Marc Desmet
email: marc.desmet@univ-tours.fr

Brice Mourier
email: brice.mourier@entpe.fr

Barbara J Mahler
email: bjmahler@usgs.gov

Peter C Van Metre

Gwenaëlle Roux
email: gwenaelle.roux@entpe.fr

Henri Persat
email: persat@biomserv.univ-lyon1.fr

Irène Lefèvre
email: irene.lefevre@lsce.ipsl.fr

Annie Peretti

Emmanuel Chapron
email: emmanuel.chapron@univ-orleans.fr

Anaëlle Simonneau
email: anaelle.simonneau@univ-orleans.fr

Cécile Miège
email: cecile.miege@irstea.fr

Marc Babut
email: marc.babut@irstea.fr

Spatial and temporal trends in PCBs in sediment along the lower Rhône River, France

Keywords: Polychlorinated biphenyl (PCB), sediment core, Rhône River, trend

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Introduction

High levels of polychlorinated biphenyls (PCBs) have been measured repeatedly in sediment and fish in the Rhône River, particularly downstream from the city of Lyon [START_REF] Babut | Correlations between dioxin-like and indicators PCBs: Potential consequences for environmental studies involving fish or sediment[END_REF][START_REF] Santiago | Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River[END_REF], the largest city in the basin (metropolitan area population of 1.7 million) and the third largest in France. PCBs are ubiquitous environmental contaminants because of their extensive use from the 1930s to the 1980s and their environmental persistence (U.S.

Environmental Protection Agency, 2004Agency, , 2011)). In France, PCB use was banned in 1975 for open systems (e.g., pesticides, coatings) and in 1986 for closed systems (e.g., transformers, capacitors) [START_REF] Chevreuil | Polychlorobiphenyl behavoir in the water/sediment system of the Seine River, France[END_REF]. Contamination of sediment and fish by PCBs in the Rhône River system was reported as early as the 1980s [START_REF] Andre | Qualité du fleuve Rhone, Synthese des connaissances[END_REF][START_REF] Brodhag | PCB livre blanc, ou le Pyralène dans tous ses états[END_REF].

Since 2005, however, concern for human exposure to PCBs through fish consumption has risen greatly as sampling continues to identify streams in the Rhône basin with PCB-contaminated fish. Currently, commercial fishing and consumption of selected species of fish are banned as a result of PCB contamination in the main channels of the Rhône and Saône Rivers (the largest tributary to the Rhône) from upstream from Lyon to the Mediterranean Sea, and there are full bans of all fish species along reaches of numerous tributaries [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF]. All of these bans have been imposed since 2005 and most were imposed since 2009, including one in 2011, on the basis of new or continued identification of fish with elevated concentrations of PCBs (Saônor.fr, 2011; Agence national de sécurité sanitaire, 2011).

PCBs are strongly hydrophobic and sediments therefore are the primary reservoir of PCBs in aquatic systems [START_REF] Wong | National-scale, field-based evaluation of the biota-sediment accumulation factor model[END_REF]. Most mechanistic studies of food webs consider sediment ingestion as a major exposure route for PCBs for aquatic biota (e.g., [START_REF] Gewurtz | Use of a food web model to evaluate the factors responsible for high PCB fish concentrations in Lake Ellasjøen, a high Arctic Lake[END_REF][START_REF] Morrison | Development and verification of a benthic/pelagic food web bioaccumulation model for PCB congeners in western Lake Erie[END_REF]. [START_REF] Lopes | PCB contamination pathways of freshwater fish: When spatial contamination gradients drive fish consumption advisories[END_REF] demonstrated that PCB concentrations in fish in the Rhône near Lyon were significantly related to PCB concentrations in Rhône bed sediment.

Using a statistical model, [START_REF] Babut | Transferts de contaminants hydrophobes du sediment au biote: construction de modèles dans un perspective de gestion (Transfer of hydrophobic contaminants from sediment to biota: construction of models for environmental management)[END_REF] determined that the maximum PCB I (sum of concentrations of seven indicator PCBs [START_REF] Order | Concerning the maximum dioxin and polychlorinated biphenyl content in certain foodstuffs[END_REF]; non-detections treated as zeros) in sediment that corresponds to an acceptable PCB level in 90% of Rhône River fish is 5.9 g/kg, a concentration well below concentrations of PCBs reported historically (1989) for the Rhône [START_REF] Santiago | Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River[END_REF]. Knowledge of long-term trends in PCBs might contribute greatly to an improved understanding of long-term risks and of management options in the basin. Longterm data sets (e.g., 20-30 y) for PCBs in water, sediment, and fish, however, are not available for most large rivers, including the Rhône. Sedimentary archives such as sediment cores have been used to reconstruct PCB trends in many systems (e.g., [START_REF] Alcock | Long-term changes in the polychlorinated biphenyl (PCB) content of United Kingdom soils[END_REF][START_REF] Eisenreich | Accumulation and diagenesis of chlorinated hydrocarbons in lacustrine sediments[END_REF][START_REF] Grimalt | Persistent organochlorine compounds in soils and sediments of European high altitude mountain lakes[END_REF][START_REF] Van Metre | Trends in hydrophobic organic contaminants in lake sediment across the United States, 1970-2001[END_REF], but because sediment-core reconstructions require long-term depositional settings (e.g., lakes), with the exception of a few studies in large fluvial reservoirs [START_REF] Rosen | Assessment of multiple sources of anthropogenic and natural chemical inputs to a morphologically complex basin, Lake Mead, USA[END_REF][START_REF] Van Metre | Historical trends in organochlorine compounds in river basins identified using sediment cores from reservoirs[END_REF], river deltas [START_REF] Mai | Abundances, depositional fluxes, and homologue patterns of polychlorinated biphenyls in dated sediment cores from the Pearl River Delta, China[END_REF], and oxbow lakes [START_REF] Bábek | Contamination history of suspended river sediments accumulated in oxbow lakes over the last 25 years, Morava River (Danube catchment area), Czech Republic[END_REF], this approach is rarely attempted in rivers.

In terms of mean annual discharge, the Rhône River is the second largest river in France and the third largest river in Europe [START_REF] Pekarova | Long-term trends and runoff fluctuations of European rivers. Climate Variability and Change-Hydrological Impacts[END_REF]. The mean daily discharge in the Rhône just downstream from the confluence with the Saône is 1,040 m 3 /s (1966 to 2009) (Le Rhône à Ternay, streamflow-gauging station V3130020 (http://www.hydro.eaufrance.fr/)) (fig. 1; SI fig.

S-1). The watershed of the Rhône is largely mountainous and covers 97,800 km² with marked climatic and geological heterogeneity [START_REF] Desmet | Holocene Climate in the Alps: towards a common framework -an introduction[END_REF]. The Rhône is a complex hydrologic system and several factors contribute to the distribution of sediment contamination, including:

(i) multiple tributaries and complicated watershed morphology, (ii) urban areas and industrial zones providing historical and current potential sources of contamination, (iii) extensive anthropogenic modification of the hydrology, and (iv) frequent flooding and sediment-flushing events that redistribute sediment. Since the Second World War, flow and sediment transport in the Rhône have been affected by construction of numerous dams (18 in France between Lake Geneva and the Mediterranean Sea) accompanied by structures for navigation, dike construction, and channel dredging [START_REF] Andre | Qualité du fleuve Rhone, Synthese des connaissances[END_REF][START_REF] Pont | Medium-term changes in suspended sediment delivery to the ocean: Consequences of catchment heterogeneity and river management (Rhône River, France)[END_REF].

Accumulation of sediment behind the dams has led to periodic "flushing" (releases of impounded water) to clear sediment deposits, carried out approximately triennially from 1965 to 2003.

There are numerous potential sources of PCBs to the Rhône and its tributaries. These include industrial facilities with probable historical PCB discharges, PCB-contaminated sites and soils, and reported PCB spills (Robin des [START_REF] Des | Atlas des sites terrestres pollués aux PCB[END_REF]. A synoptic survey of suspended sediments in the Rhône in 1989 found that PCB concentrations were much higher downstream from Lyon than upstream [START_REF] Santiago | Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River[END_REF], which the authors attributed to urban nonpoint sources and to inputs from the heavily industrialized zone downstream from Lyon. Major tributaries also might contribute to PCBs in the Rhône: the Bourbre, the Saône, and the Gier Rivers (fig. 1) all have partial or full commercial fishing and fish consumption bans as a result of PCB contamination [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF]. A hazardous-waste treatment facility about 40 km upstream from Lyon (fig. 1) also has been identified as a potential PCB source [START_REF] Babut | Correlations between dioxin-like and indicators PCBs: Potential consequences for environmental studies involving fish or sediment[END_REF][START_REF] Santiago | Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River[END_REF]. This hazardous-waste treatment facility is one of two PCB incineration facilities in France and is authorized to release small amounts of PCBs into the Rhône [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF].

Concerns about PCB contamination in the Rhône basin are prompting major sampling efforts to characterize PCB occurrence, identify sources, and understand trends [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF]. As one of these efforts, the study presented here evaluated trends in PCBs in Rhône River sediments in the area of Lyon relative to land use, river-management practices, and restrictions on PCB releases. Recent (20-50 y) trends in PCBs in sediment were evaluated on the basis of sediment cores collected from depositional zones adjacent to the channel of the Rhône upstream and downstream from the PCB treatment facility.

Methods

Sampling locations

Sediment-core collection sites were chosen to evaluate the upstream-downstream distribution and temporal trends of PCB contaminated sediment and to assess the potential effects of the PCB treatment facility, the city of Lyon, and major industrial areas in the watershed on the sediment quality of the Rhône (fig. 1). Sediment cores were collected during May to July 2008 at three sites from 90 km upstream from Lyon to 40 km downstream (in upstream to downstream order, followed by their short names: La Morte, MTE; Crépieux-la-Pape, CPX; and Ile du Beurre, BRE), and in 2010 at a rural reference site in the interior of the Rhône basin (Lake Paladru, PAL). Additional site information, maps, and aerial photos are provided in Supporting Information.

Core collection and sediment description

Sediment cores were collected with a UWITEC® gravity corer fitted with a 1.20-m long, 63-mm diameter plastic barrel with a core catcher at the bottom. Using an extended rod, the corer was pushed gently into the sediments from a boat. Multiple cores were collected at sites MTE, CPX, and BRE and one core was collected at site PAL. The cores ranged in length from 52 to 112 cm. At the laboratory, cores were sub-sampled at a 1-cm interval with the exception of the core from site PAL, which was sampled at a 0.5-cm interval. For PAL and MTE cores, subsamples from one core were analyzed for PCBs, total organic carbon (TOC), grain-size distribution, and radionuclides. At sites CPX and BRE, sub-samples from one core (herein the primary core) were analyzed for PCBs, TOC, and grain-size distribution and samples from a second core (herein the radionuclide core) were analyzed for grain-size distribution and radionuclides. Grain-size distribution was used at sites CPX and BRE to help correlate the primary and radionuclide cores so that dates could be assigned to the primary core.

Dry bulk density (g/cm 3 ) was determined as the difference between wet and dry mass divided by the volume of the container. High-resolution grain-size distributions were determined by sonicating and then analyzing each sample with a Mastersizer 2000® laser mounted with a hydro SM small volume dispersion unit. Grain-size mean, mode, sorting, and skewness were computed. Cumulative volumetric percentages of sand (>63 µm), silt (4-63 µm), and clay (<4 µm) were determined for each depth interval.

Analytical methods

Sediment-core samples were analyzed for radionuclides at the Laboratoire des sciences du climat et de l'environnement, Gif sur Yvette, France. Following drying, sub-samples from each core were analyzed for radionuclides by counting for at least 24 hours using gamma spectrometry. Gamma emissions were detected with a germanium detector and used to quantify 226 Ra, 210 Pb, and 137 Cs [START_REF] Pinglot | Radioactivity measurements applied to glaciers and lake sediments[END_REF].

Samples were prepared for measurement of total organic carbon by drying, sieving to 2 mm, and grinding to <200 m, in accordance with standard NF ISO 11464 (Groupement d'Etudes Méthodologiques pour l'Analyse des Sols, 2011), and acidifying with aqua regia. Total organic carbon was analyzed by high-temperature combustion (1,250°C) in accordance with standard NF ISO 10694 (Groupement d'Etudes Méthodologiques pour l'Analyse des Sols, 2011).

PCBs were analyzed by the EUROFINS laboratory, Orleans, France (www.eurofins.fr). PCBs were extracted in accordance with the certified method QMA 504-192 (DIN ENISO/IEC 17025:2000) (http://www.graie.org/osr/IMG/pdf/RS2_web.pdf). In brief, 5 g of dried, homogenized sediment were extracted for a minimum of 8 hours with toluene/acetone (90/10 v/v) in a Soxhlet apparatus. After addition of PCB-155 as an internal standard, the sample extract underwent a clean-up step using gel permeation chromatography. Clean-up consisted of sequential passes through silica, aluminium oxide, Florisil, and active carbon columns. The sample extract was analyzed by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) in selected ion monitoring (SIM) mode. For the seven indicator PCBs, quantification limits ranged from 0.042 g/kg (PCBs 118 and 180) to 0.146 g/kg (PCB 153). Laboratory quality control consisted of periodic analysis of blanks and certified reference materials. Analytical results for environmental samples were not blank-corrected.

Eighteen PCB congeners were quantified (SI table S-1). Interpretations herein are based on the sum of the seven so-called indicator PCBs (PCB I ): International Union of Pure and Applied Chemistry (IUPAC) numbers 28, 52, 101, 118, 138, 153, and 180, the sum of which is commonly used in European national legislation to ensure food safety [START_REF] Order | Concerning the maximum dioxin and polychlorinated biphenyl content in certain foodstuffs[END_REF]. These congeners are generally considered persistent and are thought to be good markers for human exposure to PCBs through ingestion [START_REF] Wingfors | Identification of markers for PCB exposure in plasma from Swedish construction workers removing old elastic sealants[END_REF]

Age dating of sediment

The date of deposition of sediment intervals within each core (SI figs. S-6 to S-9) was estimated on the basis of radionuclide profiles and, for Rhône sediment cores, on correlation of changes in grain-size distribution in the sediment cores to the timing of major floods and to major changes in river hydrology. The 137 Cs profile was used to determine one or two age-date markers in each core. 137 Cs was released into the atmosphere by nuclear weapons testing from 1952 to 1963 [START_REF] Beck | Estimates of fallout in the continental U.S. from Nevada weapons testing based on gummed-film monitoring data[END_REF]. In 1986, the accident at the Chernobyl nuclear power plant in Ukraine resulted in relatively large releases of 137 Cs, and about 79% of the total fallout occurred over Europe [START_REF] Anspaugh | The global impact of the Chernobyl reactor accident[END_REF]. Two date-depth markers thus can be identified in European sediment cores: the weapons testing peak in 1963 and the Chernobyl peak in 1986. The radionuclide 210 Pb was not used as a primary age-dating tool because the riverine setting of the Rhône coring sites leads to violation of the principle assumptions of 210 Pb dating models [START_REF] Appleby | Application of lead-210 to sedimentation studies[END_REF]. However, the 210 Pb profiles provided some corroboration of the dating and the chronological integrity of the cores. The occurrence of floods and associated changes in grain-size distribution in the cores provided support for the 137 Cs dating, provided additional depth-date markers at site CPX, and were the basis for correlating the primary and radionuclide cores at sites CPX and BRE for the purposes of age-dating (SI figs. S-8 and S-9). Dates were assigned to intervals in the cores between date markers assuming a constant sediment mass accumulation rate (MAR; g/cm 2 -yr) between the date markers.

Results

Sediment age-dating

Primary date markers used to estimate sediment deposition dates in the cores were the date of site construction or of connection to the Rhône (sites CPX and BRE), peaks in 137 Cs, and changes in grain-size distribution corresponding to major flood events (SI figs. S-6 to S-9). A large or sudden increase in grain size (i.e., a sudden decrease in the percentage of fine-grained fraction of material, i.e., less than 63 microns diameter, herein referred to as fines) followed by a gradual increase in the percentage of fines was assumed to be the result of a flood event, although only a few very pronounced changes in grain size were used as date markers in cores.

At the two sites where radionuclides and PCBs were analyzed in separate cores (sites CPX and BRE), the cores were correlated for dating purposes on the basis of changes in grain size. Age dates for each sediment sub-sampling interval were based on assumption of a constant sediment MAR between date markers.

Age dates for core PAL were based on two distinct peaks in 137 Cs at depths of 8-8.5 and 3.5-4 cm (SI fig. S-6), interpreted as corresponding to nuclear-weapons testing (1963) and the Chernobyl accident (1986). The MAR during 1964-86 was 0.30 g/cm 2 -yr, and during 1986-2010 was 0.27 g/cm 2 -yr. Use of the 137 Cs peaks for age dating resulted in slightly older dates (about 2 y for most intervals) than those estimated using the 210 Pb constant-flux, constant-supply model [START_REF] Appleby | Application of lead-210 to sedimentation studies[END_REF], but because the 137 Cs peaks were so marked, this age-dating approach was preferred.

Age dates for core MTE were based on a 137 Cs peak at 25-26 cm interpreted as the Chernobyl accident and a rise in 137 Cs at the bottom of the core interpreted as being associated with the weapons-testing peak in 1963 (SI fig. S-7). The Chernobyl peak is well defined, but the weapons-testing peak is indicated only by an increase to a maximum at the bottom of the core.

The deepest sample in the core has a 137 Cs activity about 5 times greater than do samples in the middle of the core, but the 137 Cs peak may have been at a depth deeper than that sampled by the core. The MAR during 1964-1986 was computed as 1.1 g/cm 2 -yr, and during 1986-2008 as 0.48 g/cm 2 -yr. The decrease in sedimentation rate after 1986 might have resulted from the construction of the dam at Champagneux in 1984 upstream from site MTE (fig. 1).

For site CPX, age dates for the radionuclide core (CPX 08-02) were estimated on the basis of the 137 Cs profile and on variations in grain size that were matched with floods (SI fig. S-8). A single pronounced 137 Cs peak at 85-86 cm was interpreted as resulting from the Chernobyl accident.

A 137 Cs peak corresponding to nuclear weapons testing was not expected because the site was not formed until the early 1980s. Episodic large decreases in percentage of fines between about 57 and 77 cm in the core, reaching minima of about 20% (80% sand), were interpreted as 1984-1986 was computed as 7.1 g/cm 2 -yr. The higher MAR from 1984-1986 might be caused by the channel-clearing operations in 1984. Prior to 1984, the BRE site was not directly connected to the Rhône, the sediments were uniformly fine-grained, and no date markers were evident in either core. The primary core (BRE 08-01) was correlated to the radionuclide core (BRE 08-03) using grain-size distribution, specifically by matching the rapid transition from the thick sequence of silt and clay deposits deep in the cores to coarser and much more variable sequences above (SI fig. S-9). PCBs were detected to the bottom of the primary core (deepest sample at 104-105 cm), but the samples from before 1984 to the bottom of the core are not presented here because they are assumed to represent sediment originating elsewhere than the Rhône.

Recent trends in PCBs

PCB I concentration profiles at the four sites differ both in timing and magnitude of peak concentrations (fig. 2; SI Table 1). In general, PCB I concentrations at the rural (PAL) and upstream (MTE) reference sites peaked in the 1970s and decrease to present. Although PCB I concentrations for the 1970s are not known at the downstream sites (CPX and BRE), at these sites there are large peaks in PCB concentrations in about 1990, followed by a rapid decrease in concentration in the early 1990s; PCB concentrations have remained relatively stable since the mid-1990s (fig. 2). Peak and current PCB I concentrations are lowest at the PAL rural reference lake site and increase in the Rhône in a downstream direction. PCB I concentrations in sediment deposited at site PAL peaked in 1976 at a concentration of 13 g/kg (fig. 2). Concentrations decreased rapidly from 1976 to 1985 before leveling off for about 10 years, then decreased steadily from 1998 to 2.2 g/kg at the top of the core (2010). Organic carbon-normalized concentrations (PCB I /TOC) were used to determine rates of change in concentrations (fig. 3); the normalization was done to exclude variations associated with the textural properties of the particles and enable between-site comparison. An exponential curve fit to PCB I /TOC from the peak to present (r 2 =0.90) indicates a half-life (t 1/2 ) of 12.6 years. The half-life determined here is not a rate of chemical decay but rather is a measure of the rate of change in PCB inputs to the coring site. The decrease in PCB I from 1998 to 2008 is more rapid and linear than the long-term trend, with a t 1/2 of 6.2 y (r 2 =0.94). The reason for a change in the rate of decrease in about 1998 is not known, but might be related to sediment dilution caused by anthropogenic activities in the watershed, such as construction, that mobilized deeper, uncontaminated sediment.

At site MTE, PCB I concentrations are elevated and variable in sediment deposited during the 1960s and 1970s, and the maximum concentration of 78 g/kg is 6 times higher than at the PAL reference site (fig. 2). Carbon-normalized PCB I concentration decreases exponentially from 1964 to the top of core MTE ( 2008), with a half-life of 8.5 years (r 2 =0.87) (fig. 3). The concentration in the most recently deposited sediment is 3.5 g/kg. The decrease in PCB I since 2001 is slightly more rapid than for the whole period of the core, with a t 1/2 for this 8-year period of 6.4 years (r 2 =0.99).

In contrast to PCB I profiles at sites PAL and MTE, concentrations at site CPX are elevated and variable in the early 1990s, followed by a very rapid decrease in the mid-1990s and relatively stable concentration since (fig. 2). Maximum PCB I concentrations (72 to 84 g/kg) occur in sediment deposited during 1991-93. Since the decrease in the mid-1990s, PCB I concentration varies around a central tendency of 15 g/kg to the top of the core. Concentrations in Rhône sediment along this reach of the river prior to 1990 are not known. Although there is no significant trend in PCB I concentrations from about 1993 to the top of the core in 2008 (Kendall's tau test of correlation, p=0.13), there are two brief increases followed by longer (~6y) decreases within this time period (fig. 2). The rate of decrease from 2003 to 2008 following the small increase in 2003 is virtually identical to that of the longer-term rate of change during 1991-2000 (t 1/2 of 2.4 and 2.5 y, respectively; fig. 3). The minimum PCB I concentration (8.9 g/kg) occurs in the most recently deposited sediment.

Similar to the profile from site CPX, PCB I concentrations at site BRE are elevated into the early 1990s then decrease rapidly (fig. 2). Concentrations reach a maximum of 282 g/kg in 1991, about 3 times higher than that at site CPX, then decrease to concentrations that vary between 20 and 55 g/kg. Also similar to site CPX, the sediment core does not record concentrations in Rhône sediments prior to the early 1980s. There was no significant change in concentrations of PCB I from 2001 to 2008 (fig. 3).

PCBI congener profiles

Profiles of the seven indicator PCBs at the three Rhône sites were constructed for three time windows chosen to reflect periods of peak concentrations at site MTE (1965MTE ( -1980) ) and at sites CPX and BRE (1986-93) and recent concentrations (2000-08) (fig. 4). The profiles represent the mean proportion of each congener contributing to PCB I for the samples in the time window.

Core profiles for the time windows were relatively similar at the three sites (fig. 4). In all cases, PCB 153 made up the highest proportion (typically 25-30%) and PCB 28 made up the lowest proportion (typically <5%) of PCB I . Profiles for site PAL, however, differ somewhat from those at the other three sites in that proportions of the less chlorinated congeners (PCBs 52, 101, 118) are more elevated and those of the more chlorinated congeners (PCBs 153 and 180) are less elevated (fig. 4), consistent with preferential volatilization and atmospheric transport of the less chlorinated congeners [START_REF] Wania | Global fractionation and cold condensation of low volatility organochlorine compounds in polar regions[END_REF]. In contrast, the profiles for sites MTE, CPX, and BRE fall within nearly identical ranges. Similar differences between the profiles for the 1986-93 and 2000-08 time windows at sites CPX and BRE (lower proportions of PCB 28 and higher proportions PCB 138 during the 2000-08 time window) and slightly higher proportions of PCB 180 relative to those at sites PAL and MTE suggest that sites CPX and BRE might have one or more PCB I sources that changed over time and(or) are different from those at sites PAL and MTE. The differences, however, are relatively subtle, and a thorough forensic analysis of PCB profiles would entail use of a much larger suite of congeners.

Discussion

The use of sedimentary archives to reconstruct contaminant trends has a long history [START_REF] Davis | The scope of Quaternary paleolimnology[END_REF]. With the exception of studies using cores from reservoirs (e.g., [START_REF] Callender | Transport and accumulation of radionuclides and stable elements in a Missouri River reservoir[END_REF][START_REF] Van Metre | Historical trends in organochlorine compounds in river basins identified using sediment cores from reservoirs[END_REF], however, the approach is rarely applied to riverine settings because of the requirement that sediments remain undisturbed at the timescale of interest, often decades. The reservoirs created by dams on the Rhône were flushed approximately triennially to remove deposited sediment, and therefore are assumed to be unsuitable for coring. However, in a large river system such as the Rhône, sediment may accumulate over long periods in settings other than reservoirs. Here we have located three such sites that are demonstrated, on the basis of their radionuclide profiles, to have accumulated sediment over about 25-50 years. Two of the sites (MTE and BRE) are in abandoned channels (oxbows) of the Rhône that remain connected to the main channel and one site (CPX) is in an abandoned sand and gravel quarry that is open to an active channel of the Rhône. Compared with cores from undisturbed natural lakes, the age dating of the riverine cores is not ideal, as indicated by the comparison of radionuclide profiles at site PAL to those at the other three sites (SI figs. S-6 to S-9). We contend, however, that because the dating is constrained by known date markers, it is adequate for the reconstruction of recent (25-50 y) PCB concentration trends, and that this reconstruction provides a valuable perspective where historical data do not exist.

Differences in trends among sites and implications for sources

PCB trends vary considerably among the sites sampled here (fig. 2). The temporal trends at the rural (PAL) reference lake site and upstream (MTE) Rhône reference site are similar to those of global PCB emissions [START_REF] Breivik | Towards a global historical emission inventory of selected PCB congeners --a mass balance approach 2[END_REF] and widely reported patterns in PCB contamination, with the highest concentrations coinciding with maximum use and environmental release in the 1960s and 1970s and systematic decreases since (e.g., [START_REF] Eisenreich | Accumulation and diagenesis of chlorinated hydrocarbons in lacustrine sediments[END_REF][START_REF] Van Metre | Historical trends in organochlorine compounds in river basins identified using sediment cores from reservoirs[END_REF][START_REF] Van Metre | Trends in hydrophobic organic contaminants in lake sediment across the United States, 1970-2001[END_REF][START_REF] Jung | Temporal evolution of urban wet weather pollution: Analysis of PCB and PAH in sediment cores from Lake Bourget, France[END_REF][START_REF] Iozza | Historical profiles of chlorinated paraffins and polychlorinated biphenyls in a dated sediment core from Lake Thun (Switzerland)[END_REF]. The marked decreases in PCBs at these two sites beginning in the late 1970s are consistent with the interdiction of PCB use in open systems in France in 1975 (Journal Officiel, 1975).

The two sites downstream from Lyon (CPX and BRE) had high and variable concentrations in the late 1980s and early 1990s followed by rapid decreases into the mid-1990s, after which they remained relatively stable (fig. 2). The shape of the PCB profiles at sites CPX and BRE differ from the monotonic downward trend recorded at sites PAL and MTE from the mid-1980s on and are inconsistent with widely reported downward trends in PCBs beginning in the 1970s (e.g., [START_REF] Van Metre | Similar rates of decrease of persistent, hydrophobic contaminants in riverine systems[END_REF][START_REF] Iozza | Historical profiles of chlorinated paraffins and polychlorinated biphenyls in a dated sediment core from Lake Thun (Switzerland)[END_REF]. The rapid decreases in PCBs at sites CPX and BRE following peak concentrations in about 1990 indicate point-source releases likely occurred contemporaneous with those peaks. PCB I concentrations at sites CPX and BRE decreased by 75% or more in the early to mid-1990s, and remained low thereafter (with the exception of one questionable sample collected from a depth of 36.5 cm in ~1997 at site BRE [fig. 2,). Such a sudden and substantial decrease in the 1990s, 15 or more years after peak use, would be expected only if a relatively large source had been recently discontinued. Although flooding in the early 1990s might have played a role in variations in PCB concentrations at these two sites, it had little apparent effect at site MTE upstream, and subsequent floods in 2001 and 2002 did not cause substantial changes in concentration at either of sites CPX or MTE.

Although the trends are anomalous relative to those at many sites, industrial releases of PCBs might have resulted in a later initiation of downward trends following regulation of production and use. Elevated concentrations of PCBs into the 1990s were reported for a sediment core from Lake Maggiore in northern Italy, which has a heavily industrialized and densely populated watershed [START_REF] Guzzella | Contamination by polybrominated diphenyl ethers of sediments from the Lake Maggiore basin (Italy and Switzerland)[END_REF], and a lack of a downward trend was reported for PCBs in sediment cores from an oxbow in the Morava River (Czech Republic), the basin of which has historical industrial land use [START_REF] Bábek | Contamination history of suspended river sediments accumulated in oxbow lakes over the last 25 years, Morava River (Danube catchment area), Czech Republic[END_REF]. One limitation to this interpretation, which is a consequence of the difficulty in finding long-term depositional sites in a river system, is that the cores of Rhône sediment do not extend back in time to the 1960s and 1970s. It therefore is not known if the concentrations in the Rhône in and downstream from Lyon were even greater before the 1980s.

There at least two potential PCB sources in the reach of the Rhône between sites MTE and CPX.

The first is the Bourbre River (fig. 1), for which an elevated sediment PCB I concentration (~200 g/kg) in a sediment sample collected in 1997 was reported [START_REF] Babut | Contamination des poissons et des sédiments du Rhône par les polychlorobiphényles[END_REF], and which currently has a ban on fish consumption [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF]. The watershed of the Bourbre includes several semi-industrial towns. A second potential PCB source is the hazardous-waste treatment facility upstream from Lyon (fig. 1). Because PCBs are treated and disposed of at this facility, its regulation is different from that of PCB use in France. It is not known how actual discharges from the hazardous-waste treatment facility compared to permitted limits from 1987-97, but the magnitude and timing of the permitted limits from 1987 to present is consistent with temporal trends in PCBs at sites CPX and BRE. The hazardouswaste treatment facility began operating in 1985, but PCB releases were not regulated until 1987; the first limit established in 1987 was 1.5 kg/d, which was reduced to 500 g/d in 1992, 200 g/d in 1995, and to 10 g/d in 2007 (Préfecture de la Région Rhône-Alpes, 2007). Actual releases, not reported until 1997, are very low (2-8 g/d in 1997 reduced to 0.17 g/d in 2010) [START_REF] Fruget | Acceptabilité écologique du rejet dans le Rhône de l'usine TREDI Saint Vulbas en ce qui concerne les PCB[END_REF]. An additional consideration is the construction of the dam at Villebois in 1986, downstream from site MTE and upstream from the hazardous-waste treatment facility and the confluence of the Rhône and Bourbre Rivers (fig. 1). The dam could have trapped relatively uncontaminated sediment from upstream, resulting in less dilution of sedimentassociated PCB sources in the Rhône downstream after 1986.

Releases of PCBs to the Rhône upstream from site CPX, however, cannot fully explain the trends observed at site BRE, which indicates some unknown source in the city of Lyon or the adjacent industrial corridors. Mean PCB I concentrations during 1990-1993 in the BRE core were 165 g/kg, about 3 times greater than those for the same period in the CPX core (59 g/kg). Also, recent trends between sites CPX and BRE differ: There is a systematic decrease at site CPX since 2003 but an increase at site BRE since 2006 (fig. 2). The watershed of the Rhône at site BRE is much larger than at site CPX, as it is downstream from the confluence with the Saône and other tributaries (fig. 1), thus, considerable dilution of a point source upstream from site CPX is expected at site BRE. Mean flow in the Rhône 2 km upstream from the confluence with the Saône is 593 m 3 .s -1 (Le Rhône à Lyon (Perrache), station V3000015) compared with 1,040 m 3 .s -1 in the Rhône downstream from Lyon at Ternay (station V3130020) (http://www.hydro.eaufrance.fr/). The substantial increase in PCB I concentrations during 1986-1993 in the downstream direction and the difference in recent trends can only be explained by additional PCB sources in the intervening reach. Potential sources include the city of Lyon, the industrial corridor downstream from Lyon, and the Saône and Gier River watersheds (fig. 1), consistent with sources evoked by [START_REF] Santiago | Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River[END_REF].

Hydrologic controls on PCB concentrations

Sediment flushing events and floods likely have some effect on PCB distribution and trends in the Rhône watershed. Possible effects of the triennial flushing events are evident at site MTE, where episodic increases in PCBs (fig. 2) and organic carbon (not shown) from 1965 to the mid-1980s occur at intervals similar to those of the flushing events. Effects of major floods are evident at site CPX: A large decrease in percentage of fines in about 1990, assumed to be caused by the 1990 flood, corresponds to a sharp decrease in PCB I concentration, followed by a large increase in the fine fraction, from 20 to 80% (SI fig. S-8), and a correspondingly large increase in PCB I concentration, from 18.8 to 83.5 g/kg (fig. 2,SI table 1). This pattern appears to repeat to a lesser degree with the 1992 and 1993 floods (decrease in grain size and PCB I concentrations, although grain-size data are not available for several intervals in this part of the core). The large increase in PCB I concentrations following the return to finer sediment size, however, does not recur, possibly because a localized source was eliminated. Smaller shifts in grain size and PCB I concentration after 1994 appear to be related, but the relation between the two is inconsistent.

Spatial and temporal PCB concentrations in sediment and implications for fish consumption

Sediment cores from multiple locations in a river system can indicate spatial and temporal variations in contamination [START_REF] Callender | The urban environmental gradient: Anthropogenic influences on the spatial and temporal distributions of lead and zinc in sediments[END_REF]. Concentrations of PCBs in sediments in the Rhône increase substantially in the downstream direction as the river passes through industrial and urban areas for all time windows analyzed (fig. 5). The median PCB I concentration during 1986-1993 was 9 times higher at site BRE than at site MTE, and from 2000-2008 was 6 times higher. Temporally, the decrease in PCB I concentration from one time window to the next is approximately proportional between sites (fig. 5). The relevance of these spatial and temporal patterns from the standpoint of fish contamination is reflected in PCB I concentrations in bream (Abramis brama) [START_REF] Babut | Transferts de contaminants hydrophobes du sediment au biote: construction de modèles dans un perspective de gestion (Transfer of hydrophobic contaminants from sediment to biota: construction of models for environmental management)[END_REF], which follow a similar upstreamdownstream pattern (fig. 5). The fish were collected in 2008 at sites MTE and BRE, and at a third site, the Grand Large, which is in another channel of the Rhône a few kilometers from site CPX and a similar distance upstream from Lyon (fig. 1). PCB I concentrations are similar in surface sediments from Grand Large (median 18.5 g/kg) [START_REF] Babut | Transferts de contaminants hydrophobes du sediment au biote: construction de modèles dans un perspective de gestion (Transfer of hydrophobic contaminants from sediment to biota: construction of models for environmental management)[END_REF] and recent sediments (past 10 y) in the CPX core (median 13.7 g/kg). The consistent relation between recent sediment and fish concentrations and the temporal trends in sediment concentrations indicate that PCB concentrations in fish 20 to 40 years ago likely were much higher than they are now.

Long-term downward trends in PCB concentrations at reference sites PAL and MTE and rapid decreases after highs in the early 1990s at sites CPX and BRE indicate that regulatory changes likely have contributed to a reduction in PCB contamination in the Rhône. Decreases in PCB concentrations at the downstream sites, however, appear to have slowed or stopped in the past ~15 years, and current concentrations (2011) are above the concentration of 5.9 g/kg identified by [START_REF] Babut | Transferts de contaminants hydrophobes du sediment au biote: construction de modèles dans un perspective de gestion (Transfer of hydrophobic contaminants from sediment to biota: construction of models for environmental management)[END_REF] as the maximum sediment concentration corresponding to acceptable concentrations in 90% of fish. The results of this study indicate that PCB contamination in the lower Rhône basin is likely to remain of concern for years to come. [START_REF] Babut | Transferts de contaminants hydrophobes du sediment au biote: construction de modèles dans un perspective de gestion (Transfer of hydrophobic contaminants from sediment to biota: construction of models for environmental management)[END_REF]. Also shown are locations of two urban areas relative to the main stem of the Rhône and con uences with major tributaries ( g. 1), and locations of known or suspected PCB sources (Robin des Bois, 2011). The site is in a small side channel in a forested wetland adjacent to the Rhône River, 40 km downstream from Lyon (Fig. 1, full text). This site was chosen because of its location downstream from Lyon and the industrial corridor that extends from south of Lyon to the confluence with the Gier River, which, like the Bourbre River, has a full fish consumption ban as a result of PCB contamination [START_REF] Eaufrance | L'eau dans le bassin Rhône-Méditerranée[END_REF]. Before 1977, the side channel was not connected to the Rhône River. In 1977, construction of the dam at Saint Pierre de Boeuf downstream from lle Beurre raised the water level in the river to that of the side channel, and in 1984 a debris dam separating the side channel and the Rhône was removed, allowing connection between the two water bodies (Eric Doutriaux, Départment du Patrimoine Fluvial, Pôle Hydraulique et Environnement, Compagnie Nationale du Rhône, 2011, written communication).

  corresponding to three large floods in the early 1990s (SI fig. S-1). More modest decreases in percentage of fines occurred in the upper part of the core, the largest of which was from 14 to 19 cm and was interpreted as corresponding to floods in 2001 and 2002. These date markers indicate an MAR of 2.3 g/cm 2 -yr during the 1980s, 5.0 g/cm 2 -yr during the early 1990s flooding, and 1.8 g/cm 2 -yr after the 2001-2002 flooding. No major floods have occurred on the Rhône in the reach investigated since 2002 (SI fig. S-1). The river has been building a natural levee at the mouth of the site CPX quarry since the quarry was abandoned (SI fig. S-4), consistent with the observed decrease in MAR and increase in fines over time. The interval near the bottom of the radionuclide core with a dip in percentage of fines (to 22%) is dated as 1983.9, coinciding with a large flood in 1983. The primary core (CPX 08-01) was correlated to the radionuclide core (CPX 08-02) using grain-size distribution (measured in both cores), specifically by matching the thick sequence of sand deposits that occurred in both cores at about 70-cm depth and a series of maxima and minima in percent fines that occurred in the upper 40-45 cm of the cores (SI fig. S-8). For site BRE, age dates for the radionuclide core (BRE 08-03) were based on a 137 Cs peak at 53 cm depth and the pronounced change in grain-size distribution at 70-cm depth (SI fig. S-9). The 137 Cs peak was interpreted as corresponding to the Chernobyl accident. The change in grainsize distribution at 70 cm was interpreted as corresponding to removal of debris in 1984 that established a connection to the Rhône at the upper end of the side channel where the BRE core was collected. The MAR during 1986-2008 was computed as 3.1 g/cm 2 -yr and the MAR from

Figure 1 .Figure 2 .FishFigure 5 .

 125 Figure 1. Map of the study area, Rhône basin, France.

Figure 4 .

 4 Figure 4. Pro les of the seven indicator PCBs at the three sediment coring sites along the Rhône River, France, for three time windows. Each congener is expressed as its proportional contribution the sum (PCB I ). The grey area indicates the range of pro les for the rural reference site Lake Paladru.

Fig

  Fig. S-1. Rhône River daily mean discharge, as measured at the streamflow-gaging station at Ternay, near the city of Lyon

Fig. S- 3 .

 3 Fig. S-3. Map and aerial image of the coring site at La Morte (MTE). La Morte is the most upstream of the three coring sites on the Rhône River (Fig.1, full text). It is located in a pond at the downstream end of a cutoff meander of the Rhône, which is open to the river at its downstream end. La Morte is in a relatively non-industrialized part of the Rhône basin upstream from the hazardous-waste treatment facility and Lyon. It was chosen to represent reference concentrations for the lower Rhône basin (i.e., downstream from Lake Geneva). There are, however, urban and industrial areas in the upper Rhône valley in Switzerland (upstream from Lake Geneva) and smaller urban areas and a few historical contamination sites between Lake Geneva and La Morte that might affect PCB concentrations at La Morte (Robin des Bois, 2011).

Fig

  Fig. S-4. Map and aerial image of the coring site at Crépieux-la-Pape (CPX). The site is a former gravel quarry adjacent to the old channel of the Rhône River. It was formed at the beginning of the 1980s by dredging of alluvial deposits. The harbor-shaped feature left by quarrying is about 3 m deep at the center. Since quarrying ended in the early 1980s, flood deposits have formed a submerged levee that

Fig. S- 3 .

 3 Fig. S-3. Map and aerial image of the coring site at La Morte (MTE). La Morte is the most upstream of the three coring sites on the Rhône River (Fig.1, full text). It is located in a pond at the downstream end of a cutoff meander of the Rhône, which is open to the river at its downstream end. La Morte is in a relatively non-industrialized part of the Rhône basin upstream from the hazardous-waste treatment facility and Lyon. It was chosen to represent reference concentrations for the lower Rhône basin (i.e., downstream from Lake Geneva). There are, however, urban and industrial areas in the upper Rhône valley in Switzerland (upstream from Lake Geneva) and smaller urban areas and a few historical contamination sites between Lake Geneva and La Morte that might affect PCB concentrations at La Morte (Robin des Bois, 2011).

Fig

  Fig. S-4. Map and aerial image of the coring site at Crépieux-la-Pape (CPX). The site is a former gravel quarry adjacent to the old channel of the Rhône River. It was formed at the beginning of the 1980s by dredging of alluvial deposits. The harbor-shaped feature left by quarrying is about 3 m deep at the center. Since quarrying ended in the early 1980s, flood deposits have formed a submerged levee that

Fig. S- 5 .

 5 Fig. S-5. Map and aerial image of the coring site at lle du Beurre (BRE).The site is in a small side channel in a forested wetland adjacent to the Rhône River, 40 km downstream from Lyon (Fig.1, full text). This site was chosen because of its location downstream from Lyon and the industrial corridor that extends from south of Lyon to the confluence with the Gier River, which, like the Bourbre River,

Fig. S- 6 .

 6 Fig. S-6. Grain size and radionuclide data by depth (A) and grain size and PCB I profiles by date (B) for a sediment core from Lake Paladru (PAL), Rhône basin, France.

Fig

  Fig. S-7. Grain size and radionuclide data by depth (A) and grain size and PCB I profiles by date (B) for the sediment core from La Morte (MTE), Rhône basin, France.

Acknowledgements

This project was funded by the Rhône-Mediterranean and Corsica Water Agency, the Rhône-Alpes Region, the Provence-Alpes-Côte d'Azur Region, and the Compagnie Nationale du Rhône (CNR) in the context of the Rhône River ecological restoration plan, and ONEMA (National Office of Water and Aquatic Environments). Beyond financial support, these agencies had no role in the study design or in the preparation, review, or approval of this article for publication.


Ph. D Gaoussou Karamoko
email: karamgao@yahoo.fr

Sabine Danthine

Gilles Olive

Christophe Blecker

Danthine Sabine

Olive Christophe Giles

Blecker

Interfacial and foaming properties of two types of total proteose-peptone fractions

Keywords: Proteose-peptone, whey protein concentrate, kinetic adsorption, monolayer film, foaming properties, foamscan Proteose-peptone, whey protein concentrate, kinetic adsorption, monolayer film, 23 foaming properties, foamscan 24 25

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction 26

Foams are a subject of strong interest in a large number of industries where they play a 27 crucial role. In the food industry, a foaming formulation involves the use of surface-active 28 agents. During the foaming processes, these agents adsorb rapidly and form a film at the 29 surface of gas bubbles. Surface-active agents must simultaneously give abundant and stable 30 foams. Many researches are focused on the development of novel ingredients, including 31 proteins which can be used as foaming agents. Food processing industries use these protein 32 ingredients to improve the functional properties of a wide range of formulated food (Cassiani 33 et al., 2011). Among food proteins, whey proteins are considered as important and valuable 34 ingredients due to their high nutritional and functional properties [START_REF] Blecker | Improved emulsifying and foaming of whey proteins after enzymic fat hydrolysis[END_REF] 35 Mladen et al., 2009). These features confer to whey proteins ingredients high foaming and 36 stabilizing properties. 37 Among the whey components the total proteose-peptone fraction (TPP) could be of 38 interest. Indeed the TPP is a minor fraction of the heat-stable and acid-soluble protein which 39 is found in whey [START_REF] Blecker | S'adapter aux exigences du marché, innover dans les productions animales et 343 maîtriser la qualité[END_REF]. According to the literature, this fraction has 40 interesting functionalities such as surface properties, emulsifying properties and foaming 41 properties [START_REF] Shimizu | Emulsifying properties of the proteose-414 peptone fraction obtained from bovine milk[END_REF]Innocente et al., 1998a[START_REF] Innocente | Emulsifying properties 376 of the total fraction and hydrophobic fraction of bovine milk proteose-peptone[END_REF].

42

However, previous works on the foaming property of the milk's TPP fraction are 43 controversial. Some authors such as [START_REF] Zhu | Proteose Peptones and Physical Factors Affect 416 Foaming Properties of Whey Protein Isolate[END_REF] have shown a negative effect 44 of the TPP on the foam property (foam stability) produced by whey due to certain competitive 45 actions of other proteins. On the other hand, the TPP fraction is considered responsible of 46 milk foaming capacity due to its component PP3 [START_REF] Girardet | PP3 component of bovine milk: a 366 phosphorylated whey glycoprotein[END_REF]. Moreover, 47 most of studies reported in the literature concerns the TPP extracted from milk.

48

In the light of these considerations, this study attempted to provide new information 49 about the foaming properties of TPP, especially the TPP extracted from whey. To reach this 50 goal, two types of TPP fractions were extracted in this work. The first one was extracted from 51 skimmed milk using a classic method according to [START_REF] Pâquet | Study of hydrophobic protein fraction of 397 isolated from milk proteose-peptone[END_REF] and the second was 52 extracted from whey protein concentrate according to a procedure which is feasable on an 53 industrial scale.

54

The interest of this work is to study the TPP extracted from whey protein concentrate 55 (78 % of protein), a commercially available by-product of cheese processing. The use of this 56 alternative source could allow an increased TPP production for industrial applications. Foam 57 properties were characterized for possible use in food applications requiring foam formation 58 such as whipped cream, ice cream, cakes and desserts.

59

In the literature, SDS-PAGE electrophoresis was used to characterize the TPP fraction's 60 components according to Laemmli's method [START_REF] Pâquet | Study of hydrophobic protein fraction of 397 isolated from milk proteose-peptone[END_REF]. However several others 61 separation techniques were also used including liquid chromatography which allow a good 62 separation of the TPP's components. The hydrophobic interaction was most often used to 63 separate the TPP due to the hydrophobicity of some major components. In our case, Tricine 64 SDS-PAGE electrophoresis according to schägger (2006) was used to characterize the TPP, 65 due to it's high resolution, especially for the small proteins.

66

The aim of this study was to determine the ability of TPP fractions to form and stabilize 67 foams at several protein concentrations, at various pH, and to highlight the influence of the 68 extraction method on these properties. The TPP fractions obtained were analyzed for ash (AOACI, 2000), lipid by modified 93 mojonnier method (AOACI, 2000) and lactose (Miller, 1959). The determination of protein 94 content was performed by the Lowry method as described by [START_REF] Lowry | Protein 392 measurement with the folin phenol reagent[END_REF] tension as a function of the drop formation time [START_REF] Innocente | Dynamic surface 373 properties of the proteose-peptone fraction of bovine milk[END_REF]. All experiments were 119 performed at 25 ± 0.5°C. Each measurement was repeated three times. Equilibrium interfacial 120 tension (γ e ) and the initial adsorption rate (v) were determined by extrapolated values of γ vs.

121 t -1/2 at t→ ∞ and the tension (γ) versus time (t) curve slope at initial time (t 0 ), respectively 122 (Blecker et al., 2002;[START_REF] Innocente | HPLC profile and dynamic 379 properties of the proteose-peptone fraction from bovine milk and whey protein concentrate[END_REF].

139 π = γ 0 -γ, ( 1
)
140
where π is the surface pressure, γ 0 and γ are the surface tension of the clean surface and the 141 surface containing the monolayer, respectively. The elasticity (ε) of the protein film at the air-142 water interface, defined as the variation in surface pressure for a variation in the surface unit 143 area, was then calculated for every point of the isotherm π/A by means of the following 144 equation:

145 ε = -A (dπ/dA),
(2)

146
where dπ/dA is the surface pressure derivative in surface area [START_REF] Zhu | Proteose Peptones and Physical Factors Affect 416 Foaming Properties of Whey Protein Isolate[END_REF]. All

147 experiments were performed at 25 ± 0.5°C. 159

FC= V f /V g , (3
).

187

The protein analysis on milk TPP by Tricine SDS-PAGE (Figure 1), presented the same 188 bands as described in the literature, notably component PP3 also named LP28 (28 kDa)

189 according to [START_REF] Kanno | Purification and separation of multiple forms of lactophorin from 386 bovine milk whey and their immunological and electrophoresis properties[END_REF], LP18 (high hydrophobic component of 18 kDa) (Sorensen and 190 60 Petersen, 1993), component PP5 (14.3 kDa) [START_REF] Kollar | Proteose-peptone fraction of bovine milk: 389 lacteal serum component 5 and 8 casein associated glycoprotein[END_REF] and component 191 PP8S (9.9 kDa) [START_REF] Kollar | Proteose-peptone fraction of bovine milk: 389 lacteal serum component 5 and 8 casein associated glycoprotein[END_REF]. The band corresponding to component PP8 F as 192 referred to by [START_REF] Kollar | Proteose-peptone fraction of bovine milk: 389 lacteal serum component 5 and 8 casein associated glycoprotein[END_REF] was not observed on the gel due to its very low properties and surface hydrophobicity (Innocente et al., 1998a;Piquet et al., 1988). Therefore, 217 the relative percentage of PP3 in each fraction might certainly explain the lower interfacial 218 activity of WPC TPP compared to milk TPP.

219

The discussions hereafter are focused on the 1 % (w/w) solutions because the 220 difference between the samples was not significant at 0.1 % and 0.5 % (P 0.05). It appeared 221 that the milk TPP lowered the surface tension when the pH increased. Conversely, for WPC

222

TPP the lowest value of surface tension was observed at pH 4.0 or native pH (4.7). These shown that the adsorption rate is correlated with the foaming capacity of proteins (German et 246 al., 1985;[START_REF] Dagorn-Scaviner | Etudes des proprieties tensioactives des gliadines du pois. 359 Application aux isolats protéiques utilisés comme agents d'émulsification ou de moussage[END_REF] and the development of an important surface pressure is 247 required to obtain good stability of dispersed systems [START_REF] Nakai | Relationships between hydrophobicity 406 and emulsifying properties of some plant proteins[END_REF]. Overall, these 248 findings suggested a better foaming capacity for milk TPP compared to WPC TPP. [START_REF] Gaines | Insoluble Monolayers at Liquid-gaz Interface[END_REF]. Maximum elasticity values obtained 276 at the different pH are shown in Table 3. The highest values were observed for the TPP WPC 277 spread at pH 4 (13.15 mN.m -1 ), whereas milk TPP obtained its highest value at pH 7.0 (11.62 278 mN.m -1 ). This trend inversement has already been mentioned for adsorption kinetics and 279 compression isotherms. In view of the results presented in Table 3, WPC TPP films were 280 more elastic than milk TPP film except at pH 7.0 (P < 0.05). The molecules of WPC TPP film

281

were more resistant to conformational change compared to those of milk TPP film except at 282 pH 7.0. Such a property is required for good foam stability [START_REF] Poré | Dispersion Aqueuses, Suspension, Emulsions, Mousses[END_REF]. The differences 283 between milk TPP and WPC TPP films could again be explained by considering differences 284 in their compositions. (see paragraph 2.1). This is in accordance with [START_REF] German | Film forming and foaming behavior 364 of food proteins[END_REF] and Dagorn-Scaviner

293

(1986) which showed a correlation between the adsorption rate and the foaming capacity.

294

The foaming inability of milk TPP can be explained by the quantity of fat in its 295 composition. The quantity of lipid is greater in milk TPP than in WPC TPP (Table 1). The 296 presence of small lipid quantities can have a strong destabilizing effect on foaming (Huppertz, 297 2010). A lower FC of milk TPP induced a collapse of the air bubble during foam formation.

298

This is probably due to the weak mechanical strengh of the lipid-protein interaction.

299 Foam stability (FS) was characterized by the half-life of foam volume. The effect of pH 300 on this parameter is represented in Figure 5. As in the case of foaming capacity, the effect of 301 pH on foam stability was correlated with the surface pressure obtained in Adsorption kinetics 302 study. This was mentioned by [START_REF] Nakai | Relationships between hydrophobicity 406 and emulsifying properties of some plant proteins[END_REF] which say that the development of an 303 important surface pressure lead to a good stability of dispersed systems.

304

In general, the increasing concentration of TPP improved foam stability (data not 305 shown). WPC TPP capacity to stabilize foam was greater compared to milk TPP (at all pH), 306 as the time needed to reduce the foam volume to half of the maximum volume (T 1/2 ) was 307 higher. This indicates the higher resistance of WPC TPP film to the coalescence of bubbles.

308

The evolution of foam volume of TPP fraction at 1 % during the first 450 seconds as function

  -peptone fraction (milk TPP) was extracted from UHT skimmed milk 74 according to the procedure described by[START_REF] Pâquet | Study of hydrophobic protein fraction of 397 isolated from milk proteose-peptone[END_REF] with some modifications. Three 75 batches of ten litres of commercial skimmed milk (Everyday, Colruyt, Belgium) were used to 76 extract the TPP. The process consisted in heating one litre of skimmed milk at 90 -95 °C 77 during 30 min and then cooling it down in an ice bath to 20 °C during 40 min. pH was then 78 adjusted at 4.6 with 1M HCl to induce isoelectric precipitation. Samples were centrifuged at 79 5000 g for 30 min at 20 °C (Centrifuge Beckman Model J2-21, Indianapolis, USA).

  -time dependence of TPP solutions was measured with the 125 automatic drop tensiometer TRACKER (IT Concept, Longessaignes, France) forming a rising 126 protein solution bubble of at the tip of 5 μL a curved capillary in an empty cell (containing 127 air). The surface tension was measured and recorded every second. All measurements were 128 prepared by dissolving the lyophilized material in ultra-pure 131 water (ultra-pure water system Milli.Q50, Millipore). Protein solution (1 mg.mL -1 ) were 132 characterized at various pH values: 4.0, 4.6-4.7 (native pH) and 7.0. Measurements of the 133 surface pressure (π) versus surface area were performed on Langmuir-type film balance 134 LFW2 (LAUDA GmbH, Königshöfen, Germany) of which the principle and the experimental 135 condition were described by Innocente et al. (1998). Schematically this technique involved 136 the measurement of the surface tension of an aqueous surface under different compression 137 conditions in the presence and absence of a surface monolayer, and may be described by the 138 following equation:

  foam stability of total proteose-peptone were determined with a 150 Foamscan (IT Concept Instrument, Longessaignes, France). The system includes a transparent 151 column of glass, with at its base a porous frit through which the gas is injected. The volume of 152 liquid and foam are measured with a conductivity meter, composed of a series of two 153 electrodes located at the base of the column and four electrodes placed at the top of the 154 column. 25 ml of protein solution was aerated at a flow of 100 mL.min -1 . The evolution of the 155 foam was monitored to assess foam stability and its height was continuously measured by 156 image analysis. A fritted glass with a porosity of 4 (P4) was used and the aeration time was 60 157 seconds. Measurements were performed at 25 °C. The foam capacity (FC) was determined 158 by:

193

  molecular weight (4 kDa). The LP18 (18kDa) was the high hydrophobic component of the 194 FHPP of TPP. LP18 hydrolyzed by plasmin from LP28 in milk was absent in the extract of 195 WPC (WPC TPP). However, a difference in thickness and intensity was observed with regard 196 to the bands PP5 and PP3. Thus, the PP3 band in milk TPP appears stronger and more intense 197 than the PP3 band in WPC TPP. The PP5 band was thinner in milk TPP than WPC TPP. In 198 view of these observations, it could be said that milk contained more PP3 and less PP5 199 compared to WPC TPP, this depends on milk's evolution before undergoing heat treatments 200 such as UHT or whey processing. the air-water interface of TPP fractions extracted from skimmed 203 milk and WPC were compared by using dynamic surface tension technique. Many factors 204 may affect this property at the interface such as concentration, pH, and temperature. Among 205 these, the concentration and pH were specifically considered. Therefore, Figure 2 represents 206 the surface tension as a function of drop formation time for both different fractions at 1 % 207 (w/w), and at various pH. Surface tension value decrease with the increase in drop formation 208 time, because molecules have more time to position themselves uniformly at the interface 209where they can develop their surfactant activity[START_REF] Innocente | HPLC profile and dynamic 379 properties of the proteose-peptone fraction from bovine milk and whey protein concentrate[END_REF]. At all tested 210 concentrations and pH, the surface tension reduction was more pronounced for milk TPP in 211 comparison to WPC TPP. This suggested that milk TPP had a higher surface activity than 212 those extracted from WPC. This difference in behavior could be related to the hydrophobic 213 fraction commonly called HFPP which contains LP18 and LP28 in milk TPP according to the 214 electrophoresis. Moreover, it was demonstrated that the surface-active properties of the TPP 215 fractions are in part governed by the physicochemical properties of PP3 such as aggregation 216

  223differences are due to the composition of each fraction. The LP18 observed in milk TPP 224 composition could be at the origin of these behaviors with regards to pH. Indeed, LP18 225 comprises four isolectric isoforms (pI 5.0 -8.0)[START_REF] Inagaki | The 370 multiplicity of n-glycan structures of bovine milk 18 kDa lactophorin (milk glyCAM-1)[END_REF]. At pH 7.0 milk TPP 226 presented a net charge because this pH is in this range, which explained the lowest surface 227 tension value at pH 7.0. Whereas, the pI of TPP is 4.0 to 5.0 according to[START_REF] Pâquet | Study of hydrophobic protein fraction of 397 isolated from milk proteose-peptone[END_REF]. 228 So, WPC TPP presented a net charge at pH 4.0 and native pH (4.70), which explained the 229 lowest surface tension values obtained at these pH compared to pH 7.0. 230 The analysis of adsorption kinetic curves was quantified by determining the initial 231 adsorption rate and equilibrium surface pressure (Table2) to explain the reduction of surface 232 tension. Thus, the initial adsorption rate is equivalent to the slope of the tangent to the curve at 233 the start of the kinetics; it characterizes the velocity at which molecules lower the surface 234 tension at the beginning adsorption. This initial adsorption rate has been determined using the 235 automatic drop tensiometer tracker (Table2) which was used as a complement to TVT. The 236 initial surface tension value at t = 0 corresponding to the water surface tension, was equal to 237 72.00 mN.m -1 . Equilibrium surface pressure value (π e = γ 0 -γ e , where γ 0 is the initial surface 238 tension and γ e is the equilibrium surface tension) characterizes the maximum reduction of 239 surface tension induced by the surfactant molecules, in our case the dispersion of the 240 proteose-peptone. The highest values of initial adsorption rate and equilibrium surface 241 pressure were observed for milk TPP (P < 0.05). Equilibrium pressure values obtained 242 correspond to a reduction of the initial surface tension value of 32 to 35 % for milk TPP and 243 29 to 30 % for WPC TPP. This indicated that milk TPP caused a rapid reduction of interfacial 244 tension that reached the equilibrium values faster compared to WPC TPP. Many authors have 245

  Fuente[START_REF] Rodriguez-Patino | Destabilization of monogleceride 402 monolayers at the air-water interface: structure and stability relationships[END_REF] allowing to explain the stability mechanism of film. The elasticity was

  FC) of proteose-peptone fractions at 1 % (w/w) were compared at 287 various pH as depicted in Figure4. Generally, the foaming capacity increased with increasing 288 concentration of protein. The foaming capacity of milk TPP was lower than that of WPC TPP 289 at all pH studied. The milk TPP foaming power increased with the increasing of pH, whereas 290 the WPC TPP foaming power decreased with increasing pH. This effect of pH on foaming 291 capacity of each fraction was correlated with the adsorption rate obtained for each fraction 292

309

  of pH is represented in Figure6. The difference in foam behavior was demonstrated by the 310 speed of decrease after switching off the bubbling (

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 1 :Figure 2 :

 12345612 Figure 1: Tricine-SDS-PAGE electrophoresis of proteose-peptones fractions,WPC and milk.

Figure 3 :

 3 Figure 3: Isotherms of compression of total proteose-peptone fractions ( Milk TPP) and ( WPC TPP) monolayers spread on aqueous solutions at (A) pH 4.0, (B) native pH and (C) pH 7.0. Registered at 25 ± 0.5°C. Spread 50 µL of solution. Average of three repetitions.

Figure 4 :

 4 Figure 4: Foaming capacity at various pH of total proteose-peptone fractions 1%. ( ) Milk TPP and ( ) WPC TPP at various pH.

Figure 4

 4 Figure 4 Click here to download Figure: Figure 4.doc

Figure 5 :

 5 Figure 5: half-life of the foam related to various pH of total proteose-peptone fractions at 1%: ( ) Milk TPP and ( ) WPC TPP.

Figure 5

 5 Figure 5 Click here to download Figure: Figure 5.doc

Figure 6 :

 6 Figure 6: Evolution of total proteose-peptone fractions foam ( Milk TPP) and ( WPC TPP) at 1% (w/w) for the first 450s at (A) pH 4.0, (B) native pH and (C) pH 7.0.

є

  = -A (dπ/dA).

.2. Chemical composition of TPP 92

	88
	89	dispersion. Whey total proteose-peptone (WPC TPP) was then extracted from the dispersion
	90	using the same procedure described for extraction from milk without the ammonium sulfate
		addition. The TPP yield was stored at 4 °C until analysis.

80

Ammonium sulfate (304 g) was added to the supernatant (800 mL) to obtain a half-saturated 81 solution that was then shaked and centrifuged (5000 g for 10 min at 20 °C). The pellet was 82 dispersed, exhaustively dialyzed and finally lyophilized. The TPP yield was stored at 4 °C 83 until analysis. 84 1.1.2 Extraction from whey protein concentrate (WPC) 85 Whey protein concentrate (WPC) (Lacprodan 80) was obtained from Arla Foods 86 Ingredients (Viby J, Denmark). 2.85 Kg of commercial WPC were used. WPC was dispersed 87 into distilled water (95 g.L -1 ) by stirring at room temperature for 2 hours to ensure good 91 1

. 95 1.3. Electrophoretic Analysis using Tricine-SDS-PAGE

	96
	97	TPP molecular weight distribution was evaluated by Tricine-SDS-PAGE
	98	electrophoresis following the method of Schägger (2006). The Tricine-SDS-PAGE was
	99	performed with a vertical electrophoresis apparatus model 2001 (Amersham-Pharmacia,
	100	Uppsala, Sweden) with a 16 % polyacrylamide separating gel (16 % T, 3 % C where T is the
	101	total concentration of acrylamide/bis-acrylamide monomers and C is the amount of
	102	crosslinker relative to the total concentration) and thickness of 1.5 mm. Tricine-SDS-Page
	103	uses two buffers and offers a high resolution, especially in the small protein and peptide
	104	range. The electrophoresis buffers were tris-HCl buffer (anode buffer) and Tricine-tris buffer
	105	(cathode buffer). 500 µl of sample (1-3 % w/v of proteins) were treated with a equivalent
	106	volume of sample buffer (150 mM Tris-HCl, pH 7.0, 12 % SDS; 6 % mercaptoethanol, 30 %
	107	glycerol, 0.05 % Coomassie blue), and then heated at 37 °C for 15 min before electrophoresis.
	108	Electrophoresis was performed at an initial voltage of 30 V for stacking and at a constant
	109	voltage of 90 V for separation. The protein bands were fixed in a solution consisting of 50 %
	110	methanol, 10 % acetic acid, and 100 mM ammonium acetate for 60 min, before staining by
	111	0.025 % coomassie dye in 10 % acetic acid for 24 h. The gel destaining was performed in 10
		% acetic acid.

112 1

.4. Dynamic Drop Volume Method

	118	measurements at air-water interface. The experiments resulted in the measurement of surface
	113
	114	TPP samples were prepared in ultra-pure water (ultra-pure water system Milli.Q50,
	115	Millipore). TPP solutions of 0.1, 0.5 and 1% (w/w) were prepared at various pH values: 4.0,
	116	4.6-4.7 (native pH) and 7.0. Automated drop volume tensiometer TVT1 (LAUDA,
	117	Königshöfen, Germany) was used to perform dynamic measurements for surface tension

Table 1 :

 1 Table4). The highest value of decrease 311 speed was observed for milk TPP foam; this indicated a greater ability to stabilize for WPC 312 TPP. The better foaming properties of WPC TPP were essentially due to the lower quantities 313 of lipids in WPC TPP composition, as it is well known that lipids destabilize foam (Huppertz, Whereas the best foaming properties were exhibited by WPC TPP. This best performance of 321 WPC TPP compared to milk TPP is due to the lipid fraction of milk TPP which prevents them 322 to foam. It is conceivable they could both be used as surface-active agents in food Composition, expressed in percentage of dry material, of powders of total proteose-

	314
	315	2010).
	316	3. Conclusion
	317	Variations in functional and surface properties of the two TPP fractions are due to
	318	several factors (heat treatments, UHT or whey processing), extraction source, extraction
	319	method and the composition of each fraction (lipids and proteins). Milk TPP exhibited a
		higher surface activity and a better monolayer film resistance to mechanical disturbances.
	323
	324	applications requiring foam formation. On the other hand, the extraction from WPC would be
	325	interesting because it is advantageous in terms of high TPP production yield and economical
	326	compared to TPP extracted from milk.
	327

320 422

Table 2 :

 2 Equilibrium surface pressure (π e ) and Initial adsorption rate (V) at air-water interface

	423
	424	for total proteose-peptone fractions at 1 % (w/w) of protein at various pH.
	425

Table 3 :

 3 Maximum elasticity value of total proteose-peptone fractions films at various pH.

	426
	427

Table 4 :

 4 Speed of decreasing (cm 3 .min -1 ) after switching off the bubbling of total proteose-

	428
	peptone fractions foams at 1 % (w/w) at various pH.

Table 1 :

 1 Composition (% of dry material) of freeze dried TPP fractions extracted from WPC and milk. Values within the same line followed by the same superscript letters do not differ significantly at P < 0.05 (Duncan test). Mean of triplicates. Click here to download Table: Table 1.doc

	Protein Ash Lipid Lactose a Table 1	WPC TPP (Dry matter : 91.40 % ± 0.04) 65.20 % ± 0.03 a 3.50 % ± 0.15 a 4.60 % ± 0.08 a 26.22 % ± 0.09 b	Milk TPP (Dry matter : 91.75 % ± 0.10) 79.15 % ± 0.10 b 2.85 % ± 0.13 a 6.55 % ± 0.05 b 10.52 % ± 0.07 a

Table 2 :

 2 Equilibrium surface pressure (π e ) and Initial adsorption rate (V) at air-water interface for total proteose-peptone fractions at 1 % (w/w) of protein at various pH. Values within the same column followed by the different superscript letters are differ significantly at P < 0.05 (Duncan test). Mean of triplicates.

		pH	V (mN.m -1 .s -1 )	π e (mN.m -1 )
	Milk TPP	4.00 4.67 7.00	11.39 ± 0.12 d 15.60 ± 0.15 e 17.37 ± 0.19 f	23.39 ± 0.24 d 24.11 ± 0.03 e 24.88 ± 0.12 f
	WPC TPP	4.00 4.70 7.00	9.61 ± 0.18 c 8.80 ± 0.09 b 7.95 ± 0.26 a	21.66 ± 0.09 c 21.29 ± 0.13 b 20.94 ± 0.06 a
	a

Table 2

 2 Click here to download Table: Table 2.doc

Table 3 :

 3 Maximum elasticity value (є) of total proteose-peptone fractions films at various pH. Values within the same column followed by the same superscript letters do not differ significantly at P < 0.05 (Duncan test). Mean of triplicates.

		pH	є (mN.m -1 .s -1 )
	Milk TPP	4.00 4.67 7.00	10.19 ± 0.01 b 10.22 ± 0.27 b 11.62 ± 0.12 c
	WPC TPP	4.00 4.70 7.00	13.15 ± 0.09 e 12.32 ± 0.14 d 9.05 ± 0.09 a
	a

Table 3

 3 Click here to download Table: Table 3.doc

Table 4 :

 4 Speed of decreasing (cm 3 .min -1 ) after switching off the bubbling of total proteose-peptone fractions foams at 1 % (w/w) at various pH.

		pH	Speed of decreasing (cm 3 .min -1 )
	Milk TPP	4.00 4.67 7.00	8.84 ± 0.72 9.16 ± 1.06 12.06 ± 1.55
	WPC TPP	4.00 4.70 7.00	5.75 ± 0.77 4.15 ± 2.99 7.40 ± 0.31
	Means of triplicates.

Table 4

 4 Click here to download Table: Table 4.doc

ABSTRACT

Total proteose-peptone (TPP) fractions were extracted from skimmed milk (UHT) and whey protein concentrate (WPC) on a laboratory scale. Protein solutions (0.1, 0.5 and 1 % w/w) were characterized as function of pH: 4.0, 4.6-4.7 (native pH) and 7.0. Their foaming capacities and stabilities were studied. Beforehand, the surface properties that govern them were investigated, notably the kinetics of adsorption and mechanical properties of monolayer films at the air-water interface involved in the formation and the stability of foams respectively. The TPP extracted from skimmed milk showed the lowest values as well as a significant reduction in surface tension and presented a good mechanically resistant film. The TPP extracted from WPC presented a better foaming capacity and stability which was unexpected. However, foaming properties and surface properties of TPP fractions depended on the pH. The considerable influence of extraction source and method on proteose-peptone's properties were highlighted.

Abstract