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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Integrated pest management of ‘Golden Delicious’ apples a
a
a
a
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A. Simončič , M. Stopar , Š. Velikonja Bolta , D. Bavčar , R. Leskovšek & H. Baša Česnik a
Agricultural Institute of Slovenia, Department for Agroecology and Natural Resources, Ljubljana, Slovenia Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: A. Simončič, M. Stopar, Š. Velikonja Bolta, D. Bavčar, R. Leskovšek & H. Baša Česnik (2015): Integrated pest management of ‘Golden Delicious’ apples, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2015.1035765 To link to this article: http://dx.doi.org/10.1080/19393210.2015.1035765
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part B DOI: 10.1080/19393210.2015.1035765
Integrated pest management of ‘Golden Delicious’ apples
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A. Simončič, M. Stopar, Š. Velikonja Bolta , D. Bavčar , R. Leskovšek, H. Baša Česnik*
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Abstract
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Monitoring of plant protection product (PPP) residues in ‘Golden Delicious’ apples was performed in 2011– 2013, where 216 active substances were analysed with 3 analytical methods. Integrated pest management production (IPM) and improved IPM production were compared. Results were in favour of improved IPM production. Some active compounds determined in IPM production (boscalid, pyraclostrobin, thiacloprid and thiametoxam) were not found in improved IPM production. Besides that, in 2011 and 2012 captan residues were lower in improved IPM production. Risk assessment was also performed. Chronic exposure of consumers was low in general, but showed no major differences for IPM and improved IPM production for active substances determined in both types of production. Analytical results were compared with the EU report of 2010 where 1.3 % of apple samples exceeded MRLs, while MRL exceedances were not observed in this survey.
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Keywords: apples; Golden Delicious; pesticide residues; integrated pest management; Slovenia.
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Introduction Agricultural production in Europe has been intensified in recent decades. Intensive agriculture is characterised by high productivity, high inputs (i.e. pesticides, fertilisers and water), increased mechanisation and more simplified cropping sequences where the crop choice is often market driven. However, the long-term sustainability and environmental consequences of the intensification of agricultural systems have raised concerns in terms of negative consequences, such as increased soil erosion, decreased soil fertility, reduced farmland biodiversity, pollution of groundwater and eutrophication of rivers and lakes, and negative global consequences including the emission of greenhouse gasses (Vasileiadis et al. 2011). Reduction of pesticide use and risk is one of the key issues of the European Union’s (EU) agenda for agriculture. In 2009 EU has adopted Directive 2009/128/EC regarding the sustainable use of pesticides. The objective of this Directive is to reduce dependence on, as well as the risks and adverse impacts of pesticide use on human health and the environment, and a key element to reach this goal is to promote the implementation of IPM, which will become compulsory in the EU by the end of 2014. Finally, fruit growers as well as other exporters of agricultural products are facing strict food safety standards. For product marketing 2 aspects need to be considered: cheaper food production, with the aim to reduce final price of the product and fulfilling trading standards. The customers and merchants are demanding and producers need to earn their trust by offering safe and healthy products at reasonable price. According to Directive2009/128/EC, IPM means careful consideration of all available plant protection methods and subsequent integration of appropriate measures that discourage the development of populations of harmful organisms and keep the use of plant protection products and other forms of intervention to levels that are economically and ecologically justified and reduce or minimise risks to human health and the environment. IPM
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emphasises the growth of a healthy crop with the least possible disruption to agro-ecosystems and encourages natural pest control mechanisms. In broad terms, IPM is the co-ordination of all known chemical, cultural and biological control methods in such a way as to maximise total benefits and minimise harmful side efects that can arise from the exclusive use of chemical pesticides. In simple terms, IPM is applied ecology (Finch and Collier 2000), but it may include chemical control. However, pesticides are only allowed to be used as a last resort when other control methods have failed to achieve satisfactory control. IPM approaches have succeeded in reducing the use of a number of broad-spectrum pesticides and have also been shown to be more economical than conventional methods (Timprasert et al. 2014). In our orchard Brdo pri Lukovici (46 °19 ' N, 14 °67 ' E), IPM has been introduced in 1994. In recent years new groups of pesticides have been developed for plant protection of fruits, which usually have less unfavorable impacts on human health and the environment. For this reason different pesticides as well as spraying programs have been tested at our institute to reduce the residues of plant protection products (PPPs) and dependence on PPPs. In this study IPM and improved IPM have been studied and evaluated (Ministry of Agriculture Forestry and Food, MAFF 2011-2013). Improved IPM includes spraying programs according to IPM guidelines until the end of July and then only PPPs allowed in organic production are used. This leads to lower production costs, lower burden to the environment and lower PPP residues in fruit. The apple (Malus × domestica Borkh.) cultivar ‘Golden Delicious’ is one of the most important apple varieties in fruit growing areas of the world. It is widely cultivated for the export (Buron-Moles et al. 2014) and is one of the main produce in the Brdo pri Lukovici orhard as well. ‘Golden Delicious’ was a chance seedling, perhaps of ‘Golden Reinette’ and ‘Grimes Golden,’ introduced about 1914. Flavor is sweet, spicy and moderately acidic. It is firm at harvest, with a tendency to soften in storage. ‘Golden Delicious’ has tender golden yellow to greenish yellow skin with a tendency to russet, yellowish white flesh and has relatively low browning potential (Abbott et al. 2004). ‘Golden Delicious’ apples are appreciated for their productivity and can be stored while maintaining good quality for a long time after harvest. Moreover, they are preferred by consumers because they are known to be sweet and crisp (Ciesa et al. 2013). ‘Golden Delicious’ is very resistant to apple powdery mildew (Podosphaera levcotricha), but very sensitive to scab (Venturia inaequalis). This variety is also attacked by numerous pests like codling moth (Laspeyresia pomonella), apple aphid (Aphis pomi), fruit tree red spider mite (Panonychus ulmi) and apple sawfly (Hoplocampa testudinea). Their occurence depend on region where apples are grown and climatic conditions, mainly solar radiation and rainfall. To produce quality fruit, use of PPPs is inevitable. Besides that, comparison of ‘Golden Delicious’ production systems showed that ‘Golden Delicious’ apples from IPM and improved IPM production had higher weight and sugar content than organically produced ones (Jakopič et al. 2012). IPM production ensures low incidence and low levels of residues in fruits, but consumers wish further reduction of PPP residues to minimum level possible, for which improved IPM should be applied. From consumer point of view, monitoring PPP residues in products offered on the market are inevitable. In literature different methods for PPP residues determination in apples are described, consisting of 3 steps: extraction, cleanup and determination. Mainly there are 3 approaches for extraction and cleanup: (i) The first one is liquid-liquid extraction with ethyl acetate (Štěpánet al. 2004; Ticha et al. 2008), acetone (Szpyrka and Walorczyk 2013), acetonitrile (Prasad et al. 2013; Ticha et al. 2008), a mixture of acetone-dichloromethanehexane (50:20:30, v/v/v) (Lacassie et al. 1999) or a mixture of methanol-water (1:1, v/v) (Sanagi et al. 2013). The extraction is usually followed by cleanup using solid phase extraction cartridges (SPE) packed with florisil (Szpyrka et al. 2013) or C 18 (Sanagi et al. 2013) or using gel permeation chromatography (GPC) (Štěpánet al. 2004; Ticha et al. 2008; Zrostlíková et al. 2002); (ii) The second approach is the QuEChERS method (Quick Easy Cheap Effective Rugged and Safe method) which includes extraction with small volumes of acetonitrile and dispersive solid phase extraction (dSPE) cleanup with primary secondary amine (PSA) (Bakirci et al. 2014; Camino-Sánchez et al. 2011; Cervera et al. 2012; Han et al. 2014; Kwon et al. 2012; Malhat et al. 2013; Poulsen et al. 2012; Sinha et al. 2012; Walorczyk 2012), graphitized carbon black (GCB) (Kwon et al. 2012; Malhat et al. 2013) and/or C 18 (Kwon et al. 2012); (iii) The third approach is solid phase microextraction (SPME), where extraction and cleanup are performed at the same time (Abdulra'uf and Tan 2013; Song et al. 2013). The final step, determination, can be performed by gas chromatography (GC) or by liquid chromatography (LC). Gas chromatograph can be coupled with electron-capture detector (ECD) (Bakirci et al. 2014; Han et al. 2014; Szpyrka and Walorczyk 2013; Štěpánet al. 2004), nitrogen-phosphorus detector (NPD) (Szpyrka and Walorczyk 2013; Štěpánet al. 2004), mass spectrometer (MS) (Abdulra'uf and Tan2013; Bakirci et al. 2014; Kwon et al. 2012; Štěpánet al. 2004; Ticha et al. 2008), time-of-flight mass spectrometer (TOF-MS) (Cervera et al. 2012) or tandem mass spectrometer (MS/MS) (Camino-Sánchez et al. 2011; Poulsen et al. 2012; Walorczyk 2012). Liquid chromatography can be coupled with UV detection (Prasad et al. 2013), photodiode array detection (DAD) (Malhat et al. 2013; Song et al. 2013), mass spectrometry (Lacassie et al. 1999) or MS/MS (Bakirci et al. 2014; Kwon et al. 2012; Poulsen et al. 2012; Sinha et al. 2012; Ticha et al. 2008; Zrostlíkováet al. 2002). Mass spectrometry is the only one giving unequivocal identification and tandem mass spectrometry means a further step providing greater sensitivity. In our laboratory a method is used, which includes determination of a wide range of active substances with different polarities: from very polar (e.g. methamidophos) to non-polar ones (e.g.
DDT). This is why extraction was performed with a mixture of three organic solvents: acetone, petroleum ether and dichloromethane. Clean up was performed with GPC, which separates molecules according to their size and is therefore universal. Finally, determination was performed with GC/MS and GC/MS/MS. Both instruments enable unequivocal qualitative and quantitative determination at the same time. In this study monitoring of PPP residues in ‘Golden delicious’apples was performed with the aim to present how improved IPM leads to production of fruit which contains lower PPP residues and is therefore safer for consumers.
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Materials and Methods Plant material and pesticides used The study started on 3-year-old ‘Golden Delicious’ apple trees grafted on M.9 rootstock grown in the experimental orchard Brdo of Agricultural Institute of Slovenia in central Slovenia (46 °19 ' N, 14 °67 ' E). Each experimental plot consisted of 15 trees grown in accordance with IPM and improved IPM productions. Trees from both production systems were planted on the same field at a distance of 20 m between rows to prevent spray drift in between. In 2011-2013 ‘Golden Delicious’ apples were attacked by scab (Venturia inaequalis), codling moth (Cydia pomonella), apple aphid (Aphis pomi), fruit tree red spider mite (Panonychus ulmi) and apple sawfly (Hoplocampa testudinea). Numerous PPPs were used each year. Improved IPM included spraying programs according to IPM guidelines until the end of July and then only Madex (1 % granulosis virus) as insecticide and low dose rates of copper together with sulfur as fungicides were used for pest and disease control. Sampling In 2011, 2012 and 2013 apples were harvested at technical maturity. Fruits from IPM and improved IPM harvest were sampled at different dates and PPP residues were analysed at the time of harvest and after 14 days of storage at 4 °C. Apples were taken from the middle of 5 trees in each of these treatments (5 fruits from each tree, resulting in 25 fruits per laboratory sample). Six samples were taken in 2011, 2 in 2012 and 6 in 2013. All apples included in this study were of first quality, healthy and free of pests and diseases at the time of sampling. Upon arrival in the laboratory the apples were cut into quarters. Two opposite quarters were mixed and stirred in a homogeniser Stephan Mill (Stephan Machinery GmbH, 31789 Hameln, Germany). Homogenised laboratory samples were stored at -20 °C prior to chemical analyses. Analytical methods Apple samples were analysed for 216 different residues, using 3 methods: 1. Multiresidual GC/MS method for the determination of 97 compounds, as listed in Baša Česnik et al. (2015). Extraction was performed by a mixture of acetone, petroleumether and dichlorometane, cleaned-up by gel permeation chromatography and determined by GC/MS (Baša Česnik and Gregorčič 2003; Baša Česnik et al. 2006). 2. GC/MS method for the determination of the dithiocarbamates maneb, mankozeb, metiram, propineb and zineb. The sum is expressed as carbon disulfide. Samples were heated in a two-phase system isooctane/stannous (II) chloride in diluted hydrochloric acid. The produced carbon disulfide was dissolved in the organic phase (iso-octane) and determined by GC/MS (Baša Česnik and Gregorčič 2006; Baša Česnik et al. 2006). 3. Multiresidual LC/MS/MS method for the determination of 118 compounds as listed in Baša Česnik et al. (2015). Extraction was performed by a mixture of acetone, petroleumether and dichlorometane, cleaned-up by gel permeation chromatography and determined by LC/MS/MS (Bossi et al. 2002; Lehotay et al. 2005; Ortelli et al. 2004). Trueness of methods was verified from recoveries in each batch of analyses: two spiked samples of apples at a level of 0.2 mg kg-1 for GC/MS analyses and one spiked sample of apples at a level of 0.05 mg kg-1 for LC/MS/MS analyses. Recoveries had to be 70 % - 110 %, except for active compounds which were not found. Trueness was also verified by participation in the inter-laboratory proficiency testing scheme of BIPEA (Bureau InterProfessionnel d´Etudes Analytiques), with z-values from 0.0-1.7 in apple matrix in 2011 and with z-values from -1.1 to 0.6 in 2013 (BIPEA 2011 and 2013). In January 2005, a range of analyses covering pesticide residues was accredited by the French accreditation body COFRAC (Comité Français d'Acréditation) which was in 2012 transferred to the Slovenian accreditation body. Limits of detection (LODs) and of quantification (LOQs) were calculated from signal to noise ratios (S/N) of matrix match standards. LOD is defined as S/N=3 and LOQ as S/N=10. For determination of precision (ISO 5725 1994) repeatability and reproducibility of spiked apple samples were analysed. Within a 10 days period 2 spiked samples were prepared each day. Both were injected twice. The standard deviation of repeatability of the level (s r ) and standard deviation of reproducibility of the level (s R ) were both calculated. Uncertainty of repeatability (U r ) and uncertainty of reproducibility (U R ) were calculated by multiplying the standard deviation of repeatability and standard deviation of reproducibility by Student’s t factor for 9 degrees of freedom and 95%
confidence level (t 95;9 = 2.262) as U r = t 95; 9 x s r and U R = t 95; 9 x s R . Uncertainty was calculated for a range of active substances (mainly accredited ones) for pome fruits. Risk assessment The risk assessment (chronic exposure) was calculated with EU EFSA PRIMo model version 2 (EFSA 2009).
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Results and discussion In 2011-2013 active substances determined in apple samples were: acetamiprid, boscalid, captan, cyprodinil, dithiocarbamates, methoxyfenozide, pirimicarb, pyraclostrobin, spirodiclofen, thiacloprid and thiamethoxam. Limits of quantification (LOQs), recoveries and data about used PPPs which contain active substances found are presented in Table 1. MRL values were never exceeded. Among active substances found, in 2011 in apple samples active substances methoxyfenozide, spirodiclofen and thiamethoxam were not found in improved IPM production, contrary to IPM production (Table 2). In the 2012 samples captan and thiacloprid were not determined in improved IPM production in contrast to the IPM production (Table 3). In the 2013 samples the same pattern was repeated for boscalid and pyraclostrobin (Table 4). In 2011 and 2012 captan residues diminished with improved IPM. The most persistent pesticide during this 3 year monitoring period was pirimicarb. Cool storage did not influence significantly the content of PPP residues for all period. Comparing number of samples in certain concentration ranges for IPM and improved IPM production in the years 20112013 showed that for captan lower number of samples with residues in ranges 0.1 - 0.5 mg kg -1 and 0.51 - 1.0 mg kg -1 were observed for improved IPM. The same applies for spirodiclofen in ranges < 0.1 mg kg -1 and 0.1 0.5 mg kg -1 and for cyprodinil, methoxyfenozide, pyraclostrobin, thiacloprid, thiamethoxam
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Integrated pest management of ‘Golden Delicious’ apples a
a
a
a
a
a
A. Simončič , M. Stopar , Š. Velikonja Bolta , D. Bavčar , R. Leskovšek & H. Baša Česnik a
Agricultural Institute of Slovenia, Department for Agroecology and Natural Resources, Ljubljana, Slovenia Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: A. Simončič, M. Stopar, Š. Velikonja Bolta, D. Bavčar, R. Leskovšek & H. Baša Česnik (2015): Integrated pest management of ‘Golden Delicious’ apples, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2015.1035765 To link to this article: http://dx.doi.org/10.1080/19393210.2015.1035765
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis Journal: Food Additives & Contaminants: Part B DOI: 10.1080/19393210.2015.1035765
Integrated pest management of ‘Golden Delicious’ apples
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A. Simončič, M. Stopar, Š. Velikonja Bolta , D. Bavčar , R. Leskovšek, H. Baša Česnik*
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Abstract
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Monitoring of plant protection product (PPP) residues in ‘Golden Delicious’ apples was performed in 2011– 2013, where 216 active substances were analysed with 3 analytical methods. Integrated pest management production (IPM) and improved IPM production were compared. Results were in favour of improved IPM production. Some active compounds determined in IPM production (boscalid, pyraclostrobin, thiacloprid and thiametoxam) were not found in improved IPM production. Besides that, in 2011 and 2012 captan residues were lower in improved IPM production. Risk assessment was also performed. Chronic exposure of consumers was low in general, but showed no major differences for IPM and improved IPM production for active substances determined in both types of production. Analytical results were compared with the EU report of 2010 where 1.3 % of apple samples exceeded MRLs, while MRL exceedances were not observed in this survey.
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Keywords: apples; Golden Delicious; pesticide residues; integrated pest management; Slovenia.
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Introduction Agricultural production in Europe has been intensified in recent decades. Intensive agriculture is characterised by high productivity, high inputs (i.e. pesticides, fertilisers and water), increased mechanisation and more simplified cropping sequences where the crop choice is often market driven. However, the long-term sustainability and environmental consequences of the intensification of agricultural systems have raised concerns in terms of negative consequences, such as increased soil erosion, decreased soil fertility, reduced farmland biodiversity, pollution of groundwater and eutrophication of rivers and lakes, and negative global consequences including the emission of greenhouse gasses (Vasileiadis et al. 2011). Reduction of pesticide use and risk is one of the key issues of the European Union’s (EU) agenda for agriculture. In 2009 EU has adopted Directive 2009/128/EC regarding the sustainable use of pesticides. The objective of this Directive is to reduce dependence on, as well as the risks and adverse impacts of pesticide use on human health and the environment, and a key element to reach this goal is to promote the implementation of IPM, which will become compulsory in the EU by the end of 2014. Finally, fruit growers as well as other exporters of agricultural products are facing strict food safety standards. For product marketing 2 aspects need to be considered: cheaper food production, with the aim to reduce final price of the product and fulfilling trading standards. The customers and merchants are demanding and producers need to earn their trust by offering safe and healthy products at reasonable price. According to Directive2009/128/EC, IPM means careful consideration of all available plant protection methods and subsequent integration of appropriate measures that discourage the development of populations of harmful organisms and keep the use of plant protection products and other forms of intervention to levels that are economically and ecologically justified and reduce or minimise risks to human health and the environment. IPM
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emphasises the growth of a healthy crop with the least possible disruption to agro-ecosystems and encourages natural pest control mechanisms. In broad terms, IPM is the co-ordination of all known chemical, cultural and biological control methods in such a way as to maximise total benefits and minimise harmful side efects that can arise from the exclusive use of chemical pesticides. In simple terms, IPM is applied ecology (Finch and Collier 2000), but it may include chemical control. However, pesticides are only allowed to be used as a last resort when other control methods have failed to achieve satisfactory control. IPM approaches have succeeded in reducing the use of a number of broad-spectrum pesticides and have also been shown to be more economical than conventional methods (Timprasert et al. 2014). In our orchard Brdo pri Lukovici (46 °19 ' N, 14 °67 ' E), IPM has been introduced in 1994. In recent years new groups of pesticides have been developed for plant protection of fruits, which usually have less unfavorable impacts on human health and the environment. For this reason different pesticides as well as spraying programs have been tested at our institute to reduce the residues of plant protection products (PPPs) and dependence on PPPs. In this study IPM and improved IPM have been studied and evaluated (Ministry of Agriculture Forestry and Food, MAFF 2011-2013). Improved IPM includes spraying programs according to IPM guidelines until the end of July and then only PPPs allowed in organic production are used. This leads to lower production costs, lower burden to the environment and lower PPP residues in fruit. The apple (Malus × domestica Borkh.) cultivar ‘Golden Delicious’ is one of the most important apple varieties in fruit growing areas of the world. It is widely cultivated for the export (Buron-Moles et al. 2014) and is one of the main produce in the Brdo pri Lukovici orhard as well. ‘Golden Delicious’ was a chance seedling, perhaps of ‘Golden Reinette’ and ‘Grimes Golden,’ introduced about 1914. Flavor is sweet, spicy and moderately acidic. It is firm at harvest, with a tendency to soften in storage. ‘Golden Delicious’ has tender golden yellow to greenish yellow skin with a tendency to russet, yellowish white flesh and has relatively low browning potential (Abbott et al. 2004). ‘Golden Delicious’ apples are appreciated for their productivity and can be stored while maintaining good quality for a long time after harvest. Moreover, they are preferred by consumers because they are known to be sweet and crisp (Ciesa et al. 2013). ‘Golden Delicious’ is very resistant to apple powdery mildew (Podosphaera levcotricha), but very sensitive to scab (Venturia inaequalis). This variety is also attacked by numerous pests like codling moth (Laspeyresia pomonella), apple aphid (Aphis pomi), fruit tree red spider mite (Panonychus ulmi) and apple sawfly (Hoplocampa testudinea). Their occurence depend on region where apples are grown and climatic conditions, mainly solar radiation and rainfall. To produce quality fruit, use of PPPs is inevitable. Besides that, comparison of ‘Golden Delicious’ production systems showed that ‘Golden Delicious’ apples from IPM and improved IPM production had higher weight and sugar content than organically produced ones (Jakopič et al. 2012). IPM production ensures low incidence and low levels of residues in fruits, but consumers wish further reduction of PPP residues to minimum level possible, for which improved IPM should be applied. From consumer point of view, monitoring PPP residues in products offered on the market are inevitable. In literature different methods for PPP residues determination in apples are described, consisting of 3 steps: extraction, cleanup and determination. Mainly there are 3 approaches for extraction and cleanup: (i) The first one is liquid-liquid extraction with ethyl acetate (Štěpánet al. 2004; Ticha et al. 2008), acetone (Szpyrka and Walorczyk 2013), acetonitrile (Prasad et al. 2013; Ticha et al. 2008), a mixture of acetone-dichloromethanehexane (50:20:30, v/v/v) (Lacassie et al. 1999) or a mixture of methanol-water (1:1, v/v) (Sanagi et al. 2013). The extraction is usually followed by cleanup using solid phase extraction cartridges (SPE) packed with florisil (Szpyrka et al. 2013) or C 18 (Sanagi et al. 2013) or using gel permeation chromatography (GPC) (Štěpánet al. 2004; Ticha et al. 2008; Zrostlíková et al. 2002); (ii) The second approach is the QuEChERS method (Quick Easy Cheap Effective Rugged and Safe method) which includes extraction with small volumes of acetonitrile and dispersive solid phase extraction (dSPE) cleanup with primary secondary amine (PSA) (Bakirci et al. 2014; Camino-Sánchez et al. 2011; Cervera et al. 2012; Han et al. 2014; Kwon et al. 2012; Malhat et al. 2013; Poulsen et al. 2012; Sinha et al. 2012; Walorczyk 2012), graphitized carbon black (GCB) (Kwon et al. 2012; Malhat et al. 2013) and/or C 18 (Kwon et al. 2012); (iii) The third approach is solid phase microextraction (SPME), where extraction and cleanup are performed at the same time (Abdulra'uf and Tan 2013; Song et al. 2013). The final step, determination, can be performed by gas chromatography (GC) or by liquid chromatography (LC). Gas chromatograph can be coupled with electron-capture detector (ECD) (Bakirci et al. 2014; Han et al. 2014; Szpyrka and Walorczyk 2013; Štěpánet al. 2004), nitrogen-phosphorus detector (NPD) (Szpyrka and Walorczyk 2013; Štěpánet al. 2004), mass spectrometer (MS) (Abdulra'uf and Tan2013; Bakirci et al. 2014; Kwon et al. 2012; Štěpánet al. 2004; Ticha et al. 2008), time-of-flight mass spectrometer (TOF-MS) (Cervera et al. 2012) or tandem mass spectrometer (MS/MS) (Camino-Sánchez et al. 2011; Poulsen et al. 2012; Walorczyk 2012). Liquid chromatography can be coupled with UV detection (Prasad et al. 2013), photodiode array detection (DAD) (Malhat et al. 2013; Song et al. 2013), mass spectrometry (Lacassie et al. 1999) or MS/MS (Bakirci et al. 2014; Kwon et al. 2012; Poulsen et al. 2012; Sinha et al. 2012; Ticha et al. 2008; Zrostlíkováet al. 2002). Mass spectrometry is the only one giving unequivocal identification and tandem mass spectrometry means a further step providing greater sensitivity. In our laboratory a method is used, which includes determination of a wide range of active substances with different polarities: from very polar (e.g. methamidophos) to non-polar ones (e.g.
DDT). This is why extraction was performed with a mixture of three organic solvents: acetone, petroleum ether and dichloromethane. Clean up was performed with GPC, which separates molecules according to their size and is therefore universal. Finally, determination was performed with GC/MS and GC/MS/MS. Both instruments enable unequivocal qualitative and quantitative determination at the same time. In this study monitoring of PPP residues in ‘Golden delicious’apples was performed with the aim to present how improved IPM leads to production of fruit which contains lower PPP residues and is therefore safer for consumers.
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Materials and Methods Plant material and pesticides used The study started on 3-year-old ‘Golden Delicious’ apple trees grafted on M.9 rootstock grown in the experimental orchard Brdo of Agricultural Institute of Slovenia in central Slovenia (46 °19 ' N, 14 °67 ' E). Each experimental plot consisted of 15 trees grown in accordance with IPM and improved IPM productions. Trees from both production systems were planted on the same field at a distance of 20 m between rows to prevent spray drift in between. In 2011-2013 ‘Golden Delicious’ apples were attacked by scab (Venturia inaequalis), codling moth (Cydia pomonella), apple aphid (Aphis pomi), fruit tree red spider mite (Panonychus ulmi) and apple sawfly (Hoplocampa testudinea). Numerous PPPs were used each year. Improved IPM included spraying programs according to IPM guidelines until the end of July and then only Madex (1 % granulosis virus) as insecticide and low dose rates of copper together with sulfur as fungicides were used for pest and disease control. Sampling In 2011, 2012 and 2013 apples were harvested at technical maturity. Fruits from IPM and improved IPM harvest were sampled at different dates and PPP residues were analysed at the time of harvest and after 14 days of storage at 4 °C. Apples were taken from the middle of 5 trees in each of these treatments (5 fruits from each tree, resulting in 25 fruits per laboratory sample). Six samples were taken in 2011, 2 in 2012 and 6 in 2013. All apples included in this study were of first quality, healthy and free of pests and diseases at the time of sampling. Upon arrival in the laboratory the apples were cut into quarters. Two opposite quarters were mixed and stirred in a homogeniser Stephan Mill (Stephan Machinery GmbH, 31789 Hameln, Germany). Homogenised laboratory samples were stored at -20 °C prior to chemical analyses. Analytical methods Apple samples were analysed for 216 different residues, using 3 methods: 1. Multiresidual GC/MS method for the determination of 97 compounds, as listed in Baša Česnik et al. (2015). Extraction was performed by a mixture of acetone, petroleumether and dichlorometane, cleaned-up by gel permeation chromatography and determined by GC/MS (Baša Česnik and Gregorčič 2003; Baša Česnik et al. 2006). 2. GC/MS method for the determination of the dithiocarbamates maneb, mankozeb, metiram, propineb and zineb. The sum is expressed as carbon disulfide. Samples were heated in a two-phase system isooctane/stannous (II) chloride in diluted hydrochloric acid. The produced carbon disulfide was dissolved in the organic phase (iso-octane) and determined by GC/MS (Baša Česnik and Gregorčič 2006; Baša Česnik et al. 2006). 3. Multiresidual LC/MS/MS method for the determination of 118 compounds as listed in Baša Česnik et al. (2015). Extraction was performed by a mixture of acetone, petroleumether and dichlorometane, cleaned-up by gel permeation chromatography and determined by LC/MS/MS (Bossi et al. 2002; Lehotay et al. 2005; Ortelli et al. 2004). Trueness of methods was verified from recoveries in each batch of analyses: two spiked samples of apples at a level of 0.2 mg kg-1 for GC/MS analyses and one spiked sample of apples at a level of 0.05 mg kg-1 for LC/MS/MS analyses. Recoveries had to be 70 % - 110 %, except for active compounds which were not found. Trueness was also verified by participation in the inter-laboratory proficiency testing scheme of BIPEA (Bureau InterProfessionnel d´Etudes Analytiques), with z-values from 0.0-1.7 in apple matrix in 2011 and with z-values from -1.1 to 0.6 in 2013 (BIPEA 2011 and 2013). In January 2005, a range of analyses covering pesticide residues was accredited by the French accreditation body COFRAC (Comité Français d'Acréditation) which was in 2012 transferred to the Slovenian accreditation body. Limits of detection (LODs) and of quantification (LOQs) were calculated from signal to noise ratios (S/N) of matrix match standards. LOD is defined as S/N=3 and LOQ as S/N=10. For determination of precision (ISO 5725 1994) repeatability and reproducibility of spiked apple samples were analysed. Within a 10 days period 2 spiked samples were prepared each day. Both were injected twice. The standard deviation of repeatability of the level (s r ) and standard deviation of reproducibility of the level (s R ) were both calculated. Uncertainty of repeatability (U r ) and uncertainty of reproducibility (U R ) were calculated by multiplying the standard deviation of repeatability and standard deviation of reproducibility by Student’s t factor for 9 degrees of freedom and 95%
confidence level (t 95;9 = 2.262) as U r = t 95; 9 x s r and U R = t 95; 9 x s R . Uncertainty was calculated for a range of active substances (mainly accredited ones) for pome fruits. Risk assessment The risk assessment (chronic exposure) was calculated with EU EFSA PRIMo model version 2 (EFSA 2009).
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Results and discussion In 2011-2013 active substances determined in apple samples were: acetamiprid, boscalid, captan, cyprodinil, dithiocarbamates, methoxyfenozide, pirimicarb, pyraclostrobin, spirodiclofen, thiacloprid and thiamethoxam. Limits of quantification (LOQs), recoveries and data about used PPPs which contain active substances found are presented in Table 1. MRL values were never exceeded. Among active substances found, in 2011 in apple samples active substances methoxyfenozide, spirodiclofen and thiamethoxam were not found in improved IPM production, contrary to IPM production (Table 2). In the 2012 samples captan and thiacloprid were not determined in improved IPM production in contrast to the IPM production (Table 3). In the 2013 samples the same pattern was repeated for boscalid and pyraclostrobin (Table 4). In 2011 and 2012 captan residues diminished with improved IPM. The most persistent pesticide during this 3 year monitoring period was pirimicarb. Cool storage did not influence significantly the content of PPP residues for all period. Comparing number of samples in certain concentration ranges for IPM and improved IPM production in the years 20112013 showed that for captan lower number of samples with residues in ranges 0.1 - 0.5 mg kg -1 and 0.51 - 1.0 mg kg -1 were observed for improved IPM. The same applies for spirodiclofen in ranges < 0.1 mg kg -1 and 0.1 0.5 mg kg -1 and for cyprodinil, methoxyfenozide, pyraclostrobin, thiacloprid, thiamethoxam
