Klaus Wallner

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Erratum to: Neonicotinoid insecticides translocated in guttated droplets of seed-treated maize and wheat: a threat to honey bees?

The immune system of bees is influenced by a diversity of factors, some of which have changed in the last 10 years such as the application of pesticides. In addition to pollen, nectar and dust, guttated water of seed-dressed plants might be a new source of contamination to bees. Our experiments demonstrated that guttated water of plants germinated from seeds dressed with neonicotinoids contains neonicotinoids. Maize seeds treated with clothianidin (Poncho® 0.5 mg/seed and Poncho® Pro 1.25 mg/seed) resulted in neonicotinoid concentrations up to 8,000 ng mL−1 in the guttated fluid. This concentration decreases rapidly, but remained detectable over several weeks. Seeds treated with Poncho® Pro did not result in higher concentrations in guttated droplets in the first stages of plant development, but the concentration decreased more slowly. Triticale seed treated with imidacloprid contained small quantities of this active agent (up to 13 ng mL−1) in the guttated fluid the following spring after overwintering. During the sampling of guttation fluid, no bees were observed collecting these droplets from triticale or maize. To evaluate the attractiveness of guttation fluid exuded from seed-treated plants under field conditions, more studies are required.

Effects on Varroa jacobsoni from acaricides in beeswax

SUMMARYThe effect from acaricide residues in beeswax on mortality and fertility of Varroa jacobsoni was investigated. Amitraz, coumaphos and fluvalinate were added to acaricide-free wax at 0, 1, 10, 100 ppm each and the wax subsequently was used to produce comb foundation. Amitraz could not be recovered from the foundation, but foundation where coumaphos and fluvalinate had been added to the wax contained approximately the added residue levels. When mites were allowed to enter brood in combs made from this foundation, the effect from coumaphos residues was dramatic. Almost all mother mites died in combs where 100 ppm coumaphos had been added to the foundation and increased mother mite mortality occurred at the 10-ppm level. An effect from fluvalinate was only seen in combs drawn from foundation with 100 ppm fluvalinate. If one brood generation had been present prior to the brood attacked by the mites, no harmful effects to mites from wax contaminants were found, even at the highest contamination levels. T...

A case report of a honey bee colony poisoning incident in France

A case report of a honey bee colony poisoning incident in France Marie-Pierre Chauzat, Anne-Claire Martel, Philippe Blanchard, Marie-Claude Clément, Frank Schurr, Cosette Lair, Magali Ribière, Klaus Wallner, Peter Rosenkranz and Jean-Paul Faucon Agence Française de Sécurité Sanitaire des Aliments, Unit of honey bee Pathology, 105 route des Chappes, BP 111, 06 902 Sophia Antipolis cedex, France. Apicultural State Institute, University of Hohenheim, August-von-Hartmannstrasse 13, D-70599 Stuttgart, Germany.

Uptake of Neonicotinoid Insecticides by Water-Foraging Honey Bees (Hymenoptera: Apidae) Through Guttation Fluid of Winter Oilseed Rape.

Abstract The water-foraging activity of honey bees (Apis mellifera L.) on guttation fluid of seed-coated crops, such as winter oilseed rape (WOR; Brassica napus L.), has not yet been evaluated. We analyzed the uptake of active substances (a.s.) in guttation fluid by evaluating residues of honey-sac contents. In autumn, insecticide residues of up to 130 µg a.s. per liter were released in WOR guttation fluid; this concentration is noticeably lower than levels reported in guttation fluid of seed-coated maize. Until winter dormancy, the concentrations declined to <30 µg a.s. per liter. In spring, residues were linked to prewintered plants and declined steadily until flowering. The maximum release of residues in guttation fluid of seed-coated WOR occurs on the first leaves in autumn when the colonieś water demand decreases. For the first time, proof for the uptake of guttation fluid from seed-coated WOR by honey bees was provided by measuring residues in individual honey-sac contents. In total, 38 out of 204 samples (19%) showed residues of thiamethoxam at concentrations ranging from 0.3 to 0.95 µg per liter while the corresponding concentrations in guttation fluid of WOR varied between 3.6 to 12.9 µg thiamethoxam per liter. The amounts of thiamethoxam we found in the honey sacs of water-foraging honey bees were therefore below the thresholds in nectar and pollen that are considered to have negative effects on honey bees after chronic exposure.

Chronic exposure of honeybees, Apis mellifera (Hymenoptera: Apidae), to a pesticide mixture in realistic field exposure rates

Pollen might be contaminated by multiple pesticides representing a risk for long-term contamination of honeybees when collected. Standardized methodology to assess the effects of pesticide mixtures under field conditions is lacking. We conducted an experiment on chronic feeding of a diet contaminated with a field-realistic pesticide mixture on free-flying honeybee colonies. Pesticide residues in larvae and adult tagmata were detected in trace amounts. In colonies treated with a pesticide mixture, larval weight was higher and acini diameters of the hypopharyngeal glands of nurse bees were smaller than in the untreated control. Brood termination and adult lifespan did not differ between both groups. Our study offers a reproducible and applicable approach for testing effects of pesticides on bee health. As feeding of a field-realistic pesticide mixture did not elicit acute bee toxic effects, the described differences might be explained by sub-lethal effects or joint action of single compounds.

Control of the mite Varroa destructor in honey bee colonies

Klaus Wallner from Universitat Hohenheim in Stuttgart, Germany and Ingemar Fries from the Swedish University of Agricultural Sciences in Uppsala, Sweden discuss the control of V. destructor in honey been colonies by management methods and organic and traditional methods of chemical control.

An optimised technique for the preparation of honey sacs of Apis mellifera L.

In the past, significant questions about the foraging behaviour of honey bees (Apis mellifera L.) as well as morphological aspects have been studied by analysing the honey sac (honey stomach or crop) extract. Various techniques have been used for the preparation of the honey-sac. Gary and Lorenzen (1976) as well as Sylvester et al. (1983) have reviewed earlier techniques used for honey sac preparation. The earliest technique of honey sac preparation for the discrimination between nectar and water foragers was described as killing and manually dissecting the honey bee to reveal the honey sac, or to lead the honey bee to regurgitate a drop of honey sac contents on a filter paper by gently pressure on the abdomen (Park, 1929) or squeezing the abdomen of living bees (Lindauer, 1954). To evaluate the intake of foraging goods into honey bee colonies, Markosyan (1955, cited in Gary and Lorenzen, 1976) differentiated by weighing in and outgoing foraging honey bees. Previous studies have demonstrated possible exposure of honey bees to active substances in pesticides based on residue analysis of honey-sac extracts (Wallner and Potyka, 2006; Wallner, 2009) and honey bee products such as honey, pollen and beebread (Kubik et al., 1999, 2000). However, the enormous exclusion rate of unusable extracts meant that large numbers of honey sacs had to be prepared. Until now, the honey sacs have usually been prepared individually by hand (Medrzycki et al., 2013), which is a very time-consuming technique. In the context of the discussion about the impact of guttation from seed-treated plants on the vitality of honey bee colonies, the focus has been shifted to water foraging honey bees. Guttation is an active process occurring in many monoand dicotyledonous plants when the transpiration rate of the plant is reduced or eventually ceases because the surrounding air reaches the dew point (Harries, 1999). When the air is saturated by water vapour, the plants secrete surplus water by specialised cells called hydathodes. Previous experiments with xylem-mobile fungicides (Harries, 1999) as well as insecticides such as neonicotinoids (Girolami et al., 2009, Reetz et al., 2011) revealed that significant quantities of the active substances can be excreted through guttation. In addition to pollen and nectar, guttation of plants treated with systemic insecticides might therefore be an additional pathway of contamination for honey bee colonies. Specific observations and sampling of water foraging honey bees under field conditions are, however, nearly impossible due to the wide foraging range. Therefore returning foragers may be gathered using a vacuum cleaner (RosyTec GmbH, Germany) in front of the closed entrance of the hive. After sampling, bees are frozen with dry ice within the vacuum cleaner and stored in the freezer at -20°C until laboratory investigation of the honey-sac extracts. Early research on nectar and water foragers has already been based on the analysis of honey-sac contents (Park, 1929). Previous studies on the water foraging behaviour of honey bees have shown that a large number of honey sacs have to be prepared for analysis of sugar concentration and residues. Gary and Lorenzen (1976) have described a simplified technique for collecting the honey-sac contents under field conditions by fixation of CO2-anaesthetized honey bees within a special apparatus allowing reproducible pressures on each abdomen. We have now developed an optimised technique for preparing honey-sac extracts by centrifugation in the laboratory.

Pesticide residue survey of pollen loads collected by honeybees (Apis mellifera) in daily intervals at three agricultural sites in South Germany

In agricultural landscapes honeybees and other pollinators are exposed to pesticides, often surveyed by residue analysis of bee bread. However, bee bread is a mixture of pollen pellets of different plants collected over a longer time period. Therefore, pesticide content in the hive varies with plant species and time of pollen collection. Hence, the analysis of bee bread is an approximate approach to gain information on detailed pesticide exposure during the agronomic active season. As high-resolution data is missing, we carried out a pesticide residue survey over five years (2012–2016) of daily collected pollen pellets at three agricultural distinct sites in southern Germany. 281 single day pollen samples were selected and subjected to a multi-pesticide residue analysis. Pesticide contaminations of pollen differed between the sites. Intensive pesticide exposure can be seen by high pesticide concentrations as well as a high amount of different pesticides detected. During the five years of observation 73 different pesticides were found, of which 84% are characterized as non-harmful to honeybees. To estimate pesticide risks for honeybees, the pollen hazard quotient (PHQ) was calculated. Even though pesticides were detected in sublethal concentrations, we found substances not supposed to be exposed to honey bees, indicating the necessity for further improvement of seed treatments and increasing awareness of flowering shrubs, field margins and pesticide drift. Additionally, an in-depth analysis of nine pollen samples, divided into sub-fractions dominated by single plant species, revealed even higher concentrations in single crops for some pesticides. We give precise residue data of 1,657 single pesticide detections, which should be used for realistic laboratory and field tests.

Orientating experiments on guttation fluid of seed treated maize ( Zea mays L.) in relation to the water collecting behaviour of honey bees ( Apis mellifera L.)

The major task of this study was to examine the honey bee’s (Apis mellifera L.) water collection behaviour in relation to the process of guttation in a corn plot treated with clothianidin. As shown in experiments 2009 under field conditions, guttation fluid of seed coated maize contains concentrations up to 8000 ng ml. Based on these results, further investigations were conducted to examine the honey bee’s water collection behaviour in relation to designated water collection stations and varying water quality under tent conditions. In the ‘corn tent’ a high number of maize seed coated with clothianidin (Poncho and Poncho Pro, Bayer CropScience) was sown to create an extreme situation for the bees. Beside the guttation no alternative water sources were offered. In the ‘mesh tent’ the soil was covered with texture to exclude natural water sources so defined water sources were placed at varying distances and with varying qualities of water. Pollen and sugar dough were offered on feeding stations Observations on bees drinking at the water sources, their behaviour and reaction on water quality were induced in both tents. The instances of death in the experimental bee colonies were regularly noted, and the dead bees collected. These tent experiments showed, that bees, which collected guttation droplets in seed dressed corn or clothianidin-spiked water at the artificial water source return to the colony and get damaged after a certain time with the known symptoms. Dead bees can be found in the colony as well as in front of the hive. The number of affected bees in the colony is limited but under the chosen conditions the consumption of contaminated water led to a reduced colony development.