Spatiotemporal assessment and monitoring of hydrocarbons contamination of water and sediments in skikda bay (algeria)

ABSTRACT The current study aims to the spatiotemporal assessment and monitoring of hydrocarbons contamination in water and sediments of Skikda bay (Algeria), as a result of the presence of industrial wastewater rejected by a petroleum refinery complex in the area. Water and sediment samples were obtained from six different sites during the fourth seasons in the studies area. Total Hydrocarbons (THC) were determined by a gravimetric method, while polycyclic aromatic hydrocarbons (PAHs)(Anthracene, Pyrene and Benzo (a) pyrene) were determined by using High-Performance Liquid Chromatography (HPLC-UV/Vis). The results obtained indicateda high contamination by hydrocarbons in water and sediment samples from the sites exposed to the industrial refinery.The concentrations of THC, Anthracene, Pyrene and Benzo (a) pyrene recorded in water during the four seasons were in this order: 78–9457 µg/L; < LOD Anthracene −157.1 µg/L; < LOD Pyrene- 188.6 µg/L; < LOD Benzo (a) pyrene −2224.45 µg/L respectively, with averages of: 1209.14 µg/L; 16.03 µg/L; 22.98 µg/L; 119.03 µg/L respectively. While the concentrations of THC, Anthracene, Pyrene and Benzo (a) pyrene recorded in sediments during the four seasons were in this order: 323–185,450 µg/g dw;< LOD Anthracene – 87,624.7 µg/g dw;< LOD Pyrene- 17,485.5 µg/g dw;< LOD Benzo (a) pyrene −39,555.5 µg/g dw respectively,With averages of: 13,201.264 µg/g dw; 4199.966 µg/g dw and 1524.478 µg/g dw; 2464.815 µg/g dw respectively.An important seasonal impact in the water was demonstrated by the threecompounds selected for this analysis (Anthracene, Pyrene, and Benzo (a) pyrene). While in sediments, just THC showed significant seasonal variations. This variation can be attributed to the influence of physicochemical parameters, microbial load, reject flow and the type of hydrocarbon studied. Thereby, Wastewater in the refinery should be well treated to protect marine life and human safety.


Introduction
The technological development of human society has greatly increased the requirements for energy. Petroleum is the main source of energy for industry and daily life. It is a mixture of chemicals compounds that are made mainly from hydrogen, carbon and so-referred to as hydrocarbons [1]. Nowadays, the contamination resulting from the activities related to the petrochemical industry and the coal-refining process that is become a serious environmental problem because of their wastewaters, which commonly contain substances considered as persistent organics pollutants (POPs) such as polycyclic aromatic hydrocarbons (PAHs), Phenols, Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) [2]. These pollutants reach marine water and sediments in different ways, which include wastes burn out, accidental oil spill during transportation, deposition from municipal and industrial effluents [3]. Hydrocarbons are persistent environmental pollutants under their recalcitrant nature to biodegradation, bioaccumulation in the environment and immense health effects associated with its exposure [4]. POPs, especially PAHs cover a wide range of environmentally relevant properties, including semi to low volatility and intermediate to low water solubility [5]. They are generally toxic compounds, some of them presented carcinogenic, genotoxic, and/or mutagenic potential for several organisms; they can be transported over long distances and bioaccumulate in the food chain where can reach high concentrations in living organisms [6][7][8][9][10]. Aside from carcinogenicity, mutagenicityandgenotoxicity,PAHs have also been reported to cause cardiovascular diseases [11,12] asthma and other respiratory diseases [13,14], birth defects [15] And other diseases like renal, neuro, immuno, reproductive and developmental toxicities in humans and laboratory animals [16].
The majority of the PAHsmight be introduced in the aquatic environment such as rivers, lakes, reservoirs, and estuaries through anthropogenic inputs. They might also affect the aquatic biota, as well as wildlife and humans health via the food chain [17]. Several studies have indicated that mostsusceptible environments regarding anthropogenic activities are coastal areas including estuaries and have been frequently reported as contaminated by PAHs [18,19]. In Algeria, there are five oil refineries with a total production capacity of 652,500 Barrels perDay (BPD). The Skikda refinery, which occupies 183 hectares, produces more oil than all otherfourrefineries. It is also ranked as a number one in Africa with 352,700 BPD and in the number three over the world [20]. Due to the location of this refinery on the coastal area of the Skikda region (Algeria), the discharges of wastewaters constitute an important pollution hazard in the Mediterranean Sea and the environmental fate of total hydrocarbons and PAHs contained in their wastewaters is of great concern.
The objectives of this study are:(1) Perform a Spatiotemporal Assessment and Monitoring of THC and three PAHs (Anthracene, Pyrene, and Benzo (a) pyrene) concentrations in two different compartments (water and sediments) of the aquatic ecosystem exposed to the petroleum refinery rejects.(2) Perform a physicochemical and microbial characterisation of the aquatic ecosystem that surrounds the petroleum refinery and their effects on THC and PAHs behaviour and fate between the ecosystem compartments.(3) Tryto comprehend the seasonal and site effects on physicochemical microbialparameters and THC and PAHs distribution.

Description of the sampling sites
The Gulf of Skikda is located in the northwestern region of Algeria, about 470 km east of the capital Algiers. The study area is in contact with a massive manufacturing petrochemical complex, where raw petroleum and refined hydrocarbon products contaminate the surrounding areas via atmospheric pollution as well as effluents, which are dumped into the aquatic ecosystems. For all analyses, the sediments, the river water, the estuary water and the seawater were collected from 6 sites located nears to the petroleum refinery of Skikda. Thelocalizationssampling sites are showed in the Table 1.The first site St1 (chosen as the reference site) is located before the rejection point of the refinery in the confluence point of 'Safsaf' and 'Zramna' rivers, the second one is located after the refinery rejection point in the estuary zone, the rest of the sites are located on both sides of the estuary area the third and the fourth are located in 'Ben Mhidi' and 'Flifla' beaches respectively. Finally the fifth and the sixth points are located in 'the paradise' and 'the career' beaches respectively, as shown in Figure 1.

Samples collection
Water and sediments samples were obtained in October 2016, January 2017, April 2017 and July 2017. Three Sediment and Water samples were collected from each site and in each season and then we proceed to homogenisation and have an average sample for  each site and in each season. Water samples were collected from0.3 m below the air-water interface in amber glass bottles of 1 L. Sediment samples were taken using cylindrical amber glass bottles of 0.5L. Samples intended for microbiological analysis are taken in sterilised glass bottles of 0.25 L. All samples were transported to the laboratory for analysis in a cooler at 4-6°C. All tools used for sampling are previously cleaned and rinsed with acetone.

Water
The Physicochemical parameters, such as pH, Conductivity, Salinity, Dissolved Oxygen (DO), Temperature and Oxidation-Reduction Potential (ORP) were measured in situ by using a portable multi-parameter apparatus (HANNA Hl9829 model). Suspended Matter (SM) was determined by filtration of the water with a vacuum pump on cellulose nitrate filters with a porosity of 0.45 µm as described in [21].

Sediments
For the sediments the following characteristics were determined: Electrical Conductivity (EC) and pH were measured by using multi-parameters apparatus (HANNA Hl9829 model). Organic carbon and matter were measured by the chromic acid wet oxidation method as described by Walkley and Black [22]. Phosphorus was determined by Olsen method [23].

Enumeration of total viable heterophilic bacteria
This enumeration was done on ordinary nutrient agar, thus allowing revivable heterotrophic bacteria to develop. Decimal dilutions were firstly prepared for each water sample. Two Petri dishes of nutritive agar were inoculated on their surface with 0.1 ml of each dilution. The dishes are then incubated at 25°C for 72 hours [24].
To calculate the microbial load, we apply the following formula:N ¼ P

Total hydro carbons (THC) were determined as described in the Gravimetric method (for water and sediment)
Total hydrocarbons (for water and sediment) were determined using the Gravimetric method as described by Fuseyet al [25,26]. This method can be simply done by evaporating the extraction solvent and the residue weighing. The abiotic (evaporation process) loss was previously estimated in the marked sterile vials. For the extraction from the soil, the technique was slightly modified, according to this technique we could determine the quantity of hydrocarbonspresent into the sample to be analysed. The principle of the method consists of the following points: (1) Volume of (100 mL) of the sample to be analysed was poured into a separating funnel. (2) Few drops of Methyl Orange and 0.2 mL of 50% HCl were added in order to acidify the sample. (3) Chloroform (best solvent for extraction of organic phases) was added for a volume equal to half of the sample volume. (4) The mixture was well shaken with opening from time to time to let the gas escape and then it was settled for some minutes. Finally, the upper phase was filtered through Na 2 SO 4 to eliminate all traces of humidity. The solvent containing the organic phase was recuperated in a beaker previously weighed and placed into a stove until total evaporation. The final sample was weighed and the obtained quantity of the residue was calculated using the formula below: Q Total (ppm) = W f -W i /V s Where: Q Total is the quantity of the total hydrocarbon, W f the final weigh of the sample, W i the initial weigh of the sample and V S the volume of the sample.

Polycyclic aromatic hydrocarbons (PAHs)(anthracene, pyrene, benzo (a) pyrene) analysis
They were determined as described by Tadashi et al [27] and the Technical Committee CEN/TC BT [28]. PAHs selected for this study (Anthracene, Pyrene, and Benzo (a) pyreneare)are among the 16 PAHs most environmentally hazardous PAHsand listed as priority control pollutants according to the United States Environmental Protection Agency (USEPA) [29,30]. They represent also the three categories of PAHs; light 2-3 rings 152-178 g/mol, intermediate 4 rings 202 g/mol, and heavy > 5 rings 228-278 g/mol(fig. S1). Anthracene, Pyrene and Benzo (a) pyrene concentrations in sediments and water were determined using HPLCsystem with a UV/Visdetector according to their maximum absorption and retention time ( fig.S3 and table S.1).For the extraction procedure 20 mL of water samples and 20 g dry weight of sediment was acidified with 1 N HCl, shaken for 3 min with an equal volume of 1:1 (v:v) n-hexane:ethyl acetate, and centrifuged (9600 rpm; 4 •C; 10 min). The organic layer was then collected. The extract was dried under flowing nitrogen and the dry extract was dissolved in 1 mL acetonitrile and analysed [26]. The HPLC analysis was conducted using an Agilent Technology HPLC system (Agilent 1260 Infinity Quaternary) by 20 μL sample injection, a UV/Vis detector and an analytical column (StableBond C18 (Zorbax) (4.6 × 150 mm × 15 µm). The system was controlled by use of Chem Station for LC 3D software (Agilent Technologies). The mobile phase was a gradient flow (1 mL/min) with acetonitrile and water with detection at wavelengths of 253 nm, 239 nm, and 295 nm for Anthracene, Pyrene, and Benzo (a) pyrene respectively according to their maximum absorption ( fig.S2). The temperature in the column oven was set to 25°C. Mobile phase with multi-step gradient elution conditions was used with a total run time of 60 min per sample extract injection (Table 2).
For the sediments the residuals concentrations were calculated using:  3) The recovery of Anthracene, Pyrene and Benzo (a) pyrenewas determined by comparing the response of the method to the reference material and the individual PAH compounds spiked and analysed as real sample. The average recovery of Anthracene, Pyrene and Benzo (a) pyrene in water was 91%, 95% and 99% respectively and in the solid phase was 84.68%, 79.50% and 97.05% for Anthracene, Pyrene and Benzo (a) pyrene respectively. The average percent recovery was calculated using thefollowing equation: Where, Concentration (observed) is the concentration observed in the samples and Concentration (spiked) is the initial concentration spiked to the sample.
To assess the sensitivity of the instrument detection Limit of determination and limit of quantification (LOD and LOQ)were calculated using the following formula: Where:S a is the standard deviation of the response and b isthe slope of the calibration curve. The standard deviation ofthe response can be estimated by the standard deviation ofeither y-residuals, or y-intercepts, of regression lines [31].The calculated LOD and LOQ ranged between 0.028-0.044 mg/L and 0.044-0.147 mg/L (table S.1).

Statistical analysis
Statistical analyses were performed using XLSTAT (Inc. corporation. 2014) software. Data normality was checked using the Shapiro-wilk test. Since data sets were not normally distributed Kruskal-Wallis analysis was applied for investigating the seasons and site variations of physicochemical characteristics, microbial load, and hydrocarbons concentrations. If significant results were found, Dunn's test was used to detect significant differences between different sites and reference site St1 (p < 0.05). In addition, Principal Component Analysis (PCA) and Spearman (n-1) matrix correlation test were applied to highlight the main factors that influence the hydrocarbons distribution,investigate the relationship dependency between them and physicochemical parameters and microbial load. Finally, the linear Regression between water and sediment was applied in order to highlight the behaviour and fate of hydrocarbons in the studied compartments. The critical level of significance was set at (p < 0.05) for all tests.

Physicochemical and microbial parameters
Water physicochemical parameters measured over four seasons are presented in Table 3.
Obtained results allowed distinguishing a very high effect of season on microbial load and physicochemical parameters of water (T°, pH, Conductivity, Dissolved Oxygen (DO), Salinity, Total Dissolved Solids (TDS) and Suspended Mater (SM)) at (p < 0.001), except for the Redox Potential (RP) ( Table 3).
From a spatial point of view, except T°, all other studied parameters showed also a very highly significant effect at (p < 0.0001). Although the highest temperatures were recorded at St2 (estuary area after the discharge of wastewater from the refinery) and this is due to the high temperature of the effluent water that is probably used in the cooling process. Sediment Physicochemical parameters measured over four seasons and reported in (Table 4) showed a highly season variations of pH and Organic Matter (OM) contents at (p < 0.01), very highly significant for AssimilablePhosphorus (A. Phosphorus) concentration and microbial load at (p < 0.0001). Besides, very highly significant variation at (p < 0.0001) was found in the case of pH, Organic Matter (OM), A. Phosphorus, Conductivity and Microbial Load, from one site to another.
Spatiotemporal variations recorded in physicochemical parameters values of water and sediments samples (Tables 3 and Tables 4) can be attributed to the heterogeneity of sampling sites and wastewater releases of the petroleum refinery.Regarding Microbial Load, obtained results (Tables 3 and Tables 4) showed values ranged between (95 and 58 × 10 9 UFC/mL) in water and between (70 and 837 × 10 5 UFC/mL) in sediments samples. Kruskal-Wallis test results showed an important spatiotemporal variation of microbial load at (p < 0.05). In both of water and sediments compartment, the highest load is recorded at St1 during autumn, spring and summer. However, in winter, in St2 a high microbial load was distinguished comparing with other sites, this result can be attributed to the drop in temperature which slows down microbial growth. However, the temperature in St2 remains relatively high compared to other stations because of the hot rejection from the refinery. In general, the stations are classified in

Water
The concentrations of THC, Anthracene, Pyrene and Benzo (a) pyrene recorded in water during the four seasons were in this order: 78-9457 µg/L; < LOD Anthracene −157.1 µg/L; < LOD Pyrene -188.6 µg/L; < LOD Benzo (a) pyrene −2224.45 µg/L respectively, with averages of: 1209.14 µg/L; 16.03 µg/L; 22.98 µg/L; 119.03 µg/L respectively (Figure 2). From a seasonal point of view, an important effect was observed in the case of Pyrene (p < 0.05) and Benzo (a) pyrene (p < 0.05). The highest concentrations were recorded in winter compared with other seasons. This result is probably due to the decrease in temperature that causes an increase of hydrocarbons viscosity, which slows down their dilution in water. Volatilisation rates and biodegradation by surrounding microorganisms are also less efficient under lower temperatures. This agrees with what has been reported by Zheng et al that in cold environments, the persistence of petroleum hydrocarbons is greater [32].
Regarding the spatial evolution of THC and PAHs studied, very high concentrations were reported in St2 directly exposed to the discharge of industrial wastewater released by the petroleum refinery, compared with other sampling sites at (p < 0.0001) (Figure 2). The presence ofPAHsin the reference site (located before the refinery reject) can be attributed to other anthropogenic sources such as household waste, car traffic, agriculture and different industrial activities [33,34] and they can even originate from natural sources

Statistical results.
The principal component analysis was performed to determinate the main factors influencing the distribution of physicochemical parameters (T°, pH, Conductivity, DO, Salinity, RP, TDS, and SM), Microbial Load and hydrocarbons concentrations, it reveals also the dependencies and relationships the similarities between these parameters (Figure 3). The PC1 and PC2 together accounted for 71.52% (44.29% for PC1 and 27.23% for PC2) of the total variance. Obtained results showed three major groups of studied parameters following the main factors F1 and F2. F1represente water salinity which is in a positive correlation with conductivity, dissolved oxygen, pH and redox potential, andin negative correlation with TDS and microbial load which is mainly concentrated in St1 (freshwater of confluence point of'Safsaf' and 'Zramna' rivers) relatively loaded with organic matter and less oxygenised compared to other sampling stations. On the other hand, F2 represents hydrocarbons concentrations, mainly higher in St2, directly exposed to the industrial wastewater rejected by the petroleum refinery.
Spearman's correlation analysis results (Table 5) showed a significant positive correlation between hydrocarbons (p < 0.05) R THC-Anthracene = 0.92; R THC-Pyrene = 0.68; R THC-B(a)pyrene = 0.77; R Anthracene-Pyrene = 0.76; R Anthracene-B(a)Pyrene = 0.81; R Pyrene-B(a)Pyrene = 0.79. This result can be attributed to the same origin of hydrocarbons in the ecosystem (industrial wastewater of petroleum refinery). On another hand, the obtained results did not show a significant correlation between hydrocarbons and different water physicochemical parameters except for pH, which has a significant negative correlation (p < 0.05) with R pH-Anthracene = −0.35; R pH-Pyrene = −0.24; R pH-B(a)Pyrene = −0.23. This result agrees with the study of Caiyun et al [39]. who showed that water properties including pH and temperature have been frequently reported to influence behaviour and fate of PAHs in the water.
Regarding Microbial Load, significant positive correlation was found with THC (R = 0.29); Anthracene (R = 0.53); Pyrene (R = 0.35), B(a) Pyrene (R = 0.36). As regards temperature is in a positive correlation with the microbial load and the suspended matter because the growth of the total mesophilic flora is slowed down at low temperature (<15° C) whereas it is favoured in relatively high temperatures (20-37°C). This microbial growth leads to the formation of microbial mass in water, which will increase the rate of suspended matter. On the other hand, the temperature is inversely proportional to the rate of dissolved oxygen in the water. We can also note that the dissolved oxygen rate is inversely proportional to the SM rate which makes sense since the purity of water plays an important role in the solubility of oxygen [21]. Finally, the microbial load is negatively influenced by pH, Cond., DO, Salinity, RP,and positively by TDS and temperature, while MS has no significant influence.

Sediments
In this study, THC, Anthracene, Pyrene and Benzo (a) pyrene concentrations recorded during the four seasons were in this order: 323-185,450 µg/g dw;<LOD Anthracene -87,624.7 µg/g dw;<LOD Pyrene -17,485.5 µg/g dw;<LOD Benzo (a) pyrene -39,555.5 µg/g dw respectively (Figure 4). With averages of 13,201.264 µg/g dw; 4199.966 µg/g dw and 1524.478 µg/gdw; 2464.815 µg/g dw respectively. According to Baumard et al [41]., PAHscontamination in sediments can be classified into four different levels: low (<0.1 µg/g dw), moderate (0.1-1 µg/g dw), high (1-5 µg/g dw) and very high (> 5 µg/g dw). Thus, we can consider PAHs (Anthracene, Pyrene, and Benzo (a) pyrene) concentrations in Skikda bay sediments as very high. This result can be attributed to the presence of petroleum refinery.  Table 5. Spearman (n-1) correlation matrix (n-1) between studied parameters of water (p < 0.05). Compared to other studies, the concentrations of studied PAHs recorded in Skikda bay sediments, especially at the level of St2, greatly exceed the concentrations of PAHs recorded in other studied area through the world; 0.024 to 4.710 µg/g dw in Brighton marina of United Kingdom [36], 0.062 to 840.5 µg/g dw in Daliao River watershed of China [37], 0.016 to 1.358 µg/g dw in Yellow River of middle China [38], 0.090 to 0.218 µg/g in Guan River estuary of China [42], 0.0002 to 0.498 µg/g dw in estuary area in Northeast of Brazil [19], 1 to 5.750 µg/g dw in Yinma River Basin of China [39], 0.895 to 5.678 µg/g dw in Yinma River Basin of China [40], to 0.156 µg/g dw in Goiana estuary in the Northeastern coast of Brazil [5]. The abundance of PAHs in sediments was in the following order: Anthracene > Benzo (a) pyrene > Pyrene. Recorded concentrations do not follow the same order as in water. According to National Institute for Industrial Environment and Risks (NIIER), the ubiquitous concentrations of Anthracene and Benzo (a) pyrene in the soil and sediment are in order of 0.01 µg/gdw and 0.002 µg/gdw respectively [43], which can explain our results. Furthermore, the difference between Anthracene and Pyrene abundance can be attributed to their solubility in water (Anthracene: 0.076 mg/L; Pyrene 0.135 mg/L) [44].Possibly this makes theAnthracene less accessible to biodegradation by the microorganisms in the environment and tends to adhere to suspended solids to be finally found in sediments with higher concentrations compared to Pyrene.
From a seasonal point of view, statistical analysis showed a significant seasonal effect (p < 0.001) on THC concentrations in Skikda bay sediments. Dunn's test allowed distinguishing very high concentrations of THC in winter compared to other seasons. This is probably caused by the drop in temperature in the winter season. On the other hand, no seasonal effect was recorded on PAHs concentration in sediment; this is probably due to the no effect of volatilisation and photo-degradation. Regarding spatial evolution, the highest concentrations of THC, Anthracene, Pyrene, and Benzo (a) pyrene were reported in St2 at (p < 0.0001) directly exposed to the discharge of industrial wastewater released by the petroleum refinery, compared with other sampling sites (Figure 4). Dunn's test groups the stations into three different groups, the first contains stations 3, 4, and 6 the second contains stations 1 and 5 and in the third group is classified station 2.

Statistical results.
The principal component analysis was performed to highlight the main factors influencing the distribution of physicochemical parameters (OM, A. Phosphorus, pH, Cond), Microbial Load and hydrocarbons concentrations in different sediments sampling sites and seasons ( Figure 5). The PC1 and PC2 together accounted for 75.39% (56.46% for PC1 and 18.93% for PC2) of the total variance. Obtained results showed three major groups of studied parameters following the main factors F1 and F2. F1 represents OM positively correlated with hydrocarbons rejected by the petroleum refinery, especially in St2. F2 represents the microbial load negatively correlated with pH.

Linear regression
The linear regression between the concentrations of hydrocarbons in the two ecosystem's compartments (water and sediments) shows a positive correlation, with a linear coefficient of determination R 2 around: 0.84, 0.71, and 0.90 for THC, Anthracene, and Benzo (a) pyrene, respectively ( Figure 6). This means that the concentrations in the sediments are proportional to the concentrations in the water. According toArruda-Santos et al [5]. andCaiyun et al [39].,PAHs in the sediment-water system were in an unsteady state and prefer to beadsorbed onto a Suspended Particulate Matter (SPM) then rush and accumulate in the sediments, where slow chemical and biochemical transformations may occur which agree to whit our results. It should be noted that the strongest correlation between water and sediments was recorded in the case of Benzo (a) pyrene, which has the longest half-life (708 days) in soil and sediment compared to the others PAHs [45] and it is also concerned as the most carcinogen and toxic PAH [39]. On the other hand, this is not the case for the Pyrene contents where we find that the correlation between its concentration in water and sediments was positive but weak with R 2 = 0.25 ( Figure 6).

Conclusion
This study provides valuable knowledge on the physicochemical characteristics of the Skikda gulf aquatic ecosystem, the spatiotemporal distribution of THC and PAHs, and their environmental activity and fate. THC andPAHsstudied are mainly concentrated in the estuary zone (St2) just after the discharge point of wastewater from the petroleum refinery considered as the main source of pollution of this ecosystem by hydrocarbons.
Then this pollution spreads out on both sides of this zone to reach St3 and St5 with concentrations relatively weakened by the effect of the dilution but relatively high, because of maritime activities, in St5 compared to the St3. External factors such as temperature and microbial can have a powerful influence on contaminant behaviour. Besides, several factors such as molecular weight, the number of rings and solubility of PAHs play a very important role in their persistence and dissipation in the aquatic ecosystem studied.
Contamination in sediment is always higher than that in water due to the hydrophobic nature of hydrocarbons and their tendency to be adsorbed on the organic and suspended matter and accumulated finally in the sediment. This study warns us that the industrial wastewater, rejected by the petroleum refinery of Skikda, is poorly treated and can affect fauna and flora in the ecosystem. It can also represent a great threat to human health via the trophic chain.