Real-time monitoring of honeybee colony daily activity and bee loss rates can highlight the risk posed by a pesticide.

Information on honeybee foraging performance and especially bee loss rates at the colony level are crucial for evaluating the magnitude of effects due to pesticide exposure, thereby ensuring that protection goals for honeybee colonies are met (i.e. threshold of acceptable effects). However, current methods for monitoring honeybee foraging activity and mortality are very approximate (visual records) or are time-limited and mostly based on single cohort analysis. We therefore assess the potential of bee counters, that enable a colony-level and continuous monitoring of bee flight activity and mortality, in pesticide risk assessment. After assessing the background activity and bee loss rates, we exposed colonies to two concentrations of sulfoxaflor (a neurotoxic insecticide) in sugar syrup: a concentration that was considered to be field realistic (0.59 µg/ml) and a higher concentration (2.36 µg/ml) representing a worst-case exposure scenario. We did not find any effect of the field-realistic concentration on flight activity and bee loss rates. However, a two-fold decrease in daily flight activity and a 10-fold increase in daily bee losses were detected in colonies exposed to the highest sulfoxaflor concentration as compared to before exposure. When compared to the theoretical trigger values associated with the specific protection goal of 7 % colony-size reduction, the observed fold changes in daily bee losses were often found to be at risk for colonies. In conclusion, the real-time and colony-level monitoring of bee loss rates, combined with threshold values indicating at which levels bee loss rates threaten the colony, have great potential for improving regulatory pesticide risk assessments for honeybees under field conditions.

Pesticide risk assessment: honeybee workers are not all equal regarding the risk posed by exposure to pesticides.

Toxicological studies in honeybees have long shown that a single pesticide dose or concentration does not necessarily induce a single response. Inter-individual differences in pesticide sensitivity and/or the level of exposure (e.g., ingestion of pesticide-contaminated matrices) may explain this variability in risk posed by a pesticide. Therefore, to better inform pesticide risk assessment for honeybees, we studied the risk posed by pesticides to two behavioral castes, nurse, and forager bees, which are largely represented within colonies and which exhibit large differences in their physiological backgrounds. For that purpose, we determined the sensitivity of nurses and foragers to azoxystrobin (fungicide) and sulfoxaflor (insecticide) upon acute or chronic exposure. Azoxystrobin was found to be weakly toxic to both types of bees. However, foragers were more sensitive to sulfoxaflor than nurses upon acute and chronic exposure. This phenomenon was not explained by better sulfoxaflor metabolization in nurses, but rather by differences in body weight (nurses being 1.6 times heavier than foragers). Foragers consistently consumed more sugar syrup than nurses, and this increased consumption was even more pronounced with pesticide-contaminated syrup (at specific concentrations). Altogether, the stronger susceptibility and exposure of foragers to sulfoxaflor contributed to increases of 2 and tenfold for the acute and chronic risk quotients, respectively, compared to nurses. In conclusion, to increase the safety margin and avoid an under-estimation of the risk posed by insecticides to honeybees, we recommend systematically including forager bees in regulatory tests.

Toxicity of the Pesticides Imidacloprid, Difenoconazole and Glyphosate Alone and in Binary and Ternary Mixtures to Winter Honey Bees: Effects on Survival and Antioxidative Defenses.

To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.

Delayed effects of a single dose of a neurotoxic pesticide (sulfoxaflor) on honeybee foraging activity.

Pesticide risk-assessment guidelines for honeybees (Apis mellifera) generally require determining the acute toxicity of a chemical over the short-term through fix-duration tests. However, potential long-lasting or delayed effects resulting from an acute exposure (e.g. a single dose) are often overlooked, although the modification of a developmental process may have life-long consequences. To investigate this question, we exposed young honeybee workers to a single sublethal field-realistic dose of a neurotoxic pesticide, sulfoxaflor, at one of two amounts (16 or 60 ng), at the moment when they initiated orientation flights (preceding foraging activity). We then tracked in the field their flight activity and lifespan with automated life-long monitoring devices. Both amounts of sulfoxaflor administered reduced the total number of flights but did not affect bee survival and flight duration. When looking at the time series of flight activity, effects were not immediate but delayed until foraging activity with a decrease in the daily number of foraging flights and consequently in their total number (24 and 33% less for the 16 and 60 ng doses, respectively). The results of our study therefore blur the general assumption in honeybee toxicology that acute exposure results in immediate and rapid effects and call for long-term recording and/or time-to-effect measurements, even upon exposure to a single dose of pesticide.

Pollen nutrition fosters honeybee tolerance to pesticides.

A reduction in floral resource abundance and diversity is generally observed in agro-ecosystems, along with widespread exposure to pesticides. Therefore, a better understanding on how the availability and quality of pollen diets can modulate honeybee sensitivity to pesticides is required. For that purpose, we evaluated the toxicity of acute exposure and chronic exposures to field realistic and higher concentrations of azoxystrobin (fungicide) and sulfoxaflor (insecticide) in honeybees provided with pollen diets of differing qualities (named S and BQ pollens). We found that pollen intake reduced the toxicity of the acute doses of pesticides. Contrary to azoxystrobin, chronic exposures to sulfoxaflor increased by 1.5- to 12-fold bee mortality, which was reduced by pollen intake. Most importantly, the risk of death upon exposure to a high concentration of sulfoxaflor was significantly lower for the S pollen diet when compared with the BQ pollen diet. This reduced pesticide toxicity was associated with a higher gene expression of vitellogenin, a glycoprotein that promotes bee longevity, a faster sulfoxaflor metabolization and a lower concentration of the phytochemical p-coumaric acid, known to upregulate detoxification enzymes. Thus, our study revealed that pollen quality can influence the ability of bees to metabolize pesticides and withstand their detrimental effects, providing another strong argument for the restoration of suitable foraging habitat.

Physiological effects of the interaction between Nosema ceranae and sequential and overlapping exposure to glyphosate and difenoconazole in the honey bee Apis mellifera.

Pathogens and pollutants, such as pesticides, are potential stressors to all living organisms, including honey bees. Herbicides and fungicides are among the most prevalent pesticides in beehive matrices, and their interaction with Nosema ceranae is not well understood. In this study, the interactions between N. ceranae, the herbicide glyphosate and the fungicide difenoconazole were studied under combined sequential and overlapping exposure to the pesticides at a concentration of 0.1 µg/L in food. In the sequential exposure experiment, newly emerged bees were exposed to the herbicide from day 3 to day 13 after emerging and to the fungicide from day 13 to day 23. In the overlapping exposure experiment, bees were exposed to the herbicide from day 3 to day 13 and to the fungicide from day 7 to day 17. Infection by Nosema in early adult life stages (a few hours post emergence) greatly affected the survival of honey bees and elicited much higher mortality than was induced by pesticides either alone or in combination. Overlapping exposure to both pesticides induced higher mortality than was caused by sequential or individual exposure. Overlapping, but not sequential, exposure to pesticides synergistically increased the adverse effect of N. ceranae on honey bee longevity. The combination of Nosema and pesticides had a strong impact on physiological markers of the nervous system, detoxification, antioxidant defenses and social immunity of honey bees.

Toxicological status changes the susceptibility of the honey bee Apis mellifera to a single fungicidal spray application.

During all their life stages, bees are exposed to residual concentrations of pesticides, such as insecticides, herbicides, and fungicides, stored in beehive matrices. Fungicides are authorized for use during crop blooms because of their low acute toxicity to honey bees. Thus, a bee that might have been previously exposed to pesticides through contaminated food may be subjected to fungicide spraying when it initiates its first flight outside the hive. In this study, we assessed the effects of acute exposure to the fungicide in bees with different toxicological statuses. Three days after emergence, bees were subjected to chronic exposure to the insecticide imidacloprid and the herbicide glyphosate, either individually or in a binary mixture, at environmental concentrations of 0.01 and 0.1 µg/L in food (0.0083 and 0.083 µg/kg) for 30 days. Seven days after the beginning of chronic exposure to the pesticides (10 days after emergence), the bees were subjected to spraying with the fungicide difenoconazole at the registered field dosage. The results showed a delayed significant decrease in survival when honey bees were treated with the fungicide. Fungicide toxicity increased when honey bees were chronically exposed to glyphosate at the lowest concentration, decreased when they were exposed to imidacloprid, and did not significantly change when they were exposed to the binary mixture regardless of the concentration. Bees exposed to all of these pesticide combinations showed physiological disruptions, revealed by the modulation of several life history traits related mainly to metabolism, even when no effect of the other pesticides on fungicide toxicity was observed. These results show that the toxicity of active substances may be misestimated in the pesticide registration procedure, especially for fungicides.

Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic physiological disturbances in winter Apis mellifera honey bees.

Multiple pesticides originating from plant protection treatments and the treatment of pests infecting honey bees are frequently detected in beehive matrices. Therefore, winter honey bees, which have a long life span, could be exposed to these pesticides for longer periods than summer honey bees. In this study, winter honey bees were exposed through food to the insecticide imidacloprid, the fungicide difenoconazole and the herbicide glyphosate, alone or in binary and ternary mixtures, at environmental concentrations (0 (controls), 0.1, 1 and 10 µg/L) for 20 days. The survival of the honey bees was significantly reduced after exposure to these 3 pesticides individually and in combination. Overall, the combinations had a higher impact than the pesticides alone with a maximum mortality of 52.9% after 20 days of exposure to the insecticide-fungicide binary mixture at 1 µg/L. The analyses of the surviving bees showed that these different pesticide combinations had a systemic global impact on the physiological state of the honey bees, as revealed by the modulation of head, midgut and abdomen glutathione-S-transferase, head acetylcholinesterase, abdomen glucose-6-phosphate dehydrogenase and midgut alkaline phosphatase, which are involved in the detoxification of xenobiotics, the nervous system, defenses against oxidative stress, metabolism and immunity, respectively. These results demonstrate the importance of studying the effects of chemical cocktails based on low realistic exposure levels and developing long-term tests to reveal possible lethal and adverse sublethal interactions in honey bees and other insect pollinators.