**2. Materials and Methods**

The model samples were fired bricks from brickyards situated in the towns of: (a) Vysoké Mýto and (b) Holešov-Žopy (Czech Republic) (Figure 1). Dry brick samples in the shape of discs (d = 5 cm, h = 1 cm) represented three layers from the surface to the depth of the brick (0–1 cm, 1–2 cm and 2–3 cm). The composition of brick samples is described in previous literature [17,18]. Pore size distribution, bulk density, total porosity and the specific surface area, determined by Mercury Intrusion Porosimetry, are described in Figures 2 and 3 and in Table 1.

**Figure 1.** Cutouts of bricks: Vysoké Mýto (left) and Holešov-Žopy (right).

**Figure 2.** Pore size distribution of individual layer of the brick: Vysoké Mýto (0–1 cm, 1–2 cm, 2–3 cm from the cover).

**Figure 3.** Pore size distribution of individual layer of the brick Holešov-Žopy (0–1 cm, 1–2 cm, 2–3 cm from the cover).

**Table 1.** Selected properties of the tested bricks—specific surface area (m/g), total cumulative volume (cc/g), total porosity (%) and bulk density (g/cm3). The mean values from three replicates are used in the table.


CaCl2 (99% purity) from Lach-Ner Ltd. (Prague, Czech Republic) was used in the experiment. The concentration of the CaCl2 solution was chosen to be 10 g/L based on previous laboratory experiments. The solution was prepared by dissolving appropriate amounts of CaCl2 powder in distilled water.

A two-compartment box made of plexiglass was used (see Figure 4). Initially, dry brick discs were sealed with silicone in a circular aperture cut in the plexiglass panel, splitting the box into two chambers. One of the chambers (chamber 1) was filled with 500 mL of distilled water, while the other chamber (chamber 2) was filled with calcium solution of the desired concentration (500 mL).

**Figure 4.** One of the model plexiglass boxes for experiments with calcium diffusion.

The box was closed with a plexiglass lid. The box was placed on a table at the ambient temperature of 20 ± 2 ◦C for a period of 240 h. The amount of calcium diffused through the sample was measured in chamber 1 by titration in selected time periods (0–240 h). When the accumulation started, the distilled water was replaced by the same new water and these replacements were conducted after each measuring of calcium concentrations.

A 0.05 M solution of chelaton III was used to measure the calcium concentration. Namely 500 μL of a 5 M solution of KOH was added to a specific amount of the monitored aquatic sample from chamber 1 and colored using a murexide indicator. Chelaton III was then dripped into the sample by an automatic burette [12]. A specific amount of titration reagent was used to calculate the calcium concentration c(Ca) in mg/L, according to the following Equation (1):

$$\text{c(Ca)} \left(\frac{\text{mg}}{\text{L}}\right) = \frac{\text{V(ch).M(ch).M(Ca).1000}}{\text{V(sample)}} \tag{1}$$

where:

V(ch) is the volume of used chelaton (mL); M(ch) = 0.05 is its molarity; M(Ca) = 40 g/mol is the calcium molar mass; V(sample) is the volume of the analyzed aquatic sample (mL).

The disks from all of the studied brick layers (0–1 cm, 1–2 cm and 2–3 cm) from the two bricks were used. Every disc was separately placed in distilled water (V = 100 mL) in a glass vessel and left at a temperature of 20 ± 2 ◦C and under the illumination of 5000 LUX intensity in a light–dark period of 12:12. No organisms were added to the water. The samples were left in order for natural bio-colonization to occur on their surface. The samples were immersed in water for 30 days. They were then pulled out of the solutions and photographed. The biofilm scraped from the samples was then examined under the light microscope Olympus BX43 (Olympus, Prague, Czech Republic) with a CMOS camera (magnification 400×). The brick disks of the individual layers were then put into the glass test vessels into a 100 mL volume of distilled water. The test vessels were left at a temperature of 20 ± 2 ◦C and under the illumination of 5000 LUX intensity in a light-dark period of 12:12 for a time period of 168 h. The biomass of the biofilm was measured using a VIS spectrometer (under 680 nm of wavelength) (Thermo Fisher Scientific, Prague, Czech Republic).
