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

 

Authors
Summary
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

         3.1.3.1. Active ingredient and chemical formulation
         3.1.3.2. Physical formulation
         3.1.3.3. Temperature and hygrometry
         3.1.3.4. Exposure features
         3.1.3.5. Sex, age and caste
         3.1.3.6. Weight and diet
         3.1.3.7. Health
         3.1.3.8. Genetics and resistance
         3.1.3.9. Density of subjects
         3.1.3.10. 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

         3.2.1.1. Introduction
         3.2.1.2. General principle
         3.2.1.3. Experimental conditions and modalities
            3.2.1.3.1. Establishing the hoarding cages
            3.2.1.3.2. Identifying and replicating the treatment modalities
            3.2.1.3.3. Substance administration
         3.2.1.4. Mortality assessment
         3.2.1.5. Extension to other tests
      3.2.2. Topical application
         3.2.2.1. Introduction
            3.2.2.1.1. Field simulated contact toxicity
            3.2.2.1.2. Contact LD50
         3.2.2.2. Description of the method
            3.2.2.2.1. Outline of the test
            3.2.2.2.2. Collection of bees
            3.2.2.2.3. Test cages
            3.2.2.2.4. Handling and feeding conditions
            3.2.2.2.5. Preparations of bees
            3.2.2.2.6. Preparation of doses
         3.2.2.3. Procedure
            3.2.2.3.1. Test and control groups
            3.2.2.3.2. Replicates
            3.2.2.3.3. Toxic reference
            3.2.2.3.4. Administration of doses
            3.2.2.3.5. Test conditions
            3.2.2.3.6. Duration and observations
         3.2.2.4. Calculation of the LD50
         3.2.2.5. Limit test
         3.2.2.6. Validity of the test
         3.2.2.7. Data and reporting
            3.2.2.7.1. Data
            3.2.2.7.2. Test report
               3.2.2.7.2.1. Test substance
               3.2.2.7.2.2. Test bees
               3.2.2.7.2.3. Test conditions
               3.2.2.7.2.4. Results
         3.2.2.8. Recommendation
      3.2.3. Toxicity of residues on foliage
         3.2.3.1. Testing toxicity of contaminated dust from pesticide-dressed seed by indirect contact
            3.2.3.1.1. Introduction
            3.2.3.1.2. Test procedures
               3.2.3.1.2.1. Background
               3.2.3.1.2.2. Dust extraction
               3.2.3.1.2.3. Dosages
               3.2.3.1.2.4. Contaminated dust preparation
               3.2.3.1.2.5. Substrate
               3.2.3.1.2.6. Dust application
               3.2.3.1.2.7. Control
               3.2.3.1.2.8. Exposure to test substance
               3.2.3.1.2.9. Number of animals tested
               3.2.3.1.2.10. Number of replicates
               3.2.3.1.2.11. Duration of the test
               3.2.3.1.2.12. Test conditions
               3.2.3.1.2.13. Endpoints
         3.2.3.2. Testing contact toxicity on bees exposed to pesticide-contaminated leaves
            3.2.3.2.1. Introduction
            3.2.3.2.2. Methodology
   3.3. Field methods for testing toxicity of chemical substances on individual adult bees
      3.3.1. In-field exposure to dust during sowing

         3.3.1.1. Introduction
         3.3.1.2. The management of the bees after exposure
         3.3.1.3. Study conditions
         3.3.1.4. Capturing the bees
            3.3.1.4.1. Inducing the bees to visit the dispenser
            3.3.1.4.2. Collecting bees for use during the study
         3.3.1.5. Trials in mobile cages
         3.3.1.6. Trials in free flight
         3.3.1.7. 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

         4.3.1.1. Introduction
         4.3.1.2. Replicates
         4.3.1.3. External factors
         4.3.1.4. Application of treatment
         4.3.1.5. Colonies
         4.3.1.6. Level of exposure
         4.3.1.7. Mode of assessment and recording
         4.3.1.8. Interpretation of results
            4.3.1.8.1. Simultaneous trials
            4.3.1.8.2. Consecutive trials
            4.3.1.8.3. Data processing
      4.3.2. Forced in-hive nutrition
         4.3.2.1. Introduction
         4.3.2.2. Methods
            4.3.2.2.1. The use of test syrups
               4.3.2.2.1.1. For pesticide studies
               4.3.2.2.1.2. For antibiotic studies
            4.3.2.2.2. The use of pollen patties
      4.3.3. Dust dispersion during sowing
         4.3.3.1. Introduction
         4.3.3.2. Methods and general requirements for dust exposure field studies
            4.3.3.2.1. Requirements for establishment of field trials
               4.3.3.2.1.1. Set up and location of bee hives
               4.3.3.2.1.2. Seeds
               4.3.3.2.1.3. Amount of seeds used per hectare
               4.3.3.2.1.4. Machinery and modifications of sowing machines
               4.3.3.2.1.5. Location of fields
               4.3.3.2.1.6. Soil conditions
               4.3.3.2.1.7. Wind conditions, direction, weather conditions
               4.3.3.2.1.8. Sowing
               4.3.3.2.1.9. Foraging conditions during full bee flight
               4.3.3.2.1.10. Crop for sowing
               4.3.3.2.1.11. Flowering adjacent crops
               4.3.3.2.1.12. Residue samples (plants, bees, bee matrices) proof of exposure
            4.3.3.2.2. Setup of field trials using other devices for a direct dust application
      4.3.4. Foraging on a treated crop
         4.3.4.1. Returning foragers as a tool to measure the pesticide confrontation and the transport into the bee colony
            4.3.4.1.1. Reasons for collection of forager bees
            4.3.4.1.2. Collection of forager bees in tunnel tents or in the field
               4.3.4.1.2.1 Preparation of the honey stomachs
               4.3.4.1.2.2. Preparation of the pollen loads
      4.3.5. Systemic toxins expressed in plant matrices
         4.3.5.1. Introduction
         4.3.5.2. Application of systemic products as seed and soil treatment (SSST), bulbs or root bathing
            4.3.5.2.1. Introduction
            4.3.5.2.2. Principle of the trial
            4.3.5.2.3. Preliminary steps
            4.3.5.2.4. Environment of the trial
            4.3.5.2.5. Trial plots: experimental and control
               4.3.5.2.5.1. Crops planted in the trial plots
               4.3.5.2.5.2. Size of the trial plots
               4.3.5.2.5.3. Location of the colonies at the trial plots
               4.3.5.2.5.4. Distance among trial plots
            4.3.5.2.6 Colonies used
               4.3.5.2.6.1. Colony health status
               4.3.5.2.6.2. Number of colonies/replicates – statistical power
               4.3.5.2.6.3. Colony placement and equipment
            4.3.5.2.7. Duration of the test
            4.3.5.2.8. Bees’ exposure
               4.3.5.2.8.1. Pollen analyses
               4.3.5.2.8.2. Residue analyses
               4.3.5.2.8.3. Reserves of the colonies at the beginning of the trial
            4.3.5.2.9. Observations
               4.3.5.2.9.1. Controls
               4.3.5.2.9.2. Brood and reserves content
               4.3.5.2.9.3. Interpretation of residual information
               4.3.5.2.9.4. Toxicological endpoints
                  4.3.5.2.9.4.1. Mortality trend
                  4.3.5.2.9.4.2. General evolution of the colony during the test
                  4.3.5.2.9.4.3. Behavioural observations
                  4.3.5.2.9.4.4. Colony health
                  4.3.5.2.9.4.5. Brood surface and quality
            4.3.5.2.10. 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

         5.2.2.1. Introduction
         5.2.2.2. Material and method of a semi-field brood test
            5.2.2.2.1. Design of the test
            5.2.2.2.2. Preparation of the colonies
            5.2.2.2.3. Test conditions
            5.2.2.2.4. Application
            5.2.2.2.5. Assessments The total observation period of the colonies is at least 28days.
               5.2.2.2.5.1. Meteorological data
               5.2.2.2.5.2. Mortality of honey bees
               5.2.2.2.5.3. Flight activity and behaviour
               5.2.2.2.5.4. Brood assessments
                  5.2.2.2.5.4.1. Condition of the colonies
                  5.2.2.2.5.4.2. Development of the bee brood in single cells
         5.2.2.3. Evaluation of the results of the semi-field test
         5.2.2.4. Discussion and conclusion
      5.2.3. Evaluation of honey bee brood development by using digital image processing
         5.2.3.1. Introduction
         5.2.3.2. Material and methods
            5.2.3.2.1. Photographing of the brood combs at the field site
            5.2.3.2.2. Evaluation of the brood combs at the laboratory
         5.2.3.3. 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

         7.1.4.1. Discriminating dose bioassay
         7.1.4.2. 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

         7.2.2.1. Honey bees
         7.2.2.2. Pesticide
         7.2.2.3. 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

         8.2.1.1. Honey bee brood development
         8.2.1.2. LD50 determination
      8.2.2. Brood development index (numerical example)
         8.2.2.1. Analysis of variance for numerical example
         8.2.2.2. Interaction statistical analysis
   8.3. Conclusion
   8.4. Formulas and procedures frequently used in toxicological studies
      8.4.1. Correction of the mortality rates

         8.4.1.1. Example correction for control mortality
      8.4.2. Calculation of the HQ and RQ
         8.4.2.1. Hazard Quotient HQ (EPPO, 2010b)
         8.4.2.2. Risk Quotient RQ (EPHC, 2009)
      8.4.3. NOAEL and NOAEC
      8.4.4. Power of a test
9. Acknowledgements
10. References