Evaluation of a battery of biotests to improve waste ecotoxicity assessment (HP 14), using incineration bottom ash as a case study

The assessment of waste ecotoxicity (hazardous property HP14 in the European Union) is fundamental for proper waste classification and safe application/disposal. Biotests are relevant for evaluating waste complex matrices, but their efficiency is crucial to encourage their adoption at the industrial level. This work aims at evaluating possibilities of improving the efficiency of a biotest battery previously suggested in the literature, regarding test selection, duration, and/or laboratory resources optimization. Fresh incineration bottom ash (IBA) was the case study. The test battery analysed included standard aquatic (bacteria, microalgae, macrophytes, daphnids, rotifers, fairy shrimp) and terrestrial (bacteria, plants, earthworms, collembolans) organisms. The assessment followed an Extended Limit Test design (three dilutions of eluate or solid IBA) and the Lowest Ineffective Dilution (LID-approach) for ecotoxicity classification. The results emphasize the importance of testing different species. It was also evidenced that tests with daphnids and earthworms may be shortened to 24 h; the miniaturization of tests is suitable as e.g. differential sensitivity of microalgae and macrophytes was captured with low variability; alternative testing kits can be used when methodological difficulties are found. Microalgae were more sensitive than macrophytes. Similar results were found for the Thamnotoxkit and daphnids test for eluates with natural pH, so the former may be used as an alternative. B. rapa was the most sensitive organism, suggesting that it may be tested as the only terrestrial plant species and that minimum test duration is appropriate. F. candida does not appear to add information to the battery. The differences in sensitivity of A. fischeri and E. fetida compared to the remaining species were not significant enough to exclude them from the battery. Thus, this work suggests a biotest battery to test IBA comprising aquatic tests -Aliivibrio fischeri, Raphidocelis subcapitata (miniaturised test), and Daphnia magna (24 h when clear deleterious effects are observed) or Thamnocephalus platyurus (toxkit) - and terrestrial tests - Arthrobacter globiformis, Brassica rapa (14 d), and Eisenia fetida (24 h). Testing waste with natural pH is also recommended. The Extended Limit Test design considering the LID-approach seems useful in waste testing, particularly for the industry, involving low effort, test material requirements, and few laboratory resources. The LID-approach allowed for differentiating ecotoxic from non-ecotoxic effects and captured different sensitivities between species. Ecotoxicological assessment of other waste may benefit from these recommendations, but caution should be taken given the properties of each waste type.