Detection method for rapidly evaluating soil toxicity

Abstract

The invention belongs to the field of agricultural safety production early warning and environmental pollutant toxicity monitoring, and provides a detection method for rapidly evaluating soil toxicity, which comprises the following steps: planting disinfected and germinated rape seeds in to-be-detected soil, culturing under a dark condition to obtain rape etiolated seedlings, and carrying out early warning on the rape etiolated seedlings; detecting the germination rate of the rape seeds and the growth and development state of the rape yellowing seedlings, and evaluating the soil toxicity according to the germination rate and the growth and development state of the rape yellowing seedlings. The method does not need to measure the soil toxicity like a traditional monitoring method, so that the monitoring cost is greatly reduced, and the fastest monitoring time can be shortened to 4 days.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural production safety early warning and environmental pollutant toxicity monitoring, and provides a detection method for rapidly evaluating soil toxicity, and rapidly judging whether the test soil has toxic or adverse effects on plant seed germination and seedling growth. The toxic and harmful substances judged include residual pesticides, heavy metals, harmful microorganisms, uncomposted organic fertilizers, etc. in the soil. Background Art

[0002] Plant growth retardants play an important role in sweet potato production. The rational use of such agents can increase the yield and improve the quality of sweet potatoes to achieve the maximum benefits in production. However, many plant growth retardants will remain in the soil after use, posing a threat to cultivated crops and their successor crops and human health. In order to reduce the toxicity of such agents to soil and crops in agricultural production, it is first necessary to understand their critical threshold of toxicity to crops. However, according to the "Soil Environmental Quality-Agricultural Land Soil Pollution Risk Control Standard" (GB15618-2018) issued by the state in 2018, only the total amount of hexachlorocyclohexane, the total amount of DDT and benzo[a]pyrene in the soil are limited, while the limit values ​​of other organic pollutants are not recorded, and there is no data reference for plant growth retardants. Therefore, it is very important to explore the toxic threshold of plant growth retardants in the soil. Such research can provide certain data support for the writing of standards.

[0003] Usually, to understand the toxic threshold of a soil pollutant to crops, it is necessary to dynamically monitor the content of the pollutant, analyze and monitor the effective state of the pollutant that is closely related to the plant absorption, and observe and check whether the growth and development of crops are inhibited, because the causal relationship between the content of pollutants in the soil and the growth and development of crops is very complicated. This greatly increases the time and cost consumption of the formulation of soil pollution standards. Therefore, there is a need for a method to quickly evaluate the degree of soil pollution, which can shorten the toxicity observation time and reduce the difficulty of pollution evaluation.

[0004] In addition, the residue of plant growth retardants in the soil environment depends on their degradation efficiency in the soil environment. The more difficult it is to degrade the pesticide in the natural environment, the longer the impact on the soil environment will last. In order to prevent the residue of plant growth retardants applied during the sweet potato planting period from toxicating the subsequent crops of sweet potatoes, it is particularly important to understand their degradation efficiency in the natural soil environment. The degradation efficiency information can provide agricultural production with a suitable planting time for subsequent crops corresponding to their toxic threshold to avoid the toxic effects of their residues in the soil.

[0005] In agricultural growth, we often encounter problems such as seeds not germinating normally or delayed emergence after sowing, long seedling recovery time after transplanting, delayed and stagnant growth of seedlings, and even death of seedlings, which seriously affect agricultural production. These phenomena may be related to the presence of too many toxic and harmful substances in the soil, such as toxic pesticides remaining in the soil, high concentrations of heavy metals, a large number of soil-borne harmful microorganisms, and uncomposted fertilizers applied before planting. These factors are often difficult to judge their toxicity with the naked eye, and the detection target range is wide, the detection targets are scattered, and the instrument detection cost is high and takes a long time. Rapidly judging whether the soil has an adverse effect on seed germination and seedling growth has the advantages of early warning and disaster prevention and mitigation for agricultural safety production, improving the safety of agricultural production, and reducing losses. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a detection method for rapidly evaluating soil toxicity, which does not require the determination of soil toxicity like traditional monitoring methods, thereby reducing monitoring time and cost.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] A method for rapidly evaluating soil toxicity comprises the following steps: planting germinated rapeseed seeds in soil to be tested, culturing in dark conditions to obtain yellow rapeseed seedlings, detecting the growth and development status of the yellow rapeseed seedlings, and evaluating soil toxicity according to the growth and development status of the yellow rapeseed seedlings.

[0009] Furthermore, the detecting of the growth and development status of the etiolated rapeseed seedlings includes: detecting the bud length of the etiolated rapeseed seedlings.

[0010] Furthermore, the method for cultivating yellow rapeseed seedlings comprises: sowing the germinated rapeseed seeds in a pot containing the soil to be tested, and placing the pot in a dark incubator for cultivation for 4 to 7 days.

[0011] Furthermore, after the rapeseed seeds are sown, water is poured into the tray at the bottom of the pot, and water is infiltrated from the bottom. After the water reaches the surface soil, the remaining water in the pot is poured out.

[0012] Furthermore, the germinated rapeseed seeds were sown at a sowing density of 2 seeds per square centimeter, and after sowing, they were covered with 4-mesh test soil.

[0013] Furthermore, the culture conditions of the yellow rapeseed seedlings are: 23-27 degrees Celsius and 35-45% humidity.

[0014] Furthermore, the rapeseed germination method includes: screening the rapeseed seeds before sowing, selecting seeds of relatively uniform size, soaking the seeds in water for 1 hour, removing the seeds floating on the water surface, rinsing with tap water twice, disinfecting with 75% alcohol for 1 minute, sterilizing the seeds with 3% NaClO for 15 minutes (keeping the seeds in a shaking state during this period), and then rinsing with deionized water for 5-6 times to achieve the disinfection effect. Finally, the seeds are transferred to an empty culture dish padded with moist filter paper for 16-24 hours (depending on the vitality of the rapeseed seeds, most of them can be taken out for experiments when they are exposed to white), so as to achieve the germination effect and activate the seeds, and reduce the growth difference error caused by different seed activities.

[0015] The present invention also provides the application of the above detection method in detecting residual pesticides, heavy metals, harmful microorganisms or uncomposted organic fertilizers in soil.

[0016] The present invention also provides application of the above detection method in detecting the content of plant growth retardant in soil.

[0017] Furthermore, the growth retardant includes paclobutrazol, clofosamide, prohexadione-calcium and meclocyclamide.

[0018] Beneficial Effects

[0019] The method uses yellow rape seedlings as indicator plants for soil environmental pollution. The rapid growth characteristics of yellow rape seedlings can be observed to be inhibited in a short time under the influence of a certain concentration of plant growth retardants, reflecting the toxicity of plant growth retardants in the soil. The method of the present invention does not need to measure soil plant growth retardants like traditional monitoring methods, which reduces the time and cost of monitoring and can provide early warning for agricultural production safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an analysis of the differences in shoot length of rapeseed after 4 days of growth in the test soil in Jiangxi;

[0021] Figure 2 This is an analysis of the differences in shoot length of rapeseed after 4 days of growth in the test soil of Nanchong;

[0022] Figure 3 The growth of yellow rape seedlings treated with paclobutrazol in the test soils with pH values ​​of 5.77 (a), 6.78 (b), and 7.52 (c);

[0023] Figure 4 The growth of yellow rape seedlings treated with uniconazole in the test soils with pH values ​​of 5.09 (a), 6.50 (b), and 7.56 (c);

[0024] Figure 5The growth of yellow rapeseed seedlings treated with prohexadione-calcium in the test soils with pH values ​​of 5.65 (a), 6.19 (b), and 7.27 (c);

[0025] Figure 6 The growth potential of yellowing rapeseed seedlings treated with chlorpyrifos in the test soils with pH values ​​of 5.65 (a), 6.19 (b) and 7.15 (c). DETAILED DESCRIPTION

[0026] Example 1

[0027] 1. Materials and Methods

[0028] 1.1 Test crops in potted experiments

[0029] The test plant for the pot experiment is the oil crop rapeseed (Brassica napus L.). In the pot experiment, rapeseed seeds are small, the growth and germination of yellow seedlings are fast, the cycle is short, and the growth rate is obvious. It can clearly reflect whether there are toxic substances that inhibit growth in the soil, and it is a very suitable experimental material. The variety selected in this embodiment is Fengyou 737, a representative variety that has been well promoted nationwide.

[0030] Rapeseed seeds were screened before sowing, and seeds of relatively uniform size were selected. The seeds were first soaked in water for 1 hour, and the seeds floating on the water surface were removed. They were rinsed with tap water twice, disinfected with 75% alcohol for 1 minute, and sterilized with 3% NaClO for 15 minutes (keep shaking during the period), and then rinsed with deionized water for 5-6 times to achieve the disinfection effect. Finally, the seeds were transferred to an empty culture dish with moist filter paper for 16-24 hours (depending on the vitality of the rapeseed seeds, most of them can be taken out for experiments when they are white), so as to achieve the effect of germination and activate the seeds, and reduce the growth difference error caused by different seed activity.

[0031] 1.2 Soil preparation:

[0032] The test soil was air-dried in advance, large stones or debris were removed, large clods were crushed, and mixed evenly. About 350 grams of soil was taken, passed through a 4-mesh sieve, and about 50 grams of sieved soil was kept (for covering the seeds to be sown later). The rest of the soil was thoroughly mixed evenly.

[0033] 1.3 Experimental pots:

[0034] A plastic or other material pot, about 8cm in diameter, 10cm high, with holes at the bottom. Place a piece of filter paper or 2 layers of toilet paper at the bottom of the plastic pot.

[0035] 1.4 Filling the pot with soil:

[0036] The mixed test soil was placed in a pot, with about 100g of soil in each pot, and three replicates were performed. The rapeseed seeds after the above germination were sown at a sowing density of about 2 seeds per square centimeter, and 20 seeds were sown in each pot. After sowing, 4-mesh fine soil (the same test soil) was used for covering (about 50 grams of fine soil, basically used for covering 3 pots). Clean uncontaminated soil was used as a control. Also, 4-mesh uncontaminated soil was reserved for covering the seeds.

[0037] 1.5 Cultivation of yellow seedlings:

[0038] After sowing rapeseed seeds, pour water into the tray at the bottom of the pot, and use the method of water seepage from the bottom. After the water reaches the surface soil, pour out the remaining water in the pot. Put the pot into a dark incubator for 3-5 days (25 degrees Celsius, humidity 35-45%). (Because the yellow seedlings grow fast, if there are toxic pollutants in the soil that inhibit growth, the length of the yellow seedlings will be significantly lower than that of the control group in 3-5 days).

[0039] 1.6 Data collection and analysis:

[0040] Gently pull out the etiolated seedlings from the control and test soils and shake off the soil. Arrange them in order of shoot length (excluding root system) from long to short (those without emergence are considered to have shoot length of 0). Remove the longest 5 plants and the shortest 5 plants (including those without emergence), select the middle 10 plants, measure the shoot length, calculate the average value, and perform a significance analysis of the difference in shoot length.

[0041] 2 Results and analysis

[0042] 2.1 Report on the biological testing of five soil samples in Jiangxi

[0043] Five soil samples from Jiangxi were subjected to a rapeseed germination bioassay to estimate the extent of soil contamination. The results are as follows:

[0044] Table 1 Germination rate of rapeseed seeds in the test soil of Jiangxi

[0045]

[0046] like Figure 1 As shown, the mean sprout lengths of yellowed rapeseed seedlings planted in the soils of Jiangxi 001, Jiangxi 002 and Jiangxi 003 were 2.56 cm, 0.42 cm and 4.06 cm, respectively, which were significantly lower than those of the control group (sprout length 7.96 cm); and as shown in Table 1, the germination rate of rapeseed seeds sown in the soil of Jiangxi 001 was 58.33%, and the germination rate of rapeseed seeds sown in the soil of Jiangxi 002 was only 18.33%, which was significantly lower than that of the control group (germination rate 98.33%).

[0047] 2.2 Biological test report of 9 soil samples in Nanchong

[0048] Nine soil samples from Nanchong were subjected to a rapeseed germination bioassay to estimate the extent of soil contamination. The results are as follows:

[0049] Table 2 Germination rate of rapeseed seeds in the test soil of Nanchong

[0050]

[0051] like Figure 2 As shown in Table 2, the average length of yellowed rape seedlings planted in soil samples from Peng'an 3 and Shuangdian was 3.61 cm and 3.38 cm, respectively, which were significantly lower than the control group (sprout length 7.96 cm). As shown in Table 2, although there was no significant difference in the germination rate of rape seeds planted in each soil sample compared with the control, the germination rate of rape seeds sown in soil samples from Peng'an 3 and Shuangdian was the lowest, at 70.00% and 73.33%, respectively.

[0052] Example 2

[0053] Different concentrations of paclobutrazol, clofosinate, calcium proteolytic enzyme and mepiquat were added to the soil respectively. The experimental dosages of paclobutrazol and calcium proteolytic enzyme were 0, 0.05, 0.075 and 0.1 mg / kg, the experimental dosage of clofosinate was 0, 0.005, 0.0075 and 0.01 mg / kg, and the experimental dosage of mepiquat was 0, 0.05, 0.1 and 0.25 mg / kg. Rapeseed was planted and its growth was observed. Add the plant growth retardant solutions required to meet the set concentration gradient to the soil and mix them with the soil. The plant growth retardant content in the solution is added according to the actual amount of soil used (for example, if the paclobutrazol content is set to 0.1 mg / kg, if there is 100g of soil in the potted plant, add a solution containing 0.01 mg of paclobutrazol that can mix 100g of soil). The pots used in the experiment are small pots with a diameter of 7.5 cm, and each pot has 100g of soil. After mixing with the solution, the crop seeds are sown on the soil surface. When watering, the soil moisture content is controlled at 20%. Three replicates are made for each treatment group, and 15 to 20 seedlings are retained in the end. After placing the potted plants in a dark environment at 25 degrees for 4 to 5 days, the yellowed seedlings are gently taken out of the soil, the soil is shaken off, and the roots are taken out. After 5 days, samples are taken to investigate their growth. The results are as follows:

[0054] Potted plants treated with paclobutrazol ( Figure 3 ) showed that among the three tested soils, the critical threshold of paclobutrazol toxicity to rapeseed was greater than 0.1 mg / kg when the soil pH was 5.77, and the critical thresholds were 0.075 mg / kg and 0.1 mg / kg when the soil pH was 6.78 and 7.52, respectively.

[0055] Potted plants treated with uniconazole ( Figure 4) showed that the critical threshold of chlorpyrifos to rapeseed was 0.01 mg / kg when the soil pH was 5.09 and 7.56, and the critical threshold was 0.0075 mg / kg when the soil pH was 6.50.

[0056] Potted results of prohexadione-calcium treatment ( Figure 5 ) showed that the critical threshold of toxicity of calcium cyclohexadione to rapeseed was 0.075 mg / kg when the soil pH was 5.65, and the critical threshold of toxicity of calcium cyclohexadione to rapeseed was 0.1 mg / kg when the soil pH was 6.19 and 7.27.

[0057] Potted plants treated with chlorpyrifos ( Figure 6 ) showed that the critical threshold of mepiquat to rapeseed was 0.1 mg / kg when the soil pH was 5.65, the critical threshold was 0.25 mg / kg when the soil pH was 6.19, and the critical threshold was higher than 0.25 mg / kg when the soil pH was 7.15.

[0058] In summary, for paclobutrazol and calcium cyclamate, the minimum soil threshold for toxicity is 0.075 mg / kg; for clofosamide and mepiquat, the toxicity thresholds are 0.0075 mg / kg and 0.1 mg / kg, respectively.

[0059] Plant growth retardants in the soil will significantly slow down the growth rate of the plants affected by them. In the etiolated seedling pot experiment, if the growth of the etiolated seedlings in the treatment group is significantly inhibited compared with the blank control, it means that at the concentration of the treatment group, the plant growth retardants will seriously affect the normal growth of the plants, which can reflect that the concentration of plant growth retardants in the soil has reached the pollution level.

[0060] In the potted experiment, the growth of rapeseed crops in the soil with different concentrations of plant growth retardants well reflects the threshold concentration of plant growth retardants in the soil to produce toxicity. Judging from the results of several threshold concentrations, rapeseed has well demonstrated its sensitivity to toxic residues in the environment. In a smaller concentration range of the same retardant treatment, rapeseed growth showed significant differences. Among them, the toxic threshold concentration of rapeseed in the soil under paclobutrazol treatment was about 10 times that of clobutrazol treatment. This may be because the biological activity of clobutrazol is much higher than that of paclobutrazol, and its effect on controlling plant growth is more efficient than paclobutrazol. The soil toxicity threshold of the two crops under the treatment of meclocycline is higher than that of the other three plant growth retardants, which also indirectly reflects that meclocycline requires a higher concentration to produce an inhibitory effect on crop growth. Since 2021, the "National Food Safety Standard Maximum Limits of Pesticide Residues in Food" formulated by the state

[0061] (GB2763-2021) stipulates the maximum residue limits of four plant growth retardants in 376 kinds of food, but there is still no clear standard for its residue limit in soil. This may be due to the different soil properties in different regions and the different crops planted, which makes it impossible to better unify the standards. The potted experiment in this embodiment uses farmland soil collected from sweet potato planting areas as test soil to simulate the soil environment for planting sweet potatoes, which can provide a reference for the safe residue limit range of four plant growth retardants in sweet potato farmland soil. Using rapeseed crops as an indicator of toxicity can better show the concentration of growth retardants that are toxic to subsequent crops. These data can provide a reference for the risk assessment of soil contamination in sweet potato fields, including the detection of the content of growth retardants in the soil of sweet potato fields after the sweet potatoes are harvested to determine whether it is suitable for planting subsequent crops.

[0062] In summary, using yellow rapeseed seedlings as indicator crops to determine the degree of growth inhibition can quickly estimate the critical threshold of plant growth retardants in the soil. At the same time, due to the rapid growth characteristics of yellow rapeseed seedlings and their sensitivity to growth inhibition, the degree of soil contamination by plant growth retardants can be quickly determined, which greatly reduces the time and testing costs compared to traditional methods of detecting and evaluating suspected contaminated soil samples.

Claims

1. A method for rapidly evaluating soil toxicity, characterized in that: The following steps are involved: The rapeseed seeds after germination are planted in the soil to be tested and cultured in dark conditions to obtain yellow rapeseed seedlings. The growth and development status of the yellow rapeseed seedlings is detected, and the soil toxicity is evaluated based on the growth and development status of the yellow rapeseed seedlings.

2. The method for rapidly assessing soil toxicity according to claim 1, characterized in that: The detecting the growth and development status of the yellow rapeseed seedlings includes: detecting the bud length of the yellow rapeseed seedlings.

3. The method for rapid soil toxicity assessment according to claim 1, characterized in that: The method for cultivating yellow rape seedlings comprises: sowing the germinated rape seeds in a pot containing soil to be tested, and placing the pot in a dark incubator for cultivation for 4 to 7 days.

4. The method for rapidly assessing soil toxicity according to claim 3, characterized in that: After sowing the rapeseed seeds, pour water into the tray at the bottom of the pot, using the method of seeping water from the bottom. After the water reaches the surface soil, pour out the remaining water in the pot.

5. The method for rapid soil toxicity assessment according to claim 3, characterized in that: The germinated rapeseed seeds were sown at a sowing density of 2 seeds per square centimeter. After sowing, they were covered with 4-mesh test soil.

6. The method for rapid soil toxicity assessment according to claim 3, characterized in that: The culture conditions of the yellow rape seedlings are: 23-27 degrees Celsius and 35-45% humidity.

7. The method for rapid soil toxicity assessment according to claim 1, characterized in that: The method for accelerating germination of rapeseed seeds comprises: firstly soaking the seeds in water, removing the seeds floating on the water surface, rinsing with tap water and disinfecting with alcohol and NaClO in sequence, and after rinsing, transferring the seeds to an empty culture dish padded with moist filter paper for 16-24 hours.

8. Use of the detection method according to any one of claims 1 to 7 in detecting residual pesticides, heavy metals, harmful microorganisms or uncomposted organic fertilizers in soil.

9. Use of the detection method according to any one of claims 1 to 7 in detecting the content of plant growth retardants in soil.

10. The use according to claim 9, characterized in that: The growth retardants include paclobutrazol, uniclobutrazol, prohexadione-calcium and meclocyclamide.

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