CN114705647A - Method for detecting acute toxicity of water body - Google Patents
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Abstract
The invention belongs to the technical field of water body detection, and discloses a method for detecting acute toxicity of a water body. The method comprises the steps of adding a substance containing a mercaptan group into a water body to be detected to enable the substance to react with toxic substances in the water body, quantifying the residual mercaptan by using an Elman spectrophotometry, and representing the content of the toxic substances in a water sample by using a positive reference substance with a definite toxicity mechanism through an equivalent concentration method, so that the acute toxicity of the water quality of a complex water sample can be rapidly detected and analyzed.
Description
Technical Field
The invention belongs to the technical field of water body detection, and particularly relates to a method for detecting acute toxicity of a water body.
Background
Water is a life source, and water quality safety is a foundation for ecosystem safety and is related to human survival and development. In recent years, as human activities have increased, more and more pollutants are directly or indirectly discharged into water bodies, causing pollution of the water bodies. The toxicity and biosafety of water bodies are receiving increasing attention. Due to the existence of a large amount of unknown pollutants and the coexistence of a plurality of pollutants, compound toxins such as antagonism, synergism and the like can be generatedDue to sexual function, the traditional water quality physical and chemical indexes and the conventionally monitored pollutant concentration cannot comprehensively reflect the toxicity level of the polluted water body. At present, the toxicity of the polluted water body is comprehensively evaluated by mainly adopting a biological monitoring method, wherein an acute toxicity test is widely applied to the rapid detection of the water body, and mature water quality acute toxicity measuring methods comprise a luminous bacteria method, a flea (daphnia magna) method, a freshwater fish (zebra fish) method and a zebra fish roe method. The luminous bacteria method is based on the fact that the relative luminosity of luminous bacteria is obviously and negatively correlated with the total concentration of toxic components in a water sample, so that the relative luminosity of the water sample can be measured through a bioluminescence photometer to show the acute toxicity level of the water sample. The flea method, freshwater fish method and zebra fish roe method are implemented by measuring half inhibitory concentration and half lethal concentration (EC) of polluted water body on test organisms50) To judge the toxicity degree.
The flea method, the freshwater fish method and the zebra fish roe method belong to biological behavior reaction monitoring methods and have the following common defects:
1) low sensitivity and easy interference to test organism
Only after toxic substances in the water environment are accumulated to a certain concentration, the poisoning reaction of the fishes or the fleas can be generated, and the response speed and the sensitivity are lower for the water quality with lower pollutant concentration. In addition, the behavior of the test organism is easily interfered by conditions such as external environment, climate and the like, and the accuracy of the result is influenced.
2) Need to train the professional
The biological behavior reaction monitoring method is carried out through biological behavior reaction when determining the half inhibitory concentration and the half lethal concentration of the polluted water body on test organisms, and can be completed after trained professionals accumulate experience, so that the requirement on experimenters is higher.
3) Long monitoring period and complex operation
Freshwater fish needs to be domesticated, zebra fish roes also need to lay eggs by culturing breeding fish, and then available fish roes are screened out for experiment, so that a large amount of time and experimental materials are consumed in the process, the operation is complicated, and the cost is high.
In addition, the biological behavior monitoring method consumes a lot of time for testing biological culture, and has long operation time in the actual detection process, so that the monitoring period is long, and the method is not suitable for field monitoring and needs to be operated in a laboratory.
The luminescent bacteria method has the following defects: the activity of the luminescent bacteria is unstable, the effective test time is short, and the difference of the luminous intensity of different batches of strains is large, so that the reproducibility of the luminescent bacteria method is low. In addition, the luminescent strains are not easy to culture, need to be purchased and used, and the required instruments and equipment are expensive and have high cost.
In general, although the biotoxicity detection technology overcomes the limitations of traditional physicochemical detection and instrument analysis, can judge the single toxicity and comprehensive toxicity of pollutants in water and quickly judge the water quality condition, and has important significance in the aspect of guaranteeing the drinking water safety of people, the biotoxicity detection technology still has the problems of low detection sensitivity, long monitoring period, complex operation, high cost, poor result reproducibility and the like.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for detecting the acute toxicity of the water body, which has the advantages of high detection sensitivity, simple and convenient operation, short detection time, good stability and repeatability and no need of expensive instruments.
In order to achieve the above object, the present invention provides the following specific technical solutions.
A method for detecting acute toxicity of a water body comprises the following steps:
preparing an Elemann reagent (5, 5' -dithiobis- (2-nitrobenzoic acid, DTNB) solution as a color developing agent;
preparing a solution of a thiol group-containing substance;
preparing a positive reference substance solution;
establishing response relation between positive reference substance solutions with different concentrations and absorbance: after the positive reference substance solutions with different concentrations react with the solution of the substance containing the thiol group with the same concentration, adding a developer solution, measuring the absorbance at 412nm, and determining the response relation between the absorbance and the concentration of the positive reference substance;
detecting the acute toxicity of the water body to be detected: and (3) after the water body to be detected reacts with the solution of the thiol group-containing substance, adding a developer solution, measuring the absorbance at 412nm, and determining the equivalent concentration of the positive reference substance of the water body to be detected according to the response relation between the determined absorbance and the concentration of the positive reference substance.
In some preferred embodiments of the invention, the developer is formulated by: firstly, preparing a potassium phosphate buffer solution, and then adding an Eleman reagent into the potassium phosphate buffer solution to obtain the color developing agent.
Further, potassium phosphate buffer solution was prepared using dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
Further, the pH value of the potassium phosphate buffer solution is 7 to 9.
In a partially preferred embodiment of the present invention, the thiol group-containing substance is at least one selected from Glutathione (GSH), N-acetyl-L-cysteine (NAC), Cysteamine (CY), L-Homocysteine (HCYS), Thioglycolic acid (TGA).
In some preferred embodiments of the present invention, the positive reference is selected from at least one of soluble salts of mercury, arsenic, cadmium, lead, and chromium.
According to the invention, a substance containing a mercaptan group is added into a water body to be detected to react with toxic substances in the water body, then the residual mercaptan is quantified by using an Elmann spectrophotometry, a positive reference substance with a definite toxicity mechanism is used for representing the content of the toxic substances in a water sample by using an equivalent concentration method, and the rapid detection and analysis of the acute toxicity of the water quality of a complex water sample can be carried out.
Compared with the prior art, the invention has the following characteristics and advantages:
1) and (3) the speed is high. The detection result can be obtained within 1 hour. In actual detection, only a substance solution containing thiol groups needs to be prepared at present, and a buffer solution, a color developing agent and a positive reference substance can be prepared into a stock solution in advance and used directly or after being diluted according to needs on site. From the preparation of the solution to the acquisition of the detection result, only 50 to 60 minutes are needed, so that the detection time is greatly saved, the rapid detection of the acute toxicity of the water quality on site can be realized, and the safety conditions of the river water quality, the urban water supply system and the drinking water sold in the market can be pre-warned.
2) Simple operation and strong practicability. The method for detecting the acute toxicity of the water body is simple, the prepared solution of the substance containing the mercaptan group and the water sample are reacted, then the color developing agent is added to test the absorbance, and meanwhile, a positive reference substance concentration-absorbance standard curve is drawn to obtain a result, so that the operation and the principle are simple, and the requirement on experimenters is not high. During on-site detection, a high-end special instrument is not needed, the detection time is short, the operation is convenient, and the on-site detection of the acute toxicity of the water sample can be realized. In addition, the available thiol group-containing compound and the positive reference substance are various in types and can be flexibly selected according to actual conditions.
3) The sensitivity is high. When the concentration of the water body pollutants is low, the biological monitoring method has slow toxic response and low sensitivity to the pollutants, and the water body with low concentration of the pollutants monitored by the thiol group-containing substance used in the method is very high in sensitivity. In addition, the substance containing the thiol group belongs to a chemical reagent, and is slightly interfered by external environment, climate and the like when in use.
4) The result is good in reproducibility. The method of the invention represents the water toxicity by using the substance containing the thiol group as a detection probe to react with the toxic substances in the water body, and is different from a biological monitoring method, the reagent used in the method belongs to a chemical reagent, the property is stable, the preservation is easy, the difference of the monitoring results of reagents in different batches is very small, and the using process is not influenced by external environment, climate conditions and the like, so the reproducibility of the monitoring results obtained by using the method of the invention is good.
5) The cost is low. The reagents used in the method mainly comprise a thiol group-containing substance, a buffer reagent and a positive reference substance (such as cadmium chloride, mercury chloride and the like), and the required reagents are low in price, long in storage time and capable of being used for a long time; the required instruments mainly comprise a shaking table, an ultraviolet spectrophotometer and the like, expensive instruments and equipment and special reagents are not required, and the cost is low.
6) Can detect the comprehensive toxicity of the poison in the water sample to be detected. The method can judge the single toxicity and the comprehensive toxicity of pollutants in the water body and quickly judge the water quality condition, which is the same as the biological monitoring method. Even poisons that have not been discovered by man or new chemically synthesized poisons that have never appeared are capable of reacting with thiol group-containing substances, thereby affecting absorbance. The method of the invention can also be used for detecting the toxicity.
7) The application range is wide. The present invention can be applied to: firstly, detecting the toxicity of surface water; detecting the toxicity of the industrial wastewater; thirdly, detecting the effluent of the sewage plant; fourthly, detecting the safety of the urban water supply system; detecting the drinking water pollution caused by leakage accidents, environmental pollution, natural disasters and artificial damages; sixthly, detecting the toxicity of water quality of rivers, lakes and the like.
Drawings
FIG. 1 is a diagram showing the reaction process of thiol with Elemann reagent.
FIG. 2 shows CdCl2Concentration-absorbance standard curve.
FIG. 3 shows HgCl2Concentration-absorbance standard curve.
FIG. 4 is NaAsO2Concentration-absorbance standard curve.
Detailed Description
The invention is described in detail below, and the description in this section is merely exemplary and explanatory and should not be construed as limiting the scope of the invention in any way. Furthermore, those skilled in the art can combine features from the embodiments of this document and from different embodiments accordingly based on the description of this document.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
The invention uses the substance containing cysteine thiol as a detection probe to detect the acute toxicity of water quality. Cysteine thiol is a main reducing agent in organisms against active toxicants, can cause adverse biological reactions if thiol is overloaded or exhausted, is very sensitive to trace toxic and harmful substances existing in water, and can react with thiol groups to consume thiol when harmful substances including heavy metals, inorganic chemical pollutants, organic pollutants and the like exist in water. Adding a quantitative substance containing cysteine thiol groups into a water sample, and evaluating the acute toxicity of the water sample by measuring the content of the residual thiol in the water sample after a period of time. Thiol can react with an Elemann reagent (5, 5' -dithiobis- (2-nitrobenzoic acid, DTNB) 1:1 to generate a yellow product NTB (2-nitro-5-thiobenzoate), as shown in figure 1, the NTB can measure the absorption peak at 412nm by spectrophotometry, that is, the residual thiol can be used for measuring the absorbance at 412nm for quantification, but because the measured absorbance has no reference value, the relationship between the absorbance and the acute toxicity of water quality can not be judged, the invention uses an equivalent concentration method, uses pollutants with a clear toxicity mechanism as a positive reference, generates a concentration-absorbance standard curve by measuring the absorbance of the positive reference with different concentrations after reacting with a thiol group-containing substance, and judges the equivalent concentration of the positive reference by using the curve and the absorbance actually measured by a water sample, further, the acute toxicity of the water quality can be judged.
Furthermore, the thiol group-containing substance for acute toxicity detection of water quality includes five kinds, i.e., reduced Glutathione (gshi), acetylcysteine (N-acetyl-L-cysteine, NAC), Cysteamine (CY), L-Homocysteine (HCYS), Thioglycolic acid (TGA), and the positive reference substance may be soluble salts of mercury, arsenic, cadmium, lead, chromium, and the like.
Example 1
Detection of acute toxicity of effluent of sewage plant by acetylcysteine (CdCl)2Concentration expression):
1) obtaining a water sample: effluent water samples of sewage plants are collected from effluent water samples of three sewage treatment plants in Guangzhou city.
2) Solution preparation
Step 1: preparation of a buffer: 100 mL of a 10 mmol/L potassium phosphate buffer solution was prepared using 1 mol/L potassium phosphate dibasic and potassium phosphate dibasic solutions, and the pH was adjusted to 8 using 0.1 mol/L phosphoric acid.
Step 2: preparing a color developing agent: 4.0 mg of Eleman reagent (5, 5' -dithiobis (2-nitrobenzoic acid), DTNB) was weighed out and prepared in a potassium phosphate buffer solution at a concentration of 100. mu. mol/L.
And 3, step 3: preparation of thiol group-containing material solution: acetylcysteine (NAC) is selected as a detection probe, NAC mother liquor with the concentration of 10 mmol/L is prepared, 1mL of NAC mother liquor is diluted into 49 mL of ultrapure water, and the diluted NAC mother liquor is diluted to 200 mu mol/L and used as NAC application liquid.
And 4, step 4: preparation of positive reference substance solution: CdCl is selected2As a positive reference, 10mL of 200 mg/L stock solution was prepared, and 6 mL of CdCl was taken2The stock solution was diluted into 24 mL of ultrapure water to obtain 30 mL of 40 mg/L CdCl2The liquid is applied.
3) Drawing CdCl with different concentrations2And the standard curve of the absorbance measured after reaction with NAC: CdCl in the system2Final concentrations were 0, 1, 2, 4, 6, 8 mg/L: CdCl 0, 0.125, 0.25, 0.5, 0.75 and 1mL are measured respectively2The application liquid is added into a 6-piece 10mL centrifuge tube, and then 1.5 mL, 1.375 mL, 1.25 mL, 1mL, 0.75 mL and 0.5 mL of ultrapure water are respectively added until the total volume is 1.5 mL. Add 1mL of NAC application solution to the centrifuge tubes. Placing the centrifuge tube in a shaking table, and incubating for 30 min in the dark, wherein the rotation speed of the shaking table is 180 r/min. Taking out after the incubation is finished, adding 2.5 mL of color development agent DTNB, measuring the absorbance at 412nm by using an ultraviolet-visible spectrophotometer, and determining CdCl2Standard curve of concentration versus absorbance of the solution.
CdCl at each different concentration when determining the standard curve2Solution samples were run in 3 replicates.
4) Detection of acute toxicity of water sample to be detected
The detection steps of the acute toxicity of the water sample to be detected are basically the same as the step 3), and only CdCl added into a centrifugal tube is added2The solution is changed into a water sample to be detected, the color developing agent is added to determine the absorbance after the reaction with the NAC is finished, the reaction is repeated for three times, the average value of the determined absorbance is compared with the standard curve drawn in the step 3), and the CdCl of the water sample to be detected can be obtained2And (5) determining the acute toxicity of the water quality by equivalent concentration.
CdCl of different concentrations2The standard curve against absorbance is shown in FIG. 2. The absorbance values measured after the three effluent water samples of the sewage plant react with NAC are 0.388, 0.393 and 0.391 respectively, and CdCl of the three effluent water samples of the sewage plant can be obtained by calculating a standard curve2The equivalent concentrations were 0.955 mg/L, 0.793 mg/L, 0.858 mg/L, respectively.
The reference poison HgCl is measured by adopting a method for measuring the acute toxicity of water quality by using a luminescent bacteria method described in the national standard GB/T15441-19952By conversion to CdCl2The water samples are tested, and the results show that the CdCl of the effluent water samples of the three sewage plants2The equivalent concentrations are respectively 0.949 mg/L, 0.790 mg/L and 0.859 mg/L, and the CdCl measured by the method of the invention2The relative errors of the equivalent concentration and the result measured by the national standard are 0.63%, 0.38% and 0.12%, which shows that the method of the invention is consistent with the detection result of the national standard method and has high accuracy.
Example 2
Detection of acute toxicity of industrial wastewater by using acetylcysteine (HgCl)2Concentration expression):
1) obtaining a water sample: the industrial waste water samples are respectively collected from the waste water of a certain food processing factory, a certain paper mill and a certain smelting plant in Guangzhou city.
2) Solution preparation
Steps 1 to 3 were the same as those in steps 1 to 3 of example 1.
And 4, step 4: preparation of positive reference substance solution: selecting HgCl2As a positive control, 20 mg/L of HgCl was prepared2Stock solution, take 6 mL HgCl2The stock solution was diluted into 24 mL of ultrapure water to obtain 30 mL of HgCl at a concentration of 4 mg/L2The liquid is applied.
3) Drawing HgCl of different concentrations2And the standard curve of the absorbance measured after reaction with NAC: HgCl in the system2Respectively at 0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6 mg/L: 0, 12.5, 62.5, 125, 250, 375, 500 and 750 mu L of HgCl are respectively measured and measured2The application liquid is added into a 6-piece 10mL centrifuge tube, and then 1.5 mL, 1.4875 mL, 1.4375 mL, 1.375 mL, 1mL and 0.75 mL of ultrapure water are respectively added until the total volume is 1.5 mL. Add 1mL of NAC application solution to the centrifuge tubes. Placing the centrifuge tube in a shaking table, and incubating for 30 min in the dark, wherein the rotation speed of the shaking table is 180 r/min. After the incubation is finished, taking out the cells, adding 2.5 mL of color developing agent DTNB, measuring the absorbance at 412nm by using an ultraviolet-visible spectrophotometer, and determining HgCl2Standard curve of concentration versus absorbance of the solution.
HgCl at each different concentration when determining the standard curve2Solution samples were run in 3 replicates.
4) Detection of acute toxicity of water sample to be detected
The acute toxicity detection step of the water sample to be detected is basically the same as the step 3), except that the HgCl added into the centrifuge tube is added2The solution is changed into a water sample to be detected, the color developing agent is added to determine the absorbance after the reaction with the NAC is finished, the reaction is repeated for three times, the average value of the determined absorbance is compared with the standard curve drawn in the step 3), and the HgCl of the water sample to be detected can be obtained2And (5) determining the acute toxicity of the water quality by equivalent concentration.
HgCl of different concentrations2The standard curve against absorbance is shown in FIG. 3. The absorbance values measured after the industrial wastewater water samples of the food processing plants, the paper mills and the smelting plants react with NAC are 0.208, 0.185 and 0.138 respectively, and the HgCl of the industrial wastewater water samples of the food processing plants, the paper mills and the smelting plants can be calculated by a standard curve2The equivalent concentrations were 0.429 mg/L, 0.474 mg/L, and 0.566 mg/L, respectively.
The water sample is tested by adopting the method for determining the acute toxicity of the water quality by the luminescent bacteria described in the national standard GB/T15441-1995, and the result shows that the HgCl of the industrial wastewater water sample of the food processing plant, the paper mill and the smelting plant2The equivalent concentration is 0.427 mg/L, 0.478 mg/L and 0.563 mg/L respectively, which are measured by the method of the inventionTo obtain HgCl2The relative errors of the equivalent concentration and the result measured by the national standard are 0.47%, 0.84% and 0.53%, which shows that the method of the invention is consistent with the detection result of the national standard method and has high accuracy.
Example 3
Detection of acute toxicity of tap water by reduced glutathione (NaAsO)2Concentration expression):
1) obtaining a water sample: tap water samples were collected from tap water in three regions of Guangzhou City.
2) Solution preparation:
And 3, step 3: preparing a thiol group-containing substance solution: selecting reduced Glutathione (GSH) as a detection probe, preparing GSH mother liquor with the concentration of 10 mmol/L, taking 1mL of GSH mother liquor to dilute into 49 mL of ultrapure water, and diluting to 200 mu mol/L to be used as GSH application liquid.
And 4, step 4: preparation of positive reference substance solution: selecting NaAsO2As a positive reference, 200 mg/LNaAsO was prepared26 mL of NaAsO was taken as the stock solution2The stock solution was diluted into 24 mL of ultrapure water to obtain 30 mL of 40 mg/L NaAsO2The liquid is applied.
3) Plotting NaAsO at different concentrations2And the absorbance standard curve measured after the reaction with GSH: NaAsO in the system2Are 0, 1, 2, 4, 6, 8 mg/L: respectively measuring NaAsO with different concentrations of 0, 0.125, 0.25, 0.5, 0.75 and 1mL2The solution was added to 6 10mL centrifuge tubes, followed by 1.5, 1.375, 1.25, 1, 0.75, and 0.5 mL of ultrapure water, respectively, to a total volume of 1.5 mL. 1mL of GSH application solution was added to each centrifuge tube. Placing the centrifuge tube in a shaking table, and incubating for 30 min in the dark, wherein the rotation speed of the shaking table is 180 r/min. After the incubation is finished, taking out the cells, adding 2.5 mL of color developing agent DTNB, measuring the absorbance at 412nm by using an ultraviolet-visible spectrophotometer, and determining NaAsO2Standard curve of concentration versus absorbance of the solution.
Determination of the Standard Curve, NaAsO at each of the different concentrations2Solution samples were run in 3 replicates.
4) Detection of acute toxicity of water sample to be detected
The detection steps of the acute toxicity of the water sample to be detected are basically the same as the step 3), and NaAsO added into a centrifugal tube is added2The solution is changed into a water sample to be tested, the color developing agent is added to determine the absorbance after the reaction with GSH is finished, the reaction is repeated for three times, the average value of the determined absorbance is compared with the standard curve drawn in the prior art, and the NaAsO of the water sample to be tested can be obtained2And (5) determining the acute toxicity of the water quality by equivalent concentration.
Different concentrations of NaAsO2The standard curve against absorbance is shown in FIG. 4. The absorbance values measured after the tap water samples in the three areas react with GSH are respectively 0.484, 0.488 and 0.485, and NaAsO of the tap water samples can be obtained by calculating a standard curve2The equivalent concentration is 0.262 mg/L, 0.183 mg/L, 0.241 mg/L.
The reference poison HgCl is measured by adopting a method for measuring the acute toxicity of water quality by using a luminescent bacteria method described in the national standard GB/T15441-19952By conversion to NaAsO2The water samples are tested, and the results show that the NaAsO of tap water samples in three regions2The equivalent concentration is 0.264 mg/L, 0.182 mg/L and 0.242 mg/L respectively, and the NaAsO measured by the method of the invention2The relative errors of the equivalent concentration and the result measured by the national standard are respectively 0.76%, 0.55% and 0.41%, which shows that the method of the invention is consistent with the detection result of the national standard method and has high accuracy.
The above-mentioned embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical scope of the present invention, and equivalents and modifications of the technical solutions and concepts of the present invention should be covered by the scope of the present invention.
Claims (6)
1. A method for detecting acute toxicity of a water body is characterized by comprising the following steps:
preparing an Elemann reagent solution as a color developing agent;
preparing a solution of a thiol group-containing substance;
preparing a positive reference substance solution;
establishing response relation between positive reference substance solutions with different concentrations and absorbance: after the positive reference substance solutions with different concentrations react with the solution of the substance containing the thiol group with the same concentration, adding a developer solution, measuring the absorbance at 412nm, and determining the response relation between the absorbance and the concentration of the positive reference substance;
detecting the acute toxicity of the water body to be detected: and (3) after the water body to be detected reacts with the solution of the thiol group-containing substance, adding a developer solution, measuring the absorbance at 412nm, and determining the equivalent concentration of the positive reference substance of the water body to be detected according to the response relation between the determined absorbance and the concentration of the positive reference substance.
2. The assay of claim 1, wherein the color-developing agent is formulated by: firstly, preparing a potassium phosphate buffer solution, and then adding an Eleman reagent into the potassium phosphate buffer solution to obtain the color developing agent.
3. The detection method according to claim 2, wherein the potassium phosphate buffer solution is prepared using dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
4. The detection method according to claim 2 or 3, wherein the pH of the potassium phosphate buffer solution is 7 to 9.
5. The method according to claim 1, wherein the thiol group-containing substance is at least one member selected from the group consisting of glutathione, acetylcysteine, cysteamine, L-homocysteine, and thioglycolic acid.
6. The detection method of claim 1, wherein the positive reference is selected from at least one of soluble salts of mercury, arsenic, cadmium, lead, and chromium.
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| CN117092274A (en) * | 2023-07-14 | 2023-11-21 | 国药集团致君（深圳）坪山制药有限公司 | Method for determining sevelamer carbonate content |
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