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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)
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
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
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
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
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
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
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
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
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
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
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
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
Assessment:
Mortality
Mortality assessment - example

plastic sheet

14
Assessment:
Foraging activity
 Checked daily inside the tunnels

 Only foraging bees
 Three randomly chosen locations of 1 m² each
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
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
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
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
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
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
Muito Obrigado!
Thank you very much!

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Apresentação Christoph Schneider - BASF

  • 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
  • 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