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The Effects of Selected Pyrethroids on Embryos of Bombina bombina during different Culture and Semi-field Conditions

Contents

Preface

Summary

Dansk sammendrag

PART I : The effects of esfenvalerate on embryos of Bombina bombina during different culture conditions and prolonged studies for identifying effects after recovery of a short embryological sublethal exposure

1 Introduction

2 Materials and methods

• 2.1 Principles and methods for maintaining and breeding the fire-bellied toads, Bombina bombina
  • 2.1.1 Principles and methods for maintaining adults Bombina bombina
  • 2.1.2 Principles and methods for breeding Bombina bombina
• 2.2 Toxicity tests using embryos of Bombina bombina
  • 2.2.1 Principle and design of the toxicity test
  • 2.2.2 Test substances
  • 2.2.3 Preparation of test solutions
  • 2.2.4 Embryos
  • 2.2.5 Culture medium
  • 2.2.6 Experimental design
  • 2.2.7 Determination of the effects of esfenvalerate on embryos of Bombina bombina
  • 2.2.8 Replicates and controls
  • 2.2.9 Data treatment and reporting
• 2.3 Principle and design of recovery experiments using embryos of Bombina bombina
  • 2.3.1 Exposure of the embryos to esfenvalerate
  • 2.3.2 Principles and methods for maintaining embryos and larvae (tadpoles) of Bombina bombina during the recovery period
  • 2.3.3 Principles and methods for maintaining Bombina bombina just before and during metamorphosis

3 Results

• 3.1 Embryo development in control groups at different culture condition
  • 3.1.1 Embryo development in FETAX solutions at two different temperatures
  • 3.1.2 Embryo development in two different media
• 3.2 Tests with reference substance
• 3.3 Effects of esfenvalerate on Bombina bombina at two different temperatures
  • 3.3.1 In vivo observations
  • 3.3.2 Recordings of malformation, growth inhibition and mortality at the end of the test (120 h and 216 h for 24°C and 20°C, respectively)
• 3.4 Effects of esfenvalerate on Bombina bombina in different culture media
  • 3.4.1 In vivo observations
  • 3.4.2 Recordings of malformation, growth inhibition, and mortality at the end of the test
• 3.5 Recovery experiments using embryos of Bombina bombina
  • 3.5.1 The effects of esfenvalerate on Bombina bombina during the toxicity test
  • 3.5.2 Embryo and larval development during the first part of the recovery period
  • 3.5.3 Development before and during the metamorphosis of Bombina bombina

4 Discussions

• 4.1 The use of amphibians as an ecotoxicological test organism
• 4.2 Effects of esfenvalerate during the exposure period
• 4.3 Recovery and delayed effects
• 4.4 Effects of different media used
• 4.5 Effects of different temperatures
• 4.6 Developmental effects of esfenvalerate in relation to other studies
• 4.7 Further aspect

PART II : The effects of selected pyrethroids on embryo development of Bombina bombina under semi-field conditions

5 Introduction

6 Materials and methods

• 6.1 Principles and methods for maintaining and breeding the fire-bellied toads, Bombina bombina
  • 6.1.1 Principles and methods for maintaining adults Bombina bombina
  • 6.1.2 Principles and methods for breeding Bombina bombina
• 6.2 Semi-field toxicity tests using embryos of Bombina bombina
  • 6.2.1 Principle and design of the semi-field toxicity test
  • 6.2.2 Establishing of experimental ponds, mesocosms
  • 6.2.3 Embryo selection for the test
  • 6.2.4 Experimental design of the semi-field toxicity test
  • 6.2.5 Test substances
  • 6.2.6 Spraying method and estimated initial concentrations
  • 6.2.7 Physical state of the ponds
  • 6.2.8 Determination of the effects of pesticide application on embryos of Bombina bombina

7 Results

• 7.1 Embryo development of Bombina bombina
  • 7.1.1 Embryo development in laboratory control
• 7.2 Effects of selected pyrethroids on Bombina bombina in a semi-field test
  • 7.2.1 In vivo observations
  • 7.2.2 Recordings of malformation, growth inhibition and mortality at the end of the test (nine days)

8 Discussions

• 8.1 The use of amphibians as an ecotoxicological test organism
• 8.2 Development in the controls and in the sprayed pond

9 References

Appendix A: Score sheet of malformations after 96 hours

Appendix B: The composition of the culture media

Appendix C: The effect of esfenvalerate on malformation, growth inhibition, and mortality

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Version 1.0 September 2004, © Danish Environmental Protection Agency