Description of the study fields, study field subareas and study locations
The study was conducted at two neighbouring study sites in the vicinity of Sternberg, northern Germany. Each study site covered an area of approximately 65 km² with a diameter of 9 km. Both sites together provided in total nearly 1,800 ha of OSR crops (27 % of arable land). During OSR flowering, no other bee attractive crops were present. The selection of the project area and the study sites took place in summer 2013 and is described in detail in Heimbach et al. (2016). Before drilling, soil samples were collected from all study fields for the analysis of clothianidin residues and soil characterisation. In addition, clothianidin loadings of the OSR seeds were analysed and the entire development of OSR from drilling to harvest was monitored. The results of these analyses are reported in Heimbach et al. (2016). Farmers cultivated OSR and other crops at both study sites according to their common procedures and at their own discretion. They were allowed to compensate for the missing insecticidal dressing in OSR fields at the R site as necessary by foliar spray applications of pyrethroids according to their own selection. Except for the clothianidin dressing of OSR seeds at the T site, no further neonicotinoid was used from autumn 2013 until summer 2014 at the study fields (for details of farming and PPP applications in the study area, see: Heimbach et al. (2016).
In autumn 2013, Elado® (10 g clothianidin & 2 g β-cyfluthrin/kg seed)-dressed OSR seeds were drilled at all 18 study fields (total area approximately 792 ha) at the T site, whereas Elado®-free OSR seeds were drilled at all 17 study fields (total area approximately 615 ha) at the R site (Fig. 1). The median area of study fields was 35.3 ha (1.22–198.0 ha) at the T site and 33.5 ha (9.0–97.3 ha) at the R site. On average, 3.4 ± 1.1 kg/ha of OSR seeds were drilled in the study fields. 1 kg of OSR seeds from the T site contained on average 8.0 ± 1.2 g clothianidin, which amounts to 28.8 ± 10.0 g/ha when the seeds were drilled in the T fields, and this in turn amounts to 19.2 ± 6.7 μg clothianidin per kg soil in the uppermost 10 cm of the soil after drilling assuming an equal distribution of clothianidin in the top 10 cm soil and a soil density of 1.5 kg/l. OSR seeds in the R fields exhibited a median loading of 0.02 g clothianidin per kg seeds. In the R site, the resulting amount of clothianidin per unit area was 0.19 ± 0.25 g/ha that was 0.7 % of the analysed concentration of the T site. For a detailed description of the seed treatment, OSR fields and planting, see Heimbach et al. (2016). Each study field was divided into equally sized sampling plots, called subareas, of approximately 10 ha by means of the GIS programme QGIS. The main aim of subdividing the study fields into subareas was to achieve an appropriate number of samples related to the field size. Examples of the subdivision of two study fields are shown in Fig. 2. Study field T13 was an OSR variety demonstration field and each of the 22 OSR varieties was represented by a subarea, which was smaller than 1 ha. In accordance to the given criteria, the study fields of the R site were subdivided into 58 subareas, and the 18 study fields of the T site were divided into 96 subareas.
For free flying bees, six study locations were selected each at the R and T sites (Fig. 1). Three out of the six study locations per study site were established at the edge of an OSR field, whereas the other three were situated 400 m (for honey bees and bumble bees) or 100 m (for mason bees) distant from the nearest OSR field. Honey bees and bumble bees were placed at the same study locations, whereas mason bees were positioned at different locations taking into account their maximum flight distances. For detailed descriptions of the study locations, see Rolke et al. (2016), Sterk et al. (2016), and Peters et al. (2016).
Estimation of OSR plant density at tunnel tent positions
The density of emerged OSR plants was estimated before stem elongation in March 2014 to compare between the densities of drilled seeds (data provided by Heimbach et al. 2016) and developed plants at the later tunnel tent positions. At each position, a frame of 1 × 1 m was randomly placed 10 times on the ground within a radius of 5 m. The number of emerged OSR plants was counted within the frame. Mean numbers of OSR plants were calculated for each tunnel tent position.
Tunnel tents and tunnel tent arrangement
Tunnel tent experiments were conducted to sample nectar and pollen from OSR plants in the study fields via honey bees under semi-field conditions. The tunnel tents (10 × 5 × 2.5 m) used in this study were semi-circular in cross-section and constructed out of a tubular steel frame, covered with synthetic gauze (mesh size ca. 2 mm). The tunnel tents had a sampling area of approximately 50 m² (at least 45 m² covered with OSR) and were placed in subareas of the study fields for 3 days in a north-south direction, with a minimum distance of 20 m to the edge of the OSR field (Supplementary Figure S1). To ensure that samples of nectar and pollen originated from OSR flowers of the study field subarea, honey bees were enclosed in the tunnel tents at least 1 day before sampling. Each honey bee colony (see 2.4) was used only once and was removed after successfully collecting pollen and nectar.
Preparation and management of bees
Honey bee colonies
Honey bee colonies, Apis mellifera carnica (Apidae), bred according to normal beekeeping practice, disease-free and queen-right were used. All queens were offspring (F1) of the same mother queen. Two different types of honey bee hives were used in this study. For semi-field tunnel tent experiments, small hives (“Mini Plus”, 30 × 30 × 30 cm, 6 combs, and approximately 2,500 bees) were used. These hives were placed inside the tunnel tents at the northwest corner with south-facing entrances.
Hives for free flying honey bees were larger in size and consisted of one brood chamber and 1–2 honey supers (“Zander”, measures of one level (brood chamber and honey supers): 50 × 43.5 × 23 cm, 10 combs per level). Eight hives were placed with south-facing entrances at each of the 12 study locations at the beginning of OSR full flowering on 22/23 April 2014. Rolke et al. (2016) give a detailed description of the preparation, placement and management of the honey bee colonies.
Bumble bee colonies
The buff-tailed or large earth bumble bee, Bombus terrestris dalmatinus (Apidae), was used, as this subspecies is commercially available and widely used for pollination indoors and outdoors. At each study location, one single hive was established for the sampling of returning bumble bees loaded with pollen on 25 April 2014 (in addition to 9 hives established for assessment of colony development, see, Sterk et al. 2016). All hives were positioned south-facing to be protected against wind and rain at the start of OSR flowering. They were set up on top of concrete blocks about 30 cm above the ground. To achieve comparability, every colony contained a mother queen from the same hibernation batch. A detailed description of the preparation, placement and management of the bumble bee colonies can be found in Sterk et al. (2016).
In total, 18,000 cocoons of the red mason bee, Osmia bicornis (Megachilidae), were used in this study. The cocoons were kept at −2 °C to +4 °C over the winter period 2013/2014. Before the cocoons were placed in the study area, they were incubated for 5 days at 8 °C followed by 2 days at 11 °C. At each study location, two cardboard boxes with 750 cocoons each were placed into nesting shelters at the start of OSR flowering on 21 April 2014. Nesting shelters were positioned south-east-facing so that they were exposed to direct sunlight but protected against rain. In all cases, the nesting shelters were placed in front of a forest, a hedge or large shrubs to ensure similar protection from wind. Inside the nesting shelters, nesting blocks composed of 20 medium-density fibreboards (16 × 16 cm) were provided. Each board contained 10 rows of nesting holes arranged in parallel and each row contained 10 nesting holes (8 mm in diameter). See Peters et al. (2016) for a more detailed description of preparation and placement of the mason bees.
In the tunnel tents, sampling started on 23 April 2014 and lasted until 19 May 2014. On 21/22 April, all OSR fields appeared to be in full flower and by 22 May 2014 the flowers in the majority of study fields had withered (Heimbach et al. 2016). During these 4 weeks, samples from 34 out of 58 subareas in the R site and 41 out of 96 subareas in the T site were collected, with collection of at least one sample per study field. The colonies were set up inside the tunnel tents 2 days before sampling and bees were allowed to fly and habituate to the test conditions. For the collection of pollen samples, pollen traps (a punched plate through which pollen-carrying bees must crawl to separate the pollen pellets from the bees’ legs and a fine meshed grid to store these pellets) were attached in front of the entrance of the honey bee hives the evening before sampling. One sample with a minimum amount of 100 mg pollen was collected once per tent location until noon of the sampling day.
Colonies of free flying honey bees were placed at the study locations on 21/22 April 2014, with 21 April 2014 set as day after placement (DAP) 0. Pollen samples were taken twice from all 96 colonies (8 colonies per study location) at two different time points during OSR flowering, on DAP 15 and DAP 19/23. Pollen traps (a grid through which pollen-carrying bees must crawl to separate the pollen pellets from the bees’ legs and a fine meshed grid to store these pellets) were introduced into the bottom board of the hives the day before sampling. On the following day (sampling day), pollen traps were removed and the entire pollen yield was transferred to bottles and shaken in order to mix the pollen properly. Out of this mixture, subsamples of a minimum of 300 mg were taken.
Pollen samples from bumble bees were taken once at every study location during OSR flowering (DAP 19). At each bumble bee hive, 11–21 returning workers with pollen loads were caught and immediately transferred to dry ice. At least 230 mg of pollen was collected from the legs of the bumble bees.
Pollen samples from mason bees were taken once at every study location during OSR flowering (DAP 23). Accordingly, the nesting blocks were opened and 10 subsamples were collected from the rear end of an active, provisioned nesting cell by retrieving the pollen with a micro spoon and combined in a pooled sample with a minimum of 200 mg pollen.
All pollen samples were transferred separately into 15 ml tubes and stored frozen (between −10 °C and −43 °C) until analysis.
For practical reasons, nectar samples were taken from honey bees only, both in tunnel tents and from free flying individuals. About 200 returning honey bee foragers per sample were caught at the hive entrances by using a vacuum collector and immediately transferred to dry ice and stored frozen (between −10 °C and −43 °C) until dissection. The honey stomachs of the collected honey bees were dissected in the laboratory (according to Carreck et al. 2013) and the nectar (minimum of 200 mg) was discharged into a 15 ml tube. All nectar samples were stored frozen (between −10 °C and −43 °C) until analysis.
Honey was only harvested from colonies of free flying honey bees. The harvest of spring honey took place immediately after the end of OSR flowering (DAP 32). Combs from individual colonies were uncapped and honey from each colony was extracted separately by the use of a radial extractor 42000 Voll (Carl Fritz Imkereitechnik Mellrichstadt, Germany). Honey samples (minimum 5 g) were transferred into 15 ml tubes and stored frozen (between −10 °C and −43 °C) until analysis.
Residue analyses were performed by Eurofins Agroscience Services Chem GmbH (Hamburg, Germany). The analytical method was based on the multi-residue sample preparation technique QuEChERS (“Quick Easy Cheap Effective Rugged Safe”, see Lehotay 2006) according to the European Standard EN15662:2008 (2009). Around 100 mg each of a homogenised specimen of pollen, nectar or honey was weighed into a 50 ml centrifuge tube. The exact weight of each sample was documented and used for calculating the residue concentration. To adjust the water content, 10 ml of water was added. For extraction, 10 ml acetonitrile was added to each tube. The centrifuge tube was capped and shaken by hand for at least 2 min. Thereafter, 4.0 g of MgSO4, 1.0 g of NaCl, 1.0 g of trisodium citrate dihydrate and 0.5 g of disodium hydrogen citrate sesquihydrate were added. The centrifuge tube was capped again and immediately shaken by hand for ≥1 min. The sample tube was centrifuged for 4 min at 4,000 rpm. Thereafter, an aliquot of exactly 6 ml of the acetonitrile phase was transferred to a 15-ml centrifuge tube and evaporated to dryness using a nitrogen stream and water bath at 40 °C. The residue was carefully taken up in exactly 1 ml of mixed internal standard solution (water/acetonitrile/formic acid, 75/25/0.1 v/v/v, final concentration: 0.3 ng/ml). The final solvent was equal to the solvent used for preparation of solvent-based standard solutions. If necessary, the uptake was supported by vortex and/or ultrasonic bath, and brief centrifugation was carried out afterwards to separate insoluble particles. Extracts were transferred to HPLC vials for analysis. Calibration solutions were prepared in mixed internal standard solutions ranging from 0.015 ng/ml – 2.0 ng/ml.
For clothianidin and its metabolites TZNG and TZMU, the quantification was performed by internal standardisation using stable-labelled internal standards in pure solvent. A calibration curve was established with at least six concentration levels and used for quantification. For each calibration curve, the coefficient of determination R² was >0.979. The chromatographic system used for the determination of clothianidin and its metabolites TZNG and TZMU was a high performance liquid chromatograph with reversed phase chromatography (Zorbax RRHD Eclipse Plus C18, 50 × 2.1 mm, 1.8 µm column) coupled with tandem mass spectrometry (MS/MS) with electrospray ionisation (AB Sciex API 6500 Triple Quadruple Mass Spectrometer, Analyst version 1.6.2). The limit of quantification (LOQ) was 1.0 µg/kg and the limit of detection (LOD) 0.3 µg/kg for all three compounds.
Average residue concentrations were calculated using 0.0 µg/kg for individual concentrations “<LOD” and 0.65 µg/kg (= mean value for range between LOD and LOQ) for concentrations “<LOQ” to account for the fact that residues were detected and provide a reasonable estimate regarding potential biological effects. No separate average concentrations were calculated in cases where a minimum of 90 % of values were < LOD or < LOQ. The results are presented both as mean ± standard deviation (SD) as well as median values. The mean plant densities were compared between R and T site study fields were compared using unpaired t-test (GraphPad Prism version 6.04, GraphPad Software, La Jolla, California, USA).