Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Reeve, John


Neonicotinoids are primarily used in agriculture where they are applied as seed coatings, foliar sprays, and soil drenches or through drip irrigation. In urban areas neonicotinoids are used in home garden products and tree treatments. The maximum foraging ranges of honey bees are usually 10 – 15 km (median distances are 1 – 6 km) from the hive. Hence bee exposure to neonicotinoids is dependent upon the land use type within limited foraging distances from the hive. However, there are virtually no data showing levels of neonicotinoid use in urban areas and few studies have been done to compare urban and agricultural exposure. Several neonicotinoids have shown various toxic effects on pollinators and particularly honey bees. Honey bees have a limited arsenal of detoxification proteins to withstand neonicotinoid exposure, which makes them more sensitive and less able to develop tolerance to these insecticides compared to other insects. Sublethal exposure of honey bees to neonicotinoids can cause behavioral disturbances, orientation difficulties, impairment of social activities, and respiratory pattern changes. These behavioral changes can cause insufficient foraging behavior in honey bees due to the sublethal effect of neonicotinoids, thus putting the colony at risk of food shortage and eventually collapse. My objectives were to (1) develop a highly sensitive and selective, multi-residual analytical method for neonicotinoids in honey bee and pollen samples, (2) investigate the impacts of land use type (agriculture vs. urban) on the exposure of honey bees to neonicotinoid, (3) investigate the sublethal effect of imidacloprid on honey bees’ behavioral performance, and (4) investigate the sublethal effect of field-realistic concentrations of imidacloprid on honey bees’ metabolism at different ambient temperatures.To address my first objective (Chapter 2), I tested three sample cleanup methods (silica SPE, NH2-silica SPE, and Z-Sep SPE) based on solid phase extraction (SPE), which were investigated for determination of neonicotinoid insecticides and selected metabolites in honey bee and pollen samples by LC-MS/MS. Samples were extracted by hexane and ethyl acetate and then cleaned up with a SPE cartridge packed with silica gel, which showed a better cleanup efficiency compared to the aminopropyl silica SPE and zirconium-based sorbents method. Matrix effects of the three cleanup methods were evaluated and compared. Silica gel showed the highest analyte recoveries and method detection limit for this method were 2.0 to 9.1 μg/kg for honey bees and 2.4 to 4.7 μg/kg for pollen. Recovery studies were performed at three spiking levels (10, 60, and 120 μg/kg) and ranged from 78 to 140% with RSDs between 3 to 18% in honey bees and 83 to 124% with RSDs between 3 to 17% in pollen. The silica gel SPE cleanup method was then applied using honey bee and pollen samples that were collected from different apiaries. To address my second objective (Chapter 3), I analyzed honey bee and beebread (pollen) samples from apiaries in agricultural, developed, and undeveloped areas that were collected during two years in Virginia to assess if landscape type or county pesticide use were predictive of honey bee colony exposure to neonicotinoid insecticides. Trace concentrations of the neonicotinoid imidacloprid were detected in honey bees (3 out of 84 samples, 2.02 – 3.97 ng/g), while higher levels were detected in beebread (5 out of 84 samples, 4.68 – 11.5 ng/g) and pollen (3 out of 5 pollen trap samples, 7.86 – 12.6 ng/g). Imidacloprid was only detected in samples collected during July and August and were not detected in honey bees from hives where neonicotinoids were detected in pollen or beebread. Number of hives sampled at a site, county pesticide use, and landscape characteristics were not predictive of neonicotinoid detections in honey bees or beebread (all P>0.05). Because of the low detection rates, field surveys may underestimate honey bee exposure to field realistic levels of pesticides or the risk of exposure in different landscapes. Undetectably low levels of exposure or high levels of exposure that go undetected raise questions with regard to potential threats to honey bees and other pollinators. To address my third objective (Chapter 4), I investigated the effects of sub-lethal concentrations of imidacloprid on late fall forager honey bees’ behavior by accessing their activity levels and walking performance after being fed ad libitum with six different concentrations (2 – 125 μg/kg) of imidacloprid-dosed syrup for up to 48 hours in laboratory. Honey bee activity levels and motivation to move after being released into a UV light illuminated tunnel decreased significantly as dosages of neonicotinoid in their diet increased. However, their walking speeds were not significantly affected by imidacloprid. The behavioral changes I observed in honey bees chronically exposed to neonicotinoid via diet could negatively affect individual honey bee performance of their hive duties and consequently, colony survival during late fall and winter. To address my fourth objective (Chapter 5), I measured honey bee (Apis mellifera) foragers’ CO2 production rates at different temperatures (25, 30, or 35°C) after they consumed syrup dosed with a field realistic (5 μg/L) or high (20 μg/L) concentration of a neonicotinoid insecticide (i.e. imidacloprid) for 48h. We found that imidacloprid exposure significantly disrupted honey bees’ non-flight metabolic rates and there was a significant interaction between imidacloprid dosage and ambient temperature. Honey bee foragers dosed with 5 μg/L imidacloprid displayed higher average metabolic rates and those dosed with 20 μg/L imidacloprid displayed similar average metabolic rates compared to the corresponding control group across all temperatures. Exposure to field realistic concentrations of neonicotinoid may have a higher energetic cost for honey bees at 25℃ than at higher ambient temperatures. Disrupted energy costs in honey bees fed imidacloprid might be due to the thermoregulation, nerve excitation, or detoxification processes. Metabolic rate changes caused by pesticide exposure could result in less available energy for honey bees to perform hive duties and forage, which could negatively affect colony health.




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