Detection of Pesticide Residues from the APIStrip: Results from the nine countries participating in the INSIGNIA study in 2020

The practical sampling phase of the INSIGNIA study took place over the beekeeping seasons of 2019 and 2020. In 2019, three sets of data were available for statistical analysis, resulting from the lab analyses of samples collected via the four matrices trialled for pesticide, biocide, veterinary medical product and varroacide monitoring via bee colonies , i.e. collected pollen, beebread, the Beehold tube and the APIStrip, from Austria, Denmark and Greece. The UK had also participated in 2019 but the samples proved to be unsuitable for chemical analysis. The study extended to include nine countries in the 2020 beekeeping season: Austria, Denmark, Greece, the UK, Ireland, Belgium, France, Italy and Latvia. Statistical analysis of the data from 2019 identified the in – hive passive APIStrip sampler as the most effective matrix for the monitoring of pesticide residues via honey bees, and two to three colonies as a suitable number to use per apiary. Therefore the APIStrip was used in two colonies per beekeeper in the extended study in 2020, and nine apiaries were involved per country. Selection of the apiaries participating in 2020 was arranged between national coordinators and beekeepers primarily on a pragmatic basis, such as ease of provision of practical advice and transfer of APIStrips from the beekeeper to the national coordinator, commitment and reliability of the beekeeper, rather than on the basis of land use around the apiary or other environmental criteria. Sampling was again carried out biweekly (fortnightly) as in 2019, with ten sampling rounds.  

Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at -20°C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P-value<0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ sample> assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

Environmental monitoring study of pesticide contamination in Denmark through honey bee colonies using APIStrip-based sampling

Due to their extensive use in both agricultural and non-agricultural applications, pesticides are a major source of environmental contamination. Honey bee colonies are proven sentinels of these and other contaminants, as they come into contact with them during their foraging activities. However, active sampling strategies involve a negative impact on these organisms and, in most cases, the need of analyzing multiple heterogeneous matrices. Conversely, the APIStrip-based passive sampling is innocuous for the bees and allows for  long-term monitorings using the same colony. The versatility of the sorbent Tenax, included in the APIStrip composition, ensures that comprehensive information regarding the contaminants inside the beehive will be obtained in one single matrix. In the present study, 180 APIStrips were placed in nine apiaries distributed in Denmark throughout a six-month sampling period (10 subsequent samplings, April to September 2020). Seventy-five pesticide residues were detected (out of a 428-pesticide scope), boscalid and azoxystrobin being the most frequently detected compounds. There were significant variations in the findings of the sampling sites in terms of number of detections, pesticide diversity and average concentration. A relative indicator of the potential risk of pesticide exposure for the honey bees was calculated for each sampling site. The evolution of pesticide detections over the sampling periods, as well as the individual tendencies of selected pesticides, is herein described. The findings of this largescale monitoring were compared to the ones obtained in a previous Danish, APIStrip-based pilot monitoring program in 2019. Samples of honey and wax were also analyzed and compared to the APIStrip findings.

15. Dissipation and cross-contamination of miticides in apiculture. Evaluation by APIStrip-based sampling

he active substances coumaphos, tau-fluvalinate and amitraz are among the most commonly employed synthetic miticides to control varroa infestations in apiculture. These compounds can persist inside the beehive matrices and can be detected long time after their application. The present study describes the application of a new passive sampling methodology to assess the dissipation of these miticides as well as the cross-contamination in neighboring beehives. The APIStrips are a recently developed sampling device based on the sorbent Tenax, which shows a remarkable versatility for the sorption of molecules onto its surface. This avoids the need of actively sampling apicultural matrices such as living bees, wax or reserves (honey and pollen), therefore allowing to obtain representative information of the contamination in the beehive environment in one single matrix. The results show that the amitraz-based treatments have the fastest dissipation rate (half-life of 11–14 days), whereas tau-fluvalinate and coumaphos remain inside the beehive environment for longer time periods, with a half-life up to 39 days. In the present study, tau-fluvalinate originated an intense cross-contamination, as opposed to coumaphos and amitraz. This study also demonstrates the contribution of drifting forager bees in the pesticide crosscontamination phenomena. Moreover, the sampling of adult living bees has been compared to the APIStrip-based sampling, and the experimental results show that the latter is more effective and consistent than traditional active sampling strategies. The active substances included in this study do not migrate to the honey from the treated colonies in significant amounts.

14. Beebread consumption by honey bees is fast: results of a six-week field study

Due to their foraging behavior, honey bees interact with the landscape. As a result, honey bees and their brood will be exposed to pesticides through nectar and pollen entering the hive. Although these pathways seem rather straightforward, there are several steps between the entry of nectar and pollen and its consumption by the colony. One of the aspects involved here is the time between collection and consumption of pollen in the hive. This is of importance for the actual exposure of nurse bees and larvae to pesticides in pollen. Although lab and short-term field studies indicated that bees prefer to consume freshly stored pollen, this has to our knowledge not been verified in a long-term field study under realistic  environmental and apiculturist conditions. To study pollen consumption dynamics, influx and consumption were recorded at 3 or 4 day intervals over a six-week period in two colonies. It was demonstrated that throughout the experimental period, beebread consumption was high in the first 3 to 5 days after  collection, over which approximately 70% was consumed. The remaining 30% was consumed within a 2 to 3 week period. Pollen consumption is swift and indicates that only limited time is available for potential degradation processes. As actual data on degradation of pesticides in stored pollen are not available, a justified worse case assumption would be that the actual expos[1]ure concentrations consumed by the nurse bees and larvae are the same as the concentrations in collected pollen.

13. Varroa Control: A Brief Overview of Available Methods

Adequate varroa control is a tool to reduce winter losses (Le Conte et al., 2010; Dainat et al., 2012; Rosenkranz et al., 2010). As beekeepers still fail to choose and implement varroa control methods, there is a need for a better understanding of the mode of action and consequences of the various control methods. This article provides an overview of varroa treatment and provides beekeepers with background information to make varroa control an integral part of beekeeping management. Each control has its efficacy, depending on in-hive conditions. Generally, a combination of control methods appears to be the most effective for year-round low varroa infestation pressure.