NEONICOTINOID RESIDUES IN WILDFLOWERS, A POTENTIAL ROUTE OF CHRONIC EXPOSURE FOR BEES

Transcription

1 1 2 NEONICOTINOID RESIDUES IN WILDFLOWERS, A POTENTIAL ROUTE OF CHRONIC EXPOSURE FOR BEES Cristina Botías 1*, Arthur David 1, Julia Horwood 1, Alaa Abdul-Sada 1, Elizabeth Nicholls 1, Elizabeth Hill 1, Dave Goulson 1 1 School of Life Sciences, Sussex University, Falmer BN1 9QG, UK. *Correspondence to: c.botias@sussex.ac.uk. Phone: +44(0) CONTENT (28 pages): - Supporting information for materials and methods. - Figure S1 - Tables S1a-S1g - Tables S2a-S2j - Tables S3a-S3b - Table S4 - Table S5a-S5b - Tables S6a-S6b - Table S7 - Tables S S1

2 Supporting Information: - Collection of pollen and nectar from oilseed rape flowers. To obtain pollen samples, flowers of oilseed rape ( 1,000 flowers) were gathered and stored on ice in coolers in the field and then frozen immediately at -80 C until further handling. Flowers were then gently defrosted and dried in an incubator at 37 C for 24 hours to facilitate pollen release from the anthers. After drying, flowers were brushed over food strainers to separate pollen from anthers and sifted through multiple sieves of decreasing pore size (pore sizes from 250 to 45 µm; oilseed rape pollen grain: Ø = µm). Collection of nectar from flowers was performed in situ through capillary action into glass calibrated micropipettes (5 µl, Blaubrand intramark, Ref ), and once they were filled, both ends were sealed with putty (Cristaseal; Hawksley& Sons Ltd., Ref ). Due to small quantities of nectar available per flower, nectar had to be extracted from several hundred flowers to collect the required quantities for analysis (50 µl/sample). When nectar sample volumes were not high enough for analyses, replicates gathered from the same field were pooled and analysed as a single sample. Both pollen and nectar samples were stored at -80 C prior to pesticide extraction and analysis. Analyses were performed within 10 months after collection Chemicals and reagents Certified standards of thiamethoxam, thiamethoxam-d3, clothianidin, clothianidin-d3, imidacloprid, imidacloprid-d4 and thiacloprid, formic acid, ammonium formate, magnesium sulphate, sodium acetate and Supel TM QuE PSA/C18/ENVI-Carb were obtained from Sigma Aldrich UK. All pesticide standards were > 99% compound purity and deuterated standards > 97% isotopic purity. HPLC grade acetonitrile, hexane, methanol and water were obtained from Rathburns UK. Individual standard pesticide (native and deuterated) stock solutions (1 mg/ml) were prepared in acetonitrile (ACN). An additional internal standard mixture of the three deuterated pesticides at 100 ng/ml was also prepared. Calibration points in H 2 0:ACN (90:10) were prepared weekly from the stock solutions. All stocks were stored at -20 o C in the dark GC-MS/MS analyses Enough sample was available for extracts of four wildflower pollen samples to be analysed by gas chromatography tandem mass spectrometry (GC-MS/MS) as an additional confirmation of the high thiamethoxam concentrations detected by UHPLC-MS/MS in these extracts. Sample extracts were evaporated to dryness using a gentle flow of nitrogen and reconstituted in 10 μl of toluene and 2 μl was injected in splitless mode on a gas chromatograph system (Trace GC Ultra, Thermo Scientific) linked to an ion trap mass spectrometer (ITQ1100, Thermo Scientific). Compounds were separated on an Agilent DB- 5MS UI column (30 m 0.25 mm, 0.25-µm film thickness) using helium as the carrier gas (99.996% purity) at a flow rate of 1.2 ml/min. The injector and transfer line were set at 250 C and 290 C respectively, the source at 270 C. The column was held at 60 C for S2

3 min after injection, then programmed at 40 C/min to 170 C, and then at 10 C/min to 310 C which was held for 1 min. The mass spectrometer was operated in the electron ionization mode (EI, 70 ev) and thiamethoxam and thiamethoxam-d3 were detected using the MS/MS mode. The precursor ions at m/z 212 and 215 for respectively thiamethoxam and thiamethoxam-d3 were trapped and subjected to collisionally induced fragmentation with helium to obtain characteristic product ions at m/z 125 and 182 for thiamethoxam and 125 and 185 for thiamethoxam-d3. Retention times for thiamethoxam and thiamethoxam-d3 were respectively of and min. Solvent blanks were injected between each sample to monitor analyte carryover. GC-MS/MS spectra were analyzed on Xcalibur v1.2 software (Thermoquest-Finningan) and concentrations were determined using a least-square linear regression analysis of the peak area ratio versus the concentration ratio (native to deuterated). Five calibration points between 0.1 and 2 µg/ml were used for the calibration curve (R 2 > 0.99). Comparison of thiametoxam concentrations in pollen obtained using either UHPLC- MS/MS or GC-MS/MS analyses showed good agreement and confirmed the high concentrations of thiamethoxam measured in wildlflower pollen samples (SI Table S8). S3

4 Figure S1. Concentration of thiamethoxam in pollen of oilseed rape flowers as a function of the levels present in the soil in which the plants were growing S4

5 Tables S1a-S1g. List of products used in the year of sampling (2013) and in the two previous years in the 7 winter oilseed rape fields and 5 winter wheat fields where the sampling was performed (The size of the fields in hectares (ha) are indicated in each case). Table S1a. Field 1 (13.9 ha). FIELD 1 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide (Ins.)/Fungicide (Fung.) Cruiser 25/08/2012 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Alert 14/10/2012 Alpha-cypermethrin Mavrik 26/03/2013 Tau-fluvalinate 25/08/2012 Fungicides Genie 25 14/10/2012 Flusilazole Juventus 05/01/2013 Metaconazole OILSEED RAPE Filan 17/04/2013 Boscalid Flanker 17/04/2013 Picoxystrobin Propulse 31/05/2013 Fluopyram Prothioconazole Herbicides Insecticide /Fungicide Samurai Redigo Deter 27/06/ /09/2011 Glyphosate Clothianidin (Ins.) Strada 27/06/2013 Orthosulfamuron Prothioconazole Insecticide Alert 30/12/2011 Alpha-cypermethrin Herbicides Crystal 30/12/2011 Pedimethalin Flufenacet Fidox 20/12/2011 Prosulfocarb Atlantis WG 01/03/2012 Iodosulfuron-methyl-sodium Mesosulfuron-methyl Shadow 22/08/2012 Quinmerac Dimethenamid-P Metazachlor 25/09/2011 Dictate 22/08/2012 Bentazone as sodium salt Fungicides Bravo /03/2012 Chlorothalonil WHEAT Menara 17/03/2012 Propiconazole Cyproconazole Branched alkyl esters C 6 - C 8 Kingdom 06/04/2012 Boscalid Epoxiconazole Bassoon EC 06/04/2012 Epoxiconazole Cello 17/04/2012 Tebuconazole Spiroxamine Prothioconazole Covershield 17/04/2012 Epoxiconazole Kresoxim-methyl Pyraclostrobin Insecticide /Fungicide Redigo Deter 28/09/2010 Clothianidin (Ins.) Prothioconazole Fungicides Kingdom 06/04/2011 Boscalid Epoxiconazole Menara 06/04/2011 Propiconazole Cyproconazole Branched alkyl esters C 6 - C 8 28/09/2010 Sparticus Xpro 09/05/2011 Tebuconazole Bixafen WHEAT Prothioconazole Cello 31/05/2011 Tebuconazole Spiroxamine Prothioconazole Fandango 31/05/2011 Fluoxastrobin Prothioconazole Firefly 31/05/2011 Fluoxastrobin Prothioconazole Odin 19/06/2012 Tebuconazole S5

6 Table S1b. Field 2 (6.6 ha). SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide (Ins.)/Fungicide (Fung.) Cruiser 23/08/2012 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Gandalf 30/10/2012 Beta-cyfluthrin Alert 26/05/2013 Alpha-cypermethrin Fungicides Genie 25 30/10/2012 Flusilazole Crawler 16/02/2013 Carbetamide 23/08/2012 Caramba 26/05/2013 Metconazole Delsene 50 Flo 26/05/2013 Carbendazim OILSEED RAPE Herbicides Shadow 07/09/2012 Quinmerac Dimethenamid-P Metazachlor Fox 15/11/2012 Binefox Cogent 15/02/2013 Glyphosate Beltstone 15/02/2013 Binefox Pilot Ultra 16/02/2013 Quizalofop-P-ethyl Insecticide /Fungicide Redigo Deter 01/03/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticides Alert 02/05/2012 Alpha-cypermethrin 01/03/2012 Fungicides Bravo /05/2012 Chlorothalonil Bontima 17/05/2012 Cyprodinil BARLEY Isopyrazam Herbicides Agritox 50 02/05/2012 MCPA Oxytril Cm 02/05/2012 Ioxynil Bromoxynil Simba SX 02/05/2012 Metsulfuron-methyl Insecticide /Fungicide Redigo Deter 29/09/2010 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticides Alert 16/11/2010 Alpha-cypermethrin Fungicides Cherokee 14/04/2011 Cyproconazole Chlorothalonil Propioconazole 29/09/2010 Basson 28/04/2011 Epoxiconazole Bravo /04/2011 Chlorothalonil WHEAT Covershield 13/05/2011 Epoxiconazole Kresoxim-methyl Pyraclostrobin Herbicides Agena 08/10/2010 Chlorotoluron Diflufenican Defy 08/10/2010 Prosulfocarb FIELD 2 S6

7 Table S1c. Field 3 (13.5 ha). FIELD 3 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide (Ins.)/Fungicide (Fung.) Cruiser 23/08/2012 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Gandalf 05/11/2012 Beta-cyfluthrin Alert 17/05/2013 Alpha-cypermethrin Hallmark Zeon 05/06/2013 Lambda-cyhalothrin 1,2-Benzisothiazolin-3-one Mavrik 05/06/2013 Tau-fluvalinate Fungicides Fiddle 28/08/2012 Clomazone 25/08/2012 Crawler 10/10/2012 Carbetamide Genie 25 05/11/2012 Flusilazole OILSEED RAPE Filan 17/05/2013 Boscalid Flanker 17/05/2013 Picoxystrobin Propulse 05/06/2013 Fluopyram Prothioconazole Herbicides Shadow 28/08/2012 Quinmerac Dimethenamid-P Metazachlor Dictate 28/08/2012 Bentazone as sodium salt Pilot Ultra 16/02/2013 Quizalofop-P-ethyl Molluscicide TDS Major 08/09/2012 Metaldehyde Slug pelleter Insecticide /Fungicide Redigo Deter 01/03/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticides Alert 27/04/2012 Alpha-cypermethrin Fungicides Bravo /04/2012 Chlorothalonil 01/03/2012 Bontima 17/05/2012 Cyprodinil Isopyrazam Herbicides Samurai 23/11/2011 Glyphosate BARLEY Strada 23/11/2011 Orthosulfamuron Agritox 50 27/04/2012 MCPA Oxytril Cm 27/04/2012 Ioxynil Bromoxynil Simba SX 27/04/2012 Metsulfuron-methyl Insecticide /Fungicide Redigo Deter 21/09/2010 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticides Alert 22/11/2010 Alpha-cypermethrin Fungicides Cherokee 13/04/2011 Cyproconazole Chlorothalonil Propioconazole Basson 26/04/2011 Epoxiconazole Bravo /04/2011 Chlorothalonil 21/09/2010 Covershield 13/05/2011 Epoxiconazole Kresoxim-methyl Pyraclostrobin WHEAT Herbicides Shooter 21/09/2010 Flufenacet Pendimethalin Champion 500 SC 21/09/2010 Diflufenican Dictate 21/09/2010 Bentazone as sodium salt Upright 13/04/2011 Imazaquin Chlormequat Hunter 13/04/2011 Fluroxypyr Florasulam Hatchet Xtra 26/04/2011 Fluroxypyr S7

8 154 Table S1d. Fields 4 (15.6 ha), 5 (7.1 ha), 6 (12.4 ha) and 7 (11.5 ha) FIELDS 4-7 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide (Ins.)/Fungicide (Fung.) Cruiser 31/08/2012 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Toppel 100 EC 14/11/2012 Cypermethrin Mavrik 18/05/2013 Tau-fluvalinate Fungicides Proline /12/2012 Prothioconazole Genie 25 08/01/2013 Flusilazole Delsene 50 Flo 18/05/2013 Carbendazim 31/08/2012 Herbicides Glyfos Dakar 23/08/2012 Glyphosate Rosate 36 23/08/2012 Glyphosate OILSEED RAPE Novall 03/09/2012 Quinmerac Metazachlor Mazone /09/2012 Clomazone Falcon 15/09/2012 Propaquizafop Kerb Flo /01/2013 Propyzamide Fusilade Max 08/01/2013 Fluazifop-P-butyl Fox 19/02/2013 Binefox Molluscicide Insecticide /Fungicide Attract Redigo Deter 01/10/ /09/2011 Metaldehyde Clothianidin (Ins.) Allure 01/11/2012 Metaldehyde Prothioconazole (Fun.) Slug pelleter Insecticide Hallmark Zeon 20/10/2011 Lambda-cyhalothrin Fungicides Cherokee 06/04/2012 Cyproconazole Flexity 06/04/2012 Metrafenone Aviator 235 Xpro 02/05/2012 Bixafen Prothioconazole Ignite 18/06/2012 Epoxiconazole Odin 18/06/2012 Tebuconazole 13/09/2011 Bravo /06/2012 Chlorothalonil Proline /06/2012 Prothioconazole WHEAT Herbicide Glyphosate /08/2011 Glyphosate Crystal Pendimethalin Flucenacet Hurricane SC 23/09/2011 Diflufenican Atlantis WG 17/02/2012 Iodosulfuron-methyl-sodium Mesosulfuron-methyl Starane XL 02/05/2012 Florasulam Fluoxypyr Reglone 19/08/2012 Diquat Fungicides Plover 15/11/2010 Difenoconazole Icon 09/04/2011 Tebuconazole Herbicides Clipper 28/08/2010 Glyphosate 15/09/2010 Defy 15/09/2010 Prosulfocarb Falcon 25/10/2010 Propaquizafop LINSEED Lexus SX 15/11/2010 Flupyrsulfuron-methyl Chekker 09/04/2011 Amidosulfuron Iodosulfuron-methyl-sodium Glyphosate /07/2011 Glyphosate S8

9 160 Table S1e. Field 8 (35.9 ha). 161 FIELD 8 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide /Fungicide Redigo Deter 30/09/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticide Alert 30/08/2013 Alpha-cypermethrin Fungicides Cello 24/04/2013 Tebuconazole Spiroxamine Prothioconazole Vortex 18/05/2013 Fluxpyroxad Epoxiconazole 30/09/2012 Pyraclostrobin Basoon EC 18/05/2013 Epoxiconazole WHEAT Herbicides Sprinter 30/08/2013 Pyroxsulam Florasulam Cloquintocet-mexyl Broadway Star 16/04/2013 Pyroxsulam Clonquitocet-mexyl Florasulam Congent 16/04/2013 Glyphosate Mircam Plus 04/05/2013 MCPA Molluscicide TDS Major 26/09/2012 Methaldehyde Slug pelleter Insecticide/Fungicide Cruiser 23/08/2011 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Alert 12/10/2012 Alpha-cypermethrin 23/08/2011 Mavrik 26/03/2012 Tau-fluvalinate Fungicides Genie 25 12/10/2011 Flusilazole OILSEED RAPE Juventus 05/01/2012 Metaconazole Filan 12/04/2012 Boscalid Flanker 19/04/2013 Picoxystrobin Herbicides Insecticide /Fungicide Samurai Redigo Deter 23/06/ /03/2011 Glyphosate Clothianidin (Ins.) Strada 23/06/2012 Orthosulfamuron Prothioconazole (Fun.) Fungicides Mobious 09/05/2011 Trifloxystrobin Prothioconazole Cello 09/05/2011 Tebuconazole Spiroxamine 05/03/2011 Prothioconazole Bontima 06/06/2011 Cyprodinil BARLEY Isopyrazam Herbicides Chimera SX 01/03/2011 Metsulfuron-methyl Thifensulfuron-methyl Oxytril Cm 01/03/2011 Ioxynil Bromoxynil Agritox 50 01/03/2011 MCPA S9

10 166 Table S1f. Fields 9 (2.3 ha) and 10 (21.9 ha). 167 FIELDS 9 & 10 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide /Fungicide Redigo Deter 15/10/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticide Gandalf 09/11/2012 Beta-cyfluthrin Fungicides Basoon EC 07/05/2013 Epoxiconazole Kingdom 07/05/2013 Boscalid Epoxiconazole Bravo /05/2013 Chlorothalonil Adexar 27/05/2013 Epoxiconazole Fluxapyroxad 15/10/2012 Cello 19/06/2013 Prothioconazole Spiroxamine WHEAT Tebuconazole Herbicides Dictate 09/11/2012 Bentazone as sodium salt Fidox 09/11/2012 Prosulfocarb Liberator 09/11/2012 Flufenacet Diflufenican Quintacel 5C 07/05/2013 Chlormequat chloride Scitec 23/04/2013 Trinexapac-ethyl Oxytril Cm 07/05/2013 Ioxynil Bromoxynil Molluscicide TDS Major 10/01/2012 Methaldehyde Slug pelleter Insecticide /Fungicide Redigo Deter 01/03/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticide Alert 27/04/2012 Alpha-cypermethrin 01/03/2012 Fungicides Bravo /04/2012 Chlorothalonil Bontima 17/05/2012 Cyprodinil BARLEY Isopyrazam Herbicides Agritox 50 27/04/2012 MCPA Oxytril Cm 27/04/2012 Ioxynil Bromoxynil Simba SX 27/04/2012 Metsulfuron-methyl Insecticide /Fungicide Redigo Deter 03/03/2011 Clothianidin (Ins.) Prothioconazole (Fun.) 03/03/2011 Fungicides Bravo /04/2011 Chlorothalonil Bontima 11/05/2011 Cyprodinil BARLEY Isopyrazam Herbicides Asset 12/04/2011 Metsulfuron-methyl Agritox 50 12/04/2011 MCPA Hatchet Xtra 12/04/2011 Fluroxypyr S10

11 174 Table S1g. Fields 11 (4.1 ha) and 12 (20.1 ha). 175 FIELD 11 & 12 SOWING TYPE OF PESTICIDE PRODUCT DATE OF APPLICATION ACTIVE INGREDIENTS APPLICATION METHOD Insecticide /Fungicide Redigo Deter 20/09/2012 Clothianidin (Ins.) Prothioconazole (Fun.) Fungicides Crafter 01/05/2013 Chlorothalonil Tebuconazole Talius 01/05/2013 Proquinazid Fielder SE 16/05/2013 Proquinazid Tracker 16/05/2013 Epoxiconazole Boscalid Aviator 235 Xpro 03/06/2013 Bixafen Prothioconazole 20/09/2012 Odin 18/06/2013 Tebuconazole Proline /06/2013 Prothioconazole WHEAT Herbicides Rosate 36 03/09/2012 Glyphosate Crystal 07/10/2012 Pendimethalin Flucenacet Hurricane SC 07/10/2012 Diflufenican Atlantis WG 25/04/2013 Iodosulfuron-methyl-sodium Mesosulfuron-methyl Oxytril Cm 25/04/2013 Ioxynil Bromoxynil Starane XL 03/06/2013 Florasulam Molluscicide Carakol Plus 05/10/2012 Methaldehyde Gusto 3 12/10/2012 Methaldehyde Slug pelleter Decoy Wetex 12/10/2012 Methiocorb Insecticide/Fungicide Cruiser 25/08/2011 Thiamethoxam (Ins.) Fludioxonil (Fung.) Metalaxyl-M (Fung.) Insecticides Toppel 100 EC 14/09/2011 Cypermethrin Permasect C 19/11/2011 Cypermethrin Mavrik 27/03/2012 Tau-fluvalinate Fungicides Genie 25 19/11/2011 Flusilazole Sunorg Pro 19/11/2011 Metconazole Proline /11/2011 Prothioconazole Capitan 25 16/03/2012 Flusilazole 25/08/2011 Delsene 50 Flo 12/04/2012 Carbendazim Herbicides Centium 360 CS 26/08/2011 Clomazone OILSEED RAPE Novall 26/08/2011 Quinmerac Metazachlor Falcon 14/09/2011 Propaquizafop Fox 06/01/2012 Binefox Galera 08/03/2012 Picloram Clopyralid Laser 16/03/2012 Cycloxydim Glyphosate /03/2012 Glyphosate Glyfos Dakar 12/07/2012 Glyphosate Rosate 36 12/07/2012 Glyphosate Molluscicide Gusto 3 27/08/2011 Methaldehyde Slug pelleter Insecticide /Fungicide Redigo Deter 06/03/2011 Clothianidin (Ins.) Prothioconazole (Fun.) Insecticide Hallmark Zeon 11/06/2011 Lambda-cyhalothrin Fungicides Kinto 06/03/2011 Triticonazole Prochloraz Fandango 03/05/2011 Fluoxastrobin Prothioconazole 25/08/2011 Bravo /06/2011 Chlorothalonil Comet 11/06/2011 Pyraclostrobin BARLEY Tracker 11/06/2011 Epoxiconazole Boscalid Herbicide Optica 09/04/2011 Mecoprop-P Oxytril CM 09/04/2011 Ioxynil Bromoxynil Harmony M SX 03/05/2011 Metsulfuron-methyl Thifensulfuron-methyl Agritox 03/05/2011 MCPA RoundUp 25/07/2011 Glyphosate S11

12 Tables S2a S2j. Residues of neonicotinoids (ppb) detected in pollen from wildflowers growing in the margins of 5 OSR and 5 WW fields. When the weight of pollen samples was not big enough to be analysed separately, pollen from different species growing in the same margins were pooled and analysed as a single sample. MDL =Method detection limit; MQL = Method Quantification Limit Table S2a. Field 1 (OSR margins) FIELD 1 MARGIN M1 M2 M3 M4 DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC 18/06/2013 Lamium purpureum 18/06/2013 Glechoma hederacea 18/06/2013 Lamium album 18/06/2013 Vicia sativa 25/06/2013 Trifolium pratense /06/2013 Cardamine pratensis 18/06/2014 Papaver rhoeas /06/2014 Ranunculus repens /06/2013 Cardamine pratensis 03/07/2013 Trifolium repens 26/07/2013 Trifolium repens 03/08/2013 Rubus fruticosus agg. 03/08/2013 Papaver rhoeas /08/2013 Glechoma hederacea 03/08/2013 Viola arvensis 15/06/2014 Silene latifolia 03/07/2013 Calystegia sylvatica 03/07/2013 Malva sylvestris /07/2013 Matricaria recutita 03/07/2013 Sonchus oleraceus S12

13 Table S2b. Field 2 (OSR margins) FIELD MARGIN DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC 09/07/2013 Cirsium vulgare M1 12/07/2013 Rubus fruticosus agg. 12/07/2013 Asteraceae 12/07/2013 Sonchus arvensis M2 M3 M4 05/07/2013 Rubus fruticosus agg. 24/06/2013 Silene vulgaris 05/07/2013 Cirsium vulgare 05/07/2013 Anthriscus sylvestris 05/07/2013 Heracleum sphondylium 05/07/2013 Stachys sylvatica /07/2013 Matricaria recutita 12/07/2013 Matricaria recutita 12/07/2013 Cirsium vulgare 12/07/2013 Papaver rhoeas 23/07/2013 Veronica persica 23/07/2013 Senecio jacobaea 23/07/2013 Sonchus oleraceus /07/2013 Matricaria recutita 23/07/2013 Sonchus oleraceus 23/07/2013 Cirsium vulgare 23/07/2013 Fumaria officinalis 23/07/2013 Carduus sp. 23/07/2013 Lamium purpureum 2.38 S13

14 197 Table S2c. Field 3 (OSR margins) 198 FIELD MARGIN DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC 24/06/2013 Matriaria recutita 05/07/2013 Fumaria officinalis M1 24/06/2013 Matriaria recutita 07/07/2013 Sonchus arvensis 07/07/2013 Cirsium arvense M2 M3 M4 07/08/2013 Anthriscus sylvestris 07/08/2013 Matricaria recutita 13/08/2013 Pimpinella saxifraga 12/08/2013 Senecio jacobaea 12/08/2013 Convolvulus arvensis 12/08/2013 Solanum dulcamara 12/08/2013 Ballota nigra 12/08/2013 Calystegia sepium 12/08/2013 Rubus fruticosus agg. 12/08/2013 Cirsium arvense 12/08/2013 Convolvulus arvensis 12/08/2013 Matriaria recutita MQL MQL 199 S14

15 200 Table S2d. Field 4 (OSR margins) 201 FIELD DATE OF NEONICOTINOID RESIDUES MARGIN SPECIES COLLECTION TMX IMC CLO THC 08/07/2013 Malva moschata MQL 25/06/2013 Matricaria discoidea MQL MQL 08/08/2013 Rubus fruticosus agg. M1 02/08/2013 Rosa arvensis /08/2013 Silene latifolia 02/08/2013 Cirsium vulgare 4 M2 M3 M4 02/08/2013 Heracleum sphondylium /08/2013 Epilobium hirsutum MQL MQL 02/08/2013 Epilobium hirsutum 15/08/2013 Clematis vitalba 02/08/2013 Cirsium vulgare 02/08/2013 Sonchus arvensis /08/2013 Epilobium hirsutum 20/08/2013 Agrimonia eupatoria 20/08/2013 Senecio jacobaea 20/08/2013 Centaurea nigra 20/08/2013 Sonchus arvensis 15/08/2013 Heracleum sphondylium 15/08/2013 Silene latifolia 15/08/2013 Cirsium vulgare MQL MQL S15

16 Table S2e. Field 5 (OSR margins) 215 FIELD DATE OF NEONICOTINOID RESIDUES MARGIN SPECIES COLLECTION TMX IMC CLO THC 22/08/2013 Cirsium vulgare M1 22/08/2013 Brassica rapa 22/08/2013 Heracleum sphondylium M2 M3 M4 26/06/2013 Viola arvensis /06/2013 Myosotis arvensis /08/2013 Silene latifolia 08/07/2013 Papaver rhoeas 27/06/2013 Rubus fruticosus agg. 08/07/2013 Senecio jacobaea MQL 08/07/2013 Silene latifolia MQL MQL 01/07/2013 Matricaria recutita MQL MQL 08/07/2013 Silene dioica 01/07/2013 Papaver rhoeas MQL 22/08/2013 Hieracium sp. 22/08/2013 Rubus fruticosus agg. 22/08/2013 Senecio jacobaea 01/08/2013 Matricaria recutita S16

17 Table S2f. Field 8 (WW margins). DATE OF NEONICOTINOID RESIDUES FIELD MARGIN SPECIES COLLECTION TMX IMC CLO THC 10/07/2013 Lamium purpureum 10/07/2013 Matricaria recutita M1 10/07/2013 Papaver rhoeas 15/07/2013 Arctium sp. 8 M2 M3 M4 15/07/2013 Heracleum sphondylium /07/2013 Trifolium repens 15/07/2013 Viola arvensis 08/08/2013 Senecio jacobaea 16/07/2013 Papaver rhoeas 16/07/2013 Heracleum sphondylium 16/07/2013 Fumaria officinalis 16/07/2013 Centaurea scabiosa 26/07/2013 Viola arvensis 26/07/2013 Fumaria officinalis 10/07/2013 Heracleum sphondylium 10/07/2013 Calystegia sylvatica 10/07/2013 Ligustrum vulgare 10/07/2013 Papaver rhoeas 10/07/2013 Rubus fruticosus agg. S17

18 234 Table S2g. Field 9 (WW margins). 235 FIELD MARGIN DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC 09/07/2013 Rosa canina /07/2013 Ligustrum vulgare 29/07/2013 Malva sylvestris M1 09/07/2013 Rosa canina 29/07/2013 Epilobium hirsutum 29/07/2013 Calystegia sepium 9 M2 M3 M4 26/07/2013 Epilobium hirsutum 29/07/2013 Cirsium vulgare 26/07/2013 Malva sylvestris 26/07/2013 Senecio jacobaea 29/07/2013 Heracleum sphondylium 29/07/2013 Calystegia silvatica 29/07/2013 Heracleum sphondylium 29/07/2013 Cirsium arvense 29/07/2013 Epilobium hirsutum 29/07/2013 Senecio jacobaea 05/08/2013 Fumaria officinalis 05/08/2013 Hieracium sp S18

19 Table S2h. Field 10 (WW margins). FIELD MARGIN DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC 13/08/2013 Senecio jacobaea 14/08/2013 Pimpinella saxifraga M1 13/08/2013 Papaver rhoeas 14/08/2013 Fumaria officinalis 14/08/2013 Hieracium sp. 14/08/2013 Chaenorhinum minus 10 M2 M3 14/08/2013 Senecio jacobaea 14/08/2013 Aethusa cynapium 14/08/2013 Matricaria recutita 13/08/2013 Knautia arvensis 13/08/2013 Silene latifolia 13/08/2013 Convolvulus arvensis 13/08/2013 Cirsium vulgare 13/08/2013 Ballota nigra Table S2i. Field 11 (WW margins). FIELD DATE OF NEONICOTINOID RESIDUES MARGIN SPECIES COLLECTION TMX IMC CLO THC 19/08/2013 Heracleum sphondylium 19/08/2013 Cirsium arvense M1 19/08/2013 Cirsium vulgare 19/08/2013 Viola arvensis 19/08/2013 Sonchus arvensis M2 28/08/2013 Clematis vitalba /08/2013 Solanum dulcamara 28/08/2013 Brassica rapa 28/08/2013 Rubus fruticosus agg. 28/08/2013 Ballota nigra 27/08/2013 Heracleum sphondylium M3 27/08/2013 Heracleum sphondylium M4 27/08/2013 Silene latifolia 27/08/2013 Cirsium arvense 27/08/2013 Veronica persica 27/08/2013 Brassica rapa 27/08/2013 Bryonia dioica S19

20 252 Table S2j. Field 12 (WW margins) FIELD DATE OF NEONICOTINOID RESIDUES MARGIN SPECIES COLLECTION TMX IMC CLO THC 21/08/2013 Matricaria recutita 21/08/2013 Heracleum sphondylium 21/08/2013 Matricaria recutita M1 21/08/2013 Cirsium vulgare 21/08/2013 Arctium sp. 21/08/2013 Veronica persica 12 M2 M3 M4 21/08/2013 Matricaria recutita 21/08/2013 Hypericum hirsutum /08/2013 Heracleum sphondylium 28/08/2013 Matricaria recutita 28/08/2013 Matricaria recutita 21/08/2013 Epilobium hirsutum 28/08/2013 Senecio jacobaea 28/08/2013 Sonchus arvensis 28/08/2013 Cirsium arvense 28/08/2013 Convolvulus arvensis 28/08/2013 Senecio jacobaea 28/08/2013 Brassica rapa 21/08/2013 Brassica rapa 28/08/2013 Matricaria recutita 21/08/2013 Chaenorhinum minus 21/08/2013 Datura stramonium 28/08/2013 Sonchus oleraceus S20

21 Tables S3a-S3b. Residues of neonicotinoid insecticides (ppb) detected in nectar from wildflowers growing in the margins of 5 OSR (7a) and 4 WW (7b) fields. When the volume of nectar samples was not big enough to be analysed separately, nectar from different species growing in the same fields were pooled and analysed as a single sample. Table S3a. Fields 1 5 (OSR margins). S21

22 FIELD MARGIN DATE OF NEONICOTINOID RESIDUES SPECIES COLLECTION TMX IMC CLO THC Lamium purpureum M1 18/06/2013 Glechoma hederacea Lamium album 1 Trifolium repens Malva sylvestris M2 + M4 03/07/2013 Sonchus oleraceus Calystegia silvatica MQL 2 M1 M2 M3 M4 12/07/2013 Hieracium sp. MQL MQL Sonchus arvensis 24/06/2013 Silene vulgaris MQL MQL 05/07/2013 Rubus fruticosus agg. MQL MQL 12/07/2013 Cirsium vulgare MQL 23/07/2013 Sonchus oleraceus Sonchus oleraceus 1.80 MQL 23/07/2013 Cirsium vulgare MQL Carduus sp M1 07/07/2013 Cirsium arvensis MQL MQL M4 12/08/2013 Cirsium arvensis MQL 08/08/2013 Rubus fruticosus agg. 02/08/2013 Silene latifolia MQL M1 08/07/2013 Malva moschata M2 + M3 +M4 02/08/2013 Rosa arvensis 02/08/2013 Cirsium vulgare 02/08/2013 Cirsium vulgare 15/08/2013 Cirsium vulgare 20/08/2013 Sonchus arvensis 15/08/2013 Clematis vitalba M4 15/08/2013 Silene latifolia M2 + M4 02/08/2013 Epilobium hirsutum Sonchus arvensis MQL Brassica rapa M1 22/08/2013 Cirsium vulgare M3 08/08/2013 Silene dioica Rubus fruticosus agg. M4 22/08/2013 MQL Hieracium sp. S22

23 272 Table S3b. Fields 8-12 (WW margins). DATE OF NEONICOTINOID RESIDUES FIELD MARGIN SPECIES COLLECTION TMX IMC CLO THC Arctium sp. 8 M1 + M2 15/07/2013 Trifolium repens Viola arvensis 9 09/07/2013 Rosa canina M1 29/07/2013 Malva sylvestris 29/07/2013 Epilobium hirsutum 29/07/2013 Calystegia sepium M3 29/07/2013 Epilobium hirsutum Chaenorhinum minus M4 05/08/2013 Viola arvensis Hieracium sp. Knautia arvensis 10 M3 13/08/2013 Convolvulus arvensis Cirsium vulgare Cirsium arvensis 11 M1 19/08/2013 Cirsium vulgare Viola arvensis Sonchus arvensis Sonchus arvensis 12 M3 28/08/2013 Cirsium arvensis Convolvulus arvensis Brassica rapa S23

24 Table S4. Multiple reaction monitoring conditions used for UHPLC MS/MS analysis of neonicotinoids (ESI, positive mode) and their respective retention times. TMX = thiamethoxam, TMX-d3 = thiamethoxam-d3, CLO = clothianidin, CLO-d3 = clothianidin-d3, IMC = imidacloprid, IMC-d4 = imidacloprid-d4, and THC = thiacloprid Pesticide Transition mass (m/z) a Dwelltime CV (V) CE (ev) Rt (min) 290 TMX 292.2> > TMX-d3 295> > CLO 250.0> > CLO-d > > IMC 256.2> > IMC-d > > THC * 253.0> > * IMC-d4 was used as internal standard for THC S24

25 Table S5a. Method detection limits (MDLs) and method quantification (MQLs) limits of four neonicotinoids for nectar, pollen and soil samples extracted using the QuEChERS method and analysed by UHPLC-MS/MS. TMX = thiamethoxam, CLO = clothianidin, IMC = imidacloprid, and THC = thiacloprid Nectar Pollen Soil MDL MQL MDL MQL MDL MQL ppb ww ppb ww ppb dw TMX CLO IMC THC Table S5b. Absolute recoveries (%) of four neonicotinoids from spiked nectar, pollen and soil extracted with the QuEChERS method. TMX = thiamethoxam, CLO = clothianidin, IMC = imidacloprid, and THC = thiacloprid Nectar (n=4) Pollen (n=4) Soil (n=4) 1 ppb dw 1.2 ppb ww 10 ppb dw Av SD Av SD Av SD TMX CLO IMC THC S25

26 Tables S6a-S6b. Percentage of pollen types collected by honey bees during oilseed rape flowering period (June 2013) and after it (August 2013). The diversity of plant taxa represented in pollen collected by honey bees was lower in June (Simpson s index of Diversity (1-D) = 0.54) than in August (1-D = 0.85) Table S6a. Pollen types collected by honey bees during oilseed rape flowering period (June 2013). POLLEN TYPE DURING OILSEED RAPE BLOOMING PERIOD (JUNE 2013) % OF POLLEN BY WEIGHT Crataegus monogyna (Hawthorn) Anthriscus sylvestris (Cow Parsley) Brassica napus (Oilseed Rape) 9.86 Papaver rhoeas (Common Poppy) 6.77 Vicia sativa (Common Vetch) 3.19 Salix sp. (Willow) 1.13 Carduus sp. (Thistle) 1.01 Taraxacum officinale agg. (Dandelion) 0.28 Plantago sp. (Plantain) 0.26 Asteraceae 0.19 Ligustrum vulgare (Wild Privet) 0.11 Rosaceae 0.05 Rubus fruticosus agg. (Bramble) 0.04 Rosa sp. (Rose) 0.01 Aesculus hippocastanum (Horse Chesnut) 0.01 Unknown S26

27 Table S6b. Pollen types collected by honey bees during oilseed rape flowering period (June 2013). POLLEN TYPE AFTER OSR BLOOMING PERIOD (AUGUST 2013) % OF POLLEN BY WEIGHT Epilobium hirsutum(hairy Willowherb) Rubus fruticosus agg. (Bramble) Trifolium repens (White Clover) Clematis vitalba (Old man s beard) Sonchus sp. (Sow Thistle) 9.85 Papaver rhoeas (Common Poppy) 9.79 Ligustrum vulgare (Wild Privet) 6.57 Cirsium sp. (Thistle) 2.93 Melilotus sp. (Sweet Clover) 1.31 Buddleja davidii (Buterfly Bush) 0.70 Heracleum sphondylium (Hogweed) 0.57 Rosa sp. (Rose) 0.38 Plantago sp. (Plantain) 0.35 Centaurea sp. (Knapweed) 0.29 Scabiosa columbaria (Small Scabious) 0.24 Asteraceae 0.19 Linaria sp. (Toadflax) 0.07 Trifolium pratense (Red Clover) 0.05 Senecio jacobaea (Ragwort) 0.04 Arctium sp. (Burdock) 0.03 Taraxacum officinale agg. (Dandelion) 0.02 Verbascum sp. (Common Mullein) 0.01 Carduus sp. (Thistle) 0.01 Unknown S27

28 Table S7. Concentrations of neonicotinoid insecticides detected in commercial seeds used in conventional farms. Thiamethoxam and clothianidin concentrations have been calculated by external dilution in the highly concentrated samples. 361 TREATED WITH NEONICOTINOIDS TREATED WITH FUNGICIDES ng/g seed SEED TREATMENT (a.i.) TMX CLO IMC THC OSR Thiamethoxam (24.5 % w/w) 1,288, WW Clothianidin (21.4% w/w) , OSR Prochloraz (12.6% w/w) + Thiram (27.9% w/w) 1, OSR Fludioxonil (2.34% w/w) + Flutriafol (2.34% w/w) 2, Barley Triticonazole (1.94% w/w) + Prochloraz (5.65% w/w) S28

29 Table S8. Comparison of thiamethoxam (TMX) concentrations in pollen from wildflowers collected in OSR margins analysed by UHPLC-MS/MS and GC-MS/MS FIELD MARGIN 2 M3 3 M3 4 M1 4 M3 DATE OF COLLECTION SPECIES 12/07/2013 Matricaria recutita 12/07/2013 Cirsium vulgare 12/07/2013 Papaver rhoeas 23/07/2013 Veronica persica 23/07/2013 Senecio jacobaea 23/07/2013 Sonchus oleraceus 12/08/2013 Senecio jacobaea 12/08/2013 Convolvulus arvensis 12/08/2013 Solanum dulcamara 12/08/2013 Ballota nigra 08/08/2013 Rubus fruticosus agg. 02/08/2013 Rosa arvensis 02/08/2013 Silene latifolia 02/08/2013 Cirsium vulgare 02/08/2013 Epilobium hirsutum 20/08/2013 Agrimonia eupatoria 20/08/2013 Senecio jacobaea 20/08/2013 Centaurea nigra 20/08/2013 Sonchus arvensis TMX CONCENTRATIONS ppb dw UHPLC- MS/MS GC- MS/MS S29