Occurrence and Characteristics of Microplastics Contamination in Different Intensive Aquaculture Systems Nearby the Yangtze Estuary, China.

Microplastics (MPs) pollution has been extensively investigated in natural fishery waters, but studies on intensive aquaculture systems are scarce. Here, the occurrence and properties of MPs were investigated and compared between four different aquaculture systems nearby the Yangtze Estuary. The average MPs concentration was in order of recirculating aquaculture system (RAS, 1.67 particles/L) < aquarium (2.47 particles/L) < cement pond (10.09 particles/L) < earthen pond (13.81 particles/L). Compared to fragment MPs, fiber was the more abundant shape in aquarium (85.88%), RAS (77.61%) and earthen pond (68.13%). A total of six colors were found in four systems. The black MPs accounted for 56.86% and 47.45% in aquarium and RAS system, respectively. The high proportion of blue MPs was found in cement pond (37.65%) and earthen pond (40%). The most MPs sizes observed in the four systems were 43% of 50-300 µm MPs in aquarium; 44% and 30.19% of 300-1000 µm MPs in RAS and cement pond, respectively; and 30.19% of 3000-5000 µm MPs in earthen pond. For polymers, polypropylene occupied 47.83% in aquarium and RAS, 41.46% in cement pond and 27.79% in earthen pond. Proportion of rayon was highest in RAS (60.87%) and 34.04% of nylon was found in earthen pond. These results could provide scientific reference for further traceability and removal of MPs in different aquaculture systems.

Characteristics and risks of microplastic contamination in aquaculture ponds near the Yangtze Estuary, China.

Microplastic pollution has been frequently reported in natural water environments, but studies on the occurrence and characteristics of microplastic in aquaculture environments especially in pond production system are relatively scarce. Herein, we investigated the abundance and characteristics of microplastic pollution in aquaculture ponds that farm different species (fish, prawn and crab) near the Yangtze Estuary, China. The average abundance of microplastic in pond water and sediment was 36.25 ± 6.79 items/L and 271.65 ± 164.83 items/kg, respectively. Compared to fish ponds (208.43 ± 57.82 items/kg), microplastic abundance was significantly higher in sediment of crab and prawn ponds (312.02 ± 38.76 and 248.87 ± 36.51 items/kg respectively). Across all ponds, transparent, white and black microplastic were the common colors. Fiber was the most common type, accounting for 40.9% and 58.6% in pond water and sediment, respectively. The size of microplastic was mainly distributed between 300 and 1000 µm. For microplastic polymer composition, polyethylene (PE) was predominant in pond water, accounting for 55%, followed by polyamide with 15%. The predominant polymer in sediment was PE with 34%, followed by polypropylene with 18%. As for the ecological risk assessment of microplastic, the pollution load index was 7.6 (risk level I) and 8.9 (risk level I) for pond water and sediment, respectively. The polymer hazard index was 85.3 (risk level II) and 12.1 (risk level II) for pond water and sediment, respectively. Taken together, the pollution risk index was rated as high and very high for pond sediment and water, respectively. These results provide a basis for the comprehensive evaluation and developing practical approaches to deal with microplastic in aquaculture pond, which is of great significance to the healthy development of pond aquaculture.

Reshaping the plastisphere upon deposition: Promote N2O production through affecting sediment microbial communities in aquaculture pond.

Microplastics (MPs) could provide vector for microorganisms to form biofilm (plastisphere), but the shaping process of MPs biofilm and its effects on the structure and function of sedimentary microbial communities especially in aquaculture environments are not reported. For this, we incubated MPs biofilm in situ in an aquaculture pond and established a sediment microcosm with plastisphere. We found that the formation of MPs biofilm in surface water was basically stable after 30 d incubation, but the biofilm communities were reshaped after deposition for another 30 d, because they were more similar to plastisphere communities incubated directly within sediment but not surface water. Moreover, microbial communities of MPs-contaminated sediment were altered, which was mainly driven by the biofilm communities present on MPs, because they but not sediment communities in proximity to MPs had a more pronounced separation from the control sediment communities. In the presence of MPs, increased sediment nitrification, denitrification and N2O production rates were observed. The K00371 (NO2-⇋NO3-) pathway and elevated abundance of nxrB and narH genes were screened by metagenomic analysis. Based on structural equation model, two key bacteria (Alphaproteobacteria bacterium and Rhodobacteraceae bacterium) associated with N2O production were further identified. Overall, the settling of MPs could reshape the original biofilm and promote N2O production by selectively elevating sedimental microorganisms and functional genes in aquaculture pond.

Astaxanthin mitigates oxidative stress caused by microplastics at the expense of reduced skin pigmentation in discus fish.

Microplastics (MPs) exposure generally triggers oxidative stress in fish species and vertebrate pigmentation is commonly influenced by oxidative stress, but MPs-induced oxidative stress on fish pigmentation and body color phenotype has not been reported. The aim of this study is to determine whether astaxanthin could mitigate the oxidative stress caused by MPs but at the expense of reduced skin pigmentation in fish. Here, we induced oxidative stress in discus fish (red skin color) by 40 or 400 items/L MPs under both astaxanthin (ASX) deprivation and supplementation. We found that lightness (L*) and redness (a*) values of fish skin were significantly inhibited by MPs under ASX deprivation. Moreover, MPs exposure significantly reduced ASX deposition in fish skin. The total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity in fish liver and skin were both significantly increased with the increase of MPs concentration, but content of glutathione (GSH) in fish skin showed a significant decrease. For ASX supplementation, the L*, a* values and ASX deposition were significantly improved by ASX, including the skin of MPs-exposed fish. The T-AOC and SOD levels changed non-significantly in fish liver and skin under the interaction of MPs and ASX, but ASX significantly reduced GSH content in fish liver. Biomarker response index indicated that ASX could improve the moderately altered antioxidant defense status of MPs-exposed fish. This study suggests that the oxidative stress caused by MPs was mitigated by ASX but at expense of reduced fish skin pigmentation.

Microplastics inhibit biofloc formation and alter microbial community composition and nitrogen transformation function in aquaculture.

Biofloc technology, extensively used in intensive aquaculture systems, can prompt the formation of microbial aggregates. Microplastics (MPs) are detected abundantly in aquaculture waters. This study explored the effects of MPs on biofloc formation, microbial community composition and nitrogen transformation function in simulated biofloc aquaculture production systems. The formation process and settling performance of bioflocs were examined. High-throughput sequencing of 16S and 18S rRNA genes was used to investigate the microbial community compositions of bioflocs. Nitrogen dynamics were monitored and further explained from functional genes and microorganisms related to nitrogen transformation by metagenome sequencing. We found that the aggregates consisting of bioflocs and MPs were formed and the systems with MPs had relatively weak settling performance. No significant differences in bacterial diversity (p > 0.05) but significant differences in eukaryotic diversity (p < 0.05) were found between systems without and with MPs. Significant separations in the microbial communities of prokaryotes (p = 0.01) and eukaryotes (p = 0.01) between systems without and with MPs were observed. The peak concentration of nitrite nitrogen (NO2--N) in systems with MPs was lower than that in systems without MPs (pControl/MPs Low = 0.02 and pControl/MPs High = 0.03), probably due to the low abundance of hao and affiliated Alphaproteobacteria_bacterium_HGW-Alphaproteobacteria-1 and Alphaproteobacteria_bacterium, but the high abundance of nxrA and affiliated Alphaproteobacteria_bacterium_SYSU_XM001 and Hydrogenophaga_pseudoflava that related to nitrification. The low concentration of NO2--N in systems with MPs suggested that the presence of MPs might inhibit ammonia oxidation but promote nitrite oxidation by altering the microbial community structure and function. These results indicated that aggregates consisting of bioflocs and MPs could be formed in aquaculture water, and thus, inhibiting their settlement and altering nitrogen transformation function by affecting the microbial community composition.

Microplastics drive nitrification by enriching functional microorganisms in aquaculture pond waters.

The plastisphere refers to biofilm formation on the microplastic (MP) surface, but its subsequent functions, especially driving the nitrogen biogeochemical cycle, are rarely studied. Here, MPs were incubated in the pelagic water and benthic water-sediment interface of an aquaculture pond, and the two corresponding microcosms amended with incubated plastisphere were simulated. The results showed decreased ammonia concentrations and increased nitrification rates in microcosms with either pelagic or benthic plastispheres. To uncover the possible mechanisms, the community structure and function of the plastisphere were investigated. As clarified by 16S rRNA, the community diversity of the pelagic plastisphere was significantly higher than that of the corresponding hydrosphere. Plastisphere communities, especially those incubated in pelagic water, were separated from the hydrosphere. Moreover, the abundance of Proteobacteria increased while the abundance of Cyanobacteria decreased in both plastispheres. Metagenome further revealed that the abundance of amoA and annotated Nitrososphaeraceae_archaeon and hao and affiliated Nitrosomonas_europaea, which contributed to ammonia oxidation to nitrite, was higher in the benthic plastisphere. Comparing the pelagic plastisphere with the corresponding hydrosphere, however, the abundance of nxrA and annotated Nitrobacter hamburgensis and nxrB and the affiliated Nitrospira moscoviensis, which are involved in nitrite oxidation, was more abundant in the plastisphere. These findings suggest that the plastisphere might selectively enrich functional microorganisms and genes in a habitat-dependent manner to promote nitrification in aquaculture ponds.

Micro/nano-plastics cause neurobehavioral toxicity in discus fish (Symphysodon aequifasciatus): Insight from brain-gut-microbiota axis.

Numerous studies have investigated neurobehavioral toxicity of microplastics, but no studies have illustrated mechanism via brain-gut axis. Here, juvenile discus fish (Symphysodon aequifasciatus) were exposed for 96 h to microfibers (900 µm, fiber, MFs) or nanoplastics (~88 nm, bead, NPs) with three concentrations (0, 20 and 200 µg/L). Accumulation in fish gut was independent of plastics type and concentration. MFs reduced growth performance while NPs weakened swimming and predatory performance of post-exposed discus. For brain cholinesterase activity, acetylcholinesterase was activated by NPs while NPs/MFs exposure inhibited butyrylcholinesterase. Concentrations of neurotransmitters (acetylcholine, dopamine and γ-aminobutyric acid) increased in brain but decreased in gut after NPs or MFs exposure. For gut microbiota, increased richness under MFs exposure was observed. At phylum level, Proteobacteria proportion was lower in NPs but higher in MFs. Abundance of Clostridia and Fusobacteriia (Bacillus), potentially secreting neurotransmitters, increased in NPs but decreased in MFs. Brain transcriptomics revealed seven upregulated and four downregulated genes concerning neural-activities. Pathways of neuroactive ligand-receptor interaction and serotonergic synapse were enriched in both MFs and NPs, but dopaminergic synapse pathway was enriched only in MFs. These results established a novel mechanism by which microplastics might cause behavioral toxicities via brain-gut-microbiota axis.

Microplastics: A tissue-specific threat to microbial community and biomarkers of discus fish (Symphysodon aequifasciatus).

As detriments in aquatic environments, microplastics (MPs) have been commonly studied on organisms, but tissue-scale effects of MPs were poorly understood. Discus fish (Symphysodon aequifasciatus), herewith, were exposed to polystyrene MPs (0/20/200 µg/L) for 28 d. We found that MPs significantly inhibited growth performance. MPs were observed in skin, gill and intestine after 14/28-d exposure. MPs bioaccumulation was independent of exposure time, but increased with MPs concentrations. Microbial community diversity of fish gill, but not skin and intestine, in MPs treatments was significantly increased. Bacterial community of MP-treated skin and gill were obviously separated from control. Skin dominant phyla changed from Actinobacteriota to Proteobacteria and Firmicutes. Proteobacteria gradually occupied dominance in gill after exposure. Furthermore, MPs-induced skin oxidative stress was demonstrated by the activation of superoxide dismutase and catalase. Skin malondialdehyde also increased and showed significant correlations with four bacterial phyla, e.g., Proteobacteria. Gill Na+/K+-ATPase activity decreased, strongly correlating to microbial community changes caused by MPs. Intestinal digestive enzymes activity (pepsin, lipase and α-amylase) reduced, revealing correlation with bacterial community especially Fibrobacterota. These results suggest a tissue-specific effect of MPs to microbial community and biomarkers in aquatic organism.

Ecological stoichiometric and stable isotopic responses to microplastics are modified by food conditions in koi carp.

Microplastics (MPs) can be easily taken up by a wide range of aquatic animals and cause blockage of the digestive tract leading to starvation. Meanwhile, aquatic organisms are facing threats posed by food restriction in both wild and cultured environment. Little knowledge, however, exists on how MPs interact with food conditions to affect aquatic animals. Here, koi carp were exposed to polystyrene MPs (0, 100 or 1000 µg/L) under controlled feeding (satiated or starved) for 30 or 60 days. MPs reduced and interacted synergistically with food conditions on growth after 30 days but antagonistically after 60 days. MPs reduced crude lipid and carbohydrate but increased and antagonistically interacted with feeding conditions on crude protein. Food conditions interacted with MPs on C, N and P but stoichiometric responses were decoupled with macromolecules changes. Food conditions antagonistically interacted with MPs on δ13C after 60 days. Linear discriminant analysis revealed that C:P and N:P were the two most important measured parameters accounting for the response of koi towards MPs and food restriction, presenting an antagonistic interaction of MPs and food status with the prolonged exposure duration.

Microplastics intake and excretion: Resilience of the intestinal microbiota but residual growth inhibition in common carp.

Aquatic animals can be influenced by exposure to microplastics (MPs), but little is known about their recovery capacity following MPs excretion. Here, common carp were exposed to environmentally relevant concentrations of MPs for 30 days and followed by MPs excretion for another 30 days. Growth, isotopic and elemental compositions and intestinal microbiota were investigated. We found that fish growth was not influenced by exposed to MPs but was significantly reduced following MPs excretion, indicating a delayed effect on growth. MPs intake and excretion, however, had no obvious effects on isotopic and elemental compositions. MPs altered the community structure and composition of intestinal microbiota and might reduce functional diversity. After MPs excretion, interestingly, bacterial community structures of MPs treatments were grouped together with the control, suggesting the general resilience of fish intestinal microbiota. Nevertheless, high abundance of pathogenic Shewanella, Plesiomonas and Flavobacterium was observed in MPs treatments but did not affect the functional potential of intestinal microbiota. The results of this study provide new information for the application of adverse outcome pathway (AOP) in MPs, suggesting the necessity of paying attention to recovery assay following MPs intake in the development of AOP frameworks.

Integrated response of growth, antioxidant defense and isotopic composition to microplastics in juvenile guppy (Poecilia reticulata).

Microplastics (MPs) pollution becomes a research hotspot and many studies focus on threats of MPs, but few have integrated multi-level indicators to assess response to MPs of organisms. Here we exposed guppy (Poecilia reticulata) to MPs (polystyrene; 32-40 µm diameter) with two concentrations (100 and 1000 µg/L) for 28 days. We found that higher accumulation of MPs appeared in guppy gill than that in gut. MPs had no obvious effect on guppy growth but significantly inhibited the condition factor. Oxidative stress presented in guppy viscera with activated antioxidants. The decline of Na+/K+-ATP activity in guppy indicated that MPs might interfere with the osmotic balance of gills. MPs reduced body molar ratio of C:N and δ13C value, but no apparent impact on δ15N. It implied that MPs probably altered elemental transition. Eventually, through integrated biomarkers response index (IBR) of guppy, we found that catalase activity was the highest index in response to MPs, and the response of growth performance to MPs was lower than that of oxidative stress and element alteration. Risks of MPs aggravated in a concentration-dependent manner. These findings suggested that multi-level IBR approach should be adopted to quantify effects of MPs on aquatic organisms, especially on fish.

Exposure to microplastics impairs digestive performance, stimulates immune response and induces microbiota dysbiosis in the gut of juvenile guppy (Poecilia reticulata).

Microplastics (MPs) are widely distributing in aquatic environment. They are easily ingested by aquatic organisms and accumulate in digestive tract especially of intestine. To explore the potential effects of MPs on intestine, here we, using juvenile guppy (Poecilia reticulata) as experimental animal, investigated the response characteristics of digestion, immunity and gut microbiota. After exposure to 100 and 1000 µg/L concentrations of MPs (polystyrene; 32-40 µm diameters) for 28 days, we observed that MPs could exist in guppy gut and induce enlargement of goblet cells. Activities of digestive enzymes (trypsin, chymotrypsin, amylase and lipase) in guppy gut generally reduced. MPs stimulated the expression of immune cytokines (TNF-α, IFN-γ, TLR4 and IL-6). Through high throughput sequencing of 16S rRNA gene, decreases in diversity and evenness and changed composition of microbiota were found in guppy gut. PICRUSt analysis revealed that MPs might have effects on intestinal microbiota functions, such as inhibition of metabolism and repair pathway. Our findings suggested that MPs could retain in the gut of juvenile guppy, impair digestive performance, stimulate immune response and induce microbiota dysbiosis in guppy gut. The results obtained here provide new insights into the potential risks of MPs to aquatic animals.

Single and combined effects of microplastics and cadmium on the cadmium accumulation, antioxidant defence and innate immunity of the discus fish (Symphysodon aequifasciatus).

Microplastics (MPs) have the potential to interact with the toxicity of other common environmental contaminants, such as heavy metals. Here, we investigated the impacts of polystyrene-MPs (32-40 µm), cadmium (Cd) and their combination on early juveniles of the discus fish (Symphysodon aequifasciatus) in relation to Cd accumulation, antioxidant defence and innate immunity. Animals were exposed to three concentrations of MPs (0, 50 or 500 µg L-1) crossed with two levels of Cd (0 or 50 µg L-1) for 30 days. Our findings showed that MPs and Cd had no adverse effects on growth and survival. Under exposure to Cd, however, accumulation of Cd in the body of fish decreased with increasing MP concentrations as supported by a reduced metallothionein content. The activities of superoxide dismutase and glutathione peroxidase increased with MPs but decreased with Cd. MPs, Cd or the mixture increased catalase activity, despite an antagonistic interaction between the two stressors. Glutathione levels increased when exposed to high MP concentrations but decreased when co-exposed to Cd. Malondialdehyde content was only influenced by MPs and increased with elevated MPs. MPs or Cd alone did not increase protein carboxyl content but showed a synergistic effect and increased content. MPs or Cd alone showed no effect on lysozyme activity but had a synergistic effect and activated activity. Activities of both acid phosphatase and alkaline phosphatase were enhanced by MPs, Cd or their mixture, although there was an antagonistic interaction between the two stressors. In contrast, MPs, Cd or their mixture decreased complement 3 content, despite an antagonistic interaction between the two stressors. Collectively, this study suggests that exposure to Cd led to reduced Cd accumulation in the presence of MPs. Nevertheless, co-exposure could induce severe oxidative stress and stimulate innate immunity in the juvenile S. aequifasciatus.

Microplastics have a more profound impact than elevated temperatures on the predatory performance, digestion and energy metabolism of an Amazonian cichlid.

Knowledge on the impacts of microplastics (MPs) pollution on freshwater environments and biota remains limited. Meanwhile, freshwater ecosystems have been threatened by elevated temperatures caused by climate change. To date, no information exists on how MPs-especially under elevated temperature conditions-affect predatory performance, digestive processes and metabolic pathways in freshwater organisms. Here, we examined MPs, elevated temperature and their combined effects on juveniles (0+ group) of an Amazonian cichlid, the discus fish (Symphysodon aequifasciatus). For 30 days, fish were exposed to ambient or elevated temperatures (i.e., 28 or 31 °C) in the absence or presence of MPs (i.e., 0 or 200 µg/L). The following metrics were quantified: MPs accumulation; predatory performance; and biomarkers involved in neurotransmission, digestion and energy production. The results showed that survival rate and body length were not affected by MPs, elevated temperatures or their combination. Elevated temperatures resulted in an increase in MP concentrations in fish bodies. Exposure to MPs decreased the post-exposure predatory performance (PEPP) at ambient temperatures but not at elevated temperatures. Elevated temperatures, however, had no effect on the PEPP but antagonistically interacted with MPs, leading to similar predatory performances under present and future conditions. Acetylcholinesterase (AChE) activity was only affected by MPs and decreased in the presence of MPs, indicating adverse effects in nervous and neuromuscular function and, thus, potentially in predatory performance. Trypsin activity was only influenced by MPs and decreased during exposure to MPs. Elevated temperatures or MPs alone increased the amylase activity but interacted antagonistically. Lipase activity was not influenced by either of the two stressors. In contrast, alkaline phosphatase (ALP) activity was affected by MPs or elevated temperatures alone and decreased with both stressors. Such results indicate deficits in the digestive capabilities of early-stage S. aequifasciatus under elevated temperature conditions and especially during exposure to MPs. Electron transport system (ETS) activity was not influenced by either of the two stressors. Both elevated temperatures and MPs alone increased LDH activity; however, the interaction between the two stressors cancelled activity but was still higher than activity in present conditions. Citrate synthase (CS) activity decreased with elevated temperature but increased during exposure to MPs. Cytochrome c oxidase (COX) activity was only influenced by MPs and increased in the presence of MPs. Thus, S. aequifasciatus juveniles exposed to elevated temperatures and MPs not only relied on anaerobic glycolysis for energy production but also depended on aerobic metabolism in the presence of MPs. Overall, these findings suggested that MPs showed a greater impact than elevated temperatures on the predatory performance, digestion and energy production of S. aequifasciatus. Nevertheless, juvenile survival and growth were minimally impacted, and thus, S. aequifasciatus could cope with near-future temperature increases and MP exposure.