Non-negligible Toxicity to Fish in the Early Life Stages Triggered by Aqueous Leachate of Takeaway Plastic Containers.

Ordering takeout is a growing social phenomenon and may raise public health concerns. However, the associated health risk of compounds leaching from plastic packaging is unknown due to the lack of chemical and toxicity data. In this study, 20 chemical candidates were tentatively identified in the environmentally relevant leachate from plastic containers through the nontargeted chemical analysis. Three main components with high responses and/or predicted toxicity were further verified and quantified, namely, 3,5-di-tert-butyl-4-hydroxycinnamic acid (BHC), 2,4-di-tert-butylphenol (2,4-DTBP), and 9-octadecenamide (oleamide). The toxicity to zebrafish larvae of BHC, a degradation product of a widely used antioxidant Irganox 1010, was quite similar to that of the whole plastic leachate. In the same manner, RNA-seq-based ingenuity analysis showed that the affected canonical pathways of zebrafish larvae were quite comparable between BHC and the whole plastic leachate, i.e., highly relevant to neurological disease, metabolic disease, and even behavioral disorder. Longer-term exposure (35 days) did not cause any effect on adult zebrafish but led to decreased hatching rate and obvious neurotoxicity in zebrafish offspring. Collectively, this study strongly suggests that plastic containers can leach out a suite of compounds causing non-negligible impacts on the early stages of fish via direct or parental exposure.

Diel Pattern of Microplastic Residues in Zebrafish.

As one of the emerging pollutants, microplastics (MPs) can be taken up by aquatic organisms through ingestion. However, little is known about the uptake pattern in organisms over time and the associated mechanisms of retention patterns. The present study aims to elucidate these patterns in fish, their relationship with light/dark conditions, and examine the uptake kinetic process of small-sized plastic pollutants, especially during the long-neglected dark period. Zebrafish were sampled every 2 h during the light and dark periods after exposure to an environmentally relevant concentration (100 items/L) of MPs. The results demonstrated that MP residues in zebrafish decreased during the dark period rather than increased over time. The MP retention rhythm and the swimming behavior of exposed zebrafish displayed a statistically significant light/dark variation. Moreover, a very strong and statistically significant positive correlation was found between the swimming speed of zebrafish and the number of MP residues in the gastrointestinal tracts of zebrafish. These results clearly demonstrate that fibrous MP residues in the fish have a discernible diel pattern. This work improves the understanding of the dynamic residual process of MPs in organisms and calls for further in-depth circadian toxicokinetic studies to better suit particle pollutants.

Plastic debris in coastal macroalgae.

Macroalgae are the most productive marine macrophytes in the coastal ecosystem. Although plastic debris has been ubiquitously detected in marine animals, little is known about plastic pollution in macroalgae and how they interact with each other. In this study, the occurrence of plastic debris including microplastics was investigated in 5 macroalgae species that are commonly found along the Chinese shorelines. These species consisted of Gracilaria lemaneiformis, Chondrus ocellatus, Ulva lactuca, Ulva prolifera and Saccharina japonica. We categorized the plastic debris into 3 size classes, i.e., macroplastics (>25 mm), mesoplastics (5-25 mm), and microplastics (1 µm-5 mm). It was shown that there were 5 loading patterns of plastic debris interaction with the macroalgae. The 5 patterns included entanglement, adherence, wrapping, embedment, and entrapment by epibionts. According to direct observations through the non-digestion method, all 3 size classes of plastics were found in the macroalgae. The abundances were 0-201.5 (macroplastics), 0-1178.0 (mesoplastics) and 0-355.6 (microplastics) items/kg dry weight, respectively. These plastics were dominated by fibers (52.2%) in shape, 1-5 mm (39.6%) in size, and polystyrene (36.5%) in composition. According to indirect observations through the digestion method, only 2 size classes of plastics were identified in the macroalgae: mesoplastics, 0 to 888.9 items/kg dry weight; microplastics, 148.1 to 5889.0 items/kg dry weight. These plastics were prevailing by fibers (71.5%) in shape, 1-5 mm (52%) in size, and polyester (29.3%) in composition. Furthermore, plastic characteristics in the detected macroalgae were related to their species, sampling regions, and beach types based on the results of similarity and principal component analysis. This study indicated that macroalgae utilized diverse pathways for loading plastics in the coastal environment. Meanwhile, environmental factors significantly influenced the distribution of plastics loaded by macroalgae.

Distribution and translocation of micro- and nanoplastics in fish.

Microplastics (MPs) and nanoplastics (NPs) are regarded as emerging particulate contaminants. Here, we first summarize the distribution of plastic particles in fish. Field investigations verify the presence of various kinds of fibrous, spherical, and fragmentary MPs in fish gastrointestinal tract and gills, and specifically in muscle and liver. Laboratory works demonstrate that NPs even penetrate into blood vessels of fish and pass onto next generations. Second, we systematically discuss the translocation ability of MPs and NPs in fish. MPs can enter early-developing fish through adherence, and enter adult fish internal organs by intestine absorption or epidermis infiltration. NPs can not only penetrate into fish embryo blastopores, but also reach adult fish internal organs through blood circulation. Third, the cellular basis for translocation of plastic particles, NPs in particular, into cells are critically reviewed. Endocytosis and paracellular penetration are two main pathways for them to enter cells and intercellular space, respectively. Finally, we compare the chemical and physical properties among various particular pollutants (MPs, NPs, settleable particulate matters, and manufactured nanomaterials) and their translocation processes at different biological levels. In future studies, it is urgent to break through the bottleneck techniques for NPs quantification in field environmental matrix and organisms, re-confirm the existence of MPs and NPs in field organisms, and develop more detailed translocating mechanisms of MPs and NPs by applying cutting-edge tracking techniques.

Fish Ingest Microplastics Unintentionally.

Microplastics (size of plastic debris <5 mm) occur in various environments worldwide these days and cause detrimental effects on biota. However, the behavioral responses of fish to microplastics in feeding processes are not well understood. In the present study, juveniles from four fish species and two common shapes of microplastics were used to explore fish feeding responses. We found swallowing-feeding fish ingested more pellets than filtering- and sucking-feeding fish. With high-definition and high-speed observational experiments, we found that all species did not actively capture microfibers; instead, they passively sucked in microfibers while breathing. Surprisingly, fish showed a rejective behavior, which was spontaneously coughing up microfibers mixed with mucus. Nevertheless, some of the microfibers were still found in the gastrointestinal tracts and gills of fish, while abundances of ingested microfibers were increased in the presence of food. Our findings reveal a common phenomenon that fish ingest microplastics inadvertently rather than intentionally. We also provide insights into the pathways via which microplastics enter fish and potential strategies to assess future ecological risk and food safety related to microplastics.

Process-oriented impacts of microplastic fibers on behavior and histology of fish.

Microplastic pollution has raised global concern for its hazards to biota. To determine the direct impact of microplastics during their contact with fish, we exposed goldfish (Carassius auratus) to 100 and 1000 items/L waterborne microplastic fibers in the short- and long-term. In the presence of 1000 items/L of microplastic fibers, the coughing behavior of fish increased significantly after 2 h of exposure. Predatory behaviors decreased significantly by 53.0% after 45 d of exposure, and the reduction in daily food intake was negatively related to exposure duration in the 1000 items/L group. In addition, microplastic fibers stimulated dynamic mucus secretion across different fish tissues during the different processes evaluated in this study, with 30.0% and 62.9% overall increases in the secretory capacity of mucus cells in the 100 and 1000 items/L groups, respectively. These behavioral and histological alterations were derived from the ventilation, feeding, and swimming processes of goldfish. We regarded these changes as process-oriented impacts, suggesting the effects of microplastics on fish and how fish cope with microplastics.

Concurrent water- and foodborne exposure to microplastics leads to differential microplastic ingestion and neurotoxic effects in zebrafish.

Organisms constantly ingest microplastics directly from the environment or indirectly via trophic transfer due to the pervasiveness of microplastic pollution. However, most previous studies have only focused on waterborne exposure at the individual level, while few studies have investigated the contribution of trophic transfer to the exposure in organisms. We comprehensively evaluated the differences in microplastic ingestion and toxic effects in zebrafish exposed to microplastics via two concurrent routes (waterborne and foodborne). The polyethylene microplastics (40-47 µm, 0.1-10 mg/L) concentration used here was set in a range closed to the environmentally relevant microplastic concentrations, especially considering the extreme high concentration scenarios in wastewater. The concentration of microplastics resulting from foodborne exposure (0.01±0.01 µg/mg; 0.1±0.1 particles/mg) was significantly lower than that through waterborne exposure (0.06±0.02 µg/mg; 0.8±0.3 particles/mg), suggesting the ingestion of microplastics in their tissues occurs mainly through direct environmental uptake rather than food chain transfer (though the initial microplastic concentration was 1000 folds lower). However, more sublethal impacts, including the significant abnormal hyperactive swimming behaviour (107±5% induction; p< 0.05), were observed in the foodborne group than waterborne group. Additionally, ingenuity pathway analysis predicted both exposure routes caused obvious nervous system interference but through opposite modes of action. This was further verified by the alteration of neurotransmitter biomarkers that neurotoxicity mechanisms were completely different for the two exposure routes. The neurotoxic effects of microplastics are non-negligible and can exert together through both water- and foodborne exposure routes, which deserves further attention.

Effects of microplastics and food particles on organic pollutants bioaccumulation in equi-fugacity and above-fugacity scenarios.

Microplastics (MPs), as emerging contaminants, sorb organic pollutants from the environment or leach out additives, thereby altering the fate of co-existing pollutants to organisms. We chose equi-fugacity and above-fugacity concentrations of polychlorinated biphenyls (PCBs) as background contamination and plastic additive concentrations, respectively, to investigate the effects of MPs on PCB bioaccumulation; we compared the effects of MPs with those of food-borne particles (FBPs). Co-exposure to MPs and FBPs at both the equi-fugacity and above-fugacity PCB concentrations had no obvious toxic effects (ROS generation and cyp1a expression) on zebrafish. When the zebrafish were exposed to the equi-fugacity PCB concentrations, the PCB concentrations reached 177.7-400.5 ng/g after a 7-d uptake; the presence of MPs did not significantly enhance PCB bioaccumulation. The remaining PCB concentrations in the fish after a 4-d depuration were 58.4-125.1 ng/g; the effects of MPs were the same as those during the uptake period. However, at the above-fugacity PCB concentrations, the MPs markedly increased the PCB bioaccumulation (by 1.8-fold) to 712.9 ng/g. This is because at above-fugacity concentrations, PCBs on MPs migrate to organisms as there were high fugacity gradients. The FBPs enhanced PCB bioaccumulation in zebrafish more effectively than the MPs, even after depuration. In the presence of FBPs, PCB bioaccumulation increased by 2.8- and 4.2- fold after uptake in the equi-fugacity and above-fugacity scenarios, respectively, both of which were significantly higher than that observed for the MPs. This is probably because FBPs are easily assimilated by fish, making the associated PCBs more bioavailable. Finally, during the co-existence of MPs and FBPs, MPs facilitate the depuration of PCBs accumulated via FBP vectors; conversely, FBPs did not affect PCB accumulation via MP vectors. Thus, this study elucidated the effects of MPs and FBPs on the bioaccumulation of pollutants at equi-fugacity or above-fugacity concentrations in aquatic environments.

Application of internal persistent fluorescent fibers in tracking microplastics in vivo processes in aquatic organisms.

Tracking the in vivo transportation and localization of microplastics is the prerequisite for a better understanding of the toxicity but has not been reported yet. Fluorescent microbeads are widely used for tracking in the laboratory, whereas the most abundant microplastic (e.g., fiber) found in the field is rarely adapted. In this study, we used fiber slicer and blades to cut spinning materials into fluorescent micro-sized fibers with various lengths. Fiber slicer and blades are efficient tools to produce specific lengths of fibers. The microfibers showed a specific color under the ultraviolet irradiation; therefore, these microfibers can be clearly visualized when they are within aquatic organisms, such as water flea, shrimp, medaka, and zebrafish, which enables us to observe the whole biological processes of microfibers directly. Thus, the present study provides a novel method of microfibers preparation, and this kind of fluorescent fiber is applicable to be an efficient and convenient tracer for in vivo process observation and potential toxicological analysis in the future.

Microplastics in the commercial seaweed nori.

Microplastics have been reported to attach to the marine macroalgae which act as the vector for microplastic transfer in the marine food web. In this study, the edible seaweed nori (Pyropia spp.) was chosen as a target species. The microplastic contaminant situations in nori were analyzed in both its final commercial products and the intermediate products across different processing stages. The abundance of microplastics ranged from 0.9 to 3.0 items/g (dw) among 24 brands of commercially packaged nori samples. With the development of nori processing stages, an enlarged size fraction of greater microplastics (1-5 mm) was observed. Compared with commercially packaged nori samples, the proportions of polypropylene, polyethylene and poly (ethylene-propylene) copolymers increased, whereas that of polyester decreased in factory-processed nori. Additionally, we further simulated and quantified the number of fluorescent polyester fibers (concentrations: 0, 1000, 5000, 10,000 fibers/L) attach to the algal pieces of Pyropia yezoensis under laboratory conditions. The average abundance of microfibers on the nori was positively and quantitatively related to their abundances in seawater (p < 0.01). To our best knowledge, this is the first work that shows the prevalence of microplastics in the commercial seaweed nori and relates to their potential sources during the processing phase.