V Niderkorn

D P Morgavi

E Pujos

A Tissandier

simulating corn silage

Keywords: zearalenone, Fumonisins, Mycotoxins -fusarium, trichothecenes Food Types: Animal feedingstuffs Fusarium, mycotoxins, fermentative bacteria, probiotic, binding, 33 biotransformation, corn silage 34 35 Running headline: Fusarium toxins removal by fermentative bacteria

Screening of fermentative bacteria for their ability to bind and biotransform deoxynivalenol, zearalenone and fumonisins in an in vitro model

Introduction 37

Whole-plant corn silage and high moisture, fermented corn grain are widely utilized as 38 ruminants and swine feeds, respectively. Under unfavourable climatic conditions, 39

Fusarium toxins including deoxynivalenol (DON), zearalenone (ZEN), and fumonisins 40 (FB) can be produced on corn plants in the field [START_REF] Lepom | Occurrence of Fusarium species and their mycotoxins in maize. 6. Formation of zearalenone and trichothecenes type A by indigenous Fusarium isolates[END_REF]) and be present in 41 silage [START_REF] Scudamore | Occurrence and significance of mycotoxins in forage crops and silage: a review[END_REF][START_REF] Orsi | Mycoflora and occurrence of fumonisins in freshly harvested and stored hybrid maize[END_REF]. Ingestion of mycotoxin-42 contaminated corn by animals affects performances and health. Among farm animals, 43 swine appear to be most susceptible to the toxic effects of Fusarium toxins, especially 44 to ZEN [START_REF] Diekman | Mycotoxins and reproduction in domestic livestock[END_REF]. Although ruminants are considered more resistant, 45 the risk should not be underestimated as data of toxicity after chronic exposure or due to 46 interactions among mycotoxins are limited [START_REF] Seeling | Relevance of the Fusarium toxins deoxynivalenol and zearalenone in ruminant nutrition. A review[END_REF]. ZEN is 47 associated with estrogenic syndrome and DON causes vomiting in pigs, as well as 48 decreased feed intake and digestive disorders in both pigs and ruminants, leading to 49 losses of weight gain (D' Mello et al. 1999;[START_REF] Eriksen | Toxicological evaluation of trichothecenes in animal feed[END_REF]. Fumonisins 50 cause pulmonary edema in swine [START_REF] Harrison | Pulmonary edema and hydrothorax in swine produced by fumonisin B1, a toxic metabolite of Fusarium moniliforme[END_REF]) and hepatotoxicity in ruminants 51 [START_REF] Diaz | Effect of fumonisin on lactating dairy cattle[END_REF]. 52 53 In spite of the progress made in preventive approaches such as breeding of resistant corn 54 varieties [START_REF] Mesterhazy | Progress in breeding of wheat and corn genotypes not susceptible to infection by fusaria[END_REF]) and improvement in agronomic practices (Trenholm et al. 55 1989) to decrease Fusarium contamination, hazardous concentrations of Fusarium 56 toxins may occur in corn justifying the use of detoxification methods. Physical and 57 chemical methods such as ammonia treatment or the addition of adsorbents [START_REF] Scott | Industrial and farm detoxification processes for mycotoxins[END_REF]58 Huwig et al. 2001) are generally expensive, drastic, not specific, or simply not adapted 59 to the treatment of corn destined to livestock. Inorganic adsorbents, although they have 60 a high affinity to bind aflatoxins, are not efficient for Fusarium toxins. In addition, these 61 agents can also bind important micronutrients like minerals or vitamins [START_REF] Guerre | Interest of the treatments of raw materials and usage of adsorbents to decontaminate animal food containing mycotoxins[END_REF]. 62

Practical methods for the detoxification of silage are currently not available. A 63 promising approach is the use of selected microorganisms to remove Fusarium toxins 64 [START_REF] Bata | Detoxification of mycotoxin-contaminated food and feed by microorganisms[END_REF]. Detoxification can be achieved by microbial binding and/or 65 biotransformation of mycotoxins into less toxic compounds (Styriak and Conkova 66 2002). Due to their role in the ensiling process, fermentative bacteria are appropriate 67 candidates for the biological detoxification of corn silage. The bioconversion of 68 Fusarium toxins by rumen microbes and intestinal microflora was reported (Kiessling et 69 al. 1984;[START_REF] Kollarczik | In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs[END_REF][START_REF] Caloni | In vitro metabolism of fumonisin B1 by ruminal microflora[END_REF] but the bacteria responsible for their 70 biotransformation were not identified. In contrast, binding of DON and ZEN to the cell 71 wall of probiotic strains in vitro has been recently reported (El-Nezami et al. 2002a;72 2002b). This property can decrease the bioavailability of these compounds by reducing 73 absorption and limit their toxic effects [START_REF] El-Nezami | Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B1, from the chicken duodenum[END_REF]. However, to date, only 74 three strains were thoroughly investigated. In addition, they were effective at about 10 10 75 CFU ml -1 , a high bacterial concentration that is difficult to obtain in fermented corn 76 feeds. 77

78

In this study, a large number of strains of fermentative bacteria were screened for their 79 ability to remove DON, ZEN, FB 1 and FB 2 from a medium that simulated silage in term 80 of substrate, pH, and bacterial concentration. [START_REF] Malik | An evaluation of the taxonomy of Propionibacterium[END_REF], was used for the culture of 102 Propionibacteria. A corn infusion was prepared by steeping dry whole-plant corn in 103 water (6% w/v) at 60°C for 2 h. The infusion was then filtered through filter paper 104 (Durieux, no. 120, VWR, Fontenay-sous-Bois, France), the filtrate was centrifuged 105 (8500 g, 5 min) and the decanted supernatant was filtered through a 0.45 µm membrane. 106

The filtered infusion was stored at 4°C and adjusted to pH 4 with lactic acid before 107 Sigma-Aldrich Chemie Gmbh (Steinheim, Germany). The most common derivative of 113 DON, deepoxy-deoxynivalenol (DOM) was purchased from Biopure (Tulin, Austria). 114 Derivatives of fumonisins, aminopentols (HFB 1 and HFB 2 ), were obtained by 115 hydrolysis of FB 1 and FB 2 according to [START_REF] Pagliuca | Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography-fluorescence detection[END_REF]. DON and ZEN (and 116 derivatives) were dissolved in acetonitrile and methanol, respectively. Concentration 117 was determined by measuring the absorbance at 218 nm for DON (ε = 6406 mmol -1 118 cm 2 ) and at 274 nm for ZEN (ε = 13 909 mmol -1 cm 2 ). Since spectrophotometry is not 119 possible with FB 1 and FB 2 , these compounds were dissolved in an exact volume of 120 acetonitrile-water 1:1(v/v) to achieve the desired concentration. Mycotoxin solutions of 121 DON (5 µg ml -1 ), ZEN (5 µg ml -1 ), FB 1 + FB 2 (5 µg ml -1 each) in corn infusion were 122 prepared by evaporating solvents with nitrogen gas, redissolving in water (DON, FB 1 , 123 FB 2 ) or ethanol (ZEN) and adding corn infusion to reach the desired concentration. The 124 water or ethanol solvent represented 5% (v/v) of the final mycotoxin solution. 125 126

Mycotoxin removal test 127

Bacteria screened for their ability to bind and/or biotransform Fusarium toxins were 128 prepared as follow. For each strain, one tube containing 20 ml of medium was 129 inoculated with 0.1 ml of an overnight culture and was incubated at optimal temperature 130 of growth (30 or 37°C) for 24 h. Propionibacteria strains were incubated at 30°C for 72 131 h. At the end of incubation, the bacterial concentration of cultures was estimated by 132 measuring the absorbance at 600 nm as described previously [START_REF] Niderkorn | Binding of Fusarium mycotoxins by fermentative bacteria in vitro[END_REF]. A 133 volume of culture containing 35 × 10 8 CFU was centrifuged (3000 g, 10 min, 5°C) and 134 the supernatant was removed. The bacterial pellet was washed twice with 5 ml of 135 phosphate-buffered saline (PBS 0.01 M, pH 7.4). After the second wash, the bacterial 136 pellet was resuspended in 7 ml PBS to obtain a concentration of 5 × 10 8 CFU ml -1 , and 137 transferred to six polypropylene tubes (1 ml per tube). Tubes were centrifuged (3000 g, 138 10 min, 5°C), supernatants were removed, and bacteria were re-suspended in 1 ml of 139 one of the three mycotoxin solutions. Positive controls containing no bacteria and a 140 negative control per bacterial genus containing no mycotoxin were included. All the 141 tubes were incubated at 25°C in microaerophilic conditions for 1 h with shaking (480 142 rpm), then for 23 h without shaking. At the end of the incubation period, tubes were 143 centrifuged (3000 g, 10 min, 5°C) and supernatants were analysed for mycotoxins by 144 reversed-phase HPLC. Assays and positive controls were performed in duplicate. 145

The percentage of mycotoxin removed was calculated by using the following 146

HPLC mycotoxins analysis 150

Separation and quantification of mycotoxins were performed by the method described 151 previously [START_REF] Niderkorn | Binding of Fusarium mycotoxins by fermentative bacteria in vitro[END_REF] with slight modifications in order to remove 152 interferences. The HPLC system consisted of a P1000XR pump (SpectraSYSTEM, San 153 Jose, California, USA) and an automatic sampler (SpectraPhysics, San Jose, California, 154 USA). Separation was performed on C 18 reversed-phase columns (Macherey-Nagel, 155

France); Nucleodur (125 × 4.6 mm, 5 mm) was used for DON and ZEN and Prontosil 156 (120 × 3.0 mm, 3 µm) was used for FB 1 and FB 2 . The mobile phase used to separate 157 DON consisted of a water-acetonitrile solution (95:5 v/v) and the flow rate was 1 ml 158 min -1 . Detection was set at 220 nm and retention time was 12.1 min. After 14 min of 159 run, the column was washed with a water-acetonitrile solution (80:20 v/v) to remove 160 were mixed with 90 µl of borate buffer pH 10, then 100 µl of derivatizing reagent were 167 added. The preparation was mixed and allowed to react for 2 min before injection. 168 Separation of FB 1 and FB 2 was performed with a gradient elution using acetonitrile (A) 169 and a mixture of 0.05 mol l -1 dihydrogen phosphate-methanol (1:1 v/v) acidified at pH 170 3.35 (B). The gradient program was as follows: 10% to 50% A in 6 min, 50% A for 7 171 min, and 50% to 10% A in 1 min. The flow rate was 1 ml min -1 and detection was set at 172 336 nm excitation and 440 nm emission. The retention times were 10.0 and 12.7 min for 173 FB 1 and FB 2 , respectively. 174 175

HPLC-ESI-MS/MS analysis 176

A HPLC-MS/MS (TSQ Quantum Ultra AM, Thermo Electron, San Jose, California, 177 USA) equipped with an electrospray ionization source (ESI) interface operating in the 178 negative mode was used to identify the ZEN derivative produced by some strains. 179 Chromatographic separation and MS analysis was performed on a 125 × 4.6 mm 180 Prontosil C 18 column with particle size of 3 µm (Bischoff, Leonberg, Germany), using a 181 The most intense transitions from the fragmentation of ZEN, α ZOL and ZAN were 187 determined. Six product ions were selected at different collision energies (CE) so as to 188 characterize each molecule of interest (Table I). For analysis, 4 ml of corn infusion 189 sample were extracted with 4 ml dichloromethane. The mixture was mixed for 15 min 190 then centrifuged at 3000 g for 10 min. One ml of the organic layer was transferred into a 191 tested (about 5.5%), an additional peak was observed in the chromatograms. This peak 214 had the same retention time for all 11 strains suggesting that it was the same metabolite 215 (Figure 2). Eight out of the 11 strains were Lactobacilli and the other three were 216

Leuconostoc. These biotransforming Lactobacilli removed approximately twice more 217 ZEN from the incubation media than strains from the same genus that only bound the 218 mycotoxin (Table II andIII which was set at 0.8 ml min -1 . Under these conditions, the Rt of the unknown metabolite 226 was 9.56 min, similar to α ZOL (9.50 min), while Rts of β ZOL, α and β ZAL, and ZAN 227 standards were 6.10, 7.74, 4.35, and 9.84 min, respectively. This result indicated that α 228 ZOL was most likely the metabolite produced. The Rt of ZAN was also near to that of 229 the unknown metabolite. However, ZAN, at a concentration of 5 µg ml -1 had a peak 230 area considerably lower than the peak area of the metabolite produced by active 231 bacterial strains incubated with an equal concentration of ZEN and hence ZAN was 232 unlikely the searched compound. Additional HPLC-MS/MS analyses were performed 233 on ZEN-containing corn infusion extracts after incubation with biotransforming strains. 234 The LC-MS showed that the new peak had the same mass transitions characteristic (m/z 235 = 319) as α ZOL (Figure 3) and thus unequivocally identified the metabolite. [Insert 236

Figure 3 about here] 237 238

Binding of zearalenone, deoxynivalenol and fumonisins from acidified corn infusion 239

Table III shows the mycotoxin-binding capacity of the tested fermentative bacteria 240 grouped by genera. Across all genera, FB 2 was the mycotoxin most efficiently removed 241 followed by ZEN, DON, and FB 1 . The average fraction of DON bound by the different 242 genera ranged from 15 to 22% but these differences were not significant (p > 0.05). In 243 contrast, large differences among genera were observed for the binding of ZEN and 244 fumonisins (p < 0.05). It is interesting to note that, independently of the molecular 245 structure of these mycotoxins, the genera Streptococcus and Enterococcus ranked 246 always as the most efficient binders under the conditions of the assay. In particular, 247 strains of Streptococcus thermophilus, which bound up to 49%, 33%, 24%, and 62% of 248 ZEN, DON, FB 1 and FB 2 , respectively. Out of the 202 strains tested about two third 249 were Lactobacilli. This apparent bias has a biotechnological explanation because 250 species of this genus are very active during the ensiling process and hence widely used 251 as inoculants. Lactobacilli are dominant members of the bacterial population found in 252 grass and corn silages [START_REF] Langston | A study of the microorganisms from grass silage. II. The Lactobacilli[END_REF][START_REF] Dellaglio | DNA-DNA homology, physiological characteristics and distribution of lactic acid bacteria isolated from maize silage[END_REF]. L. 253 plantarum, of which we tested 46 isolates, is a particularly common epiphytic specie, 254 which is also found in many commercial silage additives [START_REF] Weinberg | New trends and opportunities in the development and use of inoculants for silage[END_REF]. 255 All the other selected strains used in this work are representative of genera of 256 fermentative bacteria regularly associated with silage [START_REF] Moon | Lactic acid bacteria active during the fermentation of wheat silage in small scale silos[END_REF]Driehuis and 257 Elferink 2000). The binding activities of Lactobacillus spp. are shown in Table IV The ability of fermentative bacteria to biotransform or bind Fusarium toxins in whole-264 plant corn silage and fermented corn grain is a promising way to reduce their toxic 265 effects on livestock. In this work, the conditions used to screen fermentative bacteria 266 permitted to observe simultaneously binding and biotransformation in a liquid medium 267 representative of corn silage. 268

269

The reduction of ZEN into α-ZOL can be carried out in the liver and also by the flora 270 harboured in the digestive tract of animals [START_REF] Olsen | Species differences in zearalenone-reducing activity in subcellular fractions of liver from female domestic animals[END_REF]Kiessling et al. 271 1984;[START_REF] Kollarczik | In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs[END_REF]). However, it had never been observed with pure strains of 272 lactic acid bacteria (LAB). In this study, this biotransformation was observed in 11 273 strains but it cannot be considered as a detoxification because α-ZOL is three to four 274 times more estrogenic than ZEN [START_REF] Mirocha | Incidence of zearalenol (Fusarium mycotoxin) in animal feed[END_REF]. All the positive bacterial strains 275 detected in this work were capable to produce large quantities of α-ZOL from ZEN. 276 Even if strains possessing this property represent a relatively low percentage of the total 277 fermentative bacteria, they could form part of the epiphytic population present in 278 fermented feeds and convert significant amounts of ZEN into α-ZOL. This means that 279 the absence of ZEN or its presence in non toxic concentrations does not guarantee feed 280 innocuity, and therefore, it could be advisable to check for the presence of α-ZOL in 281 fermented feeds. It could also be recommended to check if this property is not present in 282 strains used as fermentation starters. The biotransformation of ZEN to α-ZOL might 283 explain the results of a recent study where a significant decrease in the concentration of 284 [START_REF] Mokoena | Reduction of fumonisin B1 and zearalenone by lactic acid bacteria in fermented maize meal[END_REF]. 286 287 Binding of mutagens and mycotoxins by LAB was reported in numerous studies 288 [START_REF] Morotomi | In vitro binding of potent mutagenic pyrolysates to intestinal bacteria[END_REF][START_REF] El-Nezami | Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1[END_REF]. Binding of DON and ZEN by three 289 probiotic strains was also recently reported, but they were not effective at the low 290 densities found in fermented corn feeds (El-Nezami et al. 2002a;2002b). The results of 291 the present work showed that significant quantities of Fusarium toxins including 292 fumonisins are bound by most species of fermentative bacteria at a density of 5 ×10 8 293 CFU ml -1 . We also confirmed previous observations [START_REF] Niderkorn | Binding of Fusarium mycotoxins by fermentative bacteria in vitro[END_REF] showing 294 that FB 2 was removed more efficiently than FB 1 in spite of their similarity in chemical 295 structure. 296 297 Since most strains of fermentative bacteria tested seem to have the ability to bind DON,298 ZEN and fumonisins, it is probable that these Fusarium toxins are also partially bound 299 by the epiphytic microflora of silage or by inoculants already utilized to improve silage 300 fermentation or aerobic stability. Although Lactobacilli are widely utilized as silage 301 additives, none of the strains tested was particularly efficient at binding Fusarium 302 toxins. From the isolates tested in this work, the use of fermentative bacteria to bind 303 Fusarium toxins in silage appear to be more advantageous for Streptococci and 304

Enterococci, which bound more ZEN and fumonisins than other genera. Consequently, 305 an advantageous use of fermentative bacteria to bind Fusarium toxins in silage would 306 be limited to these two genera. The higher capacity to bind Fusarium toxins by 307

Streptococci and Enterococci cannot be explained by their size as they are smaller than 308 other genera with lower binding capacity such as Lactobacillus. The composition of the 309 The mechanism of binding of mycotoxins by bacteria is still not clear. In the case of 311 ZEN, it was recently suggested that this mycotoxin predominantly binds to 312 carbohydrate moieties of the cell wall of LAB by means of hydrophobic interactions 313 [START_REF] El-Nezami | Chemical moieties and interactions involved in the binding of zearalenone to the surface of Lactobacillus rhamnosus strains GG[END_REF]). As hydrophobic interactions are relatively weak, this 314 bacterium-mycotoxin complex could be unstable. Nevertheless, these authors also 315 suggested that ZEN binding is not limited to hydrophobic links and other type of 316 interactions could be important. 317 318

Conclusions 319

The use of selected strains of fermentative bacteria could be a method to limit the toxic 320 effects of Fusarium toxins present in corn silage. Our results have shown that the 321 biodegradation of mycotoxins is possible. However, the resulting compound is not 322 always less toxic than the parent mycotoxin. Binding seems to be the most promising 323 mode of action, but for the practical application of this technology, it is essential that the 324 bacterium-mycotoxin complex remain stable during the digestion process. Further 325 studies are planned to evaluate the stability of the complex in the different parts of the 326 gastrointestinal tract of farm animals. 327

Figure captions

Figure 1. HPLC chromatograms of corn infusion supernatants incubated with bacteria (5 × 10 8 CFU ml -1 ) at 25°C for 24 h without and with zearalenone (ZEN, 5 µg ml -1 ) (a), without and with fumonisins B 1 and B 2 (FB 1 and FB 2 , 5 µg ml -1 each) (b), without and with deoxynivalenol (DON, 5 µg ml -1 ) (c).

Figure 2. HPLC chromatograms of corn infusion supernatants containing zearalenone (ZEN, 5 µg ml -1 ) incubated at 25°C for 24 h without bacteria (a) and with Lactobacillus brevis R0002

(5 × 10 8 CFU ml -1 ) (b). ZEN (retention time = 10.4 min) and metabolite (retention time = 9.2 min) showed a similar UV spectrum (inset). *Bacteria (5 × 10 8 CFU ml -1 ) were incubated for 24 h at 25°C in corn infusion, pH 4, containing ZEN, 5 µg ml -1 , DON, 5 µg ml -1 , and a mixture of FB 1 and FB 2 , 5 µg ml -1 each.

DON

Figure 1. HPLC chromatograms of corn infusion supernatants incubated with bacteria (5 × 10 8 CFU ml -1 ) at 25°C for 24 h without and with zearalenone (ZEN, 5 µg ml -1 ) (a), without and with fumonisins B 1 and B 2 (FB 1 and FB 2 , 5 µg ml -1 each) (b), without and with deoxynivalenol (DON, 5 µg ml -1 ) (c).

  1 , FB 2 , ZEN and its main derivatives αand β-zearalenols (α and β ZOL), α-111 and β-zearalanols (α-and β-ZAL), and zearalanone (ZAN) were purchased from 112

  formula: Mycotoxin removed (%) = 100 × [1 -(Peak area of mycotoxin in the 147 supernatant / Peak area of mycotoxin in the positive control)]. 148 149

  equilibrated with the mobile phase for 6 min before the following 161 injection. The mobile phase used to separate ZEN consisted of a water-methanol 162 solution (40:60 v/v) and the flow rate was 1.2 ml min -1 . ZEN was detected by photo-163 diode-array (scan 200 -380 nm) and fluorescence (λ exc = 274 nm, λ em = 440 nm) placed 164 in series. The retention time of ZEN was 10.7 min. Fumonisins were derivatized before 165 injection using ortho-phtalaldehyde (OPA) plus mercaptoethanol. Ten µl of sample 166

  methanol -1% acetic acid (65:35 v/v) mobile phase at a flow rate of 0.8 ml min -1 . 182 Parameters of the MS were optimised using standard solutions of α ZOL in mobile 183 phase. The test responses were recorded with electrospray voltage 3.0 kV, capillary 184 temperature 400°C, sheath flow and auxiliary flow 50 and 10 arbitrary units, 185 reaction monitoring (MRM) was used to identify the metabolite. 186

  new tube and evaporated to dryness under a stream of N 2 . The dried residue was 192 redissolved in 200 µl of mobile phase, and 5 µl of this solution were injected into the 193 HPLC-MS/MS system. MS-analysis of extract was compared withZEN, α ZOL and 194 ZAN standards. [Insert Table I about here] 195 196 Statistical analysis 197 Data was subjected to the analysis of variance (ANOVA). A significant difference (p < 198 0.05) between means was determined by Duncan's multiple range test using the 199 Statistical Analysis System software package (SAS Institute Inc., Cary, NC). 200 201 Results202The experimental model used in this work allowed us to observe at the same time 203 biotransformation and binding of Fusarium toxins by fermentative bacteria. The HPLC 204 method used, separated each mycotoxin as well as their main derivatives from the corn 205 infusion medium matrix without an extraction step (Figure1) and thus facilitated the DON and fumonisins no biotransformation products were 210 observed. There were not peaks corresponding to DOM and aminopentol-derivatives 211 of DON and fumonisins, respectively-or any other unknown peak in the HPLC 212 chromatograms. In contrast, in incubations containing ZEN for 11 out of the 202 strains 213

  ). [Insert Figure2and TableII abouthere] 219 220 The ZEN metabolite showed a UV spectrum similar to ZEN with absorption maxima at 221 236, 274, and 316 nm (Figure 2, inset). Retention time (Rt) of this peak was compared 222 to those of main ZEN derivatives using modified chromatographic conditions to 223 improve separation. The changes in the chromatography were in the mobile phase, 224 which consisted in methanol -1% acetic acid mixture (65:35 v/v), and the flow rate, 225

  during the fermentation of corn meal by LAB but without a 285 reduction in toxicity

  confer the Streptococci and Enterococci their superior binding property. 310

  of Microorganisms, University Louis Pasteur/CNRS FRE 2326, 335 Strasbourg, France, the Cheese Research Laboratory, INRA, Aurillac, France, and 336 Lallemand SAS, Blagnac, France, for providing the strains of fermentative bacteria. 337 They also thank Henri Durand, Frederique Chaucheyras-Durand, and Corinne Pouyet 338 for the scientific assistance in this project. 339

Figure 3 .

 3 Figure 3. HPLC-MS spectra of zearalenone, ZEN (a) and α zearalenol, α ZOL (b) standards

Figure 2 .

 2 Figure2. HPLC chromatograms of corn infusion supernatants containing zearalenone (ZEN, 5 µg ml -1 ) incubated at 25°C for 24 h without bacteria (a) and with Lactobacillus brevis R0002 (5 × 10 8 CFU ml -1 ) (b). ZEN (retention time = 10.4 min) and metabolite (retention time = 9.2 min) showed a similar UV spectrum (inset).

Figure 3 .

 3 Figure 3. HPLC-MS spectra of zearalenone, ZEN (a) and α zearalenol, α ZOL (b) standards alone, and the extract from corn infusion supernatant containing ZEN and the produced metabolite (c). Spectra of peaks 1 and 2 showed parent mass of m/z 319.08 and 317.06, respectively.

  Streptococcus and Enterococcus strains the binding capacity of the Lactobacilli was less 260 important. [Insert Table III and Table IV about here] 261

	262
	263	Discussion
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	258	.
	259	Significant differences were observed among strains; however, compared to
		12

Table captions Table I .

 captionsI Product ions of zearalenone (ZEN), α zearalenol (α ZOL), and zearalanone (ZAN) for HPLC-ESI-MS/MS analysis.

Table II .

 II Lactic acid bacteria biotransforming zearalenone (ZEN) to α zearalenol (α ZOL).

Table III .

 III In vitro binding of zearalenone (ZEN), deoxynivalenol (DON), and fumonisins B 1 and B 2 (FB 1 , FB 2 ) by different genera of fermentative bacteria.

Table IV .

 IV In vitro binding of zearalenone, deoxynivalenol, and fumonisins B 1 and B 2 by

	Lactobacillus species.

Table I .

 I Product ions of zearalenone (ZEN), α zearalenol (α ZOL), and zearalanone (ZAN) for HPLC-ESI-MS/MS analysis

	Compound	Retention time (min)		Transition	CE (V)
	ZEN	11.01		317 → 130	46
				317 → 131	35
				317 → 149	35
				317 → 175	34
				317 → 187	30
	α-ZOL	9.59 F o r	317 → 273 319 → 130 319 → 131 319 → 160 319 → 174	27 54 55 37 13
		P	319 → 275 319 → 301	31 27
	ZAN	9.90	319 → 137 319 → 161 e 319 → 163 319 → 205 e r	52 35 38 31
				319 → 259 319 → 275 R	37 28
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Table II .

 II Lactic acid bacteria biotransforming zearalenone (ZEN) to α zearalenol (α ZOL) *

	Taxon	Source	Used in	ZEN biotransformed (%)
	Lactobacillus
	L. brevis	LGMPA -C11	Cheese making	41 (1)
	L. brevis	Lallemand -R0002	Animal nutrition	47 (2)
	L. brevis	Lallemand -L62	Baking	24 (1)
	L. casei casei	LGMPA -B1	Cheese making	49 (4)
	L. fermenti	LGMPA -34	Cheese making	19 (0)
	L. plantarum	LGMPA -J1	Cheese making	41 (2)
	L. plantarum	Lallemand -R1113	Baking	50 (3)
	L. spp.	LGMPA -F7	Cheese making	23 (1)
	Leuconostoc Ln. mesenteroides or Ln. pseudomesenteroides Ln. mesenteroides or Ln. pseudomesenteroides Ln. mesenteroides F o r	URF -171 URF -365 URF -1118	Cheese making Cheese making Cheese making	16 (2) 8 (0) 9 (0)
	*Bacteria (5 × 10 8 CFU ml -1 ) were incubated for 24 h at 25°C in corn infusion, pH 4, containing ZEN (5 µg ml -1 ). Data shown are the means (SD) of duplicate. P show only binding, strains biotransforming ZEN were not included. e
	e r
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Table III .

 III In vitro binding of zearalenone (ZEN), deoxynivalenol (DON), and fumonisins B 1 and B 2 (FB 1 , FB 2 ) by different genera of fermentative bacteria *

	Genus	Number of strains		Fraction bound (%) †
			ZEN ‡	DON	FB1	FB2
	Enterococcus	4	35 (2) c	22 (8)	14 (7) b	43 (8) b
	Lactobacillus	137	20 (4) b	15 (9)	10 (6) ab	24 (11) a
	Lactococcus	17	17 (4) ab	18 (8)	11 (4) ab	19 (8) a
	Leuconostoc	6	28 (7) bc	18 (2)	4 (3) a	18 (5) a
	Pediococcus	2	17 (2) ab	18 (3)	4 (3) a	23 (10) a
	Propionibacterium	5	10 (3) a	18 (6)	10 (7) ab	15 (7) a
	Streptococcus	31	40 (4) c	18 (7)	15 (5) b	48 (9) b

Table IV .

 IV Each strain was tested for each mycotoxin in duplicate.† Values are the means (SD) of mycotoxin bound for all strains belonging to the same genus. Data within a column with unlike letter are not significantly different (p < 0.05). ‡ To show only binding, strains biotransforming ZEN were not included. In vitro binding of zearalenone, deoxynivalenol, and fumonisins B 1 and B 2 by Lactobacillus species * Values are the means (SD) of mycotoxin bound for strains belonging to the same species. Data within a column with unlike letter are not significantly different (p < 0.05). ‡ To show only binding, strains biotransforming ZEN were not included.

																	Volts Volts Volts
	Lactobacillus species				Number of strains				Fraction bound (%) †
														ZEN ‡		DON	FB1	FB2
	L. acidophilus							7			19 (4)	22 (4)	c	13 (6)	b	22 (10)	abc
	L. brevis									5			20 (1)	19 (5) c	9 (4) ab	27 (9) bcd
	L. buchneri								3			20 (2)	4 (4) ab	11 (1) ab	28 (12) cd
	L. casei . subsp. alactosus					1			18 (0) ZEN	10 (0) abc	3 (0) a	9 (0) a
	L. casei . subsp. casei						21			18 (4)	17 (9) c	11 (5) ab	25 (11) bcd
	L. casei . subsp. rhamnosus					4			18 (6)	19 (6) c	12 (7) ab	13 (3) ab
	F o r L. delbruekii . subsp. bulgaricus L. delbruekii . subsp. lactis L. fermentum L. helveticus L. jugurti L. paraplantarum r o Minutes Minutes F 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8	5 3 10 8 7 7 9 9	10 10	11 11	28 (4) 22 (5) 21 (7) 19 (2) 19 (4) 22 (2) 12 13 12 13	14 14	15 15	11 (9) abc 19 (9) c 13 (8) abc 17 (7) c 20 (9) c 13 (8) abc	12 (5) ab 9 (4) ab 13 (12) b 11 (6) ab 12 (7) ab 5 (4) ab	26 (7) bcd 23 (9) abc 25 (11) bcd 31 (9) cd 34 (9) cd 10 (6) a
	L. pentosus L. plantarum L. reuteri L. unidentified					P e 1 46 1 P 8 e		23 (0) 19 (3) 17 (0) 20 (2)	2 (0) a 13 (9) abc 15 (0) bc 19 (9) c	4 (0) ab 9 (5) ab 21 (0) c 8 (5) ab	32 (0) cd 22 (10) abc 37 (0) d 26 (6) bcd
											e r e r FB 1
													R R FB 2
															e e
	0 0	1 1	2 2	3 3	4 4	5 5	6 6	7 7	Minutes Minutes 8 8	9 9	10 10	11 11	12 12	13 13	14 14	v i e e i v 15 15
																	w w
																	O n l O n l
																	y y
	0 0	1 1	2 2	3 3	4 4	5 5	6 6	7 7	8 8	9 9	10 10	11 11		12 12	13 13		14 14	15 15
									Minutes Minutes

*Test conditions were similar to those shown in table

I

.

†

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

Acknowledgements 330 V. Niderkorn is the recipient of a CIFRE Lallemand SAS research fellowship. The 331 authors are thankful to the Research Unit for Food Process Engineering and 332 Microbiology, INRA Thivernal-Grignon, France, the Research Laboratory for Animal


O ' Keeffe

Michael O'keeffe
email: michael.okeeffe@teagasc.ie

Emiliana Capurro

Martin Danaher

Katrina Campbell

Christopher T Elliott

Investigation of the causes for occurrence of residues of the anticoccidial feed 1 additive nicarbazin in commercial poultry 2 3

Keywords: nicarbazin, poultry, commercial production, residues, feed, liver 37 38

Investigations were undertaken to identify causes for the occurrence of high levels of the 13 zootechnical feed additive nicarbazin in broiler liver at slaughter. The first investigation 14 on 32 commercial broiler flocks involved sampling and analysis for nicarbazin (as 15 dinitrocarbanilide, DNC) in liver from birds during a 3-10 day period after withdrawal 16 of nicarbazin from their feed and prior to commercial slaughter. DNC residues in liver 17 samples of broilers scheduled as being withdrawn from nicarbazin for ≥ 6 days ranged 18 from 20 to > 1600 µg/kg (the specified withdrawal period for nicarbazin is 5 days and 19 the JECFA MRL is 200 µg/kg liver). Further on-farm investigations on 12 of these flocks, 20 selected on the basis of the feeding system in use and the levels of DNC residues 21 determined in liver, identified issues in feed management contributing to elevated 22 residues in broiler liver. A significant correlation (0.81, p < 0.01, n = 10) between DNC 23 residues in liver samples and in feed samples from the feeding pans was observed. The 24 second investigation on 12 commercial broiler flocks involved sampling and analysis for 25 DNC in liver samples and feed samples from feeding pans and from the feed mill at the 26 three thinnings of birds for commercial slaughter. In the case of one flock, a clear 27 relationship between nicarbazin in feed from the feed mill (10.5 mg/kg DNC), in feed 28 from the feeding pans (6.6 mg/kg DNC) and in liver (583 µg/kg DNC) at first thinning (9 29 days scheduled withdrawal from nicarbazin) was observed. Such a clear relationship was 30 not observed in other cases, particularly at second and third thinnings, pointing to re-31 exposure of birds to nicarbazin late in the flock production cycle, probably from the 32 litter. Guidelines outlining best farm practice to eliminate nicarbazin residues in poultry 33 have been published in booklet and poster format for broiler producers and deal with 34 feed system cleaning, feed bin management, feed deliveries, feed usage and records.

compliant poultry liver samples in recent years, there is still a substantial problem. While 77 residues of nicarbazin in muscle (poultry meat), as distinct from liver, tend to be of the 78 order of 10-20 times lower, thereby reducing the food safety issue, the occurrence of 79 residues in tissues have given rise to consumer concerns [START_REF] Young | Too Hard to Swallow -the truth about drugs and poultry[END_REF]) and a 80 negative image for the broiler industry. Previous research has identified contamination of 81 ostensibly nicarbazin-free feed during feed manufacturing as a potential source for 82 residues in eggs and in broiler liver [START_REF] Dorn | Zur problematic der 414 wirkstoffverschleppung bei der mischfutterherstellung am beispiel nicarbazin[END_REF], Cannavan et al. 2000, Cannavan 83 and Kennedy 2000). In particular, [START_REF] Mcevoy | Contamination of animal 423 feedingstuffs with nicarbazin: investigations in a feed mill[END_REF] identified practices in feed 84 manufacturing related to recycling of fines and of surplus material from the pelleting 85 process as factors contributing to nicarbazin contamination of withdrawal feed. Another 86 important source for nicarbazin residues in broiler tissues is faeces recycling from litter; 87 [START_REF] Castelli | Nicarbazina: deplezione 405 della componente DNC dal tessuto muscolare ed epatico di broilers[END_REF] determined the main source of nicarbazin contamination resulting in 88 residues in the livers of broilers, four weeks after withdrawal of medicated feed, to be the 89 litter on which they were housed. [START_REF] Penz | Nicarbazin residues in broiler tissue 439 and litter[END_REF] demonstrated that DNC was 90 detectable in the liver of birds at slaughter from the last of three crops of birds reared on 91 the same litter, where nicarbazin had only been given to the first crop of birds. Cannavan 92 and Kennedy (2000) found that where broilers were fed with nicarbazin according to the 93 product licence conditions, DNC residues in liver exceeding the JECFA MRL of 200 94 µg/kg were found in broilers housed on deep litter but not in broilers housed on wire 95 flooring, pointing to faecal recycling from litter as the probable cause. the results of the liver sample analyses (including both low and high levels of DNC) and 130 the type of feeding system(s) in use on the farms. During these visits aspects of the 131 poultry production system were studied and recorded, including, in particular, the feeding 132 system used, feed delivery records and feed management. of nicarbazin-containing feed from the birds. On each occasion that the birds were 143 removed at the three thinnings, a sample of feed was collected from the feeding pans (a 144 composite feed sample from 10 feed pans). In addition, the feed samples retained by the 145 company's feed mill for all finisher and withdrawal feeds supplied by the company to the 146 flocks under study were made available for analysis. All samples of liver and of feed 147 from feeding pans and from the feed mill were analysed for DNC content by HPLC. Table 2 provides additional information on those flocks/farms selected for further 236 investigation; selection of flocks/farms for further investigation was based on the results 237 of the pre-slaughter sample analysis (including both low and high levels of nicarbazin) 238 and the type of feeding system(s) in use on the farms. Data for the 12 poultry flocks, on 8 239 farms, selected for further study, showed that 10 of these poultry flocks were scheduled 240 as being on nicarbazin-free feed for between 6 and 10 days, i.e. 1 to 5 days longer than 241 the specified withdrawal period of five days (Table 2). Liver samples from 4 of these 10 242 flocks contained nicarbazin residues in excess of 200 µg/kg DNC (Figure 3), indicating 243 that for these flocks, in particular, the withdrawal period was not effective and that on 244 these farms there was continued exposure of birds to nicarbazin for extended periods after 245 feed changeover. Samples of feed taken from the feeding pans on which the birds were 246 feeding at pre-commercial slaughter sampling were analysed for nicarbazin (Table 2). For 247 birds that were scheduled as being withdrawn from nicarbazin-containing feed for periods the same batch of feed from the feed mill was supplied to another flock on this farm 297 (EE1), into a split bin system. In this case, the birds were slaughtered one day earlier and 298 nicarbazin residue in liver (124 µg/kg DNC) and feed in the feeding pans (0.36 mg/kg 299 DNC) were considerably lower. These differences in nicarbazin content between birds 300 (liver samples) and feeding pan feeds for the two flocks on the same farm may be due to The results for samples from the third thinnings show no further decrease in liver 319 nicarbazin concentration overall, ranging from ND to 195 µg/kg DNC. Samples of the 320 feed supplied by the feed mill show generally similar nicarbazin contents as were found 321 in feed being fed to birds at the time of second thinnings, i.e. ND to 0.24 mg/kg DNC. 322 However, the nicarbazin content in feed samples from feeding pans was relatively high in 323 some cases, such as farm/flock AA1 (7.1 mg/kg DNC) and farm/flock BB1 (0.60 mg/kg 324 DNC), and, overall, was higher than the levels found in feeding pan samples at the 325 second thinnings. These elevated nicarbazin levels in feeding pan samples may be caused 326 by older, nicarbazin-medicated feed in the feeding system and at the bottom of feeding 327 pans becoming available when the cycle of poultry production is finishing. For a number 328 of farms/flocks, such as AA1, FF1, JJ1, there appears to be a re-contamination of poultry 329 with nicarbazin between the second and third thinnings, not always related to the 330 nicarbazin level determined in the feed samples from the feeding pans. As was observed 331 for the second thinnings, these latter findings suggest an alternative source of nicarbazin 332 contamination to the birds. The second study, involving analysis for nicarbazin of poultry liver and feed samples 357 from the feeding pans and from the feed mill at the three commercial thinnings for 12 358 farms/flocks, gives some interesting insights into the potential causes for nicarbazin 359 residue in broilers at slaughter. In one case (farm/flock EE2, first thinning), there was a 360 clear "cause and effect" relationship between contaminated feed supplied from the feed 361 mill, contaminated feed on farm and relatively high nicarbazin residue levels in liver.

362

Relatively high nicarbazin residue levels were determined in poultry liver from some 363 farms/flocks at each of the three thinnings but there does not appear to be a consistent 364 pattern for individual farms/flocks across the three thinnings, nor any clear relationship 365 with feeding system used (single bin, split bin, double bin). The results obtained at the 366 second and, especially, at the third thinnings are particularly difficult to explain and 367 appear to point to issues such as (a) re-contamination of birds with nicarbazin and (b) 368 feed samples taken from feeding pans not being representative of what the birds were 369 consuming at time of third thinning. These latter results suggest that birds may be 370 exposed to alternative sources of nicarbazin close to slaughter, such as older nicarbazin-371 medicated feed from the feeding system or from the litter (Cannavan and Kennedy, 372 2000). Clearly number all feed bins, including each side of split bins.

2. Empty and clean the total feeding system -feed bins, hoppers, feed lines, feed pans -before delivery of Nicarbazin-free feed.

3. Check that there is no lodging of feed in bins, hoppers and feed pans.

4. Clearly specify the feed bin that is to receive the feed delivery and lock all other bins.

5. Take and store a sample of each feed delivered.

6. Follow a planned rotation of feed bins to prevent mixing of Nicarbazin-free feed with Nicarbazin-containing feed.

7. Prevent feed spillage into litter and clean up any Nicarbazincontaining feed that has spilled.

8. Ensure that all Nicarbazin-containing feed in feed pans is consumed by withdrawing feed from the birds for a period before the changeover to Nicarbazin-free feed. 9. Ensure that birds do not receive Nicarbazin-containing feed again after they have been changed to Nicarbazin-free feed.

10. Ensure that thinning of birds for slaughter is scheduled so that the full withdrawal period of 5 days off nicarbazin-containing feed is observed.

11. Maintain accurate records on feed deliveries, feed usage and thinnings.

  , nicarbazin, is widely used as a zootechnical feed additive in the 43 earlier stages of poultry (broiler) production to prevent coccidiosis caused by species of 44 Eimeria. The commercial product, Maxiban®, is a premix product marketed by the (Veterinary Residues Committee 2001 to 2005) and of 19% to 7% of 72 samples tested (O'Keeffe and Coen 2005, O'Keeffe et al. 2006), respectively. Similarity 73 in the incidence of MRL exceedances determined in the monitoring programmes in the 74 UK and in Ireland suggest similar usage patterns for nicarbazin by the poultry industries 75 in both jurisdictions. Although there have been decreases in the incidence of non-76

  practices applied in feed manufacturing, feed delivery, feed storage and 98 use and poultry housing are all potential contributors to the problem of nicarbazin 99 residues in broilers in commercial poultry production. A comprehensive study was 100 developed to address this problem on the island of Ireland. Much of the previous research 101 in this area had been undertaken on research flocks in highly-designed and controlled 102 contrast, the research reported here was undertaken with commercial 103 flocks, representing the conditions applying in normal commercial production of broilers. 104 As part of this research, studies with commercial poultry flocks were designed to 105 investigate poultry production practices, particularly feed management, that might be 106 contributing to the high incidence of DNC-positive liver samples at slaughter. These 107 studies encompassed (a) pre-commercial sampling of poultry from commercial flocks 108 together with on-farm visits to some of these flocks to investigate feeding practices and 109 (b) sampling of poultry from selected flocks at each of the commercial thinnings to track 110 the occurrence of nicarbazin residues in poultry at the later stages of the flock production 111 cycle. The aim of the research was to develop knowledge that would be used to produce 112 practical guidelines for poultry producers to assist them in reducing or eliminating the 113 incidence of nicarbazin residues at levels above the JECFA MRL in broiler livers. company was used for this study. In conjunction with routine testing for 120 Salmonella, representative samples of poultry (livers of five birds) and of feed were 121 obtained from 32 commercial flocks. Each feed sample consisted of a composite of 122 individual feed samples taken from 10 feeding pans in the house at the same time as the 123 poultry livers were sampled. The flocks were selected to be representative of different 124 feeding systems (single bin, split bin, double bin) and to be at that stage of the production 125 cycle where nicarbazin-medicated feed had been withdrawn. The samples of liver and 126 feed were analysed for DNC content by high performance liquid chromatography 127 (HPLC). Investigative visits to 12 selected flocks were made within one week of testing 128 the liver and feed samples. The selection of flocks for further investigation was based on 129

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  168 separation was carried out on a stainless-steel analytical column (250 mm × 4.6 mm i.d.) 169 equipped with a Securiguard™ pre-column, both packed with Hypersil BDS C 18 material 170 (Phenomenex, Cheshire, UK). The column temperature was maintained at 40°C. The 171 mobile phase, consisting of water/acetonitrile (55/45, v/v), was pumped at 1 ml/min. 172 Under these conditions, the retention time for DNC was approximately 13.5 min. A 173 Waters 746 data processing module was used for recording and processing 174 chromatograms. The injection volume for standards and sample extracts was 25 µl. liver (comprising the livers from 5 individual birds) were chopped 178 and mixed thoroughly and a representative subsample taken for analysis. Liver samples 179 were analysed for DNC content according to the method described by Capurro et al 180 (2005). This method involves extraction of a 2 g liver sample in a 30 ml polypropylene 181 tube by homogenising the sample with 10 ml acetonitrile, using a Polytron™ 182 homogeniser, vortexing (2 min), sonicating (3 min) and shaking (15 min) before 183 centrifuging (2500 rpm, 10 min, 4°C) and transferring the supernatant to a clean 184 polypropylene tube. The sample was re-extracted with 5 ml acetonitrile plus 1 ml water 185 and the supernatants combined and defatted using n-hexane (2 × 10 ml). The acetonitrile 186 layer was evaporated to dryness and reconstituted in acetonitrile/water (70/30, v/v, 500 187 µl). Sample extracts were purified by passing through C 18 SPE cartridges and eluting with 188 2.5 ml acetonitrile/water (70/30, v/v). The purified extracts were evaporated to dryness 189 under nitrogen (60°C) and reconstituted in acetonitrile/water (80/20, v/v, 500 µl) for 190 HPLC analysis. Representative chromatograms for liver samples are shown in Figure 1. 191 192 Performance of the method during sample testing was evaluated by fortifying control 193 liver samples, in duplicate, with DNC at 25 and 250 µg/kg. These quality control samples 194 were included with each batch of test samples; mean recovery of added DNC was 195 for both fortification levels. The limit of determination of the method 196 was 12.5 µg/kg, based on the lowest standard in the calibration curve. mixed thoroughly and representative 20 g subsamples were ground in 200 a swinging hammer mill (Glen Creston) through a 1 mm sieve. Following further manual 201 mixing of the ground feed sample, 2.5 g was weighed into a 50 ml screw-cap 202 polypropylene tube. Feed samples were analysed for DNC content according to the 203 method described by De Jong et al (2004). Water (5 ml) was added to the sample which 204 was mixed and allowed to stand for 10 min, ensuring that the total sample was wetted. 205 Methanol/acetonitrile (50/50, v/v, 35 ml) was added to the sample and the contents of the 206 tube were mixed thoroughly by hand. The tube was placed in a water bath (50°C) for 15 207 min, with the contents of the tube being mixed thoroughly by hand mid-way through the 208 incubation period. The tube was removed from the water bath and mixed on an end-to-209 end shaker (15 min). Following extraction of the DNC, the tube contents were allowed to 210 settle and the supernatant was removed and filtered through paper filters (Whatman), 211 prior to final filtration through an HPLC sample filter (0.45 µm, Acrodisc LC 13 mm 212 PVDF). An aliquot (3 ml) of the filtered feed sample extract was evaporated to dryness 213 under nitrogen (60°C) and reconstituted in acetonitrile/water (80/20, v/v, 300 µl) for 214 HPLC analysis. Representative chromatograms for feed samples are shown in Figure 2. 215 216 Performance of the method during sample testing was evaluated by fortifying control 217 feed samples, in duplicate, with DNC at 1.6 and 8.0 mg/kg. These quality control samples 218 were included with each batch of test samples; mean recovery ± standard deviation (n = 219 14) of added DNC was 95 ± 13.3 % and 83 ± 10.1 % for fortification at 1.6 and 8.0 220 mg/kg, respectively. The limit of determination of the method was approximately 0.1 221 mg/kg, based on the lowest standard in the calibration curve.

  data on the samples of poultry liver taken from pre-slaughter flocks, 227 representing 32 individual flocks on 23 different farms; the data comprise results for 228 testing for nicarbazin residues in liver and details of the feeding system used for each 229 flock. Measurable nicarbazin residues were determined in all samples of liver taken from 230 the 32 flocks included in the initial study. Nicarbazin residue content of liver samples 231 varied from approximately 20 to 5000 µg/kg DNC. 15 of the 32 liver samples contained 232 nicarbazin residues at concentrations exceeding the JECFA MRL value of 200 µg/kg

  10 days, residue levels in liver were generally related (correlation coefficient 0.81, 249 p < 0.01, n = 10) to drug concentration in the feed from feeding pans; in the exceptional 250 case of farm/flock O2, the relatively high nicarbazin content in the feeding pan was not 251 reflected by a high liver content of nicarbazin. Elevated drug concentration in feed at 6 to 252 10 days after planned feed changeover, resulting in elevated nicarbazin residue in liver, 253 indicates significant feed contamination in these feeding systems. For farm/flock C1 and 254 L2, where the scheduled times off nicarbazin-medicated feed were 4 and 3 days, 255 respectively, only in the case of farm/flock L2 was a high liver nicarbazin residue content 256 high feed nicarbazin. For farm/flock C1, the elevated liver nicarbazin 257 residue may have been related to drug persistence. 258 259 The on-farm investigations did not show clear relationships between the feeding system 260 used (5 double bin, 5 single bin, 2 split bin), including the presence and type of hopper, 261 and the nicarbazin residue levels determined in flocks. There was a tendency for liver 262 samples from flocks with single bin systems to have a greater incidence of relatively high 263 concentrations of nicarbazin residue, but high levels of nicarbazin residue in pre-264 slaughter liver samples were associated, also, with double bin and split bin systems. The 265 on-farm investigations strongly suggested that the feed management system used on the 266 particular farm may be the most important issue in determining whether birds remain 267 exposed to nicarbazin following feed changeover. 268 269 Studies on poultry at commercial slaughter 270 Complete details for the farms/flocks included in the study are shown in Table 3, 271 including type of feeding system used and, for each of the three commercial thinnings, 272 scheduled number of days withdrawal from nicarbazin-containing feed and nicarbazin 273 content determined in samples of feed from the feed mill, feed from the feeding pans and 274 liver from the slaughtered birds. Of the 36 samples of feed (both feed mill and feeding 275 pan samples) and liver targeted for sampling, in total 32 samples of liver, 30 samples of 276 feed from feeding pans and 25 samples of feed from the feed mill were received determined in all samples taken at the first thinning, ranging in 281 concentration from 25 to 600 µg/kg DNC. These samples were from birds that had been 282 scheduled as being withdrawn from nicarbazin for 6 days or more. No pattern of 283 nicarbazin residue in liver being related to feeding system was discernible; the three 284 samples containing nicarbazin residue in excess of 200 µg/kg DNC were each from 285 single, split and double bin systems. Where samples of feed from the feeding pans were 286 available, there was a relationship between nicarbazin residue in liver and nicarbazin in 287 two highest liver samples, 583 µg/kg DNC (farm/flock EE2) and 292 µg/kg 288 DNC (farm/flock HH1) were associated with feed nicarbazin levels of 6.6 mg/kg DNC 289 and 0.81 mg/kg DNC, respectively. All other liver samples (containing 25 -128 µg/kg 290 DNC) were associated with feed nicarbazin levels of not detectable (ND) to 0.42 mg/case (farm/flock EE2) was a potential linkage from nicarbazin residue in 294 liver (583 µg/kg DNC) through nicarbazin in feed in the feeding pans (6.6 µg/kg DNC) to 295 nicarbazin in feed supplied by the feed mill (10.5 µg/kg DNC) discernible. Interestingly, 296

  301 the different feeding systems used for the two flocks and/or may indicate that the feed 302 mill sample, containing 10.5 mg/kg DNC, may not be representative of the total feed 303 delivery, particularly the feed delivered to farm/flock EE1. 304 305 Results for second and third thinnings 306 Nicarbazin residue levels in liver from birds taken at the second thinnings were lower, 307 generally, than levels in birds from the first thinnings, ranging in concentration from ND 308 to 208 µg/kg DNC. These samples were from birds that had been scheduled as being 309 withdrawn from nicarbazin for 11-19 days. The associated feed from feeding pans 310 showed corresponding lower nicarbazin levels, also, ranging from ND to 0.25 mg/kg 311 DNC. Some relatively high levels of nicarbazin residue in liver from birds taken in the 312 second thinnings, for example farm/flock BB1 (208 µg/kg DNC), CC1 (135 µg/kg DNC) 313 and GG1 (129 µg/kg DNC), could not be explained by the levels of nicarbazin 314 determined in the corresponding feed samples. These results suggest some alternative 315 source of nicarbazin to the birds, particularly since birds from these flocks showed lower 316 liver nicarbazin residue levels at first thinning.

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  commercial slaughter sampling and the follow-on investigations on farms 337 identified the following issues as important contributors to exposure of birds to 338 nicarbazin following feed changeover: 339 (a) poor understanding by some farmers of the different feed types (nicarbazin-medicated 340 feed, ionophore-medicated feed and non-medicated withdrawal feed) and the 341 necessity not to mix them 342 (b) poor communication between feed supply and farms; in some cases it was found that 343 feed deliveries were made before bins were empty, and identification of bins in 344 double bin systems or of compartments in split bin systems appeared to be deficient 345 (c) poor management of feed bins on farms; in some cases rotation of bins and/or 346 compartments for the different deliveries of feed did not appear to be based on the 347 feed additive contents and the need to avoid cross-contamination 348 deficient cleaning and checking (that is, visual inspection of feed bins/hoppers for 349 lodged feed) of feeding equipment between feed changeovers; in some cases even 350 where bins were emptied before feed changeover, hoppers and feeding lines might not 351 be emptied -the importance of this aspect was identified by the thorough cleaning 352 practice observed on one farm with a single bin system (farm/flock E1) for which the 353 pre-slaughter sampling, at 6 days off nicarbazin-medicated feed, showed a very low 354 level of nicarbazin residue in liver. 355 356

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  the investigations was to obtain comprehensive knowledge in commercial 375 poultry production systems on the causes for nicarbazin occurring in poultry at slaughter 376 and to use this knowledge to develop guidelines for poultry producers on prevention of 377 nicarbazin residues in broilers. Multiple potential causes for nicarbazin residues occurring 378 in edible tissues of birds at slaughter were identified, including contamination of feed at 379 or during transport, supply of incorrect feed type, delivery of feed into 380 incorrect feed bin, inadequate cleaning of feeding system on farm prior to feed delivery 381 and feed changeover, inadequate withdrawal periods being observed, poor management 382 of feed on farm leading to re-exposure of birds to nicarbazin close to slaughter, and 383 recycling of nicarbazin by birds from litter. Guidelines have been developed in two 384 formats to address the observed deficiencies in management of feed on poultry farms. 385 One format is a leaflet that provides poultry producers with information on how to 386 manage their enterprise to prevent, or at least reduce, the occurrence of nicarbazin 387 residues in poultry meat (O'Keeffe 2006). The other format is a poster giving an 11-388 point plan for proper feed management on farm (Figure 4); the material is provided on a 389 laminated poster, 60 cm × 85 cm, for display in poultry production units.

Figure 3 .

 3 Figure 3.Nicarbazin residue levels in poultry liver samples as a function of scheduled time of withdrawal from medicated feed

Figure 4 .

 4 Figure 4.Poster with 11-point plan to prevent nicarbazin residues in poultry

Figure 4 .

 4 Figure 4. Safefood 11-point plan to prevent nicarbazin residues in poultry

148 149 Analysis of liver and feed samples 150

	165
	166	HPLC conditions
	167	The HPLC system consisted of a model 600E HPLC pump with a model 717 autosampler
		and model 484 UV detector (set at 350 nm), all from Waters (Milford, MA, USA). The
		F o
		r
		P
		e e r
	151 152 153 154	Reagents and chemicals DNC standard material was from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol (both HiPerSolv grade), dimethylsulphoxide and n-hexane (Analar grade) were obtained from BDH (Merck, Poole, Dorset, UK). Bond Elut™ cartridges (C 18 , 500 mg, 3 R e v i e w
	155 156 157 158 159	ml) were from Varian (Harbor City, CA, USA). The primary standard stock solution (1 mg/ml) was prepared in dimethylsulphoxide. Secondary standard stock solutions (100 O n and 10 µg/ml were prepared by dilution of the primary standard stock in methanol. Calibration standards (50-2000 ng/ml), used to quantify the DNC content in liver and feed samples, were prepared by diluting the 10 µg/ml solution in acetonitrile/water l y
	160	(80/20, v/v). The primary stock standard solution was prepared every 3 months and was
	161	stored in a glass test-tube covered in aluminium foil at room temperature in the dark;
	162	storage at room temperature prevented solidification of the stock solution. Secondary
	163	standard stock solutions (in methanol) and calibration standards for HPLC (in
	164	acetonitrile/water) were prepared monthly and stored at 4°C.
		6

Table 1 .

 1 Results of pre-commercial slaughter testing for nicarbazin in poultry flocks by HPLC

	Farm/Flock	Feeding system	Nicarbazin
			(DNC, µ µ µ µg/kg)
	A1	double bin	86
	B2	double bin	68	*
	C1	single bin	865	*
	D1	single bin	1119	*
	D2	split bin	106	*
	D3	double bin	33	*
	E1	single bin	19	*
	F1	single bin	139
	F2	double bin	51
	G1	double bin	31
	G2	double bin	97
	H1	double bin	354
	I1	double bin	210
	J1	double bin	109
	K1	single bin	419	*
	L1	double bin	48
	L2	single bin	1592	*
	M1	single bin	241
	N1	double bin	85
	O2	double bin	47	*
	O3	double bin	50	*
	P2	double bin	243
	Q1	double bin	251
	R2	double bin	112
	S1	double bin	354
	T2	double bin	4897
	U1	double bin	44
	U2	double bin	359
	V1	double bin	609	*
	V2	split bin	1624	*
	W1	single bin	121
	W2	double bin	224
	* Farms selected for further investigation.

Table 2 .

 2 Details for farms/flocks subjected to further investigation

	Farm/ Flock	Nicarbazin residue in liver (DNC, µ µ µ µg/kg)	Feeding system details	feed Scheduled days withdrawal from medicated	Nicarbazin in feed (DNC, mg/kg)
	B2	68	double bin (separate hopper)	10	0.26
	C1	865	single bin (no hopper)	4	1.2
	D1	1119	single bin (separate hopper, old feeders)	7	4.9
	D2	106	split bin (separate hopper)	7	0.67
	D3	33	double bin (separate hopper)	8	0.14
	E1	19	single bin (no hopper)	6	0.48
	K1	419	single bin (hopper in/under bin)	6	9.2
	L2	1592	single bin (hopper in/under bin)	3	22.6
	O2	47	double bin (hopper in/under bin)	9	2.9
	O3	50	double bin (hopper in/under bin)	6	0.70
	V1	609	double bin (hopper in/under bin)	9	3.4
	V2	1624	split bin (hopper in/under bin)	9	11.0

Table 3 .

 3 Details of samples taken and nicarbazin content in feed and liver samples

	Farm/ Flock	Feeding system (bin type)	Scheduled days off Nicarbazin	First thinning Nicarbazin (DNC) Feed mill (mg/kg) Feed pan (mg/kg) Liver (µg/kg)	Scheduled days off Nicarbazin	Second thinning Nicarbazin (DNC) Feed mill (mg/kg) Feed pan (mg/kg) Liver (µg/kg)	Scheduled days off Nicarbazin	Third thinning Nicarbazin (DNC) Feed Feed Liver (mg/kg) (mg/kg) mill pan (µg/kg)
	AA1	Split	9	ND	-	293	16	ND	0.12	44	21	-	7.1	97
	BB1	Split	6	ND	-	101	11	0.70	0.25	208	21	ND	0.60	ND
	CC1 Double	6	ND	ND	45	13	ND	ND	135	19	0.24	0.17	55
	DD1	Single	7	-	ND
	DD2	Double	7	-	0.17

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

Acknowledgements

project. The co-operation of the two poultry companies, and their associated 469 poultry producers, with the investigations is gratefully acknowledged. Dr. thanked for her assistance with the project.


P Béjot

E Hertz

B Lavorel

J Kasparian

J.-P Wolf

O Faucher

From higher-order Kerr nonlinearities to quantitative modeling of 3rd and 5th harmonic generation in argon

The recent measurement of negative higher-order Kerr effect (HOKE) terms in gases has given rise to a controversial debate, fed by its impact on short laser pulse propagation. By comparing the experimentally measured yield of the third and fifth harmonics, with both an analytical and a full comprehensive numerical propagation model, we confirm the absolute and relative values of the reported HOKE indices.

In a recent experiment, we have shown that the electronic optical Kerr effect in Ar, N 2 , O 2 , and air exhibits a highly nonlinear behavior versus the applied intensity [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF], resulting in a saturation of the nonlinear refractive index observed at moderate intensity, followed by a sign inversion at higher laser intensity. This observation has a substantial impact on the propagation of ultrashort and ultra-intense laser pulses, especially in the context of laser filamentation [START_REF] Chin | The propagation of powerful femtosecond laser pulses in optical media: physics, applications, and new challenges[END_REF][START_REF] Bergé | Ultrashort filaments of light in weakly-ionized, optically-transparent media[END_REF][START_REF] Couairon | Femtosecond filamentation in transparent media[END_REF][START_REF] Kasparian | Physics and applications of atmospheric nonlinear optics and filamentation[END_REF] where the higher-order Kerr effect (HOKE), rather than the defocusing contribution of the free electrons, can play a key role in the self-guiding process [START_REF] Béjot | Higherorder kerr terms allow ionization-free filamentation in gases[END_REF], especially at long wavelengths [START_REF] Ettoumi | Spectral dependence of purely-kerr-driven filamentation in air and argon[END_REF] and for short pulses [START_REF] Loriot | On negative higher-order kerr effect and filamentation[END_REF]. However, this issue is still controversial [START_REF] Kolesik | Femtosecond filamentation in air and higher-order nonlinearities[END_REF][START_REF] Polynkin | Experimental tests of the new paradigm for laser filamentation in gases[END_REF][START_REF] Kolesik | On the higher-order kerr effect in femtosecond filaments[END_REF]. Therefore, an independent confirmation of our measurement of the HOKE is still needed. Recently, Kolesik et al. [START_REF] Kolesik | Femtosecond filamentation in air and higher-order nonlinearities[END_REF] have proposed such test, based on the comparison of the yields of the third harmonic (TH) and the fifth harmonic (FH) radiations generated by the nonlinear frequency up-conversion of a short and intense laser pulse in air. Based on numerical simulations, they suggested that, considering the HOKE indices, "the relative strength of the FH to the TH should reach values of the order of 10 -1 " while, if omitting them, "this ratio should be about 4-5 orders smaller" [START_REF] Kolesik | Femtosecond filamentation in air and higher-order nonlinearities[END_REF].

So far, no measurement of the yield of FH versus the TH have been achieved in air. However, Kosma et al. [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF] measured the yields of TH and FH produced by a short and intense laser pulse in argon. The present paper aims at confronting the results of this experiment to predictions based on the HOKE in argon [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF].

In the first part, we confirm the ratio of the recently measured non-linear indices [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF] based on the analytical description of the harmonic generation. In the second part, a comprehensive model including linear and nonlinear propagation effects such as dispersion, self-phase modulation, ionization, and Kerr effect, is presented.

For a focused laser beam propagating linearly, the harmonic power of the qth harmonic in the perturbative regime is given by

P q = A q N 2 |J q (b∆k)| 2 , ( 1
)
where N is the atomic density of the medium and

A q = qω 2 1 4n ℓ q (n ℓ 1 ) q (ǫ 0 π) q-1 c q w 2q-2 0 χ (q) 2 P q 1 , (2)
with P 1 , ω 1 , and w 0 the power, the angular frequency, and the beam waist of the incident beam, respectively [START_REF] Boyd | Nonlinear Optics[END_REF][START_REF] Reintjes | Comparaison of fifth and third harmonic conversion in helium[END_REF]. χ (q) is the qth-order microscopic nonlinear susceptibility (q = 3, 5) given in SI units, n ℓ j are the linear refractive indices at the fundamental (j = 1) and harmonic frequencies (j = 3, 5), ǫ 0 is the permittivity of vacuum, and c is the speed of light. J q is a dimensionless function that accounts for the phase matching

J q = 2(L-f )/b -2f /b dξ exp (-ib∆kξ/2) (1 + iξ) q-1 , (3)
with ∆k = k q -qk 1 = 2πq λ1 n ℓ q -n ℓ 1 the phase mismatch, with n ℓ q -n ℓ 1 proportional to the pressure, and k j (j = 1, q) the wave vectors, b the confocal parameter, L the length of the static cell, and f the position of the focus with respect to the entrance of the static cell [START_REF] Bjorklund | Effects of focusing on third-order nonlinear processes in isotropic media[END_REF]. According to Eqs. ( 1) and ( 2), the ratio of the FH to the TH power is

P 5 P 3 ≈ 5 3ǫ 2 0 π 2 c 2 w 4 0 χ (5) χ (3) 2 N 5 |J 5 | N 3 |J 3 | 2 P 2 1 , (4)
where n ℓ j have been approximated to unity in Eq. 2. N 3 and N 5 refer to the different atomic densities at the pressures maximizing the harmonic conversion for the 3rd and 5th orders, respectively. This equation provides a direct relationship between the power ratio of the harmonics and the ratio of the corresponding non-linear susceptibilities. The latter are related to the nonlinear refractive indices through the relation [START_REF] Ettoumi | Spectral dependence of purely-kerr-driven filamentation in air and argon[END_REF] so that

n 2j = (2j + 1)! 2 j+1 j! (j + 1)! 1 (n ℓ 1 ) 2 ǫ 0 c j χ (2j+1) Kerr . (5)
P 5 P 3 ≈ 3 5π 2 w 4 0 n 4 n 2 2 N 5 |J 5 | N 3 |J 3 | 2 P 2 1 , (6)
In the experiment by Kosma el al., b=7.8 cm, w 0 = 100 µm, L=1.8 cm, f =L/2, and the wavelength λ 1 =810 nm [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF]. The fundamental power, calculated from the input energy E 1 =710 µJ and the pulse duration τ 1 =12 fs, is P 1 = 59 GW. They observed that the pressure maximizing the TH power ranged between 160 mbar [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF] and 250 mbar [START_REF] Kosma | Cyclohexadiene ring opening observed with 13 fs resolution: coherent oscillations confirm the reaction path[END_REF], for similar experimental conditions. One single maximum, around 50 mbar, is observed for the FH. The maximum energies of the TH and FH measured at the respective optimal pressures reported in [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF] are 140 and 4 nJ, respectively, while the pulse duration was estimated to be 11 fs for both harmonics [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF][START_REF] Kosma | Cyclohexadiene ring opening observed with 13 fs resolution: coherent oscillations confirm the reaction path[END_REF]. This leads to a power ratio P 5 /P 3 = 0.028. According to Eq. ( 6), where J q of Eq. ( 3) has been calculated using n ℓ 1 = 1.00028, n ℓ 3 = 1.00030, and n ℓ 5 = 1.00035 for the values of the refractive index of argon at 1 bar at 810, 270, and 162 nm, respectively [START_REF] Bideau | Higher-order kerr terms allow ionization-free filamentation in gases[END_REF], the corresponding ratio of the HOKE indices is |n 4 /n 2 | = 6.8 × 10 -19 m 2 /W. This value confirms, within a factor of 2 compatible with the experimental error, the ratio of the experimental HOKE indices

n 2 = 10 -23 m 2 /W 1 and n 4 = -3.6 × 10 -42 m 4 /W 2 [1], resulting in |n 4 /n 2 | = 3.6 × 10 -19 m 2 /W.
The agreement is remarkable, especially considering the simplicity of the analytical model used.

Further comparison with the experiment was performed by computing the value of N 2 |J q | 2 as a function of the argon pressure relying on Eq. (3) (Fig. 1). This function should reflect the pressure dependence of the harmonic powers. The analytical model predicts a maximum at about 300 mbar for TH, in line with the experimental results. It yields three maxima between 0 and 400 mbar for FH, the first of them close to the observed optimum pressure for the FH. This oscillatory structure, which is due to the periodic phase matching, was not observed in the experiments [START_REF] Fuß | Private Communication[END_REF] probably due to nonlinear propagation effects which are not considered in the analytical model.

To overcome these limitations and take into account the perturbations of the fundamental pulse during its propagation through the gas sample, as well as the effect of the HOKE indices on the phase matching, we have solved the unidirectional pulse propagation equation for the experimental conditions of Kosma et al. More precisely, assuming a cylindrical symmetry around the propagation axis z, the angularly resolved spectrum E(k ⊥ , ω) of the real electric field E(r, t) follows the equation [START_REF] Kolesik | Nonlinear optical pulse propagation simulation: From maxwell's to unidirectional equations[END_REF] ∂

z E = ik z E + 1 2k z iω 2 c 2 P NL - ω ǫ 0 c 2 J , (7)
where k z = k 2 (ω) -k 2 ⊥ , P NL (resp., J ) is the angularly resolved nonlinear polarization (resp., free charge induced current) spectrum, and k(ω) = n(ω)ω c . The nonlinear polarization P NL is evaluated in the time domain as P NL = χ (3) E 3 + χ (5) E 5 + χ (7) E 7 + χ (9) E 9 + χ (11) E 11 . Since the nonlinear polarization is defined from the real electric field, Eq. 7 captures without any modifications all frequency-mixing processes induced by the total field. For numerical stability concerns, we considered only the part responsible for the refractive index change around ω 0 , neglecting harmonics generation induced by the terms proportional to E 7 , E 9 , and E 11 . The current induced by the free charges is calculated in the frequency domain as J = e 2 me νe+iω ν 2 e +ω 2 ρε, where e (resp., m e ) is the electron charge (resp., mass), ν e is the effective collisional frequency, and ρ is the electron density which is evaluated as

∂ t ρ = W (I) (ρ at -ρ) + σ U i I -βρ 2 , ( 8
)
where W (I) is the ionization probability evaluated with the Keldysh-PPT (Perelomov, Popov, Terent'ev) model [START_REF] Bergé | Ultrashort filaments of light in weakly-ionized, optically-transparent media[END_REF], ρ at is the atomic number density, σ is the inverse Bremsstrahlung cross-section, β is the recombination constant (negligible on the time scale investigated in the present work), and I is proportional to the time-averaged E 2 . Figure 2 displays the harmonics intensity as a function of argon pressure for an input pulse and a detection geometry matching the experimental parameters: 12 fs pulse duration (FWHM), 700 µJ input energy, and a beam radius of 4 mm before focusing. In order to mimic the experiment, the pulse first propagates in vacuum up to the position of the cell (99.1 cm after the f =1 m lens). After this focusing step, the pulse propagates over 1.8 cm in the argon cell. The optimal pressure for the FH is 50 mbar, in full agreement with the experiment [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF]. The reduction of the second and third maxima of the FH, as compared to Fig. 1, results from the phase mismatch introduced by the HOKE at large pressure. The TH yield is maximal at 260 mbar, similar to the value reported in [START_REF] Kosma | Cyclohexadiene ring opening observed with 13 fs resolution: coherent oscillations confirm the reaction path[END_REF]. In full agreement with the experiment by Kosma al. [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF], the ratio at 50 mbar is about 0.1 and becomes even larger at reduced pressures. Furthermore, the total FH and TH energies at their respective optimum pres- (open red circles) harmonics in argon integrated over the full radial distribution. To be compared with the Fig. 3 of [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF]. The spectrum calculated at 50 mbar is shown in the inset.

sures are 6 and 218 nJ, in good agreement with the experimental values of 4 and 140 nJ, where losses due to the setup lead to a slight underestimation of the output energies [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF].

If the HOKE are not considered in the model, the ratio of the FH to the TH at a pressure of 50 mbar drops to 0.017, and the FH and TH energies are respectively 1.7 and 584 nJ: These values are inconsistent with the experimental results of Kosma et al. Furthermore, contrary to the experimental observations [START_REF] Fuß | Private Communication[END_REF], the FH would exhibit strong maxima at 160 and 250 mbar. These discrepancies show that the HOKE are necessary to reproduce the experimental results [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF][START_REF] Kosma | Cyclohexadiene ring opening observed with 13 fs resolution: coherent oscillations confirm the reaction path[END_REF], further validating their measured values [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF]. Note that the ratio of 0.017 strongly depends on the propagation distance, so that it cannot be directly compared to that of 10,000 predicted by Kolesik et al. for the "classical" model over an unspecified propagation distance. For a propagation length of 220 µm, 80 times shorter than in our work but consistent with neglecting the phase matching, our calculation indeed predicts a ratio of 10,000.

In conclusion, as recently suggested in [START_REF] Kolesik | Femtosecond filamentation in air and higher-order nonlinearities[END_REF], we have compared the recent experimental measurements of the TH and FH yields in argon [START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF] with both analytical and numerical simulations. These results agree quantitatively with the measured high-order Kerr indices [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF]. This conclusion is supported by the following findings. First, the harmonic yield reported in argon by Kosma et al. at the pressure that optimized the 5th harmonic leads to a ratio of about 0.1 between the fifth and the third harmonics. This ratio implies a ratio of the Kerr indices consistent with our measurement of the HOKE indices within their uncertainty range [START_REF] Loriot | Measurement of high order kerr refractive index of major air components[END_REF]. Second, the analytical model based on our HOKE indices reproduces the pressure maximizing the TH, as well as the first pressure maximum of the 5th harmonic yield. Third, a full numerical propagation model accounting for the dispersion and nonlinear effects such as ionization and higher-order Kerr effects quantitatively reproduces the ratio of the harmonic yields observed in the experiment, as well as the pressure dependence of both the 3rd and 5th harmonics. It even reproduces the absolute harmonics intensity within a fairly good accuracy.

6 )Fig. 1 .

 61 Fig. 1. (Color online) Analytical calculation of the pressure dependence of the 3rd (solid blue line) and 5th (dashed red line) harmonics in argon.

Fig. 2 .

 2 Fig. 2. (Color online) Numerical calculation of the pressure dependence of the 3rd (dotted blue line) and 5th(open red circles) harmonics in argon integrated over the full radial distribution. To be compared with the Fig.3of[START_REF] Kosma | Vacuum ultraviolet pulses of 11 fs from fifth-harmonic generation of a ti:sapphire laser[END_REF]. The spectrum calculated at 50 mbar is shown in the inset.

Acknowledgments

The authors are grateful to Werner Fuß and Kyriaki Kosma for fruitful discussions and critical reading of the manuscript. This work was supported by the Conseil Régional de Bourgogne, the ANR COMOC, the FASTQUAST ITN Program of the 7th FP and the Swiss NSF (contract 200021-125315).