KR20170089544A

KR20170089544A - Method for evaluating aquatic ecotoxicity using the root abscission of lemnoideae plant

Info

Publication number: KR20170089544A
Application number: KR1020160009895A
Authority: KR
Inventors: 한태준; 박지혜
Original assignee: 인천대학교 산학협력단
Priority date: 2016-01-27
Filing date: 2016-01-27
Publication date: 2017-08-04
Also published as: WO2017131281A1

Abstract

The present invention provides a method for evaluating water toxicity, comprising the steps of: introducing Spirodela polyrhiza and plants into a culture container containing a water sample and culturing the same under light irradiation conditions of 0 (dark condition) to 1 mol photon/m^2s; and measuring the number of abscised roots and the root abscission ratio of the Spirodela polyrhiza and the plants after cultivation. Since the method for evaluating water toxicity according to the present invention uses the number of abscised roots and the root abscission ratio of Spirodela polyrhiza and plants as parameters for evaluating the water toxicity and the Spirodela polyrhiza and the plants are cultured under the condition promoting the root abscission, it is possible to evaluate the water toxicity visually without a certain image analysis device, shorten the evaluation time of the water toxicity to about 2 days or less, have high sensitivity to water toxicity sources, and evaluate water contamination by various toxic substances.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for assessing toxicity of water using a method for assessing toxicity of a water-

The present invention relates to a method for evaluating a water quality toxicity, and more particularly, to a method for assessing toxicity of a water quality using a deviation of a frog and a root of a plant.

With the development of human activities and industry, new and potentially harmful chemicals are being produced and entering the Suez ecosystem. The development of modern chemistry not only contributes to the production of known useful chemicals, but it also produces about 1,200 new substances every day. Unless efforts are made to evaluate the risks to these toxic substances and to take legal action, the nature of the Suseo ecosystem, where material movement and conversion occurs rapidly, is not enough to prevent crises by industrial wastewater and city sewage containing thousands of chemicals each year And is exposed to the risk that it will face.

Contaminants that pollute rivers and lakes include industrial heavy metals and VOCs, agricultural herbicides, pesticides, and nitrogen and phosphorous compounds in vast domestic wastewater from populated cities. Traditionally, physicochemical methods have been used for this purpose. The physicochemical method is to quantitatively measure nitrates and phosphates that cause eutrophication such as dissolved oxygen (DO), biochemical oxygen demand (BOD), chemical oxygen demand (COD), turbidity, electrical conductivity and pH, Water pollution level. However, this method can not provide information on bioavailability, complex effects (such as elevation or antagonism) and further ecological meaning, along with the disadvantage of requiring long time, high cost, and expertise.

In order to overcome these limitations, international attention has been paid to the method of pollution diagnosis using surface organisms such as aquatic microorganisms, algae, underwater invertebrates and aquatic vascular plants. Recently, In evaluating the toxicity, it can be seen that the toxicity evaluation data by the indicator organism is borrowed. The method of pollution diagnosis using indicator organisms is based on the concept of bioindicator, which conventionally determines the concentration of pollutants in living organisms or judges the existence of species according to the pollution level of a habitat. Currently, the characteristic of biological organisms Which is a biomonitor method for the diagnosis of pollutants. The latter method can quantify the risk of individual or mixed substances present in the environment through various physiological changes of the organism or evaluate the potential influence of the substance, so that the signs can be grasped before the environmental pollution is wide-spread It has the advantage of finding an ecological meaning.

In recent years, the use of aquatic plants in aquatic ecosystems has been highlighted, and interest in methods for assessing toxicity using aquatic plants is increasing. Aquatic plants are the primary producers of aquatic ecosystems and are the habitats of plant and plankton, invertebrates and fish, and have the ability to absorb and purify many organic materials.

Among the aquatic plants in particular, frogs are recommended as testosterone toxicity and phototoxicity test models. Plants belonging to the family Lepidoptera ( Lemnoideae ) are the smallest perennial aquatic plants. They live in water, lakes, ponds and other water of constant flow rate or slow flow rate. They live on the water surface or live in water. Duckweed plants are divided into five genera, such as in duckweed (Spirodela), in some duckweed (Lemna), jombun in duckweed (Wolffia), Landoltia and Wolffiella, is believed to be about 30 paper distribution throughout the world.

To evaluate the water toxicity evaluation method using conventional frog rice registered in the International Organization for Standardization (ISO), the US Environmental Protection Agency (EPA), and the Organization for Economic Cooperation and Development (OECD), the frog rice was cultured in a water sample, frond number, frond area, population growth rate, fresh weight, dry weight, or pigment content of a plant. However, the method of evaluating the water quality toxicity using the conventional frog rice has a problem that the incubation time is about 7 days in order to secure an effective change amount, sensitivity to toxic source is low, and it needs to be improved.

The present invention has been made in view of the background of the prior art, and it is an object of the present invention to provide a new water toxicity evaluation method having a short evaluation time and high sensitivity.

The inventors of the present invention have continuously studied water quality toxicity evaluation methods using diverse characteristics of frogs and plants, and have found that ecological toxicity, especially water quality It was confirmed that the time required for toxicity evaluation is short, sensitivity is excellent, and various toxic substances can be evaluated. Thus, the present invention has been completed.

In order to solve the above-mentioned problems, the present invention provides a method for culturing a human body, comprising the steps of: feeding frozen rice and plants into a culture container containing a water sample and culturing under light irradiation conditions of 0 (dark condition) to 1 μmol photon / m 2 s; And a step of measuring the number of roots removed or the rate of root removal of the frozen rice and the plant after cultivation is completed.

The water sample is preferably composed of at least two diluting raw water different in raw water and dilution water, and the diluted raw water is prepared by diluting the raw water with Steinberg medium. In addition, the water sample preferably further comprises a culture medium containing no frog and a toxic substance which inhibits the removal of roots of the plant as a control for raw water. The toxic material may be selected from the group consisting of silver (Ag), aluminum (Al), arsenic (As), cadmium (Cadmium), cobalt (Co), chromium (Cr) And may be at least one metal selected from the group consisting of copper, copper (Fe), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) . In addition, the control group is preferably Steinberg medium. The pH of the water sample is preferably 5 to 11.

In addition, the incubation temperature of the step of culturing is preferably 20 to 35 占 폚.

The method for evaluating the water quality toxicity according to the present invention uses frog rice and the number of roots leaving the plant or the rate of roots removal of the plant as parameters for evaluating the water quality toxicity and cultivates the frog and the plants under the condition promoting the root removal, It is possible to evaluate the toxicity of water by the naked eye, shorten the time to judge the toxicity of water quality within about 2 days, have high sensitivity to the water toxicity source, and can evaluate the water contamination by various toxic substances.

Figure 1 shows the root abandonment rate of the mulberry plants according to the passage of culture days under various light irradiation conditions.
Fig. 2 shows the root removal rates of the plants in the mulberry forest according to the culture days under various culture temperature conditions. In the experiment of FIG. 2, the light irradiation condition was a dark condition (light irradiation amount: 0 μmol photon / m 2 · s), and the pH of the culture was about 7.0.
Fig. 3 shows the root removal rate of the mulberry plants according to the passage of culture days under various pH conditions. In the experiment of FIG. 3, the light irradiation conditions were as follows (light irradiation amount: 0 μmol photon / m 2 · s) and the incubation temperature was 25 ° C.

The present invention relates to a water toxicity evaluation method having a short discrimination time, simple operation, and high sensitivity to various toxic substances. The water toxicity evaluation method of the present invention comprises the steps of: And culturing under a predetermined light irradiation condition; And a step of measuring the number of roots removed or the rate of root removal of the frozen rice and the cultivated plant. Hereinafter, the method for evaluating the water toxicity according to the present invention will be described by dividing it into components.

A water sample

The water body sample refers to a sample in which water occupies a major volume. The water body sample according to the present invention is collected from seawater, a lake, a wastewater, discharged water, sewage, sludge leached water, soil leached water, Include one sample.

The first water sample (hereinafter referred to as "raw water") was prepared by diluting the raw water with a liquid not containing a toxic substance such as artificial pond or water and diluting it with at least two or more, preferably four or more concentrations. . In this case, the dilution method to be used is not particularly limited. For example, the dilution method used is, for example, a semi-dilution method [100% (raw water), 50% (diluted with 1/2 concentration of raw water), 25% ), 12.5% (diluted with 1/8 concentration of raw water), 6.25% (diluted with 1/16 concentration of raw water)]. The liquid for diluting the raw water is not limited to the kind that can be compatible with the frog rice and the cultivation of the frog, and does not contain toxic substances that inhibit the separation of the frog and the root of the plant, and specifically, water, Steinberg medium, and Hoagland's medium. Among these, Steinberg medium is preferable in consideration of smooth cultivation of frog rice and plants.

In addition, the water sample preferably further comprises a culture medium containing no frog and a toxic substance that inhibits the removal of roots of the plant as a control of the raw water. The culture medium constituting the control group can be selected from a variety of known liquid mediums and the like. Specifically, there are Steinberg medium or Hoagland's medium, and considering the smooth cultivation of frog rice and plants It is preferably a Steinberg medium. Examples of the poisonous substances that inhibit the removal of roots of the frogs and plants include silver (Ag), aluminum (Al), arsenic (As), cadmium (Cad), cobalt (Co) , Chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) &Lt; RTI ID = 0.0 > and / or < / RTI >

In addition, the water sample including the raw water, the diluted raw water or the control water of the raw water may be adjusted before the water sample is accommodated in the culture container or after being accommodated in the culture container and then the pH is adjusted to a predetermined range . Specifically, the pH of the water sample is preferably adjusted to 5 to 11, more preferably adjusted to 6 to 11. When the pH of the water sample is less than 5, the separation of frog and the root of the plant during the cultivation process may be due to the degradation of the frog and the plant due to the acidity of the culture medium, which may deteriorate the reliability of the evaluation of the water quality toxicity.

A culture container

The culture container is used for storing a water sample, a frog and a plant, and then putting it in an incubator to cultivate a frog and a plant. The shape of the culture container is not limited. For example, a well plate, an Erlenmeyer flask ). Preferably, the well plate includes at least six wells. In one well, an artificial medium containing no toxic substance is added as a control group to cultivate frogs and plants. In the remaining five wells, raw water and four Add diluted water and cultivate frozen rice and plants. Further, considering that the water plate toxicity of various raw water can be evaluated at one time, it is more preferable that the well plate includes at least 12 wells, most preferably at least 24 wells. The size of the Erlenmeyer flask is not particularly limited, but is preferably 200 ml to 100 ml.

Ducks and Plants

Duckweed and plants are subspecies of the genus Agate , widely distributed throughout the world, and about 30 species of 6 genera are known. polyrhiza) and some duckweed (Lemna paucicostata) 2 paper grows. In addition, the species belonging to the mulberry is Lemna gibba , Lemna minor , Lemna minuta , Lemna trisulca , Lemna valdiviana, Lemna and perpusilla . Ducks and plants consist entirely of three parts: fronds (2 to 3 long split leaves), connective roots and roots. In the present invention, as a biomarker for evaluation of water toxicity, frog rice and plants belonging to aquatic plants, preferably Lemna gibba , Lemna minor , and Lemna paucicostata .

Cultivation of frog rice and plants

The culture container containing the frozen rice and the plant and the water sample is transferred to an incubator, and then the frog and the plant are cultured under the predetermined light irradiation condition. At this time, the incubation time of the frozen rice and the plant is not limited but is preferably at least 24 to 72 hr, more preferably 36 to 60 hr in order to ensure the reliability of the evaluation of the water quality toxicity. It is preferable that the light irradiation amount in the cultivation of frogs and plants is 0 (dark condition) to 1 mu mol photon / m 2 · s and 0 (dark condition) to 0.5 μmol photon / m 2 , More preferably 0 (dark condition) to 0.1 mu mol photon / m < 2 > s. The separation of frogs and roots from plants is carried out smoothly under the conditions of darkness. When the amount of irradiation is more than 1 μmol photon / ㎡ · s when cultivating frogs and plants, irrespective of the kind and amount of toxic substances present in the water sample It is difficult to assess the toxicity of water because the removal of frogs and roots of the plants is not significant. The culture temperature at the time of cultivation of frozen rice and plants is preferably 20 to 35 ° C, more preferably 25 to 30 ° C, from the viewpoint of ensuring smooth root separation. However, optimal culture conditions (eg, incubation time, light dose, incubation temperature, etc.) for the removal of roots of frogs and plants may be somewhat changed depending on the species of frogs and plants.

Ducks and roots of plants Number of exits Or root bounce rate and assessment of water quality toxicity

When frog rice and plants are cultured for 48 hours under a specific culture condition in a culture container containing a water sample, the number of leaves of the frog and the removal rate of the root of the frog or the plant varies depending on the kind or content of the toxic substance in the water sample. After the cultivation was completed, the number of roots removed from the foliage and plants was measured, and the roots removal rate can be calculated by the following equation.

In the above formula, Rt represents the total number of roots of the frog and the plant contained in the culture container before culturing, and Ra represents the number of roots removed from the frog and the plant contained in the culture container after culturing.

Water quality toxicity of the water sample can be evaluated by using the number of leaves of the frog and the number of leaves of the plant or the rate of removal of the roots. Specifically, the water toxicity of a water sample is evaluated by comparing the number of roots removed from frog and plant cultivated in raw and diluted raw water to the number of roots removed from frog and plant cultivated in a control without toxic substances. In addition, the water quality toxicity of the water sample is evaluated by comparing the rate of removal of frog and plant roots from raw and diluted raw water with that of frogs and plants that were cultivated in a control without toxic substances. At this time, quantitative values for water quality toxicity are expressed as Half maximal effective concentration (EC ₅₀ ) to No Observed Effect Concentration (NOEC) values.

Half maximal effective concentration (EC ₅₀ ) is the ratio of the number of roots leaving the roots of the frog and the plant or the rate of roots leaving the roots of the frogs and plants that have been removed in the control group without toxic substances by 50% Means the concentration of the sample, expressed as the dilution rate of the raw water in the case of an unknown sample containing a single toxic substance and in the case of an unknown source containing a plurality of toxic substances, expressed as a specific concentration of a single toxic substance. The no effect concentration was the concentration of the water sample which was maintained at a level that did not significantly differ from the number of leaves of the frog and the number of leaves of the root of the frog or the plant or the rate of removal of the root of the frog which was cultivated in the control group which did not contain toxic substances Meaning, as in the case of half effective concentration, in the case of a water sample containing a single toxic substance, it is expressed as a dilution rate in the case of an unknown water indicated by a specific concentration and containing a plurality of toxic substances. On the other hand, since it means that the larger the size of the half effective concentration and the larger the toxicity of the actual water sample is, the more toxic unit (Toxic Unit, TU) .

TU = 100 / half effective concentration (%)

In addition, the toxicity of the water sample can be evaluated by the following equation.

In the above formula, A represents the number of leaves of the frog and the number of roots leaving the plant or the rate of removal of the roots in the experimental group (the water sample containing the toxic circle), and B represents the number of roots of the frog in the control group Or root abandonment rate.

Use of the method for evaluating the water toxicity according to the present invention

Toxic substances that can be diagnosed by the method of the present invention include silver (Ag), aluminum (Al), arsenic (As), cadmium (Cad), cobalt A heavy metal such as chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) There is. The water quality toxicity assessment method of the present invention can also be useful for quickly determining the sludge dilution factor to be taken in order to prevent negative impacts on the ecosystem before throwing the sewage and wastewater sludge. The method of the present invention can not detect an unknown toxic substance in a water body when the toxic substance in the water pollution measurement method depending on the conventional chemical analysis method is introduced into the water body and further, Which can not be predicted at all by the effect of water pollution.

Hereinafter, the present invention will be described more specifically by way of examples. However, the following embodiments are intended to clearly illustrate the technical features of the present invention, and do not limit the scope of protection of the present invention.

1. Establishment of optimal culture conditions for the removal of roots of frogs and plants

(1) Pretreatment culture of frog rice and plants

Lemna minor , a frog rice and a frog rice plant, was placed in a water tank of 25 cm width, 10 cm length and 15 cm height containing 1.5 L Steinberg medium (pH 7 ± 0.2), transferred to an incubator, The culture was stopped at a temperature of 25 ° C while irradiating light with a dose of 20-30 μmol photon / ㎡ · s (light cycle is 24 hours continuous light condition). At this time, a white fluorescent lamp was used as a light source. To prevent contamination with the outside, the slides were covered with a clear plastic lid and the whole amount of Steinberg medium (pH 7 ± 0.2) was changed every 7 days.

Table 1 shows the components and concentrations included in the Steinberg artificial medium used in the examples of the present invention. As shown in Table 1, the artificial medium contains a total of 5 stock solutions, each of which consists of a solution containing an inorganic component or an organic component necessary for culturing the frog and the plant.

Stock solution Constituting the stock solution
Types of Ingredients The spheres in the stock solution
Concentration of gender component (g / l) One liter per artificial badge
Tok solution occupies
Volume (ml / l) Ⅰ KNO ₃ 17.5 20 K ₂ HPO ₄ 4.5 KH ₂ PO ₄ 0.63 Ⅱ MgSO ₄ 7H ₂ O 5 20 Ⅲ _{Ca (NO 3) 2 4H 2} O 14.75 20 IV H ₃ BO ₃ 0.12 One ZnSO ₄ 7H ₂ O 0.18 Na ₂ MoO ₄ 2H ₂ O 0.044 MnCl ₂ 4H ₂ O 0.18 V FeCl ₃ 6H ₂ O 0.76 One Na ₂ -EDTA ₂ H ₂ O 1.5

(2) Culture of frog rice and plants

Pretreatment Cultured mugwort rice plants were selected from healthy individuals with roots growing more than 10 mm and 2-3 fronds and 1 root. The selected individuals were put into a 6-well plate containing the culture solution, 10 per well, covered with the well, and wrapped around the 6-well plate with a paraffin film to seal. Subsequently, the bryophytes were incubated for 5 days under various conditions of light exposure, various incubation temperatures and various pH conditions. At this time, Steinberg medium (pH 7 ± 0.2) was used as a culture medium, and the pH of the culture medium was adjusted using 1 M aqueous HCl solution or 1 M aqueous NaOH solution. Then, the dark condition was formed by irradiating with a predetermined light irradiation amount using a white fluorescent lamp or by using aluminum foil. Then, the number of roots removed from the plants of a bryophyta according to the days of culture was visually measured, Respectively.

Figure 1 shows the root abandonment rate of the mulberry plants according to the passage of culture days under various light irradiation conditions. In the experiment of FIG. 1, the light cycle was 24 hours continuous light condition, the incubation temperature was 25 ° C, and the pH of the culture was about 7.0. As shown in Fig. 1, when the light irradiation condition was dark, that is, when the light irradiation amount was 0 mu mol photon / m < 2 > s, the root removal rate of the rice dwarf plants was increased in proportion to the day of cultivation, Showed a root ablation rate of 90% or more. On the other hand, when the light dose is very weak (for example, when the light dose is 5 μmol photon / ㎡ · s), the rate of root abandonment of the duckweed plant is drastically decreased and the light dose is 25 μmol photon / ㎡ · S, the roots of the plants in the mulberry borealis did not substantially escape even though the incubation time increased.

Fig. 2 shows the root removal rates of the plants in the mulberry forest according to the culture days under various culture temperature conditions. In the experiment of FIG. 2, the light irradiation condition was a dark condition (light irradiation amount: 0 μmol photon / m 2 · s), and the pH of the culture was about 7.0. As shown in FIG. 2, when the incubation temperature was 15 ° C., the rate of removal of roots from the rice plants was not greatly increased even when the incubation time was increased. On the other hand, when the incubation temperature was 25 to 30 ° C., The rate of root ablation was increased in proportion to the incubation time.

Fig. 3 shows the root removal rate of the mulberry plants according to the passage of culture days under various pH conditions. In the experiment of FIG. 3, the light irradiation conditions were as follows (light irradiation amount: 0 μmol photon / m 2 · s) and the incubation temperature was 25 ° C. As shown in FIG. 3, when the pH of the culture medium was 3, the root removal rate of the mulberry plants was highest until the third day of cultivation, because the pH of the mulberry juice was lowered due to the acidity of the culture medium. When the pH of the culture was 5 ~ 11, it showed almost similar root removal rate on the 3rd day of culture and showed the highest root removal rate in the culture solution having pH 7 on the fourth day of culture.

Optimum cultivation conditions for the removal of roots of the plants belonging to the genus Fagopyrum were found to be dark condition at 25 ± 2 ℃ and 7 ± 0.2 at the light condition.

2. Assessment of water toxicity of toxic substance standard solution using frozen rice and plant roots removal rate

(1) Preparation of toxic substance standard solution

A single metal toxic substance arsenic (As), cadmium (Cd), 6 to prepare a chromium (Cr ⁶ ^+), copper (Cu), mercury (Hg), and are toxic, including silver (Ag), each substance standard solution. In addition, a toxic substance standard solution was diluted with a Steinberg medium by a half-dilution method to prepare a toxic substance standard solution diluted to 50%, 25%, 12.5%, and 6.25% of the initial concentration. At this time, the pH of the toxic substance standard solution and diluted toxic substance standard solution was adjusted to 7.0 ± 0.1 using 1 M aqueous hydrochloric acid solution or 1 M sodium hydroxide aqueous solution.

(2) Toxic substances In the standard solution,

Pretreatment Cultured mugwort rice plants were selected from healthy individuals with roots growing more than 10 mm and 2-3 fronds and 1 root. Then, 10 ml of each of the control solution (Steinberg medium), the toxic substance standard solution and the diluted toxic substance standard solution was added to each well of a 6-well plate, and 10 selected wells were added to the wells. The plate was wrapped around a 6-well plate with a paraffin film and sealed. Then, the 6-well plate was transferred into an incubator, and cultured at a temperature of 25 ° C for 48 hours under a dark condition in which light irradiation was interrupted. The experiment under the same condition was repeated three times in total.

(3) Determination of the rate of removal of roots from the plants of aquaculture and evaluation of water toxicity of single metal toxic substances

When 24hrs and 48hrs were elapsed after the cultivation of duckweed rice plants, the number of roots removed from duckweed rice plants was visually observed and the rate of root removal of duckweed rice plants was calculated. Then, the half effective concentration (EC ₅₀ ) of each single metal toxic substance was calculated by using point estimation techniques based on the root removal rate of the plants in the mulberry. Table 2 below shows the half-effective concentration (EC ₅₀ ) of each of the single metal toxins calculated on the basis of the root removal rate of the mulberry leaves after culturing the mulberry leaves for 24 hours and 48 hours.

Metal toxic substance EC ₅₀ (mg / L) based on Lemna minor root removal rate after 24 hours of incubation EC ₅₀ (mg / L) based on Lemna minor root ablation rate after 48 hours of incubation AS 4.630 3.950 CD > 5 0.127 Cr ⁶ ⁺ > 5 2.419 Cu 0.106 0.125 Hg 0.098 0.070 Ag 0.043 0.018

As shown in Table 2, the time required for assessment of water toxicity was greatly shortened and the response sensitivity to single metal toxicants was very high when the roots removal rate of the duckweed plant was used as a reference parameter for evaluation of water quality toxicity.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments but should be construed as including all embodiments belonging to the claims attached hereto.

Claims

Introducing frozen rice and plants into a culture container containing a water sample and culturing under light irradiation conditions of 0 (dark condition) to 1 mu mol photon / m < 2 > And
And measuring the number of roots removed or the rate of root removal of the frozen rice that has been cultivated and the plant.

The water sample according to claim 1, wherein the water sample is composed of at least two dilution raw water having different raw water and dilution multiples,
Wherein the dilution raw water is prepared by diluting raw water with Steinberg medium.

3. The method according to claim 2, wherein the dilution raw water is diluted by a half-dilution method.

[Claim 3] The method according to claim 2, wherein the water sample further comprises a culture medium containing no frog and a toxic substance that inhibits the removal of roots of plants as a control group of raw water.

5. The method of claim 4, wherein the toxic material is selected from the group consisting of Ag, Al, Arsenic, Cadmium, Co, Cobalt, ), Copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb), and zinc Wherein the at least one metal is one or more metals.

5. The method according to claim 4, wherein the control group is Steinberg medium.

The method according to claim 1, wherein the pH of the water sample is 5 to 11.

The method according to claim 1, wherein the culture temperature of the culturing step is 20 to 35 占 폚.