Working group of AG Bienenschutz* INTERNATIONAL COMMISSION FOR PLANT-BEE RELATIONSHIPS Bee Protection Group   11th International Symposium hazards of pesticides to bees   WAGENINGEN, THE NETHERLANDS November 2-4, 2011 Working group of AG Bienenschutz* Effectiveness of method improvements to reduce variability of brood termination rate in honeybee brood studies under semi-field conditions *Pistorius, J.; Becker R.; Lückmann, J.;  Schur, A.; Barth, M.; Jeker, L.; Schmitzer, S.; von der Ohe, W.

Background Increased focus on brood studies led to an increased number of requested brood studies according to OECD 75 and Oomen & de Ruijter 4 years of experiences indicated problems with e.g. high brood mortality, low effect of the reference item, high variability between the replicates need of improvement for the OECD Guidance Document 75 in order to decrease control brood mortality and variabilty between the replicates. 2 meetings of WG AG Bienenschutz took place in 2011. Some possible improvements of the method were discussed as basis for studies in 2011

Status Quo According to average data until 2011 control termination rate (mortality of bee brood on combs e.g. eggs) is approx. 30-40% (including bad and invalid studies) Effects in reference item group not stable High variation between replicates Critical time for an interrupted brood development is between BFD 0 to BFD 5 Difficulties in tunnel studies more present than under field conditions Variability complicates interpretation of the results Quality of studies need to be improved in order to decrease high termination rate in control colonies

Test Method according to Guidance Document OECD 75 (2007) Colony size: Small test colony consist of approximately 3000 brood cells respectively 750 cm2 with brood in all stages, 1 food comb with honey and pollen and approximately 800 g worker bees (approximately 6000 worker bees) Crop area: At least 40 m2 per tunnel Reference item: Insegar to be applied at a rate of at least 600 g/ha, corresponding to 150 g fenoxycarb/ha Assessment of brood development: Marking brood stage of cells with acetate sheets

Test Method according to Guidance Document OECD 75 (2007) --> Brood termination-rate, brood-index, compensation-index BFD = Brood area fixing day

Improvements for Studies in 2011 Colonies: Colonies with 10 frames (Zander), 3-5 brood combs, high proportion of capped cells Crop : Large tunnels (crop size area > 80 m² Timing of trials: Early start of trials was recommended, irrigation of test fields when dry conditions Reference item: Single of double rate? Assessment of brood development: Photo documentation of the brood development (at least 200 cells per colony)

Data used 2011 For control data 13 studies from 6 laboratories, total of 50 replicates (3 to 4 replicates per study) For toxic reference item data 12 studies, all done with fenoxycarb (one with dimethoate and therefore not considered), total of 43 replicates (2 to 4 replicates per study) Historical data 21 studies carried out between 2002 and 2010 (data analysis presented by Becker & Lückmann at the 58th Jahrestagung der Arbeitsgemeinschaft der Institute für Bienenforschung e.V., Berlin-Dahlem 29. - 31.3.2011). - only studies carried out on Phacelia tanacetifolia were considered

Brood termination rate (historical data, 2002 to 2010): Control: Ø 34.7% ± 24.8% (min: 4.9%, max. 100%, median: 25.9%) Tox: Ø 76.8% ± 24.2% (min: 20.9%, max. 100%, median: 83.4%) Brood termination rate (2011): Control: Ø 21.7% ± 14.8% (min: 2.0%, max. 66.8%, median: 18.4%) Tox: Ø 63.7% ± 21.1% (min: 11.5%, max. 100%, median: 65.1%)

Colonies: colony strength and brood termination rate Historical and recent control data from 2011 no real tendency but colonies with 7.000 to 9.000 bees show higher probability to achieve BTR ≤30%

Timing: study initiation in the year and brood termination rate Historical and recent control data from 2011 June July August May overall conclusion: study initiation before ~ day 181 (end of June) with increased probability to achieve BTR ≤ 30% !

Crop: effective crop area and brood termination rate Historical and recent control data from 2011 overall increased crop area resulted in higher probability to obtain BTR ≤ 30%

Timing and crop: study initiation and crop area and BTR Historical and recent control data from 2011 early: ≤ day 181, small: ≤ 80m²; late: > day 181, large: > 80 m² June July August May studies with eff. crop area ≤80 m² & performed end of June or in July with high probability of BTR > 30%

Timing and crop: study initiation and crop area and BTR Historical and recent control data from 2011 early: ≤ day 181, small: ≤ 80m²; late: > day 181, large: > 80 m² June July August May performance of studies until mid of June or in July and / or increase of effective crop area enhances probability of BTR ≤30%

Reference item: sum of dead pupae in dead bee traps and brood termination rate (data from 2011) replicates with low BTR often with increased pupal mortality, indicating exposure of the bees to the test item and thus suitability of test system to detect potential effects on the bee brood!

No or only limited improvements of brood termination rate Duration and condition during assessments, when combs are outside colony Marking of one connected brood area vs. marking of several areas on one comb (because these factors were optimized already in the previous years)

Our Recommendations Colonies: Big(ger) colonies should be used for the test (3-4 brood combs, high amount of capped cells, avoid major modifications of colonies) Use 4 instead of 3 replicates (better interpretation of data) Timing: Start studies early in the season, if possible. Crop: Use large tunnels (crop area in tunnels > 60 m², preferably > 80 m²) Watering of crop if dry conditions

Our Recommendations Reference Item: Evaluation of termination rate and pupal mortality Single or double field rate can be used

Our Recommendations Assessment method: No correlation between BTR and use of acetate sheets and digtial ass. method No correlation between BTR and higher numbers of larvae Nevertheless, use of digital photo assessment and observation of 200 to 400 cells is recommended. (several advantages, e.g. quicker, reduced stress for colonies) Photo: Frommberger/Lückmann Photo: Jeker

Conclusions Progress was achieved: Better results observed in 2011 compared with historical data (different factors improved) Work will be continued and more data evaluated from 2012 Recommendations for for improvement of OECD GD 75 intended to be developped till end of 2012

Acknowledgements Thanks for valuable contributions to: Claasen, C.; Bocksch, S.; Hampel, K.; Lüken, D.; Scazzari, S.; Thorbahn, D.; Zenker, K. Thanks to the different companies for supplying data! Thank you, dear audience! You got data to contribute and/or brood studies planed for 2012 ? Contact me! jens.pistorius@jki.bund.de Your data will be a very important contribution!