1. The honey bee (Apis mellifera) is essential for the pollination of wild flowers and agricultural crops. Around one third of our diet is reliant on pollinators and any threat to these may threaten the biodiversity of wild plants and our food security. There is an ongoing debate as to whether we are facing a ‘global pollinator crisis’ but there is a significant decline in wild and domesticated bee populations across the US, Canada and Europe.. Neonicotinoids have become one of the most widely used class of pesticides, and many studies have implicated that these may be a contributing factor in increased colony losses. In 2010 approximately 20,000 tonnes of active ingredient was used globally making up ~1/3 of all insecticide treatments. In Ontario Canada, >50% of all arable crops are treated with neonicotinoids. Almost 100% of corn and canola seeds, 80% of soybean and 35% of wheat are treated. Of the remaining arable land 23% is hay production. The Prairies of Canada (Saskatchewan, Manitoba and Alberta), use 60% of all pesticides in Canada. With Saskatchewan being the greatest user of pesticides. Saskatchewan is home to some 128’000 bee hives and accounts for around 23% of the honey sold in Canada. These bees are vital to the economy of the province providing pollination for many crops as well as some 12000 tons of honey. December 2015 Neonicotinoid insecticides in Honey, Pollen and Honey Bees (Apis mellifera L.) in Central Saskatchewan, Canada As the largest agricultural region in Canada Saskatchewan's bee population is essential to the economy of the region. Samples of worker bees, pollen and honey were collected from 3 hives at 7 apiaries and analyzed for neonicotinoid insecticides and their metabolites. A health assessment based on published LD 50 values was used to identify any samples that may be impacted by these compounds. Project Aim Garry Codling 1, Yahya. Al Naggar 1, 2, John. P. Giesy, 1,2, Albert J. Robertson 3 1 Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, S7N 5B3, Canada. 2 Department of Zoology, Faculty of Science, Tanta University 31527, Tanta, Egypt. 3 Meadow Ridge Enterprises LTD, Saskatoon, Saskatchewan, S7K 3J9.Canada. Email contact: garrycodling@yahoo.co.uk INTRODUCTION LD 50 BeesHoneyPollen Directly measured in Bee Winter Exposure Compound HoneyPollen ng bee -1 % detectionng bee -1 Parent Compounds Clothianidin 18.0 (2.5-44) 52.967.957.9 2.5 (0.1-7.1) 6.7, (0.7-20.0) 5.2 (<0.01-15.7) Imidaclorpid 116 (3.7-490) N.D.32.15.3N.D. 1.1, (0.2-6.2) 4.2 Thiacloprid 23300 (12600- 38800) N.D.3.6N.D. 14.4N.D. Thiamethoxam 11.8 (3.5-30) N.D.75.021.1N.D. 19.4, (2.5-41.1) 28.7 (13.2-62.5) Flonamidid>200017.6N.D. 0.6 (0.2-1.0) N.D. Nitenpyram 80.5 (23-138) N.D. 26.3N.D. 4.48, (1.2-10.5) Imidaclorpid Metabolites Imi olefin 32 (28-36) 11.846.410.5 0.3 (0.3-0.5) 6.0 (1.4-16.4) 1.0 (0.99-1.1) Imi 5-hydroxy 153.5 (49-258) 11.871.410.5 5.6 (0.1-11.1) 21.4 (8.1-41.2) 28 (0.15-0.41) Imi urea>1000N.D.7.1N.D. Imi-desnitro HCL>1000N.D.3.610.5N.D. 0.28 (0.15-0.41) 6-Chloronictonic ACID>100011.8N.D. 0.2 (0.1-0.3) N.D. Table 1: Summary of Neonicotinoid exposure to the honey bee, values in brackets are the min and max. Discussion The frequency that neonicotinoids were found in this study was greater than that reported in previous studies. Lethal dose values (LD 50 ) are based upon previously published concentrations for individual compounds and those highlighted in yellow exceeded the minimum reported LD 50. Directly measured concentrations for the bee are the actual concentrations found in this project, (Table 1) Winter exposure levels take into account concentrations in honey and pollen measured in this study and assuming there are 10’000 worker bees living for 6 months and consuming the honey and pollen stored with no loss of compound, (i.e. a worst case exposure) Conclusions Concentrations of Nis in most samples of pollen and honey did not exceed or even come close to the oral LD 50 values for bees. There are a wide range of LD50 concentrations predicted as bees vary in their tolerance, genes and how they are exposed but based on this study some hives may have been impacted by neonicotinoid exposure at the time of sampling and over the winter. Clothianidin was the most commonly detected parent compound though we hypothesized that imidaclorpid would be based on use patterns expected The metabolites of imidaclorpid were more frequently detected than the parent compound Further investigation into metabolites of other neonicotinoids is needed along with a more comprehensive survey around Canada to identify if the chosen apiaries represent an impacted site or are indicative of the whole region/countries exposure. Figure 3: Concentrations of parent neonicotinoids (a) and the metabolites of imidaclorprid (b) identified in individual samples of bees, pollen and honey. a) b) This work was published in Chemosphere 2015: http://authors.elsevier.com/a/1S4MoAOM9Z-cH Images ued here are taken with permission from www.micro2macro.net http://authors.elsevier.com/a/1S4MoAOM9Z-cH Figure 2: A standard neonicotinoid chromatogram with a typical honey chromatogram and identifying two of the compounds investigated Identifying NIs Samples were extracted using a method called QuEChERs Identification of compounds was by LC-MS/MS using multiple parent and daughter ions Mass labeled ( 13 C), blanks, and spiked samples were used to validate results Figure 1: Summary of Detected concentrations Highlights Honey, Bees and Pollen were collected from 7 Apiaries within 15km of Saskatchewan. 3 Hives were sampled at each location 7 neonicotinoid insecticides were tested Metabolites of Imidaclorpid were also investigated