KR101663665B1 - Kit for testing aquatic ecotoxicity usig turion separated from Spirodela polyrhiza - Google Patents

Abstract

The present invention provides a water quality toxicity diagnostic kit comprising a sleeping state separated from frogs, a container for culturing frozen rice, a water pollutant sample, and a medium for culturing frozen rice of frog. The water quality toxicity test according to the present invention uses a dormant sleeping louse isolated from the thrips of the frog rice as an index substance for the evaluation of water quality toxicity and is used as a parameter for the evaluation of the water quality toxicity by the roots length, Or thallus area, it takes about 3 days to determine the toxicity of the water, has a high sensitivity to the toxicity of water quality, and it is possible to evaluate the water pollution by various toxic substances.

Description

{Kit for testing aquatic ecotoxicity usig turion separated from Spirodela polyrhiza}

More particularly, the present invention relates to a kit for diagnosing or evaluating water quality toxicity or water pollution using the root length of the frog, germination rate of the throat, or leaf area of the frog, .

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. There are physico-chemical and biological methods to do this. Examples of the physicochemical method include a gas chromatography (GC) method and an inductively coupled plasma method. However, these methods require expensive analytical equipments such as GC-MS (gas chromatography-mass spectroscopy) and ICPMS (inductively coupled plasma-mass spectroscopy) and highly skilled analytical personnel. There is a problem to be performed. In addition, when the analysis time is long and the components of the toxic substances are unknown, analysis is difficult. In addition, when the sample is transported over a long distance, there is a problem that the components and the concentration of the sample may be changed due to the reaction in the sample.

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 general, when fish or invertebrates are used, it is judged by the mortality, the inhibition of movement, and the biological characteristic analysis. In the case of using microalgae, the growth inhibition is measured. Daphnia (Daphnia) Daphnia magma and Ceriodaphnia dubia are very sensitive to environmental toxic substances, easy to grow, have a short life span, are small in size and can be used for many experiments. . However, the method of evaluating the water toxicity using the fish, invertebrate or protozoa requires the expertise of the measurer and expensive equipment, takes a long time, and has a problem that the sensitivity is not high.

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. Hydrophytes have the flexibility to become morphologically diverse by the transformation of the various organs of the plant to the habitat environment. Some aquatic plants undergo structural deformation into very shrunken plants that do not form roots, stems, leaves, and other organs to survive and adapt to the particular underwater environment they are in.

Extremely reduced plant populations ( Spirodela polyhriza ) develops into a very small individual consisting of three parts: lobes, connective roots, and roots (Lee, 1996). They are infertile aquatic plants of 5 to 8 mm in size, reproductive reproducting with frond, and multiply the plants at very high speeds, with the plants doubling in a few days (Landolt, 1986; Appenroth, 2003). When an aquatic plant finishes its life cycle, it forms a seed as in land plants, or it forms a special structure that can keep the growth stopped, and enters a dormancy in the winter season. A perennial herb plant, frog, is a dormant structure that forms a turion, a kind of winter snow, and maintains its dormancy in winter. During the growing period, the thalli increase rapidly by rapid division and multiply the individual to occupy most of the plant volume. These lobsters stop nutrient breeding and form turion primordium inside the leaf tissue when the temperature starts to fall (Kwak & Kim, 2008). Afterwards, the sleeping is separated from the mother frond and sinks to the bottom of the water. When the temperature starts to rise in the next year, it floats on the surface of the water and repeats the life cycle that develops as a new plant. In their sleep, vegetative sheath and frond primordium, which were enclosed in the lyophilized tissues, germinate due to the rise in temperature after the dormant period, resulting in the thallus. This characteristic of frogbill sleeping to maintain dormancy at the time of temperature drop has been proposed as a representative model system species in the study of the dormant structure formation and development process of aquatic plants (Smart, 1996). Extremely reduced frog rice plants consisting of only thallus, connective roots and roots have the structural features of resuming the sleeping and thyroid gland in the thighs and lining tissues, allowing them to resume their growth rapidly after the dormant period or directly into the dormant state (Kwak & Kim, 2008), which has a significant role in the circulation of life in reduced plants (Newton et al., 1978; Sibasaki & Oda, 1979; Kim & Kim, 2000;

It is an object of the present invention to provide a novel method for assessing or diagnosing water quality toxicity using the germination characteristics of a latte separated from a thallus of frogs.

Another object of the present invention is to provide a novel kit for diagnosing or evaluating water quality toxicity or water quality contamination using immobilization separated from frogs.

The inventors of the present invention used sleeping slugs isolated from the thrips of frog rice as an indicator substance for evaluation of ecotoxicity and found that the parameters such as roots length, , The present inventors confirmed that ecotoxicity, particularly the time required for evaluating the toxicity of water, is short, sensitivity is excellent, and various toxic substances can be evaluated. Thus, the present invention has been completed.

In order to solve one object of the present invention, the present invention provides a method for producing a culture medium, comprising the steps of: (a) (b) injecting a louse separated from the frog into a culture container containing a water sample; (c) culturing the gill inserted into the culture container and germinating; And (d) measuring the leaf area of the frog or the root length of the frog formed by the germination of the frog formed by germination of the frog. At this time, the water sample of the step (a) is composed of at least two dilution raw water having different raw water and dilution multiples, and the diluted raw water is used as the raw water for the incubation of the submerged slice separated from the frog, for example, (Steinberg ' s medium or Hoagland ' s medium). It is preferable that the dilution raw water is diluted by a half-dilution method. In addition, the water sample further comprises a culture medium for incubation of the slug separated from the frog, for example, Steinberg's medium or Hoagland's medium as a control for the raw water, And preferably does not contain a toxic substance which inhibits the germination of sleeping. The toxic substance may be at least one selected from the group consisting of silver (Ag), aluminum, arsenic, cadmium, cobalt, chromium (Cr), copper (Cu) Iron, iron, mercury, nickel, lead, zinc, acetone, chloroform, dimethylsulfate, DMSO, Dimethyl sulfoxide, ethyl alcohol, formaldehyde, methanol, and phenol. [0028] The term " a " In addition, the water sample of the step (a) preferably has a pH of 5 to 8. In addition, the slug separated from the frog in step (b) is preferably in a dormant state. In addition, the slug separated from the frog in step (b) is preferably stored for at least 2 months under the refrigerated condition. It is preferable that the incubation temperature in the step (c) is in the range of 15 to 35 ° C, the irradiation amount is 50 to 150 μmol photon / m 2 · s, and the incubation time is 2 to 4 days.

In order to solve the other object of the present invention, an example of the present invention provides a water quality toxicity evaluation kit comprising a sleeping state separated from frogs. At this time, the kit may further include a container for incubation, a artificial medium for diluting a water sample, or a toxic substance standard solution. The standard toxic substance is a substance which inhibits the germination of a sleeping child and is composed of silver (Ag), aluminum, arsenic, cadmium (Cad), cobalt (Co), chromium (Cr) , Copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb), zinc (Zn), acetone, chloroform And may be composed of at least one member selected from the group consisting of chloroform, dimethyl sulfoxide (DMSO), ethyl alcohol, formaldehyde, methyl alcohol and phenol. In addition, the artificial medium for diluting the water sample is preferably Steinberg's medium or Hoagland's medium.

In order to solve the other object of the present invention, another example of the present invention includes a dormant sleeping state separated from a frog; A container for cultivating a frog's mouth; Water pollutant samples; And a culture medium for culturing a frozen duck fish. At this time, it is preferable that the sleeping state of the frog separated from the frog is provided in the form of a biowear in which the frog is stored in the culture medium for the frog's nest, and the culture medium for the frog nest, It is preferably selected from Steinberg's medium or Hoagland's medium. In addition, the frog-covered biowares are preferably stored under refrigeration conditions. Also, it is preferable that the container for culturing the frozen rice is selected from a well plate or a test tube. For example, the container for culturing the frozen duck can be a test tube, and the kit preferably further comprises test tube fixing means. In addition, the water pollutant sample may be composed of at least one selected from copper sulfate solution, silver nitrate solution, ammonia water, soapy water or brine. In addition, the medium for culturing the frozen duck can be selected from Steinberg's medium or Hoagland's medium, wherein the above-mentioned Steinberg's medium or Hoagland's medium ) Is preferably provided in the form of a stock solution constituting it. In addition, the water quality toxicity test kit according to another example of the present invention may further include at least one selected from the group consisting of tweezers, a syringe, a petri dish, or a coloring paper for pH measurement.

The water quality toxicity assessment method or the water quality toxicity evaluation kit according to the present invention uses sleeping ducks separated from the fronds of frog rice as an index substance for evaluation of water quality toxicity and is used as a parameter for evaluation of water quality toxicity, It is possible to evaluate the water pollution by various toxic substances because it takes about 3 days to determine the toxicity of the water due to its root length, degree of whiteness or thorn area.

FIG. 1 is a graph showing changes in the area of cytoplasmic area of frog rice formed by germination of a slug according to the concentration of silver, which is a poisonous substance. FIG. FIG. 3 shows changes in the area of cytoplasmic area of the frog prepared by germination of the frog according to the concentration of chromium which is a poisonous substance. FIG. 4 shows the change in the area of the frog rice formed by germination of the frog according to the concentration of copper, Fig. 5 shows changes in the area of cytoplasmic area of frog rice formed by the germination of the louse according to the concentration of mercury which is a toxic substance.
FIG. 6 shows changes in root length of frog rice formed by germination of a slug according to the concentration of silver, which is a toxic substance, and FIG. 7 shows changes in root length of frog rice formed by germination of slug according to the concentration of the toxic substance cadmium FIG. 8 is a graph showing changes in the root length of the frog prepared by germination of the submersion according to the concentration of chromium, which is a toxic substance, and FIG. 9 is a graph showing changes in the root length of the frog And Fig. 10 shows changes in the root length of the frog prepared by germination of the submersion according to the concentration of mercury which is a toxic substance.
11 is a photograph schematically showing constituent elements included in the aquatic toxicity test kit of the present invention.
FIG. 12 shows changes in root length of frog rice formed by germination of the subspecies according to the addition amounts of copper sulfate solution, silver nitrate solution, ammonia water, soapy water and salt water.

One aspect of the present invention relates to a water toxicity evaluation method having a short discrimination time, a simple operation, and a high sensitivity to various toxic substances. The water toxicity evaluation method of the present invention comprises the steps of placing a water sample into a culture vessel ; Injecting a lump separated from the frog into a culture container containing a water sample; Culturing a gill inserted into a culture container and germinating; And measuring the leaf area of the frog or the root length of the frog or the frog formed by germination of the frog. Hereinafter, the method for evaluating the water toxicity according to the present invention will be described by dividing it into components.

Water body  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 in its kind as long as it is compatible with the cultivation of the slug isolated from the frog, without containing the toxic substance which inhibits germination of the slug separated from the frog, And a medium used for culturing the immersed slice. The type of medium used for culturing the subspecies isolated from frogs is not limited to a wide variety of known media and the like. For example, in consideration of smooth cultivation and germination of frogs separated from frogs, Steinberg's medium ) Or Hoagland's medium.

In addition, the water sample may further comprise a medium used for culturing a slug separated from frog, preferably as a control for raw water, which does not contain a toxic substance that inhibits the germination of the slug separated from the frog desirable. The kind of the toxic substance which inhibits the germination of the immature frog separated from the frog is not limited to a wide range and is, for example, silver (Ag), aluminum, arsenic (As), cadmium (Cadmium) Co, Cobalt, Cr, Chromium, Copper, Iron, Mercury, Nickel, Pb, Selected from the group consisting of Zinc, acetone, chloroform, dimethyl sulfoxide (DMSO), ethyl alcohol, formaldehyde, methanol and phenol. There is more than one kind. The culture medium for slug isolated from frog rice constituting the control group is selected from a variety of known media and the like, and the type thereof is not particularly limited. For example, in consideration of smooth cultivation and germination of slug isolated from frog, (Steinberg ' s medium or Hoagland ' s medium).

In addition, the water sample containing the raw water, the diluted raw water or the control water of the raw water is put into a container for culture before putting it in a container for culture or in a container for culture, and then it is necessary when considering the smooth cultivation and germination of the slit separated from the frog it is preferable that the pH is adjusted to 5 to 8, preferably 6 to 8.

A culture container

The culture container is a container for storing a water sample and frozen rice separated from the frozen rice, and then putting it in an incubator to cultivate the frozen food. The shape of the culture container is not limited. For example, a well plate, Erlenmeyer flask ). It is preferable that the well plate includes at least six wells. In the wells, artificial medium containing no toxic substance is added as a control group to cultivate the embryo. In the remaining five wells, raw water and four dilution enemies Put the slices to incubate. 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.

A frog separated from a frog

The method for evaluating the water toxicity according to the present invention uses a slug separated from frog rice as an evaluation index substance. At this time, the slug is specifically separated from the mother lobes of the frog. In the present invention, the mother lobes are relative concepts of the hypothalamus of the frog prepared by germination of the louse. The frozen slices separated from the mother lobes of the frozen rice used in the present invention are preferably stored under refrigeration conditions (for example, 0 to 5 ° C) to maintain a dormant state. In addition, when stored at a low temperature such as a refrigeration condition, it usually takes a certain time for smooth germination. The water quality toxicity evaluation method according to the present invention uses various characteristics after inducing germination of frog separated from frog rice as a parameter for water quality evaluation. The frog separated from frog rice used in the present invention is at least 2 months Or more, more preferably from 2 months to 6 months or less.

Frog Sleepy  culture

The incubation vessel containing the submerged and water sample separated from the frog is transferred to an incubator and then incubated for a predetermined time. At this time, the culture time is not limited, but is preferably at least 2 days, more preferably from 2 to 4 days, most preferably from 3 to 4 days, from the viewpoint of securing the reliability of the evaluation of water quality toxicity. In addition, it is preferable that the amount of light irradiation is 50 to 150 mu mol photon / m < 2 > s, more preferably 75 to 150 mu mol photon / m < / M < 2 > The incubation temperature for incubation of frogs separated from frogs is preferably 15 to 35 ° C, more preferably 20 to 30 ° C.

Sleepy  Measurement of leaf area or root length of frog prepared by germination

When a slit separated from frogs is placed in a culture container containing a water sample and cultured for 72 hours under the above-mentioned specific culture condition, the slit germinates and the hypothalamus of the frog is formed and the roots grow. After the cultivation is completed, the leaf area or the root length of the frog prepared by the germination of the slug can be measured and the water quality toxicity can be evaluated therefrom. Specifically, the water toxicity was evaluated by comparing the area of cytoplasm of frog rice formed by the germination of frozen rice cultivated in raw water and diluted raw water with that of frog rice formed by germination of frozen rice cultivated in a control group containing no toxic substance . In addition, the water toxicity was evaluated by comparing the root growth length of the frog prepared by the germination of the submerged root cultured in the raw water and the diluted seeds with the root growth length of the frog prepared by the germination of the submerged cultured in the control group containing no toxic substance . 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.

The half-maximal effective concentration (EC ₅₀ ) was calculated as the area of the cytoplasm of the frog or the root length of the frog formed by the germination of the frog. The leaf area of the frog formed by the germination of the frog cultured in the control group, Or a concentration of a water sample which is reduced by 50% of the root growth length, in the case of a water sample containing a single toxic substance, in the case of an unknown water indicated by a specific concentration of a single toxic substance and containing a plurality of toxic substances, Of the dilution rate. The no effect concentration was significantly different from the area of cytoplasmic area of the frog rice formed by the germination of the slug or the root growth length of the frog rice formed by the germination of the slug cultivated in the control group without the toxic substance Means the concentration of a bodily sample which is maintained at an undetectable level. As with the half effective concentration, in the case of a water sample containing a single toxic substance, in the case of an unknown water indicated by a specific concentration and containing a plurality of toxic substances, do. 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 (%)

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.

Another aspect of the present invention relates to a water quality toxicity assessment or diagnostic kit for implementing a water toxicity evaluation method having a short discrimination time, simple operation, and high sensitivity to various toxic substances, The water quality toxicity assessment kit according to the present invention includes sleeping dormant detached from frog. In addition, the water quality toxicity evaluation kit according to an exemplary embodiment of the present invention may further include a container for incubation, an artificial medium for diluting a water sample, or a toxic substance standard solution. The submerged culture container is a container for accommodating a sleeping state of a dormant state separated from a water sample and a frog, and cultivating the submerged fish. Examples of the container include a well plate, a Erlenmeyer flask, and the like. In addition, the artificial culture medium for diluting a water sample is diluted water used for producing diluted raw water from raw water, for example, Steinberg's medium or Hoagland's medium. In addition, the toxic substance standard solution is prepared so as to have a predetermined concentration of a substance inhibiting germination of the subspecies separated from the frog. In the present invention, poisonous substances which inhibit germination of frogs separated from frogs are silver (Ag), aluminum, arsenic (As), cadmium (Cad), cobalt (Co) Chromium, Copper, Iron, Hg, Nickel, Lead, Zinc, and Acetone are the main components of the system. , Chloroform, dimethyl sulfoxide (DMSO), ethyl alcohol, formaldehyde, methyl alcohol or phenol, and toxic substance standard solutions are selected from them And may include one or more species. The toxic substance standard solution can be used to test the state of the immune system separated from the frog, that is, the response sensitivity to toxic substances, before evaluating the water toxicity of the water sample. Specifically, when the standard solution of the toxic substance according to the present invention is a solution containing copper as a toxic substance, data on the throat area or root growth length of the frog prepared by germination of the submerged in various copper solutions and the control group are obtained in advance Which can then be provided with a water quality toxicity assessment kit. The user measures the leaf area of the frog or the root growth length of the frog formed by the germination of the frog in a copper solution and judges whether or not the frog separated from the frog is normally reacting with the toxic substance by comparing with the data provided in advance, (For example, 80% or more of the original response sensitivity), the evaluation of the water quality of an unknown water sample can be carried out.

In addition, the water quality toxicity test kit according to another example of the present invention can be provided in a form that can observe the influence of typical water pollutants on water quality. For example, the water-toxicity test kit according to another embodiment of the present invention includes a dormant state of diving separated from frog, a container for cultivating frozen ducks, a water pollutant sample, and a medium for culturing frozen ducks . At this time, the sleeping state separated from the frog is preferably provided in the form of a bio-ware stored in a medium such as Steinberg's medium or Hoagland's medium. The type of the container for culturing the frozen rice is not limited, and may be selected from, for example, a well plate or a test tube. When the container for culturing the frozen rice is a test tube, the water toxicity test kit according to another example of the present invention may further comprise test tube fixing means (for example, a test tube fixing box) capable of fixing the test tube have. In addition, the type of the water pollutant is not limited to a specific type, and may be composed of at least one selected from copper sulfate solution, silver nitrate solution, ammonia water, soapy water or salt water, which are typical water pollutants. In addition, the medium for culturing the frozen duck can be selected from a variety of known media, and preferably an artificial medium such as Steinberg's medium or Hoagland's medium. Also, in the case where the medium for culturing the frozen rice is Steinberg's medium, Steinberg's medium may be provided in the form of a stock solution constituting the same. Also, when the medium for culturing the frozen duck is Hoagland's medium, Hoagland's medium may be provided in the form of a stock solution constituting it. Table 1 below shows the composition of Hoagland's medium and the stock solutions constituting it. In addition, the water quality toxicity diagnostic kit according to another embodiment of the present invention may further comprise a tweezers for transferring the frog's mouth to the culture container, a syringe for injecting the water pollutant sample into the culture container, a petri dish, And coloring paper for pH measurement for use in the present invention, but the present invention is not limited thereto.

Stock solution The kind of constituent constituting the stock solution The concentration of constituents in the stock solution (g / l) The stock solution per liter of artificial medium
Volume (ml / l) Ⅰ MgSO ₄ 7H ₂ O 246 1.0 Ⅱ K ₂ HPO ₄ 236 2.3 Ⅲ _{Ca (NO 3) 2 4H 2} O 136 0.5 IV KNO ₃ 101 2.5 V H ₃ BO ₃ 2.86 0.5 MnCl ₂ 4H ₂ O 1.82 ZnSO ₄ 7H ₂ O 0.22 _{Cu (SO 4) 2 6H 2} O 0.09 Na ₂ MoO ₄ 2H ₂ O 0.09 VI FeCl ₃ 6H ₂ O 0.484 20 Na ₂ -EDTA ₂ H ₂ O 1.5

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.

One. Steinberg  badge( Steinberg medium )

Table 2 below shows the components and concentrations contained in the stain bug medium used as the artificial medium used in the examples of the present invention. As shown in Table 2, the artificial medium contains a total of 5 stock solutions, and each stock solution is composed of a solution containing an inorganic component or an organic component necessary for culturing the frog. The pH of the Stainberg medium is 7 ± 0.2.

Stock solution Constituting the stock solution
Types of Ingredients The concentration of constituents in the stock solution (g / l) The stock solution per liter of artificial medium
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. Preparation of toxic substance standard solutions

A toxic substance standard solution containing silver (Ag), cadmium (Cd), chromium (Cr), copper (Cu) and mercury (Hg) was prepared as a single metal toxic substance. 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 is adjusted to 7 ± 0.2 using 1M hydrochloric acid solution or 1M sodium hydroxide solution.

3. Separated from the frog in the toxic substance standard solution Sleepy  culture

To each well of a 24-well plate, 3 ml of each of the control solution, the toxic substance standard solution and the diluted toxic substance standard solution was added to each well, and one slice separated from the frog was added to each well. At this time, the slime was separated from the mother lobes of the mature domestic frog populations and stored in a refrigerator at 4 ° C for 2 months or more under the condition of blocking light. Then, the lid of the well was covered, and the periphery of the well was wrapped with a parafilm and sealed. The 24-well plate was then transferred to an incubator and incubated for 72 hours at a light dose of 90-100 mu mol photon / m < 2 > s (light period was 24 hours continuous light condition) The experiment under the same condition was repeated three times in total.

4. Jim's  Measurement of leaf area and roots growth length of frog prepared by germination

When about 48 hours have elapsed after the initiation of incubation, the submerged surface floats to the surface. When 72 hours have elapsed, the submerged thorns of the frog bud are formed by the germination of the submerged root and the root is grown. When 72 hours had elapsed after the initiation of the incubation, the germinated germinated in each well was taken out and placed on a slide glass and photographed. Then, the area of the foliage of the frog and the root growth length in the photographs were measured using the image analysis program.

FIG. 1 is a graph showing changes in the area of cytoplasmic area of frog rice formed by germination of a slug according to the concentration of silver, which is a poisonous substance. FIG. FIG. 3 shows changes in the area of cytoplasmic area of the frog prepared by germination of the frog according to the concentration of chromium which is a poisonous substance. FIG. 4 shows the change in the area of the frog rice formed by germination of the frog according to the concentration of copper, Fig. 5 shows changes in the area of cytoplasmic area of frog rice formed by the germination of the louse according to the concentration of mercury which is a toxic substance.

FIG. 6 shows changes in the root length of the frog prepared by germination of the slug according to the concentration of silver, which is a toxic substance. FIG. 7 shows changes in the root length of the frog prepared by germination of the slug according to the concentration of the toxic substance cadmium FIG. 8 shows the root length change of frog rice formed by the germination of the molasses depending on the concentration of chromium which is a toxic substance, FIG. 9 shows the root length of frog rice formed by germination of the molasses according to the concentration of copper, FIG. 10 shows changes in the root length of frog rice formed by the germination of the subspecies according to the concentration of mercury which is a toxic substance.

5. Assessment of water toxicity of single metal toxic substances

The root of the frog, formed by the germination of the frog according to the change in the area of the self-lobular body of the frog, formed by the germination of the frog, according to the concentration of the toxic substance shown in Figs. 1 to 5 or the concentration of the toxic substance shown in Figs. From the change in length, the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of each of the single metal toxicity substances was calculated. The EC ₅₀ values were calculated using point estimation techniques, and the NOEC values were calculated using hypothesis testing methods such as the Dunnett procedure.

Table 3 shows the half-effective concentration of each single metal toxic substance when the leaf area of the frog rice formed by the germination of the slug and the root length of the frog formed by the germination of the slug were regarded as a reference parameter for evaluation of the water quality toxicity EC ₅₀ ) to no effect concentration (NOEC).

Metal toxic substance Based on the leaf area of the frog, Based on the length of the frog root formed by germination NOEC (占 퐂 / L) EC ₅₀ (占 퐂 / L) NOEC (占 퐂 / L) EC ₅₀ (占 퐂 / L) Ag 15.6 23.2 15.6 23.4 CD 125.0 204.9 31.3 121.9 Cr 312.5 507.1 156.3 219.2 Cu 125.0 630.2 125.0 365.4 Hg 31.3 92.8 31.3 109.1

As shown in Table 3, when the root length of the frog was determined as a reference parameter for evaluating the water toxicity, the maximum quantum yield (Fv / Fm) of the frog rice formed by the germination of the frog was measured as water toxicity It was found that the sensitivity of the single metal toxicity substance was higher than that of the standardized parameter.

6. Preparation of aquatic ecotoxicity test kit and measurement of water toxicity of various water pollutant samples

(1) Preparation of aquatic ecotoxicity test kit

Frogblower, frozen food, glassware test tube, test tube fixation box, copper sulfate solution, silver nitrate solution, ammonia water, soapy water and so on, provided in a steinberg's medium in a falcon tube, A water pollutant sample provided in the form of a container in which salt water is individually stored, and a water ecotoxicity diagnostic kit (or a water toxicity test kit) containing 5 stock solutions constituting Steinberg's medium as a medium for cultivating frozen rice, Diagnostic kit). Preferably, the aquatic toxicity test kit further comprises a petri dish, tweezers, a syringe, a coloring paper for pH measurement, and the like. Further, the aquatic toxicity test kit further preferably includes a manual recording a usage method of a parafilm, a rubber glove, a water test chart, and a water toxicity test kit. 11 is a photograph schematically showing constituent elements included in the aquatic toxicity test kit of the present invention.

(2) How to use aquatic toxicity diagnostic kit

① Preparing Test Solution

- Steinberg's medium is prepared using 5 stock solutions and distilled water constituting Steinberg's medium.

- Fix a number of test tubes in a test tube fixed box, and put 3 ml of Steinberg's medium in each test tube.

- With the exception of the control test tube, drop the water pollutant sample into each test tube using the dropper. In this case, one drop of the water pollutant sample in the test tube corresponding to the lowest concentration water pollutant test solution, and 4 drops of the water pollutant sample in the test tube corresponding to the highest concentration water pollutant test solution , But it is not necessarily limited thereto.

② Preparing the frog for sleeping

- Frozen frozen rice Please keep refrigerated frozen food until just before the test so that frozen rice frozen can stay dormant.

- Frying pan frozen Put frozen rice frozen in bioware in a Petri dish, and use a tweezers to put frozen rice frozen in a test tube in a test tube containing test solution. Then, the test tube inlet is sealed with a parafilm.

③ Cultivating a frog's sleep under investigation of sunlight

- The test tube with the entrance sealed by the parafilm is placed in the window where the sunlight is best, and it is allowed to stand for a predetermined time to cultivate the frog's mouth.

④ Measuring the root length of frog rice formed by sprouting germination

- It is not necessary to measure the initial root length because the frog's sleeping is germinated and only the final root length is measured.

- Specifically, use a tweezers to take out the frog's gum germinated and measure the root length using a ruler under the background of white paper.

(3) Evaluation of effect of water pollutant samples on root length of frog prepared by germination

Effects of copper sulfate solution, silver nitrate solution, ammonia water, soapy water and salt water on the root length of frog prepared by germination of the submerged paddy were evaluated based on the method of using the aquatic toxicity test kit. FIG. 12 shows changes in root length of frog rice formed by germination of the subspecies according to the addition amounts of copper sulfate solution, silver nitrate solution, ammonia water, soapy water and salt water. In Fig. 12, the control was cultured in the frozen rice frozen state without any sample of the water pollutant added. As shown in FIG. 12, the salt water did not significantly inhibit the root growth of the frog prepared by the germination of the slug compared with other water pollutants. However, when one drop was added, the root growth was inhibited by about 50% based on the control. However, even when the addition amount of the brine was increased, the thallus of the frog formed by the germination of the louse did not whiten or die. In the case of the copper sulfate solution, when 4 drops were added, the roots of the frog did not grow at all and when the 3 drops were added, the fronds of the frog formed by the germination of the frog were whitened. In the case of soapy water, the leaf throat of the frog was formed by germination of the submerged flora even at the lowest concentration of 1 drop, and showed similar toxicity to the copper sulfate solution as a whole. Silver nitrate solution and ammonia water showed the highest toxicity. Specifically, at the lowest concentration of one drop added, all the thallus was whitened and the root did not grow at all. In addition, at the high concentration of 4 drops of silver nitrate solution, whitening of the thallus occurred and the thallus turned slightly black.

Although the water toxicity of a toxic substance identified by a component and a concentration is evaluated in the above example, it can be modified and applied to a water sample including an unknown toxic substance, and the EC ₅₀ value or the NOEC value at that time It will be apparent to those skilled in the art that the effect of unknown toxic substances on water quality can be known to those skilled in the art.

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 (10)

A dormant sleep separated from frogs; A container for cultivating a frog's mouth; Water pollutant samples; And a culture medium for culturing a frozen duck,
Wherein the root length or the throat area of the frog is formed by the germination of the slip as a parameter for evaluation of the water quality toxicity.
2. The kit for diagnosing aquatic toxicity according to claim 1, wherein the sleeping state separated from the frog is provided in a culture medium for the culture of the frog.
3. The method according to claim 2, characterized in that the culture medium for the frog slug used for storing the dormant sleeping state separated from the frog is selected from Steinberg ' s medium or Hoagland ' s medium Water quality toxicity diagnostic kit.
[Claim 3] The water quality toxicity diagnosis kit according to claim 2, wherein the frozen sleeping state separated from the frozen frozen food is stored in a refrigerated condition.
[Claim 7] The water quality toxicity diagnostic kit according to claim 1, wherein the container for culturing the frozen rice is selected from a well plate or a test tube.
6. The kit of claim 5, wherein the container for culturing the frozen rice is a test tube, and the kit further comprises a test tube fixing means.
The water quality toxicity diagnosis kit according to claim 1, wherein the water pollutant sample is composed of at least one member selected from copper sulfate solution, silver nitrate solution, ammonia water, soapy water or salt water.
The water quality toxicity diagnostic kit according to claim 1, wherein the medium for culturing the frozen duck is selected from Steinberg's medium or Hoagland's medium.
[Claim 9] The water quality toxicity diagnostic kit according to claim 8, wherein the Steinberg's medium or Hoagland's medium is provided in the form of a stock solution constituting the same.
10. The kit according to any one of claims 1 to 9, wherein the kit further comprises at least one member selected from the group consisting of tweezers, a syringe, a petri dish, and a coloring paper for pH measurement.

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