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!