Guidance for experimental design and statistical analysis of ecotoxicological community effect studies (field studies) Report to UK Chemicals Regulation Directorate (CRD) CRD/DEFRA project code PS2363 Report Authors: Alan Lawrence (Cambridge Environmental Assessments, ADAS UK Ltd) Kevin Brown (Independent Consultant) Geoff Frampton (University of Southampton) Paul J. Van den Brink (Alterra & Wageningen University) CEA report no. 1088 CONTENTS Executive summary ............................................................................................................... 3 1 Introduction .................................................................................................................... 7 1.1. Aims, objectives and limitations .................................................................................. 7 1.2. Project team ............................................................................................................... 7 1.3. Report structure .......................................................................................................... 7 1.4. Definitions .................................................................................................................. 8 1.5. Funding ...................................................................................................................... 9 2 Summary of Guidance Documents............................................................................... 10 2.1. Section summary ...................................................................................................... 16 3 The context of community studies ................................................................................ 17 3.1. Introduction .............................................................................................................. 17 3.2. Types of community studies and place in tiered testing ............................................ 17 3.3. Advantages and limitations of replicated manipulative field studies .......................... 18 3.4. Population versus community; protection goals ........................................................ 18 3.5. Representation of communities by test systems ....................................................... 20 3.6. Section summary ...................................................................................................... 21 4 Experimental design .................................................................................................... 23 4.1. Study layouts and randomisation of plots ................................................................. 23 4.2. Pseudoreplication ..................................................................................................... 26 4.3. Grouping data for analysis ........................................................................................ 27 4.4. Section summary ...................................................................................................... 29 5 General introduction to statistical testing ...................................................................... 30 5.1. Introduction to statistics in community ecotoxicology studies .................................... 30 5.2. Hypothesis testing .................................................................................................... 30 5.3. Probability within statistical hypothesis testing .......................................................... 31 5.4. Repeated two sample testing ................................................................................... 33 5.5. Importance of assumptions – normality, independence, randomisation .................... 34 5.6. Replication ............................................................................................................... 35 5.7. Section summary ...................................................................................................... 36 6 Initial data interpretation and univariate approaches .................................................... 37 6.1. Introduction .............................................................................................................. 37 6.2. Initial investigation .................................................................................................... 37 6.3. Outliers ..................................................................................................................... 38 6.4. Screening prior to analysis ....................................................................................... 38 6.5. Data pre-requisites for analysis ................................................................................ 39 6.6. Univariate tests and statistical power ........................................................................ 39 1 6.7. Consecutive significant events ................................................................................. 40 6.8. Percent reduction measures ..................................................................................... 41 6.9. Section summary ...................................................................................................... 43 7 Multivariate approaches ............................................................................................... 44 7.1. Introduction to multivariate analyses ......................................................................... 44 7.2. Background to ordination techniques ........................................................................ 46 7.3. Introduction to Principal Response Curves analysis ................................................. 47 7.4. PRC mechanics and terminology.............................................................................. 50 7.5. Interpretation of first PRC axis .................................................................................. 51 7.6. Construction and interpretation of the second PRC axis ........................................... 53 7.7. Other PRC outputs, significance testing, explained variance .................................... 60 7.8. Summary output statistics from PRC analyses conducted in CANOCO .................... 61 7.9. Data transformations ................................................................................................ 62 7.10. Analysing a reference item with PRC .................................................................... 62 7.11. Other uses for multivariate approaches in ecotoxicological field studies ............... 63 7.12. Section summary .................................................................................................. 64 8 Application of PRC analysis to example scenarios ....................................................... 65 9 Summary of data examination and analysis ................................................................. 72 10 Monitoring studies for effects of plant protection products on terrestrial invertebrates .. ................................................................................................................................. 73 10.1. Background........................................................................................................... 73 10.2. Setting the research question ............................................................................... 74 10.3. Spatial scale ......................................................................................................... 74 10.4. Size and siting of study plots ................................................................................. 76 10.5. Sampling locations within study plots .................................................................... 76 10.6. Temporal scale ..................................................................................................... 77 10.7. Sampling considerations ....................................................................................... 78 10.8. Statistical analysis................................................................................................. 78 11 References ............................................................................................................... 79 Appendix I ........................................................................................................................... 84 Appendix II .......................................................................................................................... 90 2 Executive summary Introduction Ecotoxicological field studies conducted under EC Regulation 1107/2009 may be designed by industry notifiers in association with external consultants and contract research organisations (CROs). The results may then be interpreted and summarised before inclusion in a regulatory submission in support of a product or active substance registration. The submission will be reviewed by representatives from Member State (MS) Competent Authorities, in terms of both study quality and meaning. Not all parties involved may be familiar with the statistical design and interpretation requirements of such studies, which are complex. This project was conceived in order to provide background information and guidance to specialists and non-specialists alike on the statistical design and interpretation of ecotoxicological community effect studies. The specific objectives of this project were to: a. Develop a guide to best practice for experimental design of ecotoxicological community studies (aquatic mesocosms, non-target