Ecotoxicology Day 2. Adam Peters and Graham Merrington 2017

What is a ecotoxicology? Endpoints Test principles and interpretation Hypothesis testing Regression modelling/curve fitting Reliability and relevance 2017

What is a ecotoxicology? (ECHA 2011, R7b) again Typically standardised tests aimed at comparing different chemicals Simple tests on organisms which are readily cultured in the lab Standardised test procedures Performance of control animals critical for a valid test Tests usually performed under optimum conditions for the test species 2017

Endpoints? What counts as an effect? Acute – Mortality Chronic – endpoints relating to development, growth, or reproduction What kinds of plants and animals get tested? Unicellular green algae Water fleas (Daphnia) Fish 2017

Testing? (OECD 201, 202, 203, etc.) Acute Daphnia test 10 water fleas in plastic cups Are they alive or not? Prod with a pencil, does it move? If it doesn’t move it’s immobilised (presumed dead) Endpoint is immobilisation Assumed equivalent to mortality 2017

Results? 10 Water fleas (Daphnia) All alive – no effects All dead – 100% effects Lethal Concentration LC100, Effective Concentration EC100 5 dead, 5 alive – 50% effects LC50 or EC50 2017

Statistical Analysis ANOVA – Analysis of variance Requires replication at each test concentration level Identifies a difference from the control Used to identify the highest test concertation with no effects No Observed Effects Concentration (NOEC) Lowest test concentration at which a significant effect was observed Lowest Observed Effect Concentration (LOEC) 2016

Alternative Approach Curve fitting Uses data from the entire experiment Allows more test concentrations with fewer replicates for each one Same total number of replicates Allows calculation of any effect level EC10, EC25, EC50, EC90 EC10 usually treated as equivalent to a NOEC for PNEC and EQS derivation in Europe 2016

Test Quality and Standardisation Most tests conducted to OECD standard guidelines Many also conducted to GLP Reliability criteria specific to each test Information in test guidelines Control performance? Minimum performance required for a valid test Usually based on endpoint used Standardised water chemistry common Different for different test species 2016

Chronic tests Usually same test species For deriving longer term thresholds more reliably Relevant endpoints growth or reproduction Algae population growth Daphnia reproduction (# of offspring) Fish growth (biomass, length) or development Rotifer population growth Plant root growth Molluscs growth (biomass, shell length) Insect growth (biomass, length) 2016

Data Sources Study summaries e.g. IUCLID/ECHA Study reports – if available Open literature Other reviews – OECD, ICCA, ESR, NICNAS, ECETOC Thresholds may not be directly usable, but data should have been reviewed and summarised Different interpretation of the same data may be required NOEC vs EC10 vs EC25, SSD vs deterministic, AF? 2017

What Data Counts Test data for a species which is not locally or regionally relevant is taken as being representative of other untested locally and regionally relevant species Include all reliable and relevant data Relevant endpoints should have a direct link to population viability Many behavioural endpoints are not relevant! 2017

Non standard tests Usually from research laboratories Different species and taxonomic groups which might be required to represent an ecosystem Insects, molluscs, plants, worms, etc. Can be difficult to assess adequacy of control performance without guidance Difficulties in drawing conclusions about the test Could also be standard species under non-standard test conditions, or in natural waters 2017

Summary Standardised species Standardised tests Standardised endpoints Existing regulatory summaries 2017

2016