Luc Pussemier

Michel Hoenig

Ludwig De Temmerman

Vincent Hanot

Ilse Van Overmeire

Leo Goeyens

Chemical contamination in eggs of free-range hens in Belgium

Keywords: free-range hens, eggs, pesticides, heavy metals, CALUX, dioxins, POPs, PCBs, environmental contamination, food safety, home-produced food

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Introduction

Currently consumers increasingly prefer "free-range" eggs over battery eggs due to the general perception that these products are attributed a healthier nature and better nutritional qualities. Therefore, private owners (PO) often keep and raise hens in a freerange system. Free-range is a term that brings to mind idyllic barnyard scenes, whereby hens can run free and have access to pasture for foraging. Collecting the flock's eggs is considered an enjoyable and profitable business. However, since the hens live in close contact with the outside environment they are exposed to contaminants. Moreover, contaminants' levels in the eggs from free-range hens are considered good indicators of the contamination of the environment the hens live in [START_REF] Chang | Foraging animals as biomonitors for dioxin contamination[END_REF]. Eggs are fat rich food items and, hence, humans consuming free-range eggs are eventually exposed to the lipophilic contaminants that accumulated in these eggs. It is generally accepted 3 that fatty food intake constitutes a predominant exposure route for polychlorinated dibenzo-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs) and other persistent organic pollutants (POPs). On the other hand, the general population is also exposed to heavy metals through both food intake as well as inhalation.

According to the literature background total TEQ levels for dioxins and dioxin-like PCBs in commercially available eggs sampled in several European countries range from 0,69 to 2.76 pg TEQ per g egg fat [START_REF] Hamm | Levels of polychlorinated dibenzo(p)dioxins, dibenzofurans and dioxin-like PCBs in milk, milk products and eggs from West European countries[END_REF][START_REF] Eljarrat | Toxic potency of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls in food samples from Catalonia (Spain)[END_REF][START_REF] Gallani | Levels of dioxins and dioxin-like PCBs in food and feed in Europe[END_REF][START_REF] Baars | Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuffs: occurrence and dietary in take in The Netherlands[END_REF][START_REF] Focant | Levels and congener distributions of PCDDs, PCDFs and non-ortho PCBs in Belgian foodstuffs Assessment of dietary intake[END_REF]. The data do not accurately distinguish between free-range and battery farm eggs, however, and often only very few samples were analysed. For free-range hens that forage on soil higher levels up to 88 pg PCDD/F WHO TEQ per g fat were observed at different occasions. These elevated values have been linked to environmental parameters, e.g. incinerator ash contaminated soils, wood shavings in chicken litter, contaminated environmental feed ingredients such as ball clay and kaolin and the proximity of chemical plants. [START_REF] Pless-Mulloli | Transfer of PCDD/F and heavy metals from incinerator ash on footpaths in allotments into soil and eggs[END_REF][START_REF] Diletti | A case of eggs contamination by PCDD/Fs in Italy: analytical levels and contamination source identification[END_REF][START_REF] Hayward | Elevated TCDD in chicken eggs and farm-raised catfish fed a diet with ball clay from a southern United States mine[END_REF][START_REF] Schmid | Levels of polychlorinated dibenzo-p-dioxins and dibenzofurans in food of animal origin. The Swiss dioxin monitoring program[END_REF][START_REF] Harnly | Polychlorinated dibenzo-pdioxin and polychlorinated dibenzofuan contamination in soil and home-produced chicken eggs near pentachlorophenol sources[END_REF][START_REF] Schuler | The transfer of polychlorinated dibenzo-pdioxins and dibenzofurans from soil into eggs of foraging chicken[END_REF]. Alarmingly high values of ∼120 pg PCDD/F TEQ per g fat were found in eggs sampled nearby an old municipal solid waste incinerator in France [START_REF] Pirard | Environmental and human impact of an old-timer incinerator in terms of dioxin and PCB level: a case study[END_REF].

Home produced foodstuffs are generally not submitted to any compliance control although they are consumed in large quantities by their producers. Therefore, it was decided to investigate the contamination of PO free-range eggs. Additionally, the present study was triggered by an earlier concern of the Belgian Federal Agency for the Safety of the Food Chain (FASFC). In 2002 the FASFC was informed about elevated dioxin levels in eggs from free-range hens kept by PO living north of Antwerp (Belgium) [START_REF] Pussemier | Enhanced levels of dioxins in eggs from free range hens: a fast evaluation approach[END_REF]. Observed levels were considerably higher than those found in free-range eggs from commercial egg production farms. In its advice (2002/35 www.favv.be) the FASFC committee emphasized the risks associated with regular consumption of such eggs as well as the need for more elaborate studies to estimate the impact of the problem and its possible health effects.

The design of this study focuses on contaminations by POPs, including dioxin-like chemicals, PCBs, OCPs, as well as heavy metals, in eggs from free-range hens, sampled in all regions of Belgium. The objectives of this study are (1) a better insight in the occurrence patterns, (2) an evaluation of the measured levels and/or excesses, and (3) a prudent risk assessment.

Materials and methods

Sample collection

Egg samples (free-range) from different regions in Belgium were acquired during the months April-July 2004 (spring 2004), both from free-range hens held by private owners and from commercial egg production farms. Egg samples were obtained from 22 private owners residing in different parts of Belgium. Additionally, samples from 19 commercial egg production farms were collected with the kind help of the Belgian Federal Agency for the Safety of the Food Chain. Both organic (n = 8) and traditional farms (n = 11) were included. Each sample was made up by collecting 12 eggs from one breeding. Eggs were kept at 4 °C until analysis. The investigated analytes comprehend seven marker PCBs (IUPAC numbers 28,52,101,118,138,153,180), polychlorinated dioxins and furans, dioxin-like PCBs and the OCPs hexachlorobenzene (HCB), α-, βand γhecachlorocyclohexane (HCH) isomers, heptachlor, heptachlor epoxide, aldrin, dieldrin, αand βendosulfan, p,p'-DDD, p,p'-DDE, o,p'-DDT, p,p'-DDT, methoxychlor and endrin. The elements analysed are Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Sb, Tl, Pb and Hg.

Analytical procedures

Elements. The elements were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Two of the 12 eggs obtained by each breeder were used for duplicate analyses of the elements (i.e. two separate analyses on two different eggs from the sample).

The external surface of eggs was washed and dried before the separation of yolk and white that were subsequently analyzed. Subsamples were weighed (0.2-0.5 g) and mineralized by acid digestion (4 mL nitric acid + 4 mL demineralised water) in pressure vessels of a X-PRESS (CEM Corporation, NC, U.S.A.) microwave digestion system.

The obtained solutions were diluted (to a factor 400 which allows avoiding matrix effects) directly in tubes for ICP-MS autosampler. The ICP-MS system used was a VG PQ-ExCell (TJA, U.S.A.).A calibration curve (eight aqueous standards between 0.1 and 10 µg/L, corresponding to 40-4000 µg/kg in the egg) was established with freshly prepared solutions of the same acidity as samples. Under standard analytical conditions, Generally around 25% of fat was obtained from the egg yolks. A part of the obtained fat was used for the analyses of the seven marker PCBs and the organochlorinated pesticides and another part was used for the determination of dioxins and dioxin-like PCBs.

Analysis of marker PCBs. Marker PCBs were determined by gas chromatography with electron capture detection (GC-ECD) and GC/MS/MS (for confirmation). The method is based on the prEN1528-1-1996E method [START_REF] En | Fatty food-determination of pesticides and polychlorinated biphenyls (PCBs). General Part, European Committee for Standardization[END_REF] and the recommendations of the Belgian Bureau of Accreditation Beltest [START_REF] Beltest | Determination of polychlorobiphenyls in feed, fats of animal origin, eggs, egg products, diary and other food stuffs, I014[END_REF]. 0.4 g of fat was dissolved in 2 7 mL of hexane and 50 µl of a 1 mg/mL PCB 143 solution was added to control the purification step. The resulting solution was loaded on an acidified silicagel column composed of 6 g of acidified silica gel (60/40 silica/H 2 SO 4 ), 1 g of Al 2 O 3 containing 8 % of water and on the top, 0.5 g of anhydrous Na 2 SO 4 . The tube containing the fat solution was rinsed twice with 2.5 mL of hexane, and the column was further eluted with 20 mL of hexane. After concentration under a gentle dried-air stream to ca 1 mL, 50 µl of a 1 mg/mL mirex solution was added as internal standard and 3 µl was injected into the gas chromatography (GC) system. Analysis was performed on Trace 2000 (Thermo-Finnigan, Interscience, Belgium) equipped with ECD at 300 °C, an auto sampler AS2000, a HT-8 capillary column (25 m, 0.22 mm x 0.25 µm) (SGE, Achrom, Belgium). Argon/methane was used as make-up gas and Helium was used as carrier gas (1 mL/min). Injection was done at 220 °C in a split/splitless mode. The temperature program was set at 100 °C for 0.5 min, then increased by 25 °C/min to 270 °C and held for 8 min. PCBs results above the MRL were confirmed by GC-MS/MS, an ion Trap Polaris-Q from Thermo-Finnigan (Interscience, Belgium) equipped with a HT-8 capillary column (25 m, 0.22 mm x 0.25 µm). The flow of helium was 1 mL/min and the temperature programme was set at 90 °C for 0.5 min and then increased by 25 °C/min to 220 °C, the temperature was then increased by 5 °C/min to 265 °C and held for 6 min. As a quality control, a blank solvent, a blank procedure and a control sample were run within each series. The limits of quantification are 10 ng/g of fat.

Analysis of pesticides. OCPs were analysed by GC-ECD and GC-MS/MS (for confirmation). The method is also based on the prEN1528-1-1996E method [START_REF] En | Fatty food-determination of pesticides and polychlorinated biphenyls (PCBs). General Part, European Committee for Standardization[END_REF]. 0.2 g of fat was dissolved in 2 mL of hexane and 50 µl of a 1 mg/mL containing 8 % of water and on the top, 0.5 g of anhydrous Na 2 SO 4 . The tube containing the fat solution was rinsed twice with 1 mL of hexane, and the column was further eluted with 80 mL of hexane. After concentration under a gentle dried-air stream to ca 1 mL, 50 µl of a 1 mg/mL mirex solution was added as internal standard and 1 µl was injected into the gas chromatography (GC) system. Analysis was performed on the same system as for PCBs but the Helium flow is 1.4 mL/min and the temperature of the column was set at 80 °C for 2 min, then increased by 4 °C/min to 180 °C and finally by 6 °C/min to 270 °C and held for 10 min. The identity and concentration of all pesticides exceeding the MRL (Maximum Residue Level) (Royal Order, 2000a) were confirmed on a DB1701 capillary column (60 m x 0.32 mm x 0.25 µm) (J&W, Alltech, Belgium).

The Helium flow was set at 3 mL/min and the program temperature was: 100 °C for 2 min, then increased by 20 °C/min to 180 °C, then increased by 1.8 °C/min to 245 °C and finally by 20 °C/min to 265 °C and held for 5 min. The identity and concentration for unusual ratio of DDT isomers were confirmed by GC-MS, an ion Trap Polaris-Q from Thermo-Finnigan (Interscience, Belgium) equipped with a HT-8 capillary column (25 m, 0.22 mm x 0.25 µm). The flow of Helium was 1 mL/min and the temperature program was set at 80 °C for 2 min and then increased by 12 °C/min to 270 °C and held for 15 min. As a quality control, a blanco solvent, a blanco procedure and a control sample were run within each series. The limits of quantification are 10 ng/g of fat.

CALUX assay. Dioxins and dioxin-like compounds were determined by the chemically activated luciferase gene expression (CALUX) bioassay provided by Xenobiotic compounds that are able to activate the aryl hydrocarbon receptor (AhR) and AhRdependent gene expression. The bioassay result can be considered as a TCDD toxicity or TEQ value since the calibration in this assay is performed with 2,3,7,8-TCDD. 1 g of fat (ASE extracted) was purified on an acidified silica gel column and dioxin-like compounds were isolated by passing the eluate over an activated carbon column (X-Carb from XDS). A PCB fraction containing the dioxin-like PCBs and a dioxin fraction containing the polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans were separately obtained by elution of the carbon column with a mixture of hexane/ethyl acetate/toluene (8:1:1) and with toluene, respectively. Both fractions were analysed with the CALUX assay yielding a dioxin TEQ result and a PCB TEQ result. Details of the in vitro assay were previously described [START_REF] Van Overmeire | Interpretation of CALUX results in view of the EU maximal TEQ level in milk[END_REF]).

Briefly, mouse hepatoma H1L6.1.cells, grown in 96 well plates, were exposed to the purified extracts for 20 h and the produced luciferase was quantified by measuring the luminescence with a Berthold Orion luminometer after adding of luciferin. A standard curve of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (range: 0.098-50 pg /well) is included on each plate. The CALUX method has been validated for the screening of egg samples. The precision meets the requirements of the EU [START_REF] Eu | Council Directive 2001/102/EC of 27 November 2001 amending Directive 1999/29/EC on the undesirable substances and products in animal nutrition[END_REF]. The mean coefficients of variation for repeatability and for intralaboratory reproducibility were 10 % and 21 %, respectively. With each series of purification a quality control sample at 3 pg TEQ/g fat and a procedural blank were included. Quality control of the plates with cells and extracts was performed as described (Windal et al. 2005a). The method has also been evaluated by comparison with GC-HRMS results during the EU (Carbonnelle et al., 2004a).

Statistical analysis

For the statistical calculations, the concentration was considered as zero when specific compounds gave a result below the LOQ. Pearson correlation coefficients were calculated with STATISTICA version 6.0 (1998).

Results

Heavy metals

Levels of 13 elements (mostly belonging to the group of so-called "heavy metals") were determined in eggs obtained from private owners (PO) and in eggs from commercial farms (CF). Descriptive statistics (including medians and range) of the results are shown in Table I (PO) and in Table II (CF). Figure 1 shows the ratios (PO:CF) of median concentrations determined in the eggs. This ratio is close to one for most substances except for Mo (ratio = 0.54) and for some toxic elements (Pb, Tl, Hg and Co) for which the ratio ranges from 2 to 6.

Insert Table I andTable II andFigure 1 Organochlorinated compounds Several pesticides, PCBs as well as dioxin-like compounds have been determined in PO eggs and in CF eggs. Descriptive statistics for the compounds for which the mean levels were higher than the quantification limit are summarised in Table III (Royal Order, 2000a and2000b). In addition, dioxin and PCB TEQ values are given.

Insert Table III andTable IV Marker PCBs. The median of the sum of the seven marker PCBs (PCB 28,52,101,118,138,153,180) is 32.30 ng /g fat for the PO eggs with levels ranging from 0 to 351 ng/g fat. The median PCB level in eggs from commercial farms is zero and levels range rom 0 to 63.20 ng/g fat. In 8 of the 22 PO egg samples none of the marker PCBs was detected while in 95% of the CF eggs no marker PCB congeners were detected. The congener profile for the eggs from hens of PO is shown in Figure 2. The most abundant PCB congeners are PCBs 153, 138 and 118. For all samples the mean contribution of these three congeners to the sum of the seven marker PCBs is 82 %, of which PCB 153 accounts for 49 %.

Insert Figure 2 OCPs. The values for the sum of aldrin and dieldrin are in general low but elevated levels were found in PO eggs as compared to CF eggs. The median values for PO and for CF eggs are 14,35 and 0 ng/g fat, respectively. The maximal values amount to 115.40 and 18.10 ng/g fat, respectively. The levels for the sum of DDT, DDE and DDD in PO eggs range from 26.40 to 5250 ng/g fat with a median value of 125.45 ng/g fat.

Six samples of PO eggs exceed the norm level of 500 ng/g fat. These elevated values result in a mean level for all private owners of 770,71 ng/g fat (above the norm level). In CF eggs lower levels were found ranging from 0 to 171.90 ng/g fat (median: 10.70 ng/g fat). They were calculated for PO eggs, for the six PO egg samples that exceed the norm value and for the CF eggs. In the farms' eggs only p,p'-DDE and p, p'-DDD contribute to the sum with median values of 100 and 91 % respectively, while in the eggs obtained from private owners there is, in addition to p,p'-DDE and p,p'-DDD also a considerable contribution of p,p'-DDT to the sum of DDT, DDE and DDD. The contribution of p,p'-DDE to the sum DDT/DDE/DDD ranges from 13 to 97% (median: 52 %) and the contribution of p,p'-DDT to the sum DDT/DDE/D ranges from 2 to 56 % with a median of 32 %. In addition, the profile for all egg samples analysed is shown in Figure 3. p,p'-DDE was found in all PO egg samples, p,p'-DDT in 91 % of the samples, p,p'-DDD in 77 % of the samples and o,p'-DDT in 27 % of the samples (Figure 3A). In CF egg samples p,p'DDD and p,p'DDE were detected in 63 % and 47 %, respectively while p,p'-DDT and o,p'-DDT were not detected in CF eggs (Figure 3B).

Insert Figure 3 Dioxin-like chemicals. Evaluation of the dioxin like activity revealed higher TEQ levels in PO eggs than in CF eggs. The dioxin TEQ values range from 2.28 to 23 pg /g fat for PO eggs (median 8.53 pg TEQ/g fat) and from 0.5 to 4.21 pg/g fat for CF eggs (median 1.04 pg TEQ/g fat).

The dioxin like activity for the PCB fraction obtained after purification of the egg fat is generally low for both types of eggs but here is a difference visible between the levels in the different types of eggs. The PCB TEQ levels in PO eggs are higher than the levels in

Discussion

Contamination levels

Heavy Metals. For many elements such as Cu, Zn, Mn, Ni, As, Se, Cd and Sb the median values are similar whatever the origin of the eggs (OP or CF) (see Table 1 and Figure 1). Overall, it can be said that the levels determined in this study are in agreement with those documented elsewhere (FSA, 2004[START_REF] Leblanc | The 1st French Total Diet Study. Mycotoxins, minerals and trace elements[END_REF]. For Mo, it appears that eggs from PO have a lower median concentration (ratio PO: CF = 0.54).

This can be explained by the fact that the feed used by PO cannot provide the same intake level as can be obtained with commercial feed. The latter is supplemented with mineral elements and, more specifically, with Mo. For Pb, Tl, Hg and Co, the ratios of median concentrations range from 2 to 6, indicating much higher levels of these contaminants in PO eggs. For these elements, the agreement with published data (FSA, 2004[START_REF] Leblanc | The 1st French Total Diet Study. Mycotoxins, minerals and trace elements[END_REF] is not always satisfactory but it must be added that the mode of egg production (battery or free-range) and the method of determination of the very low concentrations of elements are not fully documented in the above mentioned studies. Our results can also be compared with the data obtained on eggs taken from markets in Huelva, a Spanish city with strong industrial activity. In our study, the levels determined in the eggs from the commercial farms are lower for Cd and Hg but higher for As and Pb than the levels determined in Spain [START_REF] Bordajandi | Survey of persistent organochlorine contaminants (PCBs, PCDD/Fs, and PAHs), heavy metals (Cu, Cd, Zn, Pb, and Hg), and arsenic in food samples from Huelva (Spain): levels and health implications[END_REF]. On the other hand, the levels that we found for Cu and Zn are comparable with the levels published 14 by these authors. Moreover, the Cd, Hg and Pb levels that we obtained for both egg types were clearly higher than the levels found in free-range eggs in Ireland (FSAI, 2004). This suggests a higher background soil contamination with these toxic elements in Belgium as compared to Ireland.

Marker PCBs. For the eggs from the egg production farms no significant contamination with indicator PCBs was observed. On the other hand, the norm level of 200 ng/g fat for the sum of the seven indicator PCBs (Royal Order, 2000b) was exceeded in two PO egg samples. In PO eggs the most abundant PCB congeners were PCBs 118, 138, 153, and 180. The low levels of PCBs 52 and 101 are in accordance with literature data demonstrating metabolism of these two congeners in hens (De [START_REF] Vos | Digestibility, retention and incorporation of low-level dietary PCB contents in laying hens[END_REF][START_REF] Maervoet | Accumulation and tissue distribution of selected polychlorinated biphenyl congeners in chickens[END_REF]. The low levels of PCBs 52 and 101 in the eggs that were analysed in our study are therefore linked to either a low intake of these congeners or to metabolism of the ingested amounts. The mean level of the sum of the seven marker PCBs for the CF eggs is in the same range as the level for indicator PCBs in Irish freerange eggs [START_REF] Tlustos | Investigation into levels of dioxins, furans and PCBs in battery, free range, barn and organic eggs[END_REF] and the level in commercial eggs from Catalonia in Spain [START_REF] Eljarrat | Toxic potency of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls in food samples from Catalonia (Spain)[END_REF]. The levels that we found in PO eggs are approximately twice as high as the levels for total PCBs (13 congeners including the seven indicator PCBs that we analysed) in eggs from markets in Huelva, a Spanish city situated in an industrial area [START_REF] Bordajandi | Survey of persistent organochlorine contaminants (PCBs, PCDD/Fs, and PAHs), heavy metals (Cu, Cd, Zn, Pb, and Hg), and arsenic in food samples from Huelva (Spain): levels and health implications[END_REF].

OCPs. The results for most OCPs are well below the Belgian tolerable levels. Only for the sum of DDT, DDE and DDD some high exceedings, up to 10 times the tolerated level, were oberved for PO eggs. In the present study a mean of 770,71 ng/g fat for the performed a study on a limited number of egg samples in Brazil, where DDT has been used to control leishmaniasis. These authors report average ΣDDT concentrations in chicken eggs of 1983 ng/g fat (n = 3) and 730 ng/g fat (n = 6) with 82 % of p,p'-DDE. It is interesting to note that these eggs were sampled in the vicinity of places where DDT had been used 7 and 9 years before the sampling [START_REF] Vieira | DDT environmental persistence from its use in a vector control program: a case study[END_REF].

DDT, and more particularly, the main compound in the technical pesticide product, was found in all PO eggs. Knowing that the use of DDT in Belgium has been banned more than 25 years ago, the mean DDT/DDE ratio of 0.68 that was found in eggs obtained from private breeders might indicate recent use of DDT (to decontaminate the breeding place). This is illustrated by one case of contaminated PO eggs (4200 ng/g fat) that we studied in more detail. The p,p'-DDT/p,p'-DDE ratio was unusually high in this case (1,42). We investigated a dust sample taken inside the hens' house as well as a soil sample from the land on which the chickens of this breeder were allowed to forage.

Remarkably, in the dust sample, both the ΣDDT (5942 ng/g) and the percentage of p,p'-DDT (85.2%) were higher than in the soil sample (3416 ng/g and 40.9%, respectively), which pointed to the dust inside the hens' house as the primary source of DDT contamination in this breeding. This suggests that DDT was formerly used inside the hens' house (most probably before the ban on DDT, according to the breeder) and because of the limited degradation of DDT under indoor conditions a high proportion of untransformed DDT has been found in this case. This phenomenon seems typical for the Dioxin-like activity. The European Community has promulgated strict maximum levels for food and feed (EU, 2001a andEU, 2001b). They are expressed in TEQ units of 7 PCDDs and 10 PCDFs with an assigned TEF value (Van den [START_REF] Van Den Berg | Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife[END_REF]. It must be noticed that recently new maximum levels for the sum of dioxins and dioxin-like PCBs have been set [START_REF] Eu | Commission Regulation (EC) No 199/2006 of 3 February 2006 amending Regulation (EC) No 466/2001 setting maximum levels for certain contaminants in foodstuffs as regards dioxins and dioxin-like PCBs[END_REF]. For a transitional period the existing levels for dioxins will continue to apply in addition to the newly set levels for the sum of dioxins and dioxin-like PCBs. Typically, concentrations are determined by capillary gas chromatography (GC) in combination with high-resolution mass spectroscopy (HRMS), whereas the data presented in this study are based on the CALUX technology [START_REF] Garrison | Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8tetrachlorodibenzo-dioxin-like Chemicals[END_REF], Van Overmeire et al., 2001, Baeyens et al., 2004 and references herein).

While CALUX assay derived TEQs and GC-HRMS TEQs have been observed to be relatively close for numerous sample matrices [START_REF] Denison | Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals[END_REF], they can differ too and a variety of factors contribute hereto (Windal et al., 2005). Conceptually, CALUX analyzes the overall biological activity or induction of gene expression caused by all AhR ligands (agonists and antagonists) present in the sample extract, while a chemical analysis focuses on selected numbers of compounds only. In the present study CALUX dioxin TEQ values are compared to the compliance concentration for dioxins of 3 pg TEQ per g fat for eggs (including free-range) (EU, 2001a[START_REF] Eu | Commission Regulation 684/2004 of 13 April 2004 amending regulation (EC) No 466/2001 as regards dioxins[END_REF]). An estimate of the TEQ values for dioxin-like PCBs, obtained with the CALUX approach, is also presented in this paper. For eggs the maximal level for the sum of dioxins and dioxinlike PCBs is 6 pg WHO TEQ/g fat [START_REF] Eu | Commission Regulation (EC) No 199/2006 of 3 February 2006 amending Regulation (EC) No 466/2001 setting maximum levels for certain contaminants in foodstuffs as regards dioxins and dioxin-like PCBs[END_REF].Over 75 % of the observed dioxin TEQ obtained in this study is 9.66 pg TEQ/g fat for PO eggs and 1.87 pg TEQ/g fat for CF eggs. Even when CALUX results are considered non-quantitative or semi-quantitative [START_REF] Hoogenboom | Determination of dioxins and planar PCBs in fish using the GC/MS and CALUX bioassay; introduction of the screening approach for control purposes[END_REF] the median value for CALUX dioxin TEQ of 8.53 pg TEQ per g fat definitely indicates non-compliance with the norm level. Van Overmeire et al.

(2004) calculated that the decision limit or CC α corresponding to the norm level of 3 pg PCDD/F TEQ per g fat for milk samples amounts to 4.53 [START_REF] Van Overmeire | Interpretation of CALUX results in view of the EU maximal TEQ level in milk[END_REF].

The CC α concept takes into account the measurement uncertainty. Hence, assuming a similar measurement uncertainty for egg fat analyses, all results above the 1 st quartile significantly exceed the EU norm value. This illustrates the extent of the dioxin contamination problem for PO eggs. Additionally, the observed values range from 2 to 23 pg TEQ per g fat; a very broad range that suggests different contamination sources, presumably in combination with varying foraging regimes. In general, these measurement results compare well with previously published data. An average and maximal value of, respectively, ∼10 and ∼13 pg TEQ per g fat were obtained for few GC-HRMS analyses of PO eggs sampled in the vicinity of Antwerp, Belgium [START_REF] Pussemier | Enhanced levels of dioxins in eggs from free range hens: a fast evaluation approach[END_REF]. Several studies carried out in neighboring countries do also confirm our data. Maximum values of ∼23 pg I-TEQ per g fat and ∼8 pg PCDD/F TEQ per g fat were mentioned by two German studies [START_REF] Furst | PCDD/PCDF in commercial chicken eggs-Dependence on the type of housing[END_REF][START_REF] Malisch | Update of PCDD/PCDF-intake from food in Germany[END_REF] and one Dutch study [START_REF] Traag | Residues of dioxins and coplanar PCBs in eggs of free range chickens[END_REF], respectively. The PCB TEQ values measured during the present study are low. However, one must keep in mind that these are CALUX data and that the cells used in this assay are less sensitive to dioxin-like PCBs than to PCDDs and PCDFs [START_REF] Brown | Determination of REP values for the CALUX bioassay and comparison to the WHO TEF values[END_REF]. A comparison of GC-HRMS Carbonnelle et al., (2004b) suggests a threefold underestimation by the CALUX values (Carbonnelle et al., 2004b). This factor was confirmed by validation results (data not shown). Assuming an underestimation by a factor of three, the PCB TEQ values for PO eggs would range from 0.2 to 35 pg TEQ per g fat and the maximal total sum of PCDDs, PCDFs and dioxin-like PCBs would amount to ∼58 pg TEQ per g fat.

All together, the data obtained during this study illustrate unmistakable contamination with dioxins and dioxin-like chemicals in PO eggs. The more that CF eggs revealed much lower concentrations. Their median and maximum dioxin TEQ values are eight and five times lower, whereas their median and maximum PCB TEQ values are three and seven times lower, respectively.

Correlations between contaminants and differences between PO and CF eggs

Environmental contaminants. For PO breeding, the most probable contamination source of the hens and their eggs with heavy metals and persistent organic compounds is the environment (contaminated soil, grit, grass, earthworms, soil-dwelling insects, etc) [START_REF] Stephens | Biotransfer and accumulation of dioxins and furans in soil: chickens as a model for foraging animals[END_REF][START_REF] Schuler | The transfer of polychlorinated dibenzo-pdioxins and dibenzofurans from soil into eggs of foraging chicken[END_REF][START_REF] Kijlstra | The role of organic and free-range poultry production systems on the dioxin levels in eggs[END_REF]. In PO eggs, indeed, significant (p > 0.01) correlations were found between some typical environmental contaminants such as Tl and Hg (r = 0.76), indicator PCBs and dioxins (r = 0.59), As and DDT/DDE/DDD (r = 0.53), indicator PCBs and DDT/DDE/DDD (r = 0.51), Tl and DDT/DDE/DDD (r = 0.50), and between dioxins and dioxin-like PCBs (r = 0.48). When considering eggs from CF, it appears that the correlations between these environmental contaminants are no longer significant (p > 0.01). This suggests that the origin of PO eggs contamination is environmental and that the contamination of the environment is 19 rather widely disseminated for a large number of contaminants. The significant correlation between the levels of various contaminants in PO eggs is in agreement with good correlations found for dioxins and the indicator PCBs in serum and plasma samples in Belgium [START_REF] Fierens | PCB body burden in the vicinity of different sources of environmental pollution in Belgium[END_REF], Van Wouwe et al., 2004). Pauwels et al. also found a correlation between CALUX TEQ values (obtained with rat cell assay) and the sum of four indicator PCBs in serum and in follicular fluid [START_REF] Pauwels | Comparison of Chemical-Activated Luciferase gene expression bioassay and gas chromatography for PCB determination in human serum and follicular fluid[END_REF].

Recent data of concentrations of heavy metals determined in cord blood in Belgium indicated that Cd levels in Flanders were enhanced, compared to levels found in other countries (Centre for environment and health, 2005). Also, a recent study [START_REF] Goemans | Rapport IBW: Instituut voor Bosbouw en Wildbeheer. The Flemish network on contaminants in eel, 1994-2001: polychlorinated biphenyls, organochlorinated pesticides and heavy metals in eel[END_REF] has shown that eels caught in rivers from Flanders were largely contaminated by various chemicals such as PCBs, heavy metals and, to a lesser extent, organochlorinated pesticides such as DDT and metabolites. This supports the idea of a widely distributed environmental contamination of these contaminants in Belgium.

Metal elements. On the other hand, the CF eggs are characterized by very significant (p < 0.001) correlations between some metal elements such as Cu and Zn (r = 0.94), Zn and Co (r = 0.85) and between Cu and Co (0.79). Such very significant correlations between these three metals are not found anymore in PO eggs where the correlation coefficients dropped to 0.58, 0.44 and 0.43, respectively. This suggests, once again, that hens from PO are fed in a less controlled way whilst commercial breeders use exclusively commercial feedstuffs supplemented with specific metal elements in very precise concentration ratios. Differences between PO and CF free-range eggs. Considering all the results together, it appears that the lower contamination observed in CF eggs (although free-range) is probably due (i) to the less extensive contact of these hens with the environment (high number of birds and, hence, less space available per hen) and (ii) to the exclusive use of (supplemented) commercial feed. It is also worth mentioning that within the commercial free-range farms it was not possible to find out any significant distinction between the organic and conventional modes of production considering either the quantitative analysis (contaminant levels determined) or the qualitative analysis (correlations found between groups of contaminants) (results not shown).

Health consequences due to the consumption of home-produced free-range eggs

The consumption of home-produced free-range eggs could lead to unusual effects due to both the elevated contamination of the eggs and their possible unusual consumption rate. To estimate the dietary intake of the examined contaminants via the consumption of eggs, we considered three scenarios: A) "normal consumption" whereby one egg per day is consumed and the contaminants' concentration is the median value for that compound that is obtained in this study, scenario B) a case of "extreme use" whereby three eggs with the obtained median concentration are consumed daily and scenario C) the case of "extreme contamination" whereby one egg daily is consumed and the contaminants' concentration is the maximal value for that compound that is obtained in this study. We compared these intakes to the tolerable weekly intake (TWI) for the specific contaminant when a TWI was available. For the heavy metals we only focused on Pb, Cd and Hg because for these metals the ratio between the median levels in PO eggs to the median levels in CF eggs was higher than one and we used the obtained Insert Figure 4 The estimation of the exposure to dioxins and dioxin-like PCBs for the same three scenarios is shown in Figure 5. The results are shown for the two types of eggs (PO and CF). To be able to compare the results with the TWI the total TEQ (dioxin TEQ+ PCB TEQ) values were used. The results are compared to the tolerable weekly intake of 14 pg WHO TEQ/kg bw for dioxins and dioxin-like PCBs as determined by the Scientific Committee on Food of the EU (EU, 2001c). The contamination with dioxin-like compounds is elevated in such a way that it has pronounced effects on the TEQ intake.

A 'normal egg consumption '(scenario A) leads to 48 % of the TWI whereas the same consumption of eggs from commercial farms does not exceed 10 % of the TWI (Figure 5). For extreme use and extreme contamination of PO eggs (scenarios B and C) the TWI is exceeded. The present results indicate that consumption of home-produced eggs leads to a TEQ intake almost five times as high as the intake by the same consumption of free-range eggs from commercial farms. It must be stressed that regular consumption of such eggs (e.g. a daily consumption of one egg) increases the total TEQ intake with almost 50 % of the tolerated intake. This finding leads to concerns. Dioxin intake occurs predominantly via consumption of fatty food from animal origin, with meat, dairy products and fish being the major contributors [START_REF] Focant | Levels and congener distributions of PCDDs, PCDFs and non-ortho PCBs in Belgian foodstuffs Assessment of dietary intake[END_REF]. This means that, in addition to eggs, there is already considerable dioxin TEQ intake. It is thus important that consumption of such eggs is not exaggerated in order to keep the TEQ intake as low as possible. Moreover, it has already been shown that PCBs and PCDD/F concentrations in serum of people in Flanders are higher than in other countries. [START_REF] Koppen | Persistent organochlorine pollutants in human serum of 50-65 years old women in he Flanders Environmental and Health Study (FLEHS). Part 1: concentrations and regional differences[END_REF]. This suggests that Belgian people are subjected to higher background exposure to dioxin-like compounds as compared to other countries.

Finally, it has already been noted that private owners of free-range hens might consume large quantities of the same food type: their own produced eggs [START_REF] Stephens | Biotransfer and accumulation of dioxins and furans in soil: chickens as a model for foraging animals[END_REF].

If these food items are contaminated these individuals may have different exposures from what is considered 'background' and they should, therefore, be considered as a special sub-population [START_REF] Stephens | Biotransfer and accumulation of dioxins and furans in soil: chickens as a model for foraging animals[END_REF].

Conclusion

The most striking finding of this study is the clear distinction between the results obtained for the eggs from private owners and those for free-range eggs obtained from professional farms. The difference is the most visible for environmental contaminants like dioxins, PCBs, DDT (and metabolites) and toxic heavy metals (Tl, Pb, Hg).

Apparently several types of contaminants are present in the environment and they Consumers of free-range eggs obtained from private owners should be aware of the possible health risks associated with the use of these products especially because eggs add to the total diet and other food items also contribute to a large degree to the total intake of contaminants. Currently, we believe that the rearing conditions and the influence of the environment in which the hens live play a major role to explain the difference between levels of compounds in the two types of eggs that were investigated.

The fact that home-produced eggs are significantly more contaminated with dioxins, DDT, Pb, Tl and Hg than free-range eggs from farms calls for serious attention. It is of great importance that the involved consumers are aware of the health consequences associated with excessive intake of their obtained eggs.

The results obtained in this study were passed to the Ministry of Public Health and the Federal Agency of the Security of the Food Chain. The latter one installed a working group to forward suggestions. This working group is currently investigating the present and additional results. The Belgian Federal Agency for the Safety of the Food Chain (FASFC) is acknowledged for its support in the sampling of commercial farms. We are also grateful for the active participation of the private owners in this study. extreme contamination: intake of 1 egg daily and maximal contamination) (assumed levels are levels obtained for eggs from private owners).

F

Figure 5: Percentage of tolerable weekly intake (TWI) for dioxins and dioxin-like PCBs that is obtained for different intake scenarios of eggs from private owners (PO) and eggs from commercial farms (CF) (normal use: intake of 1 egg daily and median contamination; extreme use: intake of 3 eggs daily and median contamination; extreme contamination: intake of 1 egg daily and maximal contamination).

F

  Mo, 114 Cd, 121 Sb,205 Tl and 208 Pb. Mercury was determined using a separate analytical method based on the atomic absorption of mercury vapour after combustion of the raw sample in a stream of oxygen. The released mercury was first trapped on a gold amalgamator. It was then released by heating and its vapour conducted to the absorption cell. The system used was AMA-254 (Altec, Czech Republic).Fat extraction. The yolks of 10 of the 12 eggs obtained from each breeding were pooled for the analyses of organohalogenated contaminants. The pooled yolks were frozen before analysis.They were lyophilised during 48 h and then extracted with hexane (pestanal, for residue analysis grade) by pressurized liquid extraction (PLE) using a Dionex (USA) ASE extractor. Cells of 33 mL were filled with freeze-dried samples and anhydrous Na 2 SO 4 .The extraction conditions were: 125 °C, 1500 PSI, two static cycles of 5 min. The extracts were dried afterwards under a stream of nitrogen until constant weight.

  added to control the purification step. The resulting solution was loaded on an alumina column composed of 10 g of Al 2 O 3

  .(XDS) (USA). This assay does not provide information about the presence of individual PCDD/F congeners. It gives an overall response for all

  different sample matrices among which eggs

  eggs. The median levels for PO eggs and CF eggs are 1.73 and 0.69 pg TEQ/g fat, respectively. In PO eggs the PCB TEQ values range from 0.06 to 11.84 pg/g fat and in CF eggs they range from 0.31 to 1.69 pg TEQ/g fat. The ratio of the median PCB TEQ value for PO eggs to the median PCB TEQ value for CF eggs is 2.5.

  , DDE and DDD was obtained for the eggs from private owners. To put the data of our study in perspective they can be compared with literature data.Vieira et al.

  chickens as in eggs from commercial farms no traces of p,p'-DDT were detected.

  eggs exceed the current norm concentration for dioxins of 3 pg TEQ per g fat. The median of the sums of CALUX dioxin TEQ and CALUX PCB TEQ values

  PO eggs. In Figure4the fractions of the provisional tolerable weekly intakes (PTWI), as established by the WHO that are reached are shown for the three intake scenarios. The fractions are all below 1 %, except for Pb for which an egg intake according to scenarios B and scenario C results in 3,6 % and 5,9 % of the PTWI, respectively.

  that consumption of PO eggs will increase the body burden of the consumers.

  the eggs via soil-egg transfers. This is to be expected from their long residence time in the environment and their bioaccumulating potential. The threat for human health increases by the presence of a mixture of different toxic compounds at elevated levels, which leads to an increased risk for additive or synergetic effects between compounds.

Figure 2 :

 2 Figure 2: Congener profile of marker PCBs in eggs from private owners.

Figure 3 A

 3 Figure 3 A: Percentage contribution of different DDT forms to sum DDT/DDE/DDD in

Figure 3B :

 3B Figure 3B: Percentage contribution of different DDT forms to sum DDT/DDE/DDD in

Fig 4 :

 4 Fig 4: Percentage of provisional tolerable weekly intake (PTWI) for Pb, Cd and Hg that

  for the eggs from hens of PO and in table IV for CF eggs. Results are shown, for the sum of seven marker PCBs, for the sum of aldrin and dieldrin and for the sum of DDT, DDE and DDD because the Belgian legislation foresees a norm level (maximal allowed level) for these

	sums of PCBs and pesticides
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	11

  Figure 1: Ratio of median concentrations in eggs from private owners (PO) to median concentrations in eggs from commercial farms (CF) for different elements.

	FSA Food Standards Agency. 2004. Total Diet Study 2000 of 12 elements -
	aluminium, arsenic, cadmium, chromium, copper, lead, manganese, mercury, nickel,
	selenium,	tin	and	zinc.	Food	Survey	Information	Sheet	48/04.
		o r F o r F o r F o r F o r F o r F o r F o r F o r F o r F o r
			P P P P P P P P P P P
			e e e e e e e e e e e
				e r e r e r e r e r e r e r e r e r e r e r
					R R R R R R R R R R R
					e e e e e e e e e e e
					v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e
						w w w w w w w w w w w
							O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l
							y y y y y y y y y y y
									28

Table I :

 I Descriptive statistics of concentrations of elements (µg/kg egg) in eggs from private owners (n = 22).

	Page 41 of 46				Food Additives and Contaminants Food Additives and Contaminants	Page 40 of 46
	100%				Pb Cd Hg
	100% 7 7%		120,0
		80% 6% 6		100,0		PO CF
		5 5% 80% 180		80,0
		4 60% 160	ng/g fat	60,0			p,p'-DDT
	% PTWI	3 4% 60% 140		40,0			p,p'-DDD o,p'-DDT p,p'-DDE
	o r P e e r F o r 0,0 20,0 sample F o r F o r F o r F o r F o r mean min quartile1 313.98 87.00 272.29 5.15 1.73 3.59 36.58 13.78 26.54 F o r Mn Element 1 2 20% 40% 40% 1% 2% 3% 100 120 %TWI Mn Co Ni 0 Co Ni Cu Zn As Se P e e r P e e r 0% sample P e e r 0% 20% sample P e e r 0% normal use extreme use P e e r 20 40 60 80 P e e r Cu 603.64 430.17 554.58 Zn 11543.64 7655.00 10702.50 As 15.95 8.94 12.86 P Se 272.97 109.42 205.18 e Mo 26.35 5.93 21.02 Cd 0.53 0.07 0.18 Sb 4.23 0.89 2.32 e r	median 312.51 4.43 31.86 Mo Cd extreme contamination Sb 597.5 11237.5 16.09 295.2 26.06 0.45 3.54	quartile3 349.18 5.75 44.60 Tl Pb pcb 28 pcb 52 pcb 101 pcb 118 pcb 138 pcb 153 pcb 180 646.88 12554.17 17.65 325.42 31.68 0.93 5.61	p,p'-DDD DDE 620.67 max 12.93 85.00 Hg 829.17 15093.33 24.26 477.33 52.45 1.12 9.02
	Tl Pb Hg	0	normal use 68.56 0.69 2.05	0.08 19.06 0.70	R e R e R e R e R e R e extreme use R e 0.30 0.53 33.67 48.83 1.09 1.58 R e	extreme contamination 0.74 2.54 90.54 239.58 2.33 6.19
						v i e v i e v i e v i e v i e v i e v i e v i e
							w w w w w w w w
							O n l O n l O n l O n l O n l O n l O n l O n l
							y y y y y y y y

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Table II :

 II Descriptive statistics of concentrations of elements (µg/kg egg) in eggs from commercial farms (n = 19). Table III: descriptive statistics of levels of organochlorinated compounds in eggs from private owners (n = 22).

					Food Additives and Contaminants				Page 44 of 46
	Element contaminants mean mean	min min	quartile1 quartile 1 median quartile 3 median quartile3	max max norm
	Mn Sum 7 marker 280.25	159.00	198.75	282	313.00			502.00
	Co	PCBs	2.35	58,72	1.17 0	2.02 0	2.32 32,30	2.73 73,80		351	3.60 200
	Ni	(ng/g fat)	22.41	1.67	5.10	24.88	32.89			77.81
	Cu Zn As Se Mo aldrin/dieldrin 507.40 9743.95 13.42 197.1 49.52 F o r Sum (ng/g fat) 27,29 F o r	329.20 6005.00 8957.50 483.75 3.03 9.53 107 172.56 9.35 28.47 0 0	511.58 9930.83 14.26 213.36 48.38 14,35	551.57 11102.90 17.53.00 228.60 58.66 40,55 115,40 200 622.17 13285.00 20.41 270.00 158.83
	Cd Sb Tl Pb DDT/DDE/DDD 0.27 2.53 0.22 8.91 Sum (ng/g fat) Hg 0.51 Dioxins (pg CALUX TEQ/g fat)	0.07 0.33 0.03 1.03 P e 770,71 26,40 P e 0.17 e r 0.13 1.55 0.07 2.46 50,25 0.26 10,18 2,28 5,57 e r	0.33 3.30 0.11 7.94 125,45 0.37 8,53	0.33 3.33 0.23 12.58 519,53 0.66 12,01	0.49 4.01 1.08 5250 500 32.03 1.29 23 3
	Dioxin-like PCBs (pg CALUX TEQ/g fat)	2,05	0,06	R e v i e 0,50 1,73 R e v i e	2,68	11,84
							w w
							O n l O n l
								y y

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Table IV :

 IV Descriptive statistics of levels of organochlorinated compounds in eggs from commercial farms (n = 19).

	contaminants	mean	min	quartile 1 median quartile 3	max norm
	Sum 7 marker
	PCBs	3,33	0	0	0	0	63,20 200
	(ng/g fat)
	Sum aldrin/dieldrin (ng/g fat) F o 3,33 r	0	0	0	3,75	18,10 200
	Sum DDT/DDE/DDD (ng/g fat)	17,30 P e 0	0	10,70	14,15	171,90 500
	Dioxins (pg CALUX TEQ/g fat)	1,44	0,50 e r 0,62	1,04	1,94	4,21	3
	Dioxin-like PCBs (pg CALUX TEQ/g fat)	0,77	0,31	0,53 R e v 0,69 i e	0,91	1,69
					w
					O n l
						y

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Acknowledgements


Sara C Cunha

José O Fernandes

M Beatriz
email: beatoliv@ff.up.pt

P P Oliveira

Comparison of matrix-solid phase dispersion and liquid-liquid extraction for the chromatographic determination of fenthion and its metabolites in olives and olive oils

Keywords: Food Additives and Contaminants Chromatographic analysis, Chromatography -GC, Chromatography -GC/MS, Clean-up Additives/Contaminants: Environmental contaminants, Pesticide residues, Pesticidesorganophosphorous Food Types: Oils and fats, Olive oil, Vegetables Fenthion, metabolites, matrix-solid phase dispersion, olive oil, GC/NPD

Comparison of matrix-solid phase dispersion and liquid-liquid extraction for the chromatographic determination of fenthion and its metabolites in olives and olive oils

Sara Cunha

Intoduction

Fenthion is an organophosphorus insecticide commercialised since 1960, used in the control of several fruit flies. In olive trees, fenthion is employed to control olive fly (Batrocera oleae, Gmel.), a pest that widely decreases the quality and amount of oil produced [START_REF] Lentza-Rizos Ch | Pesticide residues in olives and olive oil from a European perspective[END_REF]. Olive oil is a product obtained from olives solely by mechanical or other few physical means and which the presence of pesticides such as fenthion or other lipophilic organosphosphorus can occur.

The toxicity of fenthion and other pesticides has encouraged the establishment of MRLs (maximum residual levels) to control their presence in foods. Therefore, the European Commission (EC) and the Codex Alimentarius of the Food Agriculture Organization of the United Nation (FAO) have specified the MRL of 1 mg/kg of fenthion residues in olive oils and olives (EC 2001, FAO/WHO 1998). Moreover, pesticide metabolites show often higher toxicity than the parent molecule as occurs with fenthion. Fenthion is rapidly oxidised to sulfoxide and sulfone in plants, both with a lethal dose (LD 50 ) of 125 mg/kg, lower than the LD 50 of the parent compound: 220 mg/kg [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF].

Another oxidative process caused by the plant enzymes can involve the other fenthion moiety, thus leading to another three substances, fenoxon, fenoxon sulfoxide and fenoxon sulfone, with a LD 50 of 125 mg/kg, 50 mg/kg and 30 mg/kg, respectively (FAO/WHO 1998).

Several studies carried out by [START_REF] Lentza-Rizos Ch | Determination of residues of fenthion and its oxidative metabolites in olive oil[END_REF], [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF],

and [START_REF] Molinari | Trattamenti all' olivo con fenthion e rischio di residui in olive ed olio[END_REF], for chromatographic determination of fenthion and its metabolites in olive and olive oils, commonly used direct LLE as the sample preparation method. Organic solvents such as ethyl acetate, hexane saturated with acetonitrile, acetonitrile saturated with hexane, chloroform and acetone, are employed.

These methods of sample preparation have the advantage to be simple and easy to execute, but they require the use of large sample sizes and appreciable amounts of solvents. Moreover lipids are always present in the final extracts at high amounts. These lipids are generally not volatile and tend to coat the column and internal parts of the GC injector, decreasing the chromatographic performance [START_REF] Fong | Extraction, clean-up, and fractionation methods[END_REF]. In order to minimise these problems a number of post-extraction cleanup methodologies based on solid phase extraction (SPE), using adsorbents such as alumina [START_REF] Molinari | Trattamenti all' olivo con fenthion e rischio di residui in olive ed olio[END_REF](Molinari et al. , 1998)), Extralut (Di Muccio et al. 1999) and Florisil [START_REF] Rotunno | Decay of fenthion in green table olives[END_REF]) have been used for determination of fenthion and its oxidised metabolites in olive oils and olives. Afterwards, more sophisticated instrumentation such as supercritical fluid extraction (SFE) [START_REF] Poustka | Application of supercritical fluid extraction in multi-residue pesticide analysis of plant matrices[END_REF], pressurised liquid extraction (PLE) and gelpermeation chromatography (GPC) [START_REF] Jongenotter | Automated on-line GPC-GC-FPD involving co-solvent trapping and on-column Kirivanta A[END_REF], are also being applied in samples with high fat content for multi-residue pesticides determination.

An attractive alternative introduced recently for sample preparation of complex matrixes is the so called matrix solid-phase dispersion (MSPD) (Barker 2000a;2000b). This technique was successfully applied for the determination of pesticides in several food samples such as honey [START_REF] Fernández | Rapid screening of organophosphorus pesticides in honey and bees by liquid chromatography-mass spectrometry[END_REF], fruit [START_REF] Torres | Matrix solid-phase dispersion extraction procedure for multiresidue pesticide analysis in oranges[END_REF], vegetables (Kristenson et al. 2003[START_REF] Chu | Determination of 266 pesticide residues in apple juice by matrix solid-phase dispersion and gas chromatography-mass selective detection[END_REF]) and olive oil [START_REF] Ferrer | Determination of pesticide residues in olives and olive oil by matrix solidphase dispersion followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry[END_REF]. MSPD is a sample preparation methodology that combines aspects of several techniques for sample disruption, whilst generating a material with unique dispersed character for extraction of compounds (Barker 2000 a). MSPD also helps to prevent emulsion in liquid distribution and to reduce organic solvents disposals and analysis time.

The application of MSPD involves the mixture of the sample with a solid support, such as octadecylsilyl (C 18 ), octyl (C 8 ) or aminopropyl (NH 2 ), packing the extraction/cleanup column and eluting the target compounds with solvents. The extraction and cleanup can be completed in one step simplifying the sample preparation procedure (Barker 2000b).

In general, the analysis of organophosphorus residues is carried out by gas chromatography coupled with selective detection of analytes, such as flame photometric detector (FPD), mass spectrometry (MS), atomic emission detector (AED) and nitrogen phosphorus detector (NPD) [START_REF] Poustka | Application of supercritical fluid extraction in multi-residue pesticide analysis of plant matrices[END_REF].

The aim of this study was to develop an analytical procedure that combines MSPD and GC/NPD for the determination of the fenthion and its metabolites in olive oils and olives. Louis, MO, US), silica (pore size 45 µm) and alumina (pore size 60-325 mesh) from Supelco (Bellefonte, PA, US) and Extralut 3 from Merck (Darmstadt, Germany).

Standards

Stock solutions of 20 mg/l of each analyte were prepared in acetonitrile and keep at -20 ºC. Working standard pesticide mixtures of 5 mg/l was prepared in acetone from the stock standard solution. Internal standard solutions of TPP (4 mg/l in acetone) and TBP (0.5 mg/l in acetone) were used in quantification and in monitorization of chromatographic conditions, respectively.

Samples

The trial was carried out in 2 pilot Cv. Cobrançosa olive groves with 2.4 ha and 1.9 ha, respectively, located in the northeast of Portugal. were registered every day during this period (data not shown). Samples of 50 fruits per tree were collected and stored at -20 ºC until analysis.

Olive oils were obtained following the method described by [START_REF] Pereira | Influence of Olive Storage Period on Oil Quality of Three Portuguese Cultivars of Olea europea, Cobrançosa, Madural, and Verdeal Transmontana[END_REF].

Briefly, olive fruits were hand-picked and processed through a mill, a thermo beater and a pulp centrifuge, after which the oil was separated from the pulp by decantation, and kept in dark glass bottles, at 4ºC, in the absence of light.

Sample extraction MSPD extraction

Olives: 25 g of olive pulp were homogenized in Ultra-turrax (2 min at 8000 rpm). An aliquot (1 g) was placed in a mortar with 1 g NH 2 , added with 25 µl of TPP solution (4 mg/l) and gently blended for a few minutes using a pestle to obtain a dry-powder-like homogeneous mixture. Then, the mixture was introduced into a column containing 1 g of Florisil. This column was attached to a vacuum manifold (Visaprep, Supelco, PA, US) coupled with a small vacuum pump, and the flow adjusted to 3 ml/min. The elution step was carried out with 3 x 5 ml of acetonitrile. The eluent was dried with anhydrous MgSO 4 , centrifuged at 3500 g for 1 min and decanted. The extracts were evaporated until dryness, then re-dissolved in 1 ml of acetone containing TBP (0.5 mg/l).

Olive oil: 0.2 g of sample were placed in a mortar and gently blended with 1 g NH 2 and 25 µl of TPP solution (4 mg/l) using a pestle. The other steps of the method were the same described above for olives.

Liquid-liquid extraction (LLE)

LLE was based on the method of [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF] with minor modifications. Briefly, in olives: 10 g of olive pulp were mixed with 10 ml of n-hexane, 25 µl of TPP solution (4 mg/kg) and 1 g of anhydrous Na 2 SO 4 . The mixture was blended for 30 min and centrifuged at 3500 g for 2 min. The decanted phase was evaporated until a final volume of about 1.5 ml. For olive oil, the same described procedure was used with 1 g of sample. But another method published by [START_REF] Ferrer | Determination of pesticide residues in olives and olive oil by matrix solidphase dispersion followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry[END_REF] has also been adopted for olive oil samples. The procedure consists in liquid-liquid partitioning with 15 ml of ether petroleum saturated with acetonitrile followed by 35 ml acetonitrile saturated with ether petroleum of sample (5 g) with 25 µl of TPP solution (4 mg/l). Finally, an aliquot (7 ml) of the acetonitrile fraction was evaporated until about 1 ml.

Equipment and chromatographic conditions

Chromatographic analysis was performed in a ThermoFinnigan Trace GC gas chromatograph (Milan, Italy) equipped with a nitrogen phosphorus detector (NPD) and a split-splitless injector. A fused silica capillary column, ZB-5 (30 m x 0.32 mm I.D., 0.25 µm film thickness-Phenomenex, Torrance, CA, US), was used for compounds separation. GC conditions were as follows: splitless injection (period: 0.80 min), carrier gas flow rate (helium) 1 ml/min (constant flow), injector temperature: 250 ºC, detector temperature: 300 ºC, oven temperature: 70 ºC (1 min held), 10 ºC/min to 230 ºC, 5 ºC/min to 280 ºC (2 min held). 1 µl of the extract was injected, and quantification was based on the internal standard method.

Results and Discussion

MSPD procedure for olive fruits

In sample preparation of olives several parameters were studied including, sample size, sorbents, sample/sorbents ratio, solvents and sample/solvents ratio. The evaluation of these parameters involved, in most of the cases, recovery determinations. With this purpose blank samples were spiked with appropriate volumes to achieve 1 mg/kg of analytes before extraction and compared with a mixture standard solution with the same concentration.

a) Sample size

The sample preparation methods described in current published papers for determination of fenthion and its metabolites often use large amounts of sample (i.e 25 to 50 g). However, the trend is clearly to reduce sample size to minimum amounts providing statistically reliable results and to scale methods accordingly (Anastassiades Therefore, particular care should be taken during extraction to recover all pesticides from the bulk of lipidic material. Table I shows the recoveries obtained using three different types and amounts of sorbents: C 18 , PSA and NH 2 . Accordingly to the literature these sorbents have been successfully used in extraction of pesticide residues in fatty matrixes [START_REF] Gillespie | Multicolumn solid-phase extraction cleanup of organophosphorus and organochlorine pesticide residues in vegetable oils and butterfat[END_REF], Lehotay et al. 2005b, Ferrer et al. 2005). To select the most appropriate sorbent for fenthion and its metabolites, the elution was performed with 15 ml of acetonitrile trough the extract/sorbent. C 18 was tested in this study with and without previous activation, being washed with methanol and water to activate. However, both experiments presented poor recoveries of all the compounds, probably due to a strong apolar interaction with the alkyl chain of the sorbent. In contrast, NH 2 and PSA interact with analytes by hydrogen bonding and dipole-dipole interactions. In this study, NH 2 was found to be the best olive pulp dispersant, as can be seen in Table I.

[Insert Table I about here]

Table I also shows the results concerning the influence of the amount of NH 2 (from 0.5 g to 2 g) in recovery of fenthion and its metabolites. The best recoveries are obtained using 1 g of the solid phase .

The effect of several salts on phase separation using the so called QuEChERS (quick, easy, cheap, effective, rugged and safe) method has been studied by [START_REF] Anastassiades | Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "Dispersive Solid-Phase Extraction" for determination of pesticide residues in produce[END_REF] for multiresidue determination. Their results have demonstrated that better recoveries were obtained with a combination of anhydrous MgSO 4 and NaCl. Thus, the applicability of different salts (MgSO 4 , NaCl and Na 2 SO 4 ) on MSPD sample preparation was evaluated. As shown in Table II, the recoveries obtained without salts were slightly higher than the obtained with any salt or salt combination. In conclusion, these salts are not a good option to be used as dispersant for this kind of matrix. With polar sorbents, like NH 2 , the eluting power increase with the solvent polarity (nhexane < chloroform < toluene < ethyl acetate < acetone < acetonitrile) [START_REF]Principles of extraction and the extraction of semivolatile organics from liquids[END_REF].

The effects of these different solvents were compared in a series of experiments to determine their ability to elute adequately fenthion and its metabolites on NH 2 . The only parameter changed was the type of solvent maintaining the volume constant (15 ml).

Results were presented in Figure 1.

[Insert Figure 1 about here]

The best results were obtained with acetonitrile. This is in according with Kirivanta (2004). The large vaporisation expansion volume of acetonitrile that tends to limit amount that can be injected in GC injection can also lead to the deterioration of the NPD. Thus we re-dissolved the extract in acetone after elution with acetonitrile.

The dependence of recoveries upon solvent volume was also evaluated (Figure 2).

Starting from the initial conditions mentioned above (addition of 1 g of sample and 1g

NH 2 ) recoveries were carried out at several elution volumes, i.e. 5, 10, 15 and 20 ml.

When elution volumes of 20 ml were applied no significant increase in the peak area of the compounds was observed. Decreasing the elution volumes to 10 ml or less had an adverse effect on the recovery. Thus, 15 ml was the elution volume adopted.

[Insert Figure 2 In order to eliminate some of the lipid material that co-eluted with the compounds in study, the extract were submitted to a further clean-up procedure before analysis by GC/NPD. In this study, several types of clean-up were used namely, graphitized carbon black (GCB), alumina, silica, Extralut 3 and Florisil. GCB has resulted very efficient from standpoint of visual appearance, but unfortunately it tends to remove the analytes studied. In fact, GCB strongly retains some chemicals namely planar pesticides as observed by Lehotay et al. (2005 a, b). Silica, alumina and Extralut 3 were discarded, due to low recoveries and low clean-up efficiencies. Thus, a cartridge column of 1 g of Florisil was found to be the best system to retain interfering compounds without significant losses of the compounds in study as can seen in Figure 3.

[Insert Figure 3 about here]

To prevent negative effects in chromatographic separation caused by the presence of water traces in the eluate, MgSO 4 was added before injection in GC/NPD.

MSPD procedure for olive oils

Fenthion and its metabolites are efficiently extracted from olive oil with the MSPD procedure developed for olive fruits. However, taking into account that to obtain one litre of olive oil, 5 kg of olives were usually required, the sample size was reduced to an aliquot of 0.2 g, which showed to be a adequate.

Comparison between MSPD and LLE procedures a) Olive fruits

Table III summarises the limits of detection, linearity and reproducibility of the method to determine fenthion and its metabolites in olives, by MSPD under the established conditions and by LLE. Linearity experiments were carried out with blank olive fruits.

The extraction procedures presented good linearity at least over the investigated concentration ranging from 0.03 to 1 mg/kg by MSPD and from 0.1 to 1 mg/kg in LLE.

The detection limit was estimated on the basis of signal-to-noise (3:1). The MSPD procedure presented higher sensitivity for all the compounds than LLE with detection limits from 0.002 to 0.01 mg/kg and from 0.006 to 0.04 mg/kg for MSPD and LLE respectively (Table III). This advantage is especially useful for the determination of sample compounds present in small amounts, such as the metabolites of fenthion. The To determine the reproducibility, the proposed extractions were applied six times on the same blank sample spiked with working standard solutions (to achieve 1 mg/kg of each analyte). As can be seen in Table III, repeatability values (measured by the relative standard deviation) ranged from 1.7 % to 15.6 % in MSPD and from 4.3 % to 15.9 % in LLE, which are considered acceptable for most validation procedures.

[Insert Table III about here]

The recoveries of the fenthion and its metabolites in olive fruits at different spiking levels (0.2, 0.3 and 0.5 mg/kg), obtained by the MSPD and LLE procedures, are summarized in Table IV. The results for the MSPD procedure were slightly higher to those of LLE procedure. The recoveries for all compounds were higher than 85% and 68% on the MSPD and LLE procedures, respectively. As can been observed in Table IV, when the spiking level was lower than 0.3 mg/kg the recoveries for LLE were significantly worse than those of the MSPD procedure.

[Insert Table IV about here]

b) Olive oils

For olive oil, the MSPD procedure was compared with two different LLE procedures used by [START_REF] Ferrer | Determination of pesticide residues in olives and olive oil by matrix solidphase dispersion followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry[END_REF] and [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF]. The determinations were similar to the described for the olive fruits. Thus, blank samples were spiked with appropriate volumes to achieve 1 mg/kg of analytes before extraction and compared with a mixture standard solution with the same concentration. Both LLE procedures assayed, presented satisfactory recoveries (Table V). However, they involve the consumption of large amounts of solvents and large samples size, becoming more expensive and unclean. As shown in the

Method application

The proposed method has been applied in the analysis of olive pulp obtained a previously described in Experimental section. All samples (along the pre-harvest interval) had a common qualitative pattern, showing four identified compounds, the levels are depicted in Table VI. The fenthion residue levels herein reported were lower than the established by legislation (1 mg/kg) ranging from 0.01 to 0.03 mg/kg. Fenthion sulfoxide ranged from 0.03 to 0.01 mg/ kg, fenoxon ranged from 0.01 to 0.02 mg/ kg, and fenoxon sulfoxide ranged from 0.03 to 0.05 mg/ kg. Notwithstanding the concentration values, all compounds had a decreasing tendency along the pre-harvest interval. Fenthion and three metabolites were still present in the olives at harvest time. [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF] described the existence of other metabolites of fenthion in olives namely fenthion sulfone and fenoxon sulfone, but those compounds were not detected in this study. The observed difference could be ascribed to the number of phytossanitary treatments used in that work, which was three times higher than was the applied in this research. In addition, fenthion showed a slow degradation in olive fruits as previously referred in literature [START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF][START_REF] Lentza-Rizos Ch | Determination of residues of fenthion and its oxidative metabolites in olive oil[END_REF][START_REF] Tsatsakis | Three-year study of fenthion and dimethoate pesticides in olive oil from organic and conventional cultivation[END_REF] which contributed to the different levels of fenthion and its metabolites.

[Insert Table VI about here]

Conclusions

MSPD is a good alternative to the LLE procedures for the analysis of fenthion and its metabolites in olive fruits and olive oils. The proposed method involves the use of NH 2 as dispersant followed by elution with acetonitrile, clean-up with Florisil and finally chromatographic analysis by GC/NPD. The results of this study show that the proposed method is simple and sensitive, requiring small volumes of solvents. Additionally, the proposed method offers good recovery and low detection limits when compared to other conventional methods. The method was applied to evaluate fenthion and its metabolite residue levels during the pre-harvest interval in olive pulp. Fenthion residual levels, in all periods, are lower than the MRL established by legislation. Fenthion sulfone, fenoxon and fenoxon sulfoxide were the only metabolites detected in the olive fruits.

  Comparison of matrix-solid phase dispersion and liquidliquid extraction for the chromatographic determination of fenthion and its metabolites in olives and olive oils

  al. 2003). Considering the published literature and taking into account that sample size need to be representative and appropriate to sorbent amounts, amounts of olive pulp ranging from 1 to 5 g, were tested. The best results were obtained with 1g. b) Type of sorbent and sample/sorbent ratio Fenthion is intimately associated with lipid components of the sample matrix.

  of solvent, sample/solvent ratio Solvents are characterized by their polarity and elution strength for a specific sorbent.

(

  1983) which referred that the best solvents for extraction of pesticide residues are simultaneously miscible with water and with apolar solvents. The solvents that follow this premise are acetone and acetonitrile. Compared with acetone, acetonitrile extracts less lipophilic material, e.g. waxes, fat and lipophilic pigments. Advantages and disadvantages of these and other solvents in extraction of pesticides are well explained by Maštovská et al.

  for each type of procedure were well-below of MRLs set by legislation.

Figure 1 -

 1 Figure 1 -Recoveries (%) of fenthion and its metabolites in olives samples extracted 1 mg/kg with different solvents in a MSPD method with 1 g NH 2 and 15 ml of eluent. The errors bars represent relative standard deviation (n= 3).

Figure 2 -

 2 Figure 2 -Recoveries (%) of fenthion and its metabolites spiked at 1 mg/kg in olives samples extracted with different volumes of acetonitrile used in the MSPD proposed method in. The errors bars represent relative standard deviations (n= 3).

Figure 3 -

 3 Figure 3 -Chromatograms of olive extracts obtained with different types of clean-up procedures after extraction with NH 2 and elution with acetonitrile.

  Table V best recovery results were again obtained with MSPD, as previously observed for olives. Linearity in MSPD method was checked with

	[Insert Table V about here]
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	12

calibration curves in a range of concentrations from 0.05 to 1 mg/kg. Regression analysis was performed to generate the linear equations and coefficients of determination (R 2 ) that ranged from 0.9640 to 0.9951.

Table I -

 I Recoveries (Rec. %) and relative standard deviations (RSD %) obtained with different sorbents in the extraction of fenthion and its metabolites, after elution of 15 ml of acetonitrile through extract/sorbent (n= 3).

	Page 21 of 26	Food Additives and Contaminants
	Rec. (%) 93.2 ± 53.3 ± 54.2 ± 73.1 ± 39.6 ± 57.8 ± C 18 a) RSD (%) Rec. (%) 6 72.4 9 31.2 7 64.2 5 70.5 12 35.5 11 55.0 1 g C 18 b) RSD (%) Rec. (%) ± 7 100.8 ± ± 10 67.6 ± ± 8 77.5 ± ± 5 102.4 ± ± 16 57.5 ± ± 15 78.8 ± 1 g RSD (%) Rec. (%) 8 95.0 9 77.0 9 87.5 7 91.0 18 75.2 12 79.5 1 g NH 2 RSD (%) Rec. (%) ± 6 82.0 ± 8 65.3 ± 7 68.2 ± 5 72.3 ± 12 55.7 ± 9 60.2 1 g 0.5 g RSD (%) Rec. (%) ± 6 88.4 ± 7 70.8 ± 6 71.6 ± 6 73.9 ± 15 65.8 ± 11 63.6 NH 2 NH 2 RSD (%) ± 8 ± 9 ± 8 ± 6 ± 14 ± 10 2 g PSA a) With previous activation with methanol/water Fenthion Fenthion sulfoxide Fenthion sulfone Fenoxon Fenoxon sulfoxide Fenoxon sulfone Fenthion and its metabolites b) Without previous activation F o r P e e r R e v i e w O n l y
		2

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Table II -

 II Recoveries (Rec. %) and relative standard deviations (RSD %) obtained with 1 g NH 2 and with different salts in the extraction of fenthion and its metabolites in olives, after elution of 15 ml of acetonitrile through extract/sorbent (n = 3).

	Page 23 of 26		Food Additives and Contaminants Food Additives and Contaminants			Page 22 of 26
	Rec. (%) 95.0 ± 77.0 ± 87.5 ± 91.0 ± 75.2 ± 79.5 ± Without salt RSD (%) Rec. (%) 6 70.6 8 58.4 7 48.6 5 70.6 12 46.4 9 41.4 NaCl RSD (%) Rec. (%) ± 8 74.1 ± ± 8 109.2 ± ± 8 54.8 ± ± 7 74.1 ± ± 11 51.8 ± ± 12 51.1 ± MgSO 4 RSD (%) Rec. (%) 7 84.1 5 76.6 7 75.5 5 81.2 13 64.8 11 70.0 MgSO 4 + NaCl RSD (%) Rec. (%) ± 7 61.8 ± 8 75.0 ± 6 79.6 ± 6 56.8 ± 11 50.2 ± 10 62.3 NaSO 4 + NaCl RSD (%) ± 9 ± 8 ± 7 ± 6 ± 12 ± 11 0.125 + 0.125 g 0.25 g 0.25 g 0.125 + 0.125 g F o r P e e r R e v i e w O n l y Table III -Limits of detection (LODs), linear range and repeatability (Relative standard deviations %) by MSPD and LLE procedures in Fenthion Fenthion sulfoxide Fenthion sulfone Fenoxon Fenoxon sulfoxide Fenoxon sulfone Fenthion and its metabolites olives. MSPD LLE Fenthion and its metabolites LODs (mg/kg) Linear range (0.03 -1 mg/kg) R 2 Repeatability (n=6) RSD (%) LODs (mg/kg) Linear range (0.1 -1 mg/kg) R 2 Repeatability (n=6) RSD (%) Fenthion 0.009 0.9959 8.51 0.02 0.9898 11.30 Fenthion sulfoxide 0.003 0.9977 1.66 0.009 0.9931 4.34 Fenthion sulfone 0.006 0.9940 2.92 0.02 0.9937 6.34 Fenoxon 0.002 0.9982 9.42 0.006 0.9884 9.52 Fenoxon sulfoxide 0.01 0.9953 14.08 0.04 0.9870 15.91 F o r P e e r R e v i e w O n l y
	Fenoxon sulfone	0.009	0.9996	15.58	0.02	0.9962	12.51
							3 4

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Table IV -

 IV Recoveries (Rec. %) and relative standard deviations (RSD %) obtained with MSPD and LLE procedures in olive fruits (n = 3).

		Food Additives and Contaminants	Page 24 of 26
	Fenthion Fenthion sulfoxide Fenthion sulfone Fenoxon Fenoxon sulfoxide Fenoxon sulfone Fenthion and its metabolites	Rec. (%) 110 98 112 96 85 85 MSPD RSD (%) Rec. (%) ± 4 108 ± 7 91 ± 8 102 ± 5 104 ± 10 88 ± 9 96 LLE RSD (%) Rec. (%) ± 6 101 ± 7 92 ± 8 105 ± 6 102 ± 15 87 ± 14 95 0.5 mg/kg MSPD RSD (%) Rec. (%) ± 6 102 ± 8 83 ± 9 97 ± 5 95 ± 11 68 ± 10 92 0.3 mg/kg LLE RSD (%) Rec. (%) ± 7 95 ± 9 88 ± 10 102 ± 6 93 ± 12 86 ± 11 92 MSPD RSD (%) Rec. (%) ± 5 84 ± 8 79 ± 7 85 ± 5 98 ± 13 73 ± 12 72 0.2 mg/kg LLE RSD (%) ± 7 ± 8 ± 8 ± 6 ± 13 ± 14 F o r P e e r R e v i e w O n l y
		5

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Table V -

 V Recoveries (%) and relative standard deviations (RSD %) obtained with 3 extraction procedures applied to olive oil and linearity obtained with MSPD procedure (n = 5).

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	Linearity (R 2 ) 0.9945 0.9872 0.9910 0.9951 0.9640 0.9798 F o r P e e r R e v i e w Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) Fenthion 79 ± 6 62 ± 7 68 Fenthion sulfoxide 96 ± 8 91 ± 8 115 Fenthion sulfone 90 ± 9 84 ± 8 105 Fenoxon 78 ± 5 71 ± 6 65 Fenoxon sulfoxide 67 ± 11 68 ± 13 63 Fenoxon sulfone 98 ± 11 79 ± 10 81 LLE (2) RSD (%) ± 6 ± 8 ± 7 ± 6 ± 12 ± 14 Fenthion and its metabolites MSPD LLE (1) O n l y
	6

(1) Method based in

[START_REF] Cabras | Persistence of insecticide residues in olives and olive oil[END_REF]

(2) Method based in

[START_REF] Ferrer | Determination of pesticide residues in olives and olive oil by matrix solidphase dispersion followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry[END_REF]

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Table VI -

 VI Pesticide residues (mg/kg) determined olives, after treatment with the insecticide mixture.

	Days after			Residues of pesticides (mg/kg)
	treatment Fenthion Fenthion sulfoxide Fenthion sulfone Fenoxon Fenoxon sulfoxide	Fenoxon Sulfone
	7	0.02	0.03	n.d.	n.d.	n.d	n.d.
	14	0.01	0.02	n.d.	0.02	0.03	n.d.
	21	0.02	0.03	n.d.	0.01	0.05	n.d.
	28	0.02	0.02	n.d.	0.01	0.05	n.d.
	35	0.02	0.02	n.d.	0.01	0.04	n.d.
	42	0.02	0.01	n.d.	0.01	0.03	n.d.

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Acknowledgments S. C. Cunha is grateful to "Subprograma Ciência e Tecnologia do 3º Quadro Comunitário de Apoio (BD 8822/2002)" and to "Acção 8.1. PO AGRO Nº 482" for financial support.

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