A fish heavy metal pollution risk grading detection method

Abstract

The application discloses a fish heavy metal pollution risk grading detection method, relates to the technical field of aquatic product safety and environmental monitoring, and comprises the following steps: taking muscle, enrichment indicator organs and exposure contact organs of fish in the same breeding water area, taking not less than five fish of the same kind to form a group sample, detecting the heavy metal concentration of each marked part, individual and water batch respectively, comparing the muscle concentration with the food safety limit benchmark to determine the edible risk, comparing the enrichment indicator organ concentration and the ratio of the muscle concentration to determine the breeding exposure indicator benchmark to determine the breeding risk, comparing the exposure contact organ concentration, the group average concentration, the detection rate and the proportion of individuals exceeding the background to determine the regional background benchmark to determine the regional pollution risk, and three sets of benchmarks are respectively used for one dimension and are not borrowed from each other.

Description

Technical Field

[0001] This invention relates to the field of aquatic product safety and environmental monitoring technology, specifically a method for risk classification and detection of heavy metal pollution in fish. Background Technology

[0002] In fish farming and aquatic product distribution, the detection of heavy metals such as cadmium, lead, mercury, and arsenic in fish is a common technical foundation supporting batch disposal, aquaculture site management, and regional environmental monitoring. The three types of management work mentioned above have fundamentally different interpretations of the test results: food safety supervision, based on the "National Food Safety Standard Limits for Contaminants in Food" (GB 2762), makes decisions on whether the heavy metal content in edible parts is up to standard; aquaculture management focuses on whether the accumulation of heavy metals in fish viscera indicates continuous heavy metal exposure in the aquaculture water, requiring early warning signals before edible parts exceed the limits; regional environmental monitoring focuses on the overall pollution pattern of the same water area, needing to distinguish between systemic pollution at the water level and occasional individual exceedances.

[0003] Regarding detection methods, anodic stripping voltammetry, inductively coupled plasma mass spectrometry, and atomic fluorescence spectrometry are all known methods for heavy metal detection. Publication number CN113092564A discloses a rapid method for heavy metal detection in water quality, which uses anodic stripping voltammetry, indicating that anodic stripping voltammetry is already known as a rapid screening method for heavy metals. For fish samples, existing studies typically employ tissue-specific sampling and testing, followed by single-dimensional evaluation using pollution indices and health risk models. For example, the study on heavy metal pollution status and health risk assessment of recirculating aquaculture redfin pufferfish (Journal of Dalian Ocean University, 2022) tested liver, muscle, and gill tissues separately, and used single-factor pollution indices, the Nemerow comprehensive pollution index, and THQ indicators to assess health risk; the enrichment and safety assessment of heavy metals in fish in the Wucheng Poyang Lake Nature Reserve (Acta Hydrobiologica Sinica, 2017) also used tissue-specific determination combined with THQ for safety assessment. Therefore, it can be seen that the aforementioned representative existing technical routes are already able to complete the detection of heavy metals in different tissues of fish and summarize the detection results into a single evaluation conclusion such as consumption or health risk.

[0004] However, the conclusions drawn from the aforementioned evaluation approaches typically focus only on a single dimension: food safety or health risk. Applying the same set of food safety limits to all test data can only determine whether edible parts are compliant; it cannot directly determine whether there is ongoing exposure in the aquaculture water body, nor can it distinguish whether the pollution pattern of the water body constitutes regional systemic pollution. When it is necessary to simultaneously conduct food safety risk management, aquaculture early warning, and regional pollution assessment, it is often necessary to conduct supplementary testing or re-evaluation for each of the three categories. It is difficult to provide three independent and separately manageable classification results in one step, thus limiting the efficiency of data interpretation and the direct applicability of the test data.

[0005] Therefore, how to use the same set of fish heavy metal detection data to derive three risk levels with independent calibers that can be used for food safety disposal, aquaculture risk warning, and regional pollution assessment, and how to improve the efficiency of the test results interpretation to a level that can directly support risk warning, sampling and grading, and regulatory disposal, has become a technical problem that needs to be solved in this field. Summary of the Invention

[0006] The technical problem this invention aims to solve is that fish heavy metal detection data is difficult to use for three different purposes: food safety, early warning of exposure in aquaculture, and regional pollution assessment, each requiring a separate risk assessment. Existing detection methods typically converge the concentration measurements of various parts and metals into a single-dimensional comprehensive risk conclusion. When the same batch of test results needs to be used separately for risk warning, sampling grading, and regulatory disposal, manual comparison against standards and cross-dimensional analysis is still required, making the interpretation inefficient in practice. The purpose of this invention is to simultaneously provide risk levels for food safety, aquaculture, and regional pollution from fish batches within the same aquaculture area, each directly applicable to disposal.

[0007] The overall technical concept of this invention lies in corresponding the sampling sites and population designs to three risk dimensions, and then using three independent sets of benchmarks to determine the corresponding dimensions, thereby splitting a batch of test data into three categories of risk results that are independent of each other. Edible muscle is directly related to human intake, and using food safety limits to measure consumption risk is most appropriate; the liver and kidneys are the main accumulation sites of heavy metals in fish, and their concentration changes often precede muscle responses, so organ accumulation can identify continuous exposure in aquaculture water earlier; gills are in direct contact with water for a long time, and combined with population statistics of multiple fish in the same water area, they can better reflect the overall condition of the water area rather than the occasional situation of individual fish. Because the indicative significance of the three types of sites is different, corresponding each to an independent benchmark ensures that the determination results of each dimension are not affected by the caliber of other dimensions. On the testing side, by sampling by site and population and establishing site markers, individual markers, and water area batch markers, it is ensured that different sites and populations within the same batch correspond one-to-one in terms of heavy metal detection items, so that subsequent three-dimensional classification is implemented on specific test objects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: Fish samples were collected from the same aquaculture area. For each individual fish, muscle tissue, enrichment indicator organs, and exposed contact organs were collected to prepare test samples. Simultaneously, a population sample of the same species, consisting of at least five fish, was collected from the same area. Site markers, individual markers, and batch markers for each test sample were established. The concentrations of heavy metals, including at least cadmium, lead, mercury, and arsenic, were measured. Heavy metal concentration data were obtained by site and individual, belonging to the same batch within the same water area. The muscle tissue, enrichment indicator organs, and exposed contact organs within the same batch maintained the same heavy metal correlation with the population sample. These three types of test samples, along with the population sample, constituted the batch-level testing data for the same water area. It was not required that all samples for each dimension come from the same fish to avoid conflating regional population statistics with individual site-specific testing.

[0009] Based on the aforementioned concentration data, three independent benchmarks are used to determine the risk levels in three dimensions: First, the heavy metal concentration in muscle tissue is compared with food safety limits to form a comprehensive contamination assessment metric, determining the food risk level. Second, the heavy metal concentration in enrichment indicator organs is compared with aquaculture exposure indicator benchmarks, and the ratio of enrichment indicator organ concentration to muscle tissue concentration is used as the aquaculture exposure assessment metric, determining the aquaculture risk level. Third, the heavy metal concentration in exposed organs, along with the population average concentration, detection rate, and proportion of individuals exceeding the regional background benchmark, are compared with the regional background benchmark. Whether the proportion of individuals exceeding the regional background benchmark reaches the regional contamination proportion threshold is used as the distinguishing factor between regional contamination and occasional individual occurrences, forming a regional contamination assessment metric, determining the regional contamination risk level. The fact that these three benchmarks are independent and each only serves the corresponding dimension's level determination is the core technical difference between this invention and single-dimensional comprehensive evaluation.

[0010] The risk levels for food consumption, aquaculture, and regional pollution, along with their respective baseline categories, judgment quantities, excessive metals, corresponding locations, water batches, and tiered treatment recommendations, will be linked and output.

[0011] Furthermore, the aforementioned muscle tissue is taken from edible muscle of the fish, enriched with indicator organs including the liver and kidneys, and exposed contact organs including the gills.

[0012] Furthermore, the ratio of the concentration of each heavy metal in the muscle tissue to its corresponding food safety limit can be used as the single exceedance multiple for that heavy metal. The maximum and average values ​​of each single exceedance multiple are combined to form a comprehensive food contamination index. Food risk is then classified into at least four progressively higher levels based on the comprehensive food contamination index. When the concentration of any heavy metal in the muscle tissue exceeds the corresponding food safety limit, the food risk level is raised to at least level three to prevent individual high-concentration metals from being masked by the average effect of the comprehensive index. For mercury and arsenic used for food safety limit comparison, the corresponding forms specified in the benchmark can be used. When the total mercury or total arsenic screening value obtained by rapid detection reaches 90% or more of the corresponding food safety limit, the confirmatory detection result of the corresponding form is used as the basis for determining the food risk level, avoiding directly equating the total screening value with the limit form. The 90% trigger threshold is a stricter setting based on the GB 2762 screening rules, used to detect potential form risks in advance within the limit values.

[0013] Furthermore, the ratio of the concentration of the enrichment indicator organ to the concentration in the muscle can be defined as the organ enrichment coefficient. When the concentration of heavy metals in the muscle has not exceeded the food safety limit but the organ enrichment coefficient reaches 5 or above, or reaches the warning range determined by historical monitoring data of the same species of fish, the aquaculture risk level is raised to the warning level, giving an early warning result of continuous exposure in the aquaculture water. When there is no historical monitoring data, an initial baseline can be established by using multiple batches of test values ​​of the same species of fish in the control water area, and the upper quantile, mean plus twice the standard deviation or organ enrichment coefficient of 5 can be used as the initial warning boundary.

[0014] Furthermore, the population average concentration, the proportion of individuals exceeding the regional background benchmark, and the detection rate can be statistically analyzed for the population sample. The condition for determining regional pollution is that the proportion of individuals exceeding the regional background benchmark reaches 30% or more, or reaches the proportion threshold determined by multiple batches of historical sampling data of this water area. The multiple of the population average concentration relative to the regional background benchmark and the detection rate are used as auxiliary verification quantities for the regional pollution risk level. When the proportion of individuals exceeding the regional background benchmark does not reach the proportion threshold, it is determined to be an isolated incident, and the corresponding regional pollution risk level is output as lower than the level corresponding to regional pollution.

[0015] Furthermore, to ensure the reliability of the judgment results for each dimension, when the judgment value of any dimension falls within the critical range of 10% above or below the threshold of the adjacent level, or when the judgment value of comprehensive food pollution falls within the critical range of 0.1 above or below the threshold of the adjacent level, the corresponding part of the sample to be tested is repeatedly measured and the average value is taken before re-judgment; when the required part of the sample to be tested for a certain dimension is missing, only the dimension level that can be obtained is output, and the risk dimension corresponding to the missing part is marked as insufficient data and not graded; when the number of fish in the group sample is less than five or less than the higher sample lower limit determined according to the historical fluctuation of the water area, the output of the regional pollution risk level is stopped and the insufficient sample is marked; when any dimension reaches a high risk level, a return mark for quantitative arbitration by a qualified laboratory is attached.

[0016] Furthermore, the detection process can employ a temperature-controlled digestion method using neutral protease and lipase in combination with citric acid and hydrogen peroxide to first dissociate the sample matrix. The digestion solution is then adjusted to pH 2 to 4, and multiple heavy metals are chelated and enriched in a single step using thiol-modified cotton or thiol-modified diatomaceous earth. After uniform elution with acidic thiourea hydrochloric acid elution solution, the concentration of heavy metals is determined by electrochemical stripping voltammetry. When electrochemical measurement equipment is unavailable, cadmium, lead, mercury, and arsenic are treated separately using corresponding colorimetric systems for individual color development, blank correction, and standard curve conversion, enabling the pretreatment and detection to be stably completed with lower equipment requirements.

[0017] The beneficial effects of this invention are: This invention separates samples from multiple fish populations in the same water area, sampling muscle, liver, kidney, and gills separately, and establishes batch markers for each part, individual, and water area. The three risk dimensions—consumption, aquaculture, and regional—are each mapped to a specific sample source from the starting point of the detection process. While tissue-specific testing is already used in existing fish heavy metal assessments, its results typically only lead to a single health risk conclusion, lacking a stable correlation between the sampling site and the risk trajectory. This invention binds the sampling site to the dimension at the sampling stage, allowing subsequent grading to directly target specific samples rather than relying on general statistical definitions. This improves the applicability of the detection results to the three applications: consumption disposal, aquaculture early warning, and regional determination.

[0018] Food safety limits, aquaculture exposure indicators, and regional context are set as three independent judgment criteria, with each criterion serving only its corresponding risk dimension, no longer using a single exceedance line to simultaneously handle multiple purposes. Existing schemes often use whether or not the limit is exceeded as a unified entry point, causing different regulatory objectives to be compressed into the same interpretation. By separating the sources and scope of application of the three criterions, batch data from the same water area are not intertwined when making judgments across different dimensions, thus reducing ambiguities in interpretation caused by the mixed use of cross-dimensional criteria.

[0019] The method calculates the exceedance multiple of individual metal concentrations in muscle tissue, and combines the maximum and average values ​​to form a comprehensive food contamination index. It stipulates that if any metal exceeds its corresponding limit, the food risk is raised to a level no lower than the upper-middle range. This approach combines comprehensive judgment with individual safety baselines. While comprehensive index evaluation is susceptible to averaging effects, where individual metal exceedances may be masked by the overall level, incorporating the individual priority upgrade rule ensures that any real risk in edible parts is not smoothed out by the comprehensive index, thus reducing the probability of missed detections at the food safety level.

[0020] The ratio of liver and kidney concentrations relative to muscle concentration is defined as the organ enrichment coefficient. When this coefficient reaches a warning threshold before muscle concentration exceeds the limit, an early warning of aquaculture risk is generated, allowing fish body accumulation information to directly serve the early identification of continuous exposure in aquaculture water. Currently, aquaculture assessments largely rely on individual water or bottom sediment measurements, which are relatively slow to respond to continuous exposure in live fish. By utilizing the physiological characteristic that organ accumulation changes precede muscle manifestations, warnings can be triggered before edible parts exceed the limit, enhancing the early detection capability at the aquaculture level.

[0021] Regional pollution is determined by considering the population average concentration, the proportion of individuals exceeding the background concentration, and the detection rate. The primary criterion is whether the proportion of individuals exceeding the background concentration reaches the regional pollution threshold, while the population average concentration and detection rate serve as supplementary verification. This ensures that regional judgment is based on population distribution rather than single-tail anomalies. Individual fish concentrations fluctuate significantly due to factors such as feeding, body length, and metabolism. A single fish exceeding the threshold may reflect a problem in the water area or be purely accidental. Only when a large number of individuals exceed the threshold, and the population detection status corroborates this, can regional pollution be identified. This distinguishes systemic pollution from sporadic individual anomalies, thus reducing misjudgments of regional pollution.

[0022] The risk levels across three dimensions are linked to their baseline category, judgment quantity, excessive metal content, corresponding location, water area batch, and tiered disposal recommendations. Once the test results are generated, they can directly enter the risk warning, sampling grading, and regulatory disposal processes. Current practices often still require manual verification of standards item by item, cross-dimensional comparisons, and separate determination of disposal direction. With the risk level and disposal recommendations provided along with the results, concentration data can be directly converted into actionable regulatory information, simultaneously improving interpretation efficiency and result usability.

[0023] The pretreatment process employs temperature-controlled digestion using a combination of a complex enzyme, a weak acid, and hydrogen peroxide. Multiple metals are then enriched through a single chelation process using thiol-based materials. Readings are obtained via either electrochemical dissolution or colorimetric analysis, enabling stable determination of sensitive metals such as mercury and arsenic with relatively low equipment requirements. High-temperature, strong acid digestion easily introduces mercury and arsenic volatilization losses, while separate pretreatment steps increase operational burden and matrix interference. By controlling the digestion temperature within a range where mercury and arsenic are less likely to escape, and by incorporating the step-by-step pretreatment into a single enrichment step, negative bias is reduced, and the effective response to low-content metals is simultaneously increased, thus improving the stability and applicability of the detection chain.

[0024] For abnormal situations such as retesting of critical intervals, downgrading of missing parts, suspension of regional classification due to insufficient population samples, and high-risk return for arbitration, quantitative triggering criteria and recovery conditions are set respectively. The three-dimensional classification maintains controlled output even when data boundaries are unstable or samples are incomplete. If a fixed process is used to forcibly give complete conclusions, it is easy to make misleading judgments under conditions of critical values, missing samples, or small samples. After making the review and rollback into an action chain triggered by criteria, abnormal samples are identified separately and processed according to the corresponding path, and the stability of the results in grassroots sampling and regulatory applications is improved. Attached Figure Description

[0025] Figure 1 The process of this invention Figure 1 ; Figure 2 The process of this invention Figure 2 ; Figure 3 The process of this invention Figure 3 . Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1 like Figure 1As shown, this embodiment presents the basic scheme of the method, independently carrying out sampling and labeling by part and group, itemized detection, and graded interpretation and correlation output of the three dimensions of edible, aquaculture, and regional pollution under three independent benchmarks. The target heavy metals processed include at least cadmium, lead, mercury, and arsenic, and chromium, copper, and zinc are added if necessary. In this text, muscle part refers to the edible muscle of the fish, enrichment indicator organs are the liver and kidney, the exposed contact organ is the gill, and the group sample is a collection of no less than five fish of the same species from the same aquaculture water area. Each of the three benchmarks serves only one dimension and does not borrow from each other. The food safety limit benchmark is taken from the current "National Food Safety Standard for Limits of Contaminants in Food" (GB 2762) on the application date, which corresponds to the food category limit of the tested fish species, edible parts, and contaminant forms; when the tested fish species have differences in carnivorous fish, freshwater fish, or other subcategories, the limit values ​​of methylmercury, inorganic arsenic, etc., are selected according to the corresponding food category. This embodiment illustrates the determination criteria, using lead 0.5 mg / kg, cadmium 0.1 mg / kg, methylmercury 0.5 mg / kg, and inorganic arsenic 0.1 mg / kg. When obtaining the total mercury or total arsenic screening value in the rapid detection stage, this screening value is used to identify potential risks in advance. This method uses 90% of the corresponding food safety limit as the tightened trigger threshold for morphological confirmation—when the screening value reaches 90% or more of the limit, further confirmatory testing is performed according to the limited forms such as methylmercury or inorganic arsenic, and the confirmatory test results are used as the basis for determining the edible risk level. When the screening value does not reach 90%, GB standards are referenced. The screening rules of 2762 use screening values ​​as a reference record; the aquaculture exposure indicator benchmark is the exposure warning threshold of heavy metals in the liver and kidneys, which is determined by combining the historical organ monitoring values ​​and organ enrichment patterns of this water area. The representative liver and kidney warning thresholds used in this embodiment are 0.5 mg / kg for cadmium, 1.0 mg / kg for lead, 0.3 mg / kg for total mercury, and 0.5 mg / kg for inorganic arsenic. When historical organ monitoring values ​​of this water area are missing, the upper quantile or mean of the liver and kidney test values ​​of multiple batches of the same species of fish in the control water area plus twice the standard deviation is taken as the initial warning threshold, and updated in subsequent sampling. The regional background benchmark is the upper quantile of the historical background content of the same species of fish in this water area or the control water area. The representative background upper limit used in this embodiment is 0.05 mg / kg for cadmium, 0.2 mg / kg for lead, 0.1 mg / kg for total mercury, and 0.05 mg / kg for inorganic arsenic.

[0028] The process begins with sampling and labeling. Grass carp are first collected from the same aquaculture area. For each fish, muscle, liver, kidney, and gill samples are homogenized. At least five more grass carp are then collected and individually numbered to form a group sample. Each sample is marked with a location, an individual, and a batch / area label, ensuring a one-to-one correspondence between muscle, liver, kidney, gill, and group samples within the same batch for heavy metal testing. Subsequent three-dimensional grading uses batch testing data from the same water area. The edible and aquaculture dimensions are derived from the samples from different locations, while the regional dimension is derived from gill and group sample statistics. It is not required that all three dimensions be completed independently by the same fish. Subsequently, the concentrations of cadmium, lead, mercury, and arsenic were measured separately for each part of the sample. Concentration data for each part, individual, and batch belonging to the same water area were obtained. In this example, the representative results for the muscle of the tested fish were cadmium 0.06, lead 0.40, total mercury 0.08, and inorganic arsenic 0.08; for the liver and kidneys, cadmium 0.42, lead 0.55, total mercury 0.18, and inorganic arsenic 0.12; and for the gills, cadmium 0.09, lead 0.30, total mercury 0.10, and inorganic arsenic 0.06. All units are milligrams per kilogram. After obtaining the concentration, the edible dimension is determined: the concentration of each metal in the muscle is compared with the corresponding limit to obtain the single-item exceedance multiple. The square root of the sum of the square of the maximum value and the square of the average value is taken as the comprehensive edible contamination index. The edible risk is divided into four levels according to the index from low to high: Level 1 is below 0.7, Level 2 is between 0.7 and 1.0, Level 3 is between 1.0 and 2.0, and Level 4 is above 2.0. When any metal in the muscle exceeds the limit, the edible risk is raised to at least Level 3. The speciation of mercury and arsenic is determined as follows: In this embodiment, the total mercury screening value of 0.08 is 16% of the methylmercury limit of 0.5 in food, which is far below the 90% (i.e., 0.45) threshold for speciation confirmation set by this method. Therefore, it does not trigger speciation confirmation. The total mercury screening value is used as a reference record in this embodiment and is not included in the single-item exceedance multiple of the comprehensive edible contamination index. Inorganic arsenic is already a specified speciation and is directly included in the calculation. For cadmium (0.06 / 0.1=0.60), lead (0.40 / 0.5=0.80), and inorganic arsenic (0.08 / 0.1=0.80), the individual exceedance multiples were obtained, with the highest value being 0.80 (lead and inorganic arsenic) and the average value being approximately 0.73. The comprehensive pollution index for food was approximately 0.77, falling within the secondary range, and the food risk was assessed as secondary. In terms of aquaculture, liver and kidney concentrations were first compared with the aquaculture exposure indicator baseline, and then the ratio of liver and kidney concentrations to muscle concentrations was used as the organ enrichment coefficient. When liver and kidney concentrations approached or reached the warning threshold, or the organ enrichment coefficient reached 5 or higher, the aquaculture risk increased accordingly. In this example, the liver and kidney cadmium concentration was 0.42, approaching the warning threshold of 0.5, and the organ enrichment coefficient was approximately 7, thus the aquaculture risk was assessed as warning level.At the regional level, the population average concentration, the proportion of individuals exceeding the regional background benchmark, and the detection rate of each metal were calculated tail-by-tail for the population sample, and the gill concentration was compared with the regional background benchmark. The proportion of individuals exceeding the background benchmark (reaching 30%) was used as the primary criterion for distinguishing between regional pollution and sporadic occurrences. The multiple of the population average concentration relative to the regional background benchmark and the detection rate served as auxiliary verification measures for the risk level. In this embodiment, only 20% of the individuals in the population exceeded the cadmium background benchmark, and neither the detection rate nor the population average concentration showed a sustained regional upward trend. Therefore, it was treated as sporadic occurrences, and the regional pollution risk was classified as low. Finally, the risk levels of the three dimensions, along with their respective benchmark categories, judgment quantities, excessive metals, corresponding locations, water batches, and treatment recommendations, were output in a correlated manner. In this embodiment, the output includes recommendations for routine sampling and periodic retesting of secondary feed for edible products, recommendations for early warning of aquaculture water bodies and bottom sediment investigation and feed water source verification, and recommendations for routine monitoring of lower-risk regional feeds.

[0029] The meanings and sources of the variables for each judgment quantity are explained as follows: The single-item exceedance multiple is the ratio of the measured concentration of a metal in a specified location to its corresponding benchmark, derived from the measured concentration in that location and the benchmark value; the comprehensive food pollution index comprehensively reflects the most severe single-item and overall levels, derived from the maximum and average of the single-item exceedance multiples; the organ enrichment coefficient is the ratio of the concentration of the same metal in the liver, kidney, and muscle, derived from the measured concentrations in these two locations, with a default early warning threshold of 5, which can also be corrected from historical monitoring data of the same fish species; the proportion of individuals exceeding the background concentration in the population is the share of individuals in the population whose concentration exceeds the regional background benchmark, derived from tail-by-tail testing results, with a default regional pollution proportion threshold of 30%, which can also be corrected from historical sampling data of multiple batches in this water area. The values ​​of the three benchmarks are derived from the national food safety standards, historical organ monitoring values ​​of this water area, and regional historical background values, respectively. The concentrations and judgment quantities listed in this embodiment are representative recorded values ​​used to illustrate the judgment criteria and ensure the smooth operation of the process. They only reflect the recording method and judgment process of this batch and are not extrapolated to general statistical conclusions or comparative conclusions with other schemes.

[0030] The reason why the same set of test data can be simultaneously divided into three categories of risks that can be handled separately is that the physiological significance of the three parts in terms of enrichment is different. This is the technical principle and causal mechanism of this method. Edible muscle directly corresponds to the human intake route, and measuring the risk of consumption by food limits is the most appropriate. The liver and kidneys are the main accumulation sites of heavy metals in fish, and their concentration changes often occur earlier than those in muscle. By using organ concentration and organ enrichment coefficient, continuous exposure in aquaculture water can be identified earlier. The gills are in long-term contact with water, and combined with population statistics, they can better reflect the overall condition of the water area rather than the occasional occurrence of individual fish. After each of the three parts is assigned to an independent benchmark, the judgment of each dimension is no longer affected by the standards of other dimensions. The data that originally required manual comparison with standards and cross-dimensional comparisons is transformed into a unified standard with a clear purpose, thereby improving the efficiency of interpretation. Individual exceedances are prioritized to safeguard the bottom line of food safety and avoid the average effect of the comprehensive index from masking individual exceedances. Population proportions distinguish between regional pollution and individual occasional occurrences, reducing misjudgments and omissions.

[0031] Abnormal situations are handled according to preset criteria and prioritized accordingly. When the grade determination falls within a 10% threshold range of the adjacent grade, or the comprehensive food contamination index falls within a 0.1% threshold range of the adjacent grade, a critical review is triggered. The recovery condition is to repeat the measurement of the corresponding part of the sample, take the average, and then re-determine. When the required part of the sample for a certain dimension is missing, only the available dimension grade is output. The missing dimension is not graded due to insufficient data. The threshold is the minimum requirement for the completeness of the part data for that dimension. The recovery condition is to replenish the missing sample and then re-grade. When the population sample is less than five, the output of the regional contamination grade is stopped and the sample is marked as insufficient. The threshold is the minimum population size required for regional statistics. The recovery condition is to replenish the population to above the lower limit and then re-statistically analyze. The priority of the three is as follows: first determine the availability of data and whether the sample is sufficient, then review the critical grade, that is, first confirm whether grading is possible, then verify whether the grading is accurate; when any dimension reaches level three or higher, a return tag for quantitative arbitration by a qualified laboratory is attached to connect with subsequent laboratory quantification.

[0032] Example 2 like Figure 2 As shown, this embodiment, based on Embodiment 1, details the early warning of organ enrichment coefficients in the aquaculture dimension, and also provides a sample pretreatment and enrichment process with lower equipment requirements, as well as a dual-mode readout switching method. Sampling labeling, sub-item detection, and the three-dimensional judgment main chain are all the same as in Embodiment 1. The definition of target heavy metals, muscle, and organs, the independence of the three sets of benchmarks, and the algorithm for the comprehensive food contamination index are also used and will not be repeated here. The following only focuses on the newly added early warning judgment and optimized process.

[0033] In the process, pretreatment is performed first. A complex enzyme consisting of neutral protease and lipase is added to each sample (muscle, liver, kidney, gills), along with citric acid and a low concentration of hydrogen peroxide, and digestion is carried out under controlled temperature. For areas with less fat, digestion at 45-55 degrees Celsius for 30-45 minutes is sufficient; for areas with more fat, the lipase action time is extended and the temperature is increased to 55-65 degrees Celsius, with the total digestion time controlled between 30-90 minutes. However, the temperature is not allowed to rise to the point where mercury and arsenic will significantly volatilize. Digestion continues until the liquid is clear or nearly clear. A feasible initial formulation is: for every 1 gram of homogenate sample, add 10-50 mg of neutral protease, 5-30 mg of lipase, 5-20 ml of 1%-5% citric acid solution, and 0.5-3 ml of 0.5%-3% hydrogen peroxide solution. When there are significant differences in fat content among different fish species, adjustments are made within this range using blank and spiked samples. The digestion solution was then adjusted to a weakly acidic pH of 2 to 4 to allow cadmium, lead, mercury, and arsenic to be simultaneously chelated and enriched on the same thiol-functionalized material. The material used was thiol-modified cotton or thiol-modified diatomaceous earth. 50 to 300 mg of thiol-functionalized material was added to every 10 mL of digestion solution, and the mixture was shaken for 10 to 30 minutes. After enrichment, 1 to 5 mL of an acidic thiourea-hydrochloric acid eluent (0.05 to 0.5 mol / L thiourea and 0.1 to 1.0 mol / L hydrochloric acid) was used to elute the metals all at once. The volume ratio before and after elution was recorded as the enrichment factor. The concentration of each metal in the eluent was then measured, and the concentration was calculated back to the original fish sample concentration based on the enrichment factor and sample dilution. Electrochemical stripping voltammetry is the preferred method for reading absorbance. The responses of cadmium, lead, mercury, and arsenic are read sequentially according to the preset potential window of the target metal. When electrochemical equipment is unavailable, a colorimetric method is used to determine absorbance. During colorimetric analysis, the four metals are not combined into a single colorimetric result. Instead, equal portions of eluent are taken for cadmium, lead, mercury, and arsenic, and their respective colorimetric systems and necessary masking agents are added before separate colorimetric analysis. Blank correction and standard curve conversion are performed separately. Both reading methods convert concentrations using the same batch of blank samples, spiked samples, and standard curves. When the total mercury or total arsenic screening value reaches 90% or more of the corresponding food safety limit, a separate morphological confirmation test is conducted. This 90% threshold is the morphological confirmation trigger line for this method, which is stricter than the GB 2762 screening rule, and is used to detect potential morphological risks in advance within the limit value. The determination of aquaculture risk is based on this: the ratio of liver and kidney concentration to muscle concentration for each metal is calculated as the organ enrichment coefficient. When a metal in the muscle has not yet exceeded its limit, but its organ enrichment coefficient reaches 5 or above, or reaches the warning range formed by historical monitoring data of the same fish species, or the liver and kidney concentration reaches the aquaculture exposure indicator, the aquaculture risk is raised to the warning level, and an early warning result of continuous exposure in the aquaculture water is output. The input here is the concentration converted for each part, and the output is the aquaculture risk level and its early warning marker.In this embodiment, the representative boundary of the warning interval is set at an organ enrichment coefficient of 5 or higher. When the cadmium concentration in the muscle of the tested fish is 0.07, still within the limit of 0.1, while the cadmium concentration in the liver and kidney is 0.45, and the organ enrichment coefficient is approximately 6.4, which has entered the warning interval, an aquaculture risk warning is issued, indicating that there is continuous cadmium exposure in the water. The edibility dimension remains the same as in Example 1. The concentration of each metal in the muscle is compared with the limit to obtain the multiple of each exceeding the limit. Then, the square root of the sum of the square of the maximum value and the square of the average value is taken to obtain the comprehensive pollution index. The edibility level is determined according to the threshold zone it falls into.

[0034] The meanings and sources of variables involved in the new process are explained here: Digestion temperature refers to the control temperature during enzymatic hydrolysis and weak oxidation to release bound metals, and is taken within two ranges depending on the fat content of the part; the enrichment factor is the ratio of the volume before and after elution, obtained from the actual volume records of the enrichment and elution steps, and the concentration conversion method is the same as in Example 1; the organ enrichment coefficient is consistent with Example 1, still being the ratio of liver and kidney concentration to muscle concentration, derived from the detection concentration of these two parts. The amount of compound enzyme, the concentration of citric acid and hydrogen peroxide, the amount of thiol material, the volume of elution buffer, the potential window, and the colorimetric wavelength can be calibrated within the above range using blank samples, spiked samples, and standard curves; recalibration is required when changing fish species or substrate types. The criteria for successful calibration can be set as a spiked recovery rate of 70% to 130%, a blank value below one-third of the lowest standard point, and a correlation coefficient of the standard curve not less than 0.99; if these criteria are not met, pretreatment should be repeated or the dilution factor adjusted. The temperature ranges, enrichment factors, and organ enrichment coefficients listed in this embodiment are representative recorded values ​​illustrating process conditions and early warning judgments, and are not extrapolated to general statistical conclusions.

[0035] The technical principle and causal mechanism of this process lies in controlling the digestion temperature within the range where mercury and arsenic are not easily volatilized: some forms of mercury and arsenic are lost at high temperatures. Digestion can be completed at a lower temperature by first enzymatically decomposing the fat and protein matrix, and then releasing the bound metals through weak oxidation. This reduces the negative bias of mercury and arsenic, and consequently decreases the dependence on strong acids and equipment. After adjusting to a weakly acidic environment, thiol-based materials are used for enrichment. This relies on the chelating ability of thiol groups for soft or interfacial soft ions such as cadmium, lead, mercury, and arsenic. By combining the pretreatment of each component into a single adsorption-elution system, the relative concentration of low-content metals is increased, matrix interference is suppressed, and detection capability is correspondingly enhanced. Using organ enrichment coefficients as an early warning when muscle does not exceed the limit is crucial. Since the liver and kidneys are the main accumulation organs, their relative enrichment ratio to muscle often increases before muscle exceeds the limit. This increase in the ratio becomes an early signal of continuous exposure in the water, allowing warnings at the aquaculture end to appear before problems occur in edible parts.

[0036] Anomalies are also triggered and handled according to criteria and priorities. If the digestion solution is turbid, the reading baseline drifts, or the color development is abnormal, retesting is performed first. The threshold is derived from the allowable limits set by the reading device for baseline drift and absorbance anomalies. The recovery condition is to dilute and retest or supplement with enzyme digestion before rereading. If the enrichment factor is less than 5 times, or the spiked recovery rate is less than 70% or greater than 130%, it indicates that enrichment or matrix correction is inadequate. The recovery condition is to redo the enrichment and elution and then retest. If the organ enrichment coefficient is within 10% of the warning interval, the liver and kidney samples are repeatedly measured, the average is taken, and the judgment is recalculated. The threshold is derived from the boundary of the warning interval in the aquaculture dimension. The recovery condition is to re-determine the aquaculture level after the average of the retests stabilizes. In terms of priority, first check whether the enrichment and recovery rate are sufficient, then retest the readings, and finally verify the criticality of the warning interval to avoid hastily proceeding to subsequent judgments when enrichment is insufficient.

[0037] Example 3 like Figure 3 As shown, this embodiment follows embodiment 1, refining the population statistical discrimination at the regional pollution dimension, and running the entire anomaly backoff closed loop through a more complex sampling scenario. The sampling labeling, sub-item detection, and three-dimensional judgment main chain are the same as in embodiment 1. The method of collecting and numbering population samples and the independence of the three sets of benchmarks are also used. Repeated content will not be elaborated here; this section focuses on the refined implementation of population discrimination and anomaly backoff at the regional dimension.

[0038] The process begins with the composition of the population sample. Multiple fish of the same species are collected from the same aquaculture area to form a population sample, and each fish is individually numbered. Individual markers and batch markers for the aquaculture area are recorded. In this embodiment, the lower limit for the population sample size is set at 5 fish, aiming to cover individuals of different body lengths. When historical sampling data for this area shows significant fluctuations, the lower limit can be increased to 8 or 10 fish. Subsequently, each metal in the population is tested individually for each fish. The population average concentration, the proportion of individuals exceeding the regional background benchmark, and the detection rate are calculated. Simultaneously, the gill concentration of each fish is compared with the regional background benchmark to examine the extent of exposure of the contact organs beyond the background. During the determination, if the proportion of individuals exceeding the background for a certain metal reaches 30% or more, or reaches a higher threshold determined by historical sampling data from multiple batches in this area, it is considered regional pollution. The regional pollution level is determined based on the multiple of the population average concentration relative to the regional background benchmark combined with the detection rate level. If the proportion does not reach the threshold, it is treated as an occasional occurrence, and a regional pollution level one level lower than regional pollution is output. In this embodiment, the representative value of the proportion threshold is set at 30%: when 40% of the individuals in the population with cadmium background exceedance and the detection rate remains at a high level, it is considered regional pollution; if they only account for 10%, it is considered an isolated individual pollution. Based on this, an abnormal fallback closed loop is then run. When the judgment quantity falls within the critical range of 10% above and below the adjacent level threshold, or the comprehensive pollution index falls within the critical range of 0.1 above and below the adjacent level threshold, the corresponding part of the test sample is repeatedly measured, the average value is taken, and the dimension is re-judged. In this embodiment, the cases where the comprehensive pollution index of food is near the level threshold and the population exceeding the background ratio is near the threshold are included in the review. When the test sample of the required part of a certain dimension is missing, only the available dimension is output. The missing dimension is not graded due to insufficient labeled data. For example, when gills are not collected and the population is insufficient, the regional pollution dimension is labeled as insufficient data and is not graded. The food and aquaculture dimensions are output as usual. When the population tail is less than the lower limit, the insufficient sample mark is output, the formal regional pollution level is stopped, and only the population statistical overview is given in reference form. When the risk of any dimension reaches level three or higher, a return mark for quantitative arbitration by a qualified laboratory is attached.

[0039] The meanings and sources of variables for each quantity in the regional dimension are explained as follows: The population average concentration is the arithmetic mean of the tail-by-tail concentrations of a certain metal in the population, derived from the tail-by-tail detection results, covering all tested individuals within the batch in this water area; the proportion of individuals exceeding the regional background benchmark is the share of the total population with concentrations higher than the regional background benchmark, obtained by comparing the tail-by-tail concentration with the regional background benchmark one by one; the detection rate is the proportion of individuals with concentrations higher than the method detection limit, derived from comparing the tail-by-tail detection results with the method detection limit. In this embodiment, it serves as an auxiliary verification quantity for the regional pollution level, used to determine whether population anomalies have a persistent population detection characteristic. The value of the regional background benchmark is defined in Example 1. The proportion threshold is set to 30% by default, and the lower limit of the population sample size is set to no less than 5 tails by default. If there are multiple batches of population sampling records in this water area, the regional background benchmark can be corrected using the upper quantile or mean of the normal fluctuation range in the historical records plus twice the standard deviation, and the proportion threshold should be increased according to the historical false positive control requirements. The individual proportions and thresholds listed in this embodiment are representative recorded values ​​illustrating the regional discrimination criteria. They only reflect the recording method of this water area and are not extrapolated to general statistical conclusions.

[0040] The key to this dimension's technical principle and causal mechanism lies in basing regional judgment on population statistics rather than individual fish. The heavy metal concentration in a single fish is influenced by feeding, body length, and metabolism, resulting in significant fluctuations. An excessive concentration in one fish could reflect a problem in the water area or be purely accidental; a single fish's presence alone is insufficient to draw regional conclusions. By introducing the proportion and detection rate of individuals exceeding the background threshold, the judgment criteria expand from a single point to the distribution of the entire population. Only when a large number of individuals exceed the threshold is regional pollution identified, thus distinguishing systemic pollution at the water level from sporadic, occasional exceedances by individual fish, reducing the probability of misjudging a single fish anomaly as regional pollution. Gill concentration, combined with population statistics, and given the long-term contact between gills and water, its exceeding of the background threshold can indirectly confirm water exposure, making regional judgments more robust.

[0041] Anomalies are still handled according to criteria and priorities. When a required sample for a certain dimension is missing, only the available dimension is output with insufficient data. The threshold is the minimum requirement for the completeness of the sample data for each dimension. The recovery condition is to replenish the missing sample and then reclassify. When the population size is below the lower limit of 5, the output of the formal regional pollution level is stopped and the sample size is insufficient. The threshold is the minimum population size required for regional statistics. The recovery condition is to replenish the population to above the lower limit and then re-statistically analyze. When the judgment quantity falls within the critical range of 10% above and below the threshold of the adjacent level, a critical review is initiated. The threshold is the boundary of the threshold band for the level division of that dimension. The recovery condition is to repeat the measurement, take the average, and then re-judge. In terms of priority, first confirm whether the sample data is complete and whether the population size is sufficient to support the classification, and then review the critical level. When any dimension reaches level three or higher, the laboratory quantitative arbitration is connected according to the aforementioned feedback marking.

[0042] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be modified within the scope of the concept described herein by means of the above teachings or the technology or knowledge in related fields.

Claims

1. A method for risk classification and detection of heavy metal pollution in fish, characterized in that, Includes the following steps: Fish samples were collected from the same aquaculture area. For each individual fish, muscle tissue, enrichment indicator organs, and exposed contact organs were extracted and prepared as test samples. Simultaneously, a population sample of the same species, consisting of at least five fish, was collected from the same aquaculture area, meeting the minimum population sample size requirement. Site markers, individual markers, and batch / area markers were established for each test sample. The concentrations of at least cadmium, lead, mercury, and arsenic were measured in each test sample to obtain heavy metal concentration data for each site, individual fish, and corresponding batch / area. The muscle tissue, enrichment indicator organs, exposed contact organs, and population / area samples within the same batch / area maintained the same correspondence for heavy metals. The heavy metal concentrations in the muscle tissue were compared with food safety limits to form a comprehensive contamination assessment metric and determine the food safety risk level. The concentration of heavy metals in enrichment indicator organs is compared with the aquaculture exposure indicator benchmark, and the ratio of the concentration in enrichment indicator organs to the concentration in muscle tissue is used as the aquaculture exposure criterion to determine the aquaculture risk level. The concentration of heavy metals in the exposed organs, along with the population average concentration, detection rate, and proportion of individuals exceeding the regional background benchmark in the population sample, are compared with the regional background benchmark. Whether the proportion of individuals exceeding the regional background benchmark reaches the regional pollution proportion threshold is used as the distinguishing condition between regional pollution and occasional individual occurrences, forming a regional pollution criterion and determining the regional pollution risk level. The food safety limit benchmarks, aquaculture exposure indication benchmarks, and regional background benchmarks are each used independently for determining the level of the corresponding risk dimension; The system outputs the risk levels for food consumption, aquaculture, and regional pollution, and links the baseline category, judgment quantity, excessive metals, corresponding location, water area batch, and graded treatment recommendations for each risk level.

2. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, The enrichment indicator organs include the liver and kidneys, the exposed contact organs include the gills, and the muscle portion is edible muscle of the fish.

3. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, In the process of obtaining the food safety risk level, the ratio of the concentration of each heavy metal in the muscle part to the corresponding food safety limit is used as the single exceedance multiple of that heavy metal. The comprehensive food safety pollution index is obtained based on the maximum and average values ​​of the single exceedance multiples. The food safety risk level is divided into at least four levels that increase sequentially according to the comprehensive food safety pollution index. When the concentration of any heavy metal in the muscle part exceeds the corresponding food safety limit, the food safety risk level is raised to a level no lower than the third level. Mercury and arsenic used for food safety limit comparison adopt the mercury form and arsenic form specified in the food safety limit benchmark, respectively. When the total mercury or total arsenic screening value obtained by rapid detection reaches more than 90% of the corresponding food safety limit, the confirmatory test result of the corresponding form is used as the basis for determining the food safety risk level.

4. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, In the step of obtaining the aquaculture risk level, the ratio of the concentration of the enrichment indicator organ to the concentration of the muscle tissue is used as the organ enrichment coefficient. When the heavy metal concentration in the muscle tissue does not exceed the food safety limit, and the organ enrichment coefficient reaches 5 or above or falls into the warning range determined by historical monitoring data of the same species of fish, the aquaculture risk level is raised to the warning level, thus obtaining an early warning result of continuous exposure to the aquaculture water.

5. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, In the step of obtaining the regional pollution risk level, the population average concentration of heavy metals, the proportion of individuals exceeding the regional background benchmark, and the detection rate are calculated for the population sample. The proportion of individuals exceeding the regional background benchmark reaching 30% or more, or reaching the proportion threshold determined by multiple batches of historical sampling data of this water area, is used as the condition for determining regional pollution. The multiple of the population average concentration relative to the regional background benchmark and the detection rate are used as auxiliary verification quantities for the regional pollution risk level. When the proportion of individuals exceeding the regional background benchmark does not reach the proportion threshold, it is determined to be an isolated incident, and the regional pollution risk level is output as lower than the corresponding level of regional pollution.

6. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, When the determination of any risk level falls within the critical range of 10% above or below the threshold of the adjacent level, or when the determination of comprehensive food contamination falls within the critical range of 0.1% above or below the threshold of the adjacent level, the corresponding part of the sample to be tested is measured repeatedly and the average value of the test results is taken to re-determine the risk level.

7. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, When the required sample for a certain risk level is missing, only the available risk level is output, and the risk dimension corresponding to the missing part is marked as insufficient data and not graded.

8. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, When the number of fish in the group sample is less than five or below the higher sample lower limit determined by the historical fluctuations of the water area, an insufficient sample marker is output, and the output of the regional pollution risk level is stopped.

9. The method for risk classification and detection of heavy metal pollution in fish according to claim 1, characterized in that, In the process of determining the heavy metal concentration of samples from various parts, a complex enzyme consisting of neutral protease and lipase, citric acid, and hydrogen peroxide are first added to the sample. The sample is then digested at a controlled temperature of 45 to 65 degrees Celsius for 30 to 90 minutes to obtain a digestion solution. The digestion solution is then adjusted to pH 2 to 4 and the heavy metals are simultaneously chelated and enriched using thiol cotton or thiol-modified diatomaceous earth. Subsequently, the enriched heavy metals are uniformly eluted with acidic thiourea hydrochloric acid elution solution before the heavy metal concentration is determined.

10. The method for risk classification and detection of heavy metal pollution in fish according to claim 9, characterized in that, The concentration of the eluted heavy metals was determined by electrochemical stripping voltammetry. Under conditions where the electrochemical measurement equipment was unavailable, cadmium, lead, mercury and arsenic were respectively measured by colorimetric determination using corresponding colorimetric systems. The colorimetric determination included separate colorimetric determination of the metals to be tested, blank correction and standard curve conversion.

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