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Varroasis control: stability of homemade oxalic acid water sugar solution

Luciana PRANDIN, Nicoletta DAINESE, Barbara GIRARDI, Ornella DAMOLIN, R. PIRO, F. MUTINELLI Centro Regionale per l’Apicoltura c/o Istituto Zooprofilactico Sperimentale delle Venezie, Via Romea 14/A, 35020 Legnaro (PD), ITALY Tel.: +39 049 8084344, Fax: +39 049 8830572, E-mail: 1. Introduction
Since the paper by RADETZSKI et al. (1994) on the use of oxalic acid for controlling varroasis, sev- eral reports have been published providing the efficacy and tolerability of this organic acid when applied by spraying or trickling. Tests have been carried out by spraying water-diluted oxalic acid (TAKEUKI and HA-RADA, 1983; NANETTI et al., 1995, NANNETTI and STRADI, 1997) or by trickling a solution of oxalic acid, water and sugar (MUTINELLI et al., 1997; CHARRIÈRE, 1997; IMDORF et al., 1997, NANETTI 1999) in the colonies during a broodles period. The oxalic acid, water and sugar solution and the water-diluted oxalic acid (HIGHES et al., 1999) have occasionally been considered responsible for honeybees’ losses after the treat-ment and no data are currently available on its long-term stability. Since the oxalic acid, water and sugar solution is widely used by beekeepers for controlling varroasis in broodless time, out aim was to evaluate the characteristics and the stability of a home-made oxalic acid, water and sugar solution in different storage conditions in order to detect the modifications of the active in-gredients possibly related to the toxicity to honeybees. 2. Materials and methods
2.1. Oxalic acid solution preparation The homemade oxalic acid / water / sugar solution (OAWS) prepared in the laboratory for the pre- sent study, is the same used for treating beehives against varroosis. It is composed of 100 g oxalic acid di-hydrate (Sigma), 1000 g commercial sugar, and 1000 ml drinkable water. The characteristics of water were as follows: pH 7.5, solids 320 mg/l, hardness 27.1 oF, chloride 8.0 mg/l, nitrate 17.5 mg/l, sulphate 21.5 mg/l, iron 5µ/l. This solution was distributed in 10 ml tubes that were stored at different conditions: -20oC, +4 oC, room temperature (RT) in the dark and in the light. This solution was tested for the following parameters soon after its preparation: oxalic acid, colour, pH, hydroxymethylfurfural and sugar. The same parameters were determined 3, 7, 15 and 30 days, 2, 3, 4, 5, 6, 7, 8 and 16 months after preparation for each storage condition. 2.2. Determination of oxalic acid Oxalic acid was determined using a commercially available kit (Oxalate SIGMA Diagnostics kit, Cat. N. 591-D) adapted to honey (MUTINELLI, 1997). This enzymatic method is based on the oxidation of ox-alate-by-oxalate oxidase followed by measurement of hydrogen peroxide produced by a peroxidase-catalyzed reaction (CHIRIBOGA, 1963). 100 µl of OAWS solution was diluted to 100 ml and 10 µl added to a mixture of oxalate oxidase, peroxidase and the substrates (3-methyl-2-benzothiazolinone and 3-dimethylamino benzoic acid) that react with hydrogen peroxide to yield an indamine dye which has an ab-sorbance maximum at 590 nm. Quantification of oxalic acid was extrapolated from a standard curve pre-pared using multi-level oxalate standard (22.5, 45, 90 mg/l). The oxalic acid content is expressed in mg/kg. The colour of the OAWS solution was examined by means of a 2000 Comparator (Lovibond) used for honey colour determination. A cuvette was filled with the solution, inserted into the Comparator and its colour was compared with the different standard colours of the disk. The range of colours is between 5 and 100 mm of the Pfund Scale. 2.4. Determination of hydroxymethylfurfural Hydroxymethylfurfural (HMF) was determined using reverse phase HPLC equipped with UV detec- tion (JEURING, 1980). One ml of OAWS solution was diluted to 50 ml and injected in a C18 reversed phase column. A water/methanol (90+10 by volume) mobile phase was used in isocratic condition. Detection of HMF peaks was performed at 285 nm, the sample signal was compared with those from the standard (1000 µg/l). The HMF content is expressed in mg/l. 2.5 Determination of sugars (fructose, glucose, sucrose) Sugars were determined by an HPLC method using a Dionex DX 500 Chromatograph. Carbohy- drates (pKa between 12 and 13) behave as very weak acids at high pH (12-14) and are partially or totally ionized so they can be separated by an ion exchange mechanism using a non-porous pellicular resin column (Carbopac PA 100, Dionex). OAWS solution was diluted (1:1000) and injected into the loop of the chromato-graph. The sugars were eluted with a sodium hydroxide solution (50 mM) in isocratic condition and detected by a pulsed amperometric system with a stable and sensitive response due to the continuous elimination of the sugar oxidation products. The quantification of fructose, glucose and sucrose was calculated by comparing the peak area of a mix standard solution containing 50 mg/l glucose, 50 mg/l fructose, 10 mg/kg sucrose. The sugars content is expressed in mg/l. 3. Results
The fresh OAWS solution is a water white strongly acid (pH 0-1) solution containing 4.2 mg/l oxalic acid, 18.9 mg/l sucrose, 25.6 mg/l glucose, 24.0 mg/l fructose and a low content of HMF (1,7 mg/l) (Table I). Oxalic acid was very stable in samples stored at –20 oC and 4 oC, in fact after 16 months was 92.9% and 95.2% of the initial concentration respectively. A light decrease in oxalic acid content was detected in OAWS solution stored at RT. A decrease of 10% was registered after 3 month in the solution stored in the light and after 5 months in that one stored in the dark. Both showed 3.5 mg/l oxalic acid content (83%) at the end of the 16-month study. The pH value was determined using a pH test paper and remained constant at 0-1 dur-ing the whole test period. The initial colour of the solution was water white (≤5 mm) and remained stable until the last determination (after 16 months) in the sample stored at –29oC and 4oC. When stored at RT, the samples colour became darker 60 (in the light) and 90 (in the dark) days after preparation. Over the 16 month study the colour changed from water white to white (18-34 mm), to amber extra light (35-50 mm), to light amber (51-85 mm), to amber (86-114 mm) and finally, after 16 months, to dark amber (> 115 mm). Composition of the fresh and 16-month-old OAWS solution
The HMF content of the solution remarkably increased. HMF is spontaneously produced by degrada- tion of sugars, particularly fructose, in acid condition. HMF was very low in the sample stored at –20 oC. In fact after 16 months it was as high as the fresh solution (4.2 mg/kg); in the sample stored at 4oC it increased up to 50.6 mg/kg, due to a slow hydrolysis of sugar. The degradation of the sugar was more evident in the sample stored at RT. After 15 days HMF was about 100 mg/kg, after 2 months 5 times higher and after 6 months exceeded 1000 mg/kg. At the end of the study HMF was 2107 mg/kg in the sample stored in the light and 1945 mg/kg in the sample stored in the dark. No difference between sugar degradation in the light and in the dark was observed. Fresh pre- pared OAWS solution contained approximately the same ratio of three analysed sugars, but after only few days the ratio changed radically. Sucrose is a disaccharide composed of fructose and glucose and, in acid condition; it breaks into the two sugars spontaneously. In the sample stored at –20 oC this degradation developed progressively and after 16 months the sucrose decreased from 19.9 mg/l to 13.9 mg/l. This degradation is less gradual in the sample stored at 4 oC. In fact after 30 days sucrose was 1.8 mg/l and after 3 months it was stable at 0.3 mg/l level. Sucrose hydrolysis is immediate in sample stored at RT, 3 days later the concentration of this sugar was 0.5 mg/l independently to be stored in the light or in the dark. On the contrary if sucrose decreases, fructose and glucose increase. In fresh prepared OAWS solu-tion fructose and glucose concentration was similar (24 and 25.6 mg/l respectively). At –20 oC the increase of fructose and glucose concentration is less evident than in the sample stored in the other conditions. After 16 months the samples stored at 4 oC showed that the fructose and glucose concentration in- creased to 32.6 and 32.9 mg/l, the same amount of the decrease of sucrose. In the sample stored at RT the fructose concentration increased initially, but after 3 months a slight decrease was registered. An increase of the HMF concentration was registered contemporarily. 4. Discussion
This study demonstrated that the concentration of oxalic acid and pH value remained constant in all the tested storage condition and over a long time, as opposed to the changes affecting the other parameters. Colour changes observed in samples stores at RT were related to the numerous possible condensation reac-tions (Maillard reaction), which produce many polycyclic compounds absorbing light in the visible region. The main modification is the production of a high amount of HMF that was formed in the samples stored at RT. HMF is toxic for honeybees and at high concentrations causes ulceration of the digestive system ( JACHIMOWICZ and EL SHERBIN (1975) demonstrated that a HMF level lower than 30 mg/l is safe while above 150 mg/l is toxic and it can cause an increase of honey-bees mortality. As a consequence the treatment of hives with an old preparation of OAWS solution stored at RT is still effective in order to control varroa mites, but HMF could remain in honey stored for feeding the lar-vae and this could have lethal effects on them. Another expected modification was the hydrolysis of sucrose into fructose and glucose because of the acid condition. A particular situation was registered in the samples stored at RT. The fructose concentra-tion increased initially, but after 3 months decreased. This can be explained by the contemporary increase of the HMF concentration. In fact HMF is produced mainly by degradation of fructose. According to the results of this long-term study, we recommend to use fresh preparation of OAWS solution for the treatment of hon-eybees against varroosis in broodless time. Moreover, if any storage is required it is advisable to keep the OAWS solution at 4 oC. R E F E R E N C E S
Charrière J.D., Potentiel et limites de l’emploi des acides organiques, Sanitaire 161 (1997): 219-227 Chiriboga J., Some properties of an oxalic oxidase purified from barley seedlings, Biochem. Biophys. Res. Commun 11 (1963): 277 Higes M., Meana A., Suárez M., Llorente J., Negative long-term effects on bee colonies treated with acid against Varroa jacobsoni Oud., Imdorf A., Charrière J.D., Bachofen B., Efficiency checking of the Varroa jacobsoni control methods by means of oxalic acid, Apiacta 32 Jachimowich T., El Sherbiny G., Zür Problematik der verwendung von Invertzucker für die Bienenfüttering, Apidologie 6 (1975): 121-143 Jeuring J., Kuppers F., High performance liquid chromatographic of furfural and hydroxymethylfurfural in honey, J. Assoc. Off. Anal. Mutinelli F., Baggio A., Capolongo F., Piro R., Prandin L., Biasion L., A scientific note on oxalic acid by topical application for the control of varroasis , Apidologie 28 (1997): 461-462 Nanetti A., Massi A., Mutinelli F., Cremasco S., L’acido ossalico nel controllo della varroasi: note preliminari, Apitalia 22 (1995): 29-32 Nanetti A., Stradi G., Varroasi: trattamento chimico con acido ossalico in sciroppo zuccherino, L’Ape Nostra Amica 19 (1997): 6-14 Nanetti A., Oxalic acid for mite control – Results and review, In: Proceedings of the Concerted Action 3686 meeting “Coordination in Europe of research on integrated control of Varroa mites in honey bee colonies”, Merelbeke, Belgium, November 13-14, 1999, pp. 7-14 Radetzki T., Reiter M., von Negelein B., Oxalsäure zur Varroabekämpfung, Schweiz Bienen-Zeitung 117 (1994): 263-267 Takeuchi K., Harada K., Control of Varroa jacobsoni mites with oxalic acid spray, Honeybee Sci. 4 (1983): 113-116 (in Japanese)


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