# Species Identification and Effects of Aromatic Hydrocarbons on the Fluorescence Spectra of Different Oil Samples in Seawater

Authors: Yongchao Hou; Ying Li; Guannan Li; Mingfei Xu; Yunpeng Jia
Journal: Journal of Spectroscopy
 (2021)

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## Abstract

The identification of oil species and their characteristics in oil spills is particularly important for their efficient disposal. Since aromatic hydrocarbons (AHs) with various characteristics are the major fluorescent components of oils in seawater, they can be used to detect different oil species in seawater. Here, we developed a composition method using the fluorescence spectra of eight AH categories analyzed by gas chromatography-mass spectrometry (GC-MS) to evaluate the contribution of AHs to the total fluorescence of six different oil species. Qualitative and quantitative difference analysis of the experimental and composite fluorescence spectra was performed based on the redshift of the main peak position, the fluorescence intensity distance, and the generalized included angle cosine, while correlation analysis was used to establish the relationship between the different fluorescence spectral parameters and the American Petroleum Institute gravity and viscosity of the oil species. The fluorescence spectra recorded for heavy oil samples indicated a reduction in the fluorescence signal of fluorene series and an increase in the contribution of acenaphthene and pyrene series, indicating that the currently developed composition method by fluorescence spectroscopy combined with GC-MS can be used to distinguish and identify oil species in seawater samples.


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## Body

## 1. Introduction

Oil spills are one of the most common contaminants in marine environments that seriously affect all aspects of marine ecology and transport [1, 2]. Early determination and characterization of oil spills in terms of oil species, oil film thickness, and oil distribution can significantly facilitate decision-making for effective emergency disposal and cleanup programs [3, 4]. To date, remote sensing techniques, such as microwave, infrared, fluorescence, and visible remote sensing, as well as chemical analytical methods, including gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS), have been most frequently used to detect oil slicks and identify oil species in seawater [5–7].Fluorescence spectroscopy is a useful diagnostic tool used to evaluate petroleum hydrocarbons in natural seawater either separated by extraction and fractionation or not separated using direct detection methods [8, 9]. Most petroleum products emit light at longer wavelengths than the excitation light on their own or in organic solvents, as they consist of aromatic hydrocarbons (AHs) that have aromatic structures and bear conjugated double bonds with intrinsic fluorescence properties in the ultraviolet-visible (UV-vis) region (Figure 1) [10, 11]. Furthermore, AHs are the major fluorescent components in petroleum and predominate in the fluorescence spectra of sea surface oil slicks [12–14]. Thus, petroleum products containing different AH groups exhibit different fluorescence characteristics, which have been recently used to successfully detect and identify oil species in seawater [15, 16]. In addition, the development of reliable and relatively inexpensive instruments by fluorescence spectroscopy has enabled real-time detection of petroleum hydrocarbons, identification of oil species, and measurement of oil thickness in oil slicks [17–19].Figure 1
Fluorescent and nonfluorescent substances found in oil slicks.Due to their carcinogenic and toxic properties, AHs can be easily transferred and accumulated in food chains, thereby harming living organisms [20]. Fluorescence spectroscopy has been employed to analyze AHs in the marine environment [21, 22], while several studies have used GC-MS and stable isotope analysis to distinguish and quantify the AH content in petroleum-based products such as diesel, fuel, and crude oils [23–25]. In addition, the United States Environmental Protection Agency (US-EPA) has indicated a number of AHs as priority pollutants and summarized some of their key photophysical properties, which reflect their absorption and fluorescence behavior on sea surface or in seawater [26]. It has been reported that the presence of AHs in different oil species can significantly affect their fluorescence spectra, as they can generate characteristic fluorescence emissions [27]. However, the analyses of relationship between the chemical components and fluorescence spectra of different oils are yet to be developed. Therefore, the effect of AHs on the fluorescence spectra of oil species should be particularly considered during qualitative and quantitative analysis, especially when fluorescence spectroscopy is used to accurately distinguish oil species. To the best of our knowledge, the fluorescence spectroscopy method has never been applied to analyze the contribution of AHs fluorescence to the fluorescence spectra of different oils; we believe that this method can better detect and identify oil species.Here, we studied the effects of AHs on the fluorescence spectra of crude, fuel, and diesel oil seawater samples. To that end, we established a composition method using the fluorescence spectra of individual AHs bearing 2–4 rings to evaluate their contribution to the fluorescence spectra of the different oil species. More specifically, the relative content of each AH in each sample was measured by GC-MS, and the fluorescence spectroscopic data of the individual AHs were obtained from the US-EPA. In addition, the fluorescence spectra of the oil samples were recorded using a spectrofluorometer, and the differences between the experimental and composite fluorescence spectra were evaluated based on the spectral intensity and overall shape characteristics. The ultimate goal of this study was to assess the relevance of the relative content of AHs in the detection and identification of oil species in seawater based on their fluorescence signatures.


## 2. Materials and Methods

### 2.1. Oil Samples

Seawater samples were collected in October 2020 at the port of Lingshui, northern Yellow Sea, China. Kuwait and Iranian crude oil, 380# and 180# fuel oil, and 0# and −10# diesel oil samples were used as representatives of light crude oil, heavy fuel oil, and refined oil (diesel), respectively (Table1). For laboratory measurements, small amounts of the oil samples were weighed, added to a seawater sample, and shaken vigorously to reach the desired oil concentration in seawater (0.02 mg/L).Table 1
Characteristics of the tested oil samples.
NumberOil sampleAPI gravity1 (°)Viscosity (50°C, mm2/s)Oil species1Kuwait crude31.86.96Light crude oil2Iranian crude35.05.2Light crude oil3180# fuel11.3180.0Heavy fuel oil4380# fuel11.3380.0Heavy fuel oil50# diesel38.23.35Refined oil6−10# diesel38.23.40Refined oil1American Petroleum Institute (API) gravity = (141.5/specific gravity (60°F))−131.5.The fluorescence emission spectra of the samples were measured using a Cary Eclipse Varian spectrofluorometer using a 1 × 1 cm quartz cuvette. The fluorescence wavelength ranged between 260 and 700 nm with a 5 nm emission sampling interval, a 10 nm excitation slit, and a 10 nm emission slit. An excitation wavelength of 266 nm was selected to measure the fluorescence emission, as it was in the range where oil samples show high absorption and corresponded to a typical excitation wavelength of a Nd : YAG laser used for laser-induced fluorescence measurements applied directly in oil spill detection [28, 29].


### 2.2. GC-MS Analysis

The content of AHs in oil samples was analyzed based on our previous GC-MS studies [30, 31]. Each oil sample (∼0.2 g) was weighed and dissolved in 8 mL n-hexane, and the aromatics were fractionated using a chromatographic column (0.47 cm i.d. × 12 cm). The column was dry-packed with 3 g of silica gel (activated at 180°C for 20 h, 100–200 mesh, AR) and was topped with about 1 cm anhydrous sodium sulfate (activated at 350°C for 4 h, AR). The column was eluted with hexane, and the eluent was discarded before exposing the sodium sulfate layer to air. Approximately 200 μL of the concentrated extract was quantitatively transferred for GC-MS analysis. The aromatics were eluted with n-hexane/dichloromethane mixture (15 mL, 1 : 1 v/v) and concentrated to 1 mL under N2 flow prior to GC-MS analysis. GC-MS (Thermo Fisher Scientific, Waltham, MA, USA) was performed such that 1 μL of each sample was injected in a DB-5MS quartz chromatographic column (60 m × 0.25 mm × 0.25 μm) in the splitless mode. The GC temperature program involved heating from 60°C to 100°C at a rate of 20°C/min, where it was maintained for 6 min. Then, the temperature was increased from 100°C to 300°C at 4°C/min and isothermally maintained at 300°C for 35 min. For the MS measurements, the injector, interface, and ion source were maintained at 280°C, 250°C, and 200°C, respectively. The electron bombardment source for electron impact was 70 eV.


### 2.3. Fluorescence Composition Method

A fluorescence composition method was developed using the fluorescence spectra of individual AHs to evaluate their contribution to the total fluorescence of the oil samples. Moreover, the sum of these contributions was used to prepare a composite spectrum, which was further used to determine the total contribution of AHs bearing 2–4 rings to the fluorescence of different oil species following already reported studies [32, 33]. The fluorescence composition method of the identified AHs could be expressed in matrix/vector notation as follows:(1)Ifoil=IfAHXFXC,where Ifoil is the nλ × 1 vector of the computed or measured fluorescence intensity (nλ = number of wavelengths between 260 and 700 nm), IfAH is the nλ × nAH matrix of the AH fluorescence based on the fluorescence spectra data obtained from the US-EPA (nAH = number of AHs), XF is the nAH × nAH diagonal matrix representing the contribution rate of each AH to the total fluorescence spectra (when XF = E obtaining the composite spectrum, E is the identity matrix), and XC is the nAH × 1 vector of the relative content of each AH (determined by GC-MS) in the total identified AHs extracted from each oil sample.


## 2.1. Oil Samples

Seawater samples were collected in October 2020 at the port of Lingshui, northern Yellow Sea, China. Kuwait and Iranian crude oil, 380# and 180# fuel oil, and 0# and −10# diesel oil samples were used as representatives of light crude oil, heavy fuel oil, and refined oil (diesel), respectively (Table1). For laboratory measurements, small amounts of the oil samples were weighed, added to a seawater sample, and shaken vigorously to reach the desired oil concentration in seawater (0.02 mg/L).Table 1
Characteristics of the tested oil samples.
NumberOil sampleAPI gravity1 (°)Viscosity (50°C, mm2/s)Oil species1Kuwait crude31.86.96Light crude oil2Iranian crude35.05.2Light crude oil3180# fuel11.3180.0Heavy fuel oil4380# fuel11.3380.0Heavy fuel oil50# diesel38.23.35Refined oil6−10# diesel38.23.40Refined oil1American Petroleum Institute (API) gravity = (141.5/specific gravity (60°F))−131.5.The fluorescence emission spectra of the samples were measured using a Cary Eclipse Varian spectrofluorometer using a 1 × 1 cm quartz cuvette. The fluorescence wavelength ranged between 260 and 700 nm with a 5 nm emission sampling interval, a 10 nm excitation slit, and a 10 nm emission slit. An excitation wavelength of 266 nm was selected to measure the fluorescence emission, as it was in the range where oil samples show high absorption and corresponded to a typical excitation wavelength of a Nd : YAG laser used for laser-induced fluorescence measurements applied directly in oil spill detection [28, 29].


## 2.2. GC-MS Analysis

The content of AHs in oil samples was analyzed based on our previous GC-MS studies [30, 31]. Each oil sample (∼0.2 g) was weighed and dissolved in 8 mL n-hexane, and the aromatics were fractionated using a chromatographic column (0.47 cm i.d. × 12 cm). The column was dry-packed with 3 g of silica gel (activated at 180°C for 20 h, 100–200 mesh, AR) and was topped with about 1 cm anhydrous sodium sulfate (activated at 350°C for 4 h, AR). The column was eluted with hexane, and the eluent was discarded before exposing the sodium sulfate layer to air. Approximately 200 μL of the concentrated extract was quantitatively transferred for GC-MS analysis. The aromatics were eluted with n-hexane/dichloromethane mixture (15 mL, 1 : 1 v/v) and concentrated to 1 mL under N2 flow prior to GC-MS analysis. GC-MS (Thermo Fisher Scientific, Waltham, MA, USA) was performed such that 1 μL of each sample was injected in a DB-5MS quartz chromatographic column (60 m × 0.25 mm × 0.25 μm) in the splitless mode. The GC temperature program involved heating from 60°C to 100°C at a rate of 20°C/min, where it was maintained for 6 min. Then, the temperature was increased from 100°C to 300°C at 4°C/min and isothermally maintained at 300°C for 35 min. For the MS measurements, the injector, interface, and ion source were maintained at 280°C, 250°C, and 200°C, respectively. The electron bombardment source for electron impact was 70 eV.


## 2.3. Fluorescence Composition Method

A fluorescence composition method was developed using the fluorescence spectra of individual AHs to evaluate their contribution to the total fluorescence of the oil samples. Moreover, the sum of these contributions was used to prepare a composite spectrum, which was further used to determine the total contribution of AHs bearing 2–4 rings to the fluorescence of different oil species following already reported studies [32, 33]. The fluorescence composition method of the identified AHs could be expressed in matrix/vector notation as follows:(1)Ifoil=IfAHXFXC,where Ifoil is the nλ × 1 vector of the computed or measured fluorescence intensity (nλ = number of wavelengths between 260 and 700 nm), IfAH is the nλ × nAH matrix of the AH fluorescence based on the fluorescence spectra data obtained from the US-EPA (nAH = number of AHs), XF is the nAH × nAH diagonal matrix representing the contribution rate of each AH to the total fluorescence spectra (when XF = E obtaining the composite spectrum, E is the identity matrix), and XC is the nAH × 1 vector of the relative content of each AH (determined by GC-MS) in the total identified AHs extracted from each oil sample.


## 3. Results and Discussion

### 3.1. Distribution and Categories of AHs

Τhe relative content of AHs in Kuwait crude, Iranian crude, 380# fuel, 180# fuel, 0# diesel, and −10# diesel oil samples was determined by GC-MS after extraction and fractionation (Figure2), and the types of AHs included in each oil sample were identified by matching the retention times to 16 standard AHs [34, 35]. Specifically, the AHs detected in the six oil samples were naphthalene, methylnaphthalene, dimethylnaphthalene, trimethylnaphthalene, biphenyl, methylbiphenyl, 2, 2' + 4, 4'-dimethylbiphenyl, acenaphthene, fluorene, methylfluorene, dibenzothiophene, methyldibenzothiophene, 2, 4 + 4, 6-dimethyldibenzothiophene, phenanthrene, methylphenanthrene, dimethylphenanthrene, trimethylphenanthrene, pyrene, methylpyrene, and chrysene. The AH contents in the oil samples were determined by analyzing a standard mixture containing known concentrations of each AH. Moreover, the alkyl aromatic hydrocarbon compounds were identified by comparing the mass spectra and retention times obtained in this study with existing data [36, 37].Figure 2
Relative content of AHs in six oil samples determined by GC-MS analysis. Naphthalene (N); methylnaphthalene (MN); dimethylnaphthalene (DMN); trimethylnaphthalene (TMN); biphenyl (B); methylbiphenyl (MB); dimethylbiphenyl (DMB); acenaphthene (A); fluorene (F); methylfluorene (MF); dibenzothiophene (DBT); methyldibenzothiophene (MDBT); dimethyldibenzothiophene (DMDBT); phenanthrene (P); methylphenanthrene (MP); dimethylphenanthrene (DMP); trimethylphenanthrene (TMP); pyrene (PY); methylpyrene (MPY); and chrysene (C).Petroleum, a complex mixture containing multiple fluorescent substances, and the differences in photophysical properties and content of each component affect the overall fluorescence characteristics and intensity of the oil samples in seawater. It is, therefore, clear that the composition of the oil samples in AHs is complex, making it difficult to analyze the effect of the individual ingredients on the fluorescence spectra. Moreover, the effect of alkane substituents on the fluorescence spectra is generally weak, suggesting that AHs bearing alkyl substituents would exhibit similar fluorescence spectra [38, 39]. Therefore, AHs were divided into eight categories, namely, naphthalene, biphenyl, acenaphthene, fluorene, dibenzothiophene, phenanthrene, pyrene, and chrysene series (Table 2). Dominant AHs with a relative content of 60.88%, 60.90%, and 65.93% in Kuwait crude oil, Iranian crude oil, and 180# fuel oil, respectively, belonged to the naphthalene series. In contrast, the AHs in 380# fuel oil were almost equally distributed, and the highest contents were detected in the naphthalene (33.15%), dibenzothiophene (16.85%), and phenanthrene series (27.46%). Furthermore, the diesel oil samples included mainly AHs with two or three benzene rings. Hence, the relative contents of the eight AH categories in the six oil samples were used as the values of the XC parameter in the fluorescence composition method with nAH = 8.Table 2
Relative content of eight AH categories in Kuwait crude, Iranian crude, 380# fuel, 180# fuel, 0# diesel, and −10# diesel oil samples.
AHsaCategoriesKuwait crude (%)Iranian crude (%)180# fuel (%)380# fuel (%)0# diesel (%)−10# diesel (%)NNaphthalene series60.8860.9065.9333.1527.9528.7MNDMNTMNBBiphenyl series2.784.913.9414.3118.7322.46MBDMBTMBAAcenaphthene series1.180.800.331.333.562.84FFluorene series4.165.512.945.7023.8322.25MFDBTDBT series19.2110.809.1316.8517.5513.53MDBTDMDBTPPhenanthrene series10.4415.7515.3627.468.3810.22MPDMPTMPPYPyrene series0.770.752.371.2000MPYCChrysene series0.580.580000aNaphthalene (N); methylnaphthalene (MN); dimethylnaphthalene (DMN); trimethylnaphthalene (TMN); biphenyl (B); methylbiphenyl (MB); dimethylbiphenyl (DMB); acenaphthene (A); fluorene (F); methylfluorene (MF); dibenzothiophene (DBT); methyldibenzothiophene (MDBT); dimethyldibenzothiophene (DMDBT); phenanthrene (P); methylphenanthrene (MP); dimethylphenanthrene (DMP); trimethylphenanthrene (TMP); pyrene (PY); methylpyrene (MPY); and chrysene (C).


### 3.2. Composite and Experimental Fluorescence Spectra

To analyze the fluorescence contribution of AHs, the composite fluorescence spectra of the six oil samples were determined using equation (1) based on the fluorescence spectra of the individual AHs obtained from the US-EPA, which were distributed over a wavelength range of 285–525 nm with a resolution of 1 nm (Figure 3). Hence, the nλ × nAH matrix of the individual AHs fluorescence spectra represented the IfAH parameter in the composition method with nλ = 241 and nAH = 8. Moreover, due to the unknown contribution of the individual AHs to the fluorescence spectra of the oil samples, XF was calculated as a 8 × 8 identity matrix (equation (2)) to obtain the composite fluorescence spectra via the composition method. As shown in Figure 4(a), the composite fluorescence spectra of all oil samples exhibited one main peak, which ranged between 320 and 350 nm and was centered at 330 nm. However, the fluorescence intensity of the 380# fuel oil composite between 350 and 450 nm was higher compared to the other oil samples and exhibited several small peaks in this wavelength range. Moreover, the fluorescence intensity of the 0# and −10# diesel oil composites in the 300–320 nm region was higher than that of the other oil samples. These results were used as comparative data to analyze the differences of the experimental fluorescence spectra and verify the contribution of different AHs to the fluorescence of the examined oil samples.(2)XF=E=10…001…0⋮⋮⋱⋮10…18×8.Figure 3
Fluorescence spectra of the individual AHs obtained from the US-EPA.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 4
(a) Composite (XF = E) and (b) experimental fluorescence spectra (260–700 nm) of Kuwait crude, Iranian crude, 380#, 180# fuel, 0#, and −10# diesel oil samples. (c) Comparison of the composite and experimental fluorescence spectra of the six oil samples between 285 and 525 nm.The experimental fluorescence spectra of the six oil samples were recorded using UV-excited (λexc = 266 nm) and normalization and adjacent average smoothing to eliminate environmental and instrumental errors during the measurement process (Figure 4(b)) [40]. The experimental spectra were then directly compared with the respective composite fluorescence spectra of the six oil samples at a wavelength range of 285–525 nm, to better understand the effects of AHs on the fluorescence of oil seawater samples (Figure 4(c)). As shown in Figure 4(c), the experimental spectra were redshifted compared to the composite fluorescence spectra and exhibited significant fluorescence intensity in the long-wavelength region (350–500 nm). Moreover, the experimental fluorescence spectra of the crude and diesel oil were very similar to the respective composite spectra. In contrast, great deviations were observed for the 380# and 180# fuel oil samples, while the small peaks in the composite fluorescence spectra, especially those of 380# fuel oil at 350–400 nm, could not be detected in the experimental fluorescence spectra (Figure 4(c)). These results were similar to the fluorescence spectral patterns reported in previous studies on oil-contaminated seawater samples [41–43].These significant differences could be explained considering the effect of heavy petroleum hydrocarbons (high API gravity and viscosity) or the possible interaction between AHs and other aromatic ring systems in the oil samples. In particular, fuel oils, and especially 380# fuel oil, are the heaviest samples among the six oils examined in this study and contain heavy petroleum hydrocarbons (e.g., unidentified AHs, resins, and asphaltenes) with strong fluorescence emission in the long-wavelength range. In contrast, a reduction of the experimental spectra between 300 and 320 nm compared with the composite spectra is evident in diesel oil samples. Therefore, we concluded that, in order to successfully quantify the effect of AHs on the fluorescence spectra of the six oil samples, the differences between the experimental and composite fluorescence spectra and their correlation with the physical properties (API gravity and viscosity) of the oil samples should be first analyzed, as they reflect the content of heavy petroleum hydrocarbons.


### 3.3. Fluorescence Spectra Difference Analysis

To further analyze the effects of heavy petroleum hydrocarbons on the fluorescence of different oil species in seawater, the differences between the composite and experimental fluorescence spectra were quantified to establish the relationship between the physical parameters and the spectral differences. To that end, we used the redshift of the main peak position (Δf), which was determined by the wavelength of the maximum fluorescence intensity in the experimental and composite fluorescence spectra, and the fluorescence intensity distance (DF) within the composite and experimental fluorescence spectra [44], which represented the average difference between the composite and experimental spectra and could be calculated as follows:(3)DF=1n∑i=1nai−bi2,where n is the spectrograph sample number and ai and bi are the fluorescence intensities of the composite and experimental spectra, respectively.In addition, the overall shape similarity between the experimental and composite fluorescence spectra of the six oil samples was analyzed using the spectral angle mapping (SAM) method [45]. SAM is a supervised classification technique that quantifies the angle between the target and the reference spectral vector using spectra as high-dimensional vectors. Small angles indicate high similarity between the two spectra. Moreover, the SAM method was selected, as it uses all the dimensional information of the spectrum, emphasizes its shape characteristics, and reduces the feature information.In this study, each composite fluorescence spectrum was used as the high-dimensional vector of the reference spectrum, while the experimental fluorescence spectra were used as the high-dimensional vectors of the target spectra. The generalized included angle cosine (θ) of the six oil samples was calculated as follows:(4)θ=cos−1∑i=1ntiri∑i=1nti2∑i=1nri2,where n is the spectrograph sample number, while T = (t1, t2, …, ti) and R = (r1, r2, …, ri) represent the experimental and composite fluorescence spectra, respectively. The Δf, DF, and θ values determined for the experimental and composite fluorescence spectra of the six oil samples are summarized in Table 3.Table 3
Redshift of the main peak position (Δf), fluorescence intensity distance (DF), and generalized included angle cosine (θ) determined for the experimental and composite fluorescence spectra.
Oil samplesΔf (nm)DF (a.u.)θ (°)Kuwait crude oil80.0790.2186Iranian crude oil90.1640.3871180# fuel oil70.2580.5221380# fuel oil200.3040.50030# diesel oil50.0510.3416−10# diesel oil40.0950.3970Correlation analysis was also performed to examine the correlation of the physical properties (API gravity and viscosity) and the spectral difference parameters (Δf, DF, and θ). In particular, the API gravity was negatively correlated with Δf, DF, and θ (R2 = 0.44, 0.85, and 0.52, respectively) (Figure 5(a)), while the viscosity showed a positive correlation with Δf, DF, and θ with R2 values of 0.74, 0.81, and 0.51, respectively (Figure 5(b)). Based on the API gravity and viscosity data (Table 1), the 380# fuel oil was the heaviest among the six oil samples, as it contained the highest amount of heavy petroleum hydrocarbons. Given also the correlation of the API gravity and viscosity of the six oil samples and their fluorescence spectral difference, we concluded that heavy petroleum hydrocarbons can affect the fluorescence spectra, leading to differences between the experimental and composite fluorescence spectra.Figure 5
Correlation analysis of the relationship between the (a) API gravity and (b) viscosity of the six oil samples and the main peak position (Δf), fluorescence intensity distance (DF), and generalized included angle cosine (θ).
(a)(b)In addition, the fluorescence spectral difference and correlation analysis implied that the six oil samples can be distinguished and identified in the scattered plots. Consequently, fluorescence spectroscopy in combination with GC–MS is an effective strategy to study the effects of heavy petroleum hydrocarbons on the fluorescence spectra of different oil species in seawater and determine the presence of different oil species.


### 3.4. Contribution of Individual AHs

Considering the AHs relative content information (Section3.1), the characteristic diagonal matrix (XF) was constructed to estimate the contribution rate of the eight AH categories (i.e., naphthalene, biphenyl, acenaphthene, fluorene, DBT, phenanthrene, pyrene, and chrysene series) using the fluorescence composition method (equation (5)).(5)XF=IfoilIfAHXC=ωNS0⋯00ωBS⋯⋮⋮⋮⋱00⋯0ωCS8×8.The corresponding results (Table4) confirmed the complex contribution of AHs in the six oil samples. More specifically, it was clear that the AHs of the fluorene series contributed negatively to the experimental fluorescence spectra of all oil samples regardless of their relative content. Moreover, except for 0# diesel oil, the AHs of the acenaphthene series had the greatest positive contribution to the experimental spectra, while the pyrene series also showed strong positive contribution to the crude and fuel oil samples despite their low individual relative contents. It is worth noting that these AH contributions were particularly obvious in fuel and crude oils, which contain high amounts of heavy petroleum hydrocarbons. In contrast, the contribution of AHs to the fluorescence spectra of diesel oils was more balanced.Table 4
Contribution of eight AH series in Kuwait crude, Iranian crude, 180# fuel, 380# fuel, 0# diesel, and −10# diesel oil samples.
Kuwait crude oilIranian crude oil180# fuel oil380# fuel oil0# diesel oil−10# diesel oilNaphthalene series1.050.960.720.963.51.82Biphenyl series9.946.3519.134.52.391.52Acenaphthene series33.7361.05137.8647.442.8210.64Fluorene series−1.39−1.67−5.55−2.91−0.70−0.03DBT series0.981.621.431.04−0.671.96Phenanthrene series1.762.313.873.211.064.04Pyrene series18.4852.6917.6653.48——Chrysene series−11.33−12.75————Furthermore, it has been reported that UV-induced fluorescence of oils in seawater involves a multidimensional complicated process based on the electronic energy level for the transition of the specific structure of organic matter [46]. The combination of complex composition and multiplicity along with energy transfer and intermolecular interactions can also lead to a very complex pattern of energy levels [47]. In addition, the energy transfer between fluorene and other fluorescing AHs can be explained by the occurrence of an external heavy atom effect in oil samples containing high amounts of heavy petroleum hydrocarbons [16, 48], indicating that AHs affect the fluorescence spectra of oils by quenching signals and generating other peaks. These literature reports further supported our findings that oil samples in seawater can be distinguished based on the fluorescence spectral difference and the contribution of individual AHs. However, this complex effect of external heavy atom required further detailed study on the components of petroleum oils.


## 3.1. Distribution and Categories of AHs

Τhe relative content of AHs in Kuwait crude, Iranian crude, 380# fuel, 180# fuel, 0# diesel, and −10# diesel oil samples was determined by GC-MS after extraction and fractionation (Figure2), and the types of AHs included in each oil sample were identified by matching the retention times to 16 standard AHs [34, 35]. Specifically, the AHs detected in the six oil samples were naphthalene, methylnaphthalene, dimethylnaphthalene, trimethylnaphthalene, biphenyl, methylbiphenyl, 2, 2' + 4, 4'-dimethylbiphenyl, acenaphthene, fluorene, methylfluorene, dibenzothiophene, methyldibenzothiophene, 2, 4 + 4, 6-dimethyldibenzothiophene, phenanthrene, methylphenanthrene, dimethylphenanthrene, trimethylphenanthrene, pyrene, methylpyrene, and chrysene. The AH contents in the oil samples were determined by analyzing a standard mixture containing known concentrations of each AH. Moreover, the alkyl aromatic hydrocarbon compounds were identified by comparing the mass spectra and retention times obtained in this study with existing data [36, 37].Figure 2
Relative content of AHs in six oil samples determined by GC-MS analysis. Naphthalene (N); methylnaphthalene (MN); dimethylnaphthalene (DMN); trimethylnaphthalene (TMN); biphenyl (B); methylbiphenyl (MB); dimethylbiphenyl (DMB); acenaphthene (A); fluorene (F); methylfluorene (MF); dibenzothiophene (DBT); methyldibenzothiophene (MDBT); dimethyldibenzothiophene (DMDBT); phenanthrene (P); methylphenanthrene (MP); dimethylphenanthrene (DMP); trimethylphenanthrene (TMP); pyrene (PY); methylpyrene (MPY); and chrysene (C).Petroleum, a complex mixture containing multiple fluorescent substances, and the differences in photophysical properties and content of each component affect the overall fluorescence characteristics and intensity of the oil samples in seawater. It is, therefore, clear that the composition of the oil samples in AHs is complex, making it difficult to analyze the effect of the individual ingredients on the fluorescence spectra. Moreover, the effect of alkane substituents on the fluorescence spectra is generally weak, suggesting that AHs bearing alkyl substituents would exhibit similar fluorescence spectra [38, 39]. Therefore, AHs were divided into eight categories, namely, naphthalene, biphenyl, acenaphthene, fluorene, dibenzothiophene, phenanthrene, pyrene, and chrysene series (Table 2). Dominant AHs with a relative content of 60.88%, 60.90%, and 65.93% in Kuwait crude oil, Iranian crude oil, and 180# fuel oil, respectively, belonged to the naphthalene series. In contrast, the AHs in 380# fuel oil were almost equally distributed, and the highest contents were detected in the naphthalene (33.15%), dibenzothiophene (16.85%), and phenanthrene series (27.46%). Furthermore, the diesel oil samples included mainly AHs with two or three benzene rings. Hence, the relative contents of the eight AH categories in the six oil samples were used as the values of the XC parameter in the fluorescence composition method with nAH = 8.Table 2
Relative content of eight AH categories in Kuwait crude, Iranian crude, 380# fuel, 180# fuel, 0# diesel, and −10# diesel oil samples.
AHsaCategoriesKuwait crude (%)Iranian crude (%)180# fuel (%)380# fuel (%)0# diesel (%)−10# diesel (%)NNaphthalene series60.8860.9065.9333.1527.9528.7MNDMNTMNBBiphenyl series2.784.913.9414.3118.7322.46MBDMBTMBAAcenaphthene series1.180.800.331.333.562.84FFluorene series4.165.512.945.7023.8322.25MFDBTDBT series19.2110.809.1316.8517.5513.53MDBTDMDBTPPhenanthrene series10.4415.7515.3627.468.3810.22MPDMPTMPPYPyrene series0.770.752.371.2000MPYCChrysene series0.580.580000aNaphthalene (N); methylnaphthalene (MN); dimethylnaphthalene (DMN); trimethylnaphthalene (TMN); biphenyl (B); methylbiphenyl (MB); dimethylbiphenyl (DMB); acenaphthene (A); fluorene (F); methylfluorene (MF); dibenzothiophene (DBT); methyldibenzothiophene (MDBT); dimethyldibenzothiophene (DMDBT); phenanthrene (P); methylphenanthrene (MP); dimethylphenanthrene (DMP); trimethylphenanthrene (TMP); pyrene (PY); methylpyrene (MPY); and chrysene (C).


## 3.2. Composite and Experimental Fluorescence Spectra

To analyze the fluorescence contribution of AHs, the composite fluorescence spectra of the six oil samples were determined using equation (1) based on the fluorescence spectra of the individual AHs obtained from the US-EPA, which were distributed over a wavelength range of 285–525 nm with a resolution of 1 nm (Figure 3). Hence, the nλ × nAH matrix of the individual AHs fluorescence spectra represented the IfAH parameter in the composition method with nλ = 241 and nAH = 8. Moreover, due to the unknown contribution of the individual AHs to the fluorescence spectra of the oil samples, XF was calculated as a 8 × 8 identity matrix (equation (2)) to obtain the composite fluorescence spectra via the composition method. As shown in Figure 4(a), the composite fluorescence spectra of all oil samples exhibited one main peak, which ranged between 320 and 350 nm and was centered at 330 nm. However, the fluorescence intensity of the 380# fuel oil composite between 350 and 450 nm was higher compared to the other oil samples and exhibited several small peaks in this wavelength range. Moreover, the fluorescence intensity of the 0# and −10# diesel oil composites in the 300–320 nm region was higher than that of the other oil samples. These results were used as comparative data to analyze the differences of the experimental fluorescence spectra and verify the contribution of different AHs to the fluorescence of the examined oil samples.(2)XF=E=10…001…0⋮⋮⋱⋮10…18×8.Figure 3
Fluorescence spectra of the individual AHs obtained from the US-EPA.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 4
(a) Composite (XF = E) and (b) experimental fluorescence spectra (260–700 nm) of Kuwait crude, Iranian crude, 380#, 180# fuel, 0#, and −10# diesel oil samples. (c) Comparison of the composite and experimental fluorescence spectra of the six oil samples between 285 and 525 nm.The experimental fluorescence spectra of the six oil samples were recorded using UV-excited (λexc = 266 nm) and normalization and adjacent average smoothing to eliminate environmental and instrumental errors during the measurement process (Figure 4(b)) [40]. The experimental spectra were then directly compared with the respective composite fluorescence spectra of the six oil samples at a wavelength range of 285–525 nm, to better understand the effects of AHs on the fluorescence of oil seawater samples (Figure 4(c)). As shown in Figure 4(c), the experimental spectra were redshifted compared to the composite fluorescence spectra and exhibited significant fluorescence intensity in the long-wavelength region (350–500 nm). Moreover, the experimental fluorescence spectra of the crude and diesel oil were very similar to the respective composite spectra. In contrast, great deviations were observed for the 380# and 180# fuel oil samples, while the small peaks in the composite fluorescence spectra, especially those of 380# fuel oil at 350–400 nm, could not be detected in the experimental fluorescence spectra (Figure 4(c)). These results were similar to the fluorescence spectral patterns reported in previous studies on oil-contaminated seawater samples [41–43].These significant differences could be explained considering the effect of heavy petroleum hydrocarbons (high API gravity and viscosity) or the possible interaction between AHs and other aromatic ring systems in the oil samples. In particular, fuel oils, and especially 380# fuel oil, are the heaviest samples among the six oils examined in this study and contain heavy petroleum hydrocarbons (e.g., unidentified AHs, resins, and asphaltenes) with strong fluorescence emission in the long-wavelength range. In contrast, a reduction of the experimental spectra between 300 and 320 nm compared with the composite spectra is evident in diesel oil samples. Therefore, we concluded that, in order to successfully quantify the effect of AHs on the fluorescence spectra of the six oil samples, the differences between the experimental and composite fluorescence spectra and their correlation with the physical properties (API gravity and viscosity) of the oil samples should be first analyzed, as they reflect the content of heavy petroleum hydrocarbons.


## 3.3. Fluorescence Spectra Difference Analysis

To further analyze the effects of heavy petroleum hydrocarbons on the fluorescence of different oil species in seawater, the differences between the composite and experimental fluorescence spectra were quantified to establish the relationship between the physical parameters and the spectral differences. To that end, we used the redshift of the main peak position (Δf), which was determined by the wavelength of the maximum fluorescence intensity in the experimental and composite fluorescence spectra, and the fluorescence intensity distance (DF) within the composite and experimental fluorescence spectra [44], which represented the average difference between the composite and experimental spectra and could be calculated as follows:(3)DF=1n∑i=1nai−bi2,where n is the spectrograph sample number and ai and bi are the fluorescence intensities of the composite and experimental spectra, respectively.In addition, the overall shape similarity between the experimental and composite fluorescence spectra of the six oil samples was analyzed using the spectral angle mapping (SAM) method [45]. SAM is a supervised classification technique that quantifies the angle between the target and the reference spectral vector using spectra as high-dimensional vectors. Small angles indicate high similarity between the two spectra. Moreover, the SAM method was selected, as it uses all the dimensional information of the spectrum, emphasizes its shape characteristics, and reduces the feature information.In this study, each composite fluorescence spectrum was used as the high-dimensional vector of the reference spectrum, while the experimental fluorescence spectra were used as the high-dimensional vectors of the target spectra. The generalized included angle cosine (θ) of the six oil samples was calculated as follows:(4)θ=cos−1∑i=1ntiri∑i=1nti2∑i=1nri2,where n is the spectrograph sample number, while T = (t1, t2, …, ti) and R = (r1, r2, …, ri) represent the experimental and composite fluorescence spectra, respectively. The Δf, DF, and θ values determined for the experimental and composite fluorescence spectra of the six oil samples are summarized in Table 3.Table 3
Redshift of the main peak position (Δf), fluorescence intensity distance (DF), and generalized included angle cosine (θ) determined for the experimental and composite fluorescence spectra.
Oil samplesΔf (nm)DF (a.u.)θ (°)Kuwait crude oil80.0790.2186Iranian crude oil90.1640.3871180# fuel oil70.2580.5221380# fuel oil200.3040.50030# diesel oil50.0510.3416−10# diesel oil40.0950.3970Correlation analysis was also performed to examine the correlation of the physical properties (API gravity and viscosity) and the spectral difference parameters (Δf, DF, and θ). In particular, the API gravity was negatively correlated with Δf, DF, and θ (R2 = 0.44, 0.85, and 0.52, respectively) (Figure 5(a)), while the viscosity showed a positive correlation with Δf, DF, and θ with R2 values of 0.74, 0.81, and 0.51, respectively (Figure 5(b)). Based on the API gravity and viscosity data (Table 1), the 380# fuel oil was the heaviest among the six oil samples, as it contained the highest amount of heavy petroleum hydrocarbons. Given also the correlation of the API gravity and viscosity of the six oil samples and their fluorescence spectral difference, we concluded that heavy petroleum hydrocarbons can affect the fluorescence spectra, leading to differences between the experimental and composite fluorescence spectra.Figure 5
Correlation analysis of the relationship between the (a) API gravity and (b) viscosity of the six oil samples and the main peak position (Δf), fluorescence intensity distance (DF), and generalized included angle cosine (θ).
(a)(b)In addition, the fluorescence spectral difference and correlation analysis implied that the six oil samples can be distinguished and identified in the scattered plots. Consequently, fluorescence spectroscopy in combination with GC–MS is an effective strategy to study the effects of heavy petroleum hydrocarbons on the fluorescence spectra of different oil species in seawater and determine the presence of different oil species.


## 3.4. Contribution of Individual AHs

Considering the AHs relative content information (Section3.1), the characteristic diagonal matrix (XF) was constructed to estimate the contribution rate of the eight AH categories (i.e., naphthalene, biphenyl, acenaphthene, fluorene, DBT, phenanthrene, pyrene, and chrysene series) using the fluorescence composition method (equation (5)).(5)XF=IfoilIfAHXC=ωNS0⋯00ωBS⋯⋮⋮⋮⋱00⋯0ωCS8×8.The corresponding results (Table4) confirmed the complex contribution of AHs in the six oil samples. More specifically, it was clear that the AHs of the fluorene series contributed negatively to the experimental fluorescence spectra of all oil samples regardless of their relative content. Moreover, except for 0# diesel oil, the AHs of the acenaphthene series had the greatest positive contribution to the experimental spectra, while the pyrene series also showed strong positive contribution to the crude and fuel oil samples despite their low individual relative contents. It is worth noting that these AH contributions were particularly obvious in fuel and crude oils, which contain high amounts of heavy petroleum hydrocarbons. In contrast, the contribution of AHs to the fluorescence spectra of diesel oils was more balanced.Table 4
Contribution of eight AH series in Kuwait crude, Iranian crude, 180# fuel, 380# fuel, 0# diesel, and −10# diesel oil samples.
Kuwait crude oilIranian crude oil180# fuel oil380# fuel oil0# diesel oil−10# diesel oilNaphthalene series1.050.960.720.963.51.82Biphenyl series9.946.3519.134.52.391.52Acenaphthene series33.7361.05137.8647.442.8210.64Fluorene series−1.39−1.67−5.55−2.91−0.70−0.03DBT series0.981.621.431.04−0.671.96Phenanthrene series1.762.313.873.211.064.04Pyrene series18.4852.6917.6653.48——Chrysene series−11.33−12.75————Furthermore, it has been reported that UV-induced fluorescence of oils in seawater involves a multidimensional complicated process based on the electronic energy level for the transition of the specific structure of organic matter [46]. The combination of complex composition and multiplicity along with energy transfer and intermolecular interactions can also lead to a very complex pattern of energy levels [47]. In addition, the energy transfer between fluorene and other fluorescing AHs can be explained by the occurrence of an external heavy atom effect in oil samples containing high amounts of heavy petroleum hydrocarbons [16, 48], indicating that AHs affect the fluorescence spectra of oils by quenching signals and generating other peaks. These literature reports further supported our findings that oil samples in seawater can be distinguished based on the fluorescence spectral difference and the contribution of individual AHs. However, this complex effect of external heavy atom required further detailed study on the components of petroleum oils.


## 4. Conclusion

In this study, a composite method and correlation analyses of fluorescence spectra characteristics and the API gravity and viscosity of the oil samples were carried out. Moreover, we combined the GC-MS and fluorescence analyses to identify and quantify the contribution and effects of eight categories of AHs in oil samples. A detailed database of the AH content of different oil species is needed to apply the composition method we developed. Our study revealed that the effect of heavy petroleum hydrocarbons as well as the relative content of AHs and their potential interactions should be taken into account to sufficiently interpret the fluorescence spectra of different oil species in seawater. Although the composition method we developed by both fluorescence spectroscopy and GC–MS analysis cannot replace the standard chromatographic techniques, it can be used as a low-cost and efficient screening method for the detection and identification of oil species in seawater. According to the fluorescence spectra characteristics of different oil species, in future, it would help the selection of the characteristic band of the oil spillvia the fluorescence detection sensor.



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