A. G. Sabatini

Classification of Italian honeys by 2D HR-NMR.

The importance of honey has been recently increased because of its nutrient and therapeutic effects, but the adulteration of honey in terms of botanical origin has increased, too. The floral origin of honeys is usually determined using melisso-palynological analysis and organoleptic characteristics, but the application of these techniques requires some expertise. A number of papers have confirmed the possibility of characterizing honey samples by selected chemical parameters. In this study high-resolution nuclear magnetic resonance (HR-NMR) and multivariate statistical analysis methods were used to identify and classify honeys of five different floral sources. The 71 honey samples (robinia, chestnut, citrus, eucalyptus, polyfloral) were analyzed by HR-NMR using both 1H NMR and heteronuclear multiple bond correlation spectroscopy (HMBC). Spectral data were analyzed by application of unsupervised and supervised pattern recognition and multivariate statistical techniques such as principal component analysis (PCA) and general discriminant analysis (GDA). The use of 1H-(13)C HMBC coupled with appropriate statistical analysis seems to be an efficient technique for the classification of honeys.

Nectar Flavonol Rhamnosides Are Floral Markers of Acacia (Robinia pseudacacia) Honey

With the objective of finding floral markers for the determination of the botanical origin of acacia (robinia) honey, the phytochemicals present in nectar collected from Robinia pseudacacia flowers were analyzed by high-performance liquid chromatography-tandem mass spectrometry. Eight flavonoid glycosides were detected and characterized as kaempferol combinations with rhamnose and hexose. Acacia honey produced in the same location where the nectar was collected contained nectar-derived kaempferol rhamnosides. This is the first time that flavonoid glycosides have been found as honey constituents. Differences in the stability of nectar flavonoids during honey elaboration and ripening in the hive were shown to be due to hydrolytic enzymatic activity and to oxidation probably related to hydrogen peroxide (glucose-oxidase) activity. Acacia honeys contained propolis-derived flavonoid aglycones (468-4348 microg/100 g) and hydroxycinnamic acid derivatives (281-3249 microg/100 g). In addition, nectar-derived kaempferol glycosides were detected in all of the acacia honey samples analyzed (100-800 microg/100 g). These flavonoids were not detected in any of the different honey samples analyzed previously from different floral origins other than acacia. Finding flavonoid glycosides in honey related to floral origin is particularly relevant as it considerably enlarges the number of possible suitable markers to be used for the determination of the floral origin of honeys.

Influence of brood rearing temperature on honey bee development and susceptibility to poisoning by pesticides

Summary Adult honey bees (Apis mellifera) usually maintain colony brood rearing temperature between 34–35°C by thermoregulation. The brood may, however, also be subjected to suboptimal temperature. Here we investigated whether a decrease of brood rearing temperature may have effects on larval mortality, adult emergence, longevity, morphology and susceptibility to poisoning by pesticides (dimethoate). Using the in vitro rearing protocol of Aupinel (2005), we were able for the first time to control the brood temperature not only during the pupal stage but also during the larval stage. Honey bee larvae were reared in vitro at 35°C (optimal) and 33°C (suboptimal) from 12 h after hatching for 15 days. Dimethoate was tested by ingestion either on 4-day old larvae or on 7-day old adults. Our results showed that lower rearing temperature had no significant effects on larval mortality and adult emergence, but adult bee mortality was strongly affected. Moreover, adult workers emerging at 33°C were significantly more susceptible to dimethoate. Larval LD50 (48 h) was, however, 28 times higher at 33°C than at 35°C. The striking differences between larvae and adults may be explained by differential larval metabolism at 33°C and resulting slower active ingredient absorption. We conclude that adult honey bees reared at even slightly suboptimal brood temperature may be more susceptible to pesticide poisoning and be characterised by reduced longevity. Thus, low temperature brood rearing could be another stress factor for colonies.

Acaricide residues in beeswax after conversion to organic beekeeping methods

Beekeepers interested in converting their honey farms to organic management must replace old combs with organic foundations. The experiment described in this paper compares two methods of replacement of old combs, “fast” (5 combs per year) and “slow” (2 combs per year), by measuring the levels of acaricide residues in the newly built combs. Tested acaricides were coumaphos (Perizin and Asuntol), fluvalinate (Apistan), and chlorfenvinphos (Supona). Significant differences between the two replacement groups were observed only for the Apistan group in the third year, confirming high lipophilicity of fluvalinate. The residue levels in the new combs three years after beginning the conversion were significantly lower than initial levels for all products. Direct contamination of the combs was highest in Asuntol-treated hives and lowest in Perizin-treated hives. Residues in honey exceeding EU Maximum Residue Limit were found only in the case of Asuntol.ZusammenfassungDie ökologische Bienenhaltung hat in Italien seit Einführung der Richtlinien der Europäischen Gemeinschaft zur ökologischen Tierhaltung (Reg. N. 1804/1999, nun ersetzt durch Reg. N. 834/2007) zugenommen. Zur Umstellung müssen Bienenhalter alte Waben innerhalb von drei Jahren durch rückstandsfreie Mittelwände ersetzen. Auf Grund der fettlöslichen Natur der meisten Milbenbehandlungsmittel reichern sich Rückstände im Wachs an und neue Waben sind dem Risiko einer indirekten Kontamination ausgesetzt. In dem in diesem Artikel beschriebenen Experiment wurden zwei Umstellungsmethoden verglichen, bei denen der Ersatz der alten durch neue Waben über zwei oder mehr Jahre hinweg stattfand. Die von den italienischen Imkern am häufigsten verwendeten Akarizide wurden in Betracht gezogen, darunter auch kommerzielle Produkte, die für die Anwendung bei Honigbienen nicht zugelassen sind: Coumaphos (Perizin und Asuntol), Fluvalinat (Apistan), Chlorfenvinphos (Supona) (Tab. I). Das rasche Auswechseln der Waben führte im dritten Jahr der ökologischen Bienenhaltung nur bei Apistan zu einer verringerten insgesamten Kontamination (Tab. IV), dies ist vermutlich auf die hohe Lipophilie von Fluvalinat zurückzuführen. Die erhaltenen Daten wurden zusätzlich genutzt, um direkte und indirekte Kontamination durch die untersuchten Akarizide zu vergleichen. Die Analyse der während der nichtökologischen Bienenhaltung in den Völkern vorhandenen Waben zeigte, dass die Kontamination bei Asuntol am höchsten war, während sie in den mit Perizin behandelten Völkern am geringsten war (Tab. III). Die indirekte Kontamination von neu gebauten Waben war bei Perizin am höchsten (∼ 83 %) und lag bei den anderen Akariziden zwischen 20 % und 30 % (Tab. III). Der Rückstandsgehalt der frisch gebauten Waben drei Jahre nach der Umstellung war bei allen Produkten signifikant geringer als der Anfangsgehalt, in den meisten Fällen lag er unterhalb der von den ökologischen Kontrollinstanzen übergangsweise zugelassenen Grenzwerten. Die indirekte Kontamination des Honigwabenwachses während der nichtökologischen Bienenhaltung war in den mit Asuntol behandelten Völkern am höchsten. Neue, während des Experiments auf rückstandsfreien Mittelwänden gebaute Waben wurden ebenfalls kontaminiert, allerdings war der Rückstandgehalt im dritten Jahr nach Beginn der Umstellung sehr gering oder nicht mehr nachweisbar. Im Honig wurden die EU MRL überschreitenden Rückstandsgehalte ausschließlich in dem mit Asuntol behandelten Bienenstand und nur im ersten Jahr des Experimentes gefunden.

Use of Quinoline Alkaloids as Markers of the Floral Origin of Chestnut Honey

To identify potential floral markers of chestnut honey, the phytochemicals present in chestnut floral nectar collected by bees were analyzed. Two nitrogen-containing compounds were detected, isolated, and identified as 4-hydroxyquinaldic acid (kynurenic acid) and 4-quinolone-2-carboxylic acid by (1)H NMR and (13)C NMR. In addition, chestnut nectar contained the monoterpene 4-(1-hydroxy-1-methylethyl)cyclohexa-1,3-diene-1-carboxylic acid, its gentiobioside ester, and the flavonol quercetin 3-pentosylhexoside. These nectar markers were found in different chestnut unifloral honey samples, although the flavonol was not detected in all samples analyzed. The terpenoid derivatives had previously been found in linden and tilia honeys. These results show that quinoline alkaloids are potentially good markers of chestnut honey, as they were not detected in any other unifloral honey analyzed so far. They are present at concentrations ranging from 34 to 65 mg/100 g of honey in the samples analyzed. In addition, the terpenoid and flavonoid derivatives present in nectar, although not exclusively characteristic of this floral origin, are good complementary markers for the determination of the floral origin of chestnut honey.