Keywords: Heavy metals -arsenic, Heavy metals -cadmium, Lead Food Types: Animal, Meat cadmium, lead, arsenic, kidney, liver, cattle, age, maximum level

The European maximum level for cadmium in bovine kidneys is in Belgium only realistic

Introduction

Cadmium is an environmental contaminant that is readily taken up by plants. Through feed intake it is further transferred to animals where it, because of its slow excretion, mainly accumulates in kidneys and liver [START_REF] Lehman | Dosage-dependent disposition of cadmium administered orally to rats[END_REF]. Age-related increases in renal Cd concentrations have been observed in experimental studies [START_REF] Lindén | Cadmium levels in feed components and kidneys of growing/finishing pigs[END_REF] as well as in biomonitoring studies on cattle [START_REF] Andersen | Cadmium and zinc in kidneys from Danish cattle[END_REF][START_REF] Vos | Arsenic, cadmium, lead and mercury in meat, livers and kidneys of cattle slaughtered in The Netherlands during 1980-1985[END_REF][START_REF] Antoniou | Cadmium concentrations in beef consumable tissues in relation to age of animals and area of breeding[END_REF]López Alonso et al., 2000), hares [START_REF] Pedersen | Heavy metal accumulation in arctic hares (Lepus acticus) in Nunavut, Canada[END_REF] and horses [START_REF] Teilmann | Cadmium and selenium levels in kidneys from Danish horses[END_REF][START_REF] Baldini | Cadmium in organs and tissues of horses slaughtered in Italy[END_REF]. Regression models relating renal Cd concentrations to the age of animals are, however, less available. [START_REF] Andersen | Cadmium and zinc in kidneys from Danish cattle[END_REF] developed a regression model relating the logarithmic transformed Cd concentrations in kidneys of Danish cattle to the age of the animals and the geographical location of their site of rearing. A linear relation between age and Cd concentration in both kidneys and livers of cattle from The Netherlands was found by [START_REF] Vos | Arsenic, cadmium, lead and mercury in meat, livers and kidneys of cattle slaughtered in The Netherlands during 1980-1985[END_REF]. Regression models are not only interesting for modelling purposes, but also for policy makers. The models can be used to determine the age at which maximum levels (MLs) could be reached in a certain animal tissue. The European ML of Cd in bovine kidneys is 1.0 mg kg -1 fresh weight [START_REF] Eu | Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs[END_REF]. This value was exceeded in 75% of the kidneys sampled in contaminated agricultural areas in Belgium, and in 47% of kidneys sampled in uncontaminated areas (Waegeneers et al., 2009). The age of the animals might play a role in the large number of samples exceeding the ML, but in previous studies this factor was not taken into account. The objective of this study was therefore to analyze the effect of age on trace element concentrations in Belgian cattle tissues and, in case of Data of contaminated areas as well as reference areas will be considered.

Materials and methods

Sample collection

Kidneys, livers and meat tissue of 150 bovine animals were sampled in 2005 by the Belgian Federal Agency for the Safety of the Food Chain (FASFC) (Waegeneers et al., 2009). The samples were collected on the one hand in three metal contaminated areas (A, B & C), and on the other hand in rural areas away from trace element point sources.

The latter areas are further called reference areas. The contaminated areas were (i) site A -a rural area in the north-eastern part of the Kempen region in the vicinity of three (former) primary zinc smelters, (ii) site B -a rural area south of the city of Antwerp near a precious metals refining facility, which processes secondary raw materials and wastes to extract and refine non-ferrous metals such as lead, copper and precious metals, and (iii) site C -a rural area in the south of the Antwerp Kempen region near a copper refining facility and a cobalt production plant. Trace element concentrations (cadmium, lead, arsenic, zinc and copper) in tissues of 97 animals from the reference areas and 53 animals from the contaminated areas were investigated in this study. The concentrations have been published in Waegeneers et al. (2009). Together with each sample, a copy of the bovine passport was received, containing information on the date of birth and of slaughtering of the animal. These data were used to determine the age of each animal. (UNISTAT Ltd, London, England). For concentrations larger than 0.001 mg kg -1 but below the quantification limit (LOQ), measured values were used in the statistical analyses to minimise the distortion of distributions. Values below 0.001 mg kg -1 were set equal to 0.001 mg kg -1 . The normal distribution of data was verified by the Kolmogorov-Smirnov test with Lilliefors correction. Data that were log-normally distributed, were log-transformed before further analysis. Linear regression analysis (Y = b 0 + b 1 ⋅X) was used to determine relationships between age and tissue (kidney, liver) metal concentrations. The 1-α confidence intervals around the mean predicted tissue concentrations were calculated as

Statistical analysis

F

Y pred ± { } pred Y s 2) - n /2; - t(1 ⋅ α [1]
with Y pred the mean predicted tissue concentration and s{Y pred } the estimated standard deviation [START_REF] Neter | Applied linear statistical models: regression, analysis of variance, and experimental designs -3 rd ed[END_REF]. The 95% confidence interval around the slope b 1 was calculated as

b 1 ± { } 1 s 2) - n t(0.975; b ⋅ [2]
with s{b 1 } the standard error of b 1 [START_REF] Neter | Applied linear statistical models: regression, analysis of variance, and experimental designs -3 rd ed[END_REF]. More than half of the meat samples had Cd and Pb concentrations below 0.001 mg kg -1 , which made it impossible to perform a linear regression analysis on these samples.

Results and discussion

Age effects in reference areas

Analysis of variance indicated that Cd concentrations in meat samples showed an increasing trend with age (Figure 1), but even in the oldest animals the Cd concentration remained well below the European ML of 0.05 mg kg -1 fresh weight [START_REF] Eu | Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs[END_REF]. Meat-Pb concentrations did not show an age-related trend, although the average Pb concentration in animals older than 8 years was significantly larger than in most of the younger age groups (Figure 1). There was no significant linear relation between log(Age) and log(As), Cu or Zn concentrations in meat. Muscles are generally not a storage organ for trace elements.

[insert Fig. 1 here]

After log-transformation, a significant linear relation was found between the age of the animals from the reference area and their renal or hepatic Cd levels (Table 1).

After gastrointestinal uptake, Cd is transported in blood and preferentially taken up by the liver, where it induces the synthesis of metallothioneins (MT's), a family of cysteine rich, low molecular weight proteins (Sharma et al, 1979). They are known to act as detoxifying agents for Cd and are involved in the transport of Cd from liver via blood to kidneys [START_REF] Nordberg | General aspects of cadmium: transport, uptake and metabolism by the kidney[END_REF]. Because of their low molecular weight, Cd-MT's are efficiently filtered in the kidneys through the glomeruli and reabsorbed by the tubules.

Only a small portion of the gastrointestinally adsorbed Cd is finally excreted, which results in time in an accumulation of Cd in kidney and liver, even at low intake levels.

Although the regression equations ( unexplained variability in renal (R² = 0.56) and hepatic Cd (R² = 0.39). This is due to individual differences in dietary Cd intake, absorption and excretion.

The regression equations for the logarithms of renal and hepatic Pb against log(Age) are both very similar (Table 1). This similarity is related to the strong correlation between renal and hepatic Pb levels (r = 0.88; Waegeneers et al., 2009). The slope of the regression lines is low, indicating that mean Pb concentrations in kidney and liver only slowly increase with age. Furthermore, little of the variation between renal or hepatic Pb levels is explained by the age of the animals (R² = 0.12). Although absorbed Pb is rapidly distributed to soft tissues (e.g. kidney, liver, brain), in a second phase, it is redistributed from the soft tissues to bone. In all phases, Pb is excreted through faeces, urine and, in lactating cows, through milk [START_REF] Rumbeiha | A retrospective study on the disappearance of blood lead in cattle with accidental lead toxicosis[END_REF].

When soft tissue Pb is depleted, Pb is mobilised again, circulated and excreted. As a consequence, the relation between the age of the animals and the renal or hepatic Pb content is blurred.

Copper levels in kidney and Zn levels in liver did not increase with age, while there was a small but significant decrease in hepatic Cu concentrations and an increase in renal Zn concentrations with age (Table 1; R² < 0.1). These are probably anomalies due to the negative correlation between the Cu and Cd levels in liver, and the positive correlation between Zn and Cd levels in kidneys (Waegeneers et al., 2009). Indeed, Cu and Zn are not expected to accumulate in the body with age because they are essential elements that are homeostatically regulated by alterations in the rate and amount of absorption, distribution and rate of excretion (EU, 2003;ATSDR, 2004).

Studies in mice, hamsters and humans have shown that internal As is widely distributed throughout the body without a clear organ preference (Liebscher and [START_REF] Lindgren | Autoradiographic studies on the distribution of arsenic in mice and hamsters administered 74 As-arsenite or -arsenate[END_REF][START_REF] Hughes | Accumulation and metabolism of arsenic in mice after repeated oral administration of arsenate[END_REF]. Furthermore it is easily removed from the body by urinary excretion [START_REF] Vahter | Metabolism of 74 As-labeled trivalent and pentavalent inorganic arsenic in mice[END_REF]. This explains why no relation can be found between tissue As levels in cattle and the age of the animals.

[insert Table 1 here]

Renal Cd in contaminated versus reference areas

In the reference areas, the exposure to Cd (i.e. Cd concentration in feed, drinking water, …) is less variable than in polluted areas, since there are no Cd point sources in the reference areas. In polluted areas on the other hand, the exposure to Cd varies enormously from location to location: there is not only a gradient in Cd pollution between sites A, B and C, but also between farms located within the same area.

Quantifying the difference due to differences in Cd exposure between internal Cd levels of animals from reference areas and those from contaminated areas, is not evident because of the age-dependency of the data. A young animal at a more polluted farm might have higher internal Cd levels than an older animal at a less polluted farm (Figure 2). The regression equation in

        ⋅ = = [3]
with [Cd kidney ] at age = X the kidney Cd concentration (in mg kg -1 fresh weight) at a chosen age X, [Cd kidney ] at slaughtering the measured Cd concentration in the kidney of the animal and b 1 the slope of the regression line (Table 1). The age is expressed in days.

Although Eq. [3] uses the slope of animals from the reference area, it was used to recalculate the kidney Cd levels of all animals (both from the reference area and the contaminated areas) to the age of 1434 days, which is approximately 4 years and corresponds to the geometric mean age of the animals in the reference area. The underlying hypothesis is that each individual renal Cd concentration of an animal from the contaminated areas shows the same relative trend (i.e. same slope) with age as that of the animals on which the model is based. Due to the age correction, the variance caused by age in kidney Cd levels of different areas, can be eliminated. Multiple range testing after the age correction and log-transformation shows significant differences (α = 0.05) between kidney Cd levels in the reference area and the contaminated areas, and between the contaminated site C and the other contaminated sites (Table 2). Compared to the reference area, geometric mean kidney Cd levels are 1.6-fold larger at site C, 3fold larger at site B and 5-fold larger at site A. No significant differences could be found between Cd concentrations in kidneys from site A and B.

[insert Fig. 2

        ⋅ = = [4]
with [Cd liver ] at age=X the liver Cd concentration (µg kg -1 fresh weight) at a chosen age X, [Cd liver ] at slaughtering the measured Cd concentration in the liver of the animal and b 1 the slope of the regression line (Table 1). Equation [4] was used to recalculate the liver Cd levels of all animals (both from the reference area and the contaminated areas) to the age of 1434 days (Table 2). Multiple range testing after the age correction and logtransformation shows significant differences (α = 0.05) between hepatic Cd levels in the reference area and in the contaminated areas, and between the contaminated site C and the other contaminated areas (Table 2). Compared to the reference area, geometric mean liver Cd levels are 1.4-fold larger at site C, 3.3-fold larger at site B and almost 4-fold larger at site A. No significant differences could be found between Cd concentrations in livers from site A and B.

Exceedance of the European MLs

The European ML of Cd in bovine kidneys is 1.0 mg kg -1 fresh weight. As that resided at least 18 months in the contaminated area, into the food chain for human consumption. However, also 47% of the kidneys from the reference area exceeded the European maximum Cd level (Waegeneers et al., 2009). The age of the 150 investigated animals varied between 7.5 months and 12 years. The geometric mean age of the animals in the reference area was 1434 days, which is approximately 4 years, for the contaminated areas it was 1455 days. Use of the regression equation between the logarithm of the renal Cd concentration and log(Age) and of its confidence intervals (Table 1 andFigure 3) provides more insight in the relation between the age of bovines and the chance of exceeding the European ML for Cd in kidneys. As shown in Figure 3, the Cd concentration in the kidney of an average bovine animal from uncontaminated areas in Belgium will exceed the European ML at the age of 4 years and 10 months (1750 days), 10 % of the animals will exceed the ML at approximately 2.5 years (845 days) and 5% of the animals will exceed the ML when they are 1 year and 9 months old (645 days).

[insert Fig. 3

here]

To have an idea about the percentage of animals that exceed the European ML at any age, the measured kidney Cd concentration of each animal from the reference area was recalculated to different ages (1-10 years), by means of Eq. [3], using (1) the mean slope b 1 , (2) the lower 95% confidence level of the slope and (3) the upper 95% confidence level of the slope (Eq.[2]). Each time, the percentage of samples exceeding the European ML was calculated, resulting in a mean exceedance percentage, lower and upper limits of exceedance (Figure 4). The advantage of such calculations is that at each age level the whole database (97 animals) can be used instead of the limited number of animals that were slaughtered at that age (Table 3). As such it is predicted that no kidneys of one-year old animals from uncontaminated areas would exceed the ML, and 0 to 5% of kidneys from two-year old animals would exceed the ML (Figure 4). Once the animals reach the age of five, 52-58% of the kidneys are predicted to exceed the European maximum level. In contaminated areas the ML is more rapidly exceeded.

While none of the kidneys of one-year old animals from site C are predicted to exceed the maximum level, the chance of exceeding the ML is 9 to 30% at sites A and B at the age of one (Figure 4). At the age of two, the chance of exceeding the maximum level is 10-17% at site C and 57-74% at sites A and B.

[insert Fig. 4 here] [Insert Table 3 here] Equation [4] was also used to recalculate the measured hepatic Cd concentration of each animal to different ages (Figure 5). At the age of five, less than 5% of the animals from the reference area and from site C have hepatic Cd levels above the ML of 0.5 mg kg -1 (EU 2006). In contrast, at sites A and B already 9 to 13% of the 1-year old animals have a Cd level in their liver above the ML.

[insert Fig. 5 here] Within the European Union it is the aim to lay down MLs at a level that is reasonably achievable following good agricultural practices and taking into account the risk related to the consumption of the food product [START_REF] Eu | Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs[END_REF]. In accordance with this ALARA (As Low As Reasonably Achievable) principle it is the aim to have a rejection percentage for a certain food product of 5% or less in all member states. At the current ML for Cd in bovine kidneys, this rejection percentage can only be obtained for Belgian cattle if the kidneys of animals older than two years from the reference areas and of all animals from the contaminated areas are not considered. To obtain the rejection percentage of 5% for all kidneys from the reference areas, irrespective of the age of the animal, the ML should be increased to 3 mg kg -1 . Given the low consumption rate of offal by the general population, this would not considerably increase the risk related to Cd intake. The current estimated average daily Cd intake due to the consumption of kidneys is only 0.03 µg at a mean contamination level of 1.25 mg Cd kg -1 , i.e. including the kidneys from reference areas that exceeded the European ML (Vromman et al., 2008). This estimation is based on a mean consumption of 0.02 g kidney per day, which was based on both kidney consumers and non-consumers. Selected groups such as highlevel kidney consumers or young children deserve special attention as they might be more exposed to Cd than the average population. Cadmium intake of high-level kidney consumers can be estimated by taking the 97.5 th percentile intake for consumers of this food item (excluding non-consumers) and adding this to the population average Cd intake from the rest of the diet [START_REF] Tennant | Modelling consumer exposures to nutrients and additives in animal products[END_REF]. Data for high-level kidney consumption are not available for Belgium, therefore values presented by [START_REF] Tennant | Modelling consumer exposures to nutrients and additives in animal products[END_REF] based on UK food consumption data, were used to calculate a theoretical estimated daily Cd intake (TEDI) for high-level kidney consumers (Table 4). Furthermore, different age groups were included in the calculations. Mean Cd intake data for different age groups were taken from [START_REF] Brussaard | Dietary intake of food contaminants in The Netherlands (Dutch nutrition surveillance system)[END_REF] as their data for adults (15-19 µg Cd d -1 ) correspond well with the value of 17 µg d -1 presented by Vromman et al. (2008). For high-level kidney consuming children between 1 and 4 4). It should be stressed, however, that a 97.5 th percentile bovine kidney consumption of 13.95 g d -1 is very high. Food recommendations advice children between 8 months and 3 years to eat 30-50 g of meat per day (FOD VVL, 2009). This implies that every other fourth day a 50-g portion of meat would be replaced by an equal portion of kidney to get at a kidney consumption rate of 13.95 g d -1 . Such a high kidney consumption would not only lead to undesirable Cd intake levels but also to an unfavourable cholesterol intake of ~ 180 mg [START_REF] Nubel | Belgische voedingsmiddelentabel[END_REF]. Pre-school children who consume mean levels of kidneys, and mean-or high-level kidney consumers of other age groups are not expected to exceed the provisional tolerable daily Cd intake (Table 4). Whatever the age of the animals, bovine kidneys from the contaminated sites A and B do not comply with the ML and the ALARA principle, even when the ML would be increased to 3 mg kg -1 . Hence kidneys from the contaminated sites A and B should be considered as not suitable for human consumption.

[Insert Table 3 here]

Conclusions

Of the trace elements studied, Cd showed the most obvious increase in tissue concentrations as a function of age. Taking into account this age effect, significant higher renal and hepatic Cd levels were found in bovines reared at contaminated rural areas compared to reference areas. In the vicinity of some (former) zinc smelters and a precious metals refining facility, 9 to 30% of the one-year old animals were predicted to exceed the European maximum level for kidneys. In unpolluted reference areas, 0 to 5% of two-year old animals would exceed this ML. To obtain a rejection percentage of maximum 5% for all kidneys from these reference areas, which would be desirable according to the ALARA-principle 'As Low As Reasonably Achievable' of EU policy, then (i) kidneys from animals older than two years from the reference areas should a priori not be considered suitable to be placed on the market, or (ii) the maximum level for Cd should be increased to 3 mg kg -1 . The latter would bear negligible risks for the general population and the majority of high-level kidney consumers. Bovine kidneys from the most contaminated areas do not seem fit for the human food chain, even if the ML would be increased to 3 mg kg -1 . Figure 3. Predicted Cd concentration in bovine kidneys from the reference area (in mg kg -1 fresh weight) as a function of age, including confidence intervals (Eq [1]). The prediction is based on the regression equation presented in Table 1. The horizontal dotted line represents the maximum level of 1 mg kg -1 . Figure 4. Predicted percentage of kidneys, at different animal ages, exceeding the maximum level of 1 mg kg -1 in the reference areas (top) and in the contaminated areas (bottom). Equation [3] and the 95% confidence limits around the slope b 1 (Eq. [2] and Table 1) were used for these predictions.

  to determine the age at which the European MLs are exceeded.

  Statistical analyses were performed with UNISTAT Statistical Package, Version 5.5.06

  shown byWaegeneers et al. (2009) this value was exceeded in 75% of the bovine kidneys sampled in contaminated areas. Being aware of the problem, the Belgian federal government has already taken actions, involving e.g. a ban on bringing kidneys of cattle

  , the TEDI exceeds the provisional tolerable daily Cd intake(WHO, 2001; Table

  Saegerman, C, Pussemier L, Huyghebaert A, De Temmerman L, Pizzolon J-C, Waegeneers N. 2008. Cadmium in the food chain near non-ferrous metal production sites. Food Addit Contam. 25: 293-301. Waegeneers N, Pizzolon J-C, Hoenig M, De Temmerman L. 2009. Accumulation of trace elements in cattle from rural and industrial areas. Food Addit Contam. 26: 326-332. WHO 2001. Cadmium. In: Safety Evaluation of Certain Food Additives and Contaminants. Fifty-fifth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Geneva/ WHO Food Additives Series 46. pp 247-312.

Figure 1 .

 1 Figure 1. Cadmium and lead concentrations in meat samples of cattle at age ≤ 2 (n =

Figure 2 .

 2 Figure 2. Renal and hepatic Cd concentrations (in mg kg -1 fresh weight) as a function of

Figure 5 .

 5 Figure 5. Predicted percentage of livers, at different animal ages, exceeding the maximum level of 0.5 mg kg -1 in the reference areas (top) and in the contaminated areas

  Figure 1.

F

  Figure 2.

  Figure 3.

F

  The animals were grouped into seven different age groups, and the effect of age on the Cd and Pb concentration in meat

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	was assessed by one-way analysis of variance (ANOVA) followed by Duncan's y
	multiple range test (α = 0.05).

Table 1

 1

	) are significant, there is still a lot of

Hepatic Cd in contaminated versus reference areas

  Hepatic Cd levels can also be corrected for age as was done for renal Cd concentrations although the strength (R²) and significance of the relation between Cd and age is less for liver than for kidneys. The slope of the regression equation in Table1is significant (p < 0.001) hence each liver Cd concentration can be recalculated to a

	chosen age following the equation:
	[ Cd	liver	] at	F r o [ ] slaughteri at liver X age Cd	ng	age	slaughteri X age at	ng	1 b
				P
				e
				e r
						R
							e
							v i e
									w
									O n l
									y
									here]
						[Insert Table 2 here]

Table 1 )

 1 were used for these predictions.

Table 1 .

 1 Linear regression equations of tissue metal concentrations (mg kg -1 fresh weight) against animal age (in days), based on data collected in reference areas. All data

	were log-transformed. Y = b 0 + b 1 ⋅X
	Y	b 0 ± s{b 0 }	b 1 ± s{b 1 }	X	R²
	log([Cd kidney ])	-4.12 *** ± 0.37	1.27 *** ± 0.12	log(Age)	0.56
	log([Cd liver ]) o -3.28 *** ± 0.31 r log([Pb kidney ]) -2.59 *** ± 0.48	0.78 *** ± 0.10 0.55 *** ± 0.15	log(Age) log(Age)	0.39 0.12
	log([Pb liver ]) log([Zn kidney ])	-3.00 *** ± 0.49 P e 0.79 *** ± 0.16	0.55 *** ± 0.15 0.14 ** ± 0.05	log(Age) log(Age)	0.12 0.08
	log([Cu liver ]) p < 0.05 *	3.02 *** ± 0.55 e r -0.40 * ± 0.17	log(Age)	0.05
	** p < 0.01	R
	*** p < 0.001		e e i v
			w
				O n l
				y
					1

Table 2 .

 2 Geometric mean cadmium concentration (mg kg -1 fresh weight) in kidneys and liver of bovines after recalculation to the age of 1434 days (~ 4 years). The recalculation is based on the slope of the equations presented in Table1. Numbers within the same row followed by the same superscript character, are not significantly different (α = 0.05) following Duncan's multiple range test on log-transformed data. The data are retransformed into natural numbers.

	Page 27 of 29		Food Additives and Contaminants
		Reference area	Site C	Site B	Site A
	[Cd kidney ]	0.777 a	1.271 b	2.280 c	3.678 c
	[Cd liver ]	0.152 a	0.210 b	0.504 c	0.560 c

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Table 3 .

 3 Number of animals that were analysed, number of kidney samples that exceeded the ML for Cd in kidneys and number of liver samples that exceeded the ML for Cd in liver per area and per age category.

			Food Additives and Contaminants						Page 28 of 29
	Age (years)	1	2	3	4	5	6	7	8	9	10	11	12
				Reference areas
	# animals	1	13	16	17	13	13	9	3	6	2	2	2
	# samples > 1 mg kg -1 (ML kidney)	0	0	2	9	5	10	8	2	5	1	2	2
	# samples > 0.5 mg kg -1 (ML liver)	0	0	0	1	0	0	0	0	1	0	1	1
					Site A & B
	# animals	0	3	5	6	3	3	1	2	0	0	0	0
	# samples > 1 mg kg -1 (ML kidney)	-	2	3	6	3	2	1	2	-	-	-	-
	# samples > 0.5 mg kg -1 (ML liver)	-	1	1	2	2	2	1	1	-	-	-	-
					Site C
	# animals	1	1	2	5	10	4	2	3	1	0	1	0
	# samples > 1 mg kg -1 (ML kidney)	0	0	0	3	8	4	1	3	1	-	1	-
	# samples > 0.5 mg kg -1 (ML liver)	0	0	0	0	1	0	0	1	1	-	1	-

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Table 4 .

 4 Results of Cd intake estimations for mean and high-level kidney consumers of different age classes if the European ML would be increased to 3 mg kg -1 . The theoretically estimated daily Cd intake (TEDI) for bovine kidney consumers is calculated by multiplying their kidney intake with the mean Cd concentration in Belgian bovine kidneys excluding values > 3 mg kg -1 , i.e. 1.038 mg kg -1 . PTDI is the provisional tolerable daily intake(WHO, 2001).

	Age class General Population	Kidney consumers

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Food Additives and Contaminants

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Susana Lorán

Pilar Conchello

Susana Bayarri

Antonio Herrera

Evaluation of daily intake of PCDD/Fs and indicator PCBs in formulafed Spanish children

Keywords: Dioxins -TEQs, PCBs PCDD/Fs, PCBs, infant formulae, dietary intake, children, exposure assessment

Human exposure to polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) occurs predominantly via food intake.

In this study, the exposure assessment of these contaminants has been estimated for infant formula fed children up to one year of age. PCDD/Fs concentration in the infant formulae was low, ranging between 0.09 and 0.17 pg WHO-TEQ g -1 fat and between 0.30 and 0.46 pg WHO-TEQ g -1 fat when results were calculated with the lower and medium bound values, respectively. Indicator PCBs contamination level were below 1 ng g -1 fat in all cases. Thus, the estimated PCDD/Fs and indicator PCBs daily intake of infants has been assessed taking into account the above mentioned contamination levels as well as different scenarios of body weight and food consumption data for babies aged 0 to 12 months. The results vary in the different scenarios considered, but on the whole, the daily estimated dioxin and indicator PCBs intake of the average infant population due to the consumption of infant formulae does not exceed the tolerable daily intake (TDI) of 2 pg WHO-TEQ kg -1 b.w. day -1 (SCF, 2001) nor the threshold of 10 ng kg -1

Introduction

Organochlorine compounds such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) represent a class of widely distributed environmental contaminants which exhibit a potential risk for human health. Dioxins are mainly by-products of industrial processes, being waste incineration, especially if combustion is incomplete, the largest contributor of dioxin release into the environment. But dioxins can also result from natural processes like volcanic eruptions and forest fires (SCF, 2001;[START_REF] Baars | Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuffs: occurrence and dietary intake in The Netherlands[END_REF]). Unlike dioxins, PCB mixtures were commercially produced for about five decades for its use in a wide scale of applications such as electronic appliances, heat transfer systems, hydraulic fluids and many other different industrial purposes [START_REF] Baars | Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuffs: occurrence and dietary intake in The Netherlands[END_REF]EFSA, 2005). Although PCB production was banned in industrialized countries in the 1970s, residues can still enter the environment due to their lipophilic nature, persistence and ample use in the past.

Previous reports have revealed that dietary intake is the main route (> 90%) for non-occupational human exposure to these toxicants; foods of animal origin usually being the main contributors of the total intake [START_REF] Baars | Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuffs: occurrence and dietary intake in The Netherlands[END_REF]EFSA, 2005). In many countries concentrations of PCDD/Fs and PCBs have regularly been monitored in a great variety of food matrices. Available data indicate a reduction in the past decades 3 due to measures implemented to control the emissions of these compounds and so exposures and body burden are lower now than years before [START_REF] Llobet | Significant decreasing trend in human dietary exposure to PCDD/PCDFs and PCBs in Catalonia, Spain[END_REF]). Yet, despite these extensive efforts, foods are still contaminated [START_REF] Fernández | Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxinlike polychlorinated biphenyls in Spain[END_REF]EFSA, 2005;[START_REF] Gómara | Levels and trends of polychlorinated dibenzo-pdioxins/furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) in Spanish commercial fish and shellfish products, 1995-2003[END_REF][START_REF] Bordajandi | Organochlorine compounds (PCBs, PCDDs and PCDFs) in seafish and seafood from the Spanish Atlantic Southwest Coast[END_REF][START_REF] Bocio | Concentrations of PCDD/PCDFs and PCBs in fish and seafood from the Catalan (Spain) market: Estimated human intake[END_REF][START_REF] Llobet | Significant decreasing trend in human dietary exposure to PCDD/PCDFs and PCBs in Catalonia, Spain[END_REF].

Like adults, children's primary source of exposure to these contaminants is food.

Exposure assessments of PCDD/Fs and PCBs for the general population have been made in several industrialized countries but most of them have no consideration of young children [START_REF] Lázaro | Estimated dietary intake of polychlorinated biphenyls and organochlorine pesticides from Aragonese (NE Spain) diet[END_REF][START_REF] Tard | Dioxins, furans and dioxinlike PCBs: Occurrence in food and dietary intake in France[END_REF], although many studies have also been made for breast-fed infants [START_REF] Focant | Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk. Estimation of infant intake[END_REF][START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF][START_REF] Szyrwińska | Exposure to specific polychlorinated biphenyls and some chlorinated pesticides via breast milk in Poland[END_REF]. This is probably due to the fact that as these compounds accumulate in fatty tissues, breast milk has been one of the preferred matrices to evaluate human background contamination. Additionally, there has been a particular concern in recent years about the characterization of breast-fed exposure to these substances and the associated potential health risk.

Infant formulae are an alternative to human milk which is recommended for infants who are not breast-fed or during the weaning period and they can play an important role in the infant diet. However, only a few reports are available on the PCDD/Fs and PCB levels in infant formulae, usually showing low concentrations of these pollutants [START_REF] Ramos | Determination of polychlorinated biphenyls in soybean infant formulas by gas chromatography[END_REF]FSA UK, 2004;[START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF]).

Related to the daily PCDD/Fs intake, World Health Organization (WHO) recommended in 1998 a tolerable daily intake (TDI) of PCDD/F in a range of 1 to 4 pg WHO-TEQ kg -1 b.w.day -1 (WHO, 1998). In later revisions, a tolerable weekly intake of 14 pg WHO-TEQ kg -1 b.w.week -1 (SCF 2001) and a tolerable monthly intake of 70 pg WHO-TEQ kg -1 b.w.month -1 (JECFA 2002) have been established. For practical reasons these maximum intakes can be handled as a tolerable daily intake of 2 pg TEQ per kg b.w. per day. In addition, the European Commission recently set maximum levels of PCDD/Fs and dioxin-like PCBs permitted in food (EC, 2006). For non-dioxin like PCBs neither a tolerable daily intake nor a maximum limit in feed and food have been established for these compounds in the European Union, which is why some countries have established reference values that can be used in order to evaluate food contamination or human exposure to these compounds.

Current PCDD/Fs exposure of breast-fed infants is about 13 pg WHO-TEQ kg -1 b.w. day -1 for one year old children in Taiwan [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF], 51 pg WHO-TEQ kg -1 b.w. day -1 in Portugal [START_REF] Reis | Infant exposure to PCDD/Fs in Portugal: first results from an environmental health survey program near Lisbon[END_REF] or 76 pg WHO-TEQ kg -1 b.w. day -1 in Belgium [START_REF] Focant | Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk. Estimation of infant intake[END_REF]. Mean exposure to the seven indicator PCBs has been calculated between 364 and 375 ng kg -1 b.w. day -1 for first and second breast-fed infants in Poland, respectively [START_REF] Szyrwińska | Exposure to specific polychlorinated biphenyls and some chlorinated pesticides via breast milk in Poland[END_REF], and 1249 ng kg -1 b.w. day -1 in the European Union (EFSA, 2005). There are few surveys which have studied the exposure of non breast-fed children to these pollutants revealing PCDD/Fs intake levels of 2.1 pg WHO-TEQ kg -1 b.w. day -1 in one year old children [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF] or varying between < 0.01 and 0.3 pg WHO-TEQ kg -1 b.w. day -1 in 6 month old babies and between < 0.01 and 0.2 pg WHO-TEQ/kg. b.w./day in 9 and 12 month old English children (FSA UK, 2004).

One of the main difficulties when comparing and evaluating infant babies dietary exposure to these compounds is the children's different ages, body weights and food consumption data used in these estimations, which are not consistent in the published literature. Milk consumption intakes between 600 and 800 ml/day have been considered for 5-7 kg of body weight children [START_REF] Focant | Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk. Estimation of infant intake[END_REF][START_REF] Reis | Infant exposure to PCDD/Fs in Portugal: first results from an environmental health survey program near Lisbon[END_REF]EFSA, 2005). In Taiwan, PCDD/Fs dietary intake was estimated for breast and non- Taiwanese Department of Health [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF]). However, this information is not available in many countries. Concerning this point, it is important to mention the Euro-Growth study which is a multi-center, longitudinal cohort study performed in different European regions. The main objectives of this study were to evaluate: a) the longitudinal growth patterns of European children and to develop reference growth charts, b) the dietary habits of European infants and toddlers and c) the influence of nutritional, socioeconomic and geographical factors on growth and health (Haschke and van't Hof, 2000;[START_REF] Van't Hof | The Euro-Growth Study: Why, Who, and How[END_REF]. In this survey, the patterns of milk and food intake in infants from birth to age 36 month were evaluated [START_REF] Freeman | Patterns of Milk and Food Intake in Infants from Birth to Age 36 Months: The Euro-Growth Study[END_REF]Haschke et al. 2000a).

When estimating the dietary exposure to potential dangerous substances contained in commercial infant food we should also consider the DONALD study (Dortmund Nutritional and Anthropometrical Longitudinally Designed). This is a longitudinal study collecting detailed data on diet, growth, development and metabolism between infancy and adulthood in Germany. As a part of this study dietary records from 3-, 6-, 9 and 12-month old infants regarding consumption of commercial infant food were evaluated [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: results from the DONALD study[END_REF].

The main goal of the present study is to estimate the dietary intake of PCDD/Fs and indicator PCBs of Spanish infant babies up to one year of age through the consumption of commercial infant formulae. In so doing we have analyzed the presence of these contaminants in a number of samples of initial, follow-on and lactose-free formulae. Thus, a congener-specific analysis of the 17 toxic PCDD/Fs congeners and the seven indicator PCBs (PCB 28,52,101,118,138,153 and 180) values of these pollutants depending on the scenarios considered. This survey is one of the scarce studies in which the exposure of formula-fed children to these pollutants is assessed.

Materials and Methods

Sampling collection

Samples of infant formulae were acquired from local markets, big supermarkets and pharmacies in Zaragoza (Spain) between 2004 and 2005. Trade marks were selected among the most popular in Spain. A total of 70 samples (5 different batches from the same manufacturer) was studied. Infant formulae were divided in three groups, 25 infant formula, 25 follow-on formula and 20 special lactose-free formulas. Infant formulae are those for children from 0 to 6 months of age and follow-on formulas are for infants 6 months and older. Among special formulae, lactose-free formulas are specially adapted to children with lactose intolerance. These commercial infant foods contain skimmed cow milk and mixtures of vegetable oils which are added to these products in order to reach the nutrient requirements for children at this age.

As for the sample preparation, an aggregate sample (1 kg) was made by mixing proportional pooled samples of 5 different batches from the same commercial trade.

Finally, 14 analytical samples were made up, 5 samples of infant formula, 5 samples of follow-on formula and 4 samples of special lactose-free formulas. The methodology used for PCDD/Fs analysis has been described in detail elsewhere [START_REF] Lorán | Evaluation of GC-ion trap-MS/MS methodology for monitoring PCDD/Fs in infant formulas[END_REF]. In brief, about 20 grams of the analytical samples were mixed with sodium sulphate anhydrous and extracted with acetone-n-hexane (1:1, v/v) in an automated Soxhlet extractor (Soxtec System®, Foss Tecator Cat. No. 1045 HT2, Höganäs, Sweden). For identification and quantification, samples were spiked with 13 C 12 -PCDD/Fs labelled internal standard prior to extraction. Each extract was purified in a sequence that comprises purification on a column with sodium sulphate and diatomaceous earth (Isolute®) with sulphuric acid and then by using a multilayer chromatography column (with sodium sulfate anhydrous , silica gel, a mixture of sodium sulphate and sodium hydrogen carbonate, diatomaceous earth, a mixture of diatomaceous earth and sulfuric acid and a mixture of sodium sulfate and sodium chloride). Finally, the organic extract was subjected to a chromatographic filtration on activated alumina. Fraction containing PCDD/Fs was eluted with dichloromethane. For indicator PCBs analysis, an aliquot of approximately 0.5 g of the extracted fat was dissolved in hexane and purified on alumina chromatographic column partially deactivated. Elution was carried out with n-hexane. Prior to use, sodium sulphate anhydrous (Na 2 SO 4) and sodium sulphate anhydrous mixtures were kept at 120 °C overnight, and alumina was activated in a furnace at 500 °C for 12-24 hours. Determination of PCDD/Fs and indicator PCBs was carried out after adding the internal standard solution, EPA 1613-ISS (PCDD/Fs) and PCB 209 (indicator PCBs).

Analytical procedure

The eluting fractions containing the compounds of interest were analyzed by high resolution gas chromatography (HRGC) coupled to ion trap mass spectrometry (Varian, Saturn 2000) in the MS/MS mode. This is a three step process where analytes undergo electron ionization and then, a parent ion is selectively stored and fragmented into characteristic product ions through collision induced dissociation (CID). The product Chromatographic separation was achieved with a DB-5 fused-silica capillary column (60 m x 0.25 mm ID, 0.25 µm film thickness) with helium as the carrier gas at a linear velocity of 1 ml/min. As for dioxin analysis, injection was carried out in the splitless injection mode (2 µl) at a constant temperature of 300 ºC during 1.50 min with pressure pulse of 45 psi for 1.60 min. The oven temperature program was 120 ºC and held for 2 min, then to 200 ºC (held for 3 min) at 30 ºC/min, to 230 ºC (held 15 min) at 3 ºC/min and to 280 ºC (held for 12 min) at 5 ºC/min and finally to 310 ºC/min ( held for 3 min) at 10 ºC/min. PCBs injection was also made in the splitless injection mode (2 µl) at 280 ºC and with pressure pulse of 40 psi. The oven temperature program was 65 ºC and held for 2 min, then to 235 ºC (held for 10 min) at 25 ºC/min, to 310 ºC at 10 ºC/min and finally 4 minutes at 310 ºC.

The quantification of PCDD/Fs was carried out by the isotopic dilution method and methodology was validated according to EPA Method 1613 by doing an initial and ongoing precision and recovery study. Instrumental limit of detection (LOD) ranged between 0.05 ng ml -1 for tetra-chloro substituted congeners to 5.0 ng ml -1 for OCDF, depending on the specific congener. PCBs methodology was validated according to international recommendations (EC, 2002) and detection limits for the different PCB congeners were set at 0.5 ng ml -1 . In both cases, samples were analyzed in batches constituted by a calibration verification standard solution, a method blank, one spiked sample to check both the ongoing precision and recovery, along with a set of four to six analytical samples. To assess the reliability of our results we have participated in interlaboratory studies related to dioxins and PCBs (Interlaboratory Comparison on As for PCDD/Fs global concentrations, toxic equivalents (TEQ) were calculated using the toxic equivalent factors (TEFs) reported by the World Health Organisation in 1998 [START_REF] Van Den Berg | Toxic Equivalency Factors (TEFs) for PCBs, PCDDs, PCDFs for Humans and Wildlife[END_REF]. The total concentrations of PCDD/Fs and indicator PCBs have been calculated assuming that non-detected congener concentration is equal to zero (lower bound) and equal to half the limit of detection (medium bound). In order to express the results on a lipid basis, fat lipid content was determined by the Gerber Method modified for powdered milk [START_REF] Egan | Pearson's Chemical Analysis of Foods[END_REF].

Intake estimation

PCDD/Fs and indicator PCBs daily dietary exposure has been calculated multiplying the concentrations of the pollutants found in the samples analyzed by the consumed amount of infant formulae in the different considered periods during the first year of life. In order to calculate the exposure per kilogram of body weight, the estimated daily exposure has been divided by the body weight of children at those ages.

Food consumption data of children up to one year of age have been reviewed in order to measure the intake of these pollutants by Spanish infant babies. Although many nutritional recommendations and studies related with the food consumption habits of the infant babies can be found in the literature [START_REF] Franch | Recomendaciones de ingesta durante el primer año de vida[END_REF][START_REF] Bueno | Nutrición en Pediatría[END_REF]) no surveys which quantify the food consumption for Spanish children at these ages have been found. Average daily milk intake has then been obtained from two main sources: the Euro-Growth [START_REF] Freeman | Patterns of Milk and Food Intake in Infants from Birth to Age 36 Months: The Euro-Growth Study[END_REF] and the DONALD study [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: results from the DONALD study[END_REF].

Consequently, two different scenarios of exposure have been evaluated in this work. In the scenario 1, in order to estimate the daily exposure to PCDD/Fs and indicator PCBs we have used data from the Euro-Growth study. Is interesting to note that 25% of these data come from Spanish children. In this case, the food consumption quantities considered correspond to children who are not breast-fed and the volume of milk consumed is for the mean population [START_REF] Freeman | Patterns of Milk and Food Intake in Infants from Birth to Age 36 Months: The Euro-Growth Study[END_REF]. Body weight data, which has also been obtained from the Euro-Growth study (Haschke et al. 2000b), have been given for the mean population as well as for different percentiles. Some of the extreme body weight values have been considered to estimate the exposure of the highest and lowest exposed children to these compounds, that is the 95 and 5 percentile of body weight respectively. The milk consumption quantities and body weight data used for the intake estimations carried out in the scenario 1 are shown in Table I.

For the scenario 2, food consumption quantities as described in the DONALD Study were used (Table II). These values are given for the mean population but also for those children with different consumption patterns: minimum, maximum and percentiles 5 and 95. However, weight data of infant babies were just described for the mean population [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: results from the DONALD study[END_REF]). These P 95 and P 5 values together with the mean intake of commercial infant food were used in this case for estimating the PCDD/Fs and indicator PCBs dietary intake.

"[Insert Table I about here]" "[Insert Table II about here]"

Regarding other considerations and according to nutritional recommendations and food consumption habits for infant babies we have considered that initial formula is only consumed by 3-month old children while 6 month children and older drink followon formulas. Besides, milk consumption quantities as described in the Euro-Growth study are referred in volume, however all samples analyzed were powdered infant given in the information nutritional label. In addition, infants' weight data used in these estimations have been given separately for boys and girls in both studies and so we have made in our calculations.

Results and discussion

The PCDD/Fs (pg WHO-TEQ) concentrations of initial and follow-on formulae have been reported elsewhere [START_REF] Lorán | Evaluation of GC-ion trap-MS/MS methodology for monitoring PCDD/Fs in infant formulas[END_REF]. Results for the sum of the 17 toxic compounds at the lower bound values are 0.09 pg WHO-TEQ g -1 fat (0.023 pg WHO-TEQ g -1 product) for the former and 0.17 pg WHO-TEQ g -1 fat (0.039 pg WHO-TEQ g - 1 product) for the latest. Besides, 4 composite samples of lactose-free formulae have also been studied with lower bound values of 0.11 pg WHO-TEQ g -1 fat (0.030 pg WHO-TEQ g -1 product). When infant formulae contamination have been calculated with the medium bound values these concentrations add up to 0.30 pg WHO-TEQ g -1 fat or 0.08 pg WHO-TEQ g -1 product (initial formulae); 0.46 pg WHO-TEQ g -1 fat or 0.11 pg WHO-TEQ g -1 product (follow-on formulae) and 0.43 pg WHO-TEQ g -1 fat or 0.11 pg WHO-TEQ g -1 product (lactose-free formulae).

The contamination pattern of the three kind of infant formulae analyzed is represented in Figure 1. The mean concentration of each PCDD/F has been expressed as pg g -1 fat and calculated as medium bound values. "[Insert Figure 1 about here]"

The pattern in follow-on and lactose free formulae is characterized by the dominance of PCDFs while PCDDs predominate in initial formulae. For most of the samples, those congeners present in higher concentration are those with a high- 12 chlorination degree, especially octachlorinated congeners followed by 1,2,3,6,7,8-HxCDD, 1,2,3,4,7,8-HxCDD and 1,2,3,4,7,8,9-HpCDF in initial formulae and 1,2,3,4,6,7,8-HpCDD and 1,2,3,4,7,8,9-HpCDF in the others. However, main contributors to the total TEQ values are 2,3,4,7,8 PCDF, 1,2,3,7,8 PCDD and2,3,7,8 TCDD. This contamination pattern is similar to that found by [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF] in the analysis of infant formulae commercialized in Taiwan and also similar to the contamination pattern of soybean infant formulae analyzed by [START_REF] Schecter | Levels of polychlorinated dibenzodioxins and dibenzofurans in cow's milk and in soy bean derived infant formulas sold in the United States and other countries[END_REF]. [START_REF] Schecter | Levels of polychlorinated dibenzodioxins and dibenzofurans in cow's milk and in soy bean derived infant formulas sold in the United States and other countries[END_REF] also found a high percent of non detected congeners; besides, those present in higher concentrations were: OCDD > OCDF > 1,2,3,4,6,7,8-HpCDD > 1,2,3,4,6,7,8-HpCDF. Nonetheless, it is important to point out that in this work the higher concentration of those congeners with a high chlorination degree is also due to the fact that these compounds have a lower detection limit than those with less chlorine content.

The overall contamination of the infant formulae analyzed, expressed in pg WHO-TEQ g -1 fat and considering the medium bound value is shown in Table III. For comparative purposes, levels found in the analysis of similar products in other countries are also shown.

"[Insert Table III about here]"

PCDD/Fs concentrations calculated in this work, both with the lower and medium bound values, are higher in follow-on than in lactose-free formulae; these values are also higher than those observed in the initial formulae studied. However, the differences are not significant when results are calculated with the lower bound values.

When this contamination is expressed with the medium bound values PCDD/Fs levels in the initial formulae analyzed are significantly lower (p < 0,05) than those measured in the other two kind of samples studied. This difference has previously been found for As it can be seen in Table III, infant formulae contamination is similar to that reported for this kind of products in the UK (FSA UK, 2004) with values for the sum of PCDD/Fs and dioxin-like PCBs of 0.2-0.4 pg-TEQ g -1 fat and lower than those measured in infant formulae in Slovakia for the sum of PCDDs and PCDFs of 0.64 pg WHO-TEQ g -1 fat [START_REF] Chovancová | PCDDs, PCDFs and dioxin-like PCBs in food of animal origin (Slovakia)[END_REF] or the ones analyzed in Taiwan (0.713 ± 0.163 pg WHO-TEQ g -1 fat) by [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF]. However, in these two last studies sample acquisition was carried out years before: in 2001 [START_REF] Chovancová | PCDDs, PCDFs and dioxin-like PCBs in food of animal origin (Slovakia)[END_REF]) and between 2000 and 2001 [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF]. Besides, as these compounds accumulate in fatty tissues, levels found in the infant formulae analyzed are lower than the PCDD/Fs concentrations found in breast-milk samples. Therefore, concentrations of 7.77 pg WHO-TEQ g -1 fat have been recently found in samples of breast-milk analyzed in Spain [START_REF] Bordajandi | Occurrence of PCBs, PCDD/Fs, PBDEs and DDTs in Spanish breast milk: Enantiomeric fraction of chiral PCBs[END_REF]). In addition, other studies on the levels of these pollutants in human milk carried out in Spain in recent years, have revealed concentrations which have declined from 11.82 pg I-TEQ g -1 fat in samples collected in 1996 [START_REF] Schuhmacher | PCDD/F concentrations in milk of nonoccupationally exposed women living in southern Catalonia, Spain[END_REF]) to 9.1 pg WHO-TEQ g -1 fat [START_REF] Schuhmacher | Monitoring dioxins and furans in a population living near a hazardous waste incinerator: levels in breast milk[END_REF]). The current levels of PCDD/Fs found in human milk samples in other countries, with values of 14.85 pg WHO-TEQ g -1 fat in Japan [START_REF] Guan | Congener profiles of PCDDs, PCDFs, and dioxin-like PCBs in the breast milk samples in Tokyo, Japan[END_REF]); 14.7 pg WHO-TEQ g -1 fat in Taiwan [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF] or 29.4 pg WHO-TEQ g -1 fat in Belgium [START_REF] Focant | Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk. Estimation of infant intake[END_REF], are slightly higher than the results reported for Spanish breast milk samples and clearly higher than those found in formula milk samples.

The indicator PCB concentration in the samples analyzed is summarized in Table IV. Results are presented for the seven indicator congeners; the sum of all of them is also shown both in a fat and product basis. PCB concentration is below 1 ng g -1 fat for the three kinds of samples analyzed and the differences between them are not significant. These values are usually lower than those found in the literature for this kind of food. [START_REF] Schecter | Levels of polychlorinated dibenzodioxins and dibenzofurans in cow's milk and in soy bean derived infant formulas sold in the United States and other countries[END_REF] analysed PCBs 28, 138, 153 and 180 in one soybean infant formula and found concentrations below detection limits (2 ng g -1 of lipids). [START_REF] Ramos | Determination of polychlorinated biphenyls in soybean infant formulas by gas chromatography[END_REF] studied the presence of 15 congeners of PCBs in 8 soya based infant formulae collected in Spain. Levels found for the sum of the seven indicator PCBs were 7.36 ng g -1 fat. The indicator PCBs concentrations, as well as the analysis of PCDD/Fs, are much higher in breast milk samples than in infant formulae: 149.81 ng g -1 fat in Sweden [START_REF] Glynn | Polychlorinated Biphenyl Congeners as Markers of Toxic Equivalents of Polychlorinated Biphenyls, Dibenzo-p-dioxins and Dibenzofurans in Breast Milk[END_REF], 55.42 ng g -1 fat in Taiwan [START_REF] Wang | Infant exposure to polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls (PCDD/Fs, PCBs)-correlation between prenatal and postnatal exposure[END_REF] or 108.3 ng g -1 fat in Poland (Szyrwinska y Lulek, 2007).

"[Insert Table IV about here]"

The most frequently detected congeners are PCB 28 and 52 (86% of the samples analyzed) have also been dominant in the three kind of samples analyzed. In fact, the average concentration of PCBs gets lower as the chlorination grade goes up from trichloro to heptachloro substituted congeners. This contamination pattern is similar to that of the soya based infant formulae analyzed in Spain by [START_REF] Ramos | Determination of polychlorinated biphenyls in soybean infant formulas by gas chromatography[END_REF], where PCB 101, 28 and 77 are the most abundant congeners followed in this case by PCB 153, 138 and 180. This profile is usually different to that observed in cow or breast milk samples where the most abundant congeners are the highest chlorinated ones [START_REF] Paumgartten | PCDDs, PCDFs, PCBs and other organochlorine compounds in human milk from Rio de Janeiro, Brazil[END_REF][START_REF] Glynn | Polychlorinated Biphenyl Congeners as Markers of Toxic Equivalents of Polychlorinated Biphenyls, Dibenzo-p-dioxins and Dibenzofurans in Breast Milk[END_REF]). However, it is important to take into account that mixtures of vegetable oils are added to these skimmed-milk based products in order to meet the nutrient requirements for children at this age. Unlike to animals, plants lack the ability to metabolise the lower chlorinated congeners and usually have PCB profiles different from other food categories (EFSA, 2005).

Assessment of dietary exposure is vital to obtain fundamental data concerning both food safety and trends in the intake of chemicals as well as to identify the sources of unusual It is important to take into account that in scenario 1, the highest PCDD/Fs and indicator PCBs intakes correspond to those children with a mean food intake and lower body weight (Percentile 5) whereas those children with a mean food intake and higher body weight (Percentile 95) have the lowest intake of these contaminants. However, in scenario 2, P 5 stand for those children with the lowest food consumption (Percentile 5) and mean body weight, and so the lowest PCDD/Fs and indicator PCBs intake and P 95 represents those children with the highest food consumption (Percentile 95) and mean body weight, and so the highest pollutants intake.

Estimated PCDD/Fs daily dietary intake during the first year of life due to the consumption of commercial infant formulae goes from 0.22 pg WHO-TEQ kg -1 b.w. day -1 to 0.56 pg WHO-TEQ kg -1 b.w. day -1 in the first scenario and from 0.05 pg WHO-TEQ kg -1 b.w. day -1 to 0.65 pg WHO-TEQ kg -1 b.w. day -1 in the second one, when results are calculated with the lower bound values. The intake of indicator PCBs in children at this age goes from 0.92 to 2.32 ng kg -1 b.w. day -1 and from 0.20 to 2.69 ng kg -1 b.w. day -1 in the first and second scenario respectively. These values go up to 0.61-1.93 pg WHO-TEQ kg -1 b.w. day -1 (scenario 1) and 0.13-2.08 pg WHO-TEQ kg -1 b.w. day -1 (scenario 2) in the case of PCDD/Fs exposure when these estimations are calculated with the medium bound values of the infant formulae analyzed. In this case, As it can be seen in Figures 2 and3, mean exposures to PCDD/Fs and indicator PCBs are higher in the first scenario than in the second one. This is due to the fact that milk consumption quantities used in the first scenario are higher than those described in the DONALD study [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: results from the DONALD study[END_REF], which is the reference used in the second situation. However, infant body weight data are very similar in the references used in both scenarios. Besides, in order to use the milk consumption quantities referred in the Euro-Growth study it was necessary to convert the values given in ml into grams of powdered infant formulae. Because of this transformation, the second scenario, where milk consumption quantities are given in grams of powdered infant formulae, is thought to provide a better estimation of these contaminant intakes. However, the highest and lowest PCDD/Fs and indicator PCBs intakes are found in the second scenario in which remarkable differences were observed between the different percentiles considered. In this case, P 95 and P 5 represent those children with a respectively higher and lower food intake than the average population but with the same body weight data. As important variations in food consumption quantities are observed between these two percentiles, so the estimated contaminant intakes also do. Though, it would be reasonable to think that the higher the food intake is, the more the body weight data increase. Since this circumstance has not been taken into account, it is possible that an underestimation (P 5) and an overestimation (P 95) of the estimated intake values have been produced when considering those extreme cases. Conversely, in the first scenario differences between the different percentiles considered are just due to the variations in the body weight which are not as significant as those mentioned for the second scenario. In all the cases girl's exposure to these compounds is higher than those observed in boys of the same age. This is only due to the fact that for the same food intake in both scenarios girls have a lower body weight than boys of the same age have. In addition, important differences are observed in these estimations when calculations are made with the lower or the medium values of the samples analyzed. These variations are higher in PCDD/Fs exposure than in those calculated for indicator PCBs. This is due to the higher L.O.D of the MS/MS methodology in relation to the HRMS methodology and the elevated percentage of non-detected congeners in the samples analyzed.

Although levels of these compounds are higher in follow-on than in infant formulae, a decrease of the estimated daily intake in the second semester of the year was observed in all cases. This is due to the lower food consumption of this kind of products in relation to the body weight as children get older. In most cases, the highest PCDD/Fs exposure for the mean population occurs in 3 month old babies while the highest indicator PCBs intake was observed in 6 month old babies. As far as we know, the introduction of complementary feeding, especially meat and dairy products, which occurs from 4-6 months, usually contributes in a higher proportion of the total dietary intake of these pollutants in children at these ages [START_REF] Sasamoto | Estimation of daily intake of PCDDs, PCDFs and Co-PCBs from baby foods. Absract[END_REF][START_REF] Weijs | Dioxin and dioxin-like PCB exposure of non-breastfed Dutch infants[END_REF]. This is why the decline with age observed in these intake estimations would not occur in a total diet study.

When children are fed with lactose-free formulae, an increase in PCDD/Fs and indicator PCBs exposure was observed particularly for 3 month old infants (Figure 4).

In this case PCDD/Fs intakes for the average 3 month old population and with the lower bound values of the samples analyzed reach values up to 0.53 pg WHO-TEQ kg -1 b.w. day -1 and 0.58 pg WHO-TEQ kg -1 b.w. day -1 for boys and girls respectively in the first scenario and 0.49 pg WHO-TEQ kg -1 b.w. day -1 and 0.54 pg WHO-TEQ kg -1 b.w. day -1 in the second. Indicator PCB exposure in children fed with lactose-free formulae also increase in 3 month old infants while an important decrease is observed in 6, 9 and 12 month old babies. This decrease is just due to the decline in the consumption of this product with age.

"[Insert Figure 4 about here]" Similarly to our results, previous reports which have studied the PCDD/Fs exposure of non breast-fed children show regular intake levels in the range of the TDI of 1-4 pg WHO-TEQ kg -1 b.w. day -1 (WHO, 1998) and near to the TDI of 2 pg WHO-TEQ kg -1 b.w. day -1 (SCF, 2001) as it can bee seen in the estimations calculated by [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF] of 2.1 pg WHO-TEQ kg -1 b.w. day -1 in one year old children. Lower exposure levels have been also seen in the UK with values varying between < 0.01 and 0.3 pg WHO-TEQ kg -1 b.w. day -1 in 6 month old babies and between < 0.01 and 0.2 pg WHO-TEQ kg -1 b.w. day -1 in 9 and 12 month old English children (FSA UK, 2004).

Nonetheless, PCDD/Fs intakes by breast-fed infants, which usually have a higher exposure to these pollutants than non breast-fed children, habitually exceed the tolerable daily intake values. This can be seen in the results reported in previous works with exposure levels of 13 pg WHO-TEQ kg -1 b.w. day -1 [START_REF] Hsu | A comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk feeding[END_REF], 51 pg WHO-TEQ kg -1 b.w. day -1 [START_REF] Reis | Infant exposure to PCDD/Fs in Portugal: first results from an environmental health survey program near Lisbon[END_REF] or 76 pg WHO-TEQ kg -1 b.w. day -1 [START_REF] Focant | Levels and profiles of PCDDs, PCDFs and cPCBs in Belgian breast milk. Estimation of infant intake[END_REF].

Regarding indicator PCBs intake, estimated exposure levels for breast-fed infants are also usually high. Considering 5 kg babies which consume 800 ml of milk per day the intake of PCBs has been estimated in the European Union in 1249 ng kg -1 b.w. day -1 (EFSA, 2005); in Poland they have calculated a lower intake with values of 364 ng kg -1 b.w. day -1 for the first breast-fed infants and 375 ng kg -1 b.w. day -1 for the second [START_REF] Szyrwińska | Exposure to specific polychlorinated biphenyls and some chlorinated pesticides via breast milk in Poland[END_REF]. These values are both higher than those [START_REF] Baars | Re-evaluation of human toxicological maximum permissible risk levels[END_REF]. We should consider that since this Maximum Permissible Risk level (MPR) was estimated on the basis of an experiment with a commercial PCB mixture it has to be appreciated as provisional and limited in its applicability [START_REF] Bakker | Indicator PCBs in foodstuffs: occurrence and dietary intake in The Netherlands at the end of the 20 th century[END_REF]. As far as we know, there are no studies estimating indicator PCBs exposure for children at this age fed with commercial infant formulae but, in any case, these exposures are expected to be similar to our results and lower than those observed in breast fed children.

The fact that the highest PCDD/Fs and PCBs human exposure occurs in infants via breast-feeding has been indicated by several authors [START_REF] Guan | Congener profiles of PCDDs, PCDFs, and dioxin-like PCBs in the breast milk samples in Tokyo, Japan[END_REF][START_REF] Szyrwińska | Exposure to specific polychlorinated biphenyls and some chlorinated pesticides via breast milk in Poland[END_REF] who think this fact is due to the elevated concentrations of these compounds in breast milk together with the higher food consumption in relation to body weight. As a consequence and due to the dioxins and PCBs bioaccumulation capability the accumulated doses of these contaminants are also higher in breast fed children. [START_REF] Patandin | Dietary exposure to polychlorinated biphenyls and dioxins from infancy until adulthood: a comparison between breast-feeding, toddler, and long-term exposure[END_REF] found that breast feeding during a period of six months increases the PCDD/Fs and PCBs TEQ body burden by 12% in boys and 14% in girls until the age of 25 years. However, evidence for the health advantages of breastfeeding and recommendations for practice have continued to increase over the past decades. Breast feeding provides all the energy and nutrients that the infant needs for the first months of life, promotes sensory and cognitive development, protects the infant against infectious and chronic diseases and reduces infant mortality due to common childhood illnesses [START_REF] León-Cava | Quantifying the benefits of breastfeeding: a summary of the evidence[END_REF][START_REF] Horta | Evidence on the long-term effects of breastfeeding: systematic reviews and meta-analyses[END_REF]. Consequently, despite the presence of PCDD/Fs and PCBs in breast milk and the risk those contaminants pose to the nursing infant WHO supports and encourages breast-feeding due to their demonstrated benefits. low and similar to those found in the analysis of this kind of food in other countries, but yet they are lower than the levels found in breast-milk samples. Moreover, PCDD/Fs content is in all cases below the maximum limits established for the EU for the presence of these contaminants in dairy products. Estimated mean daily intake of PCDD/Fs and indicator PCBs as calculated in this work shows that exposure to these contaminants through the consumption of infant formulae is also low and below the tolerable daily intake values for the exposure to these contaminants. Although consumption of infant formulae is not supposed to be a risk due to the low levels of these contaminants found in this study, Health Departments, WHO and FSA encourage breastfeeding for the overall health and development of the infant on the basis of the convincing evidence. However the introduction of complementary feeding can increase this exposure to significant values. Furthermore, the observed differences between the diverse scenarios and percentiles studied show the importance of data selection when estimating exposure to chemicals. In fact, it is essential to know the uncertainties concerning intake estimation. In order to improve this situation it is necessary to publish guides collecting data about the food consumption of the infant population similar to those published by the Spanish government and the European Union regarding the consumption of food items of the average population.

  children by using body weight and food consumption data reported by the

  has been performed. Estimated intakes have been calculated by using different scenarios of body consumption data. The present study focus on the complexity of finding those suitable referential values as well as on the different estimated intake

  ejected from the trap according to their mass/charge (m/z) ratio and further detected by an electron multiplier.

  Dioxins in Food, 2003/2006, Division of Environmental Medicine, Norwegian Institute of Public Health, (Folkehelse, Norway).

  transformation of these values given in ml of milk consumed to grams of powdered infant formulae has been made taking into account the recommendations

ortho

  PCBs by the FSAUK (2004) in the analysis of this kind of food and it is explained by the different nutrients content.

  estimated intake of PCDD/Fs and indicator PCBs in the two scenarios considered and calculated with the contamination of the samples previously described are depicted in Figure 2 (PCDD/Fs) and Figure 3 (PCBs). In these figures the estimated mean intake of these pollutants has been represented together with the 5 and 95 percentiles. "[Insert Figure 2 about here]" "[Insert Figure 3 about here]"

  exposure reaches values of 0.97-2.43 ng kg -1 b.w. day -1 in the first scenario and from 0.21 to 2.82 ng kg -1 b.w. day -1 in the second one.

  work for non-breast fed children and higher than the TDI of 10 ng kg -1 b.w. day -1 , value proposed by the Dutch National Institute of Health and Environment (RIVM), for the sum of the seven indicator PCBs

  /Fs and indicator PCBs in the samples of infant formulae analyzed are

FFigure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Estimated intake of PCDD/Fs (pg WHO-TEQ/kg -1 b.w. day -1 ) in children up to one year of age through the consumption of infant formulae calculated with the lower (A) and medium (B) bound values of the samples analyzed. Whiskers above and below the mean value indicate the 5th and 95th percentiles.

Table I .

 I References of milk consumption quantities and body weight data used in the Scenario 1 obtained from the Euro-Growth Study[START_REF] Freeman | Patterns of Milk and Food Intake in Infants from Birth to Age 36 Months: The Euro-Growth Study[END_REF] Haschke et al.

	2000b)
	Age (months)	Infant formulae intake (ml)	P 5 Boys	Body weight (kg) Mean Girls Boys Girls	P 95 Boys Girls
	3		841	5.143	4.817 6.216 5.708 7.340 6.752
	6		626	6.701	6.310 8.051 7.491 9.477 8.911
	9		500	7.715	7.194 9.284 8.677 11.003 10.444
	12	485 o F r	8.586	8.023 10.268 9.648 12.196 11.616
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Table II .

 II References of milk consumption quantities and body weight data used in the scenario 2 obtained from the DONALD Study[START_REF] Kersting | Measured consumption of commercial infant food products in German infants: results from the DONALD study[END_REF]

	Age (months)	Infant formulae intake (g of dry wet) P 5 Mean P 95	Body weight (kg) Boys Girls
	3	38.4	105.3	143.9	6.4	5.8
	6	22.1	67.1	124.6	8.1	7.5
	9	17.6	46.8	90.2	9.3	8.6
	12	12.9	44.9	90.4	10.3	9.4
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Table III .

 III PCDD/Fs (pg WHO-TEQ g -1 fat) concentration in the infant formulae analyzed in this work and in samples analyzed in other countries.

	Country	No. of samples	PCDD/Fs	Year of collection	Reference
	Brazil	4	0.22-0.45*	1998	Päpke and Tritscher, 2000.
	UK	96	0.2-0.4**	2003	FSA UK, 2004
	Slovakia	4	0.64	2001	Chovancová et al. 2005
	Taiwan Spain F 5 (Initial formula) 10 5 (Follow-on formula) 4 (Lactose-free formula) o r	0.71 0.30 0.46 0.43	2000-2001 2004-2005	Hsu et al. 2007. This study
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Table IV .

 IV Indicator PCBs in samples of infant formulae analyzed in this study. Figure 1. Contamination pattern of the three kind of infant formulae analyzed. White bar represents follow-on formula. Grey bar represents lactose-free formula. Black bar represents initial formula.

		Initial (n=5)	Follow-on (n=5)	Lactose-free (n=4)
		(ng g -1 fat)	(ng g -1 fat)	(ng g -1 fat)
	PCB	Lower	Medium	Lower	Medium	Lower	Medium
		bound	bound	bound	bound	bound	bound
	28	0.17	0.17	0.53	0.53	0.35	0.35
	52	0.05	0.05	0.09	0.09	0.06	0.06
	101	0.01	0.02	0.02	0.02	0.01	0.01
	118	0.01	0.02	0.03	0.04	n.d.	0.01
	153 138 180 TOTAL Σ PCBs ngg -1 fat F o n.d. n.d. n.d. 0.23 r	0.01 0.01 0.01 0.29	n.d. 0.01 0.01 0.70	0.01 0.02 0.02 0.73	n.d. n.d. 0.01 0.42	0.01 0.01 0.01 0.46
	Σ PCBs ng g -1 product n.d.= non detected 2 , 3 ,7 ,8 -T C D 0.06 P e 0.08 e r D 2 ,3 ,7 , 8 -T C D F 1 ,2 ,3 ,7 ,8 -P e C D D 1 ,2 ,3 ,7 ,8 -P e C D F 2 ,3 ,4 ,7 ,8 -P e C D F 1 ,2 ,3 , 4 ,7 ,8 -H x C D D 1 , 2 ,3 ,6 ,7 ,8 -H x C D D 1 ,2 ,3 ,7 , 8 ,9 -H x C D D 0.16 1 ,2 ,3 ,4 ,7 ,8 -H x C D F 1 , 2 ,3 ,6 ,7 ,8 -H x C D F 1 ,2 ,3 ,7 ,8 , 9 -H x C D F 2 ,3 ,4 ,6 ,7 ,8 -H x C 0.17 D F 1 ,2 , 3 ,4 ,6 ,7 ,8 -H p C D D 1 ,2 ,3 ,4 ,6 , 7 ,8 -H p C D	0.12	0.13
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

This work has been supported by the Spanish Ministry of Science and Technology (Project AGL 2001-2533) and Gobierno de Aragón (Group A01/2003(Group A01/ -2008)). Author S.