Welcome in Aix enProvence Scientific Committee

70593 Stuttgart, Germany

Bound pesticides in plants are usually referred to as residues getting non-extractable by metabolic pathways including activation, conjugation, and incorporation into biopolymers, e.g. lignin or cellulose [1].

However, in fruit peels formation of bound residues may also occur by reactions between pesticides and cuticle constituents (as the cutin polymer or lipid transfer proteins), directly or after photochemical activation.

Additionally, bound residues can be formed by thermal and enzymatic reactions during fruit processing, reactions which sometimes are also responsible for low recoveries in residue analyses.

Using radiolabelled pesticides in metabolism studies, bound and non-extractable residues can easily be localized and quantified, but structural information on the residue’s nature is rather low.

If possible, liberating methods hydrolyzing the covalent link can be used successfully, as was shown for citrus peel bound 2,4-D [2]. Using suitable antibodies we were able both to localize (dot blot immunoassay) and to quantify (ELISA) bound residues, structurally specific. Furthermore, if the linking bond is resistant to acid conditions, plant polymers containing the residue can be depolymerized, and the monomer/pesticide conjugate can be analyzed by immunochemical (ELISA) or chromatographic (TLC, LC) methods.

For chlorothalonil [3], and parathion [4] as examples of halogenated and nitroaryl pesticides, respectively, the reactions principles of bound residue formation in fruits (apples, tomatoes) and the analytical strategies used in our group will be presented in a brief overview.

[1] Skidmore, M.W. et al. (1998) Pure Appl. Chem. 70, 1423-1447

[2] Anastassiades, M.; Schwack, W. (1998) J. Chromatogr. A, 825, 45-54

[3] Jahn, C.; Schwack, W. (2001) J. Agric. Food Chem., 49, 1233-1238

[4] Wettach, J.; Rung, B.; Schwack, W. (2002) Food Agric. Immunol. 14, 5-13


com. 23
IMPROVING THE EFFICIENCY OF PESTICIDE RESIDUE ANALYSIS
Michelangelo Anastassiades and Ellen Scherbaum
CVUA Stuttgart, Schaflandstrasse 3/2, 70736 Fellbach, Germany
The expectations and requirements of pesticide residue laboratories in terms of analytical quality and sample throughput are continuously rising. Analysts around the world are thus seeking for ways to improve the overall efficiency and cost effectiveness of pesticide residue analysis so as to achieve the highest possible impact and benefit for the consumer. Considering the limited financial and personnel resources available, the attempt to keep pace with the growing demands is a challenging task. Various measures to improve the efficiency of pesticide residue laboratories will be discussed in this presentation including organizational restructuring of laboratories, strategies for targeted sampling and analysis but also the need for coordination and information exchange between laboratories.

a) Organizational restructuring: In recent years there has been a growing trend for a better coordination and a more centralized organization of pesticide residue analysis. Following this trend many small laboratories have been merging into bigger ones which have more capabilities to buy and efficiently use expensive instrumentation and to perform praxis-oriented research activities.

b) Analytical methodology: There is a general trend for faster and more cost effective methods which require less manual labour and organic solvents than traditional approaches and at the same time can cover even a broader spectrum of pesticides.

c) Targeted analysis: The time and cost of analysis increases with the number of targeted analytes. For more efficiency analysis should be therefore more targeted giving more emphasis to analytes which are relevant to the specific matrices. This is especially important when analysing samples with mass spectrometric methods in the Single Ion monitoring (SIM) mode. This “matrix oriented” approach, however, requires the collection of data about the use of pesticides in agriculture and data on residue findings in various laboratories. However, this kind of data is often difficult for residue analysts to access which arises the need for more coordination and information exchange between laboratories. At the CVUA Stuttgart we have been collecting such data for the last 7 years and have developed a database which will be soon accessible via internet for search and data input.

Com. 24
PERSISTANCE AND RESIDUES OF A NEW FORMULATION OF THE FENAMIPHOS IN SOIL AND TOAMTOES GROWN IN GREENHOUSES

A. Hormatallah¹., R. Salghi² E., Ait Addi³ E. Zine³ ., and A. Faouzi^1
1 Department of Plant Protection – Institut Agronomique and Veterinaire Hassan II, BP: 18/S Agadir – Morocco – E-mail: [e-mail] or [e-mail]

² Laboratoire d'Analyse Agro-alimentaire Ecole Supérieure de Technologie d'Agadir. BP: 33/S Agadir – Morocco

³ Etablissement Autonome de Contrôle et de Coordination des exportations

BP: 1710 Ancien Post – Agadir – Morocco

Nemacur 240CS, a new formulation of the fenamiphos nematocide, is used in tomato grown in greenhouses in Morocco especially for the Meloidogyne spp control.

Fenamiphos persistence in sandy soil and residues level of fenamiphos at the first time of tomatoes fruit harvest has been studied under the typical horticultural practices and climatic conditions in Souss Massa Valley. Souss Massa is the major region for the tomato production in Morocco with 90% is exported to the European Union. Fenamiphos is applied at the seventeenth (17th) day after plantation of tomato crop in greenhouses by drip irrigation at 1.6 g a.i/plant.

Soil samples were randomly taken from 0-10 cm and 10-30 cm layers and collected at 4 hours, 5 days, 10 days and 75 days after application. 120 tomatoes fruits were taken 75 days after application of the product. The samples were extracted by the acetone and clean up with silica column. Residues level of fenamiphos were determined by CPG with NPD Detector. The fenamiphos residues found represented 68.8% and 9.8% of the applied dose in the 0-10 cm and 10-30 cm soil layers at 4 hours after a treatment. These values are declined rapidly to achieve 8.7% and 2.8% at 9 days after application.

After 75 days, the fenamiphos residues were 0.5% and 0.1% of applied. These data can be explained by the fenamiphos leaching through the soil to larger depth. The fenamiphos residues in tomatoes fruit harvested were equivalent to the detection limit (0.01 ppm). This value is far bellow the tolerance established for the fenamiphos.

Keywords : fenamiphos , persistence, Residues, soil, tomatoes, leaching.

Com. 25
Determination of fenitrothion and its main metabolites fenitrothion-oxon and 3-methyl-4-nitrophenol in kaki fruits.
Fernández-Cruz, M.L., Villarroya, M., Alonso-Prados, J.L. and García-Baudín, J.M.
Departamento de Protección Vegetal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Ctra. de la Coruña km 7.5. 28040 Madrid. Spain. [e-mail]
The establishment of maximum residue levels (MRLs) of pesticides in plant products in Europe is a continuous process regulated by Directives 76/895/EEC, Directive 86/362/EEC and Directive 90/642/EEC. European Commission has initiated a peer review program of all the existing active substances of pesticides in the market regulated by Directive 91/414/EEC. Only active substances included in the Annex I of this Directive could be authorized. After all this review program, it is expected that some minor crops will not be protected against pests and diseases.

The main producers of the minor crop kaki (Diospyros kaki) in the European market are Italy, Israel and Spain. The income-producing regions in Spain are Andalusia and Valencia, the 70% of the production being exported to the European market. The organophosphate insecticide Fenitrothion, a cholinesterase inhibitor, is effective against a wide range of chewing, sucking and boring insects on some raw agricultural commodities. Fenitrothion is oxidized by mono-oxygenases in animals, insects and plants to the metabolite fenitrothion-oxon, which is a more powerful inhibitor of cholinesterase. The major excretion product is the metabolite 3-methyl-4-nitrophenol which potential mutagenic effect is not clear. The literature does not describe analytical methods for the determination of pesticide residues in kaki fruits. An analytical method for the simultaneous determination of these compounds in kaki fruits has been validated in our laboratory by gas chromatography with TSD detector, after extraction with ethyl acetate. The compounds could be determined with sufficient sensitivity and reproducibility. The concentrations of analytes in the sample extracts were obtained by comparing peak areas from samples with those recorded for the mixed standard solutions. Satisfactory linearity, recoveries and repeatability were obtained.

Com. 26
Pesticide Residues in viticulture and enology.
Barba, A.
Department of Agricultural Chemistry, Geology and Pedology. University of Murcia, Campus de Espinardo, 30100, Murcia, Spain.*e-mail: [e-mail]
The use of insecticides and fungicides in vine growing may lead to toxicological problems if the residues in the grape exceed RML. Commercial consequences may also arise if the residues affect wine-making processes or its final organoleptic quality.
Winegrowers therefore need to be informed both about the problems that the use of the most traditional/classical chemically based phytosanitary products supposes and about the alternatives that are currently available (integrate pest management). It is also clear that the winegrowers should be aware of the problems that may arise from an irrational use of some pesticides, such as fungicides, which, given their physical and chemical characteristics, may interfere in fermentation processes by acting on the microflora of the wine yeasts. It is also important to study the effect of pesticide residues on the quality of the wine, and in particular their influence on its organoleptic characteristics. Likewise requiring study is their role in wine-making processes, with special attention to the influence of enotechnical processes on the disappearance of pesticide residues.
In this paper the problems arising from the traditional use of pesticides in winegrowing will be outlined,and alternative methods currently employed in Murcia (SE,Spain) in the cultivation of the Monastrell grape (Appellation d´Origine Jumilla) are studied with special attention to the control of berry moth (Lobesia botrana), powdery mildew (Uncinula necator) and downy mildew (Plasmopara viticola). We also review works performed with different insecticides and fungicides, both classical and more modern ones, with respect to their disappearance during wine-making and to the influence of their residues on the final quality of the wine.


Com. 27
organochlorine pesticides residues in food web organisms from a camargue lagoon

PERSIC Ana, ROCHE Hélène, RAMADE François
Université de Paris-Sud XI,

CNRS UMR 8079

Écologie, Systématique et Evolution, Bât 442,

F91450 ORSAY cedex.

[e-mail]
The Reserve of Biosphere of Camargue is a strictly protected area frequently exposed to anthropogenic perturbation related to agricultural and industrial activities. In particular, organochlorine (OC) pesticides sprayed on the adjacent rice fields get transferred, mainly via the irrigation ditches, into the Vaccarès lagoon resulting in a chronic contamination of this aquatic ecosystem.

The objective of this study was to determine the contamination by prohibited (or strongly restricted) OC pesticides - lindane, dieldrin, pp’-DDE, HCB - and permanently used as diuron and fipronil, in the Vaccarès lagoon food-web. In order to establish its complex trophic structure the stable isotopes method was used. The concentration of ¹⁵N and ¹³C (¹⁵N, ¹³C), as well as the tissue OC amounts were measured on organisms from different trophic levels (bivalves, crustaceans, prey and predator fishes) collected from the lagoon in March, May and April 2002.

The main goal was to develop pesticide - trophic level relationships in order to estimate the extent of the biomagnification phenomenon eventually occurring in this protected ecosystem.

The results demonstrate the presence of pesticides in all the species involved and through all the trophic compartments. In the up to date research, the infrequency of positive relation between ¹⁵N, ¹³C and contaminant concentration does not suggest a trophic level biomagnification of the investigated pesticides. In fact, the tissue impregnation tends to decrease with increasing trophic level, especially within the benthic species. Therefore we suggest that the accumulation of pesticides in the Vaccarès lagoon biota occurs primarily by a passive diffusion process (at the body surface via the gills and teguments) directly from water.

Com. 28
Determination of new pesticide residues in processed fruit products by LC/MS/MS and GC/MS .
Anna Sannino, Luciana Bolzoni and Mirella Bandini
SSICA- Stazione Sperimentale per l’ Industria delle Conserve Alimentari

Viale F.Tanara, 31/A- 43100 Parma- Italy, e-mail: [e-mail]

Agrochemicals belonging to carboxamide, quinazolin,phenoxypyrazol, strobilurin, pyrimidine,triazol, carbamate, chloronicotinin, classes are representive of the newly introduced molecules that are extensely used in fruit and vegetable treatements.

Analytical methods must be developed and validated to monitor these new pesticides in food commodities at low level.

Many of these pesticides are very polar respect to older ones and consequently less amenable to determination by the existing methods.

Liquid chromatography coupled with mass spectrometry ( LC/MS) has proven to be a very useful technique to detect polar, ionic or heavyweight compounds. In particular, LC/MS/MS technique allows a higher selectivity even at the concentration required by current regulator laws for baby and organic foods.

In this work a simple and sensitive method based both on LC/MS/MS and GC/MS is described for the determination of about 27 new pesticides in processed fruit products. The analytical procedure involved an extraction with acetone and a liquid- liquid partition with ethyl acetate- cyclohexane (50/50, v/v) combined in one step. An aliquot of extract was evaporated and the residue taken up with 1mL of LC mobile phase. The solution was analysed by a triple quadrupole spectrometer without a further clean-up. As for MS conditions , Applied Biosystem Sciex API 2000 instrument was used with the standard turbo-ion spray source operating in positive ion mode. At least two transitions were monitored for each compound . The following pesticides were determined by LC/MS/MS: Imidacloprid, Dimethomorph, Benfuracarb, Fenothiocarb, Hexythiazox, Tebufenpyrad, Tebufenozide,Azoxystrobin, Trifloxystrobin, Kresoxim-methyl, Indoxacarb, Fenpyroximate, Fenazaquin, Flusilazole, Difeconazole, Fenbuconazole, Tebuconazole, Tetraconazole, Paclobutrazole. Recovery at two concentration levels ( 10 - 50 ppb) on apple puree and citrus juice were > 70% with limits of quantitation < 10ppb.

An other aliquot of extract was vortexed with PSA sorbent and then centrifuged. The purified extract was analysed by GC/ITMS. The following active compounds were determined: Ciprodinil, Buprofezin, Pymetanil, Mepanipyram, Fludioxonil, Fenhexamid, Pyiridaben, Etofenprox, Bupirimate, Kresoxim-methyl. Limits of quantitation ranged from 5 to 20 ppb depending on compounds.

Because of insufficient residue data of these new pesticides about 100 samples of fruit processed products ( nectars, purees and juices ) purchased from markets were analysed.

Com. 29
Applications récentes du couplage LC-MS à l’analyse de contaminants
Eric Génin – ThermoFinnigan
Le couplage CLHP-API-MS est aujourd’hui une technique de routine et son utilisation dans le domaine de l’analyse et du contrôle des contaminants dans l’environnement complète de plus en plus, sinon remplace, la GC-MS. Cette réalité doit être reliée directement à l’évolution très rapide des performances instrumentales ; ainsi, en se limitant aux deux technologies les plus utilisées – les analyseurs quadripolaires et les trappes ioniques - on peut noter les faits marquants suivants :

• 
  en moins de dix ans, les limites de détection LC-MS des pesticides courants ont été abaissées d’un facteur 100
  
• 
  l’amélioration et la meilleure connaissance des sources d’ionisation à pression atmosphérique, d’une part, les développements logiciels et électroniques , d’autre part, ont permis la mise au point de méthodes multi-résidus robustes, sensibles et rapides.
  
• 
  L’édification de bibliothèques de spectres MS/MS facilite la confirmation de composés cibles en sortie d’une colonne CLHP, tandis que la technologie MSn aide à l’identification de composés inconnus
  

Plus récemment,

• 
  l’introduction des sources de photo-ionisation à pression atmosphérique – APPI – a élargit le champ d’application de la LC-MS à des molécules difficilement ionisables (organo-chlorés, HAP) par électro-nébulisation ou APCI
  
• 
  l’amélioration considérable des performances résolutives d’un instrument triple-quadripolaire – le TSQ Quantum – permet d’abaisser encore les seuils de détection et de quantification, notamment dans les matrices les plus complexes et notamment pour des molécules chlorés dont le défaut de masse est notable.
  
• 
  Enfin les capacités de mesure de masse précise de cet instrument sont un outil de choix par exemple pour l’identification structurale de métabolites de pesticides.
  

Parallèlement à ces évolutions technologiques, de nouveaux défis sont nés qui dépassent le seul contrôle des substances bio-accumulables et toxiques. Ainsi en est-il de l’intérêt porté à toutes ces substances susceptibles de perturber le système endocrinien : oestrogènes naturels ou de synthèse, alkylphénols, pesticides chlorés, tri-butyl étain, etc… La diversité des classes chimiques concernées nous permettra d’illustrer différents aspects des récentes améliorations technologiques évoquées.

Com. 30
PHOTOCHEMISTRY OF SOME SULFONYLUREA HERBICIDES
Sabino A. Bufo,¹ Laura Scrano,¹ Maurizio D’Auria ² and Jean Marc Chovelon ^3
1 Dipartimento di Produzione Vegetale, and ² Dipartimento di Chimica, Università della Basilicata, Via N. Sauro 85, I 85100 Potenza, Italy.

³ Lace, Université Claude Bernard, Boulevard du 11 novembre 1918, 69622 Villeurbanne Cedex, France.

The most important degradation pathways of sulfonylurea herbicides are chemical hydrolysis and microbial breakdown. ^[1] Since they are generally sprayed on soil and leaves surfaces and can undergo to photochemical degradation due to solar irradiation, more information on the degradation time of active ingredients, and consequently, on their activity and environmental fate, can be obtained by studying the photochemical properties of these herbicides. ^[2] This work deals with the study of photochemical behaviour of two sulfonylureas, triasulfuron and thifensulfuron methyl. Triasulfuron is utilised for the treatment of wheat, barley, rye, and oats and thifensulfuron methyl is used to control dicotyledonous weeds in maize cultures.

Photochemical reactions were carried out by using a high-pressure mercury arc and a solar simulator. The photochemical reactions were also carried out in the presence of either a singlet or a triplet quencher. Kinetic parameters and quantum yields were determined. The identification of photoproducts was performed by mass spectrometry. Substances used as inhibitors of the excited levels T₁ and S₁ showed that photodegradation of both herbicides begins from a triplet state T₁. The photolysis process occurred through a first order kinetic reaction. The main metabolites identified in acetonitrile were 2-chloroethoxybenzene, for triasulfuron, and a monosubstituted urea, common to triasulfuron and thifensulfuron-methyl.
References

[1] Frederikson D.R. and P.J. Shea (1988) Weed Science, 34, 328-334.

[2] Scrano L., S.A. Bufo, P. Meallier and M. Mansour (1999) Pestic. Sci., 55, 955-961.


Com. 31
Photodegradation of carbaryl and metabolites in the aquatic environment
O. Brahmia and C. Richard
Laboratoire de Photochimie Moléculaire et Macromoléculaire, UMR CNRS-Université Blaise Pascal n°6505, 63177Aubière Cedex
Photochemistry is a way of pollutant degradation in the aquatic environment. Two types of reactions may occur. Organic compounds that absorb solar light undergo photolysis. Phototransformation of pollutants mediated by chromophoric constituents of natural waters may also take place. Humic substances that are the main absorbing compounds contained in waters are known to exhibit photoinductive properties : under light excitation they generate reactive species capable of degrading organic pollutants.

In this work, we investigated the photochemical degradation of carbaryl, a carbamate insecticide, in conditions close to environmental ones. At first, carbaryl (10^-4 M) was irradiated in pure water using lamps emitting at wavelengths longer than 290 nm. 1,2- and 1,4-naphthoquinone, 2 and 7-hydroxy-1,4-naphthoquinone were found as photoproducts. Naphthol was also detected but its formation was attributed to thermal hydrolysis of carbaryl. Then, carbaryl was irradiated in the presence of humic substances (25 mg/l) : no photoinduced reactions were observed.

In a second step, we investigated the photochemical transformation of naphthol and 1,4-naphthoquinone (10^-4 M) in pure water and in the presence of humic substances. In pure aqueous solution, naphthol was found to be photolysed into 1,2-and 1,4-naphthoquinone, 2 and 7-hydroxy-1,4-naphthoquinone while 1,4-naphthoquinone yielded mainly 2 and 7-hydroxy-1,4-naphthoquinone. The phototransformation of naphthol was drastically enhanced in the presence of humic substances whereas that of 1,4-naphthoquinone not affected.

These results show that photochemistry should contribute to transformation of carbaryl in natural waters. Both photoreactions through absorption of light by pollutant and photoinduced reactions mediated by humic substances should be involved.