Abstract
Diarrhetic shellfish toxins (DST) are a group of phycotoxins that include Okadaic acid
(OA) and structurally related toxins. In Japan, the regulatory limit of DST in shellfish
for human consumption is a total OA equivalent of 0.16 mg per kg of edible tissue.
Distribution and individual differences of DST in scallops collected in Aomori Prefecture
were investigated. Fourteen to 20 individual scallops were divided into hepatopancreas,
gonads, mantles, gills, adductor muscles, and the concentrations of diarrhetic shellfish
poisoning (DSP) in each tissue were quantified by LC/MS/MS after hydrolysis. The dominant
toxin in the scallops was Dinophysis toxin 1 (DTX1). More than 97% of the observed DTX1 in
the scallop tissue was detected in the hepatopancreas and the average level of DTX1 was
higher in mussels than the scallops. The number of individual scallops or mussels required
to correctly reflect the DTX1 content of a sample group was estimated by resampling. In
scallops, using 10 individuals fell within ± 20% of 30 individual’s average with a
probability of 99.8%. On the other hand, in the blue mussel, an average of 19 individuals
fell within ± 20% of 30 individual’s average with 98% probability. In addition, the
analysis of the DST in ascidians collected from Miyagi Prefecture was carried out. The
muscles, gills, hepatopancreas and intestines were analyzed. High concentration of both
DTX1 and OA were detected in the hepatopancreas after hydrolysis. Low levels of DST were
detected from other tissues, indicating that DST are primarily accumulated in the
hepatopancreas in the ascidians.
1. Introduction
Diarrhetic shellfish toxins (DST) are a group of phycotoxins that include Okadaic acid (OA)
and structurally related toxins. In Japan, the regulatory limit of DST in shellfish for
human consumption is a total OA equivalent of 0.16 mg per kg of edible tissue as described
in CODEX Standards1). Testing
for DST using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been officially
authorized from 2015 in Japan2).
It is expected that the risk management of diarrhetic shellfish poisoning (DSP) using
LC/MS/MS will benefit from the significantly improved sensitivity and the accuracy of
LC/MS/MS compared to the conventional mouse bioassay. In addition to Japanese scallop
Patinopecten yessoensis, which is important species for fishery industry,
the distribution of DST (OA and DTX1) in the mussel Mytilus edulis was
analyzed. Because little information is available, DST accumulation in the edible ascidian
Halocynthis roretzi was also investigated.
2. Materials and Methods
2-1. Bivalves and Ascidian
Scallops and mussels were collected from Nonai station of Mutsu Bay, Aomori Prefecture,
Japan in 2014. Aomori Prefecture is located at the northern end of Honshu Island.
Specimens of the ascidian were harvested from Iwaizaki station of Kesennuma Bay, Miyagi
Prefecture, Japan in 2016.
2-2. Extraction of DSTs from Samples and Hydrolysis of Esterified DSTs
Each dissected tissue was homogenized with 9 volumes of methanol-distilled water (9:1,
v/v), and the homogenates were centrifuged at 3000 rpm for 5 min3). Alkaline
hydrolysis of OA group was carried out according to the EU harmonized standard operating
procedure for lipophilic marine biotoxins in Molluscs by LC-MS/MS. Ver. 54). For the hydrolysis, 125 μL of
2.5M NaOH solution was added to a 1 mL aliquot of a methanolic extract of each sample. The
mixture was kept at 80°C for 30 min and neutralized with 125 μL of 2.5M HCl. The
hydrolyzed samples were analyzed by LC/MS/MS without further purification.
2-3. LC/MS/MS Analysis of DSTs
Authentic standards of OA, dinophysistoxin-1 (DTX1) were prepared according to previous
methods5). Toxins were
dissolved in HPLC grade methanol to prepare the calibration standards. OA, DTX1, in sample
extracts were analyzed and quantified by LC/MS/MS as reported in our previous
methods3,6). Triplicate analysis was carried
out for each sample extract. Multiple reaction monitoring (MRM) LC/MS/MS analysis for
toxins was carried out using [M-H]- as the target parent ions in Q1 and
particular fragment ions of each toxin in Q3, with a dwell time of 100 msec for each
analog as follows. OA: m/z 803.5>255.3; DTX1: m/z
817.5>255.3. LOD of OA and DTX1 < 0.01 mg/kg.
2-4. Statistical Analyses
Statistical analysis program R with the boot
(https://cran.r-project.org/web/packages/boot/index.html) and MASS
(https://cran.r-project.org/web/packages/MASS/index.html) package was employed for
resampling analysis. The values repeatedly calculated 10,000 times with 5-25 random
sampling without overlap from 30 scallops or mussels were compared with the average value
of 30 scallops or mussels. The program is a kind of bootstrap estimation that has been
modified not to allow overlapping. This provides an estimate of the shape of the
distribution of the mean, from which solutions are presented about how much the mean
varies.
3. Results
Distribution and individual differences of DST in scallops collected from Nonai station,
Mutsu Bay Aomori were investigated. The 14-20 scallops were dissected to separate the
hepatopancreas, gonads, mantles, gills, and adductor muscles, and combined for each part.
The concentrations of OA group in each tissue were quantified by LC/MS/MS after hydrolysis
(Table 1).
Table 1.
Concentration of DTX1 and OA in hepatopancreas of scallops
| Date (2014) |
May 26 |
Jun 2 |
Jun 9 |
Jun 30 |
Jul 14 |
Jul 22 |
Jul 28 |
| Individuals |
16 |
18 |
17 |
18 |
15 |
20 |
14 |
| DTX1 (ng/g) |
472 |
556 |
381 |
831 |
207 |
147 |
121 |
| OA (ng/g) |
17 |
14 |
13 |
28 |
20 |
7 |
6 |
Fourteen to 20 individuals were combined into each sample set used for analysis. The
highest DTX1 was recorded at Jun 30.
The dominant toxin in the scallops was DTX1, the highest concentration was found on June 30
and was equivalent to about half of the regulation value. By multiplying the measured
concentration by the tissue weight, the ratio of the DTX1 quantity included in each tissue
was calculated. (Table 2). More than 97% in
total amounts of DTX1 were harbored in the hepatopancreas. Regardless of DSTs amount, DSTs
were hardly detected in the adductor muscles. The concentration of DTX1 included in the
hepatopancreas of 30 individuals (scallop and mussel) collected at the Mutsu Bay was
quantified for each individual (Table 3). The
concentration of OA was not described due to the overall low amounts found in the individual
samples. The average value of DTX 1 differs even for the same date in mussel and scallop,
and the DTX1 value of mussel was higher (Fig. 1,
Table 3 Jun 2).
Table 2.
Content ratio (%) of DTX1 in each scallop tissue
|
May 26 |
Jun 2 |
Jun 9 |
Jun 30 |
Jul 14 |
Jul 22 |
Jul 28 |
| Hepatopancreas |
97.1 |
98.5 |
97.5 |
97.8 |
98.2 |
97.8 |
98.6 |
| Gonad |
0.8 |
0.5 |
0.8 |
1.0 |
1.2 |
1.5 |
1.4 |
| Mantle |
1.0 |
0.3 |
0.8 |
0.6 |
0.0 |
0.0 |
0.0 |
| Gill |
1.2 |
0.7 |
0.9 |
0.6 |
0.6 |
0.7 |
0.0 |
| Adductor muscle |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
In all cases, hepatopancreas contained almost of all DTX1 whether high or low.
Table 3.
DTX1 concentration (ng/g) in hepatopancreas of 30 individual samples (scallop and
mussel)
| Date (2014) |
May 26 |
Jun 2 |
Jun 9 |
Jun 16 |
Jul 7 |
Aug 8 |
| Scallop |
489 ± 127
(26%) |
628 ± 168
(27%) |
521 ± 110
(21%) |
321 ± 82
(25%) |
- |
- |
| Mussel |
- |
735 ± 450
(61%) |
- |
- |
805 ± 307
(38%) |
227 ± 164
(72%) |
The average value of 30 samples, standard deviation (SD) and relation ratio of SD
(%)
By statistical resampling, the number of individuals that are necessary to correctly
reflect the DST content of collected samples were estimated. Using the measured data set of
30 individuals at June 2nd for scallops or blue mussels, both were high and highly variable,
the average value of 5 to 25 samples were calculated repeatedly by 10,000 times with random
sampling without overlap (Fig. 1, Table 4). In the scallop, using 10 individuals fell
within ± 20% of average value of 30 individuals with a probability of 99.8% (Table 4 left underlined number). On the other hand,
in the blue mussel, average of 19 individuals or 15 individuals fell within ± 20% of average
value of 30 individuals with 98% or 90% probability, respectively (Table 4 right underlined number).
Table 4.
The resampling analysis of DTX1 concentration in hepatopancreas of scallop and
mussel
The percentage; each 0.1 to 99.9th percentile value takes on the average value of 30
individual data set.
The N vertical columns represent extracted sample numbers 5 to 25 samples. The
horizontal 0.1 to 99.9 represent each percentile. Gray back bold character and gray back
white character corresponding ± 20 and ± 10% respectively.
Analysis of the DST of ascidians collected from Miyagi Prefecture was carried out when the
toxification of blue mussel was confirmed at the same monitoring station (Table 5). The muscles, gills, hepatopancreas and
intestines of ascidians were analyzed (Fig. 2,
Table 6).
Table 5.
DTX1 concentration of mussel from Nonai station
| Date (2016) |
mg/kg |
| June 7 |
0.61 |
| June 14 |
0.18 |
The DTX1 concentration in edible part of mussel.
Table 6.
The tissues and weight (g) of ascidian samples at June 13
| Sample |
Muscle |
Gill |
Hepatopancreas |
Intestin |
| 1 |
56.8 |
4.4 |
2.8 |
5.0 |
| 2 |
49.5 |
2.7 |
3.0 |
2.5 |
| 3 |
56.2 |
2.9 |
2.5 |
2.5 |
| 4 |
34.8 |
1.5 |
1.8 |
1.5 |
| 5 |
53.6 |
2.4 |
3.3 |
2.7 |
| 6 |
45.7 |
1.4 |
1.9 |
2.3 |
| 7 |
42.2 |
1.5 |
2.8 |
2.0 |
| 8 |
36.2 |
2.0 |
1.6 |
1.8 |
| 9 |
27.8 |
1.9 |
1.7 |
1.9 |
| 10 |
50.3 |
2.4 |
2.6 |
1.5 |
Numbers in the table represent weights of tissues.
High concentration of DTX1 and OA were detected in the hepatopancreas after hydrolysis
(Fig. 2D). Low levels of DST were detected from
other tissues (Fig. 2A–C).
4. Discussion
Since there was no report that DTX2 has been detected in Japan, we analyzed OA and DTX1 in
this study. In the case of scallops and mussels, it is impractical to sample 30 individuals.
The results of the calculation simulated how much reasonable sampling size reflects the mean
value of impractical 30 samples. Approximately 10 samples were considered to be adequate for
the scallops, as the variation in DTX1 level was not large. It is assumed that a greater
number of samples are necessary for the mussel, due to the larger variation in the DTX1
level. Ultimately, the required sample number will vary depending on the degree to which
desired risk management level.
The concentration of DSTs in ascidians increased after hydrolysis. Thus, DSTs are
accumulated in ascidians as the esterified form
7-O-acyl-OA/DTX1 (DTX3)7,8), like in many bivalves9). Most of the DST are accumulated in the hepatopancreas of
the ascidians. This portion accounts for just 5% of the edible parts and thus the risk of
diarrhetic shellfish poisoning is not considered to be very high. However, there is a
personal preference for consumption of hepatopancreas in Japan. To consume internal organs
that have been highly contaminated will increase the risk of poisoning. Although these
preliminary studies provide useful information for improving DST risk management, more
detailed investigations of both contamination and consumption patterns will be required.
Acknowledgments
The series of studies was conducted and supported by Ministry of Agriculture, Forestry and
Fisheries.
Conflict of interest
The authors have no conflict of interest.
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