Reductive Transformation of Hexavalent Chromium in Ice Decreases Chromium Toxicity in Aquatic Animals.

In this study, the toxicity of hexavalent chromium [Cr(VI)] reduced by citric acid in ice was measured using representative aquatic model invertebrates (i.e., rotifer, water flea, amphipod, and polychaete) and a vertebrate (zebrafish) by analyzing short- and/or long-term endpoints that are frequently applied to each animal. Cr(VI) reduction in the presence of citric acid was markedly enhanced in the ice phase compared to that in an aqueous solution through the freeze concentration effect. The highly concentrated Cr(VI) and citric acid in ice grain boundaries were also confirmed using in situ cryogenic confocal Raman spectroscopy. Overall, exposure to Cr(VI) resulted in higher acute and/or chronic effects on aquatic animals, such as drastic mortality, growth inhibition, and decrease in offspring number, whereas the animals were increasingly tolerant to Cr(VI) that was reduced in the ice phase. Sublethal concentrations of Cr(VI) significantly decreased the antioxidant capacity in the aquatic animals. However, when the same concentrations of Cr(VI) were reduced in ice, these treatments showed no modulation or increase in the antioxidant defense system. Taken together, our results suggest that Cr(VI) reduction into Cr(III) was successfully achieved in ice and that this methodology can decrease the actual toxicity of Cr(VI) in aquatic animals.

Physiological and molecular responses of the Antarctic harpacticoid copepod Tigriopus kingsejongensis to salinity fluctuations - A multigenerational study.

Since Antarctica and the surrounding Southern Ocean are facing global climate change, biota inhabiting those coastal regions is now challenged by environmental fluctuations including coastal freshening. In this study, the effects of salinity range of 0-75 (practical salinity unit, PSU) on the Antarctic harpacticoid copepod Tigriopus kingsejongensis was investigated by measurement of 96 h survival rate, lifespan, and sex ratio with further analysis of multigenerational growth parameters and mRNA expressions under salinity of 15-45. Different stages of the copepods (i.e., nauplius, male, and female) generally showed tolerance to hypo- and hypersalinity, wherein female copepods were more tolerant than males when exposed to salinity fluctuations. Lifespan was significantly shortened by hypo- and hypersalinity compared to control salinity (34), but there was no significant difference in the sex ratio between salinity treatments. Multigenerational experiments across five generations revealed that exposure to salinities of 15 and 45 reduced body length compared to that in control salinity and the first generation of each salinity group. Our results provide evidence regarding T. kingsejongensis on their preferred salinity ranges, physiological limit to salinity fluctuations, and population dynamics in future salinity.

Validation of Direct Boiling Method for Simple and Efficient Genomic DNA Extraction and PCR-based Macroalgal Species Determination.

A rapid, simple, and cost-effective total DNA extraction method referred to as a direct boiling method for PCR-based algal species determination was validated using representative species of green, brown, and red algae. Each sample was briefly minced in two buffers, Tris-EDTA (TE) or PCR buffer, and transferred to a heat block for boiling at 98°C for 5 min. No detergent was used in this experiment. The entire DNA isolation procedure was completed within 10 min. After brief centrifugation, the supernatant was directly used as a template for PCR. As a result, all genomic DNA markers were successfully amplified and sequenced from each algal taxon. Regardless of DNA quality, the direct boiling method is very suitable to identify unknown species of algae from a large amount of samples in a limited time and can be applied broadly to routine seasonal or annual monitoring of algae. DNA from an Undaria pinnatifida sporophyte, however, was not successfully extracted by this direct boiling method, probably due to a high concentration of polysaccharides.

Constant and intermittent hypoxia modulates immunity, oxidative status, and blood components of red seabream and increases its susceptibility to the acute toxicity of red tide dinoflagellate.

Hypoxia is an increasing threat to aquatic ecosystems and its impact on economically and ecologically important marine fish species needs to be studied. Especially, the consequences of hypoxia when occurring along with harmful algal blooms (HABs) are currently not well documented. In this study, we investigated the effect of constant and intermittent (daily and weekly) hypoxia on respiration, immunity, hematological parameters, and oxidative status of red seabream for 2, 4, and 6 weeks. Under constant and daily intermittent hypoxia, respiration rate significantly increased in 2 weeks compared to the control. Constant and daily intermittent hypoxia caused significant decreases in the activity of alternative complement pathway, lysozyme, and the level of total immunoglobulin (Ig), as well as significant increases in the concentrations of cortisol, hemoglobin, red blood cells, and white blood cells. A significantly higher level of malondialdehyde was measured for all hypoxia-exposed groups, indicating lipid peroxidation and oxidative stress. At 4 and 6 week, the level of glutathione and enzymatic activities of glutathione reductase and glutathione peroxidase were significantly decreased after constant and daily intermittent hypoxia challenge. The enzymatic activities of superoxide dismutase and catalase were significantly increased at 2 and 4 weeks, but they were decreased after 6 weeks by constant and daily intermittent hypoxia. Constant and daily intermittent hypoxia with subsequent non-toxin producing dinoflagellate Cochlodinium polykrikoides treatment significantly reduced the respiration rate in 3 and 24 h exposure and survival rate of red seabream. Taken together, the red seabream can be vulnerable to HABs under hypoxia condition through inhibition of immunity and antioxidant defense ability. Our findings are helpful in better understanding of molecular and physiological effects of hypoxia, which can be used in aquaculture and fisheries management.

Inorganic nitrogen compounds reduce immunity and induce oxidative stress in red seabream.

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m-3). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m-3, while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m-3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m-3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition.

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.

Benzo[a]pyrene constrains embryo development via oxidative stress induction and modulates the transcriptional responses of molecular biomarkers in the marine medaka Oryzias javanicus.

Embryos from the marine medaka fish Oryzias javanicus were treated with eight concentrations of benzo[a]pyrene (BaP) (0.001, 0.01, 0.1, 1, 2, 5, 10, or 20 µg L--1) after they had been fertilized. Significant mortality and hatching delays were detected in embryos that had been exposed to 10 and 20 µg L-1 BaP for 4 weeks. The mortality rate after hatching was higher in the medaka that had been previously exposed to > 2 µg L-1 BaP. Significant elevations in intracellular reactive oxygen species and malondialdehyde contents were measured and the mRNA expressions of the antioxidant defense system genes (gst, sod, cat, and gpx) increased in the embryos exposed to 10 and 20 µg L-1 BaP for 1 week. The hsp70, ahr, and cyp1a transcriptional responses were also significantly upregulated in the exposed groups after 1 week. The alterations to the in vivo parameters and molecular components suggested that waterborne BaP had a toxic effect on marine medaka embryos. Finally, fin defects, spinal curvature, and cardiac edema were highly induced when the embryos were exposed to > 5 µg L-1 BaP.

Waterborne manganese modulates immunity, biochemical, and antioxidant parameters in the blood of red seabream and black rockfish.

Immunotoxic effects of manganese (Mn) were investigated in the blood of the economically important marine fish, red seabream (Pagrus major) and black rockfish (Sebastes schlegelii) when exposed to different concentrations of Mn (0, 0.5, 1, and 2 mg L-1) for 14 days. During exposure, the levels of alternative complement activity in both fish were significantly lowered at 2 mg L-1 of Mn of exposure. Lysozyme activity was significantly decreased in black rockfish in all concentrations of Mn after 14 days, while in red seabream, the decrease was significant with concentrations of 1 and 2 mg L-1 of Mn after 7 and 14 days of exposure. A significantly low level was observed only in the 2 mg L-1-exposed red seabream on day 14 of exposure. The concentrations of hemoglobin, red blood cells, white blood cells, and total serum proteins were significantly decreased in both fish under exposure to 1 and 2 mg L-1 of Mn, while cortisol, alanine transferase, aspartate transaminase, and alkaline phosphatase levels were significantly increased compared to the levels of control groups. No significant change was found in serum glucose and albumin except in red seabream exposed to 2 mg L-1 of Mn for 14 days. The responses of the antioxidant defense system were significantly induced in both fish after exposure to 1 and 2 mg L-1 of Mn on day 7 and 14 of exposure. Taken together, alterations of these parameters suggest the immunotoxicity of waterborne Mn produced by the modulation of hematological components and the induction of oxidative stress in the blood of these marine fish.

Waterborne zinc pyrithione modulates immunity, biochemical, and antioxidant parameters in the blood of olive flounder.

In this study, potential immunological and hematological effects of different concentrations (0, 1, 10, and 50 µg L-l) of waterborne zinc pyrithione (ZnPT) were studied in the blood of the olive flounder Paralichthys olivaceus over 30 days. Reduced alternative complement activity (ACH50) and lysozyme activity were measured in fish exposed to 10 and/or 50 µg L-l of ZnPT for 20 days. Decreased levels of total Ig were also observed in response to 10 and/or 50 µg L-l ZnPT during the exposure period. Levels of cortisol, a marker of stress, were significantly increased by 10 and 50 µg L-l ZnPT from day 10, and by 1 µg L-l exposure on day 30. The levels of red blood cells (RBCs) and white blood cells (WBCs) decreased following exposure to 10 and/or 50 µg L-l ZnPT, while no significant change was observed in hemoglobin level. Concentrations of total protein and albumin were significantly reduced with 50 µg L-l ZnPT at day 20. Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities were significantly increased following exposure to 10 and/or 50 µg L-l ZnPT. Lipid peroxidation was induced by ZnPT, and higher concentrations (10 and 50 µg L-l) significantly increased intracellular malondialdehyde levels during exposure. Regarding the subsequent antioxidant response, intracellular glutathione levels increased significantly in response to 10 and 50 µg L-l ZnPT on days 20 and 30. Similarly, catalase and superoxide dismutase activity was significantly increased in response to 10 and 50 µg L-l ZnPT after day 10. Taken together, changes in the studied parameters suggested the immunotoxicity of ZnPT, with modulations observed in hematological homeostasis and oxidative stress induction in the blood of olive flounder.

De novo transcriptome assembly of brackish water flea Diaphanosoma celebensis based on short-term cadmium and benzo[a]pyrene exposure experiments.

To develop a brackish water flea as a promising model for marine monitoring, Diaphanosoma celebensis were exposed to two pollutants, cadmium (Cd) and benzo[a]pyrene (BaP), which have different chemical characteristics and distinct modes of metabolic action on aquatic animals. Twenty-four hours after exposure to Cd (2 mg/L) or BaP (25 µg/L), whole body transcriptomes were analyzed. In total, 99.6 Mbp were assembled from nine libraries, resulting in 98,458 transcripts with an N50 of 1883 bp and an average contig length of 968 bp. Functional gene annotations were performed using Gene Ontology, Eukaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Cd significantly modulated endocrine and digestive enzyme system. Following BaP treatment, DNA repair and circadian rhythm related metabolisms were significantly modulated. Both the chemicals induced stress response and detoxification metabolism. This brackish water flea genomic information will be useful to monitor estuaries and coastal regions, as water fleas have been confirmed as promising sentinel models in freshwater ecosystems.

Genomic organization and transcriptional modulation in response to endocrine disrupting chemicals of three vitellogenin genes in the self-fertilizing fish Kryptolebias marmoratus.

Vitellogenin (Vtg) is the precursor of egg yolk proteins, and its expression has been used as a reliable biomarker for estrogenic contamination in the aquatic environment. To examine the biomarker potential of the self-fertilizing killifish Kryptolebias marmoratus Vtgs (Km-Vtgs), full genomic DNAs of Km-Vtgs-Aa, Km-Vtgs-Ab, and Km-Vtgs-C were cloned, sequenced, and characterized. Three Vtg genes in K. marmoratus are tandemly placed in a 550 kb section of the same chromosome. In silico analysis of promoter regions revealed that both the Km-Vtgs-Aa and Km-Vtgs-Ab genes had an estrogen response element (ERE), but the Km-Vtgs-C gene did not. However, all three Km-Vtgs genes had several ERE-half sites in their promoter regions. Phylogenetic analysis demonstrated that the three deduced amino acid residues were highly conserved with conventional Vtgs protein, forming distinctive clades within teleost Vtgs. Liver tissue showed the highest expression of Km-Vtg transcripts in all tested tissues (brain/pituitary, eye, gonad, intestine, skin, and muscle) in response to endocrine disrupting chemical (EDC)-exposed conditions. Km-Vtg transcripts were significantly increased in response to 17ß-estradiol (E2), tamoxifen (TMX), 4-n-nonylphenol (NP), bisphenol A (BPA), and octylphenol (OP) over 24hr exposure. The Km-Vtg-A gene was highly expressed compared to the control in response to NP and OP. EDC-induced modulatory patterns of Km-Vtg gene expression were different depending on tissue, gender, and isoforms.

ß-Naphthoflavone induces oxidative stress in the intertidal copepod, Tigriopus japonicus.

ß-Naphtoflavone (ß-NF) is a flavonoid and enhances oxidative stress in vertebrates with little information from aquatic invertebrates as yet. In this study, we investigated the effects of ß-NF on the antioxidant defense systems of the intertidal copepod Tigriopus japonicus. To measure the ß-NF-triggered changes in oxidative stress markers, such as intracellular reactive oxygen species (ROS), glutathione (GSH) concentration, residual glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) activity, T. japonicus were exposed to ß-NF (0.5 and 1 mg/L) for 72 h. Significant (P < 0.05) induction of the intracellular ROS content (%) was observed in 1 mg/L of ß-NF exposed T. japonicus, compared to the negative control and H2O2-exposed group. The GSH levels were significantly increased in the 0.5 mg/L of ß-NF-exposed group for 12 h and 1 mg/L of ß-NF-exposed groups for 12-24 h. GPx, GST, and GR activities showed a significant increase in the 1 mg/L ß-NF-exposed group, indicating that ß-NF induces oxidative stress in T. japonicus. To understand the effects of ß-NF at the level of transcript expression, a 6K microarray analysis was employed. Transcript profiles of selected antioxidant-related genes were modulated after 72 h exposure to 1 mg/L of ß-NF. From microarray data, 10 GST isoforms, GR, GPx, PH-GPx, and Se-GPx were chosen for a time-course test by real-time RT-PCR. T. japonicus GST-S, GST-O, GST-M, and GST-D1 were significantly increased in a 1 mg/L ß-NF-exposed group. T. japonicus GPx, GR, and Se-GPx mRNA levels were also significantly increased at both concentrations. Our results revealed that oxidative stress was induced by ß-NF exposure in T. japonicus.

Genome-wide identification of whole ATP-binding cassette (ABC) transporters in the intertidal copepod Tigriopus japonicus.

BACKGROUNDS: The ATP-binding cassette (ABC) transporter superfamily is one of the largest transporter gene families and is observed in all animal taxa. Although a large set of transcriptomic data was recently assembled for several species of crustaceans, identification and annotation of the large ABC transporter gene family have been very challenging. RESULTS: In the intertidal copepod Tigriopus japonicus, 46 putative ABC transporters were identified using in silico analysis, and their full-length cDNA sequences were characterized. Phylogenetic analysis revealed that the 46 T. japonicus ABC transporters are classified into eight subfamilies (A-H) that include all the members of all ABC subfamilies, consisting of five ABCA, five ABCB, 17 ABCC, three ABCD, one ABCE, three ABCF, seven ABCG, and five ABCH subfamilies. Of them, unique isotypic expansion of two clades of ABCC1 proteins was observed. Real-time RT-PCR-based heatmap analysis revealed that most T. japonicus ABC genes showed temporal transcriptional expression during copepod development. The overall transcriptional profile demonstrated that half of all T. japonicus ABC genes were strongly associated with at least one developmental stage. Of them, transcripts TJ-ABCH_88708 and TJ-ABCE1 were highly expressed during all developmental stages. CONCLUSIONS: The whole set of T. japonicus ABC genes and their phylogenetic relationships will provide a better understanding of the comparative evolution of essential gene family resources in arthropods, including the crustacean copepods.

Genome-wide identification of nuclear receptor (NR) superfamily genes in the copepod Tigriopus japonicus.

BACKGROUND: Nuclear receptors (NRs) are a large superfamily of proteins defined by a DNA-binding domain (DBD) and a ligand-binding domain (LBD). They function as transcriptional regulators to control expression of genes involved in development, homeostasis, and metabolism. The number of NRs differs from species to species, because of gene duplications and/or lineage-specific gene losses during metazoan evolution. Many NRs in arthropods interact with the ecdysteroid hormone and are involved in ecdysone-mediated signaling in arthropods. The nuclear receptor superfamily complement has been reported in several arthropods, including crustaceans, but not in copepods. We identified the entire NR repertoire of the copepod Tigriopus japonicus, which is an important marine model species for ecotoxicology and environmental genomics. RESULTS: Using whole genome and transcriptome sequences, we identified a total of 31 nuclear receptors in the genome of T. japonicus. Nomenclature of the nuclear receptors was determined based on the sequence similarities of the DNA-binding domain (DBD) and ligand-binding domain (LBD). The 7 subfamilies of NRs separate into five major clades (subfamilies NR1, NR2, NR3, NR4, and NR5/6). Although the repertoire of NR members in, T. japonicus was similar to that reported for other arthropods, there was an expansion of the NR1 subfamily in Tigriopus japonicus. The twelve unique nuclear receptors identified in T. japonicus are members of NR1L. This expansion may be a unique lineage-specific feature of crustaceans. Interestingly, E78 and HR83, which are present in other arthropods, were absent from the genomes of T. japonicus and two congeneric copepod species (T. japonicus and Tigriopus californicus), suggesting copepod lineage-specific gene loss. CONCLUSIONS: We identified all NR receptors present in the copepod, T. japonicus. Knowledge of the copepod nuclear receptor repertoire will contribute to a better understanding of copepod- and crustacean-specific NR evolution.

Immune gene discovery in the crucian carp Carassius auratus.

The crucian carp Carassius auratus (Cyprinidae) is one of the important fish species in aquaculture. Although the crucian carp has several economic benefits, their immune system and gene information have not been investigated in depth as yet. Here, we performed the transcriptome analysis of C. auratus using the pyrosequencing method and selected several immune-related genes. Of unigenes obtained in this species, we identified a number of immune system-related genes (e.g. adhesive protein, antimicrobial protein, apoptosis- and cell cycle-related protein, cellular defense effector, immune regulator, pattern recognition protein, protease, protease inhibitor, reduction/oxidation-related protein, signal transduction-related protein and stress protein) that are potentially useful for studies on fish immunity. To be of public and practical use, we designed primer pairs of each gene from the crucian carp for real-time RT-PCR application and tested the amplicon identity of entire gene sets with the total RNA sample. For comparative analysis, we measured tissue-preferential transcript profiles of selected genes. This study will be helpful to extend our knowledge on the immune system of the crucian carp in comparative aspects and to develop the crucian carp as a potential model organism for aquatic quality monitoring in fish farming.

Chromosomal-level genome assembly data from the pale chub, Zacco platypus (Jordan & Evermann, 1902).

The pale chub, Zacco platypus (Cypriniformes; Xenocyprididae; homotypic synonym: Opsariichthys platypus; Jordan & Evermann, 1902), is widely distributed in the freshwater ecosystems throughout East Asia, including South Korea. In this study, we constructed a de novo genome assembly of Z. platypus to serve as a reference for fundamental and applied research. The assembly was generated using a combination of long-read Pacific Bioscience (PacBio) sequencing, short-read Illumina sequencing, and Hi-C sequencing technologies. The draft genome of Z. platypus consisted of 16,422,113 reads from the HiFi library, 702,143,130 reads from the Illumina TruSeq library, and 250,789,660 reads from the Hi-C library. Assembly with Hifiasm resulted in 336 contigs, with an N50 length of 31.9 Mb. The final assembled genome size was 838.6 Mb. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis indicated that 3,572 (98.1 %) of the expected genes were found in the assembly, with 3,521 (96.7 %) being single-copy and 51 (1.4 %) duplicated after searching against the Actinopterygii database. Of the 319 Hi-C scaffolds, 24 exceeded 10 Mb were thus classified as chromosome-level scaffolds. The assembled genome comprises 41.45 % repeat sequences. Gene annotation was performed using Illumina RNA-Seq and PacBio Iso-Seq data, based on repeat-masked genome sequences. The final annotation resulted in 34,036 protein-coding genes. This chromosomal-level genome assembly is expected to be a valuable resource for future health assessments in aquatic ecosystems, providing insights into the developmental, environmental, and ecological aspects of Z. platypus.

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.

Acute and chronic effects of triclosan on the behavior, physiology, and multigenerational characteristics of the water flea Moina macrocopa.

Triclosan, a chlorinated biphenyl ether is widely used in industrial products and cosmetics due to its antibiotic activity. Although relatively levels of triclosan have been detected in aquatic ecosystems, limited information is available regarding the acute and chronic impacts of triclosan on aquatic invertebrates, especially planktonic crustaceans. In this study, we analyzed the acute (24 h) and chronic (14 days exposure across three generations) effects of different concentrations of triclosan [1/10 of the no observed effect concentration (NOEC), the NOEC, and 1/10 of the LC50] calculated from the 24 h acute toxicity value, on the water flea Moina macrocopa. In the acute exposure experiment, the 1/10 LC50 value of triclosan significantly reduced survival, feeding rate, thoracic limb activity, heart activity, and acetylcholinesterase activity. In response to the 1/10 LC50 value, intracellular reactive oxygen species increased along with elevated levels of malondialdehyde and glutathione. Enzymatic activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase were significantly increased by the 1/10 LC50 value, suggesting active protection of the antioxidant defense system against oxidative stress. Chronic exposure to the 1/10 NOEC and NOEC values revealed multigenerational adverse impacts of triclosan. The second generation was found to be the most sensitive to triclosan, as the NOEC value significantly reduced the survival rate, body length, and the number of neonates per brood, along with a delayed hatching period. Taken together, these results indicate that even sublethal levels of triclosan can have detrimental effects on the water flea population's maintenance through intergenerational toxicity.

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.