KR101278945B1 - Storage solution of Ulva as biomarker for evaluating water toxicity and method for storing Ulva - Google Patents

Abstract

The present invention is a solution formed by dissolving an alkali metal salt of hydrazonic acid in a brine having a salt concentration of 25 ~ 45 ‰, the concentration of the alkali metal salt of hydrazonic acid in the solution is 0.01 ~ 1 It provides a blue storage solution, characterized in that the ppb. In addition, the present invention is a method for storing the seaweed used as a biomarker of water toxicity evaluation, comprising the steps of (a) preparing the seaweed; And (b) storing the prepared seaweed in a container containing the storage solution of the seaweed and storing it under refrigerated conditions at 0 ° C to 10 ° C.
In the case of storing the seaweed using the storage solution of the seaweed according to the present invention, unlike the conventional storage method, it does not require the light irradiation dose, the air generator and the exchange of a certain range of intensity range, and thus continuously maintain the cultured seaweed as a test organism There is an advantage to solve the economic cost and labor shortage. In addition, the experiment can be performed at any time the water toxicity evaluator desired date, saving time. As a result, it is possible to increase the utilization of the method for evaluating water toxicity using already developed seaweed.

Description

Storage solution of Ulva as biomarker for evaluating water toxicity and method for storing Ulva}

The present invention relates to a storage solution and a storage method of the green sea used as a biomarker of water toxicity evaluation, more specifically, a storage solution of green sea containing an alkali metal salt of hydrazonic acid in a specific concentration range and The present invention relates to a method of storage of greenery, characterized in that the storage solution is treated with greenery.

Recently, as a method for improving the water pollution problem, which is mentioned as one of the environmental problems, there is increasing interest in the method of improving the water quality and monitoring the leakage of harmful substances in rivers and the like. As a method of detecting toxic substances that may enter the water system, there are generally physicochemical methods and biological methods using living organisms as biomarkers of toxic substances. Among them, physicochemical methods use expensive physicochemical equipment, etc. There are inherent shortcomings, and the development of biological toxicity measurement techniques and devices has been required to compensate for them, and in particular, research on biological species, which is the core of biological toxicity measurement techniques and devices, is being actively conducted. However, previously developed organisms absolutely require more advanced methods for storing species, due to limitations in the distribution of test organisms, difficulties with continuous cultivation, supply and maintenance of test organisms.

On the other hand, among the species used in biological water quality measurement techniques, the green sea has a variety of habitat environment, and has the advantage of easy sample acquisition and cultivation, and in this regard, Korean Patent Nos. 10-0653100 and 10-0653101 A method for evaluating water toxicity using a spore formation rate of green seaweed is disclosed. However, the storage method for the use of the green sea for the measurement of water toxicity is shown in a 48-hour cycle with a 24-hour flow using an air generator at a temperature of 30 to 200 μmol / m 2 · s and a temperature of 10 ° C. There was a need to exchange the entire culture.

SUMMARY OF THE INVENTION The present invention was derived to solve the conventional problems, and an object of the present invention is to obtain a seaweed in a state capable of reacting stably and repetitively to water toxic substances without requiring a medium exchange and using an air generator. It is to provide a blue storage solution that can be stored.

It is another object of the present invention to provide a method for storing the seaweed in a state that can be used for the assessment of water toxicity using the storage solution of the seaweed.

The inventors of the present invention screened a variety of bioactive inhibitors in the seaweed, and studied the effect of the storage of the seaweed, as a result, when the seaweed treated with an alkali metal salt of hydrazonic acid (hydrazonic acid) in a specific concentration range in the seaweed The present invention was completed, stating that it reacts stably and reproducibly to toxic substances in water.

In order to solve the above object of the present invention, the present invention is a solution formed by dissolving an alkali metal salt of hydrazonic acid in saline having a salt concentration of 25 ~ 45 ‰, hydrazonic acid in the solution It provides a blue storage solution, characterized in that the concentration of the alkali metal salt of) 0.01 ~ 1 ppb.

In addition, in order to solve the other object of the present invention, the present invention is a method for storing the seaweed used as a biomarker of water toxicity evaluation, (a) preparing the seaweed; And (b) storing the prepared seaweed in a container containing the above-described seaweed storage solution and storing it under refrigerated conditions at 0 ° C to 10 ° C.

In the case of storing the seaweed using the storage solution of the seaweed according to the present invention, unlike the conventional storage method, it does not require the light irradiation dose, the air generator and the exchange of a certain range of intensity range, and thus continuously maintain the cultured seaweed as a test organism There is an advantage to solve the economic cost and labor shortage. In addition, the experiment can be performed at any time the water toxicity evaluator desired date, saving time. As a result, it is possible to increase the utilization of the method for evaluating water toxicity using already developed seaweed.

Figure 1 is a photograph showing a state in which the seaweeds in the form of circular slices are stored in a plastic storage container.
2 is a graph showing the change of the spore formation rate according to the storage conditions of the greenery.
FIG. 3 is a graph showing the results of toxic reactions against green standard stored in 0.1 ppb sodium azide solution for 4 weeks and blue used immediately without storage as a control.

Hereinafter, the present invention will be described in detail.

Green storage solution

The storage solution of the green according to the present invention is a solution formed by dissolving an alkali metal salt of hydrazonic acid (HN ₃ ) in brine.

Alkali metal salts of hydrazonic acid, which is one component of the green stock solution according to the present invention, are generally known to inhibit physiological activity, and alkali metal salts of hydrazonic acid used in the present invention are Lithium azide (LiN ₃ ), sodium azide (NaN ₃ ), potassium azide (potassium azide, KN ₃ ), etc. are not greatly limited, but the greening environment and the brine component In consideration of the compatibility with the sodium azide (sodium azide, NaN ₃ ) is preferred. Alkali metal salt of hydrazonic acid (hydrazonic acid, HN ₃ ) in the storage solution of the green according to the present invention is a concentration of 0.01 ~ 1 ppb (parts per billion), preferably a concentration of 0.05 ~ 0.5 ppb, most preferably 0.05 ~ It is limited to a concentration of 0.2 ppb. When the concentration of the alkali metal salt of hydrazonic acid (HN ₃ ) is less than 0.01 ppb, the effect of addition of the alkali metal salt of hydrazonic acid (HN ₃ ) is insufficient, and 1 ppb If it exceeds, it damages the green sea due to the toxicity of alkali metal salt of hydrazonic acid (HN ₃ ).

Brine, which is a component of the storage solution of the green sea according to the present invention, has a salt concentration of 25 to 45 ‰, preferably a salt concentration of 30 to 40 ‰. It should be suitable for keeping the seaweed intact. In addition, the brine is preferably sodium chloride (NaCl), magnesium sulfate (MgSO ₄ ), magnesium chloride (MgCl ₂ ), calcium chloride (CaCl ₂ ), potassium chloride (KCl), sodium bromide, sodium nitrate (NaNO ₃ ), Sodium hydrogen carbonate (NaHCO ₃ ), boric acid (H ₃ BO ₃ ), sodium silicate (Na ₂ SiO ₃ ), strontium nitrate [Sr (NO ₃ ) ₂ ], and disodium hydrogen phosphate (Na ₂ HPO ₄ ). Seawater not contaminated with the brine used in the present invention may be used, and artificial seawater may be preferably used. Artificial seawater (artificial seawater) refers to salt solutions reorganized in the same proportion to the proportion and concentration of a large amount of inorganic components by analyzing the seawater components. In the present invention, various commercial products known as artificial seawater may be used, and the kind thereof is not limited to a large extent, and double OTT's artificial seawater is preferable for the storage of the green sea.

Storage method of green

The method of storage of the seaweed according to the present invention is characterized by maintaining its reproductive function to act as a biomarker when the seaweed is used in the evaluation of water toxicity, specifically (a) preparing the seaweed; And (b) storing the prepared green sea in a container containing the above-described storage solution and storing it under refrigerated conditions of 0 to 10 ° C, preferably 2 to 6 ° C.

In the present invention, the seaweed ( Ulva ) used as a biomarker in the evaluation of water toxicity is not greatly limited in its type, for example, the intestinal seaweed ( Ulva) intestinalis ), flat blue ( Ulva compressa ), green leaf ( Ulva linza ), spiny blue ( Ulva prolifera ), lattice blue ( Ulva clathrata ), green onion ( Ulva lactuca), hole galparae (Ulva pertusa ), among them the hole reddish ( Ulva pertusa ) is preferred in view of its sensitivity to toxic substances. In addition, when evaluating the actual water toxicity, the green leaves are used in the leaf part corresponding to the leaves, and are cut into specific shapes such as round and square to facilitate the measurement of spore formation rate. It is preferable to have a shape cut into a circle in the step (a) to avoid and to facilitate storage.

In the method of storage of seaweed according to the present invention, the shelf life of the seaweed is not greatly limited, but in order for the seaweed to be used as a reliable biomarker in the evaluation of water toxicity, it is generally used at 80% or more of the spore formation rate exerted at the initial state of extraction. The storage period of the step (b) is 4 weeks or less, preferably 3 weeks or less, since the corresponding sporulation rate should be exhibited. When the seaweed is stored for three weeks by the method of storage of the seaweed according to the present invention, the seaweed may exhibit a spore formation rate corresponding to 90% or more of the original spore formation rate.

Method for Evaluating Water Toxicity Using Archived Laver

To understand how to use seaweed as a biomarker to assess the toxicity of water quality, you first need to look at the life history of the seaweed. Life cycle of the green sea "① spore formation (asexual reproduction) and spore release at the edge of the spores (green) ⇒ ② spores are released as spores and spouse formation (oil reproduction) ⇒ ③ male and female spouses are joined Formation and splicer growth to form a spore body (green sea) ", such as, the water quality toxicity evaluation method using the spore formation rate of the seagrass is using the step ① above. More specifically, in a green, uncontaminated environment, ① stage of reproduction (formation and release of spores) proceeds, and the color change of the leaf body called leaf appears. At first, the light greenish leaf body begins to reproduce (asexual reproduction). It turns dark olive and finally the spores turn white. However, in contaminated or toxic waters, fertility decreases and the degree of color change is reduced. Based on this principle, after exposing the green leaf to the toxic source, the leaf (leaf) is visually observed or the image is captured by a camera, and a scale magnifier is used to measure the area of the leaf showing the color change to white. In addition, the degree of toxicity of the toxin is indicated by comparison with the area of white leaf in the control group that does not contain the toxin.

The water toxicity of the water sample is assessed by comparing the spore formation rate of the green seedlings cultured in raw water and the diluted water sample with the rate of spore formation of green seedlings cultured in a control solution that does not contain toxic substances. sporulation inhibition concentration (EC ₅₀ ) to No Observed Effect Concentration (NOEC). The median sporulation inhibition concentration (EC ₅₀ ) refers to the concentration of a water sample that shows a 50% reduction in sporulation rate compared to the spore formation rate in a control solution that does not contain toxic substances. For water samples, it is expressed as a specific concentration of a single toxic substance, and for unknown raw water containing multiple toxic substances, it is expressed as the dilution rate of the raw water. The no-affected concentration refers to the concentration of water samples that show a level of spore formation that is not significantly different from the rate of spore formation in a control solution that does not contain toxic substances. For water samples, it is expressed at a certain concentration, and for unknown raw water containing a large number of toxic substances, it is expressed as the dilution rate.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the protection scope of the present invention is not limited by the following examples.

Table 1 shows the components and concentrations of salts contained in OTT's artificial seawater, which is artificial brine used in the following Preparation Examples, Examples, and Comparative Examples of the present invention, and the salt concentration of OTT's artificial seawater is 35 ‰. Has

Salt Concentration (g / L) Salt Concentration (g / L) NaCl 21.00 NaNO ₃ 0.20 MgSO ₄ 7H ₂ O 6.00 NaHCO ₃ 0.20 MgCl ₂ 6H ₂ O 5.00 H ₃ BO ₃ 0.06 CaCl ₂ 2H ₂ O 1.00 Na ₂ SiO ₃ 9H ₂ O 0.01 KCl 0.80 Sr (NO ₃ ) ₂ 0.03 NaBr 0.10 Na ₂ HPO ₄ 0.02

1.Preparation of green storage solution

Production Example 1

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 0.01 parts per billion (ppb) to prepare a blue storage solution.

Production Example 2

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 0.1 ppb to prepare a green storage solution.

Production Example 3

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 1.0 ppb to prepare a blue storage solution.

Comparative Production Example 1.

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 10.0 ppb to prepare a green storage solution.

Comparative Preparation Example 2.

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 100.0 ppb to prepare a green storage solution.

Comparative Preparation Example 3.

Sodium azide was dissolved in OTT's artificial seawater to a concentration of 1000.0 ppb to prepare a green storage solution.

2. Changes in Spore Formation Rate of Seagrass According to Storage and Storage Conditions

(1) custody

Example 1.

Ulva in the water near Ahnin, Korea pertusa Kjellman) were collected, and the leaves (leaves) of the perforated rinds were cut into coins of 4 mm in diameter to form a circular section. Thereafter, 24 seaweeds in the form of circular sections were placed in a plastic storage container containing 15 ml of the blue storage solution prepared in Preparation Example 1, and stored for 6 weeks under refrigerated conditions at 4 ° C. Figure 1 is a photograph showing a state in which the seaweeds in the form of circular slices are stored in a plastic storage container.

Example 2.

The seaweed was stored in the same manner as in Example 1 except that the seaweed storage solution prepared in Preparation Example 2 was used as the seaweed storage solution.

Example 3.

The seaweed was stored in the same manner as in Example 1 except that the seaweed storage solution prepared in Preparation Example 3 was used as the seaweed storage solution.

Comparative Example 1

The green seaweed was stored in the same manner as in Example 1 except that the blue seaweed storage solution prepared in Comparative Preparation Example 1 was used as the blue seaweed storage solution.

Comparative Example 2

The green seaweed was stored in the same manner as in Example 1 except that the blue seaweed storage solution prepared in Comparative Preparation Example 2 was used as the blue seaweed storage solution.

Comparative Example 3

The green seaweed was stored in the same manner as in Example 1 except that the blue seaweed storage solution prepared in Comparative Preparation Example 3 was used as the blue seaweed storage solution.

Comparative Example 4

The seaweed was stored in the same manner as in Example 1 except that OTT's artificial seawater itself, which did not contain sodium azide, was used as the seaweed storage solution.

(2) Measurement of Spore Formation of Seagrass

As in Examples 1 to 3 and Comparative Examples 1 to 4, the greens were taken out on a weekly basis while the greens were kept for 6 weeks to induce spore formation of the greens, and the spore formation rate of the greens was measured. First remove the greens from the storage vessel and place them in a 24-well plate containing OTT's artificial seawater, a photon irradiance of 100 μmol / m 2 · s, a light cycle of 12: 12h, and a 15 ° C Incubation for 96 hours at the temperature conditions induced the formation of green spores.

The formation of spores on the green leaves is indicated by the change of the color of the green leaf. The normal leaves have green color, but gradually yellow, brown, and white as the spore formation progresses. This color change is regarded as the area of spore formation.

Using the image analyzer (Model: MW-200; Manufacturer: Samsung Co., Korea), the total area of the cultured green leaf and the spore formation area of the double color were measured, and then the sporulation area of the total leaf area was measured. The relative percentage of is expressed as the rate of spore formation in the greens. 2 is a graph showing the change of the spore formation rate according to the storage conditions of the greenery. As shown in FIG. 2, when the green seedlings were stored in a storage solution having an OTT's artificial seawater or sodium azide concentration of 10 to 1000 ppb, the rate of spore formation of the greens rapidly decreased as the storage period elapsed. After 4 weeks of storage, no spores formed. On the other hand, in case of storing the green seaweed in the storage solution of sodium azide concentration of 0.01 ~ 1.0 ppb, the spore formation rate was over 90% after 3 weeks storage period and 4 weeks storage period. Even after about 80% of the spore formation rate was shown. Particularly, when the green seaweed was stored in a storage solution having a sodium azide concentration of 0.1 ppb, the spore formation rate was higher than 90% even after 4 weeks storage period.

3. Assessment of Water Toxicity of Stored Seagrasses As a biomarker  Stability and Reproducibility

The storage solution and the method of storage of the present invention was investigated whether the seagrass stored as a biomarker for evaluating the toxicity of a water sample containing a toxic substance has stability and reproducibility. In this case, a copper standard toxic solution was used as a water sample containing a toxic substance. The copper toxic substance solution (initial concentration 0.4 mg / L, salinity concentration is 35 ‰) was half-diluted with OTT's artificial seawater (salin concentration was 35 ‰) and the copper concentration was 0.4 mg / L, 0.2 mg / L, 0.1 A copper standard toxic solution was prepared, mg / L, 0.05 mg / L, 0.05 mg / L, 0.025 mg / L. In addition, OTT's artificial seawater was used as a standard solution containing no toxic substances.

First, the prepared standard solutions were put in a 24-well plate, and then, the seaweed stored for 4 weeks in Example 2 was taken out and placed in a 24-well plate containing standard solutions. Afterwards, the spore formation was induced by incubating for 96 hours at 100 μmol / m 2 · s photon irradiance, 12: 12h contrast period, and 15 ° C. temperature conditions. Thereafter, the spore formation rate of the cultured green ginseng was measured using an image analyzer (model name: MW-200; manufacturer: Samsung Co., Korea).

As a control, a leaf of a perforated green larch ( Ulva pertusa Kjellman) directly extracted from Ahnin, South Korea was used, and the same method as described above was used to induce spore formation and determine the rate of spore formation.

FIG. 3 is a graph showing the results of toxic reactions against green standard stored in 0.1 ppb sodium azide solution for 4 weeks and blue used immediately without storage as a control. As shown in FIG. 3, the greens stored by the storage method of the present invention and immediately used greens showed almost the same sporulation rate regardless of the copper concentration of the copper standard toxic solution. In addition, in the case of the effective sporulation inhibition concentration (EC ₅₀ , median sporulation inhibition concentration), the value measured by the seaweed stored with the storage method of the present invention and the seaweed used immediately without storage were 0.088 mg Cu / L, 0.103, respectively. Mg Cu / L. There was no statistically significant difference in the results of the toxic reactions on the copper toxic substances stored by the storage method of the present invention and immediately used without storage, and the seagrass stored by the storage method of the present invention was bio-based for water toxicity evaluation. It turned out to have stability and reproducibility as a marker.

Although the present invention has been described through the above embodiments as described above, the present invention is not necessarily limited thereto, and various modifications can be made without departing from the scope and spirit of the present invention. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, the protection scope of the present invention should not be construed as being limited to the particular embodiments disclosed as the best mode contemplated for carrying out the invention but to cover all embodiments falling within the scope of the appended claims.

Claims (10)

As a method for storing green seaweed used as a biomarker for water toxicity evaluation,
(a) preparing a green onion; And
(b) putting the prepared green sea into a container containing a solution of sodium azide dissolved in a brine having a salt concentration of 25 ~ 45 ‰ at a concentration of 0.01 ~ 1 ppb and stored under refrigerated conditions of 0 ~ 10 ℃; Blue storage method to include.
The method of claim 1,
The method of claim 1, wherein the step of (a) is a green sea cucumber (Ulva pertusa) storage method of the sea.
The method of claim 1,
The storage method of the green sea, characterized in that the sea of step (a) is in the shape of a circular cut.
The method of claim 1,
The method of storage of the seaweed, characterized in that the seaweed of step (a) is a leaf body part.
The method of claim 1,
Shelf life method of the step (b) is characterized in that less than four weeks.
The method of claim 1,
The brine is sodium chloride (NaCl), magnesium sulfate (MgSO ₄ ), magnesium chloride (MgCl ₂ ), calcium chloride (CaCl ₂ ), potassium chloride (KCl), sodium bromide, sodium nitrate (NaNO ₃ ), sodium hydrogen carbonate ( NaHCO ₃ ), a green sea characterized by comprising boric acid (H ₃ BO ₃ ), sodium silicate (Na ₂ SiO ₃ ), strontium nitrate [Sr (NO ₃ ) ₂ ], and disodium hydrogen phosphate (Na ₂ HPO ₄ ) How to store.
The method of claim 1,
The storage method of the blue sea, characterized in that the brine is artificial sea water.
8. The method of claim 7,
The artificial seawater is OTT's artificial seawater (OTT's artificial seawater) characterized in that the storage method of the blue. delete delete

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