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1. Regulatory testing with bees –
Experience from other regions
Relação Produtiva entre Agricultura e Apicultura
Campinas, SP
Dr. Christof Schneider
BASF SE
Crop Protection
Global Ecotoxicology
(christof.schneider@basf.com)

2. Environmental Impact
 Crop protection products are
intentionally introduced into the
environment
 They show biological activity
(toxicity) towards target organisms
 Side effects that may occur should
be minimized and kept at an
acceptable level
2

3. Environmental Risk Assessment
Key Concepts
Key concepts:
 Risk = Hazard x Exposure
Risk Quotients
Toxicity Exposure Ratio
 At its simplest:
Exposure < Hazard = No Risk
Toxicity
TER
=
Exposure
Therefore:
(Exposure / Hazard < 1) = No Risk
(Hazard / Exposure > 1) = No Risk
!
Large numbers are good!
Risk Quotient
Exposure
Hazard may be referred to as
„toxicity“ or „effects“
RQ
=
Toxicity
Small numbers are good!
 The risk assessment integrates both toxicity and exposure data.
3

4. The Hazard Quotient (HQ) Approach
References:
Aldridge CA & Hart ADM (1993) Validation of the EPPO/CoE Risk assessment scheme for honeybees. Proceedings of the 5th International Symposium on the
hazard of pesticides to bees. October 26-28, 1993. Wageningen, The Netherlands.
Mineau P, Harding KM, Whiteside M, Fletcher MR Garthwaite D & Knopper LD (2008) Using reports of bee mortality in the field to calibrate laboratory-derived
pesticide risk indices. Environmental Entomology 37(2): 546-554
Defra (2009) Systemic pesticide risk assessment for honeybees – PS2335. SID 5 – Research project final report. Study by the Central Science Laboratory (CSL) for
the Department for Environment, Food and Rural Affairs, London, UK.
 Crucial part of risk assessment is setting of appropriate trigger values
 The currently used trigger values were validated with field data

5. The Hazard Quotient (HQ) Approach
Scenarios:
Scenario 1: LD50 of 1 µg a.i./bee and an application rate of 100 g a.i./ha
100 g a.i./ha
1 µg a.i./bee
HQ = 100  risk cannot be excluded
Higher tier studies triggered
Scenario 2: LD50 of 1 µg a.i./bee and an application rate of 40 g a.i./ha
40 g a.i./ha
1 µg a.i./bee
HQ = 40  No higher tier study
required

6. Determine the Effect (Hazard)
From Simple to Complex
Exceeding a Hazard Quotient does not mean that a risk exists
Tier I
Complexity
Realism
Uncertainty
Laboratory
Extended
Laboratory
Semi-Field
Field
 Start with simple tests first then go more complex
6
Higher Tier

7. Test Systems
Honey bees – contact and oral test
Test species
Apis mellifera (honey bee)
Test design
 Duration of the test: up to 96 h
Acute oral test:
 10 bees per replicate are exposed to a
water-sugar solution with dissolved test item
Acute contact test:
 10 bees per replicate are exposed
to the test item via a droplet on their back
Result evaluation
 Determination of mortality and behavioral signs
 Determination of LD/LC50

8. Test Systems
Honey bees – higher tier tests
Further test systems are:
 Semi-field test: e.g. Tunnel Test
(EPPO 170, OECD GD 75)
 Field Tests (EPPO 170)
Observations:
 mortality, foraging activity, behaviour,
colony development, specific issues
 optional: detailed brood assessments

9. Closer look on test designs in the area of
bees
1. Established:
Semi-field testing on colony level
2. Close to be implemented:
In vitro larval testing

10. Semi-field testing on colony level
 Current guidance: EPPO 170, OECD GD 75
 OECD puts special emphasis on marked cells
filled with eggs
 Semi-field conditions (tunnel tent) as
realistic worst case situation
 Three treatment groups:
 Water control
 Test item
 Toxic reference
 Crop attractive to honeybees
e.g. Phacelia, oilseed rape

11. Honey bee colonies
One healthy colony per tunnel
 sister queens of the same year
 approximately 6,000 worker bees
 approximately 3,000 brood cells
 one food comb

12. Application
 Application rate: realistic field relevant AR
 Into a flowering of crop
 Foraging activity (≥ 10 bees/m²)
worst case conditions
 Variable exposure of the foraging bees possible
 Exposure of the whole colony by contaminated
pollen/nectar
Direct exposure of the foraging bees
Exposure of the individuals inside
the hive by contaminated food

13. Space and Time
BFD
28 m
Hive with bee trap
2.5m
24 m
Water supply
0
6.5m
Linen sheets
~ 80
m²
5
Phacelia area
10
16
22
BFD = Brood Fixing Day

14. Assessment:
Mortality
Mortality assessment - example
plastic sheet
14

15. Assessment:
Foraging activity
 Checked daily inside the tunnels
 Only foraging bees
 Three randomly chosen locations of 1 m² each

16. Assessment:
Condition of the colony
 Estimation according to
Imdorf et al. (1987)
 Area covered with:
 Bees (strength)
 Eggs
 Larvae
 Sealed brood
 Pollen cells
 Nectar cells
 Six assessments over 28 days

17. Things to keep in mind regarding the
semi field test




Well established higher tier test in EU and US
In theory, an easy to use test design
However, intensive effort and many complex assessments necessary
In general, colonies used in field realistic tests can be subject to great variability
even in the untreated control group
 Often expert judgment is necessary for interpretation of the results

18. Closer look on test designs in the area of
bees
1. Established:
Semi-field testing on colony level
2. Close to be implemented:
In vitro larval testing

19. Lower-Tier In vitro Larval Toxicity Test
 New study type to assess toxicity to larvae under
artificial laboratory condition (OECD 237, recently
published)
 New method, technically more challenging than
laboratory studies with adult bees
 Reliability and practicability of method is not clear yet
(especially regarding full brood development cycle)
 Will be part of the future risk assessment schemes and
testing requirements in EU and US, if method proves to
be feasible and reliable

20. Effort necessary to develop a reliable test
system
Data generated in order to produce a
guideline

53 acute tests

involving 5 institutes (2008 – 2012)

just data from Germany

Countries involved in the OECD process:
Germany, UK, France, Italy, Greece, and US
2005:
First publication of an in vitro
larvae rearing test by Aupinel et
al.
2013:
Publication of finalized OECD
Guideline (OECD 237)
 Development of reliable and robust test systems is requiring time and effort

21. Conclusions
 Bees are an important part of the ecosystem and adequate risk assessment
tools are needed
 Currently, established laboratory test systems are in place which are
complemented with internationally agreed higher tier testing options
 In order to address future data requirements, time and combined effort is
needed to validate new test methodologies.
 The aim must be to create a tiered system that is protective on the one hand
but avoids unnecessary testing on the other

22. Muito Obrigado!
Thank you very much!