## 5 Results and Discussion
### Ecotoxicological Characterization (EC) of RA, FA, A1, A2 and A3 Eluate Samples
Based on the TU values \(>10\) for the most sensitive ecotoxicity test used, i.e., 48 h mobility for _Daphnia magna_ (Table 8), the samples A2 and A3 were classified as moderate-highly toxic (class IV) (Table 6). Comparatively, both the A3 and A2 samples were found to exert higher levels of ecotoxicity towards the freshwater model crustacean than the A1 sample, and the order of potential ecotoxicity is: A2 \(>\) A3 \(>\) A1 (Tables 6 and 8). Given the results obtained, there was also no evidence to classify the eluate samples of RA, FA and A1 as ecotoxic (Table 6), since the results of CC and EC for these materials (Tables 7 and 8) comply with the limit values established in the French proposal CEMWE [25]. However, the results obtained for samples A2 and A3 allow classifying these materials as potentially ecotoxic based on the fact that the limit value of 10% for 48 h mobility \(EC_{50}\) in _Daphnia magna_ (corresponding to TU = 10 in Table 5) defined in the French proposal CEMWE [25] was exceeded (Table 6). There are reports by other authors of a considerable sensitivity of _Daphnia magna_ invertebrates when suspended in alkaline media (pH values as high as 11.3) [26]. In our work, despite the high pH values of all the eluate samples tested (Table 8), pH is not anticipated to be the only relevant factor contributing to the negative effects of the samples of this model invertebrate. This is because eluates from RA (A1) with a pH value of 12.5 were found much less toxic to the crustacean than the A2 and A3 eluates with pH values equal to 12.4 and 12.1, respectively (Table 8). Therefore, other factors, for instance the release of chemical compounds, including metals, from these materials into water due to leaching, may be influencing the biological response of the test-organism to the eluates' samples, as reported by others [13; 19]. Of the three short-term ecotoxicity tests used (with _Vibrio fischeri, Daphnia magna_, and _Saccharomyces cerevisiae_), the most sensitive one in analysing the ecotoxicity of cement-based raw and construction materials was the standard acute mobility test with the freshwater crustacean _Daphnia magna_. On the contrary, the bioluminescent bacterium test was the least sensitive to all eluates' samples, while the microplate susceptibility test with _Saccharomyces cerevisiae_ showed an intermediate response (except for the FA eluate sample). In this study, the yeast-based test is proposed as a suitable test for preliminary screening of the potential ecotoxicity of raw and construction materials. Even though a relatively low sensitivity of the yeast test, compared with the _Daphnia magna_ one, should be recognized, the yeast test provides some relevant advantages as test system, namely: sparing of higher eukaryotes in toxicity testing (required by regulators, e.g., REACH); meaningfulness of data for eukaryotic microorganisms that provide relevant services in ecosystems; simplicity; low-cost; small-scale (96-well microplate format) and reproducibility [21]. The potential leaching of metals and other chemicals from FA, A2 and A3 was very low for almost all analysed metals, including the ones in the European Union list of priority substances in the field of water policy (e.g., Cd, Hg, Pb, and Ni) [44]. Nevertheless, regarding landfill deposition and based on the CC alone, FA eluate was classified as hazardous (due to the Se level) while the eluates of A2 and A3 were considered non-hazardous (due to Mo and TDS levels) (Table 6). On the contrary, based on EC, the former was NOE or class III (according to CEMWE or TCS, respectively) while the latter were classified as class IV based on ecotoxicity towards _Daphnia magna_ (Table 6). These changes can be due to possible differences in the chemical composition of the eluates and/or in other parameters not analysed in this work.