阅读说明：本技术 一种测算污染物生态毒性效应半效浓度的方法 (Method for measuring and calculating half-effect concentration of pollutant ecotoxicity effect ) 是由 王长友 于 2019-10-08 设计创作，主要内容包括：本发明公开了一种测算污染物生态毒性效应半效浓度的方法,基于种群水平上的毒性效应实验结果,利用种群间的生态关系构建群落毒性效应模型,将若干种群水平上的反应终点与群落水平上的反应终点关联起来,由种群水平上的污染物浓度与毒性效应之间的定量关系构造出群落水平上的污染物浓度与毒性效应之间的定量关系,以一定污染物浓度下群落中各种群共同周期积分生物量中值偏离对照群落共同周期积分生物量中值的程度作为半效浓度的判据,计算污染物生态毒性效应半效浓度。本发明不仅提高了半效浓度的生态相关性,还可以充分利用现有的大量单物种种群毒性效应实验数据,节约社会资源。(The invention discloses a method for measuring and calculating the half-effect concentration of the ecological toxic effect of a pollutant, which is characterized in that a community toxic effect model is constructed by utilizing the ecological relationship among populations based on the toxic effect experimental result on the population level, reaction end points on a plurality of population levels are associated with reaction end points on the community level, the quantitative relationship between the pollutant concentration on the population level and the toxic effect is constructed to form the quantitative relationship between the pollutant concentration on the community level and the toxic effect, and the half-effect concentration of the ecological toxic effect of the pollutant is calculated by taking the degree that the common period integral biomass median of each population in the community deviates from the common period integral biomass median of a control community under a certain pollutant concentration as the criterion of the half-effect concentration. The invention not only improves the ecological relevance of the half-effect concentration, but also can fully utilize a large amount of existing single species population toxicity effect experimental data and save social resources.)

1. A method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of pollutants is characterized by comprising the following steps:

step 1), performing a toxicity effect experiment of a single-algae culture system, and calculating the intrinsic growth rate and the environmental tolerance of microalgae populations under different pollutant concentrations;

step 2), carrying out a toxicity effect experiment of the zooplankton population, calculating the survival rate, the reproduction rate, the intrinsic growth rate and the generation time of the zooplankton under different pollutant concentrations, and the female oviposit rate and the population proliferation rate of the zooplankton population under different pollutant concentrations, and analyzing the toxicity effect of the pollutants on the zooplankton population by taking the female oviposit rate and the population proliferation rate as reaction endpoints;

step 3), calculating EC at each reaction end point based on the toxicity effect experiment result of plankton population₁₀、EC₅₀、EC₉₀And 95% confidence interval thereof, analyzing the sensitivity, reliability and stability of the ecotoxicity effect at different reaction end points, and determining the half-Effect Concentration (EC) of the ecotoxicity effect₅₀Constructing a population toxicity effect model at the reaction end point of (1);

step 4), performing a double-algae competition experiment, and calculating competition inhibition parameters of the microalgae;

step 5), performing an ingestion experiment of the zooplankton on the microalgae, and calculating the water drainage rate and the ingestion rate of the zooplankton on the microalgae;

step 6), carrying out a feeding behavior experiment of the zooplankton on the microalgae, and calculating a feeding selectivity coefficient of the zooplankton on the microalgae;

step 7), carrying out a feeding experiment of the large and medium zooplankton on the small zooplankton, and calculating the water filtration rate and the feeding rate of the large and medium zooplankton on the small zooplankton;

step 8), carrying out a feeding behavior experiment of the large and medium zooplankton on the small zooplankton, and calculating the feeding selectivity coefficient of the large and medium zooplankton on the small zooplankton;

step 9), constructing a community model with a feeding-competition relationship, establishing a quantitative relationship between the concentration of pollutants and the toxic effect on the population level through a population toxicity effect model based on the reaction end point on the population level, establishing the community model through a Logistic growth model and a Lotka-Voterra predation model, embedding the reaction end point on the population level into the community model as a community model parameter, and establishing a community toxicity effect model through the toxicity effect function of the reaction end point on a plurality of population levels according to the variation relationship between the concentration of pollutants and the model parameter;

step 10), simulating and calculating the change period of biomass of each biological population in the community based on a community toxicity effect model, further calculating the minimum common multiple of the change period and the minimum common multiple of the change period to obtain the common period of biomass change of each biological population in the community, and calculating the integral biomass and the median value thereof in the common period of the community under the preset concentration of each pollutant;

and 11) calculating the ecotoxicity effect half-effect concentration of the pollutant by taking the deviation of the common period integral biomass median value of any population in the community from the common period integral biomass median value of the control community by 50% as the criterion of the ecotoxicity effect half-effect concentration under each preset pollutant concentration, comprehensively using model calculation and mathematical statistics methods, wherein the pollutant ecotoxicity effect half-effect concentration is the minimum pollutant concentration of the deviation of the common period integral biomass median value of each population in the community from the common period integral biomass median value of the control community by 50%, and carrying out uncertainty analysis.

2. The method for calculating the semi-effective concentration of the ecotoxic effect of the pollutant according to claim 1, wherein the specific steps of the step 1) are as follows:

step 1.1), taking microalgae bred in a laboratory as an object, filtering seawater, adding a proper amount of nutrient salt as a culture solution, placing the culture solution in a culture bottle, and culturing in an illumination incubator according to preset culture temperature, salinity, illumination intensity, microalgae initial density and photoperiod;

step 1.2), setting 3 parallel samples in each group according to a preset pollutant concentration interval; performing species identification and counting under a microscope every day, and finishing the experiment when the population of the experimental object shows decay;

and 1.3) utilizing Logistic growth model fitting to obtain the intrinsic growth rate and the environmental tolerance of the microalgae, and analyzing the toxic effect of the pollutants on the microalgae population.

3. The method for calculating the semi-effective concentration of the ecotoxic effect of the pollutant according to claim 1, wherein the specific steps of the step 2) are as follows:

step 2.1), zooplankton monomer culture:

step 2.1.1), selecting healthy and active mature females of zooplankton, and placing 1 in each spawning bottle containing culture solution;

step 2.1.2), setting 10 parallel samples in each group according to the preset concentration interval of pollutants, and culturing at constant temperature according to the preset culture temperature; checking and counting the number of spawned eggs, the number of hatched larvae and the survival condition of a parent every day, removing the larvae, simultaneously changing culture solution water according to the proportion of about 1/2, and adding bait algae; the experiment was carried out until the female died;

step 2.1.3), calculating survival rate, reproduction rate, intrinsic growth rate and generation time of zooplankton, and analyzing toxic effect of pollutants on zooplankton population;

step 2.2), carrying out accumulative culture of zooplankton population:

step 2.2.1), quantitatively moving a plurality of lively and strong zooplankton which are pre-cultured under the same conditions to be placed in a beaker filled with culture solution;

step 2.2.2), setting 3 parallel samples in each group according to the preset concentration interval of pollutants, culturing at constant temperature until the population growth reaches a peak and begins to decline; feeding bait once every 24h during the experiment; counting the total individual number, the egg-carrying individual number and the number of fallen summer eggs of the zooplankton every day;

and 2.2.3) calculating the egg laying rate and the population proliferation rate of female zooplankton, and analyzing the toxic effect of pollutants on zooplankton populations.

4. The method for calculating the semi-effective concentration of the ecotoxic effect of the pollutant according to claim 1, wherein the specific steps of the step 4) are as follows:

step 4.1), mixing the experimental microalgae two by two, and setting the initial inoculation biomass of the microalgae according to the density of algae cells in a culture solution and the biological volume of a single algae cell so that the biomass ratio of the co-cultured microalgae is 1: 1;

and 4.2) fitting by using a Lotka-Volterra competition model to obtain a competition inhibition parameter.

5. The method for calculating the semi-effective concentration of the ecotoxic effect of the pollutant according to claim 1, wherein the specific steps of the step 5) are as follows:

step 5.1), domesticating and culturing the pre-cultured zooplankton in corresponding microalgae to be ingested for 3-5d, and starving for 24 h;

step 5.2), carrying out experiments in beakers, wherein the microalgae density is a preset optimal feeding density threshold, a plurality of zooplanktons are added into each beaker, each experimental group is provided with 3 parallel samples, and a control group without the zooplanktons is additionally arranged; covering the experimental beaker with black cloth and culturing for 24h under a dark condition;

step 5.3), adopting a bait concentration difference method, counting and calculating the density of algae cells by a blood ball counting plate under a microscope after 24 hours;

and 5.4) calculating the water drainage rate and the food intake rate of the zooplankton to the microalgae.

6. The method for calculating the semi-effective concentration of the ecotoxic effect of the pollutant according to claim 1, wherein the specific steps of the step 6) are as follows:

step 6.1), mixing the microalgae two by two, feeding the selected zooplankton, determining the mixing ratio according to the biological volume of a single algae cell to ensure that the biomass ratio of the two microalgae is 1:1, adding a plurality of zooplanktons into each beaker, arranging 3 parallel samples in each experimental group, and arranging a control group without the zooplanktons; covering the experimental beaker with black cloth and culturing for 24h under a dark condition;

step 6.2), calculating the ingestion rate of the zooplankton to the microalgae by adopting a bait concentration difference method;

and 6.3) determining the feeding selectivity coefficient according to the feeding degree of the zooplankton to different microalgae.

Technical Field

The invention relates to the field of water environment protection, in particular to a method for measuring and calculating the semi-effective concentration of the ecotoxic effect of pollutants, and specifically relates to a method for measuring and calculating the semi-effective concentration of the ecotoxic effect of the pollutants in the environment by utilizing the common period of biomass change of various groups in a community and the median value of integrated biomass in the time range of the common period.

Background

The core of the water quality standard is a dose effect relationship, and the basic link of the water quality standard is a method for determining the threshold concentration of the ecological toxicity effect of the pollutants. More complete pollutant threshold concentration measuring and calculating methods are researched by international influential organizations such as the United States Environmental Protection Agency (USEPA), the european union integrated research center (EUC), the world economic and cooperative Organization (OECD) and the like. The technical guide issued by organizations such as USEPA, EUC and OECD mainly adopts toxicity data of individual level reaction end points when calculating water quality standards. Although the more microscopic the level of the reaction endpoint, the stronger the causal relationship of the experimental results, the less significant the ecological significance, and the reaction endpoint at the individual level and below is difficult to meet the need for deriving the water quality benchmark. Even multiple reaction endpoints of the same ecological receptor tend to differ in the ability to characterize the adverse effects of pollutants. It is now common practice to select the most sensitive reaction endpoint as the reaction endpoint for determining the threshold concentration. However, the toxic effects determined by the most sensitive end-point of the response at the level of an individual do not necessarily reflect the population toxic effects, since the population effects are not necessarily determined by the most sensitive life cycle characteristics. Likewise, the toxic effects identified by the most sensitive reaction endpoint at a population level do not necessarily reflect community or ecosystem toxic effects, since ecological relationships such as competition, feeding, etc. in the ecosystem affect the toxic effects of the contaminant. Although the higher the level of the life building of the ecoreceptors, the greater the capacity of the corresponding reaction end points to comprehensively reflect the toxic effects of the ecosystem, only the reaction end points at the population level can be quantitatively measured at present due to the technical condition limitations. Therefore, the current research on the threshold concentration of the ecological toxicity effect of the pollutants is mostly limited to single species toxicity experiments, and ecological correlation among ecological system populations is not considered, so that the authenticity and reliability of research results are reduced.

With the development of ecotoxicology, community construction technology containing multiple populations in the field of ecology is introduced into the measurement and calculation of the ecological toxic effect half-effect concentration of pollutants, and an ecological system constructed manually is used for measuring and calculating the environmental pollutant toxic effect, so that the reliability of the ecological toxic effect half-effect concentration is improved to a certain extent. However, because of the existence of multiple competition and feeding relationships, a community comprising multiple populations is a complex nonlinear system, the biomass of each population in the community changes periodically, and the change period is different. Under the condition that the group is in the presence of pollutants, the biomass change of various groups is violently oscillated, and the oscillation period and the oscillation intensity of the biomass of the groups are different under different pollutant concentrations. Therefore, the biomass comparability of each population of the community oscillation system at the same specific time is very low, the community oscillation system is used as a toxicity reaction end point to analyze toxicity effects of different dye concentrations, and the reliability and the repeatability are very low; the calculated half-effect concentration of the ecological toxic effect of the pollutant based on the biomass of the population at a specific time also has larger uncertainty. Namely, the comparability and the representativeness of the periods of biomass of the populations at specific time of each treatment group are reduced due to the different oscillation periods, and the reliability and the repeatability of the calculated ecotoxicity effect are also reduced.

At present, no relevant report for improving the reliability of the measurement result of the ecological toxicity effect half-effect concentration of the pollutants by using the community common period integral biomass median exists. The measuring and calculating method can improve the reliability of measuring and calculating the ecological toxic effect half-effect concentration of the community containing multiple populations, is favorable for perfecting the derivation method of the ecological toxic effect half-effect concentration of pollutants, is favorable for compacting the scientific basis of environmental management, and promotes the harmonious development of industrial and agricultural production and ecological environment.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of pollutants aiming at the defects involved in the background technology.

The invention adopts the following technical scheme for solving the technical problems:

the invention discloses a method for measuring and calculating the half-effect concentration of the ecological toxic effect of a pollutant, which is characterized in that based on the experimental result of the toxic effect on a population level, a community toxic effect model is constructed by utilizing the ecological relationship among populations, reaction end points on a plurality of population levels are associated with reaction end points on the community level, the quantitative relationship between the pollutant concentration on the population level and the toxic effect is constructed to form the quantitative relationship between the pollutant concentration on the community level and the toxic effect, the half-effect concentration of the ecological toxic effect of the pollutant is calculated by taking the degree that the mean value of the integrated biomass of each population in the community in a certain pollutant concentration in a common period deviates from the mean value of the integrated biomass of a control community in a common period as the criterion of the half-effect:

step 1), performing a toxicity effect experiment of a single-algae culture system, and calculating the intrinsic growth rate and the environmental tolerance of microalgae populations under different pollutant concentrations;

step 2), carrying out a toxicity effect experiment of the zooplankton population, calculating the survival rate, the reproduction rate, the intrinsic growth rate and the generation time of the zooplankton under different pollutant concentrations, and the female oviposit rate and the population proliferation rate of the zooplankton population under different pollutant concentrations, and analyzing the toxicity effect of the pollutants on the zooplankton population by taking the female oviposit rate and the population proliferation rate as reaction endpoints;

step 3), calculating EC at each reaction end point based on the toxicity effect experiment result of plankton population₁₀、EC₅₀、EC₉₀And 95% confidence interval thereof, analyzing the sensitivity, reliability and stability of the ecotoxicity effect at different reaction end points, and determining the half-Effect Concentration (EC) of the ecotoxicity effect₅₀Constructing a population toxicity effect model at the reaction end point of (1);

step 4), performing a double-algae competition experiment, and calculating competition inhibition parameters of the microalgae;

step 5), performing an ingestion experiment of the zooplankton on the microalgae, and calculating the water drainage rate and the ingestion rate of the zooplankton on the microalgae;

step 6), carrying out a feeding behavior experiment of the zooplankton on the microalgae, and calculating a feeding selectivity coefficient of the zooplankton on the microalgae;

step 7), carrying out a feeding experiment of the large and medium zooplankton on the small zooplankton, and calculating the water filtration rate and the feeding rate of the large and medium zooplankton on the small zooplankton;

step 8), carrying out a feeding behavior experiment of the large and medium zooplankton on the small zooplankton, and calculating the feeding selectivity coefficient of the large and medium zooplankton on the small zooplankton;

step 9), constructing a community model with a feeding-competition relationship, establishing a quantitative relationship between the concentration of pollutants and the toxic effect on the population level through a population toxicity effect model based on the reaction end point on the population level, establishing the community model through a Logistic growth model and a Lotka-Voterra predation model, embedding the reaction end point on the population level into the community model as a community model parameter, and establishing a community toxicity effect model through the toxicity effect function of the reaction end point on a plurality of population levels according to the variation relationship between the concentration of pollutants and the model parameter;

step 10), simulating and calculating the change period of biomass of each biological population in the community based on a community toxicity effect model, further calculating the minimum common multiple of the change period and the minimum common multiple of the change period to obtain the common period of biomass change of each biological population in the community, and calculating the integral biomass and the median value thereof in the common period of the community under the preset concentration of each pollutant;

and 11) calculating the ecotoxicity effect half-effect concentration of the pollutant by taking the deviation of the common period integral biomass median value of any population in the community from the common period integral biomass median value of the control community by 50% as the criterion of the ecotoxicity effect half-effect concentration under each preset pollutant concentration, comprehensively using model calculation and mathematical statistics methods, wherein the pollutant ecotoxicity effect half-effect concentration is the minimum pollutant concentration of the deviation of the common period integral biomass median value of each population in the community from the common period integral biomass median value of the control community by 50%, and carrying out uncertainty analysis.

As a further optimization scheme of the method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of the pollutant, the specific steps of the step 1) are as follows:

step 1.1), taking microalgae bred in a laboratory as an object, filtering seawater, adding a proper amount of nutrient salt as a culture solution, placing the culture solution in a culture bottle, and culturing in an illumination incubator according to preset culture temperature, salinity, illumination intensity, microalgae initial density and photoperiod;

step 1.2), setting 3 parallel samples in each group according to a preset pollutant concentration interval; performing species identification and counting under a microscope every day, and finishing the experiment when the population of the experimental object shows decay;

and 1.3) utilizing Logistic growth model fitting to obtain the intrinsic growth rate and the environmental tolerance of the microalgae, and analyzing the toxic effect of the pollutants on the microalgae population.

As a further optimization scheme of the method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of the pollutant, the step 2) comprises the following specific steps:

step 2.1), zooplankton monomer culture:

step 2.1.1), selecting healthy and active mature females of zooplankton, and placing 1 in each spawning bottle containing culture solution;

step 2.1.2), setting 10 parallel samples in each group according to the preset concentration interval of pollutants, and culturing at constant temperature according to the preset culture temperature; checking and counting the number of spawned eggs, the number of hatched larvae and the survival condition of a parent every day, removing the larvae, simultaneously changing culture solution water according to the proportion of about 1/2, and adding bait algae; the experiment was carried out until the female died;

step 2.1.3), calculating survival rate, reproduction rate, intrinsic growth rate and generation time of zooplankton, and analyzing toxic effect of pollutants on zooplankton population;

step 2.2), carrying out accumulative culture of zooplankton population:

step 2.2.1), quantitatively moving a plurality of lively and strong zooplankton which are pre-cultured under the same conditions to be placed in a beaker filled with culture solution;

step 2.2.2), setting 3 parallel samples in each group according to the preset concentration interval of pollutants, culturing at constant temperature until the population growth reaches a peak and begins to decline; feeding bait once every 24h during the experiment; counting the total individual number, the egg-carrying individual number and the number of fallen summer eggs of the zooplankton every day;

and 2.2.3) calculating the egg laying rate and the population proliferation rate of female zooplankton, and analyzing the toxic effect of pollutants on zooplankton populations.

As a further optimization scheme of the method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of the pollutant, the specific steps of the step 4) are as follows:

step 4.1), mixing the experimental microalgae two by two, and setting the initial inoculation biomass of the microalgae according to the density of algae cells in a culture solution and the biological volume of a single algae cell so that the biomass ratio of the co-cultured microalgae is 1: 1;

and 4.2) fitting by using a Lotka-Volterra competition model to obtain a competition inhibition parameter.

As a further optimization scheme of the method for measuring and calculating the semi-effective concentration of the ecological toxicity effect of the pollutant, the specific steps of the step 5) are as follows:

step 5.1), domesticating and culturing the pre-cultured zooplankton in corresponding microalgae to be ingested for 3-5d, and starving for 24 h;

step 5.2), carrying out experiments in beakers, wherein the microalgae density is a preset optimal feeding density threshold, a plurality of zooplanktons are added into each beaker, each experimental group is provided with 3 parallel samples, and a control group without the zooplanktons is additionally arranged; covering the experimental beaker with black cloth and culturing for 24h under a dark condition;

step 5.3), adopting a bait concentration difference method, counting and calculating the density of algae cells by a blood ball counting plate under a microscope after 24 hours;

and 5.4) calculating the water drainage rate and the food intake rate of the zooplankton to the microalgae.

As a further optimization scheme of the method for measuring and calculating the half-effect concentration of the ecological toxicity effect of the pollutant, the specific steps of the step 6) are as follows:

step 6.1), mixing the microalgae two by two, feeding the selected zooplankton, determining the mixing ratio according to the biological volume of a single algae cell to ensure that the biomass ratio of the two microalgae is 1:1, adding a plurality of zooplanktons into each beaker, arranging 3 parallel samples in each experimental group, and arranging a control group without the zooplanktons; covering the experimental beaker with black cloth and culturing for 24h under a dark condition;

step 6.2), calculating the ingestion rate of the zooplankton to the microalgae by adopting a bait concentration difference method;

and 6.3) determining the feeding selectivity coefficient according to the feeding degree of the zooplankton to different microalgae.

Step 7), step 8) may be detailed with reference to step 5) and step 6).

Compared with the prior art, the invention adopting the technical scheme has the following technical effects:

according to the invention, based on the periodic change of the biomass of various groups in the community, a community toxicity effect model is constructed through the ecological relationship among the biological populations, the common period of the change of the biomass of various groups in the community is calculated, and then the integral biomass and the median value of each population in the common period are calculated, so that the problem of poor comparability caused by the phase difference of the periods of the biomass of various groups at the same specific moment is solved, the problem of poor comparability of the biomass between different treatments caused by the oscillation period and the oscillation intensity difference of the change of the biomass of various groups due to different concentrations of pollutants is solved, and the reliability and the accuracy of the calculation of the half-effect concentration of the ecological toxicity effect of the pollutants are improved. In addition, the method can utilize the toxicity effect data on the population level to measure and calculate the ecological toxicity effect half-effect concentration of the pollutants on the population level, not only improves the ecological relevance of the half-effect concentration, but also can fully utilize the existing large amount of single-species population toxicity effect experimental data, and saves social resources. The biotoxicity experiments related to the method are all routine experiments, and a community model designed for monitoring biology can be applied to different pollutants, so that the method is convenient to popularize and use in a wide range of water quality environment monitoring departments.

Drawings

FIG. 1 is a schematic diagram of biomass integrated over a common period and biomass at specific times for various groups in a community;

FIG. 2 is a graph showing the variation of oscillation period and oscillation intensity of various group biomass caused by different concentrations of contaminants;

FIG. 3 is a graph showing the variation of the biomass median value (. zeta.) integrated in the common period of various groups and the biomass median value (TB) quantity integrated in the common period of the groups with the concentration of petroleum hydrocarbon.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the attached drawings:

the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, components are exaggerated for clarity.

The invention discloses a method for measuring and calculating the half-effect concentration of the ecological toxicity effect of a pollutant, which is characterized by taking the toxicity effect experiment of plankton populations as a basis, obtaining model parameters, constructing a community toxicity effect model, calculating the integral biomass and the median value of each population in a community in a common period time range under a certain pollutant concentration, and further calculating the half-effect concentration of the pollutant. The scientificity of the method can be evaluated by comparing the experimental results with the communities, analyzing the goodness of fit of the model and verifying the calculation result of the half-effect concentration, as shown in fig. 1, wherein t1 and t2 are two specific times respectively; t is a common period; the relative sizes of the areas of P1, P2, Z2 and Z1 represent the relative sizes of biomass of the populations P1, P2, Z2 and Z1.

1. Ecological toxicity effect experiment and parameter acquisition:

(1) toxicity Effect test of Single microalgae

Using laboratory breeding to obtain large red algae of Qingdao (Pandalia glauca L.) (Platymonas helgolandica) And dinoflagellates such as Strongylocentrotus (Haematococcus)Isochrysis galbana) Filtering seawater to adjust salinity to 9, adding a proper amount of nutrient salt as a culture solution, placing the culture solution in a 1000mL conical flask, and culturing in an illumination incubator. The culture temperature is 15 deg.C (or 23 deg.C, respectively matched with suitable culture temperature of two zooplankton in later experiment), pH is 8.0, and illumination intensity is 60 μmol m^-2s^-1Initial densities were all 1X 10⁴cells mL^-1And a light period 12L: 12D. The petroleum hydrocarbon contaminants were set at 6 concentration gradients, including the control. Each set was set with 3 replicates. 5mL of the culture medium was taken every 24 hours, and the fixed solution was added thereto to carry out species identification and counting under a microscope. And finishing the experiment when the experimental object population shows decay. And fitting by utilizing a Logistic growth model according to the experimental result to obtain parameters such as the intrinsic growth rate, the environment accommodation capacity and the like of the microalgae.

(2) Competition experiment with diatom

Mixing the large Platymonas subcordata, the Isochrysis galbana and the like, setting the initial inoculation biomass of the microalgae according to the cell density of the algae in the culture solution and the biological volume of a single algae cell, ensuring that the biomass ratio of the co-cultured microalgae is 1:1, and ensuring that other culture conditions and detection indexes are the same as those of a single algae toxicity effect experiment. And fitting by using a Lotka-Volterra competition model according to the experimental result to obtain a competition inhibition parameter.

(3) Zooplankton population toxicity effect experiment

Selecting healthy and active daphnia magna (daphnia magna) by monomer cultureD.magna) (or Brachionus plicatilis: (or Brachionus plicatilis)B.plicatilis) ) mature females were placed in 15mL 6-well plates, respectively. The concentration of the contaminant was set up with 6 concentration gradients, including control samples, with 10 replicates per set. The experiment is carried out at a constant temperature of 23 ℃, the number of eggs laid, the number of hatched larvae and the survival condition of the parent are checked and counted every day, the larvae are removed, meanwhile, the seawater is changed according to the proportion of about 1/2, and bait algae is added until the females die. According to the experimental result, the survival rate, the reproduction rate, the intrinsic growth rate, the generation time and the like of the zooplankton can be calculated.

Quantitatively moving active and robust daphnia magna (or Brachionus plicatilis) pre-cultured under the same condition through colony accumulated culture (the initial density is 10ind mL)^-1) Placed in a beaker containing 300mL of culture medium. The concentration of the contaminant was set with 6 concentration gradients, including control samples, with 3 replicates per group. The experiment was incubated at 23 ℃. The culture time is until the population reaches a peak and begins to decline. Baits were fed every 24h during the experiment. Counting the total number of individual zooplankton, the number of egg-carrying individual and the number of fallen summer eggs (putting back to the original culture after counting) every day, repeating for three times continuously, and calculating the average number. And calculating the egg laying rate of female zooplankton, the population proliferation rate and the like according to the experimental result.

(4) Feeding and choking behavior experiment of zooplankton

Separately feeding Daphnia magna (or Brachionus plicatilis) such as Pandalis island and Dictyotanus globulus. The zooplankton is obtained by laboratory breeding, and before the experiment, the pre-cultured zooplankton is domesticated and cultured in the corresponding microalgae to be ingested for 3-5d respectively, and then starved for 24 h.The experiment is carried out in 150mL beakers, the volume of the experimental algae liquid is 50mL, the density of the microalgae is set as the optimal feeding density according to the preliminary experiment, the temperature is 23 ℃, and 10ind mL is added into each beaker^-1Daphnia magna (or Brachionus plicatilis) each experimental group is provided with 3 parallel samples, and a control group without zooplankton is additionally arranged. The experimental beaker was covered with black cloth and incubated in the dark for 24 h. Counting after 24 hours by adopting a bait concentration difference method, fixing algae liquid by using the Rogowski fluid, counting by using a blood ball counting plate under a microscope, calculating the cell density of algae, and calculating the water filtration rate and the ingestion rate of the microalgae by using daphnia magna (or Brachionus plicatilis) according to an experimental result. The steps of the experimental method for the food intake and feeding selection behaviors of the ragworm by the daphnia magna are the same as the steps of the experimental method.

Mixing the green island large flat algae, the dinoflagellate such as the green island and the like, feeding daphnia magna (or the Brachionus plicatilis), determining the mixing ratio according to the biological volume of a single algae cell, and enabling the biomass ratio of the two microalgae to be 1:1, wherein the other experimental conditions and steps are the same as the above. And calculating the ingestion rate of daphnia magna (or Brachionus plicatilis) on the microalgae by adopting a bait concentration difference method, and calculating the ingestion selectivity coefficient of the daphnia magna (or Brachionus plicatilis) according to the ingestion degree of the daphnia magna on different microalgae.

(5) Community toxicity effect experiment

Mixing the green island large flat algae, the dinoflagellate such as the ball and the like to ensure that the biomass ratio of the co-cultured microalgae is 1:1, inoculating the daphnia magna and the Brachionus plicatilis into the microalgae mixed solution, and adding pollutants. The initial inoculation of microalgae biomass, zooplankton biomass and concentration interval of added contaminants were as above. Selecting an active, robust and pre-cultured individual under the same condition for zooplankton, performing acclimation culture in corresponding microalgae mixed liquor for 3-5d before experiment, and starving for 24h to empty intestinal tract. The experiment was carried out in a 10L glass vessel and incubated at 23 ℃. Each experimental group was provided with 3 replicates and a control group without added contaminants. Other culture conditions were the same as those in the single algae toxicity effect experiment. The change of the species and the number of the counted plankton is observed every day, and the counting method is the same as the corresponding experiment. And determining the end time of the experiment according to the stable duration of the number of the plankton populations in the experiment system. As shown in fig. 2, t1 and t2 are two specific times respectively; t1 is the common cycle under the condition of pollutant concentration C1; t2 is the common cycle under the condition of pollutant concentration C2; t1> T2.

2. Acquisition of population toxicity effect parameters:

according to the result of the population toxicity test, a Log-logistic model is used for calculating EC under different population toxicity reaction end points (parameters such as intrinsic growth rate, environment accommodating amount and the like of microalgae population, survival rate, reproduction rate, intrinsic growth rate, generation time, female oviposition rate, population proliferation rate, drainage rate, ingestion rate and the like of zooplankton population)₁₀、EC₅₀、 EC₉₀And 95% confidence intervals thereof, and the sensitivity, reliability and stability of the toxic effect at different reaction endpoints.

And (4) conclusion: through reaction end point analysis, parameters such as the intrinsic growth rate and the environmental tolerance of the microalgae population and parameters such as the survival rate, the intrinsic growth rate, the water filtration rate and the ingestion rate of the zooplankton population are found to have higher sensitivity, reliability and stability, and can be used as a reaction end point on the population level to establish a population toxicity effect model to represent the pollutant ecotoxicity effect on the population level.

3. Construction of a community toxicity effect model:

according to parameters measured by a diatom competition experiment and a zooplankton feeding and food choosing behavior experiment, a community model is established based on a Logistic growth model and a Lotka-Voterra predation model, wherein density constraints are considered for microalgae growth, the density constraints are not considered for zooplankton growth, and the variation relationship between pollutant concentration and model coefficients is constructed through a plurality of toxicity effect functions based on reaction endpoints on a population level, so that the population toxicity effect model is embedded into the community model to construct the community ecotoxicity effect model.

And (4) analyzing results: and (3) associating the reaction end points on a plurality of population levels with the reaction end points on the population level by utilizing a community model established by ecological relations of competition-food intake and the like, and constructing a quantitative relation between the pollutant concentration and the toxic effect on the community level by using the quantitative relation between the pollutant concentration and the toxic effect on the population level.

4. Calculation of common period integral biomass of various groups in community

Under the experimental conditions, the biomass of each biological population in the community changes periodically, and the change period is different (T)_Z1,T_Z2,T_P1,T_P2). Simulating and calculating the change period (minimum positive period) of the biomass of each group in the community by using a community toxicity effect model, calculating the minimum common multiple of the change period and the minimum positive period to obtain the common period (T) of the biomass change of each group of the whole community, and further calculating the common period integral biomass of the whole community and the median value M thereof under a certain pollutant concentration_T(Z1_T,Z2_T,P1_T,P2_T)。

And (4) analyzing results: the biomass can be characterized by the periodic variation of the biomass of the community. The biomass median value of the whole community common period integration under a certain pollutant concentration is taken as a reaction end point of toxic effect, and the method not only has higher ecological relevance than the reaction end point on the population level, but also has higher representativeness, comparability and reliability compared with the time biomass taken as the reaction end point.

5. Measurement and verification of half-effect concentration:

the median biomass integrated over the community co-period is the end of the toxic effect reaction at the community level. Integrating biomass median M of community common period under the condition of certain pollutant concentration_T(Z1_T,Z2_T,P1_T,P2_T) Changes are generated and deviate from the prior community common period integral biomass median value M_T0*(Z1_T0*,Z2_T0*,P1_T0*,P2_T0Here we integrate the biomass median ξ over a common period for any population in the community_Tc(ξ_Tc= Z1_Tc, Z2_Tc, P1_Tc, P2_Tc) Deviation from common period integral biomass median xi of corresponding population of original population_T0*(ξ_T0*=Z1_T0*,Z2_T0*,P1_T0*,P2 _T050%) as the biotoxicity effect half-effect concentration and applying the Monte-Carlo method for threshold concentration reductionDeterministic analysis, as shown in fig. 3.

Determining the biomass of the planktonic organism population at each pollutant concentration in the biotoxicity effect experiment (Z1)_t,Z2_t, P1_t, P2_t) Corresponding result to model calculation(Z1 _t , Z2 _t , P1 _t , P2 _t )Comparing, calculating to obtain model goodness of fitR ²>0.9 inspection of the model-calculated median biomass values integrated over a common period of the communityM _T (Z1 _T ,Z2 _T ,P1 _T ,P2 _T )Integration of median biomass M with experimentally determined communities over a common period_T(Z1_T,Z2_T,P1_T,P2_T) And no significant difference exists, so that the reliability of the model calculation result is verified.

And (4) conclusion: the degree that the mean value of the biomass of the common period integral of any population in the community deviates from the mean value of the biomass of the common period integral of the corresponding population of the community of a control experiment under a certain pollutant concentration is taken as a criterion of half-effective concentration, the pollutant concentration of 50% of the mean value of the biomass of the common period integral of any population in the control experiment and the minimum half-Effective Concentration (EC) of each population are calculated by an iterative approximation method₅₀) As the median concentration of the colonies, the confidence intervals were obtained by the Monte-Carlo method. The result of the calculation of the half-effect concentration of the ecotoxicity effect of the petroleum hydrocarbon is 3.6 +/-0.2 mg/L.

By applying the method, based on population toxicity effect experimental data of the Pantoea glaucus, the Isochrysis galbana, the Brachionus plicatilis, the daphnia magna and the like, the measured pollutant ecotoxicity effect half-effect concentration measured by taking the common period integral biomass median deviation degree calculated by the community model as a criterion is basically consistent with the measured result of an experimental community formed by the Pantoea glaucus, the Isochrysis galbana, the Brachionus plicatilis and the daphnia magna. The result further verifies the operability and reliability of measuring and calculating the toxicity effect half-effect concentration of the pollutants by using the community common period integral biomass median as a reaction end point, and simultaneously shows that the method has certain application value in measuring and calculating the toxicity effect half-effect concentration of the pollutants in the water environment.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.