OECD 203, Regulation (EC) No 440/2008, C.1, DIN EN ISO 7346-1, DIN EN ISO 7346 Ospar PARCOM Proceeding with marine species
Determination of acute toxicity on fish (96h) of a test substance.
The fish are exposed to the test substance by means of a serial dilution for a period of 96 h. In parallel, a control test line is run without test substance. After 24, 48, 72, and 96 h, the mortalities and the test conditions (temperature, pH, and oxygen content) are recorded. The LC50 (lethal concentration for 50% of the test fish) is calculated as test result from the dose-effect relationship. From the test data, also the LC0 (highest concentration at which all fish survive) and the LC100 (lowest concentration at which all fish die) is estimated.
According to OECD 203 or EU 92/69/EEC, C.1, various test fish species can be used: Danio rerio (zebrafish), Pimephales promalas (fathead minnow), Cyprinus arpio (common carp), Poecilia reticulata (guppy), Lepomis macrochirus (bluegill), Oncorhynchus mykiss or Salmo gairdneri (rainbow trout).
For chemicals that can enter the marine environment (e. g. chemicals used in offshore petroleum industry), the OSPAR Commission for the protection of the marine environment of the North-East Atlantic as well as the European Union considers a risk assessment approach based on marine tests. According to the "OSPAR PARCOM Protocols on Methods for the Testing of Chemicals used in the Offshore Oil Industry", the turbot (Scophthalmus maximus) or the sheepshead minnow (Cyprinodon variegatus) are used as marine test fish.
Test substance properties
Dependent on the characteristic of the test substance the design of the test will be adapted: static test design without renewal of the test solution for soluble substances and semistatic test design with renewal of the test solution after 24, 48 and 72 hours for unstable substances.
Foranimal protection reasons, the number of test fish has to be restricted to a minimum. Therefore, according to the OECD Guidance No. 126, the implementation of a "limit test" with the lowest EC50 concentration achieved in the algae or daphnia test is considered before performing main test to determine exact LC50.
As part of the amendment of the Wastewater Ordinance from June 17, 2004, the fish toxicity test (DIN 38412-31) has been replaced by the fish egg test for wastewaters. Only in exceptional and well motivated cases, e. g. to address specific questions and problems, the fish test with golden orfs (Leuciscus idus) can still be used.