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General enquiries on this form should be made to:Defra, Procurements and Contracts Division (Science R&D Team)Telephone No. 0207 238 5734E-mail: [email protected]

SID 5 Research Project Final Report

SID 5 (Rev. 07/10) Page 1 of 7

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code VM02156

2. Project title

Investigation into the experimental protocols required to determine Maximum Residue Limits (MRLs) in honey: Preliminary study using organic acids as model compounds to propose ways in which withdrawal times might be calculated.

3. Contractororganisation(s)

The Food and Environment Agency (Fera)Sand HuttonYorkYO41 1LZ

54. Total Defra project costs £ 287,083(agreed fixed price)

5. Project: start date................ 01/05/2007

end date................. 31/03/2010


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

1. Background

Pollination by bees is essential for the sustainability of many sectors of agricultural production so maintaining the health of bee colonies is of great economic importance. Bee colonies are susceptible to a number of diseases including Varroa and Foulbrood but, worldwide, there are relatively few drugs legally permitted to control infestations. For example, whilst the United States has recently authorised the use of the antibiotic tylosin to treat American Foulbrood (AFB), the EU has yet to licence veterinary medicines for the treatment of beehives.

Consideration is being given within the EU, and more widely in international fora, to setting Maximum Residue Limits (MRLs) and withdrawal periods for veterinary medicines in honey production. The withdrawal period is the time after the last administration of the veterinary medicinal product during which the treated product/animal must not be taken for human consumption, thus ensuring that residues will not exceed the MRLs.

Whilst comprehensive guidelines exist for calculating the withdrawal periods of drugs in most of food producing species, the analytical variables to be studied have not been that well defined for bees/honey. Therefore research was needed to determine the most important factors that should be considered when withdrawal periods are to be calculated for honey.

In this three-year research project a labelled organic acid (13C2 oxalic acid) was to be used as a model compound to establish which external factors have an effect on the depletion of residues in honey. Labelled oxalic acid was employed so that the measured concentrations of this chemical could be differentiated from natural oxalic acid that is naturally present in honey. The initial project proposal included the following phases:

A. The development and validation of a suitable analytical method for the accurate quantification of 13C2 oxalic acid in honey.

B. Experimental Phase No.1: A statistically valid number of beehives (n=10) were to be dosed under controlled conditions. Samples of honey from both the dosed hives and two control (untreated) hives kept at one geographical location in the UK were to be analysed to determine the 13C2 oxalic acid concentrations in honey.


C. Statistical evaluation of the data from Phase 1: This evaluation was to (a) produce draft guidelines as to which experiments are required when setting reliable withdrawal periods in honey and (b) generate recommendations for Phase 2 of the project.

D. Experimental Phase No.2: The initial plan for Experimental Phase 2 was to conduct an additional (second–year) dosing with 13C2 oxalic acid at a different geographical location, but it was recognised that amendments to the study plan may be required after the assessment of Phase 1 data.

2. Method

2.1 Experimental Phase No.1

A method based on ion-chromatography-tandem mass spectrometry (IC-MS/MS) was successfully developed for the determination of low concentrations of labelled 13C2 oxalic acid in honey.

Ten individual hives were treated with 13C2 oxalic acid in the winter of 2007 and the plan was for samples of honey to be collected for ten weeks, following the start of the honey flow in the Spring of 2008.

Sampling of Phase 1 honey was restricted due to poor weather conditions (unseasonally cold and wet Spring in 2008). Only five of the treated hives produced any super honey flow during the first three weeks of sampling. Consequently there was insufficient data from Phase 1 for a full statistical analysis and the project officer at the Veterinary Medicines Directorate (VMD) agreed that this trial should be repeated in Experimental Phase No.2:

2.2 Experimental Phase No.2

Ten individual hives were again treated with 13C2 oxalic acid in January 2009. As a contingency, the same hives were treated with a second “backup” drug (the fluoroquinolone antibiotic, ciprofloxacin) in Spring 2009, directly before the honey flow. From May 2009, honey samples were taken from the hives at weekly intervals for ten weeks. Two further samples were also taken at the end of the summer and an “over-winter” sample in the Spring of 2010. Samples of honey were collected from the ten hives so that data on both the intra- and inter-hive variation in residue concentrations could be assessed.

3. Results and key findings

Oxalic acid (13C2-labelled), which was applied as a winter treatment, depleted rapidly and therefore did not prove to be a suitable ‘model compound’ for other treatments. The high level of censoring in the data (i.e. the amount of data less than the limit of detection of 10 µg/kg) meant that standard statistical models could not be fitted to these data and this information was therefore of limited value.

By contrast ciprofloxacin which was applied as a Spring treatment did produce measurable residues for all time points. Ciprofloxacin was detected in the over-winter sample, 283, days after treatment which indicates that the parent compound is a suitable marker for the detection of (ab)use of ciprofloxacin in apiculture.

Because of the large inter-hive variability of residues of ciprofloxacin, and the fact that residue concentrations increased slightly in some hives near the end of the study, it was not possible to calculate a robust withdrawal period for ciprofloxacin at theoretical (i.e.’ arbitrary’) MRLs between 25 and 500 µg/kg. For a veterinary medicine with less persistent residues it may however be possible to calculate a more robust withdrawal time using a suitable statistical model.

The observed large inter-hive variation in concentrations of residues of ciprofloxacin, not unexpected in a biological system, indicates that the validity of the current EU guidelines for bees/honey, which requires five samples from five hives to calculate a withdrawal period, is questionable and may require revision. The results of this study indicate that increasing the number of hives is more influential in decreasing the relative standard uncertainty in the statistical assessment than simply the increasing the number of samples taken per hive.


4. Recommendations

Although the results of the current study provide valuable information, this investigation was undertaken using only two model compounds during one season at one geographical location. Ideally, further studies need to be conducted to assess other potential variables such as; the effects of the use of other model compounds with different application regimes, different geographic locations, different climatic/seasonal variables, different bee husbandry practices and different sampling strategies.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

The scientific objectives as set out in the contract, and comments on delivery/issues are presented in the Table below. The initial objectives are shown in black text. Revisions to milestones and objective that were agreed with Defra VMD in November 2008 are shown in red.

OriginalTarget date

AdjustedTargetDate

Scientific objective Comments

1 30/10/2007 Finalise Study Plan Milestone met in full2 30/11/2007 Validated analytical method

for the quantification oforganic acids in honey

Milestone met in full

3 30/09/2008 Statistical assessment andreport of data from Phase 1and Proposals for Phase 2 ofstudy

Bees adversely affected bypoor weather - Insufficientdata for statistical analysis proposals for Phase 2incorporated into SID 4interim report

4 31/10/2008 Stop/Go decision (If project isto continue a revised shouldbe available

The consensus at the jointFera/VMD meeting held on08/07/2008 was that theoxalic acid treatmentshould be repeated in phase2 but with a dual treatment approach. e.g. to also treat


with an antibiotic

Revised milestones agreed with Defra (shown in red)

5 30/09/2009 31/02/2010 Completion of Phase 2experiments.


6 31/03/2010 30/03/2010 Statistical assessment of theoverall results and productionof final project report in SID5 format.

Replaced by milestone 11.

7 New forPhase 2

30/12/2008 To reduce the LoD/LoQ for quantification of oxalic acid in honey.


8 New forPhase 2

30/04/2009 To validate LC-MS/MS method for the target antibiotic in honey.


9 New forPhase 2

31/01/2010 Investigation (treatment,sampling, and analysis) of oxalic acid


10 New forPhase 2

31/01/2010 Investigation (treatment,sampling and analysis) of the the target antibiotic


11 New forPhase 2

30/03/2010 Statistical assessment of oxalic acid and antibioticresults and produce report inSID 5 format.

Milestone met, but submission of final report was delayed.

The full scientific report, in pdf format, can be found here [double icon click to open]

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

A lecture on this project was given at the Sixth International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent in June 2010 (see http://www.vdra.ugent.be/ ). A copy of the presentation can be found below:

In additions, a scientific publication titled, Investigation into the Experimental Protocols Required to Determine Maximum Residue Limits (MRLs) in Honey, was submitted to the conference journal.


http://www.vdra.ugent.be/


Documents

General enquiries on this form should be made to: - GOV.UKrandd.defra.gov.uk/Document.aspx?Document=10667_Fera_VM... · Web view3. Contractor organisation(s) The Food and Environment