Rapid Mass Conversion for Environmental Microplastics of Diverse Shapes.

Rivers have been recognized as the primary conveyors of microplastics to the oceans, and seaward transport flux of riverine microplastics is an issue of global attention. However, there is a significant discrepancy in how microplastic concentration is expressed in field occurrence investigations (number concentration) and in mass flux (mass concentration). Of urgent need is to establish efficient conversion models to correlate these two important paradigms. Here, we first established an abundant environmental microplastic dataset and then employed a deep neural residual network (ResNet50) to successfully separate microplastics into fiber, fragment, and pellet shapes with 92.67% accuracy. We also used the circularity (C) parameter to represent the surface shape alteration of pellet-shaped microplastics, which always have a more uneven surface than other shapes. Furthermore, we added thickness information to two-dimensional images, which has been ignored by most prior research because labor-intensive processes were required. Eventually, a set of accurate models for microplastic mass conversion was developed, with absolute estimation errors of 7.1, 3.1, 0.2, and 0.9% for pellet (0.50 ≤ C < 0.75), pellet (0.75 ≤ C ≤ 1.00), fiber, and fragment microplastics, respectively; environmental samples have validated that this set is significantly faster (saves ∼2 h/100 MPs) and less biased (7-fold lower estimation errors) compared to previous empirical models.

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.

The processes and transport fluxes of land-based macroplastics and microplastics entering the ocean via rivers.

Approximately 80% of marine plastic waste originates from land-based sources and enters oceans through rivers. Hence, to create effective regulations, it is crucial to thoroughly examine the processes by which land-based plastic waste flows into marine environments. To this end, this review covers the complete journey of macro- and microplastics from their initial input into rivers to their ultimate release into oceans. Here, we also discuss the primary influencing factors and current popular research topics. Additionally, the principles, applicability, accuracy, uncertainty, and potential improvement of the standard methods used for flux estimation at each stage are outlined. Emission estimates of land-based macro- and microplastics are typically assessed using the emission factor approach, coefficient accounting approach, or material flow analysis. Accurately estimating mismanaged plastic waste is crucial for reducing uncertainty in the macroplastic emission inventory. In our review of the processes by which land-originating plastics enter rivers, we categorized them into two major types: point-source and diffuse-source pollution. Land surface hydrological models simulate transport from diffuse sources to rivers, necessitating further research. Riverine (micro)plastic flux to the ocean is often estimated using monitoring statistics and watershed hydrological models at the watershed scale; however, standardized monitoring methods have not yet been established. At the global scale, algorithms based on river datasets are often used, which require further improvements in river data selection and microplastic number-mass conversion factors. Furthermore, the article summarizes the accuracy and sources of uncertainty of various methods. Future research efforts should focus on quantifying and mitigating uncertainties in resultant projections. Overall, this review deepens our understanding of the processes by which land-based plastic waste enters the ocean and helps scholars efficiently select or improve relevant methods when studying land-ocean transport fluxes.

Swimming behavior affects ingestion of microplastics by fish.

Microplastics (< 5 mm) are widely found in organisms and have the potential harm to ecosystems. Despite their widespread prevalence in environments, there is high individual varation in the abundance of microplastics found in individuals of the same species. In the present study, juvenile cichlid fish (Chindongo demasoni) were chosen to determine the ingestion personality for microplastics in the laboratory. The visible implant fluorescent tags were used for individual recognition. The fish were fed with microplastic fiber, pellet, and food for comparison. Our results showed that the observation of the behaviors of fish could be successfully matched with subsequent measurements for each individual through the tag method in microplastic research. The difference in the abundance of fiber (0-27 items/ind.) among fish individuals was also observed in our study. Meanwhile, the abundance of fiber showed a positive correlation with the average speed and covered area of fish, which indicates the degree of activity of fish. Moreover, fish with higher speed or a front position had higher capturing times for pellet. Our results suggest that the swimming behaviors of fish affect their ingestion of microplastics, and active fish had a higher likelihood of ingesting microplastics, which might be one of the reasons for the common phenomena, i.e., great individual differences observed in microplastic studies.

The hidden risk of microplastic-associated pathogens in aquatic environments.

Increasing studies of plastisphere have raised public concern about microplastics (MPs) as vectors for pathogens, especially in aquatic environments. However, the extent to which pathogens affect human health through MPs remains unclear, as controversies persist regarding the distinct pathogen colonization on MPs as well as the transmission routes and infection probability of MP-associated pathogens from water to humans. In this review, we critically discuss whether and how pathogens approach humans via MPs, shedding light on the potential health risks involved. Drawing on cutting-edge multidisciplinary research, we show that some MPs may facilitate the growth and long-range transmission of specific pathogens in aquatic environments, ultimately increasing the risk of infection in humans. We identify MP- and pathogen-rich settings, such as wastewater treatment plants, aquaculture farms, and swimming pools, as possible sites for human exposure to MP-associated pathogens. This review emphasizes the need for further research and targeted interventions to better understand and mitigate the potential health risks associated with MP-mediated pathogen transmission.

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.

Exposure protocol for ecotoxicity testing of microplastics and nanoplastics.

Despite the increasing concern about the harmful effects of micro- and nanoplastics (MNPs), there are no harmonized guidelines or protocols yet available for MNP ecotoxicity testing. Current ecotoxicity studies often use commercial spherical particles as models for MNPs, but in nature, MNPs occur in variable shapes, sizes and chemical compositions. Moreover, protocols developed for chemicals that dissolve or form stable dispersions are currently used for assessing the ecotoxicity of MNPs. Plastic particles, however, do not dissolve and also show dynamic behavior in the exposure medium, depending on, for example, MNP physicochemical properties and the medium's conditions such as pH and ionic strength. Here we describe an exposure protocol that considers the particle-specific properties of MNPs and their dynamic behavior in exposure systems. Procedure 1 describes the top-down production of more realistic MNPs as representative of MNPs in nature and particle characterization (e.g., using thermal extraction desorption-gas chromatography/mass spectrometry). Then, we describe exposure system development for short- and long-term toxicity tests for soil (Procedure 2) and aquatic (Procedure 3) organisms. Procedures 2 and 3 explain how to modify existing ecotoxicity guidelines for chemicals to target testing MNPs in selected exposure systems. We show some examples that were used to develop the protocol to test, for example, MNP toxicity in marine rotifers, freshwater mussels, daphnids and earthworms. The present protocol takes between 24 h and 2 months, depending on the test of interest and can be applied by students, academics, environmental risk assessors and industries.

Chemical features and biological effects of degradation products of biodegradable plastics in simulated small waterbody environment.

A plethora of research has focused on the biosafety of biodegradable plastics (BPs), including their microplastic formation and additives leaching; however, relatively fewer studies have explored biodegradation products. This study aims to investigate the biological effects and chemical features of degradation products from three kinds of BPs, namely polyglycolic acid (PGA), poly (butylene adipate-co-terephthalate) (PBAT), and the blends of PGA/PBAT without the addition of additives, in a simulated small waterbody environment with extracted soil solution for three months. Results showed that exposure to the whole degradation remnants of three BPs had no lethal effects on zebrafish at the current BP environmental concentrations (from 0.24 to 12.72 mg plastic/L) in small waterbodies. However, from the calculated BPs environmental concentrations (from 0.57 to 43.82 mg plastic/L) in 2026, PGA and PGA/PBAT blends may cause adverse effects on the cardiovascular system such as heartbeat rate suppression in zebrafish embryos, and also lead to reduced body length and pericardial edema and spinal curvature in fish larvae. We further qualitatively analyzed the composition of degradation products, and quantitatively measured four dominant degradation monomers (glycolic acid (GA), adipic acid (A), 1,4-butanediol (B), and terephthalic acid (T)) in the degradation remnants. It was found that the observed toxicities were probably due to the presence of GA, A, and T monomers, and their concentrations can reach 0.776, 0.034, and 0.6 mg/L under the calculated future scenario, respectively. It is worth mentioning that either GA or T monomers at the above concentrations were found to cause suppressed heartbeat rate in zebrafish embryos. Collectively, though the degradation products of BPs are temporarily safe at current environmental concentrations, they may lead to non-negligible toxicity with increasing production and continual improper recycling and/or BP waste management.

Proceeding the categorization of microplastics through deep learning-based image segmentation.

Microplastics (MPs) have been recognized as prominent anthropogenic pollutants that inflict significant harm to marine ecosystems. Various approaches have been proposed to mitigate the risks posed by MPs. Gaining an understanding of the morphology of plastic particles can provide valuable insights into the source and their interaction with marine organisms, which can assist the development of response measures. In this study, we present an automated technique for identifying MPs through segmentation of MPs in microscopic images using a deep convolutional neural network (DCNN) based on a shape classification nomenclature framework. We used MP images from diverse samples to train a Mask Region Convolutional Neural Network (Mask R-CNN) based model for classification. Erosion and dilation operations were added to the model to improve segmentation results. On the testing dataset, the mean F1-score (F1) of segmentation and shape classification was 0.7601 and 0.617, respectively. These results demonstrate the potential of proposed method for the automatic segmentation and shape classification of MPs. Furthermore, by adopting a specific nomenclature, our approach represents a practical step towards the global standardization of MPs categorization criteria. This work also identifies future research directions to improve accuracy and further explore the possibilities of using DCNN for MPs identification.

Holes on surfaces of the weathered plastic fragments from coastal beaches.

The surface morphology of weathered plastics undergoes a variety of changes. In this study, 3950 plastic fragments from 26 beaches around the world, were assessed to identify holes. Holes were identified on 123 fragments on 20 beaches, with the highest frequency (10.3 %) being identified at Qesm AL Gomrok Beach in Egypt. The distribution of holes could be divided into even, single-sided, and random types. The external and internal holes were similar in size (37 ± 15 µm) of even type fragments. The external holes were larger than the internal holes in single-sided (516 ± 259 µm and 383 ± 161 µm) and random (588 ± 262 µm and 454 ± 210 µm) fragment types. The external hole sizes were positively correlated with the internal hole sizes for each type. This study reports a novel deformation phenomenon on the surface of weathered plastics and highlights their potential effects on plastics.

Insight into chemical features of migrated additives from plastics and associated risks to estuarine ecosystem.

Distinct hydrodynamic conditions created a hotspot of plastic and associated additive pollution within estuaries, which is of considerable scientific interest. However, the effects of specific estuarine weathering (severe mechanical wear, constant turbulence, and strong ultraviolet radiation) on migration of additives remain unclear. Therefore, we investigated the release of migrated plastic additives (MPAs) from three representative plastics, namely floating foam, fishing nets, and packaging bags, under simulated estuarine conditions. Sixty-seven MPAs leached out under the wave scenario, greater than those under the ultraviolet radiation (62) and shoal (40) scenarios. We detected forty MPAs in the plastic bag leachates, whereas fewer MPAs were released from the foam and nets. Several MPAs were peculiar to specific plastics, e.g., antistatic and curing agents in the bag and foam leachates, respectively. Particularly, a suite of nonionic surfactants, octylphenol polyethoxylates (OPEOn), exhibited outstanding responses in the packaging bag leachates and had elevated toxic potential. OPEOn significantly inhibited the hatching of zebrafish and caused cardiovascular system disorder and morphological distortions even at environmentally relevant concentrations as in estuaries. Collectively, the leaching of MPAs was significantly enhanced by wave actions, and the plastic leachates, particularly those of plastic bags, can cause detrimental risks to the estuarine ecosystem.

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.

Polystyrene microplastics alleviate adverse effects of benzo[a]pyrene on tissues and cells of the marine mussel, Mytilus galloprovincialis.

As two major ubiquitous pollutants, microplastics (MPs) and polycyclic aromatic hydrocarbons (PAHs) coexist in the marine environment. However, the role of MPs in altering the toxicity of PAHs to marine organisms is poorly understood. We therefore investigated the accumulation and toxicity of benzo[a]pyrene (B[a]P, 0.4 nM), in the marine mussel Mytilus galloprovincialis over a 4-day of exposure with or without the presence of 10 µm polystyrene microplastics (PS MPs) (10 particles/mL). The presence of PS MPs significantly decreased B[a]P accumulation in soft tissues of M. galloprovincialis by approximately 6.7%. Single exposure of PS MPs or B[a]P decreased the mean epithelial thickness (MET) of digestive tubules and enhanced reactive oxygen species (ROS) levels in haemolymph, while upon co-exposure the adverse impacts were alleviated. Real-time q-PCR results showed that most selected genes involved in stress response (FKBP, HSP90), immune (MyD88a, NF-κB) and detoxification (CYP4Y1) were induced for both single exposure and co-exposure. The co-presence of PS MPs down-regulated the mRNA expression of NF-κB in gills compared with of B[a]P alone. The uptake and toxicity reductions of B[a]P might result from the decrease of its bioavailable concentrations caused by the adsorption of B[a]P by PS MPs and the strong affinity of B[a]P to PS MPs. Adverse outcomes for the co-existence of marine emerging pollutants under long-term conditions remain to be further validated.

Burrowing invertebrates induce fragmentation of mariculture Styrofoam floats and formation of microplastics.

Secondary microplastics originate from the fragmentation of large plastics, and weathering is supposed to be the main cause of fragmentation. In this study, we investigated burrows and burrowing invertebrates on Styrofoam floats from the mariculture areas of China's coastal waters. Various burrows were found on the submerged surface of Styrofoam floats and could be divided into 'I', 'S', 'J', and 'Y' types based on the burrow entrance number and passage curvature. Different invertebrate species, including 5 isopods, 8 clamworms, and 12 crabs, were found inside the burrows. Micro-foams were found in the bodies of these burrowers, with an average abundance of 4.2 ± 0.3 (isopod), 6.9 ± 2.0 (clamworm), and 3.0 ± 0.5 (crab) micro-foams per individual. In the laboratory, we observed the boring process of crabs in abandoned floats. Field and laboratory evidence suggested that these invertebrates bored various burrows. The total volume of crab burrows on a 3-year-used float was estimated to be 2.6 × 103 cm3, producing 4.1 × 108 microplastics. This study highlights the critical role of bioerosion in destroying man-made substrates and prompting microplastic pollution.

High accumulation of microplastic fibers in fish hindgut induces an enhancement of triphenyl phosphate hydroxylation.

Fiber shedding from artificial textiles is among the primary sources of pervasive microplastics in various aquatic habitats. To avoid molten drop burning, triphenyl phosphate (TPhP), a typical flame retardant additive, is commonly incorporated into textile fibers. However, the role of microplastic fibers (MFs) as a vehicle for TPhP remains largely unknown. In this study, we investigated the effects of MFs on the bioaccumulation and metabolism of TPhP in zebrafish. We applied the compound spinning technique for a non-disruptive in situ measurement of fluorescent MFs in fish, and the desorption electrospray ionization mass spectrometry (DESI-MS) to display the tissue distribution of TPhP and its metabolites vividly. Laboratory results showed that ingested MFs did not change the TPhP distribution in fish; however, they statistically increased the metabolite p-OH-TPhP concentration in the fish hindgut, which was probably because the high accumulation of MFs there enhanced the TPhP hydroxylation. Field investigation further supported the lab-based analyses. Higher concentrations of MFs did cause a higher ratio of [p-OH-TPhP]/[TPhP] in the wild fish gut, particularly in the hindgut. Collectively, our results demonstrated that MFs can change the distribution and bioavailability of TPhP metabolites, which was confirmed by both laboratory and fieldwork. Therefore, the ingestion of MFs can indirectly but substantially influence the bioaccumulation and biotransformation of co-existing pollutants.

The co-occurrent microplastics and nano-CuO showed antagonistic inhibitory effects on bacterial denitrification: Interaction of pollutants and regulations on functional genes.

The wide occurrence of microplastics (MPs) and nanoparticles resulted in their inevitable coexistence in environment. However, the joint effects of these two types of particulate emerging contaminants on denitrification have seldomly been investigated. Herein, non-biodegradable polyvinyl chloride, polypropylene, polyethylene and biodegradable polyhydroxyalkanoate (PHA) MPs were chosen to perform the co-occurrent effects with nano copper oxide (nano-CuO). Both the nano-CuO and MPs inhibited the denitrification process, and biodegradable PHA-MPs showed severer inhibition than non-biodegradable MPs. However, the presence of MPs significantly alleviated the inhibition of nano-CuO, suggesting an antagonistic effect. Other than MPs decreasing copper ion release from nano-CuO, MPs and nano-CuO formed agglomerations and induced lower levels of oxidative stress compared to individual exposure. Transcriptome analysis indicated that the co-occurrent MPs and nano-CuO induced different regulation on denitrifying genes (e. g. nar and nor) compared to individual ones. Also, the expressions of genes involved in denitrification-associated metabolic pathways, including glycolysis and NADH electron transfer, were down-regulated by nano-CuO or MPs, but exhibiting recovery under the co-occurrent conditions. This study firstly discloses the antagonistic effect of nano-CuO and MPs on environmental process, and these findings will benefit the systematic evaluation of MPs environmental behavior and co-occurrent risk with other pollutants.

Microplastics in global bivalve mollusks: A call for protocol standardization.

A growing body of evidence shows that microplastic pollution is ubiquitous in bivalve mollusks globally and is of particular concern due to its potential impact on human health. However, non-standardized sampling, processing, and analytical techniques increased the difficulty of direct comparisons among existing studies. Based on 61 peer-reviewed papers, we summarized the current knowledge of microplastics in bivalve mollusks globally and provided an in-depth analysis of factors affecting the outcome of microplastic data, with the main focus on the effects of different species and methodologies. We found no significant differences in microplastic abundance among genera from the same family but significant differences among bivalve families, indicating habitats play an important role in microplastic ingestion by bivalve mollusks. This also provided foundational knowledge for using epifaunal and infaunal bivalves to monitor microplastic pollution in water and sediment, respectively. Recommendations for microplastic monitoring protocol in bivalve mollusks were proposed according to the results of this review, covering (i) a sample size of at least 50 bivalves in the study area, (ii) the use of 10 % KOH as the digestion solution, and (iii) the pore size of a filter membrane of < 5 µm. Acknowledging the need for a standard procedure, more efforts towards protocol standardization used in long-term and large-scale microplastic monitoring programs in bivalve mollusks are needed.

Surface water, sediment, and biota: The first multi-compartment analysis of microplastics in the Karnafully river, Bangladesh.

The Karnafullly River, which flows through Chattogram and falls into the Bay of Bengal, Bangladesh, is vulnerable to microplastic contamination. In this study, we looked at microplastics in the Karnafully River's surface water (5 sites), sediment (9 sites), and biota (4 species). Microplastic concentrations ranged from 0.57 ± 0.07 to 6.63 ± 0.52 items/L in surface water, 143.33 ± 3.33 to 1240 ± 5.77 items/kg dry weight in sediment, and 5.93 ± 0.62 to 13.17 ± 0.76 items/species in biota. A significant difference (P < 0.05) was found in the concentration of MPs in the Karnafully River's sediment, biota, and surface water. High percentage of fiber-shaped and small-sized MPs (<1 mm) were detected throughout the samples. Water and sediment MPs were often transparent/white and blue, whereas biota MPs were mostly black and red, indicating a color preference during biological uptake. The Bay of Bengal received 61.3 × 109 microplastic items per day. The feeding zone of biota influenced the level of microplastics, with a trend of pelagic > demersal > benthic > benthopelagic. Polyethylene and polyethylene terephthalate were the most abundant polymer. Using the average fish intake rate in Bangladesh, we computed a possible consumption of 4015-7665 items of MPs/person/year.

Crack Patterns of Environmental Plastic Fragments.

Secondary microplastics usually come from the breakdown of larger plastics due to weathering and environmental stress cracking of plastic wastes. In the present study, 5013 plastic fragments were collected from coastal beaches, estuary dikes, and lake banks in China. The fragment sizes ranged from 0.2 to 17.1 cm, and the dominant polymers were polypropylene and polyethylene. Cracks were observed on the surfaces of 49-56% of the fragments. Based on the extracted crack images, we proposed a general crack pattern system including four crack types with specific definitions, abbreviations, and symbols. The two-dimensional spectral analysis of the cracks suggests that the first three patterns showed good regularity and supported the rationality of the pattern system. Some crack metrics (e.g., line density) were closely correlated with the carbonyl index and additives (e.g., phthalate esters) of fragments. For crack investigation in field, we proposed a succinct protocol, in which five crack ranks were established to directly characterize the degree of cracking based on the line density values. The system was successfully applied to distinguish the differences in crack features at two representative sites, which indicates that crack pattern is a useful tool to describe the morphological changes of plastic surfaces in the environment.

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.