A multi-level bioindicator method for effective phytotoxicity of heavy / heavy metals

Abstract

This invention belongs to the field of ecological environment monitoring technology and relates to a multi-level bioindicator method for the effective phytotoxicity of heavy metal-like substances / heavy metals. The method includes the following steps: S1. Obtaining plant samples affected by the soil or water body to be tested; S2. Measuring the activity of superoxide dismutase (SOD), gibberellin (GA), and chlorophyll content in the plant samples; S3. Based on the measured results of SOD activity, plant hormone content, and chlorophyll content, and their changes over time, classifying and assessing the effective phytotoxicity effects of heavy metal-like substances or heavy metals. This invention is the first to propose a unified three-level bioindicator method (SOD activity, GA hormone, and chlorophyll) that can effectively reflect soil pollution levels for typical heavy metal-like substances / heavy metals present in the actual environment, enabling a simpler assessment of the degree of heavy metal pollution in actual soil.

Description

Technical Field

[0001] This invention belongs to the field of ecological environment monitoring technology and relates to a multi-level biological indicator method for effective plant toxicity of heavy / heavy metals. Background Technology

[0002] With the increasing severity of environmental pollution and the continuous improvement of environmental protection requirements, assessing the suitability of soil and its surrounding area for crop production has become particularly important. This assessment typically involves detecting several known major pollutants in the soil, such as heavy metals and pesticides. However, with the acceleration of industrialization, the types of toxic pollutants in the environment are increasing, making traditional detection methods complex and cumbersome. Especially when facing new pollutants, the time and cost required to update and improve detection methods increase significantly, undoubtedly reducing the efficiency of traditional methods. Therefore, discovering a comprehensive bioindicator system that can reflect the soil pollution status is of great significance for guiding human activities and agricultural production, which is also the core objective of this study.

[0003] Current research primarily focuses on the toxicity response mechanisms under single pollutant stress, without establishing a unified system. A 2006 study proposed using the biology of nanoparticle-induced oxidative stress as an important predictive model for nanoparticle toxicity. This model divides oxidative stress into three layers: in the first layer (lower doses), cells primarily restore redox homeostasis by inducing phase II antioxidant enzymes through Nrf-2 transcriptional activation of antioxidant response elements; in the second layer (moderate doses), activation of the MAPK and NF-κB cascade induces pro-inflammatory responses; and in the third layer (higher doses), perturbation of mitochondrial PT pores and disruption of electron transfer lead to apoptosis or necrosis. This provides an opportunity to reflect soil pollution by utilizing plant response mechanisms to pollutants. However, current research trends both domestically and internationally indicate a significant lack of unified model data for summarizing plant responses to pollutant stress.

[0004] Soil heavy metal pollution is mostly complex pollution, but the phytotoxic effects of different heavy metals vary significantly. Although various evaluation indicators for the toxicity effects of single heavy metals exist, these indicators are diverse and yield inconsistent results, lacking a unified evaluation indicator for the toxicity effects of different heavy metals. Secondly, the bioavailability or effective toxicity of different heavy metals differs from their pollution concentration. This means that traditional physicochemical monitoring cannot be used to determine the risk of heavy metals; instead, it is necessary to analyze the biomarkers that cause phytotoxic effects. Therefore, establishing a unified bio-evaluation indicator for the phytotoxic effects of different heavy metals is a critical technical challenge that urgently needs to be addressed. The establishment of a unified bio-evaluation method for the phytotoxicity of different heavy metals will provide important technical support for pollution control in the fields of ecological environment, food contamination, and health.

[0005] Constructing a practically applicable three-tiered biomarker system requires substantial experimental data. While numerous related studies have been reported, the number of publications demonstrating different response levels corresponding to different pollution levels remains limited. Furthermore, many laboratories select pollutant types and concentrations based on effective concentrations when exposing plants, leading to lower practical environmental significance and inconsistencies among various plant effect data.

[0006] Therefore, using the actual environmental concentration and type of pollutants to stress plants and conducting a complete test of the plant response system will provide more practically valuable plant effect data. In the future, the current soil pollution situation can be judged by measuring the degree of plant effect response, which will significantly reduce the time and economic costs of measuring soil pollution. Summary of the Invention

[0007] To address the problems and shortcomings of existing technologies, this invention uses lettuce as a model plant to systematically study the effects and response mechanisms of typical heavy metals (As / Pd, Cd, Cr(III)) on its growth. Through detailed analysis of the effects of different concentrations and exposure times of various pollutants on lettuce, key physiological indicators that can indicate the degree of pollution stress under these conditions were successfully identified, including changes in the antioxidant enzyme system, hormone level adjustments, and chlorophyll content. The current soil pollution status can be assessed by systematically measuring this set of multi-level biomarkers.

[0008] The technical solution provided by this invention is as follows: A multi-level bioindicator method for effective phytotoxicity of heavy / heavy metals, comprising the following steps: S1. Obtain plant samples affected by the soil or water body to be tested; S2. Determine the SOD activity, the content of at least one plant hormone, and the chlorophyll content in the plant sample; S3. Based on the measurement results of the SOD activity, the plant hormone content, and the chlorophyll content, and their change patterns over time, the phytotoxicity effects of heavy metal-like substances or heavy metals are classified and uniformly assessed.

[0009] Further, in step S2, the SOD activity, the plant hormone content, and the chlorophyll content have a time-response order, wherein: The response time window for SOD activity is 18-36 hours after exposure; The response time window for plant hormone levels is 108–132 hours after exposure; The response time window for chlorophyll content was 228–252 hours after exposure; Furthermore, the plant hormone response occurs after SOD activity has already produced a response, while the chlorophyll response occurs after both SOD activity and plant hormones have already produced a response.

[0010] Furthermore, in step S2, the plant hormone is gibberellin (GA).

[0011] Further, in step S2, the chlorophyll is chlorophyll a and / or chlorophyll b.

[0012] Further, in step S3, the hierarchical unified assessment includes: A significant increase in SOD activity indicates the presence of pollutant stress. When the plant hormone GA content was further detected to show a pattern of first increasing and then decreasing, it indicated an increase in the degree of pollutant stress. When a significant decrease in chlorophyll content is detected, it indicates that the level of pollutant stress has reached a high level.

[0013] Furthermore, the heavy metal-like substance includes arsenic, and the heavy metal includes at least one of cadmium, chromium, and lead.

[0014] Furthermore, the plant in question is lettuce.

[0015] Compared with existing technologies, this invention proposes for the first time a three-level bioindicator (SOD enzyme activity, GA hormone, and chlorophyll) that can effectively reflect soil pollution levels for typical heavy metals (As, Cd, Cr(III), Pb) present in the real environment. When lettuce is subjected to pollutant stress, the most significant characteristic is the initial increase in SOD. As the pollution intensity increases, GA hormone levels first rise and then fall, followed by a decrease in chlorophyll. This allows for a simpler assessment of the actual soil pollution level. The bioindicators provided by this invention are uniform, meaning that the degree of pollution from a single typical heavy metal can be evaluated using the three-level indicators of this invention. Attached Figure Description

[0016] Figure 1 The values ​​represent the SOD levels of lettuce under stress from three typical heavy metals and arsenic for 1, 5, and 10 days. All plants exposed to 0.8 mM As died before 10 days, making data unavailable. The values ​​for AD were As, Cd, Pb, and Cr, respectively. Figure 2 The changes in GA and IAA hormone levels in lettuce under 5 days of single stress from three typical heavy metals and arsenic compared to the control group; A-D represent As, Cd, Pb, and Cr, respectively. Figure 3 The data represents the changes in GA and IAA hormone levels in lettuce under single stress of three typical heavy metals and arsenic for 10 days compared to the control group. All plants exposed to 0.8 mM As died before 10 days, making it impossible to obtain data. A-D represent As, Cd, Pb, and Cr, respectively. Figure 4 The values ​​represent the chlorophyll a, chlorophyll b, and carotene levels in lettuce under 1 day of stress from three typical heavy metals and arsenic; A-D represent As, Cd, Pb, and Cr, respectively. Figure 5 The values ​​represent the chlorophyll a, chlorophyll b, and carotene levels in lettuce under 5 days of stress from three typical heavy metals and arsenic; A-D represent As, Cd, Pb, and Cr, respectively. Figure 6 The values ​​represent the chlorophyll a, chlorophyll b, and carotene levels in lettuce under 10-day stress from three typical heavy metals and arsenic. All plants exposed to 0.8 mM As died before 10 days, making data unavailable. A-D represent As, Cd, Pb, and CrS, respectively. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0018] Example 1: Primary biomarker for lettuce under stress from a single pollutant Superoxide dismutase (SOD), a key enzyme that dismutates superoxide anion free radicals into hydrogen peroxide and oxygen, plays an important role in protecting cells from oxidative damage. Its activity level can reflect the level of oxidative stress on plants.

[0019] 1. Preparation of Hogrange Nutrient Solution (25%) Weigh out 1.26 g of Hogland nutrient solution powder and 0.945 g of calcium salt, and dissolve them in 1 L of ultrapure water by sonication. If not completely dissolved, add a stirrer to promote dissolution. Finally, dilute to a 4 L container to obtain a 25% Hogland nutrient solution.

[0020] 2. Plant incubation and cultivation The plant used in this study was lettuce, and the seeds were Hong Kong Glass Crisp Lettuce from Hebei Qingfeng Agriculture. First, a packet of lettuce seeds was disinfected in 30 ml of 10% hydrogen peroxide solution for 10 minutes. After disinfection, the seeds were washed three times with ultrapure water to remove as much hydrogen peroxide solution as possible from the seed surface. Then, 100 ml of ultrapure water was added, and the seeds were placed in a 30°C water bath for eight hours to germinate. Filter paper was moistened with ultrapure water and placed on a germination tray, with the seeds evenly distributed on top. Aluminum foil was covered, and the seeds were kept moist and in the dark at 25°C, sprayed with ultrapure water every six hours to replenish moisture. After three days, the filter paper was removed, and 50% Hoagland's solution was added to provide sufficient nutrients. The lettuce was then placed in an incubator under 16 hours of 24°C light and 8 hours of 22°C darkness, with the culture medium changed every two days. After seven days, the solution was replaced with 25% Hoagland's solution to facilitate later experimental exposure.

[0021] 3. Plant exposure After 14 days of growth, lettuce seedlings with relatively consistent growth were selected for an exposure experiment, with the exposure light conditions remaining consistent with the cultivation stage. The experimental group consisted of lettuce exposed to 25% Hogrange nutrient solution containing different concentrations of As, Cd, Pb, and Cr, while a control group was set up with lettuce exposed only to the 25% Hogrange nutrient solution. The concentration settings are shown in Table 2. Three replicates were set up for each concentration, and samples were taken for relevant testing at 1, 5, and 10 days after exposure.

[0022] Table 1 Experimental Materials .

[0023] Table 2 Pollutant Exposure Concentration .

[0024] 4. Preparation of phosphate buffer solution (0.1 M) Dissolve 8.5 g of sodium chloride, 30.8 g of disodium hydrogen phosphate dodecahydrate and 2.8 g of sodium dihydrogen phosphate dihydrate in a beaker containing 800 ml of Wahaha water. After complete dissolution, transfer the solution to a 1 L volumetric flask and make up to volume to obtain 1 L of 0.1 M phosphate buffer solution.

[0025] 5. Determination of MDA, SOD, and POD Fresh lettuce leaf tissue (0.2 g each, weight error controlled within ±5 mg) was homogenized at low temperature on ice using a grinding pestle in 1.8 ml of 0.1 M phosphate buffer (pH=7.8). The homogenate was centrifuged at 10000 rpm at 4 °C for 15 min. After centrifugation, the tissue was processed according to the corresponding instructions and measured using a microplate reader at wavelengths of 532 nm, 600 nm, 450 nm, and 420 nm.

[0026] 6. Data Analysis All experimental results were processed in triplicate, and the results were expressed as mean ± SD (standard deviation). One-way ANOVA was performed on the data using IBM SPSS Statistics 26 statistical software.

[0027] 7. Results and Discussion Experimental results are as follows Figure 1 As shown, SOD under heavy metal stress (As, Cd, Pb) exhibits high response levels at lower concentrations and does not continuously increase with increasing exposure concentration or time. Meanwhile, SOD levels under Cr stress also show concentration dependence after a short period (1 day) of stress, with a gradient increase over extended exposure time. Furthermore, its level reaches a steady state by the fifth day of exposure. Under individual stress from different concentrations of As, Cd, and Pb, the SOD activity level in lettuce leaves is around 180 U / g FW. While the SOD activity level in lettuce leaves under Cr stress is lower, it remains around 100 U / g FW and 90 U / g FW under high concentration and long-term stress, respectively.

[0028] Although SOD activity is a powerful indicator under current single exposure conditions, its ability to differentiate between medium to high concentrations of pollutants or long-term stress is weakened, suggesting that more indicators need to be explored to comprehensively assess plant responses to different environmental stresses, so as to classify the environmental stresses on plants more finely.

[0029] Example 2: Higher-level biomarkers for lettuce under single pollutant stress 1. Plant incubation, cultivation, and exposure The experimental methods used in this section are consistent with those in Example 1.

[0030] 2. Determination of plant hormones The specific extraction and concentration steps for plant hormones IAA and GA are as follows: Take 0.5 g of fresh lettuce leaf tissue, controlling the weight error within ±5 mg, and homogenize it in 5 ml of 80% methanol solution pre-cooled to -20 ℃ (diluted with Wahaha purified water) using a pestle. After extraction in the dark at 4 ℃ for 24 h, centrifuge at 8000 rpm for 15 min. Add the supernatant from centrifugation to an activated extraction column. After complete filtration, add 5 ml of water to remove ions from the extraction column, and finally add 1 ml of 80% ice-cold methanol to a liquid chromatography vial. The temperature will be strictly controlled throughout the extraction process to prevent thermal decomposition of the hormones.

[0031] The activation process of the solid phase extraction column is as follows: Add 5 ml of pure methanol, 5 ml of water, and 5 ml of 80% ice methanol to the solid phase column in sequence. The activation is complete after the filter is completely removed. The column should be activated immediately before use.

[0032] Exploration of detection wavelengths for plant hormones IAA and GA. Both were dissolved in 80% ice-cold methanol, and a full-wavelength scan was performed using a UV spectrophotometer. The closest detection wavelength was found to be 254 nm.

[0033] The determination of plant hormones IAA and GA was performed using high-performance liquid chromatography (HPLC). The instrument used was an LC-2030 HPLC system; the mobile phase A was 0.6% glacial acetic acid (diluted with Wahaha purified water), and the mobile phase B was pure methanol; the column oven was set to 25 ℃; the detector wavelength was 254 nm; the flow rate was 1 ml / min; the analysis time was 20 min; the injection volume was 50 μL; and the analytical column used was a C18 column (250*4.6 mm, 5 μm).

[0034] 3. Data Analysis All experimental results were processed in triplicate, and the results were expressed as mean ± SD (standard deviation). One-way ANOVA was performed on the data using IBM SPSS Statistics 26 statistical software.

[0035] 4. Results and Discussion In this invention, long-term stress durations (5 days and 10 days) were selected to more comprehensively assess the physiological and biochemical responses of plants to sustained environmental stress. Experimental results are as follows: Figure 2 and Figure 3 As shown, Figure 2The changes in hormone levels in lettuce under 5 days of different single pollutant stress compared to the control group showed a certain regularity. Under all pollutant stresses, GA hormone levels, even if they were inhibited, were somewhat increased relative to the decrease in IAA. At the same time, it was found that GA showed an overall promoting trend, and this was somewhat concentration-dependent.

[0036] Lettuce under 10 days of stress Figure 3 As shown, under 10 days of stress, the levels of IAA and GA hormones generally stabilized. IAA hormone was significantly suppressed, and this suppression became more pronounced with increasing pollutant concentration. GA hormone expression generally increased with increasing pollutant concentration gradient. This indicates that as pollutant concentration increased, IAA concentration gradually decreased while GA concentration gradually increased, showing a clear concentration dependence. This suggests that there may be a certain correlation between the expression of GA and IAA hormones in plants under pollutant stress.

[0037] In summary, under stress from three heavy metals (Cd, Pb, and Cr) and a metalloid (As), the expression of IAA hormones in lettuce leaves was generally downregulated and suppressed, showing a continuous decreasing trend with increasing pollutant concentration and exposure time. In contrast, GA hormone levels were generally higher than IAA hormone levels, and its dominant expression tended to expand further with prolonged exposure time. Under prolonged stress (10 days) with high concentrations of pollutants, GA hormone expression showed a significant upregulation trend, possibly indicating that the plant needs an increased expression of GA to enhance its resistance to external abiotic stresses.

[0038] Example 3: Tertiary biomarkers under single pollutant stress The inhibition of photosynthesis in plants under heavy metal ion stress has been widely reported. This is because heavy metal ions enhance the activity of chlorophyllase, and some heavy metal cations, due to their similarity, can replace the central magnesium ion in the porphyrin ring, promoting the degradation of chlorophyll molecules. Simultaneously, heavy metal stress also inhibits chlorophyll production by damaging chlorophyll-producing enzymes, ultimately leading to a decrease in chlorophyll concentration. Excessive heavy metal ions in the photosynthetic system can also affect photosynthetic electron transport, the Calvin cycle, enzyme activity, and the function of the thylakoid membrane.

[0039] Therefore, the contents of chlorophyll a, chlorophyll b, and carotene in lettuce under single stress from different pollutants for 1 day and 5 days were first determined. The determination method is as follows: Take 0.5 g of fresh lettuce leaf tissue, with a weight error controlled within ±5 mg, cut it into small pieces, and immerse it in a centrifuge tube containing 5 ml of 95% ethanol solution. Under complete darkness, allow it to stand at room temperature for 24 h to extract chlorophyll. After extraction, take 200 μL of each sample and place it in a transparent 96-well plate. Use a fluorescence microplate reader to read the absorbance in batches at 665.2, 652.4, and 470 nm. The formulas for calculating the content of chlorophyll a, chlorophyll b, and carotenoids are as follows: ; ; ; The final chlorophyll content is expressed in mg·g. -1 FW stated.

[0040] Experimental results are as follows Figure 4 and Figure 5 As shown, the former represents the chlorophyll level of lettuce under one day of different single pollutant stress, while the latter represents the chlorophyll level of lettuce under five days of different single pollutant stress. The chlorophyll levels under single heavy metal stress did not show a concentration gradient trend or even a time gradient trend. This is likely because chlorophyll, as the most important photosynthetic pigment in plants, is affected by various factors. Its expression level may not show a significant decreasing trend until the regulation among signaling molecules in the plant reaches a steady state. Furthermore, plants may require more energy under pollutant stress and thus increase chlorophyll concentration to counteract external stress.

[0041] Since chlorophyll levels did not show a clear gradient dependence or trend in a short period of time, the chlorophyll levels of lettuce under 10 days of stress from a single pollutant were then measured. The experimental results are as follows: Figure 6 As shown, under prolonged (10 days) low-concentration single pollutant stress, chlorophyll concentration showed a certain increasing trend compared to the control group, or remained at a relative level. Furthermore, except for lead ion stress, all other single pollutants showed a significant decrease in chlorophyll under high-concentration stress, with the lowest decrease reaching 0.1 mg / g FW. The increase in chlorophyll content under high-concentration lead ion stress may be due to the use of lead nitrate as the lead compound, which contains a high nitrogen content in its nitrate ions. Nitrogen is an important component of chlorophyll; when its concentration increases, the chlorophyll content also increases accordingly, ultimately leading to a higher chlorophyll content under lead stress. Therefore, the chlorophyll content of lettuce significantly decreases under prolonged high-concentration single pollutant stress, and it can also serve as a biomarker.

[0042] By integrating the above two indicators, it was found that IAA hormones show a relatively direct inhibitory effect under pollutant stress, and changes in concentration and time do not significantly affect their trend level, thus not meeting the criteria for being a biomarker. GA hormones show a relatively low inhibitory level under low-concentration pollutant stress for a short period (5 days), but as the pollutant concentration increases or the exposure time prolongs, GA hormones begin to show a significant increasing trend. Therefore, when GA hormone levels rise, it can be used to infer that the plant is currently experiencing a certain degree of pollution stress, meaning that GA hormones can be used as a secondary biomarker in the current exposure combination. Chlorophyll levels do not show significant regularity under short-term stress (1 day and 5 days), but the chlorophyll concentration in lettuce leaves only significantly decreases under high-concentration pollution and long-term stress. Therefore, under the current exposure combination, it can be designated as a tertiary biomarker.

Claims

1. A multi-level biological indicator method for effective phytotoxicity of heavy / heavy metals, characterized in that, Includes the following steps: S1. Obtain plant samples affected by the soil or water body to be tested; S2. Determine the SOD activity, the content of at least one plant hormone, and the chlorophyll content in the plant sample; S3. Based on the measurement results of the SOD activity, the plant hormone content, and the chlorophyll content, and their change patterns over time, the effective phytotoxicity of heavy metal-like substances or heavy metals is classified and uniformly assessed.

2. The method according to claim 1, characterized in that, In step S2, the SOD activity, the plant hormone content, and the chlorophyll content have a time-response order, wherein: The response time window for SOD activity is 18-36 hours after exposure; The response time window for plant hormone levels is 108–132 hours after exposure; The response time window for chlorophyll content was 228–252 hours after exposure; Furthermore, the plant hormone response occurs after SOD activity has already produced a response, while the chlorophyll response occurs after both SOD activity and plant hormones have already produced a response.

3. The method according to claim 1 or 2, characterized in that, In step S2, the plant hormone is gibberellin (GA).

4. The method according to claim 1 or 2, characterized in that, In step S2, the chlorophyll is chlorophyll a and / or chlorophyll b.

5. The method according to claim 1, characterized in that, In step S3, the hierarchical unified assessment includes: A significant increase in SOD activity indicates the presence of pollutant stress. When the plant hormone GA content was further detected to show a pattern of first increasing and then decreasing, it indicated an increase in the degree of pollutant stress. When a significant decrease in chlorophyll content is detected, it indicates that the level of pollutant stress has reached a high level.

6. The method according to claim 1, characterized in that, The heavy metals include arsenic, and the heavy metals include at least one of cadmium, chromium, and lead.

7. The method according to any one of claims 1-6, characterized in that, The plant in question is lettuce.

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