A Sea Mud Feed Matrix Shapes Short-Term Dietborne Glyphosate Exposure in the Sea Cucumber (Apostichopus japonicus): Tissue Residues, Buffered Enzyme Responses, and Dominance-Structured Gut Microbiota Shifts

Deposit-feeding sea cucumbers ingest sediment-like particles, making substrate-associated exposure pathways ecologically relevant in coastal aquaculture. In this study, a sea mud feed matrix was used to evaluate short-term dietborne/substrate-linked glyphosate exposure in Apostichopus japonicus over 72 h, with the aim of characterizing early residue formation, short-term sublethal biomarker responses, and gut microbiota shifts under a benthic feeding scenario. Analytical verification confirmed a clear glyphosate gradient in the prepared feed matrices, with no glyphosate detected in the control matrix and measured concentrations of 8.66 ± 1.59 mg/kg, 1330 ± 390 mg/kg, and 6960 ± 1710 mg/kg in the low-, medium-, and high-dose groups, respectively. No mortality or obvious external lesions were observed during the exposure period. Tissue analysis confirmed measurable internal glyphosate residues and compartment-specific distribution, indicating successful internal exposure under the matrix-linked route. Most digestive and immune/antioxidant biomarkers remained relatively stable within the 72 h window; however, amylase showed a marked response in the low-dose group, and superoxide dismutase showed dose-associated changes in the medium- and high-dose groups, indicating selective sensitivity among enzyme endpoints. Gut microbiota analysis revealed a dominance-structured community with limited alpha-diversity variation among groups, whereas community composition showed subtle treatment-related shifts that were more evident at finer taxonomic resolution. Predicted functional profiles remained broadly similar across treatments. Overall, the 72 h exposure design was effective for identifying early internal exposure and short-term biological responses under a sea mud-associated feeding route, while host physiological responses remained largely buffered over this time scale and the gut microbiota provided a more sensitive interface-level signal of exposure-associated change. These findings support the value of a route-specific, gut-centered framework for evaluating early herbicide exposure responses in benthic mariculture species and suggest that matrix-associated feeding conditions may modify the apparent magnitude of short-term responses.