# Novel Aromatic Estolide Esters from Biobased Resources by a Green Synthetic Approach
## 2 Materials and Methods
### Biodegradation Studies  
Biodegradation studies of the oligoester were carried out in accordance with measurements of the OECD 306 protocols and the OxiTop(r) system equipped with measuring units (amber glass bottles (510 mL) and self-check measuring units), an inductive stirring platform and magnetic stirrer bars. In each bottle, there was 327.5 mL of the salt solution (prepared in accordance with OECD 306); 1 mL of DMSO or sample dissolved in DMSO; and 36.5 mL of sea water (inoculum) to reach a final concentration of 100 mg/L. The BOD measurements were performed at 21 \(\pm\) 1 \({}^{\circ}\)C. The BOD and Dt (biodegradability) were calculated as previously reported [26; 38].  
Biodegradability was assessed through the biochemical oxygen demand (BOD) consumed by aerobic microorganism in order to metabolize the oligomers containing ricinoleic acid and caffe, determined using an OxiTop(r) system from Xylem Analytics, Weilheim, Germany. This system is equipped with a sensor to measure the BOD. The biodegradability assessment for the synthesized oligomers was performed based on their solubility in DMSO, following the standardized protocol ISO 17556:2019 [39]. The measurements were taken in an aqueous culture medium using an inoculum sourced from natural river water, specifically the Bega River in Timisoara, Romania. The methodology described by [PERSON] et al. [40] was used in the preparation of the aqueous culture medium, which included a salt solution and liquid inoculum. The sample was dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 100 mg/L in the liquid medium and was tested in duplicate. The sample was then incubated at 21 \({}^{\circ}\)C under magnetic stirring for 21 days and compared against a control sample that contained the same amount of DMSO but did not include the oligomeric material. Measurements of the dissolved oxygen were taken every 24 h.  
The BOD values for the oligoester sample (BODs) and the control samples (\(BOD_{c}\)) were initially expressed in milligrams per liter (mg/L) and later converted to a weight-based measure in the mg/mg unit (\(BOD_{d}\)) for the biodegradability analysis, as defined in Equation (1):  
\[BOD_{d}=\frac{BOD_{s}-BOD_{c}}{c}(\text{mg/mg}) \tag{1}\]  
In this equation, \(BOD_{d}\) represents the biochemical oxygen demand required to degrade the sample over a specified number of days (d) in mg/mg; \(BOD_{s}\) is the biochemical oxygen demand measured for the OxiTop(r) bottle containing the sample in mg/L; \(BOD_{c}\) is the biochemical oxygen demand measured for the control bottle without the sample in mg/L; and \(c\) is the sample concentration in the aqueous medium in mg/L.  
The \(BOD_{d}\) values were then compared with the theoretical oxygen consumption (TOD) for each sample, calculated using Equation (2):  
\[TOD=\frac{16\cdot(2C+0.5H-O)}{M_{n}}(\text{mg/mg}) \tag{2}\]  
In this equation, 16 represents the molecular weight of oxygen in mg/mmol, while C, \(H\), and \(O\) denote the weight fractions of carbon, hydrogen, and oxygen present in the chemical structure of the sample, \(M_{n}\) is the molecular weight of the sample expressed in mg/mmol.  
Finally, the ultimate degree of biodegradability (\(D_{t}\)) was expressed as a percentage using Equation (3):  
\[D_{t}=\frac{BOD_{d}}{TOD}\cdot 100(\%) \tag{3}\]