This document provides guidance for the testing of substances, and mixtures, classed as “difficult to test” for the purposes of determining their aquatic toxicity. Whilst the guidance given is targeted towards tests described by the OECD Guidelines identified it may also be relevant to other tests. The guidance relates to the practical aspects of carrying out valid tests with “difficult” test substances and mixtures and presenting the results. The guidance is considered to reflect good practice but it is important to recognise that some test substances and mixtures will present specific scientific and technical issues that may fall outside its scope. This document is divided into five substantive sections covering guidance for carrying out a preliminary assessment of test substance stability; general considerations on selection of exposure regimes; development of media preparation and exposure systems; sampling of test media for analysis, and calculating and reporting test results.