Here we present results of a three-year study to determine the fate of imidacloprid residues in hive matrices and to assess chronic sublethal effects on whole honey bee colonies fed supplemental pollen diet containing imidacloprid at 5, 20 and 100 μg/kg over multiple brood cycles. Various endpoints of colony performance and foraging behavior were measured during and after exposure, including winter survival. Imidacloprid residues became diluted or non-detectable within colonies due to the processing of beebread and honey and the rapid metabolism of the chemical. Imidacloprid exposure doses up to 100 μg/kg had no significant effects on foraging activity or other colony performance indicators during and shortly after exposure. Diseases and pest species did not affect colony health but infestations of Varroa mites were significantly higher in exposed colonies. Honey stores indicated that exposed colonies may have avoided the contaminated food. Imidacloprid dose effects was delayed later in the summer, when colonies exposed to 20 and 100 μg/kg experienced higher rates of queen failure and broodless periods, which led to weaker colonies going into the winter. Pooled over two years, winter survival of colonies averaged 85.7, 72.4, 61.2 and 59.2% in the control, 5, 20 and 100 μg/kg treatment groups, respectively. Analysis of colony survival data showed a significant dose effect, and all contrast tests comparing survival between control and treatment groups were significant, except for colonies exposed to 5 μg/kg. Given the weight of evidence, chronic exposure to imidacloprid at the higher range of field doses (20 to 100 μg/kg) in pollen of certain treated crops could cause negative impacts on honey bee colony health and reduced overwintering success, but the most likely encountered high range of field doses relevant for seed-treated crops (5 μg/kg) had negligible effects on colony health and are unlikely a sole cause of colony declines.

Introduction

Honey bee (Apis mellifera) colony losses and declines in native pollinators have caused much concern worldwide [1–7]. In the United States, annual surveys conducted since the appearance of the syndrome known as colony collapse disorder (CCD) in 2006 continue to show consistent losses of colonies exceeding 30%, although the incidence of CCD has declined in recent years [8–10]. These losses threaten the economic viability of the beekeeping industry and have serious implications to pollination services for both cultivated and wild plants [11,12]. The consensus among bee scientists is that honey bee colony declines are the result of multiple stressors, working independently, in combination, or synergistically to impact honey bee health. Many stress factors have been identified, including parasitic mites (predominantly Varroa destructor), pathogens (viruses and Nosema spp.), interaction between mites and viruses, poor nutrition, pesticide exposure, management stress, and loss of foraging habitat [13–17]. While the specific causal pathways and relative contribution of these stressors are still unknown, beekeepers and many scientists assert that the extensive use of pesticides has had negative impacts on the health of honey bees and other pollinators.

Honey bees are exposed to pesticides used within the hive by beekeepers to control parasitic mites and pathogens, as well as to pesticides used to control pests and diseases of cultivated plants on which bees visit for nectar and pollen. Multiple studies conducted in Europe and the U.S. showed that both healthy and unhealthy colonies contained a diverse range of pesticides in pollen, honey, beewax, and bees [14,18–23]. In U.S. hive surveys, miticides (fluvalinate and coumaphos) used by beekeepers were the most frequently found, followed by pyrethroids, organophosphates, fungicides (mainly chlorothalonil), carbamates, and herbicides. In a recent study that collected pollen from bee hives in seven major crops, 35 different pesticides were detected with a total residue load ranging from 23.6 to 51,310 μg/kg from an average of 9.1 pesticides per pollen sample [24]. Honey bees have probably been exposed to these pesticide loads for many years prior to 2006, yet there has been no evidence linking hive residues of an individual chemical or combination of chemicals to recent honey bee declines, particularly the rapid colony depopulation that is characteristic of CCD [14,16,19]. Some studies have actually shown that residue levels of coumaphos and the pyrethroid esfenvalerate were lower in CCD-affected colonies [14], and expression of genes involved in pesticide detoxification in collapsed colonies was not different compared to control colonies [25].

Despite the lack of evidence implicating pesticides as a major causal factor, neonicotinoid insecticides have been widely implicated in adversely affecting honey bee health due to their extensive use worldwide, systemic activity, and presence in pollen and nectar. These insecticides are very effective on a broad spectrum of insect pests [26] but also moderately to highly toxic to honey bees depending on the particular active ingredient. Six neonicotinoids, including imidacloprid, thiamethoxam, clothianidin, acetamiprid, thiacloprid, and dinotefuran, are applied as systemics on many crops that require managed honey bee colonies and non-Apis bees to attain economic yields. Neonicotinoids bind agonistically to the post-synaptic nicotinic acetylcholine receptors in the insect central nervous system, causing spontaneous discharge of nerve impulses and eventual failure of the neuron to propagate any signal [27–29]. In this study, we focused on imidacloprid because it is the most widely used and has drawn more attention to bee health issues than other neonicotinoids. As the first neonicotinoid registered in the U.S. in 1994, imidacloprid is now generic and currently used in over 400 products, accounting for about one-fifth of the global insecticide market [30]. Imidacloprid is also used on home gardens, turf, ornamental shrubs and trees at application rates much higher than label rates for agricultural crops. For many labeled uses on agricultural crops, imidacloprid can be applied at planting as seed coatings or soil treatments but also by chemigation, side-dress treatment, or foliar spray during the crop cycle (including during flowering if bees are not actively foraging) [31]. Due to their systemic activity, imidacloprid and other neonicotinoids are absorbed by the roots or leaves and then xylem transported in the vascular system through the plant, where they may persist for weeks or months following application depending on the application rate and abiotic conditions. Generally, these chemicals are less likely to move translaterally from leaves to the fruiting structures; however, there is increasing evidence that they move to some extent into pollen and nectar [32–35]. For these reasons, many reports claim that neonicotinoids, particularly imidacloprid, are the major causal factor affecting honey bee health and also may act to trigger other stresses on bees. However, there is no scientific evidence to link neonicotinoids as the major cause of colony declines [36]. A recent workshop of bee experts evaluated the relationship of 39 candidate causes to colony declines and judged neonicotinoids to be a possible contributing factor but unlikely the sole cause [37].

Several review papers [38–40] present comprehensive accounts of the available data on residue levels and exposure risks of neonicotinoids and other pesticides to bees. Of the exposure routes outside the hive, residue studies have detected imidacloprid at average levels of 2–3.9 μg/kg in pollen and less than 2 μg/kg in nectar of seed-treated corn, sunflowers and rape [41–43]. More recent studies of treated cucurbit crops revealed higher residues of imidacloprid and other neonicotinoids in field-collected pollen and nectar, particularly when insecticides were applied at higher rates and closer to flowering [34,35]. Residue levels of imidacloprid ranged 24 to 101μg/kg in pollen and 7 to 16 μg/kg in nectar in pumpkin plants receiving the high label rate, delivered as a transplant water application and later by drip irrigation during bloom [35]. Other routes of exposure to foraging bees include residues of neonicotinoids in surface water, guttation droplets exuded from treated corn seedlings, and contaminated talc dust from planter exhaust [44–47]. Of the imidacloprid concentrations detected inside the hive, residues in bees, bee bread and other hive matrices have been consistently lower than residues in pollen collected directly from flowers and also lower and much less frequent than other pesticides. The highest levels in pollen collected at the hive entrance have been reported in France, where imidacloprid residues were detected in 40.5% of the samples, with levels ranging from 0.9 to 3.1 μg/kg [48,49]. In these studies, levels were either below the limit of detection or <2 μg/kg. Other studies in Europe reported lower detection frequencies in bee-related matrices [50], and less than 3% of the pollen samples collected from U.S. colonies contained neonicotinoid residues, of which most were the less toxic acetamiprid and thiacloprid [21]. The reported concentrations of imidacloprid in pollen and nectar from seed-treated crops (<5 μg/kg) are not acutely lethal to honey bees based on dietary LD 50 values. However, these residues are near to levels shown to cause sublethal effects in laboratory studies [38]. In general, published results suggest that exposure levels above 20 μg/kg of imidacloprid can lead to subtle physiological and behavioral abnormalities in honey bees, including reductions in associative learning, queen fecundity and foraging activity, as well as increased susceptibility to other stresses [38,51–54]. For example, some studies have shown interactions of sublethal doses of imidacloprid with other chemicals [55] and with the gut parasite Nosema [56], resulting in increased susceptibility to pesticides and increased disease infection levels, respectively. While there are demonstrated side effects on honey bees from sublethal doses of imidacloprid, laboratory results are conflicting and some disagreed with the no effects observed in field studies [37]. For example, chronic exposure of honey bee hives for 39 days to field realistic concentrations of imidacloprid (2–20 μg/kg) in sunflower nectar did not result in increased worker mortality or overwintering loss [39,57]. Furthermore, most laboratory studies measured sublethal effects on individual bees (or larvae) or small cohorts of workers by exposing them orally or topically to single doses of pesticides in sucrose solution or contaminated pollen. These effects are most likely less disruptive to the overall health of a functional colony than the direct effects on individual bees. The honey bee colony, as a superorganism, can compensate for many stress factors as a result of the social interactions and feedback mechanisms between individual bees. A meta-analysis reported that the results of impaired learning effects from neonicotinoids exposed to individual bees in laboratory tests cannot be extrapolated to a real exposure scenario under field conditions [58]. Thus, chronic lethal and sublethal effects of pesticide exposure need to be assessed at the colony level.

Few field studies using honey bee colonies have examined sublethal effects of dietary exposure to imidacloprid or other neonicotinoids over multiple brood cycles. One study fed replicate colonies with repeated imidacloprid doses of 0.5 and 5 μg/kg in sucrose syrup and showed no immediate or delayed mortality effects [57]. Several long-term studies found no detrimental effects on colonies placed during flowering in fields of clothianidin-seed treated and untreated canola [33], in oilseed rape fields treated with foliar-applied and seed-treated neonicotinoids [59], and in tunnel cages enclosed over thiamethoxam-seed treated oilseed rape and maize [60]. A similar study investigated the potential impact on honey bee mortality in 16 apiaries surrounded by variable land use of imidacloprid seed-treated corn fields [50]. They reported a negative correlation between colony mortality rate and the acreage of treated fields, suggesting imidacloprid had no adverse effect on colony health. Two whole colony experiments involving sublethal exposure of neonicotinoids to honey bees showed adverse impacts on forager longevity, homing activity, and winter survival of colonies, but these studies used unrealistically high field doses and routes of exposure [61,62]. More recently, Sandrock et al. [63] reported that sublethal dietary exposure to field relevant concentrations of thiamethoxam and clothianidin had significant negative short-term and long-term effects on colony performance and queen health.

To date, no field study has shown that imidacloprid adversely affects honey bee colony health when directly exposed to field realistic dietary doses. Here we present results of a study examining the chronic sublethal effects on whole honey bee colonies fed supplemental pollen diet containing imidacloprid at field realistic doses for 12 weeks. Various endpoints of colony performance and foraging behavior were measured during and after exposure, including winter survival. We also present data from a related experiment that addressed the fate of imidacloprid within colonies and the quantification of the actual exposure dose to worker bees, brood and the queen via honey, beebread and royal jelly.