Alscher, Annika

Classen, Christian

Exeler, Nina

Franke, Lea

Fricke, Julian

Frommberger, Malte

Hecht-Rost, Sabine

Kimmel, Stefan

Klein, Olaf

Knaebe, Silvio

Lückmann, Johannes

Molitor, Claire

Peters, Britta

Schneider, Christof

Sorli Guerola, Juan

English

JKI Open Journal Systems (Julius Kühn-Institut)

Hazards of pesticides to bees - 13th international symposium of the ICP-PR Bee protection group, October 18 – 20 2017, Valencia (Spain)  142  Julius-Kühn-Archiv, 462, 2018 Section 4 – Testing methodologies for non-Apis bees 4.1 Progress of working group Non-Apis testing N. Hanewald1, N. Exeler2, Olaf Klein3, Silvio Knäbe3, Roberta Nocelli4, Thaisa Roat4, Sjef van der Steen5 1BASF SE; Ivo Roessink, Wageningen Environmental Research 2Bayer 3Eurofins Agroscience Services 4Universidade Federal de Sao Carlos 5Alveus AB Consultancy See summary of progress of the Non-Apis group on page 8 Thomas Steeger:  Working Groups of the ICP-PR Bee Protection Group – Developments and Progress 4.2 Summary of an ICPPR Non-Apis workshop – Subgroup higher tier (bumble bees and solitary bees) with recommendations for a semi-field experimental design Silvio Knaebe1, Annika Alscher2, Christian Classen2, Nina Exeler3, Lea Franke1, Julian Fricke1, Malte Frommberger4, Juan Sorlí Guerola5, Sabine Hecht-Rost2, Stefan Kimmel6, Olaf Klein1, Johannes Lueckmann2, Claire Molitor7, Britta Peters8, Christof Schneider9  1Eurofins Agroscience Services Ecotox GmbH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany 2Rifcon GmbH, Goldbeckstr. 13, 69493 Hirschberg, Germany 3Bayer CropScience, 40789 Monheim, Germany 4Institut für Bienenschutz, Julius Kühn-Institut (JKI), Messeweg 11-12, 38104 Braunschweig, Germany 5Trialcamp SLU, Avda. Antic Regne de Valencia 25, 46290 Alcasser, Spain 6Innovative Environmental Services (IES) Ltd, Benkenstrasse 260, 4108 Witterswil, Switzerland 7Testapi, 464 Sarré, Gennes, 49350 Gennes-Val-de-Loire, France 8tier3 solutions GmbH, Kolberger Strasse 61-63, 51381 Leverkusen, Germany 9BASF, 67117 Limburgerhof, Germany   E-mail contact: silvioknaebe@eurofins.com  DOI 10.5073/jka.2018.462.041 Introduction The publication of the proposed EFSA risk assessment guidance document of plant protection products for pollinators [1] highlighted that there are no study designs for non-Apis pollinators available. Since no official guidelines exist for semi-field testing at present, a protocol was proposed and a ringtest was conducted in 2016 to develop a general test set-up. The ringtest design was based on the draft EFSA guidance document [1], OEPP/EPPO Guideline No. 170 [2] and results of discussions regarding testing solitary bees during the meetings of the ICPPR non-Apis workgroup in 2015, 2016 and 2017 [3, 4, 5] and an hand on workshop in May 2017 [6].  Materials and Methods Ring-tests were conducted with two different test organisms, one representative of a social bumble bee species (Bombus terrestris L; Hymenoptera, Apidae) and one representative of a solitary bee species (Osmia bicornis L; Hymenoptera, Megachilidae). Both are polylectic and foraging on a diverse spectrum of flowering crops. In addition, they are common species in Europe, commercially available and widely used for pollination services.  Several laboratories participated in the higher-tier ring test. Seven semi-field tests were conducted with B. terrestris and 8 semi-field tests were done with O. bicornis in 2016. In 2017 8 semi-field tests with bumble bees and 8 semi-field tests with solitary bees were run.  Two treatment groups were always included in the ringtest: an untreated control (water treated) and dimethoate as a toxic reference item (optional other i.e. brood affecting substances (fenoxycarb, diflubenzuron)). The toxic reference items were chosen based on their mode of action and long term experience in honey bee testing.  Hazards of pesticides to bees - 13th international symposium of the ICP-PR Bee protection group, October 18 – 20 2017, Valencia (Spain)  Julius-Kühn-Archiv, 462, 2018 143 In the solitary bee study design adult bees (both sexes) were caged in tunnels containing a bee attractive flowering crop and exposed during their reproductive period. After the application of the respective reference items, the adult female bees collected the relevant food items from the treated crop, providing their offspring with exposed pollen and nectar as the only food source during brood development. The final result on developing and hatching success of the progeny was assessed in the following year.  In the bumble bee study design only the early part of the colony development took place during the exposure phase in the tunnels. At the end of flowering, the bumble bee colonies were transferred to a monitoring site until they produced queens and drones (“switch-point”). Results and discussion Test design  A general test design was developed for a solitary bee and a bumble bee semi-field study based on results of the first year of testing 2016.  For the solitary bees the aim of the second run of the ring-test was to define a more standardized test design to reduce the variability between study results and to guarantee reproducible test conditions (e.g. nesting material, latest study start, assessments, overwintering procedure). The replication was under discussion since MDDs (Minimum Detectabel Differences) calculated from ring-test results of 2016 were high and there was no information what are expected variations for this kind of tests. Furthermore, immature mortality of the bee brood needed to be lowered by reducing parasitation and improving handling of the sensitive eggs and larvae. Also, the optimal timing of spray applications was under discussion. For the bumbe bees the aim of the second ringtest run was also to define a more standardized test design to reduce the variability between study results and to guarantee reproducible test conditions (e.g. worker number per m² crop, colony composition at study start, assessment of endpoints, determination of switch point, timing of deep-freezing). The replication was under discussion since one of the most important endpoints, i.e. the queen reproduction, showed high variability [7]. To reduce variability between replicates a special focus was upon the origin of the hives and the selection of colonies for the test. One further challenge was and is the best timing for the termination of a study allowing the assessment of the most important endpoint, queen reproduction, which was discussed in detail. The basis requirements for studies after the first ring test of 2016 and discussions are given in the following table.  Buff-tailed bumble bee (Bombus terrestris L.) Red mason bee        (Osmia bicornis L.) Replicates 6 4 Size of tunnels ≥ 30 m² Number of test organisms Initial colony size 10 bumble bees, approx. colony size 20 bumble bees after 14 days in laboratory 1 ♀ /m² / 1.5 ♂ /m² Nests Commercial bumble bee hives with queen excluder Chipboard units MDF   (100 cavities) Test itema Dimethoate (600 g a.i./ha) Dimethoate (75 g a.i./ha) Exposure Flowering period of crop Flowering period of crop after first cells are produced Test duration 6 - 15 weeks 10 - 12 months Time of testing April - August April – May (- July) Crop Oil seed rape, Phacelia Oil seed rape, Phacelia                                                  aoptional additional test of other substances Table 1 Test design of semi-field studies with solitary bees and bumble bees for 2017 Hazards of pesticides to bees - 13th international symposium of the ICP-PR Bee protection group, October 18 – 20 2017, Valencia (Spain)  144  Julius-Kühn-Archiv, 462, 2018 All ring test participants agreed on the design for studies run in 2017. For the ring test in 2017 bumble bee colonies from one distributor were used to reduce the variability.  Endpoints It was agreed on the most important, obligatory endpoints to be recorded for the tests.  In the test with solitary bees hatching success (1st generation) will be established since it will be the basis for later calcualtions of reproductive success and gives an information of the quality of the coccons. The next endpoints are the establishment at the nesting units (nest occupation), flight activity, reproduction and hatching success (2nd generation). The latter is the most important information that needs to be observed.  In the bumble bee trials endpoints are brood development, colony weight and colony reproduction (production of sexuals). It was agreed that the trial should only stopp when first queens have been hatched.  Test performace  For the solitary bee test will consist of two reatment groups, the untreated control C and the test item treatment group T (applied with Dimethoate). Bee cocoons, e.g. O. cornuta or O. bicornis, need to be placed in the tunnel when the first flowers are open (approx. BBCH 60). Nesting units are placed in each tunnel where the bees will establish their brood nests. The adult bees and their larvae will be exposed to the nectar and pollen of the crop throughout the flowering period. After the end of exposure the development of their progeny will be followed through to the following spring and the reproduction success will be determined by the number and vitality of hatched individuals. For bumble bees the test will consist of two treatment groups, the untreated control C and the test item treatment group T (applied with Dimethoate). Additionally, brood-affecting substances can be added as further treatment groups, if required (i.e. Diflubenzuron). The application will take place as spray application during bee flight at least 3-6 days after set-up of the bumble bee colonies in the tunnels. Exposure will last until the end of flowering. After the exposure phase in the tunnels, the bumble bee colonies will be transferred to a remote site (natural area with foraging resources and minimal pesticide exposure) location in order to assess the development of the colonies and the reproduction of young queens and drones. Outlook Based on the results of the ringtest main open questions will be adressed and the aim will be to propose a guidance for the performance of semi-field studies. The open points at the moment are:   …for bumble bees:  • how many replicates are needed to see possible effects? • how can minimal variation of endpoints be achieved and specifically what are realistic variations in queen number and size/weight? • how can the “switch-point” be defined reliably for a test protocol? • how can the assessment of hatched queens be handled? …for solitary bees:  • how can cocoon incubation and hatching of bees be synchronised with the onset of flowering? • how fit are solitary bees out of season (tests in summer)? • which substance can be used as reference item for brood studies?  References [1] EFSA. [EFSA] European Food Safety Authority. 2013. Guidance on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees). EFSA Journal 11(7):3295, 268 pp.        Hazards of pesticides to bees - 13th international symposium of the ICP-PR Bee protection group, October 18 – 20 2017, Valencia (Spain)  Julius-Kühn-Archiv, 462, 2018 145 [2] OEPP/EPPO. [OEPP/EPPO] European and Mediterranean Plant Protection Organization. 2010. Efficacy evaluation of plant protection products – Side effects on honeybees. OEPP/EPPO PP 1/170 (4), EPPO Bulletin 40, 313-319. [3] ICPPR. [ICPPR] International Commission for Plant Pollinator Relationships. 2015. Short Overview of the ICPPR Non-Apis Workshop - Subgroup Higher Tier (Bumble bees and Solitary bees). Non-Apis Workshop. Limburgerhof, BASF Agrarzentrum, Germany, 19-20 February 2015. [4] ICPPR. [ICPPR] International Commission for Plant Pollinator Relationships. 2016. Short Overview of the ICPPR Non-Apis Workshop - Subgroup Higher Tier (Bumble bees and Solitary bees). Non-Apis Workshop. Braunschweig, Julius-Kühn Institute (JKI), Germany, 29 February - 01 March 2016. [5] ICPPR. [ICPPR] International Commission for Plant Pollinator Relationships. 2017. Short Overview of the ICPPR Non-Apis Workshop - Subgroup Higher Tier (Bumble bees and Solitary bees). Non-Apis Workshop. Wageningen, Netherlands, 14 February - 15 February 2017. [6] ICPPR . [ICPPR] International Commission for Plant Pollinator Relationships. 2017. Subgroup Higher Tier (Bumble bees and Solitary bees). Non-Apis Semi-field Hands on Workshop. IBACON Rossdorf Germany 5 April 2017. [7] Cabrera, A.R., Almanza, M.T., Cutler, G.C., Fischer, D.L., Hinarejos, S., Lewis, G., Nigro, D., Olmstead, A., Overmyer, J., Potter, D.A., Raine, N.E., Stanley-Stahr, C., Thompson, H., van der Steen, J.: Initial recommendations for higher-tier risk assessment protocols for bumble bees, Bombus spp. (Hymenoptera: Apidae), Integrated Environmental Assessment and Management DOI 10.1002/ieam.1675 June 2015   4.3 An international workshop on pesticide exposure assessment for non-Apis bees Silvia Hinarejos1, Richard Bireley2, Jordi Bosch3, Natalie Boyle4, Wayne Hou5, Theresa Pitts-Singer6, Rajwinder Singh7, Thomas Steeger8, Neil Williams9 1Valent USA LLC, Dublin, CA, U.S.A. 2California Department of Pesticide Regulation, Sacramento, CA, U.S.A. Richard.Bireley@cdpr.ca.gov 3Centre for Ecological Research and Forestry Applications, Bellaterra, Spain, jordi.bosch@uab.cat  4United States Department of Agriculture-ARS, Logan, UT, U.S.A., Natalie.Boyle@ars.usda.gov 5Health Canada Pest Management Regulatory Agency, Ottawa, Canada, wayne.hou@hc-sc.gc.ca 6United States Department of Agriculture-ARS, Logan, UT, U.S.A., Theresa.Pitts-Singer@ars.usda.gov 7BASF Corporation, Research Triangle Park, NC, rajwinder.singh@basf.com 8United States Environmental Protection Agency, Washington, DC, U.S.A., steeger.thomas@epa.gov 9University of California Davis, Davis, CA, U.S.A., nmwilliams@ucdavis.edu DOI 10.5073/jka.2018.462.042 Abstract The honey bee (Apis mellifera) is typically used as a surrogate to evaluate the risk of pesticides to all bee species. However, there is uncertainty regarding the extent to which honey bees can serve as surrogates for solitary bees, bumble bees and stingless bees given differences in their life history traits (e.g., body size, feeding, sociality, flight/activity season, nesting materials, behavior, overwintering strategy, etc.). Lack of basic knowledge of non-Apis bee exposure scenarios has been among the biggest challenges in determining whether honey bees are sufficient surrogates for non-Apis bees. As a result of a tripartite effort between regulatory agencies, academia and agrochemical industry, an international workshop was organized in Washington D.C. on 10th-12th January 2017. Forty bee researchers and risk assessors from ten different countries gathered to discuss the current state of science on pesticides exposure to non-Apis bees, and to determine how well honey bee exposure estimates used by different regulatory agencies may be protective for non-Apis bee species. There was a general consensus that the current honey bee exposure assessment paradigm is highly conservative. However, several data gaps were identified that hindered a complete analysis of various routes of exposure between Apis and non-Apis bees, especially when non-Apis bees may be exposed via nesting materials such as soil (e.g., blue orchard bees; Osmia spp., alkali bees; Nomia spp.), leaves (e.g., alfalfa leafcutting bees, Megachile rotundata), or a combination of soil and leaves (e.g., stingless bees; tribe Meliponini). Basic conceptual models and preliminary exposure equations were discussed that could help to quantify these exposure routes, allowing for future comparisons with honey bee exposure estimates. The workshop proceedings, along with a list of critical research needs identified to quantify non-Apis bee exposure routes, will be published as a series of peer-reviewed journal articles. 

4.2 Summary of an ICPPR Non-Apis workshop – Subgroup higher tier (bumble bees and solitary bees) with recommendations for a semi-field experimental design

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4.2 Summary of an ICPPR Non-Apis workshop – Subgroup higher tier (bumble bees and solitary bees) with recommendations for a semi-field experimental design

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