V Rossi

V Terzi

F Moggi

C Morcia

P Faccioli

M Haidukowski

M Pascale

Assessment of Fusarium infection in wheat heads using a quantitative PCR assay

Keywords: Fusarium head blight, real-time qPCR, deoxynivalenol, Triticum, Fusarium culmorum, Fusarium graminearum

Accuracy of a qPCR assay in quantifying the DNA of trichothecene-producing F. culmorum and F. graminearum within harvested wheat grains and head tissue was evaluated in comparison with incidences of infected kernels and deoxynivalenol (DON) levels. In a first experiment six durum and bread wheat varieties were grown in randomized plots for a 2-year period, and inoculated with Fusarium macroconidia at six growth stages between heading and dough ripening, to obtain a wide range of Fusarium head blight (FHB) incidences. There was a close relationship between fungal DNA and amount of DON, and this relationship was consistent over Fusarium species, wheat species and varieties, and over a wide range of FHB infection. In a second experiment potted wheat plants were grown under environment-controlled conditions and inoculated with the two Fusarium species at full flowering; head samples were collected before inoculation and after 6 hours to 12 days, and processed by the qPCR assay. This assay made it possible to detect the dynamic of fungal invasion in planta after infection had occurred, and to single out the presence of infection before onset of the disease symptoms: a robust detection of the infection occurred within 18 to 24 hours for F. culmorum, and within 2 to 9 days for F. graminearum.

Introduction

Fusarium head blight (FHB) is a relevant disease of small-grain cereals worldwide [START_REF] Parry | Fusarium ear blight (scab) in small grain cereals-a review[END_REF], McMullen et al. 1997). Several causal organisms have been associated with the disease, but

Fusarium avenaceum (Corda ex Fries) Sacc., F. graminearum Schw. (Gibberella zeae Petch), F. culmorum (Wm. G.) Sacc., F. poae (Peck) Wollenw, F. cerealis Cooke (syn. F. crookwellense), and

Microdochium nivale (teleomorph, Monographella nivalis) are the most common species [START_REF] Parry | Fusarium ear blight (scab) in small grain cereals-a review[END_REF].

Damage caused by FHB is multifold: reduced yields, discoloured and shrivelled kernels, reduced grain weight, lowered market grade, reduced seed quality due to reduced germination, seedling blight and poor stand [START_REF] Bechtel | The effects of Fusarium graminearum infection on wheat kernels[END_REF][START_REF] Tuite | Wheat scab in soft red winter wheat in Indiana in 1986 and its relation to some quality measurements[END_REF]). FHB also is of great concern since several species that cause the disease can produce mycotoxins [START_REF] Bottalico | Fusarium species and their mycotoxins in infected corn in Italy[END_REF]) and particularly trichothecenes, a group of sesquiterpenoid mycotoxins that cause a wide range of acute and chronic effects in humans and animals [START_REF] Iarc | Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins[END_REF], D'Mello et al. 1999).

Deoxynivalenol (DON), also known as vomitoxin, is the predominant trichothecene found in cereal grains in Europe and North America produced by Fusarium culmorum, F. graminearum, and F. poae (Placinta et al. 1999, Bottalico and[START_REF] Bottalico | Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe[END_REF]. The Scientific Committee for Food (SFC) of the European Commission has recently established a TDI (Tolerable Daily Intake) of 1 µg/kg bodyweight/day for DON in humans. In addition, the Commission of the European Community has recently fixed the maximum levels of DON in cereals and cereal products (EC Regulation No 856/2005). Therefore, accurate methods for early detection of both Fusarium infection and DON contamination are needed to be applied along the production chain, from growing plants to raw materials and processed food, to ensure efficient consumer protection.

Methods for quantifying FHB include visual assessments of disease severity and incidence of affected ears, spikelets or damaged kernels at harvest [START_REF] Hilton | Relationship between cultivar height and severity of Fusarium ear blight in wheat[END_REF][START_REF] Lacey | Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat[END_REF]. All these methods estimate disease symptoms and not pathogen populations. In contrast, methods based on plate counts of kernels infected by Fusaria (Henriksen and Elen 2005) determine incidence of the different FHB causing fungi. Identification of the fungal species involved in a FHB epidemic can be determined by subculturing colonies growing from the infected kernels onto agar plates and examining their morphological characters [START_REF] Nelson | Fusarium species. An illustrated manual for identification[END_REF]; it requires taxonomical expertise and is time expensive.

DNA-based methods for identification of Fusarium species have been developed and applied as an alternative to morphological approaches. Assays based on SCAR (sequence-characterized amplified region) markers for the differential detection of F. culmorum, F. graminearum, F.

avenaceum, and F. poae have been developed [START_REF] Nicholson | Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays[END_REF][START_REF] Llorens | Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)[END_REF]. Advances in the molecular characterization of genes from "toxin clusters" had provided the potential to develop PCR assays based upon the mycotoxin biosynthetic gene sequences for identification and characterization of fungal species and isolates (Edwards et al. 2002, Seifert and[START_REF] Seifert | Phylogeny and molecular diagnosis of mycotoxigenic fungi[END_REF]. In particular, the molecular characterization of the Tri-5 gene cluster, which contains most of the genes involved in trichothecene biosynthesis, has been exploited in the development of several PCR-based assays to target Fusarium species and chemotypes [START_REF] Lee | Identification of deoxynivalenol and nivalenol producing chemotypes of Gibberella zeae by using PCR[END_REF][START_REF] Ward | Ancestral polymorphism and adaptative evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium[END_REF][START_REF] Kimura | Trichothecene non producer Gibberella species have both functional and non functional 3 O-acetyltransferase genes[END_REF]. On these sequences, both generic assays that target all tricothecene producers [START_REF] Li | Development of a generic PCR detection of deoxynivalenol and nivalenol-chemotypes of Fusarium graminearum[END_REF] as well as species-specific assays have been developed (Demeke et al. 2005). Generic primers designed on tri5 gene sequence, that amplify all thricothecene producers have been developed by [START_REF] Schnerr | Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring[END_REF], by [START_REF] Doohan | Fusarium ear blight of wheat: the use of quantitative PCR and visual disease assessment in studies of disease control[END_REF] and by [START_REF] Edwards | Quantification of trichothecenetroducing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat[END_REF]. Sequences from the Tri3, Tri5 and Tri7 genes have been used to develop multiplex PCR assay for the chemotype identification of several European Fusarium isolates [START_REF] Quarta | Multiplex PCR assay for the identification of nivalenol, 3-and 15-acetyl-deoxynivalenol chemotypes in Fusarium[END_REF]. Chemotype determination of Fusarium isolates from China and Nepal and from England and Wales trough a PCR assays based on Tri7 and Tri13 sequences have been reported respectively by [START_REF] Chandler | Development of PCR assay to Tri7 and Tri13 trichothecene biosynthetic genes, and characterization of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis[END_REF] and [START_REF] Jennings | Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assay[END_REF]. Primers specific to Tri6

have been designed and can be used in conjunction with assays for either Tri5 or Tri4 as part of a robust detection system for trichothecene producers [START_REF] Mulè | Advances in molecular diagnosis of toxigenic Fusarium species: a review[END_REF].

Real-time quantitative PCR is a sensitive, rapid and versatile tool that has opened new opportunities for quantitative diagnosis of phytopathogenic fungi [START_REF] Schena | Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi[END_REF]). [START_REF] Waalwijk | Quantitative detection of Fusarium species in wheat using TaqMan[END_REF] developed a quantitative assay based on TaqMan technology to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals. [START_REF] Bluhm | Multiplex real-time PCR detection of fumonisinproducing and trichothecene-producing groups of Fusarium species[END_REF] developed a 5' fluorogenic real-time PCR assay for group specific detection of trichothecene and fumonisin producing Fusarium ssp. using primers and fluorogenic probes designed from genes directly involved in mycotoxin biosynthesis (tri6 and fum1), whereas Sarlin et al. (2006) evaluated the applicability of this technique for quantification of toxigenic Fusarium species in barley and malt. In a recent work [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF]), a qPCR-based analytical method has been developed targeted at the quantification of the biomass of thricothecene producing F. graminearum and F. culmorum strains, using a pair of primers designed on the Tri5-Tri6 intergenic sequence, that is involved in DON synthesis. This intergenic sequence has been already used [START_REF] Bakan | Identification by PCR of Fusarium culmorum strains producing large and small amount of deoxynivalenol[END_REF][START_REF] Li | Development of a generic PCR detection of deoxynivalenol and nivalenol-chemotypes of Fusarium graminearum[END_REF], but the analytical approach developed by [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF] is quantitative and can be used also in samples characterized by high level of DNA fragmentation, as those derived from food and feed, because of the small size of the amplicon. For this reason it can be potentially used along the entire production chain. The objectives of this work were (i) to test accuracy of the qPCR assay to quantify the biomass of trichothecene-producing F. culmorum and F. graminearum within harvested grains of different bread and durum wheat varieties; (ii) to determine if there is a correlation between the DNA content of F. culmorum and F. graminearum, incidence of infected kernels as determined by classical plating methods, and DON levels in these grain samples; (iii) to investigate the potential of the qPCR assay for an early in planta diagnosis of Fusarium infection.

Materials and methods

Plant and fungal materials

Three durum and three bread wheat varieties were used, characterised by increasing susceptibility to FHB: Simeto, Duilio and S. Carlo for durum wheat; Bilancia, Centauro and Sagittario for bread wheat. These varieties were selected to ensure different levels of Fusarium infection and kernel colonization and therefore to constitute a representative data set for robust results.

Three monosporic strains of F. culmorum (named MPVP/69, MPVP/70 and MPVP/71) and two of F. graminearum (ITEM 6477 and ITEM 6484) were used. F. culmorum strains have been used in previous works [START_REF] Rossi | Influence of temperature and humidity on the infection of wheat spikes by some fungi causing Fusarium head blight[END_REF][START_REF] Rossi | Effect of constant and fluctuating temperature regimes on sporulation of four fungi causing head blight of wheat[END_REF] and maintained in our culture collection repository, while F. graminearum strains were kept at the Institute of Sciences and Food Production (CNR, Bari, Italy). All fungal strains were able to produce DON in vitro when grown on autoclaved wheat.

Strains were stored on PDA (Potato Dextrose Agar) at 4°C.

To produce conidia for artificial inoculations, PDA 'starter' plates were inoculated with each fungal strain from stock cultures and incubated for one week at 20°C. Plugs (0.5 cm 2 wide) were then extracted from the periphery of these colonies with a cork borer, transferred to the centre of a PDA plate (9 cm diameter), and incubated for 15 days at 25°C, with a photoperiod of 12 hours light and 12 hours dark. Fungal colonies were washed two times with 10 ml of sterile water and gently shaken; these suspensions were filtered with a double layer of sterile cheesecloth and suspended again in sterile water, streptomycin sulphate and neomycin sulphate (both at 50 mg per l), and Tween 20 (0.1%), until a concentration of 1x10 4 conidia/ml. The suspensions used for inoculation were finally prepared by mixing equal amounts of the three suspensions produced for each strain of the same Fusarium species.

Field experiments

In plots (1 m 2 ) according to a completely randomized design, with three replicates. Seeds were treated with tebuconazol (3 g/q seed) before sowing to prevent foot rots and then cropped using the common practice, with exclusion of fungicide applications.

Plots were inoculated with either F. graminearum or F. culmorum conidia. Considering that susceptibility of wheat plants to Fusarium infection changes with the growth stage [START_REF] Andersen | The development of Gibberella zeae headblight of wheat[END_REF][START_REF] Diehl | Weizenfusariosen -Einfluss von Infektionstermin, Gewebeschadigung und Blattlausen auf Blatt-und Ahrenbefall[END_REF][START_REF] Lacey | Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat[END_REF] One-hundred spikes were collected randomly from the edge of each plot at maturity and threshed. Kernels were split in three sub-samples to determine: i) proportion of infected kernels; ii) amount of Fusarium DNA; iii) DON contamination.

To determine Fusarium infection, kernels were washed in running tap water for 20 min, sterilised with ethyl alcohol (70%) for 15 sec and then in sodium hypochlorite (2%) for 2.5 min, rinsed three times in sterile water and dried on absorbent paper under a sterile air flow. Kernels were placed in Petri plates on water agar (1.2%) adjusted to pH 5.2. After 6 to 8 days of incubation at 25°C, fungal colonies growing from the kernels were transferred to other Petri plates on PDA with streptomycin sulphate (50 mg per l) and incubated at 25°C, with an artificial day length of 12 hours. Fusarium species were then identified according to [START_REF] Nelson | Fusarium species. An illustrated manual for identification[END_REF] and [START_REF] Burgess | Laboratory manual for Fusarium research[END_REF]. When necessary, isolates where transferred onto carnation-leaf agar, as described by [START_REF] Nelson | Fusarium species. An illustrated manual for identification[END_REF].

Environment-controlled experiments

In 2006, four wheat varieties (Duilio, S. Carlo, Bilancia, Sagittario) were sown in pots (50 cm in diameter, 120 seed per pot, 4 pots per variety) and grown under environment-controlled conditions.

These plants were inoculated with the two Fusarium species at full anthesis (65 BBCH).

Inoculations were performed by distributing 0.3 l of spore suspension per pot by a nebulizer; an Five spikes per pot were collected before inoculation and after 6, 12, 18 hours, 1, 2, 3, 6, 9 and 12 days. Spikes were washed under running tap water using a brush to remove epiphytic fungal parts, and then washed again for 30 min. Afterwards, spikes were dried on absorbent paper, frozen with liquid nitrogen and stored at -80°C until determination of the amount of Fusarium DNA.

Fusarium DNA analyses

DNA was extracted from lyophilised Fusarium mycelium, prepared as reported in [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF], according to the method described by [START_REF] Al-Samarrai | A simple method for extraction of fungal genomic DNA[END_REF]. 2007) is of 4 pg of fungal DNA, corresponding to about 100 F. graminearum haploid genomes [START_REF] Kullman | Fungal Genome Size Database[END_REF]. The specificity of the assay was previously evaluated on a panel of different Fusarium species [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF].

DNA

DON analyses

DON levels were determined by HPLC/UV after immunoaffinity column clean-up [START_REF] Haidukowski | Effect of fungicides on Fusarium head blight, yield and deoxynivalenol accumulation in wheat inoculated under field conditions with Fusarium graminearum and Fusarium culmorum[END_REF]. Twenty-five g of ground samples were added to polyethylene glycol (PEG-8,000) and extracted with 100 mL water by blending at high speed for 2 min with a Sorvall Omnimixer (Sorvall Instruments, Norwalk, CT, USA). Extracts were filtered through both filter paper (Whatman no. 4) and glass microfibre filter (Whatman GF/A) and cleaned up by DonTest™ HPLC immunoaffinity column (VICAM, Watertown, MA, USA). The toxin was determined by an Agilent 1100 Series HPLC system equipped with a diode-array UV detector set at 220 nm (Agilent Technologies, Palo Alto, CA, USA). The column was a Waters C18 Symmetry Shield, 150 × 4.6 mm, 5 µm (Waters, Milford, MA, USA) preceded by a 0.5 µm Rheodyne guard filter. The mobile phase was a mixture of acetonitrile:water (10:90) eluted at a flow rate of 1.0 ml/min. Appropriate dilutions of sample extracts before loading on immunoaffinity columns were necessary to avoid saturation of the DON-antibody binding sites. The detection limit of the method was 0.05 ppm (signal-to-noise ratio 3:1).

Data analysis

All the statistical analyses of this work were performed using the SPSS package ver. 11.5 (SPSS Inc., Chicago, USA). Data on Fusarium DNA (expressed in pg) and of DON (in ppm) collected in the field experiments were transformed to make variances homogeneous, using the natural logarithm in the following way: ln(x+1), where x is value in the original scale. In a similar way, incidence of infected kernels (in %) were transformed using the arcsin of the square rot of the percent value.

A preliminary data analysis was performed by calculating summary statistics and by drawing box-plots to explore data variability. Afterwards, a factorial analysis of variance (AOV) was made to test significant effects of the main factors considered (year, Fusarium species, wheat growth stages and varieties) as well as their interactions. The Fisher protected least square difference (LSD)

was applied at P=0.05 to separate means. Effects of wheat species and varieties were not analysed in detail because this was not within aims of the present work.

Pearson's coefficient of correlations were calculated to determine the degree of association between variables and a regression analysis was performed to determine quantitative relationships between Fusarium DNA and DON in grain samples, using the nonlinear regression procedure of SPSS.

Also Fusarium DNA data obtained with the qPCR assays performed in the second experiment were ln-transformed before applying a factorial AOV and the LSD test at P=0.05.

Results

Fusarium infection, DNA and DON in wheat kernels

Colonisation of kernels by the two Fusarium species was higher in 2004 than in 2003, with an overall average of 876 pg of DNA and 46 pg, respectively. DNA of F. culmorum was 871 pg on average against 54 pg for F. graminearum. Spikes inoculated at anthesis produced more colonised kernels (1012 pg at stage 61 and 1470 pg at stage 65), than these inoculated at heading (214 pg), water (49 pg), milk (24 pg) and dough (4 pg) ripening (Figure 1). AOV showed a significant effect of all the above mentioned factors (at P<0.001); year accounted for 21% of total variance, Fusarium species for 6% and growth stage for 32%. Wheat variety was also significant (at P<0.001) and accounted for 4% of total variance. Interactions 'year x growth stage' (5% of total variance), 'Fusarium species x growth stage' (4%), and 'year x variety' (8%) were also significant (at P<0.01).

Incidence of kernels infected by F. culmorum was 1.7 times higher than incidence of F. graminearum (19.0% and 12.3% on average, respectively), but magnitude of this difference changed depending on different growth stages when spikes have been inoculated (interaction 'Fusarium species x growth stage' was significant at P<0.05) (Figure 1). For inoculations made during ripening differences between the two species were not significant, frequency of infected kernels ranging between 7.0% and 13.2%. At full anthesis (stage 65), 35.4% of kernels were infected by F. culmorum but only 16.6% of them were infected by F. graminearum; at early anthesis (stage 61) infection incidences were 26.2% and 17.0% for the two fungi, respectively. For the inoculations made at heading (stage 59) incidences were of 16.7% and 9.8% of kernels, respectively. Maximum incidence of infected kernels was observed when F. culmorum was inoculated at full anthesis in 2004 (51.0% of kernels).

DON contamination was higher in kernels that have been inoculated with F. culmorum than with F. graminearum (1.34 ppm and 0.27 ppm on average respectively); this difference was significant (at P<0.001) and accounted for 5% of total variance. Year and growth stages were also significant, and accounted for 11% and 17% of variance, respectively. Contamination was higher in 2004 (1.44 ppm on average) than in 2003 (0.18 ppm). DON was higher in kernels from spikes that have been inoculated at anthesis (1.65 ppm at stage 61 and 2.19 ppm at stage 65) than at heading (0.69 ppm)

and especially during ripening (from 0.21 at stage 71 to 0.02 at stage 85). Because a significant (P<0.001) interaction 'Fusarium species x growth stage', differences between growth stage were higher for F. culmorum than for F. graminearum (Figure 1).

A significant correlation was observed between Fusarium DNA and DON contamination of kernels (r = 0.797, P<0.0001, n = 144) and this correlation was consistent over years and fungal species: correlation coefficients varied between 0.617 for F. graminearum in 2003 and 0.848 for F.

culmorum in 2004 (Table I). When DNA was lower than 54 pg (a value of 4 on the ln-scale) about 70% of grain samples had a DON content lower than the detection limit; when DNA was higher than this value 93% of samples contained measurable DON, when DNA was greater than 1096 pg (7 in the ln-scale) all samples were contaminated (Figure 2). Quantitative relationship between the ln-transformed values of these two variable was fitted by a power equation that accounted for 88% of data variability (Figure 3). Back-transformation of the fitted data showed that increases in Fusarium DNA resulted in nonlinear increases of DON: DON accumulation per unit of fungal DNA was greater for severely infected grain samples than for the less infected ones.

Fusarium DNA was also correlated with incidence of infected kernels, but this correlation was lower and less significant (r = 0.195, P=0.019, n = 144); furthermore, the coefficient of correlations between these two variables were significant only in 2004 (Table I), when the incidence of infection was higher. In a similar way, infected kernels were also correlated with DON, but less closely than DNA did (r = 0.411, P<0.001, n = 144) and only in 2004 (Table I).

Fusarium DNA detection after infection

AOV made on Fusarium DNA contents detected in spike tissues of four wheat varieties at different times after inoculation showed a significant effect for all the factor tested and for their interactions, .001; only 'Fusarium species x variety' interaction was significant at P = 0.017. Time elapsed after infection and Fusarium species were the more influential factors, accounting for 72% and 12% of total variance, respectively, with their interaction accounting for 7%.

Using the qPCR assay the Fusarium DNA was always detected in the inoculated spikes before the appearance of first disease symptoms. Considering a level of 4 pg of DNA (1.61 in the ln-scale)

as the minimum content of DNA for robust detection of the fungi in the spike tissue, it was observed that F. culmorum in wheat spikes was detected for first time 18 hours after infection in three varieties and after 24 hours also in the bread wheat Bilancia (Table II). After this time amount of DNA progressively increased until 9 days and then did not significantly increased in the following 3 days. First robust detection of F. graminearum DNA occurred 48 and 72 hours after inoculation in the durum wheat S. Carlo and Duilio, respectively. In bread wheat Sagittario first detection occurred after 6 days and only after 9 days in Bilancia (Table II). On average of different varieties, the DNA content of spikes progressively increased until the last sample collected 12 days after inoculation.

Discussion

This work was planned in such a way to obtain a data set characterised by high variability in both Fusarium infection and DON contamination to draw representative and robust results. For this reason, artificial inoculations with F. culmorum and F. graminearum were made on three durum and three bread wheat varieties characterised by different susceptibility to FHB, at six different growth stages between heading and dough ripening, for a 2-year period.

The degree of Fusarium infection was determined as incidence of infected kernels and amount of fungal biomass measured as Fusarium DNA, and these variables were correlated with the level of DON contamination. In this work we did not perform a visual disease assessments of FHB, but literature demonstrates that there is no robust association between FHB symptoms and DON contents in grains [START_REF] Boyacioglu | Effect of three systemic fungicides on deoxynivalenol (vomitoxin) production by Fusarium graminearum in wheat[END_REF][START_REF] Lemmens | Head blight (Fusarium spp.) on wheat: investigations on the relationship between disease symptoms and mycotoxin content[END_REF][START_REF] Liu | Comparison of visual head blight ratings, seed infection levels, and deoxynivalenol production for assessment of resistance in cereals inoculated with Fusarium culmorum[END_REF][START_REF] Martin | Effects and control of Fusarium diseases of cereal grains in the Atlantic Provinces[END_REF][START_REF] Doohan | Fusarium ear blight of wheat: the use of quantitative PCR and visual disease assessment in studies of disease control[END_REF][START_REF] Edwards | Quantification of trichothecenetroducing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat[END_REF][START_REF] Birzele | Epidemiology of Fusarium infection and deoxynivalenol content in winter wheat in the Rhineland, Germany[END_REF] because some fungi causing the disease do not produce this mycotoxin. Furthermore, no correlation was found between visual FHB assessments and concentration of trichothecene-producing F. culmorum and F.

graminearum measured with a PCR-based assay based on primers derived from Tri5 [START_REF] Edwards | Quantification of trichothecenetroducing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat[END_REF].

Correlation and regression analyses of trichothecene-producing Fusarium DNA and DON in harvested grain showed a close correlation between biomass and the amount of DON produced. Similar results had been previously found by other authors [START_REF] Edwards | Quantification of trichothecenetroducing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat[END_REF][START_REF] Schnerr | Correlation between DNA of thricotecene-producing Fusarium species and deoxynivalenol concentrations in wheat samples[END_REF], Waalwiijk et al. 2004), but the present work showed that this relationship is consistent over Fusarium species, wheat species and varieties, and over a wide range of epidemiological conditions due to different meteorological conditions and times of infection occurrence. In a previous work, different correlations were found between Fusarium exoantigens and DON in harvested grain from field samples of hard red spring wheat in Manitoba, Canada, and of soft white winter wheat in Ontario, Canada, indicating that the relationship between amounts of Fusarium biomass and DON in harvested grain may be affected by the host and by its interactions with environmental conditions [START_REF] Abramson | Relationships among deoxynivalenol, ergosterol and the Fusarium exoantigens in Canadian hard and soft wheat[END_REF]. In this study, correlation between Fusarium DNA and DON concentrations occurred in experimental fields that have been artificially inoculated with trichothecene-producing strains of F. culmorum and F. graminearum. This relationship could probably be weaker for field samples, where the mycoflora infecting kernels may differ for the capability to produce DON or, in the case of F. graminearum, in the relative amounts of DON and NIV they produce. Nevertheless, good correlations were found with the qPCR assay in field samples showing a high variability in both DNA and DON contents [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF]).

This work also showed that correlation between Fusarium DNA and DON is closer and more consistent than correlation between incidence of Fusarium-infected kernels and DON. Significant relationships between the incidence of particular Fusarium species and the concentrations of fusariotoxin have been previously found. For instance, [START_REF] Krisinska-Traczyk | Levels of fungi and mycotoxins in samples of grain and grain dust collected on farms in eastern Poland[END_REF] found that concentration of F. culmorum in total examined grain and grain dust samples collected in Poland was significantly correlated with the concentration of DON, at P<0.05. A significant correlation between the incidence of F. graminearum and DON has been also found by [START_REF] Dalcero | Occurrence of deoxynivalenol and Fusarium graminearum in Argentinian wheat[END_REF] in Argentina, while [START_REF] Contreras | Determination of deoxynivalenol (DON) in wheat, barley and corn and its relationship with the levels of total molds, Fusarium spp., colonization percentage and water activity[END_REF] did not show any significant correlation between the presence of Fusarium spp. and DON in grain samples collected in Venezuela.

The qPCR assay made it possible to detect differences between levels of kernel infection better than fungal isolation from kernels with classical mycological methods. Some reasons can support this result. Kernel infection was determined by plating samples of kernels and by determining percent incidence of grains with developing colonies of F. culmorum or F. graminearum. This procedure can only distinguish between healthy and infected grains but can't define the biomass of mycelia invading an infected kernel. For instance, a kernel which was infected in an advanced stage of ripening may result as infected using the plating method because a Fusarium colony will grow from it, but invasion of grain tissue may have a little extent as well as the amount of DON produced because of a few time will be available between infection and the drop of available water below the minimum for fungal growth and mycotoxin production, respectively [START_REF] Rossi | Factors influencing deoxynivalenol production by Fusarium culmorum and F. graminearum in wheat kernels[END_REF]. Furthermore, the plating method can only detect living mycelia in Fusarium-infected kernels while the qPCR assay provides a measure of biomass taking in account living mycelia actively producing DON but also dead mycelia, which formerly had contributed to the DON content actually found.

The qPCR assay also proved to be an useful tool to detect the dynamic of fungal invasion in planta after infection had occurred, and to single out the presence of infection before onset of the disease symptoms. A robust detection of the infection occurred within 18 to 24 hours for F.

culmorum, and within 2 to 9 days for F. graminearum. This result is very promising for his potential impact on tactical disease management: a detection of infection occurrence within 2 or 3 days may result in an efficient application of curative fungicides [START_REF] Boyacioglu | Effect of three systemic fungicides on deoxynivalenol (vomitoxin) production by Fusarium graminearum in wheat[END_REF][START_REF] Homdork | Effects of field application of tebuconazole on yield, yield components and the mycotoxin content of Fusarium-infected wheat grain[END_REF][START_REF] Matthies | Effect of tebuconazole (Folicur®) and prochloraz (Sportak®) treatments on Fusarium head scab development, yield and deoxynivalenol (DON) content in grains of wheat following artificial inoculation with Fusarium culmorum[END_REF][START_REF] Siranidou | Chemical control of Fusarium head blight on wheat[END_REF][START_REF] Suty-Heinze | Fusarium head blight: an additional strength of prothioconazole[END_REF]). It must be considered that this result was obtained by inoculating wheat spikes at full flowering with a high inoculum dose, under optimum environment conditions for infection to occur. It can be then supposed that under these conditions the disease spread rapidly within the spike tissue and that the minimum DNA content for robust Fusarium detection (i.e. 4 pg) may have reached early in comparison with natural infections. For this reason, further works will be carried out to confirm possibility to early detect presence of trichothecene-producing Fusaria in wheat spikes under field conditions.

The qPCR assay based on Tri5-Tri6 intergenic sequence [START_REF] Terzi | Fusarium DNA traceability along the bread production chain[END_REF]) provides a valuable tool for detection of trichothecene-producing Fusaria both in planta and within harvested grains.

The assay has many potential uses both for research purposes, such as in selection of wheat cultivars and in testing efficacy of fungicides, and for practical disease management, such as timing fungicide applications and early estimating risk for trichothecene mycotoxins within grains at harvest.

F

  Assessment of Fusarium infection in wheat heads using a quantitative PCR assay

  , plots were inoculated at six growth stages to obtain different levels of infection: heading (stage 59 of the BBCH scale; Meier, 2001), beginning of anthesis (stage 61), full anthesis (stage 65), water (stage 71), milk (stage 75) and dough (stage 85) ripening. Inoculations were performed in late afternoon, by distributing 0.6 l of spore suspension per plot by a nebulizer. After inoculation plots were managed in such a way to favour infection: in 2003, plants were wetted every 2 to 3 hours during daytime by distributing water with a handoperated nebulizer, taking care that plants did not drip water; in 2004, a permanent nebulizer was applied over the plants, misting water for 40 sec every 35 min during daytime and every 3 hours in the dark. An uninoculated test was included in the experimental design to determine the natural occurrence of Fusarium infection.

  included. After inoculation pots were maintained at 25°C, 100% relative humidity, for 72 hours and then put outdoor.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Relationships between Fusarium DNA (as natural logarithm of pg over 25 ng of total DNA) and deoxynivalenol (as natural logarithm of DON in ppm) found in wheat kernels that have been inoculated with F. culmorum and F. graminerum at six growth stages in 2003 (A and B, respectively) and in 2004 (C and D, respectively). Coefficients of correlation are shown in TableI.

Figure 3 .

 3 Figure 3. Relationships between Fusarium DNA (as natural logarithm of pg over 25 ng of total DNA) and deoxynivalenol (as natural logarithm of DON in ppm) found in wheat kernels that have been inoculated with F. culmorum and F. graminerum at six growth stages in 2003 and 2004; ---represents the regression line.

  forty amplification cycles were carried out with the following conditions for each cycle: 95°C for 20 sec, 60°C for 1 min. The PCR reactions were subjected to a heat dissociation protocol and analysed by AB5700 software (Applied Biosystems, Foster City, CA, USA) for melting curve analysis. For the quantification of Fusarium DNA, a standard curve was generated by plotting the Ct values vs. the Log 10 amount of pure F. graminearum or F. culmorum DNA (10-fold dilution series). The quantification of fungus DNA in wheat samples was derived from the standard curve run in parallel reactions and the results were expressed as pg of fungal DNA starting from 25 ng of total DNA (of plant and fungal origin) extracted from a sample. A melting curve analysis step was always included in each run to control false positive results that can be arose from primer-dimers hybridization and non-specific amplifications. The sensitivity of the assay, as reported inTerzi et al.

	(	F o r
			P
				e
				e r
					R
					e
						v i e
						w
	extracted from all wheat samples. From the Tri5-Tri6 intergenic sequence (AY134892 accession O n l
	number),	the	primer	pair	22F	(AATATGGAAAACGGAGTTCATCTACA)/122R y
	(ATTGCCGGTGCCTGAAAGT) was designed to amplify a 100 bp fragment of the Fusarium
	genome (Terzi et al. 2007).

extraction from wheat samples. Samples of wheat grains (field experiment) and of spike tissues (environment-controlled experiment) were respectively milled with Ika M20 Universal Mill and finely ground with mortar and pestle in liquid nitrogen. Three-hundred milligrams of this vegetal material were mixed with 860 µl of extraction buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% SDS) and 100 µl of 5M guanidinium chloride. After incubation at 56-60°C for 3 h, the samples were centrifuged for 10 min at 14000 rpm. Five µl of RNase (500 µg/ µl ) were added to 500 µl of the supernat and incubated at 37°C for 10 min, to digest contaminating RNA. The extracted DNAs were purified according to the Wizard protocol and eluted with 50 µl buffer (10 mM Tris-HCl, pH 9.0). DNA concentrations were determined at 260 and 280 nm.

PCR primers. GLUDF (GGT TGG CAC CGG AGT TG )/GLUDR (TCA CCG CTG CAT CGA CAT) primers, designed on wheat seed storage protein sequences, have been previously demonstrated to be genus-specific, able to produce a 101 bp amplicon in genotypes belonging to Triticum genus

[START_REF] Terzi | Development of analytical systems based on real time PCR for Triticum species-specific detection and quantitation of bread wheat contamination in semolina and pasta[END_REF]

. This primer pair was used to check the amplifiability of DNAs Real time PCR. Real time PCR reactions were prepared with 6.25 µl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 900 nM forward and reverse primers, 25 ng of DNA template and water to 12.5 µl. All PCR samples and controls were prepared in duplicate. The PCR mixture was held at 50°C for 5 min and denatured at 95°C for 10 min. After this initial step,

Table I .

 I Pearson's coefficient of correlation between incidence of Fusarium infection, amount of Fusarium DNA and content of deoxynivalenol (DON) in wheat kernels which have been inoculated with F. culmorum and F. graminearum in two years.

	Fusarium species	Variables a	2003	Years 2004	Both
	o r P e e r R e F o r P e e r R e F o r P e e r R e F o r P e e r R e F o r P e e r R e F o r P e e r R e -0.042 Inc vs DNA ns b Inc vs DON 0.189 ns F. culmorum 0.799 F o r DNA vs DON <0.0001 Inc vs DNA -0.061 ns b P e 0.019 F. graminearum e r Inc vs DON ns 0.617 R DNA vs DON 0.0001 e	0.836 <0.0001 b 0.844 <0.0001 0.848 <0.0001 0.608 0.0001 b 0.722 <0.0001 0.816 <0.0001	0.371 0.0013 c 0.549 <0.0001 0.818 <0.0001 -0.104 ns c 0.118 ns 0.779 <0.0001
	F. culmorum + F. graminearum	Inc vs DNA Inc vs DON	v i e w v i e w v i e w v i e w v i e w v i e w -0.065 ns c <0.0001 c 0.774 0.108 ns 0.832 <0.0001 v i e w	0.195 0.019 d 0.411 <0.0001
	O n l y O n l y O n l y O n l y O n l y O n l y 0.828 <0.0001 O n l a Inc = percent incidence of infected kernels (arcsen % ); DNA (ln pg); DON (ln ppm) DNA vs DON 0.757 <0.0001 0.797 <0.0001 b n = 36 (6 wheat varieties x 6 times of Fusarium inoculation, between heading and ripening) y
	c n = 72 (6 varieties x 6 times x 2 years or 2 fungi)
	d n = 144 (6 varieties x 6 times x 2 years x 2 fungi)

Table II .

 II Biomass (expressed as natural logarithm of DNA in pg) of F. culmorum and F. graminearum within spike tissue of two bread (Bilancia and Sagittario) and two durum (Duilio and S. Carlo) wheat varieties at different times after inoculation.

	Hours		Wheat varieties
	after					Average
	inoculation Bilancia Sagittario Duilio S.Carlo
			F. culmorum
	0	0.00 a	0.21	0.00	0.00	0.05 c
	6	0.35	1.59	1.55	1.08	1.14
	12	0.79	1.43	1.55	0.96	1.18
	18	1.51	1.67	2.71	2.81	2.17
	24	1.64 d	2.44	2.24	2.04	2.09
	48	2.57	3.03	1.64	3.48	2.68
	72	4.02	4.92	3.33	5.65	4.48
	144	5.68	6.83	7.39	7.53	6.86
	216	7.37	8.10	8.80	8.81	8.27
	288	6.84	7.10	8.04	8.07	7.51
	Average	3.08 b	3.73	3.72	4.04	3.64
			F. graminearum
	0	0.00 a	0.00	0.023	0.00	0.06 c
	6	0.24	0.00	0.00	0.00	0.06
	12	0.00	0.52	0.79	0.52	0.46
	18	0.27	1.42	1.06	0.76	0.88
	24	0.00	1.58	1.52	0.96	1.02
	48	0.00	1.12	0.64	2.78	1.13
	72	1.52	1.59	2.31	2.88	2.07
	144	1.59	2.71	4.18	3.45	2.98
	216	1.96	4.14	5.16	5.01	4.07
	288	4.86	5.74	5.34	5.37	5.33
	Average	1.04 b	1.88	2.12	2.17

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

Acknowledgements

This work has been partially supported by MIUR (project: SINSIAF), Emilia-Romagna Region (project: Fusariosi e micotossine ), and MiPAF (project: BURAFOCOBANIMIC).


A R Fernandes
email: a.fernandes@csl.gov.uk

M Rose

C Charlton

4-Nonylphenol (NP) in Food Contact Materials: Analytical Methodology and Occurrence

Keywords: 4-nonylphenol, food packaging, materials, GC-MS, migration, APEOs, TNPP

Nonylphenol (NP) is a recognised environmental contaminant but it is unclear whether its occurrence in food arises only through environmental pathways or also during the processing or packaging of food, as there are reports that indicate that materials in contact with food such as rubber products and polyvinylchloride (PVC) wraps can contain NP. A review of the literature has highlighted the scarcity of robust analytical methodology or data on the occurrence of NP in packaging materials. This paper describes methodology for the determination of NP in a variety of packaging materials which includes plastics, paper and rubber. The method uses either Soxhlet extraction or dissolution followed by solvent extraction (depending on the material type), followed by purification using adsorption chromatography. Procedures were internally standardised using 13 carbon-labelled NP and the analytes were measured by GC/MS. The method has been validated and data relating to quality parameters such as limits of detection (LODs), recovery, precision and linearity of measurement are provided. Analysis of a range of 25 food contact materials found NP at concentrations of 64-287 µg/g in some polystyrene and PVC samples. Far lower concentrations (<0.03 -1.4 µg/g) were detected in the other materials. It is possible that occurrence at the higher levels has the potential for migration to food.

Introduction

Nonylphenol (NP) is a term commonly used to describe a complex commercial mixture of mainly nonyl-substituted phenol. The complexity arises from the production of the compound from commercial nonene that is used to alkylate the phenol. Commercial nonene is a complex mixture of C9 branched chain alkenes and the resultant product is predominantly 4-nonylphenol (>90%, because alkylation favours the para-position) with varying amounts of 2-nonylphenol (5% in the purer grades), and smaller amounts of dinonylphenol and decylphenol (Kirk 1992, KEMI 1994). Most NP produced commercially is thus 4-nonylphenol with varied alkyl chain branching.

NP is a recognised environmental contaminant, occurring principally as a hydrolytic breakdown product of the most widely used non-ionic surfactants -the alkylphenol ethoxylates (APEOs) (Thiele et al 1997). Recently, there have also been reports on the occurrence of NP in food [START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF], Nemoto et al 2000[START_REF] Guenther | Endocrine disrupting nonylphenols are ubiquitous in food[END_REF] and although APEOs are used as dispersing or stabilising agents in food packaging plastics, rubber and papers, it is less clear whether NP in food might also derive from the breakdown of the compounds used in this way. Another potential source of NP in food may be hydrolysis of the antioxidant tris(nonylphenyl)phosphite (TNPP) which is used as a heat stabiliser in the manufacture of a range of polymeric food packaging materials such as, styrenes, vinyl polymers, rubber polyolefins, etc.

TNPP is a phosphite stabilizer used to maintain the colour stability, processing stability and performance integrity of the polymers in which it is incorporated (TNPP product description). NP is used as a starting material in the manufacture of TNPP and so it may be present as an impurity as well as a hydrolysis product, formed either within the food or after gastric digestion. NP has been shown to have biological effects in a number of animal studies.

Reviewed data shows that the lowest aquatic toxicity (LC50) is in the range of 0.0033 mg/L to 0.41mg/L (freshwater algae) and 0.0074 mg/L to 0.31 mg/L (fresh and saltwater fish) (EU Risk Assessment on NP). However, it is concern about the estrogenic activity of these compounds that has resulted in many studies on endocrine disruption. Subsequent investigative work showed that the proliferation was caused by NP. It has also been shown in laboratory studies that the no-observed effect concentration of NP for vitellogenin (a fish egg protein normally produced in large amounts by female fish) production in male rainbow trout is approximately 5 µg/L with inhibition of testicular growth at concentrations greater than 30 µg/L [START_REF] Jobling | Inhibition of testicular growth in Rainbow Trout exposed to Estrogenic Alkylphenolic Chemicals[END_REF]. Similar studies [START_REF] Lye | Estrogenic Alkylphenols on Fish Tissues, Sediments and Waters from the UK Tyne and Tees Estuaries[END_REF][START_REF] Gimeno | Feminisation of Male Carp[END_REF][START_REF] Gray | Induction of Testis-Ova in Japanese Medaka exposed to p-Nonylphenol[END_REF] have confirmed that alkylphenols are capable of inducing vitellogenin production and inhibiting testicular growth in a number of fish species. In rats, a single 50 ppm dose was seen to elicit a uterotrophic response [START_REF] Lee | In vivo estrogenic action of nonylphenol in immature female rats[END_REF], CSTEE opinion 2000), and increased uterine vascular permeability was observed in ovariectomized mice at 70 mg/kg bodyweight (Milligan et al 1986, CSTEE opinion 2000). Similar effects such as increased uterine weight and acceleration in vaginal openings at 30-100 mg/kg bw/day have also been reported [START_REF] Chapin | Multigenerational study of 4-nonylphenol in rats[END_REF]. Within the European Union, the risk assessment for alkylphenols and their ethoxylates is covered under the EU existing Substances Regulation (EEC 793/93). Based upon the Draft Risk Assessment Report (Draft EU Risk Assessment on NP), a specific opinion was issued on the evaluation and control of these chemicals (CSTEE 2000(CSTEE , 2001)). EC Directive 2003/53/EC came into force on 17 January 2005 and significantly limited the use of nonylphenol/nonylphenol ethoxylates. In addition, the CSTEE opinion agreed with the view that there were low concerns for carcinogenicity arising from these compounds; rather, the focus of concern was interaction with the endocrine system.

The aim of this study was the development of methodology for the measurement of 4-NP in order to investigate occurrence in a range of commonly used food contact materials. The first stage of this process was the identification of suitable materials. It is possible that some of these materials could contain NP as a result of the presence of NP ethoxylate additives or in particular, TNPP. There have recently been reports (Ozaki and Baba 2003, TNO 2003, Inoue et al 2001, Noriko 1999, Nemoto et al 2000[START_REF] Kawamura | Nonylphenol in food contact plastics and toys[END_REF][START_REF] Loyo-Rosales | Migration of Nonylphenol from plastic containers to water and a milk surrogate[END_REF] of positive identification and quantification of NP in some materials either directly or as a result of migration studies. The results from these reports on the measurements of NP in materials that may come in contact with food are summarised in Table 1. However, it is difficult to anticipate exactly which materials may contain nonylphenols. For example, in a Japanese study [START_REF] Kawamura | Nonylphenol in food contact plastics and toys[END_REF] high levels of NP were found in a plasticised PVC cling film, even though PVC itself does not require an antioxidant stabiliser. The presence was attributed to the possible use of tris(nonylphenyl)phosphite to stabilise the plasticisers used. Similarly, NP was found in high-impact polystyrene (HIPS) [START_REF] Kawamura | Nonylphenol in food contact plastics and toys[END_REF] but it was thought to be present from the use of alkylphenol ethoxylate detergents in the suspension-polymerisation of the butadiene rubber component of the HIPS. Consequently, for method development purposes, a wide range of food contact materials were considered.

Insert Table 1 here

Selection of food-contact materials

The range of materials used for food contact is wide. The following 10 types of materials are in general use and have the potential to contain NP: The choice of materials for the development of the analytical methodology was based on the following criteria:

• Widespread current usage as food contact material.

• Potential to contain NP either because of the suspected presence of tris(nonylphenyl) phosphite or nonylphenol ethoxylates (NPEO).

• Availability of unused (uncontaminated by food) material.

On the basis of these criteria, 20 sub-types of the above listed materials were obtained. For 5 of the 20 sub-types another example was obtained (the same material sub-type obtained at a different time and from different sources) in order to Noriko 1999) and in rubber products (Ozaki and Baba 2003). In a few cases it was not possible to obtain unused material and so external portions of the packaging that were not in contact with the food were used.

Materials and methods

The identity of the materials obtained was confirmed either by the embossed recycling code or, where this was absent, by Fourier Transform-Infra Red spectroscopy of the material.

A procedure using internal standardisation was used for the analysis. NP was isolated from the material matrix either by sample dissolution (polystyrene and polyvinyl chloride) in cyclohexane or by Soxhlet extraction using methanol (all other materials).

The extracts were purified by open column chromatography using deactivated neutral alumina. The purified extracts were concentrated and analysed by gas chromatography-mass spectrometry. A schematic representation of the methodology is given in Figure 1.

Reagents and standards

4-Nonylphenol technical mixture was obtained from ChemService, Pa. USA, 13 C 6 labelled 4-n-NP (purity -99%) and 13 C 12 labelled PCB 52 were from Cambridge Isotope Laboratories Mass, USA. The labelled standards were used as the internal standard and internal sensitivity standard, respectively. Methanol, cyclohexane, dichloromethane and petroleum ether (boiling range 40-60 o C), were from Rathburn Chemicals Ltd, Scotland, UK. Other reagents included alumina (Brockmann Grade 1, Fluka) sulphuric acid (98 %) and anhydrous sodium sulphate, from Fisher Chemicals Ltd. PVC film and polystyrene foam and film samples (~ 0.1 -0.5 g) were extracted ultrasonically for 30 min at 40 0 C using 20 ml of cyclohexane giving either partial or complete dissolution. The extracts were partitioned against 25 ml of acidified methanol and the partitioning repeated two times. The combined methanol extract was partitioned into cyclohexane as described above, and concentrated to ~0.5 ml.

Method

The crude extracts were purified chromatographically using alumina that was deactivated with 15 % (w/w) water after heating in a muffle furnace at 450 0 C for a minimum of four hours. Glass columns (300 x 8mm pre-soaked in methanol for 3-4 hours and dried) were packed with 5.6 g of this reagent and topped with a 5 mm layer of anhydrous sodium sulphate. The columns were conditioned with 10 ml of cyclohexane. The concentrated extracts were eluted successively with 50 ml petroleum ether (discarded) followed by 50 ml dichloromethane. The dichloromethane fraction was evaporated down to a final volume of approximately 100 µl, with addition of the internal sensitivity standard (25 µl of 13 C PCB 52).

Insert Figure 1 here

The extract was analysed by GC-MS carried out in selected ion monitoring mode (SIM), using a 0.22 mm x 60m DB-5 (5% phenyl methyl polysiloxane, 0.25µ) column. A full description of the GC-MS measurement procedure has been reported [START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF]. In brief, 2 µl injections of the sample extracts were made using a Programmed Temperature Vaporisation (PTV) injector. The GC oven was

Results and Discussion

Methodology considerations

Two specific stages of the analytical process were considered for development -the analyte isolation or extraction stage, and the extract purification stage. The measurement stage by GC-MS used the same procedures developed earlier for the determination of NP in food [START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF]. The methodology developed here uses internal standardisation with 13 C labelled 4-n-NP to validate the measurement of each individual sample analysis. and in part due to the levels of NP detected in some of these samples.

The concentrated extracts were purified prior to GC-MS analysis because the potential chemical background arising from co-extractives could obscure or interfere with analyte signals. Deactivated alumina has been used successfully in the past [START_REF] Lye | Estrogenic Alkylphenols on Fish Tissues, Sediments and Waters from the UK Tyne and Tees Estuaries[END_REF][START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF], Van Ry et al 2000) for purification of food and environmental samples. However methodology that has been used to investigate levels of NP in packaging material has generally not used (or used minimal) further purification, probably because the LODs for these methods has generally been higher

-of the order of µg/g levels, rather than the sub-µg/g levels achieved in this work. As observed in previous work on food matrices [START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF], good recoveries were observed using alumina deactivated to 15%, compared to other adsorbents, with generally low level of chemical interference from co-extractives. [START_REF] Lye | Estrogenic Alkylphenols on Fish Tissues, Sediments and Waters from the UK Tyne and Tees Estuaries[END_REF][START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF] The basic principle of the extraction and purification procedure described here is similar to other procedures described in the literature for the analysis of NP and NPEOs [START_REF] Lye | Estrogenic Alkylphenols on Fish Tissues, Sediments and Waters from the UK Tyne and Tees Estuaries[END_REF][START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF], Ozen and Floros 2001) where good recoveries were obtained for both NP and NPEOs, demonstrating the stability of these analytes to the extraction and purification techniques used. Recoveries for NP using this procedure are described in the following section.

The GC-MS method has been described in detail [START_REF] Fernandes | Determination of 4-octylphenol and 4nonylphenol congeners in composite foods[END_REF]. A number of ions corresponding to major mass fragments of the analytes were monitored for quantification or confirmation purposes. These ions correspond to cleavage of the branched nonyl chain and depending on the degree of branching, different ions are observed, e.g. m/z 135 corresponds to the loss of the C 6 H 13 group, m/z 149 to loss of C 5 H 11 group, etc. Quantification was based on the molecular mass ions m/z 220 for NP, and m/z 226 for the 13 Carbon labelled internal standard. Although these ions give a lower response than some of the fragment ions, the higher mass is more selective and thus less prone to interference. Ions corresponding to 13 C labelled PCB52 -m/z 302 and m/z 304 as internal sensitivity standard were also monitored in order to estimate analytical recovery.

Method validation

A number of steps were taken to ensure validity of the developed methodology. The results of these are summarised in Table 2.

The analytical methodology described here has considerable potential to be compromised due to NP background levels, as the chemical may be commonly used as a stabiliser in standard laboratory equipment such as tubing, plastic shelving, detergents, etc. Careful selection of solvents and conditioning of the reagents together with scrupulously clean glassware has resulted in low blank levels (0.01 -0.02 µg/g).

The average LOD (defined as mean + 3 times SD of blank determinations) thus achieved was 0.03 µg/g.

The linearity of NP response to the SIM GC-MS conditions was investigated particularly with respect to the ions used for quantification (molecular ions for NP).

Measurements covering a practical range from ~0.01 µg/g -1000 µg/g showed a linear GC-MS response (regression analysis value of 0.9995) for NP normalised to the 13 Carbon labelled internal standard.

Analytical recovery was assessed for each sample as a measure of the efficacy of the methodology used and gives an indication of the losses incurred during the extraction, purification and measurement stages. The recovery calculated relative to the internal sensitivity standard ( 13 Carbon labelled PCB 52) gave an average value of ~75% and typically ranged from ~ 50 to 100% (Table 3). As the methodology uses internal standardisation, concentration values reported for the materials are corrected for recovery. Analyte recovery from the sample matrix was confirmed by analysing a fortified (with native NP) sample. PET was chosen as the matrix as preliminary investigations showed no detectable levels of NP. The measurement showed that 94 % of the added NP was recovered and also confirmed that the analyte was not degraded during the extraction and purification process.

A number of replicate sub-samples of PVC and HIPS were analysed to investigate the reproducibility of the method. The two sets of analyses were separated in time by a few weeks. The precision (% CV) of the analysis was ~8% for the HIPS and ~10% for the PVC. The % CV was also estimated from the individual recovery measurements This approach was therefore used to estimate precision as %CV across the whole range of materials from recovery data for each of the individual materials. This value was estimated to be of the order of 24%. As this was an externally calibrated measurement the %CV for the internally standardised method would be expected to be lower than this value (as observed in the case of HIPS and PVC).

Precision was also investigated by analysing 4 materials in duplicate. As the replicate analyses described above were based on materials where higher concentrations were detected, materials that showed lower concentrations (PP, HDPE, PC and latex) were investigated. Good agreement was observed between duplicates with differences ranging from 0 -16%.

Analytical Results

The results are given in Table 3 and have been corrected for the blank contribution.

As the measurement was internally standardised the concentrations are inherently corrected for recovery losses. The table also shows data for the 5 duplicate sub-types (these were the same material sub-types but obtained at a different time and from different sources).

The levels detected in the 20 material sub-types ranged from <0.03 -287 µg/g with the majority of samples (84%) showing sub-µg/g concentrations. The materials therefore fall into 2 broad categories -those in which NP was detected at high µg/g levels (64 -287µg/g) and those in which NP was either not detected or was detected at around or below the 1 µg/g level. A study of the duplicate sub-types shows large variation between the levels of NP detected. This is particularly true for the PS butadiene co-polymer, where the ice-tray sample contained 64µg/g compared to the filter coffee holder where NP was not detected. This may indicate the use of different ingredients or different processes in the production of the same material sub-type. There are a limited number of reports in the literature that discuss concentrations or identification of NP in similar materials to those investigated here and a comparison with Table 1 shows that the results of these studies accord well with those presented here, particularly in the types of material that show the presence of NP. There are also similarities between the concentrations detected in this study and those detailed in Table 1, although the differences in the units used for reporting concentrations (concentration/area) make comparison difficult in some cases. The studies listed in Table 1 also record considerable variation in NP content even within material subtypes. This agrees well with the variation seen between the duplicate sub-types listed in Table 2 and supports the view that this variation could arise from the use of different ingredients or different processes in the production of the same material subtype.

Migration potential

The detection of NP in some of the materials studied here raises the possibility that the chemical could migrate to food contained in these materials.

Migration depends on the concentration of NP available in a material coupled with the nature of the food in contact and the contact conditions of temperature and duration.

(e.g. as NP is lipophilic, it is likely to migrate more strongly into material with a high lipid content). It should also be noted that in this work, the combination of methanol as extracting solvent and soxhlet extraction on finely divided material (in order to exploit larger surface areas) used here to analyse for NP represents a fairly aggressive extraction, particularly when compared to the conditions likely to be prevalent when most migration to food occurs. In this context, the levels detected in the materials

probably represent an extreme migration potential.

Inoue et al (2001) reported, not surprisingly, that NP migrates more strongly (62.5%) into n-heptane than into water (0.23%) or acetic acid (4%). Investigating cooked rice wrapped in PVC film (containing NP up to 3300 µg/g) and reheated in a microwave for 1 min or left at room temperature for 30 min, they found maximum concentrations up to 410 ng/g in the surface of the rice sample and 171.8 ng/g for the whole sample.

This value corresponded to the film with the highest NP concentration -3300 µg/g.

Since the concentration in the food is 5% of the initial concentration in the film, even assuming 100% migration, the tests clearly used at least 5g of film per 100g portion of rice. This is unrealistic since at a typical film thickness of 20 µm this means that the ratio would have been 1700 cm 2 film contacting 100 g rice. Their dietary intake estimation from this data, of 0.7 µg /kg bw/day, gave a safety margin of 70000, based on a NOAEL of 50mg/kg bw/day [START_REF] Cunny | Subchronic Toxicity (90 day) study with para-nonylphenol in rats[END_REF][START_REF] Chapin | The effects of 4-nonylphenol in rats: a multigeneration reproduction study[END_REF] Although not directly studying migration into food, a similar study (Ozaki and Baba 2003) found that NP in rubber products that could come in contact with food such as spatulas, migrated more strongly into n-heptane (6.6%) when compared to 20% ethanol (0.7%) and water (0.03%). The results indicated that migration of NP to food can occur during cooking and that food is affected at different rates depending on its hydrophobic content; e.g. if 60ml fatty food were cooked with a spatula with an NP migration level of 1.5 µg/ml it would lead to 1.5 µg/g intake per 60-kg adult per day.

The study concluded that the migration amounts observed could be regarded as having no influence on an adult's endocrine system.

The Greenpeace study (TNO 2003) referred to above also looked at migration from poultry and cheese wrappers into the top layer of the food that they were in contact with. Although only 8 samples were studied, (with undetected levels of NP in 3 of the wraps), it appeared that in most cases, more NP was detected in foods that were in contact with wraps that showed higher NP content. The report concluded that migration of NP from the wrapper to food does take place, and indicated that the migration level of NP to the outer layer of food contained in the wraps was roughly 1% for meat products and 0.1 % for cheese products. Nemoto et al 2000, studied the migration of NP from wrapping film, HIPS trays and expanded polystyrene trays (as described above), as well as the concentrations of NP present in the fish that were contained in these packaging materials. Their results suggested that most of the NP detected in the fish was due to migration from the wrapping film.

It would be useful to supplement the literature described above with some more systematic studies of migration from a range of food contact materials into foods under realistic conditions of use.

F

  The first observations were made in 1991 when a medical researcher observed a proliferation of breast cancer cells stored in plastic containers(Soto et al 1991, Renner This behaviour is normally observed only in the presence of an oestrogen.

•

  Polyolefins and co-polymers • Polystyrene and co-polymers • Polyesters and co-polymers • Chlorinated polymers and co-polymers • Polyamides • Polycarbonate • Cellulose and cellulose esters • Thermoset phenolic polymers • Paper • Rubber products

  Of the material types listed above the first four polymeric groups cover the most common contact materials and therefore made up the majority of the sample set. Additionally there have been findings of NP in PVC(Inoue et al 2001,

  ) the materials were extracted as follows. Approximately 2 g of sample was fortified with 50 µl of internal standard, 13 C 6 4-n-NP (1 ng/µl) and Soxhlet extracted for ~ 20 h in methanol. Prior to extraction the soxhlet thimbles used were similarly extracted in order to minimise any NP contribution from the thimble material. Method blanks were included with every analytical batch. The extracts were concentrated to around 50 ml using a Turbo Vap II TM (Zymark Corporation) apparatus. The extracts were acidified with four drops of concentrated sulphuric acid and partitioned into 65ml of cyclohexane by shaking for 3 min. The partitioning was repeated a further two times and the cyclohexane fractions were combined and concentrated to ~0.5 ml.

  0 C for 3 min to 160 0 C at 25 0 C /min for 2 min, then 0.8 0 C /min to 180 0 C, then 30 0 C /min to 300 0 C for 5 min. The mass spectrometer was operated in SIM mode and a number of fragment and molecular ions corresponding to NP, 13 C NP and 13 C PCB 52 were monitored in the same group.

  Samples were size reduced by cutting, using scissors or metal cutters in order to minimise the possibility of degradation of either the materials or the analytes by the use of electric grinding equipment or filing that could raise the temperature of the material. The reduction in particle size also resulted in maximisation of the surface area available for extraction. This, combined with the use of methanol and the higher temperature (65°C) resulting from the Soxhlet extraction promoted effective extraction. The use of methanol for NP extraction due to the strong solubility of the compounds in this solvent has been reported in the literature (Thiele et al 1997,Fernandes et al 2003, Marcomini and[START_REF] Marcomini | Determination of Linear Alkylbenzenesulphonates, Alkylphenol Ethoxylates and Nonylphenol by HPLC[END_REF] and it was therefore used for the extraction of most of the contact materials.Polystyrene and PVC gave high levels of co-extractives which interfered with the measurement stage. They were therefore extracted ultrasonically using cyclohexane in which they dissolved either completely (PS, HIPS) or partially (PVC). Smaller amounts of sample were taken for analysis (~2 g) because the detection limit of the GC-MS method used for identification of the NP was sufficiently low. More importantly however, the large amount of co-extractives such as plasticizers, from bigger sample sizes have the potential to modify the efficacy of the later purification stages. For sample types where dissolution or partial dissolution of the sample was observed (expanded EPS, HIPS and PVC samples) it was necessary to take a yet (0.1 -0.5 g), in part due to the level of co-extractives observed,

  figures (recoveries) are based on external calibration they nonetheless reflect the precision of measurement of replicates of the same sample measured at the same time.

  Figure 1 Schematic representation of NP analysis

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

Table 1 :

 1 Concentrations of nonylphenol reported in potential food contact materials

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

Table 2 :

 2 Summary of Method Validation Parameters.

	Parameter	Value
	Method Limit of detection across all materials	0.03 µg/g
	Measurement Linearity. Regression analysis value across the	0.9995
	range 10 ng/g -100 µg/g
	Average recovery across all materials	78%
	Recovery from Fortified Sample Material (PET)	94%
	Precision as % Coefficient of Variation for individual materials	∼ 10%
	(PVC and HIPS)
	Precision as % Coefficient of Variation across all materials	24%

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Acknowledgements

This work was funded by the UK Food Standards Agency (FSA). The views expressed in this paper are those of the authors and do not necessarily represent those of the FSA, DEFRA or any other organisation.

Nemoto S, Takatsuki S, Sasaki K and Toyoda M (2000) Determination of nonylphenol in fish on the market. Journal of the Food hygiene Soc of Japan 41(6),

377-380

Noriko S (1999) Detecting Nonylphenol in PVC food wrap, Japan Offspring Fund Newsletter 117-118 (Feb 1999). Ozaki A and Baba T, (2003), Alkylphenol and Bisphenol A in rubber products, Food Additives and Contaminants,. 20(1), 92-98 Ozen, B. F., and Floros, J. D., Trends in Food Science and Tech, 2001, 12, 60-67 Renner R (1997) European Bans on Surfactants trigger Transatlantic debate, Environmental Science and Technology, 31(7), 316A-320A Soto A.M., Justica H., Wray J.W., Sonnenschein C. (1991): p-Nonyl-phenol: An estrogenic xenobiotic released from "modified" polystyrene. Environ. Health Perspect. 92: 167-173 The determination of additives in food products (2003) TNO Report TR 2003/725 available at: www.greenpeace.org.uk/MultimediaFiles/Live/FullReport/6120/pdf The Innovation Group. Nonylphenol. Chemical profile. (2001): Chem. Mark. Rep. 260 (2). Available at : http://www.aperc.org/nonylphenol.pdf Thiele B, K Gunther and M J Schwuger, Alkylphenol Ethoxylates: Trace Analysis and Environmental Behaviour, (1997), Chemical Reviews, 97(8), 3247 -3272. Tris (nonylphenyl) phosphite. Product description. Available at: http://www.chemicalland21.com Van Ry D, J Dachs, C Gigliotti, P Brunciak, E Nelson and S Eisenreich, Atmospheric Seasonal trends and Environmental Fate of Alkylphenols in the Lower Hudson River, (2000), Environmental Science and Technology, 34, 2410 -2417. Page 17 of 21 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Conclusions

The analytical methodology developed and validated for the determination of NP has proved to be robust enough to investigate the occurrence of this chemical in a range of food contact materials. NP was detected at concentrations of up to 1.4µg/g in 85% of the materials studied. However 3 of the material sub-types, two polystyrenes and one PVC, showed concentrations of NP in the range 64 -287µg/g. The results of this study are in broad agreement with the limited literature available. It is possible that the higher levels of NP detected in some of the materials have the potential for migration to food and some migration testing into foods would be informative.