Assessment of toxic normal saturated hydrocarbons in the Arabian seawater and Omani freshwater samples using liquid chromatography–mass spectrometry

The NHs contaminated water samples can cause several health problems such as skin irritation, acne, irregular breathing, lung irritation, cardiac arrest, itching, central nervous system (CNS), and cancer. Therefore, this current study aims to develop extraction and detection methods for normal hydrocarbons (NHs) in water samples with dichloromethane (DCM) and to detect and quantify the level of NHs in the seawater and freshwater samples by using liquid chromatography-mass spectrometry (LC–MS). Sixty-four water samples were collected from five different ports of Oman, including, Matrah, Al Mouj Marina, Salalah, Duqm, and Sohar ports, and one fresh water from Falaj Daris. The solvent–solvent extraction method was used to extract the NHs of seawater and freshwater sample with DCM. The DCM layer was passed through the silica gel column and evaporated DCM by using a rotary evaporator at low temperature and pressure. The samples were analyzed using liquid chromatography-mass spectrometry (LC/MS). The results showed that all collected seawater and freshwater samples contain NHs, but the concentration is below the detection limit. The water samples of those selected areas were not polluted by NHs toxicants. In conclusion, the low concentration of NHs could be due to the effective treatment of effluents discharged by industries, refineries, ships, and other marine vehicles. So far, it is the first report on extracting and detecting NHs in seawater and freshwater samples using LC/MS.


Introduction
70% of the earth's surface is covered by water, and it is the most vital liquid that exhibits anomalous behavior and distinct properties [1]. Therefore, water is needed for the survival of life. Recently, water quality has been a major challenge humanity faces as a consequence of the dramatic increase in water demand in most countries worldwide. However, rising human consumption of water, industrialization, marine dumping, wastewater, agriculture, oil leaks, and radioactive waste all speed up water pollution [2]. The most common ways to pollute water are industrial waste, toxic waste, petroleum, and disease-causing microorganisms [3][4][5][6]. Dumping trash in seawater and discharging domestic wastes, toxic chemicals, pesticides, and fertilizers will make the water unusable [7]. Moreover, the toxic heavy metals may also pollute the underground water [7].
Oman has long coastal areas, which have a large number of seaports, mainly in the dockyard areas [8]. All the ships and other water vehicles dump or discharge their chemical waste in the water. As a result, the water is polluted continuously. Hydrocarbons are a class of chemical constituents consisting of carbon and hydrogen. Petroleum compounds are the most discharged chemicals in seawater. Hydrocarbons are dangerous chemicals. The hydrocarbon-contaminated water can cause several health problems such as skin irritation, acne, irregular breathing, lung irritation, choking, cardiac arrest, itching, neurologic, central nervous system (CNS) and cancer diseases [9,10]. All different factors involved in rapid urbanization cause pollution of water resources. Each factor plays a foremost part in polluting the water. Certain pollutants have become a high environmental risk in the past two decades. The most frequent pollutants are the phenols and their derivatives, Polycyclic Aromatic Hydrocarbons (PAHs) and other hydrocarbons [11]. In addition, PAHs also have adverse effects on both the ecosystem and marine biodiversity. Several reports showed that most petroleum compounds continuously pollute the water and are common water pollutants [12][13][14].
Several research groups worked on normal and aromatic hydrocarbons for public health benefits. Normal hydrocarbons represent a group of organic non-polar stable compounds. Their properties deliver useful information on the biological and petrogenic sources of pollution. One report showed that the level of total hydrocarbon in seawater was within the range of 46 and 76 mg/L. However, the range in freshwater was between 28 and 102 mg/L [11]. Statistical data from the Ministry of Health in the Sultanate of Oman have shown that about 3% of the population suffers from various health conditions linked to exposure to natural saturated hydrocarbons [15]. They also showed that the nearest areas to oil refineries were highly contaminated by hydrocarbons based on aliphatic hydrocarbons. The data have shown that in the past few years, people exposed to saturated hydrocarbons have developed skin diseases, respiratory diseases, cardiac diseases, and different types of cancer. However, the study has estimated that the percentage of the infected population might rise to 6% in 2029 [15]. The primary source of exposure to saturated hydrocarbons in Oman is water.
All over the world, there has been a fast expansion in population, housing building, heavy industry, and agricultural activities over the last four decades [16]. This fast expansion shows no signs of slowing down and has resulted in an increasing rate of water pollution. For this reason, several studies in many countries have been conducted to detect the presence of chemical compounds in water samples. Hossain et al. [17] published a study to determine the level of NHs in one of the Bangladeshi rivers by Gas-Liquid Chromatography. This report showed that a high concentration of hydrocarbons is present in the river water samples. The presence of NHs components was analyzed through Flame Ionization Detection coupled with Gas Chromatography (GC-FID). The result of this study showed that different concentrations of hydrocarbons were found in river water samples within the range of 1.04-6135 parts per billion (ppm) [18]. A group of researchers from Sultan Qaboos University conducted another study on sewage effluents in Oman. The results showed the presence of different types of halogenated saturated hydrocarbons in the groundwater samples contaminated by sewage water. There was a high concentration of halogenated alkane presence in the sewage samples, such as; bromodichloromethane, which is considered a derivative of NHs. Several standard techniques were used for sample collection, handling and analysis [18]. The samples were collected from different coastal areas in Oman and Ion chromatography was used to detect anions and cations in a complex [19]. Various sensitive analytical instruments and detection methods were proposed to determine the organic contaminants in natural freshwater and wastewater samples worldwide. Most researchers widely used gas chromatography and gas chromatography-mass spectrometry to detect these organic contaminants [20]. All those methods are prevalent and sensitive, but there are drawbacks as the collection of samples, the process and the extraction of the organic contaminants are timeconsuming and expensive [9,10]. Since the old time, several analytical methods have been used to analyze NHs in various water and biological samples [21,22]. The literature searches showed that there is no work conducted on NHs in the seawater and freshwater samples in the Arabian seawater samples. In addition, nobody has yet used the LC-MS method to detect NHs in the water samples. Therefore, a new approach has been developed for the extraction, detection and continuous monitoring of the toxic NHs in the water samples by using Liquid Chromatography-Mass Spectrometry (LC-MS).

Chemicals and reagents
Several chemicals and agents were used in this experiment. Copper sulfate (purity 99.5%) and silica gel GF 254 for column chromatography were obtained from E. Merck, UK. Dichloromethane and acetone (HPLC grade) were obtained from Fluka, Japan. Acetonitrile (HPLC grade) was obtained from Fisher Company, Germany. All the obtained chemicals are analytical grade.

Equipment and glassware
A rotary evaporator (Model WEV-1001V, DAIHAN, China) was used to evaporate the samples. Some of the glassware, including graduated cylinders, beakers, test tubes, separatory funnel, conical and round bottom flasks, and other glassware was purchased from Borosil, India.

LC-MS analysis
LC-MS (Q-TOF, Agilent technologies 6530, Singapore) was used to analyze the sea and freshwater samples collected from different ports. The pre-concentration samples (10.00 µL) were injected using Hamilton syringes into the injector. Agilent C 18 column (4.6 × 150 mm, 5 μm) was used to analyze the pre-concentrated samples. The oven temperature was 30 °C, and the gradient mobile phase consisted of A) 100% H 2 O + 0.1% formic acid + 10 mM AF B) 100% acetonitrile + 0.1% formic acid + 10 mM AF. The flow rate was 0.2 ml/min. The software program used belongs to Agilent Technologies Company.

Sample collection
Sixty-four water samples were collected from five different ports of the Arabian Sea in Oman in December 2021. Freshwater samples were collected from Falaj Daris, Al Dakhiliyah Governorate. The seawater samples were collected from five coastal points: Al Duqm Port, Al Mouj Port, Matrah Port, Salalah Port and Sohar Port (Fig. 1). The water samples were collected in one-liter capacity amber plastic bottles. Twelve bottles of seawater were collected from each port and four bottles of fresh water were collected from Falaj Daris. The method of the collection focused on two levels in each port. The first level was one kilometer from the shore, and the second was 100 m further into the sea. Two samples were collected from each point, one from surface water and the second from a one-meter depth (Fig. 2) [18]. The total number of collected samples from each level was six. Then 5 ml of copper sulfate was added immediately to the samples, and they were shaken to stabilize the NHs following the process described by Hossain et al. [23]. Similarly, four freshwater samples were collected from Falaj. Finally, all the collected seawater and freshwater samples were carried to the Pharmacy Research Lab for further extraction.

Extraction of hydrocarbons
For the extraction of hydrocarbons, 100 ml from each collected water sample was transferred to a separatory funnel. Then, DCM (20 ml) was added to the separatory funnel and shaken vigorously by hand for 20 min. The separatory funnel was kept at room temperature until the two layers of DCM and water were formed. The DCM layer was collected in a pre-cleaned conical flask. The extraction process was repeated twice, and the DCM layers were combined [18]. The combined DCM layer was passed through a column containing silica gel GF 254 (mesh size 120-260 mm) to remove any interfering chemicals. The column was packed with 10 gm of silica gel GF 254 with petroleum ether. The collected DCM layer was passed through to the column, and the eluent was collected in a pre-cleaned conical flask. Then, the DCM was evaporated using a rotary evaporator at a low temperature until 2-3 ml was left. Finally, the evaporated samples were collected in vials for analysis. All 64 water samples were processed, isolated and evaporated for the analysis of NHs [24].

Quantification of NHs
All the isolated pre-concentrated samples were analyzed by the LC-MS. The analysis method was developed for NHs from the water samples. The samples were analyzed using a C 18 column (4.6 × 150 mm, 5 μm, Agilent) at room temperature. Ten microliters (10 µl) samples from each pre-concentrated vial were injected into the LC-MS using a Hamilton syringe [18]. The oven temperature was maintained at 30 °C, and the gradient mobile phase was used. Finally, the presence of NHs in each sample was determined based on their molecular mass from the chromatograms [18].

Statistical data analysis
All data obtained from LC-MS were expressed as means of triplicate measurements. The NHs concentration of the sea and freshwater samples were used as input variables for recognized discriminant analysis. All LC-MS data were processed via SPSS 15 (SPSS, Chicago, United States). Differences at P < 0.05 are considered to be significant.

Results
Water pollution is defined as the contamination of water bodies, usually as a consequence of different human activities. Fuel combustion is one of the main activities contributing to water contamination by hydrocarbons [24]. NHs are one of the most well-known hydrocarbons that cause severe problems for living organisms and the environment. For this reason, the current study seeks to detect the presence of normal saturated hydrocarbons by LC-MS in fresh and seawater samples from six locations, as mentioned earlier.  7) showed that all the collected water samples contained NHs. Nevertheless, concentration of every NHs (C 10 to C 24 ) in the studied regions were below detection level.

Discussion
Water plays a vital role on earth. Without water, living organisms will no longer survive. In the past two decades, water pollution has increased tremendously in a threatening way. Safe and contaminants-free water is needed for future generations. Water resources are often contaminated by several industries such as cosmetics, leather, textile and paper [14]. Saturated hydrocarbons are dangerous to human health, marine organisms, and biological ecosystems. This study is designed to identify and analyze the level of saturated hydrocarbons in the collected sea and freshwater samples from coastal regions and groundwater in Oman [14]. Furthermore, it aims to determine whether it is adhering to international standard guidelines in order to ensure the safety and quality of water in Oman. Finally, to recommend solutions to prevent the toxicants and treat the pollution. This experimental data could raise awareness concerning the adverse outcomes of water pollution among citizens and inspire researchers to work on future studies on water pollution. They are found in trace amounts in the marine environment, including water, suspended solids, organisms, and sediments [14]. The sources of hydrocarbons are both natural, meaning they occur irrespective of human interference or artificial caused by a multitude of human activities. The analysis of hydrocarbons in the marine environment stems from the often-repeated observation that raised concentrations of non-biosynthetic hydrocarbons have detrimental effects on many marine life forms. Normal hydrocarbons are one of the organic toxicants that continuously contaminate seawater in different ways. The use of contaminated water is a significant cause of human diseases such as irregular breathing, lung irritation, choking, cardiac arrest, itching, skin cancer, and neurologic problems [25]. Therefore, the fundamental demand of the worldwide population today is to control water contamination in any possible way. Most scientists have used gas chromatography and gas chromatography-mass spectroscopy to determine NHs in water, sediments, wastewater samples, and the like [18,25,26]. Previous studies using GC-MS showed that high concentrations of NHs were about 3940 μg/L obtained in the water samples [18]. In general, several studies showed that all the analyzed sea and freshwater samples contain a high amount of NHs, which is above the permissible concentration [27]. These kinds of abnormal results are due to the disposal of waste, urban runoffs, automobile wastes, stormwater, industrial effluents, domestic wastes, and oil spills [28][29][30].
In this experiment, 60 seawater and four freshwater samples were collected to measure the status of NHs levels. During the collection of the water samples, CuSO 4 solution was added to the samples immediately to stabilize the NHs derivatives. The extraction and detection methods of NHs by using GC and GC-MS analytical techniques were described by several authors [12][13][14][15][16][17][18][19]. However, the method for extraction and detection of NHs by using LC-MS is still pending. Nobody has developed the method of extraction and detection of NHs using LC-MS. Therefore, in our present LC-MS experiment, we used the solvent-solvent extraction method to extract NHs from the water samples. As a solvent, DCM was used twice for complete extraction. After extraction, the DCM layer was passed through the solid phase silica gel column to remove the other interfering contaminants [18]. The eluent from the column was concentrated by the usual method, and the samples were analyzed by LC-MS. The programs for the detection and analysis by LC-MS were described in the experimental section. By these developed extraction and detection methods, the results showed that all the collected sea and freshwater samples contained a good number of NHs. However, we cannot detect their levels because the concentration of all NHs derivatives is lower than the LC/MS detection limit. The 1 3 LC-MS detection limit of NHs is the low pg/µL level in the water samples. In addition, due to the lack of NHs standards, we are unable to quantify the NHs in the collected seawater and freshwater samples. The literature showed that there is no report on the detection of NHs by using LC-MS Q-TOF. So far, it is the first report for determining NHs in the sea and freshwater samples using LC/MS Q-TOF. Therefore, we cannot compare our experimental results to the reported values of NHs. According to the experimental findings, all the seawater samples from five seaports and the freshwater from Falaj Daris contain NHs. However, the concentration is below the detection limit, which means the water samples from collected areas are not polluted by NHs. Moreover, the low concentration of NHs could be attributed to the well-managed effluents discharged by industries, refineries, ships, and other marine vehicles, all of which adhere to international and local governmental rules and regulations.

Conclusion
NHs contaminated water and poor sanitation are linked to the transmission of diseases such as skin irritation, acne, irregular breathing, lung irritation, choking, cardiac arrest, itching, neurologic, central nervous system (CNS) and cancer diseases. Therefore, this study aims to modify the extraction  method for NHs from water samples, and a new approach was developed to analyze NHs using LC-MS. The newly discovered method was used to analyze NHs in the water samples for the first time. All the collected water samples contain NHs, which were detected by LC-MS. The results showed that the amount of NHs in the sea and freshwater samples was below the LC-MS detection limit. It means the seawater and freshwater samples are free from organic contaminants of NHs. Therefore, the Government of Oman manages water pollution, and Oman's citizens and residents are aware of the significance of beach hygiene to avoid the negative outcomes of water pollution. The present study could raise awareness concerning the adverse outcomes of water pollution among citizens and inspire researchers to work on future studies on water pollution. This study may help the country guide the competent authorities to impose strict laws and regulations to avoid water contamination, which could increase people's cognition of the importance of water and its conservation. In the future, this study can be conducted by developed methods to get more precise results, like using the Kundu-Danish apparatus instead of a rotary evaporator to preserve the volatile substances from evaporation. In addition, the study could be expanded to include more freshwater resources, such as reservoirs, waterfalls in Salalah, hot springs, and more coastal areas, such as the Al Sharqya Governorate and Musandam Governorate.