A Breton

Caroline Sablayrolles
email: caroline.sablayrolles@ensiacet.fr

C Vignoles

M Montréjaud-Vignoles

Lauryl Alkylbenzene Sulfonates in the Urban Water Cycle (Toulouse, France)

Keywords: Surfactant, Pollution, Quantification, liquid chromatography, mass spectrometry

Application of the European Water Framework Directive requires Member States to have better understanding of the quality of surface waters in order to improve knowledge of priority pollutants. Xenobiotics in urban receiving waters are an emerging concern. This study proposes a screening campaign of laurylalkylbenzene sulfonates in a separated sewer system. An analytical method by solid-phase extraction and liquid chromatography coupled with mass spectrometry detection was developed providing satisfactory detection and quantification limits. Ten sites and seven types of waters (wastewaters, treated wastewaters, ground water, rainwaters, roof collected waters, run-off waters and carwash wastewaters) were investigated over one year in Toulouse (France) using quantitative monitoring. Ground, rain and roof collected water concentrations are similar to treated wastewater levels. Run-off water was the most polluted and impacted the aquatic environment. Washing station were responsible for 75% of the LAS load in stormwaters. The wastewater treatment plant reduced laurylalkylbenzene sulfonates concentrations by 90% before discharge into the environment. The results showed that laurylalkylbenzene sulfonates concentrations were variable over time and space in all urban water compartments. No correlation between laurylalkylbenzene sulfonates concentrations and global water pollution parameters was observed.

Introduction

Linear alkylbenzene sulfonates (LAS) are synthetic anionic surfactants, produced since the 1960s. They were introduced to substitute highly branched alkyl benzene sulfonates which were slowly biodegradables [START_REF] Jensen | Fate and effects of linear alkylbenzene sulphonates (LAS) in the terrestrial environment[END_REF]. In 2003, worldwide consumption of LASs has been estimated at about 18 million tonnes [START_REF] Mungray | Fate o linear alkylbenzene sulfonates in the environment: A review[END_REF]. Thus, it is the most surfactants used in detergents and cleaning products (shampoos, personal care products …). They consist of a mixture of homologues differing in the number of carbons in the aliphatic chain. For each homologous, positional isomer exist depending on the position of the benzene sulfonate group on the aliphatic chain. Commercially, produced LASs are mixtures containing homologues with alkyl chains ranging from 10 to 13 carbon atoms [START_REF] León | Handling of marine and estuarine samples for the determination of linear alkylbenzene sulfonates and sulfophenylcarboxylic acids[END_REF]. Their detergency depends mainly on the position of the benzene sulfonate group from the alkyl chain and the length of this channel [START_REF] Ding | Analysis of linear alkylbenzenesulfonates by capillary zone electrophoresis with large-volume sample stacking[END_REF]. These surfactants enter into wastewater treatment plants (WWTP) and are dispersed into the environment through effluent discharges into surface waters. They could also be discharged directly in surface waters [START_REF] Jensen | Fate and effects of linear alkylbenzene sulphonates (LAS) in the terrestrial environment[END_REF]. LASs were found in range of concentrations of 1.6 to 3.7 µg.L -1 in drinking waters and between 14 and 155 µg.L -1 in river water [START_REF] Eichhorn | -Incomplete degradation of linear alkylbenzene sulfonate surfactants in Brazilian surface waters and pursuit of their polar metabolites in drinking waters -The[END_REF]. In WWTP, reported values of LASs are between 104 and 1920 µg.L -1 in wastewaters and between 11 and 872 µg.L -1 in treated wastewaters [START_REF] Gonzalez | Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)[END_REF]. Evaluation of LAS levels in environmental samples were principally performed by high-performance liquid chromatography (HPLC) coupled with UV-detection ( [START_REF] Wangkarn | Determination of linear alkylbenzene sulfonates in water samples by liquid chromatography-UV detection and confirmation by liquid chromatography-mass spectrometry[END_REF]; [START_REF] Batarseh | Fate of linear alkylbenzene sulfonates in wastewater treatment plants[END_REF]) or fluorescence detection ( [START_REF] León | Handling of marine and estuarine samples for the determination of linear alkylbenzene sulfonates and sulfophenylcarboxylic acids[END_REF]; ([9]; [START_REF] Holt | AIS/CESIO environmental surfactant monitoring programme[END_REF]; [START_REF] Crescenzi | Simultaneous determination of alkylbenzenesulfonates and dialkyltetralinsulfonates in water by liquid chromatography[END_REF]). Methods using mass spectrometric detection were more rare [START_REF] Gonzalez | Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)[END_REF]. Thus, the aim of this study was to develop and optimize an analytical method to quantify the LASs in different water samples by liquid chromatography coupled with mass spectrometry detection in order to determine LAS concentrations in the urban water cycle of Toulouse (France).

Experimental

Reagents and standards

All chemicals used were analytical quality. Methanol, acetonitrile and hydrochloric acid 37% Multisolvent HPLC grade were purchased from Scharlau (France). N-hexane Suprasolv and ammonium acetate were obtained by VWR Merck (France). Condea Chimie SARL supplied the Marlon ARL which is a commercial surfactant powder containing 80% of C 10 -C 13 LAS. This commercial homologue LAS mixture has the following homologue mass distribution: C 10 (14.3%), C 11 (35.7%), C 12 (30.8%), and C 13 (19.2%). C 8 -LAS from Alfa Aesar (France) was used as internal standard at 10 mg.L -1 in each extract. Sodium DodecylSulfate (SDS; 99% purity) was purchased from Acros Organics (France). It was added in extract at the concentration of 2 mM. GF/C filters (glass microfibre; 1.2 µm) from Whatman (France) were used for filtration of water samples. Lichrolut RP-18e SPE cartridges (500 mg; 3 mL) were obtained by VWR Merck (France) and SAX (Strong Anionic eXchange) SPE cartridges (500 mg; 3 mL) were purchased from Varian (France). Syringe filters in PTFE with a 0.45 µm pore size from VWR Merck (France) were used to filtrate extract before analysis.

Sample collection

Nine sites were investigated in Toulouse in order to evaluate the LASs contamination. Ground water was collected under a normally frequented urban road. Rainwaters were collected in a zone free from any overhanging interference. Roof collected waters were taken from buildings in the town centre. The town of Toulouse is equipped with a separated sewer system where pollutants in wastewater system could not mix with the stormwater. The two main outlets of Toulouse (France) were selected in order to evaluate the stormwater quality. Outlet 1 fed from an urbanised catchment area of 439 hectares. Outlet 2 drains water of a low urbanised area which corresponds to a catching area of 1428 hectares. Three carwashes were investigated: truck carwash which trucks were washed manually, self-service carwash for car and motorcycles equipped with self-service high-pressure water jet and petrol station carwash equipped with self-service high-pressure water jet and washing roller brushing. These three sites discharged wastewaters into the stormwater network after a pre-treatment process composed of a scrubber and an oil separator. The Toulouse wastewater treatment plant (WWTP) was also investigated. This treats about 125 000 m 3 per day of wastewaters and discharges its effluent into the River Garonne. It is composed of a pre-treatment grid, sand trap and degreaser plus three treatment units: G1 (400 000EH), G2 (150 000EH), G3 (250 000EH), followed by a nitrification unit G4 (800 000EH) which treats all water from G1, G2 and G3, before discharging it into the River Garonne. In this study, wastewater entering unit G1 (biological treatment using activated sludge) and treated wastewaters (after unit G4) were investigated. Figure 1 shows the locations of the sampling sites.

FIGURE 1: Location of sampling sites

Sampling method

Four samples were collected from ground water, rainwater and roof collected water between November 2008 and November 2009. For ground water, samples were taken in dry weather in order to limit road scrubbing and were made in a sealed manhole chamber. For rainwater, forty basins were distributed on the ground in order to cover a large area. For roof collected water, samples were taken from the gutter down pipe. Each time, around 15L of water was collected and then homogenized in order to obtain a representative sample. For the two outlets studied, an automatic sampler was used for ten events over 24 hours. Concerning carwashes, five samplings were made. Samplings were realised manually from a conveyance at the pre-treatment process exit. Outlet and carwashes samples were collected between December 2006 and December 2007. For wastewaters and treated wastewaters, two automatic samplers were used to sample four dry events and four rainy events over 24 hours. WWTP sampling was carried out between March 2008 and March 2009. Amber glass bottles of 1L were filled with samples and stored at -25°C prior to analysis.

Sample extraction

LASs determination in environmental samples was carried out according to a protocol of several determinative steps, that is, pretreatment, extraction, and analysis (Figure 2). Water samples were filtered through GF/C filters to remove suspended solids in order to avoid clogging of the cartridge. Water sample extractions were performed with Lichrolut RP-18e SPE cartridges (500mg; 3mL) pre-conditioned with 10 mL of methanol followed by 10 mL of milliQ-water. 1000 mL of water sample was transferred to the SPE cartridge using a vacuum manifold system (Supelco). SPE cartridge was rinsed with 5 mL of milliQ-water followed by 5 mL of methanol/milliQwater mixture (20/80; v/v). LAS were eluted with 10 mL of methanol. This extract was then transferred to a SAX SPE cartridge pre-conditioned with 3mL of n-hexane followed by 10 mL of methanol. LAS were eluted with 2 mL of hydrochloric acid/methanol mixture (20/80; v/v). The final extract was concentrated under a gentle stream of nitrogen and redissolved in 1mL of methanol. The extract was filtered through a 0.45 µm syringe filter. Then, 10 µL of internal standard C 8 -LAS at 1 g.L -1 and 20 µL of SDS at 28.8 g.L -1 were added to the extract.

Sample analysis

Chromatographic analysis was preformed on a Dionex high performance liquid chromatography (Dionex, France) equipped with a SOR100 degasser, a P680 HPLC pump, a ASI 100 autosampler, a TCC 100 thermostated column compartment and a UVD340U ultraviolet diode array. A mass spectrometer MSQ Surveyor (Thermo Finnigan, France) was connected on line. The system was computer-controlled with data acquisition and processing using Chromeleon (Dionex) software. LASs separation was carried out using a Lichrospher® 100 RP-18e (250x4 mm id, 5µm) column protected by Lichrospher® 100 RP-18e (4x4 mm id, 5µm) guard column (VWR Merck, France). The injection volume was set at 30 µL, the column was thermostated at 25°C and the flow rate was 0.8 mL.min -1

. The elution was performed with a gradient composed of milliQ-water (A) and a solution of acetonitrile/milliQ-water (80/20; v/v) containing 2 mM of ammonium acetate (B). The gradient programme for the first 3min was 30% B and was increased linearly to 100% B over 10 min, then kept isocratic for 2 min and decreased linearly to 30% B over 2 min (Figure 3).

Multivariate data analysis

The multivariate data analysis simulation was performed using the commercial software XL stat. The objectives of Principal Component Analysis (PCA) are to find and interpret hidden complex and casually determined relationships between dataset. The key idea is to study the data structure in a reduced dimension while retaining the maximum amount of variability present in the data. A matrix of pairwise correlations among compounds concentrations is decomposed into eigenvectors, which, are sorted in descending order of their corresponding eigenvalues. In this work, the variables were standardized in order to ensure that they have equal weights in the analysis (mean is equal to zero and the standard deviation is equal to the unit. Then, the calculation of the covariance matrix by identifying the eigenvalues and their corresponding eigenvectors was carried out.

Results and discussion

Chromatographic separation, calibration and limits of detection Figure 6 shows the chromatogram resulting from the methodology described above applied to a standard mixture of LASs. LASs were separated in 17 min, with retention time of 11.1 min, 11.9 min, 12.6 min and 13.3 min for C 10 , C 11 , C 12 , C 13 -LAS, respectively. The quantitative calculations are made from the peak area corrected with C 8 peak area. LASs were determined as the sum of homologous C 10 to C 13 LASs. The calibration curve was obtained with standard solution of LASs. The linear range was set between 0.01 µg.L -1 and 3 mg.L -1 for the sum of LASs (C 10 -C 13 ), with regression coefficient (r²) above 0.99. The precision of the chromatographic determination was evaluated by standard solution of 5 mg.L -1 . The standard solution was analysed ten times and the relative standard deviation (%) was calculated. The repeatability of the chromatographic determination was found to be 7% for the sum of LASs. Reproducibility of the method was tested on EVIAN water spiked with three concentrations of the sum of LASs (0.7, 10 and 300 µg/L). Extraction was repeated three times for each concentration at different days. The Σ LAS (C 10 -C 13 ) percentages of recovery for each concentration were respectively in the 120 ± 5 %, 84 ± 6 % and 60 ± 3 %. The percentages of recovery were good for the concentration of 0.7 and 10 µg.L -1

. For the concentration of 300 µg.L -1 , the recovery was lower than other due to the possible saturation of the cartridge during the extraction time. In an other hand, the matrix which is moderately mineralized could also interact with the sorbent and induce a decrease of the percentages of recovery. Limit of detection, determined as 3 standard deviations above the mean blank signal, was 0.01 µg.L -1 for the sum of LASs. Limit of quantification was determined as 10 standard deviations above the blank signal and was equal to 0.03 µg.L -1 . The method developed in this paper allows to obtain limits lower than those obtained by other works ([3]; [START_REF] Holt | AIS/CESIO environmental surfactant monitoring programme[END_REF]; [START_REF] Alzaga | Determination of linear alkylbenzensulfonates in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gas chromatography-mass spectometry[END_REF]; [START_REF] Crescenzi | Simultaneous determination of alkylbenzenesulfonates and dialkyltetralinsulfonates in water by liquid chromatography[END_REF]).

Concentrations in environmental samples

LAS concentration was determined for the ten types of water investigated in Toulouse. The results of analysis were summarized in Table 1. The minimum, maximum, mean and standard deviation (SD) values were calculated. Values less than the quantification limit were taken as zero for statistical calculations. According to Figure 7 and Table 1, wastewaters from self-service carwash were the most polluted water and presented LAS values higher than those observed in literature for wastewater ([13]; [START_REF] Gonzalez | Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)[END_REF]).

The petrol station carwash studied discharges 700 µg.L -1 of LASs in mean into the stormwater network. LAS concentrations observed from petrol station carwash were in order of magnitude of values observed for surface waters in England [START_REF] Fox | Removal of linear alkylbenzene sulfonate from a small Yorkshire stream: contribution to GREAT-ER project #7[END_REF] and in Spain [START_REF] Alzaga | Determination of linear alkylbenzensulfonates in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gas chromatography-mass spectometry[END_REF]. LAS concentrations found in wastewater from truck carwash are low compared with data from surface waters in England (mean=147 µg.L -1

) [START_REF] Fox | Removal of linear alkylbenzene sulfonate from a small Yorkshire stream: contribution to GREAT-ER project #7[END_REF] and in Spain (mean=44µg.L -1 ) [START_REF] Alzaga | Determination of linear alkylbenzensulfonates in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gas chromatography-mass spectometry[END_REF]. The difference of LAS concentrations between self-service carwash and petrol station carwash could be explained by the difference between carwash processes, perhaps high-pressure water jet consumes more detergent with LASs than washing roller brushing. Moreover, the self-service carwash could be more used than the petrol station carwash. The wastewater treatment plant (WWTP) reduced LAS concentrations by 90% before discharge into the environment. LAS concentrations in wastewaters from WWTP were lower than those found in the literature for wastewaters from WWTP in Spain (mean=837 µg.L -1 ) [START_REF] Gonzalez | Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)[END_REF], in Jordan (mean=10.21 µg.L -1 ) [START_REF] Batarseh | Fate of linear alkylbenzene sulfonates in wastewater treatment plants[END_REF] and in Italy (mean=6329 µg.L -1

) [START_REF] Crescenzi | Simultaneous determination of alkylbenzenesulfonates and dialkyltetralinsulfonates in water by liquid chromatography[END_REF]. The levels observed in WWTP entry are lower than those of wastewaters from carwash. A Spanish study reported LAS mean concentrations for treated wastewaters (590 µg.L -1 ) higher than those observed in this study [START_REF] Gonzalez | Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain)[END_REF]. Two other studies reported LAS values of 2.59 µg.L -1 [START_REF] Batarseh | Fate of linear alkylbenzene sulfonates in wastewater treatment plants[END_REF] and 68 µg.L -1 [START_REF] Crescenzi | Simultaneous determination of alkylbenzenesulfonates and dialkyltetralinsulfonates in water by liquid chromatography[END_REF] which were higher than values found in treated wastewaters in Toulouse. Mean values observed for the two outlets were comparable to concentrations found for surface water in Turkey (mean=224µg.L -1 ) [START_REF] Akyüz | Ion-pair extraction and GC-MS determination of linear alkylbenzene sulphonates in aqueous environmental samples[END_REF] and in England [START_REF] Fox | Removal of linear alkylbenzene sulfonate from a small Yorkshire stream: contribution to GREAT-ER project #7[END_REF]. Ground water, rainwaters and roof collected waters have low concentrations of LASs. Rejected waters by the outlets to the environment were more polluted than those rejected by WWTP. Wastewaters entering in unit treatment G1 of WWTP were less concentrated in LASs than stormwaters. As carwashes are allowed to reject wastewaters into the stormwater network after a pretreatment, a comparison of loads from carwashes to pollution flows at outlets was performed. Thanks to an inventory of the organizations owning discharges licences and a modelisation scenario, we manage to establish the composition of the two outlets: two self-service carwashes, two petrol station carwashes and one truck carwash. Thus, for run-off waters (outlet 1 and 2), 75% of the LAS pollution could attributed to the carwash discharges [START_REF] Crescenzi | Simultaneous determination of alkylbenzenesulfonates and dialkyltetralinsulfonates in water by liquid chromatography[END_REF].

Correlation study

Principal Component Analysis was carried out in order to find relationships between LAS and global parameters of water pollution. More than LAS levels, samples were measured for pH, conductivity, turbidity, chemical oxygen demand (COD), nitrogen (Nt), phosphorus (Pt), suspended solid (MES) and volatile suspended solid (MVS). A data matrix, with columns representing the different samplings (observations) and rows corresponding to the measured parameters (variables), was constructed. A total of 48 complete observations were selected for analysis. The Principal Component Analysis showed that of the 9 components, the first component (F1) accounted for about 59.6 % of the total variance, the second component (F2) accounted for about 16.7 % of the total variance and the third component (F3) accounted for about 9.9 % of the total variance of the dataset. The loadings for the three first components and square cosines are presented in Figure 8. A variable is increasingly well represented by a component as the corresponding value of the square cosine approaches the unit. Almost all variables are well represented by the first three components, F1, F2 or F3, that collectively explain 86.2 % of the total variance of the dataset. The variables that primarily contributed to the first eigenvector were principally turbidity, chemical oxygen demand, total phosphorous, total nitrogen, suspended solid and conductivity. The second eigenvector was mainly related to volatile suspended solid. The third eigenvector was mainly related to LAS (Figure 8). It is clear that turbidity, chemical oxygen demand, total phosphorous, and suspended solid are correlated (R 2 >0.90). Orthogonal variables are significantly not correlated; it seems to be the case between all the global pollution parameters and the level of LAS in waters. Moreover, it is important to note that LAS and MVS are not negatively correlated (R 2 = -0.40) even if they seem to be symmetrically opposed regarding the center of the circle.

Conclusion

An analytical method for determination of LAS contents in water samples by SPE extraction and HPLC-UV/MS quantification was developed. The method provided satisfactory detection and quantification limits (LOD=0.01 µg.L -1 and LOQ=0.03 µg.L -1

). This method was applied to water sampled in Toulouse (France). Wastewaters, treated wastewaters, ground water, rainwaters, roof collected waters, run-off waters and carwash wastewaters were sampled and analysed. Determination of LAS levels in these waters showed that run-off waters were more polluted than treated wastewaters or wastewaters. LAS concentrations observed in carwash discharges were not negligible. These wastewaters were discharged into the stormwater network and impacted stormwater quality. The results showed that laurylalkylbenzene sulfonates concentrations were variable over time and space in all urban water compartments. A multivariable analysis was performed and no correlation between LAS levels and global water pollution parameters was observed.

FIGURE 2 :

 2 FIGURE 2: Outline of LASs analysis

FIGURE 3 :

 3 FIGURE 3: Diagram of gradient used for the separation of LAS homologues. Flow-rate=0.8 mL.min -1

FIGURE 4 :FIGURE 5 :

 45 FIGURE 4: UV spectrum of LAS

FIGURE 6 :

 6 FIGURE 6: HPLC-MS chromatogram of LASs standard mixture at 5 mg.L -1

FIGURE 8 :

 8 FIGURE 8: Box plots of LAS concentrations by sampling site, noting the minimum and maximum values, the median () and the mean (+)

FIGURE 8 :

 8 FIGURE 8: The square cosines for all variables in a) components F1 and F2 and b) components F1 and F3

TABLE 1 :

 1 Statistical data on LAS concentrations found in studied waters

	Type of water	n	Minimum (µg.L -1 )	Maximum (µg.L -1 )	Mean (µg.L -1 )	SD (µg.L	-1 )
	Ground water	4	0.01	0.03	0.02	0.01
	Rainwaters	4	0.03	0.21	0.09	0.08
	Roof collected waters	4	0.02	0.09	0.04	0.03
	Run-off waters	20	6.20	920	252	244
	Truck carwash	5	0.005	53.0	14	23.0
	Self-service carwash	5	8 100	64 000	20 120	24 540
	Petrol station carwash	5	46.0	3 000	719	1 280
	Wastewaters	8	2.70	6.80	3.84	1.36
	Treated wastewaters	8	0.08	0.14	0.11	0.02
	A schematic representation and box plot representation of LASs concentration in the different waters
	was shown in Figure 7 and 8 respectively.

Table 2

 2

	present the statistical data on the

Acknowledgment

This work was supported by Veolia Water Technical Direction (Toulouse, France) with the grant n° 20040101. We would like to thank Bernard Sentenac and Isabelle Dos Santos Meira for their technical support.


Caroline C Sablayrolles
email: caroline.sablayrolles@ensiacet.fr

Claire Vialle
email: claire.vialle@ensiacet.fr

Jérôme Silvestre
email: silvestre@ensat.fr

Michel Treilhou
email: michel.treilhou@univ-jfc.fr

Claire Lhoutellier
email: claire.lhoutellier@veolia.com

Violaine Brochier
email: violaine.brochier@veolia.com

Mireille Montrejaud Vignoles
email: mireille.vignoles@ensiacet.fr

M Montréjaud-Vignoles

PCBs uptake by carrots after sludge composts application: worst-case

Keywords: priority pollutants, organic waste products, plant growth, chemical analysis, food chain

come

INTRODUCTION

Polychlorinated biphenyls (PCBs) are the most important class of ubiquitous priority pollutants whose mutagenic/carcinogenic and endocrine disrupting effects on biota have been reported [START_REF] Winneke | PCB-induced neurodevelopmental toxicity in human infants and its potential mediation by endocrine dysfunction[END_REF][START_REF] Fischer | Symposium Overview: Toxicity of Non-Coplanar PCBs[END_REF][START_REF] Gan | Disappearance and crop uptake of PCBs from sludge-amended farmland[END_REF]. They have been included in the "priority pollutants" listings implemented by the United State Environmental Protection Agency and by the European Commission. PCBs general formula is C 12 H (10-n) Cl n where n is the number of chlorine atoms, from 1 to 10 [START_REF] Waid | PCBs and the environment[END_REF]. These molecules have an extremely low solubility in water especially the more chlorinated members [START_REF] Jones | Organic contaminants in sewage sludge applied to agricultural land[END_REF]. They are soluble in oils and most organic solvents and moreover have a very high environmental mobility due to their high vapour pressure [START_REF] Stringer | Chlorine and the environment: an overview of the chlorine industry[END_REF]. Starting in the thirties, PCBs were widely synthesized as part of different industrial compounds such as plastifying agents, inks or resins and in dielectric or coolant fluids [START_REF] De Alencsatro | PCBs concentration in wastewater treatment plants[END_REF]. They were available for sale mixed, under such trade names as Arochlor, Kanechlor, Clophen, Phenachlor, Pyralene. Although gradually banned since the eighties due to their toxicity, they are still present in the environment owing to their high physical, chemical and biological stability and the continued presence of various sources. According to Mhiri and Tandeau de Marsac [START_REF] Mhiri | Bioremediation of sites polluted by commercial PCBs: problematical questions and perspectives[END_REF], 400 000 tons of PCBs would have been spread in the environment. The most important problem with PCBs is their potential for transmission within the food chain [START_REF] Schecter | Levels of dioxins, dibenzofurans, PCB and DDE congeners in pooled food samples collected in 1995 at supermarkets across the United States[END_REF][START_REF] Kannan | Persistent Organochlorine Residues in Foodstuffs from Australia, Papua New Guinea and the Solomon Islands: Contamination Levels and Human Dietary Exposure[END_REF]. They are carried in mammalian milk and accumulate in adipose tissues, increasing the risks of bioaccumulation and transfer along food chains [START_REF] Borja | Polychlorinated biphenyls and their biodegradation[END_REF]. However, plants are the first step in the food chain.

Organic-farmed crops are grown in soils that may be contaminated with persistent organic pollutants at low concentrations from past applications of agrochemicals or byproducts, or from atmospheric deposition of volatile and semi-volatile organic compounds. In France, the production of sewage sludge composts is around 430 000 tons dry matter [START_REF] Ademe | Organic micro-pollutants in sewage sludge from wastewater treatment plant[END_REF]. The compost which follows the norm NFU 44-095 [START_REF] Afnor | NFU 44-095 Organic amendments -composts with agronomic matter from wastewater treatment plant[END_REF] can be recycled on arable crops where wheat and maize may be cultivated. However, sewage sludge compost could be applied soon on market gardening and notably carrots. In Europe, PCBs levels (Σ 7 PCBs) from 0.03 to 0.11 mg.kg -1 dry matter in sewage sludge composts were reported [START_REF] Vergé-Leviel | Organic micro-pollutants in composts: study during composting process and bioavailability after land application[END_REF][START_REF] Berset | Organic Micropollutants in Swiss Agriculture: Distribution of Polynuclear Aromatic Hydrocarbons (PAH) and Polychlorinated Biphenyls (PCB) in Soil, Liquid Manure, Sewage Sludge and Compost Samples; a Comparative Study[END_REF].

There are a lot of studies on PCBs uptake by terrestrial plants after sewage sludge land application [3] [16] but only few after compost application [START_REF] Webber | Plant Uptake of PCBs and Other Organic Contaminants from Sludge-Treated Coal Refuse[END_REF]. A lack of quantitative data was identified. Moreover, the most part of publications were focused on trace metal elements and not on trace organic compounds [START_REF] Sims | Chemical Fractionation and Plant Uptake of Heavy Metals in Soils Amended with Co-Composted Sewage Sludge[END_REF]. Numerous publications deal with the efficiency of this type of compost to improve organic matter and fertilizing elements contents of market gardening soils [START_REF] Casado-Vela | Evaluation of composted sewage sludge as nutritional source for horticultural soils[END_REF][START_REF] Perez-Murcia | Use of composted sewage sludge in growth media for broccoli[END_REF]. Therefore, knowledge on PCBs behaviour in environment and plants is essential in elucidating their potential bioaccumulation in the food chain.

Accordingly, the objective of this study was to investigate potential PCBs transfer from organic amendment (sewage sludge composts) amended sand/soil into a food-chain crop under real operational practice. Carrots (Daucus carota L. var. Amsterdam A.B.K. Bejo) were chosen as the test crop because they have been reported to be the crop having the greatest potential for organic uptake due to their high lipid content [START_REF] Wild | Organic chemicals in the environment: polynuclear aromatic hydrocarbon uptake by carrots grown in sludge-amended soil[END_REF]. In addition, carrots can be easily separated in peel, core and leaves which allow the determination of the organic compounds transfer path.

EXPERIMENTAL

Materials

Plant

The experiment was conducted on carrot (Daucus carota) Amsterdam A.B.K. Bejo variety. This variety was chosen because: (1) It is fully developed after two and a half months, [START_REF] Fischer | Symposium Overview: Toxicity of Non-Coplanar PCBs[END_REF] The carrots are dwarf carrots commonly used for human consumption, (3) It is a model plant, identified as having the greatest absorption potential of trace organic compounds due to its high lipid content [START_REF] Javorskam | Ability of carrot (Daucus carota L.) to accumulate selected polychlorinated biphenyl congeners[END_REF].

Sand and soil substrat

Crops were grown on sand and soil, in order to compare the influence of substrate on transfer. Sand was chosen because it has no organic matter content, thus applied organic compounds may be more bio-available. So a root crop growing in sand can be considered the worst-case scenario to study the PCBs behaviour [START_REF] Morard | Les cultures végétales hors sol[END_REF]. The quartz-siliceous sand used (medium diameter 0.5 mm) was carefully washed in order to release organic matter (hydrochloric acid and demineralised water). A density of 1.6 kg.L -1 was calculated and 3 kg of sand per pot were used. The water holding capacity of sand, that is to say the mass percentage of water which keeps sand, is around 15 %. The water holding of sand + compost mixtures in different application rates remains included between 12 and 14 %.

The soil came from a field belonging to an organic farmer with no fertilizers application since 1996. Geographically, this field is located between Saint Suplice-sur-Lèze (31, France) and Lézat-sur-Lèze (09, France) (Longitude: 01°20'24'' E; Latitude: 43°18'17'' N). The soil was sampled in the upper horizon (0-20 cm), air-dried and sieved at 5 mm diameter in order to fill the pots. The soil had a loamy texture and slimy-loamy type: clay (< 2µm) 36% ; fine loam (2-20 µm) 30% ; rude loam (20-50 µm) 18% ; fine sand (50-200 µm) 12% ; rude sand (200-2000 µm) 5%. The soil was rather basic with an alkalinity degree rather low (pH 8.3). This soil showed a good level of organic matter (23.2 g.kg -1 dry matter). It was also rich in major elements. Trace metal elements levels were less than limit values (table 1). Soil density was 1.1 kg.L -1 and 2 kg fresh matter of soil was put in containers. The water holding capacity of soil is higher than sand, in the order of 18 %.

Organic amendment

The sludge compost (Compost) was obtained from a composting facility processing sewage sludge mixed with crushed green waste and riddling refusal. The sampling of compost was accomplished following norm NF EN 12579 [START_REF] Afnor | NF EN 12579 Soil improvers and growing media -Sampling[END_REF]. Then, the sample was sieved at 5 mm before use. Table 1 presents information about nutrient composition and PCBs concentrations. The acquired compost answers the norm NF U 44-095 [START_REF] Afnor | NFU 44-095 Organic amendments -composts with agronomic matter from wastewater treatment plant[END_REF].

PCBs standards

PCBs standards (PCBs in pure form) were introduced for the pure substances experiments: tri-, tetra-, penta-, hexa-, hepta-chlorinated biphenyls (IUPAC numbers: PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, PCB180) at 10 mg.L -1 in acetone from Cluzeau Info Labo (France).

Experimental set-up

A total of 42 pots were used for the experiment (table 2). Four treatments were applied to carrots: (1) carrots grown in sand with added PCBs pure substances; (2) carrots grown in sand with an agronomic compost application (25 t/ha), which corresponds to the classical application dose of compost in gardening, (3) carrots grown in sand with an extreme compost application (60 t/ha), (4) carrots grown in soil with an agronomic compost application (25 t/ha). Soil cultures closer to reality were carried out in order to evaluate the real impact on transfers of an agronomic use of organic waste products. Control pots specific for each treatment (mineral content) were added to check growth conditions.

Cultivation technique

Carrot plants were grown on sand or soil in pots. The culture was conducted under greenhouse conditions (day: mean temperature 24°C, 14 hours light, 50% relative humidity / night: temperature 19°C, 10 hours light, 50% relative humidity). Carrot seeds were germinated on filter paper moistened with deionised water for 5 days at 23°C in the dark. Carrot seedlings were transplanted into glass pots (2 L volume ; 15 10 -2 m high ; 13 10 -2 m diameter) with bottom hole. Six pots, each containing 7 carrots, per treatment were used. Pots were randomly arranged in the greenhouse. All plants were watered with nutrient solution (KNO 3 5, KH 2 PO 4 2, Ca(NO 3 ) 2 5, MgSO 4 1.5 mM for macronutrients, and Fe 268.6, Mn 8.9, Cu 0.9, Zn 1.7, Mo 0.1, B 24.1 µM for micronutrient). The drain water contained in the saucer under the pots is poured back onto the sand. Carrots were irrigated daily to maintain the moisture content at 66% of the field capacity. Carrots were harvested after 90 days, carefully washed, divided into peel, core and leaves and weighted.

Analytical procedure

No standard reference material (SRM) was available for PCBs quantification in carrots. The analytical procedure evaluation was performed with control carrot (spiked concentration 10 µL PCBs at 10 ng.µL -1 acetone). Seven PCB isomers (IUPAC codes: 28, 52, 101, 118, 138, 153 and 180) were determined.

The plant matter was freeze-dried and ground up using a household grinder. 2 g sample, 1 g Fontainebleau sand (particle size 150-300 µm) to control boiling, 1g powdered Florisil (Florisil PR particle size 60-100 mech) to adsorb grease and the extraction standard PCB209 (10 µL of a solution at 200 µg.mL -1 in n-hexane (Cluzeau Info Labo, France)) were introduced in cellulose extraction cartridge (30 x 100 cm) (Schleicher & Schuell, France). An extraction with 100 mL of n-hexane Suprasolv (VWR, France) for 3 hours (2 hours in boiling mode and 1 hour in rinsing mode) is performed with a Soxtec System HT 1045 (Tecator, France). Then, a rotary evaporator (Rotavapor, Büchi) and a 30°C temperature controlled bath were used to concentrate the sample down to 10 mL. Concentration of the extract to 1 mL before purification was performed by a nitrogen stream (Alpha 1, Air Liquide, France). Clean up is done by 1g Florisil Solid Phase Extraction cartridges (Supelco, France) placed on a manifold (Supelco, France) and rinsed with 10 mL of n-hexane. A 10 mL graduated tube is placed under the manifold to collect the extract coming out of the cartridge. The 1 mL concentrated extraction sample is placed at the top of the cartridge. An elution at a rate of 1-2 drops per second is carried out with 8 mL of n-hexane to recover the PCBs. Each purified extract is then concentrated to 1 mL in a stream of nitrogen.

PCBs were analyzed by high resolution gas chromatography coupled with low resolution mass spectrometry (HRGC-LRMS) on electron impact mode. The apparatus is a Finnigan Trace 2000 series with a quadrupole type analyzer, entirely computer-controlled with data acquisition and processing using XCalibur software. The chromatograph is fitted with a Restek RTX-5MS (5% diphenyl; 95% dimethylpolysiloxane) column 30 meters long, 0.25 mm in diameter and 0.25 µm film thickness. Helium (Alpha 2, Air Liquide, France) carrier gas is used at a flow rate 1.2 mL.min -1 . A 1 µL sample is injected into the split/splitless inlet in splitless mode at 250°C. The temperature of the HRGC-LRMS interface is 250°C, and the oven temperature program starts at 60°C for 2 minutes. Then, the temperature increases to 230°C at 16°C.min -1 , reaches 282°C at 5°C.min -1 and levels off at this temperature for 1 minute. The full scan electron impact data is obtained under the following conditions: solvent delay 5 minutes, electron impact energy 70 eV, source temperature 250 °C, emission current 150 µA, detector voltage 350 V. Separation has been set up using a standard mixture of 7 PCBs at 10 µg.mL -1 in isooctane. Tetrachlorometaxylene (TCMX at 20 µg.mL -1 of acetone), an organo-chloride is used as internal standard and is added to the purified extract just before the gas chromatography analysis. Quantification is performed in Multiple Ion Monitoring (MIM) mode. Seven retention time windows have been used, each corresponding to the ions selected per compound: ( 1 1 shows a chromatogram for a standard mixture of 7 PCBs, the internal standard (TCMX) and the extraction standard (PCB 209). Each extract has been analyzed three times.

Analytical quality assurance

Analytical quality control and quality assurance programs were run in the laboratory during sample analysis in order to get reliable data [START_REF]AFNOR XPT 90-210 Evaluation protocol for an alternative analytical method development[END_REF]. Internal calibration curves have been obtained for each compound by linear regression of the peak area against the concentration injected. In each case, the regression coefficient ranged from 0.990 to 0.996. The calibration range of PCBs scans the concentrations from 10 to 100 ng.mL -1 .

Limit of detection (LOD) and limit of quantification (LOQ) were evaluated from standard deviation of ten replicates of carrot sample spiked. LOD (3 standard deviations) is 0.3 µg.kg -1 fresh mater for each PCB and LOQ (10 standard deviations) is 1 µg.kg -1 fresh mater for each PCB. These values are compatible with the levels found in plants tissues [START_REF] Bobovnikova | The influence of a capacitor plant in Serpukhov on vegetable contamination by polychlorinated biphenyls[END_REF].

The repeatability, expressed as the relative standard deviation (in %), is an evaluation of the overall extraction -purification -analysis procedure. It is calculated from 5 replicates of 5 carrot peel samples and was less than 5% per compounds.

The HRGC-LRMS apparatus gives concentration results in µg.L -1 . Knowing the mass of the sample and taking into account all the analytical steps, the results can be expressed in terms of µg.kg -1 of fresh matter (Figure 2).

Statistics

Variance analysis of data and a Newman-Keuls multiple range test at 0.05 probability level were performed (Statistical Software, Sigma Stat 2.00). The same letter in a column means that there is not significant difference at a probability equal to 0.05. On the other hand, a different letter means that there is a significant difference between control and treatments.

RESULTS AND DISCUSSION

Biomass production

For each modality, carrot fresh weight of controls and treatments were compared (Table 3). Plant masses were comprised between 7.5 and 53.0 g fresh matter according to compartments and experiment. No phytotoxicity symptoms were observed for plants regardless of the PCB concentration in pot. First, there is not significant difference between treatments and control in terms of the number of carrots per pot and the average number is always between 6 and 7. Secondly, there were no significant differences in growth between the carrots on the sand only (C1) and those on the sand with PCBs pure substances (T1). Concerning the compost experiment, a decrease in peel production was observed in treatment pots whatever dose of compost applied.

PCBs distribution in the pure substance experiment

PCBs average levels in peel, core and leaves of the carrots were calculated. PCBs were grouped according to chlorine number in the molecule: tri-chlorinated biphenyls (PCB28), tetra-chlorinated biphenyls (PCB52), penta-chlorinated biphenyls (PCB101 and PCB118), hexa-chlorinated biphenyls (PCB138 and PCB153), hepta-chlorinated biphenyls (PCB180). Distribution percentages of PCBs in carrot compartment were calculated according to equation presented in figure 3. Distribution profiles in the different compartment, presented in figure 4, clearly show that PCBs accumulated in carrot peel: 85% of ΣPCBs transferred were found in the peel. This result is consistent with other studies showing more than 80% of PCBs contamination was associated with peels [16] [27]. This behaviour can be explained with partition phenomena between lipophilic properties of PCBs and high lipid content of carrots peel (presence of oil channels). Moreover, root parts (peel + core) have absorbed 7 till 10 times more PCBs than the foliar parts. Besides, we observed that the more PCB is chlorinated, the more PCB is fixed in peel. Indeed, lipophilic properties of PCBs which are correlated to octanol-water partition coefficient (Kow) increase with the chlorine atoms number. The more lipophilic the chemical is, the greater is the association with the root.

Around 5% of ΣPCBs were found in the core. In theory, the lower the degree of chlorination is, the more the transfer in the core takes place [START_REF] Iwata | Translocation of the polychlorinated biphenyl Aroclor 1254 from soil into carrots under field conditions[END_REF]). In our case, this tendency is respected.

Around 10% of ΣPCBs were found in the leaves. The lower chlorinated biphenyls are generally reported to be more labile in soils, more soluble, less absorbed, more quickly degraded and more volatile than the higher chlorinated biphenyls. This phenomena is clear for leaves where more the degree of chlorination is weak, more the transfer takes place. We noticed that the lightest PCBs (tri-and tetra chlorinated) are found predominantly in the leaves. Volatilisation of these PCBs from the sand would allow explaining this presence [START_REF] Bobovnikova | The influence of a capacitor plant in Serpukhov on vegetable contamination by polychlorinated biphenyls[END_REF]. This hypothesis was verified with experimentation without carrots (Figure 5). Tri and tetrachlorinated PCBs are present at lower levels that high chlorinated PCBs in the upper part of the sand. The origin of these PCBs in leaves can be owned to a volatilisation and adsorption of PCB in the air. Indeed, Henry's law constant of these compounds are upper than 10 -4 [START_REF] Cousins | Measured and predicted volatilisation fluxes of PCBS from contaminated sludge-amended soils[END_REF].

Two pathways by which PCBs can enter a carrot were identified: (1) uptake and transport in oil channels (2) foliar uptake from surrounding air.

PCBs uptake in the compost experiment

PCBs concentrations in sludge compost were comprised between 1 and 13 µg.kg -1 fresh matter according to the congener (Table 1). PCBs concentrations in plants (leaves, core and peel) for treatments and controls pots were lower than the limit of quantification equal to 1 µg.kg -1 fresh matter (Table 4). No correlation between concentration of PCBs in soil and their uptake by carrots was observed. Apparently PCBs are so strongly adsorbed on organic matter from the soil or the compost indeed bioavailability of PCBs is independent of organic carbon. Moreover, a level of 1 µg.kg -1 fresh matter do not give cause for concern in relation to the maximum EU tolerable daily intake limit of 10 pg.kg -1 .body weight -1 .day -1 [START_REF] Zohair | Residues of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides in organically-farmed vegetables[END_REF].

CONCLUSION

First, PCBs concentrations were determined in a pure substance experiment. Distribution of PCBs has shown that compounds with high octanol-water partition coefficient are most likely to be sorbed by the peel. They accumulate on root surface and remain there bound to lipids in cells walls. The trend for preferential contamination of leaves with more soluble and lower chlorinated biphenyls has been reported.

Then, uptake of polychlorinated biphenyls from compost amended soil was measured at real conditions: no PCBs uptake was observed. Indeed, PCBs levels were lower than the limit of quantification in all cases. Consequently, this experiment in real operational practice conditions shows that NFU 44-095 sludge compost could be applied on carrots culture even on sand substrate.

  ) 9.0-12.5 min m/z = 244 ; 178 (2) 13.0-13.5 min m/z = 256 ; 186 (3) 13.5-14.5 min m/z = 292 ; 220 (4) 14.5-16.4 min m/z = 326 ; 184 (5) 16.4-18.0 min m/z = 360 ; 290 ; 254 (6) 18.0-22.0 min m/z = 394 ; 324 (7) 22.0-24.0 min m/z = 498 ; 428.

Figure

Figure 2 .Figure 3 .•

 23 Figure 2. Equation to calculate the concentration of PCBs in the plant matter (µg.kg -1 fresh matter) as a function of the PCBs concentration in the extract in µg.mL -1 (C extract ), the initial volume of the extract in mL (V initial ), the final volume of the extract in mL (V final ), the mass of the plant matter introduced into the extraction cartridge in g fresh matter (m), the extraction yield (the extraction is considered to be valid when the extraction yield is > 80 %) and the concentration factor (= initial volume of extract / final volume of extract) (CF).

Figure 4 .

 4 Figure 4. PCBs distribution in peel, core and leaves of carrots as a function of chlorine numbers (pure substances experiment).

Figure 5 .

 5 Figure 5. PCBs average levels in sand at the end of the experimentation (pure substances experiment without carrots)

Table 1 . Physical and chemical characterisation of the compost and soil used in this study

 1

		Unit	Number of	Compost	Soil
			repetitions
	pH		5	8.3 ± 0.0	8.3 ± 0.0
	Dry matter	% fresh matter	5	68.8 ± 0.0	82.9 ± 1.0
	Organic matter	g.kg -1 dry matter	5	538 ± 26	463 ± 4
	Total nitrogen	g.kg -1 dry matter	5	38.8 ± 1.1	-
	C/N ratio	-	5	6.9 ± 0.1	-
	P2O5	g.kg -1 dry matter	5	60.7 ± 4.4	146 ± 10
	K 2 O	g.kg -1 dry matter	5	-	400
	MgO	g.kg -1 dry matter	5	-	525
	CaO	g.kg -1 dry matter	5	-	11210
	Cu	mg.kg -1 dry matter	5	244 (300*)	28,24 (100*)
	Zn	mg.kg -1 dry matter	5	295 (600*)	86,13 (300*)
	Cd	mg.kg -1 dry matter	5	0,82 (3*)	0,32 (2*)
	Cr	mg.kg -1 dry matter	5	21,7 (120*)	33,71 (150*)
	Hg	mg.kg -1 dry matter	5	1,41 (2*)	0,042 (1*)
	Ni	mg.kg -1 dry matter	5	16,7 (60*)	28,74 (50*)
	Pb	mg.kg -1 dry matter	5	31,2 (180*)	19,98 (100*)
	PCB 28	µg.kg -1 fresh matter 5	<1	<1
	PCB 52	µg.kg -1 fresh matter 5	4.1 ± 1.7	<1
	PCB 101	µg.kg -1 fresh matter 5	6.4 ± 2.5	<1
	PCB 118	µg.kg -1 fresh matter 5	5,6 ± 5,9	<1
	PCB 138	µg.kg -1 fresh matter 5	8,5 ± 3,6	<1
	PCB 153	µg.kg -1 fresh matter 5	10,0 ± 2,7	<1
	PCB 180	µg.kg -1 fresh matter 5	1,7 ± 2,0	<1
	* Threshold value in compost and soil

Table 4 . PCBs levels (µg.kg -1 fresh maater) in each compartment for the compost experiments

 4

	Treatment		Number of repetitions Peel	Core	Leaves
	C2	PCB 28	5	<1	<1	<1
		PCB 52	5	<1	<1	<1
		PCB 101	5	<1	<1	<1
		PCB 118	5	<1	<1	<1
		PCB 138	5	<1	<1	<1
		PCB 153	5	<1	<1	<1
		PCB 180	5	<1	<1	<1
	C3	PCB 28	5	<1	<1	<1
		PCB 52	5	<1	<1	<1
		PCB 101	5	<1	<1	<1
		PCB 118	5	<1	<1	<1
		PCB 138	5	<1	<1	<1
		PCB 153	5	<1	<1	<1
		PCB 180	5	<1	<1	<1
	C4	PCB 28	5	<1	<1	<1
		PCB 52	5	<1	<1	<1
		PCB 101	5	<1	<1	<1
		PCB 118	5	<1	<1	<1
		PCB 138	5	<1	<1	<1
		PCB 153	5	<1	<1	<1
		PCB 180	5	<1	<1	<1

Chromatogram of a standard solution (7 PCBs at 10 µg.mL -1 , the TCMX at 20 µg.mL -1 and PCB 209 at 1 200 µg.mL -1 ) in SIM mode.

	11	12	13	14	15	16	17	18	19	20	21	22	23	24
						Time (min)

Acknowledgement

This research was supported by Veolia Environnement (Paris, France) and the French national agency of research and technology (ANRT) grant.