Anne-Antonella Serra

Amélie Miqueau

Fanny Ramel

Ivan Couée
email: ivan.couee@univ-rennes1.fr

Cécile Sulmon

Gwenola Gouesbet

Species-and organ-specific responses of agri-environmental plants to residual agricultural pollutants

Keywords: Ecotoxicity, Field margin plants, Heavy metals, Persistent organic pollutants, Pesticides, Soil pollution 5 Highlights

Soil pollution by anthropogenic chemicals is a major concern for sustainability of crop production and of ecosystem functions mediated by natural plant biodiversity. The complex effects on plants are however difficult to apprehend. Plant communities of field margins, vegetative filter strips or rotational fallows are confronted with agricultural pollutants through residual soil contamination and/or through drift, run-off and leaching events that result from chemical applications. Exposure to xenobiotics and heavy metals causes biochemical, physiological and developmental effects. However, the range of doses, modalities of exposure, metabolization of contaminants into derived xenobiotics, and combinations of contaminants result in variable and multi-level effects. Understanding these complex plantpollutant interactions cannot directly rely on toxicological or agronomical approaches that focus on the effects of field-rate pesticide applications. It must take into account exposure at root level, sublethal concentrations of bioactive compounds and functional biodiversity of the plant species that are affected. The present study deals with agri-environmental plant species of field margins, vegetative filter strips or rotational fallows in European agricultural landscapes. Root and shoot physiological and growth responses were compared under controlled conditions that were optimally adjusted for each plant species. Contrasted responses of growth inhibition, no adverse effect or growth enhancement depended on species, organ and nature of contaminant. However, all of the agricultural contaminants under study (pesticides, pesticide metabolites, heavy metals, polycyclic aromatic hydrocarbons) had significant effects under conditions of sublethal exposure on at least some of the plant species.

The fungicide tebuconazole and polycyclic aromatic hydrocarbon fluoranthene, which gave highest levels of responses, induced both activation or inhibition effects, in different plant species or in different organs of the same plant species. These complex effects are discussed in terms of dynamics of agri-environmental plants and of ecological consequences of differential root-shoot growth under conditions of soil contamination.

Introduction

Extensive use of chemicals in agriculture and industry has resulted in widespread environmental contamination with potential impact on environmental quality, human wellbeing and planetary sustainability [START_REF] Arias-Estévez | The mobility and degradation of pesticides in soils and the pollution of groundwater resources[END_REF][START_REF] Persson | Confronting unknown planetary boundary threats from chemical pollution[END_REF][START_REF] Macleod | Identifying chemicals that are planetary boundary threats[END_REF][START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF][START_REF] Silva | Pesticide residues in European agricultural soils -A hidden reality unfolded[END_REF]. On the basis of expected environmental concentration calculations taking into account a 15-cm depth of affected soil or waterbody [START_REF] Peterson | Aquatic phytotoxicity of 23 pesticides applied at expectedenvironmental concentrations[END_REF], worldwide consumption of 2.6-4.6 Tg of pesticides per year [START_REF] Wilson | Why farmers continue to use pesticides despite environmental, health and sustainability costs[END_REF][START_REF] Zhang | Global pesticide consumption and pollution: with China as a focus[END_REF][START_REF] Bringel | Plant-pesticide interactions and the global chloromethane budget[END_REF] could theoretically result in yearly accumulations of 34-60 µg L -1 by dispersion over a global Earth surface of 510x10 6 km 2 . On a regional scale, average application rates of pesticides per hectare of arable land can attain 6.5-60 kg ha -1 in Asia and South America [START_REF] Carvalho | Pesticides, environment, and food safety[END_REF], which may result in very high levels of expected environmental yearly accumulations of 4,333-40,000 µg L -1 .

Actual measurements in pesticide-contaminated soils show that residual persistence of pesticides [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Arias-Estévez | The mobility and degradation of pesticides in soils and the pollution of groundwater resources[END_REF]Jablonowsksi et al., 2010;[START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF] can fall within these ranges of theoretical values of potential accumulation. In Argentinian agrosystems, expected environmental concentration calculation [START_REF] Peterson | Aquatic phytotoxicity of 23 pesticides applied at expectedenvironmental concentrations[END_REF] on the basis of annual applications [START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF] yields a glyphosate concentration of 2,600 µg L -1 . This theoretical value is lower than the average measurement of 6,433 µg.kg -1 for glyphosate and its metabolite aminomethylphosphonate (AMPA) in soils of these Argentinian agrosystems [START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF], thus emphasising the importance of persistence and pluri-annual accumulation.

Moreover, agricultural and livestock activities are not only sources of agrochemicals, but also of heavy metals and polycyclic aromatic hydrocarbons (PAHs) that can accumulate in the environment as agriculture-related contaminants in parallel with pesticides and pesticide residues [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF].

Root-level exposure to such residual levels of xenobiotics and heavy metals causes a variety of biochemical, physiological and developmental effects on agricultural or natural plant species [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015aSerra et al., , 2015b;;[START_REF] Zhang | Accumulation and toxicological response of atrazine in rice crops[END_REF]. Herbicides such as atrazine, glyphosate or imazethapyr are known to act through a major biochemical target [START_REF] Holländer | The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. Inhibition by glyphosate of phenylpropanoid synthesis in buckwheat (Fagopyrum esculentum moench)[END_REF][START_REF] Padgette | Site-directed mutagenesis of a conserved region of the 5enolpyruvylshikimate-3-phosphate synthase active site[END_REF][START_REF] Rutherford | Herbicide-induced oxidative stress in photosystem II[END_REF][START_REF] Qian | Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity[END_REF][START_REF] Sammons | Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS[END_REF], that can be considered to be the canonical target mediating their mechanism of action [START_REF] Piya | Canonical and noncanonical ethylene signaling pathways that regulate Arabidopsis susceptibility to the cyst nematode Heterodera schachtii[END_REF]. However, root-level effects of herbicides have often been shown to be disconnected from usual effects on canonical targets, thus suggesting alternative and noncanonical mechanisms of action under such conditions of exposure [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF][START_REF] Qian | Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015aSerra et al., , 2015b)).

Moreover, exposure to sublethal doses, the diverse modalities of contaminant exposures, environmental and plant metabolization of contaminants into novel metabolites that can also be harmful and the presence of contaminant mixtures in the environment result in variable and multi-level effects [START_REF] Armendáriz | Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots[END_REF][START_REF] Busi | Evolution of glyphosate resistance in a Lolium rigidum population by glyphosate selection at sublethal doses[END_REF][START_REF] Reeves | Evaluation of Methods for Predicting the Toxicity of Polycyclic Aromatic Hydrocarbon Mixtures[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]. Soil pollution by anthropogenic chemicals is therefore a major concern for the sustainability of crop production [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF][START_REF] Silva | Pesticide residues in European agricultural soils -A hidden reality unfolded[END_REF]. Soil pollution is also a major concern for the efficiency of ecosystem services mediated by natural plant biodiversity [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF][START_REF] Silva | Pesticide residues in European agricultural soils -A hidden reality unfolded[END_REF], such as the maintenance of pollinator diversity [START_REF] Kuussaari | Pollinator insects benefit from rotational fallows[END_REF], soil stabilization of field margins or pollution buffering [START_REF] Mench | Successes and limitations of phytotechnologies at field scale: outcomes, assessment and outlook of COST Action 859[END_REF][START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF].

However, the range of soil pollution effects on plants is difficult to apprehend and remains to be fully understood [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF]. Agri-environmental plant communities in field margins, vegetative filter strips (VFS) or rotational fallows are necessarily confronted with agricultural pollutants through residual soil contamination and/or through drift, run-off and leaching events that result from chemical applications [START_REF] Gove | Effects of herbicide spray drift and fertilizer overspread on selected species of woodland ground flora: comparison between short-term and long-term impact assessments and field surveys[END_REF][START_REF] Helander | Glyphosate in northern ecosystems[END_REF][START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF]. On the one hand, in the context of VFS at the edge of agricultural fields, the confrontation of plant communities with pesticides and other agricultural contaminants is designed to mitigate the dispersion and the impact of pollutants in terrestrial and aquatic environments [START_REF] Collins | Mitigating diffuse pollution from agriculture: international approaches and experience[END_REF][START_REF] Krutz | Reducing herbicide runoff from agricultural fields with vegetative filter strips : a review[END_REF][START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF][START_REF] Stehle | Pesticide risk mitigation by vegetated treatment systems: a metaanalysis[END_REF]. On the other hand, plant biodiversity and functional group composition in agriculturally-intensive landscapes are affected by land-use practices and application rates of fertilizers and pesticides [START_REF] Billeter | Indicators for biodiversity in agricultural landscapes: a pan-European study[END_REF][START_REF] Liira | Plant functional group composition and large-scale species richness in European agricultural landscapes[END_REF]. Understanding these complex plant-pollutant interactions cannot directly rely on toxicological or agronomical studies that focus on the effects of field-rate applications of pesticides on selected plant species. It must take into account the specificities of environmental exposure, such as exposure at root level, sublethal concentrations of bioactive compounds, exposure to derived metabolites and the functional biodiversity of the plant species that are affected.

Application of such conditions has revealed that root growth was particularly sensitive to supposedly non-phytotoxic levels of xenobiotics or of their metabolized derivatives (Serra et al., 2013(Serra et al., , 2015a(Serra et al., , 2015b;;[START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]. In parallel, major agricultural pollutants such as atrazine and glyphosate [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF][START_REF] Silva | Pesticide residues in European agricultural soils -A hidden reality unfolded[END_REF] that are commonly found in field margins, VFS or rotational fallows in European agricultural landscapes [START_REF] Billeter | Indicators for biodiversity in agricultural landscapes: a pan-European study[END_REF][START_REF] Liira | Plant functional group composition and large-scale species richness in European agricultural landscapes[END_REF][START_REF] Kuussaari | Pollinator insects benefit from rotational fallows[END_REF][START_REF] Stehle | Pesticide risk mitigation by vegetated treatment systems: a metaanalysis[END_REF][START_REF] Toivonen | Environmental fallows as a new policy tool to safeguard farmland biodiversity in Finland[END_REF][START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF]. Root and shoot physiological and growth responses were compared under controlled conditions that were optimally adjusted for each plant species, with the parallel analysis of the unrelated model plant species Arabidopsis thaliana as a reference species. The herbicide glyphosate, the glyphosate metabolite AMPA, the herbicide atrazine, the atrazine metabolite hydroxyatrazine, the fungicide tebuconazole, copper, zinc and the PAH fluoranthene all had significant effects under conditions of sublethal exposure on at least two of the plant species under study. However, this diversity of agricultural contaminants (pesticides, pesticide metabolites, heavy metals, PAHs) revealed contrasted responses of growth stimulation, no adverse effect or growth inhibition, depending on plant species, plant organ and nature of contaminant. Tebuconazole and fluoranthene, which gave the highest level of responses, could induce both activation or inhibition effects, in different plant species or in different organs of the same plant species. These complex effects are discussed in terms of plant dynamics in agri-environmental contexts and of ecological consequences of differential root-shoot growth under conditions of soil contamination.

Materials and methods

Plant material

Seeds of Arabidopsis thaliana (Columbia, Col-0) were obtained from the Nottingham Arabidopsis Stock Center, and propagated under laboratory-controlled conditions. Seeds of Agrostis stolonifera (cv. Penncross), Anthemis tinctoria (bulk seeds), Centaurea cyanus (bulk seeds), Fagopyrum esculentum (bulk seeds), Festuca rubra (cv. Herald), Lolium perenne (cv. Brio), Lotus corniculatus (cv. Leo), Phleum pratense (cv. Kaba), and Trifolium pratense (cv. Violetta) were obtained from the Phytosem (Gap, Hautes-Alpes, France) seed company.

These seeds did not have any pesticide treatment coating.

Growth conditions

rinsed in absolute ethanol, and dried overnight. Germination and growth were carried out under axenic conditions in square Petri dishes. After seed sowing, Petri dishes were placed in the dark at 4 °C for 72 h in order to break dormancy and homogenize germination, and were then transferred to a control growth chamber at 22 °C/20 °C under a 16 h light (100 µmol m -2 s -1 )/8 h dark regime. Growth medium consisted of 0.8% (w/v) agar in Hoagland basal salt mix (H2395, Sigma-Aldrich) adjusted to pH 6, in the absence of any additional sucrose or soluble carbohydrate (Ramel et al., 2009b;[START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015a;;[START_REF] Nuttens | Sucrose modifies growth and physiology in axenically grown Myriophyllum spicatum with potential effects on the response to pollutants[END_REF].

Seeds of Agrostis stolonifera, Anthemis tinctoria, Centaurea cyanus, Fagopyrum esculentum, Festuca rubra, Lolium perenne, Lotus corniculatus, Phleum pratense, and Trifolium pratense were surface-sterilized in ethanol (1 min) and bayrochlore:tween at various concentrations of bayrochlore (20 min), and rinsed in sterile distilled water. Germination and growth were carried out under axenic conditions in square Petri dishes (Agrostis stolonifera, Anthemis tinctoria, Festuca rubra, Lotus corniculatus, Phleum pratense, Trifolium pratense) or in 30-mL Falcon tubes (Centaurea cyanus, Fagopyrum esculentum, Lolium perenne). Growth medium in Petri dishes consisted of 0.8% (w/v) agar in Hoagland basal salt mix (H2395, Sigma-Aldrich) adjusted to pH 6. Growth medium in 30-mL Falcon tubes consisted of 0.3% (w/v) agar in Hoagland basal salt mix (H2395, Sigma-Aldrich) adjusted to pH 6. Xenobiotics (glyphosate, AMPA, atrazine, hydroxyatrazine, tebuconazole) and heavy metals (Cu 2+ added as CuSO4, Zn 2+ added as ZnSO4) were axenically added to cooled-down melted agar-Hoagland medium prior to pouring into Petri dishes or Falcon tubes. Xenobiotic and heavy metal treatments were carried out by direct exposure starting at early development by seed sowing on chemical-containing growth medium.

After seed sowing, Petri dishes or Falcon tubes were placed in the dark at 4 °C for 3 to 8 days (according to plant species) in order to break dormancy and homogenize germination. Seeds of Centaurea cyanus did not undergo any treatment of dormancy break. Sown Petri dishes or Falcon tubes were then transferred to a control growth chamber at 22 °C/20 °C under a 16 h light (100 µmol m -2 s -1 )/8 h dark regime. Analysis of physiological and growth parameters was carried out after 7 (Agrostis stolonifera, Anthemis tinctoria, Festuca rubra, Lotus corniculatus, Phleum pratense, Trifolium pratense), 10 (Fagopyrum esculentum, Lolium perenne), 12 (Centaurea cyanus) or 14 (Arabidopsis thaliana) days of growth in the absence or presence of xenobiotics or heavy metals.

Analysis of growth responses

Plantlets in Petri dishes were directly photographed. Seedlings in Falcon tubes were aligned on glass plates and photographed. Lengths of main root and elongating leaf were measured using ImageJ software. Results were expressed as root or leaf length or as percentage of inhibition of growth [(Growthcontrol -Growthtreatment)/Growthcontrol] relatively to the control in the absence of xenobiotic or heavy metal. The action of xenobiotic and heavy metal treatments on Arabidopsis thaliana root growth was characterized by median effective concentrations (EC50) resulting in a 50% inhibitory response. The nine agri-environmental plant species (Agrostis stolonifera, Anthemis tinctoria, Centaurea cyanus, Fagopyrum esculentum, Festuca rubra, Lolium perenne, Lotus corniculatus, Phleum pratense, Trifolium pratense) were exposed to the eight xenobiotics and heavy metals at the EC50 inhibitory concentrations determined for Arabidopsis thaliana root growth.

Analysis of physiological parameters

Pigments were extracted by grinding shoots of seedlings in 80% (v/v) acetone, and the absorbance of the resulting extracts was measured at three wavelengths: 663, 646, and 470 nm. Levels of chlorophylls and total carotenoids (xanthophylls and carotenes) in these extracts were determined from the equations given by [START_REF] Lichtenthaler | Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents[END_REF], as previously described [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF].

Physiological and growth parameters were measured on five to thirty independent biological replicates. Results were given as the mean (± SEM) of these determinations. Statistical analysis was carried out with the R version 3.1.3 software. Pairwise comparisons of means used the non-parametric Mann-Whitney-Wilcoxon test. In order to test and visualize relationships between treatments and response parameters, principal component analysis (PCA) based on the correlation matrix (Ramel et al., 2009a) was carried out using the FactoMineR package of R.

Results

Characterization of sublethal effects of root-level agricultural contaminants on the model plant species Arabidopsis thaliana

In line with the molecular effects of soluble sugars on xenobiotic stress responses [START_REF] Ramel | Genome-wide interacting effects of sucrose and herbicidemediated stress in Arabidopsis thaliana : novel insights into atrazine toxicity and sucroseinduced tolerance[END_REF](Ramel et al., , 2009a(Ramel et al., , 2009b)), the toxicological impact of xenobiotics and heavy metals on Arabidopsis plantlets was significantly modified in the presence of additional sucrose (data not shown). Whereas the inhibitory effects of atrazine were lifted by exogenous sucrose, glyphosate showed the same level of toxicity in the absence or presence of exogenous sucrose (data not shown). Cultivation in the absence of exogenous soluble sugars was therefore essential to reflect a realistic evaluation of comparative chemical stress sensitivity, as advocated by [START_REF] Nuttens | Sucrose modifies growth and physiology in axenically grown Myriophyllum spicatum with potential effects on the response to pollutants[END_REF].

Since exposure was carried out at root level, and given the sensitivity of roots to xenobiotic and heavy metal stress [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al. , 2015a)), dose-response relationships were derived from measurements of primary root growth (Fig. 1). All of the xenobiotics and heavy metals under study, glyphosate (Fig. 1), AMPA, atrazine, hydroxyatrazine, tebuconazole, copper, zinc and fluoranthene (data not shown), had inhibitory effects on the root growth of Arabidopsis plantlets, with typical hyperbolic or sigmoid inhibition curves. EC50 values (Table 1) were estimated graphically from these hyperbolic or sigmoid curves of inhibition. The EC50 values for glyphosate, AMPA, atrazine, and hydroxyatrazine agreed with previous analyses under similar modalities of exposure (Serra et al., 2013;[START_REF] Sulmon | Sugar-induced tolerance to atrazine in Arabidopsis seedlings: interacting effects of atrazine and soluble sugars on psbA mRNA and D1 protein levels[END_REF].

In contrast, in the case of tebuconazole, the EC50 value was much higher than the inhibitory concentrations described by [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]. In the case of Zn, Cu and fluoranthene, direct comparisons with previous studies could not be carried out because of major discrepancies in the modalities of exposure. The much lower EC50 value for Zn relatively to that described by [START_REF] Richard | Diversity analysis of the response to Zn within the Arabidopsis thaliana species revealed a low contribution of Zn translocation to Zn tolerance and a new role for Zn[END_REF] reflected a higher sensitivity to Zn toxicity that could be ascribed to the absence of exogenous sucrose in the present study. In contrast, the EC50 for Cu was very similar to that resulting from exposure of 3-week-old Arabidopsis plants under hydroponics conditions in the absence of exogenous sucrose [START_REF] Lequeux | Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile[END_REF]. As previously described for the analysis of phenanthrene effects on Arabidopsis [START_REF] Alkio | Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms[END_REF], the relatively high range of fluoranthene concentrations (up to 550 µM) exceeded water solubility. However, the phytotoxicity of PAHs is also induced by contact (Paškova et al., 2009). Thus, in line with the effects of phenanthrene [START_REF] Alkio | Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms[END_REF], fluoranthene had a significant effect on primary root growth [START_REF] Alkio | Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms[END_REF], with an EC50 value (532 µM) that was within the range of inhibitory PAH concentrations [START_REF] Alkio | Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms[END_REF].

Finally, the fungicide tebuconazole and Cu were the most highly effective non-herbicide chemical stressors, in agreement with previously-described side effects of tebuconazole treatment [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF] and the high toxicity of copper [START_REF] Lequeux | Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile[END_REF] in plants.

Effects of sublethal levels of root-level agricultural contaminants on root growth of agrienvironmental plant species

Xenobiotic and heavy metal exposure of the agri-environmental plant species was carried out in the absence of exogenous soluble sugars, under growth conditions that were similar to those of the Arabidopsis study. Certain conditions of seed sterilisation (percentage of bayrochlore; duration of treatment) and stratification (duration of low temperature treatment) were adjusted for each plant species in order to optimise germination efficiency and axenic growth (data not shown). In order to take into account the growth characteristics of each species, experiments were carried out either in Petri dishes or in 30-mL Falcon tubes as described in Materials and Methods. The nine agri-environmental plant species were subjected during germination and early growth to xenobiotic and heavy metal concentrations corresponding to the EC50 values of impact on root growth of Arabidopsis plantlets (Fig. 1, Table 1), except in the case of glyphosate and AMPA, which were applied at concentrations of respectively 1 µM (instead of EC50 = 0.75 µM) and 50 µM (instead of EC50 = 30 µM).

All of the nine agri-environmental species showed significant root responses to at least some of the xenobiotics or heavy metals (Fig. 2), except red fescue, which showed responses that were not significantly different from control. Treatment with herbicide compounds and metabolites, glyphosate, AMPA, atrazine and hydroxyatrazine, resulted in either a no observable adverse effect (NOAE) situation or inhibition of root growth. Tebuconazole, Zn, Cu or fluoranthene showed significant inhibitory effects on root growth, for instance in the case of yellow chamomile (Anthemis tinctoria) and cornflower (Centaurea cyanus) (Fig. 2). However, in parallel, tebuconazole, Zn, Cu or fluoranthene enhanced root growth of bird'sfoot trefoil (Lotus corniculatus), creeping bentgrass (Agrostis stolonifera), and English ryegrass (Lolium perenne) (Fig. 2; supplementary data 1), thus suggesting the induction of hormetic effects [START_REF] Belz | Herbicides and plant hormesis[END_REF][START_REF] Dyer | Stress-induced evolution of herbicide resistance and related pleiotropic effects[END_REF][START_REF] Velini | Glyphosate applied at low doses can stimulate plant growth[END_REF].

Tebuconazole and fluoranthene induced the highest level of responses, with highly positive effects on root growth of Lolium perenne and highly negative effects on root growth of Anthemis tinctoria, relatively to all or most of the other plant species (supplementary data 1).

Zn gave the lowest level of responses, with NOAE interactions in Trifolium pratense, esculentum. On the other hand, species-specific behaviours were significantly contrasted.

Root growth of yellow chamomile (Anthemis tinctoria) and cornflower (Centaurea cyanus)

was negatively affected by all or most of xenobiotic or heavy metal treatments. Root growth of bird's-foot trefoil (Lotus corniculatus), creeping bentgrass (Agrostis stolonifera), English ryegrass (Lolium perenne) and red fescue (Festuca rubra) showed contrasted chemicalspecific responses, with a mixture of negative and positive responses.

Effects of sublethal levels of root-level agricultural contaminants on shoot growth of agri-environmental plant species

The impact of xenobiotics and heavy metals on shoot growth was characterized under the same conditions of cultivation and treatment as those described in subsection 3.2. Root-level exposure can result in xenobiotic or heavy metal translocation throughout the plant and targeting of shoot and leaf mechanisms and/or root-level interactions or perturbations can lead to modifications of shoot growth and development [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF](Serra et al., ,, 2015a;;[START_REF] Sulmon | Sucrose amendment enhances phytoaccumulation of the herbicide atrazine in Arabidopsis thaliana[END_REF]. In all of the nine species, shoot growth significantly responded to at least some of the xenobiotics or heavy metals (Fig. 3), thus showing the involvement of root-shoot interactions in the response to root-level exposure.

Treatment with herbicide compounds and metabolites, glyphosate, AMPA, atrazine and hydroxyatrazine, resulted in either a NOAE situation or inhibition of shoot growth. AMPA gave the lowest level of responses, with NOAE shoot growth interactions in all of the nine agri-environmental plant species. In contrast with their effects on root growth (Fig. 2), tebuconazole and fluoranthene did not show any enhancement effets on shoot growth (Fig. 3), but induced either a NOAE situation or inhibition of shoot growth (Fig. 3). In contrast, Zn and Cu had significant enhancement effects on shoot growth of respectively white Dutch clover (Trifolium pratense) and red fescue (Festuca rubra) (Fig. 3; supplementary data 1), thus suggesting the induction of hormetic effects [START_REF] Belz | Herbicides and plant hormesis[END_REF][START_REF] Dyer | Stress-induced evolution of herbicide resistance and related pleiotropic effects[END_REF][START_REF] Velini | Glyphosate applied at low doses can stimulate plant growth[END_REF]. Tebuconazole induced the highest level of shoot growth inhibition, especially in bird's-foot trefoil (Lotus corniculatus) and cornflower (Centaurea cyanus) (Fig. 3; supplementary data 1). Moreover, tebuconazole was the only contaminant that affected shoot growth of nearly all of the nine agri-environmental plant species (Fig. 3). Zn treatment gave the most contrasted responses with highly positive effects on shoot growth of Trifolium pratense and highly negative effects on shoot growth of Lotus corniculatus and Centaurea cyanus (Fig. 3; supplementary data 1).

On the other hand, species-specific behaviours were significantly contrasted. Shoot growth of bird's-foot trefoil (Lotus corniculatus) and cornflower (Centaurea cyanus) was negatively affected by a range of xenobiotic or heavy metal treatments (Fig. 3). Shoot growth of white Dutch clover (Trifolium pratense) and red fescue (Festuca rubra) showed contrasted chemical-specific responses, with a mixture of negative and positive responses (Fig. 3).

Moreover, in bird's-foot trefoil (Lotus corniculatus) and English ryegrass (Lolium perenne), significant inhibition of shoot growth by tebuconazole, Zn or fluoranthene did not correlate with inhibition of root growth (Fig. 3), thus demonstrating root-shoot translocation of toxic compounds or root-shoot crosstalk of root-level exposure.

Species-specific analysis of the effects of sublethal levels of root-level agricultural contaminants on growth and development of agri-environmental plant species

The comparison of root and shoot responses (Table 2) highlighted strikingly different behaviours of the nine agri-environmental plant species towards to the different xenobiotics and heavy metals, with cases of general tolerance (Festuca rubra, Lolium perenne) and cases of general sensitivity (Lotus corniculatus, Centaurea cyanus, Anthemis tinctoria). Analysis of the effects of xenobiotics and heavy metals on the levels of carotenoids and chlorophylls did not reveal any clear relationship between the decrease or increase of carotenoids and chlorophylls and growth responses (data not shown). For example, both pollutant-sensitive species, such as Centaurea cyanus and Anthemis tinctoria, and the pollutant-tolerant species Festuca rubra showed stable levels of chlorophylls and carotenoids across the different xenobiotic and heavy metal treatments. Moreover, negative or positive variations of chlorophylls and carotenoids were found to be xenobiotic-specific. Another pollutantsensitive species, Lotus corniculatus, showed a 30% (± 5, SEM) decrease of carotenoid levels in the presence of atrazine. In the case of pollutant-tolerant species, chlorophyll levels showed a 55% (± 6, SEM) increase in Lolium perenne under conditions of tebuconazole treatment and a 67% (± 15, SEM) increase in Agrostis stolonifera under conditions of glyphosate treatment. This differential behaviour was further analysed by PCA of root and shoot growth responses (Fig. 4). Distribution along the two axes on the first plane (Dim1 and Dim2) identified five types of plant-contaminant interactions (Fig. 4A): (i) highly-tolerant red fescue (Festuca rubra), which was negatively affected exclusively by the effects of tebuconazole on shoot growth (Fig. 3), (ii) a central cluster comprising creeping bentgrass (Agrostis stolonifera), timothy grass (Phleum pratense) and English ryegrass (Lolium perenne), which maintained significant growth in the presence of the different agricultural contaminants (Fig. 3), (iii) a cluster comprising white Dutch clover (Trifolium pratense), yellow chamomile (Anthemis tinctoria) and common buckwheat (Fagopyrum esculentum), which showed significant sensitivity of root growth to glyphosate and tebuconazole (Fig. 3), (iv) cornflower (Centaurea cyanus), which showed high sensitivity of root growth to AMPA and copper (Fig. 3), (v) bird's-foot trefoil (Lotus corniculatus), which showed high sensitivity of shoot growth to glyphosate, hydroxyatrazine, tebuconazole and zinc (Fig. 3). Hierarchical classification (Fig. 4B) revealed a slightly different clustering of four types of responses: (i) highly-tolerant red fescue (Festuca rubra), (ii) a central cluster comprising moderately tolerant English ryegrass (Lolium perenne), creeping bentgrass (Agrostis stolonifera), timothy grass (Phleum pratense), yellow chamomile (Anthemis tinctoria), white Dutch clover (Trifolium pratense) and common buckwheat (Fagopyrum esculentum), (iii) cornflower (Centaurea cyanus), (iv) bird's-foot trefoil (Lotus corniculatus). Cornflower (Centaurea cyanus) and bird's-foot trefoil (Lotus corniculatus) showed the highest level of shoot growth sensitivity.

Festuca rubra and the Agrostis stolonifera-Phleum pratense-Lolium perenne cluster showed a common response pattern towards the 8 different agricultural contaminants (Fig. 4C). In contrast, the Trifolium pratense-Anthemis tinctoria-Fagopyrum esculentum cluster, Centaurea cyanus and Lotus corniculatus showed patterns of responses that were strongly driven by glyphosate, AMPA or hydroxyatrazine (Fig. 4C), thus highlighting the potentially important action of herbicide metabolites on plant community dynamics in agricultural landscapes.

Whereas most root-related and shoot-related vectors showed parallel effects, Zn and glyphosate showed strikingly different effects on root and shoot growth (Fig. 4C), thus suggesting differential modes of action at root and shoot level. This would agree with the multiple roles and multiple sites of action of Zn [START_REF] Richard | Diversity analysis of the response to Zn within the Arabidopsis thaliana species revealed a low contribution of Zn translocation to Zn tolerance and a new role for Zn[END_REF][START_REF] Rouached | Recent developments in plant zinc homeostasis and the path toward improved biofortication and phytoremediation programs[END_REF] and with the involvement of glyphosate in other processes [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF][START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF][START_REF] Vivancos | Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration[END_REF] than its canonical inhibition of EPSPS [START_REF] Padgette | Site-directed mutagenesis of a conserved region of the 5enolpyruvylshikimate-3-phosphate synthase active site[END_REF], as described in subsection 3.1.

Discussion

Agri-environmental plant species can be affected by a large array of root-level agricultural contaminants at residual concentrations

The nine agri-environmental plant species of the present study were generally less sensitive to xenobiotics and heavy metals than the reference species Arabidopsis thaliana, except for Anthemis tinctoria and Centaurea cyanus which were oversensitive to respectively tebuconazole and AMPA. In Arabidopsis thaliana, atrazine and glyphosate were the most effective chemical stressors with EC50 values lower than 1 µM, which could be ascribed to the impact of their herbicide activity. The canonical targets of their mode of action are well characterized, with atrazine targeting the D1 protein of photosystem II (PSII) [START_REF] Rutherford | Herbicide-induced oxidative stress in photosystem II[END_REF], and glyphosate targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) [START_REF] Holländer | The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. Inhibition by glyphosate of phenylpropanoid synthesis in buckwheat (Fagopyrum esculentum moench)[END_REF][START_REF] Padgette | Site-directed mutagenesis of a conserved region of the 5enolpyruvylshikimate-3-phosphate synthase active site[END_REF][START_REF] Sammons | Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS[END_REF].

The present EC50 value for atrazine (0.5 µM) was within the range of half maximal inhibitory concentrations (IC50=0.4-0.8 µM) for inhibition of PSII fluorescence in isolated pea thylakoids [START_REF] Jursinic | Changes in [ C]Atrazine Binding Associated with the Oxidation-Reduction State of the Secondary Quinone Acceptor of Photosystem II[END_REF], and in agreement with previously-described correlations between PSII inhibition and root growth inhibition [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]. In contrast, the present EC50 value for glyphosate (0.75 µM) was lower than the 6-37 µM IC50 values or the 16.1 (± 2.6) µM IC50 value reported for inhibition of purified 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) respectively from different plant species [START_REF] Padgette | Site-directed mutagenesis of a conserved region of the 5enolpyruvylshikimate-3-phosphate synthase active site[END_REF] or from Arabidopsis thaliana [START_REF] Sammons | Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS[END_REF]. This discrepancy suggested that glyphosate affected additional noncanonical targets in parallel with its action on EPSPS. Indeed, glyphosate toxicity in plants has been hypothesised to involve multiple mechanisms in addition to EPSPS inhibition [START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF][START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF][START_REF] Vivancos | Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration[END_REF]. In contrast with Arabidopsis thaliana, many of the agri-environmental species under study showed no decrease of root growth in response to these EC50 levels of atrazine and glyphosate (Fig. 2).

Moreover, the effects of tebuconazole, Zn, Cu and fluoranthene on root growth involved both inhibition and activation rather than mere inhibition as in the case of Arabidopsis, thus highlighting major species-specific differences of mechanisms and responses. However, all of these conditions affected root growth of at least two of the nine plant species under study, with wide-ranging impacts for tebuconazole and hydroxyatrazine and narrow impacts for atrazine, Zn and Cu. Moreover, glyphosate, atrazine, hydroxyatrazine, tebuconazole, Zn and fluoranthene affected both root growth and shoot growth. All of the conditions of exposure 20 resulted in sublethal effects, in accordance with the general effects of residual contaminants in the environment.

The eight xenobiotic and heavy metal compounds of the present study are found in agricultural landscapes and field margins [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] He | Trace elements in agroecosystems and impacts on the environment[END_REF][START_REF] Hildebrandt | Occurrence and fate of organochlorinated pesticides and PAH in agricultural soils from the Ebro River basin[END_REF][START_REF] Jiang | Ecological risk evaluation of combined pollution of herbicide siduron and heavy metals in soils[END_REF][START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF][START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF].

The parallel analysis of pesticide-related compounds (glyphosate, AMPA, atrazine, hydroxyatrazine, tebuconazole), heavy metals (Cu, Zn) and polycyclic aromatic hydrocarbons (fluoranthene) thus gave a more realistic view of potential agriculture-related contaminations than the exclusive study of pesticide contaminants [START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF]. The levels of exposure were lower than or similar to the theoretical values of potential accumulation described in section 1. The concentration of atrazine (100 µg L -1 ) corresponded to environmental concentrations commonly found in soils, waters and sediments [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]Jablonowski et al., 2010). The concentration of glyphosate (169 µg L -1 ) was lower than observed environmental values in soils and suspended particulate matter [START_REF] Ghanem | Glyphosate and AMPA analysis in sewage sludge by LC-ESIMS/MS after FMOC derivatization on strong anion-exchange resin as solid support[END_REF][START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF] and than experimental concentrations used in mechanistic [START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF] or ecotoxicological [START_REF] Saunders | Root-Zone Glyphosate Exposure Adversely Affects Two Ditch Species[END_REF][START_REF] Soares | Is soil contamination by a glyphosate commercial formulation truly harmless to non-target plants? -Evaluation of oxidative damage and antioxidant responses in tomato[END_REF] studies of root-zone impact of residual concentrations. It was for instance much lower than the average concentration of glyphosate in Argentinian agrosystems [2,229 (± 476) µg.kg -1 ] [START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF] and than the lowest experimental concentrations used by [START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF] (53 mg L -1 ) or [START_REF] Saunders | Root-Zone Glyphosate Exposure Adversely Affects Two Ditch Species[END_REF] (10 mg kg -1 ). The concentration of fluoranthene was also lower than observed PAH contaminating levels in the environment [START_REF] Rodríguez-Eugenio | Soil Pollution: a hidden reality[END_REF]. The concentration of AMPA (5,550 µg L -1 ) corresponded to environmental values that have been measured in agricultural landscapes [START_REF] Ghanem | Glyphosate and AMPA analysis in sewage sludge by LC-ESIMS/MS after FMOC derivatization on strong anion-exchange resin as solid support[END_REF][START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF], such as Argentinian agrosystems, which show an average concentration of 4,204 (± 2,258) µg.kg -1 . The concentration of tebuconazole corresponded to high environmental levels that have been described for individual fungicides [START_REF] Rial-Otero | Parameters affecting extraction of selected fungicides from vineyard soils[END_REF]. The concentrations of Cu or Zn also corresponded to high environmental levels that have been described in industrial polluted soils [START_REF] Dazy | Ecological recovery of vegetation on a cokefactory soil: role of plant antioxidant enzymes and possible implications in site restoration[END_REF].

In contrast, the concentration of hydroxyatrazine (5,000 µg L -1 ) was significantly higher than currently-described (up to 640 µg.kg -1 ) environmental values [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]Jablonowski et al., 2010;[START_REF] Lerch | Hydroxyatrazine in soils and sediments[END_REF]. Moreover, none of the nine agri-environmental plant species under study showed any oversensitivity to hydroxyatrazine (Fig. 2) relatively to that of Arabidopsis thaliana.

Thus, the present results indicated that glyphosate, AMPA, atrazine, tebuconazole, Zn, Cu and fluoranthene could not be considered as innocuous in terms of plant-contaminant interactions under environmental conditions. Residual environmental levels of both glyphosate and its metabolite AMPA were likely to affect Trifolium pratense, Anthemis tinctoria, Centaurea cyanus and Fagopyrum esculentum, and residual environmental levels of atrazine were likely to affect Lotus corniculatus and Anthemis tinctoria. The significant sensitivity of Trifolium pratense, Anthemis tinctoria, Centaurea cyanus and Fagopyrum esculentum to low levels of glyphosate suggested the possibility that effects under environmental conditions are more drastic than observed in the present study. The significant sensitivity of Centaurea cyanus to AMPA (Fig. 2) and the influence of AMPA and hydroxyatrazine on the patterns of responses of Trifolium pratense, Anthemis tinctoria, Fagopyrum esculentum and Lotus corniculatus (Fig. 4) underlined the potential environmental impact of herbicide metabolites., Such an impact of chemically-distinct compounds, with established bioactivity or supposed inactivation, and such a diversity of sensitivities emphasise the need to expand the range of pesticide tests in order to improve predictive power on the environmental impacts of pesticides [START_REF] Milner | Toward pesticidovigilance[END_REF]. Moreover, cumulative exposures can result in growth disruption that is not reducible to the sum of individual effects because of potential synergistic or antagonistic interactions between contaminants [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]. There are therefore environmental situations where the combined effects of mixtures of xenobiotics and/or heavy metals on plant growth and development may be more drastic than the predicted effects construed from single contaminant exposure experiments.

4.2.. Modes of action of root-level agricultural contaminants and risk assessment of differential root-shoot growth under conditions of soil contamination

The present results under controlled conditions emphasised the direct action of chemical stressors on plant tissues. The effects of chemical stressors have been experimentally characterized in a limited number of plant species, and their targets and mechanisms of action remain to be fully investigated [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF][START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015;;[START_REF] Vivancos | Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration[END_REF]. Even widely-used herbicides with well-known chloroplast-localized canonical sites of action, such as glyphosate and atrazine, have additional noncanonical effects that particularly affect signaling mechanisms, hormone dynamics, developmental processes and root growth [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF][START_REF] Couée | Physiology and toxicology of hormonedisrupting chemicals in higher plants[END_REF][START_REF] Dogramaci | Glyphosate's impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching[END_REF][START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF][START_REF] Ramel | Xenobiotic sensing and signalling in higher plants[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015;;[START_REF] Vivancos | Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration[END_REF]. Moreover, the corresponding metabolites, AMPA and hydroxyatrazine, can significantly affect plant metabolism and growth in the absence of observed effects on the canonical targets of parent molecules, thus implying alternative mechanisms of action [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF][START_REF] Gomes | Differential effects of glyphosate and aminomethylphosphonic acid (AMPA) on photosynthesis and chlorophyll metabolism in willow plants[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015)).

The present comparative study confirmed the importance of noncanonical patterns of responses to glyphosate and atrazine. The significant effects on root growth in the absence of effects on shoot growth in several plant species (Trifolium pratense, Anthemis tinctoria, Fagopyrum esculentum) could be related to the root-specific effects that have been discovered in Arabidopsis thaliana and Lolium perenne [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF][START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015)), and showed that root-specific effects of glyphosate and atrazine through root-level exposure can affect natural plant communities. The exclusive effects of AMPA on root growth, rather than on shoot growth, were coherent with a similar differential impact previously described in Lolium perenne (Serra et al., 2015). The effects of AMPA were usually similar to or weaker than the effects of glyphosate (Fig. 2, Fig. 3). However, AMPA decreased root growth in the same plant species (Trifolium pratense, Anthemis tinctoria, Centaurea cyanus, Fagopyrum esculentum) that were affected by glyphosate at root level (Fig. 2), thus suggesting potential additive effects on natural plant communities given that the co-occurrence of both compounds appears to be ubiquitous in soil [START_REF] Primost | Glyphosate and AMPA, « pseudo-persistent » pollutants under real-world management practices in the Mesopotamic Pampas agroecosystem, Argentina[END_REF][START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF].

In contrast, the effects of atrazine and hydroxyatrazine did not exactly follow the same pattern, with parallel effects of both compounds on root and shoot growth of Lotus corniculatus and exclusive effects of hydroxyatrazine on root and shoot growth of Agrostis stolonifera and Phleum pratense (Fig. 2, Fig. 3). Moreover, in several cases (Fig. 2, Fig. 3), hydroxyatrazine showed greater toxicity than atrazine. These important and specific effects of hydroxyatrazine on root growth, especially in Agrostis stolonifera, Phleum pratense, and Centaurea cyanus, were coherent with root-level effects [START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF] and regulation effects on root development genes [START_REF] Alberto | Low doses of triazine xenobiotics mobilize ABA and cytokinin regulations in a stress-and low-energy-dependent manner[END_REF] that have been previously described in Arabidopsis thaliana. The case of Trifolium pratense, where hydroxyatrazine decreased shoot growth in the absence of root growth inhibition, suggested that hydroxyatrazine may also have a shoot-related target. The mechanisms involved in the high toxicity of hydroxyatrazine remain to be characterized. They may be related to interferences of the triazine structure with cytokinin signaling [START_REF] Couée | Physiology and toxicology of hormonedisrupting chemicals in higher plants[END_REF][START_REF] Alberto | Root-level exposure reveals multiple physiological toxicity of triazine xenobiotics in Arabidopsis thaliana[END_REF]. The effects of tebuconazole, fluoranthene, Cu and Zn on plant growth are not related to actions on specific biochemical targets, and must involve a mixture of nutritional, biochemical, oxidative stress and regulatory modes of action [START_REF] Alkio | Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms[END_REF][START_REF] Lequeux | Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile[END_REF][START_REF] Paškova | Toxic effects and oxidative stress in higher plants exposed to polycyclic aromatic hydrocarbons and their N-heterocyclic derivatives[END_REF][START_REF] Rouached | Recent developments in plant zinc homeostasis and the path toward improved biofortication and phytoremediation programs[END_REF]Serra et al., 2015;[START_REF] Shishatskaya | Toxic effects of the fungicide tebuconazole on the root system of fusarium-infected wheat plants[END_REF][START_REF] Sverdrup | Toxicity of eight polycyclic aromatic compounds to red clover (Trifolium pratense), ryegrass (Lolium perenne), and mustard (Sinapis alba)[END_REF].

The diverse pattern of responses (Fig. 2, Fig. 3) could not therefore be ascribed to a specific mechanism. Moreover, in species-specific cases, all of these compounds could induce enhancement of root or shoot growth (Fig. 2, Fig. 3), thus emphasising a complex action on plant growth mechanisms. The root growth enhancement effect of fluoranthene in Lolium perenne has been shown to occur in parallel with accumulation of fructose, glucose and the cell-wall metabolite arabinose (Serra et al., 2015). On the other hand, strong negative effects on Lotus corniculatus, Anthemis tinctoria and Centaurea cyanus emphasised the potentially high toxicity of tebuconazole and Zn for natural plant communities.

In spite of the diversity of modes of action, all of the chemical stressors were found to induce, in at least one plant species, a disconnection of shoot and root growth. Thus, tebuconazole and fluoranthene significantly decreased shoot growth and increased root growth in Lolium perenne, and glyphosate significantly decreased shoot growth without affecting root growth in Lotus corniculatus. Such whole-plant modifications are likely to influence carbon and nitrogen status of the plant. Metabolomics and transcriptomics analysis of plant-xenobiotic interactions has revealed a significant impact of xenobiotics on carbon and nitrogen metabolisms [START_REF] Armendáriz | Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots[END_REF][START_REF] Orcaray | The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF]Serra et al., , 2015aSerra et al., , 2015b;;[START_REF] Vivancos | Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration[END_REF][START_REF] Zulet | Proteolytic pathways induced by herbicides that inhibit amino acid biosynthesis[END_REF]. In Lolium perenne, besides affecting root-shoot balance (Fig. 2, Fig. 3), fluoranthene treatment induces carbon metabolism modifications and nitrogen metabolism disturbances in both leaves and roots (Serra et al., 2015), thus suggesting that xenobiotic-induced enhancement of root growth may not be necessarily adaptive. Further work will determine whether carbon and nitrogen metabolites could be robust biomarkers of exposure to subtoxic chemical stress in the field.

Moreover, given the effects of carbon status on xenobiotic stress responses [START_REF] Ramel | Genome-wide interacting effects of sucrose and herbicidemediated stress in Arabidopsis thaliana : novel insights into atrazine toxicity and sucroseinduced tolerance[END_REF](Ramel et al., , 2009a(Ramel et al., , 2009b)), modifications of carbon/nitrogen balance may affect exposure to additional xenobiotics, as occurs during sequential or concomitant contaminations. The responses to constraints associated with climate change, such as temperature, elevated CO2 and salinity depend on adjustments of the levels of soluble sugars [START_REF] Bigot | Pivotal roles of environmental sensing and signaling mechanisms in plant responses to climate change[END_REF].

Differential responses of agri-environmental plant species and plant community dynamics under conditions of agriculture-related chemical contaminations

The comparative analysis of the nine plant species established that the dynamics of plantcontaminant interactions was highly contrasted. The plant cultivars studied in the present work are commonly used in the design and establishment of VFS and rotational fallows, and as such, could be considered to be reference cultivars in this context. However, comparative studies have shown differences of responses to abiotic stresses within cultivars, for instance in Agrostis stolonifera [START_REF] Xu | Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance[END_REF] and Festuca rubra [START_REF] Davies | The effects of Zinc on cell viability and on mitochondrial structure in contrasting cultivars of Festuca rubra L. -a rapid test for zinc tolerance[END_REF]. Beyond interspecific differences, further work on cultivars or populations of the plant species under study will be necessary to reveal the determinants of root-shoot responses to agricultural contaminants.

The three most tolerant species (Festuca rubra, Lolium perenne, Agrostis stolonifera) were Poaceae that develop deep rooting systems [START_REF] Roumet | Root traits and taxonomic affiliation of nine herbaceous species grown in glasshouse conditions[END_REF] and are used for phytostabilization and pollutant buffering of VFS and for phytoremediation of polluted soils [START_REF] Bidar | Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity[END_REF][START_REF] Mench | Successes and limitations of phytotechnologies at field scale: outcomes, assessment and outlook of COST Action 859[END_REF][START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF]. Festuca rubra and Lolium perenne have thus been found to colonize highly-contaminated soils containing Cu, Zn and fluoranthene [START_REF] Dazy | Ecological recovery of vegetation on a cokefactory soil: role of plant antioxidant enzymes and possible implications in site restoration[END_REF]. Moreover, Festuca rubra, Lolium perenne and Agrostis stolonifera are able to adjust carotenoid and chlorophyll levels under conditions of xenobiotic and heavy metal exposure (subsection 3.4; Serra et al., 2015a). Given that vegetation characteristics are important for the efficiency of VFS [START_REF] Hénault-Ethier | Herbaceous or Salix miyabeana 'SX64' narrow buffer strips as a means to minimize glyphosate and aminomethylphosphonic acid leaching from row crop fields[END_REF] or rotational fallows [START_REF] Kuussaari | Pollinator insects benefit from rotational fallows[END_REF][START_REF] Toivonen | Environmental fallows as a new policy tool to safeguard farmland biodiversity in Finland[END_REF], these plant species are likely to be useful candidates for designing agri-environmental schemes [START_REF] Ma | Plant functional diversity in agricultural margins and fallow fields varies with landscape complexity level: conservation implications[END_REF] and sustaining related ecosystem functions [START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF] in agriculturally-intensive landscapes under a wide range of agricultural pollution conditions. However, the effects of agricultural contaminants on plant growth and development through indirect effects on plantplant competition or plant-microbe associations should also be taken into account [START_REF] Damgaard | The effect of glyphosate on the growth and competitive effect of perennial grass species in semi-natural grasslands[END_REF][START_REF] Helander | Glyphosate decreases mycorrhizal colonization and affects plant-soil feedback[END_REF][START_REF] Van Bruggen | Environmental and health effects of the herbicide glyphosate[END_REF].

Fagopyrum esculentum, Lotus corniculatus, Phleum pratense, and Trifolium pratense have often been used for designing VFS (Krutz et al., 2015;[START_REF] Serra | Implications des communautés végétales péri-agricoles dans la dynamique environnementale des pollutions par les pesticides[END_REF][START_REF] Stehle | Pesticide risk mitigation by vegetated treatment systems: a metaanalysis[END_REF]. However, their intermediary sensitivity and tolerance to xenobiotics and heavy metals indicated that their establishment and growth in agricultural landscapes, especially as part of pesticide-exposed VFS, were likely to be strongly influenced by pollution and environment conditions. Shoot growth of Lotus corniculatus was highly sensitive to glyphosate, hydroxyatrazine, tebuconazole and Zn, although these compounds have very distinct mechanisms of action on plants [START_REF] Richard | Diversity analysis of the response to Zn within the Arabidopsis thaliana species revealed a low contribution of Zn translocation to Zn tolerance and a new role for Zn[END_REF][START_REF] Rouached | Recent developments in plant zinc homeostasis and the path toward improved biofortication and phytoremediation programs[END_REF][START_REF] Serra | Low environmentally-relevant levels of bioactive xenobiotics and associated degradation products cause cryptic perturbations of metabolism and molecular stress responses in Arabidopsis thaliana[END_REF].

Root growth of Fagopyrum esculentum was significantly affected by glyphosate, which is intensively applied [START_REF] Silva | Distribution of glyphosate and aminomethylphosphonic acid (AMPA) in agricultural topsoils of the European Union[END_REF][START_REF] Silva | Pesticide residues in European agricultural soils -A hidden reality unfolded[END_REF], thus suggesting that the phytostabilization role of Fagopyrum esculentum in VFS may be limited under conditions of agricultural pollution.Establishment and growth of Asteraceae Anthemis tinctoria and Centaurea cyanus, which were the most sensitive plant species, were likely to be impaired under a wide range of agricultural pollution conditions. Anthemis tinctoria and Centaurea cyanus, which show a high degree of plant-insect interactions, are important components of wild flower seed mixtures [START_REF] Ditner | Effects of experimentally planting non-crop flowers into cabbage fields on the abundance and diversity of predators[END_REF][START_REF] Stehlik | Floral free fall in the Swiss lowlands: environmental determinants of local plant extinction in a peri-urban landscape[END_REF][START_REF] Toivonen | Environmental fallows as a new policy tool to safeguard farmland biodiversity in Finland[END_REF][START_REF] Warzecha | Attractiveness of wildflower mixtures for wild bees and hoverflies depends on some key plant species[END_REF] for field margin sowing, intersowing or VFS in order to increase pollinator biodiversity and regulate predator dynamics. Their sensitivity to all or most xenobiotics and heavy metals may hamper such ecological engineering in agriculturally-intensive landscapes. Conversely, plant species with greater sensitivity to xenobiotics and heavy metals may be useful bioindicators of soil pollution in agroecosystems. Impairment of Lotus corniculatus, Anthemis tinctoria and Centaurea cyanus establishment and growth is thus likely to reflect contaminating levels of glyphosate and tebuconazole. Given the complex interactions between the multiple effects of diverse pesticides, bioindication of residual contaminating levels in soils may be useful to prevent crop injury or weed control failure due to excessive pesticide treatment of crops grown on pesticide-contaminated soil [START_REF] Alberto | Herbicide-related signaling in plants reveals novel insights for herbicide use strategies, environmental risk assessment and global change assessment challenges[END_REF].

Moreover, the general sensitivity of Anthemis tinctoria and Centaurea cyanus to xenobiotics and heavy metals entails that their cultivation as specialty crops should be carried out in a clean environment requiring at least a restricted use of pesticide treatments.

  act on shoot chloroplastic targets. The present study therefore aims to characterize the potential perturbations of root and shoot growth under conditions of exposure to agricultural contaminants, taking into account the chemical diversity of contaminants and the diverse responses of plants [bird's-foot trefoil (Lotus corniculatus), common buckwheat (Fagopyrum esculentum), cornflower (Centaurea cyanus), creeping bentgrass (Agrostis stolonifera), English ryegrass (Lolium perenne), red fescue (Festuca rubra), timothy grass (Phleum pratense), white Dutch clover (Trifolium pratense), yellow chamomile (Anthemis tinctoria)]

  Simultaneous or sequential exposure to xenobiotic and climate-change-related abiotic stresses might elicit complex modifications of soluble sugar dynamics. Besides immediate impacts on the plant, xenobiotic-induced changes of carbon and nitrogen status could therefore have cascading effects on abiotic stress and climate change responses, and thus more generally on plant ecosystem functioning under climate change.

Figure 1 .

 1 Figure 1. Effects of root-level exposure to glyphosate on primary root growth of Arabidopsis

Figure 2 .

 2 Figure 2. Effects of root-level exposure to agricultural xenobiotics and heavy metals on main

Figure 3 .

 3 Figure 3. Effects of root-level exposure to agricultural xenobiotics and heavy metals on shoot

Figure 4 .

 4 Figure 4. Principal component analysis and hierarchical clustering of species-specific plant

  Figure 1

Table 1 .

 1 Median effective concentrations (EC50) of the impact of agricultural xenobiotics and heavy metals on primary root growth in Arabidopsis thaliana seedlings. Concentrations used in subsequent experiments are given.

	Agricultural contaminant	EC50 (µM) Experimental	Experimental
			Concentration (µM) Concentration (mg L -1 )
	Glyphosate	0.75	1	0.169
	AMPA	30	50	5.550
	Atrazine	0.5	0.5	0.1
	Hydroxyatrazine	25	25	5
	Tebuconazole	4	4	1.28
	Zn	15	15	0.98
	Cu	6	6	0.38
	Fluoranthene	532	532	107.5

Acknowledgements

Our research on plant-herbicide interactions, plant-pesticide interactions and xenobiotic stress has been funded, in part, by the "Ecosphère continentale et côtière" and "Ingénierie écologique" interdisciplinary programmes from the Centre National de la Recherche Scientifique (CNRS, France), and by the Fondation pour la recherche sur la biodiversité (FRB, France). The Kuzul-rannvro Breizh/Conseil régional de Bretagne (France) is acknowledged for a doctoral scholarship to AAS. Funding sources had no involvement in the design of the study, in the collection, analysis or interpretation of data, in the writing of the report, nor in the decision to submit this article for publication.

Conflict of interest

The authors do not have any commercial or financial conflict of interest regarding the present article.