Method for evaluating total mercury concentration

Abstract

Provided is a method for evaluating the total mercury concentration included in an edible part of a fish or cetacean, the method comprising measuring the total mercury concentration of a sample which is the flesh of a blood-releasing part or a ventricle of the fish or cetacean by a plasma spectroscopic analysis method.

Description

Evaluation method for total mercury concentration

[0001] The present disclosure relates to methods for assessing total mercury concentration.

[0002] Mercury, which is naturally present in fish and cetaceans, especially large fish such as tuna, accumulates in their bodies through the food chain. Mercury is a toxic heavy metal, and organic mercury, such as methylmercury, in particular, can damage the central nervous system. Therefore, Japan has set provisional regulatory limits for mercury in seafood at 0.4 ppm for total mercury and 0.3 ppm (mercury equivalent) for methylmercury. In Hong Kong, one of the export destinations for tuna, the Food Safety Center of the Food and Environmental Hygiene Bureau tests mercury in fillet samples of imported products, and if the limit (methylmercury concentration 0.5 mg / kg) is exceeded, a notice is issued and sales are suspended.

[0003] Because measuring mercury concentrations by cutting out portions of edible parts of edible fish or whales leads to a decrease in commercial value, methods for assessing mercury concentrations in edible parts without using edible parts have been investigated. For example, Japanese Patent Application Laid-Open No. 2007-225582 describes a method for assessing the amount of mercury contained in fish, in which the mercury concentration in the edible parts of the fish is assessed by analyzing the mercury concentration in blood collected during a fish bleeding operation or in the muscle of a part of the fish (particularly the tail) that will be discarded, using a known method. Specifically, a method for measuring mercury levels in tuna is disclosed in which the mercury concentration in the blood or tuna tail meat is measured using the official method of reduction vapor-cold atomic absorption spectrometry (CV-AAS), and the mercury concentration in the edible parts is assessed from the measured value.

[0004] Meanwhile, plasma spectroscopy is known as a method for measuring the concentration of metals such as mercury in a sample. For example, Japanese Patent Application Laid-Open Nos. 2016-130734, 2019-15560, and 2019-15562 describe a method in which an analyte is concentrated near at least one of a pair of electrodes, a voltage is applied to the pair of electrodes to generate plasma, and the analyte in the sample is analyzed based on the amount of light emitted.

[0005] Since the total mercury concentration varies depending on the part of the edible part of fish or whales, the method described in JP 2007-225582 A may not reflect the total mercury concentration in the blood or tail muscle of fish in some cases. As a result, the mercury concentration in some edible parts may exceed the value estimated from the total mercury concentration measured in the blood or tail muscle.

[0006] In view of these circumstances, the present disclosure relates to a method for evaluating the total mercury concentration contained in the edible parts of fish or cetaceans, which can evaluate the total mercury concentration in the entire edible parts using parts other than the edible parts of fish or cetaceans.

[0007] Means for solving the above problems include the following aspects. <1> A method for evaluating the total mercury concentration in an edible portion of a fish or cetacean, comprising measuring the total mercury concentration in a sample that is meat from the ventricle or bleeding site of a fish or cetacean by plasma spectrometry. <2> The evaluation method according to <1>, in which the evaluation of the total mercury concentration comprises estimating the maximum total mercury concentration in the edible portion of the fish or cetacean. <3> The evaluation method according to <2>, in which the maximum total mercury concentration in the edible portion is estimating to be 1.00 to 1.25 times the measured total mercury concentration of the sample. <4> The evaluation method according to <2> or <3>, in which the maximum total mercury concentration in the edible portion is estimating to be 1.00 to 1.10 times the measured total mercury concentration of the sample. <5> The evaluation method according to any one of <1> to <4>, in which the fish or cetacean is a tuna. <6> The evaluation method according to <5>, in which the tuna is bluefin tuna. <7> The evaluation method according to any one of <1> to <6>, further comprising treating the meat of the ventricle or the bleeding site of the fish or cetacean with an alkaline solution to prepare a sample solution for plasma spectroscopic analysis. <8> The evaluation method according to <7>, wherein the alkaline solution contains a lithium hydroxide solution. <9> The evaluation method according to any one of <1> to <8>, wherein the plasma spectroscopic analysis method comprises: a concentration step of concentrating mercury in the sample in the vicinity of at least one of the electrodes by applying a voltage to the pair of electrodes; and a detection step of generating plasma by applying a voltage to the pair of electrodes and detecting the light emission of the mercury generated by the plasma.

[0008] According to the present disclosure, a method for evaluating the total mercury concentration contained in the edible parts of fish or cetaceans is provided, which can evaluate the total mercury concentration in the entire edible parts using parts of fish or cetaceans other than the edible parts.

[0009] 1 is a schematic perspective view of an example of a plasma spectrometer; 2 is a schematic cross-sectional view of the plasma spectrometer as viewed from the II direction in FIG. 1; 3 is a schematic diagram showing the ventricle, atrium, edible portion, tail, and bled portion of meat collected in the examples; 4 is a graph plotting the relative total mercury concentrations of the edible portion (portions A to D) and tail (portion E) of three farmed bluefin tuna, with the total mercury concentration of the ventricle set to 1; 5 is a graph showing the relationship between the total mercury concentration of the ventricle and the total mercury concentration of the edible portion (a mixture of portions A to D) for each individual of three farmed bluefin tuna and two young wild bluefin tuna; 6 is a graph showing the results of comparing the total mercury concentrations of the ventricle, atrium, and bled portion of meat; and 7 is a graph showing the correlation between the total mercury concentrations of the ventricle and atrium, and the total mercury concentration of the ventricle and bled portion of meat. 1 is a graph showing total mercury concentrations in the ventricle and lean meat measured by plasma spectrometry and reduction vapor-cold atomic absorption spectrometry (CV-AAS).

[0010] Hereinafter, embodiments of the present disclosure will be described in detail. However, the embodiments of the present disclosure are not limited to the following embodiments. In the following embodiments, components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the embodiments of the present disclosure.

[0011] In the present disclosure, the term "process" includes not only processes that are independent of other processes, but also processes that cannot be clearly distinguished from other processes as long as the purpose of the process is achieved. In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another staged numerical range. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, each component may contain multiple corresponding substances. When multiple substances corresponding to each component are present in a composition, the content or amount of each component means the total content or amount of the multiple substances present in the composition, unless otherwise specified. In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment. In the present disclosure, even when an element is described in the singular, this does not exclude the presence of multiple substances unless technical contradiction arises, unless otherwise specified.

[0012] <Method for Evaluating Total Mercury Concentration> In one embodiment of the present disclosure, a method for evaluating the total mercury concentration in the edible portion of fish or cetaceans (hereinafter also referred to as the "evaluation method of the present disclosure") is provided, which includes measuring the total mercury concentration in a sample of meat from the ventricle or exsanguination portion of a fish or cetacean by plasma spectrometry. According to the evaluation method of the present disclosure, meat from the ventricle or exsanguination portion of a fish or cetacean is used, eliminating the need to collect edible portions for mercury concentration measurement. Furthermore, mercury concentrations vary depending on the part of a fish or cetacean. Therefore, when measuring the mercury concentration in the edible portion based on the mercury concentration in a part other than the edible portion, the mercury concentration in the edible portion may be underestimated depending on the part measured. On the other hand, according to the evaluation method of the present disclosure, by measuring the mercury concentration in the meat from the ventricle or exsanguination portion, the total mercury concentration in the entire edible portion can be evaluated without underestimating the total mercury concentration in the edible portion. Therefore, the evaluation method of the present disclosure enables evaluation of the total mercury concentration in a manner that is safer for consumers.

[0013] According to data on beaked whales published by the Ministry of Health, Labor and Welfare in 2003, total mercury and methylmercury concentrations were higher in muscle than in the heart (i.e., the ventricles and atria). Therefore, it was thought that mercury concentrations in the heart of cetaceans and fish were not higher than in other parts of the body. However, the inventors evaluated total mercury concentrations in various parts of fish using plasma spectrometry and found that total mercury concentrations in the ventricles were higher and lower in the atria. The reason for the higher total mercury concentrations in the ventricles of fish is unclear, but one possible explanation is the difference in the susceptibility of mercury accumulation, such as the higher blood inflow, which increases exposure to mercury in the blood, and the possible difference in the concentrations of proteins and amino acids that easily bind to mercury between the ventricles and atria. Furthermore, similar to the ventricles, high levels of mercury were found to accumulate in the flesh of the bleeding area. The reason for the higher total mercury concentrations in the bleeding area is unclear, but one possible explanation is that the flesh of the bleeding area is closer to the surface and heart, making it more susceptible to mercury accumulation. Furthermore, differences in analytical methods may result in different measurements of total mercury concentrations in each tissue, and the same is thought to apply to cetaceans.

[0014] As described above, it has been found that the total mercury concentration in the ventricle or bled part of fish meat is higher than that in the edible part, and that the total mercury concentration in the edible part is generally lower than that in the ventricle or bled part. Therefore, the evaluation of total mercury concentration in the evaluation method of the present disclosure may be an estimation of the maximum total mercury concentration contained in the edible part of fish or cetaceans. Note that, in the present disclosure, "estimating the maximum total mercury concentration contained in the edible part" does not necessarily mean that the total mercury concentration measured using the ventricle or bled part is the maximum total mercury concentration measured in the edible part, but rather means estimating a possible value for the total mercury concentration measured in the edible part based on the total mercury concentration measured using the ventricle or bled part. Based on the above findings, it is highly likely that the total mercury concentration measured in the edible part is equal to or lower than that measured using the ventricle or bled part, but it is also possible that the total mercury concentration in a small portion of the edible part exceeds that measured using the ventricle or bled part. In one aspect, "estimating the maximum total mercury concentration in the edible parts of fish or cetaceans" can mean estimating the total mercury concentration in the edible parts as the total mercury concentration measured using the ventricle or bled meat, or a value with a margin of error. For example, the "Guidelines for Validation of Analytical Methods" (Ministry of Agriculture, Forestry and Fisheries, October 2019, https: / / www.maff.go.jp / j / syouan / seisaku / pdf / guide_validation.pdf) allows a recovery rate of 80-110% for quantitative analytical methods of chemical substances at concentrations of 0.1 mg / kg or higher (Table 1 of the guidelines). Other factors, such as individual differences and measurement errors, may also occur. In light of these factors, the maximum possible total mercury concentration in the edible part may be estimated to be 1.00 to 1.25 times, 1.00 to 1.20 times, 1.00 to 1.10 times, or 1.00 to less than 1.10 times the total mercury concentration measured using meat from the ventricle or the bled part. Alternatively, the total mercury concentration measured using meat from the ventricle or the bled part may be estimated to be the maximum possible total mercury concentration in the edible part.

[0015] The type of fish is not particularly limited, and may be either a wild fish or a farmed fish. Examples of fish include tuna such as bluefin tuna, southern bluefin tuna, albacore, bigeye tuna, yellowfin tuna, Atlantic tuna, and longfin tuna; sharks such as blue shark and leopard shark; swordfish, bonito, short-finned pilot whale, alfonsino, swordfish, yellow bream, marlin, dream scorpionfish, black porgy, salmon, trout, yellowtail, yellowtail, sea bass, mackerel, and red sea bream. Examples of cetaceans include dolphins such as bottlenose dolphins, short-finned pilot whales, and Dall's porpoises, sperm whales, and beaked whales.

[0016] It is preferable to measure the total mercury concentration using only the ventricle or the flesh of the bled portion of a fish or cetacean as a sample. It has been found that almost no mercury accumulates in the atrium, so when measuring the total mercury concentration in the ventricle, it is preferable to measure the total mercury concentration using only the ventricle separated from the atrium. Similarly, when measuring the total mercury concentration in the flesh of the bled portion, it is preferable to measure the total mercury concentration in only the flesh of the bled portion without mixing it with other parts (e.g., edible parts).

[0017] The bleeding area refers to the area around the cut area when the fish or cetacean is bled. In other words, the meat from the bleeding area refers to meat harvested from the cross section cut with a knife or the like when the fish or cetacean is bled. Any tool such as a knife or punch can be used for harvesting. From the standpoint of ease of harvesting, the harvested area may be near the surface of the fish or cetacean, for example, within 200 mm or within 100 mm from the surface.

[0018] The amount of sample is not particularly limited as long as the total mercury concentration can be measured, and a larger amount of sample is desirable from the viewpoint of homogeneity. From the viewpoint of ensuring a sufficient amount of sample, the amount of sample used for measurement is preferably 0.1 g or more, more preferably 0.2 g or more, and even more preferably 0.3 g or more. Furthermore, plasma spectroscopy including the concentration step and detection step described below allows the total mercury concentration to be evaluated even with a relatively small amount of sample. From this viewpoint, the amount of sample used for measurement may be 10.0 g or less, 5.0 g or less, or 1.0 g or less. From this viewpoint, the amount of sample used for measurement may be 0.1 to 10.0 g, 0.2 to 5.0 g, or 0.3 to 1.0 g.

[0019] As described below, samples can be pretreated with an appropriate solution and used as a sample solution for analysis. Sample solutions prepared by mixing the sample and a sample treatment solution (e.g., an alkaline solution such as lithium hydroxide solution) at the same concentration and ratio will yield the same analytical luminescence in plasma spectrometry. For example, a sample solution treated with 2.5 mL of 4 M lithium hydroxide aqueous solution per 0.25 g of sample and a sample solution treated with 5 mL of 4 M lithium hydroxide aqueous solution per 0.5 g of sample will yield the same results in plasma spectrometry. For example, if 0.5 g of sample is treated with 2.5 mL of 4 M lithium hydroxide aqueous solution to increase the sensitivity of the obtained total mercury concentration, the analytical luminescence of the resulting sample solution will be approximately twice that of a sample solution treated with 2.5 mL of 4 M lithium hydroxide aqueous solution per 0.25 g of sample. Conversely, if the analytical luminescence is high, the amount of sample can be reduced. Users can adjust the sample amount and the concentration of the treatment solution as appropriate. By creating a calibration curve using samples, standards, and standard samples of known concentrations and using the sample and the solution used to treat the sample at a specified ratio, it becomes possible to measure the sample in a sample solution of any ratio.

[0020] Generally, methods for measuring mercury concentration include measuring total mercury, which includes inorganic and organic mercury, and measuring methyl mercury. However, the evaluation method disclosed herein is suitable for measuring total mercury concentration. In plasma spectrometry, the total mercury concentration can be measured by measuring the mercury concentration without distinguishing between inorganic and organic mercury in the sample.

[0021] [Plasma Spectroscopy] In the evaluation method disclosed herein, plasma spectroscopy is used to measure total mercury concentration. Conventional methods for measuring total mercury include plasma spectroscopy, absorptiometry (dithizone colorimetry), activation analysis, and cold atomic absorption spectrometry (including thermal vaporization-cold atomic absorption spectrometry and reduction-vaporization-cold atomic absorption spectrometry), with cold atomic absorption spectrometry being widely used. For example, Japanese Patent Application Laid-Open No. 2007-225582 measures the mercury concentration of fish using reduction-vaporization-cold atomic absorption spectrometry. In contrast, the evaluation method disclosed herein uses plasma spectroscopy to appropriately evaluate the total mercury concentration in edible portions using meat from the ventricle or bled portion. The analytical procedure is described in detail below.

[0022] For the measurement of total mercury concentration by plasma spectrometry, the sample is pretreated by an appropriate method to prepare a sample solution, such as by cutting the sample, homogenizing it with a homogenizer, and then adding and mixing an appropriate solution.

[0023] Examples of solutions for pretreatment include alkaline solutions and acid solutions. Among these, alkaline solutions are preferred from the viewpoint of protein denaturation and ease of metal ion detection. In one aspect, the evaluation method of the present disclosure includes treating meat from the ventricle or bleeding site of a fish or cetacean with an alkaline solution to prepare a sample solution for plasma spectroscopic analysis.

[0024] Examples of alkalis include sodium hydroxide, lithium hydroxide, potassium hydroxide, and ammonia. Of these, lithium hydroxide is preferred as the alkali from the viewpoints of protein denaturation, ease of detection of metal ions, and background stability. Examples of alkaline solutions include those obtained by diluting alkali with water or a buffer solution. The concentration of alkali in the alkaline solution is not particularly limited and may be, for example, 0.01 to 10 mol / L, and preferably 0.1 to 5 mol / L.

[0025] Examples of acids include hydrochloric acid, sulfuric acid, acetic acid, boric acid, phosphoric acid, citric acid, malic acid, succinic acid, and nitric acid. Examples of acid solutions include those obtained by diluting an acid with water or a buffer solution. The concentration of the acid in the acid solution is not particularly limited and may be, for example, 0.01 to 5 mol / L.

[0026] In one aspect, the plasma spectrometry method includes a concentration step of concentrating mercury in a sample near at least one of a pair of electrodes by applying a voltage to the pair of electrodes, and a detection step of generating plasma by applying a voltage to the pair of electrodes and detecting the light emission of the mercury generated by the plasma.

[0027] Examples of the electrode include solid electrodes, such as rod electrodes. The electrode material is not particularly limited as long as it is a solid conductive material, and may be a metal, a nonmetal, or a mixture thereof. When the electrode material contains a nonmetal, the electrode material may contain one type of nonmetal or two or more types of nonmetal. Examples of nonmetals include carbon. When the electrode material contains a metal, the electrode material may contain one type of metal or two or more types of metal. Examples of metals include gold, platinum, copper, zinc, tin, nickel, palladium, titanium, molybdenum, chromium, iron, etc. When the electrode material contains two or more types of metal, the electrode material may be an alloy. Examples of alloys include brass, steel, Inconel (registered trademark), nichrome, stainless steel, etc. The materials of a pair of electrodes may be the same or different.

[0028] The size of the electrode is not particularly limited, and may be any size that allows contact with the sample. When the electrode is a rod electrode, the diameter of the electrode may be 0.02 to 50 mm, or 0.05 to 5 mm. When the electrode is a rod electrode, the length of the electrode may be 0.1 to 200 mm, or 0.3 to 50 mm. The sizes of the pair of electrodes may be the same or different.

[0029] (Concentration step) In the concentration step, mercury in the sample is concentrated near at least one of the electrodes by applying a voltage to the pair of electrodes. The pair of electrodes is, for example, in contact with (liquid-contacted with) the sample solution. "Concentrating mercury in the sample near at least one of the electrodes" means that the mercury concentration in the sample solution is concentrated near the electrode compared to a portion farther from the electrode. In one aspect, "near the electrode" refers to the range in which plasma is generated in the detection step described below. Note that "near the electrode" also includes on the electrode.

[0030] In the concentration step, a portion of the mercury in the sample may be concentrated near the electrode, or all of the mercury may be concentrated near the electrode.

[0031] In the concentration step, it is preferable to set the charge conditions of the electrode so that mercury is concentrated on the electrode used for mercury detection in the detection step described below, i.e., the electrode where plasma is generated. Because mercury has a positive charge, it is preferable to set the charge conditions so that the electrode where plasma is generated has a negative charge.

[0032] The concentration of mercury can be adjusted, for example, by voltage. The voltage at which concentration occurs (hereinafter also referred to as "concentration voltage") can be set appropriately. The concentration voltage may be, for example, 1 mV or more, or 400 mV or more. The concentration voltage is not particularly limited and may be 2000 V or less, or 1000 V or less. Therefore, the concentration voltage may be 1 mV to 2000 V, or 400 mV to 1000 V. The concentration voltage may be constant throughout the concentration process, or may be varied during the concentration process. The concentration voltage may be a voltage at which plasma is not generated.

[0033] The time for applying the concentration voltage can be set appropriately depending on the concentration voltage, etc. The time for applying the concentration voltage may be, for example, 0.2 to 40 minutes, or 1 to 5 minutes. The concentration voltage may be applied continuously or discontinuously. An example of discontinuous application is pulse application. When the voltage is applied discontinuously, it is preferable that the total time for applying the concentration voltage is within the above range.

[0034] The voltage can be applied to the electrodes by a voltage application means. The voltage application means is not particularly limited as long as it can apply a voltage between the electrodes, and a voltage generator or the like can be used. In the concentration step, the current between the electrodes may be, for example, 0.01 to 200 mA, 10 to 60 mA, or 10 to 40 mA.

[0035] (Detection Step) In the detection step, plasma is generated by applying a voltage to a pair of electrodes, and light emitted from mercury produced by the plasma is detected.

[0036] The detection step may be performed continuously or discontinuously with the concentration step. In the former case, the detection step is performed simultaneously with the completion of the concentration step. In the latter case, the detection step is performed within a predetermined time after the completion of the concentration step. The predetermined time may be, for example, 0.001 to 1000 seconds or 1 to 10 seconds after the concentration step.

[0037] In the detection step, "generating plasma" means generating plasma that exhibits detectable light emission. As a specific example, it can be said that plasma has essentially been generated when plasma light emission can be detected by a plasma light emission detector.

[0038] The generation of plasma can be adjusted, for example, by voltage. The voltage for generating plasma that exhibits detectable light emission (hereinafter also referred to as "plasma voltage") can be set appropriately. The plasma voltage may be, for example, 10 V or higher, or 100 V or higher. The plasma voltage is preferably 50,000 V or lower, more preferably 5,000 V or lower, even more preferably 2,000 V or lower, particularly preferably 1,000 V or lower, extremely preferably 1,000 V or lower, and even more preferably 500 V. Therefore, the plasma voltage may be 10 to 50,000 V. The voltage at which plasma is generated is, for example, a voltage relatively high relative to the voltage at which concentration occurs. For this reason, the plasma voltage is preferably a voltage higher than the concentration voltage. The plasma voltage may be constant throughout the detection process, or may be varied during the detection process.

[0039] The time for applying the plasma voltage can be set appropriately depending on the plasma voltage. The time for applying the plasma voltage may be, for example, 0.001 to 0.02 seconds, or 0.001 to 0.01 seconds. The plasma voltage may be applied continuously or discontinuously. An example of discontinuous application is pulse application. When the voltage is applied discontinuously, it is preferable that the total time for applying the plasma voltage is within the above range.

[0040] In the detection step, the electrodes at which plasma is generated can be adjusted, for example, by making the liquid contact areas of the pair of electrodes different. Specifically, by making the liquid contact area of ​​one electrode smaller than the liquid contact area of ​​the other electrode, plasma can be generated on the former. For this reason, the pair of electrodes are preferably a pair of electrodes with different liquid contact areas with the sample solution, and the electrode of the pair with the smaller liquid contact area with the sample solution is preferably the electrode that analyzes mercury by generating plasma. When the liquid contact areas of the pair of electrodes are different, the difference in the liquid contact areas of the pair of electrodes is, for example, 0.001 to 300 cm. ２ 1 to 10 cm ２The "liquid contact area" refers to the area of ​​the electrode that comes into contact with the sample solution. The method for adjusting the liquid contact area is not particularly limited, and examples include varying the length of the electrode immersed in the sample solution, or covering a portion of the electrode that comes into contact with the sample solution with an insulating material. Examples of insulating materials include resin, silicone, glass, paper, ceramics, rubber, etc. Examples of resins include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyethylene terephthalate, polymethacrylate, polyamide, saturated polyester resin, acrylic resin, polybutylene terephthalate (PBT), polyether ether ketone (PEEK), and polymethylpentene (e.g., TPX®); thermosetting resins such as urea resin, melamine resin, phenolic resin, epoxy resins such as fluororesin glass epoxy, and unsaturated polyester resin. Examples of silicones include polydimethylsiloxane, etc.

[0041] In the detection step, the light emission from the generated plasma may be detected continuously or discontinuously. Examples of detecting light emission include detecting the presence or absence of light emission, detecting the intensity of light emission, detecting a specific wavelength, and detecting a spectrum. Examples of detecting a specific wavelength include detecting a unique wavelength emitted by mercury when it emits plasma light. There are no particular limitations on the method for detecting light emission, and optical measuring devices such as a CCD (Charge Coupled Device) and a spectroscope can be used.

[0042] The voltage can be applied to the electrodes by a voltage application means. Examples of the voltage application means include the voltage application means described above as being used for applying a concentration voltage. In the detection step, the current between the electrodes may be, for example, 0.01 to 100,000 mA, or 50 to 2,000 mA.

[0043] The evaluation method of the present disclosure may further include a calculation step of calculating the total mercury concentration in the sample from the detection results of the detection step. Examples of the detection results include the aforementioned luminescence intensity. In the calculation step, the total mercury concentration can be calculated, for example, based on the detection results and the correlation between the detection results and the mercury concentration in the sample. The correlation can be determined, for example, by plotting the detection results obtained by the analytical method of the present disclosure for a standard sample with a known mercury concentration against the mercury concentration of the standard sample. The standard sample is preferably a dilution series of mercury. Performing the calculation in this manner enables highly reliable quantification.

[0044] The pair of electrodes may be disposed in a container including a light-transmitting portion. In this case, in the detection step, the emitted light is detected by a light-receiving portion disposed so as to be able to receive the emitted light of mercury through the light-transmitting portion. The description of the analyzer described below can be used for the description of the container, the light-transmitting portion, the light-receiving portion, etc.

[0045] (Analytical Apparatus) Plasma spectroscopic analysis can be performed using a plasma spectroscopic analyzer. The plasma spectroscopic analyzer preferably includes a pair of electrodes, a container, and a light-receiving unit, the container including a light-transmitting unit, the pair of electrodes being disposed within the container, and a light-receiving unit disposed outside the container that is capable of receiving light emitted by application of a voltage to the pair of electrodes and the light emitted by the analyte (mercury in the evaluation method of the present disclosure) through the light-transmitting unit. Using such an analytical apparatus, the evaluation method of the present disclosure can be easily performed.

[0046] An example of a plasma spectrometer will be described with reference to the drawings. In the drawings, for the sake of convenience, the structure of each part may be shown in a simplified manner, and the dimensional ratios of each part may be shown schematically and different from the actual size.

[0047] FIG. 1 is a schematic perspective view of an example of a plasma spectrometer (hereinafter also referred to simply as "analyzer"). FIG. 2 is a schematic cross-sectional view of the plasma spectrometer as viewed from the II direction in FIG. 1. As shown in FIGS. 1 and 2, the analyzer 10 includes a pair of electrodes 1 and 2, a container 4, and a light-receiving unit 5. The container 4 includes a light-transmitting unit 3. The light-receiving unit 5 is disposed outside the container 4 and is configured to receive light emitted by application of a voltage to the pair of electrodes 1 and 2 and the light emitted by the analyte through the light-transmitting unit 3. The electrode 1 is disposed perpendicular to the bottom surface of the container 4, with its tip positioned so as to abut against the light-transmitting unit 3. The electrode 2 is disposed from the side of the container 4 toward the interior. The electrode 1 is coated with an insulating material 6. In the analyzer 10, a sample is introduced, for example, into the cylindrical container 4 so as to contact the electrodes 1 and 2. 1 and 2, the analysis device 10 is a vertically placed analysis device, but the analysis device 10 is not limited to this embodiment and may be, for example, a horizontally placed analysis device.

[0048] 1 and 2, the electrode 1 has a portion of its surface covered with an insulating material 6, but the insulating material 6 may or may not be present. Also, in Fig. 1 and 2, the electrodes 1 and 2 are arranged on different surfaces of the container 4, but the positions of the electrodes 1 and 2 are not particularly limited and they may be arranged in any positions.

[0049] 1 and 2, the electrode 1 and the light-transmitting portion 3 are in contact with each other, but the analysis device is not limited to this, and the electrode 1 may be disposed away from the light-transmitting portion 3. The distance between the electrode 1 and the bottom surface of the container 4 is not particularly limited, and may be, for example, 0 to 2 cm, or 0 to 0.5 cm.

[0050] The material of the light-transmitting portion 3 may be any material that transmits light emitted by application of a voltage to the pair of electrodes 1 and 2, and may be appropriately selected depending on the wavelength of the emitted light. Examples of materials for the light-transmitting portion 3 include quartz glass, acrylic resin (PMMA), borosilicate glass, polycarbonate (PC), cycloolefin polymer (COP), and methylpentene polymer (TPX (registered trademark)). The size of the light-transmitting portion 3 is not particularly limited, and may be any size that allows light emitted by application of a voltage to the pair of electrodes 1 and 2 to pass through.

[0051] 1 and 2, the container 4 is cylindrical with a bottom, but the shape of the container 4 is not limited to this and may be any shape. The material of the container 4 is not particularly limited, and examples thereof include acrylic resin (PMMA), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polystyrene (PS). The volume of the container 4 is, for example, 0.3 to 0.5 cm ３ When the container 4 is cylindrical with a bottom, the diameter of the container 4 may be, for example, 0.4 to 50 cm, or 1 to 5 cm. The height of the container 4 may be, for example, 0.3 to 50 cm, or 0.7 to 2 cm.

[0052] The light receiving unit 5 is not particularly limited, and examples thereof include known optical measuring devices such as a CCD, a spectroscope, etc. The light receiving unit 5 may be, for example, a transmission means for transmitting emitted light to an optical measuring device disposed outside the analysis device 10. Examples of the transmission means include a transmission path such as an optical fiber.

[0053] The method for manufacturing the container 4 is not particularly limited, and for example, a molded body may be manufactured by injection molding or the like, or the container 4 may be manufactured by forming a recess in a substrate such as a plate. Other methods for manufacturing the container 4 and the like are not particularly limited, and examples thereof include lithography and cutting. Details of the plasma spectroscopy and the analytical device can be found in JP 2016-130734 A.

[0054] Next, embodiments of the present disclosure will be described in detail using examples, but the embodiments of the present disclosure are not limited to these examples.

[0055] 1. Test Method 1-1. Sample Collection - Obtaining Three Farmed Bluefin Tuna - At a farm in Japan, several farmed bluefin tuna (Thunnus orientalis), including three fish whose edible parts would be used in subsequent tests, were fished from their cages, immediately bled, and gutted on board. The farmed bluefin tuna were tagged with an identification tag. The hearts were collected and placed in polyethylene bags labeled with the corresponding identification number. The gutted fish were immediately placed in ice water.

[0056] At the port, farmed bluefin tuna were removed from the ice water, washed, and weighed. Next, a biopsy punch with a plunger (BPP-40F, Kai Corporation) was inserted into the area where the blood had been drawn (the bleeding site), and a piece of fish meat weighing 0.5 g or more (meat from the bleeding site) was collected (Figure 3). After blood stains were removed from the heart with saline, the heart and meat from the bleeding site were immediately refrigerated and transported to the laboratory.

[0057] After delivery to the laboratory, the hearts were cut into ventricles and atria (Figure 3). The ventricles were weighed. These samples were stored in a freezer at -30°C. After collecting the hearts and the meat from the bled areas of three farmed bluefin tuna, the remaining parts were transported to a processing plant.

[0058] At the processing plant, farmed bluefin tuna halves were separated into edible parts A to D and discarded tail part E (Figure 3), and then separated into 200g pieces. These were placed in individual polyethylene bags, vacuum-packed using a vacuum packing machine, and flash-frozen in an ethanol bath at -40°C. The vacuum-packed samples were delivered to the laboratory and stored in a freezer at -60°C.

[0059] - Sample collection from two juvenile wild bluefin tuna - To obtain bluefin tuna with low mercury levels for subsequent testing to evaluate the correlation between total mercury concentrations in the ventricles and edible parts, two juvenile wild bluefin tuna were purchased and sent to the laboratory. At the laboratory, the fish were assigned identification numbers and their internal organs were removed. The hearts were collected and placed in individual polyethylene bags labeled with the corresponding identification numbers. Because wild bluefin tuna juveniles are light in weight (approximately 2 kg), the flesh of one half of the body was divided into only two parts. The fish flesh was placed in individual polyethylene bags and vacuum-packed using a vacuum packing machine. The vacuum-packed samples were stored in a freezer at -30°C. The samples were used for measurements within one week.

[0060] 1-2. Alkaline Treatment of Fish Samples First, 0.5 g of homogenized fish sample was weighed using a calibrated balance within an error of ±0.0009 g. Next, the sample was transferred to a disposable homogenizer tube, BioMasher® SP (Nippi Corporation). Next, 2.5 mL of 4 M lithium hydroxide solution was added to the tube. The sample was mashed and mixed with the 4 M lithium hydroxide solution for approximately 5 seconds using a masher rod connected to a Power Masher II (Nippi Corporation). Next, this tube was placed on a rack installed in the bath of an ultrasonic cleaner (ASU-10D; AS ONE Corporation). The water temperature in the ultrasonic bath was maintained at approximately 20°C to 25°C, and the ultrasonic bath oscillation frequency was set to a high frequency of 23 kHz. After ultrasonic treatment, the sample was thoroughly stirred using the masher rod. Finally, the masher rod was removed from the tube.

[0061] 1-3. Measurement of total mercury concentration by plasma spectrometry Approximately 1 mL of the treated sample was dispensed into the sample reservoir of a SillBe Kit Hg, Pb (ARKRAY, Inc.) reagent pack. Next, the chip was placed in the SillBe LB-5410 automated metal analyzer, and Start was pressed. All subsequent steps were performed automatically.

[0062] 238 μL of the sample in the reservoir was mixed and diluted in a dilution well containing 4.4 M lithium hydroxide solution (195 μL), 86 mM thallium (I) nitrate solution (21.6 μL), and ethanol (EtOH) (45.5 μL). After mixing, the diluted sample was transferred to a light-emitting well in which an electrode was placed. A carbon electrode (Φ4.0 mm diameter, liquid contact area ≧33 mm) was used. ２ A nichrome electrode (0.1 mm diameter x 0.5 mm length) was used. A constant current of 20 mA was applied for 180 seconds to concentrate the metal, and a DC pulse voltage of 500 V (50 μs period, 50 times) was used for plasma emission. Plasma emission was performed twice, and the two emission intensity measurements were averaged to calculate the total mercury value from the mercury and thallium.

[0063] - Preparation of mercury calibration curve - To prepare a calibration curve, six mercury solutions with different concentrations were prepared using a 1000 mg / L methylmercury standard solution (Thermo Scientific Chemicals, Massachusetts, USA). The calibration curve showed linearity in the mercury concentration range of 0 to 1.5 mg / L. A regression line was prepared to calculate the mercury concentration (slope = 0.6100; Y-intercept = -0.003889; Pearson r = 0.9998; R ２ =0.9961; p<0.0001).

[0064] - Calculation of Total Mercury Value - As described in Japanese Patent No. 6754326, the analytical luminescence intensity of mercury in a sample was calculated by subtracting 1 from the ratio of the signal intensity of the sample to the signal intensity of the baseline at a wavelength (253.7 nm) specific to mercury (mercury signal intensity / baseline mercury signal intensity - 1). Similarly, the analytical luminescence intensity of thallium in a sample was calculated by subtracting 1 from the ratio of the signal intensity of the sample to the signal intensity of the baseline at a wavelength (276.8 nm) specific to thallium added as an internal standard (thallium signal intensity / baseline thallium signal intensity). Finally, the analytical luminescence intensity of mercury was divided by the analytical luminescence intensity of thallium (formula (1) below) to calculate the mercury concentration from the calibration curve.

[0065]

[0066] The terms in formula (1) have the following meanings: Value: mercury concentration in the sample Hg signal intensity: mercury signal intensity in the sample at 253.7 nm Hg base signal intensity: mercury signal intensity at the baseline at 253.7 nm Tl signal intensity: thallium signal intensity in the sample at 276.8 nm Tl base signal intensity: thallium signal intensity at the baseline at 276.8 nm

[0067] 1-4. Measurement of total mercury concentration by reduction vaporization-cold atomic absorption spectrometry Frozen tuna meat samples were sent to an inspection laboratory (IDEA Co., Ltd.), where total mercury was measured in accordance with the Ministry of the Environment's guidelines (Mercury Analysis Manual, http: / / nimd.env.go.jp / kenkyu / analysis_manual.html).

[0068] 2. Evaluation Results 2-1. Total Mercury Concentration in Edible Portions and Tail Portions Relative to Total Mercury Concentration in Ventricles Figure 4 plots the relative total mercury concentrations of 200g of fish meat from 109 edible portions (A-D portions) and six discarded tail portions (E portions) for three farmed bluefin tuna, where the total mercury concentration in the ventricles is set to 1. Because total mercury concentrations vary among individuals, the ventricles and each portion of each individual were linked, and the relative values ​​of each portion relative to the total mercury concentration in the ventricles were evaluated. The results showed that the relative values ​​of the edible portions relative to the ventricles for the three farmed bluefin tuna ranged from 0.39 to 1.08, with only two exceeding 1.05. The relative values ​​of the total mercury concentrations of more than 95% of the edible portions (A-D portions) and tail portions (E portions) were generally below 1. Therefore, it was found that the total mercury concentrations in the edible parts (parts A to D) and tail part (part E) were generally lower than that in the ventricles. It was observed that the total mercury concentration in the ventricles showed values ​​close to the maximum of the total mercury concentration in the edible parts. From these results, it is believed that the maximum total mercury concentration in the edible parts can be estimated to be, for example, about 1.00 to 1.10 times the total mercury concentration measured in the ventricles.

[0069] 2-2. Correlation between total mercury concentration in the ventricle and total mercury concentration in the edible part Figure 5 shows a graph showing the relationship between the total mercury concentration in the ventricle and the total mercury concentration in the edible part (a mixture of parts A to D and averaged) for each individual for three farmed bluefin tuna and two young wild bluefin tuna. As the graph shows, although there is variation in total mercury concentration between each individual, a positive correlation is observed between the total mercury concentration in the ventricle and the total mercury concentration in the edible part for each individual.

[0070] 2-3. Total mercury concentrations in the ventricles, atria, and bled areas of the body We compared the total mercury concentrations in the ventricles, atria, and bled areas of the body. The results showed that there was no significant difference between the ventricles and the bled areas, and that the total mercury concentration in the bled areas was as high as in the ventricles. On the other hand, it was found that almost no mercury accumulated in the atria (Figure 6).

[0071] When we investigated the correlation between the total mercury concentrations in the ventricles and atria, and between the ventricles and the bled meat, we found a positive correlation in both cases (Figure 7). Based on these results, the total mercury concentrations in the ventricles and the bled meat may be used as representative values.

[0072] 2-4. Comparison of plasma spectrometry and CV-AAS When the total mercury concentrations in the ventricles and lean meat measured by plasma spectrometry and CV-AAS were compared, differences in the total mercury concentrations were observed between plasma spectrometry and CV-AAS (Figure 8). In particular, the total mercury concentration in the ventricles was higher when measured by plasma spectrometry. This suggests that plasma spectrometry is suitable for measuring the total mercury concentration in the ventricles to evaluate the total mercury concentration in edible parts.

[0073] The disclosure of Japanese Patent Application No. 2024-084354, filed on May 23, 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

[0074] 1, 2 Electrode 3 Transparent part 4 Container 5 Light receiving part 6 Insulating material 10 Analyzer

Claims

1. A method for assessing the total mercury concentration in edible parts of fish or cetaceans, which comprises measuring the total mercury concentration in a sample that is the flesh of the ventricle or bled part of a fish or cetacean by plasma spectrometry.

2. The evaluation method according to claim 1, wherein the evaluation of the total mercury concentration includes estimating the maximum total mercury concentration contained in the edible portion of the fish or cetacean.

3. The evaluation method according to claim 2, which comprises estimating the maximum total mercury concentration contained in the edible portion to be 1.00 to 1.25 times the measured total mercury concentration of the sample.

4. The evaluation method according to claim 2, which comprises estimating the maximum total mercury concentration contained in the edible portion to be 1.00 to 1.10 times the measured total mercury concentration of the sample.

5. The evaluation method according to claim 1, wherein the fish or cetacean is a tuna.

6. The evaluation method according to claim 5, wherein the tuna is bluefin tuna.

7. The evaluation method according to claim 1, further comprising treating the flesh of the ventricle or exsanguination site of the fish or cetacean with an alkaline solution to prepare a sample solution for plasma spectroscopic analysis.

8. The evaluation method according to claim 7, wherein the alkaline solution comprises a lithium hydroxide solution.

9. The evaluation method according to any one of claims 1 to 8, wherein the plasma spectroscopic analysis method comprises: a concentration step of concentrating mercury in the sample in the vicinity of at least one of a pair of electrodes by applying a voltage to the pair of electrodes; and a detection step of generating plasma by applying a voltage to the pair of electrodes and detecting the light emitted from the mercury generated by the plasma.

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