Francisco M Valle-Algarra

Eva M Mateo

Angel Medina-Vayá

Fernando Mateo

Vicente Gimeno-Adelantado

Misericordia Jiménez
email: misericordia.jimenez@uv.es

Á Medina

J V Gimeno-Adelantado

Jiménez

Changes in ochratoxin A and type B trichothecenes contained in wheat flour during dough fermentation and bread baking processes

Keywords: Mycotoxins -trichothecenes, Mycotoxins -ochratoxin fermented dough, wheat flour, mycotoxin reduction, baking process. 34 35 36 37

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 2 3 Food such as cereals, are susceptible to be contaminated with mycotoxins (Scott et al. 4 1984, [START_REF] Samar | Distribution of 14 deoxynivalenol in wheat, wheat flour, bran, and gluten, and variability associated with the test 15 procedure[END_REF]. Mycotoxin-producing fungi are mainly Fusarium spp., Aspergillus 5 spp. and Penicillium spp., which are frequently isolated from the whole kernel (González et 6 al. 1996[START_REF] González | Relationship between Fusarium 15 graminearum and Alternaria alternata contamination and deoxynivalenol occurrence on 16 Argentinian durum wheat[END_REF], Medina et al. 2006a). It has been demonstrated that different 7 cereals by-products of massive consumption in Europe and other countries, like bakery 8 products and beer, are often contaminated with ochratoxin A (OTA) and trichothecenes 9 [START_REF] Quiroga | Natural 5 occurrence of trichothecenes and zearalenone in Argentine wheat[END_REF], Pacin et al. 1997[START_REF] Moltó | Occurrence of trichothecenes in 19 Argentinean beer A preliminary exposure assessment[END_REF][START_REF] Samar | Effect of fermentation on naturally 18 occurring deoxynivalenol (DON) in Argentinean bread processing technology[END_REF], Medina et al. 10 2006b). 11 OTA formation presents a risk in cereals because grain is often harvested at high 12 moisture contents, sometimes above 20%. Drying is often necessary to prevent the 13 development of storage moulds, such as Penicillium verrucosum, Aspergillus ochraceus and 14

Aspergillus section Nigri, species considered to be the usual OTA producers in cereals 15 [START_REF] Frisvad | Toxin and secondary metabolite production by Penicillium species[END_REF]Lund 1993, Valero et al. 2006). However, this procedure is sometimes not 16 carried out quickly enough or the cereals are not dried sufficiently for safe, long-term storage. 17 OTA has been found to be a potent renal toxin in all of the animal species tested. [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF] induces a typical karyomegaly and a progressive nephropathy. Previous National Toxicology 19

Program studies in United States showed that OTA can induce renal tumours in rodents at 20 high dosages (EFSA 2006). [START_REF] El-Banna | Fate of mycotoxins during processing. III. Ochratoxin A 22 during cooking of faba beans (Vicia faba) and polished wheat[END_REF] Type B trichothecenes, produced by some Fusarium species such as F. graminearum 22 and F. culmorum [START_REF] Llorens | Influence of 32 environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium 33 spp. from Spanish crops[END_REF]) are frequent contaminants of cereal crops worldwide 23 [START_REF] Scott | Trichothecene Mycotoxicosis: Pathophysiologic Effects[END_REF][START_REF] Scott | Effects of processing and detoxification treatments on ochratoxin A: 27 introduction[END_REF]. They affect animal and human health and present various toxicological 24 characteristics, such as skin irritability, hemorrhagic syndrome, decrease in bone marrow, 25 depression, diarrhoea, nausea, feed refusal and vomiting. They can inhibit protein synthesis, 26 alter brain neurochemicals and may be immunosuppressive [START_REF] Ueno | Trichothecenes: Chemical, Biological and Toxicological Aspects[END_REF], Tryphonas et al. 27 1984[START_REF] Pacin | Las micotoxinas como factor de riesgo en la salud humana. Santafe de Bogota 25 (Colombia): International Seminar Memories about Mycotoxins[END_REF][START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF][START_REF] Sugita-Konishi | Effect of trichothecenes on 4 host defense against infectious diseases[END_REF]). The WHO presented a 28 comparative estimate of human exposure in Europe, the Far East, Latin America and the 29

Middle East. This comparison demonstrated that in Europe, approximately 80 % of the total 30 deoxynivalenol (DON) intake is associated with the consumption of wheat, whereas in the Far 31 East, wheat and rice are equally important as source of DON exposure (WHO 1998, 32 FAO/WHO 2001b). 33 Tolerable Weekly Intake of 100 ng kg -1 body weight (b.w.) per week. The European Food 5 Safety Authority (EFSA) recommended a Tolerable Weekly Intake of 120 ng kg -1 b.w. per 6 week for OTA (EFSA 2006). In Europe, the SCF (1999,2002) and the recent SCOOP task 7 [START_REF] Gareis | SCOOP task 3.2.10. Collection of Occurrence Data of Fusarium Toxins in Food and 11 Assessment of Dietary Intake by the Population of EU Member States[END_REF]) have concluded that an exposure of consumers close to the TDI of 1 and 8 0.7 µg kg -1 b.w. day -1 for DON and nivalenol (NIV), respectively, is possible (Samar et al. 9 2007). 10

The European Commission (EC) has recently established maximum levels in cereals 11 intended for direct human consumption to reduce the risk to the consumer for OTA and DON, 12 which are 3 µg kg -1 and 750 µg kg -1 , respectively (Commission Regulation 2006). 13 Dietary exposure to OTA and type B trichothecenes varies considerably depending on 14 different factors, among which food-processing systems must be considered. These systems 15 are often traditional and characteristic of the different geographical regions and, up to date, 16 their influence on the mycotoxin content of the food finished products ready for consumption 17 has been scarcely studied. [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF] Thus, an effective treatment for eliminating these mycotoxins from cereal products is 19 essential to minimize human exposure to the toxins. The results from the literature suggest 20 that during food processing (cleaning, milling, baking), fractionation or reduction occurs 21 [START_REF] Abbas | Effect of cleaning, milling and baking 10 on deoxynivalenol in wheat[END_REF], Charmley and Prelusky 1994[START_REF] Schollenberger | 25 Trichothecene toxins in different groups of conventional and organic bread of the German 26 market[END_REF], Bullerman and 22 Bianchini 2007). Some physical and chemical methods have been used to reduce mycotoxin 23 contamination of foodstuffs and feedstuffs. These methods include segregation of 24 contaminated from non-contaminated kernels in water and saturated sodium chloride, milling, 25 cleaning or washing, sieving and dehulling [START_REF] Scott | Cereal grains. Mycotoxins. fungi and quality in drying and storage[END_REF][START_REF] Trenholm | Two physical methods for 15 decontamination of four cereals contaminated with deoxynivalenol and zearalenone[END_REF]. 26

Few studies have been carried out on the reduction of OTA during bakery processing 27 (El-Banna and Scott 1984, [START_REF] Osborne | The effects of milling and processing on wheat contaminated with ochratoxin A[END_REF][START_REF] Scudamore | Fate of ochratoxin A in the processing of 33 whole wheat grains during milling and bread production[END_REF]. It has been reported 28 that temperatures above 250ºC applied during several minutes are required to reduce OTA 29 concentration [START_REF] Boudra | Thermostability of ochratoxin A in wheat under two 19 moisture conditions[END_REF], and that under certain conditions of high temperature, 30 acidic or alkaline conditions or in the presence of enzymes breakdown can occur (Baxter 31 1996[START_REF] Scott | Effects of processing and detoxification treatments on ochratoxin A: 27 introduction[END_REF][START_REF] Scudamore | Ochratoxin A in animal feed -effects of processing[END_REF], Subirade 1996). 32

In the case of DON, reports have shown that boiling could be expected to be an 33 effective means of reducing the toxin level because the secondary metabolite is water-soluble 34 [START_REF] Ragab | Fate of deoxynivalenol in contaminated 8 wheat grain during preparation of Egyptian "balila[END_REF]). In other study, the presence of DON in the 1 different steps of the manufacture of turnover pie dough covers prepared for baking and for 2 frying was studied with the objective to evaluate the industrial processing effect on DON 3 reduction (Del Pelo 2002, [START_REF] Samar | Deoxynivalenol 22 reduction during the frying process of turnover pie covers[END_REF]. 4 DON has been reported to be highly stable to bakery processes due to its thermal 5 stability (El [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF], Young et al. 1986[START_REF] Scott | Cereal grains. Mycotoxins. fungi and quality in drying and storage[END_REF][START_REF] Boyacioglu | Additives affect deoxynivalenol 22 (vomitoxin) flour during breadmaking[END_REF], Neira et 6 al. 1997) On the other hand, other authors have reported that bakery processing reduce DON 7 contamination [START_REF] Neira | The effects of bakery processing 22 on natural deoxynivalenol contamination[END_REF][START_REF] Samar | Effect of fermentation on naturally 18 occurring deoxynivalenol (DON) in Argentinean bread processing technology[END_REF], 2007). The effects of various food 8 additives, such as oxidizing (potassium bromate) and reducing (sodium hydrogensulfite, L-9 cysteine and L-ascorbic acid) agents, and ammonium phosphate, at varying levels, on DON 10 in whole wheat flour during breadbaking were also investigated. Fermentation of the dough, 11 sodium hydrogensulfite and L-cysteine and ammonium phosphate were moderately effective 12 in reducing DON level in bread [START_REF] Young | Reduction in levels of 4 deoxynivalenol in contaminated wheat by chemical and physical treatment[END_REF][START_REF] Young | Reduction in levels of deoxynivalenol in contaminated corn by chemical 8 and physical treatment[END_REF][START_REF] Boyacioglu | Additives affect deoxynivalenol 22 (vomitoxin) flour during breadmaking[END_REF]. 13

Due to the culinary customs and considering that appreciable amounts of wheat based 14 foods, among which bread must be highlighted, are consumed worldwide, it is important to 15 establish how processes such as baking can reduce the contamination by OTA and 16 trichothecenes B in bread ready to be consumed. This research is complicated because baking 17 is a handmade process characteristic of each geographic region and the levels of mycotoxins 18 in the wheat used in each region are also different. 19

The aim of this study was to evaluate how the different bread making conditions usual 20 in Spain affect the levels of OTA, DON, NIV and 3-acetyldeoxynivalenol (3-ADON) that 21 occur initially in the wheat flour used to make bread. 22 23

Materials and methods 24 25

Chemicals and reagents 26

OTA and trichothecene standards, including NIV, DON, and 3-ADON were supplied 27 by Sigma (Sigma-Aldrich, Alcobendas, Spain). Toluene and sodium hydrogen carbonate 28 were from Panreac (Montcada i Reixac, Barcelona, Spain). Acetonitrile, methanol, were from 29 J.T. Baker (Deventer, Holland). All solvents were HPLC grade. Standardized 70-230 mesh 30 aluminium oxide 90 (0.063-0.2 mm particle size) was purchased from Merck (Darmstadt, 31 Germany). Activated charcoal (Norit) was from Fluka (Sigma-Aldrich). Glass microfibre 32 filters (GF/C) and filter papers (Whatman No 4) were from Whatman (Maidstone, UK). 33 Phosphate-buffered saline (PBS) was prepared with potassium chloride (0.2 g, Panreac), 3 potassium dihydrogen phosphate (0.2 g, Sigma), anhydrous disodium hydrogen phosphate 4

(1.16 g, Panreac) and sodium chloride (8.0 g, J.T. Baker) in 1 l of pure water; the pH was 5 brought to 7.4. Pure water was obtained from a Milli-Q apparatus (Millipore, Billerica, MA, 6 USA) and was used when water was required. Compressed yeast cake was purchased in a 7 local bakery and was stored at 4 ºC. 8 9

Preparation of standard solutions 10

A stock solution of OTA was prepared by solving 1 mg of OTA in 2 ml of toluene-11 acetic acid (99:1, v/v) and stored at -20 ºC in a sealed vial until use. Working standards (100, 12 25.0, 10.0, 5.0, 1.0, 0.5, 0.2, 0.1, 0.05 ng ml -1 ) was prepared by evaporation of known aliquots 13 of the stock solution and dissolution in filtered LC mobile phase. 14 Each standard of NIV, DON and 3-ADON was dissolved in acetonitrile at a 15 concentration of 1.0 mg/ml and stored at -20 ºC in a sealed vial until use. Working standards 16 (500, 100, 25, 10.0, 2.0, 1.0, 0.5, 0.25, 0.1, 0.05 and 0.02 µg ml -1 ) were prepared by 17 appropriate dilution of known volumes of the stock solution with acetonitrile. [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF] 19

Spiking process 20

The wheat flour batch used in the experiments was obtained from the retail market. It 21 was previously analyzed for OTA and trichothecenes and found to contain these toxins below 22 the limits of detection of the methods used in this work. Part of the wheat flour was 23 contaminated with 2, 5, and 10 µg of OTA kg -1 , 200, 750, and 1500 µg of both DON and 3-24 ADON kg -1 , and 200, 500 and 1000 µg of NIV kg -1 by addition of the appropriate volumes of 25 standard solutions of the mycotoxins. These levels fell within the linear range of the 26 calibration lines. Sample handling was carried out in a hood and laboratory glasses, masks 27 and gloves were worn for safety purposes. After being spiked, each portion of contaminated 28 flour was thoroughly homogenized and used to prepare the dough once the mycotoxin 29 solvents were evaporated. Three combinations were assayed: a) 2 µg of OTA kg -1 , 200 µg of 30 DON kg -1 , 200 µg of 3-ADON kg -1 and 200 µg of NIV kg -1 ; b) 5 µg of OTA kg -1 , 750 µg of 31 DON kg -1 , 750 µg of 3-ADON kg -1 and 500 µg of NIV kg -1 ; and c) 10 µg of OTA kg -1 , 32 1500 µg of DON kg -1 , 1500 µg of 3-ADON kg -1 and 1000 µg of NIV kg -1 . These spiked 33 samples were analyzed before fermentation and the levels were consistent with the recovery 1 rates found for the method. 2 3

Dough preparation and bread making 4

Dough was made with mycotoxin-spiked wheat flour, compressed yeast cake, water 5 and sodium chloride. (50:2:30:1, w/w/v/w). Firstly, flour was mixed with sodium chloride 6 then, moistened with water and, finally, yeast cake (suspended in water) was incorporated. 7 Dough was manually mixed until held together with a non-sticky, smooth and satiny 8 appearance and optimum handling properties. 9

Rounded pieces of about 8 cm diameter each weighing 80 g were prepared. They were 10 covered with a damp cloth and fermentation was carried out at 29-30 ºC, for 1 hour. 11 After fermentation, dough was baked in a muffle furnace (Gallur, Valencia, Spain) at 12 190, 207, 223, and 240ºC for 50, 40, 35 and 30 min, respectively. These conditions were 13 established on the basis of previous experiments to obtain well-baked bread. After baking, 14 bread pieces were stored at -20 ºC until determination of the mycotoxins. [START_REF] Baxter | The fate of ochratoxin A during malting and brewing[END_REF] The baking process to obtain bread was repeated under the same temperature-time 16 conditions previously indicated, but now each piece of bread was separated in two portions, 17 the inner part and the outer part (or crust) and both parts were stored at -20ºC until analysis. [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF] At each mycotoxin-spiking level, dough fermentation and baking for analysis of the 19 whole piece or the inner/outer part of bread were performed in triplicate. [START_REF] Ueno | Trichothecenes: Chemical, Biological and Toxicological Aspects[END_REF] 21

Extraction and clean-up for OTA 22

Extraction and clean-up was performed according to the Vicam technical note and 23 suggestions for the analysis of wheat [START_REF] Vicam | OchraTest instruction manual[END_REF]) with slight modifications. Bread was 24 finely ground using an IKA A10 laboratory mill (IKA, Stauffen, Germany) and homogenized. 25

Seven and half grams of ground bread or dough (was extracted with 25 ml of 26 acetonitrile/water (60:40, v/v) in a high-speed blender (Ultraturrax T25, IKA) for 5 min. The 27 suspension was centrifuged at 6000 rpm for 6 min at 4 °C (Heraeus Multifuge 1 s-r, Hanau, 28 Germany). Supernatant (10 ml) was diluted with 40 ml of pure water. Diluted solution (10 ml) 29 was loaded into the immunoaffinity column and passed at a flow rate at 1-2 drops/s. The 30 column was washed sequentially with 10 ml of PBS and 10 ml of pure water. Then, it was 31 eluted with 1.5 ml of methanol at a rate of about 1 drop/s in a 4-ml vial. The purified extract 32 was concentrated to dryness at 45ºC under gentle stream of nitrogen and solved in 0.5 ml of 33

Liquid chromatographic analysis of OTA 2

The LC system consisted of a Waters 600 pump, a Waters 717 automatic injector and 3 a Waters 474 scanning fluorescence detector (FLD). Millennium 32 software, version 3.05.01 4 (Waters Co., Milford, MA, USA) was used to control the system and to process signals. LC-5 FLD conditions were used as described by [START_REF] Medina | 5 Determination of ochratoxin A in beer marketed in Spain by liquid chromatography with 6 fluorescence detection using lead hydroxyacetate as a clean-up agent[END_REF]. 6

The solution (100 µl) was injected into the liquid chromatograph. Separation was 7 performed on a Gemini C18 column (150mm×4.6 mm, 5 µm particle size) connected to a 8 Gemini guard column (12.5 mm×4.6 mm, 5 µm particle size) (Phenomenex Inc., Torrance, 9 CA, USA) filled with the same phase. The column was kept at 40 ºC. A mixture of 10 acetonitrile-water-acetic acid (44:56:1, v/v/v) at 1.0 ml/min flow-rate was used as mobile 11 phase. Quantification of OTA was performed by measuring its peak area with the help of a 12 calibration curve calculated by regression from standard solutions. Excitation and emission 13 wavelengths were 333 and 460 nm, respectively. Five injections per extract were performed 14 and the average concentration was used for calculations. [START_REF] Baxter | The fate of ochratoxin A during malting and brewing[END_REF] The limit of detection (LOD) for OTA in ground bread was 0.015 µg kg -1 (Signal-to 16 noise ratio = 3). The limit of quantification (LOQ) was considered to be 3 times the LOD. 17

Recovery in ground bread contaminated with 5 µg of OTA kg -1 was 78% ± 5% (n = 3). Made-in-laboratory cartridges were prepared using 5-ml sterile plastic syringes. A 26 glass microfibre filter was placed at the bottom. Then, a mixture of packing bed was poured 27 on it. It was made of 1.16 g of alumina-charcoal-C18 silica (75:1:40, w/w/w). Another glass 28 microfibre filter was placed on the bed top. Then, it was pressed tightly but carefully with a 29 plunger. Three ml of sample extract was passed through the prepared cartridge and collected 30 in a vial. The cartridge was rinsed with 2 ml of acetonitrile-water (84:16, v/v). The eluate was 31 collected in the same vial and the purified extract was concentrated to dryness at 45 ºC under 32 gentle stream of nitrogen. 33

Derivatization for GC-ECD determination for trichothecenes 1

A 2 mg/ml solution of DMAP (100 µl) in toluene-acetonitrile (80:20, v/v) and 50 µl 2 of PFPA were added to each dry extract in a screw cap vial. After capping tightly, the reaction 3 mixture was heated at 60 ºC for 60 min in aluminium heater block (Lab-Line 2050-1 Multi-4 Block Heater, Melrose Park, IL, USA). After the mixture had cooled, 0.4 ml toluene and 1 ml 5 of a 3% (w/v) aqueous solution of NaHCO 3 were added and the vial was vortexed for 15 s. 6

The two layers were allowed to separate. The top (organic phase) layer was transferred to a 7 GC autoinjector vial and analyzed by GC-ECD. 8 9

GC-ECD analysis of trichothecenes 10

The GC system was composed of a HP-6890 Plus gas chromatograph, equipped with 11 a 63 Ni ECD (Hewlett-Packard, Avondale, PA, USA) and an Agilent 7683 Series injector 12 (Agilent Technologies, Waldbronn, Germany). Signals were processed by HP GC 13 ChemStation software Version A.07:01(682) (Hewlett-Packard). 14

The solution (1 µl) was injected in splitless mode. The temperatures of the injection 15 and detection ports were 250 and 300 ºC, respectively. Separation was carried out in a HP-5 16 fused-silica capillary column [5% methyl phenyl siloxane (30 m×0.32 mm i.d., 0.25 µm film 17 thickness, Agilent Technologies)]. The oven temperature program was: 90 ºC held for 1 min, 18 40 ºC/min to 160 ºC, 1.5 ºC/min to 182 ºC, 5 ºC /min to 240 •C, and then 40 ºC/min to 275 ºC, 19 held for 2 min. Helium at a constant pressure of 42.1 kPa was used as carrier gas. 20

LODs for DON, NIV and 3-ADON were 8 µg kg -1 , 15 µg kg -1 and 11 µg kg -1 , 21 respectively (signal-to-noise ratio = 3). The limit of quantification (LOQ) was considered to 22 be 3 times the LOD. Recoveries of DON, NIV and 3-ADON, added to milled bread at a level 23 of 500 µg of each trichothecene kg -1 and expressed as percentages, were 76 ± 8, 86 ± 9 and 77 24 ± 8, respectively. 25 26 Results and discussion 27 28 The effect of different baking conditions on the levels of OTA, DON, NIV, and 3-29 ADON present in the flour used to bake bread has been studied. The results indicate that 30 during dough fermentation a significant reduction (p < 0.05) of the OTA level takes place, 31 which ranged from 29.8% to 33.5%, depending on the OTA amount added to the what flour 32 (Figure 1). Some authors [START_REF] Cecchini | Influence of yeast strain on 31 ochratoxin A content during fermentation of white and red must[END_REF]) have reported that during alcoholic 33 fermentation of wine, OTA level in the medium decreases significantly. However, Scudamore OTA reduction during fermentation caused by yeasts is dependent on the particular strain 2 involved in fermentation [START_REF] Cecchini | Influence of yeast strain on 31 ochratoxin A content during fermentation of white and red must[END_REF][START_REF] Westby | Review of the effect of fermentation on naturally 14 occurring toxins[END_REF], which could explain the 3 controversial results reported by different authors. Therefore, it is important to highlight that, 4 on the basis of the results obtained in our work and those from other researchers, the use of 5 yeasts selected in industrial fermentations could be an excellent tool to control OTA in food 6 and beverages that are common in diet, like bread, beer and wine, where a high occurrence of 7 OTA has been reported [START_REF] González-Osnaya | Dietary intake of ochratoxin A 22 from conventional and organic bread[END_REF][START_REF] Mateo | An overview of ochratoxin A in 2 beer and wine[END_REF]). 8

Under the conditions employed in the present study, fermentation does not decrease 9 the level of any of the assayed trichothecenes. Although very few papers concerning this topic 10 appear in the literature, some authors have analyzed the effect of fermentation on the level of 11 DON in Egyptian bread (El [START_REF] Banna | Fate of mycotoxins during processing of foodstuffs 19 II-Deoxynivalenol (vomitoxin) during making of Egyptian bread[END_REF]) and in French bread and bridge roll (Samar et 12 al. 2001). None of these studies showed a reduction of DON in dough during fermentation at 13 30 ºC, the temperature commonly used for fermentation in bread making. However, it was 14 found that a significant reduction of the DON level in the fermented dough of a bridge roll 15 took place if fermentation was performed at 50 ºC during periods of 60 min (Samar et al. 16 2001). Studies on the effect of fermentation on the stability of type B trichothecenes in bread 17 dough and other matrices are necessary to determine under what conditions this process could 18 be effective as a tool to eliminate this mycotoxin from fermented by-products ready for 19 consumption such as bread and beer, a beverage where DON has been detected (Anselme et 20 al. 2006). [START_REF] El-Banna | Fate of mycotoxins during processing. III. Ochratoxin A 22 during cooking of faba beans (Vicia faba) and polished wheat[END_REF] The effect of fermentation on 3-ADON and NIV levels in bread dough has been 22 studied in this present work for the first time. Figure 1 shows that fermentation of the dough 23

does not affect the level of any of these trichothecenes under the tested conditions. 24

After fermentation, the bread was baked at different temperature/time combinations 25 (190 ºC/50 min, 207 ºC/40 min, 223 ºC/35 min and 240 ºC/30 min). These baking conditions 26 were chosen because they are commonly used in bread production in Spain. The results for 27 the reduction percentage and standard deviation of the different toxins under the tested baking 28 conditions are shown in Figure 2. ANOVA of the data showed no significant differences (p > 29 0.05) in the reduction percentages of each toxin in relation to the temperature/time 30 combination used, but there were significant differences (p < 0.05) among the different 31 mycotoxins. The average reduction percentages for NIV, 3-ADON, DON and OTA during 32 baking were 76.9%, 65.6%, 47.9% and 32.9%, respectively. Reduction rate during 33 fermentation was calculated as the percentage of decrease in toxin content during 34 fermentation with respect to the initial toxin level in wheat flour. Reduction rate during the 1 whole process was calculated in the same way but in this case the decrease refers to the final 2 toxin content (after bread baking). The difference between the latter and the former is the 3 reduction rate due only to the baking process. 4 OTA reduction rate was higher than that detected by other authors (Scudamore et al. 5 2003[START_REF] Osborne | The effects of milling and processing on wheat contaminated with ochratoxin A[END_REF], especially if the process is considered as a whole (fermentation 6 plus baking) and was lower than that detected by [START_REF] Boudra | Thermostability of ochratoxin A in wheat under two 19 moisture conditions[END_REF]. These authors tested 7 treatments of 200ºC/24 minutes, 200ºC/48 min and 250ºC/16 min, and detected OTA 8 reductions in wheat flour of 80%, 94% and 88%, respectively. 9

Conflicting results have been obtained by different authors for the percentage 10 reduction in DON during the bread making process. Some authors [START_REF] Tanaka | Residues of Fusarium 11 mycotoxins, nivalenol, deoxynivalenol and zearalenone, in wheat and processed food after 12 milling and baking[END_REF], Scott 11 et al. 1983[START_REF] Sugita-Konishi | Effect of cooking process on the deoxynivalenol content and its subsequent 8 cytotoxicity in wheat products[END_REF]) detected no significant reductions whereas Abbas et al. 12 (1985) and [START_REF] Neira | The effects of bakery processing 22 on natural deoxynivalenol contamination[END_REF] found reduction rates ranging from 19.1-69.4% and 16.8%-13 96.6%, respectively. According to FAO/WHO (2001b) trichothecenes are stable at 120ºC, 14 moderately stable at 180ºC and decompose within 30-40 min at 210ºC. In this study, a 15 reduction rate of 48.6% was found during baking but not during fermentation. As noted 16 above, this is the first study to examine NIV and 3-ADON stability during baking. The results 17

show that of the four mycotoxins tested, NIV and 3-ADON show the greatest reduction rate 18 during the process under the conditions used. 19

In addition to temperature, other factors, such as the strain of yeast used in the 20 fermentation, baking time, bread size, etc, influence the bread making process and can 21 influence the stability of the studied mycotoxins. 22

The European Union has set up maximum levels for OTA and DON in cereals for 23 human consumption of 3 µg kg -1 and 750 µg kg -1 , respectively (European Commission 2006). 24

Moreover, it has been established in Europe that exposure of consumers close to the TDI of 1 25 µg kg -1 body weight per day for DON and to the Tolerable Weekly Intake of 120 ng kg -1 26 body weight per week for OTA is possible [START_REF] Gareis | SCOOP task 3.2.10. Collection of Occurrence Data of Fusarium Toxins in Food and 11 Assessment of Dietary Intake by the Population of EU Member States[END_REF], EFSA 2006, Samar et al. 27 2007). To reach these values, for a person of 70 kg weight it would be necessary an intake of 28 8.4 µg of OTA per week and 70 µg of DON per day. Taking into account that during baking 29 average reduction rates of OTA and DON are 60 and 48%, respectively, the maximum levels 30 cited above will be reduced during the baking process to 1.2 µg OTA and 390 µg DON per kg 31 of wheat flour. To reach the tolerable intakes, a person of 70 kg weight must consume in the 32 form of bread 7000 g of wheat flour contaminated at the maximum levels per week in the case 33 of OTA and 179 g of wheat flour per day in the case of DON. The European Union has 34 established maximum levels of 500 µg of DON kg -1 in bread (including small bakery wares), 1 pastries, biscuits, cereal snacks and breakfast cereals, which limits the daily consumption of 2 bread to a maximum of 140 g. It is then possible to exceed the TDI for DON under the current 3 European Regulation because not only bread but also other products made from cereals that 4 can be contaminated with DON are consumed regularly. Therefore, a revision of the current 5

European Union regulation concerning the maximum levels for these toxins seems to be 6 necessary to protect consumers' health. 7

In order to examine for differences in the reduction percentage of the different 8 mycotoxins on the inside and outside of the bread, when the baking process finished, samples 9 were taken from the outer part (crust) and the inner part of the pieces of bread and the four 10 mycotoxins were determined in both parts. Figure 3 shows that the decrease in the different 11 mycotoxins was greater in the bread crust than in the centre. ANOVA of the data showed that 12 there were significant differences (p ≤ 0 .05) in the stability of the different toxins inside and 13 outside the bread. 14

The ranges of reduction percentages for OTA, NIV, 3-ADON and DON inside the 15 bread, taking into account all the assayed conditions, were 7. . The observed reduction indicates that the product is not detected with the methods 26 employed but does not proof any degradation of the toxins. Binding of toxins to matrix 27 compounds such as proteins or carbohydrates may be possible and bear the risk that during 28 digestion the toxin is released as reported for fumonisins [START_REF] Humpf | Effects of thermal food processing on the chemical structure 29 and toxicity of fumonisin mycotoxins[END_REF]

  few years, different international committees considered the question about 1 risk assessment and tolerable daily intake (TDI) of OTA and trichothecenes [Eriksen and 2 Alexander 1998, Scientific Committee on Food (SCF) 1999, 2000, FAO/WHO 2001a]. In 3 case of OTA, the FAO/WHO (2001a) retained the previously established Provisional 4

  [START_REF] Sugita-Konishi | Effect of cooking process on the deoxynivalenol content and its subsequent 8 cytotoxicity in wheat products[END_REF]

  Sigma. C 18 -silica was from Waters (Milford, MA, USA). Immunoaffinity clean-up 1 columns (OchraTest TM ) were purchased to Vicam (Vicam Science Technology, MA, USA). 2

18 19

 18 Extraction and SPE clean-up for trichothecenes 20Extraction and clean-up for trichothecenes were performed as described by Valle-21 Algarra et al.(2005). Five grams of dough or milled bread sample was poured into a 100-ml 22 Erlenmeyer flask. After adding 40 ml of acetonitrile-water (84:16, v/v), the mixture was 23 blended in a high-speed blender for 10 min. After filtering through Whatman No. 4 filter, the 24 extraction mixture was stored in a tightly closed glass bottle at -20 ºC until use. 25

  al.(2003) did not find toxin reduction during dough fermentation. It has been found that 1

  3 -38.2, 46.4 -83.4, 42.5 -64.7 16 and 18.1-62.6 %, respectively. In the bread crust the same ranges for OTA, NIV, 3-ADON 17 and DON were 20.4 -51.3, 72.0 -93.1, 58.3 -83.4, and 41.0 -70.7%, respectively. 18 Bread size may have a relevant effect on the level of mycotoxin reduction during 19 baking and may be one of the factors behind the conflicting results reported by different 20 authors. 21 It is important to take into account the fact that this reduction in mycotoxin level 22 would result in equally reduced harmful effects to health because food processing methods 23 such as heating and boiling can themselves produce new toxic compounds (Bullerman and 24 Ryu 2002, Howard et al. 2002, Yumbe-Guevara and Yoshizawa 2003, Sugita-Konishi et al. 25 2006)

Figure 1 . 8 Figure 2 .Figure 1 .Figure 2 .Figure 3 .

 182123 Figure captions 1 2 Figure 1.-Average reduction percentages and standard deviations of OTA, DON, 3-ADON 3 and NIV during dough fermentation at 30 ºC for 60 min. Spiking levels (in µg kg -1 wheat 4 flour): (C1) 200 for each DON, NIV and 3-ADON and 2 for OTA; (C2): 750 for each DON 5 and 3-ADON, 500 for NIV, and 5 for OTA; (C3): 1500 for each DON and 3-ADON, 1000 6 for NIV and 10 for OTA. Experiments were performed in triplicate. 7 8 Figure 2.-Average reduction percentages and standard deviations of (a) OTA, (b) DON, (c) 9 NIV and (d) 3-ADON during the baking stage at different temperature/time combinations 10 (190 ºC/50 min, 207 ºC/40 min, 223 ºC/35 min and 240 ºC/30 min). Spiking levels (in µg kg -1 11 wheat flour) OTA: 2, 5, and 10; DON and 3-ADON: 200, 750, and 1500; NIV: 200, 500, and 12 1000. Experiments were performed in triplicate. 13 14 Figure 3.-Average reduction percentages and standard deviations of (a) OTA, (b) DON, (c) 15 NIV, and (d) 3-ADON at the inside and the outside parts of the bread during the baking stage 16 at 190 ºC/50 min, 207 ºC/40 min, 223 ºC/35 min and 240 ºC/30 min. Spiking levels (in µg kg - 17 1 wheat flour) OTA: 2, 5, and 10; DON and 3-ADON: 200, 750, and 1500; NIV: 200, 500, 18 and 1000. Experiments were performed in triplicate. 19 20 21

  The authors wish to thank financial support from the Spanish Government "Ministerio 1 de Educación y Ciencia" (Projects AGL-2004-07549-C05-02/ALI and AGL2007-66416-2 C05-01/ALI and for two research grants) and from the Valencian Government (Conselleria 3 d'Empresa, Universitat i Ciencia) (Project ACOMP/2007/155). Finally, the authors thank Ms 4 Scientific Committee on Food. 2000. Opinion on Fusarium toxins. Part 4: Nivalenol 1 expressed on 19 October 2000. European Commission SCF/CS/CNTM/MYC/26 Final. 2 Available from http://europa.eu.int/comm/food/fs/sc/scf/out74_en.pdf 3 4 Scientific Committee on Food. 2002. Opinion of the Scientific Committee on Food on 5

	1	Charmley LL, Prelusky DB. 1994. Mycotoxins in Grain-Compounds other than Aflatoxins.
	2	St. Paul (MN, USA): Miller JD, Trenholm HL; Eagan Press. Decontamination of Fusarium
	3	mycotoxins, p. 421-435.
	4
	5 5	Esther Gamero, Ms Teresa Liberia, Ms Gema Garcia, Ms Maria Jose Aparisi, Ms Carmen European Commission. 2006. Commission Regulation No 1881/2006 of 19 December 2006
	6 6 6	Garcia, and Ms Elena Sanchis for her technical assistance. setting maximum levels for certain contaminants in foodstuffs. Official Journal of the Fusarium toxins. Part 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and
	7 7 7	European Union of 20.12.2006, L 364, p. 5-24. deoxynivalenol, adopted on 26 February	2002.	European	Commission
	8 9 10 11 12 13 14 8 9 10 11 12 13 14 15	F o r P e e r F o r P e e r Del Pelo DH. 2002. Modificaciones en el proceso de elaboración de tapas de empanadas tendientes a disminuir el contenido de micotoxinas [Thesis in Food Engineering]. [Luján (Argentina)]: Universidad Nacional de Luján. EFSA, 2006. Question N° EFSA-Q-2005-154. Opinion of the scientific panel on contaminants in the food Chain on a request from the commission related to ochratoxin A in o r F o r F o r F o r SCF/CS/CNTM/MYC/27 Final. Available from http://europa.eu/comm/food/fs/sc/scf/out123_en.pdf Scott PM, Kanhere SR, Lau PY, Dexter JE, Greenhalgh R. 1983. Effects of experimental food. Adopted on 4 April 2006. Available from F P e e r P e e r P e e r flour milling and breadbaking on retention of deoxynivalenol (vomitoxin) in hard red spring P wheat. Cereal Chem. 60: 421-424. e e r Scott PM, Kanhere SR, Dexter JE, Brennan PW, Trenholm HL. 1984. Distribution of the
	16 17	R R R R R trichothecene mycotoxin deoxynivalenol (vomitoxin) during the milling of naturally R contaminated hard red spring wheat and its fate in baked products. Food Addit Cont. 1: 313-
	18	323.	e e e e e e
			v i e v i e v i e v i e v i e v i e
			w w w w w w
				O n l O n l O n l O n l O n l O n l
					y y y y y y
	29					. These are
	30	little studied aspects of processed foods.
	31
			13

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


Keith A Scudamore

Harry Baillie

Sue Patel

Simon G Edwards

The occurrence and fate of Fusarium mycotoxins during the commercial 1 processing of oats in the UK 2 3 4

Keywords: Oats, deoxynivalenol, HT-2 toxin, T-2 toxin, nivalenol, milling, co-5 products

come

The occurrence and fate of fusarium mycotoxins during the commercial processing of oats in the UK

Cereals can be invaded by a range of fungi both in the field before harvest and 3 during storage. Some species of these moulds can produce toxic metabolites 4 called mycotoxins that exhibit a wide range of toxic effects. Legislation setting 5 maximum admissible limits for certain mycotoxins in cereals has been agreed or 6 is under consideration for a number of these contaminants within the EU. Limits 7 have been set for deoxynivalenol (DON) and zearalenone (ZON) in cereals 8 including oats and will be set for HT-2 toxin (HT2) and T-2 toxin (T2) possibly 9 from 1 July 2007 or later if considered necessary taking into account the latest 10 information available on occurrence and toxicology. Current limits relevant to 11 oats are given in Table I. 12 Some data for oats are shown in an EC SCOOP report on Fusarium 13 mycotoxins and includes limited data for HT2 and T2 [START_REF] Gareis | Collection of Occurrence Data of Fusarium Toxins in Food and 10 Assessment of Dietary Intake by the Population of EU Member States[END_REF]. High 14 concentrations of DON were shown to occur in oats in Finland (Hietaniemi & 15 Kumpulainen 1991) and in Canada [START_REF] Tekauz | Fusarium head blight of oat in western Canada-preliminary 20 studies on the fusarium species involved and levels of mycotoxin (s) in 21 grain[END_REF]2004) while even food-grade [START_REF] Hietaniemi | Contents of fusarium toxins in Finnish and 17 imported grains and feeds[END_REF] oats are often contaminated with trichothecenes in Norway, particularly with 17 HT2, T2, DON and nivalenol (NIV) [START_REF] Langseth | The occurrence of HT-2 toxin and other 14 trichothecenes in Norwegian cereals[END_REF]Langseth and 18 Elen 1996). [START_REF] Müller | Fusarium toxins in 1 wheat harvested during six years in an area of southwest Germany[END_REF] determined the occurrence of Fusarium 19 mycotoxins in wheat over the years 1987-1993 and in oats over a five-year period 20 in southwest Germany [START_REF] Müller | Natural occurrence 4 of fusarium toxins in oats harvested during five years in an area of 5 southwest Germany[END_REF]. They observed that the incidence and 21 levels of toxins varied from year-to-year and were not correlated with 22 precipitation levels. The occurrence and concentrations of Fusarium mycotoxins 23 in oats and other cereals in Europe were reviewed by [START_REF] Pettersson | Trichothecene occurrence in European cereals-A Review[END_REF] who 24 observed that the relative frequency and concentrations of HT2 and T2 occurring 25 in oats were comparable to other cereals although data available for oats were 1 limited. HT2 and T2 were much higher in some samples of oats than are usually 2 found in wheat. Only small differences in the susceptibility of oat cultivars to 3 DON were shown in a study of different cultivars in Norway (Langseth et al. 4 1995) but it seems there were considerable geographical differences. [START_REF][END_REF] The concentrations of trichothecenes in oats during official variety, 6 organic cultivation and fertilization trials in Finland were determined (Hietaniemi 7 et al. 2004) and DON was the dominant trichothecene found. The highest 8 concentrations of DON occurred in the dryer and warm summers of 1997 and 9 1999 rather than in the cold wet summer of 1998. In addition there was little 10 effect due to variety, nitrogen fertilization levels or conventional/organic 11 cultivation methods. A number of reports have stressed the importance of 12 background factors. For example the incidence of rain during heading time 13 represents a significant factor (Döll et al. 2000; [START_REF] Oldenburg | Risikoabschätzung und 7 Vermeidungstrategien bei der Futtermittelerzeugung Landbauforschung 8 Volkenrode[END_REF]; Langseth 14 et al. 2001). [START_REF] Langseth | Occurrence of toxigenic fungi and 17 mycotoxins in plants, food and feed in Europe[END_REF] A four-year investigation of Fusarium mycotoxins in UK barley and oats 16 during 2002-2005 by one of the current authors shows a high incidence of HT2 17 and T2, with combined concentrations of these two mycotoxins of more than 18 1000 µg/kg in 16 per cent of raw oat samples collected at harvest (Edwards 19 unpublished). However, DON concentrations were low and less than 300 µg/kg in 20 every sample. The levels of HT2 and T2 raised concern in the oat industry with 21 its possible implications for consumer health and led the UK Food Standards 22 Agency to commission a survey of oat-based food products [START_REF] Anon | Food Standards Agency, UK. Mycotoxins in oats survey[END_REF]). The 23 results from this survey showed that the concentrations reaching the consumer 24 were in fact low and probably of minimal toxicological concern. However, the 25 reason for the considerable difference in concentrations between raw oats and 1 oat-based foods needed explanation. In addition, if the answer should lie in 2 processing and the removal of co-products containing concentrated levels, the use 3 of these products would need careful consideration. It could thus be concluded 4 that a large reduction in mycotoxin contamination was being achieved during 5 processing. 6

There appears to be no work reported on the effect of commercial oat 7 processing except for de-hulling studies of trichothecene-contaminated oats that 8

shows most of the mycotoxin concentrates in the hull [START_REF] Pettersson | Controlling mycotoxins in animal feed[END_REF]). When 9 the mycotoxicolological quality parameters of naked and covered oats with regard 10 to the production of bran and flakes were investigated, de-hulling brought about a 11 large reduction in the contamination of oat kernels [START_REF] Adler | Mycotoxicolological quality parameters of naked and 16 covered oats with regard to the production of bran and flakes[END_REF]). In the 12 same way a large reduction in DON was shown to occur in heat-treated groats 13 compared to the levels in raw oats [START_REF] Tekauz | Fusarium head blight 23 of oat-current studies in western Canada[END_REF]. [START_REF] Pettersson | Controlling mycotoxins in animal feed[END_REF] Approximately 700,000 tonnes of oats are produced in the UK each year 15 (DEFRA 2007) with around 40% of the total being used in UK food production . 16 This is similar in quantity to the amount of imported maize used for human food. 17

The balance is exported or used in animal feed. In addition, oats are imported 18 from Scandinavia and Canada. [START_REF] Tekauz | Fusarium head blight of oat in western Canada-preliminary 20 studies on the fusarium species involved and levels of mycotoxin (s) in 21 grain[END_REF] The oat grain consists of the groat (caryopsis) and the surrounding hull 20 (husk). Only the groat is required for milled products while the indigestible hull 21 must be separated and removed in a de-hulling (shelling) stage. The groat has a 22 lipid content which is 2-5 times that of wheat. It also has an active lipase that is 23 separated from the lipid in the intact seed, which can lead to hydrolysis resulting 24 in a 'soapy' taste in final products. This means that the lipase needs inactivation 25 by a process called stabilisation to avoid these undesirable effects in processed 1 products. This is usually achieved by kilning or cooking. An excellent description 2 of the chemistry of whole oat milling and product manufacture is available (Kent 3 and Evers 1994). 4

Oats products for human consumption are mainly based on oat flakes. 5

These are produced in different physical forms depending on the final use. In 6 general, oats are cleaned and de-hulled before kilning as used in this study. 7

However in some mills the oats may be kilned (drying with hot air to about 4-8% 8 moisture) before de-hulling. After the kilning stage, the whole particles are 9 separated, with large particles being used for muesli and the groats or kernels are 10 mainly cut into smaller pinhead oatmeal. To make the flakes in each case the 11 particles are steam treated, rolled and cooled. The various types or cuts of 12 oatmeal are produced by grinding particles in a stone mill or break roll. Oat bran 13 and oat flour is produced from either the whole groat or oat flake by grinding and 14 separation. Figure 1 shows the complexity of the complete oat milling procedure 15 used in a typical UK oat mill and the steps at which samples were taken. [START_REF] Hietaniemi | Contents of fusarium toxins in Finnish and 17 imported grains and feeds[END_REF] Oats are rolled rather than crushed during processing. Coarse, medium 17 and fine grades of oatmeal are all used for porridge and oatcakes, while rolled 18 oats are also used for porridge, and oat flour for baby foods and for ready-to-eat 19 breakfast cereals. 20 Small-scale laboratory or pilot scale studies of cereals mimicking true 21 industrial situations do not always reflect accurately what happens in the practice. 22 Studies of commercial oat processing have mostly been ignored because of the 23 resources required and commercial sensitivity. The operating scale, mill set up 24 and commercial product will also differ from mill to mill. For oats specifically, 25 the end product can usually be defined as the oat flake that is then used to 1 manufacture the majority of products that the customer buys at the retail outlet. 2

This study followed the changes in concentrations of the Fusarium 3 mycotoxins HT2, T2, DON and related trichothecenes and F2 occurring in raw 4 oats during commercial processing and was carried out in close cooperation with 5 the UK oat industry. It forms part of a more extensive project studying the fate of 6

Fusarium mycotoxins occurring during cereal processing funded within the UK. In this study oat milling was carried out at one site on 12 separate consignments 14 of oats. Each consignment consisted of two lorry loads of 25 tonne of raw oats 15 giving a bulk of 50 tonnes. An industry standard vacuum sampling probe was 16 used to withdraw samples from both lorries to provide a 10 kg aggregate. This 17 type of probe samples at various locations in the vehicle and draws material from 18 the bottom to the top of the load at each location. The sampling was carried out 19 twice per vehicle. The probe was set to sample at 6 separate points on each 25 20 tonne load of oats. The samples drawn at each probing point are deposited into 21 the one receiver and the two sample quantities were then bulked together. Each 22 sampling contributed 5 kg to the final sample quantity giving a total of 10 kg 23 from 12 separate points. For the various products and by-products, 500 g samples 24 of material were drawn from the processing streams at 10-minute intervals. These 25 samples were then bulked to give a total of 9 kg per sampling point. 26 Biopharm Rhone) under gravity. The column was washed with 10 ml water. ZON 7 was eluted under gravity with 2 ml of acetonitrile. The eluate was evaporated to 8 dryness and transferred to a vial with chloroform. The chloroform was evaporated 9 to dryness and dissolved in 1 ml of mobile phase of which 20 µl were used for the 10 detection of mycotoxins. The equipment consisted of a Gilson 307 pump, Gilson 11 231/401 auto-injector and a Jasco fluorescence detector. Liquid chromatography 12 was performed on a Kromasil C8 (Hichrom) 5 µ particle size 25 cm x 3.2 mm i.d., 13 operated at 0.5 ml min -1 with 1 % acetic acid: acetonitrile (55:45) as mobile phase. 14

The excitation wavelength of the fluorescence detector was set at 274 nm and the 15 emission wavelength was 440 nm. [START_REF] Hietaniemi | Contents of fusarium toxins in Finnish and 17 imported grains and feeds[END_REF] 17 Method validation and quality control 18 19 UKAS accreditation ensures that methods are valid for the tasks they are 20 performing, but do not provide an absolute measure of accuracy. Under the 21 Laboratory Quality System a protocol for internal and external proficiency test 22 schemes participation is defined and the laboratory demonstrates on-going 23 precision and accuracy. In this laboratory analysis includes in-house reference 24 material, participation in sample testing schemes such as FAPAS 25 (www.fapas.co.uk/fapas.cfm) and collaborative projects. Because of the cost and 1 limited availability of Certified Reference Materials (CRM's), on-going control 2 of the methods was monitored using in-house reference materials prepared from 3 naturally contaminated materials received at this laboratory, checked for 4 homogeneity and packaged for on-going use. CRM's are only used for 5 intermittent checks for the robustness of the methodology used. Typical mean 6 recoveries for each toxin ranged from 70 to 110 % with coefficient of variation of 7 less than 8 % for 10 replicates. 8

For ZON, maize in house reference material contaminated at 29.7 µg/kg 9 of ZON was spiked at 30 µg/kg (in the absence of suitable oat reference sample). 10

Typical mean recovery was 85% with a coefficient of variability of 6.3% for 6 11 replicates. 12 For trichothecenes wheat in house reference material contaminated at 35 13 µg/kg of DON was spiked with 200 µg/kg of each trichothecene. Typical mean 14 recovery was 92% with a coefficient of variability of 6.3% for 6 replicates. 15 16 Recovery, Limit of Detection and Determination 17 18 All analyses were conducted with a spiked sample, i.e. a known amount of toxin 19 was added to a sample of ground oats each day prior to extraction, clean-up and 20 detection for each batch of 1-5 samples, these samples are used to access 21 recovery, and recoveries of 70-110% were considered acceptable. The spike 22 level was 200 µg/kg for each trichothecene and 50 µg/kg for ZON. 23

The limit of detection and limit of determination was similar for each 24 trichothecene and 5 µg/kg and 10 µg/kg respectively were used in this study while 25 values of 3 and 6 µg/kg were used for ZON. The limit of detection is defined as 3 1 times the electronic baseline noise and the limit of determination as 6 times 2 baseline noise. After analysis samples were retained and stored at -20°C. 3 4

Measurement uncertainty 5 6

Calculation of the measurement uncertainty was not carried out specifically for 7 this study but was based on historical in-house data, performance in international 8 collaborative trials and FAPAS trials incorporating repeatability and 9 reproducibility data. The expanded measurement of uncertainty was calculated 10 using a standard coverage factor of 2, equivalent to a confidence of 11 approximately 95% that the actual level of the mycotoxin being measured lies 12 within the quoted range. The assessment of measurement uncertainty was 13 targeted at samples containing mycotoxins at a range of levels. The standard 14 measurement of uncertainty for these analyses was assessed to be ± 12% at a 15 level of 320 µg/kg for DON. The expanded measurement of uncertainty was 16 assessed to be ± 24 % at a level of 320 µg/kg for DON. These calculations are 17 product fell well inside the 70 and 110 % considered as acceptable and results 8 were corrected accordingly. 9

Oat consignments examined in this study were from "unprocessed" 10 parcels i.e. grain that had been dried and cleaned on farm to meet purchase 11 specification, as far as the legislation is concerned these are unprocessed samples. 12 These include both home grown and imported samples. the 12 raw oat consignments were within the existing maximum permissible 5 limits, Table 1. 6 NIV occurred in oats from both regions although there is currently no plan 7

to include this mycotoxin in future legislation. NEO and T23 were present in the 8 samples containing the highest concentrations of HT2 and T2. 9

Mean concentration values for the four most commonly occurring 10 trichothecene mycotoxins calculated for the two geographical regions are given in 11

Table III. During this period there was a notable difference in concentration of 12 DON between the two regions and much greater than the uncertainty factor of the 13 analytical methods. However, the number of samples was small with only eight 14 consignments from the UK/Ireland area and four from Scandinavia so it is unclear 15 whether this is a true geographical and/or climatic difference or whether due to 16 another undefined factor. Mean HT2 and T2 concentrations were higher in UK-17 grown oats while mean values of NIV were higher in samples from Sweden and 18

Finland. 19

Oat processing involves many different operations as seen in Figure 1 but 20 the three most important material components in the context of this study are the 21 raw oats, the finished oat flake (used as the basis of many consumer products) 22 and the co-product 'pellet'. This latter consists of the hulls and other co-products 23 produced during de-hulling and other operations such as cleaning, aspiration and 24 length separation. Figure 2 shows the concentration of HT2 in these three 25 materials measured in the 12 consignments studied using a logarithmic scale. The 1 histogram bar for oat flakes is very small for all the consignments while that for 2 the pellet by-product is considerably higher than that for the raw oats in every 3 case. Histograms drawn for T2, DON and NIV follow a very similar pattern and 4 are not presented here. These results show that a large reduction in toxin levels 5 occurs at the de-hulling stage so that the amounts of mycotoxins entering the 6 human food chain from the flakes are greatly reduced from those initially 7 indicated by the levels in raw oats. The results explain the apparent contradictory 8 results obtained from recent studies of raw oats and of oat-based foods discussed 9

earlier. 10

The downside to this situation is the high concentrations that occur in the 11 pellet by-product that reached more than 20,000 µg/kg and 6000 µg/kg of HT2 12 and T2 respectively in sample 7. This co-product is currently used as a ruminant 13 feed because of the high cellulose content. Use for animal feed thus needs to be 14 considered carefully to ensure that the risk is considered acceptable for the 15 species in receipt. [START_REF] Hietaniemi | Contents of fusarium toxins in Finnish and 17 imported grains and feeds[END_REF] The relative concentrations of Fusarium mycotoxins found here in oats 17 appear to be considerably different to those in wheat and maize with HT2, T2 18 NIV usually present while DON is more variable in its occurrence. This contrasts 19 with infected wheat and maize in which DON (and fumonisins in maize) is 20 commonly the major Fusarium mycotoxin with HT2 and T2 in much lower 21 concentrations (Edwards & Ray 2005). In Canada, the occurrence and levels of 22 DON in oats is reported as similar to wheat and barley [START_REF] Tekauz | Fusarium head blight of oat in western Canada-preliminary 20 studies on the fusarium species involved and levels of mycotoxin (s) in 21 grain[END_REF].

Reduction of mycotoxins during oat processing 1 2

As discussed earlier, the principle end material used for manufacture of oat-based 3 foods is the oat flake (Figure 1). The flaked material may be subjected to further 4 processing before retailing or by the consumer, e.g. preparation of porridge from 5 porridge oats. Table IV shows the amounts of DON, HT-2 toxin, T-2 toxin and 6

NIV in the raw oats and the flakes and the reduction that occurred for each 7 sample. It is not possible to calculate the reduction in those consignments for 8 which no quantitative result was obtained for the oat flake. The mean values for 9 reduction of DON and T2 are similar but are higher for HT2 and NIV. However, 10 when the percentage reduction that occurred is plotted against the mycotoxin 11 concentration in the raw oats, the reduction achieved appears to depend on the 12 initial level of contamination being greatest in the most highly contaminated 13 samples (Figure 3). 14

A cursory look at the results for DON and T2 in Table IV suggests that 15 the same effect occurs for these mycotoxins although the concentrations are 16 usually less than for HT2. However, NIV seems to be lost to the greatest extent 17 and this may be because it is less heat stable and thus that remaining after de-18 hulling may be destroyed during the high-temperature kilning stage. 19 These findings have implications when considering setting statutory limits 1 for HT2 and T2 in particular so that it may be unjustified to set too stringent 2 limits for raw oats. However, the concentration of mycotoxins in the pellet by-3 product must be considered and deemed suitable for its end use. 4 5

Mass balance calculation 6 7

When raw oat grain is de-husked, the material removed is used to produce the by-8 product referred to here as the oat pellet. On average approximately 28-30% of 9 the total weight of raw oats ends up in this product. After kilning, groats are 10 colour sorted so that discoloured groats are separated and removed leaving a 11 product that is used for production of a range of food products. The dark 12 (discoloured) groats make up between 10 and 15% of the kilned groat. In order to 13 carry out a mass balance calculation mean values of 29% and 12.5% respectively 14 for the quantity of pellet by-product and reject groats have been used here 15 throughout and a diagram is shown (Figure 4). Typical moisture contents for the 16 various materials are 14% for raw oats, 11% for oat flake, 11% for dark groats 17 and 10.5% for pellets and all analytical results are adjusted to a dry weight basis 18 using these values. [START_REF] Tekauz | Fusarium head blight of oat in western Canada-preliminary 20 studies on the fusarium species involved and levels of mycotoxin (s) in 21 grain[END_REF] In addition, a small amount of bran may be collected by scouring the outer 20 layer of the final groat. This fraction of oat bran produced for human 21 consumption is normally extracted from the oat after hulling, kilning and cutting 22 and as such is produced at the end of the milling process. It consists of the 23 pericarp layer of the oat kernel and the first few layers of cells under the pericarp. [START_REF] Krska | The State-of-the-art in the Analysis of Estrogenic 1 Mycotoxins in Cereals[END_REF] The amount of material involved and mycotoxin levels detected in a couple of 25 samples examined showed the quantity of this bran removed and any mycotoxin 1 levels to be insignificant and were excluded from the calculations. 2

Results for the 4 consignments in which all the relevant components were 3 analysed are given in Table VI. The mean for all values suggest that the quantity 4 of all the four mycotoxins present in the raw oats can be accounted for within the 5 variability inherent in such studies. However, the results are quite variable (being 6 particularly low for HT2 and T2 in sample 11). This may be due to a combination 7 of sampling and analytical errors, differences in the relative weights of oat groats 8 to pellets and of sound sorted groats to rejected ones. In addition, as 9 approximately 95% of the mycotoxins are found in the pellet by-product that can 10 be quite variable in composition an error in their measurement will strongly affect 11 the recovery figures. 12

In the only study here in which samples were taken at other stages during 13 processing, analysis suggests that only a very small loss occurs during kilning. 14 Colour sorting of groats shows higher concentrations present in the dark coloured, 15 particles. These are used in other products in which discolouration is masked or 16 not otherwise a problem. 17 In these studies, HT2 is always present with and at greater level than T2 so that if 21 monitoring of industrial consignments shows the presence of T2, HT2 would also 22 be expected to be present in approximately three-fold concentration. The numeric 23 values for ratios of HT2:T2 are given in Table VII for the 12 raw oat samples 24 examined. The ratio of the concentrations of these toxins are quite consistent with 25 a mean value and median value each of 3.5 and a standard deviation of 22.8 % 1 and this could be useful, particularly in for example simple rapid test procedures 2 for indicating the approximate concentration of a related mycotoxin other than 3 that measured. 4 A variation of this value is within that expected just from sampling and 5 analysis so this implies a strong relationship during secondary metabolism within 6 the fungal species involved. This is consistent with previous literature reports that [START_REF] Langseth | Differences between barley, oats and wheat in the 8 occurrence of deoxynivalenol and other trichothecenes in Norwegian 9 grain[END_REF] show they are usually formed by the same fungal species, normally with HT2 8 present in higher concentrations. Even though these toxins occur less widely in 9 wheat the same relationships during formation occurs (personal observations). 10 infrequent and only at low levels although the study indicates that this would be 1 reduced to a similar extent. There are some initial data that suggest the processing 2 of heavily contaminated raw oats is more effective in reducing contamination 3 than the less contaminated consignments. 4

These results explain why studies in the UK have shown high levels of 5 mycotoxins in oats at harvest but generally low concentrations in consumer 6 products. In the consignments studied, no DON, HT2 or T2 exceeded a 7 concentration of 100 µg/kg in the final oat flakes. The European Regulations for 8 DON in unprocessed oats should ensure that strictest limits set for cereal-based 9 food for infants and young children should be effectively achieved by a 10 substantial margin. Indeed a level of 3500 µg/kg in raw oats should still be 11 reduced below this level after processing. However, the mycotoxins are 12 concentrated in the pellet product formed from hulls etc. and are likely to be 13 between 4 and 10 times the concentration in the raw oats. Hence the appropriate 14 use of this product should be carefully considered. The maximum concentration 15 of ZON found in the consignments studied was 22 µg/kg in raw oats and <3 16 µg/kg in the flakes. This suggests that concentrations of approximately 300 µg/kg 17 will be reduced below the levels required for infant foods. 18 Examination of further consignments are to be studied to confirm these 19 initial findings. The effects of the kilning stage and colour sorting should be 20 examined further. Planned studies aim to confirm the extent by which the 21 proportions of mycotoxins that are transferred to each commercial product vary 22 from consignment to consignment. If the factors that cause this variability can be 23 understood it should be possible to provide the oat processing industry with a 24 guide to the concentrations of each mycotoxin that can be expected in the 25 Results obtained using rapid tests that currently only measure either T2 or 2 HT2 could be used to estimate the combined concentration of T2 + HT2 although 3 a test for HT2 would be preferable for such a purpose as this mycotoxin is the 4 major component of the pair. 5

7

 7

( 15 16 9 Macdonald

 159 All the bulked samples were then mixed and quartered to reduce the 1 sample size to about 2 kg per point. Samples were taken at the points shown 2 -product pellets consisting of all the husk and other material removed 6 during de-hulling, 7 4. Kilned groats-colour sorter accepts 8 5. Kilned groats-colour sorter rejects (discoloured groats) 9 6. Oat flakes.10 11 All these samples comprised ~9 kg bulked portions except the discoloured oats 12 that form only a small fraction of the whole kilned groats. Only samples 1, 3 and 13 6 were collected for all consignments. A few samples of oat bran were also 14 collected.Extraction, clean-up and determination of mycotoxins 17 18On receipt in the laboratory, the whole of each oat sample or product was ground 19 through a 0.8 mm screen and mixed for 30 minutes in a Gardner horizontal mixer to 20 ensure homogeneity. Samples were stored at -20°C if not analysed immediately. 21 All reagents were purchased from commercial sources and were of 22 analytical grade. Solvents were of HPLC grade. Mycotoxin standards of DON, 23 NIV, 3-acetyldeoxynivalenol (3AcDON), 15-acetyldeoxynivalenol (15AcDON), 24 fusarenone X (FUSX), T2, HT2, diacetoxyscirpenol (DAS), neosolaniol (NEO), 25 (T23) and ZON were purchased from Sigma-Aldrich Company Ltd., 1 Gillingham, Dorset, UK. DON, NIV and ZON were quantified prior to use by 2 UV spectrometry. 3 The methods used for the trichothecenes and ZON are UKAS (ISO 4 17025) accredited and followed the UK Food Standards Agency Requirements of 5 External Contracts (Thompson and Wood, 1995). Such methods have been used 6 successfully in a number of surveys, collaborative and inter-comparison trials in 7 the UK and EC, e.g. trichothecenes (Gilbert et al. 1992; Langseth & Rundberget 8 1998) and ZON (Scudamore & Patel 2000; Krska & Josephs 2001; Anderson & determined following the method of Patel et al. 141996. In summary, ground samples (20 g) were extracted with 100 ml 15 acetonitrile/water (84:16) by shaking for 2 hours on a wrist action shaker. A 5 ml 16 aliquot of the extract was applied to a pre-washed charcoal/alumina column and 17 this then washed with 40 ml acetonitrile: water (84:16). The total eluate was 18 taken to dryness and transferred to a vial with acetonitrile. The residue was 19 derivatised to form the trichothecene -trimethyl silyl (TMS) derivatives and 20 determined by GC/MS operating in selected ion mode, using 4 25 g) were extracted with 125 ml of acetonitrile/water (75/25) 3 by shaking for 30 minutes. The extract was filtered. Ten ml of the extract was 4 diluted with 40 ml of Phosphate Buffered Saline (PBS) solution. The diluted 5 extract was passed through EASI-EXTRACT ZON immunoaffinity column (R-6

19 20Food 5 6

 195 only intended as a guide to the uncertainty associated with measurements in this 18 study.Results from check trials 21 22Acceptable results have been achieved in the FAPAS Scheme for deoxynivalenol 23 and ZON in cereals during rounds 1 to 24 (August 2000 -April 2006) as they[START_REF] Krska | The State-of-the-art in the Analysis of Estrogenic 1 Mycotoxins in Cereals[END_REF] were well inside the limits for Z scores of ± 2 considered acceptable. In an initial 25Page 12 of 39 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk organised by FAPAS for T2 and HT2, Z-SCORES were = -0.1 and 1Analytical recovery of spiked mycotoxins from raw oats, oats flakes and the by-7

13 14

 13 Occurrence of mycotoxins in raw oats15 16 The concentrations of DON, NIV, HT2, T2, related compounds and ZON in 12 17 consignments of raw oats received by the commercial oat mill participating in this 18 study are presented in TableII. Samples processed originated from the 2004 and 192005 harvests eight originated from the UK or Ireland while 4 were imported 20 from Scandinavia. Concentrations of ZON in raw oats were very low or 21 undetected, the highest being 22 µg/kg in one sample from Sweden. HT2 and T2 22 occurred in every sample and NIV in all but one sample. The combined levels of 23 HT2 and T2 in the two most contaminated samples of the 12 were approximately 24 2500 µg/kg and 3500 µg/kg. 25 DON was detected in 8 out of 12 consignments but the 4 samples with the 1 highest levels were all grown in Scandinavia and 3-acetyl DON was detected in 2 each of these although 15-acetyl DON was undetected. DON occurred only in 3 very low levels in UK samples during this period. Concentrations of DON in all 4

  Table V shows the extent to which Fusarium mycotoxins are concentrated 20 into the pellet by-product. Mean values are similar for each mycotoxin with the 21 concentration factors ranging from 1.7 to 15 fold. The biggest concentrations 22 values tended (but not always) to occur when mycotoxin concentrations were 23 relatively high in the raw oats. 24

18

  Relative occurrence of Fusarium mycotoxins 19 20

17 18 19

 1718 By comparison, the values of HT2 to DON show no correlation, which is 11 expected from current knowledge that the two mycotoxins are formed by 12 different Fusarium species. However, DON and NIV are produced by different 13 chemotypes of the same fungus, and calculated values for NIV and DON indicate 14 no interdependence. In a final scenario there is poor relationship for the ratios of 15 HT2 and T2 to NIV.[START_REF] Hietaniemi | Contents of fusarium toxins in Finnish and 17 imported grains and feeds[END_REF] ConclusionsIn summary, the concentrations of all mycotoxins remaining in the oat 20 flakes are approximately 5-10 % of those of intake oats. Thus the industrial 21 processing of raw oats used in the UK is very effective in reducing the amount of 22 Fusarium mycotoxins that exist in consumer foods at the retail point. Ninety to 23 ninety five percent of all Fusarium mycotoxin contamination in the raw oats is 24 removed during the production of oat flakes. The occurrence of ZON was 25

  from each consignment of raw oats. 1

Figure 3 .

 3 Thompson M, Wood R. 1995. Guidelines on Internal Quality Control in 1 Analytical Chemistry Laboratories. Pure and Applied Chemistry 67: 649Relationship between HT2 concentration in raw oats and amount 3

Figure 2 .Food

 2 Figure 1. Oat Processing Scheme

Figure 3 .Figure 4

 34 Figure 3. Relationship between HT2 concentration in raw oats and amount removed during processing

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	1 1 1
	2 2 2			RAW OAT (100%)
	3 3 3	Table I. EC, Maximum permissible limit for fusarium mycotoxins in cereals.
	4 4 4	Table VI. Total mass balance calculation for mycotoxins in recovered oat
	5 5 5	GROAT (71%) materials			PELLET (29%)
	6		Mycotoxin, µg/kg Mycotoxin weights, µg, in each fraction
	7 8	KILNED Cereal	DON analysed based on 1 kg of raw oats, calculated ZON on a dry weight basis
	9	Unprocessed durum wheat and oats Oat sample	1750	100
	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 6 7 8 9 10 11 12 13 6	F o r P e e r R e v i e w COLOUR SEPARATION GROAT (87.5 %) = 62 % of raw oat DISCOLOURED GROAT (12.5%) = 9% OAT FLAKE (62 %) Figure 4 Relative proportions of each oat product used to carry out mass balance calculations F o r P e e r R e v i e w Cereal flour, including maize flour, 750 Sample Mycotoxin Raw Oat Pellet Rejects Total Recovery, 75 no. oats flakes processed % maize grits and maize meal Bread, pastries, biscuits, cereal snacks and breakfast cereals 500 50 Processed cereal based foods for 200 20 DON <10* <10* 7 <10* <10* -F NIV 124 <10* 147 2 149 120 HT2 578 16 739 40 795 137 4 o T2 176 10 281 9 300 170 r infants and young children and baby food Table III. Mean and median mycotoxin concentration in oats from two different geographical origins DON 1230 43 1356 47 1446 118 P NIV 111 <10* 77 2 79 71 HT2 487 20 378 19 417 86 8 e T2 125 8 111 6 125 100 e DON 833 54 529 37 620 74 r NIV 204 7 182 7 196 96 HT2 588 28 500 22 550 94 9 R T2 184 11 149 6 166 90 Mycotoxin, concentration, µg/kg Mycotoxin UK/Ireland (n=8) Scandinavia (n=4) mean median mean median DON 13 <10 628 e DON 288 33 338 3 374 130 v NIV 181 <10* 208 <10 208 115 HT2 832 27 407 9 443 53 11 i T2 252 12 111 3 126 50 e 514 NIV 67 56 131 135 Mean all 99.3 w *= <10 in the analysed fraction
	14 7 8 9 10 11 12 13	T2 HT2	246 718	28 115	O n l y O n l y 135 128 466 471 O n l y
	14
	15
	16
	17
	18
	19
	20
	21
	22
	23
				29 30 34

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	1
	2
	3
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	35

Table VII .

 VII Ratio of concentrations of various pairs of mycotoxins in the raw oats 4based on results from the first 12 consignments examined. 5

			Mycotoxin, concentration, µg/kg		Mycotoxin ratios
		Sample DON NIV	T2	HT2	HT2/T2 HT2/DON HT2/NIV
		1	<10 *	66	53	257	4.8	no DON	3.9
		2	15	23	41	196	4.8	13.1	8.5
		3	20	142	103	386	3.7	19.3	2.7
		4	<10 *	109	154	504	3.3	no DON	4.6
		5	298	90	50	193	3.9	0.6	2.1
		6	<10 *	36	105	230	2.2	no DON	6.4
		7	26	105	958	2570	2.7	137.3	24.5
		8	1230	97	110	427	3.9	0.3	4.4
		9	731	179	161	516	3.2	0.7	2.9
		10	<10 * <10 *	73	217	3.0	no DON	no NIV
		11	253	159	221	730	3.3	2.9	4.6
		12	<10 * <10 *	527	1940	3.7	no DON	no NIV
		Mean	216 *	85 *	213	764	3.5	25	6.5
		Median	20	93	107	456	3.5	-
		SD	-	-	-	-	0.80	50	6.61
		% SD	-	-	-	-	23	201	102 +
	6	* Half the value of the limit of quantitation is included in the calculation of mean
	7	values of the mycotoxin levels.
	8	+ But omitting ratio value for sample 7 then % SD is reduced to 43.5
	9
	10
	11
	12
	13
	14
	15
	16
	17
	18
	19
	20

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Acknowledgements 8

These studies were funded by the UK Food Standards Agency, the UK DEFRA, 9

Food Quality and Safety LINK Programme and the UK oat industry. 10 11 12


Maria Vass

Iva Diblikova

Eloise Kok

Kamil Stastny

Karla Frgalova

Karel Hruska

Milan Franek

In-house validation of an ELISA method for screening of semicarbazide in eggs

Keywords: Extraction, Immunoassays, Method validation, Screening -ELISA Additives/Contaminants: Veterinary drug residues -semicarbazide Nitrofurazone, semicarbazide, egg homogenate, ELISA, LC-MS/MS, 21 validation, incurred samples, SEM depletion 22

Nitrofurazone (NFZ) belongs to the nitrofuran family of broad spectrum antibiotics which 3 are banned in the EU due to concerns about their carcinogenic and mutagenic effects on 4 human health. The global nitrofuran crisis which came to light during 2002/03 revealed a 5 frequent presence of nitrofuran residues in poultry and aquaculture products imported 6 into Europe, especially from many Asian countries and Brazil [START_REF] Diblikova | Monoclonal antibody-based 18 ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in 19 tissues using a simplified sample preparation[END_REF]. [START_REF] Kumar | Determination of Nitrofuran Residues in Poultry 8 Muscle Tissues and Eggs by Liquid-Chromatography[END_REF] This led to import restrictions and mandatory testing. Member states of the European 8

Union are now required to monitor compliance of the prohibition of nitrofuran antibiotic 9 use through their annual national residue control plans. The minimum required 10 performance limit (MRPL) for nitrofurans (and respective residue markers) in poultry 11 muscle and shrimp has been set at 1 µg kg -1 by the EU (Commission Decision 12 2002/181/EC 2003). The presence of protein bound metabolites in eggs after the 13 administration of nitrofuran drugs was demonstrated by [START_REF] Mccracken | Evaluation of the residues of 14 furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs[END_REF]. [START_REF] Cooper | Enzyme 15 immunoassay for semicarbazide-the nitrofuran metabolite and food contaminant[END_REF] However, an MRPL for nitrofurans in eggs has not been laid down within the EU. Some 15 countries, such as Germany, have translated the MRPL of 1 µg kg -1 from muscle and 16 aquaculture products to egg as stated in the predefined German National Residue Control 17 Plan [START_REF] Bock | Validation of a confirmatory method for the 3 determination of residues of four nitrofurans in egg by liquid chromatography-4 tandem mass spectrometry with the software InterVal[END_REF]. 18

Effective control of nitrofuran drug use in livestock is based on measuring the 19 concentration of tissue bound nitrofuran metabolites after their release from tissues and 20 derivatisation [START_REF] Hoogenboom | Depletion 4 of Protein-Bound Furazolidone Metabolites Containing the 3-Amino-2-5 Oxazolidinone Side-Chain from Liver, Kidney and Muscle Tissues from Pigs[END_REF]). Semicarbazide (SEM) is the tissue bound 21 metabolic product of NFZ persisting in animal tissues for considerable periods after 22 administration of the parent drug to livestock and was therefore established as a marker 23 for nitrofurazone detection. Recently, the presence of SEM was confirmed in jars of 24 processed baby food, bread and other commodities. The source of this contamination was 25 azodicarbonamide, which is used in the manufacture of the plastic gaskets inside the lids 26 of glass jars. It was found that SEM is a breakdown product of this commonly used 27 foaming agent. As a consequence, the use of SEM as a definitive marker for the 28 monitoring of illegal NFZ administration has become questionable [START_REF] Pereira | Implications of the use of semicarbazide as a 17 metabolic target of nitrofurazone contamination in coated products[END_REF]. 29 However, according to EU legislation, SEM remains the marker residue for NFZ in foods 30 of animal origin [START_REF] Cooper | Enzyme 15 immunoassay for semicarbazide-the nitrofuran metabolite and food contaminant[END_REF]. 31 The common method of SEM analysis involves acid hydrolysis for the release of 1 tissue bound residues in a sample, followed by derivatisation with o-nitrobenzaldehyde 2 (o-NBA). The formed structure is the derivatised analyte nitrophenyl semicarbazide 3 (NPSEM). Analysis based on this approach provides data on total SEM content (bound 4 and free) in a tested sample. Chromatographic methods, predominantly LC-MS/MS, used 5 for the determination of nitrofuran residues in poultry tissues have been well established 6 for monitoring marker metabolites in chicken eggs [START_REF] Kumar | Determination of Nitrofuran Residues in Poultry 8 Muscle Tissues and Eggs by Liquid-Chromatography[END_REF] [START_REF] Bock | Validation of a confirmatory method for the 3 determination of residues of four nitrofurans in egg by liquid chromatography-4 tandem mass spectrometry with the software InterVal[END_REF]). These methods are highly sensitive and specific, 9 however, they are also time consuming and quite expensive. Therefore, a method offering 10 efficient routine analysis which enables the pre-selection of positive samples prior to 11 confirmatory analysis is required. ELISA technology provides a very convenient way to 12 reach the sample frequency requirements with a reduced cost in terms of both time and 13 resources. 14 A recently developed ELISA method was validated as a screening procedure for 15 the determination of SEM in chicken muscle [START_REF] Cooper | Enzyme 15 immunoassay for semicarbazide-the nitrofuran metabolite and food contaminant[END_REF]). In our study, a 16 competitive ELISA for SEM, previously developed in this laboratory, was validated for 17 its suitability for the screening of egg samples, a more available matrix for the monitoring 18 of NFZ abuse on chicken farms. Validation was performed using analysis of fortified 19 blank samples and by the comparison of results obtained by ELISA and LC-MS/MS 20 using incurred egg samples. In addition, ELISA was used to monitor levels of SEM in 21 eggs for long term periods after medication. Sigma (St. Louis, USA). 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from 1 Serva (Heidelberg, Germany). Ethyl acetate, n-hexane, methanol and all other chemicals 2 (HPLC or gradient grade) were purchased from Dorapis (Brno, Czech Republic). The 3 internal standard ( 13 C-15 N 2 -) SEM and derivatised analyte NPSEM used for LC-MS/MS 4 analysis were supplied by Sigma-Aldrich, Czech Republic, and by Riedel-de Haen from 5 VETRANAL, Czech Republic, respectively. Rabbit antibody MVK31 (IgG) raised 6 against NPSEM and 3[(3-Carboxyphenyl)methylene]-hydrazinecarboxamide-horse radish 7 peroxidase (CPSEM-HRP) conjugate were prepared by this laboratory and described in 8 detail elsewhere [START_REF] Vass | ELISA for semicarbazide 24 determination: Characterisation of antibodies and assay development[END_REF]

Reagents and calibration standards 25

The following buffers and solutions were used: (1) 10 mmol L -1 phosphate buffered saline 26 4) 50 mmol L -1 carbonate buffer (pH 9.6) as a coating buffer. (5) PBS 1 with 0.5 % (w/v) BSA (pH 7.2) for HRP conjugate dilution. (6) PBS with 0.1 % Tween-2 20 as washing buffer. ( 7) Substrate buffer was 0.1 mol L -1 sodium acetate adjusted to pH 3 5.5 by addition of 1 mol L -1 citric acid. (8) Substrate solution was prepared by the 4 addition of substrate buffer (1 mL), 1 % (w/v) solution of TMB in DMSO (200 µL) and 5 20 µL of 6 % H 2 O 2 to 20 ml MilliQ-water. (9) 2 mol L -1 H 2 SO 4 was used as stopping 6 reagent. ( 10) LC stock SEM ( 13 C-15 N 2 -) internal standard (1 mg mL -1 ) was prepared in 7 methanol and stored at 2 to 8 ºC for a maximum of three months. 8

Stock NPSEM solution in methanol (1 mg mL -1 ) was diluted with PBS to obtain a 9 100 µg L -1 NPSEM working solution. Serial dilution with PBS yielded NPSEM 10 calibration concentrations of 0, 0.09, 0.19, 0.37, 0.75, 1.49, 1.87 and 7.47 µg L -1 . To 11 account for the mass increase arising from the derivatisation of SEM to NPSEM 12 concentrations were divided by the coefficient 1.867 to obtain SEM equivalent 13 concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1 and 4 µg L -1 . 14 15

Preparation of incurred egg samples 16

Twelve clinically healthy chickens were randomly divided into three groups and housed 17 in individual wire cages. Drinking water and feed was given ad libitum throughout the 18 experiment. All chickens were fed a non-medicated ration of a non-granular conventional 19 layer diet for 3 days prior to medication, and their eggs collected. Group 1 (n = 2) was fed 20 the non-medicated ration for the entire course of the experiment, whereas Group 2 (n = 5) 21 and Group 3 (n = 5) were fed medicated rations throughout a 10 day medication period, 22 containing 30 mg kg -1 and 400 mg kg -1 of NFZ, respectively, followed by the non-23 medicated ration for the remainder of the 90 day experiment. Eggs were collected 24 regularly, homogenised within a week of sample collection, and the homogenates stored 25 at -20 º C until use. 26 27

Sample preparation for ELISA 28

Egg homogenate was thawed and 1 g aliquots weighed into 15 mL plastic tubes. When 29 required, blank samples were fortified by the addition of SEM. Distilled water (3 mL) 30 was added to the sample homogenate and vortexed thoroughly. Then, 1 mol L -1 HCL 31 (0.5 mL) and o-NBA solution (50 µL) were added successively to the homogenate. The 1 mixture was vortexed for 30 sec and incubated overnight for 16 hr at 37 ºC. After cooling 2 to room temperature, 2 mol L -1 NaOH in 0.1 mol L -1 PBS (0.3 mL) was added into each 3 tube to neutralise the pH level and vortexed thoroughly for 20 sec. Ethyl acetate (5 mL) 4 was then added gently to prevent foaming, shaken horizontally on a low setting (10 min), 5 and the mixture centrifuged (1330 x g, 10 min, 4 ºC). The upper ethyl acetate layer (4 6 mL) was transferred into a glass test tube and evaporated under nitrogen at 60ºC. Hexane 7

(2 mL), was added to the evaporate, vortexed thoroughly, followed by the addition of 8 PBS (0.8 mL). Samples were vortexed again, centrifuged (1330 x g, 5 min, 4ºC) and 9 stored at -80 ºC for 10 min (or 25 min at -20 ºC). The hexane layer was removed under 10 vacuum, and the solid phase residue left to thaw. To remove remaining lipid components 11 from sample extract, a further 1 mL hexane was added, the sample was centrifuged and 12 the hexane layer separated as described above. The thawed sample extract in PBS was 13 pipetted directly into the wells of the microtitre plate for analysis by ELISA. 14

15

ELISA detection 16

Microtitre wells were coated with antibody diluted with coating buffer 1:8000 17 (200 µL per well) and left to incubate overnight (16 hr, 4 ºC). Plates were washed three 18 times with washing buffer (0.3 mL per well) and then 100 µL of standard analyte or 19 sample, followed by 100 µL of CPSEM-HRP (diluted to 1:40000) was pipetted to each 20 well. Plates were incubated (1 hr, 4 ºC) and the washing step repeated. Substrate solution 21 (200 µL per well) was added and after 15 min the enzymatic reaction stopped by the 22 addition of 2 mol L -1 H 2 SO 4 (100 µL per well). Absorbance was measured at 450 nm. samples were fortified with SEM and carried out to investigate the detection capability of 1 the method at different false positive rates. The limit of detection (LoD) was calculated 2 on the basis of standard deviation of the blank response by the equation B + 3 SD B where 3 B is the mean response of blanks and SD B is standard deviation for 20 blank samples 4 taken for the investigation. Variation coefficients were calculated from the repeated 5 measurement of recovery using blank samples fortified at 0.3, 1 and 3 µg Kg -1 . 6 Specificity of the assay towards NPSEM and potential cross-reactants is described 7 elsewhere [START_REF] Vass | ELISA for semicarbazide 24 determination: Characterisation of antibodies and assay development[END_REF]). 8 9

Sample preparation for LC -MS/MS analysis 10

A modified procedure according to McCracken and Kennedy, 1997 was carried out as 11 follows: Homogenised eggs (1 g) were transferred into a 15 mL centrifuge tube and an 12 aliquot of internal standard dissolved in methanol ( 13 C-15 N 2 -SEM, 40 µL) was added to 13 the homogenates while vortexing thoroughly. After an equilibration period (15 min), 14 0.1 mol L -1 HCL (10 mL) and o-NBA solution (250 µL) were added to samples, followed 15 by an overnight incubation at 37 ºC under gentle mixing. Samples were cooled to room 16 temperature, neutralised with 0.3 mol L -1 tri-sodium phosphate (500 µL) and pH was 17 adjusted to 7 (± 0.5) using 2 mol L -1 NaOH. Samples were then centrifuged (1800 x g, 18 15 min, 24 ºC) and the supernatant (10 mL) applied to cation exchange SPE columns 19 previously conditioned with methanol (2 mL) and water (2 mL). The SPE columns were 20 dried (2 min) under a vacuum and eluted with ethyl acetate (3 mL) into glass test tubes. 21

The sample eluent was evaporated under nitrogen at 45 ºC and re-dissolved in 0.5 mL of 22 water:acetonitrile (1:1 v/v). The final solution was filtered through a 0.45 µm syringe 23 filter into a HPLC vial and injected into the LC-MS/MS equipment (10 µL). A calibration 24 curve was prepared using blank samples fortified with a SEM standard solution and 25 internal standard ( 13 C-15 N 2 -SEM) over the range of 0.2 to 5.0 µg Kg -1 . 26

27

LC -MS/MS conditions 28

The mobile phase consisted of 0.1 % acetic acid in water (A) and acetonitrile (B) using a 29 gradient run at a flow rate of 0.2 mL per minute at 25ºC. Chromatographic separation was 30 performed on an XTerra MS, C 18 column (3.5 µm, 2.1 x 150 mm), in combination with a 31 was analysed using positive polarity (APCI +). The mass spectrometer was operated 2 using the selected reaction monitoring mode (SRM) and settings were optimised for 3 sensitivity as follows: spray voltage = 8.5 kV, collision gas pressure = 0.5 mTorr, scan 4 time = 0.30 sec, scan width = 0.500 m/z, sheath gas flow = 30 units, aux gas flow 5 = 5 units and capillary temperature = 220 ºC. 6 7

Results and discussion 8

Calibration curve 9

The representative ELISA curve for SEM (equivalents) in assay buffer is shown in Figure 10 1. Dose responses are expressed as B/B 0 ratio, where B = absorbance at a given 11 concentration of the analyte and B 0 = absorbance in the absence of the analyte in the 12 reaction mixture. The ELISA was performed 6 times over a two month period to assess 13 calibration stability and repeatability. The variation coefficients (CV) calculated for 14 individual calibration points of the standard curve ranged from 7.2 to 14.5 % which 15 indicated good calibration stability. The mean IC 50 value (corresponding to 50 % binding 16 inhibition) for the 6 repeated assays was 0.18 ± 0.035 µg L -1 . Calibration curve generated 17 in buffer was used for all SEM determinations carried out in egg samples in this study. 18 ELISA investigation of 20 blank samples fortified with SEM at a 95 % confidence limit. 28

Based on these experiments, CC β derived from the standard curve generated in assay 29 buffer was determined to be 0.3 µg kg -1 . At this level of fortification, the assay recovered 30 the analyte at values between 0.12 to 0.39 µg kg -1 , fulfilling the maximum permissible 1 false negative rate (5 %). The LoD obtained by ELISA investigation of 20 blank samples 2 was calculated to be 0.13 µg Kg -1 with values ranging from 0.09 to 0.15 µg kg -1 . 3 4 [Insert Figure 2] 5 6

Assessment of assay recovery 7

To ascertain information on matrix interferences and assay variability, a set of blank 8 samples were fortified with the analyte (SEM) at concentrations of 0.3, 1.0 and 3.0 9 µg kg -1 and carried through the ELISA procedure. The results of this study are shown in 10 Table 1. The mean recovery values were calculated from six individual sample 11 measurements at each concentration level. The recovery for 0.3 µg kg -1 of added SEM to 12 egg homogenates was found to be approximately 110 %, whereas recovery values for 1.0 13 and 3.0 µg kg -1 were lower (around 80 %). This difference in recovery can be caused by 14 non-linearity of peripheral parts of calibration curve. Intra-assay CV, calculated from six 15 recovery measurements carried out using one microtitre plate simultaneously, ranged 16 from 7.4 to 28.2 %. Inter-assay CV, obtained from six repeated analysis carried out over a 17 one month period, were calculated to be 13.7 to 18.2 %. 18

[Insert Table 1] 20 21

Specificity 22

Specificity of ELISA detection is determined by the binding properties of the antibody 23 incorporated into the assay system. High specificity of the antibody towards NPSEM 24 allows reduction of interference with structurally related chemicals in a sample, without 25 use of additional clean up steps. Cross-reactivity (CR) values were calculated using 26 equation CR (%) = (50 % binding inhibition by standard (NPSEM) / 50 % binding 27 inhibition by related compound) x 100. CR results for the antibody used in this assay 28 against 18 potential cross-reactants are presented elsewhere [START_REF] Vass | ELISA for semicarbazide 24 determination: Characterisation of antibodies and assay development[END_REF]. No 29 measurable CR was found with SEM, nitrofuran antibiotics, their metabolites, their NP 30 derivatives, pesticides or nonylphenol. Additionally, the negligible assay response to 31

Comparison of ELISA and LC-MS/MS analysis 4

In order to compare the analytical performance of the screening ELISA against the 5 LC-MS/MS reference method, the concentration of derivatised SEM was determined by 6 analysing extracts from eggs using the respective methodologies. Data concerning the 7 LC-MS/MS fragmentation of SEM are presented in Table 2. 8

[Insert Table 2] 10 11

A total of 14 incurred egg samples from 5 different chickens were selected at 12 random, analysed by both ELISA and LC-MS/MS procedures, and compared using the 13 correlation graph in Figure 3. A strong correlation between the ELISA and LC-MS/MS 14 results within the concentration range 0.12 to 20.3 µg kg -1 was found. The determined 15 regression equation for these samples was y = -0.055 + 1.038x (r = 0.992, n = 14). A 16 good correlation between the two methods was also obtained when the results were 17 correlated using low values within the concentration range 0.1 to 1.3 µg Kg -1 . The 18 respective equation y 1 = 0.12 + 0.1513x (r = 0.882, n = 10) shows an underestimation by [START_REF] Szilagyi | Development and validation of an analytical method for 20 the determination of semicarbazide in fresh egg and in egg powder based on the 21 use of liquid chromatography tandem mass spectrometry[END_REF] ELISA in comparison with the broader concentration range. The validation results of the 20 introduced ELISA demonstrated its use as a reliable screening tool for semi-quantitative 21 analysis of SEM in real egg samples. the detection capability of the method. A similar experiment was also performed using 2 five chickens fed with feed containing 30 mg of NFZ per kilogram (Group 2, Data not 3 shown). On average, the time span of contamination in eggs after ingestion of NFZ in low 4 dose chickens was detectable above and including 0.3 µg kg -1 , up to day 40 of the 5 experiment, whereas contamination in high dose chickens was detectable as late as day 6 80 of the experiment. 7 8 [Insert Figure 4] 9 10 Surprisingly, ELISA also detected the presence of SEM (0.32 to 1.0 µg kg -1 ) in 11 five egg homogenates which were collected during and after the medication period (days 12 6 to 16) from the control group of birds (group 1, Figure 4b). Analysis of these samples 13 using LC-MS/MS confirmed the validity of the ELISA findings. The results suggest that 14 a low nitrofurazone concentration could circulate in the air. Ingestion of NFZ by the 15 control chickens that were located in the same room resulted in the bioavailability of 16 SEM in the egg homogenates. Other mechanisms of contamination (i.e. feed, water, 17 faeces or physical contact) were ruled out as possibilities. 18 19 Conclusions 20

The ELISA method described in this study offers a reliable screening tool for the 21 determination of total (extractable and tissue-bound) SEM content in egg homogenates. 22 Sensitivity of the immunoassay was comparable to confirmatory LC-MS/MS technique. [START_REF] Vass | ELISA for semicarbazide 24 determination: Characterisation of antibodies and assay development[END_REF] The method was validated using fortified egg homogenates at levels of 0.3, 1.0 and 24 3.0 µg kg -1 of SEM, satisfying the 1 µg kg -1 EU MRLP requirement for nitrofuran 25

  ), semicarbazide (SEM, hydrazinecarboxamide) hydrochloride 26 and o-nitrobenzaldehyde (o-NBA) were obtained from Sigma-Aldrich (Steinheim, 27 Germany). 3[(2-Nitrophenyl)methylene]-hydrazinecarboxamide (NPSEM) was obtained 28 from WITEGA (Berlin, Germany). Dimethyl sulphoxide (DMSO), horse-radish 29 peroxidase (HRP) and polyethylene-sorbitan-monolaurate (Tween20) were obtained from 30

(

  PBS) containing 145 mmol L -1 NaCl (pH 7.2) for antibody dilution, sample extract and 27 calibration standard preparation. (2) o-NBA solution comprised of 50 mmol L -1 o-NBA in 28 DMSO and 100 mmol L -1 o-NBA in methanol for the derivatisation reaction in ELISA 29 and LC-MS/MS, respectively. (3) 2 mol L -1 NaOH in 0.1 mol L -1 PBS for sample 30

23 24

 23 ELISA validation procedure 25The study was performed in accordance with Commission Decision 2002/657/EC, 26 Chapter 3 of technical Annex (Commission Decision 2002/657/EC 2002), applying 27 selected performance characteristics to validate the immunoassay screening method. 28 Twenty egg samples, 10 from farms in the Brno area and 10 from retail outlets, were 29 collected for validation experiments. Because no measurable SEM residues were found 30 by ELISA and LC-MS/MS analysis, the samples were considered free of SEM. The blank 31

  2.1 x 10 mm), both purchased from Waters. The injection volume (10 µL) 1

19 [

 19 need not be fully quantitative, they should measure the 23 presence or absence of the target analyte at or below regulatory levels. Thus, performance 24 data including the detection capability of the assay were assessed with respect to the 25 declared MRPL (1 µg kg -1 ) for nitrofuran metabolites in edible tissues (Commission 26 Decision 2002/181/EC 2003). Figure 2 demonstrates the assessment of the CC β by 27

  (CR < 0.01 %) eliminated the need for separation of this reagent from the 1 reaction mixture after derivatisation. 2 3

Food

  was utilised to study the depletion of SEM in incurred egg samples 27 collected from experimental chickens for a 90 day period. Results obtained from five 28 chickens fed with feed containing 400 mg of NFZ per kilogram of feed (Group 3) during 29 the medication period (day 1 to 10) are shown in Figure4a. As expected, SEM 30 Page 10 of 22 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk plateau around day 12, followed by a successive decline toward 1

7 PageFigure 1 .

 71 FIGURES LEGEND

Figure 2 .

 2 Figure 2. Determination of CC β in egg homogenates.

Figure 3 .

 3 Figure 3. Correlation of the results for the extracted incurred egg samples analysed by ELISA and LC-MS/MS, respectively. a) Within the concentration range 0 -20 µg kg -1 , n = 14, b) within low analyte concentrations, n = 10.

Figure 4 .Food

 4 Figure 4. Depletion of SEM measured by ELISA in eggs from a) Five chickens fed with meal containing NFZ at a concentration of 400 mg kg -1 (Group 3), b) Un-medicated Group 1 control chickens (n = 2). Bars show the standard deviations for the groups of experimental birds. Days 1 to10 indicated represent the medication period.

  Figure 3

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

spectrometry. Journal of Chromatography B 824:30-5.