Within the EU, ash should be classified by its inherent hazardous effects under criterion H-14 (ecotoxic) in the Directive on waste (2008/98/EC). Today, however, there are no harmonized quantitative criterions for such a classification, but it is stated that biological test systems can be used. In this study seven ash materials were leached and characterized, both biologically and chemically. The objectives were to evaluate if (a) clear concentration-response relationships could be achieved for the selected toxicity tests (bacteria, algae, crustacean and fish), (b) some test(s) are generally more sensitive and (c) the toxic responses were consistent with the chemical analyzes. Interestingly, our results indicate that high concentrations of non-hazardous components (Ca, K) influenced the toxicity of almost all ash eluates, whereas hazardous components (e.g. Zn, Pb) only influenced the toxicity of the eluates ranked as most hazardous. If considering both hazardous and non-hazardous substances, the observed toxic responses were relatively consistent with the chemical analyzes. Our results further showed that the (sub)chronic tests were much more sensitive than the acute tests. However, the use of extrapolation factors to compensate for using the less sensitive acute tests will likely lead to either over- or underestimations of toxicity. Our recommendation is therefore that classification of waste according to H-14 should be based on (sub)chronic test data. Finally, given that treatment of the eluates prior to toxicity testing has a major significance on the concentration and speciation of released substances, further studies are needed in order to propose a relevant testing scheme.