Toxic effects of fragmented polyethylene terephthalate particles on the marine rotifer Brachionus koreanus: Based on ingestion and egestion assay, in vivo toxicity test, and multi-omics analysis.

Microplastics (MPs) are a major concern in marine ecosystem because MPs are persistent and ubiquitous in oceans and are easily consumed by marine biota. Although many studies have reported the toxicity of MPs to marine biota, the toxicity of environmentally relevant types of MPs is little understood. We investigated the toxic effects of fragmented polyethylene terephthalate (PET) MP, one of the most abundant MPs in the ocean, on the marine rotifer Brachionus koreanus at the individual and molecular level. No significant rotifer mortality was observed after exposure to PET MPs for 24 and 48 h. The ingestion and egestion assays showed that rotifers readily ingested PET MPs in the absence of food but not when food was supplied; thus, there were also no chronic effects of PET MPs. In contrast, intracellular reactive oxygen species levels and glutathione S-transferase activity in rotifers were significantly increased by PET MPs. Transcriptomic and metabolomic analyses revealed that genes and metabolites related to energy metabolism and immune processes were significantly affected by PET MPs in a concentration-dependent manner. Although acute toxicity of PET MPs was not observed, PET MPs are potentially toxic to the antioxidant system, immune system, and energy metabolism in rotifers.

Pathway-dependent toxic interaction between polystyrene microbeads and methylmercury on the brackish water flea Diaphanosoma celebensis: Based on mercury bioaccumulation, cytotoxicity, and transcriptomic analysis.

Given their worldwide distribution and toxicity to aquatic organisms, methylmercury (MeHg) and microplastics (MP) are major pollutants in marine ecosystems. Although they commonly co-exist in the ocean, information on their toxicological interactions is limited. Therefore, to understand the toxicological interactions between MeHg and MP (6-µm polystyrene), we investigated the bioaccumulation of MeHg, its cytotoxicity, and transcriptomic modulation in the brackish water flea Diaphanosoma celebensis following single and combined exposure to MeHg and MP. After single exposure to MeHg for 48-h, D. celebensis showed high Hg accumulation (34.83 ± 0.40 µg/g dw biota) and cytotoxicity, which was reduced upon co-exposure to MP. After transcriptomic analysis, 2, 253, and 159 differentially expressed genes were detected in the groups exposed to MP, MeHg, and MeHg+MP, respectively. Genes related to metabolic pathways and the immune system were significantly affected after MeHg exposure, but the effect of MeHg on these pathways was alleviated by MP co-exposure. However, MeHg and MP exhibited synergistic effects on the expression of gene related to DNA replication. These findings suggest that MP can reduce the toxicity of MeHg but that their toxicological interactions differ depending on the molecular pathway.

Microplastics disrupt energy metabolism in the brackish water flea Diaphanosoma celebensis.

Energy metabolism is crucial for normal biological processes, such as growth, development, and reproduction. Microplastics disrupt energy homeostasis by modulating the digestive capacity and contents of energy reserves to overcome stress. This study investigated the modulation of digestive enzyme activity and energy reserves in the brackish water flea Diaphanosoma celebensis exposed to polystyrene (PS) beads (0.05-, 0.5-, 6-µm) for 48 h, and examined transcriptional changes in digestive enzyme-coding genes and AMP-activated protein kinase (AMPK) signaling pathway genes. PS particle size differentially modulated digestive enzyme activity, energy molecule content (glycogen, protein, and lipids), and metabolism-related gene expression. In particular, the 0.5-µm PS had the most significant effect on digestive enzyme activity. In contrast, the 0.05-µm PS caused significant metabolic disorder following a decrease in total energy budget (Ea). These findings suggest that PS beads can modulate energy metabolism through different modes depending on the bead size.

Effect of heavy metals on the energy metabolism in the brackish water flea Diaphanosoma celebensis.

Heavy metals such as lead (Pb), cadmium (Cd), and arsenic (As) are of great concern in aquatic ecosystems because of their global distribution, persistence, and biomagnification via the food web. They can induce the expression of cellular protective systems (e.g., detoxification enzymes and antioxidant enzymes) to protect organisms from oxidative stress, which is a high-energy-consuming process. Thus, energy reserves (e.g., glycogen, lipids, and proteins) are utilized to maintain metabolic homeostasis. Although a few studies have suggested that heavy metal stress can modulate the metabolic cycle in crustaceans, information on changes in energy metabolism under metal pollution remains lacking in planktonic crustaceans. In the present study, the activity of digestive enzymes (amylase, trypsin, and lipase) and the contents of energy storage molecules (glycogen, lipid, and protein) were examined in the brackish water flea Diaphanosoma celebensis exposed to Cd, Pb, and As for 48 h. Transcriptional modulation of the three AMP-activated protein kinase (AMPK) and metabolic pathway-related genes was further investigated. Amylase activity was highly increased in all heavy metal-exposed groups, whereas trypsin activity was reduced in Cd- and As-exposed groups. While glycogen content was increased in all exposed groups in a concentration-dependent manner, lipid content was reduced at higher concentrations of heavy metals. The expression of AMPKs and metabolic pathway-related genes was distinct among heavy metals. In particular, Cd activated the transcription of AMPK-, glucose/lipid metabolism-, and protein synthesis-related genes. Our findings indicate that Cd can disrupt energy metabolism, and may be a potent metabolic toxicant in D. celebensis. This study provides insights into the molecular mode of action of heavy metal pollution on the energy metabolism in planktonic crustaceans.

Metabolomic analysis of combined exposure to microplastics and methylmercury in the brackish water flea Diaphanosoma celebensis.

Owing to their widespread distribution and high bioaccumulation, microplastics (MPs) and mercury (Hg) are considered major threats to the ocean. MP interacts with Hg because of its high adsorption properties. However, their toxicological interactions with marine organisms, especially combined effects at the molecular level, are poorly understood. This study investigated the single and combined effects of MP and Hg on the metabolic profile of the brackish water flea Diaphanosoma celebensis. A total of 238 metabolites were significantly affected by MP, Hg, or MP + Hg. Metabolite perturbation patterns showed that toxicity of Hg and MP + Hg was similar and that of MP was not significant. Among the 223 metabolites affected by Hg, profiles of 32 unannotated metabolites were significantly different from those of MP + Hg, and combined effects of MP + Hg decreased the effect of Hg on 25 of these metabolites. Only 11 annotated metabolites were significantly affected by Hg or MP + Hg and were related to carbohydrate, lipid, vitamin, and ecdysteroid metabolism. Ten metabolites were decreased by Hg and MP + Hg and were not significantly different between the exposure groups. Enrichment analysis showed that galactose, starch, and sucrose metabolism were the most affected pathways. These findings suggest that MP has negligible toxic effect, and Hg can induce energy depletion, membrane damage, and disruption of growth, development, and reproduction. Although the impact of MP was negligible, the combined effects of MP + Hg could be metabolite specific. This study provides better understanding of the combined effects of MP and Hg on marine organisms.

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.

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.

Combined effects of heavy metals (Cd, As, and Pb): Comparative study using conceptual models and the antioxidant responses in the brackish water flea.

The combined effect of toxic inducers has emerged as a challenging topic, particularly due to their inconsistent impacts on the environment. Using toxic unit (TU) based on LC50 value, we investigated the 48 h acute toxicities of the following combinations: Cd + As, Cd + Pb, As + Pb, and Cd + As + Pb, and binary and ternary combined effects were interpreted using concentration addition (CA) and independent action (IA) model. The molecular effects of these combinations were further examined on the basis of gene expression (four GST and two SOD isoforms) and antioxidant enzymes activity (SOD and GST). The CA-predicted LC50 was similar to the observed results, indicating that the CA model is more applicable for evaluating the combined effects of the metal mixtures. Synergistic effects (ΣTULC50 < 0.8) were observed for the mixtures As + Pb and Cd + Pb, while additive effects (0.8 < ΣTULC50 < 1.2) were observed for the mixtures Cd + As + Pb and Cd + As. No antagonistic effects were observed in this study. Molecular biomarkers for oxidative stress caused by metals, as well as traditional endpoints such as lethality, have shown a clear response in assessing the toxicity of binary and ternary mixtures. This study opens up a new avenue for the use of biomarkers to assess the combined effects of metals in aquatic environments.

Oxidative stress responses in brackish water flea exposed to microcystin-LR and algal bloom waters from Nakdong River, Republic of Korea.

Microcystis blooms and the impact of their toxins, particularly microcystin (MC), in coastal ecosystems is an emerging threat, but the species-specific effects of MC and the potential for bioconcentration are not fully understood. We exposed the brackish water flea, Diaphanosoma celebensis, to MC-LR, which showed antioxidant responses measured at the molecular to enzyme levels but no acute toxicity. We extended our experimental investigation to measure the released MC and its uptake by D. celebensis exposed to river water. In a short-term exposure (48 h) experiment, D. celebensis exposed to water from an algal bloom (approximately 2 µg L-1 MC) assimilated more than 50 pg MC per individual. The significant increase of MCs suggests the potential for the species to accumulate MCs. The dose-dependent increase in the antioxidant response observed in the mRNA levels also showed that D. celebensis exposed to diluted algal bloom waters were affected by toxins from cyanobacteria.

Acute toxicity of bisphenol A and its structural analogues and transcriptional modulation of the ecdysone-mediated pathway in the brackish water flea Diaphanosoma celebensis.

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.