Persistence of Azadirachtin Residues on Olives after Field Treatment PIERLUIGI CABONI, † MARCO CABRAS, † ALBERTO ANGIONI, † MARIATERESA RUSSO, ‡ AND PAOLO CABRAS* ,† Dipartimento di Tossicologia, Universita ` di Cagliari, via Ospedale 72, 09124 Cagliari, Italy, and Dipartimento STAFA, piazza S. Francesco di Sales, 89061 Gallina (R.C.) Italy In this work an HPLC method for the determination of azadirachtin residues on olives was developed, and the field degradation kinetics of the pesticide was studied. In field trials the active ingredient (a.i.) decay had a half-life time of 0.8 days, which was too short to show a good efficacy of treatment. The mechanism of disappearance of the pesticide studied with model systems showed that it was unrelated to evaporation, thermodegradation, and co-distillation, but it was related to photodegradation. The high photodegradation rate of commercial formulations calls for the need to test different formulates in order to increase the persistence of the residue and thus the pesticide’s efficacy. KEYWORDS: HPLC; analysis; azadirachtin; residues; olives; photodegradation INTRODUCTION Azadirachtin is a limonoid of the tetranortriterpenoid type (Figure 1) extracted from the oil obtained from seeds of the neem tree (Azadirachta indica). The neem tree is original to the Union of Myanmar (Burma). It is widespread in India, and to a lesser extent in Africa and Asia. Neem extracts show an insecticidal activity and antifeedant and repellent properties. Neem extracts and pure azadirachtin are used to control Lepidoptera and Diptera both by contact and ingestion. This active ingredient (a.i.) seems to be selective, easily degradable, and nonmutagenic. Moreover, it has a low toxicity against nontarget and beneficial organisms and causes less disruptance to ecosystems than conventional insecticides (1). The azadirach- tin content in the seed extracts and in commercial formulates was determined by HPLC (2-4), by enzyme-linked immun- osorbent assay (5), and by supercritical liquid chromatography (6). Analytical methods for determining azadirachtin residues on leaves and soils are reported (5), but residues on fruits have not been described. In Italy this insecticide is registered for many crops with a maximum residue level (MRL) of 0.5 mg/kg and a pre-harvest interval (PHI) of 3 days for all crops. In this work an HPLC method for the determination of azadirachtin residues on olives was developed, and the field degradation kinetics of azadirachtin was studied. This paper also reports the results of the disappearance mechanism of this insecticide obtained on a model system. MATERIALS AND METHODS Field Trials. A field trial was carried out in an olive grove at Elmas, in the neighborhood of Cagliari, Italy. The grove was planted in 1960 with a tree spacing interval of 7 × 7 m (200 plants/ha); the cultivar was Tonda di Cagliari. A random-block design with four replications was used, and each block contained three trees in a single row. Treatments were carried out with an F-320 portable motorized sprayer (Fox Motori, Reggio Emilia, Italy). The commercial formulation was Oikos (azadirachtin A 3.2%, Sipcam, Milan, Italy), and was used at the doses recommended by the manufacturer (3.8 L/ha). Four treatments were carried out every 14 days, from August 8 to September 19, 2001. Sampling for residue control was carried out at 1, 2, 3, and 7 days after the last treatment. Random 2-kg samples were collected from each block (ca. 0.7 kg per plant). The weather conditions were continuously recorded with an SM 3800 automatic weather station (SIAP, Bologna, Italy). Rainfall was continu- ously recorded with an AD-2 automatic weather station (Silimet, Modena, Italy). No rainfall was recorded after the last treatment and during the experiments. Maximum and minimum average temperatures were 26.5 and 18.7 °C, respectively. Chemicals and Materials. Acetonitrile, acetone, and methanol were HPLC grade (Merck, Milan, Italy); water was distilled and filtered through a Milli-Q apparatus before use (Millipore, Milan, Italy). Azadirachtin (95-98% purity) was purchased from Sigma Aldrich (Steinheim, Germany), Na2SO4 was analytical grade. Stock standard solutions of the pesticides (ca. 200 mg/kg) were prepared in methanol. Working standard solutions of the pesticides were prepared by diluting with the mobile phase (acetonitrile/water; 15:85, v/v). Membranes of * Corresponding author (phone +39 70 6758604; fax +39 70 6758612; e-mail pcabras@unica.it). † Universita ` di Cagliari. ‡ Dipartimento STAFA. Figure 1. Structure of azadirachtin. J. Agric. Food Chem. 2002, 50, 3491-3494 3491 10.1021/jf020076+ CCC: $22.00 © 2002 American Chemical Society Published on Web 05/10/2002