## 2 Materials and Methods
### Biodegradation of Formulations by Bacterial Cultures
#### 2.11.2 Method for Determining the Potential for Aerobic Biodegradation of Formulations  
The only sources of carbon and energy were H-B, H-E, H-S, O-B, O-E, O-S, and SDS (sodium dodecyl sulfate as reference compound) formulations with organic carbon concentration of 40 mg/L, which were tested in duplicate, and the results are presented as mean + SD. Two tests were conducted independently (in two measuring vessels). Figure 2 presents the arrangement of the test vessels (1.1, 1.2, 1.3, 1.4, and 1.5) and the placement of a magnetic stirrer (1.6) connected with tubes. The test apparatus was aerated by air (I) passing through carbon dioxide (CO\({}_{2}\)) absorbers (B and C). The velocity of the air was regulated by a valve, which was routed to a carbon dioxide absorber (1.1), followed by a CO\({}_{2}\) indicator (1.2), with the intention of indicating the presence of carbon dioxide in the air via turbidity. A total of 250 mL of the test medium, 25 mg of dried active sludge, and formulations corresponding to 40 mg/L of organic carbon were deposited in test vessel 1.3. The concentrations of the starting formulations were 69.78 mg for H-B, 70.37 mg for H-E, 71.53 mg for H-S, 13.66 mg for O-B, 13.68 mg O-E, 13.87 mg for O-S, and 84.11 mg for SDS, respectively. Figure 2: The carbon dioxide measurement method. The formulation's capacity for biodegradation resulted in the production of CO\({}_{2}\) in vessel 1.3, which reacted with sodium hydroxide (NaOH) to produce sodium carbonate (Na\({}_{2}\)CO\({}_{3}\)):  
\[2\;\mathrm{NaOH}+\mathrm{CO}_{2}=\mathrm{Na}_{2}\mathrm{CO}_{3}+\mathrm{H}_{2} \mathrm{O} \tag{4}\]  
To determine the CO\({}_{2}\) concentration in vessel 1.3, 10 mL of the solution from vessel 1.4 was moved into a 25 mL volumetric flask. Deionized water was added to the flask until the designated maximum level was reached. Next, the sample was analyzed (twice) with total organic carbon analyzer (TOC-LCSH/CSN Shimadzu Corporation) as follows:  
\[ \begin{array}{l}3\;\mathrm{Na}_{2}\mathrm{CO}_{3}+2\;\mathrm{H}_{3}\mathrm{PO }_{4}=\mathrm{CO}_{2}+2\;\mathrm{Na}_{3}\mathrm{PO}_{4}+3\;\mathrm{H}_{2} \mathrm{O}\\ 3\;\mathrm{NaHCO}_{3}+\mathrm{H}_{3}\mathrm{PO}_{4}=\mathrm{CO}_{2}+\mathrm{Na} _{3}\mathrm{PO}_{4}+3\;\mathrm{H}_{2}\mathrm{O}\end{array}  \tag{5}\]  
The test sample comprises carbonates (Na\({}_{2}\)CO\({}_{3}\)) and acidic carbonates (NaHCO\({}_{3}\)) that were acidified with orthophosphoric acid (H\({}_{3}\)PO\({}_{4}\)) to achieve a pH of 2-3. Na\({}_{2}\)CO\({}_{3}\) and NaHCO\({}_{3}\) acidified with H\({}_{3}\)PO\({}_{4}\) are converted to CO\({}_{2}\). First, sodium bicarbonate (NaHCO\({}_{3}\)) and sodium carbonate (Na\({}_{2}\)CO\({}_{3}\)) calibration curves were created. In order to achieve this, 4.415 g of Na\({}_{2}\)CO\({}_{3}\) (which had been dried for two hours at 285 \({}^{\circ}\)C in a muffle furnace) and 3.500 g of NaHCO\({}_{3}\) (which had been dried for two hours over silica gel) were added to a 1000 mL flask. The flask was then filled with deionized water (previously boiled). From the starting solution thus prepared, dilutions of sodium carbonate and sodium bicarbonate were prepared in the concentration range of 0-100 mg/L inorganic carbon (IC), and a calibration curve was prepared. The volume of the dispensed sample was 50 \(\mu\)L. Utilizing the calibration curve (y = 4.1187x + 7.1718; R\({}^{2}\) = 0.999), the inorganic carbon (IC) content in the test specimens was determined.