Using the DEBkiss model to integratively assess effects of tributyltin on the freshwater gastropod Lymnaea stagnalis

Alpar Barsi, Tjalling Jager, Laurent Lagadic,

Virginie Ducrot

• An endocrine disrupter (EDC) is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations (WHO/IPCS 2002)

• The endocrine system regulates the metabolism and function of the body

Background

Which chemicals are EDCs? • certain pesticides/biocides (DDT) • industrial chemicals (PCBs, bisphenol A) • plant hormones (phytoestrogens)

• alterations of hormonal balance (energy) • reproductive abnormalities (early puberty, affect

fertility and fecundity, male/female ratio) • developmental impairment • diseases • behavioural changes • teratogenic effects…

Why EDCs are of concern?

Background

scienceclarified.com

Noriega et al. 2000

• Directive on PPP (EC 1107/2009): marketing and use of chemical products shall be approved only if they do not have endocrine disrupting properties that may cause adverse effects on populations of non-target organisms under realistic conditions

• Constrains for practical application of the directive: - scientific criteria for identifying EDs properties are not agreed yet - terms “adverse effects” and “realistic conditions” are vague - the population effects need to be assess - standard guidelines are needed

Background

OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals (revised in 2012)

• Standardisation of toxicity tests with molluscs is on the way

Background

Effects of TBT in molluscs: • imposex – superimposition of male sex organs on a

female, registered in more than 200 gastropod species (Europe, US, Japan, South America)

• intersex • sterilisation of females • shell abnormal thickening in oysters • decreased fecundity in freshwater snails • egg abnormalities in freshwater snails

• Tributyltin (TBT) has been used in antifouling paints

Background

Giusti et al. 2013 Higuera-Ruiz et al. 2000 Schulte-Oehlmann et al. 2004

Study objectives: • to assess relevance of the test protocol (OECD) for L. stagnalis • to provide data suitable for mechanistic modelling of effects of EDCs • to parameterize and calibrate a mechanistic model for individuals in control

and contaminated conditions

Aims of the study: to develop test methods and data analysis tools to evaluate toxic effects of TBT on a freshwater snail Lymnaea stagnalis

Aim and study objectives

Materials and methods

Test animal The pond snail Lymnaea stagnalis • A freshwater hermaphroditic species, common in the northern hemisphere • It has been proposed as a candidate species for OECD standard molluscs

reproduction toxicity tests

Photo: M.Collinet – INRA A.Barsi – INRA

L. stagnalis – the pond snail

Feeding: ad libitum by fresh organic lettuce

Test duration: 28 days

Photoperiod: 14:10 h light:dark

⁰C Water temperature: 21⁰C

Semi-static conditions. Renewal of the test medium: twice a week

Materials and methods

Adult test setup (OECD standardisation)

TBT exposures (ng/L):

325 1300 2600 650

Endpoints followed over time: • survival – twice a week • shell length – once a week • body dry mass – end of the test • cumulative number of eggs –

collected twice a week

Control treatment

6 X 5 adult snails

Exposure treatment

6 X 5 adult snails

Solvent: acetone 2 µl/L

Feeding: ad libitum by fresh organic lettuce

Test duration: 35 days

Photoperiod: 14:10 h light:dark

⁰C Water temperature: 21⁰C

Exposure treatment

4 isolated juvenile and adult snails

Control treatment

16 isolated juvenile and adult snails

Semi-static conditions. Renewal of the test medium: twice a week

Materials and methods

Juvenile test setup

TBT exposures (ng/L):

11 53 117 258 24

587 1995 2743 907 1247

Endpoints followed over time: • survival – twice a week • shell length – once a week • body dry mass – end of the test • cumulative number of eggs –

collected twice a week

Solvent: acetone 20 µl/L

• Toxic effects modelled by implementing a toxicokinetic-toxicodynamic (TK-TD) model • One-compartment first-order toxicokinetic model • Accounting for concentration dilution by growth and by losses due to reproduction • Toxicodynamics modelled using a process model for the organism

Mechanistic approach as a basis for modelling toxic effects

Materials and methods

• Survival

• Dry mass of egg clutches

• Shell length

Input data Model outputs…

• Survival

• Reproduction • Growth

…predictions as mean values of responses

toxicological parameters

physiological parameters

TBT exposures

external concentration

(in time) toxico-kinetic

model internal

concentration in time

toxicokinetics

process model for the organism

effects on endpoints

in time

toxicodynamics

TK-TD model

Conceptual approach for the “process model for organisms”

Dynamic Energy Budget (DEB) theory

Materials and methods

The illustration taken from the previous presentation of Elke Zimmer

Simplified DEB model – DEBkiss (poster no. 18, Jager et al.)

• an explicit mass balance for an animal over its entire life cycle • less parameters than standard DEB model • convenient model for ecotox studies on invertebrates

Process model for L. stagnalis

Materials and methods

0 5 10 15 20 25 30 35 1

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Fitting the data for the control groups - juvenile and adult snails -

Results and discussion

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0 ng/L

325 ng/L

650 ng/L

1300 ng/L

2600 ng/L

0 5 10 15 20 25 30 35 1

1.5

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0 ng/L

11 ng/L

24 ng/L

53 ng/L

117 ng/L

258 ng/L

567 ng/L

907 ng/L

1247 ng/L

1995 ng/L

2743 ng/L

Ad

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s Ju

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Fitting the data for the exposure groups - juvenile and adult snails -

Results and discussion

• Toxicokinetics differs between two tests - different concentrations of the carrier solvent used

Mode of action (DEB framework) of TBT:

Decrease in energy assimilation from food

Results and discussion

Conclusions (I – model calibration): • Using only snapshots from the life cycle we can successfully

parameterise and calibrate the DEBkiss model • Our test designs provided data needed for parameterisation and

calibration of the model for TBT • The model allowed estimation of biologically relevant model

parameters for both control and exposed snails • Model can be validated (independent data exist)

Conclusions

Conclusions (II - test design): • An additional endpoint, the dry mass of the animal body, provides

information on body shrinking • Further recommendations for adapting test design for use of

mechanistic models (like DEBkiss): - measuring the dry body mass (at several time points if possible) - both dry mass of egg clutches and egg numbers should be included

in analyses - full life-cycle test

Thank you for your attention!

Alpar.Barsi@rennes.inra.fr

This research has been financially supported by the European Union under the 7th Framework Programme project acronym CREAM, contract number PITN-GA-2009-238148 and by French funds under the ”Programme Environnement-Santé-Travail de l’ANSES avec le soutient de l’ITMO cancer dans le cadre du plan cancer 2009-2013” project acronym MODENDO.

References: 1. WHO/IPCS (World Health Organization/International Programme on Chemical Safety). 2002. Global Assessment of the State-of-the-

science of Endocrine Disruptors. WHO/PCS/EDC/02.2, 180 pp. 2. Noriega NC, Hayes TB. 2000. DDT congener effects on secondary sex coloration in the reed frog Hyperolius argus: a partial evaluation of

the Hyperolius argus endocrine screen. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 126:231-237.

3. OECD (Organisation for Economic Cooperation and Development). 2010. Series on testing and assessment N° 121 Detailed Review Paper (DRP) on molluscs life-cycle toxicity testing. OECD, Paris, 182 p.

4. Giusti A, Barsi A, Dugué M, Collinet M, Thomé J-P, Joaquim-Justo C, Roig B, Lagadic L, Ducrot V. 2013. Reproductive impacts of tributyltin (TBT) and triphenyltin (TPT) in the hermaphroditic freshwater gastropod Lymnaea stagnalis. Environmental Toxicology and Chemistry 32:1552-1560.

5. Schulte-Oehlmann U, Oetken M, Bachmann J, Oehlmann J. 2004. Effects of Ethinyloestradiol and Methyltestosterone in Prosobranch Snails. In Kümmerer K, ed, Pharmaceuticals in the Environment. Springer Berlin Heidelberg, pp 233-247.

6. Higuera-Ruiz R, Elorza J. 2009. Biometric, microstructural, and high-resolution trace element studies in Crassostrea gigas of Cantabria (Bay of Biscay, Spain): Anthropogenic and seasonal influences. Estuarine, Coastal and Shelf Science 82:201-213.

7. Jager T, Martin BT, Zimmer EI. 2013. DEBkiss or the quest for the simplest generic model of animal life history. Journal of Theoretical Biology 328:9-18.