Maria D Fumi
email: daria.fumi@unicatt.it

Daria

M Lambri

D M De Faveri

Off-flavours in wines through indirect transfer of volatile organic compounds (VOCs) from coatings

Keywords: Exposure, Risk assessment, Sensory analysis Additives/Contaminants: Environmental contaminants, Volatiles indoor VOCs, coatings, wines, cork stoppers, off-flavours

This paper assesses the impact of VOCs from the drying of coatings on the sensory characters of corks and wines. According to UNI 11021:2002 a small-scale chamber was used 1) to expose wines to the drying of coatings with both low and high VOCs and 2) to expose corks to the same coatings. After exposure to the coatings the corks were then placed in direct contact with wine.

Different styles of white, red and rosé wines were tested. In both directly exposed wines and in wines after contact with the exposed cork, the taste and smell off-flavour perception and intensity were assessed by a panel of 8 experienced wine tasters using a 5 point numerical scale according to ISO standard methods. The results showed that the sensory characters of wines, especially taste, were influenced by the VOC content of the coatings. The taste off-flavour perception was found to be higher than the smell in wines exposed to coatings with either high or low VOCs contents.

ANOVA and Duncan analysis prove that: a) Panellist's answers were significantly different, b) It was difficult to differentiate the off-flavour perception on the high level scale c) The panellist offflavour perceptions versus wine style discriminated the wines into two groups (red and white/rosè).

For all the wine styles, the Pearson's test showed no-significant correlation between off-flavour perception levels and the main chemical characters of the wines. For the wines which were in direct contact with the exposed corks, the panellists detect the off-flavours according to the levels of VOCs in the coating and wine styles and they assessed the highest levels of alteration were to the taste.

Introduction

Over the past decade it has become evident that volatile organic compounds (VOCs) are the major contributors to the pollution of the indoor environment. VOCs can originate from a variety of sources including building and coating materials such as paints and varnishes [START_REF] Brown | Concentration of organic compounds in indoor air-a review[END_REF][START_REF] Clausen | Emission of volatile and semi-volatile organic compounds from water-borne paints, the effect of the film thickness[END_REF]; [START_REF] Rothweiler | Emission of Degradation Products of Building Materials in the Indoor Environment-Case studies[END_REF]Seltze 1997;[START_REF] Wolkoff | Volatile Organic Compounds-Sources, measurements, emissions and the impact on indoor air quality[END_REF][START_REF] Yu | A review of the emission of VOCs from Polymeric Materials used in buildings[END_REF] and they can have an adverse effect on human health as well as a sensory impact on the occupants of buildings [START_REF] Berglund | Effect of indoor air pollution on human health[END_REF]Harrison 1997;Holcomb and Seabrook 1995;[START_REF] Jones | Indoor air quality and health[END_REF]Knudsen et al.1999;[START_REF] Mølhave | Indoor climate, air pollution, and human comfort[END_REF][START_REF] Otto | Exposure of humans to a volatile organic mixture I. Behavioural assessment[END_REF][START_REF] Stranger | Comparative overview of indoor air quality in Antwerp, Belgium[END_REF][START_REF] Woolkoff | Organic compounds in indoor air-their relevance for perceived indoor air quality ?[END_REF][START_REF] Woolkoff | Organic compounds in office environments-sensory irritation, odour, measurements and the role of reactive chemistry[END_REF].

The sensory impact bound to the presence of VOCs is also more important in places where food are manufactured, stored, packed and consumed; because organoleptic alterations can occur, and the consumers' reaction is to reject foods and beverages (Fumi et al. 2004). Moreover evaluating the risks originating from coating materials is a fundamental requirement since the E.U. Directives 43/1993 and 852/2004 on ensuring safety of foodstuffs through the application of the Hazard Analysis Critical Control Points (HACCP) came into force.

A National Standard Method (UNI 11021:2002) has been in place in Italy since 2002 where requirements and test methods are specified for coatings used in environments where foods are likely to be present. In addition to the physical-chemical properties of coatings this standard takes two specific areas into consideration: a) resistance to fungi growth and b) off-flavour release. Offflavour release is one of the essential tests for paints and varnishes intended for use as coatings in indoor environment were foods are handled.

The VOCs emissions from coatings are continuous and last for many years in a building (Yu and Crum 1998) so it can have a long-term sensory impact on food present in the environment. Consequently it is recommended to use coating products which have a low impact on the air quality from the moment they are applied [START_REF] Knudsen | Sensory and chemical characterization of VOC emissions from building products: impact of concentration and air velocity[END_REF]. The determination of off-flavours due to VOCs emission during the initial period of paints or varnishes application is useful to predict the impact of a coating materials during their entire life on the occupant's wellbeing and on the quality of the foods .

Sensory characteristics are important in food as well as in beverages and above all in wine. In fact wine sensory properties are the result of the interaction of several factors or conditions: geographic origin, biodiversities, cultural traditions, local know-how, technological processes [START_REF] Clarke | Factors influencing sensory perceptions[END_REF]. Wine tasting has become a pleasant and fashionable activity. Professionally tasting is used to identify wine variety, age, complexity, whether typical and to evaluate the overall wine quality, in the context of consumers' experience the sensory perceptions have to fulfil hedonic expectation. The organoleptic quality of any bottled wine is directly related to its flavour components but indirectly related to compounds released by the cork stoppers. The physical properties of corks such as waterproof, hydrophobicity, and elasticity have encouraged their use for sealing wine bottles especially high-quality wines. Nevertheless, some negative aspects can appear.

In fact corks absorb many volatile compounds from the environment [START_REF] Barker | Absorption of 2,4,6-trichloroanisole by wine cork via the vapour phase in the enclosed environment[END_REF][START_REF] Pollnitz | The analysis of 2,4,6-trichloroanisole and otrher chloroanisoles in tainted wines and corks[END_REF][START_REF] Skurray | Factor affecting cork permeability[END_REF]Strauss et al.1985). So when faulty corks are used for sealing wine bottles, unpleasant substances can migrate into the wine and and off-flavours can occur [START_REF] Chatonnet | Identification and respondability of 2,4,6,tribromoanisole in musty, corked odors in wine[END_REF][START_REF] Sefton | Compound causing cork taint and the factor affecting their transfer from natural cork closures to wine-a review[END_REF]. It is therefore important to ensure that the properties of wine are not altered by environmental contamination such as VOCs from coatings. This paper assesses the influence of the drying of coatings with different VOCs levels on wine and cork stopper sensory characters.

Wines and cork stoppers

White wines (Chardonnay from neutral grapes and sweet Malvasia from aromatic grapes), red wines (Cabernet Sauvignon and Montepulciano bottle-Aged Red Wine), a rosé table wine (from Pinot noir grapes) and agglomerated double diaphragm cork stoppers were used to evaluate the sensory impact of paints. The wines were chosen according to differences in the level of some important compounds for the sensory perception of wines e.g. pH, total acidity, ethanol and phenolic compounds (Table I). Ethanol and sugar content, pH, total acidity and total phenols were determined in all the wines according to EU Official methods (1990).

Paints

In-can low VOCs (LV) and high VOCs (HV) water-based paints containing respectively ≤ 30 g l -1 and ≤ 100 g l -1 of VOCs according to EU Directive 2004/42 were chosen.

Experimental design

The coating impact test on wine flavour was carried out according to UNI 11021:2002.

The tests were performed in a Glass gas-tight chamber with a loading ratio = S V -1 (Coated surface/Chamber volume) = 3 m -1 , simulating a common room. The paint was applied on a glass plate carrier (non adsorbent surface) 100 mm x 60 mm x 2 mm.

Immediately after the application of the paint we put the wet specimen (without conditioning) in the chamber where 50 ml of wine in a 100 ml beaker was kept. After 48 hours of exposure at 25°C the sensory characters of the wine were estimated.

The corks were exposed, at first in the gas-tight chamber to the same coatings during 48 hours at 25°C , then placed in direct contact with 50 ml wine for 48 hours at 25°C. The surface cork area to wine volume ratio was 0.75 cm -1 . A blind sample was carried out exposing the wine in the gas-tight chamber in the presence of nocoating carrier in order to exclude eventual modifications due to the exposure equipment.

From each lot of wines we took 3 times 50 mL of each and put them into 3 different gas-tight chambers under the same experimental conditions (wet coating, exposure time, temperature). The same procedure was carried out on the blind sample, too.

The sensory alterations were detected: a) in exposing wines directly to fumes from coating and b) in wines after direct contact with the cork previously exposed to fumes from coatings.

Sensory evaluation

The panellists were experts in wine tasting and were selected and tested according to the ISO 8586 (Part I: 1993;Part II :2008), andISO 6658 (2005). Eight 30-50 year old panellists (5 females, 3 males) sensory assessors were chosen according to their ability to perceive eventual wine offflavours. The threshold of VOCs off-flavours was determined by ISO 13301 (2002) using solutions of 2-buthoxyethoxyethanol (the main VOCs component of the analysed water-based paints) in tasteless and odourless water. To determine the candidates' ability to discriminate graded levels of intensity of a given attribute a ranking/rating test for intensity by a 5 point numerical category scale anchored with words was used (Meilgaard et al. 2007a, b).

The sensory analysis of wines directly exposed to VOCs fumes and wines kept in contact with corks previously exposed to VOCs fumes were performed by a triangle test according to ISO 41201 (2004). The result was considered significant for α ≤ 0.05 and β ≤ 0.01, the statistical power of the test was p > 95%. The sensory analysis laboratory met all the basic criteria regarding equipment and application of the suitable environmental condition according to ISO 8589 (2007).

Panel screening and training

The 8 selected panellists were screened for their ability to perceive the paint off-flavours stimulus in water exposed in the gas-tight chamber to 2-buthoxyethoxyethanol spaced concentrations (3; 9; 27 µg m -3 ) close to the threshold reported in literature [START_REF] Knudsen | Sensory and chemical characterization of VOC emissions from building products: impact of concentration and air velocity[END_REF]. With this aim in mind a 3-AFC test (ISO 13301:2002) was followed.

In order to discriminate graded levels of intensity of VOCs off-flavour, the panellists were asked to rate on a 5 point numerical category scale anchored with words (Meilgaard et al. 2007b) the water samples exposed to ascending 2-buthoxyethoxyethanol concentration: 2; 4.5; 9; 18; 27 µg m -3 . The panel members were informed on how to use the scale to evaluate the intensity where 0 is = notperceptible; 1= perceptible; 2= clearly perceptible; 3 = strongly perceptible; 4= very strongly perceptible.

The volume for sample was 50 ml, served in clear wine glasses at 20 + 1°C. A control sample of non-exposed water was provided for both detection and ranking/rating tests. The panellist with the highest sensitivity to VOCs off-flavour performed the wine testing.

Wine testing

According to ISO 41201 (2004) and UNI 11021(2002) the triangle test were carried out on wines exposed and non-exposed to coatings and on wines kept in contact with corks previously exposed to VOCs fumes. In each phase of the test, one or two of the samples for each wine were exposed to applied coating and the position of the exposed sample was randomised. The volume of sample was 50 ml, served in clear wine glasses at 20 + 1°C. The panellists were asked to identify the samples that were different by sniffing and by tasting. After the identification of the different samples the panellists were asked to mark the intensity of the off-flavour perception on the above mentioned scale.

Results and discussion

Impact on the wines of the different VOC levels of the coatings

As we can see in Figure 1 and Figure 2 the taste coating impact is higher than smell coating impact in wines having been exposed to either high or low VOCs content.

As far as the wines exposed to low VOCs (LV) are concerned (Figure 1), the panellists indicate the smell alteration, from not-perceptible (intensity 0) to slightly perceptible (intensity 1) except for Chardonnay where 10% of them identified the alteration as clearly perceptible (intensity 2).

After tasting (Figure 2), the off-flavour is identified from perceptible (intensity 1) to strongly perceptible (intensity 3). Most panellists assessed the highest taste alteration in Rosé wine.

As for the sensory analysis of wines exposed to high VOCs (HV) (Figure 2), the panellists' answers indicated the smell alterations up to clearly perceptible (intensity 2) except for Cabernet and Aged-wine where a strongly perceptible off-flavour (intensity 3) was assessed. As regards taste alteration, most panellists rated the wines alteration from strongly perceptible (intensity 3) to very strongly perceptible (intensity 4) except for Chardonnay where an intensity 2 was given.

When comparing high VOC with low VOC samples (Figures 1 and2), we can see that the panellists' assessed the highest off-flavour levels to high VOCs.

Impact on panellist off-flavour perceptions of the different VOC levels of the coatings

The ANOVA analysis of the panellists' answers shows that the differences between the smell and taste perception levels for both coatings were highly significant as far as smell was concerned (Table II). Considering high VOC impact on smell, the Duncan's test (Table III) showed that the panellists' answers were significantly different, in fact their answers can be divided into 4 groups (a,b,c,d reported as superscript letters), but we noted no significant differences between strongly perceptible alteration (intensity 3) and very strongly perceptible alteration (intensity 4) nor between clearly perceptible (intensity 2) and strongly perceptible alteration (intensity 3). However clearly perceptible alteration was distinguishable from very strongly perceptible alteration (intensity 4) and from the others (0 and 1 intensity), and in fact we can see that the superscript letters are different.

Perceptible alteration (intensity 1) is distinguishable from not-perceptible alteration (intensity 0) and both from the others. Therefore it means that it is difficult for the panellists to distinguish a strongly perceptible alteration from a very strongly perceptible one and a strongly perceptible alteration from a clearly perceptible one.

In coating with low VOCs (Table III) smell alteration at 0 level has been significantly distinguished by the panellists from intensity 1 and both from 2, 3, 4 intensity level, and in fact we can see that the superscript letters are different. Alterations at 2, 3, 4 intensity level were not easily distinguishable from each other, in fact we get common super-script letter. That means that it was difficult for the panellists to identify the alteration above 1 intensity level. III). Even in this situation it was difficult for the panellists to distinguish strongly perceptible and very strongly perceptible as well as clearly perceptible from strongly perceptible, perceptible and not-perceptible. As far as the taste of high VOCs coating was concerned there are no difference between the intensity levels, the off-flavour was detected as very strongly perceptible whatever the type the wine (data not shown).

Panellist off-flavour perceptions versus wine style

When comparing panel answers versus the wines, ANOVA analysis divides the wines into two groups. In the first group (Table IV) which includes white (Chardonnay and Malvasia) and rosé wines, the panellists clearly perceived different levels of smell alteration, while they identified in Chardonnay also different levels of taste alterations due to the exposure to low VOCs. The second group (Table V) was composed of the two red wines (Cabernet sauvignon and Montepulciano bottle-aged wine), and the panellists perceived a significant olfactory alteration in Cabernet only when VOCs were at a low concentration.

In aged-wine they perceived a significant smell alteration in presence of both low and high VOCs and taste off-flavour according to low VOCs.

Impact on cork of the different VOC levels of the coatings

In relation to coating impact on cork sensory characters the panellists detected off-flavours in the wine in contact with cork stoppers previously exposed to coating specimens according to VOC levels and wine styles. In fact, after sniffing, we can note in wines exposed to low VOCs (Figure 3), that: in Malvasia all the panellists indicated the alteration as clearly perceptible (intensity 2); in Chardonnay most of them (70%) did not assess any alteration (0 = not-perceptible) and the other Even in this test, the panellists assessed the highest alteration level to taste in comparison to smell and when comparing high VOCs (Figure 4) and low VOCS coatings (Figure 3) they confirmed that higher VOCs coating have the most important impact on wine flavour.

Our results point out that the panellists are more sensitive to taste than smell alteration. We can suppose that mouth temperature enhanced the volatility of some VOCs and that chemical feeling factors stimulate the taste panellist perceptions. The difficulty for the panellists to distinguish significantly high levels of off-flavour intensity was probably because the mouth off-flavour persistence makes it difficult to compare high alteration levels. Furthermore, the VOCs can interact with the sensory characters of wines having a strong "masking" effect.

Moreover the wine style affected the panel's ability to perceive off-flavour: for example among the white wines the panellists clearly perceived smell alteration in Malvasia, while in Chardonnay they even perceived taste modifications. This suggests that varietal flavour present in Malvasia interacts with the panelists'ability to feel the different levels of taste alteration.

Compared to white and rosé wines, Cabernet Sauvignon and aged -wine were characterized by a strong complex olfactory and gustatory structure which makes the identification of the different alteration levels even more difficult. The Pearson's test (data not reported) showed no-significant correlation between the level of off-flavour perception and the main chemical characters of wines (ethanol, sugar total acidity and phenolic compounds). So this leads us to think that off-flavour perception due to VOCs contamination is mostly influenced by the totality of the volatile part of the The low VOC content does not seem to be enough to guarantee good indoor air quality due to the fact that coatings are complex mixtures of chemical substances some of which are present at so low a percentage that it is not possible to quantify them by analysis, but their low threshold strongly influences the quality of indoor air. Moreover, some of them are not considered VOC according to 200/4 E.U. Directive, but they also strongly influence the indoor air quality.

In Europe, a voluntary labelling scheme for building material according to their impact on the indoor air quality has been developed and introduced in Denmark and Scandinavian countries [START_REF] Yu | A review of the emission of VOCs from Polymeric Materials used in buildings[END_REF]). The Danish labelling scheme includes the use of pre-determined odour and irritation thresholds to ensure building occupant comfort . The Italian Standard Method UNI 11021:2002 gives information on coating impact on foods to assure food quality and genuine sensory perceptions and fulfil consumers' hedonic expectations.

In conclusion, the evaluation of the impact of coatings having different VOC levels on wine and on cork stopper sensory characters showed that:

-coatings modify sensory characters of wine (mainly taste); -off-flavour perceptions are correlated to VOC level and wine style; -cork stoppers are confirmed to be good absorbents of indoor paint contaminants.

These results lead us to conclude that the UNI 11021:2002 method is a great help to easily predict the coating impact on indoor air quality during the different coating manufacturing steps. Moreover these outcomes are helpful to advise manufacturers on how to develop low-polluting coating with low impact on air sensory characters in order to ensure food and beverage quality. The panel was constituted of 8 tasters.

For each parameter 3 replicate sets of each wine were presented to each taster.

For each parameter the Fisher Test (F) and the significance level (p) are reported. The panel was constituted of 8 tasters.

F

For each parameter 3 replicate sets of each wine were presented to each taster.

For each parameter the Duncan test at p < 0.05 is reported.

Different super-script letters (a, b, c, d) mean significant differences between data. Table IV. Impact of VOCs Coating on flavours of rosè and white wines: significance of panellist's answers versus the wines.

The panel was constituted of 8 tasters.

For each parameter 3 replicate sets of each wine were presented to each taster.

For The panel was constituted of 8 tasters.

For each parameter 3 replicate sets of each wine were presented to each taster.

For each parameter the Fisher Test (F) and the significance level (p) are reported.

n.s.= not significant

  tested the same day, the panellists analysed 3 replicates of each wine at 30 min intervals. The number of samples that have been evaluated in one sensory session was decided on the basis of the result of preliminary tests at the end of training of assessors. The first wines to be tested were the wines exposed to low VOCs first white wines having low ethanol contents and proceeding until red wines having high ethanol contents. Then the panellists tasted the wines exposed to high VOCs following the same protocol.Data analysisAll the data have been subjected to Microsoft Excel 2003 and to one-way analysis of variance (ANOVA). Correlations between panel sensory perceptions and wine chemical parameters were performed by Pearsons' test. Statistical elaboration was carried out by SPSS software version 13.0 (SPSS Inc., Chicago, Illinois).

  in wine exposed to low VOCs we can note that the panellists clearly distinguished the lowest and the highest alteration levels only (Table

  (intensity 1) to clearly perceptible (intensity 2); in Cabernet the answers were 50% not-perceptible and 50% perceptible.

  regard paints it is not possible to set a correlation between the quantity of VOCs present in paints and the alteration level in wines.

  working on the correlation of indoor VOCs decay kinetics with off-flavour perception intensity in wine and cork stoppers to estimate the shortest time necessary before manufacturing or consuming foods, after coating. Correlation between the off-flavour thresholds in foods and beverages and VOCs content in paints is also still being worked on.

  /42. Limitation of emissions of VOCs due to the use of organic solvents in paints and varnishes and vehicle refinishing products. E.U. Regulation n° 2676/90 (1990). Metodi di analisi Comunitari da utilizzare nel settore del Vino. G.U.C.E. L272, 33. Fumi M.D., Galli R., Pagella C., De Faveri D.M. 2004. Problematiche alimentari da inquinamento indoor. Tecnica Molitoria. 55:215-222. Harrison P.T.C. 1997. Health impacts of indoor air pollution. Chem Ind. 17:677-681 ISO 4120:2004. Sensory analysis-Methodology-triangle test. ISO 6658:2005. Sensory analysis-Methodology-General guidance. ISO 8586-1:1993. Sensory analysis-General guidance for the selection, training and monitoring of assessors. -Part I: Selected assessors. ISO 8586-2:2008. Sensory analysis -General guidance for the selection, training and monitoring of assessors --Part 2: Expert sensory assessors ISO 8589:2007 Sensory analysis-General guidance for design of test rooms. ISO 13301:2002. Sensory analysis -Methodology -General guidance for measuring odour, flavour and taste detection thresholds by a three-alternative forced-choice (3-AFC) procedure.

Figure 1 . 5 Figure 2 . 8 Figure 3 .Figure 4 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1528341234 Figure captions1

Table I .

 I Wine denomination and analytical characters of the wines used for the tests.

	Wines	Ethanol (% v v -1 )		Sugars (g l -1 )	pH	Total acidity (g tartaric acid l -1 )	Total Phenols (Folin-Ciocalteu index)
	Chardonnay	11.70		7.0	3.50	5.7	7
	Malvasia Rosè	7.55 11.20 F o r	65.0 5.0	3.50 3.50	6.0 5.3	6 20
	Cabernet Sauvignon	13.90 P	3.0	3.70	5.0	67
	Bottle-Aged Red	11.50	2.5 e e r	3.85	5.6	46
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Table II .

 II Impact of VOCs Coating on flavours of wines: significance of panellist's answers.

	Parameter	F	p
	HV_Smell	16.58	0.001
	HV_Taste	4.25	0.05
	LV_Smell	45.95	0.001
	LV_Taste	3.49	0.05

Table III .

 III Impact of VOCs coating on smell and taste of wines: panellist response frequencies %.

	Alteration intensity level	High VOCs Smell	Low VOCs Smell	Low VOCs Taste
	4 (very strongly perceptible)	0.0 a	0.0 a	1.9 a
	3 (strongly perceptible)	2.4 ab	0.0 a	10.7 ab
	2 (clearly perceptible)	16.2 b	7.6 a	22.5 bc
	1 (perceptible)	30.9 c	35.9 b	28.4 c
	r o 0 (not perceptible)	50.6 d	56.3 c	36.5 c
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Table V .

 V each parameter the Fisher Test (F) and the significance level (p) are reported. Impact of VOCs Coating on flavours of red wines: panellist's answers versus the wines.

				Aged -Wine				Cabernet sauvignon
	Parameter		Rosè F		p	Malvasia	F	Chardonnay p
	HV_Smell		4.32		0.05			1.79		n.s
	Parameter HV_Taste	F	1.04	p	F n.s.		p	0.73	F	n.s	p
	HV_Smell LV_Smell	20.42	8.72	0.001	5.15 0.01		0.05 77.56	7.90	0.01 0.001
	HV_Taste LV_Smell LV_Taste F r o LV_Taste	0.72 23.37 1.14	3.53	n.s. 0.001 n.s.	1.73 49.01 2.07 0.05	n.s. 0.001 n.s. 2.67	0.75 8.53 8.78	n.s. 0.01 0.01 n.s.
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n.s.= not significant

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


R Mladenova
email: rmladenova@gmail.com

D Shtereva

Investigation of pesticide residues in apple fruits from conventional and integrated pest management

Keywords: GC/MS Chromatography, Pesticide residues, apple fruit

This paper describes method validation for determination of more than 40 pesticides in apples using GC technique. Target compounds belong to the groups of organochlorine, organophosphorus, carbamates, pyrethroids, triazoles, dicarboximides, strobilurins etc. The sample preparation consists of acetone extraction and subsequent cleanup/concentration by SPE with polymer-based sorbent. Single quadrupole GC-MS operating in SIM mode and electron impact ionization was used for identification and quantification of the pesticides. The average recoveries obtained for analytes ranged between 70 and 110 % at three fortification levels -0.01, 0.1 and 0.2 mg kg -1 . The relative standard deviation was lower than 20 % for all tested compounds. The calculated limits of detection and limits of quantification were below 0.01 mg kg -1 which were sufficiently low compared to the maximum residue levels (MRLs) established by European legislation. The proposed method was applied for determination of pesticide residue in four selected apple varieties after harvesting. Whole and processed fruits (peel and peeled fruits) were analyzed from different schemes of treatment: two conventional, one based on integrated pest management (IPM) and two variants based on organic production (controls). Higher level of pesticide residues were found in apple fruits under conventional conditions. Fenitrothion and chlorpyrifos residues were detected frequently in apple peel at concentrations up to 0.45 and 0.77 mg kg -1 , respectively. The levels found in the whole fruits of the same samples were much lower than in peels and below the respective MRLs (0.5 mg kg -1 for both pesticides). In the apples from IPM were found only measurable residues of triadimenol, concentrated in the peels up to 0.05 mg kg -1 .

Introduction

Apple production in Bulgaria is one of the most vulnerable crops to pest damage due to serious problems caused by a number of pests namely apple scab (Venturia inaequalis), powdery mildew (Phodoshpera leucotricha), fire blight (Erwinia amylovora) , codling moth (Cydia pomonella Linnaeus), leopard moth borer (Zeuzera pyrina) and aphids [START_REF] Bobev | Fireblight in Bulgariacharacteristics of E. Amylovora isolates[END_REF]; [START_REF] Kutinkova | The leopard moth borer, Zeuzera pyrina L. (Lepidoptera: Cossidae) -important pest in Bulgaria[END_REF]Velcheva et al. 2006). In most cases the use of plant protection products (PPPs) is the only possibility for reducing these losses. Usually, the number of pesticide applications in conventionally treated orchards is about 10-15 times per year [START_REF] Andreev | Resistance to aphids and scale insects in nine apple cultivars[END_REF]. Nearly all of these treatments contain broad spectrum insecticides and fungicides.

In Bulgaria in 2007, 35 fungicides, 51 insecticides and 22 herbicides, containing a total of 62 active substances are in the list of authorized PPPs for placing on the market and use for apple production (National Service for Plant Protection. 2007). The large number of pesticide applications associated with apple production and the diversity of PPPs increases the potential risk for human exposure and harmful impact to the environment. To ensure food safety for consumers, the European Commission has established maximum residue limits (MRLs) for pesticides in foodstuffs, which are also regulated in Bulgarian legislation (European Parliament, 2005;Ministry of Health, Ordinance No 31. 2003). Adequate, reliable and rapid analytical methods for determination of pesticide residues are needed to provide control of the safety of production and PPPs usage.

Common procedure for analysis of pesticide residues consists of extraction from food commodity, cleaning of co-extracted materials and determination by instrumental techniques that separate analytes as gas chromatography (GC) or liquid chromatography (LC) [START_REF] Alder | Residue analysis of 500 high priority pesticides: Better by GC-MS or LC-MS/MS[END_REF]. Multiresidue methods (MRMs) became a great opportunity for simultaneous determination of large number of pesticides and therefore are preferable because of their speed, low cost and high sample throughput. The main difficulty in development of MRMs is setting up optimum conditions in order to obtain satisfactory metrological parameters for analysis of pesticides with various physicochemical properties [START_REF] Bolanos | Development and validation of a multiresidue method for the analysis of 151 pesticide residues in strawberry by gas chromatography coupled to a triple quadrupole mass analyzer[END_REF].

Experienced residue analysts are aware that no validation report prepared in another In order to minimize the negative effects that conventional apple production pose to the environment and human health, some alternative cultivation systems were developed (Tsakiris et al. 2004). Integrated pest management (IPM) is a sensitive approach that relies on current, comprehensive information on the life cycles of pests and their interaction with the environment. This information is used for drawing up of predictive models which allow growers to apply chemicals when is absolutely necessary. During last decades application of different IPM programs has started in many Bulgarian orchards (Andreev et al. 2003). The obtained results have showed that in orchards where the IPM approach was used the number of pesticides applications can be reduced considerably.

The aims of the present study were:

• to develop and validate GC-MS method for simultaneous determination of 42 pesticides frequently used in the apple growing process

• to estimate residual amounts of PPPs applied in different schemes of treatment: conventional agriculture and integrated pest management (IPM)

• to study the effectiveness of IPM approach for minimization of pesticide residues in apple production

• to investigate the pesticide residues distribution in apple fruit through analysis of residual pesticides in whole and processed fruits (peeled apples and peels).

Materials and methods

Samples

Totally fourteen representative samples of four different varieties of apples (Florina, Granny Smith, Erwin Baur and Prima) were collected in amount about 10 units (2.5 kg) during the harvest time and the sampling was performed according to Commission Directive 2002/63/EC (Comission Directive 2002/63/EC). The trees were treated by different schemes of application of PPPs: two conventional, one based on integrated pest management (IPM) and two variants based on organic production (controls). In order to obtain data about the pesticide distribution in the fruits from each treatment scheme, the laboratory sample from each variant was divided into three parts -a half of the sample was peeled and produce peeled fruits and peels, and remained half of whole fruits was processed unpeeled. All three derived samples for each variant were chopped with kitchen robot and after homogenization, an analytical sample for each of them was stored at -18°C until their analysis.

Reagents

Analytical standards of the pesticides and ethion as internal standard (IS) with certified purity were obtained from Dr. Ehrenstorfer (Augsburg, Germany) and Riedel-de Haen (Seelze, Germany). Acetone, ethyl acetate and methanol (gas chromatography grade) were purchased from Merck KGaA (Darmstadt, Germany). The list of analytes with selected physicochemical properties (selected ions monitored, water partition coefficients and water solubility) are given in Table I. Stock solutions of each pesticide were prepared by dissolving in ethyl acetate or acetone at concentration ranged between 960-1420µg ml -1 and was stored in a freezer at -18°C. Mixed standard solution of all target pesticides was prepared in acetone at concentration 10 µg ml -1 and was used for the preparation of working standards and fortification. Matrix-matched standard solutions were prepared by adding appropriate amounts of standard solutions to pesticide-free apple extract before the solid phase extraction step. Acetone solution of ethion with concentration 100 µg ml -1 was used as IS in order to compensate sample and injection volume changes. An equal volume of IS was added to the final extracts immediately prior to GC analysis. Polymer-based sorbent LiChrolut® EN with surface area 1200 m 2 g and particle size 40-120 µm, and weight 500 mg was commercially prepacked in 6 ml cartridges and was purchased from Merck KGaA (Darmstadt, Germany).

Apparatus

A Thermo Finnigan Trace GC Ultra (Thermo Finnigan, Milan, Italy) coupled to Trace DSQ MSD (Thermo Finnigan, Austin, Texas, USA), equipped with split/splitless injector and Varian FactorFour capillary column VF 5ms, 30 m × 0.25 mm ID × 0.25 µm film thickness was applied. A Thermo Finnigan AI 3000 autoinjector (Thermo Finnigan, Milan, Italy) was used to perform automatically 1µl injections. Xcalibur 1.3 data system software was used for data acquisition and processing. The carrier gas He was of purity 99.9995 % (SIAD, Bulgaria). Vortex mixer V-1 plus (Boeco, Germany) and centrifuge Rotofix 32A (Hettich GmbH&Co.KG, Germany) were used in the sample preparation process.Vacuum manifold Vac-Elut (Varian Inc., The Netherlands) coupled to vacuum pump 2012С (Welch Rietschle Thomas, USA) was used for SPE. Ultrapure water was prepared with Water pro PS (Labcono, USA).

Sample preparation

The following procedure was previously described by Stajnbaher (Stajnbaher et al. 2003) but some changes were made in order to adjust and optimize the methodology for present study.

The used sample preparation procedure for the three types of sample (peels, peeled apples and whole fruits) was as follows: 10 g of analytical samples were weighed into 50 ml centrifuge polypropylene tubes with screw caps, 20 ml of acetone were added, cap was tightened and the mixture was vigorously shaken by Vortex for 5 min. Then centrifugation was performed at 3000 rpm for 5 min and the supernatant was transferred in a glass cylinder in order to measure the volume. The exact half of the raw extract was diluted with water to 70 ml and was allowed to pass through the LiCrolut EN cartridge with maximum flow rate 8 ml min -1 .

Before loading the cartridge was conditioned with 6 ml ethyl acetate, followed by 6 ml methanol and 8 ml ultra pure water. After loading the cartridge was washed with 10 ml water and the sorbent was dry under vacuum (about -20 mmHg) for approximately 30 min. Then the retained pesticide residues were eluted with 2 ml ethyl acetate followed by 7 ml mixture ethyl acetate:acetone 9:1, after that the eluates were evaporated under gentle nitrogen stream to final volume of 5 ml (1 g matrix in each ml extract). Finally 50 µl of IS were added and 1µl of the extract was injected in the GC-MS system.

GC-MS conditions

Splitless injection of 1 µl was carried out at 230°C and splitless time 1 min. The temperature program applied was as follows: 70°C hold 1 min, then increased at rate 30°C min -1 to 190°C, hold 1 min again increased to 235°C with rate 5°C min -1 and finally increased at rate 15°C min -1 to 280°C, hold 8 min. The carrier gas flow rate was 1.0 ml min -1 .The mass spectrometer was operated in electron impact ionization mode (ionization energy 70 eV) at transfer line and ion source temperatures set on 250°C and 220°C, respectively. Selected ion monitoring (SIM) mode by detecting minimum 3 ions for each analyte was used (Table I). Depending on the number of monitored ions in one segment dwell time was set from 60 to 10 ms which resulted to scan times between 0.24 and 0.34 s. Performance criteria for estimation of analytical

Results and discussion

Analytical parameters were calculated for the multi-residue method proposed (Table II). The accuracy of the method was expressed as recovery by fortified blank samples at three concentration levels -0.01, 0.1 and 0.2 mg kg -1 with three replicates for each level and three injections for each replicate (totally 9 injections per level). Recoveries obtained were in the range 70.2-109 % which complies with the requirements of European Commission concerning pesticide residues analysis in food (European Commission, DG SANCO, Document N° SANCO/2007/3131). Precision was calculated in terms of relative standard deviation (RSD) and was ranged between 2.8-20.4 %. Matrix matched standards at 5 concentrations 0.01, 0.05, 0.10, 0.25 and 1.0 mg kg -1 and internal standard approach were used for calibration. The corresponding pesticides were added to the blank apple extract before SPE step, instead to the final extract as the custom proposes due to low elution degree of some pesticides (as pyrethroids) from the SPE cartridge. Prior addition and passing the matrix matched standards through the cartridge standardize SPE conditions both for the samples and the matrix matched standards and eliminate the differences.

The calibration curves for all investigated pesticides were linear with coefficients of determination higher than 0.98 in the range 0.01-1.0 mg kg -1 with exception for endosulfan II for which the concentration range was 0.05-1.0 mg kg -1 . Limits of quantification (LOQs) calculated as the lowest concentration of compound that give a response ten times higher than background noise, ranged from 0.002 to 0.01 mg kg -1 (with exception for endosulfan II -0.011 mg kg -1 ) and were lower than established MRLs. The obtained results showed that the proposed analytical method can be used for effective control of pesticide residues in apples.

The analysis of processed apple samples (whole fruits, peeled apples and peels)has showed presence of some pesticide residues (Table III) mostly in the peels and smaller amounts in the whole fruits of conventionally treated apples. Residues which exceed the set MRL were detected in only one sample -0.060 mg kg -1 fenitrothion in the whole fruits of variety Florina.

The level of fenitrothion in the peels of the same sample was 7.5 times higher -0.45 mg kg -1 .

Additional calculations were made in order to find whether the level in the whole fruits exceed the toxicological limit for fenitrothion -acute reference dose (ARfD) of 0.013 mg kg -1 bw d -1 (Document 3010). The calculations made according to EU guidance (European Commission, DG SANCO, Document N° SANCO/3346/2001 rev 7) showed that the predicted short term intake (PSTI) represent 6.1 % of the ARfD which means that the found violation does not present any risk for consumers as far as concern the acute toxicity. The highest amount of residues was found for chlorpyrifos in the peels of variety Florina -0.77 mg kg -1 (Figure 1). In the whole fruits of the same sample the residue level of chlorpyrifos was 0.11 mg kg -1 which is nearly 5 times lower than the established MRL of 0.5 mg kg -1 .

Measurable residues of tebuconazole, difenoconazole, myclobutanyl and hexaconazole were found only in apple peels. In whole fruits sub samples the residues found were below the lowest calibration level (LCL).

In the apples grown by the IPM approach only in a few samples residues of triadimenol and fenitrothion were found in the peels at concentrations ranged between 0.019 and 0.049 mg kg - 1 . In the whole fruits triadimenol and pirimicarb were detected but below the LCL. In the peeled fruits neither conventionally nor IPM treated was found any pesticide residues.

Conclusions

The proposed analytical method possesses satisfactory metrological and economic characteristics and can be used for effective control of the most frequently applied PPPs. The analyses of real samples indicated that the IPM approach allows much safer production of apples compared to conventional application of PPPs. Obtained data related to the distribution of residues in apple fruit showed that the presence of pesticide residues can be minimized almost completely by peeling of the apple fruits. Pesticides-online data pool. Available online from: http://pesticides-online.com/ (cited 2008 Dec 18).

Caption of figures

  accepted without verification that the method does indeed work in the analyst's own laboratory (FDA Pesticide Analytical manual. 1999).

  method were based on current European recommendations (European Commission, DG SANCO, Document N° SANCO/2007/3131; European Commission, Commission decision of 12 August 2002).

  Zupancic-Kralj L. 2003. Multiresidue method for determination of 90 pesticides in fresh fruits and vegetables using solid-phase extraction and gas chromatography-mass spectrometry. J. Chromatogr. A. 1015: 185-198. Tsakiris IN, Danis T G, Stratis IA, Nikitovic D, Dialyna I A, Alegakis AK, Tsatsakis AM. 2004. Monitoring of pesticide residues in fresh peaches produced under conventional and integrated crop management cultivation. Food Addit. Contam. 21(7): 670-677. Velcheva N, Peeva P. 2006. Studies on The Composition and Dynamics of the Lepidopterian Families in Trophic Relations with Apples in Sofia District. Plant Sci. (in Bulgl.). 43: 494-498.

Figure 1 .

 1 Figure 1. Chromatograms of chlorpyrifos in a) matrix-matched standard solutionconcentration 1.0 mg kg -1 and b) peels of apple sample Florina variety contained 0.77 mg kg -1 of the analyte.

Formatted

Table III .

 III Levels (in mg kg -1 ) of pesticide residues found in peels and whole fruits of the analyzed apple samples.

											Formatted: Width: 792 pt, Height:
											612 pt
		Apple varieties	Prima	Erwin Baur	Florina	Granny Smith	Formatted
		MRL	Peels	Whole	Peels	Whole	Peels	Whole fruits	Peels	Whole fruits
		mg kg -1		fruits		fruits
				Conventional treatment
	Chlorpyrifos	0.5	0.15	0.023	0.26	0.035	0.074÷0.77	0.010 ÷ 0.11	0.62	0.079
	Tebuconazole	1*	<0.01**	-***	-	-	0.038	< 0.01	0.013	< LCL
	Hexaconazole	0.1	0.028	0.010	< 0.01	-	< 0.01	-
	Myclobutanil	0.5	-	-	-	-	0.026	< 0.01	0.018	< 0.01
	Difenoconazole	0.5*	-	-	-	-	0.040	< 0.01	0.014	< 0.01
	Fenitrothion	0.01	-	-	-	-	0.45	0.06
				Integrated Pest Management
	Pirimicarb	2*	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	-	-
	Triadimenol	0.2	0.045	< 0.01	.049	< 0.01	0.019	< 0.01	-	-
	Fenitrothion	0.01	-	-	-	-	0.030	< 0.01	-	-
	* -Temporary MRLs
	** -Detected but below the lowest calibration level (LCL)
	*** -Not detected

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Food Additives and Contaminants

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Acknowledgements

The authors acknowledge the financial support of the Scientific Research Department at Ministry of education and science in Bulgaria for project No AC-1612-2006. The authors exceptionally thank to Assoc. Prof. Ph.D. M. Borovinova from Institute of Agriculture, Kyustendil, Bulgaria for providing of apple samples.


Lara La Pera

Alfredo Liberatore

Giuseppe Avellone

Serena Fanara

Giacomo Dugo

Pasquale Agozzino

C Avellone

La Pera Lara

Liberatore Alfredo

Avellone Giuseppe

Fanara Serena

Dugo Giacomo

Agozzino Pasquale

ANALYSIS OF FURAN IN COFFEE FROM DIFFERENT PROVENIENCE BY HEAD-SPACE SOLID PHASE MICROEXTRACTION GAS CHROMATOGRAPHY-MASS SPECTROMETRY: EFFECT OF BREWING PROCEDURES

Keywords: brewing procedures, coffee, furan, SPME-GC/MS

whether they are published or not. The documents may come

Experiments were carried out using 12 lots of 650 kg of roasted coffee coming from different 88 countries (2 from India, 4 from Vietnam, 2 from Cameroun, 1 from Ethiopia, 2 from Brazil, 1 from 89 Salvador). All the studied coffee samples are of "Arabica" type. Furthermore 16 pods made of 90 miscellaneous coffee from two different lots (8 pods from each lot), named A and B, were also 91 analyzed. The degree of grinding of coffee used for the expresso machine (pods) is extra fine (200-92 250 microns particle size), whereas that used for moka pot and infusion is fine (300-350 microns 93 particle size). The sampling was conducted by the provider; several aliquots of each sample was 94 taken at different site in each lot (ISO 4072-1982).

95

Sample preparation procedure: cold extraction

96

To determine the content of furan powdered coffee, both magnetic assisted extraction (MAE) and 97 ultrasound (USAE) extraction were tested, using cold water. For MAE, 20 g of grounded roasted 98 coffee were mixed with 100 ml of distilled water and placed under magnetic stirring at 20 C° for 20 99 min. For USAE, 20 g of grounded roasted coffee were mixed with 100 ml of distilled water and 100 placed in a ultrasound bath for 10 min. at 20 C°. In both cases, 20 µl of the mixture were placed in a 101 4 ml vial together with 10 µl of 0.6 ngg -1 d 4 -furan and brought to 1.5 ml with distilled water. placed in a 4 ml vial together with 10 µl of 0.6 ngg -1 d 4 -furan and brought to 1.5 ml with distilled 106 water.

107

Brewing with an Italian moka pot: 100 mL of water were poured in the boiler, then the funnel-108 shaped metal filter is inserted. 20 g of ground coffee is thereupon added, and the upper part is 109 tightly screwed on the base. By placing the pot on a heat source, the water is brought close to 110 boiling point creating steam in the boiler. The espresso is created when the steam reaches a high 111 enough pressure: it gradually forces the surrounding water up the funnel through the coffee powder 112 and into the upper chamber, where the coffee is collected. The brew was transferred into a 100 mL 113 graduated cylinder, made up to the mark with water and analysed as described earlier 114 Expresso machines Coffee was prepared according to the manufacturer instructions using 7 g coffee 115 pods. After coffee preparation, the dilution ratio has been calculated using a graduate cylinder.

116

Samples fortification

117 Recovery experiments were performed by spiking 20 g of coffee samples containing low amount of 118 furan, with 100 µl of furan standard solutions (prepared by appropriate dilution of the stock 119 solution), to achieve the final spiked amounts of 30, 60, 90 ng g -1 . Spiked samples were left at least 120 1 h at 4°C prior to extraction. To study the between-day precision of the method, the furan sample 121 spiked with 60 ng g -1 , was analysed in triplicate for 3 days; the between-day precision was 122 expressed as relative standard deviation of the obtained measurements. The overall analytical method was validated according to the guidelines reported by Green (1996) 165 in terms of specificity, linearity, sensitivity, inter and intra day precision and accuracy. The 166 comparison of the chromatogram of a standard solution of furan with that of a coffee sample 167 (Figure 2), gave evidence that no co-elution occurred and a good resolution and peak purity were 168 obtained in the chromatographic conditions described.

169

A seven points calibration curve (y= 33.28x; R 2 =0.994) covering the range 0.0075-0.486 ngg -1 was 170 obtained. The limit of detection (LOD) and the limit of quantitation (LOQ), calculated as 3-and 10-171 fold the signal to noise ratio, were 0.002 and 0.006 pgg -1 , respectively. The best way to assess accuracy is by analyzing standard reference matrices, but coffee standards 174 certified for furan were not available. The second approach is to compare test results from the new 175 method with results from an existing alternate method that is known to be accurate. Again, for furan 176 analysis, such an alternate method is not available. The third approach, which is the most widely showed that the accuracy, expressed as recovery factor, ranged between 92.8 and 102%; no 183 significant differences were observed between the two extraction methods. USAE has been used for 184 further analysis. Intra-day precision was within 8%, expressed as RSD% of nine measurements 185 performed on a standard solution and on a diluted coffee sample both containing 0.05 ngg -1 of 186 furan. The between-day precision achieved was within 10%, expressed as RSD% . 192 193 The method validated in this study (test method) for furan analysis in coffee was compared with the 194 method proposed by Altaki et all (2007). The mathematical relation between methods was estimated 195 by a scatterplot with identity line (Fig 3) (Bland and Altman, 1986). The regression line obtained 196 showed a good agreement between the two methods. In particular the 7.14 % of the 14 lots of 204 grounded coffee presented a mean concentration of furan lower than 100 ng g -1 , the 50 % ranging 205 from 100 to 200 ngg -1 , the 21.43% from 200 to 400 ng g -1 and the 21.43% higher than 400 ng g -1 .

206

The highest mean levels was measured in coffee lots coming from Brazil (527±85 ng g -1 ), the 207 lowest in those coming form Cameroun (142±119 ng g -1 ). The concentration of furan found in Our study aimed to give further insight into the effect of infusion in hot water (Turkish coffee), 222 moka express and expresso machines brewing (Italian coffee) on the content of furan in coffee. To 223 determine the significance of furan reduction by brewing coffee, a paired t-test was performed.

224

The results (Table 1) gave evidence that furan in contaminated coffee, was reduced by 57.0±6.9% 225 (t<0.0001) after infusion in hot water (80 C°) for 5 minutes (Turkish coffee); stronger furan 226 reductions were observed by brewing coffee using a moka pot (67.5±15.3%; t<0. 0001) or an

  , roasted, cooled, ground, and packaged by a Sicilian company (ZiCaffè, 83 Marsala, Italy). Bags of green coffee beans are machine-opened, dumped into a hopper, and 84 screened to remove debris. The green beans are then transferred by belt to storage hoppers. From 85 the storage hoppers, the green beans are conveyed to the roaster. 240 kg of green coffee were 86 simultaneously roasted. Roasters operated at temperatures of 200°C for 3 min (full roast).

  87

  10 g of grounded roasted coffee were mixed with 100 mL of hot water (about 80 C°).104After five minutes the mixture was filtered trough a metal filter; then 20 µl of the filtrate were 105

  by[START_REF] Altaki | Analysis of furan in foods by head-space solid phase 281 microextraction gas chromatography-ion trap mass spectrometry[END_REF] was optimized for coffee analysis. 75 µm 125 carboxen/polydimethylsiloxane fibers (CAR-PDMS) (Supelco, Milan, Italy) were used for the 126 extraction of furan by using a manual device. The fiber was exposed to the head space of the 4 ml 127 sample vial, adsorption time and temperature were optimized in the ranges 10-30 min. and 20-35 128 C°, respectively. A constant magnetic stirring was applied. The fiber was conditioned in the 129 injection port of the gas chromatograph at 295 C° under helium flow for 2 h. Desorption was 130 performed at 290 C° for 5 min. furan in coffee samples were performed using a GC 2010 (Shimadzu, Milan, Italy) 133 equipped with a GCMS QP 2010 mass spectrometer. For furan separation a SGE BPX-VOL 134 column (cyanopropylphenyl polysilphenilenel-siloxane), 30 m length, 0.25 mm ID, 1.4 µm film 135 thickness (SGE Europe, Villebon, France) was used. The carrier gas was helium at a flow rate of 136 0.95 ml/min; the GC oven temperature programme was 35C° (held for 2 min) to 230C° at 20 °C 137 min -1 , the injector temperature was 290°C and the splitless injection mode was used. Interface and 138 ion source temperature were 220 and 250°C, respectively. The MS spectrometer automatically 139 tuned using perfluorotrybutilamine (PFTBA) according to the manufacturer instruction; after 140 automatic tuning the electron multiplier voltage was set at 1000 V. First, EI full scan data were 141 acquired over the range m/z 35-100 at 0.73 sec per scan to confirm appropriate masses for the 142 selected-ion monitoring mode (SIM). In the SIM mode the current of the following ions was 143 recorded: m/z 68 and 39 for furan, m/z 72 and 39 for d 4 -furan; the ionization energy and emission 144 current were 70 eV and 250 µA, respectively. 145 HS-SPME optimization 146 As Altaki et al reported (2007), a 0.75 µm CAR-PDMS fiber and a SGE BPX-VOL capillary 147 column were chosen for furan analysis in coffee. HS-SPME optimization experiments were carried 148 out using a fortified coffee sample. Three parameters of the SPME procedure were re-valuated: 149 extraction temperature, extraction time and ionic strength. The optimum conditions reported by 150 Altaki et al for furan analysis in food were the following: sampling temperature of 20 °C, sampling 151 time 30 min. In this study, the temperature was set at 35 C° and sampling time from 10 to 45 min 152 were tested; the same experiments were performed at 30, 25 and 20 C°. The best analytical response 153 for furan extraction was obtained by fixing sampling temperature at 25C° for 15 min. Furthermore154[START_REF] Altaki | Analysis of furan in foods by head-space solid phase 281 microextraction gas chromatography-ion trap mass spectrometry[END_REF] performed furan analysis in food increasing the ionic strength of the medium by 155 adding NaCl in the concentration of 20% (w.w). To confirm this result also for furan analysis in 156 coffee, different amounts of NaCl in the range 0-30% (w.w.) were added to the sample vial. In this 157 were obtained (Fig.1): by increasing NaCl amount from 0 to 10% no 158 significant difference were noticed, whereas by increasing NaCl concentration from 10% to 30% a 159 remarkable decrease of the analytical response (expressed as furan/ d 4 -furan area ratio) was 160 observed. Therefore further analysis has been carried out with no addition of salt. As Altaki 161 reported, a desorption time of 5 min at 290 C° in the injection port ensured a complete removal

  day coefficient of variation, is a very important parameter when establishing routine 189 methods to be used over extended periods. It was determined by performing extraction and analysis 190 of furan on a coffee sample three consecutive days; five measurements were performed each day.191

  furan has been performed on 12 lots of coffee from different producing countries 201 and on 16 pods made of miscellaneous coffee from two different lots (A and B). The concentration 202 of furan found in the powder ranged from 53 to 587 ng g -1 and no correlation was observed 203 between furan levels and the zone of provenience (Fig 4).

  this study were lower than those reported byAltaki et al (2279±53 ng g -1 ) and 209 within the range reported by EFSA in 2004 (3-5050 ng g -1 ) and higher than those reported by 210Hasnip et al (2006), 28±7 ngg -1 .211Effect of brewing procedures212 There have been a few studies to investigate if furan persists in food and particularly in coffee, 213 during normal preparation processes leading to consumption.Kuballa et al. (2005) reported that 214 automatic coffee machines produced brews with the highest levels of furan, because the closed 215 system favours retention of furan; much lower levels were produced by standard home coffee-216 making machines and by manual brewing. Unfortunately, the data provided by this study were too 217 limited to draw any firm conclusions.Hasnip et al., (2006) gave evidence that furan can be lost 218 during food cooking, presumably by evaporation and by entrainment in the large volumes of steam 219 that are released.

Figure 1 Figure 2 PageFigure 3 Figure 4 Figure 5

 12345 Figure1Effect of ionic strength on furan extraction from coffee (t sampling =15 min; T sampling =25°C)

Acknowledgements

represent a poor alimentary source of furan: 100 mL of brew prepared by infusion with 10 g of 261 powder, provides about 0.3-3 µg/day of furan; the intake of 3 cups of coffee (60 mL total) prepared 262 with 20 g of powder using a moka, provide from 0.72 to 2.6 µg/day of furan, whereas 3 cups of 263 expresso (60 mL total) each prepared using the pods of this study provide about 1-1.9 µg/day of Furan, ng/g (reference method) Furan, ng/g (test method)


Keywords: Exposure assessment, GC/MS, Risk assessment Additives/Contaminants: Process contaminants, Volatiles Food Types: Baby food, Cereals, Potatoes, Vegetables Furan, carcinogens, gas chromatography, mass spectrometry, food 27 contamination, food analysis, baby food, infant food, risk assessment 28

Risk assessment of furan in commercially jarred baby foods,

1 insights into furan occurrence and formation in freshly home-2 cooked foods for infants and young children

Introduction 29

Furan (C 4 H 4 O), a very volatile and colourless liquid, has been classified by the 30 International Agency for Research on Cancer (IARC 1995) as a possible human 31 carcinogen (group 2B). However, it is still a matter of debate whether the carcinogenic 32 effects are caused by a genotoxic mechanism [START_REF] Durling | Furan is not genotoxic in the 452 micronucleus assay in vivo or in vitro[END_REF]. Other chronic effects 33 in humans may include hepatotoxicity [START_REF] Jun | Correlation of urinary furan 480 with plasma gamma-glutamyltranspeptidase levels in healthy men and women[END_REF]. Due to its widespread 34 occurrence in many types of food products, concerns have been raised that furan might 35 become a serious problem, similar to that seen for acrylamide [START_REF] Yaylayan | Precursors, Formation and Determination of Furan in Food[END_REF]. 36 37 Furan generation in food is multi-factorial [START_REF] Yaylayan | Precursors, Formation and Determination of Furan in Food[END_REF]. A number of model tests 38 have shown that furan is produced through heating of reducing carbohydrates through 39 the Maillard reaction [START_REF] Maga | Furan in foods[END_REF][START_REF] Additives | Origin and mechanistic pathways of formation of 506 the parent furan -A food toxicant[END_REF]. Furan generation 40 has also been determined in the presence of ascorbic acid [START_REF] Fan | Formation of furan from carbohydrates and ascorbic acid following 467 exposure to ionizing radiation and thermal processing[END_REF]Becalski and 41 Seaman 2005) or certain amino acids [START_REF] Additives | Origin and mechanistic pathways of formation of 506 the parent furan -A food toxicant[END_REF]Märk et al. 42 2006;[START_REF] Limacher | Formation of furan and 493 methylfuran by Maillard-type reactions in model systems and food[END_REF]. The pathway from ascorbic acid is of special importance as 43 many foods are fortified with vitamin C as part of processing. Possible intermediates in 44 the formation from ascorbic acid are 2-deoxyaldotetroses, 2-furoic acid and 2- for furan appear to have been resolved [START_REF] Senyuva | Analysis of furan in foods. Is headspace sampling a fit-516 for-purpose technique?[END_REF]Crews et al. 2007;52 Wenzl et al. 2007;Crews and Castle 2007b). 53

In our current study, we have focused on furan in foods intended to be consumed by 54 infants, because they are the most susceptible consumer group [START_REF] Murray | Environmental hazards to children's health in 518 the modern world[END_REF] and 55 because relatively high amounts of furan were previously detected in commercial baby 56 foods. [START_REF] Heppner | Data requirements for risk assessment of furan in 475 food[END_REF] summarized the data from a number of sources 57 (including data from the US FDA) and found that the concentration of furan in baby 58 foods ranged from non-detectable to as much as 112 µg kg -1 . Taking into consideration 59 a consumption of 234 g per day of canned baby food [START_REF] Kersting | Measured 483 consumption of commercial infant food products in German infants: results from the 484 DONALD study[END_REF], Heppner and 60 Schlatter (2007) assumed an exposure range of <0.03-3.5 µg per kg body weight (bw) 61 per day, assuming a body weight of 7.5 kg for a 6-month-old baby. Yoshida et al. (2007) 62 reported levels of furan in a similar range of 1.4 to 90 µg kg -1 in 15 baby foods from the 63 Japanese market. Most recently, the US FDA reported furan in ready-to-eat baby foods 64 from the USA in ranges between 2.5 and 112 µg kg -1 were found (Morehouse et al. 65 2008). 66 67 The European Food Safety Authority's (EFSA) Scientific Panel on Contaminants in the 68 Food Chain noted an absence of information on freshly prepared foods in home cooking 69 and catering in general [START_REF] Heppner | Data requirements for risk assessment of furan in 475 food[END_REF]. A first indication of the effect of 70 consumer cooking on furan in convenience food showed that the levels decreased in 71 most canned and jarred foods after heating due to simple evaporation (Roberts et al. 72 2008). In contrast, other researchers found furan to be relatively persistent in food 73 (Crews and Castle 2007a) and that it is not lost to a very significant extent when the 74 foods are simply warmed to be ready to eat [START_REF] Hasnip | Some factors affecting the formation of furan in 473 heated foods[END_REF]. At this point in time, 75 only one study has researched furan in home-prepared baby foods and it showed that all 76 seven preparations examined had significantly lower levels of furan in comparison with 77 commercial baby foods [START_REF] Bianchi | Development and validation of a solid 435 phase micro-extraction-gas chromatography-mass spectrometry method for the 436 determination of furan in baby-food[END_REF]). However, the results were described to be 78 insufficient to make a definite conclusion regarding furan content and home cooking. 79

In the context of EFSA's open call for scientific data on furan in food and beverages 80 (EFSA 2006), we have analyzed a large collection of commercial jarred baby food 81 products from the German market and have evaluated the influence of domestic heating 82 procedures on the furan content of the jars. In addition, we have analyzed freshly 83 prepared baby foods prepared according to typical baby food recipes using fresh fruits 84 and vegetables. Finally, we provide some insight into the pathway of formation of furan 85 in baby foods. 86 87

Materials and Methods

88

Quantitative determination of furan 107

For the survey, a headspace-gas chromatography-mass spectrometry (HS-GC/MS) 108 method with deuterated furan as internal standard and standard addition for 109 quantification was used, which was based on a FDA method (FDA, 2004). The HS-110 GC/MS method was previously described in detail [START_REF] Kuballa | Furan concentrations in coffee and coffee 488 beverages[END_REF][START_REF] Kuballa | Furan in coffee and other foods[END_REF]. 111 112The HS-GC/MS method was validated for the determination of baby foods and 113 accredited to ISO 17025. The precision of the method never exceeded 6.4% (relative 114 standard deviation, n=5) and the average recovery was 105% (n=8). Determined 115 according to DIN 32645, the limit of detection (LOD) was 0.20 µg kg -1 and the limit of 116 quantification (LOQ) was 0.60 µg kg -1 . 117 118 Because of the extremely high time effort of the HS-GC/MS method due to standard 119 addition, the home preparation and formation experiments were evaluated using purge-120 and-trap-GC/MS procedure. Deuterated furan was used as internal standard, but the 121 quantification was conducted using a more efficient external calibration. 122 The purge-and-trap system used for analysis was an Eclipse 4660 Sample Concentrator in combination with a model 4552 water/soil autosampler (OI 125 Analytical, College Station, TX, USA). The trap was #10 (8 cm, tenax, silica gel, carbon 126 molecular sieve) provided by the manufacturer. The sample temperature was 40°C. The 127 sample was purged for 5 min. The desorption was conducted for 4 min at 190°C, 128 followed by a bake time of 20 min at 210°C to avoid carry over. 129 130

The purge-and-trap system was combined with an Agilent model 6890 Series Plus gas 131 chromatograph and an Agilent 5973N mass selective detector (Agilent,Waldbronn,132 Germany). Data acquisition and analysis were performed using standard software 133 supplied by the manufacturer. Substances were separated on a fused silica capillary 134 column (HP Plot Q, 30 m x 0.32 mm I.D., film thickness 0.25 µm). Temperature 135 program: 90°C hold for 1 min, 10°C min -1 up to 225°C, hold for 15 min. The 136 temperatures for the ion source, quadrupole and interface were set at 230°C, 150°C, and 137 225°C, respectively. Helium with a constant flow rate of 1.5 ml min -1 as carrier gas was 138 used. For quantification, the mass fragments m/z 68 and m/z 72 were used for furan and 139 furan-d 4 , respectively. 140 141

The purge-and-trap method was validated similar to the HS-GC/MS method. The 142 precision of the purge-and-trap method never exceeded 3.7% (relative standard 143 deviation, n=8) and the average recovery was 106 % (n=8). The LOD was 0.15 µg kg -1 144 and the LOQ was 0.44 µg kg -1 . No artifactual furan formation was observed during the 145

Experiments for home preparation of baby foods 149

The home preparation experiments were aimed to simulate typical home cooking as 150 closely as possible. We used only standard kitchen equipment, e.g. a kitchen stove and a 151 household blender. The recipes were taken from a cookbook intended for parents 152 preparing foods for babies between five and 10 months of age, i.e. the time period of 153 adjustment from breast feeding to normal solid foods [START_REF] Von Mandelsloh | Babybreie selbst gemacht[END_REF]. These 154 foods are generally prepared as a puree (so-called 'Babybrei' in German) using hand or 155 table-top blenders to achieve a consistency that allows easy ingestion by the babies. In 156 Germany, potatoes are typical ingredients, alone or in combination with carrots and 157 other vegetables or meat. In general, we cooked the ingredients in boiling water until 158 done, then the ingredients were blended, with addition of water to adjust the consistency 159 to correspond with that of typical commercial baby foods. No other additives were used. 160

The food groups under investigation are shown in Table 1. Besides the direct analysis of 161 the freshly prepared food, we have stored the foods overnight at 8°C, followed by a re-162 heating in closed vessels for 1 h in a water bath at 70°C. Finally, we have added 2-furan 163 carboxylic acid (80 mg kg -1 , purity 98%) and re-heated similarly. 164 165 As it became evident in the first experiment that potatoes, in particular, appeared to 166 have the capacity to form furan, we conducted a second experiment with potato purees 167 specifically. Potatoes of different origins and agricultural production methods were used 168 2). In this experiment, we also performed additional tests involving the addition 169 of ascorbic acid (500 mg kg -1 ) to the ingredients. 170 171

Preparation experiments involving commercial baby foods 172

To assess possible changes in furan content during normal home preparation of 173 commercial baby foods, we chose 10 products of which we had more than one jar of the 174 same production lot, and analyzed them as opened, as well as following heating with a 175 baby food warmer. The warmer was a PTC baby food warmer type 702 by Reer-176 Kindersicherheit, Leonberg, Germany. The baby food jar is placed into the device, 177 which is then filled with water, so that the jar is indirectly heated by the water. To assess 178 the maximal furan formation capacity, we have used extreme conditions for the heating, 179 including a maximum temperature setting of the device (80°C water temperature, own 180 measurement), a 1 h heating time, and closed jars. In two cases, we had three jars of one 181 lot, so we were also able to assess the effect of heating in an open jar, with regular 182 stirring during the heating process. 183 184

Experiment on influences on furan formation in potato puree baby foods 185

In order to evaluate possible furan formation mechanisms in potato puree baby foods, 186 with a minimum number of experiments, the information gleaned from each experiment 187 and the relationships between the experiments have to be fully exploited. This was done 188 using a D-optimal design type [START_REF] Box | 439 Codex alimentarius. Codex general standard for contaminants and toxins in foods 440 (CODEX[END_REF][START_REF] Montgomery | Design and Analysis of Experiments[END_REF]). The D-optimal 189 algorithm was used because it chooses an ideal subset of all possible combinations and 190 significantly reduces the number of required experiments when compared to standard 191 design types. 192 The potato puree used in this experiment was freshly prepared from organic potatoes 194 (Nicola from Egypt) using the same methods described above for the home preparation 195 of baby foods. The preparations were then re-heated using the following conditions. The 196 heating temperature and heating time were studied at two levels (50°C, 100°C; 1 h, 2 h) 197 whereas the ascorbic acid concentration was assessed as numeric factor between 0 and 198 1000 mg kg -1 . 199 200 Approach for risk assessment 201 Risk assessment was conducted according to the harmonised approach of the European 202 Food Safety Authority (EFSA) for the risk assessment of substances which are 203 genotoxic and carcinogenic [START_REF] Efsa | Opinion of the Scientific Committee on a request from EFSA related to a 454 harmonised approach for risk assessment of substances which are both genotoxic and 455 carcinogenic[END_REF]. The EFSA has developed and recommends 204 an approach known as the margin of exposure (MOE). This approach uses the value of 205 doses of substances that have been observed to cause low but measurably harmful 206 responses in animals as a reference point, which is then compared with relevant 207 substance-specific dietary intake estimates in humans, taking into account differences in 208 consumption patterns. For this case of commercial ready-to-eat baby foods, we used the 209 consumption data provided by [START_REF] Kersting | Measured 483 consumption of commercial infant food products in German infants: results from the 484 DONALD study[END_REF].

Results and Discussion

245

Furan in freshly homeprepared baby foods 246

A major finding of our study is the fact that self-prepared baby food does not contain 247 furan. Furan was below the limit of detection in all of the ten different foods prepared 248 (Table 1). Our results are in agreement with those of [START_REF] Bianchi | Development and validation of a solid 435 phase micro-extraction-gas chromatography-mass spectrometry method for the 436 determination of furan in baby-food[END_REF] who detected 249 none or only very low concentrations in seven home-made baby foods. We think that the 250 available literature now conclusively supports the assumption of a very low risk of furan 251 in home prepared baby foods. 252 253 In our study, only after storage and re-heating was furan detectable, in three cases in 254 concentrations between 4.5 and 10.0 µg kg -1 (Table 1). However, it must be stressed that 255 our re-heating conditions (70°C, 1 h, closed vessel) were extreme compared to normal 256 household conditions. Furan was formed in five cases, at concentrations between 1.8 257 and 51.8 µg kg -1 , after the addition of 2-furan carboxylic acid, a possible precursor of 258 furan [START_REF] Yaylayan | Precursors, Formation and Determination of Furan in Food[END_REF][START_REF] Limacher | Formation of furan and 490 methylfuran from ascorbic acid in model systems and food[END_REF]). As the formation capability appears to 259 have been highest in samples containing potatoes, we have studied potato baby foods in 260 more detail (Table 2). Again, furan was below the limit of detection in all freshly 261 prepared samples. After re-heating, amounts between 2.3 and 29.2 µg kg -1 were formed. 262 No significant differences could be detected between organically and conventionally 263 produced potatoes (p=0.1306) or between potatoes with origins in Germany and Egypt 264 (p=0.8600). If ascorbic acid was added to the potato baby foods, the furan content 265 increased in every case, ranging between 8.3 and 58.3 µg kg -1 . The furan contents of the 266 re-heated samples before and after ascorbic acid addition show a significant linear 267 been formed in every sample, had ascorbic acid been the only factor involved. This 270 finding is in agreement with [START_REF] Limacher | Formation of furan and 490 methylfuran from ascorbic acid in model systems and food[END_REF], who also suggested that other 271 compounds must be functioning as precursors and who deduced that transformation of 272 these into furan is being accelerated by the presence of ascorbic acid. 273 274 A more detailed insight into the furan formation in potato baby foods is provided by our 275 formation experiment (Figure 1). The ANOVA for the response surface two factor 276 interaction model implies that the model is highly significant (p<0.0001). Significant 277 influences on furan formation were temperature (p<0.0001), ascorbic acid content 278 (p<0.0001) and heating time (p=0.0093). Significant interactions occurred between 279 ascorbic acid and temperature (p<0.0001) and between temperature and heating time 280 (p=0.0212). Figure 1 shows that, at 50°C, the furan content stays almost constant and 281 there are no differences between 1 and 2 h heating nor between the different ascorbic 282 acid additions. The picture changes at 100°C, when furan shows a linear increase with 283 rising ascorbic acid concentrations, and higher concentrations were found at 2 hours of 284 or act itself as a precursor. We can only stress the warning of Limacher et al. (2007) 292 who concluded that it is not recommended to fortify canned and jarred food products 293 with vitamin C before thermal treatment. 294 295

Behaviour of furan during heating of commercial baby food jars 296

The heating of commercial baby food jars in a typical baby food warmer showed no 297 consistent trend (Table 3). Only two products containing potatoes (potato-carrot puree, 298 cream potatoes) again showed increases in furan, at 7.4 and 6.1 µg kg -1 . The eight other 299 products showed no significant increase or decrease due to heating, but results were 300 identical within the method uncertainty. Heating of opened jars with stirring also 301 showed no consistent trend. In the case of a potato-carrot-puree, a slight decrease of 4.9 302 µg kg -1 was detectable, and in the case of a vegetable puree a slight, but not significant, 303 increase of 0.7 µg kg -1 was noted. Further research is required to evaluate the influence 304 of stirring and other influences during heating, with the aim of determining the best 305 methodology for reducing furan during household preparation of baby foods. 306 307 There is still an ongoing discussion concerning to what extent consumer cooking may 308 influence the furan levels in foods. Some researchers have found that furan persists 309 during the normal heating practices that precede consumption [START_REF] Hasnip | Some factors affecting the formation of furan in 473 heated foods[END_REF], 310 whereas other studies reported losses of 29-55% in vegetable purees during different 311 warming procedures in microwave ovens [START_REF] Zoller | Furan in food: Headspace method and product 529 survey[END_REF]). Losses of up to 85% were 312 reported during heating of opened jars over a period of 5.5 h in boiling water, and a 313 reduction of ca. 50% if the baby food jar was opened but not heated (Goldmann et al. 314 ). However, such significant furan losses were not evident at more moderate 315 heating or storage conditions. 316

Our results are in agreement with those of Crews et al. (2007a), which indicate that the 317 removal of furan is not as easy as has been sometimes assumed on the basis of its 318 volatility. Furan appears to be well dissolved within the matrix of the baby food and 319 opening of the jars exposes only a relatively small surface area. Therefore, despite its 320 volatility, the evaporation of furan appears to be hindered due to its apparently slow 321 diffusion inside the food matrix. The same observations were made by Roberts et al. 322 (2008), who demonstrated larger declines of furan in baby foods heated in a saucepan 323 under stirring. A preliminary recommendation could therefore be to transfer the baby 324 foods into a larger pan or bowl to increase the surface area for furan evaporation. 325 However, due to the unknown conditions of household preparation, we currently cannot 326 assume a significant reduction of risk due to evaporation. We agree with Crews et al. 327 (2007a) that these initial thoughts proved to be naïve, and our results show, in 328 accordance with [START_REF] Hasnip | Some factors affecting the formation of furan in 473 heated foods[END_REF], that there is even the possibility of additional 329 formation, which could balance the potential evaporative losses. 330 331

Survey of commercial ready-to-eat baby foods 332

Table 4 shows the results of our survey of 230 jarred baby foods, which is so far the 333 largest overview of furan in this food group in Germany. We have classified the baby 334 foods into five broad groups and ANOVA shows that the groups have highly significant 335 differences in furan content (p<0.0001), as shown in the distribution patterns in 336 Figure 2. The highest average content was found in pasta products (i.e. spaghetti or 337 other pasta meals), followed by vegetable meals and meals with meat. Lower 338 Preparations containing only fruits had the lowest furan content and the majority of 340 samples in this group were below the detection limit. Our results correspond well to the 341 ranges reported in previous surveys in Europe [START_REF] Heppner | Data requirements for risk assessment of furan in 475 food[END_REF], Japan 342 [START_REF] Yoshida | Rapid and 526 improved determination of furan in baby foods and infant formulas by headspace 527 GC/MS[END_REF], Switzerland [START_REF] Zoller | Furan in food: Headspace method and product 529 survey[END_REF], and the USA (Morehouse et al. 343 2008). 344

As the classification of the foods into the five broad groups only allows limited 345 conclusions regarding the ingredients attributable to furan formation (e.g. most meals 346 with meat also contain vegetables and potatoes), we have also separately evaluated the 347 ingredients lists of all products as to whether they contain fruits, cereals, carrots, 348 potatoes, meat or other vegetables. The PLS correlation loadings plot in Figure 3 shows 349 that furan is closely correlated with the presence of carrots, potatoes, meat and other 350 vegetables, while cereals and fruits appear to have no such influence. As our PLS 351 evaluation is based only on qualitative data (we had no information regarding the 352 quantity of each ingredient in each jar), this result can only be interpreted as 353 preliminary, but it does allow the setting of a research focus and of possible preventive 354 measures on foods containing these suspect ingredients. Currently, we cannot point 355 specifically to other vegetables besides potatoes and possibly carrots, because the 356 sample size for each vegetable was too low to acquire statistically significant 357 information. This type of analysis would also be confounded by the multi-component 358 vegetable mixtures that occur in commercial baby foods. Further research, with the aim 359 of studying each vegetable type separately, possibly in a realistic pilot plant setting, 360 would be required to identify vegetable groups that may be especially prone to furan 361 formation. 362

Preliminary risk assessment of furan in baby foods 364

The EFSA (2005) recommends that risk assessments provide different exposure 365 scenarios (e.g. for entire, or specific groups of populations) along with their inherent 366 uncertainties. Other than the mean and median, intakes from highly exposed individuals 367 (due to high consumption or to average consumption of highly contaminated foods) 368 should be considered as represented by the 90 th , 95 th , 97.5 th and 99 th percentiles. 369

The intake of commercial food products intended for infants in Germany is available 370 from the DONALD (Dortmund Nutritional and Anthropometrical Longitudinally 371 Designed) study [START_REF] Kersting | Measured 483 consumption of commercial infant food products in German infants: results from the 484 DONALD study[END_REF]. The DONALD study also provided separate data 372 for ready-to-eat or -drink baby foods (without consideration of infant formulas, follow-373 on formulas and dry cereal-based foods), so we can directly take the DONALD intake 374 data to evaluate our commercial ready-to-eat baby foods. We selected four different 375 exposure scenarios (3 months, 6 months, 9 months and 12 months). Because of their 376 lower body weights, we chose the data for girls from the DONALD study. The four 377 scenarios, corresponding body weights, and intake of ready-to-eat baby foods are shown 378 in Table 5. This table also shows the calculated exposures to furan for mean and median 379 concentrations and the percentiles. The statistical data for this exposure evaluation were 380 calculated from all 230 commercial baby foods investigated in our study. 381

382

The exposure data from Table 5 were then used to characterize risk using the margin of 383 exposure (MOE) calculated from T25 (table 6). MOEs can be used by risk managers for 384 setting priority, with a small MOE representing a higher risk than a larger one. 385 In our case of furan in baby foods, the MOEs were below 10,000 in all cases, a finding 386 which signifies a potential public health concern for this contaminant [START_REF] Efsa | Opinion of the Scientific Committee on a request from EFSA related to a 454 harmonised approach for risk assessment of substances which are both genotoxic and 455 carcinogenic[END_REF]. 387

These values would still be below this threshold even if we were to assume a 30% 388 evaporation during food preparation. In addition, an additional safety factor of 10 for 389 exposures to children between 0 and 2 years of age could be required (Barton et al. 390 2005) because the MOE threshold of 10,000 was deduced for adults. 391 392 Uncertainties in the evaluation include the T25 as reference point, as it is calculated 393 from only one data point in an animal experiment, and there is no clear information of 394 the raw data (i.e. study type, animal species, exposure route, endpoint) from which the 395 T25 of furan was derived in the [START_REF] Sanner | A simple method for quantitative 511 risk assessment of non-threshold carcinogens based on the dose descriptor T25[END_REF] study. Nonetheless, we believe that 396 our preliminary MOEs more than justify priority research with the aim of finding 397 measures to reduce furan in baby foods. This would be required anyway, according to 398 the ALARA-principle ("As low as reasonably achievable"), introduced into the Codex 399

Alimentarius standards for dealing with contaminants and toxins (Codex alimentarius 400 1997) and the corresponding EU legislation (Council of the European Communities 401 1993). The European baby food directive also demands that baby foods shall not contain 402 any substance in such quantity as to endanger the health of infants and young children 403 (European Commission 2006). As the EFSA is still in the process of toxicological 404 evaluation of furan, we cannot finally decide at the present time if the levels detected in 405 our baby foods endanger the health of the children. In our view, baby foods from other 406 European countries should be included as priority food group in the data collection of 407 EFSA mentioned in the introduction, in order to provide exposure data for a final risk 408 assessment of foods available to this vulnerable consumer group. 409 = T25 / Exposure). Calculated with T25 of 1.4 mg kg bw -1 day -1 from Sanner et al. 24 (2001)

  commercial ready-to-eat baby foods included 230 products sampled and 90 analyzed between 2004 and 2007. As part of official food control in the German federal 91 state of Baden-Württemberg, our institute covers the district of Karlsruhe in North 92 Baden (Germany) with a population of approximately 2.7 million. The sampling was 93 carried out by local authorities directly at the food producers or at retail trade 94 establishments including pharmacies and organic food stores. The samples were 95 randomly selected and collected by government food inspectors. Only products intended 96 for use as foods specifically for infants and young children, in the sense of Commission 97 Directive 2006/125/EC, were included in the survey (European Commission 2006)home preparation of baby foods were purchased in May and June 100 2008 at local farmer's markets and organic food stores. In the initial experiment 101 concerning different product groups, only ingredients classifiable as organic according 102 to Council Regulation (EEC) No 2092/91 were chosen (European Council 2001). The 103 second experiment, which focused on potato-based baby foods, was conducted with 104 organic as well as conventionally produced ingredients. 105 106

FoodFoodFood

  210 211To obtain the MOE, the Benchmark Dose Lower Confidence Limit (BMDL) of 10% 212 was suggested. In cases where the data are unsuitable for obtaining a BMDL, the EFSA 213 recommends the use of the T25 calculation, wherein the dose corresponding to a 25% 214 incidence of tumours, after correction for spontaneous incidence, is represented. Since 215 the dose-response modelling of a BMDL would have gone beyond the scope of this 216Page 10 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk therefore chose the T25 of 1.4 mg kg bw -1 day -1 , as tabulated bySanner et al. 217 (2001), to provide a preliminary risk assessment. 218219Statistics 220The experimental designs and calculations were done using the Software Package 221 Design Expert v7 (Stat-Ease Inc., Minneapolis, MN, USA). The experiments were 222 evaluated using Analysis of Variance (ANOVA) to find the significance of variables and 223 their interactions in the models. The models were checked for consistency by looking at 224 the lack of fit and possible outliers. 225The survey data were evaluated using Origin Pro v7.5(OriginLab Corporation, 226 Northampton, MA, USA). Statistical significance was assumed at below the 0.05 227 probability level. Groups of two cases were compared using t-tests. One-way ANOVA 228 was used to test whether three or more cases had the same mean, including the 229 Bonferroni post hoc means comparison. Pearson's test was used to evaluate the 230 significance of linear relations. Box and whisker plots were used for visualization of 231 data (box 25 th -75 th percentile, line in the box: median, whiskers: minimum and 232 maximum (max. 1.5 times the length of the inner quartiles), data points outside are 233 outliers). 234235To assess the influence of ingredients in commercial jarred baby foods on furan content, 236 the 230 products in the survey were categorized according to their ingredients lists as to 237 whether they contained potatoes, carrots, other vegetables, meats, fruits or cereals. A 238 data matrix was compiled containing the binary classification (0 if the ingredient was 239 not contained, and 1 if it was contained) as well as the furan content of the product. A 240Page 11 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk squares (PLS) analysis was conducted using the binary classifications as x-241 variables and the furan content as y-variable, with the aim of visualizing correlation 242 between the variables. A multivariate PLS analysis was conducted using the software 243 Unscrambler v9.7 (CAMO Process AS, Oslo, Norway). 244Page 12 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  .7+1.8x; p=0.0001), which proves that other factors besides ascorbic acid 268 are involved in the formation mechanism. Otherwise, the same amount should have 269

Page

  our results that furan content in freshly home-cooked baby foods is 287 not an issue. The problem may only arise if self-prepared foods are stored and then re-288 heated. Especially when they contain potatoes, baby foods should only be mildly 289 reheated to avoid furan formation. The experiments also show that ascorbic acid, a 290 common ingredient and additive in foods, has the potential to increase furan formation 291

  in beverages and in meals that were comprised of cereals. 339

Figure 1 .

 1 Figure captions

Food

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

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	146	purge-and-trap step, as e.g. proven by results below LOD in unheated samples
	147	containing furan precursors.
	148
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y

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	537 538	F o r	).
	539 540	Figure 3. PLS correlation loadings plot of a dataset of 230 commercial baby food products analyzed for furan content. Furan is predominantly found in products P e
	541	containing carrots, potatoes, other vegetables or meat. e r
		R
		e
		v i e
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			O n l
			y
		1

Table 1 .

 1 Furan in freshly homeprepared vegetable, cereal, and fruit-based baby foods 1

	2	(all ingredients from organic production).
				Furan [µg kg -1 ]
		Product	Freshly prepared,	Re-heated (1 h,	Re-heated (1 h,
			directly analysed	70°C) in closed jar	70°C) in closed jar
				after storage over	after storage over
				night at 8°C	night at 8°C and
					addition of 2-furan
					carboxylic acid (60
					mg kg -1 )
		Potato Puree	n.d.	10.0	51.8
		Carrot Puree	n.d.	n.d.	n.d.
		Potato-Carrot Puree	n.d.	6.7	3.3
		Spinach Puree	n.d.	n.d.	n.d.
		Broccoli Puree	n.d.	4.5	2.5
		Spaghetti, mashed	n.d.	n.d.	2.7
		Semolina-milk pudding	n.d.	n.d.	n.d.
		Rice porridge	n.d.	n.d.	n.d.
		Apple puree	n.d.	n.d.	1.8
		Chicken puree	n.d.	n.d.	n.d.
	3	n.d. not detectable (< 0.15 µg kg -1 )

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	F o r
	P
	e
	e r
	R
	e
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	y
	2

Table 2 .

 2 Furan in freshly homeprepared potato puree baby foods.

	4
						Furan [µg kg -1 ]
		Potato	Potato	Method of	Freshly	Re-heated (1	Re-heated (1
		variety	origin	agricultural	prepared,	h, 70°C) in	h, 70°C) in
				production *	directly	closed jar	closed jar after
					analysed	after storage	storage over
						over night at	night at 8°C
						8°C	and addition
							of ascorbic
							acid (500 mg
							kg -1 )
		Rosetta	Egypt	organic	n.d.	4.1	11.6
		Nicola	Italy	organic	n.d.	6.0	19.7
		Nicola	Egypt	organic	n.d.	6.2	32.1
		Astoria	Germany organic	n.d.	8.6	15.8
		Nicola	Egypt	organic	n.d.	10.1	17.3
		Christa	Germany organic	n.d.	11.9	21.7
		Gloria	Germany organic	n.d.	16.1	40.0
		Nicola	Egypt	organic	n.d.	29.2	58.3
		Nicola	Egypt	conventional	n.d.	2.3	10.6
		Princess	Egypt	conventional	n.d.	2.8	8.3
		Princess	Germany conventional	n.d.	4.6	14.2
		Berber	Germany conventional	n.d.	6.2	19.3
		Marabel	Israel	conventional	n.d.	7.0	16.4
		Atlas	Egypt	conventional	n.d.	7.2	19.3
		Annabelle	Cyprus	conventional	n.d.	7.6	15.9
		Monalisa	Germany conventional	n.d.	7.9	15.1
		Berber	Germany conventional	n.d.	16.8	37.2
		Convenience	(unknown) conventional	n.d.	6.5	18.4
		pre-boiled
		potatoes
	5	* Accordingt to label. Organic production was assumed when labelling requirements
	6	were in accordance with Council Regulation (EEC) No 2092/91 (European Council
	7	2001). Conventional production was assumed when no information on production
	8	method was provided on the label.

Table 3 .

 3 Furan in commercial baby food jars analyzed without preparation and heating 9

	10	in a typical baby food warmer.
				Furan [µg kg -1 ]
		Product	unprepared	Heated with	Heated with
				baby food	baby food
				warmer,	warmer,
				closed (1 h,	opened jar,
				80°C)	periodical
					stirring
		Potato-Carrot Puree	39.6	47.0	34.7
		Mixed Vegetable Puree	66.8	67.0	67.5
		Vegetables with	28.7	29.7	-*
		chicken and pasta
		Muesli	10.0	8.5	-
		Cream potatoes with	73.5	79.6	-
		cauliflower and veal
		Rigatoni Napoli	92.0	89.5	-
		Vegetables with beef	25.4	27.6	-
		Spaghetti Bolognese	44.0	45.9	-
		Mixed vegetables with	27.5	28.5	-
		chicken
		Potato-vegetables with	47.5	48.0	-
		pork
	11	* experiment not conducted
	12

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Table 4 .

 4 Incidence of occurrence of furan in commercial ready-to-eat babyfoods 13(analyzed as bought without prior heating) 14

	Sample	Numbers of samples in the range [µg kg -1 ]			Furan concentrations [µg kg -1 ] a
	matrix
		< 2 2-5 5-10 10-20 20-30 30-40 40-50 >50 Mean ± SD Median 95 th
											percentile
	Baby	7	4	1	0	2	1	1	0	9.6 ± 14.8	2	45
	beverages
	Fruit-only	30	3	15	1	0	0	0	1	3.5 ± 8.4	0.2	9
	dishes
	Meals	5	9	5	4	1	1	0	0	6.6 ± 7.2	4	20
	with
	cereals
	Pasta	0	0	0	1	3	4	1	2 34.8 ± 14.5	35	63
	Meals	0	0	2	15	21	8	6	5 28.2 ± 15.0	26	59
	with meat
	Vegetable	3	0	1	13	17	18	8	11 31.2 ± 17.3	30	63
	meals
	(without
	meat)

a Samples below LOD were calculated as zero 15

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Table 5 .

 5 Exposure with furan from commercial ready-to-eat baby foods. The exposure 16 scenarios were derived using the mean, median and percentiles of the furan 17 concentrations in all ready-to-eat baby foods and beverages in our survey (n=230) 18 Data from[START_REF] Kersting | Measured 483 consumption of commercial infant food products in German infants: results from the 484 DONALD study[END_REF]. Only ready-to-eat baby foods and beverages were 19 included in the consumption estimate (dry infant formulae and dry processed cereal-20 based foods were not included in this evaluation) 21 22

	Intake scenarios a		Exposure scenarios for different furan concentrations in the foods
				[µg kg bw -1 day -1 ]
	Age (months)	Body weight (kg)	Intake of baby foods (g day -1 )	Mean (20 µg kg -1 )	Median (18 µg kg -1 )	90th percentile (44 µg kg -1 )	95th percentile (54 µg kg -1 )	99th percentile (68 µg kg -1 )
	3	5.8	67	0.2	0.2	0.5	0.6	0.8
	6	7.5	195	0.5	0.5	1.1	1.4	1.8
	9	8.6	234	0.5	0.5	1.2	1.5	1.9
	12	9.4	208	0.4	0.4	1.0	1.2	1.5

a

Table 6 .

 6 Margin of Exposure (MOE) for furan in the different exposure scenarios (MOE 23

Acknowledgments 424 H. Mann is thanked for excellent technical assistance. 425 426