## 4 Testing Methods  
The tests performed are summarized in Table 4. The leaching test and further CC and EC of the obtained eluate samples were carried out in the Analytical Laboratory of Instituto Superior Tecnico (LAIST, Tecnico-Lisboa, Portugal) following recommended guidelines as indicated (Table 4), except for the yeast growth inhibition test, which used an in-house microplate susceptibility test (Table 4). The leaching test was intended to simulate the behaviour of materials when they come into contact with water as leaching agent under given conditions. This test allows predicting the behaviour of the waste if it is deposited in a landfill or in contact with an ecosystem, assuming this process may occur similarly in the environment. In this study, eluate samples were obtained in accordance with the requirements of EN 12457-4 [31]. The test consists of leaching a portion of the solid raw materials with a liquid/solid ratio of 10 over a period of 24 h at \(20\pm 2\)\({}^{\circ}\)C. Deionized water (ultrapure) through the Millipore system (Merck KGaA, Darmstadt, Germany) was used as a leaching agent, with practically zero electrical conductivity. The pH of this water was close to that of rainwater (pH 5-7). The leaching test according to EN 12457-4 [31] required the materials to be fragmented and sieved to obtain particles finer than 10 mm. The mixture of the obtained solid material with the deionized water was placed in a vessel and mechanically agitated for a period of \(24\pm 1\) h in a shaking equipment, with orbital motion. Subsequently, the samples were subjected to vacuum filtration through 0.45 \(\upmu\)m porosity filter paper and placed in a sterile container at 4 \({}^{\circ}\)C until further analysis (CC and EC). The CC of the eluate samples aimed at classifying the materials in relation to their potential danger. Therefore, the parameters analysed for CC (Table 4) were selected from the ones established in Directive 1999/31/EC [23] and in EC Decision 2003/33/EC [24] that regulate the acceptance of solid waste in landfills; most of them are also present in the French proposal on Criteria on the Evaluation Methods for Waste Ecotoxicity (CEMWE) [25]. The EC of the eluates allows detecting the effect of chemical components that may be present in solution, translating this information into an effect on a test organism used as a bioindicator. In this study, the EC of the samples was made using a battery of three short-term acute toxicity tests with the following test organisms: _Vibrio fischeri_, _Daphnia magna_, and _Saccharomyces cerevisiae_ (Table 4), as described before [21]. Briefly, the degree of toxicity was determined from the measurement of the harmful effects caused by various concentrations of each sample, comprising the undiluted eluate sample (100% sample) and a dilution series of it (nominal concentrations: 75%, 50%, 25%, 12.5%, 6.25%, and 3.125%), on the test organisms. For each undiluted or diluted sample, the endpoint measured was bioluminescence upon 30-min exposure for _Vibrio fischeri_, mobility after 24- and 48-h exposure for _Daphnia magna_, or growth after 16-h exposure for the yeast (Table 4). The toxicity index \(EC_{50}\), namely the median-effective concentration of each eluate sample (an eluate concentration that causes a response decrease in the exposed organism population to 50% of that measured in a non-toxic control sample), was determined [21]. These toxicity indexes were further processed following the Toxicity