Exposure to hull cleaning wastewater induces mortality through oxidative stress and cholinergic disturbance in the marine polychaete Perinereis aibuhitensis.

While wastewater and paint particles discharged from the in-water cleaning process of ship hulls are consistently released into benthic ecosystems, their hazardous effects on non-target animals remain largely unclear. In this study, we provide evidence on acute harmful effects of hull cleaning wastewater in marine polychaete Perinereis aibuhitensis by analyzing physiological and biochemical parameters such as survival, burrowing activity, and oxidative status. Raw wastewater samples were collected during ship hull cleaning processes in the field. Two wastewater samples for the exposure experiment were prepared in the laboratory: 1) mechanically filtered in the in-water cleaning system (MF) and 2) additionally filtered with a 0.45 µm filter in the laboratory (LF). These wastewater samples contained high concentrations of metals (zinc and copper) and metal-based booster biocides (copper pyrithione and zinc pyrithione) compared to those analyzed in seawater. Polycheates were exposed to different concentrations of the two wastewater samples for 96 h. Higher mortality was observed in response to MF compared to LF-exposed polychaetes. Both wastewater samples dose-dependently decreased burrowing activity and AChE activity. Drastic oxidative stress was observed in response to the two wastewater samples. MDA levels were significantly increased by MF and LF samples. Significant GSH depletion was observed with MF exposure, while increased and decreased GSH contents were observed in LF-exposed polychaetes. Enzymatic activities of antioxidant components, catalase, superoxide dismutase, and glutathione S-transferase were significantly modulated by both wastewater samples. These results indicate that even filtered hull cleaning wastewater can have deleterious effects on the health status of polychaetes.

Hull-cleaning wastewater poses serious acute and chronic toxicity to a marine mysid-A multigenerational study.

We conducted a comprehensive assessment involving acute effects on 96-hour survival and biochemical parameters, as well as chronic effects on growth and reproduction spanning three generations of the marine mysid Neomysis awatschensis exposed to filtered wastewater to evaluate the potential impact of ship hull-cleaning wastewater on crustaceans. The analyzed wastewater exhibited elevated concentrations of metals, specifically zinc (Zn) and copper (Cu) and metal-based antifoulants, i.e., Cu pyrithoine (CuPT) and Zn pyrithoine (ZnPT). The results revealed dose-dependent reductions in survival rates, accompanied by a notable increase in oxidative stress, in response to the sublethal values of two wastewater samples: 1) mechanically filtered using the cleaning system (MF) and 2) additionally filtered in the laboratory (LF) for 96 h. Mysids exposed to MF displayed higher mortality than those exposed to LF. Furthermore, mysids subjected to continuous exposure of 0.001% LF across three generations exhibited significant inhibition of the feeding rate, more pronounced growth retardation along with an extended intermolt duration, and a diminished rate of reproduction compared to the control. A noteworthy inhibition of the feeding rate and growth was observed in the first generation exposed only to the LF sample. However, although the reproduction rate was not significantly affected. Collectively, these findings underscore the potential harm posed by sublethal concentrations of wastewater to the health of mysid populations under consistent exposure.

Detrimental effects of hull cleaning wastewater on oxidative status, life cycle parameters, and population growth of the monogonont rotifer Brachionus manjavacas.

While wastewater discharged from in-water cleaning process of ship hulls on rotifer consistently released into aquatic ecosystem, its detrimental effects on non-target animals are largely unclear. In this study, we provide evidence on detrimental effects of hull cleaning wastewater in the monogonont rotifer Brachionus manjavacas by analyzing biochemical and physiological parameters in its oxidative status, survival, lifespan, growth, fecundity, and population. The wastewater contained high concentrations of metals (Zn and Cu) and metal-based antifoulants (CuPT and ZnPT). Significant oxidative stress was observed in response to two wastewater samples [1) raw wastewater (RW) and 2) mechanical filtrated in the cleaning system (MF)]. Higher detrimental effects in survival, lifespan, fecundity, and population growth for 10 days were measured in the RW-exposed rotifers than those results analyzed in the MF-exposed rotifers. Two growth parameters, lorica length and width were also significantly modulated by both wastewater samples. These results indicate that even filtered hull cleaning wastewater would have deleterious effects on the maintenance of the rotifer population when they exposed constantly.

Metals and suspended solids in the effluents from in-water hull cleaning by remotely operated vehicle (ROV): Concentrations and release rates into the marine environment.

An increase in the use of ROVs for in-water hull cleaning (IWC) has led to the need to understand the risks to the marine environment posed by the release of IWC effluents. The primary objective of this research is to investigate the characteristics of wastewater generated during IWC, specifically concerning suspended solids (SS) and metal concentrations, and their release rates and total load to the environment. The IWC effluents contain substantial amounts of SS and metals, with Cu and Zn being the most prevalent. These metals are predominantly associated with fine antifouling paint particles, posing a potential risk of secondary pollution upon release into the marine environment. While the treatment systems demonstrated effectiveness in reducing SS and particulate metals, achieving complete removal of dissolved and particulate metals below ambient levels proved to be challenging. To mitigate environmental risks, this study proposes, based on the particle size analysis, the implementation of multistage filtration systems with an optimal filtration pore size for the effluent treatment. In conclusion, we highlight the potential environmental risks of IWC activities. As most metals have a strong affinity towards particles in wastewater, effective removal of particles is essential to alleviate environmental stress at IWC sites.

The effects of environmental Microplastic on wharf roach (Ligia exotica): A Multi-Omics approach.

This is the first report to evaluate the potential effects of microplastics (MPs) on wild wharf roaches (Ligia exotica) in a shoreline habitant. L. exotica is an important plastic detritus consumer in coastal area. A survey was conducted from May to June in the years 2019 and 2020 in two South Korean nearshore sites: Nae-do (as MPs-uncontaminated) and Maemul-do (as MPs-contaminated). MPs (>20 µm in size) were detected highly in gastrointestinal tracts of the L. exotica from Maemul-do, at an average level of 50.56 particles/individual. They were detected in much lower levels in the L. exotica from Nae-do. at an average rate of 1.00 particles/individual. The polymer type and shape were dominated by expanded polystyrene (EPS, 93%) and fragment (99.9%) in L. exotica from Maemul-do. Especially, Hexabromocyclododecanes, brominated flame retardants added to EPS, have been detected highly in L. exotica from Maemul-do (630.86 ± 587.21 ng/g l. w.) than those of Nae-do (detection limit: 10.5 ng/g l. w). Genome-wide transcriptome profiling revealed altered expression of genes associated with fatty acid metabolic processes, the innate-immune response-activating system and vesicle cytoskeletal trafficking in L. exotica from Maemul-do. The activation of the p53 signaling pathway (which is related to proteasome, ER regulation and cell morphogenesis) is likely to be involved in the EPS-uptake of wild L. exotica. Four neurosteroids were also detected in head tissue, and cortisol and progesterone concentrations differed significantly in L. exotica from Maemul-do. Our findings also suggest that resident plastic detritus consumer might be a useful indicator organism for evaluating pollution and potential effects of environmental microplastics.

Toxic effects of the wastewater produced by underwater hull cleaning equipment on the copepod Tigriopus japonicus.

Unmanaged disposal of wastewater produced by underwater hull cleaning equipment (WHCE) is suspected to induce toxic effects to marine organisms because wastewater contains several anti-fouling compounds. To investigate the effects of WHCE on marine copepod, we examined the toxicity on life parameters (e.g. mortality, development, and fecundity) and gene expression changes of Tigriopus japonicus as model organism. Significant mortality and developmental time changes were observed in response to wastewater. No significant differences in fecundity were observed. Transcriptional profiling with differentially expressed genes from WHCE exposed T. japonicus showed WHCE may induce genotoxicity associated genes and pathways. In addition, potentially neurotoxic effects were evident following exposure to WHCE. The findings suggest that wastewater released during hull cleaning should be managed to reduce physiological and molecular deleterious effects in marine organisms.

Chemical hazard of robotic hull in-water cleaning discharge on coastal embryonic fish.

In-water cleaning (IWC) involves the removal of biofilms and foulants from the hull of a ship using brush or water jet. During IWC, several factors associated with the harmful chemical contaminants release to the marine environment, which can create "hotspots" of chemical contamination in coastal areas. To elucidate the potential toxic effects of IWC discharge, we investigated developmental toxicity in embryonic flounder, which are sensitive life stage to chemical exposure. Zinc and copper were the dominant metals, while zinc pyrithione was the most abundant biocide associated with IWC discharge in two remotely operated IWC. Discharge from IWC carried by both remotely operated vehicles (ROVs) produced developmental malformations including pericardial edema, spinal curvature, and tail-fin defects. In an analyses of differential gene expression profiles (fold-change of genes with a cutoff < 0.05) as assessed by high-throughput RNA sequencing, genes associated with muscle development were commonly and significantly changed. The gene ontology (GO) of embryos exposed to IWC discharge from ROV A activities highly enriched muscle and heart development, while cell signaling and transport were evident in embryos exposed to IWC discharge of ROV B. We analyzed the gene network by significant GO terms. In the network, TTN, MYOM1, CASP3, and CDH2 genes appeared to be key regulators of the toxic effects on muscle development. In embryos exposed to ROV B discharge, HSPG2, VEGFA, and TNF genes related to the nervous system pathway were affected. These results shed light on the potential impacts of muscle and nervous system development in non-target coastal organisms exposed to contaminants found in IWC discharge.

The single and combined effects of mercury and polystyrene plastic beads on antioxidant-related systems in the brackish water flea: toxicological interaction depending on mercury species and plastic bead size.

Plastics are considered as a major threat to marine environments owing their high usage, persistence, and negative effects on aquatic organisms. Although they often exist as mixtures in combination with other pollutants (e.g., mercury (Hg)) in aquatic ecosystems, the combined effects of plastics and ambient pollutants remain unclear. Therefore, in the present study, we investigated the toxicological interactions between Hg and plastics using two Hg species (HgCl2 and MeHgCl) and different-sized polystyrene (PS) beads (diameter: 0.05, 0.5, and 6-µm) in the brackish water flea Diaphanosoma celebensis. The single and combined effects of Hg and PS beads on mortality were investigated, and changes in the antioxidant system and lipid peroxidation were further analyzed. After 48-h exposure to single Hg, HgCl2 induced a higher mortality rate than MeHgCl. The combined exposure test showed that 0.05-µm PS beads can enhance the toxicity of both the Hg species. The expression of GST-mu, glutathione S-transferease (GST) activity and malondialdehyde (MDA) content increased significantly after exposure to Hg alone (HgCl2 or MeHgCl) exposure. Combined exposure with PS beads modulated the effects of Hg on the antioxidant system depending on bead size and the Hg species. In particular, the 0.05-µm beads significantly increased the expression level of GST-mu, GST activity and MDA content, regardless of Hg species. These findings suggest that toxicological interactions between Hg and PS beads depend on the type of Hg species and the size of PS beads; nano-sized 0.05-µm PS beads can induce synergistic toxicity with Hg.

Phenotypic toxicity, oxidative response, and transcriptomic deregulation of the rotifer Brachionus plicatilis exposed to a toxic cocktail of tire-wear particle leachate.

Tire-wear particles (TWPs) are potential source of microplastic (MP) pollution in marine environments. Although the hazardous effects of MPs on marine biota have received considerable attention, the toxicity of TWPs and associated leachates remain poorly understood. Here, to assess the toxicity of TWP leachate and the underlying mechanisms of toxicity, the phenotypic and transcriptomic responses of the rotifer Brachionus plicatilis were assessed with chemistry analysis of a TWP leachate. Although acute toxicity was induced, and a variety of metals and polyaromatic hydrocarbons were detected in the leachate, levels were below the threshold for acute toxicity. The results of particle analysis suggest that the acute toxicity observed in our study is the result of a toxic cocktail of micro- and/or nano-sized TWPs and other additives in TWP leachate. The adverse effects of TWP leachate were associated with differential expression of genes related to cellular processes, stress response, and impaired metabolism, with further oxidative stress responses. Our results imply that TWPs pose a greater threat to marine biota than other plastic particles as they constitute a major source of nano- and microplastics that have synergistic effects with the additives contained in TWP leachate.

Comparative toxicity study of waterborne two booster biocides (CuPT and ZnPT) on embryonic flounder (Paralichthys olivaceus).

A new generation of booster biocides that include metal pyrithiones (PTs) such as copper pyrithione (CuPT) and zinc pyrithione (ZnPT) are being used as tributyltin alternatives. In the marine environment, ZnPT can easily transchelate Cu to form CuPT, and the environmental fate and persistence of these two metal pyrithiones are closely related. Although some data on the toxicity of biocides on marine fish are available, little is known about their toxicity and toxic pathway. We thus compared the toxic effects of CuPT and ZnPT on embryonic olive flounder (Paralichthys olivaceus) by investigating their adverse effects based on developmental morphogenesis and transcriptional variation. In our study, the toxic potency of CuPT was greater with respect to developmental malformation and mortality than ZnPT. Consistent with the developmental effects, the expression of genes related to tail fin malformation (including plod2, furin, and wnt3a) was higher in embryonic flounder exposed to CuPT than in those exposed to ZnPT. Genes related to muscle and nervous system development exhibited significant changes on differential gene expression profiles using RNA sequencing (cutoff value P < 0.05). Gene ontology analysis of embryos exposed to CuPT revealed affected cellular respiration and kidney development, whereas genes associated with cell development, nervous system development and heart development showed significant variation in embryonic flounder exposed to ZnPT. Overall, our study clarifies the common and unique developmental toxic effects of CuPT and ZnPT through transcriptomic analyses in embryonic flounder.

Seawater contamination associated with in-water cleaning of ship hulls and the potential risk to the marine environment.

In-water cleaning can clear-off foulants from ship hulls to prevent transportation of non-indigenous species and reduce hull friction and consequent fuel use. However, during cleaning, antifouling paint residues containing toxic substances can be released into the environment. To understand the potential risks of in-water hull cleaning, cleaning effluents were collected and analyzed for total suspended solid (TSS), particle size distribution, and metal concentrations. TSS concentrations were 97.3-249 mg/L, corresponding to release rates of 12.9-37.5 g/m2 from the hull surface. Particles with sizes of ≥8 µm contributed 75-94% of the TSS. Average Cu and Zn concentrations in the effluents were 209 µg/L and 1510 µg/L, respectively, which were used for risk assessment in two port scenarios. Although the risks vary with the scale of the hull cleaning and the ports, in-water cleaning poses clear risks to marine environments, unless the effluents are recovered or treated before being released.

Sediment quality assessment combining chemical and biological (non)target analysis.

Aquatic sediments act as a storage for diverse mixtures of organic and inorganic contaminants. Nevertheless, most evaluations of contaminated sediments have been limited to the assessment of concentrations of target compounds and lethal effects on some test species. To identify the organic contaminants causing sub-lethal effects of contaminated sediment, this study combined chemical and biological (non)target analysis involving comprehensive two-dimensional gas chromatography coupled with a time-of-flight Mass Spectrometer (GCxGC/ToF-MS) analysis, embryonic malformation and high-throughput sequencing (RNA-seq) analysis on developing flounder. Polycyclic aromatic hydrocarbons were more abundant in the sediment extract of Yeongil Bay (SEY), while Jinhae Bay (SEJ) was contaminated with a large amount of unidentified chemicals. The unidentified chemicals of SEJ included branched alkanes, oxygenated cycloalkanes, heterogeneous hydrocarbons, and other unknown compounds. Percentage of pericardial edema was the highest in embryonic flounder exposed to SEY. Consistent with the morphogenesis results, the expression level of genes related to heart formation including the nkx2.5 and robo1 was greater in embryonic flounder exposed to SEY. In the analyses of differential gene expression profiles (cutoff P < 0.05), by RNA-seq, embryos exposed to SEJ showed changes related to cell differentiation, cell part morphogenesis, neurogenesis, and neuron development. Genes related to neurogenesis and positive regulation of molecular functions variated significantly in embryos exposed to SEY. These results demonstrated the advantages of combining target and non-target analysis to accurately evaluate the major chemical groups causing sediment toxicity. Therefore, this work provided a useful approach to tracking and revealing the causes of toxic effects and identifying potential toxic mechanisms.

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.

Effects of polystyrene in the brackish water flea Diaphanosoma celebensis: Size-dependent acute toxicity, ingestion, egestion, and antioxidant response.

Owing to the increasing usage of plastics, their debris is continuously deposited in marine environments, resulting in deleterious effects on aquatic organisms. Although it is known that microplastics disturb the cellular redox status, knowledge of molecular in marine cladocerans is still lacking. In the present study, we investigated the acute toxicity of different-sized polystyrene (PS) beads (0.05, 0.5, and 6-µm diameter), ingestion and egestion patterns, their distribution in the tissues, and their effects on the antioxidant systems in the brackish water flea Diaphanosoma celebensis. All different-sized PS beads showed no mortality at the concentrations used in this study. After 48 h of exposure to PS beads of different sizes, all microbeads were retained in the digestive tract, but the retention time varied according to the bead size. In particular, the group that was exposed to 0.05-µm beads showed widely distributed fluorescence (e.g., in the embryo, and probably in lipid droplets as well as the digestive tract). The transcriptional level and enzyme activities of antioxidants were modulated depending on the size of the PS beads, and lipid peroxidation was induced in groups exposed to 0.05 and 0.5-µm beads. These findings suggest that the size of PS beads is an important factor for cellular toxicity, and can induce size-dependent oxidative stress in this species. This study provides a better understanding of the molecular modes of action of microplastics in marine zooplankton.

Characterization of hazards and environmental risks of wastewater effluents from ship hull cleaning by hydroblasting.

Hydroblasting is used to remove biofouling and exhausted antifouling paints from ship hulls. Effluents generated from this process contain paint particles, metals, and booster biocides that may have toxic effects on organisms. To understand the potential risks of effluent discharge on marine environments, we analyzed the concentrations of metals in effluents collected during the dry-dock cleaning of ship hulls by hydroblasting. Copper and zinc were the principal metals, with concentrations ranging from 1440 to 9110 µg/L and 1800 to 22,600 µg/L, respectively. These concentrations are sufficiently high to cause harmful effects to most marine organisms. Model predictions suggested that the effluent discharge from hydroblasting posed risks to the wider marine environment of a hull-cleaning site, depending on the scale of the hull-cleaning operations and the size of the receiving environment, as well as various hydrodynamic factors. These effluents are inevitably hazardous, and their environmental release should be managed and regulated on the basis of site-specific risk assessments.

Alkylated phenanthrene distributions in black rockfish (Sebastes schlegelii) and biotransformation into hydroxylated metabolites after intragastric administration.

Marine organisms such as fish are at risk of exposure to petrogenic polycyclic aromatic hydrocarbons (PAHs) released in oil spills. PAH toxicities are affected by the rates of PAH biotransformation and elimination in fish tissues, but little information on these rates is available. In this study, the biotransformation and tissue distribution of methylated phenanthrenes-typical petrogenic PAHs found after oil spills-in black rockfish (Sebastes schlegelii) were investigated. Two groups of fish were used. Each fish in one group was given a single intragastric dose of 3-methylphenanthrene, and each fish in the other group was given a single intragastric dose of 3,6-dimethylphenanthrene. The fish were allowed to recover in purified sea water for 196 h. Methylated phenanthrenes were detected in only blood and liver for 24 h after dosing, but the concentrations decreased over time and > 98% had been eliminated by the end of the study. Four mono-hydroxylated metabolites of 3,6-dimethylphenanthrene and six mono-hydroxylated metabolites of 3-methylphenanthrene were tentatively identified for the first time from tandem mass spectrometry analyses of fish bile. The concentrations of these metabolites in bile remained constant for 192 h, suggesting that the metabolites could be used as biomarkers of rockfish exposure to petrogenic PAHs.

Development and Evaluation of Olive Flounder cyp1a1-Luciferase Assay for Effective Detection of CYP1A-Inducing Contaminants in Coastal Sediments.

Flounders have been widely used as indicator species for monitoring the benthic environment of marine coastal regions owing to their habitat and feeding preferences in or on sandy sediments. Here, a single-step, sensitive, specific, and simple luciferase assay was developed, using the olive flounder cyp1a1 gene, for effective detection of CYP1A-inducing contaminants in coastal sediments. The developed cyp1a1-luciferase assay was highly sensitive to the widely used CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (B[a]P), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). In the case of TCDD, significant dose-dependent increases in luciferase activity (0.3-300 ng/L) were detected. The assay was more sensitive to PCB 126 than to B[a]P. The assay also involved the highly sensitive expression of luciferase to extracted mixtures of PCBs and polycyclic aromatic hydrocarbons (PAHs) collected from coastal sediments. PCBs were more capable of cyp1a1 induction in the assay system at small doses than PAHs in environmental samples. Using the cyp1a1-luciferase assay along with water or sediment chemistry will certainly aid in diagnosing CYP1A-inducing contaminants in coastal environments.

Is hull cleaning wastewater a potential source of developmental toxicity on coastal non-target organisms?

Chemical contaminants can be discharged by vessel hull cleaning processes, such as scraping, jet spraying, and painting, all of which produce readily transportable contaminants into the marine environment, where they are referred to as 'hotspots' of contamination in coastal areas. However, many countries have not yet established effective evaluation methods for disposal of waste mixtures or management guidelines for areas of hull cleaning. To define the toxic effects of wastewater from vessel hull cleaning in dry docks on resident non-target organisms, we investigated the chemical concentrations and developmental toxicity on embryonic flounder, which is an organism sensitive to chemical contamination. In this study, the dominant inorganic metal discharged was zinc when cleaning Ship A (300 tons) and copper for Ship B (5,000 tons). The wastewater from high-pressure water blasting (WHPB) of Ship A (300 tons) and Ship B (5,000 tons) produced a largely overlapping suite of developmental malformations including pericardial edema, spinal curvature, and tail fin defects. Forty-eight hours after exposure, the frequency percentage of malformation began to increase in embryos exposed to a 500-fold dilution of WHPB from Ships A and B. We performed transcriptome sequencing to characterize the toxicological developmental effects of WHPB exposure at the molecular level. The results of the analysis revealed significantly altered expression of genes associated with muscle cell differentiation, actin-mediated cell contraction, and nervous system development (cutoff P < 0.01) in embryonic flounder exposed to high-pressure cleaning effluent from Ship A. Genes associated with chromatin remodeling, cell cycling, and insulin receptor signaling pathways were significantly altered in embryonic flounder exposed to WHPB of Ship B (cutoff P < 0.01). These findings provide a greater understanding of the developmental toxicity and potential effects of WHPB effluent on coastal embryonic fish. Furthermore, our results could inform WHPB effluent management practices to reduce impacts on non-target coastal organisms.

Formation and distribution of phenanthrene and its metabolites (monohydroxy-phenanthrenes) in Korean rockfish (Sebastes schlegelii).

This study investigated the tissue distribution of phenanthrene (PHE) and the formation of monohydroxy-phenanthrene (OH-PHE) metabolites in Korean rockfish (Sebastes schlegelii). PHE was intragastrically administered to two groups of rockfish. The first group was exposed to PHE at a low dose (10 mg/kg body weight) and the second group was exposed at a high dose (30 mg/kg body weight). The rockfish were analyzed and the levels of PHE were higher in the liver, followed by muscle, and then bile. PHE concentrations in the liver, muscle, and bile were 1.4-26, 0.10-2.01, and not detected (ND)-0.13 µg/g wet weight, respectively. All five monohydroxylated PHE metabolites (1-OH-PHE, 2-OH-PHE, 3-OH-PHE, 4-OH-PHE, and 9-OH-PHE) were detected only in bile. Among these OH-PHE metabolites, 3-OH-PHE was found at the highest concentration from all fish bile samples in both PHE exposure groups, indicating that regioselective OH-PHE formation occurs in rockfish and 3-OH PHE could be a good biomarker of exposure of Korean rockfish to PHE. Suspect screening analysis of the rockfish bile was performed by LC-QTOF/MS, and the formation of two OH-PHE-DNA adducts (thymine-OH-PHE and cytosine-OH-PHE) were identified in the bile sample collected 6 h after rockfish were exposed to the high PHE dose, indicating that OH-PHE metabolites may be toxic to fish. This is the first report on the formation characteristics of OH-PHE metabolites in rockfish and their use as biomarkers of exposure of rockfish to parent PHE.

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.