First Report of Pinnatoxins in Bivalve Molluscs from Inhaca Island (South of Mozambique)—South of the Indian Ocean

Abstract

The objective of this work was to screen the EU-regulated lipophilic and cyclic imine toxins in four bivalve species (Atrina vexillum, Pinctada imbricata, Anadara antiquata, and Saccostrea Cucculata) from the Mozambican coast in the Indian Ocean. Toxins were extracted and analyzed according to the EU reference method for the determination of lipophilic toxins in shellfish via LC–MS/MS, but no regulated toxins were found in the analyzed species. However, pinnatoxins (PnTX G, E, and F) were detected in A. vexillum, P. imbricata, and A. antiquata. Higher levels of the PnTX G were determined for A. vexillum (7.7 and 14.3 µg·kg⁻¹) than for P. imbricata (1.6 and 2.4 µg·kg⁻¹), and for A. antiquata (4.5 and 5.9 µg·kg⁻¹) with both hydrolyzed and non-hydrolyzed extracts, respectively. The higher levels of PnTX G determined in the hydrolyzed extracts indicate the high potential of this species to esterify pinnatoxins, in particular PnTX G.

1. Introduction

Lipophilic marine toxins (LMTs) are produced by several harmful algae species that proliferate in marine environments worldwide [1,2]. They constitute one of the great threats to public health since they can be accumulated in marine organisms for human consumption such as bivalves, crustaceans, and pufferfishes [2]. The most reported LMTs include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), yessotoxins (YTXs), and azaspiracids (AZAs). Currently, at least 1000 metabolites from marine microorganisms are LMTs, including the class of cyclic imines (CIs), such as pinnatoxins (PnTXs), pteriatoxins (PtTXs), gymnodimines (GYMs), spirolides (SPXs), prorocentrolides, spiro-prorocentrimine, and portimine [3]. CIs are an interesting group of LMTs (emerging toxins group), with its toxicological profile being poorly understood [4]. They are macrocyclic compounds with imine and spiro-linked ether moieties and are produced by several species of dinoflagellates (Alexandrium spp., Gymnodium spp., Vulcanodinium rugosum), except PtTXs, which are products of biotransformation from PnTXs via shellfish metabolic and hydrolytic transformation [2,4,5]. Among CIs, PnTXs (Figure 1), which were discovered in 1990 in extracts of the bivalve mollusk Pinna attenuate, have received special attention due to their increased occurrence worldwide overtime [6]. PnTXs are emerging toxins, and their toxicological data are very limited; however, they act as potent neurotoxins inhibiting both the nicotinic and muscarinic acetylcholine receptors in the central and peripheral nervous system and at the neuromuscular junction [4,7], which are kept even after cooking procedures [2]. There are no reports of PnTXs in humans yet, but the symptoms observed in animals (mice) include respiratory arrest, mobility decreasing, hind limb paralysis, breathing difficulties, tremors, and jumps [8].

The prevalence and occurrence of LMTs were already reported in several species of marine organisms for human consumption as well as human intoxication worldwide. Fortunately, some LMTs are already monitored, and a maximum limit in seafood was fixed in many parts of the world depending on the prevalence and incidence of a given toxin group [2]. Although harvesting restrictions are imposed when shellfish present levels of toxins above the safety limit, cases of human intoxication are still reported nowadays, possibly due to the lack of monitoring programs in some regions (mainly African countries) or due to disrespecting of the health authorities’ regulations [1,2]. In African countries of the Indian Ocean, including Mozambique, where this study was focused, data regarding LMTare very limited. Few studies reported the occurrence of OA in Haliotis asinine, Crassostrea gigas, and Choromytilus meridionalis from Europa Island, Mayotte, and Reunion Island, South Africa, Mauritius [2,9,10]. Cases of human intoxication caused by ciguatoxins (CTXs), another class of algal toxins, were already recorded in Madagascar involving 124 (2 deaths) and 500 (100 deaths) people in 2013 and 1993, respectively [11,12]. On the other hand, cases of human intoxication may be attributed to non-legislated LMTs (emerging toxins) in countries where traditional toxins are already monitored [13,14]. In Mozambique, due to the lack of marine toxin monitoring programs coupled with the increasing demand for shellfish for human consumption, further investigations to guarantee the consumption of safe bivalve mollusks are required. This study aims to investigate the presence of both EU-legislated (okadaic acid, azaspiracid, and yessotoxin group toxins [15]) and non-legislated (toxins whose maximum limit has not yet been set in the EU) lipophilic toxins in four bivalve species—Atrina vexillum, Pinctada imbricata, Anadara antiquata, and Saccostrea cucullate—collected in the Inhaca Island, south Mozambique.

2. Material and Methods

2.1. Sampling

Four local bivalve species—Atrina vexillum, Pinctada imbricata, Anadara antiquata, and Saccostrea cucculata (

Table 1. Number of individuals and weights of the sample used in this study.

Species	Individuals	Weigh (g)
Atrina vexillum	5	17.2–43.1
Pinctada imbricata	28	30.9–51.4
Anadara antiquata	3	23.5–27.4
Saccostrea cucculata	40	34.4–63.8

)—were collected in Inhaca Island, south of Mozambique (26°03′28.9″ S 32°57′20.7″ E) (Figure 2) which is the growing area of these species.

The sampling was carried out in January and April 2020, which corresponds to the summer season in this region. According to the local population, these species are among the most consumed bivalves locally. The species were dissected and stored at −20 °C in the laboratory of the chemistry department of Eduardo Mondlane University (Maputo, Mozambique) and later were transported to Portugal for toxins analysis in the National Reference Laboratory for Marine Biotoxins Monitoring at IPMA.

2.2. Chemicals

Ammonium formate (LC–MS grade, Fluka Analytical, Steinheim, Germany), acetonitrile (LC–MS grade, Merck, Darmstadt, Germany), water (LC–MS grade, J.T. Baker, Center Valley, PA, USA), formic acid (LC–MS grade), methanol (LC–MS grade). OA, AZA1-3, YTX, PTX and related reference standard solutions were purchased from CIFGA (Lugo, Spain). PnTXG, GYM, and SPX1 reference standard solutions were purchased from the Certified Reference Materials Program of the Institute for Marine Biosciences, National Research Council (NRC, Ottawa, ON, Canada).

2.3. Extraction of the Toxins

The extraction of EU-regulated and cyclic imines toxins was carried out according to the method proposed by the European Union Reference Laboratory for Marine Biotoxins (EURLMB) [15]. Two g of homogenized tissues of pooled samples (Table 1) were mixed with 9 mL of absolute methanol using vortex (Vortex Genie 2) for 3 min at the maximum speed level. The resultant mixture was centrifuged for 10 min at 2000× g, 20 °C, and the supernatant was transferred to a 20 mL volumetric flask. This procedure was repeated by adding another 9.0 mL of methanol to the remaining tissue pellet, and it was subsequently vortexed for 1 min and then centrifuged under the same conditions while combining both supernatants, and the final volume was made up to 20 mL with methanol. An aliquot was filtered through a 0.2 μm syringe filter, and 5 μL was injected into the LC–MS/MS system.

An alkaline hydrolysis step was carried out to convert acylated compounds, which may result from shellfish metabolism, into their respective parental toxin. The hydrolysis was started by adding 313 μL of 2.5 M NaOH to a 2.5 mL aliquot of the sample extract in a test tube, which was homogenized for 30 s in the vortex and heated at 76 °C for 40 min in a heating block. The sample was allowed to cool down until reaching room temperature and neutralized with 313 μL of 2.5 M HCl. The sample was vortexed for 30 s, and an aliquot was filtered through a 0.2 μm syringe filter, and 5 μL was injected into the LC–MS/MS system.

2.4. LC–MSMS Analysis

Determination of lipophilic toxins in both hydrolyzed and non-hydrolyzed extracts was carried out via liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection following the standardized operating procedure (SOP) for the determination of marine lipophilic biotoxins in bivalve mollusks of the EURLMB [15]. The LC–MS/MS equipment consisted of an Agilent 1290 Infinity chromatograph coupled to a triple quadrupole mass spectrometer Agilent 6470 (Agilent Technologies, Böblingen, Germany). The chromatographic separation was conducted with a Zorbax SB-C8 RRHT column (2.1 × 50 mm, 1.8 μm) protected with a guard column (2.1 × 5 mm, 1.8 μm). Mobile phase A was water with 2 mM ammonium formate and 50 mM formic acid, and mobile phase B was 95% acetonitrile with 2 mM ammonium formate and 50 mM formic acid. An elution gradient at a flow rate of 0.4 mL·min⁻¹ was used as follows: 0–3 min, gradient from 88 to 50% eluent A; 3–6.5 min, gradient 50 to 10, 183% eluent A; 6.5–8.9 min, 10% eluent A; 8.9–10 min, gradient 10 to 88% eluent A. The detection was carried out in Multiple Reaction Monitoring (MRM) acquisition mode. Two MRM transitions were monitored, one being used for quantification and the other for confirmation (Supplementary Materials).

For PnTX G quantification, a six-point calibration curve (Signal = 2330.8927C–24.6694; R² = 0.9993) with a concentration of PnTX G ranging from 0.5 to 24.0 ng·mL⁻¹ was set up for quantification purposes. The lowest calibration point was considered as the quantification limit. The level of esterification was calculated using the formula % esterified = 100 × (1−NH/H), where NH and H mean concentration of the PnTX G in non-hydrolyzed and hydrolyzed extracts, respectively.

3. Results

The screening of EU-legislated lipophilic toxins did not reveal the presence of these toxins in any of the analyzed species. These results may not be conclusive for risk assessments of lipophilic toxins since the samples were collected in a single location and in one time frame period. Regarding non-EU legislated lipophilic toxins, PnTX G, E, and F were found in Atrina vexillum, Pinctada imbricata, and Anadara antiquata.

PnTXG was confirmed using commercial standards available in the lab. PnTX E and F were deduced by comparing spectral data of the ion product of m/z 784.6 and m/z 766.3, respectively, with available data in the literature [16]. Figure 3 shows chromatograms of the PnTX E and F in the samples and PnTX G both standard and in the samples. The spectral data of the PnTX E and F are illustrated in Figure 4, with the fragments [M+H]⁺, [M+H-H₂O]⁺, [M+H-2H₂O]⁺, [M+H-3H₂O]⁺, and diagnostic fragments at m/z 164.1 and 446.0.

The highest levels of PnTX G were observed in the hydrolyzed extracts, and this suggests that these species easily esterify PnTX G. Among species, Atrina vexillum presented higher levels of PnTX G in both non-hydrolyzed and hydrolyzed extracts (7.7 and 14.3 µg·kg⁻¹) followed by Anadara cucculata (4.5 and 5.9 µg·kg⁻¹) and Pinctada imbrica (1.6 and 2.4 µg·kg⁻¹). Regarding esterification levels, Atrina vexillum showed 46% of the compounds in the esterified form, and contrarily to the levels of PnTX G in the extracts, Pinctada imbrica (33%) presented higher levels of esterification than Anadara cucculata (24%). PnTX G was detected in both hydrolyzed and non-hydrolyzed extracts, while PnTX A and E were found in non-hydrolyzed and hydrolyzed extracts, respectively.

4. Discussion

Reports of PnTXs date since 1990 and were discovered in the extracts of the bivalve mollusk Pinna attenuata by Chinese investigators [6]. Nowadays, there have been reports of PnTXs in other species of bivalves for human consumption [16,17,18,19,20,21,22,23,24,25], putting at risk public health despite no confirmed reports of human intoxication involving this group of toxins. In this study, three PnTXs were detected, PnTX G, E, and F, in three species Anadara antiquata, Pinctada imbricata, and Atrina vexillum. As proposed in the previous studies, all PnTXs are formed from PnTX G and F as precursors since they are primary toxins produced by Vulcanodinium rugosum [16]. PnTX E is formed readily from PnTX F, in which the lactone ring of PnTX F is opened by hydrolysis forming PnTX E via metabolic and hydrolytic transformations in shellfish and water, and they are also available to be taken by bivalve species [26,27,28,29]. This means that the PnTX E detected in this study could be formed from PnTX F produced by an algae species present in seawater or by shellfish metabolism, or both. The rate of conversion of PnTX F to E may vary from species to species. In this study, it was not possible to quantify PnTX F and E due to the lack of reference standards. However, their detection was deduced from product ion spectral data analysis by the screening of m/z 784.7, which corresponds to PnTX E, and m/z 766.3, which was attributed to PnTX F, and their spectral data were similar to data available in the literature [16,29].

PnTXs below quantification limits found in Saccostrea cucullata may suggest a very low ability or even inability to bioaccumulate PnTXs. For PnTX G, the high level found in Atrina vexillum when compared with other species, suggests that this species could be considered very suitable to be used as bio-indicator of PnTXs, among the three analyzed species, on the Mozambican coast, but further study is required.

A higher content of PnTX G in hydrolyzed extracts appears to be in agreement with findings reported from extracts of mussel (Mytilus edulis) samples from Eastern Canada, in which higher levels of PnTX G were found in hydrolyzed (0.7 to 108 µg·kg⁻¹) than in non-hydrolyzed samples (0.3 to 3 µg·kg⁻¹) [19]. The notable difference in PnTX G levels between the hydrolyzed and non-hydrolyzed samples suggests that these species may contain considerable amounts of esters of PnTX G.

PnTXs are emerging toxins that are not regulated yet worldwide [5], and this complicates the associated risk assessment for public health based on the PnTX G levels found in this study. Previous studies focused on PnTXs in species used in this study are very limited. However, the occurrence of PnTXs in Atrina vexillum was expected since Atrina sp. are closely related to Pinna sp. [30], for which PnTXs were reported for the first time (P. attenuate, P. murica, and P. biclor) in China, Japan, and Australia [16,31,32,33,34,35]. Comparing this study with others, the levels of PnTX G found in this study are not different from those found in previous studies in other species in some parts of the world. Similar levels were reported in 35% of European commercial seafood (flat oyster: Ostrea edulis, clams: Ruditapes decussatus, mussels: Mytilus galloprovincialis, blue mussels: Mytilus edulis) collected in Spain, Slovenia, Italy, Ireland, and Norway, which were contaminated by PnTX G at levels up to 12 µg·kg⁻¹) [23]. In Chile, one of the major mussel producers worldwide, PnTX G at concentrations ranging from 2.9 to 5.2 µg·kg⁻¹ was found in the cooked mussel Mytilus chilensis [18]. Samples of Mytilus edulis from six locations in Eastern Canada were also contaminated by both PnTX G and A, with levels varying from 0.6 to 108 and 0.3 to 2.5 μg·kg⁻¹, respectively, with PnTX G being the major toxin in all locations studied [19]. Contrary to this study, high levels of PnTX G were recorded in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from In Ingril, a French Mediterranean lagoon, during a period between 2009 and 2012 [21]. In that study, the concentration of PnTX G varied from 40 to 1200 µg·kg⁻¹ and 17 to 95 µg·kg⁻¹ for Mytilus galloprovincialis and Venerupis decussata, respectively, and in a recurring way during the study period. The higher levels of PnTX G found in Mytilus galloprovincialis (than Venerupis decussata, with the ratio of mussels/clams varying from 3 to 16 during all 4 years of the study) may suggest this species as a good candidate to act as a sentinel species for PnTX G. Based on these findings, the French Agency for Food Safety (ANSES) recommend the implementation of a monitoring program for PnTXs [36]. Blue mussels (Mytilus galloprovincialis) and Pacific oysters (Crassostrea gigas) from the shellfish harvesting areas of Catalonia, Spain (NW Mediterranean Sea) were tested for PnTX G at concentration ranging from 2 to 60 μg·kg⁻¹ [17]. In Mozambique, to date, there are no reports of PnTX occurrence in bivalves, neither are there confirmed cases of human intoxication involving PnTXs. This is the first study of PnTXs in bivalve species from Mozambique, although it is very preliminary due to the reduced number of specimens analyzed, and sampling was performed at a single point.

5. Conclusions

PnTX G, E, and F were found in the local Atrina vexillum, Pinctada imbricata, and Anadara antiquata collected in the Mozambican coast in the Indian Ocean. No EU-regulated lipophilic marine toxins were found in all analyzed species, and no PnTXs were found in Saccostrea Cucculata. On the other hand, PnTX G was determined to be at considerably high levels in Atrina vexillum, followed by Pinctada imbricata and Anadara antiquata in both hydrolyzed and non-hydrolyzed extracts, respectively. In addition, PnTX E and PnTX F were also detected. The high level of PnTX G found in Atrina vexillum, when compared with other species, suggests that this species could be used as a bio-indicator of PnTXs, among the three analyzed species, on the Mozambican coast, but further study is required. This is the first study showing PnTXs in bivalve species from the Mozambican coast.