Dietary Exposure Assessment of Paralytic Shellfish Toxins through Shellfish Consumption in Shenzhen Population, China

doi:10.21203/rs.3.rs-630710/v1

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Dietary Exposure Assessment of Paralytic Shellfish Toxins through Shellfish Consumption in Shenzhen Population, China

https://doi.org/10.21203/rs.3.rs-630710/v1

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published 13 Sep, 2021

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Paralytic shellfish toxins (PSTs) produced by certain marine dinoflagellates accumulate in filter-feeding marine bivalves. We used LC-MS/MS to detect and quantify 13 PSTs in 188 shellfish samples of 14 species collected from Shenzhen city’s Buji seafood wholesale market from March 2019 to February 2020. Twenty-six of 188 shellfish samples (13.8%) were PST- positive, with highest values in samples of the Noble clam Chlamys nobilis (10/34, 29.4%). Samples originating from Nan’ao island among 11 source sites in China recorded the highest detected rate (7/17, 41.2%). Samples containing PSTs were concentrated in Spring and Winter, with the highest levels in March > December > January. Among PSTs detected, C1 was dominant. Acute dietary exposure assessments for Shenzhen residents were based on P₉₉ consumption data (139.2g/day) and maximum PST concentration for each shellfish species. The outcome for Chlamys nobilis was 2.4 ~ 3.7-fold higher than recommended ARfDs (0.5 ~ 0.7 µg STX eq./kg bw/day). Mean PST concentration (10.9 ~ 134.1 µg STX Eq. /kg), mean shellfish consumption (4.8 g/day) and P₉₉ consumption data were used to assess chronic dietary exposure. The results were lower than the recommended ARfDs. In conclusion, residents in Shenzhen are at risk for acute PST poisoning, while relatively safe from chronic PST exposure.

Environmental Engineering

Environmental Policy

Paralytic Shellfish toxins

Saxitoxin

dietary exposure assessment

The escalating incidence of paralytic shellfish poisoning (PSP) is associated with increasing harmful algal blooms linked to global ocean warming and anthropogenic activity that promotes eutrophication (Visciano et al., 2016). Paralytic shellfish toxins (PSTs) are mainly produced by marine dinoflagellates, such as Alexandrium, Gymnodinium, and Gonuaulax spp, and accumulated in filter-feeding bivalves (Liu et al., 2004; Asakawa et al.,2006; Shin et al., 2018). Bivalves commonly contaminated with PSTs include saltwater clams (Meretrix lyrata, Siliqua patula, Paphi subtriangulata, Saxidomus giganteus), mussels (Perna viridis, Mytilus edulis), scallops (Amusium pleuonectes, Mimachlamys nobilis, Argopecten irradians), oysters Crassostrea rivularis), sea snail (Haliotis tuberculate) and crab (Zosimus aeneus), among other edible marine organisms (Bricelj & Shumway, 1998; Tan & Ransangan, 2015).

PST intoxication results from ingestion of seafood containing potent algal toxins, the parent molecule of which is saxitoxin (STX), which acts by blocking sodium channels in excitable membranes of cells, notably neurons (Pellegrino et al., 1984). Clinical symptoms of PST intoxication in mild cases begin with circumoral paresthesia that slowly spreads to the face, neck, fingers, and other parts, and dizziness, nausea. Symptoms usually begin 2~12 hours after ingestion of the contaminated products, with gradual resolution over 24 hours (FAO/IOC/WHO, 2004). Severe poisoning results in muscle paralysis, dyspnea, and death from respiratory paralysis (FAO/IOC/WHO, 2004), with symptom onset 2-12 hours after consumption of contaminated shellfish. An antidote for PST intoxication has yet to be found.

STX is a trialkyl tetrahydropurine derivative (Figure 1) that is soluble in water and has thermal and acid stability (Wang, 2008). Wiese et al. (2010) collected 57 PST derivatives, ~20 of which can be extracted from shellfish. The toxic potencies of individual PSTs are expressed in relation to STX, and the relevant toxicity-equivalence factors (TEFs) are shown in Table 1 (ESFA, 2009; Van de Riet et al.,2009). International regulation limits for PSTs in shellfish are set at 800μg STX eq./kg shellfish meat (Vale & Taleb, 2005; EFSA, 2009).

In recent years, outbreaks of human PSP have occurred in coastal areas around the world, particularly in countries with Atlantic and Pacific coastlines. More than 2100 cases of PSP with 120 deaths were reported in the Philippines between 1983 and 2002 (Visciano et al., 2016.Ching et al., 2015). Hundreds of other PSP cases have been reported in Asia over the past decade (Nicolas et al., 2017, Ching et al., 2015). China has experienced many PSP cases and fatalities in coastal cities. Between 1991 and 2003, the coastal city of Lianyungang City in China’s Jiangsu Province reported 11 PSP events, which together sickened 40 individuals and resulted in 8 fatalities, and coastal Fujian Province in S.E. China reported a PSP incident involving 164 cases in 2017 (Xiang-Tian et al. ,2005; Zhou et al., 2013; Chen et al., 2018). Additionally, the southern coast of China has experienced sporadic outbreaks of PSP (Anderson, 1996) and, at the turn of the 21st century, up to 30% of shellfish samples containedalgal toxins in the Shenzhen area of the South China Sea (Jiang et al., 2003), where the predominant PSP source was the dinoflagellate Alexandrium pacificu (Liu et al., 2021). In March 2005, Shenzhen’s neighbor Hong Kong experienced 36 clusters of shellfish poisoning related to consumption of fresh scallops (Atrina vexillum). Since Shenzhen and Hog Kong have a combined a population of 20 million residents, all of whom are at risk for exposure to PSP, the present study assesses the current level of toxin contamination of edible shellfish and the extent of residential dietary exposure to these biologically dangerous substances.

2.1 Sampling

Every month from March 2019 to February 2020, shellfish samples were collected from the Buji seafood wholesale market in Luohu, which supplies 90% of shellfish consumed in Shenzhen. Other shellfish were collected from local retail stores. In total, 188 shellfish samples of 14 species were collected, including: Chlamys nobilis (scallop), Crassostrea rivularis (oyster), Perna viridis (mussel), Paphia undulata (clam), Atrina pectinate (bivalve), Babylonia areolate (sea snail), Scapharca broughtoni (clam), Scapharca subcrenata (bivalve), Meretrix meretrix (clam), Sinonovacula constricta (razor clam), Ruditapes philippinara (clam), Patinopecten yessoensis (scallop), Solen gouldi (bivalve), and Mytilus edulis (mussel).

Since the year-round availability of three shellfish species (Solen gouldi, Mytilus edulis and Scapharca subcrenata) was limited, their sample sizes were small relative that of other species.

2.2 Reagents

Methanol, acetonitrile, and formic acid were purchased from Merck (Darmstadt, Germany‎).

Methylene chloride was obtained from Damao Chemical Reagent Factory (Tianjin, China). Water was distilled and passed through a MilliQ water purification system (Millipore, Burlington, Mass. USA). Ammonium formate was purchased from Sigma (Bangalore, India). PST standard reagents (C1, C2, dcGTX2, dcGTX3, dcNEO, dcSTX, GTX1, GTX2, GTX3, GTX4, GTX5, STX, NEO) were purchased from Bedford Institute of Oceanography (Dartmouth, Nova Scotia, Canada).

2.3 Toxin extraction

Shellfish samples were cleaned with fresh water, emoved from their shells, and rinsed again with MiliQ water to remove silt from the flesh. Shellfish meat and viscera (200g of each batch) was removed and homogenized. An 5 g aliquot of homogenized sample was transferred to a centrifuge tube, 8 mL of 0.5% formic acid solution added, for vortex-mixed for 5 min, ultrasonically extracted for 10 min, followed by centrifugation at 9500 rpm/min for 10 min. The suspension was immersed in 0.5% formic acid solution to 10 mL. Two mL of this mixture were added to 5 mL dichloromethane, vortex-mixed for 3 min, and then centrifuged for 5 min at 9500 rpm. Then, 1 mL suspension was passed through a C-18 solid-phase extraction cartridge (Waters, Millford, Mass., USA) that had been activated by adding 3 mL acetonitrile and 3 mL 0.5% formic acid solution in that order, and then immersed in 1.5 mL 0.5% formic acid solution. The eluate was collected, adjusted to 6 mL acetonitrile solution, and vortexed. The resulting solution was allowed to rest for 30 min in a -4℃ refrigerator, then centrifuged at 9500 t/min for 5 min, passed through 0.22 μm nylon syringe filter and collected in a brown test tube.

2.4 Detection and quantification of PSTs

Firstly, a chromatographic analysis was performed with a Nexera X2 HPLC system (Shimadzu, Kyoto, Japan) comprising a degassing unit, an auto-sampler and a micro binary pump and equipped with a Waters Amide column (2.1*100 mm，3.5 μm, Waters, USA). The temperature of the column was set at 40℃. The injection volume was 5 μL, and the flow rate was set to 0.5 mL/min in all time. The mobile phase consisted of two eluates: A (1 mmol/L ammonium formate solution) and B (acetonitrile), both of which contained 1.3 mmol/L formic acid. The elution was performed starting with 5% A for 2 min, increasing to 15%-30% for 16 min, and finally 5% A for 2.5 min for column re-equilibration. PSP toxins were detected by mass spectrometry analysis using a Shimadzu 8050 series mass spectrometer equipped with an electrospray interface set in the positive ionization mode (ESI+). PSP toxins (and their corresponding PubChem CID) included: neo-dc saxitoxin (dcNEO, 100962170), gonyautoxin 5 (GTX5, 49789073), neosaxitoxin (NEO, 135562690 ), dc saxitoxin (dcSTX ,101936522), saxitoxin (STX, 56947150). A negative ionization mode (ESI－) was used to detect: N-sulfocarbamoyl-gonyautoxin-2 and -3(C1,2, 49789085), and other gonyautoxins, including dcGTX2 (101034662), GTX1 (135061918), GTX2 (101650338), GTX4 (440699577), GTX3 (46217347), and dcGTX3(101034664). The multiple reaction monitoring (MRM) mode was used, with specific transition parameters as shown in Table 2. The capillary voltage was 4000 V, the nebulizer gas flow rate was 3 L/min, the drying gas flow rate was 10 L/min, and the temperature of the ion source was 300℃.The limits of detection (LOD, μg/kg) were dcNEO (24), GTX5 (60), NEO (30), dcSTX (30), STX (30), C1(40.8), C2 (24), dcGTX2 (42), GTX1 (38.4), GTX2 (28.8), GTX4 (48), GTX3 (48, dcGTX3(48).

2.5 Dietary exposure assessment

Dietary exposure to shellfish toxins was performed by multiplying shellfish PST value and amount of shellfish consumed divided by the target population mean body weight (WHO, 2009; Wong, 2013).

2.5.1 Consumption data

The consumption data and mean body weight of the target population were obtained from the individual-based Shenzhen Food Consumption Survey 2008 (SZFCS 2008), which was conducted by the Shenzhen Center for Disease Control and Prevention (SZCDC). A three-stage cluster sampling was performed taking account of local population flow and density, together with geographic and economic characteristics (Yang et al. 2014). Referring to the sampling principles of China National Health and Nutrition Survey (2002), 244 households were selected throughout 4 urban regions and 2 rural regions, a total of 853 individuals was included. Food consumption data were assessed by a continuous 3-day door-to-door interview, which included weighing that was consumed at home and using a 24-h dietary-recall questionnaire to record food intake outside of the home.

2.5.2 PSTs contamination data

Sample PST contamination data were assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since the data were left-censored, the protocol for assigning concentration values to ND results was applied in this study. In a lower bound (LB) scenario, 0 was used to substitute for ND results (FAO/WHO,2009; EFSA,2010); while, in an upper bound (UB) scenario, 1/2 LOD was used to substitute for ND results.

3.1 Detection of PSTs in shellfish

Among 188 shellfish samples, 26 samples (13.8%) detected positive for PSTs (Table 3). Samples of the Noble scallop Chamys nobilis had the highest positive rate: 10 of 34 samples (29.4%) were PST-positive. High-positive rates were also recorded for the oyster Crassostrea rivularis (5/18, 21.7%) and the Asian green mussel Perna viridis (4/20, 20%). PSTs were not detected in samples of 7 shellfish species (Meretrix meretrix, Sinonovacula constricta, Ruditapes philippinara, Patinopecten yessoensis, Solen gouldi and Mytilus edulis.

Nan'ao or Nanao, formerly romanized Namoa, is an island and county of the prefecture-level city of Shantou in Guangdong Province, China.

Shellfish PST contamination varied by the source location in China (Figure 2). Most contamination was detected in samples taken from waters around Nan’ao island (7/17, 41.2%), followed by Huidong (6/31, 19.4%) and Zhanjiang (6/31,19.4%). Shellfish samples from coastal Dianbai, Shantung, and Yangjiang were PST-negative. Only samples distributed in spring and winter were positive for PSTs: the highest detected rate was in March (5/15, 33.3%), followed by December (5/16, 34.3%) and January (5/16, 31.3%). Samples collected from June to October 2019 were free from PSTs contamination. Detected rates among the time-sampled groups differed significantly by Fisher exact test (p<0.01).

3.2 Concentration of PSTs in shellfish

The concentration PSTs (n=13) was quantified for each seafood sample. The number of samples tested and the distribution of individual PSTs is shown in Figure 3. Most samples were C1 positive (19/188, 10.1%), with the widest concentration range of 65.0~1596.5 μg/kg, followed by C2 (14/188, 7.5%), GTX5 (8/188, 4.3%) and others. NEO, STX, CTX3, GTX4 was not detected in any sample. The concentration ranges for individual PSTs are summarized in Figure 4. Individual PST concentration was converted to the STX-equivalent by multiplying the appropriate toxic equivalent factors (Shin et al., 2018) accordingly to obtain the total PST concentration/sample.

Among all samples, mean PST concentration ranged from 10.85(LB)~134.06(UB)μg STX eq./kg, with maximum values ranging from 715.60(LB) to 796.00(UB) μg STX eq./kg. Both mean and maximum PST concentrations were highest in Chlamys nobilis and samples obtained from Nan'ao. Samples collected in March and April contained similar mean and maximum PSTs concentrations, and these values were higher than in samples collected in other months. Detailed PST concentration sets for all samples, by shellfish species, sources, and time of sampling, are integrated in Table 4.

3.3 Acute Dietary Exposure Assessment

International guidance dictates that large portions of shellfish should be used to conduct an acute dietary exposure assessment (WHO, 2009). A large portion was estimated as 139.2 g/day in the present study, which corresponded to P₉₉ shellfish consumption data of individuals included in SZFCS 2008. Consumption data and adult mean body weight (60.2 kg) were acquired from SZFCS 2008. Both upper and lower bounds of maximum PST concentrations were used to assess the acute dietary exposure level. In studies conducted by the IOC/WHO Expert Consultation on Biotoxins in Molluscan Bivalves, the PST toxicological reference value of provisional Low Observed Adverse Effect Level( LOAEL) was established as 2.0μg/kg bw. A safety factor of 3 was used to drive an Acute Reference Dose (ARfD ) of 0.7μg STX eq./kg bw because a wide spectrum of people report shellfish poisoning, and mild illness is readily reversible (FAO/IOC/WHO, 2004; Picot et al.,2011; Toyofuku, 2006). On the other hand, the European Food Safety Authority (EFSA) established a lower LOAEL (1.5μg/kg bw) based on ~500 reported PSP cases; an ARfD of 0.5μg STX eq./kg bw was proposed subsequently using a safety factor of 3 (EFSA, 2009).

Data for the acute dietary exposure level with respect to individual seafood species are summarized in Figure 5. The highest acute dietary exposure value was 1.84μg STX eq./kg bw/day using the upper bound of the maximum contamination value of Chlamys nobilis, which corresponded to approximately 2.6~3.7 times the ARfD established by FAO/IOC/WHO and ESFA respectively. In the lower bound scenario, the acute dietary exposure value for Chlamys nobilis was 1.65μg STX eq./kg bw/day, a value corresponding to 2.4~ 3.3 times as ARfDs established by FAO/IOC/WHO and ESFA respectively. The acute dietary exposure values of other species were lower than 0.5μg STX eq./kg bw.

3.4 Chronic Exposure Assessment

For the chronic exposure assessment of PSTs in shellfish, both lower and upper bounds (LB, UB) of mean concentrations were used. According to SZFCS 2008, the mean shellfish consumption was 4.82 g/day. The PST chronic dietary exposure value of all samples ranged from 0.009(LB) to 0.0107(UB)μg STX eq./kg bw/day. To estimate an extreme situation, chronic dietary exposure using P₉₉ shellfish consumption data(139.2 g/day) were also assessed, with results ranging from 0.0251(LB) to 0.31(UB)μg STX eq./kg bw/day. Tolerable daily intake (TDI) is considered to be the standard reference for chronic exposure assessment (Shin et al., 2018); however, no such reference has been established by reliable authorities or formal organizations. Therefore, in this study, chronic exposure values were compared to ARfDs by EFSA and FAO/IOC/WHO. Chronic dietary exposure values using mean shellfish consumption data were much lower than recommended ARfD. In the estimated extreme scenario, even the upper-bound chronic dietary exposure value was 0.44~0.62 times the EFSA ARfD. Chronic dietary exposure assessment was also analyzed for different species as summarized in Table 5.

The present study used mass spectrometry to detect and quantify PSTs in marine species sold for human consumption in Shenzhen city in southern China. Seafood samples included the viscera, human consumption of which was identified in neighboring Hong Kong as the highest risk factor for shellfish poisoning (Chung et al., 2006). We found that most seafood samples had lower concentrations of PSTs than the corresponding limits of detections. Among 13 PST derivatives, C1, C2, GTX1, GTX2, dcGTX2, dcGTX3, GTX5, dcNEO, and dcSTX were detected in seafood samples sold in Shenzhen, with C1 and C2 the most predominant components. These results are similar to those of Liu and colleagues (2017), who studied PSTs in phytoplankton and shellfish samples from the Bohai Sea in northern China. They found that toxic Alexandrium spp. and Gymnodinium catenatum were potential producers of PSTs, and most low-PST-content phytoplankton samples were C1/C2- positive.

The concentration of PSTs differs in shellfish species (Brieelj et al., 1990) and sources. We found that 8 of 14 species were contaminated, particularly Chlamys nobilis (Noble scallop) with PST concentration of 715.6(LB) ~ 796(UB)µg STX eq./kg, a value at the edge of the international regulation limit of 800µg STX eq./kg (Vale & Taleb, 2005; EFSA,2009). Studies by Fan and Xiao-Yan (2006) and Jiang and colleagues (2000) showed PST accumulation in Chlamys nobilis was greater than in Perna viridis (Asian green mussel). We found that shellfish collected from Nan’ao contained higher than PST concentrations than samples obtained from other locations. PST-positive shellfish from all locations were highest from December to May match months with those reported in the Results. These findings are comparable with seasonal variation in shellfish concentrations of PSTs (Taleb et al., 2001; Harley et al., 2020), which are mostly detected in spring, early summer, and autumn in the South China Sea (Yao et al., 2019).

Acute dietary exposure of Chinese citizens to shellfish PSTs was determined in the present study to assess the risk of paralytic shellfish poisoning (PSP). Expert assessment of several PSP case series showed that mild cases generally had ingested PST levels of 2–30 µg/kg bw, while more severe cases resulted from intake levels of > 10–300 µg/kg bw (FAO/IOC/WHO, 2004). Based on ARfDs of 0.5–0.7µg STX eq./kg, we show here that Shenzhen residents are at risk for PSP, especially after ingestion of Chlamys nobilis (1.65–1.84µg STX eq./kg bw/day). Consumers might suffer from PSP mild case symptoms such as dizziness, nausea and a pricking sensation of the tongue and lips. Consumption of other shellfish species on sale in Shenzhen requires ongoing PST monitoring but presently appears to be safe.

The Tolerable Daily Intake (TDI) for PSTs has not been established because chronic toxicity studies are unavailable (Visciano P, et al., 2016); thus, we used the Acute Reference Dose (ARfD) as a reference limit for chronic dietary exposure assessment. The chronic exposure value ranged from 0.0009 ~ 0.0107µg STX eq./kg bw/day using mean shellfish consumption data, a level much lower than the ARfD. To assess the chronic dietary exposure in a more conservative way, we used P₉₉ shellfish consumption data to exaggerate the chronic dietary exposure level, the results of which were 0.0251 ~ 0.31µg STX eq./kg bw/day, values that were also lower than the recommended ARfDs.

There are limitations in this study, including outdated (2008) local dietary survey data and population data (9.5 million vs. 12.5 million today); uncertainties associated with dietary exposure assessment, such as the accuracy of consumption data; the limited amount of sample contamination data; using 0 and 1/2 LOD to substitute ND values as the lower and upper bounds; and the lack of a proper reference for chronic exposure assessment, which could cause the outcomes to be under- or over-estimated.

Ongoing studies should be conducted on the diet, nutrition and health status of the Shenzhen population, with dietary PST exposure assessments based on contemporary population figures and stratified consumption data. Since species like Solen gouldi and Mytilus edulis are marketed in only specific months, periodic long-term toxin screening studies are needed to develop an in-depth picture of temporal changes in shellfish contamination. Studies to understand the habitats of shellfish species prone to high concentrations of PSTs are also needed.

We found that none of the seafood samples contained levels of total PSTs that exceeded international regulation limits. However, dietary exposure results indicated that Shenzhen residents are at risk for acute paralytic shellfish toxins poisoning. Local authorities should strengthen the screening of shellfish for PSTs before they are placed on the market, especially in spring and winter, thereby ensuring product quality and control of high-risk species, notably Chlamys nobilis, Crassostrea rivularis, Perna viridis, among other species. Meanwhile, the Shenzhen PST exposure levels determined in in this study might serve as a basis for establishing local limit values for shellfish consumption.

ARfD: Acute Reference Dose; EFSA: European Food and Safety Association; FAO: Food and Agriculture Organization; IOC: Intergovernmental Oceanographic Commission of UNESCO; LB: Lower bound; LOD(s): Limits of detection; LOAEL: Low Observed Adverse Effect Level; PSP: Paralytical shellfish poisoning; PST(s): Paralytic shellfish toxin(s); STX：saxitoxin; SZCDC：Shenzhen Center for Disease Control and Prevention；SZFCS 2008:Shenzhen Food Consumption Survey 2008; TEF(s): Toxicity-equivalence Factors; TDI: Tolerable daily intake; UB：Upper bound; WHO: World Health Organization.

Ethical approval and consent to participate

No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with an unregulated invertebrate species. Not applicable.

Consent to publish

Not applicable.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Shenzhen Basic Research Plan [JCYJ20180508152311822], the National Key Research and Development Program of China (2018FY100201, 2019YFC407900), Shenzhen Key Medical Discipline Construction Fund (SZXK069), and Sanming Project of Medicine in Shenzhen (SZSM201611090).

Authors' contributions

Yan Zhou: Writing-Original draft preparation, Visualization, Formal analysis. Shenpan Li: Methodology. Jianying Zhang: Data curation. Jinzhou Zhang: Project administration, Methodology. Zhou Wang: Methodology, Data curation. Liubo Pan: Methodology, Data curation. Baiqiang Huang: Conceptualization, Methodology. Ke Huang: Data curation. Xiao Chen: Methodology. Qionghui Zhao: Data curation. Tianjiu Jiang: Methodology, Funding acquisition. Jianjun Liu: Project administration, Conceptualization, Methodology, Supervision, Funding acquisition.

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Table 1

The toxic factor and standard limit of PSTs

PST	TEF	Limit
dcNEO	0.4	800 μg STX eq./kg
GTX5	0.1
NEO	1
dcSTX	1
STX	1
C1	0.01
C2	0.1
dcGTX2	0.2
GTX1	1
GTX2	0.4
GTX4	0.7
GTX3	0.6
dcGTX3	0.4

Table 2

Mass spectrum parameters, limits of detection and limits of quantification for PSTs

PST	Precursor-product ions	Positive/ Negative	Linear range (ng/mL)	Limit of detection (μg/kg)	Limit of quantification (μg/kg)
dcNEO	273.1-126.0	ESI+	4~80	24	48
GTX5	380.1-300.1	ESI+	10~500	60	120
NEO	316.0-220.0	ESI+	10~500	30	60
dcSTX	257.1-126.0	ESI+	5~200	30	60
STX	300.0-204.0	ESI+	5~200	30	60
C1	474.0-357.0	ESI－	6.8~270	40.8	81.6
C2	474.0-121.9	ESI－	4~80	24	48
dcGTX2	351.1-333.1	ESI－	7~275	42	84
CTX1	410.1-367.1	ESI－	6.4~254	38.4	76.8
GTX2	394.0-351.1	ESI－	4.7~187	28.8	56.4
GTX4	410.1-367.1	ESI－	8~160	48	96
GTX3	394.0-351.1	ESI－	8~160	48	96
dcGTX3	351.1-333.1	ESI－	8~160	48	96

Table 3

PST presence and absence of detected PST contamination in shellfish samples (n=188)

	Detected (n=26,13.83%)		Not detected (n=162,86.17%)		Ƿ^*
	n	%	n	%
*Species*					0.053
Chlamys nobilis	10	29.41	24	70.59
Crassostrea rivularis	5	21.74	18	78.26
Perna viridis	4	20.00	16	80.00
Paphia undulata	3	7.50	37	92.50
Atrina pectinate	1	8.33	11	91.67
Babylonia areolata	1	10.00	9	90.00
Scapharca broughtoni	1	33.33	2	66.67
Scapharca subcrenata	1	50.00	1	50.00
Meretrix meretrix	0	0	18	100
Sinonovacula constricta	0	0	11	100
Ruditapes philippinara	0	0	6	100
Patinopecten yessoensis	0	0	5	100
Solen gouldi	0	0	2	100
Mytilus edulis	0	0	2	100
Sources					0.074
Nan’ao	7	41.18	10	58.82
Huidong	6	19.35	25	80.65
Zhanjiang	6	19.35	25	80.65
Aotou	2	9.52	19	90.48
Guangxi	2	12.50	14	87.50
Fukien	1	5.88	16	94.12
Hainan	1	10.00	9	90.00
Zhuhai	1	8.33	11	91.67
Dianbai	0	0	6	100
Shantung	0	0	9	100
Yangjiang	0	0	18	100
Time of sampling					＜0.01
2019	March	5	33.33	10	66.67
	April	2	13.33	13	86.67
	May	4	28.57	10	71.43
	June	0	0	14	100
	July	0	0	17	100
	August	0	0	15	100
	September	0	0	16	100
	October	0	0	16	100
	November	1	0	16	94.12
	December	5	31.25	11	68.75
2020	January	5	31.25	11	68.75
2020	February	4	23.53	13	76.47
^* Fisher exact test p value comparing detected and not detected groups

Table 4

Concentration data set of PSTs of all samples and by shellfish species, sources, time of sampling

		Number of samples		Mean (μg/kg)		P₉₅(μg/kg)		Max (μg/kg)
		n	n%	LB	UB	LB	UB	LB	UB
	Total species	188	100	10.85	134.06	91.09	195.09	715.60	796.00
*Species*
	Paphia undulata	40	21.28	1.29	125.42	0.99	124.95	49.81	172.78
	Chlamys nobilis	34	18.09	50.84	170.90	710.26	790.66	715.60	796.00
	Crassostrea rivularis	23	12.23	5.88	129.09	54.42	168.42	60.79	174.79
	Perna viridis	20	10.64	1.64	125.41	12.37	134.23	13.68	134.64
	Meretrix meretrix	18	9.57	0.00	124.16	0.00	124.16	0.00	124.16
	Atrina pectinate	12	6.38	0.06	124.20	0.69	124.65	0.69	124.65
	Sinonovacula constricta. constricta	11	5.85	0.00	124.16	0.00	124.16	0.00	124.16
	Babylonia areolate	10	5.32	6.09	129.77	60.86	180.23	60.86	180.23
	Ruditapes philippinara	6	3.19	0.00	124.16	0.00	124.16	0.00	124.16
	Patinopecten yessoensis	5	2.66	0.00	124.16	0.00	124.16	0.00	124.16
	Scapharca broughtoni	3	1.60	0.27	124.36	0.80	124.74	0.80	124.76
	Solen gouldi	2	1.06	0.00	124.16	0.00	124.16	0.00	124.16
	Mytilus edulis	2	1.06	0.00	124.16	0.00	124.16	0.00	124.16
	Scapharca subcrenata	2	1.06	14.61	137.27	29.22	150.38	29.22	150.38
*Sources*
	Huidong	31	16.49	2.49	126.20	13.68	134.64	60.79	174.79
	Zhanjiang	31	16.49	6.24	129.19	54.42	168.42	78.02	181.58
	Aotou	21	11.17	0.24	124.33	0.55	124.51	4.54	127.30
	Yangjiang	18	9.57	0.00	124.16	0.00	124.16	0.00	124.16
	Fukien	17	9.04	0.03	124.18	0.52	124.48	0.52	124.48
	Nan’ao	17	9.04	97.12	214.29	715.60	796.00	715.60	796.00
	Guangxi	16	8.51	3.16	127.23	49.81	172.78	49.81	172.78
	Zhuhai	12	6.38	0.10	124.25	1.18	125.14	1.18	125.14
	Hainan	10	5.32	6.09	129.77	60.86	180.23	60.86	180.23
	Shantung	9	4.79	0.00	124.16	0.00	124.16	0.00	124.16
	Dianbai	6	3.19	0.00	124.16	0.00	124.16	0.00	124.16
*Time of sampling*
2019	March	15	7.98	49.45	170.46	710.26	790.66	710.26	790.66
	April	15	7.98	47.75	168.99	715.60	796.00	715.60	796.00
	May	14	7.45	0.66	124.60	4.54	127.30	4.54	127.30
	June	14	7.45	0.00	124.16	0.00	124.16	0.00	124.16
	July	17	9.04	0.00	124.16	0.00	124.16	0.00	124.16
	August	15	7.98	0.00	124.16	0.00	124.16	0.00	124.16
	September	16	8.51	0.00	124.16	0.00	124.16	0.00	124.16
	October	16	8.51	0.00	124.16	0.00	124.16	0.00	124.16
	November	17	9.04	0.07	124.22	1.18	125.14	1.18	125.14
	December	16	8.51	15.06	136.13	78.02	181.58	78.02	181.58
2020	January	16	8.51	14.08	136.49	137.41	242.98	137.41	242.98
	February	17	9.04	6.19	129.95	49.81	172.78	49.81	172.78

Table 5

Chronic PST exposure assessment using Shenzhen resident mean and P₉₉ shellfish consumption data

species	mean contamination data (μg STX eq./kg)		chronic exposure values (μg STX eq./kg bw)
	mean contamination data (μg STX eq./kg)		mean consumption data		P₉₉ consumption data
	LB	UB	LB	UB	LB	UB
Total species	10.85	134.06	0.0009	0.0107	0.0251	0.3100
Paphia undulata	1.29	125.42	0.0001	0.0100	0.0030	0.2900
Chlamys nobilis	50.84	170.90	0.0041	0.0137	0.1175	0.3952
Crassostrea rivularis	5.88	129.09	0.0005	0.0103	0.0136	0.2985
Perna viridis	1.644	125.41	0.0001	0.0100	0.0038	0.2900
Meretrix meretrix	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Atrina pectinate	0.06	124.20	0.0000	0.0099	0.0001	0.2872
Sinonovacula constricta	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Babylonia areolata	6.07	129.77	0.0005	0.0104	0.0141	0.3001
Ruditapes philippinara	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Patinopecten yessoensis	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Scapharca broughtoni	0.27	124.36	0.0000	0.0100	0.0006	0.2876
Solen gouldi	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Mytilus edulis	0.00	124.16	0.0000	0.0099	0.0000	0.2871
Scapharca subcrenata	14.61	137.27	0.0023	0.0120	0.0338 0.	0.3174

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Dietary Exposure Assessment of Paralytic Shellfish Toxins through Shellfish Consumption in Shenzhen Population, China

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Abstract

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1. Introduction

2. Material And Methods

2.1 Sampling

2.2 Reagents

2.3 Toxin extraction

2.4 Detection and quantification of PSTs

2.5 Dietary exposure assessment

2.5.1 Consumption data

2.5.2 PSTs contamination data

3. Results

3.1 Detection of PSTs in shellfish

3.2 Concentration of PSTs in shellfish

3.3 Acute Dietary Exposure Assessment

3.4 Chronic Exposure Assessment

4. Discussion

5. Conclusions

Abbreviations

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