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By Joana F. Leal Joana F. Leal Scilit Preprints.org Google Scholar 1, Gabriel Bombo Gabriel Bombo Scilit Preprints.org Google Scholar 2, Patrícia S. M. Amado Patrícia S. M. Amado Scilit Preprints.org Google Scholar 1, Google Scholar 1, Hugopirras. Scilit Google Cristiano Google Scholar 1, *
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Center for Marine Sciences (CCMAR) and Department of Chemistry and Pharmacology, Faculty of Science and Technology, University of Algarve, Campus de Campelas, 8005-139 Faro, Portugal
Received: January 18, 2023 / Revised: February 6, 2023 / Accepted: February 7, 2023 / Published: February 10, 2023
Accumulation of marine biotoxins in shellfish and their consumption are serious food safety problems, threaten human health and damage the availability of protein-based food. Therefore, it is urgent to develop ways to detoxify live bivalves, avoiding their economic and nutritional damage. In this context, we tested the adsorption mechanism of paralytic shellfish toxins (PST) based on cation-exchange resin. The first studies using the cultures of Gymnodinium catenatum (natural producers of PST) showed approximately 80% reduction in total eros after 48 hours. Interestingly, we found that the toxins are different, with the features of the toxins playing a role in the adsorption capacity through steric hindrance, electronic effects, or good charge ratio (for example DCSTX). The positive effect of resin on the acceleration of PST elimination in live mussels ( Mytilus edulis ) is not clear when compared to elimination without resin; However, relevant information can be obtained that will facilitate further in vivo studies. Several factors are at play, such as competition of natural substances (eg, salt, organic matter) for the same binding sites, blocking of pores by intermolecular interactions, and/or difficulties in resin absorption by mussels. In addition, the present work demonstrates the ability of mussels to neutralize pH and proposed biotransformation reactions within PST materials.
Paralytic shellfish toxins (PST) are a large group of highly potent neurotoxins and goniatoxins produced by the harmful phytoplankton Alexandrium, Kymonodinium and Pyrodinium [1]. Accumulation of PST in marine species such as mussels, clams, and fish in the food chain causes fatal disease in marine mammals and humans [2, 3]. Cases of paralytic shellfish poisoning (PSP) are reported annually in many coastal areas of the world [4, 5, 6]. These toxins can cause food poisoning and are associated with severe economic losses worldwide [5, 7]. PST is a group of saxitoxin (STX) derivatives, consisting of about 50 analogs, which have been reported in many species (Figure 1) [5, 8]. Naturally occurring PST can be modified in a way by many biological factors.
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In order to protect human consumption, it is a legal requirement in many countries to sell fresh shellfish. Different strategies for PST induction in shellfish have been investigated, including chemical, physical and physical methods [9]. Among these, the use of adsorbents to remove PST seems to be promising, such as chitin shell powder [10], clay [11, 12], carrageenan [13], activated charcoal [14, 15], and chitosan derivatives [14, 15] and chitosan derivatives (18).
Ion-exchange resins are polymeric particles or beads with multiple functional groups capable of binding ions with opposite charges [19]. They are ion-exchange resins (CER) or anion-exchange resins, CER has been found to adsorb compounds that have a positive effect including tricyclic antidepressants [20] and monosaccharides [21]. Despite the versatility and flexibility of cation-exchange resins, their interaction with PST has not been explored.
PST is a highly polar and hydrophilic molecule due to the tricyclic structure of 3, 4-propinoperhydropurine and the presence of two guanidine groups in their structure [22], and they are usually in a chargeable form at seawater pH [15]. We decided to investigate the effect of CER by considering the molecular structure of BST and their charge nature, as well as their susceptibility to adsorption by different polymers and clays. 48-hour period. PST was analyzed using high performance liquid chromatography with fluorescence detection (HPLC-FLD) and their profile was compared between matrices and throughout the adsorption process.
Gymnodinium catenatum IO13-26-02 was obtained from the ALISU Culture Collection (University of Lisbon) and was previously characterized for its toxicity profile [23]. This is a Dinoflagellate producing PST grown under the following culture conditions: 19.0 ± 1.0 °C, 14 h under: light 10 h: dark and 80 µmol m
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Light fire. Cultures are grown in L1-medium [24], prepared using sea salts (Extraordinary Sea Salt, Tropic Marine) with synthetic seawater to a final concentration of 33 ppt, which is sterilized at 121 °C and 15 psi for 20 minutes. To assess the cell concentration, samples are taken from confluent cultures and fixed in an acidified solution of Lucolin. Cell counts were performed in quadruplicate using an inverted microscope (Zeiss Primovart, Carl Zeiss Microscope GmbH, Jena, Germany) with 40× magnification in a Sedgewick-Rafter reading chamber [ 25 ].
In the initial phase of the experiments, cultures of G. catenatum were used for the first investigations of PST removal tests. For that, G. catenatum cultures yielded at the end of the maximum phase with a concentration of 1.38 × 10
, Cytiva, Global Life Sciences Solutions, USA LLC, Marlborough, MA, USA). Biomass was frozen at -80 °C until use. In subsequent experiments, cultures of G. catenatum were prepared to feed live mussels. For this reason, cultures are grown in 6 L flat bottom balloon flasks [26], up to 10 starting concentrations.
A strong acidic resin was used to evaluate the ability of G. catenatum cultures and toxin removal in contaminated bivalves: ambergrom.
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50WX8 hydrogen form (100-200 mesh, Thermo Fisher Scientific, Waltham, MA, USA, CAS 69011-20-7). It is a cation-exchange resin of styrene-divinylbenzene (ST-DVB) with sulfonic acid functional groups as aryl substituents (Figure 2). For culture and detoxification studies in bivalves, the resin concentration is 1 g/L.
Before its use, the resin was cleaned and activated as follows [27]. Deionized water – 10 times the volume (mL) of the resin mass (g) – is added to the resin, in a beaker. The mixture was placed with a glass rod and allowed to settle for 20 minutes. Then, fine particles were washed. To activate the resin, the same amount of 1 MH
(95%, Fisher Chemicals, Fisher Scientific UK, Loughborough, UK) is added and the mixture is left for 15-20 min, after which the fine particles are re-dispersed. This activation process is repeated three times. Then, the resin was washed with deionized water until pH > 5, using a Buchner funnel and a PVDF membrane filter paper 43-48 µm (Filter-Lab, Filtros Anoya SA, Barcelona, In addition, a form of sodium (Na
The resin form is prepared from the acid form (H-form) as follows: deionized water – 10 times the amount of resin (g) volume (mL) – is added to the resin in the beaker. The mixture was placed with a glass rod and allowed to settle for 20 minutes. Then, fine particles were washed. To convert the resin to sodium form, the same amount of 4% NaOH (pa, Chem-Lab NV, Zedelgem, Belgium) was added and the mixture was left for 15-20 min, after which the fine particles were re-dispersed. Then, 4% NaCl (p.a., ≥99.8%, Chem-Lab) solution is added, the mixture is left for 15-20 minutes and then removed. This last step is repeated twice. The resin was washed with deionized water until pH = 7