Method for quantitatively evaluating offshore ecological risk of modified clay

Abstract

The invention discloses a method for quantitatively evaluating the offshore ecological risk of modified clay. The method comprises the following steps: culturing three tested organisms; carrying out an exposure experiment based on the on-site use concentration of the modified clay; detecting a biomarker of each species; calculating the IBR value of each species by using an improved integrated biomarker response (IBR) evaluation method; adding to obtain a total IBR value; performing statistical analysis on the total IBR value to obtain a significance P value; and jointly representing the ecological risk level by using the total IBR value and the P value. On the basis of a multi-species biomarker index system, an offshore ecological risk quantitative evaluation method suitable for modified clay is established by improving assignment and calculation methods of an IBR evaluation method.

Description

Technical Field

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

[0002] Modified clay technology is a guiding method for harmful algal bloom control that is widely accepted and widely used internationally. It is also the national standard method for harmful algal bloom control in my country (GB / T 30743-2014). Modified clay uses natural kaolin or montmorillonite as raw materials. After inorganic or organic modification, it removes and controls harmful algal blooms through flocculation and sedimentation. Modified clay is taken from natural materials and is not a synthetic chemical material. In theory, it has the characteristics of being green, non-toxic and environmentally friendly. However, the ecological risks caused by any material added to natural water bodies cannot be ignored. At present, the offshore ecological risk assessment of modified clay is still at the level of lethal effects and physiological activities on single marine organisms, and there is no substantial evidence to prove whether it has ecological risks.

[0003] According to the technical standards for ecological risk assessment and water quality benchmark derivation formulated by environmental protection departments at home and abroad, the current common ecological risk assessment methods include assessment factor method (AF), risk quotient method (RQ), species sensitivity step method (SSD) and probabilistic risk assessment method (PERA). The main evaluation targets of these methods are pollutants in the ecological environment, and the data acquisition of quantitative evaluation parameters depends on the lethal effect of pollutants on the test organisms or a certain effect endpoint. However, modified clay may not have a lethal effect on the test organisms, and will not produce other biological effects in a short period of time. Therefore, traditional evaluation methods are not suitable for the offshore ecological risk assessment of modified clay.

[0004] With the deepening of ecotoxicology 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 series of 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 used for 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: Step 1, culturing the test organism; Step 2, setting the exposure concentration according to the field use dosage 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, using the improved integrated biomarker response evaluation method to calculate the IBR values ​​of each species in the control group and each exposure group; Step 5, sum the IBR values ​​of each species to obtain the total IBR value; Step 6, statistically analyze the total IBR value at each exposure concentration with 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.

[0007] In the above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, the test organisms in step 1 are Brachionus plicatilis rotifer, Penaeus vannamei shrimp, and Mytilus edulis.

[0008] In the above method for quantitatively evaluating the offshore ecological risk of modified clay, the biomarkers detected in step 3 are specifically: The biomarkers detected for Brachionus plicatilis were 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 detected in 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.

[0009] In the above method for quantitatively evaluating the offshore ecological risk of modified clay, step 4 is specifically as follows: Step 4.1, calculate the mean value 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, 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; Step 4.4, the S value is given by S = Z + |Z min |Get, Z min It is the minimum value among the Z values ​​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.

[0010] The above-mentioned method for quantitatively evaluating the offshore ecological risk of modified clay, the step 6 specifically comprises: using Kolmogorov-Smirnov to test the normal distribution of IBR data, and using Levene to test the homogeneity of variance of data; using one-way analysis of variance and Waller-Duncan comparison to calculate the significant P value for IBR data that conform to the normal distribution and have homogeneous variance, and using Kruskal-Wallis test to calculate the significant P value for data that do not conform to the normal distribution and homogeneity of variance.

[0011] The beneficial effect of the present invention is that, based on a multi-species biomarker index system, the present invention establishes a nearshore ecological risk assessment method suitable for modified clay by improving the value assignment and calculation methods of the IBR assessment method. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the process of the present invention; FIG. 2 is the total IBR value and significant P value of each group in the embodiment of the present invention. DETAILED DESCRIPTION

[0013] 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 in conjunction with the accompanying drawings and specific implementation methods.

[0014] In view of the problems that existing ecological risk assessment methods are not applicable and cannot be quantified, this embodiment discloses a method for quantitatively evaluating the offshore ecological risk of modified clay. The specific process is as follows: Figure 1As shown, firstly, the test organisms are selected. In this embodiment, the test organisms selected are Brachionus plicatilis, Penaeus vannamei, and Mytilus edulis.

[0015] Test organisms Brachionus plicatilis belongs to the phylum Rotifera, order Monogenea, family Brachionidae, and genus Brachionus. It 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.

[0016] Litopenaeus vannamei belongs to the genus Litopenaeus, family Penaeidae, order Decapoda, class Crustacea, and is an important aquaculture species in the world. It is also often used in marine ecological risk assessment because it is sensitive to environmental stress and easy to cultivate.

[0017] Blue mussel (Mytilus edulis) belongs to the phylum Mollusca, class Bivalvia, family Mytilidae, genus Mytilus. It is a benthic bivalve widely distributed around the world and one of the most commonly farmed shellfish in my country. At the same time, due to its sessile lifestyle and filter-feeding feeding method, blue mussel is widely used as a sentinel organism for environmental monitoring and a model organism for ecological risk assessment.

[0018] Step 1: Cultivate the test organism.

[0019] Brachionus plicatilis was hatched from dormant eggs. After successful hatching, active and healthy individuals were selected under a stereo microscope and continued to be cultured in a 2L beaker filled with sterilized seawater. During the culture period, Chlorella was fed once every 24 hours, and the residue in the beaker was filtered out with a 100-mesh sieve. At the same time, the temperature of the intelligent light incubator was set to (25±1)℃, the light cycle was 12h: 12h, and the light intensity was 60μmol / (m 2 ·s). Before the experiment, the females with eggs were taken out separately and placed in a six-well plate for culture. The culture conditions were the same as above. The hatching of eggs was observed every 2 h. If the newly hatched larvae within 2 h met the experimental number, the subsequent experiment was carried out. If not, all the larvae were removed and the observation was continued.

[0020] Vannamei shrimp were taken from the farm and temporarily raised in a laboratory glass tank. The raising temperature was 26±1℃, the salinity was 30±1‰, and the pH was 8.1±0.1. Dead individuals were removed in time to avoid water pollution. The water was changed by 50%-100% daily depending on the water quality. Commercial feed was fed at 5% of the shrimp body weight at 8:00, 14:00 and 20:00 every day.

[0021] The blue mussels were collected from the farm and temporarily kept in the laboratory water tank before the experiment. The shell length was (4.5±1.0) cm. After the collection was completed, it was quickly transferred to the laboratory, and the excess sediment was washed away with artificial seawater. The dead individuals were cut off and the attached organisms were removed. They were temporarily kept in the water tank to adapt to the laboratory environment and observe the survival of the blue mussels. During the temporary culture period, the seawater temperature was maintained at (15±1)℃, the pH was 7.9±0.2, and the salinity was (30±1)‰. It was continuously aerated and oxygenated, and the natural light cycle was maintained. Dead individuals were removed in time to avoid water pollution. Chlorella was fed and the seawater was replaced every 48 hours.

[0022] Step 2: Conduct exposure experiments.

[0023] This implementation selected aluminum sulfate modified clay to evaluate its ecological risk. A 0.1g / L modified clay exposure group was set according to the on-site use dosage of aluminum sulfate modified clay. In order to more realistically and effectively evaluate its ecological risk, a simulated algae removal group was set up to remove harmful algae species using 0.1g / L modified clay. At the same time, a single exposure group of harmful algae species was set up to compare the changes before and after algae removal. The blank control group had no additions.

[0024] The harmful algae species selected were Heterosigma akashiwo, which is a common harmful algal bloom species in coastal waters around the world and can produce toxins, and has negative effects on a variety of marine organisms. Heterosigma akashiwo was cultured in an intelligent light incubator, the temperature was set to (20±1)℃, the light cycle was 12h: 12h, and the light intensity was 60 μmol / (m 2 ·s). The conical flask needs to be cleaned in advance, soaked in an acid tank for 24 hours, then rinsed with distilled water and sterilized, and used after cooling. The formula of f / 2 medium mother solution is shown in Table 2-1. After dissolving and fixing the volume, it is sterilized and stored at 4℃ in the dark after cooling. The seawater used for culture is filtered through a 0.45μm pore size filter membrane, sterilized at 121.3℃ and 0.105MPa for 30min, and added to the f / 2 medium mother solution at a ratio of 1000:1 after cooling. During the algae culture period, shake the conical flask 3-4 times a day to prevent the algae from adhering to the wall and settling. At the same time, use a cell counting plate to count the algae density and expand the algae in time to ensure the optimal growth state of the microalgae. The formula of f / 2 medium mother solution is shown in Table 1.

[0025] Table 1 Formula of f / 2 culture medium stock solution

[0026] The exposure experiment of Brachionus plicatilis was carried out in a well plate, and the exposure experiment of Penaeus vannamei and Mytilus edulis was carried out in a glass tank. The exposure time was 7 days. The settings of each exposure group are shown in Table 2, in which the algae removal group was first added with Heterobacter akashiwo and then with modified clay.

[0027] Table 2. Settings of the exposure group experiment

[0028] Step 3: Biomarker detection.

[0029] Biomarker detection of Brachionus plicatilis (1) Determination of the time of first spawning and first larval laying The exposure and test experiments were conducted in 24-well plates with an experimental volume of 1 mL. One rotifer larva (worm age < 2 hours) was inoculated in each well, and 12 parallels were set up in each group. From the beginning 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 recording the time of the first spawning and first larvae laying of rotifers.

[0030] (2) Measurement of body length and width 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 to measure their body length and width.

[0031] (3) Determination of population density and population growth rate The exposure and test experiments were conducted in 12-well plates with an experimental volume of 2 mL. Five rotifer larvae (worm age < 2 h) were inoculated in each well. The rotifers were observed every 24 h, and dead individuals were removed in time. 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, the experiment lasted for 7 days, and each group was set up with 4 parallels. The population growth rate was calculated as follows:

[0032] 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).

[0033] (4) Determination of maternal survival rate and larval hatching rate The exposure and test experiments were conducted in 24-well plates with an experimental volume of 1 mL. Five rotifer larvae (worm age < 2 h) were inoculated in each well as the initial mothers. The rotifers were observed every 24 h, and the newborn larvae and dead individuals were removed in time. The number of rotifer eggs, larvae, and dead mothers in each well was recorded. The culture medium was replaced and the feeding density was 2 × 10 5 cells / mL of Chlorella, the experiment lasted for 7 days, and each group was set up with 4 parallels. The formula for calculating the maternal survival rate and larval hatching rate is as follows:

[0034] Where M represents the maternal survival rate, L represents the larval hatching rate, M0 is the initial number of rotifer maternal bodies, 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.

[0035] (5) Determination of life cycle indicators The exposure and test experiments were conducted in 24-well plates with an experimental volume of 1 mL. Five rotifer larvae (worm age < 2 h) were inoculated in each well. The number of rotifer eggs, larvae, and deaths was recorded every 24 h. Newborn larvae and dead individuals were removed in time and the culture medium was replaced. The feeding density was 2 × 10 5 cells / mL of Chlorella, until all rotifers died, with 4 parallels in each group. The following formula was used to calculate the corresponding index:

[0036] Where x is the age of the rotifer, l x and m x represent the 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.

[0037] The changes in biomarkers of Brachionus plicatilis after exposure to modified clay are shown in Table 3.

[0038] Table 3 Changes in biomarkers of Brachionus plicatilis after exposure to modified clay

[0039] Biomarker detection of Penaeus vannamei (1) Determination of molting rate, fatness, meat yield, intestine-to-body ratio and liver-to-body ratio The number of molting shrimps was counted every day during the exposure period. At the end of the exposure, 6 shrimps were randomly selected from each group and randomly divided into 3 groups. They were placed in an ice bath to slow down the movement of the shrimps. After measuring their weight and length, the hepatopancreas, intestines, and muscle tissues of the shrimps were quickly dissected and weighed. The calculation formula of the index is as follows:

[0040] Among them, 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 length of shrimp (cm), W0 represents the weight of shrimp (g), W1 represents the muscle weight of shrimp (g), W2 represents the liver weight of shrimp (g), and W3 represents the intestine weight of shrimp (g).

[0041] (2) Determination of muscle water loss rate Boil a piece of shrimp meat (W1) in boiling water for 3 minutes, take it out, cool it to room temperature, wipe off the surface water with absorbent paper and weigh it (W2). When measuring freezing loss, take a piece of muscle (W1) and place it in a -20℃ refrigerator for 24 hours, take it out and thaw it at room temperature, wipe off the surface water with absorbent paper and weigh it (W2). Water loss is expressed as the weight loss during boiling or freezing. The formula for calculating water loss rate is as follows:

[0042] (3) Measurement of muscle hardness and elasticity Three shrimps were randomly selected from each group, and the shrimp shells were peeled off. The central muscle of the second abdominal segment was taken and the muscle hardness and elasticity were measured using a texture analyzer TMS-Touch muscle texture analyzer (Food Technology Corporation, USA). A 25 mm × 25 mm cylindrical probe was used to contact sense 5 gf, the test speed was 1 mm / s, the target mode was deformation, the deformation was 40%, and the time was 2 s.

[0043] (4) Determination of liver alanine aminotransferase activity and aspartate aminotransferase activity Liver alanine aminotransferase activity and aspartate aminotransferase were measured using biochemical kits (BC1555 and BC1565, Beijing Solebow Technology Co., Ltd.). Alanine aminotransferase activity was defined as the amount of 1 μmol of pyruvate produced per gram of sample per hour as one unit of enzyme activity. Aspartate aminotransferase activity was defined as the amount of 1 μmol of pyruvate produced per gram of sample per hour as one unit of enzyme activity.

[0044] The changes in biomarkers of Penaeus vannamei after exposure to modified clay are shown in Table 4.

[0045] Table 4 Changes in biomarkers of Litopenaeus vannamei after exposure to modified clay

[0046] Biomarker detection of blue mussel (1) Determination of state index and moisture content After the exposure, 9 blue mussels were randomly selected from each group and divided into 3 groups. The shell and soft tissue were separated, and the moisture on the surface of the sample shell was absorbed with absorbent paper. The wet weight was weighed using an electronic balance, and the soft tissue was placed in a blast drying oven at 65°C to a constant weight, and the dry weight was weighed again. The state index and moisture content were calculated as follows:

[0047] 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).

[0048] (2) Determination of water filtration rate The water filtration rate can be defined as the ability to filter suspended particles (such as bait algae) in the water body per unit time. After the exposure, the test was carried out in 500 mL beakers, with 3 parallel groups in each group. Three blue mussels were randomly placed in each beaker, and 500 mL of 2×10 6 cells / mL of Chlorella, start timing for 30 minutes after the purple mussels open their mouths, take out the purple mussels, mix the Chlorella thoroughly and take samples, and use a cell counting plate to count the final Chlorella density. The following formula is used to calculate the filtration rate and feeding rate:

[0049] 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 ) (3) Oxygen consumption rate measurement Oxygen consumption rate is defined as the amount of oxygen consumed per unit wet weight per hour. After the exposure, 9 blue mussels were randomly selected from each group and randomly divided into 3 groups. The 3 blue mussels were placed in a measuring cylinder with water to measure their volume and then placed in a conical flask filled with sterilized seawater. The conical flask was sealed with a sealing film to prevent leakage and bubble generation. The experimental time was 2 hours. The dissolved oxygen content in the seawater was measured at the beginning and end of the experiment using a DO-200 (YSI, USA) dissolved oxygen meter. The oxygen consumption rate was calculated as follows:

[0050] 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).

[0051] (4) Determination of total blood cell count, phagocytic ability, and acid and alkaline phosphatase activity After the exposure, 9 blue mussels were randomly selected from each group, and the hemolymph of blue mussels was extracted using the method authorized by the research group's patent No. CN204705520U. The hemolymph of three mussels was mixed into one sample, and each group had three samples. An equal amount of blood cell preservation solution (Aldrich's solution) was added in time to mix, and the mixture was placed on ice and filtered using a single layer of 400 mesh silk sieve. The main components of Aldrich's solution are: sodium citrate (dihydrate), citric acid (monohydrate), glucose and sodium chloride, with concentrations of 8.0g / L, 0.55g / L, 20.5g / L and 4.2g / L, respectively. 100μL of the above hemolymph was taken from each sample in each group and counted under an optical microscope using a hemocytometer to calculate the total number of blood cells.

[0052] 1000 μL of hemolymph was taken from each sample in each group and placed in a 1.5 mL centrifuge tube. After centrifugation at 500 g for 10 min at 4°C to obtain the precipitated blood cells, 1000 μL of PBS containing 50-fold dilution of fluorescent microspheres was added to resuspend the blood cells. The ratio of 50-fold dilution of fluorescent microspheres to PBS was 1:5. After incubation at 18°C ​​in the dark for 1 h, the supernatant was removed by centrifugation, and 1000 μL of PBS was added to resuspend the blood cells again. The phagocytosis of blood cells was analyzed by flow cytometry. The percentage of cells that phagocytized microspheres to the total number of blood cells was used to indicate the phagocytic ability of blue mussel blood cells.

[0053] The activity units of acid phosphatase and alkaline phosphatase can be defined as the production of 1 μmol of phenol per minute per gram of tissue. The activity of acid and alkaline phosphatase was detected using biochemical kits (BC2135 and BC2145, Beijing Solebow Technology Co., Ltd.) according to the instructions.

[0054] (5) Determination of digestive enzyme activity The activities of protease, lipase and amylase were measured using biochemical kits (BC2325, BC2345, BC2045, Beijing Solebow Technology Co., Ltd.) according to the instructions. The protease activity unit was defined as the hydrolysis of hemoglobin to produce 1 μmol tyrosine per gram of tissue per minute. The lipase activity unit was defined as the hydrolysis of olive oil to produce 1 μmol fatty acid per gram of tissue per minute. The amylase activity unit was defined as the production of 1 mg reducing sugar per gram of tissue per minute.

[0055] The changes in biomarkers of blue mussel after exposure to modified clay are shown in Table 5.

[0056] Table 5 Changes in biomarkers of blue mussel after exposure to modified clay

[0057] Step 4: Calculate the IBR values ​​of the control group and each exposure group using the improved integrated biomarker (IBR) analysis method.

[0058] The IBR value was calculated for each parallel experiment. The calculation method is as follows (1) Calculate the mean value m and standard deviation s of each biomarker X at different exposure concentrations.

[0059] (2) Calculate the standard value Y of each biomarker X: Y = (Xm) / s.

[0060] (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.

[0061] (4) The S value is given by S = Z + |Z min |Get, Z min It is the minimum Z value of the same biomarker.

[0062] (5) IBR value = {[(S1×S2) / 2]+[(S2×S3) / 2]+…[(Sn-1×Sn) / 2]+ [(Sn×S1) / 2]}.

[0063] Step 5: Add the IBR values ​​of each species to get the total IBR value.

[0064] The total IBR values ​​of each group after calculation are shown in Figure 2.

[0065] Step 6: Statistical analysis was performed on the total IBR values ​​at each exposure concentration compared with the control group.

[0066] 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 conformed to 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, and the data that did not conform to the normal distribution and homogeneity of variance were calculated using the Kruskal-Wallis test to calculate the significant P value. The P values ​​of each group after statistical analysis are shown in Figure 2.

[0067] Step 7: Use IBR value and P value to jointly characterize ecological risk.

[0068] The characterized ecological risk levels are shown in Table 6.

[0069] Table 6 Ecological risk levels represented by IBR values ​​and P values

[0070] 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.

[0071] The above embodiments are only 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 essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope 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 dosage 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, calculate the IBR values ​​of each species in the control group and the exposed group using the improved integrated biomarker response evaluation method; Step 5, summing up 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 up 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.

2. The method for quantitatively evaluating the offshore ecological risk of modified clay according to claim 1, characterized in that: The tested organisms in step 1 are Brachionus plicatilis, Penaeus vannamei and Mytilus edulis.

3. A method for quantitatively evaluating the offshore ecological risk of modified clay according to claim 2, characterized in that: The biomarkers detected in step 3 are specifically: The biomarkers detected for Brachionus plicatilis were 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 detected in 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.

4. 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 value 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, 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; Step 4.4, the S value is given by S = Z + |Z min |Get, Z min It is the minimum value among the Z values ​​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.

5. 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 IBR data is tested using Kolmogorov-Smirnov, and the variance homogeneity of data is tested using Levene; the significant P value is calculated using one-way analysis of variance and Waller-Duncan comparison for IBR data that conform to the normal distribution and have homogeneous variance, and the significant P value is calculated using the Kruskal-Wallis test for data that do not conform to the normal distribution and homogeneous variance.

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