KR101136039B1 - Method for evaluating aquatic ecotoxicity using the growth area change rate or fluorescent property of Lemna paucicostata - Google Patents

Abstract

PURPOSE: A method for evaluating water toxicity with high sensitivity is provided to shorten evaluation period and to ensure simplicity. CONSTITUTION: A method for evaluating water toxicity comprises: a step of adding a water sample to a container; a step of adding Lemna perpusilla Torr. to the container; a step of culturing Lemna perpusilla Torr.; a step of measuring growth area change rate of Lemna perpusilla Torr. The water sample is diluted by at least five different concentrations.

Description

Method for evaluating aquatic ecotoxicity using the growth area change rate or fluorescent property of Lemna paucicostata

The present invention relates to a method for evaluating water toxicity, and more particularly, to a method for evaluating water toxicity using the growth rate or fluorescence characteristics of a frog.

Human activities and industry developments produce new and potentially hazardous chemicals and enter aquatic ecosystems. The development of modern chemistry not only contributed to increasing the production of known and useful chemicals, but also created more than 1200 new substances every day. Due to the nature of the aquatic ecosystem, where the movement and transformation of substances occurs rapidly, industrial wastewater and urban sewage containing thousands of chemicals are threatened every year unless efforts are made to assess the risks and take legal action against these toxic substances. You are exposed to the risk of being in danger.

Pollutants that contaminate rivers and lakes include heavy metals from industrial sources, VOCs, agricultural herbicides, pesticides, and nitrogen and phosphorus compounds in the vast domestic wastewater discharged from densely populated cities. Traditionally, physicochemical methods have been used. The physicochemical method is used to quantitatively measure the amount of dissolved oxygen (DO), biochemical oxygen demand (BOD), chemical oxygen demand (COD), turbidity, electrical conductivity and pH, and nitrates and phosphates that cause eutrophication or to analyze heavy metals. Refers to the method of measuring the degree of water pollution. However, this method does not provide information on bioavailability, compound effects (such as synergistic or antagonistic action) and further ecological significance, with the disadvantage of requiring a long time, high cost, and expertise.

In order to overcome these limitations, there is an increasing interest in the method of diagnosis of contamination using indicator organisms such as aquatic microorganisms, algae, aquatic invertebrates, and aquatic plants. In evaluating toxicity, it is understood that the data of toxicity evaluation by indicator organisms are borrowed. The pollution diagnosis method using indicator organisms is a bioindicator concept that directly measures the concentration of pollutants in a living organism or determines whether there is a species by pollution grade in a habitat. It is evolving into a biomonitor method for diagnosing contaminants using The latter method can quantify the risk or assess the potential impact of a single or mixed substance in the environment through various physiological changes in the organism, so that the signs can be identified before environmental pollution becomes widespread. It has the advantage of finding ecological meaning.

In the past, the species used for toxicology studies in most cases were aquatic vertebrates or invertebrates, but recently, the role of aquatic plants in aquatic ecosystems has been highlighted, and interest in aquatic toxicity assessment methods has been increasing. Aquatic plants, the primary producers of aquatic ecosystems, are home to flora and fauna, invertebrates and fish, and have the ability to absorb and purify many organic substances.

Among the aquatic plants, duckweed is recommended as aquatic and phototoxic test models. Plants belonging to the Lemnaceae family are the smallest annual aquatic plants of the monocotyledon. They live in paddy water, lakes, ponds, etc. In particular, the Frogweed is divided into two subfamilies, Lemnoideae and Wolffioideae, and Lemnoideae has two genera, Spirodela and Lemna. It is very useful as a biological material for assaying activity or studying mechanism of action.

To date, the water toxicity evaluation method using the conventional frog rice registered with ISO or OECD consists of cultivating the frog rice in a water sample and measuring the change in the number of colonies. However, the conventional method for evaluating water toxicity using frog rice has to be improved because it takes about 7 days of incubation time and inferior sensitivity to toxic sources in order to secure an effective population change.

The present invention was derived to solve the conventional problems, the present invention is more convenient and the time required for the evaluation by using the growth area change rate or fluorescence characteristics among the characteristics appearing in the growth process of the frog frog rice The aim is to provide a method for assessing water toxicity that is significantly shortened and highly sensitive.

In order to solve the above object of the present invention, the present invention comprises the steps of (a) placing a water sample in a measuring container; (b) injecting Frogweed into a measuring vessel containing a water sample; (c) culturing the little frog rice put in the measuring container; And (d) measuring the rate of change of growth area of the cultivated rice Frogweed rice. In addition, the present invention comprises the steps of (a) placing a water sample in a container for measurement; (b) injecting Frogweed into a measuring vessel containing a water sample; (c) culturing the little frog rice put in the measuring container; And (e) measuring the chlorophyll fluorescence characteristics of the cultured rice paddy frog rice. In this case, the chlorophyll fluorescence characteristics of step (e) include the characteristics detected by fluorescence, specifically, the maximum electron transfer rate (Maximum electron transfer rate).

In addition, the water sample of step (a) preferably comprises dilution with Steinberg artificial medium and gradientd to at least five concentrations. At this time, the water sample is more preferably diluted by a concentration gradient at regular intervals by a half-water dilution method.

In addition, the water sample of step (a) is preferably characterized in that adjusted to the salinity concentration of 0 ~ 2 ‰. In the case of cultivating the frog in the salt concentration range of the above can be obtained more significant growth area change rate or chlorophyll fluorescence characteristics.

In addition, the water sample of step (a) is preferably characterized in that it is adjusted to a pH of 5.5 ~ 9.5. In the case of cultivating the frog frog rice in the above pH range, a more significant growth area change rate or chlorophyll fluorescence characteristics can be obtained.

In addition, the culture conditions of step (c) is characterized in that the light irradiation amount is 30 ~ 150 μmol photon / ㎡ · s and the culture temperature is 20 ~ 30 ℃. In the case of cultivating the little frog in the above culture conditions, a more significant growth area change rate or chlorophyll fluorescence characteristics can be obtained.

The method for evaluating water toxicity according to the present invention uses the rate of change of growth area or chlorophyll fluorescence characteristics (particularly the maximum electron transfer rate) of zombie frog rice, rather than the number of colonies in the number of colonies, as an indicator of water toxicity. It takes about three days and is more than two times shorter than the conventional water toxicity assessment time (about 7 days). In addition, since the growth rate or chlorophyll fluorescence characteristics are used as indicators of water toxicity evaluation rather than the change in the number of colonies, it is simpler to measure and has a high sensitivity to water toxins.

Figure 1 shows the change in growth area according to the dilution multiple of raw water containing toxic substances in cultivated rice frog frog (100% in Figure 1 is raw water itself, 50% is the raw water diluted to 1/2 concentration, 25 % Means dilute raw water to 1/4 concentration, 12.5% dilute raw water to 1/8 concentration, 6.25% dilute raw water to 1/16 concentration, and control to culture with Steinberg artificial medium. One control group).
Figure 2 shows the chlorophyll fluorescence characteristics according to the dilution multiples of raw water containing toxic substances in cultivation of the frog frog (in Figure 2, raw water, dilute the raw water to 1/2 concentration, raw water 1 Dilute to / 4 concentration, dilute raw water to 1/8 concentration, dilute raw water to 1/16 concentration, and control.)
Figure 3 shows the growth rate of the growth area of jokjeokbap in various culture conditions, Figure 4 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of joggapbob in various culture conditions.
Figure 5 shows the growth rate of the growth area of the little green frog rice according to the concentration of a single metal toxic substance, 6 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of the little frog tree according to the concentration of a single metal toxic substance.
Figure 7 shows the growth rate of the growth of the frog tree according to the concentration of volatiles of single volatile organic compounds, Figure 8 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of the rice frog according to the concentration of toxic substances of the single volatile organic compounds will be.
FIG. 9 shows the growth rate of the growth of the black-and-white frog rice according to the concentration of DCMU [3- (3,4-dichlorophenyl) -1,1-dimethylurea] which is a single herbicide toxic substance, and FIG. 10 shows DCMU [3 which is a single herbicide toxic substance. It shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of jog frog rice with concentration of-(3,4-dichlorophenyl) -1,1-dimethylurea].

The present invention relates to a method for evaluating water toxicity, which is simple for a water sample, has a short discrimination time, and has a high sensitivity to response to toxic substances (metal toxic substances, volatile organic compound toxic substances, herbicides, insecticides, etc.). Water toxicity evaluation method according to the water toxicity evaluation method of the present invention comprises the steps of placing a water sample in a measuring container; Injecting a little frog into a measuring container containing a water sample; Culturing the little frog rice put in a measuring container; And measuring a growth area change rate or chlorophyll fluorescence characteristics of the cultured rice paddy frog rice. Hereinafter, the water toxicity evaluation method according to the present invention will be described by dividing by component.

Waterbody  Sample

A water body sample refers to water in which water occupies a major volume, and the water body sample according to the present invention is collected from seawater, rivers, lakes, wastewater, effluent, sewage, sludge effluent, soil effluent, and sediment effluent. One sample is included.

The original water sample (hereinafter, raw water) is preferably diluted with artificial medium, gradientd to at least five or more concentrations, and placed in a measuring vessel. The dilution method used is not particularly limited, for example half dilution method [100% (raw water itself), 50% (diluted to 1/2 of raw water), 25% (diluted to 1/4 concentration of raw water) ), 12.5% (diluted to 1/8 concentration of raw water), 6.25% (diluted to 1/16 concentration of raw water)]. The artificial medium for diluting raw water does not include a toxic substance against the rice frog, and if the type is compatible with the culture of rice frog, the type is not limited to a large, and specifically, there is a Steinberg artificial medium.

The aqueous sample diluted with raw water and artificial medium is adjusted to have a salt concentration in the range of 0-5 ‰, preferably 0-2 ‰, for smooth cultivation of Frogweed before being placed in the measuring vessel and the pH is 4.5- 9.5, preferably 5.5 to 7.5.

In the present invention, in the case of cultivating the rice frog frogs with four or more diluted water samples in addition to the raw water whose salt concentration and pH are adjusted as the culture medium, the growth rate or the chlorophyll fluorescence characteristics (particularly maximum Electron transfer rate), and based on this, when assessing the water toxicity of a water sample, the exact half-effective concentration (50% reduction in growth rate or growth rate of chlorophyll fluorescence in the control frogs without toxic substances) The concentration of toxins with area change rate or chlorophyll fluorescence, expressed as a specific concentration for a single toxic substance and as a dilution rate for unknown raw water; Half maximal effective concentration (EC ₅₀ ) to no effect concentration (including toxic substances) Changes in Growth Area or Chlorophyll Fluorescence of Rice Frog Rice And significant as the concentration of the toxic substance that the difference is maintained at a level that for a single display to a particular toxic substance concentration, and the case of the unknown raw water indicated by dilution; can be evaluated in the No Observed Effect Concentration, NOEC).

Measuring vessel

The measuring container accommodates the water sample and the rice frog, and cultivates the rice frog, the form is not limited to a large, for example, there is a well plate (Well plate). Well plate is preferably composed of at least six wells (Well), one well is added to the artificial medium containing no toxic substances as a control to cultivate little frog rice, the remaining five wells dilute raw water and raw water Add four water samples, and incubate the frog frog rice.

Little Frog Rice

The moth frog rice is a perennial plant with a monocotyledonous plant Chunnamseongmok frog rice. It is aquatic plant that floats on water. The present invention utilizes the growth rate or chlorophyll fluorescence properties (particularly the maximum electron transfer rate) of the growth areas of aquatic plants belonging to aquatic plants, more specifically, as a biomarker for evaluation of water toxicity. Since the growth area is visually changed within 72 hours, it can be observed visually, and the maximum electron transfer rate, which is the chlorophyll fluorescence property of the rice frog, can be measured quickly within 5 minutes. As the water toxicity increases during the same time, the growth area of the Frogweed rice tends to decrease gradually, and the chlorophyll fluorescence properties tend to change from purple to green, yellow and red. Therefore, it is possible to quickly and easily determine the quality of water readings when using the growth rate or chlorophyll fluorescence characteristics of juvenile frog rice as a standard for evaluating the water toxicity.In particular, the chlorophyll fluorescence characteristics (particularly the maximum electron transfer rate) of juvenile frog rice are indicators of water toxicity evaluation. When used as, the response sensitivity to toxic substances can be greatly improved than when using the change rate of growth area, and at the same time, there is an advantage that visual display such as fluorescence is possible.

Little frog  culture

The light irradiation amount for growth of Lemna paucicostata belonging to an aquatic plant is preferably 30 to 150 mol mol of photon / m 2 s and the culture temperature is preferably 20 to 30 ° C. In addition, the pH of the culture medium is 4.5 to 9.5, preferably 5.5 to 7.5, and the salt concentration of the culture solution is 0 to 5 ‰, preferably 0 to 2 ‰. In the culture conditions and culture medium characteristics of the above cultures when cultivation of the little frog, more significant growth area change rate or chlorophyll fluorescence characteristics can be obtained.

Little frog  growth

 Area change rate and chlorophyll fluorescence characteristics

After 72 hours of cultivation of aquatic plants belonging to aquatic plants, visible area changes occurred according to growth, and the rate of change in the growth area of cultivated rice frogs was increased after the completion of the cultivation of the area of the cultivated rice plants. It can be calculated as a ratio and can be represented by various equations as follows. At this time, if the toxicity of the water sample constituting the culture solution is so strong that whitening or whitening of yellow frog frog turns white, it is excluded from the measurement of area.

In Equation 1, A represents the area of Jogakbabbap at the time of the first culture, and B represents the area of Jogakbabbap after completion of the culture.

Equation 1 is obtained by dividing the difference in the area at the time of the initial culture from the area after the completion of the culture by the total culture days, and multiplying by 100, and the value is treated the same as the growth area change rate. Toxicity of the water sample can be evaluated by comparing the growth area increase or growth area change rate value calculated by Equation 1 with the growth area increase or growth area change rate value of the frogs cultured in the control group containing no toxic substances. At this time, the growth area measurement of the frog frog rice is measured using an image analysis device.

On the other hand, the chlorophyll fluorescence characteristics measurement is carried out using a fluorescence measuring device after adapting the forged frog rice cultured for 72 hours in the dark state for about 15 minutes. The fluorescence measurement device measures chlorophyll fluorescence properties, and the final value may be displayed in the form of a visible value such as fluorescence color, and may also be expressed by a value such as an electron transfer rate (ETR). For example, in the case of measuring the change in electron transfer rate due to the increase in the amount of light irradiation due to the photosynthesis of plants using a fluorescence measuring device, the data obtained by the fluorescence measuring device is entered into a statistical program to maximize the maximum electron transfer rate, ETRmax) value. In addition, the value obtained by the fluorescence measuring device can be visually measured when converted into color. This is an image of the fluorescence value, which indicates that the health state of Jog Frog is best as 1, and the health state of Narrow Frog is the worst. Is expressed as 0 and then the above value is converted to color. In this case, 1 is converted into purple and 0 is converted into red. The color converted from 1 to 0 changes from purple to green to yellow to red. The chlorophyll fluorescence measurement device is designed to detect changes in photosynthetic activity, and is known as a device capable of detecting the photosynthetic mechanism activity of plants under environmental stress in a quick and non-destructive manner.

Figure 1 shows the change in growth area according to the dilution multiple of raw water containing toxic substances in cultivated rice frog frog (100% in Figure 1 is raw water itself, 50% is the raw water diluted to 1/2 concentration, 25 % Means dilute raw water to 1/4 concentration, 12.5% dilute raw water to 1/8 concentration, 6.25% dilute raw water to 1/16 concentration, and control to culture with Steinberg artificial medium. One control group). As shown in FIG. 1, the growth of the little frog increases as the concentration of toxic substances increases.

Figure 2 shows the chlorophyll fluorescence characteristics according to the dilution multiples of raw water containing toxic substances in cultivation of the frog frog (in Figure 2, raw water, dilute the raw water to 1/2 concentration, raw water 1 Dilution to / 4 concentration, dilution of raw water to 1/8 concentration, dilution of raw water to 1/16 concentration, and control group.) In Fig. 2, the vertical axis shows the increase of incubation time from the top to the bottom. it means. As shown in FIG. 2, the fluorescence image of zombie frog rice cultured in a control group containing no toxic substance shows violet color, and the fluorescence image of zombie frog rice incubated in raw water with the highest concentration of toxic substance is close to red. The fluorescence image shown in Figure 2 is obtained from the chlorophyll fluorescence measurement device (0 at the bottom of Figure 2 indicates the worst health condition of the frog frog, specifically, when the concentration of toxic substances in the culture environment is relatively high Also, 1 means that the health status of the frog is the best, and specifically, when the concentration of toxic substances in the culture environment is not present or low enough to not affect the growth of the frog.) It corresponds to a color bar and converts it to color.

Assessment of Water Toxicity

The water toxicity of the water sample was determined by the change in the growth area of the green frog rice cultured in the raw water and the diluted water sample or the chlorophyll fluorescence characteristics (especially the maximum electron transfer rate). It is evaluated in comparison with chlorophyll fluorescence properties (particularly the maximum electron transfer rate) and is specifically expressed as a half maximal effective concentration (EC ₅₀ ) to no observable effect concentration (NOEC) value. In this case, when using the chlorophyll fluorescence characteristics (particularly, the maximum electron transfer rate) of the jog frog rice as a standard for evaluating the water toxicity, the response sensitivity to the toxic substances is improved than the use of the change in the growth area of the jog frog rice, so that the water toxicity can be evaluated more accurately. .

The half maximal effective concentration (EC ₅₀ ) is the change in growth area or chlorophyll fluorescence properties (particularly the maximum electron transfer rate) of the growth area change or chlorophyll fluorescence properties (particularly the maximum electron transfer rate) in a control that does not contain toxic substances. Implying a concentration of water samples effective at 50% reduction), for water samples containing a single toxic substance, expressed as a specific concentration of a single toxic substance, and dilution of the raw water for unknown raw water containing multiple toxic substances. It is expressed as a rate. The effectless concentration is such that the growth area change or chlorophyll fluorescence properties (particularly the maximum electron transfer rate) are not significantly different from the growth area change rate or chlorophyll fluorescence properties (particularly the maximum electron transfer rate) in the control group without toxic substances. Means the concentration of a water sample that is effective to maintain, such as a half effective concentration, for a water sample containing a single toxic substance at a specific concentration and for an unknown raw water containing a large number of toxic substances at a dilution rate. On the other hand, since the toxicity of the actual water sample is relatively small as the size and size of the half-water effective concentration are relatively small, the Toxic Unit (TU) can be expressed as follows to convert the half-water effective concentration into actual water sample toxicity. Can be.

TU = 100 / half effective concentration (%)

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

The water toxicity evaluation method of the present invention may be carried out by a water toxicity evaluation system including a water sample, a rice frog, and an incubator (measurement container), and further, by measuring the growth area to chlorophyll fluorescence characteristics of the cultured rice frog It may be configured as a ubiquitous system (remote coordination system) that transmits it, returns it and returns the result. Toxic substances that can be evaluated by the water toxicity evaluation method of the present invention include silver (Ag; Silver), arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), Toxic substances in metals such as copper (Cu), mercury (Hg), mercury (Ni) and nickel (Ni; Nickel), acetone, acetic acid, chloroform, dimethylsulphate (DMSO) toxic substances of volatile organic compounds (VOC) such as sulfoxide, formalin, phenol, phenol, toluene, xylene, and the like, herbicides (DCMU), and the like.

The water toxicity evaluation method of the present invention can also be usefully used to quickly determine the sludge dilution drainage to be taken so as not to adversely affect the ecosystem before throwing sewage and wastewater sludge. The method of the present invention cannot detect unknown toxic substances when introduced into water bodies among the problems inherent in water pollution measurement methods that rely on the conventional chemical analysis method, and furthermore, the actual ecosystem only has the result value by chemical analysis. It is a practical technique that makes up for the drawback that there is no prediction about the effects of water pollution.

Another aspect of the present invention relates to a water toxicity test kit capable of implementing the water toxicity test method according to the present invention, wherein the water toxicity test kit according to the present invention is frog rice; A measuring container for injecting the water sample and the spores of the seaweed and culturing the spores of the seaweed; And artificial media to dilute the water sample; It includes. In addition, the water toxicity test kit according to the present invention is silver (Ag; Silver), arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Metals such as copper, mercury (Hg) and nickel (Ni), acetone, acetic acid, chloroform, dimethyl sulfoxide (DMSO), and formalin any one selected from the group consisting of toxic substances of volatile organic compounds (VOCs) such as formulaal, phenol, toluene, xylene, and herbicide (DCMU) It may further comprise a standard toxic solution prepared by dissolving the toxic material in the artificial medium, which tests the condition of the frog frog rice, i.e. the response sensitivity to the toxic material, before assessing the water toxicity of the water sample. It can be used to Specifically, when the standard toxic substance solution according to the present invention is a solution containing copper as a toxic substance, in advance the data on the growth area change rate of chlorophyll rice and chlorophyll fluorescence characteristics (especially the maximum electron transfer rate) in various copper solutions and controls Can be obtained and provided with the water toxicity assessment kit. The user measures the growth rate of chlorophyll rice to the copper solution to chlorophyll fluorescence characteristics (especially the maximum electron transfer rate) and compares it with the data provided in advance to determine whether or not the frog will respond normally to toxic substances, and to a certain extent If there is a response sensitivity (eg, 80% or more of the original response sensitivity), an assessment of water toxicity can be performed on an unknown body of water sample.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only intended to clarify the contents of the present invention and do not limit the protection scope of the present invention.

One. Little frog  Establish optimal culture conditions

(1) pretreatment culture of rice frog

Lemna paucicostata ( Lmna paucicostata ) was placed in a 25 cm wide, 10 cm long, 15 cm high water bath containing 1.5 L of artificial medium and transferred to the incubator under a continuous light condition of 20-30 μmol photon / ㎡? S light dose and The stop culture was carried out under the temperature condition of 15 ~ 20 ℃ and the pH was adjusted to 6.5 or more. At this time, the artificial medium was replaced with a full amount every 7 days.

Table 1 shows the components and concentrations included in the Steinberg artificial medium which is the artificial medium used in the embodiment of the present invention. As shown in Table 1, the artificial medium includes a total of five stock solutions, and each stock solution is composed of a solution containing inorganic to organic components required for cultivation of frogs.

Stock solution division Constituting Stock Solution
Type of ingredient Sphere in Stock Solution
Concentration of the active ingredient (g / ℓ) Per 1 liter of artificial medium
Tok solution occupies
Volume (ml) 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 BO₃ 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) Cultivation of Little Frog Rice

After measuring the area at the time of initial culture, the lid of the 24-well plate containing the frog frog rice was closed and transferred to the incubator, and cultured under various conditions of light irradiation, pH of the culture solution, salt concentration of the culture solution, and culture temperature conditions. After 72 hours of incubation, the growth area of the Frogweed was measured in each well using an image analyzer (Moticam 2000, Motic Instruments Inc., BC, Canada). Figure 3 shows the growth rate of the growth area of jokjeokbap in various culture conditions, Figure 4 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of joggapbob in various culture conditions.

Looking at the optimum culture conditions of the rice frog, which maximizes the change in the growth area and the chlorophyll fluorescence properties (particularly the maximum electron transfer rate) of the rice frog, the culture medium has a salt concentration of 0 to 2 ‰, preferably 0 to 1 ‰, and a pH of 4.5. ˜9.5, preferably 5.5 to 7.5, the culture conditions were 30 to 150 μmol photon / m 2 s of light irradiation, preferably 90 to 120 μmol photon / m 2 s, and the culture temperature was 20 to 30 ° C. It was.

2. Evaluation of Water Toxicity of Single Metal Toxic Solution

(1) Preparation of single metal toxic substance solution

As a single metal toxic substance, silver (initial concentration 8 mg / L), mercury (initial concentration 8 mg / L), cadmium (initial concentration 8 mg / L), cobalt (initial concentration 8 mg / L), and chromium (initial concentration) 8 mg / L), copper (initial concentration 8 mg / L), mercury (initial concentration 8 mg / L), nickel (initial concentration 8 mg / L) solution of toxic substances (pH adjusted to 6.5), and Dilution with half dilution using a Steinberg artificial medium (pH adjusted to 6.5) produces a toxic substance solution diluted to 50%, 25%, 12.5%, 6.25% of the initial concentration and the water toxicity of these toxic substance solutions Evaluated. As a control solution, Steinberg artificial medium (pH adjusted to 6.5) containing no toxic substances was used.

(2) Cultivation of Little Frog Rice

The control solution and the toxic substance solution were placed in the wells of the 24-well plate, and the green frog rice ( Lemna pauaicostata ) was added thereto , and the 24-well plate was transferred to the incubator. Thereafter, the culture was incubated for 72 hours at a temperature of 90 ℃ mol photon / m 2 s light irradiation and 25 ℃.

(3) Measurement of growth area change to chlorophyll fluorescence characteristics (maximum electron transfer rate) of rice frog frog

Using an image analyzer (Moticam 2000, Motic Instruments Inc., BC, Canada), the growth area of the frog was measured during the initial culture and after 72 hours of incubation. In addition, chlorophyll fluorescence characteristics (maximum electron transfer rate) was measured by using a fluorescence measuring device (Imaging pulse-amplitude-modulated fluorometer; Imaging PAM; Walz Co., Erlangen, Germany) after adapting the frog frog rice for 15 minutes in the dark state .

Figure 5 shows the growth rate of the growth area of the little green frog rice according to the concentration of a single metal toxic substance, 6 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of the little frog tree according to the concentration of a single metal toxic substance. In FIG. 5, the RGR (relative growth area change rate (% / d)) of the vertical axis is a value calculated by Equation 1 described above, and in FIG. 6, the ETRmax of the vertical axis represents the maximum electron transfer rate. As shown in FIGS. 5 to 6, the growth rate to chlorophyll fluorescence characteristics (maximum electron transfer rate) of the frog frog rice showed a linear relationship with the concentration of metal toxic substances in a range.

(4) Water Toxicity Determination of Single Metallic Toxic Substances Based on Growth Rate of Chromium Frog Rice to Chlorophyll Fluorescence Characteristics (Maximum Electron Transfer Rate)

Based on the growth area change rate of chlorophyll rice shown in FIGS. 5 to 6 to the chlorophyll fluorescence characteristics (maximum electron transfer rate), the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single metal toxic substance was calculated. Based on the water quality toxicity evaluation criteria, the sensitivity of the reaction to a single metal toxic substance was determined based on the change rate of growth area and the chlorophyll fluorescence characteristics (maximum electron transfer rate) of the rice frog. EC ₅₀ values were calculated using point estimation techniques, and NOEC values were calculated using hypothesis testing methods such as Dunnett's procedure. Table 2 shows the half-effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single metal toxic substance based on the change rate of growth area of chlorophyll rice to chlorophyll fluorescence characteristics (maximum electron transfer rate) as the criteria for evaluation of water toxicity.

Metal Toxic Substances Growth area change rate indication Based on maximum electron transfer rate NOEC (mg / L) EC ₅₀ (mg / L) NOEC (mg / L) EC50 (mg / L) As 2 4.1533 2 3.79 Ag <0.5 0.4594 <0.5 0.25 CD 2 3.1106 2 3.78 Co One 2.6483 One 1.97 Cr <0.5 0.9516 <0.5 1.08 Cu <0.5 3.1345 <0.5 2.84 Hg <0.5 0.3798 <0.5 0.3 Ni 2 2.9460 2 3.27

As shown in Table 2, when the chlorophyll fluorescence characteristics (maximum electron transfer rate) were used as the evaluation criteria for water toxicity, the response sensitivity to the single metal toxic substance was higher than when the area growth rate was used as the evaluation criteria for water toxicity.

3. Single Volatility Organic Compounds  Water Toxicity Evaluation of Toxic Solution

(1) Preparation of a single volatile organic compound toxic substance solution

Single volatile organic compounds toxic substances, acetone (initial concentration: 80 ml / L), acetic acid (initial concentration: 0.05 ml / L), chloroform (initial concentration: 1 ml / L), dimethyl sulfur oxide (initial concentration: 40 ml / L) PH solution containing formalin (initial concentration 0.005 mL / L), phenol (initial concentration 0.5 mL / L), toluene (initial concentration 10 mL / L), xylene (initial concentration 10 mL / L) 6.5) and Steinberg artificial media (pH is adjusted to 6.5) to dilute by half dilution to prepare a solution of toxic substances diluted to 50%, 25%, 12.5%, 6.25% of initial concentration and these toxicities. The water toxicity of the material solution was evaluated. As a control solution, Steinberg artificial medium (pH adjusted to 6.5) containing no toxic substances was used.

(2) Cultivation of Little Frog Rice

The control solution and the toxic substance solution were placed in the wells of the 24-well plate, and the green frog rice ( Lemna pauaicostata ) was added thereto , and the 24-well plate was transferred to the incubator. Thereafter, the culture was incubated for 72 hours at a temperature of 90 ℃ mol photon / m 2 s light irradiation state and 25 ℃.

(3) Measurement of growth area change to chlorophyll fluorescence characteristics (maximum electron transfer rate) of rice frog frog

Using an image analyzer (Moticam 2000, Motic Instruments Inc., BC, Canada), the growth area of the frog was measured during the initial culture and after 72 hours of incubation. In addition, chlorophyll fluorescence characteristics (maximum electron transfer rate) was measured by using a fluorescence measuring device (Imaging pulse-amplitude-modulated fluorometer; Imaging PAM; Walz Co., Erlangen, Germany) after adapting the frog frog rice for 15 minutes in the dark state .

Figure 7 shows the growth rate of the growth of the frog tree according to the concentration of volatiles of single volatile organic compounds, Figure 8 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of the rice frog according to the concentration of toxic substances of the single volatile organic compounds will be. As shown in FIG. 7 to FIG. 8, the growth rate to chlorophyll fluorescence characteristics (maximum electron transfer rate) of the green frog rice showed a linear relationship with the concentration of volatile organic compounds toxic substances in a certain range.

(4) Water Toxicity Determination of Single Volatile Organic Compounds Toxic Substances Based on Growth Rate and Chlorophyll Fluorescence Characteristics (Maximum Electron Transfer Rate)

Based on the growth area change rate of chlorophyll rice shown in FIGS. 7 to 8 to chlorophyll fluorescence characteristics (maximum electron transfer rate), the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single volatile organic compound toxic substance was calculated. On the basis of the results, the sensitivity of the reaction to the single volatile organic compounds toxic substances was determined when the water quality toxicity criteria were determined as the growth rate of the frogs and the chlorophyll fluorescence characteristics (maximum electron transfer rate). EC ₅₀ values were calculated using point estimation techniques, and NOEC values were calculated using hypothesis testing methods such as Dunnett's procedure. Table 3 shows the half-effective concentration (EC ₅₀ ) to no-effect concentration (NOEC) of single volatile organic compounds toxic substances based on the change rate of growth area of chlorophyll rice to chlorophyll fluorescence characteristics (maximum electron transfer rate). It is shown.

Volatile Organic Compounds Toxic Substances Growth area change rate indication Based on maximum electron transfer rate NOEC (mg / L) EC ₅₀ (mg / L) NOEC (mg / L) EC50 (mg / L) Acetone <5 7.5049 <5 4.772 Acetic acid 0.00625 0.0259 0.025 0.035 Chlororoform 0.03125 0.3322 <0.0625 0.0543 DMSO 2.5 15.295 10 18.920 Formalin <0.00125 0.0007 0.000625 0.00077 Phenol 0.03125 0.0797 <0.03125 0.0324 Toluene 5 6.3152 0.625 > 10 Xylene 5 7.5797 <5 7.928

As shown in Table 3, when the chlorophyll fluorescence characteristics (maximum electron transfer rate) were used as the evaluation criteria for water toxicity, the reaction sensitivity to the single volatile organic compounds toxic substances was higher than when the area growth rate was used as the evaluation criteria for water toxicity. In phenol the reaction sensitivity difference was very noticeable.

4. Evaluation of Water Toxicity of Single Herbicide Toxic Solution

(1) Preparation of a Single Herbicide Toxic Solution

Toxic solution containing DCMU [3- (3,4-dichlorophenyl) -1,1-dimethylurea] (initial concentration 0.2 mg / L) as a single herbicide toxicant (pH was adjusted to 6.5 and DCMU dissolution was used with Acetone) And dilution by half dilution using Steinberg artificial media (pH adjusted to 6.5) to prepare toxic substance solutions diluted to 50%, 25%, 12.5%, 6.25% of initial concentration and water toxicity of these toxic substance solutions. Was evaluated. As a control solution, Steinberg artificial medium (pH adjusted to 6.5) containing no toxic substances was used.

(2) Cultivation of Little Frog Rice

The control solution and the toxic substance solution were placed in the wells of the 24-well plate, and the green frog rice ( Lemna pauaicostata ) was added thereto , and the 24-well plate was transferred to the incubator. Thereafter, the culture was incubated for 72 hours at a temperature of 90 ℃ mol photon / m 2 s light irradiation and 25 ℃.

(3) Measurement of growth area change to chlorophyll fluorescence characteristics (maximum electron transfer rate) of rice frog frog

Using an image analyzer (Moticam 2000, Motic Instruments Inc., BC, Canada), the growth area of the frog was measured during the initial culture and after 72 hours of incubation. In addition, chlorophyll fluorescence characteristics (maximum electron transfer rate) was measured by using a fluorescence measuring device (Imaging pulse-amplitude-modulated fluorometer; Imaging PAM; Walz Co., Erlangen, Germany) after adapting the frog frog rice for 15 minutes in the dark state .

Figure 9 shows the growth rate of the growth area of jokjeokbap in accordance with the concentration of a single herbicide toxic substance, Figure 10 shows the chlorophyll fluorescence characteristics (maximum electron transfer rate) of zombie frog rice in accordance with the concentration of a single herbicide toxicant. As shown in FIGS. 9 to 10, the growth rate and chlorophyll fluorescence characteristics (maximum electron transfer rate) of the frog were shown in a linear relationship with the concentrations of herbicide toxic substances in a certain range.

(4) Determination of Water Toxicity of Single-Product Toxic Substances Based on the Growth Rate of Chromium Frog Rice to Chlorophyll Fluorescence Characteristics (Maximum Electron Transfer Rate)

Based on the growth area change rate of chlorophyll rice shown in FIGS. 9 to 10 to the chlorophyll fluorescence characteristics (maximum electron transfer rate), the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single product toxic substance was calculated. Based on the results, the sensitivity of the response to a single toxic substance was determined when the evaluation criteria of water quality were based on the growth rate of chlorophyll rice and the chlorophyll fluorescence characteristics (maximum electron transfer rate). EC ₅₀ values were calculated using point estimation techniques, and NOEC values were calculated using hypothesis testing methods such as Dunnett's procedure. Table 4 shows the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single herbicide toxic substance based on the change rate of growth area of chlorophyll rice to chlorophyll fluorescence characteristics (maximum electron transfer rate) as the criteria for evaluation of water toxicity.

Herbicide toxic substances Growth area change rate indication Based on maximum electron transfer rate NOEC (mg / L) EC ₅₀ (mg / L) NOEC (mg / L) EC50 (mg / L) DCMU 0.0125 0.0295 0.0125 0.0082

As shown in Table 4, when the chlorophyll fluorescence characteristics (maximum electron transfer rate) were used as the water toxicity evaluation criteria, the response sensitivity to a single herbicide toxic substance was about 4 times higher than when the growth rate was based on the water toxicity evaluation criteria.

In the above examples, although the toxicity of the water due to the specific toxic substance and the concentration of the substance was evaluated, it may be modified and carried out on a water sample containing an unknown toxic substance, and then the EC ₅₀ value or the NOEC value is used. It will be apparent to those skilled in the art that the effects of unknown toxic substances on water quality can be grasped. In addition, it is apparent to those skilled in the art that the technical idea of using the growth area itself of the rice frog is the equivalent range of the technical idea using the rate of change of the growth area of the rice frog. In the case of using the growth area of the frog itself, the growth area in the control culture and the method of using the growth area ratio in the raw water (including dilute raw water) culture.

The numbers shown at the bottom of FIG. 2 are numerical values obtained from the fluorescence spectrometer according to the health status of the little frog, 0 to 1 are numerical values of the fluorescence characteristics of the little frog, and the health status of the little frog is increased as the number increases. Good means 0, where the health status of the frog is the worst (specifically, the concentration of toxic substances in the culture environment is relatively high), 1 means the health status of the frog is the best (specifically the culture environment) The fluorescence property is when the concentration of toxic substance is not present or low enough to not affect the growth of Frogweed. In FIG. 3 to FIG. 10, the RGR (growth area change rate,% / d) of the vertical axis refers to the growth area change rate calculated by Equation 1, and in FIG. 6, the ETRmax of the vertical axis refers to the maximum electron transfer rate.

Claims (8)

delete (a) placing a water sample in a measuring vessel;
(b) injecting Frogweed into a measuring vessel containing a water sample;
(c) culturing the little frog rice put in the measuring container; And
(e) measuring the chlorophyll fluorescence characteristics of the cultured rice paddy frog; water quality toxicity evaluation method comprising a.
The method of claim 2, wherein the chlorophyll fluorescence property of step (e) is a maximum electron transfer rate.
4. The method of claim 2 or 3, wherein the water sample of step (a) is diluted with Steinberg artificial medium and gradientd to at least five or more concentrations.
5. The method of claim 4, wherein the water sample is diluted by half dilution.
5. The method of claim 4, wherein the water sample of step (a) is adjusted to a salinity concentration of 0 to 2 ‰.
7. The method of claim 6, wherein the water sample of step (a) is adjusted to a pH of 5.5 to 7.5.
8. The method of claim 7, wherein the culture conditions of step (c) have a light irradiation amount of 30-150 μmol photon / m 2 · s and a culture temperature of 20-30 ° C.

Images