Detection of Bactrocera oleae (Diptera: Tephritidae) DNA in the gut of the soil species Pseudoophonus rufipes (Coleoptera: Carabidae)

Pest control service provided by natural enemies of Bactrocera oleae, the key pest of the olive tree, is nowadays recognized as fundamental. B. oleae has developed resistance to common insecticides, and negative effects both on consumers' health and non-target species arc the major drawbacks of conventional control strategics. Carabid beetles are potential B. oleae pupae predators, but their predation on field still need to be assessed. We tested adult Pseudoophonus rufipes, a species known to be active in olive orchard when pest pupae are abundant in the soil, in order to detect B. oleae pupae consumption at different post feeding times for both male and female carabids. An already existing protocol was used for detecting B. oleae mtDNA sequences of the cytochrome oxidase subunit I gene in carabids' gut, and its versatility improved. B. oleae mtDNA was detected up to 20 h after pupa ingestion with a high percentage of success, without significant differences between sexes and pair primers used. Prey DNA extraction was tested from both dissected and non-dissected carabids, obtaining comparable results. The trapping system used to collect carabids for molecular assays and the new elements introduced in the protocol represent cost-effective solutions that may be beneficial for future laboratory trials and, mostly, for the analysis of field-collected predators. Fostering the investigation of soil predators in olive orchard may increase the design of conservation control strategies against B. oleae.Scuola Superiore Sant’Anna, Pisa, Italy (PhD Programme in Agrobiodiversity)

Development of an analyttical method and its application for the determination of selected pesticides in olives and olive oil on the transformation process of olives into oil

In the present study a multiresidue determination for the analysis of 35 pesticides from different chemical families in olive oil, olives and olive mill waste waters was developed and validated. The analytical methods for the multiresidue determination were stepwise developed, optimized and validated. For this purpose different liquid-liquid extraction (LLE) procedures were tested whereas different experimental tests were performed in order to study the efficiency of different clean up procedures with N-Alumina, Florisil, C-18, ENVI-Carb, phenyl bonded silica (Ph), diol bonded silica (Diol), cyanopropyl bonded silica (CN), and amino propyl bonded silica (NEL) solid phase extraction (SPE) cartridges with respect specific detection of analytics by GC-NPD and GC-ECD. The methods developed feature good analytical characteristics (high recoveries, low LOD and high precision) and were applied with success in the determination of selected pesticides in real olive oil, olive and olive mill waste water samples from different olive oil production areas in Greece. A selected ion monitoring method was also developed for the confirmation of results by GC-MS. Processing factors of the pesticides persist on raw olives in olive oil production as well as the influence of water addition during olive oil extraction in these processing factors was the sight of the last part of the present study.Στην παρούσα διατριβή αναπτύχθηκαν μέθοδοι προσδιορισμού 35 φυτοφαρμάκων στο ελαιόλαδο, τον ελαιόκαρπο και τα υδατικά απόβλητα ελαιοτριβείου (ΥΑΕ) χρησιμοποιώντας την τεχνική της υγρού-υγρού εκχύλισης (LLE) και τον ακόλουθο καθαρισμό του λιπαρού εκχυλίσματος με την τεχνική της εκχύλισης δια της στερεάς φάσης (SPE) σε συνδυασμό με αέρια χρωματογραφία σε δύο εκλεκτικούς ανιχνευτές (GC-NPD, GC-ECD), δοκιμάζοντας συνολικά οκτώ προσροφητικά υλικά (ENVI-Carb, N-Alumina, Florisil, C18, Ph, Diol, CN, ΝΗ₂). Η LLE του εξανικού διαλύματος ελαιολάδου με διαλύτη εκχύλισης ακετονιτρίλιο και ο καθαρισμός του εκχυλίσματος στην μικροστήλη ENVI-Carb, έδωσε τα ικανοποιητικότερα αποτελέσματα για όλες τις επιλεχθείσες ενώσεις. Για τον προσδιορισμό των ενώσεων με GC-ECD στη μέθοδο ενσωματώθηκε και ένα επιπλέον στάδιο καθαρισμού κατά το οποίο το εκχύλισμα μετά τον καθαρισμό σε μικροστήλη ENVI-Carb υποβάλλεται σε νέο καθαρισμό με τη χρήση μικροστήλης Diol. Οι μέθοδοι που αναπτύχθηκαν παρουσιάζουν χαμηλά όρια ανίχνευσης, υψηλές ανακτήσεις και υψηλή ακρίβεια για όλους τους αναλυτές και εφαρμόστηκαν με επιτυχία για τη μελέτη της υπολειμματικότητας των 35 δραστικών ουσιών στο ελαιόλαδο των Δ.Ο. Σε δείγματα ελαιολάδου διαφορετικών κατηγοριών και από διαφορετικές ελαιοπαραγωγικές περιοχές της Ελλάδας, εκτίμηση του κινδύνου των καταναλωτών από τα υπολείμματα που ανιχνεύτηκαν και προσδιορισμός των συντελεστών συγκέντρωσης επιλεγμένων ενώσεων στο ελαιόλαδο κατά τις πιο διαδεδομένες και σύγχρονες διεργασίες παραλαβής του ελαιολάδου: με φυγοκέντρηση σε οριζόντιο φυγοκεντρικό σύστημα τριών (Π3), δύο (Π1) και δυόμιση (Π2) φάσεων

Chemometric study and optimization of extraction parameters in single-drop microextraction for the determination of multiclass pesticide residues in grapes and apples by gas chromatography mass spectrometry

A simple and rapid single-drop microextraction method coupled with gas chromatography and mass spectrometry (SDME-GC/MS) for the determination of 20 pesticides with different physicochemical properties in grapes and apples was optimized by the use of a multivariate strategy. Emphasis on the optimization study was given to the role of ionic strength, sugar concentration and pH of the donor sample solution prepared from the fruit samples. Since all three variables were found to affect negatively SDME (a lower extraction efficiency was observed as the values of variables were increased for most of the pesticides studied), donor sample solution was optimized using a central composite design to evaluate the optimum pH value and the optimum dilution of the sample extract. With some exceptions (chlorpyrifos ethyl, alpha-endosulfan, beta-endosulfan, pyriproxyfen, gamma-cyhalothrin and bifenthrin), the optimum method included the dilution of the analytical sample by 12.5-fold with a buffered acetone/water solution at pH = 4 and exhibited good analytical characteristics for the majority of target analytes (pyrimethanil, pirimicarb, metribuzin, vinclozolin, fosthiazate, procymidone, fludioxonil, kresoxim methyl, endosulfan sulfate, fenhexamid, iprodione, phosalone, indoxacarb and azoxystrobin) by providing high enrichment factors (14-328), low limits of detection (0.0003-0.007 mu g/g), and good precision (relative standard deviations below 15%). (C) 2009 Elsevier B.V. All rights reserved

Application of single-drop microextraction coupled with gas chromatography for the determination of multiclass pesticides in vegetables with nitrogen phosphorus and electron capture detection

In the present work the single-drop microextraction (SDME) technique coupled with GC-NPD and GC-ECD was evaluated for the determination of multi-class pesticides in vegetables. The donor sample solution preparation was optimized by testing different mixtures of solvents and dilutions with water. The SDME procedure was optimized by controlling drop organic solvent, drop volume, agitation, and exposure time. The optimum sample preparation was achieved with the use of a mixture of acetone/H(2)O (10/90, v/v) in donor sample Solution preparation and the consequent SDME using a toluene drop under mild stirring for 25 min. The efficiency of the extraction process was studied in fortified tomato and courgette samples and matrix effects were further estimated. The proposed method showed good linearity, limits of detection at the sub-mu g kg(-1) level and high precision (RSD <15%) and was applied with success in real vegetable samples showing that SDME can be a promising way for sample preparation in pesticide residue analysis. (C) 2008 Elsevier B.V. All rights reserved

Determination of Pesticide Residues in Honey by Single-Drop Microextraction and Gas Chromatography

A novel, simple, and rapid single-drop microextraction (SOME) procedure combined with GC has been developed, validated, and applied for the determination of multiclass pesticide residues in honey samples. The SDME was optimized using a Plackett-Burman screening design considering all parameters that may influence an SOME procedure and a consequent central composite design to control the parameters that were found to significantly influence the pesticide determination. The developed analytical method required minimal volumes of organic solvents and exhibited good analytical characteristics with enrichment factors ranging from 3 for alpha-endosulfan to 10 for lindane, procymidone, and captan and method quantification limits ranging from 0.03 mu g/kg for phosalone to 10.6 mu g/kg for diazinon. The relative recoveries obtained ranged from 70.8% for captan to 120% for fenarimol, and the precision (RSD) ranged from 3 to 15%. The proposed SDME procedure followed by GC with an electron capture detector for quantification and GC/MS for identification was applied with success to the analysis of 17 honey samples. Monitoring results indicated a low level of honey contamination by diazinon, chlorpyrifosethyl, procymidone, bromopropylate, and endosulfan (alpha-, beta-, and endosulfan sulfate) residues that were far below the maximum residue limit values specified by the European Union for endosulfan (10 mu g/kg) and bromopropylate (100 mu g/kg) in honey samples

Determination of diphenylamine in contaminated air of agricultural buildings using active sampling on solid sorbents

This paper reports the results of a study carried out with solid sorbents in order to establish the optimum procedure for sampling and determination of diphenylamine (DPA), the most widely used post-harvest chemical in apples, in the indoor air of apple storage buildings. Different sorbents (Amberlite XAD-2, Amberlite XAD-4, Supelpak 2, Florisil, and the octadecyl silica bonded sorbent, C-18) were evaluated for their capacity to efficiently retain DPA under different air sampling and storage conditions, whereas a desorption study of all sorbents tested was also performed to optimise a simple extraction procedure using low volumes of organic solvents. In general all sorbents produced acceptable results for DPA air sampling whereas DPA was recovered easily by the use of low volumes of both ethyl acetate and acetone from all sorbents studied thus making DPA a suitable analyte to be used in methods of indoor air analysis for multi-organic pollutants. However, the best results (analytical features, recovery results, and stability results during storage) were obtained by the use of Supelpak 2 as a sorbent for DPA active sampling. Limits of Quantification (LOQs) for the GC-NPD system ranged from 1.0 to 2.0 mu g m(-3) for 120 and 60 L air sampled, respectively. The developed air sampling procedure and analytical methodology was applied with success in the field to measure DPA residues in indoor air of two apple storage plants in Greece and results were further used to calculate the occupational inhalation exposure to DPA and consequently risk characterisation. Since DPA was detected in indoor air (at concentrations ranged from 1.6 to 580 mu g m(-3)), there is no zero occupational risk for workers. However, the inhalation exposure of workers to DPA estimated in this study is far below the Acceptable Operator Exposure Level recently reviewed by the European Union and far below the critical exposure level for haematotoxicity systemic effect observed in carcinogenicity studies in rats for long-term inhalation exposure to DPA