A selected organosilicone spray adjuvant does not enhance lethal effects of a pyrethroid and carbamate insecticide on honey bees.

As part of the agricultural landscape, non-target organisms, such as bees, may be exposed to a cocktail of agrochemicals including insecticides and spray adjuvants like organosilicone surfactants (OSS). While the risks of insecticides are evaluated extensively in their approval process, in most parts of the world however, authorization of adjuvants is performed without prior examination of the effects on bees. Nevertheless, recent laboratory studies evidence that adjuvants can have a toxicity increasing effect when mixed with insecticides. Therefore, this semi-field study aims to test whether an OSS mixed with insecticides can influence the insecticidal activity causing increased effects on bees and bee colonies under more realistic exposure conditions. To answer this question a pyrethroid (Karate Zeon) and a carbamate (Pirimor Granulat) were applied in a highly bee attractive crop (oil seed rape) during bee flight either alone or mixed with the OSS Break-Thru S 301 at field realistic application rates. The following parameters were assessed: mortality, flower visitation, population and brood development of full-sized bee colonies. Our results show that none of the above mentioned parameters was significantly affected by the insecticides alone or their combination with the adjuvant, except for a reduced flower visitation rate in both carbamate treatments (Tukey-HSD, p < 0.05). This indicates that the OSS did not increase mortality to a biologically relevant extent or any of the parameters observed on honey bees and colonies in this trial. Hence, social buffering may have played a crucial role in increasing thresholds for such environmental stressors. We confirm that the results of laboratory studies on individual bees cannot necessarily be extrapolated to the colony level and further trials with additional combinations are required for a well-founded evaluation of these substances.

Potential Risk of Residues From Neonicotinoid-Treated Sugar Beet Flowering Weeds to Honey Bees (Apis mellifera L.).

In 2018 the European Union (EU) banned the three neonicotinoid insecticides imidacloprid, clothianidin (CLO), and thiamethoxam (TMX), but they can still be used if an EU Member State issues an emergency approval. Such an approval went into effect in 2021 for TMX-coated sugar beet seeds in Germany. Usually, this crop is harvested before flowering without exposing non-target organisms to the active ingredient or its metabolites. In addition to the approval, strict mitigation measures were imposed by the EU and the German federal states. One of the measures was to monitor the drilling of sugar beet and its impact on the environment. Hence we took residue samples from different bee and plant matrices and at different dates to fully map beet growth in the German states of Lower Saxony, Bavaria, and Baden-Württemberg. A total of four treated and three untreated plots were surveyed, resulting in 189 samples. Residue data were evaluated using the US Environmental Protection Agency BeeREX model to assess acute and chronic risk to honey bees from the samples, because oral toxicity data are widely available for both TMX and CLO. Within treated plots, we found no residues either in pools of nectar and honey crop samples (n = 24) or dead bee samples (n = 21). Although 13% of beebread and pollen samples and 88% of weed and sugar beet shoot samples were positive, the BeeREX model found no evidence of acute or chronic risk. We also detected neonicotinoid residues in the nesting material of the solitary bee Osmia bicornis, probably from contaminated soil of a treated plot. All control plots were free of residues. Currently, there are insufficient data on wild bee species to allow for an individual risk assessment. In terms of the future use of these highly potent insecticides, therefore, it must be ensured that all regulatory requirements are complied with to mitigate any unintentional exposure. Environ Toxicol Chem 2023;42:1167-1177. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Large-scale study investigating the effects of a tank mixture containing thiacloprid-prochloraz on honey bees (Apis mellifera).

Risk assessment of plant protection products (PPPs) will be conducted before authorization for their possible effects on non-target organisms, including honey bees. Tank mixtures are often common practice by farmers, and mostly their effects on honey bees are not routinely assessed. To enable a realistic assessment of laboratory-reported effects of a combination of the insecticide thiacloprid and fungicide prochloraz on honey bees, a large-scale field study with spray application in winter oilseed rape was conducted in four regions in Germany. Several parameters were investigated, including mortality, flight activity, and colony development. Residue analysis of various materials (e.g., dead bees, nectar, and pollen) was conducted to assess exposure level. We observed several intoxication symptoms 2 h after application, including a high number of moribund bees and dead bees on the first day after application (DAA +1) compared to the control. Adverse effects were observed on the number of open brood cells, with a significant reduction of approximately 22% compared to control over the experimental period. High residue concentrations were detected on flowers and dead bees on the day of application, which decreased rapidly within six days. The residue concentrations detected were higher in bee-collected materials than in materials stored in the hive. In conclusion, exposure to a combination containing thiacloprid-prochloraz poses a high risk to honey bees. Thus, the application of such a mixture on flowering crops is restricted in Germany.

Transfer of xenobiotics from contaminated beeswax into different bee matrices under field conditions and the related exposure probability.

Beeswax is known to have a high capacity to accumulate different contaminants due to its fat-soluble properties. Many surveys in Europe and the USA have shown high levels of contamination in beeswax especially with acaricides used for varroa treatment. In this study, we investigated the transfer pathways of various active substances from beeswax into different matrices under field conditions. Honey, bee bread, larvae, and pupae samples were collected 6-8 weeks after building the experimental colonies on different charges of wax foundations. Identification and quantification of the target substances were performed with an established and validated multi-residue method using LC-MS/MS and GC-MS systems. Nine out of 19 active substances in wax could be detected in the analyzed matrices. Our results confirm the migration of different contaminants from wax into different bee matrices including honey, bee bread, and bee brood. The concentration of detected residues in the different matrices was significantly increased by increasing residue concentration in wax. Therefore, the maximum detected residues in the matrices were almost in wax containing high residual concentrations. Bee bread can be considered as the most important matrix due to relatively high detected concentrations and transfer ratios of the most contaminants. A significant effect of the lipophilicity of active substances on the transfer ratio into bee bread was found, which means that increasing the Log P values has positive effects on the transfer ratio. In conclusion, our results provide the first detailed information regarding the migration of active substances from wax into various matrices under realistic field conditions and are fundamentally important for assessing potential exposure and risks for honey bees.

High nutritional status promotes vitality of honey bees and mitigates negative effects of pesticides.

Honey bee health is affected by multiple stressors, such as the exposure to plant protection products (PPPs), dietary limitation, monofloral diets and pressure of diseases and pathogens and their interactions. Here, we analysed the interacting effects of plant protection products and low nutritional pollen source on honey bee health under semi-field conditions. We established a healthy honey bee colony in each of 24 tents, planted either with monofloral maize, maize with a diverse flower strip or with monofloral Phacelia tanacetifolia. To evaluate the interaction between exposure to PPPs and nutritional status, a mixture of the insecticide thiacloprid and the fungicide prochloraz was applied. For each colony, we investigated brood capping rate as well as adult longevity, body and head weight, and enzyme activity of acetylcholinesterase and P450 reductase of newly hatched worker bees. We found a significant reduced capping rate in treated maize compared to flowering strips and Phacelia, but no interaction effect between pesticide treatment and nutritional status on capping rate. The response to treatment on the longevity of adults differed significantly between maize and Phacelia, with flower strips being intermediate, indicating interaction effects of PPP treatment and low pollen quality in maize compared to Phacelia and flowering strip treatments. Head weight of newly hatched worker bees showed significant interaction of nutritional status and treatment of PPPs. PPPs slightly increased body weight in all nutritional statuses, except for Phacelia. Enzyme activity of acetylcholinesterase and P450 reductase showed significant different responses between maize and Phacelia to PPP exposure, but not between maize and flowering strip. Our results support the hypothesis that higher pollen quality promotes development of larvae and pupae, longevity of adults and detoxification of PPPs.

Deposition of dust with active substances in pesticides from treated seeds in adjacent fields during drilling: disentangling the effects of various factors using an 8-year field experiment.

The side effects from the use of plant protection products and their potential effects on non-target arthropods (NTAs) such as honey bees, other insects within the vegetation layer and epigeic arthropods nowadays receive more attention. However, uncertainties about the factors driving the deposition of active substances (a.s.) into off-crop areas persist, in particular during sowing of treated seeds. Analysing a highly standardised 8-year field experiment, we assessed the importance of various factors potentially affecting dust drift and deposition of a.s., emitted during the sowing process of treated seeds and deposited on fields adjacent to the drilling field, i.e. on the ground, on flowers, and on nonflowering plant parts. Regarding a.s. deposition, the Heubach a.s. value has a predictive capability, which is independent from all other factors taken into account in this study, and can thus be considered as a scenario-independent measure of potential dust deposition. Petri dish samplers, an established standard method for measuring a.s. deposition, were representative of the results from the plant samplers for a given combination of drilling technique and adjacent crop type. Adjacent crop type is likely to impact on a.s. deposition. The present work will enable a more field-realistic exposure assessment for bees and other NTAs.

Floral resource diversification promotes solitary bee reproduction and may offset insecticide effects - evidence from a semi-field experiment.

Pollinator declines in agricultural landscapes are driven by multiple stressors, but potential interactions of these remain poorly studied. Using a highly replicated semi-field study with 56 mesocosms of varying wild plant diversity (2-16 species) and oilseed rape treated with a neonicotinoid, we tested the interacting effects of resource diversity and insecticides on reproduction of a solitary wild bee. Compared to mesocosms with oilseed rape monocultures, availability of resources from wild plants complementing oilseed rape doubled brood cell production. In addition, bee reproduction increased due to plant diversity and identity effects. Exposure to neonicotinoid-treated oilseed rape reduced bee larval to adult development by 69%, but only in mesocosms with oilseed rape monocultures. Availability of complementary flower resources can thus offset negative effects of neonicotinoid-treated oilseed rape on wild bee reproduction. Policy should encourage the implementation of diverse floral resources mitigating negative effects of crop monocultures and insecticides, thereby sustaining solitary bee populations in agricultural landscapes.

Chronic High Glyphosate Exposure Delays Individual Worker Bee (Apis mellifera L.) Development under Field Conditions.

The ongoing debate about glyphosate-based herbicides (GBH) and their implications for beneficial arthropods gives rise to controversy. This research was carried out to cover possible sublethal GBH effects on the brood and colony development, adult survival, and overwintering success of honey bees (Apis mellifera L.) under field conditions. Residues in bee relevant matrices, such as nectar, pollen, and plants, were additionally measured. To address these questions, we adopted four independent study approaches. For brood effects and survival, we orally exposed mini-hives housed in the "Kieler mating-nuc" system to sublethal concentrations of 4.8 mg glyphosate/kg (T1, low) and 137.6 mg glyphosate/kg (T2, high) over a period of one brood cycle (21 days). Brood development and colony conditions were assessed after a modified OECD method (No. 75). For adult survival, we weighed and labeled freshly emerged workers from control and exposed colonies and introduced them into non-contaminated mini-hives to monitor their life span for 25 consecutive days. The results from these experiments showed a trivial effect of GBH on colony conditions and the survival of individual workers, even though the hatching weight was reduced in T2. The brood termination rate (BTR) in the T2 treatment, however, was more than doubled (49.84%) when compared to the control (22.11%) or T1 (20.69%). This was surprising as T2 colonies gained similar weight and similar numbers of bees per colony compared to the control, indicating an equal performance. Obviously, the brood development in T2 was not "terminated" as expected by the OECD method terminology, but rather "slowed down" for an unknown period of time. In light of these findings, we suggest that chronic high GBH exposure is capable of significantly delaying worker brood development, while no further detrimental effects seem to appear at the colony level. Against this background, we discuss additional results and possible consequences of GBH for honey bee health.

Comparing response of buff-tailed bumblebees and red mason bees to application of a thiacloprid-prochloraz mixture under semi-field conditions.

Recent studies have reported interspecific differences in how bee species respond to various stressors. Evaluating the exposure and responses of different bee species to plant protection products is considered an essential part of their risk assessment. This study was conducted to assess the impacts of thiacloprid-prochloraz mixture on buff-tailed bumblebees (Bombus terrestris) and red mason bees (Osmia bicornis) in a worst-case scenario under semi-field conditions. Bumblebee colonies or solitary bee trap nests were confined in tunnels with flowering oilseed rape. The recommended maximum application rates of 72 g thiacloprid/ha and 675 g prochloraz/ha were applied as a tank mixture during bee flight in full flowering oilseed rape. Several parameters such as flight and foraging activity, population parameters, and exposure level were investigated. Our results show adverse effects of the combination of thiacloprid and prochloraz on the reproductive performance of red mason bees. The number of cocoons produced by O. bicornis was significantly reduced in the treatment compared to the control group. Regarding bumblebees, we found no effects of the thiacloprid-prochloraz mixture on any observed parameters of colony development. The maximum detected concentrations of both active substances three days after application were higher in O. bicornis pollen mass compared to B. terrestris stored pollen. We conclude that this worst-case scenario of thiacloprid-prochloraz exposure poses a high risk to solitary bees and thus the use of such mixture should be restricted.

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.

Data on three-year pesticide monitoring in ditches of the apple orchard region of Altes Land, Germany.

The data presented in this article are related to the research article 'Chemical and biological monitoring of the load of plant protection products and of zoocoenoses in ditches of the orchard region Altes Land' (Süß et al., 2006) [1], which is only available in the German language. The pesticide data presented here were acquired from four ditches (three ditches were located in apple orchards, and one ditch was located in a grassland region) between 2001 and 2003 (Lorenz et al., 2018) [2]. Two different monitoring strategies were applied: event-driven sampling after pesticide applications and weekly integrated sampling using automatic water samplers. A total of 70 active substances were monitored while farmers applied 25 active substances. This article describes the study sites and the analytical methods used to quantify the pesticides in the water samples. The field data set is publicly available at the OpenAgrar repository under https://doi.org/10.5073/20180213-144359 (Lorenz et al., 2018) [2].

Temporal dynamics of whole body residues of the neonicotinoid insecticide imidacloprid in live or dead honeybees.

In cases of acute intoxication, honeybees often lay in front of their hives for several days, exposed to sunlight and weather, before a beekeeper can take a sample. Beekeepers send samples to analytical laboratories, but sometimes no residues can be detected. Temperature and sun light could influence the decrease of pesticides in bee samples and thereby residues left for analysis. Moreover, samples are usually sent via normal postal services without cooling. We investigated the temporal dynamics of whole-body residues of imidacloprid in live or dead honeybees following a single-meal dietary exposure of 41 ng/bee under various environmental conditions, such as freezing, exposure to UV light or transfer of individuals through the mail system. Immobile, "dead" looking honeybees recovered from paralysis after 48 hours. The decrease of residues in living but paralysed bees was stopped by freezing (= killing). UV light significantly reduced residues, but the mode of transport did not affect residue levels. Group feeding increased the variance of residues, which is relevant for acute oral toxicity tests. In conclusion, elapsed time after poisoning is key for detection of neonicotinoids. Freezing before mailing significantly reduced the decrease of imidacloprid residues and may increase the accuracy of laboratory analysis for pesticides.

Secondary biomarkers of insecticide-induced stress of honey bee colonies and their relevance for overwintering strength.

The evaluation of pesticide side-effects on honeybees is hampered by a lack of colony-level bioassays that not only are sensitive to physiological changes, but also allow predictions about the consequences of exposure for longer-term colony productivity and survival. Here we measured 28 biometrical, biochemical and behavioural indicators in a field study with 63 colonies and 3 apiaries. Colonies were stressed in early summer by feeding them for five days with either the carbamate growth regulator fenoxycarb or the neurotoxic neonicotinoid imidacloprid, or left untreated. Candidate stress indicators were measured 8-64 days later. We determined which of the indicators were influenced by the treatments, and which could be used as predictors in regression analyses of overwintering strength. Among the indicators influenced by fenoxycarb were the amount of brood in colonies as well as the learning performance and 24h-memory of bees, and the concentration of the brood food component 10HDA in head extracts. Imidacloprid significantly affected honey production, total number of bees and activity of the immune-related enzyme phenoloxidase in forager bee extracts. Indicators predictive of overwintering strength but unrelated to insecticide feeding included vitellogenin titer and glucose oxidase-activity in haemolymph/whole body-extracts of hive bees. Apart from variables that were themselves components of colony strength (numbers of bees/brood cells), the only indicator that was both influenced by an insecticide and predictive of overwintering strength was the concentration of 10HDA in worker bee heads. Our results show that physiological and biochemical bioassays can be used to study effects of insecticides at the colony level and assess the vitality of bee colonies. At the same time, most bioassays evaluated here appear of limited use for predicting pesticide effects on colony overwintering strength, because those that were sensitive to the insecticides were not identical with those that were predictive of colony overwintering. Our study therefore illustrates the difficulties involved in evaluating the economic/ecological significance of pesticide-induced stress in honey bee field studies.

Honey Bees' Behavior Is Impaired by Chronic Exposure to the Neonicotinoid Thiacloprid in the Field.

The decline of pollinators worldwide is of growing concern and has been related to the use of plant-protecting chemicals. Most studies have focused on three neonicotinoid insecticides (clothianidin, imidacloprid, and thiamethoxam) currently subject to a moratorium in the EU. Here, we focus on thiacloprid, a widely used cyano-substituted neonicotinoid thought to be less toxic to honey bees and of which use has increased in the last years. Honey bees (Apis mellifera carnica) were exposed chronically to thiacloprid in the field for several weeks at a sublethal concentration. Foraging behavior, homing success, navigation performance, and social communication were impaired, and thiacloprid residue levels increased both in the foragers and the nest mates over time. The effects observed in the field were not due to a repellent taste of the substance. For the first time, we present the necessary data for the risk evaluation of thiacloprid taken up chronically by honey bees in field conditions.

The neonicotinoid clothianidin interferes with navigation of the solitary bee Osmia cornuta in a laboratory test.

Pollinating insects provide a vital ecosystem service to crops and wild plants. Exposure to low doses of neonicotinoid insecticides has sub-lethal effects on social pollinators such as bumblebees and honeybees, disturbing their navigation and interfering with their development. Solitary Hymenoptera are also very important ecosystem service providers, but the sub-lethal effects of neonicotinoids have not yet been studied well in those animals. We analyzed the ability of walking Osmia to remember a feeding place in a small environment and found that Osmia remembers the feeding place well after 4 days of training. Uptake of field-realistic amounts of the neonicotinoid clothianidin (0.76 ng per bee) altered the animals' sensory responses to the visual environment and interfered with the retrieval of navigational memory. We conclude that the neonicotinoid clothianidin compromises visual guidance and the use of navigational memory in the solitary bee Osmia cornuta.