HYGIENIC WATER SUPPLY FOR THE BEES

Za pravilan razvoj i opstanak pčelama je neophodno osigurati kontinuiranu i dostatnu opskrbu kvalitetnom vodom. Ima raznih vrsta pojilica kojima se koriste pčelari. Kako u praksi gotovo svaka od njih ima nedostataka (nedostatna zapremina, otežana kontinuirana opskrba vodom, nedovoljna higijena), razvijena je nova higijenska pojilica od nehrđajućeg lima. U uvjetima 6 izoliranih pokusnih pčelinjaka na otoku Unije praćena je djelotvornost novog modela higijenske pojilice glede kontinuirane opskrbe, higijenske ispravnosti i potrošnje vode. Tijekom cijelog trajanja pokusa snaga pčelinjih zajednica bila je ujednačena. U razdoblju od 26. srpnja do 29. rujna 2006. godine 83 zajednice su potrošile ukupno 374,10 litara vode. Prosječna potrošnja vode za promatrano razdoblje po zajednici bila je 5,19±2,88 litara vode. Prosječna dnevna potrošnja vode po zajednici iznosila je 0,12±0,07 litara vode.The bees must have continuous and adequate supply of good quality water for normal development and survival. In their work, the beekeepers use various water supply containers that usually have many disadvantages ( inadequate loading capacity, inconstant supply of water, unsatisfactory sanitary conditions). In order to study permanency, consumption and hygiene of water supply, a new model of hygienic water supply container has been constructed and applied in the experiment on the island of Unije. The trial took place from 26 July to 29 September 2006 and it included six isolated apiaries of equal strength of colonies. During that period, 83 beehives consumed 374,10 litters of water, the average water consumption per beehive being 5,19 ± 2,88 liters. The average daily water consumption per beehive was 0,12 ± 0,07 liters

STANJE PČELARSTVA U REPUBLICI HRVATSKOJ

In this paper analysis of beekeeping conditions, number and distribution of beekeepers and honeybee colonies, annual colony losses as well as honey production, export, import and consumption are given. Beekeeping in Croatia has a long tradition. In comparison to pasture potential and necessity for pollination of cultivated and wild plants, present beekeeping is inadequately developed and distributed in different regions. According to data of the Croatian Livestock Center (2008) there are 313 978 honeybee colonies registered in the 2007 in Croatia on professional and sideline apiaries. The majority of honeybee colonies is placed in Pannonian region with 238 300 (76%), second in representation is in Mediterranean region with 59 763 (19%) and less represented is in Mountain region with 15 915 (5%) honeybee colonies. Yearly honey production is approximately 5 000 t and 2 000 t of national honey consumption (0.4 kg per capita) gives possibility for honey export. Croatia exported 1 051 in 2003, but 274 t of honey in 2007, which indicates on significant export decreasing trend. Normal 10 % of winter colony losses till 2007 increased in 2007/2008 on 55 615 (41.71 %) in Pannonian region, 13 892 (37.46 %) in Mediterranean and 5 359 (32.71 %) in Mountain region. There is a need to make beekeeping much more flexible to fit into an integrated agricultural system, as well as more oriented to the consumers’ demands, to be able to increase competitiveness.U ovom je radu prikazana analiza stanja pčelarstva u Republici Hrvatskoj, broj i raspodjela pčelara i pčelinjih zajednica, godišnji gubitci pčelinjih zajednica, kao i analiza proizvodnje i potrošnje meda, te uvoza i izvoza meda. Pčelarstvo u Hrvatskoj ima dugu tradiciju. U odnosu na pašne potencijale i potrebu za oprašivanjem kultiviranog i samoniklog bilja, sadašnje je pčelarstvo nedekvatno razvijeno i raspodjeljeno u različitim regijama. Prema podatcima Hrvtaskog stočarskog centra (2008.), u Hrvatskoj je 2007. godine registrirano 313.978 pčelinjih zajednica [2]. Većina pčelinjih zajednica, odnosno 238.300 (76 %) smješteno je u panonskoj regiji. Druga po zastupljenosti je mediteranska regija sa 59.763 pčelinjih zajednica (19 %), a najmanji broj zajednica, odnosno 15.915 (5 %) nalazi se u gorskoj regiji. U Hrvatskoj se godišnje proizvede približno 5.000 tona meda [8], a potroši tek 2.000 t. Potrošnja meda u Hrvatskoj je vrlo niska, samo 0,4 kg po stanovniku godišnje. Takav omjer proizvodnje i potrošnje meda pruža mogućnost za izvoz. Hrvatska je u 2003. godini izvezla 1.051 t meda, a u 2007. godini 274 t, što ukazuje na značajan opadajući trend izvoza meda. Normalnih 10 % zimskih gubitaka pčelinjih zajednica u Hrvatskoj do 2007. godine, poraslo je u razdoblju 2007./2008. na 55.615 (41,71 %) u panonskoj regiji, 13.892 (37,46 %) u mediteranskoj i 5.359 (32,71 %) u gorskoj regiji. Pčelarstvo bi u Hrvatskoj trebalo biti fleksibilnije kako bi se uklopilo u integrirani poljiprivredni sektor, te usmjerenije zadovoljavanju potreba potrošača sa ciljem postizanja bolje konkurentnosti

POLLEN SPECTRUM AND VOLATILE COMPOUNDS IN THE HONEY SAC CONTENT AND MINT (MENTHA SPP.) HONEY

Bioraznolikost medonosnih biljnih vrsta iz različitih klimatsko-zemljopisnih područja Hrvatske pruža veliki potencijal za proizvodnju specifičnih vrsta meda. Međutim, peludni spektar, kao i kemijske značajke većine hrvatskih vrsta meda nedovoljno su poznati. U jednu od vrsta meda, koja nije sustavno istraživana, ubraja se i med od metvice (Mentha spp.). Stoga je cilj istraživanja bio utvrditi botaničko podrijetlo i profil isparljivih spojeva iz sadržaja mednog mjehura u odnosu na med od metvice te skupljačku aktivnost pčelinje zajednice na paši metvice. Istraživanje je bilo provedeno na pokusnoj lokaciji Mahovo (područje Parka prirode Lonjsko polje) na pet pokusnih pčelinjih zajednica sive pčele (Apis mellifera carnica Pollmann, 1879), gdje je praćena skupljačka aktivnost pčelinje zajednice i posjećenosti pčela na paši metvice. Analiza sadržaja mednog mjehura i peludnog tereta provedena je prema metodi Soehngen i Jay (1974). Na prikupljenim je uzorcima meda, zatim, sadržaju mednog mjehura i peludnom teretu provedena kvalitativna melisopalinološka analiza. Od fizikalno-kemijskih analiza bilo je provedeno utvrđivanje udjela vode, električne provodnosti i pH vrijednosti. Analiza isparljivih spojeva provedena je pomoću mikroekstrakcije vršnih para na krutoj fazi (HS-SPME) i ultrazvučne ekstrakcije organskim otapalima (USE) na uzorcima meda i sadržaju mednih mjehura. Od analiziranih skupljačica najveći je udio bio onih koje su skupljale nektar (1 774), zatim one koje su skupljale pelud (1 190), a najmanje onih kod kojih je utvrđen puni medni mjehur i peludni teret. U ranim popodnevnim satima (14:00) je utvrđen statistički značajno veći broj skupljačica s punim mednim mjehurom, kao i s peludnim teretom u usporedbi sa skupljačicama prikupljenim u 9:00 i 18:00. Prosječna se masa sadržaja mednog mjehura kretala od 9,58 do 12,22 mg s rasponom od 0,20 do 50,60 mg. U uzorcima pčela skupljenih u 9:00 sati bila je utvrđena statistički značajno niža prosječna masa sadržaja mednog mjehura (9,02 mg) u odnosu na uzorke prikupljene u 18:00 (11,06 mg) i one prikupljene u 14:00 (12,04 mg). Prosječna se masa peludnog tereta kretale od 4,77 mg do 9,60 mg, a raspon se kretao od 0,10 mg do 31,70 mg. U uzorcima pčela skupljenih u 9:00 sati utvrđena je statistički značajno niža masa peludnog tereta (7,73 mg) u odnosu na uzorke prikupljene u 14:00 (8,63 mg), dok između skupljačica prikupljenih u 9:00 i 18:00 te 14:00 i 18:00 nije bila utvrđena statistički značajna razlika. Obzirom na posjećenost pčela paši metvice utvrđeno je da je statistički značajno manji broj pčela bio utvrđen u 18:00 sati (20,08) u odnosu na broj pčela u 10:00 (30,22) i 13:00 sati (36,50). Prema utvrđenom peludnom spektru u analiziranim uzorcima meda, uz mirisnu metvicu (M. pulegium), kao prateće biljne vrste bile su utvrđene zlatošipka (S. gigantea), amorfa (A. fruticosa), pitomi kesten (C. sativa) i smiljkita (L corniculatus). Iz analiziranih uzoraka skupljačica utvrđeno je da je u njihovim peludnim teretima utvrđen udio od 37,82% metvice, od kojih je veći udio bio podrijetlom od mirisne metvice (20,42%) u odnosu na samo 1,93% peludi od vodene metvice (M. aquatica). U uzorcima meda, prosječni je udio vode iznosio 17,3 % s rasponom od 14,5 do 18,8%, zatim je električna provodnost iznosila 0,62 mS/cm s rasponom od 0,37 do 0,75 mS/cm te se je pH vrijednost kretala u rasponu od 3,52 do 4,15 s prosjekom od 3,80. Na osnovu analize isparljivih spojeva u uzorcima meda su po intenzitetu izdvojeni metil-siringat i vomifoliol, dok je u ekstraktu mednih mjehura, uz navedene spojeve, utvrđen još i 3,7-Dimetilokta-1,5-dien-3,7-diol (terpendiol I). Također, analizom vršnih para, kao najzastupljeniji spojevi, utvrđeni su hotrienol, 2-metoksi-4metilfenol, cis- i trans-linalol oksidi, linalol i nerol oksid.Nectariferous plants biodiversity from different climatic and geographical areas of Croatia (Pannonian, Mountain and Adriatic) offers a great potential for the production of specific types of honey. The diversity that we have in climazonal and biocenological schedule represents a unique national treasure that is in Europe rarely found. However, due to nonsystematic research, pollen spectrum and chemical properties of most Croatian honeys are poorly understood. One of the honey types which is not systematically researched is mint (Mentha spp.). According to available literature on European unifloral honeys, mint honey belongs to rare types. Therefore the aim of this study was to characterize the mint honey through determination of botanical origin (pollen spectrum) and content of the honey sac and certain physical and chemical parameters (water content, electrical conductivity and pH value). The second goal was to determine the profile of volatile compounds in the honey sac content, as well as the end product, honey. At the same time, one of the study goals was to investigate the behaviour of bees on mint nectar flow through the foraging activity of bee colonies. The research was conducted at the experimental site Mahovo, which is located in the Lonjsko Polje Nature Park. Five experimental colonies of Carniolan (Apis mellifera carnica Pollmann, 1879) bees in standard Langstroth-Root (LR) colonies were located at the selected location. Gathering activity of honey bee colonies was conducted by sampling from each colony three times a day (9:00, 14:00 and 18:00) for six days. Monitoring of the foraging on the mint field was conducted on the five separate plots of 1 m2 by counting bees for 1 min on the either side of the plot three times a day (10:00, 13:00, and 18:00) for 30 minutes during six days. Analysis of the honey-sac content and pollen loads was carried out according to the method of Soehngen and Jay (1974). Qualitative melisopalinological analysis of collected samples of honey, honey-sac content and the pollen loads was conducted according to the methodology of von der Ohe et al. (2004). From the physicochemical analyses, water content, electrical conductivity and pH were performed. Analysis of volatile compounds was performed using solvent-free headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) in the honey samples and the honey sac content. From the totally analysed 8 721 workers, 2 992 have brought nectar, pollen, or both, while 5 471 returned to the hive without load. From analysed foragers that delivered load, the main portion was of those who have gathered nectar (1 774), then those who have gathered pollen (1 190), but the least those with honey and pollen load, which implies that most foragers are specialized in collecting either nectar or pollen. Statistically higher number of foragers with nectar or pollen load were determined in the early afternoon (14:00) compared with foragers collected in 9:00 and 18:00. The average weight of honey-sac content ranged from 9.58 to 12.22 mg with a range of 0.20 to 50.60 mg. Significantly lower average weight of honey-sac content (9.02 mg) was determined in collected samples of bees at 9:00 o’clock compared to samples collected at 18:00 (11.06 mg) and 14:00, when average weight of 12.04 mg was determined. In the largest group of foragers weight load was ≤ 10 mg, followed with group with load from 11 to 20 mg, and at the least number of bees collected ≥ 31 mg load. Average weight of pollen load was from 4.77 to 9.60 mg, with range of 0.10 to 31.70 mg. In the group of foragers collected at 9:00 o’clock statistically lower pollen load weight (7.73 mg) was determined in comparison to samples collected at 14:00 (8.63 mg). Contrary, between groups collected at 9:00 and 18:00 as well as at 14:00 and 18:00 o’clock difference was not significant. Regarding attendance of bees to mint flowers, statistically lowest number of bees was determined at 18:00 o’clock (20.08) in comparison to count at 10:00 (30.22) and 13:00 knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin. Keywords: honeybee colony foraging activity, honey sac, pollen load, mint (Mentha spp.) honey, pollen spectrum, volatile compounds (36.50) o’clock. According to pollen spectrum, accompanying plants to Mentha pulegium were Solidago gigantea, Amorfa fruticosa, Castanea sativa and Lotus corniculatus. From pollen loads of collected foragers it was determined that 37.82% samples contain mint pollen, from which most part (20.42%) is from pennyroyal (M. pulegium) in comparison to 1.93% of water mint (M. aquatica). Apart from mint pollen, additional 13 plant species were determined in the pollen loads, and the most frequent were Daucus carota, Salvia spp, Ranunculus spp., Inula britanica, Plantago lanceolata, Solidago gigantea, Amorfa fruticosa and Centaurea spp. The average water content was 17.3% with a range from 14.5 to 18.8%. Electrical conductivity was 0.62 mS/cm and the range from 0.37 to 0.75 mS/cm. pH value was in range from 3.52 to 4.15 with the average of 3.80. Based on the volatile compounds analysis of the honey-sac content, the most intensive were 3 compounds: 3.7-dimethylocta-1.5-dien-3.7-diol (terpendiol I), followed by methyl syringate and vomifoliol. In the extract of analysed honey samples were also 3.7dimethylocta-1.5-dien-3.7-diol (terpendiol I). Also, by the analysis of major honey headspace compounds hotrienol, cis- and trans-linalool oxides, linalool and neroloxide were determined. Valuable scientific contribution of the dissertation results from the first time connection of the honey-sac content and the end product of honey, from monitoring of pollen spectrum changes and the knowledge of volatile compounds fate. Analysis of mint honey was performed using two complementary techniques, namely solvent-free headspace solidphase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). It was found that both methods are suitable, since did not cause the formation of thermal artefacts during the extraction of volatile compounds. Also, these methods are complementary because the HS-SPME method enables extraction of the most volatile compounds which are in equilibrium with a honey solution, while the USE method allows the isolation of volatile and semi-volatile compounds. These methods are applied to mint honey for the first time and enabled the preparation of chemical profiles, which represents an original contribution to this work. The organic extract of the foragers’ honey sac content on mint nectar flow was analysed for the first time and methyl-siringat, terpendiol I and vomifoliol found that can be attributable to plant origin. The other major compounds analysed in the honey sac content were close to the part of cuticular waxes and less to bee pheromones. Methyl syrnigate was the most common compound in ultrasonic assisted organic honey extracts together with vomifoliol. Comparison of the organic honey extracts and extracts of the honey sac content indicated that the methyl-syringate and vomifoliol are transferred to honey, while terpendiol was partially transformed into hotrienol during maturation of honey, which is also the original contribution to knowledge about the transport of volatile compounds in relation nectar - honey sac - honey. Also, study results, based on exact data, represent scientific contribution related to the biology of honeybee colony, i.e., knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin

POLLEN SPECTRUM AND VOLATILE COMPOUNDS IN THE HONEY SAC CONTENT AND MINT (MENTHA SPP.) HONEY

Bioraznolikost medonosnih biljnih vrsta iz različitih klimatsko-zemljopisnih područja Hrvatske pruža veliki potencijal za proizvodnju specifičnih vrsta meda. Međutim, peludni spektar, kao i kemijske značajke većine hrvatskih vrsta meda nedovoljno su poznati. U jednu od vrsta meda, koja nije sustavno istraživana, ubraja se i med od metvice (Mentha spp.). Stoga je cilj istraživanja bio utvrditi botaničko podrijetlo i profil isparljivih spojeva iz sadržaja mednog mjehura u odnosu na med od metvice te skupljačku aktivnost pčelinje zajednice na paši metvice. Istraživanje je bilo provedeno na pokusnoj lokaciji Mahovo (područje Parka prirode Lonjsko polje) na pet pokusnih pčelinjih zajednica sive pčele (Apis mellifera carnica Pollmann, 1879), gdje je praćena skupljačka aktivnost pčelinje zajednice i posjećenosti pčela na paši metvice. Analiza sadržaja mednog mjehura i peludnog tereta provedena je prema metodi Soehngen i Jay (1974). Na prikupljenim je uzorcima meda, zatim, sadržaju mednog mjehura i peludnom teretu provedena kvalitativna melisopalinološka analiza. Od fizikalno-kemijskih analiza bilo je provedeno utvrđivanje udjela vode, električne provodnosti i pH vrijednosti. Analiza isparljivih spojeva provedena je pomoću mikroekstrakcije vršnih para na krutoj fazi (HS-SPME) i ultrazvučne ekstrakcije organskim otapalima (USE) na uzorcima meda i sadržaju mednih mjehura. Od analiziranih skupljačica najveći je udio bio onih koje su skupljale nektar (1 774), zatim one koje su skupljale pelud (1 190), a najmanje onih kod kojih je utvrđen puni medni mjehur i peludni teret. U ranim popodnevnim satima (14:00) je utvrđen statistički značajno veći broj skupljačica s punim mednim mjehurom, kao i s peludnim teretom u usporedbi sa skupljačicama prikupljenim u 9:00 i 18:00. Prosječna se masa sadržaja mednog mjehura kretala od 9,58 do 12,22 mg s rasponom od 0,20 do 50,60 mg. U uzorcima pčela skupljenih u 9:00 sati bila je utvrđena statistički značajno niža prosječna masa sadržaja mednog mjehura (9,02 mg) u odnosu na uzorke prikupljene u 18:00 (11,06 mg) i one prikupljene u 14:00 (12,04 mg). Prosječna se masa peludnog tereta kretale od 4,77 mg do 9,60 mg, a raspon se kretao od 0,10 mg do 31,70 mg. U uzorcima pčela skupljenih u 9:00 sati utvrđena je statistički značajno niža masa peludnog tereta (7,73 mg) u odnosu na uzorke prikupljene u 14:00 (8,63 mg), dok između skupljačica prikupljenih u 9:00 i 18:00 te 14:00 i 18:00 nije bila utvrđena statistički značajna razlika. Obzirom na posjećenost pčela paši metvice utvrđeno je da je statistički značajno manji broj pčela bio utvrđen u 18:00 sati (20,08) u odnosu na broj pčela u 10:00 (30,22) i 13:00 sati (36,50). Prema utvrđenom peludnom spektru u analiziranim uzorcima meda, uz mirisnu metvicu (M. pulegium), kao prateće biljne vrste bile su utvrđene zlatošipka (S. gigantea), amorfa (A. fruticosa), pitomi kesten (C. sativa) i smiljkita (L corniculatus). Iz analiziranih uzoraka skupljačica utvrđeno je da je u njihovim peludnim teretima utvrđen udio od 37,82% metvice, od kojih je veći udio bio podrijetlom od mirisne metvice (20,42%) u odnosu na samo 1,93% peludi od vodene metvice (M. aquatica). U uzorcima meda, prosječni je udio vode iznosio 17,3 % s rasponom od 14,5 do 18,8%, zatim je električna provodnost iznosila 0,62 mS/cm s rasponom od 0,37 do 0,75 mS/cm te se je pH vrijednost kretala u rasponu od 3,52 do 4,15 s prosjekom od 3,80. Na osnovu analize isparljivih spojeva u uzorcima meda su po intenzitetu izdvojeni metil-siringat i vomifoliol, dok je u ekstraktu mednih mjehura, uz navedene spojeve, utvrđen još i 3,7-Dimetilokta-1,5-dien-3,7-diol (terpendiol I). Također, analizom vršnih para, kao najzastupljeniji spojevi, utvrđeni su hotrienol, 2-metoksi-4metilfenol, cis- i trans-linalol oksidi, linalol i nerol oksid.Nectariferous plants biodiversity from different climatic and geographical areas of Croatia (Pannonian, Mountain and Adriatic) offers a great potential for the production of specific types of honey. The diversity that we have in climazonal and biocenological schedule represents a unique national treasure that is in Europe rarely found. However, due to nonsystematic research, pollen spectrum and chemical properties of most Croatian honeys are poorly understood. One of the honey types which is not systematically researched is mint (Mentha spp.). According to available literature on European unifloral honeys, mint honey belongs to rare types. Therefore the aim of this study was to characterize the mint honey through determination of botanical origin (pollen spectrum) and content of the honey sac and certain physical and chemical parameters (water content, electrical conductivity and pH value). The second goal was to determine the profile of volatile compounds in the honey sac content, as well as the end product, honey. At the same time, one of the study goals was to investigate the behaviour of bees on mint nectar flow through the foraging activity of bee colonies. The research was conducted at the experimental site Mahovo, which is located in the Lonjsko Polje Nature Park. Five experimental colonies of Carniolan (Apis mellifera carnica Pollmann, 1879) bees in standard Langstroth-Root (LR) colonies were located at the selected location. Gathering activity of honey bee colonies was conducted by sampling from each colony three times a day (9:00, 14:00 and 18:00) for six days. Monitoring of the foraging on the mint field was conducted on the five separate plots of 1 m2 by counting bees for 1 min on the either side of the plot three times a day (10:00, 13:00, and 18:00) for 30 minutes during six days. Analysis of the honey-sac content and pollen loads was carried out according to the method of Soehngen and Jay (1974). Qualitative melisopalinological analysis of collected samples of honey, honey-sac content and the pollen loads was conducted according to the methodology of von der Ohe et al. (2004). From the physicochemical analyses, water content, electrical conductivity and pH were performed. Analysis of volatile compounds was performed using solvent-free headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) in the honey samples and the honey sac content. From the totally analysed 8 721 workers, 2 992 have brought nectar, pollen, or both, while 5 471 returned to the hive without load. From analysed foragers that delivered load, the main portion was of those who have gathered nectar (1 774), then those who have gathered pollen (1 190), but the least those with honey and pollen load, which implies that most foragers are specialized in collecting either nectar or pollen. Statistically higher number of foragers with nectar or pollen load were determined in the early afternoon (14:00) compared with foragers collected in 9:00 and 18:00. The average weight of honey-sac content ranged from 9.58 to 12.22 mg with a range of 0.20 to 50.60 mg. Significantly lower average weight of honey-sac content (9.02 mg) was determined in collected samples of bees at 9:00 o’clock compared to samples collected at 18:00 (11.06 mg) and 14:00, when average weight of 12.04 mg was determined. In the largest group of foragers weight load was ≤ 10 mg, followed with group with load from 11 to 20 mg, and at the least number of bees collected ≥ 31 mg load. Average weight of pollen load was from 4.77 to 9.60 mg, with range of 0.10 to 31.70 mg. In the group of foragers collected at 9:00 o’clock statistically lower pollen load weight (7.73 mg) was determined in comparison to samples collected at 14:00 (8.63 mg). Contrary, between groups collected at 9:00 and 18:00 as well as at 14:00 and 18:00 o’clock difference was not significant. Regarding attendance of bees to mint flowers, statistically lowest number of bees was determined at 18:00 o’clock (20.08) in comparison to count at 10:00 (30.22) and 13:00 knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin. Keywords: honeybee colony foraging activity, honey sac, pollen load, mint (Mentha spp.) honey, pollen spectrum, volatile compounds (36.50) o’clock. According to pollen spectrum, accompanying plants to Mentha pulegium were Solidago gigantea, Amorfa fruticosa, Castanea sativa and Lotus corniculatus. From pollen loads of collected foragers it was determined that 37.82% samples contain mint pollen, from which most part (20.42%) is from pennyroyal (M. pulegium) in comparison to 1.93% of water mint (M. aquatica). Apart from mint pollen, additional 13 plant species were determined in the pollen loads, and the most frequent were Daucus carota, Salvia spp, Ranunculus spp., Inula britanica, Plantago lanceolata, Solidago gigantea, Amorfa fruticosa and Centaurea spp. The average water content was 17.3% with a range from 14.5 to 18.8%. Electrical conductivity was 0.62 mS/cm and the range from 0.37 to 0.75 mS/cm. pH value was in range from 3.52 to 4.15 with the average of 3.80. Based on the volatile compounds analysis of the honey-sac content, the most intensive were 3 compounds: 3.7-dimethylocta-1.5-dien-3.7-diol (terpendiol I), followed by methyl syringate and vomifoliol. In the extract of analysed honey samples were also 3.7dimethylocta-1.5-dien-3.7-diol (terpendiol I). Also, by the analysis of major honey headspace compounds hotrienol, cis- and trans-linalool oxides, linalool and neroloxide were determined. Valuable scientific contribution of the dissertation results from the first time connection of the honey-sac content and the end product of honey, from monitoring of pollen spectrum changes and the knowledge of volatile compounds fate. Analysis of mint honey was performed using two complementary techniques, namely solvent-free headspace solidphase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). It was found that both methods are suitable, since did not cause the formation of thermal artefacts during the extraction of volatile compounds. Also, these methods are complementary because the HS-SPME method enables extraction of the most volatile compounds which are in equilibrium with a honey solution, while the USE method allows the isolation of volatile and semi-volatile compounds. These methods are applied to mint honey for the first time and enabled the preparation of chemical profiles, which represents an original contribution to this work. The organic extract of the foragers’ honey sac content on mint nectar flow was analysed for the first time and methyl-siringat, terpendiol I and vomifoliol found that can be attributable to plant origin. The other major compounds analysed in the honey sac content were close to the part of cuticular waxes and less to bee pheromones. Methyl syrnigate was the most common compound in ultrasonic assisted organic honey extracts together with vomifoliol. Comparison of the organic honey extracts and extracts of the honey sac content indicated that the methyl-syringate and vomifoliol are transferred to honey, while terpendiol was partially transformed into hotrienol during maturation of honey, which is also the original contribution to knowledge about the transport of volatile compounds in relation nectar - honey sac - honey. Also, study results, based on exact data, represent scientific contribution related to the biology of honeybee colony, i.e., knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin

POLLEN SPECTRUM AND VOLATILE COMPOUNDS IN THE HONEY SAC CONTENT AND MINT (MENTHA SPP.) HONEY

Bioraznolikost medonosnih biljnih vrsta iz različitih klimatsko-zemljopisnih područja Hrvatske pruža veliki potencijal za proizvodnju specifičnih vrsta meda. Međutim, peludni spektar, kao i kemijske značajke većine hrvatskih vrsta meda nedovoljno su poznati. U jednu od vrsta meda, koja nije sustavno istraživana, ubraja se i med od metvice (Mentha spp.). Stoga je cilj istraživanja bio utvrditi botaničko podrijetlo i profil isparljivih spojeva iz sadržaja mednog mjehura u odnosu na med od metvice te skupljačku aktivnost pčelinje zajednice na paši metvice. Istraživanje je bilo provedeno na pokusnoj lokaciji Mahovo (područje Parka prirode Lonjsko polje) na pet pokusnih pčelinjih zajednica sive pčele (Apis mellifera carnica Pollmann, 1879), gdje je praćena skupljačka aktivnost pčelinje zajednice i posjećenosti pčela na paši metvice. Analiza sadržaja mednog mjehura i peludnog tereta provedena je prema metodi Soehngen i Jay (1974). Na prikupljenim je uzorcima meda, zatim, sadržaju mednog mjehura i peludnom teretu provedena kvalitativna melisopalinološka analiza. Od fizikalno-kemijskih analiza bilo je provedeno utvrđivanje udjela vode, električne provodnosti i pH vrijednosti. Analiza isparljivih spojeva provedena je pomoću mikroekstrakcije vršnih para na krutoj fazi (HS-SPME) i ultrazvučne ekstrakcije organskim otapalima (USE) na uzorcima meda i sadržaju mednih mjehura. Od analiziranih skupljačica najveći je udio bio onih koje su skupljale nektar (1 774), zatim one koje su skupljale pelud (1 190), a najmanje onih kod kojih je utvrđen puni medni mjehur i peludni teret. U ranim popodnevnim satima (14:00) je utvrđen statistički značajno veći broj skupljačica s punim mednim mjehurom, kao i s peludnim teretom u usporedbi sa skupljačicama prikupljenim u 9:00 i 18:00. Prosječna se masa sadržaja mednog mjehura kretala od 9,58 do 12,22 mg s rasponom od 0,20 do 50,60 mg. U uzorcima pčela skupljenih u 9:00 sati bila je utvrđena statistički značajno niža prosječna masa sadržaja mednog mjehura (9,02 mg) u odnosu na uzorke prikupljene u 18:00 (11,06 mg) i one prikupljene u 14:00 (12,04 mg). Prosječna se masa peludnog tereta kretale od 4,77 mg do 9,60 mg, a raspon se kretao od 0,10 mg do 31,70 mg. U uzorcima pčela skupljenih u 9:00 sati utvrđena je statistički značajno niža masa peludnog tereta (7,73 mg) u odnosu na uzorke prikupljene u 14:00 (8,63 mg), dok između skupljačica prikupljenih u 9:00 i 18:00 te 14:00 i 18:00 nije bila utvrđena statistički značajna razlika. Obzirom na posjećenost pčela paši metvice utvrđeno je da je statistički značajno manji broj pčela bio utvrđen u 18:00 sati (20,08) u odnosu na broj pčela u 10:00 (30,22) i 13:00 sati (36,50). Prema utvrđenom peludnom spektru u analiziranim uzorcima meda, uz mirisnu metvicu (M. pulegium), kao prateće biljne vrste bile su utvrđene zlatošipka (S. gigantea), amorfa (A. fruticosa), pitomi kesten (C. sativa) i smiljkita (L corniculatus). Iz analiziranih uzoraka skupljačica utvrđeno je da je u njihovim peludnim teretima utvrđen udio od 37,82% metvice, od kojih je veći udio bio podrijetlom od mirisne metvice (20,42%) u odnosu na samo 1,93% peludi od vodene metvice (M. aquatica). U uzorcima meda, prosječni je udio vode iznosio 17,3 % s rasponom od 14,5 do 18,8%, zatim je električna provodnost iznosila 0,62 mS/cm s rasponom od 0,37 do 0,75 mS/cm te se je pH vrijednost kretala u rasponu od 3,52 do 4,15 s prosjekom od 3,80. Na osnovu analize isparljivih spojeva u uzorcima meda su po intenzitetu izdvojeni metil-siringat i vomifoliol, dok je u ekstraktu mednih mjehura, uz navedene spojeve, utvrđen još i 3,7-Dimetilokta-1,5-dien-3,7-diol (terpendiol I). Također, analizom vršnih para, kao najzastupljeniji spojevi, utvrđeni su hotrienol, 2-metoksi-4metilfenol, cis- i trans-linalol oksidi, linalol i nerol oksid.Nectariferous plants biodiversity from different climatic and geographical areas of Croatia (Pannonian, Mountain and Adriatic) offers a great potential for the production of specific types of honey. The diversity that we have in climazonal and biocenological schedule represents a unique national treasure that is in Europe rarely found. However, due to nonsystematic research, pollen spectrum and chemical properties of most Croatian honeys are poorly understood. One of the honey types which is not systematically researched is mint (Mentha spp.). According to available literature on European unifloral honeys, mint honey belongs to rare types. Therefore the aim of this study was to characterize the mint honey through determination of botanical origin (pollen spectrum) and content of the honey sac and certain physical and chemical parameters (water content, electrical conductivity and pH value). The second goal was to determine the profile of volatile compounds in the honey sac content, as well as the end product, honey. At the same time, one of the study goals was to investigate the behaviour of bees on mint nectar flow through the foraging activity of bee colonies. The research was conducted at the experimental site Mahovo, which is located in the Lonjsko Polje Nature Park. Five experimental colonies of Carniolan (Apis mellifera carnica Pollmann, 1879) bees in standard Langstroth-Root (LR) colonies were located at the selected location. Gathering activity of honey bee colonies was conducted by sampling from each colony three times a day (9:00, 14:00 and 18:00) for six days. Monitoring of the foraging on the mint field was conducted on the five separate plots of 1 m2 by counting bees for 1 min on the either side of the plot three times a day (10:00, 13:00, and 18:00) for 30 minutes during six days. Analysis of the honey-sac content and pollen loads was carried out according to the method of Soehngen and Jay (1974). Qualitative melisopalinological analysis of collected samples of honey, honey-sac content and the pollen loads was conducted according to the methodology of von der Ohe et al. (2004). From the physicochemical analyses, water content, electrical conductivity and pH were performed. Analysis of volatile compounds was performed using solvent-free headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) in the honey samples and the honey sac content. From the totally analysed 8 721 workers, 2 992 have brought nectar, pollen, or both, while 5 471 returned to the hive without load. From analysed foragers that delivered load, the main portion was of those who have gathered nectar (1 774), then those who have gathered pollen (1 190), but the least those with honey and pollen load, which implies that most foragers are specialized in collecting either nectar or pollen. Statistically higher number of foragers with nectar or pollen load were determined in the early afternoon (14:00) compared with foragers collected in 9:00 and 18:00. The average weight of honey-sac content ranged from 9.58 to 12.22 mg with a range of 0.20 to 50.60 mg. Significantly lower average weight of honey-sac content (9.02 mg) was determined in collected samples of bees at 9:00 o’clock compared to samples collected at 18:00 (11.06 mg) and 14:00, when average weight of 12.04 mg was determined. In the largest group of foragers weight load was ≤ 10 mg, followed with group with load from 11 to 20 mg, and at the least number of bees collected ≥ 31 mg load. Average weight of pollen load was from 4.77 to 9.60 mg, with range of 0.10 to 31.70 mg. In the group of foragers collected at 9:00 o’clock statistically lower pollen load weight (7.73 mg) was determined in comparison to samples collected at 14:00 (8.63 mg). Contrary, between groups collected at 9:00 and 18:00 as well as at 14:00 and 18:00 o’clock difference was not significant. Regarding attendance of bees to mint flowers, statistically lowest number of bees was determined at 18:00 o’clock (20.08) in comparison to count at 10:00 (30.22) and 13:00 knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin. Keywords: honeybee colony foraging activity, honey sac, pollen load, mint (Mentha spp.) honey, pollen spectrum, volatile compounds (36.50) o’clock. According to pollen spectrum, accompanying plants to Mentha pulegium were Solidago gigantea, Amorfa fruticosa, Castanea sativa and Lotus corniculatus. From pollen loads of collected foragers it was determined that 37.82% samples contain mint pollen, from which most part (20.42%) is from pennyroyal (M. pulegium) in comparison to 1.93% of water mint (M. aquatica). Apart from mint pollen, additional 13 plant species were determined in the pollen loads, and the most frequent were Daucus carota, Salvia spp, Ranunculus spp., Inula britanica, Plantago lanceolata, Solidago gigantea, Amorfa fruticosa and Centaurea spp. The average water content was 17.3% with a range from 14.5 to 18.8%. Electrical conductivity was 0.62 mS/cm and the range from 0.37 to 0.75 mS/cm. pH value was in range from 3.52 to 4.15 with the average of 3.80. Based on the volatile compounds analysis of the honey-sac content, the most intensive were 3 compounds: 3.7-dimethylocta-1.5-dien-3.7-diol (terpendiol I), followed by methyl syringate and vomifoliol. In the extract of analysed honey samples were also 3.7dimethylocta-1.5-dien-3.7-diol (terpendiol I). Also, by the analysis of major honey headspace compounds hotrienol, cis- and trans-linalool oxides, linalool and neroloxide were determined. Valuable scientific contribution of the dissertation results from the first time connection of the honey-sac content and the end product of honey, from monitoring of pollen spectrum changes and the knowledge of volatile compounds fate. Analysis of mint honey was performed using two complementary techniques, namely solvent-free headspace solidphase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). It was found that both methods are suitable, since did not cause the formation of thermal artefacts during the extraction of volatile compounds. Also, these methods are complementary because the HS-SPME method enables extraction of the most volatile compounds which are in equilibrium with a honey solution, while the USE method allows the isolation of volatile and semi-volatile compounds. These methods are applied to mint honey for the first time and enabled the preparation of chemical profiles, which represents an original contribution to this work. The organic extract of the foragers’ honey sac content on mint nectar flow was analysed for the first time and methyl-siringat, terpendiol I and vomifoliol found that can be attributable to plant origin. The other major compounds analysed in the honey sac content were close to the part of cuticular waxes and less to bee pheromones. Methyl syrnigate was the most common compound in ultrasonic assisted organic honey extracts together with vomifoliol. Comparison of the organic honey extracts and extracts of the honey sac content indicated that the methyl-syringate and vomifoliol are transferred to honey, while terpendiol was partially transformed into hotrienol during maturation of honey, which is also the original contribution to knowledge about the transport of volatile compounds in relation nectar - honey sac - honey. Also, study results, based on exact data, represent scientific contribution related to the biology of honeybee colony, i.e., knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of the results, this study provides a contribution in practical terms, because they indicate preliminary actions to be undertaken for the timely preparation of colonies for the main pasture. At the same time, the original data on the profile of mint honey volatile compounds can be used as potential biomarkers to determine its botanical origin

THE STATE OF BEEKEEPING IN CROATIA

In this paper analysis of beekeeping conditions, number and distribution of beekeepers and honeybee colonies, annual colony losses as well as honey production, export, import and consumption are given. Beekeeping in Croatia has a long tradition. In comparison to pasture potential and necessity for pollination of cultivated and wild plants, present beekeeping is inadequately developed and distributed in different regions. According to data of the Croatian Livestock Center (2008) there are 313 978 honeybee colonies registered in the 2007 in Croatia on professional and sideline apiaries. The majority of honeybee colonies is placed in Pannonian region with 238 300 (76%), second in representation is in Mediterranean region with 59 763 (19%) and less represented is in Mountain region with 15 915 (5%) honeybee colonies. Yearly honey production is approximately 5 000 t and 2 000 t of national honey consumption (0.4 kg per capita) gives possibility for honey export. Croatia exported 1 051 in 2003, but 274 t of honey in 2007, which indicates on significant export decreasing trend. Normal 10 % of winter colony losses till 2007 increased in 2007/2008 on 55 615 (41.71 %) in Pannonian region, 13 892 (37.46 %) in Mediterranean and 5 359 (32.71 %) in Mountain region. There is a need to make beekeeping much more flexible to fit into an integrated agricultural system, as well as more oriented to the consumers’ demands, to be able to increase competitiveness

Organic Extractives from Mentha spp. Honey and the Bee-Stomach: Methyl Syringate, Vomifoliol, Terpenediol I, Hotrienol and Other Compounds

The GC and GC/MS analyses of the solvent organic extractive from the stomach of the bees, having collected Mentha spp. nectar, revealed the presence of methyl syringate (6.6%), terpendiol I (5.0%) and vomifoliol (3.0%) that can be attributed to the plant origin. Other major compounds from the bee-stomach were related to the composition of cuticular waxes and less to pheromones. Organic extractivesfrom Mentha spp. honey were obtained by solvent-free headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) and analyzed by GC and GC/MS. The major honey headspace compounds were hotrienol (31.1%–38.5%), 2-methoxy-4-methylphenol (0.5–6.0%), cis- and trans-linalool oxides (0.9–2.8%), linalool (1.0–3.1%) and neroloxide (0.9–1.9%). Methyl syringate was the most abundant compound (38.3-56.2%) in the honey solvent extractives followed by vomifoliol (7.0–26.6%). Comparison of the honey organic extractives with the corresponding bee-stomach extractive indicated that methyl syringate and vomofoliol were transferred to the honey while terpendiol I was partially transformed to hotrienol in ripened honey