Abstract
Oil spill risk assessment mainly focus on the removal of hydrocarbons from the oil phase, especially polycyclic aromatic hydrocarbons (PAHs). However, as no compound completely disappear, there is an incentive to look further into the fate of the “removed” hydrocarbons.
The aim of this thesis is to understand degradation of a crude oil in a warm, pre-exposed marine environment, and especially the role of formed degradation products present in the water phase.
The environment of the Persian Gulf is interesting in regards to oil degradation and formation of degradation products due to several factors. Petroleum pollution is high in the area, the microbial population is highly adapted to oil compounds, and high degradation rates are leading to significant formation of degradation products.
For the main experiment, oil spiked microcosms with water from the Musa estuary in Iran, were incubated in dark at 30 °C for up to 27 days. Crude oil degradation in the microcosms was described by oil hydrocarbon fingerprinting of the oil phase through gas chromatography - mass spectrometry selected ion monitoring (GC-MS SIM). The water phase was first fractionated into acids and neutrals by solid-phase extraction (SPE). The acid fraction was derivatized with borontrifluoride in ethanol (BF3·EtOH) following a method optimized in manuscript III. Chemical analysis of both acid- and neutral fractions was done by comprehensive two-dimensional gas chromatography - high resolution mass spectrometry (GC×GC-HR MS) analysis. The applied combination of sample preparation procedures and analytical methods was suggested based on experience from the work performed in paper I-II and manuscript III.
The acidic degradation products were further tested for activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) to assess increase in oxidative stress and AhR-mediated responses, respectively.
Highly weathered beached oil samples was found in paper III, indicating that the warm environment of the Persian Gulf affect oil degradation. Furthermore, the main experiment showed that degradation of nC10-nC21 alkanes started at day 2 of the degradation period, and all nC10-nC35 alkanes were completely degraded at day 15. Parent and alkylated PAHs with two to three aromatic rings were also degraded within the degradation period, but no degradation of four ring PAHs was observed. C0- to C3-naphthalene (N) were not detected at day 27, whereas C4-N were degraded to 48 % of the value at day 1. The parent compounds of fluorene (F) and phenanthrene (P) were not detected after 27 days, whereas dibenzothiophene (DBT) were degraded to only 12 % of the initial value. The alkylated homologues were more recalcitrant, and C1-F, C2-F, and C3-F were degraded to 48 %, 73 %, and 94 % compared to day 1, respectively. P and DBT were degraded up to C2-P and C2-DBT. After ending of the degradation period, 6 % and 77 % of C1- and C2-phenathrene, and 35 % and 78 % of C1- and C2-DBT were present compared to day 1.
A super-complex mixture of acidic degradation products were seen in the water phase of the microcosms including tentatively identified compound groups of aliphatic- (AAs), monocyclic aromatic- (MAAs), and polycyclic aromatic acids (PAAs). The benzoic acids (BAs) (only MAAs) were present at highest levels at day 15 for C1-BA, day 4 for C2- to C4-BA, and at day 8 for C5- BA, before they were removed again from the water phase. The PAAs took longer to reach the highest level in the water phase with naphthoic acid and benzothiophene carboxylic acid reaching the highest level at day 8, and the C1- homologues reaching highest levels at day 15. Presence of the larger, two to three ring PAAs and their alkylated homologues increased over the entire degradation period.
Activation of the AhR, potentially causing formation of DNA adduct via upregulation of CYP1, was increased from 3.97 ± 0.57 at day 1 to 23.02 ± 3.7 at day 27 for the acid fraction of the sample microcosms. Acid fractions from the autoclaved control microcosms also showed increased AhR-activity during the degradation period, but at lower levels than for the samples (4.18 ± 1.4 at day 1 to 8.19 ±1.7 at day 27). No activity of Nrf2 were observed in any of the samples.
A correlation between the increased levels of PAAs in the water phase, and the increased AhR activity was evident from modelling by orthogonal projection to latent structures (o-PLS).
This study showed formation of water-soluble acidic degradation products during biodegradation of crude oil, more specifically, formation of aliphatic- and aromatic acids. The MAAs and smaller PAAs (parent- and C1-naphthoic acids and -benzothiophene carboxylic acids) might be intermediate degradation products, as these were seen to decrease again in the water phase after reaching the highest values. Larger PAAs could be dead-end products of the degradation pathway, and hence be persistent in the marine environment. Furthermore, the mobility of polar degradation products give rise to concerns regarding increased external exposure and spreading of this compound group. European legislation has previously focused mainly on persistent, bioaccumulative, and toxic (PBT) substances, which are generally very lipophilic. In the last couple of years, however, the concern for more polar, mobile substances has led to the suggested classification of equally environmental important persistent, mobile, and toxic (PMT) substances, or very persistent, very mobile substances (vPvM). Further studies on the properties of MAAs and PAAs (half-life, solubility, and toxic responses) are necessary to evaluate whether these compounds classify as PMT or vPvM substances. The formation of these degradation products can potentially have a large environmental impacts and should not be neglected in oil spill risk assessments.
The aim of this thesis is to understand degradation of a crude oil in a warm, pre-exposed marine environment, and especially the role of formed degradation products present in the water phase.
The environment of the Persian Gulf is interesting in regards to oil degradation and formation of degradation products due to several factors. Petroleum pollution is high in the area, the microbial population is highly adapted to oil compounds, and high degradation rates are leading to significant formation of degradation products.
For the main experiment, oil spiked microcosms with water from the Musa estuary in Iran, were incubated in dark at 30 °C for up to 27 days. Crude oil degradation in the microcosms was described by oil hydrocarbon fingerprinting of the oil phase through gas chromatography - mass spectrometry selected ion monitoring (GC-MS SIM). The water phase was first fractionated into acids and neutrals by solid-phase extraction (SPE). The acid fraction was derivatized with borontrifluoride in ethanol (BF3·EtOH) following a method optimized in manuscript III. Chemical analysis of both acid- and neutral fractions was done by comprehensive two-dimensional gas chromatography - high resolution mass spectrometry (GC×GC-HR MS) analysis. The applied combination of sample preparation procedures and analytical methods was suggested based on experience from the work performed in paper I-II and manuscript III.
The acidic degradation products were further tested for activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) to assess increase in oxidative stress and AhR-mediated responses, respectively.
Highly weathered beached oil samples was found in paper III, indicating that the warm environment of the Persian Gulf affect oil degradation. Furthermore, the main experiment showed that degradation of nC10-nC21 alkanes started at day 2 of the degradation period, and all nC10-nC35 alkanes were completely degraded at day 15. Parent and alkylated PAHs with two to three aromatic rings were also degraded within the degradation period, but no degradation of four ring PAHs was observed. C0- to C3-naphthalene (N) were not detected at day 27, whereas C4-N were degraded to 48 % of the value at day 1. The parent compounds of fluorene (F) and phenanthrene (P) were not detected after 27 days, whereas dibenzothiophene (DBT) were degraded to only 12 % of the initial value. The alkylated homologues were more recalcitrant, and C1-F, C2-F, and C3-F were degraded to 48 %, 73 %, and 94 % compared to day 1, respectively. P and DBT were degraded up to C2-P and C2-DBT. After ending of the degradation period, 6 % and 77 % of C1- and C2-phenathrene, and 35 % and 78 % of C1- and C2-DBT were present compared to day 1.
A super-complex mixture of acidic degradation products were seen in the water phase of the microcosms including tentatively identified compound groups of aliphatic- (AAs), monocyclic aromatic- (MAAs), and polycyclic aromatic acids (PAAs). The benzoic acids (BAs) (only MAAs) were present at highest levels at day 15 for C1-BA, day 4 for C2- to C4-BA, and at day 8 for C5- BA, before they were removed again from the water phase. The PAAs took longer to reach the highest level in the water phase with naphthoic acid and benzothiophene carboxylic acid reaching the highest level at day 8, and the C1- homologues reaching highest levels at day 15. Presence of the larger, two to three ring PAAs and their alkylated homologues increased over the entire degradation period.
Activation of the AhR, potentially causing formation of DNA adduct via upregulation of CYP1, was increased from 3.97 ± 0.57 at day 1 to 23.02 ± 3.7 at day 27 for the acid fraction of the sample microcosms. Acid fractions from the autoclaved control microcosms also showed increased AhR-activity during the degradation period, but at lower levels than for the samples (4.18 ± 1.4 at day 1 to 8.19 ±1.7 at day 27). No activity of Nrf2 were observed in any of the samples.
A correlation between the increased levels of PAAs in the water phase, and the increased AhR activity was evident from modelling by orthogonal projection to latent structures (o-PLS).
This study showed formation of water-soluble acidic degradation products during biodegradation of crude oil, more specifically, formation of aliphatic- and aromatic acids. The MAAs and smaller PAAs (parent- and C1-naphthoic acids and -benzothiophene carboxylic acids) might be intermediate degradation products, as these were seen to decrease again in the water phase after reaching the highest values. Larger PAAs could be dead-end products of the degradation pathway, and hence be persistent in the marine environment. Furthermore, the mobility of polar degradation products give rise to concerns regarding increased external exposure and spreading of this compound group. European legislation has previously focused mainly on persistent, bioaccumulative, and toxic (PBT) substances, which are generally very lipophilic. In the last couple of years, however, the concern for more polar, mobile substances has led to the suggested classification of equally environmental important persistent, mobile, and toxic (PMT) substances, or very persistent, very mobile substances (vPvM). Further studies on the properties of MAAs and PAAs (half-life, solubility, and toxic responses) are necessary to evaluate whether these compounds classify as PMT or vPvM substances. The formation of these degradation products can potentially have a large environmental impacts and should not be neglected in oil spill risk assessments.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2019 |
