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Regulations & Guidance Document Excellent basic

scientific research

Scientific / Public / Politics awareness of the problems and meaningful endpoints

Pesticide Risk Assessment

ring-testing

… we don’t need …

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B L P

B ad L aboratory P ractice B L P

«Breaking Bad» / © Ben Leuner/AMC

We need: compliant methods

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G L P

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The new EFSA requirements: what changed?

Honeybees Apis mellifera

Bumblebees Bombus ssp.

Solitary bees Osmia cornuta Osmia bicornis

New testing requirements honeybees

• Acute and Chronic Lab Toxicity Test OECD 213 (oral) / 214 (contact) / EFSA / CEB

• In-vitro Larvae Lab Test OECD

• Brood Test Oomen & de Ruijter 1992 / EFSA

• Tunnel Test OECD ENV/JM/MONO (2007) 22 and under consideration of the current recommendations of the “AG Bienenschutz” and CEB Method No. 230 / EFSA

• Field Test OEPP/EPPO (2010): Guideline for the efficacy evaluation of plant protection products – Side effects on honey bees. OEPP/EPPO, PP 1/170(4) / EFSA

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• Tunnel Test or Greenhouse Study

• Field Test

New testing requirements bumblebees

• Acute and Chronic Lab Toxicity Test

• In-vitro Larvae Lab Test

• Brood Test

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For chronic and larvae toxicity tests, honeybee studies and associated endpoints are proposed to serve as surrogates until internationally agreed and adopted guidelines are available.

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Acute contact & oral toxicity bumblebees

- average sized workers / young colonies - 30 bees/treatment - anaesthetized, ventral thorax application (contact) - 5 doses test item, +/- control, reference - 48 h exposure - 2 tests replicated in time - Mortality, behaviour

Endpoint: contact & oral LD50 (µg a.i./bee)

30 bumblebees per treatment,

two test replicates in time

filter paper

micro-applicator

Test design for the contact treatment

Based on OECD 213/214, van der Steen et al. (1996, 2001), Marletto et al. (2003)

bumblebees together

in holding cage

hole for transfer

sealed with a rubber plug

black plastic container

(1 starved bumblebee / jar)

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EFSA Guidance document Bumblebee higher tier studies

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Mommaerts et al., 2010; Laycock et al., 2012

Second step laboratory test, recommended when HQ or ETR is breached, or when potential for accumulative effects is indicated.

EFSA Guidance document Bumblebee higher tier studies

Unclear: - treatment replication (10)?

Queenless Bombus terrestris microcolonies • 5 workers per ‘nest box’ • chronic dietary exposure • nest box connected to ‘foraging box’ • up to 60 days exposure period • 2 test replicates using different colonies Parameters: worker mortality larval development / mortality food uptake / collection reproductive output (male progeny only) Endpoint: NOEC

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“The OECD semi-field study for honeybees can be readily adapted to bumble bees. Owing to the limitations of semi-field tests (....) , as for honeybees, it is proposed that semi-field studies have a limited use in the risk assessment and decision-making process.”

OECD 75 & Tasei et al. (1993)

EFSA Guidance document Bumblebee higher tier studies

Endpoints

Foraging activity

Adult and larval mortality

Colony strength

Amount of brood

“The number of replicates must be high enough to account for normal inter-colony variability and allow statistical analyses with adequate power of the test.”

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EFSA Guidance document Bumblebee higher tier studies

Endpoints

Foraging activity

Adult and larval mortality

Colony strength

Amount of brood

The replication issue: Tunnel partitioning?

③ ② ① ③ ②

①

?

No, whole tunnel replication!

Whole tunnel !?

In-hive, tunnel,

overall (mark and recapture)?

Weighing, (indoor) counting?

Impossible without dissection!

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EFSA Guidance document Bumblebee higher tier studies

Colony subset setup for tunnel studies:

Step 1: Weigh colonies, count workers (~100); run exposure phase.

Step 2: Stop tunnel phase (~7-10 days), weigh colonies.

Step 3: Stop monitoring phase (~21 days), Weigh, freeze and dissect colonies Set A (repeat Step 2b).

Step 2b: freeze and dissect colonies Set B, count workers, eggs, young larvae, old larvae & pupae (evtl. drones and gynes).

Step 2a: count workers Set A and run monitoring phase (comparable to honeybees).

Set A Set B

Treatment Y

1 3 2 4 4 1 2 3 5 5

Set A Set B

Treatment X

1 3 2 4 4 1 2 3 5 5

Set A Set B

Treatment Z

1 3 2 4 4 1 2 3 5 5

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EFSA Guidance document Bumblebee higher tier studies

Endpoints inferred by colony freezing and dissecting:

relative effects on brood development (number of eggs, young / old larvae, pupae), and investment in worker, male and gyne production

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Whitehorn et al. (2012), Gill et al. (2012)

EFSA Guidance document Bumblebee higher tier studies

Lab exposure of entire colonies (including ‘foraging challenge’) with subsequent field monitoring...

Taken from Gill et al. 2012 Supp. Mat.

… or ‘field-lab’ approach.

See also Larson et al. (2013)

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‘Transgenerational effect’ enpoints: queen hibernation and colony founding success

Successful hibernation requires previous mating.

... time needed to reach the ~8-10 worker stage.

Initiation of egg-laying and…

Problem: non-synchronized queen production…

EFSA Guidance document Bumblebee higher tier studies

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As long as a new protocol to study the effect of pesticide on bumblebees is not available and validated, the combined field-to-laboratory studies should be used.

‘… weak study design and methodology’ - conclusions impossible…

EFSA Guidance document Bumblebee higher tier studies

New testing requirements solitary bees

• Acute and Chronic Lab Toxicity Test

• In-vitro Larvae Lab Test

• Brood Test

• Tunnel or Cage Study

• Field Test

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For chronic and larvae toxicity tests, honeybee studies and associated endpoints are proposed to serve as surrogates until internationally agreed and adopted guidelines are available.

“At the moment official test protocols are not available for solitary bees.”

Acute contact & oral toxicity solitary bees

19 Taken from Ladurner et al. 2003

Unclear: repeatability?

Based on OECD 213/214 (see also Biddinger et al. (2013)) & Ladurner et al. (2003)

• 30 female bees / treatment (1 day old)

• starved over night before exposure

• chilled bees, dorsal thorax application (contact)

• individual feeding, ‘flower method’ (oral)

• 5 doses test item + control + reference

• 2 tests replicates in time

• Mortality, behavior

Endpoint: Oral & contact LD50 (µg a.i./bee/48 h)

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?

EFSA Guidance document Solitary bees higher tier studies

Method: - collection of ‚non-treated‘ food provisions and eggs - sexing and transfer to lab - pesticide application on / into provisions (without moving egg) - mortality / development / emergence

see EFSA PPR (2012a)

Uncertainties: - synchronization - homogenization - replication - repeatability

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Ladurner et al. (2008) , EFSA PPR (2012a)

EFSA Guidance document Solitary bees higher tier studies

Method: - 40 m2 tunnels (Phacelia) - nesting shelter with sufficient holes - 10-15 newly emerged bees of both sexes - sufficient tunnel replicates/treatment - subsequent progeny maintaining

Assessments: 1. day 0, 1, 2 & 4: nesting behavior, foraging activity & cell production rate 2. day 0, 1, 2 & 4: adult & larval mortality 3. offspring emergence, body weights & sex ratios (<7%)

? phenology?

? more (~30-50)?

?

? 1.+2. more robust replication of overall reproductive success (covering: 1-(Emergence / Cell production) = Mortality)?

Unclear: exposure duration adjusted to limited crop flowering? Offspring vigour?

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EFSA Guidance document Solitary bees higher tier studies

Semi-field tunnel versus indoor cage

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EFSA Guideline Solitary bees higher tier studies

Torchio (1973)

Method: -Nesting shelters in the middle of each field providing sufficient holes - ≤ 400 females, ≤ 200 males per field - sufficient field replicates/treatment - assessments similar to tunnel approach (with similar problems…)

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Validation Working Groups

INTERNATIONAL COMMISSION FOR PLANT-BEE RELATIONSHIPS

Non Apis Group

AG BIENENSCHUTZ

sjef.vandersteen@wur.nl

IngoTornier@eurofins.com

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Thank you for your attention!