What toxicants can teach us
about metabolic organisation …
Tjalling Jager
Dept. Theoretical Biology
Contents
 “There’s something rotten in the field of
ecotoxicology” …
 Toxic effects in a DEB context
 ‘DEBtox’: an incomplete history and recent
developments
 Challenges for further research
Talking about toxicants …
Ecotoxicology:
Study of effects of chemicals on organisms
 In practice …
•
•
•
•
exclude humans, mice, rats, rabbits, etc.
exclude other mammals and birds
all levels of organisation (molecule to ecosystem)
stronger link to risk assessment than to science
 What is “rotten” in ecotoxicology?
• basic tool: the standard toxicity test
Reproduction test
Reproduction test
Reproduction test
Reproduction test
wait for 21 days …
Range of Concentrations
total offspring
Dose-response plot
EC50
log concentration
What use is an EC50?
 The EC50 …
•
•
•
•
changes in time and differs between endpoints
time patterns are chemical-dependent
depends on test conditions (e.g., pH, temp, food)
depends on definition of response (e.g., total offspring or
reproduction rate)
 And therefore …
• tests are highly standardised by OECD, ISO, ASTM
EC50
“Something rotten …”
Ecotoxicologists are asking the wrong question
 Which concentration leads to 50% effect under highly
standardised test conditions?
Species: Daphnia magna
Age:
less than 24 hours
Duration: 21 days
Endpoint: total # offspring
Temp.:
18-22 °C
Food:
0.1-0.2 mg C/ind./day
OECD (1998)
toxic
effects
“Something rotten …”
Ecotoxicologists are asking the wrong question
 Which concentration leads to 50% effect under highly
standardised test conditions?
Better questions
 What is the process by which a toxicant affects an
organism’s life history?
 How can we use test data to predict population
response under field-relevant conditions?
Look closer at individual
Look closer at individual
Look closer at individual
Look closer at individual
Natural role for DEB
Toxic effects have energetic consequences
Understanding effects on reproduction requires
understanding how food is turned into offspring
(start of DEB theory 30 years ago!)
Natural role for DEB
Toxic effects have energetic consequences
Understanding effects on reproduction requires
understanding how food is turned into offspring
(start of DEB theory 30 years ago!)
Assumptions for toxicant effects
1. Effects are caused by internal concentrations
2. Internal concentrations affect DEB parameter(s)
DEBtox concept
external
concentration
(in time)
toxicokinetics
internal
concentration
in time
DEB
parameters
in time
DEB
model
effects
in time
Potential targets
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
body length
cumulative offspring
Potential targets
time
Jager et al. (2004)
TPT
time
Potential targets
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
Potential targets
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
body length
cumulative offspring
Potential targets
time
Alda Álvarez et al. (2006)
Pentachlorobenzene
time
Potential targets
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
Potential targets
body length
cumulative offspring
Chlorpyrifos
time
Jager et al. (2007)
time
DEBtox concept
external
concentration
(in time)
Affected DEB parameter has
specific consequences for life cycle
toxicokinetics
time-varying
concentrations
internal
concentration
in time
DEB
parameters
in time
temperature
Extrapolation!
food limitation
DEB
model
effects
in time
A brief history of ‘DEBtox’
egg
Corresponds with
origin of DEB in
1979
A brief history of ‘DEBtox’
egg
The 80’s …
 Kooijman (1981)
• toxicokinetics determines
survival pattern
 Kooijman & Metz (1984)
• toxicants affect energy
budgets and thereby
population response
A brief history of ‘DEBtox’
egg
The early 90’s …
 Parallel to OECD trajectory
• review test guidelines with
respect to statistical analysis
• 1996: “analyse time course of
effects” and “study
mechanistic models”
A brief history of ‘DEBtox’
Birth in 1996 …
 Windows software and
booklet (Kooijman &
Bedaux, 1996)
 Series of papers
• Bedaux & Kooijman (1994)
• Kooijman & Bedaux (1996)
• Kooijman et al (1996)
A brief history of ‘DEBtox’
And 10 years later …
 ISO/OECD (2006)
• DEBtox next to methods for
NOEC and EC50
 ECB workshop (2007)
• presenting DEBtox to EU
risk assessors
A brief history of ‘DEBtox’
The 2000’s …
 Péry et al (2002, 2003)
• modifications for midges
 Ducrot et al (2004, 2007)
• midges and snails
 Lopes et al (2005)
• link to matrix models
 Billoir et al (2007, 2008)
• matrix models, Bayes, new
derivation
division
A brief history of ‘DEBtox’
In our group …
 Jager et al (2004), Alda
Álvarez et al (2005, 2006)
• multiple endpoints and
ageing
• population (Euler-Lotka)
 Baas et al (2007, 2009)
• mixtures: lethal effects
• (sub-lethal subm.)
division
A brief history of ‘DEBtox’
Embryo division …
 Klok & De Roos (1996),
Klok et al (1997, 2007)
• earthworm matrix model,
Bayesian approach
A brief history of ‘DEBtox’
The near future …
‘DEB 3’
Limits of ‘DEBtox’
To work with standard test data …
‘DEBtox’ applied a simplified DEB model
• constant reserve density
• constant length at puberty
• constant cost for an egg
Constant reserve density …
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
Constant length at puberty …
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
Constant egg costs …
food
faeces
assimilation
reserves
somatic maintenance
maturity maintenance

structure
1-
maturity
offspring
‘DEB 3’
 Explicit calculation of maturation
• maturity level → hatching and onset reproduction
•
→ maturity maintenance
 Explicit calculation of egg costs
• may change under (toxic) stress
 Disadvantages
• data requirements
• computation more complex
• parameter interpretation
Kooijman et al (2008)
Toxicants and DEB 3
Example:
• Nematode Acrobeloides nanus
• Data from Alda Álvarez et al (2006)
Pentachlorobenzene
65
200
cumulative offspring per female
60
55
body length
50
45
40
35
30
25
20
15
180
160
140
120
100
80
60
40
20
0
5
10
15
time
20
25
30
0
0
 mode of action: decrease of ingestion rate
 allows estimation of all basic parameters
 somatic maint. coeff. = maturity maint. coeff.
5
10
time
15
20
Cadmium
180
cumulative offspring per female
65
60
55
body length
50
45
40
35
30
25
140
120
100
80
60
40
20
20
15
160
0
5
10
15
20
25
30
35
time
 MoA: increase costs for structure
 decrease maturity maintenance?
 .
0
0
5
10
time
15
20
Cadmium
180
cumulative offspring per female
65
60
55
body length
50
45
40
35
30
25
140
120
100
80
60
40
20
20
15
160
0
5
10
15
20
25
30
35
time
 MoA: increase costs for structure
 decrease maturity maintenance?
 increase ageing?
0
0
5
10
time
15
20
Cadmium
180
cumulative offspring per female
65
60
55
body length
50
45
40
35
30
25
140
120
100
80
60
40
20
20
15
160
0
5
10
15
20
25
30
35
time
 MoA: increase costs for structure
 decrease maturity maintenance?
 increase ageing?
0
0
5
10
time
15
20
Lessons and questions
 Using ‘DEB 3’ allows more flexibility
• any parameter can be affected by toxicant
• fully consistent consequences of effects
• but … user-friendliness is decreased
 Pentachlorobenzene:
• effect through ingestion
• basic parameters can be estimated
Lessons and questions
 Cadmium:
• effect on costs for structure
• and … effect on maturity maintenance and ageing?
 maturity maintenance linked to structural costs?
 maturity maintenance trading off with ageing?
Lessons and questions
 Cadmium:
• effect on costs for structure
• and … effect on maturity maintenance and ageing?
 maturity maintenance linked to structural costs?
 maturity maintenance trading off with ageing?
 How to address these questions:
• other endpoints, e.g., length at birth (30% reduction)
• other chemicals/species with this mode of action
Where do we go?
DEB 3 allows to
address new
questions
Avenues for further research
Challenges opened up by ‘DEB 3’
 Interaction toxicants and egg development
• egg costs determine reproduction rates
• hatching time and size are essential for population
 Consistent analysis when puberty length is affected
• e.g., cadmium in some nematodes
• compounds that affect kappa …
Natural ‘toxicants’
Suspected kappa effects:
 Trematodes in snails
• Gorbushin and Levakin (1999)
 Microsporidian in Daphnia
• Chadwick and Little (2005)
 Fish infochemicals on Daphnia
• Stibor (1992)
Natural ‘toxicants’
Man-made endocrine
disruptors?
 Daphnia and ibuprofen?
• Heckmann et al (2007)
 Nematodes and nonyl-phenol?
• Höss et al (2002)
Avenues for further research
Challenges opened up by ‘DEB 3’
 Nature of maturation and maturity maintenance
• is maturity maintenance ‘optional’? at what costs?
• why is constant length at puberty so common?
 Interaction between ageing and toxicants
• both on survival and reproduction
Toxicants influence ageing
0.7
1
0.6
fraction surviving
volumetric body length (mm)
Folsomia candida
and cadmium
0.5
0.4
0.3
0.2
0.8
0.6
0.4
0.2
0.1
0
0
20
40
60
80
time (days)
Jager et al. (2004)
100
120
0
0
20
40
60
80
time (days)
100
120
Toxicants influence ageing
Acrobeloides nanus
and carbendazim
70
cumulative offspring
500
body length
60
50
40
30
400
300
200
100
20
0
5
10
15
time
Alda Álvarez et al. (2006)
20
0
0
10
20
30
time
40
50
60
Where do we go?
Birth of new
embryonic
ideas?
Avenues for further research
New embryonic idea
 Link between metabolic and molecular level
• enormous development in molecular biology
Molecule to metabolism
toxicant
target site
metabolic process
?
genes
maintenance
assimilation
maturation
…
gene products
?
life history
Swain et al. (subm.)
Final words …
 Understanding toxic effects requires energy budgets
• started DEB development 30 years ago
• essential for ecotoxicology and risk assessment
 Understanding energy budgets requires toxicants
• shed light on e.g., maturation and ageing
• often requires ‘DEB 3’ formulation
 Toxicants stress organisms in very specific ways
• some 100,000 man-made chemicals registered
• plus whole range of natural ‘toxicants’
 However, DEB analyses raise new questions …
• modellers must have close links to the lab