A method for assessing stress levels in shellfish wasting syndrome

Abstract

This invention discloses a method for assessing the stress level of shellfish wasting syndrome, belonging to the field of aquaculture technology. The method utilizes respiration rate as an indicator of the stress level of shellfish wasting syndrome. By comparing the respiration rate of shellfish under natural conditions in aquaculture areas with that under normal feeding conditions, the stress level of shellfish wasting syndrome can be predicted, facilitating farmers to take preventative measures against shellfish wasting syndrome. This invention uses the shellfish's own physiological indicators as indicators, avoiding the low accuracy, difficulty, and inability to directly reflect the nutritional stress level of shellfish in marine nutrient monitoring. It fills the gap in the accurate evaluation of wasting syndrome in filter-feeding shellfish and provides important technical support for healthy shellfish aquaculture.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a method for assessing the stress level of shellfish wasting syndrome. Background Technology

[0002] Marine shellfish are a high-quality source of "blue protein," yet frequent large-scale mortality events seriously threaten the healthy development of the industry. The biological characteristics of open-water aquaculture and filter-feeding make shellfish highly susceptible to nutritional stress caused by insufficient food in the sea. In recent years, nutritional conditions in major near-shore shellfish farming areas have faced immense pressure, with farmed shellfish generally in a sub-healthy state of "emaciation syndrome." Emaciation syndrome in shellfish is a systemic disease caused by insufficient nutrient intake, resulting in emaciation, reduced metabolic and immune function, dysbiosis of the symbiotic flora, and decreased ability to resist external stimuli. Reports indicate that "during high summer temperatures, emaciation syndrome caused by insufficient food is a significant cause of large-scale mortality in farmed shellfish." Assessing the stress level of emaciation syndrome in shellfish can facilitate the prediction of the risk of developing the syndrome, enabling farmers to take preventative measures in advance. This aligns with the current basic principle of "prevention first, prevention is more important than treatment" in shellfish disease control and can also provide important reference for the formulation of aquaculture strategies such as translocation farming.

[0003] However, since shellfish diets include not only unicellular algae but also organic particles and other substances, accurate quantification is difficult, making it impossible to truly reflect the nutritional status of the sea area, let alone directly reflect the nutritional stress level of shellfish. Therefore, there is an urgent need to find a method that can accurately reflect the nutritional stress level of shellfish. Summary of the Invention

[0004] The purpose of this invention is to provide a method for assessing the stress level of shellfish wasting syndrome, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of the present invention is the application of the respiratory rate value of shellfish in assessing the stress level of shellfish wasting syndrome.

[0007] The second technical solution of the present invention: a method for assessing the stress level of shellfish wasting syndrome, comprising the following steps:

[0008] Obtain the respiration rate (A) of shellfish samples in the aquaculture area;

[0009] Obtain the respiration rate (B) of shellfish samples after normal feeding;

[0010] A t-test was performed on the respiratory rates A and B, and the p-value was recorded. The stress level of shellfish wasting syndrome was assessed based on the p-value.

[0011] Furthermore, when p≥0.05, there is no coercion, and when p<0.05, there is coercion.

[0012] Normal feeding refers to feeding 6-8 times a day, maintaining the density of algae feed in the culture seawater at 2×10⁻⁶. 5 ~4×10 5 cell / mL.

[0013] The third technical solution of the present invention: a method for assessing the stress level of shellfish wasting syndrome, comprising the following steps:

[0014] Obtain the respiration rate A of shellfish in the aquaculture area;

[0015] Obtain the respiration rate B of shellfish after normal feeding;

[0016] Perform a t-test on the respiratory rates A and B, and record the p-values;

[0017] Calculate the mean respiratory rate B and the mean respiratory rate A, and the fold difference FC between the two means;

[0018] Assessment of stress levels in shellfish wasting syndrome based on p-value and FC;

[0019] Wherein, FC = mean respiratory rate B / mean respiratory rate A.

[0020] Furthermore, p≥0.05 indicates no coercion;

[0021] p < 0.05 and FC < 1.4, primary stress;

[0022] p < 0.05 and 1.4 ≤ FC < 1.8, moderate stress;

[0023] p<0.05 and FC≥1.8 indicate severe stress.

[0024] Furthermore, the number of shellfish samples is no less than 30; after obtaining the respiration rate A, the shellfish samples are fed normally in an indoor aquarium temporary holding system, and then the respiration rate B of the shellfish after normal feeding is obtained. The water temperature and salinity of the aquarium system are consistent with those of the aquaculture area.

[0025] Furthermore, the normal feeding period is 10 days (during which the respiration rate is relatively stable), and the feed is unicellular algae, with the density of unicellular algae in the culture seawater maintained at 2 × 10⁻⁶. 5 ~4×10 5 cell / mL.

[0026] Furthermore, the unicellular algae include one or more of the following: *Rhizophora microcresticata*, *Chaetoceros*, and *Phaeodactylum tricornutum*.

[0027] Furthermore, the method for obtaining the respiratory rate A and respiratory rate B includes:

[0028] Shellfish samples were placed in sealed test bottles containing seawater for testing. Dissolved oxygen data in the sealed test bottles were measured every 3 to 10 seconds using a fiber optic oxygen analyzer. The measurements were taken continuously for 2 to 2.5 hours. The slope of all dissolved oxygen values ​​was calculated to obtain the respiration rate of the shellfish samples.

[0029] The water temperature and salinity of the seawater were measured to be consistent with those of the aquaculture area.

[0030] Furthermore, when calculating the slope of all dissolved oxygen values, non-changing dissolved oxygen values ​​of shellfish that do not breathe with their mouths closed are removed, as are outliers above the upper quartile and below the lower quartile, to obtain the respiration rate of the shellfish samples.

[0031] Furthermore, before obtaining respiration rate A and respiration rate B, the process also includes cleaning the shellfish samples, removing surface deposits, and ensuring that the shellfish samples are out of water for no more than 2 hours.

[0032] Furthermore, a fiber optic oxygen analyzer (model: OXY-10ST, brand and place of origin: PreSens, Regensburg, Germany) was used to obtain the respiratory rate.

[0033] Furthermore, the device used to obtain the respiration rate consists of a circulating water system, a sealed measurement bottle, and a fiber optic oxygen analyzer. The sealed measurement bottle contains shellfish samples and seawater (from the aquaculture area), is vented and sealed, and has a magnetic rotor at the bottom rotating at 200 rpm to ensure uniform dissolved oxygen distribution within the bottle. The sealed measurement bottle is placed in the circulating water system, with the temperature set to match that of the aquaculture area. The dissolved oxygen probe of the fiber optic oxygen analyzer is inserted into the sealed measurement bottle, recording the dissolved oxygen data every 3 seconds for 2–2.5 hours.

[0034] The fourth technical solution of the present invention: a system for assessing the stress level of shellfish wasting syndrome, comprising a shellfish respiratory rate data acquisition module and a shellfish wasting syndrome stress level assessment module;

[0035] The shellfish respiration rate data acquisition module is used to collect the respiration rate A of shellfish samples in the aquaculture area and the respiration rate B of shellfish samples after normal feeding.

[0036] The shellfish wasting syndrome stress level assessment module is used to assess the shellfish wasting syndrome stress level based on p-value and FC;

[0037] The p-value is obtained by performing a t-test on the respiratory rates A and B, and FC is the multiple of the difference between the mean respiratory rate B and the mean respiratory rate A.

[0038] The present invention discloses the following technical effects:

[0039] The method of the present invention for assessing the stress level of shellfish wasting syndrome uses the respiratory rate as an indicator of the stress level of shellfish wasting syndrome. By comparing the respiratory rate of shellfish under natural conditions in aquaculture areas with the respiratory rate of shellfish under normal feeding conditions, the stress level of shellfish wasting syndrome can be estimated, which helps aquaculture farmers to take measures in advance to prevent and control shellfish wasting syndrome.

[0040] This invention uses the physiological indicators of shellfish themselves as indicators, avoiding the problems of low accuracy, high difficulty and inability to directly reflect the nutritional stress level of shellfish in marine nutrition monitoring. It fills the gap in the accurate evaluation of filter-feeding shellfish wasting syndrome and provides important technical support for the healthy aquaculture of shellfish. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 A system for assessing stress levels in shellfish wasting syndrome;

[0043] Figure 2 Respiration rate of oysters subjected to starvation stress;

[0044] Figure 3 The mortality rate of heat-stressed oysters after starvation stress. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] In a first aspect, the present invention provides the application of respiratory rate values ​​of shellfish in assessing the stress level of shellfish wasting syndrome.

[0051] In a second aspect, the present invention provides a method for assessing the stress level of shellfish wasting syndrome, comprising the following steps:

[0052] Obtain the respiration rate (A) of shellfish samples in the aquaculture area;

[0053] Obtain the respiration rate (B) of shellfish samples after normal feeding;

[0054] A t-test was performed on the respiratory rates A and B, and the p-value was recorded. The stress level of shellfish wasting syndrome was assessed based on the p-value.

[0055] Furthermore, when p≥0.05, there is no coercion, and when p<0.05, there is coercion.

[0056] In a third aspect, the present invention provides a method for assessing the stress level of shellfish wasting syndrome, comprising the following steps:

[0057] Obtain the respiration rate A of shellfish in the aquaculture area;

[0058] Obtain the respiration rate B of shellfish after normal feeding;

[0059] Perform a t-test on the respiratory rates A and B, and record the p-values;

[0060] Calculate the mean respiratory rate B and the mean respiratory rate A, and the fold difference FC between the two means;

[0061] Assessment of stress levels in shellfish wasting syndrome based on p-value and FC;

[0062] Wherein, FC = mean respiratory rate B / mean respiratory rate A.

[0063] Furthermore, p≥0.05 indicates no coercion;

[0064] p < 0.05 and FC < 1.4, primary stress;

[0065] p < 0.05 and 1.4 ≤ FC < 1.8, moderate stress;

[0066] p<0.05 and FC≥1.8 indicate severe stress.

[0067] In a specific embodiment of the present invention, the sample quantity of shellfish is not less than 30 pieces;

[0068] After obtaining the respiration rate A, the shellfish samples were fed normally in an indoor aquarium temporary holding system, and then the respiration rate B of the shellfish after normal feeding was obtained. The water temperature and salinity of the aquarium system were kept consistent with those of the aquaculture area.

[0069] The normal feeding period is 10 days, and the feed consists of single-celled algae, with a density of not less than 2 × 10⁻⁶. 5 ~4×10 5 cell / mL; unicellular algae include one or more of the following: small crescent-shaped algae, chaete algae, and triangular brown finger algae.

[0070] In a specific embodiment of the present invention, the method for obtaining respiratory rate A and respiratory rate B includes:

[0071] Shellfish samples were placed in sealed test bottles containing seawater for measurement. Dissolved oxygen data in the sealed test bottles were measured every 3 to 10 seconds using a fiber optic oxygen analyzer. The measurements were taken continuously for 2 to 2.5 hours. The slope of all dissolved oxygen values ​​was calculated to obtain the respiration rate of the shellfish samples. The water temperature and salinity of the seawater were kept consistent with those of the aquaculture area.

[0072] In a specific embodiment of the present invention, when calculating the slope of all dissolved oxygen values, non-changing dissolved oxygen values ​​of shellfish that do not breathe with their mouths closed are removed, and outliers above the upper quartile and below the lower quartile are removed to obtain the respiration rate of the shellfish samples.

[0073] In a specific embodiment of the present invention, before obtaining respiration rate A and respiration rate B, the process further includes cleaning the shellfish sample to remove surface deposits, and the shellfish sample is out of water for no more than 2 hours.

[0074] In a specific embodiment of the present invention, a fiber optic oxygen analyzer (model: OXY-10ST, brand and place of origin: PreSens, Regensburg, Germany) is used to obtain the respiratory rate.

[0075] In a specific embodiment of the present invention, the device for obtaining the respiration rate consists of a circulating water system, a sealed measuring bottle, and a fiber optic oxygen analyzer. The sealed measuring bottle contains shellfish samples and seawater (from the aquaculture area), is sealed with an vent, and has a magnetic rotor at the bottom rotating at 200 r / min to ensure uniform dissolved oxygen distribution within the bottle. The sealed measuring bottle is placed in the circulating water system, with the temperature set to match that of the aquaculture area. The dissolved oxygen probe of the fiber optic oxygen analyzer is inserted into the sealed measuring bottle, recording dissolved oxygen data every 3 seconds for 2–2.5 hours.

[0076] In a fourth aspect, the present invention provides a system for assessing the stress level of shellfish wasting syndrome, comprising a shellfish respiratory rate data acquisition module and a shellfish wasting syndrome stress level assessment module;

[0077] The shellfish respiration rate data acquisition module is used to collect the respiration rate A of shellfish samples in the aquaculture area and the respiration rate B of shellfish samples after normal feeding.

[0078] The Shellfish Emaciation Syndrome Stress Level Assessment Module is used to assess the stress level of shellfish emaciation syndrome based on p-value and FC.

[0079] The p-value is obtained by performing a t-test on the respiratory rates A and B, and FC is the multiple of the difference between the mean respiratory rate B and the mean respiratory rate A.

[0080] Systematic assessment of stress levels in shellfish wasting syndrome is described in [link to relevant documentation]. Figure 1 .

[0081] Methods for defining FC values:

[0082] 700 oysters were placed in an indoor aquarium holding system, with water temperature, salinity, pH, dissolved oxygen, ammonia nitrogen, and nitrite levels maintained at the same levels as in the aquaculture area. They were held in the aquarium for 10 days, fed with *Nyctaginosa* (small crescent-shaped algae) six times daily, maintaining a food density of 2 × 10⁶. 5 ~4×10 5 cell / mL. On day 10, 30 oysters were taken, their surface organisms were cleaned, and their respiration rate was measured and recorded as respiration rate B. After the respiration rate measurement, the oysters were returned to the aquarium system, and the oysters were marked and recorded.

[0083] Then, all oysters were subjected to a 44-day starvation treatment (without feeding). On days 0 (i.e., day 10 of the feeding treatment), 3, 5, 7, 10, 13, 16, 20, 23, 30, 34, 37, and 44, the respiration rate of 30 marked oysters was measured and recorded as A0, A3, A5, A7, ..., A44. When oysters died, 30 live replacement oysters (replacement oysters that had undergone simultaneous starvation treatment) were added for subsequent respiration rate measurements and marked. After removing oysters with unchanged dissolved oxygen values ​​(those not respiring) and outliers, the respiration rate results are shown below. Figure 2 Respiratory rate A0 (respiratory rate B) is the respiratory rate under non-starvation stress, with a mean respiratory rate of 3.5333 × 10⁻⁶. -4 The respiratory rate was measured in mg / L·g·3s; A3, A5, A7, and A10 were the respiratory rates at the initial stage of starvation stress, with an average respiratory rate of 2.6298 mg / L·g·3s; A13, A16, and A20 were the respiratory rates at the middle stage of starvation stress, with an average respiratory rate of 2.2466 mg / L·g·3s; A23, A30, A34, A37, and A44 were the respiratory rates at the later stage of starvation stress, with an average respiratory rate of 1.8865 mg / L·g·3s. The respiratory rate under non-starvation stress was significantly different from that at the latter three stages (p<0.05) according to the t-test. The fold differences (FC) between respiratory rate B and respiratory rates A3, A5, A7, and A10 were 1.27, 1.37, 1.34, and 1.39, respectively; the fold differences (FC) between respiratory rate B and respiratory rates A13, A16, and A20 were 1.58, 1.61, and 1.73, respectively; and the fold differences (FC) between respiratory rate B and respiratory rates A23, A30, A34, A37, and A44 were 1.82, 1.85, 1.81, 1.91, and 1.99, respectively.

[0084] At days 0, 3, 5, 7, 10, 13, 16, 20, 23, 30, 34, 37, and 44 of the starvation treatment, 30 oysters were collected at each time point and subjected to heat shock in seawater at 42°C for 1 hour. They were then exposed to air until room temperature and placed in separate tanks within the original aquarium holding system. Mortality was recorded over the following 15 days. Oysters used for heat shock were not included in subsequent starvation experiments or used for respiration rate measurements. Oyster mortality after heat shock is shown in [link to relevant documentation]. Figure 3 The mortality rate in the early stage of hunger stress was 5.11%–23.09%, with an average of 15.75%; the mortality rate in the middle stage of hunger stress was 39.18%–51.27%, with an average of 44.67%; and the mortality rate in the late stage of hunger stress was 59.13%–87.17%, with an average of 75.8%.

[0085] Based on the three typical stages of respiratory rate changes during starvation stress and the corresponding differences in heat shock mortality, and combined with the threshold selection principle of "tightening rather than loosening", we define primary stress as FC < 1.4, intermediate stress as 1.4 ≤ FC < 1.8, and severe stress as FC ≥ 1.8.

[0086] Example 1

[0087] A method for assessing stress levels in shellfish wasting syndrome:

[0088] (1) In November 2022, 30 diploid long oysters cultured in the Xiaoqingdao sea area of ​​Rushan were taken. The surface of the oysters was cleaned and the attached substances were removed (the time of the oysters out of the water was no more than 2 hours). The respiration rate A of the shellfish samples was obtained by using a fiber optic oxygen meter (model: OXY-10ST, brand and place of origin: PreSens, Regensburg, Germany).

[0089] The apparatus used to obtain the respiration rate consisted of a circulating water system, a sealed measurement bottle, and a fiber optic oxygen analyzer. The sealed measurement bottle contained the shellfish to be tested and seawater (from the aquaculture area), was vented and sealed, and had a magnetic rotor at the bottom rotating at 200 rpm to ensure uniform dissolved oxygen distribution within the bottle. The sealed measurement bottle was placed in the circulating water system, with the temperature set to match that of the aquaculture area (seawater temperature 16.1℃, salinity 29‰). The dissolved oxygen probe of the fiber optic oxygen analyzer was inserted into the sealed measurement bottle, and dissolved oxygen data was recorded every 3 seconds for 2 hours, recording the respiration rate A.

[0090] (2) After the respiration rate A measurement was completed, 30 oysters were placed in an aquarium temporary holding system, maintaining a water temperature and salinity of 16.1℃ and a salinity of 29‰. They were fed with *Nyctaginosa* to maintain a *Nyctaginosa* density of 2×10⁻⁶ in the culture seawater. 5 ~4×10 5 After feeding continuously for 10 days, the oysters to be tested were removed, the surface of the oysters was cleaned, and the attached substances were removed (the time of oysters out of water should not exceed 2 hours). The respiration rate was measured using a fiber optic oxygen analyzer (model: OXY-10ST, brand and place of origin: PreSens, Regensburg, Germany).

[0091] The apparatus used to obtain the respiration rate consisted of a circulating water system, a sealed measurement bottle, and a fiber optic oxygen analyzer. The sealed measurement bottle contained the shellfish to be tested and seawater (from the aquaculture area), was vented and sealed, and had a magnetic rotor at the bottom rotating at 200 rpm to ensure uniform dissolved oxygen distribution within the bottle. The sealed measurement bottle was placed in the circulating water system, with the temperature set to match that of the aquaculture area (seawater temperature 16.1℃, salinity 29‰). The dissolved oxygen probe of the fiber optic oxygen analyzer was inserted into the sealed measurement bottle, and dissolved oxygen data was recorded every 3 seconds for 2 hours, recording the respiration rate B.

[0092] Table 1. Results of respiratory rate measurement

[0093]

[0094]

[0095] (3) Remove the non-changing dissolved oxygen values ​​of shellfish that do not breathe with their mouths closed, and remove outliers (the outlier is calculated as: Cmax=Q3+k(Q3-Q1), Cmin=Q1-k(Q3-Q1);

[0096] Where Cmax is the estimated maximum value, Cmin is the estimated minimum value, Q3 is the upper quartile, Q1 is the lower quartile, and k is the outlier parameter, where k = 1.5.

[0097] Values ​​higher than Cmax or lower than Cmin are outliers;

[0098] The respiratory rate A-values ​​were: 0.0009, 0.0006, 1.3029, and 1.4296.

[0099] The respiratory rate B values ​​after data removal were: 0.0008, 3.0256, and 3.9882. A t-test was performed on the respiratory rates A and B after data removal, and the result was p < 0.05.

[0100] Mean respiratory rate B / Mean respiratory rate A = 1.61;

[0101] That is, p < 0.05 and 1.4 ≤ FC < 1.8, the oyster under test is at a moderate stress level.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for assessing the stress level of shellfish wasting syndrome, characterized in that, Includes the following steps: Obtain the respiration rate (A) of shellfish samples in the aquaculture area; Obtain the respiration rate (B) of shellfish samples after normal feeding; A t-test was performed on the respiratory rates A and B, and the p-values ​​were recorded. The stress level of shellfish wasting syndrome was assessed based on the p-values. When p≥0.05, there is no coercion; when p<0.05, there is coercion.

2. The method for assessing the stress level of shellfish wasting syndrome according to claim 1, characterized in that, The number of shellfish samples shall not be less than 30; and / or, After obtaining the respiration rate A, the shellfish samples were fed normally in an indoor aquarium holding system, and then the respiration rate B of the shellfish after normal feeding was obtained. The water temperature, salinity, pH, dissolved oxygen content, ammonia nitrogen content, and nitrite content of the aquarium holding system were kept consistent with those of the aquaculture sea area.

3. The method for assessing the stress level of shellfish wasting syndrome according to claim 2, characterized in that, The normal feeding period is 10 days, and the feed is unicellular algae. The density of unicellular algae in the culture seawater is maintained at 2 × 10⁻⁶. 5 ~4×10 5 cell / mL.

4. The method for assessing the stress level of shellfish wasting syndrome according to claim 1, characterized in that, The methods for obtaining respiratory rate A and respiratory rate B include: Shellfish samples were placed in sealed test bottles containing seawater for testing. Dissolved oxygen data in the sealed test bottles were measured every 3 to 10 seconds using a fiber optic oxygen analyzer. The measurements were taken continuously for 2 to 2.5 hours. The slope of all dissolved oxygen values ​​was calculated to obtain the respiration rate of the shellfish samples. The water temperature and salinity of the seawater were measured to be consistent with those of the aquaculture area.

5. The method for assessing the stress level of shellfish wasting syndrome according to claim 4, characterized in that, When calculating the slope of all dissolved oxygen values, non-changing dissolved oxygen values ​​of shellfish that do not breathe with their mouths closed are excluded, as are outliers. The outlier is calculated as follows: Cmax = Q3 + k(Q3 - Q1), Cmin = Q1 - k(Q3 - Q1); Where Cmax is the estimated maximum value, Cmin is the estimated minimum value, Q3 is the upper quartile, Q1 is the lower quartile, and k is the outlier parameter, where k=1.5; Values ​​higher than Cmax or lower than Cmin are outliers.

6. A system for assessing the stress level of shellfish wasting syndrome, used in the method of any one of claims 1-5, characterized in that, Includes a shellfish respiratory rate data acquisition module and a shellfish wasting syndrome stress level assessment module; The shellfish respiration rate data acquisition module is used to collect the respiration rate A of shellfish samples in the aquaculture area and the respiration rate B of shellfish samples after normal feeding. The shellfish wasting syndrome stress level assessment module is used to assess the shellfish wasting syndrome stress level based on the p-value; The p-value is obtained by performing a t-test on the respiratory rates A and B.

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