Claudia Walburga Heppner

Josef Rudolf Schlatter

Data needs for a risk assessment on furan in food

Keywords: Furan, data for risk assessment, carcinogenicity

come

Introduction

In May 2004, the U.S. Food and Drug Administration (FDA) published results on furan in food. The European Food Safety Authority's (EFSA) Scientific Panel on Contaminants in the Food Chain (CONTAM) recognised the importance of furan in food and published an initial report in December 2004. Furan (C 4 H 4 O) (CAS-Nr. 110-00-9) is an organic, lipophilic, volatile compound which serves as an intermediate in some chemical manufacturing industries such as for the preparation of temperature-resistant structural laminates or in the preparation of copolymers used in machine dishwashing products (NTP 1993). The presence of furan in canned meat was already described by [START_REF] Maga | Furans in Food[END_REF], however the report of the U.S. FDA outlined the occurrence of furan in a much broader variety of commonly used foods that undergo heat treatment such as canned or jarred foods including baby foods (U.S. FDA

2004).

Furan is classified by the International Agency for Research on Cancer (IARC) as group 2B, possibly carcinogenic to humans, and is carcinogenic in rodents (NTP 1993;IARC, 1995).

The purpose of this paper is to briefly summarize the conclusions of the CONTAM report and to highlight which data are essential to enable a comprehensive risk assessment of furan in food and providing scientific arguments to risk managers for possible regulatory measures and/or consumer advice. Unless otherwise stated, the primary source has been the CONTAM report (EFSA 2004). No new information since this time has been included in this paper on analytical methodology as this will be dealt with in the subsequent papers from the Workshop in this journal. Also no new data on exposure was included as the CONTAM Panel awaits EFSA´s collection of data from a European monitoring programme on furan in foods.

Analytical methodology

Furan is a small volatile molecule which can be determined by headspace gas chromatography (GC). This is a relatively simple and well-established methodology in which a food sample in liquid or slurry form is heated in a sealed vial to achieve partition equilibrium between the liquid phase and the gaseous headspace. The headspace gas is sampled and the vapour injected into a GC. Detection can be by non-selective means such as flame ionisation detector or by mass spectrometry (MS). The U.S. FDA developed a GC-MS method which involves heating the sample at 60°C for a minimum of 30 min, followed by sampling the headspace gas and GC/MS monitoring of m/z 39 and 68 (U.S. FDA 2006). Quantification is by internal standardisation using isotopically labelled furan (monitoring m/z 72 for d4-furan). The FDA method claims a limit of quantification (LOQ) of 2.0 -5.0 ng/g depending on the food type. The advantage of this approach is, that there is no need for a sample clean-up and it can be automated for high sample throughput. The disadvantages are that the foodstuff is heated, meaning that furan might be formed during analysis, although this is unlikely at the low temperatures used; that sample sizes are small (typically 5 to 10 g), meaning good homogenisation is required; and that because furan has a low molecular weight and even with MS detection there may not be adequate specificity.

For good quantification, the labelled internal standard is essential but there seem to be problems of availability of the standard, meaning that laboratories may be undertaking this analysis with less ideal internal standards or using external standardisation.

There are no precision data given in terms of repeatability, and neither of the methods have been validated, so there is no available insight into the robustness of the method.

There has been no proficiency testing undertaken to date, but it is foreseen to perform a European proficiency testing on furan in diverse foods in the future, which will establish the comparability of data obtained by different laboratories.

Occurrence

Furan occurs widely in different foods such as roasted coffee, canned and jarred foods including baby food containing meat and various vegetables. This suggest that there are multiple routes of formation rather than a single mechanism such as carbohydrate thermal decomposition.

In 2004, there was only a limited set of data available for occurrence of furan in food.

Therefore, the CONTAM Panel decided not to calculate mean, median or standard deviations for intake, but to present the range of estimated exposure using consumption data from the GEMS/Food Regional Diets (WHO 2003) as at that time no European food consumption database was available. Such data can allow ranking of food categories as a function of their contribution to the overall exposure via food. [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: Results from the DONALD study[END_REF], this would result in an exposure of < 0.03 to 3.5 µg/kg body weight (b.w.) per day assuming a body weight of 7.5 kg for a 6 month old baby [START_REF] Ec | Nutrient and energy intakes for the European Community[END_REF].

For a sound exposure assessment, the CONTAM Panel concluded that the following information is needed: (i) Occurrence data from a broad variety of food categories including beverages. (ii) Data on variability between and within brands. The European Commission supports this activity by preparing a recommendation on the monitoring of furan levels in foodstuffs which is expected to be published early 2007.

Toxicokinetics and Metabolism

Furan has a low polarity and can pass biological membranes and is readily absorbed from the lung or intestine, probably also from the skin. In a two compartment physiologically based pharmacokinetic (PBPK) model, [START_REF] Kedderis | Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro[END_REF] predicted 84 % metabolism agreement with other studies [START_REF] Burka | Disposition of [14C]-furan in the male F344 rat[END_REF][START_REF] Kedderis | Prediction of furan pharmacokinetics from hepatocyte studies: Comparison of bioactivation and hepatic dosimetry in rats, mice and humans[END_REF] it can be concluded that the amounts of furan reaching body tissues are limited by the high capacity of the liver to eliminate furan from the blood stream by first-pass metabolism catalysed by cytochrome P450 (CYP2E1). The major primary metabolite of furan is a reactive dialdehyde, cis-2-butene-1,4-dial, which has been found to bind irreversibly to nucleophiles such as proteins and nucleotides [START_REF] Burka | Disposition of [14C]-furan in the male F344 rat[END_REF][START_REF] Byrns | Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan[END_REF].

Toxicology

The toxicity database of furan is incomplete. Only qualitative data are available on the acute toxicity (vapours are narcotic, anaesthetic and convulsant; U.S. National Research Council NRC 2000), no data are available on reproductive and developmental toxicity of furan and there are no data from human studies.

Toxicology and carcinogenicity studies were conducted by the National Toxicology Program (NTP) by administering furan (purity > 99 %) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 16 days, 13 weeks, and 2 years (NTP 1993).

These studies are summarized in detail in the CONTAM report (EFSA 2004) and are briefly reviewed here.

In the 16-day studies, furan resulted in increased mortality in both species and both sexes at 80 mg/kg b.w. per day and higher, in male mice already at 40 mg/kg b.w. per day Final mean body weights of male rats that received 20 mg/kg b.w. per day and of male and female rats that received 40 mg/kg b.w. per day were significantly lower than controls (National Research Council, 2000).

In the 13-week studies, nine male and four female rats that received furan doses of 60 mg/kg b.w. per day died before the end of the studies (National Research Council, 2000).

There were no chemical-related deaths in mice. Final mean body weights of male rats that received 15 mg/kg b.w. per day or more, female rats that received 60 mg/kg b.w. per day and male mice that received 30 mg/kg were significantly lower than controls. A dosedependent increase in relative liver-and kidney weights was found in both sexes of rats that received 15 mg/kg b.w. per day or more. Toxic lesions of the liver (bile duct hyperplasia, cholangiofibrosis, cytomegaly and degeneration of hepatocytes, and nodular hyperplasia of hepatocytes) were associated with furan administration in all dose groups of rats; the severity of the lesions increased with dose. Other effects such as decreased thymus weights and kidney lesions were also seen in rats that received 30 or 60 mg/kg b.w. per day. Relative and absolute liver weights in mice of both sexes were dosedependently increased in male mice that received 15 or 30 mg/kg b.w. per day and in females that received 30 or 60 mg/kg b.w. per day. Toxic liver lesions (cytomegaly, degenerationnecrosis) were present in all groups of furan-exposed mice. Bile duct hyperplasia and cholangiofibrosis were observed in groups of mice receiving 30 or 60 mg/kg b.w. per day.

In the 2-year rat study, animals of each sex were administered furan at 2, 4, or 8 mg/kg b.w. per day five days per week. Mean body weights of male rats that received 8 mg/kg b.w. per day were lower than controls from approximately week 73 to the end of the study. controls from approximately week 85 to the end of the studies as a result of moribund condition associated with liver and biliary tract neoplasms and mononuclear cell leukaemia (see below). Increased incidences of numerous non-neoplastic liver and kidney lesions were present in treated rats. The incidence of forestomach hyperplasia was increased in male and female rats and the incidence of sub-acute inflammation of the forestomach was increased in female rats. Cholangiocarcinoma of the liver occurred in all groups of dosed rats (males: 0/50; 43/50; 48/50; 49/50; females: 0/50; 49/50; 50/50; 48/50) and was present in many rats of each sex at the 9-and 15-month interim evaluations. Hepatocellular adenomas or carcinomas (combined) were significantly increased in male rats after 2 years of administration (1/50, 5/50, 22/50, 35/50) and hepatocellular adenomas were significantly increased in female rats (0/50, 2/50, 4/50, 7/50); hepatocellular neoplasms were not observed at the 9-or 15-month interim evaluations. The incidence of mononuclear cell leukaemia was increased in male and female rats that received 4 or 8 mg/kg furan (males: 8/50, 11/50, 17/50, 25/50; females: 8/50, 9/50, 17/50, 21/50); the incidence in the 8 mg/kg groups of each sex exceeded the historical control ranges for corn oil gavage studies.

In the 2-year mice study, animals of each sex received doses of furan of 8 or 15 mg/kg b.w. per day five days per week. Mean body weights of male and female mice that received 15 mg/kg b.w. per day were lower than controls during the studies. Survival of low-and high-dose male and high-dose female mice was lower than controls from approximately week 80 to the end of the study as a result of moribund condition associated with liver neoplasms (see below). The incidences of squamous papilloma, focal The incidences of benign pheochromocytoma and focal hyperplasia of the adrenal medulla were increased in low-and high-dose male and in high-dose female mice. The incidence of hepatocellular lesions was increased in dosed mice, including hepatocyte cytomegaly, degeneration, necrosis, multifocal hyperplasia, and cytoplasmic vacuolization and biliary tract dilatation, fibrosis, hyperplasia, and inflammation. The incidences of hepatocellular adenomas and carcinomas were significantly increased (males: adenoma 20/50, 33/50, 42/50; carcinoma 7/50, 32/50, 34/50; females: adenoma 5/50, 31/50, 48/50; carcinoma 2/50, 7/50, 27/50).

A separate 2-year study was conducted in which 50 male rats were administered 30 mg/kg furan in corn oil by gavage five days per week for 13 weeks and then maintained for the remainder of the 2 years without additional furan administration (NTP 1993). Groups of 10 animals were evaluated for the presence of treatment-related lesions at the end of the 13-week period of furan administration and at 9 and 15 months. Cholangiocarcinoma of the liver occurred with an overall incidence of 100 % (40/40) and hepatocellular carcinoma occurred with an overall incidence of 15 % (6/40) in stop-exposure male rats that survived at least 9 months. Cholangiocarcinoma was observed in all 10 males at both the 9-month and 15-month interim evaluations. Hepatocellular carcinoma was first observed in 2 males at the 15-month interim evaluation.

According to FDA (2004), a preliminary report from a second 2-year bioassay in female mice found an increased incidence and multiplicity of hepatic tumours and a decreased tumour latency in mice dosed with 4 or 8 mg furan/kg b.w. per day, but not in mice dosed with 0.5, 1.0, or 2.0 mg furan/kg b.w. per day [START_REF] Goldsworthy | Dose response relationships between furan induced cytotoxicity and liver cancer[END_REF].

Several other non-standard carcinogenicity studies were performed, all confirming the carcinogenic potential of furan: Preweaning male B6C3F1 mice were treated by intraperitoneal administration of furan in tricaprylin either as a single dose of 400 mg/kg b.w. per day (LD 10 ) or six doses of 200 mg/kg b.w. per day. The mice were sacrified between 28 and 95 weeks after dosing [START_REF] Johansson | Frequency of Ha-ras gene mutations inversely correlated with furan dose in mouse liver tumours[END_REF]). In the single-dose group the furan-treated mice showed a statistically non-significant increase of the overall frequency of hepatocytic neoplasia and of the overall liver tumour multiplicity, compared with the vehicle control group. No treatment-related histopathological lesions other than development of liver tumours were found. In mice given six doses of 200 mg/kg b.w. per day, there was a statistically significant increased incidence of hepatocellular neoplasms (adenomas and carcinomas) and tumour multiplicity compared with the vehicle control group [START_REF] Vesselinovitch | Kinetics of diethyl nitrosamine hepatocarcinogenesis in the infant mouse[END_REF][START_REF] Wiseman | Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3F1 mouse[END_REF].

In male F344 rats, furan (30 mg/kg b.w. per day by gavage, five days/week) for 9, 12, and 13 weeks induced primary hepatic adenocarcinomas exhibiting small intestine mucosal cell differentiation at 70 to 90 % incidences by 16 months [START_REF] Elmore | Intestinal-type of adenocarcinoma preferentially induced in right/caudate liver lobes of rats treated with furan[END_REF]. The incidences of primary hepatocellular carcinomas ranged from 0 to 20 % (two hepatocellular carcinomas).

Metaplastic small intestinal-like glands were selectively derived from putative hyperplastic bile ductule-like progenitor structures of young adult F344 male rats given furan by gavage at a daily dose of 30-45 mg/kg b.w. per day five times/week for 2 to 6 weeks [START_REF] Sirica | Biliary proliferation and adaptation in furan induced rat liver injury and carcinogenesis[END_REF]. Administration of furan at 30 mg/kg b.w. per day for 9 to 19 weeks resulted in the preferential development of primary hepatic adenocarcinomas, which were characterized by small intestine mucosal cell differentiation.

Genotoxicity

In vitro

Furan was not mutagenic in the Salmonella typhimurium strains TA100, TA1535, TA1537 and TA98 with and without S9 metabolic activation (NTP 1993). In another study [START_REF] Lee | Genotoxicity of 1,3-dithiane and 1,4-dithiane in the CHO/SCE assay and the Salmonella microsomal test[END_REF] furan was weakly mutagenic in strain TA100 with and without S9 activation.

Furan was negative in the sex-linked recessive lethal mutation test in Drosophila melanogaster [START_REF] Foureman | Chemical mutagenesis testing in Drosophila, IX. Results of 50 coded compounds tested for the National Toxicology Program[END_REF] but was mutagenic in the thymidine kinase locus of L5178Y mouse lymphoma cells in the absence of S9 activation [START_REF] Mcgregor | Responses of the L5178Y tk+/tk-mouse lymphoma cell forward mutation assay: Mouse Lymphoma Cell Forward Mutation Assay. III.72 coded chemicals[END_REF] and, in another study, with and without S9 activation (NTP 1993). Furan induced chromosomal aberrations and sister chromatid exchanges (SCE) in Chinese hamster ovary cells (CHO) both with and without S9 activation (NTP 1993). In another study on CHO cells [START_REF] Stich | Clastogenicity of furans found in food[END_REF] furan caused chromosome aberrations only in the presence of S9 metabolic activation. Furan produced DNA double-strand breaks in isolated rat hepatocytes [START_REF] Mugford | Furan-mediated DNA double-strand breaks in isolated rat hepatocytes[END_REF].

The furan metabolite cis-2-butene-1,4-dial was directly mutagenic at non-toxic concentrations in the S. typhimurium strain TA104, a strain sensitive to aldehydes [START_REF] Peterson | A reactive metabolite of furan, cis-2butene-1,4-dial is mutagenic in the Ames assay[END_REF]. The mutagenic activity of furan in TA104 is similar to that of crotonaldehyde, acrolein and glyoxal. Cis-2-butene-1,4-dial was negative in strains TA97, TA98, TA100 and TA102; its mutagenic activity was inhibited by glutathione. Cis-2butene-1,4-dial caused DNA single-strand breaks and DNA cross-links in CHO cells [START_REF] Marinari | DNAdamaging activity of biotic and xenobiotic aldehydes in Chinese hamster ovary cells[END_REF] and produced DNA adducts in vitro [START_REF] Byrns | Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan[END_REF].

In vivo

Furan (250 mg/kg b.w. by i.p. injection) induced structural chromosome aberrations in mice bone marrow cells, but not sister chromatid exchange (SCE) up to the dose of 350 mg/kg b.w. i.p. (NTP 1993). Furan did not induce unscheduled DNA synthesis (UDS) in mouse or rat hepatocytes in vivo after a single oral dose of 200 or 100 mg/kg b.w., respectively (NTP 1993).

In the study with [2,5-14 C]-furan by [START_REF] Burka | Disposition of [14C]-furan in the male F344 rat[END_REF] which was discussed earlier, the liver contained more radioactivity than other tissues and 80 % was associated with proteins, while no radioactivity was associated with DNA. Repeated daily administration of the radiolabelled furan at four dose levels resulted in an approximately linear increase in covalent protein binding. There was either no binding to DNA or the furan-DNA adduct was not stable to the isolation procedure. The results of this study were considered inconclusive by the CONTAM Panel as the specific activity of [2,5-14 C] furan was probably too low to detect DNA adducts and because the labelled carbon atoms at position two and five are the labile carbons giving rise to CO 2 , further reducing the sensitivity of the radioactivity determinations on DNA in addition to the potential instability to the isolation method used. A study performed with 14 C-furan labelled on carbons three and/or four, using a DNA extraction method different from that based on phenol and possibly the Page 14 of 22 mg/kg), but did find evidence of cytotoxicity and secondary enhanced cell proliferation after exposure to 15 mg/kg b.w. furan for up to 6 weeks. These authors postulated that furan-induced hepatocellular proliferation secondary to cytotoxicity (necrosis/apoptosis), rather than in vivo genotoxicity, was primarily responsible for the production of liver tumours. Supporting evidence available at that time for an indirect mechanism was the observation that radiolabelled furan given orally to rats produced metabolites that covalently bound to proteins but not to DNA [START_REF] Burka | Disposition of [14C]-furan in the male F344 rat[END_REF]. However, the results of Burka et al. were considered to be inconclusive by the CONTAM Panel because of limitations in the experimental protocol. Furthermore, the CONTAM Panel noted that negative results of the liver UDS cannot be taken as a proof of absence of genotoxicity of furan, because this assay detects only bulky DNA adducts in heptacytes and is unable to detect mutagenicity resulting from misrepair and non-repair. [START_REF] Kedderis | The biochemical toxicology of furan[END_REF] found that furan-treated rat hepatocytes show 95 % loss of ATP, due to irreversible uncoupling mitochondrial oxidative phosphorylation [START_REF] Mugford | Furan-mediated uncoupling of hepatic oxidative phosphorylation in Fischer-344 rats-an early event in cell death[END_REF]. This depletes the energy stores, with activation of cytotoxic enzymes, including endonucleases that produce DNA double-strand breaks prior to cell death. The endonuclease inhibitor aurinotricarboxylic acid reduced the formation of DNA doublestrand breaks. These authors postulated that erroneous repair of the DNA double-strand breaks may lead to mutations that are involved in tumour development and supported an indirect mechanism for the formation of DNA double-strand breaks.

Taking into account all the presently available data on the mode of action of furan, the CONTAM Panel concluded that the weight of evidence indicates that furan-induced

Conclusions

In summary the CONTAM Panel noted that for a reliable risk assessment on furan in food data in the following areas are needed:

• Analytic: Need to validate the performance of analytical methods through interlaboratory trials to allow comparability of data between laboratories.

• Exposure: For a sound exposure assessment occurrence and formation data of furan in a wide variety of food and food products including prepared foods from home cooking and catering is needed.

• Toxicology: Furan is clearly carcinogenic in rats and mice, showing a dosedependent increase in hepatocellular adenomas and carcinomas in both sexes and in rats also a dose-dependent increase in mononuclear leukaemia as well as a very high incidence of cholangiocarcinomas of the liver in both sexes. The CONTAM Panel concluded that the weight of evidence indicates that furan-induced carcinogenicity is probably attributable to a genotoxic mechanism. However, dose-response data at lower dose-levels combined with mechanistic studies and investigations of the role of species differences in metabolism in the generation of toxic metabolites are needed for clarification of the formation and significance of the cholangiocarcinomas in rats, which occur at a very high incidence at the lowest dose tested. To further improve the toxicity database of furan, studies on reproductive and developmental toxicity are also needed.

F

  (iii) Information on freshly prepared foods from home cooking and catering (iv) Release of furan from food due to volatilisation in foods. (v) Other routes of furan exposure such as via inhalation during cooking. (vi) Identification of suitable biomarkers to clarify internal exposure. (vii). Mechanism of formation and stability in foods. As it is within EFSA´s remit to collect data (Article 33, EC 2002) a monitoring database on furan in foodstuffs with inputs from the European Member States was developed. The online submission of data via the Authority's website is possible since 1 November 2006.

  attributable to a genotoxic mechanism. However, chronic toxicity with secondary cell proliferation may indirectly amplify the tumour response.

Table 1

 1

	gives an overview

Table 1 .

 1 Furan in food. Data derived from the U.S. FDA Laboratory, the Swiss Federal Office of Public Health, the Kantonales Laboratorium Basel-Stadt Switzerland, the Confederation of the Food and Drink Industries of the EU, and the Chemische und Veterinäruntersuchungsamt Stuttgart Germany.

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

Acknowledgment

We would like to acknowledge the contributions of the members of the EFSA´s CONTAM Panel (period April 2003 to May 2006) Jan Alexander, Herman Autrup, Denis Bard, Angelo Carere, Lucio Guido Costa, Jean-Pierre Cravedi, Alessandro Di Domenico, Roberto Fanelli, Johanna Fink-Gremmels, John Gilbert, Philippe Grandjean, Niklas Johansson, Agneta Oskarsson, Andrew Renwick, Jiri Ruprich, Josef Schlatter, Greet Schoeters, Dieter Schrenk, Rolaf van Leeuwen and Philippe Verger as well as the Chemische und Veterinäruntersuchungsamt Stuttgart, Germany (CVUA), the Confederation of the Food and Drink Industries of the EU (CIAA), the Kantonales Laboratorium Basel-Stadt, Switzerland, the Swiss Federal Office of Public Health, and the U.S. Food and Drug Administration (FDA) for providing occurrence data on furan.

Mutation of proto-oncogenes in liver tumours induced in mice by furan

Ras proto-oncogene activation was studied in hepatocellular tumours (both adenomas and carcinomas) induced in mice by furan [START_REF] Reynolds | Activated oncogenes in B 6 C 3 F 1 mouse liver tumors: implications for risk assessment[END_REF]. The frequencies of activated H-ras and K-ras oncogenes were similar in liver tumours from 12/29 treated mice and 15/27 vehicle controls, but the spectrum of mutations in the H-ras gene differed significantly. In addition to finding five tumours with commonly occurring point mutations in codon 61 of Hras1, Reynolds et al. found novel mutations in eight liver tumours, including G-T and G-C transversions in codon 117 of Hras1 in four tumours and activation of Ki-ras. The unique ras oncogene mutational profile was interpreted by the authors as a suggestion that the novel mutations could have been due to a genotoxic effect of furan.

Mode of Action

The mechanism of carcinogenic activity of furan in rodents has not yet been fully elucidated. Hypotheses of both direct (genotoxic) and indirect mechanisms have been formulated.

While several pieces of evidence available suggest that furan or its proximate metabolite cis-2-butene-1,4-dial can react directly with DNA in target cells and can play a role in furan-induced tumours [START_REF] Wilson | Evaluation of genotoxicity, pathological lesions, and cell proliferation in livers of rats and mice treated with furan[END_REF] found no evidence of unscheduled DNA synthesis (UDS) after short-term exposure of male mice to furan by gavage (up to 200


Lars O Jorhem

Christina Åstrand

Birgitta Sundström

Malcolm Baxter

Penny Stokes

John Lewis

Kierstin Petersson Grawé

Elements in rice on

Keywords: Rice, heavy metals, inorganic arsenic, trace elements, cadmium, lead, arsenic

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 [WHO, 2003].

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 environmental contaminants, e.g. metals, in rice is therefore of global interest. Of toxicological importance are mainly cadmium (Cd), lead (Pb) and arsenic (As). The FAO/WHO Joint Expert Committee (JECFA) established a provisional tolerable weekly intake (PTWI) for Cd of 0.007 mg kg -1 bodyweight, based on the effects Cd may exert on 3 kidney function [START_REF] Who | Cadmium. In: Safety evaluation of certain food additives and contaminants[END_REF]. For Pb, JECFA established a PTWI of 0.025 mg kg -1 bodyweight , corresponding to 0.20-0.25 mg/day for adults [START_REF] Who | Lead. In: Safety evaluation of certain food additives and contaminants[END_REF]. This is based on the neurodevelopmental effects of Pb, and covers all sources of exposure, i.e. not only dietary exposure. Inorganic arsenic (In-As) is carcinogenic in humans and a PTWI of 0.015 mg kg -1 bodyweight was established by JECFA in 1988 [START_REF] Who | Arsenic. Joint Expert WHO/FAO Expert Committee on Food Additives and Contaminants[END_REF].

As the consumption of rice has increased markedly in Sweden it was decided to carry out a survey of the content of cadmium (Cd), lead (Pb) and arsenic (As) on a number of different rice brands available on the Swedish market.

In addition to the total concentration of an element in foods, the chemical form in which it is present is important when assessing the potential threat to human health. This is particularly important for As where, in its inorganic forms (As (III) and As (V) ), it is genotoxic and carcinogenic, whereas in its organo-forms, e.g., arsenobetaine, it is considered to be of no health concern [START_REF] Iarc | IARC Monographs on the evaluation of carcinogenic risks to humans. Some drinking-water disinfectants and contaminants, including arsenic[END_REF].

Rice is imported to the Swedish market from different parts of the world, e.g. Thailand and USA. Occurrence data could be used to calculate the Swedish dietary intake, and furthermore, by using the WHO/GEMS Regional Food Diets [WHO, 2003] to estimate the intake of Cd, Pb and As 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. 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 US, 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). To reduce cost As was analysed in 40 of the 49 samples.

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.

Total and inorganic As, were measured at the Central Science Laboratory (CSL), York, UK.

Lead and cadmium were measured at the National Food Administration (NFA), Uppsala, Sweden.

The rice samples were analysed as received, i.e. air dry (fresh weight).

Sample preparation

Preparation of samples for the analysis of Cd and Pb

An aliquot of sample (10 g) was weighed into Pt-crucibles and ashed overnight at a maximum temperature of 450°C, following a temperature increase of approximately 50°C/hour. [START_REF] Jorhem | Determination of metals in foodstuffs by atomic absorption spectrophotometry after dry ashing: NMKL interlaboratory study of lead, cadmium, zinc, copper, iron, chromium and nickel[END_REF][START_REF] Jorhem | Determination of metals in foods by atomic absorption spectrometry after dry ashing: NMKL collaborative study[END_REF]]. After wetting the sample ash with water and drying, the ashing cycle was repeated until the ash was free from visible carbon particles. The ash was then dissolved in 30 ml of 0.1 M HNO 3 . Every 10 th sample was analysed in duplicate, and the rest as single determinations. One Certified Reference Material (CRM) and two procedural blanks accompanied each set of samples and were taken through the complete ashing procedure.

Preparation of samples for the analysis of total As

An aliquot of sample (0.5 g) was quantitatively digested in concentrated HNO 3 (5 ml) using a microwave-assisted digestion system (Multiwave, Perkin-Elmer Ltd, Beaconsfield, Bucks, UK). Approximately every 10 th sample was analysed in duplicate, and the rest as single determinations. The power settings and timings were as per the manufacturer's preset program. The digest solution was quantitatively transferred to a graduated test tube and made up to volume with water (10 ml). An aliquot (1 ml) of the resulting solution was quantitatively transferred to a test-tube, and an aqueous solution containing Rhenium, as internal standard, was added (4 ml).

Preparation of samples for inorganic As analysis

An aliquot of sample (0.25 g) was quantitatively transferred to a PTFE centrifuge tube. Since the samples were dry solids, one ml of water was added and the tube shaken so as to ensure that the sample was thoroughly wetted. An aliquot (10 ml) of concentrated HCl (nominally 37 vol%) was added, and allowed to stand overnight. Hydrobromic acid (1 ml, 47 vol%) and aqueous hydrazine sulphate (0.5 ml, 15 wt %) were added to the resulting solution and shaken for 30 seconds). An aliquot of chloroform (4.8 ml) was added to the centrifuge tube and shaken for 3 minutes. The mixture was centrifuged (15 minutes, 14,000 rpm) and the chloroform removed (lower layer). This extraction procedure was repeated a further 2 times, with the chloroform layers being collected and combined in a second centrifuge tube.

An aliquot of 1.0 M hydrochloric acid (4.8 ml) was added to the combined chloroform solution, and shaken (3 minutes). The mixture was centrifuged (15 minutes, 14,000 rpm) and the acid solution removed (upper layer). This extraction procedure was repeated one more time, with the acid layers being combined and collected into a volumetric flask (10 ml) and made up to volume with 1.0 M HCl. An aliquot (5 ml) of this solution was quantitatively transferred to a plastic test-tube and an aliquot of diluent containing gallium, as an internal standard, added.

Measurement procedures

Measurement of Cd and Pb 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 both metals. The method parameters were previously optimised by establishing ash-and atomisation curves for each metal. Matrix modifiers were not used. The background correction systems was regularly checked for function. The operating conditions are given in Table 2.

Measurement of total As 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.

The concentration of 'inorganic' arsenic in the hydrochloric acid back-extractant solutions was measured using a high resolution ICP-MS instrument (Axiom, Thermo Elemental, Winsford, Cheshire, UK)), operating at a resolution setting of 11 000 in order to resolve the arsenic signal from the ArCl + interferent produced in the plasma of the ICP. The operating conditions are given in Table 3.

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 was unacceptably high 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 dilution. For Cd and Pb the LOD was 0.0003 and 0.001 mg kg -1 , respectively. For total and inorganic As the LOD was 0.03 and 0.005 mg kg -1 , respectively.

Certified Reference Materials (CRMs)

The results of the CRMs and Standard RMs (SRMs) analysed throughout this survey are shown in Table 4. There were no CRM's available for inorganic As at the time of analysis, but BCR 627 'Arsenic in Tuna Fish' reference material provides data for total arsenic, arsenobetaine and DMA, allowing a calculation of the 'inorganic' portion, to be made. The CRM DORM-2 had an indicative value of 0.122 mg kg -1 , based on a collaborative trial involving CSL and several other laboratories (unpublished results).

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.

Measurement uncertainty

Results from samples analysed in official control should be accompanied by their measurement uncertainty (U), according to ISO standard [ISO/IEC 17025, 2005]. The U include all known sources of uncertainty and a coverage factor of two (= u × 2). Lead and Cd are the only elements in this study for which Maximum Limits are established within the EU, and thus targets for official control. The U for Pb and Cd in this study were 0.005 -0.009 mg/kg for the concentration range 0.002 -0.016 mg/kg , and 0.001 -0.019 mg/kg for the concentration range 0.001 -0.088 mg/kg, respectively. The results from the duplicate analysis of every 10 th rice sample showed that the repeatability SD was consistently lower than U/2 (= u). The calculated intakes of Cd, Pb and As were based on the average consumption of dry rice (11 g day -1 ) by Swedish adults aged 19-54 years, according to the Swedish consumption survey "Riksmaten" that was performed in 1998 [START_REF] Becker | Riksmaten 1997-1998, Kostvanor och näringsintag I Sverigemetod och resultatanalys[END_REF]. However, it is known from the per capita statistics [START_REF] Sjv | Statistik från Jordbruksverket, Statistikrapport 2006[END_REF]] 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, and a total average daily consumption of 15 g rice.

In addition, the WHO/FAO GEMS Food Regional Diets [WHO. 2003] were used to estimate the contribution of rice to the intake of Cd, Pb and As. The intake was related to PTWI's established by JECFA [START_REF] Who | Arsenic. Joint Expert WHO/FAO Expert Committee on Food Additives and Contaminants[END_REF], WHO. 200, WHO. 2004].

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 of rice 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

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.

Cadmium

The grand mean Cd-level of 0.024 ± 0.020 (SD) mg kg -1 (range <0.001 -0.088 mg kg -1 ) was much lower than the current European ML of 0.2 mg kg -1 [EC Directive 466/2001]. See Table 6. No sample exceeded the ML.

The mean Cd concentration was similar in brown, white and parboiled white rice (0.021, 0.020, 0.025 mg kg -1 , respectively) but the variation in Cd within each group of rice was considerable, possibly indicating influence of other parameters like e.g. variation in Cd , 1997] or differences in Cd uptake between genotypes [START_REF] Cui | Cadmium uptake by different rice genotypes that produce white or dark grains[END_REF]. In a previous survey on rice on the Swedish market including both brown and white rice, a mean of 0.029 mg kg -1 was reported [START_REF] Jorhem | Lead, cadmium and certain other metals in food on the Swedish Market[END_REF].

Several studies carried out in recent years have found mean levels of the same magnitude, ranging between <0.006 -0.050 mg kg -1 (range of individual samples <0.006-0.070) [START_REF] Phuong | Elemental content of Vietnamese rice Part 1. Sampling, analysis and comparison with previous studies[END_REF][START_REF] Shimbo | Cadmium and lead contents in rice and other cereal products in Japan in 1998-2000[END_REF][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][START_REF] Lin | Heavy metal content of rice and shellfish in Taiwan[END_REF]]. These results indicate that the Cd levels in rice, grown on presumably uncontaminated soil, rarely exceed 0.1 mg kg -1 . [START_REF] Shindoh | Changes in cadmium concentration in rice during cooking[END_REF] concluded that washing and soaking of polished rice reduced the cadmium content by about 7 % indicating that Cd intake is overestimated when using data on Cd concentration in rice as traded.

In Table 8, the contribution of Cd in rice in relation to the PTWI is given using the average, minimum and maximum Cd level found in this survey, and the lowest and highest average consumption of rice according to GEMS/Food Regional Diets [WHO, 2003]. In Sweden rice contributes to 0.6 % (0-2.2 %) of PTWI for Cd. In an average European diet rice contributes to a mean of 0.4 % (range 0.0 -1.3 %), while in regions with higher consumption of rice like Far East, the corresponding values are on average 11 % (range 0.5 % -41 %) of the PTWI, depending on the Cd concentration in the rice consumed. Thus it can be concluded that longtime large consumption of rice, and concomitantly high Cd levels in rice will result in a large part of the PTWI being covered by rice alone. On the other hand, in regions where the consumption of rice is low, like in Sweden, it is not likely that rice will contribute to more than a few percentages of the PTWI for Cd.

Lead

The Pb levels ranged from <0.002 to 0.016 mg kg-1, with a mean level of 0.004 mg kg -1 (Table 6). This was markedly lower than the current European ML of 0.2 mg kg -1 [EC Directive 466/2001]. An earlier survey [START_REF] Jorhem | Lead, cadmium and certain other metals in food on the Swedish Market[END_REF]] found a similar range in imported brown and white rice (0.003-0.066 mg kg -1 ). Comparable results were recently reported by [START_REF] Shimbo | Cadmium and lead contents in rice and other cereal products in Japan in 1998-2000[END_REF] and [START_REF] Lin | Heavy metal content of rice and shellfish in Taiwan[END_REF] with means of 0.002 and 0.01 mg kg -1 , respectively.

Others have, however, reported much higher levels: Al-Saleh et al. 2003) reported a mean of 1.58 mg kg -1 , [START_REF] Bennet | Heavy metals in wild rice from northern Wisconsin[END_REF] reported a mean for wild rice of 0.96 mg kg -1 and [START_REF] Alam | Metal concentrations in rice and pulses of Samta village, Bangladesh[END_REF] found a mean as high as 7.71 mg kg -1 dry wt (range 2.60-15.89). Such high Pb levels are remarkable, considering that the rice samples were not reported as coming from contaminated areas.

However, we conclude that in the present study, rice from several regions of the world contain very low Pb levels. Even calculated as a worst-case scenario, i.e. using the maximum Pb concentration in rice and the highest daily consumption according to GEMS Regional Food Diets [WHO, 2003] Pb intake from rice is only 2 % of PTWI (Table 8).

Arsenic, total and inorganic

The mean level of total arsenic (T-As) in long grain brown rice (0.24 mg kg-1; range 0.09-0.35 mg kg-1) was similar to that of parboiled white rice (0.21 mg kg-1; range 0.09-0.40 mg kg-1), whereas the white rice contained considerably less (0.10 mg kg-1; range 0.04-0.16 mg kg-1) as can be seen in Table 7. Whether the lower level in white rice is due to the polishing process, area of production or rice variety is not possible to elucidate. It is noteworthy, however, that most of the parboiled rice (both brown and white) in this study originates from the USA and that the white rice is imported from Southeast Asia. The T-As levels found were similar to those reported previously of 0.286 mg kg -1 , 0.074 mg kg -1 and 0.05-0.26 mg kg -1 [START_REF] Park | A survey of trace elements in fresh-water fish and rice along the Han River by neutron activation analysis[END_REF][START_REF] Dabeka | Survey of arsenic in total diet food composites and estimation of the dietary intake of arsenic by Canadian adults and children[END_REF][START_REF] Schoof | A market basket survey of inorganic arsenic in food[END_REF].

Rice is, besides fish and seafood, one of the major contributors to dietary As intake [START_REF] Tao | Dietary arsenic intakes in the United States: FDA Total Diet Study, September 1991-December 1996[END_REF]. From a toxicological point of view the amount of inorganic As (In-As) is considered the most important. However, the analytical methods for speciation of As have only recently been developed and therefore there are only limited information on the content of In-As in rice and other foods [START_REF] Bohari | Speciation of arsenic in plants by HPLC-HG-AFS: extraction optimisation on CRM materials and application to cultivated samples[END_REF][START_REF] Lamont | Concentration of inorganic arsenic in samples of white rice from the United States[END_REF]D`Amato et al. 2004;[START_REF] Vela | Total arsenic determination and speciation in infant food products by ion chromatography-inductively coupled plasma-mass spectrometry[END_REF][START_REF] Williams | Variation in arsenic speciation and concentration in paddy rice related to dietary exposure[END_REF].

The content of In-As averaged 0.11 mg kg -1 (range 0.04-0.19 mg kg -1 )and can be seen in Table 7. These results are in good agreement with others [START_REF] Lamont | Concentration of inorganic arsenic in samples of white rice from the United States[END_REF], D`Amato et al. 2004] who reported mean levels of In-As of 0.112 and 0.132 mg kg -1 , respectively. In Figure 1, in which the rice samples were compiled into groups different from those in table 7, the ratio of In-As/T-As is shown for different types of rice. An average of 64 % (range 17-100 %) of the T-As in rice in the present study consisted of In-As. The lowest proportion of organic As was seen in rice from the south East Asia (i.e. basmati and jasmine). Similarly, [START_REF] Williams | Variation in arsenic speciation and concentration in paddy rice related to dietary exposure[END_REF] found on average 64 % In-As in European rice, 80 % in rice from Bangladesh and 81 % in Indian rice. However, the numbers of rice varieties are in some cases low in our study, and furthermore the results for the T-As in Basmati rice is in some cases below the limit of quantification (10 σ for the blank). Therefore no firm conclusions can be made.

The current PTWI for In-As is 0.015 mg kg -1 body weight [START_REF] Who | Arsenic. Joint Expert WHO/FAO Expert Committee on Food Additives and Contaminants[END_REF]. In Table 8, the dietary intake of In-As from rice is shown for different consumption scenarios. In Sweden, rice contributed to 1.3 % of the PTWI for In-As. In a WHO European diet, rice contributes on average 0.8 % (range 0.3 -1.3 %) to the current PTWI for In-As, while in the WHO Far East diet, the corresponding values are considerably higher with a mean of 24 % (range 8.9 -41 %). The latter is in line with previous reports [START_REF] Williams | Variation in arsenic speciation and concentration in paddy rice related to dietary exposure[END_REF][START_REF] Meharg | Arsenic contamination of Bangladesh paddy field soils: implications for rice contribution to arsenic consumption[END_REF].

Conclusions

Cd levels were low in rice on the Swedish market (average 0.024, range <0.001-0.088 mg kg -1 ), as were indeed the Pb levels (average 0.004 mg kg -1 ). The level of In-As in rice was on average 0.11, range 0,04-0,19 mg kg -1 . Rice can be a large contributor in relation to the PTWI for Cd and In-As, if the consumption of rice is high. On the opposite, Pb intake from rice remains low even if the rice consumption is comparatively high. . Total and inorganic arsenic (mg kg -1 ) in rice purchased on the Swedish market in 1999-2001. The height of the bars applies to the concentration of total As, the black bars show the concentration of inorganic As in brown (n = 3), brown parboiled (n = 5), white parboiled (n = 7), white parboiled organic (n = 3), basmati (n = 4) and jasmine rice (n = 7). Brown and white parboiled rice were imported mainly from the USA, basmati from India/Pakistan and jasmine rice from Thailand.

  135 mg kg -1 .Gorbunov et al. (

  arsenic, lead and cadmium was part of an IAEA Co-ordinated Research Project (SWE 12004).

F

  Figure 1.

Table 1 .

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

	Grain	Type	Other information Imported from
	Long grain Brown		Not known
	(Indica)	Parboiled brown		Not known
				USA
		White (polished) Asian	Thailand
			Basmati	India/Pakistan
			Jasmin	Thailand
			Ecological Jasmin	Thailand
		Parboiled white Instant	Not known
		(Polished after		Not known
		parboiling)		USA
			Ecological	Italy
				USA/Italy
			Wild rice mix	Not known
				USA
	Short grain Brown	Ecological brown	Australia/Italy
	(Japonica) White (polished) Instant flakes	Not known
				Not known
				USA
			Ecological	Italy
		Parboiled white Parboiled Avorio	Not known

Table 2 .

 2 Graphite Furnace AAS operating conditions.

				Ash temp.	Atomize temp.	Sample	Diluent
	Element Wavelength Slit	°C /time s.	°C/ time s.	volume	volume
	Cd	228.8 nm	0.7 Low 300	1500	5 µl	5 µl
	Pb	283.3 nm	0.7 Low 500	1700	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. Values in bracket are indicative only.

		Element n Found mean	Certified value
	NIST SRM	Cd	2 0.019	0.022
	Rice flour 1568a	Pb	2 <0.014	(<0.010)
		As	1 0.39	0.29
	NRCC DORM-2	As	1	20.8	18
	Dogfish muscle	As inorg.	1	0.13	(0.122)
	BCR 627 Tuna fish	As inorg. 1 0.081	(0.081)
	muscle

Table 5 .

 5 Results from proficiency tests for certain metals. All results in mg kg -1 dry wt

	PT-provider Round, date	Test sample	Metal Found	Assigned	z-score
					result	value
	FAPAS 1	7-31, 2001-06 Sunflower seed Cd	0.612 0.711	-0.8
	FAPAS	7-31, 2001-06 Sunflower seed Pb	0.105 0.124	-0.7
	NFA 2	T-6, 2001-11	Potato powder Cd	0.126 0.140	-0.5
	NFA	T-6, 2001-11	Potato powder Pb	0.003 0.025	-1.1
	Euromet 565 3 2002-02	Rice	Cd	1.68	1.618	2,2
	FAPAS	07-43, 2003-06 Milk powder	Cd	0.045 0.0442	0.1
	FAPAS	07-43, 2003-06 Milk powder	Pb	0.198 0.188	0.3
	FAPAS	7-46, 2003-10 Canned Fish	As	0.88	0.74	1.1
	FAPAS	7-48, 2004-04 Canned crab	As	9.9	9.83	0.1
			meat
	1 FAPAS Secretariat, Central Science Laboratory, York, UK
	2 National Food Administration, Uppsala, Sweden
	3 Institute for Reference Materials and Measurement, JRC, Geel, Belgium. Result converted
	from nmol/g.

Table 6 .

 6 Concentration of Cd and Pb in different types of rice on the Swedish market (mg kg -1 fresh weight).

	Cd	Pb

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Table 7 .

 7 Concentration of total and inorganic As, in different types of rice on the Swedish market in mg kg -1 fresh weight.

	Total As	Inorganic	As

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uk Food Additives and Contaminants

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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 inorganic and total arsenic was financed by the Swedish National Food Administration. The part concerning

 8. The contribution of consumption of rice in relation to WHO/FAO Provisional Tolerable Weekly Intake (PTWI) [START_REF] Who | Arsenic. Joint Expert WHO/FAO Expert Committee on Food Additives and Contaminants[END_REF]2000;2004) Cd, Pb and Inorganic As. The calculations are made using the average and min-max values reported in Tables 6 and7.

% PTWI mean (range) Consumption of rice, grams/day 1 15 1 (Sweden) 1 estimated average daily consumption in Sweden based on consumption survey [START_REF] Becker | Riksmaten 1997-1998, Kostvanor och näringsintag I Sverigemetod och resultatanalys[END_REF] and updated per capita statistics 2 average consumption of raw rice according to GEMS/Food Regional Diets [WHO, 2003].

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Food Additives and Contaminants


Sarah Pastorelli
email: sarah.pastorelli@jrc.it

L Torri

A Rodriguez

S Valzacchi

S Limbo

C Simoneau

Solid phase micro-extraction (SPME-GC) and sensors as rapid 2 methods for monitoring lipid oxidation in nuts 3 4

Keywords: Multi-sensor system, electronic nose, SPME, hexanal, lipid oxidation, 9 oxygen scavengers, shelf-life 10 11 12 13

Dry foods with high fat content are susceptible to lipid oxidation, which involves a 22 quality deterioration of the product, since this process is responsible for the generation 23 of off-flavours. Hexanal is considered to be a good shelf-life indicator of such oxidation 24 products. In addition, due to its high volatility, hexanal can be easily determined by fast 25 headspace analytical techniques. For this reason an electronic nose comprising 10 metal 26

 Stashenko 5 et al. 2002). Different storage variables such as light and temperature can influence the 6 off-flavours development from the products generated by lipid oxidation (Jensen et al. 7 2005). 8 9Hexanal is the main aldehyde formed during the oxidation of unsaturated fats (Shahidi 10 2001). For this reason hexanal is a representative marker of the oxidative rancidity . 11 The purpose of the present paper was the development of a simple, efficient and fast 12 method to determine hexanal and to monitor its presence in hazelnuts samples stored 13 under controlled conditions. 14

15

A multi-sensor system (electronic nose) and solid phase micro extraction (SPME) were 16 compared in order to evaluate the effect of storage conditions on packaged food. 17

According to the Gardner and Bartlett's definition [START_REF] Gardner | A brief history of electronic noses[END_REF]), an 18 electronic nose is "an instrument, which comprises an array of electronic chemical 19 sensors with partial specificity and an appropriate pattern-recognition system, capable 20 of recognising simple or complex odours". Different kinds of gas sensors have been 21 developed and in function of their ability, the e-nose can be fit for various applications 22 in food industry [START_REF] Schaller | Electronic noses' and their application to food[END_REF]. It was shown to be suitable both to evaluate the 23 odour of packaging materials such as paper [START_REF] Holmberg | Identification of 6 paper quality using a hybrid electronic nose[END_REF] and to determine the sensory contamination of packaged foodstuffs [START_REF] Heiniö | Monitoring of taints related to printed solid boards 4 with an electronic nose[END_REF]. 2

The aroma evaluation of foods can be performed in different steps of production from 3 the raw material stage right to the final product in order to monitor the quality and 4 freshness [START_REF] Bartlett | Electronic noses and their 23 application in the food industry[END_REF]). In the last years, the use of an e-nose was tested both on 5 Commercial pouches for food active packaging were tested for their efficiency and the 7 eventuality of an undesiderable absorption of flavours generated from the food spoilage 8 was also investigated. 9 10

Materials and methods 11

Samples 12

Powdered hazelnuts purchased from a local supermarket were packed in transparent 13 plastic pouches with high oxygen barrier properties. For each time-storage condition 3 14 different sachets were prepared: pouch with oxygen scavenger, pouch with 7 g of nuts, 15 pouch with 7 g of nuts and an oxygen scavenger. 16 17 Samples were then stored at room temperature, at 40 o C and under UV light. After each 18 exposure period (0, 2, 5, 16, 31 days), pouches were opened and 6 head space vials (20 19 mL) were prepared for each package containing hazelnuts: 3 vials with 2 g of hazelnuts 20

for e-nose evaluation and 3 vials with 0.1 g of hazelnuts for SPME analysis. 21 22 In the same way the oxygen scavenger powder was removed from the pouches, 23 transferred in a 20 mL headspace vials and analysed by SPME/GC-FID technique. 24

Reagents and Analytical Standards 2

Hexanal with purity of 98% and triacetin for standard solutions preparation were 3 purchased from Sigma-Aldrich (Steinheim, Germany). 4

5

E-Nose 6

A commercial portable electronic nose (PEN 2 model) from WMA Airsense Analitics 7

Inc. was used. The sensor array system comprises 10 metal oxide semiconductors 8 (MOS) of different chemical composition and thickness to provide selectivity towards 9 volatile compound classes as indicated by the instrument supplier even if typically the 10 sensors react broadband-like on organic and inorganic gases (Table 1). These devices, 11 also called oxide or ceramic gas sensors, rely on changes of conductivity induced by the 12 adsorption of odour molecules. The elevated operative temperature (200-500°C) allows 13 avoiding interference from water and to aid rapid response and recovery times ( Kohl 14 1992). The detection limit of hot sensors was in the range of 1 mg kg -1 . 15 16 [Table 1, please insert here] 17

18 For e-nose evaluations, 2 g of nut powder was placed in a 20 mL glass vial, sealed with 19 a PTFE/silicone septum and a crimp top, stored at 25°C for 1 hour to equilibrate and 20 analysed at the same temperature. 21 22 The measurement system permitted suction of the gaseous compounds from the 23 headspace of sample through the sensor array at 400 ml/min for 180 secs. Then a 24 second pump transported the filtered reference air to the sensor array (the flow rate was 1 600 ml/min) for 400 secs to rinse the system between two following samples. The 2 dimensionless ratio between the conductivity change induced by the volatile compounds 3 from the headspace sample and the conductivity variation due to the interaction between 4 the reference air and the metal oxide semiconductor (analysing air sample the ratio for 5 each gas sensor is equal to 1) has been considered as response furnished by the single 6 gas sensors. 7 8

SPME-GC-FID-MS 9

Details concerning the development of the method and the analytical conditions are 10 reported in a previous paper [START_REF] Pastorelli | Solid-phase microextraction 1 method for the determination of hexanal in hazelnuts as an indicator of the interaction of 2 active packaging materials with food aroma compounds[END_REF]). 11 12Results and discussion 13 14 E-nose and SPME have been used in this experimental work in order to monitor the 15 shelf-life and oxidation state of hazelnuts during different storage conditions. Both 16 techniques are rapid, convenient and solventless methods for the determination of 17 volatiles compounds from sample materials. 18 19 The possibility of using an electronic nose with 10 metal oxide semiconductor sensors 20 to evaluate the quality of nuts stored under different conditions was explored and 21 compared with the SPME-GC-FID results. Figure 1 The two first principal components (PCs) accounted 89% of the total variability (71% 3 and 18% respectively). The dominant effect was given by the first principal component 4 (PC1) and a minor part by the second (PC2). The biplot (a scatter plot for scores and 5 factor loadings) illustrated the mutual relationships between samples and sensors 6 (Figure 1). In fact, the position of the samples in a part of the graph was determined by 7 the influence of the response of the sensors collocated in the same area of the plot. In 8 particular, in the left part of the graph the samples that have induced a higher interaction 9 between volatile compounds and some metal oxide semiconductors (W5S, W6S, W1S, 10 W1W, W2S, W2W, W3S) are shown. On the contrary, the position of the samples in 11 the right part of the biplot was determined by a lower response of the above cited 12 sensors. Moreover, the raw data used to perform the PCA (data not shown) indicated 13 that the sensors coded as W1C, W3C and W5C provided the lowest electronic signals 14 for all samples, similar to those obtained analysing the reference air. Therefore the 15 samples resulted distributed along the PC1 (from positive to negative values) according 16

to their increasing emission of volatile compounds. 17 The new score plot (Figure 2) did not show significant differences in distribution of 2 samples in comparison with previous results obtained considering all 10 sensors (Figure 3 1). On the basis of that, it is possible to affirm that the W1C, W3C and W5C sensors did 4 not provide any essential information to discriminate nuts, because probably nut 5 degradation did not produce aromatic compounds able to react with these metal oxide 6 semiconductors (Table 1). In other words, satisfactory results could be achieved 7 performing analysis with only the seven selected sensors (W5S, W6S, W1S, W1W, 8 W2S, W2W, W3S). 9 10 PCA data could be used to differentiate sample degradation as function of different 11 storage conditions (Figure 2). As can be seen, nuts stored under UV light and without 12 oxygen scavenger in the pack were well separated from the others. The action of UV 13 light associated with the presence of oxygen represented the worst storage condition 14 that favoured the highest production and accumulation of volatile compounds in nuts, as 15 demonstrated by the position of these samples in the part of score plot that reveals the 16 highest quantity of volatile compounds detected from the electronic nose. 17 In figure 2, it was also possible to appreciate a minor detection of volatile compounds in 1 hazelnuts packed with oxygen scavenger (empty symbol) with respect to ones packed 2 without the active device (full symbol) at the same storage time and condition. In fact, 3 in all cases, the samples with oxygen scavenger were positioned in the plot more to the 4 right than the nuts without oxygen scavenger. 5 6 For the development of a fast screening method the systems would need to calibrate 7 more samples and a greater variety of conditions but the discrimination appeared to be 8 feasible. The multi-sensor system provided a good differentiation among samples 9 stored at different time-conditions. Anyway e-nose did not permit an identification of 10 odour compounds responsible for the shelf-life state of the product. For this reason we 11 used a SPME/GC-FID method for the identification of hexanal as indicator of hazelnuts 12 oxidation state. 13 14 The results obtained with the SPME/GC-FID confirmed the findings of the investigation 15

with the e-nose. The different hexanal content obtained in the SPME/GC-FID analysis 16 was correlated to the results of the sensor array measurements. The hexanal content 17 profile of the mean results out of triplicate measurements of different samples is 18 described in the figure 3. at room temperature and at 40°C. However no big differences have been detected 1 among samples stored at room temperature and at 40°C so that temperature seemed to 2 have a minor effect on oxidation process. 3 4 With samples stored under UV radiation hexanal content increased during storage time 5 while in all the other hazelnuts without oxygen scavenger the hexanal content remained 6 mostly constant or slightly decreased along exposure time. In the samples with oxygen 7 scavenger it was possible to observe a clearly reduction of hexanal content compared to 8 hazelnuts without active packaging under the same storage conditions. These results of 9 samples stored under UV showed the efficiency of oxygen scavenger in reducing 10 hazelnuts oxidation during storage. 11 12 In the case of samples stored at room temperature and at 40°C the hexanal content in the 13 hazelnuts with an oxygen scavenger decreased progressively with time. Due to the fact 14 that in hazelnuts without oxygen scavenger under these storage conditions there was no 15 increase in hexanal content during exposure, the data obtained seemed to indicate that 16 active packaging could have absorbed hexanal from package headspace. In order to 17 check this phenomenon the oxygen scavenger content was analysed by SPME. 18 19 Hexanal was always found in the powder content in oxygen scavenger pouches and, 20 with the exception of 40°C, hexanal concentration increased along storage time (figure 21 4 shows the mean results out of triplicate measurements).

Foods 3 (

 3 with high fat content like nuts are very good model products for shelf-life studies Del Nobile 2001). These products are susceptible to rancidity because of the lipid 4 oxidation (Brunton et al. 2000; Goodridge et al. 2003; Lee and Krochta 2002;

2 3

 2 different foods such as olive oil (Ulmer et al. 1997; Gonzáles Martín et al. 1999; García-6 Gonzáles et al. 2004), tomato (Berna et al. 2004), tea (Dutta et al. 2003), coffee (Ulmer 7 et al. 1997; Singh et al. 1996), fruits (Di Natale et al. 2001; Saevels et al. 2003), bakery 8 products (Needham et al. 2005) and on animal foods, e.g. meat (Berdagué and Talou 9 1993), poultry (Rajamäki et al. 2006), fish (Di Natale et al. 2001) and milk (Capone et 10 al. 2001; Labreche et al. 2005). Moreover, the correlation of the sensor data with 11 classical analytical techniques was investigated by various authors (Frank et al. 2001; 12 Annor-Frempong et al. 1998; Bleibaum et al. 2002; Aishima 2004; Strathmann et al. 13 2005). 14 15 Solid phase micro extraction was selected as sampling technique to compare to e-nose 16 data because it has been proven to be a simple, fast, solventless technique and it does 17 not require sample preparation. Volatile profiles in different foodstuffs have been 18 successfully characterised by SPME. Many SPME applications can be found in the 19 literature for fruit (Augusto et al. 2000; De Lourdes-Cardeal et al. 2005), meat (Ramirez 20 et al. 2004), vinegar (Natera-Marin et al. 2002), olive oil (García-Gonzáles et al. 2004), 21 soybean (Boué et al. 2003), truffle (Diaz et al. 2002). The analysis of hexanal in several 22 matrices has also been performed by SPME in turkey (Brunton et al. 2000), powder 23 milk formula (Fenaille et al. 2003) and freeze-dried chicken (Goodridge et al. 1 2003).Due to the fact that both e-nose and SMPE has been successfully applied in food 4 analysis, in the present work specific methods for hexanal determination in nuts have 5 been carried out. The experimental design included also the use of oxygen scavengers. 6

[

  shows the principal component 22 analysis (PCA) visualization of sensor array measurement performed on hazelnuts 23 stored at room temperature, at 40°C and under UV light. 24 Figure 1, please insert here] 2

18 An 23 [

 1823 examination of the sensor correlation matrix and the overlapping of the W1C,W3C 19 and W5C sensors revealed that those sensors were strongly correlated and so 20 contributed little to the discrimination process. In order to evaluate the usefulness of 21 these three sensors in data exploration, a PCA excluding them was performed. 22 Figure2, please insert here] 24

18 With

 18 regard to the storage temperature differences were not observed among samples 19 stored at 40°C and at room temperature. In fact the response of the array of the7 20 sensors did not permit separation of nuts into two groups as function of the storage 21 temperature, on the contrary the samples were grouped in only one cluster.

19 20[Food

 19 Figures 3, please insert here] 21 22 UV radiation seems to be the most critical factor for product oxidation. In fact, samples 23 stored under UV light showed a higher hexanal content compared with the ones stored 24 Page 10 of 23 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

22

 22

23 [Food 8 EFigure 1 . 6 Figure 2 . 9 10Figure 3 .

 23816293 Figure legends1

Figure 4 .

 4 Figure 4. Hexanal content in scavenger powder at room temperature, 40 o C and under 13

  et al. 1998) and plastic films (Frank et al. 2001; Van Deventer and Mallikarjunan 2002) 1

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