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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
2
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
3
Risk = Hazard x Exposure
At its simplest:
Exposure < Hazard = No Risk
Therefore:
(Exposure / Hazard < 1) = No Risk
(Hazard / Exposure > 1) = No Risk
Environmental Risk Assessment
Key Concepts
Risk Quotients
Toxicity
Exposure TER =
Exposure
Toxicity RQ =
Toxicity Exposure Ratio
Risk Quotient
Key concepts:
Hazard may be referred to as „toxicity“ or „effects“
!
Small numbers are good!
Large numbers are good!
The risk assessment integrates both toxicity and exposure data.
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
Scenario 2: LD50 of 1 µg a.i./bee and an application rate of 40 g a.i./ha
1 µg a.i./bee
40 g a.i./ha HQ = 40 No higher tier study
required
Scenarios:
Scenario 1: LD50 of 1 µg a.i./bee and an application rate of 100 g a.i./ha
1 µg a.i./bee
100 g a.i./ha HQ = 100 risk cannot be excluded
Higher tier studies triggered
6
Determine the Effect (Hazard)
From Simple to Complex
Start with simple tests first then go more complex
Uncertainty
Complexity Realism
Higher Tier
Tier I
Semi-Field Field Extended Laboratory
Laboratory
Exceeding a Hazard Quotient does not mean that a risk exists
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
Test Systems
Honey bees – contact and oral test
Apis mellifera (honey bee)
Determination of mortality and behavioral signs
Determination of LD/LC50
Test species
Test design
Result evaluation
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 conditions (tunnel tent) as
realistic worst case situation
Crop attractive to honeybees
e.g. Phacelia, oilseed rape
Three treatment groups:
Water control
Test item
Toxic reference
Semi-field testing on colony level
Current guidance: EPPO 170, OECD GD 75
OECD puts special emphasis on marked cells
filled with eggs
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 rate: realistic field relevant AR
Into a flowering of crop
Foraging activity (≥ 10 bees/m²)
Variable exposure of the foraging bees possible
Exposure of the whole colony by contaminated
pollen/nectar
worst case conditions
Direct exposure of the foraging bees Exposure of the individuals inside
the hive by contaminated food
Application
Space and Time
Hive with bee trap
Water supply
Linen sheets
28 m
Phacelia area
6.5
m
2.5
m
24 m
~ 80 m²
BFD
0
5
10
16
22
BFD = Brood Fixing Day
14
Mortality assessment - example
plastic sheet
Assessment:
Mortality
Checked daily inside the tunnels
Only foraging bees
Three randomly chosen locations of 1 m² each
Assessment:
Foraging activity
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
Assessment:
Condition of the colony
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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