Liliana J G Silva

Celeste De Matos

Angelina Lopes

Simões Pena

C M Lino
email: cmlino@ci.uc.pt

A Pena

J C Moltó

Occurence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended

Keywords: Canned foods, Cereals, Processed foods, Snack products fumonisins B 1 and B 2, maize, maize derivatives, LC

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fumonisins (FBs) are a group of toxinsfumonisin B 1 (FB 1 ), B 2 (FB 2 ), B 3 (FB 3 ), and other analogues, structurally similar to sphingosine [START_REF] Lerda | Fumonisins in foods from Córdoba (Argentina), presence and genotoxicity[END_REF], mainly produced by Fusarium verticillioides, Fusarium proliferatum and other fungal species of the section Liseola [START_REF] Thiel | Survey of fumonisin production by Fusarium species[END_REF]. According to Bezuidenhout et al. (1988), FB 1 has the structure 2S-amino-12S,16R-dimethyl-3S,5R,10R,14S,15R-penta-hydroxy-eicosane, being C 14 and C 15 hydroxyl groups sterified by a terminal carboxyl group of propane-1,2,3-tricarboxylic acid [START_REF] Sydenham | Preparation and isolation of the partially hydrolysed moiety of fumonisin B1[END_REF][START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF]. The other analogues have different hydroxylation patterns [START_REF] Seo | Natural occurrence of the C series of fumonisins in moldy corn[END_REF].

FBs occur mainly in maize and maize-based foods. FB 1 , followed by FB 2 , is the most abundant and the most toxic [START_REF] Labuda | Identification and enumeration of Fusarium species in poultry feed mixtures from Slovakia[END_REF]. They may affect animal and human health. FB 1 is known to cause equine leukoencephalomalacia (ELEM) and porcine pulmunary edema (PPE). It is also a kidney and liver carcinogen in rats and mice, respectively, causes DNA damage in human fibroblasts, and is immunotoxic [START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF]. The risk of oesophageal cancer in humans in the Transkei region of South Africa and China [START_REF] Lerda | Fumonisins in foods from Córdoba (Argentina), presence and genotoxicity[END_REF], and the possibility that they are connected with neural tube defects (NTD) in South Texas, USA [START_REF] Stack | Analysis of fumonisin B1 and its hydrolysis product in tortillas[END_REF]) have been reported. The International Agency for Research on Cancer (IARC) classified FB 1 as a possible carcinogen to humans, Group 2B (IARC 2002).

Exposure to FBs is worldwide. In general, high levels of FBs are reported in maize or derivatives. Studies from Morocco [START_REF] Zinedine | Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco[END_REF], Brazil [START_REF] Bittencourt | Mycotoxin occurrence in corn meal and flour traded in São Paulo, Brazil[END_REF], Nigeria [START_REF] Bankole | Occurrence of aflatoxins and fumonisins in preharvest maize from south-western Nigeria[END_REF], South Africa [START_REF] Sydenham | Evidence for the natural occurrence of fumonisin B 1 , a mycotoxin produced by Fusarium moniliforme, in corn[END_REF] [START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom[END_REF][START_REF] Cirillo | Natural co-occurrence of deoxynivalenol and fumonisins B 1 and B 2 in Italian marketed foodstuffs[END_REF][START_REF] Molinié | Analysis of some breakfast cereals on the French market for their contents of ochratoxin A, citrinin and fumonisin B1: development of a method for simultaneous extraction of ochratoxin A and citrinin[END_REF]) have been recently published.

In Portugal the area on which maize is cultivated and maize production are 126, 000 ha and 665, 000 Mt, respectively (FAO 2003), moreover between 2002 and 2003 the annual consumption of maize reached 10 kg per capita (INE 2004). Here, as far as we know, only a single investigation of FB 1 and FB 2 in naturally contaminated maize hybrids was reported, revealing a contamination frequency of 100% [START_REF] Doko | Incidence and levels of fumonisin contamination in maize genotypes grown in Europe and Africa[END_REF].

The majority of the reported methods for fumonisin analysis involve an extraction with mixtures of polar solvents [START_REF] Mo | Fumonisins and fungal species in corn from Sonora, Mexico[END_REF][START_REF] Lombaert | Mycotoxins in infant cereal foods from the Canadian retail market[END_REF], a cleanup processes through solid phase extraction (SPE), especially using immunoaffinity columns (IAC) [START_REF] Mo | Fumonisins and fungal species in corn from Sonora, Mexico[END_REF][START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF]) that show higher specificity, or other sorbents [START_REF] Castro | Fumonisins in Brazilian corn-based foods for human consumption[END_REF][START_REF] Bittencourt | Mycotoxin occurrence in corn meal and flour traded in São Paulo, Brazil[END_REF], and derivatization, usually using o-phthaldialdehyde (OPA) [START_REF] Bittencourt | Mycotoxin occurrence in corn meal and flour traded in São Paulo, Brazil[END_REF][START_REF] Bankole | Occurrence of aflatoxins and fumonisins in preharvest maize from south-western Nigeria[END_REF], and naphthalene-2,3-dicarboxaldehyde (NDA) [START_REF] Dombrink-Kurtzman | Fumonisin content in masa and tortillas from Mexico[END_REF][START_REF] Lombaert | Mycotoxins in infant cereal foods from the Canadian retail market[END_REF]. Detection and quantification are mainly accomplished through liquid chromatography (LC) with spectrofluorimetric detection (LC-FD) [START_REF] Bittencourt | Mycotoxin occurrence in corn meal and flour traded in São Paulo, Brazil[END_REF][START_REF] Castro | Fumonisins in Brazilian corn-based foods for human consumption[END_REF]), the most used, or mass spectrometry (LC-MS) [START_REF] Cirillo | Natural co-occurrence of deoxynivalenol and fumonisins B 1 and B 2 in Italian marketed foodstuffs[END_REF][START_REF] Plattner | HPLC/MS analysis of Fusarium mycotoxins, fumonisins and deoxynivalenol[END_REF].

The present study aims to provide data on natural occurrence of FB 1 and FB 2 in maize and maize-based foods for human consumption from the central zone of Portugal, using LC-FD, with pre-column derivatization and confirmation by LC-ESI-MS and to assess

Materials and methods

Chemicals

FB 1 and FB 2 standards, and NDA were obtained commercially from Sigma Chemicals Co (St. Louis, USA). HPLC grade acetonitrile and methanol were purchased from Carlo Erba (Milan, Italy). Acetic acid, hydrochloride acid, sodium hydroxide, potassium chloride, potassium dihydrogenphosphate, anhydrous disodium hydrogenphosphate, and sodium chloride were obtained from Merck (Darmstadt, Germany). Formic acid was purchased from Scharlau Chemie (Barcelona, Spain). Immunoaffinity columns FumoniTest TM were from Vicam (Watertown, USA). Water was prepared from a Milli Q system (Millipore, Bedford, MA, USA).

Phosphate buffer solution (PBS) was prepared from 0.2 g potassium chloride, 0.2 g potassium dihydrogen-phosphate, 1.2 g anhydrous disodium hydrogen-phosphate, and 8.0 g sodium chloride to 990 mL distilled water, adjusted to pH 7.0 with 25% HCl, and the solution was made to 1 L.

Stock solutions, prepared in the FB 1 and FB 2 vials, were made in 1 mL acetonitrilewater (50:50) at 1000 µg mL -1 . Intermediate solutions were prepared at 50 µg mL -1 , diluting 250 µL of stock solution with 5 mL acetonitrile-water (50:50). For fortification assays, one work solution was prepared with acetonitrile-water (50:50) at 5 µg mL -1 for both FBs. For calibration curve with LC-FD, work solutions for calibration were prepared at 50 µg mL -1 and 5 µg mL -1 . The others were prepared by diluting this solution at the following concentrations: 5.0, 2.5, 1.0, 0.5, and 0.25 µg mL -1 . For calibration curve with LC-MS, work solutions for calibration were prepared at 50 µg mL -1 and 25 µg mL -1 . The others were prepared by diluting this solution at the following concentrations: 10, 2.5, 1.0, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 µg mL -1 . All solutions were kept in amber flasks to protect from light.

Sampling

A total of 67 samples were purchased in commercially available size from March to July 2005 from shops, health food stores, and supermarkets located in the city of Coimbra. The following commodities were collected: yellow maize (n=9), white maize (n=2), maize flour (n=3), maize semolina (n=3), maize starch (n=3), sweet maize (n=11), cornflakes (n=16), mixed breakfast cereals (n=4) and maize snacks (n=16).

Except for maize flour, maize semolina and maize starch, the samples were finely milled.

Recoveries

Recoveries of FBs from maize and maize starch were determined by spiking the ground sample of each food, in triplicate, with known amounts of FBs at final concentrations between 100 µg kg -1 and 250 µg kg -1 . Ground samples (25 g) were extracted with 40 mL methanol:water (80:20, v/v), and centrifuged for 15 min at 2500 g. The remaining solid was extracted twice with 30 mL methanol: water (80: 20, v/v) each time. The three extracts were combined and filtrated (Whatman Nº 1 paper). For cleanup, 10 mL of filtrate diluted with 40 mL PBS were filtrated through glass microfiber. Twenty mL were added to a FumoniTest TM IAC attached onto a vacuum manifold. The column was washed with 10 mL PBS, and FBs were eluted with 2 x 1.5 mL methanol, and evaporated under one gentle nitrogen stream at 60ºC.

Extraction and clean-up procedures

LC-FD Analysis

For LC-FD analysis, the residue was reconstituted in 50 µL methanol-water (50:50, v/v) and determination and quantification were carried out on the NDA-derivatives of fumonisins, accordingly to Chu & Li 1994. Therefore, 500 µL 0.05M sodium borate buffer (pH 9.5 adjusted with 1 N NaOH), 500 µL sodium cyanide reagent, and 150 µL NDA reagent (0.5 mg mL -1 in ACN) were added to the reconstituted residue. The mixture was heated for 15 min at 60ºC in a heating bath and cooled to room temperature. The mobile phase (acetronitrile/water/acetic acid 61:38:1 v/v/v) was maintained at a flow rate of 1 mL min -1 . Isocratic analysis under the conditions described above allowed the elution of FB 1 and FB 2 with a mean retention time of 7.36 min and 13.35 min, respectively.

LC apparatus used consisted of a 307 Gilson (Gilson

LC-ESI-MS Confirmation

For LC-ESI-MS confirmation of the presence of fumonisins, the residue was reconstituted in 500 µL methanol-water (50:50, v/v). A Hewlett Packard (Palo Alto, CA, USA) HP-1100 Series LC-MS system equipped with a binary solvent pump, an autosampler, and a MS detector coupled with an analytical work-station was used. The MS detector consisted of a Standard API source that can be configured as APCI (atmospheric pressure chemical ionization) or ES (electrospray ionization). A C 18 -5 µm (150 mm x 4.6 mm i. d.) (Phenomenex, USA) stainless steel column and a guard column LiChrosorb RP-8 (10 x 4.6 mm, 5 µm) were used. The solvents examined as a mobile phase were methanol, and water with acetic or formic acid, using isocratic and gradient conditions.

The analytical separation for LC-ESI-MS was performed using the following gradient:

(a) methanol:0.5% formic acid 75% and (b) water:0.5% formic acid 25%, isocratic for 4 min, then increased to 95% (a) and 5% (b) in 4 min and held for 7 min. Flow rate was maintained at 0.5 mL min -1 . The ESI-MS interface was operated in positive ion mode under the conditions of 350ºC gas temperature, 13.0 L min -1 drying gas flow, 30 psi nebulizer gas pressure and 4000 V of capillary voltage. Mass spectra were obtained by scanning from m/z 300 to 800.

FB 1 (positive ion m/z = 722) and FB 2 (positive ion m/z = 706) were monitored by selected ion monitoring (SIM) with the cone voltages ranging from 60 to 180 V. The highest responses were obtained at 160V for both compounds, thus, this value was chosen for identification and quantification purposes. These conditions allowed the elution of FB 1 and FB 2 with a retention time of 7.5 min and 12.5 min, respectively.

Results and discussion

The calibration curves were obtained using the linear least squares regression procedure of the peak area versus the concentration. The linearity using LC-FD for FB 1 and FB 2 , in the working standard solutions at four determinations of five concentration levels, between 0.25 and 5.0 µg mL -1 , which corresponds to 0.52 ng and 10.4 ng injected, was good as shown by the fact that the correlation coefficients (R 2 ) were 0.984 and 0.994 for FB 1 and FB 2 , respectively. The limit of detection obtained by the NDA derivatization procedure, at a signal-to-noise of 3:1, was 20 µg kg -1 for FB 1 and 15 µg kg -1 for FB 2 .

The proposed analytical method was optimised in order to obtain good accuracy and precision. Recovery values were between 79.0 % for FB 1 , and 99.6 % for FB 2 at fortification levels of 150 and 200 µg kg -1 , respectively. The repeatability varied between 2.7, and 10.4 % for fortification levels at 200 and 100 µg kg -1 , respectively. Samples revealed high contamination levels, between ND and 1569 µg kg -1 for FB 1 and between ND and 457 µg kg -1 for FB 2 . In all groups of studied commodities, FB 2 was present at levels lower than FB 1 , which is usual in other studies [START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom[END_REF][START_REF] Domijan | Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia[END_REF]. Quantitative data for FB 1 and FB 2 determined in the commodities referred above are reported in Table I together with FB 1 /FB 2 concentration ratios.

[Insert Figures 1 and2 about here]

[Insert Table I about here]

In our study, 72.7 % for all maize samples were contaminated with FBs (Table I). This corroborates the only known study in Portugal for FB 1 and FB 2 in nine naturally contaminated maize hybrids, originated from the 1992 crop from Agricultural School of Coimbra [START_REF] Doko | Incidence and levels of fumonisin contamination in maize genotypes grown in Europe and Africa[END_REF]. The mean concentrations in the present study were, for FB 1 , 322 µg kg -1 and 363 µg kg -1 , in yellow and white maize, respectively. For FB 2 , the mean concentrations were 99 µg kg -1 and 275 µg kg -1 , in yellow and white maize, respectively. These values were much lower when compared to those obtained in 1992, 1031 µg kg -1 for FB 1 and 1077 µg kg -1 for FB 2 [START_REF] Doko | Incidence and levels of fumonisin contamination in maize genotypes grown in Europe and Africa[END_REF]. II).

[Insert Table II about here] In maize from Spain the incidence was 87.3 % with a mean contamination level for FB 1 of 4800 µg kg -1 [START_REF] Castellá | Surveillance of fumonisins in maize-based feeds and cereals from Spain[END_REF]. In Morocco, the percentage of positive samples for FB 1 was 50%, being the highest value 5960 µg kg -1 [START_REF] Zinedine | Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco[END_REF]. In maize imported into United Kingdom, the maximum concentration found was 3406 µg kg -1 for FB 1 , and 1268 µg kg -1 for FB 2 [START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom[END_REF]. In Nigeria, FBs were detected in 78.6 % of the maize samples, being FB 1 the predominant toxin [START_REF] Bankole | Occurrence of aflatoxins and fumonisins in preharvest maize from south-western Nigeria[END_REF]). The same happened in Brazil, being FB 1 , and FB 2 present in all of the maize samples [START_REF] Westhuizen | Fumonisin contamination and Fusarium incidence in corn from Santa Catarina, Brazil[END_REF].

In this study, maize flour samples had the highest mean concentration of FB 1 contamination, 822 µg kg -1 with a maximum value of 1569 µg kg -1 being the maximum for total FBs 2026 µg kg -1 . [START_REF] Molinié | Analysis of some breakfast cereals on the French market for their contents of ochratoxin A, citrinin and fumonisin B1: development of a method for simultaneous extraction of ochratoxin A and citrinin[END_REF] also found 1113 µg kg -1 of FB 1 in breakfast cereals from the French market made exclusively from maize flour. In Italy, [START_REF] Cirillo | Natural co-occurrence of deoxynivalenol and fumonisins B 1 and B 2 in Italian marketed foodstuffs[END_REF] found in flour and wholemeal cereals ranges between 10 and 2870 µg kg -1 for FB 1, and 10 and 420 µg kg -1 for FB 2 . It was also observed that all the samples of maize flour from São Paulo, Brazil were positive for FB 1 and FB 2 . Mean values were 2100 and 700 µg kg -1 , respectively for FB 1 and FB 2 [START_REF] Bittencourt | Mycotoxin occurrence in corn meal and flour traded in São Paulo, Brazil[END_REF]. In Argentina, [START_REF] Broggi | Distribution of fumonisins in dry milled corn fractions in Argentina[END_REF] reported mean values of 358 and 122 µg kg -1 for FB 1 and FB 2 , respectively. In this study FB 1 ranged from 109 to 1600 µg kg -1 . In respect to maize semolina, 67 % of the samples were positive for FB 1 , with a mean contamination level of 118 µg kg -1 . [START_REF] Piñeiro | Fumonisins levels in Uruguayan corn products[END_REF] found an average of 105 µg kg -1 for FB 1 , whereas FB 2 was not detected in maize semolina samples from Uruguay.

In Switzerland, [START_REF] Pittet | Occurrence of fumonisin B 1 and B 2 in cornbased products from the Swiss market[END_REF] reported that 61.8 % of maize semolina samples were contaminated with 260 µg kg -1 of FB 1 and 23.6 % contaminated with 100 µg kg -1 of FB 2 . In a study conducted by [START_REF] Broggi | Distribution of fumonisins in dry milled corn fractions in Argentina[END_REF], in Argentina, it was found 135 µg kg -1 of FB 1 and 39.1 µg kg -1 of FB 2 in the 12 analysed maize semolina samples.

FBs were not detected in maize starch. The wet-milling process leads to losses that explain these results. Analysis of the fractions obtained from laboratory-scale wet milling of maize contaminated with fumonisins revealed that the starch fractions did not contain measurable amounts of FB 1 (Fumonisins -JECFA 2001) because of leaching of FBs into aqueous solutions (EMAN Basic Fact Sheet 5 Fumonisins). [START_REF] Piñeiro | Fumonisins levels in Uruguayan corn products[END_REF] also could not detect either FB 1 or FB 2 in the maize starch samples from Uruguay.

Sweet maize samples had the lowest contamination, 64 µg kg -1 in 18 % of the analysed samples, with no FB 2 being detected. The reduction in FBs levels might be due to the migration of fumonisins to the liquid owing to their water solubility [START_REF] Truckess | Immunoaffinity column coupled with liquid chromatography for determination of fumonisin B1 in canned and frozen sweet corn[END_REF], the high temperature used during canning [START_REF] Arranz | Review: HPLC determination of fumonisin mycotoxin[END_REF]) or a more rigorous control in the selection of this maize. [START_REF] Truckess | Immunoaffinity column coupled with liquid chromatography for determination of fumonisin B1 in canned and frozen sweet corn[END_REF] determined the presence of FB 1 in canned and frozen maize and reported that only 38% of the samples were positive, with an average level of 70 µg kg -1 for canned maize and 98 µg kg -1 for frozen maize. The analysis of the sweet maize from the Swiss market showed that FB 1 was detected in 14.3 % of the samples with mean concentration of 70 µg kg -1 while FB 2 was not found [START_REF] Pittet | Occurrence of fumonisin B 1 and B 2 in cornbased products from the Swiss market[END_REF]). In 1997, Piñeiro et al. only detected FB 1 in one of the analysed samples of frozen and canned maize with a contamination level of 65 µg kg -1 .

In respect to breakfast cereals, none of cornflakes and maize-based breakfast cereals samples contained FBs. These results are in agreement with those published by [START_REF] Sydenham | Fumonisins contamination of commercial corn-based human foodstuffs[END_REF] and [START_REF] Pittet | Occurrence of fumonisin B 1 and B 2 in cornbased products from the Swiss market[END_REF]. The first authors analysed, among other samples, cornflakes from South Africa and USA in order to study the FBs contamination of commercial maize-based human foodstuffs and did not find contaminated samples. [START_REF] Pittet | Occurrence of fumonisin B 1 and B 2 in cornbased products from the Swiss market[END_REF] studied the occurence of FB 1 and FB 2 in maize-based products from the Swiss market. Among the 12 cornflakes samples analysed, FB 1 was detected in only one in a contamination level of 55 µg kg -1 , while FB 2 was not detected.

More recently different authors have reported higher contamination levels of FBs in breakfast cereals. The FB 1 range in cornflakes from Uruguay was not detectable -218 µg kg -1 , while FB 2 was not detected [START_REF] Piñeiro | Fumonisins levels in Uruguayan corn products[END_REF]. [START_REF] Park | Analysis of heat-processed corn foods for fumonisins and bound fumonisins[END_REF] found that 14 out of 15 samples of cornflakes and other maize-based breakfast cereals analysed, contained detectable levels of FB 1 with a mean of 67 µg kg -1 (13-237 µg kg -1 ). Two samples also had detectable levels of FB 2 (21-23 µg kg -1 ). [START_REF] Tseng | Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market[END_REF] reported an incidence of FB 1 of 3.5% with a range of 140-1281 µg kg -1 , whereas for FB 2 the incidence was of 17.6% with a range of 120-466 µg kg -1 . [START_REF] Molinié | Analysis of some breakfast cereals on the French market for their contents of ochratoxin A, citrinin and fumonisin B1: development of a method for simultaneous extraction of ochratoxin A and citrinin[END_REF] analysed breakfast cereals, with several ingredients including maize, alone or mixed. The range In general, the levels of fumonisin contamination found in cornflakes cereals in different parts of the world are regarded as low, although occasional samples with high concentrations have been reported [START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF]. One reason for the low levels found in cornflakes and other breakfast cereals is that the grits have the lowest concentration of fumonisins of any other fraction produced in commercial dry-milling of maize [START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF]. Another important reason is that free fumonisins are substantially lost during extrusion cooking of maize grits, extrusion cooking of maize flour, flaking and roasting of maize grits and the overall processing of maize flour to cornflakes [START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF][START_REF] Girolamo | Comparison of different extraction and clean-up procedures for the determination of fumonisins in maize and maizebased food products[END_REF][START_REF] Castells | Fate of mycotoxins in cereals during extrusion cooking: A review[END_REF]. Addition of glucose results in greater reduction of FB 1 on extrusion of maize grits, and during heatprocessing fumonisins are also reduced because of protein binding [START_REF] Kim | Hidden fumonisin in corn-flakes[END_REF].

Regarding snacks, the contamination levels reported by different authors are usually low, which is in agreement with our results. In our study from the 16 analysed samples, only one was found to be contaminated with 170 µg kg -1 of FB 1 and 90 µg kg -1 of FB 2 in a total of 260 µg kg -1 for both fumonisins.

In the USA the total contamination levels of FBs in snacks was lower than 250 µg kg -1 with an incidence of positive samples of 16.6% [START_REF] Pohland | Occurrence of fumonisins in the U.S. food supply[END_REF]. In snacks from Spain, FB 1 was found in a range of 0-200 µg kg -1 , being only 2 samples positive (Sanchis et 2001) reported the presence of 2-65 µg kg -1 of FB 1 in 6 out of 10 maize snacks samples and of 4-8 µg kg -1 of FB 2 in 3 out of 10 samples. Only [START_REF] Tseng | Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market[END_REF] reported higher contamination levels in maize snacks of the Taiwan market. FB 1 , with an incidence of 33%, was found in a range of 0-2395 µg kg -1 , and FB 2 , with an incidence of 21 % was in a ramge of 0-715 µg kg -1 .

As for cornflakes these results might be explained by the extrusion processes that are the base of snacks manufactures [START_REF] Castells | Fate of mycotoxins in cereals during extrusion cooking: A review[END_REF] as under the temperatures reached in normal commercial conditions, FB 1 and FB 2 losses are small [START_REF] Jackson | Effects of baking and frying on the fumonisin B1 content of corn-based foods[END_REF].

Loss of fumonisins may also result from a non-enzymatic browning reaction involving the primary amine in fumonisins and reducing sugars in the food and not from the hydrolysis of FBs [START_REF] Jackson | Effects of baking and frying on the fumonisin B1 content of corn-based foods[END_REF].

One maize flour sample exceeded 1000 µg kg -1 and one sweet maize sample exceeded 400 µg kg -1 , limits proposed by European Commission (Nº856/2005). Two maize samples exceeded the maximum allowable concentration of FB 1 +FB 2 established by Switzerland, 1000 µg kg -1 [START_REF] Shephard | Natural occurrence of fumonisins in corn from Iran[END_REF].

One of the most important aspects of the risk assessment of chemical substances is the determination of the degree of human exposure. Considering the results obtained in the present study, for all samples collected at central zone of Portugal, the average sample contamination of FB 1 was 110 µg kg -1 , and for FB 1 + FB 2 was 140 µg kg -1 . On the other place in the EU rank of total consumption of cereals (INE, 2004). Assuming these, and that an adult body weights 60 kg, the estimated daily intake (EDI) in the Portuguese population for FB 1 and FB 1 +FB 2 in this study was 51 and 65 ng kg -1 body weight per day, respectively, corresponding to 26 and 33 %, respectively, to the estimated total intake of FB 1 in the European diet, 200 ng kg -1 body weight per day [START_REF] Soriano | Intake, decontamination and legislation of fumonisins in foods[END_REF]. The TDI, consumption, and EDIs based on mean level of FB1 and FB 1 +FB 2 of the different commodities are shown in Table III. The EDIs are much lower than the TDI established by European Commission (Nº856/2005).

The tolerable daily intake (TDI) for FB 1 +FB 2 is 2 µg kg -1 body weight per day (EC Nº856/2005). This intake was not exceeded for the most contaminated sample, representing 47 % of the TDI reported by the European Commission (Nº856/2005).

Maize flour made the main contribution to FB 1 ingestion, being the percentage of the estimated daily intake (EDI) related to the estimated total intake (ETI) of the European data of 178 % (Table III).

[Insert Table III about here]

Conclusions

Fumonisins were not detected in samples of maize starch, cornflakes and mixed cereals.

Every other commodity was positive for FB 1 +FB 2 , with mean levels between 16 and 995 µg kg -1 . Only two samples exceeded the limits proposed by European Commission.

Although none of the analysed samples would lead to exceedance of the TDI

  of maize and maize products to Portuguese consumers' dietary exposure to FBs.

  Medical Electronics, Villiers-le-Bel, France) pump model, one 50 µL Rheodyne 7125 injector (Cotati, CA, USA), a C 18 -5 µm Nucleosil 120 KS (30 mm x 4 mm i.d.) guard column, and a C 18 -5 µm Nucleosil 120 (250 mm x 4.6 mm i.d.) column. A Perkin Elmer LS45 spectrofluorimeter (Perkin Elmer, Beaconsfield, UK) operated at an excitation wavelength of 420 nm, and an emission wavelength of 500 nm was used.

  recorded on a 3390 integrator (Hewllet-Packard, Philadelphia, PA).

  Both recovery and RSD values are in accordance with the performance characteristics for FB 1 and FB 2 established by CE 2005/38/EC of 6 June 2005.

  was successfully applied to 67 samples of maize and maizebased foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks. The presence of FBs was verified in 15 samples (22.4%). All the contaminated and suspected samples were re-analysed by LC-ESI-MS, and the presence of FB 1 and FB 2 was confirmed. Figures1, and 2show chromatograms of standards solutions and contaminated samples analysed by LC-FD and LC-MS, respectively.

  in cereals from different countries shows high incidence and contamination levels (Table

  1-1110 µg kg -1 for FB 1 . The most contaminated sample was made exclusively from maize flour. In two samples FB 2 was also detected but not quantified.

  ).[START_REF] Patel | countries and occurrence of fumonisins (FB1 and FB2) in corn-based foods on the Danish market[END_REF] determined a range of 11-220 µg kg -1 of total fumonisins in maize snacks from UK with an incidence of 31/40. In Nordic countries, Peterson & Thorup (

  to data from Portuguese Food Balance, the maize consumption reached, in 2003, 10.19 kg per person per year, which means 27.90 g per person per day, second

  , some samples had contamination levels of concern. Consequently, additional efforts are required to prevent contamination of maize and maize-based products by Fusarium species and subsequent formation of FBs in order to minimize human exposure.

c

  Figure 1 -Liquid chromatography-spectrofluorimetric chromatogram of fumonisin B1 and B2: a) standard; b) contaminated sample (retention time 7.36 and 13.34 min for FB1 and FB2, respectively).

Figure 2 -

 2 Figure 2 -Liquid chromatography-mass detection chromatogram of fumonisin B1 and B2: a) standard; b) contaminated sample (retention time 7.5 and 12.5 min for FB1 and FB2, respectively).

Table II -

 II Occurrence of fumonisins in maize and maize-based foods (continued)

	Sample type	Country	Fumonisins		References
			Incidence	Range (µg kg -1 )
	Breakfast cereals			FB 1 : 1 -1110	Molinié et al. 2005
	Heat-processed maize		HFB1: 100%	HFB 1 : 24 -418	Park et al. 2004
	Cornflakes		FB1: 22/25	FB 1 : 68	Kim et al. 2003
			FB2: 12/25	FB 2 : 8
	Snacks	USA	Total: 1/6	Total: 250	Pohland 1996
	Snacks	Spain		FB 1 : 0-200	Sanchis et al. 1994
	Snacks	UK	Total: 31/40	Total: 11 -220	Patel et al. 1997
	Snacks	Nordic	FB1: 6/10	FB 1 : 2-65	Peterson & Thorup 2001
		countries	FB2: 3/10	FB 2 : 4 -8
	Snacks	Taiwan	FB1: 33%	FB 1 : 0-2395	Tseng & Liu 1997
			FB2: 21%	FB 2 : 0 -715

Table III -

 III Estimated daily intakes (EDIs)ª of fumonisins found in the different studied

		the mean body weight, which was considered 60 kg. b TDI proposed by European Commission
		(2005).
			F o r
			P
			e
		commodities	e r
	Food Maize	TDI b (µg kg -1 bw day -1 ) 2	Consumption (g person -1 day -1 ) 27.9	Estimated daily R intake (EDI) for FB 1 (ng kg -1 bw day -1 ) e v related to % EDI Tolerable daily intake (TDI) from European i e diet 153 7.65 w	% EDI related to Estimated total intake (ETI) from European diet 76.5	Estimated daily intake (EDI) for FB 1 +FB 2 (ng kg -1 b.w day -1 ) 214
	Maize flour Maize semolina	2 2	25.9 c 1.36 d	356 2.7	17.8 0.135 O n 1.35 178 l	431 2.7
	Sweet	2	15.83 e	16.9	0.8	8.4 y	16.9
	maize
	Maize	2	1.99	0.34	0.017	0.17	0.52
	snacks
		ª EDI was calculated using the equation EDI = (∑c) (CN -1 D -1 K -1 ), where ∑c is the sum of
							4

fumonisins residue concentration in the analysed samples (µg kg -1 ), C is the mean annual intake per person, N is the total number of analysed samples, D is the number of the days in a year, and K is

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

Acknowledgements

This work was carried out with the financial support, which is gratefully acknowledged, from the FCT and FEDER/POCTI.


Dr Tanja Goyarts
email: tanja.goyarts@fal.de

Sven Dänicke

Hana Valenta

Karl- Heinz Ueberschär

Carry over of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from naturally contaminated wheat to the pig

Keywords: carry over, deoxynivalenol, Fusarium, pigs, zearalenone 30

The frequent contamination of grains with the Fusarium toxins deoxynivalenol (DON) 2 and zearalenone (ZON) is an important issue in animal and human nutrition. However, 3 data on the exposure of humans to these toxins through consumption of tissues of 4 http://mc.manuscriptcentral.

animals exposed to Fusarium toxins (carry over) are still fragmentary. Therefore, the 5 residues of DON, ZON and their metabolites were determined in tissues and body fluids 6 of pigs (female and castrated male) from a fattening trial which were fed a control (n = 7 6, 0.24 mg DON and 0.009 mg ZON per kg diet as fed) or a Fusarium toxin 8 contaminated diet (n = 12, 6.68 mg DON and 0.056 mg ZON per kg diet as fed) either 9 for ad libitum or restrictive consumption for 12 weeks. After slaughter (96.3 ± 11.6 kg 10 live weight), the concentrations of DON and its metabolite de-epoxy-DON were 11 measured in serum, bile, liver, kidney, musculus longissimus and back fat, while ZON 12 and its metabolites αand β-zearalenol (α-/β-ZOL) were determined in serum, bile and 13 liver. The mean carry over factor of DON + de-epoxy-DON, defined as the 14 concentration of both substances in the tissue/fluid divided by the DON concentration in 15 the diet, for all pigs decreased from bile (0.1046 ± 0.0653) >> kidney (0.0151 ± 0.0070) 16 > liver (0.0057 ±0.0043) > serum (0.0023 ± 0.0018) > muscle (0.0016 ± 0.0016) >> 17 back fat (0.0002 ± 0.0004). The time interval between the end of feeding and slaughter 18 had no consistent effect on DON + de-epoxy-DON concentrations in the analyzed 19 specimen of Fusarium toxin exposed pigs fed restrictively. No transfer of ZON and its 20 metabolites could be observed into serum of pigs, while the mean carry over factors of 21 ZON + α-ZOL + β-ZOL were 0.0094 ± 0.0123 and 4.0 ± 2.2 for liver and bile, 22 respectively. Therefore, it can be concluded that serum is a reliable indicator for a DON 23 exposure, but an inappropriate parameter to deduce on a ZON exposure, which might be 24 better represented by the bile concentration of ZON + α-ZOL + β-ZOL. However, the 25

Introduction

31

In temperate climate zones, the Fusarium toxins deoxynivalenol (DON) and 32 zearalenone (ZON) are common contaminants of cereal crops such as wheat and maize. 33

Due to their stability at high temperatures and during storage, milling, processing and 34 cooking of food and feed, humans and animals are always exposed to both mycotoxins 35 to a certain extent (EFSA 2004a;b). 36

Besides a direct exposure of humans by Fusarium toxin contaminated food, a potential 37 carry over might be possible from tissues of animals which consumed these Fusarium 38 toxins. Among farm animals, pigs are regarded as particularly susceptible to these 39 mycotoxins (Pestka and Smolinski 2005;[START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF]) and may therefore be a 40 good indicator for human health risk. Furthermore, DON was shown to be rapidly and 41 nearly completely absorbed, highly distributed, but poorly metabolized in pigs (Goyarts 42 and Dänicke 2006;[START_REF] Prelusky | Pharmacokinetic fate of 14 C-438 labeled deoxynivalenol in swine[END_REF], resulting in a high systemic availability. ZON 43 and its metabolites are known to undergo an extensive enterohepatic cycling (Dänicke et 44 al. 2005b;[START_REF] Biehl | Biliary 391 excretion and enterohepatic cycling of zearalenone in immature pigs[END_REF]) and may therefore accumulate in the body. As in naturally 45 with Fusarium contaminated foods and feeds both mycotoxins occur concomitantly, the 46 transmission or a possible retention of DON and ZON and its metabolites into edible 47 tissues or body fluids has to be examined. For this reason, we determined the residues of 48 DON and ZON as well as their metabolites in tissues and body fluids of female and 49 castrated male fattening pigs which were exposed chronically (12 weeks) to a Fusarium 50 Furthermore, it was of interest whether the feeding regimen (ad libitum or restricted) or 52 the feeding interval between end of feeding and slaughter had an influence on the tissue 53 toxin concentrations. 54

Materials and methods

55

Animals, diets and experimental procedure 56

The results presented here are part of a study described in more detail by Goyarts et al. 57 (2005). In brief, a diet with a total wheat content of 400 g/kg was formulated to meet or 58 exceed all nutritional requirements of fattening pigs as recommended by the 59 "Gesellschaft für Ernährungsphysiologie" (GfE 1987; Table I). The wheat proportion, 60 either uncontaminated control wheat (control) or wheat infected naturally with 61 Fusarium and contaminated mainly with deoxynivalenol (contaminated; Table II), was 62 constant over the course of the study. 48 crossbred German Landrace x Pietrain pigs 63 were divided into four groups (n = 12, female and barrows one half each) and fed the 64 control or naturally with Fusarium contaminated diet either for ad libitum consumption 65 or restrictively over a fattening period of 12 weeks (study design see Table III). Each 66 animal was housed in a separate pen with free access to water from drinking nipples and 67 was fed individually. The restrictive feeding regimen was characterized by an equal 68 feed intake of all pigs (control and contaminated diet) and was therefore based on the 69 pig with the lowest feed consumption to exclude feed refusal. The daily feed amount 70 was given in two equal portions at 7 a.m. and 2 p.m., while the ad libitum fed pigs had 71 free access to their feed. Grower diets were fed for the first 7 weeks (average live 72 weight range 26 -65 kg) of the experiment, and finisher diets thereafter until slaughter. 73

As the proportion of the contaminated wheat was constant over the course of the study, 74 the toxin concentrations did not vary noteworthy. Therefore, only the finisher diet is slaughter and the end of feed intake (when the through was totally empty) was recorded 79 for the restrictively fed pigs, while the ad libitum fed pigs had free access to their feed 80 until slaughter. Serum, bile, liver, kidney, muscle (M. longissimus 13 th rib) and 81 corresponding back fat were prepared, frozen and stored at -20°C until analysis of 82 mycotoxin residues. 83

Treatments and experiments were conducted according to the European Community 84 regulations concerning the protection of experimental animals and the guidelines of the 85 Regional Council of Braunschweig, Lower Saxony, Germany (File Number 86 509.42502/09-02.02). 87 Samples from wheat batches and diets were analyzed for crude nutrients according to 89 the methods of the "Verband Deutscher Landwirtschaftlicher Untersuchungs-und 90

Forschungsanstalten" (VDLUFA) [START_REF] Naumann | Die chemische Untersuchung von Futtermitteln[END_REF]. Deoxynivalenol in 91 experimental diets was determined by high performance liquid chromatography (HPLC) 92 with diode array detection (DAD) after clean-up with immuno-affinity columns (IAC,93 DONtest HPLC, VICAM, Klaus Ruttmann GmbH, Hamburg, Germany) according to a 94 modified VDLUFA-method described by [START_REF] Valenta | Vergleich einer HPLC-und einer ELISA-461 Methode zur Bestimmung von Deoxynivalenol in Mühlenstäuben, Kleien und Getreide. 462 VDLUFA-Kongreßband[END_REF]. The detection limit was 95 30 ng/g and the recovery was approximately 90 %. Freeze-dried liver, kidney, muscle 96 and fresh back fat samples as well as bile and serum samples were analyzed for DON 97 and de-epoxy-DON by HPLC with UV detection after incubation with β-glucuronidase 98 (EC 3.2.1.31; Type H-2, Sigma-Aldrich Chemie, Taufkirchen, Germany) according to 99 [START_REF] Valenta | Vergleich einer HPLC-und einer ELISA-461 Methode zur Bestimmung von Deoxynivalenol in Mühlenstäuben, Kleien und Getreide. 462 VDLUFA-Kongreßband[END_REF] with modifications as described by Dänicke et al. (2005a) for DON and de-epoxy-DON, respectively. 103 ZON, α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in diets, liver and bile were 104 determined by HPLC with fluorescence detection after overnight treatment with 2 U β-105 glucuronidase [START_REF] Dänicke | Excretion kinetics 407 and metabolism of zearalenone in broilers in dependence on a detoxifying agent[END_REF][START_REF] Ueberschär | Einfluß von Zearalenon auf Wachstum und Rückstände in den 455 Geweben von Mastkaninchen[END_REF]. The detection limit of ZON, α-106 ZOL and β-ZOL was 1, 1 and 4 ng/g, respectively. The mean recovery for ZON, α-ZOL 107 and β-ZOL was 82, 81 and 74 % for feedstuffs; 88, 76 and 66 % for liver; and 92, 81 108 and 104 % for bile, respectively. 109 Further Fusarium mycotoxins were analyzed in wheat samples by the Institute of 110 Animal Nutrition of the University of Hohenheim using a gas chromatography-mass 111 spectrometry (GC-MS) method (Schollenberger et al. 1998). 112 Results of analyses of DON, ZON and its metabolites were not corrected for recovery. 113

Mycotoxin concentrations in physiological samples, which were lower than the above 114 mentioned detection limits, were considered to be zero. Therefore, calculated mean 115 values might be lower than the detection limit. 116

Calculations and statistics 117

Mycotoxin residues of tissues were given as the concentration in fresh samples by 118 considering their dry matter content. The carry over factor was calculated as follows: 119

] kg / g [ diet of ion concentrat toxin ] kg / g [ tissue of ion concentrat

124

Mycotoxin contamination in wheat and diets 125

The control wheat contained only low levels of DON, ZON and further trichothecenes 126 (Table II). In contrast, the wheat contaminated naturally with Fusarium included 127 predominantly DON (16.6 mg/kg) and only small amounts of ZON (0.114 mg/kg) and 128 other trichothecenes such as 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, 129 nivalenol, scirpentriol, and HT-2 toxin (Table II). The Fusarium contaminated diet 130 contained mainly deoxynivalenol (6.68 mg/kg) and little zearalenone (0.056 mg/kg, < 1 131 % of the DON concentration), while the control diet was contaminated to a much lesser 132 extent (0.24 mg DON/kg; 0.009 mg ZON/kg; Table I). 133

Performance and intake of DON and ZON 134

The mean live weight (LW) on the day of slaughter was significantly higher in the ad 135 libitum control (109.8 ± 18.6 kg) and Fusarium contaminated wheat (98.9 ± 9.2 kg) fed 136 pigs as compared to the restricted Fusarium contaminated wheat fed pigs (88.8 ± 2.5 137 kg), while the pigs fed the control diet restrictively had an intermediate live weight of 138 92.2 ± 3.3 kg (Table III). 139

The daily feed intake of pigs in the last week (Week 12, Table III) ranged between 2.9 -140 4.7 kg/d and 3.1 -4.8 kg/d for pigs fed the control or Fusarium contaminated diet ad 141 libitum and was significantly higher than in the restrictively fed pigs (both control and 142 Fusarium contaminated), which showed no variation in their daily feed intake of 2.4 143 kg/d (1.2 kg/meal). For that reason, the daily DON and ZON intake per kg LW of pigs 144 fed the Fusarium toxin contaminated diet ad libitum (Group 2) was significantly 145 increased compared to that of restrictively fed pigs (Group 4, Table III), although the 146 live weight of Group 2 was significantly higher (Table III). The DON intake of the 147 control pigs (Groups 1 & 3) was only 3 %, and the ZON intake only 14 % compared to 149 III). In restrictively fed pigs, the consumption of the control diet appeared to be more 150 rapid than of the Fusarium contaminated diet, which resulted in a greater time interval 151 between the end of feed intake and slaughter for the control compared to the pigs fed 152 contaminated wheat (6.1 h versus 4.8 h, Table III). However, the effect of the time after 153 the end of feeding the Fusarium contaminated diet restrictively on DON + de-epoxy-154 DON concentrations of tissues and fluids was rather inconsistent (Figure I). When the 155 time interval between the end of feeding and slaughter increased, the toxin 156 concentrations of serum, kidney, muscle and back fat appeared to decrease, but to 157 increase in bile, while liver toxin concentrations kept constant (Figure I). However, it 158 has to be noted that the relation of time interval and DON + de-epoxy-DON 159 concentrations of tissues and fluids of Fusarium toxin exposed pigs showed very low 160 correlations (r 2 = 0.011 -0.33). The toxin concentrations of the restricted fed control 161 animals were rather low and were therefore not included in Figure I to maintain the 162 clearness, but they showed also no significant decrease with increasing time interval. 163

Therefore, it has to be emphasized that the toxin concentrations were still rather high 164 after 6-7 hours. 165

Residues of DON, ZON and metabolites 166

DON was only detected in serum samples of pigs exposed to the Fusarium 167 contaminated diet as the parent toxin (Table IV). The DON concentrations in serum of 168 pigs fed the Fusarium contaminated diet were significantly higher than of control 169 animals (Figure III, Table IV). However, there was a high deviation between the 170 individuals at higher daily DON intake (Figure III), indicating that someone could not 171 predict on the level of exposure after analyzing a serum sample. In muscle and back fat 172 no or only marginal de-epoxidation of DON was found (Table V). In contrast, the mean 173 the toxin concentrations were significantly higher in bile, liver, kidney and muscle of 176 pigs fed the Fusarium contaminated diet, the carry over of the sum of DON + de-epoxy-177 DON into bile, liver and kidney was significantly higher in control pigs (Figure II,178 Tables IV &V). The carry over of DON + de-epoxy-DON decreased in following 179 order: bile >> kidney > liver > serum > muscle >> back fat, as only negligible amounts 180 of DON could be found in back fat (Table V). The DON + de-epoxy-DON 181 concentrations of liver and bile were positively correlated (r = 0.847), whereby the liver 182 concentration increased by 0.04 µg/kg with an increase of 1 µg/l bile. 183

No ZON, α-ZOL or β-ZOL was detectable in serum of pigs independent of their 184 exposure to Fusarium contaminated feed. Residues of ZON and its metabolite α-ZOL 185 were mainly found in bile, while β-ZOL could not be detected (Table VI). The mean 186 carry over factor (averaged over all groups) for α-ZOL + ZON in bile was 4.0 ± 2.2. In 187 liver, only the metabolite α-ZOL was detected at low amounts with a mean carry over 188 factor of 0.0094 ± 0.0123 (Table VI). In any case, the increase of ZON + α-ZOL + β-189 ZOL in bile was accompanied by an increase of liver toxin concentrations (r = 0.644). 190

The variations in toxin intake and toxin concentrations in physiological matrices caused 191 by all pigs were used for a regressive evaluation of the data. When the daily DON 192 intake increased by 1 µg/kg LW, the DON concentration increased by 0.11 µg/kg fresh 193 liver and 1.95 µg/l bile, respectively (Figure IV A). After an increase of the daily ZON 194 intake by 1 µg/kg LW, the ZON, α-ZOL and β-ZOL residues in liver and bile will be 195 increased even more by 0.33 µg/kg and 61.78 µg/l, respectively (Figure IV B). 196

However, it has to be emphasized that the low control values contribute too much to the 197 significance of the correlation and that therefore the high variation at higher toxin intake 198 levels may be overlooked. 199 positively correlated in liver and bile, respectively. However, it has to be noted that the 201 mean carry over factor of ZON + metabolites into bile and liver was 38.6 and 1.6 times 202 higher, respectively, compared to the DON + de-epoxy-DON carry over. 203

Discussion

204

DON and de-epoxy-DON 205

In the present study, the carry over factor of DON + de-epoxy-DON decreased from bile 206 >> kidney > liver > serum > muscle >> back fat of fattening pigs fed a control (0.24 mg 207 DON/kg diet) or a Fusarium contaminated diet (6.68 mg DON/kg diet) over 12 weeks. 208

In agreement with our results, [START_REF] Schneeweis | 447 Influence of organically or conventionally produced wheat on health, performance and 448 mycotoxin residues in tissues and bile of growing pigs[END_REF] found decreasing DON residues 209 in the bile > liver > muscle of growing pigs following dietary exposure to different 210 varieties of wheat cultivated under organic and conventional conditions. Accordingly, 211 tissue distribution of pigs following an intravenous DON bolus of 1 mg/kg LW showed 212 the ranking order: urine > bile > kidney > plasma > liver >> other tissues (Prelusky et 213 al. 1991). Nervertheless, it has to be pointed out that oral and intravenous DON 214 exposure are only limited comparable due to the differences in absorption and 215 metabolism [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol 427 (DON) from naturally contaminated wheat for the pig[END_REF]. For example, [START_REF] Prelusky | Pharmacokinetic fate of 14 C-438 labeled deoxynivalenol in swine[END_REF] collected 216 blood, urine, bile and faeces from pigs over 24 h after an intragastric dose of 0.6 mg/kg 217 LW or an intravenous dose of 0.3 mg/kg LW of radio-labeled [ 14 C]deoxynivalenol. 218 Following the intravenous dosing, an average of 3.8, 93 and <0.3 % of the administered 219 dose were recovered in bile, urine and faeces, respectively. After oral dosing, only 68 % 220 of the recovered dose was found in the urine, but 20 % in the faeces, while the bile 221 contained still 2.5% [START_REF] Prelusky | Pharmacokinetic fate of 14 C-438 labeled deoxynivalenol in swine[END_REF]. 222 It has to be mentioned that the carry over factor calculated in the present investigation 223 might be higher as calculated from preceding studies, as the total DON (unconjugated (Goyarts and 227 Dänicke 2006). Furthermore, the less toxic metabolite de-epoxy-DON (EFSA 2004a) is 228 included into the carry over estimation, although it might not be toxicological important 229 for consumers. On the other hand, it was shown that the de-epoxidation is probably an 230 acquired ability of pigs [START_REF] Eriksen | Absorption, metabolism and excretion of 423 3-acetyl DON in pigs[END_REF][START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol 427 (DON) from naturally contaminated wheat for the pig[END_REF]. Therefore, the 231 carry over of DON in pigs lacking this ability might be at least as high as the calculated 232 carry over factor including the metabolite de-epoxy-DON as the total recovery of DON 233 in urine and faeces decreased when de-epoxy-DON was detected (Goyarts and Dänicke 234 2006). 235

Although the acetylated derivates of DON can be rapidly converted to DON in pigs 236 [START_REF] Eriksen | Absorption, metabolism and excretion of 423 3-acetyl DON in pigs[END_REF]), these derivates have not been included in the calculations on carry 237 over because the proportions of 15-acetyl-DON and 3-acetyl-DON amounted just 0.6 238 and 0.2 % of the total DON intake, respectively. 239 Because of the low correlation of the time interval between the end of feeding and 240 slaughter, and the DON + de-epoxy-DON concentrations in the analyzed specimen of 241 pigs fed the Fusarium contaminated diet restrictively, a reliable conclusion for fasting 242 before slaughter can not be made (Figure I). However, in practice the fasting interval 243 before slaughter is probably longer (fasting before transport, transport (up to 6 h), 244 slaughter) than in the present study (1.7-6.9 h), and may result in a greater influence on 245 the toxin levels in tissues of pigs. Nevertheless, pigs which fasted up to 6-7 h in the 246 present study showed still rather high toxin values (Figure I). In the view of human risk 247 assessment, further research is necessary to estimate the impact of fasting interval. 248

Regarding the serum DON concentrations of piglets fed Fusarium contaminated maize 249 [START_REF] Döll | Effects of graded levels of Fusarium toxin contaminated maize in 411 diets for female weaned piglets[END_REF]2005), the carry over factor was in the same range as in the present concentrations resulted in a decreased carry over factor. In the present study, this was 252 also observed for the carry over into serum of pigs, but the maximum carry over factors 253 of DON and de-epoxy-DON into bile, liver and kidney seemed to be higher in the 254 control pigs. The question of why the DON transfer may be higher in control pigs can 255 not be sufficiently answered from the present data, but it may be the misleading result 256 of relatively higher failures in analyses at low mycotoxin levels. However, calculated 257 from the data of Dänicke et al. (2005a), the carry over factors of bile and liver (0.0338 258 and 0.0017) of gilts fed 6 mg DON were considerably lower than in the present study. 259Accordingly, the concentrations and median carry over factors of DON + de-epoxy-260 DON in bile and liver of sows in the last third of gravidity after consuming a Fusarium 261 contaminated diet of 9.57 mg DON/kg for 35 days [START_REF] Dänicke | On 394 the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from 395 the sow to the piglet during the last third of gestation[END_REF] were about 10 262 times lower compared to the current results, indicating that the systemic availability, 263 metabolism, distribution and retention of this toxin are influenced not only by species 264 but also by sex, age and category. Since no [START_REF] Dänicke | On 394 the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from 395 the sow to the piglet during the last third of gestation[END_REF] or only negligible 265 (present study, [START_REF] Prelusky | Nonaccumulation of residues in swine tissue 442 following extended consumption of deoxynivalenol-contaminated diets[END_REF] amounts of DON were detected in back 266 fat, a considerable displacement of toxin to this deeper compartment as discussed by 267 [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol 427 (DON) from naturally contaminated wheat for the pig[END_REF] can probably be ruled out. Furthermore, the intoxication of 268 humans with DON by back fat, muscle or liver from exposed animals appeared to be 269 unlikely, as a person of 70 kg would have to eat more than 8.7, 3.4 or 1.9 kg per day of 270 each tissue, respectively, considering the highest amount measured in the present study 271 and the tolerable daily intake (TDI) of 1 µg DON/kg LW. On the other hand, the TDI of 272 1 µg DON/kg LW would be achieved after consumption of 572 g kidney or 4.2 g of the 273

Fusarium contaminated wheat. 274

The high concentrations of DON in kidneys in the present study are in accordance with 275 data after an intravenous DON application [START_REF] Prelusky | Tissue distribution of deoxynivaleol in swine dosed 440 intravenously[END_REF] 14 metabolite in all specimen of gilts analyzed and reported that the proportion of de-303 epoxy-DON of the sum of DON + de-epoxy-DON appeared to be higher in liver (~ 28 304 %) and bile (~ 35 %) compared to serum (~ 16 %) and urine (~ 15 %). Thereby, the 305 degree of metabolization of DON to de-epoxy-DON was not influenced by increasing 306 dietary DON concentrations (0.21, 3.07, 6.1, 9.57 mg DON/kg, Dänicke et al. (2005a)). 307 However, it should not be overlooked that the toxin concentrations in serum, bile and 308 tissues varied considerably even in the restrictively fed pigs, indicating that beyond the 309 DON intake the individual variation in absorption, distribution, metabolism and 310 excretion of DON has a major impact on the toxin concentrations in analyzed specimen. 311 Therefore, the serum DON concentration of several pigs can give an advice for a DON 312 exposure which exceeds a "threshold", but it seems unlikely to predict the level of toxin 313 exposure. This threshold with higher tissue and body fluid DON concentrations than the 314 normal background should be determined in further dose-response residue studies. 315

ZON and metabolites 316

In the present experiment, ZON and its metabolites were transferred into the bile and 317 liver with mean carry over factors of 4.0 and 0.0094, respectively, while no toxins could 318 be found in the serum of pigs. The accumulation in the bile is probably caused by 319 enterohepatic cycling of ZON and its metabolites [START_REF] Biehl | Biliary 391 excretion and enterohepatic cycling of zearalenone in immature pigs[END_REF]Dänicke et al. 320 2005b). ZON administered orally to 10-to 14-week-old pigs was rapidly and efficiently 321 absorbed and metabolized, reaching maximum plasma concentrations by 2-3 h. 322 Furthermore, the elimination half-life of ZON in plasma of pigs dosed intravenously 323 with 5 mg [³H]ZON/kg LW resembled that of pigs exposed orally to 10 mg [³H]ZON/kg 324 LW, while after bile removal the half-life decreased to < 4 % [START_REF] Biehl | Biliary 391 excretion and enterohepatic cycling of zearalenone in immature pigs[END_REF]). On the 325 other hand, the route of ZON exposure appeared to affect the way of excretion, as the 326 urinary recovery was 57.4 and 45.4 and the faecal recovery 6.6 and 21.7 after 327 metabolite pattern varies not only between the specimen examined, but also between the 356 experiments or pig categories, as the proportions of ZON, α-ZOL and β-ZOL were 46, 357 52 and 2 %, respectively, in the bile of exposed gravid sows [START_REF] Dänicke | On 394 the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from 395 the sow to the piglet during the last third of gestation[END_REF], and 358 71, 25 and 4 %, respectively, in the bile of piglets [START_REF] Accordingly | found high variations in the DON content of bile fluids 292 but strong dose-response relationships in the serum of piglets fed graded levels of 293 Fusarium contaminated maize. 294 In the present study, no de-epoxy-DON was detected in serum, muscle and back fat of 295 pigs. Furthermore, the degree of metabolization of DON to de-epoxy-DON was 296 relatively low with approximately 15, 8 and 7 % in bile, liver and kidney of Fusarium 297 toxin exposed pigs, respectively. Accordingly, de-epoxy-DON was not detected in 298 serum and liver, but in bile (20 %) and urine (14 %) of Fusarium toxin exposed (9.57 299 mg DON/kg) gravid sows (Dänicke et al. 2006)[END_REF]. While the 359 conversion to β-ZOL is considered to be a detoxification of ZON, α-ZOL showed a 360 higher binding activity to the oestrogen receptors. Therefore, the high susceptibility of 361 pigs to ZON is ascribed to the high conversion to α-ZOL in this species (EFSA 2004b). 362

Conclusions

363

In fattening pigs, the carry over of DON + de-epoxy-DON decreased from bile >> 364 kidney > liver > serum > muscle >> back fat, with higher carry over factors into bile, 365 liver and kidney of control pigs compared to the toxin exposed. Serum appeared to be 366 the most reliable indicator of a chronic DON exposure, as only the Fusarium toxin 367 exposed pigs showed DON values in serum. The time interval between the end of 368 feeding and slaughter had no consistent influence on the DON + de-epoxy-DON 369 concentrations of the analyzed specimen of pigs fed the Fusarium contaminated diet 370 restrictively, but it has to be emphasized that after 6-7 hours the DON + de-epoxy-DON 371 concentrations were still rather high. From the perspective of human risk assessment, no 372 reliable advice can be made from the present data for the fasting time before slaughter. 373 However, the low carry over factors of far below 1 indicated that there is no 374 accumulation of DON in these tissues. In contrast, ZON + α-ZOL + β-ZOL were 375 accumulated in the bile, which can therefore be indicative of a dietary ZON exposure,376 but it has to be taken into account that these toxins were detected in the bile of control 377 animals as well. It has to be noted that the considerable variation in toxin concentrations 378 and retention as indicated by [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol 427 (DON) from naturally contaminated wheat for the pig[END_REF]. However, the risk for human 381 exposure by consumption of edible tissues of animals exposed to a Fusarium toxins 382 seemed to be negligible compared to the direct consumption of grain-based food. 383

F

  (6.68 mg DON and 0.056 mg ZON per kg diet as fed). 51

  Fusarium contaminated n = 12) in the afternoon. The time between 78

  as mean and standard deviation or range (min -max). Differences in 121 mycotoxin residual data and carry over factors were evaluated by the non-parametric 122 Kruskal-Wallis-test (p < 0.05), as the data were not normally distributed. 123

  the naturally Fusarium contaminated diet (Groups 2 & 4; Table

  residues of DON + de-epoxy-DON and ZON + α-ZOL + β-ZOL were 200

  concentration of tissues and body fluids was determined by 225 incubation with β-glucuronidase. Up to now, the toxicological relevance of 226 glucuronidated DON and the degeneration to DON are not examined

  003) when the diets exceeded 1 mg DON per kg diet, while lower dietary DON 251

FusariumFigure

  Figure I. Deoxynivalenol (DON) + de-epoxy-DON concentration of bile ( ), kidney ( 502

Table I .

 I Composition of the experimental diets (g/kg, 88 % dry matter) 465

				22
				Control diet	Contaminated diet
		Ingredients
		Control wheat			400	0
		Contaminated wheat			0	400
		Barley			446	446
		Soybean meal			110	110
		Soybean oil			15	15
		Dicalcium-phosphate		1	1
		o r F o r F o r F o r F o r Sodium chloride L-lysine-HCl Premix 1 Calculated composition F o r		0.5 2.5 25	0.5 2.5 25
		Crude ash Crude protein Crude fat Crude fibre N-free extractives Starch ME (MJ/kg)	P e e r P e e r P e e r P e e r P e e r P e e r	57.6 149.3 35.0 34.8 612.6 477.0 13.2	58.9 150.9 33.9 37.9 607.7 470.8 13.2
		Lysine Methionine + Cystine	R R R R R R	8.0 5.0	8.1 5.1
		Threonine Tryptophan Calcium Total phosphorus Sodium Deoxynivalenol (µg/kg) 2 Zearalenone (µg/kg) 2	e v i e w e v i e w e v i e w e v i e w e v i e w 5.1 1.9 e 6.8 5.9 1.8 158.0 v i e 18.0 w	5.1 1.9 6.8 5.7 1.9 6657.0 46.0
	466 467	O n l y O n l y O n l y O n l y O n l y 142.4 6683.0 56.0 O n l 1 Provided per kg diet: Ca 6.1 g; P 1.5 g; Na 1.4 g; Mg 0.3 g; Fe 100 mg; Cu 25 mg; Mn 50 Analyzed composition Crude protein 140.3 Deoxynivalenol (µg/kg) 3 236.0 Zearalenone (µg/kg) 3 9.0 mg; Zn 100 mg; I 1.3 mg; Se 0.4 mg; Co 0.5 mg; vitamin A 10 000 IU; vitamin D 3 1000 IU; y
	468	vitamin E 30 mg; vitamin B 1 18.8 µg; vitamin K 3 1.3 mg; nicotinic acid 12.5 mg; pantothenic
	469	acid 8.4 mg; choline chloride 125 mg.
	470	2 calculated on the basis of the analyzed concentrations of the contaminated wheat (Table II)
	471	3 measured by HPLC methods according to Valenta et al. (2002)
	472

Table IV .

 IV Concentrations of deoxynivalenol (DON) and de-epoxy-DON (DOM) and the carry over factor of the sum of DON + de-epoxy-DON in 482

	483	serum (µg/l), bile (µg/l) and liver (µg/kg) of pigs fed a control (n = 6; 0.24 mg DON/kg) or Fusarium contaminated diet (n = 12; 6.68 mg DON/kg)
	484	either ad libitum or restrictively (means ± range)
		Group	Feeding regimen 1	DON/ZON 2	DON	Serum DOM Carry over	DON	Bile DOM Carry over	Liver DON DOM Carry over
				(mg/kg diet)	(µg/l)	(µg/l)	factor	(µg/l)	(µg/l)	factor	(µg/kg) (µg/kg)	factor
					0.0 b	0.0	0.0 b	41.2 b	3.3 b	0.1886 a	2.7 b	0.0 b	0.0116 a
		1	ad libitum	0.24/0.009	-22.6 a	-0.0	-0.0034 a	(30-52) 427.6 a	(0-10) 80.2 a	(0.1271-0.2627) 0.0761 b	(0-4.6) 26.0 a	-1.9 a	(0-0.0196) 0.0042 ab
		2		6.68/0.056	(13-30)	-	(0.0019-0.0040)	(270-610) (40-120)	(0.0506-0.1072)	(16.8-36.3) (0-3.9)	(0.0028-0.0059)
					0.0 b	0.0	0.0 b	31.5 b	6.0 b	0.1589 a	1.7 b	0.0 b	0.0073 ab
		3	restrictive 3	0.24/0.009	-22.6 a	-0.0	-0.0034 a	(8-48) 367.6 a	(0-14) 60.4 a	(0.0339-0.2627) 0.0641 b	(0-3.2) 20.5 a	-1.9 a	(0-0.0134) 0.0034 b
		4		6.68/0.056	(14-38)	-	(0.0021-0.0057)	(160-630) (24-106)	(0.0276-0.1055)	(13.7-26.4) (0-3.1)	(0.0023-0.0044)
	485	abc data in one column with various letters are significantly different (Kruskal-Wallis-test, p < 0.05); 1 feeding regimen: ad libitum = 3.8 ± 0.6 kg
	486	diet/d, restrictive = 2.4 ± 0 kg diet/d; 2 toxin concentration of the diet (DON, deoxynivalenol; ZON, zearalenone); 3 time since the end of feeding of
	487	restrictive fed pigs (control = 6.1 h, contaminated = 4.8 h)

12, pigs were fed their diet at 7 a.m. (restrictive, 1200 g/meal) and were slaughtered

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± 5.3 and 5.2 ± 4.2 % in bile, liver and kidney, respectively (TablesIV & V). Although

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route of DON, while only low amounts were found in faeces[START_REF] Dänicke | On the effects of increasing 401 deoxynivalenol (DON) concentrations in pig feed on growth performance, utilization of 402 nutrients and metabolism of DON[END_REF]

therefore depend on individual differences in the absorption, metabolism, distribution

Schweigert FJ, Flachowsky G. 2005. The efficacy of a modified aluminosilicate as a

humans. Journal of Toxicology and Environmental Health-Part B-Critical Reviews

deoxynivalenol in animal tissues by liquid chromatography after clean-up with an

Table II. Mycotoxin pattern (µg/kg) of control and Fusarium toxin contaminated wheat

(88 % dry matter)

Acknowledgements

384

The financial support of the 'Deutsche Forschungsgemeinschaft' 558/1-1) is gratefully acknowledged. 386

The authors would like to thank the co-workers of the abc data in one column with various letters are significantly different (Kruskal-Wallis-test, p < 0.05); 1 feeding regimen: ad libitum = 3.8 ± 0.6 kg 497 diet/d, restrictive = 2.4 ± 0 kg diet/d; 2 toxin concentration of the diet (DON, deoxynivalenol; ZON, zearalenone); 3 time since the end of feeding of 498 restrictive fed pigs (control = 6.1 h, contaminated = 4.8 h) 499


Torben ; Leth
email: fac@tandf.co.uk

Keywords: Chromatography -HPLC, Risk assessment, Total diet studies Estimated intake, intense sweeteners, Denmark, cyclamate, acesulfame-K, aspartame, saccharin, children

In 1999 116 samples of non-alcoholic beverages were analysed for the intense sweeteners cyclamate, acesulfame-K, aspartame and saccharin. High contents of cyclamate close to the maximum permitted level in 1999 of 400 mg/L were found in many soft drinks. The estimated intake of the sweeteners was calculated using the Danish Dietary Survey based on 3098 persons aged 1-80 years. The estimated intake with 90 th percentiles of 0.7, 4.0 and 0.2 mg/kg bw/day for acesulfame-K, aspartame and saccharin respectively was much lower than the ADI values of 15 mg/kg bw/day, 40 mg/kg bw/day and 2.5 mg/kg bw/day for acesulfame-K, aspartame and saccharin respectively. However, the 90 th percentile of the estimated cyclamate intake in 1-3 year olds was close to the ADI value of 7 mg/kg bw/day and the 99 th percentile in the 1-10 year olds far exceeded the ADI value. Boys aged 7-10 year had a significantly higher estimated intake of cyclamate than girls. The 90 th percentile for the whole population was 1.8 mg/kg bw/day. After the reduction in the maximum permitted level in EEC in 2004 from 400 to 250 mg cyclamate/L the exposure in Denmark can be expected to be reduced. A new investigation in 2007 should demonstrate whether the problem with high cyclamate intake is now solved.

Introduction

Artificial sweeteners have been in use for many years for their sweet taste with less calories (Corti 1999). Due to increasing problems with obesity in developed countries and a general growing awareness of health implications connected to a high calorie diet, the use of sweeteners is increasing [START_REF] Fry | Low-Calorie Sweeteners: Present and Future[END_REF]. The worldwide increase in the number of people suffering from diabetes also contributes to an increased use of artificial sweeteners, as they have higher intake than non-diabetics (Food Standards Australia and New Zeeland 2004). Finally sweeteners reduce tooth decay.

The food safety of the consumer needs to be assured. Thus in 1994 and1995 the European Commission announced three directives, regarding sweeteners, colours and miscellaneous additives, setting the maximum permitted levels (MPL) with later adjustments. The directives also demand that all member states report intake of food additives, including sweeteners every year. The Commission is advised by the European Food Safety Authority (EFSA). EFSA is allocating the Acceptable Daily Intake (ADI) of all approved food additives, which is an intake that can be ingested daily over a lifetime without appreciable health risk. The MPL are adjusted to avoid consumer intake in excess of the ADI.

The ADI of sweeteners are 40 mg/kg body weight/day for aspartame, 7 mg/kg bw/day for cyclamate, 5 mg/kg bw/day for saccharin and 15 mg/kg bw/day for acesulfame-K.

The Danish Dietary Survey [START_REF] Andersen | The Danes dietary habits 1995. Main results[END_REF] provides extensive information on the intake of foodstuffs in a representative part of the population aged 1 to 80 years. The food survey can provide general information on what the Danes eat and in which quantities. An The Danish monitoring programme for food additives samples foodstuffs for chemical analysis. The primary objective is to check that the additives are used legally and within the maximum permitted levels, but the analyses are also utilized to survey the intake of food additives. Non-alcoholic beverages are the major source of the intake of intense sweeteners [START_REF] Bergsten | Intake of artificial sweeteners. Acesulfame-K, aspartame, cyclamate and saccharin[END_REF] .

In a recent review of intake estimations of intense sweeteners [START_REF] Renwick | The intake of intense sweeteners -an update review[END_REF]) it appears that very few intake estimations based on large representative parts of the population have been carried out and only one for people under 12 years of age. Thus, it was the purpose of this investigation to determine the intake of intense sweeteners with non-alcoholic beverages in different age groups of the Danish population.

Materials and methods

Monitoring program

In 1999 the project: "Monitoring of additives: Intense sweetening agents in non-alcoholic beverages" was carried out as part of the The Danish Veterinary and Food Administration's programme for monitoring of additives [START_REF] Andersen | Monitoring of food additives: Intense sweeteners in non-alcoholic beverages[END_REF]). An initial market survey of non-alcoholic beverages was carried out to establish the number of products relevant for analysis. 246 products were found on the market. The content was calculated from standard curves with six standard concentration levels also derivatised like the samples (external standardisation). All chemicals were analytical grade and the solvents were HPLC grade. The laboratory was accredited to perform the methods according to ISO 17025.

In every series of samples was included a duplicate divided between two series and a recovery experiment with addition of the sweeteners to the original sample in the same amount as judged to be in the sample. From the 30 duplicate determinations the relative internal reproducibilities were calculated to be 2.2%, 2.5%, 2.9% and 1.9% for acesulfame-K, aspartame, cyclamate and saccharin, respectively. From the 15 recovery experiments recoveries of 100%, 103%, 98% and 100% for acesulfame-K, aspartame, cyclamate and saccharin, were calculated respectively. The results showed that the analytical methods had been under control at all times with no values exceeding the control limits for double determinations or for recoveries.

Dietary survey

The survey was conducted in 1995 on a random sample of Danes 1-80 yrs drawn from the Central Population Register, stratified by sex and age [START_REF] Andersen | The Danes dietary habits 1995. Main results[END_REF] The food record was validated against urinary nitrogen as part of a pilot study [START_REF] Groth | Organisation, implementation and data quality of the Dietary Survey[END_REF]. In all 1261 children 1-14 yr and 1837 adults 15-80 yr participated in the survey. For children below 10 yr of age parents recorded the food intake for the child. For children aged 10-12 yr parents decided whether the child was capable of filling in the records or not. 39 adults reported that they suffered from diabetes, but no information about diabetes in the children was reported. However, the prevalence of diabetes among children is considered much lower than for adults.

Estimated intake calculation

The 116 samples of beverages analysed were aggregated into four different types of beverages for which intake data was present: with carbon dioxide, light (average intake 24 ml/day) and sugar sweetened (average intake 108 ml/day) and without carbon dioxide, light (average intake 71 ml/day) and sugar sweetened (average intake 136 ml/day). For each person the registered average daily intake of the four categories was multiplied with the content of intense sweetener, and a calculation of basic statistic parameters of the intake distribution was carried out in the form of mean, standard error of mean and percentiles with the persons divided in age groups, males, females and the total population. For the content of intense sweetener was used two scenarios: One with the analytical determined mean value for all the light samples with content of the specific sweetener called "high intake" and one with the analytical determined mean value for all the light samples in the category called "average intake", which will be lower than "high intake", since many of the light samples were not sweetened with all the four artificial sweeteners. The variability in the levels of sweeteners inside the four categories of soft drinks has not been used in the estimated intake calculation.

Results

The maximum permitted levels (MPL) for the sweeteners were at the time of this study 350 mg acesulfame-K/L, 600 mg aspartame/L, 400 mg cyclamate/L and 80 mg saccharin/L.

Table I shows the distribution of the sweeteners in the different types of beverages with average for all the samples in the aggregated four categories of beverages used for an average intake calculation. Table II shows the average, range and number of samples with content of the specific sweetener used for a high intake calculation.

[Insert table I and II about here]

No illegal use of the four examined sweetening agents was found in the analysis of 62 different products of soft drinks without carbon dioxide. One soft drink exceeding the maximum permitted level for cyclamate was found (454 mg/L). 13 faulty declarations were found in 12 samples, most often a sweetener found which was not declared, amongst others, three samples of Ice Tea with added aspartame, and a sample of a sugar sweetened product with a small content of aspartame. No illegal use of the four examined sweetening agents was found in the analysis of 18 different products of juice. The maximum permitted level was not exceeded in any product.

Six faulty declarations were detected in three samples (more than one sweetener per sample not declared). One sugar sweetened product also contained acesulfame-K and aspartame. The sum total of the 62 samples of soft drinks and the 18 juice samples equals the 80 samples in the category of beverage without carbon dioxide.

No illegal use of the four examined sweetening agents was found in the analysis of 36 different products of soft drinks with carbon dioxide. The maximum permitted level was not exceeded in any product. Two faulty declarations were detected in two samples. One sugar-sweetened product also contained a high amount of aspartame.

The sum of all the intense sweeteners in the products is shown in Figure 1 and 2 for soft drinks without carbon dioxide (including juice) and with carbon dioxide respectively and where beverages without intense sweeteners are excluded from the figures. It appears that if added at all, the level of cyclamate is often very close to the maximum permitted level. For the other sweeteners the content is usually much lower than the maximum permitted levels.

[Insert fig. 2 and3 about here]

In the present study representative for the Danish market, most often a mixture of sweeteners is used with cyclamate as the dominating sweetener, especially in beverages without carbon dioxide. In soft drinks with carbon dioxide a mixture of aspartame and acesulfame-K is more often used to sweeten the products. Two intake calculations were 10 carried out. One using an average only of the samples with a content of sweetener called high intake (Figure 3), and another with an average of all samples in each of the four categories of beverages called average intake (Figure 4). Estimated intake in mg/kg bw/day was calculated from an average for each age group of the actual body weights registered during the food survey.

[Insert Fig. 3 and 4 about here]

Both high intake and average intake of all the sweeteners for all persons are shown in

Discussion

To take into account brand loyalty [START_REF] Leclercq | Stochastic modelling of human exposure to food chemicals and nutrients within the Montecarlo project. An exploration of the influence of brand loyalty and market share on intake estimates of intense sweeteners from sugar-free soft drinks[END_REF] we have found it relevant to carry out a high intake calculation by averaging only the samples with the analytical determined content of intense sweeteners. Even higher estimated intakes could be found under the assumption that a person only drinks a beverage with the highest found content of intense sweetener, but that would be a different brand for each of the four sweeteners and therefore not a very likely scenario. However, it could also be argued that the average for all samples in each of the four categories of beverages is a more reasonable figure to put into the intake calculation, so that extremes do not dominate the intake calculation.

Therefore this calculation has also been carried out. The results for cyclamate are presented in Figure 3 and for all the sweeteners in Table III.

For cyclamate the differences between the high and average intake are not so great, because most of the soft drinks both with and without CO 2 contained cyclamate and none of the sugar sweetened drinks contained cyclamate (Table I andII). For aspartame the differences are much greater (Table III) due to the fact that a few sugar sweetened beverages also contained aspartame. Therefore just two samples increase the intake of aspartame significantly, since they get to represent the whole category of sugar sweetened soft drinks. This shows very well the limitations of the high intake calculation, but since intake of acesulfame-K, aspartame and saccharin under all circumstances are much lower than the ADI values, further refinement of the intake calculation is not warranted.

However, the estimated cyclamate intake is much higher than the ADI value for groups of children. For the high intake calculation (Figure 3) the 90 th percentile in the cyclamate intake distribution for 1-3 year olds of 7.1 mg/kg bw/day is close to the ADI value, and for 1-10 year olds the 99 th percentile is much higher than the ADI value, and more than double the ADI value for the group 1-3 year with 14.3 mg/kg bw/day (Table III), and the child with the highest intake gets 18 mg cyclamate/kg bw/day. Boys have a significantly higher estimated intake of cyclamate than girls in the age group 7-10 yr both on the 90 th and 99 th percentile but not on the median value (Table III), and also for boys in the age group 11-14 yr the 99 th percentile is higher than the ADI value The average intake calculations in Figure 4 give a slightly lower intake, but the 99 th percentiles from 1-10 yr are still much higher than the ADI value of 7 mg/kg bw/day, i.e. 13.1 mg for the age group 1-3 yr, and the child 12 with the highest intake gets 17 mg/kg bw/day. Apparently small children have a much higher risk of high cyclamate intakes with their low body weights and the often reported widespread use of dilutable artificial sweetened soft drinks in homes, kindergartens as well as in day nurseries. It seems also that boys at a certain age have a higher cyclamate intake than girls perhaps due to a higher activity level and therefore a higher consumption of soft drinks.

This means that with a maximum permitted level of 400 mg cyclamate/L relatively large groups of Danish healthy children had an intake of cyclamate higher than the new ADI value of 0-7 mg/kg bw/day established in March 2000 by the revised opinion on cyclamic acid and its sodium and calcium salts from the Scientific Committee on Food (SCF 2000).

Therefore Denmark presented these data for the EEC Commission in 2001 and argued for a lower permitted level of cyclamate in beverages, and in January 2004 the permitted level was reduced from 400 mg to 250 mg cyclamate/L. However, FAO/WHO is still recommending a maximum level of 400 mg/L and also the high ADI value of 11 mg/kg bw/day (Codex 2006).

Most studies dealing with intake of sweeteners have focused on high consuming subgroups e.g. diabetes patients or teenagers (Baer A. and Biermann C. 1992;[START_REF] Arcella | Dietary estimated intake of intense sweeteners by Italian teenagers. Present levels and projections derived from the INRAN-RM-2001 food survey[END_REF][START_REF] Garnier-Sagne | Calculation of the intake of three intense sweeteners in young insulin-dependent diabetics[END_REF]Ilback et al. 2003), and cyclamate intakes above the ADI value is found in some cases, especially for children. The calculation of intake of sweeteners based on analytical determined contents of all brands on the market and in a large representative part of the population is well suited to identify high consuming groups in the general population. As seen in this investigation children may be high consumers, for cyclamate intake in a high consumer group aged 12 to 39 years of 9.3 mg/kg bw/day and in a group of diabetics of 11.6 mg/kg bw/day, which exceed the EEC ADI value of 7 mg/kg bw/day. Furthermore, the Food Standards Agency in UK (Food Standards Agency 2003) has conducted an investigation of the intake of aspartame, acesulfame-K, saccharin and cyclamate in soft drinks with similar conclusions as in the present investigation. High consumers (97.5 th percentile) from 1½ to 4½ years old had an intake of 14 mg cyclamate/kg bw/day apparently calculated as the high intake calculation in the present investigation, i.e. an average only for the children, who consumed the sweeteners in question. However, values for content of sweeteners were not determined analytically, but based on information from the manufacturers. Based on these results FSA advised parents to give young children no more than three beakers (about 180 ml each) a day of dilutable soft drinks.

The other significant source of intense sweeteners is tabletop sweeteners. High consumers of cyclamate based tabletop sweeteners ate 230 mg/day corresponding to 35% of the ADI [START_REF] Bergsten | Intake of artificial sweeteners. Acesulfame-K, aspartame, cyclamate and saccharin[END_REF]). However, tabletop sweeteners are typically not used by healthy children, so they are without significance for the high cyclamate intake found in this investigation in groups of small children.

Although from 1999 this investigation has still a value as the only investigation with analytical data for all artificial sweetened soft drinks on the market and with a food survey from 400 mg/L to 250 mg/L. However, Codex still recommends 400 mg/L and an ADI of 11 mg/kg bw/day, thus the cyclamate intake in the rest of the world probably remains high.

A new investigation of soft drinks on the Danish market will be started in 2007 with food survey data from 2000 to 2005 to ascertain whether also a maximum permitted level of 250 mg cyclamate/L will lead to higher cyclamate intakes in children than the ADI value of 7 mg/kg bw/day.

Conclusions

In an investigation from 1999 it was demonstrated that almost 10% of Danish children aged from 1 to 10 years had an estimated cyclamate intake higher than the ADI value of 7 mg cyclamate/kg bw/day. Boys from 7-10 year had a significantly higher intake of cyclamate than girls of the same age group. The intake of acesulfame-K, aspartame and saccharin was far below their respective ADI-values.

  intake of specific compounds is possible if the level of the compound in all potential foodstuffs is known.

  photos of different portion sizes) were used to get the best estimate of amounts

  207 food items, including 20 beverages (e.g. different types of milk, beer, wine, soft drinks and fruit juices). Sweetened beverages included two types of beverages with carbon dioxide: soft drinks light and soft drinks sugar sweetened and two types of beverages without carbon dioxide: soft drinks sugar sweetened and soft drinks light including fruit juices.

  are not diabetics. Very few other similar investigations on a large representative part of the population exist. However, an investigation from Australia and New Zealand (Food Standards Australia and New Zeeland 2004) showed a 95 th percentile

  representative part of the population demonstrating a high estimated intake of cyclamate in children. The intake of cyclamate in EEC countries could be expected to have decreased after the reduction of the maximum permitted level in 2004

Table III

 III

for four of the age groups of the intake distribution and for boys and girls in one age group. [Insert Table III about here].

Table I .

 I After the reduction in the maximum permitted level in EEC in 2004 from 400 to 250 mg cyclamate/L the exposure in Denmark and other EEC member states can be expected to be reduced. A new investigation in 2007 shall demonstrate whether the problem with high cyclamate intake is now solved. Intense sweeteners in non-alcoholic beverages, average of all samples in each category (for average intake calculation) T-test shows significant difference between boys and girls on the 99.9% probability level

	Sex	Age	Percentile Acesulfame-K	Aspartame	Cyclamate	Saccharin
	F o r P e e r F o r P e e r F o r P e e r F o r P e e r Table II. Intense sweeteners in non-alcoholic beverages, average of samples with content of sweetener (for high intake calculation), (range) and (number of samples with content of sweetener) Table III. Intake of intense sweeteners in the whole population and in children from 1-10 years, mg/kg bw/day Figure 1. Intense sweeteners in soft drinks without carbon dioxide F o r Type of sample Number of samples Acesulfame-K mg/L Aspartame mg/L Cyclamate mg/L years Av. High Av. High Av. High Av. High Saccharin mg/L Soft drinks (with CO 2 ) Light 21 49 152 152 15 Soft drinks (with CO 2 ) With sugar 15 -30 --Soft drinks (no CO 2 ) Light 60 42 36 305 26 Soft drinks (no CO 2 ) With sugar 20 2 35 --F o r Type of sample Acesulfame-K mg/L Aspartame mg/L Cyclamate mg/L mg/kg bw/day mg/kg bw/day mg/kg bw/day mg/kg bw/day Saccharin All 1-80 50 0.1 0.1 0.1 0.8 0.1 0.1 0.01 0.01 mg/L Soft drinks (with CO 2 ) Light 129 (32-179) (n=8) 159 (26-460) (n=20) 245 (203-404) (n=13) 90 0.3 0.7 0.7 4.0 1.6 1.8 0.1 0.2 29 99 0.9 2.0 1.8 9.4 6.1 6.9 0.5 0.8 (7-58) All 1-3 50 0.3 1.0 0.8 3.7 1.6 1.8 0.1 0.2 (n=11) Soft drinks (with CO 2 ) With sugar -450 -90 0.9 2.3 1.9 8.8 6.3 7.1 0.6 0.8 -99 1.9 3.9 2.8 16.9 13.1 14.3 1.1 1.6 (n=1) Soft drinks (no CO 2 ) Light 62 (20-161) (n=39) 56 (4-226) (n=43) 333 (84-454) (n=55) 37 (15-65) (n=42) Soft drinks (no CO 2 ) With sugar 43 (n=1) 124 (n=1) --All 4-6 50 0.2 0.8 0.7 3.4 1.1 1.2 0.1 0.1 90 0.7 1.7 1.4 7.3 5.3 5.8 0.5 0.7 F 99 1.6 3.7 2.6 12.4 10.2 11.7 0.9 1.3 Boys 7-10 50 0.2 0.6 0.7 3.8 1.1 1.2 0.1 0.1 o 90 0.7 1.6 1.3 7.3 5.2* 5.8* 0.3 0.6 r 99 1.5 3.1 2.5 13.0 10.5* 11.7* 0.5 1.3 Figure 2. Intense sweeteners in soft drinks with carbon dioxide Figure 3. High estimated intake of cyclamate from non-alcoholic beverages -All Figure 4. Average estimated intake of cyclamate from non-alcoholic beverages -All P e e r P e e r Girls 7-10 50 0.2 0.6 0.6 3.4 0.9 1.0 0.1 0.1 P 90 0.6 1.3 1.1 6.1 3.9* 4.2* 0.4 0.5 99 1.0 2.5 1.9 9.7 6.1* 6.9* 0.7 0.8 e r e ADI 15 40 7 2.5
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