Different Sensitivity of Flower-Visiting Diptera to a Neonicotinoid Insecticide: Expanding the Base for a Multiple-Species Risk Assessment Approach.

Insects play an essential role as pollinators of wild flowers and crops. At the same time, pollinators in agricultural environments are commonly exposed to pesticides, compromising their survival and the provision of pollination services. Although pollinators include a wide range of species from several insect orders, information on pesticide sensitivity is mostly restricted to bees. In addition, the disparity of methodological procedures used for different insect groups hinders the comparison of toxicity data between bees and other pollinators. Dipterans are a highly diverse insect order that includes some important pollinators. Therefore, in this study, we assessed the sensitivity of two hoverflies (Sphaerophoria rueppellii, Eristalinus aeneus) and one tachinid fly (Exorista larvarum) to a neonicotinoid insecticide (Confidor®, imidacloprid) following a comparative approach. We adapted the standardized methodology of acute contact exposure in honey bees to build dose-response curves and calculate median lethal doses (LD50) for the three species. The methodology consisted in applying 1 µL of the test solution on the thorax of each insect. Sphaerophoria rueppelli was the most sensitive species (LD50 = 10.23 ng/insect), and E. aeneus (LD50 = 18,176 ng/insect) the least. We then compared our results with those available in the literature for other pollinator species using species sensitivity distribution (SSD). Based on the SSD curve, the 95th percentile of pollinator species would be protected by a safety factor of 100 times the Apis mellifera endpoint. Overall, dipterans were less sensitive to imidacloprid than most bee species. As opposed to most bee species, oviposition and fecundity of many dipteran species can be reliably assessed in the laboratory. We measured the number of eggs laid following exposure to different insecticide doses and assessed the potential trade-off between oviposition and survival through the sublethal sensitivity index (SSI). Exposure to imidacloprid had a significant effect on fecundity, and SSI values indicated that oviposition is a sensitive endpoint for the three dipteran species tested. Future studies should integrate this information related to population dynamics in simulation models for environmental risk assessment.

Bees exposed to climate change are more sensitive to pesticides.

Bee populations are exposed to multiple stressors, including land-use change, biological invasions, climate change, and pesticide exposure, that may interact synergistically. We analyze the combined effects of climate warming and sublethal insecticide exposure in the solitary bee Osmia cornuta. Previous Osmia studies show that warm wintering temperatures cause body weight loss, lipid consumption, and fat body depletion. Because the fat body plays a key role in xenobiotic detoxification, we expected that bees exposed to climate warming scenarios would be more sensitive to pesticides. We exposed O. cornuta females to three wintering treatments: current scenario (2007-2012 temperatures), near-future (2021-2050 projected temperatures), and distant-future (2051-2080). Upon emergence in spring, bees were orally exposed to three sublethal doses of an insecticide (Closer, a.i. sulfoxaflor; 0, 4.55 and 11.64 ng a.i./bee). We measured the combined effects of wintering and insecticide exposure on phototactic response, syrup consumption, and longevity. Wintering treatment by itself did not affect winter mortality, but body weight loss increased with increasing wintering temperatures. Similarly, wintering treatment by itself hardly influenced phototactic response or syrup consumption. However, bees wintered at the warmest temperatures had shorter longevity, a strong fecundity predictor in Osmia. Insecticide exposure, especially at the high dose, impaired the ability of bees to respond to light, and resulted in reduced syrup consumption and longevity. The combination of the warmest winter and the high insecticide dose resulted in a 70% longevity decrease. Smaller bees, resulting from smaller pollen-nectar provisions, had shorter longevity suggesting nutritional stress may further compromise fecundity in O. cornuta. Our results show a synergistic interaction between two major drivers of bee declines, and indicate that bees will become more sensitive to pesticides under the current global warming scenario. Our findings have important implications for pesticide regulation and underscore the need to consider multiple stressors to understand bee declines.

Are Botanical Biopesticides Safe for Bees (Hymenoptera, Apoidea)?

The recent global decline in insect populations is of particular concern for pollinators. Wild and managed bees (Hymenoptera, Apoidea) are of primary environmental and economic importance because of their role in pollinating cultivated and wild plants, and synthetic pesticides are among the major factors contributing to their decline. Botanical biopesticides may be a viable alternative to synthetic pesticides in plant defence due to their high selectivity and short environmental persistence. In recent years, scientific progress has been made to improve the development and effectiveness of these products. However, knowledge regarding their adverse effects on the environment and non-target species is still scarce, especially when compared to that of synthetic products. Here, we summarize the studies concerning the toxicity of botanical biopesticides on the different groups of social and solitary bees. We highlight the lethal and sublethal effects of these products on bees, the lack of a uniform protocol to assess the risks of biopesticides on pollinators, and the scarcity of studies on specific groups of bees, such as the large and diverse group of solitary bees. Results show that botanical biopesticides cause lethal effects and a large number of sublethal effects on bees. However, the toxicity is limited when comparing the effects of these compounds with those of synthetic compounds.

Pesticide residues in nectar and pollen of melon crops: Risk to pollinators and effects of a specific pesticide mixture on Bombus terrestris (Hymenoptera: Apidae) micro-colonies.

Residues detected in pollen collected by honey bees are often used to estimate pesticide exposure in ecotoxicological studies. However, for a more accurate assessment of pesticides effect on foraging pollinators, residues found directly on flowers are a more realistic exposure approximation. We conducted a multi-residue analysis of pesticides on pollen and nectar of melon flowers collected from five fields. The cumulative chronic oral exposure Risk Index (RI) was calculated for Apis mellifera, Bombus terrestris and Osmia bicornis to multiple pesticides. However, this index could underestimate the risk since sublethal or synergistic effects are not considered. Therefore, a mixture containing three of the most frequently detected pesticides in our study was tested for synergistic impact on B. terrestris micro-colonies through a chronic oral toxicity test. According to the result, pollen and nectar samples contained numerous pesticide residues, including nine insecticides, nine fungicides, and one herbicide. Eleven of those were not applied by farmers during the crop season, revealing that melon agroecosystems may be pesticide contaminated environments. The primary contributor to the chronic RI was imidacloprid and O. bircornis is at greatest risk for lethality resulting from chronic oral exposure at these sites. In the bumblebee micro-colony bioassay, dietary exposure to acetamiprid, chlorpyrifos and oxamyl at residue level concentration, showed no effects on worker mortality, drone production or drone size and no synergies were detected when pesticide mixtures were evaluated. In conclusion, our findings have significant implications for improving pesticide risk assessment schemes to guarantee pollinator conservation. In particular, bee pesticide risk assessment should not be limited to acute exposure effects to isolated active ingredients in honey bees. Instead, risk assessments should consider the long-term pesticide exposure effects in both pollen and nectar on a range of bees that reflect the diversity of natural ecosystems and the synergistic potential among pesticide formulations.

Biochemical responses, feeding and survival in the solitary bee Osmia bicornis following exposure to an insecticide and a fungicide alone and in combination.

In agricultural ecosystems, bees are exposed to combinations of pesticides that may have been applied at different times. For example, bees visiting a flowering crop may be chronically exposed to low concentrations of systemic insecticides applied before bloom and then to a pulse of fungicide, considered safe for bees, applied during bloom. In this study, we simulate this scenario under laboratory conditions with females of the solitary bee, Osmia bicornis L. We studied the effects of chronic exposure to the neonicotinoid insecticide, Confidor® (imidacloprid) at a realistic concentration, and of a pulse (1 day) exposure of the fungicide Folicur® SE (tebuconazole) at field application rate. Syrup consumption, survival, and four biomarkers: acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), and alkaline phosphatase (ALP) were evaluated at two different time points. An integrated biological response (IBRv2) index was elaborated with the biomarker results. The fungicide pulse had no impact on survival but temporarily reduced syrup consumption and increased the IBRv2 index, indicating potential molecular alterations. The neonicotinoid significantly reduced syrup consumption, survival, and the neurological activity of the enzymes. The co-exposure neonicotinoid-fungicide did not increase toxicity at the tested concentrations. AChE proved to be an efficient biomarker for the detection of early effects for both the insecticide and the fungicide. Our results highlight the importance of assessing individual and sub-individual endpoints to better understand pesticide effects on bees.

Effects of chronic exposure to the new insecticide sulfoxaflor in combination with a SDHI fungicide in a solitary bee.

The recent EU ban of the three most widely used neonicotinoids (imidacloprid, thiamethoxam and clothianidin) to all outdoors applications has stimulated the introduction of new insecticides into the market. Sulfoxaflor is a new systemic insecticide that, like neonicotinoids, acts as a modulator of nicotinic acetylcholine receptors. In agro-environments, bees can be exposed to this compound via contaminated pollen and nectar for long periods of time. Therefore, it is important to assess the potential effects of chronic exposure to sulfoxaflor, alone and in combination with fungicides, on pollinators. In this study, we tested the effects of chronic exposure to two field concentrations of sulfoxaflor (20 and 100 ppb) alone and in combination with four concentrations of the fungicide fluxapyroxad (7500, 15,000, 30,000 and 60,000 ppb) on syrup consumption and longevity in females of the solitary bee Osmia bicornis L. Exposure to 20 ppb of sulfoxaflor, alone and in combination with the fungicide, stimulated syrup consumption, but did not affect longevity. In contrast, syrup consumption decreased in bees exposed to 100 ppb, all of which died after 2-6 days of exposure. We found no evidence of synergism between the two compounds at any of the two sulfoxaflor concentrations tested. Comparison of our findings with the literature, confirms that O. bicornis is more sensitive to sulfoxaflor than honey bees. Our results highlight the need to include different bee species in risk assessment schemes.

Evaluation of the risks for animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees.

The European Commission has asked the EFSA to evaluate the risk for animal health related to the presence of hydroxymethylfurfural (HMF) in honey bee feed. HMF is a degradation product of particular sugars and can be present in bee feed. HMF is of low acute toxicity in bees but causes increased mortality upon chronic exposure. A benchmark dose lower limit 10% (BMDL10) of 1.16 µg HMF per bee per day has been calculated from mortalities observed in a 20-day study and established as a Reference Point covering also mortality in larvae, drones and queens for which no or insufficient toxicity data were available. Winter bees have a much longer lifespan than summer bees and HMF shows clear time reinforced toxicity (TRT) characteristics. Therefore, additional Reference Point intervals of 0.21-3.1, 0.091-1.1 and 0.019-0.35 µg HMF/bee per day were calculated based on extrapolation to exposure durations of 50, 90 and 180 days, respectively. A total of 219 analytical data of HMF concentrations in bee feed from EU Member States and 88 from Industry were available. Exposure estimates of worker bees and larvae ranged between 0.1 and 0.48, and between 0.1 and 0.51 µg HMF/per day, respectively. They were well below the BMDL10 of 1.16 µg HMF/bee per day, and thus, no concern was identified. However, when accounting for TRT, the probability that exposures were below established reference point intervals was assessed to be extremely unlikely to almost certain depending on exposure duration. A concern for bee health was identified when bees are exposed to HMF contaminated bee feed for several months.

Assessment of the Vulnerability to Pesticide Exposures Across Bee Species.

In many countries, the western honey bee is used as surrogate in pesticide risk assessments for bees. However, uncertainty remains in the estimation of pesticide risk to non-Apis bees because their potential routes of exposure to pesticides, life histories, and ecologies differ from those of honey bees. We applied the vulnerability concept in pesticide risk assessment to 10 bee species including the honey bee, 2 bumble bee species, and 7 solitary bee species with different nesting strategies. Trait-based vulnerability considers the evaluation of a species at the level of both the organism (exposure and effect) and the population (recovery), which goes beyond the sensitivity of individuals to a toxicant assessed in standard laboratory toxicity studies by including effects on populations in the field. Based on expert judgment, each trait was classified by its relationship to the vulnerability to pesticide exposure, effects (intrinsic sensitivity), and population recovery. The results suggested that the non-Apis bees included in our approach are potentially more vulnerable to pesticides than the honey bee due to traits governing exposure and population recovery potential. Our analysis highlights many uncertainties related to the interaction between bee ecology and the potential exposures and population-level effects of pesticides, emphasizing the need for more research to identify suitable surrogate species for higher tier bee risk assessments. Environ Toxicol Chem 2021;40:2640-2651. © 2021 SETAC.

A dataset for sustainability assessment of agroecological practices in a crop-livestock farming system.

This article presents data designed by European researchers who performed a literature review and interpreted the results to determine impact factors of many agroecological practices on a wide variety of sustainability indicators. The impact factors are represented in a matrix that connects practices to indicators. The indicators are related to environmental, economic and social sustainability of a typical European integrated crop-livestock farm. The data are included in the serious game SEGAE to learn agroecology, as described in "SEGAE: a serious game to learn agroecology" [1]. The data can be modified to adapt the game to other agricultural systems. Finally, the data can be re-used in research projects as a basis to assess impacts of agroecological practices.

Use of a Managed Solitary Bee to Pollinate Almonds: Population Sustainability and Increased Fruit Set.

Osmia spp. are excellent orchard pollinators but evidence that their populations can be sustained in orchard environments and their use results in increased fruit production is scarce. We released an Osmia cornuta population in an almond orchard and measured its population dynamics, as well as visitation rates and fruit set at increasing distances from the nesting stations. Honeybees were 10 times more abundant than O. cornuta. However, the best models relating fruit set and bee visitation included only O. cornuta visitation, which explained 41% and 40% of the initial and final fruit set. Distance from the nesting stations explained 27.7% and 22.1% of the variability in initial and final fruit set. Of the 198 females released, 99 (54.4%) established and produced an average of 9.15 cells. Female population growth was 1.28. By comparing our results with those of previous O. cornuta studies we identify two important populational bottlenecks (female establishment and male-biased progeny sex ratios). Our study demonstrates that even a small population of a highly effective pollinator may have a significant impact on fruit set. Our results are encouraging for the use of Osmia managed populations and for the implementation of measures to promote wild pollinators in agricultural environments.

Seek and you shall find: An assessment of the influence of the analytical methodologies on pesticide occurrences in honey bee-collected pollen with a systematic review.

Honey bee mortality and colony losses have been reported worldwide. Although this phenomenon is caused by a combination of factors, agrochemicals have received special attention due to their potential effects on bees. In agricultural and urban environments bees are exposed to several compounds that may interact in unexpected ways, but information on the extent of pesticide exposure remains unclear. Several monitoring studies have been conducted to evaluate the field-realistic exposure of bees to pesticides after their release on the market. However, their outputs are difficult to compare and harmonize due to differences in the analytical methodologies and the sampling protocols (e.g. number of screened compounds and analysed samples, and detection limits (LODs)). Here, we hypothesize that the analytical methodologies used in the monitoring studies may strongly affect the pesticide occurrences in pollen underestimating the real pesticide exposure. By mean of a systematic literature review, we have collected relevant information on pesticide contaminations in the honey bee-collected pollen. Our findings showed that the pesticide occurrences were associated with the analytical methodologies and the real pesticide exposure has likely been underestimated in some monitoring studies. For four highly toxic compounds, the LOD used in these monitoring studies exceeded the doses that cause toxic effects on honey bees. We recommend that, especially for the highly toxic compounds, the LODs used in the monitoring studies should be low enough to exclude lethal or sublethal effects on bees and avoid "false negative" samples.

Immunosuppression response to the neonicotinoid insecticide thiacloprid in females and males of the red mason bee Osmia bicornis L.

Solitary bees are frequently exposed to pesticides, which are considered as one of the main stress factors that may lead to population declines. A strong immune defence is vital for the fitness of bees. However, the immune system can be weakened by environmental factors that may render bees more vulnerable to parasites and pathogens. Here we demonstrate for the first time that field-realistic concentrations of the commonly used neonicotinoid insecticide thiacloprid can severely affect the immunocompetence of Osmia bicornis. In detail, males exposed to thiacloprid solutions of 200 and 555 µg/kg showed a reduction in hemocyte density. Moreover, functional aspects of the immune defence - the antimicrobial activity of the hemolymph - were impaired in males. In females, however, only a concentration of 555 µg/kg elicited similar immunosuppressive effects. Although males are smaller than females, they consumed more food solution. This leads to a 2.77 times higher exposure in males, probably explaining the different concentration thresholds observed between the sexes. In contrast to honeybees, dietary exposure to thiacloprid did not affect melanisation or wound healing in O. bicornis. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of O. bicornis, possibly leading to an impaired disease resistance capacity.

Chronic oral exposure to field-realistic pesticide combinations via pollen and nectar: effects on feeding and thermal performance in a solitary bee.

Pesticide use is one of the main causes of pollinator declines in agricultural ecosystems. Traditionally, most laboratory studies on bee ecotoxicology test acute exposure to single compounds. However, under field conditions, bees are often chronically exposed to a variety of chemicals, with potential synergistic effects. We studied the effects of field-realistic concentrations of three pesticides measured in pollen and nectar of commercial melon fields on the solitary bee Osmia bicornis L. We orally exposed females of this species throughout their life span to 8 treatments combining two neonicotinoid insecticides (acetamiprid, imidacloprid) and a triazole fungicide (myclobutanil) via pollen and sugar syrup. We measured pollen and syrup consumption, longevity, ovary maturation and thermogenesis. Pesticide intake was three orders of magnitude higher via syrup than pollen. At the tested concentrations, no synergistic effects emerged, and we found no effects on longevity and ovary maturation. However, all treatments containing imidacloprid resulted in suppressed syrup consumption and drastic decreases in thoracic temperature and bee activity. Our results have important implications for pesticide regulation. If we had measured only lethal effects we would have wrongly concluded that the pesticide combinations containing imidacloprid were safe to O. bicornis. The incorporation of tests specifically intended to detect sublethal effects in bee risk assessment schemes should be an urgent priority. In this way, the effects of pesticide exposure on the dynamics of bee populations in agroecosystems will be better assessed.

Pesticide Exposure Assessment Paradigm for Solitary Bees.

Current pesticide risk assessment for bees relies on a single (social) species, the western honey bee, Apis mellifera L. (Hymenoptera: Apidae). However, most of the >20,000 bee species worldwide are solitary. Differences in life history traits between solitary bees (SB) and honey bees (HB) are likely to determine differences in routes and levels of pesticide exposure. The objectives of this review are to: 1) compare SB and HB life history traits relevant for risk assessment; 2) summarize current knowledge about levels of pesticide exposure for SB and HB; 3) identify knowledge gaps and research needs; 4) evaluate whether current HB risk assessment schemes cover routes and levels of exposure of SB; and 5) identify potential SB model species for risk assessment. Most SB exposure routes seem well covered by current HB risk assessment schemes. Exceptions to this are exposure routes related to nesting substrates and nesting materials used by SB. Exposure via soil is of particular concern because most SB species nest underground. Six SB species (Hymenoptera: Megachilidae - Osmia bicornis L., O. cornifrons Radoszkowski, O. cornuta Latreille, O. lignaria Say, Megachile rotundata F., and Halictidae - Nomia melanderi Cockerell) are commercially available and could be used in risk assessment. Of these, only N. melanderi nests underground, and the rest are cavity-nesters. However, the three Osmia species collect soil to build their nests. Life history traits of cavity-nesting species make them particularly suitable for semifield and, to a lesser extent, field tests. Future studies should address basic biology, rearing methods and levels of exposure of ground-nesting SB species.

Combined exposure to sublethal concentrations of an insecticide and a fungicide affect feeding, ovary development and longevity in a solitary bee.

Pollinators in agroecosystems are often exposed to pesticide mixtures. Even at low concentrations, the effects of these mixtures on bee populations are difficult to predict due to potential synergistic interactions. In this paper, we orally exposed newly emerged females of the solitary bee Osmia bicornis to environmentally realistic levels of clothianidin (neonicotinoid insecticide) and propiconazole (fungicide), singly and in combination. The amount of feeding solution consumed was highest in bees exposed to the neonicotinoid, and lowest in bees exposed to the pesticide mixture. Ovary maturation and longevity of bees of the neonicotinoid and the fungicide treatments did not differ from those of control bees. By contrast, bees exposed to the pesticide mixture showed slow ovary maturation and decreased longevity. We found a synergistic interaction between the neonicotinoid and the fungicide on survival probability. We also found an interaction between treatment and emergence time (an indicator of physiological condition) on longevity. Longevity was negatively correlated to physiological condition only in the fungicide and the mixture treatments. Delayed ovary maturation and premature death imply a shortened nesting period (highly correlated to fecundity in Osmia). Our findings provide a mechanism to explain the observed dynamics of solitary bee populations exposed to multiple chemical residues in agricultural environments.

Lethal effects of Cr(III) alone and in combination with propiconazole and clothianidin in honey bees.

Several anthropogenic contaminants, including pesticides and heavy metals, can affect honey bee health. The effects of mixtures of heavy metals and pesticides are rarely studied in bees, even though bees are likely to be exposed to these contaminants in both agricultural and urban environments. In this study, the lethal toxicity of Cr alone and in combination with the neonicotinoid insecticide clothianidin and the ergosterol-biosynthesis-inhibiting fungicide propiconazole was assessed in Apis mellifera adults. The LD50 and lowest benchmark dose of Cr as Cr(NO3)3, revealed a low acute oral toxicity on honey bee foragers (2049 and 379 mg L-1, respectively) and the Cr retention (i.e. bee ability to retain the heavy metal in the body) was generally low compared to other metals. A modified method based on the binomial proportion test was developed to analyse synergistic and antagonistic interactions between the three tested contaminants. The combination of an ecologically-relevant field concentration of chromium with clothianidin and propiconazole did not increase bee mortality. On the contrary, the presence of Cr in mixture with propiconazole elicited a slight antagonistic effect.

Neonicotinoid pesticides and nutritional stress synergistically reduce survival in honey bees.

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (-50%). Nutritional and pesticide stressors reduced also food consumption (-48%) and haemolymph levels of glucose (-60%) and trehalose (-27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.

Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species.

BACKGROUND: Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. RESULTS: We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin. CONCLUSION: Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes. © 2016 Society of Chemical Industry.