Abstract:Harmful algal blooms (HABs), particularly those caused by diatoms of the genus Pseudo-nitzschia, pose a significant and escalating threat to marine ecosystems and human health worldwide. These phytoplankton species are capable of producing domoic acid (DA), a potent neurotoxin that can bioaccumulate in filter-feeding shellfish, leading to amnesic shellfish poisoning (ASP) in humans. Over the past two decades, the diversity and geographical distribution of Pseudo-nitzschia species have expanded considerably, making them a global environmental concern. China, a major aquaculture producer, has witnessed increasing reports of DA contamination in various bivalve mollusks across its coastal regions. Pacific oysters (Crassostrea gigas) are a commercially important species, and studies have indicated they can accumulate higher levels of DA compared to other bivalves like mussels and scallops. Understanding the intricate processes of DA accumulation, metabolism, and its subsequent toxicological impacts within C. gigas is crucial for effective risk assessment, monitoring strategies, and safeguarding both consumer health and aquaculture sustainability. While research has explored DA distribution in various tissues and the influence of species and size on accumulation, a comprehensive understanding of its metabolic kinetics and detailed toxicological mechanisms, particularly concerning oxidative stress, remains limited in Pacific oysters. This study aimed to comprehensively investigate the metabolic kinetic processes and toxic effects of domoic acid (DA) in Pacific oysters (Crassostrea gigas) following exposure to the toxic diatom Pseudo-nitzschia cuspidata. Specifically, the research sought to: Quantify the dynamic accumulation and depuration of DA in key tissues (visceral mass, gonads, gills, adductor muscle, and mantle) of Pacific oysters under different exposure densities of P. cuspidate; Elucidate the tissue-specific distribution and metabolic clearance rates of DA in C. gigas; Assess the impact of DA exposure on oxidative stress biomarkers in the visceral mass of Pacific oysters, including lipid peroxidation levels and the activity of key antioxidant enzymes; Evaluate the overall toxicological response of C. gigas to DA using an integrated biomarker response (IBR) index to capture the combined effects of multiple biomarkers; Provide critical data to inform the development of effective monitoring programs and risk management strategies for DA contamination in Pacific oysters. A controlled laboratory experiment was conducted over 24 days, comprising a 14-day accumulation phase followed by a 10-day metabolism phase. Pacific oysters (Crassostrea gigas) were acquired and acclimated. Three experimental groups were established: a control group (CK) fed with Chlorella vulgaris, a low-density exposure group (L) fed with P. cuspidata at a density of 5×10? cells/L, and a high-density exposure group (H) fed with P. cuspidata at a density of 2×10? cells/L. Both exposure groups received P. cuspidata during the accumulation phase, while during the metabolism phase, all groups were fed C. vulgaris. Water temperature was maintained at 20 ℃ and salinity at 31 ‰. Samples of oysters were collected at specific time points throughout the experiment (days 0, 1, 3, 5, 7, 10, 14, 15, 17, 19, 21, and 24). For DA analysis, five different tissues were dissected: visceral mass, gonads, gills, adductor muscle, and mantle. DA extraction was performed using a 50% methanol solution followed by solid-phase extraction (SPE) purification. Quantitation of DA and its isomers was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To assess oxidative stress, the visceral mass of oysters was analyzed for the following biomarkers: malondialdehyde (MDA) as an indicator of lipid peroxidation, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), glutathione-S-transferase (GST), and total glutathione (GSH) as key components of the antioxidant defense system. Cytochrome P450 (CYP450) activity was also measured, which is involved in xenobiotic metabolism. Enzyme activities were determined using commercially available assay kits according to the manufacturer's instructions. The integrated biomarker response (IBR) index was calculated to provide a comprehensive assessment of the overall toxicological impact. This involved standardizing the individual biomarker responses and then calculating the area of a polygon formed by these standardized responses on a radar plot. Statistical analysis, including one-way ANOVA and LSD post-hoc tests, was performed using SPSS software (version 28) to determine significant differences between groups (P < 0.05). The study demonstrated that Pseudo-nitzschia cuspidata exhibited optimal growth and toxin production during its exponential and early stationary phases, with peak cell densities reaching approximately 1.33×10? cells/L and an average single-cell toxin production of 2.86 ± 0.865 fg/cell. DA was the predominant toxin, with minor contributions from isomers like epi-DA and DA-D, consistent with previous findings. During the 14-day accumulation phase, DA levels significantly increased in all tested tissues of the Pacific oysters in both the low (L) and high (H) density groups compared to the control. The visceral mass emerged as the primary target organ for DA accumulation, with concentrations reaching (9.41 ± 1.40) μg/kg in the L group and (15.91 ± 0.59) μg/kg in the H group after 14 days. Gonads and mantle showed lower accumulation, while adductor muscle and mantle exhibited the lowest DA levels in the L and H groups, respectively. Following the 10-day metabolism phase, a significant reduction in DA levels was observed across all tissues in both exposure groups. The visceral mass showed the highest clearance rates, reaching 84% and 85% in the L and H groups, respectively. Tissue-specific differences in DA clearance were evident, with the order of clearance rate being visceral mass > mantle > gills > gonads > adductor muscle. Despite depuration, DA remained detectable in the visceral mass and gills, indicating persistent accumulation. Analysis of DA distribution revealed that the visceral mass consistently showed the highest percentage of DA content throughout the accumulation phase. In contrast, gill DA percentage decreased during accumulation. Post-exposure (day 15), the percentage of DA in the visceral mass significantly decreased, while it increased in the mantle, gills, and gonads, suggesting potential redistribution. During the metabolism phase, DA percentage in the visceral mass showed fluctuations, while gills, mantle, and gonads generally followed an initial increase and subsequent decrease. The exposure to P. cuspidata significantly impacted the oxidative stress biomarkers in the visceral mass. Compared to the control group, both L and H groups exhibited significantly increased MDA levels and CAT activity after 14 days of exposure and 10 days of metabolism, indicating heightened lipid peroxidation and oxidative damage. The H group consistently showed higher MDA and CAT levels than the L group. SOD activity was initially suppressed in both L and H groups at 7 days, but then increased significantly in the L group at 14 days, suggesting a potential adaptive response. However, the H group experienced sustained suppression of SOD activity throughout the experiment, even intensifying during the metabolism phase. GSH, GST, and GSH-PX activities were significantly reduced in both exposure groups after 14 days of exposure, with the H group showing more pronounced inhibition. During the metabolism phase, GST and GSH-PX activities recovered and even surpassed control levels in the L group, while GSH remained significantly lower. CYP450 content was higher in the H group than in the L group at earlier time points. Principal Component Analysis (PCA) of these enzyme activities revealed distinct clustering of the control group from the exposure groups, with significant changes observed at 14 days of exposure, and a trend towards recovery after the metabolism phase. The Integrated Biomarker Response (IBR) index analysis underscored the dose-dependent toxic effects. The H group exhibited a significantly higher IBR value (50.33) compared to the L group (approximately 1.7 times higher), indicating a more pronounced comprehensive stress response at higher DA exposure levels. The L group showed a recovery trend in IBR values during the metabolism phase, whereas the H group indicated a more persistent stress burden. This study provides critical insights into the accumulation, metabolism, and toxic effects of domoic acid (DA) in Pacific oysters (Crassostrea gigas) exposed to the toxic diatom Pseudo-nitzschia cuspidata. The results confirm that the visceral mass is the primary site of DA accumulation in C. gigas, and that higher algal densities lead to significantly greater DA burden. While Pacific oysters demonstrate a capacity for DA depuration, particularly from the visceral mass, the toxin can persist in certain tissues, suggesting a potential for chronic exposure even after the cessation of bloom events. The exposure to DA induces significant oxidative stress in the visceral mass of Pacific oysters. This is evidenced by increased lipid peroxidation (MDA) and altered activities of key antioxidant enzymes. The response is dose-dependent, with higher DA concentrations causing more severe and persistent oxidative damage. While low-level exposure may trigger adaptive responses, such as compensatory upregulation of SOD and CAT in the L group, high-level exposure can overwhelm the antioxidant defense system, leading to chronic inhibition of crucial enzymes like SOD and GSH-PX, and depletion of GSH. These disruptions in the antioxidant defense system suggest a compromised ability to mitigate cellular damage and potentially impair physiological functions, growth, reproduction, and survival. The Integrated Biomarker Response (IBR) index effectively integrates the responses of multiple biomarkers, providing a robust measure of the overall toxicological impact. The higher IBR values in the high-density exposure group highlight the significant cumulative stress imposed by higher DA levels, emphasizing the importance of considering the cumulative effects of toxins. The observed recovery in the low-density group during the metabolism phase underscores the resilience of oysters to moderate stress, while the persistent stress indicators in the high-density group suggest impaired recovery potential.
