animal behaviour as a biomarker of chemical...
TRANSCRIPT
Ecotoxicology
Animal behaviour as a biomarker
of chemical stress
Scientific objectives
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
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
RedGreenBlueSizeShape
0 255
0 2550 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
14 C-Dimethoate0, 140, 280, 560
g ha-1
14C
Exposure (22 hrs)Control activity (22 hrs)
Residual uptake of Dimethoate in woodlouseat three application rates
nga.
i. /m
g w
oodl
ouse
0 100 200 300 400 500 600 700
0
1
2
3
4
5
6
meters
140 g / ha240 g / ha560 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
RecoveryControl Exposure (140 g a.i./ha)
Control
140 g a.i./ha corresponds to 1/10 of the LD20 – 48 hours
Prolonged effect of Dimethoate onwoodlouse locomotor parameters
1 2 3 246080
10012014016018040
60
80
100
120
140
60
70
80
90
100
110
60708090
100110120130140
Time in activity
Turning rate
Path
Average velocity
Perc
enta
geac
tivity
(Nig
htn/
Nig
ht1)
Night number
Exposed
Controls
Tim
e in
vel
ocity
inte
rval
s
Velocity intervals
Prolonged effect of an organophosphate onwoodlouse velocity frequency distribution
0100020003000400050006000
010002000300040005000
1 2 3 4 5 6 7 8 9 100
10002000300040005000
Seco
nds
in e
ach
velo
city
inte
rval
Velocity interval
Control
34 hrs exposure
21 days recovery
ControlsExposed (140 g / ha)
Exposure of a carabid beetle to copper during larval development
Cu
Cu
32 days 10 days 9 days
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
Averagevelocity
mm
/ se
c
6
8
10
12
14
16
18
Movementdisruption
Sto
ps /
wal
ked
met
er60
80
100
120
Turningrate
Deg
rees
/ sec
20
25
30
35
40
45
50
55
60
ControlsExposed
AChE inhibition and locomotor behaviour
Dimethoate application:- 0% - 7% - 15% - 26% - 59%
of LD50 (48H)
AChE24 hours
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-a
ktiv
ity( µ
mol
/ m
in /
g fw
beet
le)
0,0
0,1
0,2
0,3
0,4
0,5
Males Females
Relationship between AChE activity andlocomotor behaviour in a carabid beetle
Males
Path
leng
th(m
)50
100150200250300350
Tim
e in
act
ivity
( hou
rs)
0
1
2
3
4A
vera
geve
loci
ty(m
m/s
ec)
05
1015202530
0,0 0,1 0,2 0,3 0,4 0,5 0,601234567
Females
50100150200250300350
0
1
2
3
4
51015202530
AChE aktivity (µmole/min/g fw)0,0 0,1 0,2 0,3 0,4 0,5
Turn
ing
rate
(deg
rees
/mm
)
01234567
Path
leng
th(m
)Ti
me
in a
ctiv
ity( h
ours
)A
vera
geve
loci
ty(m
m/s
ec)
Turn
ing
rate
(deg
rees
/mm
)
Control, Mean ± SE5% LD50 (48 h), Mean ± SE
10% 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
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 (sec)0 200 400 600 800 1000 1200 1400 1600 1800
Dis
tanc
e (m
m)
0
5
10
51
20
Path
leng
th(m
m)
0
100
200
300
400
500
600
700
Collembola
Mite
Kaplan-Meier analysis of collembolan survival- females are more efficient hunters than males
Time (min)
0 5 10 15 20 25 30
Cum
ulat
ive
surv
ival
of C
olle
mbo
la
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Females
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
Effect of dimethoate on the survival of collembolain a Mite-Collembola predator-prey system
0 500 1000 1500 2000
Time (sec)
0.2
0.4
0.6
0.8
1.0C
umul
ativ
esu
rviv
al
Kaplan-Meier analysis
0.75 mg dimethoate / kg soil Controls
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
Sampling of woodlice at the plastics recycling factory in Thetford, UK
perimete
r fence
Edge
Reference
Plastics recycling factory
Plastic
N
100 m
October 1991
June 1995
Reference Edge Plastic
:g
met
al /g
dry
wei
ght
0
50
100
150
200
250
300
350
400
Body-burden of heavy metals in woodlicefrom the three sampling sites
PbCdZnCu
Time in activity
mm
/s
**
seco
nds
x 10
0
met
erde
gree
s/m
m
degr
ees/
mm
mov
es/m
Turning rateTurn bias Movement rate
Average velocityPath length
R E P0
15
30
45
60
R E P0
15
30
45
60
75
R E P0
2
4
6
8
10
12
R E P0,0
0,5
1,0
1,5
2,0
R E P0,0
0,1
0,2
0,3
0,4
0,5
R E P0
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 collectedat 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
BackgroundlevelsZn 5.8 - 59.7 ppmPb 4.5 - 19.2 ppmCr 2.7 - 30.4 ppmNi 0.9 - 15.1 ppm
(5-95% Fractile)
100 Km
Path= ⋅ + ⋅0 01. .f AvV0.33 el− ⋅. 0.26 AVlog( Move TurnRate+ ⋅. ) 5.55 Max. Vel0.04 .− ⋅ −1107.
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.
Zn PbPooled control groupCarcass 41.3 ± 1.2 (19) 2.12 ± 0.3 (17)Hepatopancreas 542 ± 114 (19) 243 ± 53 (16)Foundry groupCarcass 70.7 ± 5.6 (19) 13.1 ± 3.8 (19)Hepatopancreas 15770 ± 1093 (19) 205 ± 19 (19)
µg metal / g dry wt. tissue ± S.E.
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 inwoodlice from polluted sites
a
a
a
a
a
b
b
Applicability of the behavioural biomarker
● 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
● Can be run by technical personnel with only little training