Sara Lee De

N V Gerhard-Rieben

Elke ; Nestec

Helmut Guenther
email: hguenther@krafteurope.com

Katrin Hoenicke

Steven Biesterveld

Elke Gerhard-Rieben

Ingo Lantz

Furan in coffee: Pilot studies on formation during roasting and losses during production steps and consumer handling

Keywords: furan, coffee, formation, analysis, exposure

The occurrence of furan in some food products has been known for a few decades already and it has been re-confirmed in more recent investigations that furan is present in a variety of foodstuffs. This list of products includes roasted coffee which has been shown to generate furan as a result of the heat treatment during the roasting reactions which are applied to achieve the desired aroma and flavor profile of a roasted coffee. The objective of this study was to provide data to better understand the available data of furan in coffee, to understand the kinetics of furan generated during roasting and to estimate the reduction of furan levels afterwards due to subsequent processing steps and consumer handling. Finally, the study was meant as a contribution to establish exposure data on the basis of scientific data at the stage of coffee consumption. This paper shows that the formation of furan during roasting is dependent on roasting conditions and is therefore directly linked to achieving targeted flavor profiles. Furthermore it is demonstrated that modifications in process conditions to potentially reduce furan levels may have the opposite effect on other undesired reaction products of the roasting chemistry like e.g. acrylamide. Due to the high volatility of furan any subsequent processing step or consumer handling has an impact on the level of furan. As a guidance from this study and in consideration of the identified losses of each process and handling step on basis of the trial conditions it is estimated that only approximately 10% of the initially generated furan during roasting gets into the cup of coffee for consumption.

Introduction

It has been known to the scientific community for a number of years that furan is present in coffee as part of the coffee volatile aroma components which are generated during roasting.

More recent risk assessments have been initiated by a report from the US Food and Drug Administration in 2004. Amongst others due to improved analytical methodology its surveys have identified the occurrence of furan in a number of food products which have undergone a heat treatment. This range of foodstuffs includes among others coffee products. The presence of furan is a potential concern as, based on animal trials, furan is considered possibly carcinogenic to humans (IARC 1995).

There has been little knowledge available on the relationship between coffee roasting conditions and resulting furan formation. Moreover, the impact of subsequent processing steps and consumer handling of the product on levels of furan is not well understood.

Finally, due to the high volatility of furan, sampling has been shown to be crucial to obtain reliable results. Therefore it has been difficult to interpret individual data on furan in coffees which have been produced under different conditions and sampled at different process steps.

As such, the contribution of coffee as consumed to the human exposure to furan has been very difficult to assess from analysis data for roasted whole beans or roast & ground coffee and only been possible with analysis of coffee brews ready for consumption. This paper is meant to increase knowledge on formation and disappearance of furan in different stages of coffee roasting and handling. Intentionally the investigations have been limited to bench top or pilot plant screening trials to estimate the magnitude of impact from individual process steps. The trials have been designed to simulate typical commercial plant conditions and average consumer handlings but are not covering the full range of all process variables and conditions. Overall, it is meant as contribution to establish exposure data on basis of scientific data at the stage of coffee consumption.

Materials and methods

Green coffee

The roasting experiments to investigate furan formation during roasting were performed with the three major types of green coffees: wet processed Arabica coffee (Coffea arabica) (represented by a sample of a Colombian green coffee); dry processed Arabica coffee (C.

Arabica, represented by a sample of a Brazilian green coffee); and dry processed Robusta coffee (Coffea canephora robusta, represented by a Vietnamese green coffee). Studies to investigate the losses of furan in subsequent processing steps were typically performed by using a sample of a Brazilian green coffee. The roasting experiments were executed on a Neuhaus Neotec RFB-10 pilot plant roaster with a batch size of typically 1.5 kg.

Analysis of degree of roast

The degree of roast was measured by light reflectance using a Lange colorimeter. No harmonized methodology across the European coffee industry is available and the coffee industry is applying individual calibration curves taking the roast color as characterization of the degree of roast. LRUs (Lange reflectance units) as used for this study would be 100-110

LRUs for a very light roast and 40-50 LRUs for a very dark roast.

Furan analysis

Furan was analyzed according to the FDA-method "Determination of furan in foods" using headspace gas chromatography mass spectrometry (GC/MS). Here, furan is quantified by using a standard addition curve, where the concentration of furan in fortified test portions is plotted versus the furan/d4-furan response factors. The amounts of furan and/or d4-furan added to the test portions are based on an estimate (x 0 ) of the furan concentration in the food.

Chemicals

Furan was purchased from Acros Organics (Niederau, Germany) and d4-furan from Isotec (Miamisburg, USA). Methanol was from Sigma-Aldrich (Seelze, Germany). All other chemicals used were of analytical grade.

Pre-treatment

Whole coffee beans are frozen at -20 °C before milling. The homogenization is performed under cooling with liquid nitrogen using a Retch mill, mesh size 0.5 µm.

Estimation of the furan level in the coffee sample

For the one-point estimate of the concentration of furan in the sample 0.5 mg of coffee is weight into a 20 ml headspace vial. After addition of 4.5 ml water the headspace vial is sealed. The sample is fortified through the septum of the seal with 50 µl of internal standard (IS) solution (d4-furan, c = 30 µg ml -1 ). After shaking, the sample is analyzed by headspace GC/MS as described below. By using the integrated response ratio for furan/d4-furan (m/z 68/72) and the ng amount of the IS, the ng amount of furan in the coffee sample is estimated. 0.5 g of sample is weight into seven 20 ml headspace vials respectively. To every vial 4.5 ml of water is added and the vials are than sealed. IS solution is added (c = 30 µg ml -1 ) to all of the seven vials at the level of the estimated furan level (x 0 ) in the sample. To two of the samples furan (furan standard, c = 30 µg ml -1 ) is added at a level of 0.5 times of the estimated furan level (0.5 x 0 ) through the septum of the seal. Two further vials are fortified with furan at 1 time (1 x 0 ) and 2 times (2 x 0 ) the estimated furan level of the coffee sample, respectively. Three of the seven vials were not fortified with furan (x 0 ). After shaking all seven vials are transferred to GC/MS analysis as described below.

Standard addition procedure

GC/MS analysis

GC/MS analysis is performed using a Varian Saturn 2100 GC/MS (Varian, Darmstadt, Germany) coupled to a CTC Analytics Combi PAL Autosampler-Headspace with incubator oven (CTC Analytics AG, Zwingen, Switzerland).

The vials are incubated for 30 min in the headspace oven at 50 °C before injection (transfer line: 230 °C, injection volume: 1000 µl, injector temperature: 200 °C, 1 min splittless, split ratio 1:20, carrier gas: helium). The chromatographic separation is made on a HP-PLOT Q column (15 m x 0.32 mm ID x 20 µm) (Agilent Technologies, Waldbronn, Germany). The following temperature profile is used: 0.1 min at 50°C, 10°C/min to 190°C, 30°C/min to 225 °C, hold for 1 min. The detection is made in the scan mode, scan range: m/z 35 to 85, MSD delay time: 5 min, trap temperature: 150 °C. The following ions are used for quantification: D4-furan: m/z 72 and 42, furan: m/z 68 and 39.

For calculation of the furan content of the coffee sample the integrated responses for m/z 68 (for furan) and m/z 72 (for d4-furan) are determined and the response ratio is calculated, m/z 68 divided by m/z 72. All test portions (0 x 0 ) and calibration standards (0.5 x 0 , 1 x 0 , 2 x 0 ) are subjected to a linear regression analysis where the x-axis equals the ng amount of furan added to the test portion and the y-axis equals the response ratios. The slope and intercept of the calibration curve is determined and the equation is solved for 0 x 0 at y equal to zero. In order to determine the µg kg -1 amount of furan in the sample 0 x 0 is divided by the test portion amount in grams.

Validation data

The expanded measurement uncertainty (level of confidence 95%, coverage factor k=2) for the determination of furan in coffee (x = 3200 µg/kg) using the described method is calculated to be 20 %. The recovery rate of furan spike to a sample is usually between 90 and 120 %.

In a ring trial organised by the German § 64 working group "furan analysis", a z-score of 0.48 was achieved for the analysis of furan in coffee (x = 2840 µg/kg) using the described method.

The repeatability and the reproducibility of the analysis of furan in coffee was calculated to be 9 and 19 %. Artifacts of the furan analysis Sample weight. According to the FDA method "Determination of furan in foods" 5 g of sample is used for the analysis. However, it could be shown that for samples containing higher amounts of furan, like coffee, the sample weight has to be reduced to 0.5 g in order to receive robust and comparable results.

Isotope dilution method versus standard addition procedure. It could be shown that for coffee the calculation of the furan content can lead to significant higher or lower amounts (up to 50 %) when using the simple isotope dilution method with d4-furan as internal standard. It seems that for the analysis of samples with high furan content (higher than 500 µg kg -1 ), like coffee, the standard addition procedure is essential to obtain reliable results.

Choose of the analytical column. Besides the HP-PLOT Q column the use of a HP-INNOWax column (Agilent Technologies, Waldbronn, Germany) was tested. For some samples, significant higher furan levels (up to 50%) were calculated when using the HP-INNOWax column. The background of this phenomenon could not be identified. In order to obtain comparable results, the column proposed by the FDA for the analysis of furan was used in our experiments.

Results and discussion

Formation of furan at roasting

Green Coffee

The selected green coffees representing the major types of green coffee have been roasted under varying roasting conditions to result in a range of roast colors for a constant roast time.

The resulting furan levels in the roasted coffees are depending on the roast colors (Figure 1).

In contrast, the results do not indicate that the furan formation potential is significantly varying for different green coffee types. The results for the different green coffee types at constant roast times are within a range which represents the measurement uncertainty (95% confidence level) of the analytical methodology of +/-20%. The conclusion is consistent with the investigations of La [START_REF] Pera | Analysis of furan in coffee of different provenance by head space solid phase microextraction gas chromatography-mass spectrometry: effect of brewing procedures[END_REF]. This study has covered green coffees from 6 producing countries and no correlation was observed between furan levels after roasting and provenance of green coffee. On this basis no further studies have been made to investigate in differentiation among green coffee origins of the same type.

Here Figure 1 Roast conditions European roast coffees cover a broad range of flavor profiles according to the different regional preferences and are designed to meet specific consumer groups' acceptances. The individual flavor profiles are controlled by specifying the green coffee blend, the degree of roast (typically measured by light reflectance or color measurements) and roast time. When analyzing pilot plant samples which have been roasted to cover qualities from light to dark roasted and from fast to long roasted coffees it can be seen that darker roast colors (indicated by lower LRU-values) and longer roast times in tendency result in higher levels of furan (Figure 2). Statistical analysis through modeling shows that both the roast colour effect and the roast time effect are statistically significant at 99% confidence level. Options to modify process conditions with the intent to reduce to furan levels after roasting are very limited because from one side the mentioned parameters need to be set in tight ranges to maintain the product identity of an existing product and to achieve its typical flavor profile. On the other side it needs to be considered that modifying process conditions to reduce one undesired constituent may increase at the same time the level of another undesired constituent. [START_REF] Lantz | Studies on Acrylamide levels in Roasting, Storage and Brewing of Coffee[END_REF] have shown that acrylamide levels in roast coffee are decreasing with increasing degree of roast and with longer roast time. These results have been confirmed in this study (Figure 3). Acrylamide is showing the opposite relationships with the same process parameters compared to those which have been found for furan in this study.

Here Figure 2 und Figure 3 Accordingly, the indications from this study are that process conditions to result in low acrylamide levels tend to lead to higher furan levels (Figure 4)

Here Figure 4 The full range of furan levels for roast whole beans from this study of 2000-7000 µg kg -1 is consistent with published data which are reporting levels of 3510-6100 µg kg -1 [START_REF] Kuballa | Furan in Kaffee und Kaffeegetränken[END_REF].

Plant processing steps

The purpose of this study was to assess the impact of coffee production processes and consumer handling on furan levels in a cup of coffee. Therefore pilot scale trials have been conducted, simulating typical conditions of commercial plant processes and consumer handling. In the scope of these investigations it was not intended to cover the full range of all variables and technology configurations. Additional investigations may be required for specific cases and to fully understand the levels of furan in all commercial coffee products. Roasting in commercial plants is typically followed by storage of the roasted whole beans of up to several hours before grinding -with maximum tolerable storage time depending on individual quality criteria. For the purposes of these tests the effect of this storage was simulated by storing freshly roasted coffee in open bags at defined temperatures and sealing the bags after defined periods to stop potential furan losses. Analyzing samples with up to 96 hours of storing at ambient temperatures and at 40 °C shows that results are constant over time within a range of +/-20% which represents the measurement uncertainty (95% confidence level) of the analytical methodology. This indicates that storage of whole beans of up to 96 hours seems not influenced by temperature. Moreover, such a storage does not seem to significantly impact the furan levels in an open system (Figure 5).

Here Figure 5 Grinding In order to ensure a consistent brew performance and extractability the roast whole beans are ground to controlled mean particle sizes. This grinding process opens the cell structures of the beans and it is known that grinding potentially causes significant losses of volatile aroma compounds. The level of aroma loss depends on specific process conditions. It was therefore to be expected that furan levels will be reduced as well during grinding. Tests to estimate the magnitude of furan losses at grinding have been done with two different types of laboratory grinders (Mahlkönig: Model DK5LS; Modern Process Equipment Inc: Model MPE 4555). It is shown that in parallel to achieving smaller mean particle size the furan loses are increasing (Figure 6). At typical European drip filter grind sizes of 350-500 µm, furan losses are estimated to be in the order of magnitude of 40%. With starting levels of ca. 2000-7000 µg kg -1 in roast whole beans depending on roasting conditions this would lead to levels of 1200-4200 µg kg -1 after grinding. This range is in line with levels as published previously [START_REF] Kuballa | Furan in Kaffee und Kaffeegetränken[END_REF][START_REF] Zoller | Furan in Food: Headspace method and product survey[END_REF]) with 790-3390 µg kg -1 and 959-5938 µg kg -1 respectively.

Here Figure 6 Degassing CO 2 is generated in coffee beans as part of the roasting reactions. Roast & ground coffees which are intended to get packed in vacuum packs need to degas before packing. This is to allow the CO 2 to escape from the beans in order to avoid that this takes place inside the vacuum bag which would result in a pressure build up and softening of the pack. The required degassing times are mainly depending on bean type, roast conditions, moisture and particle size. For this study, two roast and ground samples were placed into open plastic bags directly after roasting. After defined degassing periods at ambient temperatures the bags were sealed and prepared for analysis. Results show that degassing of up to four hours resulted in a furan reduction of app. 20% (Figure 6).

The degree of reduction for commercial plant degassing processes will additionally depend on product temperature after grinding. Furthermore and depending on specific facilities configurations the effect of the transport from the degassing silo to the packaging machine needs to be assessed as a potential basis for additional furan losses.

Shelf life

It was investigated if furan levels in packed coffee products would change during shelf life.

For this, vacuum packed roast & ground coffee was stored at 4 °C and at ambient temperatures (Figure 7). As shown all results are within a range of +/-20% which represents measurement uncertainty (95% confidence level) of the analytical methodology. It therefore can be concluded that levels of furan for roast coffee are not significantly changing during the first three month of the shelf life.

Here Figure 7 Consumer handling Kitchen life Typically, in Europe roast coffee is packed and sold in vacuum packs or soft packs with pack quantities for more than one brewing / serving. The packs are being used up over a longer time period. Therefore a product sample taken out of a freshly opened product should not be seen as representative for an exposure estimate. It needs to be understood, how the 'kitchen life' -the typical house hold use up period-impacts the furan levels. There are different consumer practices in place from leaving packs open to tightly closing packs during the use-up period. Specifically and for quality preservation reasons it is often recommended to store opened pack in a refrigerator to maintain the initial and desired aroma level.

In a simulation of a typical consumer handling a 500 g pack of a roast coffee the product was opened, followed by taking a defined quantity of product (25 g) out of the pack, reclosing the pack tightly (with a clip) and storing under different conditions (ambient or in a refrigerator). This procedure was repeated once every day.

The furan level in the refrigerated coffee remained almost constant over the usage period. In contrast, the products which have been stored at ambient temperatures (both whole beans and ground coffee) are showing a significant decrease in furan levels in the order of magnitude of 20-25% over a seven days usage period. Brewing Furan exposure estimates require realistic brews as made for consumption. It has to be understood that preparing a cup of coffee for consumption is not harmonized at all and a number of different aspects need to be considered, especially as furan is very volatile and has low water solubility properties. The most important aspects cover:

-Type of preparation. Brewing of roast & ground coffee on drip filter machines is still typical for the majority of the European coffee consumption. Other methods like fully automatic machines (which include grinding of roast whole beans), espresso brewing (with typically higher coffee usage per beverage volume) and machines based on coffee pads, disks or capsules are preparing single cups for immediate consumption.

For these coffees storage / warming of the beverages on hot plates until consumption does not need to be considered.

-Brew recipe. The brew recipe is varying significantly from consumer to consumer and typically consumers are preparing their coffees according to individual preferences.

There may also be regional trends to be considered. Espresso beverages are typically based on higher brew recipes (i.e. same amount of coffee powder is used for a lower beverage volume) resulting in higher levels of coffee solubles (aroma, volatile compounds etc.) per volume of beverage. For exposure estimates of furan these higher Espresso levels should not be seen as typical for the total coffee intake.

Furan levels should only be considered together with the corresponding consumption rates and volumes of the specific type of coffee preparation.

The effect of brewing and the corresponding transfer of furan from a roast & ground coffee product into the beverage have been investigated intensively by [START_REF] Kuballa | Furan in Kaffee und Kaffeegetränken[END_REF] and La [START_REF] Pera | Analysis of furan in coffee of different provenance by head space solid phase microextraction gas chromatography-mass spectrometry: effect of brewing procedures[END_REF]. Both studies showed significant losses for furan at the brewing step. On basis of this existing data base for the effect of brewing, in this study only one brewing experiment per type of brewing equipment has been performed to confirm the magnitude of the effect . On basis of the data from this study together with the data from the other studies it can be assumed that in average and across different brewer types 50-60% of the furan in the roast & ground coffee is lost when brewing coffee.

Here Table 1 Our results are within the reported variation from earlier studies (Table 1). Variability of results for data from different brewing methodologies are probably due to differences in design of equipment in terms of brewing temperatures, brewing time and how closed the system is to retain volatile components, including furan. For a specific brewing system the sampling procedure needs to be specified in detail to allow reproducible data. In order to get relevant data for consumer exposure estimates it is recommended to take the brew samples after a defined cooling period to get to typical drinking temperature of the beverage.

Published data for furan in the prepared beverage from roast coffee [START_REF] Kuballa | Furan in Kaffee und Kaffeegetränken[END_REF][START_REF] Zoller | Furan in Food: Headspace method and product survey[END_REF][START_REF] Morehouse | Survey of furan in heat processed foods by headspace gas chromatography/mass spectrometry and estimated adult exposure[END_REF] show a range from 8-199 µg l -1 . The upper end of the range has been analyzed from espresso type beverages which -due to the higher recipes of typically 100-140 g coffee per liter compared to 30-60 g coffee per liter for a standard cup of coffee-results in higher levels of furan. For an exposure estimate from an European perspective it has to be considered though that the average consumption rate and volume for espresso coffee is much lower than the consumption rate of a standard cup of coffee.

Hot plate / cup

Coffee beverages brewed on drip filter machines are typically prepared to be consumed over a longer time period after brewing while the beverage is kept hot on a heating plate. This may cause furan losses depending on time, temperature and design of the coffee machine.

For this study a trial was conducted to analyze the change in furan level in the brew within the first 60 min. This period covers the timeframe which is typically provided as guidance on how long coffee brew can be kept without significantly compromising in quality. After 60 min the furan level in the brew has decreased to app. 50% of the initial level as measured immediately after brewing. Assuming that brews in average are kept hot for 30 min, this would lead to reduction rates of 35%.

The effect on furan loss by pouring the beverage into a cup including the cooling of the beverage in the cup to normal drinking temperatures was estimated in a separate trial. This showed that furan in a cup is reduced by 25% after 30 min. As coffee is typically consumed within the first few minutes, a reduction could be assumed in the order of magnitude of 10%.

Here Figure 9 These findings are consistent with results reported from other studies. [START_REF] Goldmann | Rapid determination of furan in heated foodstuffs by isotope dilution solid phase micro-extraction-gas chromatography-mass spectronomy[END_REF] and [START_REF] Zoller | Furan in Food: Headspace method and product survey[END_REF] have analysed brews when exposed to the atmosphere at room temperature. With higher losses in the beginning and diminishing losses with time after 1 hour the furan content in the brew was reduced to 30% respectively 50% of the initial level after brewing. Higher losses have been analyzed by La [START_REF] Pera | Analysis of furan in coffee of different provenance by head space solid phase microextraction gas chromatography-mass spectrometry: effect of brewing procedures[END_REF] when maintaining the temperature of the brew. Depending on the temperature 50% losses of the initial furan content have been achieved after 10 min or less.

Figure 1 .

 1 Figure captions

Furan in Beverage (µg/kg)

	F o r
	P
	e
	Coffee on a hot plate r e
	Coffee poured into a cup R
	e
	v i e
	w
	O n l
	y
	1

Table 1 .

 1 Effect of brewing method on furan extraction using different brewing techniques.Data presented show the amounts of furan in the brew expressed as percentage of the initial furan levels in roast and ground respectively instant coffee.

		This study (n=1)	Kuballa et al. 2005	La Pera et al. 2009
	Drip Filter	50%	Mean: 26%
			Range: 11-91%
	Cafetière/ Infusion in	65%		43.0 +/-6.9%
	hot water
	Pads	50%
	Espresso	33%	Mean: 40%	36.7 +/-7.9
			Range: 23-57%
	Instant Coffee	81%	Mean: 70%
			Range: 32-122%

Table 2 .

 2 Summary of influencing factors on furan from roasting to coffee as consumed.

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

Conclusions

In order to provide consumers with products which they can enjoy due to their levels of volatile aroma compounds the industry has implemented technologies and processes to protect products against aroma losses. As furan is a volatile component as well, all these measures will also reduce furan losses during production

The studies described in this paper have shown that furan is generated as a function of the process parameter 'degree of roast' and 'roast time' with no significant options to modify these process parameters to achieve lower furan levels. Firstly, these parameters have to be kept in narrow ranges to maintain the quality/flavor profile of a product. Secondly, these same parameters are known factors to influence the levels of acrylamide in coffee in an opposite direction. Acrylamide is another, undesired component generated when roasting coffee.

During subsequent processing and consumer handling the furan levels will decrease significantly and all factors need to be assessed when trying to estimate typical levels of furan in coffee when consumed. For this assessment it needs to be noted that 'best practices' for commercial plant processes and advice to consumers to retain the desired volatile aroma component in the coffee product will also reduce the losses for furan in parallel. As a guidance from this study and in consideration of the estimated typical impact factors of each process and handling step the total furan losses from roasting to final cup for consumption are indicated to be around 90%.

Here Table 2 On basis of the full range of furan levels after roasting of this study and assuming a brew recipe of 50 g coffee per liter it would calculate to in-cup furan levels of 10-35 µg l -1 , which is at the low end of published data (ranging from 8-199 µg l -1 ). The discrepancy can be explained as the published data includes brews based on higher brew recipes and may not consider all impacting factors from a typical consumer handling in their results as outlined in this study.

This project has been financed by the members of the European coffee working group


Dinoraz Velez

Silvia Torres-Escribano

Rosa Montoro

Dinoraz Vélez

Mercury and methylmercury bioaccessibility in swordfish 1 2 3

Keywords: Metal Contamination Montoro, Rosa Metals -bioavailability, Metals analysis Additives/Contaminants: Heavy metals -mercury mercury, methylmercury, bioaccessibility, swordfish, intake, risk 31 assessment

problem for many countries. This study analyzes total Hg concentrations in 27 samples 16 of swordfish marketed in Spain in 2005 and in their bioaccessible fractions (soluble 17 concentration in gastrointestinal medium), obtained after applying an in vitro digestion 18 method. Methylmercury (MeHg) was also determined in the bioaccessible fractions. 19 Total Hg concentrations in the samples were 0.41-2.11 mg kg -1 wet weight (ww), with a 20 mean value of 0.96 ± 0.47 mg kg -1 ww. 37% of the samples exceeded the Hg limit set 21 by Spanish legislation (1.0 mg kg -1 ww). Bioaccessible total Hg concentrations were 22 0.17-1.72 mg kg -1 ww (0.63 ± 0.4 mg kg -1 ww), corresponding to 38-83% (64 ± 14%) 23 of total Hg. Bioaccessible MeHg concentrations, representing 94% of the bioaccessible 24 total Hg concentrations, were 0.16-1.53 mg kg -1 ww, with a mean value of 0.49 ± 0.32 25

INTRODUCTION 35
Consumption of fish offers an excellent source of protein and essential nutrients such as 36 iodine and selenium. Also, some species of oily fish provide significant amounts of 37 long-chain, polyunsaturated omega w-3 fatty acids, eicosapentaenoic acid (EPA), and 38 docosahexaenoic acid (DHA), which help to reduce the risk of cardiovascular disease 39 [START_REF] Fussenegger | What kind of fish on the table? Omega-3 fatty acids versus mercury contamination[END_REF]. However, fish can also be a source of substances harmful to 40 the body, as they contain polychlorinated biphenyls, dioxins, and methylmercury 41 (MeHg) [START_REF] Virtanen | Mercury as a risk factor for cardiovascular diseases[END_REF]. 42

It is currently considered that consumption of fish is the main path for human 43 exposure to mercury (Hg) [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to mercury and methylmercury in food[END_REF]. In predatory marine fish, about 90% of 44 mercury is in the methylated form (methylmercury, MeHg) [START_REF] Who | Guidance for identifying populations at risk from mercury exposure[END_REF], while the 45 remainder consists of small or undetectable quantities of inorganic mercury [Hg(II)], 46 ethylmercury, and phenylmercury [START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF][START_REF] Chang | Speciation analysis of mercury and lead in fish samples using liquid chromatography-inductively coupled plasma mass spectrometry[END_REF]. The 47 MeHg/Hg ratio is lower in freshwater fish [START_REF] Who | Guidance for identifying populations at risk from mercury exposure[END_REF]. Methylmercury compounds 48 are considered by the International Agency for Research on Cancer as possibly 49 carcinogenic to humans, Group 2B (IARC, 1993). Research has shown that MeHg 50 hearing loss, delayed development, language disorders, and memory loss [START_REF] Who | Exposure to mercury: a major public concern[END_REF]. 52

Prenatal mercury exposure may produce alterations affecting children's 53 neurodevelopment [START_REF] Gao | Prenatal exposure to mercury and neurobehavioral development neonates in Zhoushan City, China[END_REF][START_REF] Jedrychowski | Fish consumption in pregnancy, cord blood mercury level and cognitive and psychomotor development of infants followed over the first three years of life psychomotor development of infants followed over the first three years of life. Karkow epidemiological study[END_REF]. 54

Predatory fish such as swordfish, shark, and tuna have the highest concentrations 55 of MeHg. Therefore, the European Food Safety Agency (EFSA), the Food and Drug 56 Administration (FDA), and the Environmental Protection Agency (EPA) have advised 57 vulnerable population groups (women who may become pregnant, pregnant women, 58 nursing mothers, and young children) to avoid some types of fish that might accumulate 59 high levels of MeHg, such as large predators, and eat fish and shellfish that are lower in 60 mercury [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to mercury and methylmercury in food[END_REF][START_REF] Fda | What you need to know about mercury in fish and shellfish[END_REF]. Hg and MeHg have been quantified in various 61 species of fish [START_REF] Mira | Avaliação da composição centesimal, aminoácidos e mercúrio contaminante de surimi[END_REF][START_REF] Morgano | Mercury levels in freshwater fishes from piscicultures established in São Paulo State[END_REF]Burger and 62 Gochfeld, 2006;[START_REF] Sivaperumal | Heavy metal concentrations in fish, shellfish products from internal markets of India vis-a-vis international standards[END_REF][START_REF] Afonso | Contaminant metals in black scabbard fish (Aphanopus carbo) caught off Madeira and the Azores[END_REF][START_REF] Cortes | Mercury contents in Chilean fish and estimated intake levels[END_REF]; 63 the influence of the physiological characteristics of the fish (sex, age) on the Hg 64 concentrations has been evaluated [START_REF] Monteiro | Mercury content of swordfish, Xiphias gladius, in relation to length, weight, age, and sex[END_REF][START_REF] Afonso | Contaminant metals in black scabbard fish (Aphanopus carbo) caught off Madeira and the Azores[END_REF]65 Storelli et al., 2007), and intakes of Hg have been calculated for child and adult 66 populations [START_REF] Llobet | Concentrations of arsenic, cadmium, mercury, and lead in common foods and estimated daily intake by children, adolescents, adults, and seniors of Catalonia, Spain[END_REF]Wilhelm, 2003;[START_REF] Muñoz | Estimation of the dietary intake of cadmium, lead, mercury, and arsenic by the population of Santiago (Chile) using a Total Diet Study[END_REF][START_REF] Falcó | Daily intake of arsenic, cadmium, mercury, and lead by consumption of edible marine species[END_REF]67 Farias et al., 2006;[START_REF] Marti-Cid | Intake of chemical contaminants through fish and seafood consumption by children of Catalonia, Spain: Health risks[END_REF][START_REF] Sahuquillo | Methylmercury determination in fish and seafood products and estimated daily intake for the Spanish population[END_REF][START_REF] Rubio | Total dietary intake of mercury in the Canary Islands, Spain[END_REF]. 68

To make a more realistic evaluation of the toxicological risk involved in intake of 69 Hg from consumption of fish, it is necessary to estimate not only the concentration in 70 the product as consumed but also its bioavailability (the fraction of the intake that is 71 absorbed and reaches the systemic circulation and is thus available to exercise its effect 72 on the receiving organism). For an element to be absorbed by the intestinal epithelium, 73 the first requisite is that it should be soluble. It is therefore interesting to know the 74 bioaccessible content, i.e., the maximum soluble content in simulated gastrointestinal 75 Bioaccessibility can be used as an indicator of maximum oral bioavailability 79 [START_REF] Versantvoort | Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food[END_REF]. Static and dynamic in vitro gastrointestinal models can be 80 used to determine bioaccessibility of nutrients. The static models simulate transit 81 through the human digestive tract by sequential exposure to simulated mouth, gastric, 82 and small intestinal conditions (pH, temperature, time, enzymes, etc.) (Oomen et al., 83 2002), and they are the ones that have been most used in studying the bioaccessibility of 84 trace elements from food samples (Intawongse and Dean, 2006). 85

Very little research has been done on Hg bioaccessibility [START_REF] Cabañero | Selenium and mercury bioaccessibility in fish samples: an in vitro digestion method[END_REF](Cabañero et al., , 86 2007;;[START_REF] Shim | Impact of phytochemical-rich foods on bioaccessibility of mercury from fish[END_REF]. It is therefore of great interest to investigate how consideration 87 of the bioaccessible content of total Hg (t-Hg) and MeHg instead of the content in the 88 raw product affects exposure assessments. The aims of the present work were a) to 89 quantify concentrations of t-Hg in raw swordfish and concentrations of t-Hg and MeHg 90 in the bioaccessible fraction obtained after an in vitro gastrointestinal digestion method 91 and b) to make risk assessments by estimating daily intake of Hg and MeHg resulting 92 from consumption of this top predator. 93

94

MATERIALS AND METHODS 95

Equipment. A microwave accelerated reaction system (MARS) from CEM (Vertex,96 Spain) operating at a power of 800 W was used for digestion of samples prior to 97 quantification of t-Hg. Teflon perfluoroalkoxy (PFA) vessels of 55 ml inner volume 98 were employed. For t-Hg quantification, a continuous flow cold vapor generation-99 For Hg speciation analysis, the HPLC system employed (Hewlett Packard Model 102 1100, Spain) was equipped with a quaternary pump, an on-line degassing system, an 103 automatic injector and a thermostated column compartment. Separations were 104 performed on a Hamilton PRP-X100 anion-exchange column (10 µm, 250 mm × 4.1 105 mm i.d., Teknokroma, Barcelona, Spain). A guard column packed with the same 106 stationary phase (12-20 µm; 25 mm × 2.3 mm i.d.) preceded the analytical column. A 107 heated bath (Julabo model HC, Merck, Spain) was used to thermooxidize the outlet 108 from the HPLC column. After thermooxidation the Hg content was quantified by CV-109

AFS. An analog-digital converter (Model 35 900 C, Hewlett Packard) was used to 110 acquire the AFS signal, which was processed by the chromatographic software. 111

Other equipment used included a lyophilizer (FTS Systems, USA), a mechanical 112 shaker (KS 125 Basic, IKA Labortechnik, Merck, Spain), a pH meter (pH 526,Multical,113 Spain), an ultrasonic bath (J.P. Selecta, Spain), a heating bath (HC Julabo,Merck,114 Spain), an orbital shaking water bath (Unitronic Orbital C, J. P. Selecta, Spain), and 115 various centrifuges (Superspeed refrigerated centrifuge Du Sorvall,116 Sorvall Pont;Eppendorf 5810,Merck;Heraeus Biofuge Pico,Merck). 117 118 Reagents. In the determination of t-Hg and MeHg, deionized water (18.2 MΩ cm), 119 obtained with a Milli-Q water system (Millipore Inc., Millipore Ibérica, Madrid, Spain), 120 was used for the preparation of reagents and standards. All glassware was treated with 121 10% v/v HNO 3 for 24 h, and then rinsed three times with deionized water before use. 122

All chemicals used were of analytical or reagent grade. A standard solution of 123 1000 mg L -1 Hg (Merck) was employed. A standard solution of 1000 mg L -1 MeHg was 124 Samples. In Spain, consumption of frozen swordfish is much higher than consumption 139 of fresh swordfish (94% as opposed to 6% in 2003, according to data provided by the 140 Ministry of the Environment and Rural and Marine Environment's General Secretariat 141 of the Sea). Reflecting the habits of the Spanish population, we analyzed 27 samples of 142 frozen swordfish muscle. The swordfish muscle samples were provided in 2005 by 143 companies that market this product in Spain. The samples were thawed, the skin was 144 removed, and the edible portions were frozen, lyophilized, ground, and stored at 4 ºC 145 until analysis. A reference material with certified contents for t-Hg and MeHg, DORM-146 2 sample (muscle of dogfish, National Research Council of Canada), was also analyzed. 147

Each sample was analyzed in triplicate, for both t-Hg and MeHg and their respective 148

bioaccessibilities. 149 150

In vitro gastrointestinal digestion. Samples of seafood products were digested using a 151 simulated digestion process developed by our group in an earlier study (Laparra et al., 152 2003). A quantity of lyophilized swordfish sample, equivalent to 10 g of fresh sample, 153 was weighed and cellular-grade water (90 mL) was added. The pH was adjusted to 2.0 154 with 6 mol L -1 HCl. After 5 min, the pH value was checked and if necessary readjusted 155 to pH 2.0. Freshly prepared pepsin solution (1 g of pepsin in 10 mL of 0.1 mol L -1 HCl) 156 was added to provide 0.01 g of pepsin/10 g fresh sample. The sample was made up to 157 100 g with water, and incubated in a shaking water bath (stroke rate 120 min -1 ) at 37 ºC 158 for 2 h to emulate the gastric stage of digestion. 159

Then, for the intestinal digestion, the pH value was raised to pH 5.0 by drop-wise 160 addition of 1 mol L -1 NaHCO 3 . The pancreatin-bile extract mixture (0.2 g of pancreatin 161 and 1.25 g of bile extract in 50 mL of 0.1 mol L -1 NaHCO 3 ) was added to provide 162 0.0025 g of pancreatin/10 g fresh sample, and 0.015 g of bile extract/10 g fresh sample. 163 3). Only in samples acquired in Taiwan much 217 higher mean concentrations have been found (3.6 ± 0.5 mg kg -1 ww) (Chen et al., 2007). 218 Some authors have found a linear correlation between the concentrations of Hg 219 and the length and age of the fish, as most of the Hg is in the form of MeHg bound to 220 the thiol groups of proteins, which increase with age [START_REF] Monteiro | Mercury content of swordfish, Xiphias gladius, in relation to length, weight, age, and sex[END_REF]221 Storelli and Marcotrigliano, 2001;[START_REF] Branco | Total and organic mercury concentrations in muscle tissue of the blue shark (Prionace glauca L. 1758) from the Northeast Atlantic[END_REF][START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF]. A 222 correlation has also been found between the concentration of Hg in the muscle and the 223 area where the fish was caught [START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF]. The size of the liver of predatory 224 fish is another physiological variable that researchers have attempted to correlate with 225 Hg concentrations. However, further studies must be conducted in this regard as the 226 results differ depending on the species of fish considered. Concentrations have been 227

found in the liver of swordfish and bluefin that are between 2 and 3 times greater than 228 the levels in muscle (Storelli et al., 2005;[START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF], attaining 9.8 µg g -1 ww 229 in the liver of swordfish from the area of Ecuador [START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF]. In samples of 230 shark, however, Hg concentrations in the liver have been shown to be less than those of 231 muscle [START_REF] Branco | Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean[END_REF]. 232

With regard to the food safety of the samples analyzed, 37% exceed the value of 1 233 µg g -1 ww, the maximum limit of Hg permitted by Spanish legislation in swordfish 234 (European Commission, 2006). It is important to emphasize that food alerts concerning 235 high Hg concentrations in fish have increased in Spain and other countries in recent 236 years. This is shown in a report of the Rapid Alert System for Food and Feed of UE 237 (RASFF, 2009), which gives details of the notification of 128 alerts concerning mercury 238 For this purpose, we evaluated the analytical characteristics (detection limit, 248 precision, and recovery) of the t-Hg method, which includes in vitro digestion, 249 microwave acid digestion of the soluble fraction, and quantification by CV-AFS. The 250 results obtained endorse the suitability of the method for the aims proposed, and the 251 absence of matrix interference: limit of detection = 0.02 ng g -1 , ww; precision = 10%; 252 recovery = 98%. The quantification of MeHg in the bioaccessible fraction entails a 253 more complex treatment, because after the in vitro gastrointestinal digestion it is 254 necessary to lyophilize the soluble fractions and then perform an ultrasonic acid 255 extraction of MeHg. The analytical characteristics of the method endorse its use for the 256 aims proposed: limit of detection = 0.11 ng g -1 , ww; precision = 5%; recovery = 87-257

104%. 258

Bioaccessible t-Hg. The concentrations of bioaccessible t-Hg (Table 2) varied 259 over a wide range: 0.17-1.72 mg kg -1 ww (mean value = 0.63 ± 0.4 mg kg -1 ww). In the 260 study that was conducted, there was not a proportional relationship between the Hg 261 concentration in the samples and their gastrointestinal solubility. This can be seen in 262 various samples, including samples 17 and 22, where the t-Hg concentrations in the 263 Recommended safe intake. The results presented indicate that there is a small 361 percentage of the child and adult population in Spain, corresponding to consumers of 362 swordfish, that is at risk as a result of exposure to Hg from this product. This is the case 363 even when the swordfish has a concentration of Hg below the maximum limit 364 established by the legislation (1 mg/kg ww). This should lead the authorities to make 365 recommendations of maximum consumption of this product by children and adults. In 366 view of the neurotoxic nature of MeHg and its transmission through the placenta and 367 breast milk, some countries have adopted recommendations for groups at risk: women 368 who may become pregnant, pregnant women, nursing mothers, and young children. 369 This is the case in the UK, where the FSA recommends that babies and pregnant women 370 should not consume swordfish and that breastfeeding women should not consume more 371 than one portion per week [START_REF] Fsa | Food Standards Agency of UK[END_REF]. Another example is Canada, where Health 372 Canada recommends a maximum consumption of 150 g of swordfish per month for 373 certain women (those that are or may become pregnant or are breastfeeding), 125 g per 374 month for children 5-11 years old, and 75 g per month for children 1-4 years old 375 (Health Canada, 2008). In Spain, there are no recommendations in this regard. As Table 376 6 shows, even if all the Hg quantified in the fish analyzed were in the form of MeHg, 377 the weekly consumption of 25 g swordfish/week for Spanish children and 50 g/week for 378 adults would give MeHg intakes lower than the PTWI recommended by the WHO. 379 Even if the concentration of Hg in the fish (0.958 mg kg -1 ww) were very close to the 380 maximum value permitted by the legislation (1 mg kg -1 ww), consumption of these 381 quantities would not represent a health risk. Recommendations for the Spanish 382 population concerning monthly consumption could be set at values very close to those 383 stipulated in the UK and Canada. 384 the present study (0.164-1.53 mg kg -1 ww; mean = 0.495) (Fig. 1), the mean estimated 388 intake for children (51.7 g of swordfish/day) ranges between 8 and 79 µg MeHg/day 389 (mean value = 32 µg MeHg/day), exceeding the TDI values recommended by the WHO 390 by 1-10 times (mean = 3.2 times the TDI). For adult consumers of swordfish (60.3 391 g/day according to the study by the CSN mentioned earlier) the estimated intakes of 392 MeHg range between 10 and 92 µg MeHg/day (mean value = 38 µg MeHg/day), values 393 that represent from 63% of the TDI to 6 times the TDI (mean = 1.9 times the TDI). 394

When the value established by USEPA is used as the toxicological reference value, the 395 limit is exceeded for both children and adults and over the entire range of bioaccessible 396 MeHg concentrations found in the fish analyzed. 397

These results show that there would still be a situation of risk if bioaccessible 398 MeHg concentrations were considered instead of using the concentrations in the 399 product, although the TDIs are 2.5 times lower than those obtained from the contents in 400 raw swordfish. If MeHg bioaccessibility were considered, therefore, perhaps the 401 recommendations concerning consumption of swordfish could be modified by 402 increasing the quantity consumed or the frequency of consumption. 403

The results presented show the need for recommendations about swordfish 404 consumption by population groups at risk in Spain, and the desirability of broadening 405 the study of the bioaccessibility of MeHg from these products. 406 b Minimum, mean, and maximum concentrations of mercury for all the swordfish samples analyzed in this study (Table 2).

b Intakes calculated on the basis that all the Hg is present as MeHg.

  effects such as mental retardation, seizures, vision and 51

  available for subsequent processes of absorption into the intestinal 76 mucosa. The term bioaccessibility also indicates the relation between the bioaccessible 77 content of a substance and the total content of the substance present in the sample. 78

  (CV-AFS) model PSA 10.025, Millennium Merlin 100 (PS Analytical, UK), was used. 101

  a commercially available salt of MeHgCl (Sigma-Aldrich) in 125 50% (v/v) MeOH/H 2 O mixture. Working standard solutions of Hg and MeHg were 126 prepared daily by serial dilutions of standard solution. 127 Other reagents used were: hydrochloric acid (Merck); nitric acid (Merck); acetic 128 acid (Probus); sulfuric acid (Panreac); sodium hydroxide (Merck); hydrogen peroxide 129 (Prolabo); L-Cysteine (Merck); tin(II) chloride dihydrate (Scharlau Chemie S.A, Spain); 130 sodium hydrogen carbonate (Panreac), and potassium peroxodisulfate (Prolabo). 131 Enzymes and bile salts were purchased from Sigma Chemical Co. (St. Louis, MO, 132 USA): porcine pepsin (enzymatic activity 944 U/mg protein), porcine pancreatin 133 (activity equivalent to 4 x US Pharmacopoeia specifications/mg pancreatin), and bile 134 extract (glycine and taurine conjugates of hyodeoxycholic and other bile salts). Water of 135 cellular grade (B. Braun Medical, S.A., Barcelona, Spain) was used throughout the in 136

  37 ºC continued for 2 hours. The pH was then adjusted to 7.2 by 164 addition of 0.5 mol L -1 NaOH. Aliquots of 40 g of the digests were transferred to 165 polypropylene centrifuge tubes and centrifuged (15000 rpm/30 min/4 ºC) to separate 166 soluble and precipitate. The concentrations of t-Hg and MeHg were quantified in the 167 soluble fraction. 168 169 Total mercury determination. An assisted digestion in microwave oven with 170 subsequent quantification by CV-AFS was used for the determination of t-Hg in the 171 samples of swordfish and in their bioaccessible fractions. 172Lyophilized swordfish samples (0.2 g) or bioaccessible fractions (0.6 g) were 173 placed in a Teflon PFA vessel and treated with 4 mL of HNO 3 concentrate (14 N) and 1 174 mL of H 2 O 2 . The Teflon PFA vessel was irradiated at 800 W (180 ºC, 15 min). At the 175 end of the digestion program, the digest was placed in a 250 mL beaker and allowed to 176 rest all night to eliminate nitrous vapor. It was then filtered through Whatman No. 1 177 paper and made up to volume with 5% HCl (v/v). 178 Mercury contents were determined by CV-AFS using the following analytical 179 conditions: reducing agent, 2% (m/v) SnCl 2 in 15% (v/v) HCl, 4.5 mL min -1 flow rate; 180 blank reagent, 5% HCl (v/v), 9 mL min -1 flow rate; carrier gas, argon 0.3 L min -1 flow 181 rate; dryer gas, air 2.5 L min -1 flow rate. 182 Throughout the experiment, the quality assurance-quality control of Hg 183 measurement was checked by analyzing the certified reference material DORM-2 with 184 each batch of samples. 185 186 Methylmercury determination. For extraction of mercury species an ultrasonic acid 187 extraction was employed. The lyophilized swordfish samples (0.2 g) or bioaccessible 188 6 g) were weighed into a 50 mL centrifuge tube and 8 mL of 2.4 N HCl was 189 added. The mixture was sonicated for 5 min, centrifuged (2000 rpm/15 min), and the 190 supernatant was centrifuged again (12000 rpm/10 min). The resulting extract was 191 filtered through 0.45 µm Whatman Nylon prior to species quantification by Samples were injected into the PRP-X100 column and, using PTFE tubing and T-194 joints, the eluate from the column was mixed with the persulfate solution. The mixture 195 was thermooxidized by being passed through a loop of Teflon tubing placed in a heated 196 bath. After cooling in an ice bath, the eluate was mixed with a continuous flow of 197 SnCl 2 . Using a gas-liquid separator and a continuous flow of argon, the arsines 198 generated were introduced into the AFS. The hygroscopic-membrane drying tube used 199 to transport the arsines allowed elimination of moisture by circulating a counterflow of 200 air. Details of operating conditions are given in Table1. 201 Signals were identified by coincidence of sample and standard retention times. 202The quantification was obtained from the peak area by interpolation of external 203 calibration curves. Throughout the experiment, the quality assurance-quality control of 204 MeHg measurement was checked by analyzing the certified reference material DORM-205 2 with each batch of samples. 206207RESULTS AND DISCUSSION 208Total mercury in swordfish. The concentrations of t-Hg in the swordfish samples 209 analyzed (Table2) ranged between 0.41 and 2.1 mg kg -1 ww (mean ± standard deviation 210 = 0.96 ± 0.47 mg kg -1 ww; n = 27). The analysis of Hg in seafood products marketed in 211 Spain is a customary practice in Spanish food control laboratories, but few data are 212 published in scientific journals. The range of concentrations obtained overlaps the few 213

  of which were for swordfish. Of the samples that exceeded the legislated 239 values, 34% were of Spanish origin, which demonstrates the need for the application of 240 rigorous health control measures for these products. 241 242 Total Hg and MeHg bioaccessible in swordfish. The swordfish samples were 243 subjected to a gastrointestinal digestion and t-Hg and MeHg were determined in the 244 bioaccessible fraction. As the bioaccessible fraction is a very different matrix from the 245 raw seafood, it was necessary to evaluate the suitability of the application of the 246 methods for t-Hg and MeHg quantification developed previously in the laboratory. 247

  bioavailability into account would alter the risk associated with 386 consumption of this product. Assuming the quantities of bioaccessible MeHg found in 387

Figure 1 .

 1 Figure 1. Bioaccessible total mercury (t-Hg) and bioaccessible methylmercury (MeHg) (expressed as Hg) in the soluble fractions of 15 swordfish samples (mg kg -1 , wet

Table 1 .

 1 Instrumental parameters used for the determination of methylmercury by HPLC-thermooxidation-CV-AFS

	HPLC
	Column	Anionic exchange Hamilton PRP X-100; polymer base; 10 µm; 250
		mm x 4.1 mm i.d.
	Precolumn	Hamilton PRP X-100; 12-20 µm; 25 mm x 2.3 mm i.d.
	Mobile phase	0.04 mol L -1 cysteine in 0.1 mol L -1 acetic acid
	Injection volume	100 µL
	Flow	1 mL min -1
	Thermooxidation
	Oxidant	1% (m/v) K 2 S 2 O 8 in 0.5% (mol L -1 ) H 2 SO 4 . Flow 2 mL min -1
	Reaction loop	3 m x 0.5 mm i.d.
	Bath temperature	150 ºC
	CV-AFS
	Reducing agent	2% (m/v) SnCl 2 in 15% (v/v) HCl. Flow 2 mL min -1
	Carrier gas	Argon. Flow 300 mL min -1
	Dryer gas	Air. Flow 2.5 mL min -1
	Amplification range	100
	Filter	32
	Wavelength	254 nm

Table 2 .

 2 Total mercury (t-Hg) concentrations in swordfish samples (mg kg -1 , wet weight), bioaccessible t-Hg (mg kg -1 , wet weight), and t-Hg bioaccessibility.

Values are expressed as mean ± standard deviation (n = 3).

	Sample	t-Hg		Bioaccessible t-Hg	Bioaccessibility a
	1		0.440 ± 0.006		0.166 ± 0.015	38
	2		0.826 ± 0.027		0.608 ± 0.017	74
	3		0.787 ± 0.051		0.299 ± 0.022	38
	4 5 6 7	0.779 ± 0.043 0.605 ± 0.007 0.705 ± 0.073 0.748 ± 0.071 o F r		0.522 ± 0.030 0.301 ± 0.025 0.542 ± 0.025 0.371 ± 0.022	67 50 77 50
	8 9		0.650 ± 0.023 0.647 ± 0.016 P		0.517 ± 0.039 0.466 ± 0.039	80 72
	10 11 12 13		1.05 ± 0.060 1.41 ± 0.01 e 1.13 ± 0.04 0.439 ± 0.011 e r	0.665 ± 0.065 0.862 ± 0.007 0.622 ± 0.016 0.348 ± 0.006	63 61 55 79
	14 15 16		0.629 ± 0.002 0.880 ± 0.021 0.939 ± 0.049	0.479 ± 0.010 0.514 ± 0.045 0.716 ± 0.010 e R	76 58 76
	17 18 19		1.18 ± 0.04 0.687 ± 0.012 0.782 ± 0.016	0.482 ± 0.018 0.486 ± 0.045 0.502 ± 0.006 v i e	41 71 64
	20 21		1.02 ± 0.09 1.25 ± 0.12		0.624 ± 0.007 0.928 ± 0.014 w	61 74
	22 23 24 25 26 27		1.20 ± 0.04 2.11 ± 0.18 2.00 ± 0.01 2.08 ± 0.01 0.488 ± 0.031 0.413 ± 0.024		0.844 ± 0.004 1.41 ± 0.08 1.66 ± 0.13 1.72 ± 0.13 0.226 ± 0.021 O n l 0.206 ± 0.027 y	70 67 83 83 46 50
	a Bioaccessibility = (t-Hg in bioaccessible fraction of swordfish) x 100
			(t-Hg in swordfish)
					3

Table 3 .

 3 Summary of total Hg levels (mg kg -1 , wet weight) in swordfish reported in the literature since 2004.

	Origin		t-Hg (mg kg -1 , wet weight)	n	Reference
	(specified in article)	min	max	mean
	Spain	----	----	0.42 ± 0.01	1	Cabañero et al., 2004
	Canada (seafood outlets)	0.40	3.85	1.82	10	Forsyth et al., 2004
	Mediterranean Sea	0.02	0.15	0.07 ± 0.04	58	Storelli et al., 2005
	Supermarkets in USA	0.15	3.07	1.40 ± 0.18	18	Burger and Gochfeld,
						2006
	Spain	----	----	1.59-2.22	Composite of 20 samples	Falcó et al., 2006
	Réunion Island	----	----	1.24 ± 0.83	7	Kojadinovic et al., 2006
	Mozambique Channel	----	----	0.38 ± 0.26	37	Kojadinovic et al., 2006
	Azores (Atlantic Ocean)	0.031	2.4	----	29	Branco et al., 2007
	Ecuador (Atlantic	0.9	2.3	----	23	Branco et al., 2007
	Indian Ocean	0.26	2.54	1.47 ± 0.63	171	Chen et al., 2007
	Atlantic Ocean	0.06	3.97	1.20 ± 1.12	55	Chen et al., 2007
	Acquired in Taiwan	----	----	3.64 ± 0.15	1	Chang et al., 2007
	Chilean markets	1.25	1.7	1.53	6	Cortes and Fortt, 2007
	Spain	-	1.74	0.68	24	Blanco et al., 2008
	Spain	0.413	2.11	0.958 ± 0.475	27	This work

Table 4 .

 4 Estimated daily intake of total mercury from Spanish consumption of the swordfish samples analyzed and comparison with the Provisional Tolerable Weekly Intake recommended by the WHO a . Minimum, mean, and maximum concentrations of mercury for all the swordfish samples analyzed in this study (Table2). Consumption of swordfish by the Spanish population(CSN, 2002).

		Hg content in swordfish b
	Minimum	Mean	Maximum
	0.413 mg kg -1 ww	0.958 mg kg -1 ww	2.11 mg kg -1 ww
		Intake

b c

Table 5 .

 5 Estimated daily intake of methylmercury from Spanish consumption of the swordfish samples analyzed and comparison with the Provisional Tolerable Weekly Intake recommended by the WHO a . WHO recommendation for MeHg = 1.6 µg MeHg/kg body weight/week[START_REF] Who | Summary and conclusions of the sixty-first Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)[END_REF]. For Spanish children 7-12 years old, mean body weight is 34.48 kg and the WHO TDI recommendation is 8 µg/day. For Spanish adults, over 17 years old, mean body weight is 68.48 kg and the WHO TDI recommendation is 16 µg/day. Minimum, mean, and maximum concentrations of mercury for all the swordfish samples analyzed in this study (Table2). It is assumed that all of the Hg in raw swordfish is present as MeHg. Consumption of swordfish by the Spanish population(CSN, 2002).

		Hg content in swordfish b
	Minimum	Mean	Maximum
	0.413 mg kg -1 ww	0.958 mg kg -1 ww	2.11 mg kg -1 ww
		Intake

a b c

Table 6 .

 6 Weekly consumption of swordfish by Spanish children and adults that would give intakes of methylmercury below the Provisional Tolerable Weekly Intake value recommended by the WHO a . WHO recommendation for MeHg = 1.6 µg MeHg/kg body weight/week[START_REF] Who | Summary and conclusions of the sixty-first Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)[END_REF]. For Spanish children 7-12 years old, mean body weight is 34.48 kg and the WHO PTWI recommendation is 55 µg/week. For Spanish adults, over 17 years old, mean body weight is 68.48 kg and the WHO PTWI recommendation is 110 µg/day.

				Hg content in swordfish b
			Minimum	Mean	Maximum
			0.413 mg kg -1 ww	0.958 mg kg -1 ww	2.11 mg kg -1 ww
		Consumption		MeHg Intake c		MeHg PTWI a
	Children	25 g/week	10 µg/week	24 µg/week	53 µg/week	55 µg/week
	Adults	50 g/week	21 µg/week	48 µg/week	106 µg/week	110 µg/week

a

kg -1 ww[START_REF] Cabañero | Selenium and mercury bioaccessibility in fish samples: an in vitro digestion method[END_REF][START_REF] Falcó | Daily intake of arsenic, cadmium, mercury, and lead by consumption of edible marine species[END_REF][START_REF] Blanco | Mercury, cadmium and lead levels in samples of the main traded fish and shellfish species in Galicia, Spain[END_REF]. Furthermore, the

values found in the present work are similar to those reported in the last five years for

bioaccessible concentrations are very different (0.48 and 0.84 mg kg -1 ww,

respectively). The bioaccessibility, the percentage relating the bioaccessible Hg

consumption is much greater than the TDI: between 1.9 and 9.5 times. Adults face a

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


Enrico Campioli

Manuela Zavatti

Rossella Avallone

Giulia Puia

Gabriele Losi

Mario ; Baraldi

Evidence that isopropylthioxanthone (ITX

Keywords: Food contact materials, Migration, Packaging -inks Isopropylthioxanthone (ITX), benzodiazepine receptors, anxiety, sedation, electrophysiology

France, Spain and Portugal after an announcement from the Italian authorities to the European Commission via a Rapid Alert System for Food and Feed (RASFF) notification. Some batches of liquid baby milk contained up to 250 µg L -1 (ppb) of ITX (Fig. 1), a photoinitiator used in printing inks for offset printing on the outside of beverage cartons [RASFF Annual Report, 2005]. Its presence in milk was accidentally due to the manufacturing process in which the multilayer cardboard was rolled on reels and the substance was then transferred from the outer printed surface to the inner plastic surface prior to the construction of the individual cartons [RASFF Annual Report, 2005].

The European Food Safety Authority (EFSA), was requested by the European Commission to give an Opinion, and noted that levels of ITX in dairy products varied from 27 to 445 µg/L and from 5 to 249 µg/L in fruit juices, fruit nectars and drinks [START_REF] Efsa | Opinion of the Scientific Panel of Food Additives, Flavourings, Processing Aids and Materials in contact with Food on a request from the commission related to 2isopropyl thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB) in food contact materials[END_REF]. In addition a German research institute also published positive findings of ITX in TetraPack fruit juices of 405 µg/kg [Rothenbacher et al., 2007]. At the end of 2005, EFSA stated that the presence of ITX in food does not raise health concerns, so the Standing Committee decided to close the survey and to take no measures.

Little is known about ITX toxicity and its general biological activity, only a few studies refer that ITX seemed to have limited genotoxicity in vitro [START_REF] Kirkland | Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and noncarcinogens I. Sensitivity, specificity and relative predictivity[END_REF], but literature is lacking in certain proofs. ITX was tested in vivo in the mouse bone marrow micronucleus test [Notox, 2004a] and subsequently tested for its ability to induce unscheduled DNA synthesis in rat liver using an in vivo/in vitro procedure [Notox, 2004b]. In the first experiment, treatments were well tolerated with no signs of systemic toxicity, no increase in the incidence of micronucleated PCEs and no decrease in the ratio of polychromatic to normochromatic erythrocytes in treated animals compared to vehicle controls [Notox, 2004a]. Then in the second experiment, treatments did not produce clinical signs at any time, no increase in mean Net Grain Count (NNG) value and in the incidence of cells in repair (with NNG >5) was observed in rats treated with ITX compared to the vehicle group [Notox, 2004b]. Momo et al. (2007) demonstrated that ITX changes the lipid structure in multilamellar liposomes of saturated phospholipids of different length with and without cholesterol. As a matter of fact in the gel state, ITX acts as a disordering agent, increasing the mobility of the bilayer; while in the fluid state, it tends to rigidify the membrane, in a cholesterol like way [Momo et al., 2007].

During a meeting of CODACONS (private association for the defence of the consumer and the environment) in Modena (Italy) at the end of 2006, it was stated that ITX probably act as a benzodiazepine-like (BDZ) compound [START_REF] Codacons | ITX Meeting[END_REF]. In addition, at the end of February 2009, a judge of Giarre (Catania, Italy) awarded against Nestlè and Tetrapack, compensation to two families for the psychological damage caused by the concern that their daughters might be contaminated by ITX present in the milk they drank [START_REF] Della Sera | [END_REF].

The aim of the present study was to establish whether or not ITX exerts an anxiolytic-sedative effect and if it acts as a benzodiazepine-like compound. Adult male Sprague-Dawley rats (Harlan Italy, Udine, Italy) weighing 250 ± 1.9 g were used throughout the experiments. They were housed (2 rats each cage) in a controlled environment (air-conditioned room at 22 ± 1°C and 60% humidity) maintained under a 12:12 h light/dark cycle. Rats had free access to water and food, a standard pellet containing water 10.68%, crude proteins 21.12%, crude fats 4.22%, fibers 4.11%, ashes 6.61% and other components 53.26% (Mucedola s.r.l., Settimo Milanese, Milano, Italy).

All procedures were performed in accordance with the guidelines of the National (D.L. no. 116/1992) and European legislation (EEC no. 86/609) and of the National Institute of Health on the use and care of laboratory animals. Behavioral experiments were conducted from 10:00 a.m. to 2:00 p.m.

Treatments

ITX (Isopropyl-9H-thioxanthen-9-one, mixture of 2-and 4-isomers; CAS 75081-21-9, purity 97%; Sigma-Aldrich Italy, Milano, Italy) was solubilized in Tween 80 (1%) and water and then administered at the doses of 1, 10 and 25 mg/kg in a volume of 5 ml/kg body weight (b.w.), by oral gavage, 60 min before tests. The control groups received the vehicle treatment (Tween 80 1% and water).

Pentobarbital sodium (Carlo Sessa S.p.A., Milano, Italy) was injected intraperitoneally (i.p.) at the dose of 35 mg/kg in a volume of 2 ml/kg b.w., 60 min after the treatment with the tested compounds. A., Monza, Milano, Italy), used as reference drug in the elevated plus maze test, was i.p. injected at the dose of 1 mg/kg, 20 min before the test; in the open field test it was i.p. injected at the dose of 2 mg/kg, 20 min before the test.

Elevated plus maze test

The elevated plus maze was a wooden apparatus consisting of two opposite open arms (50 cm long, 10 cm wide) and two opposite closed arms of the same size, enclosed by 40 cm high walls. The apparatus was elevated 50 cm above the floor and illuminated from the top. A video camera was suspended above the maze to record trials for analysis. Following the procedure described by Pellow et al. [Pellow et al., 1985]

Central benzodiazepine receptor competition binding assay

The activity of ITX-solutions from 10 -4 M to 10 -9 M on the central benzodiazepine (BDZ) recognition binding site, was evaluated by a radioligand binding assay using [ 3 H]RO 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4-himidazo[1,5-a] previously described from Olasmaa [Olasmaa et al., 1990]. Rat cerebellar membranes (100 µl, 500-1000 µg of protein/ml), re-suspended in sodium phosphate monobasic monohydrate buffer, were incubated with 1 nM [ 3 H]RO 15-1788 (25 µl) and aliquots (100 µl) of ITXsolutions or 1 µM unlabeled diazepam (Roche S.p.A.) to assess non-specific binding. The final volume (250 µl) was obtained by adding sodium phosphate monobasic monohydrate buffer.

The mixture was incubated at 0-4 °C for 1 h. The reaction was terminated by vacuum filtration through glass fibre filters (Whatman GF/C; Whatman International Ltd, United Kingdom) followed by three washings with 3 ml of ice-cold sodium phosphate monobasic monohydrate buffer. The filters were transferred to vials with liquid scintillation cocktail (3 ml) and radioactivity was measured in an LS 1701 Rack Beta liquid scintillation counter (Beckman Coulter S.p.A., Milano, Italy). Protein concentration was determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with Bovine Serum Albumin (BSA) used as a standard.

The activity of the ITX solution was based on the displacement of [ 3 H]RO 15-1788 from cerebellar membranes. This activity was quantified by comparison with a competition curve generated with diazepam at the same concentration of ITX-solutions.

Peripheral benzodiazepine receptor competition binding assay

The activity of ITX-solutions on peripheral benzodiazepine (BDZ) receptor was evaluated by a radioligand binding assay using [ 3 H]PK 11195 (specific activity 3.18 TBq/mmol; NEN TM Life Science Products, PerkinElmer Italy) as specific ligand. Aliquots of ITX-solutions were tested for their ability to inhibit 1 nM [ 3 H]PK 11195 specific binding to rat adrenal glands membranes prepared as previously described from Anholt [START_REF] Anholt | The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane[END_REF]. Adrenal glands membranes (160 µl, 100 µg of protein/ml) resuspended in Tris-HCl buffer were unlabeled PK 11195 (Sigma-Aldrich Italy) to assess non-specific binding. The mixture was incubated at 0-4 °C for 1 h. The reaction was terminated by vacuum filtration through glassfibre filters (Whatman GF/C, Whatman International Ltd) presoaked in 0.5% polyethyleneimine (Sigma Aldrich Italy) followed by three washings with 3 ml of ice cold Tris-HCl buffer. The filters were transferred to vials with liquid scintillation cocktail (3 ml) and radioactivity was measured in an LS 1701 RackBeta liquid scintillation counter (Beckman Coulter S.p.A.). Protein concentration was determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with BSA used as a standard. The activity of the ITX solution was based on the displacement of [ 3 H]-PK11195 from adrenal glands membranes. This activity was quantified by comparison with a competition curve generated with unlabeled PK11195 at the same concentration of ITX-solutions.

Central benzodiazepine receptor saturation binding assay

In saturation binding assays, increasing concentrations of [ 3 H]RO 15-1788 (25 µl, 0.37-12 nM) were incubated with rat cerebellar membranes (100 µl, 500-1000 µg of protein/ml) prepared as previously described from Olasmaa [Olasmaa et al., 1990], with and without ITX (25 µl at the dose of 10 -4 M). The final volume (250 µl) was obtained by adding sodium phosphate monobasic monohydrate buffer. Non-specific binding was measured in the presence of diazepam 1 µM.

The mixture was incubated at 0-4 °C for 1 h. The reaction was terminated by vacuum filtration through glass fiber filters (Whatman GF/C, Whatman International Ltd) followed by three washings with 3 ml of ice-cold sodium phosphate monobasic monohydrate buffer. The filters were transferred to vials with liquid scintillation cocktail (3 ml) and radioactivity was Protein concentration was determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with BSA used as a standard. The activity of the ITX solution was based on its ability to act as an allosteric modulator to enhance or inhibit the effects of diazepam.

3 Electrophysiological studies

Primary cultures of cerebellar granule cells

Primary culture of cerebellar granule neurons were prepared from 7 days old Sprague-Dawley rats as previously described [START_REF] Gallo | The role of depolarization in the survival and differentiation of cerebellar granule cells in culture[END_REF]Luo et al 2002) following the European Community Guidelines for the use of experimental animals. Briefly, cells from cerebella were dispersed with trypsin (0.24 mg/ml; Sigma-Aldrich Italy) and plated at a density of 1.5 x 10 6 cells/ml on 35 mm Falcon dishes coated with poly-L-lisine (10 µg/ml; Sigma-Aldrich Italy). Granule cells were grown in Basal Eagle`s Medium (BME; Celbio S.p.A., Milano, Italy), supplemented with 10% foetal bovine serum (Celbio S.p.A.), 2 mM glutamine (Invitrogen s.r.l., Milano, Italy), and 100 µg/ml gentamycin (Sigma-Aldrich Italy) and maintained at 37°C in 5% CO 2 . After 24 hours in vitro the medium was replaced with a half and half mixture of BME and Neurobasal medium containing 2% B27 supplement, 1% antibiotic, and 0.25% glutamine. At second day in vitro (DIV2) cytosine arabinofuranoside (Sigma-Aldrich Italy) was added at the final concentration of 10 µM. Recordings were made from DIV 9-11 neurons in culture.

Solutions and drugs

The recording chamber was continuously perfused at 5 ml/min with an extracellular medium composed of (mM): NaCl 145, KCl 5, CaCl 2 1, Hepes 5, Glucose 5, Sucrose 20, pH 7.4 with NaOH all purchased from Sigma-Aldrich Italy. GABA (Sigma-Aldrich Italy) was dissolved in the extracellular solution, ITX in dimethyl sulfoxide (DMSO) at 1 and 10 µM, diazepam was dissolved in DMSO at 10-2M and then diluted in the extracellular medium. All drugs were applied directly by gravity through a Y-tube perfusion system. Drug application had a fast onset and achieved a complete local perfusion of the recorded cell.

Patch clamp recordings

Recordings were performed on single neuron after 7-9 days in culture of cerebellar granule cells at room temperature, under voltage -clamp in the whole-cell configuration of the patchclamp technique [START_REF] Hamill | Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches[END_REF]. Electrodes were pulled from borosilicate glass (Hidelberg, FRG) on a vertical puller (PB-7, Narishige) and had a resistance of 5-7 Ohm when filled with KCl internal solution. Currents were amplified with an Axopatch 1D amplifier (Axon Instruments, Foster City, California, USA), filtered at 5 kHz, digitised at 10kHz.

Statistical analysis

Data were analyzed using the GraphPad Prism program (GraphPad Software Inc., La Jolla, California, USA).

The behavioral data, expressed as mean ± SEM, are obtained by groups of 5 animals each.

One-way analysis of variance (ANOVA) with Dunnett's post-hoc comparisons between individual treatment groups and vehicle group was performed.

Elettrophysiological data, expressed as mean ± SD, are obtained by 10 cells each group.

Control group and diazepam group were compared with the t-test.

Results

As shown in Table 1, ITX was unable to affect animal behavior in the elevated plus maze test at any tested dose. As expected, diazepam significantly (p<0.05) increased the total number of entries, the percentage of open arms entries and the percentage of time spent in open arms in comparison with vehicle group, indicating and confirming its anxiolytic effect.

ITX, administered at the doses of 1, 10 and 25 mg/kg, altered neither the total pathway, nor the total number of rearings, as shown in table 2. Diazepam administered at the dose of 2 mg/kg, significantly reduced the total pathway (p<0.01) and the number of rearings (p<0.05) in comparison to the vehicle group.

The administration of ITX did not affect the latency to the loss of the righting reflex and the sleeping time induced by pentobarbital sodium in comparison with controls (tab.3). Furthermore ITX did not show the ability to bind the central or the peripheral benzodiazepine receptors, contrary to what diazepam or PK11195 did when used as control drugs, as clearly shown in figure 2 and3.

Considering the possibility of an allosteric modulatory effect, ITX was challenged for its ability to modify the binding of diazepam to central benzodiazepines receptor (fig. 4) and the result was negative. Moreover, by using the patch clamp technique in primary cultures of granule neurons we studied the effect of ITX on GABA-evoked currents. ITX applied at 1 and 10 µM did not affect GABA current (n=10). In the same experiments diazepam (1µM), used as reference drug, was able to potentiate GABA-evoked current of 40% (n=10) (fig. 5).

Discussion

There are mounting concern about the impact, sometimes not overt at all, of food contaminants on health and wellness of thousands of people in the world. This topic is particular stressful when one considers the worldwide distribution of food from one country to another with the modern fast transportation system. Hence it seems imperative to prevent massive event of human toxicity to know in advance or as fast as possible the safety of chemical compounds found in food for voluntary or accidental addiction. If the issue is related to infant food such as milk, the issue becomes particularly complex since the growing subjects are more sensible than the adults, especially as far as the impact of contaminants on the CNS development and in turn to behavioral disturbances are concerned. It is clear that for many chemical compounds present in food, we must accept the presence based on the so called ADI (Admissible Daily Intake) which derives from the data obtained after chronic exposure studies and which are in turn converted in the LOAEL (LOwest Adverse Effective Level), an index to calculate the risk and the MOS (Margin Of Safety).

In these last years many examples of contaminants in food from the family of colorants have emerged (i.e. the genotoxic and carcinogenic effect of the colorant Sudan I found in Europe as present in pepper and curry powder). It is imperative, however, to exercise caution before criminalizing some chemical compound present in food. First of all one has to accept traces of contaminants based on the dose-effect demonstration of real health risk. This acceptance is mainly related to the necessity to avoid unnecessary food wasting. Second, if necessary, the claimed potential toxicity of contaminants must be proved with accepted scientific methodology before alerting the public opinion. It is clear that when food contaminants are a real risk for human the law must be severely applied and the contaminant excluded drastically. In this paper we studied the potential anxiolytic or sedative effect of ITX since this photoinitiator was found in milk and someone blamed the producers for subtly administering psychoactive drugs [CODACONS]. According to our results, there was no evidence of ITX to exert any anxiolytic or sedative effect in rats as demonstrated by pharmacological, biochemical and electrophysiological experiments. These conclusions are based on the fact that the major sedative system in the CNS, the GABA receptor complex, is not affected by relevant doses of ITX.

Of course the importance of the issue will allow us to extend our studies on this topic for a better definition in terms of food safety and could avoid such type of accident in the future.

  , 30 million litres of baby milk were recalled from the market in Italy,

  , rats were individually placed in the central square facing an open arm and observed for 5 min. The number of entries and the time spent in the open and closed arms were recorded. The maze was cleaned following each trial to remove any residue or odour of the animals. For the purpose of analysis, open (or closed) arms activity was quantified as the percentage of number of entries into open (or closed) arms relative to the total number of entries into any arm and the percentage of the time spent in the open (or closed) arms relative to the total time spent in the maze.1.4. Open field testFor the test a black painted arena 100 cmx100 cm square floor and 50 cm high walls was used. The test started by gently placing each animal in the centre of arena. Rats were continuously filmed for 10 min with a camera connected to a computerized system (Biomedica Mangoni s.n.c., Pisa, Italy), which divided the field into 25 squares of equal size.Sixteen squares delimited the peripheral area and nine squares the central area. The length of in the total open field as well as in the peripheral and the central areas, and the number of rearings (when the rat reared upon its hind feet) were recorded. At the end of each trial, the open field was wiped clean to remove traces of the previous assay.1.5. Pentobarbital-induced sleeping timeA single dose of pentobarbital sodium (35 mg/kg) was i.p. injected in rats 60 min after the oral administration of the vehicle or ITX. Sleeping time was determined as the interval between the loss and the recovery of the righting reflex.2. Radioligand binding assay studies2.1. Sample preparation ITX (Sigma-Aldrich Italy) was dissolved in methanol (Carlo Erba Reagenti, Milano, Italy) to obtain the 10 -2 M ITX-standard solution. The standard 10 -2 M ITX-standard solution was diluted with sodium phosphate monobasic monohydrate buffer (Sigma-Aldrich Italy) 50 nM pH 7.40 or Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) (Sigma-Aldrich Italy) buffer 50 nM pH 7.70 to obtain the experimental ITX-solutions from 10 -4 M to 10 -9 M.

  1,4 benzodiazepine) (specific activity 3.09 TBq/mmol; NEN TM Life Science Products, PerkinElmer Italy, Monza, Milano, Italy) as specific ligand. Aliquots of ITX-solutions were tested for their ability to inhibit 1 nM [ 3 H]RO 15-1788 specific binding to rat cerebellar membranes prepared as

  [ 3 H]PK 11195 (20 µl) and aliquots (20 µl) of the ITX-solutions or 1 µM

  LS 1701 Rack Beta liquid scintillation counter (Beckman Coulter S.p.A.).

  Luo JH, Fu ZY, Losi G, Kim BG, Prybylowski K, Vissel B, Vicini S, 2002. Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture. Neuropharmacology. 42, 306-18. Momo F, Fabris S, Stevanato R, 2007. Interaction of isopropylthioxanthone with phospholipid liposomes. Biophys Chem. 127, 36-40. Notox BV, 2004a. Micronucleus test in bone marrow cells of the mouse with IGM ITX. Notox Project 411885. Notox BV, 2004b. Evaluation of DNA repair inducing ability of IGM ITX in male rat hepatocytes (in vivo rat hepatocyte DNA-repair assay). Notox Project 411874. Olasmaa M, Rothstein JD, Guidotti A, Weber RJ, Paul SM, Spector S, Zeneroli ML, Baraldi M, Costa E, 1990. Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy. J Neurochem. 55, 2015-23. Pellow S, Chopin P, File SE, Briley M, 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods. 14, 149-67. RASFF, 2005. The Rapid Alert System for Food and Feed (RASFF), No. 178/2002, Annual Report (http://ec.europa.eu/food/food/rapidalert/report2005_en.pdf) page 22. Rothenbacher T, Baumann M, Fügel D, 2007. 2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market. Food Addit Contam. 24: 438-44.

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 12345 Figure Caption

Table 1

 1 Effect of ITX on arms exploration in the elevated plus maze test The data are expressed as mean±SEM (n=5) of the total number of arms entries and the percentage of open and closed arms entries and of the time spent in open and closed arms in a 5 min test. ANOVA followed by Dunnett's test: *p<0.05 vs. vehicle.

	Treatment		Arms entries		Time spent in arms
	(mg/kg)	Total (n°)	Open (%)	Closed (%)	Open (%)	Closed (%)
	Vehicle	7.0±1.6	13.3±9.7	86.7±9.7	3.5±3.3	89.6±3.9
	Diazepam 1	15.0±1.1*	42.1±5.8*	57.9±5.8*	19.0±2.2*	69.1±3.1*
	ITX 1	3.4±1.4	0.0±0.0	100.0±0.0	0.0±0.0	97.0±2.1
	ITX 10	8.4±3.1	13.0±8.3	87.0±8.3	6.2±3.8	86.2±6.1
	ITX 25	7.8±2.3	17.7±8.8	82.3±8.8	9.7±7.2	81.2±8.2

Table 2

 2 Effect of ITX on locomotor activity of rats in the open field test

	Treatment (mg/kg)	Total pathway (cm)	Rearings (n°)
	Vehicle	5924±426.2	14.4±1.2
	Diazepam 2	2568±975.6**	3.6±1.0*
	ITX 1	5230±471.5	14.8±4.8
	ITX 10	5139±475.5	10.2±0.9
	ITX 25	5283±474.8	11.7±2.3
	Values are means±SEM (n=5). ANOVA followed by Dunnett's test: **p<0.01, *p<0.05 vs. vehicle.

Table 3

 3 Effect of ITX on the latency to the loss of the righting reflex and on the sleeping time induced by All animals received i.p. pentobarbital (35 mg/kg) 1 h after the oral administration of ITX.The data are expressed as mean±SEM (n=5).

	pentobarbital sodium in rats
	Latency to the loss of the
	Treatment (mg/kg)		Sleeping time (min)
	righting reflex (min)
	Vehicle	4.4±0.8	45.2±5.0
	ITX 1	4.6±0.5	41.6±2.9
	ITX 10	4.6±0.7	44.4±5.3
	ITX 25	4.3±0.3	40.3±5.7