Field agrochemical exposure impacts locomotor activity in wild bumblebees.

Agricultural intensification has been identified as one of the key causes of global insect biodiversity losses. These losses have been further linked to the widespread use of agrochemicals associated with modern agricultural practices. Many of these chemicals are known to have negative sublethal effects on commercial pollinators, such as managed honeybees and bumblebees, but less is known about the impacts on wild bees. Laboratory-based studies with commercial pollinators have consistently shown that pesticide exposure can impact bee behavior, with cascading effects on foraging performance, reproductive success, and pollination services. However, these studies typically assess only one chemical, neglecting the complexity of real-world exposure to multiple agrochemicals and other stressors. In the summer of 2020, we collected wild-foraging workers of the common eastern bumblebee, Bombus impatiens, from five squash (Cucurbita) agricultural sites (organic and conventional farms), selected to represent a range of agrochemical, including neonicotinoid insecticide, use. For each bee, we measured two behaviors relevant to foraging success and previously shown to be impacted by pesticide exposure: sucrose responsiveness and locomotor activity. Following behavioral testing, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) chemical analysis to detect and quantify the presence of 92 agrochemicals in each bumblebee. Bees collected from our sites did not vary in pesticide exposure as expected. While we found a limited occurrence of neonicotinoids, two fungicides (azoxystrobin and difenoconazole) were detected at all sites, and the pesticide synergist piperonyl butoxide (PBO) was present in all 123 bees. We found that bumblebees that contained higher levels of PBO were less active, and this effect was stronger for larger bumblebee workers. While PBO is unlikely to be the direct cause of the reduction in bee activity, it could be an indicator of exposure to pyrethroids and/or other insecticides that we were unable to directly quantify, but which PBO is frequently tank-mixed with during pesticide applications on crops. We did not find a relationship between agrochemical exposure and bumblebee sucrose responsiveness. To our knowledge, this is the first evidence of a sublethal behavioral impact of agrochemical exposure on wild-foraging bees.

Pesticide contamination of beeswax from managed honey bee colonies in New York State.

The New York State (NYS) beekeeping industry generated >$11M worth of honey in 2020 and >$300M in pollination services to agriculture annually. Bees are frequently exposed to pesticides through foraging and husbandry practices. Lipophilic pesticides can remain in beeswax for extended periods. We analyzed for pesticides in wax comb samples collected from NYS apiaries at the end of the growing season, comparing residue numbers and concentrations among beekeepers of different operation scales: commercial beekeepers (>300 colonies), sideliners (50-299 colonies), and hobbyists (<50 colonies). We analyzed samples collected from 72 managed honey bee colonies for 92 insecticides, herbicides, and fungicides by liquid chromatography-tandem mass spectrometry. Pesticides were detected in all samples and included 34 fungicides, 33 insecticides, and 22 herbicides. Each wax sample contained 7-35 different residues (x¯ = 17.8 residues). Wax from colonies managed by commercial beekeepers contained the most residues (x¯ = 21.9 residues), hobbyists were second (x¯ = 16.3 residues), and sideliners had the fewest (x¯ = 11.7 residues). Nearly all wax samples (98.6%) contained the pesticide synergist piperonyl butoxide, most samples (86%) contained common varroacides used to control honey bee parasites, including coumaphos and amitraz breakdown products, and 93.1% contained the fungicide difenoconazole. We detected 34 fungicides, 7 of which were found in 50% or more of the samples. We detected 22 herbicides. We found pesticide contamination of beeswax to be common, with commercial beekeepers experiencing the greatest contamination.

Wild bees are exposed to low levels of pesticides in urban grasslands and community gardens.

Globally documented wild bee declines threaten sustainable food production and natural ecosystem functioning. Urban environments are often florally abundant, and consequently can contain high levels of pollinator diversity compared with agricultural environments. This has led to the suggestion that urban environments are an increasingly important habitat for pollinators. However, pesticides, such as commercial bug sprays, have a range of lethal and sub-lethal impacts on bees and are widely available for public use, with past work indicating that managed bees (honeybees and bumblebees) are exposed to a range of pesticides in urban environments. Despite this, we still have a poor understanding of (i) whether wild bees foraging in urban environments are exposed to pesticides and (ii) if exposure differs between genera. Here we assessed pesticide exposure in 8 bee genera foraging across multiple urban landscapes. We detected 13 different pesticides, some at concentrations known to have sub-lethal impacts on pollinators. Both the likelihood of pesticides being detected, and the concentrations observed, were higher for larger bees, likely due to their greater foraging ranges. Our results suggest that restricting agrochemical use in urban environments, where the economic benefits are limited, is a simple way to reduce anthropogenic stress on wild bees.

Quantifying exposure of bumblebee (Bombus spp.) queens to pesticide residues when hibernating in agricultural soils.

Exposure to pesticides is a major threat to bumblebee (Bombus spp.) health. In temperate regions, queens of many bumblebee species hibernate underground for several months, putting them at potentially high risk of exposure to soil contaminants. The extent to which bumblebees are exposed to residues in agricultural soils during hibernation is currently unknown, which limits our understanding of the full pesticide exposome for bumblebees throughout their lifecycle. To generate field exposure estimates for overwintering bumblebee queens to pesticide residues, we sampled soils from areas corresponding to suitable likely hibernation sites at six apple orchards and 13 diversified farms throughout Southern Ontario (Canada) in fall 2019-2020. Detectable levels of pesticides were found in 65 of 66 soil samples analysed for multi-pesticide residues (UPLC-MS/MS). A total of 53 active ingredients (AIs) were detected in soils, including 27 fungicides, 13 insecticides, and 13 herbicides. Overall, the frequency of detection, residue levels (median = 37.82 vs. 2.20 ng/g), and number of pesticides per sample (mean = 12 vs. 4 AIs) were highest for orchard soils compared to soils from diversified farms. Ninety-one percent of samples contained multiple residues (up to 29 different AIs per sample), including mixtures of insecticides and fungicides that might lead to synergistic effects. Our results suggest that when hibernating in agricultural areas, bumblebee queens are very likely to be exposed to a wide range of pesticide residues in soil, including potentially harmful levels of insecticides (e.g., cyantraniliprole up to 148.82 ng/g). Our study indicates the importance of empirically testing the potential effects of pesticide residues in soils for hibernating bumblebee queens, using field exposure data such as those generated here. The differences in potential exposure that we detected between cropping systems can also be used to better inform regulations that govern the use of agricultural pesticides, notably in apple orchards.

Pesticide risk to managed bees during blueberry pollination is primarily driven by off-farm exposures.

When managed bee colonies are brought to farms for crop pollination, they can be exposed to pesticide residues. Quantifying the risk posed by these exposures can indicate which pesticides are of the greatest concern and helps focus efforts to reduce the most harmful exposures. To estimate the risk from pesticides to bees while they are pollinating blueberry fields, we sampled blueberry flowers, foraging bees, pollen collected by returning honey bee and bumble bee foragers at colonies, and wax from honey bee hives in blooming blueberry farms in southwest Michigan. We screened the samples for 261 active ingredients using a modified QuEChERS method. The most abundant pesticides were those applied by blueberry growers during blueberry bloom (e.g., fenbuconazole and methoxyfenozide). However, we also detected highly toxic pesticides not used in this crop during bloom (or other times of the season) including the insecticides chlorpyrifos, clothianidin, avermectin, thiamethoxam, and imidacloprid. Using LD50 values for contact and oral exposure to honey bees and bumble bees, we calculated the Risk Quotient (RQ) for each individual pesticide and the average sample RQ for each farm. RQ values were considered in relation to the U.S. Environmental Protection Agency acute contact level of concern (LOC, 0.4), the European Food Safety Authority (EFSA) acute contact LOC (0.2) and the EFSA chronic oral LOC (0.03). Pollen samples were most likely to exceed LOC values, with the percent of samples above EFSA's chronic oral LOC being 0% for flowers, 3.4% for whole honey bees, 0% for whole bumble bees, 72.4% for honey bee pollen in 2018, 45.4% of honey bee pollen in 2019, 46.7% of bumble bee pollen in 2019, and 3.5% of honey bee wax samples. Average pollen sample RQ values were above the EFSA chronic LOC in 92.9% of farms in 2018 and 42.9% of farms in 2019 for honey bee collected pollen, and 46.7% of farms for bumble bee collected pollen in 2019. Landscape analyses indicated that sample RQ was positively correlated with the abundance of apple and cherry orchards located within the flight range of the bees, though this varied between bee species and landscape scale. There was no correlation with abundance of blueberry production. Our results highlight the need to mitigate pesticide risk to bees across agricultural landscapes, in addition to focusing on the impact of applications on the farms where they are applied.

Identities, concentrations, and sources of pesticide exposure in pollen collected by managed bees during blueberry pollination.

Bees are critical for crop pollination, but there is limited information on levels and sources of pesticide exposure in commercial agriculture. We collected pollen from foraging honey bees and bumble bees returning to colonies placed in blooming blueberry fields with different management approaches (conventional, organic, unmanaged) and located across different landscape settings to determine how these factors affect pesticide exposure. We also identified the pollen and analyzed whether pesticide exposure was correlated with corbicular load composition. Across 188 samples collected in 2 years, we detected 80 of the 259 pesticide active ingredients (AIs) screened for using a modified QuEChERS method. Detections included 28 fungicides, 26 insecticides, and 21 herbicides. All samples contained pesticides (mean = 22 AIs per pollen sample), with pollen collected from bees on conventional fields having significantly higher average concentrations (2019 mean = 882.0 ppb) than those on unmanaged fields (2019 mean = 279.6 ppb). Pollen collected by honey bees had more AIs than pollen collected by bumble bees (mean = 35 vs. 19 AIs detected at each farm, respectively), whereas samples from bumble bees had higher average concentrations, likely reflecting differences in foraging behavior. Blueberry pollen was more common in pollen samples collected by bumble bees (25.9% per sample) than honey bees (1.8%), though pesticide concentrations were only correlated with blueberry pollen for honey bees. Pollen collected at farms with more blueberry in the surrounding landscape had higher pesticide concentrations, mostly AIs applied for control of blueberry pathogens and pests during bloom. However, for honey bees, the majority of AIs detected at each farm are not registered for use on blueberry at any time (55.2% of AIs detected), including several highly toxic insecticides. These AIs therefore came from outside the fields and farms they are expected to pollinate. For bumble bees, the majority of AIs detected in their pollen are registered for use on blueberry during bloom (56.9% of AIs detected), though far fewer AIs were sprayed at the focal farm (16.7%). Our results highlight the need for integrated farm and landscape-scale stewardship of pesticides to reduce exposure to pollinators during crop pollination.

Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium.

Rosebay willowherb (Epilobium angustifolium) contains large amounts of polyphenolic compounds, including tellimagrandin I-based oligomeric ellagitannins (ETs). The aim of this study was to assess the interpopulational and inter-organ variability of the polyphenol fingerprint of E. angustifolium. Seven ETs, 11 flavonol glycosides and neochlorogenic acid were quantified by UHPLC-DAD-ESI-QqQ-MS in the leaves, flowers and stem parts of plants from 10 populations. Total polyphenol content of leaves and flowers ranged from 150 to 200 mg/g dry wt, of which 90% was constituted by dimeric to heptameric ETs. Flowers contained, on average, 10% more oenothein B (dimeric ET) and 2 times less oenothein A (trimeric ET) than leaves. Tetrameric and pentameric ETs exhibited rather similar levels in leaves and flowers whereas hexameric and heptameric were 3-4 times more abundant in flowers than in leaves. Quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside were specific to flower tissue and were absent from leaves. The inflorescence stem showed the highest content in total polyphenols with an average of 250 mg/g dry wt and contained remarkably large amounts of oenothein B and A. Polyphenol content steadily decreased along the inflorescence stem and reached its lowest level in the vegetative part of the stem. The interpopulational variability of most polyphenols was within a two- to threefold range across the 10 sampled populations. Myricetin-3-O-glucoside and myricetin-3-O-glucuronide, however, showed a more population-specific distribution with concentrations varying from 0 to 2.3 mg/g dry wt. Finally, this study showed that the levels of oenothein B and A in the plant are not interdependent but that their relative abundance is constant within a population.

A study of the structure-activity relationship of oligomeric ellagitannins on ruminal fermentation in vitro.

The aim of this study was to investigate how the degree of oligomerization of ellagitannins (ET) influences their ability to alter ruminal fermentation. Dimeric to heptameric ET were isolated from rosebay willowherb (Epilobium angustifolium) flowers and purified. Ellagitannins were tested in vitro on a mixture of grass silage and buffered rumen fluid. Total gas production was measured in real time using an automated pressure evaluation system. Methane production was monitored at regular interval by gas chromatography for 72h. The effect of ET was evaluated on 2 sources of rumen fluid using a randomized block design. Ammonia nitrogen, volatile fatty acid concentration, and pH were measured at the end of the experiment. Results show that oligomeric ET decreased gas production and total volatile fatty acid concentration proportionally to their degree of oligomerization. Methane production was also decreased by all the tested compounds and dimer was less effective than the larger ET, which showed similar levels of activity. Additionally, willowherb's oligomeric ET decreased ammonia-nitrogen and branched-chain volatile fatty acid concentrations, thus indicating reduced protein degradation by ruminal bacteria. This effect showed a quadratic relationship with the degree of oligomerization and was maximal with the tetramer. In conclusion, this study shows that the degree of oligomerization of ET has more than a simple linear effect on fermentation parameters in vitro. Large oligomers, in fact, have more detrimental effects on volatile fatty acid and gas production than small ones, while being similarly effective at inhibiting methane production.

Large Variability of Proanthocyanidin Content and Composition in Sainfoin (Onobrychis viciifolia).

Proanthocyanidins (PAs) in sainfoin (Onobrychis viciifolia Scop.) are of interest to ameliorate the sustainability of livestock production. However, sainfoin forage yield and PA concentrations, as well as their composition, require optimization. Individual plants of 27 sainfoin accessions from four continents were analyzed with LC-ESI-QqQ-MS/MS for PA concentrations and simple phenolic compounds. Large variability existed in PA concentrations (23.0-47.5 mg g(-1) leaf dry matter (DM)), share of prodelphinidins (79-96%), and mean degree of polymerization (11-14) among, but also within, accessions. PAs were mainly located in leaves (26.8 mg g(-1) DM), whereas stems had less PAs (7.8 mg g(-1) DM). Overall, high-yielding plants had lower PA leaf concentrations (R(2) = 0.16, P < 0.001) and fewer leaves (R(2) = 0.66, P < 0.001). However, the results show that these two trade-offs between yield and bioactive PAs can be overcome.

Isolation, characterisation and quantification of the main oligomeric macrocyclic ellagitannins in Epilobium angustifolium by ultra-high performance chromatography with diode array detection and electrospray tandem mass spectrometry.

Tannins have beneficial effects in animal nutrition as they are able to decrease methane emission in ruminants and exert anthelminthic activity against intestinal nematodes. However, tannins can have very diverse structures and therefore, different activities. In order to enhance the research in tannin-rich forages we need tools which are able to quantify tannins individually. In this study we isolated and characterised the main tellimagrandin I (TI)-based oligomeric ellagitannins (ETs) from Epilobium angustifolium (willowherb) and developed a UHPLC-DAD-ESI-MS/MS method to quantify them in plant extracts. The mass spectrometer was operated in Multiple Reaction Monitoring mode to enable the selective detection of dimeric to heptameric ETs from the plant extract. The method proved to be sensitive, with limits of detection ranging from 0.1 to 1.3µgmL(-1). The stability test showed a good repeatability with an inter-run deviation of the results from 0.1 to 5%, except for the pentamer and hexamer where it reached 8%. The method was then successfully applied to evaluate the distribution of those ETs in the plant. This work also provides the first time evidence of the presence of tetrameric to heptameric TI in willowherb.

Comparative metabolite profiling and chemical study of Ramalina siliquosa complex using LC-ESI-MS/MS approach.

A chemical study of the lichen Ramalina siliquosa complex found in Brittany was conducted. Eight chemotypes were considered and their chemical composition was elucidated for the first time by LC-MS analysis. Ten main compounds were identified: conhypoprotocetraric acid (1), salazinic acid (2), peristictic acid (3), cryptostictic acid (4), protocetraric acid (5), stictic acid (6), norstictic acid (7), hypoprotocetraric acid (8), 4-O-demethylbarbatic acid (9), (+)-usnic acid (10) and 22 minor compounds were reported. The MS/MS fragmentation patterns of each compound of R. siliquosa complex were determined and proposed.