Integrated analysis of marine biotoxins and contaminants of emerging concern in bivalve mollusks from Santa Catarina, Brazil.

Santa Catarina is the main producer state of oysters and mussels in Brazil, reaching 98 % of national production. To assure the safety of bivalve mollusks production, control programs of marine biotoxins (MBs) have been continuously performed. Herein, the co-occurrence of MBs and contaminants of emerging concern (CECs) in oyster and mussels from the main production sites of Santa Catarina was reported, covering 178 compounds. Samples of wild and non-cultivated oysters and mussels were also assessed. Chemometric tools were used to evaluate and optimize several sample preparation techniques such as solid-liquid, ultrasound assisted, and pressurized liquid extraction. The optimized protocol was based on ultrasound assisted extraction followed by liquid chromatography coupled to tandem mass spectrometry. The results showed the incidence of several CECs and MBs. In the case of MBs, all results were below the regulatory limits for both cultivated and non-cultivated samples. Wild mollusks have shown a higher number of compounds. Regarding CECs, the more frequent compounds were caffeine, diclofenac, meloxicam, and sertraline. Domoic acid and okadaic acid were the main toxins detected. The results highlighted the need of monitoring for MBs and the potential of oyster and mussels as sentinel organisms to risk analysis of CECs in coastal regions. To the best of our knowledge, this is the first method to describe a simultaneous sample preparation and analysis of CECs and MBs in bivalve mollusks, as well as the first report of meloxicam and florfenicol in mussels and oysters.

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.

Analysis of highly polar marine biotoxins in seawater by hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry.

The monitoring of marine biotoxins (MBTs) in seawater is presented as an alternative strategy to determine their presence and the possible implications in the ecosystem. For this, an analytical method based on hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry (HILIC-HRMS) has been developed to identify and quantify some hydrophilic MBTs in seawater: saxitoxin (STX), decarbamoyl-saxitoxin (dcSTX), neosaxitoxin (NeoSTX), gonaytoxin-2,3 (GTX-2,3) and tetrodotoxin (TTX), which are responsible of gastrointestinal and central nervous system distress in humans when are consumed via seafood. Particulate and filtrate portion were analyzed separately in order to characterize the extracellular toxins dissolved in the water and those present in the particulate. Ultrasound assisted solid-liquid extraction with methanol was used for the isolation of the MBTs from particulate and solid phase extraction using silica cartridges for the filtrate. Extraction procedure was the most critical step during the analytical method due to the high polarity of the toxins and the absolute recoveries obtained ranged from 15 to 47 % in the filtrate and 26 to 71 % in the particulate portions. Limits of detection of the method ranged from 0.5 to 5 µg/L in the filtrate portion and from 3.1 to 62 µg/L in the particulate portion.•Saxitoxins and tetrodotoxins have been analysed by using HILIC-HRMS.•UAE with methanol and SPE with silica cartridges have been employed for the extractions of the polar MBTs from seawater.

Development and validation of a multianalyte immunoassay for the quantification of environmental pollutants in seawater samples from the Catalonia coastal area.

Five different enzyme-linked immunosorbent assays (ELISAs) have been developed and applied for the detection of five representatives of important families of chemical pollutants in seawater: Irgarol 1051® (triazine biocide), sulfapyridine and chloramphenicol (antibiotics), 17ß-estradiol (hormone), and domoic acid (algae toxin). The assays were validated by high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (HRMS) showing good correlation between both immunochemical and chemical techniques. A process of extraction and clean-up was added prior to the analysis based on solid-phase extraction (SPE). The multianalyte platform presented good specificity for each compound and adequate sensitivity, with limits of detection (LOD) after the SPE treatment of 0.124 ± 0.006, 0.969 ± 0.09, 0.20 ± 0.05, 1.11 ± 0.012, and 1.39 ± 0.09 ng L-1 for Irgarol 1051®, sulfapyridine, chloramphenicol, 17ß-estradiol, and domoic acid, respectively. No matrix effects were noticed in working with the seawater extracts. Afterward, seawater samples from the Mediterranean Sea (coastal area of Catalonia) were analyzed by both techniques and only one sample presented one contaminant, 17ß-estradiol, in the concentration of 0.011 ± 0.04 µg L-1.

Metabolic Responses of Mytilus galloprovincialis to Fullerenes in Mesocosm Exposure Experiments.

In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed through the diet to fullerene soot at three concentrations in parallel to a control group. Their metabolomics response was assessed by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS). The experiments were conducted in marine mesocosms, during 35 days (7 days of acclimatization, 21 days of exposure, and 7 days of depuration). Real conditions were emulated in terms of physicochemical conditions of the habitat. Results confirmed the bioaccumulation of fullerenes, and the metabolome of the exposed organisms revealed significant differences in the concentrations of seven free amino acids in comparison to the control group. An increase in small nonpolar amino acids (e.g., alanine) and branched chain amino acids (leucine and isoleucine) were observed. Also, glutamine concentrations decreased significantly, suggesting the activation of facultative anaerobic energy metabolism. Branched chain amino acids, such as leucine and isoleucine, followed the opposite trend after the highest level of exposure, which can imply hormesis effects. Other significant differences were observed on lipids content, such as the general increase of free fatty acids, i.e., long-chain fatty acids (lauric, myristic, and palmitic acids) when the concentration of exposure was increased. These results were consistent with hypoxia and oxidative stress.

Analysis of lipophilic marine biotoxins by liquid chromatography coupled with high-resolution mass spectrometry in seawater from the Catalan Coast.

Marine biotoxins regularly occur along the coast, with several consequences for the environment as well as the food industry. Monitoring of these compounds in seawater is required to assure the safety of marine resources for human consumption, providing a means for forecasting shellfish contamination events. In this study, an analytical method was developed for the detection of ten lipophilic marine biotoxins in seawater: azaspiracids 1, 2, 3, 4 and 5, classified as azaspiracid shellfish poisoning toxins, and pectenotoxin 2, okadaic acid and the related dinophysistoxin 1, yessotoxin and homoyessotoxin, classified as diarrheic shellfish poisoning toxins. The method is based on the application of solid-liquid ultrasound-assisted extraction and solid-phase extraction, followed by high-performance liquid chromatography coupled with high-resolution mass spectrometry. The limits of detection of this method are in the range of nanograms per litre and picograms per litre for most of the compounds, and recoveries range from 20.5% to 97.2%. To validate the effectiveness of this method, 36 samples of surface water from open coastal areas and marinas located along the Catalan coast on the Mediterranean Sea were collected and analysed. Eighty-eight per cent of these samples exhibited okadaic acid in particulate and aqueous phases in concentrations ranging from 0.11 to 560 µg/g and from 2.1 to 1780 ng/L respectively. Samples from open coastal areas exhibited higher concentrations of okadaic acid in particulate material, whereas in samples collected in sportive ports, the particulate material exhibited lower levels than the aqueous phase. Graphical Abstract Biotoxins investigated in seawater of the Catalan coast.

Nanoparticle tracking analysis characterisation and parts-per-quadrillion determination of fullerenes in river samples from Barcelona catchment area.

In the present work, the analysis of seven fullerenes (C60 and C70 fullerenes and five functionalised fullerenes) has been performed in river samples collected in the vicinities of Barcelona (Catalonia, NE of Spain). The results of 48 samples (25 river waters, 12 river sediments and 11 wastewater effluents) are presented. Extracts of river water, river sediments and wastewater effluents were analysed by liquid chromatography (LC), using a pyrenylpropyl group bonded silica based column, coupled to a high-resolution mass spectrometer (HRMS), using a dual ion source, atmospheric pressure photoionisation/atmospheric pressure chemical ionisation source (APPI/APCI). The novel methodology presents good chromatographic separation, excellent selectivity and instrumental limits of quantification (ILOQ) in the femtogram order. Method limits of quantification (MLOQ) ranged from 2.9 to 17 pg/l and from 3.2 to 31 pg/l in surface waters and wastewaters, respectively. In wastewater effluents, the sums of C60 and C70 ranged from 0.5 to 9.3 ng/l. In surface waters, C60 fullerene was the most ubiquitous compound, being detected in 100% of the samples in concentrations from 31 pg/l to 4.5 ng/l, while C70 concentrations ranged from less than the method limits of detection (MLOD) to 1.5 ng/l. The presence of fullerenes in both the large particulate (diameter Ø > 450 nm) and the colloidal (Ø < 450 nm) fractions of surface waters should be noticed. In sediments, the concentrations of fullerenes were between the MLOD and 34.4 pg/g. In addition, nanoparticle tracking analysis (NTA) was used for the characterisation of water samples in terms of nanoparticle number concentration and size distribution. As far as our knowledge is concerned, this is the first time that NTA has been used for the characterisation of complex river waters with an environmental focus.