CROP PROTECTION: NEW STRATEGIES FOR SUSTAINABLE DEVELOPMENT Thymol as an alternative to pesticides: persistence and effects of Apilife Var on the phototactic behavior of the honeybee Apis mellifera Jean-Luc Carayon & Nathan Téné & Elsa Bonnafé & Julie Alayrangues & Lucie Hotier & Catherine Armengaud & Michel Treilhou Received: 15 July 2013 /Accepted: 6 September 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Thymol is a natural substance increasingly used as an alternative to pesticides in the fight against the Varroa destructor mite. Despite the effectiveness of this phenolic monoterpene against Varroa , few articles have covered the negative or side effects of thymol on bees. In a previous study, we have found an impairment of phototaxis in honeybees following application of sublethal doses of thymol—lower or equal to 100 ng/bee—under laboratory conditions. The present work shows the same behavioral effects on bees from hives treated with Apilife Var®, a veterinary drug containing 74 % thymol, with a decrease in phototactic behavior ob- served 1 day after treatment. Thus, thymol causes disruption of bee phototactic behavior both under laboratory conditions as well as in beehives. The bee exposure dose in treated hives was quantified using gas chromatography coupled to mass spectrometry (GC–MS), giving a median value of 4.3 μg per body 24 h after treatment, with 11 ng in the brain. The thymol level in 20 organic waxes from hives treated with Apilife Var® was also measured and showed that it persists in waxes (around 10 mg/kg) 1 year after treatment. Thus, in the light of (1) behavioral data obtained under laboratory conditions and in beehives, (2) the persistence of thymol in waxes, and (3) the high load on bees, it would appear important to study the long-term effects of thymol in beehives. Keywords Thymol . GC–MS . Derivatization TMS . Phototaxis . Apis mellifera Introduction The problems of hive weakening and depopulation encoun- tered by beekeepers over the last 10 years have driven the development of much research. A large part of this has fo- cused not only on the effects of extended exposure to sublethal doses of pesticides (Henry et al. 2012), but also on the fight against the Varroa destructor ectoparasite, which is regarded as an important scourge of bees. Originating in Asia, its geographical distribution now covers almost all continents. This parasite weakens colonies until their collapse, by feeding on the hemolymph of larvae and adults, and initial treatments were based on synthetic pesticides such as coumaphos, fluvalinate, or amitraz. However, the development of resis- tance caused by some synthetic molecules (Martin 2004) and/ or the undesired transfer to the honey (Wallner 1995; Wallner 1999) as well as the increasing demand for organic agricul- tural products has lead beekeepers to abandon these active substances in favor of natural products such as essential oils. This search for alternatives to phytosanitary compounds is also related to the preservation of the environment and to the reduction in treatment impacts on non-target organisms. Thymol, one of the main phenolic compounds from Thy- mus vulgaris , is an effective acaricide, especially concerning the Varroa ectoparasite (Damiani et al. 2009; Imdorf et al. 1995). With accurate dosage, it has been shown that this phenolic monoterpene can kill nearly 100 % of Varroa with- out noticeable loss of bees (Imdorf et al. 1995). It has been used for several years in the form of evaporating tablets (Apilife Var®) or gel boxes (Apiguard®) that beekeepers can place in hives at the end of summer, after honey harvest. Responsible editor: Philippe Garrigues Catherine Armengaud and Michel Treilhou contributed equally. J.<L. Carayon : N. Téné : E. Bonnafé : M. Treilhou (*) Centre Universitaire Champollion, Equipe VAcBio EA 4357 place de Verdun, 81012 Albi, France e-mail: michel.treilhou@univ-jfc.fr J. Alayrangues : L. Hotier : C. Armengaud CNRS-UMR 5169, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France Environ Sci Pollut Res DOI 10.1007/s11356-013-2143-6