Effects of the plastic additive 2,4-di-tert-butylphenol on intestinal microbiota of zebrafish.

Plastic additives such as the antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) have been widely detected in aquatic environments, over a wide range of concentrations reaching 300 µg/L in surface water, potentially threatening the health of aquatic organisms and ecosystems. However, knowledge of the specific effects of 2,4-DTBP on aquatic vertebrates is still limited. In this study, adult zebrafish were exposed to different concentrations of 2,4-DTBP (0, 0.01, 0.1 and 1.0 mg/L) for 21 days in the laboratory. The amplicon sequencing results indicated that the diversity and composition of the zebrafish gut microbiota were significantly changed by 2,4-DTBP, with a shift in the dominant flora to more pathogenic genera. Exposure to 2,4-DTBP at 0.1 and 1.0 mg/L significantly increased the body weight and length of zebrafish, suggesting a biological stress response. Structural assembly defects were also observed in the intestinal tissues of zebrafish exposed to 2,4-DTBP, including autolysis of intestinal villi, adhesions and epithelial detachment of intestinal villi, as well as inflammation. The transcriptional expression of some genes showed that 2,4-DTBP adversely affected protein digestion and absorption, glucose metabolism and lipid metabolism. These results are consistent with the PICRUSt2 functional prediction analysis of intestinal microbiota of zebrafish exposed to 2,4-DTBP. This study improves our understanding of the effects of 2,4-DTBP on the health of aquatic vertebrates and ecosystems.

How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Detection and dietary risk of per- and polyfluoroalkyl substances in shellfish products from the coasts of Bohai Sea and South China Sea.

Artificial per- and polyfluoroalkyl substances (PFASs) are widely distributed in the environment and are potentially harmful to human health. This study assessed the matrix effect of different shellfish on LC-MS analysis and the recoveries of PFASs in purified extracts purified by adding ENVI-Carb graphitized carbon black. Total 76 samples were collected from coastal cities of the Bohai Sea and South China Sea in China. Results showed that the signal response of perfluorocarboxylic acid increased with the length of fluorocarbon chains. ENVI-Carb can mitigate the shellfish matrix effects for analysis of PFASs. Ten PFASs components were detected in shellfish samples at concentrations ranging from 1.3 to 8.5 ng/g wet weight. The PFOA and PFHxS were the dominant components, and PFOA, PFTrDA and PFNA were detected at high rates of 58-93%. The highest levels of ∑PFASs were accumulated in clams, while the lowest levels were found in mussels. The dietary risk assessment indicated that PFASs potentially threaten human health via consumption of clam products in the Bohai Sea region. This study will improve the understanding of the contamination status and the dietary risk of PFASs in shellfish products along the coasts of Bohai Sea and South China Sea in China.

Responses of the intestinal microbiota to exposure of okadaic acid in marine medaka Oryzias melastigma.

It is still limited that how the microalgal toxin okadaic acid (OA) affects the intestinal microbiota in marine fishes. In the present study, adult marine medaka Oryzias melastigma was exposed to the environmentally relevant concentration of OA (5 µg/L) for 10 days, and then recovered in fresh seawater for 10-days depuration. Analysis of taxonomic composition and diversity of the intestinal microbiota, as well as function prediction analysis and histology observation were carried out in this study. Functional prediction analysis indicated that OA potentially affected the development of colorectal cancer, protein and carbohydrate digestion and absorption functions, and development of neurodegenerative diseases like Parkinson's disease, which may be associated with changes in Proteobacteria and Firmicutes in marine medaka. Significant increases of C-reactive protein (CRP) and inducible nitric oxide synthase (iNOS) levels, as well as the changes of histology of intestinal tissue demonstrated that an intestinal inflammation was induced by OA exposure in marine medaka. This study showed that the environmental concentrations of OA could harm to the intestinal microbiota thus threatening the health of marine medaka, which hints that the chemical ecology of microalgal toxins should be paid attention to in future studies.

Mechanistic insights into the effects of diuron exposure on Alexandrium pacificum.

Diuron (N-(3,4-dichlorophenyl)-N,N­dimethylurea, DCMU), a ureic herbicide, is extensively used in agriculture to boost crop productivity; however, its extensive application culminates in notable environmental pollution, especially in aquatic habitats. Therefore, the present study investigated the effect of diuron on the dinoflagellate Alexandrium pacificum, which is known to induce harmful algal blooms (HAB), and its potential to biodegrade DCMU. Following a four-day DCMU exposure, our results revealed that A. pacificum proficiently assimilated DCMU at concentrations of 0.05 mg/L and 0.1 mg/L in seawater, attaining a complete reduction (100 % efficiency) after 96 h for both concentrations. Moreover, evaluations of paralytic shellfish toxins content indicated that cells subjected to higher DCMU concentrations (0.1 mg/L) exhibited reductions of 73.4 %, 86.7 %, and 75 % in GTX1, GTX4, and NEO, respectively. Exposure to DCMU led to a notable decrease in A. pacificum's photosynthetic efficacy, accompanied by increased levels of reactive oxygen species (ROS) and suppressed cell growth, with a growth inhibition rate of 41.1 % at 72 h. Proteomic investigations pinpointed the diminished expression levels of specific proteins like SxtV and SxtW, linked to paralytic shellfish toxins (PSTs) synthesis, as well as key proteins associated with Photosystem II, namely PsbA, PsbD, PsbO, and PsbU. Conversely, proteins central to the cysteine biosynthesis pathways exhibited enhanced expression. In summary, our results preliminarily resolved the molecular mechanisms underlying the response of A. pacificum to DCMU and revealed that DCMU affected the synthesis of PSTs. Meanwhile, our data suggested that A. pacificum has great potential in scavenging DCMU.

Apoptosis and oxidative stress of mouse breast carcinoma 4T1 and human intestinal epithelial Caco-2 cell lines caused by the phycotoxin gymnodimine-A.

Gymnodimine-A (GYM-A) is a cyclic imine phycotoxin produced by some marine dinoflagellates. It can cause rapid death of mice via intraperitoneal administration and frequently accumulate in shellfish potentially threatening human health. In this study, four different cell lines were exposed to GYM-A for the viability assessment. Results showed that GYM-A was cytotoxic with concentration-dependent pattern to each cell type, with mean IC50 values ranging from 1.39 to 2.79 µmol L-1. Results suggested that the loss of cell viability of 4T1 and Caco-2 cells was attributed to apoptosis. Furthermore, the collapse of mitochondrial membrane potential and caspases activation were observed in the GYM-A-treated cells. Reactive oxygen species (ROS) and lipid peroxides (LPO) levels were markedly increased in 4T1 and Caco-2 cells exposed to GYM-A at 2 µmol L-1, and the oxidative stress in 4T1 cells was more obvious than that in Caco-2 cells. Additionally, unusual ultrastructure impairment on mitochondria and mitophagosomes occurred in the GYM-A-treated cells. These results suggested that an ROS-mediated mitochondrial pathway for apoptosis and mitophagy was implicated in the cytotoxic effects induced by GYM-A. This is the first report to explore the cytotoxic mechanisms of GYM-A through apoptosis and oxidative stress, and it will provide theoretical foundations for the potential therapeutic applications of GYM-A.

Adverse effects of plastic leachate and its component 2,4-DTBP on the early development of zebrafish embryos.

Plastic waste has become a global environmental problem threatening the health of aquatic organisms especially via leachate. In this study, the test of zebrafish embryo showed adverse effects of leachate from some agricultural mulching films after UV light aging for 60 h. A typical phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) was detected in the leachate and tested further for the zebrafish embryo biotoxicity. The microplastic leachate (6, 8 g/L, mass concentration measured by weight of plastic) increased the death and malformation rates, and reduced the hatching rate, heart rate, and body length of zebrafish larvae in the 96-hour early development period. Similar adverse effects were also caused by the 2,4-DTBP (0.01, 0.1, 1.0 mg/L, corresponding to 0.049, 0.49, and 4.85 µM) to some degree but could not completely explain the significant influences caused by the plastic leachate. Transcriptome analysis of zebrafish embryos exposed to the 2,4-DTBP for 96 h showed that the protein, fat, and carbohydrate digestion and absorption pathways, pancreatic secretion, PPAR signaling pathway, tryptophan metabolism, and adipocytokine signaling pathway were considerably down-regulated, but the cholesterol metabolism pathway was up-regulated in larval zebrafish. The altered transcriptional expression of mRNA at early development stage (96 h post fertilization) of zebrafish suggested that the 2,4-DTBP caused reduction of digestive capacity and pancreatic secretory function, and adversely affected processes associated with energy metabolism and glycolipid metabolism of larval zebrafish. This study helps us further understanding the effects of plastic leachate on the early development of fishes.

Effects of marine phycotoxin dinophysistoxin-1 on the growth and cell cycle of Isochrysis galbana.

The phycotoxin dinophysistoxins are widely distributed in the global marine environments and potentially threaten marine organisms and human health. The mechanism of the dinophysistoxin toxicity in inhibiting the growth of microalgae is less well understood. In this study, effects of the dissolved dinophysistoxin-1 (DTX1) on the growth, pigment contents, PSII photosynthetic efficiency, oxidative stress response and cell cycle of the marine microalga Isochrysis galbana were investigated. Growth of I. galbana was significantly inhibited by DTX1 with 0.6-1.5 µmol L-1 in a 96-h batch culture, corresponding the 96 h-EC50 of DTX1 at 0.835 µmol L-1. The maximum quantum yield of PSII (Fv/Fm), and light utilization efficiency (α) were obviously reduced by DTX1 at 1.5 µmol L-1 during 96-h exposure. Contents of most of pigments were generally reduced by DTX1 with a dose-depend pattern in microalgal cells except for diatoxanthin. The ROS levels were increased by DTX1 with 0.6-1.5 µmol L-1 after 72-h exposure, while the contents or activities of MDA, GSH, SOD and CAT were significantly increased by DTX1 at 1.5 µmol L-1 at 96 h. The inhibitory effect of DTX1 on the growth of I. galbana was mainly caused by the production of ROS in the cells. Cell cycle analysis showed that the I. galbana cell cycle was arrested by DTX1 at G2/M phase. This study enhances the understanding of the chemical ecology effects of DTX1 on marine microalgae and also provides fundamental data for deriving water quality criteria of DSTs for marine organisms.

Effects of lipophilic phycotoxin okadaic acid on the early development and transcriptional expression of marine medaka Oryzias melastigma.

The lipophilic okadaic acid (OA)-group toxins produced by some species of Dinophysis spp. and Prorocentrum spp. marine dinoflagellates have been frequently and widely detected in natural seawater environments, e.g. 2.1∼1780 ng/L in Spanish sea and 5.63∼27.29 ng/L in the Yellow Sea of China. The toxicological effects of these toxins dissolved in seawater on marine fish is still unclear. Effects of OA on the embryonic development and 1-month old larvae of marine medaka (Oryzias melastigma) were explored and discussed in this study. Significantly increased mortality and decreased hatching rates occurred for the medaka embryos exposed to OA at 1.0 µg/mL. Diverse malformations including spinal curvature, dysplasia and tail curvature were also observed in the embryos exposed to OA and the heart rates significantly increased at 11 d post fertilization. The 96 h LC50 of OA for 1-month old larvae was calculated at 3.80 µg/mL. The reactive oxygen species (ROS) was significantly accumulated in medaka larvae. Catalase (CAT) enzyme activity was significantly increased in 1-month old larvae. Acetylcholinesterase (AChE) activity significantly increased with a dose-dependent pattern in 1-month old larvae. Differentially expressed genes (DEGs) were enriched in 11 KEGG pathways with Q value < 0.05 in 1-month old medaka larvae exposed to OA at 0.38 µg/mL for 96 h, which were mainly related to cell division and proliferation, and nervous system. Most of DEGs involved in DNA replication, cell cycle, nucleotide excision repair, oocyte meiosis, and mismatch repair pathways were significantly up-regulated, while most of DEGs involved in synaptic vesicle cycle, glutamatergic synapse, and long-term potentiation pathways were markedly down-regulated. This transcriptome analysis demonstrated that a risk of cancer developing was possibly caused by OA due to DNA damage in marine medaka larvae. In addition, the neurotoxicity of OA was also testified for marine fish, which potentially cause major depressive disorder (MDD) via the up-regulated expression of NOS1 gene. The genotoxicity and neurotoxicity of OA to marine fish should be paid attention to and explored further in the future.

Cell cycle of microalga Isochrysis galbana arrested by neurotoxin ß-N-methylamino-l-alanine and corresponding molecular mechanisms.

The phycotoxin ß-N-methylamino-l-alanine (BMAA) has attracted attention due to its risks to marine organisms and human health. In this study, approximately 85 % of synchronized cells of the marine microalga Isochrysis galbana were arrested at the cell cycle G1 phase by BMAA at 6.5 µM for a 24-h exposure. The concentration of chlorophyll a (Chl a) gradually decreased, while the maximum quantum yield of PSII (Fv/Fm), the maximum relative electron transport rate (rETRmax), light utilization efficiency (α) and half-saturated light irradiance (Ik) reduced early and recovered gradually in I. galbana exposed to BMAA in 96-h batch cultures. Transcriptional expression of I. galbana analyzed at 10, 12, and 16 h disclosed multiple mechanisms of BMAA to suppress the microalgal growth. Production of ammonia and glutamate was limited by the down-regulation of nitrate transporters, glutamate synthase, glutamine synthetase, cyanate hydrolase, and formamidase. Diverse extrinsic proteins related to PSII, PSI, cytochrome b6f complex, and ATPase were influenced by BMAA at transcriptional level. Suppression of the DNA replication and mismatch repair pathways increased the accumulation of misfolded proteins, which was reflected by the up-regulated expression of proteasome to accelerate proteolysis. This study improves our understanding of the chemical ecology impacts of BMAA in marine ecosystems.

Occurrence and spatial distribution of paralytic shellfish toxins in seawater and marine organisms in the coastal waters of Qinhuangdao, China.

In recent years, paralytic shellfish toxins (PSTs) have been prevalent in the coastal waters of Qinhuangdao, the west coast of the Bohai Sea, China. The content of PSTs in shellfish often exceeded the regulatory limit of 800 µg STX equivalent (eq.) kg-1, which poses a serious threat to human health. In this study, two surveys were conducted in May 2021 and May 2022 to investigate the distribution of PSTs in the coastal waters of Qinhuangdao. Seawater, surface sediment, phytoplankton, zooplankton, and other marine organism samples were collected, and the composition and concentration of PSTs were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results showed that multiple PST components were detected in all seawater samples collected at different depths, mainly including GTX1/4, GTX2/3, dcGTX2, STX and C1/2, and the highest concentration of PSTs reached 244 ng STX eq. L-1. The sediment samples also contained low levels of C1/2 and GTX2/3. Trace amounts of C1/2 and GTX1-4 were detected in phytoplankton and zooplankton. Moreover, all bivalve shellfish samples were found to contain PSTs, and the scallop Azumapecten farreri and the ark clam Anadara kagoshimensis showed relatively high concentrations of 607 and 497 µg STX eq. kg-1, respectively. In addition, low levels of PSTs were also found in some non-traditional PST vectors, including whelk Rapana venosa, octopus Amphioctopus ovulum, goby Ctenotrypauchen chinensis, and greenling Hexagrammos agrammus. Results of this study improve the understanding of the distribution of PSTs in seawater and marine organisms and the potential risk of persistent PSTs in seawater to marine ecosystems and human health.

Putative biosynthesis mechanism of the neurotoxin ß-N-methylamino-L-alanine in marine diatoms based on a transcriptomics approach.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been presumed as an environmental cause of human neurodegenerative disorders, such as Alzheimer's disease. Marine diatoms Thalassiosira minima are demonstrated here to produce BMAA-containing proteins in axenic culture while the isomer diaminobutyric acid was bacterially produced. In the co-culture with Cyanobacterium aponinum, diatom growth was inhibited but the biosynthesis of BMAA-containing proteins was stimulated up to seven times higher than that of the control group by cell-cell interactions. The stimulation effect was not caused by the cyanobacterial filtrate. Nitrogen deprivation also doubled the BMAA content of T. minima cells. Transcriptome analysis of the diatom in mixed culture revealed that pathways involved in T. minima metabolism and cellular functions were mainly influenced, including KEGG pathways valine and leucine/isoleucine degradation, endocytosis, pantothenate and CoA biosynthesis, and SNARE interactions in vesicular transport. Based on the expression changes of genes related to protein biosynthesis, it was hypothesized that ubiquitination and autophagy suppression, and limited COPII vesicles transport accuracy and efficiency were responsible for biosynthesis of BMAA-containing proteins in T. minima. This study represents a first application of transcriptomics to investigate the biological processes associated with BMAA biosynthesis in diatoms.

Cytotoxicity and intestinal permeability of phycotoxins assessed by the human Caco-2 cell model.

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Stability and Chemical Conversion of the Purified Reference Material of Gymnodimine-A under Different Temperature and pH Conditions.

Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (-20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at -20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤-20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study.

Matrix Effect of Diverse Biological Samples Extracted with Different Extraction Ratios on the Detection of ß-N-Methylamino-L-Alanine by Two Common LC-MS/MS Analysis Methods.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.

Bioaccessibility of paralytic shellfish toxins in different cooked shellfish using the simulated digestive model.

Current regulatory limit values for paralytic shellfish toxins (PSTs) in shellfish products are not considering the bioaccessibility of PSTs in seafood matrix during the gastrointestinal digestion for human beings. In this study, the bioaccessibility of PSTs in the shellfish cooked by different ways was assessed using a static in vitro human digestion model. Results showed that the dissolution of PSTs from shellfish tissues was not significantly affected by digestion time, ratios of solid weight to liquid volume (S/L) and cooking methods, but obviously facilitated by digestive enzymes. Different cooking ways reduced the contents of PSTs in shellfish by 45% to 88%, but did not significantly change the high bioaccessibility of PSTs that ranged from 80% to 95% in four different shellfish matrices. Transformation or degradation of PSTs occurred during the simulated digestion process. This work will help us to objectively assess the potential risks of PSTs to human health.

Comparative study on the esterification of gymnodimine in different shellfish exposed to the dissolved toxin in seawater.

Some lipophilic phycotoxins dissolved in seawater can be accumulated by bivalves via the filtering process. To explore the relationship between the bioaccumulation of gymnodimine-A (GYM-A) and free fatty acids (FFAs) of shellfish, three species of bivalves (venus clam Meretrix meretrix, mussel Mytilus galloprovincialis, and ark shell Anadara kagoshimensis) were exposed to dissolved GYM-A for 7 days in the same seawater system. Results indicated that GYM-A can be accumulated by these bivalves from the dissolved phase and esterified with FFAs reaching over 90% in most tissues of bivalves. Gymnodimine-A and its esters mainly distributed in the gills of shellfish, and the highest concentration of toxins occurred in mussel, followed by ark shell and venus clam. Similar percent of different fatty acid esters occurred in the experimental shellfish, in which the C16:0, C17:0, C18:0, C18:1, C20:1, C20:2, C22:2, and C22:6-GYM-A esters were the main metabolites of GYM-A. The binding capacity of fatty acids and GYM-A varied in different FFAs, which can explain why the C20:1-GYM-A ester dominated the ester profile while C16:0 was the most abundant fatty acid in all samples. Comparing with the FFA profile of shellfish in the control groups, overexpression of some FFAs occurred in the tissues of shellfish exposed to GYM-A in the experimental groups, which suggested that biosynthesis of FFAs was affected by the accumulation and metabolism of GYM-A in bivalves. Multiple fatty acids including some valuably nutritional FFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were consumed in the esterification metabolism of GYM-A, which hinted that the lipid metabolism and nutritional quality of shellfish affected by the contamination of GYMs should be explored and assessed in future works.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Prevalence and distribution of domoic acid and cyclic imines in bivalve mollusks from Beibu Gulf, China.

Beibu Gulf is an important shellfish aquaculture area in the northwest of the South China Sea, China. In this study, the toxin profile and spatial-temporal distribution of domoic acid (DA) and 10 lipophilic phycotoxins were systematically analyzed in the bivalve mollusks collected in Beibu Gulf from October 2018 to October 2020. Neurotoxin DA was first detected in the mollusks from the investigative regions with a prevalence of 17.7%, peaking at 401 µg kg-1. Cyclic imines (CIs) including gymnodimine-A (GYM-A, 46.6%) and 13-desmethyl-spirolide-C (SPX1, 15.8%) predominated the lipophilic phycotoxins in shellfish, peaking at 10.1 µg kg-1 and 19.6 µg kg-1, respectively. Gymnodimine-A partially accompanied by SPX1 was detected in all batches of shellfish samples, suggesting that Alexandrium ostenfeldii and Karenia selliformis were possible sources of CIs-group toxins in Beibu Gulf. During the investigative period, relatively higher levels of DA occurred in shellfishes from March to August, while slightly higher contents of CIs in mollusks appeared in October and December. Spatial distribution of the targeted phycotoxins demonstrated that shellfishes tended to accumulate relatively higher contents of toxins in Lianzhou, Qinzhou and Tieshan bays.

Performance of different extraction methods for paralytic shellfish toxins and toxin stability in shellfish during storage.

Accurate analysis of paralytic shellfish toxins (PSTs) in shellfish is important to protect seafood safety and human health. In this study, the performance of different extraction protocols for PSTs from scallop tissues is compared and discussed, including regular extraction solvents hydrochloric acid (HCl) and acetic acid (AcOH) followed by heating and solid-phase extraction (SPE) purification, and a novel technique of matrix solid-phase dispersion (MSPD) without heating. The possible conversion of C1/2 and GTX2/3 standards after heating, and the stability of PSTs in wet scallop tissues stored at -20 °C for a 6-month period are also explored. Results showed that the MSPD technique could effectively mitigate matrix interference, but its recoveries of PSTs were significantly lower than those of the HCl and AcOH extraction methods followed by carbon SPE purification. The molar concentrations of M-toxins obtained by the MSPD method were generally lower than those analyzed by the HCl and AcOH extraction methods, which demonstrated a weak chemical conversion of C1/2 and GTX2/3 due to the heating process. Most of the PSTs were relatively stable in scallop tissues during 1-month storage at -20 °C, while the concentrations of PSTs in scallop tissues obviously changed after 6 months due to the degradation and transformation of PSTs during long-term storage at -20 °C. This work helps improve our understanding of the performance of different extraction methods and the stability of PSTs in scallop tissues stored at -20 °C.