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.

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.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.

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.

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.

Initial impacts of the Hebei Spirit oil spill on the sandy beach macrobenthic community west coast of Korea.

Petroleum hydrocarbon contamination and macrobenthos in the sandy tidal flats of Taean were monitored for 1 year to assess the impacts of Hebei Spirit oil on the macrobenthic community. A total of 207 macrobenthic fauna was collected, and the mean density and biomass of macrobenthic fauna continued to decrease until 12 months after the oil spill, but macrobenthic density at the most heavily affected sites increased by about twofold. In January 2008, the dominant species occurred at very low densities in strongly affected sites. The macrobenthic communities differed between oil-affected and unaffected sites. In particular, differences in community structure at Mallipo beach were larger than those at Shinduri. We suggest that long-term monitoring is needed to assess the specific effects of oil pollution on the sandy intertidal macrobenthic community.

Mesocosm study on weathering characteristics of Iranian Heavy crude oil with and without dispersants.

The environmental fate of Iranian Heavy crude oil (IHC) with and without an added oil spill dispersant (OSD) has been studied using a 1000 kL capacity in situ mesocosm. Physical weathering and chemical composition changes of the oil were monitored for 77 days. Compound-specific effects of the OSD could be observed as changes over time in the content of the total petroleum hydrocarbon (TPH), unresolved complex mixture (UCM), alkanes, polycyclic aromatic hydrocarbons (PAHs), hopanes and steranes in the oil. As oil weathers, most hydrocarbons showed a rapid decreasing phase followed by a slowdown and stabilization. Recalcitrant biomarkers, however, showed a different trend. An increase in hydrocarbon contents in the form of UCM occurred after OSD treatment. The enhanced solubility of the low molecular weight PAHs by the OSD decreased the half-life of the alkylated PAHs in the OD. After 77 days of exposure at the sea surface, both the oils with and without the OSD exhibited moderate weathering. Most of the source diagnostic indices maintained their source information, and the weathering indices indicated that evaporation, dissolution, and dispersion were the major weathering processes. The mass balance of the weathered oil was calculated using laboratory and mesocosm data and the results demonstrate the importance of using a mesocosm for the production of environmentally realistic data.

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.

Stronger impact of dispersant plus crude oil on natural plankton assemblages in short-term marine mesocosms.

To assess the effects of crude oil and dispersant on marine planktonic ecosystems, analyses were performed in 1000-L mesocosm over a period of nine days. Triplicate experiments were conducted for two different treatments, namely, addition of crude oil alone and oil plus dispersant. In the mesocosm with oil plus dispersant, high concentrations of total petroleum hydrocarbon (TPH) were soon found in the bottom layer. In addition, most planktonic communities responded drastically to the presence of dispersant acting to disperse TPH: total bacterial abundances increased for the first two days and then decreased rapidly for the remainder of the experiment. The abundance of heterotrophic flagellates increased rapidly in association with the increase in bacterial cells. The abundance of phytoplankton and zooplankton communities decreased clearly within two days. Time-delayed relationship also revealed that the TPH concentration had a significant negative relationship with phyto- and zooplankton communities within two days. However, most planktonic communities were affected less adversely in the mesocosms treated with crude oil alone than in those treated with both crude oil and dispersant. The present results demonstrate that the planktonic ecosystem was damaged more severely by the introduction of dispersant than by the harmful effects of crude oil itself. Therefore, caution should be taken when considering the direct application of dispersant in natural environments, even though it has the advantage of rapidly removing crude oil.

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.