KR101897473B1 - Method for measuring soil toxicity using mini―pipe - Google Patents

Abstract

The present invention relates to a method for measuring soil toxicity using a mini-pipe, and more particularly, to a method for measuring soil toxicity using a mini pipe, in which a mini-pipe is installed in a soil sample contaminated with silver nanoparticles in a greenhouse, ( Chlamydomonas reinhardtii ) as a soil algae test species, and the soil toxicity was examined by isolating the soil algae. As a result, the cell size of the clamidomonas reinhardtii in the soil exposed to silver nanoparticles, the chlorophyll-a Chlorophyll-a) esterase activity is decreased and lipid content is increased. Therefore, the method of measuring soil toxicity using a pipe can be applied to an environment in which a soil medium exists.

Description

[0001] The present invention relates to a method for measuring soil toxicity using a mini pipe,

The present invention relates to a method for measuring soil toxicity using a mini pipe. More specifically, a pipe is installed in the soil, the soil is exposed to the upper part of the soil in the pipe and collected, And a method for measuring the same.

Algae is widely known as a representative ecotoxicological test species and mainly lives in water. However, Algae is a type of soil which is known as a soil algae, aerial algae, Cryoalgae, Symbiotic algae, Freshwater algae, and Marine algae.

Algae is a major producer in the aquatic ecosystem or soil ecosystem and occupies a dominant position in the ecosystem nutrition stage by being spawned by small watersheds or soil biomass and medium waters or soil biota. Currently, the risk assessment for the Suseo ecosystem is based on probabilistic Ecological Risk Assessment or Deterministic Ecological Risk Assessment based on the Suseo-toxicity data extended to three nutritional stages such as algae, daphnia and fish. Risk Assessment) is applied, but the risk assessment for soil ecosystem utilizes limited soil toxicity data for some nutritional stages such as vegetation, earthworms, nematodes, and insects.

Soil algae are distributed on soil surface or under soil in almost all soil environments and are used as biomarkers for soil ecological risk assessment. However, due to limited data on soil algae, it is difficult to apply soil toxicity data of soil algae to ecological risk assessment. There is no international standard for assessing the toxicity of soil algae exposed to contaminated soil for such reasons, and the current guidelines, the American Society for Testing and Materials (ASTM), the European Community (EC), the International Organization for Standardization (ISO) , And the OECD (International Organization for Economic Co-operation and Development). Most of the toxicities of contaminated freshwater or seawater to soil algae are assessed (American Society for Testing and Materials (ASTM), 2012. D3978-04 (ISO), 2006. ISO 10253: 2006 Water quality - Marine algae growth inhibition test with Algal inhibition test (ISO), European Community (EC), 1992. C.3.Algal inhibition test, Standard practice for algal growth potential testing with Pseudokirchneriella subcapitata Skeletonema costatum and Phaeodactylum tricornutum; Organization for Economic Co-operation and Development (OECD), 2011. Guideline for testing of chemicals No. 201. In freshwater alga and cyanobacteria, growth inhibition tes.

In addition, techniques that can be used to assess soil toxicity using existing soil algae have been developed to be performed on microscales such as laboratory scale. In addition, most of the soil-related algal toxicity studies reported so far have been conducted in soil extracts, soil water, pore water, and agar medium, , Which are performed on a laboratory scale, and there are very few studies that directly use the soil ecology. In addition, when these techniques are applied to the pot scale in the greenhouse, it is difficult to collect a certain amount of soil algae to quantitatively evaluate soil toxicity.

As a result of efforts to develop an evaluation method of soil toxicity with an improved accuracy while maintaining the environment in which the soil medium exists, the present inventors extended the applicable scale of the soil toxicity evaluation, And the soil toxicity test can be carried out by collecting and separating the soil algae test species after directly inoculating the soil algae test species. Therefore, the present invention has been accomplished by developing a method for measuring soil toxicity which is applicable to a wider scale than a laboratory scale.

Korean Patent No. 10-1655654

Gorman, D.S., Levine, R.P., 1965. Cytochrome f and plastocyanin: their sequence inthe photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc. Natl. Acad. Sci. U.S.A. 54 (6). Nam, S.-H. andan, Y.-J., 2015. An efficient and reproducible method for improving alga (Chlorococcum infusionum) for toxicity assays. Journal of Microbiological methods 119: 59-65. Nam, S.-H. and An, Y.-J., 2013. Trends in soil-related algal toxicity research. J. Kor. Soc. Environ. Eng 35 (8): 607-612.

It is an object of the present invention to provide a method for measuring soil toxicity using a pipe.

Yet another object of the present invention is to provide a kit for measuring soil toxicity comprising a pipe.

In order to achieve the above object,

1) installing a pipe in the soil;

2) Inoculating soil algae on the soil inside the pipe of step 1);

3) collecting soil inside the pipe of step 2) and separating the soil algae; And

4) measuring the soil toxicity endpoint of the isolated soil alga in step 3).

In addition,

a) soil algae; And

b) pipe

And a kit for measuring soil toxicity.

The present invention provides a mini-pipe in a soil sample contaminated with silver nanoparticles (AgNPs) in a greenhouse, and a soil microalgae Chlamydomonas reinhardtii ) was exposed for a certain period of time, and it was confirmed that soil pod species could be collected easily by collecting mini pipes including soil. In addition, the soil algae test species were separated from the soil collected from the mini-pipe using the growth medium to obtain soil algae test specimens, and the cell size, chlorophyll-a ester hydrolysis The esterase activity and lipid content were analyzed to confirm the soil toxicity.

Therefore, the present invention can easily collect soil algae for the measurement of soil toxicity on a wider scale than a laboratory scale, and can isolate a certain amount of soil algae test species according to the degree of soil pollution. Therefore, Measurement is possible.

FIG. 1 is a soil algae test species for measuring soil toxicity, and includes Chlamydomonas reinhardtii ) is prepared:
(A): Clamidomonas Reinharti preculture; And
(B): Clamidomonas lane strain cell suspension.
2 is a photograph showing the process of preparing a mini pipe for measuring soil toxicity using a mini-pipe:
(A): size of mini pipe; And
(B): UV sterilization process of mini pipe.
Figure 3 is a set of four soil groups prepared to measure soil toxicity using mini pipes in soils contaminated with silver nanoparticles (AgNPs) at various concentrations:
Control group: Uncontaminated soil group (noncontaminated soil group);
Low-Layer group: AgNPs low concentration (20 mg / kg) Contaminated soil was only in the upper layer;
High-Layer: AgNPs high concentration (80 mg / kg) contaminated soil was only in the upper layer; And
Low-Mix group: AgNPs low concentration (20 mg / kg) contaminated soil.
FIG. 4 is a photograph showing a process of installing a mini-pipe in four soil groups prepared for the measurement of soil toxicity using a mini-pipe and inoculating the soil algae test species on the soil inside the mini-pipe.
FIG. 5 is a photograph showing the process of collecting the mini-pipe including soil after exposure of the soil bird test species inoculated in FIG. 4 to 4 soil groups for 1 week, 4 weeks, 6 weeks and 8 weeks.
FIG. 6 is a photograph showing the growth of soil algae formed inside and outside the mini-pipe at 1 week, 4 weeks, 6 weeks, and 8 weeks after inoculation of the soil algae test species into the mini-pipe of the 4 soil group.
FIG. 7A is a photograph of soil algae formed in a mini pipe at 1 week, 4 weeks, and 8 weeks after inoculation of a soil algae test species in a mini pipe of a 4 soil group.
FIG. 7b is a photograph of soil algae formed on the outside of a mini pipe at 1 week, 4 weeks, 6 weeks and 8 weeks after inoculation of the soil algae test species in the mini-pipe of the 4 soil group.
Figure 8 shows that the soil algae test species was inoculated into the mini-pipe of the 4 soil group and then exposed to the inner mini-pipe and the soil algae isolated from the external soil of the mini pipe, a (Chlorophyll-a) of a mini-pipe.
FIG. 9 is a view showing the cell size of the soil algae test species isolated from the soil inside the mini pipe taken in the first week after exposure to the inside of the mini-pipe of the 4 soil group.
FIG. 10 is a view showing chlorophyll-a of a soil algae test species isolated from a soil inside a mini pipe collected in the first week after exposure to the inside of a mini-pipe of 4 soil group.
FIG. 11 is a graph showing the esterase activity of the soil algae test species isolated from the soil inside the mini pipe collected in the first week after exposure to the inside of the mini-pipe of the 4 soil group.
FIG. 12 is a view showing the lipid content of the soil algae test species isolated from the soil inside the mini pipe collected in the first week after exposure to the inside of the mini pipe of the 4 soil group.

Hereinafter, the present invention will be described in detail.

The present invention

1) installing a pipe in the soil;

2) Inoculating soil algae on the soil inside the pipe of step 1);

3) collecting soil inside the pipe of step 2) and separating the soil algae; And

4) measuring the soil toxicity endpoint of the isolated soil alga in step 3).

In the method of the present invention, the pipe of step 1) may be referred to as a "pipe", "tube" or "cylinder" Mini-tube "or" mini-cylinder ". The pipe may be formed in the shape of a pipe having a generally circular cross section and an empty interior. For example, the pipe may be formed of an empty pipe having a non-metallic or metallic material. However, the present invention is not limited thereto. Further, the depth of the pipe installed in the soil is preferably 15 to 25 mm deep from the surface of the soil to be installed, more preferably 17 to 23 mm deep, even more preferably 20 mm deep, It is not limited and may vary in depth depending on the state of the soil.

In the method of the present invention, the soil alga is preferably inoculated in an amount of 0.01 to 10 ml / cm ² in step 2), more preferably in an amount of 0.1 to 1 ml / cm ² , It is not.

In the method of the present invention, the soil alga in step 2) is preferably exposed to the soil for one to eight weeks after inoculation, more preferably to the soil for three to six weeks, more preferably to the soil for one to four weeks It is more preferable to be exposed, and it is most preferable to expose to soil for one week, but is not limited thereto.

In the method of the present invention, it is preferable that the soil alga in step 3) is separated by putting the growth medium in the collected pipe internal soil and floating the soil algae. For example, the growth medium is TAP medium, BBM medium or OECD medium But it is not limited thereto.

In this method, the soil toxicity end point of step 4) is selected from the group consisting of cell size, cell density, chlorophyll-a, esterase activity, oxidative stress and lipid content And more preferably at least one selected from the group consisting of cell size, chlorophyll-a, ester hydrolytic enzyme activity, and lipid content, but is not limited thereto.

In the method, soil algae are, for example, Chlorella vulgaris (Chlorella vulgaris), no stock ring Escherichia (Nostoc linckia ), Scenedesmus bijugatus , Synechococcus elongates , Pseudokirchneriella < RTI ID = 0.0 > Subcapitata , Chlorococcum infusionum , scenedesmus subspicatus or Chlamydomonas reinhardtii , and more specifically, Clamidomonas rehartii , but is not limited thereto.

In this method, the soil toxicity is preferably metal toxicity, and the metal includes all the metals on the periodic table, for example, mercury (Hg), lead (Pb), cadmium (Cd) And may be Cu, Ni, Zn, Mn, Co, Sn, or Ag. More specifically, .

In a specific embodiment of the present invention, the present inventors have found that a mini-pipe is installed in a soil sample contaminated with silver nanoparticles (AgNPs) in a greenhouse, and a soil micro- After reinharti was inoculated and exposed for a certain period of time, it was confirmed that it was possible to collect the soil algae test species easily by collecting the mini pipe including the soil. In addition, the soil algae test species were separated from the soil collected from the mini-pipe using the growth medium, and the cell size, chlorophyll-aester hydrolyzing enzyme activity, and lipid content were analyzed as the end point, The toxicity was confirmed. Thus, the method of measuring soil toxicity using pipes can be applied directly to the environment where the soil medium exists.

In addition,

a) soil algae; And

b) pipe

And a kit for measuring soil toxicity.

The soil algae may be selected from the group consisting of, for example, Chlorella vulgaris, No Stokingia, Senedesmus bijugatus, Cynekococus elongataceus, Pseudokirchneriella subcapitata, Chlorocachum infujiumum, Tata or Clamidomonas reinhardtii, more specifically Clamidomonas reharti, but is not limited thereto.

The pipe may be marked on its surface with the depth to be installed in the soil. The depth is preferably from 15 mm to 25 mm depth from the surface of the soil, more preferably from 17 mm to 23 mm depth, even more preferably 20 mm depth, but not limited to, Depth can be different.

The soil toxicity is preferably metal toxicity and the metal comprises all metals on the periodic table and can be, for example, mercury, lead, cadmium, chromium, copper, nickel, zinc, manganese, cobalt, tin, More specifically, it may be silver, but is not limited thereto.

The soil toxicity measurement preferably measures one or more selected from the group consisting of cell size, cell density, chlorophyll-a, ester hydrolase activity, oxidative stress, and lipid content, and is preferably measured in terms of cell size, chlorophyll- Hydrolytic enzyme activity, and lipid content, but it is not limited thereto.

In the present invention, a mini-pipe is installed in a soil sample contaminated with silver nanoparticles in a greenhouse, a clamidomonas rainhyti is inoculated as a soil algae test species on the soil inside the mini pipe, exposed for a predetermined period, The mini-pipe was collected, and soil collected from the collected mini-pipe was separated from the soil algae test species using a growth medium to obtain a soil algae test species. The cell size, chlorophyll-aester hydrolyzing enzyme activity and lipid content And thus it is useful as a soil toxicity measurement kit including the soil algae and the pipeline.

Hereinafter, the present invention will be described in detail with reference to examples.

The following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.

< Example  1> Preparation of Soil and Metal Samples for Soil Toxicity Measurement

Soil samples contaminated with metals were prepared for metal toxicity. Specifically, LUFA 2.2, a natural soil, was used for the soil, and silver nanoparticles (AgNPs) were selected as metal contaminants. AgNPs were in powder form and were used as metal samples by purchasing a material with size ≤ 100 nm (Sigma-aldrich, USA) containing 0.2% polyvinyl pyrrolidone. AgNPs were added to the soil, and the soil containing 20 mg / kg of AgNPs and the soil containing 80 mg / kg of AgNPs were prepared using a roller and a hand mixer.

< Example  2> Preparation of Soil Algae for Soil Toxicity Measurement

Chlamydomonas reinhardtii was cultivated for 4 days in a tris-acetate-phosphate (TAP) medium (Gorman and Levine, 1965) (24 ° C, 100 rpm, 16h: 8h = day : Night) (Fig. 1, (A)). The cell suspension was prepared by adjusting the absorbance value to 2.0 at a wavelength of 660 nm of a spectrophotometer (Libra S32 PC, Biochrom, UK), which was cultured for 4 days before (Fig. 1, )).

< Example  3> Mini-pipe (inside the soil) Exposed  Soil toxicity measurement using soil algae

In order to apply the soil toxicity measurement performed in the conventional laboratory scale to a wider scale environment, a mini-pipe was installed in the soil, and the soil toxicity was measured by inoculating and collecting the soil algae in the mini pipe.

Specifically, a thin mini-pipe made of a plastic material having a diameter of 20 mm was cut to have a total height of 30 mm, and a mark was left on the corresponding portion of 10 mm (Fig. 2 (A)). Next, the mini-pipe was prepared by sterilizing with ultraviolet rays for 30 minutes before the soil was installed (Fig. 2 (B)). A sterilized mini-pipe was installed on the soil prepared in Example 1 above. In this case, the minipipe is installed so that 20 mm is exposed to the ground under the ground and 10 mm is exposed to the soil ground based on the mark of the mini pipe. 1 ml of the soil alga Clomidomonas reinhate prepared in Example 2 was inoculated into the mini-pipe installed in the soil, exposed to the soil for 1 to 8 weeks, and the mini pipe containing the soil was collected . On the surface of the collected mini-pipe soil, the green algae were collected as spatula and placed in a 12-well plate. 5 ml of BBM medium was added to the soil for 24 hours in an incubator (24 ° C, 100 rpm , 16h: 8h = day: night). Twenty - four hours later, the soil supernatant from the soil was collected, transferred to a 15 - ml tube, centrifuged at 2,500 rpm for 5 minutes, and washed once with BBM medium to remove pesticide soil particles to obtain soil algae. Soil toxicity was measured using the obtained soil algae.

< Comparative Example  1> Measurement of soil toxicity using algae predominating in external minipipe soil

In order to apply the soil toxicity measurement performed at the conventional laboratory scale to a wider scale environment, a mini pipe was installed in the soil and the soil algae formed outside the mini pipe was collected to measure the soil toxicity.

Specifically, when the soil algae were exposed to the soil inside the mini pipe for 1 to 8 weeks in the same manner as described in Example 3, and the mini pipe containing the soil was collected, the outer surface of the mini pipe was green Growing soil algae were harvested in spatula, placed in 12-well plates, and 5 ml of BBM medium was added to float the soil algae in an incubator (24 캜, 100 rpm, 16 h: 8 h = day: night) for 24 h . Twenty - four hours later, the soil supernatant from the soil was collected, transferred to a 15 - ml tube, centrifuged at 2,500 rpm for 5 minutes, and washed once with BBM medium to remove pesticide soil particles to obtain soil algae. Soil toxicity was measured using the obtained soil algae.

< Comparative Example  2> Identification of soil algae formed inside and outside the mini pipe

In order to determine whether soil algae exposed to various concentrations of metal contaminated soil using a mini pipe can be applied to soil toxicity measurement method, a mini pipe was installed in soil contaminated with various concentrations of AgNPs, After the birds were inoculated and exposed for a certain period of time, the inside or outside soil of the mini pipe was collected and the growth of the soil algae formed on the soil was visually observed.

To reproduce the soil environment in which various AgNPs were exposed, concretely, as shown in FIG. 3, a polluted soil sample prepared in the above Example 1, a low concentration of AgNPs (20 mg / kg) was added to a pot in a greenhouse, Low-layer, high-layer, and low-mix groups were prepared by mixing contaminated soil samples and soil samples contaminated with AgNPs at a high concentration (80 mg / kg) And aging for 7 days.

group Control group Uncontaminated soil (non-contaminated soil) Low-layer group AgNPs low-concentration (20 mg / kg) contaminated soil was only present in the upper layer High-Layer Group High concentrations of AgNPs (80 mg / kg) Low-Mix group AgNPs low concentrations (20 mg / kg) contaminated soil

In the same manner as described in Example 3, 4 mini-pipes per 4 soil groups were installed on the stabilized 4 soil groups, and 1 ml of soil algae Clamidomonas reinhaine was inoculated to the soil to be exposed (FIG. 4). One mini-pipe was collected at 1, 4, 6, and 8 parking starts (FIG. 5), and the soil algae were collected from the soil inside the mini pipe -Well plate. In addition, the soil algae were taken in spatula from the outer surface layer of the mini pipe in the same manner as described in <Comparative Example 1>, and placed in a 12-well plate. Then, soil algae in the inner and outer surfaces of the mini-pipe were obtained in the same manner as in the above-described <Examples 3> and <Comparative Example 1>, and the algae were observed under a microscope.

As a result, as shown in Fig. 6, in the case of the inside of the mini-pipe, when the soil birds were exposed, the soil birds grew green inside the mini-pipes of the control group, the low-layer group and the low- Was greenish enough to be observed with the naked eye. On the other hand, in the case of outside the mini pipe, it was confirmed that the birds of 1, 4, 6, and 8 in the control group grew green enough to be visually observed.

In addition, as shown in FIGS. 7A and 7B, it was confirmed through microscopic observation that Clamidomonas lanehtia, which was artificially inoculated for soil toxicity measurement at the first exposure of the mini-pipe internal soil, dominated, and High- In the case of the layer group, it was confirmed that the concentration of AgNPs was relatively high and the growth of Clamidomonas lane Heart was inhibited due to toxicity. However, as the exposure progressed for a long period until the 8th week, it was confirmed that the Clamidomonas lanehtia species to be measured in the mini pipe of the control group did not grow conspicuously. On the other hand, in the fourth week of exposure, the green algae were visible inside the mini-pipe. However, microscopic observation revealed that the birds were naturally produced in addition to the artificially inoculated experimental species Clamidomonas lanehati (Fig. 7A). On the other hand, in the case of the external soil of the mini pipe, it was confirmed that the soil birds were grown green in the control group, the low-layer group and the low-mix group at the time of the first exposure, and the micro- It was confirmed that various kinds of soil algae, which are morphologically different from the Reinharti species, dominate, and it was confirmed that various kinds of soil algae dominate as in the first week of the fourth exposure (Fig. 7B).

Therefore, it has been found that it is difficult to select one kind of dominant species when soil contaminated soil is collected by separating soil contaminated with metals. On the other hand, when a mini pipe is used, it is easy to install a mini pipe in a desired soil, and a mini pipe containing the same amount of soil is collected after a certain period of exposure by inoculating the soil algae test species in the mini pipe, And it was confirmed that the inoculated soil algae test species can be easily harvested. Also, in order to minimize the influence of algae of naturally grown birds in the soil, the appropriate exposure period of the inoculated soil algae test species ranged from 1 to 4 weeks, and it was confirmed that one week of the above exposure period was most appropriate .

< Comparative Example  2> of the soil algae formed in the inside or outside soil of the mini-pipe As an endpoint,  Determination of Soil Toxicity by Chlorophyll-a Analysis

In order to confirm that the soil algae exposed to the soil inside the mini pipe was applicable to the soil toxicity evaluation in the proper exposure period confirmed by the <Comparative Example 1>, the artificial inoculation of the soil algae test paper was most dominant for 4 weeks The chlorophyll - a analysis was carried out by collecting the soil algae formed in the inside or outside of the mini - pipe of the fourth group and as the end point of the soil algae.

Specifically, in the same manner as described in <Comparative Example 1>, four weeks of Clamidomonas reinhine species were exposed to the soil inside the mini pipe of the control group, the low-layer group, the high-layer group, and the low- , And then soil and alien soil inside the mini pipe were collected to separate soil algae. Then, chlorophyll-a was separated from the soil algae and analyzed by measuring the intensity of the FL3 band filter (emission> 670 nm) of the flow cytometer.

As a result, as shown in Fig. 8, in the case of the soil inside the mini-pipe, the clomidomonas lehatti species, which is an artificially inoculated soil algae test species, dominated and the chlorophyll-a peak appeared narrow and constant by the clamidomonas lehatti, It was confirmed that chlorophyll-a was decreased according to the degree of contamination of AgNPs. On the other hand, in the case of mini-pipe external soil, various species of soil algae dominate, confirming that the chlorophyll-a peak is dispersed and not constant (FIG. 8). Therefore, it was confirmed that soil toxicity can be evaluated by using the soil algae test species exposed to the soil inside the mini pipe in an appropriate exposure period through the above results.

< Experimental Example  1> Mini-pipe inside soil Exposed  Algae To the end point  Identification of soil toxicity through cell size and cell density analysis

In order to measure the soil toxicity of the soil algae exposed to the soil inside the mini pipe at the proper exposure period determined through the above <Comparative Example 1>, the artificial inoculated soil algae test paper was classified into 4 groups And the cell size and cell density were analyzed as the end point of the soil algae.

Specifically, the same method as described in Comparative Example 1 was used to expose Clamidomonas reinhaine species to the soil inside the mini pipe of the control group, the low-layer group, the high-layer group, and the low-mix group for one week , The soil was collected and the soil algae test species was separated. Then, the cell size of the separated Clamidomonas reinhine species was analyzed by measuring the intensity of the forward scattered light (FSC) of a flow cytometer.

As a result, as shown in Fig. 9, it was confirmed that the cell size of the Clamidomonas reinhane species decreased in a concentration-dependent manner in the soil contaminated with AgNPs as compared with the control group (Fig. 9).

< Experimental Example  2> Mini-pipe inside soil Exposed  Algae To the end point  Determination of Soil Toxicity by Chlorophyll-a Analysis

In order to measure the soil toxicity of the soil algae exposed to the soil inside the mini pipe at the proper exposure period determined through the above <Comparative Example 1>, the artificial inoculated soil algae test paper was classified into 4 groups Of soil algae were collected and chlorophyll-a, an indicator related to growth, was performed as the end point of soil algae.

Specifically, the same method as described in Comparative Example 1 was used to expose Clamidomonas reinhaine species to the soil inside the mini pipe of the control group, the low-layer group, the high-layer group, and the low-mix group for one week , The soil was collected and the soil algae test species was separated. Then, chlorophyll-a was separated from the separated Clamidomonas reinhaine species and analyzed by measuring the intensity of the FL3 band filter (emission> 670 nm) of the flow cytometer.

As a result, as shown in FIG. 10, chlorophyll-a was decreased in a concentration-dependent manner in the soil contaminated with AgNPs as compared with the control group, and it was confirmed that the growth of Clamidomonas lane heterotypes was inhibited as the contamination by AgNPs was severe 10).

< Experimental Example  3> Mini-pipe inside soil Exposed  Algae To the end point Esterase (esterase) activity analysis to confirm soil toxicity

In order to measure the soil toxicity of the soil algae exposed to the soil inside the mini pipe at the proper exposure period determined through the above <Comparative Example 1>, the artificial inoculated soil algae test paper was classified into 4 groups And the esterase activities were analyzed by the end point of the soil algae.

Specifically, the same method as described in Comparative Example 1 was used to expose Clamidomonas reinhaine species to the soil inside the mini pipe of the control group, the low-layer group, and the low-mix group for one week, And the soil algae test species were separated. Then, the separated Clamidomonas reinhaine species was stained with a fluorescent dye and analyzed by a flow cytometer. In the case of the high-layer group, as shown in FIG. 6, the growth of the soil algae was inhibited to such an extent that it could be observed with the naked eye, and the analysis of the ester hydrolytic enzyme activity was excluded from the analysis of the end point using the dyeing technique. Calcein acetoxymethyl ester (Calcein-AM, Sigma-Aldrich Corp., Missouri, USA) was used as a fluorescent dye for confirming the activity of the ester hydrolase. The calcein-AM is not a fluorescent dye but is hydrolyzed by an esterase to convert it to a callaine, a green fluorescence (Teplova et al., 2010). The calcein-AM was dissolved in DMSO to prepare 500 [mu] M of Calcine-AM storage solution. A total of 500 μl of sample (10 μM) was prepared by adding 10 μl of Calcine-AM storage solution to 490 μl of the soil algae test species. The samples were maintained for 30 minutes at 37 캜 in dark room conditions for calcein-AM staining. The fluorescence intensity of the converted calcein was then measured using a flow cytometer. After 488 nm excitation wavelength, fluorescence intensity was analyzed by FL1 (500-560 nm band pass filter, green fluorescence). The results were analyzed using Flowjo V10. The geometric mean of the esterase activity of the soil algae test species was normalized, and the relative changes in esterase activity were evaluated.

As a result, as shown in Fig. 11, it was confirmed that the activity of the ester hydrolase of the species of Clamidomonas reinhini was markedly decreased in the soil group (low-layer group) contaminated with AgNPs in the surface layer as compared with the control group (Fig. 11 ).

< Experimental Example  4> Mini Pipe Inside Soil Exposed  Algae To the end point  Determination of soil toxicity through lipid content analysis

In order to measure the soil toxicity of the soil algae exposed to the soil inside the mini pipe at the proper exposure period determined through the above <Comparative Example 1>, the artificial inoculated soil algae test paper was classified into 4 groups And analyzed the lipid content as the end point of the soil algae.

Specifically, the same method as described in Comparative Example 1 was used to expose Clamidomonas reinhaine species to the soil inside the mini pipe of the control group, the low-layer group, and the low-mix group for one week, And the soil algae test species were separated. Then, the separated Clamidomonas reinhine species was stained with a staining sample and analyzed by a flow cytometer. In the case of the high-layer group, as shown in FIG. 6, the growth of the soil algae was inhibited to such an extent that it could be observed with the naked eye, and the end point analysis using the dyeing technique was impossible. Nile red (5H-Benso [a] phenoxazine-5-one, 9-diethylamine, Sigma, USA) was used as a dye for the lipid content confirmation. The soil algae test specimens were mixed to a final concentration of 1 mg / l of Nile red. Then, the cells were kept at room temperature and dark room for 10 minutes, and then analyzed using an FL2 band filter (yellow emission, 564-606 nm) using a flow cytometer. The results were analyzed using Flowjo V10, and the geometric mean of the lipid content of soil algae species was normalized and then averaged.

As a result, as shown in Fig. 12, it was confirmed that the triglyceride of Clamidomonas reinhithi species was significantly increased due to AgNPs contamination in both the low-layer group and the low-mix group as compared with the control group (Fig. 12).

Claims (14)

1) installing a pipe in the soil;
2) inoculating a soil algae on top of the soil in the pipe in the step 1), exposing the soil algae to the soil for 1 week to 4 weeks after inoculation;
3) collecting soil inside the pipe of step 2) and separating the soil algae; And
4) measuring the soil toxicity endpoint of the isolated soil algae in step 3).
The method for measuring soil toxicity according to claim 1, wherein the soil alga is inoculated in an amount of 0.01 to 10 ml / cm ² in the step 2).
The method for measuring soil toxicity according to claim 1, wherein the soil alga is inoculated in an amount of 0.1 to 1 ml / cm ² in the step 2).
delete delete delete The method according to claim 1, wherein the soil alga in step (3) is obtained by putting the growth medium in the collected soil in the pipe and floating the soil algae.
2. The method of claim 1, wherein the soil toxicity end point of step 4) is selected from the group consisting of cell size, cell density, Chlorophyll-a, esterase activity, oxidative stress and lipid content Wherein the method comprises the steps of:
The method of claim 1, wherein the soil alga is selected from the group consisting of Chlorella vulgaris , Nostoc Scandinavia species such as , for example, Linckia , Scenedesmus bijugatus , Synechococcus elongates , Pseudokirchneriella Subcapitata , Chlorococcum infusionum , scenedesmus subspicatus and Chlamydomonas reinhardtii . &lt; RTI ID = 0.0 &gt; 21. & lt ; / RTI &gt;
The method of claim 1, wherein the soil toxicity is metal toxicity.
The method of claim 10, wherein the metal is selected from the group consisting of Hg, Pb, Cd, Cr, Cu, Ni, Zn, (Co), tin (Sn), and silver (Ag).

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