ECOTOXICOLOGY Translocation of Neonicotinoid Insecticides From Coated Seeds to Seedling Guttation Drops: A Novel Way of Intoxication for Bees V. GIROLAMI,1,2 L. MAZZON,1 A. SQUARTINI,3 N. MORI,1 M. MARZARO,1 A. DI BERNARDO,1 4 5 5 M. GREATTI, C. GIORIO, AND A. TAPPARO J. Econ. Entomol. 102(5): 1808Ð1815 (2009) ABSTRACT The death of honey bees, Apis mellifera L., and the consequent colony collapse disorder causes major losses in agriculture and plant pollination worldwide. The phenomenon showed in- creasing rates in the past years, although its causes are still awaiting a clear answer. Although neonicotinoid systemic insecticides used for seed coating of agricultural crops were suspected as possible reason, studies so far have not shown the existence of unquestionable sources capable of delivering directly intoxicating doses in the Þelds. Guttation is a natural plant phenomenon causing the excretion of xylem ßuid at leaf margins. Here, we show that leaf guttation drops of all the corn plants germinated from neonicotinoid-coated seeds contained amounts of insecticide constantly higher than 10 mg/l, with maxima up to 100 mg/l for thiamethoxam and clothianidin, and up to 200 mg/l for imidacloprid. The concentration of neonicotinoids in guttation drops can be near those of active ingredients commonly applied in Þeld sprays for pest control, or even higher. When bees consume guttation drops, collected from plants grown from neonicotinoid-coated seeds, they en- counter death within few minutes. KEY WORDS guttation, neonicotinoid, honey bee, seed coating Phytophagous insects occurring in soil at sowing time 2008). The reduced load of insecticide per Þeld unit, tend to concentrate around on corn, Zea mays L., allowed by conÞning it on the seed, represents main seedlings causing extensive damage. Granular insec- advantages in environmental terms compared with ticides applied to the soil have been the method of former products requiring whole-soil or furrow appli- choice for their control for a long time. More recently, cations. Elbert et al. (2008) pointed out impressive the strategy has been surpassed by the seed coating Þgures revealing the turnover toward insecticidal seed technique using neonicotinoids, which are active treatment. Starting from: a niche-level market of €155 against a broad range of pest species, including wire- million in 1990, mostly represented by carbamates, by worms (Agriotes spp.) and the rootworm Diabrotica 2005 seed coating developed into a €535 million mar- virgifera virgifera LeConte (Altmann 2003). ket, with a 77% share for neonicotinoid insecticides. Among the main reasons of success of neonicotin- The loss of pollinating bees is a worldwide crisis. In oids is their systemic action. Upon application on the particular it became manifest as colony collapse dis- seed surface, the active compound is translocated and order (CCD), characterized by a sudden disappear- distributed throughout the whole plant, conferring a ance of worker bees that do not return to the hive. substantial and long-lasting control of insects and pro- Parasitic mites and viruses have come under suspicion, tecting young plants also from sucking leafhoppers although no clear conclusions could be drawn as con- and aphids (Magalhaes et al. 2009), which are poten- cerns these biotic causes. Pesticides have been shown tial vectors of plant virus (MaienÞsch et al. 2001). to be more directly involved and in recent years the Nowadays, neonicotinoids are widely used for seed attention has been focused on neonicotinoids (imi- treatment in cotton (Gossypium spp.), sugarbeet (Beta dacloprid, clothianidin, and thiamethoxam), a class of vulgaris L.), oilseed rape (Brassica napus L.) corn (Zea insecticides among the most widely used worldwide. mays L.), and other cereals and crops (Elbert et al. The effects of neonicotinoids, such as imidacloprid on honey bees (Suchail et al. 2000, Maus et al. 2003) could 1 Dipartimento di Agronomia Ambientale e Produzioni Vegetali, be consistent with the symptoms of CCD. However, Entomologia, Universita` di Padova, Viale dellÕUniversita` 16, 35020 the blame on neonicotinoids has not yet been con- Legnaro (Padova) Italy. 2 Corresponding author, e-mail: [email protected]. clusive as the amounts detected in nectar and pollen 3 Dipartimento di Biotecnologie Agrarie, Universita` di Padova, of plants grown from treated seeds were lower than 10 Viale dellÕUniversita` 16, 35020 Legnaro (Padova) Italy. ng/g (10 ppb), whereas higher doses, as Ͼ40 ␮g/liter 4 Dipartimento di Biologia e Protezione delle Piante, Entomologia, (40 ppb) are necessary for abnormal honey bee for- Universita` di Udine, Via delle Scienze 208, 33100 Udine, Italy. Ͼ 5 Dipartimento di Scienze Chimiche, Universita` di Padova, Via aging behavior, 0.5 mg/liter (0.5 ppm) for the Þrst Marzolo 1, 35131 Padova, Italy. missing bees, and Ͼ3 mg/liter (3 ppm) for 100% of the 0022-0493/09/1808Ð1815$04.00/0 ᭧ 2009 Entomological Society of America October 2009 GIROLAMI ET AL.: GUTTATION TOXICITY OF SEEDLING FROM COATED SEEDS 1809 bees failing to return to a source of sugar offered to Þpronil (Regent 500 FS, BASF SE, Ludwigshafen, Ger- them (Yang et al. 2008). many; 1 mg/seed). The last nonsystemic compound is In Italy, a highly recurring coincidence between a member of the phenyl pyrazole class of pesticides. corn sowing time and bee death has been noticed Each of the four insecticides mentioned was a regu- previously (Greatti et al. 2003, Greatti et al. 2006), larly registered product for corn seed coating in Italy leading to the hypothesis that solid coating debris and in 2008. Seeds used (hybrid PR34N84) were from dust uplifted from sowing machines could fall over Pioneer Hi-Bred Italy (Johnston, IA), and all also were nearby vegetation and contaminate wildßowers. coated with the fungicide Celest XL (Syngenta), Within the same frame of thought, we postulated based on Fludioxonil (2.4%) and Metalaxyl-M that a different and hitherto overlooked source of (0.93%). The untreated control was also from Pioneer directly lethal doses could be present in the Þelds, and Hi-Bred (for biological agriculture) and belonged to we took into consideration the hypothesis that intox- the hybrid PR33A46. icating concentrations could accumulate in guttation In Þeld crops, we had cases treated with each of the drops of young corn plants. above-mentioned compounds. For potted plants, we Guttation (from the Latin “gutta”ϭdrop) is the for- focused essentially on imidacloprid. mation of drops of xylem sap on the tips or along the Collection of Guttation Drops. During spring edges of leaves. It is a physiological phenomenon oc- (April) corn seedlings were grown in open Þeld, curring in many vascular plants, in particular grasses, spaced 20 cm within the row and 75 cm between rows. water entering roots creates a slight pressure that In subsequent periods (May), tests were replicated by forces it to rise and be exuded through the hydatodes sowing coated seeds in pots with a diameter of 15 cm at leaf margins. This is a regular occurrence in many and growing two to Þve plants per pot in the labora- plants and is not restricted to nighttime, although in tory. In total, six to eight pots for each compound were the dark stomatal closure can lead to higher internal used and equal numbers were sown with uncoated pressure that increases guttation drop volumes, seeds as control, or with seeds coated with fungicides. thereby enhancing the visibility of the phenomenon For each seedling, we gathered all guttation drops (Goatley and Lewis 1966, Koulman et al. 2007). Gut- at all plant levels, by using Pasteur micropipettes. tation is common but often unnoticed as easily con- Collection in the Þeld was carried out from 8:00 to 9:00 fused with dew characterized by small condensation a.m. from all plants within a row, until a volume of 5 drops from atmospheric humidity. Guttation drop can ml was available. In the laboratory, because guttation roll off, evaporate or may be sucked back into the leaf occurs throughout the days and night, it was possible (Chen and Chen 2007). to collect them three times a day, yielding a volume of Bees require intense drinking activity (Visscher et Ϸ1Ð2 ml/d. Samples were stored at ϩ2ЊC. Half of the al. 1996, Ku¨ hnholz and Seeley 1997) and have been volume was sent for chemical analyses and half for reported to collect guttation water (Shawki et al. toxicity bioassays, which were normally performed 2005). within 2Ð3 d. In the current study, we wanted to verify whether Collection of guttation drops was carried out from neonicotinoids used for seed coating could be trans- corn emergence up to the Þrst 3 wk for each of the located in guttation drops and reach concentrations treatments as subsequently the phenomenon ceased toxic to bees. In parallel, we tested the toxicity of serial in its intensity both in the Þeld and in the laboratory. concentrations of these insecticides by setting up a Insecticide Content in Guttation Drops. Pure test apt to evaluate, in reasonably short time, the chemicals for the preparation of solutions of thiame- appearance of intoxication symptoms in bees upon thoxam, clothianidin, imidacloprid, and Þpronil, to be consumption of neonicotinoid aqueous solutions. used for reference toxicity curves and as analytical determination standards, were from Fluka (Sigma- Aldrich Group, Milan, Italy; Pestanal, purity Ͼ99.7% Materials and Methods for thiamethoxam, clothianidin, imidacloprid, Ͼ97.6% Insect Origin. Trials were carried out in the exper- for Þpronil). Methanol (VWR, International, Milan, imental farm of the faculty of Agriculture (University Italy), and acetonitrile (Riedel de Hae¨n, Sigma-Al- of Padova) located in Legnaro, Italy. Bees (Apis mel- drich Group) were of high-performance liquid chro- lifera L.) used for the tests were collected from dif- matography (HPLC) grade. Pure water was produced ferent colonies residing within the farmÕs Þeld facili- by Milli-Q equipment (Millipore, Billerica, MA).