DOI: 10.6084/m9.figshare.24235573
title: NicholsonKnapp_etal_2023_BombusPesticideEffects

description: In each of eight European countries (Estonia, Germany, Ireland, Italy, Spain, Sweden, Switzerland, United Kingdom) we selected 16 sites split evenly between two focal crops (Apple: N = 64, Oilseed: N = 64) following a predefined PoshBee protocol (Schweiger et al. 2019). All sites were &gt; 3 km apart to ensure spatial independence of bumblebee foraging ranges. Sites were selected to represent a gradient of agrochemical use and proportion of cropland. in the surrounding landscape The proportion of cropland coverage ranged from 3-98% within 1-km radius buffers. See Hodge et al. (2022) for setup of the study system using a multi-actor approach. Colonies were deployed between 2019-03-24 and 2019-05-12 depending on region and crop specific bloom times. Similarly, colonies were retrieved between 2019-05-06 and 2019-06-12. Colonies were weighed three times i) before installment at the field sites, ii) during the middle of flowering, iii) when the colonies are retrieved from the field sites. Total production, pesticide residue and pollen identity data collection happened after colonies were retrieved from the field.At each site we placed three colonies (N = 384) of Bombus terrestris terrestris, except in Ireland and the United Kingdom where the local subspecies Bombus terrestris audax was used. Colonies were all sourced through local providers. Before deployment colonies were checked for a natal queen and weighed (648 ± 70.9 g, mean ± SD). Colonies were deployed prior to crop bloom. Colonies were housed in protective structures. From each of these colonies we collected 1) metrics of colony performance and 2) pollen samples for pesticide residue analysis and palynological identification. For metrics of colony performance we determined the weight change and maximum weight achieved by individual colonies and colony total production. For weight, we measured each colony before, during, and after bloom and took the maximum value. For weight change, we calculated the natural-log response ratio for each colony as ln(g_max/g_initial), where g_initial is weight prior to bloom and g_max is the maximum weight achieved by a colony during its field placement. For colony production, we closed colonies after bloom and froze them (-20 C) for laboratory dissection. From dissected colonies we counted the number of intact and eclosed worker/male and queen cocoons, including the eclosed cocoons used for nectar and pollen storage. We sum these colony structures because individual counts may be unrepresentative due to absent colony members at the time of retrieval. After dissection, we extracted pollen stored in the colonies and pooled pollen evenly from all three colonies, aiming to obtain at least 15.0 g. Pollen samples were stored below -20 C. Samples were sent on dry ice to PIWet, where each site sample was homogenized and split for the analysis of pollen identity (1/5 sample amount) and pesticides residues (remaining sample amount). Analyses of residues were performed for samples in which quantity was at least 0.52 g (see Data Exclusions). For pesticide residue analysis, we used a previously described method (Kiljanek et al., 2021) that is validated according to SANTE/12682/2019 (European Commission et al., 2020) and accredited in accordance with the ISO 17025 standard. Reagent blanks and blank samples were analyzed in each batch. Recovery checks with samples spiked with pesticides at LOQ levels were performed in each analytical batch to meet SANTE/12682/2019 criteria. For pollen identification, palynological analyses were performed at the Research Centre for Agriculture and Environment (CREA) Bologna, Italy, a laboratory specialized in analyses of bees and bee products and accredited according to UNI CEI EN ISO/IEC 17025. Recognition of pollen type was based on comparison between the observed pollen forms and those present in the CREA collection of reference slides (developed using anthers of identified plant species). For each pollen type, the percentage with respect to the total number of counted pollen grains was calculated. Land use data is based on high resolution images provided by World Imagery (ESRI) land cover features were classified at a consistent scale of 1:2500. World Imagery provides one meter satellite and aerial imagery, typically within 3-5 years of currency, using a combination of imagery sources such as 2.5m SPOT imagery and 0.5m resolution imagery from DigitalGlobe. While performance metrics are replicated at the colony level, pesticide, pollen and landscape data are replicated at the site level. To accommodate this hierarchical data structure we used mixed effects models with site nested within country as a random factor References: Kiljanek, T., Niewiadowska, A., Małysiak, M., & Posyniak, A. (2021). Miniaturized multiresidue method for determination of 267 pesticides, their metabolites and polychlorinated biphenyls in low mass beebread samples by liquid and gas chromatography coupled with tandem mass spectrometry. Talanta, 235, 122721. European Commission. Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. (2020) SANTE/12682/2019 Schweiger, O., Hodge, S., Rundlöf, M., Dominik, C. (2019) WP1.1.1 Field site selection. PoshBee protocol. Hodge, S., Schweiger, O., Klein, A. M., Potts, S. G., Costa, C., Albrecht, M., ... & Stout, J. C. (2022). Design and planning of a transdisciplinary investigation into farmland pollinators: rationale, co-design, and lessons learned. Sustainability, 14(17), 10549.