated protocol to investigate massive algal mats and sample the whole fish community [START_REF] Le Luherne | Fish community responses to green tides in shallow estuarine and coastal areas[END_REF]. Concerning the ecological guilds, in spite of the moderate level of GTs investigated, the present approach provided evidence that GTs had negative consequences on marine juvenile fish. The local productivity of several coastal and estuarine nursery-dependent marine fish will be reduced with the proliferation of green macroalgae in coastal [START_REF] Pihl | Relating plaice (Pleuronectes platessa) recruitment to deteriorating habitat quality: effects of macroalgal blooms in coastal nursery grounds[END_REF], Brown et al., 2017) and estuarine areas [START_REF] Peterson | Synthesis of linkages between benthic and fish communities as a key to protecting essential fish habitat[END_REF][START_REF] Stoner | Spatially explicit analysis of estuarine habitat for juvenile winter flounder: combining generalized additive models and geographic information systems[END_REF]. The juvenile stage is a key life stage for fish population size [START_REF] Stoner | Spatially explicit analysis of estuarine habitat for juvenile winter flounder: combining generalized additive models and geographic information systems[END_REF][START_REF] Pape | The food limitation hypothesis for juvenile marine fish[END_REF], and marine populations of species that are dependent on coastal and estuarine nursery grounds and the related fisheries could be impacted by GTs, as observed in the Baltic Sea [START_REF] Baden | Eutrophication on Benthic Communities Including Fish: Swedish West Coast[END_REF][START_REF] Jokinen | Decline of flounder (Platichthys flesus (L.)) at the margin of the species' distribution range[END_REF].

615

Figure captions

616 Figure 1: a) Locations (in WGS84 coordinates) of the studied estuaries in northwestern France split into two ecoregions: north and south. The spatial protocol for the fish survey is provided 618 as an example for 2 of the thirteen estuaries studied, in (b) the Morlaix Estuary and (c) the 619 Aulne Estuary. Lines: the locations of beam trawls; hatched grid: mats of green macroalgae. 620 621 Figure 2: GLM prediction (with the 10-90% confidence intervals) of the estuarine benthic fish density (number/ha) according to the salinity and the two scores of the ecological quality ratio (moderate in grey and high in black) in an estuary located in South Brittany that was sampled 624 in spring.

625 626 Figure 3: GLM prediction (with the 10-90% confidence intervals) of the estuarine marine juvenile density (number/ha) according to the percentage of the surface that had more than 628 25% algal cover and was within 500 m, in an estuary located in South Brittany that was 629 sampled in autumn.

  FIGURE 1

1 .

 1 Validation of the GLMs on the positive density for the total density of fish (log scale) in: (a) autumn, (b) spring.

Table 1 :

 1 Total estuary area and intertidal area (ha)(Teichert et al, 2018), mean ecological 641 quality ratio (EQR) and the Estuarine and Lagoon Fish Index (ELFI,[START_REF] Delpech | Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries[END_REF], 642 Lepage et al. 2016) and the associated scores for the thirteen studied estuaries.

	Estuary	Total area (ha)	Intertidal area (ha)	EQR (mean)	EQR score	ELFI (mean)	ELFI (Score)
	Aber Wrach	699	594	0.60	Good	0.46	Moderate
	Aulne	1831	973	0.60	Good	0.59	Moderate
	Aven	166	116	0.74	Good	0.88	Good
	Belon	183	128	0.96	High	0.88	High
	Blavet-Scorff	1531	863	0.47-0.8		0.67	High
	Elorn	631	324	0.66	Good	0.96	Good
	Goyen	155	111	0.93	High	0.65	Moderate
	Laita	247	125	1.00	High	0.77	Good
	Morlaix river	1361	1146	0.48	Moderate	0.44	Good
	Odet	900	529	0.82	High	0.21	Good
	Pont l'Abbe	646	545	0.43	Moderate	0.67	High
	Trieux	777	478	0.55	Moderate	0.67	Bad
	643
	644
	645

Table 2 :

 2 Definition of the three vertical distribution guilds and the two selected ecological 646 guilds (based on[START_REF] Franco | Life strategies of fishes in European estuaries: the functional guild approach[END_REF] Potter et al., 2015).

	Guild	Criterion	Definition
	Pelagic	Vertical distribution	Species living in the water column
	Demersal	Vertical distribution	Species living in the water layer just above the bottom
	Benthic	Vertical distribution	Species living on the substratum
	Marine juvenile	Ecology	Species using the shallow coastal waters and estuaries primarily as nursery grounds
	Resident	Ecology	Species spending their whole life cycle in shallow coastal waters and estuaries
	647
	648

Table 3 :

 3 Description of the four indicators of green tide proliferation.

	Indicator	Spatial scale	Variable	Green tide indicator	Available data
	GT1	Large scale	Factor	Ecological quality ratio (EQR)	2008-2014
	GT2	Local scale	Continuous	Macroalgal mat surface ratio on the trawl haul (percentage)	2008-2014
	GT3	Local scale	Continuous	Macroalgal mat surface ratio near beam trawl hauls (percentage)	2008-2014
	GT4	Local scale	Continuous	Ulva spp. density per beam trawl haul (kg.m-²)	2013-2014

Table 4 :

 4 Statistical significance of the generalized linear models of the fish metrics for the probability of presence, the positive densities and the 654 species richness, and for the two GT indicators (large scales: GT1 and local scale: GT3). NS: nonsignificant; p-value: *≤5%, **≤1% and 655 ***≤0.1%.

	Spring

Acknowledgements

This work was supported by the French Fisheries Organization, France Filière Pêche and the Loire-Bretagne Water Agency. We would like to thank all the scientific partners involved in the estuarine fish surveys for the Water Framework Directive in Brittany (France), especially Eric Thiebaut and Caroline Broudin (Université Pierre et Marie Curie, CNRS, Roscoff, France), Elena Lucchetti (MNHN, Concarneau, France), Laurent Thieulle (Asconit, Lyon, France), and Alexandre Carpentier (Université de Rennes 1, MNHN, Dinard, France). We would also like to thank the three anonymous reviewers and the editor for their numerous helpful suggestions, improvements and advice.

Appendix


Isabelle Ziegler-Devin

Zahra Menana

Laurent Chrusciel

Michel Chalot

Valérie Bert

Nicolas Brosse
email: nicolas.brosse@univ-lorraine.fr

Steam explosion pretreatment of willow grown on phytomanaged soils for bioethanol production

Keywords: phytoremediation-borne biomass, steam explosion, bioethanol, willow short rotation coppice, trace element

A steam explosion (SE) process was evaluated as a pretreatment method to achieve simultaneously the pretreatment and the decontamination of trace elements (TE) from woody biomass for bioethanol applications. The willow biomass used in this study was obtained from short rotation coppice phyto-managed plots harvested on a TE-contaminated soils (Zn, Mn).

The influence of the SE reaction severity on the composition of the cellulosic pulp and on the TE extraction in the water effluent was investigated. SE performed at 220°C after a 2 % sulfuric acid presoaking allowed an extraction of 80 % of Mn and Zn in the water effluent.

The enzymatic hydrolysis of the resulting pulps was examined. A cellulose-to-glucose conversion of ~80 % was obtained after 75 hr of incubation of the pulp obtained after a SE treatment performed at 180 °C. The subsequent fermentation into ethanol using Saccharomyces cerevisiae was successfully performed. No significant influence of TEs on the action of the biocatalysts (enzymes and yeast) was observed.

Introduction

Soil pollution due to human activities is a world-wide issue with significant environmental impact and health risks [START_REF] Gallego | Comprehensive waste characterization and organic pollution co-occurrence in a Hg and As mining and metallurgy brownfield[END_REF][START_REF] Nagendran | Phytoremediation and rehabilitation of municipal solid waste landfills and dumpsites: A brief review[END_REF][START_REF] Medina | Crop residue stabilization and application to agricultural and degraded soils: A review[END_REF].

The presence of trace elements (TE) in high concentrations has been identified as one of the major threats to European soils. Recently, the utilization of plants and trees for the management of polluted land gained interest [START_REF] Chalot | Fate of trace elements during the combustion of phytoremediation wood[END_REF][START_REF] Suer | Biofuel or excavation? -Life cycle assessment (LCA) of soil remediation options[END_REF][START_REF] Witters | Phytoremediation, a sustainable remediation technology? II: Economic assessment of CO 2 abatement through the use of phytoremediation crops for renewable energy production[END_REF]. These phytotechnologies include plant phytoextraction using crops with TE extraction capacity or phytostabilization in order to limit TE dissemination [START_REF] Gomez | Phytoremediation for bioenergy: challenges and opportunities[END_REF]. As a consequence, a subsequent utilization of the TE-enriched biomass seems to be suitable in order to limit the pollutant dissemination in the environment. It has been demonstrated from willow wood that a direct disposal of phytoextraction crops is associated with a high leachability of toxic TE (primarily Zn and Cd) and thus a high probability of return to the environment [START_REF] Syc | Willow trees from heavy metals phytoextraction as energy[END_REF].

In this context, the utilization of contaminated land for growing industrial crops could be a promising approach not only for polluted land management but also for biomass production [START_REF] Gomez | Phytoremediation for bioenergy: challenges and opportunities[END_REF]. Willow (Salix) has been described to be a promising phytoremediation crop for TE extraction because of its capacity to accumulate in aboveground biomass compartments relatively high levels of TE such as Zn up to 1000 mg.kg -1 ) and Cd ( 30 mg.kg -1 ) [START_REF] Syc | Willow trees from heavy metals phytoextraction as energy[END_REF][START_REF] Migeon | Metal accumulation by woody species on contaminated sites in the North of France[END_REF][START_REF] Xu | Comparative transcriptome analysis of duckweed (Landoltia punctata) in response to cadmium provides insights into molecular mechanisms underlying hyperaccumulation[END_REF]. The potential of Very Short Rotation Coppice (VSRC) of willow has been investigated for phytoremediation. The repeating harvesting of biomass (every 3-5 years) has been described to maximize the biomass production and to progressively reduce the TE content in the soil (Xu et al., 2015).

Phytoremediation willow has been proposed as a potential source of biomass for bioenergy and various energy-recovery techniques such as combustion, gasification and pyrolysis [START_REF] Migeon | Metal accumulation by woody species on contaminated sites in the North of France[END_REF][START_REF] Vervaeke | Fate of heavy metals during fixed bed downdraft gasification of willow wood harvest from contaminated sites[END_REF][START_REF] Delplanque | Combustion of Salix used for phytoextraction: The fate of metals and viability of the processes[END_REF]. The mobilization of phytoremediation willow VSRC could be profitable for the production of biofuels in order to reduce the impacts on the food markets. However, some important questions about the fate of the TE during the process and their content in biofuel have to be addressed.

In a recent paper, we described the pretreatment of TE-enriched woody (Salix viminalis) and non-woody biomass (Nicotiana tabacum L.) using dilute acid, alkali catalyzed and organosolv pretreatments for the production of bioethanol and biofuel [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF]. The distribution of TE in the pretreatment fractions (pulp, liquid effluent and lignin) was investigated. It was concluded that (1) the TE extractability was low in basic conditions, (2) using a dilute acid treatment TE were mainly recovered in the liquid effluent producing a clear pulp, (3) in organosolv treatment, TE extraction in liquid phase increased with the water content and the acidity but decreased with the temperature.

Steam explosion (SE) process is one of the most valuable and cost effective pretreatment technologies for cellulosic bioethanol production [START_REF] Jacquet | Application of Steam Explosion as Pretreatment on Lignocellulosic Material[END_REF]. This technology is currently developed at a commercial scale. During SE, biomass is treated with hot steam for short residence times (~ 2-30 min) at temperatures in the range of 180 to 210 °C, followed by explosive decompression. The effect of SE on biomass combines a chemical hydrolysis, a thermal effect during the steam treatment and a mechanical effect during the explosive decompression. SE induces (1) a partial breakdown of glucosidic and lignin-hemicelluloses linkages by hydrolysis and of intramolecular hydrogen bonds (2) a thermo-mechanical defibration due to the flash evaporation of water in the cell wall [START_REF] Jacquet | Application of Steam Explosion as Pretreatment on Lignocellulosic Material[END_REF][START_REF] Sauvageon | Toward the cottonisation of hemp fibers by steam explosion -Part 1 -Defibration and morphological characterization[END_REF]. As a result, SE is described as an efficient way to decrease the particle size and to induce an extensive defibration of the biomass. This process has been the subject of many studies for decades for increasing the accessibility of carbohydrates to enzymes but to the best of our knowledge, the SE pretreatment of TE-contaminated biomass produced on phytomanaged sites has never been examined. The aim of this study was to explore the potential of SE pretreatment of SRC willow biomass produced at TEcontaminated and phytomanaged sites, for the production of bioethanol with a sound management of TE to avoid their dissemination in the environment. The influence of the severity of the SE process on the composition in TE of the cellulosic residues was examined.

The residual TE effect on the enzymatic hydrolysis of cellulose into glucose and on the fermentation step was also investigated.

Materials and methods

EtOH, H 2 SO 4 , HNO 3 and H 2 O 2 used in this study were purchased from Sigma Aldrich and used as received. The enzymes were obtained from Sigma Aldrich (St. Louis, MO).

Plants materials

Willow (Salix viminalis W) was harvested on a TE-contaminated sediment disposal site located at Deûlémont (France) at an experimental plot described previously [START_REF] Kidd | Agronomic practices for improving gentle remediation of trace-element-contaminated soils[END_REF]. Because of their young age, willow samples contain bark. Willow stems with bark (stem diameter 1-3 cm) were milled to a powder using a Wiley mill and dried for 2-3 days at 40 °C.

Steam explosion pretreatment

Biomass was first impregnated during 15 hr at room temperature with a 0.9 % or 2 % (w/w based on wood dry matter content) dilute sulfuric acid solution (water/biomass ratio : 15/1).

After pressing, the biomass was transferred into a 2 L pressure-resistant steam gun where 50 g (dry basis) of biomass were exposed to steam at varying temperatures and residence times (see Table 2 for experimental conditions, the framework of the steam explosion facility has been described in a previous publication, [START_REF] Simangunsong | Steam explosion of beech wood. Effect of the particle size on the xylans recovery[END_REF]. After the residence time, a pneumatic valve was open leading to the vapor phase exit from the reactor through a nozzle entraining the biomass. Both liquid (SE water effluent) and solid (pulp) fractions are then collected after SE pretreatment in the discharge tank. The severity factor, as shown in Eq.

(1), describes the severity of the pretreatment as a function of residence time (min) and temperature (°C) [START_REF] Jacquet | Application of Steam Explosion as Pretreatment on Lignocellulosic Material[END_REF]. The severity factor can be extended to facilitate trading off among temperature, time, and acid concentration, using Eq. ( 1) [START_REF] Chum | Pretreatment catalyst effects and the combined severity parameter[END_REF]). Severity factor and combined severity factor values for this study are grouped in Table 2. Equation 1Where Log R 0 : severity factor, t : residence time (min), T : reaction temperature (°C) Equation 2Where Log C s : combined severity factor, Log R 0 : severity factor, pH : pH measurement of the acidic pretreated hydrolysate

TE analysis

SE pretreated willow pulps were dried overnight at 105 °C and then weighted aliquots (0.2 g dry mass) were placed in a 50 cm 3 Erlenmeyer flask respectively. Then 10 cm 3 of nitric acid (65 %) were added in the flask and placed on a graphite bed at 85 °C. The flask was heated until reddish-brown fumes no more and then 4 cm 3 of hydrogen peroxide was added dropwise. If some plant particles remained in the solution, an additional 4 cm 3 hydrogen peroxide was added for total dissolution of material. After cooling, the solution adjusted to 200 cm 3 with distilled water. TE content was determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES, Radial ICAP 6500 Model, Thermo Fischer Scientific, Courtaboeuf, France), and all samples were analyzed in triplicate with certified reference materials (INCT-OBTL-5, LGC Promochem, Molsheim, France). The recovered percentages of Zn and Mn concentrations in the treated material were calculated based on the initial concentration of TEs in biomass.

Klason lignin and sugars analysis

Extractive-free samples were ground and their moisture content was measured (Kern MRS 120-3 moisture analyzer). Approximately 0.175 g (exactly weighed) of dried samples was added to centrifuge tubes (50 cm 3 tubes from Corning®) to which 1.5 cm 3 of sulfuric acid (72 %) were added. The mixture was stirred few times with a glass rod and placed in a water bath at 30°C for 1 hr. After adding 42 cm 3 of water, the tubes were autoclaved at 121 °C for 1 hr.

The samples were then cooled and filtered with glass microfibers (particle retention 1.2 µm).

The insoluble residue was washed with pure water, oven-dried at 105 °C and weighed.

Monosaccharide contents of the soluble fraction were analyzed by HAPE-PAD (ICS-3000 Dionex) equipped with a Dionex CarboPac TM PA-20 (3 x 150 mm) analytical column using a described method [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF].

Enzymatic hydrolysis

Enzymatic hydrolysis was performed on willow SE cellulosic pulp using standard literature conditions [START_REF] Pan | Role of functional group in lignin inhibition of enzymatic hydrolysis of cellulose to glucose[END_REF]. The cellulosic pulp (1.0 g) was mixed with 50 cm 3 of 50 mM acetate buffer (pH 4.8) in a 100 cm 3 Erlenmeyer flask and then incubated by shaking for 30 min.

After this preincubation step, the hydrolysis was initiated by adding 0.8 g.L -1 of cellulase (Trichoderma reesei) and prolonged by shaking (175 rpm) at 40 °C for 30 hr. The glucose contents of the aqueous phase were quantified using a Shimadzu High Performance Liquid Chromatograph (HPLC), equipped with an evaporative light scattering (ELS) detector and a Prevail Carbohydrates ES column. Acetonitrile/water (75 %, v/v) was used as the eluent.

Enzymatic hydrolysis data were reported as averages from duplicate experiments.

Fermentation tests

The aqueous phases from the enzymatic hydrolysis experiments were fermented using Bakers' yeast Saccharomyces cerevisiae). The method outlined in Söderstrom et al. [START_REF] Söderstroem | Two-step steam pretreatment of softwood by dilute H 2 SO 4 impregnation for ethanol production[END_REF] has been followed for these experiments.

Detoxification of SE water effluents

Phosphorylated kraft fibers are brought into contact with SE water effluent (0.1 w/w) at room temperature for 1 hr under stirring. TE content of the liquid effluent is measured before and after detoxification.

Results and discussion

The chemical composition of the willow samples, containing bark, examined in this study is given in Table 1. Compared to literature reports on the composition of willow wood, willow samples have a substantially lower polysaccharides content 60 %) but significant amounts of galactan and galacturonan (2.06 % and 3.57 % of the total sugars respectively). This can be rationalized by the presence of bark, which contains high levels of pectins and phenolics, lignin and tannins being recovered as acid-insoluble material using the Klason lignin method.

According to [START_REF] Han | Biobutanol production from 2-year-old willow biomass by acid hydrolysis and acetone-butanol-ethanol fermentation[END_REF], willow RSC stem and bark contain 70 % and 46 % of polysaccharides respectively, bark retaining high content of lignin (24.1 %), water extracts (24.7 %) and aceton extracts (5.1 %). Willow is described to have a high biomass production and is one of the trees that accumulates the most TE but at concentrations much lower than those found in hyperaccumulator plants [START_REF] Syc | Willow trees from heavy metals phytoextraction as energy[END_REF][START_REF] Migeon | Metal accumulation by woody species on contaminated sites in the North of France[END_REF][START_REF] Xu | Comparative transcriptome analysis of duckweed (Landoltia punctata) in response to cadmium provides insights into molecular mechanisms underlying hyperaccumulation[END_REF]. Significantly lower TE concentrations were found in the stem compared with the leaves [START_REF] Migeon | Metal accumulation by woody species on contaminated sites in the North of France[END_REF]. The dominant TE concentrations in the studied willow SRC with bark samples of this work is given in Table 1.

Steam explosion pretreatment performed on H 2 SO 4 impregnated milled willow wood has been reported in a previous paper [START_REF] Pan | Role of functional group in lignin inhibition of enzymatic hydrolysis of cellulose to glucose[END_REF]. The most favorable SE conditions regarding the sugar yields were 200 °C for 4 or 8 min using dilute acid presoaking (0.25 % and 0.50 % sulfuric acid w/w). The condition sets used in the present study were selected considering these previously described results focusing on sugars yields but also, starting from a TEenriched wood, considering the TE extractability in the water effluent. In this context, higher sulfuric acid concentrations were investigated in our study (0.9 % and 2 % w/w) for promoting the TE extraction in the water effluents. The chemical compositions of the pulp after SE treatment of willow according to experimental conditions are given in Table 2. Fig. 1 provides the effect of the combined severity (Log Cs) on the pulp yield whereas the glucose and xylose contents in the pulp are shown in Fig. 2. As seen in Fig. 1, an increase in severity leads to the solubilization of an increasing amount of the initial biomass. As expected, it appears from Fig. 2 that the mass loss was primarily due to the hemicelluloses solubilization (or degradation), resulting in a decreasing of the xylan content. In agreement with the literature [START_REF] Simangunsong | Steam explosion of beech wood. Effect of the particle size on the xylans recovery[END_REF], an increasing proportion of xylan with the severity of the treatment was extracted from the solid pulp and recovered in the aqueous effluent producing a glucan-rich pulp. Using harsh conditions (220 °C, 8 min, 2 % H 2 SO 4 ), a cellulose rich pulp containing approximately 97 % of glucose among the total sugars in the treated wood was produced (Fig. 2). [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF][START_REF] Sassner | Steam pretreatment of H 2 SO 4impregnated Salix for the production of bioethanol[END_REF]. The lignocellulosic matrix has been described to have affinity with divalent metal ions, which may be bound primarily to non-cellulosic polysaccharide components [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF]. As a result, the acid-catalyzed removal of the hemicellulosic fraction during the steaming step of SE previously mentioned could partly explain the concomitant sugars and TE extraction in the liquid stream of the process. The results recently published starting from the same biomass and using dilute acid pretreatment (similar treatment but without explosive decompression), showed that an increase in temperature resulted in a decrease of the TE extraction from the pulp [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF]. Interestingly, an opposite trend was observed in the present study using SE, the higher the operating temperature, the higher the TE extraction in the water effluents. Indeed, using comparable conditions at 200°C and 5 min residence time, ~ 37 % of Zn was recovered in the pulp using dilute acid treatment compared to ~ 21 % for SE. In the SE process, the temperature is correlated to pressure which has a direct impact on the shearing forces applied to the lignocellulosic fibers during the explosive release. [START_REF] Pielhop | Steam explosion pretreatment of softwood: the effect of the explosive decompression on enzymatic digestibility[END_REF] showed that increasing the pressure difference of the explosion led to more defibration and to smaller particle size, increasing the digestibility. As a result, at high operating temperature an extensive disruption of the fibrils during SE with a strong reduction of the particle size could increase the accessibility of the TE in the cell wall tissue facilitating their extraction in aqueous phase.

A) Zinc B) Manganese

In a previous study performed on willow wood [START_REF] Asad | Pretreatment of trace element-enriched biomasses grown on phytomanaged soils for bioethanol production[END_REF], we showed that in a basic pretreatement, TE were mostly recovered in the pulp while in an organosolv pulping ~ 30 % -40 % of Mn and Zn were solvent-extracted. [START_REF] Syc | Willow trees from heavy metals phytoextraction as energy[END_REF] studied the composting of willow contaminated with Zn and Cd. According to these authors, 24 -30 % of the total Zn amount and 8 -12 % of the total Cd were leachable at weakly acid pH with strong effect of the pH, the more acidic medium, the higher Cd extraction in water. Using a SE pretreatment performed at 220 °C during 5 min after a 2 % w/w H 2 SO 4 impregnation, ~ 80 % of Zn and Mn were removed from wood and recovered in the water effluent. Thus, steam explosion appears to be an efficient method for the water extraction of TE from phytoremediation wood.

In this context, TE removal from the water stream (water condensed in the discharge tank of the SE facility) has to be considered carefully to minimize TE dissemination and negative environmental impacts of the whole process. In the literature, efficient TE adsorbents exhibiting a high adsorption efficiency and short equilibrium time have been described via coordination and ion exchange. Graphene, zeolite, chitosane derivatives have been successfully used to remove heavy metal ions from wastewater [START_REF] Zhang | A new functionalized reduced graphene oxide adsorbent for removing heavy metal ions in water via coordination and ion exchange[END_REF]. In the present study, a phosphorylated cellulosic material [START_REF] Belosinschi | Phosphorylated lignocellulosic fibers, uses and processes of preparation thereof[END_REF] was experimented as TE adsorbent for the detoxification of water effluents recovered after SE.

Fig. 4 gives the Zn and Mn concentrations before and after a resin treatment (0.1 % of resin w/w, 1 hr, 20 °C). After resin adsorption, a reduction of ~ 80 % of Zn was observed and Mn concentration was under the limit of detection. Thus in this preliminary experiment, we demonstrated that the detoxification of the SE liquid effluents could be possible using an ion exchange resin. value is close to that previously reported starting from pretreated willow pulps [START_REF] Huijgen | Catalytic organosolv fractionation of willow wood and wheat straw as pretreatment for enzymatic cellulose hydrolysis[END_REF]. The presence of TE and the relatively high Klason lignin content in willow wood do not negatively impact the enzymatic hydrolysability of lignocellulosic substrate. At higher pretreatment temperatures (200 °C and 220 °C) slightly lower cellulose-to-glucose conversions were observed (~ 75 %). Using these more severe conditions, the degradation reactions gave rise to furfural and HMF [START_REF] Sassner | Steam pretreatment of H 2 SO 4impregnated Salix for the production of bioethanol[END_REF], which inhibited the enzymatic digestion. The sugar solutions resulting from the enzymatic hydrolysis were fermented using Saccharomyces cerevisiae to investigate their fermentability. Figure 5B shows ethanol yields after 12 hr of fermentation of the enzymatic hydrolysates derived from SE-pulps containing respectively Zn (88 mg.kg -1 , 87 mg. kg -1 , 71 mg. kg -1 ) and Mn (11 mg. kg -1 , 9 mg. kg -1 , 8 mg. kg -1 ). No significant difference was observed in the ethanol yields (~ 65 %)

demonstrating the fermentability of the liquors. It has been described that TE can exhibit a toxic effect toward microorganisms and decrease the fermentation efficiency [START_REF] Yang | Surface display of synthetic phytochelatins on Saccharomyces cerevisiae for enhanced ethanol production in heavy metal-contaminated substrates[END_REF], Gomez et al., 2002). However, Saccharomyces cerevisiae is recognized to exhibit a relatively high tolerance to inhibitors and the toxicity was primarily reported in the presence of Cd, Hg and Pb [START_REF] Buijs | Advanced biofuel production by the yeast Saccharomyces cerevisiae[END_REF]. In the present study, the relatively low concentrations of TE (< 2 mg.L -1 ) had no significant impact on the yeast productivity.

aFig. 1 .

 1 Fig. 1. Pulp yield after SE as a function of the combined severity (log Cs) for the two

  17

Fig. 2 .

 2 Fig.2. Glucan (A) and xylan (B) levels (% based on the sugar contents) in the solid residues

Fig. 3 .

 3 Fig. 3. Trace elements (expressed as percent of the total initial content of the raw material)

Fig. 4 .

 4 Fig. 4. Zn and Mn concentrations before and after detoxification by resin treatment (0.2 % of resin w/w, 1 hr, 20 °C)

Fig. 5 .

 5 Fig. 5. Enzymatic saccharification of phytoremediation SRC willow wood. A) Cellulose-to-glucose conversion (%) at different points during enzymatic hydrolysis of willow1 (W1) SE pulps. B) Ethanol yield after 12 hr of fermentation

Table 1 -

 1 Composition of willow and trace element content (mg.kg -1 )

	4
	5
	6
	7
	8
	9
	10

a % of dry wood. b mg.kg -1 determined ICP-AES

Table 1 -Composition of willow and trace element content (mg.kg -1 )

 1

	Klason lignin a	37.4%
	Cellulose a	43.2%
	Hemicellulose a	16.0%
	Extracts a	7.75%
	Zn b	223.3±12.0
	Mn b	25.8±1.9
	Cd b	<0.1

Table 2 -

 2 Experimental conditions for the SE pretreatment. Compositions of the solid residues after pretreatment.

Glc: glucose, Xyl : xylose.

Table 2 -Experimental conditions for the SE pretreatment. Compositions of the solid residues after pretreatment. Glc: glucose, Xyl : xylose.

 2

	T°C T	PS a	Log	Glc a	Xyl b % Other	Zn c	Mn c
	(mi	%	Cs	%		sugars b %
	n)
	180 2	0.9	0.0	70.4 9	22.06	7.45	55.06 ±3,43	48.29±1 3,13
	180 5	0.9	0.4	79.3 1	15.57	5.11	38.97 ±2,61	37.13±1 ,13
	180 8	0.9	0.6	80.3 9	15.50	4.11	30.43 ±1,48	40.83 ±1,14
	200 2	0.9	0.5	81.9 7	13.93	4.10	34.00 ±4,40	22.92 ± 9,85
	200 5	0.9	0.9	82.5 5	12.92	4.54	26.74 ±2,09	34.75 ±0,98
	200 8	0.9	1.1	87.4 0	9.59	3.01	30.24 ±0,58	31.32 ±0,07
	220 2	0.9	1.1	87.3 2	9.17	3.51	26.74 ±2,09	34.75 ±0,98

Acknowledgements

The authors gratefully acknowledge the financial supports of the Conseil Régional de Lorraine through the LORVER project, the ANR through the PHYTOCHEM project (ANR 13-CD2I-0005-01) and the European Commission through FEDER funds. LERMAB is

supported by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-12-LABXARBRE-01). D. Belosinschi and F. Brouillette are warmly thanked for the resin samples.