1 Introduction

Shellfish is an important nutrient-rich source of lean proteins, healthy fats, and minerals for many people living in coastal regions. Recently, shellfish, fish, and other seafoods have been called blue foods because of their nutritionally healthy ingredients (Harnedy and FitzGerald 2012). However, unfortunately, shellfish are one of the most common food allergens and contain contaminants, heavy metals, and biotoxins. Annually, a significant number of shellfish poisoning incidents occur worldwide (Gerssen et al. 2010; Mafra et al. 2023). Normally, marine biotoxins are produced by phytoplankton, particularly dinoflagellates. Shellfish consume these microalgae, and biotoxins accumulate in their bodies (Otero et al. 2011).

Cyclic imines (CIs) are a group of marine lipophilic biotoxins that originate from ocean dinoflagellates such as Alexandrium ostenfeldii, Vulcanodinium rugosum, Karenia spp. and Pteria penguin. They are classified into four subgroups: spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pterygiatoxins (PtTXs). The common structure of CIs is characterized by macrocycles, which include ring sizes between 14 and 27 carbons, related to two subunits: the spiroimine and spiroketal ring systems. They are categorized into the same group owing to the imino group functionalities as their commonly supposed pharmacophore and similarities in their mechanisms of action in animal experiments (Otero et al. 2011).

They are “fast-acting” marine biotoxins that affect muscarinic and nicotinic acetylcholine receptors (Gill et al. 2003), exert toxicity through Na+/K+ ATPase channels, and irreversibly affect weak L-type transmembrane Ca2+ channels (Sleno et al. 2004). In SPX toxicity tests using a Mouse Bioassay (MBA), symptoms such as abdominal cramps, hyperextension, and tail bending can be observed (Tubaro et al. 2012). LD50 values of SPX-E and -F are higher than those of SPX-B and -D, respectively, and the LD50 value was confirmed to be 40 μg/kg body weight (b.w.) when a mixture of SPXs was administered (Don Richard et al. 2000). For PnTXs, there are several analogs such as PnTX-A to -G. Among them, PnTX-E and -F showing the strongest toxicity as LD50 value ranged from 16 to 50 μg/kg b.w. (Rhodes et al. 2010; Selwood et al. 2010). GYM-A have shown a lethal dose of 80–96 μg/kg b.w. after i.p. injection into female rats. The symptoms of acute toxicity include hyperactive behavior, jumps, slow movement and numbness (Munday et al. 2004; Rhodes et al. 2011). PtTXs have only three analogs, PtTX-A, -B, -C. According to Takada et al. PtTXs are assumed to have the same absolute stereochemistry as that of PnTXs. In case of PtTX-A, LD50 value is 100 μg/kg b.w. and that of a PtTX-B and -C mixture is 8 μg/kg b.w. (Takada et al. 2001b). As mentioned above, CIs are potent neurotoxins; however, toxicological data for CIs are limited, except for acute toxicity studies. Therefore, the number of CIs in shellfish is not regulated in South Korea or other countries. However, with regard to the acute toxicity of SPXs and GYMs, CIs trigger high intraperitoneal toxicity in rodents. Many previous studies have shown that CIs regulate the levels of these toxins in shellfish and/or marine organisms through animal tests or instrumental analyses (Davidson et al. 2015; Otero et al. 2011; Rambla-Alegre et al. 2018; Rundberget et al. 2011).

In the present study, commercial shellfish samples from different regions of South Korea were purchased in 2021 and 2022 and evaluated for CI toxins. This study aimed to establish a database of the levels of these toxins by analyzing the presence and quantity of CIs, and consequently, to conduct a risk assessment to determine the potential health risks of CIs through shellfish consumption.

2 Material Methods

2.1 Chemicals and Standards

Certified reference material (CRM) of Pinnatoxin E (18.20 ± 1.64 µg/mL), Pinnatoxin F (18.06 ± 0.63 µg/mL), and Pinnatoxin G (18.18 ± 2.55 µg/mL) were purchased from Merck (Darmstadt, Germany), 13-desmethyl Spirolide C (5.01 ± 0.24 µg/mL), Gymnodimin-A (2.50 ± 0.13 µg/mL), were purchased from the National Research Council of Canada (NRC, Halifax, NS, Canada). 13,19-didesmethyl spirolide C (7.53 ± 0.57 µg/mL) and 20-methyl spirolide G (7.01 ± 0.61 µg/mL) were purchased from CIFGA (Lugo, Spain). Analytical grade methanol (MeOH) and hypergrade acetonitrile (ACN) for LC-MRM/MS were purchased from Merck (Darmstadt, Germany). Other materials and reagents were of analytical grade or higher unless otherwise noted below.

2.2 Collection of Dietary Shellfishes and Sample Preparation

In total, 67 samples from 2021 and 213 samples from 2022 of shellfish consumed in Korea were collected from different local fish markets (Fig. 1). To determine seasonal CIs content, samples were collected in April, June, and August 2021, and March–August 2022. Shellfish samples were washed with tap water and drained for 5 min using a sieve. All tissues were ground using a homogenizer at 15,000 rpm for 3 min.

Fig. 1
figure 1

Collection site of shellfish samples from Korean domestic market in 2021 and 2022

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2.3 Extraction and Enrichment of Cyclic Imines

Homogenized shellfish samples (10 g) were suspended in 100 ml of 50% methanol (MeOH) in water (v/v). Samples were vortexed until a homogenous mixture was obtained, and sonication was repeated 3 times for 30 min. The extracted samples were then centrifuged (10,000 rpm for 10 min at 4 °C), and the residues was removed and obtained supernatant followed by evaporated in vacuum. The dried extracts were resolubilized in 1 ml of 50% acetonitrile in water (v/v) and purified using a Supel™ swift HLB Solid Phase Extract (SPE) cartridge (60 mg, 3 ml tube) prior to LC/MRM-MS analysis. The SPE cartridge was conditioned using 12 ml of MeOH followed by 6 ml of water. The extracts were loaded onto an HLB SPE cartridge and washed with 6 ml of 20% MeOH in water (v/v) to remove salts and small lipophilic contaminants. Subsequently, the CI extracts were eluted with 6 ml of MeOH and completely dried under vacuum.

2.4 CI Toxins Analysis Using LC/MRM-MS

Seven different CI marine biotoxins were analyzed following the methods described by Seo et al. (Seo et al. 2023). Briefly, LC/MRM-MS analysis was performed using an Agilent 1260 liquid chromatography (LC) system coupled to an Agilent 6495 triple quadruple mass spectrometer (QqQ-MS) (Agilent Technologies, Santa Clara, CA, USA). CI extract samples were separated on a 2.1 × 100 mm BIOshell™ A160 Peptide C18 Column (Merck, Darmstadt, Germany). The mobile phase consisted of (A) 100% water with 2 mM ammonium formate and 50 mM formic acid and (B) 95% acetonitrile with 2 mM ammonium formate and 50 mM formic acid. The LC gradient conditions were as follows: 0‒8 min, 25‒65% B; 8‒10 min, 65‒95% B; 10‒15 min, 95% B; 15‒20 min, 25% B, followed by holding at 95% B for 5 min for equilibration to analyze the next sample run. The column temperature was maintained at 40 °C during analysis, and the capillary and nozzle voltages were set to 3.5 and 0 kV, respectively. A drying gas flow 11 L/min at 200 °C, nebulizer was operated with the dwell time of 10 ms and collision energy of 50 eV in MRM mode. The MRM transitions monitored for the CI toxins are presented in Table 1.

Table 1 Shellfish species from local fish market in 2021 and 2022
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3 Results

3.1 Occurrence of CIs in Shellfish

Considering the 67 samples in 2021, CIs were detected at low concentrations from 5.02‒41.03 pg/g for PnTX-G, 5.24‒393.34 pg/g for Gym-A and 6.62‒27.84 pg/g for 13-desMe SPX C. Other PnTX-E, -F, and other SPX analogs were not detected in any of the commercial shellfish samples in 2021. For 213 samples in 2022, PnTX-G, Gym-A and 13-desmethyl SPX C were detected at low concentrations from 2.58 to 7.35 pg/g for PnTX-G, 1.36 to 179.22 pg/g for Gym-A and 1.01 to 260.04 pg/g for 13-deMe SPX C. Other PnTX and SPX analogs were not detected in any of the samples in 2022. Figure 2 shows the total ion chromatogram (TIC) profile of seven different CI toxin-certified reference materials (CRM). The analysis results are summarized in Tables 1 and 2 for 67 and 213 samples from 2021 and 2022, respectively, including the detected CI concentration and their location of collection.

Fig. 2
figure 2

Representative total ion Chromatogram obtained from LC/MRM-MS methods for analysis seven standard mixture of cyclic imine toxins. A baseline separation (Rs > 2.9) was achieved over the entire working ranges from 10 to 2 ng/ml. PnTX-E, F, G: Pinnatoxin E, F, G; GYM-A: Gymnodimine A; 13-desMe SPX C: 13-desmethyl spirolide C; 13, 19-didesME SPX C: 13,19-didesmethyl spirolide C; 20-Me SPX C: 20-Methyl Spirolide C

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Table 2 MRM transitions and corresponding parameters for cyclic imines quantitation
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No significant toxin levels were detected for PnTX-G. However, in the 2021 samples, comparatively high concentrations of 30 pg/g or higher were observed in Mya arenaria (April), Mytilus coruscus (June), and Turbo cornutus (July). In the 2022 samples, PnTX-G was detected only in April, May, and August samples (4.63% of all species), with all values below 10 pg/g. (April: Azumapecten farreri, Turbo cornutus, Styela clava, Anadara broughtonii; May: Saxidomus purpurata, Turbo cornutus; August: Anadara broughtonii).

Conversely, Gym-A was consistently detected in all seasons. In April samples in 2021, Gym-A was detected in oysters (393.34 pg/g), mussels (243.85 pg/g), and abalones (156.21 pg/g). In June, it was detected in oysters (159.6 pg/g) and scallops (140.1 pg/g). For July, it was found in comb pen shell (28.02 pg/g), while in August, short neck clam (309.85 pg/g) and clam (143.78 pg/g) contained Gym-A. In the 2022 samples, Gym-A was detected in Tropical oyster during March (179.22 pg/g) and in April sample (123.58 pg/g). No species had Gym-A concentrations above 100 pg/g in May, June, or July; however, in August, the tropical oyster contained 101.58 pg/g of Gym-A. Gym-A demonstrates the highest quantities in both 2021 and 2022, with significant amounts observed in April and August.

As for 13-desMe SPX C, the highest concentration of 27.84 pg/g was observed in mussels in April 2021, while other species and seasons analyzed below 10 pg/g levels. In the 2022 samples, Tropical oyster from April exhibited 260.04 pg/g, whereas other species showed detections below 100 pg/g.

3.2 Differences Between Locations, Seasonal and Toxin Levels

The given data pertain to a study conducted in Korea involving the analysis of toxin levels in seafood samples obtained from local fish markets in 2021 and 2022. Samples were collected from three regions, 9 sites: the eastern, western, and southern coasts (Fig. 1). The highest frequency of Gym-A detection was observed in the South Coast, with total amounts of 2734.92 pg/g and 1960.77 pg/g in 2021 and 2022, respectively (Fig. 3). In 2022, 13-desMe SPX C will have the highest content on the southern coast. PnTX-G was most abundant in 2021 with a concentration of 176.14 pg/g in the South Coast, although it exhibited lower levels compared to Gym-A and 13-desMe SPX C.

Fig. 3
figure 3

Contents of PnTX-G, GYM-A and 13-desMe SPX C in shellfish samples each collection site differences in 2021 (A)–2022 (B). PnTX-G: Pinnatoxin G; GYM-A: Gymnodimine A; 13-desMe SPX C: 13-desmethyl spirolide C

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The detection probabilities and average toxin levels were established based on the sample sizes from each region. Gym-A exhibited the highest contamination across all three toxins, with average levels of 39.17 pg/g in the West Coast, 76.1 pg/g in the East Coast, and 69.93 pg/g in the South Coast for the year 2021. In the year 2022, Gym-A was most prevalent with 14.47, 29.63, and 7.76 pg/g in the respective coasts. 13-desMe SPX C was predominantly detected in the East and South Coasts, with average concentrations of 18.61 and 12.05 pg/g in 2021.

Seasonal analysis revealed the highest detection of Gym-A and 13-desMe SPX C in April 2021, whereas PnTX-G was most abundant in June. In 2022, PnTX-G peaked in April, Gym-A in March, and 13-desMe SPX C in April. The South Coast exhibited the highest toxin concentrations by region for both years; (PnTX-G, 176.14 pg/g; GYM-A, 2734.92 pg/g; 13-desMe SPX C, 109.54 pg/g for 2021; (PnTX-G, 37.48 pg/g; GYM-A, 1960.77 pg/g; 13-desMe SPX C, 718.01 pg/g for 2022 (Fig. 4).

Fig. 4
figure 4

Contents of PnTX-G, GYM-A and 13-desMe SPX C in shellfish samples each collection date from South Korea in 2021–2022. PnTX-G: Pinnatoxin G; GYM-A: Gymnodimine A; 13-desMe SPX C: 13-desmethyl spirolide C

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Despite low-unit detection levels (pg/g), Gym-A was widely detected, especially on the South Coast (55.31%), in contrast with lower detection probabilities on the East and West Coasts (19.44% and 19.64%, respectively; Table 3). Notably, all three toxins exhibited high detection probabilities in the samples from the South Coast. Oysters (Crassostrea nippona) were the species most affected by Gym-A was the oyster (Crassostrea nippona), while mussels, abalones, and blood clams had toxin levels exceeding 100 pg/g. These species were predominantly collected from Sites 4 and 5 in the South Sea.

Table 3 Sample numbers and statistical results of the detection rate (%) and toxin contents (pg/g whole soft tissue) in shellfish samples from coastal of South Korea during 2021–2022
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Overall, PnTX-G levels decreased significantly in 2022 compared to 2021, whereas Gym-A levels remained high in April and August 2021 but decreased afterward. Despite the low levels of 13-desMe SPX C, its detection probability increased slightly in 2022 compared to 2021. This study demonstrated the distribution and trends of these toxins in Korean seafood, highlighting the most affected regions and species.

4 Discussion

Harmful algal blooms, known as HABs, are natural phenomena in which marine phytoplankton experience excessive growth covering the ocean surface (Ferreiro et al. 2015). The occurrence of HABs is increasing due to increased sea-surface temperatures and the proliferation of nutrients along coastal waters (McCarthy et al. 2015). The expansion of HABs in specific regions is linked to the movement and practices of ballast water and aquaculture (Anderson et al. 2002; Maso and Garcés 2006; Smayda 2007). Among the thousands of known species of microalgae, approximately 100 consistently produce natural toxins that can cause poisoning or even fatality in humans and animals (Farabegoli et al. 2018; Karlson et al. 2021). Moreover, these toxic effects have significant socio-economic impacts and costs.

HABs are triggered by rising temperatures, with the period between March and June being a critical period in Korea, when sea temperatures increase (Fig. 5), leading to the proliferation of toxic phytoplankton and the concentrated occurrence of shellfish toxins. Phytoplankton-producing CI inhabit regions including Europe, southern Japan, the Philippine Sea, and the South Sea of Korea (Kim et al. 2022). A report from Korea’s National Institute of Fisheries Science (NIFS) indicates that, in areas where shellfish were sampled, the South Sea exhibited the highest annual average seawater temperature, recorded at 19–20 ℃ (Fig. 6). Plankton species, exemplified by Vulcanodinium rugosum and Alexandrium ostenfeldii, known for producing Spirolide and Pinnatoxin, demonstrate heightened growth and toxin production within the temperature range of 16–25 ℃ (Abadie et al. 2016; Jensen and Moestrup 1997; Laabir et al. 2011; Medhioub et al. 2011). The NIFS conducts toxin testing for Paralytic Shellfish Poisoning (PSP), Neurotoxin Shellfish Poisoning (NSP), Diarrheic Shellfish Poisoning (DSP), and Amnesic Shellfish Poisoning (ASP) from February to October each year, alerting the public to ensure safety (MOF 2015). These toxins, including saxitoxin, okadaic acid, brevetoxin, and azaspirate, have been tested in mouse bioassays (Chain 2010; Dickey et al. 1999; Shumway 1995; Suzuki and Okada 2018). However, toxins such as CI have been less studied, and some countries still lack regulations.

Fig. 5
figure 5

Average sea temperature and salinity in the shellfish sampling sites in 2021 and 2022

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Fig. 6
figure 6

Percent composition of shellfish samples with different detected toxins for PnTX-G, GYM-A, and 13-desMe SPX C in different sampling dates. PnTX-G: Pinnatoxin G; GYM-A: Gymnodimine A; 13-desMe SPX C: 13-desmethyl spirolide C

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Owing to factors such as rising sea levels, increased sea temperatures, and eutrophication, HAB occurrence are increasing. In Korea, the primary phytoplankton responsible for CI toxins has been identified, and this study presents the first evidence of the accumulation of these toxins in shellfish using LC–MS/MS analysis.

PnTX-G, a derivative of PnTX, is produced by the dinoflagellate Vulgaria rugosum and is found in various regions including Australia, New Zealand, Japan, and Hawaii (Kim et al. 2022). This species is predominantly found in Europe but is now present in regions such as the East China Sea, Philippines, Japan, and Korea (Kim et al. 2022). PnTX-G has been found to exhibit acute toxicity in mouse intraperitoneal tests at concentrations ranging from 35 to 68.1 μg/kg b.w. and oral LD50 at 105–199 μg/kg b.w. (Takada et al. 2001a). Similar to other toxins, the distribution of PnTX-G and its potential effects require thorough investigation. For Azaspiracid1 (AZA1), when administered using the same intraperitoneal (i.p) method, an LD50 value of 74 μg/kg b.w. was determined, and AZA2 caused 50% mouse mortality at a concentration of 117 μg/kg b.w. (Kilcoyne et al. 2014). Similarly, because PnTX-G exhibits comparable toxicity, there is a need for contamination studies of this toxin, which is progressively spreading. In the present study, PnTX-G was detected in the highest amount (41.03 pg/g) in Mya arenaria samples collected from the southern coast in April 2021. It was detected in 11 of the 179 shellfish samples. It was primarily found in shellfish harvested from the southern coast, where sea temperatures are relatively high, whereas in the East and West Seas, it was detected in only one sample each. According to the French Agency for Food, Environmental and Occupational Health and Safety (ANSES), if the accumulation of PnTX-G in shellfish exceeds 23 μg/kg, it is considered risky and warrants a warning (Servent et al. 2021). Norway, Canada, and Spain have reported quantities of 115, 83, and 59 μg/kg respectively (García-Altares et al. 2014; McCarron et al. 2012; Rundberget et al. 2011), while in the French Mediterranean region of La Garenne, levels of PnTX-G ranged from 261 to 1244 μg/kg (Hess et al. 2013).

GYM-A is a toxin produced by the dinoflagellate Gymnodinium mikimotoi and has mainly been identified in fish and oysters in Japan. It is known for its high toxicity, with an LD50 value of 80‒96 μg/kg, and its symptoms include hyperactive behavior, jumps, slow movements, and numbness (Botana 2014). Over the past 50 years, harmful algal blooms caused by Karenia mikimotoi and Karenia brevis have affected the Gulf of Mexico, leading to severe ecological impacts (Brand et al. 2012; Ninčević Gladan et al. 2019; Van Wagoner et al. 2014; Wunschel et al. 2018). In Korea, GYM-A was most abundant along the southern coast, detected in 55.31% of the 179 samples, with higher amounts in the East Sea.

Among the identified toxins, 13-desMe SPX C exhibited the highest toxicity and was the most prevalent (Don Richard et al. 2000; Gill et al. 2003; Sleno et al. 2004; Tubaro et al. 2012). Its LD50 values are 37 μg/kg for SPX A and 7‒28 μg/kg for 13-desMe SPX C (Don Richard et al. 2000). In Korea, SPX C was found in relatively higher proportions along the southern coast (23.46%) than along the eastern and western seas. Considering its extreme toxicity, the continuous monitoring of SPX C is necessary.

When examining seasonal variations in toxin detection rates, GYM-A exhibited the highest frequency (Fig. 7). Despite the seasonal variations, no discernible trends were observed. In comparison to 2021, the detection rate of SPX C surged from 0% to 4.9%, reaching a peak of 41.1% in 2022. Conversely, the presence of PnTX-G was recorded to be less than 3% by 2022, a significant decrease from 54.4% observed in July 2021. These data suggest an annual shift in the toxin types, emphasizing the need for ongoing monitoring.

Fig. 7
figure 7

Changes in sea temperature and salinity in the shellfish sampling sites in 2021 and 2022

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With the rise in average temperatures due to global warming, accompanied by an increase in sea temperatures, and the occurrence of algal blooms, the proliferation of toxin-producing microalgae is leading to an increase in new toxins in the ocean (Kim et al. 2014; Landsberg 2002; Zhang et al. 2011). Furthermore, as Korea’s waters transition from temperate to tropical zones, there is an increase in the occurrence of toxin-producing microalgae (Lim and Jeong 2021). The emergence of new toxins, such as cyclic imines, due to the proliferation of microalgae species, necessitates proactive management strategies in South Korea. The conventional mouse bioassay method, which measures the toxicity level by orally administering extracts of contaminated shellfish to mice, is significantly less sensitive than instrumental analysis and faces challenges related to reproducibility and ethical concerns (Bodero et al. 2018). Therefore, we conducted monitoring tests for CI toxins using LC/MRM-MS analysis, capable of detecting even trace amounts of substances. Starting with this study, we aim to develop testing methods for detecting new toxins and continue to ensure safe food consumption for the public.