S C Duarte

J M V Bento

A Pena

C M Lino

Estimation of ochratoxin A exposure of Lisbon inhabitants through analysis of bread and urine in winter 2007/2008

Keywords: Mycotoxins -ochratoxin Ochratoxin A, bread, urine, LC-FD, Portugal, Lisbon

A survey on the occurrence of ochratoxin A (OTA) in 41 bread samples was carried out in the Portuguese capital. Maize (5) and wheat bread (36) and 43 representative urine samples from the Lisbon region were assayed for OTA content using immunoaffinity column cleanup (IAC) and HPLC with fluorimetric detection (LC-FD). The percentage of OTA-positive samples was slightly higher for maize bread (80%) than for wheat bread (70.8%), although, due to its higher consumption, the latter contributes more to OTA exposure, featuring a higher estimated daily intake (EDI). In the urine samples analyzed, both female and male residents displayed similar high levels of OTA frequency and average contamination. Therefore, OTA is a food contaminant of concern and may constitute a hazard for public health through consumption of cereal-based products.

Introduction

The attention of public health authorities and toxicologists in the world has recently been focused on systematic control of the presence of xenogenous substances in foodstuffs which might endanger the health of the population. This is the case of mycotoxins, toxic secondary metabolites of mycelial microscopic fungi, moulds [START_REF] Malir | Ochratoxin A in the Czech Republic[END_REF]. The mycotoxin OTA is an example. Since its discovery in 1965, several authors and projects were devoted to its study. It is currently regarded as nephrotoxic, immunotoxic, and genotoxic [START_REF] Schilter | Ochratoxin A: Potential epigenetic mechanisms of toxicity and Carcinogenicity[END_REF]. Additionally, since 1993, it is included in the group 2B (possible human carcinogens) by the International Agency for Research on Cancer (IARC) of WHO, based on sufficient evidence for carcinogenicity in animal studies and inadequate evidence in humans. In several areas of Eastern Europe, where chronic exposure to OTA occurs, involvement of this mycotoxin in the cancer etiology of the urinary system, and in kidney pathologies typical of Balkan endemic nephropathy (BEN) has been suspected. Studies on the correlation between OTA and BEN [START_REF] Vrabcheva | Co-occurrence of ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy[END_REF]Puntarić et al., 2001) have shown higher OTA contamination level in cereals from endemic areas with respect to cereals from non-endemic areas [START_REF] Palermo | Occurrence of ochratoxin A in cereals from Puglia (Italy)[END_REF]. Hence most countries and organizations have established maximum permitted levels in foodstuffs to reduce the exposure of its citizens to this contaminant. Additionally, tolerable intakes were recommended, specifically of 120ng/kg bw/week [START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. (Question n° EFSA-Q-2005-154[END_REF], and 100ng/kg bw/week (JEFCA, 2007). For their part, Canadian authorities proposed a TDI of 1.2-5.7ng/kg bw/day [START_REF] Kuiper-Goodman | Risk assessment of ochratoxin A: an update[END_REF]. The Nordic Working Group on Food Toxicology and Risk Evaluation (1991) has suggested a maximum TDI of OTA by humans of 5ng/kg body weight, based on carcinogenicity studies with OTA in adult rats. The Scientific Committee on Food also proposed that exposure should be below 5ng/kg bw/day (SCF, 1998).

The importance of OTA is further underlined by its global occurrence, in a large variety of commodities, of both plant and animal origin. Cereals and their derived products, assumed to be the major dietary source of OTA, are some of the most consumed foodstuffs in the world. Of these, bread is the most popular yeast-leavened cereal product by far provides more nourishment for humans than any other food source (FAO, 2002). Even today, the importance of bread is stated by its symbolic power (solidarity), its existence in every civilization, and its major role in many religions, in many popular festivities and in most family meals worldwide (www.world-bread-day.com, 2008). This universal product can be made of different types of cereal grain, characterized by the many fermentation processes and the different ways of baking, a wide variety of sizes, shapes, textures and tastes dependent on the agricultural, cultural and feeding habits of the populations [START_REF] González-Osnaya | Dietary intake of ochratoxin A from conventional and organic bread[END_REF], www.world-bread-day.com, 2008). By far the most consumed type is wheat bread. In Portugal, the latest estimate of the average bread consumption is from 1994, at a level of 32 kg/person/year. Maize bread, a traditional Portuguese type of bread, is estimated to represent a quarter of the total nationwide bread consumption (Instituto Português do Consumidor, 2005). The famous and traditional 'Mafra' bread is a regional wheat bread type, commercialized in different shapes and sizes, but always with the same dough, which is made of wheat flour, water, yeast and salt. Although typical from a certain region of Lisbon, most of the shops, bakeries and supermarkets of the capital feature this kind of bread.

To study the OTA exposure, different approaches have been attempted. OTA levels in plasma and urine are considered as typical biomarkers of exposure, and no other appropriate effect-related biomarkers have as yet been identified [START_REF] Pacin | Survey of Argentinean human plasma for ochratoxin A[END_REF]. Although elimination in urine has been considered the main route of OTA excretion from the human body, the urinary monitoring is relatively unexplored due to the lower concentrations involved [START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF] and due to the fact that, conversely to its content in plasma, it is not possible to relate concentrations or total amount of OTA excreted in urine to exposure.

Recent developments in analytical methodology resulted in a more practical urinary monitoring, which has the advantage of being less invasive than blood monitoring. However, data on the frequency and concentration of OTA in human urine remain scarce [START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF]. In a one-month duplicate diet study in the UK OTA in urine was found to be a better indicator of OTA consumption than OTA in plasma, although levels in urine were two orders of magnitude lower; there was no good correlation between plasma concentrations and consumption of OTA [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF]. It is thus suggested that urine could provide a more useful marker of OTA intake than plasma [START_REF] Pascale | Rapid method for the determination of ochratoxin A in urine by immunoaffinity column clean-up and high-performance liquid chromatography[END_REF].

When compared to food products, urine has an additional advantage as a target for OTA exposure quantification. The large variability in contamination levels and sampling difficulties means that large numbers of individual food samples need to be analyzed.

Estimates of exposure based on determining OTA in food products will thus inevitably be vulnerable to large sources of error. Further, the fact that estimated exposure levels use methods based on food intake/analysis, which are inherently inaccurate, appears to indicate that exposures close to the TDI require a better approach for assessing OTA exposure. Initial studies have used plasma levels to estimate intake, but the study of [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF] suggests that this may not be the most suitable method. A disadvantage of urine analysis to assess OTA exposure is the need to define further the relationship between consumption and excretion to estimate actual intake levels. So, further work needs to be done to verify if urine concentrations are a short-or a long-term indicator of exposure to this mycotoxin [START_REF] Manique | Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations[END_REF]. According to [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF], the correlation might be stronger if comparisons were made between urine excretion and previous day intake. Moreover, Lisbonians' exposure to OTA was evaluated, based on bread intake and the toxin's presence in urine.

Materials and Methods

Bread and urine sampling

The present study was carried out with samples collected in the Portuguese capital city, Lisbon, located in the country's central western coast. The samples were collected during the Winter of 2007/2008, between November 2007and March 2008. The bread samples, consisting of 24 units of regular wheat bread, 12 units of regional Mafra wheat bread, and 5 units of maize bread, were commercially acquired from random bakeries and supermarkets located in the Lisbon region. After purchase, the samples were brought to the laboratory under ambient condition for milling. All of the information on the samples was obtained from the respective labels. Each milled sample, stored in sealed plastic bags, was frozen at -20ºC, and kept in this condition until analysis.

The 43 urine samples were collected from healthy individuals residing in the same area where the bread samples were commercialized. From the 43 local participants involved, 48.8% (n=21) were female, aged 18-69 years old, and 51.2% (n=22) were male, aged 23-75 years old. The sampled population is characterized in Table I.

Reagents

HPLC grade acetonitrile (Carlo Erba, Milan, Italy), methanol (Panreac Química Sau, Barcelona, Spain), toluene (Barker Analyzed, J. T. Barker, Holland) and benzene (HACH Company, USA) were used. Of analytical grade were used boron-trifluoride-methanol 14% with ≥98% purity. The OTA standard stock solution was prepared by diluting 1mg of OTA from A. ochraceus in 4mL of toluene:acetic acid (99:1) at 250µg/mL, and stored at -20ºC. The intermediate solutions were prepared at 10µg/mL and 1µg/mL, in toluene:acetic acid, and working standard solutions at 0.1µg/mL and 0.01µg/mL, in mobile phase. The calibration curve standard solutions were prepared in mobile phase, at concentrations between 0.001 and 0.01µg/mL.

Water was obtained daily from a Milli-Q System (Millipore, Bedford, MA, USA).

Equipment

Experimental Procedure

The methodologies used for OTA content determination were based on those described by [START_REF] Juan | Determination of ochratoxin A in maize bread samples by LC with fluorescence detection[END_REF] and [START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF], for bread and urine samples, respectively. were added to 20g of milled bread and then homogenized (5min) and filtered. Twenty milliliters of the resulting filtered solution were diluted in 30mL of PBS before the passage through the IAC at a vacuum-induced rate of 1 drop per second. The IAC was then washed with 10mL of water prior to the elution with 3mL of methanol.

For extraction from the urine matrix, 10mL of the sample were mixed with 10mL of NaHCO 3 , 5%, and then filtered. The filtrate was cleaned-up through the IAC at a flow rate of about 1 drop per second. The column was then washed twice with 5mL of distilled water, and afterwards OTA was eluted with 3mL methanol.

The eluates were dried in a bath at 50ºC under a gentle nitrogen flow, and the dried extract was stored at -20ºC. Before injection, the dried extract was dissolved in 250µL or 125µL of mobile phase, in the case of bread or urine samples, respectively. LC-FD analysis was performed using a vacuum-filtered solution of acetonitrile/water/acetic acid (49.5:49.5:1.0, v/v/v) as mobile phase, flowing at a 1mL/min rate. Wavelengths used were 333nm for excitation and 460nm for emission, both with a spectral bandwidth of 10nm. An OTA working standard solution of 0.01µg/mL was analyzed at the beginning of each run. A 20µL injection volume was used for both bread and urine extracts.

Analytical quality assurance

OTA method revalidation was performed by spiking an OTA-free wheat bread sample, in triplicate, during three days at fortification levels of 0.1, 0.5 and 2ng/g. After fortification, the sample was left to stand in the dark for 15min, after which the [START_REF] Juan | Determination of ochratoxin A in maize bread samples by LC with fluorescence detection[END_REF] protocol was followed.

The fortification assays performed on urine, in a triplicate of a blank sample and during three different days, were made at 0.02, 0.1 and 0.5ng/mL. After fortification the sample was left to stand in the dark for 15min, after which the [START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF] protocol was followed.

OTA presence in bread samples was confirmed as described by Guillamont et al. (2005) and Pena et al. (2005), by conversion into its methyl ester form, adding 150µL of boron trifluoride methanolic solution (BF 3 -CH 3 OH 14%) to the dried sample extracts, and evaporating the mixture at 60ºC for 10min, before reconstitution in 250µL of mobile phase.

For urine samples, a similar method was used as described by [START_REF] Castegnaro | High performance liquid chromatography determination of ochratoxin A and its 4R-4-hydroxy metabolite in human urine[END_REF], differing from the previous in its lower reconstitution volume of 150µL. The OTA methyl esters of both samples were analyzed according to the LC method described above.

RESULTS AND DISCUSSION

Analytical performance

Linearity for OTA quantification was verified by plotting OTA peak areas against calibration samples (concentrations of 1, 2, 5 and 10ng/mL). The resulting calibration curve featured a coefficient of correlation of 0.9998.

Recovery values for bread ranged from 76.7 to 103.7%, at fortification levels of 2.0 and 0.1ng/g, respectively, with intra-day repeatabilities ranging from 3.6 to 14.4%, for levels of 0.5 and 0.1ng/g, and inter-day ones from 7.2 to 12.9%, at the levels of 0.5 and 2.0ng/g. For urine, recovery values were from 90.6 to 96.0% at the 0.02 and 0.5ng/mL fortification levels. Intra-and inter-day repeatabilities varied between 2.9 and 5.4%, and 4.3 and 8.9% for fortification levels of 0.5 and 0.02ng/mL, respectively.

The above results for accuracy and precision, for both bread and urine, were in agreement with the EC directive Nº 401/2006 [START_REF] Ec | Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs[END_REF].

Determination of LOQs was performed by spiking blank samples with OTA standard solutions. Repeatable precision and trueness were achieved at minimum concentrations of 0.1ng/g and 0.008ng/mL for bread and urine samples, respectively, thus setting the LOQs at those values.

As shown by the chromatograms displayed in figure 1, there are no interfering peaks near the retention time of OTA (ca.11.30) in neither bread nor urine samples. The different retention time of OTA and its methyl ester and the almost complete disappearance of the OTA peak and the appearance of a new one, at 27.30min, permitted the confirmation of OTA presence in the sample (Figure 1).

Occurrence of OTA in bread

As shown in Table II, of the total 41 bread samples analyzed from Lisbon, 68.3% contained OTA, and 51.2% showed OTA levels above the LOQ.

Incidence was found to be high in both maize and wheat bread, and nearly all positive samples featured OTA content levels above the LOQ. In both instances, values were slightly higher, though not significantly so, in the maize bread. Similarly, average contamination 8 levels were also somewhat more elevated in maize bread than in common wheat bread (0.28 vs. 0.21ng/g). On the other hand, less than half of the Mafra bread samples contained detectable levels of OTA, and none of those featured OTA content quantifiable through this method.

Similar results were detected in studies concerning other Portuguese regions [START_REF] Juan | Determination of ochratoxin A in maize bread samples by LC with fluorescence detection[END_REF][START_REF] Bento | Determination of ochratoxin A content in wheat bread samples collected from the Algarve and Bragança regions, Portugal: Winter 2007[END_REF]. A notable exception is that of [START_REF] Juan | Levels of ochratoxin A in wheat and maize bread from the central zone of Portugal[END_REF], whose study found much lower incidence and contamination levels in wheat bread. A possible explanation for this discrepancy may lie in the fact that the samples for this study originated in a different area and period, and were thus subject to different conditions during processing and storage, as well as different edafo-climatic influences during crop growth.

Independently of study, however, data from across the world (Table III) puts Portuguese bread as the least contaminated. Since some of the data in that table refers to the cereal grain, it is possible that processing methods result in a similar OTA content in the bread of some of those countries -namely, Italy and Croatia.

Those same studies concur with the present one in that they have found that both maize grains and their derivates tend to feature higher frequencies of OTA contamination than their wheat counterparts. In the specific case of bread, [START_REF] Cengiz | Ochratoxin A Levels in Different Types of Bread and Flour[END_REF] have detected statistically significant differences between OTA content average levels in white and corn bread samples, corn and whole meal bread samples, and corn and corn whole meal samples, with the latter of each case featuring higher levels. Earlier, [START_REF] Zummo | Interaction of Fusarium moniliforme and Aspergillus flavus on Kernel infection and aflatoxin contamination in maize ears[END_REF] demonstrated that maize (Zea mays) has an ideal nutrient composition for fungi development and is thus constantly exposed to the risk of fungal contamination. Moreover, tropical and subtropical climate countries, a few of which are among the worldwide top producers of maize, have favorable environmental conditions to the development of the main types of genotoxicant fungi, Aspergillus, Fusarium and Penicillium [START_REF] Sekiyama | Aflatoxins, Ochratoxin A and Zearalenone in maize-based food products[END_REF]. However, studies of OTA quantification offer contradictory results on the subject.

A study from Brazil [START_REF] Sekiyama | Aflatoxins, Ochratoxin A and Zearalenone in maize-based food products[END_REF] found a single sample of corn flour (0.8%) with an OTA content of 64µg/kg, while another [START_REF] Machinski | Aflatoxins, ochratoxin A and zearalenone in Brazilian corn cultivars[END_REF] found two samples (1.8%) with contamination levels of 128 and 206µg/kg, values that exceed the maximum permitted legal limit. Yet another [START_REF] Caldas | Aflatoxinas e ocratoxina A em alimentos e riscos para a saúde humana[END_REF] found no samples with contamination levels above their limit of detection (25µg/kg).

In Argentina, however, a different study [START_REF] Paolo | Contaminacion de harina de maiz con ochratoxina A, aflatoxina y zearalenona[END_REF] found a high content of OTA (1250-2500µg/kg) in all the analyzed maize-derived foodstuffs, though non-processed The cited Brazilian papers results corroborate the low levels of OTA contamination in Brazilian maize. On this subject, it is worth noting that Argentina is significantly colder than Brazil, and though OTA-producing fungi favor warm and wet environments, mycotoxin production seems to be spurred by cold and wet climates [START_REF] Eskola | Study on Trichothecenes, Zearalenone and Ochratoxin A in Finnish Cereals: Occurrence and analytical techniques[END_REF]. Yet, the Ministry of Agriculture, Fishing and Food of the United Kingdom found that crude maize samples imported from Argentina -as well as from a number of European countries -featured contamination levels no higher than 1.5µg/kg, and only in 10.1% of the samples. It is, however, possible that produce destined to exportation is subjected to tighter control and the most contaminated samples are discarded. Conversely, other studies reveal higher mean levels of OTA in wheat, than in maize, irrespective of their ripening season, like in BEN endemic regions (Puntarić et al., 2001). This high contamination of wheat cereal can be explained by the fact that glutamic acid is incorporated into OTA during its production, and so a high content of this aminoacid in cereals, as typical in wheat, could be a cofactor for the presence of OTA, as suggested by [START_REF] González-Osnaya | Dietary intake of ochratoxin A from conventional and organic bread[END_REF]. Furthermore, and according to Laca et al., 2006, OTA content differences between cereals depend on multiple factors, such as the grain condition at harvest, care in grain drying, and storage conditions (Gonzalez-Osnaya et al., 2007).

Taking into account the latest data, which claims that a Portuguese citizen has an average adult bodyweight of 65kg [START_REF] Miraglia | Assessment of dietary intake of ochratoxin A by the population of EU member states[END_REF] and an average bread consumption of 32kg/year (Instituto Português do Consumidor, 2005), consisting one quarter maize bread and three quarters wheat bread, the estimated contributions to OTA daily intake are 0.09 and 0.21ng/kg bw/day for maize and wheat bread, respectively (Table IV).

It should be noted that, though maize bread tends to feature higher levels of contamination, the much higher consumption of wheat bread results in that the exposure of the Lisbonian residents to OTA is higher through the consumption of the latter, 0.21ng/kg bw/day vs. the 0.09ng/kg bw/day for maize bread, although far from the values established by the SCF in 1998 (5ng/kg bw/day) and the JECFA in 2001 (17ng/kg bw/day). These values are lower than the ones demonstrated by Leblanc et al. (2005) for the French population, and by the SCOOP report for the Spanish (0.77) and German (0.36) inhabitants [START_REF] Miraglia | Assessment of dietary intake of ochratoxin A by the population of EU member states[END_REF]. Conversely, the values of the EDI in the present study are similar to the ones calculated for the Danish residents (0.19) by the same SCOOP report. Further, the EDI from wheat bread, 0.21ng/kg bw/day, represents over a quarter of the national average, and thus Mafra bread, although also made of wheat, did not present any detectable level of OTA, and so does not contribute to the exposure to OTA. This difference is likely due to the fact that, as previously mentioned, while Mafra bread is, as a rule, baked with only wheat flour, water, yeast, and salt, the more common wheat bread also tends to include other components that generally lead to an increase of the OTA content in bread, such as seeds, bran [START_REF] Scudamore | Fate of ochratoxin A in the processing of whole wheat grains during extrusion[END_REF][START_REF] Juan | Levels of ochratoxin A in wheat and maize bread from the central zone of Portugal[END_REF], and even flour made of other cereals [START_REF] Czerwiecki | On ochratoxin A and fungal flora in Polish cereals from conventional and ecological farms. Part 2: Occurrence of ochratoxin A and fungi in cereals in 1998[END_REF].

Occurrence of OTA in urine

The present study shows an elevated incidence of OTA occurrence in the urine of the Lisbonian residents (Table V). Incidence was high, with nearly three-quarters (72.1%) of the samples being positive for OTA, and 62.8% featuring contamination levels above the LOQ, with concentrations ranging from n.d. to 0.071ng/mL. The mean concentration and standard deviation were 0.026 and 0.017ng/mL, respectively.

Except in the cases of OTA occurrence in Balkan region where the high incidence of positive cases and contamination level reflect the endemic condition of exposure to OTA, the results of most surveys are comparable with those reported by the present work (Table VI).

Previously, other studies showed similar results in some other European countries where BEN has not been observed. However, a difference in analytical methods and/or eating habits can result in far higher values of contamination [START_REF] Baydar | Aflatoxin and ochratoxin in various types of commonly consumed retail ground samples in Ankara, Turkey[END_REF], as exemplified by the values for Sierra Leone detected by [START_REF] Jonsyn | Intake of Aflatoxins and Ochratoxins by infants in Sierra Leone: possible effects on the general health of these children[END_REF] and [START_REF]Seasonal variation in exposure frequency and concentration levels of Aflatoxins and Ochratoxins in urine samples of boys and girls[END_REF].

This study also reinforces the previous results obtained for samples of central Portugal and Valencia, Spain [START_REF] Dinis | Ochratoxin A in nephropathic patients from two cities of central zone in Portugal[END_REF][START_REF] Lino | Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree[END_REF][START_REF] Manique | Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations[END_REF], demonstrating a concerning exposure of Iberian residents to this mycotoxin, corroborated by the presence of OTA in Spanish and Portuguese foodstuffs such as cereals and cereal products, rice, dried fruits, milk, and wine [START_REF] Manique | Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations[END_REF].

According to [START_REF] Scott | Biomarkers of human exposure to ochratoxin A[END_REF], the OTA level in urine does not present any correlation with age [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF], nor gender [START_REF] Fazekas | Ochratoxin A content of urine samples of healthy humans in Hungary[END_REF]. Other authors [START_REF] Manique | Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations[END_REF][START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF] also reported the absence of significant differences between genders. [START_REF] Pena | Estimation of ochratoxin A in Portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection[END_REF] observed differences between gender only in the group aged between 20 and 39 years (p=0.044). In Sierra Leone, [START_REF]Seasonal variation in exposure frequency and concentration levels of Aflatoxins and Ochratoxins in urine samples of boys and girls[END_REF] discarded the correlation with the nutritional status and the prevalence of OTA in the urine samples of the [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF] found no significant differences associated with the ethnic diet of the subjects. Vegetarians had higher OTA consumption on average, although their plasma or urine levels were not significantly higher.

The low levels of OTA observed in the study of [START_REF] Pascale | Rapid method for the determination of ochratoxin A in urine by immunoaffinity column clean-up and high-performance liquid chromatography[END_REF] are clearly indicative of the much lower amount of the toxin which is transmitted to the urine as compared to the blood. However, [START_REF] Gilbert | Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples[END_REF] observed a significant correlation between OTA human consumption through controlled diets and OTA concentration in urine (ranging from <0.01 to 0.058ng/ml) suggesting that the content of OTA in 94 urine samples could be a useful marker of OTA intake.

Conclusion

In conclusion, the high percentage of bread samples featuring significant levels of OTA, corroborated by a similarly high frequency of contamination in urine samples, suggests a real and concerning exposure of Lisbon residents to this mycotoxin. Although maize bread is more contaminated, the wheat bread, due to its higher consumption, contributes more to the exposure to OTA. An exception is the Mafra wheat bread type, which has an insignificant level of contamination.

This study contributes thus to the gathering of information about exposure to OTA, which is also important when making decisions on the regulation of chemical contaminants and the safety of foodstuffs. Further studies need to be conducted to expand this data and pursue the surveillance in cereals and derived products, since they are the major contributors to OTA intake. a EDI was calculated using the equation EDI = (∑c) (CN -1 D -1 K -1 ), where ∑c is the sum of OTA concentration in the analyzed samples, C is the mean annual intake estimated, N is the total number of analyzed samples, D is the number of days in a year, and K is the mean body weight, which was considered 65Kg (mean of body weight of the studied population)

F

b According to EFSA-established TWI (120ng/kg bw/week).

  The present study aims to estimate the OTA contamination levels of different types of bread, a widely consumed foodstuff, in the Lisbon region during the Winter of 2007/2008.

  acid purchased from Sigma-Aldrich (Laborchemikalien, Germany), hydrochloric acid, sodium hydroxide, potassium chloride, potassium dihydrogenphosphate and anhydrous disodium hydrogenphosphate supplied by Merck (Darmstadt, Germany), and sodium chloride purchased from Baker Ltd., Pagenham, England.The OTA standard was acquired from Sigma Chemical Co. (St. Louis, MO, USA)

Filter

  paper Whatman Nº4 (150mm, Whatman International Ltd. Maidstone, England) and polyamide membrane filters (0.2µm, 50mm, Whatman GmbH, Dassel, Germany) were used. Immunoaffinity columns (IAC) Ochratest TM were from VICAM (Watertown, USA). A Braun MR 5000M multiquick/minipimer 500W (220-230V, 50-60Hz, Esplungues del Llobregat, Spain), a Moulinex blender 700W (230-240V, 50-60Hz, Barcelona, Spain), a vacuum manifold of Macherey-Nagel (USA), a pump of Dinko (mol. D-95, 130W, 220V), a magnetic stirrer (Agimatic-S, Selecta, Barcelona, Spain), and a Retsh vortex mixer (Haan, Germany) were employed. A Sonorex RK 100 ultrasonic bath (Berlin, Germany) was used for 15min degassing of all chromatographic solvents prior to use, and also for cleaning and decontaminating of the material used in the experiments. The LC instrument used possessed a pump (Model 307, Gilson Medical Electronics, Villiers-le-Bel, France), and a guard column Hichrom Ltd, HI-173 (30mmx4mm i.d.) (England) preceding a Hichrom C 18 column (5µm, 250 mm x 4.6mm i.d.). Detection and quantification were performed with a Perkin-Elmer Model LS45 (Beaconsfield, UK). A Hewlett-Packard 3390A integrator (Philadelphia, PA, USA) recorded the results.

  OTA extraction from the bread matrix, 100mL of PBS:methanol (50:50)

  higher levels of contamination. Food processing, thus, does contribute slightly to decontamination, though it does so in blatantly insufficient levels.

  contribution of bread in particular, and cereals in general, as the main contributors to OTA exposure.

  On the other hand,

Figure 1 :

 1 Figure 1: LC-FD chromatograms obtained after the described experimental procedure, from (a) OTA standard solution (5ng/mL; RT 11.30), (b) bread sample naturally positive to OTA (RT 11.30), (c) urine sample naturally positive to OTA (RT 11.30) and (d) an OTA methyl ester peak after boron fluoridemethanol derivatization of a wheat bread sample (RT 27.31).

Table I -

 I Anthropometric data of the sampled population.

	Gender	Size	Weight (kg)	Age (years)	Stature (cm)
			Range	Mean±SD	Range	Mean±SD	Range	Mean±SD
	Female	48.8% (21/43)	[45;89]	65.67±11.59 [18;69] 45.95±15.63 [150;170]	157±7
	Male	51.2% (22/43) [62;108]	77.5±10.47	[23;75] 46.09±13.61 [170;180]	171±7
	Total	100% i.e. 43	[45;108] 71.72±12.44 [18;75] 46.02±14.45 [150;180]	164±10
		o r F o r F o r F o r F o r F o r
				P P P P P P
				e e e e e e
					e r e r e r e r e r e r
						R R R R R R
						e e e e e e
							v i e v i e v i e v i e v i e v i e
							w w w w w w
							O n l O n l O n l O n l O n l O n l
							y y y y y y
							1

Table II -

 II Prevalence (%) and mean levels (ng/g) of OTA in different types of bread samples.

	Sample a	Sample size	≥LOQ b (%)	≥LOD (%)	Range (ng/g)	Mean b ±SD Mean c ±SD
	Maize bread	5	4 (80)	4 (80)	n.d.-0.36	0.28±0.09	0.22±0.14
	Wheat bread	24	17 (70.8)	19 (79.2)	n.d.-0.41	0.21±0.09	0.15±0.12
	Mafra type wheat bread	12	0 (0)	5 (41.7)	<0.1	n/a	0.02±0.03
	TOTAL	41	21 (51.2)	28 (68.3)	n.d.-0.41	0.22±0.09	0.12±0.12
	a Bread type, b Samples ≥LOQ (≥0.1ng/mL) only, c All samples

Table III -

 III Compilation of some international studies and the present on OTA occurrence in maize and wheat grains and derivate bread.

		Conventional wheat bread, 67	19%	-	0.04-10.81
		Other conventional cereals bread, 7	14.3%	-	2.59
		Maize bread, 15	60%	0.43	-	Juan et al. (2007)
		Maize bread, 30	70%	0.44	-	Juan et al. (2008)
		Wheat bread, 31	12.9%	0.02	-
	Portugal	Wheat bread, 30	80%	0.2±0.1	n.d.-0.49	Bento et al. (2009)
		Wheat bread, 20	65%	0.3±0.14	n.d.-0.43
		Maize bread, 5	80%	0.28±0.09	n.d.-0.36
		Wheat bread, 24	79.2%	0.21±0.09	n.d.-0.41	Present study
		Mafra wheat bread, 12	41.7%	n/a	<0.1	Deleted: Frequency c
		F o r
		P
		e
	Country	Sample type, number	Incidence e r	Mean±SD	Range (µg/kg)	Source
	Italy Croatia	Maize, 70 (1999-2000) Wheat, 70 (1999-2000) Maize, 49	27.1% 8.6% R 1.7±1.6 0.8±0.3 38.7% 1.47±0.38 e	0.2-5.2 0.4-1.4 0.9-2.54	Palermo et al. (2002) Domijan et al. (2005)
	Ivory Coast	Maize, 16 (1998) Maize, 15 (2001) Maize, 10 (2002) Maize, 10 (2002) Corn bread, 10	100% 100% 100% 100% -	44±12 266±428 37±25 -v i 27-64 3-1738 9.8-86 0.16-0.86 e 4.94±0.20 4.08-5.28 w	Sangare-Tigori et al, (2006a) Sangare-Tigori et al. (2006b)
	Turkey Spain	Corn flour, 10 White bread, 25 White flour, 22 Whole meal bread, 28 Whole meal flour, 14 Organic wheat bread, 20	-----20%	6.39±1.10 3.36±0.73 6.89±0.46 7.84±0.39 9.30±1.33 -	3.86-15.67 n.d.-11.40 1.87-7.44 3.54-12.61 O Cengiz et al. (2007) n l 1.07-16.70 0.03-0.81 González-Osnaya et al., y
		Other organic cereals bread, 6	33.3%	-	0.39-0.81	2007
						2

Table IV -

 IV Adult estimated daily intake (EDI) and weekly intake (EWI) of OTA, according to the type of bread.

	Bread type	EDI a (ng/kg bw/day)	% TWI b
	Maize bread contribution	0.09	0.54
	Wheat bread contribution	0.21	1.22
	Mafra type wheat bread contribution	0	n/a
	Total	0.30	1.76

Table V -

 V Prevalence (%) and mean levels (µg/L) of OTA in urine

		F o r
			P
			e
				e r
					R
					e
					v i e
	Gender Females Males Total	Size 48,8% (21/43) 51.2% (22/43) 100%	Incidence 71.4% (15/21) 72.7% (16/22) 72.1% (31/43)	≥LOQ* 57.1% (12/21) 68.2% (15/22) 62.8% (27/43)	Mean±SD 0.027±0.015 n.d.-0.055 Range w 0.025±0.020 n.d.-0.071 0.026±0.017 n.d.-0.071 O n l
					y
					3

Table VI -

 VI Occurrence of OTA and/or its metabolites in urine in different countries

	Country	Biomarker	Sampled population, number	Incidence	Range (Mean) (ng/mL)	Reference
	U.K.	OTA	Volunteers, 50	92%	n.d.-0.058	Gilbert et al. (2001)
	Italy (South & North)	OTA	Healthy, 38	57.9%	0.012-0.046	Pascale & Visconti (2001)
	Italy (Southern)	OTA	Healthy	100%	0.12-2 (0.53)	Breitholtz-Emanuelsson et al.(1994)
	Hungary	OTA	Healthy, 88	61%	0.006-0.065 (0.013)	Fazekas et al. (2005)
	Bulgaria (Gorno Peshtene village)	OTA	Healthy BEN area residents , 5	100%	0.010-0.33 (0.0508)	Petkova-Bocharova et al. (2003)
	Bulgaria (Beli Izvor village)	OTA	Healthy BEN area residents , 11	100%	0.010-1.910 (0.168)
			BEN/UTT patients, 36	38.9%	0.005-0.604
			Healthy persons from BEN families, 25	48%	0.005-0.033
			Healthy persons from
	Bulgaria (1984-1990)	OTA	non-BEN families in BEN villages, 32	44%	0.005-0.043	Nikolov et al. (2002)
			Healthy persons from
			non-BEN villages in	12.9	0.017-0.041
			BEN area, 31
			Healthy persons from non-BEN area, 3	0	n.a.
	Bulgaria	OTA 4-hydroxyOTA	BEN/UTT patients and controls, 152	40% 0%	0.005-0.03 -	Castegnaro et al. (1991)
		OTA 4-hydroxyOTA	Children (<5years), 54	24% 44.4%	0.3-26.6 0.04-21	Jonsyn (1999)
	Sierra Leone	4-hydroxyOTA	Children (5-14years), 434	38.5 %	n.d.-37
		OTA	Boys (5-14years), 231	23.8 %	0.07-72	Jonsyn-Ellis (2000)
			Girls (5-14years), 203	27.1%	0.08-148
			Healthy, 60	70%	0.021-0.105 (0.038)	Pena et al. (2006)
					M:0.011-0.208
	Portugal (Coimbra)	OTA	Healthy, 30	M: 43.3% A: 46.7%	(0.019) A: 0.008-0.11
					(0.018)	Manique et al. (2008)
					M: 0.007-0.124
	Spain (Valencia)	OTA	Healthy, 31	M: 80.6% A: 83.9%	(0.032) A: 0.008-0.089
					(0.028)
			Healthy women, 21	71.4%	n.d.-0.055 (0.027)
	Portugal (Lisbon)	OTA	Healthy men, 22	72.7%	n.d.-0.071 (0.025)	Present study
			Total, 43	72.1%	n.d.-0.071 (0.026)

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

Scientific Committee on Food.

Acknowledgements

This study was supported by the FCT through the Project PTDC/AGR-ALI/65528/2006 and the grant SFRH/BD/37409/2007.


Miguel Mauricio-Iglesias

Valérie Guillard

Nathalie Gontard

Stéphane Peyron
email: peyron@univ-montp2.fr

K ; ; Hinrichs

T ; Lickly

P Mercea

O ; 2005 Piringer

Application of FTIR and Raman microspectroscopy to the study of food/packaging interactions

Keywords: Chemometrics, Method validation Additives/Contaminants: Additives general, Packaging -migration modelling

24 2005;Brandsch et al. 2002;Franz 2005;Piringer 1994;Reynier et al. 1999;Reynier et al. 25 2001b; [START_REF] Vitrac | Decision trees as applied to the robust 10 estimation of diffusion coefficients in polyolefins[END_REF]) enable a rough evaluation of these parameters that would require 26 otherwise a high number of experimental mass transfer tests, to be determined experimentally.

27

An outstanding example of such relationship is the so called "Piringer equation" used to 28 determine the diffusivity of a migrant and needs as inputs the temperature during the process, 29 the molecular weight of the migrant and five constants, two of which depend on the type of 30 polymer. All these parameters have been derived from a thorough analysis of reported 31 diffusion coefficients in order that the diffusion coefficient determined should always 32 overestimate the real value. Alternatives to the estimation of the diffusion coefficient have concentration profile in the depth of polymer sample in order to easily gather mass transfer 1 parameters. To the best of our knowledge, this has never been done up to now, partly because 2 both methods are not considered as sensitive enough to study migration, but, as it will be seen 3 subsequently, they can be successfully used to characterize mass transfer in model systems. The internal diffusion of migrant in the packaging is:

16         ∂ ∂ ∂ ∂ = ∂ ∂ < < > x C D x t C t L, x L - , 0 (1)
17 where x is the distance (m), C the packaging concentration in migrant (kg additive/kg of 18 polymer) and D the migrant diffusivity in the packaging (m²s -1 ). If D is independent of the 19 concentration of the diffusing substances (at least at the concentration range found in the 20 experiment), the system is said to follow Fickean kinetics.

21

Equation (1) was solved with the initial and boundary conditions that apply for this case 22 study i.e:

23 t = 0, -L < x < L, C = C in (2) 24 t > 0, x = +L, C = C L,∞ (3) 25 0 0, x , 0 = ∂ ∂ = > x C t (4)
where C in is the concentration initially present in the polymer, C L,∞ is the concentration of the 28 diffusing substance on the surface of the polymer required to maintain equilibrium with the 29 concentration of this substance in the liquid at time t.

( )       +       + - + - - = - - ∑ ∞ = ∞ L x n t D L n n C C C t x C n n in L in 2 ) 1 2 ( cos . 4 ) 1 2 ( exp 1 2 1 4 1 ) , ( 2 2 2 0 , π π π (6) 18 ( )       + - + - = - - ∑ ∞ = ∞ t D L n n C C C C n in L in t . 4 ) 1 2 ( exp 1 2 1 8 1 2 2 2 0 2 2 , π π (7)
where C is the concentration in diffusing substance in x at time t and t C is the average The additive diffusivity was identified from experimental data by minimizing the root mean 15 square error (RMSE) between experimental and predicted data (Equations ( 6) and ( 7)) and by 16 using an optimization method (Levenberg-Marquardt algorithm, optimization routine 17 predefine from Matlab software). A Monte Carlo sampling was applied to the input 18 parameters (concentration, thickness and time) in order to determine the 95% confidence 19 intervals as explained elsewhere (Hessler 1997;Press et al. 1989) . 2) proved to be heterogeneous and normally distributed. The test of 2 normality was applied to 186 samples and gave as a result a mean concentration of 0.41% 3 w/w with a standard deviation of 0.051%. So the coefficient of variation, i.e. the quotient 4 between the standard deviation and the mean was of 12.2%. As an illustration, this means that 5 the 95% of the samples will have concentrations comprised between 0.31% and 0.50% w/w. 6 For a migration experiment, the additive content is usually assumed to be homogeneous in 7 migration tests for all the samples. However, the distribution of additive among the samples 8 hinders the use of standard destructive methods since the variations on the initial 9 concentration of each sample could be comparable to the variations of concentrations 10 throughout the test. This obstacle can be overcome either by increasing the number of 11 samples for destructive methods (highly time consuming), either using a non-destructive 12 method such as FTIR, that can thoroughly guarantee the traceability of the samples 13 throughout the test and then, avoid the uncertainty caused by the variations in the initial 14 content of additive. Finally, it must be borne in mind that the lack of sensitivity of the 15 methods implied using a high concentration (0.4% w/w of Uvitex OB). If the additive is not 16 completely soluble in LLDPE at that concentration, it would be the responsible for the 17 heterogeneous distribution of the additive. exhibited some specific bands which could be employed to detect its occurrence in the films.

24

Regarding the higher absorbance value, Uvitex OB occurrence was specifically detected at 25 1579 cm -1 assigned to the aromatic C=C stretching band (Figure 3). In turn, olive oil sorption 26 into LLDPE was followed in the ester stretching band at 1750 cm -1 . The uptake of olive oil was considered to be equal to the increase of mass of the sample less 2 the quantity of Uvitex desorbed (quantified with FTIR measurements). The data fitted 3 satisfactorily the model (R 2 = 0.99, RMSE= 0.040% w/w). The migration of Uvitex OB in olive oil was followed during 26 days by FTIR. Spectra were 7 normalized on a specific LLDPE doublet (1369-1378 cm-1) and the decrease in absorbance at 8 1579 cm -1 showed the desorption of Uvitex OB from LLDPE film from an initial content of 9 0.4% w/w to a value below the detection limit after 26 days. Likewise, the uptake of olive oil 10 in the sample was obtained as a function of time and a plateau was determined at 1.3±0.1% 11 w/w of olive oil. From data obtained from FTIR experiments, desorption curves of Uvitex OB 12 from LLDPE in olive oil were obtained by plotting the relative concentration as a function of 13 time (Figure 4). The determination of diffusivity values for Uvitex desorption and olive oil sorption in the 16 LLDPE film was evaluated during migration kinetics in olive oil using equation ( 7). For 17 Uvitex OB, the resulting diffusivity value was of 8.0±2.5⋅10 -14 m 2 s -1 and the RMSE of 0.02% 18 w/w, lower than the experimental error evaluated as a 0.04% w/w. This results was in 19 agreement with experimental data previously reported since a value of diffusivity of 5⋅10 -14 20 m 2 s -1 was found by Dole et al (2006) (same temperature but in LDPE). For olive oil the 21 diffusivity value was 6.9±3.4⋅10 -14 m 2 s -1 and the RMSE 0.07 % w/w, slightly larger than the 22 experimental error estimated as 0.04% w/w but remaining still in an acceptable range. The use 23 of non-destructive measurement by FTIR allowed to fully trace the content of Uvitex OB of 24 the sample all throughout the experiment and thus, to determine an accurate value of 25 diffusivity which would not be feasible with other common analytical procedures with a 26 comparable accuracy. Furthermore, FTIR also provided data on the uptake of olive oil in the 27 sample that led to the determination of its diffusivity in LLDPE. Advantages of FTIR to carry 28 migration tests out include, besides the possibility to obtain non-destructive measurements, 29 supplying additional information of the polymer evolution (e.g. oxidation) and of a number of 30 substances that could be sorbed during the test. Its main disadvantage lies in its lack of 31 sensitivity compared to other analytical methods. When dealing with complex migration 32 behaviour, as for instance, when diffusivity can no longer be considered as a constant (non- The three concentration profiles obtained were treated altogether and used to determine 21 whether diffusivity of Uvitex OB was independent of its own and/or olive oil concentration.

22

Four scenarios were considered (table 1): i) diffusivity is independent of Uvitex OB and olive 23 oil (Fickean kinetics), ii) diffusivity depends only on Uvitex OB concentration, iii) diffusivity 24 depends only on olive oil concentration and iv) diffusivity depends on both Uvitex OB and 25 olive oil. The first case was solved using equation ( 6) and the last three using a modification 26 of the Crank Nicolson method as stated in the Materials and Methods section. For cases iii) 27 and iv) olive oil was considered to modify the diffusivity of Uvitex OB in LLDPE but not its 28 own diffusivity. Thus, the constant value of diffusivity obtained with the FTIR analysis using 1), it was verified that the increase of factors in the model did not 1 significantly increase the accuracy of the predictions. Figure 6 shows the experimental data of 2 concentration profiles of Uvitex OB in LLDPE as well as those predicted with the diffusivity 3 value obtained for scenario i), i.e. Fickean kinetics and how they are well correlated if 4 compared to previous works in which the concentration profile is fitted (Helmroth et al. 2003;5 Reynier et al. 2002) . Therefore, scenario i) was accepted as the most likely and simplest, i.e. It must be pointed out that the concentration at 15 µm of the interface was always 28 underestimated by the predicted values (Figure 6) what could mean that there is a non-29 negligible mass transfer resistance in the interface. This hypothesis is attractive and it would 30 make the Raman approach even more valuable since it would easily reveal whether mass 31 transfer resistance at the interface can be neglected. Unfortunately, the measurements of 32 points located so close to the interface are less reliable than those in the inside (Everall 2008). that olive oil does not modify the diffusivity of a medium molecular weight (431 g mol -1 ) 13 additive in polyolefins. In addition, it must be borne in mind that the olive oil uptake is only 14 of a 1-2% w/w in polyolefins (1.3% w/w in our study) whereas it is commonly stated that, as a rule of thumb, 15% w/w of solvent absorption accounts for an increase of ten folds of 16 diffusivity (Helmroth et al. 2002b) . With this in mind, olive oil sorption as observed in this With the scope of improving the analysis of food/packaging interactions, the application of 21 two types of vibrational spectroscopy (FTIR and Raman) was presented. The aim of the 22 approach was not to quantify migration levels but to characterize mass transfer parameters in 23 the system. Both, FTIR and Raman, were successfully used to determine diffusivity values of 24 Uvitex OB that could be further use to simulate its release, according to legislation. With the 25 example developed here, each of the methods (FTIR and Raman) proved to be a very efficient 26 mean to access to the parameters necessary for predicting the migration in realistic conditions.

  of food contact material additives into foods or food simulating liquid is 14 a crucial point of safety assessment of packaging materials. However these tests, based on 15 destructive extraction and quantification (e.g. chromatographic or spectrophotometric) 16 procedures, are expensive and time-consuming. The directive 2002/72/CE considers the use 17 of "generally recognised diffusion models based on experimental data [...] under certain 18 conditions" to determine overestimated migration levels. Most of these models, used to 19 describe migration, are based on Fick diffusion equation that involve (Crank 1980) at least 20 two key parameters : (i) the diffusion coefficient (D), a kinetic parameter, which measures the 21 average ability of molecules to diffuse across the permeable phase of polymer film, and (ii) 22 the partition coefficient (KPL) which is the ratio of additive content in the packaging material 23 and in the foodstuff at equilibrium. Empirical and semi-empirical relationships (Begley et al.

  33 used the volume of the migrant (Reynier et al. 2001b) ; the Van der Waals volume, the 34

Fick

  were used to determine the diffusivity of a migrant in a plastic 6 packaging material by two vibrational spectroscopy methods i.e. FT-IR and Raman 7 spectroscopy. Uvitex OB, an optical brightener and UV stabiliser approved for Food Contact 8 Materials, was selected as model migrant to spike LLDPE films. The samples were submitted 9 to migration test according to EC directives and analysed by FT-IR and Raman 10 microspectroscopy during immersion in the worst case food simulating liquid i.e. the olive 11 oil, with the objective to determine the diffusion coefficient on the basis of a resolution of 12

(

  In some cases, the diffusivity D is dependent on the concentration of the diffusing substance 1 as it is the case of substances that alter the structure of the polymer. According to the free 2 volume theory, D is proportional to the exponential of the free volume within the polymer 3 Duda and Zielinsky 1996) . Based on this assumption, several authors (Helmroth et al. 2003; 4 Reynier et al. 2002) have successfully modelled the dependence of D as: the zero concentration diffusivity and γ is a proportional factor. Of course, if γ is 7 equal to zero, diffusivity is a constant and kinetics follows the Fickean model. Taking into 8 account the potential dependency of diffusivity and concentration, several scenarios are 9 possible; case i) diffusivity is independent of Uvitex OB and olive oil (Fickean kinetics); case 10 ii) diffusivity depends only on Uvitex OB concentration; case iii) diffusivity depends only on 11 olive oil concentration; and case iv) diffusivity depends on both Uvitex OB and olive oil. /desorption phenomena follow Fickean kinetics, the system of equations (1-4), 15 can be solved analytically and the evolution of concentration with time is given by 16 Crank(1980) :

  17

8 9 (

 9 photobleaching for 15 s from the third day of test in order to avoid fluorescence caused by

Page

  additive distribution in the film samples, the quantification of Uvitex 24 OB and olive oil content was investigated by FTIR throughout the test and used to determine 25 the migration level of Uvitex OB as well as the diffusivity of both olive oil and Uvitex OB. 26 Likewise, Raman measurements were used to determine diffusivity of Uvitex OB in LLDPE 27 and both results were compared 28 29 Uvitex OB distribution in LLDPE samples 30 The Uvitex OB concentration in LLDPE used for the tests turned out not to be 31 homogeneously distributed as determined by the test of normality of Anderson-Darling 32 Ortiz et al. 2007) (the result was positive for an α = 0.05). The distribution of the 1 additive (Figure

  Uvitex OB and sorption of olive oil were investigated using 21 FTIR. A detailed study of the additive fingerprint allowed to determine the most suitable 22 bands for the analysis. Considering the blend LLDPE spectrum, Uvitex OB fingerprint 23

  including variable contents in Uvitex OB, a Partial Least Square (PLS) 29 model was calibrated on the basis of the intensity ratio 1579/1378 cm -1 . The regression 30 produced a linear relationship (R 2 = 0.96, RMSE= 0.034% w/w) between spectral data and 31 additives concentration measured by UV spectroscopy analysis. same procedure was carried out to relate spectral data and olive oil content.1

4 5

 4 Determination of the diffusivities of Uvitex OB and olive oil in LLDPE with FTIR 6

  Fickean kinetics), a large number of data are required that are difficult to obtain by FTIR or 1 other similar method that provided the average concentration of a sample. Raman 2 microspectroscopy was explored as a promising tool to obtain a further insight of mass 3 transfer in the polymer for it gives access to more information on migration kinetics such as 4 concentration profiles of Uvitex OB in the depth of the LLDPE sample as a function of time. determined after contact times of 4h, one day and three days, 8 shorter than those required by FTIR, proving to be an efficient method for the determination 9 of diffusivity. For each contact time, Raman intensity profile obtained was compared to the 10 total mass of Uvitex remaining in the film determined by FTIR (Figure5). The Raman profile 11 allowed the determination of the total content of Uvitex OB in the film according to equation 12 for the bulk density of the polymer (920 kg m -3 for LLDPE), S for the surface 15 and C for the concentration. A linear regression was used to successfully correlate (R²=0.96) 16 the data supplied by FTIR and the band assigned to Uvitex (1569-1614 cm -1 ) normalized with 17 the area ratio of the LLDPE specific band (1129 cm -1 ). 18 19 Determination of the diffusivity of Uvitex OB in LLDPE 20

Page

  29and equation (6) and, the concentration of olive oil in LLDPE samples was estimated as a 30 function of space and time. These values of concentration were then used to determine the 31 potential dependency of Uvitex OB diffusivity on olive oil content. On the basis of the RMSE 32

6

  Fickean kinetics and a diffusivity independent of the concentration of either olive oil or 7 Uvitex OB, at least in the concentration range found during the experiments, which confirms 8 the results obtained by FTIR. According to Raman measurements a value of diffusivity was 9 obtained of (14.6±2.5⋅10 -14 m 2 s -1 ). Although significantly different for p=0.05, it lies in the 10 same range as the one obtained by FTIR, and indeed both values were no longer different 11 when considering a value of p=0.025. It is a quite meaningful result given the high deviations 12 that can be found in the determination of diffusivity. For example, Dole et al. (2006) reported 13 differences up to 350% for the diffusivity of Uvitex OB in PP measured with the same investigation of diffusivity of a model molecule by Raman spectroscopy allowed 17 the assessment of several complex phenomena that influence migration in an integrated 18 approach. Raman microspectroscopy was used to provide large amount of data on the inside 19 of the polymer that can be used to assess diffusivity variations. Even though it is theoretically 20 possible to consider a variable diffusivity with global measurements as those performed by 21 FTIR or standard extraction/quantification methods, in reality it is often unfeasible since a 22 high number of experimental values are needed in practice to fit several parameters correctly 23 especially for non-linear models (Hessler 1997). In most cases, FTIR sensitivity is too low to 24 provide such a number of values from a desorption experiment and other conventional 25 methods turn out to be far too time-consuming. 26 27

3

  Extreme care must be taken if assumptions are made based exclusively on the measurements 1 located the closest to the interface.2Regarding the effect of olive oil in desorption, it is sometimes widely accepted that, since the 4 polarity of triglycerides in olive oil is similar to that of polyolefins, olive oil is easily absorbed 5 and greatly influences the diffusivity in polyolefins. This has been tested for several low 6 molecular solvents (Helmroth et al. 2003) but very few results in the literature support this 7 hypothesis for olive oil. Several authors found that olive oil did not modify the release of 8 medium molecular weight additives from polyolefins (Goydan et al. 1990; Helmroth et al. 9 2002b; O'Brien et al. 1999) In contrast, Reynier et al.(2002) suggested an exponential 10 dependency of diffusivity with the triglyceride content, but they used glyceril tripelargonate 11 instead of olive oil, which could explain this discrepancy. Our results underpin the hypothesis 12

  17 study is too low to carry out an important modification of diffusivity. 18 Conclusion 20

27FTIR

  was used to know the average concentration in the LLDPE sample as a function of time.28Raman microspectroscopy gave the possibility to gather the concentration profile in the depth 29 of the sample and use these data to perform a complex analysis of the factors that could 30 influence the diffusion coefficient, e.g. it was demonstrated that olive oil sorption did not 31 modify the diffusivity of Uvitex OB in LLDPE. Regarding the experimental set up, a non-32 negligible time gain could be achieved.

Figure 1 :Figure 2 :Figure 3 .Figure 4 :Figure 5 .Figure 6 :

 123456 Figure 1: Preparation of a sample for the Raman analysis. The slice is cut in order that the thickness (2L) becomes the width of the sample Figure 2: Distribution of the Uvitex OB concentration in LLDPE samples Figure 3. FTIR spectral fingerprint of LLDPE + Uvitex OB. Focused region shows the band selected to follow Uvitex OB desorption and its decrease throughout the test Figure 4: Simultaneous sorption of olive oil and desorption of Uvitex OB from LLDPE at 40°C as determined by FTIR. ( represent experimental olive oil values; □ represent experimental Uvitex OB values; continuous lines represent fitted data) Figure 5. Raman spectral fingerprint of LLDPE + Uvitex OB. Focused region shows the doublet selected to follow Uvitex OB (C=N, C=C) desorption and its variation in the depth of the film Figure 6: Concentration profiles of Uvitex OB within the half-depth of LLDPE film submitted to a contact with olive oil at 40°C. The profiles were obtained after 4 h (■), 1 day (○) and 3 days (▲). Continuous lines represent the profiles predicted with the value obtained in case i), Fickean kinetics.

Figure 1 :Figure 2 :Figure 3 .Figure 4 :Figure 5 .Figure 6 :

 123456 Figure 1: Preparation of a sample for the Raman analysis. The slice is cut in order that the thickness (2L) becomes the width of the sample

Table 1 .

 1 Values obtained after fitting of Raman data of desorption of Uvitex OB from LLDPE in olive oil at 40°C. Note: D 0 and γ values are defined in equation (5). For case i) both γ are zero and D 0 is actually the Fickean diffusivity.

		γ Uvitex OB	γ Olive oil	D 0 (10 -14 m² s -1 )	RMSE (% w/w)
	Case i (Fickean)			14.9 (2.5)	0.023
	Case ii	0.12		14.2 (1.3)	0.023
	Case iii		0	14.4 (1.3)	0.023
	Case iv	0.19	0	13.3 (1.3)	0.022

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Acknowledgements

This study has been carried out with financial support from the Commission of the European