Phenotypic and transcriptomic responses of the rotifer Brachionus koreanus by single and combined exposures to nano-sized microplastics and water-accommodated fractions of crude oil.

Sorption of organic pollutants on microplastics can be an alternative uptake route for organic pollutants in aquatic organisms. To assess the combined effects of microplastics and organic pollutants, we employed phenotypic and transcriptomic analyses to the responses of the marine rotifer Brachionus koreanus to environmentally relevant concentrations of nano-sized microplastic (0.05 µm), water-accommodated fractions of crude oil, and binary mixtures thereof. Our multigenerational in vivo experiments revealed more than additive effects on population growth of B. koreanus in response to combined exposure, while a single exposure to nano-sized microplastic did not induce observable adverse effects. Synergistic transcriptome deregulation was consistently associated with dramatically higher numbers of differentially expressed genes, and increased gene expression was associated with combined exposure. The majority of synergistic transcriptional alteration was related to metabolism and transcription, with impaired reproduction resulting from energetic reallocation toward adaptation. As further supported by chemistry analysis for polycyclic aromatic hydrocarbons sorption on microplastic, our findings imply that nano-sized microplastics can synergistically mediate the effects of organic pollutants in aquatic organisms.

Rapid recovery of coastal environment and ecosystem to the Hebei Spirit oil spill's impact.

The 2007 Hebei Spirit oil spill (HSOS), the largest in the national history, has negatively impacted the entire environment and ecosystem along the west coast of South Korea. Although many studies have reported the damages and impacts from the HSOS, quantitative assessment evaluating the recovery time and status have not been documented. Here, we first address the recovery timeline of the HSOS, by comprehensive analyses of 10-years accumulated data in quantitative manner. Concentrations of residual oils in seawater, sediments, and oysters rapidly dropped to backgrounds in 16, 75, and 33 months, respectively. Also, damaged benthic communities of intertidal and subtidal areas were fully recovered only after ~6 years. The present results collectively indicated unexpectedly fast recovery of the damaged environment and ecosystem from such a huge oil spill. The high tidal mixing (~9 m tidal height) and intensive human cleanup (~1.2 million volunteers) at the initial cleanup period might have contributed to rapid recovery; cf. 4-5 times faster than the Exxon Valdez oil spill. However, potential risk to human health remains unclear. Thus, it is warranted to conduct more in depth epidemiological studies to address chronic health effects associated with the cleanup volunteers as well as the local residents who have been living nearby the oil spill impacted sites.

Environmental significance of lubricant oil: A systematic study of photooxidation and its consequences.

Lubricant (lube) oil discharge from ships has been widely considered as normal "operational consumption", but is now deemed to be oil pollution. Despite the chronic contamination of the marine environment by lube oil, the number of studies related to its environmental impact, characteristics, and toxicity is limited. This study is the first attempt to investigate the environmental fate of lube oil subjected to photooxidation using in situ mesocosms. A tiered approach using thin-layer chromatography-flame ionization detection (TLC-FID), Fourier-transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) demonstrated compositional changes in lube oil and the water-soluble fraction (WSF). Total polycyclic aromatic hydrocarbons (ΣPAHs) in lube oil after 96 h of photooxidation were measured at 79.8 and 41 µg/g in the control (Con) and exposure (Exp) groups, respectively. Meanwhile, the ΣPAHs concentration in WSF after 96 h was very low, at 0.25 and 0.45 µg/L in Con and Exp, respectively. FTIR and GC-MS helped identify bond changes and photoproducts in WSF. A wide range of photoproducts, including carboxylic acids, esters, anhydrides, aldehydes and ketones, were identified in WSF. Toxic effects of WSF in both the Con and Exp groups obtained after 96 h of photooxidation were evaluated on olive flounder (Paralichthys olivaceus) embryos. Morphological defects, especially tail fin fold defects, were found to be significantly elevated in both the Con and Exp groups, with marginally higher frequency in Exp. The results of this study demonstrate the need for further research on lube oil weathering, including monitoring over prolonged periods of time.

Molecular level determination of water accommodated fraction with embryonic developmental toxicity generated by photooxidation of spilled oil.

In this study, developmental toxicity was increased as the oil was further degraded under natural sunlight. Detailed chemical composition of the degraded oils was examined by use of gas chromatography (GC) and (-) electrospray ionization ultrahigh resolution mass spectrometry (UHR-MS). Baseline toxicities were estimated based on chemical activities of polycyclic aromatic hydrocarbons, and it was obvious that the predicted chemical activities can not explain increased toxicity alone. However, the ultrahigh resolution mass spectral abundance of polar compounds including O3 and O4 class compounds was significantly increased as the photodegradation proceeded. Further examination of double bond equivalence values of the compounds showed that polar compounds with both non-aromatic and aromatic polar structures were increased. Statistical analysis indicates that the increased toxicity can be well explained by the increased polar compounds. Therefore, the oxygenated compounds identified in this study can play an important role in toxicity of degraded oils.

Adverse effects and immune dysfunction in response to oral administration of weathered Iranian heavy crude oil in the rockfish Sebastes schlegeli.

To demonstrate the effects of weathered crude oil residue on the immune systems of resident fish, we measured the changes in toxic chemical concentrations, apoptosis, phagocytosis, metabolism, immune-related gene expression, and cell cycle arrest in livers or kidneys for up to 96 h after the weathered Iranian heavy crude oil (WIHCO) exposure by oral gavage in juvenile rockfish Sebastes schlegeli. Parent polyaromatic hydrocarbons (PAHs) in livers increased up to 5590 ng/g after 6 h exposure and then declined rapidly within 24 h. Hepato-detoxification and immune-related gene expression were also significantly increased (P < 0.05) after 6 h exposure and then declined rapidly within 24 h. However, biliary PAH metabolites and EROD activity remained elevated throughout the test period. Flow cytometry analysis also indicated sustained apoptosis and cell cycle arrests with reduced phagocytic activity for 96 h. Taken together, these results demonstrate rapid declination of the parent PAHs, whereas PAH metabolites remained much longer in tissues with prolonged suppression of immunity in molecular and cellular level, suggesting that weathered crude oil residue is likely linked to the high incidence of immune dysfunction in residential rockfish in oil spill area.

Long-Term Monitoring of PAH Contamination in Sediment and Recovery After the Hebei Spirit Oil Spill.

Approximately 10,900 t of crude oil was released 10 km off the west coast of Korea after the collision between the oil tanker Hebei Spirit and a barge carrying a crane in December 2007. To assess the areal extent and temporal trends of PAH contamination, 428 sediment samples were collected from December 2007 through May 2015 for PAH analysis. Sedimentary PAH concentrations measured immediately after the spill ranged from 3.2 to 71,200 ng g-1, with a mean of 3800 ng g-1. Increases in PAH concentrations were observed at stations 7-23, which were heavily oiled due to tidal currents and northwesterly wind that transported the spilled oil to these locations. Mean and maximum PAH concentrations decreased drastically from 3800 to 88.5 and 71,200 to 1700 ng g-1, respectively, 4 months after the spill. PAH concentrations highly fluctuated until September 2008 and then decreased slowly to background levels. Reduction rate was much faster at the sandy beaches (k = 0.016) than in the muddy sites (k = 0.001). In muddy sediments, low attenuation due to low flushing rate in the mostly anaerobic sediment possibly contributed the persistence of PAHs. By May 2015 (~7.5 years after the spill), mean and maximum PAH concentrations decreased by 54 and 481 times, respectively, compared with the peak concentrations. The sedimentary PAH concentrations in the monitoring area have returned to regional background levels.

RNA seq- and DEG-based comparison of developmental toxicity in fish embryos of two species exposed to Iranian heavy crude oil.

To determine and compare the toxic effects of Iranian heavy crude oil (IHCO) on the embryonic development of two fish species, we examined transcriptome profiles using RNA-seq. The assembled contigs were 66,070 unigenes in olive flounder embryos and 76,498 unigenes in spotted seabass embryos. In the differential gene expression (DEG) profiles, olive flounder embryos showed different up- and down-regulated patterns than spotted seabass embryos in response to fresh IHCO (FIHCO) and weathered IHCO (WIHCO). In this work, we categorized DEG profiles into six pathways: ribosome, oxidative phosphorylation, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cardiac muscle contraction, validating the expression patterns of 13 DEGs using real-time quantitative RT-PCR. The expression of the CYP1A, CYP1B1, and CYP1C1 genes in spotted seabass embryos was higher than in olive flounder embryos, whereas genes related to cell processing, development, and the immune system showed the opposite trend. Orthologous gene cluster analysis showed that olive flounder embryos were sensitive (fold change of genes with cutoff P<0.05) to both FIHCO and WIHCO, but spotted seabass embryos exhibited higher sensitivity to WIHCO than FIHCO, indicating that species-specific differences are likely to be reflected in population levels after oil spills. Overall, our study provides new insight on the different embryonic susceptibilities of two marine fish species to FIHCO and WIHCO and a better understanding of the underlying molecular mechanisms via RNA-seq and DEGs.

Developmental toxicity in flounder embryos exposed to crude oils derived from different geographical regions.

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees.

Differential Toxicokinetics Determines the Sensitivity of Two Marine Embryonic Fish Exposed to Iranian Heavy Crude Oil.

Interspecific difference in the developmental toxicity of crude oil to embryonic fish allows the prediction of injury extent to a number of resident fish species in oil spill sites. This study clarifies the comparative developmental effects of Iranian heavy crude oil (IHCO) on the differences of biouptake and toxic sensitivity between embryonic spotted sea bass (Lateolabrax maculates) and olive flounder (Paralichthys olivaceus). From 24 h after exposure to IHCO, several morphological defects were observed in both species of embryonic fish, including pericardial edema, dorsal curvature of the trunk, developmental delay, and reduced finfolds. The severity of defects was greater in flounder compared to that in sea bass. While flounder embryos accumulated higher embryo PAH concentrations than sea bass, the former showed significantly lower levels of CYP1A expression. Although bioconcentration ratios were similar between the two species for some PAHs, phenanthrenes and dibenzothiophenes showed selectively higher bioconcentration ratios in flounder, suggesting that this species has a reduced metabolic capacity for these compounds. While consistent with a conserved cardiotoxic mechanism for petrogenic PAHs across diverse marine and freshwater species, these findings indicate that species-specific differences in toxicokinetics can be an important factor underlying species' sensitivity to crude oil.

Combined effects of Iranian heavy crude oil and bacterial challenge (Streptococcus iniae) on biotransformation and innate immune responses in rockfish (Sebastes schlegeli).

To clarify the effects of spilled crude oil on fish bacterial disease resistance, rockfish (Sebastes schlegeli) were exposed to Iranian Heavy crude oil (IHCO) and Streptomyces iniae in combination. Hepatic biotransformation enzymes (ethoxyresorufin O-de-ethylase, glutathione-S-transferase) and plasma biochemical parameters (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and glucose) in fish exposed to IHCO were not significantly different from those in unexposed fish. The level of biliary 1-OH-pyrene and cytochrome P4501A mRNA expression increased in a dose-dependent manner with IHCO exposure. The interferon stimulated gene 15, interleukin-1beta and cathepsin L were increased significantly in the liver in IHCO-exposed fish, but not dose-dependently, but the granulocyte colony stimulating factor was not related to IHCO exposure. The percentage mortality in fish following a single exposure to S. iniae was positively correlated with IHCO exposure concentration. We concluded that IHCO exposure exacerbates fish mortality following environmental bacterial infection.

Expression of three novel cytochrome P450 (CYP) and antioxidative genes from the polychaete, Perinereis nuntia exposed to water accommodated fraction (WAF) of Iranian crude oil and benzo[a]pyrene.

To report a novel CYP genes and to evaluate its potency as a biomarker for oil pollution, we cloned three CYP genes and measured their expression profiles under controlled lab conditions using real-time reverse transcription PCR (real-time RT-PCR) after exposure of the water accommodated fraction (WAF) of Iranian crude oil and benzo[α]pyrene (B[α]P) as a positive control. Of these, CYP432A1 (CYP3 clan) gene was significantly induced by B[α]P exposure, indicating that the CYP3 clan gene would play an important role in polycyclic aromatic hydrocarbon (PAH) metabolisms, particularly for B[α]P in this species. However, the Perinereis nuntia CYP431A1 mRNA, a CYP2 clan gene, was sensitively expressed to WAF exposure with other two CYP genes. As one of Phase II detoxification enzymes, the glutathione S-transferase (GST) genes also upregulated with other antioxidant genes (SOD and CAT), indicating that WAF-exposed P. nuntia was properly responding to this kind of chemical stress. Thus, three CYP genes from the polychaete, P. nuntia have a potential as a biomarker in monitoring of the marine sediment after an oil spill accident.

Acute toxic responses of the rockfish (Sebastes schlegeli) to Iranian heavy crude oil: feeding disrupts the biotransformation and innate immune systems.

To clarify the toxic effects of Iranian Heavy Crude Oil (IHCO) from the "Hebei spirit" oil spill, innate immune toxic effects defending on biotransformation pathway have been investigated on fish exposed to IHCO. Juvenile rockfish were exposed to IHCO in gelatin capsules by feeding. The effects on multiple fish biotransformation enzymes (Cytochrome P4501A and glutathione-S-transferase) and the expression level of the several immune response genes, including interleukin-1beta, granulocyte colony-stimulating factor and Cathepsin L, were measured in the liver, spleen and kidney. The tissue-specific expression patterns of these genes demonstrated that the highest expression levels of Cytochrome P4501A, glutathione-S-transferase, interleukin-1beta, granulocyte colony-stimulating factor, interferon stimulated gene 15 and Cathepsin L were found in the liver and that the TNF receptor was high in spleen. The oil-fed fish had significantly higher concentrations of biliary fluorescent metabolites and Cytochrome P4501A expression during the initial stage (12 ∼ 48 h after exposure) than those in the liver and kidney of the sham group. Similarly, the highest mRNA expression levels of interleukin-1beta and granulocyte colony-stimulating factor were detected in the liver at the early stages of exposure (12 h after exposure). Following exposure, the levels of interferon stimulated gene 15 and granulocyte colony-stimulating factor mRNA remained high at 120 h after exposure in the liver but the levels of interleukin-1beta and Cathepsin L gradually decreased to an expression level equal to or less than the sham group. Our data suggest that the innate immune and hepatodetoxification responses in oil-fed fish were induced at the initial stage of exposure to the IHCO at the same time but several immune-related genes decreased to less than that of the sham group after the initial stage of response. Therefore, immune disturbances in fish exposed to IHCO may allow the pathogens, including the infectious diseases, to more easily affect the oil exposed fish.

Geologically distinct crude oils cause a common cardiotoxicity syndrome in developing zebrafish.

Crude oils from different geological formations vary in composition, yet most crude oils contain a polycyclic aromatic hydrocarbon (PAH) fraction that would be expected to produce cardiotoxic effects in developing fish. To determine whether different crude oils or PAH compositions produce common or distinct effects, we used zebrafish embryos to directly compare two crude oils at different states of weathering. Iranian heavy crude oil (IHCO) spilled in the Yellow Sea following the 2007 Hebei Spirit accident was compared to the intensively studied Alaska North Slope crude oil (ANSCO) using two different exposure methods, water-accommodated fractions containing dispersed oil microdroplets and oiled gravel effluent. Overall, both crude oils produced a largely overlapping suite of defects, marked by the well-known effects of PAH exposure on cardiac function. Specific cardiotoxicity phenotypes were nearly identical between the two oils, including impacts on ventricular contractility and looping of the cardiac chambers. However, with increased weathering, tissue-specific patterns of aryl hydrocarbon receptor (AHR) activation in the heart changed, with myocardial AHR activation evident when alkyl-PAHs dominated the mixture. Our findings suggest that mechanisms of cardiotoxicity may shift from a predominantly AHR-independent mode during early weathering to a multiple pathway or synergistic mode with prolonged weathering and increased proportions of dissolved alkyl-PAHs. Despite continued need for comparisons of crude oils from different sources, the results here indicate that the body of knowledge already acquired from studies of ANSCO is directly relevant to understanding the impacts of other crude oil spills on the early life history stages of fish.

Evaluation of biomarker potential of cytochrome P450 1A (CYP1A) gene in the marine medaka, Oryzias melastigma exposed to water-accommodated fractions (WAFs) of Iranian crude oil.

CYP1A is involved in the metabolism of diverse chemicals, including polycyclic aromatic hydrocarbons and alkylated-PAHs, as a first line of detoxification mechanism. First, we identified and characterized the CYP1A gene from the marine medaka, Oryzias melastigma. O. melastigma CYP1A (Om-CYP1A) showed a high similarity of motifs/domains compared to those of vertebrates in their amino acid sequences. To check whether the Om-CYP1A would be inducible, we tested two strong CYP1A inducers, ß-naphthoflavone (ß-NF) and benzo[α]pyrene (B[α]P), and observed concentration-dependent transient expression on transcripts of Om-CYP1A for 96 h over a wide range of concentrations. Om-CYP1A mRNA level was significantly increased in exposure to different concentrations of ß-NF and B[α]P, and its expression was highly transcribed within 12 h upon the exposure to low concentrations of both chemicals. Inducible transcript profiles revealed that Om-CYP1A would be associated with the toxicant metabolism via AhREs/DREs/XREs in its promoter region. To uncover the effects of the water-accommodated fraction (WAF) of crude oil on transcripts of Om-CYP1A, we measured mRNA expression of Om-CYP1A towards different concentrations of WAF for 24h. As a result, WAF exposure significantly increased Om-CYP1A transcripts at all concentrations as well as during time-course experiments for 96 h. In this paper, we demonstrated that WAF would trigger up-regulation of the CYP1A gene that would be associated with the initiation of the cellular defense systems. This finding provides a better understanding of the molecular mechanism of cellular protection particularly that involved in the WAF-mediated cellular response in O. melastigma.

Petroleum hydrocarbon contaminations in the intertidal seawater after the Hebei Spirit oil spill--effect of tidal cycle on the TPH concentrations and the chromatographic characterization of seawater extracts.

In December 2007, the oil tanker Hebei Spirit released approximately 12,547,000 L of crude oil off the west coast of Korea, impacting more than 375 km of coastline. The seawater TPH concentrations immediately after the spill ranged from 1.5 to 7310 µg L⁻¹, with an average of 732 µg L⁻¹. The concentrations appeared to decrease drastically to 2.0-224 µg L⁻¹ in one month after the spill. The TPH concentrations in seawater fluctuated with time thereafter because of the remobilization of oil by continuing shoreline cleanup activities and subsequent wave/tidal actions. Seawater TPH concentrations were much higher during high tide than during low tide due to the resuspension of stranded oil. The variation of TPH levels in seawater also matched the spring-neap tidal cycle in the study areas for the first three weeks of the study. Comparisons of the gas chromatograms of the seawater with the water accommodated fraction and the cargo oil indicated that seawater samples were contaminated mainly by the dispersed droplets of spilled oil. One year of monitoring revealed that the oil content in seawater had clearly decreased at most sites, although some regional fluctuations of oil contamination were noted until June 2008.

Biomarker responses in pelagic and benthic fish over 1 year following the Hebei Spirit oil spill (Taean, Korea).

After the Hebei Spirit oil spill incident (7th December, 2007) in the west coast of Korea, contamination of biliary PAH metabolite and hepatic biomarkers in a pelagic and a benthic fish was monitored for 1 year. Concentrations of 16 PAHs and alkylated PAHs in fish muscle were highest (22.0 ng/g d.w. for 16 PAHs and 284 ng/g d.w. for alkylated PAHs) at 5 days after the spill and then decreased rapidly to background levels at 11 months after the spill. Fish from the oiled site had elevated biliary PAH metabolite concentrations immediately after the spill; these declined steadily in both species, but were still above reference site concentrations 2 months after the spill. Oiled-site fish showed hepatic CYP 1A induction whose trend closely followed those of biliary PAH metabolite concentrations, implying continuous exposure to PAHs. Brain acetylcholinesterase activity was not related to oil exposure.

Hebei Spirit oil spill monitored on site by fluorometric detection of residual oil in coastal waters off Taean, Korea.

The spatiotemporal distributions of dissolved and/or dispersed oil in seawater and pore water were monitored on site by fluorometric detection method after the Hebei Spirit oil spill. The oil concentrations in intertidal seawater, 15 days after the spill, were as high as 16,600 microg/L and appeared to decrease below the Korean marine water quality standard of 10 microg/L at most sites 10 months after the spill. Fluorometric detection of oil in pore water was introduced to eliminate the effects of grain size for the quantification of oil in sediments and to better explain spatial and temporal distribution of oil pollution at sandy beaches. The fluorescence detection method was compared with the conventional laboratory technique of total petroleum hydrocarbon analysis using gas chromatography. The method of fluorescence detection of oil was capable of generating results much faster and more cost-effectively than the traditional GC technique.

Biochemical changes in rockfish, Sebastes schlegeli, exposed to dispersed crude oil.

This paper describes the response of the ovoviviparous rockfish, Sebastes schlegeli, to hydrocarbons in the water-accommodated fraction (WAF) of crude oil, in the presence or absence of oil dispersants. Concentrations of cytochrome P-450 1A (CYP1A) and levels of its catalytic activity ethoxyresorufin O-de-ethylase (EROD) in rockfish exposed to WAF at concentrations of 0.1% and 1% were significantly increased by the addition of a dispersant, Corexit 9500 after 48 h exposure. After 72 h exposure, the levels of CYP1A and EROD activity were significantly increased in 0.1% and 0.01% chemically enhanced WAF (CEWAF) (Corexit 9500 and Hiclean II dispersant). Bile samples from fish exposed to WAF alone had low concentrations of hydrocarbon metabolites, exemplified by 1-hydroxypyrene. After 72 h exposure, hydrocarbon metabolites in bile from fish exposed to WAF in the presence of either Corexit 9500 or Hiclean II were significantly higher compared with fish exposed to WAF alone or control fish. These experiments confirm that the use of oil dispersants will increase the exposure of ovoviviparous fish to hydrocarbons in oil.