Method for obtaining effect trigger value of in-vitro acute toxicity test of water environment sample and application thereof

Abstract

The invention discloses a method for obtaining an effect trigger value of an in-vitro acute toxicity test of a water environment sample and application of the method. The method comprises the following steps: determining a photobacterium acute toxicity test positive compound; determining the chronic toxicity minimum toxicity equivalent (SAFE TEQ) of the photobacterium acute toxicity test positive compound to aquatic organisms; determining the chronic toxicity HC5TEQ of the photobacterium acute toxicity test positive compound to the aquatic organisms; determining the clean TEQ of the representative clean water environment; and determining the effect trigger value of the luminous inhibition test of the luminous bacteria in the water environment sample. According to the method, an effect trigger value of a luminous bacterium luminescence inhibition test is established, and particularly, an effect trigger value suitable for the luminous bacterium luminescence inhibition test of a water environment sample is deduced.

Description

Technical Field

[0001] This application relates to the field of ecological risk assessment, and particularly to a method for obtaining an effect trigger value for in vitro acute toxicity testing of water environment samples and its application. Background Art

[0002] The presence of organic micro-pollution in the water environment poses potential hazards to the ecosystem and human health. In vitro bioassays can directly obtain comprehensive toxicity effect information of water samples and are important supplementary means for the assessment of surface water, drinking water, and even sewage quality. What level of toxicity effect indicates that the toxicity risk is acceptable is the key issue restricting the application of in vitro testing in water quality assessment. However, there is currently a lack of a method for determining toxicity risk through in vitro testing for the acute toxicity assessment of water environment samples.

[0003] Currently, in vitro bioassay methods for water environment samples involve multiple toxicity endpoints, which are divided into specific endpoints and non-specific endpoints. According to the type of toxicity endpoint and environmental protection objectives, different in vitro tests require independent effect trigger values as the basis for toxicity determination. The representative test method for acute toxicity is the luminescent bacteria toxicity test. As an important toxicity index for evaluating water ecological risk, an EBT value with certain reference value has been derived. Among them, Escher et al. obtained an EBT for bioluminescence inhibition assay of 1.26 mg·L-1 (calculated as Phenol-EQ) based on the GV value of phenol in the EQS. Ma et al. reported an EBT of 6.04 mg·L-1 (calculated as Phenol-EQ) for the luminescent bacteria toxicity test based on the HC5 value. However, the currently reported methods do not consider the range of acute toxicity background values of clean water bodies in China, or the aquatic organism toxicity data they rely on is less, which may lead to an underestimation of the adverse effects of water quality in risk determination.

[0004] Therefore, there is an urgent need in the art to develop a new method for determining the acute toxicity risk of water environment samples. Summary of the Invention

[0005] Based on this, in view of the technical problem that there is currently a lack of an effective method for determining the acute toxicity risk of water environment samples, it is necessary to provide at least one method for obtaining an effect trigger value for in vitro acute toxicity testing of water environment samples and its application.

[0006] In the first aspect of this application, a method for obtaining an effect trigger value for in vitro acute toxicity of water environment samples based on species sensitivity distribution is provided, which includes the following steps:

[0007] Determine the positive compounds for the luminescent bacteria acute toxicity test: Collect the luminescent bacteria acute toxicity test data, select the positive compounds for the luminescent bacteria acute toxicity test, and calculate the relative effect potential (REP) relative to phenol;

[0008] Determine the minimum toxicity equivalent (SAFETEQ) of the chronic toxicity of the positive compounds in the luminescent bacteria acute toxicity test to aquatic organisms: According to the REP, screen the compounds with REP > 0.001, collect the acute and chronic lethality toxicity data SAFE of the positive compounds in the luminescent bacteria acute toxicity test to aquatic organisms, convert them into the toxicity equivalent of phenol to luminescent bacteria, and divide the collected no-effect concentration, lowest-effect concentration, 50% effect concentration, and 50% lethal concentration by the corresponding assessment factor (AF) for normalization, and select the minimum toxicity equivalent as the SAFETEQ of the positive compounds with REP > 0.001;

[0009] Determine the chronic toxicity HC5 TEQ of the positive compounds in the luminescent bacteria acute toxicity test to aquatic organisms: Make a species sensitivity curve, i.e., an SSD curve, from the above-normalized toxicity data of different aquatic organisms; Obtain the 5% species hazard concentration, i.e., HC5, as the HC5 TEQ according to the SSD curve;

[0010] Determine the clean TEQ of a representative clean water environment: Select a representative clean water environment sample and conduct a luminescent bacteria luminescence inhibition test, and take the average value of the toxicity equivalents of phenol in multiple clean water bodies as the background value, which is determined as the clean TEQ of the toxicity level of the representative clean water body; And,

[0011] Determine the effect trigger value of the luminescent bacteria luminescence inhibition test for the water environment sample: Determine the effect trigger value according to the SAFE TEQ, the ecological safety threshold HC5, and the clean TEQ of the representative clean water environment.

[0012] In some embodiments, the number of types of the positive compounds in the luminescent bacteria acute toxicity test is at least 10.

[0013] In some embodiments, the positive compounds in the luminescent bacteria acute toxicity test include formaldehyde, phenol, nitrobenzene, chloroform, benzene, toluene, chlorobenzene, ethylbenzene, m-xylene, trichloroethane, isopropylbenzene, 1,2-dichlorobenzene, and benzo(a)pyrene.

[0014] In some embodiments, the number of types of the aquatic organisms is at least 10.

[0015] In some embodiments, the aquatic organisms are selected from the group consisting of at least mites, algae, crustaceans, fish, insects, invertebrates, microorganisms, plants, protozoa, and worms.

[0016] In some embodiments, the acute and chronic toxicity data includes one or more of no-effect concentration, lowest-effect concentration, 50% effect concentration, and 50% lethal concentration.

[0017] In some embodiments, the SAFE TEQ is 4 ng phenol / L to 5 ng phenol / L.

[0018] In some embodiments, the ecological safety threshold HC5 is 35 μg phenol / L to 45 μg phenol / L.

[0019] In some embodiments, the representative clean water environment is derived from water bodies of typical regions in China, including water sources.

[0020] In some embodiments, the representative clean water environment includes at least two cities, and the average value of the toxicity equivalents of the at least two cities is used as the clean TEQ.

[0021] In some embodiments, the effect trigger value is determined by combining the SAFE TEQ, the ecological safety threshold HC5, and the clean TEQ of the representative clean water environment according to the EBT value derivation route of STOWA.

[0022] In some embodiments, the effect trigger value is 4 times the CLEAN TEQ.

[0023] In some embodiments, the effect trigger value is 1.5 mg phenol / L to 1.8 mg phenol / L.

[0024] In the second aspect of the present application, a method for determining the acute toxicity risk in a water environment is provided. The determination method includes:

[0025] Taking the water environment to be tested, measuring its toxicity equivalent relative to phenol, and comparing it with the effect trigger value measured by the method described in the first aspect.

[0026] The present application establishes an effect trigger value for the inhibition of luminescence of luminous bacteria, especially derives the effect trigger value applicable to the luminescence inhibition test of water environment samples; and proposes a method for determining the acute toxicity risk of water environment samples based on the luminescence bacteria test. This method is particularly applicable to the water environment in China and provides technical support for water quality risk assessment work. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments and implementations of the present application, and to more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or implementations. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0028] Figure 1 This is the SSD curve of acute toxicity positive compounds to aquatic organisms in one embodiment of the present application.

[0029] Figure 2 This is the test result (TEQ - phenol) of acute toxicity of luminous bacteria in source water of typical regions in China in one embodiment of the present application. Detailed implementation manners

[0030] To facilitate the understanding of the present application, the present application will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0032] In the present application, unless otherwise stated, "one or more" means any one of the listed items or any combination of the listed items. Similarly, in other cases where "one or more" and the like are used to represent "one or more", the same understanding shall be made unless otherwise stated.

[0033] The "combinations thereof", "any combinations thereof", "any combination manners thereof", etc. used in the present application include all suitable combination manners of any two or more of the listed items.

[0034] In the present application, in the "suitable combination manners", "suitable manners", "any suitable manners", etc., "suitable" is subject to being able to implement the technical solution of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application.

[0035] In the present application, terms such as "further", "furthermore", "especially", "for example", "such as", "example", "exemplification", etc. are used for descriptive purposes, indicating that there is an association in the covered content between the different technical solutions before and after, but should not be understood as a limitation on the previous technical solution, nor should it be understood as a limitation on the protection scope of the present application. In the present application, unless otherwise stated, A (such as B) means that B is a non - restrictive example of A, and it can be understood that A is not limited to B.

[0036] In this application, "optionally", "optional", and "option" mean "may or may not be", that is, any one of two alternative options of "yes" or "no". If the word "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual constraints, each "optional" is independent. Without other instructions, descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or may not include".

[0037] The terms "comprising", "containing", and "including" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0038] In this application, in a technical feature or technical solution described in an open language, it includes a closed technical feature or technical solution composed of the listed content, and also includes an open technical feature or technical solution containing the listed content.

[0039] In this application, exemplary descriptions such as "in some embodiments" and "in one embodiment" can cover but are not limited to the following meanings: These solutions can be combined with other solutions in a suitable manner to form a new technical solution.

[0040] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0041] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within the numerical interval is considered continuous, and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical values" in the numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. The "numerical interval" is allowed to broadly include numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0042] In this application, for a method process involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limit, and it can be executed in other orders than the described ones. Moreover, any one step can include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be executed at the same time, but can be executed at different times, and their execution order does not necessarily need to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0043] The presence of thousands of pollutants in the water environment poses potential hazards to aquatic organisms and human health. The transformation of water quality assessment from a single chemical analysis to an effect-based assessment method will overcome many limitations. Among them, although in vitro tests can directly obtain the comprehensive biological effects of water quality, there is a lack of an effect trigger value to define whether the risk level is acceptable. By establishing an effect trigger value, it can be compared with the bioequivalent obtained from biological tests, which helps to determine whether it is necessary to conduct further safety assessments for this specific endpoint, and at the same time will contribute to the use and promotion of in vitro biological tests in future environmental monitoring.

[0044] Based on the concept of the species sensitivity curve, this application proposes an effect trigger value for in vitro acute toxicity tests that is particularly applicable to the water environment in China, forming a method for determining the acute toxicity of water environment samples.

[0045] The Dutch Water Foundation proposed a technical route for determining whether the in vitro toxicity risk level of water environment samples is acceptable based on the HC5 value of the species sensitivity distribution (SSD) curve. For multiple in vitro tests of nuclear receptor effects, by obtaining the HC5 values of positive compounds and combining them with the background values of clean water, the effect trigger values for multiple in vitro tests of nuclear receptor effects are derived, which are used to determine what risk level the endocrine effects of water environment samples are.

[0046] The present application provides a method for obtaining an effect trigger value of in vitro acute toxicity of a water environment sample based on species sensitivity distribution, which includes the following steps:

[0047] Determine the positive compounds for the luminescent bacteria acute toxicity test: Collect the data of the luminescent bacteria acute toxicity test, select the positive compounds for the luminescent bacteria acute toxicity test, and calculate the relative effect potential (REP) relative to phenol;

[0048] Determine the minimum toxic equivalent (SAFETEQ) of the chronic toxicity of the positive compounds for the luminescent bacteria acute toxicity test to aquatic organisms: According to the REP, screen the compounds with REP>0.001, collect the acute and chronic lethality toxicity data SAFE of the positive compounds for the luminescent bacteria acute toxicity test to aquatic organisms, convert them into the toxic equivalent of phenol to luminescent bacteria, and divide the collected no-effect concentration, lowest effect concentration, 50% effect concentration and 50% lethal concentration by the corresponding assessment factor (AF) for normalization, and select the minimum toxic equivalent as the SAFETEQ of the positive compounds with REP>0.001;

[0049] Determine the chronic toxicity HC5 TEQ of the positive compounds for the luminescent bacteria acute toxicity test to aquatic organisms: Make a species sensitivity curve, i.e., an SSD curve, for the normalized toxicity data of different aquatic organisms above; Obtain the 5% species hazard concentration, i.e., HC5, as HC5 TEQ according to the SSD curve;

[0050] Determine the clean TEQ of a representative clean water environment: Select a representative clean water environment sample and conduct a luminescent bacteria luminescence inhibition test, and take the average value of the toxic equivalents of phenol in multiple clean water bodies as the background value and determine it as the clean TEQ of the toxicity level of the representative clean water body; And,

[0051] Determine the effect trigger value of the luminescent bacteria luminescence inhibition test for the water environment sample: Determine the effect trigger value according to the SAFE TEQ, the ecological safety threshold HC5, and the clean TEQ of the representative clean water environment.

[0052] In some embodiments, the number of types of the positive compounds for the luminescent bacteria acute toxicity test is at least 10. Without wishing to be bound by any theory, it is considered that when the number of types is more than 10, the influence of the number of types on the SSD curve can be reduced as much as possible.

[0053] In some embodiments, the positive compounds for the luminescent bacteria acute toxicity test include formaldehyde, phenol, nitrobenzene, chloroform, benzene, toluene, chlorobenzene, ethylbenzene, m-xylene, trichloroethane, isopropylbenzene, 1,2-dichlorobenzene, and benzo(a)pyrene.

[0054] In some embodiments, the number of species of the aquatic organisms is at least 10. Exemplarily, the aquatic organisms include mites, algae, crustaceans, fish, insects, invertebrates, microorganisms, plants, protozoa, and worms.

[0055] In the present application, the acute toxicity test data of luminescent bacteria can be sourced from conventional databases in the art. Exemplarily, it can be sourced from the luminescent bacteria toxicity data reported in the literature Vighi, M., Migliorati, S. and Monti, G. S. 2009. Toxicity on the luminescent bacterium Vibrio fischeri (Beijerinck). I: QSAR equation for narcotics and polar narcotics. Ecotoxicology and Environmental Safety 72(1), 154 - 161; or from the luminescent bacteria toxicity data predicted by QSAR toolbox v 4.7.1.

[0056] In some embodiments, the SAFE TEQ is 4 ng phenol / L to 5 ng phenol / L. Exemplarily, the SAFE TEQ is 4 ng phenol / L, 4.1 ng phenol / L, 4.2 ng phenol / L, 4.3 ng phenol / L, 4.4 ng phenol / L, 4.5 ng phenol / L, 4.6 ng phenol / L, 4.7 ng phenol / L, 4.8 ng phenol / L, 4.9 ng phenol / L, 5 ng phenol / L, or a range or value between any two values.

[0057] In some embodiments, the ecological safety threshold HC5 is 35 μg phenol / L to 45 μg phenol / L. Exemplarily, the ecological safety threshold HC5 is 35 μg phenol / L, 36 μg phenol / L, 37 μg phenol / L, 38 μg phenol / L, 39 μg phenol / L, 40 μg phenol / L, 41 μg phenol / L, 42 μg phenol / L, 43 μg phenol / L, 44 μg phenol / L, 45 μg phenol / L, 46 μg phenol / L, 47 μg phenol / L, 48 μg phenol / L, 49 μg phenol / L, 50 μg phenol / L, or a range or value between any two values.

[0058] In some embodiments, the representative clean water environment sample is sourced from the source water including typical regions or cities. It should be understood that the source water of the typical regions or cities is regionally representative and meets the requirements of clean water bodies. Exemplarily, the typical regions or cities include one or more of Harbin, Jinan, Shanghai, Wuhan, Chongqing, and Zhuhai.

[0059] In some embodiments, there are at least two representative clean water environment samples, which are from different regions, and the average value of the toxic equivalent of at least two representative clean water environment samples is used as clean TEQ.

[0060] In some embodiments, the effect trigger value is determined by combining the EBT value derivation route of STOWA, SAFE TEQ, the ecological safety threshold HC5, and the clean TEQ of the representative clean water environment.

[0061] In some embodiments, the effect trigger value is 4 times the CLEAN TEQ.

[0062] In some embodiments, the effect trigger value is 1.5 mg phenol / L to 1.8 mg phenol / L. Exemplarily, the effect trigger value is 1.5 mg phenol / L, 1.6 mg phenol / L, 1.7 mg phenol / L, 1.8 mg phenol / L, or the range or value between any two values.

[0063] This application also provides a method for determining the acute toxicity risk in a water environment sample. The determination method includes:

[0064] Using the method described above to determine the effect trigger value for the luminescence inhibition test of luminous bacteria in the water environment sample;

[0065] Taking the water environment sample to be tested, determining its toxic equivalent relative to phenol, and comparing it with the effect trigger value; and,

[0066] Using this effect trigger value to determine the acceptable level of toxicity risk when evaluating the acute toxicity of the water environment sample through the luminescence inhibition test of luminous bacteria.

[0067] Some embodiments are provided below.

[0068] The embodiments of the present application will be described in detail below in combination with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following embodiments, the guidance given in the present application is preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0069] 1. Sample pretreatment:

[0070] Collect 20 L of drinking water for pretreatment. After sampling, transport it back to the laboratory for sample pretreatment as soon as possible. Otherwise, it should be stored in the dark and sealed at 2°C to 5°C, but not exceeding 24 h.

[0071] Before the in vitro toxicity test, the sample needs to be pretreated to prepare a concentrated organic extract of the sample for testing. The collected sample is filtered using a filtration device and a microporous membrane to remove particulate matter. The filtered sample should be stored at 2°C - 5°C and subjected to solid-phase extraction as soon as possible.

[0072] The drinking water sample for in vitro toxicity test needs to be concentrated 50,000 - 100,000 times. The extraction of organic matter in the sample is carried out by solid-phase extraction. The eluent of the SPE column is further concentrated by rotary evaporation and nitrogen blowing, and finally an organic matter extract solution with DMSO as the solvent is prepared.

[0073] a) Activation of the solid-phase extraction column

[0074] Fix the SPE column on the solid-phase extraction device, and sequentially add 6 mL each of dichloromethane, methanol, and ultrapure water for pre-rinsing and activation of the SPE column, allowing it to pass through the SPE column at a flow rate of about 5 mL / min to achieve the purpose of activating the SPE column.

[0075] b) Sample enrichment

[0076] Put one end of the large-volume sampling tube into the sample after coarse filtration, and connect the other end to the activated SPE column. Turn on the vacuum pump, and adjust the air valve to make the vacuum degree of the solid-phase extraction device between 15 mm and 20 mm of mercury. The flow rate of the sample passing through the column is controlled at 10 mL / min. The SPE column with a specification of 500 mg can enrich no more than 4 L of drinking water sample, and the SPE column with a specification of 200 mg can enrich no more than 2 L of drinking water. After enrichment, drain the water, dry it with nitrogen for 20 min or use a high-speed centrifuge to spin-dry it. After dehydration, it can be stored at -20°C in the dark and should be eluted as soon as possible.

[0077] c) Elution and concentration

[0078] Place the SPE column after enrichment and drying on the solid-phase extraction device, and add 6 mL of dichloromethane / methanol (9:1) twice for each extraction column, a total of 12 mL for elution. The eluent is collected with a clean glass tube, and after draining, maintain the same vacuum degree for 5 min.

[0079] The eluent is dehydrated through an SPE column filled with anhydrous sodium sulfate. After dehydration, the eluent is collected in a round-bottom flask, rotary evaporated and concentrated to 5 mL - 6 mL on a rotary evaporator. Transfer the concentrated solution to a K-D concentrator, place it on a nitrogen blowing instrument, gently blow it with nitrogen until slightly wet, add DMSO for solvent replacement, and make the volume up to 200 μL, and store it at -20°C.

[0080] 2. Photobacterium phosphoreum luminescence inhibition test:

[0081] 1) Bacterial strain culture:

[0082] Transfer the slant culture of Q67 preserved at 4°C to a fresh slant. After culturing at 22°C for 24 h, inoculate the bacteria on the fresh slant into a liquid medium (the medium formula is shown in Table 1). Under the condition of 22°C, shake (180 r·min -1 ) and culture for 16 h - 18 h, centrifuge at 2000 r·min -1 for 10 min, collect the bacterial cells, and make the bacterial cells into a bacterial suspension with simulated lake water (the formula is shown in Table 2). Adjust the density of the bacterial suspension so that the luminescence intensity of 1 ml is between 2 million RLU and 6 million RLU for standby.

[0083] Table 1

[0084]

[0085]

[0086] Note: Adjust the pH of the prepared medium to 9.0, dispense it into 100 mL conical flasks, 15 mL per flask, sterilize at 121°C by high-pressure steam for 20 min, and store in the refrigerator for standby.

[0087] Table 2

[0088] Component Concentration (mg / L) KCl 4.2 <![CDATA[CaCl 2 > 11.1 <![CDATA[MgSO 4 > 28.6 <![CDATA[NaHCO 3 > 42.0 <![CDATA[NaNO 3 > 4100

[0089] 2) Sample addition:

[0090] Add the serial dilution solutions of the sample to be tested and the blank control one by one to the 96-well white plate of the microplate reader (Infinite M200) (the same below), add 180 μl of the sample to each tube, and make 3 parallels for each dilution. Take 20 μl of the adjusted bacterial suspension, quickly add it to the 96-well white plate with 180 μl of the sample added (the test instrument is Infinite M200), shake and mix well, and the reaction time with the sample is 15 min.

[0091] 3) Testing:

[0092] After the bacterial suspension reacts with the sample for 15 min, test the luminescence on the tester (Infinite M200 microplate reader). Calculate the relative luminescence rate of the sample from the measured luminescence value RLU (Relative Light Units):

[0093] Relative luminescence rate (%) = (RLU of the sample / RLU of the control) × 100%

[0094] 1.2 Toxicity equivalent:

[0095] The acute toxic effect of the sample on the luminescent bacteria can be converted into the toxicity equivalent of the acute toxicity positive substance phenol, and the phenol equivalent is calculated according to the following formula:

[0096] TEQ phenol = EC 50,phenol × REC 50,样品 ……………………………(1)

[0097] In the formula:

[0098] TEQ phenol ——The acute toxicity effect of the test sample is equivalent to the toxicity effect of phenol at what concentration;

[0099] EC 50,phenol ——When the inhibition rate of the luminescence of the positive substance phenol is plotted against the exposure concentration and logistic fitting is performed, the concentration EC 50 ;

[0100] REC 50,样品 ——When the percentage (%) of the acute toxicity effect of the test sample to the maximum acute toxicity effect of phenol is plotted against the sample exposure concentration (i.e., dilution factor) and logistic fitting is performed, the concentration REC 50 .

[0101] Example 1

[0102] 1. Collection of aquatic ecotoxicity data of positive compounds for the acute toxicity test of luminescent bacteria

[0103] According to the luminescent bacteria toxicity data reported in the literature "Vighi, M., Migliorati, S. and Monti, G. S. 2009. Toxicity on the luminescent bacterium Vibrio fischeri (Beijerinck). I: QSAR equation for narcotics and polar narcotics. Ecotoxicology and Environmental Safety 72(1), 154 - 161." and the luminescent bacteria toxicity data predicted by QSAR toolbox v 4.7.1, the relative effect potencies (REP) were calculated. Ten positive compounds for the acute toxicity test of luminescent bacteria with REP greater than 0.001 were selected, as shown in Table 3. The chronic toxicity data of these compounds on aquatic organisms were collected through the ECOTOX database. For the 10 positive compounds, a total of 1000 toxicity data on different aquatic organisms were collected, as shown in Table 4.

[0104] Table 3

[0105]

[0106]

[0107] Note: a: Predicted EC50; b: Experimental data are from Vighi et al, 2009; "formaldehyde" is formaldehyde, "Phenol" is phenol, "nitrobenzene" is nitrobenzene, "trichloromethane" is trichloromethane, "Benzene" is benzene, "Toluene" is toluene, "chlorobenzene" is chlorobenzene, "ethylbenzene" is ethylbenzene, "m-Xylene" is m-xylene, "carbon tatrachloride" is trichloroethane, "isopropylbenzene" is isopropylbenzene, "1,2-Dichlorobenzene" is 1,2-dichlorobenzene, "Benzo(a)pyrene" is benzo(a)pyrene.

[0108] Table 4

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] 2. Determination of the chronic toxicity SAFE TEQ of aquatic organisms

[0163] For the acute and chronic toxicity data of the collected positive compounds, further screen out the minimum chronic toxicity value of each species. Considering the acute-chronic conversion parameter, the assessment factor (AF), and the conversion of the REP value of the luminescent bacteria test of each compound into the toxicity equivalent of phenol, 80 chronic toxicity data are finally obtained, and the minimum TEQ, that is, the safe TEQ, is 4.81 ng phenol / L.

[0164] Table 5 Minimum chronic toxicity of positive compounds with REP>0.001 to aquatic organisms

[0165]

[0166] Note: “Dichlofluanid” is anilazine, “Phenol” is phenol, “Water Flea” is water flea, “Japanese medaka” is Japanese medaka, “Bluegill” is bluegill sunfish; The full name of PNEC is Predicted No Effect Concentration, that is: Predicted No Effect Concentration; The full name of NOEC is No Observed Effect Concentration, that is: No Observed Effect Concentration; The full name of LOEC is Lowest Observed Effect Concentration, that is: Lowest Observed Effect Concentration; The full name of EC50 is 50% effect concentration, that is: 50% effect concentration; The full name of LC50 is 50% Lethal Concentration, that is: 50% Lethal Concentration.

[0167] 3. Determination of chronic toxicity HC5 TEQ of aquatic organisms

[0168] The statistical data takes the average value of the toxicity data of the same compound corresponding to the same species. 80 pieces of toxicity data are obtained, and the software ETX 2.3 is used to draw the species sensitivity curve and fit to obtain the HC5 value. See Figure 1 : Each point represents the average value of the toxicity equivalents of different positive compounds to a species. The fitted HC5 value is 40.6 μg phenol / L.

[0169] 4. Determination of clean TEQ of clean water bodies in China

[0170] Collect the source water of 6 typical cities in China for the luminescence inhibition test of luminous bacteria. The average value of TEQ-phenol of all samples is used as the clean water body background value CLEAN TEQ (see Figure 2 ), and the average toxicity equivalent of the six source waters is 0.424 mg phenol / L, which is CLEAN TEQ.

[0171] According to the EBT value derivation route proposed by STOWA, since CLEAN TEQ is 10 times higher than HC5-BEQ, the derivation of EBT should be based on CLEAN TEQ. Therefore, multiply CLEAN TEQ by 4 to get 1.695 mg phenol / L.

[0172] This value of 1.695 mg phenol / L is used as the effect trigger value for evaluating the acute toxicity of water environment samples in the luminescence inhibition test of luminous bacteria. Higher than this value indicates that the acute toxicity risk of the water sample is unacceptable.

[0173] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the various technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0174] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for obtaining an effect trigger value of in vitro acute toxicity of a water environment sample based on species sensitivity distribution, characterized in that: It includes the following steps: Determine the positive compounds in the luminescent bacteria acute toxicity test: collect the luminescent bacteria acute toxicity test data, select the positive compounds in the luminescent bacteria acute toxicity test, and calculate the relative effect potential (REP) relative to phenol; Determine the minimum chronic toxicity equivalent (SAFETEQ) of the positive compounds in the acute toxicity test of luminescent bacteria to aquatic organisms: According to the REP, screen the compounds of REP>0.001, collect the acute and chronic lethality toxicity data SAFE of the positive compounds in the acute toxicity test of luminescent bacteria to aquatic organisms, and convert them into the toxic equivalent of phenol to luminescent bacteria, and divide the collected no effect concentration, minimum effect concentration, 50% effect concentration and 50% lethal concentration by the corresponding assessment factor (AF) for normalization, and select the minimum toxic equivalent to determine as the SAFETEQ of the positive compound of REP>0.001; Determine the chronic toxicity HC5 TEQ of the positive compounds in the acute toxicity test of luminescent bacteria to aquatic organisms: the above normalized toxicity data of different aquatic organisms are used to make a species sensitivity curve, i.e., an SSD curve; and according to the SSD curve, the 5% species hazard concentration, i.e., HC5, is obtained as the HC5 TEQ; Determine the clean TEQ of a representative clean water environment: select a representative clean water environment sample and conduct a luminescent bacteria luminescence inhibition test, and use the average value of the toxic equivalent of phenol in multiple clean water bodies as the background value to determine the clean TEQ of the toxicity level of the representative clean water body; and, Determine the effect trigger value of the luminescence inhibition test of luminescent bacteria in water environment samples: determine the effect trigger value according to the SAFE TEQ, the ecological safety threshold HC5 and the clean TEQ of a representative clean water environment.

2. The method according to claim 1, characterized in that The number of positive compounds in the luminescent bacteria acute toxicity test is at least 10; Optionally, the positive compounds in the acute toxicity test of the luminous bacteria include formaldehyde, phenol, nitrobenzene, chloroform, benzene, toluene, chlorobenzene, ethylbenzene, m-xylene, trichloroethane, isopropylbenzene, 1,2-dichlorobenzene and benzo(a)pyrene.

3. The method according to claim 1, characterized in that The number of species of the aquatic organisms is at least 10.

4. The method according to claim 3, characterized in that The aquatic organisms include acarina, algae, crustaceans, fish, insects, invertebrates, microorganisms, plants, protozoa and worms.

5. The method according to any one of claims 1 to 4, characterized in that: The SAFE TEQ is 4 ng phenol / L to 5 ng phenol / L; And / or, the ecological safety threshold HC5 is 35 μg phenol / L to 45 μg phenol / L.

6. The method according to any one of claims 1 to 4, characterized in that The representative clean water environment samples are derived from source water including water from a typical region or city.

7. The method according to any one of claims 1 to 4, characterized in that: The representative clean water environment samples include at least two samples from different regions, and the average value of the toxic equivalents of the at least two representative clean water environment samples is taken as clean TEQ.

8. The method according to any one of claims 1 to 4, characterized in that: The effect trigger value is determined based on STOWA's EBT value derivation route combined with SAFE TEQ and the ecological safety threshold HC5 and clean TEQ of a representative clean water environment.

9. The method according to claim 8, characterized in that The effect trigger value is 4 times CLEAN TEQ; Optionally, the effect trigger value is 1.5 mg phenol / L to 1.8 mg phenol / L.

10. A method for determining acute toxicity risk in water environment samples, characterized in that: The determination method comprises: Determine the effect trigger value of the luminescence inhibition test of luminescent bacteria in water environment samples by the method described in any one of claims 1 to 9; Taking a water environment sample to be tested to determine its toxic equivalent relative to phenol, and comparing it with the effect trigger value; and, This effect trigger value is used to determine the acceptable level of toxicity risk when evaluating the acute toxicity of water environment samples through the luminescence inhibition test of luminescent bacteria.

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