Outcome of the peer review of bee study protocols submitted by Bayer CropScience AG to assess the effects of imidacloprid on bees 1

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1 EFSA supporting publication 2014:EN-601 TECHNICAL REPORT Outcome of the peer review of bee study protocols submitted by Bayer CropScience AG to assess the effects of imidacloprid on bees 1 European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The European Food Safety Authority (EFSA) was asked by the European Commission to provide scientific and technical assistance to perform a peer review of the study protocols as submitted by the applicants until 28 February 2014 to assess the effects of clothianidin, thiamethoxam, imidacloprid and fipronil on bees. The context of the evaluation was that required by the European Commission in accordance with Article 31 of Regulation (EC) No 178/2002. The current report summarises the outcome of the consultation process with Member State experts organised by EFSA on the study protocols submitted by Bayer CropScience AG on imidacloprid and will serve as recommendations for consideration by the applicant in the upcoming postapproval procedure for this active substance. European Food Safety Authority, 2014 KEY WORDS imidacloprid, bee study protocol, peer review, consultation, post-approval, confirmatory data 1 On request from the European Commission, Question No EFSA-Q , approved on 20 May Correspondence: pesticides.peerreview@efsa.europa.eu Suggested citation: European Food Safety Authority, 2014; Outcome of the peer review of bee study protocols submitted by Bayer CropScience AG to assess the effects of imidacloprid on bees. EFSA supporting publication 2014:EN pp. Available online: European Food Safety Authority, 2014

2 SUMMARY Imidacloprid was included in Annex I to Directive 91/414/EEC on 1 August 2009 by Commission Directive 2008/116/EC, and has been deemed to be approved under Regulation (EC) No 1107/2009, in accordance with Commission Implementing Regulation (EU) No 540/2011, as amended by Commission Implementing Regulation (EU) No 541/2011 and Commission Implementing Regulation (EU) No 485/2013. It was a specific provision of Commission Implementing Regulation (EU) No 485/2013 that the applicant is required to submit by 31 December 2014 further confirmatory information as regards the risk to bees. In February 2014 EFSA received a request from the European Commission to conduct a peer review of the study protocols submitted by the applicants until 28 February 2014 to assess the effects of clothianidin, thiamethoxam, imidacloprid and fipronil on bees in the context of the relevant regulatory process following approval of these substances. This peer review aims to support EFSA and Member States in assessing the quality and consistency of the protocols submitted by Bayer CropScience AG on imidacloprid in the context of the upcoming confirmatory data assessment. The current report summarises the outcome of the consultation process with Member State experts organised by EFSA on the submitted study protocols and will be considered by the relevant rapporteur Member State in the preparation of their list of recommendations that will be addressed to the applicant in support of the upcoming post-approval procedure. EFSA supporting publication 2014:EN-601 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Commission... 4 Terms of reference as provided by the European Commission... 5 Evaluation... 6 Documentation provided to EFSA... 7 References... 7 Appendix... 8 Abbreviations EFSA supporting publication 2014:EN-601 3

4 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Imidacloprid was included in Annex I to Directive 91/414/EEC 3 on 1 August 2009 by Commission Directive 2008/116/EC 4, and has been deemed to be approved under Regulation (EC) No 1107/2009 5, in accordance with Commission Implementing Regulation (EU) No 540/2011 6, as amended by Commission Implementing Regulation (EU) No 541/ and Commission Implementing Regulation (EU) No 485/ The peer review leading to the approval of this active substance was finalised on 29 May 2008, as set out in the EFSA Scientific Report (2008) 148 (EFSA, 2008). In addition, a specific Conclusion was issued on 19 December 2012 concerning the risk assessment for bees (EFSA, 2013). It was a specific provision of Commission Implementing Regulation (EU) No 485/2013 that the applicant is required to submit by 31 December 2014 further confirmatory information as regards the risk to bees. To ensure common understanding and harmonisation of the assessments between the different Member States and EFSA in the framework of the upcoming post-approval regulatory procedures related to the three neonicotinoids (clothianidin, imidacloprid, thiamethoxam) and fipronil, the European Commission requested EFSA to organise a peer review of bee study protocols related to these substances. The agreed deadline for submission of bee study protocols by the applicants was 28 February A consultation on the study protocols submitted by Bayer CropScience AG on imidacloprid was conducted with Member States and EFSA via a written procedure in March The comments received were collated by EFSA and forwarded to the relevant RMS (DE) for compilation and evaluation in the format of a Reporting Table. The applicant was invited to respond to the comments in column 3 of the Reporting Table. The comments and the applicant s response were considered by the RMS in column 3. A specialised peer review meeting with Member State experts took place in April The outcome of the expert discussions was recorded in column 4 of the Reporting Table. A final consultation on the conclusions arising from the peer review on the study protocols took place with Member States via a written procedure in May The current report summarises the outcome of the consultation process with Member State experts organised by EFSA on the submitted study protocols and will be considered by the relevant rapporteur Member State in the preparation of their list of recommendations that will be addressed to the applicant in support of the upcoming post-approval procedure. 3 Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230, , p. 1-32, as last amended. 4 Commission Directive 2008/116/EC of 15 December 2008 amending Council Directive 91/414/EEC to include aclonifen, imidacloprid and metazachlor as active substances OJ L 337, , p Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, , p Commission Implementing Regulation (EU) No 540/2011 of 25 May 2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 541/2011 of 1 June 2011 amending Implementing Regulation (EU) No 540/2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 485/2013 of 24 May 2013 amending Implementing Regulation (EU) No 540/2011, as regards the conditions of approval of the active substances clothianidin, thiamethoxam and imidacloprid, and prohibiting the use and sale of seeds treated with plant protection products containing those active substances. OJ L 139, , p EFSA supporting publication 2014:EN-601 4

5 TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION On 28 February 2014 EFSA received a request from the European Commission to perform a peer review of the study protocols submitted by the applicants until 28 February 2014 to assess the effects of clothianidin, thiamethoxam, imidacloprid and fipronil on bees in the context of the relevant regulatory process following approval of these substances. In performing this task EFSA followed the procedure and timelines agreed at the Pesticides Steering Committee meeting dedicated on the risk assessment for bees (17-18 December 2013) and organised a specialised peer review meeting with Member State experts. The outcome of the expert discussion on the study protocols is recorded in the Reporting Table which forms part of the current Technical Report. The deadline for the delivery of the Technical Report on the evaluation of the bee study protocols is 21 May The report is also made available to the relevant rapporteur Member State. EFSA supporting publication 2014:EN-601 5

6 EVALUATION The comments received on the study protocols submitted by Bayer CropScience AG on imidacloprid together with the applicant s reaction to the comments as well as the conclusions and recommendations following the consultation with Member State experts and the subsequent written procedure are presented in the format of a Reporting Table. The comments received during the consultation process are summarised in column 2 of the Reporting Table. The applicant s reaction to the comments together with the RMS considerations are provided in column 3, while the final outcome and recommendations from the experts meeting are outlined in column 4 of the table. The finalised Reporting Table is provided in the Appendix of this report. EFSA supporting publication 2014:EN-601 6

7 DOCUMENTATION PROVIDED TO EFSA Outcome of the peer review of bee study protocols submitted by Bayer on imidacloprid Bee study protocols on imidacloprid submitted in support of the upcoming confirmatory data assessment. February Submitted by Bayer CropScience AG. REFERENCES Campillo N., Viñas P., Férez-Melgarejo G. & Hernández-Córdoba M Liquid Chromatography with Diode Array Detection and Tandem Mass Spectrometry for the Determination of Neonicotinoid Insecticides in Honey Samples Using Dispersive Liquid Liquid Microextraction. J. Agric. Food Chem., 2013, 61 (20), pp DOI: /jf400669b EFSA (European Food Safety Authority), Conclusion regarding the peer review of the pesticide risk assessment of the active substance imidacloprid, finalised on 29 May EFSA Scientific report (2008) 148, doi: /j.efsa r. Available online: EFSA (European Food Safety Authority), Conclusion on the peer review of the pesticide risk assessment for bees for the active substance imidacloprid. EFSA Journal 2013;11(1): pp. doi: /j.efsa Available online: Kamel A Refined Methodology for the Determination of Neonicotinoid Pesticides and Their Metabolites in Honey Bees and Bee Products by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). J. Agric. Food Chem., 2010, 58 (10), pp DOI: /jf904120n Whitehorn P. R., O Connor S., Wackers F. L. & Goulson D Neonicotinoid Pesticide Reduces Bumble Bee Colony Growth and Queen Production. Science (2012). DOI: /science EFSA supporting publication 2014:EN-601 7

8 APPENDIX REPORTING TABLE: COLLATION OF COMMENTS FROM MEMBER STATES AND EFSA ON THE BEE STUDY PROTOCOLS SUBMITTED BY BAYER CROPSCIENCE AG ON IMIDACLOPRID AND THE CONCLUSIONS AND RECOMMENDATIONS FOLLOWING THE EXPERT CONSULTATION WITH MEMBER STATE EXPERTS Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(1) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 1(2) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 1(3) Residues of imidacloprid in Western Germany, GLP Study No. P EFSA: Generally, it is considered that the study/studies could deliver valuable information for the subsequent risk assessments. Please note however that extrapolation to other crops is not possible without additional data/argumentation since the systemic translocation is likely species dependent (not only a.s. dependent). It might be considered to involve those types of attractive crops in the study those are likely grown after the treated crop. EFSA: It is noted that this study protocol is an example of several similar studies. It is indeed suggested that if residues are taken, than a number of different fields should be investigated (the representativeness of a single/few field(s) is difficult to evaluate). EFSA: It is very difficult to follow what is going on as the Amendment (1st Study Plan Amendment) is not in line with the study plan. The references to the chapters (and the referred page numbers) do not match. In BCS: considering the available timeline focus is to ensure that a range of flowering crops in 2014 can be sampled for residue measurements. Data will be put into context of results from other studies (e.g. oilseed rape). DE: BCS comment accepted and EFSA comment is regarded as highly valuable. BCS: noted DE: noted and see comment 1(30) It should be noted that extrapolation of residue from one crop to another crop is not possible without additional data/argumentation since the systemic translocation is likely species dependent (not only a.s. dependent). See also comment in 1(32). Noted BCS: EFSA is correct that the study started in It is noted that the document 2013 and the provided study outline is not the submitted for this peer-review study plan. was not the study plan. The study outline was compiled to provide a concise overview about the overall set-up of EFSA supporting publication 2014:EN-601 8

9 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) BCS Activity ID: EBTIN110 1(4) P (succeeding crop) 1 st amendment some cases the information that had been changed in the Amendment was not presented in the Study plan (called Study outline). Also, in several cases the new information is already included in the study plan (it is unlikely that it can work under GLP). No dates of issues of the documents are indicated (neither signatures are available), however the study must already be running. Should the sponsor not be indicated somewhere? It cannot be worked out from the documents whether the sampling will be done in spring or summer EFSA has the feeling that only an early draft was given for this peer review for this already running study for which the final version of protocol (and amendments) should be available. Or is this discrepancy originating from what is called Study outline is not the Study plan? EFSA is unsure on that. FR: The 1 st amendment is unclear (numbering and pages do not correspond to the study outline). 1(5) Subsection 3.1., p.5ff AT: Why do you plan the application of a product containing a mixture of imidacloprid and clothianidin? Is this product currently used? The presence of one substance might this study which is in a similar way also performed by another contractor in France. Since the purpose of the review process is to comment on activities which are still to be performed in 2014, the draft amendment is the document to focus on. This amendment describes the envisaged sampling of bee relevant matrices which will be applied in a range of studies in DE: addressed BCS: see above DE: addressed BCS: We have no indication that uptake may be influenced. A mixture of Imidacloprid and Clothianidin was applied to keep the study set-up within feasible dimensions. Moreover Addressed See note in 1(3). Point for Expert discussion Experts to discuss the test items to be used and the EFSA supporting publication 2014:EN-601 9

10 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) alter the uptake of the other and may not reflect the worst-case residue level in nectar and pollen regarding one substance. It has to be justified that the products, rates and crops used in the study do cover the worst case situation(s) with respect to potential residues in pollen and nectar of rotational crops for all products in use. it needs to be considered that Imidacloprid and Clothianidin can be used in rotations and indeed we considered worst-case assumptions in our calculation of plateau concentrations. DE: addressed. The concerns expressed by AT contribute to the uncertainty and should be carefully considered in interpreting the results of the study regime for soil residue analysis. See also comments in 1(8), 1(9), 1(10), 1(11), 1(27), 1(37). See additional comments in 1(7), 1(35) and 1(36). Pesticides Peer Review Meeting on the review of bee study protocols (23 24 April 2014) The experts agreed that the applicant should document (supported with data) whether the mixture of imidacloprid and clothianidin may result in a different root uptake for each individual substance. Applicant will provide historical data on pesticide uses. In addition, it is recommended to have soil background concentrations of the neonicotinoids. The experts agreed that the residue analysis should be performed at least EFSA supporting publication 2014:EN

11 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) for thiamethoxam, imidacloprid, clothianidin and their pertinent metabolites. 1(6) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 Point (7) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 Point 4.2. Table 1 EFSA: How it will be achieved that the colonies will be disease-free? EFSA opinion on bees suggests that the level of at least the most important diseases (e.g. most frequently observed in the region) and Varroa should be controlled. EFSA: It is noted that in variant blue, plateau PECsoil of ca mg/kg; and in variant green, plateau PECsoil of ca mg/kg will be simulated (assuming a mixing depth of 20 cm). EFSA assumes that the seed dressing rates for the barley are meant to cover realistic worst case situations for the EU (or at least for the region where the test is conducted). Can this be confirmed? BCS: the bee colonies used in these studies are managed by professional beekeepers who apply state of the art disease control. Moreover honeybees are only used in these studies as a sampling device for nectar and pollen. DE: addressed BCS: BCS confirms that worst-case situations have been considered. DE: addressed. See comment no. 1(35) Regarding the depth of soil layers, the experts proposed that soil analyses should cover the root exposure profile EFSA assumes that some information on this issue will be available in the final report. Addressed See also note in 1(35). EFSA supporting publication 2014:EN

12 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(8) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point (9) P (succeeding crop) point (10) Imidacloprid rotational crop study, soil sampling 1(11) Imidacloprid rotational crop study, soil sampling EFSA: Historical records on the pesticide use, especially for neonicotinoids (i.e. imidacloprid) and /or residue analysis of the soil before the application would be helpful to understand the background residue level of the soil. Would a soil analysis be carried out for soil characterisation (organic matter, ph, texture)? FR: Soil core sampling before application and at the time of pollen and/or nectar sampling are recommended. DE (UBA): Soil samples should be taken before the first application to obtain information on possible background concentrations and the history of pesticide use at the location should be documented. DE (UBA): In the experiments with succeeding crops, the soil samples should cover not just the 0-15 cm layer, but also deeper soil layers (e.g. to a depth of 90 cm). As leaching of the active substance into lower layers cannot be ruled out, the potential rootavailable occurrence of residues in lower layers should be considered. BCS: Historical records will be compiled and soil characterization will be conducted. Soil samples were taken and an analysis will be made to determine the soil residues after plateau incorporation has been established. These data include all potential Imidacloprid and Clothianidin soil residues from previous treatments. Further soil samples for subsequent residue analysis will be performed in DE: addressed BCS: see above DE: addressed BCS: see above DE: addressed BCS: Any potential transport processes are integratively considered as part of the study. DE: The transport processes have to be described based on chemical analyses. See point for expert discussion 1(5). Note: thiamethoxam should be added to the analytics. See also comment in 1(37). See point for expert discussion 1(5). See point for expert discussion 1(5). See point for expert discussion 1(5). 1(12) Residues of imidacloprid EFSA: Residue trials in open field conditions DE: noted Point for Expert discussion EFSA supporting publication 2014:EN

13 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point 4.3 (where normal size colonies can be used for nectar/pollen collection) probably result in more realistic results. In terms of potential dilution, it is acknowledged that the tunnel design is a worst case. However please see next comment just below. Experts to discuss the set up of the study and the sampling regime for residue analysis for pollen and nectar. See also comments in 1(13), 1(14), 1(15), 1(16), 1(17), 1(20), 1(21), 1(22), 1(23), 1(29), 1(31). Pesticides Peer Review Meeting on the review of bee study protocols (23 24 April 2014) The experts agreed that the sampling procedures should be more detailed. The experts recommended triplicate samples at each of the 3 sampling dates (start of the flowering, middle and end of the flowering). Each sample is a pool of at least 20 plants. The triplicate samples should be analysed separately. The experts agreed that bees EFSA supporting publication 2014:EN

14 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) returning to the hive should be collected at the entrance of the hive. A pollen trap could be an option, but collection of pollen foragers is preferred. Foragers with a medium/large nectar load in the crop should be preferably used for residue analysis. It was highlighted that foragers with small load should not be used because the residue concentration might be low (e.g. no foraging activity, just left the hive). Size of colonies The experts noted that bees per sampling time might be unrealistic considering a colony of bees. The applicant indicated that the value of bees per sampling time is a maximum value. The experts noted that considering the size of the tunnel, larger colonies could be used. Although it is not an effect study, the experts considered EFSA supporting publication 2014:EN

15 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) that any observed behavioural and mortality effect could be recorded as useful additional information. The experts consider that the LOQ/LOD should be as low as possible. Literature data (Kamel 2010; Campillo et al. 2013) are available showing that the analytical precision for residues in bee products could be better than the analytical precision currently available in the dossiers. RMS clarified that even if nectar is frozen and thawed; the accuracy of the measurements is not affected. Regarding maize pollen sampling the experts proposed to take at least 5 samples of maize pollen in the flowering period, if drying of pollen is necessary. If not, 3 samples are considered sufficient. In general, the stability of EFSA supporting publication 2014:EN

16 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) residue from field to lab should be documented e.g. by analytical measurements showing the stability of the residue in the samples in different conditions (e.g. temp. light etc.). 1(13) Subsection 4.3., p.11ff AT: Generally more details would be helpful because evaluators are not familiar with this methodology. Is the target amount of nectar and pollen considered and analysed as one sample? Is there any replication planned? BCS: A detailed description of the methodology will be provided with the final report. Replication is considered for the sampling and analysis. DE: addressed The experts agreed the semifield design seems to be appropriate for residue measurement. See point for expert discussion 1(12). Note: The number of repetitions should be known. EFSA assumes that samples taken from one tunnel at one time is the basis for the repetitions. EFSA supporting publication 2014:EN

17 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(14) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point EFSA: Colonies are planned to be well fed and feed during the sample collection period. This may reduce the foraging activity. Moreover pending on the timing of the feeding this may lead to dilution, which should be avoided. In order to maximise the foraging activity thus the residue collection, the stored reserves should be kept minimal and it should be ensured that sufficient proportion of larvae is available in the hive which need feeding. It is however acknowledged that the colony collapse should also be avoided (at least till the end of the sampling period). It is also acknowledged that the high foraging activity is essential rather in effect studies which are not the case here. If good and realistic samplings are still possible with considerable food reserves in the hive and feeding than these might not influence significantly the main goals of the study. However please see next comment just below. BCS: see above DE: addressed. It is unlikely that food stores in the hive will reduce the nectar and pollen foraging intensity and a certain amount of food stored is needed for survival of the colonies. The stores should be kept minimal but as much as needed for survival. High foraging activity is ensured by the choice of phacelia and mustard as highly bee attractive crops. See point for expert discussion 1(12). EFSA supporting publication 2014:EN

18 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(15) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point (16) P (succeeding crop) point nectar sampling EFSA: Bee sampling from the crop might be avoided and the focus should be on sampling at the hive entrance, including exclusively entering bees and excluding leavers or guards. This is because in the crop plot bees just arrived and started to forage might be collected in high proportion. The crop content of these bees might contain the food from the hive which is uncontaminated (especially if the colonies are well fed and feed). This sampling regime might work properly in open field conditions, but EFSA has reservations for semi-field conditions (e.g. due to the very small distances). Also, to avoid this potential error, it might help to select only those bees for the analysis, which had a significant amount of nectar in the honey stomach and leave out those with a minimal amount. FR: It is wondered whether the bee colonies ( bees in point 4.3.4) will be sufficiently large to allow the sampling of a sufficient number of foragers for honey stomach collection. BCS: Honeybees for nectar and pollen sampling will generally be collected in front of the hive when returning. DE: addressed. BCS: we will take this into account by making sure that sufficient capped brood is available to compensate. DE: Noted. As it takes some time to develop from hatching bee to a forager bee, it seems necessary to reconsider the amount of bees sampled on each sampling date. See 1 (17) See point for expert discussion 1(12). See point for expert discussion 1(12). EFSA supporting publication 2014:EN

19 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(17) Imidacloprid rotational crop study, bee sampling 1(18) P (succeeding crop) point pollen sampling 1(19) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point DE (JKI): Flight and foraging activity, behavioural impairments and mortality in front of the hives (lines sheets) should be recorded once a day during the sampling period. Sufficent food stores should be provided before the sampling period. Amount of 800 / 1000 bees at each sampling event seems too high- there will soon be no more foragers left in the hive when colonies with bees are used. Could it be checked that this number is the one intended, or is it maybe foragers per sampling? FR: It should be discussed which of the bee pollen or pollen from traps will be preferred. EFSA: Persons collecting the guttation samples need to be familiar with distinguishing guttation droplets from other fluids (dew, rain droplets, etc). It is noted that it is not planned to force the maize plant to guttate (i.e. by irrigation). Guttation will or will not happen pending on the abiotic circumstances (meteorology, soil type). Therefore there is no guarantee that the worst case situation will be covered. Also, please note that the concentration in the guttation droplets may change during the day and the attractiveness of the droplets is BCS: bees are the absolute maximum number; please see further comments above. DE: noted, see also 1 (16) See point for expert discussion 1(12). DE: noted. It is mentioned in point that the pollen trap is the preferred method. EFSA considers that the difference between the two is negligible. DE: agree. In case of guttation, to distinguish guttation droplets from other fluids, only drops from the leaf edge should be collected. Point for Expert discussion Experts to discuss whether the guttation is sufficiently addressed. Pesticides Peer Review Meeting on the review of bee study protocols (23 24 April 2014) In order to measure concentrations in guttation EFSA supporting publication 2014:EN

20 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) dependent on the distance from permanent water bodies. These should also be considered. Maybe a kind of analyses for the link between the abiotic parameters and the guttation/residue levels in guttation droplets could be done, once the data are available. fluid, it has to be ensured that guttation occurs. It was considered whether to recommend irrigating the maize field to maximise the phenomenon. However, the experts did not consider this necessary because guttation will occur unless the environmental conditions are extremely dry. According to the standard procedures for such studies, sampling is done in the morning and variation on residue concentration in guttation droplets during the day is not considered. This could be considered as an uncertainty in the risk assessment. The experts noted that it could be technically difficult to collect guttation droplets in the afternoon. A way to address this potential uncertainty might be to collect different samples i.e. early and late morning. EFSA supporting publication 2014:EN

21 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) Seeds should be analysed to confirm the proper seed loading against the residue levels in guttation. 1(20) Imidacloprid DE (UBA): A crucial issue in assessing the rotational crop study, exposure of active substances in pollen and analytical phase nectar is the precision of the measurements. However, neither the limit of detection (LOD) nor the limit of quantification (LOQ) for the chemical analysis is indicated. For the discussion of the study results in the context of chronic effects we consider it important that the reported residue concentrations are low enough to be comparable with chronic NOEC values. DE: LOD and LOQ should be provided. This point is not addressed by BCS. Major open point for discussion. Whitehorn et al. (2012) found a 85% reduction in production of new queens when colonies were fed sugar water containing 0.7 ppb imidacloprid. Therefore, a LOQ << 0.7 ppb is seen to be necessary in order to be able to rule out significant effects. The following articles sound promising, that it might be feasible to lower the previous standard LOQ of 1 ppb: Kamel J. Agric. Food Chem., 2010, 58 (10), pp Campillo et al J. Agric. Food Chem., 2013, 61 (20), pp The experts agreed that maize is a worst-case crop for guttation on the basis of the current knowledge. However, different abiotic conditions across the EU should be covered by additional studies. See point for expert discussion in 1(12). EFSA supporting publication 2014:EN

22 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(21) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point 4 1(22) Imidacloprid rotational crop study, maize pollen sampling 1(23) P (succeeding crop) point 6 EFSA: Do we understand well that the nectars will be frozen and thawed twice until the analytical measurements? Will this have impact on the accuracy of the measurements? DE (JKI): Maize pollen sampling: History of each individual pollen sample should be stated in the report (if sample was collected in dry conditions or wet conditions and if drying was necessary). It is also recommended to increase the number of samples. It is proposed to take at least 5 samples of maize pollen in the flowering period. FR: The stability of the residue from field to the analytical laboratory should be documented. BCS: technically this cannot be avoided when using bees for sampling. BCS will take maximum care to avoid unnecessary processing steps. DE: addressed BCS: noted DE: noted BCS: BCS will take care to store and process samples appropriately. DE: addressed See point for expert discussion in 1(12). See point for expert discussion in 1(12). See point for expert discussion in 1(12). 1(24) Residues of imidacloprid in Western Germany, GLP Study No. P BCS Activity ID: EBTIN110 point 6 EFSA: It would be very nice if the analytical report could administratively absolutely clearly be linked with the report of the field phase (i.e. all the study identifications would be apparent in both reports). BCS: BCS will make sure that reports will be well linked. DE: addressed Applicant indicated he will ensure that reports will be well linked. EFSA supporting publication 2014:EN

23 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(25) Residues of imidacloprid in Western Germany 1(26) Residues of imidacloprid in Western Germany 1(27) Residues of imidacloprid in Western Germany 1(28) Residues of imidacloprid in Western Germany 1(29) Residues of imidacloprid in Western Germany UK: It is unclear whether it is barley seeds or barley plants that will be incorporated. Tables and and the text under 4.2 imply that seeds are to be incorporated but the text under section implies that it is plants grown from treated seed. This should be made clearer in the methodology. UK: The spray application was incorporated to 20cm; however, the UK considers that PEC plateau should be considered over 5cm, rather than 20cm, especially for arable crops. UK: It is unclear if the soil sample cores are to be mixed up when stored or kept in the core formation. It would be beneficial to measure the concentrations in the core at the different depths across the core sample. UK: It would be beneficial to also measure sugar content of the nectar and protein content of the pollen. BCS: will be explained appropriately in the report. DE: addressed UK: It is recommended to sample pollen and nectar in triplicate, in order to verify the certainty of the sampling method. It is unclear from the methodology if samples are DE: addressed to be kept separate or pooled for analysis. However, it is recommended to follow the guidance in this respect. BCS: we consider 20 cm incorporation to reflect realistic worst case conditions. DE: addressed. See comment no. 1(35) Applicant indicated that the issue will be properly explained in the final report. The approach for 20 cm is considered appropriate for arable crops. BCS: BCS considers a differentiation See point for expert discussion particularly after incorporation not be in 1(5). relevant for the purpose of the study. DE: Measurements in different depths are preferred. DE: Sugar and protein content are regarded as useful additional information. BCS: replication is part of the study design (3 tunnels per sampling time). Noted See also comment in 3(3). See point for expert discussion in 1(12) EFSA supporting publication 2014:EN

24 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(30) Residues of imidacloprid in Western Germany UK: Only one site is proposed to be used, whereas the guidance suggests at least 5 sites for determining the 90 th percentile residue. How is it going to be ensured that the 90 th percentile exposure level will be covered? BCS: more than one site is considered in the total study package. DE: it is still unclear whether at least 5 sites will be used. Minimum statistical requirements should be fulfilled. Noted The recommendations for 5 sites for determining the 90 th percentile residue is included in the bee Guidance Document (not in the opinion) and it refers to the regime for exposure field studies to establish the overall 90 th percentile worst-case residue level for the area of use (such as the whole EU in this case). The exposure levels of this study will be considered in the follow-up risk assessment. This study alone will not be considered to represent a realistic worst-case situation for the area of use unless it is demonstrated by using also additional data. For this, the applicant may consider to conduct separate exposure field studies or use data from existing studies (after critical review). EFSA supporting publication 2014:EN

25 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(31) Residues of imidacloprid in Western Germany 1(32) Residues of imidacloprid in Western Germany 1(33) Residues of imidacloprid in Western Germany UK: It is noted that the exposure study is planned to be carried out in tunnels, rather than in open fields. The exposure to bees in tunnels would be expected to be worst case; therefore it is questioned how the 90 th percentile exposure level determined from the exposure study would then be achieved in the effects study (as the exposure study sets the benchmark for the effects study), if this is planned for the future? UK: The crops being grown for pollen and nectar sample (i.e. mustard and Phacelia) are not expected to be the major crops to be grown as following crops, therefore, how is it going to be demonstrated that these surrogate crops are sufficiently worst case to cover the exposure resulting from growing other crops? This is in relation to the translocation of the active in the crop, rather than the attractiveness of the crop (it is accepted that mustard and Phacelia are attractive to bees). UK: Maize is known to demonstrate the phenomenon of guttation well; however, how is it going to be ensured that maize covers the worst case of guttation of all crops that may be grown as succeeding crops? DE: We do not see any reason why not to compare this exposure level to effect levels, because dominating monocultures can occur in agricultural practice. BCS: see above DE: It is assumed that Mustard and Phacelia are worst case crops for the intended scenarios to be covered, due to the short time between planting and flowering and the attractiveness of the crops for nectar and pollen Three crops give at least an indication of the inter-crop variation. BCS: data will be put into context of available studies on a range of crops. DE: addressed See point for expert discussion 1(12). It should be noted that extrapolation of residue from one crop to another crop is not possible without additional data/argumentation since the systemic translocation is likely species dependent (not only a.s. dependent). See also comment in 1(1). Applicant indicated that additional data will also be considered in the risk assessment. EFSA supporting publication 2014:EN

26 Residues of imidacloprid in Western Germany (plus amendment to cover guttation), Study-No.: P Reference to study Comments from Member States /EFSA Evaluation by (RMS) rapporteur and Conclusion protocol (e.g. point, page) 1(34) Residues of imidacloprid in Western Germany 1(35) Residues of imidacloprid in Western Germany 1(36) Residues of imidacloprid in Western Germany 1(37) Residues of imidacloprid in Western Germany UK: Section refers to pollen sampling and drying of the pollen to avoid the pollen becoming mouldy. Should the pollen not be frozen, similar to the other sampled matrices, in order to avoid both mould and residue deterioration? UK: It would be useful to have some indication in the protocol of why these application rates have been chosen and whether they reflect field usage rates or worst case scenarios and if the latter, how these are arrived at. UK: There is no justification of why the chosen application rates genuinely reflect the long-term plateau concentration in soils. UK: The protocol does not specify that the experimental site should not have been treated with soil/seed applied neonicotinoids in previous years. BCS: clarification: all pollen samples will be frozen. DE: addressed BCS: information will be provided as part of the report. DE: addressed. The estimation of realistic worst case conditions should be clearly explained. BCS: see above DE: Open point. See comment no. 1(35). This issue could be discussed in the expert meeting BCS: see above DE: addressed Applicant clarified that all pollen samples will be frozen. An estimation of the severity of the conditions of this study should be taken into account in the follow up risk assessment. See also comments 1(7) and 1(36). Addressed See also comments 1(7) and 1(35) and note in 1(35). See point for expert discussion in 1(5). See also comment in 1(8). EFSA supporting publication 2014:EN

27 A long-term field study to monitor potential effects on the honeybee (Apis mellifera L.) from exposure to guttation fluid of potato plants, grown from seed tubers treated with Monceren G in Southern Germany in 2014 and 2015 Reference to Comments from Member States / EFSA Evaluation by (RMS) rapporteur and Conclusion study protocol (e.g. point, page) 2(1) A Long-Term EFSA: It is noted that it is not planned to DE: noted See point for expert discussion in 2(5). Field Study to force the potato to guttate (i.e. by Monitor irrigation). Guttation will or will not happen Potential Effects pending on the abiotic circumstances on the (meteorology, soil type). Therefore there is Honeybee no guarantee that the worst case situation (Apis mellifera will be covered. Also, please note that the L.) from attractiveness of the droplets is dependent Exposure to on the distance from permanent water Guttation Fluid bodies. These should also be considered. of Potato Plants, Grown from Seed Tubers Treated with Monceren G in Southern Germany in 2014 and 2015 S14-xxxxa 2(2) Page 1 DE (JKI): Page 1: placed at the field sites... BCS: it is not in line with apicultural practice See point for expert discussion in 2(5). placed directly in the field to put honeybee colonies in the middle of potato fields. See also comment in 2(6). DE: BCS comment accepted. Applicant does not seem to be willing to place the hives in the field. A few meters from the edge of the field would be sufficient in this case, if the applicant would wish to address this EFSA supporting publication 2014:EN

28 A long-term field study to monitor potential effects on the honeybee (Apis mellifera L.) from exposure to guttation fluid of potato plants, grown from seed tubers treated with Monceren G in Southern Germany in 2014 and 2015 Reference to Comments from Member States / EFSA Evaluation by (RMS) rapporteur and Conclusion study protocol (e.g. point, page) comment. The logic behind is to place the colonies as close as possible to the guttation water from potato and as far as possible from other water sources in order to consider this study as worst case from this aspect. Probably it is more important to fulfil the study objective as far as possible than to be in line with the apicultural practice. 2(3) A Long-Term Field Study to Monitor Potential Effects on the Honeybee (Apis mellifera L.) from Exposure to Guttation Fluid of Potato Plants, Grown from Seed Tubers Treated with Monceren G in Southern Germany in EFSA: It seems that the test facility will not check the test item (i.e. a.s. content of the tubers). It is expected that the provider/sponsor will check and document it with appropriate certificate(s). BCS: The product is applied as an in-furrow spray. Application and planting will be documented. DE: addressed Addressed See also comment 2(18). EFSA supporting publication 2014:EN

29 A long-term field study to monitor potential effects on the honeybee (Apis mellifera L.) from exposure to guttation fluid of potato plants, grown from seed tubers treated with Monceren G in Southern Germany in 2014 and 2015 Reference to Comments from Member States / EFSA Evaluation by (RMS) rapporteur and Conclusion study protocol (e.g. point, page) 2014 and 2015 S14-xxxxa point 5.1 2(4) A Long-Term EFSA: It is important that the location of BCS: noted See point for expert discussion in 2(5). Field Study to the shared monitoring site is chosen by Monitor taking into consideration minimal DE: noted Potential Effects impact/exposure of pesticides (especially on the neonicotinoids) to the colonies. Honeybee (Apis mellifera L.) from Exposure to Guttation Fluid of Potato Plants, Grown from Seed Tubers Treated with Monceren G in Southern Germany in 2014 and 2015 S14-xxxxa points 5.2 and 5.3 2(5) A Long-Term Field Study to Monitor Potential Effects EFSA: It is noted that accuracy of the methods for the biological observations such as the mortality and the colony strength and the design of the study BCS: Technical feasibility, availability of Point for Expert discussion qualified expert and contract resources needs to be balanced against timelines. The study as Experts to discuss the design of the outlined will be performed additionally at study as commented in 2(2), 2(4), 2(5), EFSA supporting publication 2014:EN

30 A long-term field study to monitor potential effects on the honeybee (Apis mellifera L.) from exposure to guttation fluid of potato plants, grown from seed tubers treated with Monceren G in Southern Germany in 2014 and 2015 Reference to Comments from Member States / EFSA Evaluation by (RMS) rapporteur and Conclusion study protocol (e.g. point, page) on the including the number of repetitions need to different site. 2(7), 2(8), 2(9), 2(15), 2(17), 2(20), Honeybee be balanced in such a way that the set up 2(22), 2(23), 2(24) and 2(32). (Apis mellifera will allow a robust statistical analysis. The DE: noted Additionally comments in 2(12), 2(14), L.) from statistical analysis should be sensitive to 2(16), 2(19), 2(25), 2(27 and 2(33) Exposure to indicate small (e.g. 7%) differences between might be considered. Guttation Fluid the control and the treated group for at least of Potato Plants, the mortality and colony strength. Grown from Notes: It is noted that the study will be performed additionally at a different Seed Tubers site. Treated with Technical difficulties of conducting Monceren G in Southern studies cannot be taken into account in the subsequent risk assessment. Germany in 2014 and 2015 S14-xxxxa points 5.2, 5.3 and 5.12 Pesticides Peer Review Meeting on the review of bee study protocols (23 24 April 2014) The experts discussed and agreed that the hives should be placed inside the field as a worst-case for exposure. The colonies will be put in a common place after the exposure phase. It is suggested that this place is not an intensive agricultural area (e.g. pesticide use) to avoid additional stressors. The experts considered the study EFSA supporting publication 2014:EN