阅读说明：本技术 一种利用海湾扇贝d型幼体进行现场慢性毒理试验的方法 (Method for carrying out on-site chronic toxicological test by using bay scallop D-type larvae ) 是由 纪志永 李杨 武洪庆 穆迪 谭丹丹 郭小甫 李非 王士钊 袁俊生 于 2021-10-25 设计创作，主要内容包括：提供了一种利用海湾扇贝D型幼体进行现场慢性毒性试验的方法,包括：试验材料的前处理、海湾扇贝幼体的选优和喂养、慢性毒性试验及分析计算。其中,分析计算时,首先统计扇贝幼体附着率,而后对附着率与浓度进行方差分析,在确定浓度对附着率有显著影响后,绘制浓度-效应关系图,确定污染物对海湾扇贝的无效应浓度(NOEC)与低效应浓度(LOEC),最后对试验数据进行可靠性评价,结果为限制可靠及以上时为可用数据。本发明为现场试验方法,充分考虑了自然环境下污染物对水生生物的毒性效应,克服了目前国内贝类慢性试验研究方法单一、未充分考虑自然条件下水生生物对污染物的耐受能力以及试验结果未进行可靠性验证等问题。(There is provided a method for conducting on-site chronic toxicity testing using bay scallop D-larvae, comprising: pretreatment of test materials, selection and feeding of bay scallop larvae, chronic toxicity test and analysis and calculation. During analysis and calculation, firstly, the attachment rate of scallop larvae is counted, then, variance analysis is carried out on the attachment rate and the concentration, after the concentration is determined to have obvious influence on the attachment rate, a concentration-effect relation graph is drawn, the ineffective response concentration (NOEC) and the ineffective response concentration (LOEC) of pollutants on bay scallops are determined, finally, reliability evaluation is carried out on test data, and the result is available data when the limitation is reliable or above. The invention is a field test method, fully considers the toxic effect of pollutants on aquatic organisms in the natural environment, and overcomes the problems that the existing domestic shellfish chronic test research method is single, the tolerance of the aquatic organisms to the pollutants under the natural condition is not fully considered, the reliability of the test result is not verified, and the like.)

1. A method for carrying out on-site chronic toxicological tests by using D-type larvae of Argopecten irradians comprises the following steps:

(1) preparing pretreated seawater;

(2) preparing a pretreated test container and a test device;

(3) preparing bay scallop D-type larvae: selecting normally-developed D-shaped young bay scallops, domesticating the D-shaped young bay scallops by using the pretreated seawater obtained in the step (1), and starting a chronic toxicity test after the D-shaped young bay scallops eat the seawater basically normally;

(4) carrying out a chronic toxicology test:

(4.1) preparing a pollutant mother solution: when the pollutant is a pollutant dissolved in seawater, the pollutant mother liquor comprises the pollutant and pretreated seawater; when the pollutant is a pollutant insoluble in seawater, the pollutant mother liquor comprises the pollutant, a cosolvent for dissolving the pollutant and pretreated seawater;

(4.2) preparing a control group and a plurality of pollutant test groups:

respectively injecting preset water samples of a control group and a pollution test group into the pretreated test containers obtained in the step (2), and keeping the other conditions the same to form the control group and the pollution test group;

wherein, when the pollutant is a pollutant dissolved in seawater, the control group is a blank control group; when the pollutant is a pollutant which is difficult to dissolve in seawater, the control group comprises a blank control group and a cosolvent control group;

wherein the content of the first and second substances,

the water sample of the blank control group is the pretreated seawater obtained in the step (1);

the water sample of the cosolvent control group consists of the cosolvent used in the step (4.1) and the pretreated seawater obtained in the step (1);

the water samples of the plurality of pollutant test groups are respectively pollutant test water samples with different pollutant concentrations, which are prepared by the pollutant mother liquor obtained in the step (4.1) and the pretreated seawater obtained in the step (1);

(4.3) putting 8-10 million gulf scallop D-type larvae obtained in the step (3) into each control group and each pollutant test group, starting a chronic toxicological test, and putting a plurality of attaching bases into each control group and each pollutant test group after the test is carried out for n days, wherein n is a positive integer in the range of 8-12;

(4.4) the total days of the test is 14-18 days, after the test is finished, taking out the attaching bases, randomly finding 4-8 areas on each attaching base, taking 12-20 attaching points in each area, and counting the attaching rate of the larva on each attaching base by using a microscope;

(5) and (3) analysis and calculation: counting and calculating the attachment rate of the scallop larvae on the attachment base in each pollutant test group and each control group, and performing variance analysis on the pollutant concentration and the larva attachment rate to obtain the ineffective response concentration and the ineffective response concentration of the pollutants; wherein, the attachment rate of the blank control group is set as 100%, the ineffective response concentration is defined as the highest contaminant concentration in all contaminant test groups with the attachment rate of the larvae greater than 95% of the attachment rate of the larvae of the blank control group, and the ineffective response concentration is defined as the highest contaminant concentration in all contaminant test groups with the attachment rate of the larvae greater than 75% of the attachment rate of the larvae of the blank control group.

2. The on-site chronic toxicological test method according to claim 1, wherein in the step (1), the seawater to be pretreated is natural seawater.

3. The on-site chronic toxicology test method of claim 1 or 2, wherein the preparing the pretreated seawater in the step (1) comprises: taking seawater, and filtering the seawater through three-stage filtration consisting of a sand filter, 200-sand 300-mesh and 400-sand 600-mesh bolting silk to obtain the pretreated seawater.

4. The on-site chronic toxicology testing method of claim 1, wherein in the step (2), the preparing the pre-treated test receptacles and test instruments comprises: cleaning a test container, filling the pretreated seawater obtained in the step (1) with sodium hypochlorite bleaching solution in a volume ratio of not more than 1:300, standing for 20-28h, washing with the pretreated seawater for multiple times after standing, drying in the air, continuously filling the pretreated seawater, standing for 20-28h, and using after standing; wherein the test device is disposed in the test container during the pretreatment of the test container.

5. The on-site chronic toxicology testing method of claim 1, wherein in the step (4.2), the number of the pollutant test groups is 4-8.

6. The on-site chronic toxicology testing method of claim 1, wherein in the step (4.2), each control group and each pollutant test group are provided with 2-4 parallel groups.

7. The on-site chronic toxicological test method according to claim 1, wherein in the step (4.4), the attachment base is a palm curtain woven with palm ropes; 2-4 pieces of attachment base were placed in each test vessel, wherein each piece of attachment base had 1000-2000 attachment points.

8. The on-site chronic toxicological test method according to claim 1, wherein in the step (4), the whole test process is carried out in a nursery room of a farm, a semi-static exposure test is adopted, and the water change amount is 50-150L every day during the test.

9. The on-site chronic toxicological test method according to claim 1, wherein in the step (4), the temperature of the test seawater is 23 ± 1 ℃, the light is 12-15h, the dark is 9-12h, and the dissolved oxygen is maintained at more than 60% of the saturation value.

10. A method of detecting marine water ecotoxicity by the on-site chronic toxicology test method according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of environmental toxicology, in particular to a method for carrying out on-site chronic toxicology test on bay scallop D-type larvae.

Background

Coastal waters are severely threatened by increasing human activities such as shipping, fishery, tourism and industry, and the survival of aquatic organisms in the waters is also severely threatened by the discharge of various pollutants, and many pollutants can even further threaten the health of human beings through a food chain. Therefore, the management of pollutants has become a main target for realizing the sustainable utilization of resources and the sustainable development of ecosystems.

The toxicity test of aquatic organisms mainly measures the toxic effect of pollutants on organisms in a short term or a long term, and the test result provides an important evaluation basis for environmental pollutants and pollution degree.

Bay scallops, as the introduced economic species, have become one of the important species for offshore culture in the north of China due to high economic value and strong temperature and salinity tolerance, and gradually expand to the sea areas of the east and south seas. The bay scallop develops to complete the development of the shells after the D-type larvae develop, the average shell length is about 100 microns, and the bay scallop can be observed by a microscope. The eye points of the larvae grow from small to large in about ten days, the color becomes darker and darker, the larvae have the double functions of creeping and swimming, the larvae are called creeping larvae, and the average shell length of the larvae also grows to about 180 micrometers. After the larvae are completely transformed into young shellfish after 13-15 days of development, the average shell length of the young shellfish is 500 micrometers, and the young shellfish with the size of the millet grains can be seen on the attachment base. Therefore, based on high economic value and high feasibility of long-term test, bay scallops are selected as the chronic test organisms, and the physicochemical conditions of test water are comprehensively considered, so that reliable data can be obtained and used as one of data sources for deducing the water quality reference value of the seawater.

The invention aims to provide a method for carrying out on-site chronic toxicological tests by utilizing D-type bay scallops, which has wide applicability, is simple and effective, and aims to overcome the defects of an on-site test scheme in the existing test system, reduce the living conditions of organisms in the natural environment as much as possible, simulate the influence effect of pollutants on the organisms in a dynamic balance.

Disclosure of Invention

Aiming at the defects of field tests in the existing test system, the living conditions of organisms in the natural environment are restored as far as possible, the influence effect of pollutants on the organisms in a dynamic balance is simulated, the reliability of test results is evaluated, and finally, the method for carrying out the field chronic toxicological test by utilizing the D-type bay scallop larvae is wide in applicability, simple, easy and effective. The toxicity data obtained by the method can be finally used for deducing the seawater quality standard through reliability evaluation.

A method for carrying out on-site chronic toxicological tests by using D-type larvae of Argopecten irradians comprises the following steps:

(1) preparing pretreated seawater;

(2) preparing a pretreated test container and a test device;

(3) preparing bay scallop D-type larvae: selecting normally-developed D-shaped young bay scallops, domesticating the D-shaped young bay scallops by using the pretreated seawater obtained in the step (1), and starting a chronic toxicity test after the D-shaped young bay scallops eat the seawater basically normally;

(4) carrying out a chronic toxicology test:

(4.1) preparing a pollutant mother solution: when the pollutant is a pollutant dissolved in seawater, the pollutant mother liquor comprises the pollutant and pretreated seawater; when the pollutant is a pollutant insoluble in seawater, the pollutant mother liquor comprises the pollutant, a cosolvent for dissolving the pollutant and pretreated seawater;

(4.2) preparing a control group and a plurality of pollutant test groups:

respectively injecting preset water samples of a control group and a pollution test group into the pretreated test containers obtained in the step (2), and keeping the other conditions the same to form the control group and the pollution test group;

wherein, when the pollutant is a pollutant dissolved in seawater, the control group is a blank control group; when the pollutant is a pollutant which is difficult to dissolve in seawater, the control group comprises a blank control group and a cosolvent control group;

wherein the content of the first and second substances,

the water sample of the blank control group is the pretreated seawater obtained in the step (1);

the water sample of the cosolvent control group consists of the cosolvent used in the step (4.1) and the pretreated seawater obtained in the step (1);

the water samples of the plurality of pollutant test groups are respectively pollutant test water samples with different pollutant concentrations, which are prepared by the pollutant mother liquor obtained in the step (4.1) and the pretreated seawater obtained in the step (1);

(4.3) putting 8-10 ten thousand gulf scallop D-type larvae obtained in the step (3) into each control group and each pollutant test group, starting a chronic toxicological test, and respectively putting a plurality of attaching bases into each blank control group and each pollutant test group after the test is carried out for n days, wherein n is a positive integer in the range of 8-12, and preferably n is 9;

(4.4) the total days of the test is 14-18 days, after the test is finished, taking out the attaching bases, randomly finding 4-8 (preferably 5) attaching points on each attaching base, taking 12-20 (preferably 15) attaching points on each attaching base, and counting the attaching rate of the larva on each attaching base by using a microscope;

(5) and (3) analysis and calculation: and (3) counting and calculating the attachment rate of the scallop larvae on the attachment base in each pollutant test group and each control group, and performing variance analysis on the pollutant concentration and the attachment rate of the larvae to obtain the ineffective response concentration (NOEC) and the ineffective response concentration (LOEC) of the pollutants.

Wherein, in the step (1), the seawater to be pretreated is natural seawater.

Wherein, in the step (1), the preparing the pretreated seawater comprises: taking seawater, and filtering the seawater through three-stage filtration consisting of a sand filter, 200-sand 300-mesh and 400-sand 600-mesh bolting silk to obtain the pretreated seawater.

Wherein in step (2), the preparing the pretreated test container and the test device comprises: cleaning a test container, filling the pretreated seawater obtained in the step (1), adding sodium hypochlorite bleaching solution according to a volume ratio of not more than 1:300, standing for 20-28h (preferably 24h), washing for multiple times by using the pretreated seawater after standing is finished, drying in the air, continuously filling the pretreated seawater, standing for 20-28h (preferably 24h), and using after standing is finished; the test apparatus is placed in the test container for treatment in the process of pretreating the test container, and comprises a silicone tube, a gas stone, a water exchanger, an adhesive base and the like.

Wherein, the test container comprises a nylon barrel with the depth of 100-120cm and the bottom radius of 25-35cm (preferably 30cm) or a square glass cylinder with the same depth under the same volume.

In the step (3), after the fertilized eggs of the bay scallop are incubated for 22-26h (preferably 24h), 250-350-mesh (preferably 300-mesh) bolting silk is used for filtering, the un-incubated single-cell transparent eggs are discharged with water, and the rest is normally developed D-shaped larvae, namely the preferred normally developed D-shaped larvae of the bay scallop; putting the optimized D-type larvae of bay scallops which normally develop into the pretreated test container, wherein the pretreated test container is filled with more than 200L of pretreated seawater, then adding 400 plus 500mL of the Chrysophyte with the weight as bait, observing the ingestion condition of the larvae by using a microscope after 20-26h (preferably 24h), observing that most of the scallop larvae are normally ingested, starting a chronic toxicity test, adding 300mL of the Platymonas subcordanus terniflora into the bait after 3-4 days of the test, and properly reducing the feeding amount of the Chrysophyte with the weight of 200 plus 300 mL.

Wherein, in the step (4.2), the number of the pollutant test groups is 4-8, preferably 5.

In the step (4.2), each control group and each pollutant test group are provided with 2-4 parallel groups.

Wherein, before the step (4.3), scallop larvae cultured in the test container are concentrated by using a net made of 250-350 mesh (preferably 300 mesh) bolting silk, the larvae are concentrated into a beaker, and the number of the larvae in the beaker is calculated by sampling and counting for a plurality of times.

Wherein in the step (4.3), feeding and death of scallop larvae are regularly observed and recorded during the chronic toxicological test.

Wherein in the step (4.4), the attachment base is a palm curtain woven by palm ropes; 2-4 pieces of attachment base are dosed per test vessel, wherein each piece of attachment base carries 1000 and 2000 (preferably 1500) attachment points.

In the step (4), the whole test process is carried out in a nursery room of a farm, a semi-static exposure test is adopted, the water change amount is 50-150L (preferably 100L) every day during the test period, and dead golden algae and flat algae at the bottom of the test container are periodically cleaned; in the whole test process, the temperature is maintained by the integral temperature control of the seedling raising room and the large-volume water filling of the test container, the temperature of the test seawater is 23 +/-1 ℃, the illumination is 12-15 hours (preferably 14 hours) and the darkness is 9-12 hours (preferably 10 hours), the dissolved oxygen is maintained above 60% of the saturation value, and the illumination and the oxygen increasing are realized by sharing a light illuminator and an oxygen increasing pump.

In the step (5), the attachment rate of the scallop larvae on the attachment base in each pollutant test group and the blank control group is counted and calculated, variance analysis is performed on the pollutant concentration and the larva attachment rate, a concentration-effect relation graph is further drawn when the pollutant concentration has a significant influence on the larva attachment rate, and then the relation between each concentration and the blank control group is analyzed, so that the ineffective response concentration (NOEC) and the ineffective response concentration (LOEC) of the toxic substances are obtained. Wherein, the attachment rate of the blank control group is set as 100%, the ineffective response concentration (NOEC) is defined as the highest contaminant concentration in all contaminant test groups with the attachment rate of the larvae greater than 95% of the attachment rate of the larvae of the blank control group, and the ineffective response concentration (LOEC) is defined as the highest contaminant concentration in all contaminant test groups with the attachment rate of the larvae greater than 75% of the attachment rate of the larvae of the blank control group. For the pollutants which are difficult to dissolve in the seawater, the cosolvent has no obvious influence on the scallop larvae if the adhesion rate of the cosolvent control group is greater than or equal to the adhesion rate of the cosolvent with the ineffective response concentration. Finally, a reliability assessment is made on the results to determine data availability.

The invention also provides a method for detecting the ecological toxicity of the ocean water, which is realized by the field chronic toxicological test method.

The invention has the following beneficial technical effects:

1. the offshore culture economic variety is preferably selected, the bay scallops have high economic benefit and wide culture area distribution, and the pollutant tolerance of the economic variety can be well expressed. In addition, the test period of the invention covers the whole metamorphosis period from D-type larvae after fertilized eggs to creeping larvae and then to juvenile mollusks. The method specifically establishes a field test method of pollutants and an evaluation means of test results, and provides guarantee for the accuracy of subsequent water quality reference derivation.

2. The field chronic toxicology test is provided with a plurality of pollutant test groups and 1 blank group, each test group and the blank group are provided with a plurality of parallel groups, the interference of the increase of an instrument in the test process on the development of the scallop larvae is reduced as much as possible by using the temperature control and the oxygen dissolving capacity of a large-volume container, and then the attachment environment in the process of transforming the creeping larvae into the larvae under the natural condition is simulated by adding the attachment base, so the whole test reduces the growth and development process of the scallops and the situation after the interference of toxic pollutants as much as possible.

3. The test method is rigorous in thinking, after the scallop attachment rate is obtained by expanding the statistical method of a five-point counting method in biology, firstly, the variance analysis of concentration-attachment rate is carried out to determine whether the scallop attachment rate and the concentration-attachment rate have significant influence, then, required data is obtained according to the analysis of the concentration-effect relation, and then, the reliability of the obtained data is evaluated. Compared with the existing test system, the obtained result has higher reliability and is more beneficial to the improvement of the reference accuracy of the water quality in the future.

Brief description of the drawings

FIG. 1 is a flow chart of an experiment of the present invention;

FIG. 2 is a line graph showing the concentration-effect of scallop larvae in each contaminant test group and blank control group in example 1 of the present invention;

FIG. 3 is a histogram of the influence rate of concentration-effect in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The invention relates to the technical field of environmental toxicology, the result of the invention can be applied to the field of water quality benchmark, and in particular relates to a method for carrying out on-site chronic toxicology test on D-type bay scallops. The invention is utilized to explore the toxic effect of heavy metal pollutants such as copper, zinc, cadmium and the like and organic pollutants such as naphthalene, phenanthrene and the like in the natural environment. The invention is further described below by way of specific examples using copper as contaminant.

Example 1

In this example, the bay scallop D-type larvae were used for chronic toxicity test of copper, taking copper as an example of the contaminant. The method comprises the following specific steps:

1. pretreatment of seawater and test materials:

collecting seawater subjected to three-stage filtration by a sand filter and a 300-mesh and 500-mesh bolting silk as pretreated seawater for standby, cleaning a nylon barrel with the depth of 120cm and the bottom radius of 30cm, and then filling the pretreated seawater with the weight ratio of 1: adding sodium hypochlorite bleaching solution according to the volume ratio of 600, and standing for 24 h. After the standing is finished, the pretreated seawater is used for washing for multiple times and is dried in the air, the pretreated seawater is filled continuously, the standing is carried out for 24 hours, and the seawater can be used after the standing is finished. In the process of pretreating the nylon barrel, the silicone tube, the air stone, the water exchanger, the attaching base and the like are put into the barrel together for pretreatment.

2. Selecting and feeding the fertilized eggs of bay scallops:

and after the scallop fertilized eggs are incubated for 24 hours, the scallop fertilized eggs are filtered by using 300-mesh bolting silk, the scallop fertilized eggs still become single-cell transparent eggs which are discharged along with water, and the remaining scallop fertilized eggs are D-type larvae after normal development. After the selection is finished, putting the scallop larvae into a pretreated nylon barrel, adding 400mL of the chrysophyceae spherules, observing the ingestion condition of the larvae by using a microscope after 24 hours, observing that most of the scallop larvae begin a chronic toxicity test after eating normally, adding 200mL of the physcomitrella brassicae into the feeding bait 3 days after the test begins, and simultaneously properly reducing the feeding amount of the chrysophyceae spherules.

3. Chronic toxicology test:

a. preparing a copper sulfate pentahydrate mother solution by using deionized water, weighing 19.6460g of copper sulfate pentahydrate, dissolving the copper sulfate pentahydrate, and preparing the Cu with 5g/L gradient by constant volume in a 1L volumetric flask²⁺The mother liquor is ready for use.

b. The method comprises the steps of using natural seawater, using a submersible pump to extract seawater subjected to primary precipitation in a reservoir, filtering particle suspended matters and zooplankton in the natural seawater through three-stage filtration consisting of a sand filter and 300-mesh and 500-mesh bolting silk to obtain filtered natural seawater, and adding 250L of filtered seawater into all test buckets to serve as a test solution.

c. The method comprises the following steps of setting 6 test groups, wherein one blank control group and five pollutant test groups are arranged, the blank control group and the five concentration groups are respectively provided with two parallel, the 1-5 copper concentrations of the pollutant test groups are respectively 5 mu g/L, 10 mu g/L, 20 mu g/L, 40 mu g/L and 80 mu g/L toxic substance test groups, a water sample of the blank control group is filtered natural seawater, and the five pollutant test groups and the blank control group have the same conditions except that the concentrations of pollutant copper are different.

d. The scallop larvae cultured in the bucket are concentrated by using a net made of 300-mesh bolting silk, the larvae are concentrated in a 2L beaker, and the number of the larvae in the beaker is calculated by sampling and counting for many times.

e. Putting about 8 million scallop larvae which are selected in the step 2 and are finished for 24 hours in each container to perform a chronic toxicological test, putting an attachment base at the 9 th day of the test, wherein the attachment base is a palm curtain formed by weaving palm ropes, and putting two attachment bases in each test barrel, wherein each attachment base is provided with about 1500 attachment points. And (3) observing the feeding condition and the death condition of the larvae at regular time during the chronic toxicological test, taking out the attaching bases after the test, randomly selecting five different areas on each attaching base, taking 15 attaching points from each area, observing and recording the attaching number of the scallop larvae on each attaching base by using a microscope after shearing.

f. The whole test process is carried out in a nursery room of a farm, a semi-static exposure test is adopted, the water change amount is 100L every day during the test, and dead golden algae and flat algae at the bottom of a test bucket are cleaned regularly.

g. In the whole test process, the water temperature is maintained at 23 +/-1 ℃ through the integral temperature control of the seedling raising chamber and the water volume of the test barrel, the illumination is controlled for 14 hours and the darkness is controlled for 10 hours, and the dissolved oxygen is maintained to be more than 60% of the saturation value.

4. And (3) analysis and calculation:

a. analyzing the attachment condition of the scallop larvae, and counting and calculating the attachment rate of the scallop larvae on the attachment base in the pollutant test groups 1-5 and the blank control group by the following specific formula:

total number of larvae deposited N1 is total number of deposition points M/total number of deposition points M × five selected regions for counting N1;

the attachment rate Y is the total number of attached larvae N1/total number of larvae N;

b. whether the pollutant concentration has a significant influence on the larva attachment rate is determined by anova, and the flow is as follows:

the univariate analysis under a general linear model in an analysis program is selected, the copper concentration is taken as a fixed factor, the logarithm of the scallop larva attachment rate is taken as a dependent variable, the analysis model is a full-factor model, when the P value is less than 0.05, the copper with different concentrations is considered to have significant influence on the scallop larva attachment rate, the table 1 shows the analysis result of the concentration-attachment rate variance in the embodiment 1 of the invention, and the P value in the analysis result of the full-factor variance is 0.000, which indicates that the copper concentration has significant influence on the scallop adhesion rate. Table 2 shows the concentration and larval attachment rate, impact rate for each contaminant test group (where each test group includes two parallel groups).

TABLE 1 analysis of variance of concentration-attachment ratio in example 1 of the present invention

a.R²0.990 (adjusted R)²＝0.981)

TABLE 2 test groups of pollutants for attachment to larvae, influence rates

c. Further, a concentration-effect line graph is plotted after determining that the concentration has a significant effect on the attachment rate, and the effect rate graph of each concentration relative to a blank control group (as shown in fig. 2 and fig. 3) is used to obtain a non-effective concentration (NOEC) and a low effective concentration (LOEC) of the toxic substance, wherein the attachment rate of the blank control group is 100%, wherein NOEC is the highest contaminant concentration in all contaminant test groups with the larva attachment rate greater than 95% of the larva attachment rate of the blank control group, and LOEC is the highest contaminant concentration in all contaminant test groups with the larva attachment rate greater than 75% of the larva attachment rate of the blank control group. From the analyses of FIGS. 2 and 3, it can be seen that the NOEC in example 1 had no results, and the LOEC was 5. mu.g/L.

d. Further, the LOEC obtained from the test was subjected to reliability evaluation (as shown in table 3). At present, no standard of shellfish chronic tests is published by the national government department or related associations, and other aquatic organism test standards published by the country are toxicity toxicological tests under a laboratory system. There is sufficient evidence for the entire data generation process, so the LOEC obtained from this experiment is a limit reliable.

TABLE 3 data reliability evaluation rules

Since the toxicity test of various researchers can be performed according to different test protocols, the above description is only a specific description of the feasible embodiments of the present invention, and is not intended to limit the scope of the present invention, and all equivalent changes and modifications depending on the present invention should be included in the scope of the present invention.