Fatima Tamtam
email: fatima.tamtam@upmc.fr

Folkert Van Oort

Barbara Le Bot

Tuc Dinh

Sophie Mompelat

Marc Chevreuil

Isabelle Lamy

Médard Thiry

Assessing the fate of antibiotic contaminants in metal contaminated soils four years after cessation of long-term waste water irrigation

Keywords:

Spreading of urban wastewater on agricultural land may lead to concomitant input of organic and inorganic pollutants. Such multiple pollution sites offer unique opportunities to study the fate of both heavy metals and pharmaceuticals. We examined the occurrence and fate of selected antibiotics in sandy-textured soils, sampled four years after cessation of 100 years irrigation with urban wastewater from the Paris agglomeration. Previous studies on heavy metal contamination of these soils guided our sampling strategy. Six antibiotics were studied, including quinolones, with a strong affinity for organic and mineral soil components, and sulfonamides, a group of more mobile molecules. Bulk samples were collected from surface horizons in different irrigation fields, but also in subsurface horizons in two selected profiles. In surface horizons, three quinolones (oxolinic acid, nalidixic acid, and flumequine) were present in eight samples out of nine. Their contents varied spatially, but were well-correlated one to another. Their distributions showed great similarities regarding spatial distribution of total organic carbon and heavy metal contents, consistent with a common origin by wastewater irrigation. Highest concentrations were observed for sampling sites close to irrigation water outlets, reaching 22 μg kg -1 for nalidixic acid. Within soil profiles, the two antibiotic groups demonstrated an opposite behavior: quinolones, found only in surface horizons; sulfamethoxazole, detected in clay-rich subsurface horizons, concomitant with Zn accumulation. Such distribution patterns are consistent with chemical adsorption properties of the two antibiotic groups: immobilization of quinolones in the surface horizons ascribed to strong affinity for organic matter (OM), migration of sulfamethoxazole due to a lower affinity for OM and its interception and retention in electronegative charged clay-rich horizons. Our work suggests that antibiotics may represent a durable contamination of soils, and risks for groundwater contamination, depending on the physicochemical characteristics both of the organic molecules and of soil constituents.

Introduction

The elimination of antibiotics present in wastewater by sewage treatments is often incomplete. Low removal rates after wastewater treatments were reported for some compounds such as sulfamethoxazole, trimethoprim or some macrolides, ascribed to their low degradation and potential sorption on soil constituents [START_REF] Mcardell | Occurrence and fate of macrolide antibiotics in wastewater treatment plants and in the Glatt Valley Watershed, Switzerland[END_REF][START_REF] Paxeus | Removal of selected non-steroidal anti-inflammatory drugs (NSAIDs), gemfibrozil, carbamazepine, beta-blockers, trimethoprim and triclosan in conventional wastewater treatment plants in five EU countries and their discharge to the aquatic environment[END_REF][START_REF] Lindberg | Screening of human antibiotic substances and determination of weekly mass flows in five sewage treatment plants in Sweden[END_REF]. The discharge of treated wastewater into surface waters was recognized as a major source of antibiotics contamination [START_REF] Miao | Occurrence of antimicrobials in the final effluents of wastewater treatment plants in Canada[END_REF][START_REF] Siemens | Concentrations and mobility of human pharmaceuticals in the world's largest wastewater irrigation system, Mexico City-Mezquital Valley[END_REF]) and, consequently, cautious management of urban wastewater is essential.

Spreading of urban wastewater on soil surfaces is widely considered as an alternative way to decrease such an organic contamination discharged by wastewater [START_REF] Tamtam | Irrigation of treated wastewater in Braunschweig, Germany: an option to remove pharmaceuticals and musk fragrances[END_REF]). The purpose of such a proceeding is to complete in a natural way the purification of wastewater by using the specific filtering and purifying characteristics of coarse textured soils, before their recharge to the groundwater. Such land management, however, may lead to a diffuse organic or inorganic contamination of soils depending on the quality of wastewater. Frequently, mineral contamination relates to the introduction and accumulation of heavy metals in soils. Although organic contaminants generally are more degradable, the fate of their long-term input in soils is not well known. Yet, such active compounds are designed to act upon soil microorganisms at low concentrations and, therefore, represent serious environmental risk [START_REF] Westergaard | Effects of tylosin as a disturbance on the soil microbial community[END_REF][START_REF] Schauss | Analysis, fate and effects of the antibiotic sulfadiazine in soil ecosystems[END_REF]. Furthermore, heavy metals and antibiotic are suspected to act together in bacterial resistance selection or maintaining [START_REF] Baker-Austin | Co-selection of antibiotic and metal resistance[END_REF][START_REF] Matyar | Antibacterial agents and heavy metal resistance in Gramnegative bacteria isolated from seawater, shrimp and sediment in Iskenderun Bay[END_REF], and many questions remain about long-term impacts on living organism by sub-inhibitory exposure in real field situations of multi organic and inorganic soil contamination.

Such concern is particularly important for antibiotics exhibiting high adsorbing properties, and for which accumulation in soil on the surface of either mineral or organic soil constituents has been assessed [START_REF] Chang | Adsorption and intercalation of tetracycline by swelling clay minerals[END_REF][START_REF] Zhang | Impacts of soil organic matter, pH and exogenous copper on sorption behavior of norfloxacin in three soils[END_REF]). On the other hand, migration to deeper horizons of more mobile compounds represents additional risk regarding contamination of groundwater resources [START_REF] Kinney | Presence and distribution of wastewaterderived pharmaceuticals in soil irrigated with reclaimed water[END_REF][START_REF] Tamtam | Irrigation of treated wastewater in Braunschweig, Germany: an option to remove pharmaceuticals and musk fragrances[END_REF][START_REF] Heberer | Behaviour and redox sensitivity of antimicrobial residues during bank filtration[END_REF][START_REF] Siemens | Transport of pharmaceuticals in columns of a wastewater-irrigated Mexican clay soil[END_REF].

Moreover, some antibiotic compounds were shown to display relatively long half-lives, reaching several months in soils [START_REF] Marengo | Aerobic biodegradation of (C-14)-sarafloxacin hydrochloride in soil[END_REF], while others undergo a rapid biodegradation occurring within a few days [START_REF] Burkhardt | Depth distribution of sulfonamide antibiotics in pore water of an undisturbed loamy grassland soil[END_REF][START_REF] Kotzerke | Alterations in soil microbial activity and N-transformation processes due to sulfadiazine loads in pig-manure[END_REF]. The sorption of antibiotics on soil constituents was shown to play a significant role in such persistence, related to a reduced biodegradability [START_REF] Ingerslev | Biodegradability of metronidazole, olaquindox, and tylosin and formation of tylosin degradation products in aerobic soil-manure slurries[END_REF]. It has been shown that the fate and impact of such compounds depend to a large extent on the nature of the molecules [START_REF] Kinney | Presence and distribution of wastewaterderived pharmaceuticals in soil irrigated with reclaimed water[END_REF][START_REF] Tamtam | Irrigation of treated wastewater in Braunschweig, Germany: an option to remove pharmaceuticals and musk fragrances[END_REF][START_REF] Carrasquillo | Sorption of ciprofloxacin and oxytetracycline zwitterions to soils and soil minerals: influence of compound structure[END_REF][START_REF] Siemens | Transport of pharmaceuticals in columns of a wastewater-irrigated Mexican clay soil[END_REF]) and on the nature and characteristics of the main soil constituents [START_REF] Vasudevan | pHdependent ciprofloxacin sorption to soils: interaction mechanisms and soil factors influencing sorption[END_REF][START_REF] Zhang | Impacts of soil organic matter, pH and exogenous copper on sorption behavior of norfloxacin in three soils[END_REF]). However, in situ research on the distribution and accumulation of antibiotics at the field scale remains scarce, thus restricting sustainable land management regarding past or future spreading of wastewater.

Therefore, in this work we aimed at 1) investigating antibiotic contamination in agricultural soils irrigated for more than 100 years with untreated urban wastewater of the Paris area, previously studied for consequent heavy metal contamination; 2) assessing in situ the importance of the nature of the antibiotic molecules on their fate in soil profiles and 3) comparing the in situ fate of non-biodegradable trace elements to that of a priori biodegradable antibiotic molecules. We focused on two antibiotic groups exhibiting contrasting properties: i) the quinolones, known for their strong adsorption properties on soil constituents, and ii) the sulfonamides, with more mobile characteristics. Since metals and antibiotics were mentioned to be detected together [START_REF] Lee | Multiple antibiotic resistance and heavy metal resistance profile of bacteria isolated from giant freshwater prawn (Macrobrachium rosenbergii) hatchery[END_REF][START_REF] Oyetibo | Bacteria with dual resistance to elevated concentrations of heavy metals and antibiotics in Nigerian contaminated systems[END_REF], we hypothesized that some inorganic and organic pollutants added to soils by wastewater irrigation might display similar distribution patterns, according to their physicochemical characteristics. Our sampling strategy of surface and subsurface horizons was based on previous field survey on heavy metal contamination [START_REF] Lamy | Use of major-and trace-element correlations to assess metal migration in sandy Luvisols irrigated with wastewater[END_REF][START_REF] Bourennane | Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography[END_REF].

Materials and methods

Site presentation

The study site (Fig. 1) is located ≈65 km downstream of the center of Paris and approximately 6 km from the Seine Aval wastewater treatment plant, treating most of the Paris city and close suburbs wastewater [START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF], estimated at about 300 × 10 6 m 3 / day at the end of the 1990s.

Since the 1890s, and for more than one century, raw wastewater has been transported to different irrigation fields, totalizing more than 5000 ha at the beginning of the 20th century, and still ~2000 ha in 2000 [START_REF] Védry | From sewage water treatment to waste water reuse. One century of Paris sewage farms history[END_REF]. Irrigation was done by flooding, with soils being immerged for one or more days, several times a year. The soils of these fields were chosen because of their coarse texture, allowing high water infiltration rates. Such irrigation practices enhanced agricultural use for market garden of the low fertile soils by strongly increasing crop yields, recognized as soon as 1895 (Risler 1897).

The actual irrigation perimeter, approximately 1200 ha, is located at the extremity of the general emissary of the Paris conurbation sewer network, ≈20 km NE of Paris. The irrigated perimeter is divided in five irrigation sectors, delivering irrigation water to about 60 irrigation fields, each field being bordered by a series of water outlets. The irrigation by gravitational flooding of wastewater is favored by annual plowing parallel to topography. During the 1990s the soils of this area still received annual rates of about 2000 mm of urban wastewater [START_REF] Védry | From sewage water treatment to waste water reuse. One century of Paris sewage farms history[END_REF]. Several reports stressed the strong accumulation of heavy metals leading to the stop of food crop production in 2000, while ongoing irrigation with wastewater clarified by flocculation was recommended for maize growth assigned to animal breeding. On the studied irrigation fields, irrigation was stopped during 2005.

Sampling

Based on the hypothesis that multi-pollution added in the soils by wastewater would lead to comparable distribution patterns, we used previous studies on distribution of concentrations of organic carbon and heavy metals in the surface horizons of the irrigated site [START_REF] Bourennane | Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography[END_REF] to select one characteristic irrigated parcel. In this parcel, on September 21st 2008, we collected nine bulk samples (P1-P9) in the middle of the plow layer, between 15 and 25 cm depth, following a 1 km wide topographical transect (Fig. 1). Sampling sites were selected in upslope (P9), middle or down slope positions (P1), and at various distances of wastewater irrigation outlets. This sampling scheme included two different sectors of the irrigation perimeter and crossed four different fields (Fig. 1).

Surface soil samples were separated in 5 cm thick slices, and stored frozen (-20 °C) to prevent from degradation of organic compounds, prior to analysis performed within a few weeks. In addition, two sites (P3 and P9) were sampled until about 1.5 m, at different depths, following the occurrence and thickness of characteristic soil horizons.

Soil composition and metal pollutant distribution

The soils of the area were described as Luvisols developed in sandy residual formations resulting from decarbonation of different facies of Lutetian limestone. Irrigated soils generally demonstrate a sandy-textured topsoil with an approximately 30 to 50 cm thick dark-brown to black colored plow layer (Ap) and a more or less well-developed light colored eluvial (E) horizon (Fig. 2), overlying a reddish-brown illuvial clay-ironenriched (Bt) horizon, at the contact of the partially weathered whiteolive-green calcareous parent material [START_REF] Lamy | Use of major-and trace-element correlations to assess metal migration in sandy Luvisols irrigated with wastewater[END_REF]). The thickness and distinctness of soil horizons is variable (Table 1) depending on microtopography and position with regard to major irrigation water outlets.

The remarkable dark color of the Ap horizon is due to the accumulation of fine, amorphous organic matter (OM) forming coatings around the quartz grains [START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF], leading to significant high organic carbon contents (Table 1) for such sandy agricultural soils. In these coatings, numerous very fine, secondary calcite crystals occur, precipitating after evaporation of the waste water. Such coatings were highlighted to represent one of the main soil fractions involved in metal retention [START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF], explaining the strong accumulation of heavy metals in the surface horizon. The surface horizons contain low clay contents (8-9% in Ap, b5% in E) that contrast with about 20% of b2 μm fraction found in the Bt horizon (Table 1). The E and Bt horizon do not show any effervescence with 10% HCl, in contrast to the Ap (secondary carbonates) and the calcareous BC and Ck horizons. The mineralogical composition also significantly contrasts between the surface Ap and E horizons [START_REF] Dère | Reconstitution des apports en éléments trace métalliques et bilan de leur migration dans un Luvisol sableux soumis à 100 ans d'irrigation massive par des eaux usées brutes[END_REF] dominated by Al-interlayered, non-swelling clay minerals, in contrast to the Bt, BC and Ck horizons displaying a predominant composition of swelling smectite [START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF]. Such a contrasting mineralogical clay composition is consistent with fairly low CEC values observed in the surface horizon, despite its high organic carbon content, whilst the Bt horizon demonstrates the highest CEC values (Table 1).

Within the irrigation fields, a great variability of metal concentrations, particularly well-correlated with the soil's total organic carbon contents was demonstrated [START_REF] Bourennane | Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography[END_REF] to a large extent explained by the geometry and distance of soil samples with respect to the irrigation outlets. At the scale of the soil profile, a consistent migration of more mobile Zn towards depth and its accumulation in the Bt horizon (Table 1) due to interception by clay-iron complexes, was clearly demonstrated by using major and trace element correlations [START_REF] Lamy | Use of major-and trace-element correlations to assess metal migration in sandy Luvisols irrigated with wastewater[END_REF]) and microspectroscopic analytical techniques on thin sections [START_REF] Lamy | Use of major-and trace-element correlations to assess metal migration in sandy Luvisols irrigated with wastewater[END_REF][START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF]. Less mobile elements such as Pb were found predominantly immobilized in the OM-rich surface horizons.

Antibiotic analysis

A first selection of 17 compounds was made based on their use in human or veterinary medicine for monitoring in the Seine River (Tamtam et al. 2008), including quinolones (oxolinic acid, nalidixic acid, flumequine, pipemidic acid), fluoroquinolones (enrofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, ofloxacin, sarafloxacin, difloxacin, danofloxacin, and enoxacin), sulfonamides (sulfamethoxazole, sulfamethazine), diaminopyrimidine (trimethoprim) and nitroimmiazole (ornidazole). An optimization of soil extraction of the different compounds was performed. Soxwave extraction tests were made at various pH, from pH 2 to pH 10, in order to determine for each compound the optimal conditions for highest recovery. No single extraction pH gave sufficient recoveries for all the compounds from different groups, as the highest recoveries for quinolones were obtained at low extraction pH, with very low recoveries for fluoroquinones and vice-versa at a high extraction pH. Further to that, a protocol involving two successive soxwave extractions was selected, including a first extraction at acidic pH 2 and then a second one at alkaline pH 10. With this protocol it was possible to obtain satisfactory recoveries for the few following compounds that were included in our work: sulfamethoxazole (sulfonamides), diaminopyrimidine (trimethoprime), nalidixic acid, oxolinic acid, pipemidic acid, flumequine (quinolones), 2. Standard compounds of quinolones (oxolinic acid, nalidixic acid, flumequine), fluoroquinolones (norfloxacin) and sulfonamides (sulfamethoxazole) were obtained Sigma-Aldrich (Steinheim, Germany) and diaminopyrimidine trimethoprim was purchased from VWR (France). All pharmaceuticals were of analytical grade (purity N 95%).

Antibiotics were extracted from 2 g of wet soil using microwave assisted Soxhlet extraction (Soxwave 100, Prolabo, France). A 50 mL mixture of methanol/H 3 PO 4 aqueous solution (pH 2, 1 M) (90:10) was used as extraction solvent. Soil samples were extracted for 15 min (45 W). Then, samples were filtered (0.45 μm, Millipore acetate filters) and solid re-extracted using 50 mL of a methanol/NaOH (5%, pH 10) mixture (90:10), in the same conditions (15 min, 45 W). The two obtained extracts were pooled and evaporated with rotative evaporator (Büchi R-144 and R-205) at 42 °C, 280 bars for 30 min, and finally diluted in 100 mL ultra pure water (Millipore, France). Samples were then adjusted to pH 7 with few drops of orthophosphoric acid (50%), extracted and purified by Solid Phase Extraction prior to analysis by Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS, Waters™) using operating analysis parameters described by [START_REF] Tamtam | Ultra performance liquid chromatographytandem mass spectrometry performance evaluation for analysis of antibiotics in natural waters[END_REF].

Method validation and quality control

Method was validated prior to the study on Seine River sediments spiked at 100 μg kg -1 (triplicates), and left during 24 h for sorption and solvent evaporation. Some recovery tests on soils gave very similar results than on sediments, as reported in literature [START_REF] Prat | Determination of flumequine and oxolinic acid in sediments and soils by microwave-assisted extraction and liquid chromatography-fluorescence[END_REF][START_REF] Vazquez-Roig | Determination of pharmaceuticals in soils and sediments by pressurized liquid extraction and liquid chromatography tandem mass spectrometry[END_REF], thus a new validation based on soil samples was not necessary. Detection limits and quantification limits were calculated based respectively on 3 and 10 times the Signal/Noise (S/N) ratio obtained from the chromatograms of spiked sediments extracts (100 μg kg -1 ). Due to strong matrix effects observed for the fluoroquinolone norfloxacin using standard addition, the high S/N ratio did not allow a detection limit lower than 12 μg kg -1 . Recoveries for each compound and method limits are given in Table 3. These detection and quantification limits are in the same range of limits reported in literature, which are often in the low μg kg -1 range [START_REF] Prat | Determination of flumequine and oxolinic acid in sediments and soils by microwave-assisted extraction and liquid chromatography-fluorescence[END_REF][START_REF] Vazquez-Roig | Determination of pharmaceuticals in soils and sediments by pressurized liquid extraction and liquid chromatography tandem mass spectrometry[END_REF]), except for norfloxacin quantification limit, which was higher in this study than reported by [START_REF] Vazquez-Roig | Determination of pharmaceuticals in soils and sediments by pressurized liquid extraction and liquid chromatography tandem mass spectrometry[END_REF], close to 5 μg kg -1 .

Total organic carbon analysis

Total organic carbon contents were determined on air-dried, homogenized and quartered subsamples of the soils, crushed and sieved to b200 μm. Analyses were performed at the INRA -Soil Analysis Laboratory of Arras (France), according international standard methods (ISO no. 1-1380). All results are expressed on an oven-dry 110 °C basis.

Results and discussion

Spatial variability of the antibiotic distribution in the surface horizons

Contents in antibiotics are given Table 4 for the three antibiotics finally found in the soil surface samples. No antibiotic from the sulfonamide group was detected in the surface soil samples. The only antibiotics determined were from the quinolone group: nalidixic acid was always found above the detection limit except in the upslope sample, while oxolinic acid and flumequine were found in lower contents and often just detected but not quantified.

The highest concentrations were observed in the P3 and P6 samples with contents around 5-6 μg kg -1 , 21-22 μg kg -1 and 6-7 μg kg -1 for oxolinic acid, nalidixic acid and flumequine, respectively. The antibiotics detected in the surface soil samples were found Nalidixic acid 1-Ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid 0.33 d 5.9 e dnf dnf dnf Oxolinic acid 1-Ethyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid 0.004 f 6.9 f 0.68 f 7400-31,600 f N 300 g Flumequine 7-Fluoro-12-methyl-4-oxo-1-azatricyclo[7.3.1.0 5,13 ]trideca-2,5,7,9 (13)-tetraene-3-carboxylic acid 0.07 f 6.4 f 1.7 f 2750-24,500 f N 300 g Fluoroquinolones Norfloxacin 1-Ethyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid 178 h 6.26-8.85 h -1.03 h 7800-15,800 i dnf dnf: data not found. well-related one to each other (Fig. 3), consistent with a common input into soils. Furthermore, the antibiotic concentrations were also found related to the organic carbon contents in the surface horizons (Table 4), with correlation coefficients ≥0.9 for the three quinolones.

at the small region scale and at the scale of the studied irrigation fields, well-correlated distributions between organic carbon and heavy metal contents were reported [START_REF] Baize | -garden soils close to Paris (France): subsequent impacts and hazards[END_REF][START_REF] Bourennane | Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography[END_REF]). These authors assumed that such correlation between heavy metals and organic carbon represented the signature of a common origin of pollution from waste water irrigation, which is also consistent with the presence of antibiotics.

As no direct veterinary waste inputs in the field were identified and considering the cessation of irrigation practices on the studied irrigation fields since 2005, our results suggest that the antibiotic contamination aged of several years. In broad literature, only few work deals with the persistence of antibiotics in terrestrial environments [START_REF] Diaz-Cruz | Environmental behavior and analysis of veterinary and human drugs in soils, sediments and sludge[END_REF][START_REF] Kinney | Presence and distribution of wastewaterderived pharmaceuticals in soil irrigated with reclaimed water[END_REF], especially for quinolones. Quinolones antibiotics are either used in veterinary or human medicine, except for oxolinic acid, which is limited since 2006 only to veterinary use and therefore unlikely to occur in urban wastewater currently applied on the site. Unfortunately, antibiotic consumption data in France, allowing to estimate field concentration for each of the compounds, and to provide insight on the fate of the molecules over the years, are unavailable for the public. Nevertheless, in the light of the progressive abandon of first generation quinolone nalidixic acid in favor of more efficient compounds such as fluoroquinolones, we speculate for a hypothesis of either a progressive accumulation of nalidixic acid over the years, or a residual contamination from times where this compound was more widely used (in the 1960s).

Thus, our results are among the first to suggest that long-term wastewater irrigation on agricultural soil can induce long-term antibiotic contamination in soils. Persistence of several months was reported for some pharmaceuticals in similar conditions of sewageirrigated soils [START_REF] Kinney | Presence and distribution of wastewaterderived pharmaceuticals in soil irrigated with reclaimed water[END_REF].

Surprisingly, norfloxacin, a widely used fluoroquinolone, on the market since the late 80s, was not detected in our soil samples, although norfloxacin concentrations as high as 150 ng L -1 were reported in the river water downstream of the WWTP discharge providing water for the irrigation field (Tamtam et al. 2008). A first explanation may be our relatively high detection limits for norfloxacin (12 μg kg -1 ): lower concentration of norfloxacin in soils will not be detected, although such concentrations may be of the same order than those of the other compounds. Second, formation of non-extractable residues (NER) in soils, notably for sulfonamides was reported [START_REF] Heise | Chemical and biological characterization of nonextractable sulfonamide residues in soil[END_REF]. This may also be the case for other groups such as quinolones and fluoroquinolones, but not reported yet. In that case, only sorption investigation in controlled condition of the fate of norfloxacin can make it possible to estimate levels of this compound in soil.

Spatial distribution of concentrations of antibiotics was found to depend on the geometry, functioning and proximity of the irrigation network, as was already reported for heavy metal pollution [START_REF] Lamy | Etude d'un secteur agricole pollué par des épandages d'eaux usées: bilan environnemental et possibilités de reconversions végétales[END_REF][START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF][START_REF] Bourennane | Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography[END_REF]. Sampling locations close to major irrigation water outlets corresponded to the highest concentrations, either for antibiotics (this work) or for heavy metals. The highest antibiotic contamination levels were measured for the P3 and P6 samples, where heavy metal concentrations were also found the highest, i.e. ~1050 and ~1230 mg kg -1 , for Zn in P3 and P6 respectively, compared to values of some hundreds mg Zn kg -1 observed for the other sampling sites, at greater distance from the water outlets.

Antibiotic distribution vs. depth in soil profiles

The contents in antibiotics determined at different soil depths in the two soil profiles (P3, P9) are presented in Table 4 as well as the total organic carbon contents. In addition, antibiotic concentrations are plotted vs. soil depth in Fig. 4, together with total Zn concentrations determined in the bulk of the characteristic soil horizons.

In the P3 profile (Fig. 4a), the highest concentrations of quinolones were observed in the plow (Ap) horizon. In the underlying E horizon, oxolinic acid and flumequine were not detected, and the concentration of nalidixic acid decreased to values b5 μg kg -1 . At greater depth, no antibiotics of the quinolone group were detected. For the P9 profile (Fig. 4b), a different vertical distribution was observed quinolones: oxolinic and nalidixic acid and flumequine were absent in the Ap horizon, but these compounds were detected in the underlying E horizon, with concentration levels fairly similar to those found in the E horizon of the P3 profile. By contrast, sulfamethoxazole was poorly detectable in samples from the plow horizons, but its presence was detected in the Bt horizons, at ~75-cm depth for the P3 profile, and at ~120-cm depth for the P9 profile. Surprisingly, sulfamethoxazole concentrations at such depths were higher than in samples from the surface horizons, which were generally b1 μg kg -1 (Table 4). As outlined before, the low detection of sulfamethoxazole in the surface horizon may be due to the formation of NER via covalent bonding with organic matter, but its detection in the Bt horizons indicates that these compounds partially migrate to depth.

In the two studied soil profiles, the distribution of antibiotics demonstrated an opposite behavior between the two groups: quinolones were found mainly in the surface horizons, where the OM contents are highest, whereas sulfamethoxazole occurred only in deeper Bt horizons. Such a predominant immobilization of antibiotics at the soil's surface was also reported for fluoroquinolones norfoxacin and ciprofloxacin, which accumulated at concentrations of around 400 μg kg -1 in soils amended with sludge [START_REF] Golet | Environmental exposure assessment of fluoroquinolone antibacterial agents from sewage to soil[END_REF]. The latter compounds were not detected in our study, but are likely to exhibit similar behavior than quinolones. These results suggest an OM-mediated location for quinolones, limiting their downward migration. The physicochemical characteristics of quinolones explain such strong sorption properties towards soil constituents [START_REF] Tolls | Sorption of veterinary pharmaceuticals in soils: a review[END_REF][START_REF] Golet | Environmental exposure assessment of fluoroquinolone antibacterial agents from sewage to soil[END_REF]: surface adsorption to mineral constituents, complex formation with metal ions and electrostatic interactions [START_REF] Nowara | Binding of fluoroquinolone carboxylic acid derivatives to clay minerals[END_REF][START_REF] Tolls | Sorption of veterinary pharmaceuticals in soils: a review[END_REF][START_REF] Diaz-Cruz | Environmental behavior and analysis of veterinary and human drugs in soils, sediments and sludge[END_REF], although interaction with other pollutants such as surfactants was shown to modify nalidixic acid sorption behavior [START_REF] Hari | Effects of pH and cationic and nonionic surfactants on the adsorption of pharmaceuticals to a natural aquifer material[END_REF]. The nondetection of quinolones in the plow layer of the P9 profile may be related to low organic carbon and heavy metal contents at this sampling site. Located at the upper slope position (Fig. 1), P9 makes part of another irrigation sector than the other sampling sites. Different irrigation management with respect to the amounts and quality of the waste water during the last years may be invoked to explain the various patterns of quinolones in surface samples. Nevertheless, the presence of oxolinic and nalidixic acid and flumequine in the E horizon of the P9 profile (Fig. 4b) indicates that such quinolone compounds have been added once in the plow layer.

By contrast, the detection of sulfamethoxazole at depth in clay-rich Bt horizons is in agreement with reported ability to migrate in soils like other sulfonamides [START_REF] Blackwell | The dissipation and transport of veterinary antibiotics in a sandy loam soil[END_REF][START_REF] Burkhardt | Depth distribution of sulfonamide antibiotics in pore water of an undisturbed loamy grassland soil[END_REF]. These Bt horizons also intercept dissolved Zn out of the percolating soil solution by adsorption of Zn 2+ on the negative charges of clay-iron complexes occurring as coatings [START_REF] Dère | Reconstitution des apports en éléments trace métalliques et bilan de leur migration dans un Luvisol sableux soumis à 100 ans d'irrigation massive par des eaux usées brutes[END_REF][START_REF] Van Oort | Microscale Zn and Pb distribution patterns in subsurface soil horizons: an indication for metal transport dynamics[END_REF][START_REF] Van Oort | Impacts of long-term wastewater irrigation on the development of sandy Luvisols: consequences for metal pollutant distributions[END_REF], leading to a more or less marked increase in Zn concentrations. Such a concurrent presence of Zn and sulfamethoxazole in clay-rich B horizons suggests a common interception mechanism of electrostatic adsorption. The evidence for sulfamethoxazole migration is consistent with reported K oc of several sulphonamides, varying from 97 to 323 L kg -1 , characteristic for mobile molecules [START_REF] Thiele-Bruhn | Pharmaceutical antibiotic compounds in soilsa review[END_REF]. Such mobility was also invoked to explain the poor removal of sulfamethoxazole after wastewater treatment, as reported in numerous studies [START_REF] Gobel | Trace determination of macrolide and sulfonamide antimicrobials, a human sulfonamide metabolite, and trimethoprim in wastewater using liquid chromatography coupled to electrospray tandem mass spectrometry[END_REF][START_REF] Lindberg | Screening of human antibiotic substances and determination of weekly mass flows in five sewage treatment plants in Sweden[END_REF][START_REF] Batt | Comparison of the occurrence of antibiotics in four full-scale wastewater treatment plants with varying designs and operations[END_REF]). Nevertheless, significant but yet undetectable amounts of sulfonamides may be present in the surface horizons, according to different studies pointing the formation of NER for sulfonamides [START_REF] Heise | Chemical and biological characterization of nonextractable sulfonamide residues in soil[END_REF][START_REF] Kahle | Sorption of the veterinary antimicrobial sulfathiazole to organic materials of different origin[END_REF], notably due to covalent bonding. If so, their distribution pattern in the Luvisol would be rather similar to that of Zn, with a main presence in the Ap horizon and a large interception in the Bt horizon (Table 4). Since sulfamethoxazole was detected in the clay-rich horizons of both soils profiles, we hypothesize that a non-negligible part of this compound was subject to downward leaching. In addition, our findings on accumulation of sulfamethoxazole to depths higher than 1 m corroborate with other studies reporting detection of sulfamethoxazole in groundwater [START_REF] Lindsey | Analysis of trace levels of sulfonamide and tetracycline antimicrobials in groundwater and surface water using solid-phase extraction and liquid chromatography/mass spectrometry[END_REF][START_REF] Sacher | Pharmaceuticals in groundwaters: analytical methods and results of a monitoring program in Baden-Württemberg, Germany[END_REF][START_REF] Tamtam | Irrigation of treated wastewater in Braunschweig, Germany: an option to remove pharmaceuticals and musk fragrances[END_REF].

Conclusion

This study assessed soil contamination by antibiotics from longterm wastewater irrigation. Among different antibiotics screened, four compounds were detected with different rates in soil samples: oxolinic acid, nalidixic acid, flumequine and sulfamethoxazole. Relations between concentrations of antibiotics, organic carbon, and metal pollutants (Zn), were found consistent with a single and same source of contamination.

Since the input of wastewater in these was decreasing since the 2000s, and stopped in 2005 (four years before our sampling campaign) the detection of significant amounts of antibiotics in the soil surface horizons highlights their persistence in soils. Concentration profiles of antibiotics vs. depth displayed a general pattern of higher quinolone contamination in the OM-rich plow layer, but low amounts of such compounds were still observed in the underlying, sandy-textured E horizons. The predominant presence of quinolones in the surface horizons is consistent with the strong adsorption properties of these molecules with large amounts of accumulated OM, likely to limit their downward migration. At the opposite, sulfamethoxazole was detected in clay-rich horizons, at great depth (75, 120 cm) at the same depths where Zn accumulated, suggesting similar immobilization mechanisms by electrostatic adsorption.

In the view of distribution patterns, according to their nature and reactivity, the occurrence of antibiotics and of heavy metal contaminants, both added to the soils by wastewater irrigation showed similarities: either a predominant accumulation with OM in the surface horizon (like quinolones and the heavy metals Pb, Cu), or a migration to depth and interception by clay-iron complexes (like sulfamethoxazole and the heavy metals Zn, Cd).

Fig. 1 .

 1 Fig. 1. Schematic presentation of the sampling locations P1 to P9, with respect to the irrigation fields and wastewater distribution sectors along a topographical transect.

  Fig. 2. Characteristic sequence of soil horizons observed in Luvisols of the irrigated plain. (Modified after van Oort et al., 2008).

Fig. 3 .

 3 Fig. 3. Relation between antibiotic concentrations (in μg kg -1 ) in the surface soil samples : (a) nalidixic acid vs. flumequine concentrations: y=3.21x (r 2 =0.96) and (b) nalidixic acid vs. oxolinic acid concentrations: y=4.18x (r 2 =0.88). When measured concentrations were between LD and LQ, concentrations were assimilated to half of the LQ.

Fig. 4 .

 4 Fig. 4. Vertical distribution of antibiotic and total zinc concentrations in horizons of the soil profiles P3 (a) and P9 (b). Succession of horizons (Ap, E, Bt, BC, and Ck) are shown in dotted lines. Red star marks sulfamethoxazole detection under quantification limits.

Table 1

 1 Selected physicochemical data of soils irrigated with waste water. Mean values +/-SE from analyses of bulk horizons of three soil profiles, close to P3, P5, and P6; nd: not determined.

	Horizon	Thickness	Clay	Sand	Org. C	CaCO 3	pH	C/N	CEC	Bulk density	Zn	Pb
		(cm)	(g kg -1 )						(cmol + kg -1 )	(Mg m -3 )	(mg kg -1 )
	Ap	39 ± 7	86 ± 5	767 ± 14	34.5 ± 6.5	38 ± 8.5	7.1 ± 0.3	15.4 ± 3.1	10.2 ± 2.1	1.40 ± 0.14	1055 ± 74	536 ± 90
	E	15 ± 5	47 ± 12	870 ± 23	1.8 ± 0.6	b1	7.4 ± 0.1	7.1 ± 0.4	3.4 ± 0.6	1.71 ± 0.03	222 ± 37	7.7 ± 0.3
	Bt	13 ± 6	199 ± 16	719 ± 17	2.6 ± 0.2	b1	7.5 ± 0.1	nd	13.3 ± 0.6	1.65 ± 0.06	402 ± 72	11.0 ± 0.7
	BC	11 ± 1	110 ± 18	748 ± 72	1.1 ± 0.6	299 ± 139	8.5 ± 0.2	nd	4.8 ± 1.4	1.61 ± 0.14	125 ± 57	5.7 ± 1.4
	Ck	N 25	108 ± 10	716 ± 86	2.0 ± 1.0	409 ± 172	8.7 ± 0.2	nd	3.8 ± 1.5	nd	155 ± 131	4.2 ± 1.1

norfloxacin (fluoroquinolones). Physicochemical properties of the selected compounds are presented in Table

Table 2

 2 IUPAC names and physicochemical properties of the studied compounds.

	Group compound	IUPAC Name	Solubility (g L -1 ) pKa (1-2) Log K ow K oc (L/kg)	Half-life in sediment (d)
	Sulfonamides
	Sulfamethoxazole 4-Amino-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide	0.61 a	6 b	0.89 b	607 c	dnf
	Quinolones

Table 4

 4 Concentrations of the four most often found antibiotics in the surface soil samples or in the soil profiles and corresponding contents in total organic carbon in the samples.

	Depth	Soil	Sulfamethoxazole	Oxolinic	Nalidixic	Flumequine	TOC
	(cm)	horizon	(μg kg -1 )	acid	acid	(μg kg -1 )	(g kg -1 )
				(μg kg -1 )	(μg kg -1 )
	P1 15	Ap	b ld	b1.2*	3.5	b2.2*	nd
	P2 15	Ap	b ld	2.2	9.0	2.7	16.4
	P3 15	Ap	b ld	4.8	20.8	6.9	38.9
	20	Ap	b ld	4.8	18.4	6.7	26.9
	40	E	b ld	bld	3.8	bld	1.5
	60	Bt	b ld	bld	1.1	bld	1.3
	75	Bt	2.5	bld	b1*	bld	2.8
	90	Bt	b ld	bld	b1*	bld	0.7
	100	BC	b ld	bld	b1*	bld	1.6
	115	BC	b ld	bld	bld	bld	2.7
	130	Ck	b ld	bld	bld	bld	0.9
	P4 15	Ap	b ld	b1.2*	8.0	2.2	17.1
	P5 15	Ap	b ld	1.2	6.2	b2.2*	14.5
	P6 15	Ap	b ld	5.9	22.1	6.3	nd
	P7 15	Ap	b ld	1.3	7.7	3.0	13.0
	P8 15	Ap	b ld	1.2	8.1	2.9	15.9
	P9 20	Ap	b ld	bld	bld	bld	12.8
	35	E	b ld	b1.2*	4.7	b2.2*	1.2
	50	Bt	b ld	bld	bld	bld	2.1
	70	Bt	b ld	bld	bld	bld	1.3
	100	Bt	b ld	bld	bld	bld	1.8
	120	BC	b 2*	bld	bld	bld	2.0
	150	Ck	b ld	bld	bld	bld	1.4
	(b ld) lower than the detection limit; (*) detected, but under the quantification limit;
	(nd) not determined.

F. Tamtam et al. / Science of the Total Environment 409 (2011) 540-547

Acknowledgments

The Ile-de-France region is gratefully acknowledged for a doctoral grant for F. T. as well as PIREN-Seine program for financial support. The authors thank Martine Blanchard for helpful discussions.


domain: sdv sde

text:
M Fattet

Y Fu

M Ghestem

W Ma

M Foulonneau

J Nespoulous

Y Le Bissonnais

A Stokes

Effects of vegetation type on soil resistance to erosion: Relationship between aggregate stability and shear strength

Keywords:

Soil water erosion and shallow landslides depend on aggregate stability and soil shear strength. We investigated the effect of vegetation on both soil aggregate stability and shear strength (through direct shear tests) in former croplands converted to vegetated erosion protection areas within the context of China's sloping land conversion programme. Four treatments were analysed in plots comprised of (i) 4 year old crop trees, Vernicia fordii, where understory vegetation was removed; (ii) V. fordii and the dominant understory species Artemisia codonocephala; (iii) only A. codonocephala and (iv) no vegetation. Soil samples were taken at depths of 0-5 cm and 45-50 cm. Root length density (RLD) in five diameter classes was measured, soil organic carbon (SOC), hot water extractable carbon (HWEC), texture and Fe and Al oxides were also measured. We found that mean weight diameter after slow wetting (MWD SW ) in the A-horizon, was significantly greater (0.94-1.01 mm) when A. codonocephala was present compared to plots without A. codonocephala (0.57-0.59 mm). SOC and RLD in the smallest diameter class (b0.5 mm), were the variables which best explained variability in MWD SW . A significant positive linear relationship existed between MWD SW and soil cohesion but not with internal angle of friction. As herbaceous vegetation was more efficient than trees in improving aggregate stability, this result suggests that the mechanisms involved include modifications of the cohesive forces between soil particles adjacent to plant roots and located in the enriched in SOC rhizosphere, thus also affecting shear strength of the corresponding soil volume. Thus, vegetation stabilised soil under different hierarchical levels of aggregate organisation, i.e. intra-and inter-aggregate. Our results have implications for the efficacy of techniques used in land conversion programs dedicated to control of soil erosion and shallow landslides. We suggest that mixtures of different plant functional types would improve soil conservation on slopes, by reducing both surface water erosion and shallow substrate mass movement. Planting trees for cropping or logging, and removing understory vegetation is most likely detrimental to soil conservation.

Introduction

Soil degradation on steep slopes throughout the world has accelerated hugely over the last 50 years. In particular, countries such as China have experienced a rapid increase in population density, as well as development and industry. Soil degradation has largely been attributed to poor farming practices, deforestation, road and dam construction [START_REF] Liang | Soil erosion assessment in the red soil region of Southeast China using an integrated index[END_REF][START_REF] Stokes | Soil bio-and eco-engineering in China, past experience and present priorities[END_REF][START_REF] Yu | Relationship between permeability and erodibility of cultivated Acrisols and Cambisols in subtropical China[END_REF][START_REF] Zhang | Effects of vegetation restoration on soil and water erosion and nutrient losses of a severely eroded clayey Plinthudult in southern China[END_REF]. Rehabilitation programs for the reduction of soil erosion can encourage the conversion of cropland to forest and orchards [START_REF] Cao | Impact of China's Grain for Green Project on the landscape of vulnerable arid and semi-arid agricultural regions: a case study in northern Shaanxi Province[END_REF][START_REF] Trac | Reforestation programs in Southwest China: reported success, observed failure, and the reasons why[END_REF]. However, one of the assumptions of such conversion programs is that by simply planting trees, erosion processes will be arrested [START_REF] Trac | Reforestation programs in Southwest China: reported success, observed failure, and the reasons why[END_REF][START_REF] Wang | Acceleration of vegetation succession on eroded land by reforestation in a subtropical zone[END_REF][START_REF] Weyerhaeuser | Local impacts and responses to regional forest conservation and rehabilitation programs in China's northwest Yunnan province[END_REF]. Thus, trees have been planted for cropping e.g. Vernicia fordii or logging purposes, e.g. Eucalyptus sp. and Catena 87 (2011) [60][61][62][63][64][65][66][67][68][69] Abbreviations: γ, dried unit weight; Φ, internal friction; Al DCB , Aluminum oxides obtained by dithionite-citrate-bicarbonate extraction; c, cohesion; Fe DCB , Iron oxides obtained by dithionite-citrate-bicarbonate extraction; HWEC, Hot water extractable carbon; MWD, Mean weight diameter; MWD FW , Mean weight diameter after fast wetting; MWD SW , Mean weight diameter after slow wetting; MWD MB , Mean weight diameter after mechanical breakdown; RLD, total root length density; RLD b 0.5 , Root length density for diameter class equivalent or b0.5 mm; RLD 0.6-1.0 , Root length density for diameter class 0.6-1.0 mm; RLD 1.1-2.0 , Root length density for diameter class 1.1-2.0 mm; RLD N 2.1 , Root length density for diameter class equivalent to or N2.1 mm; rRL b 0.5 , relative length density for diameter class b 0.5 mm; rRL 0.6-1.0 , relative root length for diameter class 0.6-1.0 mm; rRL 1.1-2.0 , relative root length for diameter class 1.1-2.0 mm; rRL N 2.1 , relative root length for diameter class equivalent to or N2.1 mm; SLCP, Sloping land conversion program; SOC, Soil organic carbon.

⁎ Corresponding author. E-mail address: alexia.stokes@cirad.fr (A. Stokes).

Cryptomeria japonica, but may not be the best solution for soil conservation on steep terrain. Understory vegetation is often removed mechanically or cannot grow in shade conditions, resulting in increased runoff and interrill erosion [START_REF] Stokes | Soil bio-and eco-engineering in China, past experience and present priorities[END_REF]. Root biomass density is usually lower than in natural forest at an equivalent age, augmenting the risk of shallow landslides, particularly during the monsoon season [START_REF] Genet | Root reinforcement in plantations of Cryptomeria japonica D. Don: effect of tree age and stand structure on slope stability[END_REF]2010). During heavy precipitation events, splash impact and slaking causes soil aggregates to break down and produce finer, more transportable particles and micro-aggregates. These particles and micro-aggregates (20 to 250 μm diameter) affect the processes of infiltration, seal and crust development, runoff and interrill erosion [START_REF] Legout | Aggregate breakdown dynamics under rainfall compared with aggregate stability measurement[END_REF][START_REF] Martinez-Mena | An evaluation of a fragmentation fractal dimension technique to determine soil erodibility[END_REF]. Soil aggregate stability is therefore an important property that may explain, quantify and predict these processes [START_REF] Zhang | Mechanisms of aggregate stabilization in Ultisols from subtropical China[END_REF]. Aggregate stability in the topsoil layer (0-5 cm) has also been closely correlated to soil susceptibility to runoff [START_REF] Barthès | Aggregate stability as an indicator of soil susceptibility to runoff and erosion; validation at several levels[END_REF] and to soil erodibility (Le [START_REF] Bissonnais | Erodibility of Mediterranean vineyard soils: relevant aggregate stability methods and significant soil variables[END_REF], i.e. soil susceptibility to detachment and transport by erosion processes.

However, even though a superior aggregate stability may reduce runoff and erosion risk, infiltration may in turn increase, thus augmenting the risk of shallow landslides on steep slopes during heavy precipitation events (Ghestem et al., in press). The only way to resolve this apparent conflict is to ensure that aggregate stability and cohesion are improved simultaneously. A small number of studies over the past 30 years have suggested a link between shallow landslides and erosion processes, although quantitative data relating the two are limited [START_REF] Al-Durrah | New methods of studying soil detachment due to waterdrop impact[END_REF][START_REF] Frei | Quantification of the influence of vegetation on soil stability[END_REF][START_REF] Ghidey | Plant root effects on soil erodibility, splash detachment, soil strength, and aggregate stability[END_REF][START_REF] Nearing | Soil strength indexes as indicators of consolidation[END_REF][START_REF] Watson | Soil strength, slope and rainfall effects of interrill erosion[END_REF]. A major factor governing substrate mass movement is the shear strength of soil [START_REF] Terzaghi | Theoretical soil mechanics[END_REF]. Shear strength within a soil matrix is the result of resistance to movement at interparticle contacts, due to particle interlocking, physical bonds formed across the contact areas (resulting from surface atoms sharing electrons at interparticle contacts), and chemical bonds, or cementation [START_REF] Craig | Craig's Soil Mechanics[END_REF]. The two major active components in resistance of soil shear stress are i) the cohesion c, which is the summation of the effect of particle interlocking and cementation and ii) the internal angle of friction Φ. Φ is the angle measured between the normal force and resultant force that is attained when failure occurs in response to a shearing stress.

One of the earliest studies which investigated the relationship between erosion and soil shear strength was in 1981, when Al-Durrah and Bradford developed a force-resistance model for waterdrop impact detachment based on soil shear strength measured with a fallcone. Later, [START_REF] Watson | Soil strength, slope and rainfall effects of interrill erosion[END_REF] related interrill erosion losses to shear strength measured with a torvane on three soils, each with varying slope angle. [START_REF] Nearing | Soil strength indexes as indicators of consolidation[END_REF], used aggregate mean weight diameter (MWD) after disaggregating in water as an indicator of the aggregate stability and shear strength (using a fall-cone and torvane) on three soils contrasting in their texture (clay, silt loam and fine sand). These authors did not find a direct relationship between both parameters, but concluded that water stress influenced similarly the MWD and the bulk soil strength. More recently, [START_REF] Ghidey | Plant root effects on soil erodibility, splash detachment, soil strength, and aggregate stability[END_REF] studied the effect of plant roots on soil erodibility, shear strength and aggregate stability using a rainfall simulator. [START_REF] Ghidey | Plant root effects on soil erodibility, splash detachment, soil strength, and aggregate stability[END_REF] found that as the amount of dead root mass and live root length increased, aggregate stability and shear strength augmented and interrill erodibility decreased. [START_REF] Frei | Quantification of the influence of vegetation on soil stability[END_REF] also found that soil aggregate stability was correlated with soil shear strength from triaxial tests, although the sample number was limited. If a direct link between aggregate stability and soil shear strength could be determined, it would allow a better understanding of the mechanisms involved and thus how of rehabilitation measures influence soils undergoing degradation processes.

The urgent necessity to conserve soil and rehabilitate fragile land on steep slopes worldwide, has led to a number of recent studies investigating the effect of vegetation on aggregate stability and shallow landslides. With regard to the latter, the presence of plant roots physically reinforces the shear zone (see [START_REF] Stokes | Desirable plant root traits for protecting natural and engineered slopes against landslides[END_REF]. However, vegetation can also influence soil structure because of the contribution of organic matter through litter and plant root turnover [START_REF] Angers | Plant-induced changes in soil structure: processes and feedbacks[END_REF], root exudates and rhizodeposition [START_REF] Gyssels | Impact of plant roots on the resistance of soils to erosion by water: a review[END_REF][START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF]. The effect of roots on erodibility decreases with increasing root diameter [START_REF] De Baets | Root characteristics of representative Mediterranean plant species and their erosion-reducing potential during concentrated runoff[END_REF]. This effect may be due to the higher turnover (rate of root growth and decay, [START_REF] Gale | Root-derived carbon and the formation and stabilization of aggregates[END_REF] of fine roots (diameter b2 mm) compared to coarse roots (N2 mm), as well as the association of fine roots with fungal hyphae [START_REF] Jastrow | Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie[END_REF], and the physically binding dense fine root network. The soil organic carbon (SOC) input in the soil by roots corresponds to temporary binding agents which bind microaggregates into macro-aggregates (N250 μm up to several mm diameter) [START_REF] Gale | Root-derived carbon and the formation and stabilization of aggregates[END_REF][START_REF] Wander | Influence of tillage on the dynamics of loose-and occluded-particulate and humified organic matter fractions[END_REF]. Labile fraction of SOC may be assessed by hot water extractable carbon (HWEC). [START_REF] Sparling | Hot-water-soluble C as a simple measure of labile soil organic matter: the relationship with microbial biomass C[END_REF] found that HWEC after air drying was about 43% of the microbial carbon (i.e. polysaccharides) and strongly related to microaggregation [START_REF] Boix-Fayos | Influence of soil properties on the aggregation of some Mediterranean soils and the use of aggregate size and stability as land degradation indicators[END_REF][START_REF] Ghani | Hot-water extractable carbon in soils: a sensitive measurement for determining impacts of fertilisation, grazing and cultivation[END_REF][START_REF] Haynes | Changes in microbial biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under field conditions[END_REF][START_REF] He | Using light fraction and macro-aggregate associated organic matters as early indicators for managementinduced changes in soil chemical and biological properties in adjacent native and plantation forests of subtropical Australia[END_REF][START_REF] Kouakoua | La matière organique soluble à l'eau chaude et la stabilité de l'agrégation. Aspects méthodologiques et application à des sols ferrallitiques du Congo[END_REF][START_REF] Lutzow | Indications for soil organic matter quality in soils under different management[END_REF]. In addition, Fe and Al oxides may interact with organic matter in macro-aggregate stability through their flocculation capacity, the binding effect of clay particles to organic molecules [START_REF] Zhang | Mechanisms of aggregate stabilization in Ultisols from subtropical China[END_REF], and a possible precipitation as gels on clay surfaces [START_REF] Amezketa | Soil aggregate stability: a review[END_REF].

The aim of our study was to analyze how soil physical properties were modified under a tree crop, Vernicia fordii, where understory vegetation had been removed, compared to a site where the dominant understory vegetation species, Artemesia codonocephala, was still present. We also aimed at determining a relationship between soil aggregate stability and shear strength with regard to vegetation type.

Materials and methods

Study site

We studied aggregate stability and soil shear strength in the Yunnan province, Southern China, where erosion and landslides are severe [START_REF] Yang | Impacts of soils moisture content and vegetation on shear strength of unsaturated soil[END_REF][START_REF] Zhang | Sustainable land use in the hilly red soil region of South-eastern China[END_REF][START_REF] Zhou | A case study of effect of lateral roots of Pinus yunnanensis on shallow soil reinforcement[END_REF].

The study area (26°01′N, 98°50′E) was located near Da Xing Di village, north of Liuku town in the Salween river valley. This part of China is under the influence of the Indian monsoon, and described as a "warm-dry climate", being is a combination of subtropical and alpine climates. Annual mean temperature (from 1961 to 2002) is 15.2 °C, and mean annual precipitation is 1200 mm, the majority of which falls between May and October [START_REF] Ghestem | Using vegetation to stabilize steep slopes in Southern China: root biomechanics as a factor in the choice of species[END_REF]. Numerous landslides occur during the monsoon season (May-October) and soil erosion is severe, largely due to the cutting of roads through the steep slopes [START_REF] Stokes | Soil bio-and eco-engineering in China, past experience and present priorities[END_REF].

At our study site, corn (Zea mays) was cultivated from 1980, after deforestation, until 1999 when a conversion program was initiated within the framework of the Sloping Land Conversion Program (SLCP). The SLCP, or Grain for Green, aims at planting trees on existing agricultural land, concentrating on zones where slopes are N25° [START_REF] Cao | Impact of China's Grain for Green Project on the landscape of vulnerable arid and semi-arid agricultural regions: a case study in northern Shaanxi Province[END_REF][START_REF] Trac | Reforestation programs in Southwest China: reported success, observed failure, and the reasons why[END_REF]. Several species of trees and shrubs were planted at the site, including Pueraria stricta, Ricinus communis, Agava americana, Jatropha curcas, and Vernicia fordii. We focused on one area where the species V. fordii had been established. The oil from its nuts is used for agrofuel production. To avoid competition from the neighbouring biennial pioneer plant, Artemisia codonocephala, farmers often remove this latter species through scything.

At our site, two soil horizons exist: an A-horizon in the top 0-15 cm, characterized by a silty clay loam texture (17% sand, 50% silt, 33% clay) and a colour of 7.5 Yr 3/4 (dark yellowish brown); a B-horizon (15-60 cm) dominated by clay (13% sand, 39% silt, 48% clay) and with a colour of 5 Yr 4/6 (yellowish red). Source rock emergence occurred at about 60 cm Author's personal copy depth. Throughout the whole profile, soil was alkaline (pH=8.42), with a high capacity of exchangeable cations (20 cmol kg -1 ) and a high carbonate content (15 cmol of Ca 2+ kg -1 ). Slope angle ranged from 20 to 35°and the site was situated at an altitude of 1000 m.

Sampling procedure

We selected four plots (5×10 m) containing: (i) 4 year old V. fordii trees (mean± standard error diameter at breast height, DBH=18.66± 2.91 cm) in a small plantation with total plant cover of 5%, where A. codonocephala was removed through scything on an annual basis; (ii) a combination of V. fordii (20% of total plant cover) and A. codonocephala (80% of total plant cover), with total plant cover of 60%; (iii) only A. codonocephala, with total plant cover of 50% and (iv) a site without vegetation, with plant coverb 5%. In the presence of V. fordii, soil samples were collected at 30 cm from the tree base. Six blocks were established within each plot. Each block was circular and was 0.5 m in diameter. Sampling took place at the end of the dry season, from 27 February to 2 March 2010.

For both aggregate stability and direct shear tests, samples were taken from representative typical A-and at B-horizons i.e. at 0-5 cm and at 45-50 cm. Litter, rock fragments and surface crust, when present, were removed prior to sampling. For aggregate stability tests, each sample was 360 cm 3 and obtained using a cylinder corer (n=2 samples from each block, hence n=48 in total). For direct shear tests, samples of soil were removed using a small spade. Each sample was approximately 800 cm 3 and was taken from within three blocks for the plot without vegetation, and from two blocks for plots with A. codonocephala, V. fordii×A. codonocephala, and V. fordii (n=18). Samples were kept cool in freezer boxes until they were transported to the laboratory.

Biotic properties

Once in the laboratory, all roots from soil samples used for aggregate stability tests were removed. To better visualize the roots during image analysis, staining was carried out using an aqueous concentration of 0.1% (w/v) methylene blue, with a contact time of 5 min [START_REF] Costa | Root contrast enhancement for measurement with optical scanner-based image analysis[END_REF]. Stained roots were then scanned with a resolution of 600 dpi and analyzed using WinRHIZO (Pro version 3.0, Regent Instruments, Canada) [START_REF] Zobel | Hardware and software efficacy in assessment of fine root diameter distributions[END_REF]. The total root length and the root length per class of diameter were obtained (b0.5 mm; 0.6 mm-1.0 mm; 1.1 mm-2.0 mm; N2.1 mm). The total root length density (RLD, mm cm -3 ) was calculated because of its relevance for evaluating the effects of roots on erosion processes [START_REF] De Baets | Effects of grass roots on the erodibility of topsoils during concentrated flow[END_REF][START_REF] Gyssels | Impact of plant roots on the resistance of soils to erosion by water: a review[END_REF]. The RLD is the total length of the roots divided by the volume of the root-permeated soil sample. The root length density per class of diameter (denoted RLD b 0.5 , RLD 0.6-1.0 , RLD 1.1-2.0 and RLD N 2.1 , mm cm -3 ) were also calculated. The relative root length for each class of diameter (denoted rRL b 0.5 , rRL 0.6-1.0 , rRL 1.1-2.0 and rRL N 2.1 , mm cm -3 ) were calculated by dividing each root length per class of diameter by the total root length of roots present in the soil sample.

Soil organic carbon (SOC) was obtained by wet digestion (g kg -1 ) (LY/T 1237-1999 in the Forestry industry standard of the People's Republic of China, 1999). Hot water extractable carbon (HWEC, g/kg) was obtained using a modified method from [START_REF] Haynes | Changes in microbial biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under field conditions[END_REF]. 6 g of oven dry soil was placed into 100 ml polypropylene centrifuge tubes and 60 ml of distilled water added. The tubes were left for extraction in a hot-water bath at 80 °C for 16 h. At the end of the extraction period, each tube was shaken for 10 s on a vortex shaker to ensure that HWEC released from the SOC was fully suspended in the extraction medium. Samples were then centrifuged for 20 min at 3500 rpm and filtered through Tokyo 5 C paper. Soluble carbon was measured using a Shimadzu TOC-5000 analyser. All analyses were replicated twice.

Abiotic properties

Soil analyses were carried out on soil fractions finer than 2 mm. Sand (2.00-0.05 mm), silt (0.050-0.002 mm), and clay (b0.002 mm) content were determined using the pipette method (%) [START_REF] Kalra | Methods manual for forest soil and plant analysis[END_REF]. All analyses were replicated twice. Dithionite-citratebicarbonate extractable Fe and Al were measured as described by [START_REF] Mehra | Iron oxide removal from soils and clays by a dithionitecitrate system buffered with sodium bicarbonate[END_REF] (respectively Fe DCB and Al DCB , g kg -1 ).

Soil aggregate stability tests

In the laboratory, soil samples were placed in a temperate wellventilated atmosphere to be air-dried for 1 week. Soil aggregate stability was determined after oven drying at 40 °C, according to the standardized method NF X 31-515 developed by Le [START_REF] Bissonnais | Aggregate stability and assessment of soil crustability and erodibility: I. Theory and methodology[END_REF]. This method combines three disruptive tests, performed on the aggregates of 3-5 mm diameter: (i) fast wetting in water; (ii) slow wetting in water; and (iii) mechanical breakdown. For the fast wetting test, about 5 g of calibrated aggregates was rapidly immersed in 50 ml of deionized water for 10 min. For the slow wetting test, aggregates were capillary rewetted with water on a tension table at a potential of -0.3 kPa for 60 min. For the mechanical breakdown test, similar amounts of aggregates were gently immersed in ethanol. After 30 min, ethanol was eliminated and aggregates were hand agitated in 200 ml of deionized water 20 times in a fast end-over-end movement. The solution was adjusted to 250 ml and was left for 30 min for sedimentation, after which the water was eliminated. After each test, the residual aggregates were collected and transferred onto a 50 μm sieve previously immersed in ethanol, which was gently hand moved in a helical movement, five times with the same amplitude (4 cm) and the same frequency (1 s per cycle) each time. The remaining aggregates on the sieve were collected, and dried at 40 °C for 48 h, and gently dry sieved using a column of six sieves: 2.00, 1.00, 0.50, 0.20, 0.10, and 0.05 mm. Each treatment was replicated three times. Aggregate stability for each sample was expressed as the mean weight diameter (MWD, mm) for each treatment (MWD FW , MWD SW , and MWD MB , for fast wetting, slow wetting, and mechanical breakdown, respectively):

MWD = ∑d × m 100
where d is the mean diameter between the two sieves (mm); and m the weight fraction of aggregates remaining on the sieve (%).

Direct shear tests

Strain-controlled direct shear tests were carried out on reconstituted, drained 60 mm × 60 mm × 20 mm soil samples with roots removed during the reconstitution process. Samples were not saturated prior to testing, and as they were kept sealed at 4 °C after removal from the field, it can be assumed that soil moisture content was similar to that in field conditions. Samples were placed in a shear testing device (VJTech 2760A, U.K.) and normal loads of 200, 300 and 500 N were applied as weights on three separate samples taken from the same block of soil [START_REF] Schuppener | Laboratory methods for direct shear tests[END_REF]. A lateral displacement was applied at a speed of 0.8 mm min -1 until failure occurred and the peak shear force recorded. The cohesion (c) and the angle of internal friction (Φ) were obtained by the Mohr-Coulomb theory [START_REF] Schuppener | Laboratory methods for direct shear tests[END_REF].

To obtain soil moisture content, sub-samples were weighed and dried at 60 °C for 4 days and then 105 °C for 24 h, or until constant weight. Soil moisture content was expressed as gram of water per gram of dry soil. Density was measured on each samples tested, using a modified method from [START_REF] Baize | Guide des analyses en pédologie[END_REF] Sample weight was obtained by dividing the volume of water occupied by the sample in a graduated Author's personal copy cylinder. To increase accuracy, the weight of water in the sample was removed from the total sample weight, by calculating the dry unit weight:

γ = P= V ð Þ= 1-w ð Þ
where γ is the dried unit weight (kN•m -3 ); P the total sample weight (kN); V the sample volume (m 3 ); and w the soil moisture content (g of water g -1 of soil).

Statistical analysis

Variables were transformed prior to analysis to meet the assumptions required in an analysis of variance (ANOVA), using an arcsine square root transformation for percentage and a log transformation for other values (MWD FW , MWD MB , MWD SW , HWEC, RLD, RLD b 0.5 , RLD 0.6-1.0 , RLD 1.1-2.0 , RLD N 2.1 ). The influence of sampling site and horizon on soil properties and aggregate stability was investigated using an analysis of variance (ANOVA). Post-hoc Fisher LSD tests (pb 0.05) were performed to compare results. The relationship between all variables was investigated using a Pearson's correlation matrix. Linear regressions were carried out to determine the relationships between MWD SW and c and Φ in both soil horizons. Statistical analysis was performed using STATISTICA 7.1 (Statsoft Inc, Tulsa, USA).

Results

Biotic and abiotic properties

The mean RLD, RLD b 0.5 , RLD 0.6-1.0 and RLD 1.1-2.0 were significantly higher in the A-horizon compared to the B-horizon (F 1,40 =39.84, pb 0.001 for RLD, Fig. 1a, F 1,40 =36.11, pb 0.001 for RLD b 0.5 Fig. 1b, F 1,40 =20.09, pb 0.001 for RLD 0.6-1.0 , and F 1,40 =4.96, pb 0.05 for RLD 1.1- 2.0 , Table 1). In the A-horizon, RLD was significantly higher at plots with A. codonocephala (8.97±1.31 mm cm -3 ) and V. fordii×A. codonocephala (9.32±1.72 mm cm -3 ) compared to plots with V. fordii (4.45±1.17 mm cm -3 ) and soil without vegetation (2.76 ±0.78 mm cm -3 ). In the B horizon, the RLD was significantly lower at plots with A. codonocephala only (1.43± 0.20 mm cm -3 ).

Differences between plots were significantly greater with regard to rRL, compared to RLD (Table 1). In the A-horizon, rRL b 0.5 was significantly higher at plots with A. codonocephala (0.75± 0.03 mm mm -1 ) and V. fordii×A. codonocephala (0.63± 0.06 mm mm -1 ) compared to plots with V. fordii (0.33± 0.05 mm cm -3 ) and soil without vegetation (0.29 ± 0.05 mm mm -1 ). The rRL 0.6-1.0 was significantly lower at plots with A. codonocephala (0.21±0.02 mm mm -1 ) and V. fordii×A. codonocephala (0.31± 0.04 mm mm -1 ) compared to plots with V. fordii (0.56 ± 0.04 mm mm -1 ) and on soil without vegetation (0.54 ±0.04 mm mm -1 ). A similar pattern was also found with regard to rRL 1.1-2.0 , which was significantly lower at plots with A. codonocephala (0.05± Table 1. 0.01 mm mm -1 ) and V. fordii × A. codonocephala (0.06± 0.02 mm mm -1 ) compared to plots with V. fordii (0.10±0.02 mm mm -1 ) and soil without vegetation (0.16 ±0.03 mm mm -1 ). The rRL N 2.1 was lower than 0.05 mm mm -1 for all plots with no significant differences between plots (Table 2).

The SOC varied from 12.31±0.99 g kg -1 to 20.00±0.82 g kg -1 (Fig. 1c), corresponding respectively to the B-and A-horizons beneath A. codonocephala. In soil from all plots, mean SOC was significantly greater in the A-horizon compared to the B-horizon (F 1,40 =52.91, pb 0.001). Significant differences were found among different types of vegetation in soil from the A-horizon (F 3,40 =2.84, p b 0.05). Mean SOC was significantly greater beneath A. codonocephala (20.00±0.82 g kg -1 ) and V. fordii×A. codonocephala (19.34±0.43 g kg -1 ) compared to V. fordii (17.80±0.18 g kg -1 ) and soil without vegetation (16.42 ±0.55 g kg -1 ). In soil from the B-horizon, only the site with A. codonocephala had a significantly lower SOC value (12.31 ± 0.99 g kg -1 ). Mean HWEC represented 1.59-4.55% of the SOC, the significantly lowest value corresponding to that of soil without vegetation. Mean HWEC was significantly higher in the A horizon compared to the B horizon (F 1,40 =4.27, pb 0.05, Table 1). In the A-horizon only, mean HWEC was significantly greater (F 3,40 =6.60, pb 0.001) beneath A. codonocephala (0.67±0.08 g kg -1 ), compared to V. fordii×A. codonocephala (0.50± 0.04 g kg -1 ), V. fordii (0.44±0.03 g kg -1 ) and soil without vegetation (0.40±0.02 g kg -1 ).

The quantity of Fe DCB oxides ranged from 18.20 ± 0.64 g kg -1 to 24.42±0.71 g kg -1 in both horizons (Table 2). The quantity of Al DCB oxides ranged from 2.48 ± 0.11 g kg -1 to 3.66±0.21 g kg -1 in both horizons (Table 2). The low variability of these two properties did not permit us to study relationships with other variables. The range of values of biotic properties was high in the A-horizon but very low in the Bhorizon. Thus, the biotic properties reflected characteristics of the different vegetation only in the A-horizon. Therefore, an analysis of relationships between biotic and abiotic properties and aggregate stability was only possible for data from the A-horizon. In the A-horizon, SOC was found to increase significantly and linearly with an increase in mean RLD, and RLD b 0.5 . Mean HWEC also increased linearly and positively with RLD and RLD b 0.5 but with a low R 2 (Table 3).

Soil aggregate stability

Soil aggregate stability was generally low. The MWD FW ranged from 0.37 to 0.56 mm, MWD SW from 0.57 to 1.01 mm and MWD MB from 0.45 to 0.67 mm. Only the two tests of slow wetting (MWD SW ) and mechanical breakdown (MWD MB ), resulted in significant differences between plots (F 3,40 = 4.69, p b 0.05 and F 3,40 = 4.15, p b 0.05, respectively). The highest MWD SW in the A-horizon was obtained for V. fordii × A. codonocephala, followed by A. codonocephala, V. fordii, and soil without vegetation (Fig. 2a). With regard to MWD MB in the Ahorizon, the plots with A. codonocephala possessed the highest values, followed by V. fordii × A. codonocephala, V. fordii, and the soil without vegetation (Fig. 2b). Between the three tests, MWD SW was the test which best discriminated between plots. Thus, MWD SW was used as the representative parameter, for the ensuing analysis of results.

Influence of biotic and abiotic properties on soil aggregate stability

As RLD is an absolute value, compared to rRL, it was more appropriate for investigating the influence of root length on MWD SW . In the A-horizon, MWD SW was found to increase significantly and linearly with an increase in mean RLD, RLD b 0.5 , SOC, and HWEC (Fig. 3a, b, c, andd, respectively for MWD SW ). No relationships were found between any MWD value for the three treatments and abiotic properties in the A-horizon (Table 3).

Shear strength characteristics and relationship with aggregate stability

Mean c was 15.84± 2.33 kPa in the A-horizon and 16.67± 0.55 kPa in the B-horizon. Mean Φ was 14.64±3.86°in the A-horizon and 7± 0.55°in the B-horizon. No significant differences in c or Φ were found between horizons. With regard to results from both horizons combined, no significant relationships were found between γ, c and Φ, and no significant Table 1 Vegetation type, soil depth (A and B horizons) and their interaction, influenced aggregate stability, biotic and abiotic variables. F and P values were obtained by factorial ANOVA, (n = 48). MWD FW : mean weight diameter after fast wetting; MWD MB : mean weight diameter after mechanical breakdown; MWD SW : mean weight diameter after slow wetting; RLD: root length density. Values following RLD indicate the roots class of diameter, i.e., b0.5: roots equivalent to or finer than 0.5 mm, 0.6-1: roots with diameter from 0.6 to 1 mm, 1.1-2: roots with diameter from 1.1 to 2 mm, N2.1: roots equivalent to or larger than 2.1 mm. SOC: soil organic carbon; HWEC: Author's personal copy relationships were found between biotic or abiotic properties and c or Φ. However, a significant and positive relationship was found between c and MWD SW (Fig. 4), using results from both horizons combined together and with a soil moisture content of 24.03 ±0.80%. This positive relationship was also found to be significant for samples from the A-horizon (c=25.73×MWD SW -0.12, R 2 =0.61, pb 0.05) and in the B-horizon (c=10.97×MWD SW +9.58, R 2 =0.48, pb 0.05).

Discussion

Variation in aggregate stability

Soil aggregate stability in the A-horizon differed significantly depending on the type of vegetation present. Variations in aggregate stability were negligible in the B-horizon, as also found by [START_REF] Eviner | The influence of plant species, fertilization and elevated CO 2 on soil aggregate stability[END_REF]. The MWD FW failed to discriminate between the different plots, because aggregate stability was generally too low to resist the high breakdown energy from this test. Inversely, the MWD SW was the best variable to discriminate between plots because it had the lowest breakdown energy. Mean MWD SW was significantly greater in plots of A. codonocephala and V. fordii×A. codonocephala compared to plots without A. codonocephala. Mean MWD SW in the A-horizon was greater than 0.8 mm in the two plots with A. codonocephala and smaller than 0.8 mm for the two plots without. Therefore, less interrill erosion should occur on plots with A. codonocephala during rain events (Le [START_REF] Bissonnais | Aggregate stability and assessment of soil crustability and erodibility: I. Theory and methodology[END_REF].

Influence of vegetation on aggregate stability

The variable best explaining soil aggregate stability in the A-horizon was RLD of the finest roots i.e. in the diameter class b0.5 mm. Similar results were found by [START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF], although these authors could Table 2 Mean value of all measured variables for the different vegetation types at different horizons and results of the post-hoc Fisher LSD tests (P b 0.05). Mean value ± standard error. Values with the same letters represent homogeneous groups under the significant level of p = 0.5.

Parameters

Species and horizon of sampling , 1997). Therefore, even when RLD is small, as in our study, it can still be a useful predictor of aggregate stability. Plant roots can promote soil aggregation by releasing material which can directly stabilize soil particles, enmesh soil particles by hyphae and roots, or can favour microbial activity in the rhizosphere which in turn will affect soil structure [START_REF] Bearden | Influence of arbuscular mycorrhizal fungi on soil structure and aggregate stability of vertisols[END_REF][START_REF] Burri | Revegetation measures improve soil aggregate stability: a case study of a landslide area in Central Switzerland[END_REF]. Very fine roots are more significantly associated Author's personal copy with fungal hyphae [START_REF] Jastrow | Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie[END_REF] and microbial activity stabilises soil aggregates through the binding action of humic substances and other microbial by-products contributing to soil organic material [START_REF] Abid | Tillage and drainage impact on soil quality I. Aggregate stability, carbon and nitrogen pools[END_REF][START_REF] Chan | Oxidizible organic carbon fractions and soil quality changes in an Oxic Paleustalf under different pasture leys[END_REF][START_REF] Goh | Carbon sequestration and stabilization in soils: implications for soil productivity and climate change[END_REF][START_REF] Jastrow | Carbon dynamics of aggregate-associated organic matter estimated by carbon-13 natural abundance[END_REF][START_REF] Li | Soil physical properties and their relations to organic carbon pools as affected by land use in an alpine pastureland[END_REF][START_REF] Liu | A study of soil surface characteristics in a small watershed in the hilly, gullied area on the Chinese Loess Plateau[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF][START_REF] Tisdall | Organic matter and water-stable aggregates in soils[END_REF]. In our study, SOC correlated significantly with MWD SW in the A-horizon, although [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF] found lower correlations between SOC content and aggregate stability in tropical soils compared to temperate soils. SOC was a better indicator of variability in MWD SW compared to HWEC. This finding contrasts with other studies [START_REF] He | Using light fraction and macro-aggregate associated organic matters as early indicators for managementinduced changes in soil chemical and biological properties in adjacent native and plantation forests of subtropical Australia[END_REF]Kouakoua et al., 1997, Von Lutzow et al.;2000), where HWEC was better related to aggregate stability than SOC. [START_REF] Kouakoua | La matière organique soluble à l'eau chaude et la stabilité de l'agrégation. Aspects méthodologiques et application à des sols ferrallitiques du Congo[END_REF]) suggested that the quantity of HWEC highly depends on the extraction method i.e. temperature and extraction time. Both SOC and HWEC decreased with increasing soil depth, largely due to the relationship with total RLD and RLD b 0.5 which were significantly higher in the A-horizon compared to the Bhorizon. Organic matter from litter input and mixing may also have contributed to higher SOC and HWEC in the shallow soil horizons [START_REF] Cerdà | Soil aggregate stability under different Mediterranean vegetation types[END_REF]. Nevertheless, mean SOC in the B-horizon was still high (12-16 g kg -1 ), probably due to past agricultural practices at the field site. Total RLD explained 53% of variability in SOC in the A-horizon and very fine roots (diameter equivalent to or b0.5 mm) explained 60% variability. Roots contribute to SOC through turnover (rate of root growth and decay, [START_REF] Angers | Plant-induced changes in soil structure: processes and feedbacks[END_REF][START_REF] Gale | Root-derived carbon and the formation and stabilization of aggregates[END_REF] and the exudation of mucilage [START_REF] Gyssels | Impact of plant roots on the resistance of soils to erosion by water: a review[END_REF][START_REF] Pohl | Higher plant diversity enhances soil stability in disturbed alpine ecosystems[END_REF], which is usually greater in very fine roots [START_REF] Jastrow | Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie[END_REF]. As SOC values were high in our study, potential relationships between Fe DCB and Al DCB oxides and aggregate stability were not evident. [START_REF] Duiker | Iron (hydr)oxide crystallinity effects on soil aggregation[END_REF] and [START_REF] Igwe | Colloidal stability in some tropical soils of southeastern Nigeria as affected by iron and aluminium oxides[END_REF], found that Fe DCB and Al DCB oxides content was similar or higher compared to our results and considered these variables responsible for aggregate stability. However, SOC was twice as low as SOC from our study. Similarly, in a study of 18 plots from tropical or subtropical plots around the world, [START_REF] Barthès | Texture and sesquioxide effects on water-stable aggregates and organic matter in some tropical soils[END_REF] found values for SOC comparable to our results, but Fe DCB and Al DCB oxides quantities were much higher than in the soil we examined.

Relationship between aggregate stability and shear strength

The values for c and Φ lay within the range for clay soils [START_REF] Magnan | Résistance au cisaillement[END_REF], although values for Φ were lower than those (11-51°) found for non-zonal soil from a debris flow in the Yunnan province [START_REF] Yang | Impacts of soils moisture content and vegetation on shear strength of unsaturated soil[END_REF]. As shear strength within a soil matrix is the result of resistance to movement at interparticle contacts, physical bonds formed across the contact areas and chemical bonds [START_REF] Craig | Craig's Soil Mechanics[END_REF], it is to some extent surface dependent and any action that will hinder or promote the cohesive and frictional forces between adjacent particles will invariably affect shear strength [START_REF] Ayininuola | Influence of calcium sulphate on subsoil cohesion and angle of friction[END_REF]. We found a significant relationship between soil c and MWD SW , the latter often depending on SOC, root exudates and microbial by-products. Shear strength is thus probably linked to some of the same bonding mechanisms as those involved in aggregation. Therefore, the bonding mechanisms which strengthen aggregates may be similar to those which strengthen interaggregate structure [START_REF] Bryan | Soil erodibility and processes of water erosion on hillslope[END_REF]. Thus, vegetation stabilised soil under different hierarchical levels of aggregate organisation (i.e. intra-and inter-aggregate). A similar result was also found by [START_REF] Frei | Quantification of the influence of vegetation on soil stability[END_REF], using triaxial tests on a limited number of samples. These authors demonstrated a significant relationship between c and aggregate stability in moraine soils. Results were attributed to modifications occurring in the soil due to the interactions between roots and fungal hyphae, and presumably subsequent changes in soil chemistry.

Soil cohesion and angle of friction can be influenced temporarily by chemical additions to the soil e.g. calcium salt, particularly in clay soils which have a large surface area that aids cation and anion exchange among clay minerals [START_REF] Ayininuola | Influence of calcium sulphate on subsoil cohesion and angle of friction[END_REF]. Nevertheless, data are scanty and significantly more research could be carried out to understand how plant root exudates alter soil chemical bonds and interparticle contacts, thus effecting shear strength. Roots can also physically modify particle contacts in soil through either loosening effects [START_REF] Yoo | Influence of soil aggregation on SOC sequestration: a preliminary model of SOC protection by aggregate dynamics[END_REF] or by compressing soil through penetration, occurring as they push their way through soil [START_REF] Bengough | Root responses to soil physical conditions; growth dynamics from field to cell[END_REF]. However, as tests in our study were on consolidated soil, the physical effect of roots was probably removed. In conclusion, aggregate stability tests cannot replace triaxial or direct shear tests on soil, but may be used as adequate indicators of a soil's stability. Such tests are easy to carry out and do not require the sophisticated equipment necessary for shear testing. However, more tests are necessary on a wide range of soils in different testing conditions.

Our results suggest that soil protection against erosion processes would be improved if understory species e.g. A. codonocephala were not removed from beneath plantation trees. Although often believed to result in competition with crop trees, the presence of herbaceous and non-woody species in agroforest systems can actually improve tree growth compared to monospecific tree cultivation [START_REF] Moreno | Driving competitive and facilitative interactions in oak dehesas through management practices[END_REF]. Our results indicate that mixtures of different functional plant types would also improve soil conservation on slopes by reducing both surface water erosion and shallow substrate mass movement.

Conclusion

We investigated how soil aggregate stability and shear strength differed between sites where the tree crop Vernicia fordii had been planted with or without the dominant understory biennial Artemisia codoncephala. Aggregate stability in the A-horizon was improved in the presence of A. codonocephala and when this species was absent, aggregates were significantly unstable. Soil organic carbon and root length density (for roots equivalent to or b0.5 mm in diameter), were the variables best explaining variations in aggregate stability. A significant positive relationship was also found between aggregate stability and soil cohesion but not internal angle of friction. Results suggest that current techniques of land conversion with removal of understory vegetation around tree crops will increase interrill erosion and soil erodibility and may actually be detrimental for slope stability. As we observed that aggregate stability and shear strength were related, our results showed that there was no contradiction between the improving of aggregate stability, thus reducing surface runoff and erosion, and the decrease in the risk of shallow substrate mass movement by improving cohesion. We explained this result by the beneficial effect of roots on aggregate stability, through rhizosphere exudates and microbial by-products, shear strength is thus probably linked to some of the same bonding mechanisms as those involved in aggregation. Therefore, the bonding mechanisms which strengthen aggregates may be similar to those which strengthen interaggregate structure, and hence cohesion.

Fig. 1 .

 1 Fig. 1. Main biotic properties measured under the different vegetation types (a): root length density (RLD), (b): relative root length diameter b0.5 mm (rRL b 0.5 ), (c): soil organic carbon (SOC) (d): hot water extractable carbon (HWEC). Ac.: Artemesia codonocephala, Vf.: Vernicia fordii, Vf.xAc.: V. fordii × A. codonocephala. Values are means ± standard error (n = 6). Where letters above bars differ, data are significantly different (P b 0.05). F and associated P values are given inTable 1.

  Artemesia codonocephala (n = 6) Vernicia fordii (n = 6)V. fordii × A. codonocephala (n = 6) No vegetation (n

Fig. 2 .Fig. 3 .

 23 Fig. 2. Mean Weight Diameter (MWD) obtained by from aggregate stability tests (Le Bissonnais, 1996) under the different vegetation types (a) MWD after the treatment by slow wetting (MWD SW ), (b) MWD after the treatment by mechanical breakdown (MWD MB ). Values are means ± standard error (n = 6). Where letters above bars differ, data are significantly different (P b 0.05). F and associated P values are given inTable 1.

5Fig. 4 .

 4 Fig. 4. Relationship between the mean weight diameter after the slow wetting treatment (MWD SW ) and the cohesion (c) obtained by direct shear testing in both horizons R 2 is the coefficient of determination at the P probability level.

  Hot Water Extractable Carbon; Fe DCB and Al DCB : dithionitecitrate-bicarbonate extractable Fe and Al.

		Vegetation		Horizon		Vegetation × Horizon
		F 3,40	P	F 1,40	P	F 3,40	P
	Biotic properties
	RLD (mm cm -3 )	5.96	**	39.84	***	2.72	0.05
	RLD b 0.5 (mm cm -3 )	11.99	***	36.12	***	2.48	0.11
	RLD 0.6-1.0 (mm cm -3 )	1.13	0.35	20.09	***	1.04	0.38
	RLD 1.1-2.0 (mm cm -3 )	0.29	0.84	4.96	*	0.96	0.42
	RLD N 2.1 (mm cm -3 )	0.71	0.55	0.19	0.67	0.77	0.52
	rRL b 0.5 (mm mm -1 )	20.10	***	0.42	0.52	2.75	0.05
	rRL 0.6-1.0 (mm mm -1 )	13.74	***	0.33	0.57	1.75	0.17
	rRL 1.1-2.0 (mm mm -1 )	4.77	*	1.50	0.23	0.94	0.43
	rRL N 2.1 (mm mm -1 )	1.14	0.35	1.20	0.28	0.79	0.51
	SOC (g kg -1 )	2.84	*	52.91	***	2.26	0.12
	HWEC (g kg -1 )	6.45	**	4.54	*	2.77	0.05
	Abiotic properties
	Sand (%)	1.91	0.14	3.98	0.05	0.89	0.45
	Silt (%)	11.68	***	15.44	***	4.32	**
	Clay (%)	8.98	***	14.44	***	3.46	**
	Fe DCB (g kg -1 )	11.23	***	2.40	0.13	1,05	0.38
	Al DCB (g kg -1 )	9.42	***	3.09	0.09	2.48	0.08
	Aggregate stability
	MWD FW (mm)	1.93	0.14	2.29	0.14	0.81	0.50
	MWD SW (mm)	3.26	*	2.44	0.13	1.93	0.14
	MWD MB (mm)	3.86	*	3.67	0.06	2.09	0.12

* Significant at 0.05 level, ** significant at 0.01 level, and *** significant at 0.001 level.

Table 3

 3 Correlation coefficients (R 2 ) for the linear relationships between the parameters of aggregate stability and the biotic and abiotic properties (n = 24) MWD FW : mean weight diameter after fast wetting; MWD MB : mean weight diameter after mechanical breakdown; MWD SW : mean weight diameter after slow wetting; RLD: root length density. Values following RLD indicate the roots class of diameter, namely, b0.5: roots equivalent to or finer than 0.5 mm, 0.6-1: roots with diameter from 0.6 to 1 mm, 1.1-2: roots with diameter from 1.1 to 2 mm, N 2.1: roots equivalent to or larger than 2.1 mm. SOC: soil organic carbon; HWEC: Hot Water Extractable Carbon; Fe DCB and Al DCB : dithionite-citrate-bicarbonate extractable Fe and Al.Ns not significant, * significant at 0.05 level, ** significant at 0.01 level, and *** significant at 0.001 level.explain only 10% of the variability in soil aggregate stability when considering roots b0.5 mm, possibly due to the wet sieving method used as an aggregate stability test. Through measurements of RLD, De Baets et al. (2007) also demonstrated the role of roots in the topsoil resistance against interrill erosion. With regard to the different types of vegetation present at our site, total RLD was highest in the plots with A. codonocephala and V. fordii×A. codonocephala and over 50% of total RLD comprised very fine roots (diameter equivalent to or b0.5 mm in these plots). In the remaining plots with V. fordii and without vegetation, total RLD was 2-5 times lower and SOC was 5-20% lower in the A-horizon than on plots where A. codonocephala was present. Nevertheless, mean RLD was low compared to values from e.g. herbaceous species where mean RLD was approximately 400 mm cm -3[START_REF] De Baets | Effects of grass roots on the erodibility of topsoils during concentrated flow[END_REF] and crop species where mean RLD values ranged from 100 to 1000 mm cm -3 (Haynes and Beare

	16 ± 0.04 a	0.06 ± 0.02 b	0.06 ± 0.02 b	0.16 ± 0.03 a	0.16 ± 0.05 a

M. Fattet et al. / Catena 87 (2011) 60-69

Acknowledgments

Thanks are due to the Xishuangbanna Tropical Botanical Garden, Kunming, China, and in particular to X Ming, Y Ming and L An, for their technical help. Advice from JL Bellotti (LISAH, France) was much appreciated. Funding was provided from INRA Jeune Equipe project. AMAP (Botany and Computational Plant Architecture) is a joint research unit which associates CIRAD (UMR51), CNRS (UMR5120), INRA (UMR931), IRD (2M123), and Montpellier 2 University (UM27).

Author's personal copy

Author's personal copy