Guest Editorial Introduction to the MYCOGLOBE Conference on "Advances in Genomics, Biodiversity and Rapid Systems for Detection of Toxigenic Fungi and Mycotoxins" and outcome of the relevant Discussion Groups*

The accumulation of mycotoxins in foods and feeds represents a major threat to human and animal health as they are responsible for several chronic health risks, including immunosuppression, cancer induction, and digestive, blood and nerve defects. Mycotoxins negatively impact agriculture and associated industries in different ways and the economic consequences of mycotoxin contamination are profound. Regulations have been established in most countries worldwide to protect consumer health and ensure fair practices in food trade.

Because of the scale of the problem several research projects have been funded by previous EU framework programmes (V and VI FP) to develop strategies for minimizing the risk of toxigenic fungi and mycotoxins in food crops. In this contest the MYCOGLOBE project was supported by the European Commission within the VI framework programme as a Specific Support Action aiming at the "Integration of Mycotoxins and Toxigenic Fungi European Research for Food Safety in the Global System" . The project was set up to share the results of EU research with other countries that have bilateral science and technology agreements with the EU, within the wider framework of a global information system on mycotoxins and toxigenic fungi. MYCOGLOBE focused its activity on the participation and cooperation between EU and other countries to gather knowledge on mycotoxin crop contamination, mapping the mycotoxin occurrence in the most widely cultivated and exported crops, networking which includes training, working and expert groups, conferences, seminars and management seminars.

The MYCOGLOBE Conference on "Advances in Genomics, Biodiversity and Rapid Systems for Detection of Toxigenic Fungi and Mycotoxins", held in Monopoli (Bari), Italy, 26-29 September 2006, was the sixth in a series of international meetings organized within the project. In keeping with the goals of the MYCOGLOBE project, the conference brought together experts from various parts of the world engaged in developing knowledge in genomics, biodiversity and novel technologies aimed to reduce and ultimately eliminate the adverse effects of mycotoxin contamination on the profitability of agriculture and related industries as well as the safety of food and feed supplies worldwide.

The Conference was organized in 3 main oral sessions, and included a poster session and Discussion Groups.

The first session on Biosynthesis of Mycotoxins and Genomics of Toxigenic Fungi included aspects relevant to EST profiling for elucidation of aflatoxin biosynthesis and the whole genome sequencing of Aspergillus flavus as well as comparative genomics of Aspergillus flavus and Fusarium graminearum. Other aspects of functional genomics in the biosynthesis of aflatoxins and ochratoxin A were also discussed.

The second session on Biodiversity of Toxigenic Fungi included biodiversity of Aspergillus, Fusarium and Penicillium in different continents as well as the role of biodiversity in plant pathogenicity and biological control.

The third session on Emerging Detection Methods for Toxigenic Fungi and Mycotoxins included a variety of molecular methods (real time PCR, microarrays, multiplex) for the detection of toxigenic fungi (producing ochratoxin A, trichothecenes or fumonisins) together with the use of electronic nose for differentiation of toxigenic and non-toxigenic fungi. Among the emerging methods for mycotoxins, capillary electrophoresis and fluorescence polarization were introduced together with microsensors and immunological flow-through devices for rapid analysis as well as new immunoaffinity columns or liquid chromatography-mass spectrometry methods for multi- Five Discussion Groups were formed among participants (about 10-15 participants per group) based on the relevant expertise to discuss specific topics of interest aiming to: i) identify priority areas within specific topics of the discussion group that may be suitable to be part of a proposal to the following call for the VII Framework Programme of the European Commission, and ii) identify at least two different fungi and toxins to be studied. The following topics were selected for the Discussion Groups: i) Genomics, ii) Biodiversity, iii) Rapid detection of toxigenic fungi, iv) Rapid detection of mycotoxins, and v) Regulation of toxin biosynthesis. Following is the list of best ideas or priorities identified by each Discussion Group:

Genomics

Create and maintain a data base resource including: Sequence information; Expression data; Metabolic profile; Evolution/phylogeny; Comparative analyses (strain/genus). Develop better tools/high throughput for functional genomics: Maintain a repository for mutants/disrupted strains; Harmonize and coordinate functional studies. Train researchers to use: Databases/bioinformatics/annotation; Functional genomics tools and technology; Special emphasis for third-world countries.

Biodiversity

Approaches: i) Understand biodiversity of fungal populations at global level; ii) Use fungal biodiversity to inform plant disease-resistance breeding programs; iii) Develop specific markers for detection of specific toxigenic/pathogenic traits and/or fungal populations; iv) Provide scientific basis for mycotoxin risk assessment; and v) Provide scientific basis for forecasting new and emerging toxigenic fungi. Specific target problems: i) Trichothecenes and Fusarium graminearum in cereals; ii) Ochratoxin A genes and Aspergillus niger group in grapes and other plant products; ii) Aflatoxin genes and Aspergillus flavus in maize and other plant products.

Rapid Detection of Toxigenic Fungi

Main priorities: i) Develop validated robust tools for trichothecene-producing fungi from biosphere to plate; ii) Develop molecular detection systems for ochratoxin A and aflatoxin producing species from spices from developing countries to avoid trade barriers; iii) Develop tools to detect ochratoxin A producing fungi in cereals; and iv) Develop quantitative multiplex methods to analyze interactions, biodiversity and epidemiology of food and feed relevant fungi.

Rapid Detection of Mycotoxins

Method development and performance validation: Global validation of methods requiring < 30 min with sample preparation or < 5 min without sample preparation; Collaborative study of point-of use methods.

Applicability and accessibility: Improve robustness of assays; Reduce cost of assays; Training; especially of third world personnel, to conduct assays. Biomarkers: Identify and validate new markers and methods; Develop analytical methods to support biomarker identification and monitoring; Utilize biomarkers to monitor exposure and perform intervention strategies. Quality assurance and risk management: Monitor contamination at critical points in the food chain; Identify and monitor breakdown products and "masked" mycotoxins; Improve/develop confirmatory assays. The MYCOGLOBE project has been an extraordinary experience of a large and fruitful cooperation at global level between of various experts and Institutions dealing with prevention and reduction of the mycotoxin problem. This led to the constitution of the International Society for Mycotoxicology that will continue the networking and promotional activity started with MYCOGLOBE. We are deeply grateful to the European Commission for allowing us to share this experience with all participants that contributed to success of the project.

This

in Genomics, Biodiversity and Rapid Systems for Detection of Toxigenic Fungi and Mycotoxins

	Title:
	Advances Guest Editors:
	Angelo Visconti and Antonio Logrieco
	Institute of Sciences of Food Production (ISPA),
	National Research Council (CNR),
	Via Amendola 122/O, 70126 -Bari, Italy
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  mycotoxin analysis. Analytical methods for biomarkers of exposure to fumonisins were also reviewed.

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  Selected ideas: i) Identify abiotic and biotic factors affecting regulation of mycotoxin biosynthesis and catabolism; ii) Identify inter-and intra-cellular signals and transduction pathways/mechanisms; iii) Identify specific and global regulatory factors at the DNA and chromatin level; iv) Identify key regulatory factors for targeting to minimize mycotoxins in food and feed.

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  special issue of Food Additives and Contaminants is a collection of selected papers which are representative of the various topics discussed at the MYCOGLOBE International Conference.Footnote* The Conference was supported by the European Commission Specific Support Action MYCOGLOBE within the VI Framework Programme. Further information on the project are available in the website http://mycoglobe.ispa.cnr.it

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	Angelo Visconti and Antonio Logrieco Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Amendola 122/O, 70126 -Bari, Italy o r P e e r R e v i e w O n l 59 F 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 y

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Lars O Jorhem

Christina Åstrand

Birgitta Sundström

Malcolm Baxter

Penny Stokes

John Lewis

Kierstin Petersson Grawé

Elements in rice on the Swedish market: Part 2. Chromium, copper, iron, manganese, platinum, rubidium, selenium and zinc

Keywords: Metals, Metals -nutritional, Metals -selenium, Trace elements Rice, trace elements, chromium, copper, iron, manganese, platinum, rubidium, selenium, zinc

come

Introduction

Rice (Oryza sativa) is one of the most important food commodities on the international market, with a world wide production of nearly 600 million tonnes in 2003, of which approximately 90 % was produced in Asia, with China producing 166 million tonnes and India 134 million tonnes. Although the USA is not among the major producers, it is nevertheless one of the biggest exporters, ranking third, with an export of 2.6 million tonnes, after Thailand (7.6 million tonnes) and Viet Nam (3.7 million tonnes) [FAO 2004].

For some regions in the world rice is a dominating staple food. According to the GEMS/Food Regional Diets [WHO, 2003] the average per capita daily consumption of raw polished rice varies between 9 grams per day in Europe to 278 grams per day in the Far East. Thus in some regions of the world rice is indeed an important staple food item. To emphasise the importance of rice the UN General Assembly declared the year 2004 the International Year of Rice, under the slogan "Rice is life" (FAO 2004). The content of trace elements and heavy metals in rice is therefore of global interest, from a nutritional point of view.

As the consumption of rice has increased markedly in Sweden it was decided to carry out a survey of the content of a number of different rice brands available on the Swedish market in 2001. The main purpose of the survey was to determine cadmium and lead, together with the results for arsenic (total and inorganic), for which the results are presented in a separate paper [Jorhem et al. 2007]. In addition there was an interest in determining the content of the essential elements copper (Cu), chromium (Cr), iron (Fe), manganese (Mn), zinc (Zn) and selenium (Se) in the different brands of rice. Furthermore the concentration of platinum (Pt) is of interest from environmental concerns, and rubidium (Rb) as it is intriguing that the concentration is so high of an element that has no known role in human nutrition. Sweden is dependent on import of rice since the climate not allows domestic production. Rice is imported to the Swedish market from different parts of the world, e.g. Far East, USA and Europe. The occurrence data can be used to calculate the Swedish dietary intake of essential elements from rice, but also by using the WHO/GEMS Regional Food Diets [WHO, 2003] estimate the intake of essential elements from rice in other regions of the world.

The rice variety as well as the treatment of the rice may affect its elemental content. Brown rice, with the bran layer intact, is considered more nutritious than white rice, in which the bran layer is removed in a milling process. Much of the white rice on the market is parboiled (i.e. soaked in water, steamed under pressure and dried) prior to the milling. One claimed effect of the parboiling is that it modifies the starch and permits the retention of much of the natural vitamins and minerals in the kernels. In this way the nutritional value of the polished white rice is maintained when compared to the brown rice.

Sampling and analysis

Forty-nine samples of imported rice were collected from retail stores in Uppsala, Sweden in 2001. These samples represent the major brands on the Swedish market. The details of the samples are shown in Table 1. The area of production was often only vaguely described, e.g. just stated as being of USA, Thai, Italian, Himalayan or Australian origin. In some cases no indication was given regarding the area of production. The types of rice sampled comprised long grain (Indica) and short grain (Japonica).

The different samples of rice are primarily grouped according to the EU customs tariff system (Taric), i.e. after grain type and treatment, as shown in Table 1. The labelling on the packages was often incomplete regarding the treatment of the rice, but it was nevertheless possible to deduce some information from the packages. It should be noted that wild rice mix only contains about 15 % wild rice.

Sample preparation

Preparation of samples for the analysis of Cr, Cu, Fe, Mn and Zn An aliquot of sample (10 g) was dry ashed at 450°C and quantitatively diluted to 30 ml. For details see: Preparation of samples for the analysis of Cd and Pb, in Jorhem et al. (2007).

Preparation of samples for the analysis of Pt, Rb and Se

An aliquot of sample (0.5 g) was quantitatively digested in concentrated HNO 3 (5 ml) using a microwave-assisted digestion system. For details see: Preparation of samples for the analysis of total As, in Jorhem et al. (2007).

Measurement procedures

Measurement of Cr was performed by graphite furnace atomic absorption spectrometry (GFAAS) using a Perkin Elmer 5100/HGA 600 instrument with Zeeman background correction (Perkin Elmer, Norwalk, Connecticut, USA). The method of standard addition was employed for all metals. The method parameters were optimised previously by establishing ash-and atomisation curves for each metal. Matrix modifiers were not used. The background correction system was regularly checked for function. The operating conditions are given in Table 2.

Measurement of Cu, Fe, Mn and Zn was performed by flame AAS on a Varian SpectrAA-300 instrument (Varian, Melbourne, Australia), using an oxidising air-acetylene flame at the wavelength of 213.9, 324.8, 248.3 and 279.5 nm, respectively. Background correction was not used.

Measurement of Pt, Rb and Se was performed on an inductively coupled plasma-mass spectrometer (ICP-MS) instrument (Elan-6000, Perkin-Elmer Ltd, Beaconsfield, Bucks, UK).

The operating conditions are given in Table 3.

Analytical quality control procedures

Procedural blanks

The mean blank value was deducted from the readings before the result was calculated. The batch blank was used to decide if the results of the batch were acceptable or not, i.e. if the blank gave a significant contribution to the result the batch was re-analysed (EN 13804).

Limit of detection

The limit of detection (LOD) is defined as three times the standard deviation (σ) of the signal from a large number of procedural blanks (n is usually >20), corrected for sample weight and

Certified Reference Materials (CRMs)

The results of the CRMs and Standard RMs (SRMs) analysed throughout this survey are shown in Table 4.

Proficiency Testing Scheme participation

The NFA and the CSL participated in several proficiency tests during the period of this study, for which the results are shown in Table 5. The z-score is an independent assessment of a laboratory's competence, and z-scores within ±2 are considered acceptable, whereas z-scores between ±2 and 3 are generally considered to be questionable, but not immediately unacceptable. For Pt and Rb in food there are no PT-programmes available, therefore the recovery of spikes was estimated. They were found to be 95.5, 105, and 108%, respectively

Intake calculations

The calculated intakes of Cu, Fe, Mn, Se, Zn, Cr, Pt and Rb were based on the average consumption of rice (11 g day -1 of air dry rice) by Swedish adults aged 19-54 years, according to the Swedish consumption survey "Riksmaten" that was performed in 1998 [Becker & Pearson, 2002]. However, it is known from the updated per capita statistics [SJV, 2006] that the rice consumption in Sweden has increased since then, and therefore the consumption data was adjusted accordingly. The latest data are derived from the year 2004 and the adjustment resulted in a 38 % increase compared to the rice consumption in 1998, i.e. a total average daily consumption of 15 g rice.

In addition, the WHO/FAO GEMS Food Regional Diets [WHO, 2004] for Europe and Far East, representing the lower and upper consumption volumes, respectively on a global scale, were used to estimate the contribution of rice to the intake of the studied elements. The intake of the essential elements Cu, Fe, Mn, Se and Zn were related to provisional tolerable intakes established by Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]

Results and discussion

All sample results are expressed on an air dry basis (i.e., fresh weight). CRMs were analysed on dry weight basis. The water content was on average 7.6 % (range 5.7 -11.6; n = 8), which means that calculated on dry matter the results would have been correspondingly higher.

Analytical quality control

There is a lack of CRMs and PT-programmes for the analysis of Pt and Rb, which is somewhat compensated by acceptable spike recoveries. These recoveries, and the results presented in tables 4 and 5 of results from the analysis of CRMs as well as participation in PT-programmes indicate that the analytical quality control is satisfactory and that the results, allowing for U, must be considered reliable.

Essential elements

Copper

The Cu levels showed little variation, ranging from 0.77-3.9 mg kg -1 , with a mean of 1.9 mg kg -1 (Table 7), and no significant difference between brown and white rice. From several recent studies similar levels have been reported, ranging between 2-5 mg kg -1 [Phuong et al.1999[START_REF] Alam | Metal concentrations in rice and pulses of Samta village, Bangladesh[END_REF][START_REF] Gorbunov | Heavy and toxic metals in staple foodstuffs and agriproducts from contaminated soils[END_REF], Lin et al. 2004]. A daily copper intake of 0.7 mg for women and 0.9 mg for men is recommended by the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]. In the Swedish diet, rice contributes to 4.1 % (range 1.7 -8.4 %). Rice in the European and Far East WHO Regional Diets contributes to 2.5 % (range 1.0 -5.1 %) and 77 % (range 31 -156 %) of the RDI for women, respectively (Table 9). It can be concluded that rice on average almost covers the copper requirements in the Far East WHO Regional Diets, if assuming that the requirements are similar worldwide.

Iron

The average Fe content in all rice samples was 4.7 mg kg-1 (range 1.0 -18 mg kg-1).

However, the mean Fe level of 10 mg kg-1 (range 7.4 -18 mg kg-1) in brown rice (Table 7), was markedly higher than the mean level of 2.5 mg kg-1 (range 1.0 -5.7 mg kg-1) in white rice (produced in Asia), and also higher than the mean level of 4.3 mg kg-1 (range 2.2 -8.6 mg kg-1) in parboiled white rice (Table 7). This is similar to that by [START_REF] Phuong | Elemental content of Vietnamese rice Part 1. Sampling, analysis and comparison with previous studies[END_REF] who reported mean levels of 6.5 and 12.06 mg kg -1 in Vietnamese white and brown rice, respectively, and [START_REF] Koivistoinen | Mineral Element Composition of Finnish Foods[END_REF] reported mean levels of 12 mg kg -1 and 36 mg kg -1 in white and brown rice, respectively. It is however not known if there are any other differences between the brown and white rice e.g. cultivars, place of origin etc., that could explain the higher Fe levels reported in brown vs white/parboiled rice. In a previous survey, mean levels of 12 mg kg -1 for both brown and white rice were reported in rice on the Swedish market [START_REF] Jorhem | Lead, cadmium and certain other metals in food on the Swedish Market[END_REF]. [START_REF] Gorbunov | Heavy and toxic metals in staple foodstuffs and agriproducts from contaminated soils[END_REF] reported a mean level of 137 mg kg -1 in rice from the Astrakhan region of Russia.

A daily iron intake of 9 -15 mg for women, where the highest is for women in their reproductive age, and 9 mg for men is recommended by the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]. Rice consumption in Sweden contributes to an average of 0.6 % (range 0.1-2.3 %) of the Nordic RDI for women. In the European and Far East WHO Regional diets rice contributes to 0.3 % (0.1 -1.4 %) and 11 % (2.3 -42 %) of the RDI for women, respectively (Table 9). For comparison, using the average Fe concentration found in brown rice for calculation of the Fe intake, rice could contribute to on average 1.3, 0.8 and 23 % of the RDI for the Swedish diet, and the European and Far East Regional WHO Diets, respectively. Iron deficiency is considered the most common worldwide micronutrient deficiency [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF], and it can be concluded that in regions relying heavily on rice as staple food, rice can be an important source of Fe.

Manganese

The overall average content of Mn in rice was 16 mg kg-1 (range 6.4 -52 mg kg-1). The mean Mn level of 42 mg kg-1 (range 22 -52 mg kg-1) (Table 7) in brown rice was on average four times as high as in white and parboiled white rice, with means of 10 and 11 mg kg-1 (range 6.4 -18). Results from Viet Nam with means of 24 -41 mg kg-1 for brown rice and 9.9 -13.6 mg kg-1 for white rice [Phuong et al.1999], together with results from Finland reporting means of 24 and 9 mg kg-1 for brown and white rice [START_REF] Koivistoinen | Mineral Element Composition of Finnish Foods[END_REF], appear to confirm these differences in concentration. The evaluations of the nutritional requirements of Mn are diverging in their conclusions. The European Scientific Committee for Food concluded that 1 -10 mg day-1 is safe and adequate [SCF, 2003], and the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF] concluded that available data did not allow daily requirements for manganese to be established. On the other hand, the US National Academy of Science calculated an adequate intake for adult men and women to 2.3 and 1.8 mg day-1, respectively [START_REF] Usnas ; Washington | Intakes (DRI) and Recommended Dietary Allowances. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium and zinc[END_REF]. In the Swedish diet rice contributes to on average 2.4 % (range 1.0 -7.8 %) of the upper safe intake proposed by SCF. Rice contributes to on average 1.4 % (range 0.6 -4.7 %) of the upper SCF 'safe and adequate intake' level in the European WHO Regional Diet (Table 9). Correspondingly, in the Far East WHO Regional Diet rice contributes to as much as 44 % (range 18 -145 %) (Table 9).

Selenium

The overall mean Se content was 0.1 mg kg-1.The mean Se level in brown rice was 0.2 mg kg-1, in white rice 0.1 mg kg-1, and in parboiled white < 0.1 mg kg-1 (Table 9). The observed levels are similar to previously reported Se concentrations in rice. [START_REF] Kelly | The application of isotopic and elemental analysis to determine the geographical origin of premium long grain rice[END_REF] found a range of 0.00 to 0.40 mg kg-1 in rice from Asia, Europe and USA. It was not shown, however, whether the results referred to brown or white rice. [START_REF] Kumar | Selenium content of common Indian cereals, pulses and spices[END_REF] reported a mean of 0.099 mg kg-1 in 1997 and Koivistoinen a mean of 0.020 mg kg-1 [START_REF] Koivistoinen | Mineral Element Composition of Finnish Foods[END_REF]. In wild rice [START_REF] Bennet | Heavy metals in wild rice from northern Wisconsin[END_REF] reported a mean level of 0.21 mg kg-1.

A daily Se intake of 0.040 mg for women and 0.050 mg for men is recommended by the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]. Rice in the Swedish diet contributes to a minor part of the Nordic RDI (3.8 -15 %) and similarly in the European Regional WHO Diet (2.3 -9 %). On the other hand, rice is a very important Se source in the Far East Regional WHO Diet, contributing to 70 -280 % of the RDI (Table 9).

Zinc

The overall mean Zn content in rice was 15 mg kg-1. In brown rice, the mean Zn level was 20 mg kg-1 (range 13 -23 mg kg-1) (Table 7), whereas the level in white (mean 17 mg kg-1; range 15 -18 mg kg-1) and parboiled white rice (mean 10 mg kg-1; range 5 -15 mg kg-1) was somewhat lower. In a previous survey [START_REF] Jorhem | Lead, cadmium and certain other metals in food on the Swedish Market[END_REF]] it was found a mean of 15 mg kg-1 and 11 mg kg-1 in brown and white rice, respectively. Several other studies have reported similar mean results ranging from 12.7 -22.9 mg kg-1 [Phuong et al.1999[START_REF] Alam | Metal concentrations in rice and pulses of Samta village, Bangladesh[END_REF][START_REF] Herawati | Cadmium, copper and zinc levels in rice and soil of Japan, Indonesia and China by soil type[END_REF][START_REF] Gorbunov | Heavy and toxic metals in staple foodstuffs and agriproducts from contaminated soils[END_REF], Lin et al. 2004]. A daily zinc intake of 7 mg for women and 9 mg for men is recommended by the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]. The Swedish diet contributes to on average 3.2 % (range 1.1 % -4.9 %), while in the European and Far East WHO Regional Diets, rice contributes to 1.9 % (range 0.7 -2.9 %) and 59 % (range 21 -91 %) of the RDI recommended by NNR, respectively.

Elements with no RDI or PTWI established

Chromium

The Cr levels in rice sold on the Swedish market ranged between <0.003-0.033 mg kg-1, average 0.008 mg kg-1, with no significant difference between brown and white rice (Table 6). This is considerably lower than what has been reported in other recent studies. Lin et al.

(2004) reported a mean Cr concentration of 0.07 mg kg-1 ± 0.20 (SD) in rice available on the Taiwanese market. [START_REF] Gorbunov | Heavy and toxic metals in staple foodstuffs and agriproducts from contaminated soils[END_REF] reported a mean Cr level of 0.16 ± 0.03 mg kg-1 in rice from the Astrakhan region of Russia. In a survey of wild rice [START_REF] Bennet | Heavy metals in wild rice from northern Wisconsin[END_REF] found a mean Cr concentration of 0.49 mg kg-1 dry wt in seeds of wild rice. These seeds had, however, been ground in a stainless steel mill, and may therefore be overestimations. The low level presented here are consistent with the range of 0.010-0.050 mg kg-1 reported from Finland for brown and white rice [START_REF] Koivistoinen | Mineral Element Composition of Finnish Foods[END_REF]).

The essentiality of chromium is debated. The European Scientific Committee on Food concluded in 1993 that data on essentiality and metabolism were so sparse that nutrirional requirements could not be established [SCF 1993]. This view was shared by Kumpulainen who found no convincing indications that Cr is essential [START_REF] Kumpulainen | Heavy metal content of rice and shellfish in Taiwan[END_REF], which is similar to the conclusions made in a later Nordic evaluation [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]]. On the other hand, other national bodies, e.g. U.K [START_REF] Evm | Expert Group on Vitamins and Minerals. Safe upper levels for vitamins and minerals[END_REF] and USA [START_REF] Usnas ; Washington | Intakes (DRI) and Recommended Dietary Allowances. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium and zinc[END_REF]] have established daily requirements for Cr.

The US National Academy of Science [START_REF] Usnas ; Washington | Intakes (DRI) and Recommended Dietary Allowances. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium and zinc[END_REF] concluded that an adequate intake of Cr is 0.025 mg day-1 for women and 0.035 mg day-1 for men. A similar view is presented in the Nordic Nutrition Recommendations [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF]. In the European and Far East WHO Regional Diets rice contributes to on average 0.3 and 8.9 % of the US recommendation, ranges 0.1 -1.2 % and 3.3 -37 %, respectively (Table 9). Correspondingly, rice in the Swedish diet contributes to 0.5 % (range 0.2 -2.0 %).

Platinum

There has been concern over potentially increased levels of Pt in foodstuffs since the global use of Pt has increased markedly during the latter third of the twentieth century. This is mainly due to the wide use of Pt as catalyst in vehicle exhaust systems, leading to increased [START_REF] Usnas ; Washington | Intakes (DRI) and Recommended Dietary Allowances. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium and zinc[END_REF][START_REF] Farago | Platinum concentrations in urban road dust and soil, and in blood and urine in the United Kingdom[END_REF]. Still, there is limited information on exposure levels and potential health effects in the general population. From occupational settings it is known that hypersensitivity to platinum salts can develop, but metallic platinum, which is considered the main form of Pt emitted from catalytic converters, is non-allergenic [IPCS, 1991].

The Pt concentration was below the LOD of 0.0003 mg kg -1 in all samples (data are therefore not shown in a table), indicating low environmental contamination. Our results were one order of magnitude lower than that reported in cooked rice purchased on the Australian market (3.48 µg kg -1 fresh weight) [START_REF] Usnas ; Washington | Intakes (DRI) and Recommended Dietary Allowances. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon, vanadium and zinc[END_REF]]. However, it was considered possible that during the cooking process Pt might have been added via the cooking water or the vessels used.

Rubidium

The Rb concentration in long grain brown rice ranged from 0.64 -12 mg kg-1 with a mean of 6.0 mg kg-1 (Table 8), whereas the white and parboiled white long grain rice had somewhat lower mean levels. Short grain rice had consistently lower levels. [START_REF] Kelly | The application of isotopic and elemental analysis to determine the geographical origin of premium long grain rice[END_REF] reported a range of 0.17 to 8.82 mg kg-1 in rice from Asia, Europe and USA. It was not shown, however, whether the results referred to brown or white rice. From Finland it was reported mean levels of 1.9 mg kg-1 for brown and 1.3 mg kg-1 for white rice, respectively [START_REF] Koivistoinen | Mineral Element Composition of Finnish Foods[END_REF]].

Rubidium belongs to the alkali metals. In biological systems Rb resembles potassium and is therefore used as a marker cation and substitute for Ka + in studies of the cellular sodium pump [START_REF] Zhen | A novel assay of cell rubidium uptake using graphite furnace atomic absorption: application to rats on a magnesium-deficient diet[END_REF]. Still, it is not known if Rb has a biological role in mammal systems, and it can be concluded that the literature on this topic is very limited. However, Rb concentration in blood in humans is reported to be in the same order of magnitude as Cu and Zn [START_REF] Bárány | Iron status influences trace element levels in human blood and serum[END_REF], indicating that the bioavailability of Rb in food in general is fairly high. Recently, correlations between Rb and Ka+ in liver autopsies were reported, suggesting that Rb might play a biological role in humans [Milman et al. 2006]. Further studies are needed to clarify if Rb has a nutritional role in human nutrition.

Conclusions

Rice may be a large contributor, covering more than 50% of the RDI for some essential elements, especially Cu, Mn, Se and Zn, if the rice consumption is high. For most elements there were no differences between concentrations in brown vs parboiled white rice. However, the levels of Fe and Mn were significantly higher in brown rice compared to white parboiled rice, indicating that the parboiling process may not be effective in maintaining the high nutritional value by all means. It cannot be ruled out, however, that the differences are due to other parameters like e.g. rice variety or production area. Iron deficiency is common worldwide, according to WHO more 30 % of the world's population is anaemic, mainly because of iron deficiency [START_REF] Who | Micronutrient Deficiencies. Iron deficiency anaemia[END_REF]. Therefore it is important that it is further elucidated if brown rice would be a better contributor to daily iron intake than other types of rice.

Although in line with rare previous reports, the mean concentration of Rb in rice was remarkably high, considering that Rb has no known function in the human body.

F

  LOD's were as follows: Cr 0.001, Cu 0.015, Fe 0.03, Mn 0.003, Pt 0.0003, Rb 0.005, Se 0.1, and Zn 0.009 mg kg -1 .

  to the environment. Concern over potentially increased human environmental exposure has therefore been raised

Table 1 .

 1 Types of rice found on the Swedish market and included in this survey.

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

Table 2 .

 2 Graphite Furnace AAS operating conditions for chromium.

			Ash temp	Atomize	Sample	Diluent
	Wavelength Slit	°C	temp.°C	volume	volume
	357.9 nm	0.7 Low 700	2200	10 µl	10 µl

Table 4 .

 4 Results from certified reference materials, analysed in parallel to the samples. All results in mg kg -1 dry wt.

		Element n Found mean	Certified value
	NIST SRM	Cr	2 <0.021	-
	Rice flour 1568a	Cu	2 2.3	2.4
		Fe	2 5.4	7.4
		Mn	2 24.4	20
		Zn	2 21.0	19.4
		Se	1 0.3	0.38
		Rb	1 7.01	6.14
		Pt	1 <0.003	-
	NRCC DORM-2
	Dogfish muscle
		Se	1.75	1.4
		Rb	5.78	-
		Pt	<0.0003	-

Table 5 .

 5 Results from proficiency tests for certain metals. Potato powder in mg kg -1 dry wt, canned fish in mg kg -1 fresh wt.

			Food Additives and Contaminants		Page 20 of 24
	PT-provider Round, date	Test sample	Metal Found	Assigned	z-score
					result	value
	NFA	T-6, 2001-11	Potato powder Cu	2.87	2.83	+0.1
	NFA	T-6, 2001-11	Potato powder Mn	3.78	3.88	-0.3
	FAPAS 2	18-32, 2004-09 Canned fish	Se	0.519 0.539	-0.2
	1 National Food Administration, Uppsala, Sweden
	2 FAPAS Secretariat, Central Science Laboratory, York, UK

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Table 6 .

 6 Concentration of Cr in different types of rice on the Swedish market in mg kg-1 fresh weight.

					Food Additives and Contaminants
	Results in bold indicate mean values
					Cr
	Type	Other information Imported from n	Mean	Min	Max
	Long grain (Indica)
	Brown		Not known	2	0.006	0.005 0.007
	Parboiled brown		Not known	2	0.024	0.015 0.033
			USA	3	0.006	0.004 0.009
	Brown mean			7	0.011	0.004 0.033
	White	Asian	Thailand	1 < 0.003
		Basmati	India/Pakistan 5	0.016	0.007 0.028
		Jasmin	Thailand	7	0.006	0.003 0.010
		Ecological Jasmin	Thailand	1 < 0.003
	White mean			14	0.009 < 0.003 0.028
	Parboiled white	Instant	Not known	2	0.003	0.003 0.004
			Not known	2	0.012	0.011 0.013
			USA	10	0.009	0.005 0.017
		Ecological	Italy	2	0.004	0.004 0.005
			USA/Italy	1	0.003
	Parboiled white mean			17 0.007	0.003 0.017
	Parboiled white	Wild rice mix	Not known	2	0.010	0.009 0.010
			USA	1	0.015
		Wild rice mix mean		3	0.011	0.009 0.015
	Short grain (Japonica)
	Brown	Ecological	Australia/Italy 1	0.004
	White	Instant flakes	Not known	2 < 0.003
			Not known	2	0.005 < 0.003 0.008
			USA	1 < 0.003
		Ecological	Italy	1 < 0.003
	White mean			6 < 0.003		0.008
	Parboiled white	Avorio	Not known	1 < 0.003
	Grand mean			49	0.008 < 0.003 0.033

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Table 7 .

 7 Concentration of Cu, Fe, Mn and Zn in different types of rice on the Swedish market in mg kg-1 fresh weight. Results in bold indicate mean values

	Cu	Fe	Mn	Zn

from n Mean Min Max Mean Min Max Mean Min Max Mean Min Max Long grain (Indica)

	Brown		Not known	2 2.1 2.0	2.2	9.0 8.2	9.7	45 39	51	21 19	23
	Parboiled brown		Not known	2 2.5 2.2	2.8	14 10	18	42 39	45	21 20	23
			USA	3 2.0 2.0	2.2	8.7 7.4	11	40 22	52	18 13	20
	Brown mean			7 2.2 2.0	2.8	10 7.4	18	42 22	52	20 13	23
	White	Asian	Thailand	1 0.77		1.9		12		15
		Basmati	India/Pakistan 5 2.1 1.6	2.5	3.8 2.0	5.7	9.0 8.7	9.5	17 16	17
		Jasmin	Thailand	7 1.2 0.92 1.5	1.8 1.0	3.4	10 9	12	17 15	17
		Ecological Jasmin Thailand	1 0.99		1.3		11		18
	White mean			14 1.5 0.77 2.5	2.5 1.0	5.7	10 9	12	17 15	18
	Parboiled white	Instant	Not known	2 1.6 1.6	1.6	2.9 2.7	3.1	8.0 7.3	8.8	8.8 8.6	9.0
			Not known	2 2.8 2.6	2.9	4.1 4.1	4.2	7.0 6.6	7.3	6.6 6.0	7.2
			USA	10 2.1 1.7	2.4	5.2 3.6	8.6	13 6.4	18	12 5.0	15
		Ecological	Italy	2 1.5 1.4	1.5	2.2 2.2	2.2	7.8 7.6	7.9	9.5 9.4	9.5
			USA/Italy	1 1.7		2.3		12		10
	Parboiled white mean			17 2.0 1.4	2.9	4.3 2.2	8.6	11 6.4	18	10 5.0	15
	Parboiled white	Wild rice mix	Not known	2 3.9 3.9	3.9	6.7 6.6	6.8	9.4 8.5	10	18 17	18
			USA	1 2.9		9.2		20		20
	Wild rice mix mean			3 3.6 2.9	3.9	7.5 6.6	9.2	13 8.5	20	18 17	20
	Short grain (Japonica)
	Brown	Ecological	Australia/Italy 1 1.6		10		39		19
	White	Instant flakes	Not known	2 1.6 1.3	1.9	1.6 1.6	1.7	8.1 7.3	9.0	16 15	16
			Not known	2 2.0 1.6	2.3	2.6 1.5	3.7	15 10	20	18 17	19
			USA	1 1.9		3.3		16		12
		Ecological	Italy	1 1.1		1.2		7.3		12
	White mean			6 1.7 1.1	2.3	2.2 1.2	3.7	11 7.3	20	15 12	19
	Parboiled white	Avorio	Not known	1 1.7		3.0		7.7		11
	Grand mean			49 1.9 0.77 3.9	4.7 1.0	18	16 6.4	52	15 5.0	23

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uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 23Table 8 .

 8 Concentration of Se and Rb in different types of rice on the Swedish market in mg kg-1 fresh weight. Results in bold indicate mean values

					Se	Rb
		Other
	Type	information	Source	n	Mean Min Max Mean Min Max
	Long grain (Indica)
	Brown		Not known	2	0.2	0.1 0.2 4.6	2.6 6.6
	Parboiled brown		Not known	2	0.3	0.2 0.4 8.8	5.3 12
			USA	3	0.2	< 0.1 0.3 5.0	0.64 7.5
	Brown mean			7	0.2	< 0.1 0.4 6.0	0.64 12
	White	Basmati	India/Pakistan	4	0.2	0.1 0.2 2.2	1.2 4.1
		Jasmin	Thailand	5 < 0.1	4.2	4.0 4.6
		Ecological Jasmin Thailand	1	0.2	5.5
	White mean			10	0.1	< 0.1 0.2 3.6	1.2 5.5
	Parboiled white	Instant	Not known	2 < 0.1	0.50	0.47 0.53
			Not known	1	0.2	5.7
			USA	6	0.2	< 0.1 0.3 4.8	0.43 6.9
		Ecological	Italy	2 < 0.1	1.5	1.4 1.6
			USA/Italy	1 < 0.1	1.1
	Parboiled white mean		12 < 0.1	0.3 3.3	0.43 6.9
	Parboiled white	Wild rice mix	Not known	2 < 0.1	0.69	0.69 0.70
			USA	1	0.3	5.4
	Wild rice mix mean		3 < 0.1	0.3 2.3	0.69 5.4
	Short grain (Japonica)
	Brown	Ecological brown Australia/Italy	1 < 0.1	1.2
	White	Instant flakes	Not known	2 < 0.1	0.62	0.57 0.67
			Not known	2 < 0.1	1.1	0.33 1.9
			USA	1 < 0.1	0.77
		Ecological	Italy	1 < 0.1	0.38
	White mean			6 < 0.1	0.76	0.33 1.9
	Parboiled white	Avorio	Not known	1 < 0.1	0.85	0.85 0.85
	Rice, grand mean			40	0.1	< 0.1 0.4 3.3	0.33 12

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Optimised to meet criteria relating to the levels of oxide (< 3%) and double charged species (<

2%) present in the plasma.

Acknowledgements

The authors wish to thank Dr. Irene Mattisson, National Food Administration, Sweden, for reading the manuscript and giving valuable comments. The analysis of rubidium, selenium and silver was financed by the Swedish National Food Administration

 9. The contribution of consumption of rice in relation to Recommended Dietary Intake [START_REF] Nnr | Nordic Nutrition Recommendations, Integrating nutrition and physical activity[END_REF] except where otherwise indicated. The calculations are made using the average and min-max concentration values reported in Tables 6, 7 and8 3.2 (1.1 -4.9) 1.9 (0.7 -2.9) 59 (21 -91) 1 estimated average daily consumption in Sweden based on consumption survey [Becker & Pearson, 2002] and updated per capita statistics 2 average consumption of raw rice according to GEMS/Food Regional Diets (WHO, 2003). 3 [FNB, 2001 ] 4 [SCF, 2003 ] Page 24 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants


Alsing Gitte

Lisbeth Krüger Pedersen

Anja Jensen

Sandra Fankhauser

Jens Højslev Biedermann

Bente Petersen

Fabech

Gitte Pedersen

Alsing

Lisbeth Krüger

Migration of epoxidized soybean oil (ESBO) and phthalates from twist closures into food

Keywords: ESBO, phthalates, gaskets, migration, enforcement

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

Introduction

Glass jars with metal twist closures are widely used for a broad range of foodstuffs, such as infant foods, pickled herrings, vegetables in oil or water, sauces, peanut butter, milk products, soft cheese products, pesto etc. Many of these products are intended for storage up to two years and in order to ensure a long shelf life, a plasticized PVC gasket is placed inside the closures to form an airtight seal against the rim of the jar. Several of these foodstuffs are pasteurized or sterilized in the glass containers at high temperatures. Several studies have reported that ESBO is frequently used as plasticizer and as a scavenger of hydrochloric acid in PVC gaskets (Hammarling et al., 1998;Fantoni et al., 2003;Fankhauser-Noti et al, 2005a). The gasket material contains typically about 25-40 % plasticizer and consequently a substantial migration to the food can take place (Biedermann-Brem et al., 2005b). The migration of ESBO into fatty foods, like food rich in edible oil, has been shown to be high (Fankhauser-Noti et al., 2005).

ESBO is on the positive list in the EU plastics directive (EC, 2002) and a specific migration limit for ESBO of 60 mg/kg is derived from the tolerable daily intake (TDI) of 1 mg/kg body weight/day (European Commission, 2005) set by the European Food Safety Authority (EFSA). This limit, which is identical to the overall migration limit, was far exceeded for many fatty and oily foods and even for food with a fat content as low as 4 % free oil or fat (Fankhauser-Noti and Grob 2006a and b). Therefore, to clarify the legal situation an interim migration limit, valid until 30 June 2008, of 300 mg ESBO/kg fatty foodis in force (EC 2007a). The Commissions risk management was based on exposure calculations from the European Food Safety Authority (EFSA, 2006). However, for infant foods the Commission has introduced a specific migration limit (SML) of 30 mg/kg food (EC, 2007a), taking into account that for infants of age 6-12 month the TDI of 1 mg/kg bw/day can easily be exceeded by a factor of up to 4 or 5 already with a limit of 60 mg/kg. A previous study (Fantoni et al., 2003) showed that some 15 % of the infant foods collected from the markets of various European Union Member States contained more than 30 mg ESBO/kg . In the study, surprisingly, no correlation between the migration of this lipofilic compound and the fat content of the samples was seen. It is well known from several studies that a migration of phthalates plasticizers from packaging materials into fatty food can take place [START_REF] Petersen | Plasticizers in total diet samples, baby food and infant formulae[END_REF][START_REF] Sharman | Levels of di-(ethylhexyl)phyhalate and total phthalate esters in milk, cream, butter and cheese[END_REF][START_REF] Page | Studies of transfer of phthalate esters from aluminium filpaper laminates to butter and margarine[END_REF]. Much public attention has been paid to phthalates since animal studies indicate that adverse effects on human reproduction could be foreseen to occur when exposed to such substances.

Therefore the use of these chemicals in the packaging industry was expected to decrease these years through substitution with other plasticizers. However, until recently only little attention has been paid to the possible use of phthalates in PVC gaskets. A Japanese study found that DEHP, DINP and DIDP were used as plasticizers in cap sealing resins of imported bottled food including foodstuffs produced in Europe [START_REF] Hirayama | Analysis of plasticizers in cap-sealing resins for bottled foods[END_REF]. Recent studies have confirmed that phthalates are used in gaskets coming into contact with different kinds of food (Fankhauser-Noti et al., 2006a;Fankhauser-Noti and Grob 2006b). From these studies it seems that the migration of phthalates (percentage of plasticizer migrating from the gasket material in food contact) is higher than for ESBO. The TDI's for di-n-butylphthalate (DBP), butylbenzylphthalate (BBP), di-2-(ethylhexyl) phthalate (DEHP), (di-isononyl-phthalate (DINP) and di-iso-decyl-phthalate (DIDP) have been re-evaluated by EFSA [START_REF] Efsa | )phthalate (DEHP) for use in food contact materials Question N° EFSA-Q-2003-191[END_REF]. The EFSA risk assessments together with considerations about human exposure from environmental sources (beside the contribution coming from food contact materials) are transformed into specific migration limits by the 4. amendment to the plastics directive .

Furthermore, this amendment, beside introducing SML's for the phthalates DBP, BBP, DEHP, DINP and DIDP, also requests that some of these shall not be used as plasticizers in single use articles in contact with fatty food (EC, 2007 b).

The aim of this project was to study the level of plasticizers migrating to selected foods packed in glass jars with twist closures. The study included: Evaluation of the quality of compliance documentation in the food industry, specific analysis of ESBO and phthalate migration (at the official food control authority of the canton of Zürich, Switzerland) and control of the overall migration limit by migration testing of unused lids (only were a high specific migration of plasticizers was seen). In order to get packed foodstuffs as well as identical new/unused lids, most of the samples came from Danish food producers/fillers. The intention was to gain a first impression of the situation with these products on the Danish market, therefore only a limited number of samples were included.

Materials and Methods

Samples

Sample material. 19 samples of different kinds of food packed in glass jars with twist closures were sampled at Danish food producers and importers by the regional food control centres. The samples included oily products like tomatoes, olives, garlic and shrimps in oil, herring in pickle and dressing, milk products, soft spreadable cheese, sauces, peanut butter and infant food (mixed dishes of vegetables, rice and meat). All samples were taken in November 2004. Best before-dates for the products ranged from December 2004 (shrimps in oil with only one month of shelf life) to November 2006. Age of the products at sampling time is given in table 1. For each sample, three identical packagings were taken. In addition the corresponding new/unused glass jars and lids of the same kind were taken when available.

Compliance documentation. Documentation containing results from migration testing or equivalent compliance testing was collected by the food inspectors when available from producers of lids and food in supplement to the sampling.

Analytical methods

Identification of plasticizers in gaskets of lids. The additives in the PVC gasket were analyzed as described by Biedermann-Brem et al., 2005.b. Briefly, some gasket material from outside food contact was dissolved in tetrahydrofurane (THF). After precipitation of the PVC, the supernatant was analysed directly by GC-FID (for phthalates, Di-(2-ethylhexyl)adipate (DEHA) and Dibutylserbacate (DBS) and after transesterification to ethyl esters (for ESBO).

Infrared spectra of gaskets. Selected samples were analysed to verify the identity of major constituents of the gaskets (the polymer and the primary plasticizer) using a Fourier Transform Infrared Spectrometer (FTIR), in supplement to the analysis of extracts by GC-FID. The IR spectra were recorded by a Perkin Elmer Spectrum One FTIR Spectrometer with an Attenuated Total Reflectance (ATR) accessory with diamond interface. The spectra were obtained at 550-4000 cm -1 using a resolution of 4 cm -1 . Digital library spectra [START_REF] Hummel-Scholl | Hummel Polymers" and "Scholl Polymer Processing Chemicals[END_REF] as well as an Atlas of Plastic Additives Spectra [START_REF] Hummel | Atlas of plastics additives. Analysis by spectrometric methods[END_REF] were used when identifying the sample spectra. Preparation of food samples. The content of three jars of a given product was pooled and homogenised for 2-5 min in a blender. For heering in pickle the fish was drained before homogenising the fish meat. For food products in oil, the oil was separated from the solid food before homogenising the solid fraction; the food and the oil were weighed and analyzed separately.

ESBO in food. ESBO was analyzed by the method described by Fankhauser-Noti et al, 2005.

Briefly, homogenised food or oil was transesterified to methyl esters without prior extraction of the ESBO, using conditions avoiding saponification. Methyl esters were analysed by on-line LC-GC-FID. Transfer of the fraction containing the methyl esters of the diepoxy linoleic acid, involved concurrent eluent evaporation and a loop-type interface. The detection limit ranged from 1-3 mg/kg food and the relative standard deviation from repeated analyses was below 10 %. The recovery was 94-103 %.

Samples with ESBO levels above the detection limit were analysed in duplicate. When the food and the oil were analyzed separately, the ESBO concentration in the product was calculated weighing the concentrations in the two fractions by the weight proportion of the fractions.

Phthalates in food. The determination of phthalates in food involved GC-MS with injector-internal thermal desorption as described by Biedermann et al., 2005a andFiselier et al, 2005. In brief, diluted oil or raw food extracts in n-butyl acetate were injected in splitless mode under conditions desorbing the phthalates from the oil layer deposited onto the wall of the injector liner. A precolumn coated with a thin film of stationary phase was backflushed towards the end of each analysis. The limit of detection was largely determined by the blanks and was below 1 mg/kg. MS involved electron impact ionization and selected ion monitoring. The recovery from spiked samples was better than 85 % and the measuring uncertainty was mostly below 20 %.

Overall migration testing of lids. Testing of overall migration from lids was done by accredited analysis using test conditions according to the relevant directives (82/711 EEC and its amendments; 85/572/EEC) (EC, 1982;EC, 1985) and the European standard EN 1186 part 1 and 8 (CEN, 2002a;CEN 2002b). Unused glass jars with lids (from identical batches) used for the fatty food samples were filled with 100 ml of olive oil (sample 11-14 and 16). For those products, where a heating step is a part of the production process, the olive oil was heated up to the relevant temperature before filling the jars with the fatty food simulant. After filling the simulant into the jars, they were closed with the corresponding lid. The lids were exposed to olive oil by inverting the samples and kept at 10 days at 40 °C. The overall migration from each lid were analysed according to EN 1186. In each analytical series at least one sample was determined by a four double determination. For samples, where quadruplicate analysis were made the results were well within the specified analytical tolerance of the method (CEN, 2002a;CEN 2002b).

Results

Composition of plasticizers in the gasket of lids

Results in table 1 show that in 5 (26 %) of the samples, ESBO was used as principal plasticizer constituting 28-33 % of the gasket material. In two of these samples (sample 18 and 19) ESBO was used alone whereas in three samples (sample 12, 13 and 14) low levels of DBS was found in addition. In the remaining 14 samples (74 %) phthalates were used as principal plasticizer in combination with ESBO. DIDP were used in 10 of the gaskets at a level of 24-29 % in combination with low levels of ESBO and in two cases (sample 6 and 10) low levels of DEHA and DEHA/DEHP. In two of the gaskets (sample 3 and 16) DINP was the principal plasticizer in combination with low levels of ESBO and DEHP (sample 3). Gaskets used with two of the dairy products (sample 1 and 2) contained DEHP at a level of 31 %.

"[Insert table 1 about here]"

Results from FTIR analysis of the unused gasket material are represented by two samples with respectively ESBO and phthalates as principal plasticizer. IR spectra of the two types of samples are shown in figure 1 where the characteristic wavenumbers for the plasticizers are annotated.

"[Insert figure 1 about here]"

Specific migration of plasticizers to foodstuffs

The plasticizer levels found in foodstuffs are presented in table 2. Three of the five samples (sample 12, 13 and 14) with a high content of ESBO in the lids gave rise to high levels of ESBO in the food. These three samples were oily foods with fatty contact to the lid. The infant food samples (sample 18 and 19) were low in ESBO with a level below the detection limit. The declared level of fat in these products was 2-3 %.

The phthalates DIDP and DINP were found in seven food samples in a level above the detection limit of 1 mg/kg. In five of these samples, the levels of DIDP or DINP in the foodstuffs were higher than the SML's of 9 mg/kg (calculated from the TDI's of 0.15 mg/kg bw/day (EFSA, 2005) using the conventional factors of 60 kg body weight and 1 kg of food consumed per day). These products included oily products (tomatoes and garlic in oil), peanut butter, sauce béarnaise, and one product of herring in aqueous pickle. The highest levels of phthalates were found in the oily food products and in peanut butter with a declared fat content above 50 %.

"[Insert table 2 about here]"

Overall migration to the food simulant olive oil For samples of oily and fatty foods showing a high migration of ESBO and/or phthalates from the lid into food, additional testing of overall migration from the lids was done. New/unused lids were however, not available for all samples. The overall migration result for the food simulant was calculated by dividing mg migrants per lid with the sample weight of a given sample. All results (in mg/kg food) were above the overall migration limit of 60 mg/kg according to table 2.

Compliance documentation

The documentation provided by the producers of lids and food included certificates of analysis from migration testing. In general the collected certificates contained information on overall migration testing of the plasticized PVC sealing. For a part of the samples additional test results from specific analysis of vinylchoride monomer or a few other specific substances were included. Only in a few cases migration testing of one or two phthalates was included in the compliance documentation, however, there were no tests for other plasticizers in the certificates. In general the testing was done on the sealing polymer and the results were expressed in mg/dm 2 of lining. Migration into food was estimated based on calculations assuming a certain surface-weight ratio.

Discussion

Compositional analysis of the gaskets by FTIR showed that due to the characteristic absorption bands of phthalates and ESBO respectively, it might be possible to distinguish between lids with ESBO as the principal plasticizer and those with phthalates as the principal plasticizers. FTIR analysis offer a quick initial semi-specific screening of the samples in order to decide which chemical analysis to be made on the food, in the food simulant or on the gasket material itself. This technique may possibly be useful in future compliance check for the presence of phthalates in the food contact material itself.

In five lids, ESBO was the principal gasket plasticizer, with a content of 28 -33 %, whereas phthalates were the principal plasticizers in a majority of the gaskets (14 samples) with a relative content of 25-31 %. This distribution is different from what has been reported by others showing that ESBO is the most commonly used plasticizer in gaskets when studying oily foods and infant food products (Biedermann-Brem et al., 2005b, Frankhauser-Noti et al. 2006b). However, this difference may partly be due to the limited number of samples in this study.

ESBO concentrations in the food were above the SML of 60 mg/kg in three of the five samples (sample 12-14) with a high content of ESBO in the gaskets. For the remaining two samples (infant foods, sample no 18 and 19), the levels in the food were below the detection limit of 3 mg/kg. This is in spite of the fact that the level of ESBO in the gaskets was in the same range for all five samples (see table 1). One explanation might be that the fat content is much higher in samples 12-14 (oily products) compared to the fat content of the infant food samples (2-3% fat). However, a number of other parameters can affect the level of ESBO migration into food, including the following: Area and thickness of the gasket, area of gasket available for direct food contact, lid surface/sample volume ratio, the applied procedure of sterilization, date of analysis in relation to the production/expiry date of the product, consistency of the food and handling of the jar on its way from producer to consumer. These are all factors strongly influencing the extent of contact between food and gasket.

The results show that ESBO is easily extracted into edible oil and that migration levels above 60 mg/kg can be found even with products that are very recently produced, as the production dates of all three products were only one week before samples were taken into analysis. This is in support of the findings in a previous study (Fankhauser-Noti et al., 2005).

As seen from table 2, the level of phthalates in food vary greatly between samples with the same relative phthalate content in the gasket, possibly due to variations in the same parameters as given for ESBO above. The highest levels of phthalates were found in samples (sample 11, 15 and 16) of oily or fatty foods in relatively small jars (180 -340 g food per jar) with rather high lid surface/sample volume ratio. In these products, the limit of 9 mg/kg for DINP/DIDP was exceeded by a factor of 10 to 19. Other products (béarnaise sauce and pickled herrings) have a lower content of phthalates in the food, but the levels were still above the limit of 9 mg/kg. As was the case for ESBO, the data show that phthalates are easily extracted into oily and fatty food within relative short time, as three of the products were taken into analysis within 1 -3 month after the production date. Phthalates have only a limited solubility in water and aqueous media [START_REF] Petersen | Plasticizers in total diet samples, baby food and infant formulae[END_REF] however, herrings contain much fat and some of this may be extracted into the aqueous pickle over time given an emulsion of water/fat. As an intimate contact between the pickle and the gasket is likely to occur, migration into the fish meat can be expected. The actual levels can be seen from table 2. Previous studies on oily products has shown that migration rate for DIDP and other phthalates far exceed migration rate of ESBO and that also migration from parts of the gasket not being in direct food contact take place due to a fast diffusion of the phthalates inside the PVCplastics constituting the gaskets (Fankhauser-Noti, 2006a).

In this study the ratio between lid surface area (in dm 2 )and sample weight (in kg) varies between samples within the range of 0.14 -1.17 (dm 2 /kg) as seen from table 1. The rather low lid surface/sample weight ratio for sample 10 (shrimps in oil, with a ratio of 0.37) is probably one important reason for the much lower concentration of phthalates in this product compared to e.g. sample 11 (garlic in oil, with a ratio of 1.00). Another observation from this study was that in samples were individual analyses of the solid food fraction and the oil fraction took place, the level of phthalates and ESBO in the oil fractions is several times higher than in the food fractions (results not shown).

Results of overall migration testing of the lids into the food simulant olive oil at 40 °C in 10 days were in the same order of magnitude as the sum of the measured specific migration of plasticisers into the food (or somewhat higher). Here the testing scheme ensures that an intimate contact between the food simulant and the gasket occurs. It is not surprising that the values can be high as the overall migration in this case is the sum of the concentration of all specific non-volatile chemicals in the gaskets. Beside additional plasticizers, this may include e.g. slip agents in high levels as found by Fankhauser et al., 2006b. An evaluation of the industry's compliance documentation indicated that the demands in Article 3 in Framework Regulation 1935[START_REF] Biedermann-Brem | Reaction of epoxidized soya bean oil (ESBO) with hydrogen chloride formed in PVC: investigation on model systems[END_REF](EC, 2004) was not respected. First of all, own-check documentation was incomplete and even misleading in many cases due to missing data on specific migrants of relevance and due to the applied principle of testing. Instead of testing the actual "ready for use" gasket, the migration testing in the gasket materials producing industry is often performed using a sample of the sealing compound without correcting for the specific lid surface-food weight ratio in actual use. Furthermore, differences in thickness of the gasket material in the final material and in the test specimen is ignored, as well as the diffusion of migrants, which is likely to occur from parts of the gasket not being in direct contact with the food. Altogether, this may lead to a serious underestimation of the migration. This observation underline the need for fulfilling the requirements in legislation saying that migration testing should be done using worst-case test conditions and preferably on the final article ready for use.

The observation that phthalates were used as principal plasticizers in a majority of the gaskets analysed in the project came as a surprise to the authors. The expectations were that these chemicals would no longer be used in materials coming into contact with fatty foods, as in Denmark much attention has been paid by authorities and the public to the negative impact of certain phthalates on reproduction. For this reason, only limited awareness has been paid from enforcement bodies to the potential use of these substances in PVC gaskets for food contact.

All samples violating the overall migration limit were withdrawn from the market by the local food inspectors. The Danish industry, including producers and importers of twist caps, food producers and retailers, were informed about the problems by the Danish Veterinary and Food Administration.

Conclusion

The lids taken into this study contained either ESBO or phthalates, like DIDP, DINP and DEHP as the principal plasticizers. When the gaskets were used with fatty or oily food in small and medium size glass jars, the level of migration of ESBO or phthalates violated several times the actual migration limits in force at the time of sampling. In samples tested for overall migration the migration level was at least in the same order of magnitude as the sum of specific migration of ESBO and phthalates.

In general the demands for safe and inert packaging materials, as required in article 3 in Regulation 1935/2004, was not fulfilled by the industry. Obviously the own-check documentation was not based on realistic worst-case test conditions.

  Moreover the European Metal Closure Group was informed about the non-compliant lids as a general problem and the immediate need for solutions. As a consequence of the observed problems, the biggest retailers in Denmark strengthened their own check control in spring 2005. Due to the the regulations it is the intension of the authorities to repeat the control campaign on lids, by taking more samples of domestic and imported origin. Results from this project (and from related projects in other European Countries) were already communicated to the EC-Commission and legislative measures will be implemented in the member states during2007 /2008 (EC 2007 a and b) a and b).

  Question N° EFSA-Q-2003-194, Adopted on 30 July 2005 • Butylbenzylphthalate (BBP) for use in food contact materials Question N° EFSA-Q-2003-190, Adopted on 23 June 2005 by written procedure (http://www.efsa.eu.int/science/afc/afc_opinions/catindex_en.html) EFSA, 2006 Opinion of the Scientific Panel AFC related to epoxidised soybean oil used in food contact materials, Adopted on 16 March 2006 by written procedure (Question No EFSA-Q-2005-219). http://www.efsa.europa.eu/en/science/afc/afc_opinions/1399.html (valid March 2007) EEC, 1982. Council Directive 82/711/ EEC of 18 October 1982 laying down the basic rules necessary for testing migration of the constituents plastics materials and articles intended to come into contact with foodstuffs. Official Journal of the European Union L 297:26 (amendments 93/8/EEC and 97/48/EC). EEC, 1985. Council Directive 85/572/ EEC of 19 December 1985 laying down the list of simulants to be used for testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs. Official Journal of the European Union L 372: 14. EC, 2002. Commission Directive 2002/72/EC (with amendments) relating to plastic materials and articles intended to into contact with foodstuffs. Official Journal of the European Union. 2002 L 220: 18. EC, 2004. Regulation (EC) NO 1935/2004 of the European Parliament and the Council of 27 October 2004 on materials and articles intended to come into contact with food and repealing directives 80/590/EEC and 89/109 EEC. Official Journal of the European Union L338: 4. EC 2007a. Commission Regulation laying down transitional migration limits for plasticizers in gaskets in lids intended to come into contact with food Official Journal of the European Union L 92: 9. EC, 2007b, Commission Directive 2007/19/EC of 2 April 2007 amending Directive 2002/72/EC relating to plastic materials and articles intended to come into contact with food and Council

17:105-112.

  Directive 85/572/EEC laying down the list of simulants to be used for testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs Official Journal of the European Union (corrigendum in) L 97: 50 EU Commission, 2005. Synoptic Document: Provisional lists of monomers and additives notified to European Commission as substances which may be used in the manufacture of plastics intended to

		Food Additives and Contaminants			Page 16 of 18
	Sample no. Sample no.	Age of product at time of sampling (month) ESBO in food Type of Food Type of Food Phthalates in food Area of lid (dm²) (mg/kg) (mg/kg)	Ratio of lid surface-to weight of Phthalates identified Sample weight (g) Overall migration Plasticizer composition of gasket polymer: ESBO DIDP DINP DEHP DEHA DBS food (dm 2 /kg) (% w/w) (% w/w) (% w/w) (% w/w) (% w/w) (% w/w) in the food (mg/kg)
	F o r P e e r R e v i e w O n l into contact with food stuffs. DG (http://europa.eu.int/comm/food/food/chemicalsafety/foodcontact/index_en.htm). Update to July come SanCO D3. 2005. Fankhauser-Noti A., Fiselier K., Biedermann-Brem S. and Grob K., 2005. Epoxidised soy bean oil migration from the gaskets of lids into food packed in glass jars analysed by on-line liquid chromatography-gas chromatography. Journal of Chromatography A, 1082, 214-219. Fantoni, L. and Simoneau C., 2003. European survey of contamination of homogenized baby food by epoxidized soybean oil migration from plasticized PVC gaskets. Food Additives and Contaminants, 20, 1087-1096. F o r P e e r R e v i e w O n l 1 Chocolate milk < 1 0.06 420 0.14 2 Cream not given 0.06 200 0.30 3 Soft cheese 1 0.28 240 1.17 4 Dressing, crème fraiche 2 0.06 387 0.15 5 Herring in dressing not given 0.25 310 0.81 6 Herring in pickle not given 0.45 619 0.73 1 Chocolate milk <3 <1 2 Cream <3 <1 3 Soft cheese <3 <1 4 Dressing, creme fraishe <3 <1 5 Herring in dressing <3 <1 6 Herring in pickle <3 8 DIDP F o r P e e r R e v i e w O n l y 4 3 10 4 25 7 24 2 25 7 Herring in pickle not given 0.45 617 0.73 6 26 8 Herring in pickle not given 0.45 467 0.96 7 25 9 Herring in pickle not given 0.25 292 0.86 7 25 10 Shrimps in oil < 1 0.62 1665 0.37 2 25 11 Garlic in oil 3 0.18 179 1.00 4 29 12 Garlic/chilli in oil < 1 0.18 184 0.98 32 13 Garlic in oil < 1 0.18 181 0.99 33 14 Olive in oil < 1 0.22 271 0.81 32 15 Tomatoes in oil 12 0.22 297 0.74 3 31 16 Peanut butter 2 0.25 340 0.74 7 17 Sauce bearnaise < 1 0.25 334 0.75 7 25 18 Infant food not given 0.15 190 0.79 29 19 Infant food 5 0.15 181 0.83 28 F 7 Herring in pickle <3 n.a. 8 Herring in pickle <3 24 DIDP 9 Herring in pickle <3 n.a. 10 Shrimps in oil <3 6 DIDP 11 Garlic in oil 6 173 DIDP 519 F o r o 12 Garlic/chilli in oil 91 n.a 464 r P e 13 Garlic in oil 91 n.a. 470 14 Olives in oil 100 n.a. P 267 15 Tomatoes in oil 6 126 DIDP 16 Peanut butter 9 99 DINP 76 17 Sauce bearnaise 6 33 DIDP 18 Infant food <3 n.a. e e r e r R e v i e 19 Infant food <3 n.a. n.a.: not analysed R e v i e w w O n O l n l y	24 20	31 31 2 1	2 3	2 2 2
			y y y
	K. Fiselier, M. Biedermann, K. Grob (2005) Injector-internal thermal desorption from edible oils.
	Part 2: chromatographic optimization for the analysis of migrants from food packaging material. J.
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. PVC plasticizers/additives migrating from the gaskets of metal closures into oily food: Swiss market survey June 2005. Eur. Food Res. Technol. 233: 447-453.

Fankhauser-Noti A., and Grob K., 2006b. Migration of plasticizer from PVC gaskets of lids for glass jars into oily food: Amount of gasket material in food contact, proportion of plasticizer migrating into food and compliance testing by simulation. Trends in Food Science & Technology. http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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