GC–MS metabolomics coupled with multi-biomarker analysis reveal toxic effects of functionalized nanoplastics in Paphia undulata

Despite the widespread occurrence of nanoplastics (NPs) in marine ecosystems, a knowledge gap persists regarding their bioaccumulation and toxic effects in marine bivalves, particularly for NPs with surface modifications. This study employed combined metabolomics and multi-biomarker analyses to investigate the bioaccumulation, elimination, and toxicity of pristine polystyrene (PS), carboxylated PS (PS-COOH), and aminated PS (PS-NH₂) NPs in marine clam Paphia undulata. Results revealed distinct tissue-specific accumulation patterns, showing highest NP uptake in intestines (109.34 μg/g), followed by digestive glands (30.36 μg/g), with minimal uptake in gills (7.47 μg/g). Following a 4-day exposure, NPs triggered tissue-specific antioxidant responses, inducing significant oxidative damage (elevated malondialdehyde) in digestive glands. PS-NH₂ activated the superoxide dismutase (SOD) and glutathione (GSH) systems, while PS and PS-COOH elicited synergistic upregulation of SOD and catalase (CAT) activities, indicating surface chemistry-dependent detoxification pathways. GC-MS-based metabolomics revealed that NPs markedly disrupted metabolic homeostasis in digestive glands. Significant alterations occurred in carbohydrates, amino acids, lipids, and organic acids, with PS-COOH exposure yielding the most differential metabolites (PS-COOH: 27, PS: 21, PS-NH₂: 20). Pathway enrichment analysis showed all NPs interfered galactose metabolism and starch/sucrose metabolism, disrupting energy homeostasis. Additionally, PS dysregulated phenylalanine, tyrosine and tryptophan biosynthesis, whereas PS-COOH perturbed cysteine and methionine metabolism, demonstrating surface chemistry-specific dysregulation of distinct amino acid pathways. This work provides mechanistic insights into how NP surface chemistry modulates toxicity in marine bivalves. The integration of biomarker responses with metabolome perturbations offers a systematic framework for evaluating NP ecotoxicity, informing ecological risk assessments for marine benthic ecosystems.