Large-scale monitoring of effects of clothianidin-dressed oilseed rape seeds on pollinating insects in northern Germany: residues of clothianidin in pollen, nectar and honey.

This study was part of a large-scale monitoring project to assess the possible effects of Elado® (10 g clothianidin & 2 g ß-cyfluthrin/kg seed)-dressed oilseed rape seeds on different pollinators in Northern Germany. Firstly, residues of clothianidin and its active metabolites thiazolylnitroguanidine and thiazolylmethylurea were measured in nectar and pollen from Elado®-dressed (test site, T) and undressed (reference site, R) oilseed rape collected by honey bees confined within tunnel tents. Clothianidin and its metabolites could not be detected or quantified in samples from R fields. Clothianidin concentrations in samples from T fields were 1.3 ± 0.9 µg/kg and 1.7 ± 0.9 µg/kg in nectar and pollen, respectively. Secondly, pollen and nectar for residue analyses were sampled from free flying honey bees, bumble bees and mason bees, placed at six study locations each in the R and T sites at the start of oilseed rape flowering. Honey samples were analysed from all honey bee colonies at the end of oilseed rape flowering. Neither clothianidin nor its metabolites were detectable or quantifiable in R site samples. Clothianidin concentrations in samples from the T site were below the limit of quantification (LOQ, 1.0 µg/kg) in most pollen and nectar samples collected by bees and 1.4 ± 0.5 µg/kg in honey taken from honey bee colonies. In summary, the study provides reliable semi-field and field data of clothianidin residues in nectar and pollen collected by different bee species in oilseed rape fields under common agricultural conditions.

Large-scale monitoring of effects of clothianidin-dressed OSR seeds on pollinating insects in Northern Germany: effects on large earth bumble bees (Bombus terrestris).

The aim of this study was to investigate the effects of Elado®-dressed winter oilseed rape (OSR, 10 g clothianidin & 2 g beta-cyfluthrin/kg seed) on the development, reproduction and behaviour of large earth bumble bees (Bombus terrestris) as part of a large-scale monitoring field study in Northern Germany, where OSR is usually cultivated at 25-33 % of the arable land. Both reference and test sites comprised 65 km2 in which no other crops attractive to pollinating insects were present. Six study locations were selected per site and 10 bumble bee hives were placed at each location. At each site, three locations were directly adjacent to OSR fields and three locations were situated 400 m distant from the nearest OSR field. The development of colonies was monitored from the beginning of OSR flowering in April until June 2014. Pollen from returning foragers was analysed for its composition. An average of 44 % of OSR pollen was found in pollen loads of bumble bees indicating that OSR was a major resource for the colonies. At the end of OSR flowering, hives were transferred to a nature reserve until the end of the study. Colony development in terms of hive weight and the number of workers showed a typical course with no statistically significant differences between the sites. Reproductive output was comparatively high and not negatively affected by the exposure to treated OSR. In summary, Elado®-dressed OSR did not cause any detrimental effects on the development or reproduction of bumble bee colonies.

Development of a new dispenser for microbiological control agents and evaluation of dissemination by bumblebees in greenhouse strawberries.

BACKGROUND: To date, in modern agriculture, biological control strategies are increasingly becoming the preferred pest management approach. However, the success of microbiological control agents (MCAs) largely depends on efficient dissemination into the crop. The pollinator-and-vector technology employs pollinating insects like bees for a better dissemination. In this study, a new dispenser for bumblebee workers of Bombus terrestris L. was developed. Binab-T-vector and Prestop-Mix were used as two typical MCA products for dissemination. RESULTS: In a first series of experiments in the laboratory for optimisation, the newly developed dispenser was a two-way type dispenser, 20 cm long, with two rectangular compartments and different entrance and exit holes. In addition, the amounts of MCA loaded on the workers were 10 times higher with the new dispenser as compared with the side-by-side passageway (SSP) dispenser. Typically, the highest amounts were recovered from the thorax and legs of the workers. In a second series of experiments under greenhouse conditions with the use of queen-right B. terrestris hives, successful dissemination in strawberry flowers was obtained at different distances from the hive (0-8 m, 8-18 m and 18-21 m), and the workers inoculated the first, second and third flowers that were consecutively visited. In addition, the new dispenser caused no adverse effects on worker foraging intensity, whereas a dramatic reduction was scored with an SSP dispenser. Finally, the data suggested that it is necessary to refill the newly developed dispenser at 3 day intervals. CONCLUSIONS: The results demonstrated that, with the use of the newly developed dispenser, bumblebee workers carried high amounts of MCA, and this resulted in a successful dissemination of MCA into strawberry flowers.

Compatibility of traditional and novel acaricides with bumblebees (Bombus terrestris): a first laboratory assessment of toxicity and sublethal effects.

BACKGROUND: This project assessed the potential hazards of different classical and novel acaricides against an important non-target and beneficial insect for the pollination of wild flowers and cultivated crops, the bumblebee Bombus terrestris (L). Twenty-three acaricides used commercially in the control of phytophagous mites (Acari) were tested in greenhouses and/or the open field. Side effects included acute mortality and also sublethal effects on nest reproduction. The different compounds were administered in the laboratory via three different worst-case field scenario routes of exposure: dermal contact and orally via the drinking of treated sugar water and via treated pollen. The compounds were tested at their respective maximum field recommended concentration (MFRC), and, when strong lethal effects were observed, a dose-response assay with a dilution series of the MFRC was undertaken to calculate LC(50) values. RESULTS: From the different acaricide classes, several chemistries caused high levels of acute toxicity in bumblebee workers, especially bifenthrin and abamectin which resulted in 100% mortality by contact. In addition, several acaricides tested were found to have a detrimental effect on drone production. For oral exposures via treated sugar water, the dose-response assay showed the LC(50) values for abamectin, bifenazate, bifenthrin and etoxazole to be 1/15 MFRC (1.17 mg AI L(-1)), 1/10 MFRC (9.6 mg AI L(-1)), 1/83 MFRC (0.36 mg AI L(-1)) and 1/13 MFRC (4.4 mg AI L(-1)) respectively, indicating that their use should be carefully evaluated. CONCLUSION: Overall, the results suggest that most of the acaricides tested are compatible with bumblebees, with the exceptions of abamectin, bifenazate, bifenthrin and etoxazole. However, the risks also depended on the type of treatment. As a result, the sugar water treatment seems to present the worst-case situation of exposure, indicating that this approach is suitable for determining the hazards of pesticides against bumblebees. Finally, it is suggested that future tier testing under more field-related conditions is required for a final decision of their risks.

Risk assessment for side-effects of neonicotinoids against bumblebees with and without impairing foraging behavior.

Bombus terrestris bumblebees are important pollinators of wild flowers, and in modern agriculture they are used to guarantee pollination of vegetables and fruits. In the field it is likely that worker bees are exposed to pesticides during foraging. To date, several tests exist to assess lethal and sublethal side-effects of pesticides on bee survival, growth/development and reproduction. Within the context of ecotoxicology and insect physiology, we report the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behavior under laboratory conditions. In brief, the experimental setup of this behavior test consists of two artificial nests connected with a tube of about 20 cm and use of queenless micro-colonies of 5 workers. In one nest the worker bees constructed brood, and in the other food (sugar and pollen) was provided. Before exposure, the worker bees were allowed a training to forage for untreated food; afterwards this was replaced by treated food. Using this setup we investigated the effects of sublethal concentrations of the neonicotinoid insecticide imidacloprid, known to negatively affect the foraging behavior of bees. For comparison within the family of neonicotinoid insecticides, we also tested different concentrations of two other neonicotinoids: thiamethoxam and thiacloprid, in the laboratory with the new bioassay. Finally to evaluate the new bioassay, we also tested sublethal concentrations of imidacloprid in the greenhouse with use of queenright colonies of B. terrestris, and here worker bees needed to forage/fly for food that was placed at a distance of 3 m from their hives. In general, the experiments showed that concentrations that may be considered safe for bumblebees can have a negative influence on their foraging behavior. Therefore it is recommended that behavior tests should be included in risk assessment tests for highly toxic pesticides because impairment of the foraging behavior can result in a decreased pollination, lower reproduction and finally in colony mortality due to a lack of food.

A laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris.

BACKGROUND: This study was undertaken to identify any potential adverse side effects of the use of seven microbiological control agents (MCAs) on the bumblebee, Bombus terrestris L., in the context of combined use in integrated pest management (IPM). AQ10 (Ampelomyces quisqualis), Binab-T-vector (Hypocrea parapilulifera + T. atroviride; 1/1), Prestop-Mix (Gliocladium catenulatum J1446), Serenade (Bacillus subtilis QST713), Trianum-P (Trichoderma harzianum T22), Botanigard (Beauveria bassiana GHA) and Granupom (Cydia pomonella granulovirus), comprising five biofungicides and two bioinsecticides, were investigated. Bumblebee workers were exposed under laboratory conditions to each MCA at its maximum field recommended concentration (MFRC) via three different routes of exposure: dermal contact and orally via either treated sugar water or pollen. RESULTS: The tested MCAs were found to be safe for workers of B. terrestris, with the exception of Botanigard and Serenade. Exposure to Botanigard via contact at its MFRC caused 92% mortality after 11 weeks, while the 1/10 MFRC killed 46% of exposed workers. For Serenade, topical contact and oral delivery via sugar water resulted in 88 and 100% worker mortality respectively. With lower concentrations (1/2, 1/5 and 1/10 MFRC) the toxicity decreased, but the effect depended on the route of exposure. In addition to lethal effects, nests were also evaluated for sublethal effects after treatment with the seven MCAs at their respective MFRCs over 11 weeks. In these bioassays, only Botanigard and Serenade gave rise to a significant (P < 0.05) decrease in drone production. Sublethal effects on foraging behaviour were also evaluated, and only Botanigard at its MFRC delivered via treated sugar water induced negative effects. CONCLUSION: The results demonstrated that most of the MCAs tested can be considered safe for use in combination with B. terrestris, based on the International Organisation for Biological Control of Noxious Animals and Plants (IOBC) classification. However, some can be harmful, such as the biofungicide Serenade and the bioinsecticide Botanigard. Therefore, it is recommended that all should be tested before use in combination with pollinators. In this context, it is also advisable that these MCAs should be evaluated in more realistic field situations for the assessment of potentially deleterious effects on foraging behaviour.

Bumblebees can be used in combination with juvenile hormone analogues and ecdysone agonists.

This study examined the lethal and sublethal effects on the beneficial insect Bombus terrestris by two classes of insect growth regulators (IGRs) that are commercially used in agriculture to control pest insects. Three juvenile hormones analogues (JHAs) (pyriproxyfen, fenoxycarb and kinoprene) and two ecdysone agonists or moulting accelerating compounds (MACs) (tebufenozide and methoxyfenozide) were tested. The bumblebee workers were exposed to the insecticides via three different routes of exposure: dermally by topical contact, and orally via the drinking sugar water or the pollen. In the first series of experiments the IGRs were applied at their respective maximum field recommended concentration (MFRC). These risk hazard tests showed that the tested IGRs caused no acute toxicity on the workers, and any compound had an adverse effect on reproduction (production of males). In addition, larval development was followed in the treated nests compared with the controls. After application of the two MACs and the JHA fenoxycarb no adverse effects were observed on larval development. However, in the nests where the workers were exposed to the JHAs pyriproxyfen and kinoprene higher numbers of dead larvae were scored. These larvae were third and fourth instars, implying a lethal blockage of development before metamorphosis. In a second test, a series of dilutions was made for kinoprene, and these results revealed that only the MFRC caused a toxic effect on the larval development. On the other hand, kinoprene at lower concentrations (0.0650 mg ai/l) had a stimulatory effect on brood production. It was remarkable that ovaries of such treated dominant workers were longer and contained more eggs than in the controls. In a last experiment, the cuticular uptake was determined for a JHA and MAC to evaluate to what extent worker bees accumulate these classes of IGRs. Cuticular uptake ranged from 34 to 83% at 24 h after topical application. Overall, the obtained results indicate that the tested IGRs at their recommended concentration are safe to be used in combination with B. terrestris.

Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris.

This research project examined the potential hazards of a major class of insect growth regulators (IGRs) to survival, reproduction and larval growth in bumblebees Bombus terrestris L. Eight chitin synthesis inhibitors (CSIs) were tested: buprofezin, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, lufenuron, novaluron and teflubenzuron. These different IGRs, which are important in the control of pest insects in greenhouses, were applied via three different routes of exposure under laboratory conditions: dermal contact, and orally via the drinking of sugar/water and via pollen. The compounds were tested at their respective maximum field recommended concentrations (MFRC) and also in dose-response assays to calculate LC(50) values. In general, none of the CSIs showed acute worker toxicity. However, there was a dramatic reduction in brood production, especially after oral treatment with pollen and sugar/water. Conspicuously, egg fertility was reduced in all treatments with diflubenzuron and teflubenzuron. In addition to egg mortality, the worker bumblebees removed larvae from the treated nest, and in most cases these individuals were dead first-second instars. Under a binocular microscope, such larvae showed an abnormally formed cuticle leading to mechanical weakness and death. In another series of experiments using (14)C-diflubenzuron and (14)C-flufenoxuron, cuticular penetration in workers was studied for a better understanding of the differences in toxicity. With (14)C-diflubenzuron, transovarial transport and accumulation in the deposited eggs supported the strong reproductive effects. Overall, the present results suggest that CSIs should be applied with caution in combination with bumblebees. The compatibility of each compound to be used in combination with B. terrestris is discussed in relation to calculated LC(50) values, routes of uptake and effects.