E Duffy

M J Gibney

The use of a food consumption database with packaging information to estimate exposure to food packaging migrants-expoxidised soya bean oil and styrene monomer

Keywords:

The use of a food consumption database with packaging information to estimate exposure to food packaging migrants-expoxidised soya bean oil

Introduction

There are numerous chemicals used in food contact materials, which includes food packaging materials, that have the potential to migrate. These chemicals include monomers and starting substances, catalysts, additives, solvents and suspension media.

European Commission Directive 2002/72/EC establishes a 'positive list' of approved monomers and starting substances (European Commission 2002), many of which have restrictions placed on their use (e.g. a specific migration limit (SML), or a maximum permitted quantity of the substance in the finished material or article (QM)). There is also an incomplete list of additives used in the manufacture of food contact plastics contained in European Commission Directive 2002/72/EC and subsequent amendments.

It is not a 'positive list', but rather a list of additives approved by the European Commission, Scientific Committee on Food (now the responsibility of the European Food Safety Authority (EFSA)). By the end of 2006, industry has to submit dossiers to EFSA for those plastics additives that have not yet been evaluated at an EU level. After this date the Commission will establish a positive list for plastics additives. EFSA is also responsible for completing exposure assessments in the EU and these assessments are performed on an ad hoc basis using available data either provided by a petitioner or obtained from various sources (European Food Safety Authority 2005a). Exposure assessments comprise one part of the risk assessment process (the other parts include hazard identification, hazard characterisation and risk characterisation). Traditionally, exposure assessments were completed using EU assumptions that 1kg of food packaged in the material of interest is consumed per person per day over a lifetime and the substance of interest migrates into the food at the maximum permitted level. There are ongoing efforts to try to refine this traditional exposure assessment methodology. In recent years there has been a need to complete safety assessments for a number of chemical migrants from food contact materials and exposure assessments have been part of these safety assessments. For example, following reports of the presence of 2isopropylthioxanthone (ITX) in foods packaged in cartons, the European Commission requested that EFSA provide scientific advice with respect to possible health risks associated with this substance by April 2006 (European Food Safety Authority 2005b).

In addition, regarding high levels of epoxidised soybean oil (ESBO) in baby foods, EFSA released an opinion on the exposure assessment of infants (European Food Safety Authority 2004) and adults (European Food Safety Authority 2006). Although exposure assessments feature prominently in the safety assessment of chemical migrants, there is no set protocol on how to complete these exposure assessments and most methodologies are determined by the availability of data.

One data source that can facilitate the exposure assessment of food packaging materials is the use of a food consumption database that also has packaging information. The Irish National Children's Food Survey (NCFS) is the first of this type of database (Irish Universities Nutrition Alliance 2005). In the NCFS information was collected on the type of food consumed and also on the packaging used for this food (e.g. glass, plastic, metal etc.). Therefore, when this data is used in the exposure assessment of packaging migrants in foods it removes the uncertainty regarding the type of food consumed, the amount of food consumed and the packaging used for this food. Because food consumption surveys are not designed to analyse the concentration of a particular migrant in a food, there will still be some uncertainty regarding the presence of the migrant in the food and the level of the migrant in the food when using this data.

However, this data permits the most accurate exposure assessment to packaging migrants currently available for Irish children as it reduces the overall uncertainty in the exposure calculation.

To demonstrate the use of the NCFS for estimating exposure to food packaging migrants two sample migrants were chosen on which to complete exposure assessments: (1) ESBO; and (2) styrene monomer. These sample migrants were chosen because their presence in foods was previously an issue of safety concern (Ministry for Agriculture, Food and Fisheries 1999a;European Food Safety Authority 2004, 2006). This use of food consumption data at an individual level with a fixed value for the concentration of the migrant present in the food is an example of a simple distribution approach to estimating exposure to a food packaging migrant.

Epoxidised Soya Bean Oil (ESBO)

ESBO is an additive used in the manufacture of food packaging. It is used as a plasticiser as well as a stabiliser for plastic polymers such as polyvinyl chloride (PVC). It is used, in particular, in closure gaskets for metal lids used to seal glass jars and bottles. It forms an airtight seal, which is needed to prevent microbial contamination and in jarred baby foods it can be employed at up to 40% of the formulation weight of the gasket (European Food Safety Authority 2004). Due to the high levels in gaskets in contact with foods there is potential for migration into the food, especially fatty food, during both sterilisation and storage. In the past two years the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) of EFSA has published two opinions on exposure to ESBO from food contact materials (European Food Safety Authority 2004, 2006). The first opinion examined exposure of infants to ESBO, while in 2006 the exposure of adults to ESBO was examined. The results of these opinions suggest that adult exposure does not exceed the tolerable daily intake (TDI) of 1 mg per kilogram body weight per day, whereas infant exposures could exceed the TDI by 4-5 fold (European Food Safety Authority 2004, 2006). As EFSA provided opinions on exposure of infants and adults to the food packaging migrant EBSO, one of the aims of the present study was to estimate exposure of Irish children to ESBO.

Styrene Monomer

Styrene is a monomer used to make polystyrene plastics and polystyrene is used to package dairy products, meats, eggs and takeaway foods [START_REF] Duffy | Estimation of exposure to food packaging materials. 1: Development of a food packaging database[END_REF]. In terms of styrene monomer, exposure occurs via inhalation and dietary intake. Styrene may be naturally present in food [e.g. cinnamon, beef, and coffee beans [START_REF] Steele | Determination of styrene in selected foods[END_REF]] or enter the food via migration from food contact materials. Migration of styrene may occur because some residual styrene can remain in the finished packaging material. Although styrene has low acute toxicity after inhalation or ingestion, it is classified as harmful by inhalation and irritating to skin and eyes. Very high exposure to styrene induces dizziness, drowsiness, headaches and potentially unconsciousness [START_REF] Filser | Estimation of a possible tumorigenic risk of styrene from daily intake via food and ambient air[END_REF]. In In practice the tainting properties of styrene monomer in foodstuffs and the pungent odour act as a restriction on the level at which styrene is tolerated by the consumer in most foods (International Life Sciences Institute 2002). An EU risk assessment of styrene is currently being completed in the UK. However, it may take some years before this assessment is completed, as it is quite extensive. Previous dietary exposure assessments have been completed for styrene. For example, in the UK the intake of styrene was calculated using the UK Total Diet Study (TDS) samples (Ministry for Agriculture, Food and Fisheries 1999a) and more recently styrene exposure was estimated using a probabilistic exposure assessment tool, which incorporated concentration data from the UK TDS [START_REF] Holmes | Dietary exposure to chemical migrants from food contact materials: A probabilistic approach[END_REF]. The results of these exposure assessments were orders of magnitude less than the provisional maximum tolerable daily intake (PMTDI) of 40 µg/kg bw/day set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (World Health Organisation 1984). The aim of the present study was to estimate exposure of Irish children to styrene.

Therefore, the overall aim of this study was to estimate exposure to one additive (ESBO) and one monomer (styrene) from food packaging materials using a food consumption database that contained food packaging information. As these exposure assessments were completed on children this study provides novel information regarding exposure in this sub group of the population who have not been previously studied for their exposure to these target migrants.

Materials and methods

A dataset that was created from information on food consumption data from the NCFS (Irish Universities Nutrition Alliance 2005), food packaging information from the IFPD [START_REF] Duffy | Estimation of exposure to food packaging materials. 1: Development of a food packaging database[END_REF]) and migration data from the literature (Flanjak and Sharrad 1984;[START_REF] Castle | Migration of epoxidised soya bean oil into foods from retail packaging materials and from plasticised PVC film used in the home[END_REF][START_REF] Lickly | A model for estimating the daily dietary intake of a substance from food-contact articles: Styrene from polystyrene food-contact polymers[END_REF][START_REF] Tawfik | Polystyrene cups and containers: styrene migration[END_REF][START_REF] Bradley | Investigation of chemical migration into takeaway snack foods[END_REF]EFSA 2006) was used to estimate exposure of Irish children aged 5-12 years. This dataset documented the types and amounts of food consumed by 594 Irish children over a seven-day period, in addition to the packaging used for these foods.

Epoxidised Soya Bean Oil (ESBO)

In the EFSA exposure assessment of ESBO for adults, the main food packaging types identified as potentially contributing to ESBO exposure were glass jars with metal lids lined with PVC gaskets and thin plasticised PVC cling films, in which ESBO is used as a stabiliser (European Food Safety Authority 2006). Foods packaged in these materials were identified in the current database and it was assumed that ESBO was present in these materials and that it migrated into the food (i.e. the probability of presence of ESBO in foods packaged in glass jars with metal lids and PVC cling films was 100%). In their exposure assessment, EFSA did not include non-fatty foods because ESBO has a low solubility in foods with no fat and therefore is not expected to migrate into such foods (EFSA 2006). However, EFSA did not clarify whether foods were classified as fatty according to European Council Directive 85/875/EC (European Council 1985), or on the amount of fat per 100g of food, or some other measurement parameter. Therefore, in the current exposure assessment, all foods that were classified as fatty according to European Council Directive 85/572/EC and packaged in one of the two materials were included in the assessment. In addition, many sauces that were classified as acidic or aqueous and packaged in glass jars with metal lids were also included in the exposure assessment as these foods contained some fat (e.g. curry sauce, tikka masala sauce and tomato based sauces).

As concentration data were not available for all foods packaged in one of the two packaging types (i.e. glass jars with metal lids or PVC cling films), foods were classified into a number of "food packaging groups". These food packaging groups were designed to reflect the food type and the packaging type used for the food. Table 1 lists these food packaging groups and the migrant concentration data used for each of these groups in the exposure assessment. In the EFSA exposure assessment, the AFC panel noted that due to the variability of the migration value in foods from glass jars with PVC lined lids, a migration value covering 90% of the samples for each category could be used as a conservative estimate of the concentration for estimating chronic exposure of adults to ESBO. Therefore these values covering 90% of samples were also used in the current exposure assessment for children (Table 1). There were few survey data available for the migration of ESBO from PVC films, and therefore, in the EFSA assessment migration values for ESBO in PVC films were extrapolated from work completed on the plasticiser di(2-ethylhexy)adipate (DEHA) (EFSA 2006). In the current study data on the migration of ESBO from PVC cling films were used [START_REF] Castle | Migration of epoxidised soya bean oil into foods from retail packaging materials and from plasticised PVC film used in the home[END_REF]. From this data, the 90 th percentile concentration for each food group was calculated and used in the exposure assessment to ensure conservative results.

[Insert table 1 about here]

Styrene monomer

For the styrene exposure assessment, polystyrene was identified as the main source of styrene monomer. All foods packaged in polystyrene were identified in the database and it was assumed that if a food was packaged in polystyrene then styrene monomer was present in the food (i.e. the probability of presence of styrene in these foods was 100%).

The migration concentration of styrene in foods was derived from the literature (Table 2). A number of food groups were developed as the migration of styrene from packaging differs according to the food type. Two conservative exposure scenarios for styrene were completed, one in which the 90 th percentile migration value for each food group was used and one in which the maximum migration value for each food group was used.

Table 2 lists the food groups that were created, the level of styrene in these groups and the data sources from which these food groups and concentration values were developed.

[Insert table 2 about here]

As some foods consumed in the NCFS did not have their exact packaging information documented, they were examined and assessed regarding the probability that they may have been packaged in one of the materials that contained one of the target migrants. The probability of whether these foods were packaged in a material that could contain the migrant was based on expert opinion, which erred on the side of conservatism. For example, sausages that could have been packaged in PVC cling film were assumed to be packaged in PVC cling film. This was similar for foods that could have been packaged in polystyrene, for example, for foods such as pot noodles, mousse desserts and fresh chickens. Once the probability of presence of ESBO and styrene monomer in the foods and the concentration at which these migrants could be present in the foods was established then the basic exposure equation was completed: Exposure = ∑amount of food consumed x amount of chemical present in the food This calculation was completed for every eating event of each child in the consumption database. The average daily intake of the migrants for each of the 594 children were calculated. These values were analysed to calculate the overall mean intake and the upper percentile intakes for children. The body weight of each child was also used to calculate intakes per kilogram body weight. Analyses were completed using the CREMe® Software v.2.0.2 (CREMe Software 2006).

Results

The mean intake of ESBO in Irish children was estimated at 0.687 mg/day with upper percentiles of 2.186 mg/day (95 th percentile) and 2.733 mg/day (97.5 th percentile) (Table 3). When actual body weights of children in the survey were incorporated into the equation the estimated mean intake was 0.023 mg/kg bw/day, while the upper percentiles were 0.077 mg/kg bw/day (95 th percentile) and 0.098 mg/kg bw/day (97.5 th percentile).

Exposure to ESBO from each of the food packaging groups is presented in Table 4.

Tomato sauce in glass jars (46.8%) and meat in PVC cling film (35.9%) were the highest contributors to ESBO intake. For some of the food groups, which were used in the EFSA exposure assessment, there were no eating events of these groups in the NCFS (e.g. fish in oil packaged in a glass jar, vegetables packaged in a glass jar, cheese in oil packaged in a glass jar). Therefore, exposure to ESBO from these groups in the current exposure assessment was zero. Figure 1 shows the distribution of ESBO intakes among the 594 children in the NCFS and the cumulative distribution.

[Insert Table 3 about here] [Insert Table 4 about here] [Insert Figure 1 about here]

In the first exposure scenario for styrene, the 90 th percentile migration concentrations were used. In this instance, the estimated mean intake was 3.7 µg/day or 0.12 µg/kg bw/day. The upper percentiles for the intake per kilogram body weight ranged from 0.33 µg/kg bw/day (95 th percentile) to 0.39 µg/kg bw/day (97.5 th percentile). When the maximum migration concentrations were used the estimated mean intake of styrene was 5.16 µg/day or 0.17 µg/kg bw/day. The upper percentiles ranged from 14.24 µg/day (95 th percentile) to 17.72 µg/day (97.5 th percentile) or 0.47 µg/kg bw/day (95 th percentile) to 0.57 µg/kg bw/day (97.5 th percentile) (Table 5). When styrene intake per food group was examined for the scenario that used the maximum migration concentrations, the food group that contributed most to styrene intake was the "other styrene group (fatty)"(71.6%) (Table 6). This group included all fatty foods (as classified in European Council Directive 85/572/EEC) packaged in polystyrene that did not belong to one of the other food groups, for example all fresh and processed meats were classified as "other styrene group (fatty)". It should be noted that for some food groups listed in Table 2 there were no records of these foods packaged in styrene in the NCFS, therefore exposure to styrene from these groups was zero (i.e. cheese, ice-cream, juice and other simulant B (acidic)). Figure 2 shows a distribution of styrene intakes in the 594 children in the NCFS and the cumulative distribution. There are two distributions on this graph, one that represents the distribution of intakes when using the 90 th percentile migration concentration values and one that represents the distribution of intakes when using the maximum migration concentration values.

[Insert Table 5 about here] [Insert Table 6 about here] [Insert Figure 2 about here]

Discussion

The use of a food consumption database, which also contains packaging information, has proved very useful in the estimation of exposure to food packaging migrants for a number of different scenarios in this study. By using a database that contains this information a large amount of the work, currently required in the exposure assessment methodology to link food consumption information with packaging information, was eliminated. It also eradicated the need to use upper percentile values for the consumption level of foods, as individual exposure assessments were completed.

Regarding ESBO, the Scientific Committee for Food (SCF) has classified it in list 2 (substances for which the committee has established a TDI or a temporary TDI (t-TDI)),

where it has a TDI of 1 mg/kg body weight. In the EU there is no SML for ESBO but the OML of 60 mg/kg food is applicable. From previous studies it was observed that the migration from ESBO alone approaches or even exceeds this limit in some samples [START_REF] Hammarling | Migration of epoxidised soya bean oil from plasticized PVC gaskets into baby food[END_REF]Ministry for Agriculture, Food and Fisheries 1999b;[START_REF] Fantoni | European survey of contamination of homogenized baby food by epoxidized soybean oil migration from plasticized PVC gaskets[END_REF]. Although ESBO is neither carcinogenic nor genotoxic the AFC panel of EFSA recommended that a SML be developed for ESBO in baby foods. They also decided not to change the TDI for ESBO as there is an inbuilt safety factor of 100fold (thereby, an intake that exceeds the TDI by 4-5 fold does not imply there will be adverse health effects in infants) (European Food Safety Authority 2004). Due to differing food consumption patterns throughout life, a significant variation in ESBO exposure is expected according to life stage. Infants are likely to have the highest exposures as the highest food intakes on a body weight basis is likely to occur within the first year of life, and also due to the consumption of commercial infant foods packaged in glass jars (European Food Safety Authority 2004). It was stated in the EFSA 2004 opinion that adult exposures are likely to be much lower than infant exposures, both due to the lower contribution from target foods, particularly jarred foods, to their total diet, and also due to the generally lower levels of ESBO in closures used in packaging adult foods (European Food Safety Authority 2004). When a conservative exposure assessment to ESBO on adults was completed the potential high exposure of adults was estimated at 0.84 mg/kg bw/day (0.64 mg/kg bw/day from glass jars + 0.2 mg/kg bw/day from cling film) (European Food Safety Authority 2006). Exposure for infants was calculated at 0.34-4.65 mg/kg bw/day (European Food Safety Authority 2004).

In the current exposure assessment it was estimated that Irish children had lower intakes of ESBO at 0.023 mg/kg bw/day compared with the EFSA estimated intake for adults and infants (EFSA 2004(EFSA , 2006)). Similar concentration datasets were used in the EFSA exposure assessment and the current assessment as the 90 th percentile concentration value for the level of ESBO present in foods was used in both studies. However, EFSA did not use actual consumption data but instead used the 95 th percentile of food intake and assumed that these food groups were packaged solely in the material of interest. By using a food consumption database, which contained precise data on the packaging used for the food, uncertainty was significantly reduced in the exposure assessment.

Conservatism was still ensured in the current study as food items that did not have their packaging identified had conservative assumptions made regarding the type of packaging used for these foods. Therefore, for Irish children exposure to ESBO is not of concern, as it does not approach the TDI of 1 mg/kg bw/day. Two scenarios of styrene exposure were completed in this study. Both scenarios used conservative migration values (i.e. the 90 th percentile migration concentration values and the maximum migration values). From Figure 2 it can be seen that by using the most conservative values (i.e. maximum migration values) a larger number of individuals have a higher exposure. This scenario analysis may provide useful information if the aim of the assessment is to establish the number of individuals exceeding a recommended upper intake level using different assumptions. From this study the mean styrene intake was 0.12 to 0.17 µg/kg bw/day, which is higher than was found in previous studies. In the UK total diet study exposure was estimated to range from 0.03 to 0.05 µg/kg bw/day for a 60kg person (Ministry for Agriculture, Food and Fisheries 1999a). Using the probabilistic exposure assessment methodology, median adult exposure in the UK ranged from 0.037 to 0.041 µg/kg bw/day while median youths exposure in the UK was 0.048 µg/kg bw/day [START_REF] Holmes | Dietary exposure to chemical migrants from food contact materials: A probabilistic approach[END_REF]. In another study, which was based on literature data, the styrene exposure from food was found to range from 0.003 to 0.0017 µg/kg bw/day for several countries [START_REF] Tang | Estimation of human exposure to styrene and ethylbenzene[END_REF]. In the US the exposure to styrene from food was estimated at 0.15 µg/kg bw/day [START_REF] Lickly | A model for estimating the daily dietary intake of a substance from food-contact articles: Styrene from polystyrene food-contact polymers[END_REF]. From these studies it can be seen that there is a large variation in estimates of styrene intake. However, the US estimate is very similar to the estimate found in the current study. The higher exposure seen in the current study as compared with the UK studies may be due to the fact that these studies were completed for adults or older children (youths) and therefore on a per kilogram body weight basis, intakes of children should be expected to be higher.

Although conservatism was built into this exposure assessment, the estimated mean intake of styrene of 0.12 to 0.17 µg/kg bw/day is still well below the PMTDI of 40 µg/kg bw/day set by JECFA (World Health Organisation 1984) and therefore styrene intake is not of concern for Irish children. By using the above methodology the exposure assessments for two food packaging migrants were completed and the results proved that the intakes of these migrants in Irish children aged 5-12 years are not of safety concern. This data lends itself to efficient use for multiple exposure scenario analysis, and future exposure assessments can be completed using this data source once the packaging in which the migrant of concern is identified and once migration data is available for this migrant.

  1987 the International Agency for Research in Cancer (IARC) classified styrene as 'group 2B' (possibly carcinogenic to humans) and maintained this classification in 1994 (Polystyrene Packaging Council 2005). The use of styrene as a monomer in food contact authorised under European Commission Directive 2002/72/EC without a SML [although an overall migration limit (OML) of 60 mg/kg still applies] (European Commission 2002).

  The work was funded by the Department of Agriculture, Food and Rural Development who provided support through the Food Institutional Research Measure and also Enterprise Ireland who provided funding through the CREMe project.

  Figure 1 Distribution of expoxidised soya bean oil (ESBO) intake among 594 Irish children (mg/ kg bw/day)

Table 1 Epoxidised Soya Bean Oil (ESBO) concentrations per food packaging group

 1

	ESBO
	Food group	ESBO	Concentration
	code	Food Group Name	mg/kg P90th	Reference
	1	Fatty sauce glass jar and metal lid	30	EFSA 2006
	2	Pesto sauce glass jar and metal lid	300	EFSA 2006
	3	Tomato sauce glass jar and metal lid	60	EFSA 2006
	4*	Fish in oil glass jar and metal lid	350	EFSA 2006
	5*	Vegetables in oil glass jar and metal lid	300	EFSA 2006
	6*	Cheese in oil glass jar and metal lid	150	EFSA 2006
	7*	Other glass jar and metal lid	30	Assumption
				Castle et al.
	8	Meat PVC cling film	10.8	(1990)
				Castle et al.
	9*	Sandwiches PVC cling film	15	(1990)
				Castle et al.
	10	Cheese PVC cling film	19.4	(1990)
				Castle et al.
	11	Other PVC cling film	22.7	(1990)

*There were no eating events of these food packaging groups in the Irish National Children's Food Survey. Therefore exposure to ESBO from these groups is zero.

Table 3 Exposure of Irish children aged 5-12 years to expoxidised soya bean oil (ESBO)

 3

		Daily ESBO exposure	Daily ESBO exposure
		(mg/day)	(mg/kg bw/day)
	Mean	0.687	0.023
	Std. Deviation	0.404	0.013
	Minimum	0.000	0.000
	Maximum	8.420	0.385
	Percentile: 90th	1.750	0.054
	Percentile: 95th	2.186	0.077
	Percentile: 97.5th	2.733	0.098
	Percentile: 99th	3.444	0.119
	Percentile: 99.9th	6.648	0.301

Table 4 Contribution of food packaging groups to expoxidised soya bean oil (ESBO) intake in Irish children

 4

	Food group	ESBO intake (mg/day)	% of total
		Mean	Std. Dev	intake
	All	0.687	0.828	100
	Fatty sauce glass jar	0.063	0.213	9.2
	Pesto sauce glass jar	0.019	0.229	2.7
	Tomato sauce glass jar	0.322	0.726	46.8
	Meat PVC cling film	0.247	0.226	35.9
	Cheese PVC cling film	0.029	0.083	4.2
	Other foods PVC cling film	0.007	0.086	1.1

Table 6 Contribution of food groups to styrene intake in Irish children when using maximum migration values

 6

	Food Group	Styrene Intake (µg /day)	% of total
		Mean	Std. Dev	intake
	All	5.163	4.906	100
	Yoghurt	1.033	1.451	20.01
	Cream	0.005	0.026	0.10
	Milk puddings, custards and desserts	0.022	0.126	0.43
	Fat spread and margarine	0.002	0.017	0.04
	Takeaway foods	0.001	0.008	0.02
	Other styrene group (Aqueous)	0.402	2.792	7.79
	Other styrene group (Fatty)	3.698	3.958	71.63

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

Scientific Committee for Food (SCF). 1999. Commission of The European

Acknowledgements


Keywords: Pesticide residues, Pesticides -spraying regimes Baby food, Fruit, Processed foods apples, pesticides, baby food, fruit production, gas-chromatography-mass 20 spectrometry, liquid-chromatography-tandem mass spectrometry 21

Safe apples for baby

Agriculture Practice (GAP), residues of pesticides can still be detected in treated crops 5 [START_REF] Hajslová | Pesticides[END_REF]. Consumers' exposure to such hazardous chemicals is of growing health 6 concern, specifically in the case of infants and young children that represent the most 7 vulnerable population group -not only because of their high food intake per body weight 8 unit but also due to yet undeveloped detoxication mechanisms. With respect to these facts, 9 a uniform Maximum Residue Limit (MRL) as low as 0.01 mg kg -1 was established by EU 10

Directive for any pesticide residue in processed cereal-based foods and baby foods for 11 infants and young children (Commission Directive 1999/39/EC). 12 13 It should be noted that EU Maximum Residue Limits (MRLs) applying to common 14 products of plant origin are often higher than 0.01 mg kg -1 . According to an internationally 15 accepted strategy, their values are based not only on toxicologically acceptable levels, but 16 also on evaluation of residue data from supervised field trials carried out according to the 17 principles of Good Agricultural Practice (GAP). Experience shows that GAP-based MRLs 18 are generally lower than MRLs derived from toxicological end-points [START_REF] Hamilton | Pesticides Residues in Food and Drinking Water[END_REF]. 19 The overview of EU legislation concerned with pesticides and their MRLs is shown at 20 http://europa.eu.int/comm/food/plant/protection/pesticides/legislation_en.htm and 21 http://ec.europa.eu/food/plant/protection/pesticides/index_en.htm, respectively. 22 23 A significant decrease in pesticide residues typically occurs during technological 24 operations such washing, steam boiling, thermal sterilisation etc. employed for 25 industrial/household fruits and/or vegetable processing [START_REF] Cano | Determination and persistence of several fungicides in 13 postharvest-treated apples during their cold storage[END_REF]Cabras et al. 26 1990; [START_REF] Mahajan | Effect of post-harvest application of fungicides and wax 27 coating on the quality of and storage of apple[END_REF][START_REF] Cabras | Persistence and fate of 5 fenthion in olives and olive products[END_REF][START_REF] Holland | Effects of storage and processing 15 on pesticide residues in plant matrices[END_REF][START_REF] Ong | Chlorine and ozone washes for 30 pesticide removal from apples and processed apple sauce[END_REF]; Cabras 27 et al. 1997;[START_REF] Burchat | The 14 distribution of nine pesticides between the juice and pulp of carrots and tomatoes after 15 home processing[END_REF]Cabras et al. 1998aCabras et al. , 1998bCabras et al. , 1998c;;Will et al. 1999 To prevent such a risk and to ensure the quality of fruit/vegetable baby food, baby 1 food producers are nowadays reluctant to process any raw materials containing residues 2 exceeding 0.01 mg kg -1 limit (even though the "common" MRL for particular crop is not 3 exceeded). To our knowledge, the current practice of major baby food producer is to check 4 all batches of components prior to processing; thus the risk of marketing a product (baby 5 food) containing violative residues is minimized. Thus, strategies relying on "dilution" of 6 contaminated batch by residues free raw material and/or degradation of occurring 7 pesticides during processing chain are not represent common approach. 8 9 Under these conditions, close collaboration between industry and farmers was established 10 to facilitate a supply of crops containing low residue or residue free requirements . 11 Although the use of organic crops might represent a conceivable option, this may not be a 12 realistic solution in view of the global demands for raw materials. Therefore, a careful 13 choice of agricultural practices namely selection of low input, non-persistent pesticide 14 preparations is a critical issue in this context. It should be noted that in addition to 15 documentation submitted by producers within the pesticides registration process, there are 16 a large number of papers documenting a decline of various pesticide residues following 17 respective food plant/crop treatment, however, due to a largely variable climatic conditions 18 as well as with respect to differences in application practices, such information is only 19 tentative and cannot be considered as fully generic [START_REF] Holland | Effects of storage and processing 15 on pesticide residues in plant matrices[END_REF], Rasmussen et al. 20 2003[START_REF] Gambacorta | Pesticide residues 6 in tomato grown in open field[END_REF][START_REF] Cengiz | Residue contents of captan and 17 procymidone applied on tomatoes grown in greenhouses and their reduction by duration of 18 a pre-harvest interval and post-harvest culinary treatmetns[END_REF]. [START_REF]Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and Environment 22 and the WHO Core Assessment Group of Pesticide Residues[END_REF]

22

In our study, extensive field experiments concerned with optimisation of apples production 23 are presented. The aim of this work was to identify the optimal strategy for apple tree 24 protection providing efficient control of pests (plant diseases) and, at the same time leaving 25 undetectable or very low pesticide residues not exceeding 0.01 mg kg -1 . 26

Materials and methods 28

Field work 29

Golden Delicious apples examined in this study for pesticide residues were obtained from 30 our project partner Research and Breeding Institute of Pomology, Holovousy Ltd., Czech 31 Republic. The field experiment was performed at the orchard Sady Rokos Petrovicky, 32 (24 years old) were situated in spacing 5 x 3 m (approximately 650 trees per ha), 3 experimental area was 0.25 ha. The overview of pesticide preparations including some 4 relevant characteristics are summarized in Table I. A tractor mounted sprayer Tifone 5

Vanguard 1070 equipped with the Albuz ATR nozzles was used for the pesticide 6 applications; the volume was 400 L per ha and the operating pressure 1.3 MPa. 7 8

In April 2004, pesticide treatment started on the whole experimental orchard. In June 2004, 9 experimental orchard was divided into six field experiments representing six differences in 10 pesticide preparation treatments (the frequency of applications was higher compared to 11 standard practice). The detailed overview of pre-harvest treatment of apple trees performed 12 in the year 2004 is summarized in Table II The scope of two multi-residue methods described below (LC-MS/MS and GC-MS) 6 covered all the pesticides involved in this study with the exception of thiram compound 7 representing the group of ethylene bisdithiocarbamates (EBDCs). These contact fungicides 8 are according to legislation requirements determined as a carbon disulphide (CS 2 ). 9

Accredited method (ISO 17025) consisting of the following steps was used for examination 10 of apples: (i) solid phase micro extraction (SPME) for absorption of CS 2 (degradation 11 product of EBDCs) from headspace of sample digested by hydrochloric acid in the 12 presence of stannous chloride and (ii) GC-MS identification/quantification of analyte 13 thermally desorbed in GC injector port. Since in none of samples residues of thiram were 14 detected (LOD of method was 0.5 µg kg -1 ), no more detailed description is provided here. 15 16

LC-MS/MS method 17

Sample preparation 18 3 kg of unwashed fresh apples (representing field sample) delivered to laboratory were 19 homogenized using a Warring Blender homogenizer. 12.5 g of homogenate were mixed 20 with 50 ml of acetonitrile and extracted for 2 min with Ultra-Turrax homogenizer. The 21 suspension was filtered under vacuum; the filtrate cake was washed with 3 x 10 ml of 22 acetonitrile and then evaporated on a vacuum rotary evaporator. 15 ml of methanol were 23 added into the evaporation flask and the volume was quantitatively transferred into 50 ml 24 volumetric flask and made-up with methanol. Samples were prior to injection filtered 25 through PTFE filters. The matrix content in crude extract was 0.25 g ml -1 . 26 27 LC-MS/MS identification/quantification 28 LC separations were carried out on a reversed phase column Discovery C 18 (150 x 3 mm, 5 29 µm). The sample temperatures as well as column temperature were maintained at 25°C. 30

The mobile phase contained water (A) and methanol (B) and the flow rate was 0.3 ml min - 31 1 . Gradient was employed, starting composition of 50% B, rising linearly 100% B over 6 32 and cone voltage) were optimised for each pesticide during infusion (5 µl min -1 ) of 7 individual pesticide solution (1-5 µg ml -1 ) into the mobile phase flow (A:B 50:50, (v/v), 0.3 8 ml ml -1 ). All experiments were realised employing the following parameters: capillary 9 voltage 3.5 kV, extractor voltage 4 V, source temperature 120°C, desolvation temperature 10 250°C, cone gas flow 100 L h -1 and desolvation gas flow 700 L h -1 (both gasses were 11 nitrogen). Argon was used as a collision gas (3.3 x 10 -3 mbar). Tuned and optimised 12 MS/MS transitions as well as specific cone voltages and collision energies are summarized 13 in Table III. Analytes were divided into time segments based on their elution 14 characteristics. The MS/MS transitions were monitored in the multiple reaction-monitoring 15 (MRM) mode at the same dwell time 0.005 s, inter-channel delays, and inter-scan delays of 16 10 ms for all transitions. 17

GC-MS method 24

Sample preparation 25 3 kg of unwashed fresh apples (representing field sample) delivered to laboratory were 26 homogenized using a Warring Blender homogenizer. A 25 g of homogenized apples were 27 weighed in a glass beaker, after addition of 100 ml ethyl acetate and 75 g of anhydrous 28 sodium sulphate the sample was extracted for 2 minutes with the Ultra-Turrax homogenizer 29 at 10 000 rpm. The suspension was filtered under vacuum through the layer of anhydrous 30 sodium sulphate, the beaker and filtrate cake were rinsed with 3 x 25 ml of ethyl acetate. 31 Combined filtrates were evaporated using vacuum rotary evaporator (temperature max. 32 transferred into volumetric flask and the final volume was made up with cyclohexane to 50 2 ml (matrix concentration 0.5 g ml -1 ). 3

HP-GPC purification 4

Samples were filtered through PTFE filters prior to purification. Crude extracts were 5 purified employing automated high performance gel permeation chromatography system. 6

The following conditions were used for sample clean-up: mobile phase ethyl acetate-7 cyclohexane (1:1), flow rate 1 ml min -1 , injection volume 2 ml, collected "pesticide" 8 fraction 14.5-31 ml. Purified pesticide fraction was evaporated using vacuum rotary 9 evaporator (temperature max. 40°C, pressure 220 mbar) almost to dryness, the residual 10 solvent was removed with a gentle stream of nitrogen. After addition of 1 ml of toluene, 11 sample was ready to GC-MS analysis. The content of original matrix was 1 g ml -1 . 12

GC-MS identification/quantification 13

All separations of GC amenable pesticides were carried out on capillary column DB-5MS 14 (60 m x 0.25 mm x 0.25 µm). Pulsed splitless injection was used (pressure pulse 60 psi, 15 pulse period 2 min, inlet temperature 250°C, injection volume 1 µl). Oven temperature 16 program started at initial temperature 90°C (hold 2 min), 5°C min -1 to 180°C then 2°C min - 17 1 to 280°C (hold 5 min). Helium was used as a carrier gas at a constant rate 19 cm s -1 . MSD 18 detector was working in electron ionization mode (EI), ion source temperature 230°C, MS 19 Quad temperature 150°C. Identification/quantification was performed in the Selected Ion 20 Monitoring mode (SIM), see Table IV. All GC-MS chromatographic data were processed 21 using ChemStation ® Software (D.02.00 SP1, Agilent, USA) 22

Matrix -matched standards 23

Matrix-matched standards used for calibration were prepared from untreated apples. Blank 24 extracts were prepared according to the above described LC-MS/MS and GC-MS 25 procedures. For LC-MS/MS, 100µl of working standard solution S 1L -S 6L were added to 26 900µl of blank apple extract prior to analysis. As for GC-MS, residue after evaporation of 27 solvent from purified blank apple extract was re-dissolved in 1ml of appropriate standard 28 working solution S 1G -S 6G for following analysis. For recovery tests, apple homogenates were spiked by pesticide mixture (spikes 1 concentration corresponded to 0.1 mg kg -1 ) and then processed as described for LC-MS/MS 2 and GC-MS analyses. Quality control procedures for pesticide residues analysis (Document 3 N° SANCO/10232/2006) were applied in setting LOD and LOQ values. The latter one was 4 the lowest calibration level (LCL) and corresponded for particular analyte to 3 x LOD. 5 Under these conditions, LOQs were the minimum concentrations of analytes that was 6 possible to quantify with acceptable accuracy and precision. 7 8 The methods described above are accredited according to ISO/IEC 17025. As a part of 9 external Quality Assurance, laboratory has been successfully participating in proficiency 10 tests -Food Analysis Performance Assessment Scheme (FAPAS ® ) and European 11 proficiency testing within the pesticide monitoring programme. Performance characteristics 12 of applied analytical methods are summarized in Table IV. [START_REF] Rasmussen | Distribution of multiple pesticide 14 residues in apple segments after home processing[END_REF]

[Insert Table IV about here] 15 16

Results and discussion 17 18

As mentioned in the introduction, where fruit is intended for baby food production, careful 19 attention has to be paid to selection of pesticide preparations matching specific 20 requirements for obtaining low or residue-free crop. Since baby food producers tolerate 21 only residues not exceeding 0.01 mg kg -1 in the raw material, fundamental changes of 22 treatment strategy might have to be adopted in this aspect to produce acceptable raw 23 material. To identify "high residue" pesticides that should be in particular case eliminated 24 from the further use, monitoring of apples grown by several farmers under common 25 conventional practices was carried out in the first phase of our experiments. The most troublesome pesticides in this respect were phosalone, chlorpyrifos-methyl, 2 captan, tolylfluanid and fenitrothion. Following the analysis of a data set generated within 3 this 3-year pilot study, farmers were advised to search for replacing preparations containing 4 these with other ones. As a consequence of modified treatment practices, significant 5 decrease of organophosphorus insecticide residues (phosalone, and fenitrothion) were 6 observed, while no changes in captan and tolylfluanid incidence were found. The search for 7 "new" preparation resulted in increased occurrence of trifloxystrobin residues. Overall, the 8 farmers` effort to improve treatment plans was documented by decrease of positive samples 9 number to 54% (i.e. only 15% exceeded baby food MRL of 0.01 mg kg - carried out 14 days after the last pesticide application, residues of fungicides (captan, 3 tetraconazole, trifloxystrobin, penconazole, tebuconazole and tolylfluanid) and insecticides 4 (pyridaben, chlorpyrifos-methyl, thiacloprid and teflubenzuron) were detected. In following 5 pre-harvest interval successive decline of residues was observed, although the rate of 6 dissipation varied largely. Since it is impossible to generalize the fate of the whole set of 7 pesticides investigated, behavior of individual compounds is discussed in separated 8 paragraphs below (Figures 23456789). Figure 10 As shown in Figure 2, the residues of captan, the main fungicide widely used for control of 14 apple scab (Venturia inaequalis), were either not detected (FE 5 and FE 6) or already fairly 15 well below the MRL (3 mg kg -1 ) at the first sampling, even in an experiment with two 16 treatments (FE 2, FE 3 and FE 4). Successive decrease of captan occurred and at the time 17 of harvest its residues at 0.01 mg kg -1 level were detected in only two samples (FE 2 and 18 FE 4). 19 20 It is worth noting that the relative decrease rate was higher in FE 2, FE 3 and FE 4 21 compared to FE 5 and FE 6 in which longer time elapsed since the last treatment with 22 Merpan 80 WG preparation. No significant differences in residue levels were found in the 23 third sampling regardless the application rate and date of application, residues were 24 between 0.01 -0.02 mg kg -1 . 25 26 Considering the data obtained from the Canadian study for JMPR (Captan residues in food: 27 1984 evaluation), we obtained significantly lower content of captan residues. Whereas 28 content of captan in Canadian apples was in the range 0.6 -2.9 mg kg -1 some 30 days after 29 its application, in apples carried out within our experiments its concentration did not 30 exceed 0.1 mg kg -1 in 33 days after captan treatment. Although applied at the same rate and the same date, pyridaben residues in apples from 3 individual experiments differed largely at the time of the first sampling. While relatively 4 rapid decrease of residues occurred in FE 1 with no detectable residues at the harvest time, 5 in FE 2 despite the lower concentration in the first sampling, traces of pyridaben were 6 detectable even in harvested apples (see Figure 3). 7 8 [Insert Figure 3 about here] 9 10

Chlorpyrifos-methyl 11

Residues of insecticide chlorpyrifos-methyl used for crop protection against codling moths 12 (Cydia pomonella) and sawfly insects (Hymenoptera, Haplocampa testudinea) dropped in 13 both FE 3 and FE 5 below the MRL (0.50 mg kg -1 ) in all experiments even before the end 14 of the safety period. As can be seen from Figure 4, further degradation of residues occurred 15 and residues were close to or below the baby food MRL. Harvested apples collected 78 16 days after the last Reldan 40 EC application in FE 3 and FE 5 did not contain any 17 chlorpyrifos-methyl residues. In apples from experiment FE 1, residues were not detected 18 even during the pre-harvest interval (61 days). Teflubezuron is an insecticide recommended against tortricid (Tortricidae) that can cause 24 mechanical damage to ripe fruits, which can consequently be a "gate" for secondary 25 putrefactive diseases (Alternaria, Nectria, Phytophtora) during apple storage. As shown in 26 Figure 5, repeated application of Nomolt 15 SC resulted in a fairly high content of 27 teflubenzuron in FE 2 contrary to single application in case of FE 3 and FE 4 in the first 28 and second samplings. In none of the experiments was the MRL (0.5 mg kg -1 ) exceeded 29 even in the first sampling. Among the first and second samplings only a slight decrease of 30 teflubenzuron content was observed, probably due to lack of precipitation that might 31 remove some surface residues (see Figure 1). In the following pre-harvest time the content 32 Recommendations for 2006). Thiacloprid was applied in all FE 1, FE 2, FE 3 and FE 4 13 once, three times in FE 5 and twice in FE 6. Since the treatment was carried out in early 14 May, in line with expectation low concentrations correlating with application rate were 15 obtained in FE 1, FE 2, FE 3 and FE 4. Interesting is that these were detected even 111 16 days after Calypso 480 SC application (i.e. 97 days after safe period elapsed). Nevertheless, 17 in time of harvest, no residues were detected. Significant differences in thiacloprid contents 18 in the first sampling date was found in the case of repeated applications carried out in FE 5 19 and FE 6 (see Figure 6). However, its content rapidly declined during pre-harvest interval 20 and only trace amounts (0.003 mg kg -1 ) were found in ripe apples. 21 Table I) against apple scab (Venturia inaequalis) in FE 5 and FE 6. Relatively high 27 persistency was recognized for tebuconazole. Residues of this fungicide exceeded the baby 28 food limit even in the third sampling (55 days beyond safety period). Nevertheless, these 29

were not detected in the harvested fruits (see Figure 7a). As compared to tolylfluanid, this 30 was found only in FE 6 at very low concentration level already in the first sampling (see 31 As shown in Figure 8, residues of tetraconazole used as a protection against apple scab and 7 powdery mildew were well below the MRL (0.5 mg kg -1 ) in the first sampling in 8 experiments with three (FE 2 and FE 3) and four (FE 4) treatments; in FE 6 tetraconazole 9 was not detected. A decline of tetraconazole residues occurred in following pre-harvest 10 time; in harvested apples only low concentrations levels (0.005 mg kg -1 ) were observed at 11 field experiments repeatedly treated (FE 2, FE 3 and FE 4). 12 13 [Insert Figure 8 about here] 14 15 Trifloxystrobin 16

As shown in Figure 9, the residues of trifloxystrobin were detected in all samples FE 1, FE 17 2, FE 3, FE 4, FE 5 and FE 6 in the first sampling. Since applied at the same date and same 18 application rate in FE 2, FE 3, FE 4 and FE 5, similar concentrations were found contrary 19 to repeated applications in FE 1 and FE 6 resulting in higher contents of trifloxystrobin in 20 the first sampling. While relatively continuous decline of residues was identified in FE 1 21 and FE 6 with no (FE 6) or trace (FE 1) residues in harvested apples, in samples with lower 22 treatment (FE 2, FE 3, FE 4 and FE 5), the reduction in residue levels was not so distinct, 23 although the baby food limit was not exceeded. 24 25

[Insert Figure 9 about here] 26 27

Penconazole 28

The fungicide penconazole applied only in FE 5, was detected in the first sampling (33 29 days after the last Topas 100 EC application) at trace concentration levels (0.005 mg kg -1 ). 30

In harvested apples no penconazole residues were found. 31 32 Field experiments were carried out aimed at identifying pesticide preparations that ensure 1 effective crop protection and yet at the same time leave very low residues. Based on 6 field 2 experiments described in this study, a set of data enabling evaluation of both the 3 degradation rate and levels of terminal residues in matured apples was obtained. The main 4 observations resulting from this study are summarized below. 5

Overview of pesticide degradation in field experiments FE 1 -FE 6 33

• In the first sampling, carried out before or immediately after the pesticides safety 6 period has elapsed, only 10 pesticides (46% of 22 active ingredients applied) were 7 detected; 8 of them exceeded or were at 0.01 mg kg -1 . Those were fungicides captan in FE 8 1, FE 2, FE 3 and FE 4, tetraconazole in FE 2, FE 3, FE 4 and FE 5, trifloxystrobin in FE 1 9 and FE 6, tebuconazole in FE 5 and FE 6; insecticides pyridaben in FE 1 and FE 2, 10 teflubenzuron in FE 2, FE 3 and FE 4, chlorpyrifos-methyl in FE 1, FE 3 and FE 5 and 11 thiacloprid in FE 5 and FE 6. On the other hand, the levels of penconazole in FE 5 and 12 tolylfluanid in FE 6 were found below baby food MRL; similarly low were thiacloprid, 13 tetraconazole and trifloxystrobin in some experiments. Residues of triflumuron, triazamate, 14 chlorpyrifos, etofenprox, fenoxycarb, kresoxim-methyl, cyprodinyl, difenoconazole and 15 thiram were not detected at all. 16

• In the second sampling, 9 pesticides were found (penconazole in FE 5 was not 17 detected anymore); 8 residues of active ingredients still exceeded 0.01 mg kg -1 in some 18 experiments (captan, pyridaben, teflubenzuron, thiacloprid, tebuconazole, trifloxystrobin, 19 tetraconazole and chlorpyrifos-methyl). Since the previous sampling, contents of 20 chlorpyrifos-methyl in FE 1 and tetraconazole in FE 3 and FE 5 declined fairly below baby 21 food MRL. 22

• Also in the third sampling, 9 pesticides were detected, nevertheless only 5 of them 23 exceeded 0.01 mg kg -1 (captan, pyridaben, teflubenzuron, thiacloprid and tebuconazole). 24

Residues of chlorpyrifos-methyl in FE 3 and FE 5, tebuconazole in FE 5, trifloxystrobin in 25 FE 1, FE 5 and FE 6 and tetraconazole in FE 2 and FE 4 dropped below this value since 26 second sampling. 27

• When assessing the residues situation at the harvest time (the fourth sampling), each 28 of the treatment regimes resulted in detectable residues (see Figure 10). In total, 6 29 pesticides i.e. 27% of active ingredients applied for the treatment of apple trees were 30 detected. 31 The lowest contamination was observed in FE 1, FE 3 and FE 6, where residues of only 1 one pesticide, well below 0.01 mg kg -1 was found (trifloxystrobin in FE 1; tetraconazole in 2 FE 3 and thiacloprid in FE 6). Field treatment FE 5 resulted in the occurrence of two 3 detectable pesticides (trifloxystrobin and thiacloprid) in matured apples. Similarly to FE 1 4 and FE 6, the concentration levels of residues found in these samples were well below the 5 baby food MRL. On the other hand, baby food producers´ requirements were not fulfilled 6 in FE 2 and FE 4, where "the worst" contamination situation was found. In both these field 7 experiments tefubenzuron exceeded 0.01 mg kg -1 and residues of captan were also present 8 close to this critical concentration level. Besides, additional pesticide residues were found; 9 these were pyridaben, tetraconazole, trifloxystrobin in FE 2 and tetraconazole and 10 trifloxystrobin in FE 4 -all of them below 0.01 mg kg - 1 11 12 Regarding the effectiveness of crop protection against pests, all six treatment regimes were 13 also evaluated for the occurrence of apple scab (Venturia inaequalis). No significant 14 differences among the quality of apples obtained from individual field experiments were 15 recognized. The extent of apple scab was only about 5% higher in our experiments 16 compared to "conventionally" treated apples (regimes conducted in accordance with GAP 17 leaving residues complying with MRL which may be, however, in some cases higher than 18 0.01 mg kg -1 ). 19 difenoconazole, etofenprox, fenoxycarb, kresoxim-methyl, penconazole, pyridaben, 1 tebuconazole, thiram, triazamate, triflumuron, and tolylfluanid in apple orchards seems -in 2 terms of contamination -free of problems, no residues were detected in harvested fruit. It 3 should be noted that employing treatment regimes aimed at minimization of residues in 4 harvested crops did not result in its lower quality in terms of damage caused by pests. 5 6

In any case, follow-up validation of these recommendations has to be carried out under 7 conditions specific for particular locality. It is believed that at the end of long-term 8 experiments specific guidelines for farmers supplying apples to baby food producers will 9 be developed. 10

2 3

 2 pests, modern pesticides are designed to dissipate due to various 1 environmental factors and detoxication processes taking place in treated plants.However, under certain circumstances, even if applied in accordance with Good 4

  ; Cabras 28 et al. 2000; Panagiotis EA et al. 2000; Rassmussen et all 2003; Yirong-Su et al. 2003). 29Despite this fact, in view of the large number of registered pesticides representing various 30 chemical classes and (consequently) a wide range of physico-chemical properties, the 31 possible transfer of some (more persistent) residues into the final product cannot be 32

  Republic. The standard agricultural practices (pruning, fertilizing, and soil 1 management) were carried out on the plots during the season. Golden Delicious apple trees 2

2 3

 2 for 11 min at 100% B followed by 10 min re-equilibration to initial mobile 1 phase composition. Injection volume 20 µl was used in all separations.Identification/quantification of target analytes was performed using a Quattro Premier 4 tandem quadrupole mass spectrometer. The detector was operated in a positive electrospray 5 (ES+) ionisation mode. Multiple reaction monitoring (MRM) conditions (collision energy 6

18 21 [

 21 Generated experimental data were processed using MassLynx software version 4.0 Service 19 Pack 4, Software Change Note #462. 20 Insert Table III about here] 22 23

  220 mbar) to volume circa 25 ml. Concentrated crude extract was 1

26 27Food

 26 Monitoring study of fresh apples 28As shown in our previous study conducted in years 2001-2003[START_REF] Stepán | Baby food production 18 chain: Pesticide residues in fresh apples and products[END_REF], almost 29 no violation of MRLs occurred. Nevertheless, 60 % from 220 batches of apples delivered 30 by farmers contained detectable pesticide residues, 48% of them exceeded baby food MRL 31 0.010 mg kg -1 and therefore had to be used for other purposes than baby food production. 32Page 10 of 34 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Figures 2 -

 2 Figures 2-10 document the dynamics of pesticides involved in our study. In the sampling 2

FE

  summarizes concentration levels of pesticides 9 found in harvested apples within all 6 field experiments (FE 1 , FE 2, FE3, FE 4, FE 5 and 10

5 [

 5 during apple maturation. No residues were detected in FE 3 1 harvested apples. However, in FE 2 and FE 4 ripe apples, teflubenzuron at concentration 2 levels exceeding the baby food MRL was found. This can be attributed to the relatively 3 high stability of this compound (The Pesticide Manual 2002). 4 the very limited group of in-season insecticides allowed to be 9 applied at the early pink stage of trees development to control beetles (e.g. Anthonomus 10 pomorum), codling moths (Cydia pomonella), rosy apple aphids, green aphids and to help 11 mite suppression (IPM Commercial Apples, Insect, Disease and Weed Control 12

  were applied together as Hattrick pesticide preparation (see 26

1 residues

 1 Figure 7b) and dissipated during the pre-harvest time; in harvested apples no tolylfluanid 1 residues were detected. 2 3 [Insert Figures 7a and 7b about here] 4 5 Tetraconazole 6

20 [Food

 20 of baby food producers for safe raw material with pesticide 25 residues not exceeding 0.01 mg kg -1 , treatment regimes FE 1, FE 3, FE 5 and FE 6 might 26 be considered as an appropriate apple protection strategy, since only traces of active 27 ingredients (tetraconazole, thiacloprid and trifloxystrobin) were contained in crops at the 28 time of harvest. Residues of captan and teflubenzuron, active ingredients of pesticide 29 preparations used for treatment in experiments FE 2 and FE 4, exceeded 0.01 mg kg -1 and 30 their use in farms supplying apples for baby food production should be carefully 31 considered. On the other hand, the use of cyprodinyl, chlorpyrifos, chlorpyrifos-methyl, 32Page 17 of 34 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  (0.4 l) Hattrick (1.125 kg) Zato 50 WG (0.15 kg) Zato 50 WG (0.15 kg) Merpan 80 WG (2.0 kg) Domark 10 EC (0.4 l) Merpan 80 (2.0 kg) Topas 100 (0.45 l) Hattrick (1.125 kg) Reldan 40 EC (1.25 l) Insegar 25 WG (0.6 kg) Nomolt 15 SC (1.0 kg) Nomolt 15 (1.0 kg) Calypso (0.2 l) Reldan 40 (1.25 l) Sanmite 20 WP (0.75 kg) Sanmite 20 WP (0.75 kg) Reldan 40 EC (1.25 l) Aztec 140 EW (0.5 l) Reldan 40 (1.25 l) Calypso 480 (0.2 l) Insegar 25 WP (0.3 kg) Nomolt 15 SC (1.0 kg)

Figure 1 :Figure 2 :

 12 Figure 1: Weather conditions in the pre-harvest period

Figure 7a :

 7a Figure 7a: The dynamics of tebuconazole decrease between samplings in FE 5 and FE 6 (error bars express the expanded uncertainty of respective results).

Figure 7b :

 7b Figure 7b: The dynamics of tolylfluanid decrease between samplings in FE 6 (error bars express the expanded uncertainty of respective results).

Figure 10 :i f l o x y s t r o b i n t e f l u b e n z u r o n c a p t a n p y r id a b e n t e t r a c o n a z o l e t r i f l o x y s t r o b i n t e t r a c o n a z o l e t e f l u b e n z u r o n c a p t a n t e t r a c o n a z o l e t r i f l o x y s t r o b i n t r i f l o x y s t r o b i n t h i a c l o p r i d t h i a c l o p r i d

 10 Figure 10: Differences in the contents of pesticide residues in FE 1, FE 2, FE 3, FE 4, FE5 and FE 6 at the day of harvest (error bars express the expanded uncertainty of respective results).

  It should be noted that only very few studies are available in the scientific literature, none 25 of them covering the spectrum of pesticides involved in our study. Actually, the only 26 available data relevant to our experiment are those summarized in reports 27 (http://www.who.int/ipcs/publications/jmpr/en/) of the Joint Meeting of the FAO Panel of 28 Experts on Pesticide Residues in Food and Environment and the WHO Core Assessment 29

	10	1 in 2004). It
	11	should be noted that climatic conditions did not differ significantly in this crop year from
	12	previous ones.
	13
	14	Considering large variation (both qualitative and quantitative) in contamination patterns
	15	among apples supplied by individual farmers, the need to harmonize treatment plans with
	16	the aim to further improve crops quality become evident.
	17
	18	Degradation of pesticide residues in pre-harvest interval
	19	To get more knowledge on the fate of individual pesticides, extensive experiments were
	20	carried out in 2004, see Table II. All 19 pesticides studied covered the range of
	21	preparations most commonly applied within apple trees treatment during the pre-harvest
	22	interval. However, there are also other registered pesticide preparations which are
	23	conceivably used for apple trees protection.
	24
	30	Group of Pesticide Residues, JMPR (similar data have to be submitted by applicants for
	31	active ingredients review process currently dealt by European Food Safety Authority,
	32	EFSA).

Table I :

 I Overview of pesticide preparations used in field experiments and physicochemical properties of their active ingredients

	Mode of action	Commercial product	Active ingredient (content)	Content in preparation	MRL (mg kg -1 )	Safety period (days)	Physico-chemical properties of active ingredient Molecular weight log K OW Water solubility (mg l -1 )
		Alsystin	triflumuron	480 g/l	1	28	358.7	4.91 (20 °C)	0.025 (20°C)
		Aztec 140 EW	triazamate	140 g/l	0.1	7	314.4	2.15 (pH 7, 25 °C)	399 (pH 7, 25°C)
		Calypso 480 SC	thiacloprid	480 g/l	0.3	14	252.7	-	185 (20 °C)
	insecticides	Nomolt 15 SC Oleoekol Trebon 10 F	teflubenzuron chlorpyrifos coleseed oil etofenprox	150 g/l 30 g/l 75% 100 g/l	0.5 0.5 -1	28 --28	381.1 350.6 376.5	4.30 (20°C) 4.7 7.05 (25°C)	0.019 (23°C) 1.4 (25°C) <1 (25°C)
		Insegar 25 Reldan 40 EC Sanmite 20 WP Discus Domark 10 EC Euparen Multi F o fenoxycarb chlorpyrifos-methyl pyridaben kresoxim-methyl tetraconazole tolylfluanid Foligreen agricultural micronutrient (N, P, K, 25% 400 g/l 20% 50% 100 g/l 50% r	0.05 0.5 0.1 0.2 0.5 1 B, Fe, Mn, Zn, Mg, Co, Cu, Mo) 60 301.3 28 322.5 42 364.9 35 313.4 14 372.1 7 347.3	4.07 (25°C) 4.24 6.37 (25°C) 3.40 (pH 7, 25°C) 3.56 (20°C) 3.90 (20°C)	7.9 (pH 7.55-7.84, 25°C) 2.6 (20°C) 0.012 (24°C) 2 (20°C) 156 (pH 7, 20°C) 0.9 (20°C)
		Hattrick	tebuconazole	10%	0.5	28	307.8	3.70 (20°C)	36 (pH 5-9, 20°C)
	fungicides	tolylfluanid Cu(OH) 2 cyprodinil captan difenoconazole Na 2 B 8 O 13 •4H 2 O Thiram Granuflo (F) thiram Champion 50W Chorus 75 WG Merpan 80 WG Score 250 EC Solubor Topas 100 EC (F) penconazole Zato 50 WG trifloxystrobin P e e 750g/kg 40% 77% 80% 250 g/l > 98 % 80% 100 g/l 500 g/kg r	1 10 1 3 0.02 -0.2 0.5	28 -28 35 49 -14 35 14	97.6 225.3 300.6 406.3 412.52 240.4 284.2 408.4	-4.0 (pH 9.0, 25°C) 2.80 (25°C) 4.20 (25°C) 1.73 3.72 (25°C) 4.50 (25°C)	2.9 mg/l (pH 7, 25°C) 20 (pH 5.0, 25°C) 3.3 (25°C) 15 (25°C) 9.7% (20°C) 18 (room temperature) 73 (20°C) 610 (25 °C)
				R e v i e w
							O n
								l y

Table II :

 II Field experiments: time-schedule of pesticide preparations use and treatment

	rates
	FE 1	FE 2	FE 3	FE 4	FE 5	FE 6
	April 7
	April 19
	April 26
	May 5
	May 11
	May 20
	May 28
	Score 250 EC (0.2 l)	Score 250 EC (0.2 l)	Merpan 80 WG (2.0 kg) Domark 10 EC (0.

Table III :

 III Optimised MS/MS transitions parameters

	Analyte	Transition (m/z)	Cone ( V )	Colision ( V )
	diflubenzuron	311 > 158	25	10
		311 > 141	25	29
	etofenprox	394 > 177	20	14
		394 > 135	20	26
	pyrimethanil teflubenzuron F o	200 > 107 200 > 82 381 > 158	54 54 23	24 24 13
	r	381 > 141	23	13
	thiacloprid triflumuron P e e r 253 > 126 253 > 186 359 > 156 359 > 139	35 35 29 29	25 13 16 30
		R e v i e w
			O n
				l y

Table IV :

 IV Performance characteristics and monitored ions (m/z) of the analytical methods employed for apples analysis

	Analyte	Method	Quantitation ion (m/z)	Confirmation ions (m/z)	LOD (mg kg -1 )	Repeatability (RSD, %) mg kg -1 at 0.100	Recovery (%)
	captan	GC-MS	149		79, 264	0.003	9	92
	cyprodinil	GC-MS	224		210, 225	0.002	17	87
	difenoconazole	GC-MS	323		207, 267, 281	0.002	4	95
	etofenprox fenoxycarb chlorpyrifos chlorpyrifos-methyl GC-MS LC-MS/MS GC-MS GC-MS F o	394 > 177 255 314 286		394 > 135 116, 186 199, 258 125, 288	0.004 0.003 0.003 0.003	8 10 8 7	94 97 94 93
	kresoxim-methyl	GC-MS r	206		116, 131	0.001	9	94
	penconazole	GC-MS	248		159, 161	0.002	10	110
	pyridaben pyrimethanil tebuconazole teflubenzuron tetraconazole thiacloprid	GC-MS LC-MS/MS P 200 > 107 147 GC-MS 250 LC-MS/MS 381 > 158 GC-MS 336 LC-MS/MS 253 > 126 e e r 125, 163, 252 117, 309 200 > 82 381 > 141.05 159, 338 253 > 186	0.003 0.004 0.002 0.004 0.001 0.004	5 9 8 7 5 6	72 87 89 86 90 94
	tolylfluanid	GC-MS	137		181, 238	0.003	5	100
	triazamate trifloxystrobin triflumuron	GC-MS GC-MS LC-MS/MS	314 131 359 > 156	227, 242, 262 116, 222 359 > 139 R e v i 0.002 0.001 0.004 e w	8 8 6	103 98 89
						O n
							l y

days e laps e d afte r the las t Sanm ite 20 WP application

  2, FE 3 and FE 4 (error bars express the expanded uncertainty of respective results). * Safety period (35 days) has not elapsed yet The dynamics of pyridaben decrease between samplings in FE 1 and FE 2 (error bars express the expanded uncertainty of respective results). The dynamics of chlorpyrifos-methyl decrease between samplings in FE 1, FE 3 and FE 5 (error bars express the expanded uncertainty of respective results).

	0 0.02 0.04 0.06 0.08 0.1 0.12 SAM PLING DATES: concentration (mg/kg) EXPERIM ENTS: Baby food MRL F FE 2 33 o r 33 FE 4 Augus t 2 54 FE 3 P e 67 e FE1 47 r Figure 3: 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 14 14 concentration (mg/kg) FE 2 FE1 Augus t 2 SAM PLING DATES: EXPERIM ENTS: MRL Baby food MRL F o r P 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 concentration (mg/kg) Augus t 2 SAM PLING DATES: Baby food Figure 4: 0 14 14 33 28 MRL e r EXPERIM ENTS: FE 3 FE1 FE 5 e	47 Augus t 16 68 28 Augus t 16 Augus t 16 28	81 28 47	61 days e laps e d afte r the las t M e rpan 80 WG application 61 82 95 96 96 130 117 Augus t 30 Octobe r 5 harve s t 42 42 78 78 Augus t 30 Octobe r 5 harve s t Augus t 30 Octobe r 5 harve s t 42 42 61 78 78 97 days e
	R e v i e w R e v i e w
				O n O n
				l y l y

* Safety period (42 days) has not elapsed yet

laps e d afte r the las t Re ldan 40 EC application

  * Safety period (28 days) has not elapsed yet

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

Figure 5 :

 5 The dynamics of teflubenzuron decrease between samplings in FE 2, FE 3 and FE 4 (error bars express the expanded uncertainty of respective results).

		0.22
		0.2
		0.18
	concentration (mg/kg)	0.04 0.06 0.08 0.1 0.12 0.14 0.16
		0.02
		0
		14	33	33	28	47	47	42	61	61	78	97	97
								days e
		FE 2	FE 3	FE 4

laps e d afte r the las t Nom olt 15 SC application

  The dynamics of thiacloprid decrease between samplings in FE 1, FE 2, FE 3, FE 4, FE 5 and FE 6 (error bars express the expanded uncertainty of respective results).

	Augus t 2 r F SAM PLING DATES: EXPERIM ENTS: Baby food MRL o P FE 5 FE 4 FE 6 EXPERIM ENTS: e r FE 3 13 33 83 83 83 83 27 47 97 97 97 97 41 61 111 111 111 111 76 96 146 146 146 146 Augus t 16 Augus t 30 Octobe r 5 harve s t days e laps e d afte r the las t Calyps o 480 SC application e 0.005 0.015 0.02 0.025 0.03 0.035 0.04 concentraton (mg/kg) Augus t 2 Augus t 16 Augus t 30 Octobe r 5 harve s t SAM PLING DATES: Baby food Figure 6: 0 0.01 MRL
	R e v i e w
	O n
	l y

days e laps e d afte r the las t Dom ark 10 EC application

  The dynamics of tetraconazole decrease between samplings in FE 1, FE 2, FE 3, FE 4 and FE 5 (error bars express the expanded uncertainty of respective results). Figure 9: The dynamics of trifloxystrobin decrease between samplings in FE 1, FE 2, FE 3, FE 4, FE 5 and FE 6 (error bars express the expanded uncertainty of respective results).

				Food Additives and Contaminants	Page 32 of 34
	035 0.04 SAM PLING DATES: concentration (mg/kg) EXPERIM ENTS: Baby food MRL F FE 5 o 55 Augus t 2 FE 6 r P e 55 e r 69 0.005 0.03 33 42 42 89 89 47 56 Augus t 16 69 56 103 103 61 days e laps e d afte r the las t Hattrick application 83 83 119 119 Augus t 30 Octobe r 5 harve s t 70 70 117 117 96 105 105 152 152 Augus t 2 Augus t 16 Augus t 30 Octobe r 5 harve s t SAM PLING DATES: EXPERIM ENTS: FE 5 FE1 FE 3 FE 4 FE 2 P r e e 0 0.005 33 42 74 74 74 74 47 56 88 88 88 88 61 70 102 102 102 102 96 105 137 137 137 137 Figure 8: 0 MRL 0.01 r 0.01 0.015 0.02 0.025 0.035 concentration (mg/kg) Baby food F o 0.015 0.02 0.025 0.03 concentration (mg/
	0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 SAM PLING DATES: concentration (mg/kg)	Augus t 2	Augus t 16 R e v i e w Augus t 30 R e v i e w O n O n	Octobe r 5 harve s t
	EXPERIM ENT:	FE 6	55	69	83 days e laps e d afte r the las t Hattrick application 119 l y l y

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kg) days e laps e d afte r the las t Zato 50 WG application

			Food Additives and Contaminants	Page 34 of 34
	SAM PLING DATES:		Augus t 2	Augus t 16	Augus t 30	Octobe r 5 harve s t
	F o
	Baby food MRL	r
		P
	EXPERIM ENTS:	FE1	FE 3 e FE 2 e FE 6 r	FE 4	FE 5
			R e v i e w
					O n
						l y

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