Mortality Pattern of Poecilus cupreus Beetles after Repeated Topical Exposure to Insecticide─Stochastic Death or Individual Tolerance?

The mortality of organisms exposed to toxicants has been attributed to either stochastic processes or individual tolerance (IT), leading to the stochastic death (SD) and IT models. While the IT model follows the principles of natural selection, the relevance of the SD model has been debated. To clarify why the idea of stochastic mortality has found its way into ecotoxicology, we investigated the mortality of Poecilus cupreus (Linnaeus, 1758) beetles from pesticide-treated oilseed rape (OSR) fields and unsprayed meadows, subjected to repeated insecticide treatments. We analyzed the mortality with the Kaplan-Meier estimator and general unified threshold model for survival (GUTS), which integrates SD and IT assumptions. The beetles were exposed three times, ca. monthly, to the same dose of Proteus 110 OD insecticide containing thiacloprid and deltamethrin, commonly used in the OSR fields. Kaplan-Meier analysis showed that the mortality of beetles from meadows was much higher after the first treatment than after the next two, indicating the IT model. Beetles from the OSR displayed approximately constant mortality after the first and second treatments, consistent with the SD model. GUTS analysis did not conclusively identify the better model, with the IT being marginally better for beetles from meadows and the SD better for beetles from OSR fields.

Effects of agricultural landscape structure and canola coverage on biochemical and physiological traits of the ground beetle Poecilus cupreus.

The intensifications in the agricultural landscape and the application of pesticides can cause adverse effects on the fitness of organisms in that landscape. Here, we investigated whether habitats with different agricultural pressures influenced acetylcholinesterase (AChE) activity - a biomarker for exposure to pesticides, respiration rate, and resistance to starvation in the ground beetle Poecilus cupreus. Two differently structured landscapes were selected for the study, one dominated by small (S) and another by large (L) fields. Within each landscape three habitat types were selected: in the S landscape, these were habitats with medium (M), small (S) and no canola (meadow, 0) coverage (i.e., SM, SS, S0), and in the L landscape habitats with large (L), medium (M) and no canola (meadow, 0) coverage (i.e., LL, LM, L0), representing different levels of agricultural pressure. The activity of AChE was the highest in beetles from canola-free habitats (S0 and L0), being significantly higher than in beetles from the SM and SS habitats. The mean respiration rate corrected for body mass was also the highest in S0 and L0 beetles, with significant differences between populations from L0 vs. SS and from S0 vs. SS. Only beetles from S0, SS, L0, and LM were numerous enough to assess the resistance to starvation. Individuals from the LM habitat showed better survival compared to the canola-free habitat in the same landscape (L0), whereas in S landscape the SS beetles survived worse than those from S0, suggesting that characteristics of L landscape may lead to developing mechanisms of starvation resistance of P. cupreus in response to agricultural pressure.

Physiological and biochemical response of the solitary bee Osmia bicornis exposed to three insecticide-based agrochemicals.

The physiological and biochemical stress induced by pesticides need to be addressed in economically and ecologically important non-Apis solitary bees, particularly at lower than field-applied concentrations. Thus, the aim of the present study was to analyse the physiological and biochemical changes in female adult Osmia bicornis bees upon continuous oral exposure to three insecticide-based agrochemicals - i.e. Dursban 480 EC (active ingredient - a.i. chlorpyrifos), Sherpa 100 EC (a.i. cypermethrin), and Mospilan 20 SP (a.i. acetamiprid), in a toxicokinetic manner (feeding with either insecticide-contaminated food or uncontaminated food (controls) for 8 d in the contamination phase followed by 8 d of decontamination (i.e. feeding with uncontaminated food)). All three tested agrochemicals altered the energetic budget of bees by the deprivation of energy derived from lipids and carbohydrates (but not proteins) and/or a decrease in respiration based metabolic rate (energy consumption) compared to the controls. The activities of acetylcholinesterase and glutathione-S-transferase enzymes were not altered by insecticides at tested concentrations. These results show that chronic exposure to at least some pesticides even at relatively low concentrations may cause severe physiological disruptions that could potentially be damaging for the solitary bees.

Different effects of Zn nanoparticles and ions on growth and cellular respiration in the earthworm Eisenia andrei after long-term exposure.

In this study, the effects of zinc nanoparticles (ZnO-NPs) and ions (ZnCl2) on the mortality, growth, maturation, and cellular respiration of the earthworm Eisenia andrei were assessed. Earthworms were individually exposed for 98 days, starting from the juvenile stage, to soils contaminated with either ZnO-NPs or ZnCl2 (125, 250, 500 and 1000 mg Zn kg-1 dry weight (dw)). Exposure to the highest-concentration ionic treatments (500 and 1000 mg kg-1) caused 100% mortality, while for other treatments, mortality did not exceed 15% at the end of exposure. Compared to the control treatment, both 125-1000 mg kg-1 ZnO-NPs and 125 or 250 mg kg-1 ZnCl2 stimulated earthworm growth, which might be due to a hormetic effect. ZnO-NPs and ZnCl2 caused different responses at medium Zn concentrations (250 and 500 mg kg-1): earthworms exposed to ionic treatment at 250 mg kg-1 were characterized by a significantly lower growth constant, lower cellular respiration rate, later inflection point, and higher final body weight than those exposed to ZnO-NPs treatments at the same (250 mg kg-1) or twice as high (500 mg kg-1) nominal Zn concentrations. However, differences were not observed in all examined parameters between the studied forms when the highest-concentration ZnO-NPs treatment was compared with the lowest-concentration ionic treatment, which was likely due to the same levels of available Zn concentrations in those treatments. Overall, different growth and maturation strategies accompanied by pronounced differences in cellular respiration were adopted by earthworms exposed to low and medium levels of either ZnO-NPs or ZnCl2.

Unravelling the ZnO-NPs mechanistic pathway: Cellular changes and altered morphology in the gastrointestinal tract of the earthworm Eisenia andrei.

A major uptake route of nanoparticles (NPs) occurs via the gastrointestinal (GI) tract. When GI tract cells are exposed, NPs cytotoxic effects are observed that subsequently adversely affect the GI tract morphology and have consequences for the whole organism. The aim of this study was to understand the mechanism of effects caused by ZnO-NPs compared to Zn ions on the earthworm Eisenia andrei. The following aspects of individually exposed earthworms were investigated: 1) qualitative structural alterations in the gut epithelium and chloragogen cells of the GI tract, 2) quantitative changes within chloragogen tissues after 48 h of exposure (using morphometric analysis), and 3) the ADP/ATP ratio in homogenized tissue of the whole organism after 21 days of exposure to contaminated soil (contamination phase) followed by 14 days of elimination in clean soil (decontamination phase) to identify possible recovery. Both ZnO-NPs and Zn ions adversely affect the gut epithelium and chloragogen tissue of earthworms after 48 h of exposure to contaminated soil. Morphometric measurements revealed that the proportions of debris vesicles in the chloragocytes were significantly lower in worms exposed to ZnO-NPs than in worms exposed to Zn ions. Moreover, numerous spherite granules were observed in the chloragocytes of ionic Zn-treated worms, but not the ZnO-NPs-treated worms, suggesting differential regulation of these Zn forms. The Zn cytotoxic effect was not reflected in ADP/ATP ratio measurements. Our study provides new insights into nano-specific effects that are distinctive from ion regulation inside the GI tract and furthers our understanding of the relationship between effects at the cellular and whole-body levels.

Effects of Cadmium Bioavailability in Food on Its Distribution in Different Tissues in the Ground Beetle Pterostichus oblongopunctatus.

In most laboratory studies with oral exposure of terrestrial invertebrates to metals an artificial food, which is easy to handle, is used. The bioavailability of metals from this artificial food may, however, be much higher than from more field relevant food sources. Such differences may affect toxicokinetic processes in different tissues. To test the effect of bioavailability of Cd in food on Cd toxicokinetics and internal distribution in terrestrial invertebrates, we performed the experiment using the ground beetle Pterostichus oblongopunctatus exposed to Cd via food differing in their soluble Cd pool. We showed that in carabids Cd accumulation and elimination pattern in different tissues is not governed by the metal availability in food.

Toxicokinetics of zinc-oxide nanoparticles and zinc ions in the earthworm Eisenia andrei.

The toxicokinetics of zinc in the earthworm Eisenia andrei was investigated following exposure for 21 days to ionic zinc (ZnCl2) or zinc oxide nanoparticles (ZnO-NPs) in Lufa 2.2 soil, followed by 21 days elimination in clean soil. Two concentrations were tested for both ZnCl2 (250 and 500µg Zn g-1) and ZnO-NPs (500 and 1000µg Zn g-1), corresponding to EC25 and EC50 for effects on reproduction. Based on the measured internal Zn concentrations in the earthworms over time of exposure, the kinetics parameters ka - assimilation rate constant (gsoil g-1body weight day-1) and ke - elimination rate constant (day-1) were estimated using a one-compartment model for either total Zn concentrations in the soil or porewater Zn concentrations. In the ZnCl2 treatments, ka was higher for total Zn concentrations in soil, whereas in the ZnO-NP treatments, ka was higher for porewater Zn concentrations. The value of ke did not differ between the two Zn forms (ZnCl2 vs ZnO-NPs) for either EC50 or EC25 when related to total Zn concentrations in soil, but for EC50, ke related to porewater Zn concentrations was significantly higher for ZnCl2 than for ZnO-NPs. It is concluded that differences in kinetic parameters between treatments were connected with exposure concentrations rather than with the form of Zn. Zinc was efficiently regulated by the earthworms in all treatments: a 2-fold increase in exposure concentration resulted in a less than 2-fold increase in internal concentration, and after transfer to uncontaminated soil the internal Zn concentrations in the earthworms returned to ca 111µgg-1 dw in all treatments.

Effect of cadmium bioavailability in food on its compartmentalisation in carabids.

Metals assimilated by organisms are sequestered in various compartments and some forms are more stable than others. Sequestration mechanisms used by invertebrates to detoxify metals and prevent interaction with important biomolecules include metal binding to proteins and other ligands, and storage in inorganic granules. The rate and extent at which metal concentrations in different compartments respond to metal concentrations in food and food characteristics has not received much attention, despite being of great relevance. We performed an experiment on the carabid beetle Pterostichus oblongopunctatus exposed to Cd via food made of ground mealworm (Tenebrio molitor) larvae, either reared on Cd contaminated medium or artificially spiked after grinding with CdCl2 solution. Thus, in both cases we used the same type of food, differing only in the soluble Cd pool available to the predators, represented by P. oblongopunctatus. Subcellular compartmentalisation of Cd into organelles, heat-sensitive and heat-stable proteins (the first supernatant, S1 fraction), cellular debris (the second supernatant, S2 fraction) and metal-rich granules (G fraction) was checked a few times during the contamination (90 d) and decontamination (24 d) phases in a toxicokinetic experiment by using different centrifugation steps. The results showed no effect of the type of food (naturally, Cd-N, vs. artificially contaminated with Cd, Cd-A) on Cd sequestration kinetics in P. oblongopunctatus, but the amount of Cd sequestered in the S1 and G fractions were in general higher in the Cd-A than the Cd-N treatment, indicating that Cd transfer in the food web depends on the speciation of the metal in the food. The proportional distribution of Cd over different fractions was, however, similar in beetles fed both food types. Most of the accumulated Cd in the beetles existed as fraction S1 (ca. 35%), which is important for the transfer of metals to higher trophic levels in a food web.

Subcellular partitioning of cadmium and zinc in mealworm beetle (Tenebrio molitor) larvae exposed to metal-contaminated flour.

By studying the internal compartmentalization of metals in different subcellular fractions we are able to better understand the mechanisms of metal accumulation in organisms and the transfer of metals through trophic chains. We investigated the internal compartmentalization of cadmium (Cd) and zinc (Zn) in mealworm beetle (Tenebrio molitor) larvae by breeding them in flour contaminated with either Cd at 100, 300 and 600mgkg(-1), or Zn at 1000 and 2000mgkg(-1). We separated the cellular components of the larvae into 3 fractions: the S1 or cytosolic fraction containing organelles, heat-sensitive and heat-stable proteins, the S2 or cellular debris fraction and the G or metal-rich granule fraction. The concentration of Cd and Zn in each fraction was measured at 0, 7, 14 and 21 days of being fed the flour. The concentration of Cd in the flour affected the concentration of Cd measured in each larval subcellular fraction (p≤0.0001), while the concentration of Zn in the flour only affected the Zn concentration in the S2 and G fractions (p≤0.02). Both Cd and Zn concentrations in mealworms remained relatively constant during the exposure (days 7, 14 and 21) in all three fractions, but the Cd concentrations were much higher than those found in larvae before the exposure (day 0). The concentration of Cd in the flour, however, did not affect the percentage of Cd in the S1 fraction. The contribution of Cd in the G fraction to the total Cd amount was similar (30-40%) in all Cd treatments. The percentage of Zn in all three fractions was not affected by the concentration of Zn in the flour and the relative contributions of each subcellular fraction to the total burden of Zn remained generally constant for both control and treated larvae. In general, larvae sequestered approximately 30% of Cd and Zn in the S1 fraction, which is important for the transport of metals to higher trophic levels in a food web.

Regulation of body metal concentrations: Toxicokinetics of cadmium and zinc in crickets.

Previous studies indicated that essential and xenobiotic metals differ substantially in terms of their toxicokinetics. Whether these differences are due to different assimilation rates, different elimination rates, or both, and whether all metals are regulated in a similar manner but with different efficiency remains unclear. To compare the mechanisms responsible for the regulation of different metals, parameters for toxicokinetic models have to be tested under exposures to the identical molar concentration of those metals. In this study, the cricket Gryllus assimilis was exposed to Zn or Cd at 2.5, 10, and 40mMkg(-1) dry food. The body concentrations of the metals were not perfectly regulated by the crickets. For Zn, a clear increase in the body concentration was found only at the highest treatment; whereas at the lowest treatment, the internal concentration remained unchanged throughout the experiment. At the lowest Zn concentration, the assimilation (kA) [day(-1)] and elimination (kE) [day(-1)] rate constants were balanced (kA=0.024, kE=0.024). When increasing the Zn exposure, kA decreased to 0.018 at 10mMkg(-1) and 0.01 at 40mMkg(-1), and kE increased to 0.05 and 0.07, respectively. Therefore, the body concentration of Zn was regulated by simultaneously changing the assimilation and elimination rate. By contrast, even at the lowest treatment, a significant increase in Cd concentration was observed in the crickets. The equilibrium Cd concentration resulted almost exclusively from increasing kE from 0.17, through 0.28 to 0.61 at 2.5, 10 and 40mMkg(-1). The kA for Cd did not reveal any clear trend. Zn was more efficiently regulated by crickets than was Cd: a 16-fold increase in exposure concentration (from 2.5 to 40mM Znkg(-1)) resulted only in a twofold increase of internal concentration, whereas the identical increase in Cd exposure concentration resulted in almost a sevenfold increase in internal concentration of this metal.

Concentration dependent toxicokinetics of copper in the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae).

To predict internal metal concentrations in animals under specific environmental exposures, the relationship between the exposure concentrations and values of toxicokinetic parameters must be known. At high exposure levels, the availability of carriers transporting metal ions through cellular membranes may become limited, thereby decreasing the assimilation rates (k A ). Furthermore, increased metal concentrations in food may result in greater damage to the gut and reduce the assimilation efficiency and/or increase the elimination rate (k E ). Therefore, k A should decrease and k E should increase with increasing metal concentrations. In fact, our study on Tribolium castaneum exposed to Cu at 500, 1000, 2000 and 4000 mg kg(-1) of dry flour showed that with increasing Cu concentrations, k A decreased from 0.0042 day(-1) at 500 mg kg(-1) to 0.0026 day(-1) at 4000 mg kg(-1) in females and from 0.0029 to 0.001 day(-1) in males and k E increased from 0.027 to 0.064 day(-1) and from 0.018 to 0.04 day(-1) in females and males, respectively. Significant differences in k A between the sexes were observed at 2000 and 4000 mg kg(-1), whereas significant differences between treatments were found for k A in males. Copper was efficiently regulated by T. castaneum: an eightfold increase in exposure concentrations resulted in only a ca. twofold increase in the internal concentration. No Cu effect on the respiratory metabolism of T. castaneum was found.

Combined effect of nickel and chlorpyrifos on the ground beetle Pterostichus oblongopunctatus.

Although terrestrial invertebrates are often exposed to mixtures of chemicals in the field, little is known about their combined effects on metabolism. We studied the effects of nickel (Ni), chlorpyrifos (CPF) and their mixtures on the respiration rate of the ground beetle Pterostichus oblongopunctatus in a toxicokinetics experiment. The respiration rate was measured six times in the uptake phase (64 days) and four times in the decontamination phase (32 days), and internal Ni concentrations were measured in parallel. The beetles׳ respiration rate in the uptake phase was especially affected by CPF. Moreover, the significant interaction of Ni with the time of exposure indicated that at certain dates Ni also affected respiration rates. Respiration rates changed significantly with time, and the most pronounced increase was observed after 2 days of exposure to a high concentration of CPF. Increased respiration rates in CPF-exposed beetles compared with controls were still observed after switching the beetles to uncontaminated food, whereas the effect of Ni disappeared in the decontamination phase altogether. In the beetles exposed to both Ni alone and Ni mixed with CPF at a low concentration, Ni reached high internal concentrations by the second day of exposure. Two days later, the internal concentrations dropped significantly, indicating efficient decontamination, even if the beetles were still exposed to Ni-contaminated food. High-concentration CPF decreased the accumulation of Ni. No effect of CPF was found in the decontamination phase. The importance of accounting for time in the mixture toxicity testing and ecological risk assessment is discussed.

Combined effects of three insecticides with different modes of action on biochemical responses of the solitary bee Osmia bicornis.

Bees are simultaneously exposed to a variety of pesticides, which are often applied in mixtures and can cause lethal and sublethal effects. The combined effects of pesticides, however, are not measured in the current risk assessment schemes. Additionally, the sublethal effects of pesticides on a variety of physiological processes are poorly recognized in bees, especially in non-Apis solitary bees. In this study, we used a full-factorial design to examine the main and interactive effects of three insecticide formulations with different modes of action (Mospilan 20 SP, Sherpa 100 EC, and Dursban 480 EC) on bee biochemical processes. We measured acetylcholinesterase (AChE), glutathione S-transferase (GST) and esterase (EST) activities, as well as a nonenzymatic biomarker associated with energy metabolism, i.e., ATP level. All studied endpoints were affected by Sherpa 100 EC, and the activities of AChE and EST as well as ATP levels were affected by Dursban 480 EC. Moreover, complex interactions between all three insecticides affected ATP levels, showing outcomes that cannot be predicted when testing each insecticide separately. The results indicate that even if interactive effects are sometimes difficult to interpret, there is a need to study such interactions if laboratory-generated toxicity data are to be extrapolated to field conditions.

Costs of living in metal polluted areas: respiration rate of the ground beetle Pterostichus oblongopunctatus from two gradients of metal pollution.

To address the question about costs of living in polluted areas, biomarkers linked to metabolism were measured in Pterostichus oblongopunctatus (Coleoptera: Carabidae) collected along two metal-pollution gradients in the vicinity of the two largest Polish zinc smelters: 'Boleslaw' and 'Miasteczko Slaskie' in southern Poland. Both gradients covered a broad range of Zn and Cd concentrations in the humus layer (109-6151 and 1.48-71.4 mg kg(-1), respectively) and body metal concentrations increased with increasing soil metal concentrations. The whole-organism respiration rate was measured as oxygen consumption with Micro-Oxymax respirometer, and cellular energy consumption-as the activity of electron transport system, which is linked to cellular respiration rate. The significant increase in the whole-organism respiration rate with the body metal concentration was found when taking into account other factors such as body mass, gradient (or year of sampling as the beetles were collected on the gradients in different years) and the interactions: body metal concentrations × collection date, body metal concentrations × body mass, and body mass × gradient/sampling year. However, no relationships between metal concentrations in soil or body metal concentrations and the whole-organism or cellular respiration rate could be detected when using mean values per site, underlining the crucial importance of incorporating individual variability in such analyses. The observed increase of the whole-organism respiration rate with increasing body contamination with metals suggests that P. oblongopunctatus incurs energetic expenditures resulting from the necessity to facilitate metal elimination or repair of toxicant-induced damage.

A toxicokinetic model for thiamethoxam in rats: implications for higher-tier risk assessment.

Risk assessment for mammals is currently based on external exposure measurements, but effects of toxicants are better correlated with the systemically available dose than with the external administered dose. So for risk assessment of pesticides, toxicokinetics should be interpreted in the context of potential exposure in the field taking account of the timescale of exposure and individual patterns of feeding. Internal concentration is the net result of absorption, distribution, metabolism and excretion (ADME). We present a case study for thiamethoxam to show how data from ADME study on rats can be used to parameterize a body burden model which predicts body residue levels after exposures to LD50 dose either as a bolus or eaten at different feeding rates. Kinetic parameters were determined in male and female rats after an intravenous and oral administration of (14)C labelled by fitting one-compartment models to measured pesticide concentrations in blood for each individual separately. The concentration of thiamethoxam in blood over time correlated closely with concentrations in other tissues and so was considered representative of pesticide concentration in the whole body. Body burden model simulations showed that maximum body weight-normalized doses of thiamethoxam were lower if the same external dose was ingested normally than if it was force fed in a single bolus dose. This indicates lower risk to rats through dietary exposure than would be estimated from the bolus LD50. The importance of key questions that should be answered before using the body burden approach in risk assessment, data requirements and assumptions made in this study are discussed in detail.

Floral resources,energetic value and pesticide residues in provisions collected by Osmia bicornis along a gradient of oilseed rape coverage.

Pollinators in agricultural landscapes are facing global decline and the main pressures include food scarcity and pesticide usage. Intensive agricultural landscapes may provide important food resources for wild pollinators via mass flowering crops. However, these are monofloral, short-term, and may contain pesticide residues. We explored how the landscape composition with a different proportion of oilseed rape (6-65%) around Osmia bicornis nests affects floral diversity, contamination with pesticides, and energetic value of provisions collected by this species of wild bees as food for their offspring. Altogether, the bees collected pollen from 28 plant taxa (6-15 per nest) and provisions were dominated by Brassica napus (6.0-54.2%, median 44.4%, 12 nests), Quercus sp. (1.2-19.4%, median 5.2%, 12 nests), Ranunculus sp. (0.4-42.7%, median 4.7%, 12 nests), Poaceae (1.2-59.9%, median 5.8%, 11 nests) and Acer sp. (0.6-42%, median 18.0%, 8 nests). Residues of 12 pesticides were found in provisions, with acetamiprid, azoxystrobin, boscalid, and dimethoate being the most frequently detected at concentrations up to 1.2, 198.4, 16.9 and 17.8 ng/g (median 0.3, 10.6, 11.3, 4.4 ng/g), respectively. Floral diversity and energetic value of provisions, but not the Pesticide Risk Index depended on landscape structure. Moreover, pollen diversity decreased, and energetic value increased with landscape diversity. Thus, even a structurally simple landscape may provide diverse food for O. bicornis if the nest is located close to a single but resource-diverse patch. Both B. napus and non-crop pollen were correlated with pesticide concentrations.

Homogeneity of agriculture landscape promotes insecticide resistance in the ground beetle Poecilus cupreus.

The intensification of agriculture leads to increased pesticide use and significant transformation from small fields towards large-scale monocultures. This may significantly affect populations of non-target arthropods (NTA). We aimed to assess whether the multigenerational exposure to plant protection products has resulted in the evolution of resistance to insecticides in the ground beetle Poecilus cupreus originating from different agricultural landscapes. Two contrasting landscapes were selected for the study, one dominated by small and another by large fields. Within each landscape the beetles were collected at nine sites representing range of canola coverage and a variety of habitat types. Part of the collected beetles, after acclimation to laboratory conditions, were tested for sensitivity to Proteus 110 OD-the most commonly used insecticide in the studied landscapes. The rest were bred in the laboratory for two consecutive generations, and part of the beetles from each generation were also tested for sensitivity to selected insecticide. We showed that the beetles inhabiting areas with medium and large share of canola located in the landscape dominated by large fields were less sensitive to the studied insecticide. The persistence of reduced sensitivity to Proteus 110 OD for two consecutive generations indicates that either the beetles have developed resistance to the insecticide or the chronic exposure to pesticides has led to the selection of more resistant individuals naturally present in the studied populations. No increased resistance was found in the beetles from more heterogeneous landscape dominated by small fields, in which spatio-temporal diversity of crops and abundance of small, linear off-crop landscape elements may provide shelter that allows NTAs to survive without developing any, presumably costly, resistance mechanisms.

Toxicokinetics of three insecticides in the female adult solitary bee Osmia bicornis.

The worldwide decline of pollinators is of growing concern and has been related to the use of insecticides. Solitary bees are potentially exposed to many insecticides through contaminated pollen and/or nectar. The kinetics of these compounds in solitary bees is, however, unknown, limiting the use of these important pollinators in pesticide regulations. Here, the toxicokinetics (TK) of chlorpyrifos (as Dursban 480 EC), cypermethrin (Sherpa 100 EC), and acetamiprid (Mospilan 20 SP) was studied for the first time in Osmia bicornis females at sublethal concentrations (near LC20s). The TK of the insecticides was analysed in bees continuously exposed to insecticide-contaminated food in the uptake phase followed by feeding with clean food in the decontamination phase. The TK models differed substantially between the insecticides. Acetamiprid followed the classic one-compartment model with gradual accumulation during the uptake phase followed by depuration during the decontamination phase. Cypermethrin accumulated rapidly in the first two days and then its concentration decreased slowly. Chlorpyrifos accumulated similarly rapidly but no substantial depuration was found until the end of the experiment. Our study demonstrates that some insecticides can harm solitary bees when exposed continuously even at trace concentrations in food because of their constant accumulation leading to time-reinforced toxicity.

Effects of agricultural landscape structure, insecticide residues, and pollen diversity on the life-history traits of the red mason bee Osmia bicornis.

Agricultural landscapes have changed substantially in recent decades, shifting from the dominance of small fields (S) with diverse cropping systems toward large-scale monoculture (L), where landscape heterogeneity disappears. In this study, artificial nests of the red mason bee, Osmia bicornis, were placed in S and L landscape types on the perimeter of oilseed rape fields representing different oilseed rape coverages (ORC, % land cover). The local landscape structure around each nest was characterised within a 100, 200, 500, and 1000 m radius using ORC and 14 landscape characteristics, which were then reduced by non-metric multidimensional scaling (nMDS) to two axes: nMDS1 characterised the dataset primarily according to land fragmentation and the main crop, whereas nMDS2 captured the prevalence of more natural areas in the landscape. Pollen diversity and insecticide risk levels in the pollen provisions collected by the bees were analysed, and their dependence on the landscape structure was tested. Thereafter, the effects of pollen diversity, insecticide risk, and landscape structure on the life-history traits of bees and their sensitivity to topically applied Dursban 480 EC were determined. Pollen taxa richness in a single nest ranged from 3 to 12, and 34 pesticides were detected in the pollen at concentrations of up to 320 ng/g for desmedipham. The O. bicornis foraging range was relatively large, indicating that the landscape structure within a radius of ~1000 m around the nest is important for this species. Pollen diversity in the studied areas was of minor importance for bee performance, but the ORC or landscape structure significantly affected the life-history traits of the bees. Contamination of pollen with insecticides affected the bees by decreasing the mass of newly emerged adults but their sensitivity to Dursban 480 EC was not related to environmental variables.