CN102226806A - A COMPATIBILITY METHOD FOR DETERMINING BINARY MIXTURES FOR OPTIMAL JOINED BIOLOGICAL EFFECTS - Google Patents

Abstract

The invention belongs to the field of environmental protection, and discloses a compatibility method for acquiring an optimal combined biological effect by testing a binary mixture. The method comprises the following steps: (1) the testing method is based on GB/T 15441-1995, and photobacterium phosphoreum is used as an indicator organism to test the single toxicity EC50-A and EC50-B of A and B compounds; (2) according to the EC50-A and the EC50-B, a mixed solution with an equivalent effect ratio is prepared by a testing method which is based on GB/T15441-1995, the biological effect concentration of the mixed solution is tested to obtain the concentration CA and CB of A and B in a mixed system when inhibition of the mixed system to the biological effect is 50 percent, and a combined biological effect index TU1:1 is calculated in the equivalent effect ratio; and (3) a combined biological effect regulation of the binary mixed system can be predicted according to the testing result TU1:1 of the combined biological effect, which is calculated in the equivalent effect ratio, and the optimal biological effect point can be acquired. The method can be applied in the aspects of environmental science, medication, pesticide and the like.

Description

A COMPATIBILITY METHOD FOR DETERMINING BINARY MIXTURES FOR OPTIMAL JOINED BIOLOGICAL EFFECTS

technical field

The invention belongs to the field of environmental protection, and in particular relates to a compatibility method for determining the optimal joint biological effect of a binary mixture.

Background technique

With the rapid development of modern science and technology and the continuous improvement of industrialization, chemicals are ubiquitous in human life, and often exist in a mixture of multiple chemicals, which have had a profound impact on the environment and human health.

In the past 40 years, the quantitative structure-activity relationship (QSAR) method has been used to study the environmental behavior and biological effects of a single compound in considerable detail, and many corresponding results have been obtained. On this basis, many scholars have studied the biological effects of mixed compounds and proposed some prediction methods.

Sprague (Sprague, JB "Lethal levels of mixed copper zinc solutions for juvenile salmon", JFish Res Board Can 1965, 22: 425-432) and others proposed to use toxic units (TU) to evaluate the combined toxic effects of organic pollutants in the environment Methods. Other scholars successively proposed additive index (AI) and mixed toxicity unit (MTI) prediction methods to study joint toxic effects (Konemann H. "Quantitative structure-activity relationships in fish toxicity studies Part 1: Relationship for 50 industrial pollutants". Toxicology 1981 , 19(3):209-221) (Marking L "Method for assessing additive toxicity of chemical mixtures". Aquatic toxicity and hazard evaluating, ASTM STP, 1977, 634: 99-108). Nirmalakhanda (Nirmalakhandan N, Arulgnanendran V, Mohsin M, Sun B, Cadena F. "Toxicity of mixtures of organic chemicals to microorganisms". Water Res 1994, 28(3): 543-551) took the lead in 1994 based on the concept of toxicity units It is proposed that, for a system containing n single compounds, under the assumption of equal toxicity, the QSAR model of a single compound is used to predict the half-lethal concentration LC _50,i of each single compound, and then calculate the mixture of equal toxicity ratios. Toxicity of any organic compound in the system. Xu (Xu S, Nirmalakhandan N. "Use of QSAR models in predicting joint effects in multi-component mixtures of organic chemicals". Water Res 1998, 32(8): 2391-2399) inherited Nirmalakhanda's theory, for the n- A mixture of an organic compound n with equal toxicity and an organic compound n with unequal toxicity proposes a method for predicting the concentration of compound n. In 1996, Prakash took another path (Prakash J, Nirmalakhandan N, Sun B, Peace J. "Toxicity of binary mixtures of organic chemicals to microorganisms". Water Res 1996.30(6): 1459-1463), proposed a similar parameter λ A method for predicting the concentration of any organic compound in a mixture system, these works make it possible to predict the joint biological effect of the mixture.

However, due to the following limitations of existing methods, their application is limited: On the one hand, none of the existing methods and researches can explain the variation of the joint biological effect of the mixed system with the composition of the compounds, which leads to the operability and inability of its practical application. Feasibility is poor. On the other hand, most of the mixed systems measured at present are equitoxic ratios, and these methods are only applicable to the ideal state of mixed systems composed of equitoxic ratios. However, in real life and production practice, the mixed system composed of equal toxicity ratio almost does not exist, and the mixed pollutants usually exist in the form of unequal toxicity ratio. Therefore, in the non-equal toxicity ratio mixed system, the quantitative prediction method of the biological effect is still a difficult problem, and there are no relevant reports. Therefore, there is an urgent need to find a convenient and quick method to predict the mixed biological effects of compounds in different proportions, and then find the appropriate optimal biological effect point to guide people to produce the best results with the smallest economic investment. effect and improve work efficiency.

Contents of the invention

The purpose of the present invention is to provide a method for determining the compatibility of a binary mixture to obtain the best combined biological effect. This method solves the complex problem of how to obtain the best combined biological effect of the binary mixed system according to the changing law of the biological effect of the mixed system for the first time. Problem, the method of the present invention can be applied to aspects such as environmental science, medicine and pesticide, and provides scientific basis for pollutant emission reduction in environmental science, ecological environment protection and medicine and pesticide to find the best joint biological effect point, has important scientific significance and practical value.

Technical scheme of the present invention is as follows:

The invention provides a method for determining the compatibility of a binary mixture to obtain the best combined biological effect, the method comprising the following steps:

(1) Determination of the biological effect concentration of a single compound

The test method is based on GB/T 15441-1995, using Photobacterium luminosa as the indicator organism, to determine the single toxicity of two types of compounds A and B, and the biological effect of a single compound is expressed by EC50, and the biological effect concentrations of these two single compounds are respectively EC _{50 -A} , EC _50-B ;

(2) Determination of the joint biological effect of the equivalent effect ratio

According to the single biological effect concentrations EC _50-A and EC _50-B of the two compounds A and B obtained through the determination, a mixed solution with an equivalent effect ratio is prepared, that is, the concentrations of the two substances A and B in the mixed solution are respectively EC _{50- A} and EC _50-B , the test method is based on GB/T 15441-1995 "Determination of Acute Toxicity of Water Quality Luminescent Bacteria Method", the concentration of the biological effect of the mixed solution is measured, and the mixed system can be obtained when the biological effect inhibition of the mixed system is 50% A and Concentrations C _A and C _B of B in the mixed system, and then calculate the joint biological effect index TU _1:1 when the equivalent effect ratio is obtained;

(3) Determination of the optimal biological effect point

According to the measurement result TU _{1 : 1} of the joint biological effect calculated at the equivalent effect ratio calculated in step (2), the change law of the joint biological effect of the binary mixed system is predicted, and the best biological effect point is obtained.

The size of the joint biological effect index TU is used to evaluate the joint biological effect:

TU _1:1 <0.80 means that the two compounds A and B have a synergistic effect;

TU _1:1 >1.20 indicates that the two compounds A and B have produced antagonistic effects;

TU _1:1 = 1.00±0.20 means that the two compounds A and B have an additive effect, also known as an additive effect (Broderius SJ, Kahl MD, Hoglund MD. "Use of joint toxic response to define the primary-mode of Toxic action for diverse industrial organic chemicals". Environ Toxicol Chem 1995, 14(9)1591-1605).

The TU _1:1 is calculated using the following formula:

$\mathrm{<span\; class="notranslate">\; TU</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

If TU _1:1 <0.80, it indicates that the mixed system presents a synergistic effect at the equivalence ratio, so it can be predicted that the combined effect (the strongest synergistic effect) of the mixed system composed of these two substances is at the equivalence point; As the concentration of the mixed system changes from the equivalent effect ratio concentration to the non-equivalent effect concentration, the corresponding TU gradually increases and is close to 1.00±0.20, that is to say, the synergistic effect gradually changes to the additive effect; In the wastewater discharge of compounds, it is necessary to avoid the discharge of equivalent concentration as far as possible to reduce ecological hazards; and in the joint use of pesticides and medical drug combinations, the joint use of two compounds with synergistic effects at the equivalent toxicity ratio point can greatly improve Medication effect.

If TU _1:1 > 1.20, it indicates that the mixed system has antagonistic effect at the equivalent effect ratio; thus it can be predicted that the mixed system composed of these two compounds has the strongest joint biological effect at the equivalent effect ratio point ( The anti-effect is the strongest); as the concentration of the mixed system changes from the equivalent specific concentration to the non-equivalent concentration, the corresponding TU gradually decreases and is close to 1.00±0.20. This suggests that when we treat wastewater containing this type of compound, we can use the equivalent toxicity ratio point discharge, and use its antagonistic effect to minimize the treatment cost and environmental hazards; the same reason, for two drugs with antagonistic effect , to absolutely avoid the drug combination at the point of equal toxicity ratio, otherwise the therapeutic effect will be greatly reduced.

If TU _1:1 = 1.00±0.20, it indicates that the mixed system has an additive effect at the equivalent effect ratio; thus it can be predicted that for the binary system with the additive effect at the equivalent effect ratio, as the two substances When the concentration of the two substances is changed from the equivalent specific concentration to the non-equivalent concentration, the size of the joint biological effect remains constant (TU between 0.80 and 1.20); that is, no matter what ratio the two substances are mixed, it is always additive effect. For the mixed system composed of these two types of compounds, when examining their joint biological effects, any ratio can be selected for joint use according to the principle of economics and practicality, so as to achieve the goal of economic input-output benefit win-win.

Further, step (1) includes the following steps:

Firstly, Photobacillus luminosa was selected as the indicator organism, and the single biological effect of the target compound (pollutant or compound) was determined according to the "National Standard for Water Quality Toxicity Detection of Photobacteria" (GB/T15441-1995), and calculated by interpolation after regression _EC50 (that is, the concentration of the compound when the luminous intensity inhibition rate is 50%);

1. Medium preparation:

500ml water, 15g sodium chloride, 2.5g peptone, 1.5g yeast extract, 1.5g glycerin, 2.5g potassium dihydrogen phosphate, 2.5g disodium hydrogen phosphate. Mix the ingredients according to the ratio, heat until the solution is clear and transparent, then use 1mol/L NaOH to adjust the pH to 6.5-7.5, divide into 100mL Erlenmeyer flasks, 40mL per bottle, wrap them in kraft paper and tie them tightly with ropes. Then, it was sterilized at 121°C for 20 minutes, cooled and stored in a refrigerator at 4°C.

2. Bacterial solution preparation:

(1) According to the national standard method, the luminescent bacteria were selected from Photobacterium phosphoreum (Photobacterium phosphoreum) T3 variant, which was purchased from Nanjing Institute of Soil Science, Chinese Academy of Sciences. Add 1 ml of sterilized 3% NaCl solution to the freeze-dried powder preparation of the luminescent bacteria, mix well, and leave it at room temperature for 2 minutes to recover the luminescence. Immediately after the recovery of the luminescent bacteria, transfer them to the slant of the test tube with an inoculation loop, incubate at a constant temperature of 20°C for 24 hours, and then transfer to the second generation.

(2) Shake flask bacteria solution: Take three spoonfuls of luminescent bacteria on an inclined plane and transfer them to a 2ml Erlenmeyer flask containing 5ml of culture solution, and shake and culture at 20°C until the logarithmic growth phase for later use. The shaking culture time was selected as 12h.

(3) Preparation of working bacteria solution: absorb 0.2ml of cultured shake flask bacteria solution into 20ml of 3% NaCl solution (pre-stirring for 20min oxygen exposure), stir for 40min before use. It is advisable to control the luminescence intensity of the blank sample at 1500,000 for the degree of dilution.

3. Test system

(1) Blank sample: the blank sample is composed of 0.8ml of 3% NaCl solution and 0.2ml of working bacteria solution. The luminous intensity of the blank is I ₀ .

(2) Test sample: Take 0.8ml of the compound standard solution in a 1ml colorimetric tube, add 0.2ml of the working bacteria solution, mix and let stand for 15min to measure the light value I _s . Key points: The test sample and the blank sample must be prepared together and measured at the same time. The testing instrument is a fluorescent immunoassay analyzer (Beijing Analytical Instrument Factory, model BH9507). Concentration gradients are set for samples at equal logarithmic intervals, and at least six gradients are made each time, and three parallel determinations are made for each concentration point. The standard deviation of the three parallel samples shall not be greater than 10%. To select an appropriate concentration range, the selection principle is that the correlation is good and the inhibition rate is included in the linear range (about 20-80%).

(3) Calculation: The inhibition rate is calculated according to the following formula:

Inhibition rate = (I ₀ -I _s )/I ₀ .

Then take the compound concentration as the x-axis (unit: mol/L) and plot the inhibition rate as the y-axis, and use the interpolation method to obtain the concentration when the inhibition rate is 50%, which is the EC ₅₀ of a single compound.

Further, step (2) includes the following steps:

According to the single biological effect concentration (EC ₅₀ ) of the two compounds A and B obtained from the measurement, a mixed solution with an equipotent ratio was prepared, that is, the concentrations of the two substances A and B in the mixed solution were EC _50-A , EC _{50- B} , the test method is based on GB/T15441-1995, the concentration of the biological effect of the mixed solution is measured, and the concentration of A and B in the mixed system (C _A , C _B ) can be obtained when the mixed system inhibits the biological effect by 50%.

1. Preparation of mixed solution

According to step (1), measure the single compound biological effect concentration (EC _50-A , EC _50-B ) of the two substances A and B obtained, and prepare a mixed solution with an equivalent effect ratio.

2. Mixed solution test series

Assuming that the depth of the mixed solution is 100%, the mixed solution is diluted according to the equilogarithmic interval, namely: 100%, 80%, 56%, 32%, 18%, 10%, 8%, . . . . Then take this as the test series, and use the Photobacterium phosphoreum T3 variant as the test organism, and use the fluorescence immunoassay analyzer to test the inhibition rate of this series.

3. Calculation: take the mixed system concentration (or dilution factor) as the x-axis, plot the y-axis with the inhibition rate, and use the interpolation method to find the concentration when the inhibition rate is 50%, and then the mixed system inhibition rate is 50%. %, the concentration of A and B substances in the mixed system (that is, C _A , C _B ). Substitute C _A , C _B and single compound biological effect concentration EC _50-A , EC _50-B into the following formula to calculate the joint biological effect index TU _1:1 at the equivalent effect ratio

$\mathrm{<span\; class="notranslate">\; TU</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

Among them, C _A , C _B represent the respective concentrations of the two compounds A and B in the mixed system when the inhibition rate of the binary mixed system of the equivalent effect ratio is 50%, and EC _50-A and EC _50-B represent the inhibition rate of the single compound The concentration when the ratio is 50%, the unit is mol/L.

Further, step (3) includes the following steps:

A mixed system that exhibits a synergistic effect (TU<0.80) for isotoxicity ratios. It can be deduced that the joint effect of the mixed system is the strongest at the equivalent effect point. When the equivalent effect concentration changes to the non-equivalent effect concentration, TU is close to 1.00, that is to say, it gradually changes from synergistic effect to additive effect. This suggests that we should try our best to avoid the discharge of equivalent concentration in the discharge of wastewater containing these compounds in order to reduce the ecological hazards. In terms of the combined use of pesticides and medical combined drugs, the combined use of two compounds with synergistic effects at the same toxicity ratio point can greatly improve the drug effect.

For the system with antagonistic effect (TU>0.80), its isotoxic ratio point joint biological effect is the weakest. This suggests that when we treat wastewater containing this type of compound, we can use the equivalent toxicity ratio point discharge, and use its resistance effect to minimize the treatment cost and environmental hazards. In the same way, for two drugs with antagonistic effects, it is absolutely necessary to avoid combining drugs at equal toxicity ratios, otherwise the therapeutic effect will be greatly reduced.

For the additive effect system (TU=1.00±0.20), the size of the joint biological effect remains constant (TU is between 0.80 and 1.20). For this type of compound, according to the principle of economics and practicality, any ratio can be selected for joint use, so as to achieve the goal of economic input-output benefit win-win.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The existing technology can only measure the joint biological effect of the binary mixture system under a certain ratio, and there is no relevant research on the law of the joint biological effect changing with the concentration. Therefore, in the existing method, if you want to understand a binary composition system At what ratio is the strongest or weakest joint biological effect, we can only test all possible composition ratios one by one to obtain a series of TU values, and then compare their sizes to obtain the best biological effect point. This method requires a lot of work, so it is necessary to find a simple and convenient method to predict the joint biological effect of the mixed system. This method aims at this problem and proposes a shortcut to find the best biological effect point.

1. The method of the present invention explains the changing law of the biological effect of the binary mixed system under different concentration conditions, points out the relationship between the best biological effect point and the equivalent effect point, and solves the problem of how the binary mixed system obtains the best joint biological effect. For complex problems, this method has the characteristics of simplicity, convenience, strong operability, and wide application range, and is suitable for large-scale promotion.

2. The method of the present invention can be applied to aspects such as environmental protection, combined use of medicine and pesticide.

Description of drawings

Fig. 1 represents the biological effect curve of the mixed system of malononitrile and p-nitrobenzaldehyde.

Fig. 2 represents the biological effect curve of the mixed system of phthalonitrile and p-dimethylaminobenzaldehyde.

Fig. 3 shows the joint biological effect curve of the mixed system of acetonitrile and terephthalaldehyde.

Detailed ways

The present invention will be further described below in conjunction with the embodiments shown in the accompanying drawings.

The preparation method of the culture medium used in the embodiment, the preparation method of the bacterial liquid and the preparation method of the experimental system

1. Medium preparation:

500ml water, 15g sodium chloride, 2.5g peptone, 1.5g yeast extract, 1.5g glycerin, 2.5g potassium dihydrogen phosphate, 2.5g disodium hydrogen phosphate. Mix the ingredients according to the ratio, heat until the solution is clear and transparent, then use 1mol/L NaOH to adjust the pH to 6.5-7.5, divide into 100mL Erlenmeyer flasks, 40mL per bottle, wrap them in kraft paper and tie them tightly with ropes. Then, it was sterilized at 121°C for 20 minutes, cooled and stored in a refrigerator at 4°C.

2. Bacterial solution preparation:

(1) Add 1 ml of sterilized 3% NaCl solution to the freeze-dried powder preparation of Luminescent bacteria, mix well, and leave it at room temperature for 2 minutes to recover the luminescence. Immediately after the recovery of the luminescent bacteria, transfer them to the slant of the test tube with an inoculation loop, incubate at a constant temperature of 20°C for 24 hours, and then transfer to the second generation.

(2) Shake flask bacteria solution: Take three spoonfuls of luminescent bacteria on an inclined plane and transfer them to a 2ml Erlenmeyer flask containing 5ml of culture solution, and shake and culture at 20°C until the logarithmic growth phase for later use. The shaking culture time was selected as 12h.

(3) Preparation of working bacteria solution: absorb 0.2ml of cultured shake flask bacteria solution into 20ml of 3% NaCl solution (pre-stirring for 20min oxygen exposure), stir for 40min before use. It is advisable to control the luminescence intensity of the blank sample at 1500,000 for the degree of dilution.

3. Experimental system:

(1) Blank sample: the blank sample is composed of 0.8ml of 3% NaCl solution and 0.2ml of working bacteria solution.

(2) Test sample: Take 0.8ml of compound standard solution in a 1ml colorimetric tube, add 0.2ml of working bacteria solution, mix and let it stand for 15 minutes to measure the light value. Concentration gradients are set for samples at equal logarithmic intervals, and at least six gradients are made each time, and three parallel determinations are made for each concentration point. The standard deviation of the three parallel samples shall not be greater than 10%. To select an appropriate concentration range, the selection principle is that the correlation is good and the inhibition rate is included in the linear range (about 20-80%).

(3) Calculation: the concentration of the compound was plotted against the inhibition rate, and the concentration when the inhibition rate was 50% was obtained by interpolation.

4. Judgment criteria for joint biological effects

The combined biological effects of the mixed system mainly include three types: synergy, resistance, and addition. The judgment criteria for these three effects are as follows:

TU<0.80 means that the two compounds A and B have a synergistic effect, TU>1.20 means antagonistic effect, TU=1.00±0.20 means additive effect, also known as additive effect. (Broderius SJ, Kahl MD, Hoglund MD." Use of joint toxic response to define the primary mode of toxic action for diverse industrial organi

Example 1

(1) Determination steps of single compound biological effect concentration

Add 0.25ml of Luminescent bacteria (purchased from Nanjing Institute of Soil Science, Chinese Academy of Sciences) solution (T3) into a volumetric flask containing 5ml of medium, and cultivate for 12 hours to make a shake flask bacteria solution; then take 0.2ml shake flask bacteria solution to 20ml 3% sodium chloride, aerated and stirred for 40 minutes to make a working bacteria solution. Take an appropriate amount of malononitrile and p-nitrobenzaldehyde to make a standard series of logarithmic gradients, with a volume of 0.8ml, add 0.2ml of working bacteria solution, shake well, expose to poison for 15 minutes, and measure the decrease in light value of the system before and after exposure The concentration at which the inhibition rate is 50% is calculated by interpolation method, which is the EC ₅₀ value of the substance (or system). The calculated EC ₅₀ values are 2.803×10 ^-3 and 5.492×10 ^-5 mol/L, respectively.

(2) Determination of the joint biological effect of the binary equivalent effect ratio mixed system

According to the single biological effect concentration (EC ₅₀ ) of the two compounds A and B obtained from the determination, and then adopt the same steps as the single compound (EC ₅₀ ) to measure the biological effect concentration of the mixed solution, the biological effect of the mixed system can be obtained. Concentrations of A and B in the mixed system (C _A , C _B ) when the inhibition is 50%.

Further, this step specifically includes the following steps (or key implementation points):

1. Preparation of mixed solution

According to the measured single compound biological effect concentration (EC _50-A , EC _50-B ) of the two substances A and B, a mixed solution with an equivalent effect ratio is prepared. Prepare a mixed solution with equivalent effect ratio, that is, the concentrations of A and B in the mixed solution are 2.803×10 ^-3 and 5.492×10 ^-5 mol/L respectively.

2. Mixed solution test series

Assuming that the depth of the mixed solution is 100%, the mixed solution is diluted according to equilogarithmic intervals, namely: 100%, 80%, 56%, 32%, 18%, 10%, 8%. Then take this as the test series, and use the Photobacterium phosphoreum T3 variant as the test organism, and use the fluorescence immunoassay analyzer to test the inhibition rate of this series.

3. Calculation: take the mixed system concentration (or dilution factor) as the x-axis, plot the y-axis with the inhibition rate, and use the interpolation method to find the concentration when the inhibition rate is 50%, and then the mixed system inhibition rate is 50%. %, the concentrations C _A and C _B of substances A and B in the mixed system are 2.102×10 ^-4 and 4.119×10 ^-6 mol/L respectively

Substitute C _A , C _B and single compound biological effect concentration EC _50-A , EC _50-B into the following formula to calculate the joint biological effect index TU _1:1 at the equivalent effect ratio

${\mathrm{<span\; class="notranslate">\; TU</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; :</span>\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

$<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 2.102</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 4</span>}}}{\mathrm{<span\; class="notranslate">\; 2.803</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 3</span>}}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 4.119</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 6</span>}}}{\mathrm{<span\; class="notranslate">\; 5.492</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 5</span>}}}$

$<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 0.15</span>}$

(3) Determination of the optimal biological effect point

The results showed that the TU _1:1 value of the mixed system of malononitrile and p-nitrobenzaldehyde was 0.15 at the equivalent effect ratio. According to the principle of joint effect judgment, TU less than 0.80 was considered as synergistic effect. It can be predicted that the combined effect (synergistic effect) of the mixed system of malononitrile and p-nitrobenzaldehyde at the equipotential point is the strongest. As the concentration of the two substances mixed changes from equivalence ratio to non-equivalence ratio, the combined effect (synergistic effect) will gradually weaken to additive effect.

This suggests that we should try our best to avoid the discharge of equivalent concentration in the discharge of wastewater containing these compounds in order to reduce the ecological hazards.

(4) Verification of method reliability

In order to verify our assay method, we measured the joint biological effect under non-equivalent ratio conditions (that is, the two substances were mixed in other concentration ratios), and compared their sizes. The results showed that the joint biological effect determined was indeed synergistic The strongest effect.

The joint biological effect test method of the mixed solution under non-equivalent effect ratio conditions is exactly the same as the equivalent effect ratio time test method, including:

1) Determination steps of single compound biological effect concentration

Add 0.25ml of luminescent bacteria solution (T3) to a volumetric flask containing 5ml of medium, and cultivate for 12 hours to make a shake flask solution; then take 0.2ml shake flask solution into 20ml of 3% sodium chloride, Gas stirring for 40min to make working bacteria liquid. Take an appropriate amount of malononitrile and p-nitrobenzaldehyde to make a standard series of logarithmic gradients, with a volume of 0.8ml, add 0.2ml of working bacteria solution, shake well, expose to poison for 15 minutes, and measure the decrease in light value of the system before and after exposure The concentration at which the inhibition rate is 50% is calculated by interpolation method, which is the EC ₅₀ value of the substance (or system). The calculated EC ₅₀ values are 2.803×10 ^-3 and 5.492×10 ^-5 mol/L, respectively.

2) Determination of the joint biological effect of the binary non-equivalent effect ratio mixed system

(1), non-equivalent ratio mixed solution preparation

According to the measured single compound biological effect concentration (EC _50-A , EC _50-B ) of the two substances A and B, a mixed solution with an equivalent effect ratio is prepared. Prepare a series of mixed solutions with non-equivalent effect ratios, assuming that the effect ratio of the two substances is n:m, that is, the concentrations of the two substances A and B in the mixed solution are respectively n×EC _50-A , m×EC 50-B (n×EC _50-B (n× 2.803×10 ^-3 , m×5.492×10 ^-5 mol/L).

(2), mixed solution test series

Assuming that the depth of the mixed solution is 100%, the mixed solution is diluted according to the equilogarithmic interval, namely: 100%, 80%, 56%, 32%, 18%, 10%, 8%. . . . Then take this as the test series, and use the Photobacterium phosphoreum T3 variant as the test organism, and use the fluorescence immunoassay analyzer to test the inhibition rate of this series.

(3), calculation: take the mixed system concentration (or dilution factor) as the x-axis, plot the y-axis with the inhibition rate, and use the interpolation method to obtain the concentration when the inhibition rate is 50% as _C'A , _C'B . Substitute C′ _A , C′ _B and single compound biological effect concentration EC _50-A , EC _50-B into the following formula to calculate the joint biological effect index TU _1:1 at the equivalent effect ratio

${\mathrm{<span\; class="notranslate">\; TU</span>}}_{\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; :</span>\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}^{<span\; class="notranslate">\; \&prime;</span>}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}^{<span\; class="notranslate">\; \&prime;</span>}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

Table 1

With log n/m as the abscissa and TU at different concentration ratios as the ordinate, draw the curve of the combined effect of the binary mixture system with the concentration.

In Fig. 1, log n/m represents the abscissa (n and m represent the concentration multiples of the two substances A and B in the mixed system, that is, the degrees of the two substances A and B in the mixed solution are respectively n×EC _50-A , m ×EC _50-B ), TU at different concentration ratios is the ordinate, and the curve of the combined effect of the binary mixture system with the concentration is drawn.

It can be seen from Figure 1 that at the equivalence ratio point (logn/m=0), TU _1:1 is the smallest, that is, the synergistic effect is the strongest. As the concentration shifted from the equivalent effect ratio point to the non-equivalent effect point, the TU value gradually increased to 1.00±0.20, that is, the synergistic effect gradually weakened, and finally weakened to the additive effect. This result shows that the prediction method used in the present invention is accurate and reliable, and can well predict the change rule of the joint biological effect and find the best biological effect point.

Example 2

(1) Determination steps of single compound biological effect concentration

Add 0.25ml of Luminescent Bacteria solution (T3) to a volumetric flask containing 5ml of culture medium, and cultivate for 12 hours to make a shake flask solution; then take 0.2ml of shake flask solution into 20ml of 3% sodium chloride and aerate Stir for 40 minutes to make a working bacteria solution. Take an appropriate amount of phthalonitrile (compound A) and p-dimethylaminobenzaldehyde (compound B) to form a standard series of logarithmic gradients, with a volume of 0.8ml, add 0.2ml of working bacteria solution, shake well, and infect for 15 minutes. Measure the reduction degree of the light value of the system before and after exposure, and use the interpolation method to calculate the concentration when the inhibition rate is 50%, which is the EC ₅₀ value of the substance (or system). The calculated EC ₅₀ values of phthalonitrile (compound A) and p-dimethylaminobenzaldehyde (compound B) are 5.860×10 ^-4 and 2.229×10 ^-6 mol/L, respectively.

(2) Determination of the joint biological effect of the binary equivalent effect ratio mixed system

According to the single biological effect concentration (EC ₅₀ ) of the two compounds A and B obtained from the determination, the same steps as the single compound (EC ₅₀ ) are used to measure the biological effect concentration of the mixed solution, and the inhibition of the biological effect of the mixed system can be obtained. is the concentration of A and B in the mixed system (C _A , C _B ) at 50%.

Further, this step specifically includes the following steps (or key implementation points):

1. Preparation of mixed solution

According to the measured single compound biological effect concentration (EC _50-A , EC _50-B ) of the two substances A and B, a mixed solution with an equivalent effect ratio is prepared. Prepare a mixed solution with equivalent effect ratio, that is, the concentrations of A and B in the mixed solution are 5.860×10 ^-4 and 2.229×10 ^-6 mol/L respectively.

2. Mixed solution test series

Assuming that the depth of the mixed solution is 100%, the mixed solution is diluted according to equilogarithmic intervals, namely: 100%, 80%, 56%, 32%, 18%, 10%, 8%. Then take this as the test series, and use the Photobacterium phosphoreum T3 variant as the test organism, and use the fluorescence immunoassay analyzer to test the inhibition rate of this series.

3. Calculation: take the mixed system concentration (or dilution factor) as the x-axis, plot the y-axis with the inhibition rate, and use the interpolation method to find the concentration when the inhibition rate is 50%, and then the mixed system inhibition rate is 50%. %, the concentrations C _A and C _B of substances A and B in the mixed system are 4.952×10 ^-4 and 1.884×10 ^-6 mol/L respectively

Substitute C _A , C _B and single compound biological effect concentration EC _50-A , EC _50-B into the following formula to calculate the joint biological effect index TU _1:1 at the equivalent effect ratio

${\mathrm{<span\; class="notranslate">\; TU</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; :</span>\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

$<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 4.952</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 4</span>}}}{\mathrm{<span\; class="notranslate">\; 5.860</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 4</span>}}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1.884</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 6</span>}}}{\mathrm{<span\; class="notranslate">\; 2.229</span>}<span\; class="notranslate">\; \&times;</span>{\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 6</span>}}}$

$<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1.69</span>}$

(3) Determination of the optimal biological effect point

The results showed that the TU _1:1 value of the mixed system of phthalonitrile and p-dimethylaminobenzaldehyde was 1.69 at the equivalent effect ratio. According to the judging principle of combined effect, TU greater than 1.20 was an antagonistic effect. It can be predicted that the joint effect (antagonism) of the mixed system of phthalonitrile and p-dimethylaminobenzaldehyde is the strongest at the equipotential point. As the concentration of the two substances mixed changes from equivalent ratio to non-equivalence ratio, the combined effect (antagonist effect) will gradually weaken to additive effect.

This suggests that when we treat wastewater containing this type of compound, we can use the equivalent toxicity ratio point discharge, and use its resistance effect to minimize the treatment cost and environmental hazards. In the same way, for two drugs with antagonistic effects, it is absolutely necessary to avoid combining drugs at equal toxicity ratios, otherwise the therapeutic effect will be greatly reduced.

(4) Verification of method reliability

In order to verify our assay method, we measured the joint biological effect under non-equivalent effect ratio conditions (that is, two substances are mixed with other concentration ratios), and compared their sizes. The results showed that the joint biological effect measured was indeed The synergy is strongest. The result is shown in Figure 2.

In Fig. 2, log n/m represents the abscissa (n and m represent the concentration multiples of the two substances A and B in the mixed system, that is, the degrees of the two substances A and B in the mixed solution are respectively n×EC _50-A , m ×EC _50-B ), TU at different concentration ratios is the ordinate, and the curve of the combined effect of the binary mixture system with the concentration is drawn.

It can be seen from Figure 2 that at the point of equivalent effect ratio (logn/m=0), TU _1:1 is the largest, that is, the antagonistic effect is the strongest. As the concentration shifted from the equivalent effect ratio point to the non-equivalent effect point, the TU value gradually decreased to 1.00±0.20, that is, the antagonistic effect gradually weakened, and finally weakened until the additive effect. This result shows that the prediction method used in the present invention is accurate and reliable, and can well predict the change rule of the joint biological effect and find the best biological effect point.

Example 3

(1) Determination steps of single compound biological effect concentration

Add 0.25ml of luminescent bacteria solution (T3) to a volumetric flask containing 5ml of medium, and cultivate for 12 hours to make a shake flask solution; then take 0.2ml shake flask solution into 20ml of 3% sodium chloride, Gas stirring for 40min to make working bacteria liquid. Take an appropriate amount of acetonitrile (substance A) and terephthalaldehyde (substance B) to form a standard series of logarithmic gradients, with a volume of 0.8ml, add 0.2ml of working bacteria solution, shake well, expose for 15 minutes, and measure the system before and after exposure. For the reduction degree of light value, the concentration at which the inhibition rate is 50% is calculated by interpolation method, which is the EC ₅₀ value of the substance (or system). The calculated EC ₅₀ values were 0.189 and 6.892×10 ^-5 mol/L, respectively.

(2) Determination of the joint biological effect of the binary equivalent effect ratio mixed system

According to the single biological effect concentration (EC ₅₀ ) of the two compounds A and B obtained from the determination, and then adopt the same steps as the single compound (EC ₅₀ ) to measure the biological effect concentration of the mixed solution, the biological effect of the mixed system can be obtained. Concentrations of A and B in the mixed system (C _A , C _B ) when the inhibition is 50%.

Further, this step specifically includes the following steps (or key implementation points):

1. Preparation of mixed solution

According to the measured single compound biological effect concentration (EC _50-A , EC _50-B ) of the two substances A and B, a mixed solution with an equivalent effect ratio is prepared. Prepare a mixed solution with equivalent effect ratio, that is, the concentrations of A and B in the mixed solution are 0.189 and 6.892×10 ^-5 mol/L respectively.

2. Mixed solution test series

Assuming that the depth of the mixed solution is 100%, the mixed solution is diluted according to equilogarithmic intervals, namely: 100%, 80%, 56%, 32%, 18%, 10%, 8%. Then take this as the test series, and use the Photobacterium phosphoreum T3 variant as the test organism, and use the fluorescence immunoassay analyzer to test the inhibition rate of this series.

3. Calculation: take the mixed system concentration (or dilution factor) as the x-axis, plot the y-axis with the inhibition rate, and use the interpolation method to find the concentration when the inhibition rate is 50%, and then the mixed system inhibition rate is 50%. %, the concentrations C _A and C _B of A and B in the mixed system are 0.103 and 3.756×10 ^-5 mol/L respectively

Substitute C _A , C _B and single compound biological effect concentration EC _50-A , EC _50-B into the following formula to calculate the joint biological effect index TU _1:1 at the equivalent effect ratio

${\mathrm{<span\; class="notranslate">\; TU</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; :</span>\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}$

$=\frac{0.103}{0.189}+\frac{3.756\&times;{10}^{-5}}{6.892\&times;{10}^{-5}}$ Formula 1

$<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1.09</span>}$

(3) Determination of the optimal biological effect point

The results showed that the TU _1:1 value of the mixed system of acetonitrile and terephthalaldehyde was 1.09 at the equivalent effect ratio. According to the judging principle of combined effect, TU=1.00±0.20 was the additive effect. It can be predicted that, for the system (acetonitrile and terephthalaldehyde) which is a mixture of additive effects in the equivalent effect ratio, the size of the joint toxic effect remains constant (TU is between 0.8 and 1.2), regardless of the concentration of the two compounds. Change and change. For this type of compound, according to the principle of economics and practicality, an appropriate ratio can be selected for joint use to achieve a win-win goal of economic input-output benefit.

(4) Verification of method reliability

In order to verify our measurement method, we measured the joint biological effect under non-equivalent effect ratio conditions (that is, the two substances were mixed at other concentration ratios), and compared their magnitudes. The results are shown in Figure 3.

In Fig. 3, log n/m represents the abscissa (n and m represent the concentration multiples of A and B in the mixed system, that is, the degrees of A and B in the mixed solution are respectively n×EC _50-A , m ×EC _50-B ), TU at different concentration ratios is the ordinate, and the curve of the combined effect of the binary mixture system with the concentration is drawn.

It can be seen from Figure 3 that the combined biological effects measured at different concentration ratios are indeed additive and do not change with concentration changes. This result shows that the prediction method used in the present invention is accurate and reliable, and can well predict the change rule of the joint biological effect and find the best biological effect point.

The above description of the embodiments is for those of ordinary skill in the art to understand and apply the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the embodiments herein. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims (3)

1. A method for determining the compatibility of a binary mixture to obtain the best joint biological effect, characterized in that: the method comprises the following steps, (1) Determination of the biological effect concentration of a single compound The test method is based on GB/T 15441-1995, using Photobacterium luminosa as the indicator organism, to determine the single toxicity of two types of compounds A and B, and the biological effect of a single compound is expressed by EC50, and the biological effect concentrations of these two single compounds are respectively EC _{50 -A} , EC _50-B ; (2) Determination of the joint biological effect of the equivalent effect ratio According to the single biological effect concentrations EC _50-A and EC _50-B of the two compounds A and B obtained through the determination, a mixed solution with an equivalent effect ratio is prepared, that is, the concentrations of the two substances A and B in the mixed solution are respectively EC _{50- A} and EC _50-B , the test method is based on GB/T 15441-1995, the concentration of the biological effect of the mixed solution is measured, and the concentration of A and B in the mixed system C A and _B can be obtained when the mixed system inhibits the biological effect by 50%. C _B , and then calculate the joint biological effect index TU _{1: 1} when the equivalent effect ratio is obtained; (3) Determination of the optimal biological effect point According to the determination result TU _{1 : 1} of the joint biological effect obtained from the calculation of the equivalent effect ratio in step (2), the change rule of the joint biological effect of the binary mixed system is determined, and the best biological effect point is obtained. 2. the method for measuring the binary mixture according to claim 1 to obtain the best joint biological effect is characterized in that: the size of the joint biological effect index TU is used to evaluate the joint biological effect, TU _1:1 <0.80 means that the two compounds A and B have a synergistic effect; TU _1:1 >1.20 indicates that the two compounds A and B have produced antagonistic effects; TU _1:1 = 1.00±0.20 means that the two compounds A and B produced an additive effect. 3. The method for measuring the compatibility of the binary mixture to obtain the best combined biological effect according to claim 1, characterized in that: the TU _1:1 is calculated using the following formula $\mathrm{<span\; class="notranslate">\; TU</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; A</span>}}}<span\; class="notranslate">\; +</span>\frac{{\mathrm{<span\; class="notranslate">\; c</span>}}_{\mathrm{<span\; class="notranslate">\; B</span>}}}{{\mathrm{<span\; class="notranslate">\; EC</span>}}_{\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; B</span>}}}<span\; class="notranslate">\; .</span>$

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