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. Author manuscript; available in PMC: 2023 Jan 4.
Published in final edited form as: Environ Sci Technol. 2021 Mar 2;55(6):3727–3735. doi: 10.1021/acs.est.0c07488

Time-related Alteration of Aqueous Phase Anthracene and Phenanthrene Photoproducts in the Presence of TiO2 Nanoparticles

Lindsey St Mary 1, Lisandra SD Trine 2, Courtney Roper 2,3, Jackson Wiley 2, Staci L Massey Simonich 2, Martin McCoustra 4, Theodore B Henry 1
PMCID: PMC9811996  NIHMSID: NIHMS1779275  PMID: 33651588

Abstract

Polycyclic aromatic hydrocarbons (PAHs) and TiO2 nanoparticles (NPs) are photoactive environmental pollutants that can contaminate aquatic environments. Aqueous-phase interactions between PAHs and TiO2-NPs are of interest due to their emerging environmental relevance, particularly with the deliberate application of TiO2-NPs to remediate pollution events (e.g., oil spills). Our objective was to investigate anthracene (ANT) and phenanthrene (PHE) photoproduct formation and transformation following UVA irradiation in the presence and absence of TiO2-NPs. ANT and PHE solutions were prepared alone or in combination with TiO2-NPs, UVA irradiated, and either exposed to early life stage zebrafish or collected for chemical analyses of diverse OHPAHs and OPAHs. Expression profiles of genes encoding for enzymes involved in PAH metabolism showed PAH and time-dependent inductions that demonstrated changes in PAH and photoproduct bioavailability in the presence of TiO2-NPs. Chemical analyses of chemical/NP solutions in the absence of zebrafish larvae identified diverse photoproducts of differing size and ring arrangements, which suggested photodissociation, recombination, and ring re-arrangements of PAHs occurred either during or following UVA irradiation. ANT and PHE solutions both showed heightened oxidative potential following irradiation, but TiO2-NP-related increases in oxidative potential were PAH-specific. The exploitation of multiple analytical methods provided novel insights into distinct PAH photoactivity; TiO2-NP influence on photoproduct formation in a PAH-specific manner; and the significant role time plays in all of these processes.

Graphical Abstract

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Introduction

Interactions between polycyclic aromatic hydrocarbons (PAHs) and TiO2 nanoparticles (NPs) can influence the environmental fate, transport, and transformation of PAHs and their photoproducts in aquatic environments. TiO2-NPs are typically released into the environment when washed from the skin or body following the use of personal care products such as sunscreen, makeup, and some toothpastes (1). The broad, expanding use of TiO2-NPs in personal care products, release into the aquatic environment during manufacturing, or the deliberate application to remediate pollution events (e.g., oil spills) all contribute to increased concentrations of TiO2-NPs in the environment (2, 3). TiO2-NP legislation restricts the amount allowed in product and remediation processes, but the focus is primarily on the TiO2-NP itself and interactions with other pollutants are rarely considered (4).

Anthracene (ANT) and phenanthrene (PHE) are three ring PAHs often produced through pyrolysis which enter surface waters through urban water run-off and atmospheric deposition. Upon absorption of ultraviolet (UV) light these PAHs can transform into parent-derived photoproducts (e.g., ANT to 9,10-anthraquinone, PHE to 9,10-phenanthraquinone) in both the absence and presence of TiO2-NPs (5, 6, 7). However, the diversity of photoproducts, time of formation, and subsequent changes in biotransformation and bioavailability in the aquatic environment has never been explored collectively. Further investigations of PAH and TiO2-NP interactions from a multi-disciplinary approach will improve our understanding of these complex photochemical processes and contribute to the reduction of adverse environmental and human health effects.

The overall aim of this study was to enhance understanding of ANT and PHE bioavailability and photoproduct formation upon interaction with TiO2-NPs following UVA irradiation in the aqueous phase. Specific objectives were: 1) to measure time-related expression profiles of specific genes (cyp1a, ephx1, ephx2, and sod1) in larval zebrafish exposed to ongoing ANT- and PHE- TiO2-NP sorption/photochemical reactions; 2) to quantify formation of diverse photoproducts that form in OECD medium during these reactions by GC-MS analyses; 3) examine changes in oxidative potential to support altered photoproduct formation in the absence or presence of TiO2-NPs; and 4) to integrate the results of these different approaches to enhance understanding of these complex biochemical and photochemical processes.

The use of the zebrafish model as a “bioindicator” of ANT and PHE photoproducts enabled unique investigations of the interplay between biotransformation and photodegradation over time. Zebrafish (Danio rerio) have proven incredibly useful in exploring molecular mechanisms of PAH metabolism due to their proven genetic specificity and sensitivity to PAH and NP exposures which have elucidated subsequent changes in the bioavailability of PAHs and their photoproducts, particularly in the presence of TiO2-NPs (8, 9, 10, 11).

In combination with gene expression analyses, diverse oxygenated PAHs (OPAHs) and hydroxylated PAHs (OHPAHs) were identified and quantified following UVA irradiation of ANT and PHE in both the absence and presence of TiO2-NPs. Additionally, the capacity for ANT and PHE to generate reactive oxygen species (ROS), or transform into reactive intermediates, was assessed through an oxidative potential assay. Time-related transitions from photodegradation to biotransformation, altered bioavailability in the presence of TiO2-NPs, and PAH-specific photoproduct formation, UVA absorption, and TiO2-NP interaction was identified as a result of combining gene expression analyses and analytical chemistry techniques.

Materials and Methods

Chemicals, Nanoparticles, and Materials

Stock of anthracene (ANT) (> 99%, SKU-141062) and phenanthrene (PHE) (98%, SKU-P11409) were purchased from Sigma Aldrich. The TiO2-NPs JRCNM01005a, formerly known as NM105, Aeroxide P25 (Evonik Degussa) from the EU nanoparticle repository were used in all experiments. TiO2-NP physical properties and overview of all materials and reagents are described in Supporting Information.

Solution Preparation and Collections

Primary stocks of ANT and PHE were made in dimethyl sulfoxide (DMSO) at 400 mg/L concentrations and TiO2-NP stock was dissolved in reverse osmosis (RO) water at 500 mg/L, which were then used to make 20 μg/L ANT, 40 μg/L PHE, 2 mg/L TiO2-NP, 20 μg/L ANT + 2 mg/L TiO2-NP, and 40 μg/L PHE + 2 mg/L TiO2-NP in OECD for exposure/chemistry preparations on the day of the experiment. Preparations for chemical analyses did not contain zebrafish larvae so as to specifically assess photoproducts resulting from photochemical processes only. Irradiated preparations were irradiated for 40 minutes at an intensity of 7.26 mW/cm2. PAH and TiO2-NP concentrations were chosen according to concentrations found in various water sources in the aquatic environment (Table S1), and UVA intensity was selected in consonance with a study done by Balasaraswathy, P., et al., 2002 (12) which found UVA intensities ranging from 4.70 to 6.59 mW/cm2 between 1200 to 1315. The overall layout for exposure and chemistry collections performed throughout the experiments is provided in Supporting Information (Figure 1), as well as solution preparations and concentrations for all PAHs and TiO2-NPs.

Figure 1.

Figure 1.

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Average (± SE, n=3) log fold change of cyp1a, ephx1, ephx2, and sod1 in zebrafish larvae following exposure to UVA irradiated (7.26 mW/cm2) and non-irradiated 20 μg/L ANT and 20 μg/L ANT+ 2 mg/L TiO2-NP at 1, 2, 4, and 6 hours post exposure (hpe). Average log fold changes were calculated using the ΔΔCT method and normalized to β-actin at each time point. Brackets indicate a significant difference between those particular time points, and star(s) above a particular time point indicates that specific time point is significantly different compared to all the time points in that exposure. Statistical differences were identified by one-way ANOVA, TukeyHSD, *= p<0.05, ** <0.01, and ***= p <0.001.

Gene Expression Analysis

Zebrafish Husbandry and Exposures

Wildtype WIK strain zebrafish (Danio rerio) were obtained from the Heriot-Watt University research facility, and the same batch was used throughout all exposure studies to avoid familial differences. The WIK adult zebrafish lines were gifted by Professor Charles Tyler at University of Exeter and housed at the Heriot-Watt University zebrafish facility in 19 L tanks filled to 14 L with OECD, on 80 L recirculating systems in a room maintained at 28°C with a 14 h light/10 h dark schedule.

All exposures were executed at 09:30, in 20 mL glass vials, to ensure all larvae were at the same developmental stage to reduce developmental-specific differences in gene expression profiles of the rapidly developing zebrafish larvae. Each exposure group (n=15) was collected at 1, 2, 4, and 6 hours post exposure (hpe), in triplicate (n=3); totaling 45 animals per exposure group. Vehicle controls, 0.1% DMSO, were collected at each time point, for every exposure day, to account for the rapidly developing larvae and the subsequent genetic changes that accompany this model and reflected the final DMSO concentration in exposures and preparations meant for chemical analyses. All larval collections were placed on ice for 20 min and immediately stored at −80°C.

RNA Isolation, cDNA Synthesis, and qPCR

Total RNA was isolated from the pooled larval samples (n=15) using the RNeasy MiniKit employing a previously described method and DNase treated to eliminate any genomic DNA contamination of the sample (13). Total RNA was then eluted into 30 μL of sterile RNase/DNase free water, and confirmation of RNA quantity and presence of impurities assessed. All samples with 280/260 ratio between 2.0–2.2 were diluted to a final concentration of 100 ng/μL and used for further analyses. cDNA was synthesized from 2 μg total RNA.

Quantitative PCR (qPCR) was performed with cDNA diluted 1 in 10 with nuclease-free water in 20 μL reactions consisting of 300 nM gene-specific primers and SYBR Green Mastermix. The conditions were as follows: 95˚C for 2 min; 40 cycles of 95˚C for 15 s, primer-specific annealing temp for 1 min; 95˚C for 15 s; 60°C for 1 min; 95°C for 15 s. All samples were analyzed in triplicate with appropriate no-template controls included in each run and with dissociation analysis to verify primer pair specificity. Primer parameters for the reference gene (β-actin) and target genes (cyp1a, ephx1, ephx2, sod1) can be found in Supporting Information (Table S2) along with the statistical analyses used in this study.

Identification and Quantification of PAH Photoproducts

Chemical solutions were prepared in OECD at the same ANT, PHE, and TiO2-NP concentrations as those used in early life stage zebrafish exposures, but did not contain zebrafish.

Sample Extraction

Solid phase extraction (SPE) of the chemical/TiO2-NP samples (in the absence of zebrafish larvae) was done using Isolute ENV+ cartridges under vacuum as previously described (14). Detailed extraction methods are available in Supporting Information.

Sample Preparation

Extracted chemical/TiO2-NP samples were prepared using a previously published method (15). Detailed preparation methods and information on the internal standards/surrogates used are found in Supporting Information (Table S3).

Chemical Analysis

Gas chromatography-mass spectrometry (GC-MS) analyses for OPAHs and OHPAHs were performed using an Agilent 7890B GC system coupled to an Agilent 5977A mass spectrometer (MS) detector with electron impact (EI) ionization. OPAH and OHPAHs were analyzed with optimized methods previously published (14, 15). All analyses were run in selected ion monitoring (SIM) mode. GC-MS analyses for all analytes were performed in triplicate using an Agilent DB-5MS (30 m × 0.25 mm I.D. × 0.25 μm film thickness) capillary column.

Oxidative Potential Analysis

Chemical and NP solutions were prepared similarly to those used for early life stage zebrafish exposures and PAH derivative screening, but did not contain zebrafish. Sample aliquots were taken from PAH and TiO2-NP, or combination, samples (n=3) and used to measure oxidative potential with the dithiothreitol (DTT) assay. The assay was performed using a previously described method (16) for measurement in 96-well plates, but with sample volume modifications containing 4 μL of experimental sample. The rate of DTT consumption was determined via quantification of DTNB by absorbance readings (412 nm) with comparison to a linear regression of known DTT standards. More information on the DTT assay is described in the Supporting Information.

Results and Discussion

Gene Expression Profiling

Irradiated ANT+TiO2-NP exposures showed significantly different cyp1a and ephx2 expression at 4 hpe, while non-irradiated ANT+TiO2-NP exposures showed reduced cyp1a expression, compared to irradiated exposures (Figure 1). Several studies have confirmed that the presence of TiO2-NPs enhances ANT photodegradation, which results in the production of bioavailable OPAH and OHPAH photoproducts (8, 17). However, this is the first instance where the time profile of biotransformation initiation was identified, and specific genes were used as “bioindicators” to predict the presence of PAHs and their photoproducts. Results suggest that either A) photodegradative processes resulting from the interactions between ANT and TiO2-NPs continued to dominate the initial 2 hours following irradiation, but adsorption of ANT to the TiO2-NP surface diminished as the time progressed (by 4 hpe) or B) initially, photoproducts formed and quickly degraded, but the continued formation caused photoproduct accumulation so biotransformation ensued (9, 17). The reduced expression of cyp1a (responsible for PAH metabolism) in non-irradiated ANT+TiO2-NP exposures, over all time points, indicates strong adsorption of ANT to the TiO2-NP surface, preventing interaction with the larvae and reducing biotransformation, which has been found in previous studies (8, 18). The predicted adsorption of ANT to the NP surface in non-irradiated exposures indicates adsorption likely did not diminish in the irradiated exposures; scenario B) from above is the most likely reason for the increase of cyp1a in irradiated exposures. These data further support photocatalytic capabilities of TiO2-NPs considering ephx2 (responsible for encoding enzymes that metabolize OPAHs) was further induced in irradiated ANT+TiO2-NP exposures, compared to exposures of ANT alone. These data show the presence of TiO2-NPs reduces the bioavailability of the ANT molecule, but upon irradiation, ANT is efficiently photodegraded and the presence of TiO2-NPs catalyzes the formation of bioavailable ANT photoproducts.

Non-irradiated PHE and PHE+TiO2-NP exposures showed significant time-dependent fluctuations in the expression of all genes assessed (Figure 2). Previous studies have shown efficient biotransformation of PAHs, particularly PHE, and their photoproducts in zebrafish (19, 20, 21). Significant induction (1.3–5.7-fold) of cyp1a, ephx1, ephx2, and sod1 at 1 hpe, with an immediate reduction in expression by 2 hpe, in non-irradiated PHE and PHE+TiO2-NP exposures further supports that efficient biotransformation of PHE occurs in the zebrafish. Studies have also shown PAHs of increasing solubility adsorb less strongly to the surface of particles, and PHE has been found to be more soluble than ANT; PHE Kow = 1.6 mg/L, ANT Kow = 0.04 mg/L (22). The induction of all genes, involved in both PAH and OPAH/OHPAH metabolism, in non-irradiated PHE+TiO2-NP exposures suggests the PHE molecule is readily bioavailable and thus, there is less adsorption of PHE to the NP surface (unlike ANT). PHE adsorption can be increased through coating of metal NPs, but desorption may occur just as quickly as the adsorption of the molecule (23, 24). The TiO2-NPs used in this study were uncoated so as to allow the NP to retain its photoactivity and environmental relevance. NP coatings have been shown to degrade quickly in the aquatic environment and it has been suggested that TiO2-NPs used for environmental remediation be preferably uncoated (2, 25, 26). Additionally, the observed increase in induction of genes in non-irradiated PHE+TiO2-NP exposures, compared to exposures of PHE alone, are indicative of non-direct catalysis in the presence of TiO2-NPs. This likely occurs through the production of •OH and •O2 radicals by TiO2-NPs. These data, in combination with previous studies, show that PHE is bioavailable and subsequently biotransformed by the larval zebrafish but the TiO2-NP elicits its catalytic effects on PHE through the production of radicals rather than direct interaction with the PHE molecule.

Figure 2.

Figure 2.

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Average (± SE, n=3) log fold change of cyp1a, ephx1, ephx2, and sod1 in zebrafish larvae following exposure to UVA irradiated (7.26 mW/cm2) and non-irradiated 40 μg/L PHE and 40 μg/L PHE+TiO2-NP at 1, 2, 4, and 6 hours post exposure (hpe). Average log fold changes were calculated using the ΔΔCT method and normalized to β-actin at each time point. Brackets indicate a significant difference between those particular time points, and star(s) above a particular time point indicates that specific time point is significantly different compared to all the time points in that exposure. Statistical differences were identified by one-way ANOVA, TukeyHSD, *= p<0.05, ** <0.01, and ***= p <0.001.

Expression of all genes, no larger than 2-fold, in irradiated PHE exposures was observed over all time points, while PHE+ TiO2-NP exposures showed delayed induction of ephx2. As stated above, studies have shown efficient biotransformation of PAHs and their photoproducts in zebrafish (8, 19, 20, 21). Reduced expression of all genes in irradiated PHE exposures, both in the absence and presence of TiO2-NPs, indicates consistent photodegradation was likely favoured over biotransformation. The delayed induction of ephx2 in PHE+ TiO2-NP exposures may be indicative of the accumulation of PHE photoproducts that resulted from photodegradation (similar to that of ANT exposures), so biotransformation ensued. In combination with previous observations, these data suggest that upon UVA irradiation, photodegradation of PHE is favoured over biotransformation and is further enhanced in the presence of TiO2-NPs.

ANT and PHE exposures showed distinct time and irradiation-dependent expression patterns of cyp1a, ephx1, ephx2, and sod1 (Figure S2). Overall, irradiation elicited the most significant effects on gene expression in ANT and ANT+ TiO2-NP exposures (Figure S2, A). However, non-irradiated PHE and PHE+ TiO2-NP exposures were all influenced by time, where all genes were significantly different through-out the time course investigated (Figure S2, B). The presence of TiO2-NPs did significantly decrease cyp1a expression in non-irradiated ANT exposures, but did not elicit any other significant gene expression changes in either irradiated ANT or PHE exposures (Figure S2, C).

PAH Photoproduct Formation

More OHPAH photoproducts smaller in size than the parent ANT molecule were identified in ANT and ANT+TiO2-NP samples, but 1-hydroxynaphthalene in particular, was identified at higher concentrations in the presence of TiO2-NPs (Figure 3). A study by Theurich, J., et al., 1997 (27) identified the smaller photoproduct, phthalic acid in ANT samples during 18 hours of UVA irradiation in the presence of TiO2-NPs. However, identification of other smaller OHPAH photoproducts at distinct, short-term time points following UVA irradiation of ANT (with or without TiO2-NPs) has not been observed prior to this study. The observed higher concentration of 1-hydroxynaphthalene is likely due to direct reactions of •OH radicals (produced from the holes left in the valence band at the surface of the TiO2-NP upon UVA absorption) with photodissociated ANT by-products and has been shown in previous studies (7). Many previous studies have shown that PAH hydroxylation is the initial photodegradation event, prior to oxygenation (7, 18, 28). In combination with previous studies, our study suggests photodissociation of ANT, to form smaller by-products, upon absorption of UVA irradiation. The photodissociated by-products of ANT are then hydroxylated, and TiO2-NPs act as •OH radical donators, increasing OHPAH formation.

Figure 3.

Figure 3.

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Concentrations (μg/L) of anthracene (ANT) photoproducts identified following UVA irradiation (7.26 mW/cm2) of 20 μg/L ANT and 20 μg/L ANT+ 2 mg/L TiO2-NP samples at 1, 2, 4, and 6 hours post irradiation (hpi). The hydroxylated PAH (OHPAH) photoproducts increase in molecular weight from left to right along the x-axis. Bars show average ± SE, (n=3) of triplicate runs of the same sample on the instrument.

The only OHPAH identified in PHE and PHE+ TiO2-NP samples identified was 1-hydroxynaphthalene, but at very high concentrations (Figure 4). Wen, S., et al., 2002 (3) identified three different phthalic acid photoproducts of PHE adsorbed to TiO2-NP surfaces, but investigations focused on photoproduct formation during UVA irradiation and concentrations of these photoproducts were not quantified. As discussed above, in regard to ANT, PAH photodissociation has been predicted using theoretically calculated spectra in studies investigating PAHs in cosmic water ice (29). In PHE + TiO2-NP samples, the initial (1 hpi) concentration of 1-hydroxynaphthalene, was 379.6% higher than PHE samples. Interestingly, PHE samples demonstrated very high concentration increases of 1-hydroxynaphthalene over time and by 6 hpi, the concentration was 635.9% higher than PHE+ TiO2-NP samples. In agreement with previous studies, the initial increase in OHPAH photoproduct concentrations in PHE + TiO2-NP samples is likely due to TiO2-NP production of •OH radicals (3, 7). In combination with previous investigations and our observations of OHPAH formation, we suggest photodissociation of PHE, forming naphthalenic molecules, upon UVA absorption. These naphthalenic by-products of PHE become directly hydroxylated with or without TiO2-NPs, but the presence of TiO2-NPs further catalyzes these reactions more efficiently through •OH donation to form 1-hydroxynaphthalene. (7, 18, 28).

Figure 4.

Figure 4.

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Concentrations (μg/L) of phenanthrene (PHE) photoproducts identified following UVA irradiation (7.26 mW/cm2) of 40 μg/L PHE and 40 μg/L PHE+ 2 mg/L TiO2-NP samples at 1, 2, 4, and 6 hours post irradiation (hpi). The hydroxylated PAH (OHPAH) photoproducts increase in molecular weight from left to right along the x-axis. Bars show average ± SE, (n=3) of triplicate runs of the same sample on the instrument.

ANT and PHE samples, with or without TiO2-NPs, were found to contain OPAH photoproducts with higher molecular weights than the original PAH molecules (Figure 5, 6). To the author’s knowledge, this is the first instance where larger photoproducts than the original ANT and PHE molecules have been identified following UVA irradiation in the aqueous phase, suggesting recombination mechanisms occur to create larger molecules. Evolution of PAHs into larger, more complex molecules has been previously observed in astronomical investigations of PAHs (30). Frequently, PAHs favour degradation into smaller by-products but if recombination into a larger molecule results in a more stable structure (increased aromatic sextets) then it is possible photoprocesses will favour the formation of larger, more stable by-products (28). The identification of larger, OPAH photoproducts such as, BaP-diones and 6H-benzo(cd)pyrene-6-one, suggests recombination of the initially photodissociated and/or hydroxylated by-products or ring additions to the parent molecules occurred since similar OHPAH derivatives of the same size and arrangement were not identified (7, 18, 28). Further to this, the presence of OPAH molecules with differing molecular arrangements, but the same size as the parent molecule suggests ring re-arrangements occurred to form more stable isomers. These results suggest that upon UVA absorption, ANT and PHE photodissociate (the smaller OHPAH photoproducts discussed previously) and recombine or rearrange to form larger molecules which has never been reported in similar conditions.

Figure 5.

Figure 5.

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Concentrations (μg/L) of anthracene (ANT) photoproducts identified following UVA irradiation (7.26 mW/cm2) of 20 μg/L ANT and 20 μg/L ANT+ 2 mg/L TiO2-NP samples at 1, 2, 4, and 6 hours post irradiation (hpi). The oxygenated PAH (OPAH) photoproducts increase in molecular weight from left to right along the x-axis. Bars show average ± SE, (n=3) of triplicate runs of the same sample on the instrument.

Figure 6.

Figure 6.

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Concentrations (μg/L) of phenanthrene (PHE) photoproducts identified following UVA irradiation (7.26 mW/cm2) of 40 μg/L PHE and 40 μg/L PHE+ 2 mg/L TiO2-NP samples at 1, 2, 4, and 6 hours post irradiation (hpi). The oxygenated PAH (OPAH) photoproducts increase in molecular weight from left to right along the x-axis. Bars show average ± SE, (n=3) of triplicate runs of the same sample on the instrument.

ANT and PHE samples both showed increased OPAH photoproduct formation over time, but had differing time-related OHPAH formation (Figure S3). The observed ANT and PHE photoproduct formation at the time points selected in this study has not been previously investigated. Previous investigations have shown that ANT absorbs UVA wavelengths more readily than PHE; ANT: 358 nm, PHE: 251 and 290 nm (31, 32). The time-related patterns of OHPAH and OPAH formation for both ANT and PHE are likely dependent on the initial hydroxylation event, which can be heavily influenced by the presence of TiO2-NPs; particularly demonstrated by the time-related formation of OHPAHs by PHE (Figure 4). The absorptive differences of ANT and PHE result in differing photodegradative efficiencies upon UVA irradiation, which is reflected in the observed time-related photoproduct formation. Increased OPAH and OHPAH concentrations over time in the PHE samples, coupled with lower concentrations of OPAHs compared to OHPAHs, may indicate delayed or continued hydroxylation of photodissociated PHE by-products. Further hydroxylation of OPAH photoproducts was considered initially, but no dihydroxy by-products were identified which would have likely appeared if this had occurred. Additionally, PHE+TiO2-NP samples demonstrated decreased OHPAH and OPAH concentrations over time, which is indicative of more efficient degradation. Over time, ANT samples demonstrated increasing concentrations of OPAH by-products while OHPAH concentrations (e.g., 1-hydroxynaphthalene) decreased and appeared at much lower concentrations than OPAHs. These data support that hydroxylation is the initial photochemical event, prior to oxygenation (18, 28). Following hydroxylation, OHPAH by-products of ANT are then oxygenated indicated by the observed increase in OPAH concentrations (Figure 5). These data suggest that UVA irradiation of ANT results in more efficient photoproduct formation and subsequent degradation than PHE in the aqueous phase, which may be a result of PAH-specific UV absorptions.

Oxidative Potential Changes

This study was the first to assess oxidative potential changes of irradiated ANT and PHE in the presence of TiO2-NPs using the DTT assay. Following irradiation, the presence of TiO2-NPs reduced the oxidative potential of PHE and maintained heightened oxidative potential of ANT, over all time points assessed (Figure 7). There is a possibility that increased oxidative potential occurred prior to the 1 hpi sample collections, upon UVA absorption, but swiftly decreased as photoproduct formation occurred. PHE is directly photoionized, thus photodegradation of PHE may occur independent of photocatalyst, while photodegradation of ANT is oxygen-dependent and favours interaction with a photocatalyst (1, 33). The heightened oxidative potential observed in ANT, but not PHE samples, may be attributed to the absorptive differences between the two PAHs. The limited UVA absorption and lesser adsorptive capabilities of PHE to the TiO2-NP surface likely contributed to the reduced oxidative potential observed in PHE+TiO2-NP samples. The absence of time-dependent fluctuations in ANT samples suggests continued ROS formation occurred, likely resulting from the interaction (adsorption) of ANT to the TiO2-NP surface. The observed oxidative potential differences in ANT and PHE samples are likely associated with their differing photodegradative mechanisms, adsorptive capabilities, and UV absorption.

Figure 7.

Figure 7.

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Oxidative potential measured by DTT consumption (nM) of non-irradiated and UVA (7.26 mW/cm2) irradiated A) 20 μg/L ANT and 20 μg/L ANT+ 2 mg/L TiO2-NP and B) 40 μg/L PHE and 40 μg/L PHE+ 2 mg/L TiO2-NP samples, 1, 2, 4, and 6 hours post irradiation (hpi). Bars show the average ± SE, (n=3) of triplicate runs of the same sample on the instrument.

Integration of Different Approaches

The appearance of numerous photoproducts over all time points in all irradiated samples confirms phototransformation of ANT and PHE both in the absence and presence of TiO2-NPs occurred in the larval zebrafish exposures, regardless of the observed gene expression changes. The significant induction of cyp1a in ANT+TiO2-NP exposures at 4 hpe, combined with the identification of increased concentrations of OHPAH and OPAH photoproducts at 4 hpi, indicates the presence of bioavailable parent PAHs over time, as well as continued recombination and/or ring re-arrangements of photodissociated molecules. In agreement with previous studies, our results indicate biotransformation of ANT and PHE by larval zebrafish occurred in the absence of UVA, but both PAHs favoured photodegradation when UVA irradiation was present (31, 34). The PAH-specific gene inductions showed that ANT photoproducts were more bioavailable than PHE photoproducts, demonstrated by the increased cyp1a and ephx2 expression of irradiated ANT exposed zebrafish (35, 36, 37).

This study suggests that there is photodissociation of ANT and PHE upon UVA absorption, followed by hydroxylation. The presence of TiO2-NPs increased the efficiency of the hydroxylation process and resulted in a higher yield of OHPAH by-products. Recombination and ring addition(s) into larger molecules was also observed which was not seen in DMSO or TiO2-NP control samples, and may have continued past the time course investigated in this study (Figure S3). Ring re-arrangements of ANT and PHE occurred and was indicated by the identification of isomeric photoproducts of the original molecules. The presence of TiO2-NPs decreased larger photoproduct concentrations suggesting the NPs promoted degradation into smaller by-products. This is the first study to observe recombination of ANT and PHE photoproducts into larger molecules in the aqueous phase.

The sustained oxidative potential of ANT in the presence of TiO2-NPs indicated ROS were produced consistently, over all time points, but sod1 was not induced significantly. The assimilation of these data suggests the radicals that were produced reacted quickly with photodissociated molecules to form OHPAH/OPAHs, which was indicated by ephx2 induction and the identification of many photoproducts in the GC-MS analyses. PHE displayed reduced oxidative potential in the presence of TiO2-NPs, which was not reflected by the gene expression analyses. Interestingly, the time-related patterns demonstrated in the oxidative potential data reflected the time-related patterns of OPAH formation observed in the GC-MS data for both ANT and PHE (particularly for the favoured photoproducts). The use of gene expression analyses and analytical chemistry techniques each provided unique perspectives, but in combination they revealed the dynamic roles that bioavailability, biotransformation, photodegradation, and TiO2-NP photocatalysis all play in PAH-specific photoproduct formation in the aqueous phase.

Regardless of adsorptive differences, both ANT and PHE produced photoproducts that inevitably became bioavailable within this study’s time course. UVA irradiated samples of both PAHs contained distinct OPAHs and OHPAHs at the final time point investigated (6 hpi), some at increased concentrations. These findings demonstrate the need for the appropriate amount of time to pass following UVA irradiation of ANT and PHE, and likely other PAHs, to ensure bioactive by-products have fully degraded during environmental remediation activities. Therefore, deliberate application of TiO2-NPs for environmental remediation methods must seriously consider the many possible photoproducts formed during these processes, not solely the degradation of the parent PAH molecule, with an in-depth assessment of the immediate and long-term environmental impacts of reactive photoproducts.

Finally, both PAHs favoured the formation of 9,10-phenanthraquinone and 1-hydroxynaphthalene, regardless of the presence of TiO2-NPs which has not been observed prior to this study. The production of the same photoproducts from two, distinct PAHs is promising for their use as environmental indicators of ANT and PHE pollution (i.e., contaminated water sources, lakes, rivers etc.), and potentially other PAHs. The employment of environmental indicators would enhance our understanding of the environmental impacts, human health implications, and distribution of PAH pollution in aquatic environments.

Supplementary Material

SI

Acknowledgements

Special thanks to Dr. Charles Tyler for gifting zebrafish from his facility at the University of Exeter and Dr. Staci Simonich for her significant contribution to the analytical chemistry work which occurred in her lab at Oregon State University. This work was supported by the European Union (EU) and Horizon 2020 awarded under the Marie-Sklodowska-Curie action to the EUROPAH consortium, grant number 722346. This publication was made possible in part by Grant Numbers AGS-1411214 from the National Science Foundation (NSF), and P42-ES016465 and P30-ES00210, from National Institute of Environmental Health Sciences (NIEHS), National Institute of Health (NIH). Its contents are the sole responsibility of the authors and do not represent the official view of the NIEHS or NIH.

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