Emmanuel Bufflier

Antonia Susca

Maryline Baud

Giuseppina Mulé
email: giuseppina.mule@ispa.cnr.it

Karen Brengel-Pesce

Antonio Logrieco

G Mulè

K Brengel

Detection of Aspergillus carbonarius and other black aspergilli from grapes by DNA OLISATM microarray

Keywords: Aspergillus, DNA arrays mycroarray, Aspergillus carbonarious, grape, oligonucleotides, ochratoxin A, 14 DNA microarray 15 16

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

Introduction 1

The presence of black aspergilli in grape is usually linked to the occurrence of ochratoxin A 3 (OTA) in grapes and wine. Aspergillus carbonarius is highlighted as the main species 4 responsible for OTA production in grapes with a high percentage of OTA-producing strains 5 [START_REF] Bau | Ochratoxigenic species 21 from Spanish wine grapes[END_REF]; [START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus section Nigri) 6 on some drying varieties of irrigated grapes[END_REF][START_REF] Serra | Black Aspergillus species as 17 ochratoxin A producers in Portuguese wine grapes[END_REF]; Sage et al. 2002[START_REF] Sage | Fungal microflora and ochratoxin A risk in french 4 vineyards[END_REF]. Ochratoxin A 6 production was also found in A. niger aggregate, but at a much lower rate, whereas A. 7 japonicus and A. aculeatus do not produce OTA [START_REF] Abarca | Taxonomy and significance of black 8 aspergilli[END_REF]Perrone et al. 2006a). 8 Recently A. ibericus also belonging to the Nigri section, not producing OTA and 9 morphologically similar to A. carbonarius, has been characterized and isolated from wine 10 grapes and/or dried vine fruit in Portugal and Spain [START_REF] Serra | 20 Aspergillus ibericus: a new species of section Nigri isolated from grapes[END_REF]). Due to the different 11 ability of the Aspergillus species to produce OTA, an accurate identification is very important 12 particularly for epidemiological studies and for toxicological risk assessment in grapes and 13 wine. 14 15 In recent years, numerous DNA-based methods have been developed to improve the 16 identification of ochratoxigenic fungi occurring in foodstuffs [START_REF] Abarca | Taxonomy and significance of black 8 aspergilli[END_REF]Niessen et 17 al. 2005;[START_REF] Castella | Molecular characterization of ochratoxin A producing strains of the genus 7 Penicillium[END_REF]: in particular techniques based on polymerase chain reaction 18 (PCR) mostly investigating on multiple gene sequences or anonymous fragments (Perrone et 19 al. 2006 a,b), on rDNA, including internal transcribed spacers (ITS) [START_REF] Accensi | New PCR method to 10 differentiate species in the Aspergillus niger aggregate[END_REF]20 Martinez-Culebras et al. 2007), or on structural genes [START_REF] Perrone | PCR assay for identification of Aspergillus 15 carbonarius and Aspergillus japonicus[END_REF], Susca et al. 2007 21 a,b). Real-time PCR systems for quantitative analysis of ochratoxigenic A. carbonarius have 22 also been developed [START_REF] Mulè | Development of quantitative real-22 time PCR assay for the detection of Aspergillus carbonarius in grapes[END_REF][START_REF] Atoui | Targeting a polyketide synthase gene for Aspergillus 18 carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR[END_REF]). These PCR detection methods were 23 designed to detect single or very few species. 24 Alternatively, microarray technology may be a useful technique for identification and 2 differentiation of high numbers of microorganisms in parallel [START_REF] Call | Detection of bacterial pathogens in environmental 4 samples using DNA microarrays[END_REF]. In arrays of 3 genus-, species-or subspecies specific oligonucleotides are immobilised on a solid support in 4 order to bind its complementary sequence, included in a DNA fragment such as a PCR 5 product, in a highly specific and reversible manner. 6 7 This technology was originally developed as a technique to screen for human genetic 8 disorders [START_REF] Saiki | Genetic analysis of amplified DNA 6 with immobilized sequence-specific oligonucleotide probes[END_REF]), but has also been successfully applied to detect and identify 9 human and animal pathogens [START_REF] Fiss | DNA amplification and reverse dot blot hybridization 13 for detection and identification of mycobacteria to the species level in the clinical 14 laboratory[END_REF] [START_REF] Kim | A simple and rapid method for 22 isolation of high quality genomic DNA from fruit trees and conifers using PVP[END_REF] and [START_REF] Loureiro | Molecular marker analysis of Vitis 11 vinifera "Albariño" and some similar grapevine cultivars[END_REF]. In 4 detail: a portion of fresh and frozen berries (300 mg) was weighed and incubated with 1.5 ml 5 extraction buffer (1M Tris-HCl Ph 8, 1.4 M NaCl, 20 mM EDTA, 3% CTAB) and 15 µl β-6 mercaptoethanol for 90 min. at 65°C under constant agitation. After incubation, samples were 7 centrifuged at 6500 rpm in a Biofuge Fresco centrifuge (Heraeus) for 5 min. at 4°C and the 8 supernatant was collected in a fresh tube. One volume of chloroform/isoamyl alcohol (24:1) 9 was added and samples were mixed and centrifuged at 6500 rpm for 20 min. at 4°C. The 10 upper aqueous phase was transferred into another tube, adding 0.1 volume of 10% CTAB. 11

Again, 1 volume of chloroform/isoamyl alcohol (24:1) was added and samples were mixed 12 and centrifuged at 6500 rpm for 20 min. at 4°C. The upper aqueous phase was transferred into 13 another tube, adding 0.1 volume of cold 2-propanol. Samples were then incubated for 60 min 14 at -80°C and centrifuged for 20 min at 13000 rpm. . 2000). This approach allows the 2 setting up of a PCR reaction with a single primer pair. An incorrect evaluation can occurs 3 with this method when target is present at lower level than others, because it could suffers 4 for primers competition and be down-amplified compared to predominant targets. 5 -The specific PCR approach: amplification of single target species, using species-specific 6 PCR primers (CARBO-1/2 and JAPO1/2, [START_REF] Perrone | PCR assay for identification of Aspergillus 15 carbonarius and Aspergillus japonicus[END_REF]). This approach necessitates 7 availability of as many primer pairs as target species to be investigated. 8

The primers sequences used are summarized in Table 1. PCR amplification conditions 9 were as folows: up to 10 ng DNA was amplified in 100µL final volume using 5'biotine-10 labelled oligonucleotides primers. The reaction mix contained 30 pmol of each primer, 11 200µM dNTPs and 2.5 U Taq polymerase (Takara Taq) from Cambrex, with the associated 12 buffer. The program was set up on the Applied Biosystem 2700 Thermocycler as follows: 10 13 min. at 94°C, followed by 35 cycles of [50 sec. at 94°C, 50 sec. at 55°C (generic 14 primers)/58°C (specific primers), 1 min. 72°C] and a final elongation of 7 min. at 72°C. [START_REF] González | Simultaneous detection of 16 marine fish pathogens by using multiplex PCR and a DNA microarray[END_REF]

16

DNA hybridization of PCR amplicons 17

The OLISA™ biochip consists of a series of 17 oligonucleotide probes deposited as spots on 18 the bottom of each of 96-wells in microtiterplate (Figure 1 biotin-labelled target (amplicon). Hybridization is detected through streptavidin-horse radish 1 peroxidase conjugate that binds to the hybridised target, and subsequently create a coloured 2 precipitate at the position of the probe (Apibio SAS, Grenoble, F). 3

The PCR products (5µL) were denaturated for 5 min. with 0.2 M NaOH (OLISA™ 4 denaturation solution), and hybridised 1 hour at 37°C on the chip together with OLISA™ 5 hybridisation buffer. OLISA TM biochips were then washed 4 times with OLISA™ washing 6 buffer using a microtiterplate washer (Colombus, Tecan, CH). After washing, the hybridised 7 probes were detected by mean of a streptavidin-HRP conjugate, combined with a colorimetric 8 substrate (TMB) which generate a blue spot where hybridisation occurred. A streptavidin-9 HRP conjugate (OLISA™ detection solution) was incubated on the chip at room temp for 10 10 min., and then washed. Finally, 50µL of TMB substrate (OLISA™ staining solution) was 11 added on the chip, and incubated for 10 min. to at 25°C. Finally images were taken using the 12

Apimager System (Apibio, Grenoble, F). Spot density is analysed by calculation of the 13 average grey level of each spot minus that of the average well-bottom background, and the 14 result is expressed in Arbitrary Unit (A.U.). 15 16 Design of specific probes 17 Probes (Table 2) were designed based on phylogenetic analyses of the calmodulin gene in 2), and adapted in OLISA TM microarray: JA1 and JA2 (positions 2 and 16, 2 figure 1) specific for the group A. japonicus/A. aculeatus, C1 and C2 (positions 6 and 7 ) 3 specific for A. carbonarius, aC1 and aC2 (positions 8 and 9 ) specific for A. ibericus, CaC1 4 and CaC2 (positions 4 and 5) specific for the group A. carbonarius/A. ibericus (Table 2). 5

Oligonucleotides for A. niger was not designed because sequences of calmodulin gene 6 showed an high grade of variability, reflecting its complex taxonomy. 7 8 Probes range from 16 to 27 nucleotides in length and present a species specific nucleotide 9 variability at different levels: unique polymorphisms (at least one mismatch) were localized 10 manually at 5'-end, 3'-end, centred, and distributed (data not shown). The length of 11 oligonucleotides was adjusted to give a Tm in the wide range 44°C-72°C (Table 2), calculated 12 using the Primer Express software (Applied Biosystems, Foster City, CA, USA). The ability 13 to form dimers and hairpin structures was checked using the same program, and 14 oligonucleotides with the lowest tendency to form such structures were chosen. Blast 15 similarity searches were conducted with Genbank program BLAST [START_REF] Altschul | Basic local alignment search 13 tool[END_REF]) by 16 using all of the probes from the array as queries against the the National Center for 17 Biotechnology Information (NCBI) database, to further ensure that the proposed probes were 18 complementary with the target species. [START_REF] Serra | 20 Aspergillus ibericus: a new species of section Nigri isolated from grapes[END_REF] 20

Specificity of DNA hybridization assay 21

Hybridizations between probes and amplicons or primers were done as previously described. 22

Hybridization were done at least three-time to confirm the result and specificity of the probes. japonicus DNA separately, using the two different approaches. Using A. carbonarius specific 3 primers (A column), typing was correct, except for probe CaC1, specific for A. carbonarius 4 and A.ibericus, which cross reacted with A. japonicus PCR product. Specifically, PCR 5 reaction in that case was set up with A. japonicus DNA as template, but no PCR product was 6 expected for it. Therefore a cross-hybridization occurred between probe CaC1 and primers 7 (Carbo-1 or Carbo-2). This is confirmed by the same result in the no template PCR control. 8

Moreover a weak signal was also observed between primers (Carbo-1 or Carbo-2) and probe 9 C2. In that case the result was not obtained in the control, giving evidence of the non 10 specificity and irreproducibility of that binding. Using A. japonicus specific primers (B 11 column), typing was correct for both probes (JA1 and JA2), but no specificity appeared for 12 probe JA2, which cross reacted with primers (Japo-1 or Japo-2), as demonstrated by the 13 signals that appeared in the hybridization with no product of PCR set up without template or 14

A. carbonarius DNA. Using generic primers (C column), DNA can be correctly typed, except 15 for the unspecific typing due to hybridization between A. japonicus specific PCR product and 16 probe aC1, specific for A. ibericus. The same signal was also observed when testing the no 17 template control, proving that the cross-hybridization was due to interaction with primers 18 (CL-1 or CL-2A). 19 20 These results are summarized in Table 3 and confirmed by the cross hybridization test 21 between each primer and probes. Each primer was tested as a single target (in an identical 22 amount to the PCR products) on the OLISA™ biochip. Primer CL-1 (generic forward primer) 23 cross hybridized with probe aC1. Primer Japo-2 (A. japonicus specific primer) cross 24 found that probe aC1 would not be suitable with generic primers, and probes JA2 and CaC1 3 would not be suitable with specific primers (Figure 3). 4 5 In conclusion, using the generic PCR approach the microarray was set up with JA2, CaC1, 6 CaC2, C1, C2 and aC2 probes; using specific PCR approach with Carbo-1/2 and Japo-1/2 7 primers, the microarray was set up with JA1, CaC2, C1, aC1 and aC2. 8 9

Interspecificity evaluation 10

Using the specific PCR approach 10 ng DNA of each species were PCR with A. carbonarius 11 specific primers and PCR product detected on the biochip. The results showed that the two 12 probes CaC2 and C1 specifically hybridized with A. carbonarius (respectively 9553 A.U. ± 13 1476, 23565 A.U. ± 3412). No hybridization signals were observed between them and A. 14 japonicus, A. niger or A. tubingiensis, confirming specificity of primers Carbo-1 and Carbo-2, 15 able to amplify only A. carbonarius DNA. These two probes were thus good candidates for 16 the A. carbonarius specific amplification approach. 17 inputs for the PCR were two different amounts: 1/20 and 1/10 of total nucleic acids isolated 2 from 300 mg of grapes. A volume 5µL of PCR product was then hybridized for both on the 3 OLISA TM microarray, as described previously. The results are shown in Figure 5. No signal 4 was detected for sample 1 (with either amount of DNA template in PCR), as expected for an 5

A. carbonarius uncontaminated sample. No A. japonicus DNA was detected in either the 6 samples. While in the sample 2 only A. carbonarius was detected , with both the generic and 7 specific PCR approaches. 8 9 In conclusion probes CaC2, C1, aC1, aC2, JA1 and JA2 are suitable for use in the A. 10 carbonarius specific PCR approach for identification of fungal DNA in pure cultures and 11 natural samples; probes JA1, CaC1, CaC2, C1, C2, aC1 and aC2,are suitable for use in the A. 12 japonicus specific PCR approach for identification of fungal DNA in pure cultures and 13 natural samples; probes JA2, CaC1, CaC2, C1, C2 and aC2 are suitable only for use in the 14 generic PCR approach for identification of fungal DNA in pure cultures, while JA2, CaC1, 15 C1 and aC2 are suitable in natural samples. 16 17

Discussion 18
The increasing use of molecular methods in fungal diagnosis has provided tools for answering 19 various taxonomic questions, that morphological procedures have left unsolved. In particular, 20 a number of PCR based methods have emerged which are sensitive, accurate and rapid for the 21 identification of toxigenic fungi. PCR identification of consensus sequences obtained from 22 specific phylogenetic information can be inferred for all taxonomic groups of mycotoxigenic 23 fungi. The most common is based on exploiting the interspecies differences between 24 Other researchers have designed PCR oligonucleotide primers for the amplification of genes 3 involved in the biosynthesis of some toxins [START_REF] Seo | Characterization of four clustered and coregulated 14 genes associated with fumonisin biosynthesis in Fusarium verticillioides[END_REF][START_REF] Sidhu | Mycotoxin genetics and gene clusters[END_REF]Chandler et al. 4 2003). These molecular markers can be used for the detection of toxigenic fungal species by 5 implementing them in DNA biochips. Microarray technology can be used for detecting 6 toxigenic species belonging to the same or a different genus. This is particularly important 7 since generally it is not a single species but a complex of species, often phylogenetically 8 closely related but toxicologically different, that causes plant disease (e.g. Fusarium head 9 blight on wheat; Fusarium ear rot on maize, Aspergillus black rot on grapes) (Nicolaisen et al. 10 2005; Schmidt-Heydt, Geisen 2007). 11 12 Oligonucleotide microarray technology is a rapid and cost-effective method that has potential 13 for parallel analysis of a large number of target organisms such as Aspergillsus spp. species 14 from grape. This is a significant improvement to other methods such as morphological, 15

ELISA and PCR based methods that are either time consuming or directed against a single or 16 few target(s). Because it is based on a preliminary PCR amplification, oligonucleotide 17 microarray technology is rapid, highly specific and can be used to detect minute quantities of 18 fungal DNA from environmental samples, allowing implementation of early control methods 19 before symptoms occur, and direct testing of environmental samples, without pure fungal pure 20 culture preparation. 21 22 OLISA TM is an example of an oligonucleotide DNA microarray which will avoid the 23 development of as many single tests as there are species to detect and identify, allowing 24 background, although the specific amplification gives a much higher signal than the generic 8 amplification, with no significant difference related to the PCR input amount. On the 9 corresponding agarose gel for PCR product analysis, the generic product could not be seen, 10 while the specific product could be seen for both amount tested. 11 12 The results of this study proved that in the specific amplification approach the sensitivity and 13 specificity are due to primers and PCR itself, while in the generic approach, the specificity 14 depends on probe sequences, and sensitivity is subjected to the prevalence of target species. In 15 conclusion, both approaches could be used, with different probes, allowing monitoring of A. 16 carbonarius (mainly ochratoxigenic species), A. japonicus/A. aculeatus and A. ibericus ( (not 17 ochratoxigenic species) DNA occurring. The assay developed in this study provides a further 18 valuable tool in multiple identification of the main Aspergillus species occurring on grapes. [START_REF] Serra | 20 Aspergillus ibericus: a new species of section Nigri isolated from grapes[END_REF] This study allowed to chose specific probes among well defined Aspergillus species occurring 20 in grapes. Since the array format allows to immobilize up to 16 oligonucleotides, further 21 probes, representing other toxigenic fungi, occurring on grapes, could be included, in order to 22

have a wide view of potential toxigenic risk. 23 24

14 Recently 22 ( 5 17and methods 8

 142258 ; Anthony et al. 2000; González et al. 2004). In 10 plant pathology, DNA microarray technology has been successfully applied to identify 11 oomycete, nematode and bacterial pathogen DNA from pure cultures (Lévesque et al. 1998; 12 Uehara et al, 1999). 13 Nicolaisen et al. (2005) and Kristensen et al. (2007) designed and tested an 15 oligonucleotide microarray, based upon Fusarium ITS2 rDNA sequences, for identification 16 and differentiation of trichothecene producing and non-producing Fusarium species occurring 17 on cereal grain and for detection of toxigenic Fusarium species, respectively. The aim of this 18 study was to set up a new microarry format to identify and detect most black aspergilli 19 occurring on grapes. To this end the Apibio biochip OLISA TM (OLIgo Sorbent Arrays), 20 usually applicated for medical diagnosis, in monitoring and testing gene expression, 21 genotyping studies (Bufflier et al. 2003; Guerniou et al. 2004) and protein identification Perrin et al. 2003), was tested for diagnosis of Aspergillus spp. mainly occurring in the 23 grapes: Aspergillus carbonarius, A. japonicus/A. aculeatus and A. ibericus. 24 In particular OLISA TM are microarrays including of up to 17 probes arrayed on the well 2 bottom of a 96-well microtiter plate, using a proprietary surface chemistry. DNA could be 3 simplex or multiplex PCR amplified to generate biotin-labelled fragments and revealed by 4 colorimetric detection, analyzed by the automated Apimager TM system (Apibio, Grenoble, 5 France), giving an easy-to-use final report of results. 6 Material Fungal strains and sequence analyses 9 All strains were obtained from the culture collection (ITEM) of the National Council of 10 Research -Institute of Sciences of Food Production. Further information about the strains 11 (year of isolation, depositor, toxin production, etc.) are available at the ITEM web site: 12 http://www.ispa.cnr.it/Collection. 13 14 Calmodulin nucleotide sequences of the thirty strains, belonging to four Aspergillus species 15 mainly occurring on grapes (ten of A. niger group, ten of A. carbonarius, seven of A. 16 japonicus and three of A. aculeatus) previously sequenced by Perrone et al. (2004), were 17 analyzed. Moreover three strains of A. ibericus (ITEM 4776, ITEM 6601, ITEM 6602) were 18 sequenced and included in this study. Total DNA extracted from pure fungal culture: ITEM 19 4500 (A. tubingensis, belonging to Niger group), ITEM 4502 (A. niger), ITEM 4503 (A. 20 carbonarius), ITEM 5019 (A. japonicus) and total DNA extracted from two natural samples 21 (red grapes) were tested for specificity and the sensitivity of the selected probes. DNA extraction from pure fungal culture, the calmodulin gene 1 amplifications and sequencing are the same as previously described by Perrone et al. (2004). 2 Purification of DNA from natural samples (grapes berries) was set up combining the 3 extraction methods of Steenkamp et al. (1994),

  ), spotted in duplicate 19 symmetrically on the right and left hand side. The specimen is the amplicon amplified with 20 biotin labelled primers, denaturated and hybridised on the chip. Short oligonucleotide probes 21 are designed and optimized to hybridize specifically to any single nucleotide polymorphism 22 of an amplified DNA target. The pattern of spots allows automated identification of the 23 species present in the specimen. Detection is based on hybridization between probe and 24

  33 18 strains belonging to different Aspergillus spp., sequenced in the present study and previously 19 by Perrone et al. (2004). As the species-specificity in the calmodulin gene of Aspergillus spp. 20 was already established in Perrone et al. (2004 and 2006 a,b), nucleotide sequences were 21 compared using the Clustal W method, in order to select appropriate species-specific 22 oligonucleotides. The visual inspection of the aligned sequences allowed identification of 23 unique regions, useful to design oligonucleotides specific at the species level. Eight probes 24 on the alignment, two for each Aspergillus species mostly occurring in 1 grapes (Table

4 5 11 12 16 17 18FoodFigure 2

 41116172 Figure 2 shows specificity test results obtained from amplifications of A. carbonarius and A. 2

18 Using 8 9 19 20

 18819 the generic PCR approach 10 ng DNA of each species was PCR amplified separately 19 using generic primers and detected on the biochip. Probes CaC1, CaC2, C1 and C2 hybridized 20 with PCR amplicon obtained by amplifying A. carbonarius DNA, while JA1 and JA2 21 hybridized with PCR amplicon obtained by amplifying A. japonicus DNA. Probes CaC2,C2 22 and JA1 weakly hybridized with the PCR amplicon obtained by amplifying A. niger DNA. 23 Probe JA1 hybridized with PCR product obtained by amplifying A. tubingensis. These results 24 should be excluded for the generic PCR approach because it hybridized with 1A. japonicus (15182 A.U. ± 1613), but also with A. niger and A. tubingensis (respectively 918 2 A.U. ± 378 and 2986 A.U. ± 253). Probes C2 and CaC2 non specifically hybridized with A. 3 niger (respectively 1227 A.U. ±384 and 448 A.U. ± 237), therefore it could be considered if a 4 threshold value is defined at about at 2000 A.U. In practice, it could be valid for pure culture 5 identifications, because in natural samples DNA levels of fungal contaminations are 6 unpredictable, thus threshold cannot be established and CaC2, C2 and JA1 should be 7 excluded.Sensitivity of A. carbonarius detection 10Using the generic primers approach the detection limit (DL) of the method was determined by 11 testing a series of 5-fold dilutions of A. carbonarius DNA in PCR reactions: 2 ng, 0.4 ng, 0.08 12 ng, 16 pg, 3.2 pg, 0.64 pg, 128 fg, 25.6 fg, 5.12 fg and 0 mixed with 2 ng of total DNA of 13 each of A. japonicus, A. niger and A. tubingiensis. A PCR negative control, without DNA 14 templates, was included. All probes tested gave a signal decreasing with the dilution factor 15 (Figure4). In particular CaC1 and C1 probes showed hybridization signals up to 3.2 pg, 16 establishing their sensitivity. Probes CaC2 and C2 gave a signal for all concentrations tested 17 and also for the negative DNA control of A. carbonarius DNA, due to the previously proved 18 cross hybridizations with A. japonicus, A. niger and A. tubingiensis DNA.Detection of A. carbonarius in environmental samples 21Qualitative microbiological analysis on the two environmental samples showed sample 1 22 uncontaminated by Aspergillus spp. and sample 2 contaminated by A. carbonarius. The two 23 samples were also tested using the generic and the specific approaches for a preliminary 24

  Aspergillus spp. OLISA TM microarray application on food samples. The DNA 1

  ribosomal intergenic spacer DNA (IGS rDNA), beta-tubulin, 1 calmodulin, elongation factor and AFLP analysis[START_REF] Schmidt | Molecular typing of 9 Aspergillus ochraceus and construction of species specific SCAR-primers based on 10 AFLP[END_REF] Mule` et al. 2004a, b). 2

5 6

 5 identification of all targeted species of Aspergillus by a molecular 1 method combining multiple amplification and multiple detection. In the generic and specific 2 PCR approaches the OLISA TM biochip allowed the subsequent detection of the PCR products 3 and the simultaneous identification of the Aspergillus. carbonarius, A. japonicus/A. aculeatus 4 and A. ibericus DNA.Both generic and specific A. carbonarius amplifications signals can be distinguished from the 7

Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 2345 Figure 2. Specificity hybridization test between probes and amplicons obtained with specific (A, B)

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

Table 1 :

 1 Primers used for specific and generic PCR approaches.

	Amplicon
	Name Sequence (5' -3')	PCR approach
	length

Table 2 .

 2 Species-specific probes designed in calmodulin gene.Probes were spotted in duplicate symmetrically on right and left part of the OLISA TM array.

	Spot # on	Probe
			Sequence (5'-3')	Length Tm (°C) Species-specificity
	OLISA TM	name
	1/17	Control (GACT) 5 -biotine	20	45	none
	2/16	JA1	TAGTGTAGTTCCTTACCCCTCAACGAA	27	61	A. japonicus and
	3/15	JA2	TAGCCACCACACTGCA	16	48	A. aculeatus
	4/14	CaC1 GATATTAACTTCTGCCAGA	19	44	A. carbonarius and
	5/13	CaC2 CTGGGCACTGTCATGCGCTCGCTC	24	72	A. ibericus
	6/12	C1	GGGCTTGTGTTCACACGAGGCAAG	24	68
						A. carbonarius
	7/11	C2	AGCCGTGATACTGATGTGACA	21	55
	8/10	aC1	TCTTCCGACCGACTTGAACTTAAT	24	59
						A. ibericus
	9	aC2	GTGTTGATATTTATAGGAAGAGGG	24	52

Table 3 :

 3 Hybridization results between probes and amplicons obtained with specific (for A. carbonarius and A japonicus) and generic PCR approach. Figure 1. DNA spots positions in each of 96 wells of microtiter plate in OLISA™ array.

	Probe A. carbonarius specific	A. japonicus specific	Generic primers
		primers	primers
	JA1	-	+	X
				(with A. niger and A. tubingensis)
	JA2	-	X	+
			(with Japo-2)
	CaC1	X	-	+
		(with Carbo-2)
	CaC2	+	-	+ Y
	C1	+	-	+
	C2	X	-	+ Y
	aC1	-	-	X
				(with CL-1)
	aC2	-	-	-
	+ corresponds to specific hybridization;
	-corresponds to none hybridization;
	X corresponds to unspecific hybridization.

Y corresponds to unspecific hybridization with A. niger, in pure culture (below the threshold value 2000A.U.) and in environmental samples identification assays.

Circles represent probes spot positions in the bottom of each well. Reciprocal relations between numbers and spotted probes are listed in table 2.

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Acknowledgements 1

Work was supported by the European Union project FP6-IST1-508774-IP. We thank 2 Vincenzo Ricci and Gaetano Stea for their valuable technical assistance. 3

4 5


Dominic Roberts

Colin Crews

Helen Grundy

Craig Mills

Wendy Matthews

Effect of

Keywords: furan, heat processing contaminants Food Types: canned food, jarred food, Cooked foods Furan, heat processing contaminants, canned food, jarred food

published or not. The documents may come

Introduction

Furan has been identified as a contaminant of food produced by the thermal degradation and rearrangement of precursors such as sugars, ascorbic acid and polyunsaturated fatty acids. It is formed in foods on heating during manufacture and accumulates (in foods sealed in cans and jars) during this process. It is thus not surprising that such foods have been the main subjects of surveys of occurrence.

Furan is highly volatile and is easily lost from foods that are heated in open vessels.

The presence of furan in food is of concern because it is regarded as a possible human carcinogen by the International Agency for Research on Cancer (IARC 1995).

It has been established that furan can be found in a range of heat-treated foods, including coffee, canned and jarred baby foods, vegetables, and soups. In a series of surveys targeted at foods likely to contain furan the United States Food and Drug Administration (USFDA) and the European Food Safety Authority (EFSA) reported that almost all of 273 baby food analysed by the USFDA contained furan, at up to 112 µg/kg (US FDA 2004, EFSA 2005). A compilation of the data submitted to EFSA and the FDA showed that of 32 convenience meals, all contained furan between 3 and 94 µg/kg with an average of 35 µg/kg, and all of 26 samples of canned baked beans contained furan between 23 and 122 µg/kg with an average of 59 µg/kg [START_REF] Crews | A review of the occurrence, formation and analysis of furan in heat-processed foods[END_REF].

There is a need to increase the database of information on the levels of furan in food and a call for such data has been issued by EFSA (2006). There is especially a 3 lack of data on levels in foods as they are purchased and prepared in the home. For the purpose of assessing the exposure of consumers to furan it is important that domestic preparation and cooking procedures are reproduced in scientific studies. There have been relatively few applications of this approach to studies of furan formation other than those applied to coffee. This paper reports the results of a study of the levels of furan present in a range of home cooked food products of various types, including canned, packeted, and home-made products. These foods were cooked by a range of techniques in a laboratory kitchen using domestic appliances. The work was carried out as part of a larger project to study the formation of heat generated food toxicants (Heatox), which was carried out under the European Union 6 th framework programme.

The objectives of this study were to report the levels of furan in a range of convenience foods as consumed in the home and the effects of various domestic cooking regimes on those levels. The outcome of this work would help in producing estimates of consumer exposure to furan, and enable advice to be given to food manufacturers regarding formulating best domestic cooking practices for their products.

The foods studied were mostly complete ready-to-eat meals (convenience foods) packed in cans or plastic trays, and soups and sauces packed in cans, cartons or jars. Attention was focused on products identified from previous work by [START_REF] Hasnip | Some factors affecting the formation of furan in heated foods[END_REF]the FDA (2004) as being likely to form furan on heating.

Materials and methods

Reagents and samples

Apparatus

Simulated domestic cooking experiments were carried out using a Sharp Carousel R-7400 domestic microwave oven, output 600 W, frequency 2450 MHz; a Victoriana HPG gas hob; and an Electrolux EOB 811 electric oven. Sample blending was carried out using an Ultra Turrax.

Cooking procedures

For convenience food samples all cooking procedures used followed the instructions or recommendations set out on the packaging. This typically involved microwave heating. Home prepared meals were cooked following the instructions given in recipes (BBC 2006).

Sample preparation

The temperatures of the foods were recorded before and after cooking or heating. On completion of cooking the foods were immediately transferred into one litre PTFE bottles, temperatures recorded and then sealed, after which they were stored in a refrigerator overnight. The samples were homogenised for analysis under chilled conditions in the one litre bottle using a laboratory blender. One sample of each of the foods was prepared in duplicate, but without heating.

Method of analysis

Sample analysis was carried out as described previously in [START_REF] Hasnip | Some factors affecting the formation of furan in heated foods[END_REF]. In brief, furan was determined by automated headspace gas chromatography-mass spectrometry (GCMS) using a ThermoFinnigan Voyager bench-top system fitted with a 15 m x 0.2 mm i.d. fused silica, HP-Plot Q column. The carrier gas was helium at a constant flow of 1.7 ml min -1 . The split/splitless injector port and mass spectrometer interface were heated to 200 and 250 °C, respectively. The oven temperature was maintained at 50°C for 1 min and then increased at a rate of 10°C min -1 to 225 °C and held at this temperature for 12.5 minutes. Split injections were made, at a ratio of 2:1, using a ThermoFinnigan HS800 static headspace autosampler with an incubation temperature of 50 °C with an incubation time of 30 minutes.

Triplicate 5 g portions of each homogenized sample were weighed accurately into 20 ml headspace vials and 5 ml chilled water were added quickly. Internal standard solution was added to each vial, and a furan spike solution was added to one of each triplicate. Vials were chilled in ice before and during the addition of internal standard to avoid loss of furan.

Furan was detected by selected ion monitoring of the molecular ion at m/z 68 and confirmed by monitoring of the fragment ion at m/z 39. The internal standard was detected by monitoring its molecular ion at m/z 72. Furan was quantified using a single point standard addition to a portion of one of the samples and corrected by the use of the internal standard. The internal standard was used to correct for any losses of furan during incubation and injection of the sample headspace. Each analytical batch included a reagent blank and a low-level tomato sauce reference material with a typical furan concentration of 7 µg/kg ± 2.

Experiments

Foods were cooked either in a saucepan on a gas hob, in a microwave oven or conventional oven as appropriate, according to the manufacturers' recommendations for temperature/power and time. Several aspects of food preparation and cooking were studied. These were (1) the changes in levels of furan present in samples of canned or jarred foods on cooking in a saucepan or microwave oven, (2) a comparison of the levels of furan in some cooked convenience foods and their home-made equivalents, (3) the changes in levels of furan present in microwave cooked foods after reheating, (4) the effect of standing and of stirring on the levels of furan in cooked foods after cooking, and (5) the changes in levels of furan in cooked foods left to stand on plates (mimicking the process of food cooling as it is consumed).

Effect of heating procedures on furan levels in canned and jarred products.

Eighteen different convenience foods were heated by microwave oven or by warming in a saucepan on a gas hob to compare the effects of the heating methods on furan levels in the prepared product. Two samples intended for consumption direct from the plastic pot in which they were supplied were warmed by the addition of boiling water to the container as specified in the manufacturers' instructions.

Comparison of furan levels in convenience meals and home made equivalents.

This study aimed to identify if furan levels differed significantly between pre-packed convenience meals and their home-made equivalents. Ten convenience meals were purchased and a home-made equivalent of each prepared in a laboratory kitchen using similar ingredients and cooking methods. The convenience meals were cooked following the manufacturers' instructions for oven or hob cooking and the home made versions were cooked as directed in the recipe. This study aimed to compare the influence of the domestic heating method on the formation of furan in foods for which alternate heating procedures were specified on the packet. Five convenience meals were cooked following the manufacturers' instructions. In addition to being unheated, the samples were cooked under four different conditions: (a) conventional oven covered, (b) conventional oven uncovered, (c) microwaved covered, (d) microwaved uncovered.

Comparison of microwave and conventional oven cooking effects on furan levels

The five convenience meals cooked previously in the microwave oven (covered from experiment (c) above) were sub-divided into two portions. One of these portions was analysed for furan and the other was stored overnight in a refrigerator. After overnight storage the second portion was re-heated in the microwave oven both covered and uncovered. The samples were warmed by reheating for one-half of the original cooking time specified by the producer, with the oven power at the maximum setting.

Changes in furan level after standing and stirring.

The aim of this study was to assess if furan levels changed significantly when convenience meals and baby foods were left to stand for up to 10 minutes between heating and consumption. It also assessed the effect of stirring during this cooling period on the furan content.

Eleven foods (three canned soups, two canned beans, three packeted convenience meals, two baby foods and one sample of fresh green beans) identified from previous experiments as possibly containing high levels of furan, were heated using a microwave oven following the guidelines listed on the packet. The soups, beans, and packeted convenience meals were heated in a microwave for the recommended time, the baby food was warmed in a covered dish in the microwave On completion of cooking the samples were allowed to stand and samples removed for analysis after 0, 5 and 10 minutes. Duplicate samples were heated and stirred with a tablespoon for 0.2, 5 and 10 minutes after heating to investigate the effects of stirring on furan loss.

A second experiment aimed to measure the levels of furan at the point of consumption. Four types of canned food, known to contain levels of furan after heating (baked beans, vegetable chilli, vegetable soup and a spinach pasta meal) were prepared following the manufacturers' microwave heating instructions. The foods were then immediately served on to a dinner plate and handled as if they were to be consumed (stirred and/or chopped as appropriate). Seven sub-samples of about 20 g were taken from the plate at time points between 0 and 10 minutes to mimic the process of food cooling as it is consumed.

Results and discussion

All reagent blank samples had responses well below those of the unheated samples, and below the reporting limit of 2 µg/kg. Analytical measurement uncertainty has previously been calculated as ± 2 µg/kg at 2 µg/kg, ± 4 µg/kg at 25 µg/kg and ± 15 µg/kg at 100 µg/kg. For clarity of data presentation individual analytical measurement uncertainty values are omitted from the tables.

Effect of heating procedures on furan levels in canned and jarred products. Table I shows the results for the twenty foods that were analysed after heating in a saucepan or a microwave oven or prepared by the addition of boiling water. Prior to cooking the canned foods had higher furan levels than those in cartons. For example, furan was only just detected (2 to 3 µg/kg) in the fresh chilled vegetable soup sold in cartons, whereas the canned equivalent contained over 20 µg/kg.

[Insert Table I about here]

When the canned foods were heated in a saucepan furan levels decreased in seven of the nine samples to varying degrees. When the canned foods were heated in a microwave oven furan levels decreased in only three of the nine samples. It remained at about the same level in four samples but increased considerably in two.

Furan decreased on saucepan heating in all of the three jarred foods. Microwave heating only decreased the furan level in the pasta sauce. The low furan levels in the cartoned foods were unchanged by any cooking procedure. Two foods, a beef and tomato noodle meal and a rice based meal, which had been prepared by adding boiling water, contained very little furan before the addition of boiling water and no detectable levels of furan after. These decreases in furan levels were possibly an effect of dilution, rather than a real loss of furan.

No overall trend was observed in the results. For example, in the canned sweet and sour chicken meal furan increased on heating, and in the jarred pasta sauce furan decreased on heating. In the canned curry sauce furan levels decreased on heating in a saucepan, but increased when cooking in a microwave. These effects cannot easily be related to the product composition on account of the large number of ingredients listed for these products. It has been proposed that the differences of furan levels between cooking methods can be explained by the rapid rate that furan evaporates once it has [START_REF] Goldmann | Rapid determination of furan in heated foodstuffs by isotope dilution solid phase micro-extraction-gas chromatography -mass spectrometry (SPME-GC-MS)[END_REF][START_REF] Hasnip | Some factors affecting the formation of furan in heated foods[END_REF][START_REF] Bianchi | Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food[END_REF]. Furan would have a greater ability to evaporate from a meal heated in an open saucepan than from one enclosed inside a microwave oven. Differences in the final furan concentration may also be due to the different kinds of energy input. Microwave heating is heterogeneous which can cause hot spots within the food being heated. This could also cause difference of furan formation within a sample.

Comparison of furan levels in convenience meals and home made equivalents.

The levels of furan found in pre-packed convenience meals and their home made equivalents are shown in Table II.

[Insert Table II about here]

There was no consistent difference in furan levels between the two sets of samples. Five of the ten samples, beef stew, beef lasagne, chicken curry, chilli beef and the vegetable soup, showed a significant difference in furan levels between the two preparation methods. The beef stew, chilli beef and the vegetable soup convenience meals had higher levels of furan than their home-made equivalents. The greatest difference was in the chilli beef, which contained 27 µg/kg in the pre-packed meal and 3 µg/kg in the home-made equivalent.

Substantial variations in the ingredients and the cooking processes may explain the differences in furan levels between the two sets of foods. In particular the convenience meals were heated at an unknown (and possibly higher) temperature and for an unknown duration during manufacture in addition to warming for consumption.

In view of this it might be expected that levels of furan in these products would exceed those of the domestic foods. When the chilli beef convenience food was than the home made equivalent heated in a saucepan at 100°C for 50 minutes. [START_REF] Bianchi | Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food[END_REF] compared the furan levels in a small range of processed baby foods. Both of two processed fruit-based foods contained furan at 3 to 4 µg/kg whereas four home made fruit-based equivalent had less than 0.03 µg/kg. All of four processed vegetable-based foods contained furan between 15 and 141 µg/kg. Furan was also detected in all of three home made vegetable-based baby foods but at much lower levels (0.1 to 1 µg/kg). These differences in furan levels were ascribed to the different cooking (processing) techniques, i.e. the processed foods were heated in closed vessels, whereas home made foods were cooked in open pans. Under the latter conditions, furan can be easily released from the matrix.

Comparison of microwave and conventional oven cooking effects on furan

Analysis of five packeted convenience foods (Irish stew, beef chilli, vegetable chilli, chicken tikka masala and sweet and sour chicken) after cooking in both conventional and microwave ovens showed that neither cooking process produced furan in any sample. The low level (~ 5 µg/kg) present in one vegetable chilli sample prior to cooking was reduced to less than the reporting limit by all of the cooking procedures.

The lack of suitable furan forming ingredients and a high surface exposure of the sample to the atmosphere is suggested as a reason for these observed low levels. A repeated cooking and analysis of the five convenience foods above with a study of the loss on reheating and standing similarly failed to produce measurable furan. It should be noted that the supplier and the packaging of the samples used in this experiment are different to those in table 1 that did show higher levels of furan when heating was done in a microwave oven. Table III shows the change in furan levels and temperatures in some canned foods that had been allowed to stand with or without stirring after having been heated in a microwave oven for the recommended time. The standing and stirring times refer to independent samples, i.e. one sample was analysed immediately after cooking, a second sample of the same food was left to stand for five minutes before analysis and a further sample of the same food was left to stand for ten minutes before analysis.

[Insert Table III about here]

Only four of the nine samples (baked beans, tomato soup, leek and potato soup and vegetable soup) showed levels of furan high enough for the loss on standing with or without stirring to be assessed (the other five samples are therefore omitted from Table III). In all cases, stirring the sample lead to decreases of furan, furan decreased in all samples at a rate that was nearly linear over the 10 minutes stirring period.

When samples were left to stand without stirring, furan levels did not change significantly. A possible explanation for this observation is that in the unstirred sample migration of furan through the matrix to the surface was slow, and consequently evaporation rates were low and the level of furan remained constant. This is analogous to the situation in headspace sampling for furan analysis, where the sample has to be made mobile by adding water and agitated in the headspace incubator in order to facilitate furan volatilisation into the headspace.

A closer examination was made of two baby food samples, canned carrot and lamb, and jarred pumpkin, swede and lamb. These were warmed in a saucepan or by standing in a bowl of hot water and then analysed for furan. Furan levels in both sample types were very similar for both warming procedures and for clarity the results IV. The results show a loss of furan for the stirred samples. Figure 1 shows the decline of furan in the carrot and lamb baby food when stirred over 10 minutes after heating by immersion in hot water.

[Insert Table IV about here] [Insert Figure 1 about here] This steady decline is a result of greater evaporation of furan from the sample than when the sample is left unstirred during cooling. Furan formation would be reduced by the disturbance of the sample causing an increased opportunity of furan evaporation. The rate of cooling would also be increased when the sample is stirred which would diminish the heat conditions required for furan to form.

In order to limit the amount of furan that is consumed it can be recommended that food is left to stand for as long as possible before consumption, with regular stirring.

[Insert Table V about here]

Table V shows the loss of furan from foods on the plate during the course of simulated consumption for four canned foods (baked beans, pasta meal, vegetable chilli, and vegetable soup). Food was cut and manipulated as if being consumed and left to stand, with sub samples being taken at given intervals and analysed for furan.

The results show that the concentration of furan did not decrease in the baked beans but fell significantly in the other samples. The greatest decrease was observed in the vegetable chilli which fell from about 60 µg/kg immediately after placing on the plate to 20-30 µg/kg after 10 minutes standing on the plate. This decline in furan levels is similar to those observed in the standing and stirring experiments reported above.

Conclusions

This work provides further confirmation that the highest levels of furan found in foods are produced during the manufacture of jarred and especially canned foods. Most domestic cooking procedures reduce the furan content of canned meals with heating in a saucepan being more efficient than microwave heating.

The quantity of furan ingested from a meal on a plate is generally less than that measured by analysis of a sample taken directly from the cooking vessel, which has implications for the estimation of dietary intake, which might otherwise be over estimated when the effects of domestic preparation are not taken into account. Indeed, consumers can contribute to a reduction in their furan intake by stirring foods prior to eating. This would be particularly pertinent and appropriate in the case of baby foods.

  ) and d 4 -furan >99 atom% were supplied by Sigma-Aldrich (Poole, Dorset). Food samples and ingredients were obtained from local retail stores.

  power for 1.5 minutes, and also by immersion in 200 ml of hot water (80 o C) for 5 minutes. The fresh green beans were trimmed and chopped, placed in a covered glass bowl with water, and heated for 4 minutes at full power in the microwave oven.

  short time (5 minutes) in the microwave oven it contained more furan

  level after standing and stirring.

  experiment for each type is provided in Table

Table I .

 I Average levels of furan (µg/kg, n=2) in canned, jarred or packeted foods unheated and after heating in a saucepan and

microwave. (n/n) are duplicate analysis of the same sample.

	Sample	Pack	Unheated	Saucepan	Microwave
				Heated	Heated
	F o r
	P
	e
	e r
		R
		e
		v i e
			w
			O n l
				y

Table II .

 II Comparison of furan levels (n=2) in convenience foods and home-cooked equivalents cooked. (

n/n) are duplicate analysis of the same sample.

	Sample		Type		Cooking	Final	Furan
						Temp	µg/kg
						°C
				Mode Temp °C/Time
					minutes
	Beef stew		Packet	O	190 / 25	87	9 / 11
			Home made	O	180 / 120	99	5 / 5
	Beef lasagne Chicken curry F o r Home made Packet Packet Home made	O O O O	200 / 45 180 / 60 190 / 25 190 / 25	99 95 91 76	2 / 2 13 / 13 7 / 8 16 / 16
	Chilli beef	Canned Home made P	S S	nm / 5 100 / 50	95 98	27 / 27 3 / 3
	Pasta with tomato Roasted vegetables	Packet Home made e Frozen bag Home made e r	M O O O	nm / 5.5 100 / 45 220 / 40 220 / 40	82 80 80 82	<2 / <2 5 / 6 <2 / <2 <2 / <2
	Shepherd's pie Vegetable baby food Vegetable curry Vegetable soup	Packet Home made Jarred R O O S Home made S e Packet O Home made O Canned S v i 190 / 30 190 / 30 Warm 5 Warm 5 190 / 25 190 / 25 Warm 5 e	84 80 80 78 83 77 95	8 / 9 5 / 6 3 / 4 6 / 6 3 / 4 3 / 4 11 / 12
	O = oven cooked		Home made	S	Warm 5 w	98	3 / 3
	M = microwave cooked S= saucepan cooked nm = not measured			O n l
						y

Table III .

 III Levels of furan (µg/kg, n=2) in foods unheated and after cooking in a microwave oven on standing or stirring. (

n/n) are duplicate analysis of the same sample.

	Sample		Treatment		Temp Furan
	Baked Beans		Unheated		48 / 50
	(canned)		Stood 0 minutes		92	14 / 18
			Stood 5 minutes		78	19 / 20
			Stood 10 minutes		70	14 / 14
			Stirred 0.2 minutes		97	9 / 9
	Stirred 5 minutes Stirred 10 minutes Leek and Potato Soup unheated (canned) Stood 0 minutes Stood 5 minutes F o r		80 59 91 75	8 / 8 6 / 6 13 / 14 5 / 5 5 / 5
	P	Stood 10 minutes Stirred 0.2 minutes		65 89	5 / 5 4 / 4
	Tomato Soup (canned)	Stirred 5 minutes Stirred 10 minutes e unheated Stood 0 minutes e r		60 54 99	3 / 3 2 / 3 37 / 39 17 / 18
			Stood 5 minutes Stood 10 minutes Stirred 0.2 minutes R Stirred 5 minutes e		65 66 98 65	13 / 13 22 / 26 19 / 19 9 / 10
	Vegetable Soup (canned)	Stirred 10 minutes unheated Stood 0 minutes Stood 5 minutes v i e Stood 10 minutes Stirred 0.2 minutes w	53 90 58 51 98	7 / 7 42 / 45 14 / 15 17 / 18 18 / 19 17 / 20
			Stirred 5 minutes Stirred 10 minutes	63 58 O n 11 / 12 6 / 6 l
					y

Table IV .

 IV Changes in furan levels (µg/kg) in baby foods on standing and stirring after heating in

a microwave and by immersion in hot water. (n/n) are duplicate analysis of the same sample.

  Figure 1. Decrease in furan concentration in a hot water heated canned carrot and lamb baby food (on standing) with stirring.

			Food Additives and Contaminants				Page 20 of 22
	25
	20
	Sample Carrot and lamb (can) 10 15 Furan ug/kg	Heating method microwave stood 0.2 minutes Treatment stood 5 minutes		Temp °C 67 51	Furan µg/kg 21 17
	5			stood 10 minutes		47		14
	0	F o r 0 2 F o r	microwave stirred 0.2 minutes stirred 5 minutes stirred 10 minutes hot water stood 0.2 minutes stood 5 minutes 4 6 8 Time minutes	10	70 53 47 45 34	12	23 10 4 26 13
		hot water P P e e e r e r	stood 10 minutes stirred 0.2 minutes stirred 5 minutes stirred 10 minutes		31 47 34 30		17 23 12 2
	Pumpkin, swede and lamb (jar)	microwave stood 0.2 minutes stood 5 minutes stood 10 minutes R R microwave stirred 0.2 minutes e e stirred 5 minutes stirred 10 minutes hot water stood 0.2 minutes v i e v i e stood 5 minutes stood 10 minutes w w		62 51 45 62 53 47 37 32 31		54 53 53 41 31 27 24 28 29
			hot water	stirred 0.2 minutes stirred 5 minutes stirred 10 minutes O n 40 29 24 l O n l	60 41 17
						y y

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Table V .

 V Mean furan levels (µg/kg) in foods unheated and at intervals after heating at the point of consumption from a dinner plate. (

n/n) are duplicate analysis of the same sample.

								Page 22 of 22
	Sample	Unheated			Standing time (minutes)
			0.2	1	2	3	4	5	10
	Canned baked beans	22 / 30	32 / 35 32 / 35 27 / 29 38 / 45 40 / 48 26 / 30 28 / 30
	Canned pasta meal	22 / 22	13 / 19 13 / 15 16 / 18 14 / 15 11 / 12	8 / 9	6 / 7
	Canned vegetable chilli	55 / 58	62 / -37 / 39 50 / 62 42 / 47 36 / 46 36 / 39 18 / 28
	Canned vegetable soup	17 / 19	17 / 18 30 / 34 23 / 23 23 / 26 21 / 31 22 / 23 13 / 13

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Acknowledgment

This work was funded by the UK Food Standards Agency.


Vaquero -Fernández

Sanz Asensio

Martínez - Soria

L Vaquero-Fernández

J Sanz-Asensio

M López-Alonso

M T Martínez-Soria

María-Teresa Martínez -Soria
email: maria-teresa.martinez@unirioja.es

penconazol fungicides from treated grapes intended for winemaking 2 3 4

Keywords: Pesticides, grapes, absorption, distribution, penetration 39

Tempranillo grapes were immersed in solutions of pyrimethanil, metalaxyl, 25 dichlofluanid and penconazol fungicides at different concentrations for several different 26 times. Determinations of the fungicide residues was carried out by GC-NPD, with an 27 additional confirmation by GC-MS. The percentage absorption was determined as well 28 as the distribution between surface, skin and pulp of the grapes. The percentage 29 absorption ranged from 7.2 to 85.5 %. These values depended on the time which grapes 30 were in contact with the fungicide solutions. Residues were mainly found in skins, with 31 percentages in skins ranged from 66.3 to 90.3 % of total residues. In pulp, these values 32 were ranged from 3.5 to 31.0 %. The overall methodology was applied to the 33 determination of pyrimethanil in Tempranillo grapes treated with the recommended 34 doses, respecting the safety period. Pyrimethanil residues found in treated grapes were 35 higher in the skin. In grapes collected within the safety period (21 days), the levels 36 found for fungicide residues were below Maximum Residues Levels. 37 38

Introduction 40

Fungicides are widely used in the treatment of grape diseases for winemaking such us 41 grey mold (Botrytis cinerea), powdery mildew (Uncinula necator) and downy mildew 42 (Plasmopara vitícola). Although the correct use of these phytosanitary products does 43 not have adverse effects for public or environmental health, indiscriminate treatment 44 applied without respect for safety periods or recommended doses can entail a risk the of 45 residues remaining in grapes used for winemaking. These fungicides may be transmitted 46 to the must and then to the wine during fermentation (Cabras et al. 2000;Fernández 47 González et al. 2003;[START_REF] Mªj | Determination of azolic 402 fungicides in wine by solid-phase extraction and high-performance liquid 403 chromatography-atmospheric pressure chemical ionization-mass spectrometry[END_REF] and affect not only the fermentation process but 48 also the final quality of the product, giving rise to off-flavors [START_REF] Cuinier | Influence eventuelle des produits phytopharmaceutiques sur les 363 fermentations et la qualite des vins[END_REF]. Moreover,49 there may also be a risk for consumer health [START_REF] Stavropoulos | Degradation of pyrazophos 418 and methidathion in fortified red and white wine under conditions of light and 419 darkness[END_REF]Navarro et al. 50 2005). Therefore, rapid and reliable analytical techniques must be developed to control 51 residue levels in grapes, in order to verify their compliance with the Maximum Residue 52 Levels (MRLs) [START_REF] Teixeira | Comparison of 423 pesticides levels in grape skin and in the whole grape by a new liquid 424 chromatographic multiresidue methodology[END_REF]. 53 54 Numerous analytical methods have been described for determining the presence of 55 fungicides in winemaking grapes. Determinations are generally carried out by GC with 56 different detectors (NPD, ECD, MS) [START_REF] Sherma | Pesticide residue analysis: 1997-1998[END_REF][START_REF] Fernández | Analytical methods for pesticide residue 373 determination in bee products[END_REF]Tadeo et al. 57 2004;[START_REF] Angioni | Gas 354 chromatographic ion trap mass spectrometry determination of zoxamide residues in 355 grape, grape processing, and in the fermentation process[END_REF] or HPLC with UV-VIS or MS detection [START_REF] Melo | New materials for solid-phase extraction 387 and multiclass high-performance liquid chromatographic analysis of pesticides in 388 grapes[END_REF]58 Nozal et al. 2005;[START_REF] De Melo Abreu | Validation and global 365 uncertainty of a liquid chromatographic with diode array detection method for the 366 screening of azoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone, 367 pyraclostrobin and fenamidone in grapes and wine[END_REF][START_REF] Vaquero-Fernández | Determination of cyprodinil and fludioxonil in the 428 fermentative process of must by high-performance liquid chromatography-diode 429 array detection[END_REF]). The MS 59 detector is commonly used as confirmatory method (de [START_REF] De Melo Abreu | Validation and global 365 uncertainty of a liquid chromatographic with diode array detection method for the 366 screening of azoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone, 367 pyraclostrobin and fenamidone in grapes and wine[END_REF]Likas et al. 60 2007) In GC analysis, the response of the system to certain pesticides may be affected by the 63 presence of co-extractives from the matrix. Recent related papers in different fields of 64 pesticide analyses report the matrix effect in calibration, and therefore carry out 65 preparation of the calibration solution using extracts from blank samples (matrix-66 matched calibration) [START_REF] Likas | Rapid gas chromatographic method for 384 the determination of famoxadone, trifloxystrobin and fenhexamid residues in 385 tomato, grape and wine samples[END_REF][START_REF] Zhu | Determination of procymidone, 431 pentachloroaniline and methil-pentachloro-phenylsulfide residues in wine by 432 MSPD-GC-ECD[END_REF]. 67 68 Numerous studies on the determination of certain pesticides in grapes at residual levels 69 have been published [START_REF] Navarro | Multiresidue method for the 396 rapid determination -In grape, must and wine -Of fungicides frequently used on 397 vineyards[END_REF][START_REF] Otero | Multiresidue method for 408 fourteen fungicides in white grapes by liquid-liquid and solid-phase extraction 409 followed by liquid chromatography-diode array detection[END_REF]. The method 70 commonly used is solid-liquid extraction, as a prior isolation step, with solvents such as 71 acetonitrile, hexane, acetone, dichloromethane, ethyl acetate or solvent mixtures as 72 dichloromethane/acetone [START_REF] Sala | Quick gas chromatographic method for determining 412 common pesticides in musts and wines[END_REF]Cabras et al. 2000b;[START_REF] Angioni | Gas 354 chromatographic ion trap mass spectrometry determination of zoxamide residues in 355 grape, grape processing, and in the fermentation process[END_REF]de 73 Melo et al. 2006;[START_REF] Likas | Rapid gas chromatographic method for 384 the determination of famoxadone, trifloxystrobin and fenhexamid residues in 385 tomato, grape and wine samples[END_REF]. Some studies also consider the use of solid phase 74 extraction (SPE) as a further purification process (Fernández González et al. 2003;Rial 75 Otero et al. 2003;[START_REF] Teixeira | Comparison of 423 pesticides levels in grape skin and in the whole grape by a new liquid 424 chromatographic multiresidue methodology[END_REF]. 76

77

The different effects of the application of fungicides to vines may explain the different 78 locations and concentrations of fungicide residues in grapes. While systemic products 79 penetrate the pulp, those in contact may be found in the skin. The adjuvants used in 80 commercial formulations are important in the penetration of fungicides in tissues 81 (Cabras et al. 2000a;[START_REF] Teixeira | Comparison of 423 pesticides levels in grape skin and in the whole grape by a new liquid 424 chromatographic multiresidue methodology[END_REF]). In previous studies, different penetration of 82 pesticides into the pulp has been observed in fruits like apples (Pérez-Clavijo et al. 83 1996;[START_REF] Sanz-Asensio | Behaviour of acephate and its metabolite methamidophos in apple 415 samples[END_REF]

Materials and chemicals 94

Pesticide analytical standards of pyrimethanil, metalaxyl, dichlofluanid and penconazol 95 were purchased from Riedel-de-Haën (Seelze, Germany). Standards were certified and 96 at least at >99% pure (Pestanal grade).. Metribuzin (a herbicide not used for vineyards) 97 from Riedel-de-Haën (Seelze, Germany) with a purity of 99.8% was used as Internal 98 Standard (IS). Scala ® (37.4 % pyrimethanil formulated as suspension concentrate) was 99 supplied by BASF Española S.A. (Barcelona, Spain) for vines treatment. The standards 100 were stored at -20ºC. HPLC grade ethyl acetate, sodium sulfate anhydrous for GC 101 residue analysis and washed glass wool were obtained from Scharlau (Barcelona, 102 Spain), Ultrapure water was obtained in a Milli-Ro plus system together with a Milli-Q 103 system from Millipore (Bedford, MA, USA

Standard solutions 118

Stock standards solution (ca. 1000 mg L -1 ) of each fungicide was prepared in methanol 119 by weighing approximately 0.0250 g of the analyte into a 25 mL volumetric flask and 120 diluting to volume. An intermediary mixed standard solution was prepared by dilution 121 in methanol of the stock standard solution to give a concentration of ca. 100 mg L -1 for 122 each compound. Stock and intermediary standard solutions of IS, metribuzin, were 123 prepared in the same way. All standard solutions were stored at -20ºC. 124

Sampling and processing 125

In 2005 and 2006, grapes of Tempranillo variety were harvested in the winemaking area 126 of La Rioja. These vines had not been treated with phytosanitary products and collected 127 grapes were considered as control grapes in order to carry out the study of the 128 compounds. The selected treated and control grapes from each bunch (top, middle and 129 bottom) were frozen at -20ºC in 150 mL flasks containing 75 grapes each. Before using the grapes, they were defrosted in a refrigerator at 4ºC for 24 hours. They 132 were kept at room temperature for one hour before handling. The grapes were weighed , 133 spiked and processed as follows: 134 a) Washing procedure: tap water (50 ml) was added to 25 g of grapes in a 250 mL 135 glass until they were completed covered to dissolve the pesticide residue 136 remaining on the exterior of the fruit. The glass was placed in an ultrasound bath 137 and shaken for 30 minutes to dissolve the residues located on the surface of the 138 grapes. The removed water was concentrated in rotavapor to 5 mL for 139 subsequent SPE analysis. SPE cartridges containing 200 mg of C18 sorbent 140 were used. The C18 cartridge was activated with 5 mL methanol and then 3 mL 141 ultrapure water. Volume sample was 5 mL. A washing step with 2 mL of 142 water/methanol (9/1) was carried out. The extract retained in the solid phase was 143 eluted with 2+2 mL of ethyl acetate. (Method 1). 144 b) Solvent extraction of fungicides in grape skin: Once the grapes had been washed 145 with water and the washing water had been removed, they were left to dry on 146 filter paper at room temperature for 10 min. The washed and dried grapes were 147 placed in 50 mL of ethyl acetate in a 250 mL glass in order to extract the 148 fungicide residues absorbed on the grape skins. The glass was placed in the 149 ultrasonic bath for 30 min. To ensure full extraction of the skin, four consecutive 150 extractions of 30 min were carried out in the ultrasonic bath for spiked grapes. 151

No signal of fungicides was observed in the third and fourth extracts. The 152 method proposed for grape skin includes two extractions with 50 ml ethyl 153 acetate for 30 min in the ultrasonic bath. The two extracts were combined, dried 154 The quantification for GC analysis was performed using metribuzin as internal standard 164 at a concentration of 1 mg L -1 . Confirmation was performed by GC-MS. 165 166

Instrumentation and chromatographic conditions 167

An Agilent Technologies GC 6890 N equipped with an NPD Nitrogen-Phosphorus 168

Detector system, an autosampler 7683 (Agilent) and a split-splitless injector connected 169 to a HP ChemStation (Hewlett-Packard) were used for determining the fungicides 170 dichlofluanid, metalaxyl, penconazole and pyrimethanil. The capillary column was a 171 HP-5MS (30m x 0.25 mm i.d. with 0.25 µm of cross-linked 5% phenylmethyl siloxane). 172

Injector and detector temperatures were 200ºC and 325ºC, respectively. Oven 173 temperature was programmed as follows: 80ºC for 0.9 min, increased to 225ºC (70ºC 174 min -1 ) for 5 min, to 275ºC (70ºC min -1 ) for 3 min. Helium was the carrier and make-up 175 gas at 1.5 mL min -1 and 3 mL min -1 , respectively. Flow ratios for the NPD detector were 176 3 mL min -1 for hydrogen and 70 mL min -1 for air. Injection was performed in splitless 177 mode with a purge time of 0.9 min and the injection volume was 2 µL. 178 The confirmation of the studied compounds was carried out on an Agilent 7890A GC 180 with MS 5975C detector system. Column, carrier flow and oven temperature program 181 were the conditions above mentioned for GC-NPD system. The volume of sample was 1 182 µL, injected in splitless mode with splitless time of 1 min. The mass spectrometer was 183 operated in electron impact (70 eV of ion energy), with 3.50 min solvent delay, the 184 interface temperature was kept at 285ºC and the ion source temperature was kept at 230 185 ºC and the dwell time was 100 ms per ion. Selected monitoring ion (SIM) mode was 186 used (198, 199, and 200

Study of absorption and distribution in grapes 191

The absorption of the fungicides was studied by soaking 25 g of untreated grapes in 50 192 mL of aqueous solution spiked with three fungicide concentration levels (1.00, 5.00 and 193 10.00 mg L -1 , corresponding to 2, 10 and 20 µg g -1 , respectively), in a 250 ml 194 beaker.The samples were kept in the dark at 4 ºC for 1, 3, 7, 13 and 21 days (the longest 195 safety period for all compounds). The spiking concentrations were similar or higher 196 than MRLs fixed by European Union (10 µg g -1 for dichlofluanid, 1 µg g -1 for 197 metalaxyl, 0.2 µg g -1 for penconazol and 5 µg g -1 for pyrimethanil).Three replicates 198 were carried out. The fungicide absorption in the whole grape was calculated as the sum 199 of the three parts: surface, skin and pulp. 200

Study in field 201

Study of fungicides absorption 240

Grapes in contact with solutions containing the fungicides at three concentration levels 241 were analyzed. The amount of residues present in whole grapes was determined by the 242 proposed methods and the percentage absorption of fungicide relative to its 243 concentration in the solution was calculated. Table 1 shows the results for each 244 compound in all the concentration and time situations. 245

246

The amount of absorbed fungicide increased with increasing initial concentration of 247 solutions for any time period. This increase was higher, the higher the contact time. 248 In eq a C t is the residue concentration at time t (µg g -1 ), C 0 is the theoretical initial 259 residue concentration at t = 0 (µg g -1 ), K is the fungicide absorption constant, and t is 260 the time that grapes have been in contact with solutions. This type of analysis, a first 261 order kinetics, allows the behavior of fungicide residues during the time to be known, 262 by showing the correlation that exists between the absorbed residues and the time. It is 263 also possible to study if the correlation existing between variables is more o less 264 significant from the statistical point of view, through a statistical demonstration with the 265 number of pairs of values used in the coefficient calculus. The following equation is 266 then used [START_REF] Miller | Estadísitica y quimiometría para Química Analítica, 4 a 393 edición[END_REF]: 267

t = |r| √ (n -2) / √ (1 -r 2 ) (c) 268
In eq c, r represents the correlation coefficient and (n -2) the degrees of freedom. From 269 this equation one obtains a value of distribution of the Student-t that can be compared to 270 t tabulated values. The fitting results are presented in Table 2. 271 As can be seen from these data, the linear correlation between Ln C t and time was good 272 for pyrimethanil, metalaxyl and dichlofluanid for all assays, with correlation 273 coefficients r > 0.906. Penconazol did not show a linear correlation, with low 274 correlation coefficients r < 0.853. However, this compound presented a linear fit from 1 275 to 7 days. The Student-t test performed to compare the slopes (K values) revealed that 276 there were no significant differences at 95 % confidence level for the three studied 277 concentrations. For the constant values, we can establish an absorption rate for the 278 fungicides as follows: metalaxyl > pyrimethanil > dichlofluanid. 279 280

Study of fungicides distribution 281

The amount of residues in the three parts of the grapes (surface, skin and pulp) was 282 determined using the proposed methods and the percentage distribution relative to the 283 total absorbed residues was calculated. Figure 1 shows this distribution of the fungicide 284 residues in the different parts of the grape as a function of time for the three studied 285 concentrations The highest content was found in the skin for all the compounds, 286 regardless of the fungicide concentration in contact with grapes, with > 66.3 % of the 287 residues for all the fungicides In pulp, the residue amounts ranged from 3.5 to 31.0 %. 288

289

A study of fungicide penetration in pulp was performed by calculating the ratio between 290 pulp and skin concentrations (Q) for all the assays. There was no significant difference 291 at 95 % confidence level between concentrations at the same time. The penetration did 292 not depend on the initial solution concentration. The average values for all 293 concentrations are shown in Table 3

Analysis of red grapes from La Rioja 301

The overall methodology was applied to determine pyrimethanil in Tempranillo 302 grapevines. The selected treated grapes from different clusters from the vineyard (top, 303 middle and bottom of the cluster) were frozen at -20ºC in 150 mL flasks containing 75 304 grapes each during one week for further analysis. The results are shown in Table 4. 305

306

The amount of pyrimethanil determined in the three parts of the fruit was higher in the 307 grapes collected after seven days of treatment. A high reduction in pyrimethanil 308 residues were observed over time. This decrease was ca. 99 % from 7 to 21 days. The 309 reduction was due to dissipation and/or a degradation by environmental conditions. 310 Pyrimethanil residues were mainly found in the skin (78.2% and 64.5% in grapes 311 collected after 7 days and 21 days, respectively). Penetration in pulp was higher for 312 grapes collected after 21 days, 15.5% of the residues vs. 10.1% in grapes collected after 313 7 days. Pyrimethanil penetration observed in field studies was comparable to that 314 observed in laboratory studies. 315 316 Conclusions 317 A simple, rapid, liquid extraction method was developed for the determination of 318 pyrimethanil, dichlofluanid, metalaxyl and penconazol in red grapes, Overall the 319 and pulp. The method gave good recoveries, linearity, precision and accuracy and was 321 highly sensitive. An additional GC-MS confirmatory method was proposed for treated 322 grapes samples. 323

324

The study of fungicides mobility in the laboratory showed that percentage absorption 325 did not depend on the initial spiked concentration and the percentage increased with 326 increasing contact time. Pyrimethanil, metalaxyl and dichlofluanid showed a first order 327 kinetics for the absorption during time. 328

329

The highest concentrations of residues were found in the skin Penetration into pulp was 330 observed for all the fungicides. The ratios between pulp and skin residues (Q) did not 331 depend on the initial spiked concentration. Penconazol had a higher penetration than the 332 other compounds. 333 334 Grapes treated with pyrimethanil and collected after 7 days had higher amounts of 335 residues than those harvested when respecting the safety period.

  anhydrous and filtered through glass wool and concentrated 155 in Turbo Vap ® to 5 mL (Method 2). 156 c) Solvent extraction of fungicides in the whole grape: Grapes were crushed after 157 removal of ethyl acetate for skin extraction. Ethyl acetate 25 mL was added. The 158 grapes were homogenized with the crusher for 2 minutes and then centrifuged at 159 6000 rpm for 15 min. The liquid phase was dried with sodium sulfate anhydrous 160 and filtered through glass wool. The extract was concentrated at 5 mL in the 161 Turbo Vap ® (Method 3). 162 163

  Rioja Baja were treated with Scala ® (37.4 % Pyrimethanil) at 202 the recommended doses (2 L ha -1 ). Scala  is a very widely-used product against Botrytis 203 cinerea. Samples were collected seven and twenty-one days after treatment. were no interferences in the extracts obtained from red grapes in GC-NPD 209 chromatogram. The proposed conditions generated narrow and reproducible 210 chromatographic peaks. Clean chromatograms were obtained with only peaks of interest 211 in fortified blank matrix. The GC-MS method allowed confirmation of the fungicides 212 with good resolution and did not show interfering peaks. 213 214 Matrix standard calibration were performed for washing water and ethyl acetate from 215 skins, and a matrix matched calibration were carried out for pulp. Linear range, limits of 216 detection (LODs), limits of quantification and repeatabilitiy were determined for the 3 217 methods and 4 fungicides. Linear range were from 10 to 1400 µg kg -1 . Good linearity 218 was obtained for all fungicides and for the entire range of studied concentrations with 219 correlation coefficients better than 0.997. LODs were ranging from 0.04 to 1.67 µg kg -1 220 in surface (washing water); from 0.01 to 1.05 µg kg -1 in skin (ethyl acetate); and from 221 2.46 to 5.64 µg kg -1 in pulp. Repeatabilities expressed as RSD were below 11.4 calibration and in solvent was also carried out for the GC-MS 225 method. In the range from 2.0 to 2000 µg kg -1 in grape and water extracts, and in ethyl 226 acetate, excellent linearity was also obtained (correlation coefficients > 0.9995). The 227 GC-MS confirmation method showed LODs and LOQs in grapes below 0.41 and 1.36 228 µg kg -1 for all compounds, values considerably below the fixed MRLs. 229 230 Recovery and accuracy were determined with spiked grapes at the three concentration 231 levels and were analyzed in triplicate. The analytical methods presented had recoveries 232 > 90.1 % and precisions (repeatability) < 7.6 % and they were within the acceptable 233 range: 70-110% with relative standard deviation (RSD) <10% (n=3). (Document Nº 234 SANCO/10232/2006). 235 236 An analysis of variance performed with the data revealed the absence of significant 237 differences (p<0.05) between the assayed levels of spiking. 238 239

  percentage absorption percentage which was found did not depend on the 249 initial fungicide concentration in contact with grapes for the same contact time. 250 Metalaxyl was the most absorbed fungicide, followed by pyrimethanil, penconazol and 251 dichlofluanid.. 252 253 To ascertain the fungicide absorption rate at each studied concentration, the 254 experimental data have been fitted to the following mathematical model (Oliva et al. 255 2005): 256 C t = C 0 e -Kt (a) 257 Ln C t = LnC 0 -Kt (b) 258

  procedures for each different part of the berry: surface, skin 320

Figure 1 .

 1 Figure 1. Distribution of fungicides in surface, skin and pulp in grapes which have been 464in contact with three spiked solutions (2 µg g -1 , 10 µg g -1 , 20 µg g -1 ) during 1, 3, 7, 13 465

  . Penconazol and dichlofluanid had the highest Q 294 values. Metalaxyl and pyrimethanil had a similar behavior, but different from the other 295

	296	compounds. The statistical analysis performed for all times showed that metalaxyl and
	297	pyrimethanil Q values did not differ at 95 % confidence level from 7 to 21 days.
	298	Dichlofluanid presented no significant differences in Q values for 1 to 7 days.
	299	Penconazol had higher Q values for the highest contact time.
	300
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
		14

Table 2 :

 2 Statistical parameters derived from the linear fit of the data for the fungicides absorption during time.

	Pyrimethanil residues

Table 3 :

 3 Average ratios (Q ± standard deviation * ) between pulp and skin concentrations for fungicides during time

	457
		Days	Pyrimethanil	Metalaxyl	Dichlofluanid	Penconazol
		1	0.056 ± 0.006 a	0.057 ± 0.002 a	0.175 ± 0.025 a	0.157 ± 0.015 a
		3	0.123 ± 0.011 b	0.078 ± 0.003 b	0.202 ± 0.021 a	0.169 ± 0.010 b
		7	0.162 ± 0.006 c	0.117 ± 0.010 c	0.226 ± 0.012 ab	0.215 ± 0.011 b
		13	0.184 ± 0.031 c	0.119 ± 0.014 c	0.254 ± 0.021 b	0.273 ± 0.022 c
		21	0.220 ± 0.028 c	0.142 ± 0.006 c	0.312 ± 0.023 c	0.410 ± 0.045 d
	458	* n = 9
	459	a b c d Different letters in the same column means significant differences (P<0.05)

Table 4 :

 4 Pyrimethanil residues in grapes from La Rioja 461

		Sample		Pyrimethanil µg kg -1 (RSD, % a )
			Surface	Skin	Pulp
		2006 (7 days)	443 (6.9)	2955 (11.3)	382 (8.6)
		2006 (21 days)	4.55 (6.3)	14.7 (13.0)	3.54 (6.5)
	462	a n = 3

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Acknowledgements

346

The authors want to thank the Government of La Rioja for the FPI scholarship granted 347 to Luis Vaquero-Fernández and the project ANGI 2004/18, the INIA for the 348 infrastructure provided (project VIN00-054-C2-01), the MEC/FEDER for the 349 AGL2005-02313/ALI project and the University of La Rioja for its support, Bodegas D. 350