1. Proposal for a new OECD Guideline for the testing of chemicals on adult honey bees (Apis mellifera L.) in a 10 day chronic feeding test in the laboratory and results of the recent ring test 2014
12th International Symposium Hazards of Pesticides to bees
Ghent, Belgium September , 2014
Annette Kling1, Stephan Schmitzer2
1 Eurofins Agroscience Services EcoChem GmbH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany, 2 IBACON GmbH, Arheilger Weg 17, Rossdorf, Germany,

2. Introduction
Recent developments in the risk assessment of plant protection products (PPP) on bees require the evaluation of potential chronic oral effects on adult honey bees (Apis mellifera L.).
For example:
EC Regulation 1107/2009: “an active substance or PPPs shall be approved only if it has no unacceptable acute or chronic effects on adult honey bees”
EFSA Guidance Document, (EFSA 2013): “ In order to determine the potential risk, the following effects data are required: ……chronic oral toxicity to adults…..”

3. Overview History of the ring test group
Recent test method described in the guideline proposal
Technical details of the ring test
First results of the ring test 2014
Additional investigations and conclusions

4. History of the ring test group
spring 2013: The national bee expert group (“AG Bienenschutz”) founded a sub-group in order to share experiences and harmonize the test method between the German labs.
summer 2013: 6 “valid” studies were conducted by 6 German labs based on a common test protocol.
autumn 2013: The results were presented at the SETAC conference in Brussels (October 2013). A report and a guideline proposal were submitted to the OECD.
spring 2014: Germany was chosen as the „lead country“ for the development of the guideline. An international ring test group was founded. A first meeting of the group took place.
summer 2014: A ring test was performed by 17 labs in 8 countries.

5. Test method and design (TG proposal)
Bees
Young adult bees (max. 4 days old )
Collection of bees
1-3 days before start one or more brood comb(s) with sealed brood are taken out of the hive(s)
Incubation of brood combs in the lab; (+ additional food comb)
brood comb(s) will be placed into an excluder box in the hive (bees will hatch in the excluder and will be fed by nurse bees)
Acclimatisation period
At least one day
Bees per test unit 10
Number of replicates
min. 3 (dose response) or 5 (limit test)
Test duration
10 days
Food
50 % (w/v) aqueous sugar solution ad libitum
will be changed daily (one feeding interval: 24 ± 2 hours)

6. Test method and design (TG proposal)
Dose-response test (5 concentration levels)
Limit test
Target concentration level
Max. 100 µg a.i./bee/day or up to the highest test item solubility
Solvents
Deionised water
Acetone (up to 5 % in the feeding solution)
Application/Treatment
Treated and untreated feeding solution ad libitum
Preparation of treated and untreated feeding solution at least every 4 days
Control(s)
One untreated control group (50 % aqueous sugar solution)
Additional solvent control (if necessary)
Reference item
Dimethoate (one concentration level)
Test conditions
Temperature: 33 ± 2°C
Rel. humidity: preferably 60 % ± 15 %

7. Test method and Design (TG proposal)
Assessments
Mortality and behavioural abnormalities (daily)
Food consumption (daily weighing of feeders)
Assessment Intervals
Daily up to day 10
Validity criteria
Control mortality ≤ 15 %
Reference item mortality ≥ 50 %
Endpoints
NOEC, NOEDD (No Observed Dietary Dose)
LC50 and LDD50 after 10 days

8. Ring test 2014: Participating Labs and Organisations
Contract Labs:
BioChem agrar GmbH, Machern (D)
Biotecnologie BT S.r.l., Todi (PG) (I)
CRA-API, Bologna (I)
Eurofins Agroscience Services, 75223 Niefern-Öschelbronn (D)
46650 Canals (E)
Prospect Hill, NC (USA)
Eurofins Mitox, 1098 XH Amsterdam (NL)
IBACON GmbH, Rossdorf (D)
ICB – VetAgroSup, Marcy L'Etoile (F)
IES Ltd, 4108 Witterswil (CH)
Testapi SARL, Gennes (F)
WBF, Zentrum für Bienenforschung, 3003 Bern (CH)
Wildlife International EAG, Easton, MD   (USA)
Industry:
BASF SE, Ludwigshafen (D)
Bayer CropScience AG, Monheim (D)
Bee Institutes:
Fera, York YO41 1LZ (UK)
LAVES, Celle (D)
Authorities (consultant; no data generated):
Julius Kühn-Institut, Braunschweig (D)
Country
Number of Labs
Germany 6
USA 2
France
Italy
Switzerland UK 1
Spain
Netherlands
Sum: 8 17

9. Ring test 2014 Start of the Ring test
First meeting of the ring test group in May 2014
A test protocol was discussed, prepared and agreed, based on the TG proposal as well as the experience of the participating labs of the ring test group.
Aim of the Ring Test
Justify the method proposed in the draft guideline
Harmonize the method over all labs in Europe and USA
Detect possible methodological weakness of the proposal
Verify the reproducibility of the method
Collect valid data
Transfer the recent knowledge into a guideline (GLP suitable)
Verify possible country or lab specific differences like e.g. bee races, unit sizes, etc.

10. Technical Details: Ring test 2014
Test method
According to TG proposal
Test item
Perfekthion / BAS I (dimethoate 400 g/L formulated product); the same batch was used by all labs
Concentration levels
0.2, 0.4, 0.6, 0.8 and 1.0 mg a.i./kg food;
Administration of feeding solutions
One stock solution was used for the whole test;
Fresh feeding solution prepared at least every 4 days
Endpoints
NOEC, NOEDD (No Effect Dietary Dose),
LC50, LDD50 (Lethal Dietary Dose50) after 10 days
Controls
50 % aqueous sugar solution (untreated control)
50 % aqueous sugar solution containing 5 % acetone (solvent control)

11. Ring test 2014: Mortality Results
Dose response curve in all labs
Low control mortality (water treated)
LC50 and LDD50 values could be determined in all labs

12. Ring test 2014: LC50 and LDD50 Values
Mean LC50

13. Ring test 2014: Mean Food Uptake among the Labs
Results
Mean food uptake: ± 9.2 mg food/bee/day
Food uptake in the first 2 days slightly reduced
Test Item treatment led to a reduced food uptake
Food uptake does not depend on the bee race or the country

14. Ring test 2014: Mean Dose among the Labs
Results
Mean calculated doses among the labs differ in the higher dose levels
Depending on the food uptake, the dose levels differ
The higher the food uptake, the lower the LDD50

15. Ring test 2014: Comparison of Control Mortalities I
Results
Untreated control values all below the validity criterion of ≤ 15 %
Mean untreated control value: 2.0 % (min: 0.0 %; max 6.7 %)
Mean solvent control value was above the validity criterion: 18.8 % (min: 0.0 %; max 90.0 %)
Reason ?

16. Ring test 2014: Comparison of Control Mortalities II
Parameters checked:
No indication of a race-related effect (carnica, ligustica, buckfast)
Acetone used was of analytical quality in all labs
Age of the bees was the same in all labs (1 to 4 days old)
No of lab 16 1 13 17 3 15 6 11 8 4 9 10 2 12 14 5 7
Mean food uptake [mg/bee/day]
19,7
32,7
33,4
34,0
34,3
34,4
36,5
39,5
39,6
40,7
42,2
42,5
44,4
50,0
50,9
58,0
Mortality [%] Untreated Control
0,0
6,7
3,3
Mortality [%] Solvent Control
23,3
33,3
16,7
80,0
20,0
90,0
Acetone purity:
>99.5%
99,8%
99,5%
~100%
>99%
99,9%
>96%
>95%
99,7%
Additional observations:
4 of 7 labs outside the validity criterion showed and increased food uptake
Mortality starts from day 6 onwards
Is there a threshold ? What is the acceptable level?
No problems in acute oral studies with up to 10 % acetone in the feeding solutions

17. Ring Test – Additional Investigations
The stability of dimethoate in the feeding solutions stored at 4 °C ± 2°C over a period of 10 days was assessed and confirmed (2 studies)
The size of the test cages has no influence on the outcome of the study (3 studies)
The test material of the test cages (steel, plastic, cardboard) has no influence on the outcome of the study (2 studies)
Sugar solution can be stored in the refrigerator for 4 days without any sign of bacterial or fungicidal contamination (several studies)
Syringes are the most suitable feeders (2 studies)
Tests with solvent mediators (e.g. Tween20, Tween 80, DMF) were done (> 3 studies)
Lower concentrated sugar solutions were tested (10, 20 and 30 %) goal: to increase the solubility of some test items

18. Conclusions
Proposed method can be used to detect chronic effects on adult honey bees
Test method is robust, reproducible and easy to establish in common laboratory practice
Method can be conducted under GLP conditions
Control mortality (untreated) is constantly low: < 10 % in all labs
“Perfekthion” (dimethoate) is a suitable reference item and can cause mortality ≥ 50 % at the concentration levels from 0.4 to 1.0 mg a.i./kg food
The stock solution for “Perfekthion” has to be prepared only once and can then be used for the whole test period
LC50 and LDD50 values are similar over the participating labs, indicating a high reproducibility
No country or bee race specific differences could be detected
The use of acetone as solvent at 5 % has to be discussed

19. Thank you for your attention!

20. Thanks to the Ring Test Participants:
Anne Sindermann and Hank Krueger (Wildlife International)
Claire Molitor (Testapi SARL)
Claudia Volles Heimo (WBF, Zentrum für Bienenforschung)
David Gladbach (Bayer CropScience AG)
Dorothee Lüken (LAVES)
Frank Bakker and Josep Roig (Eurofins MITOX)
Julie Fourrier (ICB – VetAgroSup)
Katharina Kleebaum and Saskia Ruhland (BioChem agrar GmbH)
Marcus Bicker (IBACON GmbH)
Ming Hua Huang and Guillermo Fernandez (Eurofins EAS)
Monica Colli and Simone Venturi (Biotecnologie BT S.r.l.)
Piotr Medrzycki (CRA-API)
Selwyn Wilkins (Fera)
Stefan Kimmel (IES Ltd)
Stefanie Niederdrenk and Dagmar Sack (BASF SE)