Contamination status of paralytic shellfish toxins in shellfish from Southeastern China in 2017–2021

doi:10.21203/rs.3.rs-1988631/v1

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Contamination status of paralytic shellfish toxins in shellfish from Southeastern China in 2017–2021

https://doi.org/10.21203/rs.3.rs-1988631/v1

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A total of 2,355 shellfish samples were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of the 10 paralytic shellfish toxins (PSTs) collected from the southeast coast of China from 2017 to 2021. From 2355 shellfish samples (10.91%), 257 were detected, with the highest value in samples of Perna viridis. Among the six source areas in China, the samples from Fujian recorded the highest detected rate (15.28%). PSTs were found in Fuzhou (FZ), Ningde (ND), Quanzhou (QZ), Putian (PT), Zhangzhou (ZZ), and Xiamen (XM), with QZ and FZ having the highest and lowest detection rates of 15.28% and 4.23%, respectively. Saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxin (GTX1, GTX2, GTX3, GTX4), N-sulfocarbamoyl toxin (GTX5), and decarbamoyl toxin (dcSTX, dcGTX2, dcGTX3) were detected, and GTX5 and dcGTX2 were dominant. In addition, the samples containing PSTs were mostly concentrated in May to August and occasionally detected in December. The findings of this study will serve as a solid foundation for detecting marine contaminants and preserving seafood consumers’ health.

Paralytic shellfish toxins (PSTs)

contamination status

China

UPLC-MS/MS

Paralytic shellfish toxins (PSTs) are mainly produced naturally by the neurotoxic alkaloids of dinoflagellates, which known to contain these harmful products, including the genera of Alexandrium species, Gymnodinium, and Gonuaulaxspp in the ocean (Li J, 2021). Saxitoxin analogues are a source of great concern as they are potent neurotoxins that block the voltage-gated sodium channels in excitable cells, suppressing ion permeation (Smith et al., 2019), which results in a series of poisoning symptoms, such as limb muscle paralysis, headache, salivation, and even asphyxiation and death (Woo and Bahna, 2011). At present, this toxin has no effective antidote, and regulatory testing for the presence of PSTs is a requirement in many nations, with a regulatory limit (RL) of 800 µg STXeq/kg of shellfish tissues (Dean et al., 2020). This group of toxins comprises a family of more than 50, with the most studied analogues divided into the following toxin analogues on the basis of the chemical group, namely, saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxin (GTX1, GTX2, GTX3, GTX4), N-sulfocarbamoyl toxin (GTX5), decarbamoyl toxin (dcSTX, dcGTX2, dcGTX3), and some new metabolites (M1-5) (Walker et al., 2019). Figure 1 shows the structure of PSTs.

Global incidents of PSTs mainly occurred in coastal areas, especially in countries near the coastlines of the Atlantic and Pacific oceans (Arjen et al., 2010). Outbreaks of PSTs have also been reported following the consumption of toxin bivalves in coastal areas in China. For instance, coastal cities, such as Fujian Province, reported poisoning incidents in 2017, with 164 recorded cases (Chen et al., 2018). Occurrences of PSTs-producing harmful algal blooms have been recognized along the coast of China for many years. Meanwhile, bivalves, which filter feeding organisms, may accumulate and biotransform those compounds in their tissues during toxic algal blooms, not only causing serious harm to human health but also hindering the local economic development.

Many approaches, involving mouse bioassay (MBA) (M et al., 2021) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (AS et al., 2014), have been developed to analyze PSTs in shellfish. Nevertheless, MBA cannot determine the structure and components of the toxins, and the HPLC-FLD method is often disturbed in the derivatization process. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (HN et al., 2020) addresses these defects and can provide detailed information on the toxin spectrum, which is widely and routinely applied to the monitoring of marine toxins in shellfish.

This study aims to conduct UPLC-MS/MS analysis on 2355 shellfish samples comprising of six species of bivalve mollusks harvested from the waters of Fujian from 2017 to 2021 to elucidate the contamination status of the PST analogues typically seen in bivalves. Samples were analyzed using solid phase extraction mass spectrometry to determine the individual PST concentrations and obtain the total PSTs content. The acquired data can facilitate the examination of the potential relationships to species type and the geographical and temporal variabilities throughout the coastal waters of Fujian, China. The prevalence of 10 different PSTs (STX, neoSTX, dcSTX, dcGTX2, dcGTX3, GTX1, GTX2, GTX3, GTX4, GTX5) along the Southeastern Chinese coast was studied. The objective of the present study is to access the contamination status of PSTs in the Fujian area of Southeastern China, which has great significance for establishing a highly effective marine toxin monitoring system and preventing PSTs poisoning.

2.1 Sample collection

Representative and typical seafood markets in six cities of Fuzhou (FZ), Ningde (ND), Putian (PT), Quanzhou (QZ), Xiamen (XM), and Zhangzhou (ZZ) in the south coastal areas were selected (Fig. 2). From 2017 to 2021, 2355 bivalve shellfish samples of six species were collected according to the seafood wholesale markets, including 630 samples of mussels (Perna viridis), 115 samples of razor clams (Sinonovaculla constricta), 133 samples of clams (Ruditapes philippinarum), 843 samples of oysters (Crassostyea gigas), 347 samples of scallops (Chlamys farreri), and 287 samples of mud clams (Arca granosa). The collected shellfish samples were placed in a cooler at -20°C, transported to the laboratory, and frozen before analysis and extraction.

Shellfish samples were cleaned with clean water, removing the meat from their shells, and rinsed again with Milli-Q water to remove silt from the flesh. Shellfish tissues, with meat and viscera (200 g of each batch), were removed and homogenized, placed in a kitchen blender, and then frozen at -18°C until extraction. To collect the entire soft tissue and digestive glands, the shell body was not cut, water was spread on the sieve for 5 min, and the shell meat was mixed. The edible part of the scallop sample was detected.

2.2 Chemical reagents

Acetonitrile and methanol were bought from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (≥ 99.5%), ammonium formate (≥ 99.0%), and ammonia (concentration of 25–28%) were obtained from CNW Technologies GmbH (Düsseldorf, Germany). The water used for UPLC analysis was prepared with a Millipore water purification system (Millipore Ltd, Bedford, MA, USA). The certified reference materials (CRMs) used were STX (24.68 µg/mL), dcSTX (21.40 µg/mL), neoSTX (25.47 µg/mL), GTX1 (29.25 µg/mL), GTX4 (9.54 µg/mL), GTX2 (53.48 µg/mL), GTX3 (20.32 µg/mL), GTX5 (25.19 µg/mL), dcGTX2 (49.33 µg/mL), and dcGTX3 (11.10 µg/mL). These standards were purchased from the National Research Council of Canada (Halifax, Nova Scotia, Canada)

2.3 Toxin extraction and clean up

Toxins were extracted from the shellfish following the standardized operating methods according to China’s national food safety risk monitoring manual: 2 g of homogenized specimen was vortex-mixed in a 10mL centrifuge tube, and the tissues and the solvents were vortexed for 90 s before adding the capped tubes to a boiling water bath for 5 min. The samples were subsequently cooled in cold running water for a minimum of 5 mins. After cooling, the tubes were centrifuged at 4500 r/min for 5 min prior to decanting the supernatant into a 15mL tube.

Then, 1 mL of the above extract was added into a centrifuge tube of 2 mL, added 5 µL of NH₄OH, and mixed well. An activate SupelcoENVI-Carb solid phase extraction column was adopted to clean and enrich the toxins of shellfish with 3 mL of 20% acetonitrile aqueous solution (containing 0.8% acetic acid) and 3 mL of 0.1% ammonia solution in turn, and 0.25 mL of extraction solution was added. The cartridge was washed then with 700 µL of water, dried under vacuum for approximately 5 s, and finally eluted with 2 mL of 20% acetonitrile aqueous solution (containing 0.8% acetic acid). The eluent was collected in a 5-mL centrifuge tube and filtered through a 0.22-µm syringe membrane. Ultimately, the eluent was stored in a sample injection vial at 4°C for analysis by UPLC-MS/MS.

2.4 UPLC-MS/MS analysis

Mass spectrometry detection uses a Waters Xevo TQ-XS triple quadrupole mass spectrometry detector (Waters Corporation, Milford, MA, USA) equipped with an electrostatic ionization source (ESI). Mass spectrometry was operated in both positive and negative electrostatic ionization modes for multiple reactions monitoring (MRM). The ESI conditions were optimized as follows: capillary voltage of 3.0 KV (positive), 3.0 KV (negative), offset of ion source of 30 V, desolvation temperature of 500°C, gas flow rate of 1000 L/h, conical gas flow rate of 150 L/h, and atomizer of 7 Bar. The MS/MS parameters are presented in Table 1. The MassLynx software (V4.2 Waters Corporation, Milford, MA, USA) was used for data acquisition and processing.

Table 1

Instrument parameters used in the analytical method for PSTs with UPLC-MS/MS under the mode of multiple reaction monitoring.
Toxins	Retention time (min)	ESI mode	Precursor ion (m/z)	CE (eV)	Product ion (m/z)	CE (eV)
STX	6.60	ESI⁺	300.3/204.2	30	300.0/138.2	35
dcSTX	6.63	ESI⁺	257.4/126.0	30	257.1/221.3	22
neoSTX	6.65	ESI⁺	316.2/298.1	34	2316.2/220.1	34
GTX2	6.01	ESI⁻	394.1/351.2	-16	394.1/333.1	-22
GTX3	6.19	ESI⁻	394.2/333.3	-22	394.2/351.1	-16
GTX1	6.04	ESI⁻	410.2/367.0	-15	410.1/349.2	-22
GTX4	6.22	ESI⁻	410.3/349.2	-22	410.1/367.3	-15
GTX5	6.44	ESI⁻	378.1/122.1	-25	378.1/360.1	-20
dcGTX2	6.08	ESI⁻	351.1/164.0	-30	351.1/333.1	-17
dcGTX3	6.26	ESI⁻	351.1/333.1	-17	351.1/164.0	-30

The Acquity UPLC system (Waters Corporation, Milford, MA, USA) was used for the chromatographic separation of PSTs with a TSK-Gel Amide-80 column (2.0 mm × 150 mm, 5 µm) at 40°C. The mobile phase was composed of (A) an aqueous solution containing 2 mmol/L of ammonium formate and 50 mmol/L of formic acid and (B) an acetonitrile solution containing 2 mmol/L of ammonium formate and 50 mmol/L of formic acid. The gradient program was set with an injection volume of 5 µL and a flow rate of 0.4 uL/min, which starts at 20% A.

2.5 Statistical analysis

The acquired toxin content, toxin profiles, and toxicity of the marine organism samples were processed using Microsoft Excel 2009. The figures were prepared using Origin 2021, and SPSS 22.0 was used for Pearson correlation analysis on and the linear regression model of the toxin data.

3.1 Toxin profiles

Table 2 presents the concentration data set from all the PSTs quantified in both species across the six sites. The samples collected from the Fujian coast in China were found to contain a range of PSTs analogues, including GTX1, GTX2, GTX3, GTX4, GTX5, dcSTX, STX, neoSTX, dcGTX2, and dcGTX3. In terms of toxicity proportion, the profiles were dominated by GTX5 (detection rate 4.46%), followed by neoSTX (3.48%) and dcGTX2 (2.93%). The remaining analogues were present at relatively low levels, with a detection rate of approximately 1.10–2.08%. The toxicity was higher than the European Food Safety Authority (EFSA) legal limit in 4 out of the 2355 detected samples (0.17%) (EFSA, 2009). The 2137.10 µg STXeq/kg was the strongest content, with a mean concentration from 9.59 µg/kg to 16.24 µg/kg, while the maximum content was 45.90–4080 µg/kg.

Table 2

Concentration data set of PSTs of all samples (N = 2355).
	Maximum concentration (ug/kg)	Mean concentration (ug/kg)
STX	239.0	10.01
dcSTX	440.0	9.81
neoSTX	130.0	9.66
dcGTX2	2220.0	11.72
dcGTX3	342.0	9.97
GTX1	162.0	9.76
GTX2	315.0	9.89
GTX3	103.0	9.70
GTX4	45.9	9.59
GTX5	4080.0	16.24

The shellfish species with a high detection rate were A. granosa, C. farreri, P. viridis, and C. gigas at 14.63%, 13.54%, 11.75%, and 10.79%, respectively. The highest concentrations were quantified in P. viridis and C. gigas from QZ and ZZ, with values over 800 µg STXeq/kg in both sites, with a maximum concentration of 2137.10 µg STXeq/kg in the P. viridis collected in June 2017 from ZZ. Significantly lower total PSTs concentrations were obtained in the C. gigas collected from QZ and ZZ, with the highest concentrations of 1469.46 and 1983.89 µg STXeq/kg, respectively. At the A. granosa and C. farreri sites, lower (< 800 µg STXeq/kg) levels than those of the P. viridis and C. gigas of PSTs were found between May 2017 and December 2021.

3.2 Temporal variation

Figure 3 shows the highest levels of PSTs in 2017, followed by 2018 and 2019, while almost no PSTs were detected in 2020 and 2021. The dominance of GTX5 was observed in 2017 (21.77%), followed by the detection rate of dcSTX (8.06%). The highest toxin level in 2018 was also the GTX5 (10.21%), followed by dcGTX2 (4.08%).The detection rate between 2019 and 2020 was less than 5%, and PSTs were not detected in 2021.

The distribution of toxins for each month in 2018 and 2019 is shown in Fig. 4. PSTs occurs mainly in the periods from May to July and November to December. GTX5 was present in almost every month, dcGTX2 was mainly present from May to August, while neoSTX showed high levels in November, December and January. The recent work of Wang et al. (2017) showed evidence of PSTs in the area of Shenzhen through the radial basis function difference model and concluded that the accumulation time of PSTs was from January to June (Wang et al., 2017). Yao et al. (2019) studied seven elements in China and found that it was rarely detected in spring (April to May) and detected in summer (June to August). This may be related to temperature; warm climate is conducive to the reproduction and secretion of toxins by poisonous phytoplankton (Onofrio et al., 2021).

3.3 Interspecific variation

PSTs undergo transformation from one form to another through different processes. Such processes include reduction, epimerization, oxidation, and desulfation, all of which may potentially result in changes to the overall shellfish toxicity (Andres et al., 2019). Six kinds of bivalve shellfish, namely, Perna viridis, S. constricta, R. philippinarum, Crassostyea gigas, C. farreri, Arca granosa, were collected from six sampling locations along the coastline of Fujian, China and analyzed and pretreated under optimized analytical conditions. The toxins, STX, neoSTX, GTX4, dominated the A. granosa samples throughout the observation period, with GTX5 observed every month in the C. gigas samples. The highest detection rate of 14.63% was found in the A. granosa samples, followed by the C. gigas, C. farreri, and P. viridis samples, with detection rates of 10.79%, 13.54%, and 11.75%, respectively, whereas the R. philippinarum samples exhibited only 2.26%. Moreover, differences among species were observed in the toxins content in this research. The PSTs content exceeding 800 µg STXeq/kg was recorded in 2 out of the 6 marine organism species from 6 sites, including the C. gigas and P. viridis mollusks samples.

The mean concentration of all the detected PSTs in bivalve whole soft tissues collected in five years in the study area was obtained. Figure 5 illustrates that mean concentration of GTX5 in S. constricta (12.39 µg/kg) and neoSTX, dcSTX, dcGTX2&3, and GTX1-4 have the same mean concentration (10 µg/kg). The detected samples in other shellfish A. granosa, C. gigas, C. farreri, and P. viridis were higher than that of R. philippinarum. Two toxin analogues (STX, GTX2) were rich in A. granosa, whereas the profile observed in six cockle samples appeared to have a lower mean concentration of GTX5 than that found on average in the C. gigas data set. The C. gigas samples exhibited the dominance of the GTX5 toxin (22.25 µg/kg), followed by dcGTX2 (mean concentration 14.01%). GTX5 was the highest mean concentration (14.04 µg/kg), followed by dcGTX2&3 and GTX1 (mean concentration 10.54, 10.51, and 10.83 µg/kg, respectively), however, the remainder were lower levels (9 ~ 10 µg/kg) in C. farreri. Also, mean concentration of GTX5 (13.04 µg/kg) present in the Perna viridis was dominant toxin, followed by dcSTX, dcGTX2&3. S. constricta could not be compared because of the values below the detection limit. The PST components detected in shellfish toxins are mainly carbamates, and the toxic components of shellfish are related to the species that feed on poisonous algae, which are usually similar to the PST components contained in shellfish and the toxic components of poisonous algae. However, due to the metabolism and transformation of the toxins in shellfish, the proportion and components of the toxins in shellfish will change.

3.4 Spatial variation

Significant variation in PSTs was quantified between different geographical regions within Fujian province. Figures 6 and 7 illustrate the total PSTs quantified in the sampled areas, namely, FZ, ND, PT, QZ, XM, and ZZ, each progressively exhibiting increased variation. More than half of the study samples were harvested from the most ND region of Fujian Province (21.61%). From these, the highest toxicity was 403.26 µg STXeq/kg, with no samples above the RL (800 µg STXeq/kg). The highest toxicities were observed in the sampled areas of QZ and ZZ, with the latter of the three containing the samples showing the highest toxicities out of all the samples studied (toxic equivalent = 2137.10, 1835.25, and 1983.89 µg STXeq/kg respectively), with 0.17% of the total samples above the RL. Although shellfish products in every city were contaminated with at least some of paralytic marine toxins, the toxins concentration and composition varied widely among the various cities.

The four cities (FZ, QZ, XM, and ZZ) showed a similar trend of paralytic marine biotoxin contamination, and GTX5 is the most ubiquitous toxin. Most of the paralytic marine biotoxins, except for GTX4, were present in many specimens from two cities, namely, ND and XM. The detection rate of FZ was 4.23%, and the maximum concentration was 344 µg/kg, but STX, neoSTX, dcSTX, and GTX3 were not found in the area. neoSTX was the most detected in ND, accounting for 71.74% of the total toxins, and the maximum concentration was 330 µg/kg. Furthermore, dcGTX3 was not discovered in PT, STX, neoSTX, and GTX4 were not recognized in QZ. GTX5 was the most prevalent toxins, accounting for 41.33% of the total toxins in this area. The result suggests that the detection rate of ND and QZ is high, and the difference in detection rate is insignificant, while the detection rate of FZ is much lower than that in the other areas.

Figure 8 summarizes the proportion of toxin analogues quantified in terms of STX equivalents in the bivalve mollusks harvested across each of the six regional areas along the Southeastern China coast. The figure shows that most of the toxin analogues were detected in May to August, occasionally in November to December, and never quantified in February to March. PSTs were detected in XM and PT mainly in May to June, accounting for 33–67% of the total toxicity, followed by QZ and FZ, accounting for 10–20% of the total toxicity. The mollusks from ND exhibited low relative proportions in January to November, displaying the highest relative proportion (42%).

4.1 Geographical distribution features

A comparison of the toxin levels between regional studied areas was conducted, noting the differences in the detection rate between the six geographical zones from north to south: FZ (4.23%), ND (13.36%), PT (9.18%), QZ (15.28%), XM (12.15%), and ZZ (10.13%). The spatial variability of toxicity data indicates that the highest risks from high PSTs originate from the sampled district of QZ and ZZ, given the most toxicities of, namely, 1469.46 and 2137.10 µg STXeq/kg from the two regions. The toxicity was still highly evident outside these two regions, with occasional samples exhibiting toxin levels well above the RL, and the mean values determined were notably low. Another work previously showed that the mean PSTs concentration ranged from 10.85–134.06 µg STXeq/kg, with maximum values ranging from 715.60 µg STXeq/kg to 796.00 µg STXeq/kg in Southeastern China, and the offshore banks of Guangdong Province historically exhibited low toxin levels (Zhou et al., 2022).

Based on the analysis of the geographical distribution characteristics, it was found that the PSTs were mainly distributed in QZ and ND, and the maximum toxin concentrations were 4080ug/kg and 330ug/kg, respectively. This accords with our earlier observations in the eastern coast of Guangdong Province from March 2005 to May 2006, which showed that the PSTs toxicity with 14015ug STXeq/kg higher than the safety limit (Wang et al., 2011). GTX1-4 were detected few portions which contains PSTs in PT, and only trace amounts of neoSTX were detected with a mean proportion of 32.15%. The PSTs profiles in QZ extracts were rich in GTX5 (mean 54.38% of the total) and dominated by dcSTX (mean 17.54% of the total), dcGTX2 (mean 24.56% of the total) and dcGTX2 (mean 26.31% of the total). In addition to these components, a trace great amount of GTX5 (mean 50% of the total) was detected in XM. Similarly, large amounts of GTX5 were detected in ZZ around 47.5% of the total, and GTX3 (mean 2.5% of the total) was the least proportion and the maximum toxin content in the samples collected was 3388.9ug/kg. GTX5 was the main toxin component in ZZ, QZ, XM, and FZ, while neoSTX was the main toxin component in ND and PT. The detection rate in QZ and ND was relatively high. There is little difference among the other three regions (XM, ZZ, and PT).

4.2 Seasonal variation

Temporal variability was also noted in this study given the higher toxin levels measured in the 2355 bivalve samples harvested in May 2017 to October 2021, with low concentrations in 2017 (May to June) and 2020 (June). A great prevalence of high PSTs was detected in the samples in May to August, and this result corroborates the findings of many previous works on finding obvious seasonal variation features. The most obvious finding from the analysis is that from March 2005 to May 2006, Jiang et al. (2010) detected PSTs in the bivalve samples collected from the eastern coast of Guangdong Province in spring (March to May) and then in autumn (September). This finding is consistent with that of Leng et al. (2014) who collected mollusks from the central coast of Guangdong Province, that is, 283 µg of STXeq/kg with the highest toxicity of PSTs appeared in March 2006 (Leng et al., 2014). In the study of the seasonal and multi-year trends of PSTs bivalve toxicity in Portuguese waters, Botelho et al. (2019) found that the increase of the toxicity value showed an obvious seasonal trend, reaching a peak in autumn/winter and occasionally in summer. Liu et al. (2020) researched the spatiotemporal variation of the PSTs in the adjacent waters of the Yangtze River (Liu et al., 2020). The results suggest that many PSTs were detected from May to June, and the highest seasonal distribution from July to September is similar, all concentrated on May to August, and reached the peak in summer (June to August).

The most obvious finding from the analysis is that PSTs are mainly detected in May to August and November to December. The temperature, the tidal current and wind direction of the water, the species, the quantity of toxic algae, and the cyst formation rate also affect the seasonal distribution of PSTs. PSTs are related to the production of toxic dinoflagellates. Alexandrium tamarense grows well at 15 ℃–20 ℃ and has strong cytotoxicity at low temperatures (Garcés et al., 2004; Giacobbe et al., 1996), which seem to favor the growth of Alexandrium. Meanwhile, during this period, nutrient loadings increased significantly in the bay (WANG et al., 2009), facilitating the rapid growth of phytoplankton. The cysts of Alexandrium appear in most seasons of the year, and the cyst formation rate is the highest in winter. The investigation of PSTs in the inshore waters of the East China Sea and the South China Sea by Du et al. (2013) shows that the high exposure level of shellfish in winter may be related to the upwelling and wind direction; in addition, during the cyst germination in spring, poisonous algae exist mainly in vegetative cells in water, and the high level of shellfish poisoning in spring may be related to the large amount of toxic algae in the water.

4.3 High PSTs risk species and their toxin profile

Significant differences in toxin profiles were confirmed between sampling bivalves. STX and GTX2 constituted the largest proportion of mean concentration in the A. granosa samples, whereas STX, dcGTX3, and GTX5 presented relatively low proportions in the P. viridis samples. Furthermore, differences were also observed in the mean concentration determined for each individual shellfish species; the toxins of PST were mostly detected in P. viridis between the different species, followed by A. granosa and C. gigas. The calculation of toxin profiles in terms of detection rate revealed relatively high proportions of dcGTX2 and GTX1&2&3, most notably in R. philippinarum, where they represented a 0.75% detection rate. This figure is comparable with that reported by Moroo et al. (2003) and Nielsen et al. (2016) who found that oysters, scallops, and mussels may accumulate large amounts of marine toxins, which may be related to the high feeding rate of phytoplankton due to the large size of scallops and mussels (Moroo et al., 2003; Nielsen et al., 2016). Zhou et al. investigated the bivalve shellfish in Hong Kong and detected 3200 µg STXeq/kg of toxin in the soft tissues of C. nobilis. PSTs were mainly detected in C. nobilis, P. viridis, and C. gigas. The results of the survey in the coastal waters of Guangdong Province from November 2007 to October 2008 show that PSTs were only found in C. nobilis, with a detected rate of 75% (Kemei et al., 2013). Eight species of PSTs were detected in 35 species of shellfish, including Meretrixmeretrix, mussels, and Crassostrea spp, and 11 species of PSTs were detected in 24 species of shellfish in the South China Sea, including Nassarius spp, reflecting the interspecific differences in the accumulation of PSTs (Wu et al., 2008).

Most of the toxins were detected in C. farreri and A. granosa, with maximum amounts of 365.34 and 373.52 µg STXeq/kg collected in Southeastern China. The toxins in other tissues, such as R. philippinarum and S. constricta, were very low to undetectable, except for six samples collected, with a maximum level of 246.7 µg STXeq/kg. The results show that the PSTs from both C. gigas and P. viridis are unsafe for human consumption during the summer months. The toxin levels from P. viridis (maximum value of 2137.10 µg STXeq/kg) were significantly higher than those from other species. The sharp increase in concentrations of PSTs in January was coincident with the occurrence of the Alexandrium population (both vegetative cells and cysts). This phenomenon may be partly due to the influence of the aquaculture water environment. In fact, the accumulation of shellfish toxins is also related to the number and type of toxic algal cells in the water, the neurosensitivity of individual shellfish and the different metabolism. Thus, because the shellfish samples are not entirely from the same aquaculture area, farmers directly move the shellfish products from different waters to another water area to continue the temporary culture. If the oyster is relatively clean before migration, the culture time is short after migration, the cell concentration of the toxic algae in the culture water after migration is low, or does not encounter toxic algae, then the toxicity of PSTs in shellfish may be relatively low or undetected.

PSTs were monitored for five years using the UPLC-MS/MS method based on liquid chromatography–mass spectrometry method using graphitized carbon solid phase extraction. This approach enabled the generation of data describing the total PSTs proportion together with the mean concentrations of specific PSTs congeners. Ten detected toxins were detected, namely, STX, dcSTX, neoSTX, dcGTX1&2, and GTX1-5, with a detection rate of 10.91% (257/2355); among which, four samples exceeded the standard limit of 800 µg STXeq/kg, with the highest content of 2137.10 µg STXeq/kg. Highly variable numbers of detected samples were recorded annually, together with noticeable differences in the maximum levels of PSTs reached. PSTs have obvious seasonal characteristics, and the peaks occurred in May to August and November to December. The regions where toxin events were detected were also found to vary considerably, with some areas showing high levels of PSTs above the RL. The samples that exhibited a total STXeq above the regulatory action limit were predominantly P. viridis and C. gigas. The marine biological samples were affected by the geographical distribution, seasonal changes, and differences among the species.

Funding: This work was financially supported by Construction of Fujian Provincial Scientific and Technological Innovation Platform, China (Grant No. 2019Y2001)，Health Science and Technology Program of Fujian Province, China(Grant No. 2020GGA022) and Basic Scientific Research Project of Fujian Public Welfare Scientific Research Institute , China (Grant No. 2022R1006005).

Author Contributions: Renjin Zheng:Investigation, Data curation, Yafang Yang: Investigation, Writing-Original draft preparation, Wenting Zhang: Formal analysis, Methodology Yongyou Hua: Supervision,Writing-Reviewing and Editing.

Declaration of interests: The authors declare that there is no conflict of interest.

Data availability: N/A, data supporting the results reported in the article can be found in the fgures and tables included in this paper.

Ethical Approval and Consent to Participate: Not applicable.

Consent to Publish: Not applicable.

Andres, J.K., Yñiguez, A.T., Maister, J.M., Turner, A.D., Olano, D.E.B., Mendoza, J., Salvador-Reyes, L., Azanza, R.V., 2019. Paralytic Shellfish Toxin Uptake, Assimilation, Depuration, and Transformation in the Southeast Asian Green-Lipped Mussel (Perna viridis). TOXINS. 11(8), 468.doi:10.3390/toxins11080468.
Arjen, G., Pol-Hofstad, I.E., Marnix, P., Mulder, P.P.J., Van, D.T.H.J., Jacob, D.B., 2010. Marine Toxins: Chemistry, Toxicity, Occurrence and Detection, with Special Reference to the Dutch Situation. TOXINS. 2(4), 878-904.doi: 10.3390/toxins2040878.
AS, K., RM, G., A, G., AA, K., PN, N., CJ, B., MC, B., 2014. Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: comparison of detection methods. ELECTROPHORESIS. 35(10), 1496-1503.doi:10.1002/elps.201300353.
Botelho, M.J., Vale, C., Ferreira, J.G., 2019. Seasonal and multi-annual trends of bivalve toxicity by PSTs in Portuguese marine waters. SCI TOTAL ENVIRON. 664(1095-1106.doi:10.1016/j.scitotenv.2019.01.314.
Chen, J.Z., Hong, S.P., Cai, M.R., Chen, L.I., Zhang., T.L., 2018. an outbreak of foodborne disease caused by paralytic shellfish toxin. Chinese Journal of Food Hygiene. 30(04), 445-448.doi:10.13590/j.cjfh.2018.04.022.
Dean, K.J., Hatfield, R.G., Lee, V., Alexander, R.P., Lewis, A.M., Maskrey, B.H., Teixeira Alves, M., Hatton, B., Coates, L.N., Capuzzo, E., Ellis, J.R., Turner, A.D., 2020. Multiple New Paralytic Shellfish Toxin Vectors in Offshore North Sea Benthos, a Deep Secret Exposed. MAR DRUGS. 18(8), 400.doi:10.3390/md18080400.
Du, K.M., Lei, F., Wu, N., Jiang, T.J., 2013. paralytic shellfish toxin pollution in the inshore waters of the East China Sea and South China Sea. Journal of Jinan University (Natural Science and Medicine Edition). 34(03), 343-346
EFSA, 2009. Scientific opinion of the panel on contaminants in the food chain on a request from the European commission on marine biotoxins in shellfish – Summary on regulated marine biotoxins. EFSA Journal. 1306(1-23.doi:https://doi.org/10.2903/j.efsa.2009.1306.
Garcés, E., Bravo, I., Vila, M., Figueroa, R.I., Masó, M., Sampedro, N., 2004. Relationship between vegetative cells and cyst production during Alexandrium minutum bloom in Arenys de Mar harbour (NW Mediterranean). J PLANKTON RES. 26(6), 637-645.doi:10.1093/plankt/fbh065.
Giacobbe, M.G., Oliva, F.D., Maimone, G., 1996. Environmental Factors and Seasonal Occurrence of the DinoflagellateAlexandrium minutum, a PSP Potential Producer, in a Mediterranean Lagoon. Estuarine, Coastal and Shelf Science. 42(5), 539-549.doi:https://doi.org/10.1006/ecss.1996.0035.
HN, H., JL, L., SE, L., RJ, Z., J, L., 2020. Simultaneous determination of twelve paralytic shellfish poisoning toxins in bivalve molluscs by UPLC-MS/MS and its applications to a food poisoning incident. Toxicon : official journal of the International Society on Toxinology. 174(1-7.doi:10.1016/j.toxicon.2019.11.012.
Jiang, T.J., Bao, C., Lei, F., Wu, F., Jiang, T.J., 2010. characteristics of paralytic shellfish toxins along the eastern coast of Guangdong. Chinese Aquatic Sciences. 17(01), 119-127
Kemei, D.U., Lei, F., Ni, W.U., Jiang, T., 2013. The pattern of paralytic shellfish poisoning in shellfish cultured in East China Sea and South China Sea. Journal of Jinan University(Natural Science & Medicine Edition). 34(3), 343-346
Leng, K.M., Wu, N., Du, K.M., Deng, G.Q., Jiang, T.J., 2014. characteristics of paralytic shellfish toxins in central Guangdong. Marine Environmental Science. 33(05), 666-671.doi:10.13634/j.cnki.mes.2014.05.003.
Li J, P.K.M., 2021. Quick detection method for paralytic shellfish toxins (PSTs) monitoring in freshwater - A review[J]. CHEMOSPHERE. 265(128-591
Liu, Y., Dai, L., Chen, Z., Geng, H., Lin, Z., Zhao, Y., Zhou, Z., Kong, F., Yu, R., Zhou, M., 2020. Spatiotemporal variation of paralytic shellfish toxins in the sea area adjacent to the Changjiang River estuary. ENVIRON POLLUT. 259(113-730.doi:10.1016/j.envpol.2019.113730.
M, L., B, K., J, C., U, T., 2021. AlexandriumUnknown Extracellular and Bioactive Metabolites of the Genus : A Review of Overlooked Toxins. TOXINS. 13(12), 905-944.doi:10.3390/toxins13120905.
Moroo, A., Arévalo, F., Fernández, M.L., Maneiro, J., Pazos, Y., Salgado, C., Blanco, J., 2003. Accumulation and transformation of DSP toxins in mussels Mytilus galloprovincialis during a toxic episode caused by Dinophysis acuminata. AQUAT TOXICOL. 62(4), 269-280
Nielsen, L.T., Hansen, P.J., Krock, B., Vismann, B., 2016. Accumulation, transformation and breakdown of DSP toxins from the toxic dinoflagellate Dinophysis acuta in blue mussels, Mytilus edulis. TOXICON. 117(84-93.doi:10.1016/j.toxicon.2016.03.021.
Onofrio, M.D., Egerton, T.A., Reece, K.S., Pease, S.K.D., Sanderson, M.P., III, W.J., Yeargan, E., Roach, A., DeMent, C., Wood, A., Reay, W.G., Place, A.R., Smith, J.L., 2021. Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters. HARMFUL ALGAE. 103(101993.doi:10.1016/j.hal.2021.101993.
Smith, Z.J., Martin, R.M., Wei, B., Wilhelm, S.W., Boyer, G.L., 2019. Spatial and Temporal Variation in Paralytic Shellfish Toxin Production by Benthic Microseira (Lyngbya) wollei in a Freshwater New York Lake. TOXINS. 11(1), 44.doi:10.3390/toxins11010044.
Walker, J.R., Merit, J.E., Thomas Tran, R., Tang, D.T.Y., Du Bois, J., 2019. Divergent Synthesis of Natural Derivatives of (+)‐Saxitoxin Including 11‐Saxitoxinethanoic Acid. Angewandte Chemie International Edition. 58(6), 1689-1693.doi:10.1002/anie.201811717.
Wang, Z., Nie, X., Jiang, S., Zhao, J., Cao, Y., Zhang, Y., Wang, D., 2011. Source and profile of paralytic shellfish poisoning toxins in shellfish in Daya Bay, South China Sea. MAR ENVIRON RES. 72(1-2), 53-59.doi:10.1016/j.marenvres.2011.04.007.
Wang, Z., Xiao, S.Y., Huang, W., Pan, L.B., Wan, Y., Guo, Q.F., Li, S.G., Luo, X.R., Yang, L.Q., Xiao, X.X., 2017. Spatio-temporal analysis of paralytic shellfish toxins in Shenzhen sea area. Chinese Journal of Food Hygiene. 29(01), 89-92
WANG, Z., ZHAO, J., ZHANG, Y., CAO, Y., 2009. Phytoplankton community structure and environmental parameters in aquaculture areas of Daya Bay, South China Sea. J ENVIRON SCI-CHINA. 21(9), 1268-1275.doi:https://doi.org/10.1016/S1001-0742(08)62414-6.
Woo, C.K., Bahna, S.L., 2011. Not all shellfish "allergy" is allergy!. CLIN TRANSL ALLERGY. 1(1), 3.doi:10.1186/2045-7022-1-3.
Wu, S.W., Zeng, M., Lu, D.P., Luo, Y.D., 2008. Analysis of paralytic shellfish toxins in Guangdong coastal waters in the sping of 2005. Oceanographic research. 26(04), 49-54
Yao, J., Jin, W., Li, D., Xu, D., Wen, S., Liu, R., Liang, Y., Lu, S., 2019. Geographical distribution and seasonal variation in paralytic shellfish toxins in the coastal water of the South China Sea. TOXICON. 168(67-75.doi:10.1016/j.toxicon.2019.06.221.
Zhou, M., Li, J., Luckas, B., Yu, R., Yan, T., Hummert, C., Kastrup, S., 1999. A Recent Shellfish Toxin Investigation In China. Mar. Pollut. Bull. 39(1-12), 331-334
Zhou, Y., Li, S., Zhang, J., Zhang, J., Wang, Z., Pan, L., Huang, B., Huang, K., Chen, X., Zhao, Q., Jiang, T., Liu, J., 2022. Dietary exposure assessment of paralytic shellfish toxins through shellfish consumption in Shenzhen population, China. ENVIRON SCI POLLUT R. 29(7), 10222-10234.doi:10.1007/s11356-021-16249-4.

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Reviewers agreed at journal
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Contamination status of paralytic shellfish toxins in shellfish from Southeastern China in 2017–2021

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Sample collection

2.2 Chemical reagents

2.3 Toxin extraction and clean up

2.4 UPLC-MS/MS analysis

2.5 Statistical analysis

3. Results

3.1 Toxin profiles

3.2 Temporal variation

3.3 Interspecific variation

3.4 Spatial variation

4. Discussion

4.1 Geographical distribution features

4.2 Seasonal variation

4.3 High PSTs risk species and their toxin profile

5. Conclusion

Declarations

References

Supplementary Files

Status:

Version 1