Jeremy Rohmer
email: j.rohmer@brgm.fr.

Jean-Charles Manceau

Tracing back the pressure-impact zone of the CO 2 geological storage through a cyclic injection strategy

Keywords: CO2 geological storage, Far-field pressure perturbation, cyclic injection, Time series analysis, Harmonic Pattern

Industrial-scale CO2 injection into deep aquifers might induce far-field pressure perturbations on the basin scale, potentially impacting other underground uses present within the same aquifer formation. In the present paper, we investigate an approach to trace back the area of elevated pressure induced by CO 2 storage operations through an injection strategy in a cyclic manner over time. In this manner, the CO 2 injection-induced pressure field is characterized by a low magnitude harmonic signature very specific to the considered CO 2 storage, the frequency being chosen as different to naturally fluctuating phenomena or other anthropogenic underground activities. We rely on the combination of de-trending and FFT technique to detect the harmonic pattern associated the CO 2 storage injections. We apply the methodology on synthetic pressure signal numerically generated using a typical injection scenario in the Paris basin case. On this basis, we discuss how trace back the CO 2 storage site causing the pressure impact, in particular regarding the existing sources of noise.

Introduction

CO 2 capture and geological storage is seen as a promising technology in the portfolio of measures required to mitigate the effects of anthropogenic greenhouse gas emissions (IPCC [1]). Among the available geological targets, storage in deep aquifers is recognized to offer very large potential storage capacity with a broad distribution throughout the world in all sedimentary basins. Recently, the potential large-scale hydrodynamic impacts caused by CO 2 injections have been identified as a major limiting factor to the wide-scale deployment of this newcomer in the underground space use, see e.g., IEA-GHG [2]. Numerous studies modeling CO 2 injection in deep aquifers using either large-scale single phase or multiphase flow numerical simulations have shown that as injection progresses, pressure increases within the injection formation over a relatively large region (of the order of several tens of kilometers), see e.g., Zhou and Birkholzer [3]. The pressure pulse travels much faster than the CO 2 plume, which has the potential to displace reservoir fluids, far from the CO 2 plume itself, hence potentially impacting fresh groundwater resources at the aquifer outcrops (see e.g., IEA-GHG [START_REF] Iea-Ghg | Potential Impacts on Groundwater Resources of Geological Storage[END_REF]) or potentially hampering the performance, in terms of extraction rates, pore space available, injectivity, etc., of other underground uses (e.g., hydrocarbon and geothermal activities).

However, in a context where multiple underground uses are present within the same aquifer formation, tracing back the underground activity causing the observed pressure impact can turn to be a tedious and difficult task. The deep saline aquifer of the Dogger formation in the Paris basin (France) provides a good example of an aquifer targeted for CO 2 storage (Grataloup et al. [START_REF] Grataloup | A site selection methodology for CO2 underground storage in deep saline aquifers: case of the Paris Basin[END_REF]), which is currently exploited by petroleum and geothermal activities. Another good example is provided by the Mount Simon Sandstone in the context of Illinois basin (USA), which presents multiple underground usages such as natural gas storage and potable water exploitation (e.g., Zhou and Birkholzer [3]).

A parallel can be drawn with the issue of distinguishing natural biogenic CO 2 fluxes, anthropogenic CO 2 fluxes from industrial plants and CO 2 leaking from underground sources (petrogenic). Recently, techniques based on the carbon isotopic signature of CO 2 have brought very promising results (e.g., Krevor et al. [START_REF] Krevor | Real-Time Tracking of CO2 Injected into a Subsurface Coal Fire through High-Frequency Measurements of the 13 CO2 Signature[END_REF]). In the present paper, we aim at following the idea of a signature, but from a hydrodynamic perspective, i.e. by assigning to the injection-induced pressure field a signature of hydraulic nature. This hydraulic signature should be very specific to the considered CO 2 storage so that if detected, the CO 2 storage site causing the impact can be traced back.

In this view, we describe, in a first section, a strategy relying on a cyclic injection of CO 2 over time and on the detection of the induced cyclic pattern associated to the pressure evolution. To support the study, the hydrodynamic response of a deep aquifer formation during cyclic CO 2 injection is investigated through large-scale multi-phase flow transport numerical simulations using TOUGH2/ECO2n (Pruess [START_REF] Pruess | ECO2N: A TOUGH2 Fluid Property Module for Mixtures of Water, NaCl, and CO2[END_REF]). The carbonate Dogger aquifer is used as an application case. Finally, we show in a third section how to trace back the CO 2 storage site causing the pressure impact (i.e. to monitor the pressure-impact zone of the CO 2 storage) and discuss how to use such information in case of conflict of use.

Cyclic injection strategy

Principles

The proposed methodology relies on the cyclic injection of CO 2 over time into the aquifer formation. In this preliminary study, we assume that CO 2 is injecting at a constant injection pressure P 0 . A lowmagnitude ( P 0 e.g., of a few bars), a harmonic pressure signal (of time period T) is then added, so that the pressure-controlled injection P inj over time t can be expressed as:

P inj =P 0 + P 0 .sin(2 .t/T) (1)
The harmonic (cyclic) pressure signal of equation ( 1) is expected to propagate with the pressure pulse induced by the injection within the reservoir. The evidence of such a phenomenon is empirically supported by the observations of piezometric levels in the vicinity of seasonal natural gas storages in aquifers. For instance, in the case of the natural gas storages of Lussagnet and Izaute in South-West France, cyclic patterns in the temporal evolution of the piezometric level are both reported in the vicinity of the injection zone (of magnitude of the order of several tens of bars (1 bar = 10 5 Pa)), but also several tens of kilometers away from the injection wells with magnitude of the order of 1 bar, see Figure 3.5, page 64 in IEA-GHG [START_REF] Iea-Ghg | Potential Impacts on Groundwater Resources of Geological Storage[END_REF]. Such cyclic patterns can be correlated to the cyclic injection / withdrawal operations.

The basic idea is then to detect and extract the harmonic signal from the pressure time series. The issues associated with the detection and extraction procedure are further discussed in section 4. Provided that the cyclic pattern is known to be very specific to the considered CO 2 storage site (see discussion below), then such a signal can be considered its signature.

Designing the cyclic injection

Designing such a cyclic injection implies dealing with two main issues. On the one hand, the cyclic pattern extracted from the pressure measures should be distinguishable from other fluctuating phenomena, so that the cyclic pattern can be unambiguously recognized as the specific signature of the considered CO 2 storage site. This imposes choosing a time period T different from:

1. other anthropogenic underground activities such as cyclic groundwater pumping, seasonal natural gas storage, with usually 1-year periodicity;

2. naturally-fluctuating phenomena, which can take several forms. The most usual phenomena inducing water level fluctuations are the barometric pressure changes and earth tides (see e.g., Toll and Rasmussen [START_REF] Toll | Removal of barometric pressure effects and earth tides from observed water levels[END_REF]; Chang and Firoozabadi [START_REF] Chang | Gravitational Potential Variations of the Sun and Moon for Estimation of Reservoir Compressibility[END_REF] and references therein) of periodicity ranging from 12 to 24 hours. Other more complex natural fluctuations exist such as those associated to aquifer recharge derived from snow melt and early spring rains.

On the other hand, the pressure signal magnitude P 0 during one cycle should fulfill different requirements:

1. the maximum injection pressure P 0 + P 0 should not exceed the maximum sustainable injection pressure, i.e. without geomechanical degradation (such as micro-fracturing and/or fault reactivation) of the sealing structures (Rutqvist et al. [START_REF] Rutqvist | Estimating maximum sustainable injection pressure during geological sequestration of CO2 using coupled fluid flow and geomechanical fault-slip analysis[END_REF]) or/and drive fluid through conductive features into shallow groundwater resources (such as reservoir brine, see e.g., Birkholzer et al. [START_REF] Birkholzer | Brine flow up a borehole caused by pressure perturbation from CO2 storage: static and dynamic evaluations[END_REF]). It is worth underlying that such a value should be estimated case by case.

2. the constraints on the injection process should be minimized. Imposing a time-varying injection pressure leads to a temporal variation of the total amount of injected CO 2 so that extra amount of CO 2 compared to the case of constant injection pressure is necessary. A possible option is to use a temporary storage unit (tank) at the surface. Their capacity as those commonly used in the domain of gas ship transport (see e.g., IEA-GHG [START_REF]IEA-GHG Ship Transport of CO2[END_REF]) range from 10,000 to 50,000 tonnes.

Synthetic application case

Model set-up and parameters

The described methodology is applied using the application case of the carbonate Dogger aquifer in the Paris basin. This formation has been targeted for potential host of CO 2 geological storage (Grataloup et al. [START_REF] Grataloup | A site selection methodology for CO2 underground storage in deep saline aquifers: case of the Paris Basin[END_REF]). The aquifer layer is assumed to be of very large spatial extent (>100 km). The system considers one layer with a thickness of 40 m (1D-axisymetrical model). A no-flow condition is assigned to the lateral boundary and the system is considered closed with no exchange of fluid or heat with the upper and lower layers. The grid includes series of refined zones from the injection zone with a minimum grid cell size of 0.2m, to the aquifer margins with a maximum grid cell size of 1 km. Using average values based on Andre et al. [START_REF] Andre | Numerical modeling of fluid rock chemical interactions at the supercritical CO2 liquid interface during CO2 injection into a carbonate reservoir, the Dogger aquifer (Paris Basin, France)[END_REF] and references therein, homogeneous and isotropic properties are assigned to the aquifer formation. The mean porosity is 15% and the intrinsic permeability is assumed to be spatially homogeneous at 200.10 -15 m². Note that the presence of heterogeneities (e.g. low permeable zones) are expected to have an influence on the frequency content of the pressure pulse (in a filter fashion for instance) and a future line of research will, therefore, focus on this aspect.

Capillary pressure and relative permeability model (and their parameters) are described in details in Andre et al. [START_REF] Andre | Numerical modeling of fluid rock chemical interactions at the supercritical CO2 liquid interface during CO2 injection into a carbonate reservoir, the Dogger aquifer (Paris Basin, France)[END_REF]. Initial temperature and pressure conditions respectively reach 75°C and 18.0 MPa. Salinity in the Dogger reservoir reach very low to moderate values (from 5 g of NaCl per 1000 g of water in the Southern part of the basin to about 30 g of NaCl per 1000 g of water in the Eastern part of the basin, Andre et al. [START_REF] Andre | Numerical modeling of fluid rock chemical interactions at the supercritical CO2 liquid interface during CO2 injection into a carbonate reservoir, the Dogger aquifer (Paris Basin, France)[END_REF]). The model was homogeneously assigned a mean value of 15 g of NaCl per 1000 g of water.

Numerical simulations are performed using the multi-phase, multi-component transport simulator TOUGH2 with the ECO2N Equation of State (Pruess [START_REF] Pruess | ECO2N: A TOUGH2 Fluid Property Module for Mixtures of Water, NaCl, and CO2[END_REF]), which takes into account the thermodynamic and thermophysical properties of water NaCl CO 2 mixtures. The problem is assumed to be isothermal.

Analysis of the cyclic injection

The cyclic injection is performed by imposing a time-dependent pressure evolution (following equation 1) at the grid cell representing the injection wellbore. The constant injection pressure P 0 is chosen as ~20.0 MPa (1 MPa=10 6 Pa) so that approximately 1 million tons of CO 2 per year are injected through a single injection well. Accounting for the two constraints described in section 2, the time-period T is chosen as 200 days, and the low magnitude of the harmonic signal P 0 as 2.0% of P 0 (0.4 MPa). The harmonic temporal evolution of the imposed injection pressure is depicted in Fig. 1A.

The small variation of injection pressure implies a variation in the injected amount of CO 2 (relatively to the one that would be injected with no variation in P inj ) corresponding to a maximum of about 40,000 t (see Fig. 1B), which is in good agreement with the capacity of existing storage unit (see section 2.2). . This shows that the pressure pulse can travel very far. For instance the over-pressure reaches 0.15 MPa at a distance of 20 km after 1000 days of injection. Such an order of magnitude can be judged moderately significant regarding the risk of brine uplift through potential high-permeable conduits (Birkholzer et al. [START_REF] Birkholzer | Brine flow up a borehole caused by pressure perturbation from CO2 storage: static and dynamic evaluations[END_REF]). Yet, we show that the harmonic pattern is only distinguishable through a mere visual inspection for both cases at 2 and 10 km. Over 10 km, the pattern appears to be hardly distinguishable. Logically, increasing P 0 implies detecting the harmonic pattern at farther distances, but this also means increasing the amount of temporally stored CO 2 . In the next section, we show how to extract the harmonic signal using signal processing methods.

Tracing back the pressure-impact zone

Downhole pressure transducers -coupled with electronic data-loggers -are commonly used during ground water investigations to measure and record water levels in wells for long periods at relatively short intervals. Tracing back the pressure-impact zone implies detecting the harmonic pattern associated with the cyclic injection. The issue of extracting seasonal (periodic) fluctuations from these measures has long been studied in the field of hydrology and hydrogeology (see e.g., Toll and Rasmussen [START_REF] Toll | Removal of barometric pressure effects and earth tides from observed water levels[END_REF] and references therein) resulting in the development of several post-processing techniques, which basically rely on spectral analysis of time series.

A popular approach is the Fast Fourier Transform, denoted FFT, analysis (see mathematical description provided for instance by Edge and Liu [START_REF] Edge | Comparing Power Spectra Computed by Blackman-Tukey and Fast Fourier Transform[END_REF]). This analysis relies on the representation of the data via Fourier series: instead of representing the signal amplitude as a function of time, an alternate is to represent the signal by how much information is contained at different frequencies, i.e. the coefficients of the Fourier series. The output of such an algorithm is the power spectrum of the signal representing the frequencies versus the square of the absolute value of the FFT coefficients (i.e. power of the signal).

One major difficulty associated with the considered signals is the presence of a trend, i.e. the average over time is not a constant, but a function of time, which can be non-linear (as illustrated by Fig. 2). A typical pre-processing step in spectral analysis is to de-trend the time series. This can be achieved by fitting a polynomial regression model (least-squares-fit) to the data and then remove it from the original data. More advanced techniques exist; please refer to Hamilton [START_REF] Hamilton | Time series analysis[END_REF] for a review. Fig. 3 illustrates the procedure for data where the harmonic pattern can be detected through visual inspection. Fig. 3A the original pressure signal observed at 10 km and the associated trend fitted by a polynomial regression model of order 5. Fig. 3B shows the output (extracted signal) of the de-trending procedure. Applying the FFT analysis, the power spectrum (Fig. 3C, note that it is directly presented in terms of time period versus power) clearly shows a peak at the time period of 200 days. Fig. 4 illustrates the procedure for the pressure signal at 20 km with no visual detection of harmonic pattern. Yet, the afore-described analysis is conducted on synthetic data and structures of real data are expected to be far more complex and associated with different sources of noise. On the one hand, as mentioned in section 2, fluctuations caused by gravitational Earth tides and barometric pressure fluctuations, which can be on the order of 100 1,000 Pa (see e.g., Chang and Firoozabadi [START_REF] Chang | Gravitational Potential Variations of the Sun and Moon for Estimation of Reservoir Compressibility[END_REF]), should be filtered, hence potentially introducing processing errors. On the other hand, the proposed monitoring should be conducted over long time periods (years), and p on term, which can interfere with the detection of the harmonic pattern. Based on the approach of Chabora and Benson [START_REF] Chabora | Brine Displacement and Leakage Detection Using Pressure Measurements in Aquifers Overlying CO2 Storage Reservoirs[END_REF], a cutoff of 1,000 Pa was used to categorize the detection potential as unlikely so that the harmonic pattern should be detected at 19 km away from the injection zone.

Such a distance can then be considered as the maximum spatial extent, according to the harmonic amplitude used in this study, at which it is possible to ascertain that the pressure perturbation (here of the order of 1.5 10 5 Pa after 1,000 days of injection) is due to the CO 2 storage operations. This distance should be put in perspective from a conflict of use point of view: this method can be useful to manage conflict of use from underground activities like hydrocarbon exploitation, deep geothermal activities, but at such a distance, activities related to the exploitation of drinking water may not exist. A future direction of improvement of the proposed technique is to quantify, in such a context of multiple uses of the underground, the proportion of the overpressure due to the CO 2 storage only (relatively to the overpressure due to other activities).

Concluding remarks and further work

Industrial-scale CO 2 injection into deep aquifers might induce far-field pressure perturbations on the basin scale, potentially impacting other underground uses present within the same aquifer formation. We propose an approach to assign to the injection a signature of hydraulic nature through an injection strategy in a cyclic manner over time. If detected, it would be possible to identify the CO 2 storage site causing the observed hydrodynamic impact.

Based on synthetic pressure signals numerically generated using a typical injection scenario in the Paris basin case, the present study defines the basis and discusses the difficulties (especially regarding the existing sources of noise) for such the detection of the hydraulic signature.

Further works should confront the proposed methodology to complex real pressure signals. Complexity can arise from noise, but also from the phasing of the CO 2 capture and transport process. In the present preliminary study, we have assumed that the CO 2 is injected at a constant pressure (to which a cyclic signal is added). This assumption may not be valid for industrial real injection operations. See for instance the complex pressure time series at the Ketzin site ( [START_REF] Lengler | The impact of heterogeneity on the distribution of CO2: Numerical simulation of CO2 storage at Ketzin[END_REF]: Fig. 2). Hence, as a future line of research, we can envisage taking advantage of this process-related complexity to define the hydraulic signature specific to the CO 2 storage site.

Fig. 1 .

 1 Fig. 1. (A) Harmonic temporal evolution of the imposed injection pressure with P0=20 MPa (1 MPa=10 6 Pa), T=200 days and P0= 0.4 MPa (2% of P0); (B) Temporal variation of the injected mass of CO2 due to cyclic injection, relatively to the one that would be injected with no variation in Pinj (small oscillations are due to rounding errors in the post-treatment procedure)

Fig. 2 .

 2 Fig. 2. Temporal evolution of the over-pressure (A) at 2 km away from the injection zone; (B) at 10 km; (C) at 20 km and (D) at 40 km. Note the differences in the choice of over-pressure scales used for each signal.

Fig. 3 .

 3 Fig. 3. (A) Pressure signal (black curve) at 10 km from the injection zone and the associated trend fitted by a polynomial model of the order 5 (red curve); (B) Pressure signal extracted from the original signal by removing the trend; (C) Power spectrum (expressed in time period versus power) computed through FFT analysis of the extracted signal.

Fig. 4 .

 4 Fig. 4. (A) Pressure signal (black curve) at 20 km from the injection zone and the associated trend fitted by a polynomial model of the order 7 (red curve); (B) Pressure signal extracted from the original signal by removing the trend; (C) Power spectrum (expressed in time period versus power) computed through FFT analysis of the extracted signal.

Acknowledgements

This work was funded under the BRGM's Directorate of Research project. We are grateful to T. Le Guenan, O. Bouc and H. Fabriol for useful discussion.


Floriane Delpy

Yves Lucas

Patricia Merdy

Evaluation of Roundup R effects on Chlorella vulgaris through spectral changes in photosynthetic pigments in fresh and marine water

HIGHLIGHTS

 Roundup® caused spectral changes in photosynthetic pigments of Chlorella vulgaris.

 Absorption spectra and peak areas were used to monitor toxic effects.

 Cells were stimulated from 10 to 5,000 µg L -1 in freshwater and inhibited otherwise.

 C. vulgaris adapted to seawater was more tolerant above 1 mg L -1 of glyphosate.

 The dose-response relationship depended on salinity.

INTRODUCTION

Glyphosate (N-(phosphonomethyl)glycine) is a broad-spectrum herbicide discovered in 1950 by Swiss chemist Henri Martin. It has been marketed since 1974 in different formulations such as Roundup®, first produced by the Monsanto company and then by the Bayer group, which bought the company in 2018. In 2014, 825,804 tons of glyphosate-based herbicides were sold worldwide including 113,000 tons in the United States alone [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Glyphosate is particularly used in agricultural areas where crops are genetically modified and herbicide tolerant. This organic molecule is detected in a wide variety of aquatic environments, from surface water to groundwater [START_REF] Battaglin | Glyphosate and its degradation product AMPA occur frequently and widely in U.S. soils, surface water, groundwater, and precipitation[END_REF][START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Spread by runoff, soil leaching and aerosols, it is even observed in estuarine and marine environments [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Glyphosate is also stored in soils and sediments due to its high affinity for clay and oxide particles, which induces low mobility [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Its use is now widespread in urban areas, hence its presence in the effluents of wastewater treatment plants (WWTPs) [START_REF] Grandcoin | Aminomethylphosphonic acid (AMPA) in natural waters: Its sources, behavior and environmental fate[END_REF][START_REF] Poiger | Behavior of glyphosate in wastewater treatment plants[END_REF]. Koplin et al. (2006) reported overall removal efficiencies at WWTPs ranging from 71 to 96%. Despite seasonal variations, environmental concentrations remain in the order of a few µg L -1 , suggesting chronic contamination rather than one-time acute events [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF].

Studies on the fate of glyphosate in natural environments have shown its resistance to photolysis and chemical breakdown [START_REF] Grandcoin | Aminomethylphosphonic acid (AMPA) in natural waters: Its sources, behavior and environmental fate[END_REF]. The first degradation pathway results from microbial activities in biofilms and leads to the formation of aminomethylphosphonic acid (AMPA) and glyoxylate after oxidation under aerobic conditions by glyphosate oxidoreductase [START_REF] Carles | Meta-analysis of glyphosate contamination in surface waters and dissipation by biofilms[END_REF][START_REF] Grandcoin | Aminomethylphosphonic acid (AMPA) in natural waters: Its sources, behavior and environmental fate[END_REF][START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. The second is via biodegradation by C-P lyase to sarcosine and inorganic phosphate [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. The persistence of glyphosate was determined in various environments by evaluating the dissipation half-life (DT50). A range of 7 to 130 days was estimated for soils, indicating rapid degradation [START_REF] Grandcoin | Aminomethylphosphonic acid (AMPA) in natural waters: Its sources, behavior and environmental fate[END_REF]. This duration was greater in seawater at 47-267 days, as well as in rivers at 13-301 days [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF][START_REF] Mercurio | Glyphosate persistence in seawater[END_REF]. The metabolite AMPA appears to be more mobile and persistent than its parent molecule (Koplin et al., 2006). Its presence can be detectable for several years instead of a few months for glyphosate [START_REF] Maggi | The global environmental hazard of glyphosate use[END_REF]. These processes are largely influenced by local environmental factors. [START_REF] Maggi | The global environmental hazard of glyphosate use[END_REF] showed that precipitation, pH, and soil organic carbon are the main factors controlling the distribution of contamination hotspots worldwide. Phosphorus availability is also influential as it hinders the biodegradation of glyphosate and promotes the accumulation of AMPA in water [START_REF] Carles | Meta-analysis of glyphosate contamination in surface waters and dissipation by biofilms[END_REF].

Due to strong societal demand, the ecotoxicological effects of glyphosate-based herbicides are now widely studied in terrestrial and aquatic habitats [START_REF] De Castilhos Ghisi | Glyphosate and its toxicology: A scientometric review[END_REF]. This emerging contaminant is classified as a chronic aquatic hazard (category 2) and considered toxic to aquatic organisms with long-lasting effects (hazard statement H411) [START_REF] Ineris | Valeur guide environnementale : Glyphosate (CAS n° 1071-83-6[END_REF]. Since 2015, the World Health Organization has reclassified it as probably carcinogenic to humans [START_REF] Myers | Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement[END_REF][START_REF] Van Bruggen | Environmental and health effects of the herbicide glyphosate[END_REF]. Glyphosate acts as a chelating agent that binds to micro-and macronutrients, altering many plant processes, pathogen resistance and bioavailability of elements in soils [START_REF] Mertens | Glyphosate, a chelating agent-relevant for ecological risk assessment?[END_REF]. In addition, the degradation of Roundup® significantly increases total phosphorus, which promotes the process of eutrophication [START_REF] Vera | New evidences of Roundup® (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems[END_REF]. Although toxic to some phytoplankton species, this phosphorus (P) input can be used by marine diatoms (Emiliania huxleyi, Skeletonema costatum, Phaeodactylum tricornutum) to support their growth, beyond that of any dissolved inorganic phosphorus (DIP) availability [START_REF] Wang | Differential growth responses of marine phytoplankton to herbicide Glyphosate[END_REF]. The development of these diatoms, but also of some cyanobacteria and prasinophytes, could be stimulated, potentially affecting the structure of the phytoplankton community [START_REF] Cruz De Carvalho | Glyphosate-based herbicide toxicophenomics in marine diatoms: Impacts on primary production and physiological fitness[END_REF][START_REF] Stachowski-Haberkorn | Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment[END_REF][START_REF] Vera | New evidences of Roundup® (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems[END_REF][START_REF] Wang | Differential growth responses of marine phytoplankton to herbicide Glyphosate[END_REF].

Acute toxicity tests with pure glyphosate or in commercial formulations show lethality at concentrations well above those observed in nature [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Focusing upon the foodweb base is important as this is where photoautotrophic microorganisms, essential for healthy aquatic ecosystems, are found [START_REF] Van Bruggen | Environmental and health effects of the herbicide glyphosate[END_REF]. In freshwater, green microalgae had a half maximum effective concentration (EC50) of 3.5 mg L -1 (Chlorella pyrenoidosa) and 12.5 mg L -1 (Pseudokirchneriella costatum) after a 96-hour exposure to glyphosate [START_REF] Ineris | Valeur guide environnementale : Glyphosate (CAS n° 1071-83-6[END_REF]. The noobserved-effect concentration (NOEC) for the latter species was 10 mg L -1 after 120 hours. In seawater, the data obtained often highlight much lower values. For example, the diatom Skeletonema costatum had an EC50 of 0.85 mg L -1 after a 96-hour exposure to glyphosate and a NOEC of 1.8 mg L -1 after 120 hours [START_REF] Ineris | Valeur guide environnementale : Glyphosate (CAS n° 1071-83-6[END_REF]. Moreover, chronic exposure may tend to alter the function of some fondamental metabolic processes without affecting the survival of photoautotrophic microorganisms. The mode of action of glyphosate on these microorganisms is the same as that observed on terrestrial plants. Aromatic amino acid synthesis, respiration, photosynthesis, and chlorophyll production can be disrupted [START_REF] Van Bruggen | Environmental and health effects of the herbicide glyphosate[END_REF]. Impaired electron transport in thylakoids and decreased antioxidant capacity inhibit photosynthetic activity and increase lipid peroxidation [START_REF] Cruz De Carvalho | Glyphosate-based herbicide toxicophenomics in marine diatoms: Impacts on primary production and physiological fitness[END_REF]. [START_REF] Delorenzo | Toxicity of pesticides to aquatic microorganisms: A review[END_REF] also showed negative impacts on carbon uptake in the two diatoms Nitzschia sp. and Cyclotella meneghiniana.

The green microalga Chlorella sp. is commonly used in ecotoxicological studies due to its ubiquity in marine and continental habitats, from polar to tropical environments [START_REF] Hodač | Widespread green algae Chlorella and Stichococcus exhibit polartemperate and tropical-temperate biogeography[END_REF].

Belonging to the first trophic level, this unicellular microorganism participates in the production of organic matter by photosynthesis and is a prey for herbivorous zooplankton. Contaminants such as glyphosate could cause changes in this population, including biomass and photosynthetic efficiency, which could have cascading effects on the entire ecosystem. Known for being rich in chlorophylls and carotenoids (Safi et al., 2018), the species Chlorella vulgaris Beijerinck, 1890 was selected in order to determine a dose-response relationship with Roundup® through the spectral characteristics of photosynthetic pigments. Ultraviolet/visible spectroscopy was used to follow the evolution of the cells for five days without compromising their survival. The characteristics of chlorophyll and carotenoid peaks were analyzed to seek a more informative approach than the usual determination of a single-wavelength absorbance. In addition, the resilience of this cosmopolitan species was assessed in relation to the salinity of the environment in which it occurred (freshwater / seawater).

MATERIAL AND METHODS

Contaminant characteristics

The herbicide glyphosate (C3H8NO5P, CAS Registry Number 1071-83-6) is the active ingredient in the commercial formulation of Roundup®. The solution used in this study had a glyphosate concentration of 450 g L -1 . The solubility of glyphosate is 10.5 g L -1 in water at 20°C and pH 2, and remains insoluble in most organic solvents [START_REF] Ineris | Valeur guide environnementale : Glyphosate (CAS n° 1071-83-6[END_REF]. A surfactant, such as polyoxyethylene amine (POEA), is included in glyphosate-based herbicides to facilitate penetration through the plant surface and increase effectiveness. Its main degradation product is aminomethylphosphonic acid, also known as AMPA, which is a weak organic acid with a phosphonate group (CH6NO3P, CAS Registry Number 1066-51-9).

Chlorella vulgaris culture

The phytoplankton model organism, Chlorella vulgaris Beijerinck, 1890 (5-10 µm), was used to test the potential ecotoxicological effects of Roundup® in two types of aquatic environment: inland waters and coastal marine areas. Monospecific cultures were performed in the laboratory using the « Teramer®-Start » kit. A first strain was maintained in freshwater (hereafter referred to as FW-Chlorella), while the second was adapted to a salty environment equivalent to seawater (hereafter referred to as SW-Chlorella). The organisms were kept in an air-conditioned room (~20°C), exposed to a light-dark regime (12h neon light -12h darkness), oxygenated by bubbling at a controlled air flow, and transplanted regularly. Dust deposition was limited by applying an aluminium foil to the top of the bottles but leaving an opening on the side for air bubbling.

Ecotoxicological test

Two dilution ranges were prepared from the commercial solution of Roundup®, one in ultrapure water and the other in synthetic seawater (salinity 35). The final concentrations of glyphosate to which Chlorella cells were exposed were 0, 0.1, 1, 10, 100, 1,000, 5,000, 10,000 and 25,000 μg L -1 .

A total volume of 6 mL was placed in 15 mL clear tubes at ~20°C and subjected to the same lightdark regime as for the culture. The caps of the tubes were not tightened to allow gas exchange and the water was oxygenated every day by turning them slowly. No nutrients were added during the five-day test. The concentration of 0 µg L -1 represented a control to monitor population changes under experimental conditions without exposure to the contaminant. Two experimental series, A and B, were carried out as duplicates for FW-Chlorella and for SW-Chlorella.

Spectroscopic measurements

The absorption spectrum of C. vulgaris was measured for five days by ultraviolet/visible spectroscopy (SHIMADZU®, UV-1800). Measurements were first made one hour after contact with the contaminant, and then every day at the same time. The acquisition of absorbance values was achieved over a wide range of wavelengths (200 to 900 nm) and at high resolution (every 1 nm) in order to include the peaks corresponding to the main photosynthetic pigments of this green microalga: total carotenoids (~390-540 nm) and chlorophylls (~640-730 nm). A blank containing the culture medium was measured for each strain and subtracted from the sample spectra.

Data analysis

UV Probe 2.42 software was used to process the resulting absorption spectra. Peak characteristics wavelength at maximum absorbance) were recorded for each strain, series, and concentration.

Principal component analysis (PCA) with Pearson's correlation coefficient as the similarity index was performed with XLSTAT 2019.1.1.62917 (Microsoft Excel®, Addinsoft). The objective was to assess which spectral measurements (i.e., peak area, peak height, peak width at base, wavelength at maximum absorbance for chlorophylls and carotenoids, as well as the ratio of chlorophyll to carotenoid peak areas) were most indicative of a potential effect of glyphosate on C. vulgaris. FW-Chlorella and SW-Chlorella data were analyzed together to better understand the difference in response with salinity. Variables were standardized using the skewed standard deviation to overcome the difference in units. A distance biplot was constructed to represent approximate Euclidean distances between the variables.

RESULTS

Influence of salinity on Chlorella vulgaris

Before performing the ecotoxicological tests, we evaluated the effect of salinity on the population dynamics of Chlorella vulgaris for five days without any exposure to Roundup®. Then, the results obtained in the presence of glyphosate were compared to this control experiment. The absorption spectra changed as a function of time (from day 1 to day 5), but also as a function of the culture medium. Figures 1 and2 represent the different spectral characteristics measured with the freshwater and seawater controls: absorption spectra between 350 and 800 nm (Figure 1), as well as peak areas (Figure 2A), peak widths at base (Figure 2B), peak heights (Figure 2C), and wavelengths at maximum absorbance (Figure 2D) for each photosynthetic pigment identified.

Absorption spectra had a similar appearance in freshwater and seawater with two overlapping peaks -Car 1 and Car 2 -representing total carotenoids (391-538 nm for FW-Chlorella, 391-534 nm for SW-Chlorella) and a better-defined peak representing chlorophylls (638-733 nm for FW-Chlorella, 638-720 nm for SW-Chlorella) (Figure 1). In both cases, duplicates A and B demonstrated the same spectral profile, as well as the same trend. For carotenoids, the height and wavelength at maximum absorbance were determined for each Car 1 and Car 2 peak, while the area and width at the base were evaluated for these two peaks together. Photosynthetic pigment peaks were slightly less spread out at the base for SW-Chlorella. The mean absorbance value between 350 and 800 nm was lower in freshwater, which is the natural environment of Chlorella, than in seawater (0.747 ± 0.115 and 0.992 ± 0.114, respectively). Statistics on the entire data set (series A and B) (Figure 2) showed that, as with the absorbance values, the peak areas of photosynthetic pigments were lower in FW-Chlorella (3.34 ± 0.84 for chlorophylls, 4.25 ± 1.39 for carotenoids) than in SW-Chlorella (5.56 ± 0.75 for chlorophylls, 8.52 ± 1.49 for carotenoids) (Figure 2A). The same pattern was observed for the peak height of chlorophylls (0.094 ± 0.024 in freshwater, 0.177 ± 0.023 in seawater) and carotenoids (0.048 ± 0.015 in freshwater, 0.099 ± 0.017 in seawater) (Figure 2C). However, with respect to the peak width at the base, chlorophylls exhibited higher values in freshwater (around 9 nm higher), whereas carotenoids had similar values in both environments. There was also a slight shift in the wavelength at maximum absorbance for chlorophylls and carotenoids (Figure 2B). Thus, values were higher in FW-Chlorella than in SW-Chlorella with an average deviation of 1.4 nm for chlorophylls, 1.1 nm for Car 1 and 2.9 nm for Car 2 (Figure 2D). The time course of the spectra during the five-day experiment was also different (Figure 3). Despite the significant differences observed between the two series, FW-Chlorella showed a decrease in absorbance values on the second day, followed by a stabilization on day 3, then a further decrease on the last two days. SW-Chlorella was maintained for the first two days at high absorbance values and decreased on the third day.

Evolution of C. vulgaris exposed to Roundup® contamination

The temporal evolution of the population can be determined from the average absorbance calculated from the A and B series (Figure 4). The absorption spectra exhibit a baseline drift over the measurement time. These shifts are often due to variable light scattering between samples, especially in the case of heterogeneous particle sizes. However, this does not affect the processing of the spectra.

For FW-Chlorella, a decrease was observed between day 1 and day 5 (Figures 4A and4C). At the beginning of the experiment, the spectra of the lowest and highest concentrations of glyphosate (0.1, 1, 10,000 and 25,000 μg L -1 ) were equal to or lower than the spectrum of the control (Figure 4A). Conversely, cell stimulation was observed at intermediate concentrations (10, 100, 1,000 and 5,000 μg L -1 ). Five days later, the trend was similar, but with recovery occurring with the 0.1 μg L -1 concentration (Figure 4C). SW-Chlorella appeared to maintain its population throughout the experiment with absorbance values of 1.04 ± 0.11 and close spectra for all concentrations (Figures 4B and4D). On the first day, the spectra obtained for 0.1, 1 and 10 μg L -1 were lower than the control spectrum (Figure 4B). An increase in absorbance values with increasing glyphosate concentration was then observed, with the spectra of 100 and 1,000 μg L -1 being at the same level as the control spectrum and those of 5, 10 and 25 mg L -1 exceeding it. On the fifth day, the control spectrum ended up with the lowest absorbance values (Figure 4D). The spectra of 0.1, 1, 10 and 100 μg L -1 were located just above the control spectrum, followed by those of 1, 5, 10 and 25 mg L - 1 . Furthermore, the difference between series A and B emphasized some instability for FW-Chlorella. This corresponded to 10-20% in the average absorbance values (350-800 nm) for five of the nine concentrations tested on day 1 and only one concentration on day 5. This difference reached more than 20% on four occasions on days 2, 3 and 4. In 65% of cases, it remained below 10%. For SW-Chlorella, the percentages were systematically lower than 10%, and even 5% in 68% of cases. In terms of peak area changes as a function of time, the normalized values oscillated around the control for FW-Chlorella (Figures 5A and5C), with a larger amplitude for carotenoids than for chlorophylls. The impact of glyphosate appeared to emerge at the end of the experiment with the lowest concentrations above the control (0.1, 100 and 1,000 μg L -1 ) and the highest below (5, 10 and 25 mg L -1 ). An exception was noted at 10 μg L -1 with a normalized peak area lower than the control for chlorophylls and higher for carotenoids. For SW-Chlorella, the peak areas of the contaminated solutions presented higher values than the control, especially on day 3 (Figures 5B and5D). An inverse dose-effect relationship to that observed with the absorption spectra was demonstrated, as the normalized peak areas slowly decreased with increasing glyphosate concentration, which was particularly marked on days 4 and 5. The analysis of the other spectral features allowed us to know more precisely which component of the peak area can be modified in case of environmental perturbation. For FW-Chlorella, the decrease in peak areas between the lowest concentration tested (3.52 ± 0.79 at 0.1 µg L -1 ) and the highest (2.63 ± 0.85 at 25 mg L -1 ) corresponded to a peak height crush of 23% for chlorophylls and 27-28% for carotenoids. For SW-Chlorella, the extension of peak areas for chlorophylls (from 5.75 ± 0.38 at 0.1 µg L -1 to 6.17 ± 0.59 at 25 mg L -1 ) and for total carotenoids (from 8.64 ± 0.97 at 0.1 µg L -1 to 9.62 ± 1.17 at 25 mg L -1 ) was both in height (6% for chlorophylls, 8 and 12% for Car 1 and Car 2 respectively) and in width (1.1 nm for chlorophylls, 0.9 nm for total carotenoids).

Carotenoids also showed a shift in the wavelength value of their two peaks, which decreased by 0.8-0.9 nm between the lowest and highest concentrations tested.

Use of peak areas as a bioindicator in ecotoxicology

We performed a principal component analysis (PCA) on the FW-Chlorella and SW-Chlorella data to assess which spectral variables were most relevant to consider (Figure 6). The first two factorial axis explained 71.19% of the total variance, which was related to the F1 axis (62.71%). The Pearson's correlation coefficient between Car 1 height, Car 2 height, Car peak area, Chl height, Chl peak area, and average absorbance were all higher than 0.97; so, the description of the evolution of one over time applied to the others. For the chlorophyll and carotenoid peaks, there was an anticorrelation between wavelength and height. When the peak was higher, there was a shift in wavelength at maximum absorbance to a lower value. The chlorophyll/carotenoid peak area ratio was positively correlated with the wavelength at maximum absorbance and the width at the base of the chlorophyll peak (ρ > 0.77), indicating that the variations in the chlorophyll peak were larger than those in the carotenoid peak. This analysis also confirmed the influence of water salinity on absorbance values and spectral characteristics of photosynthetic pigments, especially for chlorophylls (ρ > 0.84). Peak area anomalies of each photosynthetic pigment were then calculated (Figure 7). They corresponded to the difference between the experimental and control peak areas. In the upper right part of the graph (chlorophyll anomaly > 0 and carotenoid anomaly > 0), the increase of both values indicated a stimulation of photosynthetic cells. Conversely, in the lower left (chlorophyll anomaly < 0 and carotenoid anomaly < 0), their decrease corresponded to inhibition. In both cases, the contribution of photosynthetic pigments remained balanced. Finally, carotenoids appeared promoted in the upper left (chlorophyll anomaly < 0 and carotenoid anomaly > 0), while chlorophylls took the lead in the lower right (chlorophyll anomaly > 0 and carotenoid anomaly < 0). Regardless of the environment in which the cells evolved, chlorophyll and carotenoid anomalies were distributed along a line from a zone of inhibition (anomalies < 0) to a zone of stimulation (anomalies > 0). In our ecotoxicological test, no imbalance in the contribution of photosynthetic pigments was observed. The two inverse trends previously highlighted were confirmed by this bioindication tool.

The lowest and two highest concentrations (1, 10,000 and 25,000 μg L -1 ) negatively affected FW-Chlorella, whereas intermediate concentrations stimulated them (Figure 7A). In contrast, SW-Chlorella showed a clear increase in chlorophyll and carotenoid anomalies above 1 mg L -1 (Figure 7B). The association of chlorophyll and carotenoid peak areas in a ratio showed that the contribution of photosynthetic pigments was mainly affected by the growing environment (Figure 8). Over the whole experiment, the contribution of chlorophylls to carotenoids appeared particularly high for FW-Chlorella with an average ratio value of 0.804 ± 0.070, while it reached 0.656 ± 0.027 for SW-Chlorella. The temporal evolution of this ratio remained similar with an increase of 8.2% in freshwater and 6.8% in seawater between day 1 and day 5. The lack of imbalance in the contribution of photosynthetic pigments that was demonstrated by the analysis of peak area anomalies was confirmed by the chlorophyll/carotenoid peak area ratio. For FW-Chlorella, the ratio was slightly higher above 1 mg L -1 of glyphosate, with median values ranging from 0.816 to 0.845 (versus 0.790 for the control). For SW-Chlorella, the median value of the chlorophyll/carotenoid peak area ratio was approximately 0.653 ± 0.013 for all concentrations tested.

DISCUSSION

Standard ecotoxicology experiments on phytoplankton presented by the Organisation for Economic

Co-operation and Development (OECD) consist of a growth inhibition test (OECD, 2011). The cell biomass is evaluated by counting under a microscope equipped with a counting chamber.

Automated systems, such as flow cytometry, can also be used for pico-and nanophytoplankton (0.2 to 20 µm). An optical measurement is only carried out as a complement to appreciate the evolution of the biomass, provided that there is a good correlation between the counted biomass and the chlorophyll concentration. This type of result is intended to obtain the classical indices of ecotoxicology. The EC50 and EC10 (effective concentration) represent contaminant concentrations that result in a 50% and 10% reduction in biomass growth compared to the control [START_REF] Chemsafetypro | How to use EbC50, ErC50, ErC10 and NOEC data from algae growth inhibition test[END_REF]. The NOEC (no-observed-effect concentration) indicates the highest concentration for which no toxic effects are detected [START_REF] Chemsafetypro | How to use EbC50, ErC50, ErC10 and NOEC data from algae growth inhibition test[END_REF]. As part of the REACH regulation in Europe (Registration, Evaluation and Authorisation of CHemicals), these indices are then used to calculate the PNEC (predicted no-effect concentration) which is the highest concentration of contaminant that is safe for aquatic environments [START_REF] Chemsafetypro | How to use EbC50, ErC50, ErC10 and NOEC data from algae growth inhibition test[END_REF]. In this research work, we tried to develop an approach to assess the ecotoxicological impact on a metabolic function, such as photosynthesis, rather than on population growth. Altering this fundamental process could have negative consequences at the base of the foodweb, which can cascade to higher trophic levels and affect the functioning of aquatic ecosystems.

Pigment content is usually assayed after extraction by high performance liquid chromatography (HPLC) [START_REF] Mahmoud | Simulation of absorption spectrum of photosynthetic pigments of Chlorella vulgaris B algae[END_REF][START_REF] Picazo | Spectrophotometric methods for the determination of photosynthetic pigments in stratified lakes: a critical analysis based on comparisons with HPLC determinations in a model lake[END_REF][START_REF] Safi | Extraction of lipids and pigments of Chlorella vulgaris by supercritical carbon dioxide: influence of bead milling on extraction performance[END_REF]. Nevertheless, this technique leads to the destruction of the cells and prevents the monitoring of their physiological changes according to the studied perturbation. Microalgae were best preserved when analyzed by ultraviolet/visible spectroscopy. It should be noted that this method remains less accurate than HPLC, both in terms of quality and quantity (Dos [START_REF] Santos | Comparison of three methods for Chlorophyll determination: Spectrophotometry and fluorimetry in samples containing pigment mixtures and spectrophotometry in samples with separate pigments through high performance liquid chromatography[END_REF]. Two groups of photosynthetic pigments -chlorophylls and carotenoids -can be identified on the absorption spectrum of Chlorella vulgaris. For optimization purposes, the absorbance of photosynthetic pigments is typically measured at a single wavelength, either for biomass assessment or for calculation of spectral indices [START_REF] Kaeoboon | Toxicity response of Chlorella microalgae to glyphosate herbicide exposure based on biomass, pigment contents and photosynthetic efficiency[END_REF][START_REF] Wong | Growth medium screening for Chlorella vulgaris growth and lipid production[END_REF][START_REF] Zhou | A novel combined spectral index for estimating the ratio of carotenoid to chlorophyll content to monitor crop physiological and phenological status[END_REF]. This preliminary study proposes a compromise between fine analysis by HPLC and succinct analysis by single-wavelength spectroscopy. The influence of an environmental disturbance could then be determined by ultraviolet/visible spectroscopy on the optical characteristics of the absorption spectrum. The actual state of phytoplankton cells would be adequately represented by the spectroscopic characterization of the photosynthetic pigments. Here we used four parameters to describe a peak: area, width at base, height, and wavelength at maximum absorbance. Peak area and peak height were strongly correlated, while the wavelength at maximum absorbance can be shifted due to environmental factors. For instance, this was the case for the SW-Chlorella carotenoid peaks that shifted ~1 nm with increasing glyphosate concentration. Unlike peak areas, absorbance values could then be underestimated via their usual determination at a single wavelength and introduce a bias in data interpretation. Although it is not yet possible to link the peak width to a particular process, the shift in wavelength at maximum absorbance indicated a change that could have implications for the energy transfer within photosystems and the quantum yield of CO2 uptake [START_REF] Liu | Photosynthetic physiology of blue, green, and red light: Light intensity effects and underlying mechanisms[END_REF]. These spectroscopic parameters were used to refine the diagnosis of the impact of a contaminant on phytoplankton species. They may then be employed as a marker of an altered photosynthetic apparatus, as environmental disturbances can affect the efficiency of light-tobiomass conversion [START_REF] Vecchi | Potential and challenges of improving photosynthesis in algae[END_REF]. In this context, the measurement of the absorption spectrum by spectroscopy appeared as an accessible monitoring tool to perform daily analyses on the numerous samples needed to obtain an ecotoxicological response.

This approach was applied to evaluate the potential impact of Roundup® on the unicellular green microalga, Chlorella vulgaris Beijerinck, 1890. This species is commonly used as a model organism in ecotoxicological tests [START_REF] Kaeoboon | Toxicity response of Chlorella microalgae to glyphosate herbicide exposure based on biomass, pigment contents and photosynthetic efficiency[END_REF][START_REF] Silva | Ecotoxicity tests using the green algae Chlorella vulgaris -A useful tool in hazardous effluents management[END_REF][START_REF] Wu | Phytotoxicity comparison of organic contaminants and heavy metals using Chlorella vulgaris[END_REF]. It contains chlorophylls a and b, as well as carotenoids such as violaxanthin, neoxanthin, astaxanthin, canthaxanthin, lutein, β-carotene, and xanthophyll [START_REF] Mahmoud | Simulation of absorption spectrum of photosynthetic pigments of Chlorella vulgaris B algae[END_REF][START_REF] Mendes | Supercritical CO2 extraction of carotenoids and other lipids from Chlorella vulgaris[END_REF]. This phytoplanktonic species presents good sensitivity to contaminants and is easily cultured in the laboratory. [START_REF] Mensah | Derivation of South African water quality guidelines for Roundup using species sensitivity distribution[END_REF] found that among a panel of seven microalgae, Chlorella species were the most sensitive to Roundup® exposure. Cellular inhibition in FW-Chlorella by the highest concentrations (> 5 mg L -1 ) was consistent with the EC50 of 3.85 mg L -1 reported by [START_REF] Reno | Water polluted with glyphosate formulations: effectiveness of a decontamination process using Chlorella vulgaris growing as bioindicator[END_REF] for C. vulgaris contaminated with Roundup Ultramax®. Our study also showed an increase in absorbance values at intermediate concentrations (10 to 5,000 µg L -1 ) indicating, in this case, a stimulation of the FW-Chlorella population. Since no nutrients were added during the five-day experiment, glyphosate could be used as a source of phosphorus (P) by organisms in contact with less toxic concentrations. Indeed, bacteria convert glyphosate to inorganic phosphate (Pi) through the action of C-P lyase and make it bioavailable in water [START_REF] Matozzo | The effects of glyphosate and its commercial formulations to marine invertebrates: A review[END_REF]. Phosphorus (P) uptake and assimilation from glyphosate biodegradation were observed in freshwater cyanobacteria, with changes in gene expression and enzyme activity [START_REF] Drzyzga | Glyphosate dose modulates the uptake of inorganic phosphate by freshwater cyanobacteria[END_REF][START_REF] Qiu | Physiological and biochemical responses of Microcystis aeruginosa to glyphosate and its Roundup® formulation[END_REF]. This input is a significant problem for freshwater ecosystems where it promotes eutrophication [START_REF] Vera | New evidences of Roundup® (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems[END_REF]. However, it can be considered as a "welcome contaminant" especially in marine habitats, such as the Mediterranean Sea, where this element is often limiting for the development of primary producers [START_REF] Djaoudi | Sensitive determination of the dissolved phosphate pool for an improved resolution of its vertical variability in the surface layer: New views in the P-depleted Mediterranean Sea[END_REF].

Another objective of this study was to test the potential ecotoxicological effects of Roundup® on the same phytoplankton organism in two types of aquatic environment: inland waters and coastal marine areas. A typical freshwater strain (FW-Chlorella) was therefore successfully adapted to a salinity similar to that of seawater (SW-Chlorella). Microalgae of the genus Chlorella are known to implement tolerance mechanisms under persistent high salinity conditions [START_REF] Shetty | Salinity stress responses and adaptation mechanisms in eukaryotic green microalgae[END_REF].

Since it cannot change cell volume due to a rigid cell wall, osmoregulation occurs through the production of organic solutes (e.g., trehalose) and the accumulation of inorganic ions. The production of these osmoprotective molecules and the exclusion of Na + consume a lot of energy inducing an increase in the storage of lipids and total proteins. High salinity can then lead to stunted growth, loss of photosynthesis (i.e., chlorophyll breakdown and carotenoid accumulation), and downregulation of oxidative phosphorylation [START_REF] Almutairi | Evaluation of high salinity adaptation for lipid bio-accumulation in the green microalga Chlorella vulgaris[END_REF][START_REF] Li | Exploring stress tolerance mechanism of evolved freshwater strain Chlorella sp. S30 under 30 g/L salt[END_REF]. This loss of photosynthesis was confirmed in our study by the decrease of the chlorophyll/carotenoid peak area ratio in SW-Chlorella. Nevertheless, [START_REF] Farghl | Effect of salt stress on growth, antioxidant enzymes, lipid peroxidation and some metabolic activities in some freshwater and marine algae[END_REF] pointed out that induction of antioxidants helps cells overcome the negative effects of sodium, as shown by growth behavior. Prior to our experiment, SW-Chlorella achieved cell recovery after a few weeks of adaptation with greater growth and spectral characteristics than the original FW-Chlorella strain. These changes in metabolic functioning may contribute to improve tolerance of SW-Chlorella exposed to Roundup®.

A dose-response relationship was demonstrated with stimulation of the SW-Chlorella population with increasing glyphosate concentration. Conversely, the marine diatom Phaeodactylum tricornutum showed a decrease in cell density with only 250 µg L -1 of Roundup Pronto® (Cruz de [START_REF] Cruz De Carvalho | Glyphosate-based herbicide toxicophenomics in marine diatoms: Impacts on primary production and physiological fitness[END_REF]. Its photosynthetic activity was inhibited due to the decrease of antioxidant capacities, which were instead induced in SW-Chlorella to cope with salinity stress.

In addition, SW-Chlorella exhibited higher carotenoid content, as well as specific changes in their spectral characteristics with an increase in peak area, a shift in wavelength at maximum absorbance, and a spreading of the peak width at the base as a function of glyphosate concentration. Carotenoid accumulation has previously been reported as a defense mechanism when C. vulgaris is exposed to a salty environment [START_REF] Almutairi | Evaluation of high salinity adaptation for lipid bio-accumulation in the green microalga Chlorella vulgaris[END_REF]. These have been shown to be photoprotective antioxidant pigments used when cells are exposed to environmental stressors (Cruz de [START_REF] Cruz De Carvalho | Glyphosate-based herbicide toxicophenomics in marine diatoms: Impacts on primary production and physiological fitness[END_REF]. They increase energy dissipation by non-photochemical processes and prevent possible photoinhibitory events. Among carotenoids, lutein appears to be positively affected by glyphosate contamination [START_REF] Mysore Doddaiah | Effect of metabolic inhibitors on growth and carotenoid production in Dunaliella bardawil[END_REF]. In the carotenoid biosynthetic pathway, glyphosate inhibits the activity of lycopene β-cyclase which prevents the synthesis of β-carotene from lycopene. The synthesis of α-carotene, from which lutein is derived, is then enhanced. In addition, glyphosate can impair the chlorophyll biosynthetic pathway through magnesium (Mg) and iron (Fe) deficiency due to the formation of insoluble metal complexes [START_REF] Kaeoboon | Toxicity response of Chlorella microalgae to glyphosate herbicide exposure based on biomass, pigment contents and photosynthetic efficiency[END_REF][START_REF] Mertens | Glyphosate, a chelating agent-relevant for ecological risk assessment?[END_REF]. Electron transport, photosystem activities (PSI and PSII), and NADH and NADPH pools could also be negatively affected [START_REF] Kaeoboon | Toxicity response of Chlorella microalgae to glyphosate herbicide exposure based on biomass, pigment contents and photosynthetic efficiency[END_REF]. Exposure to Roundup® would then force a transfer of energy from photosynthesis to energy consumption. The adaptation of C. vulgaris to seawater may therefore have increased its resilience to this environmental disturbance.

CONCLUSION

The introduction of glyphosate into aquatic ecosystems constitutes a disruption of the phytoplankton compartment at the base of the foodweb. In this study, an unusual dose-response relationship was demonstrated for Chlorella vulgaris grown in freshwater, i.e. in its natural environment, with a stimulation at intermediate concentrations of Roundup® (10 to 5,000 µg L -1 ) and an inhibition otherwise. Marine-adapted C. vulgaris were stimulated in the presence of glyphosate, particularly at high concentrations (> 1 mg L -1 ). This emerging contaminant could then be used as a source of phosphorus. The defense mechanisms activated by SW-Chlorella to adapt to seawater may have increased its resilience to exposure levels that could be toxic in freshwater. It should be noted that the maximum intensity of these effects was not reached for the concentrations tested.

In addition, our exploration of spectral changes in photosynthetic pigments revealed a shift in the wavelength at maximum absorbance in response to environmental stresses. Thus, unlike the peak area, the determination of peak height could be underestimated with the usual technique of singlewavelength absorbance. Changes in peak shape (e.g., crushing with a decrease in peak height, spreading with an increase in peak width at base) may also be an indicator of altered photosynthetic efficiency. The analysis of absorption spectrum and the use of peak areas appear to be a promising approach to monitoring the evolution of ecotoxicological effects. This preliminary work must be continued and confirmed by applying this method to other emerging contaminants such as pharmaceutical substances. the data processing, the writing of original draft, and the review of manuscript.

Yves Lucas contributed to the data processing, the writing of original draft, and the review of manuscript.

Patricia Merdy contributed to the funding acquisition, the implementation of methodology, the supervision of experiments, the data processing, the writing of original draft, and the review of manuscript.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figure 1 .

 1 Figure 1. Absorption spectra of the freshwater (FW-Chlorella) and seawater (SW-Chlorella)

Figure 2 .

 2 Figure 2. Characteristics of chlorophyll and carotenoid peaks for the FW-Chlorella (white) and

Figure 3 .

 3 Figure 3. Change in peak area as a function of time. The peak area is normalized to the peak area

Figure 4 .

 4 Figure 4. Average absorption spectra for all concentrations on days 1 (A-B) and 5 (C-D) in FW-

Figure 5 .

 5 Figure 5. Change in normalized peak area as a function of time, for all glyphosate concentrations

Figure 6 .

 6 Figure 6. Principal component analysis (PCA) performed on the spectral features for the entire data

Figure 7 .

 7 Figure 7. Peak area anomalies of chlorophylls and carotenoids for FW-Chlorella (top) and SW-

Figure 8 .

 8 Figure 8. Chlorophyll/carotenoid peak area ratio for all concentrations tested in FW-Chlorella and SW-Chlorella. The center boxes show data between the 25 th and 75 th percentiles, with the median represented by a line. The whiskers extend to the 10 th and 90 th percentiles. The dots represent the 5 th and 95 th percentiles.

ACKNOWLEDGEMENTS

Funding: This research was supported by the MEDICTON program (Rhone-Mediterranean-Corsica Water Agency, Veolia Foundation, Toulon-Provence-Mediterranean Metropolis) and the MEDIMPACTON program (French Agency for Biodiversity).

KEYWORDS

Chlorella vulgaris, Glyphosate, Salinity, Absorption spectrum, Chlorophylls, Carotenoids

AUTHOR CONTRIBUTIONS STATEMENT