Ruediger ; Helling

Anja Mieth

Stefan Altmann

Thomas Joachim Simat

Determination of the overall migration from silicone baking moulds into simulants and food using 1H-NMR techniques

Keywords: Analysis -NMR, Chromatography -HPLC, Chromatography -GC Additives/Contaminants: Silicone, overall migration, Food contact materials, Food simulants Silicones, polydimethylsiloxanes, 1 H-NMR, migration, foodstuffs, cake

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Abstract

Different silicone baking moulds (37 samples) were characterized with respect to potential migrating substances using 1 H-NMR, RP-HPLC-UV/ELSD and GC techniques. In all cases cyclic organosiloxane oligomers with the formula [Si(CH 3 ) 2 -O] n were identified (n = 6…50).

Additionally, linear, partly hydroxyl-terminated organosiloxanes HO-[Si(CH 3 ) 2 -O] n -H (n = 7…20) were found in 13 samples. No other substances than siloxanes could be detected, meaning the migrants mainly consist of organopolysiloxanes. Based on this knowledge, a 1 H-NMR quantification method for siloxanes was established for the analysis of both simulants and foodstuffs. Validation of the 1 H-NMR method gave a suitable performance characteristics: limit of detection 8.7 mg kg -1 oil, coefficient of variation 7.8 % (at a level of 1.0 mg kg -1 food). Migration studies were carried out with simulants (olive oil, isooctane, ethanol (95 %), Tenax) as well as preparation of different cakes. Siloxane migration into cakes only slightly decreased in going from the 1 st to 10 th experiment with a significant dependence on fat content. Migration never exceeded a level of 21 mg kg -1 (3 mg dm -2 ) and thus migration was therefore well below the overall migration limit of 60 mg kg -1 (10 mg dm - 2 ). However, migration behaviour into simulants differed completely from these results.

Introduction

Silicone elastomers are widely used as food contact materials. Because of their non-sticking properties, high temperature resistance and flexible form, their use as baking moulds and pads is very common. Their market share among baking moulds within the last few years was in a range between 23 % and 46 % (GFK Panel Services Germany).

There are only few publications describing the migration behaviour of silicone elastomers in contact with foodstuffs or food simulants. High overall migration from silicone elastomers was reported for foodstuffs containing free available fat (Forrest andSidwell 2005, RAPEX 2007). As to be expected, no notable migration into aqueous or acidic simulants was described so far. [START_REF] Meuwly | Heat stability and migration from silicone baking moulds[END_REF] investigated the overall migration from different silicone baking moulds into modified polyphenylene oxide (Tenax) and obtained values well above the overall migration limit of 10 mg/dm 2 , for the 1 st as well as for the 3 rd migration experiment. Increasing temperatures caused higher migration values, the highest amount reported being 50.5 mg dm -2 . Characterisation of the migrate was carried out using GC-MS and MALDI-TOF and showed cyclic and linear polydimethylsiloxanes up to a molecular weight of 1500 Da as the only components.

Until 2001 silicone elastomers were covered by the Plastics Directive 90/128/EC and had to conform to the overall migration limit of 60 mg kg -1 (10 mg dm experiments despite some others using ethanol (95%), but there is a big uncertainty concerning the interpretation and legal status of these data. Furthermore, there are differences concerning the use of a fat reduction factor as described in Council Directive 85/572/EEC. To clarify this, a comparison of all the fatty food simulants and some real foodstuffs is needed.

Until now, no validated method was published allowing the determination of the overall migration from silicone elastomers into fatty foodstuffs. Hence, there was no possibility to prove the criteria of the Resolution AP ( 2004) 5 in foodstuffs. Furthermore, such a method would be of great importance to assess the suitability of migration tests with olive oil or simulants for silicone elastomers by comparing these results with the amounts measured in et al. 1996[START_REF] Mccamey | Determination of silicone in fats and oils by electrothermal atomic absorption spectrometry with in-furnace air oxidation[END_REF][START_REF] Peters | Do patients with silicone-gel breast implants have elevated levels of blood silicon compared with control patients?[END_REF][START_REF] Kennan | Methods for Detecting Silicones in Biological Matrixes[END_REF], Dorn and Skelly 1994, Biggs et al. 1987). These methods are very specific for silicon but they do not allow one to distinguish between organic and inorganic bound silicon. It is impossible to determine only the content of polydimethylsiloxanes (PDMS) as an extent of migration from silicone elastomers. Several methods based on AAS and ICP-AES techniques for PDMS determination in food after extraction with chloroform or other solvents have been described, with a recoveries from 90-95% and precision of the determination between 1 and 6% (Čavić-Vlasak et al. 1996[START_REF] Mojsiewicz-Pieńkowska | Determination of polydimethylsiloxanes by 1 H-NMR in wine and edible oils[END_REF]. The main drawbacks are possible interferences with inorganic bound silicon and the potential loss of siloxanes with low molecular mass before atomization. A useful, but quite expensive alternative might be the use of HPLC-ICP-AES. This technique would offer the possibility to separate organic and inorganic bound silicon first by liquid chromatography before quantification with ICP-AES.

Infrared spectroscopic methods are specific for organosiloxanes but are little sensitive and need massive sample enrichment. Direct analyses in the lower mg kg -1 range are not feasible (Čavić-Vlasak et al. 1996). The crux is to separate the siloxanes from the big excess of fat in the food extracts which has an almost similar polarity.

The main difficulty of chromatographic methods is the lack of analytical references for cyclic siloxanes in the mid and high molecular range which are essential for a reliable quantification.

For all this reasons, we decided to use 1 H-NMR techniques for quantification. [START_REF] Mojsiewicz-Pieńkowska | Determination of polydimethylsiloxanes by 1 H-NMR in wine and edible oils[END_REF] described a method to quantify linear polydimethylsiloxanes (food additive E 900) in edible oils after saponification and extraction with tetra chloromethane.

The limit of reliable detection was 3 mg l -1 which is well below the expected range for migrating siloxanes from silicone moulds. The advantage of 1 H-NMR quantification is the specific and isolated singlet signal for the [-Si(CH 3 ) 2 -O-] n -group in linear and cyclic siloxanes. Furthermore, even the protons of cyclic oligomers of different size are mainly chemically equivalent, resulting in only one signal for all the oligomers and thus increasing sensitivity. No analytical reference is needed. The highly specific signal for all cyclic siloxanes should also allow a direct measurement, even without prior saponification of the fat. This would make the resulting method significantly less time-consuming and provide the opportunity to use the triglycerides as a unique internal standard.

The objective of this work was therefore to show the applicability of 1 H-NMR (without prior saponification of the fat and enrichment of the siloxanes) to determine the migration from siloxane elastomers in fatty simulants and fatty foodstuffs. Migration into simulants should be compared to migration into real foodstuffs and to the overall migration limit as given in the Resolution AP ( 2004) 5. Repeated use of silicone articles was examined as well as the influence of the fat content of similar foodstuffs.

Materials and methods

Silicone moulds

The characterized silicone moulds were from various manufacturers and were all bought on the German market. More detailed investigations concerning the relation between different simulants as well as food preparation experiments were carried out with a commercial 12 cups muffin silicone mould (volume: 95.8 cm³, contact area: 0.86 dm², thickness: 1.0 mm, Pt catalyzed cross-linked) bought on the German market, too. Single cups were cut from this sample and used for the migration tests.

All samples were first washed with warm water (45-50°C) and washing-up liquid, as recommended by the manufacturers.

Chemicals

Olive oil was purchased from Fluka Germany, Tenax ® (modified polyphenylene oxide, MPPO) from Alltech Germany. Any other chemicals were obtained from Sigma-Aldrich Germany at analytical grade or higher. All food ingredients, used for the baking studies, were of commercial quality.

Silicone samples

In total 37 different silicone moulds were examined: 2 muffin pans with 15 cups, 4 muffin pans with 12 cups, 7 muffin pans with 6 cups, 7 bundform pans ('Gugelhupf'), 5 loaf pans, 3 round cake pans, 2 mini cake moulds for 12 pieces "Madeleine" and 7 motive moulds (Disney characters, Christmas tree, teddy bear). The thickness of all moulds was in the range of 1-2

Migration into olive oil

The

Migration into isooctane and 95% ethanol

The tests were carried out as a threefold determination as described in part 14 of the European Standard EN 1186:2002 with the following modifications: To determine the overall migration into isooctane, the samples were cut into stripes of 3 cm x 5 cm, put into 180 ml of the preheated solvent and then placed in a pre-heated oven at 60°C. For migration tests with ethanol/water 95/5 (v/v), the intact samples were filled to the brim with the pre-heated solvent (90 ml, 60°C). In both cases, only the inside surface area of the moulds was assumed as contact area, although during the isooctane migration the whole surface (inside and outside area) of the moulds was in contact with the simulant. Isooctane leads to an extensive, but even swelling of the complete silicone moulds, also during one-side contact. So it has to be supposed that substances may be able to migrate from the outer to the inner side of the baking moulds, resulting in nearly the same results as to be expected for a two-sided migration. A similar effect is well known e.g. for plasticizers in thin PVC-films [START_REF] Kim | DEHP Migration Behavior from Excessively Plasticized PVC Sheets[END_REF]. Again, time measurement was already started when the samples were put into the oven, although the examination temperature of 60°C was regained later. An exposure time of 50 min for isooctane and 75 min for 95% ethanol was chosen, respectively. The used solvent was refilled to a defined volume (100 or 200 ml) and divided into two portions. About 2/3 of the solvent were used to determine the overall migration by the gravimetric standard method. The remaining part was concentrated by evaporation (rotary evaporator) and refilled to 5 ml with simulant solvent. 100-200 µl of this solution were mixed with 0.5-2 g (± 0.1 mg) of sunflower oil, resulting in a mixture containing about 150-400 mg siloxane per kg sunflower oil. For 1 H-NMR analysis, 200 mg of this mixture were dissolved in 800 µl CDCl 3 , filtered and transferred into a NMR-tube. The 1 H-NMR signals of the simulant solvents, isooctane and ethanol, do not disturb the analysis of the siloxane and triglyceride signal.

Migration into Tenax® (MPPO)

The tests were carried out as described in part 13 of the European Standard EN 1186:2002.

For a threefold determination three flat pieces, each with an area of about 15 cm², were cut from the bottom of the baking moulds. The samples were put into a Petri dish and covered with 0.6 g Tenax® (4 g Tenax®/1 dm²). The Petri dish was closed and then put into the preheated oven at 175°C. As a blank sample, Tenax® was filled into a Petri dish without sample material. Quantification experiments were performed on a Hewlett-Packard 5890 GC with flame ionization detector (FID), again using a HP-5MS capillary column (30 m, 0.25 mm). Injection was splitless using a split/splitless injection system at 280°C. High temperature GC was done at an injection temperature of 330°C, expanding the oven program to 430°C and using a high temperature GC column of adequate polarity (Phenomenex ZB-5HT Inferno, 30 m, 0.25 mm, Phenomenex, Torrance, CA). Increasing the injector temperature up to 350°C resulted in a high septum bleeding but unimproved peak shape or response.

Elemental analysis

Elemental analysis was performed using a EuroVector EA 3000 (HEKAtech, Wegberg, Germany).

Migration into foodstuffs

The baking studies were performed with two different kinds of creamed cake.

Creamed cake, 21 % total fat (Das Backbuch, 1967) The dough contained 125 g sunflower oil, 100 g egg (homogenized), 125 g sugar, 250 g wheat flour, 8 g baking powder and 50 g water.

Creamed cake, 30 % total fat (Backen und Genießen 1999)

The dough consisted of 200 g sunflower oil, 200 g egg (homogenized), 150 g sugar, 200 g wheat flour and 8 g baking powder.

Baking procedure

The samples were filled to the brim with dough and then put into the pre-heated oven at 175°C. After a suitable baking time (25 min for muffins, 40 min for creamed cake in a loaf pan, 45 min for creamed cake in a round cake pan), the samples were removed from the oven.

The cakes were removed from the moulds and each one was put into a beaker.

For the blank samples, the dough was filled into a Teflon coated metal baking mould.

Soxhlet extraction

The cakes were homogenized and an equivalent (approx. 12 g of the cake) was mixed with sodium sulphate (Na 2 SO 4 ) (approx. 15 g) thoroughly in a mortar.

I M M =         ⋅ ⋅ ⋅ ⋅ = ∑ - -

Determination of the recovery rate of cyclic siloxanes in muffins

A baking study with a creamed cake (21 % fat) was carried out using siloxane spiked sunflower oil. As there were only low molecular, volatile cyclic siloxane compounds commercially available, spiking of the sunflower oil was done indirectly. Exactly, the sunflower oil was used to perform an overall migration experiment with a silicone baking mould for 25 min (see "migration into olive oil"). After this, the siloxane amount in the sunflower oil was determined by 1 H-NMR measurement (see "NMR measurement"). The sunflower oil containing the determined amount of siloxanes was then used as ingredient for a creamed cake (21 % fat). The baking study was performed as described in "migration into foodstuffs", using a Teflon® coated metal baking mould. The siloxane content was determined again by NMR in the dried fat extract.

Results and discussion

Characterization of the migration residue

Thirty-seven different baking moulds were characterized with respect to potential migrating substances. Ethanol (95%) or isooctane extracts were all analysed using RP-HPLC with UV and light scattering detection; most of the extracts were analysed by GC-MS and GC-FID, too. To compare the chromatograms of both liquid chromatography techniques Irganox 1076 (octadecyl-3-(3',5'-di(tert-butyl)-4'hydroxyphenyl)propanoate, CAS No. 2082-79-3) was used as an internal standard. Fig. 2 shows two typical corresponding chromatograms of an isooctane migration residue of a muffin mould. Cyclic oligomers below 13 siloxane units are discriminated in light scattering detection due to their relatively high volatility and are better to detect with MS techniques.

"[Insert Figure 2 about here]"

In all cases cyclic organosiloxane oligomers with the formula [-Si(CH 3 ) 2 -O-] n have been identified (n = 6…50). Additionally, linear organosiloxanes (n = 7…20) have been detected in the extracts of 13 moulds in a lower concentration (Fig. 3). This is in accordance with the migrating substances described by [START_REF] Meuwly | Heat stability and migration from silicone baking moulds[END_REF]. No other substances than siloxanes could be observed in a comparable concentration range, meaning the migrants mainly consist of organopolysiloxanes and no other substances should play an important role in the determination of the overall migration. "[Insert Figure 3 about here]" GC-chromatograms indicated the presence of low molecular weight siloxanes only in some moulds in minor concentrations, whereas cyclic siloxanes up from 14 monomer units were detected in all moulds investigated. Oligomers with more than 30 [-Si(CH 3 ) 2 -O-]-units are little volatile and hardly to be seen by GC limiting the applicability of this technique for quantification of the migration residue (Fig. 4).

"[Insert Figure 4 about here]"

To estimate the part of the overall migration which is accessible by GC a calibration was carried out with dodecamethylcyclohexasiloxane (D6) using a flame ionisation detector (FID) in the range of 0.5 to 80 mg/l. Commercial silicone oils with a mid molecular weight of 700 Da (TMS-07) or 1100 Da (TMS-11) quantified under use of the D6 calibration curve gave a recovery rate of 91% and 49% respectively. The ethanol (95%) migration residue of 4 different baking moulds all consisting only of cyclic siloxanes was analysed the same way resulting in recovery rates in the range of 13 -20% compared to the gravimetric results. Thus, determination of the overall migration from silicone moulds by GC in the common temperature range up to 340°C is impossible due to the little volatility of the oligomers.

Further experiments performed with a high temperature GC column, an injection temperature of 360°C and a final oven temperature of 410°C resulted in a recovery rate for silicone oil TMS-11 of 93 % but of only up to 56 % for the ethanolic migration residue of the tested baking moulds. This can be attributed to the high molecular weight of most of the migrating cyclic siloxanes compared to TMS-11 and it limits the applicability of even high temperature GC for determination of the overall migration from silicone food contact materials.

The isooctane extract of one typical muffin mould was more detailed characterized using 1 H-NMR and elemental analysis beside HPLC and GC. The 1 H-NMR spectrum shows a detected. As a result of two-dimensional HMBC-NMR, it could be shown that a somewhat broad peak at 0,13 ppm stands for (CH 3 ) 3 SiO 1/2 -groups (M) beside quaternary (Q) silicium atoms (Fig. 5). These MQ-units are supposed to belong to so called MQ-resins which are widely used by industry as fillers [START_REF] Vincent | Methods of making alkoxylated resin-polymer organosilicone networks and products obtainable therefrom[END_REF]. "[Insert Figure 5 about here]" Such highly branched siloxane compounds, so called MQ-resins, are widely used as an additive in the production of silicone elastomers with high tensile strength.

Elemental analysis of the isooctane migration residue showed an C:H-ratio of 1:3 indicating the presence of CH 3 -groups. The total amount of carbon and hydrogen was 32.40 % and 8.84 %, respectively, lying in the expected range for cyclic dimethylsiloxanes which theoretically contain 32.4 % and 8.2. No hints for any other compounds were found. Elemental analysis of the baking mould itself showed contents of 23.66 % carbon and 5.87 % hydrogen, also resulting in a C:H-ratio of 1:3 indicating CH 3 -groups. The much lower total amount of carbon and hydrogen in baking moulds compared to cyclic siloxanes indicates that there are highly branched siloxanes or methylized silica used as fillers.

Quantification of the 1 H-NMR signals, as described in the "materials and methods" part, resulted in a composition of the overall migration of 89 % cyclic dimethylsiloxanes and 6 % MQ-resins related to the gravimetric result. According to [START_REF] Vincent | Methods of making alkoxylated resin-polymer organosilicone networks and products obtainable therefrom[END_REF], a M:Q ratio of 0.9:1 was assumed for the calculation of the amount of MQ resins. About 5 % of the migration residue remain unexplained. In consequence of the GC-, HPLC-, NMR-and elemental analysis it seems very unlikely that this part is made of organic compounds. We suppose inorganic filling materials, e.g. pyrogenic silicic acid or chemically modified (methylated) silica gel, which are often used for silicone elastomers (BfR 2007).

Since more than 90 % of the migration residue consist of polysiloxanes, the amount of these determined by NMR can be a good approach for the determination of the overall migration. In a following migration experiment, using the same muffin baking mould as for the characterization of the residue, gravimetric results were compared with 1 H-NMR quantification. Because of their little amount and the broad signal the MQ-resins were not quantifiable in the sample solutions and therefore only the signal for the cyclic oligomers was integrated. Keeping in mind that the amount of cyclic oligomers is 89 % of the migration residue table 2 shows good compliance of gravimetric and NMR data. Determination of organosiloxanes can therefore be considered as a good approach for the sum of migrating substances from silicone baking moulds.

"[Insert Table II about here]"

Validation of the 1 H-NMR quantification method

No reference standards representing the migrating substances are commercially available so far. Cyclic silicones are only available up to hexamers (D 6 ) which are much more volatile than the cyclic silicones up to D 50 which we detected in the migrates investigated. Linear silicone oils (e.g. DMS-T07, DMS-T11) are available in higher molecular weight ranges but may exhibit a different mass distribution compared to the migrating silicones and result in additional 1 H-NMR signals for terminal groups.

1 H-NMR-signals increase linearly with the number of the excited protons. In our studies, linearity was demonstrated in the range of 20-750 mg kg -1 fat (7 levels in duplicate, r 2 = 0.9975), using decamethylcyclopentasiloxane (D5) dissolved in sunflower oil as calibrant.

Determination of the most important validation parameters was based on nine baking experiments, each done as a threefold determination, in silicone muffin moulds using a common muffin recipe with a fat content of 21 %. The intra-day standard deviation (s r ) of the method gave 0.92 mg dimethylsiloxanes kg -1 food, which corresponds to a relative standard deviation of 7.3 % (average overall migration of 12.8 mg kg -1 cake). For the uncertainty of measurement concerning the total migrate we have to take into account that the amount of dimethylsiloxanes determined by the 1 H-NMR method recovers only 83 -89 % of the total migrate determined gravimetrically (see table II). All results obtained by the 1 H-NMR method reported here only refer to the amount of dimethylsiloxanes without correction of the bias towards the gravimetry.

The uncertainty of measurement for the reference method using olive oil or one of its substitutes given in Commission Directive 2002/72/EC is set up to 20 mg kg -1 (3 mg dm -2 ). This is much higher than the sum of the estimated bias and the intra-day reproducibility. The limit of detection (quantification) was calculated based on the signal to noise ratio of 3 (9) at 8.7 (26.1) mg kg -1 fat or 1.9 (5.7) mg kg -1 cake. Because of the enrichment of siloxanes during the fat extraction step of the food, this relatively high detection limit is suitable to determine even low levels of siloxane contamination in food and is more than sufficient to prove the overall migration limit in fatty foodstuffs according to

Resolution AP ( 2004) 5.

The recovery was determined as described in the materials and methods part. In an experiment performed in triplicate, a recovery rate of (101.3 ± 6.7) % was reached for extraction of the oil and quantification of the siloxanes by 1 H-NMR. All validation parameters obtained are summarized in table 3.

"[Insert Table III about here]"

Commission Directive 2002/72/EC defines the analytical tolerance for the overall migration experiment into olive oil to 20 mg kg -1 . Compared to this criteria the described 1 H-NMR method is suitable for analysis of the overall migration from silicone baking moulds into food.

Furthermore, the method allows one to examine overall migration from silicone moulds into real foodstuffs as well as into olive oil or its substitutes. The importance is that this approach now gives the possibility of checking compliance of silicone moulds with national law or the standards described in the Resolution AP (2004) 5 as well as enabling an exposure assessment for cyclic siloxanes in prepared foodstuffs to be carried out aiding in toxicological assessment.

Siloxane migration into simulants and muffins

The overall migration into olive oil and its substitutes isooctane, ethanol (95%) and Tenax® Commission Directive 2002/72/EC the 3 rd migration is assumed of legal relevance. As to be seen both solvents overestimate the 3 rd migration value. The hypothesis that the migration test using olive oil is not suitable for silicones due to the swelling (BfR 2006) of the material could not be confirmed. Although little swelling could be observed, it was not in a way making migration results unrealistic or resulting in a scenario not to be expected for the preparation of food. Similar swelling of the material was experienced in the later repeated experiments with different foodstuffs. In contrast to olive oil, isooctane caused an obvious but reversible swelling of the silicone elastomer producing the highest migration values. Tenax®, a highly porous solid, gave a significantly lower overall migration in all 3 experiments, although this tendency weakened from 1 st to 3 rd cycle.

"[Insert figure 6 about here]"

The migration values were compared to the contamination of muffins baked in the same mould. The amounts of siloxanes detected in the muffins using 1 H-NMR are given in table IV.

As expected, we found much lower migration (about 10 mg kg -1 cake) compared to olive oil, due to the less lipophilic character and the texture of the muffin.

"[Insert Table IV about here]"

Migration was about 1/30 and 1/8 of the olive oil result for the 1 st and 3 rd experiment, respectively, demonstrating a completely different migration behaviour into the simulant compared with the food. Therefore, the applicability of the reduction factor of 5 according to Council Directive 85/572/EEC for bakery products is questionable. None of the simulants is suitable for imitating the migration processes in pastries. In a follow-up with 10 baking cycles, we surprisingly found the migration value to be almost constant (figure 7), indicating different migration kinetics compared to the liquid simulants as well as Tenax®. Further experiments with two larger moulds confirmed a nearly constant migration from 1 st to 3 rd test but were not continued to the 10 th migration. More surprisingly, despite a quite different volume/surface ratio of the three compared moulds, all tested cakes contained similar siloxane amounts. More differences could be observed comparing migration values related to the surface of the moulds (table V). These almost similar migration values into creamed cake for moulds of different shape and size may result from a somewhat longer baking time needed in case of larger moulds and thus equalizing the more advantageous volume/surface ratio. As a first conclusion, a single instead the 3 rd experiment, as claimed by law for the evaluation of plastics, could be sufficient to estimate the migration from silicone materials in muffins or cakes. Migration into cake does not differ strongly for moulds of various size making the use of small moulds as favourable as that of larger ones from the consumers point of view.

In addition, we proved the influence of the fat content in the dough using recipes with 21 % and 30 % fat. In both cases, again the results showed no significant difference between the 1 st and 3 rd migration for each fat content but gave a significantly higher migration for the muffins high in fat (figure 8). These results confirm the expected dependence of migration from the lipophilic character of the foodstuff and are therefore in good accordance with the literature (Forrest and Sidwell 2005). Muffins or plain cakes with much more than 30 % fat are hardly to imagine, meaning it is unlikely that the overall migration limit will be exceeded when using It has to be outlined that there is hardly any obvious correlation between the migration data for simulants, even when data are corrected by the reduction factor which has to be applied for cake, and the amount of siloxanes found in cake, neither for the mid nor for the high fat cake. The migration kinetics as well as the amount of migrating substances differ strongly due to the less lipophilic character and the texture of the cake. In conclusion, since the requirements of article 3 of Regulation (EC) No 1935/2004 refer to food and not to simulants and there is to date no adequate testing procedure warranting comparable results between food and simulants it is mandatory to prove compliance only with food. Intake of cyclic siloxanes from cakes was estimated to be 2 mg per portion or less, based on an assumption of a consumption of 200 g cake with 30 % of fat, made in a muffin mould with a quite adversarial volume/surface ratio of about 112 ml dm -2 . This is in the same range as expected for the typical consumption of food (e.g. fruit or vegetable preserves) containing food additive E 900 (a linear polydimethyl siloxane) at its highest legal concentration of 10 mg kg -1 (Council Directive 89/107/EEC). Insofar, there are no toxicological concerns, although a TDI of 1.5 mg kg -1 body weight was established for mid-size linear polydimethyl siloxanes as E 900 only but not for cyclic siloxanes (Synoptic Document 2005).

Conclusion

The method presented here is the first fully validated method to investigate the overall migration of silicone baking moulds into olive oil, substitutes and foodstuffs and therefore provides an essential tool to check the legal compliance of silicone moulds already placed on the market. No other suitable and validated method has so far been published.

Investigating the migration properties of silicone elastomers using gravimetric and 1 H-NMR analysis resulted in very different data depending on the food, simulant or substitute used.

Based on our data, it has to be concluded that the migration test with olive oil, as required by Council Directive 82/711/EEC, does not give a realistic scenario of the migration into cake, even when the official reduction factor is used. This concerns the amount of migration as well as variations from 1 st to 3 rd migration test. There are also big differences between the migration data for olive oil and its substitutes ethanol (95%), isooctane and Tenax® according to Directive 82/711/EEC. Neither the liquid solvents nor Tenax® give similar results to olive oil as easily to expect but presupposed by the migration directives.

Consequently, the olive oil test may act only as a convention in the sense of an inertness parameter for the investigated material. It seems more appropriate to search for a simpler and better reproducible test method for silicone elastomer materials. Based on our results we suggest the much easier and more precise extraction procedure with ethanol (95%) and to focus on the 1 st extract only (with a therefore new defined limit for the total extractives in ethanol). This follows the same consideration as German BfR recommendation XV (BfR 2005) in which limits for both volatile and extractable substances have been defined, but not a migration limit. The discussed 1 H-NMR-method is suitable for examining the legal compliance of silicone articles in food contact as well as for a reliable exposure assessment of consumers. Preparing cake in a silicone mould usually will not result in exceeding the overall migration limit of 60 mg kg -1 or in a relevant contamination of the foodstuff from the toxicological point of view.

Actual investigations of our research group indicate a much higher amount of migrating siloxanes for silicone moulds used for the preparation of meat loafs, even exceeding the overall migration limit. Further experiments are currently in preparation. EN 1186EN :2002)). Reduction factor x/5 (Council Directive 85/572/EEC) is regarded (in contrast to Fig. 6) . Data shown: mean ± confidence interval (α = 0.05), threefold determination (olive oil: single determination).

F

Figure 1 .Figure 2

 12 Figure 1. 1 H-NMR spectrum of a siloxane containing fat extract. The inset shows an expanded scale for the -0.01 to 0.12 ppm region. 1: δ = 0.05 ppm (s; 6 H; Si(CH 3 ) 2 O), 2: 0.85 ppm (m; 3 H; CH 3 aliphatic), 3: 1.18-1.38 ppm (m; 2 H; CH 2 aliphatic), 4: 1.54-1.62 ppm (m; 2 H; -O-CO-CH 2 -CH 2 -), 5: 1.92-2.08 ppm (m; 2 H; -CH 2 -CH=), 6: 2.29 ppm (m; 2 H; -O-CO-CH 2 -), 7: 2.74 ppm (m; 2 H; =CH-CH 2 -CH=), 8: 4.05-4.35 ppm (dd; 4 H; -CO-O-CH(CH 2 -O-CO-) 2 ), 9: 5.25 ppm (p; -CO-O-CH(CH 2 -O-CO-) 2 ), 10: 5.25-5.40 ppm (m; -CH=CH-)

  tests were carried out as described in part 8 of the EuropeanStandard EN 1186:2002 and Commission Directive 2002/72/EC attachment 1 N o 6 with the following modifications: The samples were filled to the brim with a pre-heated amount of olive oil (90 ml, 175°C) and then placed in a pre-heated oven at 175°C. As a blank sample, an equivalent amount of olive oil was poured into a flask and then treated identically. Time measurement was already started when the samples were put into the oven, although the examination temperature of 175°C was regained later. Contrary to repeating the migration experiment three times with one mould for 25 min at 175 °C, a different approach according to Commission Directive 2002/72/EC (Annex I No. 6) was chosen. Three moulds were incubated with olive oil at 175°C for 25, 50 (mimicking 1 st and 2 nd migration) and 75 min (mimicking 1 st , 2 nd and 3 rd migration), respectively. Quantification of olive oil in the extracts of the silicone moulds was carried out by determination of the triglycerides using normal phase HPLC and evaporative light scattering (ELS) detection. Analysis were performed on a Merck HPLC-DAD 7000 coupled with an ELS-detector Sedex 75 (Sedere, Alfortville, France) with the following parameters: chromatographic column: LiChrospher 100 Diol-5 µ, 125 x 3 mm (Merck, Darmstadt, Germany), isocratic elution: 2-methoxy-2-methyl-propane/isooctane 4/96 (v/v), run time: 12 min, flow: 0.5 ml min -1 , injection volume: 20 µl, column temperature: 25°C, nebulization temperature: 20°C, evaporation temperature: 35°C, N 2 3.5 bar, gain: 8, calibration: olive oil dissolved in isooctane. The triglyceride peak showed a retention time of about 5 min.

  Time measurement started when the examination temperature of 175°C was regained, exposure time was 25 min. Gravimetrical determination of the migration residue followed the procedure described in EN 1186:2002 part 13.Qualitative analyses of the migration residue (overall migration into isooctane)For liquid chromatography, an Agilent 1100 G HPLC-DAD (Hewlett Packard, Palo Alto, CA) coupled with an ESI-TOF-MS-detector (Biospectrometry Workstation; Applied Biosystems, Foster City, CA) and a Merck HPLC-DAD 7000 (Merck, Darmstadt, Germany) coupled with an ELS-detector Sedex 75 (Sedere, Alfortville, France) were used. Irganox 1076 (octadecyl-3-(3',5'-di(tert-butyl)-4'hydroxyphenyl)propanoate, CAS No. 2082-79-3) was added to the sample solution as a retention time standard to allow the assignment of the peaks observable in the HPLC-MS and HPLC-ELSD chromatograms. The amount of Irganox 1076 in the sample solution was about 200 mg l -1 . The measurement parameters were as follows: chromatographic column: Multospher 120 RP 18 HP 5 µm 250 x 3 mm (CS-Chromatography Service), gradient elution: isopropanol(A)/methanol(B), starting with 10% isopropanol and reaching 85% within 55 min, flow: 0.5 ml min -1 , injection volume: 10 µl (HPLC-DAD/ELSD) or 25 µl (HPLC-DAD/MS), column temperature: 40°C, ELSD parameters: nebulization temperature: 25°C, evaporation temperature: 30°C, N 2 3 bar, gain: 8, UV: 220 nm. Gas chromatography was done on a Hewlett-Packard 6890 GC (Hewlett Packard, Palo Alto, CA) equipped with a 5973 mass selective detector. A HP-5MS capillary column (30 m, 0.25 mm) was used. The MS detector was operated in full scan mode, scan range m/z 70 -800. Diallylphthalate (c = 5 mg/l) served as an internal standard.

  The dry powder was transferred quantitatively into a Soxhlet cartridge and extracted for 4 h with 150 ml diethyl ether in a Soxhlet extractor to isolate the fat matrix, including the siloxanes. At the end of the extraction, the diethyl ether was removed from the roundbottomed flask by distillation. Before and after extraction, the flask was dried repeatedly for 1h at 103°C until constant weight was reached. The weight of the lipid extract was determined accurate to 0.1 mg.Sodium sulphate had to be annealed for 3 h at 550 °C in a muffle furnace prior to use to remove siloxane impurities lasting from manufacturing. Soxhlet cartridges and filter papers used to clean the mortar and pestle were extracted thoroughly in a Soxhlet extractor with diethyl ether prior to use.For NMR measurement 400 mg of the extracted fat were dissolved in 800 µl CDCl 3, filtered through silicone free paper and transferred into a NMR tube. The 1 H-NMR spectra were recorded at 500 MHz on a DRX 500 P NMR spectrometer (Bruker). Parameter settings were as follows: temperature: 22°C, number of scans: 128, spectral width: 10330 Hz, acquisition time: 3.17 s, resolution: 0.158 Hz per point, frequency sample rotation: 20 Hz, pulse delay: 1 s, pulse angle: 30°. Tetramethylsilane (TMS) was not added as a position standard for the chemical shift. Instead of this, the solvent signal (δ(CHCl 3 ) = 7.25 ppm in relation to TMS) was used to ascertain the position of resonance bands.Calculation of the siloxane contentThe extracted fat mainly consists of triglycerides. They serve as internal standard for quantification. They form a distinct double doublet (dd) resonance signal in a range of δ = 3.95-4.45 ppm (including 13 C satellites). For quantification, the whole double doublet signal is integrated within this range. The signal is caused by the four protons at C1 and C3 of the triglyceride backbone. The cyclic dimethylsiloxanes represent the main part of the overall migrate giving a sharp singlet at 0.05 ppm, which is formed by the six equivalent protons per[-Si(CH 3 ) 2 -O-] unit."[Insert Figure1about here]" Terminal siloxane units in linear hydroxyl terminated siloxanes give a signal at 0.16 ppm (HO-Si(CH 3 ) 2 O-) and 0.11 ppm (HO-Si(CH 3 ) 2 O-Si(CH 3 ) 2 O-). Terminal groups in linear trimethylsilyl terminated siloxanes give a signal at 0.08 ppm (Si(CH 3 ) 3 O-) and 0.03 ppm (Si(CH 3 ) 3 O-Si(CH 3 ) 2 O-). Their influence on the chemical shift of other methyl protons is limited to neighbouring groups, so that most methyl protons in linear siloxanes give a signal (as do cyclic siloxanes) at approximately 0.05 ppm. The amount of siloxane was calculated from the sum of all siloxane integrals in the spectrum. By approximation, the result can be expressed in [Si(CH 3 ) 2 O]-groups, which corresponds to a cyclic siloxane. Using this method of calculation, the molar mass of linear siloxanes, and therefore also the siloxane amount, is slightly underestimated for hydroxyl terminated molecules (18 Da) and overestimated for trimethylsilyl terminated ones (30 Da). The resulting error can be disregarded, knowing that the migrating linear siloxanes range between 700 and well above 2000 Da.

  established using decamethylcyclopentasiloxane(D 5 ) in sunflower oil.Linearity was confirmed (r 2 0.9975) between the amount of siloxanes per kilogram triglyceride (abscissa) and the quotient of the integrals of the siloxane and triglyceride signals (ordinate).The overall migrate can be calculated as follows (without use of the calibration mentioned aboveis the overall migrate [mg kg -1 food], I sil and I TG are the integrals of the siloxane and the triglyceride signal, M sil is the molar mass of the [-Si(CH 3 ) 2 -O-] unit [g mol-1 ] (M sil = 74,2 g mol -1 ), and M TG is the average molar mass of the triglycerides [g mol -1 ] (M TG was calculated from the 1 H-NMR spectrum of the lipid matrix as described below), p sil and p TG are the number of protons responsible for the resonance signal of interest (p sil = 6, p TG = 4), m equ is the mass of the food equivalent [kg], which was used for Soxhlet extraction, m fat is the mass of fat [mg], which was extracted from the food equivalent.Calculation of the surface related migration: is the overall migrate [mg dm -² contact area], m food is the mass of food, which was prepared in the tested silicone mould and A O is the contact area [dm²] between the baking mould and the foodstuff during baking.Determination of M TG from the 1 H-NMR spectrum of the lipid matrix:All signals of the lipid matrix are integrated (see figure1, signal 2-10), integrals of similar subunits of the triglyceride molecule are summed up. Signals 8 and 9 are not fully separated, so they are integrated as one signal for CH-groups. The carboxy group at C2 of the triglyceride backbone is then regarded separately in the calculation (see tableI)."[Insert table I about here]"So the average molar mass of triglycerides in the used sunflower oil is:

  0.06 ppm chemical shift for the chemical equivalent protons of the cyclic oligomers [-Si(CH 3 ) 2 -O-] n . Much smaller signals for the 13 C-satellites at -0.07 and 0.18 ppm (coupling of 1 H-with 13 C-nucleusses; 1 J(C,H) = 115 Hz) and rotation side signals are

  were determined gravimetrically according to the rules set out in the migration directives (Council Directives 82/711/EEC and 85/572/EEC) from the first to the third experiment. The results are shown in figure6. Isooctane, ethanol (95%) and Tenax® are official substitutes for olive oil according to Council Directives 82/711/EEC and 85/572/EEC and therefore should give similar results. However, isooctane generated by far the highest migration giving 209 mg dm -2 in the 1 st experiment, compared with ethanol which gave 25 mg dm -2 in the 3 rd experiment. In analogy to the rules for migration from plastic materials laid down in

  silicone moulds for making a cake. Even without effective compliance control in the past, most of the silicone baking moulds placed on the market should meet the criteria of Resolution AP (2004) 5 and moreover of Article 3b of Regulation (EC) 1935/2004 prohibiting an unacceptable change of the composition of foodstuffs as long as they are used for baking pastries. "[Insert Figure 8 about here]"

  To judge a silicone article with regard to the requirements of article 3 of Regulation (EC) No 1935/2004 migration testing has to be done in food absolutely. This recognition well confirms with other findings proving migration into food to be sometimes completely different compared to migration into simulants as described for isopropylthioxanthone(BfR 2006), plasticizers in gaskets(Fankhauser-Noti 2006) and other (Foodmigrosure 2007).

Figure 3 .Figure 7 .Figure 4 .Figure 8 .

 3748 Figure 3. RP-HPLC-MS/DAD chromatogram of the migration residue from a silicone baking mould containing hydroxyl terminated dimethylsiloxanes (m/z 300-4000)

  Figure 5. 1 H-NMR spectrum of the migration residue

Table II .

 II Overall migration into isooctane and ethanol (95%) determined by 1 H-NMR and gravimetry. Data shown: mean ± confidence interval (α = 0.05), threefold determination.

					1 H-NMR	gravimetry	ratio
	simulant	migrate	w A [mg dm -²]	w B [mg dm -²]	w A /w B
	isooctane	1 st		166.7 ± 10.6	208.1 ± 3.2	0.83
		2 nd			33.3 ± 1.2	38.4 ± 2.3	0.87
	3 rd 1 st 2 nd F ethanol (95 %) o r		10.7 ± 2.9 30.2 ± 1.4 24.7 ± 3.1	12.9 ± 3.2 35.6 ± 1.3 27.7 ± 3.1	0.83 0.85 0.89
		3 rd	P	21.1 ± 3.9	24.3 ± 2.9	0.87
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Table V .

 V Overall migration into creamed cake (21% fat in the dough) from different silicone moulds, determined by 1 H-NMR. Data shown: mean (threefold determination).

	Baking dish	12-cup muffin mould	Loaf pan for cake	round cake pan
	surface/volume ratio	112 ml dm -2	171 ml dm -2	369 ml dm -2
	Baking time	25 min		40 min		45 min
	Temperature Migrate 1 st F [mg kg -1 ] 10.00 o r	175°C [mg dm -²] 1.19	175°C [mg kg -1 ] [mg dm -²] 9.97 1.86	175°C [mg kg -1 ] [mg dm -²] 8.38 2.72
	3 rd	10.11 P	1.21	9.96	1.86	7.99	2.63
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Determination of the overall migration from silicone baking moulds into simulants and food using 1 H-NMR techniques

  In consequence, the Rapid Alert System for Food and Feed (RASFF) of the European Commission shows notifications from EU member states who want to alert control authorities of silicone articles because of a too high overall migration in the olive oil migration test as well as the response of other countries (e.g. Germany) who declare such articles as conforming to national law(RAPEX 2006). Most notifications are on silicone materials exceeding the limit for volatile organic compounds, although it is not proven how far the amount of migration into food depends on the amount of volatile compounds.In the field of food contact materials made of plastics, the Council Directives 85/572/EEC, 82/711/EEC and Commission Directive 2002/72/EC define how to perform migration experiments and which food types and simulants should be used. Baking moulds which are used for making pastries containing free available fat should be tested with olive oil.

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	Resolution AP (2004) 5 on silicones used for food contact applications as well as the y
	recommendation XV on silicone articles, set up by the Federal Institute for Risk Assessment
	(BfR), Germany (BfR 2007). The Resolution AP (2004) 5 limits the overall migration to 60
	mg kg -1 food (10 mg dm -2 ), whereas in the German recommendation XV limits for the loss of
	volatile organic compounds and extractable compounds are set up, each to 0,5 g/100 g

-2 ). With Commission Directive 2001/62/EC, silicone elastomers were excluded from the plastics definition because of their physical properties, which are more similar to elastomers than to other plastics like polyolefins. Still no specific European legislation exists concerning silicone elastomers intended to come into contact with foodstuffs. This gap is filled by the Council of Europe's silicone elastomer. Although the Resolution AP (2004) 5 has only a status of recommendation in most European countries, some introduced it into national legislation. So did the Swiss authorities last in December 2006 (Swiss EDI 2005). Isooctane, ethanol (95%) and Tenax are allowed as alternative simulants if the use of olive oil is impossible for technical reasons. As required for repeat-use articles, only the result of the 3 rd migration experiment is of legal relevance (Commission Directive 2002/72/EC).

Silicone elastomers are not covered by these Directives, so the test procedures described here are not compulsory. On the other hand Resolution AP (2004) 5 declares, that migration tests should be conducted according to these Directives as appropriate, unless technically impracticable due to the nature of the material or the migration tests. Such a case of technical impracticability was stated for the migration test using olive oil. According to the Federal Institute for Risk Assessment (BfR), olive oil is not suitable for silicones due to the swelling of the material

(BfR 2006)

. For that reason, most laboratories use Tenax® for migration

  mm, maximum temperature as given by the manufacturer was from 220 °C to 260 °C. Most of the moulds were blue coloured except three red moulds shaped like Disney characters.Olive oil, isooctane, 95% ethanol and modified polyphenylene oxide (MPPO, Tenax®) were used as test media. The test conditions were based on common time-and-temperature conditions used for baking muffins and were selected according to Council Directive 82/711/EEC chapter 3 N o 1 and table 4. As baking moulds are intended for repeated usage, the first, second and third overall migrate were determined in all cases (Commission Directive 2002/72/EC).

	Migration into food simulants
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Table III .

 III Analytical parameters of the 1 H-NMR determination of dimethylsiloxanes in pastries (data refer to a creamed cake with 21% fat in the dough and are given mass related to avoid the uncertainty of surface calculation).

	Food Additives and Contaminants
	calibrated range	[mg kg -1 triglyceride]	20-750
		[mg kg -1 cake]	5-170
	coefficient of determination (r 2 )		0.9975
	recovery rate	[%]	101.3 ± 6.7*
	relative standard deviation (RSD) [%] [mg kg -1 triglyceride] standard deviation (intra day, s r ) [mg kg -1 cake] limit of detection (LOD) [mg kg -1 triglyceride] F o r	7.3 4,2 0.92 8.7
	(S/N = 3) limit of quantification (LOQ) P (S/N = 9) e e r [mg kg -1 cake] [mg kg -1 triglyceride] [mg kg -1 cake]	1.9 26.1 5.7
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*mean ± confidence interval (α = 0.05, threefold determination) Page 36 of 38 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table IV .

 IV Overall migration into creamed cake (21% fat in the dough) (test conditions: 25 min, 175°C), determined by 1 H-NMR. Data shown: mean (threefold determination). Confidence interval (α = 0.05): 0.15 mg dm -2 (1.00 mg kg -1 ) based on standard deviation s of the procedure: s = 0.11 mg dm -2 (0.92 mg kg -1 ) Comparison of the overall migration into creamed cake (determination via 1 H-NMR) and food simulants (gravimetric determination according to

				overall migration [mg dm -2
		0	2	4	6	8	40	42
	olive oil
	isooctane		siloxane migration
	Migrate ethanol (95%)	[mg dm -²]		[mg kg -1 ]
	Tenax
	1 st		1.19		10.00
	2 nd 3 rd 5 th cake (30% fat) F 1.07 1.21 1.19 r o cake (21% fat)		8.94 10.11 9.98		1 st migrate 3 rd migrate
	10 th	0	1.01 P 20	8.46 60 overall migration [mg kg 40 80 380 -1 ]	400
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] Figure 8.

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

Acknowledgment

The authors would like to thank the Wacker Co. (Burghausen, Germany) for financial support of the studies. We also gratefully acknowledge Dr. Margit Gruner and Annett Rudolph (Technical University of Dresden, Institute of Organic Chemistry) for doing numerous NMR measurements and Dr. Uwe Schwarzenbolz (Technical University of Dresden, Chair of Food Chemistry) for doing the LC-MS measurements.


S G Edwards
email: sedwards@harper-adams.ac.uk

Fusarium mycotoxin content of UK organic and conventional wheat

Keywords: Regression, Risk assessment -modelling, Survey Additives/Contaminants: Mycotoxins -trichothecenes, Mycotoxins -fusarium Food Types: Cereals trichothecenes, deoxynivalenol, nivalenol, HT-2 toxin, T-2 toxin, zearalenone, organic, conventional

Each year (2001)(2002)(2003)(2004)(2005), three hundred samples of wheat from fields of known agronomy were analysed for ten trichothecenes by GC/MS including deoxynivalenol (DON), nivalenol, 3-acetyl-DON, 15-acetyl-DON, fusarenone X, T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol and T-2 triol and zearalenone by HPLC. Of the eleven mycotoxins analysed from 1624 harvest samples of wheat only eight were detected, of these only five, deoxynivalenol, 15-acetyl-DON, nivalenol, HT-2 and zearalenone were detected above 100 µg kg -1 . DON was the most frequently detected fusarium mycotoxin, present above the limit of quantification (10 µg kg -1 ) in 86% of samples, and was usually present at the highest concentration. The percentage of samples which would have exceeded the recently introduced legal limits varied between 0.4% and 11.3% over the five-year period. There was a good correlation between DON and zearalenone concentrations although the relative concentration of DON and zearalenone fluctuated between years. Year and region had a significant effect on all mycotoxins analysed. There was no significant difference in the DON concentration of organic and conventional samples. There was also no significant difference in the concentration of zearalenone between organic and conventional samples however organic samples did have a significantly lower concentration of HT-2 and T-2. Overall, the risk of UK wheat exceeding the newly introduced legal limits for fusarium mycotoxins in cereals intended for human consumption is low, but the percentage of samples above these limits will fluctuate between years.

Introduction

The trichothecene mycotoxins are produced by some of the Fusarium head blight pathogens and head blight severity is dependant on weather conditions. High humidity during and after flowering is conducive to head blight epidemics and mycotoxin production. Deoxynivalenol (DON) and nivalenol (NIV) are Type B trichothecenes produced predominantly by F. culmorum and F. graminearum.

Isolates of both these species are either DON or NIV producers. DON producers are referred to as Type 1 chemotype, this chemotype is further divided into 1A and 1B depending on the acetylated DON that is produce as a co-contaminant, 3-or 15-acetyl DON respectively (Jennings et al. 2004a, Jennings et al. 2004b). F. poae has also been linked to high levels of NIV [START_REF] Pettersson | Nivalenol in Swedish Cereals -Occurrence, Production and Toxicity Towards Chickens[END_REF]. HT-2 toxin (HT2) and T-2 toxin (T2) are type A trichothecenes, which are thought to be produced predominantly by F. sporotrichioides and F. langsethiae [START_REF] Kosiak | The prevalence and distribution of Fusarium species in Norwegian cereals: a survey[END_REF][START_REF] Thrane | Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides[END_REF]).

The predominant fusarium mycotoxin found in UK wheat grain during previous studies was DON. An HGCA survey of mycotoxins in UK wheat grain harvested in 1999 [START_REF] Prickett | Survey of mycotoxins in stored grain from the 1999 harvest in the UK[END_REF][START_REF] Macdonald | Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK[END_REF]) showed levels of DON were below the previous EC monitoring level of 750 µg kg -1 although 58% were above 50 µg kg -1 in what was classed as a year with low head blight. Surveys of cereal products have indicated that fusarium mycotoxins are a common contaminant of human and animal diets, however they usually occur at low concentrations. DON causes reduced feed intake, reduced weight gain and vomiting in farm animals (Anon. 2004a). Nausea, vomiting, diarrhoea, abdominal pain, headache, dizziness and fever have been reported when high concentrations of DON were consumed by humans [START_REF] Anon | Opinion of the Scientific Committee on Food on Fusarium toxins. Part 1: Deoxynivalenol[END_REF]. Other trichothecenes have the same cellular activity which is disruption of protein synthesis, and have a higher cellular toxicity than DON. NIV and T2 are ca. 20 times more toxic than DON, although the relative differences are dependent on the target cell or animal studied [START_REF] Desjardins | Fusarium Mycotoxins: Chemistry, Genetics and Biology[END_REF]). HT2 and T2 were implicated in Alimentary Toxic Aluekia caused by the consumption of cereals which had overwintered in fields in Russia in the 1940s [START_REF] Desjardins | Fusarium Mycotoxins: Chemistry, Genetics and Biology[END_REF].

Although, DON is considered the predominant trichothecene mycotoxin within grain, some of the other trichothecenes have greater toxicity, so it is important that they are also monitored. Of the other trichothecenes, only HT-2 and T-2 toxins are currently being considered for legislation within the EC. There was a previous proposed combined (HT2+T2) maximum limit of 100 µg kg -1 for unprocessed wheat grains.

Zearalenone is another mycotoxin produced predominantly by F. culmorum and F. graminearum. Zearalenone has low cellular toxicity but is problematic as it has high estrogenic activity causing hyperoestrogenism in animals and humans. In animals the mycotoxin causes a range of fertility problems, with young female pigs being particularly susceptible (Anon. 2004b). There are no proven cases of human zearalenone toxicosis but the mycotoxin has been implicated in cases of premature puberty in young females [START_REF] Anon | Opinion of the scientific panel on the contaminants in the food chain on a request from the Commission related to deoxynivalenol (DON) as undesirable substance in animal feed[END_REF].

The European Commission (EC) has set legislative limits for the fusarium mycotoxins including the trichothecene, DON and ZON in cereal grains and cerealbased products intended for human consumption (Anon. 2006b). A combined limit for the trichothecenes HT2 and T2 will be introduced in the near future. The European Commission also set guideline limits in 2006 for fusarium mycotoxins in animal feed (Anon. 2006a).

The presence of relationships between mycotoxin concentrations are important as they allow assumptions for one mycotoxin based on the concentration of another.

For example, legislative limits have not been set for NIV as it is considered by the EC to be a co-contaminant of DON and as such can be controlled by controlling DON (Anon. 2006b).

There have been limited studies comparing mycotoxin concentrations in organic and conventional wheat samples. Many studies failed to identify significant differences between the two practices within Europe [START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF][START_REF] Cirillo | Evaluation of conventional and organic Italian foodstuffs for deoxynivalenol and fumonisins B-1 and B-2[END_REF][START_REF] Champeil | Influence of cropping system on Fusarium head blight and mycotoxin levels in winter wheat[END_REF][START_REF] Jestoi | Levels of mycotoxins and sample cytotoxicity of selected organic and conventional grain-based products purchased from Finnish and Italian markets[END_REF]). However, significantly lower levels of DON in organic samples was detected in some regions of Germany [START_REF] Doll | Fusarium mycotoxins in conventionally and organically grown grain from Thuringia/Germany[END_REF][START_REF] Schollenberger | Fusarium toxins in wheat flour collected in an area in southwest Germany[END_REF][START_REF] Schollenberger | Trichothecene toxins in different groups of conventional and organic bread of the German market[END_REF] although this was not the case in northern Germany in the mid-1990s [START_REF] Berleth | Mould spectrum and mycotoxins (Deoxynivalenol and Ochratoxin A) in grain samples from ecological and integrated cultivated sites[END_REF].

The aims of the present study were: i) to identify the distribution of fusarium mycotoxins in UK wheat, ii) to identify relationships between the concentrations of fusarium mycotoxins detected, iii) to identify how fusarium mycotoxin distributions vary across seasons and regions and iv) to identify if there were differences in the fusarium mycotoxin concentrations of organic and conventional wheat in the UK.

Material and methods

Sample collection

Each year, from 2001-2005, about three hundred grain samples and related agronomic data were collected by agronomists and growers. Samples were collected at harvest from specific fields either from the combine or from trailers leaving the field. Ten approx. 300 g sub-samples were taken from arbitrary points around the field and combined to provide a 3 kg sample. Growers and consultants sent these samples in cotton bags by overnight courier along with agronomic data pertaining to that field sample. A similar number of samples were requested from each of six regions within the UK. These were South, East, Midlands and North of England, Scotland and Northern Ireland. A few samples were collected from South Wales and these were included in the South of England region. A similar number of samples were requested from conventional production using different fungicide sprays at flowering; no spray, azole, strobilurin or azole/strobilurin mixture and from organic samples.

In the first year of the project the target number of samples was not achieved with 283 samples collected (Table I). This was primarily due to the outbreak of foot and mouth disease which resulted in restricted access to farms with livestock in 2001.

A lower proportion of samples was collected from organic farms in 2001 as these were predominantly livestock farms (Table II). A total of 1624 samples were analysed over the five year period (2001)(2002)(2003)(2004)(2005), including 247 organic samples.

Samples were collected from Northern Ireland from 2002 onwards. Numbers of samples collected from Scotland and Northern Ireland were lower than for other regions, this was due to the smaller areas of wheat in these regions (Table I).

On receipt of samples their moisture contents were determined; any samples above 18% moisture content were dried to ca. 15% overnight. A 500 g sub-sample of grain was removed using a ripple divider, dried to 12% moisture content and stored at room temperature for visual assessment. The remaining sample was milled with a 1 mm screen, mixed in a tumbler mixer before three 200 g laboratory samples were collected. One sample was sent to RHM Technology for GC-MS analysis of trichothecenes while a second sample was sent to the Central Science Laboratory for HPLC analysis of ZON. A third sample was held as an archive sample at -20°C.

Mycotoxin analysis

The GC-MS analysis of trichothecenes was performed by RHM Technology (High Wycombe) according to the published method [START_REF] Patel | Survey of ethnic foods for mycotoxins[END_REF]. The trichothecenes analysed for were deoxynivalenol (DON), nivalenol (NIV), 3-acetylDON, 15-acetylDON, fusarenone X, T-2 toxin (T2), HT-2 toxin (HT2), diacetoxyscirpenol (DAS), neosolaniol and T-2 triol. Spiked samples were included in each batch to determine extraction recovery. The UKAS accredited method had acceptable recovery range for each trichothecene of 70-110%. Results were adjusted according to recovery. For this study the calculation of the measurement uncertainty was carried out using in-house data, performance in international collaborative trials and Food Analysis Performance Assessment Scheme (www.fapas.co.uk/fapas.cfm) thus incorporating repeatability and reproducibility data. The expanded measurement of uncertainty was calculated using a standard coverage factor of two, equivalent to a confidence of approximately 95% that the actual level of the mycotoxin being measured lies within the quoted range. The expanded measurement of uncertainty was calculated to be ±25%. The limit of quantification (LoQ) was determined as six times the baseline noise and calculated to be 10 µg kg -1 .

The HPLC analysis of ZON was performed by Central Science Laboratory (York) according to a previously published method [START_REF] Macdonald | Determination of zearalenone in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: Interlaboratory study[END_REF]. As for the trichothecenes, the method was UKAS accredited. Spiked samples were included and results adjusted according to recovery. The recovery range for ZON was 70-110%. For this study the calculation of the measurement uncertainty was carried out using performance in an international collaborative trial [START_REF] Macdonald | Determination of zearalenone in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: Interlaboratory study[END_REF]. The expanded measurement of uncertainty was calculated using a standard coverage factor of two, equivalent to a confidence of approximately 95% that the actual level of the mycotoxin being measured lies within the quoted range. The expanded measurement of uncertainty for wheat flour was calculated to be ±34% at the concentration of 227 µg kg -1 ZON. Similar measurements of uncertainty were achieved for other cereal matrices with ZON concentration ranging from 67 to 143 µg kg -1 . The limit of quantification (LoQ) was calculated to be 5 µg kg -1 .

Statistical analysis

Summary statistics for each mycotoxin were calculated using Excel (Microsoft Office 2003). Statistics calculated included the percentage greater than 10 µg kg -1 (LoQ for trichothecenes), the mean, median, 90th percentile, 95th percentile and the maximum concentration for each mycotoxin detected. For the calculation of the mean, samples below the LoQ were assigned a value of (LoQ)/6, ie 1.667 and 0.833 µg kg -1 for trichothecenes and ZON respectively. This allows data to be directly compared to recently compiled fusarium mycotoxin occurrence data from EU member states [START_REF] Anon | Opinion of the scientific panel on the contaminants in the food chain on a request from the Commission related to deoxynivalenol (DON) as undesirable substance in animal feed[END_REF] For regression analysis samples below the LoQ were removed from the dataset and concentrations log 10 transformed to normalise the variance. For ANOVA samples below the LoQ were assigned a value of (LoQ)/2, ie 5 µg kg -1 for trichothecenes and 2.5 µg kg -1 for ZON, log 10 transformed to stabilise the variance and analysed using Genstat (version 10, Lawes Agricultural Trust). For analysis of deviance of incidence data samples below the LoQ were allocated a value of zero, those above the LoQ were allocated a value of one and analysed using a Bernoulli distribution with Genstat.

Results and discussion

Mycotoxin concentration

Of the eleven mycotoxins analysed only eight were detected, of these DON, 15-AcetylDON, NIV, HT2 and ZON were detected above 100 µg kg -1 and T2, T-2 triol and 3-AcetylDON were detected below 100 µg kg -1 . Table III shows was the next most common fusarium mycotoxin detected although it was never detected at a high concentration (maximum = 430 µg kg -1 ). This would support the decision not to set a maximum limit for this mycotoxin, although NIV should not be considered a co-contaminant with DON, as regression analysis shows the relationship between the two is complex as detailed below. Low levels detected in UK wheat would indicate that the production of NIV in UK wheat is limited. It should be noted that the relationship between pathogens and hosts is a dynamic one, and the current situation may change.

HT-2 and T-2 was detected in 31 and 16% of samples respectively, the concentration was usually low but some samples (0.5%) exceeded the EC previously proposed combined limit for consideration and discussion of 100 µg kg -1 HT2 and T2 (HT2+T2) (Table IV). ZON was quantified in 39% of samples (LoQ = 5 µg kg -1 ), 19% of samples exceeded 10 µg kg -1 . However, due to the lower legal limits for this mycotoxin, more samples exceeded the legal limit for ZON than for DON (Table IV).

It should be noted that the legal limits for DON and ZON in unprocessed cereals include a measurement of uncertainty. Therefore for a consignment of unprocessed wheat intended for human consumption to exceed the legal limit for DON and ZON the concentration as determined by the analytical procedures employed in this project would have to exceed 1563 µg kg -1 DON or 134 µg kg -1 ZON.

T-2 triol, 3-acetylDON and 15-acetylDON were detected in very few samples and always as a low concentration, co-contaminant in the presence of a high concentration of a primary contaminant (HT-2 and DON respectively). Fusarenone X, diacetoxyscirpenol and neosolaniol were not detected in any sample (LoQ = 10 µg kg -1 ).

(Insert Table 3

Regression analysis

The relationship between DON and ZON concentration was analysed by regression analysis of logarithmic transformed values, grouped by year. The regression and year were both highly significant (p < 0.01). The regression was best fitted by separate, non-parallel lines (r 2 = 0.56). Such a relationship could be expected as DON and ZON are produced by the same species, namely F. culmorum and F. graminearum, but isolates of these species may or may not produce DON and/or ZON. The major difference was the higher ratio of ZON to DON in 2004. ZON is known to be produced as the crop senesces at the end of the growing season [START_REF] Matthaus | Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum[END_REF].

It is likely that the relatively high ratio of ZON to DON in 2004 was a result of the delayed, wet harvest that year. An important point to note in Figure 1 is that samples which exceeded 1250 µg kg -1 DON may, or may not have exceeded 100 µg kg -1 ZON, and vice versa. In fact, close to 1000 µg kg -1 DON or 100 µg kg -1 ZON could occur in the absence of detectable quantities of the other. The percentage of samples which exceeded 1250 µg kg -1 DON and 100 µg kg -1 ZON fluctuated each year and the relationship between the two was not stable between years (Table IV). The consequence for the cereal industry is that a single mycotoxin test can not be used as a screen at cereal intake as samples may exceed the legal limit for DON but not ZON or vice versa. It is therefore important that both DON and ZON are analysed in samples which may exceed legal limits for fusarium mycotoxins.

There were no other positive relationships between the concentrations of other commonly detected fusarium mycotoxins detected in UK wheat. In fact both NIV and HT2+T2 show signs of mutual exclusion with DON, ie when one mycotoxin was present at high concentration then the other was low. For HT2+T2 this appears to be simple mutual exclusion (Figure 2); this is probably because HT2+T2 are produced by Fusarium species which appear to have different environmental requirements to F. culmorum and F. graminearum. As a consequence of this relationship, DON concentration cannot be used to predict HT2+T2 concentration. For NIV the relationship is more complex (Figure 3) as: i) NIV and DON are produced by different chemotypes of the same species (F. culmorum and F. graminearum), ii) NIV is produced as a low level co-contaminant by DON chemotypes (and hence there is always some NIV present in samples with high DON concentration) and iii) NIV is culmorum and F. graminearum [START_REF] Xu | Relationship between the[END_REF].

The acetylated versions of DON (3-acetyl and 15-acetyl DON) are cocontaminants of DON which occur at a low percentage of the DON concentration, and as such are only normally detected when DON is present at a high concentration [START_REF] Placinta | A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins[END_REF]). As such legislation that reduces consumer exposure to DON also reduces consumer exposure to acetylated DON in cereals and cereal products intended for human consumption. F. graminearum and F. culmorum DON chemotypes are either 3-acetyl or 15-acetyl DON producers. The plot of acetylated DON against DON (Figure 4) shows that this relationship fits for the majority of samples with no acetylated DON detected when DON concentration was low and a low concentration of acetylated DON when the DON concentration was high. For these samples, the percentage concentration of acetylated DON to DON was between 0.25 and 2.5%. However, Figure 4 shows two anomalies: i) Samples with high DON exist with no detectable acetylated DON suggesting a chemotype of F. culmorum and/or F. graminearum may exist which does not produce an acetylated DON and ii) in five samples (circled in Figure 4), acetylated DON occurred as a high proportion of the total DON concentration with a percentage acetylated DON to DON concentration of 25 to 150%. All five samples were from the 2005 harvest, four samples were from the Midlands and one from the East of England. This anomaly is of concern as legislation is set for DON alone on the assumption that acetylated forms of DON occur as a co-contaminant at a low percentage of DON concentration. Therefore there is a need to monitor acetylated DON as well as DON in cereals and cereal products to ensure that high concentrations of acetylated DON do not occur.

There was a weak relationship between HT2 and T2 concentration (Figure 5).

A relationship between these two mycotoxins would be expected as the two mycotoxins are produced by the same species on the same metabolic pathway. A good relationship over a much higher concentration range was found for HT2 and T2 in UK oats over the same period (Edwards, manuscript in preparation).

(Insert Figure 1 The DON concentration according to practice was analysed using ANOVA. Year and region were entered into the model first to account for temporal and spatial variation.

There was a highly significant interaction (p<0.001) between year and region with an overall trend of lower DON towards the North of the UK (Figure 6). There was no significant difference in the DON concentration between organic and conventional samples (p=0.147). As a consequence of the low incidence of samples with ZON and HT2+T2 above the limit of quantification then the concentration of these samples could not be analysed by ANOVA. Incidence of these two datasets were analysed by analysis of deviance using a Bernoulli distribution. Concentration of ZON and HT2+T2 was then analysed for the subset of samples with concentrations above the limit of quantification using ANOVA. As for DON analysis, year and region were entered into the models first to account for temporal and spatial variation.

For ZON there was a highly significant interaction (p=0.004) between year and region with an overall trend, as for DON concentration, of lower ZON incidence towards the North of the UK (Figure 7A). There was no significant difference in the ZON incidence of organic and conventional samples (p=0.961). For positive samples the ZON concentration was significantly different between years (p<0.001) and regions (p<0.001) although the interaction was not significant (p=0.094) (Figure 7B).

There was no also significant difference in the ZON concentration of organic and conventional positive samples (n=615, p=0.513). Consequently there was no significant difference in the overall concentration of ZON between organic and conventional samples of wheat.

For HT2+T2 there was a highly significant interaction (p<0.001) between year and region with no consistent trend across year or region (Figure 8A). There was a significant difference (p<0.001) in the HT2+T2 incidence of organic and conventional samples. The predicted incidence for HT2+T2 in organic samples was 20% compared to 36% in conventional samples. ANOVA of HT2+T2 concentration in positive samples (n=534) indicated a significant (p=0.017) difference between organic and conventional samples. The predicted concentration of positive organic samples was 19 compared to 23 µg kg -1 HT2+T2 for positive conventional samples. Consequently there was a significant difference in the overall concentration of HT2+T2 between organic and conventional samples of wheat, with lower concentrations present in organic samples.

General discussion

This research has clearly identified the extent to which UK organic and conventional wheat contains fusarium mycotoxins at harvest. Overall, levels of fusarium mycotoxins in UK wheat are lower than those frequently found in continental Europe and in North America [START_REF] Jones | Quality parameters in small grains from Minnesota affected by Fusarium head blight[END_REF][START_REF] Schaafsma | Economic changes imposed by mycotoxins in food grains: case study of deoxynivalenol in winter wheat[END_REF][START_REF] Anon | Opinion of the scientific panel on the contaminants in the food chain on a request from the Commission related to deoxynivalenol (DON) as undesirable substance in animal feed[END_REF]. The majority of samples contained fusarium mycotoxins, the predominant one being DON, however the vast majority of samples had a concentration close to the limit of quantification (10 µg kg -1 ) with only a low percentage of samples exceeding the recently introduced legal limits for DON and ZON for unprocessed wheat intended for human consumption. Numbers of samples exceeding 1250 µg kg -1 DON were between about 0.5 and 5% over the five year period. More samples, between 1 and 10% exceeded 100 µg kg -1 ZON over the five year period HT2+T2 concentrations were generally lower although until legislative limits are set the percentage of samples exceeding new limits cannot be determined. The number of samples exceeding 100 µg kg -1 HT2+T2 varied from 0 to 1% between years. The poor relationship between HT2 and T2 is important as legislation will be introduced as a combined limit for HT2+T2. ELISA assays exist for T2 but these kits cross-react to a low percentage (ca. 10%) with HT2. Consequently, with no consistent relationship between HT2 and T2 in wheat, ELISA assays for T2 are of limited use. Consequently there is no quick or cheap method to allow the industry to monitor these mycotoxins at intake or other critical control points during processing.

NIV was not detected at high concentrations. This maybe because NIVproducing isolates and species of Fusarium are less virulent on wheat than DONproducing isolates [START_REF] Wong | Pathogenicity and Mycotoxin Production of Fusarium Species Causing Head Blight in Wheat Cultivars Varying in Resistance[END_REF][START_REF] Desjardins | Patterns of trichothecene production, genetic variability, and virulence to wheat of Fusarium graminearum from smallholder farms in Nepal[END_REF]). Consequently, without very high levels of initial inoculum, infection can not cause high NIV concentrations in harvested grains. However, NIV is not a co-contaminant of DON, either based on the known chemotaxonomy or based on observational data such as this study, and as such NIV should not be treated as a co-contaminant within the legislation.

Consequently the lack of a requirement for specific legislation for NIV should be based on the estimated low exposure rather than as a co-contaminant. High NIV may occur in different cereals and/or countries and should be monitored accordingly. Acetylated versions of DON did occur as low level co-contaminants of DON in most samples, however five samples were analysed in 2005 with equivalent levels of DON and acetyl DON. These were not of concern as the combined concentration of these mycotoxins was not high. However, it is important to be aware that samples may occasionally contain equivalent concentrations of acetyl DON compared to DON and as such the acetylated versions of DON should be monitored as well as DON itself.

Highest concentrations of DON andZON occurred in 2004. In 2004, most of the UK was drier than average during June, when most wheat were in flower.

However, a wetter than average summer followed for the whole of the UK resulting in high levels of head blight reported at harvest, particularly where harvest was delayed due to continued wet weather. This indicated the importance of weather in late summer, as well as early summer on the final mycotoxin content of grain at harvest.

It is also important to note that the impact of weather late in the season varied between mycotoxins, with relatively higher concentrations of ZON in 2004 compared to DON as shown in Figure 1. Spatial variation of DON across the UK showed that higher DON occurred in the South and East of the UK. There was a consistent decline in DON towards the North in all years, which can be attributed to cooler temperatures. Fusarium graminearum, which is the most potent DON producer, is known to have an optimum temperature for sporulation, growth and infection of ca. 28°C [START_REF] Xu | Effects of environmental conditions on the development of Fusarium ear blight[END_REF]. The higher DON identified in the East may be attributable to the higher intensity of cereals in rotation (Anon. 2007a). The distribution of high DON concentrations closely matches the distribution of F. graminearum across the UK [START_REF] Anon | Planting survey 2007. Home-Grown Cereals Authority[END_REF]. The interaction of year and region is probably due to differences in rainfall distribution across the regions in different years resulting in higher average concentrations in the East in some years and in the South in others. The ZON distribution was a similar pattern to DON which is to be expected as they are produced by the same Fusarium species. The distribution of HT2 and T2 was very different with similar levels across all regions although levels fluctuated inconsistently between seasons and regions.

Previous studies comparing organic to conventional wheat have either failed to identify significant differences between the two practices [START_REF] Berleth | Mould spectrum and mycotoxins (Deoxynivalenol and Ochratoxin A) in grain samples from ecological and integrated cultivated sites[END_REF][START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF][START_REF] Cirillo | Evaluation of conventional and organic Italian foodstuffs for deoxynivalenol and fumonisins B-1 and B-2[END_REF][START_REF] Champeil | Influence of cropping system on Fusarium head blight and mycotoxin levels in winter wheat[END_REF][START_REF] Jestoi | Levels of mycotoxins and sample cytotoxicity of selected organic and conventional grain-based products purchased from Finnish and Italian markets[END_REF] or have detected significantly lower levels of DON in organic samples in some regions of Germany [START_REF] Doll | Fusarium mycotoxins in conventionally and organically grown grain from Thuringia/Germany[END_REF][START_REF] Schollenberger | Fusarium toxins in wheat flour collected in an area in southwest Germany[END_REF][START_REF] Schollenberger | Trichothecene toxins in different groups of conventional and organic bread of the German market[END_REF]. Many previous studies have been limited due to low sample number. This study with 247 organic and 1377 conventional samples distributed across the whole of the UK and over five years is the most robust study to date. Results from this study identified no significant difference in DON and ZON concentration between organic and conventional samples. There are several reasons why organic samples may have lower (eg less cereal intense rotations and less minimum tillage) or higher (eg no fungicides) DON compared to conventional samples based on known impacts of agronomic factors on DON concentration in harvested grain [START_REF] Edwards | Influence of agricultural practices on fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins[END_REF]. It is likely that these factors balance one another resulting in a similar concentration of DON in the two practices. In Germany, there is a higher acreage of maize in conventional rotations which is largely absent from organic rotations. Maize, as a previous crop to wheat, is known to be a major risk factor for high DON concentrations [START_REF] Edwards | Influence of agricultural practices on fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins[END_REF]. This maybe one reason why differences were detected in previous German studies but not in this UK study. The incidence and concentration of positive samples for HT2+T2 were both significantly lower for organic samples. This would suggest that factors that can result in lower HT2+T2 in organic practice out balance any possible negative impacts of organic practice.

However, it is important to note that the differences, although significant, were unlikely to have any relevance regarding the relative food safety of organic compared to conventional wheat and wheat products as the overall incidence and concentration of these mycotoxins were low.

Overall, the risk of UK wheat exceeding the newly introduced legal limits for fusarium mycotoxins in cereals intended for human consumption is low, but the percentage of samples above these limits will fluctuate each season.

F

  the summary statistics for each mycotoxin detected over the five year period. Combined values are provided for HT2 and T2 (HT2+T2) as these closely related mycotoxins have equivalent toxicity and European legal limits will be based on a combined concentration. DON was the most frequently detected fusarium mycotoxin and was usually present at the highest concentration. The distribution was lognormal with a left censor truncated by the LoQ. By comparison of the mean and median values for all other mycotoxins detected indicates they follow a similar lognormal distribution, however the degree of truncation of the distribution at the LoQ varies markedly. NIV

  F. poae, which has different environmental requirements to F.

Figure 1 .

 1 Figure 1. Regressions of ZON against DON concentration (log log plot) for UK wheat for each year from 2001 to 2005 (n = 1624). Regressions fitted for all samples above LoQ. Vertical line represents 1250 µg kg -1 DON and horizontal line represents 100 µg kg -1 ZON

Figure 3 .

 3 Figure 3. Relationship between NIV and DON concentration for UK wheat 2001-2005 (n=1624).

Figure 4 .

 4 Figure 4. Relationship between AcDON (3-and 15-acetyl DON) and DON concentration for UK wheat 2001-2005 (n=1624). Samples with high ratio of acetylated DON to DON are circled.

Figure 5 .

 5 Figure 5. Relationship between T2 and HT2 concentration for UK wheat 2001-2005 (n=1624). Regression fitted for all samples above LoQ.

Figure 6 .

 6 Figure 6. Predicted mean DON concentration for UK wheat from each region in each year. Bars represent 95% confidence limits for predictions.

Figure 7 .

 7 Figure 7. Predicted ZON incidence (A) and mean concentration of positive samples (B) for UK wheat from each region in each year. Bars represent 95% confidence limits for predictions.

Figure 8 .

 8 Figure 8. Predicted HT2+T2 incidence (A) and mean concentration of positive samples (B) for UK wheat from each region in each year. Bars represent 95%confidence limits for predictions.

Table I .

 I Sample distribution by year and region.

		fungal complex causing Fusarium head blight of wheat and environmental
		conditions. Phytopathology 98:69-78. Region
	Year							Total
		South	East	Midlands	North	Scotland N.Ireland
	2001	50	79	63	58	33	0	283
	2002	64	62	93	67	38	19	343
	2003	75	62	69	77	29	16	328
	2004	60	73	93	72	33	13	344
	2005	61	67	81	73	31	13	326
	Total	310	343	399	347	61	164	1624
		o r F o r F o r F o r F o r F o r
			P P P P P P
			e e e e e e
			e r e r e r e r e r e r
				R R R R R R
				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

Table II .

 II Sample distribution by year and practice.

	F o r
	Year	P		Practice		Total
	2001 2002 2003	Organic 17 e 57 61 e r	Conventional 266 286 267	283 343 328
	2004 2005		54 58 R		290 268	344 326
	Total	247	e	1377	1624
	v i e Table III. Concentrations of all fusarium mycotoxins detected in UK wheat in 2001-2005 (1624 samples). w
	DON 15AcDON 3AcDON NIV	% > 10 µg kg -1 86 2.7 1.2 67	Mean a 230 <10 <10 27	Mycotoxin concentration (µg kg -1 ) Median 90th% 95th% 42 368 722 <10 <10 <10 O n l <10 <10 <10 16 64 95 y	Max 20333 217 44 430
	T-2 triol	0.4	<10	<10	<10	<10	45
	T-2	16	<10	<10	13	17	52
	HT-2	31	<10	<10	19	28	193
	HT2+T2	34	<20	<20	30	43	214
	ZON	19	17		<5	27	61	1292

a Based on a value of 1.667 (0.833 for ZON) for all samples below the limit of quantification (10 µg kg -1 ; 5 µg kg -1 for ZON).

Table IV .

 IV Percentage of samples exceeding 1250 µg kg -1 DON, 100 µg kg -1 ZON and 100 µg kg -1 HT2+T2.

	Page 21 of 26	Food Additives and Contaminants
	Year	% > 1250 µg kg -1 DON	% > 100 µg kg -1 ZON	% > 100 µg kg -1 HT2+T2
	2001	0.4	0.7	0.7
	2002	1.7	0.6	0.0
	2003	2.4	1.2	0.6
	2004	5.5	11.3	0.9
	2005 Overall F o r	1.5 2.4	1.2 3.1	0.3 0.5
	P
	e
		e r
		R
		e
		v i e
			w
			O n l
				y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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

Acknowledgements

I would like to acknowledge all growers who supplied samples and the agronomists from the Association of Independent Crop Consultants, Agrovista and the Department of Agriculture and Rural Development in Northern Ireland who co-ordinated collection of samples. Technical support at Harper Adams was provided by Luda Ibrahim, Katerina Pirgozlieva and Rumiana Ray of the Molecular Diagnostics Laboratory and the field trial officers of the Crop and Environment Research Centre. Mycotoxin analysis was conducted by Sue Patel at RHM Technology and Susan MacDonald at CSL, York. Statistical advice was provided by Sandro Leidi at Statistical Services Centre, University of Reading. This research was funded by the UK Food Standards Agency (CO4022) and Home-Grown Cereal Authority (2452).