Dr Saba Sharif
email: saba.sharif@bwhct.nhs.uk.

Dr Rosalie Ferner
email: rosalie.ferner@kcl.ac.uk

Nalin Thakker
email: thakker.nthakker@manchester.ac.uk.

Elisa Majounuie

Andrew Shenton

Mike Baser

Meena Upadhyaya
email: meenaupadhyaya.upadhyaya@cardiff.ac.uk.

Gareth Evans
email: gareth.evans@cmft.nhs.uk.

Elisa Majounie
email: phd.majouniee@cardiff.ac.uk.

A molecular analysis of individuals with Neurofibromatosis type 1 (NF1

Keywords: NF1, OPG, genotype, phenotype

Introduction:

Neurofibromatosis Type 1 (NF1) (MIM 162200) is a common, autosomal dominant genetic disorder, affecting multiple systems in both males and females of every age, and from every ethnic background [START_REF]A pathogenetic and clinical overview[END_REF][START_REF] Majounie | Phenotype, Natural history and Pathogenesis[END_REF]. The NF1 gene is a tumour suppressor and loss of gene function due to somatic inactivation of both NF1 alleles leads to an increased risk of developing benign and malignant tumours. NF1 is a chronic, frequently progressive disorder with widespread manifestations and approximately a third of patients develop serious complications, mainly in childhood. Neurological complications include cognitive impairment and central nervous system tumours, the commonest being the optic pathway glioma (OPG) [START_REF] Listernick | Optic pathway gliomas in children with neurofibromatosis 1: consensus statement from the NF1 Optic Pathway Glioma Task Force[END_REF][START_REF] Rosser | Neurocognitive dysfunction in children with neurofibromatosis type 1[END_REF].

Optic pathway gliomas (OPGs) are detected in 15% of NF1 patients [START_REF] Lewis | von Recklinghausen neurofibromatosis. II. Incidence of optic gliomata[END_REF][START_REF] Listernick | Optic gliomas in children with neurofibromatosis type 1[END_REF][START_REF] Lund | Optic gliomas in children with neurofibromatosis type 1[END_REF], occurring predominantly in early childhood. However, they are only symptomatic in around 5% of NF1 cases [START_REF] Singhal | Neurofibromatosis type 1 and sporadic optic gliomas[END_REF][START_REF] Sharif | Second primary tumors in neurofibromatosis 1 patients treated for optic glioma: substantial risks after radiotherapy[END_REF]. Differences have been observed in the natural history and effects of treatment outcome of NF1 OPGs compared with sporadic OPGs [START_REF] Singhal | Neurofibromatosis type 1 and sporadic optic gliomas[END_REF][START_REF] Sharif | Second primary tumors in neurofibromatosis 1 patients treated for optic glioma: substantial risks after radiotherapy[END_REF], but the reasons for this discrepancy are unknown.

Debate exists about the role and nature of visual screening in the detection of NF1 OPGs and the presence of cognitive impairment makes it difficult for children to co-operate with visual testing. It is difficult to determine clinically and radiologically which tumours will behave more aggressively and require intervention. The identification of the NF1 gene alterations that predispose to OPG formation would facilitate focused screening of individuals at risk of NF1 OPG. Potentially genotype-phenotype correlation could predict disease progression and response to treatment.

We performed a long-term follow up study of 80 NF1 OPG patients to determine the natural history of these tumours, treatments outcome and psychosocial morbidity. These were largely symptomatic OPGs and therefore the ones it would be important to pick up early. This paper focuses on the molecular aspect of the study evaluating if a genotypephenotype correlation exists. If present, a genotype phenotype correlation would enable molecular genetic screening for OPGs early in childhood and potentially enable a greater understanding of the natural history of the tumour to guide management.

Methods:

The molecular study was part of a case controlled study involving patients ascertained from two NF1 clinics over an 18 month period. The Manchester NF1 clinic is based in the regional Genetics Unit at St Mary's Hospital. In addition to diagnosis and management of complications all NF1 patients are offered annual review. A database of these patients detailing their age, gender, family history and manifestations is maintained.

The second NF1 clinic is based at Guys Hospital, London. This is a neurology led multidisciplinary service which also has close links with Genetics. A database of name and DOB was available and during the study details of gender and family history were added for all patients on the database (650 notes were available). Both clinics receive referrals predominately from non-specialists, thus each represents a relatively unbiased population based group of NF1 patients. This study was performed prior to the start of the NCG funded complex NF1 service. Both the Manchester and Guys NF1 clinics now also see NF1 OPG patients referred from specialist centres as part of this NCG service. Ethical approval for our study was sought independently from each centre (Manchester 02/CM/432 and Guys 03/02/13).

Inclusion criteria for the molecular study were a confirmed diagnosis of NF1 according to the NIH diagnostic criteria, 1988 [START_REF]Conference Statement: neurofibromatosis[END_REF], suspected diagnosis of an OPG (made on clinical and/or radiological features), and aged 16 years or more. In Manchester the Manchester Children's Tumour registry (detailing cancer diagnoses from 1954 in those under 15 years) was cross-checked. The Search for OPG included 'juvenile astrocytoma' and 'neurofibromatosis tumours not specified' to achieve full ascertainment. The OPGs were diagnosed on clinical and radiological features, including visual impairment, changes on fundoscopy, pupillary alteration, proptosis or precocious puberty or radiological evidence such as thickening of the optic nerves with enhancement. Few had histological confirmation, as it is not usual clinical practice to perform biopsies for these tumours.

The OPG diagnosis was reviewed during the natural history study (details will be reported separately). An EDTA sample and Lithium heparin sample was collected from each patient for molecular and fluorescent in situ hybridisation studies (FISH) studies respectively. A two-step procedure was used to detect mutations. Firstly FISH was performed on all samples to look for any large deletions in the NF1 gene [START_REF] Tonsgard | Do NF1 gene deletions result in a characteristic phenotype?[END_REF][START_REF] Kluwe | Screening 500 unselected neurofibromatosis 1 patients for deletions of the NF1 gene[END_REF]. Then a full gene screen for mutations was carried out, using Denaturing High Performance Liquid Chromatography (DHPLC) [START_REF] Han | Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 ( NF1) gene[END_REF][START_REF] De Luca | NF1 gene analysis based on DHPLC[END_REF][START_REF] Upadhyaya | Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors[END_REF].

Interpreting the findings.

Mutations were classified as pathogenic if they resulted in a truncated protein, nonsense frameshift mutations, and splice site changes. Amino acid changes altering the protein sequence, missense changes, were also classified as pathogenic, if no SNP was detected at this position (http://www.ncbi.nlm.nih.gov/SNP/), and they were not seen in 1000 NF1 chromosomes and the same number of unaffected control chromosomes. Family studies and functional analyses were not possible during the course of the study but these may aid our interpretation of these changes further.

Polymorphisms were only considered to be non pathogenic if they were previously reported as a SNP (http://www.ncbi.nlm.nih.gov/SNP/), were previously reported as a polymorphism, had been found in normal controls or were seen in association with a pathogenic mutation in an affected individual (Upadhyaya personal communication, 2004). It was not possible to check for the presence of SNPs for the intronic changes, as sequence data was not available, and these were not looked for in the normal controls.

The location of pathogenic changes identified in the NF1 gene from the NF1 OPG patients was reviewed to evaluate possible genotype phenotype correlation. Our findings were compared to mutations identified in another cohort of NF1 OPG patients (Ars et al. [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF]). Both sets of results (our and Ars et al [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF]) were then combined with a larger series of NF1 patients (Castle et al. [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF]). This group (Castle et al. [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF]) provided a control NF1/NF1 OPG group, in whom pathogenic changes had previously been identified in a large cohort of unselected NF1 patients, predominantly from the UK (Meena Upadhyaya personal communication 2010).

A logistic regression analysis was then used to determine if the odds ratio of developing an OPG was associated with location of mutation. The outcome was presence or absence of OPG. For ease of analysis the NF1 gene was divided into tertiles of approximately equal base pair length, using the NF1 intron-exon boundaries as dividing points. The 5´ tertile included exons 1-16, the middle tertile exons 17-30 (the GAP-related domain is in exons 21-27a), and the 3´tertile exons 31-49. In the logistic regression analysis, the tertiles were represented by indicator variables. The middle tertile was the reference category in comparisons between tertiles.

Results:

Table 1 summarises patient ascertainment in the overall long-term follow up study. 80 NF1 OPG patients were identified from 1331 NF1 patients known to both centres. Of these 47 were eligible for the molecular study and 29 were recruited (subjects). There were no significant differences (in gender, age at diagnosis of NF1, or family history of NF1) between patients ascertained from the two centres. Whilst reassessing the OPGs as part of the natural history study three patients were shown not to have an OPG.

Twenty eight out of the 29 subjects had mutation analyses with DHPLC (Table 2) as one patient was identified as having a whole gene deletion by FISH. $ ? Amino acid substitution or interference with normal splicing

One patient had a whole gene deletion and a further 14 had pathogenic changes, 7 of which had not been described previously. Of these, 10 were protein truncating (Frameshift/nonsense), 2 intronic changes and the remainder were missense changes. A total of 20 non pathogenic changes were seen, 15 previously unreported. Ten were polymorphisms, 2 silent changes and 8 intronic changes. Figure 1 depicts the distribution of the pathogenic changes within the NF1 gene. Clustering of changes was seen towards the 5' end of the gene.

Discussion

Comparison to changes seen in previously reported NF1 OPG patients.

Our data suggest a clustering of mutations in the 5' region of the NF1 gene. No mutation data were available in NF1 OPG patients when we commenced this study. Subsequently, during the course of our study, Ars et al. reported 12 mutations in a cohort of 20 NF1

OPG patients but no clinical information was reported [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF].

We compared the molecular data from our patients to the data reported by Ars et al. (table 3, supplementary on line material). All of the changes described in the OPG patients by Ars et al. [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF] were shown to change mRNA processing, resulting in truncated protein. Mutation detection rate.

Mutation testing in NF1 has not proven easy, with many past studies reporting mutation detection frequencies of <50%. The large size of the gene, the lack of hotspots, the diverse spectrum of mutational mechanisms and the presence of homologous loci throughout the genome, have hindered both the previous methods available (including heteroduplex analyses, single stranded conformational polymorphism analyses and the protein truncation test) to find changes, and once found, the interpretation of their role.

DHPLC has been shown to be an effective mode of mutation detection in NF1 [START_REF] Han | Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 ( NF1) gene[END_REF][START_REF] De Luca | NF1 gene analysis based on DHPLC[END_REF][START_REF] Upadhyaya | Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors[END_REF],

and prior FISH analysis will also detect deletions, the largest group of changes missed with DHPLC.

The mutation detection rate was 52% by DHPLC, and with FISH this was increased further, to 54%, for the 28 NF1 OPG patients tested. No additional analyses such as RNA studies, functional analyses or family studies were performed. These studies, might have shown that some of the intronic changes or polymorphisms are disease causing mutations.

However, our results are comparable to those seen by Ars et al. [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF] who used SSCP/HD analyses, followed by sequencing and characterisation of the changes at the genomic level. They detected 12 mutations in 20 NF1 OPG patients, a mutation detection rate of 60%.

Assessing genotype-phenotype correlations.

Logistic regression analysis was used to determine if the relative risk of OPG was associated with the location of mutations in the NF1 gene. NF1 patients with identified mutations were ascertained from three sources: 111 NF1 patients from Castle et al. [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF],

of whom 9 had OPGs; 12 NF1 OPG patients from Ars et al. [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF]; and 15 patients with OPGs from this study. A sample from one deceased NF1 OPG patient from Manchester, ME13, was shown to have the same mutation as his monozygotic twin MS14, as would be expected. This result was also included in this analysis, as part of our NF1 OPG cohort (hence increasing our sample from 14 to 15 patients).

Thirteen people from Castle et al. [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF] were excluded from the analysis (seven with large deletions, five with non-OPGs (ie gliomas outside of the optic pathway) and one with missing information on glioma status). The resultant group was 125 patients, of whom 36 had an OPG. There were 104 families with one patient, nine families with two patients (including one pair of monozygotic twins), and one family with three patients. It was not possible to adjust for familiality due to the small number of families with more than one patient, but the type of NF1 mutation (familial or de novo) has only a minor influence on the variability of clinical expression in NF1 [START_REF] Easton | An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes[END_REF]. Therefore, each patient was treated as a separate observation.

Mutations in the OPG patients from this study (15 patients including the results on the deceased twin discussed above) were analysed with those found in the control group of nine patients from Castle et al. [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF] Clustering in the 5' region was apparent but, was not significantly different from the control population (Castle et al. dataset [START_REF] Castle | Evaluation of genotype-phenotype correlations in neurofibromatosis type 1[END_REF].

Addition of the NF1 OPG patients from Ars et al. [START_REF] Ars | Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients[END_REF], resulted in a significant distribution of mutations in the 5' third of the gene (odds ratio 6.05, p=0.003), and less so in the 3' region (odds ratio 3.30, p=0.066). These results suggest that in the NF1 OPG group, more pathogenic changes are seen 5'.

Genotype-phenotype correlations for OPGs in NF1.

Half (7 of 14), of the pathogenic changes detected in our study were previously unreported. Although no mutation hotspots have been identified, exons 4a, 4b, 10 a-c and 37 appear to harbour more mutations [START_REF] Fahsold | Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain[END_REF][START_REF] Messiaen | Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects[END_REF], and five of our mutations (including two unreported) were found in these exons. Further support for the 5' predilection is provided by a report of siblings with OPG and an exon 4b nonsense mutation (c484CAG>TAG;

Q162X) [START_REF] Kebudi | A novel mutation in the NF1 gene in two siblings with neurofibromatosis type 1 and bilateral optic pathway glioma[END_REF]. The localisation of mutations at the 5' end of the gene encompassing largely exon 1-exon 15 may indicate a new genotype-phenotype correlation in NF1 OPG patients, although further evaluation of this is required.

Earlier research into such correlations has been limited partly by the difficulty in finding mutations. Moreover, the large phenotypic variability within NF1 families has indicated that there are likely to be other modifying influences as well as the underlying NF1 gene change. These include the role of modifier genes (epistasis), epigenetic factors and environmental factors [START_REF] Easton | An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes[END_REF][START_REF] Carey | Penetrance and variability in neurofibromatosis: a genetic study of 60 families[END_REF][START_REF] Szudek | Analysis of intrafamilial phenotypic variation in neurofibromatosis 1 (NF1)[END_REF]. We did not adjust for familiality in the one family with identical twins with OPG, although clearly they will have carried any modifier genes in common [START_REF] Easton | An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes[END_REF]. However, the contribution of each of these factors in determining the NF1 phenotype is unknown, and may vary for different disease traits. Furthermore, when a genotype phenotype correlation in NF1 does exist, it may be difficult to interpret due to the pathogenic heterogeneity, the complexity of the phenotype, and the small number of patients examined. However, recently a number of correlations have been suggested including a mild phenotype of café au lait patches only in patients with an in frame deletion [START_REF] Upadhyaya | An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation[END_REF]; multiple spinal neurofibromas with few other features [START_REF] Kluwe | NF1 mutations and clinical spectrum in patients with spinal neurofibromas[END_REF][START_REF] Upadhyaya | The spectrum of somatic and germline NF1 mutations in NF1 patients with spinal neurofibromas[END_REF]; NF1 patients with a 1.4Mb microduplication with mild learning difficulties, teeth and hair characteristics [START_REF] Grisart | NF1 microduplication first clinical report: association with mild mental retardation, early onset of baldness and dental enamel hypoplasia?[END_REF] and NF1 Noonan syndrome patients with potential genotype phenotype correlations with NF1 gene alterations [START_REF] De Luca | NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome[END_REF] . It may be that the genotype is a main determinant of the development of OPGs in NF1. However, further evidence is still required to confirm our findings and the other potential genotype phenotype correlations [START_REF] Upadhyaya | An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation[END_REF][START_REF] Kluwe | NF1 mutations and clinical spectrum in patients with spinal neurofibromas[END_REF][START_REF] Grisart | NF1 microduplication first clinical report: association with mild mental retardation, early onset of baldness and dental enamel hypoplasia?[END_REF][START_REF] De Luca | NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome[END_REF].

Conclusions

The apparent localisation of mutations in the 5' end of the NF1 gene appears to be a true feature of the mutations occurring in the NF1 OPG patients when compared to NF1 patients without OPGs (mutations in 5' third of the gene, odds ratio 6.05, p=0.003). Since the delineation of the whole gene deletion group in NF1, this finding is the third molecular finding which may have a significant impact in the management of NF1 patients. However, these findings require further confirmation in larger number of NF1 OPG patients and understanding of the outcome of these changes. Corroboration of these results may allow for a more targeted approach to screening for OPGs, by first identifying those patients at risk. Potentially further clinical and molecular studies may permit the early identification of aggressive tumours and facilitate targeted treatment.

Figure 2

 2 Figure2shows the distribution of pathogenic changes in the NF1 gene in both studies

Figure 1 :Figure 2 :

 12 Figure 1: Distribution of pathogenic changes in the NF1 gene, detected by DHPLC.

Figure 3 :

 3 Figure 3: A diagrammatic representation of the location of mutations seen in a control group (Griffiths et al. [33]).

Table 1 :

 1 NF1 OPG patients identified and data collection

		Patients groups
		Manchester Guys
	Total NF1	788	543
	Total NF1 OPG	45	35
	Eligible	28	19
	Seen (Subjects)	18	11	Patient group for the molecular study
	Declined	4	8
	Unable to contact	6	0
	New diagnosis post recruitment	0	2
	Deceased	13	4
	Child	4	10

Table 2

 2 Sequence changes identified in the NF1 gene in the NF1 OPG patients

	Study number	Location Lower numbering corresponds to NCBI	DNA change	Protein Change	Type *	Pathogenic mutation	Reporte d #	Presence of SNP (not tested: NT, Not N/A) applicable	Affected first degree relative
	GS10	Ex 2	c.98-		F	Yes	No	N/A	Yes
		Ex 2	99delAA
	GS05	Ex 4b	c.574C>T	R192X	N	Yes	[18]	N/A	Yes
		Ex 5					HGMD
	MS02	Ex 7	c.1011 ins		F	Yes	No	N/A	No
		Ex 9	T
	MS06	Ex 7	c.1012G>	D338N	M	Potentially	No	No	No
		Ex 9	A			Yes
	MS11	Ex 10a	c.1306-		F	Yes	No	N/A
		Ex 12	1307delT
			C
	MS03	Ex 10a	c.1381C>	R461X	N	Yes	[18]	N/A	No
		Ex 12	T
	MS12	Ex 10c	c.1544-		F	Yes	No	N/A	Yes
		Ex 14	1545delG
			G
	GS04	IVS11	c.1721+3		I	Yes	[18,21]	NT	No
		IVS15	A>G
	MS16	Ex 12a	c.1722C>	S574R	M	Potentially	[22]$	No
		Ex 16	A			Yes
	MS07	Ex 16	c.2446	R816X	N	Yes	[18]	N/A	No
		Ex 21	C>T
	MS08	IVS 21	c.3709-		SS	Yes	No	NT	No
		IVS 27	1G>C
	MS15	Ex 34	c.6524		F	Yes	No	N/A
		Ex 42	dupGA
	GS09	Ex 37	c.6788-		F	Yes	HGMD	N/A	No
		Ex 45	6792del
			TTAC
	MS14	Ex 42	c.7411C>	Q2471X	F	Yes	No	N/A	Yes
		Ex 50	T

* Frameshift-F; Intronic-I; Nonsense-N; Splice site-SS; Missense-M # Checked if reported in

[START_REF] Han | Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 ( NF1) gene[END_REF][START_REF] De Luca | NF1 gene analysis based on DHPLC[END_REF][START_REF] Fahsold | Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain[END_REF][START_REF] Rodenhiser | A novel RsaI polymorphism within intron 39 of the neurofibromatosis type 1 (NF1) gene[END_REF][START_REF] Messiaen | Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects[END_REF][START_REF] Purandare | Characterisation of inherited and sporadic mutations in neurofibromatosis type-1[END_REF][START_REF] Mattocks | Automated comparative sequence analysis identifies mutations in 89% of NF1 patients and confirms a mutation cluster in exons 11-17 distinct from the GAP related domain[END_REF]

and the HGM (http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html).


Stefanie Marie Vandevijvere
email: stefanie.vandevijvere@iph.fgov.

Liesbeth Temme

Mirjana Andjelkovic

Marc De Wil

Christine Vinkx

Leo Goeyens

Joris Van Loco

E Temme

Wil M De

Van

Estimate of intake of sulfites in the Belgian adult population

Keywords: food consumption survey, sulphite, Tier 2, Tier 3, adults, Belgium

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.

There are 3 ways of being exposed to SO 2 : by inhalation, by skin contact or by ingestion from 79 the food chain (NPI, 2005). Sulfite (H 2 SO 3 ) is formed by sulphur dioxide (SO 2 ) gas which is 80 very soluble in water. The presence of sulphur dioxide in the environment is due to 81 combustion processes of organic compounds, such as natural combustion from forest fires or 82 combustion in energy or transformation plants. Sulfite is also present in the exhaust fumes 83 from cars and trucks. Depending on the pH of the environment, different forms of sulfite can 84 exist or coexist. 85 86 Sulfite has raised concerns because of its toxicity for the lungs and its ability to cause allergic 87 reactions, especially in asthmatic persons (Valley et al., 2009). It was estimated that an 88 exposure of 10 to 50 ppm during 5 to 15 min may have a measurable effect on humans. In the 89 UK, 2 ppm (8 hours time weighted average TWA) or 5 ppm (10 min TWA) were set as 90 maximum occupational exposure values (European Cooperation on School Education, 2004). 91

In Belgium, the maximum allowed concentration of sulfite in the air is 350 µg/m³ during 92 maximum 1 hr or 125 µg/m³ during maximum 24 hr (European Community 1999a). In a 93 review of the Health Council of the Netherlands sulfite was found to be responsible of chronic 94 urticaria, skin allergy and bronchial asthma in some humans. The NOAEL in rats was set at 95 72 mg SO 2 /kg bw/day (Health Council of the Netherlands, 2005). 96 97

The Acceptable Daily Intake (ADI) is the amount of SO 2, in food that can be ingested orally 98 over a lifetime without an appreciable health risk. The ADI is expressed per kg body weight 99 (abbreviated "bw") per day. In the case of sulfites (expressed as SO 2 ), the JECFA (Joint 100 FAO/WHO Expert Committee on Food Additives) has set the group ADI value to 0.7 mg/kg 101 bw/day (JECFA, 1999). 102 103 Sulfite can be intentionally added to food for its antioxidant or antimicrobial properties. 104 Sulfite inhibits the enzymatic activity of polyphenol oxidase, which is responsible for 105 browning in fruits, vegetables and seafood products. It also prevents oxidation of food due to 106 contact with the air and the browning effect occurring from the Maillard-type reactions. The 107 aim of adding sulfite to wine is to prevent microbial growth and oxidation of volatile odorous 108 compounds which may develop during the fermentation [START_REF] Ough | Determination of sulfur dioxide in grapes and wines[END_REF]. 109 Sulfite is allowed as a preservative (E220-E228) in a list of different foods (excluding all 111 others) under the condition of respecting the maximum use levels, as specified by the 112 European Directive 95/2/EC as amended (European Community 1995) and for wine in annex 113 V of the Council regulation (EC) No. 1493[START_REF]WHO Food Additive Series: 42[END_REF](European Community 1999b). 114

The concentration in different foods is expressed as sulphur dioxide (SO 2 ) in mg/kg or mg/l, 115 depending on the nature of the food and relates to the total quantity from all sources. The 116 legislation mentions that an SO 2 content of not more than 10 mg/kg or 10 mg/l is not 117 considered to be present. Directive 2000/13/EC on labelling of pre-packaged food requires 118 labelling of products containing sulfite, if the SO 2 concentration is higher than 10 mg/kg or 10 119 mg/l, for the benefit of people with a food intolerance for sulfite (European Community 120 2000). 121 122 Sulfite can be present in foods as free sulfite or react with a variety of organic compounds in 123 foods to form reversibly or irreversibly bound products. In our digestive tract, the free and 124 reversibly bound forms can be liberated (Wedizicha, 1992). García-Alonso et al., (2001) 125 reported that the amount of added sulfite in fresh sausage was reduced by 26 % when S-126 sulphonate bounds (irreversibly bound) were formed and by 23 % when C-O-SO 2 -bounds 127 (reversibly bound) were formed. Those percentages of loss may vary sharply from 10 to 49 % 128 depending on the conditions when adding the sulfite to the meat (time of mixing, presence of 129 oxygen, degree of mincing and fat composition of the meat). 130 131 Cooking was shown to reduce the level of sulfite in beef and chicken burgers (Pena-Egido et 132 al;[START_REF] Hansen | Conditions for use of food additives based on a budget for an acceptable 653 daily intake[END_REF][START_REF] Garcia-Alonso | S-sulfonate determination and 651 formation in meat products[END_REF] the distribution of the 3 different forms of sulfite (free, 133 reversibly bound and S-sulphonate) remained approximately the same after as before the 134 cooking process. The losses reported for meat burgers were 31 % for the S-sulphonates, 29 % 135 for the free sulfite and 28% for the total sulfite. This study also showed that the frying process 136 reduced the amount of total sulfite on average by 41 % (± 13%). A study in shrimps 137 [START_REF] Armentia-Alvarez | Residual levels of sulfite in raw 610 and boiled frozen shrimps: variability, distribution and losses[END_REF] showed that during cooking the loss of free and reversibly 138 bound sulfite varied between 45 and 55 %. The level of total sulfite was about 5 times higher 139 in the non edible parts compared to the edible parts. The authors concluded that the boiling 140 step did not change the distribution of free and total sulfite in shrimps, but a reduction of 141 about 40 % was observed for the total sulfite in the edible parts. From the boiled water it was 142 shown that the loss of SO 2 in shrimps during the boiling process was not totally recovered in 143 the water due to oxidation of sulfite and reaction of sulfite with food components. 144 145 Another study on shrimps [START_REF] Armentia-Alvarez | S-sulfonate determination in 615 shrimp[END_REF] showed that during bad freezing 146 storage (> -18°C), the free sulfite combined with the disulfide bounds of proteins denatured 147 by the freezing process. The longer the storage time, the higher the amount of combined 148 sulfite produced. Reduction of sulfite concentration after a storage period of 1 month was 149 reported to be between 25 and 50 % for fish products, potatoes and dried fruits (Leclercq et 150 al., 2000) 151 [START_REF] Finne | Effect of treatment, ice storage and freezing 647 on residual sulfite in shrimp[END_REF] studied the residual sulfite content in shrimps after different days of 152 conservation in ice. They demonstrated that, starting with a concentration of about 80 ppm, 153 the residual content was reduced to less than 10 ppm after 6 days of storage on ice. 154 155 Directive 95/2/EC (European Community, 1995) requires that Member States of the EU 156 monitor the intake and use of food additives in order to allow evaluation of intake trends. 157

Measures can be taken to amend the legislation if intake concerns are identified in one or 158 more Member States. The Report from the European Commission on Dietary Food Additive 159 Intake in the European Union (European Commission, 2001) describes a harmonized, tiered 160 method to estimate additive intakes. In the first step (Tier 1), theoretical food-consumption 161 data [START_REF] Hansen | Conditions for use of food additives based on a budget for an acceptable 653 daily intake[END_REF] are combined with the maximum permitted usage levels for the additive. 162

This conservative estimate was performed at European level. For the additives exceeding the 163 ADI in Tier 1, in a further step (Tier 2), national food consumption data are combined with 164 maximum permitted usage levels. Such intake estimate will still overestimate the real intake, 165 but can exclude additives, which do not pose a major risk from the most difficult estimate in 166 Tier 3: a combination of actual national food consumption data with actual levels of the 167 additive. The report from the Scientific Committee on Food (SCF) about Dietary Food 168 Additive Intake in the European Union (1998) showed that, based on Tier 1, there were 10 169 additives in the European Union for which a more refined intake estimate was needed. 170 Among those additives also sulfite was mentioned (Scientific Committee on Food). A Tier 2 171 exposure assessment in 7 countries (Belgium not included) showed that the range of estimated 172 intakes was 20-266% of the ADI for adults (European Commission). 173

The objective of this study was to perform a refined exposure assessment for sulfite intake in 174 the Belgian adult population, using the data from the national food consumption survey of 175

Materials and Methods

180

Study design 181 A semi-probabilistic approach was used to perform the exposure assessment. For the Tier 2 182 exposure assessment, maximum levels of sulphur dioxide in different foods were linked to the 183 food consumption data of these specific foods, derived from the Belgian food consumption 184 survey (2004). For some foods in the EU legislation, it is unlikely that they are consumed in 185 significant quantities or they were too difficult to link with the data from the food 186 consumption survey: burger meat with a vegetable or cereal content of minimum 4 %; 187 breakfast sausages; Longaniza fresca and Butifarra fresca; dried salted Gadidae fish; sago; 188 pearl barley; dried ginger; pulp of horseradish; pulp of onions, shallots and garlic; yellow 189 paprika in brine; flavourings on the basis of lemon fruit juice; concentrated grape juice for the 190 making of house wine; mostrarda di frutti; gelatinized fruit extracts or fluid pectin; glass 191 preserves of cut lemon; white syrup and molasses; orange-, grapefruit-, apple-and pineapple 192 juice for unpackaged sell in catering enterprises; concentrates on the basis of fruit juice with 193 at least 2.5 % pearl barley; capilhé groselha; unfermented grape juice for religious use; wine 194 without alcohol; fermented vinegar; marinated nuts; vacuum packaged sugar maize and 195 distilled alcoholic beverages with whole pears. Consequently these products were not taken 196 into account in the intake assessment. This could lead to an underestimation of the intake, but 197 this was supposed to be limited. As stated in the introduction, cooking and freezing can 198 decrease the sulfite concentration of foods. This was not taken into account either due to the 199 lack of reliable processing factors. Consequently this could lead to an overestimation of the 200 intake. 201

202

In order to perform a more refined estimate of the sulfite intake (partial Tier 3 exposure 203 assessment), on the basis of literature, available concentration values for products analyzed by 204

The Belgian Federal Agency for Safety of the Food Chain (FASFC) and the results from the 205 Tier 2 exposure assessment, it was decided to analyze concentrations of sulphur dioxide in 206 beers and potato products. In these samples concentrations were determined using a high 207 performance ion chromatography method with eluent conductivity detector, optimized and 208 validated for this purpose. In the partial Tier 3 exposure assessment, analyzed actual levels of 209 respondent reported the quantity of all foods and beverages consumed during the preceding 236 day. The 24-hour recall was carried out using EPIC-SOFT software [START_REF] Slimani | Perspectives of using the EPIC-SOFT programme in the context 696 of pan-European nutritional monitoring surveys: methodological and practical implications[END_REF]. 237 The selection of the matrix types to be analyzed was based on the results of the Tier 2 246 calculation, literature and on the available sulfite concentrations obtained from the FASFC in 247 different kinds of foods (Table 1). Both beers and pre-cut fresh potatoes or French fries and 248 frozen prepared potato (precooked or not) products were sampled. 249 250

The Belgian law (Belgian Monitor) specifies 4 categories of beer (S and I to III) depending on 251 the Plato degree. Beers are characterized according to the fermentation type, the bitterness, 252 the colour, the wort density and the alcohol concentration. Those characteristics were 253 important in the present study, for example in case of ale beers undergoing a second 254 fermentation in the bottle where sulfite can be released by the fermentation process. The 255 presence of fruits can also be a source of sulfite. Therefore, in this study, beers were 256 categorized as follows: beer table blond, beer table brown, beer table triple blond, beer table 257 non specified colour, beer pilsner, beer pilsner light, beer without alcohol, beer special blond, 258 beer special amber, beer special brown, beer special with fruits, beer special unknown colour, 259 beer gueuze, beer gueuze with fruits, beer white, beer white with fruits and beer unknown 260 type and colour. 261 262 From the Belgian food consumption survey 2004 the frequencies and amounts of beer 263 consumption for all beers were retrieved. Each brand was assigned to a group of beer, as 264 defined earlier; and some simple calculations were made to determine the percentage of 265 consumption for each beer category and each brand within each category. The number of 266 samples was taken proportional to the brand distribution in the respective categories. 267 Samples were only taken for the brands with a percentage of consumption higher than 0.5 %, 268 except for 1 brand of gueuze with fruits and 2 other brands of gueuze because they are 269 representative in their specific categories and they were observed in different stores. The only existing ISO method including sulfite analysis is the method ISO 10304-3:1997. The 277 HPIC/conductivity detector with eluent conductivity suppressor was used in this study to 278 perform the analyses. Ion chromatography is an appropriate technique for the separation of 279 sulfite from other anions like phosphate and sulphate, and it has also been recognised for the 280 quantification of sulfite [START_REF] Armentia-Alvarez | S-sulfonate determination in 615 shrimp[END_REF][START_REF] Kim | Comparison of the ion exclusion chromatographic method with the Monier-663 Williams method for determination of total sulfite in foods[END_REF][START_REF] Kim | Analysis of sulfites in foods by ion exclusion 666 chromatography with electrochemical detection[END_REF]Kim et al. 281 2000;[START_REF] Paino-Campa | Liquid chromatographic 685 determination of free and total sulphites in fresh sausages[END_REF][START_REF] Pena-Egido | S-sulfonate contents in raw and 688 cooked meat products[END_REF][START_REF] Pizzoferrato | Evaluation of an HPLC method for the 690 determination of sulphiting agents in foods[END_REF] Pulsed 282 amperometric detection necessitates very specific material and is not so easy to use because of 283 possible fouling of the electrode by matrix compounds [START_REF] Mcfeeters | Sulfite analysis of fruits and vegetables by high-676 performance liquid chromatography (HPLC) with ultraviolet spectrophotometric detection[END_REF]. The 284 most used detection method in ion chromatography is eluent suppressed conductivity. With 285 the carbonate/bicarbonate eluent, the produced species in the suppressor is H 2 CO 3 (which 286 RFIC analytical column type IonPac AS14A-5µm 3x150mm (particle size of 5µm) and 297 precolumn RFIC IonPac AG14A-5µm 3x30mm. 298

Reagents and solutions 299 300

All reagents were at least from analytical grade. Sodium carbonate and sodium bicarbonate 301 where purchased from VWR International. D-Mannitol, disodium salt of ethylenediamine 302 tetra acetic acid (EDTA) and formaldehyde were purchased from Sigma-Aldrich and 303 anhydrous sulfite was purchased from Fluka. 304

The aqueous solutions where prepared with a MilliQ Reagent Grade System's water with 305 conductivity lower then 18.2 MΩcm -1 (from Millipore coupled after a Millipore Elix 100 306 deionised water production system). Stock solutions NaHCO 3 0.3 M, Na 2 CO 3 0.2M and D-307 Mannitol 0.5 M were prepared. Those solutions were kept in the fridge. The eluent used for 308 the anion separation was the buffer NaHCO 3 /Na 2 CO 3 3mM/2mM. The sulfite extraction 309 bound sulfite extraction (Armentia-Alvarez A. et al. 1993;[START_REF] Wedzicha | Chemistry of sulphiting agents in food[END_REF]. The 1000 mg/l 312 sulphur dioxide standard stock solution was prepared by weighting 0.1968g anhydrous sulfite 313 and making a 100.0 ml solution with extraction buffer. 314 315

Sample collection, storage and pre-treatment 316

After collection of the samples, they were identified by a unique identification number and 317 registered in a Microsoft Excel sheet. Following characteristics were added: brand name, 318 product description, lot number, temperature of conservation in the store and any relevant 319 information for the project (presence or absence of sulfite mentioned on the label or usage of 320 ingredients containing sulfite). The liquid samples where stored in a fridge and the solid 321 samples where stored in the freezer (-18°C or less) until pre-treatment or analysis. 322

323

The pre-treatment of the solid samples consisted of grinding at least 200 g collected sample 324 with a kitchen robot (Robot Coupe model R 301 Ultra) in order to get piece cuts less than 3~5 325 mm. Afterwards the prepared samples were stored deep frozen. All analyses where conducted 326 within 3 weeks after collection of the samples. 327

Sample preparation 328

For the preparation of the liquid samples 100 µl of D-Mannitol 0.5 M was added to about 100 329 ml of the sample before degassing in an ultrasound bath for 5 minutes; 5.0 ml of sample was 330 then diluted in the extraction buffer by a factor of 5 (final volume = 25.0 ml) and the extract 331 was filtered through a PVDF syringe filter with 0.2 µm pore size before injection on the HPIC 332 system. For the preparation of the solid samples, after a short period of unfreezing, about 5 g 333 of sample (weighted with at least 3 digits after the point) was weighted in a P.E. tube of 50 ml 334 to be centrifuged; 25.0 ml of extraction buffer (measured with a graduate cylinder of 25 ml) 335 was added to the sample; the sample was then homogenated in an Ultra-Turrax for a 336 minimum of time, so that the mixture looked like creamy (10 to 15 seconds suffice). 337

The tube was closed and set in a centrifuge at 7500 rpm for 10 minutes; the surnatant extract 338 was filtered through a PVDF syringe filter with 0.2 µm pore size before injection on the HPIC 339 system. 340

Calibration curve -linearity 342

Due to the range of maximum permitted levels in the different kinds of foods it was chosen to 343 use a calibration curve ranging from 0.1 to 200 mg SO 2 /l. 344 By diluting 1000 mg SO 2 /l extraction buffer a series of eight dilutions was prepared (0.1, 0.5, 345 1. 5, 10, 50, 100, and 200 mg SO 2 /l). Blank was used as 0 mg SO 2 /l. The best fit was used in 346 order to maximize the coefficient of determination (> 98 %) and to minimise the residues at 347 each level. 348

Validation 349

The method was validated for linearity, precision (repeatability, reproducibility), accuracy and 350 limit of detection according to the procedure described by Van Loco et al (Van Loco and 351 Beernaert 91-94). After multiple tests for concentration ratios of the NaHCO 3 /Na 2 CO 3 buffer 352 it was decided to use the composition 3 mM/2 mM. This composition permits the separation 353 of phosphate, sulfite, and sulphate with a total runtime period of 25 minutes (stabilization 354 included). Different stabilization compounds (EDTA, D-Mannitol and formaldehyde) were 355 tested according to previous reports. D-Mannitol was chosen due to its best effect on the 356 sample extract. Some stability tests were made with a standard solution 10 mg SO 2 /l. The 357 responses of a standard solution prepared in water and the same amount of standard solution 358 prepared in a pH~ 9.3 buffer with 10 mM D-mannitol were compared. 359 360

The final sulfite extraction buffer chosen after pH test in mixture with the sample matrix was 361 NaHCO 3 /Na 2 CO 3 /D-Mannitol at concentration levels of 9 mM/16 mM/10 mM respectively. 362

The pH of this buffer was between 9 and 10. The extracts prepared with beer resulted in a pH 363 of 9.3 and of 8.7 when prepared with frozen French fries. NaOH/formol was tested for the 364 extraction of sulfite from beer. But the chromatographic separation gave very high 365 background noise after a few injections. The material was not able to work with an organic 366 modifier like acetone in high proportion like described in Metrohm application note S-95. 367 368 To test linearity it was opted for the weighted quadratic regression of the heights in function 369 of the extract concentration. This type of regression resulted in a determination coefficient 370 greater than 0.99 and residues lower than 15 % for the lowest concentration point. 371 The range for the calibration was 0.1 to 100 mg/l extract (The injection of the point 200 mg/l 373 was kept in order to be able to quantify higher amounts from a new regression). The range in 374 the extract corresponded to a range in the sample going from 0.5 to 500 mg/kg (or mg/l, 375 depending of the nature of sample) if the dilution factor was taken into account. 376 377 In order to test the repeatability and reproducibility a sample (table beer) was fortified at 4 378 levels of concentration corresponding to 0, 5, 10 and 15 mg/l of the sample (which 379 corresponds to 0, 1, 2 and 3mg/l in the extract). The test was conducted in triplicate for each 380 level and repeated for each level at 3 different days. The non fortified sample did not result in 381 a detectable response and the obtained result of reproducibility was lower than 12.6 %. 382

383

The apparent recovery was, on average, for the 3 levels of concentration, 73 ± 6 at 3 384 concentration levels. A lower recovery may be explained by a possible reaction of SO 2 to 385 some compounds present in the matrix and/or by oxidation after the dissolution of ambient 386 oxygen into the extracts. Therefore, it was decided to correct the results for the recovery of 387 the control standard included in the series of analysis. 388 389 In beer sample, the sulfite peak was poorly separated from matrix interferences. The 390 minimum detectable concentration in the extract was about 0.5 mg/l. This corresponds to a 391 quantification limit (LOQ) of 1 mg/l extract which corresponds to 5 mg/l (or mg/kg) in the 392 sample. This is also the lowest tested concentration. 393 394

Exposure assessment 395

Only respondents with two completed 24-hour recall interviews were included in the analyses 396 (n= 3083; 1546 men and 1537 women). 397 398The individual intake of sulphur dioxide from a certain food product was estimated using the 399 following equation: 400

i i i bw x c day kgbw mg y × = ) / / ( 401
where y i is the intake of sulphur dioxide by individual i from a particular food (in mg per kg 402 bodyweight per interview day), c is the concentration of sulphur dioxide in that food (mg per 403 kg), x i is the consumption of a certain food by individual i (kg) and bw i is the self-reported 404 body weight of individual i (kg). To estimate the total intake of sulphur dioxide per food 405 As mentioned before, two approaches were used for the calculation of sulphur dioxide intake. 408

In the Tier 2 approach, c i represents the maximum allowed concentration in each food. The 409 maximum permitted levels of sulphur dioxide used in different foods are listed in the 410 European Directive 95/2/EC (European Community, 1995). In the Tier 3 approach, c i 411 represents the actual average concentration of sulphur dioxide that is observed in a particular 412 food. 413 414The usual intake distribution for sulphur dioxide was estimated with the Nusser method 415 [START_REF] Nusser | A semiparametric transformation 680 approach to estimating usual daily intake distributions[END_REF] using the C-side software (Iowa State University, 1996). Several 416 statistical methods are available to estimate usual intake distributions with the correct mean, 417 variance and skewness. These statistical procedures adjust for within-person or day-to-day 418 variability. Of all different statistical procedures, the Nusser method [START_REF] Nusser | A semiparametric transformation 680 approach to estimating usual daily intake distributions[END_REF] is 419 highly recommended because it eliminates the intra-individual variance and additionally 420 transforms the data to obtain approximately normally distributed data. The method is suitable 421 to estimate usual intake distributions in a population both for normally and non-normally 422 distributed foods and nutrients. The usual intake distribution was weighted and adjusted for 423 the age and sex distribution of the Belgian population and adjusted for day of the week and 424 season. 425 426

Results and discussion

428

As an adult inhales about 15 m³ of air per day, this means that in the worst case the amount of 429 SO 2 intake from the environment is 215 µg/m³ x 15 m³= 3225 µg/ day = 3.2 mg/day. 215 430 µg/m³ is the highest concentration of SO 2 measured in Belgium in 2005. Since the average 431 body weight of a Belgian adult is around 70 kg, the final SO 2 intake from the environment is 432 estimated to be 0.045 mg/kg bw/day. From those results it was decided to neglect the 433 environmental contribution to SO 2 intake in Belgium. 434

435

The usual sulfite intake of the Belgian adult population estimated by the Tier 2 approach was 436 0.34 mg/kg bw/day (49 % of the ADI) at the mean level of consumption (Table 2). At the 437 97.5 th percentile of the population, the sulfite intake was estimated to be 1.1 mg SO 2 /kg 438 bw/day (157 % of the ADI). It was observed that the ADI was exceeded beyond the 90 th 439 total intake was estimated to be 71 %. Including only wine consumers (55.1%), the ADI was 441 reached at the 57.5 th percentile of population (data not shown). 442 443 Wine contributed for more than 50 % to the total intake of sulfite. Sugar and glucose-based 444 products, beers, processed fruits and processed potatoes contributed each between 7-13 % to 445 the total intake, while shellfish and processed vegetables contributed each about 4 % to the 446 total intake. Condiments and cereal products contributed to less than 1 % to the total average 447 sulfite intake (Table 2). 448 449 Concentrations of sulfite in beer samples (n=46) were all under the detection limit (LOD) of 5 450 mg/l; these results were different from the concentrations found in other countries. For 451 example, in France [START_REF] Mareschi | Estimation of consumption of sulfite 674 in France[END_REF] and Italy [START_REF] Leclercq | Dietary 670 intake exposure to sulphites in Italy--analytical determination of sulphite-containing foods 671 and their combination into standard meals for adults and children[END_REF] the average sulfite 452 concentrations found in beer were respectively 7.5 and 15 mg SO 2 /l. In these studies the 453 Monier-Williams method was used to perform the analyses. It is known that this method has 454 possible interferences with volatile compounds. The method used in the present study does 455 not have this disadvantage. With the limit of quantification of 5 mg SO 2 /l it was not possible 456 to determine any residual SO 2 levels in the beers analyzed. Provided this and the results 457 reported by other countries obtained by more sensitive electrochemical methods of analysis 458 [START_REF] Dvorak | 630 Determination of total sulphur dioxide in beer samples by flow-through chronopotentiometry[END_REF]) it was decided not to perform an intake estimation of sulfite from beer 459 using a null concentration of sulfite in beers. It is known that sulfite could be produced during 460 the natural fermentation process in the bottle. Therefore half of the LOQ concentration was 461 used (medium bound approach) instead of the null concentration to perform the intake 462 estimation. 463 464 For potato products 6 samples out of 21 analyzed samples resulted in SO 2 levels above the 465 quantification limit (LOQ) of 5 mg/l and only one sample with no sulfite declared on the label 466 resulted in a concentration higher than the LOQ. This positive sample was a fresh pre-cut 467 product in which the concentration was 9.9 mg SO 2 /kg. For the samples with a concentration 468 above LOQ, an average concentration value of 11.6 mg SO 2 /kg was found and the results 469 were spread between 9.0 and 13.5 mg SO 2 /kg. In total there were 83 samples checked for 470 sulfite on the label, while only 21 samples were analyzed. If all samples checked on the label 471 for sulfite would be considered (n=83) instead of only the analyzed ones, and if it is assumed 472 there are no frauds on sulfite usage, only 6 of 83 products were found to have a sulfite 473 This might result in an overestimation of the intake since only 7 % of selected and analyzed 476 potato products had a SO 2 concentration above the LOQ. However, some consumers might by 477 loyal to these particular kinds of brands. In addition this actual level used (11.6 mg/kg) is still 478 far below the maximum permitted levels (50-100 mg/kg SO 2 ). 479 480 From the Belgian Federal Agency for the Safety of the Food Chain (FASFC), sulfite analysis 481 results for different kinds of foods sampled between 2003 and 2006 were obtained (Table 1). 482

These results show that average actual levels found are often much lower than maximum 483 levels. 484

After considering the data from different years, it was decided to take the average results from 485 the last year only or from different previous years. For each food group, the average 486 concentration of the samples having a concentration above LOQ (5 mg/l) was used, in order 487 to account for consumers being loyal to certain brands of products. There was a drastic 488 decrease (about factor 10) in SO 2 concentration in dried tomatoes from 2004 to 2005 due to 489 the fact that in 2004 there were found levels above the maximum while no levels above the 490 maximum were found in 2005; therefore only the 2 last year's results were taken into account 491 (2005 and 2006). The available number of data for each kind of dried fruits, except dried 492 apricots, is low; therefore it was decided to use the average concentration of sulfite in dried 493 fruits (excluding dried apricots) (data from 2006) for dried fruits other than apricots. The 494 concentration assigned to dried apricots was the average of the 2005 and 2006 measurements 495 in dried apricots. For wines, the average concentration found by the FASFC in white and red 496 wines for the years 2004 and 2006 was assigned. 497 498 Measurements made in France [START_REF] Mareschi | Estimation of consumption of sulfite 674 in France[END_REF] during the period 1989-1992, showed 499 that the highest average SO 2 concentrations were found in salted fish fillets and dried fruits 500 (more than 625 mg SO 2 /kg), followed by mustard (225 mg SO 2 /kg) and peeled potatoes (100 501 mg SO 2 /kg). Wines resulted in an average concentration of 75 mg SO 2 /l (no differences where 502 made between red and white wines). Other measurements made during the same period in the 503 United States [START_REF] Daniels | Survey 624 of sulphites determined in a variety of foods by the optimized Monier-Williams method[END_REF] showed higher concentrations in dried fruits, mashed 504 potatoes and wines. More recent (2000)(2001)(2002)(2003)(2004)(2005) studies made in Italy [START_REF] Leclercq | Dietary 670 intake exposure to sulphites in Italy--analytical determination of sulphite-containing foods 671 and their combination into standard meals for adults and children[END_REF] and 505 Australia (Food Standards Australia New Zealand, 2005), showed that the sulfite level content 506 in food tends to diminish in some kinds of foods like cider, beer, died fruits and wines. 507 508

The results of the partial Tier 3 based on the analyzed products and on the maximum levels 509 for the other food groups, are given in Table 3. The mean intake of sulfites in the Belgian 510 adult population was estimated to be 0.19 mg/kg bw/day, corresponding to 27 % of the ADI. 511 At the 97.5 th percentile of the population the usual intake was 0.61 mg SO 2 /kg bw/day (87 % 512 of the ADI). It was found that for the average consumer wine contributed up to 14 % of the 513 ADI). The sugar group still had a relative significant contribution to the intake, but actual 514 concentrations were not available. It is known, however, that food producers try to avoid the 515 need for allergen labelling of sulfite, by reducing the levels below 10 mg/kg. 516 517 Some groups of the population could be more exposed than others to sulfite intake, in 518 particular wine consumers. The probability of approaching the ADI by wine consumers will 519 depend on the particular choice of wine (sulfite concentration) and the consumption level. 520 This is hard to estimate. An exceeding of the ADI can not be excluded for a limited 521 percentage of the population of wine consumers, who might consume wine with a higher 522 average concentration than the average concentration used here in the tier 3 calculations, as 523 can be shown from the tier 2 estimates (where the consumption of wine alone can reach the 524

ADI). 525 526

The fact that some food groups were not taken into account, could have led to a certain 527 underestimation of the intake. However, the reduction that takes place during further 528 preparation of foods was not taken in to account but is a factor of overestimation of the intake 529 of sulfites. Further, the fact that no illegal uses of sulfites in certain food groups were taken 530 into account, could also have led to an underestimation. In 2005 1002 tests with malachite 531 green were performed in chopped meat by the FASCA of which 54 were positive. 43 of these 532 were confirmed in the laboratory. The average sulfite content in the samples exceeding the 533 LOQ was on average 474.1 +/-487.1 mg/kg and 1782.6 mg/kg at the 97.5 percentile. 534 535 In 1999, JECFA reviewed the intake estimation of sulfite from different countries (JECFA, 536 1999): Aus-NZ, China, Finland, France, India, Japan, Spain, United Kingdom and United 537

States. The estimated range of intake of sulfite (expressed as sulphur dioxide) was between 10 538 and 1400 % of ADI. In should be noted that data modelling differed between countries. The 539 (Food Standards Australia New Zealand, 2005) showed an 578 average intake of sulfite in the population of 0.2 mg SO 2 /kg bw/day (lower bound estimation; 579 concentrations not detected set to zero). With the upper bound estimate, the intake obtained 580 was the same (30 % of the ADI). The same calculation for the 95th percentile of the 581 population showed an intake estimate of 0.9 mg SO 2 /kg bw/day (lower bound or upper 582 bound), that corresponded to 130 % of the ADI. It was shown that in Tier 2 approach the 583 intake estimated for high consumers was 1400 % of the ADI (JECFA) while with actual data, 584 the intake for the high consumers became 130 %, which is more than 10-fold difference (yet 585 still above the ADI). 586 587 In this study, actual typical levels of sulfites were much lower than the maximum levels set in 588 the legislation for several food groups. This resulted in average intake estimates going from 589 0.34 mg/kg bw/day in Tier 2 to 0.19 mg/kg bw/day in the partial Tier 3. Wine showed a 590 decrease in the sulfite intake estimate from 24 % (Tier 2) to 14 % (partial Tier 3) of the ADI 591 for the mean consumer and from 116% (Tier 2) to 69% (partial Tier 3) for the 97.5 percentile. 592 However, the choice of wine of a consumer might be different than average. 593 594

Conclusion

595

In conclusion, it was shown that the intake of sulfites in the Belgian adult population is likely 596 to be below the ADI with the possible exception for some high consumers of wines with high 597 sulfite levels. As a consequence, the intake of sulfites by wine consumers remains a point of 598 attention. Further it was shown that for several food groups actual typical levels of sulfites are 599 much lower than the maximum levels set in the legislation. 600

  objective was to identify the food groups contributing most to the total intake 176 of sulfites in Belgium. 177 178

  different foods were linked to the food consumption data of these specific 210 foods, derived from the Belgian food consumption survey(2004). For foods for which no 211 analyzed levels were available, maximum concentrations were used, as in the Tier 2 exposure 212 assessment. No illegal uses of sulfites in food groups in which no use of sulfites is authorised, 213 were taken into account in this study. 214 215 Although the regulation specifies maximum levels for specific groups, in this paper, the 216 results of the analyses were grouped to ensure a clear data presentation. The grouping was 217 performed according to the EU directive 95/2/EC (European Community, 1995) as follows: 218 beers; wines; sugars and glucose-based products; condiments and dessert sauces; gelatine and 219 meat substitutes; shellfish; cereal-based products (starches and biscuits); processed potatoes, 220 processed fruits (including dried fruits, candied fruits, jams, jellies and marmalades, lemon 221 and lime juice, fruit fillings and fruits in vinegar, brine, oil) and processed vegetables 222 (including dried tomatoes, mushrooms, vegetables in vinegar, brine and oil and white the national food consumption survey 2004 were used to perform the 228 exposure assessment. Aims, design and methods of this survey are described elsewhere (De 229[START_REF] Vriese | The Belgian food consumption survey: aims, design and methods[END_REF]. The target population comprised all Belgian inhabitants of 15 years or 230 older. The sample included 3245 participants randomly selected from the National Register, 231 using a multi-stage stratified procedure. 232 233 Information on dietary intake was collected by two non-consecutive 24-hour recalls in 234 combination with a food frequency questionnaire. During the 24-hour recall interviews the 235

  This program allows obtaining very detailed information about the foods consumed and the 238 recipes used in a standardized way. 239 240 3083 participants, of which 1537 women and 1546 men, completed two 24-hour recalls. 241 Participants were categorized into four age groups: 15-18 years (n=760), 19-59 years (n=830), 242 60-74 years (n=789) and 75 years or older (n=704). 243

  270 271 Collection of samples was performed in 2007 in 5 major supermarket chains with a good 272 market share, which are visited by approximately 77.5 % of the Belgian population in search 273for their food provisioning (CRIOC -OIVO). In total for both categories 67 samples (21 for 274 potato products and 46 for beers) were collected and analysed.

  decomposes in CO 2 and H 2 O). 287 288 Material 289 For sample preparation, a Robocoup mixer 3000, a T25 Ultra-Turrax (IKA) and a centrifuge 290 Beckman Avanti J-25 operated at 7500rpm were used. All extracts where filtered through 291 0.2µm PVDF syringe filters. The analytical system was composed of a Waters 717+ auto 292 injector, an HP 1050 pump, an Alltech Suppressor model 640 and an Alltech conductivity 293 detector model 650. The acquisition of the signal and data treatment (calibration curve and 294 extract concentration) were made by a computer equipped with an analogue/digital converter 295 and ChromQuest software version 4.1. The separation column was composed of a Dionex 296

  mixture of NaHCO 3 /Na 2 CO 3 /D-Mannitol 9mM/16mM/10mM. This resulted in a 310 final extraction pH between 8 and 10, as proposed in literature for free sulfite and reversible 311

  day, individual daily intakes of sulphur dioxide from different foods were added 406 up. 407

  population. At this percentile the contribution of wine consumption to the 440

  . For the evaluation of the sulfite intake from potato products it was 474 decided to use the average concentration of the samples having a concentration above LOQ. 475

  kg) was between 27 and 93 % of the ADI for a child of 21 kg. The study 574 was based on the actual food consumption during 4 months and maximum allowed sulfite 575 content in food. 576 577 A recent survey in Australia

Table 1 .

 1 Average sulfite concentrations * in different foods (data from the control program of the Federal Agency for Safety of the Food Chain, Belgium) Including only samples exceeding the LOQ of 5 mg/L to calculate the average

	Matrix	Year	Number of	samples	Number of	samples	exceeding the	LOQ	Average	sulphite	concentration*	(mg/kg) 1	Standard	deviation	(mg/kg)	Minimum	value (mg/kg)	Maximum	value (mg/kg)	Median	(mg/kg)	Percentile 95	(mg/kg)	Percentile 97.5	(mg/kg)
	Dried tomatoes	2005-2006	65		9				7.3		3.7		2.0	13.0	7		12.6	12.8
	All dried fruits (defined or undefined; Apricots included)	2006	40		27			384		320		9.0	917	267	876	903
	All dried fruits (defined or undefined; Apricots excluded)	2006	25		14			179		238		9.0	781
	Dried apricots	2005-2006 181		176			533		308		0.0	1448	478	1083	1182
	Wines (Red, White or undefined)	2004-2006	83		82			114		45.4	30.0	240	113	180	184
	of which Red	2004 & 2006	28		28			78.6		33.2	30.0	160	74.5	142	149
	White	2004 & 2006	39		39			139		28.1	91.0	198	136	180	181
	Raw Shrimps	2006	10		1				10.0			/		10.0	10.0	10	10.0	10.0
	Mustard	2006	4			1				18.0			/		18.0	18.0	18	18.0	18.0
	Mushrooms	2006	5			2				11.5		3.5		9.0	14.0	11.5	13.8	13.9
	Jams	2006	10		10			16.0		11.6	1.0	33.0	14	32.6	32.8
	Potato products	2006	16		9				3.8		3.4		0.3	10.0	3		9.6	9.8
	Beers	2006	9			3				1.3		0.6		1.0	2.0	1		1.9	2.0
	*

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Acknowledgements

603

The authors declare not having any conflicts of interest. The authors acknowledge the funding 604 of the study by the Federal Ministry of Health, Food Chain Safety and Environment and the

The usual dietary intakes were weighted and adjusted for the age and sex distribution of the Belgian population 2004 and adjusted for interview day and season. Total number of consumption days is 6166; total number of consumers is 3083 The acceptable daily intake (ADI) for sulphite is 0.7 mg/kg bw/day * including dried fruits (including dried apricots), candied fruits, jams, jellies and marmalades, lemon and lime juice, fruit fillings, fruits in vinegar, brine, oil. ** including dried tomatoes, mushrooms, vegetables in vinegar, brine and oil, and white vegetables The usual dietary intakes were weighted and adjusted for the age and sex distribution of the Belgian population 2004 and adjusted for interview day and season. Total number of consumption days is 6166; total number of consumers is 3083 The acceptable daily intake (ADI) for sulphite is 0.7 mg/kg bw/day * including dried fruits (including dried apricots), candied fruits, jams, jellies and marmalades, lemon and lime juice, fruit fillings, fruits in vinegar, brine, oil. ** including dried tomatoes, mushrooms, vegetables in vinegar, brine and oil, and white vegetables ° Maximum concentrations used instead of analyzed concentrations °° Combination of maximum concentrations and analyzed concentrations used depending on the availability of analyzed concentrations for the food items in the considered food groups http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

F o r P e e r R e v i e w O n l y


Antonio Logrieco
email: antonio.logrieco@ispa.cnr.

Lia Ferracane

Angelo Visconti

Alberto Ritieni

R Ferracane

Natural occurrence of fumonisin B2 in red wine from Italy

Keywords: Fumonisins, Aspergillus species, wine, mycotoxins, food safety 12

The potential risk of exposure to fumonisin B 2 (FB 2 ) in the grape-wine chain has been 3 recently pointed out after reporting Aspergillus niger in grapes and its ability to produce 4 FB 2 and FB 4 . The occurrence of these two fumonisins in wine was investigated by 5 LC/MS/MS in fifty-one market samples (45 red, 5 white and one rosè wine) produced in 6 various Italian regions. Nine samples of red wine were found contaminated by fumonisin B 2 7 at levels ranging from 0.4 to 2.4 ng/mL, while FB 4 was not detected in any of the tested 8 samples. This is the first report on the natural occurrence of FB 2 in wine indicating that, 9 although al low levels, there is a potential risk of FB 2 exposure for the wine-consumer.

Grapes and derived products such as grape juices and wines have been reported as 3 potentially contaminated with ochratoxin A [START_REF] Zimmerli | Ochratoxin A in Table wine and grape-juice: occurrence and 6 risk assessment[END_REF]Visconti et al. 4 1999). "Black aspergilli", mainly Aspergillus carbonarius followed by A. niger and A. 5 tubingensis, are the main source of contamination by ochratoxin A of grapes and wines 6 (Abarca et al. 2001; [START_REF] Battilani | Epidemiology of toxin producing fungi and ochratoxin 1 A occurrence in grape[END_REF]Belli et al. 2004; [START_REF] Perrone | Ochratoxin A production and 12 AFLP analysis of Aspergillus carbonarious, A. tubigensis and A. niger strains isolated from 13 grape in Italy[END_REF]. [START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus Section 8 Nigri) on some drying varieties of irrigated grapes[END_REF] Ochratoxin A has been shown to be nephrotoxic, immunotoxic and teratogenic to 8 several species of animals and to cause kidney and liver tumours in mice and rats 9 [START_REF] Who | Ochratoxin A. In Safety Evaluation of Certain 3 Mycotoxins in Food[END_REF]. [START_REF] Logrieco | Occurrence of fumonisin B1 11 and B2 in Fusarium proliferatum infected asparagus plants[END_REF] Recently, some industrially important strains of A. niger were reported to produce, 11 in addition to ochratoxin A, the mycotoxin fumonisin B 2 (FB 2 ) [START_REF] Frisvad | Fumonisin B 2 5 production by Aspergillus niger[END_REF]. [START_REF]Commission Regulation (EC) 1881/2006 setting maximum 13 levels for certain contaminants in foodstuffs[END_REF] Then, it has been shown that strains of A. niger from coffee beans and raisins produced 13 together with FB 2 also FB 4 [START_REF] Noonim | Fumonisin 9 B2 production by Aspergillus niger in Thai coffee beans[END_REF]; [START_REF] Mogensen | Production of fumonisin B 2 and B 4 3 by Aspergillus niger on grapes and raisins[END_REF]). Logrieco et al. 14 (2009) reported some strains of A. niger from grape producing FB 2 and the occurrence 15 of FB 2 in two must samples, and pointed out the potential mycotoxicological risk of 16 grape and derived products colonized by this species. Fumonisins have been [START_REF] Visconti | Determination of ochratoxin A and domestic and 18 imported beers in Italy by immunoaffinity column-linked HPLC[END_REF] extensively studied as mycotoxins with cancer-promoting activity and are associated 18 with a number of animal and human diseases [START_REF] Marasas | Discovery and occurrence of the fumonisins. A historical perspective[END_REF]). Mogensen

Materials and methods 3

Wine samples. Fifty-one bottled samples of red (45), white (5) and rosè (1) wine were 4 randomly purchased from the market in Apulia (Table 1). These wines orignated from most 5

Italian wine producing areas including Veneto [START_REF] Logrieco | Occurrence of fumonisin B1 11 and B2 in Fusarium proliferatum infected asparagus plants[END_REF], Apulia (6), Piedmont (6), Trentino Alto 6 Adige (5), Campania (4), Tuscany (4), Lombardy (3) Emilia Romagna (3), Sardinia (2), 7

Sicily (2), Friuli Venezia Giulia (1), Lazio (1), Umbria (1). The year of production ranged 8 from 2004 to 2008 with most in 2007. 9 10

Fumonisins analysis 11

Fumonisin B 2 standard was purchased from Sigma-Aldrich (Milan, Italy), while fumonisin 12 B 4 was kindly offered by Mark Busman (National Center for Agricultural Utilization 13 Research, Peoria, Illinois, USA). Standards were dissolved in methanol at 1 mg mL -1 14 concentration, working solution were prepared using a solution CH 3 OH/H 2 O (70:30, v/v). [START_REF]Commission Regulation (EC) 1126/2007 setting maximum 16 levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and 17 maize products[END_REF] In Figure 1 are reported the chemical structures of FB 2 and FB 4 . Fumonisin B 1 (Sigma-16

Aldrich, Milan, Italy) was also analyzed in the tested wines. 17 FB 2 and FB 4 were analysed using 7.5 mL of wine sample mixed with 2.5 mL of 18 methanol and stirred for 2 min at room temperature. The sample was acidified to pH 3 19 using 1 M hydrochloric acid and then loaded on a reversed phase Strata C18-E 500 mg 20 cartridge (Phenomenex, Torrance, CA, USA), previously conditioned with methanol (5 21 mL) and water (5 mL). The column was washed with water (3 mL), methanol/water (1:3, 5 22 mL) and then fumonisins mixture was eluted with 10 mL methanol. The eluate was dried 23 using a centrifuge evaporator (Savant Instruments Inc., Farmingdale, NY, USA), dissolved 1 in 750 µL of methanol/water (70:30), ultracentrifuged at 13,400 g for 3 min and then the 2 residue was used for LC/MS/MS analyses. Recoveries for the toxins were measured by 3 spiking 7.5 mL of wine with FB 2 and FB 4 at three different concentrations (1 ng/mL, 5 4 ng/mL and 10 ng/mL); each experiment was repeated three times on two non consecutive 5 days by different operators. FB 2 and FB 4 recoveries in spiked samples averaged 83.2 % and 6 79.4 %, respectively. The calibration curve showed good linearity in the range 1-1000 7 ng/mL. All chromatographic points of calibration curve were run in triplicate and the mean 8 values are reported. The standard deviation was less than 5%. 9

The detection limit (LOD with a signal to noise ratio of 3) for FB 2 was 0.25 ng/mL while 10 the quantification limit (LOQ with a signal to noise ratio of 9) was 0.5 ng/mL . The detection 11 limit (LOD with a signal to noise ratio of 3) for FB 4 was 0.7 ng/mL while the quantification 12 limit (LOQ with a signal to noise ratio of 9) was 1.5 ng/mL . [START_REF] Logrieco | Epidemiology of toxigenic 14 fungi and their associated mycotoxins for some Mediterranean crops[END_REF] Chromatographic separation of extracts was performed using an HPLC apparatus 14 equipped with two micropumps Series 200 (Perkin Elmer, Canada Shelton, USA) and a 15 % of all A. niger strains [START_REF] Nielsen | Review of 6 secondary metabolites and mycotoxins from the Aspergillus niger group[END_REF]).This risk is real also considering the consistent 14 presence of A. niger on grapes during the growing season, generally higher than that of 15 other black Aspergilli [START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus Section 8 Nigri) on some drying varieties of irrigated grapes[END_REF][START_REF] Chulze | Occurrence of ochratoxin A in wine and 6 ochratoxigenic mycoflora in grapes and dried vine fruits in South America[END_REF]. [START_REF] Logrieco | Fumonisin 17 B2 production by Aspergillus niger from grapes and natural occurrence in must[END_REF] Wine is also exposed to other mycotoxicological risk due to the natural occurrence of 17 to wine exposure, and assuming a contamination level (approximated per excess) equal to 1 the average of the positive samples of this study, i.e. about 1 ng/mL FB 2 , the average 2 exposure to fumonisins through wine consumption would be of about 0.003 µg/kg of body 3 weight (b.w.) per day. Such level of exposure would be far below the guideline provided for 4 risk characterization by the WHO-FAO joint expert committee for food and additives 5 (JECFA) that established the provisory maximum tolerable daily intake (PMTDI) of 2 6 µg/kg of body weight to FB 1 , FB 2 and FB 3 alone or in combination. The contribution of 7 wine to mean daily intake of fumonisins can be considered negligible in the case of people 8 drinking wine manufactured in Italy. Wine alone does not supply the diet with an amount of 9 fumonisins corresponding to PMTDI, but combined with possible fumonisins exposure 10 from the maize based staple diet, it could become a cause for concern when climatic 11 conditions are particularly favourable to the development of toxigenic A. niger strains on 12 grape-vines and the consequent transfer of FB 2 to wine. Nevertheless, the number of wine 13 samples included in this study was limited and relevant to Italy only. Additional wine 14 samples from other wine producing countries (e.g. Spain, France, Australia, South Africa, 15 etc) should be investigated for their contamination by fumonisins as well as ochratoxin A in 16 order to have a general view of mycotoxicological risk for the grape-wine chain. 17 southern regions of Europe and the lower latitude [START_REF] Visconti | Managing ochratoxin A risk in the 1 grape-wine food chain[END_REF]). Aspergillus niger, 1 which includes FB 2 producing strains, has been frequently isolated from grapes in Europe 2 [START_REF] Logrieco | Epidemiology of toxigenic 14 fungi and their associated mycotoxins for some Mediterranean crops[END_REF], Australia [START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus Section 8 Nigri) on some drying varieties of irrigated grapes[END_REF]) and South America (Chulze et al. 3 2006) and show significant differences in the physiology and ecology with respect to A. 4 carbonarius, the main responsible for wine contamination by ochratoxin A. 5

In conclusion, this report provides a new knowledge about the FB 2 occurrence in wine 6 that, added to previous data relevant to its occurrence in grapes and musts, provides a clear 7 evidence that FB 2 contamination can occur in the grape-wine chain and, under conditions 8 particularly favourable to its formation, could result in a potential risk for human and 9 animal health.

5 (

 5 Gemini 5u C 18 110 Å column (150 x 2 mm) (Phenomenex, Torrance, CA, USA). The 16 chromatographic solvents were water 0.1% formic acid (eluent A) and CH 3 CN/MeOH 17 (80:20 v/v), 0.1% formic acid (eluent B). The gradient program was as follow: 30-100 % B 18(8 min), 100 % B (3 min), 100-30% B (3 min), at a constant flow of 0.2 mL/min. The 19 injection volume was 20 µL. MS/MS analyses of FB 2 and FB 4 were performed on an API 20 3000 triple quadrupole mass spectrometer (Applied Biosystems, Canada) equipped with a 21 TurboIonspray source. The following setting were used: Capillary Voltage (IS) 5,000 V,[START_REF]Guidance for industry: Fumonisin levels 1 in human foods and animal feeds[END_REF] Declustering Potential (DP) 70 V, focus potential 340 V. Collision energy (CE) 51 V and 1 42 V generated daughter ions 336.2 and 512.2 m/z, for FB 2 ; whereas for FB 4 collision 2 energy (CE) 48V and 37V generated daughter ions 338.5 and 514.3 m/z. These mass 3 parameters were optimised by preliminary experiments with a direct infusion of pure 4 compounds (10 µg/mL) at a constant flow rate of 6 µL/min using a model 11 syringe pump Harvard Apparatus, Holliston, MA, USA) into the mass spectrometer source. Drying gas 6 (air) was heated to 350°C. Analyses were performed in the positive ion mode inMRM 7 (multiple reaction monitoring), ion mode, FB 2 showed an [M+H] + ion at m/z 706.4, FB4 8 showed an [M+H] + ion at m/z 690.4. 9In Figure2is reported the Total Ion Chromatogram (TIC) of a sample of wine 10 artificially contaminated with 1 µg/L of FB2 and FB4 been using to evaluate the 11 recovery performances of fumonisins from contaminated natural samples and to 12 identify eventual molecules interfering in the fumonisin analysis.[START_REF] Logrieco | Epidemiology of toxigenic 14 fungi and their associated mycotoxins for some Mediterranean crops[END_REF] 14Results and discussion 15This is the first report relevant to FB 2 and FB 4 surveillance in wine samples.Shephard et al. 16 (2005) reported fumonisins determination in maize beer in South Africa by reversed-phase 17 HPLC, after o-phthaldialdehyde (OPA) post-run derivatization. The analytical method 18 reported in this paper represents a real advance in the field of mycotoxin surveillance in 19 foods by using tandem mass spectrometry techniques. In particular, these minimal changes 20 made to the method of[START_REF] Shephard | Fumonisin mycotoxins in traditional xhosa maize beer in South Africa[END_REF] allow to reduce the time of the analysis and 21 handling of samples of alcoholic beverages at risk of contamination by fumonisins with 1 benefits both on the values of recovery of the analytes and cost analysis. 2Frisvad et al. (2007) reported a semiquantitative method for fumonisins analysis on agar 3 using selective ion monitoring electrospray LC-MS with a time-of-flight mass spectrometer. 4 Our work was a quantitative analysis of FB 2 and FB 4 on wine by liquid chromatography 5 coupled to electrospray ionization tandem mass spectrometry (LC/MS/MS). Acquisition 6 was in positive ion mode, FB 1 showed an [M+H] + ion at m/z 722.4, FB 2 showed an [M+H] + 7 ion at m/z 706.4, FB 4 showed an [M+H] + ion at m/z 690.4. CAD (Collision Activated 8 Dissociation) of these protonated molecules produced product ions and the two main 9 daughter peaks were selected to be monitored in the MRM mode. Previous studies on 10 fumonisins analysis by LC/MS/MS (Faberi et al. 2005; Bartók et al. 2006) showed that the 11 most abundant products ion were m/z 352.0 and m/z 336.0, respectively for FB 1 and FB 2 , 12 corresponding to the loss of water and two tricarboxylic acids (TCA) from the alkyl 13 backbone [MH-2TCA-H 2 O] + . In addition to these ions, our MS/MS spectra showed the 14 product ions at m/z 528.1 and m/z 512.2 corresponding to the loss of water and a 15 tricarboxylic acid [MH-TCA-H 2 O] + (

  ochratoxin A mainly due to the presence of A. carbonarius in field(Abarca et al. 2001; 18 Battilani et al. 2003). An European report estimated that wine is the second major source of 19 OTA intake in European populations (European Commission, 2002) and a maximum limit 20 for OTA in wine of 2 µg/kg was regulated in the EU(European Commission, 2006). 21 By assuming a daily consumption of 200 mL wine for a 70 kg person, i.e. one or two 22 glasses of wine as recommended by the World Health Organization for an healthy response 23

Further

  studies are needed to better define the environmental conditions and other 18 factors influencing the toxin production, as well as the widespread of fumonisins producing 19Aspergillus, and the fate of fumonisins during the winemaking process. Geographic 20 differences have been shown in Aspergillus and ochratoxin A contamination of grapes and 21 wines, indicating higher incidence and contamination levels in wines and grapes from the 22

10 11 12 This 16 17 AbarcaFigure 1 :Figure 2 : 5 FumonisinFigure 3 :

 101216171253 Figure 1: Chemical structures of Fumonisins B 2 (FB 2 ) and B 4 (FB 4 ) 16

  et al. (2009) 19 has recently shown that A. niger strains isolated from raisins are able to produce both 20 FB 2 and FB 4 when cultured on either grapes or raisins. 21The objective of this study was to investigate the presence of FB 2 and FB 4 in 22 market wines in order to evaluate the potential risk for wine consumers. 23

	4
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Table 1 )

 1 . For FB 4 analysis the product ions m/z 514.3 16 and 338.5 were monitored, corresponding to [MH-TCA] + and [MH-2TCA] + (Bartók et al. 172006). 18Using chromatographic conditions described in this work, fumonisins showed retention 19 times of 3.7, 4.4 and 5.1 min for FB 1 , FB 2 and FB 4, respectively. 20The occurrence of FB 2 and FB 4 was investigated in a total of 51 wine samples collected 21 in Apulia markets. The chemical identification of FB 2 and FB 4 was performed using the 22 molecular ion peaks, peak fragments and chromatographic retention time. The distribution 1 of FB 2 in wine is resumed in Table1. FB 2 was detected in 9 samples representing 17.6% of 2 the whole samples and FB 2 concentrations in the positive samples ranged from 0.4 to 2.4 3 ng/mL. TIC of wine sample #43 positive for FB 2 is reported in Figure3. This study shows 4 that there is a real risk of contamination in wine with FB 2 . No FB 4 was detected in the 5 wine samples. Likewise no FB 1 was detected, as expected due to the fact that this toxin is 6 not produced by A. niger. Fumonisins, including FB 2 , have been found in various plants 7and plant products including corn and corn based products[START_REF] Marasas | Discovery and occurrence of the fumonisins. A historical perspective[END_REF], beer (Shephard 8 et al. 2005), black tea leaves (Martins et al. 2001) and asparagus (Logrieco et al. 1998). 9 The regulatory limits in corn and corn based products in USA and Europe are of 2-4 and 10 0.2-4 µg/g total fumonisins, respectively (FDA 2001; European Commission 2007). 11

		F o
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	12 13	R Aspergillus niger represents the major fumonisin producing species among black Aspergilli e w e i v occurring on grapes (Logrieco et al. 2009) with producing strains accounting for about 75
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Table 1 .

 1 Distribution of wine samples, with reference to production region and year, and 1

	2	relevant occurrence of Fumonisin B 2
	3
		N° Origin	Wine Year	FB 2 (ng/mL)
		1	Apulia	Red	2005 n.d.*
		2	Apulia	Red	2007 n.d.
		3	Apulia	Red	2007 0.6
		4	Apulia	Red	2007 0.8
		5	Apulia	Red	2006 n.d.
		6	Apulia	Red	2005 n.d.
		7	Veneto	Red	2007 n.d.
		8	Veneto	Red	2007 n.d.
		9	Veneto	Red	2007 n.d.
		10 Veneto	Red	2006 n.d.
		11 Veneto	Red	2007 n.d.
		12 Veneto	Red	2007 1.2
		13 Veneto	Red	2006 n.d.
		14 Veneto	Red	2005 n.d.
		15 Veneto	Red	2007 n.d.
		16 Veneto	Red	2006 n.d.
		17 Piemonte	Red	2006 1.1
		18 Piedmont	Red	2007 n.d.
		19 Piedmont	Red	2007 n.d.
		20 Piedmont	Red	2007 n.d.
		21 Piedmont	Red	2007 n.d.
		22 Piedmont	Red	2008 0.5
		23 Trentino Alto Adige Red	2007 n.d.
		24 Trentino Alto Adige Red	2007 n.d.
		25 Trentino Alto Adige Red	2007 n.d.
		26 Trentino Alto Adige Red	2007 n.d.
		27 Trentino Alto Adige White 2006 n.d.
		28 Tuscany	Red	2004 0.5
		29 Tuscany	Red	2004 0.7
		30 Tuscany	Red	2006 n.d.
		31 Tuscany	Red	2007 n.d.
		32 Lombardy	Red	2006 n.d.
		33 Lombardy	Red	2007 n.d.
		34 Lombardy	Red	2007 n.d.
		35 Basilicata	Red	2007 n.d.

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