Houda ; Berrada

Mónica ; Fernández
email: monica.fernandez@uv.es

Keywords: monitoring, multiresidue analysis, fruits, pesticides, gas chromatography, mass spectrometry, Valencia, ADI and EDI

analytical procedure based on ethyl acetate extraction was used followed by gas chromatography coupled to nitrogen phosphorus detector for routine analysis and mass spectrometry for confirmation. Recovery studies with spiked samples at 0.5 mg kg -1 for each pesticide ranged from 52 % for acephate to 87 % for fenthion with standard deviation below 20 %. Limits of quantification ranged from 0.1 to 100 µg kg -1 . A total of 43 % of samples contained pesticide residues and 5 % exceeded the Maximum Residue Levels (MRLs). Nine of the pesticides studied were found.

Malathion, which was the most frequently detected, was found in 121 samples (19 %) at 0.002-4.25 mg kg -1 , followed by fenthion in 104 samples (16 %) at 0.005-2.3 mg kg -1 and methidation in 68 samples (10 %) at 0.008-1.3 mg kg -1 . Khaki showed the highest contamination rates with 60% of contaminated samples which exceeded more often the MRLs and fenthion was the pesticide more frequently detected in all the commodities studied at levels above the European MRLs. The Estimated Daily Intakes of each pesticide calculated from the results obtained were much lower than the Acceptable Daily Intakes.

Introduction

Increasing public concern in recent years about possible health risks from pesticide residues in food supply has led to establish strict regulation of pesticide tolerances and to assess dietary intakes of pesticide residues in food commodities [START_REF] Stenersen | Chemical Pesticides: Mode of Action and Toxicology[END_REF][START_REF] Ecobichon | CD editors Cassarett and Doull's Toxicology[END_REF][START_REF] Nasreddine | Food contamination by metals and pesticides in the European Union. Should we worry?[END_REF]. Monitoring programme for pesticide residues in food are a useful tool for ensuring that consumers are not exposed to unacceptable pesticide residue levels, authorised pesticides are correctly applied to food crops in terms of granted authorizations and registrations (application rates and pre-harvested intervals) and to permit the free circulation of pesticide-treated products as long as they comply with the fixed MRLs (Dogheim et al. 1997[START_REF] Fernández | Pesticide residues in oranges from Valencia (Spain)[END_REF][START_REF] Blasco | Screening and evaluation of fruit samples for four pesticide residues[END_REF]. [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market, 1998-99[END_REF], JMPR 1999, European Commission 2001). However, these results are not representative of the situation on a particular area so monitoring of the locally produced commodities is relevant to have a complete view of the pest treatment making possible to take specific measures in the contaminated area.

F

The frequent application of different types of pesticides and the large number of samples makes necessary the determination of as many compounds as possible in a single analysis at levels below the MRL. Generally monitoring analysis are accomplished with gas chromatography (GC) in combination with different detectors, such nitrogen-phosphorus detector (NPD), electron-capture detectors (ECD) or mass spectrometry (MSD) which provides a fingerprint spectra by electron ionization and allows the possibility of confirming pesticide identity [START_REF] Albero | Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography[END_REF], Puglese et al. 2004[START_REF] Lehotay | Application of gas chromatography in food analysis[END_REF], Torres et al. 1997).

When assessing the impact on consumers of pesticide exposure through diet, it is important to consider the effects of both, chronic and acute exposure. Limiting chronic exposure to a pesticide residue is managed through establishing the Acceptable Daily Intake (ADI) for the pesticide, which is an estimate of the maximum level of intake over a lifetime, judged to result without appreciable health risk. Estimations include a safety factor to ensure that the elderly, infants, children, and those whose systems are under stress because of illness are protected [START_REF] Leblanc | Estimation of the dietary intake of pesticide residues, lead, cadmium, arsenic and radionuclides in France[END_REF][START_REF] Caldas | Related chronic dietary risk for pesticide residues in food in Brazil: an update[END_REF], Chun and Kand 2003).

Material and methods

Samples and reagents

Extraction Procedure

Samples were extracted by an official procedure for routine analysis that allows the analysis of about 120 pesticides (Conselleria de Sanidad y Consumo de la Comunidad Valenciana 1995). A 50 g portion of sample previously homogenized was weighed in a 500 ml beaker, 100 ml of ethyl acetate and 75 g of anhydrous sodium sulphate were added and the mixture was blended using a stainless steelarmed blender for 5 min, the resulting mixture was filtered through 20 g of anhydrous sodium sulphate. The solid was washed with 50 ml of ethyl acetate and the organic extract was concentrated to less than 10 ml on a vacuum rotary evaporator using a water bath at 44-46ºC and 250 mBar. Finally, the extract was reconstituted to 10 ml with ethyl acetate and 2 µl was analyzed by GC. Varian gas chromatograph conditions: Injector 280°C, detector 300°C. Oven temperature program: initial temperature, 140ºC; held for 1 min, programmed to 280°C, at 5ºC/min, and held for 11 min. Helium was used as carrier gas at a flow rate of 1.9 ml/min.

Analysis of pesticide residues

Trace gas chromatograph conditions: injector and transfer line 280ºC, source temperature 230ºC. Oven temperature program: the initial temperature (50ºC) was held for 1 min, programmed to 120ºC at 30ºC/min, held for 1 min, then programmed to 275ºC at 5ºC/min, and held for a further 5 min. The mass spectrometer was used with electron impact ionization (-70 eV) in full scan mode (65-365 amu). The carrier gas was helium at 1.5 ml/min. Splitless time was 0.9 min.

Results and Discussion

Method performance Analytical parameters were calculated for the multi-residue method proposed using GC-NPD. Precision was calculated in terms of intra-day repeatability (n=5) and inter-day reproducibility (5 different days). The intra-day repeatability evaluated as relative standard deviation (RSDs) ranged from 3 to 5%. The inter-day reproducibility was lower than 8% for all instances. Calibration curves prepared with standard in ethyl acetate were linear at the studied range as shown in Table 2 with regression coefficients >0.99. Limits of quantification (LOQs) which were calculated as the lowest concentration of compound that gave a response that could be quantified as the minimum concentrations providing chromatographic signals 10 times higher than background noise ranged from 0.0001 to 0.1 mg kg -1 and were lower than the MRLs established by the EU and Spanish legislations and those recommended by the FAO/WHO for each of the commodities studied (Table 1). Accuracy of samples spiked at 0.5 mg kg -1 and 5 mg kg -1 were equivalent in the commodities studied. Except for some cases, principally at 5 mg kg -1 , results of accuracy and precision fulfilled the criteria of the European guidelines indicating that a method can be considered accurate and precise when accuracy data are comprised between 70 and 110% with relative standard deviations not higher than 20% (European Commission, 2000).

Figure 1 illustrates a NPD chromatograms corresponding to a spiked nectarine and a contaminated nectarine with 0.5 mg kg -1 of fenthion and 0.1 mg kg -1 of malathion.

Due to the presence of some interfering peaks as the one between both peaks a

Monitoring data

Table 3 outlines the data obtained after analyzed 634 samples of fruits in which 276 (43 %) of the analyzed samples were contaminated and 34 (5 %) exceeded the MRLs. In terms of commodity groups, khakis was the fruit with the highest rate of contaminated samples (61 %), followed by of tangerines (57 %), oranges (42 %), nectarines (39 %) and peaches (26 %). No residues were found in watermelons.

Most of the pesticides found were organophosphorus used as insecticides except triazole fungicides (bitertanol and flusilazole) and a chloronitrile fungicide (chlorothalonil). Malathion was the most frequently pesticide detected and at the highest concentration, 121 (19 %) samples of the commodities studied except watermelon were contaminated from 0.002 to 4.25 mg kg -1 (0.282 mg kg -1 average level). This pesticide is a wide spectrum organophosphorous insecticide suitable for the control of insects that shuck and chew on fruit and vegetables. Fenthion was detected in 104 samples (16%) at levels ranging from 0.005 to 2.3 mg kg -1 (0.179 mg kg -1 average level), this pesticide is a contact insecticide used against many sucking, biting pests, especially fruit flies, stem borers and mosquitoes.

Methidathion was found in 68 samples (10%) at levels between 0.008 and 1.3 mg kg -1 (0.2 mg kg -1 average level). Bitertanol was found in 12 samples (1.9 %) at levels that range from 0.14 and 1.9 mg kg -1 (0.52 mg kg -1 average level). Fenitrothion was found in 6 (0.9%) at levels between 0.01 to 0.53 mg kg -1 (0.05 mg kg -1 average level). Chlorpyrifos methyl and flusilazole were detected in 3 samples, the first one with a range from 0.04 mg kg -1 to 0.37 mg kg -1 (0.176 mg kg -1 average level) and the second one from 0.06 to 0.1 mg kg -1 (0.079 mg kg -1 average level). Trichlorfon and Parathion methyl were found only in one sample, both in peaches with a level around 0.02 mg kg -1 .

The pesticides detected with levels above European MRLs were fenthion ( 22samples), malathion (10 samples), bitertanol (3 samples), fenitrothion (1 sample)

and flusilazole (1 sample).

The co-ocurrence of pesticide residues are shown in Figure 3. A total of 231 samples (36% of the total) contained only one pesticide, 31 (4 %) samples of the six commodities studied contained two residues of pesticides and 7 (1%) samples of peach, tangerine, nectarine and orange were contaminated with three pesticide residues and 1 samples of nectarine contained four pesticide residues.

Furthermore, earlier monitoring data conducted in fruits contaminated with pesticide residues are similar with the levels and frequencies reported by other authors [START_REF] Andersen | Results from the monitoring of pesticide residues in fruit and vegetables on the Danish market, 1998-99[END_REF][START_REF] Blasco | Simultaneous determination of imidacloprid, carbendazim, methiocarb and hexythiazox in 13 peaches and nectarines by liquid chromatography-mass spectrometry[END_REF], , European Commission, 2001, FAO/UNEP/ WHO, 1991[START_REF] Fernández | Pesticide residues in oranges from Valencia (Spain)[END_REF], Torres 1997). The safety of fruit consumption was evaluated according to the toxicological significance of human exposure to these pesticides. The FAO/WHO have established ADIs in accordance with the evaluation of all available data relating to the substance studies conducted in human, experimental animals and in vitro system.

Daily intake

To evaluate this risk we have calculated the EDIs for a human being with a body weight of 60 kg, taking into consideration an inquire about the mean food consumption in Spain (Spanish Institute National of Statistic 2005). The average level of pesticide found, the ADIs and EDIs of each pesticide calculated for the main group of the commodities are shown in Table 4. The EDIs of pesticides is much lower than the ADIs stablished. The highest EDIs obtained for methidation in citrus fruits was 11 times lower than the ADIs. This organophosphorous is mainly used in citric fruits which are basically consumed in the Mediterranean area, therefore the combined intake of this pesticide residue from other food items is not significant and therefore, it is unlike to reach exposures above acceptable levels.

Conclusions

Results show that most of the pesticide levels detected were below than the MRLs established by the EU. Pesticide residue monitoring programmes should be recommended to be implanted for ensuring minimal residue levels in fruit commodities. Moreover, the calculation of the EDI of each pesticide from these data showed that the contribution of the fruits studied to dietary intake was much 12 lower than the ADIs published. The EDIs for calculated in this way were higher than the real values because khaki was the fruit with proportionally more contaminated samples, but their contribution of this fruit to the Spanish population diet is less than 1 kg. In general, the dietary intake of these pesticide residues through the diet for the mean adult Spanish consumer does not seem to pose a relevant risk.

F

5

  Valencia is an important citrus growing area in Europe although other fruit commodities are also produced and exported, so assessing pesticide residues in fruit intended for human consumption is necessary. The aim of this work was to analyze fifteen pesticide residues in tangerines, oranges, peaches, nectarines and khakis from agricultural cooperative of Valencia during 2001-2003. The results of the monitoring program were taken into consideration to evaluate whether the Estimated Daily Intake (EDI) of pesticides through the fruit consumed by Spanish adult population is a cause for health concern according to the recommended intake of the FAO/WHO organization.

7

  Routine analyses were carried out on a Varian Star 3400CX gas chromatograph (Varian, Walnut Creek, CA, USA) equipped with an 8200CX autosampler, an oncolumn injector (SPI 1093), and NPD system. Identification of peaks was performed in a Trace GC gas chromatograph (Thermo Electron Corporation, San José, CA, USA) with a Thermo-Finnigan AS 2000 autosampler, a split/splitless injector, and a Trace MS quadrupole mass spectrometer. Compounds were separated on DB-5 ms (J&W Scientific, Folsom, CA USA) fused-silica capillary columns (30 m × 0.25 mm i.d., 0.25 µm film thickness).

  necessary. In Figure2the total ion GC-MS chromatogram and the two peaks spectra confirmed the identity of both pesticides.

Figure 1 .

 1 FIGURES:

Figure 2 .

 2 Figure 2. Total ion chromatogram profile of the same nectarine sample of Figure 1 contained

Figure 3 .

 3 Figure 3. Number of pesticide residues found in each commodities.

  Estimated daily intakes (EDIs) (µg kg -1 b.w./day) and admissible dairy intakes (ADIs) for pesticide residues found in stone fruit (peaches and nectarines), citrus fruit (tangerines and oranges) and other fruits (khakis and watermelons) collected during 2001-2003.a EDI was calculated by the equation: EDI = (∑c) (C N1 D1 K1): ∑c is the sum of the pesticide residues concentrations in the analyzed samples (µg kg -1 ); C the mean annual intake per person. The value of the annual intake per person of stone fruit (nectarines and peaches) was 8.2 kg/person. of citrus fruit (tangerines and oranges) was 26 kg/person and of other fruit (khaki and watermelon) was 12 kg/person according with the National Inquiry of Consumption of Food, Drinks, and Tobacco performed in the years2001- 2002.

assessment of fruits contaminated with pesticide residues from Valencia, 2001-2003

  No residue One Two Three Four

		Formatted: Left, Adjust space
	Abstract	between Latin and Asian text, Adjust space between Asian text and
		numbers
	A total of 634 samples of oranges, tangerines, peaches, nectarines, khakis, and
	watermelons were collected from an Agricultural Valencia Community Cooperative
	F o during May 2001 to April 2003 campaigns and analyzed for fifteen pesticides Houda Berrada, Mónica Fernández F o among those recommended for their pest treatment. A conventional multi-residue
	r r
	P P
	e e r e e r
	R e v i e w R e v i e w
	O n O n
	l y l y
		2

1 Exposure * , Maria José Ruiz, Juan Carlos Moltó, Jordi Mañes Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain

Table 1 .

 1 Limit of quantifications (LOQs) and Maximum Residue Limits (MRLs) set by the European Union (EU), Food and Agricultural Organisation (FAO) and Spanish government for selected pesticides in the studied commodities.

	MRLs (mg kg -1 )

1 MRL for tangerine, OPPs: organophosphorous

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

Acknowledgements

This work has been financially supported by the Spanish Ministry of Science and Technology together with the European Regional Developments Funds (ERDF) (project No. AGL2003-01407).


Hez ; Hird

James Chisholm

Ana ; Sánchez

Marta Hernandez

Robert Goodier

Katrin Schneede

Claudia Boltz

Bert Popping

The effect of

Keywords: Animal feedingstuffs, Animal products -meat, Canned foods

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

There are many reports outlining the detection of meat species in food in the literature. From these reports, DNA appears to be the analyte of choice and real-time polymerase chain reaction (PCR) the most appropriate analytical tool. However, there are few reports examining the design and optimisation of real-time PCR assays, although many studies cite various significant parameters that must be considered during the design process for efficient amplification and therefore sensitive assays. Amplification efficiency is a measure of how close the amplification rates are to the theoretical doubling of template DNA with each cycle. Amplification rates are affected by many factors amongst which include, the number of target molecules, the sequence of the target, the buffer and thermostable polymerase, the cycling conditions, primer sequence and the presence of inhibitors in the sample. One of the most cited parameters, from the work of Meyer et al., (1993), is amplicon size since this group reported that the average size of DNA fragments following heat and pressure processing was approximately 300 base pairs. Consequently, most systems for the detection of DNA from highly processed matrices used amplicons of approximately 300 base pairs [START_REF] Tartaglia | Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials[END_REF][START_REF] Frezza | A competitive polymerase chain reaction-based approach for the identification and semiquantification of mitochondrial DNA in differently heat-treated bovine meat and bone meal[END_REF] Deleted: It has long been known that heat and pressure processing fragments DNA and that shorter amplicons allow greater sensitivity of PCR based assays (Meyer Candrian and Luthy 1993). [START_REF] Laube | Methods for the detection of beef and pig in foods using real-time polymerase chain reaction[END_REF] studied the effect of heat processing on the detection limit of a PCR based system using universal primers. This study used comparatively gentle cooking regimes and found that there was no effect of heating to 65 o C, 70 o C or 75 o C for 30 minutes on the detection of meat in mixtures of meat. More stringent cooking processes were analysed for their effect on the detection of cow in a variety of meat matrices by Brodman and Moor (2003). This study found that canning reduced the sensitivity of the assay ten fold, although the milder cooking regimes did not appear to affect sensitivity. ¶ The advent of real-time PCR has allowed the development of assays which utilise very small amplicons and the size of amplicon has, in some studies, appeared to override all other considerations, compromising assay specificity in some instances (Brodman and Moor 2003;[START_REF] Dooley | Detection of meat species using TaqMan real-time PCR assays[END_REF][START_REF] Mendoza-Romero | Real-time PCR detection of ruminant DNA[END_REF]).

Brodman and Moor (2003) designed assays for the smallest possible amplicons to ensure amplification of highly processed feeds. However using amplicons of 66 and 76 base pairs there was only intermittent detection of samples of cow bone meal.

These primers were also shown to cross amplify red and roe deer, moose, sheep and pig DNA. Similarly ruminant specific assays, using an amplicon of 88 base pairs, were shown to cross amplify with the non-ruminants pig, horse, chicken and turkey [START_REF] Mendoza-Romero | Real-time PCR detection of ruminant DNA[END_REF]). The interpretation of assays which cross amplify is problematic since it is not clear whether there has been detection of the target or the cross amplifying species.

The objective of the study described here was to extend the current knowledge for the design and optimisation of real-time PCR assays for the detection of meats in food products. We designed experiments to elucidate the effect of amplicon size on the sensitivity of real-time PCR assays when applied to heat and pressure processed matrices. We designed real-time PCR assays for turkey, where one primer remained constant and was paired with a series of alternative primers to give amplicons of increasing size. This primer series was used on raw turkey template DNA to determine the rate of amplification. We went on to study the effect of processing on amplification rates using preparations of meat which had been boiled, baked, canned or autoclaved. Finally we applied the turkey primer series to the detection of turkey in poultry meal to illustrate the effect of amplicon size on the detection of highly processed samples. The data presented here should allow a greater flexibility during the design of real-time PCR assays, leading to protocols for the detection of meats in food products with increased specificity. ) containing 80 ng/ml proteinase K (Sigma). After 2 hours the samples were vortexed for 10 seconds and centrifuged for 10 min at 4000 g. 1 ml of the supernatant was combined with 800 µl chloroform, vortexed briefly and centrifuged for 10 min at 14000 g. 500 µl of the aqueous phase was combined with 1 ml Wizard DNA resin and incubated for 5 min at room temperature. The DNA was then purified exactly according to the manufacturers' instructions and eluted with 100 µl Tris EDTA buffer previously heated to 65 o C.

Design of TaqMan  primer and probe sets

The primers and probe sets were designed using the mitochondrial cytochrome b gene for turkey (GenBank Accession L08381) using Primer Express  software (version 1.5). The primers were expected to have the following specific qualities: to produce an amplicon at target sizes; to have the 3' end of the primer as close as possible to the probe without overlapping it; to have a T m between 58 and 60 ºC; to contain 20-80%

G or C, to be between 9 and 40 bases long, and to have a maximum of 2 G or C bases within the last 5 residues of the 3' end. PrimerExpress  design parameters were adapted to achieve primer design. The probe was designed to have a T m 10 ºC higher than that of the primers, to contain between 20 and 80% G or C, to be between 9 and 40 bases long, have a T, C or A at the 5' end and to contain more C's than G's (Oehlmann 1999). Additionally primers were designed to be mismatched at the 3' position to negate spurious amplification with other species of farm animals and humans [START_REF] Hird | Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green[END_REF]. The turkey primers were truncated at the 5' end until specific [START_REF] Hird | Deleted: roasted Deleted: 5% admixture of all species Deleted: 312 Deleted: efficiencies Deleted: are Deleted: Page Break polymerase chain reaction[END_REF]).

Probes were 5' labelled with 6carboxyfluorescein (FAM) and 3' labelled with the fluorescent quencher dye 6carboxytetramethylrhodamine (TAMRA). Primers and probes were purchased from Applied Biosystems, UK (Table 1).

TaqMan  assay conditions

Each real-time PCR reaction contained 1 × TaqMan TM Buffer A, 25 mM MgCl 2 solution, 0.625 units AmpliTaq Gold ® DNA Polymerase (Applied Biosystems) and 0.5 µM of each deoxynucleotide triphosphate (Sigma) in 25 µL. Primers were used at 900 nM and probes at 200 nM. DNA template was diluted in water and 5 µL of either DNA or water, as a negative control, was added per reaction. Reactions were assembled in Axygen thin walled 96 well plates with optical caps and run on ABI Prism 7700 or 7900 sequence detection systems with the following thermal cycling protocol: 50 ºC for 2 min, 95 ºC for 10 min followed by 40 cycles of 95 ºC for 15 s and 60 ºC for 1 min.

Results and Discussion

The effect of amplicon length on amplification rates

It is known that food heat and pressure processing can result in DNA fragmentation which can, in turn, have implications on the sensitivity of DNA based analyses. We designed real-time PCR primers and probe systems that would enable us to determine the degree of DNA fragmentation after processing, by using amplicons of different lengths. The reverse primer remained constant (anchored in position) and was paired with a series of 8 alternative forward primers, giving amplicons of increasing size (81,116,240,317,351,427,470 and 565 base pairs) (Figure 1).

[Figure 1 Primers were identified as close as possible to target sizes, for example amplicon length of 120 base pairs was not possible for this region of turkey sequence and therefore a primer was designed which would give an amplicon as close to 120 base pairs as possible, therefore the design of the primers was largely dependant upon the sequence. Additionally, the Primer Express design parameters were relaxed for amplicons above 150 base pairs to allow the design of primers giving amplicons larger than 150 base pairs: the usual upper limit for real-time amplicons (Table 1).

[Table 1 to go about here]

These primer sets were then used to investigate the effect of amplicon length on amplification rates using raw template DNA (Figure 2). An increase in C T value (threshold cycle), indicating a reduction in amplification rate was observed with increasing amplicon size for the turkey primer series. Amplicons up to 240 base pairs had similar amplification rates, whereas for amplicon sizes above 240 base pairs the increases in C T values were more marked, particularly between amplicons of 427 and 565 base pairs, where there was an increase of 5 C T .

The results from these experiments are in accordance with the technical information supplied by Applied Biosystems. The reactions of Taq polymerase are catalysed by magnesium and the enzyme has a large tolerance for magnesium concentration with small amplicons. Amplicon sizes below 150 base pairs are recommended since the magnesium concentration can remain constant, however the data presented here suggests that amplicons above 150 base pairs can also support efficient amplification, since there was a rise of only 1.5 C T when the amplicon size was increased to over 300 bp. These data support the view that for abundant template molecules amplicon size should be kept below 150 base pairs, however for sequences which prove difficult for primer and probe design, amplicon sizes above 150 base pairs may also support efficient amplification.

Figure 2 to go about here

Assessment of the degree of DNA fragmentation during processing

Deleted: Primers were identified to give amplicons of target sizes (70, 120, 350 and 500 base pairs) although the actual lengths of the amplicons was dependant upon the sequence. Design parameters of Primer Express, the package used to design TaqMan primer and probe systems, were relaxed for amplicons above 150 base pairs: the usual upper limit for real-time amplicons Deleted: ¶ Deleted: These primer sets were then used to investigate the effect of amplicon length on amplification efficiency using raw template DNA (Figure 2). To investigate the effect of heat and pressure processing on the availability of template molecules, we used the turkey primer series to amplify DNA which had been extracted from turkey muscle which had been subjected to the following 6 different cooking conditions: light boiling, heavy boiling, light baking, heavy baking, canning and autoclaving. The DNA was extracted from duplicate samples, the DNA concentration adjusted to 17ng/µl and then 85ng/reaction analysed using the real-time PCR primer series for turkey (Figure 3).

[Figure 3 to go about here]

We found that, in general terms, there was no difference in the amplification rates of DNA extracted from samples which had been boiled or baked, in comparison to DNA from raw samples for amplicons of 81, 116, 351 or 565 base pairs. This indicated that the fragmentation of the DNA had not affected amplification for amplicons up to 565 base pairs since the amplification rates were roughly similar to those of raw samples, regardless of the boiling or baking conditions. This also indicated that amplicons of 351 and 565 base pairs are able to efficiently amplify template DNA extracted from boiled or baked samples.

There was a statistically significant increase in the C T value returned for the canned and autoclaved samples for all sizes of amplicons. This difference became more pronounced with increasing amplicon size, where amplification of DNA extracted from the autoclaved sample returned a value 1.41 C T greater than amplification of the DNA extracted from the raw sample with the 81 base pair primer set compared to an increase of 10 C T for the 351 base pair primer set and a complete failure for the 565 base pair primer set. A similar trend was observed for the canned samples, where amplification rates decreased significantly with increasing amplicon length.

Fragmentation of DNA was only observed when the meats were canned or autoclaved, with little or no fragmentation observed when the meats were cooked in the absence of pressure. The effect of DNA fragmentation on PCR based systems is two fold. Firstly, when DNA fragments, the available template molecules are reduced when a template molecule is cleaved. Secondly, small DNA fragments are not Deleted: The current method of choice for the identification of cooked or canned meat or fish usually involves the use of PCR. The degree of processing of food, and therefore the degree of DNA fragmentation, has a profound effect on the accuracy and robustness of the PCR.

There is little information on the effect of heat and pressure on DNA fragmentation. It has been determined that following autoclaving the average fragment size of pig DNA was reduced to 300 bp (Meyer Candrian and Luthy 1993), however the exact effects of processes, normally encountered in the production of commercial products, on the detection of DNA are unknown. ¶ Deleted: trends we had previously observed were again repeated for these data sets. The amplification efficiencies of the cow primer series were similar across the raw, boiled and roasted samples, however the efficiencies were lower for the canned samples. Similar data was observed for the turkey primer series where it was only in the canned samples where the effects of processing could be seen. In this study the effect of DNA extractability from heat and pressure processed matrices on amplification rates may have contributed to the increased C T values observed in the canned and autoclaved samples although this is an unlikely cause for the increased C T value since there was little effect of canning and autoclaving on the amplification rate of the 81 base pair amplicon. Had DNA extractability from the canned and autoclaved samples been a major contributor to the increased C T value returned for amplicons of 351 and 565 base pairs, we would have also observed a significant increase in the C T values returned for amplicons of 81 and 116 base pairs. These data indicated that canning and autoclaving affected the fragmentation of the DNA, rather than the DNA extractability.

To further illustrate the implication of amplicon size on the detection of meats in a mixture we used the turkey primer series to analyse a sample of poultry meal, containing approximately 15% turkey. Meal of this type is heated to 100 o C for 60 minutes, in the absence of pressure, where the final temperature reached is 120 o C resulting in fragmented DNA. Since only a proportion of the sample was derived from turkey the raw data is to show the effect of amplicon size on rates of amplification and is not for direct comparison. Nevertheless, DNA fragmentation was evident in this sample since the C T value differential between the raw and processed samples increased with increasing amplicon size indicating fragmentation of target DNA (Figure 4). However it should be noted that an amplicon of 351 base pairs was still detectable with a C T value of 33.87 in this processed sample.

Figure 4 to go about here

Conclusions

Overall this study showed that the degree of DNA fragmentation correlated with the degree of heat and pressure processing and that this could be assessed using multi- or boiled and turkey DNA extracted from autoclaved samples was easily detectable even using amplification systems spanning 351 base pairs. This study proved that amplification rates were not compromised for amplicons of more than 150 base pairs and that in future, the design of real-time PCR assays could afford greater focus on achieving specificity using a less stringent approach to limiting amplicon size. These primer sets were then used to investigate the effect of amplicon length on amplification efficiency using raw template DNA (Figure 2). An increase in threshold cycle (C T ) value, indicating a reduction in amplification efficiency was observed with increasing amplicon size for all species. For the chicken and cow primer series, amplicons up to 353 base pairs (primer sets 1, 2, & 3) had similar efficiencies, whereas amplicon sizes above 353 base pairs (primer set 4) were found to have statistically significant differences and the increase in C T values became marked. A similar pattern was observed in the data for the turkey and pig primer series where amplicons of approximately 70 and 120 base pairs (primer sets 1 & 2) had similar efficiencies, whereas amplicons with sizes of 312 base pairs for turkey and 350 base pairs for pig, and above (primer sets 3 & 4) were found to have statistically significant increases in C T value.

bp Amplicon

[Figure 2 to go about here]

The reactions of Taq polymerase are catalysed by magnesium, amplicon sizes below 150 base pairs are recommended since the magnesium concentration should remain constant for small amplicons. The data presented here suggests that larger amplicons may support efficient amplification. Any reduction in efficiency is unlikely to be critical as the sensitivity of these types of assays is rarely a limiting factor and may be negated or reduced by the optimisation of magnesium concentration.

There were two broad trends in the data from the four primer series: the cow and chicken series appeared to amplify with similar efficiencies as did the turkey and pig series. We used a primer series displaying each trend to examine the effect of diluting the target species in a meat background. Mixtures of cow in pig or turkey in pig were prepared at 95, 90, 80, 50, 20, 10 and 5% [w/w] cow or turkey. The DNA was extracted from duplicate samples and then analysed using the real-time PCR primer series for cow and turkey (Figure 3, 5%, 50% and 95% data shown). It was found that the trends in amplification efficiency, decreasing with amplicon size, were again displayed in these data sets. For cow, amplification efficiencies were similar for amplicons of 62, 109 and These data indicate that the fragmentation of DNA for the raw, boiled and roasted samples was similar and that there was significantly more fragmentation following canning, which was evident, even using the smallest primer sets, with a relatively small amplicon size of below 80 base pairs for both turkey and cow.

  Key words: real-time PCR, heat, pressure, processing, meat, DNA, detection ¶

  the need for gel visualisation meant that the amplicons were usually over 130 base pairs.

  DNA was extracted from meat mixtures using a modified version of the GenElute™ Mammalian Genomic DNA Extraction Kit (Sigma). Briefly, 2 g of tissue was finely minced with a scalpel blade, prior to incubation at 65 °C in 8 ml CTAB buffer (0.055 M CTAB, 1.4 M NaCl, 0.1 M Tris and 0.02 M EDTA, pH 8.0) containing 50 µg/ml proteinase K (Sigma). After 1 hour, the samples were cooled briefly and 400 µL of the supernatant used to extract the DNA exactly according to the manufacturer's instructions. The final elution step was repeated once and the eluates pooled. DNA was stored short term at 4 °C or long term at -20 o C. ¶ ¶ Deleted: DNA extraction from poultry and meal samples ¶ DNA was extracted from 5 g samples of meal using the Nucleospin DNA extraction kit available from Nucleon exactly according to manufacturer'

  to go about here] Deleted: chicken, Deleted: and cow Deleted: 1:4 DNA:

  We used 2 sets of meat mixtures: cow in pig and turkey in pig, at different concentrations(95, 90, 80, 50, 20, 10 and 5% [w/w]).

  These data indicate that the fragmentation of DNA for the raw, boiled and roasted samples was similar and that Deleted: The mixtures analysed during the course of this study were all prepared same efficiency as larger template molecules by some DNA extraction systems, particularly for extraction techniques which use DNA precipitation, since small fragments of DNA do not precipitate as easily as large fragments.

  DNA fragmentation was minimal for samples which had been baked

Figure 1 .

 1 Figure 1. Strategy for determining the degree of DNA fragmentation following heat and pressure processing.

Figure 2 .

 2 Figure 2. Effect of amplicon size on amplification rates of turkey primer series using raw turkey DNA as template average and standard deviations shown

  353 bp (primer set 1, 2, & 3), whereas turkey amplicons of 76 and 111 base pairs (primer set 1 & 2) had similar amplification efficiencies and larger amplicons had reduced of the percentage of the target meat species. It appeared that the presence of non-target DNA template had no effect on the amplification efficiencies of both primer series and that at the 5% level the detection of raw template DNA was achieved equally well by amplification systems using amplicons up to 350 and 111 base pairs for cow and turkey respectively.[Figure3to go about here]

  The inclusion of canned samples was instrumental in the assessment of processing conditions since we have previously found that autoclaving meat does not mimic the canning process and has little effect on the fragmentation of DNA in comparison.

Table 1 . Sequence and amplicon length of primer and probe sets used in this study.

 1

		... [5]
		Ct
	Raw A	13.24
		13.33
		13.53
	Raw B	12.82
		12.9
		13.42
	56A1	20.19
		20.28
		20.04
	56A2	20.58
		20.21
		20.07
	56B1	20.19

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


Rossella Riccio

Ettore Marco Trevisan

Keywords: Pesticide residues, chlorpyrifos-methyl chlorpyrifos-methyl residue, fruit cuticle, photodegradation .manuscriptcentral

Effect of surface waxes on the persistence of chlorpyrifos-methyl in apples, strawberries, and grapefruits

is termed the intracuticular waxes, whilst the thin film on the surface of the cutin matrix comprises epicuticular waxes [START_REF] Jetter | Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development[END_REF]. The latter forms the outermost boundary layer of the plant, representing an interface between the plant and the environment [START_REF] Koch | Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM)[END_REF] 1985).

These functionalities are due to specific chemical and physical properties. The epicuticalar waxes are a mixture of long chain compounds, such as hydrocarbons, ketones, alcohols, aldehydes, esters, and free and esterified fatty acid and related compounds [START_REF] El-Otmani | Development and topophysical effects on the nalkanes of Valencia orange fruit epicuticular wax[END_REF]. The composition and quantity of waxes vary between different vegetable species; within the same species there are also some variations in surface area and wax deposits, depending upon maturation, growth stage, environmental conditions etc. [START_REF] Angioni | Effect of the epicuticular waxes of fruits and vegetables on the photodegradation of rotenone[END_REF] ). In the epicuticular wax of pineapple, navel oranges and dancy tangerine, paraffins accounted for 30-44% and 6-12% of the wax of immature and mature fruit, respectively [START_REF] El-Otmani | Development and topophysical effects on the nalkanes of Valencia orange fruit epicuticular wax[END_REF], and during the fruit senescence a shift of nalkanes from higher to lower molecular weight components was observed [START_REF] El-Otmani | Fruit development and growth regulator effects on normal alkanes of Washinton navel orange fruit epicuticular wax[END_REF]. Physical description using microscope detection indicates a great micromorphological diversity of epicuticular waxes. Waxes may be developed as continuous layers, crusts or solitary crystalloids of varying thicknesses; the crystalloids are of differing shape and size and may appear in specific arrangements or combinations [START_REF] Barthlot | Classification and terminology of plant epicuticular waxes[END_REF]. The morphological study of epicuticular waxes was pioneered by de Bary in 1871. A more recent and refined classification, based on scanning electron microscopy (SEM) analyses of 13000 species, classified waxes into 23 types [START_REF] Barthlot | Classification and terminology of plant epicuticular waxes[END_REF].

When a pesticide reaches fruit, either via agricultural application or deposition from the atmosphere, it can interact with the cuticle and the epicutical waxes through retention and/or absorption. Such processes tend to affect the fate of the pesticide and its efficacy as well as its persistence in the fruit [START_REF] Kirkwood | Recent developments in our understanding of the plant cuticle as a barrier to the foliar uptake of pesticide[END_REF]. The higher the pesticide persistence in the fruit, the higher the potential risk to the consumer via food intake. For this reason, studies of the effects of the cuticle and waxes on pesticide persistence can provide important data for the refinement of consumer risk assessment, and inform possible options for the mitigation of pesticide residues in food. Recent studies have indicated that the diffusion pathway of solutes, such as pesticides, within the transport limiting barrier of the cuticle has a large tortuosity which is reduced or removed upon the removal of waxes [START_REF] Schönherr | Modelling penetration of plant cuticles by crop protection agents and effects of adjuvants on their rates of penetration[END_REF][START_REF] Baur | Mobilities of organic compounds in plant cuticles as affected by structure and molare volumes of chemical and plant species[END_REF].

The main aim of this paper was to evaluate the effects of the epicuticular waxes and cuticles of grapefruits, apples and strawberries on the penetration and dissipation of the pesticide chlorpyrifosmethyl. Chlorpyrifos-methyl (figure 1 A) is an organophosphate (OP) insecticide, non-systemic, poorly soluble in water, with a low persistence and low mammalian toxicity (WHO/FAO, internet accessed 13 Nov 2005), in Europe it is widely used in the treatment of fruits, as diverse as soft, citrus, and pome fruits. The European Maximum Residues Limits (MRLs) for chlorpyrifosmethyl for strawberry, apple and grapefruit are set at 0.5, 0.5, and 0.05 µg mg -1 respectively (EUROPA-Plant Health-EU MRLs, internet accessed 26 Jan 2006).

[insert figure 1]

Materials and methods

Fruit and chemicals

Apples, strawberries and grapefruits cultivated on an organic farm were purchased from a supermarket.

Analytical standards of chlorpyrifos-methyl (99.4% purity) and 3,5,6-trychloro-2 pyridinol (TCP) (99.5% purity) were purchased from Labor Dr. Ehrenstorfer and Sigma respectively (figure 1 A-B). Chloroform, ethanol, dichloromethane, hexane and acetone were HPLC grade solvents (CarloErba). Water was doubly distilled and purified with a MilliQ apparatus (Millipore, Milan) before use. Glutheraldehyde (purity 25%) was obtained from Merck. Diatomaceous earth was obtained from Sigma-Aldrich. Stock standard solution of chlorpyrifos-methyl and TCP (1000 mg L - 1 ) were prepared in acetone. More dilute solutions were prepared by dilution of the stock solution

Procedure of extraction of fruit waxes

Wax extraction was performed following the method of McDonald (1993). About 2500 g of fruits (apple, grapefruit and strawberry) were weighed then immersed in 2300 mL of chloroform for 2 min. The method was modified by: a) using warm chloroform (45-50 °C), and b) using an ultrasonic apparatus (190W, 49 kHz). The fruit was then rinsed with clean chloroform. The final volume of chloroform extract was measured and a 10 mL portion of the extract was evaporated to dryness. Fruit at the same stage of ripening were measured with a calibre and their surface area was determined. The quantity of wax on the fruit surface (µg cm -2 ) was calculated from these data.

Scanning electron microscopy (SEM) observation.

Two rind samples (1 x 1 cm) were excised with a scalpel from the equatorial zone of fruit with wax and fruit from which the wax had been removed. Samples were immediately fixed with 3% gluteraldehyde in phosphate buffer (pH 7.2) and cold-stored (4°C) prior to further preparation.

Fixed tissue was then rinsed three times in phosphate buffer (pH 7.2) followed by three washes in deionised water. Dehydration was performed using increasing concentrations of ethanol (20, 50, 70, 80, 95 and 3 x 100 % ethanol). Samples were retained for 20 min at each ethanol concentration step [START_REF] Schirra | Epicuticular changes and storage potential of cactus pear [Opuntia ficus-indica Miller (L.)] fruit following gibberellic acid preharvest sprays and postharvest heat treatments[END_REF] An aliquot (1 mL) of the chloroform wax extract was dried under a stream of nitrogen and redissolved in 1 mL of hexane. Spectra of waxes were recorded using a UV-Visible Recording Spectrophotometer UV-160 (Shimadzu) (λ from 230nm to 800nm).

Penetration studies

The overall scheme of sample treatment applied in the penetration studies with fruit samples is shown in Figure 2. Fruit, with and without wax, were weighed (average weights of apple, grapefruit, and strawberry samples are 222.86± 11.27g, 231.12± 21.12 g, and 8.34± 0.66g respectively) and immersed for 15 secs in an aqueous suspension of pesticide (1 mg L -1 ). To avoid losses, fruits were kept free of contact with surfaces, and left to dry at room temperature and in darkness. The treated fruit were divided into three aliquots. The first aliquot of fruit was weighed, milled, and extracted using diatomaceous earth in order to determine the amount of pesticide in the fortified samples 1 hr after pesticide treatment. A second aliquot was weighed, milled, and extracted 24 hours after application. After 24 hr from pesticide treatment, the third aliquot of fruit was used to obtain four fractions representing different depths, in order to perform the pesticide penetration study. The four fractions obtained were as follows:

1. Fruit was weighed and rinsed twice with 10 mL of dichloromethane. The rinses were collected and labelled fraction 1.

2. Fruits was left to dry and then peeled by a very sharpened potato peeler to avoid squashing or destroying the fruits. The thickness of the peel was approximately 1mm. The peel was weighed, milled and extracted using the diatomaceous earth. The extract was labelled fraction 2.

3. The remaining fruit was rinsed twice with 10 mL of dichloromethane. The rinses were collected and labelled fraction 3.

4. The sample was left to dry, milled and extracted with dichloromethane using diatomaceous earth; this extract was labelled fraction 4. The experiment was carried out in triplicate. Untreated fruit was used as a control.

Concentration of fractions 1 and 3. Fractions 1 and 3 were concentrated by evaporating nearly to dryness at 30 °C using a vacuum rotary evaporator Heidolph (Selecta, Düsseldorf, Germany). The residue was dissolved in 10 mL of dichloromethane (to transfer it from a round bottle into a 10mL vial), evaporated to dryness under a gentle stream of nitrogen at room temperature , dissolved in 1 mL of acetone and analysed using GC-MS.

Extraction of pesticide residues from fruit. Fruit samples (whole, peel and pulp) were milled and 5 g of the milled sample was mixed with 6.5 g of diatomaceous earth. Glass columns were filled with the mix and, after 10 minutes, were eluted with 100 mL of dichloromethane. The extract was concentrated and evaporated nearly to dryness at 30 °C using a vacuum rotary evaporator. The residue was dissolved in 10 mL of dichloromethane (to transfer it from a round bottle into a 10 mL vial), evaporated to dryness under a gentle stream of nitrogen at room temperature , dissolved in 1 mL of acetone and analysed using GC-MS. To calculate the recovery of extraction, 5 g of untreated milled apples (peel, pulp and whole fruit) were spiked with 1 mL of a 0.5 mg L -1 solution of pesticide, after which the samples were extracted as described and analysed using GC-MS.

[Insert figure 2 An aliquot of the chloroform wax mixture (the volume of which was calculated so to give concentrations in the Petri dishes identical to concentrations found on the fruit surface) and 0.5 mL of a 5 mg L -1 solution of pesticide in acetone were poured into Petri dishes (5 cm diameter, surface area approximately 20 cm 2 ). The solvents were allowed to evaporate at room temperature, leaving a uniform film on the surface of the Petri dish. The dishes were placed into the Atlas SunTest, and were exposed for 180 min at 450 W m -1 and 40°C. The residues on the plates were then dissolved in 5 mL of acetone and analysed directly to determine the chlorpyrifos-methyl residue. In order to determine TCP residue, 200 µL of sample was derivatized by adding 20 µL of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) and heating in an oven for 60 min at 60°C [START_REF] Rudel | Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrine-disrupting compounds in indoor air and dust[END_REF]. In all experiments, non-irradiated control samples were incubated in darkness at room temperature and in an oven at 40 °C. Each experiment was performed in triplicate.

A schematic diagram of the spiking and sampling procedure is shown in figure 3.

Kinetics. The kinetics of loss of the pesticide were followed using GC-MS analysis. The rate constant of the disappearance of the pesticide was calculated as a pseudo first-order rate constant by

C = C 0 e -kt
[Insert figure 3 here]

Apparatus and chromatography

GC-MS analysis was performed using an HP 6890 series gas chromatograph, equipped with an HP 5973 Mass Selective Detector and an HP 7683 series Injector (Hewlett-Packard). Chromatographic separation was performed on a ZB5 (Zebron) capillary column (30 m x 0.25 mm i.d.; 0.25 µm film thickness). The carrier gas was helium, at a flow-rate of 1 mL min -1 . The injector temperature was set at 250°C, in splitless mode. The oven temperature was programmed as follows: 90 °C for 1 min, increasing at 10°C min -1 to 140 °C, increasing at 5 °C min -1 to 270 °C with a final hold for 10 min at 270 °C. The injection volume was 1 µL. Electron impact of 1200 eV was used for the ionisation of compounds. Analyses were carried out in select ion monitoring mode using the single ions: m/z 286 and 254 for chlorpyrifos-methyl and derivatizated TCP, respectively.

Results and discussion

Extraction of waxes

The surface of grapefruit before and after chloroform treatment was examined by SEM to evaluate the extent of wax extraction. The peel of untreated fruit exhibited areas with wax layers in the form of rough, irregular platelets (figure 4-A). Wax platelets were still evident in the peel of chloroform treated grapefruit (figure 4-B). To improve wax extraction, more vigorous methods were applied:

hot chloroform (45-50°C) (figure 4-C), and using the ultrasonic apparatus (figure 4-D).Complete extraction of waxes from the grapefruit surface was achieved using ultrasound. The surface of grapefruit free of wax is shown in figure 4-D. Wax extraction was performed using this procedure for all fruit types considered in the study, including strawberries, for which the method was found to be effective but not harmful to the delicate surface of the fruit.

The amount of wax extracted from strawberry, grapefruit and apple was 22, 130 and 516 µg/cm 2 respectively (table 1). The amount of wax extracted from the apple was lower than that obtained in previous work [START_REF] Angioni | Effect of the epicuticular waxes of fruits and vegetables on the photodegradation of rotenone[END_REF]. This difference could be attributed to differences in the variety, maturation and growth stage of the fruit used in the study, and the environmental conditions to which it had been subjected.

[Insert figure 4 here]

[Insert table 1 here]

Dose-response curves and limits of quantification of chlorpyrifos-methyl and TCP

Quantification of chlorpyrifos-methyl and derivatizated TCP was performed by external standard procedure. 10 concentrations of pesticide and derivatizated metabolite (ranged from 0.001 µg mL -1 to 10 µg mL -1 , and from 0.006 µg mL -1 to 10 µg mL -1 , respectively) were injected 3 times to GC-MS. Chlorpyrifos-methyl showed a linear response with a correlation coefficient of 0.99; the repeatability value, expressed as the relative standard deviation (R.D.S.) of ten determinations, was 2.8%; the limit of quantification (LOQ) was 0.001 µg g -1 . Derivatizated TCP showed a linear coefficient of 0.99; the repeatability value, expressed as the R.D.S. of 10 determinations, was 5%;

the LOQ was 0.005 µg g -1 .

Recovery of the pesticide extraction method

Apples fortified with 0.1 µg g -1 of pesticide were extracted with diatomaceous earth. Recoveries were satisfactory for all samples of whole apple, apple peel, and apple pulp (95, 91, and 98% respectively). The R.D.S. for 3 replicates always was less than 9%.

Penetration studies

No pesticide decay was observed in fruit samples analysed 1 hour and 24 hours after pesticide treatment (tables 2,3,4).

The concentration of pesticide residues in whole fruit 24 hours after treatment were 0.022, 0.021 and 0.012 µg g -1 for strawberry, apple and grapefruit respectively (tables 2,3,4). This indicates variation in the penetration capacities of the same OP pesticide between several fruit species with different wax structures.

Residues of chlorpyrifos-methyl were below the LOQ in the third and fourth fractions of grapefruit, both with and without wax. This indicates that penetration into the grapefruit is low, probably due to the impermeable structure of its peel. The amount of residues in the peel samples from which the wax had been removed were higher than those with the wax intact (0.047 and 0.013 µg g -1 , respectively). These data indicate the low permeability of the waxes to chlorpyrifos-methyl (table 2).

24 hours after treatment, chlorpyrifos-methyl residues were detected in the first, second and third fractions of apple, but not in the apple pulp (table 3). In studies of the penetration of other OPs, residues of fenitrothion, malathion and ethiofencarb were detected in the internal fractions of apple one day after the treatment, whilst diclofluanid did not penetrate into the fruit [START_REF] Perez Clavijo | Decay study of pesticide residues in apple samples[END_REF]. As for the grapefruit samples, the amount of chlorpyrifos-methyl determined in samples of apple from which the wax had been removed was significantly higher than from samples with wax intact (0.129 and 0.095µg g -1 respectively). As shown in table 4, pesticide residues were detected in all fractions of strawberry, which indicates that penetration into the fruit is high, due to the permeable structure of the skin. In particular, the high penetration and accumulation of pesticide into the pulp of chloroform treated strawberries was observed.

[Insert table 2, 3, and4 here] The total amount of residue [measured residue (µg g -1 ) x media weight of fruit (g)] detected in the whole fruit and in the four fractions is shown in figure 5.

The total amount of pesticide residues was greater in fruit samples from which the wax was removed compared to those in which the wax was intact (figure 5 A,B,C). These results confirm the protective role of waxes against the penetration of pesticides [START_REF] El-Otmani | Fruit development and growth regulator effects on normal alkanes of Washinton navel orange fruit epicuticular wax[END_REF][START_REF] Kunst | Biosynthesis and secretion of plants cuticular waxes[END_REF] and other aerial contaminants [START_REF] Gòrna-Binkul | Modeling of the sorption and diffusion process of volatile organic air pollutants in grape fruits[END_REF] present in the atmosphere which can be taken up by plants, accumulate, and ultimately enter the human food chain, thus becoming important in terms of risk to human health.

The amount of pesticide residue detected in the waxes and in the peel was approximately100% and 91% of the total amount detected in grapefruit and apple samples respectively, both in samples with and without wax (figure 5 A and B, respectively). In strawberry samples, the total amount of residue was distributed equally between the external and internal fractions (figure 5 C).

[Insert figure 5 During the photodegradation tests reported herein, chlorpyrifos-methyl was exposed to light in the presence of a film of wax equal to that found the surfaces of the fruit species considered. The degradation rate of chlorpyrifos-methyl was calculated using pseudo first order kinetics. Table 5 shows half-lives (t 1/2 ) and correlation coefficients (r) of chlorpyrifos-methyl. The half-life of pesticide irradiated in the absence of waxes was 9.6min. The decay rate of chlorpyrifos methyl was slightly reduced by strawberry wax (t 1/2 =26 min.), and was further reduced in the presence of grapefruit wax ( t 1/2 =34 min.) and apple wax ( t 1/2 =83 min.).

During irradiation inside the Suntest apparatus, the temperature was approximately 40 °C. To investigate if this temperature influenced the persistence of the pesticide, non-irradiated control samples were held in darkness at room temperature (approximately 25 °C) and at 40 °C. No temperature effect was observed. The t 1/2 of chlorpyrifos-methyl measured on plates held in darkness at room temperature and at 40°C in the presence of strawberry waxes was 277 and 250 min, respectively. In the presence of grapefruit and apple waxes, values of t 1/2 were 385 and 577, 577 and 650 min respectively.

The presence of wax on the fruit surface affects the photodegradation of chlorpyrifos-methyl. The rate of photodegradation decreases in the presence of fruit wax. We can hypothesize that the radiation is adsorbed by wax, and only the λ that penetrate the waxes can interact with the pesticide molecule. This decrease in radiation intensity results in a decrease in the rate of photodegradation [START_REF] Pirisi | Influence of Epicuticular Waxes on the Photolysis of Pirimicarb in the Solid Phase[END_REF].

The major metabolite resulting from irradiation of chlorpyrifos-methyl is TCP (Figure 1B). This metabolite is also a degradation product of chlorpyrifos and triclopyr [START_REF] Morgan | Exposures of preschool children to chlorpyrifos and its degradation product 3,5,6-trichloro-2-pyridinol in their everyday environments[END_REF]. For this reason, control plates were assessed for the absence of TCP. During the photo-degradation experiments TCP formation was monitored. Some TCP formation was observed, and TCP formed via photolytic degradation was less photolytically stable than chlorpyrifos-methyl itself (table 5).

However, the formation of TCP may not be exclusively due to photolysis . It is possible that the primary mechanism of production of TCP, in some cases, was unrelated to the presence of sunlight. TCP is photolytically unstable and it may not be detected during aqueous photolysis studies. A 100% photo-degradation within 24 hours of an aqueous buffer (pH 8) containing 100,000 mg kg -1 of TCP has been reported. Furthermore, following photolytic exposure of a 1 mg kg -1 aqueous solution of TCP, an estimated half-life of 4.4 min was reported for a water body surface at a latitude of 40° north [START_REF] Ware | Reviews of environmental contamination and toxicology[END_REF].

[Insert table 5 here]

The data reported herein confirm results obtained by [START_REF] Angioni | Effect of the epicuticular waxes of fruits and vegetables on the photodegradation of rotenone[END_REF] who found an increase in the half-life of rotenone in the presence of waxes for irradiated eggplant, although they did not observe interference of waxes with photodegradation. [START_REF] Cabras | include chlorpyrifos, chlorpyrifos-methyl, mancozeb, maneb, and metiram as active substances[END_REF] demonstrated that wax composition, rather than film thickness, was responsible for the increase in half-life of fenthion.

The penetration rate and the dissipation of chlorpyrifos-methyl varied between the fruit species considered. This can be attributed to the chemical and physical characteristics of the waxes. SEM observations of the waxes of strawberry, grapefruit and apple suggest that the crystalloid forms of wax have different structures and shapes (figure 6). Strawberry and grapefruit waxes are organized in a granular structure, whilst the apple waxes had a more tubular structure. The UV spectra of wax extracts in the range of 230-800 nm show a rather similar absorbance for grapefruit and apple with a maximum absorbance at 230 nm, whilst the strawberry spectra show a different profile (data not shown).

[Insert figure 6 2001[START_REF]Food and Veterinary Office -Special Reports: Annual EU-wide Pesticide Residues Monitoring Report[END_REF], 2003, 2004;Annual EU-Wide Pesticide Residues Monitoring Report 2003[START_REF]Food and Veterinary Office -Special Reports: Annual EU-wide Pesticide Residues Monitoring Report[END_REF], 2001, [START_REF] Kirkwood | Recent developments in our understanding of the plant cuticle as a barrier to the foliar uptake of pesticide[END_REF], 1998, 1996;internet accessed 26 Jan 2006). In order to minimise residue levels during food processing, the rate of losses of pesticides should be assessed for the main fruit and vegetable compartments. The results reported in this paper confirm that the epicuticular waxes and cuticle influence pesticide persistence, mainly through protecting the pesticide and its transformation products from photolysis. Most of the pesticide is absorbed and only a small portion passes through the barrier of the cuticle and penetrates into the pulp. However, the extent of these processes are species dependent as the chemical and physical properties of the cuticle vary between species. In the case of this study, strawberry showed no "surface effect" compared to apple and grapefruit.

These studies can provide important information regarding options for the reduction of pesticide residues in food. Through the use of adapted food processing methods, for example, peeling, washing or brushing, residues of chlorpyrifos-methyl and its metabolites in food can be decreased, so meeting specific legislative requirements (e.g. baby food) and specific market requirements, as well as consumer needs.

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 1. A: chlorpyrifos-methyl, B: 3,4,5-trichloro 2-pyridinol (TCP).

  acetone. An aqueous suspension of chlorpyrifos-methyl, used to treat the fruit, was prepared by diluting the standard solution in distilled water, to obtain a concentration of 1 mg L -1 .

  photo-degradation test.

  Photodegradation studies

  here] Conclusion Although chlorpyrifos-methyl residues do not present a risk to the consumer (as demonstrated in the most recent European risk assessments (Commission Directive 2005/72/EC, 2005), some samples of fruit, vegetables and cereals analysed in monitoring studies performed in Italy and Europe indicate the presence of chlorpyrifos-methyl residues (Italian Pesticide Residues Monitoring Report

Table 2 .

 2 Chlorpyrifos-methyl residues on grapefruits with and without wax (µg g -1 ). P.t.:pesticide treatment.

						Deleted: Table 2. Recoveries of
	grapefruit	Wax intact	Wax removed	diatomaceous earth extraction procedure applied to apple samples fortified with
						chlorpyrifos-methyl. a Average and
		untreated	fortified	untreated	fortified	relative standard deviation, n=3. ¶
	whole 1 h after p t	Chlorpyrifos-methyl residue (µg g -1 ) < LOQ 0.012± 0.001	Chlorpyrifos-methyl residue (µg g -1 ) < LOQ 0.014± 0.001	¶	Page Break
	whole 24 h after p t	< LOQ	0.012± 0.001	< LOQ	0.013± 0.001
	fraction 1	< LOQ	0.008± 0.001	< LOQ	0.009± 0.001
	fraction 2	< LOQ	0.013± 0.001	< LOQ	0.047± 0.004
	fraction 3	< LOQ	< LOQ	< LOQ	< LOQ
	fraction 4	< LOQ	< LOQ	< LOQ	< LOQ
						Deleted: 3
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Table 3 .

 3 Chlorpyrifos-methyl residues on apples, with and without wax (µg g -1 ). P.t.: pesticide treatment.

	Deleted: 4

Table 4 .

 4 Chlorpyrifos-methyl residues on strawberries with and without wax (µg g -1 ). P.t.: pesticide treatment.

	Deleted: 5

Table 5 .

 5 Half life (t 1/2 ) and correlation coefficient (r) of chlorpyrifos-methyl and TCP in fruit wax extracts after exposure to irradiation. N.d.: not determinated.

	Deleted: 6

  . After completion in 100% ethanol, samples were critical point dried in a Baltec CPD 030 dryer and placed on aluminium stubs. Gold was sputter-coated in a Pabish Top

	F o r
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	Autocoater SC-20. SEM observations were carried out using a Philips XL30 ESEM microscope at
	20 kV and 5000x and 20000x magnification.
	UV Spectra

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