toxins Article
Anatomical Distribution of Diarrhetic Shellfish Toxins (DSTs) in the Japanese Scallop Patinopecten yessoensis and Individual Variability in Scallops and Mytilus edulis Mussels: Statistical Considerations Ryoji Matsushima 1 , Hajime Uchida 1 , Ryuichi Watanabe 1 , Hiroshi Oikawa 1 , Izumi Oogida 2 , Yuki Kosaka 2 , Makoto Kanamori 3 , Tatsuro Akamine 1 and Toshiyuki Suzuki 1, * 1
2
3
*
National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Fukuura 2-12-4, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan;
[email protected] (R.M.);
[email protected] (H.U.);
[email protected] (R.W.);
[email protected] (H.O.);
[email protected] (T.A.) Aomori Prefectural Industrial Technology Research Center, Fisheries Research Institute, Hiranai, Higashitsugarugun, Aomori 039-3381, Japan;
[email protected] (I.O.);
[email protected] (Y.K.) Hokkaido Research Organization, Fisheries Research Department, Hakodate Fisheries Research Institute, Benten-cho 20-5, Hakodate, Hokkaido 040-0051, Japan;
[email protected] Correspondence:
[email protected]; Tel.: +81-45-788-7630
Received: 20 August 2018; Accepted: 21 September 2018; Published: 27 September 2018
Abstract: Diarrhetic shellfish toxins (DSTs) are a group of phycotoxins that include okadaic acid (OA)/dinophysistoxin (DTX) analogues. At present, detailed data on the distribution of DST is insufficient, and studies of the appropriate sample sizes are lacking. This study investigated the DST frequency distribution in scallops and mussels by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and a resampling analysis of existing data was carried out. The DST population-interval and the necessary sample size were also estimated. DSTs are localized in the scallop digestive-gland, and the DST concentrations in scallops were water-depth-dependent. DST concentrations in scallops and mussels showed normal distributions, but mussels tended to contain more DSTs than scallops. In the statistical resampling analysis of the acquired data on scallops and mussels, especially that using the bootstrap method, sample size was difficult to estimate when the DST variation was large. Although the DST population-interval could be statistically estimated from the sample standard deviation of three samples, the sample size corresponded to the risk management level, and the use of 13 or more samples was preferable. The statistical methods used here to analyze individual contents and estimate population content-intervals could be applied in various situations and for shellfish toxins other than DSTs. Keywords: diarrhetic shellfish toxins; accumulation; dinophysistoxin; Japanese scallop; dinophysis; LC/MS/MS; statistical analysis Key Contribution: This is the first detailed analysis of the distribution of individual concentrations of DSTs in shellfish samples, as well as the first report of a method for analyzing and evaluating the relationship between the individual concentrations and mean population concentrations based on statistical methods.
Toxins 2018, 10, 395; doi:10.3390/toxins10100395
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1. Introduction Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by the consumption of shellfish contaminated with diarrhetic shellfish toxins (DSTs) [1]. DSTs are a group of phycotoxins that include okadaic acid (OA) and dinophysistoxin (DTX) analogues [2,3]. OA, dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are the most important DSTs because they cause severe diarrhea. These toxins have been shown to be potent protein-phosphatase inhibitors [4], a property that can cause inflammation of the intestinal tract and diarrhea [5], possibly leading to tumor promotion [6]. Okadaic acid analogues are metabolized to the esterified toxin in many bivalve species including Japanese scallops [7], and they are collectively called dinophysistoxin-3 (DTX3). In Japan, screening and quantification of DSTs are carried out on bivalves in accordance with the guidelines based on the official instrumental method [8] in production areas and markets. However, the Japanese guidelines do not provide detailed information on the distribution of DSTs between individual bivalves and have not established an appropriate sample size due to the lack of such data obtained by accurate analytical methods [9]. The Japanese scallop Patinopecten yessoensis (Mizuhopecten yessoensis), a major and important cultured species in Japan [10], has unique characteristics, including the metabolic transformation of lipophilic toxins [11]. In the present study, we analyzed the concentrations of DSTs in individuals of P. yessoensis and the mussel Mytilus edulis, and the validity of the size of sample were examined with statistical resampling analysis of the acquired DST data. Although some of our research has been presented in a previous work [12], more detailed data and novel results are provided in our present study. Furthermore, assuming conditions for investigating cultured scallops in the sea [10], estimation of the DST population-interval (interval of concentration of DST contained in population) were performed. Finally, based on our results, we consider and propose an adequate sample size. 2. Results 2.1. Concentrations and Distribution of DSTs 2.1.1. Anatomical Compartmentalization of DST in Scallops The compartmentalization of DSTs in scallops collected at Nonai Station, Mutsu Bay, Aomori prefecture was investigated. From 14 to 20 individual scallops, the digestive gland, gonad, mantle, gill, and adductor muscle were separately dissected. The pieces were then grouped together by the body part (Table 1). Table 1. Sampling information and total weight (g) of each scallop tissue. 2014
26 May
2 June
9 June
30 June
14 July
22 July
28 July
Number of Individuals
16
18
17
18
15
20
14
Digestive gland Gonad Mantle Gill Adductor muscle
72.90 42.39 155.01 95.31 301.69
72.87 39.99 151.37 93.43 315.19
72.97 40.79 163.48 106.28 311.42
70.80 44.22 175.80 92.20 355.14
60.80 48.05 163.66 87.08 318.80
75.16 54.46 213.96 116.42 434.35
58.56 39.83 169.30 85.49 340.26
The concentration of OA and DTX1 in each part was quantified by LC/MS/MS after hydrolysis.
The dominant toxin in the scallops was DTX1, the highest concentration of which was found on 30 June, corresponding to about half of the regulation value of 0.16 mg/kg of whole meat (Figure 1).
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Figure 1. Concentrations of DTX1 and OA in the digestive glands of scallops. Fourteen to twenty
Figure 1. Concentrations of DTX1 and OA in the digestive glands of scallops. Fourteen to twenty individuals were combined each sample setthe used for analysis. Black bars and white barstorepresent Figure 1. Concentrations of into DTX1 and OA in digestive glands of scallops. Fourteen twenty individuals were combined into each sample set used for analysis. Black bars and white bars represent DTX1 and OA, The concentrations of toxins in the Black samples vertical individuals wererespectively. combined into each sample set used for analysis. barsare andshown white on barsthe represent DTX1axis. and OA, respectively. The concentrations of toxins in the samples are shown on the vertical axis. DTX1 and OA, respectively. The concentrations of toxins in the samples are shown on the vertical axis.
The proportion of the DTX1 quantity toeach eachtissue tissue shown in Figure The proportion of the DTX1 quantitycorresponding corresponding to areare shown in Figure 2. 2. The proportion of the DTX1 quantity corresponding to each tissue are shown in Figure 2.
Figure Percentage (%) (%) of of DTX1 tissue. Figure 2. 2.Percentage DTX1inineach eachscallop scallop tissue. Figure 2. Percentage (%) of DTX1 in each scallop tissue.
Analysis of 30 IndividualScallops Scallops and and Mussels 2.1.2.2.1.2. DSTDST Analysis of 30 Individual Mussels
2.1.2. The DSTconcentrations Analysis of 30 Individual Scallops and Mussels DTX1ininthe thedigestive digestive glands or or mussels collected at theat the The concentrations of of DTX1 glandsofof3030scallops scallops mussels collected Nonai Station were quantified for each individual (Figure 3 and Table 2). The concentrations of DTX1 in the digestive glands of 30 scallops or mussels collected at the Nonai Station were quantified for each individual (Figure 3 and Table 2). The concentration of OA for waseach not individual described due to the overall low concentrations found in the Nonai Station were quantified (Figure 3 and Table 2). The concentration of OA was not described due to the overall low concentrations found in the individual samples. The mean values DTX1 differed for thelow same date for both found mussels Thesamples. concentration OA was not of described to even theeven overall concentrations in and the individual The of mean values of DTX1 due differed for the same date for both mussels scallops, and the DTX1 values of the mussels were higher than those of the scallops (Figure 3). The individual samples. The mean values of DTX1 differed even for the same date for both mussels and and scallops, and the DTX1 values of the mussels were higher than those of the scallops (Figure 3). distributions of the scallops and werewere closehigher to the normal distributions. scallops, and the DTX1 values ofmussels the mussels than those of the scallops (Figure 3). The
The distributions of the scallops and mussels were close to the normal distributions. distributions of the scallops and mussels were close to the normal distributions.
Table 2. Sampling information and mean weight (g) of the digestive glands of 30 scallops or mussels.
Table 2. Sampling information and mean (g)ofofthe thedigestive digestive glands 30 scallops or mussels. The mean values of 30 samples ± population standard deviation (σ).glands Table 2. Sampling information and mean weight weight (g) of 30ofscallops or mussels. The mean values of 30 samples standarddeviation deviation (σ). The mean values of 30 samples±26 ±population population standard 2014 May 2 June 9 June (σ). 16 June 7 July 8 August Scallop 2014 2014 (Digestive gland/Whole meat %) Scallop Scallop Mussel (Digestive gland/Wholemeat meat%) %) (Digestive gland/Whole (Digestive gland/Whole meat %) Mussel Mussel (Digestive gland/Whole (Digestive gland/Wholemeat meat%) %)
3.87 ± 1.07 26 26May May (10.14%) 3.87±± 1.07 1.07 3.87 (10.14%) (10.14%) --
3.62 ± 0.81 22June June (10.56%) 3.62±± 0.81 0.81 3.62 1.69 ± 0.45 (10.56%) (10.56%) (14.72%) 1.69 1.69±± 0.45 0.45 (14.72%) (14.72%)
3.70 ± 0.98 99 June June (8.78%) 3.70±± 0.98 0.98 3.70 (8.78%) (8.78%)
3.81 ± 0.72 16 June June 16 (9.18%) 3.81 ± 0.72 3.81 ± 0.72 (9.18%) (9.18%)
--
--
July 7 7July -
8 August 8 August -
1.40 -±- 0.46 (14.89%) 1.40±±0.46 0.46 1.40 (14.89%) (14.89%)
1.51 -±-0.45 (12.90%) 1.51± ± 0.45 1.51 0.45 (12.90%) (12.90%)
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Figure 3. The 30 individual distributions, DTX1inin scallops mussels. Panels Figure 3. The 30 individual distributions,means means ± ± σσofofDTX1 scallops andand mussels. Panels (a–d) (a–d) showshow the results for for scallops and (e–g) mussels. the results scallops and (e–g)show showthose those for mussels. Figure 3. The 30 individual distributions, means ± σ of DTX1 in scallops and mussels. Panels (a–d)
show the results for scallops and (e–g) show those for mussels. 2.1.3.2.1.3. Analysis of DST Concentration ininScallop Samples fromDifferent Different Water Depths Analysis of DST Concentration Scallop Samples from Water Depths
The DST concentrations of scallop digestive glands collected at different at Yakumo Station, The DST concentrations of scallop digestive glands collected at different depths at Yakumo 2.1.3. Analysis of DST Concentration in Scallop Samples from Different Waterdepths Depths the of western of Funka Bay, Hokkaido prefecture, wereSampling investigated. Samplingabout in theStation, westerninpart Funka part Bay, Hokkaido prefecture, were investigated. information The DST concentrations of scallop digestive glands collected at different depths at Yakumo information about in theTable scallops the scallops is shown 3. is shown in Table 3. Station, in the western part of Funka Bay, Hokkaido prefecture, were investigated. Sampling information about theinformation scallops isand shown inweight Table (g) 3. of scallop digestive glands. The mean value of Table 3. Sampling mean Table 3. Sampling information and mean weight (g) of scallop digestive glands. The mean value of digestive glands ± σ at each depth. digestive glands ± σ atinformation each depth.and mean weight (g) of scallop digestive glands. The mean value of Table 3. Sampling 2016depth. 18 May 28 June 11 August digestive glands ± σ at each 2016 18 May 28 15 June Number of Individuals 10 15 11 August 2016 18 May 28 June 11 August Number of Individuals5 m 10 6.45 ± 1.24 4.7715 ± 0.82 1.40 ± 0.36 15 Number of Individuals 10 15 15 10 m 6.16 ± 0.91 4.77 5.18 ± ± 1.33 5m 6.45 ± 1.24 0.82 1.38 ± 0.26 1.40 ± 0.36 5m 6.45 ± 1.24 4.77 ± 0.82 1.40 ± 0.36 15 m 5.30 ± 0.45 5.18 4.22 ± ± 0.75 10 m 6.16 ± 0.91 1.33 1.46 ± 0.42 1.38 ± 0.26 10 m 6.16 ± 0.91 5.18 ± 1.33 1.38 ± 0.26 15 m 5.30 ± 0.45 4.22 ± 0.75 1.46 ± 0.42 The vertical gradient of15 DTX1 (maximum 5 m, minimum 15 m) was reversed m distribution5.30 ± 0.45 at 4.22 ± 0.75 1.46 at ± 0.42 over the investigation period (Figure 4). Maximum levels of about half of the regulation value The vertical gradient of DTX1 distribution (maximum at 5 m, minimum at 15 m) was reversed (corresponding to 0.16 mg/kg of whole meat) were found at 15 August. at 15 m) was reversed The vertical gradient of DTX1 distribution (maximum atm5 on m,11 minimum over the investigation period (Figure 4). Maximum levels of about half of the regulation value over the investigation period (Figure 4). Maximum levels of about half of the regulation value (corresponding to 0.16 mg/kg of whole meat) were found at 15 m on 11 August.
(corresponding to 0.16 mg/kg of whole meat) were found at 15 m on 11 August.
Figure 4. Vertical distribution of DTX1 and σ in scallop digestive glands.
Figure 4.of Vertical distribution ofof DTX1 σ in in scallop scallop digestive glands. Figure 4.scallops Vertical distribution DTX1 and digestive glands. The distributions on 28 June and 11and August were close to the normal distribution (Figure 5). The distributions of scallops Juneand and11 11 August August were normal distribution The distributions of scallops onon 2828June wereclose closetotothethe normal distribution (Figure 5). (Figure 5).
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Figure 5. The5.distributions of scallop at eachatdepth 28 June 11 August. mean The concentrations Figure The distributions of scallop each on depth on and 28 June and 11The August. mean concentrations of DTX1 σ are shown (a–d) 28 June and (e–h) 11 August. of DTX1 ± σ are shown for±(a–d) 28 Junefor and (e–h) 11 August.
Environmental conditionsand and vertical vertical distribution distribution ofofDinophysis at at Yakumo Station are are Environmental conditions Dinophysis Yakumo Station represented in Table 4 and Figure respectively. represented in Table 4 and Figure 6, 6, respectively. 4. Data on environmental conditions and densities of DST producing species at Yakumo TableTable 4. Data on environmental conditions and densities of DST producing species at Yakumo Station. Station.
Transparency Date Depth Water Transparency Depth Water (m) (2016) Temperature (◦ C) Date (2016) (m) (m)
18 May 18 May
0 5 10 15 20 25 30
28 June 28 June
0 5
5.0 5.0
4.0 4.0
10 15 20 11 August 25 30
10.5
10.5
11 August
0 5 10 15 20 25 30
(m) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30
Temperature (°C) 11.2 11.2 9.79.7 9.29.2 8.18.1 7.87.8 7.47.4 7.37.3 16.6 16.6 14.8 14.8 13.7 13.7 13.4 13.0 13.4 12.8 13.0 12.3 12.8 22.6 12.3 20.7 16.9 22.6 20.7 12.7 16.9 10.8 8.6 12.7 7.9
10.8 8.6 7.9
Salinity Salinity (psu) (psu)
30.84 30.84 31.98 31.98 32.03 32.03 32.32 32.32 32.59 32.59 32.67 32.67 32.70 32.70 29.58 29.58 31.15 31.15 31.94 31.94
32.09
32.18 32.09 32.22
32.18 32.33 32.22 31.14 32.33 31.67 32.37 31.14 31.67 32.64 32.37 32.95 32.95 33.02
32.64 32.95 32.95 33.02
D. fortii D. fortii (Cells/L) (Cells/L) 00 00 00 00 00 00 00 0 30 180 180 30 40 30 0 40 0 00 00 0 0 0 10 50 0 90
10 50 90
D. acuminate
D. acuminate (Cells/L) (Cells/L) 30 30 60 60 100 100 80 80 30 30 90 90 150 150 1120 1120 650 650 740 740 490 300 490 70 300 50 070 50 0 100 00 010 0 0 20
Other Dinophysis
Other Dinophysis (Cells/L) (Cells/L) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 60 60 Dt60 Dt60 Dn150, 190 190Dn150, Dt40 Dt40 80 Dn60, Dr20 10 80 Dr10 Dn60, 10 Dn10 Dr20 10 10 Dn10 Dr10 Dn10 0 10 10 Dn10 50 Dt50 30 0 Dt30 Dt50 30 50 Dt20, Dr10 Dt30 0 30 10 Dt10 Dt20, 30 Dr10 0
0 0 20
Dinophysis tripos (Dt); Dinophysis norvegica (Dn); Dinophysis rotundata (Dr). Dinophysis tripos (Dt); Dinophysis norvegica (Dn); Dinophysis rotundata (Dr).
0 10 0
Dt10
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FigureFigure 6. Vertical distributions of Dinophysis species. (a) D. (b) D. andand other Dinophysis species. 6. Vertical distributions of Dinophysis species. (a)acuminata; D. acuminata; (b)fortii D. fortii other Dinophysis species.
D. acuminata was assumed to be main causative agent of the DST events. This species reached its maximal 28 assumed June andtodeclined on 11 August 4). Other D. density acuminataon was be main causative agent (Table of the DST events.Dinophysis This speciesspecies reachedwere its maximal density on the 28 June and declined 11 August 4). Otherbetween Dinophysis were of predominant from 10 m to surface, and thereonwas no clear(Table relationship thespecies distribution predominant from 10 m to the surface, and there was no clear relationship between the distribution cells and reversal of the vertical distribution of DTX1 in scallops on 11 August (Figures 4 and 6). of cells and reversal of the vertical distribution of DTX1 in scallops on 11 August (Figures 4 and 6).
2.2. Statistical Analysis 2.2. Statistical Analysis
2.2.1. Statistical Resampling Analysis of DSTs in Scallops and Mussels 2.2.1. Statistical Resampling Analysis of DSTs in Scallops and Mussels
The number of individuals necessary to correctly reflect the DST contents of collected samples The number of individuals necessary to correctly reflect the DST contents of collected samples was estimated by resampling analysis. For this purpose, we used a data set collected for 30 individual was estimated by resampling analysis. For this purpose, we used a data set collected for 30 individual scallops and 30 individual mussels at Aomori on 2 June (Figure 3). Both values were highly variable. scallops and 30 individual mussels at Aomori on 2 June (Figure 3). Both values were highly variable. The means of 5–25 samples were calculated samplingand andallowing allowing or not allowing The means of 5–25 samples were calculatedwith withrandom random sampling or not allowing (bootstrap method) replacement. The sample convertedtotopercentages percentages as compared (bootstrap method) replacement. The samplemeans meanswere were converted as compared withwith those those of 30 of samples (Table 5). 5). 30 samples (Table 5. Resampling analysis of scallopsand andmussels mussels without without replacement and with the the bootstrap Table Table 5. Resampling analysis of scallops replacement and with bootstrap method. The left half of the table is a resampling analysis without replacement, while the right half half method. The left half of the table is a resampling analysis without replacement, while the right shows the data using the bootstrap method. The n columns represent 5–25 samples. 1 to 99 represent shows the data using the bootstrap method. The n columns represent 5–25 samples. 1 to 99 represent percentiles. Percentage: each percentile columns represents the mean value of a data set for each mean percentiles. Percentage: each percentile columns represents the mean value of a data set for each mean of the 30 individuals. >±30;
