Methods for obtaining optimal water sample pretreatment

Abstract

This invention relates to the field of water sample pollutant detection technology, and discloses a method for obtaining an optimal water sample pretreatment method, which mainly includes the following steps: after acidification and membrane extraction, the water sample is enriched and concentrated by different solid phase extraction columns, followed by rinsing, drying, elution, nitrogen blowing and volume adjustment for detection of different biotoxicities. The detection results are ranked from worst to best based on different water sample types and different biotoxicities using the "Pareto optimality principle". Finally, the optimal water sample pretreatment method is determined by combining the two ranking results, and the optimal pretreatment method is further determined by combining the TOC before and after solid phase extraction and the recovery rate of different hydrophilic and hydrophobic spiked substances.

Description

Technical Field

[0001] This invention relates to the field of water sample pollutant detection technology, and more specifically, to a method for obtaining a preferred pretreatment method for water samples. Background Technology

[0002] Water quality assessment is crucial for protecting drinking water resources and ecosystem integrity. Therefore, water sample pretreatment is essential, especially when studying emerging contaminants and the biotoxicity of water samples. However, because pretreatment methods are often chosen based on chemical analysis and rarely optimized for biotoxicity assays, this can lead to reduced accuracy in biotoxicity detection.

[0003] Most current research focuses on optimizing pretreatment methods for chemical analysis, aiming to maximize the recovery rate of specific target compounds. When detecting biotoxicity of key toxic substances, a suitable pretreatment method is often chosen by adding a standard of the key toxic substance to the water sample and comparing the recovery rates. However, biotoxicity in water samples is often the result of the combined effects of multiple known and unknown substances. Using only the recovery rate of the key toxic substance as a criterion may lead to underestimation / overestimation or false negatives / false positives. More importantly, most biotoxicities lack a clearly defined key toxic substance. Therefore, optimizing pretreatment methods for biotoxicity detection based solely on chemical analysis has certain limitations.

[0004] Therefore, optimizing water sample pretreatment to maximize the recovery of biotoxicity is essential for bioassay studies.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for obtaining a better pretreatment method for water samples.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for obtaining a preferred pretreatment method for water samples, comprising: Provide spiked samples: Standard pollutants are added to the water sample to obtain spiked samples. The standard pollutants are a variety of different substances with an octanol-water partition coefficient between 1.49 and 6.76. Sample pretreatment: Multiple groups of spiked samples and multiple groups of water samples were acidified and filtered through membranes, and then concentrated and enriched by different activated solid phase extraction columns. Subsequently, multiple groups of pretreated spiked samples and multiple groups of pretreated water samples were obtained by rinsing, drying, elution, nitrogen blowing and volume adjustment. Biotoxicity testing: Non-specific toxicity, specific toxicity, and reactive toxicity were tested for each group of pretreated water samples; Chemical analysis: The content of standard pollutants in spiked samples and pretreated spiked samples concentrated by different solid phase extraction columns was determined, and the recovery rates of different solid phase extraction columns for various standard pollutants were calculated based on the detection results. Equal amounts of pretreated water samples concentrated by different solid-phase extraction columns were dried, the dried solids were redissolved and diluted to a fixed volume, the TOC values ​​of each group were measured, and the recovery rate of TOC by different solid-phase extraction columns was calculated. Optimal selection method: Based on the biotoxicity test results, the ranking rules of "Pareto optimality principle" are applied to rank the columns, and then the optimal solid phase extraction column is determined by combining the recovery rates of different standard pollutants and the TOC recovery rate.

[0008] In optional embodiments, different solid-phase extraction columns include HLB columns, HLB columns and Coconut columns in series, C18 columns, C18 membranes, and StrataX columns.

[0009] In an optional implementation, the sample pretreatment step involves adjusting the pH of the water sample to 2.5-3.5 using hydrochloric acid solution.

[0010] In an optional implementation, the sample pretreatment step involves passing the acidified water sample through a 0.45 μm glass fiber membrane.

[0011] In an optional implementation, during the sample pretreatment step, the water sample passes through the solid-phase extraction column at a rate of 1-2 drops / s; the column is de-pressed for 1.5-2 hours.

[0012] In an optional embodiment, in the sample pretreatment step, before solid-phase extraction, the solid-phase extraction column is activated sequentially with methanol, ethyl acetate, and hydrochloric acid solution with a pH of 2.5 to 3.5.

[0013] In an optional embodiment, in the sample pretreatment step, after solid-phase extraction is completed, the sample is rinsed with hydrochloric acid solution with a pH of 2.5 to 3.5, and then the solid-phase extraction column is dried by dry aeration for 1.5 to 2.5 hours. After that, the solid-phase extraction column is eluted with methanol and ethyl acetate, respectively. The eluent is dried with N2 and then diluted to volume with methanol.

[0014] In optional embodiments, nonspecific toxicities include cytotoxicity and acute toxicity, reactive toxicities include genotoxicity, and specific toxicities include neurotoxicity and estrogenic activity. Cytotoxicity was detected using the CCK-8 assay kit, with DLD-1 cells as the test organism. Acute toxicity was tested according to the national standard GB / T 15441-1995; Genotoxicity was determined using the SOS / umu method, with Salmonella Typhimurium as the test organism. Neurotoxicity was detected using the acetylcholinesterase inhibition method; Estrogen activity was detected using a self-luminescent recombinant yeast method, with yeast as the test organism.

[0015] In an optional implementation, the water samples are municipal influent samples, biochemical effluent samples, final effluent samples, and surface water samples.

[0016] The present invention has the following beneficial effects: The method provided by this invention employs various solid-phase extraction methods to pretreat water samples, then uses biological methods to detect the toxicity of several different types of biomolecules, and uses a TOC analyzer to detect and compare the recovery rates of TOC before and after solid-phase extraction. By applying the Pareto optimality principle, based on different water sample types and biotoxicity, and guided by the magnitude of toxicity, different pretreatment methods are ranked, and then combined with the recovery rates of standard pollutants and TOC recovery rates, the optimal pretreatment method can be determined. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A comparative graph showing the biotoxicity results of urban sewage (a) and surface water (b) samples extracted by different SPE (solid phase extraction) methods; Figure 2 A statistical chart comparing the chemical recoveries of 23 model compounds and TOC in urban wastewater and surface water samples extracted by different SPE methods; Figure 3 Figure 1 shows the comparison results of the most effective solid-phase extraction methods for extracting total organic carbon and 23 model compounds from urban wastewater and surface water using the Pareto optimization strategy. Figure 4 The image shows the detection results of 24 substances in the experimental example. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0021] This invention provides a method for obtaining a preferred pretreatment method for water samples, comprising: S1. Provide samples There are two types of samples: one is a water sample taken directly, and the other is a sample with added standard pollutants (i.e., spiked sample).

[0022] Spiked sample: Standard pollutants are added to the water sample to obtain a spiked sample. The standard pollutants are a variety of different substances with an octanol-water partition coefficient between 1.49 and 6.76.

[0023] It should be noted that the standard pollutants mentioned here are merely names of substances added to the water sample for ease of description in this case, and do not represent that they belong to conventional or standard pollutants in water bodies.

[0024] Optionally, in some embodiments of this application, the standard contaminants include 23 typical contaminants with logKow values ​​between 1.4 and 6.8, for which solid-phase extraction chemical recovery rates are evaluated. These include 16 polycyclic aromatic hydrocarbons, 5 pesticides, and 2 phenolic substances, covering polar, neutral, and non-polar chemicals. See Table 1 for details. Table 1

[0025] Optionally, the water samples may be municipal influent (0.5-1 L), biochemical effluent (1-2 L), final effluent (1-2 L), and surface water (1-2 L).

[0026] S2, Sample Pretreatment Multiple groups of spiked samples and multiple groups of water samples were acidified and filtered through membranes, and then concentrated and enriched by different activated solid-phase extraction columns. Subsequently, multiple groups of pretreated spiked samples and multiple groups of pretreated water samples were obtained by rinsing, drying, elution, nitrogen blowing and volume adjustment.

[0027] Alternatively, different solid-phase extraction columns include HLB columns, HLB columns and Coconut columns in series, C18 columns, C18 membranes, and StrataX columns.

[0028] The solid-phase extraction columns mentioned above are some of the extraction columns with better extraction effects currently known. In other embodiments of the present invention, other types of solid-phase extraction columns can also be used for extraction in order to screen out the best extraction method.

[0029] Optionally, the pH of the water sample can be adjusted to 2.5-3.5 using hydrochloric acid solution, preferably 3.

[0030] Alternatively, the membrane can be passed through a 0.45 μm glass fiber membrane after the acidified water sample has been filtered.

[0031] Optionally, the water sample passes through the solid-phase extraction column at a rate of 1-2 drops / s; the column is de-pressed for 1.5-2 hours.

[0032] Optionally, before solid-phase extraction, the solid-phase extraction column is activated sequentially with methanol, ethyl acetate, and hydrochloric acid solution with a pH of 2.5 to 3.5.

[0033] Optionally, after solid-phase extraction is complete, the solid-phase extraction column is rinsed with hydrochloric acid solution with pH 2.5-3.5, and then the solid-phase extraction column is dried by dry aeration for 1.5-2.5 hours. After that, the solid-phase extraction column is eluted with methanol and ethyl acetate, respectively. The eluent is dried with N2 and then diluted to volume with methanol.

[0034] S3, Biotoxicity testing The non-specific toxicity, specific toxicity, and reactive toxicity of each group of pretreated water samples were tested.

[0035] Specifically, non-specific toxicity includes cytotoxicity and acute toxicity, reactive toxicity includes genotoxicity, and specific toxicity includes neurotoxicity and estrogenic activity. Cytotoxicity was detected using the CCK-8 assay kit, with DLD-1 cells as the test organism. Acute toxicity was detected according to the national standard GB / T 15441-1995. Genotoxicity was detected using the SOS / umu method, with Salmonella Typhimurium as the test organism. Neurotoxicity was detected using the acetylcholinesterase inhibition method. Estrogenic activity was detected using the autoluminescent recombinant yeast method, with yeast as the test organism.

[0036] S4, Chemical Analysis (1) Determine the content of standard pollutants in spiked samples and pretreated spiked samples concentrated by different solid phase extraction columns, and calculate the recovery rate of different solid phase extraction columns for a variety of different standard pollutants based on the detection results.

[0037] Specifically, the calculation method is as follows: if the concentration of a certain pollutant in the spiked sample before solid-phase extraction is a, and the concentration of the same pollutant after solid-phase extraction and concentration is b, the recovery rate is b / a*100%. (2) Take equal amounts of each group of pretreated water samples concentrated by different solid phase extraction columns, dry them, redissolve the dried solids and make up the volume, measure the TOC value of each group, and calculate the recovery rate of TOC for different solid phase extraction columns.

[0038] Specifically, the method for calculating the recovery rate of TOC is as follows: TOC determination before SPE (solid phase extraction): The filtered water sample is directly placed on a TOC meter for measurement; TOC determination method after SPE: 100 µL of water sample extract (concentrated solution after SPE) was evaporated to dryness in air at room temperature to obtain dried organic matter. This dried organic matter was then redissolved in 100 mL of sodium bicarbonate (NaHCO3) buffer solution with the same pH as the corresponding water sample to ensure complete dissolution. The solution was then measured using a TOC analyzer. (TOC recovery rate = TOC concentration after SPE / TOC concentration before SPE).

[0039] Note: TOC recovery rate was measured using unstirred water samples.

[0040] S5, Optimal Selection Method Based on the results of biotoxicity testing, the ranking rules of the "Pareto optimality principle" were applied to determine the optimal solid phase extraction column, and then the recovery rates of different standard pollutants and TOC recovery rates were combined to determine the optimal solid phase extraction column.

[0041] Different SPE methods were ranked using the Pareto optimality principle, taking into account both biotoxicity and chemical recovery. Methods with relatively poor performance (i.e., inferior to other methods in one or more metrics and without a clear advantage) were progressively eliminated. Ultimately, the remaining methods were Pareto optimal methods that were not comprehensively superior to other methods in any of the remaining metrics.

[0042] Because the biotoxicity of water samples is relatively low, direct biotoxicity testing is not possible, necessitating enrichment and concentration of the wastewater samples. However, the key toxic substances for most of these samples are unclear. Therefore, selecting the optimal pretreatment method is crucial for maximizing substance recovery and restoring toxic effects. The method provided in this invention employs various solid-phase extraction (SPE) methods to pretreat water samples, then uses biological methods to detect the magnitude of several different types of biotoxicity. A TOC analyzer is used to compare the recovery rates before and after SPE. Applying the Pareto optimality principle, based on different water sample types and biotoxicity levels, and guided by toxicity magnitude, different pretreatment methods are ranked. This ranking is then combined with the recovery rates of standard pollutants and TOC to determine the optimal pretreatment method.

[0043] Example Provide spiked samples: Twenty-three typical pollutants with logKow values ​​between 1.4 and 6.8 (the aforementioned standard pollutants) were selected for solid-phase extraction chemical recovery evaluation, including 16 polycyclic aromatic hydrocarbons, 5 pesticides, and 2 phenolic substances, covering polar, neutral, and non-polar chemical substances (see Table 1).

[0044] 1L of municipal wastewater influent, 2L of biological effluent and 2L of final effluent, and 2L of surface water containing these 23 typical pollutants were prepared respectively. The water samples with a concentration of 5 mg / L of each pollutant were used as spiked water samples, and the water samples without any pollutants were used as unspecified water samples. Sample pretreatment: The pH of both spiked and unspecified water samples was adjusted to 3.0 using hydrochloric acid solution and then filtered through a 0.45 μm glass fiber membrane. Five commonly used solid-phase extraction (SPE) methods were selected for SPE: HLB, HLB and Coconut in series, C18 column, C18 membrane, and StrataX column. The columns were activated with 10 mL of ethyl acetate, 10 mL of methanol, and 10 mL of hydrochloric acid solution at pH 3, respectively. Subsequently, solid-phase extraction was performed on both spiked and unspecified water samples, with the flow rate controlled at 1-2 drops / s. After extraction, the samples were rinsed with hydrochloric acid solution at pH=3, and then the solid-phase extraction column was dried by dry aeration for 2 h. The solid-phase extraction column was then eluted with 10 mL of methanol and 10 mL of ethyl acetate, respectively. The eluent was dried with N2 and then diluted to 1 mL with methanol to obtain the pretreated spiked water sample and the pretreated water sample. Pretreated water samples are used for biotoxicity testing, while pretreated spiked water samples are used for chemical recovery rate assessment.

[0045] Biotoxicity testing: Non-specific toxicity (cytotoxicity, acute toxicity), reactive toxicity (genotoxicity), and specific toxicity (neurotoxicity, estrogenic activity) of pretreated water samples were detected. Cytotoxicity was detected using the CCK-8 assay kit, with DLD-1 cells as the test organism. Acute toxicity was determined according to the national standard GB / T 15441-1995 (Water Quality—Determination of Acute Toxicity—Luminescent Bacteria Method), with Photobacterium phosphoreum T3 as the test organism. Genotoxicity was detected using the SOS / umu method, with Salmonella typhimurium as the test organism. Neurotoxicity was detected using the acetylcholinesterase inhibition method. Estrogenic activity was detected using the self-luminescent recombinant yeast method, with yeast as the test organism.

[0046] Test results for different water samples, such as Figure 1 As shown in Table 2, the results of the most effective solid-phase extraction method for extracting five biotoxicities from urban wastewater and surface water using the Pareto optimization strategy analysis are presented.

[0047] Table 2

[0048] Chemical recovery rate assessment: (1) The concentrations of 23 pattern compounds in the extract were determined using an Agilent 1260 Infinity G1329B autosampler equipped with an Eclipse Plus C18 column (Agilent Technologies, 4.6 mm × 250 mm, 5 μm particle size) and a UV detector (Agilent Technologies, USA) at wavelengths of 254 nm, 220 nm, and 295 nm (the maximum values ​​obtained at the three wavelengths were taken). The mobile phase was methanol (A) and water (B), with gradient elution: 80% (V / V) A for 0–20 min, 80–95% A for 20–32.5 min, and 85% A for 32.5–50 min.

[0049] The recovery rates of 23 substances were obtained under different water samples and different extraction column treatments.

[0050] (2) 100 μL of pretreated water sample was evaporated to dryness at room temperature to obtain dried organic matter, which was then redissolved in NaHCO3 buffer solution with the same pH as the corresponding water sample to ensure complete dissolution of the organic matter. The TOC value of the water sample was determined using a TOC-L analyzer (Shimadzu Corp., Japan), and the TOC yield under different extraction column treatments was calculated.

[0051] The recovery rates of the 23 substances and the yield of TOC are as follows: Figure 2 As shown.

[0052] Optimal selection method: The detected results were ranked according to the Pareto optimality principle, and selection was based on different water sample types, biotoxicity, and recovery rates, such as... Figure 3 As shown, the tandem arrangement of HLB and Coconut columns maximizes the recovery of biotoxicity.

[0053] Experimental Example Representative estrogen disruptors of different properties were selected, and 1 L of water samples (all concentrations 100 ng / L) were accurately prepared in urban wastewater effluent containing 6 estrogenic substances (estradiol, 17β-estradiol, estriol, ethinylestradiol, 17α-estradiol, diethylstilbestrol), 5 phenolic substances (nonylphenol, octylphenol, bisphenol A, bisphenol F, p-tert-octylphenol), 4 parabens (ethylparaben, methylparaben, propylparaben, butylparaben), 4 progestins (norethindrone, norethindrone, progesterone, levonorgestrel), and 5 phytoestrogens (daidzein, genistein, chickpea sprout extract A, gentianin, equol). The pH of the water samples was adjusted to 3.0 with HCl and then... The sample was filtered through a μm glass fiber membrane. HLB and Coconut columns were selected as solid-phase extraction (SPE) columns. Before use, the extraction columns were activated with ethyl acetate, methanol, and hydrochloric acid solution at pH 3. Then, the spiked water sample was subjected to SPE extraction using the HLB and Coconut columns in series at an extraction rate of 1-2 drops / s. After extraction, the sample was washed with hydrochloric acid solution at pH 3, and then the extraction columns were vacuum dried. Elution was then performed sequentially with methanol and ethyl acetate. The eluent was dried under nitrogen and then diluted to 1 mL with methanol. Twenty-four substances in the spiked water sample were analyzed by LC-MS / MS. Figure 4 As shown, the results showed that the recovery rate of all 24 substances reached over 82%.

[0054] This demonstrates that the pretreatment method for the water sample obtained by the method provided by this invention is reliable.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for obtaining a preferred pretreatment method for water samples, characterized in that, include: Provide spiked samples: add standard pollutants to a water sample to obtain the spiked sample, wherein the standard pollutants are a variety of different substances with an octanol-water partition coefficient between 1.49 and 6.76; Sample pretreatment: The spiked samples and water samples were acidified and filtered through membranes, and then concentrated and enriched by different activated solid phase extraction columns. Subsequently, the samples were washed, dried, eluted, purged with nitrogen, and brought to a final volume to obtain the pretreated spiked samples and water samples. Biotoxicity testing: Non-specific toxicity, specific toxicity, and reactive toxicity were tested for each group of pretreated water samples. Chemical analysis: The content of the standard pollutants in the spiked sample and the pretreated spiked sample concentrated by different solid phase extraction columns was determined, and the recovery rate of different solid phase extraction columns for a variety of different standard pollutants was calculated based on the detection results. Equal amounts of the pretreated water samples from each group, concentrated by different solid-phase extraction columns, were dried. The dried solids were redissolved and brought to a final volume. The TOC values ​​of each group were measured, and the recovery rates of TOC for different solid-phase extraction columns were calculated. Optimal selection method: Based on the biotoxicity test results, the ranking rules of "Pareto optimality principle" are applied to sort the samples, and then the optimal solid phase extraction column is determined by combining the recovery rates of different standard pollutants and the TOC recovery rate.

2. The method according to claim 1, characterized in that, The different solid phase extraction columns include HLB columns, HLB columns and Coconut columns in series, C18 columns, C18 membranes and StrataX columns.

3. The method according to claim 1, characterized in that, In the sample pretreatment step, the acidification method is to adjust the pH of the water sample to 2.5~3.5 using hydrochloric acid solution.

4. The method according to claim 1, characterized in that, In the sample pretreatment step, membrane filtration involves passing the acidified water sample through a 0.45 μm glass fiber membrane.

5. The method according to claim 1, characterized in that, In the sample pretreatment step, the water sample passes through the solid phase extraction column at a rate of 1-2 drops / s; the column is dried for 1.5-2 hours.

6. The method according to claim 1, characterized in that, In the sample pretreatment step, before solid-phase extraction, the solid-phase extraction column is activated sequentially with methanol, ethyl acetate and hydrochloric acid solution with pH 2.5~3.

5.

7. The method according to claim 1, characterized in that, In the sample pretreatment step, after solid-phase extraction is completed, the solid-phase extraction column is rinsed with hydrochloric acid solution with pH 2.5~3.5, and then the solid-phase extraction column is dried by dry aeration for 1.5~2.5h. After that, the solid-phase extraction column is eluted with methanol and ethyl acetate respectively, the eluent is dried with N2 and then diluted to volume with methanol.

8. The method according to claim 1, characterized in that, The nonspecific toxicities include cytotoxicity and acute toxicity, the reactive toxicities include genotoxicity, and the specific toxicities include neurotoxicity and estrogenic activity; The cytotoxicity was detected using a CCK-8 assay kit, with DLD-1 cells as the test organism. The acute toxicity was tested in accordance with the national standard GB / T 15441-1995; The genotoxicity was detected using the SOS / umu method with Salmonella Typhimurium as the test organism. The neurotoxicity was detected using the acetylcholinesterase inhibition method; The estrogen activity was detected using a self-luminescent recombinant yeast method, with yeast as the test organism.

9. The method according to claim 1, characterized in that, The water samples included municipal influent, biochemical effluent, final effluent, and surface water.

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