Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

Mechanism of nanoplastics capture by jellyfish mucin and its potential as a sustainable water treatment technology.

The accumulation of nanoplastics (NPs) in the environment has raised concerns about their impact on human health and the biosphere. The main aim of this study is to understand the mechanism that governs the capture of NPs by jellyfish mucus extracted from the jellyfish Aurelia sp. (A.a.) and compare the capture/removal efficiency to that of conventional coagulants and mucus from other organisms. The efficacy of A.a mucus to capture polystyrene and acrylic NPs (∼100 nm) from spiked wastewater treatment plant (WWTP) effluent was evaluated. The mucus effect on capture kinetics and destabilization of NPs of different polymer compositions, sizes and concentrations was quantified by means of fluorescent NPs, dynamic light scattering and zeta potential measurements and visualized by scanning electron microscopy. A dosing of A.a. mucus equivalent to protein concentrations of ∼2-4 mg L-1 led to a rapid change in zeta potential from a baseline of -30 mV to values close to 0 mV, indicating a marked change from a stable to a non-stable dispersion leading to a rapid (<10 min) and significant removal of NPs (60 %-90 %) from a stable suspension. The A.a. mucus outperformed all other mucus types (0-37 %) and coagulants (0 %-32 % for ferric chloride; 23-40 % for poly aluminum chlorohydrate), highlighting the potential for jellyfish mucus to be used as bio-flocculant. The results indicate a mucus-particle interaction consisting of adsorption-bridging and "mesh" filtration. Further insight is provided by carbohydrate composition and protein disruption analysis. Total protein disruption resulted in a complete loss of the A.a. mucus capacity to capture NPs, while the breaking of disulfide bonds and protein unfolding resulted in improved capture capacity. The study demonstrates that natural jellyfish mucin can capture and remove NPs in water and wastewater treatment systems more efficiently than conventional coagulants, highlighting the potential for development of a new type of bio-flocculant.

Computer Analysis of the Effect of Activation Temperature on the Microporous Structure Development of Activated Carbon Derived from Common Polypody.

This paper presents the results of a computer analysis of the effect of activation process temperature on the development of the microporous structure of activated carbon derived from the leaves of common polypody (Polypodium vulgare) via chemical activation with phosphoric acid (H3PO4) at activation temperatures of 700, 800, and 900 °C. An unconventional approach to porous structure analysis, using the new numerical clustering-based adsorption analysis (LBET) method together with the implemented unique gas state equation, was used in this study. The LBET method is based on unique mathematical models that take into account, in addition to surface heterogeneity, the possibility of molecule clusters branching and the geometric and energy limitations of adsorbate cluster formation. It enabled us to determine a set of parameters comprehensively and reliably describing the porous structure of carbon material on the basis of the determined adsorption isotherm. Porous structure analyses using the LBET method were based on nitrogen (N2), carbon dioxide (CO2), and methane (CH4) adsorption isotherms determined for individual activated carbon. The analyses carried out showed the highest CO2 adsorption capacity for activated carbon obtained was at an activation temperature of 900 °C, a value only slightly higher than that obtained for activated carbon prepared at 700 °C, but the values of geometrical parameters determined for these activated carbons showed significant differences. The results of the analyses obtained with the LBET method were also compared with the results of iodine number analysis and the results obtained with the Brunauer-Emmett-Teller (BET), Dubinin-Radushkevich (DR), and quenched solid density functional theory (QSDFT) methods, demonstrating their complementarity.

Establishing a link between composition and toxicity of offshore produced waters using comprehensive analysis techniques - A way forward for discharge monitoring?

Extracts of produced waters from five mature Norwegian Sea oil fields were examined as total organic extracts (TOEs) and after fractionation into operationally-defined 'polar' and 'apolar' fractions. The TOEs and fractions were examined by gas chromatography (GC), GC-mass spectrometry (GC-MS), two dimensional GC-MS (GC × GC-MS) and liquid chromatography with high-resolution spectrometry (LC-HRMS) techniques. Low molecular weight aromatics, phenols and other common petroleum-derived hydrocarbons were characterized and quantified in the TOEs and fractions. In addition, a range of more uncommon polar and apolar constituents, including those likely derived from production chemicals, such as trithiolane, imidazolines and quaternary amine compounds (so-called 'quats'), were tentatively identified, using GC × GC-MS and LC-HRMS. The acute toxicity of the TOEs and subfractions was investigated using early life stages of the marine copepod Acartia tonsa. Toxicity varied significantly for different PW TOEs and subfractions. For some PWs, the toxicity was attributed mainly to the 'polar' components, while that of other PWs was associated mainly with the 'apolar' components. Importantly, the observed toxicity could not be explained by the presence of the commonly reported compounds only. Although, due to the vast chemical complexity even of the sub-fractions of the PW extracts, specific compounds driving the observed toxicity could be not be elucidated in this study, the proposed approach may suggest a way forward for future revisions of monitoring regimes for PW discharges.

Partitioning of PAHs between Crude Oil Microdroplets, Water, and Copepod Biomass in Oil-in-Seawater Dispersions of Different Crude Oils.

The impact of oil microdroplets on the partitioning of polycyclic aromatic hydrocarbons (PAHs) between water and marine zooplankton was evaluated. The experimental approach allowed direct comparison of crude oil dispersions (containing both micro-oil droplets and water-soluble fraction; WSF) with the corresponding WSF (without oil droplets). Dispersion concentration and oil type have an impact on the PAH composition of WSFs and therefore affect dispersion bioavailability. Higher T-PAH body residues were observed in copepods treated with dispersions compared to the corresponding WSFs. PAHs with log Kow 3-4.5 displayed comparable accumulation factors between treatments; however, accumulation factors for less soluble PAHs (log Kow = 4.5-6) were higher for the WSF than for the dispersions, suggesting low bioavailability for components contained in oil droplets. The higher PAH body residue in dispersion exposures is assumed to result mainly from copepods grazing on oil droplets, which offers an alternative uptake route to passive diffusion. To a large degree this route is controlled by the filtration rates of the copepods, which may be inversely related to droplet concentration.

Adhesion of mechanically and chemically dispersed crude oil droplets to eggs of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Crude oil accidentally spilled into the marine environment undergoes natural weathering processes that result in oil components being dissolved into the water column or present in particulate form as dispersed oil droplets. Oil components dissolved in seawater are typically considered as more bioavailable to pelagic marine organisms and the main driver of crude oil toxicity, however, recent studies indicate that oil droplets may also contribute. The adhesion of crude oil droplets onto the eggs of pelagic fish species may cause enhanced transfer of oil components via the egg surface causing toxicity during the sensitive embryonic developmental stage. In the current study, we utilized an oil droplet dispersion generator to generate defined oil droplets sizes/concentrations and exposed Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to investigate if the potential for dispersed oil droplets to adhere onto the surface of eggs was species-dependent. The influence of a commercial chemical dispersant on the adhesion process was also studied. A key finding was that the adhesion of oil droplets was significantly higher for haddock than cod, highlighting key differences and exposure risks between the two species. Scanning electron microscopy indicates that the differences in oil droplet adhesion may be driven by the surface morphology of the eggs. Another important finding was that the adhesion capacity of oil droplets to fish eggs is significantly reduced (cod 37.3%, haddock 41.7%) in the presence of the chemical dispersant.

Optimization and comparison of miniaturized extraction techniques for PAHs from crude oil exposed Atlantic cod and haddock eggs.

Two miniaturized extraction methods for a wide range of 2-6 ring polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in small lipid-rich biota samples (≤100 mg) have been developed. Both methods utilize liquid extraction (LE) prior to a clean-up step using either normal phase solid phase extraction (SPE) or mixed-phase dispersive SPE (dSPE). Optimization of the methods was achieved by comparing the type and amount of sorbents, drying agents, and solvents used. In order to improve the limits of detection (LOD) of target PAHs under high sensitivity gas chromatography-tandem mass spectrometry analysis, specific emphasis was given to minimizing lipid co-extraction. The optimized LE-SPE method comprised extraction with dichloromethane/n-hexane (1:1, v/v) and clean-up by silica SPE, whereas the optimized LE-dSPE method comprised extraction with acetonitrile and clean-up with PSA and C18 sorbents. The methods were validated and directly compared through the analysis of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) eggs exposed to oil. The LE-SPE method resulted in lower levels of co-extracted lipids (14.1 ± 1.7 ng/µL) than the LE-dSPE method (60 ± 14 ng/µL). Achieved PAH LODs for the LE-SPE method were typically an order of magnitude lower (<5 ng/g) than for the LE-dSPE method (<125 ng/g). The LE-SPE method offers the possibility for PAH analysis of small samples of fish eggs (~100 mg) exposed to small quantities of crude oil (~1-10 µg/L total PAHs).

Oil droplet interaction with suspended sediment in the seawater column: influence of physical parameters and chemical dispersants.

The interaction of dispersed oil droplets with large diameter suspended particulate materials (SPM) has been little studied. In the current study, particle size, oil characteristics and chemical dispersant significantly influence the adsorption of oil droplets to SPM in seawater. Sediments with a smaller particulate size (clay) approaching that of the oil droplets (2-20 µm) adsorbed more oil per gram than sediments with large particle size (sand). Heavier, more polar oils with a high asphaltene content adsorbed more efficiently to SPM than lighter, less polar oils. A decrease in the smaller, more water soluble oil components in the sediment adsorbed oil was observed for all oil types. Addition of chemical dispersant decreased the adsorption of oil droplets to suspended carbonate sand in an exponential-like manner. No change in the relative distribution of compounds adsorbed to the sediment was observed, indicating dispersants do not alter the dissolution of compounds from oil droplets.

Complications with remediation strategies involving the biodegradation and detoxification of recalcitrant contaminant aromatic hydrocarbons.

Environmentally persistent aromatic hydrocarbons known as unresolved complex mixtures (UCMs) derived from crude oil can be accumulated by, and elicit toxicological responses in, marine organisms (e.g. mussels, Mytilus edulis). Comprehensive two-dimensional gas chromatography time-of-flight mass-spectrometry (GCxGC-ToF-MS) previously revealed that these UCMs included highly branched alkylated aromatic hydrocarbons. Here, the effects of biodegradation on the toxicity and chemical composition of an aromatic UCM hydrocarbon fraction isolated from Tia Juana Pesado (TJP) crude oil were examined. 48h exposure of mussels to the aromatic hydrocarbon fraction (F2) resulted in tissue concentrations of 900microgg(-1) (dry wt.) and approximately 45% decrease in clearance rate. Over 90% of the hydrocarbon burden corresponded to an UCM. Following a 5day recovery period, GCxGC-ToF-MS analysis of the tissues indicated depuration of most accumulated hydrocarbons and clearance rates returned to those observed in controls. To assess the potential of biodegradation to reduce UCM toxicity, TJP F2 was exposed to bacteria isolated from Whitley Bay, UK, for 46days. Mussels exposed to the undegraded TJP F2 from the abiotic control exhibited a reduction in clearance rate comparable with values for the pure crude oil TJP F2. Clearance rates of mussels exposed to biodegraded TJP F2 were statistically similar to seawater controls, suggesting biodegradation had reduced the TJP F2 toxicity. GCxGC-ToF-MS analysis revealed the same compound groups in the tissue of mussels exposed to pure TJP F2, undegraded TJP F2 and biodegraded TJP F2 samples; however >300 fewer compounds were observed in the biodegraded (954 compounds) compared to the undegraded TJP F2 (1261). The compound distributions were markedly different, possibly accounting for the decrease in toxicity. Extraction and analysis of pelleted bacterial cell material revealed that a significant proportion of the TJP F2 had adsorbed onto the cells. Thus extreme care must be taken in interpreting biodegradation data from recalcitrant UCM hydrocarbons.

Molecular effects of diethanolamine exposure on Calanus finmarchicus (Crustacea: Copepoda).

Alkanolamines are surface-active chemicals used in a wide range of industrial, agricultural and pharmaceutical applications and products. Of particular interest is the use of alkanolamines such as diethanolamine (DEA) in the removal of CO(2) from natural gas and for CO(2) capture following fossil fuel combustion. Despite this widespread use, relatively little is known about the ecotoxicological impacts of these compounds. In an attempt to assess the potential effects of alkanolamines in the marine environment, a key species in the North Atlantic, the planktonic copepod Calanus finmarchicus, was studied for molecular effects following sublethal exposure to DEA. DEA-induced alterations in transcriptome and metabolome profiling were assessed using a suppression subtractive hybridization (SSH) gene library method and high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR), respectively. Effects were observed on transcription of genes reportedly involved in lipid metabolism, antioxidant systems, metal binding, and amino acid and protein catabolism. These effects were accompanied by altered expression of fatty acid derivates, amino acids (threonine, methionine, glutamine, arginine, alanine and leucine) and cholines (choline, phosphocholine and glycerophosphocholine). Together, SSH and HR-MAS NMR offer complementary screening tools for the assessment of molecular responses of C. finmarchicus to DEA and can be used in the study of other chemicals and organisms. Concentration-response and time-response relationships between DEA exposure and single gene transcription were investigated using quantitative PCR. Specific relationships were found between DEA exposure and the transcription of genes involved in protein catabolism (ubiquitin-specific protease-7), metal ion homeostasis (ferritin) and defence against oxidative stress (gamma-glutamylcysteine synthase, glutathione synthase and Cu/Zn-superoxide dismutase). At the lowest alkanolamine concentration used in these experiments, which corresponded to 0.5% of the LC(50) concentration, no transcriptional effects were observed, giving information regarding the lower molecular effect level. Finally, similar transcription patterns were observed for a number of different genes following exposure to DEA, which indicates analogous mechanisms of toxicity and response.

Chemical and toxicological characterization of an unresolved complex mixture-rich biodegraded crude oil.

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

Gene expression of GST and CYP330A1 in lipid-rich and lipid-poor female Calanus finmarchicus (Copepoda: Crustacea) exposed to dispersed oil.

The copepod Calanus finmarchicus is a marine ecological key species in the Northern Atlantic food web. This species was exposed to an artificially weathered North Sea oil dispersion (oil droplets and water-soluble fractions [WSF]) and a filtered dispersion (containing only WSF) in serial dilution. Female copepods were divided into lipid-rich and lipid-poor for each exposure followed by gene expression analyses of glutathione S-transferase (GST) and cytochrome P-450 330A1 (CYP330A1). Lipid-rich copepods exhibited elevated transcription of GST and reduced transcription of CYP330A1 after exposure to both dispersed oil and WSF. In contrast, lipid-poor copepods exhibited increased transcription of CYP330A1 following exposure to WSF but not the dispersion. Data suggested that small lipid storage promotes increased bioavailability of accumulated oil compounds. Variations in response in CYP330A1 gene expression indicate that oil constituents may exert different modes of toxic action in copepods depending on their reproductive stages. The contribution of oil droplets to the observed effects seemed to be low as GST gene expression was similar after exposure to both dispersed oil and WSF. However, feeding rate in copepods exposed to dispersed oil was reduced, and this may have decreased the uptake of oil constituents via the diet. Although quantitatively higher mortality was observed in copepods exposed to the highest dispersion levels, this may result from smothering of animals by oil droplets. Furthermore, increasing dilution of both the dispersions and the WSF altered their distributions and chemical composition, which may influence the bioavailability of spilled crude oil to pelagic marine organisms.