## 2 Materials and Methods
### Application of Amalgam Resorations Following Cavity Preparation  
Each tooth was prepared using a micro motor with a Tungsten Carbide 245 bur under water cooling. All occlusal cavities were standardized with dimensions of 2.5 (bucco-lingual) \(\times\) 2.5 (occlusion-pulpal) \(\times\) 3.0 mm (mesio-distal). The proximal walls of the cavities were standardized to 3.0 (bucco-lingual) \(\times\) 4.0 (occluso-gingival) \(\times\) 2.0 mm (mesio-distal). The pulpal and the axial walls of the cavity were flattened using an inverted cone bur. To ensure consistency in cavity design and dimensions throughout this study, the prepared teeth were verified using a digital caliper. The Universal Matrix system Supermat Kerr (Kerr UK, Peterborough, UK) was applied to each tooth for easy and standardized application of the amalgam. Tytin (Kerr UK, Peterborough, UK) was selected for the preparation of the dental amalgam. This product is a uncompositional, spherical alloy material that is a high copper typical restorative material. According to the manufacturer, the pre-mixed alloy powder consists of 59% silver, 28% tin, and 13% copper by weight. Upon trituration with mercury, the final amalgam contains approximately 42.5% mercury by weight. An amalgam mixer (Optimix, Kerr, Middleton, WI, USA) ensured proper mixing through a 5 s vibration. After mixing, the amalgam was placed using an amalgam carrier and condensed with a condenser. A ball burnisher was used to remove the superficial mercury-rich layer. The material was then carved to replicate the natural anatomy of the teeth using a carver. The prepared specimens were stored in plastic bottles containing 50 mL of artificial saliva at a temperature of 37 \({}^{\circ}\)C. Data were collected at predetermined time points: 2 h and 1, 2, 7, and 30 days. At the specified time intervals, samples were carefully drawn from the plastic containers to measure the concentration of the released mercury in the artificial saliva. Since the concentration is independent of the total volume, only a portion (1 mL) of the solution was required for accurate analysis. The saliva samples were not renewed between the time intervals. The mercury levels in the artificial saliva were measured using an atomic absorption spectrophotometer (GB 932 + atomic absorption spectrophotometer; Varian Techtron, SpectrAA 400; Varian Assoc., California, USA). The operating principle of the instrument is based on the measurement of the mercury signal. A blank, five calibration standards, three water samples, and two certified reference materials (Environmental Protection Agency [EPA] Trace Metals--AA Quality Control Sample, Lot TMA 989, and Environmental Resources Agency [ERA] Priority Pollution(tm)/CLP Quality Control Standard) were subjected to digestion following the EPA mercury digestion protocol. The digestion protocol consisted of the following steps: (1) Add 5 mL of conc. H\({}_{2}\)SO\({}_{4}\) and 2.5 mL of conc. HNO\({}_{3}\). (2) Add 15 mL of 5% potassium permanganate. (3) Shake and add additional potassium permanganate until purple color persists. (4) Add 8 mL of potassium persulfate. (5) Heat for 2 h at 95 \({}^{\circ}\)C. (6) Cool and add 6 mL of sodium chloride/ hydroxylamine sulfate (or hydrochloride). Then, the sample was ready for analysis. The digestion process was carried out in sealed 250 mL Nalgene PMP containers using a Precision(tm) Model 25 shaking water bath. Subsequent analysis of the blank, standards, and samples was performed using the Mercury Concentration Accessory (MCA-90) instrument. The reductant channel of the Vapor Generation Accessory (VGA-76) contained 10% SnCl\({}_{2}\) (wt/vol) in 20% HCl. The \"acid\" channel contained 1% hydroxylamine HCl. The spectrophotometer parameters were configured as follows: instrument mode set to absorbance, measurement mode to peak height, wavelength at 253.7 nm, and measurement time set to 30 s [43].