A method for quantitatively evaluating the offshore ecological risk of modified clay

Abstract

The present invention discloses a method for quantitatively assessing the offshore ecological risk of modified clay. The method comprises culturing three test organisms; conducting exposure experiments based on the field use concentration of the modified clay; detecting biomarkers of each species; calculating the IBR value of each species using an improved integrated biomarker response (IBR) evaluation method; summing the IBR values ​​to obtain a total IBR value; statistically analyzing the total IBR value to obtain a significance P value; and using the total IBR value and the P value to jointly characterize the ecological risk level. Based on a multi-species biomarker indicator system and by improving the value assignment and calculation methods of the IBR evaluation method, the present invention establishes a quantitative assessment method for offshore ecological risk of modified clay.

Description

Technical Field

[0001] The invention relates to the technical field of ecological risk assessment, in particular to a method for quantitatively assessing offshore ecological risks of modified clay. Background Art

[0002] Modified clay technology is a widely accepted and widely used guiding method for harmful algal bloom control internationally and is a national standard method for harmful algal bloom control in my country (GB / T 30743-2014). Modified clay is made from natural kaolin or montmorillonite, and after inorganic or organic modification, it removes and controls harmful algal blooms through flocculation and sedimentation. Modified clay is derived from natural materials, not synthetic chemicals, and is theoretically green, non-toxic, and environmentally friendly. However, the potential ecological risks posed by any artificial addition to natural waters cannot be ignored. Currently, offshore ecological risk assessments of modified clay remain limited to assessing its lethal effects and physiological impacts on individual marine organisms, with no substantive evidence demonstrating its ecological risks.

[0003] Based on technical standards for ecological risk assessment and water quality benchmark derivation developed by environmental protection agencies at home and abroad, currently commonly used ecological risk assessment methods include the Assessment Factor (AF) method, the Risk Quotient (RQ) method, the Species Sensitivity Stepwise (SSD) method, and the Probabilistic Risk Assessment (PERA) method. These methods primarily assess pollutants in the ecological environment, and the acquisition of data for quantitative assessment parameters relies on the lethal effect of the pollutants on the test organisms or a specific endpoint. However, modified clay may not have a lethal effect on the test organisms and will not produce other biological effects in the short term. Therefore, traditional assessment methods are not suitable for the offshore ecological risk assessment of modified clay.

[0004] With the deepening of ecotoxicological research, biomarkers have become the main entry point for studying the biological effects of environmental exogenous substances. Researchers qualitatively evaluate the impact of a substance on different organisms by detecting changes in biomarkers after exposure. On this basis, the Integrated Biomarker Responses (IBR) evaluation method assigns values ​​to the changes in the biomarkers of a species under exposure to exogenous substances, characterizing the survival pressure of this species under a range of exposure concentrations of the substance. Although IBR integrates and assigns values ​​to biomarker data, it is still a qualitative evaluation method and cannot quantitatively characterize ecological risks. At the same time, IBR is currently only applied to the integration of biomarkers of a single species and the evaluation of biological effects. The evaluation effect is relatively one-sided and cannot scientifically reflect ecological risks. Summary of the Invention

[0005] In order to overcome the above problems existing in the prior art, the present invention proposes a method for quantitatively evaluating the offshore ecological risk of modified clay.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for quantitatively evaluating the offshore ecological risk of modified clay, comprising the following steps:

[0007] Step 1, culturing the test organism;

[0008] Step 2: setting the exposure concentration according to the field use dose of the modified clay, conducting an exposure experiment, and obtaining an exposure group and a control group;

[0009] Step 3, performing biomarker detection based on the test organism selected in step 1;

[0010] Step 4, the IBR values ​​of each species in the control group and each exposure group were calculated using the improved integrated biomarker response evaluation method;

[0011] Step 5, sum the IBR values ​​of each species to obtain the total IBR value;

[0012] Step 6: Statistically analyze the total IBR values ​​at each exposure concentration with those of the control group to obtain a significant P value;

[0013] Step 7: Use the IBR value and P value to jointly characterize the ecological risk level. If the IBR value is less than or equal to the control group, there is no risk; if the IBR value is greater than the control group and P ≥ 0.05, it is a low risk; if the IBR value is greater than the control group and 0.01 ≤ P < 0.05, it is a medium risk; if the IBR value is greater than the control group and P < 0.01, it is a high risk.

[0014] In the above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, the test organisms in step 1 are Brachionus plicatilis, Litopenaeus vannamei, and Mytilus edulis.

[0015] In the above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, the biomarkers detected in step 3 are specifically:

[0016] The biomarkers tested for Brachionus plicatilis are population density, population growth rate, time to first spawning, time to first larvae laying, larval hatching rate, maternal survival rate, generation time, intrinsic growth rate, weekly growth rate, life expectancy, net growth rate, body length and body width;

[0017] The biomarkers tested for Litopenaeus vannamei were fatness, meat yield, intestine-to-body ratio, liver-to-body ratio, molting rate, muscle hardness, muscle elasticity, muscle water loss rate, liver alanine aminotransferase activity, and aspartate aminotransferase activity.

[0018] The biomarkers tested for blue mussels are water filtration rate, oxygen consumption rate, fatness, water content, total blood cell count, blood cell phagocytic ability, blood cell acid phosphatase and alkaline phosphatase activities, digestive gland amylase, protease and lipase activities.

[0019] In the above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, step 4 is specifically as follows:

[0020] Step 4.1: Calculate the mean m and standard deviation s of each biomarker X at different exposure concentrations;

[0021] Step 4.2, calculate the standard value Y of each biomarker X: Y = (Xm) / s;

[0022] Step 4.3: Calculate the Z value from the standard value Y and compare it with the control group. If the biomarker is promoted, Z = Y; if it is inhibited, Z = -Y; and the control group Z = Y;

[0023] Step 4.4, the S value is given by S = Z + |Z min |Get, Z min The minimum Z value of the same biomarker;

[0024] Step 4.5, IBR value = {[(S1×S2) / 2]+[(S2×S3) / 2]+…[(S n-1 ×S n ) / 2]+ [(S n ×S1) / 2]}, where S1 represents the S value of the first biomarker...S n represents the S value of the nth biomarker.

[0025] The above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, wherein step 6 specifically comprises: using the Kolmogorov-Smirnov test for the normal distribution of the IBR data and the Levene test for the homogeneity of variance of the data; using one-way analysis of variance and Waller-Duncan comparison to calculate the significant P value for the IBR data that conforms to the normal distribution and homogeneity of variance, and using the Kruskal-Wallis test to calculate the significant P value for the data that does not conform to the normal distribution and homogeneity of variance.

[0026] The beneficial effect of the present invention is that, based on a multi-species biomarker indicator system, the present invention establishes a nearshore ecological risk assessment method applicable to modified clay by improving the value assignment and calculation methods of the IBR evaluation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process of the present invention;

[0028] Figure 2 It is the total IBR value and significant P value of each group in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] This embodiment addresses the problem that existing ecological risk assessment methods are not applicable and cannot be quantitatively characterized, and discloses a method for quantitatively evaluating the offshore ecological risk of modified clay. The specific process is as follows: Figure 1 As shown, first, a test organism is selected. In this embodiment, the test organisms selected are Brachionus plicatilis, Litopenaeus vannamei, and Mytilus edulis.

[0031] Test organisms

[0032] Brachionus plicatilis, a rotifer belonging to the phylum Rotifera, order Monogenea, family Brachionidae, and genus Brachionus, is a widely distributed marine zooplankton with a short life cycle and is sensitive to environmental changes. It plays a connecting role in the material circulation and energy flow of marine ecosystems and is a commonly used biological model in marine ecotoxicology research.

[0033] The vannamei shrimp (Litopenaeus vannamei), belonging to the genus Litopenaeus, family Penaeidae, order Decapoda, class Crustacea, is a globally important aquaculture species. It is also frequently used in marine ecological risk assessments due to its sensitivity to environmental stresses and ease of cultivation.

[0034] The blue mussel (Mytilus edulis), belonging to the phylum Mollusca, class Bivalvia, family Mytilidae, genus Mytilus, is a globally distributed benthic bivalve and a common aquaculture species in my country. Due to its sessile lifestyle and filter-feeding habit, the blue mussel is widely used as a sentinel organism for environmental monitoring and as a model organism for ecological risk assessment.

[0035] Step 1: Cultivate the test organism.

[0036] Brachionus plicatilis was hatched from dormant eggs. After successful hatching, active and healthy individuals were selected under a stereomicroscope and continued to be cultured in a 2L beaker filled with sterile seawater. During the culture period, Chlorella vulgaris was fed once every 24 hours. The residue in the beaker was filtered out using a 100-mesh sieve. At the same time, the temperature of the intelligent light incubator was set at (25±1)℃, the photoperiod was 12h:12h, and the light intensity was 60μmol / (m 2Before the experiment began, females with eggs were individually removed and cultured in six-well plates under the same culture conditions as above. The hatching of eggs was observed every 2 h. If the number of newly hatched larvae within 2 h met the experimental number, subsequent experiments were carried out. If not, all larvae were removed and further observation was continued.

[0037] Litopenaeus vannamei were obtained from a farm and maintained in laboratory glass aquaria. The temperature was 26 ± 1°C, the salinity was 30 ± 1‰, and the pH was 8.1 ± 0.1. Dead shrimp were promptly removed to prevent water contamination. Water was exchanged 50%–100% daily depending on water quality. Shrimp were fed a commercial feed at 5% of their body weight at 8:00 AM, 2:00 PM, and 8:00 PM daily.

[0038] Mytilus edulis (Mussels) were collected from a farm and temporarily maintained in laboratory tanks prior to the experiment. Shell lengths were (4.5 ± 1.0) cm. After collection, they were quickly transferred to the laboratory, rinsed with artificial seawater to remove excess sediment, and dead individuals were removed and freed of attached organisms. They were then temporarily maintained in tanks to acclimate to the laboratory environment and observe their survival. During this period, seawater was maintained at a temperature of (15 ± 1)°C, a pH of 7.9 ± 0.2, and a salinity of (30 ± 1‰). Aeration was continuously applied, and a natural light cycle was maintained. Dead individuals were promptly removed to prevent water contamination. Chlorella vulgaris was fed and the seawater was replaced every 48 hours.

[0039] Step 2: Conduct exposure experiments.

[0040] In this study, aluminum sulfate-modified clay was used to assess its ecological risk. A 0.1g / L modified clay exposure group was established based on the field dosage of aluminum sulfate-modified clay. To ensure a more realistic and effective assessment of ecological risk, a simulated algae removal group was also established using 0.1g / L modified clay to remove harmful algae species. A separate exposure group to a single harmful algae species was also established to compare changes before and after algae removal. A blank control group was maintained without any additives.

[0041] The harmful algae species selected were Heterosigma akashiwo, which is a common species in coastal waters around the world that can produce toxins and cause harmful algal blooms, and has negative effects on a variety of marine organisms. Heterosigma akashiwo was cultured in an intelligent light incubator with a temperature of (20±1)℃, a photoperiod of 12h:12h, and a light intensity of 60 μmol / (m 2s). The Erlenmeyer flask should be cleaned in advance, soaked in an acid bath for 24 hours, then rinsed with distilled water and sterilized. Cool before use. The formula for the f / 2 medium stock solution is shown in Table 2-1. After dissolving to volume, sterilize the solution. After cooling, store at 4°C in the dark. Seawater used for culturing should be filtered through a 0.45μm pore size filter and autoclaved at 121.3°C and 0.105 MPa for 30 minutes. After cooling, add the f / 2 medium stock solution at a ratio of 1000:1. During the algae cultivation period, shake the Erlenmeyer flask 3-4 times daily to prevent algae from adhering to the wall and settling. Count the algae density using a cell counting chamber and expand the microalgae promptly to ensure optimal growth. The formula for the f / 2 medium stock solution is shown in Table 1.

[0042] Table 1 Formula of f / 2 culture medium mother solution

[0043]

[0044] The exposure experiment for Brachionus plicatilis was conducted in well plates, while the exposure experiments for Litopenaeus vannamei and Mytilus edulis were conducted in glass aquaria. The exposure duration was 7 days. The settings for each exposure group are shown in Table 2. In the algae removal group, Heterobacter akashiwo was added first, followed by the modified clay.

[0045] Table 2. Settings of the exposure group experiment

[0046]

[0047] Step 3: Biomarker detection.

[0048] Biomarker detection of Brachionus plicatilis

[0049] (1) Determination of the time of first spawning and first larval laying

[0050] Exposure and test experiments were conducted in 24-well plates with a 1 mL experimental volume. One rotifer larva (<2 hours old) was inoculated per well, with 12 replicates per group. From the start of inoculation, observations were made using a stereomicroscope every 2 hours, and the culture medium was replaced every 24 hours. The feeding density was 2 × 10 5 cells / mL of Chlorella, accurately record the time of the first egg laying and the first larvae laying of rotifers.

[0051] (2) Measurement of body length and width

[0052] During the exposure period, the morphology of rotifers in each group was observed using an inverted microscope when the rotifers laid eggs for the first time, and 9 rotifers were randomly selected and their body length and width were measured.

[0053] (3) Determination of population density and population growth rate

[0054] Exposure and test experiments were conducted in 12-well plates with a test volume of 2 mL. Five rotifer larvae (<2 hours old) were inoculated in each well. The rotifers were observed every 24 hours, and dead individuals were removed promptly. The total number of surviving rotifers in each well was recorded, and the culture medium was replaced. The feeding density was 2 × 10 5 cells / mL of Chlorella vulgaris, the experiment lasted 7 days, and each group had 4 replicates. The population growth rate was calculated as follows:

[0055]

[0056] Where R represents the population growth rate, N0 is the initial rotifer population density (ind. / mL); N t represents the rotifer population density on the tth day (ind. / mL), and t represents the number of experimental days (d).

[0057] (4) Determination of maternal survival rate and larval hatching rate

[0058] Exposure and test experiments were conducted in 24-well plates with an experimental volume of 1 mL. Five rotifer larvae (<2 h old) were inoculated in each well as the initial mothers. The rotifers were observed every 24 h, and newborn larvae and dead individuals were removed promptly. The number of eggs, larvae, and dead mothers per well was recorded, and the culture medium was replaced. The feeding density was 2 × 10 5 cells / mL of Chlorella vulgaris. The experiment lasted 7 days, with 4 replicates per group. The formulas for calculating the maternal survival rate and larval hatching rate are as follows:

[0059]

[0060]

[0061] Where M represents the maternal survival rate, L represents the larval hatching rate, M0 is the initial number of rotifer mothers, and M t is the number of surviving rotifer mothers on day t, L t-1 is the total number of eggs laid by rotifers the day before (t-1), L t is the total number of larvae produced by rotifers per day.

[0062] (5) Life cycle indicator measurement

[0063] Exposure and test experiments were conducted in 24-well plates with a test volume of 1 mL. Five rotifer larvae (<2 hours old) were inoculated into each well. The number of rotifer eggs, larvae, and deaths was recorded every 24 hours. Newborn larvae and dead individuals were removed promptly and the culture medium was replaced. The feeding density was 2 × 10 5 cells / mL of Chlorella vulgaris until all rotifers die. Each group has four replicates. The following formula is used to calculate the corresponding index:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] Where x is the age of rotifers, l x and m x represent age-specific survival rate and age-specific reproduction rate, respectively, n x and n0 are the number of rotifers surviving on day x and day 0, respectively. x is the number of rotifers produced on the xth day, R0, T, r m , λ and E0 represent the net reproductive rate, generation time, intrinsic growth rate, finite growth rate and life expectancy, respectively.

[0072] The changes in biomarkers of Brachionus plicatilis after exposure to modified clay are shown in Table 3.

[0073] Table 3 Changes in biomarkers of Brachionus plicatilis after exposure to modified clay

[0074]

[0075] Biomarker detection of Litopenaeus vannamei

[0076] (1) Determination of molting rate, fatness, meat yield, intestine-to-body ratio and liver-to-body ratio

[0077] The number of molting shrimp was counted daily during the exposure period. At the end of the exposure period, six shrimp were randomly selected from each group and divided into three groups. The shrimp were placed in an ice bath to slow their movement. After measuring their body weight and length, the hepatopancreas, intestine, and muscle tissues of the shrimp were quickly dissected and weighed. The indicators were calculated using the following formula:

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Where TP represents the molting rate, CF represents the fatness, MY represents the meat yield, HSI represents the liver-to-body ratio, ISI represents the intestine-to-body ratio, M represents the number of molts, N represents the number of shrimps, L represents the shrimp length (cm), W0 represents the shrimp mass (g), W1 represents the shrimp muscle weight (g), W2 represents the shrimp liver weight (g), and W3 represents the shrimp intestine weight (g).

[0084] (2) Determination of muscle water loss rate

[0085] A piece of shrimp meat (W1) is boiled in boiling water for 3 minutes, removed, cooled to room temperature, and the surface moisture is wiped off with absorbent paper before weighing (W2). To determine freezing loss, a piece of shrimp meat (W1) is placed in a -20°C freezer for 24 hours, removed and thawed at room temperature, and the surface moisture is wiped off with absorbent paper before weighing (W2). Water loss is expressed as the weight loss during boiling or freezing. The water loss rate is calculated as follows:

[0086]

[0087] (3) Measurement of muscle hardness and elasticity

[0088] Three shrimp were randomly selected from each group, their shells removed, and the central muscle of the second abdominal segment was examined using a TMS-Touch muscle texture analyzer (Food Technology Corporation, USA). A 25 mm × 25 mm cylindrical probe was used to measure contact force (5 gf), a test speed of 1 mm / s, a target mode of deformation, a 40% deformation change, and a duration of 2 seconds.

[0089] (4) Determination of liver alanine aminotransferase and aspartate aminotransferase activities

[0090] Liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using biochemical kits (BC1555 and BC1565, Beijing Solebold Technology Co., Ltd.). One unit of ALT activity was defined as the amount of enzyme activity required to produce 1 μmol of pyruvate per gram of sample per hour. One unit of AST activity was defined as the amount of enzyme activity required to produce 1 μmol of pyruvate per gram of sample per hour.

[0091] The changes in biomarkers of Litopenaeus vannamei after exposure to modified clay are shown in Table 4.

[0092] Table 4 Changes in biomarkers of Litopenaeus vannamei after exposure to modified clay

[0093]

[0094] Biomarker detection of blue mussel

[0095] (1) Determination of state index and moisture content

[0096] After exposure, nine blue mussels were randomly selected from each group and divided into three groups. The shells and soft tissues were separated. The surface moisture of the shells was removed with absorbent paper, and the wet weight was measured using an electronic balance. The soft tissues were then dried in a forced-air drying oven at 65°C to a constant weight, and the dry weight was re-measured. The state index and moisture content were calculated using the following formulas:

[0097]

[0098]

[0099] Where CI stands for condition index, WC stands for moisture content, DT is dry tissue weight (g), DS is dry shell weight (g), and WT is wet tissue weight (g).

[0100] (2) Water filtration rate determination

[0101] Water filtration rate can be defined as the ability to filter suspended particles (such as bait algae) in water per unit time. After the exposure, the test was carried out in 500 mL beakers, with 3 replicates per group. Three blue mussels were randomly placed in each beaker, and 500 mL of 2×10 6 cells / mL of Chlorella. After the purple mussels open, start timing for 30 minutes. After the time is up, remove the purple mussels, mix the Chlorella thoroughly, take a sample, and use a cell counting plate to count the final Chlorella density. Use the following formula to calculate the filtration rate and feeding rate:

[0102]

[0103] Where IFR represents the water filtration rate (L·ind -1 ·h -1 ), V is the volume of Chlorella used in the experiment (mL), n is the number of shellfish used in the experiment, w is the wet weight of shellfish used in the experiment (g), t is the experimental time (h), C0 and C t Represents the algal cell concentration at the start and end of the experiment (cells·mL -1 )

[0104] (3) Oxygen consumption rate determination

[0105] Oxygen consumption rate is defined as the amount of oxygen consumed per unit wet weight per hour. After exposure, nine blue mussels were randomly selected from each group and divided into three groups. Three blue mussels were placed in a graduated cylinder filled with water to measure their volume, then placed in an Erlenmeyer flask filled with sterile seawater. The flask was sealed with parafilm to prevent air leaks and bubbles. The experiment lasted for two hours. A DO-200 (YSI, USA) dissolved oxygen meter was used to measure the dissolved oxygen content in the seawater at the beginning and end of the experiment. The oxygen consumption rate was calculated as follows:

[0106]

[0107] where R O represents oxygen consumption rate (μmol·Wwg -1 ·h -1 ), D0 is the dissolved oxygen content in seawater at the beginning of the experiment (mg / L), D t is the dissolved oxygen content in seawater at the end of the experiment (mg / L), V is the volume of seawater used in the experiment (L), w is the wet weight of blue mussels (Wwg), and t is the experimental time (h).

[0108] (4) Determination of total blood cell count, phagocytic ability, and acid and alkaline phosphatase activity

[0109] After the exposure, nine blue mussels were randomly selected from each group. Hemolymph was extracted using the method authorized by the research group, patent No. CN204705520U. Hemolymph from three mussels was pooled to form a single sample, resulting in three samples per group. An equal amount of hemocyte preservation solution (Aldrich's solution) was added and mixed thoroughly. The mixture was then placed on ice and filtered through a single layer of 400-mesh silk sieve. Aldrich's solution contains sodium citrate (dihydrate), citric acid (monohydrate), glucose, and sodium chloride at concentrations of 8.0 g / L, 0.55 g / L, 20.5 g / L, and 4.2 g / L, respectively. 100 μL of hemolymph from each sample in each group was counted under an optical microscope using a hemocytometer to calculate the total number of hemocytes.

[0110] 1000 μL of hemolymph was collected from each sample in a 1.5 mL centrifuge tube and centrifuged at 500 g for 10 min at 4°C to obtain a pellet of blood cells. The cells were then resuspended in 1000 μL of PBS containing a 50-fold dilution of fluorescent microspheres (the ratio of 50-fold dilution to PBS was 1:5). After incubation at 18°C ​​in the dark for 1 h, the supernatant was removed by centrifugation, and the blood cells were resuspended in 1000 μL of PBS again. Flow cytometry was used to analyze hemocyte phagocytosis. The phagocytic capacity of blue mussel hemocytes was expressed as the percentage of cells phagocytosing microspheres relative to the total number of blood cells.

[0111] Acid phosphatase and alkaline phosphatase activity units are defined as the production of 1 μmol of phenol per minute per gram of tissue. Acid and alkaline phosphatase activities were measured using biochemical kits (BC2135 and BC2145, Beijing Solebow Technology Co., Ltd.) according to the manufacturer's instructions.

[0112] (5) Determination of digestive enzyme activity

[0113] Protease, lipase, and amylase activities were measured using biochemical kits (BC2325, BC2345, and BC2045, Beijing Solebeau Technology Co., Ltd.) according to the manufacturer's instructions. One unit of protease activity was defined as the hydrolysis of hemoglobin to produce 1 μmol of tyrosine per minute per gram of tissue. One unit of lipase activity was defined as the hydrolysis of olive oil to produce 1 μmol of fatty acids per minute per gram of tissue. One unit of amylase activity was defined as the production of 1 mg of reducing sugar per minute per gram of tissue.

[0114] The changes in biomarkers of blue mussels after exposure to modified clay are shown in Table 5.

[0115] Table 5 Changes in biomarkers of blue mussels after exposure to modified clay

[0116]

[0117] Step 4: Calculate the IBR values ​​of the control group and each exposure group using the improved integrated biomarker (IBR) analysis method.

[0118] The IBR value was calculated for each parallel experiment. The calculation method is as follows

[0119] (1) Calculate the mean m and standard deviation s of each biomarker X at different exposure concentrations.

[0120] (2) Calculate the standard value Y of each biomarker X: Y = (Xm) / s.

[0121] (3) The Z value is calculated from the standard value Y and compared with the control group. When the biomarker is promoted, Z = Y; when it is inhibited, Z = -Y; and the control group Z = Y.

[0122] (4) The S value is given by S = Z + |Z min |Get, Z min The minimum Z value of the same biomarker.

[0123] (5) IBR value = {[(S1×S2) / 2]+[(S2×S3) / 2]+…[(Sn-1×Sn) / 2]+ [(Sn×S1) / 2]}.

[0124] Step 5: Add the IBR values ​​of each species to obtain the total IBR value.

[0125] After calculation, the total IBR value of each group is as follows Figure 2 shown.

[0126] Step 6: Statistical analysis was performed on the total IBR values ​​at each exposure concentration compared with the control group.

[0127] The Kolmogorov-Smirnov test was used to analyze the normal distribution of IBR data, and the Levene test was used to test the homogeneity of variance. The IBR data that met the normal distribution and homogeneity of variance were compared using one-way analysis of variance (ANOVA) and Waller-Duncan to calculate the significant P value. The data that did not meet the normal distribution and homogeneity of variance were compared using the Kruskal-Wallis test to calculate the significant P value. After statistical analysis, the P values ​​of each group are as follows Figure 2 shown.

[0128] Step 7: Use IBR value and P value to jointly characterize ecological risk.

[0129] The characterized ecological risk levels are shown in Table 6.

[0130] Table 6 Ecological risk levels represented by IBR values ​​and P values

[0131]

[0132] After risk characterization, aluminum sulfate modified clay itself has low risk, while Bayer akashiwo has high risk. Using aluminum sulfate modified clay to remove Bayer akashiwo can reduce the ecological risk to low risk.

[0133] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the spirit and scope of protection of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present invention.

Claims

1. A method for quantitatively evaluating the offshore ecological risk of modified clay, characterized in that: The steps include: Step 1, culturing the test organism; Step 2: setting the exposure concentration according to the field use dose of the modified clay, conducting an exposure experiment, and obtaining an exposure group and a control group; Step 3, performing biomarker detection based on the test organism selected in step 1; Step 4, the IBR values ​​of each species in the control and exposed groups were calculated using the improved integrated biomarker response evaluation method; Step 5, summing the IBR values ​​of each species in the exposure group in step 4 to obtain the total IBR value of the exposure group, and summing the IBR values ​​of each species in the control group in step 4 to obtain the total IBR value of the control group; Step 6: Statistically analyze the total IBR value of the exposure group and the control group to obtain a significant P value; Step 7: Use the IBR value and P value to jointly characterize the ecological risk level. If the IBR value is less than or equal to the control group, there is no risk; if the IBR value is greater than the control group and P ≥ 0.05, it is a low risk; if the IBR value is greater than the control group and 0.01 ≤ P < 0.05, it is a medium risk; if the IBR value is greater than the control group and P < 0.01, it is a high risk. The test organisms in step 1 are Brachionus plicatilis, Litopenaeus vannamei, and Mytilus edulis; The biomarkers detected in step 3 are specifically: The biomarkers tested for Brachionus plicatilis are population density, population growth rate, time to first spawning, time to first larvae laying, larval hatching rate, maternal survival rate, generation time, intrinsic growth rate, weekly growth rate, life expectancy, net growth rate, body length and body width; The biomarkers tested for Litopenaeus vannamei were fatness, meat yield, intestine-to-body ratio, liver-to-body ratio, molting rate, muscle hardness, muscle elasticity, muscle water loss rate, liver alanine aminotransferase activity, and aspartate aminotransferase activity. The biomarkers tested for blue mussels are water filtration rate, oxygen consumption rate, fatness, water content, total blood cell count, blood cell phagocytic ability, blood cell acid phosphatase and alkaline phosphatase activities, digestive gland amylase, protease and lipase activities.

2. The method for quantitatively evaluating the offshore ecological risk of modified clay according to claim 1, characterized in that: The step 4 is specifically as follows: Step 4.1: Calculate the mean m and standard deviation s of each biomarker X at different exposure concentrations; Step 4.2, calculate the standard value Y of each biomarker X: Y = (Xm) / s; Step 4.3: Calculate the Z value from the standard value Y and compare it with the control group. If the biomarker is promoted, Z = Y; if it is inhibited, Z = -Y; the control group Z = Y; Step 4.4, the S value is given by S = Z + |Z min |Get, Z min The minimum Z value of the same biomarker; Step 4.5, IBR value = {[(S1×S2) / 2]+[(S2×S3) / 2]+…[(S n-1 ×S n ) / 2]+ [(S n ×S1) / 2]}, where S1 represents the S value of the first biomarker...S n represents the S value of the nth biomarker.

3. The method for quantitatively evaluating the offshore ecological risk of modified clay according to claim 1, characterized in that: The step 6 is specifically as follows: the normal distribution of the IBR data is tested using Kolmogorov-Smirnov, and the variance homogeneity of the data is tested using Levene; the significant P value is calculated using one-way analysis of variance and Waller-Duncan comparison for the IBR data that conforms to the normal distribution and has homogeneous variance, and the significant P value is calculated using the Kruskal-Wallis test for the data that does not conform to the normal distribution and homogeneity of variance.

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