*2.2. Eggshell Conditioning*

Eggshells with their membrane were washed under tap water, dried at room temperature, and stored at −35 ◦C in order to prevent the spoilage of the eggshell samples before using them. Before using them, eggshells were ground in a sturdy vessel in which the material was pounded with a pestle and sieved to obtain the required particle size (1–1.5 cm2). Thereafter, pieces of eggshell were used as the adsorbent in the adsorption studies.

The major constituents of the eggshell are carbonates, sulphates and phosphates of calcium and magnesium, and organic matter. Traces of Na, K, Mn, Fe, Cu, and Sr metals are also present in the eggshell [27]. The density of the eggshell is about 2.53 g/cm3, which is significantly larger than that of eggshell membrane (1.358 g/cm3). The major constituents of the eggshell are calcium carbonate (94%), organic matter (4%), calcium phosphate (1%), and magnesium carbonate (1%). The eggshell membrane possesses nearly 60% protein (collagen (35%), glucosamine (10%), chondroitin (9%), and hyaluronic acid (5%)), along with other inorganic components like Ca, Mg, Si, Zn, and so on in smaller quantities [28]. The membrane surface bears positively charged sites produced by basic side chains of amino acids. It has a very high surface area with special functional groups such as hydroxyl (−OH), thiol (−SH), carboxyl (−COOH), amino (−NH2), amide (−CONH2), and so on, which strongly interact with some chemical species present in the albumen.

### *2.3. Dye Solution Preparation*

DB78 is an azo dye (CAS 2503-73-3) whose molecular weight is 1055.91 g/mol and whose formula is C42H25N7Na4O13S4. Aqueous solutions with different concentrations of dye (25, 50, 100, 150, 200, 250, and 300 mg/L) were prepared from a 1 g/L stock solution, and used to determine the capacity of adsorption of the eggshell.

## *2.4. Analyses and Data Evaluation*

The concentration of DB78 was measured at the wavelength of 612 nm, which is the maximum absorbance of the dye [15]. A spectrophotometer was used to determine the absorbance before and after the treatment (UV-1603, Shimadzu, Kyoto, Japan).

## *2.5. Adsorption Experiments*

The adsorption experiments were conducted at different dye concentrations, ranging from 25 to 300 mg/L. The kinetic adsorption studies were performed at different doses of adsorbent, pH, and dye concentrations. The effect of contact time on the adsorption capacity of eggshell was conducted while varying dye concentrations from 25 to 300 mg/L. Furthermore, the effect of adsorbent dosage was studied to optimize the adsorption process. pH was carefully adjusted between 3 and 11 to determine the optimum pH to ensure the maximum DB78 removal. Apart from that, the effect of temperature on the adsorption of DB78 by eggshell was investigated at three different temperatures (29, 55, and 75 ◦C).

A typical experiment was conducted by adding the eggshell to 40 mL of different dye concentrations of the DB78 solution. The mixture was stirred for a predetermined period of time at a constant speed of 500 rpm. The residual dye concentration in the solution was measured at time intervals (20, 40, 60, 80, 100, 120, and 140 min) until the equilibrium conditions were attained. At the end of each interval, the samples were centrifuged at 3000 rpm for 5 min to separate the solid phase, completely removing impurities that may later affect the measure.

The amount of dye adsorbed on eggshell (*qt*) at time (*t*), in mg/g, was determined by Equation (1), as follows [29]:

$$q\_t = \frac{V \left(\mathbb{C}\_0 - \mathbb{C}\_\varepsilon\right)}{m} \tag{1}$$

where *V* is the volume of dye solution (L); *C*<sup>0</sup> and *Ce* are the initial and equilibrium concentrations of dye in liquid phase (mg/L), respectively; and *m* is the mass of eggshell (g). All the experiments were carried out in triplicate. Three adsorption kinetics models, three isotherms, and the thermodynamic study were evaluated to elucidate the mechanism of dye adsorption.
