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Using the DEBkiss model to integratively assess effects of tributyltin on the freshwater gastropod Lymnaea stagnalis
Alpar Barsi, Tjalling Jager, Laurent Lagadic,
Virginie Ducrot
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• 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
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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
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• 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
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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
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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
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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
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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
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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
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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
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• 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
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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
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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
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0 5 10 15 20 25 30 35 1
1.5
2
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3.5
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time (d)
cubic
root dry
body
weig
ht
(mg)
0 5 10 15 20 25 30 35 18
20
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time (d)
shell
length
(m
m)
0 5 10 15 20 25 30 35 0
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time (d)
cum
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tive n
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of eggs
0 5 10 15 20 25 1
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time (d)
cubic
root dry
body
weig
ht
(mg)
0 5 10 15 20 25
18
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time (d)
shell
length
(m
m)
0 5 10 15 20 0
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time (d)
cum
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tive n
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of eggs
Ad
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s Ju
ven
iles
Fitting the data for the control groups - juvenile and adult snails -
Results and discussion
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0 5 10 15 20 25 30 35 0
50
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time (d)
cum
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tive n
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of eggs
0 5 10 15 20 0
50
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time (d)
cum
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tive n
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of eggs
0 5 10 15 20 25
1
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time (d)
cubic
root dry
body
weig
ht
(mg)
0 5 10 15 20 25
18
20
22
24
26
28
30
time (d)
shell
length
(m
m)
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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time (d) cubic
root dry
body
weig
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(mg)
0 5 10 15 20 25 30 35
18
20
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24
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28
30
time (d)
shell
length
(m
m)
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
ult
s Ju
ven
iles
Fitting the data for the exposure groups - juvenile and adult snails -
Results and discussion
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• 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
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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
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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
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Thank you for your attention!
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.
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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.