Potential Risk Assessment of Different Sizes of Microplastics on the Digestive System of Hybrid Sturgeon

Potential Risk Assessment of Different Sizes of Microplastics on the Digestive System of Hybrid Sturgeon | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Potential Risk Assessment of Different Sizes of Microplastics on the Digestive System of Hybrid Sturgeon Jiayun Wu, QingQing Liao, Yingying Ren, Fei Chen, Yijun Li, Changwen Deng, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7701975/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Mar, 2026 Read the published version in Environmental Geochemistry and Health → Version 1 posted 12 You are reading this latest preprint version Abstract Microplastics are pollutants that are widely present in aquatic environments. Currently, research on the effects of microplastics of different particle sizes on freshwater fish remains relatively limited. This study utilized polyethylene microplastic particles of 1 µm and 5 µm to expose hybrid sturgeon( Acipenser baerii ♂×A. schrenckii ♀ ), analyzing changes in intestinal ultrastructure, digestive enzyme activity, and gut microbial composition (based on high-throughput sequencing of the 16S rRNA V3–V4 region). The results indicate that microplastics of both particle sizes cause changes in intestinal ultrastructure and digestive enzyme activity. The alpha and beta diversity of gut microbiota in the exposed groups were significantly higher than those in the control group. At the phylum level, the relative abundances of Bacteroidetes, Actinobacteria, and Desulfobacterota significantly increased (P < 0.01); at the genus level, the abundances of Pseudomonas , Lactobacillus , Enterobacter , Desulfovibrio, HIMB11 , and Muribaculaceae also significantly increased (P < 0.01). Furthermore, functional predictions of the microbiota indicated that the abundance of functions related to diseases, cellular processes, and organism systems increased in the 5 µm treatment group, while the abundance of functions related to genetic information processing significantly decreased (P < 0.05, FDR < 0.05). This study reveals the potential risks of microplastics to the digestive physiology and intestinal digestive system of sturgeon, providing a basis for further exploration of the mechanisms by which different particle sizes of microplastics affect freshwater fish. polyethylene microplastics Siberian hybrid sturgeon digestive enzyme activity gut microbiota 16SrRNA gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Microplastics refer to plastic particles with a diameter of less than 5 mm, which are ubiquitous in marine and terrestrial ecosystems, including various foods, drinking water, and air (Duong et al., 2025 ; Singh et al., 2025). These particles can be easily inhaled or ingested, leading to oxidative stress, inflammatory responses, and metabolic disorders (Chen et al., 2022 ). Microplastics typically originate from cosmetics, textiles, and plastic packaging (Izlal et al., 2025 ), and are often inert materials that can break down into smaller fragments due to environmental interactions. They have the capacity to adsorb a range of persistent organic pollutants and potentially toxic elements, such as polychlorinated biphenyls polycyclic aromatic hydrocarbons, Fe, and Pb, resulting in lasting impacts on the environment and living organisms (Priya et al., 2022 ; Saha et al., 2025 ; Liu et al., 2025 ). Research indicates that aged microplastics enhance their selective adsorption capacity through surface oxygen-containing functional groups, serve as a source of free radicals, increase environmental oxidative conditions, and even affect the expression of antibiotic resistance genes, thereby significantly altering their environmental behavior and ecological risks (Li et al., 2025 ). The intestine is an important digestive organ in animals, containing a variety of microbial communities, including bacteria, fungi, archaea, and viruses, collectively referred to as the gut microbiota (Antfolk and Jensen, 2020 ). The ingestion of microplastics by animals may lead to intestinal tissue damage, alterations in intestinal enzyme activity, and even changes in the composition of gut microbiota, thereby affecting digestive function (Gu et al., 2020 ). Microplastics not only pose a threat to aquatic environments but can also endanger human health through the bioaccumulation process within the food chain (Yuan et al., 2022 ). Currently, researchers have detected the presence of microplastics in various human organs, including the liver, small intestine, and kidneys (Cao et al., 2025 ). Studies have found that microplastics can damage the intestinal barrier, hepatocytes, and the central nervous system, causing irreversible harm to the human body(Parkhurst et al, 2025;Zha et al, 2024༛Liu et al, 2023). Particle size is a key factor influencing the toxicity of microplastics. Research on zebrafish indicates that exposure to polystyrene microplastics of different sizes can have varying degrees of adverse effects on the species. Specifically, the smallest microplastics (0.1 µm) significantly impact the diversity of the intestinal microbiota in zebrafish. Furthermore, compared to larger sizes (5 µm and 200 µm), smaller microplastics (100 nm) have been shown to modify the expression of genes associated with the production of reactive oxygen species (Gu et al., 2020 ; Yu et al., 2023 ). Existing studies have indicated that microplastics possess certain toxic effects on aquatic organisms, yet research on the toxic effects of microplastics of different sizes on aquatic life remains scarce. Investigating the toxicological mechanisms of microplastics of varying sizes is of great significance for understanding their ecological hazard potential and assessing risks to human health. Sturgeons primarily inhabit cold water environments. Caviar, commonly referred to as 'black gold', originates from sturgeons and is one of the most economically valuable fish products globally. The Siberian hybrid sturgeon is a hybrid offspring of the Acipenser baerii (♂) and A. schrenckii (♀). It inherits the robust disease resistance and transportability from the paternal lineage, along with the rapid growth characteristics from the maternal lineage (Zhao et al., 2020). The artificial freshwater sturgeon farming in China has seen substantial output, with extensive use of plastic products. The accumulation of microplastics within hybrid sturgeons poses a potential risk to the quality of sturgeon-derived products, ultimately leading to economic losses. Therefore, this study focuses on the hybrid sturgeons bred and raised in Sichuan Province, investigating the effects of polyethylene microplastics of different particle sizes on intestinal tissue morphology, digestive enzyme activity, and gut microbiota. The aim is to provide scientific data for further research on the toxic mechanisms of microplastics on freshwater fish such as sturgeons. Materials and Methods Experimental animals and microplastics challenge Siberian hybrid sturgeon fry were collected from April to July 2022, sourced from the healthy hybrid sturgeon cultivated at the Pengzhou base of Sichuan Runzhao Fisheries Co., Ltd. They were reared in 30-liter aquariums at the College of Life Sciences, Sichuan Agricultural University, with the water temperature maintained at 20°C for two weeks. Polyethylene microplastics were purchased from Zhengmei Plastic Products Co., Ltd. (Zhengzhou, China), and feed binders were sourced from Future Water World Biotechnology Co., Ltd. (Zhenjiang, China). Commercial sturgeon feed was crushed and mixed with microplastic powder at a ratio of 20% by weight, then dried at 60°C. According to published studies, significant potential concentrations of microplastics that pose harm to aquatic organisms have been reported (Wang Yongjin et al., 2019 ; Tang et al., 2020). A total of 90 sturgeons, weighing approximately 20 ± 5 grams and of similar weight, were randomly divided into three groups, each containing 30 fish. The sturgeons were fed with feed that did not contain polyethylene microplastics (PE0 group), feed with 1 micron polyethylene microplastics (PE1 group), and feed with 5 micron polyethylene microplastics (PE5 group) for a duration of 30 days. The daily feeding amount was equivalent to 1% of the sturgeon's body weight. The growth and mortality rates of the sturgeons were closely monitored. Subsequently, the hybrid sturgeons were anesthetized using MS-222. Measurements of intestinal morphology Samples of the intestinal tract from hybrid sturgeon were collected and prepared into paraffin sections. The intestinal samples were first fixed in 4% paraformaldehyde solution. Subsequently, a series of ethanol and xylene treatments were employed for dehydration and clearing. The tissue blocks were stained with hematoxylin and eosin (H & E) and observed under a Nikon Eclipse 80i microscope. Fold height and muscle layer thickness were measured using the Fiji ImageJ software. Assessment of α-amylase, trypsin and lipase enzyme activity The specific enzyme activity assay kits were purchased from Kemin Biotechnology Co., Ltd. α-AMS (DFMA-2-Y) α-amylase (Suzhou, China), trypsin (YPT-2-W), and lipase (LPS-2-W) were used to determine the activities of α-amylase, trypsin, and lipase in the intestine. Approximately 0.1 g of intestinal tissue from the segment was homogenized in 0.9 mL of 0.65% NaCl solution using a cold homogenizer. Following the instructions of the assay kits, enzyme activities were measured using a spectrophotometer. Data are presented as mean ± standard deviation and visualized using error bar charts. Subsequently, differences between groups were assessed using one-way analysis of variance (ANOVA) and Duncan's test with SPSS 22.0 software. Microbial composition and diversity analysis A random selection of six fish was subjected to intestinal analysis by BGI Genomics Co., Ltd. (Shenzhen, China). PCR amplification was performed using specific primers (338F-ACTCCTACGGGAGGCAGCAG and 806R-GGACTACHVGGGTWTCTAAT) targeting the V3 and V4 regions of the bacterial 16S rRNA gene, as described by Adams et al. ( 2013 ). After purification of the PCR products, they were used for library preparation. The fragment size distribution and concentration of the library were assessed using the Agilent 2100 Bioanalyzer, and qualified libraries were sequenced on the DNBSEQ-2000 sequencing platform. The Cut Adapt software was utilized to trim the raw sequencing data (He et al., 2013 ) for reads matching the primers. The denoising algorithm implemented in QIIME2, DADA2, was used to denoise the data, resulting in Amplicon Sequence Variants (ASVs). Based on the ASV distribution (Schloss et al., 2009 ), Mothur was employed to analyze the α-diversity within individual samples. QIIME was used to calculate the β-diversity values between samples, followed by Principal Coordinates Analysis (PCoA). The abundance of species at the phylum and genus levels was analyzed using R and gplots. LEfSe was utilized to identify significantly different species through the Kruskal-Wallis and Wilcoxon tests. PICRUSt2 was applied to predict the functional capabilities of the microbial community. The output functional prediction information was analyzed and visualized using methods similar to those for ASV composition and inter-group difference analysis. Results Intestinal Fold Height and Muscularis Thickness The anatomical examination revealed that all fish showed no visible pathological changes in their intestines. The intestinal contents were deep brown, uniform, and viscous, with no visible impurities. Quantitative measurements of the intestinal morphological structure indicated that the thickness of the muscular layer in the PE1 group was significantly thinner compared to the PE0 and PE5 groups (P < 0.05) (Figure 1a), while there were no significant differences in villus length among the groups. Activities of the digestive enzymes of α-amylase, trypsin and lipase To investigate whether damage to the intestinal structure affects its digestive function, this study further measured three types of digestive enzymes. The intestinal α-amylase activity in the PE1 group (0.0124 ± 0.0022 μmol/min/mg) was significantly higher than that in the PE0 group (0.0070 ± 0.0015 μmol/min/mg) and the PE5 group (0.0070 ± 0.0012 μmol/min/mg) (P < 0.01) (Figure 2a). Compared to the PE0 group (0.0332 ± 0.0222 U/mg) and the PE5 group (0.0270 ± 0.0071 U/mg), the intestinal trypsin activity in the PE1 group (0.0765 ± 0.0334 U/mg) was also significantly elevated (P < 0.05) (Figure 2b). The intestinal lipase activities in the PE0 group (0.1339 ± 0.0202 μmol/min/mg), PE1 group (0.2751 ± 0.2155 μmol/min/mg), and PE5 group (0.1786 ± 0.0810 μmol/min/mg) exhibited a consistent trend with that of α-amylase (P < 0.01) (Figure 2c). Microbial Composition and Diversity A total of 232 amplicon sequence variants (ASVs) were obtained from the co-aggregation category. Notably, the PE5 group exhibited the highest ASV count (192), including 168 unique ASVs. In contrast, the PE1 group contained 52 ASVs, of which 24 were unique, while the PE0 group had only 29 ASVs, with 8 being unique. A total of 13 ASVs were shared among the three groups (Figure 3). Alpha diversity analysis (Figure 4a-e) indicated no significant difference in the Simpson index between the PE5 and PE0 groups (P>0.05). However, significant differences were observed in the Sobs, Chao, and Shannon indices (P＞0.05). Conversely, significant differences were found in the Sob, Ace, and Chao indices (P<0.01). The PE-MPs feeding treatment enhanced the alpha diversity of the gut microbiota in hybrid sturgeon, with the PE5 group showing a more pronounced effect than the PE1 group. Beta diversity analysis (Supporting Information Fig. S1) shows that the Weighted Unifrac distance among the three groups reaches 0.4, while the unweighted Unifrac distance between the PE1 and PE0 groups is 0.7, and the Unifrac distance between the PE5 and PE0 groups is 0.8. Microbial Relative Abundance at the Phylum and Genus Levels At the phylum level (Figure 5a), the PE0 and PE1 groups are primarily composed of Fusobacteriota (75.16% and 36.10%), Firmicutes (15.21% and 38.13%), and Proteobacteria (9.60% and 25.70%). In contrast, the PE5 group exhibits a different distribution with respect to Fusobacteriota (26.42%), Firmicutes (41.56%), and Proteobacteria (31.16%). Comparative analysis of key species (Figure 5b) indicates that the abundance of Fusobacteriota in the PE0 group is significantly greater than that in the PE1 and PE5 groups (P<0.01). Moreover, significant differences are also observed among the three groups for Bacteroidota, Actinobacteriota, and Desulfobacterota (P<0.01), as well as Cyanobacteria (P<0.05). At the genus level (Figure 5c), the PE0 group was dominated by the genera Neisseria (9.55%), Clostridium (10.59%), and Bacteroides (75.16%). In the PE1 and PE5 groups, the dominant genera included Neisseria (25.55% and 28.54%), Clostridium (35.03% and 37.59%), Bacteroides (36.10% and 26.42%), and Pseudomonas (0.039% and 1.90%). A comparison of key species (Figure 5d) revealed that the abundance of Bacteroides in the PE0 group was significantly higher than that in the PE1 and PE5 groups (P < 0.01). Significant differences were also observed among the three groups for Pseudomonas (P < 0.01), Lactobacillus, Faecalibaculum, Dubosiella, HIMB11, Muribaculaceae (P < 0.01), and Clostridium (P < 0.05). There are no significant differences in the classification levels of other taxa. The LEfSe analysis results (Supporting Information Fig. S2) indicate that there are multiple statistically significant microbial communities among the three groups (LDA score > 2). Specifically, the biomarkers of the PE0 group are concentrated in Fusobacteriota. Compared to the control group, the biomarkers of the PE1 group are enriched in the phylum Firmicutes and the phylum Pseudomonas, while the biomarkers of the PE5 group are enriched in the phyla Firmicutes, Bacteroidota, Actinobacteriota, and Desulfobacterota, among others. Predicted Function and Characteristics of Microbiota According to the KEGG level 1 abundance analysis (Supporting Information Fig. S3), the primary functions of the hybrid sturgeon gut microbiota are related to metabolism, genetic information processing, cellular processes, environmental information processing, and additional categories. The KEGG level 1 functional differential analysis indicates that, compared to the PE0 group, the PE1 group exhibits higher abundances of cellular processes, organismal systems, environmental information processing, and disease-related functions, while showing lower abundances of genetic information processing and metabolism-related functions; however, these differences are not statistically significant (Figure 6a). In contrast, compared to the PE0 group, the PE5 group shows increased functional abundances related to disease, cellular processes, and biological systems, accompanied by a significant decrease in functional abundances related to genetic information processing (Figure 6b). The KEGG level 2 functional differential analysis reveals no significant changes in functional abundances between the PE0 and PE1 groups (Figure 6c). However, compared to the PE0 group, the PE5 group exhibits significantly elevated functional abundances in signal transduction, infectious diseases: bacteria, immune system, lipid metabolism, biodegradation and metabolism of xenobiotics, cellular motility, and digestive system. Conversely, the PE5 group shows significantly reduced functional abundances in translation, folding, sorting and degradation, terpenoid and polyketide metabolism, as well as nucleotide metabolism (Figure 6d). Discussion After ingesting microplastics, aquatic organisms can retain these particles in their bodies for an extended period, exhibiting characteristics of resistance to digestion. The residual microplastics not only cause physical damage to the intestine, triggering inflammatory responses, but also interfere with the normal absorption of nutrients. In a study investigating the effects of microplastic exposure on crucian carp, Mattsson et al. found that half of the individuals in the exposed group exhibited an increase in the thickness of the intestinal muscular layer, while the number of goblet cells at the tips of the villi decreased, indicating significant tissue damage (Mattsson et al., 2015 ). This study measured intestinal morphological structure indicators and conducted ANOVA analysis, revealing that the muscular layer thickness in the PE1 group was significantly lower than that in the PE0 and PE5 groups (P < 0.01). Notably, the results for the PE5 group were contrary to previous studies, which may be attributed to the fact that 1 µm microplastic particles are smaller than 5 µm particles, allowing for easier passage through the intestinal wall and deeper tissue infiltration (Stock et al., 2022 ). Microplastics can disrupt the homeostasis of the biological internal environment, affecting the composition and metabolic functions of gut microbiota, ultimately leading to dysbiosis and further interfering with digestive enzyme activity. Studies on Carassius auratus and Mytilus edulis have shown that exposure to microplastics significantly alters the gut microbiota structure and markedly inhibits the activity of serum amylase and lipase in mussels, resulting in decreased digestive capacity (Wang et al., 2021 ; Hu et al., 2022 ). This study measured and compared the activities of α-amylase, trypsin, and lipase in the intestines of hybrid sturgeon across different treatment groups. The results indicated that the activities of these three digestive enzymes in the PE1 group were significantly higher than those in the PE0 and PE5 groups (P < 0.05). Combining this with the data on the thickness of the intestinal muscular layer, we speculate that this phenomenon arises from intestinal damage induced by microplastics, which hinders the digestion and absorption of nutrients, leading to nutritional deficiency in the organism; in response to this deficiency, the organism may compensatorily increase digestive enzyme activity. In contrast, there were no significant differences in the activities of the three digestive enzymes between the PE0 and PE5 groups, a result that is inconsistent with the aforementioned studies. The reason for this discrepancy may be attributed to the smaller particle size of microplastics, which possess a larger specific surface area and adsorption capacity, allowing for more effective interaction with digestive fluids, digestive enzymes, and intestinal epithelial cells (Trestrail et al., 2021 ; Jiang et al., 2025 ). A substantial body of evidence indicates that microplastics can lead to an imbalance in the gut microbiota of animals (Lu et al., 2018 ; Jin et al., 2019 ). Studies on zebrafish, Oryzias melastigma , and brine shrimp have shown that microplastics can trigger dysbiosis in their gut microbiota, impair digestive system function, and affect normal feeding and nutrient absorption (Wan et al., 2018 ; Li et al., 2021 ; Kang et al., 2021). Consistent with previous research, this study found that microplastic treatment altered the community diversity, structural composition, and functional abundance of the gut microbiota in hybrid sturgeon. Gut microbiota plays a crucial role in enhancing host digestive capacity and immune response, which are essential for host metabolic health (Lynch et al., 2016; Karami et al., 2016). However, dysbiosis may trigger obesity and a range of harmful physiological consequences, including epithelial damage, intestinal inflammation, oxidative stress, inhibition of brain acetylcholinesterase activity, and alterations in blood biochemical indices (Chen et al., 2017; Lei et al., 2018). Research on Poecilia reticulata has shown that different concentrations (100 and 1000 µg/L) of polystyrene microplastics can inhibit the metabolic and repair pathways of gut microbiota (Huang et al., 2020 ). Based on the predictive analysis of bacterial community functional abundance, this study found that after treatment with microplastics, the functional abundance related to diseases, cellular processes, biological systems, and environmental information processing increased in the gut microbiome of hybrid sturgeon, while the abundance of functions related to genetic information processing and metabolism decreased. Additionally, the relative abundance of gut pathogens such as Desulfobacterota increased, with a corresponding enhancement in functions related to bacterial infection, the immune system, lipid metabolism, and the endocrine system. Conversely, the functional abundance associated with nucleotide metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism decreased. These changes may collectively lead to various adverse physiological responses in sturgeon (Lu et al., 2016 ). The toxic effects of microplastics exhibit size and species dependence. Studies on the Brachionus koreanus and adult male zebrafish indicate that smaller-sized polystyrene microplastics are more readily expelled by rotifers than larger-sized particles, significantly activating the mRNA and protein expression of factors such as IL1α, IL1β, and IFN in the gut. The negative effects of microplastics, such as stunted growth, decreased reproductive capacity, shortened lifespan, and prolonged reproductive cycles, are closely related to their particle size, with smaller microplastics generally exhibiting greater toxicity (Jeong et al., 2016 ; Jin et al., 2018 ). However, this study demonstrates that the 5 µm microplastic treatment group had a more significant impact on the diversity, community composition, and function of the gut microbiota in hybrid sturgeon compared to the 1 µm group. This finding contradicts the results of intestinal ultrastructure measurements and digestive enzyme activity. Such a discrepancy suggests that microplastics of different sizes may disrupt intestinal homeostasis through distinctly different pathways, warranting further investigation into the underlying mechanisms to explain this phenomenon. In summary, this study investigates the effects of microplastics of different particle sizes on the digestive system of hybrid sturgeon. The results indicate that microplastics can alter the ultrastructure of the intestine, the activity of digestive enzymes, and the composition of microbial communities. Notably, the impact of microplastics of different particle sizes on the digestive system varies significantly. The 1 µm and 5 µm particle size microplastic particles used in this study are relatively small and can easily pass through the intestinal barrier into the metabolic cycle. A limitation of the current study is its focus solely on these two small particle sizes, without considering the potential ecological toxicity of larger or smaller microplastics. Future research will expand the range of particle sizes to systematically assess the effects of a broader spectrum of microplastics on the intestinal microbiota of sturgeon and to further elucidate their toxicological mechanisms. Conclusion This study demonstrates that exposure to microplastics can alter the intestinal morphological structure, digestive enzyme activity, gut microbiota composition, diversity, and functional abundance of hybrid sturgeon. Notably, the treatment with 1 µm microplastics had a more significant impact on intestinal morphology and digestive enzyme activity, while 5 µm microplastics exhibited a more pronounced disruptive effect on gut microbiota structure and function. These findings contribute to a deeper understanding of the impact of microplastics on the gut microecology of sturgeon and provide valuable references for future exploration of the potential risks of microplastics to freshwater fish and even human health. Declarations Acknowledgements We thank Runzhao Fisheries Co., Ltd. and Xinxing Aquatic Products Co., Ltd in Sichuan Province of China for sample collection. Author’s Contribution Wu JY ,Liao QQ, Ren YY, and Senggen RB conceived and conducted the experiment. Liao QQ、Ren YY, Deng CW, Li YK, Du XG and Yang SY performed the data reduction. Liao QQ, Wang C and Yang Y analyzed the results. Liao QQ and Wu JY wrote the manuscript. The project was performed under the supervision of Yang SY. All authors reviewed the manuscript. Ethics statement The animal use protocol listed below has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval No. 20230130). Funding This study was supported by the Natural Science Foundation of Sichuan Province of China (2022NSFSC0070), Project of Sichuan Innovation Team of National Modern Agricultural Industry Technology System (SCCXTD-15), the Teaching Reform Project of Sichuan Province (JG2021-506), Science and Technology Achievement Transfer and Transformation Demonstration Project of Sichuan Province (2021ZHCG0065). Data Availability The data that support the findings of this study are available in the Additional files of this article. 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09:24:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54036,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of microplastics on digestive enzyme activity of hybrid sturgeon (*P \u0026lt; 0.05, **P \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/7f3249ebbdff062e40a2c01e.png"},{"id":93668186,"identity":"b5bee9b1-a179-4b7f-b8ac-69aa358b039c","added_by":"auto","created_at":"2025-10-16 09:24:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34971,"visible":true,"origin":"","legend":"\u003cp\u003eGut Microbial ASV Composition in Hybrid Sturgeon Under Different Treatments\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/78222cb443ad5a845cd09865.png"},{"id":93668141,"identity":"835c503f-959d-41fa-a23d-b24ef2f7d877","added_by":"auto","created_at":"2025-10-16 09:24:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81712,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of intestinal flora diversity of hybrid sturgeon after feeding different microplastics \u003cstrong\u003ea-e\u003c/strong\u003e Box plots depicting alpha diversity differences among groups\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/4249695876012454ba24aadb.png"},{"id":93668195,"identity":"7f230e8f-0297-4a63-8431-c304d79843e6","added_by":"auto","created_at":"2025-10-16 09:24:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163894,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of species composition of intestinal flora in hybrid sturgeon groups after microplastics ingestion\u003cstrong\u003e a \u003c/strong\u003eSpecies composition histogram at the phylum level \u003cstrong\u003eb \u003c/strong\u003eHistogram of key differential species at the phylum level \u003cstrong\u003ec\u003c/strong\u003e Species composition histogram at the genus level \u003cstrong\u003ed\u003c/strong\u003e Histogram of key differential species at the genus level\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/64257835d873f9f45c9de5ff.png"},{"id":93668184,"identity":"4a179615-bd3a-441e-8296-6f194ed879df","added_by":"auto","created_at":"2025-10-16 09:24:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":188901,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of functional differences in intestinal flora of hybrid sturgeon after microplastics ingestion \u003cstrong\u003ea\u003c/strong\u003e Functional difference analysis between group PE0 and group PE1 at KEGG Level 1 \u003cstrong\u003eb\u003c/strong\u003e Functional difference analysis between the PE0 and PE5 groups at KEGG Leve l \u003cstrong\u003ec\u003c/strong\u003e Functional difference analysis between the PE0 and PE1 groups at KEGG Level 2 \u003cstrong\u003ed\u003c/strong\u003e Functional difference analysis between the PE0 and PE5 groups at KEGG Level 2\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/7670bd29d52d389a479720b0.png"},{"id":104740273,"identity":"5e2a82fa-ab26-4816-bbed-beec70be0488","added_by":"auto","created_at":"2026-03-16 16:16:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":964811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/a3287eef-8a8d-4be5-8c5d-4f444cff6048.pdf"},{"id":93668672,"identity":"2cf79e15-38a1-460b-94d9-e27b3bf432f7","added_by":"auto","created_at":"2025-10-16 09:32:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":282427,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/a753a319c063beb71c8dfb6d.pdf"},{"id":93668158,"identity":"6028866e-0e13-44ed-a8fb-a237e7d90fdf","added_by":"auto","created_at":"2025-10-16 09:24:18","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":307325,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/b899b528af53535d2e5aee8b.pdf"},{"id":93668166,"identity":"8a29f42f-621e-487d-ab3a-a05c78d4208d","added_by":"auto","created_at":"2025-10-16 09:24:18","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":241589,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7701975/v1/59f9784569b77bee8b85421b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Potential Risk Assessment of Different Sizes of Microplastics on the Digestive System of Hybrid Sturgeon","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroplastics refer to plastic particles with a diameter of less than 5 mm, which are ubiquitous in marine and terrestrial ecosystems, including various foods, drinking water, and air (Duong et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Singh et al., 2025). These particles can be easily inhaled or ingested, leading to oxidative stress, inflammatory responses, and metabolic disorders (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Microplastics typically originate from cosmetics, textiles, and plastic packaging (Izlal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and are often inert materials that can break down into smaller fragments due to environmental interactions. They have the capacity to adsorb a range of persistent organic pollutants and potentially toxic elements, such as polychlorinated biphenyls polycyclic aromatic hydrocarbons, Fe, and Pb, resulting in lasting impacts on the environment and living organisms (Priya et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saha et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Research indicates that aged microplastics enhance their selective adsorption capacity through surface oxygen-containing functional groups, serve as a source of free radicals, increase environmental oxidative conditions, and even affect \u003ca class=\"FNLink\" href=\"#Fn1\" id=\"#FNLinkFn1\"\u003e\u003c/a\u003e\u003csup\u003e \u003c/sup\u003ethe expression of antibiotic resistance genes, thereby significantly altering their environmental behavior and ecological risks (Li et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The intestine is an important digestive organ in animals, containing a variety of microbial communities, including bacteria, fungi, archaea, and viruses, collectively referred to as the gut microbiota (Antfolk and Jensen, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ingestion of microplastics by animals may lead to intestinal tissue damage, alterations in intestinal enzyme activity, and even changes in the composition of gut microbiota, thereby affecting digestive function (Gu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroplastics not only pose a threat to aquatic environments but can also endanger human health through the bioaccumulation process within the food chain (Yuan et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Currently, researchers have detected the presence of microplastics in various human organs, including the liver, small intestine, and kidneys (Cao et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Studies have found that microplastics can damage the intestinal barrier, hepatocytes, and the central nervous system, causing irreversible harm to the human body(Parkhurst et al, 2025;Zha et al, 2024༛Liu et al, 2023). Particle size is a key factor influencing the toxicity of microplastics. Research on zebrafish indicates that exposure to polystyrene microplastics of different sizes can have varying degrees of adverse effects on the species. Specifically, the smallest microplastics (0.1 \u0026micro;m) significantly impact the diversity of the intestinal microbiota in zebrafish. Furthermore, compared to larger sizes (5 \u0026micro;m and 200 \u0026micro;m), smaller microplastics (100 nm) have been shown to modify the expression of genes associated with the production of reactive oxygen species (Gu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Existing studies have indicated that microplastics possess certain toxic effects on aquatic organisms, yet research on the toxic effects of microplastics of different sizes on aquatic life remains scarce. Investigating the toxicological mechanisms of microplastics of varying sizes is of great significance for understanding their ecological hazard potential and assessing risks to human health.\u003c/p\u003e\u003cp\u003eSturgeons primarily inhabit cold water environments. Caviar, commonly referred to as 'black gold', originates from sturgeons and is one of the most economically valuable fish products globally. The Siberian hybrid sturgeon is a hybrid offspring of the \u003cem\u003eAcipenser baerii\u003c/em\u003e (♂) and \u003cem\u003eA. schrenckii\u003c/em\u003e (♀). It inherits the robust disease resistance and transportability from the paternal lineage, along with the rapid growth characteristics from the maternal lineage (Zhao et al., 2020). The artificial freshwater sturgeon farming in China has seen substantial output, with extensive use of plastic products. The accumulation of microplastics within hybrid sturgeons poses a potential risk to the quality of sturgeon-derived products, ultimately leading to economic losses. Therefore, this study focuses on the hybrid sturgeons bred and raised in Sichuan Province, investigating the effects of polyethylene microplastics of different particle sizes on intestinal tissue morphology, digestive enzyme activity, and gut microbiota. The aim is to provide scientific data for further research on the toxic mechanisms of microplastics on freshwater fish such as sturgeons.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eExperimental animals and microplastics challenge\u003c/p\u003e\u003cp\u003eSiberian hybrid sturgeon fry were collected from April to July 2022, sourced from the healthy hybrid sturgeon cultivated at the Pengzhou base of Sichuan Runzhao Fisheries Co., Ltd. They were reared in 30-liter aquariums at the College of Life Sciences, Sichuan Agricultural University, with the water temperature maintained at 20\u0026deg;C for two weeks. Polyethylene microplastics were purchased from Zhengmei Plastic Products Co., Ltd. (Zhengzhou, China), and feed binders were sourced from Future Water World Biotechnology Co., Ltd. (Zhenjiang, China). Commercial sturgeon feed was crushed and mixed with microplastic powder at a ratio of 20% by weight, then dried at 60\u0026deg;C. According to published studies, significant potential concentrations of microplastics that pose harm to aquatic organisms have been reported (Wang Yongjin et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tang et al., 2020). A total of 90 sturgeons, weighing approximately 20\u0026thinsp;\u0026plusmn;\u0026thinsp;5 grams and of similar weight, were randomly divided into three groups, each containing 30 fish. The sturgeons were fed with feed that did not contain polyethylene microplastics (PE0 group), feed with 1 micron polyethylene microplastics (PE1 group), and feed with 5 micron polyethylene microplastics (PE5 group) for a duration of 30 days. The daily feeding amount was equivalent to 1% of the sturgeon's body weight. The growth and mortality rates of the sturgeons were closely monitored. Subsequently, the hybrid sturgeons were anesthetized using MS-222.\u003c/p\u003e\u003cp\u003eMeasurements of intestinal morphology\u003c/p\u003e\u003cp\u003eSamples of the intestinal tract from hybrid sturgeon were collected and prepared into paraffin sections. The intestinal samples were first fixed in 4% paraformaldehyde solution. Subsequently, a series of ethanol and xylene treatments were employed for dehydration and clearing. The tissue blocks were stained with hematoxylin and eosin (H \u0026amp; E) and observed under a Nikon Eclipse 80i microscope. Fold height and muscle layer thickness were measured using the Fiji ImageJ software.\u003c/p\u003e\u003cp\u003eAssessment of α-amylase, trypsin and lipase enzyme activity\u003c/p\u003e\u003cp\u003eThe specific enzyme activity assay kits were purchased from Kemin Biotechnology Co., Ltd. α-AMS (DFMA-2-Y) α-amylase (Suzhou, China), trypsin (YPT-2-W), and lipase (LPS-2-W) were used to determine the activities of α-amylase, trypsin, and lipase in the intestine. Approximately 0.1 g of intestinal tissue from the segment was homogenized in 0.9 mL of 0.65% NaCl solution using a cold homogenizer. Following the instructions of the assay kits, enzyme activities were measured using a spectrophotometer. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and visualized using error bar charts. Subsequently, differences between groups were assessed using one-way analysis of variance (ANOVA) and Duncan's test with SPSS 22.0 software.\u003c/p\u003e\u003cp\u003eMicrobial composition and diversity analysis\u003c/p\u003e\u003cp\u003eA random selection of six fish was subjected to intestinal analysis by BGI Genomics Co., Ltd. (Shenzhen, China). PCR amplification was performed using specific primers (338F-ACTCCTACGGGAGGCAGCAG and 806R-GGACTACHVGGGTWTCTAAT) targeting the V3 and V4 regions of the bacterial 16S rRNA gene, as described by Adams et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After purification of the PCR products, they were used for library preparation. The fragment size distribution and concentration of the library were assessed using the Agilent 2100 Bioanalyzer, and qualified libraries were sequenced on the DNBSEQ-2000 sequencing platform. The Cut Adapt software was utilized to trim the raw sequencing data (He et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) for reads matching the primers. The denoising algorithm implemented in QIIME2, DADA2, was used to denoise the data, resulting in Amplicon Sequence Variants (ASVs). Based on the ASV distribution (Schloss et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), Mothur was employed to analyze the α-diversity within individual samples. QIIME was used to calculate the β-diversity values between samples, followed by Principal Coordinates Analysis (PCoA). The abundance of species at the phylum and genus levels was analyzed using R and gplots. LEfSe was utilized to identify significantly different species through the Kruskal-Wallis and Wilcoxon tests. PICRUSt2 was applied to predict the functional capabilities of the microbial community. The output functional prediction information was analyzed and visualized using methods similar to those for ASV composition and inter-group difference analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIntestinal Fold Height and Muscularis Thickness\u003c/p\u003e\n\u003cp\u003eThe anatomical examination revealed that all fish showed no visible pathological changes in their intestines. The intestinal contents were deep brown, uniform, and viscous, with no visible impurities. Quantitative measurements of the intestinal morphological structure indicated that the thickness of the muscular layer in the PE1 group was significantly thinner compared to the PE0 and PE5 groups (P \u0026lt; 0.05) (Figure 1a), while there were no significant differences in villus length among the groups.\u003c/p\u003e\n\u003cp\u003eActivities of the digestive enzymes of \u0026alpha;-amylase, trypsin and lipase\u003c/p\u003e\n\u003cp\u003eTo investigate whether damage to the intestinal structure affects its digestive function, this study further measured three types of digestive enzymes. The intestinal \u0026alpha;-amylase activity in the PE1 group (0.0124 \u0026plusmn; 0.0022 \u0026mu;mol/min/mg) was significantly higher than that in the PE0 group (0.0070 \u0026plusmn; 0.0015 \u0026mu;mol/min/mg) and the PE5 group (0.0070 \u0026plusmn; 0.0012 \u0026mu;mol/min/mg) (P \u0026lt; 0.01) (Figure 2a). Compared to the PE0 group (0.0332 \u0026plusmn; 0.0222 U/mg) and the PE5 group (0.0270 \u0026plusmn; 0.0071 U/mg), the intestinal trypsin activity in the PE1 group (0.0765 \u0026plusmn; 0.0334 U/mg) was also significantly elevated (P \u0026lt; 0.05) (Figure 2b). The intestinal lipase activities in the PE0 group (0.1339 \u0026plusmn; 0.0202 \u0026mu;mol/min/mg), PE1 group (0.2751 \u0026plusmn; 0.2155 \u0026mu;mol/min/mg), and PE5 group (0.1786 \u0026plusmn; 0.0810 \u0026mu;mol/min/mg) exhibited a consistent trend with that of \u0026alpha;-amylase (P \u0026lt; 0.01) (Figure 2c).\u003c/p\u003e\n\u003cp\u003eMicrobial Composition and Diversity\u003c/p\u003e\n\u003cp\u003eA total of 232 amplicon sequence variants (ASVs) were obtained from the co-aggregation category. Notably, the PE5 group exhibited the highest ASV count (192), including 168 unique ASVs. In contrast, the PE1 group contained 52 ASVs, of which 24 were unique, while the PE0 group had only 29 ASVs, with 8 being unique. A total of 13 ASVs were shared among the three groups (Figure 3). Alpha diversity analysis (Figure 4a-e) indicated no significant difference in the Simpson index between the PE5 and PE0 groups (P\u0026gt;0.05). However, significant differences were observed in the Sobs, Chao, and Shannon indices (P＞0.05). Conversely, significant differences were found in the Sob, Ace, and Chao indices (P\u0026lt;0.01). The PE-MPs feeding treatment enhanced the alpha diversity of the gut microbiota in hybrid sturgeon, with the PE5 group showing a more pronounced effect than the PE1 group. Beta diversity analysis (Supporting Information Fig. S1) shows that the Weighted Unifrac distance among the three groups reaches 0.4, while the unweighted Unifrac distance between the PE1 and PE0 groups is 0.7, and the Unifrac distance between the PE5 and PE0 groups is 0.8.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicrobial Relative Abundance at the Phylum and Genus Levels\u003c/p\u003e\n\u003cp\u003eAt the phylum level (Figure 5a), the PE0 and PE1 groups are primarily composed of Fusobacteriota (75.16% and 36.10%), Firmicutes (15.21% and 38.13%), and Proteobacteria (9.60% and 25.70%). In contrast, the PE5 group exhibits a different distribution with respect to Fusobacteriota (26.42%), Firmicutes (41.56%), and Proteobacteria (31.16%). Comparative analysis of key species (Figure 5b) indicates that the abundance of Fusobacteriota in the PE0 group is significantly greater than that in the PE1 and PE5 groups (P\u0026lt;0.01). Moreover, significant differences are also observed among the three groups for Bacteroidota, Actinobacteriota, and Desulfobacterota (P\u0026lt;0.01), as well as Cyanobacteria (P\u0026lt;0.05). At the genus level (Figure 5c), the PE0 group was dominated by the genera Neisseria (9.55%), Clostridium (10.59%), and Bacteroides (75.16%). In the PE1 and PE5 groups, the dominant genera included Neisseria (25.55% and 28.54%), Clostridium (35.03% and 37.59%), Bacteroides (36.10% and 26.42%), and Pseudomonas (0.039% and 1.90%). A comparison of key species (Figure 5d) revealed that the abundance of Bacteroides in the PE0 group was significantly higher than that in the PE1 and PE5 groups (P \u0026lt; 0.01). Significant differences were also observed among the three groups for Pseudomonas (P \u0026lt; 0.01), Lactobacillus, Faecalibaculum, Dubosiella, HIMB11, Muribaculaceae (P \u0026lt; 0.01), and Clostridium (P \u0026lt; 0.05). There are no significant differences in the classification levels of other taxa. The LEfSe analysis results (Supporting Information Fig. S2) indicate that there are multiple statistically significant microbial communities among the three groups (LDA score \u0026gt; 2). Specifically, the biomarkers of the PE0 group are concentrated in Fusobacteriota. Compared to the control group, the biomarkers of the PE1 group are enriched in the phylum Firmicutes and the phylum Pseudomonas, while the biomarkers of the PE5 group are enriched in the phyla Firmicutes, Bacteroidota, Actinobacteriota, and Desulfobacterota, among others.\u003c/p\u003e\n\u003cp\u003ePredicted Function and Characteristics of Microbiota\u003c/p\u003e\n\u003cp\u003eAccording to the KEGG level 1 abundance analysis (Supporting Information Fig. S3), the primary functions of the hybrid sturgeon gut microbiota are related to metabolism, genetic information processing, cellular processes, environmental information processing, and additional categories. The KEGG level 1 functional differential analysis indicates that, compared to the PE0 group, the PE1 group exhibits higher abundances of cellular processes, organismal systems, environmental information processing, and disease-related functions, while showing lower abundances of genetic information processing and metabolism-related functions; however, these differences are not statistically significant (Figure 6a). In contrast, compared to the PE0 group, the PE5 group shows increased functional abundances related to disease, cellular processes, and biological systems, accompanied by a significant decrease in functional abundances related to genetic information processing (Figure 6b). The KEGG level 2 functional differential analysis reveals no significant changes in functional abundances between the PE0 and PE1 groups (Figure 6c). However, compared to the PE0 group, the PE5 group exhibits significantly elevated functional abundances in signal transduction, infectious diseases: bacteria, immune system, lipid metabolism, biodegradation and metabolism of xenobiotics, cellular motility, and digestive system. Conversely, the PE5 group shows significantly reduced functional abundances in translation, folding, sorting and degradation, terpenoid and polyketide metabolism, as well as nucleotide metabolism (Figure 6d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAfter ingesting microplastics, aquatic organisms can retain these particles in their bodies for an extended period, exhibiting characteristics of resistance to digestion. The residual microplastics not only cause physical damage to the intestine, triggering inflammatory responses, but also interfere with the normal absorption of nutrients. In a study investigating the effects of microplastic exposure on crucian carp, Mattsson et al. found that half of the individuals in the exposed group exhibited an increase in the thickness of the intestinal muscular layer, while the number of goblet cells at the tips of the villi decreased, indicating significant tissue damage (Mattsson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This study measured intestinal morphological structure indicators and conducted ANOVA analysis, revealing that the muscular layer thickness in the PE1 group was significantly lower than that in the PE0 and PE5 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, the results for the PE5 group were contrary to previous studies, which may be attributed to the fact that 1 \u0026micro;m microplastic particles are smaller than 5 \u0026micro;m particles, allowing for easier passage through the intestinal wall and deeper tissue infiltration (Stock et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroplastics can disrupt the homeostasis of the biological internal environment, affecting the composition and metabolic functions of gut microbiota, ultimately leading to dysbiosis and further interfering with digestive enzyme activity. Studies on \u003cem\u003eCarassius auratus\u003c/em\u003e and \u003cem\u003eMytilus edulis\u003c/em\u003e have shown that exposure to microplastics significantly alters the gut microbiota structure and markedly inhibits the activity of serum amylase and lipase in mussels, resulting in decreased digestive capacity (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study measured and compared the activities of α-amylase, trypsin, and lipase in the intestines of hybrid sturgeon across different treatment groups. The results indicated that the activities of these three digestive enzymes in the PE1 group were significantly higher than those in the PE0 and PE5 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Combining this with the data on the thickness of the intestinal muscular layer, we speculate that this phenomenon arises from intestinal damage induced by microplastics, which hinders the digestion and absorption of nutrients, leading to nutritional deficiency in the organism; in response to this deficiency, the organism may compensatorily increase digestive enzyme activity. In contrast, there were no significant differences in the activities of the three digestive enzymes between the PE0 and PE5 groups, a result that is inconsistent with the aforementioned studies. The reason for this discrepancy may be attributed to the smaller particle size of microplastics, which possess a larger specific surface area and adsorption capacity, allowing for more effective interaction with digestive fluids, digestive enzymes, and intestinal epithelial cells (Trestrail et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA substantial body of evidence indicates that microplastics can lead to an imbalance in the gut microbiota of animals (Lu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies on zebrafish, \u003cem\u003eOryzias melastigma\u003c/em\u003e, and brine shrimp have shown that microplastics can trigger dysbiosis in their gut microbiota, impair digestive system function, and affect normal feeding and nutrient absorption (Wan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kang et al., 2021). Consistent with previous research, this study found that microplastic treatment altered the community diversity, structural composition, and functional abundance of the gut microbiota in hybrid sturgeon. Gut microbiota plays a crucial role in enhancing host digestive capacity and immune response, which are essential for host metabolic health (Lynch et al., 2016; Karami et al., 2016). However, dysbiosis may trigger obesity and a range of harmful physiological consequences, including epithelial damage, intestinal inflammation, oxidative stress, inhibition of brain acetylcholinesterase activity, and alterations in blood biochemical indices (Chen et al., 2017; Lei et al., 2018). Research on \u003cem\u003ePoecilia reticulata\u003c/em\u003e has shown that different concentrations (100 and 1000 \u0026micro;g/L) of polystyrene microplastics can inhibit the metabolic and repair pathways of gut microbiota (Huang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on the predictive analysis of bacterial community functional abundance, this study found that after treatment with microplastics, the functional abundance related to diseases, cellular processes, biological systems, and environmental information processing increased in the gut microbiome of hybrid sturgeon, while the abundance of functions related to genetic information processing and metabolism decreased. Additionally, the relative abundance of gut pathogens such as Desulfobacterota increased, with a corresponding enhancement in functions related to bacterial infection, the immune system, lipid metabolism, and the endocrine system. Conversely, the functional abundance associated with nucleotide metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism decreased. These changes may collectively lead to various adverse physiological responses in sturgeon (Lu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The toxic effects of microplastics exhibit size and species dependence. Studies on the \u003cem\u003eBrachionus koreanus\u003c/em\u003e and adult male zebrafish indicate that smaller-sized polystyrene microplastics are more readily expelled by rotifers than larger-sized particles, significantly activating the mRNA and protein expression of factors such as IL1α, IL1β, and IFN in the gut. The negative effects of microplastics, such as stunted growth, decreased reproductive capacity, shortened lifespan, and prolonged reproductive cycles, are closely related to their particle size, with smaller microplastics generally exhibiting greater toxicity (Jeong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, this study demonstrates that the 5 \u0026micro;m microplastic treatment group had a more significant impact on the diversity, community composition, and function of the gut microbiota in hybrid sturgeon compared to the 1 \u0026micro;m group. This finding contradicts the results of intestinal ultrastructure measurements and digestive enzyme activity. Such a discrepancy suggests that microplastics of different sizes may disrupt intestinal homeostasis through distinctly different pathways, warranting further investigation into the underlying mechanisms to explain this phenomenon.\u003c/p\u003e\u003cp\u003eIn summary, this study investigates the effects of microplastics of different particle sizes on the digestive system of hybrid sturgeon. The results indicate that microplastics can alter the ultrastructure of the intestine, the activity of digestive enzymes, and the composition of microbial communities. Notably, the impact of microplastics of different particle sizes on the digestive system varies significantly. The 1 \u0026micro;m and 5 \u0026micro;m particle size microplastic particles used in this study are relatively small and can easily pass through the intestinal barrier into the metabolic cycle. A limitation of the current study is its focus solely on these two small particle sizes, without considering the potential ecological toxicity of larger or smaller microplastics. Future research will expand the range of particle sizes to systematically assess the effects of a broader spectrum of microplastics on the intestinal microbiota of sturgeon and to further elucidate their toxicological mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that exposure to microplastics can alter the intestinal morphological structure, digestive enzyme activity, gut microbiota composition, diversity, and functional abundance of hybrid sturgeon. Notably, the treatment with 1 \u0026micro;m microplastics had a more significant impact on intestinal morphology and digestive enzyme activity, while 5 \u0026micro;m microplastics exhibited a more pronounced disruptive effect on gut microbiota structure and function. These findings contribute to a deeper understanding of the impact of microplastics on the gut microecology of sturgeon and provide valuable references for future exploration of the potential risks of microplastics to freshwater fish and even human health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank Runzhao Fisheries Co., Ltd. and Xinxing Aquatic Products Co., Ltd in Sichuan Province of China for sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contribution\u003c/strong\u003eWu JY ,Liao QQ, Ren YY, and Senggen RB conceived and conducted the experiment. Liao QQ、Ren YY, Deng CW, Li YK, Du XG and Yang SY performed the data reduction. Liao QQ, Wang C and Yang Y analyzed the results. Liao QQ and Wu JY wrote the manuscript. The project was performed under the supervision of Yang SY. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003eThe animal use protocol listed below has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval No. 20230130).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003eThis study was supported by the Natural Science Foundation of Sichuan Province of China (2022NSFSC0070), Project of Sichuan Innovation Team of National Modern Agricultural Industry Technology System (SCCXTD-15), the Teaching Reform Project of Sichuan Province (JG2021-506), Science and Technology Achievement Transfer and Transformation Demonstration Project of Sichuan Province (2021ZHCG0065).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003eThe data that support the findings of this study are available in the Additional files of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams, RI, Miletto, M, Taylor, JW, et al. 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Progress in Fishery Sciences, 2022, 43(2): 129\u0026ndash;136.https://link.cnki.net/doi/10.19663/j.issn2095-9869.20210104001.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"polyethylene microplastics, Siberian hybrid sturgeon, digestive enzyme activity, gut microbiota, 16SrRNA gene","lastPublishedDoi":"10.21203/rs.3.rs-7701975/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7701975/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroplastics are pollutants that are widely present in aquatic environments. Currently, research on the effects of microplastics of different particle sizes on freshwater fish remains relatively limited. This study utilized polyethylene microplastic particles of 1 \u0026micro;m and 5 \u0026micro;m to expose hybrid sturgeon(\u003cem\u003eAcipenser baerii ♂\u0026times;A. schrenckii ♀\u003c/em\u003e), analyzing changes in intestinal ultrastructure, digestive enzyme activity, and gut microbial composition (based on high-throughput sequencing of the 16S rRNA V3\u0026ndash;V4 region). The results indicate that microplastics of both particle sizes cause changes in intestinal ultrastructure and digestive enzyme activity. The alpha and beta diversity of gut microbiota in the exposed groups were significantly higher than those in the control group. At the phylum level, the relative abundances of Bacteroidetes, Actinobacteria, and Desulfobacterota significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); at the genus level, the abundances of \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eDesulfovibrio, HIMB11\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e also significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, functional predictions of the microbiota indicated that the abundance of functions related to diseases, cellular processes, and organism systems increased in the 5 \u0026micro;m treatment group, while the abundance of functions related to genetic information processing significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This study reveals the potential risks of microplastics to the digestive physiology and intestinal digestive system of sturgeon, providing a basis for further exploration of the mechanisms by which different particle sizes of microplastics affect freshwater fish.\u003c/p\u003e","manuscriptTitle":"Potential Risk Assessment of Different Sizes of Microplastics on the Digestive System of Hybrid Sturgeon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 09:23:44","doi":"10.21203/rs.3.rs-7701975/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T09:12:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T02:12:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-04T16:34:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T15:00:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262533473630841364994179707235857590789","date":"2025-12-15T18:59:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73585343873892407374622246311144341850","date":"2025-12-15T18:40:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228334069827720458233121305816976833868","date":"2025-12-15T03:34:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161218844668632809014179448680957050905","date":"2025-12-11T21:56:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-03T13:35:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-27T20:43:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T14:59:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Geochemistry and Health","date":"2025-09-24T09:23:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-geochemistry-and-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"egah","sideBox":"Learn more about [Environmental Geochemistry and Health](https://www.springer.com/journal/10653)","snPcode":"10653","submissionUrl":"https://submission.nature.com/new-submission/10653/3","title":"Environmental Geochemistry and Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4474ec86-5a3a-47f2-96a2-e92c313a5c05","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:11:45+00:00","versionOfRecord":{"articleIdentity":"rs-7701975","link":"https://doi.org/10.1007/s10653-026-03028-8","journal":{"identity":"environmental-geochemistry-and-health","isVorOnly":false,"title":"Environmental Geochemistry and Health"},"publishedOn":"2026-03-14 15:59:46","publishedOnDateReadable":"March 14th, 2026"},"versionCreatedAt":"2025-10-16 09:23:44","video":"","vorDoi":"10.1007/s10653-026-03028-8","vorDoiUrl":"https://doi.org/10.1007/s10653-026-03028-8","workflowStages":[]},"version":"v1","identity":"rs-7701975","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7701975","identity":"rs-7701975","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}