1. Animal behaviour as a biomarker of chemical stress Ecotoxicology

2. 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

3. Impaired fitness Disturbed population and ecosystem stability - social behaviour - predator-prey interactions - reproduction - growth Chemical pollution - speciation - bioavailable residues Sensory interference Absorption Molecular responses Physiological responses Structural damage Exposure / effect biomarkers Effect / health biomarkers

4. x 1, y 1, time 1 x 2, y 2, time 2 x 3, y 3, time 3 x i, y i, time i

5. Red Green Blue Size Shape 0 255 0 0 0  0 1

9. Path length Velocities Turning behaviour Activity/Rest periods

14. Uptake of dimethoate in woodlice Control activity (22 hrs) Exposure (22 hrs) 14 C-Dimethoate 0, 140, 280, 560 g ha -1 14 C

15. ng a.i. /mg woodlouse Residual uptake of Dimethoate in woodlouse at three application rates 0100200300400500600700 0 1 2 3 4 5 6 meters 140 g / ha 240 g / ha 560 g / ha

16. Impaired fitness Disturbed population and ecosystem stability - social behaviour - predator-prey interactions - reproduction - growth Chemical pollution - speciation - bioavailable residues Sensory interference Absorption Molecular responses Physiological responses Structural damage Exposure / effect biomarkers Effect / health biomarkers

17. Prolonged effects of Dimethoate in woodlice Night 1 Night 2Night 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

18. 12324 60 80 100 120 140 160 180 40 60 80 100 120 140 60 70 80 90 100 110 60 70 80 90 100 110 120 130 140 Time in activity Turning rate Path Average velocity Percentage activity (Night n/Night 1) Night number Prolonged effect of Dimethoate on woodlouse locomotor parameters Exposed Controls

21. Time in velocity intervals Velocity intervals

22. Prolonged effect of an organophosphate on woodlouse velocity frequency distribution 0 1000 2000 3000 4000 5000 6000 0 1000 2000 3000 4000 5000 123456789 10 0 1000 2000 3000 4000 5000 Seconds in each velocity interval Velocity interval Control 34 hrs exposure 21 days recovery Controls Exposed (140 g / ha)

23. Exposure of a carabid beetle to copper during larval development 9 days10 days32 days Cu

24. Altered locomotor behaviour in adult female carabid beetles exposed to copper during larval development Controls Exposed

25. 24 hours Dimethoate application: - 0% - 7% - 15% - 26% - 59% of LD50 (48H) AChE inhibition and locomotor behaviour AChE

26. Correlation between organophosphate application rate and acetylcholinesterase activity in a carabid beetle Application rate (µg dimethoate / g fw beetle) 0123 AChE-aktivity ( µmol / min / g fw beetle ) 0,0 0,1 0,2 0,3 0,4 0,5 MalesFemales

27. Males Path length (m) 50 100 150 200 250 300 350 Time in activity ( hours) 0 1 2 3 4 Average velocity (mm/sec) 0 5 10 15 20 25 30 0,0 0,10,20,30,4 0,5 0,6 0 1 2 3 4 5 6 7 Females 50 100 150 200 250 300 350 0 1 2 3 4 5 10 15 20 25 30 AChE aktivity (µmole/min/g fw) 0,00,10,2 0,3 0,40,5 Turning rate (degrees/mm) 0 1 2 3 4 5 6 7 Path length (m) Time in activity ( hours) Average velocity (mm/sec) Turning rate (degrees/mm) Relationship between AChE activity and locomotor behaviour in a carabid beetle Control, Mean ± SE 5% LD50 (48 h), Mean ± SE 10% LD50 (48 h), Mean ± SE 23% LD50 (48 h), Mean ± SE

35. Predator-prey interactions in a mite-collembola system GROUP No. 12 Number of contacts: 610 Time to 1. contact : 526.5 136.9 Maximum duration : 524.6 666.6 Minimum duration : 2.4 Total duration 559.8826.5 Maximum distance : 14.7 15.3 Minimum distance: 0.0 Average distance:4.6 Time to max. meet. : 811.8293.5 Time to capture : 811.8171.8 Contacts until cap.: 4 2 ANIMAL No. 1 2 34 Walked path 9131034419 275 Walked path to cap. 830 1034 145 275 Active time 1051.6 705.4690.6167.6 Active time to cap. : 776.4705.4690.6167.6

36. Females Males Kaplan-Meier analysis of collembolan survival - females are more efficient hunters than males

37. 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 mite Size of mite and collembola Size ratio Average velocities of mite and collembola, respectively Frequency of contacts Time to first contact Parameters of importance for capture: Sex of mite Size ratio Average velocity of mite Frequency of contacts

38. 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 hunger Age of mite and collembola Time in locomotor activity (mite and collembola) Mite and collembolan average velocities Frequency of contacts Time to first contact Parameters of importance for capture: Time in locomotor activity of mite Average velocity of mite Frequency of contacts

39. 0 50010001500 2000 Time (sec) 0.2 0.4 0.6 0.8 1.0 Cumulative survival Effect of dimethoate on the survival of collembola in a Mite-Collembola predator-prey system 0.75 mg dimethoate / kg soilControls Kaplan-Meier analysis

40. 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 processes within the animal ● Provides a functional and measurable interface between individual and population disturbances ● Identifies pollutions with chemical impact on animal health

41. perimeter fence Edge Reference Plastics recycling factory Plastic N Sampling of woodlice at the plastics recycling factory in Thetford, UK 100 m October 1991

43. June 1995

44. ReferenceEdgePlastic :g metal /g dry weight 0 50 100 150 200 250 300 350 400 Pb Cd Zn Cu Body-burden of heavy metals in woodlice from the three sampling sites

45. Time in activity mm/s * * seconds x 100 meter degrees/mm moves/m Turning rate Turn bias Movement rate Average velocity Path length REP 0 15 30 45 60 REP 0 15 30 45 60 75 REP 0 2 4 6 8 10 12 REP 0,0 0,5 1,0 1,5 2,0 REP 0,0 0,1 0,2 0,3 0,4 0,5 REP 0 20 40 60 80 Locomotor behaviour of woodlice collected at Plastic layer, Edge of plastic layer and Reference site

46. Mean glycogen and total protein contents for woodlice collected at the R eference site, the E dge and the P lastic layer R EP Glycogen36.8 ± 9.9 8.1 ± 0.7 *** 8.3 ± 0.9 *** Total protein32.9 ± 1.938.0 ± 0.9 28.3 ± 1.5 µg/mg fresh weight ± standard error (n=16)

47. Foundry Zn 400 - 2000 ppm Pb 140 - 1500 ppm Cr 10 - 100 ppm Ni 11- 40 ppm Spots of tar turpentene benzene xylene petrol 100 Km Background levels Zn 5.8 - 59.7 ppm Pb 4.5 - 19.2 ppm Cr 2.7 - 30.4 ppm Ni 0.9 - 15.1 ppm (5-95% Fractile)

49. fPathAvVelAVMoveTurnRateMax. Vel  0011107.....0.330.26log()5.550.04.

51. Woodlice collected at clean and polluted field sites show differences in locomotor behaviour Discriminant value -3-20123 Silkeborg Als Hadsten Thy Hg-sludge Foundry a b a a a a

52. Mean metal concentrations in woodlice hepatopancreas and carcass. µg metal / g dry wt. tissue ± S.E. Zn Pb Pooled control group Carcass41.3 ± 1.2 (19)2.12 ± 0.3 (17) Hepatopancreas542 ± 114 (19)243 ± 53 (16) Foundry group Carcass70.7 ± 5.6 (19)13.1 ± 3.8 (19) Hepatopancreas15770 ± 1093 (19)205 ± 19 (19)

53. Rubbish dump Gas works 2500 ppm Zn 2 ppm Cd 250 ppm Pb Cyanide 25 ppt Tar 120 ppt Phenol 190 ppm Benzene 200 ppm Toluene 150 ppm Phenanthrene 8400 ppm Benzo(A)pyrene 1300 ppm

55. Discriminant value -0,6-0,4-0,2 0,0 0,20,40,60,81,0 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

56. 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