J Rose

A Bénard

S El Mrabet

A Masion

I Moulin

V Briois

L Olivi

J-Y Bottero

Evolution of Iron speciation during hydration of C4AF

Keywords: Ferro-calcium aluminate, iron oxyhydroxides, EXAFS, heavy metal fixation

Although the calcium silicate and calcium aluminate phases are good candidates for metal retention in real cement, the role of the calcium-ferric aluminate phase, shorthand C 4 AF, should not be neglected. Indeed, sorption isotherms indicated that all metals showed a strong affinity for the hydration products of C 4 AF. The C 4 AF content in Portland cement is 2-7% and the Fe 2 O 3 content is in the 1 to 3,5% range. It is therefore reasonable to assume that a non negligible proportion of the trace metals in an actual cement will be associated with Fe phases. However, the metal retention capabilities of those phases have received little attention.

Previous investigations have shown that the minerals formed during the hydration of C 4 AF are similar to those formed from C 3 A (pure tri calcium aluminate) under comparable conditions. Nevertheless no investigation was conducted at the molecular level and there is still a controversy whether Fe substitutes for Al in the hydrated minerals in whole or in part, or if it forms FeOOH clusters scattered throughout the matrix.

In this context we have conducted XAS experiments at the LURE and ELETTRA synchrotrons. It was found that the hydration of C 4 AF leads to the formation of C 3 AH 6 (hydrogarnet) in which Fe randomly substitutes for Al in the C 3 AH 6 structure, and also forms an amorphous FeOOH phase. Intermediate products like AFm (i.e. an ill organized lamellar phase) are also formed but they rapidly evolved to C 3 AH 6 and iron does not seem to be incorporated in the AFm structure.

INTRODUCTION

Although the calcium silicate and calcium aluminate phases are good candidates for metal retention in real cement, the role of the ferro-calcium aluminate phase (C 4 AF) should not be neglected. Indeed, sorption isotherms indicated that all metals showed a strong affinity for the hydrated C 4 AF phase [START_REF] Moulin | Cristallochimie des métaux dans les phases hydratées des ciments[END_REF]). The ferro-calcium aluminate content in Portland cement is 2-7% and the Fe 2 O 3 content is in the 1 to 3,5% range. It is therefore reasonable to assume that a non negligible proportion of the trace metals in an actual cement will be associated with Fe phases. However, the metal retention capabilities of those phases (either isolated or within a cement) have received only little attention. Moreover the accurate speciation of iron in cement is not fully determined. Many investigations have shown that the minerals formed during the hydration of C 4 AF are essentially similar to those formed from tri-calcium aluminate (C 3 A) under comparable conditions (Chatterji and Jeffrey (1962), Carlson (1964), Collepardi et al. (1979), De Keyser and[START_REF] De Keyser | 5 th ISCC[END_REF], Schwiete and Iwai (1964), Emanuelson and Hansen, 1997[START_REF] Meller | The paste hydration of brownmillerite with and without gypsum: a time resolved synchrotron diffraction study at 30, 70, 100 and 150[END_REF]). The first minerals formed in the absence or presence of CaSO 4 are the ill organized lamellar phaese AFm and AFt respectively. Both types of phases contain Al 3+ and Fe 3+ and tend to undergo further changes to form hydrogarnet phases. Moreover [START_REF] Teoreanu | Cement Chemistry[END_REF]), Fukuhara et al. (1981), [START_REF] Rogers | [END_REF]Aldrige (1977), and[START_REF] Brown | Early Hydration Of Tetracalcium Aluminoferrite In Gypsum And Lime-Gypsum Solutions[END_REF] indicated the possible formation of an amorphous FeOOH phase.

Nevertheless no investigation was conducted at the molecular level and a controversy still exists: does all the Fe substitute for Al or are there FeOOH clusters embedded in the formed hydrated minerals?

The aim of our work was to monitor the evolution of iron speciation during the hydration of C 4 AF in lime water. The structure of iron at the atomic scale has been investigated using X-ray Absorption Spectroscopy.

Material and methods.

C4AF hydration:

Starting materials consisted of a anhydrous ferrite (Brownmillerite) C4AF obtained from Lafarge Ciments (Vivier, France) hydrated with lime water (LW) with a water/ solid ratio of 10 and 0.5 on a rotary shaker and filtered (0.2 µm). The recovered solid was dried with acetone. The C4AF were hydrated during 24, 48 hours and 7 days. X-ray Diffraction.

Solids from the sound core and the leached layer were mechanically separated, ground to fine powder, and analyzed by X-ray diffraction. A PhilipsPW3710 X-ray diffractometer with a Co K radiation at 40 kV and 40 mA was used. The diffractograms were scanned in the 2 3.5-78° range with a counting time of 12 s per 0.02° step.

Electron Microscopy SEM-EDX images and elemental analysis were obtained using a Phillips XL30 SFEGSEM coupled to an Oxford Instruments energy dispersive spectrometer (EDS). The SEM was operated at 20 keV. X-ray Absorption Spectroscopy.

X-ray absorption spectroscopy (XAS) experiments were performed at the Laboratoire pour l'Utilisation du Rayonnement Electromagnetique (LURE, Paris-France) on the D44 beamline and at the ELLETRA synchrotron facility on the BL 11.1 beamline. The Si(111) monochromator crystals were used to scan energy from 100 eV below to 800eV above the Fe K-edge. The samples were measured in the transmission mode. EXAFS data reduction was accomplished according to a procedure described previously (Manceau and Calas, 1986) The key point to study the reactivity of the C 4 AF mineral phase in cement is to determine the dissolution kinetics of the anhydrous mineral. In order to characterize the effect of the pH, hydration experiments were performed at various liquid/solid ratio and hydration pHs (lime water, pH=11, 10 and 9 with NaOH). The figure 1-A presents the XRD pattern of C 4 AF hydrated during 48 hours with a liquid/solid ratio of 10. The different peaks shown in figure 1-A correspond to anhydrous C 4 AF and hydrogarnet (C 3 AH 6 ). After 48 hours of hydration with a Liquid/Solid ratio =10, almost all the anhydrous C 4 AF has been dissolved. The C 3 AH 6 XRD peak intensities are slightly higher at pH = 10 than at pH = 9 and lime water indicating that the formation of C 3 AH 6 is faster, or the crystallinity of the hydrogarnet is better, at pH= 10 than in lime water. However it should be noted that in the present case the relevant parameter is not the pH but the Ca concentration. [START_REF] Scrivener | [END_REF] indicated that lime water delays the C 4 AF hydration, presumably because of the sorption of calcium at the surface of C 4 AF thus creating a barrier to water. After 7 days of hydration with a L/S = 0.5 the various samples exhibit the same diffraction patterns except for a broad peak at 13.5 ° (7.59Å) that is present only for three samples (C4AF L/S = 0.5 after 7 days hydrated with lime water, NaOH pH=9 and 11). This very small and broad peak corresponds to Afm phases such as C 3 A.CaCO 3 .11H 2 O (7.56Å). Indeed, this mineral is a temporary phase that appears during the first hydration stages (after 24 h of hydration) as shown in figure 1-B. In the case of pure C 4 AF system, water and carbonate molecules occupy the inter-layer site. These AFm phases are clearly identified after 24 hours as seen in figure 1-C. During the hydration processes and in the absence of calcium sulfate Afm minerals evolve into hydrogarnet phases.

Therefore the hydration of C 4 AF leads to the formation of hydrogarnet regardless of the pH of hydration. After 7 days, some AFm phases still exist but no crystallised iron oxide or hydroxide phases have been detected. For a more in-depth investigation, EXAFS spectroscopy has been used to determine the atomic environment of iron. This technique provides complementary information to XRD since amorphous phases are also observed by EXAFS.

Iron speciation

The analysis and modelling of EXAFS spectra enable the identification of the atomic environment of the target atom, i.e. iron in our case. The very interesting point is that EXAFS is sensitive to modifications of the crystallinity and mineralogy of Fe phases. Moreover EXAFS accurately characterizes the atomic structure of amorphous phases. From XRD we know that AFm and C 3 AH 6 exist in the samples. The question is to know whether or not Fe can replace Al in the hydrogarnet and Afm structures and if other Fe phases are present.

Figure 2 displays the pseudo-radial distribution function (PRDF) of the C 4 AF hydrated during 1 week and is compared to the PRDFs of Fe-garnet, and two iron oxy-hydroxides (goethite and lepidocrocite). The various peaks of the PDRF correspond to the probability to find atoms around the central atom (Fe) at the distance (R) of the peaks. In the case of iron the first peak correspond to the presence of oxygen atoms with a Fe-O interatomic distance ≈ 2 Å.

The second peak corresponds to atoms in the next nearest coordination spheres. The differences in position and amplitude suggest that the atomic environment of Fe is different in the sample and in the reference compounds, i.e. no pure Fe-garnet or oxy-hydroxides are formed.

The modelling of the first peak indicates that each iron is surrounded by 6 oxygen atoms at 2 Å. Therefore each iron is at the center of an octahedron formed by 6 surrounding oxygen atoms.

The results of the modeling of the second peak are presented in table 1. These results indicate that each iron is surrounded by 2.1±0.4 Fe atom atoms at 3.03±0.02 Å, 0.2±0.05 Fe atoms at 3.45 ±0.02 Å and 2.1±0.4 calcium atoms at 3.59±0.02 Å. The interpretation of EXAFS structural results requires the comparison with atomic structure of reference compounds. Table 2 presents the atomic structure of different reference compounds and figure 3 illustrates the corresponding structures of Fe at the molecular scale. The comparison between structural results obtained in the case of C4AF hydrated during 1 week and the structural data of reference compounds suggests that the Fe--Fe interatomic distances determined for the hydrated C 4 AF are similar to those of goethite and amorphous FeOOH.

From the 3D structure it is possible to determine the type of linkage between Fe octahedra corresponding to Fe--Fe interatomic distances of 3.03 and 3.45 Å. The short Fe--Fe distance corresponds to edge sharing between Fe octahedra and the longer one corresponds to double corner linkages as illustrated in figure 4. The fact that Fe octahedra are linked mainly through edge indicates that Fe hydroxide or oxy-hydroxides exist in the sample. If Fe was only present in the hydrogarnet or AFm, no Fe--Fe contribution would have been detected. The Fe--Ca atomic pair determined in the hydrated C 4 AF corresponds to the hydrogarnet structure and not to the AFm phase. Indeed the Fe--Ca interatomic distance in the AFm is 3.35 Å and 3.51 Å in the case of hydrogarnet, which is what has been determined for our hydrated sample.

In the case of the C 4 AF hydrated during 24 hours, XRD analysis indicate that AFm phases are formed. EXAFS results indicate that no Fe-Ca interaction exist, thus suggesting that Fe is not associated with AFm phases and that amorphous FeOOH phases are formed.

Therefore it appears that the hydration leads to the formation of hydrogarnet in which Fe can replace Al, and the additional formation of Fe amorphous oxy-hydroxides. SEM-EDX results have also demonstrated that under our experimental conditions iron is not associated to the AFM phases (figure 5). A C B V

  . The Fourier transform yields radial distribution functions (RDF) consisting of n peaks representing n coordination spheres at Rn distances from the central atom. Distances are uncorrected for phase shift and have to be displaced toward long distances by 0.3-0.4 Å from the crystallographic position. The structural and chemical parameters Rj, Nj (N: number of atoms) and nature of atomic neighbors in the j th shell around Fe were determined by least-squares fitting of partial EXAFS spectra. The uncertainties on R and N are 0.06 Å and 10%, respectively. Results and Discussion: Hydration kinetics of C 4 AF

Figure 1 :Figure 2 :

 12 Figure 1: A) XRD pattern of anhydrous C4AF, C3AH6 and various hydrated C4AF after 48 Hours, B) Zoom of the 12-22 ° area. C) XRD pattern of C4AF hydrated after 24 and 48 Hours.

Figure 3 :

 3 Figure 3: atomic environment around Fe in different Fe minerals. These differences in term of structure lead to different interatomic distances as shown in table 2.

Figure 4 :

 4 Figure 4: Face, edge (2.97Å< Fe--Fe <3.36 Å) and double corner (3.40Å<Fe--Fe <3.66 Å) sharing between Fe octahedra.

Figure 5 :

 5 Figure 5: SEM-EDX image of C4AF hydrated during 24 hours A) Backscattered electron image B) Al image C) Fe image.

Table 1 :

 1 structural results of the analysis of the second coordination sphere of Fe in C4AF hydrated during 24 hours and 1 week. Table 2: iron atomic environment of different reference compounds

	Sample Atomic	R(Å)	(Å)	N	Residue
		pair
	C4AF	Fe-Fe	3.03	0.111 3.1	0.012
	24h	Fe-Fe	3.46	0.093	1.3
	C4AF	Fe-Fe 3.03	0.104 2.1	0.0389
	1 week Fe-Fe 3.45	0.070 0.2
		Fe-Ca 3.59	0.091 2.1

Acknowledgment:

We would like to thank the European Community for supporting this work through the INERWASTE Craft European program, and specially the YPREMA company


Wilfried Sanchez
email: wilfried.sanchez@ineris.fr

Sélim Aït-Aïssa

Olivier Palluel

Jean-Maxence Ditche

Jean-Marc Porcher

Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams

Keywords: three-spined stickleback, biomarkers, oxidative stress, field study, depuration experiment

Recently, the three-spined stickleback (Gasterosteus aculeatus L.) has been proposed as a suitable fish species for detecting both androgen-and estrogen-induced endocrine disruption by environmental pollutants. This relatively pollution-tolerant fish is present in most European streams and small rivers but also coastal and estuarian areas. The purpose of the present field study was to determine the extent to which multiple biomarkers in this fish species could distinguish between streams with different pollution levels. Sticklebacks were sampled in French rivers characterised by various urban, industrial or agricultural contaminations and in outdoor lotic mesocosms as reference site. Physiological parameters including condition factor and liver somatic index, biotransformation enzymes such as 7ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase, antioxidant enzymes including glutathione peroxidase and total glutathione (GSH) content and lipoperoxidation (as TBARS) showed several differences between sites. For example, fish from an heavily contaminated stream exhibited a 9-fold EROD induction associated to a decrease of GSH and a 3-fold increase of TBARS content in comparison to fish from uncontaminated sites. When fish were tranferred from polluted river to clean water, some of these biomarkers rapidly returned to basal levels found in fish in the reference site while others, like TBARS levels were still high after 2 weeks of depuration. Based on multivariate analyses, the battery of biomarkers proved to differentiate all sites, with a very good classification rate for highly contaminated streams. Influence of fish gender and sampling period on biomarker responses was also observed and is discussed. The results of this field study provide additional support for the use of stickleback for in situ multi-biomarker assessment.

INTRODUCTION

Biomarkers have been proposed as sensitive tools for detecting environmental exposure and adverse effects of toxic anthropogenic chemicals on aquatic organisms. Biomarkers have been defined as a change in biological response which can be related to exposure or toxic effect of environmental chemicals [START_REF] Peakall | Biomarkers: the way forward in environmental assessment[END_REF]. In recent years, the usefulness of a set of biomarkers has been demonstrated in environmental biomonitoring in several aquatic species, including freshwater fish [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF][START_REF] Triebskorn | The project VALIMAR (VALIdation of bioMARkers for the assessment of small stream pollution): objectives, experimental design, summary of results, and recommendations for the application of biomarkers in risk assessment[END_REF]. In European rivers, the fish species mostly employed to assess biological effects of contaminants using a set of biomarkers are chub and roach [START_REF] Flammarion | Multibiomarker responses in fish from the Moselle river (France)[END_REF]. However, due to the fish species diversity encountered in streams and estuaries, the assessment of various other species for biomarker-based biomonitoring is still needed.

The three-spined stickleback (Gasterosteus aculeatus L.) appears to be a promising species to assess the impact of contaminants in aquatic biota in that it is relatively tolerant to environmental pollution, and that it inhabits most streams, small rivers, estuarian and coastal areas in Europe [START_REF] Wootton | A functional biology of sticklebacks[END_REF]. Moreover, this fish species is a well-studied model for biologists since behaviour, ecology and evolution of stickleback have been extensively characterised. The responses of several biochemical parameters such as vitellogenin [START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF], spiggin [START_REF] Katsiadaki | Detection of environmental androgens: a novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterostus aculeatus) glue protein[END_REF] and 7-ethoxyresorufin-O-deethylase [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the threespined stickleback, Gasterosteus aculeatus[END_REF] have been investigated in laboratory studies and have appeared as promising biomarkers of chemical exposure to be used in environmental biomonitoring. Despite its relative ubiquity, to our knowledge, cholinesterase activity and liver histopathology are the only biomarkers investigated and validated in the stickleback [START_REF] Handy | Histopathologic biomarkers in three spined sticklebacks, Gasterosteus aculeatus, from several rivers in Southern England that meet the freshwater fisheries directive[END_REF][START_REF] Sturm | Different sensitivity to organophosphates of acetylcholinesterase and butyrylcholinesterase from three-spined stickleback (Gasterosteus aculeatus): application in biomonitoring[END_REF] and, therefore efforts are still needed to validate other biochemical biomarker responses in situ for the monitoring of fish health in freshwater ecosystems.

In previous studies, we examined a set of hepatic biotransformation and oxidative stress endpoints as potential biomarkers for various pollutants in stickleback [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF][START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF]. The purpose of the study was to validate these biomarkers in the field when this fish species are exposed to a variety of pollutants in rivers situated in Northern France. Biomarker responses in wild sticklebacks sampled in five sites subjected to different types and levels of contamination (Table I) Moreover, a first evaluation of biomarker reversibility in stickleback from one of the most contaminated streams was performed to investigate the evolution of biomarker profiles over a 15 day depuration period in clean water.

MATERIALS AND METHODS

Sampling design

During 2003 and 2004, five sites located in the North of France characterised by the presence of wild stickleback population and exhibiting various qualitative and quantitative contaminations were investigated. Table I summarises some general characteristics of the selected sites and fish sampling in each site. All sampling of adult sticklebacks were carried out by electrofishing in Spring (between June-July) and/or Autumn (September-October). All sampling sites have been impacted by urban and/or agricultural activities as determined by the Fish Based-Index (FBI) quality score based on occurrence and abundance data and reflecting the level of degradation of fish assemblage structure [START_REF] Oberdorff | Adaptation et validation d'un indice poisson (FBI) pour l'évaluation de la qualité biologique des cours d'eau français[END_REF]. Therefore they could not be considered true reference sites so, reference fish were sampled at equivalent sampling periods in uncontaminated outdoor lotic mesocosms located at INERIS (Verneuil en Halatte, France). Each mesocosm channel was 20 m long, 1 m wide with a water depth of 0.3 m in the upper section and 0.7 m in the lower section. The mesocosms were initially set-up with natural and artificial sediments, aquatic plants, phytoplankton, periphyton, benthic and pelagic invertebrates, microorganisms and one fish species that is the stickleback. The channels were fed by denitrated water from a drinking water treatment plant with a flow of 800 l/h by channel.

Depuration experiment

To determine the potential reversibility of biomarkers when fish are returned to clean water, we collected 45 fish from the Réveillon (REV) river in Spring 2003 campaign and transferred them to the laboratory in aerated tanks. The fish were divided into three groups of 15 and placed in denitrated water (alkalinity, 367 mg/L as CaCO 3 ; pH, 8.35 ± 0.05) for periods of 4, 8 and 15 days. This experiment was carried out in semi-static conditions with complete water renewal and food supply every 2 days. The water temperature was 14°C ± 0.5°C and the photoperiod corresponded to natural light/dark cycle at this period.

Sample preparation

Fish were measured, weighed and sacrificed immediately after being caught. The CF was calculated according to [START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF], while HSI was calculated as (liver weight / fish weight) x 100.

Livers were removed, weighed and frozen in liquid nitrogen prior to homogenisation and biochemical analysis. The homogenisation was in 200 µL of ice-cold phosphate buffer (100 mM, pH 7.8) with 20% glycerol and 0.2 mM phenylmethylsulfonyl fluoride using a Potter-Elvehjem homogeniser. The homogenates were centrifuged at 10,000 x g, 4°C, for 15 min and the postmitochondrial fractions were used for biochemical assays. Total protein concentrations were determined using bovine serum albumin (Sigma-Aldrich Chemicals, France) as a standard [START_REF] Bradford | A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Biochemical biomarker assays

EROD, GST, GPx and GSH assays were conducted respectively according to the methods of [START_REF] Flammarion | Statistical analysis of cyprinid ethoxyresorufin-Odeethylase data in a large french watershed[END_REF], [START_REF] Habig | Glutathione S-Transferases. The first enzymatic step in mercapturic acid formation[END_REF], [START_REF] Paglia | Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase[END_REF] and [START_REF] Vandeputte | A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol[END_REF]. These assays were adapted in microplates and optimised for stickleback to fit the linearity of each assay. The total protein concentrations used for each assay were 0.5 to 10 g/L for EROD and 0.5, 5 and 4 g/L for GST, GPx and GSH, respectively. Lipid peroxidation was estimated by adding 345 µL of TBARS reagent [START_REF] Ohkawa | Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction[END_REF] to 60 µL of a 3 g proteins/L diluted samples. Samples were then heated and TBARS were quantified by fluorimetric measurement with 515 nm wavelength excitation and 553 nm wavelength emission. Malonaldehyde (MDA) was used as standard, and results were expressed in nmol of MDA equivalent/g of proteins.

Statistical analysis

All data are reported as mean ± standard deviation and SPSS 13.0 software was used for statistical analysis. Firstly, normal distribution and homoscedasticity of data were verified using Kolmogorov-Smirnov and Levene tests respectively. Since data sets had not a normal distribution and/or homogeny of variance, the biomarker data was log-transformed, using F(x) = log(1+x), prior to parametric analysis. Secondly, a two-way analysis of variance (ANOVA) was performed for each biomarker using sites and gender as factors. When sites by gender interaction was significant ( =0.05), male and female data were treated separately. Biomarker responses measured at each sampling period in uncontaminated mesocosms, were compared using one-way ANOVA followed by Sidak test ( =0.05). To determine the differences between biomarker responses observed in contaminated sites and mesocosms, a t-test was performed ( =0.05). A standardised Principal Component Analysis (PCA) was performed to investigate relationships between investigated variables. A step by step discriminant analysis (DA) using discriminating functions and followed by cross validation was carried out to classify the sites.

RESULTS

The results for physiological and biochemical parameters are presented in Tables II andIII. In these tables, males and females are presented separately when significant differences were revealed by two-way ANOVA performed using site and gender as co-factors for each parameters (Table IV).

Influence of gender and sampling period on biomarker levels

Several biotic and abiotic factors are known to affect the biomarker responses. Our results showed that CF, HSI, phase I and phase II biotransformation enzymes, GPx activity and GSH were gender dependent parameters, while TBARS was not (Table IV). No significant interaction was noticed between biomarkers and fish weight or length (data not shown).

In uncontaminated mesocosms, morphological indexes, GPx and TBARS exhibited no significant difference between fish sampled in Spring and in Autumn (Table II andIII). In mesocosms, unexpected and relatively high EROD activity was observed in fish sampled during Spring 2003. This measure could be considered as an artefact and is unlikely to be representative of unaffected basal level since comparison to EROD levels in several laboratory controls are generally 5-10 pmol/min/mg [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the threespined stickleback, Gasterosteus aculeatus[END_REF][START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF]. No other significant differences were noticed for EROD activity measured in mesocosm fish at different sampling periods (Table III). An increase of GST was measured in mesocosm fish sampled in Autumn compared to those sampled in the Spring. Moreover, variations of GSH content were also noticed, with a weaker GSH content in those fish sampled in Spring of 2003 compared to fish in Spring 2004 (Table III).

In situ biomarker responses

Some morphological index variations were observed between fish from mesocosms and contaminated sites (Table II). CF was increased in female fish sampled in sites OUR, RHO and REV during Spring 2004. HSI was increased in all sites except in LEZ and in male fish from OUR and RHO.

EROD was induced in fish from ESC and in male fish from REV during Spring 2004. A high EROD value was also observed in REV during spring 2003, which corroborates the 2004 results, and indicates that fish were likely to have been exposed to dioxin-like compounds at these sites. A decrease of the EROD activity was measured in female fish from LEZ (Table III). Glutathione S-transferase was induced both in males and females from RHO, ESC, and in male fish from REV during Spring 2003, but was inhibited in female fish from OUR and LEZ (Table III).

The assessment of oxidative stress biomarkers showed that antioxidant defences were clearly affected in the sites characterised by moderate or high contamination (i.e. REV, OUR, RHO and ESC) as compared to the low contaminated site LEZ or mesocosms. Depletion of GSH together with increased TBARS levels (up to 4-fold the reference level) showed that pollution-exposed fish were subjected to oxidative stress. Alteration of redox liver status in REV (only in 2003), OUR and RHO, was further confirmed by strong induction of GPx, which is involved in organic and hydro-peroxide degradation.

Multi-variate analysis

To explore overall relationships between the different variables, we ran a PCA from a matrix data that included biomarkers, morphological and general indexes as variables. The figure 1A shows that 58 % of overall variance is explained by the first two principal components. The first principal component (PC1, 42 % of variance) was mainly built by the water quality index (WQI) and TBARS that were found to be strongly correlated (r = 0.8, p= 0.001, Pearson's coefficient). On this axis, fish from mesocosms and LEZ were clearly distinguished from fish sampled in other sites (not illustrated). The PC2 (16 % of overall variance) was mainly formed by GST and EROD. As expected, this axis distinguished fish from RHO, which had high GST levels, to fish from REV 2004 that had high EROD levels (not illustrated). Moreover, on the first factorial plan of the PCA, different correlation between biomarkers were suggested and further confirmed by Pearson's correlation test : negative correlation between GSH and other oxidative stress parameters : GST (r = -0.238, p<0.001), GPx (r = -0.292, p<0.001) and TBARS (r = -0.312, p<0.001), and positive correlation between TBARS and enzymatic antioxidants (GST : r = 0.429, p<0.001 and GPx : r = 0.517, p<0.001).

We then used step by step DA to further examine the discriminating power of the set of biomarkers. This analysis showed that, while all investigated biomarkers participated significantly to the discrimination, some weighted more heavily than others in site discrimination : TBARS > GST > EROD > GSH > GPx > HSI > CF. As shown in figure 1B, DA revealed a very good discrimination of the contaminated sites RHO AND ESC with respectively, 100 and 92 % of successful classification rates. The lower rates of successful classification in the contaminated sites REV and OUR were due to the misclassification of some fish in ESC, another impacted site. Likewise, the lower rate in the lesser contaminated site LEZ, were due to badly classified fish in mesocosm groups as they presented low biomarker variations as compared to this group. To overcome variations linked to the sampling period, DA were also performed within each sampling period. Overall, the results confirmed a very good discrimination characterised by successful classification rates, i.e. higher than 75 % for all sites except for the LEZ river where 30 % of fish were badly classified in mesocosm group (data not shown).

Depuration experiment

In REV, fish exhibited altered levels of all investigated biomarkers as compared to basal levels in mesocosms (Table II andIII). The depuration experiment (Table V) demonstrated that to varying degrees, some of the biomarker responses were reversed when fish were moved into clean water for 15 days. CF and HSI slightly decreased by 10 to 30% in clean water, reaching the level observed in male fish from mesocosms. This was also observed with in situ induction of EROD and GST which were completely reversed after 15 day depuration. In contrast, only slight recoveries were noted for TBARS (decreased by 26%) and GSH (increased by 34% in males), while GPx induction was still present after 15 days. Overall, these results confirmed that, in REV, fish had undergone relatively high oxidative damage in the liver that was still persistent after 2 weeks.

DISCUSSION

The aim of our research on three-spined stickleback was to determine the potential of this small fish species as a sentinel organism for in situ monitoring of aquatic contaminants. To our knowledge, this study is the first one that has explored the responses of a set of biomarkers in sticklebacks sampled in various contamination contexts. We showed that individual biomarkers such as morphological indexes (CF and HSI), biotransformation enzymes (EROD and GST) and oxidative stress parameters (GPx, GSH and TBARS) were altered in fish sampled in streams impacted by various contaminants and their combination allowed site discrimination.

The need of multi-biomarker approach for freshwater biomonitoring has been largely reported by several previous field studies [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF][START_REF] Triebskorn | The project VALIMAR (VALIdation of bioMARkers for the assessment of small stream pollution): objectives, experimental design, summary of results, and recommendations for the application of biomarkers in risk assessment[END_REF]. In our work, a noteworthy fact was that the set of selected biomarkers was able to discriminate the different sites accordingly to general indexes based on water quality and fish assemblage. Moreover, the biomarker approach enabled to distinguish between two sites that were similarly classified by general indexes. For instance, in the heavily contaminated streams RHO and ESC, biomarker response patterns were different with a strong oxidative stress in RHO compared to ESC where fish exhibited high EROD levels. These differences may probably reflect variation in the nature of chemical contaminants to which the fish were exposed. However, other sites such as OUR and REV were less discriminated than the other impacted sites, e.g. some REV fish were classified in ESC group in the DA. This may reflect a limit in the selected combination of biomarkers and the addition of other specific biomarkers could be a practical way to improve the discriminating power of the battery. Nonetheless, the multivariate analyses showed that the number of investigated biomarkers cannot be reduced without losing discriminating power. This showed that all biomarkers significantly contribute within the battery, although with different weighting.

The biomarker response patterns in stickleback suggested that fish inhabiting in polluted areas may have undergone sublethal stress, possibly due to exposure to different chemical contaminants, although chemical analyses in fish could not be performed in this study. EROD activity is a documented biomarker in stickleback and previous laboratory studies investigated its response in this fish species after exposure to polyhalogenated compounds [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the threespined stickleback, Gasterosteus aculeatus[END_REF], pesticides [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF] and metals [START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF]. In the present study, both basal levels in uncontaminated sites and EROD induction measured in REV and ESC were consistent with the known range of variations for this biomarker in stickleback. The 5-fold observed induction could be considered as moderate as compared to the 20-fold induction reported by [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the threespined stickleback, Gasterosteus aculeatus[END_REF] and probably revealed exposure of fish to aryl hydrocarbon receptor agonists in these sites.

As for EROD, baseline levels of antioxidants and oxidative damages measured in poorly impacted sites were in accordance with the levels previously reported for stickleback [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF][START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF]. Moreover, both the nature and the extent of oxidative stress biomarker responses observed in fish from contaminated streams (REV, OUR, RHO and ESC) were also in line with the range of variation reported after laboratory exposure to metals [START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF] and agricultural technical mixtures [START_REF] Sanchez | Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.)[END_REF]). An exception occurred from GPx that was characterised in situ by a strong induction (from 5 to 10 fold as compared to mesocosm fish), which had not been previously observed in laboratory experiments using model oxidative chemicals.

CF and HSI are general indicators of fish health widely used for population assessments because they integrate many levels of sub-organismal processes and are easily measured. Overall, only minor effects were observed on these parameters as illustrated by their low weight in site discrimination as compared to biochemical parameters in the DA. In stickleback, baseline data are available for comparison [START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF][START_REF] Sokolowska | Seasonal changes in brain melatonin concentration in the three-spined stickleback (Gasterosteus aculeatus): towards an endocrine calendar[END_REF] and show that only HSI of fish from heavily contaminated sites (REV and ESC) is out of the normal range of variation. This result could reflect a liver enlargement in fish exposed to environmental chemicals which corroborated findings from other biochemical biomarkers in these sites.

Biomarker responses of stickleback appear as a potential relevant tool to assess water quality. The depuration experiment provided valuable information on their reversibility in clean water and their limits for monitoring application. It demonstrated that to varying degrees, biomarker responses were generally reversed when fish were transferred into clean water. Based on this experiment, EROD and GST were characterised by a fast recovery. However, these parameters cannot reflect the time integration of contamination effects, they can only reflect a recent pollution episode. In this context, a frequent sampling could be requested in order to not underestimate pollution [START_REF] Wu | Induction, adaptation and recovery of biological responses: Implications for environmental monitoring[END_REF].

Conversely, oxidative stress biomarkers such as GPx and TBARS, appeared as parameters characterised by a slow recovery and could be considered as robust biochemical parameters providing a time-integrated estimate of pollution levels. A less frequent sampling is also required to evaluate the effects of water quality on oxidative stress parameters [START_REF] Wu | Induction, adaptation and recovery of biological responses: Implications for environmental monitoring[END_REF].

The present work suggested that stickleback biomarker responses were influenced by natural variability. Various biotic factors such as age, gender, feeding behaviour, and environmental factors including diet availability, water temperature, dissolved oxygen level and parasites can enhance or decrease the biotransformation activities [START_REF] Whyte | Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF], antioxidant defences [START_REF] Martinez-Alvarez R M | Antioxidant defenses in fish : biotic and abiotic factors[END_REF] and fish health reflected by HSI and CF [START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF][START_REF] Sokolowska | Seasonal changes in brain melatonin concentration in the three-spined stickleback (Gasterosteus aculeatus): towards an endocrine calendar[END_REF]. These confounding factors could also prevent using biomarker application for routine monitoring. Hence, it is necessary to characterise natural variability of biomarker responses. This field study provides a first indication on certain confounding factors that could influence the selected biomarkers in stickleback. Except for TBARS, all investigated indicators appeared to be gender-dependent. This effect could be explained by the important variation in physiology and biochemistry of female fish during the reproductive phase as previously described for HSI [START_REF] Sokolowska | Seasonal changes in brain melatonin concentration in the three-spined stickleback (Gasterosteus aculeatus): towards an endocrine calendar[END_REF] and EROD activity [START_REF] Flammarion | Statistical analysis of cyprinid ethoxyresorufin-Odeethylase data in a large french watershed[END_REF]. For instance, some biomarkers such as GST exhibited a seasonal variability with higher levels in Autumn than in Spring. This could be linked to its implication in the hepatic degradation of sex steroids and the variation of hormone levels after the breeding period [START_REF] Gallagher | Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameiurus nebulosus)[END_REF]. Interestingly, GPx induction were strongly gender-dependent in OUR and REV sampled in Spring. Similar trends were observed in mesocosms at the same period. To our knowledge, such gender-effect on this enzyme has not been previously reported in fish. In addition, other factors linked to seasonal variability such as predation, parasite infestation and food availability could also be the source of the variation of biomarkers in uncontaminated stickleback [START_REF] Martinez-Alvarez R M | Antioxidant defenses in fish : biotic and abiotic factors[END_REF]. As an example, [START_REF] Pascual | Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata)[END_REF] showed that susceptibility to oxidative stress in fish, as determined by oxidative stress biomarkers contents, is enhanced by food deprivation. These consideration argue for a better characterisation of stickleback biomarker's natural variability due to biotic and abiotic factors prior to their routine application for freshwater biomonitoring.

CONCLUSION

This study was designed to assess the usefulness of physiological and biochemical biomarkers in sticklebacks sampled from low and highly impacted areas. The response profiles of investigated biomarkers allowed a good discrimination between highly contaminated streams and moderately polluted or unpolluted sites. Other aspects related to using sticklebacks as a sentinel species were highlighted during this study. The abundance and distribution of stickleback within the study area facilitated sampling of several sites located in reference and polluted areas, however in large rivers, this could be limited by the availability of specific habitats. Moreover, stickleback is characterised by a stationary behaviour, hence the observed responses could reflect the local environment. This field study demonstrates the potential of stickleback as a sentinel fish species to assess sublethal stress in multipollution context. Further studies are in progress to include other specific biomarkers such as vitellogenin, spiggin and cholinesterase in this set of biochemical parameters. In addition, seasonal variability together with chemical analysis of the water will be studied more thoroughly on selected sites in order to better characterise the influence of natural and chemical factors on biomarker responses in this fish species. c Data obtained from the "Seine-Normandie" water agency for OUR and LEZ and from "Artois-Picardie" water agency for RHO and ESC.

d Fish based-index quality score based on occurrence and abundance data and reflecting the level of degration of fish assemblage structure [START_REF] Oberdorff | Adaptation et validation d'un indice poisson (FBI) pour l'évaluation de la qualité biologique des cours d'eau français[END_REF]. FBI score ranged from 1 (no disturbance) to 5 (very high disturbance).

  were examined. The studied biomarkers were related to xenobiotic metabolism (i.e. 7ethoxyresorufin-O-deethylase [EROD] and glutathione-S-transferase [GST]) and to oxidative stress (i.e. glutathione peroxidase [GPx], total glutathione content [GSH] and lipoperoxidation [TBARS]), as well as to physiological parameters (i.e. liver somatic index [HSI] and condition factor [CF]).

Figure 1 .

 1 Figure 1. Multivariate analysis carried out all data including the nine sites as samples and 7

  Figure 1.

Table I :

 I Summary of some general characteristics of the five selected sites. Strahler's rank based on structure and density of hydrographic network[START_REF] Strahler | Quantitative analysis of watershed geomorphology[END_REF]. Data obtained from the Eurowaterne database of European Environment Agency[START_REF] Eea | Reporting river quality using the water quality accounts methodology[END_REF].

		Réveillon	Ourcq	Lézarde	Rhonelle	Escaut
		(REV)	(OUR)	(LEZ)	(RHO)	(ESC)
		N 48°43'00" N 49°11'42" N 49°34'09" N 50°17'49" N 53°23'40"
	GPS coordinates	E 2°32'09" E 3°31'32" E 0°13'20" E 3°32'41" E 3°32'56"
	Strahler's rank a	2	3	1	2	4
	Pressure b	Urban dense	Intensive agriculture	Mixed	Urban dense Urban dense
	Water quality c	-	Medium	Good	Bad	Bad
	Fish based index d	5	4	3	3	3
	Sampling period	Spring 2003 Spring 2004	Spring 2003	Autumn 2004	Autumn 2004	Autumn 2004

a b

Table II :

 II Morphological parameters for three-spined sticklebacks sampled in contaminated sites and uncontaminated mesocosms. Data are presented as mean ± SD. * indicates a significant

	difference compared to mesocosm group sampled at the same period (p<0.05). Mesocosm groups
	annotated with different letters are significantly different (p<0.05).
	Sampling period	Sites	Gender	Fish number	Fish length (mm)	Fish weight (g)	CF	HSI %
		MESO	♂ ♀	11 19	43 ± 3 A 44 ± 4 A	0.7 ± 0.3 A 0.9 ± 0.2 A 2.3 ± 1.8 A 0.8 ± 0.2 A 1.0 ± 0.2 A 3.3 ± 1.1 A
	Spring 2003	REV	♂ ♀	14 15	54 ± 3* 54 ± 4*	1.6 ± 0.3* 1.6 ± 0.4*	1.0 ± 0.1 1.0 ± 0.1	4.1 ± 1.3* 6.1 ± 0.9*
		OUR	♂ ♀	9 4	54 ± 3* 55 ± 4*	1.7 ± 0.3* 1.9 ± 0.2*	1.0 ± 0.1 1.2 ± 0.1*	3.1 ± 0.7 7.2 ± 1.2*
	Spring	MESO	♂ ♀	14 10	50 ± 7 B 59 ± 6 B	4.3 ± 0.5 AB 1.0 ± 0.1 A 1.7 ± 0.6 A 2.0 ± 0.7 B 1.0 ± 0.2 A 2.5 ± 0.7 A
	2004	REV	♂ ♀	15 15	57 ± 4* 58 ± 4	2.0 ± 0.4* 2.4 ± 0.6	1.0 ± 0.09 1.2 ± 0.1*	3.5 ± 0.7* 8.2 ± 1.0*
		MESO	♂ ♀	15 15	53 ± 4 B 52 ± 7 C	1.4 ± 0.4 B 0.9 ± 0.1 A 2.4 ± 0.8 A 1.3 ± 0.7 A 0.9 ± 0.1 A 2.7 ± 0.4 A
	Autumn	LEZ	♂ ♀	10 18	39 ± 3* 39 ± 1*	0.6 ± 0.1* 0.6 ± 0.1*	1.0 ± 0.08 0.9 ± 0.06	2.4 ± 0.6 2.4 ± 0.7
	2004	RHO	♂ ♀	13 17	59 ± 3* 59 ± 5*	2.1 ± 0.4* 2.1 ± 0.5* 1.0 ± 0.08* 5.0 ± 1.1* 1.0 ± 0.09 3.0 ± 0.8
		ESC	♂ ♀	19 8	51 ± 3 50 ± 4	1.3 ± 0.2 1.3 ± 0.3	1.0 ± 0.06 1.0 ± 0.07	4.1 ± 1.4* 3.3 ± 0.6*

Table III :

 III Biomarker responses for three-spined sticklebacks sampled in contaminated sites and uncontaminated mesocosms. Data are presented as mean ± SD. * indicates a significant difference compared to mesocosm group sampled at the same period (p<0.05). Mesocosm groups annotated with different letters are significantly different (p<0.05).

	Sampling period	Sites	Gender	Fish number	EROD pmol/min/mg	GST U/g	GPx U/g	GSH µmol/g	TBARS nmol/g
		MESO	♂ ♀	11 19	23.9 ± 19.8 A 1273 ± 406 A 32.1 ± 27.2 A 1233 ± 417 A 113 ± 57 A 57 ± 14 A	41.6 ± 25.0 A 23.9 ± 18.4 A	46 ± 25 A
	Spring 2003	REV	♂ ♀	14 15	34.1 ± 15.7 20.3 ± 9.6	2131 ± 538* 1304 ± 881 583 ± 211* 88 ± 29*	18.1 ± 1.3* 20.4 ± 5.9	148 ± 41*
		OUR	♂ ♀	9 4	7.9 ± 3.9* 9.7 ± 4.7*	1458 ± 539 688 ± 147*	60 ± 25 809 ± 72*	23.5 ± 6.0* 18.4 ± 1.8	92 ± 21*
	Spring	MESO	♂ ♀	14 10	4.9 ± 1.8 B 8.4 ± 4.3 B	1259 ± 522 A 1486 ± 779 A 134 ± 116 A 82.7 ± 74.6 B 74 ± 47 A 59.7 ± 6.5 A	53 ± 29 A
	2004	REV	♂ ♀	15 15	43.3 ± 35.9* 22.7 ± 33.8	1063 ± 514 1195 ± 1226	72 ± 46 72 ± 57	42.8 ± 19.7* 47.8 ± 17.3	164 ± 46*
		MESO	♂ ♀	15 15	4.5 ± 2.1 B 5.0 ± 4.0 B	1843 ± 419 B 119 ± 113 A 36.1 ± 15.5 A 2105 ± 479 B 134 ± 53 A 46.5 ± 18.9 B	55 ± 17 A
	Autumn	LEZ	♂ ♀	10 18	4.6 ± 3.6 2.6 ± 1.7*	1572 ± 385 1151 ± 362*	160 ± 68 118 ± 69	42.2 ± 10.1 41.8 ± 21.2	48 ± 14
	2004	RHO	♂ ♀	13 17	3.2 ± 1.4 4.1 ± 3.6	3638 ± 601* 440 ± 136* 3457 ± 636* 564 ± 127*	13.6 ± 3.3* 14.0 ± 4.8*	203 ± 33*
		ESC	♂ ♀	19 8	22.5 ± 5.7* 27.6 ± 7.7*	2778 ± 675* 2782 ± 190*	84 ± 37 136 ± 40	24.6 ± 7.0 25.4 ± 5.1*	127 ± 25*

Table IV :

 IV Results of two-way ANOVA performed for physiological parameters and biochemical biomarkers in three-spined stickleback sampled in all study sites. * indicates that null hypothesis is not assumed (p<0.05).

	Biomarkers	Sites (p)	Gender (p)	Interaction (p)
	Fish length	<0.001 *	<0.001 *	0.143
	Fish weight	<0.001 *	<0.001 *	0.084
	CF	<0.001 *	0.337	0.039 *
	HSI	<0.001 *	<0.001 *	<0.001 *
	EROD	<0.001 *	0.364	0.037 *
	GST	<0.001 *	0.024 *	0.015 *
	GPx	<0.001 *	<0.001 *	<0.001 *
	GSH	<0.001 *	0.756	0.009 *
	TBARS	<0.001 *	0.549	0.355

Table V .

 V Biomarker responses in fish from REV maintained 15 days in clean water. The unit of each biomarker is indicated in the Table III and IV. Data are presented as mean ± SD. * indicates a significant difference compared to t 0 control group (p<0.05). a indicates n=1.

				Days of depuration
			0	4	8	15
	Fish	♂	14	9	12	12
	number	♀	15	6	3	3
		♂	1.0 ± 0.1	1.0 ± 0.1	0.9 ± 0.09*	0.9 ± 0.07*
	CF	♀	1.0 ± 0.1	1.0 ± 0.1	0.8 ± 0.07*	0.9 ± 0.03*
		♂	4.1 ± 1.3	2.8 ± 0.6*	2.6 ± 0.5*	2.7 ± 0.9*
	HSI	♀	6.1 ± 0.9	4.9 ± 0.9*	3.9 ± 0.05*	4.8 ± 0.5*
		♂	34.1 ± 15.7	14.2 ± 9.3*	23.4 ± 20.9	5.5 ± 4.3*
	EROD	♀	20.3 ± 9.6	8.3 ± 5.4*	4.4 ± 2.5*	4.1 ± 4.9*
		♂	2131 ± 538	1818 ± 637	1393 ± 412*	1250 ± 461*
	GST	♀	1304 ± 881	1246 ± 518	1461 ± 635	1284 ± 502
		♂	88 ± 29	98 ± 28	37.2 ± 18.0*	68 ± 88
	GPx	♀	583 ± 211	533 ± 294	689 a	588 ± 104
		♂	18.1 ± 1.3	23.2 ± 5.1*	19.1 ± 11.2	24.2 ± 6.0*
	GSH	♀	20.4 ± 5.9	17.5 ± 5.5	24 ± 13.7	17.8 ± 0.8
	TBARS		148 ± 41	147 ± 37	127 ± 29	109 ± 21*

ACKNOWLEDGEMENTS

We thank the official fishguards from the Conseil Supérieur de la Pêche and S. Lamothe for his technical assistance in fish sampling. We thank also Prof. R. Coulombe (Utah State University) and J. Sabarly for reviewing this manuscript. This work was supported by the French Ministry of Ecology and Sustainable Development (Budget Civil de la Recherche et du Développement AP-2004).


A Albinet

E Leoz-Garziandia

H Budzinski

E Viilenave

Sampling precautions for the measurement of nitrated polycyclic aromatic hydrocarbons in ambient air

Keywords: Polycyclic aromatic hydrocarbons, Nitrated polycyclic aromatic hydrocarbons, Oxygenated polycyclic aromatic hydrocarbons, Sampling artefacts

Because of the toxicity of polycyclic aromatic hydrocarbons (PAHs) and of their oxidation products, such as nitrated and oxygenated PAHs (NPAHs and OPAHs), the determination of their concentrations is of great interest in terms of atmospheric pollution control. Then, normalisation of sampling procedures appears essential. In this context, this paper presents a comparison of particulate PAH, OPAH and NPAH concentrations determined with two different samplers (cascade

impactor and conventional high volume sampler) installed in parallel during several field sampling campaigns carried out under different environmental conditions. For winter and summer periods, the PAH and OPAH concentrations determined with both sampling systems were considered as equivalent. In the summer period, NPAH concentrations quantified with both sampling devices were similar whereas in the winter period, the conventional high volume sampler underestimated their concentrations by a factor of 3 to 4. This underestimation was observed in the same proportion for all the 17 quantified NPAHs. Analytical error, NPAH formation during the sampling and NPAH degradation by reaction with gaseous oxidants associated to sampling methodology were unable to explain such differences between both samplers used in parallel. A probable hypothesis is that, the heating of the PM 10 head of the high volume sampler in the winter period generates an increase of the internal sampler temperature that could intensify the chemical degradation of the NPAHs inducing the underestimation of their concentrations in the atmosphere. Further investigations will be necessary to confirm the importance of the temperature on the chemical degradation of these compounds and to understand the different behaviour of PAHs and OPAHs. Consequently, we suggest using oxidant scrubber to prevent chemical degradation of PAHs and derivatives during their sampling. Moreover, we advise against the heating of the sampling head which could induce an increase of these reactions of degradation especially for NPAHs.

Introduction

Atmospheric aerosols are complex matrices which include different chemical species that could cause harmful effects on human health (Finlayson-Pitts and Pitts Jr, 2000). Polycyclic aromatic hydrocarbons (PAHs) are typical toxic compounds present in the particulate matter. Several PAH derivatives, such as nitrated PAHs (NPAHs) and oxygenated PAHs (OPAHs) having strong indirect-and direct-acting mutagenic properties, are also found in airborne particulate matter [START_REF] Allen | Measurement of oxygenated polycyclic aromatic hydrocarbons associated with a size-segregated urban aerosol[END_REF][START_REF] Bamford | Nitro-polycyclic aromatic hydrocarbon concentrations and sources in urban and suburban atmospheres of the Mid-Atlantic region[END_REF]. PAHs are emitted directly from combustion whereas NPAHs and OPAHs are both primarily emitted and formed in the atmosphere, by gas and heterogeneous phase reactions of PAHs induced by atmospheric oxidants (OH, NO 3 , O 3 ). PAH derivatives have a particular interest because they seem more toxic than their related parent PAHs.

For instance, NPAHs could contribute for 10% to the total mutagenicity of inhalable suspended particles in polluted areas (Arey et al., 1988b;[START_REF] Atkinson | Atmospheric chemistry of gas-phase polycyclic aromatic hydrocarbons: formation of atmospheric mutagens[END_REF]. A correct quantification of these compounds in ambient air is very important. Some surprising results obtained in this study suggest that some precautions have to be taken for the measurement of the PAHs and their derivatives in the ambient air.

Experimental section

Sampling sites and operating conditions

Experiments were carried out on four sampling sites located in the French Mediterranean and Alpine regions. 1). Traffic related site named "Les Bossons" was instrumented in the Chamonix valley and a rural one in the small hamlet of Tigny in the Maurienne valley.

The two sites of the Mediterranean region were located in the Marseilles area (city of 800 000 inhabitants) characterised by an important solar irradiation, heavy traffic and industrial activities.

Two different types of sites (urban "Cinq avenues" and sub-urban "La Penne sur Huveaune ") were instrumented for the sampling of PAHs and their derivatives.

Sampling procedure

On each site, two kinds of samplers were used in parallel. Both ambient air particulate (quartz filter) and gas (PUF: polyurethane foams) phases were collected using a modified high volume sampler equipped with a PM 10 head (Model Digitel DA-80, Hegnau, Switzerland, 30 m 3 h -1 ). As recommended by the constructor, during the winter period the PM 10 head was heated to prevent turbine overload due to moist filters because of the excessive ambient humidity. The high volume cascade impactor (Graseby Andersen, 35 m 3 h -1 ) collecting airborne particulate was the second sampling device used. The particles collected using this sampler were separated into seven size ranges and collected on six separated fritted quartz fiber filters and on a back up quartz fiber filter.

Samplings were performed every 12 h with the DA-80 sampler and every 24 h with the cascade impactor (starting at 08:00 local time).

Analytical methods

Detailed analytical procedure for PAHs, NPAHs and OPAHs has been presented previously [START_REF] Albinet | Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation[END_REF]. Filters and PUF were extracted with dichloromethane using pressurised liquid extraction. Extracts were divided into two fractions by weighting. Both fractions were evaporated under a nitrogen stream and adjusted to 1 ml with acetonitrile (PAH analysis) or dichloromethane (NPAH and OPAH analysis).

Quality assurance

PAH analytical procedure was validated during a national inter-comparison campaign (Leoz-Garziandia, 2004). Three kinds of samples were analysed in order to identify the critical steps of the analytical protocols (low and high concentrations of prepared standard solutions, extracts of natural ambient air samples and diesel standard reference material 2975). For each sample four replicate analyses were carried out by each laboratory and mean values were calculated.

Application of the OPAH and NPAH analytical procedure has been performed on standard reference material (SRM 1649a: urban dust), giving results in good agreement with the few data available in the literature [START_REF] Albinet | Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation[END_REF].

Results

Concentrations and sources of PAHs, OPAHs and NPAHs

Average levels of concentrations observed during each sampling campaign for the sum of PAHs, OPAHs and NPAHs present mainly in particulate phase (Σ PAHp, Σ OPAHp and Σ NPAHp) and for the gaseous pollutants NO, NO 2 and O 3 are presented on Table 1.

Comparison of both sampling devices

A comparison between particulate-bound individual PAH, OPAH and NPAH concentrations obtained with both samplers was made. Fig. 1 shows the results obtained by summing the concentrations of all the impactor stages and averaging the concentrations of both 12-hour total filtration samples (DA-80). Only compounds present mainly in the particulate phase were taking into account. All sampling sites were mixed and results were presented according to the sampling season (winter and summer).

In the summer period, for all compound families (PAHs, OPAHs and NPAHs) the correlation coefficients were quite good, indicating a good proportionality between both sampling methods.

The intercepts were really low, indicating no systematic bias. The slopes of the regression were close to 1, indicating minimal (or at least similar) "blowoff" or adsorption artefacts. Thus, the concentrations determined with both sampling systems were considered as equivalent with regard to the analysis for particulate PAHs, OPAHs and NPAHs.

In the winter period, the same conclusion could be drawn for PAHs and OPAHs. The differences between the PAH and OPAH concentrations determined with both samplers were not significant.

Concerning NPAHs, results were wholly different. Even if the correlation coefficient was good and the intercept quite low, the slope of the regression was significantly different from 1. Individual NPAHs concentrations determined by the cascade impactor were about 3 to 4 times higher than those determined by the DA-80.

To investigate the origin of such differences, numerous hypotheses were tested: analytical drift, production or destruction of particle-bound NPAHs during the sampling. Each of these possibilities is discussed below.

Discussion

Analytical drift

The analytical procedure used in this study was validated on standard reference material (urban dust, SRM 1649a) and results previously published [START_REF] Albinet | Simultaneous analysis of oxygenated and nitrated polycyclic aromatic hydrocarbons on standard reference material 1649a (urban dust) and on natural ambient air samples by gas chromatography-mass spectrometry with negative ion chemical ionisation[END_REF] have shown a very repeatable method with a mean coefficient of variation (standard deviation/mean) about 0.2. In this procedure, OPAHs and NPAHs were analysed simultaneously and so, if analytical drift occurred, it should be the same for both classes of compounds. Moreover, the greater discrepancies were obtained with the most concentrated samples (winter period) for which the analytical errors were minimal. The uncertainties of the analytical procedure cannot explain the important differences observed between both kinds of samplers used in winter.

Formation of NPAHs during sampling

The formation of NPAHs during sampling by heterogeneous reaction with nitrogen oxides was investigated in previous studies. During a winter high-NOx episode, the maximum percentage of artefact formation of the observed NPAHs was less than 2-3% (Arey et al., 1988a). Results from a study conducted in a tunnel showed that the formation artefact represented less than 0.1% of the studied NPAHs (Dimashki et al., 2000). Authors concluded that filter artefacts were not significant and were not problematic for NPAH ambient air samplings. In our case, differences of concentrations of the parent PAHs determined by both sampling devices were not significant and differences of concentrations between the two samplers used were the same for any primary (e.g. 1nitropyrene) or secondary (e.g. 2-nitropyrene, 2+3-nitrofluoranthene) NPAHs. Moreover, if formation during sampling were to take place, that would be easier in the summer period when atmospheric concentrations of the reaction initiators, OH and NO 3 , were maximal. The formation of NPAHs during the sampling could not explain the disparities noted in the winter period between DA-80 and impactor samplers.

Destruction of NPAHs during sampling

The major way of degradation of NPAHs is photolysis. In both cases, this parameter could not be taken into account because the collection filter, installed in the middle of the sampler, was protected from sunlight.

A second way of degradation of particulate NPAHs by reaction with gaseous oxidants such as, OH, O 3 and NO x was reported as very negligible [START_REF] Atkinson | Gas-phase atmospheric chemistry of 1-and 2-nitronaphtalene and 1,4-naphtoquinone[END_REF]Feilberg et al., 1999). Due to the difference of the sampling methodology used in this study, this way of degradation could play a role in the differences observed. Because of the conditions of contact between ambient air and particles in the impactor, there is hypothetically a greater probability of contact with the compounds (NPAHs) on the filtration unit (backup filter) of the impactor-a condition that would also exist for the DA-80 filters. However, in the cascade impactor, airflow during sampling is over the surface of the fritted filters. It is conceivable that only the upper outside surface of the top layer of the impacted particles on the fritted filters is exposed to potential oxidizing agents, whereas all sampled air must pass through the backup filter. For the summer samples from the Alpine region, the percentage of the NPAHs collected on the fritted filters reached 85% (Fig. 2). In this case, if chemical reaction with atmospheric oxidants was a major way of degradation of the NPAHs collected on the filtration unit, the NPAH concentrations determined with the impactor would be much more important than those given by the classical filter method (DA-80). As show in Table 1, it is not the case. On the opposite, when the differences of concentrations determined by both samplers were the greatest in the winter period, the NPAHs were mainly associated to the finest particles collected on the back up filter. Finally, during the summer sampling campaign in the region of Marseilles, no or slight differences of concentrations were observed between the samplers whereas the NPAH filter distribution was similar to that of the winter samples from the Alpine region. Degradation of particulate NPAHs on the filter by reaction with gaseous oxidants associated to sampling methodology does not seem to be the key factor explaining the differences of concentrations observed between both samplers used in parallel.

The difference of temperature between external and internal ambient air could play a role in the differences of NPAH concentrations observed between both samplers used. The particulate collection filters of the DA-80 are located in the body of the sampler. The heating of the PM 10 head used in winter and to a lesser extent, the heat emitted by the sampling turbine did increase the internal temperature of the DA-80 sampler (up to 25°C more). Note that the increase of the internal temperature of the DA-80 did not cause the volatilisation of the particulate compounds collected on the filter. In fact, the percentage of PAHs, OPAHs and NPAHs with molecular weight > 206 g mol -1 associated with the particulate phase was in the range of 90-100% during the winter period. In summer, the heating of the PM 10 head was not used and the difference between internal and external temperatures was lower (maximum difference observed about 10°C). In the case of the impactor, collection filters are located on the upper part of the sampler. The pump is external and the sampler is well ventilated. The external and internal temperatures at the level of the collection filters were approximately similar in wintertime. Fig. 3 shows clearly the importance of the difference of internal and external temperature of the DA-80 sampler on the determination of NPAH concentrations. For any difference between internal DA-80 and ambient air temperatures, PAH and OPAH concentration ratios were in the range of 0.5-2. For NPAHs, this ratio increased considerably until a value of 9 when differences of temperature were the greatest. At this time, we are unable to explain with certitude differences observed on the NPAH behaviour in comparison with the PAHs and OPAHs. A probable hypothesis to explain such differences is that the abrupt change of temperature between external ambient air (negative temperature in winter) and inside the DA-80 sampler could intensify the chemical degradation of the NPAHs. In the same time, the lower temperature in the impactor sampler stabilises these compounds collected on the filters.

Temperature does not seem to play a role in the degradation of the other classes of compounds namely PAHs and OPAHs. Nevertheless, as far as we know no data, showing the relation (or not) between chemical degradation of PAHs and their derivatives and the increase of the temperature permitting to confirm our hypothesis, exist.

Conclusion

This study presents a comparison of particulate PAH, OPAH and NPAH concentrations determined with two kinds of samplers (cascade impactor and conventional high volume sampler) installed in parallel during five sampling campaigns. For both winter and summer periods, the PAH and OPAH concentrations determined with both sampling systems were considered as equivalent with regard to the analysis of these compounds. In the summer period, NPAH concentrations quantified with both sampling devices were similar whereas in the winter period, the DA-80 sampler underestimated their concentrations by a factor of 3 to 4. To investigate the origin of such differences numerous hypotheses were tested: analytical drift, production or destruction of particlebound NPAHs during the sampling. Finally, a probable hypothesis is that, the heating of the DA-80 PM 10 head in the winter period generates an increase of the internal temperature of the sampler that could intensify the chemical degradation of the NPAHs inducing the underestimation of their concentrations in the atmosphere. Further investigations will be necessary to confirm the importance of the temperature on the chemical degradation of these compounds and the different behaviour of PAHs and OPAHs. As a conclusion and as suggested in a previous study (Goriaux et al., 2006), the use of oxidant scrubber would allow to prevent chemical degradation of PAHs and derivatives during their sampling. Moreover, we advise against the heating of the sampling head which could induce an increase of these reactions of degradation especially for NPAHs.

Figure Captions

  16 PAHs were quantified by HPLC with fluorescence/UV detection (acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene, indeno[1,2,3,c,d]pyrene and coronene). NPAH and OPAH concentrations were determined simultaneously using gas chromatography-mass spectrometry with negative ion chemical ionisation (GC/NICI-MS) after purification on solid phase extraction (SPE) cartridges. A total of 17 NPAHs and 8 OPAHs were quantified (1nitronaphthalene, 2-nitronaphthalene, 2-nitrofluorene, 9-nitroanthracene, 9-nitrophenanthrene, 3nitrophenanthrene, 3+2-nitrofluoranthene, 4-nitropyrene, 1-nitropyrene, 2-nitropyrene, 7nitrobenz[a]anthracene, 6-nitrochrysene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene and 6-nitrobenzo[a]pyrene; 1-naphthaldehyde, 9-fluorenone, 9-phenanthrenecarboxaldehyde, 9,10anthraquinone, benzo[a]fluorenone, benzo[b]fluorenone, benzanthrone and benz[a]anthracene-7,12dione).

Fig. 1 .

 1 Fig. 1. Comparison of particle-bound individual PAH, OPAH and NPAH concentrations measured

Fig. 2 .

 2 Fig. 2. Fraction of particle-bound NPAHs collected on the fritted filters and the back up filter

Fig. 3 .

 3 Fig. 3. PAH, OPAH and NPAH concentration ratios determined by PM 10 classic filter method (DA-

  Table 1 summarises dates, locations, main characteristics and atmospheric conditions during the campaigns.The two sites located in the Alpine region (Chamonix and Maurienne valleys) were involved in the sampling campaigns of the POVA research program (POllution des Vallées Alpines) which focused on atmospheric chemistry in the vicinity of the most important automotive traffic between France and Italy. Four campaigns of one week each were carried out (two per site), in winter 2002-2003 and in summer 2003 (Table

  sum of concentrations of 4 or 6 OPAHs mainly present in particulate phase. In the winter period: from 9phenanthrenecarboxaldehyde to benz[a]anthracene-7,12-dione. In the summer period: from benzo[a]fluorenone to benz[a]anthracene-7,12-dione. sum of concentrations of 11 NPAHs mainly present in particulate phase: from 2+3-nitrofluoranthene to 6nitrobenzo[a]pyrene.

	Σ PAHp (ng m -3 ) b	17.6 (7.6)	16.0 (4.9)	1.7 (1.6)	1.3 (1.4)	6.3 (2.3)	5.4 (1.9)	0.5 (0.8)	0.5 (0.5)	1.7 (0.5)	1.5 (0.5)	0.7 (0.5)	0.7 (0.4)
	Σ OPAHp (ng m -3 ) c	9.7 (3.4)	9.9 (3.9)	0.4 (0.2)	0.2 (0.1)	3.7 (2.4)	3.6 (0.9)	0.3 (0.7)	0.1 (0.1)	0.7 (0.3)	0.7 (0.2)	0.4 (0.3)	0.4 (0.3)
	Σ NPAHp (pg m -3 ) d	587 (247)	2492 (954)	74 (94)	61 (35)	389 (209)	1157 (523)	78 (64)	120 (80)	229 (152)	182 (34)	46 (39)	56 (42)
	Number of samples	14	7	12	7	14	6	14	7	12	7	14	7

a mean (standard deviation).

b sum of concentrations of 10 PAHs mainly present in particulate phase: from benz[a]antracene to coronene. c d

Acknowledgements

The authors wish to thank the French Ministry of Ecology and Sustainable Development (MEDD) and the ADEME agency for financial support.

 6) 32 ( 4) 29 ( 6)

O 3 (µg m -3 ) 19 ( 11) 62 ( 21) 25 ( 17) 120 ( 36) 66 ( 28) 81 ( 43)

NO (µg m -3 ) 72 ( 38) 42 ( 23) 11 ( 9)

NO 2 (µg m -3 ) 52 ( 10) 33 ( 10) 32 ( 11) 8 ( 5) 20 ( 9) 13 ( 8)