## 5 Results and Discussion
### Ecotoxicological Characterization (EC) of RA, FA, A1, A2 and A3 Eluate Samples  
The results of the EC are presented in Table 8. Despite the alkaline nature and slightly different pH of the tested eluates samples (Table 7), no corrections were made to their pH values in toxicity testing, since it was intended to simulate environmental exposure conditions as close as possible to the real ones. Therefore, the ecotoxicity indexes and TU values presented in Table 8 combine the contributions of possible harmful effects associated not only with the chemical components present in the eluate samples but also with their alkaline pH values. The eluate sample of RA (equal to A1) did not cause inhibition of _Vibrio fischeri_ luminescence. Comparatively, the toxicity of the same samples to _Daphnia magna_ and _Saccharomyces cerevisiae_ test organisms was higher (Table 8). These results suggest that the bioluminescent bacterium was not sensitive to exposure to the RA (or A1) sample. On the contrary, the crustacean and the yeast tests presented similar results between them, with TU values between 5.3 and 6.8 in the _Daphnia magna_ toxicity test (24 h and 48 h exposure, respectively) and equal to 5.1 in the yeast toxicity test (16 h exposure) (Table 8). These TU values higher than 1 but considerably lower than 10 (TCS system [41]; Table 5) in both toxicity tests with RA (or A1) indicate potentially low acute ecotoxicity (class III) of these materials (Table 6). Regarding the FA eluate sample, _Vibrio fischeri_ bioluminescencecence and _Daphnia magna_ mobility were both moderately affected by this sample, contrary to the yeast growth which was not inhibited upon exposure to the FA sample (Table 8 and [21]). The results reported in the study of [PERSON] et al. [10; 49] indicated _Daphnia magna_ among the diverse test organisms they used (bacteria, microalga, crustacean, and rotifer), to be the most sensitive test organism in the evaluation of the ecotoxicity of coal FA leachates [10; 49]. The authors attributed this result to the lower tolerance of this invertebrate to the presence of high Cr concentration in the samples [49]. This study also revealed that the presence of Cu, Ni, and Zn in the leachate samples resulted in a significant bioluminescence inhibition of _Vibrio fischeri_. In our study, the concentrations of these metals in all eluate samples are very low (Table 7), so the resulting effect is expected to be low. The TU values much lower than 10 (TCS system [41]; Table 5) in both _Daphnia magna_ and _Vibrio fischeri_ toxicity tests (TU = 3.2 and 2, respectively; Table 8), suggest very low acute ecotoxicity (class III) of FA eluate (Table 6). The eluate samples of A2 and A3 did not cause inhibition of _Vibrio fischeri_ bioluminescence. However, both the crustacean mobility and the yeast growth were considerably affected by the A2 and the A3 samples (Table 8). In both cases, the comparison of the respective TU values indicated that  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{3}{*}{**Materials**} & \multirow{3}{*}{**pH**} & \multicolumn{3}{c}{**Bacterium _Vibrio_**} & \multirow{3}{*}{**Crustacean _Daphnia magna_**} & \multicolumn{3}{c}{**Yeast**} \\  & & _fischeri_ & & & & & _Saccharomyces cerevisiae_ \\ \cline{3-8}  & & \(EC_{\text{g}}\) **(\%)** & \multirow{2}{*}{**TU**} & \(EC_{\text{g}}\) **(\%)** & \multirow{2}{*}{**TU**} & \(EC_{\text{g}}\) **(\%)** & \multirow{2}{*}{**TU**} & \multirow{2}{*}{**EC\_{g}} **(\%)** & \\  & & **[30 min]** & & **[24 h]** & & **[24 h]** & & **[48 h]** & **[16 h]** & **TU** \\ \hline RA & 12.5 & \(>\)100 & \(<\)1 & 18.8 & 5.3 & 14.6 & 6.8 & 19.8 & 5.1 \\ FA & 11.8 & 49.3 & 2.0 & 30.8 & 3.2 & 30.8 & 3.2 &  
the _Daphnia magna_ toxicity test (TU [24 h] = 7.4 and 15 for A3 and A2, respectively) was much more sensitive than the yeast one (TU [16 h] = 2.5 and 3.3, respectively) (Table 8).