Title page
Contents
Abstract 5
Foreword 6
List of Abbreviations and Chemical Formulae 11
Terminology 12
Acknowledgements 22
Section 1. Introduction 23
1.1. Background 23
1.2. Identification, Life History, and Distribution 25
1.3. Sensitivity of Frogs in Toxicity Tests 29
1.3.1. Comparison with Fish 29
1.3.2. Comparison Among Aquatic Life Stages 31
1.4. Use of Frogs in Toxicity Tests 32
Section 2. Test Organisms 36
2.1. Species and Life Stage 36
2.2. Source 41
2.3. Holding and Culturing of Embryos and Larvae 43
2.3.1. General 43
2.3.2. Facilities and Apparatus 44
2.3.3. Lighting 46
2.3.4. Temperature 46
2.3.5. Culture Water 47
2.3.6. Food and Feeding 50
2.3.7. Handling Organisms and Maintaining Cultures 51
2.3.8. Test Organism Health Criteria 51
2.3.9. Health, Quarantine, and Disease of Embryos and Larvae 52
2.4. Holding and Breeding of Adult Frogs 52
2.4.1. General 52
2.4.2. Holding of Adult Frogs 53
2.4.3. Hibernation and Breeding 56
2.4.4. Adult Frog Health, Quarantine, and Disease 56
Section 3. Test System 58
3.1. Facilities and Apparatus 58
3.2. Lighting 59
3.3. Preliminary and Definitive Tests 59
3.3.1. Preliminary Tests 59
3.3.2. Definitive Tests 59
3.4. Control/Dilution Water 60
3.5. Positive Control 61
Section 4. Universal Test Procedures 62
4.1. Preparing Test Solutions 63
4.2. Beginning the Test 68
4.3. Test Conditions and Procedures 69
4.3.1. Test Options 69
4.3.2. Test Type and Solution Renewal 69
4.3.3. Test Temperature and Lighting 70
4.3.4. Dissolved Oxygen and Aeration 70
4.3.5. pH 72
4.3.6. Food and Feeding 72
4.4. Test Observations and Measurements 73
4.5. Ending the Test 75
4.6. Test Endpoints and Calculations 77
4.6.1. Biological Endpoints 77
4.6.2. Multi-concentration Tests 79
4.6.2.1. LCp 80
4.6.2.2. ICp When a multi-con 80
4.6.2.3. Development Endpoint 84
4.6.2.4. Power Analysis 84
4.6.3. Other Test Designs 86
4.6.3.1. Test Duration 86
4.6.3.2. Suborganismal or non-apical endpoints 88
4.7. Test Validity 90
4.8. Tests with a Reference Toxicant 90
4.8.1. Multi-concentration Sublethal Tests with a Reference Toxicant 92
4.8.2. Positive Control 95
4.8.3. Multi-concentration Acute Lethality Tests with a Reference Toxicant 96
4.8.4. Warning Charts For both multi-concentration refe 97
4.9. Animal Conservation and Welfare Considerations 98
Section 5. Specific Procedures for Testing Chemicals 100
5.1. Sample Properties, Labelling, and Storage 100
5.2. Preparing Test Solutions 100
5.3. Control/Dilution Water 101
5.4. Test Observations and Measurements 102
5.5. Test Endpoints and Calculations 103
Section 6. Specific Procedures for Testing Contaminated Water Samples 104
6.1. Sample Collection, Labelling, Transport, and Storage 105
6.2. Preparing Test Solutions 106
6.3. Control/Dilution Water 106
6.4. Test Observations and Measurements 107
6.5. Test Endpoints and Calculations 107
6.5.1. Variations in Design and Analysis 107
Section 7. Reporting Requirements 110
7.1. Minimum Requirements for a Test-specific Report 110
7.1.1. Test Substance or Material 110
7.1.2. Test Organisms 110
7.1.3. Test Facilities and Apparatus 111
7.1.4. Control/Dilution Water 111
7.1.5. Test Method 111
7.1.6. Test Conditions and Procedures 111
7.1.7. Test Results 112
7.2. Additional Reporting Requirements 112
7.2.1. Test Substance or Material 113
7.2.2. Test Organisms 113
7.2.3. Test Facilities and Apparatus 113
7.2.4. Control/Dilution Water 113
7.2.5. Test Method 114
7.2.6. Test Conditions and Procedures 114
7.2.7. Test Results 114
References 115
Appendix A. Biological Test Methods and Supporting Guidance Documents Published by Environment and Climate Change Canada's Method Development and... 133
Appendix B. Environment and Climate Change Canada, Regional Environmental Testing Laboratories 136
Appendix C. Members of the Inter-Governmental Ecotoxicological Testing Group (as of June 2023) 137
Appendix D. Method Writers and Members of the Expert Peer Review Committee 140
Appendix E. Additional Procedures for Culturing Lithobates pipiens in the Laboratory and General Procedures for Frog Husbandry 141
Appendix F. Procedures for In-Laboratory Hibernation and Breeding for Lithobates pipiens Adults 146
Appendix G. Logarithmic Series of Concentrations Suitable for Toxicity Tests 152
Table 1. Approximate timing of developmental stages of Lithobates pipiens at test temperature, based on ASTM (2022a) and data from inter-laboratory studies... 40
Table 2. Checklist of required and recommended conditions and procedures for culturing Lithobates pipiens embryos and larvae for use in aquatic toxicity tests 45
Table 3. Checklist of required and recommended conditions and procedures for quarantine, holding, and breeding Lithobates pipiens adult frogs to provide test... 54
Table 4. Checklist of required and recommended conditions and procedures for conducting toxicity tests using aquatic life stages of frogs 64
Table 5. Example feeding rations for toxicity tests 73
Table 6. Checklist of required and recommended conditions and procedures for conducting reference toxicity tests using aquatic life stages of frogs 93
Figure 1. Considerations for preparing and performing toxicity tests using aquatic life stages of frogs (Lithobates pipiens) and various types of test materials or substances 26
Figure 2.1. Developmental stages (GS 1 to GS 25) of anuran embryos and hatchlings as described by Gosner (1960). (Reproduced from McDiarmid and Altig,... 38
Figure 2.2. Developmental stages (GS 26 to GS 46) of anuran larvae and metamorphs as described by Gosner (1960). (Reproduced from McDiarmid and Altig,... 39
Figure 3. The general process for the statistical analysis and selection of the most appropriate model for quantitative toxicity data (adapted and modified... 82
Table F.1. Hibernation light and temperature cycle (ALET, 2018) 148
Figure E.1. Adult frog culture tank with river rocks as terrestrial platform (ALET, 2004) 145
Figure E.2. Adult frog culture tank with Plexiglas terrestrial platform, during feeding 145
Figure E.3. Examples of spot patterns of adult northern leopard frogs (ALET, 2004) 145
Figure E.4. Frog with red-legged disease (ALET, 2004). The red coloration on the legs and abdomen is much brighter than the slight redness associated with handling frogs 145
Figure F.1. Examples of hibernation tanks with plastic plants, inverted glass dishes or rocks, and dechlorinated water (A: ALET, 2004; B: Nautilus Environmental,... 147
Figure F.2. Intraperitoneal injection of L. pipiens adult frog with GnRH-A/MET to induce breeding (ALET, 2018) 150
Figure F.3. Examples of 150-L breeding tanks for L. pipiens, with raised terrestrial platforms and plastic plants at different levels of the water column to act as... 150
Figure F.4. Frogs in amplexus (A: ALET, 2004, B: ALET, 2018) 151
Figure F.5. L. pipiens egg masses in holding containers (ALET, 2009) 151
Figure F.6. L. pipiens egg masses (A: Nautilus Environmental, 2016; B: ALET, 2004) 151