Determination of biological markers of organic substances in sediment and soil samples by gas chromatography Nemanja Koljančića, Olga Vyviurskaa, Milica Balabanb, Ivan Špánika aInstitute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, Bratislava, 812 37 Slovakia bFaculty of Natural Sciences and Mathematics, University of Banja Luka, Mladena Stojanovića 2, Banja Luka, 78000 Bosnia and Herzegovina [email protected] Abstract: Once they reach the environment, petroleum hydrocarbons undergo various chemical, physico- chemical and biochemical transformation processes. Organic compounds which are not or are very poorly subject to these processes are thermodynamically the most stable isomers and they are called biological markers (biomarkers). This paper presents the results of the determination of organic substances in twelve samples taken in the area of the city of Banja Luka (Bosnia and Herzegovina). Two soil samples were taken in the Banja Luka city heating plant area and ten river sediment and soil samples were taken in the upper and lower basin of the Vrbas river in the Banja Luka city area. The aim of this study was to determine the biomarkers of oil-type pollutants in contaminated samples as well as the type of organic substances in samples taken near the contaminated area. Assisted solvent extraction was used to isolate the total petroleum hydrocarbons (TPH) from all twelve samples. Fractionation of the extracts into saturated and aromatic hydrocarbon fractions was performed by column chromatography. The fractions were analyzed by gas chromatography-mass spectrometry (GC-MS). On basis of the obtained chromatograms, biomarkers of petroleum pollutants and specific correlation parameters of organic substances in the samples were determined. The dominance of n-alkanes with odd C atoms as well as the presence of an unresolved complex mixture (UCM) on chromatograms of saturated and aromatic hydrocarbons showed the presence of anthropogenic organic substances of petroleum origin in the analyzed samples. Based on the obtained chromatograms, it can be concluded that microbial degradation of hydrocarbons in all samples occurred. Keywords: organic substances, hydrocarbon composition, biomarkers, GC-MS analysis, correlation param- eters, assisted solvent extraction Introduction are frequently found in petroleum, coal, sedimen- tary rocks, recent sediments, and soils. Molecular Hydrocarbons found in soil and sediments reflect structures of stable geochemical products can be both natural and anthropogenic inputs as well attributed to biogenic precursor molecules. At the as diagenetic processes taking place in the water same time, conversion from biogenic and thermo- column and during transport and sedimentation dynamically less stable compounds to more stable (Shirneshan et al., 2016). Crude oil consists mainly ones is especially interesting in isomers and stereo- of a complex mixture of hydrocarbon compounds isomers (Hazra et al., 2019). From the chemical (aliphatic and aromatic) and non-hydrocarbon com- point of view, a majority of biomarker molecules pounds (resins and asphaltenes). Environmental belong to aliphatic or aromatic hydrocarbons (Wal- contamination occurs frequently and it is associated ters et al., 2018; He et al., 2018; Kao et al., 2018). with production processes, refining, transportation Thus, hydrocarbon composition of petroleum has and storage of oil and petroleum products. Oil been extensively used for the determination of undergoes various transformation processes in the hydrocarbon sources in coastal sediments and soils. environment conditions, depending on the physico- A heating plant in the Banja Luka city still uses chemical and biological factors such as evaporation, heavy oil fractions like mazut as a heating medium. dissolution, microbial degradation, photooxidation Thus, the objectives of the present study are: (i) to and interaction between oils and sediments (Faboya determine the occurrence, distribution and source et al., 2016). Lower molecular weight hydrocarbons of saturated and aromatic hydrocarbons, (ii) to normally evaporate, while heavier compounds, examine the distribution, composition, and relative such as asphaltenes and resins, partially undergo maturity levels of saturated and aromatic hydrocar- deposition in sediments (Kakhki et al., 2018; Yo- bons in sediments and soils, and (iii) to evaluate the gaswara et al., 2020). Therefore, these compounds degree of microbial degradation of organic sub- 34 Acta Chimica Slovaca, Vol. 13, No. 1, 2020, pp. 34—40, DOI: 10.2478/acs-2020-0006 stances in analyzed samples. Twelve sediment and Scientific Inc., 2011). Dichloromethane/acetone soil samples were taken for analysis in the area of (1:1) mixture was used as an extraction solvent, the city of Banja Luka within the heating plant and and the following conditions were set: extraction in the lower and upper basin of the Vrbas river. The temperature of 175 °C, static time of 5 min, flush fractions of saturated and aromatic hydrocarbons volume of 60 %, gas purging time of 100 s, 1 static were analyzed by the GC-MS method to determine cycle. The obtained extract volume was reduced the specific correlation parameters, e.g. the degree to 5 mL using a rotary evaporator. Activated cop- of maturation and origin of the organic substances. per powder was added to the extract and it was sonicated for 3 min in order to remove elemental Material and Methods sulfur from the sample extracts. Chemicals Fractionation of total petroleum hydrocarbons Dichloromethane, n-hexane, elemental copper and extracts sodium sulfate were purchased from Merck (Darm- Column chromatography was used to separate the stadt, Germany). Florisil® was obtained from Fluka TPH extract into the saturated and aromatic hydro- AG (Buchs, Switzerland) and acetone was obtained carbons fractions using 2.5 g of Florisil® and 0.2 g from Mikrochem (Pezinok, Slovakia). of sodium sulfate, both activated at 130 °C for 24 h, as adsorbents. The column was loaded with 1 mL of Sampling the extract which was previously conditioned with Exact sampling locations are shown in Fig. 1. To- 10 mL of n-hexane. The saturated hydrocarbon tally twelve samples were studied in this work, while fraction was eluted with 5 mL of n-hexane while two were taken in the Banja Luka city heating plant the aromatic hydrocarbon fraction was eluted with area — samples 1 and 2. Other ten samples represent 3 mL of dichloromethane. river sediment samples and soil samples taken in the upper and the lower basin of the Vrbas river in GC-MS analysis the Banja Luka city area. All samples were air-dried The fractions of saturated and aromatic hydrocar- for 24 hours and sieved through 0.063 mm sieves. bons were analyzed using gas-chromatography/ mass-spectrometry (GC-MS). For this purpose, an Total petroleum hydrocarbons extraction Agilent 7890A gas chromatograph with an Agilent Extraction of total petroleum hydrocarbons (TPH) 5975C Mass Selective Detector (MSD) was used. from sediment and soil samples was performed by Large volume of aliquots was injected in a pro- assisted solvent extraction using the Dionex ASE grammed temperature vaporizer (PTV) in 5 µL 150 apparatus. Extraction cells were filled with portions with a repetition of ten times (total aliquot 28 g of previously air-dried and sieved samples volume was 50 µL) onto a HP-1ms capillary column and the extraction was accomplished according to (30 m × 0.250 mm i.d. × 0.250 µm film thickness). a previously established method (Thermo Fisher The inlet temperature program was set as follows: Fig. 1. A map of Bosnia and Herzegovina (left) and a map of the sampling locations in the area of the city of Banja Luka (right). Koljančić N et al., Determination of biological markers of organic substances in sediment… 35 40 °C (2 min) up to 300 °C (5 min) at 600 °C min–1. carbons are shown in Tab. 2. n-Alkane compounds Helium (99.999 % purity) was used as a carrier gas in all samples ranged from C12 to C35. Carbon at the flow rate of 1 mL min–1.The oven temperature pre ference index (CPI) values, representing the program was set as follows: 40 °C up to 50 °C (7 min) ratio of odd to even n-alkanes, was found to be in at 100 °C min–1, and to the final temperature of the range from 1.3881 to 2.6383. CPI values close 300 °C (15 min) at 10 °C min–1. Duration of the to 1 indicate petroleum and other anthropogenic measurement was 47.1 min. The transfer line tem- substances e.g. from the combustion of fossil fuels, perature was set to 300 °C, MS source temperature wood and agricultural debris (Faboya et al., 2016). and MS quadrupole temperature were 230 °C and The dominance of hydrocarbons with an odd car- 130 °C, respectively. Mass spectra were analyzed bon number also indicates the presence of organic using the MSD ChemStation software with NIST14, substances of anthropogenic origin. Furthermore, MPW2007 and W9N11databases. organic substances in the sediment and soil samples could originate from native organic substances Results and Discussion and oil-type anthropogenic organic substances (Samelak et al., 2018). The presence of unresolved Total petroleum hydrocarbons content complex mixture (UCM) on n-alkane chromato- Results of assisted solvent extraction are shown in grams is associated with the presence of aromatic Tab. 1. Concentration of TPH extracts ranged from heterocyclic compounds formed by the biological 0.1786 mg g–1 to 25.8214 mg g–1. An increase in the degradation of hydrocarbons (n-alkanes, steranes TPH concentration in samples 1 and 2 was expected and terpanes) (Romero, et al., 2015; Di Gregorio because both sample places are located in the area et al., 2016; Catania et al., 2018; Wang et al., 2018). of the city heating plant. Similar concentration of This group of compounds is typical for samples that TPH was observed for samples 3, 4, 5, 6, 7 and 8, have undergone microbial degradation of organic from the upper basin of the Vrbas river (before the substances (Romero, et al., 2015; Di Gregorio et al., heating plant).