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Animal behaviour as a biomarker
of chemical stress
Ecotoxicology
Implement quantitative behavioural responses in the assessment of chemical stress in animals
Development of computerized video tracking systems for automated measurements of animal locomotor behaviour
To establish mechanistic links between cellular responses, behavioural changes and higher level effects of pollutants
To propose specific measurable components of animal behaviour as non-invasive health biomarkers in ecotoxicological research and environmental management
Scientific objectives
Impaired fitness
Disturbed population andecosystem stability- social behaviour- predator-prey interactions- reproduction- growth
Chemical pollution- speciation- bioavailable residues
Sensoryinterference
Absorption
Molecular responsesPhysiological responses
Structural damage
Exposure / effectbiomarkers
Effect / healthbiomarkers
x1 , y1 , time1x2 , y2 , time2x3 , y3 , time3
xi , yi , time i
Red
Green
Blue
Size
Shape
0 255
0 255
0 2550 0 1
Path lengthVelocitiesTurning behaviourActivity/Rest periods
Impaired fitness
Disturbed population andecosystem stability- social behaviour- predator-prey interactions- reproduction- growth
Chemical pollution- speciation- bioavailable residues
Sensoryinterference
Absorption
Molecular responsesPhysiological responses
Structural damage
Exposure / effectbiomarkers
Effect / healthbiomarkers
Uptake of dimethoate in woodlice
Control activity (22 hrs) Exposure (22 hrs)
14 C-Dimethoate0, 140, 280, 560
g ha-1
14C
ng
a.i.
/mg
wo
od
lou
se
Residual uptake of Dimethoate in woodlouse
at three application rates
0 100 200 300 400 500 600 700
0
1
2
3
4
5
6
meters
140 g / ha
240 g / ha
560 g / ha
Impaired fitness
Disturbed population andecosystem stability- social behaviour- predator-prey interactions- reproduction- growth
Chemical pollution- speciation- bioavailable residues
Sensoryinterference
Absorption
Molecular responsesPhysiological responses
Structural damage
Exposure / effectbiomarkers
Effect / healthbiomarkers
Prolonged effects of Dimethoate in woodlice
Night 1 Night 2 Night 3 Night 24
Control Exposure (140 g a.i./ha) Recovery
Control
140 g a.i./ha corresponds to 1/10 of the LD20 – 48 hours
1 2 3 24
60
80
100
120
140
160
180
40
60
80
100
120
140
60
70
80
90
100
110
60708090
100110120130140
Time in activity
Turning rate
Path
Average velocity
Per
cen
tag
e ac
tivi
ty (
Nig
ht
n/N
igh
t 1)
Night number
Prolonged effect of Dimethoate on woodlouse locomotor parameters
Exposed
Controls
Tim
e in
ve
loci
ty in
terv
als
Velocity intervals
Prolonged effect of an organophosphate on woodlouse velocity frequency distribution
0
1000
2000
3000
4000
5000
6000
0
1000
2000
3000
4000
5000
1 2 3 4 5 6 7 8 9 100
1000
2000
3000
4000
5000
Sec
on
ds
in e
ach
vel
oci
ty in
terv
al
Velocity interval
Control
34 hrs exposure
21 days recovery
Controls
Exposed (140 g / ha)
Exposure of a carabid beetle to copper during larval development
9 days10 days32 days
Cu
Cu
Altered locomotor behaviour in adult female carabid beetlesexposed to copper during larval development
Walkeddistance
met
er
0
50
100
150
200
250
300
Time inlocomotion
Sec
10
-3
0
5
10
15
20
Average velocity
mm
/ se
c
6
8
10
12
14
16
18
Movementdisruption
Sto
ps /
wal
ked
met
er60
80
100
120
Turningrate
Deg
rees
/ se
c
20
25
30
35
40
45
50
55
60
ControlsExposed
24 hours
Dimethoate application:- 0% - 7% - 15% - 26% - 59%
of LD50 (48H)
AChE inhibition and locomotor behaviour
AChE
Correlation between organophosphate application rate and acetylcholinesterase activity in a carabid beetle
Application rate (µg dimethoate / g fw beetle)
0 1 2 3
AC
hE
-akt
ivit
y(
µm
ol /
min
/ g
fw
bee
tle
)
0,0
0,1
0,2
0,3
0,4
0,5
Males Females
Males
Pa
th l
en
gth
(m)
50100150200250300350
Tim
e in
act
ivit
y(
ho
urs
)
0
1
2
3
4
Ave
rag
e ve
loci
ty(m
m/s
ec)
05
1015202530
0,0 0,1 0,2 0,3 0,4 0,5 0,6
01234567
Females
50100150200250300350
0
1
2
3
4
5
10
15
20
25
30
AChE aktivity (µmole/min/g fw)
0,0 0,1 0,2 0,3 0,4 0,5
Tu
rnin
g r
ate
(deg
rees
/mm
)
01234567
Pat
h le
ng
th
(m)
Tim
e in
act
ivit
y
( h
ou
rs)
Ave
rag
e ve
loci
ty
(mm
/sec
)
Tu
rnin
g r
ate
(deg
rees
/mm
)
Relationship between AChE activity andlocomotor behaviour in a carabid beetle
Control, Mean ± SE 5% LD50 (48 h), Mean ± SE10% LD50 (48 h), Mean ± SE23% LD50 (48 h), Mean ± SE
Impaired fitness
Disturbed population andecosystem stability- social behaviour- predator-prey interactions- reproduction- growth
Chemical pollution- speciation- bioavailable residues
Sensoryinterference
Absorption
Molecular responsesPhysiological responses
Structural damage
Exposure / effectbiomarkers
Effect / healthbiomarkers
Time (sec)0 200 400 600 800 1000 1200 1400 1600 1800
Dis
tan
ce (
mm
)
0
5
10
51
20
Pat
h l
en
gth
(m
m)
0
100
200
300
400
500
600
700
Collembola
Mite
Predator-prey interactions in a mite-collembola system
GROUP No. 1 2Number of contacts: 6 10Time to 1. contact: 526.5 136.9
Maximum duration: 524.6 666.6Minimum duration: 2.4 2.4
Total duration 559.8 826.5
Maximum distance: 14.7 15.3Minimum distance: 0.0 0.0Average distance: 4.6 4.6Time to max. meet.: 811.8 293.5Time to capture: 811.8 171.8Contacts until cap.: 4 2
ANIMAL No. 1 2 3 4Walked path 913 1034 419 275Walked path to cap. 830 1034 145 275Active time 1051.6 705.4 690.6 167.6Active time to cap.: 776.4 705.4 690.6 167.6
Time (min)
0 5 10 15 20 25 30
Cu
mu
lati
ve s
urv
ival
of
Co
llem
bo
la
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Females
Males
Kaplan-Meier analysis of collembolan survival- females are more efficient hunters than males
What is decisive for capture ?Size experiment
Sizes of mite and collembola: randomly paired (totally 81 cases)
Parameters considered in Cox Regression Model:Sex of miteSize of mite and collembolaSize ratioAverage velocities of mite and collembola, respectivelyFrequency of contactsTime to first contact
Parameters of importance for capture:Sex of miteSize ratioAverage velocity of miteFrequency of contacts
What is decisive for capture ?Starvation experiment
- mite starvation: 0, 4, 7, 22, 60 days (totally 131 cases)
Parameters considered in Cox Regression Model:Mite hungerAge of mite and collembolaTime in locomotor activity (mite and collembola)Mite and collembolan average velocitiesFrequency of contactsTime to first contact
Parameters of importance for capture:Time in locomotor activity of miteAverage velocity of miteFrequency of contacts
0 500 1000 1500 2000
Time (sec)
0.2
0.4
0.6
0.8
1.0C
um
ula
tive
su
rviv
al
Effect of dimethoate on the survival of collembola in a Mite-Collembola predator-prey system
0.75 mg dimethoate / kg soil Controls
Kaplan-Meier analysis
Conclusions
Unbiased measurements of changes in animal behaviour:
● Displays dose-response relationships
● Is decisive for residual uptake of xenobiotics
● Reveals long-term effects of chemical stress
● Is mechanistically linked to altered biochemical and physiological processeswithin the animal
● Provides a functional and measurable interface between individual andpopulation disturbances
● Identifies pollutions with chemical impact on animal health
perim
eter
fence
Edge
Reference
Plastics recycling factory
Plastic
N
Sampling of woodlice at the plastics recycling factory in Thetford, UK
100 m
October 1991
June 1995
Reference Edge Plastic
g m
etal
/g d
ry w
eigh
t
0
50
100
150
200
250
300
350
400
Pb
Cd
Zn
Cu
Body-burden of heavy metals in woodlicefrom the three sampling sites
Time in activity
mm
/s
**
sec
on
ds
x 10
0
met
erd
egre
es/m
m
deg
rees
/mm
mo
ves/
m
Turning rateTurn bias Movement rate
Average velocityPath length
R E P
0
15
30
45
60
R E P
0
15
30
45
60
75
R E P
0
2
4
6
8
10
12
R E P
0,0
0,5
1,0
1,5
2,0
R E P
0,0
0,1
0,2
0,3
0,4
0,5
R E P
0
20
40
60
80
Locomotor behaviour of woodlice collected atPlastic layer, Edge of plastic layer and Reference site
Mean glycogen and total protein contents for woodlice collected
at the Reference site, the Edge and the Plastic layer
R E PGlycogen 36.8 ± 9.9 8.1 ± 0.7 *** 8.3 ± 0.9 ***Total protein 32.9 ± 1.9 38.0 ± 0.9 28.3 ± 1.5
µg/mg fresh weight ± standard error (n=16)
FoundryZn 400 - 2000 ppmPb 140 - 1500 ppmCr 10 - 100 ppmNi 11- 40 ppmSpots of tarturpentenebenzenexylenepetrol
100 Km
Background levelsZn 5.8 - 59.7 ppmPb 4.5 - 19.2 ppmCr 2.7 - 30.4 ppmNi 0.9 - 15.1 ppm
(5-95% Fractile)
f Path AvVel AV Move TurnRate Max. Vel 001 1107. . . . .0.33 0.26 log( ) 5.55 0.04 .
Woodlice collected at clean and polluted field sitesshow differences in locomotor behaviour
Discriminant value
-3 -2 -1 0 1 2 3
Silkeborg
Als
Hadsten
Thy
Hg-sludge
Foundry
a
b
a
a
a
a
Mean metal concentrations in woodlicehepatopancreas and carcass.
µg metal / g dry wt. tissue ± S.E.
Zn Pb
Pooled control group
Carcass 41.3 ± 1.2 (19) 2.12 ± 0.3 (17)
Hepatopancreas 542 ± 114 (19) 243 ± 53 (16)
Foundry group
Carcass 70.7 ± 5.6 (19) 13.1 ± 3.8 (19)
Hepatopancreas 15770 ± 1093 (19) 205 ± 19 (19)
Rubbish dump
Gas works
2500 ppm Zn2 ppm Cd250 ppm Pb
Cyanide 25 pptTar 120 ppt
Phenol 190 ppmBenzene 200 ppmToluene 150 ppm
Phenanthrene 8400 ppmBenzo(A)pyrene 1300 ppm
Discriminant value
-0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0
Control 1
Control 2
Control 3
Control 4
Coal-gas
Rubbish dump
Tar-asphalt
Altered locomotor behaviour in woodlice from polluted sites
a
a
a
a
a
b
b
Applicability of the behavioural biomarker
● Can be run by technical personnel with only little training
● Provide a measurement of animal health at presumed polluted sites
● Identifies pollutions with chemical impact on animal fitness
● Includes long-term effects of chemical stress
● Fully automated data sampling and statistical calculations
● Fast (hours) and cheap (< 5.000 DK per site) screening method
