nr.1

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
• 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. 8317, 6733, 5050, 3367 og 1783 (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
• 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
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
11
ADM
X
X
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
12
ADM
X
X
X
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
13
ADM versus MSM
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
14
ADM versus MSM
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
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
16
Results Same site of action
Terrestrial plants Stellaria media
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
17
Results Same site of action
MCF7-celleprofileration assay
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
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
19
Results Different mode of action
Aquatic plants Lemna minor
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
20
Results Different mode of action
Crustacea Daphnia magna
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
21
Summary of results
Ministry of Food, Agriculture and
Fisheries Danish Institute of Agricultural
Sciences
Per Kudsk
22
Ternary mixtures
Crustacea Daphnia magna
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