Standard methods for toxicology research in Apis mellifera.

Authors: Piotr Medrzycki, Hervé Giffard, Pierrick Aupinel, Luc P Belzunces, Marie-Pierre Chauzat, Christian Claßen, Marc E Colin, Thierry Dupont, Vincenzo Girolami, Reed Johnson, Yves Le Conte, Johannes Lückmann, Matteo Marzaro, Jens Pistorius, Claudio Porrini, Andrea Schur, Fabio Sgolastra, Noa Simon Delso, Sjef van der Steen, Klaus Wallner, Cédric Alaux, David G Biron, Nicolas Blot, Gherardo Bogo, Jean-Luc Brunet, Frédéric Delbac, Marie Diogon, Hicham El Alaoui, Bertille Provost, Simone Tosi, Cyril Vidau.

Table of contents


1. Introduction
2. Common terms and abbreviations
3. Effects of toxic substances on adult worker bees: individual assays
   3.1. Introduction
      3.1.1. Definitions of poisonings and exposure
      3.1.2. Exploration of acute poisoning using the lethality criterion
      3.1.3. Factors influencing the dose-lethality relation
 Active ingredient and chemical formulation Physical formulation Temperature and hygrometry Exposure features Sex, age and caste Weight and diet Health Genetics and resistance Density of subjects Conclusion
      3.1.4. Exploration of sub-lethal poisoning
   3.2. Laboratory methods for testing toxicity of chemical substances on adult bees
      3.2.1. Oral application
 Introduction General principle Experimental conditions and modalities
   Establishing the hoarding cages
   Identifying and replicating the treatment modalities
   Substance administration Mortality assessment Extension to other tests
      3.2.2. Topical application Introduction
   Field simulated contact toxicity
   Contact LD50 Description of the method
   Outline of the test
   Collection of bees
   Test cages
   Handling and feeding conditions
   Preparations of bees
   Preparation of doses Procedure
   Test and control groups
   Toxic reference
   Administration of doses
   Test conditions
   Duration and observations Calculation of the LD50 Limit test Validity of the test Data and reporting
   Test report
      Test substance
      Test bees
      Test conditions
      Results Recommendation
      3.2.3. Toxicity of residues on foliage Testing toxicity of contaminated dust from pesticide-dressed seed by indirect contact
   Test procedures
      Dust extraction
      Contaminated dust preparation
      Dust application
      Exposure to test substance
      Number of animals tested
      Number of replicates
      Duration of the test
      Test conditions
      Endpoints Testing contact toxicity on bees exposed to pesticide-contaminated leaves
   3.3. Field methods for testing toxicity of chemical substances on individual adult bees
      3.3.1. In-field exposure to dust during sowing
 Introduction The management of the bees after exposure Study conditions Capturing the bees
   Inducing the bees to visit the dispenser
   Collecting bees for use during the study Trials in mobile cages Trials in free flight Collection and analysis of data
4. Effects of toxic substances on bee colonies
   4.1. Introduction
   4.2. Determining pesticide toxicity on bee colonies in semi-field conditions
      4.2.1. Introduction
      4.2.2. Tunnel description
      4.2.3. Mortality assessment
      4.2.4. Foraging activity assessment
      4.2.5. Hive description
      4.2.6. Treatment methodology
      4.2.7. Applications
      4.2.8. Comparison of impacts
      4.2.9. Extension to other topics in semi-field tests
   4.3. Testing toxicity on bee colonies in field conditions
      4.3.1. Problems related to the experimental design
 Introduction Replicates External factors Application of treatment Colonies Level of exposure Mode of assessment and recording Interpretation of results
   Simultaneous trials
   Consecutive trials
   Data processing
      4.3.2. Forced in-hive nutrition Introduction Methods
   The use of test syrups
      For pesticide studies
      For antibiotic studies
   The use of pollen patties
      4.3.3. Dust dispersion during sowing Introduction Methods and general requirements for dust exposure field studies
   Requirements for establishment of field trials
      Set up and location of bee hives
      Amount of seeds used per hectare
      Machinery and modifications of sowing machines
      Location of fields
      Soil conditions
      Wind conditions, direction, weather conditions
      Foraging conditions during full bee flight
      Crop for sowing
      Flowering adjacent crops
      Residue samples (plants, bees, bee matrices) proof of exposure
   Setup of field trials using other devices for a direct dust application
      4.3.4. Foraging on a treated crop Returning foragers as a tool to measure the pesticide confrontation and the transport into the bee colony
   Reasons for collection of forager bees
   Collection of forager bees in tunnel tents or in the field
      Preparation of the honey stomachs
      Preparation of the pollen loads
      4.3.5. Systemic toxins expressed in plant matrices Introduction Application of systemic products as seed and soil treatment (SSST), bulbs or root bathing
   Principle of the trial
   Preliminary steps
   Environment of the trial
   Trial plots: experimental and control
      Crops planted in the trial plots
      Size of the trial plots
      Location of the colonies at the trial plots
      Distance among trial plots
   Colonies used
      Colony health status
      Number of colonies/replicates – statistical power
      Colony placement and equipment
   Duration of the test
   Bees’ exposure
      Pollen analyses
      Residue analyses
      Reserves of the colonies at the beginning of the trial
      Brood and reserves content
      Interpretation of residual information
      Toxicological endpoints
         Mortality trend
         General evolution of the colony during the test
         Behavioural observations
         Colony health
         Brood surface and quality
   Validity of the trial
5. Effects of toxic substances on honey bee brood
   5.1. Introduction
   5.2. in vivo larval tests
      5.2.1. Oomen test
      5.2.2. Semi field test
 Introduction Material and method of a semi-field brood test
   Design of the test
   Preparation of the colonies
   Test conditions
   Assessments The total observation period of the colonies is at least 28days.
      Meteorological data
      Mortality of honey bees
      Flight activity and behaviour
      Brood assessments
         Condition of the colonies
         Development of the bee brood in single cells Evaluation of the results of the semi-field test Discussion and conclusion
      5.2.3. Evaluation of honey bee brood development by using digital image processing Introduction Material and methods
   Photographing of the brood combs at the field site
   Evaluation of the brood combs at the laboratory Discussion and conclusion
   5.3. in vitro larval tests
      5.3.1. The rearing method
      5.3.2. Toxicity testing
      5.3.3. Results
      5.3.4. Statistical analysis
      5.3.5. General discussion
6. Effects of toxic substances on queen bees and drones
   6.1. Introduction
   6.2. Mortality and poisoning signs in honey bee queens
   6.3. Reduction in egg production
   6.4. Inability to requeen
   6.5. Conclusion
7. Evaluation of synergistic effects
   7.1. Laboratory testing for interactions between agents

      7.1.1. Introduction
      7.1.2. Model synergists
      7.1.3. Response variables
      7.1.4. Experiments testing for interactions
 Discriminating dose bioassay Comparison of dose-response curves
   7.2. Laboratory approach to study toxico-pathological interactions in honey bees
      7.2.1. Introduction
      7.2.2. Materials
 Honey bees Pesticide Food supply
      7.2.3. Joint action of pathogens and pesticides
      7.2.4. Sensitization to pesticides by a previous exposure to pathogens
      7.2.5. Notes
   7.3. R script for testing synergistic interactions
8. Introduction to the use of statistical methods in honey bee studies
   8.1. Foreword
   8.2. Statistical tests and situations
      8.2.1. Honey bee tunnel study
 Honey bee brood development LD50 determination
      8.2.2. Brood development index (numerical example) Analysis of variance for numerical example Interaction statistical analysis
   8.3. Conclusion
   8.4. Formulas and procedures frequently used in toxicological studies
      8.4.1. Correction of the mortality rates
 Example correction for control mortality
      8.4.2. Calculation of the HQ and RQ Hazard Quotient HQ (EPPO, 2010b) Risk Quotient RQ (EPHC, 2009)
      8.4.3. NOAEL and NOAEC
      8.4.4. Power of a test
9. Acknowledgements
10. References