domain: sdv info shs

text:
S Firmin

P Gandia

D P Morgavi

G Houin

J P Jouany

H Boudra
email: hboudra@clermont.inra.fr

Modification of aflatoxin B 1 and ochratoxin A toxicokinetics in rats 2 administered a yeast cell wall preparation 4

Keywords: aflatoxin B 1, ochratoxin A, yeast cell wall, detoxification, adsorbing agent, 16 toxicokinetics, rat

The cell wall of Saccharomyces cerevisiae can bind mycotoxins in vitro but there is scarce 2 information on whether this property decreases the absorption of mycotoxins in vivo. The effect of a yeast cell wall preparation (YCW) on toxicokinetics and balance excretion (urine 4 and faeces) of aflatoxin B 1 (AFB1) and ochratoxin A (OTA) was tested in rats after oral administration of each toxin. The 3 H-labelled mycotoxins were used at low doses. Co-6 administration of YCW with AFB1 decreased the extent, but not the rate of absorption. Concurrently, radioactivity excreted in faeces increased by up to 55% when compared to 8 controls, whilst the excretion in urine decreased (p < 0.05). The effect of YCW on OTA was less marked, although it increased radioactivity excretion in faeces (up to 16%; p < 0.05) it 10 did not result in changes in urine and toxicokinetic parameters. The in vivo effect is in agreement with the reported in vitro binding ability for these toxins (AFB1 > OTA). In 12 conclusion, these results indicate that YCW could be used to protect monogastric animals against exposure to low dietary levels of selected mycotoxins. 14

Introduction

Mycotoxins are secondary fungal metabolites commonly found in foods and feeds 2 [START_REF] Placinta | A review of worldwide contamination of 30 cereal grains and animal feed with Fusarium mycotoxins[END_REF][START_REF] Garon | Mycoflora and multimycotoxin detection in corn silage: Experimental study[END_REF][START_REF] Binder | Worldwide occurrence of mycotoxins in commodities, feeds and feed ingredients[END_REF]). Many of these secondary metabolites are toxic to humans and animals, affecting health, reducing animal performance 4 and causing substantial economic losses. The application of good agricultural and storage practices can reduce contamination but some factors contributing to fungal development, such 6 as climatic conditions, are beyond human control and the absence of mycotoxins in the ration of farm animals cannot be fully assured. The presence of mycotoxins in feeds is of concern in 8 livestock production as it poses a risk for animals and also for consumers if toxic molecules are transferred into animal products. In most countries contamination levels of aflatoxin B 1 10 (AFB1) in feeds are regulated and thus, highly-contaminated feeds are not normally given to animals. However, contamination levels just below the legal limit may still have a negative, 12 long-term impact on production. In addition, on-farm produced feeds are seldom controlled, which may result in higher than tolerable mycotoxin ingestion. 14 Several methods have been investigated for their capacity to remove, or reduce, mycotoxins in contaminated feeds (reviewed by [START_REF] Jouany | Methods for preventing, decontaminating and minimizing the toxicity of mycotoxins in feeds[END_REF]. Some physical and chemical 16 treatments such as the use of ammonia are expensive or simply not adapted to the treatment of feeds destined for livestock [START_REF] Niderkorn | Les fusariotoxines: comment limiter leur 28 presence dans les ensilages et leur impact chez les ruminants?[END_REF]). The use of products, which could be 18 defined as mycotoxins inactivators, to reduce absorption of mycotoxins in the gastrointestinal tract of animals, is one such practical alternative which is currently receiving increased 20 attention. Among these products, organic adsorbents such as yeast cell wall (YCW) have been shown to rapidly form complexes in vitro with mycotoxins (Devegowda et al. 1998). 22 Feeds multicontaminated by mycotoxins are frequent in the field [START_REF] Scudamore | Mycotoxins in ingredients of animal feeding stuffs: II. Determination of mycotoxins in maize and maize products[END_REF][START_REF] Mansfield | Contamination of fresh and ensiled maize by multiple Penicillium mycotoxins[END_REF]) and the ability of YCW to bind several mycotoxins makes them 24 suitable for use under practical feeding conditions. In vivo studies have shown that YCW can reduce the adverse effects of mycotoxins in different animal species; swine (Swamy et al. 26 2002), horse [START_REF] Raymond | Effects of feeding a blend of grains naturally 36 contaminated with Fusarium mycotoxins on feed intake, serum chemistry, and hematology of horses, and the efficacy of a polymeric glucomannan mycotoxin 38 adsorbent[END_REF], laying hens [START_REF] Chowdhury | Effects of feed-borne Fusarium mycotoxins on hematology and immunology of laying hens[END_REF], and dairy cows [START_REF] Korosteleva | Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows[END_REF]. In these studies the detoxification efficiency of YCW has been 28 mainly evaluated on the basis of animal performance and/or the absence of toxicity, but the precise mechanism responsible for the beneficial effect of this additive has not yet been 30

clearly demonstrated. In lactating dairy cows receiving AFB1, a decrease in the excretion of aflatoxin M1 in milk was observed in some studies [START_REF] Diaz | Aflatoxin binders II: reduction of aflatoxin M1 in milk by 32 sequestering agents of cows consuming aflatoxin in feed[END_REF][START_REF] Masoero | Carryover of aflatoxin from feed to 22 milk in dairy cows with low or high somatic cell counts[END_REF] [START_REF] Stroud | The effect of feed additives on aflatoxin in milk of dairy cows fed aflatoxin-46 contaminated diets[END_REF][START_REF] Waltman | Effect of sequestering agents on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets[END_REF][START_REF] Kutz | Efficacy of Solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels 12 in milk of early to mid lactation dairy cows fed aflatoxin B1[END_REF]. A decrease in milk carryover suggests that mycotoxin absorption was impaired. 2

The purpose of this study was to evaluate the effect of a YCW-based preparation on AFB1 and ochratoxin A (OTA) toxicokinetics in rats. AFB1 and OTA were chosen as they 4 are the two most common mycotoxins found in feeds and which can cause a variety of toxic responses in animals, including nephrotoxic, hepatotoxic, teratogenic, and carcinogenic 6 effects. Mycotoxins were 3 H-labelled and radioactivity was monitored in faeces, urine and plasma following oral administration. The YCW product was tested at two different doses 8 and mycotoxins were used at relatively low concentrations and in a single dose (< 1% LD50) to prevent overt perturbations of liver and kidney functions. 10

Materials and Methods

Chemicals 12

Non-radioactive AFB1 and pentobarbital sodium were purchased from Sigma Chemical (St Quentin, France). [ 3 H]-AFB1 (two different lots were used with a specific activity of 6.9 14 and 17.5 Ci/mmol; 97% and 98.6% purity) and [ 3 H]-OTA (specific activity: 5 Ci/mmol; 99.6% purity) were supplied by Hartmann Analytic (GmbH, Braunschweig, Germany) in 16 methanol and ethanol solutions, respectively. A stock solution of non radiolabelled AFB1 was prepared in methanol at a concentration of 0.2 mg/mL. Ready Safe™ LSC cocktail was 18 supplied by Beckman Coulter (Villepinte, France), soluene-350 ® by Perkin-Elmer (Courtaboeuf, France). A preparation composed of modified cell walls extracts of 20 Saccharomyces cerevisiae strain 1026 was provided by Alltech Inc. (Yeast Cell Wall (YCW), purity 90%, batch No FR71535-1, Nicholasville, U.S.A.). YCW was used as a suspension in 22 0.9% NaCl at a concentration of 20 mg/ml. Suspensions were freshly prepared before use.

24

Animals and administration of mycotoxin

Eight week-old male Sprague-Dawley (265 ± 30 g) rats were purchased from France). Rats had free access to water and were fed ad libitum with certified 2016 Teklad Global 16% Protein Rodent Diet (Harlan Inc, WI, U.S.A.). The animal 28 room was environmentally controlled, animals were provided with 12 hours light each day with the temperature range maintained between 22 to 25 °C, and a relative humidity range of 30 Mycotoxins were administered by gavage through a gastric intubation at a constant volume of 2 ml of a dose solution per kg of BW. The exact amount administered was 4 determined by weighing the dosing syringe before and after gavage. Feed was removed from cages 12 h before gavage and rats were fed again 6 h post gavage. The dose solution 6 contained AFB1 or OTA (specific activity: 40 µCi/kg of BW) with or without YCW at one of two concentrations. Stock mycotoxin solutions were diluted to the desired dose volume with 8 0.9% NaCl and 0.1M NaHCO 3 for AFB1 and OTA, respectively. For the high dose of AFB1 (18.12 µg/kg BW) a 10-fold isotopic dilution was prepared by co-diluting [ 3 H]-AFB1 10 (specific activity: 6.9 Ci/mmol) with non radiolabelled AFB1. The YCW suspension was replaced by a solution of 0.9% NaCl in control animals. 12

Study design 14

A first assay using AFB1 was performed to optimize the protocol. In this trial a comparison between the use of blood and plasma samples for monitoring absorption was 16 made. Radioactivity in urine and faeces was monitored for up to 15 days to evaluate the minimum collection time necessary to correctly assess excretion. Two groups of rats were 18 included, each group was made up of 3 lots that received a single dose of AFB1 (40 µCi and 18.12 µg/kg BW) alone (lot 1) or in combination with YCW at 4 mg/kg BW (lot 2) and 10 20 mg/kg BW (lot 3). In group 1, 12 rats (3 per lot) were randomly and individually placed in metabolism cages (U.A.R., Epinay sur Orge, France). Urine and faeces were collected at 6, 22 12, 24, 36 and 48 h, and then every 24 h up to 15 days post dose. Following each collection cages were meticulously rinsed with water/methanol solution (1/1; v/v) and washing solutions 24 collected in order to evaluate possible loss of radioactivity due to urine remaining within the cages. Each sample (urine, faeces and recovered washings) were weighed and stored at -20° 26 C until analysis. Animals were sacrificed by cervical dislocation following the last collection of excreta. Liver, kidneys, jejunum, ileum and caecum of six animals (3 rats control and 3 28 rats YCW low dose) were dissected, weighed and aliquots were stored at -20 °C until analysis. Intestinal segments were processed without their contents. After sampling, viscera 30 from each animal were regrouped with the carcass, homogenized and analyzed for total body radioactivity. 32

In group 2 (n=117; 39 rats per lot) 3 rats were taken at each time point and anaesthetised by an intraperitoneal injection of 50 mg/kg BW sodium pentobarbital. Blood (approximately 34 [START_REF] Waltman | Effect of sequestering agents on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets[END_REF]5,[START_REF] Beauchemin | Use of Exogenous Fibrolytic Enzymes to Improve Feed Utilization by Ruminants[END_REF][START_REF] Beumer | Mass balance studies, with a focus on anticancer drugs[END_REF]10,14,24,36,[START_REF] Suzuki | The pharmacokinetics of ochratoxin A in rats[END_REF] and 72 h post dose. After sampling rats were euthanized. An aliquot of blood was immediately used for analysis as described below. The remaining blood 4 sample was centrifuged (1000 × g, 5 min at 4 °C) and the plasma fraction decanted into clean tubes and stored at -20 °C until analysis. 6 Based on this first study with AFB1 a good correlation between plasma and blood samples was established, only plasma samples were collected on the second AFB1 and OTA 8 studies. In addition, in the second study urine and faeces were collected for three days, as the bulk of the radioactivity was recovered within the first 72 h post dosing. 10

The second AFB1 experiment was conducted using a lower dose (0.716 µg/kg BW), which was provided by [ 3 H]-AFB1 alone (specific activity: 17.5 Ci/mmol). This dose, when 12 extrapolated to livestock species is in line with the European Community limitation of 0.02 mg/kg feed (directive 2003/100/EC). In a similar way, doses of YCW were 16.4 and 65.7 14 mg/kg BW, which were within the range recommended by the YCW manufacturer for different livestock species when calculated based on their inclusion in feed. For the excretion 16 study, 15 rats were used (5 per lot) and urine and faeces were collected at [START_REF] Beauchemin | Use of Exogenous Fibrolytic Enzymes to Improve Feed Utilization by Ruminants[END_REF]12,24,36,[START_REF] Suzuki | The pharmacokinetics of ochratoxin A in rats[END_REF] and 72 h post dosing. For the absorption study (n=117; 39 rats per lot), plasma was obtained 18 and stored as described above at 0 (no toxin), 1, 2, 4, 5, 6, 8, 10, 14, 24, 36, 48 and 72 h post dose. 20 The OTA study was similar to the second AFB1 study with 15 (5 per lot) and 63 rats (21 per lot) used for excretion and plasma measurements, respectively. Urine was collected for 3 22 days and faeces for 10 days. OTA was used at a dose of 3.22 µg/kg BW for a total radioactivity of 40 µCi per rat as described above. 24

Sample preparation and analysis 26

Before analysis faecal samples were homogenized in distilled water, 10% (w/w) dilution.

Faecal homogenates (50 mg) were incubated at 50 °C overnight with 1 ml of soluene ® -28 isopropanol solution (1/1; v/v) and 0.5 ml of 30% hydrogen peroxide. Blood samples (50 µl) were treated as faecal samples but were processed immediately after collection in order to 30 minimize non-radioactive scintillation counting. All samples, i.e. blood, faecal homogenate, urine (100 µl), plasma (100 µl), and wash water from metabolic cages were mixed with 10 ml 32 of Ready Safe™ scintillation fluid and stabilized overnight in the dark before counting the

Toxicokinetics and statistical data analysis 10

Non-compartmental methods were used to estimate the toxicokinetic parameters of radiolabelled mycotoxins. The parameters C max and T max were observed values defined as the 12 highest concentration achieved and the required time to achieve it, respectively. AUC 0-last was the area under plasma concentration versus time curve, calculated by the trapezoidal method 14 between the first and the last measurable concentration. AUC 0-∝ was the AUC 0-last extrapolated to infinity by the ratio of the last measurable concentration to the terminal slope 16 k e . Elimination constant (k e ) was determined by using the Kinetica software (InnaPhase, Champ-sur-Marne, France, release 4.0). T 1/2 corresponds to the terminal half-life determined 18 as the ratio of Neperian logarithm of 2 to the terminal slope (k e ). Due to the experimental design, it was not possible to conduct a statistical analysis directly on the kinetic parameters 20 [START_REF] Lellouch | Méthodes statistiques en expérimentation biologique[END_REF]. Samples for toxicokinetic parameters calculation were obtained from three different rats at each sampling time. Radioactivity values measured in plasma and blood 22 were compared at each sampling time using non-parametric, one-way-analysis of variance (Kruskal-Wallis test) using SAS version 8 (SAS Institute Inc., Cary, NC). The Kruskal-Wallis 24 test was also used to analyse excretion data (urine and faeces). Significance was declared at the 5% probability level. 26

Results

No overt toxic effects, sickness or behavioural anomalies were observed in any animals 28 in any of the studies. In the first AFB1 study radioactivity values for plasma samples were about half that of blood, but profiles were similar between both matrices (data not shown). 30

Although the recovery was lower with plasma, this matrix was used in subsequent studies because, unlike blood, plasma samples could be stored and analysed all at the same time. Radioactivity recovered in urine after 3 days and up to 15 days following the gavage was 1.3%, which did not represent a large proportion of the total. In faeces it represented a larger 2 fraction of approximately 15%. In both cases, however, no differences were observed between treatments (p > 0.05; data not shown), justifying the 72 h collection period of later 4 studies following gavage administration.

Data from all three studies are presented in Tables 1 and2, but results in the text for 6 AFB1 refer to the low dose study unless otherwise stated. Toxicokinetic parameters are summarized in Table 1 with an example of plasma profiles for [ 3 H]-AFB1 (Figure 1). 8

Radioactivity from AFB1 was detected 1 h after dosing, reaching a maximum value at 4 to 5 h post dosing and decreasing slowly thereafter with an elimination half-life (T 1/2 ) of about 64 h. 10

The combination of YCW with [ 3 H]-AFB1 altered plasma profiles, namely the absorption phase, irrespective of YCW dose. This decrease in the oral bioavailable fraction of AFB1 12 was not accompanied by modifications of the absorption rate. C max and AUC were lower in YCW treated lots when compared to values obtained in respective control groups. The 14 average decrease of AUC 0-∝ after co-administration of YCW was approximately 38% and was independent of dose, except for high YCW at the high AFB1 concentration which resulted in 16 a less marked decrease (Table 1). In contrast, the parameters of elimination phase (T 1/2 and k e )

were not influenced by YCW co-administration. [START_REF] Masoero | Effects of the procedure of inclusion of a sequestering agent in the total mixed ration on proportional aflatoxin M1 20 excretion into milk of lactating dairy cows[END_REF] The excretion balance in the presence of YCW (Table 2) was in agreement with the changes observed in the plasma toxicokinetic parameters. Urinary excretion of radioactivity 20 was lower after co-administration of YCW (p < 0.05), while faecal excretion increased (p < 0.05) (Table 2). Excretion in faeces increased by as much as 55% when compared to controls 22 without YCW. The level of YCW inclusion did not have any effect on radioactivity excretion. 24

In the OTA study this toxin reached a higher maximum concentration than AFB1 in plasma, resulting in higher calculated AUC parameters (Table 1). In contrast, the YCW 26 treatment showed a less perceptible effect during the absorption phase. In YCW treated animals OTA concentration in plasma was lower than control animals 1 h after gavage (p < 28 0.05). However, beyond 1 h post-gavage OTA concentrations were the same in all three groups (p > 0.05). Although the YCW treatment did not affect urinary excretion, it increased 30 toxin elimination in faeces (p < 0.05; Table 2). However, this increase was less marked than AFB1, approximately 15% compared to 55% for the latter. 32 Metabolic cages were rinsed and the wash water counted to address the possibility that urine was not fully collected and a proportion of urinary radioactivity was unaccounted for. 34 Cage contamination was 1.3%, 3.3% and 5.2% of the total radioactivity excreted for the low-AFB1, high-AFB1 and OTA experiments, respectively (Table 2)(data not shown). Whether 2 these values were added to the urine radioactivity or not did not affect the results. The effect of YCW on the level of radioactivity accumulating in rats was assayed using animals from the 4 first AFB1 experiment. At 15 days post-treatment the residual radioactivity in the carcasses of the control group was 2.5% ± 0.68 (mean ± SD) compared to 1.6% ± 0.41 for the low 6 YCW group (p = 0.12). Trace amounts of radioactivity (0.005%) were found in kidneys and different segments of the gastrointestinal tract. The liver accounted for the bulk of viscera 8 radioactivity with 0.31% ± 0.034 and 0.19 ± 0.082 for control and YCW group, respectively (p < 0.05). 10

Discussion

Mycotoxin sequestration in the gastrointestinal tract by adsorbing agents such as yeast 12 cell walls could be a promising strategy to protect against the toxic effect of these feed contaminants. However, there is still scarce information on their effect in vivo. Recent 14 reports indicate a positive effect on animal production performance and a reduction of mycotoxin carryover into milk [START_REF] Diaz | Aflatoxin binders II: reduction of aflatoxin M1 in milk by 32 sequestering agents of cows consuming aflatoxin in feed[END_REF][START_REF] Chowdhury | Effects of feed-borne Fusarium mycotoxins on hematology and immunology of laying hens[END_REF]Korosteleva et al. 16 2007;[START_REF] Masoero | Carryover of aflatoxin from feed to 22 milk in dairy cows with low or high somatic cell counts[END_REF]. In contrast, the use of these kind of additives do not always result in a positive effect on the parameter measured [START_REF] Stroud | The effect of feed additives on aflatoxin in milk of dairy cows fed aflatoxin-46 contaminated diets[END_REF][START_REF] Waltman | Effect of sequestering agents on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets[END_REF]Kutz et al. 18 2009). The type of yeast could be at the origin of these differences among studies. The cell wall polysaccharide responsible for mycotoxin binding such as the alkali insoluble fraction of 20 β-D-glucans (Yiannikouris et al. 2004b) varies in quality and quantity between species and strains [START_REF] Nguyen | Composition of the cell walls of several yeast 26 species[END_REF]. In addition, it has been described that for a same strain the 22 growth conditions and the preparation process influence the adsorption capacity of yeast cell wall additives [START_REF] Pradelles | Effects of yeast cell-wall characteristics on 4-ethylphenol sorption capacity in model wine[END_REF]. The inconsistency between studies, however, could also 24 be due to other reasons such as the mode of incorporation of the preparation into the diet or by the type of feed and mycotoxin present [START_REF] Battacone | The transfer of aflatoxin 4 M1 in milk of ewes fed diet naturally contaminated by aflatoxins and effect of inclusion of dried yeast culture in the diet[END_REF][START_REF] Blank | Effects of live yeast cell supplementation to high concentrate 14 diets on the toxicokinetics of ochratoxin A in sheep[END_REF]26 Masoero et al. 2009). Trials involving domestic animals involve many variables which are difficult to control between studies. The lack of a positive response reported in some trials 28 using these mycotoxin adsorbing agents has also been reported for other widely used feed additives such as probiotics or feed enzymes [START_REF] Beauchemin | Use of Exogenous Fibrolytic Enzymes to Improve Feed Utilization by Ruminants[END_REF]Robinson, Erasmus 30 2009). It is clear that more studies are needed to better understand the underlying factors inducing this variability and correctly evaluate their efficacy. 32 The present study, using a rat animal model, shows that a preparation exclusively composed of yeast cell walls extracted from Saccharomyces cerevisiae strain 1026 2 significantly reduces intestinal absorption of AFB1. It is worth mentioning that in the toxicokinetic model used, rats were in a fasted state when the mycotoxin-YCW mixture was 4 administered. Under natural conditions mycotoxins are contained in feeds and it is known that the presence of feeds in the gastrointestinal tract may affect the bioavailability of 6 different xenobiotic molecules [START_REF] Wonnemann | 12 Significant food interactions observed with a nifedipine modified-release formulation marketed in the European Union[END_REF][START_REF] Disanto | Effect of food on the pharmacokinetics of clozapine orally disintegrating tablet 12.5 mg: a randomized, open-label, crossover study in healthy 36 male subjects[END_REF].

The toxicokinetics of AFB1 and OTA were investigated using low mycotoxin doses. 8

These doses were calculated based on the maximum concentrations allowed or tolerated in animal feeds by the European legislation (2002/32/CE and 2006/576/CE). In addition, these 10 low doses are equivalent, in mg per kg BW, to the quantity that could be ingested by farm animals under normal production conditions. Although these mycotoxins have never been 12

investigated in rats at such low oral levels blood and/or plasma kinetics of both toxins were in agreement with previous reports. [START_REF] Galtier | The pharmacokinetic profile of 38 ochratoxin A in the rat after oral and intravenous administration[END_REF] and [START_REF] Coulombe | Clearance and excretion of intratracheally and orally 20 administered aflatoxin B1 in the rat[END_REF] 14 observed a biological half life of 55 h for OTA and 91.8 h for AFB1 (vs 63-65 h and 50-65h in the present study) following the oral administration of unlabeled OTA 2.5 mg kg -1 BW and 16

[ 3 H]-AFB1 600 µg kg -1 BW, respectively. The peak of radioactivity in blood was reached between 1 and 4 h following unlabeled OTA oral administration [START_REF] Suzuki | The pharmacokinetics of ochratoxin A in rats[END_REF] and that 18 of 14 C-AFB1 in plasma was reached 1 h after the same mode of administration [START_REF] Wong | Aflatoxicol: major aflatoxin B1 metabolite in rat plasma[END_REF]. Discrepancies with literature data were minor and may be due to differences in 20 protocol design.

In the current study results from toxicokinetic parameters indicate that the YCW 22 treatment modified the absorption phase. The AUC decreased in the YCW treated groups whereas the K e was similar in all groups. In addition, the increase in cumulative radioactivity 24 in faecal material was associated with a decrease in cumulative urinary excretion, mainly for AFB1 treatments. At the end of the experiment, up to 64% and 51% of total radioactivity was 26 recovered in urine and faeces for AFB1 and OTA, respectively. This amount of recovery is normally found with tritium labelled molecules administered at very low levels (Beumer et al. 28 2006). Recovery can be improved if the radioactivity recovered from cages by washing and, in the case of the first AFB1 experiment, the radioactivity fixed in the carcasses at the end of 30 the trial is included in the calculation. Residual activity in carcasses of animals 360 h after administration of AFB1 was low indicating that a significant amount was eliminated by an 32 alternative route. Other studies also reported a partial recovery of the total administrated radioactivity [START_REF] Suzuki | The pharmacokinetics of ochratoxin A in rats[END_REF][START_REF] Galtier | The pharmacokinetic profile of 38 ochratoxin A in the rat after oral and intravenous administration[END_REF][START_REF] Coulombe | Clearance and excretion of intratracheally and orally 20 administered aflatoxin B1 in the rat[END_REF]Kumagai et al. 34 1998), which could be explained by toxin retention within the kidneys in the case of OTA [START_REF] Zepnik | Toxicokinetics of the mycotoxin ochratoxin A in F 344 rats after oral administration[END_REF], or in liver in the case of AFB1 [START_REF] Wogan | Excretion and tissue distribution of radioactivity 8 from aflatoxin B1-14 C in rats[END_REF] and by exhalation of 2 tritiated water generated by exchange of the labelled 3 H [START_REF] Beumer | Mass balance studies, with a focus on anticancer drugs[END_REF]. In our study with tritiated toxins, this latter route of elimination of radioactivity was highly probable. 4

In vitro studies demonstrated the adsorption of different mycotoxins such as AFB1 and OTA to viable cultures of Saccharomyces cerevisiae [START_REF] Devegowda | A biological approach to counteract aflatoxicosis in broiler chickens and ducklings by the use of Saccharomyces cerevisiae cultures added to feed[END_REF]Meca et al. in 6 press). Preparations containing β-D-glucans portions of the specific strain 1026 exhibited a strong binding capacity in vitro for aflatoxins (up to 85%) while being poorer at sequestering 8 OTA (12.4%) [START_REF] Devegowda | Mycotoxin picture worldwide: novel solutions for their counteraction[END_REF]. Also, the extent of complexation, i.e. AFB1 > OTA, was not altered by pH values that are close to those found within the digestive tract 10 [START_REF] Yiannikouris | Chemical and conformational study of the interactions involved in 16 mycotoxin complexation with beta-D-glucans[END_REF]. The in vitro binding affinity is in agreement with the in vivo results observed here in rats, further supporting the hypothesis that binding is a key property 12 involved in the toxicity-alleviating effects of YCW.

Conversely, no dose effect of YCW was observed in the present study. A possible reason 14 for this result could be the low level of mycotoxin used. The adsorptive capacity of yeast β-D-glucans in vitro was shown to be modulated by the amount of mycotoxins added to the 16 medium according to a cooperative phenomenon. Binding of the first mycotoxin molecules induce conformation changes in β-glucans that facilitates access to new sites of fixation 18 improving binding efficiency until saturation of all sites of adsorption [START_REF] Yiannikouris | A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone[END_REF]Yiannikouris et al. 2004a). In our study, toxin molecules were highly diluted and the 20 minimum level of physical contacts capable to produce cooperative interactions was probably not fully achieved independently of YCW concentration. 22

The present study was based on measurements of radioactivity alone and provides no information on proportion of parent compounds and their metabolic products recovered in 24 blood, faeces, urine or carcasses. It has to be noted that some of the radioactivity found in faeces in the first hours following administration could have been originated from liver-26 conjugated metabolites [START_REF] Ha | Enhancement of biliary excretion of aflatoxin B1 and suppression of hepatic ornithine decarboxylase activity by 2-48 (allylthio)pyrazine in rats[END_REF][START_REF] Gross-Steinmeyer | Metabolism and lack of DNA 44 reactivity of the mycotoxin ochratoxin A in cultured rat and human primary hepatocytes[END_REF], particularly for AFB1, that are eliminated via the bile [START_REF] Bassir | Biliary excretion of aflatoxin in the rat after a single dose[END_REF][START_REF] Kumagai | Intestinal absorption and secretion of ochratoxin A in the rat[END_REF]. Nonetheless, 28 the decreased absorption and increased excretion observed in our experimental model indicates that the tested preparation may have the potential to protect monogastric animals 30 against exposure to low dietary levels of selected mycotoxins.

6

C max = maximum plasma concentration; t max = time to reach C max ; K-elimination = elimination constant; T 1/2 = terminal elimination half-life; AUC = area under the plasma concentration versus time curve from 0 to 72 h, 72 to infinite, and 0 to infinite, respectively. Pharmakinetic parameters were calculated from a concentration-time curve made 8 from samples obtained from three different rats at each sampling point except for C max and t max which are the mean of all C max and t max values obtained. Table 2. Cumulative excretion of mycotoxins after a single dose of aflatoxin B1 at two concentrations and ochratoxin A with and without coadministration of a yeast cell wall preparation in rats 2 a values are means±SD, n= 5 rats, except for aflatoxin B1 (18.12µg/kg body weigth (BW)) where 3 rats were used.

4

b Excreted at 72 h c Recovered at the end of the collection period that was 15, 3, and 10 days for aflatoxin B1 18.12 and 0.716, and ochratoxin A 3.22 µg/kg BW, respectively. NA: not

  was collected using the vacutainer ® system (Beckson Dickinson) into heparinized tubes (Beckson Dickinson) by ex-sanguination from the abdominal aortic vein at 0 (no toxin), 1, 2, 2

  in a LS 1801 scintillation counter (Beckman Coulter, Villepinte, France). All samples were analyzed in triplicate, except for blood samples that were analyzed in duplicate. 2 Tissue radioactivity was measured by liquid scintillation counting after oxidative combustion with an oxidizer (Packard 306 Oxidizer, Packard Instruments, Meridien, CT, 4 USA), using Permafluor E+ MonophaseS ® (Packard Instruments) as the scintillation cocktail.Viscera and carcase samples (200 mg) were packed into cellulose combustion cones imbibed 6 with 100 µl of an organic-based combustion aid solution Combustaid ® (Packard Instruments) and were analyzed in triplicate and quadruplicate, respectively. 8

32

 32

  wall preparation at low (YCW-L) and high (YCW-H) concentration. See Materials and Methods for details.

8 *

 8 p<0.05 between yeast cell wall treatments and controls for each column within each experiment.

Table 1 .

 1 Mean toxicokinetic parameters in plasma after a single dose of aflatoxin B1 at two concentrations and ochratoxin A with and without 2 co-administration of a yeast cell wall preparation in rats 4

		Aflatoxin B1 (18.12 µg/kg BW)	Aflatoxin B1 (0.716 µg/kg BW)	Ochratoxin A (3.22 µg/kg BW)
	Parameters	Control	YCW-L	YCW-H	Control	YCW-L	YCW-H	Control	YCW-L	YCW-H
	Cmax (ng/ml/kg BW)	3.66	1.91	2.26	0.72	0.27	0.35	28.78	20.84	21.47
		0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
	T 1/2 (h)	53.3	55.5	53.7	63.6	65.4	65.4	63.6	65.4	65.4
	AUC 0-72h(ng/ml/kg BW)	129.0	82.4	111.0	32.4	19.8	19.9	1731.3	1260.6	1337.9
	AUC 0-∞ ∞ ∞ ∞(ng/ml/kg BW)	131.4	83.9	113.1	32.9	20.1	20.2	1758.5	1279.8	1358.4
	∆ ∆ ∆ ∆AUC 0-∞ ∞ ∞ ∞(%)		36.1	13.9		38.9	38.9		27.2	22.8

Cumulative excretion (%, mean±SD) of radioactivity Experiment a Urines b Faeces b Total excreted c Cage rinse c Carcass c Average recovery of administered 3 H Aflatoxin B1 (18.12µg/kg BW)

  Figure 1. Plasma time-concentration profiles of aflatoxin B1 obtained after a single oral administration of 0.716 µg/kg body weight with ( low and high) and without ( ) co-administration of a yeast cell wall preparation in rats. * indicate p<0.05 between groups.

	Control	8.7± ± ± ±2.9		23.8± ± ± ±5.8	51.7	3.9± ± ± ±1.5	2.9	58.5
	YCW-L d YCW-H F o Aflatoxin B1 (0.716 µg/kg BW) r	4.9± ± ± ±0.8 4.8± ± ± ±1.3		31.6± ± ± ±3.5 39.6± ± ± ±27.0	55.0 58.1	2.2± ± ± ±0. 1 4.5± ± ± ±0.1	1.8 NA	59.1 63.7
	Control YCW-L YCW-H Ochratoxin A (3.22 µg/kg BW) P e 6.1± ± ± ±2.0 2.9± ± ± ±1.1 3.3± ± ± ±1.6 e r	* *	39.0± ± ± ±3.4 59.6± ± ± ±18.2 * 61.2± ± ± ±16.9 *	45.1 62.5 64.5	1.3± ± ± ±0.5 0.9± ± ± ±0. 6 0.9± ± ± ±0.4	NA NA NA	46.4 63.5 57.8
	Control YCW-L YCW-H	5.6± ± ± ±1.3 6.5± ± ± ±2.3 6.7± ± ± ±1.6 R e 16.3± ± ± ±2.6 23.8± ± ± ±4.9 * 28.7± ± ± ±6.4 * v i e w	44.1 49.3 51.8	5.1± ± ± ±1.2 2.4± ± ± ±0. 7 2.0± ± ± ±0.8	NA NA NA	49.1 51.7 53.8
				O n
					l y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgments

S. Firmin was supported by a doctoral fellowship (CIFRE 1058/2007), jointly financed


D Ruelland
email: denis.ruelland@um2.fr

L Collet

S Ardoin-Bardin

& P Roucou

How could hydro-climatic conditions evolve in the long term in West Africa? The case study of the Bani River catchment

Keywords: climatic variability, climatic scenarios, hydrological modelling, River Bani, West Africa

This paper assesses the future variability of water resources in the long term over a large Sudano-Sahelian catchment in West Africa. Flow simulations were performed with a daily conceptual model. The climate models HadCM3 and MPI-M (based on SRES-A2) were used to provide future climate scenarios over the catchment. Outputs from these models were used to generate daily rainfall and temperature series for the 21st century according to: (i) application of the unbias and delta methods, and (ii) spatial and temporal downscaling. A temperature-based formula was used to calculate present and future potential evapotranspiration (PE). The daily rainfall and PE series were introduced into the calibrated and validated hydrological model to simulate future discharge. The model correctly reproduces the observed discharge at the basin outlet with the Nash-Sutcliffe efficiency criterion over 0.89, and the volume error close to null over 1952-2000. With regard to future climate, the results show clear trends of reduced rainfall with a continuing increase in PE over the catchment. This suggests that the catchment discharge could fall in the long term to the same levels as those observed during the severe drought of the 1980s.

INTRODUCTION

Global climatic change, partly linked to anthropogenic activity, should lead to significant modifications in local and regional climatic conditions. The ability to anticipate the impacts of such changes on hydrosystems, and thus on the availability of water resources, is essential in order to face up to the adaptations of our societies. This is particularly true in West Africa, where the severe drought over the last 40 years has had significant hydrological impacts (see e.g. [START_REF] Lebel | Seasonal cycle and interannual variability of the Sahelian rainfall at hydrological scales[END_REF][START_REF] Andersen | The Niger River Basin: a vision for sustainable management[END_REF][START_REF] Ruelland | Sensitivity of a lumped and semi-distributed hydrological model to several methods of rain interpolation on a large basin in West Africa[END_REF]. These changes are extremely worrying for the local populations, especially since demographic growth has been accelerating in recent decades. The question then arises whether water resources availability can be expected to improve or deteriorate depending on the future climate in this region.

To assess the hydrological impacts of climate change, one must have both (i) a hydrological model that is adapted to various climatic conditions, and (ii) climatic scenarios in accordance with the spatio-temporal scales of the catchments considered. Where hydrological modelling is concerned, the paucity of descriptive data in West Africa militates in favour of a conceptual approach for studying large, poorly gauged catchments [START_REF] Ruelland | Modelling the long-term impact of climate change on rainfall-runoff processes over a large Sudano-Sahelian catchment[END_REF][START_REF] Ruelland | A comparison of two conceptual models for the simulation of hydro-climatic variability over 50 years in a large Sudano-Sahelian catchment[END_REF]. However, the question remains whether such an approach is adequate for simulating the long-term rainfallrunoff relationship in large catchments that are subject to significant hydro-climatic variability. Moreover, although these hydrological models are simple, they require high-resolution climate data, at least for variables such as precipitation and temperature. Global climate models (GCMs) provide such data and constitute powerful tools accounting for the complex set of processes that will produce future climate change. However, modelling the climatic system is a complex exercise, and climate projections are not easy to incorporate into hydrological impact studies [START_REF] Allen | Constraints on future changes in climate and the hydrological cycle[END_REF]. The use of GCMs' outputs currently faces three major problems: (i) the resolution of these models is not in line with that of hydrological modelling and hence requires spatial and temporal downscaling; (ii) the climatic projections produced by different GCMs and greenhouse gas scenarios (Special Report on Emissions Scenarios (SRES)) show very marked differences; (iii) precipitation projections for West Africa are generally less consistent (in comparison to projections for other regions) with large inter-model ranges for seasonal mean rainfall responses (see e.g. [START_REF] Lebel | From GCM scales to hydrological scales: rainfall variability in West Africa[END_REF][START_REF] D'orgeval | Impact du changement climatique sur la saison des pluies en Afrique de l'Ouest: que nous disent les modèles de climats actuels ?[END_REF]. As a result of these problems, direct use of the meteorological variables produced by GCMs for hydrological impact studies is a delicate matter. Climatic scenarios must thus be developed by correction of the GCMs' outputs.

This paper aims to forecast future water availability using climatic scenarios at the end of the 21st century in a large, poorly-gauged Sudano-Sahelian catchment.

STUDY AREA

The Bani River runs mainly through southern Mali (Fig. 1). Its catchment drains an area of around 100 000 km 2 at the Douna gauging station. The river flows into the Inner Niger Delta at Mopti and contributes about one-third of the annual flooding in this vast plain. Consequently, any disruption in the Bani catchment impacts on the delta and its vital economic activities. The watershed's topography is gently sloping, with elevations between 270 and 700 m. Soils are mostly ferralitic and lessivated with high sand and clay contents. Sandy hillwash is often found at the surface, while basal gravels are found in deeper parts of the profiles. Agricultural areas are growing rapidly because of demographic pressure.

The Bani catchment, located in a Sudano-Sahelian climatic regime, is characterized by a monsoon climate with a strong north-south rainfall gradient (Fig. 1) and considerable rainfall variability since the mid-20th century. As a result of this variability, the flow at the Douna gauging station fell by 68% between the 1952-1970 and 1971-2000 periods, with a decrease in the deep water recharge and baseflow contribution to the annual flood [START_REF] Ruelland | Modelling the long-term impact of climate change on rainfall-runoff processes over a large Sudano-Sahelian catchment[END_REF]. Some of the low-water periods were so severe that the river flow stopped periodically at Douna during the 1980s.

Fig. 1 The Bani catchment at Douna (104 000 km 2 ).

MATERIALS AND METHODS

Hydro-climatic data over the reference and future periods

In order to represent the hydro-climatic variability over the catchment, a 50-year period was chosen according to data availability. Daily rainfall series were derived from 72 raingauges covering the area (Fig. 1). For the 1950-2000 period, an average of 65 gauges per day (with a minimum of 39) was used to interpolate rainfall maps by the inverse distance weighted method, which proved to be optimally accurate among the classic methods available for data reconstruction in the given context [START_REF] Ruelland | Sensitivity of a lumped and semi-distributed hydrological model to several methods of rain interpolation on a large basin in West Africa[END_REF]. Potential evapotranspiration (PE) was estimated based on a 0.5° square grid from the CRU TS 2.1 World Database [START_REF] Mitchell | An improved method of constructing a database of monthly climate observations and associated high-resolution grids[END_REF]. Since the only data available for calculating PE were temperature data, a formula relying on solar radiation and mean temperature was selected [START_REF] Oudin | Which potential evapotranspiration input for a lumped rainfall-runoff model? Part 2: towards a simple and efficient potential evapotranspiration model for rainfall-runoff modelling[END_REF]. This formula was applied with a monthly temperature time step. However, solar radiation is a daily variable that depends on latitude and the Julian day of the year. Thus, PE was finally calculated with a daily time step.

Discharge data are from the Douna gauging station. This station appeared to have a highquality daily discharge series (less than 0.5% missing daily runoff values over and covers more than 75% of the entire catchment (Fig. 1).

Outputs from the GCMs HadCM3 and MPI-M were downloaded from the IPCC's Data Distribution Centre. All simulations of climate change were based on the SRES 20C3M scenario over the control period and the SRES-A2 for the future period (2071-2099).

Hydrological model: calibration and validation

In order to represent the seasonal and interannual variations in runoff from the catchment, discharge was linked to climatic series. In view of the scarcity of data on the catchment, the HydroStrahler conceptual model was chosen since it has yielded satisfactory results in West Africa (see [START_REF] Ruelland | Sensitivity of a lumped and semi-distributed hydrological model to several methods of rain interpolation on a large basin in West Africa[END_REF][START_REF] Ruelland | Modelling the long-term impact of climate change on rainfall-runoff processes over a large Sudano-Sahelian catchment[END_REF][START_REF] Ruelland | A comparison of two conceptual models for the simulation of hydro-climatic variability over 50 years in a large Sudano-Sahelian catchment[END_REF]. Using daily rainfall/PE data, this rainfall-runoff model represents, in a conceptual manner, the flow processes in a catchment and makes it possible to simulate runoff at its outlet with a daily time step. The model considers two reservoirs in the watershed (Fig. 2): (i) a shallow reservoir supplied by rainfall and feeding evapotranspiration, surface/subsurface runoff and infiltration, and (ii) a deep reservoir fed by infiltration and generating the baseflow. The model involves four parameters that need to be calibrated in order to account for three sources of runoff: immediate, rapid and delayed runoff.

Model calibration was realized based on a multi-objective function that aggregates a variety of goodness-of-fit indices (for more details on this function, see [START_REF] Ruelland | Modelling the long-term impact of climate change on rainfall-runoff processes over a large Sudano-Sahelian catchment[END_REF][START_REF] Ruelland | A comparison of two conceptual models for the simulation of hydro-climatic variability over 50 years in a large Sudano-Sahelian catchment[END_REF]:

( ) avg avg agg PE VE VE NSE 1 F + + + - = (1)
with NSE being the Nash-Sutcliffe efficiency criterion [START_REF] Nash | River flow forecasting through conceptual models, a discussion of principles[END_REF], VE the cumulative volume error, VE avg the annual average relative volume error, and PE avg the annual average peak error.

Model calibration was then performed in a 4-D parameter space by searching for the minimum value of F agg . A 10-day time step was used in order to limit problems related to time transfer. In both calibration and validation, the first two years of simulations were used as a warmup, to eliminate the influence of initial conditions in the model reservoirs. The 1952-2000 Fig. 2 Principle of the hydrological model (based on [START_REF] Ruelland | Sensitivity of a lumped and semi-distributed hydrological model to several methods of rain interpolation on a large basin in West Africa[END_REF]. simulation period was divided into three parts: calibration was performed for a 30-year period and validation was carried out in two periods of roughly 10 years (1952-1960 and 1991-2000). The two validation periods display contrasting climatic behaviours, the period 1952-1960 being wet and the period 1991-2000 being dry, which allowed us to test the model for suitability under different climatic conditions.

Building and introducing climatic scenarios in the model

Rainfall simulated by the GCMs is not sufficiently accurate to be used directly in impact studies to drive hydrological models (IPCC, 2007). This is particularly true in West Africa, as shown by [START_REF] Ardoin-Bardin | Using GCM outputs to assess impacts of climate change on runoff for large hydrological catchments in West Africa[END_REF]. Therefore, it is recommended that climatic scenarios be constructed using the ranges of variation between future climate and a baseline period in order to take climate variability changes and mean climate change into account [START_REF] Carter | Guidelines on the Use of Scenario Data for Climate Impact and Adaptation Assessment (version 1). Intergovernmental Panel on Climate Change[END_REF]. Projected climatic scenarios were thus developed based on the perturbation method. This method consists of producing future climatic scenarios by simply perturbing the observed climatic series over the control period (see e.g. [START_REF] Etchevers | Impact of a climate change on the Rhone river catchment hydrology[END_REF][START_REF] Prudhomme | Downscaling of global climate models for flood frequency analysis: where are we now?[END_REF][START_REF] Shabalova | Assessing future discharge of the River Rhine using regional climate model integrations and a hydrological model[END_REF]. For this purpose, the observed series for each hydrological model cell were simply translated and dilated so as to reproduce the changes of variance obtained between the control and future climatic simulations from both GCMs. Projected climatic scenarios were constructed in three steps. First, the average of the simulated and observed series was calculated over the reference period . The second step consisted of computing the monthly adjustments that would allow projected scenarios to be built. This was done by applying the unbiasing (for precipitation) and delta (for temperatures) methods between, respectively, the mean monthly hyetographs and thermographs simulated over the reference period and the monthly rainfall and temperatures simulated over the future period (2071-2099). The third step was to construct climatic scenarios for each hydrological model cell on a daily time step using a simple spatial and temporal downscaling. This was done by applying the adjustments calculated in step 2 to the daily mean hyetograph and thermograph calculated with the observed series over the reference period and for each cell of the hydrological model. The series generated represent simulated daily rainfall and temperature in the long term (2071-2099). PE was then calculated with the formula proposed by [START_REF] Oudin | Which potential evapotranspiration input for a lumped rainfall-runoff model? Part 2: towards a simple and efficient potential evapotranspiration model for rainfall-runoff modelling[END_REF] using monthly temperature series, yielding future daily series. This three-step method made it possible to construct climatic scenarios in the long term using a high-resolution grid and a daily time step. The future daily rainfall and PE series were used to feed the hydrological model. The latter was then run with the calibrated parameters to simulate future discharge based on those input data in the future simulation periods.

RESULTS

Model efficiency

Analysis of the fit of the hydrological model (Fig. 3) clearly shows that simulated discharge corresponds precisely to the observed values, although simulated discharge tends to end the hydrological season too abruptly. NSE values are over 0.89 in both the calibration and validation periods, and F agg close to null. Taking a closer look at the results, the 1952-1960 validation period provides better results than the calibration period, with the exception of VE. All the goodness-offit scores are quite well constrained. It can be noted that VE avg and PE avg do not exceed 0.2, which means that the interannual variability of water volume and the discharge peak are reproduced accurately in the simulations. Overall, the simulations correspond to the range of the observations. Concerning variation in water availability, the main issue of this study, it can be noted that the simulated and observed values for cumulated discharge fit particularly well. This is an important result because it shows that the model is capable of providing simulations that are useful for water resource management.

Future climatic trends

On the whole, rainfall as projected by HadCM3 and MPI-M decreases to 15-17% in the future relative to the reference observed period (Fig. 4(a)). This would reduce annual rainfall from 1081 mm/year reference period) to 898-920 mm/year in the long term, depending on the GCM. Minor changes in seasonal rainfall dynamics can be observed. While rainfall regularly increases with an annual peak in August over the reference period, future rainfall trends indicate that a peak would appear at the end of April, and another at the beginning of August. The decrease is significant at the beginning of the rainy season (which comes in May-June instead of March-April) and also during the heart of the rainy season, despite a peak at the beginning of August. Both GCMs also predict an earlier end of the rains (mid-October instead of end-October). Not only does the beginning of the rains come late, but also the appearance of peaks in April and July-August does not compensate the loss of precipitated volume in the heart of the rainy season.

Both GCMs predict a continuing rise in temperature over the 21st century that may lead to a 5.1-5.7°C increase in the long term over the catchment. This warming would lead to a large increase (16-18%) in PE over the catchment (Fig. 4(b)). This would raise annual PE from 1723 mm/year reference period) to 1995-2028 mm/year in the long term, depending on which GCM is considered. The PE values simulated by the two climatic models are similar in size, although those simulated by HadCM3 are slightly higher than those simulated by MPI-M from February to July. No modification of the seasonal dynamics of PE is observed: the lowest values are always in December-January and the highest in April-May. PE increases by a constant factor in every month of the year. This trend leads to July-October PE values that are comparable to those observed in April-May over the reference period.

The decline in rainfall accompanied by a PE increase in the long term thus shows an intensification of the trends observed since the late 1960s over the catchment.

Future hydrological trends

The projected rainfall deficit and continuing increase in PE suggest that runoff from the basin could be substantially reduced in the long term compared to the 1961-1990 reference period (Fig. 4(c)). The two models would lead to a similar decrease in discharge: when compared to the 1961-1990 reference period, the mean annual discharge would decline by 62-65% to only 120-130 m 3 /s, depending on the GCM considered. This decrease is due to some extent to the increase in PE, but more significantly to the decrease in precipitated volumes during the rainy season. The hydrological simulations predict earlier peak floods (about 15 days) than in the reference period, which can be linked to the peaks of rainfall in July simulated by the climatic scenarios. A premature depletion phase is also projected because of the loss of precipitated volume at the end of the rainy season.

The interannual variations in flows (Fig. 4(d)) simulated with both GCMs in the long term are similar and remain within the range observed over the 1961-1990 period. The hydrological situation remains overdrawn with regard to the reference period. The levels of mean annual discharge would fall to the same levels (about 100-200 m 3 /s) as those observed during the severe drought of the 1980s.

CONCLUSION

This paper aimed to assess the possible evolution of water resources in the Bani catchment and has shown that: (i) the spatial and temporal variability of GCMs' simulations in West Africa necessitated adjustment of these series for use in impact studies; (ii) daily future climatic scenarios can be constructed with simple spatial and temporal downscaling methods; (iii) a daily conceptual hydrological model yielded high-quality simulations in a large West African catchment subject to considerable hydro-climatic variability. The paper also proposes hydrological scenarios in the long term in this catchment (changes in hydrological dynamics and in interannual discharge). The results indicate that the hydrological model simulates discharge efficiently enough in the reference period to be considered an appropriate model for the Bani catchment. It should not induce substantial errors that would need to be taken into account. The future climatic trends simulated by the HadCM3 and MPI-M GCMs present the same patterns. Rainfall should decrease by 15-17% in the long term, while increasing temperature would induce a 16-18% increase in PE over the same horizon. The seasonal pattern of precipitation should change slightly, with a slower start of the rains and a loss of precipitated volume in the heart of the rainy season. In contrast, no modification of seasonal PE variability is expected. These conditions lead to the simulation of future discharge levels that are critical in terms of water availability for the end of the 21st century. Both GCMs predict a large reduction in runoff in the long term (62-65%), with a later beginning of the floods and an earlier depletion phase. Simulated flows were as low as the values observed during the severe drought of the 1980s.

Fig. 3

 3 Fig. 3 Comparison of the observed and simulated hydrographs at the Douna gauging station over the 1952-2000 period.

Fig. 4

 4 Fig. 4 Future climatic and hydrological trends in the long term (2071-2099) over the catchment: (a) changes in seasonal rainfall cycle; (b) changes in seasonal PE cycle; (c) changes in seasonal regime of river flows from the catchment; (d) interannual variations in flows during the late 21st century (bold lines indicate a five-year moving average of the values). Percentages indicate the future variations in rainfall, PE and discharge when compared, respectively, to mean annual precipitation (1081 mm/year), mean annual PE (1723 mm/year) and mean annual discharge (340 m 3 /s) over the 1961-1990 period.

Acknowledgements This work was supported by an internal project at HydroSciences Montpellier and was carried out as part of the French National Research Agency's RESSAC programme: Vulnerability of surface water resources to anthropogenic and climate changes in the Sahel.


Francis Lancelot

Enzymatic reactor using high activity immobilized enzymes during exothermic reaction

Canh Tran Minh, V.


Jerome Cornillon
email: jerome.cornillon@icloire.fr

Marie Bouteloup

teaching and research institutions in France or abroad, or from public or private research centers.

Evaluation of procalcitonin and CRP as sepsis markers

in 74 consecutive patients admitted with prolonged febrile neutropenia

Fever is a major event in critically ill patients. Its causes are difficult to identify and among them, bacterial infections must be rapidly identified and treated before sepsis reach a life threatening stage. CRP is a very sensitive marker of inflammation of whatever causes including sepsis. This is particularly crucial in immunocompromised patients. On the other hand, PCT has been extensively shown to be strongly and rapidly produced in bacterial infections only. Furthermore, PCT decreases very soon after bacterial resolution, and is thus a very useful in monitoring adequacy and efficiency of antibiotics. The utility of this marker in sever sepsis is now widely admitted. Recently, Limper et al. described biomarkers in discriminating infectious from non-infectious fever [START_REF] Limper | The diagnostic role of procalcitonin and other biomarkers in discriminating infectious from noninfectious fever[END_REF]. In new intensive chemotherapy strategies for the treatment of hemopathies as acute leukemia or bone marrow transplantation, profound and prolonged neutropenia (>7 days) expose the patients to life threatening infections. In these high risk patients, infections are the main causes of fever and bacterial origins are particularly life threatening (septic shock, pneumonia, fungal infectious…). Immediate empirical broad-spectrum antimicrobial therapy improves the survival but is not always optimal and ineffective treatment must be changed as soon as possible [START_REF] Hughes | Guidelines for the use of antimicrobial agents in neutropenic patients with cancer[END_REF][START_REF] Penack | Bloodstream infections in neutropenic patients: early detection of pathogens and directed antimicrobial therapy due to surveillance blood cultures[END_REF]. A recent review identifies PCT as a valuable tool in early evaluation of antibiotherapy efficacy [START_REF] Sakr | The role of procalcitonin in febrile neutropenic patients: review of the literature[END_REF]. But this meta analysis also underlined that published data only come from small, retrospective and/or heterogeneous study groups and do not bring any proof of high standard of evidence. The only conclusive results have shown interest of PCT in short term neutropenia. But regarding more particular populations (i.e.: stem cell transplantation) no univocal conclusion is possible from the few published studies [START_REF] Blijlevens | Procalcitonin does not discriminate infection from inflammation after allogeneic bone marrow transplantation[END_REF][START_REF] Ortega | Prospective evaluation of procalcitonin in adults with febrile neutropenia after haematopoietic stem cell transplantation[END_REF]. In this study, we intended to evaluate the interest of PCT versus CRP in early evaluation of antibiotherapy efficacy. We have analyzed prospectively serum levels of PCT and CRP every morning from the entrance into the intensive care unit for predicted adverse outcome. The study was performed in the intensive care unit of hematology of the Oncology's Institute of St-Etienne between September 2004 and July 2006. The monitoring concerned all consecutive patients who started an intensive chemotherapy program that induces prolonged neutropenia (>7 days). 74 patients were included. Among them, 44 were treated for leukemia's induction or consolidation with high dose of aracytin and 32 for bone marrow transplantation. PCT was measured by immunoluminometric LACE ® technique on Kryptor ® instrument according to manufacturer instructions (Brahms Diagnostica, Berlin, Germany) (normal value<0.5 μg/l; detection limit was 0.10 μg/l), CRP was measured by nephelemetrie on BN ProSpec instrument according to manufacturer instructions (Dade Behring, Siemens Germany) (reference value is <10 mg/L). At admission, the mean granulocyte count was 3200/mm3 (40-16600). Only 9 patients presented granulocyte count below 500/mm3. All patients have developped a neutropénia and the median duration of neutropenia was 17 days (from 4 to 113). All patients had at least one fever episode, for a median duration of 2 days (1-14). Thirty-eight (51%) episodes of fever were documented and considered to be related to bacteriema for 16 (22%) patients or local infection such as urinary tract (n = 11; 15%), lung (n = 3; 4%), gut (n = 4; 5%), skin (n = 1; 1%) and ear or nose (n = 3; 4%). In these cases, bacteria were a Gram-positive 28% or Gramnegative 19% bacteria and other (4%). coagulase negative Staphylococcus (14.8%) (2 cultures were necessary for diagnosis), Streptococcus (12.1%) and Escherichia Coli (9.5%) were the most frequent bacteria involved. At patient entrance, PCT and CRP levels were respectively 0.13 μg/l (range: 0.02-1.14) and 22.7 mg/L (range: 3-271). The PCT or CRP levels at the first day of fever were not significantly raised compared to the levels at admission and were not significantly different between types of infections (Table 1). We did no find any significant cut-off using Roc curve analyses predicting the types of infections. Only 15 patients (21%) reached a PCT level above 0.5 μg/L at first day of fever. In these patients, 12 patients presented documented bacterial infections. Majority of these patients presented a second febrile event (91%). At level of 0.5, PCT was positive in 38% of bacteriema and in 27% of localized or undocumented bacteria. On the other hand, CRP at admission was significantly higher in patients who had serious events later (death or transfert in ICU) at entrance (63.7 vs. 16.3, p = .001) Forty percents of the patients with CRP higher than 45 mg/l at admission presented serious event later (required intensive care with mechanical ventilation and/or eventually death) compared to 8% of the patients who had an initial CRP below this threshold. ENT: Ear-Nose-Throat.

T Ta ab bl le e 1 1. . P PC CT T a an nd d C CR RP P l le ev ve el ls s o of f p pa at ti ie en nt ts s, , c co or rr re el la at te ed d w wi it th h t th he e c ca at te eg go or ry y i in n i in nf fe ec ct ti io on n ( (d da ay ys s+ +1 1) ). .

No difference was noted between categories, but pulmonary's infections are presented more elevated level of PCT.

It is the first time to our knowledge, that PCT predictive values have been analyzed in a prospective and exhaustive study. Because we have evaluated PCT values every day during neutropenia, we could analyze the dynamic of PCT during the very first 24 h of fever in this period. Interestingly, we could not find in these patients any significant raise and then any significant predictive value of PCT that could help in the guidance of the treatment as it was repeatedly reported in sepsis of patients with normal count of neutrophils. The poor rise of PCT levels could be related to the neutropenia and subsequently the lack of source and production [START_REF] Oberhoffer | Procalcitonin expression in human peripheral blood mononuclear cells and its modulation by lipopolysaccharides and sepsis-related cytokines in vitro[END_REF]. Furthermore, because the diagnosis was made very early and the probabilistic treatment started immediately, it is possible that the PCT induction was killed in the egg, the infectious process being rapidly interrupted. This is in accordance with the short duration of fever events and previous reports [START_REF] Hughes | Guidelines for the use of antimicrobial agents in neutropenic patients with cancer[END_REF]. And, fortunately, we had no real antibiotherapy failure that should have let the PCT raising. It is most probable that prophylaxis including gut decontamination (non absorbable by colymicin and gentamycin) and Fluconazole could have help in limiting the occurrence of serious events.

In order to improve the specificity, manufacturer has proposed to use a cut-off at 2.0 ng/mL instead of 0.5 ng/ml. This strategy was not relevant in our early treated patients, in accordance with Massaro [START_REF] Massaro | Procalcitonin (PCT) and Creactive protein (CRP) as severe systemic infection markers in febrile neutropenic adults[END_REF] but this remains controversial [START_REF] Engel | Diagnostic value of procalcitonin serum levels in neutropenic patients with fever: comparison with interleukin-8[END_REF][START_REF] Svaldi | Procalcitoninreduced sensitivity and specificity in heavily leucopenic and immunosuppressed patients[END_REF], probably due to the heterogeneity of the populations that have been studied. The basic PCT level was never significantly raised and the patient had no inflammatory syndrome as shown by low CRP levels. In our experience, PCT above 0.5 μg/mL was significantly associated with clearly documented bacterial infection (12/15 total patients) with a good positive predictive value (80%). However, only 21% of patients were concerned and other fever events could either be related to a short-cut bacterial infection or other infectious causes that rapidly resolved with or without the help of the treatment. Finally, incidentally, our study has shown a negative predictive value of CRP raise at the admission of the patient. This can be related to an underlying inflammatory process possibly related to the disease or some incidental pathology. This fact could justify more studies to identify possible incident pathologies that could cause a high co-morbidity that could be treated in order to reduce the risks for these patients. So, at admission, theses patients should be managed with great care and we should promptly anticipate any events in neutropenic period. Thus, our study shows that early treatment of bacterial infection with probabilistic antibiotherapy could strongly reduce morbidity of febrile neutropenic patients according to the mean duration of the febrile episode and thus limit the PCT of PCT raise that was otherwise extensively demonstrated to be correlated to severe sepsis in non neutropenic patients.

Disclosures

None declared.

Conflict of interest

The authors had no conflicts of interest with any of the industrials cited in this report.