User Guide to Thresholds and Classification - Environmental ...

311User Guide for Thresholds and ClassificationsCrustacea testsAcute testingAcute tests with crustacea generally begin with first instar juveniles. For daphnids, a test duration of 48 hoursis used. For other crustacea, such as mysids or others, a duration of 96 hours is typical. The observationalendpoint is mortality or immobilisation as a surrogate to mortality. Immobilisation is defined as unresponsiveto gentle prodding. Tests consistent with OECD Test Guideline 202 Part 1 (Daphnia acute) or US-EPAOPPTS 850.1035 (Mysid acute toxicity) or their equivalents should be used for classification.Chronic testingChronic tests with crustacea also generally begin with first instar juveniles and continue through maturationand reproduction. For daphnids, 21 days is sufficient for maturation and the production of 3 broods. Formysids, 28 days is necessary. Observational endpoints include time to first brood, number of offspringproduced per female, growth, and survival. It is recommended that tests consistent with OECD TestGuideline 202 Part 2 (Daphnia reproduction) or US-EPA 850.1350 (Mysid chronic) or their equivalents beused in the classification scheme.Algae/plant testsTests in algaeAlgae are cultured and exposed to the test substance in a nutrient-enriched medium. Tests consistent withOECD Test Guideline 201 (Algal growth inhibition) should be used. Standard test methods employ a celldensity in the inoculum in order to ensure exponential growth through the test, usually 3 to 4 days‘ duration.The algal test is a short-term test and, although it provides both acute and chronic endpoints, only the acuteEC 50 is used for classification in the harmonised system. The preferred observational endpoint in this study isalgal growth rate inhibition because it is not dependent on the test design, whereas biomass depends bothon growth rate of the test species as well as test duration and other elements of test design. If the endpoint isreported only as reduction in biomass or is not specified, then this value may be interpreted as an equivalentendpoint.Tests in aquatic macrophytesThe most commonly used vascular plants for aquatic toxicity tests are duckweeds (Lemna gibba and Lemnaminor). The Lemna test is a short-term test and, although it provides both acute and sub-chronic endpoints,only the acute EC 50 is used for classification in the harmonised system. The tests last for up to 14 days andare performed in nutrient enriched media similar to that used for algae, but may be increased in strength.The observational endpoint is based on change in the number of fronds produced. Tests consistent withOECD Test Guideline 221 on Lemna and US-EPA 850.4400 (aquatic plant toxicity, Lemna) should be used.January 2012 EPA0109