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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

ADM X Per Kudsk Ministry of Food, Agriculture and Fisheries Danish Institute of Agricultural Sciences Per Kudsk 24-04-2019

ADM X X Per Kudsk Ministry of Food, Agriculture and Fisheries Danish Institute of Agricultural Sciences Per Kudsk 24-04-2019

ADM X X X Per Kudsk Ministry of Food, Agriculture and Fisheries Danish Institute of Agricultural Sciences Per Kudsk 24-04-2019

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 24-04-2019

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 24-04-2019

ADM versus MSM ADM isobole MSM isobole Per Kudsk Ministry of Food, Agriculture and Fisheries Danish Institute of Agricultural Sciences Per Kudsk 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

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 24-04-2019

Summary of results Additivity Antagonism Synergism Same site of action 19 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 24-04-2019

Ternary mixtures Crustacea: Daphnia magna Per Kudsk Ministry of Food, Agriculture and Fisheries Danish Institute of Agricultural Sciences Per Kudsk 24-04-2019

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 24-04-2019

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 24-04-2019