Salmonid smolt caudal fin and liver transcriptome responses to low sulfur marine diesel and high sulfur fuel oil water accommodated fractions for assessing oil spill effects in marine environments.

The environmental impact of oil spills is a critical concern, particularly pertaining to low sulfur marine diesel (LSMD) and high sulfur fuel oil (HSFO) that are commonly involved in coastal spills. Although transcriptomic biomonitoring of sentinel animals can be a powerful tool for assessing biological effects, conventional methods utilize lethal sampling to examine the liver. As a non-lethal alternative, we have previously shown salmonid caudal fin cyp1a1 is significantly responsive to LSMD-derived toxicants. The present study further investigated the transcriptomic biomonitoring potential of coho salmon smolt caudal fin in comparison to liver tissue in the context of LSMD and HSFO seawater accommodated fraction (seaWAF) exposure in cold-water marine environments. Assessing the toxicity of these seaWAFs involved quantifying polycyclic aromatic hydrocarbon (tPAH50) concentrations and generating gene expression profiles. Initial qPCR analyses revealed significant cyp1a1 response in both liver and caudal fin tissues of both genetic sexes to all seaWAF exposures. RNA-Seq analysis, focusing on the highest LSMD and HSFO seaWAF concentrations (28.4±1.8 and 645.08±146.3 µg/L tPAH50, respectively), revealed distinct tissue-specific and genetic sex-independent transcriptomic responses with an overall enrichment of oxidative stress, cell adhesion, and morphogenesis-related pathways. Remarkably, the caudal fin tissue exhibited transcriptomic response patterns comparable to liver tissue, particularly consistent differential expression of 33 gene transcripts in the liver (independent of sex and oil type) and 44 in the caudal fin. The present work underscores the viability of using the caudal fin as a non-lethal alternative to liver sampling for assessing and tracking oil spill exposure in marine environments.

Method development for forensic oil identification by direct analysis in real time time-of-flight mass spectrometry.

The current well-established chromatography and mass spectrometry based oil spill identification procedures, such as those outlined by the European Committee for Standardization, are highly reliable as methods, highly defensible in the court of law, and widely applicable to the majority of oil spill situations. Nevertheless, the methodology is time consuming and labour intensive, which may not be ideal when dealing with an emergency oil spill situation. In this study, direct analysis in real time time-of-flight mass spectrometry (DART/TOFMS) was used to successfully develop an efficient oil identification method. To confirm the accuracy of this method spilled oil samples were tested from five previous years of blind round robin testing organized by the oil spill identification network of experts (OSINET) under the Bonn Agreement. Heatmap inspection, principal component analysis and finally discriminant analysis of principal components were used to arrive at final predictions regarding the identities of the spilled oil samples. The results were compared with the results of previous gas chromatography flame ionization detection (GC/FID) and gas chromatography triple quadrupole mass spectrometry (GC/MS/MS) analyses of the same oils. While taking only about a tenth of the time, the DART/TOFMS analysis produced results similar to those of classical GC/FID and GC/MS/MS (EI+) procedures. The ability of DART/TOFMS to display this level of validity exemplifies its potential to be a new tool for supplementing classical analyses for oil spill forensics.

Enhancement of oil forensic methodology through the addition of polycyclic aromatic nitrogen heterocycle biomarkers for diagnostic ratios.

Current oil spill forensic identification of source oils relies upon hydrocarbon biomarkers resistant to weathering. This international technique was developed by the European Committee for Standardization (CEN), under EN 15522-2 Oil Spill Identification guidelines. The number of biomarkers have expanded at pace with technological advances, while distinguishing new biomarkers becomes more challenging due to interference of isobaric compounds, matrix effects, and high cost of weathering experiments. Application of high-resolution mass spectrometry enabled exploration of potential polycyclic aromatic nitrogen heterocycle (PANH) oil biomarkers. The instrumentation showed reduction in isobaric and matrix interferences, allowing for identification of low-level PANH and alkylated PANHs (APANHs). Weathered oil samples, obtained from a marine microcosm weathering experiment, enabled comparison with source oils to identify new, stable forensic biomarkers. This study highlighted eight new APANH diagnostic ratios that expanded the biomarker suite, increasing the confidence for identifying highly weathered oils back to their source oil.

Dynamic cyp1a1 transcript responses in the caudal fin of coho salmon (Oncorhynchus kisutch) smolts to low sulfur marine diesel water accommodated fraction exposures and depuration.

Oil spills that occur in high traffic coastal environments can have profound consequences for the health of marine ecosystems and the commercial and social interests that are dependent upon these habitats. Given that the global reliance on marine fuels is not abating, it is imperative to develop sensitive and robust tools to monitor oil contamination and remediation in a timely manner. Such tools are increasingly important for ascertaining the immediate and long-term effects of oil contamination on species of interest and local habitats as water-soluble components of oils, such as polycyclic aromatic hydrocarbons (PAHs), can persist post-remediation. We previously demonstrated that 3-methylcholanthrene responsive cytochrome P450-1a (cyp1a1) transcript abundance in the liver and caudal fin of coho salmon smolts (Onchorhynchus kisutch) was sensitive to exposure to low sulfur marine diesel (LSMD) seawater accommodated fractions (seaWAF) in cold water. We expanded upon this paradigm by assessing the utility of the cyp1a1 transcript to track both exposure to LSMD seaWAF and recovery from exposure by measuring cyp1a1 abundance in coho smolts using quantitative polymerase chain reaction (qPCR). Smolts were exposed to either 100 mg/L LSMD seaWAF or clean seawater (control) for 4 days. Fish were then transferred to clean seawater for depuration and tissues sampled at 0, 1, 2, 4, and 8 days from both treatments. Livers and caudal fins were dissected from 40 smolts per group (ntotal = 400 smolts). The LSMD seaWAF-induced cyp1a1 transcript levels significantly decreased one day after depuration in the liver and caudal fin in a sex-independent manner in genotyped females and males. After four days of depuration, cyp1a1 transcript abundance decreased to baseline control levels, regardless of tissue or sex. The present study demonstrates the value of using the caudal fin as a reliable, sensitive, and non-lethal sampling and monitoring tool.

Advancement in oil forensics through the addition of polycyclic aromatic sulfur heterocycles as biomarkers in diagnostic ratios.

In current oil spill forensics, diagnostic ratios of hydrocarbon biomarker responses are commonly used to compare oil spill samples to source materials in order to determine the identity of the oil. This well recognized procedure was developed by the European Committee for Standardization (CEN) with corresponding published EN 15522-2 Oil Spill Identification guidelines. However, it is further recognized that weathering can have a negative effect on some of the biomarkers used in the analysis, leading to decreased confidence in the result. In this study, polycyclic aromatic sulfur heterocycles (PASHs) and their alkylated forms (APASHs) were assessed for their potential as additional biomarkers. With the aim of identifying stable PASHs and APASHs useful as weathered oil biomarkers, the superior specificity of gas chromatography with high resolution mass spectrometry was exploited to determine chromatographic peak responses for sixteen petroleum oil samples. Extensive study, involving microcosm extreme weathering and spreadsheet development, led to the identification of 19 new diagnostic ratios based on newly discovered stable PASH and APASH biomarkers. Application of the extended diagnostic ratio suite showed high potential to improve the forensic attribution of post-spill weathered oil back to its original source.

A rapid gas chromatography quadrupole time-of-flight mass spectrometry method for the determination of polycyclic aromatic hydrocarbons and sulfur heterocycles in spilled crude oils.

Spilled crude oil samples contain various toxic compounds including polycyclic aromatic hydrocarbons (PAHs) as well as sulfur heterocycles (PASHs) and their related alkylated forms (APAHs and APASHs). In this study, a method was successfully developed employing a gas chromatography quadrupole time-of-flight (GC-QToF) mass spectrometer to quantitatively analyze both PAHs/APAHs and PASHs/APASHs in these samples. With GC-QToF, the monoisotopic mass of the compounds is distinguished, allowing the PASHs/APASHs to be extracted separately from the PAHs/APAHs in crude oil. A gas chromatography triple quadrupole (GC-MS/MS) mass spectrometer was also used to confirm that a GC-QToF is the preferred instrument for analyzing these compounds. With the use of PASH/APASH standards to determine response correction factors (RCFs) in relation to PAH standards, the developed method is capable of analyzing PAHs, APAHs, PASHs, and APASHs in a single injection. The use of RCFs allowed for the development of a practical polycyclic aromatic carbon (PAC) method for analyzing a total of 77 compounds of the 2 groups in crude oil. This newly developed method was applied to spilled crude oils, demonstrating its potential in toxicological study as well as oil spill forensic investigation.

Enhanced analysis of weathered crude oils by gas chromatography-flame ionization detection, gas chromatography-mass spectrometry diagnostic ratios, and multivariate statistics.

Artificially weathered crude oil "spill" samples were matched to unweathered suspect "source" oils through a three-tiered approach as follows: Tier 1 gas chromatography-flame ionization detection (GC/FID), Tier 2 gas chromatography-mass spectrometry (GC/MS) diagnostic ratios, and Tier 3 multivariate statistics. This study served as proof of concept for a promising and new method of crude oil forensics that applies principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA) in tandem with traditional forensic oil fingerprinting tools to confer additional confidence in challenging oil spill cases. In this study, weathering resulted in physical and chemical changes to the spilled oils, thereby decreasing the reliability of GC/FID and GC/MS diagnostic ratios in source attribution. The shortcomings of these traditional methods were overcome by applying multivariate statistical tools that enabled accurate characterization of the crude oil spill samples in an efficient and defensible manner.

Tiered approach to long-term weathered lubricating oil analysis: GC/FID, GC/MS diagnostic ratios, and multivariate statistics.

Frequent small-scale environmental releases of lubricating (lube) oils have deleterious effects on aquatic ecosystems. In the event of a spill, oil fingerprinting is critical to source attribution, clean-up procedures, and liability assignment. Oil forensic investigations are particularly challenging when oils are weathered over an extended period of time, as a large number of biomarkers become lost and the chemical composition changes significantly from its source. This study simulated an environmental case in which long-term weathered lube oil "spill" samples were matched to unweathered suspect "source" oils. While traditional oil fingerprinting techniques including GC/FID and GC/MS diagnostic ratios were insufficient for reliably attributing the source, a comprehensive and systematically tiered approach proved successful. The proposed methodology featured three tiers: Tier 1 GC/FID, Tier 2 GC/MS diagnostic ratios, and Tier 3 multivariate statistics. This novel approach provided environmental chemists with a powerful tool for dealing with an otherwise extremely challenging lube oil forensic investigation.

Evaluation of Gene Bioindicators in the Liver and Caudal Fin of Juvenile Pacific Coho Salmon in Response to Low Sulfur Marine Diesel Seawater-Accommodated Fraction Exposure.

Low sulfur marine diesel (LSMD) is frequently involved in coastal spills and monitoring ecosystem damage, and the effectiveness of cleanup methods remains a challenge. The present study investigates the concentration and composition of polycyclic aromatic hydrocarbons (PAHs) dispersed in LSMD seawater accommodated fractions (WAFs) and assesses the effects of exposure on juvenile coho salmon ( Onchorhynchus kisutch). Three WAFs were prepared with 333, 1067, and 3333 mg/L LSMD. The sum of 50 common PAHs and alkylated PAHs (tPAH50) measured by gas chromatography/triple quadrupole mass spectrometry showed saturation at ∼90 mg/L for all WAFs. These WAFs were diluted 30% for 96 h fish exposures. qPCR was performed on liver and caudal fin from the same genotypically sexed individuals to evaluate PAH exposure, general and oxidative stress, estrogenic activity, and defense against metals. Excluding metal response, our analyses reveal significant changes in gene expression following WAF exposure on juvenile salmon with differential sensitivity between males and females. The 3-methylcholanthrene responsive cytochrome P450-1a ( cyp1a) transcript exhibited the greatest increase in transcript abundance in the caudal fin (10-18-fold) and liver (6-10-fold). This demonstrates that cyp1a is a robust, sex-independent bioindicator of oil exposure in caudal fin, a tissue that is amenable to nonlethal sampling.

Superhydrophobic Substrates from Off-the-Shelf Laboratory Filter Paper: Simplified Preparation, Patterning, and Assay Application.

Off-the-shelf laboratory filter paper of different pore sizes and thicknesses can be modified with fluorine-free organosilanes to be superhydrophobic, patternable, and ready for quantitative assay applications. In particular, we have demonstrated that the cellulose filter paper treated with a binary hexane solution of short (methyltrichlorosilane) and long (octadecyltrichlorosilane) organosilanes exhibits remarkably high water contact angles (>150°) and low wetting hysteresis (∼10°). Beyond the optimized ratio between the two organosilanes, we have discovered that the thickness rather than the pore size dictates the resulting superhydrophobicity. Scanning electron microscopy images showed that silanization does not damage the cellulose microfibers; instead, they are coated with uniform, particulate nanostructures, which should contribute to the observed surface properties. The modified filter paper is chemically stable and mechanically durable; it can be readily patterned with UV/ozone treatment to create hydrophilic regions to prepare chemical assays for colorimetric pH and nitrite detections.

Facile Preparation of Nanostructured, Superhydrophobic Filter Paper for Efficient Water/Oil Separation.

In this paper, we present a facile and cost-effective method to obtain superhydrophobic filter paper and demonstrate its application for efficient water/oil separation. By coupling structurally distinct organosilane precursors (e.g., octadecyltrichlorosilane and methyltrichlorosilane) to paper fibers under controlled reaction conditions, we have formulated a simple, inexpensive, and efficient protocol to achieve a desirable superhydrophobic and superoleophilic surface on conventional filter paper. The silanized superhydrophobic filter paper showed nanostructured morphology and demonstrated great separation efficiency (up to 99.4%) for water/oil mixtures. The modified filter paper is stable in both aqueous solutions and organic solvents, and can be reused multiple times. The present study shows that our newly developed binary silanization is a promising method of modifying cellulose-based materials for practical applications, in particular the treatment of industrial waste water and ecosystem recovery.