Pesticide exposure of honeybees (Apis mellifera) pollinating melon crops Idalécio Pacífico da Silva, Fabiano Aurélio S. Oliveira, Heloísa P. Pedroza, Ivana Cristina N. Gadelha, Marília M. Melo, Benito Soto-Blanco To cite this version: Idalécio Pacífico da Silva, Fabiano Aurélio S. Oliveira, Heloísa P. Pedroza, Ivana Cristina N.Gadelha, Marília M. Melo, et al.. Pesticide exposure of honeybees (Apis mellifera) pollinating melon crops. Apidologie, Springer Verlag, 2015, 46 (6), pp.703-715. 10.1007/s13592-015-0360-3. hal-01302262 HAL Id: hal-01302262 https://hal.archives-ouvertes.fr/hal-01302262 Submitted on 13 Apr 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2015) 46:703–715 Original article * The Author(s), 2015. This article is published with open access at Springerlink.com DOI: 10.1007/s13592-015-0360-3 Pesticide exposure of honeybees (Apis mellifera ) pollinating melon crops 1 2,3 3 Idalécio PACÍFICO DA SILVA , Fabiano Aurélio S. OLIVEIRA , Heloísa P. PEDROZA , 1 3 3 Ivana Cristina N. GADELHA , Marília M. MELO , Benito SOTO-BLANCO 1Programa de Pós-graduação em Ciência Animal, Universidade Federal Rural do Semi-Árido (UFERSA), BR 110 Km 47, Mossoró, RN 59625-900, Brazil 2Laboratório Nacional Agropecuário (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Av. Rômulo Joviano s/n, Caixa Postal 35/50, Pedro Leopoldo, MG 33600-000, Brazil 3Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil Received 3 December 2014 – Revised 12 February 2015 – Accepted 4 March 2015 Abstract – The decline of honeybee (Apis mellifera L.) populations impacts global agricultural production and affects both food production and the economy. One of the probable causes for this decline is the indiscriminate use of pesticides. Here, we compare the levels of pesticide exposure among honeybees that are used to pollinate melon (Cucumis melo L.) crops, honeybees that forage in the forest, and stingless bees, Melipona subnitida , that forage in the forest. The level of pesticide exposure was determined by measuring residual pesticide levels of 152 compounds in the honey. Honey samples from the present study contained 19 different pesticides, 13 of which were present in honey from bees pollinating melon crops. The levels of some compounds were sufficiently high to promote toxic effects in the bees. Thus, crop pollination presents a toxicological risk to bees that may reduce their life span. environmental contamination / insecticides / acaricides / herbicides / fungicides / nematicides / multiresidue analysis 1. INTRODUCTION both food production and the economy (Potts et al. 2010). One of the probable causes for the Flower pollination by animals is critical for population declines of pollinators, including agriculture; approximately 35 % of crops are honeybees, is the indiscriminate use of pesti- dependent on pollinators for sexual reproduc- cides (Klein et al. 2007;Pottsetal.2010; tion (Klein et al. 2007). The worldwide value of Nakasu et al. 2014). pollination in 2005 was estimated to be €153 Individual bees can be exposed directly billion (Gallai et al. 2009). Honeybees (Apis through bodily contact with pesticides or indirect- mellifera L.) are the most economically valu- ly by consuming pesticide residue in the nectar able pollinators for agriculture (Klein et al. and pollen of flowers (Rortais et al. 2005). An 2007; Potts et al. 2010). However, recent de- entire colony may be exposed to pesticides clines in pollinator populations have affected through the collection and transportation of con- global agricultural production and impacted taminated pollen by forager bees (Villa et al. 2000). Bees foraging on melon crops may also be exposed to pesticides via guttation fluid, a Corresponding author: B. Soto-Blanco, xylem sap exudate that is eliminated through leaf [email protected] hydathodes (Thompson 2010; Hoffmann and Manuscript editor: Monique Gauthier Castle 2012). 704 I.P. da Silva et al. Pesticides can kill bees at sufficiently high thestandardsusedwereofhighpuritygrade doses (Rortais et al. 2005). However, pesticide (>98.0 %) and were purchased from Riedel-de doses that do not cause immediate death often Haën (Selze, Germany) or Sigma-Aldrich (Saint have other deleterious effects and may interfere Louis, USA). Individual stock solutions were pre- with the cognitive capacities and behavior of the pared at 1000 μgL−1 in either acetonitrile and bees. The potential negative consequences include stored at −20±2 °C. The working solutions were impaired learning, orientation, and food collection prepared as appropriate dilutions of the stock abilities; affected bees may therefore have a re- solutions. duced ability to collect food and navigate back to their hive (Rortais et al. 2005; Desneux et al. 2.2. Samples 2007; Godfray et al. 2014). Furthermore, some pesticides can reduce the resistance of bee to the Honey samples were collected from 23 colonies of intracellular parasite Nosema (Microsporidia) honeybees (A. mellifera ) used to pollinate melon crops, (Alaux et al. 2010;Pettisetal.2012;Wuetal. 20 colonies of honeybees (A. mellifera )thatforagein 2012; Aufauvre et al. 2012;DiPriscoetal.2013) the forest (caatinga ), and 10 colonies of stingless bees and the immune response against viruses (Di (M. subnitida Ducke) that forage in the forest Prisco et al. 2013). (caatinga ). Samples were collected directly from two The aim of the present study was to determine frames of each colony. All colonies were raised at the the levels of pesticide exposure among honeybees Mossoró (05° 11′ 16″ S and 37° 20′ 38″ W) and (A. mellifera ) that are used to pollinate melon Baraúna (05° 04′ 48″ S and 37° 37′ 01″ W) municipal- (Cucumis melo L.) crops and compare the pesti- ities, Rio Grande do Norte state, northeastern Brazil. All cide exposure levels to those of honeybees that the colonies of bees that forage in the forest were at a forage in the forest (caatinga , a xeric shrubland minimal distance of 8 km from melon crops. No pesti- and thorn forest in northeastern Brazil) and sting- cide was used in the beehives to control parasites such less bees, Melipona subnitida Ducke (tribe as Varroa spp. Meliponini from the family Apidae), that forage in the forest (caatinga ). The level of pesticide exposure was determined by measuring residual 2.3. Sample preparation pesticide levels of 152 compounds in the honey. The stingless bee M. subnitida was in addition to Sample extraction and clean up (Rissato et al. 2006; honeybees that forage in the forest because the Pittella 2009) were performed as follows. Honey sam- colonies kept in the study region did not collect ples (10.0 g) were transferred to polypropylene centri- pollen from muskmelons (Maia-Silva 2013). fuge tubes (50 mL) with 10.0 mL of deionized water. Then, 10.0 mL of ethyl acetate was added, and the tubes 2. MATERIAL AND METHODS were shaken at 3000 rpm for 1 min. The tubes were then centrifuged at 4 °C, 2700×g for 9 min. The supernatants 2.1. Chemicals and materials were transferred to clean polypropylene centrifuge tubes (50 mL) and samples re-extracted three times with All reagents were of analytical grade. Florisil, LC- 5.0 mL of ethyl acetate. The combined ethyl acetate MS grade acetonitrile, and glacial acetic acid were extracts (25 mL) were filtered using Florisil (1 g packed supplied by Merck (Darmstadt, Germany). Methanol in 6 mL cartridge) followed by magnesium sulfate was obtained from Baker (Xalostoc, México). Analyti- (4.0 g in paper filter). Florisil and magnesium sulfate cal reagent grade anhydrous magnesium sulfate (purity were used after heating overnight at 100 °C. The ex- ≥97 %) was purchased from Sigma-Aldrich, and anhy- tracts were dried at room temperature, resuspended in drous sodium acetate and ammonium acetate (purity 1.0 mL of acetonitrile, transferred to vials, and analyzed ≥98 %) were purchased from Vetec (Rio de Janeiro, using a UFLC-MS/MS system to identify different clas- RJ, Brazil). Formic acid was purchased from Tedia ses of pesticides (Table I). All identified pesticides were (Ohio, USA). Ultrapure water was generated using a evaluated by UFLC-MS/MS using a multiresidue anal- Millipore Milli-Q system (Milford, MA, USA). All of ysis technique. Pesticide exposure of honeybees 705 Table I. Pesticides surveyed in the honey samples. Pesticide Use Chemical group Molecular formula 2,4-D (2,4-Dichlorophenoxyacetic acid) H Phenoxy acid C8H6Cl2O3 2,4-DB (4-(2,4-Dichlorophenoxy) butyric acid) H Phenoxy acid C10H10Cl2O3 2,4,5-T (2,4,5-Trichlorophenoxyacetic acid) H Phenoxy acid C8H5Cl3O3 3-Hydroxycarbofuran I Carbamate C12H15NO4 Acephate I Organophosphorus C4H10NO3PS Acetamiprid I Neonicotinoid C10H11ClN4 Aldicarb I Carbamate C7H14N2O2S Aldicarb sulfone (metabolite) I Carbamate C7H14N2O4S Aldicarbe sulfoxide (metabolite) I Carbamate C7H14N2O3S Amitraz A/I Amidine C19H23N3 Aramite A Sulfite ester C15H23ClO4S Azinphos-ethyl A/I Organophosphorus