CN118028418A - Method for detecting biotoxicity of soil in tanning site - Google Patents

Abstract

A method for detecting biotoxicity of the soil in the leather field by leaching the soil in the leather field with a special leaching agent is to leach the easily-migrated chemical substances in the soil in the leather field by using a NaCl and AEO mixed solution as the leaching agent to obtain a soil leaching solution. Adding NaCl to the leaching solution to make the concentration of the NaCl to be 2.5 percent by weight, and adjusting the pH to 7.0; or diluting the leaching solution with 2.5% wt NaCl solution to a proper concentration gradient, and adjusting pH to 7.0; samples were assayed for toxicity. Taking cultured luminous bacteria, adding a sample solution, uniformly mixing, standing, and measuring luminous intensity. Expression of sample toxicity: the relative luminous intensity and half relative luminous intensity concentration EC ₅₀ of the mixture after standing were measured with a 2.5% wt NaCl solution as a blank. According to the invention, the mixed solution of sodium chloride and AEO is used as the leaching agent, toxic substances in the soil sample of the tanning site are leached, and the ecotoxicity of the leaching solution is directly detected by using a toxicity detector, so that a convenient method for detecting the ecotoxicity of the soil of the tanning site is constructed, and the defects of the traditional method are avoided.

Description

A method for detecting soil biological toxicity in tanning sites

Technical Field

The invention relates to the field of soil biological toxicity detection methods for leather-making sites, and in particular to a soil biological toxicity detection method for leather-making sites based on a special extractant.

Background technique

The pollution characteristics of the soil in the tanning site are the combined pollution of chromium and organic matter, among which the total chromium content is high, up to 59400mg/kg, and it is mainly trivalent chromium, but it also contains high hexavalent chromium up to 827mg/kg in some areas. More than 90% of the tanning process uses trivalent chromium, and a large amount of chromium-containing waste liquid and chromium-containing waste will be generated after tanning and post-processing; at the same time, the leather making process uses a wide variety of large quantities of organic matter, such as oil, resin, tannin, protein, etc.; waste liquid dripping and solid waste storage leaching have become the main sources of site pollution. These pollutants not only have their own toxicity, but also the mixing and interaction of various pollutants will also produce potential toxicity; therefore, a comprehensive toxicity evaluation is essential. The existing detection technology for the soil in the tanning site is limited to the determination of the content of some harmful components in the soil, such as ammonia nitrogen, chromium (VI), and sulfide. For each harmful component, the extraction method is different. For example, ISO/TS 14256-1-2003 uses potassium chloride solution to extract nitrate, nitrite and ammonium from soil; HJ 833-2017 uses strong acid to convert sulfide into hydrogen sulfide and blow it out to determine its content; ISO 15192-2010 uses strong alkaline extraction solution to extract chromium (VI) from samples, without considering the impact of chromium (III) on the test results when reducing or oxidizing the waste matrix. These soil extraction methods do not fully consider the risk of further transformation of various components in the soil during the extraction process under changes in ambient temperature, pH and soil matrix, which in turn affects the test results of soil toxicity.

my country is a major leather producer. The adjustment of the industrial structure has promoted the formation of an industrial cluster with upstream, midstream and downstream products that complement each other in the leather industry, while some small and medium-sized leather enterprises have gradually closed down. The "Action Plan for Soil Pollution Prevention and Control" issued in 2016 clearly stated that the leather industry is one of the key industries for chromium pollution monitoring. Before the closed leather sites are reused, risk assessment is required. Therefore, the establishment of accurate, reliable and highly sensitive soil ecotoxicity detection technology for leather sites will improve the overall soil ecotoxicity detection capabilities in the leather industry environment in my country, and provide important theoretical basis and guidance for the reuse of green leather chemicals and land resources in my country, which has very important theoretical and practical significance.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art that the Cr(III) and Cr(VI) contents cannot be accurately tested, and the eco-toxicity screening operation is complex, time-consuming, and the detection cost is high, and to provide a method for detecting soil biological toxicity in leather-making sites that is easy to operate, economical, and practical based on luminescent bacteria.

The present invention provides a method for detecting soil ecotoxicity in a tanning site based on a special extractant, comprising the following steps:

(1) Pretreatment: air-dry, crush, screen, and homogenize the soil, then take samples and treat them at a constant temperature and humidity;

(2) Extraction: The pretreated soil is added to an extractant, which is a solution of a certain pH value containing 0.1-0.3% wt NaCl and 1% AEO, and horizontally oscillated at a constant temperature for a certain period of time, and the extract is filtered to obtain the extract;

(3) Toxicity test: Sodium chloride was added to the extract to make the concentration of sodium chloride 2.5% wt, and the pH was adjusted to 7.0±0.2. Then, fresh luminescent bacterial solution with the test bacterial density was added, mixed and allowed to stand, and the luminescence intensity was measured.

(4) Toxicity expression: Taking the luminescence intensity of 2.5% wt NaCl solution as the reference, calculate the relative luminescence intensity of the extract after standing.

Furthermore, the pretreatment method is specifically as follows: the soil is air-dried and then sieved into a 2 mm sieve and sampled, dried at 45°C for 48 hours, placed in standard air at 20±2°C and a relative humidity of 65±5% for 48 hours, and stored for later use.

Furthermore, the soil is soil from a tannery site.

Furthermore, in the step (2), the liquid-to-solid ratio (mL/g) of the extractant to the soil is 2:1-10:1, the temperature is constant at 25-30°C, the horizontal oscillation is 60-100 rpm for 18-48 h, and the filtration method is through a 0.45 um filter membrane.

Furthermore, the leaching agent is a mixed solution of 0.1-0.3% wt NaCl and 1% AEO, and the pH is adjusted to 4-9 with H ₂ SO ₄ and NaOH.

Furthermore, the luminescent bacteria in the luminescent bacteria liquid is Photobacterium luminescens T3.

Furthermore, the luminescent bacteria liquid is cultured by:

(1) Prepare luminescent bacterial culture medium: take 5.0 g/L tryptone, 5.0 g/L yeast powder, 5.0 g/L Na ₂ HPO ₄ 5.0 g/L, 5.0 g/L K ₂ HPO ₄ 5.0 g/L, 30 g/L NaCl, and 3.0 g/L glycerol, dissolve in deionized water, and adjust the pH to 7.0;

(2) Cultivation process: Take the bright photobacterium T3 and inoculate it into a conical flask filled with culture medium, place it in a shaker at 20℃ and 200rpm. After culturing for 18-20 hours, immediately take the strain with the best luminescence effect for a second inoculation, and then place it in a shaker at 20℃ and 200rpm. Measure the luminescence intensity of the fresh bacterial solution every 2 hours. Cultivate it for 18-20 hours to reach the logarithmic stable growth period for testing.

Furthermore, the method for determining the test bacterial density is as follows: take a fresh bacterial solution of luminescent bacteria that has been cultured for 18 to 20 hours and has reached the logarithmic stable phase, dilute it to different bacterial densities with 2.5% wt. NaCl, take 50 uL of each solution and add 950 uL1.23 mg/L _ZnSO4 solution containing 2.5% wt. NaCl in turn, let it stand for 15 min, and use a LumiFox6000 biotoxicity tester to measure the luminescence intensity. The measurement is repeated 3 times in parallel, using a 2.5% wt. NaCl solution as a blank, so that the bacterial solution with a relative luminescence intensity of 50±5% is the test bacterial density.

Furthermore, in step (4), the relative luminescence intensity R of the extraction solution is calculated according to R=I/I ₀ ×100% based on the average luminescence intensity I ₀ of the 2.5% wt. NaCl control sample and the average luminescence intensity I of the sample to be tested; the greater the relative luminescence intensity R, the lower the toxicity.

The present invention has the following beneficial effects:

Sodium chloride is used to adjust the ionic strength, sulfuric acid or sodium hydroxide to adjust the pH, and AEO to adjust the surface activity to extract weakly bound heavy metals and organic matter in the soil. This effectively avoids the reaction between the extractant and the extractable components in the soil, accurately evaluates the toxic effects of mobile organic matter and Cr in the soil, and is simple and effective to operate.

The luminescent bacteria detection method adopted by the present invention is based on the bacterial luminescent biosensor technology. Luminescent bacteria are marine bacteria. In a certain salt concentration and pH range, the bacteria in the logarithmic growth phase have strong luminescence ability and stable luminescence. When the environment changes or toxic substances are present, the bacterial luciferase is inactivated or the cell respiration is inhibited, and the luminescence is weakened. The degree of weakening is positively correlated with the toxicity of the toxic substances. The luminescent bacteria method is low in cost and has the advantages of rapid detection, high sensitivity, and strong adaptability. Therefore, this method can be fully used for soil ecotoxicity detection in leather making sites.

The present invention uses a low-toxic standard toxicity reference substance 1.23 mg/L ZnSO ₄ to determine the density of the tested fresh bacterial liquid, so that the detection result is stable and has good repeatability.

This method uses the luminescent bacteria method for detection. The reaction time between the sample and the luminescent bacteria is short, and the test results can be obtained quickly. It is suitable for rapid detection of large quantities of samples, greatly shortens the detection time of comprehensive toxicity, and improves detection efficiency and accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Flowchart of the invention.

Fig. 2 Dose-toxicity effect curve of soil leachate.

Fig. 3 Comparison of extraction effects of extraction agents.

FIG4 shows the dose-effect curves of Example 2 and the comparative example.

Detailed ways

The present invention is further described in detail below in conjunction with embodiments. The following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned invention content.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

Example 1

A method for detecting soil ecotoxicity in a tanning site based on a special extractant, referring to FIG1 , the method comprises the following steps:

(1) Soil sample pretreatment: The soil samples were obtained from a fur and leather company in China. The soil was air-dried and sieved through a 2 mm mesh and sampled. After drying at 45 °C for 48 hours, the soil was placed in standard air at 20 ± 2 °C and a relative humidity of 65 ± 5% for 48 hours for storage.

(2) Sample extraction: Weigh 5.0 g of sample and place it in a 100 mL conical flask, add 25 mL of a mixed solution of 0.1% wt. NaCl and 0.1% wt. AEO adjusted to pH 8.0 by 0.1 MH ₂ SO ₄ , place in a ZWY-2102C constant temperature oscillator, keep the temperature at 25°C, rotate at 28 rpm, oscillate horizontally for 8 hours, and filter with a 0.45 um filter membrane. The filtrate is diluted with 2.5% wt. NaCl solution to 10 concentration gradients, containing leaching solution (V/V) of 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%, respectively, and the pH of all diluted samples is adjusted to 7.0±0.2 with 0.1 M NaOH, and toxicity evaluation is immediately carried out;

(3) Toxicity test of the extract: The present invention uses fresh bacterial solution of Photobacterium luminescens T3 to determine the comprehensive toxicity of the crust leather. The preparation of the luminescent bacterial culture medium is as follows: 5.0 g/L tryptone, 5.0 g/L yeast powder, 5.0 g/L Na ₂ HPO ₄ 5.0 g/L, 5.0 g/L K ₂ HPO ₄ , 30 g/L NaCl, 3.0 g/L glycerol, dissolved in deionized water, and adjusted to pH 7.0. The process of the culture operation is as follows: prepare 1L of culture medium, divide it into 250 mL conical bottles at 100 mL per bottle, wrap it with cotton, gauze, and newspaper, and sterilize it in a high-temperature sterilizer at 121 ° C for 20 min for use. Take the lyophilized powder of Photobacterium luminescens T3, add 2.5% sodium chloride solution and resuscitate it at 20-25 ° C for 15 min. Take 1 mL of revived Photobacterium luminescens and inoculate it into a conical flask, place it in a shaker at 20°C and 200 rpm, and culture it for about 18 to 20 hours until the luminescence intensity reaches the maximum value, then it can be harvested. Take 1.5 mL of the strain with the best luminescence effect and inoculate it for the second time, place it in a shaker at 20°C and 200 rpm, and measure the luminescence intensity of the bacteria every 2 hours. Specific measurement method: 50 uL of fresh bacterial solution, add 950 uL 2.5 wt.% NaCl, mix well and let it stand for 15 min, and measure the luminescence intensity with LumiFox6000 biological toxicity tester. Under the culture conditions of this embodiment, the luminescent bacteria do not emit light in the first 11 hours of the culture stage. After entering the logarithmic growth period, the luminescence intensity increases rapidly, and the luminescence intensity tends to the maximum and the luminescence is stable when the culture reaches 18 to 20 hours. Therefore, this experiment uses 18 to 20 hours of fresh bacterial solution for toxicity testing;

(4) Take 5 portions of 1.0 mL of luminescent bacterial solution cultured for 19 hours, add 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL and 4.0 mL of 2.5% wt. NaCl solution in turn and mix well to obtain luminescent bacterial dilutions with different bacterial densities. Take 50 uL of luminescent bacterial dilutions with different bacterial densities, add 950 uL of 1.23 mg/L ZnSO ₄ solution (containing 2.5% wt. NaCl) in turn, let it stand for 15 minutes, and use LumiFox6000 biotoxicity tester to measure the luminescence intensity. The measurement was repeated 3 times, and the 2.5% wt. NaCl solution was used as the blank to calculate the relative luminescence intensity. Among them, the bacterial solution with a relative luminescence intensity of 50±5% is the test bacterial density;

(5) Take 50 uL of the luminescent bacterial solution calibrated with ZnSO ₄ and add 950 uL of the series of concentration gradient samples of the leaching solution in step (2) in sequence, mix well and let stand for 15 min, and measure the luminescence intensity using the LumiFox6000 biotoxicity tester. The results are measured three times in parallel, and the relative deviation does not exceed 10%. The average value of the three measurements is taken;

(6) Various indicators of sample extract: extract pH: 8.49, Eh: 38, TOC content: 4562 mg/L, Cr(III) content: 0.285 mg/L, no Cr(VI);

(7) Expression of sample toxicity: Draw a dose-effect curve with the concentration of the extract as the horizontal axis and the relative luminescence intensity of the luminescent bacteria as the vertical axis (see Figure 2). If the luminescence intensity of the original solution is still less than 50%, the toxicity is very low.

Example 2

A method for detecting soil ecotoxicity in a tanning site based on a special extractant, referring to FIG1 , the method comprises the following steps:

(1) Soil sample pretreatment: The soil was obtained from a Chinese leather company. The soil was air-dried and sieved through a 2 mm mesh and sampled. After drying at 45 °C for 48 hours, the soil was placed in standard air at 20 ± 2 °C and a relative humidity of 65 ± 5% for 48 hours for storage.

(2) Sample extraction: Weigh 5.0 g of sample and place it in a 100 mL conical flask, add 25 mL of a mixed solution of 0.1% wt. NaCl and 0.1% wt. AEO adjusted to pH 8.0 by 0.1 MH ₂ SO ₄ , place in a ZWY-2102C constant temperature oscillator, keep the temperature at 25°C, rotate at 28 rpm, oscillate horizontally for 8 hours, and filter with a 0.45 um filter membrane. The filtrate is diluted with 2.5% wt. NaCl solution to 10 concentration gradients, containing leaching solution (V/V) of 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%, respectively, and the pH of all diluted samples is adjusted to 7.0±0.2 with 0.1 M NaOH, and toxicity evaluation is immediately carried out;

(3) Toxicity detection of the extract: The present invention uses fresh bacterial solution of Photobacterium luminescens T3 to determine soil ecotoxicity. The preparation of the luminescent bacterial solution in this embodiment is the same as that in Example 1. Take 50 uL of the bacterial solution with a determined bacterial density and add 950 uL of the steps in sequence; (4) The prepared series of diluted extract samples are mixed and allowed to stand for 15 minutes, and the luminescence intensity is measured using a LumiFox6000 biological toxicity tester. The measurements are performed three times in parallel, and the relative deviation does not exceed 10%. The result is the average of the three measurements;

(5) Expression of sample toxicity: Taking 2.5%wt.NaCl solution as reference, the concentration of the extract dilution as the horizontal axis, and the relative luminescence intensity as the vertical axis, a dose-effect curve was plotted, as shown in Figure 3. The EC ₅₀ value of the soil extract was 69.21%±0.56%, which was converted to EC _50(ppm) of soil = EC _50(%) * (5/25)*10 ⁶ = 138420± 1120 ppm, which was extremely low in toxicity.

Comparative Example

Weigh 5.0 g of the sample from Example 2 and place it in a 100 mL conical flask, add 25 mL of 0.1% wt. NaCl solution adjusted to pH 8.0 by 0.1 MH ₂ SO ₄ , place it in a ZWY-2102C constant temperature oscillator, keep the temperature at 25°C, rotate at 28 rpm, oscillate horizontally for 8 hours, and then filter with a 0.45um filter membrane. The filtrate was diluted with 2.5% wt. NaCl solution to 10 concentration gradients, containing leaching solution (V/V) of 6%, 12%, 18%, 24%, 30%, 36%, 42%, 48%, 54% and 60%, respectively, and the pH of all diluted samples was adjusted to 7.0±0.2 with 0.1M NaOH, and toxicity evaluation was performed immediately. The dose-toxicity effect curve of the leaching solution is shown in Figure 3 for comparison. The remaining steps are the same as in Example 2;

The corresponding extraction effect tests were performed on Example 2 and the comparative example, and the test results are shown in Table 1. It can be seen that the total amount of extracted substances and biological toxicity of the leaching agent provided by the present invention (Example 2) are significantly different from those of deionized water (Comparative Example), and the deviation is greater than 10%.

Claims (10)

1. The method for detecting the biotoxicity of the soil of the tanning site is characterized by comprising the following steps of:

(1) Pretreatment: air-drying, crushing, screening and homogenizing soil, sampling and performing constant temperature and humidity treatment;

(2) Leaching: adding a leaching agent into the pretreated soil, horizontally oscillating for a certain time at constant temperature, and filtering to obtain leaching solution;

(3) Toxicity detection: adding sodium chloride into the leaching solution to enable the concentration of the sodium chloride to be 2.5 wt%, adjusting the pH value to 7.0+/-0.2, then adding the cultured fresh luminous bacterial liquid with the bacterial density for testing, mixing uniformly, standing, and measuring the luminous intensity;

(4) Toxicity expression: the relative luminescence intensity of the leaching solution after standing was calculated with the luminescence intensity of a 2.5% wt NaCl solution as a control.

2. The method for detecting the comprehensive toxicity of the soil of the tanning site is characterized by comprising the following steps of:

(1) Pretreatment: air-drying, crushing, screening and homogenizing soil, sampling and performing constant temperature and humidity treatment;

(2) Leaching: adding a leaching agent into the pretreated soil, regulating the pH value of the leaching agent by H ₂SO₄ and NaOH, adding NaCl with the weight of 0.1-0.3% and AEO with the weight of 1% to extract organic matters in the soil, oscillating for a certain time at constant temperature and level, and filtering to obtain leaching liquor;

(3) Diluting the leaching solution by using 2.5 wt% NaCl to form a concentration gradient, so that the relative luminous intensity range is 0% -100%, and regulating the pH value to 7.0+/-0.2; then adding fresh luminous bacterial liquid with the cultured bacterial density for testing, uniformly mixing, standing and measuring luminous intensity;

(4) Toxicity expression: the relative luminescence intensity of the leaching solution after standing was calculated with the luminescence intensity of a 2.5% wt NaCl solution as a control.

3. The detection method according to claim 1 or 2, wherein the pretreatment method specifically comprises: the soil is air-dried, sieved to a 2mm screen, sampled, dried at 45 ℃ for 48 hours, placed in standard air, and kept at 20+/-2 ℃ and constant temperature and humidity with relative humidity of 65+/-5% for 48 hours for standby.

4. The detection method according to claim 1 or 2, wherein in the step (2), the leaching agent is adjusted to have a pH value of 5.5 to 10.5 by H ₂SO₄ and NaOH, and a liquid-solid ratio (mL/g) of soil is 2: 1-10: 1, carrying out oscillation leaching for 18-48 hours at the constant temperature of 25-30 ℃ and the horizontal speed of 60-100 rpm, wherein the filtering method is to pass through a filter membrane of 0.45 um.

5. The method according to claim 1 or 2, wherein the leaching agent is a NaCl solution of 0.1 to 0.3% by weight and contains 1% AEO.

6. The method according to claim 1 or 2, wherein the luminescent bacteria in the luminescent bacteria liquid is luminescent bacteria T3.

7. The method according to claim 1, 2 or 7, wherein the method for culturing the luminescent bacterial liquid comprises:

(1) Preparation of a luminescent bacterial culture medium: taking 5.0g/L of tryptone, 5.0g/L of yeast powder, 5.0g/L of Na ₂HPO₄ 5.0g/L,K₂HPO₄, 30g/L of NaCl, 3.0 g/L of glycerol and deionized water for dissolution, and adjusting the pH to 7.0;

(2) The culture process comprises the following steps: inoculating the luminous bacillus T3 into a conical flask filled with a culture medium, placing the conical flask in a shaking table with oscillation at 20 ℃ and 200 rpm, culturing for 18-20 hours, immediately inoculating the strain with the best luminous effect, placing the strain in a shaking table with 200 rpm at 20 ℃ again, measuring the luminous intensity of fresh bacterial liquid every 2h, and culturing for 18-20 hours to reach a growth log stabilization period for testing.

8. The method according to claim 1,2 or 8, wherein the method for determining the bacterial density for testing is: taking fresh bacterial liquid of luminous bacteria which grows for 18-20 h to a logarithmic stationary phase, diluting the bacterial liquid to different bacterial densities by using 2.5 wt% NaCl, sequentially adding 950 uL of 1.23mg/L ZnSO ₄ solution containing 2.5 wt% NaCl, standing the bacterial liquid for 15min, measuring the luminous intensity by using a LumiFox6000 biotoxicity tester, and carrying out parallel measurement for 3 times, wherein the bacterial liquid with the relative luminous intensity of 50+/-5% is made blank by using the 2.5 wt% NaCl solution, and is the bacterial density for testing.

9. The method according to claim 1, wherein the step (4) calculates the relative luminescence intensity R of the leaching stock solution as r=i/I ₀ x 100% with the luminescence intensity average I ₀ of the 2.5% wt. NaCl control sample and the luminescence intensity average I of the sample to be detected; the greater the relative luminous intensity R, the less toxic.

10. The method according to claim 2, wherein the step (4) is to make a dose-response curve with the serial concentration of the sample to be tested diluted as an abscissa and the relative luminous intensity R as an ordinate, and to read the corresponding concentration from the curve when the relative luminous intensity is 50%, i.e. the greater the half relative luminous intensity concentration EC ₅₀;EC₅₀, the smaller the toxicity.