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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. Although bees consumed larger amounts of syrup than pollen, pesticide intake via syrup and pollen were similar. 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.

Bee honey possess various nutritional and medicinal functions, which are the result of its diverse chemical composition. The numerous bioactive compounds in honey come from flower nectar; thus, the identification of the specific chemical profiles of honey samples is of great importance. The lipophilic compounds from eight monofloral honeys (rape, buckwheat, clover, willow, milk thistle, dandelion, raspberry and sweet yellow clover) were investigated. Analyses of the lipophilic fractions obtained by UAE (ultrasound assisted extraction) and SPE (solid phase extraction) extractions were performed using high-performance thin layer chromatography (HPTLC). Chromatographic and cluster analyses allowed the identification of a unique, colorful pattern of separated compounds with specific Rf values on the HPTLC plate for each type of monofloral honey. HPTLC is a simple and effective visual method of analysis, and it can serve as a basis for authenticating different types of honey.

The geographical origin of honey affects its composition, which is of key importance for the health-promoting properties and safety of the product. European regulations clearly define the physicochemical requirements for honey that determine the microbiological quality. On the other hand, legislation abolishes microbiological criteria. In the study 40 honey samples originating from two different climatic zones were analyzed. The water content, pH, water activity analysis and the microbiological quality of honey samples have been tested using the reference plate method (total viable count, yeast and molds, lactic acid bacteria, Bacillus spp.). The cluster classification showed that total viable count of bacteria could be used as a measure alternative to the count of Bacillus spp. and 70% of honeys from the tropical climate zone had different microbiological quality than honeys from the temperate climate zone but still under the level 3.0 log cfu/g. The study has revealed that geographical origin of honey may significantly affect the quality and safety of honey. It was considered that water content can be the most informative and handy marker of the microbiological quality of honeys. Analysis of lactic acid bacteria showed temperate climate zone honeys as a source of beneficial bacteria in the diet.

We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June. Concentrations/activities of (enzymatic and non-enzymatic) antioxidant and proteolytic systems, as well as phenoloxidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were constant during the diapause, usually lower in the males than the females. Prior to the diapause/overwintering, the concentrations/activities of all the compounds were higher in the fat bodies than in the hemolymph. Later in the spring and in the summer, they increased in the hemolymph and on the body surfaces, while decreasing in the fat bodies. The global DNA methylation levels increased with age. Higher levels were always observed in the males than in the females. The study will promote better understanding of bee evolution and will be useful for the protection and management of solitary bees, with benefits to the environment and agriculture.

An increasing number of studies show that urbanized areas are habitats of high biological value and ecological significance. Most bee species live in areas altered by man, either in cities - fragmented urban habitats - or in large rural monocultures. Our research is based on three-year observations of population development of the solitary bee L. in three habitat types: city, suburbs and villages. We compared reproductive parameters and diversity of accompanying nest fauna. Population growth rate was high in all habitats, exceeding five times the number of cocoons placed in the previous year. We found no significant differences in the number of cocoons and brood mortality between areas with different urbanization levels. In nests located in suburbs, parasites and cleptoparasites occupied almost three times more nest chambers than in other habitats. Changes in the habitat structure had a significant impact on the diversity and abundance of accompanying fauna. Our study shows that red mason bees are flexible and easily adapt to new conditions, despite changes caused by urbanization and agriculture.

The risk exposure of bee colonies to the toxicity of systemic neonicotinoid insecticides was assessed. Various methods of chemical prevention of commercial winter and spring oilseed rape crops in field-realistic conditions were taken into account in the assessment. Pesticides were applied in accordance with the actual agricultural practice. Commercial crop protection products with thiamethoxam, clothianidin or imidacloprid were used as seed treatment. Formulations containing acetamiprid or thiacloprid were used for spraying. Fifteen healthy bee colonies were placed in close proximity to each of the oilseed rape fields throughout the blooming period. During florescence, the samples of nectar (directly from flowers and nectar flow from combs) and pollen loads were collected repeatedly. Samples of honey, bee bread and adult bees were taken one week after the end of plants flowering. To ensure high specificity and sensitivity of analysed pestcicides modified QuEChERS extraction method and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used. The five of neonicotinoid insecticides (imidacloprid, clothianidin, thiametoxam, acetamiprid and thiacloprid) were analyzed in multi-residue method with 0.1 - 10 ng/g limits of detection. Palynological analysis was done to determine the botanical origin of the nectar, honey and pollen. Development of bee colonies (brood area, worker biomass, colony health) was assessed every 3 weeks until the end of the beekeeping season. The amount of pollen collected by bees per hive, bee bread area and rape honey yield was also measured. The long-term effects of insecticides on bees were estimated using the same methods in April of the following year. All the neonicotinoid insecticides applied to control oilseed rape pests were present in the samples of nectar and pollen. Their residue levels were lower than the acute oral and contact LD50 values. Among five examined neonicotinoids, the most frequently detected were: thiamethoxam, thiacloprid and acetamiprid. These substances were present in 65, 64, and 51% of the nectar samples and in 37, 62, and 45% of the pollen samples, respectively. The highest level of residues were noted after the thiamethoxam seed treatment; on average, 4.2 and 3.8 ng/g in the nectar and pollen samples. In the nectar and pollen samples from winter rape fields, lower levels of neonicotinoid residues were found in comparison to spring rape samples. The contaminations of neonicotinoids applied as seed dressing in nectar samples were significantly higher in comparison to the pollen samples. No negative effects of neonicotinoids on the bee mortality, brood development, strength, and honey yield of healthy bee colonies were found throughout the study period. However, the risk exposure of bee colonies on adverse impact of pesticide residues is high in areas of intensively cultivated oilseed rape. Celem badań była ocena narażenia rodzin pszczelich na toksyczne oddziaływanie pozostałości systemicznych insektycydów neonikotynoidowych zastosowanych w warunkach polowych do chemicznej ochrony upraw rzepaku. Badania polowe przeprowadzone zostały przy współpracy z Rolniczym Zakładem Doświadczalnym Instytutu Uprawy, Nawożenia i Gleboznawstwa w Puławach na 2 plantacjach rzepaku ozimego w roku 2010 i 3 plantacjach rzepaku jarego w roku 2012. Do zwalczania szkodników zastosowano w formie zapraw nasiennych preparaty zawierające tiametoksam, chlotianidynę lub imidachlopryd oraz w formie oprysku dolistnego, preparaty z acetamiprydem lub tiachloprydem. Na wszystkich uprawach wykonane zostały także zabiegi chwastobójcze i grzybobójcze. Na okres kwitnienia roślin rzepaku, w pobliżu każdej plantacji umieszczono 15 rodzin pszczelich, z czego 5 rodzin w każdej grupie przeznaczonych było jedynie do pozyskiwania obnóży pyłkowych. Grupę kontrolną stanowiły rodziny usytuowane w terenie rolniczym wolnym od dużych upraw rzepaku. Od chwili wywiezienia rodzin pszczelich na rzepak, aż do okresu ich zazimowania (w roku 2012 do września) monitorowano śmiertelność pszczół oraz cyklicznie oceniano parametry świadczące o kondycji i rozwoju rodzin (liczbę ramek obsiadanych przez pszczoły oraz powierzchnię czerwiu krytego i otwartego). Do badań laboratoryjnych pobrano próbki nektaru z kwiatów rzepaku, próbki nektaru, miodu i pyłku (obnóży pyłkowych i pierzgi) zgromadzonego przez pszczoły w plastrach oraz próbki pszczół. Pochodzenie botaniczne próbek materiału roślinnego określono na podstawie analizy palinologicznej. Analizę pozostałości insektycydów w zebranym materiale wykonano metodą QuEChERS z wykorzystaniem chromatografu cieczowego sprzężonego z podwójnym detektorem masowym (LC-MS/MS). W badanych próbkach nektaru i pyłku wykryto pozostałości wszystkich substancji neonikotynoidowych aplikowanych zarówno w formie zapraw nasiennych, jak i oprysku. Znacząca liczba próbek zanieczyszczona była dodatkowo substancjami, których nie stosowano w czasie zabiegów. Największa liczba próbek skażona była tiametoksamem, tiachloprydem i acetamiprydem. Obecność tych substancji wykryto odpowiednio w 65, 64 i 51% ogółem przebadanych próbek nektaru i miodu oraz w 37, 62 i 45% próbek pyłku. We wszystkich rodzajach próbek stężenie neonikotynoidów było niższe od ich doustnej i kontaktowej dawki letalnej (LD50) dla pszczół, ale w ponad 50% próbek obecne były co najmniej 2 substancje z tej grupy, a w ponad 25% co najmniej 3. Najwyższy poziom skażenia nektaru i pyłku odnotowano w przypadku zaprawiania nasion rzepaku jarego preparatem insektycydowym zawierającym tiametoksam. Insektycydy stosowane w formie zapraw nasiennych powodowały istotnie wyższe skażenie nektaru i miodu niż pyłku. W próbkach nektaru i pyłku pochodzących z rzepaku ozimego stwierdzono niższy poziom pozostałości neonikotynoidów w porównaniu do próbek z rzepaku jarego. W okresie całego sezonu pszczelarskiego 2010 i 2012 roku nie stwierdzono zwiększonej śmiertelności pszczół ani zaburzeń w rozwoju, kondycji i produkcyjności rodzin pszczelich. Po okresie zimowania 2010/2011 kondycja i rozwój rodzin pszczelich były także prawidłowe. Pozostałości insektycydów neonikotynoidowych znajdujące się w nektarze i pyłku zbieranym przez pszczoły z upraw rzepaku chronionego insektycydami neonikotynoidowymi stwarzają wysokie ryzyko ich toksycznego oddziaływania na rodziny pszczele, szczególnie dla rodzin osłabionych innymi czynnikami (np. obecnością patogenów, pasożytów), ze względu na możliwość wystąpienia zjawiska interakcji i/lub synergizmu.

A method was elaborated to determine phenolic compounds (vanillin, caffeic, p-coumaric and salicylic acids, and flavonoids: rutin, hesperetin, quercetin, pinocembrin, apigenin, kaempferol, isorhamnetin, chrysin, and acacetin) in bee pollen loads using highperformance liquid chromatography with a diode array detector (HPLC-DAD). Phenolic compounds from bee pollen were isolated on Cleanert C18-SPE columns (500 mg/6 mL, Agela Technologies). Polyphenols were identified by comparing the retention times and spectra of compounds found in pollen load samples with the ones of the standard mixture. Quantitative analysis was conducted using the external standard method. In addition, basic validation parameters for the method were determined. For the identified compounds (except for the salicylic acid), satisfactory (≥0.997) linear correlations were obtained. The elaborated method showed high repeatability and inter-laboratory reproducibility. Variability coeffcients of the majority of phenolic compounds did not exceed 10% in conditions of repeatability and inter-laboratory reproducibility, and for the total polyphenolic content they were 1.7 and 5.1%, respectively. The pollen load samples (n = 15) differed in qualitative and quantitative composition of the phenolic compounds. In all the samples, we identified the p-coumaric and salicylic acids and flavonoids rutin, hesperetin, and apigenin nevertheless, these compounds’ contents significantly differed among individual samples. The total phenolic content in the tested samples of pollen loads ranged from 0.653 to 5.966 mg/100 g (on average 2.737 mg/100 g).

The pollen analysis is currently the only reliable test to determine honey variety, but the results are sometimes burdened with error. The main reason for this is additional pollen that got into honey in a way other than with nectar collected by bees but through the centrifugation of combs containing bee bread cells. Studies were conducted in 2012 - 2013 on how different numbers of bee bread cells placed in the honey super influence lime honey pollen analysis. Bee bread pollen getting into honey during extraction in centrifugal-force honey extractors was proven to significantly influence the results of pollen analysis. In some extreme cases, it might skew the results so much that correct determination of honey variety by pollen analysis is no longer possible.

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.