A rice field urea safety early warning method and a rice field urea fertilization guidance method

Abstract

The application provides a rice field urea safety early warning method and a rice field urea fertilization guiding method, which comprises the following steps: step S1, culturing protozoa Ciliatus; step S2, collecting field surface water samples from multiple points of a to-be-tested rice field, mixing the samples, extracting a to-be-tested solution, then adding 8-12 Ciliatus per mL into the to-be-tested solution, observing the survival individual number of the Ciliatus after 24 hours, calculating the survival rate, or taking a live photo of the Ciliatus, and calculating the generation time of the Ciliatus; and step S3, if the survival rate of the Ciliatus is less than 100%, or the Ciliatus appears morphological changes and the generation time is increased, it is prompted that the urea content in the rice field is excessively high, and early warning is performed. By adopting the technical scheme, whether the nitrogen fertilizer applied to the rice field is excessive can be early warned, further, the fertilization amount and the fertilization mode can be determined according to the early warning value, 95% of the organisms in the farmland can be protected, and the environment and the biodiversity can be better protected.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, and in particular relates to an early warning method for the safety of urea in paddy fields and a method for guiding the application of urea fertilizer in paddy fields. Background Technology

[0002] Urea, with a nitrogen content of 46%, is a common elemental nitrogen fertilizer. It is popular among farmers due to its rapid effect, high application rate, and noticeable crop improvements. It is also a crucial nitrogen fertilizer in rice cultivation, used as a base fertilizer, top dressing, tillering fertilizer, heading fertilizer, and even foliar fertilizer. However, improper application can lead to fertilizer waste and "fertilizer harm," damaging crops and the native organisms in the paddy field. Addressing the current issues of excessive nitrogen fertilizer application and unreasonable fertilization techniques in paddy fields, the 2022 Guidelines for Scientific Fertilization of Rice emphasize controlling nitrogen fertilizer application and achieving rational fertilization. Therefore, the application rate of urea must be carefully considered. However, there are currently no clear regulations regarding the safe limit for urea usage. Summary of the Invention

[0003] To address the above technical problems, this invention discloses an early warning method for urea safety in paddy fields and a guidance method for urea fertilization in paddy fields, which can better protect the environment and biodiversity.

[0004] The technical solution adopted by this invention is as follows:

[0005] A method for early warning of urea safety in paddy fields includes the following steps:

[0006] Step S1: Culture the protozoan ochrefibrillaria;

[0007] Step S2: Collect surface water samples from multiple points in the paddy field to be tested, mix them well, extract the solution to be tested, and then add ochre fibrillator to the solution at a rate of 8-12 individuals / mL. After 24 hours, observe the number of surviving individuals of ochre fibrillator and calculate the survival rate, or take a live photo of the ochre fibrillator and calculate the generation time of the ochre fibrillator.

[0008] Step S3: If the survival rate of ochre fibrillator is less than 100%, or if the ochre fibrillator shows morphological changes or an increased generation time, it indicates that the urea content in the paddy field is too high, and an early warning is issued.

[0009] This technical solution utilizes a sensitive protozoan to quickly provide early warning of urea overload and assess warning levels, protecting over 95% of protozoa and better safeguarding the environment and biodiversity. This protozoan is relatively large, pinkish in color, easy to observe, easy to cultivate, and reacts rapidly, making it suitable for widespread application.

[0010] As a further improvement of the present invention, step S1 includes isolating ochre fibrillator from rice rhizosphere soil, feeding on rice extract, and culturing in a constant humidity incubator at 25°C.

[0011] As a further improvement of the present invention, step S2, collecting surface water samples from multiple points in the paddy field to be tested, includes: dividing the paddy field to be tested into several sampling areas, dividing each sampling area into 3 to 5 sampling points using an "S"-shaped sampling method, collecting surface water from different water layers at each sampling point, mixing them thoroughly, and then placing them into a water quality sample container; collecting 3 parallel samples from each sampling area. Further, the sample container is a sample box or a centrifuge tube.

[0012] As a further improvement of the present invention, in step S2, 1 mL of the test solution is extracted and 10 ochre fibrillatids are added.

[0013] As a further improvement of the present invention, in step S3, the indication that the urea content in the paddy field is too high means that it exceeds the urea warning value for paddy fields; the urea warning value for paddy fields is calculated using the following steps:

[0014] Acute toxicity tests were conducted on ochratoxin ciliates, and their growth curves under different urea concentrations were determined. The median lethal concentration (LC50) of ochratoxin ciliates at 24 h and 48 h was calculated. 50 48hLC 50 ), half-maximal inhibitory concentration (EC50) 50 The minimum effective concentration (LOEC) and minimum no-effect concentration (NOEC) were used to calculate the effect threshold (PNEC) using the evaluation factor method and the SSD probability method. The maximum permissible toxic concentration (MATC) was calculated using formula (1), and the safe concentration (SC) was calculated using formula (2). The maximum warning value for the safety of ochre fibrillator was selected as the warning value for urea in paddy fields.

[0015]

[0016]

[0017] Among them, 24hLC 50 This indicates the median lethal concentration (LC50) at 24 hours; 48 hLC. 50 This indicates the median lethal concentration after 48 hours.

[0018] This invention also discloses a method for guiding urea fertilization in paddy fields. It employs the aforementioned early warning method for urea safety in paddy fields to assess the urea safety of the paddy field under test. If the urea content in the paddy field does not exceed the early warning value, the application rate and method of urea are determined based on the early warning value, combined with the normal nitrogen fertilizer concentration and water volume in the paddy field. This technical solution can better protect over 95% of protozoa while meeting the nitrogen fertilizer requirements of paddy fields.

[0019] As a further improvement of the present invention, the application method is one or more applications of urea. The amount of urea used each time is determined based on the urea warning value and the water volume of the paddy field. The number of applications of urea is calculated based on the ratio of the normal nitrogen fertilizer concentration in the paddy field to the urea warning value. In the case of multiple applications, the fertilization interval is determined comprehensively by considering the time required for urea hydrolysis and the nutrient requirements of rice at different stages.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] By employing the technical solution of this invention, early warning can be issued regarding whether nitrogen fertilizer application in paddy fields is excessive. Based on the concentration that ochre fibrillator can tolerate, a safe warning value for nitrogen fertilizer urea can be determined, thereby determining the amount and method of fertilizer application. This approach can protect 95% of the organisms in farmland while meeting the nitrogen fertilizer requirements of paddy fields, thus better protecting the environment and biodiversity. Attached Figure Description

[0022] Figure 1 These are growth curves of four organisms exposed to urea solutions of different concentrations according to embodiments of the present invention; wherein Figure A is Ochracidium, Figure B is Nephroticosa, Figure C is Paramecium, and Figure D is Tetrahymena.

[0023] Figure 2 These are magnified microscopic images of ochratocerci in different states according to embodiments of the present invention, wherein Figure A is a live ochratocerci, Figure B is an ochratocerci under nitrogen fertilizer stress, and Figure C is an ochratocerci cyst.

[0024] Figure 3 This is a diagram showing the generation results of ochrefibrillaria in an embodiment of the present invention.

[0025] Figure 4 These are short-term SSD curves of different protozoa in embodiments of the present invention. Detailed Implementation

[0026] The preferred embodiments of the present invention will be described in further detail below.

[0027] A method for early warning of urea safety in paddy fields includes the following steps:

[0028] Step S1, culturing the protozoan ochratofiliis, specifically includes: isolating ochratofiliis from rice rhizosphere soil, feeding on rice extract, culturing in a constant humidity incubator at 25°C, and cleaning metabolic waste every 24 hours.

[0029] Step S2: Collect 10 mL of surface water samples (from different water layers) from multiple points in the paddy field to be tested. After mixing, take 1 mL as the test solution. Then add 10 ochre fibrillates to the test solution. After 24 hours, observe the number of surviving ochre fibrillates and calculate the survival rate. At the same time, take live photos of the ochre fibrillates and calculate the generation time of the ochre fibrillates. Perform 4 parallel experiments (with the surface water sample before fertilization as the control group).

[0030] Step S3: If the survival rate of ochre fibrillator is <100%, or if the ochre fibrillator shows morphological changes or an increased generation time, it indicates that the urea content in the paddy field is too high, prompting an early warning. The increased generation time is compared with the field surface water sample before fertilization.

[0031] Furthermore, in step S2, collecting surface water samples from multiple points in the paddy field to be tested includes: dividing the paddy field to be tested into several sampling areas, dividing each sampling area into 3 to 5 sampling points according to the "S" shaped sampling method, collecting surface water from different water layers at each sampling point, mixing them, and then placing them into a water quality sample box or centrifuge tube; collecting 3 parallel samples from each sampling area.

[0032] In step S3, "excessively high urea content" means exceeding the urea warning value for paddy fields. The urea warning value for paddy fields is calculated using the following steps:

[0033] Acute toxicity tests were conducted on ochratocelia, and the growth curves of ochratocelia under different urea concentrations were determined to calculate the median lethal concentration (LC50) of ochratocelia. 50 ), half-maximal inhibitory concentration (EC50) 50 The toxicity effect indicators, such as the lowest effective concentration (LOEC) and the lowest no-effect concentration (NOEC), were used to calculate the effect threshold (PNEC) using the evaluation factor method and the SSD probability method. The maximum permissible toxic concentration (MATC) was calculated using formula (1), and the safe concentration (SC) was calculated using formula (2). The maximum warning value for the safety of ochre fibrillator was selected as the warning value for urea in paddy fields.

[0034]

[0035]

[0036] Among them, 24hLC 50 This indicates the median lethal concentration (LC50) at 24 hours; 48 hLC. 50 This indicates the median lethal concentration after 48 hours.

[0037] To verify the rationale for selecting ochratoxin ciliates for early warning, the following acute toxicity experiment was conducted, including the following steps:

[0038] Urea, a nitrogen fertilizer, was selected for the experiment. Different concentration gradients of urea were set up, and an acute toxicity test was conducted on the experimental materials for 96 hours. A 24-well plate was used for the toxicity test. Initially, 10 ochre fibrillifers were inoculated into each well with a solution volume of 1 mL. The control group consisted of distilled water. After 24 hours, the number of surviving protozoa in each group was observed and recorded, growth curves were plotted, and the median lethal concentration (LC50) was calculated. 50 ), half-maximal inhibitory concentration (EC50) 50 The study included toxicity indicators such as the lowest effective concentration (LOEC) and the lowest no-effect concentration (NOEC). Live photographs of ochre fibrillifers treated with nitrogen fertilizer were taken using a Zeiss microscope.

[0039] Acute toxicity tests were conducted on ochre fibrillaria to determine its growth curve under the influence of nitrogen fertilizer urea, with *Nephroplasma gondii*, *Paramecium*, and *Tetrahymena* used as control groups. The results are as follows: Figure 1 As shown, low concentrations of urea promote the growth of ciliates, while increased urea concentrations inhibit their growth. Urea concentrations greater than 100 mg / L inhibit the growth of *Ochratus ciliata*; greater than 500 mg / L inhibits the growth of *Paramecium*; greater than 3500 mg / L inhibits the growth of *Nephroticosa*; and greater than 20000 mg / L inhibits the growth of *Tetrahymena*. Therefore, among these four organisms, *Ochratus ciliata* is the most sensitive species to nitrogen fertilizer urea and can be used as an early warning species.

[0040] according to Figure 1 The curves show that, for *Ochratus ochraceus*, a certain concentration of nitrogen fertilizer urea solution promotes the growth and reproduction of *Ochratus ochraceus*. At this point, the observed live *Ochratus ochraceus* appear as follows: Figure 2 As shown in (A). When the urea concentration reaches 100 mg / L and after 24 hours of exposure, the ochrefibrillaria cannot develop tolerance to nitrogen fertilizer urea. Ochrefibrillaria under nitrogen fertilizer stress are shown in 2(B); the ochrefibrillaria cannot perform normal physiological metabolic functions, and some of the worms enter the cyst, as shown in... Figure 2 As shown in (C). After treatment with 100 mg / L for 72 h, the morphology of ochratofibrosis changed, and vacuoles appeared at the posterior end of the worms. Once vacuoles appeared, the ochratofibrosis died. After treatment with 500 mg / L urea for 96 h, all ochratofibrosis died.

[0041] The generation times of *Ochracidia* under treatment with 4, 50, 100, and 300 mg / L urea were calculated using the formula G = (t2 - t1) / 3.322 (lgx2 - lgx1) (where x1 = the number of ciliates at the start of treatment, i.e., t1; x2 = the number of ciliates at a certain point after the start of treatment, i.e., t2). The results are as follows. Figure 3As shown, after 24 hours of exposure, when the urea concentration was between 4 mg and 300 mg, the time taken for the logarithmic growth phase of *Ochratus ochraceus* to reproduce one generation was longer than that of the control group, indicating a certain inhibitory effect on its growth rate. When the concentration reached 500 mg / L, some *Ochratus ochraceus* died and ceased reproductive division. Furthermore, calculations of the generation time of *Ochratus ochraceus* showed that the warning generation time was approximately 24 hours, which can provide relatively rapid and immediate feedback in actual agricultural production.

[0042] The effect threshold (PNEC) was calculated using the evaluation factor method and the probability method (SSD). The maximum permissible toxic concentration (MATC) was calculated using formula (1), and the safe concentration (SC) was calculated using formula (2). Species sensitivity distribution curves were plotted using the CADDIS SSD Generator (ver. 1.0) software provided by the US Environmental Protection Agency (USEPA, 2005a, b; https: / / www.epa.gov / cadddis-vol4). A reasonable urea threshold was obtained by comprehensively comparing the warning thresholds obtained from different methods, and urea warnings were issued accordingly.

[0043] Short-term SSD curves as follows Figure 4 As shown, protozoa are sensitive species, with ochre fibrillaria being the most sensitive and serving as early warning organisms.

[0044] Using the aforementioned assessment methods, the same experiments were also conducted on *Ochracidia*, *Paramecium*, *Larva*, *Nephroplasma*, *Hydra*, *Tetrahymena*, loach, guppy, and tilapia. The urea effect threshold PNEC range, determined by the evaluation factor method, was 0.16–34.97 mg / L, with a median of 15.75 mg / L. The short-term HC5 of nitrogen fertilizer urea was calculated to be 217.46 mg / L using the SSD probability method (HC5 value refers to the concentration at which 95% of species in the ecosystem are protected). Dividing this by evaluation factor 3 (source: HJ 831-2022 Technical Guidelines for Derivation of Freshwater Biological Water Quality Standards), the short-term PNEC for the eight species was calculated to be 72.49 mg / L. According to formula (2), the safe concentration (SC) range for nine species (Ochracidia, Paramecium, Megasaur, Nephroticosa, Hydra, Tetrahymena, Loach, Guppy, Tilapia) was calculated to be 0.016–2800 mg / L, with a median value of 117.57 mg / L.

[0045] Using the above assessment method, four warning thresholds were derived, and the highest warning value for the safety of ochre fimbriae was selected as the warning value for urea nitrogen fertilizer in paddy fields. Combined with... Figure 1It is known that the safety warning threshold for urea against ochratoxin is 72.49 mg / L. If this value is half of the normal nitrogen fertilizer concentration applied to paddy fields, two applications of this amount can be made to meet the growth needs of both protozoa and plants. The interval between the two applications should be determined comprehensively based on the time required for urea hydrolysis and the fertilizer requirements of rice at different stages. Under normal circumstances, the time for urea to completely hydrolyze into ammonium carbonate is: approximately 10 days at 10℃, 4-5 days at 20℃, and approximately 2 days at 30℃.

[0046] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for early warning of urea safety in paddy fields, characterized in that: Includes the following steps: Step S1: Culture protozoan ochrefibrillaria; Step S2: Collect surface water samples from multiple points in the paddy field to be tested, mix them well, extract the solution to be tested, and then add 8-12 individuals / mL of ochratocerci to the solution to be tested. After 24 hours, observe the number of surviving individuals of ochratocerci and calculate the survival rate, or take live photos of the ochratocerci and calculate the generation time of ochratocerci. Step S3: If the survival rate of ochre fibrillator is <100%, or if the ochre fibrillator shows morphological changes or an increased generation time, it indicates that the urea content in the paddy field is too high, and an early warning is issued. Step S1 includes isolating ochre fibrillosa from rice rhizosphere soil, feeding on rice extract, and culturing in a constant humidity incubator at 25°C.

2. The early warning method for urea safety in paddy fields according to claim 1, characterized in that: In step S2, the collection of surface water samples from multiple points in the paddy field to be tested includes: dividing the paddy field to be tested into several sampling areas, dividing each sampling area into 3 to 5 sampling points according to the "S" shaped sampling method, collecting surface water from different water layers at each sampling point, mixing them, and then placing them into a water quality sample container; collecting 3 parallel samples from each sampling area.

3. The early warning method for urea safety in paddy fields according to claim 2, characterized in that: In step S2, 1 mL of the test solution is extracted and 10 ochre fibrillatids are added.

4. The early warning method for urea safety in paddy fields according to claim 1, characterized in that: In step S3, the prompt that the urea content in the paddy field is too high means that it exceeds the warning value for urea in paddy fields; The urea warning value for paddy fields is calculated using the following steps: Acute toxicity tests were conducted on ochre fibrillator, and the growth curves of ochre fibrillator under different urea concentrations were measured. The median lethal concentration, median inhibitory concentration, minimum effective concentration, and minimum ineffective concentration of ochre fibrillator were calculated. The effect threshold, maximum permissible toxic concentration, and safe concentration were calculated using the evaluation factor method and the SSD probability method. The maximum warning value for the safety of ochre fibrillator was selected as the urea warning value for paddy fields.

5. A method for guiding the application of urea fertilizer in paddy fields, characterized in that: The urea safety warning method for paddy fields as described in claim 4 is used to assess the urea safety of the paddy field under test. If the urea content in the paddy field does not exceed the urea warning value, the urea application amount and application method are determined based on the urea warning value, combined with the normal nitrogen fertilizer concentration and water volume of the paddy field.

6. The method for guiding urea fertilization in paddy fields according to claim 5, characterized in that: The application method is to apply fertilizer once or multiple times. The number of times urea is applied is calculated based on the ratio of the normal nitrogen fertilizer concentration in the paddy field to the urea warning value in the paddy field.

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