1. Combination effects of pesticides
Per Kudsk, DIAS
Helle Raun Andersen, SDU
Nina Cedergreen, Jens C. Streibig, RVAU
Solvejg Mathiassen, DIAS
Flemming Møhlenberg, DHI
Anne Marie Vinggaard, DIFVR

2. Background Why should one study combination effects of pesticides?
Pesticides are often used in mixture with other pesticides
In most cases more than one pesticide is found when analysing for pesticide residues in e.g. water and food
Simultaneuos exposure to more than one pesticide can change the response of human beings, animals and other organisms compared to their response to the pesticides applied individually
Interest to incorporate knowledge on combination effects into the risk assessment of pesticides (”pesticide cocktails”)
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

3. Background
Problems associated with studies on combination effects of pesticides
Number of potential pesticide mixtures is very large
Number of relevant organisms is large
No consencus on either the terminology or statististical models (reference models)
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

4. Methods: Pesticides Pesticides Mixtures Number: 22
Chosen according to mode of action and site of action
Mixtures
3 classes of mixtures:
Same site of action
Same mode of action but different site of action
Different mode of action
101 binary mixtures
20 ternary mixtures
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

5. Methods: Test systems Ecotoxicological test systems
Terrestrial plants (Tripleurospermum inodorum/Stellaria media)
Aquatic plants (Lemna minor)
Algae (Pseudokirchneriella subcapitata)
Aquatic bacteria (Vibrio fisheri)
Crustacea (Daphnia magnia)
Human toxicological test systems
MCF7-celleproliferation assay
AR reportergene assay
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

6. Methods: Experimental design
Binary mixtures
2 herbicides (6-8 dose rates)
3 to 5 fixed-ratio mixtures of the 2 herbicides e.g. 83:17, 67:33, 50:50, 33:67 og 17:83 (6-8 dose rates of each mixture)
Ternary mixtures
3 herbicides (6-8 dose rates)
3 fixed-ratio mixtures of each of the 3 binary mixtures (6-8 dose rates of each mixture)
4 fixed-ratio mixtures of the 3 herbicides (6-8 dose rates of each mixture)
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

7. Metode: Statistiske analyser
Logistic dose response curve
D: Upper asymptote (=control)
C: Lower asymptote (=highest dose rate)
B: Slope around ED50
ED50: Dose rate resulting in a 50% growth reduction
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

8. Terminology Additivity Antagonism Synergism
The effect of the mixture is as predicted by the reference model
Antagonism
The effect of the mixture is significantly lower than predicted by the reference model
Synergism
The effect of the mixture is significantly higher than predicted by the reference model
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

9. Referencemodeller Additive Dose Model (ADM)
Compares the dose rates of the pesticides applied alone and in mixture required to obtain the same effect
Multiplicative Survival Model (MSM)
Compares effects of dose rates of pesticides applied alone and in mixture
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

10. ADM X Per Kudsk Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

11. ADM X X Per Kudsk Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

12. ADM X X X Per Kudsk Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

13. ADM versus MSM B1=B2=0.5 B1=B2=1 B1=B2=2 B1=B2=5 ADM Herbicide B
Herbicide A
Herbicide B
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

14. ADM versus MSM B1=B2=0.5 B1=B2=1 B1=B2=2 B1=B2=5 ADM X X X X X X
Herbicide A
Herbicide B X X X X X X
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

15. ADM versus MSM ADM isobole MSM isobole Per Kudsk
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

16. Results: Same site of action
Terrestrial plants: Stellaria media
ADM isobole
Estimated isobole
Observations
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

17. Results: Same site of action
MCF7-celleprofileration assay
ADM isobole
Estimated isobole
Observations
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

18. Results: Different site of action, same mode of action
Terrestrial plants: Tripleurospermum inodorum
ADM isobole
Estimated isobole
Observations
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

19. Results: Different mode of action
Aquatic plants: Lemna minor
ADM isobole
Estimated isobole
Observations
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

20. Results: Different mode of action
Crustacea: Daphnia magna
ADM isobole
Estimated isobole
Observations
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

21. Summary of results Additivity Antagonism Synergism Same site of action19 1
Samme mode of action, different site of action 5
Different mode of action 41 8
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

22. Ternary mixtures Crustacea: Daphnia magna Per Kudsk
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

23. Conclusions
The response of the majority of the 101 binary mixtures followed ADM, i.e. the effect was additive
Deviations from ADM were more frequent for mixtures of pesticides with different mode of action and/or site of action
Synergy was only observed with 8 binary mixtures and in all cases prochloraz was one of the components in the mixture (same effect with 25 to 50% af the dose rates expected according to ADM)
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk

24. Conclusions
Generally ADM predicts higher effects of pesticide mixtures than MSM and hence ADM is more suitable for risk assessment of pesticide mixtures because it is less likely that the effect of a mixture is underestimated
ADM could by combined with the use of a safety factor to comprise synergistic mixtures
Good agreement between resultats obtained with comparable test systems (terrestrial versus aquatic plants; plants versus algae)
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
Per Kudsk