Inmaculada Sahuquillo

María Maria Jesus Lagarda

Dolores Silvestre

Rosaura Farre Rovira

María Silvestre

Cardenal Dolores

Herrera

Methylmercury determination in fish and seafood products and estimated daily intake for the Spanish population

spectrometry was used to separately determine inorganic and 10 organic mercury. In all samples, inorganic mercury content was 11 below 50 µg kg -1 . There was wide variability, not only among 12 the mercury levels of different fish species, but also among 13 different samples of the same species -with methylmercury 14 content ranging from below 54 to 662 µg kg -1 . The highest mean 15 methylmercury content was found in fresh tuna. Based on an 16 average total fish consumption of 363 g/person/week, the 17 methylmercury intake was estimated to be 46.2 µg/person/week. 18 Therefore, the mercury intake of Spanish people with a body 19 weight ≤ 60 kg is lower than the JECFA PTWI of 1.6 µg/kg body 20 weight, but exceeds the US-NRC limit of 0.7 µg/kg bw/week 21 based on a benchmark dose. 22 The toxicity of mercury is highly dependent on its 13 chemical form, organomercury being more toxic than the 14 inorganic forms. The high toxicity of methylmercury (MeHg) 15 lies mostly in its neurotoxicity ( WHO, 1990 andEFSA, 2004). 16

It blocks the binding sites of enzymes, and interferes with 17 protein synthesis as well as thymidine incorporation into DNA 18 (Sanchez Uria and Sanz-Medel, 1998). Organomercury toxicity is 19 ascribed to its affinity for thiol groups to form mercapto 20 compounds [START_REF] Hansen | Organic mercury: an 25 environmental threat to the health of dietary-exposed 26 societies?[END_REF] levels for total mercury in fish of 0.5 mg kg -1 wet weight, 14 except for some species (for instance swordfish and tuna), 15 where the maximum level was raised to 1.0 mg kg -1 wet weight. 16

For physiological and ecological reasons, predatory fish which 17 are at the top of the food chain concentrate mercury more 18 easily in their tissues. The maximum levels set for total 19 mercury by Commission Regulation 466/2001/EC are under 20 revision, and in the European Union assessment is required of 21 the risk from dietary exposure to mercury (EFSA, 2004). 22

From the analysis of the SCOOP (scientific co-operation on 23 questions relating to food) data within the European Union 24 In numerous studies high mercury contents in fish have been 1

reported (Bosnir et al., 1999). One of the fish species with 2 higher mercury contents was tuna, though mercury contents 3 depended also on their origin. For instance, Thunnus thynnus 4 from the Eastern Mediterranean had mean total mercury contents 5 of 1.18 mg kg -1 (maximum 2.59 mg kg -1 ), whereas in the Atlantic 6

Ocean the mean content was 0.485 mg kg -1 (maximum 0.76 mg kg -1 ) 7 (Storelli et al., 2002). More recently, Yamashita et al. 8 (2005) reported a methylmercury content of 0.49 ± 0.31 mg kg -1 9 (maximum 0.90 mg kg -1 ) (Pacific Ocean) vs. 0.29 ± 0.04 mg kg -1 10 (maximum 0.34 mg kg -1 ) (Atlantic Ocean) for this species. 11

The opinion of the Scientific Panel on Contaminants in the 12 Food Chain (EFSA, 2004) is that the consumption of large 13 predatory fish, which are at the top of the food chain, such 14 as swordfish and tuna, and which all contain higher levels of 15 methylmercury, may be significantly greater in countries in 16 southern Europe. 17 and added to a tuna digest (standard addition method). 10

The suitability of the method was evaluated by determining 11 the analytical parameters (limit of detection, limit of 12 quantification, precision and accuracy). 13

The limits of detection (LOD) and quantification (LOQ) 14 defined as the mercury concentration corresponding to three or 15 ten times the standard deviation of blanks, respectively, and 16 were determined with nine reagent blanks. 17 The application of the standard addition method revealed 30 matrix interferences: the values of the slopes obtained for 31 inorganic and methylmercury standards in aqueous solution were 32 0.00147 and 0.00225, respectively, while in tuna digest were 33 0.00106 and 0.00144 for inorganic mercury and methylmercury, 34 respectively. The slopes obtained in aqueous and digested 1 media were significantly different (p < 0.05); therefore, the 2 standard addition method was used in all determinations. [START_REF]Summary and conclusions of the sixty-first meeting 5 of the Joint FAO[END_REF] The values obtained for the limit of detection, precision 4 and accuracy are reported in Table I. There is good agreement 5 between the certified and measured values, so the method is 6 accurate. The precision for inorganic mercury was not 7 evaluated because in the analyzed samples the inorganic 8 mercury contents were lower than the limit of detection of the 9 method. 10

The analytical parameters obtained confirm the usefulness 11 of the method in relation to the study objective. 12 13

Mercury determination 14

The methylmercury contents referred on a dry and wet matter 15 basis are reported in Tables II and III, corresponding to 16 fresh and frozen fish and to canned products, respectively. 17

Total mercury contents in fish analyzed in this study did not 18 exceed the maximum levels established by the European Union 19 (reported above). 20

In all analyzed samples inorganic mercury contents were 21 lower than the limit of detection. In this respect, a study on 22 total mercury and methylmercury determination in different 23 seafood characterized as frequently consumed species in Spain 24 concluded that methylmercury content is the major mercury 25 species in seafood samples (Gomez-Ariza et al., 2004). In the 26 same sense, mercury was present almost completely in the 27 methylated form in the muscle of hake and striped mullet from 28 the Mediterranean (Storelli et al.

, 2005). 29

There was broad variability not only among the mercury 30 levels of different fish species, but also among different 31 samples of the same species. A mean value of total mercury 32 concentration in fish and seafood products of 109 ± 845 µg kg -1 33 food was reported by the Member States by scientific co-34 In the study carried out, the highest mercury levels 5 corresponded to predatory fish species located at the highest 6 level of the food chain (tuna, swordfish), these being the 7 species for which the European Union allows the highest 8 mercury contents. In the case of mollera (Poor cod, 9

Trisopterus minutes) and pagre (Common sea bream, Pagrus 10 pagrus), despite their small size compared to tuna, salmon and 11 swordfish, they came from Valencian coastal waters at the 12 mouth of the Albufera lake -a fact that could explain their 13 relatively high mercury contents. 14

Values found for methylmercury contents are in the upper 15 level of the ranges reported in the literature. 16

The comparison of methylmercury contents in fresh and 17 canned tuna show a mean difference of 17%. In samples of the 18 same brand of canned tuna, differences between batches, 19 perhaps ascribable to differences in the origin of the tuna 20 were found. The most surprising observation corresponded to 21 tuna in vegetal oil brand B, where in one of batches (a) the 22 mercury content was lower than the limit of detection of the 23 method, while in the other total MeHg was > 400 µg kg -1 wet 24 weight. [START_REF]International program 26 on chemical safety[END_REF] Differences in mercury contents depending on tuna origin 26 have been reported. Accordingly, bluefin tuna (Thunnus 27 thynnus) caught in the Mediterranean had 4-to 5-fold higher 28 mercury contents than tuna from the same species caught in the 29 Atlantic (Storelli et al., 2002). Tuna migration and the fact 30 that the Mediterranean Sea is part of the Circumpacific-31

Mediterranean-Himalayan mercurifeous belt, which contains 32 mercury ferrous rocks, could explain this difference 33 [START_REF] Bernhard | Mercury in the Mediterranean[END_REF]EEA, 1999) 34 In addition, the variation in mercury levels among tuna 1 samples may be largely dependent on both species and body 2 size, and the mercury levels found in the muscle of cultured 3 tuna in the Mediterranean were higher than the average levels 4 in the muscle of wild tuna. This may be due to mercury intake 5 from large predatory fish species, such as mackerels, used as 6 feed in tuna aquaculture (Yamashita, Omura and Okazi, 2005). 7 No significant differences have been reported in mercury 8 levels in canned tuna packed in oil compared to water. 9

Inorganic mercury was below limit of detection and about 90% 10 of the mercury in fish was methylmercury (Burger and Gochfeld, 11 2004). In the present study, in canned natural tuna 12 methylmercury contents were, in the two analyzed batches, 13 higher than those found in tuna in vegetal oil. The 14 differences can not be ascribed to the brine, considering the 15 high variability in methylmercury content in the same fish 16 species, the possibility that the difference could be due to 17 the fish origin can not be ruled out. However, the fact that 18 the lower content corresponded to tuna in oil (with values 19 even lower than those corresponding to the analyzed fresh 20 tuna) could also be explained by partial dissolution of 21 organic mercury in the oil. 22

23

Intake estimation 24

To evaluate the contribution of fish consumption to mercury 25 intake, data from the Spanish Ministerio de Agricultura, Pesca 26 y Alimentación (MAPA) (Martín, 2002) were used. The dietary 27 intake (DI) of methylmercury was modeled as: 28 DI = ∑ I sw C s 29 where I sw was the daily intake (in kg) of fish in weight w of 30 species s, and C s was the methylmercury content (µg kg -1 ) in 31 species. In Table IV, estimated seafood consumption and its 32 contribution to mercury intakes in Spain are reported. 33 Both PTWI were calculated with the absence of appreciable 5 adverse effects in children born of mothers with mercury hair 6 contents of 14 or 12 µg g -1 hair, respectively. Estimation of 7 the methylmercury intake is complex, because it is based in 8 the conversion of biomarker data, such as hair levels, into 9 daily intake. To obtain the US-NRC limit a composite 10 uncertainty factor higher of 10 to take into account 11 interindividual variability and incompleteness of the data 12 base was applied, while in the estimation of JECFA PTWI the 13 incertainity factor was 10. Imprecision in intake estimates 14 may lead to underestimation of the true mercury effect, and to 15 an overestimation of the benchmark dose level (EFSA, 2004). 16

Considering the species analyzed in this study, their 17 daily contribution to the methylmercury intake of the Spanish 18 population was 6.6 µg. It has to be noted that only fish 19 products frequently consumed or with a potential contribution 20 to mercury intake have been analyzed. As shown in Figure 1, a 21 Spanish individual with body weight ≤ 60 kg could have mercury 22 dietary intakes in excess of the U.S.-NRC limit. This is the 23 case of children, who consume more fish than adults, when 24 intake is expressed on a body weight basis (EFSA, 2004; Crépet 25 et al., 2005). 26 Therefore, the probability of exceeding the methylmercury 27 limit is much higher in small children. On the other hand, 28 considering that intrauterine exposure is believed to 29 represent the critical period for methylmercury 30 neurodevelopmental toxicity, pregnant women must also control 31 methylmercury, and therefore our advice is to limit the intake 32 of large predators, as they are the real main sources of 33 methyl mercury. 34 In agreement with the literature, population groups with 1 frequent consumption of large predatory fish such as tuna or 2 swordfish are at risk of high methylmercury intake. 3 Data from the SCOOP report indicate that in some countries 4 the average mercury intake from fish and seafood products may 5 be at the US-NRC limit, and some average intake levels may 6 exceed this limit (EFSA, 2004). 7

In 1999, taking into account the mean annual per capita 8 fish consumption of the Spanish population, it was estimated 9 that the mean weekly mercury intake from fish would be 49.8 µg 10 (of which 45 µg corresponds to organic mercury) (Bosnir et 11 al., 1999). The estimation of the present study was 46.2 12 µg/week. However, it must be noted that the sum of fish 13 intakes reported in Table IV is about 52 g per individual, 14 while daily fish and seafood consumption by the Spanish 15 population was 73 g (Martin, 2002); as a result, the estimated 16 weekly methylmercury intake could be slightly underestimated. 17

In addition, the contribution of locally consumed fish species 18 such as chucla, mollera, llisa, etc. -some of them with 19 relevant methylmercury contents -was not considered, because 20 their consumption is unknown. 21

It should be noted that daily fish and seafood consumption 22 by the Spanish population is above the average fish 23 consumption range in the European Union -from 10 g per person 24

in The Netherlands to 80 g per person in Norway (EFSA, 2004). [START_REF]International program 26 on chemical safety[END_REF] Consequently, control of the methylmercury contents in fish is 26 required. 27

If total daily fish and seafood consumption by the Spanish 28 population of 73 g) (Martin, 2002) and the mean concentration 29 for mercury in fish and seafood products submitted by the 30 Member States of 109 µg kg -1 )(EFSA, 2004) are considered, then 31 dietary exposure to mercury should be 7.8 µg -this value 32 being higher than that estimated in the present study. It 33 should be taken into account that 109 µg kg -1 corresponds to 34 Salonen, J.T., Seppänen, K., Nyyossoen, K., Korpela, H., 13 Kauhanen, J., Kantola, M., Tuomilehto, J., Esterbauer, 14 H., Tatzber, F., Salonen, R. 1995. Intake of mercury from 15 fish, lipid peroxidation, and the risk of myocardial 16

  is calculated as three times the standard deviation of 9 reagent blanks. **Limit of quantification is calculated as ten times the standard deviation. n = number of samples n.d.= non-detectable

  . = Detection limit. * D.L. has been considered zero in the calculation of the mean value.

  . detection limit * D.L. has been considered zero in the calculation of the mean value. "a" and "b" corresponded to samples from different batches. (A), (B) and (C) corresponded to different commercial brands.

  of 25 samples of fish and seafood products 7 most frequently consumed in Spain were determined.

  products can be important sources of energy, 3 protein, ω-3 fatty acids, trace elements and vitamins, but 4 also of mercury, an environmental contaminant. 5 Some studies report that mercury from fish could mask the 6 beneficial effects of fish consumption on coronary diseases 7 derived from the presence ω-3 fatty acids (Rissanen et al., 8 2000;[START_REF] Guallar | Mercury, fish oil, 22 and the risk of myocardial infarction[END_REF] Salonen et al., 2003), since 9 high mercury intakes are associated with an increased risk of 10 acute coronary events, cardiovascular disease and coronary 11 disease mortality[START_REF] Virtanen | Mercury, fish 21 oils and risk of acute coronary events and cardiovascular 22 disease, coronary heart disease and all-cause mortality 23 in men in eastern Finland[END_REF] 12

(

  EFSA, 2004), it can be concluded that there is high 25 variability in the mean fish consumption in different in exposures that vary by a factor of 28 10. In addition, the weighted mean contamination, which was 29 based on all data for the mercury concentration in fish and 30 seafood products submitted by the Member States, was 109 ± 845 31 µg/kg;

66

  and the risk associated with mercury 19 contamination in this kind of product explain our interest in 20 determining mercury in fish commercially available in Spain. 21 A total diet study carried out in the Basque Country 22 (Spain), revealed a mean and maximum daily mercury intake of 23 12 and 18 µg day -1 was found, fish being the main dietary 24 source of mercury (Urieta et al. 1996). The study did not 25 include the speciation of mercury. Laterly, Sanzo et al. 26 (2001) estimated mercury intake due to fish consumption among 27 individuals from Gipuzkoa participating in the European 28Prospective Investigation into Cancer (EPIC), and found an 29 average intake of 48.3 µg week -1 . Recently, an estimation of 30 the daily intake of mercury from the consumption of marine 31 species has been made in Catalonia (Spain): mercury intakes 32 ranging from 5.6 (female children) to 9.89 (male adults) µg 33 day -1 have been reported. 34The techniques most frequently used to release mercury 1 species from solid samples are acid leaching(Hardisson et 2 al., 1999; Ortiz et al., 2002; Storelli et al., 2002, Cabañero 3 et al., 2004) or alkaline digestion (Slaets et al., 1999), 4 with the option of applying ultrasonic (Río and Bendicho, 5 1999) or microwave energy (Gebersmann et al., 1997; Berzas et 6 al., 2005) to assist in the procedure. In alkaline media 7 methylmercury appears to be more stable than in acid media, 8 the proteins being easily hydrolyzed (Hintelman et al., 1997). 9 To detect organomercurial compounds, selective techniques such 10 as atomic absorption spectrometry (AAS), atomic fluorescence 11 spectrometry (AFS)(Jókai et al, 2005), microwave-induced 12 plasma atomic emission spectrometry (MIP-AES) and inductively 13 coupled plasma mass spectrometry (ICP-MS) (Cai et al., 2000) 14and electron capture detection (ECD) coupled to GC methods 15 have been used. However, such advanced instrumentation is not 16 always available in the laboratories of some countries with 17 important fish catches, as for instance some African andSouth 18 American countries. This is the reason why a simple device was 19 used in the determination of organic mercury in this study. 20With the ultimate aim of estimating the contribution of fish 21 and seafood to mercury intake, mercury content was measured in 22 between inorganic and organic mercury, by 24 applying a simple method comprising cold vapor and AAS, 25 previously used and validated in our laboratory (Ubillús et 26al., 2000). 27Materials and methods 29Instrumentation 30For mercury determination by cold vapor generation atomic 31 absorption spectrometry, a Perkin-Elmer (PE) 2380 atomic 32 absorption spectrophotometer equipped with a quartz absorption 33 mm long, 17 mm i.d. with quartz windows), a System 2 1 Electrodeless Discharge Lamp (PE) and a discharge mercury lamp 2 (PE) were used. A hydride generation vessel (25 ml capacity) 3 and a small fish tank air pump were also used. A Heraeus model 4 D-6450 Hanau forced air oven was used to dry samples. 5Samples and sample preparation 7The analyzed samples were species of fish and seafood most 8 frequently consumed in Spain, and arbitrarily selected from 9 local markets. Overall 25 different fish and seafood products, 10 mainly in the fresh state, were analyzed. Two different 11 samples of each fish species (with the exception of chucla 12 (Blotched picarel, Spicara maena), serrano and pagre (Common 13 sea bream, Pagrus pagrus (Linné 1758), from which only one 14 sample could be obtained), bought in different stores or in 15 the case of fresh fish on different days were analyzed. Among 16 the available canned products, the most frequently consumed 17 were selected, and from each of them two samples from two 18 different batches were analyzed. In all cases, three aliquots 19 of each sample were analyzed. 20 Fish samples were prepared by dissection of the edible 21 parts, which were dried at 80ºC for 24 h and homogenized by 22 grinding in a glass mortar.[START_REF] Aceto | Mercury speciation in biological 25 samples[END_REF] The preparation of the samples was based on the method for 24 mercury speciation proposed by Oda et al.(1981), adapted to 25 the conditions of our laboratory (Ubillús et al., 2000are: 253.7 nm of wavelength, 31 0.7 nm of spectral bandwidth; for measurement a discharge 32 170 mA with a deuterium lamp as background 1 correction, and the peak height mode were used. the range from 0 to 40 ng ml -1 , were plotted; the 4 standard addition method was applied. 5Analytical Quality Assurance 7Matrix interference was checked by comparing the slopes of the 8 calibration curves obtained with standards in aqueous solution 9

  The precision of the method was estimated from the values 18 obtained in the independent analysis of 9 aliquots of tuna 19 fish. The values were expressed as percentage of the relative 20 standard deviation (RSD %). 21Eight aliquots (0.10-0.20g) of Tuna Certified Reference 22 Material (CRM 463, BCR) were analyzed by the method, and 23 measured values of mercury were compared with certified

  ) task 3.2.11.; the high standard deviation of 1 the mean reflects the variability of mercury contents in fish 2 (EFSA, 2004). Mercury speciation is not performed routinely by 3 national control laboratories. 4

  Fig 1, where these values are compared to the provisional 2 tolerable weekly intake (PTWI) limits set by the JECFA and by 3 the NRC. 4

  mercury content in fish, the variability in the 1 contents being very high. All this corroborates the need for 2 studies on real fish and seafood consumption (differentiating 3 among fish species) for different population groups, together 4 with routine estimation of methylmercury in products of this 5 kind -in order to obtain more reliable estimations of 6 methylmercury intakes. 7As reported by the Scientific Panel Members (EFSA, 2004), 8 other possible sources of mercury for humans, such as might 9 arise from the consumption of meat products of animals fed 10 methylmercury-containing fishmeal, have not been considered 11 but would need to be taken into account in any comprehensive 12 evaluation of methylmercury intake. 13In agreement with Sanzo et al. (2001), it can be concluded 14 that methylmercury in fish must continue to be monitored 15 closely in order to assess the risk for the population. diseases derived from the presence of 18 ω-3 fatty acids, together with the low mercury contents in 19 some species (sardine and salmon) makes fish consumption microwave-assisted extraction. Journal of 1 Chromatography A 1093 (1-2): 21-28. 2 Bosnir, J., Puntaric, D., Smit, Z., Capuder, Z. 1999. Fish as 3 an indicator of eco-system contamination with mercury. 4Croatian Medical Journal 40:546-459. 5

  Río, S., Bendicho, C. 1999. Ultrasound-assisted extraction for 1 mercury speciation by the flow injection-cold vapour 2 technique. Journal Analytical Atomic Spectrometry 14:263-3 268. 4 Rissanen, T., Voultilainen, S., Nyyssönen, K., Lakka, T.A., 5 Salonen, J.T. 2000. Fish oil-derived fatty acids, 6 docosahexaenoic acid and docosapentaenoic acid, and the 7 risk of acute coronary events. The Kuopio Ischaemic Heart 8 Disease Risk Factor Study. Circulation 103:2677-2679. 9 Sakamoto, M., Kubota, M., Matsumoto, S., Nakano, A., Akagi, H. 10 2002. Declining risk of methylmercury exposure to infants 11 during lactation. Environmental Research 90:185-189. 12

9 MFigure 1 .

 91 Figure 1. Estimated weekly methylmercury intake compared with provisional tolerable weekly intake (PTWI) limits set by the JECFA in the European Union and by the NRC in the United States.

Table II .

 II Methylmercury contents (mean ± standard deviation) in fresh fish and seafood.

			Dry weight	Wet weight
	Seafood		Methylmercury	Mean	Methylmercury	Mean
			µg kg -1	value	µg kg -1	value
	Fresh tuna	a	2269 ± 240	2294±266	530 ± 56	596±103
	(Thunnus thynnus)	b	2318 ± 218		662 ± 63
	Anchovy	a	246 ± 38	246±38	58 ± 56.08	58±56
	(Engraulis
	encrasicholus)
	Mackerel	a	306 ± 23		67 ± 5	64±5
	(Scomber scombrus)	b	279 ± 4	293±25	61 ± 1
	Chucla	a	233 ± 27	233±27	59 ± 7	59±7
	(Spicara maena)
	Swordfish	a	927 ± 152		195 ± 52
	(Xiphias gladius)	b	3370 ± 229	2148±1428	763 ± 52

Table III .

 III Methylmercury concentration in canned seafood.

	Sample		Dry weight	Wet weight
			Methylmercury	Mean	Methylmercury	Mean
			µg kg -1	value	µg kg -1	value
	Natural tuna (A)	a	2770 ± 109	2692±147	627 ± 25	609±33
	(Thunnus thynnus)	b	2614 ± 91		592 ± 21
	Tuna in vegetal oil	a	879 ± 22		289 ± 7

Table IV .

 IV Estimated mean fish and methylmercury intakes by the Spanish population.

	Species	Methylmercury	Mean daily	Methylmercury
		(µg kg -1 )	consumption for	intake
			fish and seafood	(µg/person/day)
			(kg/person/day)*	Spain
			Spain
	Tuna	596	0.0013	0.775
	Canned tuna	443	0.0052	2.301
	Anchovy	58	0.0077	0.447
	Fresh mackerel	64	0.0011	0.064
	Canned mackerel	94	0.0003	0.028
	Swordfish	479	0.0021	1.006
	Prawn	<54	0.0043	-
	Sole	56	0.0023	0.128
	Hake	143	0.0120	1.717
	Fresh mussel	<54	0.0036	-
	Canned mussel	<54	0.0006	-
	Perch	70	0.0015	0.105
	Canned squid	<54	0.0001	-
	Salmon	<54	0.0021	-
	Sardine	<54	0.0077	-
	Pagre	674	Data not found	-
	Chucla	233	Data not found	-
	Serrano	618	Data not found	-
	Mollera	908	Data not found	-
	Llisa	<54	Data not found	-
	Total methylmercury		6.571
	µg/person/day

*Total consumption is considered: both fresh and frozen product.

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Pedro ; Burdaspal

Teresa Mª Legarda

Occurrence of ochratoxin A in sweet wines produced in Spain and other countries

Keywords: Ochratoxin A, sweet wine, occurrence, analysis, risk assessment, dietary intake

A survey for the presence of ochratoxin A (OTA) was undertaken from 2001 to 2005 in 188 samples of sweet wines produced in Spain and 102 originating from other countries : France (49), Austria (9), Chile (9), Portugal (9), Greece (6), Italy (5), Germany (3), Hungary (2), Slovenia (2), Switzerland (2), Canada (1), Japan (1), New Zealand (1), Ukraine (1), South Africa (1) and USA (1). The analytical method was based on immunoaffinity chromatography clean-up and HPLC with fluorescence detection. The limit of detection (defined as signal / noise = 3) was estimated to be 0.01 µg L -1 . The limit of quantification (0.02 µg L -1 ) was checked as being the lowest measurable concentration. Ochratoxin A was detected in 281 out of 290 samples analysed (96.9 % positive), at concentrations ranging from 0.01 to 4.63 µg L -1 . The overall mean and median levels were estimated to be 0.50 µg L -1 and 0.14 µg L -1 . In Spanish sweet wines, OTA was found in 99 % of the samples, with mean and median values of 0.65 µg L -1 and 0.19 µg L -1 , respectively. The mean value obtained in this study for OTA in the Spanish sweet wines would result in an intake of ca. 37.5 ng / day and 3.2 ng /day of OTA for regular consumers and for the overall population, respectively. These figures represent a minor contribution to the PTWI or TWI established by JECFA and the European Food Safety Authority : 3.8 % and 3.1 % for regular consumers; 0.4 and 0.3 % for the whole adult population, respectively.

Ochratoxin A (OTA) is a toxic fungal secondary metabolite synthetised by Penicillium verrucosum, P. nordicum and some species of the genera Aspergillus, Petromyces and Neopetromyces [START_REF] Frisvad | Neopetromyces gen. Nov. and an overview of telemorphs of Aspergillus subgenus Circumdati[END_REF][START_REF] Frisvad | Mycotoxin production by common filamentous fungi[END_REF][START_REF] Larsen | Biochemical characterisation of ochratoxin A producing strains of the genus Penicillium[END_REF][START_REF] Castella | Molecular characterization of ochratoxin A producing starins of the genus Penicillium[END_REF]. Ochratoxin A is a potent nephrotoxic mycotoxin which is carcinogenic to rodents and possesses teratogenic, immunotoxic and possibly neurotoxic properties (Scientific Committee on Food 1998). Some evidence exists in support of both genotoxic and non-genotoxic mechanisms by which OTA causes carcinogenicity [START_REF] Mally | DNA adduct formation by ochratoxin A: Review of the available evidence[END_REF][START_REF] Pfohl-Leszkowicz | Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation[END_REF]. The Internacional Agency for Research on Cancer has classified OTA in Group 2B as possibly carcinogenic to humans based on sufficient evidence for carcinogenicity in experimental animals and inadequate evidence in humans (IARC 1993). The Joint Expert Committee on Food Additives (JECFA) of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) established at its 44th meeting (JECFA 1995) a Provisional Tolerable Weekly Intake (PTWI) rounded to100 ng / kg of body weight (b.w.) (approximately 14 ng / kg b.w. per day); this PTWI was retained at the 56th meeting, the last time JECFA evaluated OTA (JECFA 2001).

Recently, the Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) has derived a Tolerable Weekly Intake (TWI) of 120 ng / kg b.w. for OTA, which is equivalent to a daily intake of ca 17 ng / b.w. (Scientific Panel on Contaminants in the Food Chain 2006).

Ochratoxin A has been frequently detected as a contaminant in a number of foodstuffs of vegetable or animal origin [START_REF] Van Egmond | Survey of data on the incidence and levels of ochratoxin A in food and animal feed worldwide[END_REF]. Particularly, the presence of OTA in wines of different types, origins and designations has been reported worldwide since 1996 by many authors [START_REF] Zimmerli | Ochratoxin A in table wine and grape-juice: occurrence and risk assessment[END_REF][START_REF] Burdaspal | Ocratoxina A en vinos, mostos y zumos de uva elaborados en España y en otros países europeos[END_REF][START_REF] Visconti | Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography[END_REF][START_REF] Festas | OchratoxinA in some portuguese wines : Method validation and screening in port wine and vinho verde[END_REF], Otteneder & Majerus 2000, Majerus et al. 2000[START_REF] Tateo | Survey on ochratoxin A in wines. Data concerning the market of table wines in brik[END_REF][START_REF] Woese | Ochratoxin A in grape juice and wine[END_REF][START_REF] Filali | Ochratoxin A in beverages from Morocco :a preliminary survey[END_REF][START_REF] Pietri | Occurrence of ochratoxin A in italian wines[END_REF][START_REF] De Cerain | Contribution to the study of ochratoxin A in Spanish wines[END_REF][START_REF] Hocking | Occurrence of ochratoxin A in Australian wine[END_REF][START_REF] Shephard | Quantitation of ochratoxin A in south african wines[END_REF][START_REF] Soufleros | Occurrence of ochratoxin A in Greek wines[END_REF], Stefanaki et al. 2003[START_REF] Rosa | Occurrence of ochratoxin A in wine and grape juice marketed in Rio de Janeiro, Brasil[END_REF][START_REF] Ng | Ochratoxin A in wine and grape juice sold in Canada[END_REF][START_REF] Brera | Exposure assessment to ochratoxin A from the consumption of Italian and Hungarian wines[END_REF][START_REF] Sugita-Konishi | Occurrence of Aflatoxins, Ochratoxin A, and Fumonisins in Retail Foods in Japa[END_REF]. The presence of OTA in wines seems to be due exclusively to the colonization of grapes by OTA-producing moulds. At least in Europe, Aspergillus carbonarius seems to be by far the species with the highest OTA production potential in grapes, followed by other species of the section Nigri, such as A. niger or A. tubingensis [START_REF] Abarca | Current importance of ochratoxin A-producing Aspergillus spp[END_REF][START_REF] Battilani | Ochratoxin A in grapes and wine[END_REF][START_REF] Sage | Fungal flora and ochratoxin A production in grapes and musts from France[END_REF][START_REF] Battilani | Occurrence of ochratoxin A-producing fungi in grapes grow in Italy[END_REF] [START_REF] Medina | Study of Spanish Grape Mycobiota and Ochratoxin A Production by Isolates of Aspergillus tubingensis and Other Members of Aspergillus Section Nigri[END_REF][START_REF] Perrone | Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy[END_REF]). These moulds only become established after the crop has been infected by pathogenic organisms or if physical or physiological damage has occurred. The OTA levels in wines can differ widely depending on a number of factors : location of vineyards, climatic conditions such as rainfall, environmental temperature and relative humidity at the vineyards, date of harvesting, pesticide treatments, different winemaking techniques, use of fining agents, etc [START_REF] Castellari | Removal of ochratoxin A in red wines by means of adsorption treatments with comercial fining agents[END_REF][START_REF] Lo Curto | Ochratoxin A occurrence in experimental wines in relationship with different pesticide treatments on grapes[END_REF][START_REF] Roset | Survey on ochratoxin A in grape juice[END_REF][START_REF] Gambuti | Influence of Enological Practices on Ochratoxin A Concentration in Wine[END_REF] . In most of the occurrence studies, the levels of OTA found in red wines were higher than those in rosé wines, followed by white wines [START_REF] Bellí | Review: ochratoxin A (OTA) in wines, musts and grape juices: occurrence, regulations and methods of analysis[END_REF]. Some studies also have pointed out the relatively high concentrations of OTA found in sweet (dessert) wines, frequently higher in average than those found in red wines [START_REF] Zimmerli | Ochratoxin A in table wine and grape-juice: occurrence and risk assessment[END_REF][START_REF] Burdaspal | Ocratoxina A en vinos, mostos y zumos de uva elaborados en España y en otros países europeos[END_REF], Stefanaki et al. 2003).

Wine was identified as the second most important source of OTA after cereals in the European diet, accounting for as much as 13 % of total contribution, according to the conclusions of the Report of the second Scientific Cooperation Task promoted by the European Commission in 1999, concerning the assessment of dietary intake of Ochratoxin A in the population of the EU Member States [START_REF] Miraglia | Task 3.2.7 Assessment of Dietary Intake of Ochratoxin A by the population of EU Member States[END_REF]). In the referred Report, it was also stated that among the different types, red and sweet wine seemed to be the most contaminated, even if on basis of a rather low number of sweet wines.

Given the significant contribution of wine to the OTA human exposure, the European The huge variety of types of wines and winemaking operations involved in their production, make any attempt at classification a very complex task. Simplifying, it is possible to distinguish some differences between liqueur and non-liqueur wines, where wines from overripened grapes can be included as established in the Council Regulation 1493/1999 (Council of the European Union, 1999). Liqueur wine is the product having an actual alcoholic strength by volume of not less than 15 % vol. and not more than 22 % vol.; or an overall alcoholic strength by volume of not less than 17,5 % vol., except for certain liqueur wines produced in specified regions. The so-called natural sweet wines belong to that category of wines. On the other hand, "wines of overripe grapes" are those produced without enrichment, both their alcoholic content and sweetness coming exclusively from grapes, with a natural alcoholic strength of more than 15 % vol. and having an actual alcoholic strength of not less than 12 % vol. This type of wines include different ways of production: late harvest, sun drying, freezing of grapes ("ice wines"), etc. The long stay of grapes on the vines for overripening before harvest and the exposure to sun drying for a number of days after harvest, might favour fungal growth, particularly when the temperature and the humidity are high [START_REF] Soufleros | Occurrence of ochratoxin A in Greek wines[END_REF].

Taking into account both the scarcity of data on levels of OTA in sweet wines and their potential capacity for being significantly contaminated with OTA, the objectives of this work were to contribute to fill that gap in knowledge by providing a set of data on the levels of OTA in samples of this particular type of wines produced in Spain and 16 other countries. 9), Greece (6), Italy (5), Germany (3), Slovenia (2), Switzerland (2), Canada

Materials and methods

Samples

(1), Hungary (2), Japan (1), New Zealand (1), Ukraine (1), South Africa (1) and USA (1). The distribution according to the year of collection is shown in Table I. Most of the samples were purchased from retail outlets, in some cases they were obtained in the same country of origin, some samples were donated by wine producers and a few were from wines consumed privately by institute staff members (staff at Centro Nacional de Alimentación). All information on the samples was taken from the bottle labels.

[Insert Table I about here] Chemicals and reagents.

All organic solvents employed were HPLC grade or glass-distilled. A 0.5 M orthophosphoric acid and 2 M sodium chloride solution were prepared in deionized water.

Deionized water was obtained from a Milli-Q water purification system (Millipore Corporation, Bedford, MA, USA).

Immunoaffinity columns

Ochraprep and Ochratest immunoaffinity columns were purchased from R-Biopharm Rhône Ltd. (Glasgow, UK) and Vicam (Watertown, MA, USA), respectively.

Standards

Procedure

The analytical procedure was based on the use of immunoaffinity columns for clean-up and reversed phase liquid chromatography with enhanced fluorescence detection by post column addition of ammonia as previously described by [START_REF] Zimmerli | Ochratoxin A in table wine and grape-juice: occurrence and risk assessment[END_REF], with slight modifications as follows.

Extraction and immunoaffinity clean-up. A 5 mL wine sample was mixed with 10 mL of 0.5 M ortho-phosphoric acid and 2 M sodium chloride solution. After addition of 5 mL of chloroform and stirring for 2 min, the mixture was centrifuged. The organic phase at the bottom was carefully transferred to a conical flask. The extraction was repeated with another 5 mL of chloroform and the combined extracts were rotary evaporated to dryness at 40 ºC. The residue was dissolved in 5 mL of PBS solution containing 15 % (v/v) methanol and transferred to an immunoaffinity column which had been previously washed with 20 mL of PBS solution. This operation was repeated 3 times with 5-mL portions of PBS-methanol each one. The column was washed with 10 mL water, and OTA was eluted with 4 mL methanol.

The eluate was evaporated to dryness under nitrogen in a Turbovap (Zymark Corporation, Hopkinton, MA, USA) and the residue immediately redissolved in 400 µL methanol / 9 % acetic acid (72:28). An aliquot of 25 µL was injected into the LC column Liquid Chromatography. The LC system consisted of a Waters 600 solvent delivery Quantitation and confirmation. The concentration of OTA in the injected extract was determined from the calibration curve on the basis of peak areas using the Millennium 32 v.4 software. Generally, each sample was analysed singly. The results were not corrected for recovery. Approximately, in 10 % randomly selected positive samples OTA peaks were qualitatively confirmed during the study by its methyl ester preparation according to the method of [START_REF] Zimmerli | Determination of ochratoxin A at the ppt level in human blood serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column clean-up: methodology and swiss data[END_REF]. In summary, this confirmatory method was applied as follows: 50 µL of the final extract were taken to dryness. After the addition of 2.5 mL of methanol and 0.1 mL of concentrated HCl, the solution was left overnight at room temperature. The solution was evaporated and the residue redissolved in 100 µL methanol / 9 % acetic acid (72:28). This solution was used for LC analysis in the same conditions as described above. An amount of OTA standard was also esterified in the same manner. The loss of the peak corresponding to OTA and the similarity in retention times (at a longer retention time) both of the esterified standard and the sample was used as criteria for confirmation of OTA identity.

In addition, the identity of OTA was confirmed in the most contaminated samples

Method performance characteristics and quality assurance

Validation. The method for OTA in wines was initially in-house validated for the analysis of table wines by using white and rosé wines in the validation study (Burdaspal [START_REF] Van Trijp | Model for the calculation of calibration curves[END_REF]. Both characteristics were achieved in all the calibration curves obtained during the study. Chromatograms were always satisfactory in terms of separation, absence of interferences and quality of peaks for OTA and its methyl ester (Figure 1).

Limits of detection and quantification. The limit of detection was estimated as equivalent to 0.01 µg L -1 , based on a signal / noise of 3:1. The limit of quantification was established at the level of 0.02 µg L -1 (defined as signal / noise = 6). That level was tested in the validation study and confirmed in practise as being the lowest validated concentration for OTA.

Quality assurance, performance assessment and accreditation. The analysis in duplicate of a blank sample (< 0.01 µg L -1 ) and the same blank spiked at 0.05 µg L -1 were included in each batch of samples. As a part of the internal Quality Assurance Programme, the laboratory also participated three times in the Food Analysis Performance Assessment Scheme (FAPAS) rounds for the analysis of OTA in wine samples using the method employed here. Satisfactory z-scores within the range of | z | [Insert Table II andFigure1 about here]

Results and discussion

The analytical method used in this study was shown to be effective, reliable and suitable for the quantitative determination of OTA in wines at levels as low as 0.02 µg L -1 . This procedure was basically similar to the one already used in one of the first reports where the presence of OTA in wine was demonstrated [START_REF] Zimmerli | Ochratoxin A in table wine and grape-juice: occurrence and risk assessment[END_REF], and it was inhouse re-validated in this study for its specific application to sweet wines. Figures 2 show typical chromatograms from a sample of naturally contaminated sweet wine and its methyl ester. The identity of OTA was further confirmed by LC/MS/MS in the samples where levels > 2.0 µg L -1 were found (Figure 2).

The method described was used to determine the OTA content in 290 sweet wines produced in Spain and sixteen other countries. The results of the analysis are summarized in Table III.

[Insert Figure 2 about here]

Ochratoxin A was detected in 281 samples (LOD = 0.010 µg L -1 ), corresponding to a total incidence rate of OTA contamination of 97%. This high incidence rate reflects the widespread presence of OTA in this type of wines all over the world. Ochratoxin A concentrations in positive samples ranged from 0.01 to 4.63 µg L -1 with an overall median (50th percentile) of 0.14 µg L -1 . The arithmetic mean concentration was found to be 0.50 µg L -1 . The results have also been grouped in Table III according to their country of origin, as indicated on the bottle. The scarce number of samples analysed from some countries avoid in most cases any comparison of the results among them. However, it is noteworthy that the highest median and mean values obtained in this study, were for the set of 6 wines originating from Greece : 0.466 µg L -1 and 0.833 µg L -1 , respectively. Such high levels were similar to values previously reported for Greek sweet wines [START_REF] Soufleros | Occurrence of ochratoxin A in Greek wines[END_REF], Stefanaki et al. 2003).

With respect to sweet wines produced in Spain, 186 out of 188 samples contained OTA at levels > 0.01 µg L -1 , which means 99 % of the samples analysed, the median and mean values being 0.19 µg L -1 and 0.65 µg L -1 , respectively. Although there is not yet a specific regulatory level for OTA in this type of wines, eighteen samples of wines produced in Spain (9.6 %) were found to exceed the level of 2.0 µg kg -1 (or µg L -1 ), which is the current statutory limit in the legislation of the European Union for OTA in all kind of wines, except liqueur wines (Commission of the European Communities 2006). The influence of climatic conditions on the levels of OTA in wines, may be responsible at least in part, for the reported geographical differences among OTA levels in wines from different origins even in the same country [START_REF] Pietri | Occurrence of ochratoxin A in italian wines[END_REF], Stefanaki et al. 2003). This makes the global comparison of mean values among different countries a symplistic approach of limited value. The OTA contamination depending on the region of production in the Spanish and French wines was investigated (Figure 3) and it is shown in Table IV.

[Insert Table III]

Wines originating from the areas of Valencia -Alicante and Tarragona-Penedés (designations of origin) showed the highest contents in OTA when compared to the rest of Spanish wines analysed in this study, in terms of both their mean and median values.

Both types of wines are produced in the mediterranean coast of eastern Spain. This fact is in agreement with findings suggesting that wines from the area of the Mediterranean basin appear to be more affected than wines produced in other European areas (Codex Alimentarius Commission 2006). Several authors have proposed that the wines of the Mediterranean region may contain high concentrations of OTA due to the climate being characterized by high humidity and high temperature, stating that vineyards very close to the sea are also more susceptible to OTA contamination [START_REF] Roset | Survey on ochratoxin A in grape juice[END_REF]. The analysis of differences showed that median and mean values for OTA from both zones were statistically different from values for OTA corresponding to the whole set of wines from the rest of Spain (p > 0.05) although they were found not being significantly different between themselves. Similarly, the mean and median values for OTA in twenty four French wines from the zone of Languedoc were the highest of all the French wines and they were statistically different in average from the rest (p> 0.05). The vine-growing area of Languedoc has been considered as a zone of significant risk for OTA contamination by some authors [START_REF] Roset | Survey on ochratoxin A in grape juice[END_REF]. This region is located in the Mediterranean coast of the South of France just north of the Spanish border, with most of vineyards placed at a distance of less than 50 km from the sea. Values obtained in this study for OTA in wines produced in Languedoc were statistically comparable to those produced in Málaga, which is located in Southern Spain. The influence of climatic factors rather than mere proximity of vineyards to the sea was clearly shown when examining values for OTA in eight wines from Canary Islands, characterized by mild climatic conditions all over the year. In this case the mean an median values were not significantly different than those for the rest of wines produced in Spain after excluding those wines produced in Tarragona and Valencia -Alicante zones. Data referred to 37 wines produced in Navarra are also shown in Table IV just as an example of wines produced in a region located far from the sea, and characterized by typical continental climatic conditions, with sunny, hot and rather dry days in the summer season, which may be affected by some sporadic storms or rainfalls during the preharvest and harvest time. The mean and median values for Navarra produced wines were 0.037 and 0.054 µg L -1 respectively, after excluding some wines where the use of blends with musts coming from the zone of Valencia, was identified.

[Insert Table IV and Figure 3 about here]

As far as we know, this is the first survey specifically designed to determine the levels of OTA in dessert wines. Limited information is available in the literature on OTA occurrence in this type of wines. Some published papers on OTA in wines include data referred to sweet wines, but according to our review of the literature never more than 20 samples were analysed. Additionally, the variability among the limits of detection and quantification of the operating procedures used, makes it very difficult to compare the results. For the purpose of the assessment of dietary intake of OTA, it must be taken into account that consumption of this type of wines is normally occasional and the ingested quantities at individual level rarely may exceed an amount of ca. 50 mL/day. Actually, the mean consumption of sweet wine by adult consumers of this type of wine according to a recently published model for the Spanish diet is 58.21 mL/per capita/day and only 4.93 mL/per capita/day when considering the whole population (Spanish Food Safety Agency, 2006). Thus, the mean value of 0.65 µg L -1 obtained in this study for OTA in the Spanish sweet wines would mean an absolute intake of ca. 37.5 ng / day and 3.2 ng /day of OTA for adult consumers and the general population, respectively. Therefore, their contribution to the overall daily OTA intake in an adult consumer of sweet wines weighing 60 kg in average, could represent a 3. In conclusion, this survey provides a background of data for OTA in sweet wines and it confirms that OTA levels are higher in wines produced in areas with specifically favourable climatic conditions such as the Mediterranean region. This analytical information is also necessary for risk assessment which at least, in the case of sweet wines produced in Spain indicate, according to our estimations, that both exposure levels for the overall population as well as the sweet wine consumers are far below the a Some samples where use of blends with musts coming from the zone of Valencia was identified, were excluded.

List of figure captions

A

  total of 290 samples of sweet wines were collected in the period 2002 -2005. The samples comprised 188 samples originating from Spain and 102 samples from other countries (85 from European Union Member States): France (49), Austria (9), Chile (9),

  Ohratoxin A was purchased from Sigma (St. Louis, MO, USA) (catalogue number : O-1877). A stock standard solution of OTA of 40 µg/mL approximately, was made in benzene: acetic acid (99:1) and the concentration was established by spectrophotometry as described in the Official Methods of Analysis of the International Association of Official Analytical Chemists (AOAC International 2000) using the ε value of 5550. An intermediate standard solution of 10 µg/mL in toluene: acetic acid (99 : 1) was prepared. The intermediate standard solution was diluted to standard working solutions for liquid chromatography (LC) calibration. A series of 6 standard solutions covering a range of 0.3 -6.2 ng/mL was prepared representing 15.0 -313 pg OTA injected on HPLC columns. Stock, intermediate and standard solutions were stored in the freezer at less than -18ºC.

  system and system controller, Waters 717 autosampler, Waters 470(up to October, 2003) and Waters 2475 fluorescence detectors set at excitation and emission wavelengths of 390 nm and 440 nm, respectively. Separation was achieved on a 250 x 4.6 mm i.d. Spherisorb reversed-phase column (Waters) packed with 5µm ODS-1, preceded by a C18 guard column. A Waters temperature control module set at 30 ºC for the columns was used. The mobile phase was 9 % acetic acid; / methanol (40:60, v/v) Waters 510 pump was used for post column addition (0.2 -0.4 mL/min) of 25 % (w/w) ammonium hydroxide solution to the column eluate. A zero dead volume T-piece and 10 cm x 0.25 mm i.d. stainless steel reaction tube was used. The system was controlled by Waters Millennium 32 v.4 Chromatography Manager.

(

  containing > 2.0 µg L -1 ) by liquid chromatography coupled with tandem mass spectrometry -detection (LC/MS/MS). Mass spectra were obtained on a Quattro Micro triple quadrupole Z-spray mass spectrometer from Waters, and MassLynx  software was used for control and data processing. The instrument was operated in the positive ionization electrospray mode with capillary voltage 3.5 kV, cone voltage 21 V, source temperature 120 ºC, cone gas flow 50 L/h, desolvation temperature 300 ºC and desolvation gas flow 450 L/hr. The collision energy was 25 eV and the pressure of the argon used for collision-induced dissociation was 3.20 x 10 -3 mBar. The multiple reaction monitoring (MRM) mode was used for acquiring data. The ions monitored were m/z 358 and 239, daughter ions of the OTA protonated molecule [M + H] + (m/z 404).

  1999). The analytical procedure was validated again on sweet wines for the present study. Six replicate ´blank´ samples of a liqueur wine and three sets of six replicates each, of samples of the same liqueur wine spiked by addition of OTA at 0.020 µg L -1 , 0.5 µg L -1 and 2.0 µg L -1 , respectively, were prepared. Each set of ´blank´ and spiked samples was divided in two groups of three replicates each and analysed in different days and using different calibration curves every day in order to get an estimation of the internal reproducibility. The recoveries and precision values are given in TableII. They were always in agreement with the requirements expressed in the Directive 2002/26/CE of the European Commission (Commission of the European Communities 2002), which has been repealed and updated in the Regulation (CE) nr 401/2006 (Commission of the European Communities 2006) at the moment of writing this paper. The recovery percentages obtained from spiked samples were in the range 87.2 -91.4 %. Two six-point calibration curves were built for the validation study. The regression coefficients always were > 0.999 and the linearity checks in terms of the individual deviation of the response mass ratio (Yi/Xi) for each calibration point with respect to the mean of the response mass ratios of all points (n = 6) were always < 10 %

  Series number 17, Round 20, + 0.6 in Series number 17, Round 28, and + 0.1 in Series number 17, Round 45, were achieved on each occasion. The Laboratory is currently accredited by the Spanish Accreditation Board (ENAC) under the requirements of the Standard ISO/IEC 17025 for the determination of OTA in foodstuffs.

  8 % or 3.1 % fraction of the PTDI or TDI established by JECFA and the Scientific Panel on Contaminants in the Food Chain of EFSA at levels equivalent to ca.14 and 17 ng / kg b.w. /day, respectively (JECFA 2001, Scientific Panel on Contaminants in the Food Chain 2006). Those contributions represent only a 0.4 and 0.3 % fraction of both weekly intakes established by JECFA and the Scientific Panel on Contaminants in the Food Chain of EFSA respectively,when considering the whole population formed by regular sweet wine consumers and not consumers. Obviously, the contribution to the dietary intake of OTA could represent a higher fraction of both tolerable weekly intakes in some possible, although less probable, situations, such as the worst case condition formed by a consumer, weighing less than 60 kg, and drinking regularly sweet wine containing the maximum level detected in this study.

  tolerable weekly intakes established by JEFCA and the Scientific Panel on Contaminants in the Food Chain of EFSA, respectively.

Figure 1 .

 1 Figure 1. Chromatogram of : (a) an OTA standard solution (28.75 ng/mL), equivalent to 0.719 ng of OTA injected, (b) a naturally contaminated sweet wine sample with 2.196 µg L -1 OTA (injected amount equivalent to 0.312 mL wine), (c) OTA methyl ester formed from the final extract solution of the same sample (b).

Figure 2 .

 2 Figure 2. LC/MS/MS chromatograms of a naturally contaminated sweet wine sample with 2.196 µg L -1 OTA.

Figure 3 .

 3 Figure 3. Location of some specific wine-growing regions included in this study; number of analysed samples in parenthesis

  ,

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

Table I .

 I -Distribution of samples according to the year of collection

	Origin	2002	2003	2004	2005	2002 -2005
	Spain	54	16	61	57	188
	Other countries Total F o 44 98 r	5 21	31 92	22 79	102 290
		P
		e
		e r
			R
			e
			v i e
				w
				O n l
					y

Table II .

 II Ochratoxin A in-house validation recoveries and internal reproducibility (RSD R )

	Level	Number of	Mean recovery	Internal
	(µg L -1 )	replicates	± coefficient of	Reproducibility
			(n)	variation	(%)
	0.020 a		6	91.4 % ± 4.2 %	1.1
	0.500 a F o r	5	91.1 % ± 2.3 %	2.7
	2.000 a	P	6	87.2 % ± 1.4 %	1.2
	0.114 b a Spiking level	6 e e r	-	8.9
	b Naturally contaminated sample	R
			e
			v i e
				w
				O n l
					y

Table IV .

 IV Occurrence and levels of OTA contamination in sweet wines originated from different Spanish and French regions

	Country	Region	Positives/Total	Mean (µg L -1 )	Median (µg L -1 )	Range (µg L -1 )
		Valencia -Alicante	49/49	1.070	0.573	0.052 -4.630
		Tarragona	15/15	0.956	0.567	0.010 -3.972
		Málaga		18/18	0.445	0.328	0.056 -1.360
	Spain	Navarra	a	35/37	0.073	0.054	0.005 -0.334
		Canary Islands	8/8	0.206	0.122	0.058 -0.823
		Other regions	54/54	0.600	0.169	0,010 -3.976
		South (Languedoc)	23/24	0.386	0.298	0.005 -1.218
	France
		Other regions	24/25	0.044	0.016	0.005 -0.242

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

Acknowledegments

The authors are grateful to the Spanish Food Safety Agency, which supported the study.

The authors also wish to thank very much to Ladislao Montiel, winemaker of the winery "Camilo Castilla", Navarra, Spain, for providing technical advice and knowledge on sweet wines. Finally, the authors are specially grateful to Ir. H. Van Egmond for his kind review of the manuscript.


Keywords: Chromatographic analysis, Chromatography -GC, Clean-up, Extraction Additives/Contaminants: Pesticide residues deltamethrin, wheat grain, post-harvest treatment, residues, processed products, 19

Study on the effect of grain storage and processing on deltamethrin residues in post-harvest treated wheat with regard to baby food safety requirements

Anna Balinova, Rosica Mladenova, Deyana Shtereva

Post-harvest treatment for protection of raw grain against stored-product pests has been 14 reported as a source of contamination of cereals (JMPR 1991, JMPR 1992). A summary of 15 residue monitoring data sorted by frequency of incidents showed that grain protectants used 16 on cereals accounted for most of the positive findings (Hamilton et al. 1997). Studies on the 17 effect of storage on insecticides residues following post-harvest treatments have generally 18 shown that residues decline rather slowly (Holland et al. 1994). For refining of dietary intake 19 estimates of pesticide residues a generation of food processing studies data has been required.

20

Milling is one of the 10 processing methods identified as deserving main attention (World 21 Health Organization 1997). Studies on the fate of pesticide residues on grain subjected to 22 milling and processing showed that the bulk of the insecticides deposited on the grain 23 remained in the epidermis and was removed from the bran during milling. Most residues were 24 found in the outer portion of grains [START_REF] Hajslova | Pesticides[END_REF]. The concentration on the bran was from (JMPR 1988, JMPR 1989, JMPR 1991, JMPR 1992). Generally the concentration of 3 insecticide in flour was 10% or less of the concentration on the whole wheat. For the determination of residues at ultra-trace levels considerable attention has to be paid to 16 the problems of assurance the high quality of analytical methods both in respect of 17 quantitative parameters (sensitivity, trueness, precision, and range) and of qualitative 18 assessment (identification and confirmation). Implementation of analytical methods capable 19 to determine the pesticide studied at or below 0.01 mg kg -1 was mandatory in the study. All processes applied to the study for processing effects assessment were planned in a way to 6 correspond as closely as possible to those that occur in actual practices. The initial grain moisture was 14.0±0.5%. The commercial insecticide formulation was Decis 11 2.5 EC containing 25 g.l -1 active ingredient (a.i). It was applied at two rates -the 12 recommended rate of 20 ml.t -1 and the rate assessed to give highest effect in stored grain 13 protection -160 ml.t -1 (Obretenchev, D., personal communication). 40 kg wheat grain were 14 spread in about 1 cm thick layer and sprayed with 40 ml of working solution. The application 15 was accomplished by means of a hand-operated spraying device used in the practice. In order 16 to achieve uniform distribution of the pesticide the working solution was divided into three A sample of 0.5 kg of treated grain was taken 1 hour after treatment, when the grain became 1 dry, for determination of the initial concentrations of pesticide in the whole grain. The 2 following samplings were made after 7, 90, 180 and 270 days storage periods. 3 kg of the 3 treated grain and of untreated control were taken on particular sampling dates and submitted 4 to milling in laboratory mill, simulating the industrial processing.

Gas-chromatographic determination.

15 GC-ECD operating conditions: carrier gas nitrogen at flow rate 4 ml.min -1 ; make up nitrogen 16 30 ml.min -1 ; injector temperature 270°C; detector temperature 320°C. Oven temperature 17 programme: 150°C, hold 1 min; rate 30°C/min to 290°C final temperature, hold 8 min. The method was validated for each matrix studied. [START_REF] Desmarchelier | The effect of milling and processing on the level and fate 7 of grain protectant insecticide residues on wheat and oats[END_REF] The ECD response for deltamethrin was linear in the range of 0.02 ng -0.5 ng and the 7 correlation coefficient r 2 was 0.9897 (five-point calibration curve). The concentrations of the 8 analytes in the samples were calculated by using a single-level calibration method. This 9 approach is recommended in case the detector response is variable with time (Document 10 SANCO/10476/2003). In our studies it was confirmed that the single-level calibration gave 11 more accurate results than multi-level calibration when the analyte concentrations in the 12 samples were adjusted in an interval ± 10% of the calibration level used. The mean recoveries and the repeatability expressed as relative standard deviation (RSD), determined at three fortification levels (number of samples n=5) are presented in table 1. The 16 limit of quantification (LOQ), determined as the lowest concentration of the analyte that yield 17 signal-to-noise ratio of 10:1 was established at 0.005 mg.kg -1 . The LOQ was specified at this treated post harvest with 0.5 mg kg -1 deltamethrin are presented in table 2 and of grain, treated 1 with 4 mg kg -1 deltamethrin -in table 3. Deltamethrin residues were persistent under the 2 storage conditions studied. No decrease was observed of the average residue levels on the 3 whole grain and in the processed fractions after storage periods of 180 and 270 days. The 4 measured slight increase of deltamethrin residues after 180 days storage period in the higher 5 dose variant is within the estimated uncertainty of the method. This result confirms that no 6 measurable degradation of deltamethrin residues on stored wheat grain occured during the 7 storage period. These findings correspond with the results of deltamethrin residues evaluation 8 by FAO (Food and Agriculture Organization 1983). The data reported by several countries 9 confirmed that deltamethrin was stable on stored wheat grain for more than a year. Some 10 increase of the concentrations on grain after 6 -9 months of storage was also observed (Food The distribution of deltamethrin residues in fractions from processed wheat is presented in 14 tables 2 and 3. The bulk of the insecticide deposited on the grain remained in the bran. The 15 concentrations of deltamethrin in the bran were from 3.3 to 4.7 times as high as those present 16 on the whole wheat grain. A considerable part of the insecticide was distributed in the 17 semolina fractions. The residues determined in semolina were 1.8 -2.7 times as high as those 18 on the whole grain. Generally the residue levels in flour were 3 -7 times lower than the 19 concentration on the whole grain. The reduction of residues in flour and groats is mostly due 20 to the removal of the insecticides with bran and partly with semolina fractions. 4), shows that deltamethrin undergoes negligible loss of residues 2 (0.7 %) during milling. Taking into consideration that volatilization is one of the basic 3 processes acting on pesticide residues during grinding, screening etc., the negligible losses Table 2. Decline of deltamethrin residues in whole grain and processed products with time after post-harvest treatment at range 0.5 mg.kg -1 (n = 3) Table 3. Decline of deltamethrin residues in whole grain and processed products with time after post-harvest treatment at range 4 mg.kg -1 (n = 3) Table 4 Residue balance of deltamethrin in processed products after storage intervals of 180 days (application rate 0.5 mg.kg -1 )

Introduction 23 Food 6 Furthermore

 236 safety is one of the most serious problems of the modern society. Contamination of 24 plant production with pesticide residues is a source of risk for human health. Obtaining a 25 effects of processing on the fate of residues is one of the basic pre-1 conditions for refinement of pesticide risk assessment and especially of potential risk to 2 consumers of high vulnerability (Regulation (EC) No 396/2005). Special attention has to be 3 paid to the health protection of infants and young children as they represent the most 4 vulnerable group of population. Infants and young children have been shown to be very 5 sensitive to certain toxic effects (Matsumura F. 2004, National Research Council 1993). , babies and young children are exposed to relatively higher doses of toxicants 7 per unit body weight, compared to adults because of their proportionally higher consumption 8 of food. Based on FDA data, children receive 3 -5 times more cereals than adults per body 9 weight. For better protection of the health of infants and young children a general MRL of 10 0.01 mg kg -1 for any individual pesticide in processed cereal-based foods and baby foods has 11 been established by EC (European Commission 2003 a , European Commission 2003 b ).

4 5

 4 Deltamethrin has been used as grain protectant for more than 30 years. The MRL of 1 mg kg -1 6 is established for wheat grain intended for general nutritional uses and this value 7 accommodates post-harvest treatment of the commodity (European Commission 2002). The 8 MRL established by EC (European Commission 2003 a , European Commission 2003 b ). for 9 residues in baby food is 100 times below the deltamethrin MRL for food of general 10 consumption. The enforcement of this very stringent limitation of residues in processed 11 cereal-based baby food imposes the necessity of careful tuning of all technological parameters 12 in order to select procedures that ensure baby food production in compliance with the safety 13 requirements from potentially contaminated raw materials.14

20 21 The

 21 study was aimed at assessment of the risk of baby food MRL violation due to utilization 22 of contaminated wheat grain for processing and production of baby food. The objectives of 23 this work were: to study the influence of the wheat grain storage intervals and of milling on 24 the decline and distribution of residues of deltamethrin applied post-harvest on wheat grain; to 25 balance in processed products; to provide scientific data on compliance 1 of the processed post-harvest treated wheat grain products with the safety requirements

7 8

 7 Wheat treatment, sampling and processing. 9 High grain quality wheat (Triticum estivum) cultivar "Momchil" was used in the experiments.

  10

Food

  17 portions. After uniform spraying of each portion the bulk of the grain was mixed by 18 quartering, then spread again and the spraying procedure was repeated consecutively with the 19 portions left. Finally the treated wheat grain was mixed thoroughly and kept in open 20 containers in a storehouse at conditions simulating the practical grain storage. Untreated grain 21 was stored under the same conditions as control variant. The room temperature was 20±2 0 C 22 and the air moisture -70±5%. 23 Page 4 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

5 6 MillingFood

 6 was carried out in a fractionating laboratory mill Buhler model MLU 202 M/s 7 (Buhler, Uzwil, Switzerland). After milling the following fractions were collected: bran, 8 semolina, 3 fractions of groats, and 3 fractions of flour. These processed products fully 9 resemble the industrial processing products. The collected fractions as well as the whole grain 10 were subjected to analysis. deltamethrin, purity 99.9% Pestanal was obtained from Sigma-Aldrich 15 Laborchemicalien GmbH (Seelze, Germany). Stock solution (1.0 mg.ml -1 ) was prepared in 16 ethyl acetate and stored in a freezer at -23 0 C. Working standard solutions at concentration 17 rates of 10 ng.ml -1 to 500 ng.ml -1 were prepared by diluting of stock solution with ethyl 18 acetate and stored at 4°C in the dark. 19 20 Solvents and reagents. 21 All organic solvents were of residue analysis grade and were obtained from Merck KGaA 22 (Darmstadt, Germany). Double distilled water was used when needed in experiments. Solid 23 phase extraction (SPE) sorbent Bond Elute SAX 250 mg-PSA 250 mg was from Varian Inc. 24 (Lake Forest, CA 92630 USA). 25 Page 5 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk gas chromatograph Model 5890 Series II equipped with 63 Ni electron 3 capture detector (ECD) was used. Split-splitless injector operated in the splitless mode. 4 Capillary column AT™-5ms, (30 m × 0.25 mm × 0.25 µm) was obtained from Alltech 5 Associates, Inc. (Lokeren, Belgium). 6 Nitrogen of purity 99.9995 % was used for gas chromatography. 7 Vacuum manifold (Varian BV) was used for SPE.

  The weight of the samples taken of different products varied depending on the 12 expected levels of the residue concentrations. The sample: solvent ratios varied because of the 13 different swelling capacity of the matrices. The whole wheat grain laboratory samples were 14 homogenized by mixing, analytical samples of 20 g were weighed, 40 ml ethyl acetate and 10 15 g anhydrous sodium sulfate were added and samples were extracted by shaking for 30 min on 16 a shaking machine Labortechnik HS 501 (IKA Werke GmbH & Co. KG, Staufen, Germany). 17 Analytical samples of 20 g were weighed of bran and semolina, 10 g anhydrous sodium 18 sulfate and 100 ml of the solvent were added for extraction. 40 g analytical samples of flour 19 and groats were extracted with 80 ml of the solvent after adding of 10 g anhydrous sodium 20 sulfate. The raw extracts were filtered through paper filter, 5 ml aliquot of each extract was 21 taken in a graduated tube and the volume of the extract was adjusted at 4 ml by evaporation 22 under gentle stream of air. 1 ml methanol was added to the extracts. The samples dissolved in Sample clean-up. 1The mixed mode SAX-PSA sorbent combines the hydrogen bonding and weak anion-2 exchange capability of primary/secondary amines of PSA sorbent with the strong anion-3 exchange capability of quaternary amines of SAX sorbent. This SPE product possesses 4 powerful ability to remove polar interferences such as fatty acids, other organic acids, various 5 sugars. A pre-packed cartridge SAX-PSA was washed with 2 ml methanol and conditioned 6 with 2 ml ethyl acetate-methanol (4:1). The samples were passed through the sorbent at a rate 7 of 1 ml.min -1 . The eluate was collected in a graduated tube. After the passage of the sample 8 the elution of the pesticides continued with ethyl acetate-methanol (4:1). A total volume of 10 9 ml of the eluate was collected. 1 µl was injected in GC system and if the obtained detector 10 response was out of the calibration range, the sample volume was adjusted by dilution or 11 evaporation under gentle stream of air in order to obtain analyte concentrations appropriate to 12 quantitative determination. 13 14

18Food

  Injection volume was 1 µl for all samples. Quantification was performed by using external 19 standards. The recovery studies were performed by using blank samples of untreated wheat 20 grain. The weights of blanks were the same as the weights of the analytical samples of treated 21 grains. The fortification was done by addition of calculated volumes of working standard 22 solutions in order to obtain 3 concentration levels of the insecticide studied: at MRL (0.01 mg 23 kg -1 ), below MRL (0.005 mg kg -1 ) and ten times MRL (0.1 mg kg -1 ). Five parallel samples 24 were prepared for each fortification level. After waiting time of 1 h for evaporation of the 25 Page 7 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food

  18 concentration level because it ensured compliance with the European Commission criteria for 19 quantitative residue methods (Document SANCO/10476/2003). mill is a conventional device used for simulated fractionation that 23 corresponds closely to the fractionation under the real industrial process. The results of 24 residue analysis obtained from processing experiments carried out on stored wheat grain, 25 Page 8 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

21 22

 21 In order to determine the percentage loss of residues during processing the residual amounts 23 (mg) in the separate fractions were calculated by multiplication of the weight of each fraction 24 by the corresponding residue concentration. The balance of the residual amounts in all 25 the amount on whole grain before milling, determined in samples taken 1 after 180 days storage (table

4

  could be explained by the very low vapor pressure of deltamethrin -V.p.(25 0 C)=1.25x10 -5 5 mPa (Tomlin 2000).

6 78

 6 ConclusionsThe analytical method used was sensitive enough for reliable determination of deltamethrin in 9 wheat at concentration 0.01 mg kg -1 . The storage time and milling of wheat grain, treated 10 post-harvest against stored-product pests with deltamethrin at recommended application rate, 11 are not effective enough to reduce the residues in milled products to levels at or below the 12 general MRL of 0.01 mg kg -1 established by EC for any individual pesticide in processed 13 cereal-based foods and baby foods. The bulk of the insecticide deposited on the grain is 14 removed with the bran and partly with the semolina fractions during milling but yet the flour 15 and groats fractions, used directly for preparing of baby foods, could contain deltamethrin in 16 quantities which do not comply with the infants and young children safety requirements. The 17 results of this study indicate that processed products (flour, groats) of wheat, treated post-18 harvest with deltamethrin at the recommended rate are in compliance with the safety 19 requirements for general population. The increase of the application rate beyond the 20 recommended rate should be avoided because it resulted in exceeding the MRL of 1 mg.kg -D.S., editor 2000. The Pesticide Manual, Twelfth Edition, British Crop 1 Protection Council, Farnham, Surrey, UK 2 World Health Organization 1997. Guidelines for predicting dietary intake of pesticide 3 residues. Document WHO/FSF/FOS/97.7, 2nd rev. ed.

Table 1 .

 1

	Fortification level, mg.kg -1	Recovery ± RSD, %
	0.005	97.0 ± 7.1
	0.01	89.6 ± 16.6
	0.1	97.6 ± 15.3

Table 1 .

 1 Mean recoveries and standard deviations of deltamethrin in wheat (n=5)

Table 2 .

 2 Decline of deltamethrin residues in whole grain and processed products with time after post-harvest treatment at range 0.5 mg/kg (n=3)

Table 3 .

 3 Decline of deltamethrin residues in whole grain and processed products with time after post-harvest treatment at range 4 mg/kg (n=3) Table4Residue balance of deltamethrin in processed products after storage intervals of 180 days (application rate 0.5 mg/kg)

	Captions to tables
	F o r
	P
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	e r
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Table 1 .

 1 Mean recoveries and standard deviations of deltamethrin in whole grain (n = 5)

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

Acknowledgments 2 The authors acknowledge the financial support of the NATO Program "Science for Peace" for 3 Project № SfP 977983.

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