KR101415951B1 - Estimation Method for ecotoxicity of copper and mercury using inhibition of Iodonitrotetrazolium-dehydrogenase - Google Patents

Abstract

A method for measuring ecotoxicity using INT-dehydrogenase inhibition is disclosed. The present invention provides a method for producing an enzyme, comprising: preparing an enzyme solution; Feeding a plurality of filter disks, each of which has been absorbed by the enzyme solution, into a plurality of reaction vessels one by one; Introducing a phosphate buffer solution into each of the reaction vessels provided with the filter disk; Injecting a toxic standard solution having a different toxicity level into each of the reaction vessels; Injecting a sample to be measured into one reaction vessel; Leaving the reaction vessels at room temperature; Introducing a substrate solution to which INT is added into a reaction vessel to which the toxicity standard solution is injected and a reaction vessel into which a sample is injected, and culturing at a room temperature; And comparing the degree of redness of each of the reaction vessels to which the toxicity standard solution has been added with the degree of red coloration of the reaction vessels into which the measurement sample has been introduced to determine the degree of toxicity visually. It is possible to obtain a simple, economical, rapid, repeatable result, accurate and reliable result in detection.

Description

[0001] The present invention relates to a method for measuring ecotoxicity using an iodo-nitrotetrazolium-dehydrogenase inhibitor,

The present invention relates to a method for measuring ecotoxicity using the inhibition degree of Iodonitrotetrazolium (INT) -dehydrogenase, more specifically, by using the change in dehydrogenase activity of a bacterium inhibited by a toxic substance, The present invention relates to a method for measuring ecotoxicity using an INT-dehydrogenase inhibitor which enables evaluation of toxicity without the use of special equipment.

The Ministry of Environment has adopted the revised Water Environment Protection Act Enforcement Regulation, which introduces the ecotoxicant emission allowance standard, The ecotoxicant emission allowance standard is a system for managing the toxicity of the whole substance unlike the existing standard for permitting the emission of individual substances, and is set as a standard by comprehensively quantifying the effect of harmful chemical substances contained in industrial wastewater on organisms .

The toxicity detection method using organisms is widely used because it can directly evaluate the risk of chemical substances to the ecosystem and can build a system that can manage the toxicity of harmful substances in an integrated manner.

Methods that have been validated to evaluate ecotoxicity include freshwater crustacean, microtox, Toxi-chromo, and U-kit methods.

The freshwater crustacean method is a kind of freshwater crustacean ( Daphnia magna ) as a method for measuring ecotoxicity, which is designated by the OECD Standard Law and has an advantage of directly evaluating ecotoxicity. However, the measurement time is as long as 24 to 48 hours, and it is difficult to cultivate daphnia. Especially daphnia is not long-term preservation, so it is difficult to make kit. A patent for this is disclosed in Korean Patent No. 10-0407753.

The microtox method is a technique developed by SDI in the United States. It uses a marine luminescent bacterium, Vibrio fischeri , and evaluates ecotoxicity using the decrease in luminescence when exposed to toxic substances. It is being used as a standard method to confirm the inclusion of toxic substances in food and sediment in Australia and Europe, including the US EPA. However, since it is a marine bacterium, it is known that the salt added to maintain the activity may affect the toxicity evaluation and affect the reproducibility of the experiment. In addition, the cost of analysis is as high as $ 150 ~ 250 per sample.

The Toxi-chromo test was developed by Canadian EBPI. It evaluates the toxicity of a bacterium containing β-galactoxidase when it is exposed to a toxic substance. It is fast and has the advantage of visually confirming the measurement results. However, there are disadvantages such as the small number of measurable toxic substances and the high cost of analysis.

The U-kit was developed by Prof. Tae-joon Han, professor of biology at the University of Incheon, and was used as a target organism. The parasites exposed to toxic substances were developed using parasitic colors while the parasites without toxic substances were discolored with time. Although the U-kit is the first toxic evaluation kit developed in Korea, it requires 96 hours of measurement time longer than the daphnia toxicity evaluation kit and it is known that it is very difficult to keep the physiological condition of the virus constant It is judged that it is difficult to use in the field. A patent for this is disclosed in " Method for evaluating water toxicity using parasites "in Patent No. 10-0653100.

This technique uses a method of evaluating ecotoxicity using the inhibition of INT-dehydrogenase, which utilizes changes in dehydrogenase activity of bacteria that are inhibited by metabolism by toxic substances. Under optimized conditions, the toxicity evaluation time is within 2 hours, the evaluation result is red, and the evaluation result can be verified without using special equipment.

DISCLOSURE Technical Problem It is an object of the present invention to provide a method for measuring ecotoxicity using an INT-dehydrogenase inhibiting degree, which enables easy and quick evaluation of ecotoxicity of wastewater containing copper or mercury .

Another object of the present invention is to provide a method for measuring ecotoxicity using an INT-dehydrogenase inhibition degree, which enables a non-specialist to easily confirm an evaluation result without using a special instrument.

In order to solve the above object, the present invention provides a method for measuring ecotoxicity using an INT-

Preparing an enzyme solution; Feeding a plurality of filter disks, each of which has been absorbed by the enzyme solution, into a plurality of reaction vessels one by one; Introducing a phosphate buffer solution into each of the reaction vessels provided with the filter disk; Injecting a toxic standard solution having a different toxicity level into each of the reaction vessels; Injecting a sample to be measured into one reaction vessel; Leaving the reaction vessels at room temperature; Introducing a substrate solution to which INT is added into a reaction vessel to which the toxicity standard solution is injected and a reaction vessel into which a sample is injected, and culturing at a room temperature; And comparing the degree of redness of each of the reaction vessels into which the toxicity standard solution is injected with the degree of red coloration of the reaction vessel into which the measurement sample has been injected to determine the intensity of the toxicity visually.

The step of preparing the enzyme solution comprises the step of inoculating and cultivating bacteria susceptible to copper and mercury into a nutrient medium; Placing the culture on a filter disk and placing it in a cryocooler to freeze; And a step of completely drying in a freeze-drier, and then storing it in a drier at a low temperature.

The bacteria susceptible to copper and mercury are Enterobacter sp. CBEB 20-1.

According to the present invention, it is possible to measure the presence of ecotoxic substances in wastewater by using a minimum of an experimental apparatus (a spectrophotometer, a constant temperature water tank, and a small centrifuge) within one hour, .

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain a simple, economical, rapid, repeatable result, accurate and highly reliable result in detecting ecotoxicity.

Further, according to the present invention, there is an effect that a field workforce, which is a non-expert in the field of toxicity, can be easily applied.

In addition, according to the present invention, it is possible to obtain an effect of preventing destruction of ecosystem through inflow of ecosystem of pollutants by quickly discriminating heavy metal toxic substances contained in wastewater.

1 is a view showing an example of a multi-well (1.1 ml 96 Well Deep Well Plates, Axygen) used for measurement.
Figure 2 is a toxicity standard solution used in the measurement.
Figure 3 shows the results of measurements after using a toxicity standard solution.

Hereinafter, a method for measuring ecotoxicity using INT-dehydrogenase inhibition according to the present invention will be described in detail with reference to the drawings.

The method for measuring ecotoxicity according to the present invention is a method for measuring the ecotoxicity of Enterobacter sp., A bacterium susceptible to copper or mercury. CBEB 20-1 is identified and used for evaluation of ecotoxicity.

At this time, when the enzyme solution is prepared, a certain amount of the bacterial solution is injected using a filter disk, and storage characteristics can be facilitated.

In order to measure ecotoxicity according to the present invention, the enzyme solution must first be prepared, and the enzyme solution must be activated to measure the toxicity.

<Preparation step of enzyme solution>

1. Inoculate and cultivate copper and mercury-sensitive bacteria in nutrient media.

In the present invention, Enterobacter sp., A bacterium susceptible to copper and mercury, CBEB 20-1 is inoculated into nutrient broth and incubated overnight at 37 ° C.

2. Place the culture on a filter disk and freeze in a cryogenic freezer.

0.2 ml of the culture is placed on a filter disc (Paper Disc Filter, Advantec), waited for absorption, and then placed in a cryogenic freezer and frozen at about -70 ° C.

3. After thoroughly drying in a freeze dryer, store it in a drier at low temperature.

<Toxicity measurement method>

1. A filter disk having the enzyme solution absorbed therein is put into a reaction vessel.

The filter disks prepared in the form of tweezers are inserted into the reaction containers A1 to A5 shown in FIG. 1 one by one.

2. Add the phosphate buffer solution to the reaction vessel equipped with the filter disk.

To the reaction vessels A1 to A5 to which the filter disks have been added, 0.2 ml of phosphate buffer solution (0.1M, pH 7.6) is added to activate the enzymes in the filter disk.

That is, the phosphate buffer solution is the solution required to activate the enzymes in the dried state.

3. Add toxic standard solutions with different strengths to reaction vessels A1, A2, A3 and A4.

To the reaction vessels A1, A2, A3 and A4, 0.25 ml of the toxic standard solutions 1 to 4 shown in Fig.

4. Put the sample to be measured in reaction vessel A5.

5. Leave at room temperature for 30 minutes.

6. Add 0.2 ml of a substrate solution containing Iodonitrotetrazolium (INT) to the reaction vessels A1 to A5, and incubate at room temperature for 30 minutes.

7. The intensity of toxicity is judged visually by comparing the red color of reaction containers A1, A2, A3 and A4 to which the toxicity standard solution has been added and the red color of the reaction container A5 into which the measurement sample is introduced.

As shown in FIG. 3, the toxicity measurement method described above shows the red color (TU0) of the non-toxic standard solution (the first standard solution of FIG. 2) and the red color It becomes thin. That is, the stronger the toxicity, the closer the color from TU0 to TU1, TU2, TU4.

Therefore, the degree of toxicity can be measured by comparing the degree of redness of the reaction vessel A5 into which the sample to be measured is injected, with the degree of redness of the reaction vessels A1 to A4.

If the red color of the reaction vessel A5 is the same as or similar to the red color of TU0, the measurement sample put into the reaction vessel A5 can be evaluated as not toxic. On the other hand, if the red color of the reaction vessel A5 is the same as or similar to the red color of TU4, the measurement sample put into the reaction vessel A5 can be evaluated as being highly toxic.

The INT-dehydrogenase used in the present invention will be described below.

Dehydrogenase is an intracellular enzyme that catalyzes the redox reaction required for the respiration of organic compounds. Since the dehydrogenase is not active outside the cell, this assay is considered a measure of microbial activity. The reaction of the dehydrogenase can be detected using a water-soluble, nearly colorless tetrazolium salt, which when redified produces a red formazan that can be detected in many ways. The tetrazolium salt competes with other electron acceptors for the reducing ability of the electron transport system. Therefore, the measurement of the reduction of the tetrazolium salt reflects the activity of the electron transport system. That is, these measurements are indicative of the general level of activity of most microbial communities, but they are not a direct measure of microbial growth by the synthesis of new biomass.

The tetrazolium salt is INT [2- ( p- iodophenyl) 3- ( p- nitrophenyl) -5-phenyltetrazolium chloride], which is converted to a dark, water insoluble INT-formazan. INT is used to measure microbial activity in surface water, soil and sediment samples, underground sediments, and biofilm. The formation of INT-formazan is detected by microscopic observation of red stocks produced in cells or by quantification of total INT-formazan production.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the present invention is not limited to the disclosed exemplary embodiments. It will also be appreciated that many modifications and variations will be apparent to those skilled in the art without departing from the scope of the present invention.

Claims (3)

Inoculating and cultivating copper and mercury-sensitive bacteria in a nutrient medium;
Placing the culture on a filter disk and placing it in a cryocooler to freeze;
Drying it completely in a freeze dryer, storing it in a drier and storing it at low temperature to prepare an enzyme solution;
Feeding a plurality of filter disks, each of which has been absorbed by the enzyme solution, into a plurality of reaction vessels one by one;
Introducing a phosphate buffer solution into each of the reaction vessels provided with the filter disk;
Injecting a toxic standard solution having a different toxicity level into each of the reaction vessels;
Injecting a sample to be measured into one reaction vessel;
Leaving the reaction vessels at room temperature;
Introducing a substrate solution in which iodonitrotoluenol (INT) is added into a reaction vessel into which the toxicity standard solution is injected and a reaction vessel into which a sample is injected, and culturing at a room temperature; And
Comparing the degree of redness of each of the reaction vessels into which the toxicity standard solution has been injected with the degree of red coloration of the reaction vessels into which the measurement sample has been introduced to determine the intensities of the toxicity visually, (INT) - method for measuring ecotoxicity using dehydrogenase inhibition.
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