Environmental assessment of PAHs through honey bee colonies – A matrix selection study
Polycyclic aromatic hydrocarbons (PAHs) are a group of over 100 different chemicals that are formed during the incomplete burning of coal, oil and gas, garbage, or other organic substances like tobacco or charbroiled meat. Animal studies have also shown that PAHs can cause harmful effects on the skin, body fluids, and ability to fight disease after both short- and long-term exposure. But these effects have not been seen in people. Some people who have breathed or touched mixtures of PAHs and other chemicals for long periods of time have developed cancer. Some PAHs have caused cancer in laboratory animals when they breathed air containing them (lung cancer), ingested them in food (stomach cancer), or had them applied to their skin (skin cancer).
Creating country scale predictive pollen and pesticide maps using bee hives as monitoring tools
The widespread decline of pollinators in the agricultural landscape, resulting from agricultural intensification and consequent increase in stress from pesticides and lack of floral resources, has led to serious concerns . Honeybees exploit mass – flowering crops and can cover long distances to meet their energy requirements. Consequently, they are exposed to contaminants present in their local environment and honeybees involuntarily collect these contaminants during foraging. Thus the specific pollen found in a colony reflect the foraging landscape and the pollutants found in the colony reflect the environmental pollution status.
Layman's version of “ Guideline for Apicultural citizen science to apply the honey bee colony for bio - monitoring of the environment”
The INSIGNIA study The INSIGNIA study is a grant funded proposal submitted to the call f the European Commission (EC) on “Environmental monitoring of pesticides use through honey bees ”. The study aimed to develop a best practice protocol for Apicultural citizen science and to conduct “ A pilot study on the best practices for a European wide monitoring program with honey bee colonies in an Apicultural citizen science (CS) setting to study pesticide use and exposure of honey bees and investigation of pollen sources ”. The study has been completed in 2021.
LAYMAN’S SUMMARY OF THE INSIGNIA BEE-STUDY
There has been much concern in recent years about pollution of land, both in urban and countryside areas, including by chemicals used in agriculture as pesticides of various types to treat pests and diseases of agricultural crops, as veterinary medicines to treat animal pests and diseases, as biocides to protect timber and other materials from decay and as herbicides used in domestic gardens and amenity areas.
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.
Guideline for apicultural citizen science to apply the honey bee colony for bio-monitoring of the environment
This guideline consists of: 1.Rationale with the background of the application of the honey bee colony as a monitoring tool, and the key outcomes of the INSIGNIA study. 2.Study set-up. This chapter describes the outline of the study, linking relationships, research integrity, data integrity, and communication processes. 3.Protocols for the beekeeper in the role of Apiculturalist Citizen Scientist. In this edition 1, April 2021,of the guideline, the protocols for bio-monitoring for pesticides and bio-monitoring for pollen diversity, with their specific toolboxes, are presented. The format of the protocols for different subjects is identical, to facilitate combining protocols for specific monitoring subjects in a study plan. 4.Methodsapplied in the laboratories for pesticide residue analysis, pollen ITS2 metabarcoding, the statistical methods, and the description of the exposure risk and pollen availability models.
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 exposure 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.
11. INSIGNIA: Beekeepers as citizen scientists investigate the environment of their honey bees
Beekeepers can be valuable volunteers in large scale research studies. They own honey bee (Apis mellifera) colonies, have basic equipment, and are knowledgeable about apiculture. In the INSIGNIA project, citizen science beekeepers collected samples from their colonies over two seasons. Several protocols and different sampling devices for beekeeper participation in monitoring the environment for pesticides and bee forage sources were developed and tested, including all steps until sample analysis in the laboratory. The study was accompanied by studies investigating the motivations and skills of beekeepers volunteering as citizen scientists. The samples and information collected with the help of citizen scientists will be used to model the biodiversity of pollen sources, risk mapping for pollinators and environmental pollution in different environments.
10. Storage methods of mixed pollen samples collected from traps: impact on botanical identification and estimation of relatives abundances as determined by ITS2 metabarcoding
To facilitate storage at the CS premises of a large number of pollen samples collected across the bee active season, while at the same time assuring sample integrity for downstream molecular analyses, here we compared four different storage methods. The objective of this experiment was to assess whether the method of storing freshly collected pollen for long-time periods would affect botanical identification and relative abundances of mixed pollen samples, as determined by ITS2 metabarcoding, The ultimate goal of this experiment is to facilitate future Citizen Science projects by finding cheaper and easier methods for long-term storage of pollen samples that do not compromise the accuracy of downstream laboratorial analyses.
9. How and why beekeepers participate in the INSIGNIA citizen science honey bee environmental monitoring project
Citizen science is becoming more and more popular these days, although no universal definition of the term exists. The most important aspects include the recruitment and training (or instruction) of volunteers – so-called citizen scientists. Even though citizen science has a long tradition in entomology and especially in the research on honey bees and bumblebees, the term has only been used for a relatively short time. Researchers from the University of Graz for the first time ever investigated what motivates beekeepers to voluntary participate in a citizen science study like INSIGNIA. This specific group of citizen scientists was also asked what expectations they had from participating, which study aspects were easy or difficult for them and whether they saw added value from their participation.
8. APIStrip storage
Maria Murcia -Morales & Amadeo R. Fernández-Alba, University Almeria Spain Degradation of pesticides is mainly affected by light and moisture. This is generally true and it also occurs for pesticides accumulated in the APIStrip. In preliminary studies of INSIGNIA in 2019, it was shown that APIStrip storage under frozen conditions and at room temperature (under dry and dark conditions in both cases) had virtually the same impact of the pesticide stability, i.e. when adsorbed onto the APIStrip surface, the pesticide residues remained stable under both conditions……
7. Publication. APIStrip, a new tool for environmental contaminant sampling through honeybee colonies.
2020. APIStrip, a new tool for environmental contaminant sampling through honeybee colonies. María Murcia-Morales, Jozef J.M. Van der Steen, Flemming Vejsnæs, Francisco José Díaz-Galiano, José Manuel Flores, Amadeo R. Fernández-Alba. Science of the Total Environment 729 (2020) 138948
6. Publication. Honeybees as active samplers for microplastics
2020. Honeybees as active samplers for microplastics. Carlos Edo, Amadeo R. Fernández-Alba, Flemming Vejsnæs, Jozef J.M. van der Steen, Francisca Fernández-Piñas, Roberto Rosal. Science of The Total environment. Volume 767, 1 May 2021, 144481
5. Publication. An innovative home-made beebread collector as a tool for sampling and harvesting.
Loglio, G., Formato, G., Pietropaoli, M., Jannoni-Sebastianini, R., Carreck, N., & van der Steen, J. (2019). An innovative home-made beebread collector as a tool for sampling and harvesting. Bee World, 96(1), 16-18