Assessment of effects: laboratory level - methods and...

Assessment of effects: laboratory

level - methods and relationship to

protection goals

8th SETAC Europe Special Science Symposium

Brussels, Belgium

October 16-17, 2013

Stephan Schmitzer1, Jens Pistorius2

1 IBACON GmbH, Arheilger Weg 17, 64380 Rossdorf, Germany 2 Julius Kühn-Institut (JKI), Messeweg 11-12, 38104 Braunschweig, Germany

8th SETAC Europe Symposium, 16-17 October, Brussels

Introduction

• The implementation of the EU Regulation 1107/2009, Publication of

the EFSA Guidance Document, (EFSA 2013) as well as requirements

of US-EPA/PMRA require further Efforts in Method Development and

Validation of Methods.

• This session will give an overview of:

• recently validated guidelines/methods

• new methods

• new guidelines/Guidance Documents

• current status in method development

• limitations of the requirements

• linking endpoints to Protection Goals


Test methods

I. Acute Contact and Oral Toxicity Test in the Laboratory (OECD

213/214)

II. Chronic oral 10 days Feeding Test

III. Honey Bee Brood/Larval Testing

single exposure larval toxicity test (OECD TG 237)

repeated exposure larval toxicity test (Guidance Document)

Oomen Brood Feeding Test (EPPO 1992)

IV. Bumble Bee Acute Toxicity Laboratory Test (Glasgow, 2013)


Acute Contact and Oral Toxicity Test (OECD 213/214)

• Validated Guideline available since 1998

• Standard Laboratory Test subdivided in an

• acute contact toxicity exposure test and

• acute oral toxicity exposure test

• Measurements: Mortality and behavioural abnormalities

• Endpoint: Contact LD50 (48 hrs) and Oral LD50 (48 hrs)

Start of the experiment:


Oral toxicity (OECD 213)

Contact toxicity (OECD 214)

bees will share food

no anaesthesitation required

Anaesthesitation with CO2

Alignment

Application


Acute Contact and Oral Toxicity Test (OECD 213/214)

Recent Development/Requirements

• Honeybee subspecies of the relevant authorisation zone should be

used

• All sublethal behavioural abnormalities should be noted [quantitative

and qualitative]

• Notes of Report: Age, health status of colonies, subspecies, etc.

• Endpoint: Contact and oral LD50 [µg/bee] at 48 hrs


Linking endpoints to protection goals

Data from tests on acute oral and contact, chronic oral, larval toxicity used

for calculation of trigger values (combining exposure and effects)

• Worst-case conservative approaches are used for oral and contact exposure:

Based on contact exposure data, food consumption and default Residue per

Unit Dose (RUD) figures: Shortcut-values (SV) are derived (express worst

case theoretical residue intake via pollen and nectar)

Oral: nectar from the treated crop, weeds in the field, plants in field margin,

the adjacent crop or succeeding crop/permanent crop the following year

Contact: either from spray deposits (i.e. overspray or spray drift) or from dust

particles


Linking endpoints to protection goals

Link to protection goal calculated

using of Khoury model to

determine acceptable mortality

levels – Instead of background

Mortality (M = 0.153) in K. the

relative increase in mortality (=

factor of increase of background

mortality) was used derive trigger

values

EFSA, Dr. F. Streissl, 9.9.2013


Further optional refinements

• Refinement of SV-values with measured values (residue

measurements, sugar content measurements, pollen consumption?)

• Exposure factor (Ef)- values, considered in the exposure estimation

for relevant scenarios (e.g. spray drift for field margin, will lower

exposure of risk assessment. Used e.g. for calculation of deposition

onto weeds, dose deposited off-crop (spray or dust drift))

• Further refinement of Guidance possible as more information is

available:

• Background mortality rates of foragers

• Larvae mortality and effects on colony size

• Effects on development of HPG and colony size

• Calibration of trigger values with field studies


Chronic Adult 10 Days Oral Toxicity Test

No guideline available, but some publications (CEB 230, Decourtye et

al., 2005, Suchail et al. 2001)

• Young worker bees will be fed continuously over 10 days

• Measurements: Food Uptake per bee/day and Mortality

• Endpoint: LC50 and NOEC after 10 days (LD50 and NOED)

• Observation of behavioural abnormalities

• Proposal to determine effects on the hypopharyngeal glands (HPG`s)

• Test Design similar to OECD 213; difference:

• young bees

• prolonged exposure phase

• adopted climatic conditions


Chronic Adult 10 Days Oral Toxicity Test - Ringtest

A ring test* was initiated in Spring 2013 by the German National Bee

Protection Group („AG Bienenschutz“)

• harmonising the current test procedure

• adopting the current knowledge into a „GLP-like“ test method

• Exchange of experiences from different laboratories

• Collecting validated data

• Creating a method based on the results of a standardised ring test

• Evaluation and Publication of the Results in the near Future

• Proposal of a Guidance/Guideline to OECD intended

* Eurofins, IBACON, BioChem Agrar, BASF SE, BAYER CropScience, LAVES


Chronic Adult 10 Days Oral Toxicity Test - Ringtest

Finding solutions of recent „problems“:

A. best method to obtain fresh emerged worker bees

B. control mortality, and appropriate rate of the reference item,

C. keeping of the young bees, climatic conditions throughout the test

D. preparation of the test solutions (daily or once for the whole test)


Ringtest – Results and Recommendations

A. best method to obtain fresh emerged worker bees

Comb brought directly to an incubator (bees will hatch in the incubator)

1-3 days before start of test one or more brood combs with

sealed brood (ideally containing pollen) is taken out from the

hive.

during the following days, freshly

emerged bees will be taken out

and used for the test.

or, brood comb will be placed into an

excluder box, brought back to the hive

(bees will hatch in the excluder and will

be fed by nurse bees)



B. control mortality, and appropriate rate of the reference item

Table: Results of the Ring Test started in Summer 2013 *

Treatm Conc.

[mg

a.i./kg]

Mort.

[%]

Lab 1

Mort.

[%]

Lab 2

Mort.

[%]

Lab 3

Mort.

[%]

Lab 4

Mort.

[%]

Lab 5

Mort.

[%]

Lab 6

Control - 0.0 % 0.0 % 0.0 % 3.0 % 6.7 % 0.0 %

Dimeth

oate

0.2 0.0 % 0.0 % 20 % 7.0 % 23.3 % 3.3 %

0.4 3.3 % 13.3 % 5 % 6.0 % 100 % 23.3 %

0.6 30.0 % 60.0 % 5 % 13.0 % 100 % 78.3 %

1.0 100 % 100 % 100 % 85 % 100 % -

LC50 0.65 0.55 0.71 0.77 0.39 Mean: 0.61

LD50 0.023 0.016 0.02 0.018 0.009 Mean: 0.017

* Eurofins, IBACON, BioChem Agrar, BASF SE, BAYER CropScience; LAVES



C. keeping of the young bees, climatic conditions throughout the test:

Recommendation: 33 °C ± 1 °C.

best method to obtain fresh emerged worker bees:

Recommendation: using 1 – 3 days old freshly emerged bees via sealed

brood

D. preparation of test feeding solutions:

depends on the stability of the test substance; reference item

[dimethoate] can be prepared once throughout the test (reproducible and

reliable results).



• Young and fresh emerged bees can be obtained by using combs with

sealed brood and transferred to the lab

• On average a bee consumes 20-30 mg feeding solution/day

• For the first 2 days, food uptake is lower compared to latter days

• Control Mortality of 15 % is manageable

• A reference item concentration of 1 mg a.i./kg shows sufficient effect

• 10 bees per cage should be used (instead of 20 or more)

• Temperature during hatch and keeping of the bees should be 33 °C

• High relative humidity should be considered

• No additional pollen and water is necessary during the test

This all is proven by the very low control mortality of the data of the

ringtest


Hypopharyngeal Glands

• Preparations of the Glands


Chronic Oral Toxicity Test - Linking endpoints to protection

goals

• Use of LC50/NOEC values

• Use and need of chronic toxicity test is clear

• HPG-”to cover potential effects on brood care”. But: quantitative

relationship to assessment endpoints (e.g., brood development,

colony strength and survival) has not yet been developed.

Significance of findings is uncertain and unclear. Cost-benefit?

(For example, depending on the administered dose of dimethoate (insecticide) the

protein content is increased or decreased. Moreover, the protein content is also

influenced by a number of external factors.

• link with protection goals is not fully clear for HPG. Which Trigger

values need to be used, justification?

• Further investigation needed to establish link between test endpoint

and protection goals as well as test methodology before

implementation!


Larval Toxicity Test

• Common sense that data package was sufficient for an acute larval test method until day 7

• another expert meeting was found to be necessary

• Data of the assessment period from day 7 until hatch are insufficient

• Further ringtesting is required for the chronic larval toxicity test

Meeting of expert group on honey bee toxicity testing (October 2012,

OECD)

• no ring test was started between October 2012 and now

• no new data were obtained

Guideline for acute (single exposure) larval toxicity

finalised July 2013 (OECD TG 237)

• The document was commented

• a meeting is planned for October 30-31, 2013

new document provided by the OECD on the

chronic larval toxicity test (repeated exposure) as a

„Guidance Document“



TG 237 – acute exposure on day 4, end on day 7

New GD– chronic exposure on day 3-6, end on day 22



TG 237 – exposition phase from day 1 to day 7

<<

isolation of queen grafting single test unit

Application on day 4 assessments


Larval Toxicity Test - Linking endpoints to protection goals

• Larval toxicity - no quantitative link to protection goals- thus, NOEC

values are used

• Endpoint should be hatch; so far a lot of labs do not achieve

acceptable results yet. Problem is the period pupation up to hatch.

• By using only a toxicity endpoint at D7, e.g. IGR effects would be

missed

• Repeated exposure reflects better the situation in the hive, thus is

considered to provide data which supply the risk assessors with

data of higher value compared to TG 237

• Yet the methodology proposed in TG 237 from pupation to hatch

seems not yet sufficiently robust, further work is urgently needed

to establish hatch as endpoint


Oomen et al. in-hive Feeding Method

• Since implementation of EU Regulation 1107/2009 (Annex II & III) and

EFSA Guidance Document on Risk Assessment on honeybees, the

test should be used on a routine base if products fail the larvae test

trigger value

• Method to detect effects of chemicals on different honey bee

development stages

• In-hive feeding Test

• Published in 1992, only rough description of the method available

• Actually aimed for IGR`s

• Method not yet validated

• Test is conducted since many years

• EFSA provides specific proposals for “improvements” for the method


Oomen et al. in-hive Feeding Method

• Full field test, with free flying honey bees

• Exposure via spiked sugar solution provided directly to the colony

• 10.000 to 15.000 bees

• limit test with 3 Replicates

• Test duration: 21 Days

• Mortality assessments: daily until test end (day 21)

• marking and assessment of the development of 100 Eggs, 100 young

larvae, 100 old larvae via Acetate Sheet Method

• Marking of development stages on BFD 0, 7, 14, 21

• End Point: Mortality, Termination Rate


Oomen et al. in-hive Feeding Method - Ringtest

* IBACON, Eurofins, BioChem Agrar, BASF SE, BAYER CropScience, IES

A ring test was initiated in Spring 2011 by the German national Bee Protection Group („AG Bienenschutz“*)

• Group consists ~ 10 Members; Initiation in 2011

• 2 Meetings in 2012;

• Collection and Evaluation of historical Data

• 17 Studies were evaluated over a testing period from 1997 to 2012, publication of the Results at the SETAC GLB Conference in Essen, September 23 - 26, 2013

• Creation of a Ring-Test Protocol (April 2013) and Start of a new Ring Test in 2013 (August 2013)

Outlook:

• Collection and Evaluation of the Results of the Ring-Test started in 2013

• Presentation of the Results at the ICBPR Meeting in Gent 2014


Oomen et al. in-hive Feeding Method – Ringtest Results • Control BTR`s can be

considered in the context of

the historical data

• Development stages

showed different sensitivity

• Study less sensitive to

climatic conditions

• Big colonies should be used

Eggs yL2 oL3 Eggs yL2 oL3

1 / 1997 19.9 11.4 6.8 99.8 100.0 33.4

2 / 2002 10.2 6.7 6.9 90.0 30.0 13.0

3 / 2008 5.8 3.6 2.0 42.8 49.2 3.9

4 / 2010 20.0 - - 24.2 - -

5 / 2011 15.6 32.5 7.9 99.8 99.8 74.8

6 / 2011 41.2 13.7 8.8 100.0 99.8 32.5

7 / 2011 31.6 33.0 6.9 85.6 20.2 1.3

8 / 2011 8.3 3.5 3.7 38.8 12.4 3.6

9 / 2011 32.0 38.0 10.4 67.5 72.2 61.9

10 / 2012 16.0 18.9 2.7 97.4 83.1 16.2

11 / 2012 18.9 14.6 3.3 100.0 97.8 54.3

12 / 2012 60.4 51.0 10.0 94.9 72.1 15.8

13 / 2012 31.7 16.7 13.7 99.7 81.7 17.3

14 / 2012 9.3 7.0 24.7 17.7 9.3 12.3

15 / 2012 25.3 12.7 1.7 64.0 14.3 10.3

16 / 2012 41.1 7.7 5.0 85.4 43.9 51.8

17 / 2012 16.7 6.7 8.0 89.8 39.8 52.7

Mean 23.8 17.4 7.7 76.3 57.9 28.4

SD 14.4 14.0 5.6 28.5 34.3 23.61 = at test end on day 21-22 - = not assessed

2 = young larvae;

3 = old larvae

Study No. /

Year of

performance

Termination Rate [%] 1

Control Reference Item

• Control BTR`s can be considered as: Eggs: 25 %; YL: < 20 %; OL: 10 %)

• Control colonies with > 10,000 bees displayed more frequent BTRs <20 % than smaller ones (Fisher`s exact test, p=0.0217)

• Replicates with lower BTR showed frequently increased pupae mortality: verification of exposition of the bee brood to PPP can be either done by a high BTR and/or high pupae mortality

• Photo-method should be used

• Assessments should be adopted to OECD 75 (BFD 0, 5, 10, 16, 21)

• Chronic Feeding over 9 days is possible at a rate of 75 mg a.i.fenoxycarb/L.

Proposal:


Oomen et al. – Linking endpoints to protection goals

• considered as intermediate between lower (Lab) and higher tier tests

(Semi-field and Field).

• no direct quantitative link to protection goals possible

• Possibly to be used if concern is raised only regarding the potential

effects on larvae (i.e. only the ETRlarvae trigger value breached)

• EFSA recommends run under semi-field or field conditions

• assessment of the effects after exposure to (a) defined

concentration(s) of the test item in the sugar solution fed to bee

colonies

• designed for investigation of effects following oral exposure of adult

bees and especially of oral exposure of bee brood

• Classification of the results as „black-or-white“

• If effects are observed, further higher tier tests e.g. OECD 75 method

in semi-field and field are deemed necessary


Laboratory Toxicity Test on Bumble Bees (Bombus terrestris L.)

• High Importance

• Validated Test methods are not available

• Adaptions to OECD 213/214 possible

• Contact Test • relatively easy to conduct

• Oral test • individual feeding is necessary

• Bumble bees do not show trophalaxis

• no active feeding behaviour

• New oral toxicity method for bumble bees was presented by BASF SE on the SETAC Conference in Glasgow, 2013

• Suitable and useful method

• Reproducible

• Comprehensive results


Laboratory Oral Toxicity Test on Bumble Bees (Bombus

terrestris L.)


Results of Laboratory Toxicity Test on Bumble Bees

00%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

control 0.25 0.5 0.75 1 2

Mo

rta

lity

[%

]

Treatment [µg a.i./bee]

IBACON

BASF 1. Trial

BASF 2. Trial

• The first trial with the new method resulted in reliable and comparable results

• Daily food uptake was 406 mg/bumble bee (196-447 mg/bee)

• All introduced individuals consumed the offered test solutions

• Provided contaminated dimethoate solution (40 µL/bumble bee) was ingested within 5 minutes!

• LD50 values [48 hrs] in all trials were between 0.5 and 2.5 µg dimethoate/bumble bee (IBACON: 1.25 µg a.i./bee)


Bumble Bees – Linking endpoints to Protection Goals

• LD 50 oral/contact values μg/bumble bee

• Due to inavailability of specific tests, other endpoints from Honey bee

tests (chronic, larvae, pot. accumulative effects) are used with bumble

bee specific shortcut values (SV) for bumble bee risk assessment

• SVs were calculated for time of larval development: 5 days Honey

bee larva, Bumble Bee larva for 1 day and Solitary Bee larva for 30

days

• proposed safety factor of 5 is intended to cover the uncertainties of

higher ecological sensitivity

• proposed safety factor of 10 is intended to cover the uncertainties of

higher toxicological sensitivity (in 95% of the cases the difference in

sensitivity was less than a factor of 10)


Conclusions

• Acute toxicity tests (OECD 213/214) on honey bees are well

established

• Chronic 10 days oral feeding tests are manageable and a test method

or guideline should soon be implemented.

• Implementing data on hypopharyngeal glands into regulatory

framework should not be done before standardised test methods

have been established

• Acute Larval toxicity testing up to day 7 established- can be used in

the risk assessment

• Emphasis should be done on the development of a chronic toxicity

guideline on honey bee larvae from pupation to hatch

• The Oomen method can be adapted to current needs

• Acute contact and oral bumble bee tests in the laboratory are possible


Conclusions- linking endpoints

• Data from tests on acute oral and contact (LD50) in combination with

shortcut values used for calculation of trigger values (combining

exposure and effects) to ensure achievement of Protection goals

• For chronic and larval tests, no quantitative link is available, NOEC data

are used for calculations

• Due to unavailability of some tests, high and conservative safety factors

are included, using data from hb tests for extrapolation to other species

• Urgent need of further development of suitable standardized studies to

provide endpoints which can be directly related to assessment

endpoints,subsequent effects on survival, growth or reproduction and

frequently assumptions are made regarding potential relationships


Thank you for your attention!

Thanks to the Ring Test Participants:

• Johannes Lückmann (RIFCON)

• Marcus Barth (BioChem Agrar)

• Anja Wehner (Eurofins)

• Roland Becker (BASF SE)

• Nicole Hanewald (BASF SE)

• Annette Kling (Eurofins)

• Jenny Krämer (IBACON GmbH)

• Lukas Jeker (Knoell AG)

• David Gladbach (BAYER CropScience)

Pictures by Roland Kuhl, IBACON GmbH

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