Italian Journal of Food Safety 2020; volume 9:8678

Occurrence of antibiotic honey, which corresponds to the detection residues in Apulian honey: limit of the analytical method used. Correspondence: Alessandra Emilia Savarino, Limits of detection may vary depending Department of Veterinary Medicine, potential risk of environmental on the detection method adopted, and there University of Aldo Moro, Strada pollution by antibiotics may therefore be different interpretations Provinciale 62 per Km 3, 70010 Valenzano, Bari, . between European member states. In Italy, Tel.: +39 0805443878; Fax: +39 0805443855. Alessandra Emilia Savarino,1 the National Residue Control Plan (Italian E-mail: [email protected] 1 1 Ministry of Health, 2017), established in Valentina Terio, Roberta Barrasso, accordance with Council Directive Edmondo Ceci,1 Sara Panseri,2 Key words: Antibiotic residues, honey, envi- 96/23/EC, provides for the detection of ronmental pollution, multi-array. Luca Maria Chiesa,2 1 chloramphenicol, nitrofuran metabolites, Elisabetta Bonerba nitroimidazoles, tetracyclines, sulphamides, Contributions: The authors contributed equally. 1Department of Veterinary Medicine, aminoglycosides and macrolides in order to Conflict of interest: The authors declare no University of Bari Aldo Moro, Bari; detect the illegal use of antibiotics in bee- potential conflict of interests. 2Department of Health, Animal Science keeping. Specifically, the detection limits and Food Safety, University of Milan, set by the national plan are 1.3-1.6 g/kg for Funding: This research was financially sup- Milan, Italy aminoglycosides and 5 g/kg for sul- ported by Regione Puglia (Department of phamides, tetracyclines and macrolides. Agriculture, Rural Development and Even though antibiotic drugs are not Environment - Section of competitiveness of authorized for the treatment of bees, many food supply chains): Research and studies show the presence of residues in Experimentation Project in Agriculture “Ape e Abstract Ambiente: Biomonitoraggio e Valorizzazione The presence of antibiotic residues in honey, raising the suspicion that this is dei Prodotti dell’alveare Pugliesi” honey is widely documented and is attribut- caused mainly by illegal use in beekeeping. (AP.A.Bi.Va.P.P.) (Cod PSR_115); Regulation ed almost exclusively to improper beekeep- During flight, as bees are exposed to vari- (EU) No 1308/2013 of the European ing practices, due to the frequent use of ous pollutants dispersed in the environment, Parliament and of the Council of 17 December 2013, financing of aid applications 2018. drugs for the treatment of beehive diseases. the antibiotic contamination could result Therefore, the aim of our research was to from their presence in the territory. Indeed, several studies show the presence of phar- Availability of data and materials: The data evaluate the presence of antibiotics in hon- that support the findings of this study are avai- macological substances in different envi- eycomb using the Anti-Microbial Array II lable from the corresponding author upon rea- ronmental compartments such as soil and (AM II) and IV (AM IV) method and to sonable request. water (Chung et al., 2017). Currently, there assess the relationship between environ- have been only very limited studies examin- mental context and antibiotic residues in Ethics approval and consent to participate: ing the relationship between the presence of This research was conducted in accordance honey. The results show the presence of antibiotic residues and the environmental with all relevant guidelines and procedures. antibiotic residues in 26/50 honey from context in which bees collect food. brood nests samples, confirming the impact In order to understand the potential for Consent for publication: The manuscript does of environmental contamination on the not contain any individual person’s data in any drug contamination in honey by environ- health quality of this food product. In addi- form. mental antibiotic pollutants, the aim of the tion, subsequent analyses conducted on pos- present work was to evaluate the presence itive samples reveal the instability over time Received for publication: 14 November 2019. of antibiotics in honeycomb using the Anti- Accepted for publication: 21 November 2019. of antimicrobial molecules in honey. These Microbial Array II (AM II) and IV (AM IV) results highlight the need for further studies method, a competitive chemiluminescent This work is licensed under a Creative in order to understand all likely sources of assay. Commons Attribution-NonCommercial 4.0 contamination and to implement a compre- International License (CC BY-NC 4.0). hensive safety management plan for honey. ©Copyright: the Author(s), 2020 Licensee PAGEPress, Italy Materials and Methods Italian Journal of Food Safety 2020; 9:8678 doi:10.4081/ijfs.2020.8678 Introduction Chemicals and reagents Honey is a natural food product, much All solvents (HPLC/analytical grade) appreciated by consumers for its special were purchased from Fluka (Sigma- cillin, benzylpenicillin, cefalexin, functional properties. In Europe, bees are Aldrich, St. Louis, MO, USA). cefquinome, ceftiofur, chloramphenicol, considered food-producing animals and Trichloroacetic acid (TCA) crystals and the therefore beehive products must comply reagents to prepare an EDTA-McIlvaine chlortetracycline, ciprofloxacin, with legislation on residues of pharmaco- buffer solution, pH 4 (disodium hydrogen danofloxacin, dimetridazole, doxycycline, logically active substances. phosphate dihydrate, citric acid monohy- enrofloxacin, florfenicol, florfenicol amine, Currently, European regulation No. drate and EDTA) were purchased from flumequine, furaltadone, furazolidone, lin- 37/2010 (Commission Regulation (EU) No. Fluka. comycin, lomefloxacin hydrochloride, mar- 37/2010) has not established MRLs for Formic acid (98–100%) was obtained bofloxacin, nalidixic acid, nitrofurazone, antimicrobial substances in honey and from Riedel-de Haën (Sigma-Aldrich, St. oxolinic acid, oxytetracycline, ronidazole, therefore the use of antibiotics in beekeep- Louis, MO, USA). spyramicin, sulphadiazine, sul- ing is not allowed in the European commu- The extraction cartridges (Oasis HLB 3 phadimethoxine, sulphadimidine, sulpham- nity. The absence of MRLs therefore means mL, 60 mg) were provided by Waters erazine, sulphathiazole, tetracycline “zero tolerance” for antibiotic residues in (Milford, MA, USA).Amoxicillin, ampi- hydrochloride, thiamphenicol, tilmicosine,

[page 14] [Italian Journal of Food Safety 2020; 9:8678] Article tinidazole, trimethoprim, tylosin, and cally detects 12 different types of antibiotics All materials were balanced at room tem- enrofloxacin d5 as the internal standards such as amikacin/kanamicin (AMK), perature and the solutions required for the (IS) (purity >98%) were used and pur- apramycin (APA), neomycin/paromomycin test were prepared according to the instruc- chased from Fluka (Sigma-Aldrich, St. (NEO), spectinomycin/dihydrostrepto- tions provided. All analyses were carried Louis, MO, USA). mycin (SPT), streptomycin (STR), out in accordance with the manufacturer’s tobramycin (TOB), spiramycin (SPR), ery- recommendations. Standard solutions thromycin (ERY), tylosin B (TYB), baci- For each standard, stock solutions were Assay protocol −1 tracin (BCT), lincosamides (LIN), and vir- prepared (1 mg mL ) in methanol and kept giniamycin (VIR), simultaneously. The procedure for the analysis of sam- at −20°C. Working solutions at 10 and 100 ples was as follows: 100 mL of test dilution −1 The core technology is the Randox ng mL were prepared daily and main- biochip, a solid substrate containing a series solution was inserted into the biochip wells, tained at 4°C during the assay. of discrete test regions of immobilized anti- then 100 mL of the calibrator or sample was pipetted into the biochip wells. To mix the Sampling bodies specific for various antimicrobials. A reagents, all edges of the handling tray were Sampling was carried out by taking competitive chemiluminescent immunoas- tapped gently. The handling tray was fixed samples from various areas to obtain infor- say is applied. Increased antimicrobial lev- to the base plate of the thermal stirrer and mation on the impact of different environ- els in a sample result in a reduction in the incubated for 30 minutes at 25°C and 370 mental contexts on the quality of honey. binding of the antimicrobial labelled with rpm. 100 mL of the working strength conju- The study included placing a total of 25 horseradish peroxidase (HRP) and thus a gate was pipetted into the biochip wells and hives on the Apulian territory; 15 hives decrease in the emitted chemiluminescence the plate was incubated for an additional 60 were positioned in five different geographi- signal. The emission of light occurs by minutes at 25°C and 370 rpm. The handling cal areas of the , of which 3 chemical reaction, with an enzyme being tray containing the media was removed hives were placed in Toritto, 3 in used to catalyse the chemical reaction on , 3 in , 3 in the biochip, which generates the chemilu- from the thermal stirrer. Reagents were dis- and 3 in . In addition, 10 minescent signal. Luminol and Peroxide are carded by sharply shaking the handling tray. hives were placed at the experimental api- the signal reagents that create a luminous Two rapid wash cycles were immediately ary of the University of Bari A. Moro, reaction. The light emitted by the chemilu- performed, using approximately 350 mL of Department of Veterinary Medicine. minescent reaction that occurs in each dis- wash buffer per well. The handling tray was In total, from the 25 apiaries considered crete test region (DTR) is simultaneously, gently tapped to release any reagents and in which no pharmacological treatment detected and quantified by a camera with a trapped under the biochip. The wash cycle has been carried out, 50 honeycomb sam- charge-coupling device (CCD) at -50°C. was performed four more times. For each ples were taken: 6 from the apiaries of The CCD camera simultaneously records cycle, all four edges of the manipulation Toritto, 6 from Conversano, 6 from the light output from all discrete test sites on plate were gently tapped for approximately Castellana Grotte, 6 from Monopoli, 6 from each biochip on the biochip vector. The 10-15 s; the biochips were then soaked in Altamura, with another 20 samples from the concentration of the analytes detected in the Wash Buffer for 2 min. After final washing, hives at the experimental apiary of the sample is calculated from the calibration the wells were filled with Wash Buffer and University of Bari A. Moro. The sampling curve. left to soak until image capture. Shortly was carried out according to the guidelines before the signal was added, the wash Honey sample preparation of the National Reference Centre for buffer was removed; 250 mL of the working The honey sample (1 g) was added with Beekeeping, which provides for the collec- signal reagent was added to each biochip 9 mL of diluted wash buffer warmed to tion of about 100 g of honey from brood well and covered to protect it from light. 37°C. Subsequently, the tubes (Falcon ™ nests comb without operculum. In short, a The working signal reagent consisted of Round-Bottom Polypropylene, Fisher portion of the comb (10x10 cm) was taken Luminol (Luminol-EV 805 for AMII or Scientific Company, Ottawa, Ontario) con- with a sterile scalpel and the sample was Luminol-EV841 for AMIV) (1 x 10 mL) taining the samples were placed on a roller closed in sterile bags and stored at −20°C and Peroxide (1 x 10 mL) mixed in a ratio for 10 minutes or until dissolution. The until the day of analysis. of 1:1. After exactly 2 minutes, the holder preparation was diluted with an equal was inserted into the Evidence Evidence Investigator™ system amount of diluted wash buffer (1 mL + 1 Investigator™. The captured images and The honeycomb samples, collected in the mL). After these simple steps, the sample results were processed by a dedicated soft- year 2018, were tested by Evidence was ready for application to the biochip. TM ware application, an integrated graphical Investigator™ Anti-Microbial Array II and IV The Evidence Investigator analyser does user interface and an e-touch software, in order to reveal the presence of antibiotics. not automatically evaluate the sample dilu- respectively. tion; therefore, the results were multiplied Principle by 20 to obtain the final concentration of the Sensitivity The Evidence Investigator™ Anti- sample. For the application of the Honey The limits of detection (LOD) for the Microbial Array II and The Evidence protocol, the calibrators and the control Evidence™ analytes for the honey matrix is Investigator™ Anti-Microbial Arrays IV are were reconstituted with the reconstitution shown in Table 1. commercially available kits for the simulta- buffer provided by the kit. neous quantitative detection of multiple Evaluation of antibiotic depletion groups of antibiotics. Anti Microbial Array Calibration and materials In order to verify antibiotics depletions II kit (AM II) is for the simultaneous detec- A nine-point calibration was performed in honey, further, where honeycomb samples tion of quinolones, ceftiofur, thiamphenicol, using the AM II and AM IV calibrators, were detected to be positive, the samples streptomycin, tylosin and tetracyclines. covering the measuring range of all tests. A were retested, after approximately 12 Anti Microbial Array IV kit (AM IV) is maximum of 6 carrier biochips can be anal- months, with Evidence Investigator™ AMII for the detection of groups of aminoglyco- ysed simultaneously and a new calibration and AMIV system and confirmatory testing side and macrolide antibiotics and specifi- curve is suggested for each series of tests. is implemented by LC-Orbitrap method as

[Italian Journal of Food Safety 2020; 9:8678] [page 15] Article described by Chiesa et al. (2018). During this centrifuge tube. HPLC-MS/MS analyses period, positive samples were stored at 35°C The obtained extracts were purified by The simultaneous detection of the in an incubator to simulate the temperature of SPE Oasis HLB cartridges under vacuum. antibiotics was performed by LC-Orbitrap. the beehive, until the time of analysis. The SPE cartridges were precondi- For the chromatographic separation a tioned with 3 mL methanol and 3 mL Milli- LC-HRMS Orbitrap were used Surveyor MS quaternary pump Q water. The samples were loaded, and then with a degasser, a Rheodyne valve with a Extraction and clean up washed with 2 × 3 mL methanol:water (5:95 20-μL loop and a Surveyor AS autosampler An aliquot (1 g wet weight) of honey, v/v). with a column oven (Thermo Fisher spiked with the IS at a final 5 ng mL−1, 100 Finally, the analytes were eluted with 5 Scientific, San Jose, CA, USA), equipped µl of 20% TCA for protein precipitation, mL methanol. The eluate was evaporated in with a Synergi Hydro-RP reverse-phase and 5 mL McIlvaine buffer (pH 4.0), were a rotary vacuum evaporator at 40 °C. HPLC column (150 × 2.0 mm, i. d. 4 µm), homogenized and were vortexed and soni- 200 µL methanol:water (10:90 v/v) with a C18 guard column (4 × 3.0 mm; cated for 15 min. After centrifugation (2500 were uses to reconstitute the dried extract, Phenomenex, Torrance, CA, USA). g, 4°C, 10 min), the supernatant was trans- and then transferred to an auto-sampler vial. The mobile phase was a binary mixture ferred to a clean polytetrafluoroethylene The injection volume was 10 µL. of aqueous formic acid 0.1% (solvent A)

Table 1. Detection limits for the Evidence Investigator™ AM IV and for the Evidence Investigator™ AM II. Randox AM IV Randox AM II Analyte Honey LOD (ppb) Analyte Honey LOD (ppb) Spiramycin (SPR) 2.0 Quinolones (QNL) 3.0 Apramycin (APA) 2.0 Ceftiofur (CEFT) 2.0 Bacitracin (BCT) 1.2 Thiamphenicol (TAF) 1.0 Neomycin (NEO) 1.0 Streptomycin (STR) 5.0 Tobramycin (TOB) 4.0 Tylosin (TYL) 1.0 Tylosin B (TYB) 1.0 Tetracycline (TCN) 5.0 Spectinomycin (SPT) 2.6 Amikacin (AMK) 6.0 Lincosamides (LIN) 8.0 Erytromycin ERY 2.5 Streptomycin (STR) 4.0 Virginiamycin (VIR) 2.0

Table 2. Mean (±sd) antibiotic concentration in samples of honeycomb. Toritto Conversano Castellana Grotte Monopoli Altamura Experimental Apiary (Na=6) (Na=6) (Na=6) (Na=6) (Na=6) (Na=20) Mean ± Mean ± Mean ± Mean ± Mean ± Mean ± SD (ppb) nb SD (ppb) nb SD (ppb) nb SD (ppb) nb SD (ppb) nb SD (ppb) nb QNL n.d. − n.d − n.d − n.d − 9.14±0.28 6 n.d − CEFT n.d − n.d − n.d − n.d − 7.1±1.77 6 n.d − TAF n.d − n.d − n.d − n.d − 1.08±0.88 4 n.d − STR n.d − n.d − n.d − n.d − n.d − 23.3±8.6 20 TYL n.d − n.d − n.d − n.d − n.d − n.d − TCN n.d − n.d − n.d − n.d − n.d − n.d − SPR n.d − n.d − n.d − n.d − n.d − n.d − APA n.d − n.d − n.d − n.d − 14.3±1.94 6 n.d − BCT n.d − n.d − n.d − n.d − 1.67±0.37 4 n.d − NEO n.d − n.d − n.d − n.d − 1.3±0.31 3 n.d − TOB n.d − n.d − n.d − n.d − 10.2±0.98 5 n.d − TYB n.d − n.d − n.d − n.d − n.d − n.d − SPT n.d − n.d − n.d − n.d − 5.1±2.7 6 n.d − AMK n.d − n.d − n.d − n.d − 23.9±1.3 6 n.d − LIN n.d − n.d − n.d − n.d − 30.6±0.96 6 n.d − ERY n.d − n.d − n.d − n.d − 7.3±0.73 6 n.d − VIR n.d − n.d − n.d − n.d − n.d − n.d − aTotal number samples; bTotal number of positive samples; n.d. not detected.

[page 16] [Italian Journal of Food Safety 2020; 9:8678] Article and methanol (solvent B). The run was per- accuracy, speed and ease of use (Popa et al., formed at 0.3 mL min−1 for a total of 25 2012). The results of the analysis performed Discussion and Conclusions min: started with 5% B, increased to 95% B with Evidence Investigator™ Anti- Food safety is a priority to be pursued in 10 min and remained constant up to the Microbial Arrays II and IV during the year all over the world and the presence of chem- 14th min; then, after an equilibration time 2018 showed the presence of antibiotic ical substances in food is a widespread of 8 min, the initial conditions were reached residues in 26 of the 50 honey samples anal- problem. Antimicrobial drugs in honey are a again at the 17th min. ysed. Twenty-four samples showed levels serious health risk as they can cause toxic, All analytes were detected with a not detectable by the Evidence allergic and hypersensitivity reactions Thermo Q-Exactive Plus Orbitrap (Thermo Investigator™ Anti-Microbial Arrays II and (Reybroeck, 2014); in addition, they can Scientific, San Jose, CA, USA), equipped IV and, therefore, were reported as N. D. promote the spread of resistant microbial with a heated electrospray ionization In relation to the geographical position strains (Wegener et al., 1999). Different source. Set in the positive (ESI+) mode, of the hives, only honey samples from the studies have shown the presence of antibi- except chloramphenicol, florfenicol and thi- hives located in Altamura and the experi- otics in European and non-EU honey amphenicol, which were detected in the mental apiary at Bari University contained (Saridaki-Papakonstadinou et al., 2006; negative (ESI-) mode. residues of antimicrobial drugs. Baggio et al., 2009; Barrasso et al., 2018) The instrumental analytical conditions Briefly, out of fifty honey samples anal- and, in some cases, mixing with Chinese and validation parameters were the same ysed, streptomycin was detected in only 20 honeys has been thought to be the reason for reported by Chiesa et al. (2018). samples of honey from the experimental the findings of streptomycin, tetracycline Detection of the analytes was based on apiary at the University of Bari. Other class- and chloramphenicol in European honey the calculated exact mass of the protonat- es of antibiotics were detected exclusively (Reybroeck, 2003). ed/deprotonated molecular ions and at least in honeys from Altamura. The results According to the scientific bibliogra- one specific and typical fragment. obtained are shown in detail in Table 2. phy, improper beekeeping practices are The exact mass of the compounds was The samples that contained antimicro- responsible for antibiotics in honey and in calculated using Xcalibur 3.0 software bial residues were retested in the year 2019 beehive products. Our research is therefore (Thermo Fisher Scientific). with the Evidence Investigator™ Anti- one of the first studies that relates the pres- Microbial Arrays II and IV. The results ence of chemotherapeutic residues to the showed the absence of any of the detectable environmental production context and high- antibiotic molecules in any honey samples lights the possible impact of agricultural Results (Table 3). In addition, simultaneous detec- activities on the health quality of honey. The Evidence Investigator™ system is tion of antibiotics was performed by LC- The locations of the hives are characterized an adequate analytical method for screening Orbitrap on the same honeycomb samples, by different agricultural practices. The hon- analyses for the detection of antibiotics in confirming the results obtained by the eys contaminated with antibiotics came honey, demonstrating good specificity and screening method adopted. from areas where animal husbandry is

Table 3. Comparison of the antibiotic concentrations detected in the year 2018 and in the year 2019. Altamura (Na=6) Experimental Apiary (Na=20) Analysis 2018 Analysis 2019 Analysis 2018 Analysis 2019 Mean ± SD nb Mean ± SD nb Mean ± nb Mean ± (ppb) (ppb) SD (ppb) SD (ppb) nb QNL 9.14±0.28 6 n.d − n.d − n.d − CEFT 7.1±1.77 6 n.d − n.d − n.d − TAF 1.08±0.88 4 n.d − n.d − n.d − STR n.d − n.d − 23.3±8.6 20 n.d − TYL n.d − n.d − n.d − n.d − TCN n.d − n.d − n.d − n.d − SPR n.d − n.d − n.d − n.d − APA 14.3±1.94 6 n.d − n.d − n.d − BCT 1.67±0.37 4 n.d − n.d − n.d − NEO 1.3±0.31 3 n.d − n.d − n.d − TOB 10.2±0.98 5 n.d − n.d − n.d − TYB n.d − n.d − n.d − n.d − SPT 5.1±2.7 6 n.d − n.d − n.d − AMK 23.9±1.3 6 n.d − n.d − n.d − LIN 30.6±0.96 6 n.d − n.d − n.d − ERY 7.3±0.73 6 n.d − n.d − n.d − VIR n.d − n.d − n.d − n.d − aTotal number samples; bTotal number of positive samples; n.d. not detected.

[Italian Journal of Food Safety 2020; 9:8678] [page 17] Article widespread. In the other geographical areas stances. However, the presence of residues activity but suspected to cause gastrointesti- assessed, livestock farming is uncommon. was not confirmed by tests carried out more nal disorders (Pariza, 2006). In agreement Different classes of antibiotics were than one year after sampling. The results of with previous studies carried out on honey detected in honeys from Altamura and the analysis conducted by Evidence (Anon., 2002; Kochansky 2004; Martel et indeed, according to the latest censuses Investigator™ Anti-Microbial Arrays II and al., 2006; Adams et al., 2009; Thompson & reported in the database of the National IV and LC-Orbitrap at a distance of months van den Heever, 2012), this study confirms Livestock Register, this geographical area showed the absence of antibiotics in honey- the degradation of antibiotic molecules over holds numerous livestock farms with vari- comb positive at the first screening assay. time, probably caused by the acidity of the ous species of animal. In honey samples The failure to detect any antibiotic sub- honey. In addition to the chemical degrada- from this area, some antibiotics were found stance in our samples could be caused by tion of antibiotics in honey, as reported by at high concentrations; in particular, the degradation of drugs in honey. Many Reybroeck et al. (2012) a factor influencing amikacin concentrations were in the range studies have shown the degradation of the presence of residues in honey at harvest of 22.48 to 26.15 ppb, while lincosamide antimicrobials in acid media such as honey time is the yield of the honey, i.e. the dilu- contents ranged between 29.45 and 31.65 (Kochansky, 2004; Thompson et al., 2007; tion effect. It is affected by weather condi- ppb. Both substances are widely used to Thompson and Van de Heveer, 2012). Some tions during flowering and by the geograph- treat bacterial disease and some protozoa in pharmacological compounds remain stable ical context of production. In fact, the high- cattle, sheep and pigs. The presence of in honey, while other compounds are modi- est concentrations of residues in honey are streptomycin in honeycomb from the exper- fied to yield a variety of by-products detected within one week of treatment imental apiary at the University of Bari (Thompson and Van de Heveer, 2012). (Reybroeck et al., 2012). Subsequently, the probably originates from local sheep farms, The stability of beta-lactams and concentration of residues in the honey where it is used to treat ovine mastitis. quinolones have been studied in muscles decreases by a dilution effect of honey flow The pharmacological substances inves- and frozen stock solutions. Quinolones or degradation of the active compound in tigated in our study are widely used in vet- were stable for about 3 months in chicken other metabolites (Reybroeck et al. 2012). erinary medicine which makes environmen- muscle kept at -20°C, however this result Considering that the degradation of tal contamination likely. Antibiotics are cannot be extended to honey (Bailac et al., antibiotics in our honey samples was evalu- administered in different ways in animal 2006). Unfortunately, few data on the sta- ated after a period of about 365 days after husbandry for therapeutic or preventive bility of beta-lactamic and quinolone antibi- the first screening analysis had been per- purposes, thus contributing to their spread otics and in honey are available in the liter- formed, the negative results may be attribut- in the environment (Chung et al., 2017). ature. However, concerning fluoro- ed to the above factors. Some drugs are eliminated via faeces, urine, quinolones, one study showed a reduction In summary, our study demonstrates the and spreading manure from treated animals in ciprofloxacin concentrations from values relationship between the presence of antibi- increases their distribution in the environ- of >10 mg kg-1 to 622-1370 µg kg-1 after 18 otics in honey and the related production ment (Broekaert et al., 2011). Several stud- weeks (Fussell et al., 2010). The study men- areas involved in intensive breeding, thus ies have shown that plants can absorb drugs tioned could support the results of our excluding improper beekeeping practices. from the soil, which then spread to different investigation, considering that the highest Moreover, our research confirms that bees plant compartments. The presence of some initial value of quinolones we found was and beehive products are excellent bio-indi- classes of antibiotics such as tetracyclines, 0.00965 µg kg-1, significantly lower than cators for environmental monitoring. sulfonamides, enrofloxacin, amoxicillin and the initial values reported by Fussell et al. Honey is a product that is greatly influenced tylosin in such plant products for human (2010), and the reduction in concentration by the environment and, consequently, its consumption as lettuce, carrots, tomatoes, was determined after a longer period. contamination might be caused by many vetch and maize has been documented Regarding thiamphenicol (TAP) and flor- factors. Our results show that the location of (Ahmed et al. , 2015). fenicol (FFC) in honey, no data are avail- apiaries in areas far away from husbandry The uptake of drugs and their distribu- able. Their stability has been assessed in activities reduces the risk of contaminants tion in various vegetable compartments is fish samples stored at -18°C in the absence in honey and therefore this approach may conditioned by many factors, such as the of light (Evaggelopoulou and Samanidou, be useful to beekeepers. The presence of species and physiological phase of the 2013); both antibiotics showed stability for contaminants in honey is constantly plant, soil characteristics, and the chemical two weeks. increasing due to environmental pollution composition and concentration of the drug The stability of streptomycin is consid- and new agricultural practices and repre- (Chung et al., 2017). In addition, environ- ered to be about four or five months in sents a serious risk to consumer health. As mental studies also show the presence of honey stored at room temperature (Pang et several studies have found different chemi- antibiotics in deep, waste, surface (Kolpin al., 2004; Anon, 2006), whereas for lin- cal contaminants in honey, consumers may et al., 2002) and drinking waters (Barnes et comycin, this period appears to be 28 days be exposed to several chemicals at a time. al., 2008). under the same temperature conditions. In conclusion, understanding the source Some drugs such as sulfanimide and However, Adams et al. (2009) showed that of pollution is important to preserve the tetracycline are administered in drinking after treatment with 1.2 g lincomycin quality of the product and to ensure its safe- water on farms, so water can become a hydrochloride per hive, antibiotic was per- ty; moreover, in relation to the high pres- source of pollution for hive products. The sistent in the hive 290 days after dosing. ence of antibiotic residues in honey, the bee explores territories even 6 km away Alippi et al. (1999) showed that a period of application of rapid, simple screening meth- from the hive in order to forage food and 35 to 40 days after administration of ery- ods for the detection of multiple analytes is water or take honey from other apiaries and thromycin is required to obtain residue lev- essential. by doing so they contaminate their own. els below the limit of detection (50 µg kg-1). Considering the lack of studies and the The results of the present study show In addition, under acidic conditions, ery- chemical complexity of honey, further anal- that honey fresh from the hive may contain thromycin degrades to form by-products yses will have to be performed and proper various residues of antimicrobial sub- (Volmer & Hui, 1998) with no antimicrobial monitoring plans and safety management

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